**Meet the editor**

Professor Kaneez Fatima Shad is an Australian neuroscientist, and she did her PhD degree studies in 1994 from the School of Physiology and Pharmacology, Faculty of Medicine, UNSW. She is an academic, with more than 35 years of experience in teaching Medical and Health Sciences in various universities including Australia, the USA, UAE, Bahrain, Pakistan, and Brunei. She wrote 50

peer-reviewed papers, 3 text book chapters, and 2 invited reviews along with more than 90 international conference abstracts mostly as a result of her 22 successful research grants and 35 postgraduate students. She also edited 3 books. Her research passion is to identify peripheral markers for cerebro and cardiovascular disorders by using skills such as patch clamping, tissue culture, MRI, platelet electro-pharmacology, as well as molecular biological and biochemical techniques.

## Contents

## **Preface XI**


**X** Contents

#### **Section 3 Metabolism 171**

Chapter 7 **Application of 5-HT-SO4 in Biomarker Research 173** Raimond Lozda

## Chapter 8 **Energy Homeostasis by the Peripheral Serotonergic System 185** Hitoshi Watanabe, Michael Rose, Yoshinori Kanayama, Hitoshi Shirakawa and Hisashi Aso

Chapter 9 **Serotonin Effects on Expression of the LDL Receptor Family Member LR11 and 7-Ketocholesterol–Induced Apoptosis in Human Vascular Smooth Muscle Cells 203** Daiji Nagayama and Ichiro Tatsuno

### **Section 4 Systems 217**


## Preface

**Section 3 Metabolism 171**

**VI** Contents

**Section 4 Systems 217**

Raimond Lozda

Chapter 8 **Energy Homeostasis by the Peripheral Serotonergic System 185**

Shirakawa and Hisashi Aso

Chapter 7 **Application of 5-HT-SO4 in Biomarker Research 173**

Hitoshi Watanabe, Michael Rose, Yoshinori Kanayama, Hitoshi

**Member LR11 and 7-Ketocholesterol–Induced Apoptosis in**

Jolanta Dorszewska, Jolanta Florczak-Wyspianska, Marta Kowalska,

Elmina Mammadova-Bach, Maximilian Mauler, Attila Braun and

Marcin Stanski, Alicja Kowalewska and Wojciech Kozubski

Chapter 11 **The Role of Serotonin in Aggression and Impulsiveness 241** Fatih Hilmi Çetin, Yasemin Taş Torun and Esra Güney

Chapter 12 **Immuno-Thrombotic Effects of Platelet Serotonin 253**

Chapter 13 **Production and Function of Serotonin in Cardiac Cells 271** Joachim Neumann, Britt Hofmann and Ulrich Gergs

Chapter 9 **Serotonin Effects on Expression of the LDL Receptor Family**

**Human Vascular Smooth Muscle Cells 203**

Daiji Nagayama and Ichiro Tatsuno

Chapter 10 **Serotonin in Neurological Diseases 219**

Daniel Duerschmied

In this book, we are fortunate to have chapters written by the experts in their field covering from phylogenic distribution, types of receptors, to metabolism of serotonin and its effect on different systems ranging from neurological, immunological, to cardiovascular system.

We divided this book into four sections according to the contents of the chapters included. Under *Phylogeny*, we have three very interesting chapters encompassing from the origin of serotonin to its function in bivalves, followed by the three chapters in the *Types* section, in which two very upright papers by different authors focusing on 5-HT1A receptor, which is the most widely spread serotonin receptor controlling almost all systems including endo‐ crine and neuromodulation, and one paper exquisitely describe the influence of ligand-gat‐ ed serotonin receptor 5-HT3R in carving brain. In *Metabolism* section, three decent papers discuss the outcomes of different serotonergic metabolic products on both brain and body. In the *System* section, we selected four brilliant papers on the effects of serotonin on neuro‐ logical, immunological and cardiovascular systems.

This book is for a wide range of audience and we tried to include a variety of interesting chapters to cater all types of scientific taste buds.

#### **Professor Kaneez Fatima Shad**

Chronic Disease Solutions Team School of Life Sciences Faculty of Science Centre for Health Technologies University of Technology Sydney, Australia

**Section 1**

## **Phylogeny**

## **Introductory Chapter: Serotonin - The Most Ancient Neurotransmitter, Hormone and Trophic Factor**

Kaneez Fatima Shad

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70146

## **1. Introduction**

This book, as its title indicates, is about "Serotonin: a chemical messenger for all living cells", which is not only present in every tissue of human body but also detected in all aerobic organism including plant and bacteria. In humans, serotonin acts as a trophic factor starts soon after conception and is directly related to the production of serotonin by mother's enterochromaffin cells in the gut and its transfer into the platelets in mother's blood. At the same time from a very early stage of gestation, fetus also starts synthesizing its own serotonin in a special group of nuclei of the midbrain. Soon after serotonergic neurons distribute it throughout the brain and body of the fetus in turn increases division, migration and maturation of both central and peripheral tissues.

Serotonin, which is also known as 5-hydroxytryptamine (5-HT), acts both as neurotransmitter and hormone and is mainly found in the brain, bowels and blood platelets. In 1948, Rapport identified a serum agent that affected vascular tone and called it serotonin. Later, in 1950, he identified chemical composition of serotonin and named it 5-hydroxytryptamine. Serotonin, a biogenic amine is produced by the conversion of amino acid tryptophan in the presence of an enzyme tryptophan hydroxylase (TPH) that exists both in brain and bowel. Till now this enzyme is of two types: TPH1 (found in peripheral organs and CNS) and TPH2 (present only in the brain).

Enteroendocrine cells derived 5-HT acts also as a hormone, which perform multiple functions including inhibition of osteoblast proliferation and promotion of hepatic regeneration. The chemical formula of 5-HT is N2 OC10H12, such that 15.8970% nitrogen, 9.0793% oxygen, 68.1598% carbon and 6.8638% hydrogen.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Effect of serotonin on brain and body**

In the central nervous system, serotonin is synthesized by the neurons of the Raphe nuclei from the amino acid tryptophan through a short metabolic pathway that contains two enzymes: tryptophan hydroxylase and amino acid decarboxylase, and is distributed along the length of the brainstem. Serotonin is released from the varicosities along the axon into the extra-neuronal space this provides a larger area for serotonin to activate its receptors that exist on the dendrites, the cell bodies and the presynaptic terminals of the adjacent neurons. Thus serotonin not only stimulates postsynaptic serotonin receptors but also present on the extrasynaptic neurons.

Thus, it is not a surprise to find significant role of serotonin in the modulation of many behavioral and psychotic disorders such as mood, sleep, appetite, vomiting, sexuality, memory, learning, temperature, cardiovascular and endocrine regulation.

Both high and low levels of serotonin have harmful effect. High serotonin levels cause severe toxicity termed as "serotonin syndrome", which can be fatal in some cases, whereas low levels of serotonin have been associated with migraines, bipolar disorders, apathy, fear, feelings of worthlessness, insomnia, fatigue, anxiety and depression. These pathologies may be explained by the fact that 10% of large dorsal raphe nuclei (largest source of serotonin synthesis) are projected to amygdala and other medium raphe nuclei project to caudate, putamen and olfactory bulb. It is important to mention here that serotonin is required for the metabolism of stress hormone.

Scientific evidence confirms that genetic polymorphisms in the enzyme tryptophan hydroxylase in both TPH1 and TPH2 forms can affect the susceptibility to depression and anxiety. Furthermore, ovarian hormones can affect the expression of tryptophan hydroxylase, triggering postnatal depression and premenstrual stress syndrome, and expression of abnormal serotonergic neurons in infants may lead to high possibility of having sudden infant death syndrome (SIDS). Serotonin is also involved in the regeneration of organs such as liver and bone and induces cell division throughout the body.

Large diversity of serotonin receptors creates its pharmacological complexity. There are at least seven types and eight subtypes of serotonin receptors that have been identified in different areas of the body, and they all have diverse effects. Serotonin receptors are activated by psychoactive drugs such as ecstasy (MDMA), LSD, DMT and psilocybin (a substance found in psychedelic mushrooms). A small dose of ecstasy, for example, stimulates a big release of serotonin in the body causing feelings of well-being and comfort but with many side effects.

5-HT receptors (1–7) are mainly second messenger-gated receptors of which only serotonin type-3 receptor is Ligand-gated ion channel and is involved in nausea and emesis as well as a therapeutic target for depression and other mental conditions. 5-HT1 receptor has five subtypes (5-HT1A–1F, no 1E subtype) which are potential site for anxiolytics and antidepressants as these receptors are mainly responsible for regulating emotions and proprioception. Activation of 5-HT1B and 1D receptors cause vasoconstriction and their antagonists are used for the treatment of schizophrenia and migraines and their partial agonist acts as therapeutic targets for anxiety and depression.

Similarly, 5-HT2 receptor has three subtypes (5-HT2A–2C) and responsible for sleep, pain and motor regulation and are targeted for conditions such as anxiety, migraine and eating disorders. There are certain psychiatric medications that modulate the levels of serotonin in the human body. These drugs are classified into four general categories: (1) monoamine oxidase inhibitors (MAO), (2) tricyclic (TCA) antidepressants, (3) atypical antipsychotics and (4) selective serotonin reuptake inhibitors (SSRIs). SSRIs are prescribed for the treatment of social phobia, anxiety disorders, panic disorders, obsessive-compulsive disorders (OCD), major depression, irritable bowel syndrome (IBS) and eating disorders.

It is vital to know the mechanism of neuronal communication to understand the mode of action of SSRIs. Briefly, two neurons talk to each other by releasing their neurotransmitters in a space (known as synapse) between them. These neurotransmitters travel from presynaptic neurons via synapse to their specific receptors present on the postsynaptic neurons and stimulate them. Once the postsynaptic neurons receive this signal and get activated, these neurotransmitters go back through the transporters present on the presynaptic neurons. SSRIs are the class of drugs that inhibit these serotonin transporter's activities and let serotonin to halt in synapse for a longer period of time. Whereas TCA will let both serotonin and norepinephrine to stay in the gap for extended period of time than normal so both types of postsynaptic neurons can stimulate completely.

As mentioned earlier, all neurotransmitters, especially biogenic amines (serotonin, norepinephrine and dopamine) regulate each other, for example, stimulating 5-HT2 and 5-HT3 receptors by using SSRIs and result in the decrease in levels of dopamine released from the Substantia Nigra, leading to serious mental health problems. Patients on SSRIs or TCA for a longer period of time or in combination with MAOs become very agitated, having tremor and involuntary muscle contraction leading to impaired respiration, increased carbon dioxide pressure and hypoxia.

The role of 5-HT receptors is a topic of intense research, so more therapeutic applications may be discovered in the future. Concisely, role of serotonin in central nervous system is to control appetite, vomiting, sleep, mood, hallucinations and pain perception, and peripherally, responsible for the contraction of vascular and non-vascular smooth, platelet aggregation, increased capillary permeability and modulation of the release of other neurotransmitters.

## **Author details**

**2. Effect of serotonin on brain and body**

4 Serotonin - A Chemical Messenger Between All Types of Living Cells

learning, temperature, cardiovascular and endocrine regulation.

bone and induces cell division throughout the body.

targets for anxiety and depression.

extrasynaptic neurons.

lism of stress hormone.

In the central nervous system, serotonin is synthesized by the neurons of the Raphe nuclei from the amino acid tryptophan through a short metabolic pathway that contains two enzymes: tryptophan hydroxylase and amino acid decarboxylase, and is distributed along the length of the brainstem. Serotonin is released from the varicosities along the axon into the extra-neuronal space this provides a larger area for serotonin to activate its receptors that exist on the dendrites, the cell bodies and the presynaptic terminals of the adjacent neurons. Thus serotonin not only stimulates postsynaptic serotonin receptors but also present on the

Thus, it is not a surprise to find significant role of serotonin in the modulation of many behavioral and psychotic disorders such as mood, sleep, appetite, vomiting, sexuality, memory,

Both high and low levels of serotonin have harmful effect. High serotonin levels cause severe toxicity termed as "serotonin syndrome", which can be fatal in some cases, whereas low levels of serotonin have been associated with migraines, bipolar disorders, apathy, fear, feelings of worthlessness, insomnia, fatigue, anxiety and depression. These pathologies may be explained by the fact that 10% of large dorsal raphe nuclei (largest source of serotonin synthesis) are projected to amygdala and other medium raphe nuclei project to caudate, putamen and olfactory bulb. It is important to mention here that serotonin is required for the metabo-

Scientific evidence confirms that genetic polymorphisms in the enzyme tryptophan hydroxylase in both TPH1 and TPH2 forms can affect the susceptibility to depression and anxiety. Furthermore, ovarian hormones can affect the expression of tryptophan hydroxylase, triggering postnatal depression and premenstrual stress syndrome, and expression of abnormal serotonergic neurons in infants may lead to high possibility of having sudden infant death syndrome (SIDS). Serotonin is also involved in the regeneration of organs such as liver and

Large diversity of serotonin receptors creates its pharmacological complexity. There are at least seven types and eight subtypes of serotonin receptors that have been identified in different areas of the body, and they all have diverse effects. Serotonin receptors are activated by psychoactive drugs such as ecstasy (MDMA), LSD, DMT and psilocybin (a substance found in psychedelic mushrooms). A small dose of ecstasy, for example, stimulates a big release of serotonin in the body causing feelings of well-being and comfort but with many side effects. 5-HT receptors (1–7) are mainly second messenger-gated receptors of which only serotonin type-3 receptor is Ligand-gated ion channel and is involved in nausea and emesis as well as a therapeutic target for depression and other mental conditions. 5-HT1 receptor has five subtypes (5-HT1A–1F, no 1E subtype) which are potential site for anxiolytics and antidepressants as these receptors are mainly responsible for regulating emotions and proprioception. Activation of 5-HT1B and 1D receptors cause vasoconstriction and their antagonists are used for the treatment of schizophrenia and migraines and their partial agonist acts as therapeutic

Kaneez Fatima Shad

Address all correspondence to: ftmshad@gmail.com

Chronic Disease Solutions Team, School of Life Sciences, Faculty of Science, Centre for Health Technologies, University of Technology Sydney, Australia

## **Pharmacology and Molecular Identity of Serotonin Receptor in Bivalve Mollusks**

Sayyed Mohammad Hadi Alavi, Kazue Nagasawa, Keisuke G. Takahashi and Makoto Osada

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69680

#### **Abstract**

It is now known that 5-HT regulates several neurobehavioral systems such as mood, appetite, sleep, learning, and memory. It also plays critical roles in the physiological functions of peripheral organs involved in stress, growth, and reproduction in the animal kingdom. 5-HT content has seen to be higher in the nervous system of bivalves than those of other examined invertebrates and vertebrates. Thus, bivalves have been considered as an excellent model to investigate 5-HT functions in neurological and peripheral systems. The present study reviews knowledge on 5-HT signaling mediated through 5-HT receptor and its physiological contribution to regulate reproduction in bivalves. Two G-protein-coupled 5-HT1 -like receptors have been cloned in bivalve species. However, binding affinities of the 5-HT agonists and antagonists to the isolated plasma membrane proteins and their effects on spawning in bivalves suggest the presence of a single or mixed 5-HT1 -, 5-HT2 -, and 5-HT3 -like receptors. It has suggested that the 5-HT-like receptors in bivalves are distinct from those of mammalian 5-HT receptors due to pharmacological properties. The present review pays a special attention to future research perspectives to better understand 5-HT regulation of reproduction in bivalves, which can provide us with satisfactory knowledge to elucidate reproductive disorders associated with dysfunctions of the neurotransmitter system.

**Keywords:** gonad, nervous system, oocyte, serotonin biosynthesis, serotonin metabolism and reuptake, serotonin receptor, sperm

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

5-hydroxytryptamine called serotonin (5-HT) is a transmitter substance of the nervous system in animal kingdom. 5-HT has also been identified in bivalves from the period of its first discovery and earlier studies on these animals have led to convince the neurobiologist that it acts as a neurotransmitter.

A brief bibliography of discovery for 5-HT receptor and its physiological functions is provided in **Table 1**. Gaddum and Picarelli [6] were the first who demonstrated that 5-HT acts through a receptor-mediated pathway. Further studies have then directed toward pharmacological characterization of the 5-HT receptors in the nervous system and peripheral organs using radiolabelled ligands [7, 8] until the first molecular identity of the 5-HT receptor [9]. In 1960–1980s, 5-HT neurons have localized in the nervous system and peripheral organs (including gonad) of bivalves. Then, Sugamori et al. [10] and Tanabe et al. [11] cloned the 5-HT receptors in the nervous system and reproductive system of pond snail (*Lymnaea stagnalis*) and Yesso scallop (*Patinopecten yessoensis*), respectively. Taken together, bivalves and mammals become model organisms to investigate receptor-mediated mechanism of 5-HT physiological function because of small size, a simple nervous system and a high content of 5-HT in the nervous system.


**Table 1.** Bibliography of 5-hydroxytryptamine (serotonin, 5-HT) receptor: from discovery to physiological characterization.

Serotonin regulates various neurobehavioral systems (such as mood, appetite, sleep, learning, and memory). However, studies have revealed that it also plays critical roles in physiological functions of peripheral organs such as stress and growth [1–3]. One of the major system that 5-HT contributes to its regulation is reproduction. In both mammals and bivalves, it has observed that 5-HT regulates reproductive endocrine system, oocyte maturation, and sperm motility [12–23].

**1. Introduction**

acts as a neurotransmitter.

8 Serotonin - A Chemical Messenger Between All Types of Living Cells

5-HT in the nervous system.

5-hydroxytryptamine called serotonin (5-HT) is a transmitter substance of the nervous system in animal kingdom. 5-HT has also been identified in bivalves from the period of its first discovery and earlier studies on these animals have led to convince the neurobiologist that it

A brief bibliography of discovery for 5-HT receptor and its physiological functions is provided in **Table 1**. Gaddum and Picarelli [6] were the first who demonstrated that 5-HT acts through a receptor-mediated pathway. Further studies have then directed toward pharmacological characterization of the 5-HT receptors in the nervous system and peripheral organs using radiolabelled ligands [7, 8] until the first molecular identity of the 5-HT receptor [9]. In 1960–1980s, 5-HT neurons have localized in the nervous system and peripheral organs (including gonad) of bivalves. Then, Sugamori et al. [10] and Tanabe et al. [11] cloned the 5-HT receptors in the nervous system and reproductive system of pond snail (*Lymnaea stagnalis*) and Yesso scallop (*Patinopecten yessoensis*), respectively. Taken together, bivalves and mammals become model organisms to investigate receptor-mediated mechanism of 5-HT physiological function because of small size, a simple nervous system and a high content of

**Year Scientists Contribution to discovery of identification, localization,** 

1957 Gaddum and Picarelli Suggestion of two types of 5-HT receptors (5-HTM and

1978 Fillion et al. Identification of 5-HT receptors in the bovine brain using

1979 Peroutka and Snyder Evidence for the presence of two distinct 5-HT (5-HT1 and 5-HT2

1982 Matsutani and Nomura Serotonin stimulates spawning in Yesso scallop (Bivalvia, Mollusca)

1985 Osanai 5-HT regulation of the oocyte signaling required to

1991 Bandivdekar and Koide Pharmacological identification of serotonin receptor in surf clam

1993 Sugamori and Van Tol Molecular identity of 5-HT receptor in pond snail

1984 Hirai and Koide 5-HT stimulates oocyte maturation in surf clam [27]

1988 Fargin et al. Molecular identity of 5-HT1A receptor [9]

(Gastropoda, Mollusca)

**Table 1.** Bibliography of 5-hydroxytryptamine (serotonin, 5-HT) receptor: from discovery to physiological characterization.

2010 Tanabe and Osada Molecular identity of 5-HT receptor in Yesso scallop [11]

Species: pond snail, *Lymnaea stagnalis*; surf clam, *Spisula solidissima*; Yesso scallop, *Patinopecten yessoensis*.

[ 3

**and characterization of 5-HT**

5-HTD) in the guinea-pig ileum

H]-lysergic acid diethylamide

recognition by radiolabelled ligands

undergo germinal vesicle breakdown

H]-5-hydroxytryptamine and

) in the rat brain derived from their selective

radiolabelled ligands: [3

**References**

[6]

[7]

[8]

[18]

[28]

[29]

[10]

Although 5-HT biosynthesis and its receptor structure have been reviewed in bivalves [24– 26], however, there is a gap of review on physiological signaling of 5-HT in these animals. The present study reviews the biology of 5-HT in bivalves, particularly, its contribution to reproduction. Particular attention has then paid to pharmacological characteristics of the 5-HT receptor and 5-HT-stimulated spawning through a receptor-mediated mechanism. This study provides future perspectives that await investigation to better understand 5-HT network and signaling in bivalve reproduction.

## **2. Molecular identity and pharmacological characteristics of the 5-HT receptors**

Since the time Gaddum and Picarelli [6] suggested the presence of two kinds of tryptamine receptor, further studies have been conducted to identify and localize the 5-HT receptors to elucidate serotonergic signaling in biological systems. Fargin et al. [9] were the first who reported that the protein product of an orphan receptor (G21) encoding a G-protein-coupled receptor (GPCR) transiently expressed in monkey kidney cells possesses all the typical ligand-binding characteristics of the 5-HT1A receptor. Molecular identity of 5-HT receptors has revealed that there are, so far, a total of 14 structurally and pharmacologically distinct mammalian 5-HT receptors which are classified into seven groups. Except of the 5-HT<sup>3</sup> receptor that is a ligand-gated ion channel [35, 36], the 5-HT1 , 5-HT2 , 5-HT4 , 5-HT5 , 5-HT6 , and 5-HT7 belong to GPCR superfamily [4, 5, 37–41]. In invertebrates, pharmacological properties of the 5-HT receptors do not allow us to classify them in mammalian categories, although some signal transduction characteristics are similar [26].

### **2.1. Pharmacological characteristics of 5-HT receptors in bivalves**

In bivalves, primary studies have used pharmacological 5-HT agonists and antagonists to investigate their binding affinities onto isolated membrane proteins of the oocytes and sperm using radiolabelled [3 H]5-HT [29, 42–45]. The results showed that only 5-HT and its analogs are capable of inhibiting [3 H]5-HT-specific binding to the isolated plasma membrane proteins of the oocytes in surf clam, whereas other monoamines (such as acetylcholine, haloperidol, carbachol, pyrilamine, and so on) are without effects [43, 44].

In surf clam, 1 μM ICS 205930, 5-HT, 5-CT, mianserin, methysergide, 8-OH-DPAT, 2-methyl-5-HT, BMY 7378, α-methyl-5-HT, ketanserin, quipazine, and PBG inhibit [<sup>3</sup> H]5-HT binding to the isolated proteins of the oocyte plasma membrane by 49, 46, 40, 40, 37, 35, 33, 28, 26, 25, 22, and 11%, respectively [29]. The authors suggested that 5-HT receptors in the oocyte of surf clam possess sites that interact with the 5-HT1 and 5-HT3 receptor analogs, because of the binding affinity of the 5-HT<sup>1</sup> receptor (5-CT, mianserin, methysergide, and 8-OH-DPAT) and the 5-HT3 receptor (ICS 205930 and 2-methyl-5-HT) analogs. However, current pharmacological characterization of 5-HT receptor analogs reveals that 5-CT is a non-selective agonist, and mianserin and methysergide are particularly selective antagonists of the 5-HT2 receptor (**Table 2**). These may suggest that the 5-HT2 receptor also exist on the membrane of the oocytes in surf clam, in addition to the 5-HT1 and 5-HT3 receptors [29, 46].

Krantic et al. [43, 44] studied dose-dependent effects of the 5-HT analogs and observed that 5-HT, 8-OH-DPAT, metoclopramide, MDL 72222, mianserin, ICS 205930, ritanserin, imipramine, propranolol, and TFMPP inhibit specific [<sup>3</sup> H]5-HT binding to the isolated membrane


α-methyl-5-HT, α-methyl-5-hydroxytryptamine; 1-m-c-b, 1-methyl-chlorophenyl biguanide; 2-methyl-5-HT, 2-methyl-5-hydroxytryptamine; 1-NP, 1-(1-naphthyl)piperazine; 5-CT, 5-carboxamidotryptamine; 8-OH-DPAT, 7-(dipropylamino)-5,6,7,8-tetrahydronaphthalen-1-ol; mCPP, *meta*-chlorophenylpiperazine; MDL-72222 (Bemesetron) PBG, 1-phenylbiguanide; and TFMPP, 3-trifluoromethylphenylpiperazine.

8-OH-DPAT also acts as a 5-HT7 receptor agonist [76] and possesses serotonin reuptake blocking property [77]. TFMPP binds to SERT and evokes 5-HT release [78]. *mCPP acts as 5*-*HT reuptake inhibitor/releasing agent* [79]. Unlike mCPP, TFMPP has insignificant affinity for the 5-HT<sup>3</sup> receptor [80]. BMY-7378 is a weak partial 5-HTlA agonist compared to 8-OH-DPAT that is a full 5-HT1A agonist [81, 82] and is a selective antagonist of α1D-adrenoceptors [83]. PBG and mCPBG have dopamine releasing properties [84]. Methysergide also acts as a 5-HT1A, 1B, 1D receptors' partial agonist. 5-HT and methysergide appear not to compete for the same site, whereas ketanserin and methysergide do appear to compete for the same site [56, 66, 85]. Quipazine also acts via 5-HT2 receptor as an agonist [86, 87] or antagonist of 5-HT3 receptor [88, 89]. Metoclopramide acts as antagonist of dopamine D2 receptors [90] and as a 5-HT4 receptor agonist [91].

**Table 2.** Pharmacological agonists and antagonists of the 5-hydroxytryptamine (serotonin, 5-HT) receptors.

proteins of the oocytes in surf clam by 100, 67, 63, 61, 57, 57, 55, 49, 47, and 12% with IC50 of 0.52, 0.05, 0.06, 0.13, 0.45, 3.05, 0.42, 4.2, 1.32, and >100 μM, respectively. Hence, these results showing affinities of the 5-HT analogs to the 5-HT<sup>1</sup> , 5-HT2 , and 5-HT3 receptors in the oocyte of surf clam; however, the receptor possesses distinct 5-HT binding sites from 5-HT1 , 5-HT2 , 5-HT3 , and 5-HT4 receptors in mammals and *Drosophila*. For instance, the 5-HT1A receptor is more sensitive to 8-OH-DPAT than 5-HT, insensitive to ritanserin, and relatively sensitive to TFMPP in mammals. 8-OH-DPAT is a weak agonist on the *Drosophila* 5-HT receptors. Ritanserin, but not TFMPP, inhibits [3 H]5-HT binding to the isolated membrane protein of the oocyte in surf clam, although isolated 5-HT receptor is highly sensitive to 8-OH-DPAT more than that of 5-HT. The 5-HT receptor in the oocyte of surf clam does not possess pharmacological 5-HT2 receptor characteristics in mammals, as it is not equally sensitive to TFMPP and 8-OH-DPAT. The pharmacological characteristics of the isolated 5-HT receptor also differ from the 5-HT<sup>3</sup> receptor. In mammals, the 5-HT3 receptor is at least 100-fold more sensitive to 8-OH-DPAT than to metoclopramide; however, 8-OH-DPAT and metoclopramide are equipotent in inhibition of [ 3 H]5-HT binding to the 5-HT receptor in the surf clam. Based on these different responses of the isolated membrane protein of the surf clam oocytes to the 5-HT analogs, the authors suggested the presence of a novel 5-HT receptor in the plasma membrane of the surf clam oocytes.

surf clam possess sites that interact with the 5-HT1

10 Serotonin - A Chemical Messenger Between All Types of Living Cells

tor (**Table 2**). These may suggest that the 5-HT2

mine, propranolol, and TFMPP inhibit specific [<sup>3</sup>

oocytes in surf clam, in addition to the 5-HT1

binding affinity of the 5-HT<sup>1</sup>

the 5-HT3

and 5-HT3

receptor (ICS 205930 and 2-methyl-5-HT) analogs. However, current pharmaco-

and 5-HT3

logical characterization of 5-HT receptor analogs reveals that 5-CT is a non-selective agonist,

Krantic et al. [43, 44] studied dose-dependent effects of the 5-HT analogs and observed that 5-HT, 8-OH-DPAT, metoclopramide, MDL 72222, mianserin, ICS 205930, ritanserin, imipra-

**Receptor Agonists Reference Antagonist Reference** 5-HT1 8-OH-DPAT (5-HT1A) [47] Propranolol (5-HT1B) [49, 50]

TFMPP (5-HT1A, 1B, 1D ) [48] NAN-190 (5-HT1A)

5-HT2 TFMPP (5-HT2A, 2C) [49] Ketanserin (5-HT2A) [50]

5HT3 1-m-c-b (mCPBG) [52] Metoclopramide [67, 68]

Non-selective α-Methyl-5-HT (5-HT1, 2) [55] Methiothepin (5-HT1A, 1B, 1D, 5A) [75]

α-methyl-5-HT, α-methyl-5-hydroxytryptamine; 1-m-c-b, 1-methyl-chlorophenyl biguanide; 2-methyl-5-HT, 2-methyl-5-hydroxytryptamine; 1-NP, 1-(1-naphthyl)piperazine; 5-CT, 5-carboxamidotryptamine; 8-OH-DPAT, 7-(dipropylamino)-5,6,7,8-tetrahydronaphthalen-1-ol; mCPP, *meta*-chlorophenylpiperazine; MDL-72222 (Bemesetron)

binds to SERT and evokes 5-HT release [78]. *mCPP acts as 5*-*HT reuptake inhibitor/releasing agent* [79]. Unlike mCPP,

to 8-OH-DPAT that is a full 5-HT1A agonist [81, 82] and is a selective antagonist of α1D-adrenoceptors [83]. PBG and mCPBG have dopamine releasing properties [84]. Methysergide also acts as a 5-HT1A, 1B, 1D receptors' partial agonist. 5-HT and methysergide appear not to compete for the same site, whereas ketanserin and methysergide do appear to compete

**Table 2.** Pharmacological agonists and antagonists of the 5-hydroxytryptamine (serotonin, 5-HT) receptors.

5-CT (5-HT1A, 1B, 1D, 5A, 7) [56–60]

PBG, 1-phenylbiguanide; and TFMPP, 3-trifluoromethylphenylpiperazine.

8-OH-DPAT also acts as a 5-HT7

TFMPP has insignificant affinity for the 5-HT<sup>3</sup>

for the same site [56, 66, 85]. Quipazine also acts via 5-HT2

[88, 89]. Metoclopramide acts as antagonist of dopamine D2

mCPP (5-HT2B, 2C) [50] Spiperone (5-HT2A) [50] PBG [51] 1-NP (5-HT2A, 2B, 2C) [63, 64]

2-methyl-5-HT [53] ICS 205-930 (Tropisetron) [53, 69–71] Quipazine [54] LY-278584 [72, 73]

Ondansetron

receptor agonist [76] and possesses serotonin reuptake blocking property [77]. TFMPP

receptors [90] and as a 5-HT4

receptor [80]. BMY-7378 is a weak partial 5-HTlA agonist compared

receptor as an agonist [86, 87] or antagonist of 5-HT3

receptor agonist [91].

and mianserin and methysergide are particularly selective antagonists of the 5-HT2

receptor (5-CT, mianserin, methysergide, and 8-OH-DPAT) and

receptor analogs, because of the

receptor also exist on the membrane of the

H]5-HT binding to the isolated membrane

BMY 7378 [61, 62]

Mianserin (5-HT2A, 2B, 2C) [50] Ritanserin (5-HT2A, 2B, 2C) [65] Methysergide (5-HT2B, 2C) [66]

MDL-72222 (Bemesetron) [69, 74]

Cyproheptadine (5-HT2A, 2B)

receptors [29, 46].

recep-

receptor

In Yesso scallop, Osada et al. [45] observed that [3 H]5-HT binding to the oocyte plasma membrane is inhibited to 93, 83, 70, 44, 41, and 36% in the presence of 100 μM metoclopramide, 8-OH-DPAT, 5-HT, ritanserin, α-methyl-5-HT, and methiothepin, respectively. In the Pacific oyster, [3 H]5-HT binding to the oocyte plasma membrane is inhibited to 96, 83, 58, 49, 21, and 16% in the presence of 100 μM metoclopramide, 8-OH-DPAT, 5-HT, α-methyl-5-HT, ritanserin, and methiothepin respectively [45]. Ritanserin-, α-methyl-5-HT-, and methiothepin-inhibited [3 H]5-HT binding to the 5-HT receptor isolated from the oocyte of Yesso scallop suggest that mixed 5-HT1 and 5-HT2 receptors function in this species. However, the authors suggested that a single 5-HT1 receptor functions in the Pacific oyster as methiothepin acts mainly as a 5-HT1 antagonist (**Table 2**). In addition, this study shows that metoclopramide does not influence [3 H]5-HT binding to 5-HT receptor isolated from the oocyte of Yesso scallop and the Pacific oyster and 8-OH-DPAT is also a weak agonist, suggesting that 5-HT signaling is not mediated by 5-HT3 receptor and is distinct from mammalian 5-HT1A receptors in these species.

Pharmacological characteristics of the 5-HT receptor in sperm have only studied in surf clam [42]. The results have shown that 1 μM ICS 205930, 2-methyl-5-HT, 8-OH-DPAT, BMY 7378, 5-HT, 5-CT, mianserin, methysergide, α-methyl-5-HT, PBG, and ketanserin inhibit 45, 43, 37, 32, 31, 31, 30, 26, 13, 4, and 1% of [3 H]5-HT binding to the sperm plasma membrane, respectively. Considering current pharmacological characterization of 5-HT receptors, analogs of 5-HT3 , 5-HT1 , and 5-HT2 receptors are more potent to compete with 5-HT to inhibit [3 H]5-HT binding to the sperm plasma membrane.

#### **2.2. Molecular identity and cellular localization of 5-HT receptors in bivalves**

In mollusks, the 5-HTLym and 5-HT2Lym are first identified in the central nervous system of the pond snail (*L. stagnalis*). They display some pharmacological characteristics of the 5-HT1 and 5-HT2 receptors in mammals, and thus are currently considered as the 5-HT1 -like receptor and the 5-HT2 -like receptor, respectively [10, 92]. The Ap5-HTB1 and Ap5-HTB2 [93], 5-HT1AP[94], and 5-HT2AP [95] are identified in California sea slug (*Aplysia californica*). The Ap5-HTB1 and Ap5-HTB2 (79.5% homologous to each other) are expressed in the reproductive system and the nervous system, respectively; however, they are not classified into any 5-HT receptor subtypes in mammals due to differences in their amino acid sequences [93]. The 5-HT1AP is distributed in most organs, including the nervous system, kidney, gills, and heart, and its amino acid sequence and pharmacological profiles suggest that it is a 5-HT<sup>1</sup> receptor subfamily [94]. The 5-HT2AP shares 68 and 34% of its amino acid sequence identity with the 5-HTLym and 5-HT1 receptor in mammals, its pharmacological characteristics is very similar to those of the 5-HTLym receptor, and it is only expressed in the nervous system [95].

In bivalves, the 5-HT receptors are cloned in the ovary of the Yesso Scallop [11], and Pearl oyster, *Pinctada fucata* [96] (**Figure 1**). Molecular identity of the 5-HT receptor is also predicted for the Pacific oyster (5-HTcg) [97]. In the Yesso scallop, an 1818 bp cDNA encodes a putative 5-HTpy receptor that includes a 232-bp 5′-untranslated region (UTR), a 1362-bp open reading frame (ORF) encoding a putative protein of 454 amino acids, and a 224-bp 3′-UTR. In the Pearl oyster, a 2541 bp cDNA encodes a putative 5-HTpf receptor that includes a 296-bp 5′-UTR, a 1416-bp ORF encoding a putative protein of 471 amino acids, and an 829-bp 3′-UTR. The 5-HTpf is calculated to have a molecular weight of 53.55 kDa. The hydrophobicity analysis of the deduced amino acid sequence revealed seven putative transmembrane domains, which are highly conserved between 5-HTpy, 5-HTpf, and other 5-HT1 receptors coupled with Gi/o. The 5-HTpy contains two potential sites for N-linked glycosylation in the extracellular N-terminal region and the third intracellular domain. The 5-HTpf receptor contains five potential sites for N-linked glycosylation in the extracellular N-terminal region. There are 12 and 8 sites for phosphorylation by protein kinase A or C in the Yesso scallop and Pearl oyster, respectively, among which 7 sites are located in the third cytoplasmic loop. A relatively long third cytoplasmic loop and a short fourth inner terminal domain (C-terminal tail) are present in the 5-HTpy and 5-HTpf sequence.

An amino acid sequence alignment of 5-HT receptor homologs from different species reveals that a relatively high level of amino acid sequence identity exists between 5-HTpy and 5-HTpf (52%) and between 5-HTpy and 5-HTcg (48%). The amino acid sequence identity is between 5-HTpf and 5-HTcg (71%). There are conserved amino acid regions when the 5-HTpy and 5-HTpf are aligned to 5-HT1 subtypes in human (**Figure 1**). The 5-HTpy amino acid sequence is 40, 40, 37, 38, and 38% identical to the human 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F receptor, respectively. The 5-HTpf amino acid sequence is 42, 39, 39, 40, and 40% identical to the human 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F receptor, respectively. The 5-HTcg was not considered in alignment analysis as it is a predicted sequence. The amino acid sequence identity is higher within the transmembrane domains, compared to those of the intracellular and extracellular region. However, lower amino acid sequence identity exists between the 5-HT receptors in bivalves with the other 5-HT receptors (5-HT2 , 5-HT5 , 5-HT6 , and 5-HT7 ) in vertebrates. The phylogenetic analysis of the 5-HT receptors in invertebrates suggests that the 5-HT receptors of bivalves resemble the 5-HT receptors in the California sea slug (*A. californica*), pond snail (*L. stagnalis*), and air-breathing snail (*Planorbella trivolvis*), which are known to be as 5-HT1 -like receptor (**Figure 2**). These known 5-HT receptors are differentiated into a major branch, compared to the other known invertebrate 5-HT receptors. Four 5-HT receptors Pharmacology and Molecular Identity of Serotonin Receptor in Bivalve Mollusks http://dx.doi.org/10.5772/intechopen.69680 13

and 5-HT2AP [95] are identified in California sea slug (*Aplysia californica*). The Ap5-HTB1 and Ap5-HTB2 (79.5% homologous to each other) are expressed in the reproductive system and the nervous system, respectively; however, they are not classified into any 5-HT receptor subtypes in mammals due to differences in their amino acid sequences [93]. The 5-HT1AP is distributed in most organs, including the nervous system, kidney, gills, and heart, and its

ily [94]. The 5-HT2AP shares 68 and 34% of its amino acid sequence identity with the 5-HTLym

In bivalves, the 5-HT receptors are cloned in the ovary of the Yesso Scallop [11], and Pearl oyster, *Pinctada fucata* [96] (**Figure 1**). Molecular identity of the 5-HT receptor is also predicted for the Pacific oyster (5-HTcg) [97]. In the Yesso scallop, an 1818 bp cDNA encodes a putative 5-HTpy receptor that includes a 232-bp 5′-untranslated region (UTR), a 1362-bp open reading frame (ORF) encoding a putative protein of 454 amino acids, and a 224-bp 3′-UTR. In the Pearl oyster, a 2541 bp cDNA encodes a putative 5-HTpf receptor that includes a 296-bp 5′-UTR, a 1416-bp ORF encoding a putative protein of 471 amino acids, and an 829-bp 3′-UTR. The 5-HTpf is calculated to have a molecular weight of 53.55 kDa. The hydrophobicity analysis of the deduced amino acid sequence revealed seven putative transmembrane domains, which

5-HTpy contains two potential sites for N-linked glycosylation in the extracellular N-terminal region and the third intracellular domain. The 5-HTpf receptor contains five potential sites for N-linked glycosylation in the extracellular N-terminal region. There are 12 and 8 sites for phosphorylation by protein kinase A or C in the Yesso scallop and Pearl oyster, respectively, among which 7 sites are located in the third cytoplasmic loop. A relatively long third cytoplasmic loop and a short fourth inner terminal domain (C-terminal tail) are present in the 5-HTpy

An amino acid sequence alignment of 5-HT receptor homologs from different species reveals that a relatively high level of amino acid sequence identity exists between 5-HTpy and 5-HTpf (52%) and between 5-HTpy and 5-HTcg (48%). The amino acid sequence identity is between 5-HTpf and 5-HTcg (71%). There are conserved amino acid regions when the 5-HTpy and 5-HTpf

40, 37, 38, and 38% identical to the human 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F receptor, respectively. The 5-HTpf amino acid sequence is 42, 39, 39, 40, and 40% identical to the human 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F receptor, respectively. The 5-HTcg was not considered in alignment analysis as it is a predicted sequence. The amino acid sequence identity is higher within the transmembrane domains, compared to those of the intracellular and extracellular region. However, lower amino acid sequence identity exists between the 5-HT

tebrates. The phylogenetic analysis of the 5-HT receptors in invertebrates suggests that the 5-HT receptors of bivalves resemble the 5-HT receptors in the California sea slug (*A. californica*), pond snail (*L. stagnalis*), and air-breathing snail (*Planorbella trivolvis*), which are known

major branch, compared to the other known invertebrate 5-HT receptors. Four 5-HT receptors


subtypes in human (**Figure 1**). The 5-HTpy amino acid sequence is 40,

, 5-HT5

, 5-HT6

, and 5-HT7

) in ver-

receptor in mammals, its pharmacological characteristics is very similar to those of

receptor subfam-

receptors coupled with Gi/o. The

amino acid sequence and pharmacological profiles suggest that it is a 5-HT<sup>1</sup>

12 Serotonin - A Chemical Messenger Between All Types of Living Cells

the 5-HTLym receptor, and it is only expressed in the nervous system [95].

are highly conserved between 5-HTpy, 5-HTpf, and other 5-HT1

receptors in bivalves with the other 5-HT receptors (5-HT2

and 5-HT1

and 5-HTpf sequence.

are aligned to 5-HT1

to be as 5-HT1


**Figure 1.** A schematic representation of the G-protein-coupled 5-hydroxytryptamine (serotonin, 5-HT) receptor showing seven transmembrane domains (A). (B) Multiple alignment of deduced amino acid sequence of 5-HT receptors of the Yesso scallop (*Patinopecten yessoensis*, py5-HT) and pearl oyster (*Pinctada fucata*, pf5-HT) with the 5-HT1A-F receptors in human. The marked amino acids indicate seven transmembrane regions. Sequences are aligned with MUSCLE configured for highest accuracy (www.phylogeny.fr).

**Figure 2.** Phylogenetic analysis of the 5-hydroxytryptamine (serotonin, 5-HT) receptor known from invertebrates (A) and from invertebrates and vertebrates (B). Filled circles indicate bivalve species. Open circles or dark background indicate mollusk species. Note that the 5-HT3 receptors are excluded in this analysis, as they are ligand-gated ion channel. Phylogeny trees are constructed using the maximum likelihood method implemented in the PhyML program. The amino acid sequences of the 5-HT receptors are aligned with MUSCLE configured for highest accuracy (MUSCLE with default settings). After alignment, ambiguous regions (i.e. containing gaps and/or poorly aligned) are removed (www.phylogeny.fr). Accession numbers of applied 5-HT receptors are as follows: **invertebrates** dm5-HT (AAA28305, 5HT-dro), dm5-HT2 (CAA57429, 5-HT2-dro), dm5HT-2A (CAA77570, 5HT-dro2A), dm5-HT2B (CAA77571, 5HT-dro2B), ae5-HT7 (AAG49292), am5-HT (NP-001164579), px5-HT (BAD72868), rm5-HT (AAQ89933), bm5-HT (CAA64862), hv5-HT (CAA64863), dj5-HT1 (BAA22404), dj5-HT4 (BAA22403), 1ce5-HT (AAC15827), 2ce5-HT (NP-491954), 3ce5-HT (NP-497452), as5-HT (AAC78396), hc5-HT (AAO45883), ac5-HTB1 (Q16950, Ap5HTB1), ac5-HTB2 (Q16951, Ap5HTB2), ac5-HT2 (AAM46088, Ap5-HT2), ac5-HT (AAC28786, Ap5-HT), ls5-HT2 (AAC16969, Lym5-HT2), ls5-HT (AAA29290, Lym5-HT), pt5-HT1 (AAQ95277), pt5-HT7 (AAQ84306), py5-HT (BAE72141), pf5-HT (AIW04132), cg5-HT (EKC38511), pi5-HT2 (AAS57919, 5-HT type 2), me5-HT (AAS05316), aa5-HT (BAA12013), and **vertebrates** tr5-HT1Aa (CAA65175, 5-HT1Aalpha), tr5-HT1Ab (CAA65176, 5-HT1Abeta), om5-HT1A (AAP83427), xl5-HT1A (CAA69208), gg5-HT1A (NP-001163999), rn5-HT1A (NP-036717), mm5-HT1A (NP-032334), hs5-HT1A (NP-000515), gg5-HT1B (NP-001166252), rn5- HT1B (NP-071561), mm5-HT1B (NP-034612), hs5-HT1B (AAH69065), tr5-HT1D (CAA58745), om5-HT1D (AAP83428), rn5-HT1D (NP-036984), mm5-HT1D (NP-032335), hs5-HT1D (NP-000855), hs5-HT1E (NP-000856), rn5-HT1F (NP-068629), mm5-HT1F (NP-032336), hs5-HT1F (NP-000857), rn5-HT2A (NP-058950), mm5-HT2A (NP-766400), hs5-HT2A (NP-000612), tf5-HT2B (CAC85912), xl5-HT2B (CAD71264), rn5-HT2B (NP-058946), mm5-HT2B (NP-032337), hs5- HT2B (NP-000858), rn5-HT2C (NP-036897), mm5-HT2C (NP-032338), hs5-HT2C (NP-000859), rn5-HT4 (NP-036985), mm5-HT4 (CAA70775), hs5-HT4 (CAC22248), rn5-HT5A (NP-037280), mm5-HT5A (NP-032340), hs5-HT5A (NP-076917), rn5-HT5B (NP-077371), mm5-HT5B (NP-034613), rn5-HT6 (NP-077341), mm5-HT6 (NP-067333), hs5-HT6 (NP-000862), gg5-HT7 (NP-001165240), rn5-HT7 (NP-075227), mm5-HT7 (NP-032341), hs5-HT7 (NP-000863). First letters of the genus and species are used to construct the phylogenetic analysis; fruit fly (*Drosophila melanogaster*, dm); mosquito (*Aedes aegypti*, ae); honey bee (*Apis mellifera*, am); butterfly (*Papilio xuthus*, px); tick (*Rhipicephalus microplus*, rm); silkworm (*Bombyx mori*, bm); moth (*Heliothis virescens*, hv); planarian flatworm (*Dugesia japonica*, dj); nematode roundworm (*Caenorhabditis elegans*, ce); nematode roundworm (*Ascaris suum*, as); nematode (*Haemonchus contortus*, hc); California sea slug (*Aplysia californica*, ac); pond snail (*Lymnaea stagnalis*, ls); air-breathing snail (*Planorbella trivolvis*, pt); scallop (*Mizuhopecten yessoensis*, py); Pearl oyster (*Pinctada fucata*, pf); Pacific oyster (*Crassostrea gigas*, cg); lobster (*Panulirus interruptus*, pi); shrimp (*Metapenaeus ensis*, me); barnacle (*Amphibalanus amphitrite*, aa); pufferfish (*Takifugu rubripes*, tr); pufferfish (*Tetraodon fluviatilis*, tf); Tilapia (*Oreochromis mossambicus*, om); frog (*Xenopus laevis*, xl); chicken (*Gallus gallus*, gg); rat (*Rattus norvegicus,* rn); mouse (*Mus musculus*, mm); and human (*Homo sapiens*, hs).

of mollusks (5-HT2 in pond snail, 5-HT7 in the air-breathing snail, 5-HTB1 and 5-HTB2 in the California sea slug) are differentiated into different branch. Except of two latter case which display difficulties to be classified in terms of 5-HT receptors in vertebrates [26], the 5-HT2 in pond snail and the 5-HT7 in the air-breathing snail are considered as the 5-HT2 -like and the 5-HT7 -like receptors, respectively [92, 98].

The 5-HTpy and 5-HTpf are expressed in most of the organs, including the ovary, testis, mantle, adductor muscle, gill, the nervous system (cerebral-pedal ganglia and VG), digestive gland, or kidney [11, 96]. *In situ* hybridization has shown that the 5-HTpy mRNA is localized in the oocytes and epithelium of the gonoducts in the ovary and in the spermatids and epithelium of the gonoduct in the testis [11]. It has histologically observed that, at spawning, mature oocyte and sperm are collected and evacuated from the acini into the surrounding aquatic environment via gonoducts in the great scallop [99]. Real-time PCR analyses of the 5-HTpf mRNA transcription reveals that the order of decreasing is as follows: mature ovary > mature testis, VG, and digestive gland > mantle, gills, and adductor muscle. In addition, the testicular and ovary 5-HTpf mRNA transcription does not differ among resting, developmental, and mature stages, however, increases in the ovary at spawning stage [96].

## **3. Receptor-mediated 5-HT stimulation of spawning in bivalves**

**Figure 2.** Phylogenetic analysis of the 5-hydroxytryptamine (serotonin, 5-HT) receptor known from invertebrates (A) and from invertebrates and vertebrates (B). Filled circles indicate bivalve species. Open circles or dark background

channel. Phylogeny trees are constructed using the maximum likelihood method implemented in the PhyML program. The amino acid sequences of the 5-HT receptors are aligned with MUSCLE configured for highest accuracy (MUSCLE with default settings). After alignment, ambiguous regions (i.e. containing gaps and/or poorly aligned) are removed (www.phylogeny.fr). Accession numbers of applied 5-HT receptors are as follows: **invertebrates** dm5-HT (AAA28305, 5HT-dro), dm5-HT2 (CAA57429, 5-HT2-dro), dm5HT-2A (CAA77570, 5HT-dro2A), dm5-HT2B (CAA77571, 5HT-dro2B), ae5-HT7 (AAG49292), am5-HT (NP-001164579), px5-HT (BAD72868), rm5-HT (AAQ89933), bm5-HT (CAA64862), hv5-HT (CAA64863), dj5-HT1 (BAA22404), dj5-HT4 (BAA22403), 1ce5-HT (AAC15827), 2ce5-HT (NP-491954), 3ce5-HT (NP-497452), as5-HT (AAC78396), hc5-HT (AAO45883), ac5-HTB1 (Q16950, Ap5HTB1), ac5-HTB2 (Q16951, Ap5HTB2), ac5-HT2 (AAM46088, Ap5-HT2), ac5-HT (AAC28786, Ap5-HT), ls5-HT2 (AAC16969, Lym5-HT2), ls5-HT (AAA29290, Lym5-HT), pt5-HT1 (AAQ95277), pt5-HT7 (AAQ84306), py5-HT (BAE72141), pf5-HT (AIW04132), cg5-HT (EKC38511), pi5-HT2 (AAS57919, 5-HT type 2), me5-HT (AAS05316), aa5-HT (BAA12013), and **vertebrates** tr5-HT1Aa (CAA65175, 5-HT1Aalpha), tr5-HT1Ab (CAA65176, 5-HT1Abeta), om5-HT1A (AAP83427), xl5-HT1A (CAA69208), gg5-HT1A (NP-001163999), rn5-HT1A (NP-036717), mm5-HT1A (NP-032334), hs5-HT1A (NP-000515), gg5-HT1B (NP-001166252), rn5- HT1B (NP-071561), mm5-HT1B (NP-034612), hs5-HT1B (AAH69065), tr5-HT1D (CAA58745), om5-HT1D (AAP83428), rn5-HT1D (NP-036984), mm5-HT1D (NP-032335), hs5-HT1D (NP-000855), hs5-HT1E (NP-000856), rn5-HT1F (NP-068629), mm5-HT1F (NP-032336), hs5-HT1F (NP-000857), rn5-HT2A (NP-058950), mm5-HT2A (NP-766400), hs5-HT2A (NP-000612), tf5-HT2B (CAC85912), xl5-HT2B (CAD71264), rn5-HT2B (NP-058946), mm5-HT2B (NP-032337), hs5- HT2B (NP-000858), rn5-HT2C (NP-036897), mm5-HT2C (NP-032338), hs5-HT2C (NP-000859), rn5-HT4 (NP-036985), mm5-HT4 (CAA70775), hs5-HT4 (CAC22248), rn5-HT5A (NP-037280), mm5-HT5A (NP-032340), hs5-HT5A (NP-076917), rn5-HT5B (NP-077371), mm5-HT5B (NP-034613), rn5-HT6 (NP-077341), mm5-HT6 (NP-067333), hs5-HT6 (NP-000862), gg5-HT7 (NP-001165240), rn5-HT7 (NP-075227), mm5-HT7 (NP-032341), hs5-HT7 (NP-000863). First letters of the genus and species are used to construct the phylogenetic analysis; fruit fly (*Drosophila melanogaster*, dm); mosquito (*Aedes aegypti*, ae); honey bee (*Apis mellifera*, am); butterfly (*Papilio xuthus*, px); tick (*Rhipicephalus microplus*, rm); silkworm (*Bombyx mori*, bm); moth (*Heliothis virescens*, hv); planarian flatworm (*Dugesia japonica*, dj); nematode roundworm (*Caenorhabditis elegans*, ce); nematode roundworm (*Ascaris suum*, as); nematode (*Haemonchus contortus*, hc); California sea slug (*Aplysia californica*, ac); pond snail (*Lymnaea stagnalis*, ls); air-breathing snail (*Planorbella trivolvis*, pt); scallop (*Mizuhopecten yessoensis*, py); Pearl oyster (*Pinctada fucata*, pf); Pacific oyster (*Crassostrea gigas*, cg); lobster (*Panulirus interruptus*, pi); shrimp (*Metapenaeus ensis*, me); barnacle (*Amphibalanus amphitrite*, aa); pufferfish (*Takifugu rubripes*, tr); pufferfish (*Tetraodon fluviatilis*, tf); Tilapia (*Oreochromis mossambicus*, om); frog (*Xenopus laevis*, xl); chicken (*Gallus gallus*,

gg); rat (*Rattus norvegicus,* rn); mouse (*Mus musculus*, mm); and human (*Homo sapiens*, hs).

receptors are excluded in this analysis, as they are ligand-gated ion

indicate mollusk species. Note that the 5-HT3

14 Serotonin - A Chemical Messenger Between All Types of Living Cells

Matsutani and Nomura [18] observed that injection of homogenates of CG, PG, or VG into the gonad of Yesso scallop induces spawning in 100% of males; however, they are without effects on females. In another experiment, they observed that 5-HT induces spawning in 100% of males and 73.3–80% of females. No other neurotransmitters, including adrenaline, noradrenaline (NA), and ϒ-aminobutyric acid, induced spawning [100–103]. Acetylcholine and dopamine (DA) induce spawning in males (40%), however they are without effects on females. Similarly, further studies have shown that neurotransmitters except of 5-HT are not potent to induce spawning in the surf clam [40], Zebra mussel [104], and Peruvian scallop [33, 105]. It is worth to note that DA at high dose (2 × 10−3 M) is capable of inducing spawning in males of Peruvian scallop [105] and in both males and females of Lion's paw scallop (*Nodipecten nodosus*) and Nucleus scallop (*Argopecten nucleus*) [106]. Omitting these exceptions, it has been accepted that 5-HT is the most potent neurotransmitter that induce spawning in bivalves at physiological concentration (**Table 3**). Other studies also show that injection of 0.4 mM 2–20 × 10−4 M 5-HT induces spawning in bivalve species, including the Atlantic deep-sea scallop, butter clam (*Saxidomus gigantea*), Gaper clam (*Tresus capax*), Manila clam (*Ruditapes philippinarum*), Pacific geoduck (*Panopea generosa*), Pacific littleneck clam (*Protothaca staminea*), Pacific oyster, Pacific razor clam (*Siliqua patula*), Pink scallop (*Chlamys rubida*), Rock scallop (*Hinnites multirugosus*), Weathervane scallop (*Patinopecten caurinus*), and Yesso scallop [107, 108]. It has also observed that 10−4 to 10−6 M 5-HT stimulates the release of the oocytes from the ovary tissues and sperm from the testicular tissues following a 90-min incubation, *in vitro* [109–112]. These are in agreement with identification of 5-HT and localization of nerve fibers transferring 5-HT from nervous system to gonad, which are observed around acini or gamete collective tubules. Both males and females response to exogenous 5-HT in a dose-dependent manner. However, it seems that females usually require higher amount of 5-HT than that of a male to release the oocytes. The observed sex-specificity might be related to inter-sex differences in the concentration of 5-HT, which are shown to be higher in males than in females [32, 34]. Moreover, studies show that 5-HT fully stimulates spawning in ripe individuals.

As 5-HT fibers are localized in the gonad of bivalves, these observations pioneered further research to elucidate mechanism through which 5-HT induces spawning. In Zebra mussel, methiothepin, a non-selective 5-HT1 receptor antagonist (**Table 2**), decreases 5-HT-induced spawning when it is added into the aquarium 5 min after addition of 5-HT. However, it is without effects on 5-HT-induced spawning when it is added into the aquarium 10 min after addition [120]. A 2 h pre-treatment of the Zebra mussel with 10−4 M methiothepin decreases parturition from 65 to 8% and from 82 to 1% in the individuals treated with 10−4 and 10−3 M 5-HT, respectively. These suggest that 5-HT-induced spawning requires a certain period of time and that 5-HT-induced spawning is irreversible.

To better understand which type of 5-HT receptor is involved in 5-HT-induced spawning, further experiments have conducted using 5-HT receptor analogs. It has observed that 10−4 M 8-OH-DPAT, 5-HT, and TFMPP induce 80, 70, and 56% spawning in Zebra mussel; however,



manner. However, it seems that females usually require higher amount of 5-HT than that of a male to release the oocytes. The observed sex-specificity might be related to inter-sex differences in the concentration of 5-HT, which are shown to be higher in males than in females [32,

As 5-HT fibers are localized in the gonad of bivalves, these observations pioneered further research to elucidate mechanism through which 5-HT induces spawning. In Zebra mussel,

spawning when it is added into the aquarium 5 min after addition of 5-HT. However, it is without effects on 5-HT-induced spawning when it is added into the aquarium 10 min after addition [120]. A 2 h pre-treatment of the Zebra mussel with 10−4 M methiothepin decreases parturition from 65 to 8% and from 82 to 1% in the individuals treated with 10−4 and 10−3 M 5-HT, respectively. These suggest that 5-HT-induced spawning requires a certain period of

To better understand which type of 5-HT receptor is involved in 5-HT-induced spawning, further experiments have conducted using 5-HT receptor analogs. It has observed that 10−4 M 8-OH-DPAT, 5-HT, and TFMPP induce 80, 70, and 56% spawning in Zebra mussel; however,

**Species Notes Spawning of female (%) Spawning of male (%) References**

: 87.5Te :

20: 02: 1.10.2: 12.20.02: 2.2

91.7Te : 100

011.1 2: 73.3, 800.2: 1000.02: 200.002: 0

12.5 Te

20: 02: 00.2: 00.02: 0

Control 5-HT (mM) Control 5-HT (mM)

0 0 0 100 <sup>2</sup>

33.3 3.5 66.7 96.6 <sup>2</sup>

0 15.3 0 84.7 <sup>2</sup>

20: 02: 00.2: 00.02: 0

receptor antagonist (**Table 2**), decreases 5-HT-induced

0 2: 1000.2: 800.02:

– 100 [30]

20:23.32: 40.00.2: 36.60.02: 14.4

1000.002: 800.0002: 400.00002: 0

[18]

[113]

[113]

[113]

[114]

34]. Moreover, studies show that 5-HT fully stimulates spawning in ripe individuals.

methiothepin, a non-selective 5-HT1

T: 6.7–10.5M: Injection to gonadD: 0.4 ml of 5-HT solution C: FSW

T: 17–19M: Injection to gonadD: 0.4 ml of 0.1 mM 5-HT C: ASW

T: 25M: Injection to gonadD: 0.4 ml of 2 mM 5-HT C: FSW

T: 20–21M: Injection to gonadD: 0.4 ml of 2 mM 5-HT C: FSW

T: 28–29M: Injection to muscleD: 0.4 ml of 2 mM 5-HT C: FSW

T: 20M: Injection to muscleD: 0.4 ml of 5-HT solution C: FSW

Yesso scallop *Patinopecten yessoensis*

Yesso scallop *Patinopecten yessoensis*

American oyster *Crassostrea virginica*

Bay scallop *Argopecten irradians*

Hard clam *Mercenaria mercenaria*

Hard clam *Mercenaria mercenaria*

time and that 5-HT-induced spawning is irreversible.

16 Serotonin - A Chemical Messenger Between All Types of Living Cells



*Abbreviation*: ASW, artificial seawater; C, injection to control; D, dose; FSW, filtered seawater; M, method; ND, not determined; T, temperature (°C), Te , experimental trial.

1 Values for control are 0% as no individual injected with filtered seawater exhibited spawning behavior [117].

2 Numbers of female and male injected with 5-HT are not determined. Values show percentage of spawned females and males from total number of individuals that spawned following injection of 5-HT. Total percentage of spawning are 27.1% (Ocean quahog), 82.9% (Bay scallop), 70% (American oyster), 45.0% (Ribbed mussel), 41.6% (Hard clam), and 60.0% (Surf clam). In the control group of Bay scallop, Ribbed mussel, and Surf clam, 8.6, 5.0, and 2.2% spawned, respectively. Individual in the control group of American oyster, Hard clam, and Ocean quahog did not spawn.

3 Numbers of female and male injected with 5-HT are not determined. Values show percentage of spawned females and males from total number of individuals that spawned following injection of 5-HT. Total percentage of spawning are 17.1, 22.5, 37.1, and 35.5% in individual spawning trail 1 (Te1), individual spawning trial 2 (Te1), mass spawning trial 1 (Te3), and mass spawning trial 2 (Te4), respectively. Individual spawning represents spawning of a specimen placed in a glass dish (1 l FSW). Mass spawning represents placing of all individuals in troughs (140 l FSW). Individual in any control group did not spawn.

4 Induction of spawning in the male phase of hermaphrodite scallop.

**Species Notes Spawning of female (%) Spawning of male (%) References**

Feb.: 0Mar.: 0Apr.: 0

1000.1: 1001: 10010: 100

0 0.001: 00.01:

0 1 M: 1000.1: 560.01:0

0 2: 1001:

1000.1: 93.30.05: 1000.02: 1000.01: 26.70.001: 0

Feb.: 0Mar.: 0Apr.: 0

0 52.5 0 100 <sup>1</sup>

Feb.: 0Mar.: 0Apr.: 0

0 1: 1000.1: 48.7 0 1: 1000.1: 65.4 [120]

0 0–20 0 100 [105]

0 0 0 100 [123]

40 67 20 90 [106]

0 8.8 0 10: 801: 600.1:

0 2: 1001: 1000.1:

0

93.30.05: 1000.02: 1000.01: 26.70.001:

86.70.01: 1000.001: 500.0001: 0

Feb.: 55Mar.: 100Apr.: 90

600.1: 601: 10010:100

20 0.001: 200.01:

[117]

[118]

[119]

5 [121]

[122]

[124]

Bear paw clam *Hippopus hippopus*

Zigzag scallop *Pecten ziczac*

Doughboy scallop *Mimachlamys asperrima*

Zebra mussel *Dreissena polymorpha*

Fingernail clam *Musculium transversum*

Peruvian scallop *Argopecten purpuratus*

Japanese clam *Mactra chinensis*

Catarina scallop *Argopecten ventricosus*

Manila clam *Ruditapes philippinarum*

Nucleus scallop *Argopecten nucleus*

T: 27.8–30.5M: Injection to gonadD: 1–5 ml of 2 mM 5-HT C: FSW

18 Serotonin - A Chemical Messenger Between All Types of Living Cells

T: 20M: Injection to muscle and gonadD: 0.4 ml of 2 mM 5-HT C: FSW

T: 15M: Injection to gonadD: 0.05 ml of 5-HT solutionC: Saline solution (Instant Ocean, Sarrebourg, France)

T: 12M: 5-HT has added into aquarium, *in vivo*

T: 23M: 5-HT has added into aquarium, *in vivo*

T: NDM: Injection to gonadD: 0.4 ml of 0.02–2 mM 5-HT

T: NDM: Injection to footD: 0.4 ml of 0.001–2 mM 5-HT C: FSW

T: 23M: Injection to gonadD: 0.025–2.5 mM 5-HTC: ND

T: NDM: Injection to footD: 0.2 ml of 5-HT solution C: FSW

T: 22M: Injection to gonadD: 0.2 ml of 1 mM 5-HT solution C: FSW

C: FSW

5 Animals are exposed, and the percentage of parturition is evaluated based on the number of the release of juveniles.

**Table 3.** 5-hydroxytryptamine (serotonin, 5-HT) stimulates spawning in various species of bivalve mollusks.

2-methyl-5-HT and α-methyl-5-HT are without effects (4.1 and 0%) [104]. None of these 5-HT receptor agonists induce spawning at 10−5 M. A 2 h pre-treatment of Zebra mussel with 10−4 M cyproheptadine and mianserin results in 50 and 30% inhibition of 10−3 M 5-HT-induced spawning, respectively, whereas propranolol, 1-NP, NAN-190, and ketanserin are without effects. In addition, cyproheptadine is the only effective analog that totally inhibits 10−4 M 5-HT-induced spawning. A 2 h pre-treatment of Zebra mussel with 10−4 M cyproheptadine or mianserin totally suppress spawning at 10−4 or 10−3 M 8-OH-DPAT-induced Zebra mussel. In addition, 10−4 and 10−3 M 8-OH-DPAT-induced spawning are inhibited by 30 and 60% in the presence of 10−4 M NAN-190, respectively. These results may suggest that 5-HT1 receptor agonists are potent to induce spawning. Antagonists of 5-HT2 receptor are strongly potent to interfere with spawning induced by 5-HT1 receptor agonist; however, they are capable of partially inhibiting 5-HT-induced spawning. The latter note, itself, represents interaction between 5-HT binding sites [104] or suggests the presence of more than one type 5-HT receptor to regulate 5-HT-induced spawning.

In Japanese clam [122], 1, 10, 20, 50, 100, and 1000 μM α-methyl-5-HT injected into the foot induces spawning in 0, 25, 31, 63, 75, and 100% of specimens, respectively, compared to 0% in control and 100% in ≥20 μM 5-HT. In addition, Japanese clams injected with 10, 100, and 1000 μM 8-OH-DPAT into the foot spawns 15, 33, and 100%, respectively. In this species, neither TFMPP nor mCPBG induces spawning in Japanese clam. Injection of mianserin into the foot of Japanese clam decreases spawning to 25 and 0% at 100 and ≥500 μM, respectively. The mianserin-inhibited spawning can be partially overcome by the second injection of 20 μM 5-HT, resulting in 60 and 50% spawning at 100 and 500 μM, respectively. Based on the rank order of potency of the 5-HT agonists, the authors suggested that a mixed 5-HT1 /5-HT2 receptor mediates 5-HT-induced spawning in this species. However, spawning of the individual pre-treated with mianserin may also suggest that 5-HT binding sites to induce spawning are different from those of mianserin. On the other hand, there might be more than one 5-HT receptor in the Japanese clam; however, 5-HT signaling seems to be mediated via a 5-HT1 receptor.

## **4. Conclusion and future research perspectives**

A few studies exist that investigate the characteristics of 5-HT binding site in the plasma membrane of the oocyte and sperm. Pharmacological profiles of binding sites in competition experiments suggest the presence of a single or mixed 5-HT1 , 5-HT2 , and 5-HT3 receptors in bivalves. The phylogenetic analysis of 5-HT receptor suggests that classification of the bivalve 5-HT receptors based on available mammalian 5-HT receptor classification is not successful. It might be due to sensitivity and insensitivity of 5-HT binding sites to 5-HT analogs. On the other hand, the 5-HT receptor(s) in bivalves is distinct from those of other organisms. However, molecular identity of 5-HT receptor shows that the 5-HT receptor in bivalve seems to be a homolog of 5-HT1 receptors in mammals.

Tissue distribution of the 5-HT receptor has shown that it is widely expressed in various organs, although its mRNA transcription is relatively high in the ovary and testis. This suggests multifunctional characteristics of 5-HT in bivalves. In addition, transcription of the 5-HT receptor undergoes seasonal variation. Studying 5-HT content and expression of 5-HT receptor in the nervous system and the gonad of bivalves will help us to better understand 5-HT signaling in reproduction.

To better understand receptor-mediated 5-HT signaling, it requires to produce genetic models of bivalves that do not express 5-HT receptor(s). Another valuable biological tool is to use bivalves that show natural alternations in 5-HT biosynthesis or natural disruption of reproduction. Bivalves host some parasites that particularly infect the reproductive system. For instance, Garnerot et al. [31] observed histopathological changes in the gonad of softshell clam infected with a trematode *Prosorhynchus squamatus*. In infected individual, the follicles and genital follicles are not surrounded by 5-HT-IR fibers around, and 5-HT staining is clearly visible inside the parasite. Another example is protozoan *Marteilioides chungmuensis* that become mature in the oocyte of the pacific oyster [126]. The parasites affect the reproductive follicles causing irregular enlargement of the infected gonadal tissues [127]. Although infected female oysters produced oocytes continuously and spawned repeatedly, however the parasites cause nutritional wasting and mortality, and affect the reproductive output of infected female oyster [127, 128]. Ngo et al. [129] also reported that *M. chungmuensis* delays spawning and cause damages to ripe oocytes. These biological examples of parasite-infected bivalves can provide us with model organisms to study 5-HT regulation of gonadal development and gamete maturation.

## **Conflict of interest**

1000 μM 8-OH-DPAT into the foot spawns 15, 33, and 100%, respectively. In this species, neither TFMPP nor mCPBG induces spawning in Japanese clam. Injection of mianserin into the foot of Japanese clam decreases spawning to 25 and 0% at 100 and ≥500 μM, respectively. The mianserin-inhibited spawning can be partially overcome by the second injection of 20 μM 5-HT, resulting in 60 and 50% spawning at 100 and 500 μM, respectively. Based on the rank

tor mediates 5-HT-induced spawning in this species. However, spawning of the individual pre-treated with mianserin may also suggest that 5-HT binding sites to induce spawning are different from those of mianserin. On the other hand, there might be more than one 5-HT receptor in the Japanese clam; however, 5-HT signaling seems to be mediated via a 5-HT1

A few studies exist that investigate the characteristics of 5-HT binding site in the plasma membrane of the oocyte and sperm. Pharmacological profiles of binding sites in competition

bivalves. The phylogenetic analysis of 5-HT receptor suggests that classification of the bivalve 5-HT receptors based on available mammalian 5-HT receptor classification is not successful. It might be due to sensitivity and insensitivity of 5-HT binding sites to 5-HT analogs. On the other hand, the 5-HT receptor(s) in bivalves is distinct from those of other organisms. However, molecular identity of 5-HT receptor shows that the 5-HT receptor in bivalve seems

Tissue distribution of the 5-HT receptor has shown that it is widely expressed in various organs, although its mRNA transcription is relatively high in the ovary and testis. This suggests multifunctional characteristics of 5-HT in bivalves. In addition, transcription of the 5-HT receptor undergoes seasonal variation. Studying 5-HT content and expression of 5-HT receptor in the nervous system and the gonad of bivalves will help us to better understand 5-HT

To better understand receptor-mediated 5-HT signaling, it requires to produce genetic models of bivalves that do not express 5-HT receptor(s). Another valuable biological tool is to use bivalves that show natural alternations in 5-HT biosynthesis or natural disruption of reproduction. Bivalves host some parasites that particularly infect the reproductive system. For instance, Garnerot et al. [31] observed histopathological changes in the gonad of softshell clam infected with a trematode *Prosorhynchus squamatus*. In infected individual, the follicles and genital follicles are not surrounded by 5-HT-IR fibers around, and 5-HT staining is clearly visible inside the parasite. Another example is protozoan *Marteilioides chungmuensis* that become mature in the oocyte of the pacific oyster [126]. The parasites affect the reproductive follicles causing irregular enlargement of the infected gonadal tissues [127]. Although infected female oysters produced oocytes continuously and spawned repeatedly, however the parasites cause nutritional wasting and mortality, and affect the reproductive output of

, 5-HT2

, and 5-HT3

/5-HT2

recep-

receptors in

order of potency of the 5-HT agonists, the authors suggested that a mixed 5-HT1

**4. Conclusion and future research perspectives**

20 Serotonin - A Chemical Messenger Between All Types of Living Cells

experiments suggest the presence of a single or mixed 5-HT1

receptors in mammals.

receptor.

to be a homolog of 5-HT1

signaling in reproduction.

The authors declare no conflicts of interest, financial or otherwise.

## **Acknowledgements**

This study was supported by Tohoku Ecosystem-Associated Marine Sciences (TEAMS) grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Japan, JSPS KAKENHI (16H04978), JSPS postdoctoral fellow (23-01404), and JAMBIO (23-02) to M.O.

## **Author details**

Sayyed Mohammad Hadi Alavi, Kazue Nagasawa, Keisuke G. Takahashi and Makoto Osada\*

\*Address all correspondence to: makoto.osada.a8@tohoku.ac.jp

Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University, Aramaki, Aoba-ku, Sendai, Japan

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[121] Fong PP, Warner M. Serotonin-induced parturition in the fingernail clam *Sphaerium (Musculium) transversum* (Say). Journal of Experimental Zoology. 1995;**272**:163-166 [122] Fong PP, Deguchi R, Kyozuka K. Serotonergic ligands induce spawning but not oocyte maturation in the bivalve *Mactra chinensis* from central Japan. Biological Bulletin.

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30 Serotonin - A Chemical Messenger Between All Types of Living Cells

Aquaculture. 1985;**47**:321-325


## **Structure-Function of Serotonin in Bivalve Molluscs**

Sayyed Mohammad Hadi Alavi, Kazue Nagasawa,

Keisuke G. Takahashi and Makoto Osada

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69165

#### **Abstract**

It has been observed that 5-HT excites the heart nerves in hard clam and regulates contraction and relaxation of the anterior byssus retractor muscle in the blue mussel. It is now known that 5-HT regulates several neurobehavioral systems such as mood, appetite, sleep, learning, and memory. It also plays critical roles in the physiological functions of peripheral organs involved in stress, growth, and reproduction in the animal kingdom. The present study reviews conserved 5-HT biosynthesis and its localization in the nervous system, and its physiological contribution to regulate reproduction in bivalves. In the cytosol of neurons, tryptophan hydroxylase catalyzes hydroxylation of l-tryptophan to 5-hydroxytryptophan, which is converted to 5-HT by aromatic l-amino acid decarboxylase. A 5-HT transporter and a monoamine oxidase reuptakes and metabolizes 5-HT to control the amount of released 5-HT in the nervous system and peripheral organs. Perikarya and fibers of 5-HT neurons are mostly located in the cortices and neuropil of ganglia, respectively, and innervate the gonad. However, distribution and 5-HT content differ among species and sexes and undergo seasonal variations associated with gonadal development. The present review pays a special attention to future research perspectives to better understand 5-HT regulation of reproduction in bivalves.

**Keywords:** gonad, nervous system, oocyte, serotonin biosynthesis, serotonin metabolism, reuptake, sperm

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

5-Hydroxytryptamine called serotonin (5-HT) is a transmitter substance of the nervous system in animal kingdom. From its first discovery in the 1940s, many laboratories have been directing their studies toward understanding the biology of 5-HT and its physiological functions on various biological systems especially on mammals as model organism [1–15]. However, 5-HT has been also identified in bivalves from the period of its first discovery and earlier studies on these animals have led to convince the neurobiologist that it acts as a neurotransmitter.

A brief bibliography of 5-HT discovery and its physiological functions is provided in **Table 1**. Rapport et al. [16] was the first who isolated a vasoconstrictor substance from the blood serum in a crystalline form and tentatively identified it as 5-HT in a creatinine sulfate complex [17].



*yessoensis*.

**1. Introduction**

34 Serotonin - A Chemical Messenger Between All Types of Living Cells

5-Hydroxytryptamine called serotonin (5-HT) is a transmitter substance of the nervous system in animal kingdom. From its first discovery in the 1940s, many laboratories have been directing their studies toward understanding the biology of 5-HT and its physiological functions on various biological systems especially on mammals as model organism [1–15]. However, 5-HT has been also identified in bivalves from the period of its first discovery and earlier studies on these animals have led to convince the neurobiologist that it acts as a neurotransmitter.

A brief bibliography of 5-HT discovery and its physiological functions is provided in **Table 1**. Rapport et al. [16] was the first who isolated a vasoconstrictor substance from the blood serum in a crystalline form and tentatively identified it as 5-HT in a creatinine sulfate complex [17].

**References**

[16]

[17]

[19]

[21]

[20]

[22]

[23]

[25]

[45]

[46]

[47]

**Year Scientists Contribution to discovery of identification, localization, and characterization of 5-HT**

1947 Rapport et al. Isolation of a substance from the blood serum that constricts blood

1949 Rapport Identification of chemical structure of 5-HT as a creatinine sulfate

1953 Welsh 5-HT, in contrast to acetylcholine, excites the heart nerves in hard

1954 Amin et al. Localization of 5-HT in the central nervous system (brain) of mammals (dog)

1954 Twarog 5-HT-mediated relaxation of byssus retractor muscle in the blue

1956 Hoyle and Lowy 5-HT is a putative neurotransmitter controlling contraction and

1957 Welsh Identification of 5-HT in the extract of nervous system of various

1962 Falck et al. Development of Falck-Hillarp method to visualize monoamine-

1968 Sweeney Identification and localization of 5-HT in whole body extract and in

complex

and rabbit)

the muscle

bivalve mollusks

spinal cord

1964 Dahlström and Fuxe

vessels and contracts isolated intestinal strips

1948 Rapport et al. The substance contains an indole ring [42]

1951 Hamlin and Fisher Synthesis of 5-HT [18] 1953 Twarog and Page Identification of 5-HT in the extract of the brain of mammals (dog, rat,

1953 Gaddum Assigning a role for 5-HT in normal cerebral function in mammals [43]

1954 Wooley and Shaw Human schizophrenia might be due to 5-HT deficiency [44]

1957 Brodie and Shore Assigning 5-HT function as a neurotransmitter [24]

containing cells as intense yellow-green fluorescence

clam (Bivalvia, Mollusca) originating from visceral ganglion

mussel (Bivalvia, Mollusca) is antagonist of acetylcholine contracting

relaxation of the anterior byssus retractor muscle in the blue mussel

Identification of 5-HT cell bodies in the pons and midbrain, from where they project with their axons to the forebrain, medulla, and

the nervous system of blue mussel using Falck-Hillarp's method

**Table 1.** Bibliography of 5-hydroxytryptamine (serotonin, 5-HT): from discovery to physiological characterization.

Within next 5 years, 5-HT has been synthesized [18], identified in the extract of mammalian brain [19], and localized in the brain of mammals [20]. Along with these studies on mammals, Welsh [21], Twarog [22], and Hoyle and Lowy [23] demonstrated that 5-HT excites the heart nerves in hard clam (*Mercenaria mercenaria*), and regulates contraction and relaxation of the anterior byssus retractor muscle in the blue mussel (*Mytilus edulis*) that both belong to Bivalvia, Mollusca. These observations resulted in identification of 5-HT as a neurotransmitter in the nervous system of mammals [24]. In the same year, Welsh [25] identified 5-HT in the nervous system of bivalves and demonstrated that 5-HT content in these animals is higher than other invertebrates and vertebrates [26]. Moreover, bivalves have served some advantages to be used as experimental model: (A) they are small which is a great opportunity to conduct serial examinations on the whole organism, (B) they have a simple nervous system, (C) the nervous system is relatively large in size and easy to dissect ganglia and connectives, and (D) the nervous system contains high amount of 5-HT.

Serotonin regulates various neurobehavioral systems (such as mood, appetite, sleep, learning, and memory). However, studies have revealed that it also plays critical roles in physiological functions of peripheral organs such as stress and growth [3–5]. One of the major systems that 5-HT contributes to is the regulation of reproduction. In both mammals and bivalves, it has been observed that 5-HT regulates reproductive endocrine system, oocyte maturation, and sperm motility [27–38].

Although 5-HT biosynthesis and its receptor structure have been reviewed in bivalves [39–41], there is a gap of review on physiological signaling of 5-HT in these animals. The present study reviews the biology of 5-HT in bivalves; particularly its contribution to reproduction. Biosynthesis pathway of 5-HT in the nervous system and cellular localization of 5-HT neurons in the nervous system are studied. Particular attention has then paid to 5-HT content and distribution of 5-HT neurons in the gonad. This study provides future perspectives that await investigation to better understand 5-HT network and signaling in bivalve reproduction.

## **2. Biosynthesis, metabolism, and reuptake of 5-HT in the nervous system**

Hamlin and Fisher [18] were the first who synthesized 5-HT from tryptophan. A year later, Blaschko [50] suggested that 5-hydroxytryptophan (5-HTP) is the substrate for 5-HT. This suggestion led to the discovery of an enzyme in mammalian kidney [51], later called aromatic L-amino acid decarboxylase (AADC) [52] that mainly decarboxylates 5-HTP to 5-HT [53]. In parallel, studies have shown that the extract of mammalian brain contains 5-HT [19], and administration of exogenous 5-HTP or tryptophan increases 5-HT level in the brain and peripheral organs [54, 55]. A year later, Welsh and Moorhead [56] observed that homogenates of ganglia of hard clam are capable of synthesizing 5-HT from 5-HTP, *in vitro*. Further studies using the blue mussel (*Mytilus edulis*) demonstrated presence of precursors of 5-HT (either tryptophan or 5-HTP) [57–59], and decarboxylation of 5-HTP to 5-HT [60, 61]. Thus, 5-HT biosynthesis in bivalves is similar to those of higher vertebrates. Although aforementioned studies have shown biosynthesis pathway of 5-HT and demonstrated that both nervous system and peripheral organs contain 5-HT; however, it was still unknown where the 5-HT biosynthesis takes place and how it gets transferred to other organs.

In 1960s, Bertaccini [62] and Gal et al. [63] demonstrated that the brain contains 5-HT even after partial or complete removal of 5-HT in the gastro-intestinal tissues and the brain produces 5-HT after intracerebral injection of radioactive labeled tryptophan. It is worth noting that it has previously been shown that the intestine contains large amount of 5-HT [64]. These studies provided the scientists with very important information that the brain independently synthesizes 5-HT from L-tryptophan, and suggested that exogenous 5-HT administration incorporates to 5-HT contents in the nervous system. Next studies resulted in molecular identity of two major enzymes in 5-HT biosynthesis pathway: tryptophan hydroxylase (TPH) and AADC [6, 14, 65, 66] (**Figure 1**). In the cytosol of the nerve cells, TPH catalyzes hydroxylation of l-tryptophan to produce 5-HTP by incorporation of an atom of atmospheric oxygen into l-tryptophan and the other is reduced to water, in the presence of the cofactor agent, tetrahydrobiopterin. The pathway is rate-limiting step meaning that suppression of TPH activity results in stopping 5-HT biosynthesis. The AADC catalyzes conversion of 5-HTP to 5-HT which is not rate-limiting step. It has also been shown that the rate at which 5-HT is produced in the central nervous system highly depends on availability of tryptophan, tryptophan uptake into the brain, and dietary contents of tryptophan and other amino acids (such as tyrosine and phenylalanine) that compete with tryptophan uptake or transport carrier into the brain [8, 14, 67].

In the snail, it has been observed that certain nerves are capable of accumulating radioactive labeled 5-HT [68]. Using bivalves, Stefano and Aiello [69] observed that fluorescence intensity of 5-HT-immunoreactive (5-HT-IR) neurons increases in the blue mussel after administration of exogenous 5-HT. Thus, as in mammals, 5-HT biosynthesis in bivalve mollusks also takes place in the nervous system.

Further studies have shown that there are biological systems through which external amounts of the released 5-HT is regulated, as its rise may cause abnormal physiological functions or might be lethal for cells. Reuptake and metabolism of 5-HT are key determinants to remove and/or inactivate significant amount of released 5-HT, respectively. Metabolism of 5-HT is mediated by monoamine oxidase (MOA) located in the outer membrane of mitochondria, and catalyzes the oxidative deaminative of 5-HT to 5-hydroxy-3-indolacetaldehyde (5-HIAL), which is further metabolized into 5-hydroxy-3-indolacetic acid (5-HIAA) by an NAD+ -dependent aldehyde dehydrogenase. In addition, an NADH-dependent aldehyde reductase or an NADPH-dependent alcohol-dehydrogenase converts 5-HIAL to 5-hydroxytryptophol (5-HTOL) [6, 70] (**Figure 1**). In mollusks, small amount of MOA has been reported [71]. Boutet et al. [72] cloned MOA molecular structure in the Pacific oyster. Administration of MAO inhibitor leads to increase in the number and intensity of 5-HT-IR neurons in the blue mussel [69]. Thus, metabolism of 5-HT is active in bivalve mollusks. However, studies have demonstrated that 5-HT action at the synapse is mostly terminated by its reuptake across the presynaptic membrane [73–77].

suggestion led to the discovery of an enzyme in mammalian kidney [51], later called aromatic L-amino acid decarboxylase (AADC) [52] that mainly decarboxylates 5-HTP to 5-HT [53]. In parallel, studies have shown that the extract of mammalian brain contains 5-HT [19], and administration of exogenous 5-HTP or tryptophan increases 5-HT level in the brain and peripheral organs [54, 55]. A year later, Welsh and Moorhead [56] observed that homogenates of ganglia of hard clam are capable of synthesizing 5-HT from 5-HTP, *in vitro*. Further studies using the blue mussel (*Mytilus edulis*) demonstrated presence of precursors of 5-HT (either tryptophan or 5-HTP) [57–59], and decarboxylation of 5-HTP to 5-HT [60, 61]. Thus, 5-HT biosynthesis in bivalves is similar to those of higher vertebrates. Although aforementioned studies have shown biosynthesis pathway of 5-HT and demonstrated that both nervous system and peripheral organs contain 5-HT; however, it was still unknown where the 5-HT bio-

In 1960s, Bertaccini [62] and Gal et al. [63] demonstrated that the brain contains 5-HT even after partial or complete removal of 5-HT in the gastro-intestinal tissues and the brain produces 5-HT after intracerebral injection of radioactive labeled tryptophan. It is worth noting that it has previously been shown that the intestine contains large amount of 5-HT [64]. These studies provided the scientists with very important information that the brain independently synthesizes 5-HT from L-tryptophan, and suggested that exogenous 5-HT administration incorporates to 5-HT contents in the nervous system. Next studies resulted in molecular identity of two major enzymes in 5-HT biosynthesis pathway: tryptophan hydroxylase (TPH) and AADC [6, 14, 65, 66] (**Figure 1**). In the cytosol of the nerve cells, TPH catalyzes hydroxylation of l-tryptophan to produce 5-HTP by incorporation of an atom of atmospheric oxygen into l-tryptophan and the other is reduced to water, in the presence of the cofactor agent, tetrahydrobiopterin. The pathway is rate-limiting step meaning that suppression of TPH activity results in stopping 5-HT biosynthesis. The AADC catalyzes conversion of 5-HTP to 5-HT which is not rate-limiting step. It has also been shown that the rate at which 5-HT is produced in the central nervous system highly depends on availability of tryptophan, tryptophan uptake into the brain, and dietary contents of tryptophan and other amino acids (such as tyrosine and phenylalanine) that compete with tryptophan uptake or transport carrier into

In the snail, it has been observed that certain nerves are capable of accumulating radioactive labeled 5-HT [68]. Using bivalves, Stefano and Aiello [69] observed that fluorescence intensity of 5-HT-immunoreactive (5-HT-IR) neurons increases in the blue mussel after administration of exogenous 5-HT. Thus, as in mammals, 5-HT biosynthesis in bivalve mollusks also takes

Further studies have shown that there are biological systems through which external amounts of the released 5-HT is regulated, as its rise may cause abnormal physiological functions or might be lethal for cells. Reuptake and metabolism of 5-HT are key determinants to remove and/or inactivate significant amount of released 5-HT, respectively. Metabolism of 5-HT is mediated by monoamine oxidase (MOA) located in the outer membrane of mitochondria, and catalyzes the oxidative deaminative of 5-HT to 5-hydroxy-3-indolacetaldehyde (5-HIAL), which is further metabolized into 5-hydroxy-3-indolacetic acid (5-HIAA) by an

synthesis takes place and how it gets transferred to other organs.

36 Serotonin - A Chemical Messenger Between All Types of Living Cells

the brain [8, 14, 67].

place in the nervous system.

**Figure1.** Biosynthesis, metabolism and reuptake of 5-hydroxytryptamine (serotonin, 5-HT) in bivalves. In the cytosol of the 5-HT neurons, tryptophan hydroxylase (TPH) catalyzes hydroxylation of l-tryptophan to produce 5-hydroxytryptophan (5-HTP) that becomes converted to 5-HT by aromatic l-amino acid decarboxylase (AADC). Conversion of l-tryptophan to 5-HTP is rate-limiting step meaning that suppression of TPH activity results in stopping 5-HT biosynthesis, however AADC-catalyzed conversion of 5-HTP to 5-HT is not rate-limiting pathway. The 5-HT vesicles are transferred to axon terminal and released to synaptic cleft. Reuptake and metabolism of 5-HT are key determinants to inactivate significant amount of the released 5-HT. In mollusks including bivalves, 5-HT reuptake from synaptic cleft is more than the enzymatic destruction. It is an ionic-coupled system and mediated by a serotonin transporter (SERT) that transports 5-HT from synaptic cleft to the presynaptic 5-HT neuron. However, enzymatic destruction of 5-HT also exists which is mediated by monoamine oxidase (MOA) located in the outer membrane of mitochondria (Mt). The MOA catalyzes the oxidative deaminative of 5-HT to 5-hydroxy-3-indolacetaldehyde (5-HIAL) that is metabolized into 5-hydroxy-3 indolacetic acid (5-HIAA) by aldehyde dehydrogenase (ALDH). Released 5-HT binds to its receptor(s) on the surface of a postsynaptic cell or postsynaptic neuron (not shown in the figure) to trigger intracellular signaling required for a cellular response, e.g., stimulation of oocyte and sperm maturation. The 5-HT receptors are mainly G-protein coupled receptor (5-HT1, 2, 4, 6, 5, and 7 receptors), which induce adenylate cyclase (AC) or phospholipase C signaling (PLC). However, the 5-HT<sup>3</sup> receptor is a ligand-gated ion channel and regulates ionic influx.

The 5-HT reuptake is also similar between mollusks and mammals. It is an ionic-coupled pathway mediated by a serotonin transporter (SERT) that transport 5-HT from synaptic cleft to the presynaptic neuron [9, 12, 78]. SERT first binds a Na<sup>+</sup> ion, followed by 5-HT, and then a Cl– ion in the synaptic cleft and transport to presynaptic neuron. After releasing 5-HT, K+ efflux is involved in the translocation mechanism of SERT. This is an energy dependent process and a Na+ /K+ ATPase maintains the extracellular Na+ concentration as well as the intracellular K+ concentration [79]. This mechanism results in the inactivation of 5-HT by removing it from the synaptic cleft. Studies have also shown that a 5-HT reuptake inhibitor (SRI) interferes with SERT function to inhibit or suppress 5-HT reuptake [80, 81].

## **3. Anatomy of the nervous system in bivalves**

#### **3.1. Nervous system**

In bivalves, the nervous system is bilaterally symmetrical, decentralized, and consists of cerebral ganglia (CG), pedal ganglia (PG), and visceral ganglia (VG). The ganglia are joined by a cerebral commissure, a visceral commissure, and cerebral-pedal, cerebral-visceral, and cerebralvisceral-pedal connectives [82–86] (**Figure 2**). Each ganglion is surrounded by a perineurium.

**Figure 2.** Anatomy of the nervous system in bivalves. It is decentralized and consists of bilaterally symmetrical cerebral ganglia (CG), pedal ganglia (PG), and visceral ganglia (VG). The locations of ganglia highly differ among species; however, they are connected by nerve connectives. The PG are absent in oysters (e.g., Pacific oyster, *Crassostrea gigas*, A). All parts of the nervous system exist in scallops (e.g., Yesso scallop, *Patinopecten yessoensis*, B) and clam species (e.g., Manila clam, *Ruditapes philippinarum*, C). Panels a, b, and c are representative schematics of intercommunicating ganglia in Pacific oyster [83], Yesso scallop (the authors), and soft-shell clam, *Mya arenaria* [89], respectively. In most bivalves, VG innervates the gonad. AMa and p, anterior and posterior adductor muscle; AMN, adductor muscle nerves; BN, bronchial nerves; CC, cerebral commissure; CPC, cerebral-pedal connective; CVC, cerebral-visceral connective; DG, digestive gland; F, foot; G, gonad; GN, gonad nerves; Gi, gills; K, kidney; M, mantle; P, labial palp; PN, pallial nerve; Sin, incurrent siphon; Sex, excurrent siphon.

The neuronal cell bodies "perikarya" are located at the cortices and the axonal processes lie at central core called "neuropil".

The 5-HT reuptake is also similar between mollusks and mammals. It is an ionic-coupled pathway mediated by a serotonin transporter (SERT) that transport 5-HT from synaptic cleft

it from the synaptic cleft. Studies have also shown that a 5-HT reuptake inhibitor (SRI) inter-

In bivalves, the nervous system is bilaterally symmetrical, decentralized, and consists of cerebral ganglia (CG), pedal ganglia (PG), and visceral ganglia (VG). The ganglia are joined by a cerebral commissure, a visceral commissure, and cerebral-pedal, cerebral-visceral, and cerebralvisceral-pedal connectives [82–86] (**Figure 2**). Each ganglion is surrounded by a perineurium.

**Figure 2.** Anatomy of the nervous system in bivalves. It is decentralized and consists of bilaterally symmetrical cerebral ganglia (CG), pedal ganglia (PG), and visceral ganglia (VG). The locations of ganglia highly differ among species; however, they are connected by nerve connectives. The PG are absent in oysters (e.g., Pacific oyster, *Crassostrea gigas*, A). All parts of the nervous system exist in scallops (e.g., Yesso scallop, *Patinopecten yessoensis*, B) and clam species (e.g., Manila clam, *Ruditapes philippinarum*, C). Panels a, b, and c are representative schematics of intercommunicating ganglia in Pacific oyster [83], Yesso scallop (the authors), and soft-shell clam, *Mya arenaria* [89], respectively. In most bivalves, VG innervates the gonad. AMa and p, anterior and posterior adductor muscle; AMN, adductor muscle nerves; BN, bronchial nerves; CC, cerebral commissure; CPC, cerebral-pedal connective; CVC, cerebral-visceral connective; DG, digestive gland; F, foot; G, gonad; GN, gonad nerves; Gi, gills; K, kidney; M, mantle; P, labial palp; PN, pallial nerve; Sin, incurrent siphon; Sex, excurrent siphon.

 ion in the synaptic cleft and transport to presynaptic neuron. After releasing 5-HT, K+ efflux is involved in the translocation mechanism of SERT. This is an energy dependent pro-

concentration [79]. This mechanism results in the inactivation of 5-HT by removing

ion, followed by 5-HT, and then

concentration as well as the intracel-

to the presynaptic neuron [9, 12, 78]. SERT first binds a Na<sup>+</sup>

38 Serotonin - A Chemical Messenger Between All Types of Living Cells

**3. Anatomy of the nervous system in bivalves**

ATPase maintains the extracellular Na+

feres with SERT function to inhibit or suppress 5-HT reuptake [80, 81].

a Cl–

lular K+

cess and a Na+

**3.1. Nervous system**

/K+

The pairs of CG lie on the sides of esophagus and are connected by a cerebral commissure in bivalves. In oyster species, CG are less developed and positioned at the sharp angle anterior to the labial palp, gills, and digestive gland [83]. In mussel and clam species, CG are located anterior to the digestive gland, and beneath the anterior adductor muscle [82, 84]. In freshwater pearl mussel (*Hyriopsis bialata*), CG are fused [87]. In scallop species, the foot is positioned anterior to CG, and adductor muscle and digestive gland are located posterior to CG [82, 86]. Each CG consists of an anterior lobe and a posterior lobe [88]. The CG innervate the palps, anterior adductor muscle, and parts of mantle [83, 84, 86].

In most bivalves, the pairs of PG lie on the foot and are connected by a pedal commissure [84–86]. However, PG are absent in oyster species [83]. In soft-shell clam (*Mya arenaria*), the PG are fused [89]. In freshwater pearl mussel, PG are positioned in the visceral mass [87]. The PG innervate the foot [84, 86].

The paired VG are located on the ventral side of the adductor muscle, usually posterior to foot. In most bivalves, ganglia of VG are fused into a single organ [83, 89–91]. In scallop species, VG consist of five lobes; two anterior lobes, a posterior lobe, and two lateral lobes [88, 90]. There is an accessory ganglion that locates at the point of the lateral lobes. The CG and VG are joined by a pair of cerebral-visceral connective that pass through the digestive gland or gonad. The VG innervate various organs, including gonads, gills, hearts, sensory organs, posterior adductor muscle, and parts of mantle [83, 84, 86].

## **3.2. Anatomy and annual cycle of neurosecretory cells in bivalves**

Rawitz [92] seems to be first who isolated pear- or club-shaped neurons from the European flat oyster (*Ostrea edulis*). The neurons are classified into unipolar, bipolar, and multipolar neurons (**Figure 3**) [93]. Illanes-Bucher [94] classified the neurosecretory cells into A1, A2, A3, and A4 in the blue mussel. The A1-type neurons are small (6–15 μm), unipolar, and nucleus is located opposite to the axonal cone. The A2-type nerve cells are large (20–30 μm), multipolar, and nucleus is eccentric. The A3-type nerve cells are large (20–25 μm), unipolar, and nucleus is eccentric. The A4-type nerve cells are medium in size (12–15 μm), apparently unipolar, and contain numerous vacuoles surrounded by neurosecretory granules. Blake [95] observed that the neurosecretory cycle of neurons in the CG of the Bay scallop (*Argopecten irradians*) appeared identical to that of the VG. The neurosecretory cells also undergo distinct annual cycle [96–99]. Seasonal changes in the activity of neurosecretory cells are also associated with gonadal development, and the cells release their products at maturity stage [96]. Moreover, number of active neurosecretory cells positively correlates with progress of the gonad development in the Bay scallop [95], clam (*Katelysia opima*) [100], blue mussel [101], and greenlipped mussel (*Perna canaliculus*) [102].

### **3.3. Identification and cellular localization of 5-HT**

Cellular localization of 5-HT neurons and its quantitative bioassay in the nervous system and gonads provide us with highly satisfactory knowledge to elucidate ontogeny and developmental

**Figure 3.** Cellular localization of 5-hydroxytryptamine (serotonin, 5-HT) in the nervous system (A–F) and gonad (G–J) of bivalves. (A) The 5-HT immunoreactive (5-HT-IR) cell bodies (arrows) and fibers (arrowheads) in the cortex (C) and neuropil (N) of cerebral ganglia (CG) (135×). (B) A few 5-HT-IR unipolar neurons with cell bodies (arrows) and their process in the CG (360×). (C) 5-HT-IR neurons (arrows) and fibers (arrowheads) in the visceral ganglion (380×). (D) a 5-HT-IR multipolar neuron with its processes (arrows) in pedal ganglion (PG) (800×). (E) Pear-shaped unipolar 5-HT-IR neurons and fibers in cortex (C) and neuropil (N) of PG. The arrowheads show long process of (the axon) of a 5-HT-IR neuron that runs toward commissure (CM) (315×). CVPC is cerebral-visceral-pedal connective. (A)–(C) [103], (D) and (E) [104] show localization of the 5-HT neurons in *Mytilus galloprovincialis*. (F) A schematic of localization of the 5-HT neurons in Yesso scallop, *Patinopecten yessoensis* (■) [105] and great scallop, *Pecten maximus* (Δ) [90]. (G) and (H) the 5-HT-IR fibers in the testis of *Mya arenaria* and *Venus verrucosa*, respectively. The 5-HT-IR fibers (Sf) are seen in the testis containing spermatogonia (Spg) at early stage II of the development (I). The 5-HT-IR fibers (arrows) originated from cerebral-visceral connective (yellow asterisk) surround acini full of sperm (black asterisk) (H). (I) and (J) The 5-HT-IR fibers in the ovary of *M. arenaria* and *V. verrucosa*, respectively. The 5-HT-IR fibers (Sf) surround the ovary containing post-vitellogenic oocytes (Ov) (I). The 5-HT-IR fibers (arrow) surround the wall of the follicles filled with mature oocytes (asterisk) (J). Scale bar G and I = 100 μm [106] and H and J = 20 μm [91].

biology of 5-HT biosynthesis, release, and reuptake, and to understand 5-HT regulation of reproduction in bivalves.

#### *3.3.1. 5-HT in the nervous system of bivalves*

**Figure 3.** Cellular localization of 5-hydroxytryptamine (serotonin, 5-HT) in the nervous system (A–F) and gonad (G–J) of bivalves. (A) The 5-HT immunoreactive (5-HT-IR) cell bodies (arrows) and fibers (arrowheads) in the cortex (C) and neuropil (N) of cerebral ganglia (CG) (135×). (B) A few 5-HT-IR unipolar neurons with cell bodies (arrows) and their process in the CG (360×). (C) 5-HT-IR neurons (arrows) and fibers (arrowheads) in the visceral ganglion (380×). (D) a 5-HT-IR multipolar neuron with its processes (arrows) in pedal ganglion (PG) (800×). (E) Pear-shaped unipolar 5-HT-IR neurons and fibers in cortex (C) and neuropil (N) of PG. The arrowheads show long process of (the axon) of a 5-HT-IR neuron that runs toward commissure (CM) (315×). CVPC is cerebral-visceral-pedal connective. (A)–(C) [103], (D) and (E) [104] show localization of the 5-HT neurons in *Mytilus galloprovincialis*. (F) A schematic of localization of the 5-HT neurons in Yesso scallop, *Patinopecten yessoensis* (■) [105] and great scallop, *Pecten maximus* (Δ) [90]. (G) and (H) the 5-HT-IR fibers in the testis of *Mya arenaria* and *Venus verrucosa*, respectively. The 5-HT-IR fibers (Sf) are seen in the testis containing spermatogonia (Spg) at early stage II of the development (I). The 5-HT-IR fibers (arrows) originated from cerebral-visceral connective (yellow asterisk) surround acini full of sperm (black asterisk) (H). (I) and (J) The 5-HT-IR fibers in the ovary of *M. arenaria* and *V. verrucosa*, respectively. The 5-HT-IR fibers (Sf) surround the ovary containing post-vitellogenic oocytes (Ov) (I). The 5-HT-IR fibers (arrow) surround the wall of the follicles filled with mature oocytes

(asterisk) (J). Scale bar G and I = 100 μm [106] and H and J = 20 μm [91].

40 Serotonin - A Chemical Messenger Between All Types of Living Cells

Welsh [25] was the first who identified 5-HT in the nervous system of the hard clam using a paper chromatography method. Then, Welsh and Moorhead [26, 56, 107] used a spectrofluorometric method to measure 5-HT in over 60 species from 11 different phyla that includes 7 bivalve species (**Table 2**) [108]. They reported that (A) the nervous system of bivalves contains much higher 5-HT than that of other invertebrates. In the phylum Annelida, 5-HT is measured 0.1–10.4 μg/g wet in the nerve cords. In the phylum of Arthropoda, 5-HT is measured from <1.0 μg/g wet in the nerve cords, ventral ganglia, and green ganglia. In vertebrates, 5-HT is measured 0.3–2.6 μg/g wet in different parts of cat brain [109]. (B) Content of 5-HT is higher in the nervous system than the peripheral organs. (C) Content of 5-HT differs among various parts of the nervous system. It is higher in the ganglia than the connective nerves. In addition, they observed that 5-HT content is slightly lower in VG than those of CG and PG (10 vs. 15 μg/g wet) in the blue mussel. (D) The blood does not contain 5-HT. The authors suggested that 5-HT is produced in the nervous system: in cell bodies or synaptic region of neurons.




**Species Notes Nervous system Gonad Reference**

5-HT CG + PG + VG

release)

release)

VG

29.4 ± 4.3 (before spawning) 17.9\* ± 0.6 (after sperm

22.5 ± 0.5 (after oocyte

21.3\* ± 2.3 (24 h after spawning) CG + PG

107.3 ± 12.9 (**b**efore spawning) 63.6 ± 2.1\* (spawned) 100.0 ± 16.3 (unspawned)

CG + PG + VG 5-HTP: 1650 ± 715 5-HT: 1150 ± 525 5-HIAA: 180 ± 90

CG + PG + VG 5-HT: 1483 ± 828

5-HT CG + PG + VG 48.3 ± 7.2 (0 d) 46.2 ± 9.7 (0.5 d) 40.0 ± 5.6 (1 d) 37.9 ± 3.5 (7 d) 44.5 ± 5.7 (14 d) 39.0 ± 6.0 (21 d) 47.2 ± 6.2 (28 d) 63.3 ± 12.6 (35 d)

50.7 ± 4.3 (before spawning) 51.8 ± 5.1 (spawned) 53.3 ± 12.4 (unspawned)

5-HT

100 (T)

65 (T)

150 (T)

400 (T)

5-HT

release)

release)

release)

release)

5-HT

(0 d)

(0.5 d)

(1 d)

(7 d)

(14 d)

(21 d)

(28 d)

(35 d)

(T) portion

I: 625 ± 100 (O), 550 ±

[112]

[113]

[114]

[116]

II: 175 ± 50\* (O), 225 ±

III: 350 ± 95\* (O), 500 ±

IV: 1050 ± 250 (O), 575 ±

Ovary portion of gonad 1.0 ± 0.03 (before spawning) 0.6\* ± 0.02 (after sperm

0.5\* ± 0.05 (after oocyte

0.7 ± 0.15 (24 h after spawning)

Testis portion of gonad 1.7 ± 0.15 (before spawning)

0.8\* ± 0.05 (after sperm

0.7\* ± 0.09 (after oocyte

5-HT: 791 ± 408 [115]

Gonad ovary (O) or testis

1.3 ± 0.02 O, 6.8 ± 0.5 T

0.7 ± 0.03 O, 2.2 ± 0.7 T

0.7 ± 0.02 O, 2.5 ± 0.5 T

1.5 ± 0.34 O, 3.0 ± 0.5 T

1.6 ± 0.02 O, 4.8 ± 0.4 T

1.4 ± 0.03 O, 4.6 ± 1.3 T

1.0 ± 0.04 O, 4.4 ± 0.4 T

1.1 ± 0.01 O, 4.9 ± 0.9 T

1.2 ± 0.05 (24 h after spawning)

5-HTP: 2035 ± 520 5-HT: 1000 ± 180 5-HIAA: 90 ± 15

Surf clam *Spisula solidissima*

Peruvian scallop *Argopecten purpuratus*

Atlantic deep-sea

Atlantic deep-sea

Peruvian scallop *Argopecten purpuratus*

scallop *Placopecten magellanicus*

scallop *Placopecten magellanicus* M: HPLC

SEM) I. Active stage II. Ripe stage III. Spawning stage IV: Spent stage

SEM)

species

gonad

of the gonad \* shows *P* < 0.05 compared to before spawning

M: HPLC-ED V: pg/mg wet (mean ±

M: HPLC-ED V: pg/mg wet (mean ±

Samples of March

Samples of March–May

M: Spectrofluorometer V: ng/mg wet (mean ±

N.D.)

N.D.)

SEM)

V: ng/g wet (mean ±

42 Serotonin - A Chemical Messenger Between All Types of Living Cells

\* shows *P* < 0.05 compared to stages I and IV

M: Spectrofluorometer V: ng/mg wet (mean ±

It is a hermaphroditic

VG innervates mainly the female portion of the

CG and PG innervate mainly the male portion



**Species Notes Nervous system Gonad Reference**

0.20 ± 0.06 (CG) 0.15 ± 0.004 (VG)

2 CG + 2 PG + 2 VG 25.10 ± 2.71 (Jan.) 26.96 ± 2.11 (Feb.) 32.17 ± 3.85 (Mar.) 41.98 ± 1.22\* (Apr.) 48.15 ± 1.02\*\* (May) 53.13 ± 1.71\*\* (Jun.) 51.74 ± 3.14\*\* (Jul.) 57.28 ± 2.49\*\* (Aug.) 48.90 ± 1.13\* (Sep.) 44.80 ± 1.51\* (Oct.) 35.71 ± 2.70\*\*\* (Nov.) 28.97 ± 2.64 (Dec.)

5-HT

5-HT

extracts)

5-HT CG + PG + VG

5-HT CG + PG + VG 21-39

5-HT CG + PG + VG

5-HT CG + PG + VG 30–40

5-HT CG + PG + VG 8.0–14.3

2.2

5-HT 36 (VG)

Ganglia connectives

22

20

123 ± 12 – 252 ± 34 (PG)

13.4 ± 2.5 (whole body

5.4–8.6 (PG, Mar.) 26.2-42 (PG, Apr.) [117]

[118]

[119]

[120]

[47]

[26]

[26]

[26]

[26]

[26]

[26, 107]

Bent-nose clam *Macoma nasuta*

Blue mussel *Mytilus edulis*

Blue mussel *Mytilus edulis*

Blue mussel *Mytilus edulis*

Fingernail clam *Sphaerium sulcatum*

Ocean quahog *Arctica islandica*

Atlantic jackknife

Soft-shell clam *Mya arenaria*

Hard clam *Venus mercenaria*

Atlantic surf clam *Spisula solidissima*

Atlantic deep-sea

scallop *Placopecten magellanicus*

clam *Ensis directus* M: HPLC-ED

44 Serotonin - A Chemical Messenger Between All Types of Living Cells

SEM)

respectively

V: nM/g.p. (mean ± SEM) Samples of mature individuals in March–May

M: Spectrofluorometer V: μg/g wet (mean ±

\*, \*\*, and \*\*\* show *P* < 0.005, *P* < 0.001, and *P* < 0.05 compared to Jan,

M: Spectrofluorometer V: ng/ganglion pair (mean ± SD)

M: Spectrofluorometer V: μg/g wet (mean ±

M: Spectrofluorometer V: ng/individual (mean

M: Spectrofluorometer

M: Spectrofluorometer

M: Spectrofluorometer

M: Spectrofluorometer

M: Spectrofluorometer

M: Spectrofluorometer

N.D.)

± N.D.)

V: μg/g wet

V: μg/g wet

V: μg/g wet

V: μg/g wet 26 assays during 16

V: μg/g wet

V: μg/g wet

months

*Abbreviation*: CG, cerebral, cerebral-pleural or cerebroid ganglion; d, day; g.p., ganglia pair; HPLC-ED, high-performance liquid chromatography coupled with electrochemical detection; M, methods; N.D., not determined; O, ovary; PG, pedal ganglion; T, testis; SD, standard deviation; SEM, standard error of mean; V, values; VG, visceral ganglion.

**Table 2.** Identification of 5-hydroxytryptophan, (5-HTP), serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the nervous system and gonad of bivalve mollusks.

Following development of cellular and molecular methods, 5-HT has been localized in the nervous system and gonad of several bivalve species (**Table 3**). Firstly, Falck-Hillarp's method has been used to localize 5-HT in fingernail clam (*Sphaerium sulcatum*) [47], blue mussel [69], and Yesso scallop [88]. In this method, histological sections are exposed to gaseous formaldehyde or glyoxylic acid to visualize monoamine containing neurons [45, 121, 122]. In all examined bivalve species, 5-HT-IR neurons are observed in CG, PG, and VG (**Table 3**). However, the Falck-Hillarp's method is not always useful as 5-HT fluorescence tends to faint rapidly. In addition, catecholamines neurons show similar intensity to that of 5-HT neurons at high concentrations [123]. In 1978, Steinbusch et al. [124] developed a rat monoclonal antibody against a 5-HT-bovine serum albumin conjugate to localize 5-HT in nervous system. Further studies have used monoclonal or polyclonal antibody against 5-HT to localize 5-HT-IR neurons in the nervous system and peripheral organs of bivalves (**Table 3**). The advantage of immunohistochemistry method using antibodies against 5-HT is to describe morphology of 5-HT neurons, and to localize 5-HT distribution within different parts of nervous system, precisely. The 5-HT-containing neurons are mostly unipolar, although their sizes may differ among species (**Table 3**). Using an electron microscopy, it has been observed that 5-HT-IR neurons are often in close connection with each other, but without indication of gap junctions or other specialized junctions. The neurons possess numbers of granular vesicles (100–180 nm in Mediterranean mussel) containing 5-HT that concentrated at the cell periphery [104, 125]. It has confirmed that 5-HT-IR fibers are the axon or axon terminals of 5-HT containing neurons that transport 5-HT to peripheral organs. Within the nervous system, 5-HT-IR fibers seem to be synaptic region, an area where release and reuptake of 5-HT occur.

In general, studies on bivalves show that 5-HT-IR neurons are mostly located in the cortices, and 5-HT-IR fibers are located in the neuropil of CG, PG, and VG (**Table 3**). In Yesso scallop, 5-HT-IR neurons are located in the cortices of the right side of the left lobe and in the left side of the right lobe in anterior lobe (AL) of CG, while they are located throughout their cortices in PG and the posterior lobe (PL) of CG [105] (**Figure 3**). In the great scallop [90], distribution of 5-HT-IR neurons in the posterior lobe of CG slightly differs compared to Yesso scallop. In VG, 5-HT-IR neurons are restrictively scattered in the accessory lobe of scallop species [90, 105, 115] or at the roots of branchial nerves in clams [89]. Large numbers of 5-HT-IR fibers have also been observed in the cerebral-pedal, and cerebral-visceral-pedal connectives [90, 103], suggesting that 5-HT transports from CG to VG [69, 89, 90, 105]. Comprehensive overview of



**Species** Fingernail clam

Histochemistry using

a paraformaldehydeinduced fluorescence

method

*Sphaerium* 

*sulcatum*

**Methods**

**Cerebral ganglia**

5-HT-IR unipolar

cells (μm length) are

located in the cortices

at the dorsal and

anteriomedial surfaces

of the ganglion. 5-HT-IR fibers are located in the anterior pallial

nerve, the CVC, CC,

and CPC

5-HT-IR neurons

A few 5-HT-IR neurons

[69]

46 Serotonin - A Chemical Messenger Between All Types of Living Cells

(11–14 μm d.) are

located in the cortex

and neuropil. 5-HT-IR

fibers are observed in

the CVC

(9–14 μm d.) are

only located in the

cortex. Fluorescence

is observed in the

perikarya

Fluorohistochemical

Fluorohistochemical

Fluorohistochemical

Muscles of the

[88]

gonoduct stretched

under the epithelium

in the gonad

reaction is detected

in the neuropil close

to CPC

tendency is high in the

accessory ganglia

reaction is detected in

the neuropil, and its

tendency is higher than

PG and VG

5-HT-IR neurons are

ND

5-HT-IR neurons are

[105]

distributed throughout

the cortex

distributed in the AL

(right side of the left

lobe and left side of

the right lobe), and

throughout the cortex

in PL

Blue mussel

Histochemistry using

a paraformaldehydeinduced fluorescence

method

Yesso scallop

Histochemistry using a

glyoxylic acid-induced

fluorescence method

*Patinopecten* 

*yessoensis*

Yesso scallop

Immunohistochemistry

using a rat monoclonal

5-HT antibody against

a 5-HT-bovine serum

albumin conjugate (coded

YC5/45 HL, Sera-Lab, UK)

Mediterranean

Immunogold labeling

5-HT-IR unipolar

5-HT-IR neurons are

Large numbers of

[103, 104, 125,

126]

5-HT-IR unipolar

neurons and a few

bipolar or multipolar

are clustered in the

cortex. 5-HT-IR

fibers are observed in

neuropil

unipolar and located

in the cortex. Number

of 5-HT-IR neurons is

lower than CG.5-HT-IR

fibers are seen in the

visceral commissure

and CVC

neurons are mostly

located in the cortex

with a few numbers in

the neuropil. 5-HT-IR

fibers are seen in the

CC and CVPC

of nerve cells using an

anti-5-HT raised in rabbits

against formaldehyde

cross-linked 5-HT-bovine

serum albumin

(Immunonuclear, Incstar

Co, Stillwater, MN)

mussel

*Mytilus* 

*galloprovincialis*

*Patinopecten* 

*yessoensis*

*Mytilus edulis*

**Visceral ganglia**

No traces of 5-HT-IR

neurons are observed

in the VG. 5-HT-IR

fibers are observed

**Pedal ganglia** 5-HT-IR fluorescences

are uniformly

distributed in the

cytoplasm of unipolar

neurons (10–25

μm length). Greenyellow fibers extend throughout neuropil

and across the PC

**Gonad**

**Reference**

[47]


**Table3.**Cellular localization of 5-hydroxytryptamine (serotonin 5-HT) in the nervous system and gonad of bivalve mollusks.

visceral connective; CVPC, cerebral-visceral-pedal connective; PC, pedal commissure; d., diameter; ND, no 5-HT-IR neurons or fibers are detected; PL, posterior lobe.

cellular localization of 5-HT indicates that localization and distribution of 5-HT-IR neurons may differ among subclasses of bivalve, for instance between Heterodonta (genus *Mya, Ruditapes*, and *Venus*) and Pteriomorphia (genus *Pecten, Patinopecten*, and *Mytilus*) (**Table 3**). It might be due to differences in location of various parts of nervous system in the body to innervate peripheral organs.

Using histochemistry or immunohistochemistry methods, studies have shown that a few 5-HT-IR neurons are located in the cortex and neuropil of VG compared to those of the CG or PG, for instances in the blue mussel [47, 69, 128], Mediterranean mussel (*Mytilus galloprovincialis*) [103], great scallop [90], Atlantic deep-sea scallop (*Placopecten magellanicus*) [115], and soft-shell clam [89]. Matsutani and Nomura [105] reported no 5-HT-IR neurons in the VG of the Yesso scallop. Although VG contain a few 5-HT-IR neurons, they are usually rich in 5-HT-IR fibers. These studies confirm the Welsh and Moorhead's observation that 5-HT content differs among various parts of the nervous system.

Studies used spectrofluorometric method [26, 47, 56, 118–120] or electrochemical detection coupled with a high-performance liquid chromatography (HPLC-EC) to study 5-HT content in the nervous system of bivalves [90, 110, 114, 115, 117] (**Table 2**). Results confirm aforementioned differences in 5-HT content among various parts of the nervous system, for instance it is higher in the CG than the VG of gaper clam (*Tresus capax*) and bent-nose clam (*Macoma nasuta*) [117]. In addition, the metabolite of 5-HT (5-HIAA) is detected in the nervous system of the brown mussel (*Perna perna*) [110] and Atlantic deep-sea scallop [114], suggesting that metabolism of 5-HT takes place in the nervous system.

Welsh and Moorhead [56] observed that *in vitro* 5-HT synthesis by the nerve tissues undergoes a seasonal variation and suggested seasonal variation of amine oxidase. Further studies have shown that 5-HT content in the nervous system undergoes seasonal variation along with gonadal development in bivalves (**Table 2**). Content of 5-HT increases in the nervous system from early gonadal development to maturity stage in the brown mussel [110] and decreases following spawning in Peruvian scallop (*Argopecten purpuratus*) [113]. York and Twarog [120] reported that 5-HT in the PG of blue mussel is higher in April than March. It has also observed that 5-HT content in the whole nervous system of the blue mussel increases from April to October [118]. As the blue mussel spawns from late spring to late summer [129, 130], these data suggest that 5-HT content increases during spawning. 5-HT content also correlates with the content of its metabolite (5-HIAA), suggesting that metabolism of 5-HT is in parallel to its biosynthesis in the nervous system [110].

### *3.3.2. 5-HT in the gonad of bivalves*

**Species** Freshwater pearl

Immunohistochemistry

using a rabbit polyclonal

anti-5-HT IgG (Zymed

Laboratories, San

Francisco, CA or Sigma-Aldrich Co. LLC.)

mussel

*Hyriopsis bialata*

**Methods**

**Cerebral ganglia**

5-HT-IR neurons are

large (10 × 30 μm d.)

and located at the

periphery of CG.

5-HT-IR fibers are

occasionally detected

**Visceral ganglia**

5-HT-IR perikarya

are large (10 × 30

μm d.) and located

in the cortex of VG.

5-HT-IR fibers are

mostly observed in the

neuropil. Expression

of 5-HT-IR fibers or

neurons is higher in

females than males

48 Serotonin - A Chemical Messenger Between All Types of Living Cells

*Abbreviations*: 5-HT-IR, serotonin-immunoreacted; ACL, accessory lobe; AL, anterior lobe; CC, cerebral commissure; CPC, cerebral-pedal connective; CVC, cerebralvisceral connective; CVPC, cerebral-visceral-pedal connective; PC, pedal commissure; d., diameter; ND, no 5-HT-IR neurons or fibers are detected; PL, posterior lobe.

Cellular localization of 5-hydroxytryptamine (serotonin 5-HT) in the nervous system and gonad of bivalve mollusks.

**Table 3.**

**Pedal ganglia** 5-HT-IR neurons are

large (10 × 30 μm d.)

and located at the

periphery of PG

**Gonad**

**Reference**

[87, 127]

Localization of 5-HT in the gonad has studied in a few species of bivalves (**Table 3**). Using method of Falck-Hillarp, Sweeney [47] and Matsutani and Nomura [88, 105] observed the 5-HT-IR fibers in the gonoduct and epithelium around gonad in the Fingernail clam and Yesso scallop, respectively, and suggested that the 5-HT-IR fibers originate from CVC to innervate the gonad. Further studies using antibodies against 5-HT confirmed existence of 5-HT-IR fibers in the gonad of Yesso scallop [105], great scallop [90], Atlantic deep-sea scallop [115], surf clam [112], warty venus [91], and soft-shell clam [106]. These studies clearly indicated that the nervous system innervation of the gonads is mostly emerged from VG or derived from CVC. The 5-HT-IR fibers surround periphery of collecting tubes and of gonadal lobules (acini) in males and females filled with sperm and oocytes, respectively (**Figure 3**).

As seasonal-dependent 5-HT content in the nervous system, distribution of 5-HT fibers also changes in the gonad throughout reproductive cycle [91, 106, 112, 115] (**Figure 3**; **Tables 2** and **3**). Generally, the 5-HT-IR fibers are occasionally observed around the germinal acini, and extensively distributed around the collective tubes at early developmental stage. However, the 5-HT-IR fibers around the acini are more frequent at maturity stage [112]. After spawning, the 5-HT-IR fibers still exist around collecting tubes, and are abundant around gamete empty acini.

Using spectrofluorometric or HPLC-EC method, 5-HT content has been measured in the gonad of the Atlantic deep-sea scallop [114, 115], surf clam [112], Pacific lion's paw scallop (*Nodipecten subnodosus*) [111], and brown mussel [110]. Matsutani [131] reported a tendency toward an increase and a decrease of 5-HT content in the testis and ovary of Japanese scallop (*Chlamys farreri nipponensis*) during spawning, respectively. It has shown that 5-HT content increases from early developmental stage of the gonad to maturity stage in males and females [110, 111]. In surf clam, Masseau et al. [112] reported that changes in 5-HT content are uncertain in males during testicular development and after spawning. However, in females, 5-HT is high at early development stage, decreases at maturity stage and spawning, and then increases after spawning. They also reported that 5-HT content does not differ between males and females when they are compared at similar gonadal development stage. Klouche et al. [110] pooled the data of males and females in brown mussel, as there are no differences between sexes, and observed that 5-HT content increases toward maturation of gonad. In Peruvian scallop, 5-HT content decreases in the male and female portions of gonad following spawning [113, 116]. Observed differences in 5-HT content among studies may represent inter-species differences associated with 5-HT regulation of reproduction that might also be different between sexes. Klouche et al. [110] reported that the gonadal content of 5-HT metabolite (5-HIAA) in brown mussel is high at early development and become decreased at maturity stage. As 5-HT content is high at maturity, these suggest that 5-HT-dependent reproduction associates with decreasing 5-HT inactivation mediated by its metabolism.

A few studies show 5-HT content in both nervous system and gonad, for instance in the Peruvian scallop [113, 116] and brown mussel [110]. Results show higher 5-HT content in the nervous system than gonadal tissue as 5-HT content is lower in connective nerves than 5-HT neurons [26, 56].

Croll et al. [115] observed that distribution of 5-HT-IR neurons and fibers is similar between juvenile and adult in the Atlantic deep-sea scallop or between sexes in the surf clam [112]. However, abundance or distribution of 5-HT neurons and 5-HT content may differ between sexes. Martínez and Rivera [116] observed that 5-HT content is higher in the male portion than female portion of the gonad of the Peruvian scallop. Expression of 5-HT-IR fibers or neurons has been seen to be higher in the VG of females than that of males [127]. These studies may suggest inter-sex difference in 5-HT biosynthesis or inter-sex difference in 5-HT regulatory function of reproduction.

## **4. Conclusion and future research perspectives**

that the nervous system innervation of the gonads is mostly emerged from VG or derived from CVC. The 5-HT-IR fibers surround periphery of collecting tubes and of gonadal lobules

As seasonal-dependent 5-HT content in the nervous system, distribution of 5-HT fibers also changes in the gonad throughout reproductive cycle [91, 106, 112, 115] (**Figure 3**; **Tables 2** and **3**). Generally, the 5-HT-IR fibers are occasionally observed around the germinal acini, and extensively distributed around the collective tubes at early developmental stage. However, the 5-HT-IR fibers around the acini are more frequent at maturity stage [112]. After spawning, the 5-HT-IR fibers still exist around collecting tubes, and are abundant around gamete empty acini.

Using spectrofluorometric or HPLC-EC method, 5-HT content has been measured in the gonad of the Atlantic deep-sea scallop [114, 115], surf clam [112], Pacific lion's paw scallop (*Nodipecten subnodosus*) [111], and brown mussel [110]. Matsutani [131] reported a tendency toward an increase and a decrease of 5-HT content in the testis and ovary of Japanese scallop (*Chlamys farreri nipponensis*) during spawning, respectively. It has shown that 5-HT content increases from early developmental stage of the gonad to maturity stage in males and females [110, 111]. In surf clam, Masseau et al. [112] reported that changes in 5-HT content are uncertain in males during testicular development and after spawning. However, in females, 5-HT is high at early development stage, decreases at maturity stage and spawning, and then increases after spawning. They also reported that 5-HT content does not differ between males and females when they are compared at similar gonadal development stage. Klouche et al. [110] pooled the data of males and females in brown mussel, as there are no differences between sexes, and observed that 5-HT content increases toward maturation of gonad. In Peruvian scallop, 5-HT content decreases in the male and female portions of gonad following spawning [113, 116]. Observed differences in 5-HT content among studies may represent inter-species differences associated with 5-HT regulation of reproduction that might also be different between sexes. Klouche et al. [110] reported that the gonadal content of 5-HT metabolite (5-HIAA) in brown mussel is high at early development and become decreased at maturity stage. As 5-HT content is high at maturity, these suggest that 5-HT-dependent repro-

(acini) in males and females filled with sperm and oocytes, respectively (**Figure 3**).

50 Serotonin - A Chemical Messenger Between All Types of Living Cells

duction associates with decreasing 5-HT inactivation mediated by its metabolism.

neurons [26, 56].

tory function of reproduction.

A few studies show 5-HT content in both nervous system and gonad, for instance in the Peruvian scallop [113, 116] and brown mussel [110]. Results show higher 5-HT content in the nervous system than gonadal tissue as 5-HT content is lower in connective nerves than 5-HT

Croll et al. [115] observed that distribution of 5-HT-IR neurons and fibers is similar between juvenile and adult in the Atlantic deep-sea scallop or between sexes in the surf clam [112]. However, abundance or distribution of 5-HT neurons and 5-HT content may differ between sexes. Martínez and Rivera [116] observed that 5-HT content is higher in the male portion than female portion of the gonad of the Peruvian scallop. Expression of 5-HT-IR fibers or neurons has been seen to be higher in the VG of females than that of males [127]. These studies may suggest inter-sex difference in 5-HT biosynthesis or inter-sex difference in 5-HT regulaThe essential components of 5-HT biosynthetic pathway are highly conserved in the animal kingdom. The 5-HT biosynthesis from the essential amino acid L-tryptophan is catalyzed by TPH, which convert L-tryptophan to 5-HTP, and by AADC, which convert 5-HTP to 5-HT. All precursors of 5-HT are identified in the nervous system of bivalves. In mammals, there are two isoforms of TPH (TPH1 and TPH2), which are predominantly expressed in the peripheral organs and in the nervous system, respectively. However, TPH1 is the primary form and expresses earlier in neural development [132, 133]. Molecular sequence of the gene encoding AADC has also been identified and localized in mammals [134, 135]. It has a non-specific tissue distribution and is expressed in wide range of cell types [66]. In bivalves, molecular identity, localization, and characterization of TPH and AADC are unknown. These studies will provide us with satisfactory information to better understand ontogeny of 5-HT neurons in the nervous system and to elucidate developmental biology of 5-HT regulation of reproduction.

It has been seen that the first 5-HT-IR neurons appearing within the nervous system correspond to the location of the CG and apical ganglion (AG) during the late trochophore stage: 30–32 h postfertilization in blue mussel [136], 24 h postfertilization in surf clam [137], and 27 h postfertilization in the Bay mussel (*Mytilus trossulus*) [138]. Kreiling et al. [137] reported that the 5-HT-IR neurons appear in VG of surf clam at 48 h postfertilization. Following 72 h postfertilization, the 5-HT-IR neurons emerging from the CG and AG extend their processes to the VG, through which connections of the 5-HT-IR neurons between CG/AG and VG are formed at 96 h postfertilization. During the embryonic development, the size of the 5-HT area in the CG/AG and VG increases from 24 h to 96 h postfertilization, which is associated with an increase in 5-HT content. Cann-Moisan et al. [139] reported that 5-HT content undergoes variation throughout the larval and postlarval stages. It rises from 2 d to 27 d postfertilization (15–50 pg/μg of protein, respectively); however, it decreases to less than 1 pg/μg of protein after 55 d postfertilization. These indicate that 5-HT neurons form at the embryonic stage, and 5-HT content increases from embryonic development to metamorphosis, and decreases after metamorphosis. Voronezhskaya et al. [138] observed that 5-HT-IR neurons innervate the peripheral organs in the postmetamorphic stage, suggesting that 5-HT biosynthesis undergoes developmental variation. This might be related to the availability of the 5-HT precursors or inactivation mechanisms of 5-HT. However, further studies are required to investigate development of 5-HT fibers in the gonad through developmental stage.

As animals lost the ability to synthesize tryptophan, there possess developed biological mechanisms through which animals obtain tryptophan from their diets. Thus, 5-HT biosynthesis highly depends on dietary factors including availability of tryptophan and competitive uptake or transport of tryptophan with other amino acids (such as tyrosine and phenylalanine) into the 5-HT neurons. Studying nutritional effects on 5-HT biosynthesis will lead to better understanding of physiological relationships between seasonal variation in 5-HT content and gonadal development. In addition, it can help us to investigate the impacts of parental nutrition on gamete maturation and fertility in bivalves. These studies can provide us with knowledge to better understand 5-HT controls of feeding behaviors such as appetite and satiety, which have been demonstrated in mammals [140].

Mechanisms of 5-HT inactivation in the nervous system and peripheral organs of bivalves are poorly understood. It requires molecular identity, localization, and characterization of SERT and MOA. In this regard, several types of SERT and MOA inhibitors are available [80, 114, 141] that provide us with useful tools to elucidate molecular signaling that control 5-HT reuptake and metabolism. A few studies show that selective 5-HT reuptake inhibitors modulate 5-HT-induced spawning in bivalves. Fong [142] and Fong et al. [143, 144] reported spawning of Zebra mussel treated with selective 5-HT reuptake inhibitors (fluvoxamine, fluoxetine, zimelidine, and paroxetine). Both males and females are capable of releasing their gametes after treatment with fluvoxamine at 10−7 and 10−6 M, respectively. Following treatment with fluoxetine, 100% of males have spawned at 10−4 to 10−5 M, however spawning has induced in 50–60% of females at 10−5 M. Zimelidine induces spawning in 100 and 60–70% of males and females at 10−4 M. Paroxetine induces spawning in 50 and 20% of males and females at 10−6 and 10−5 M, respectively. Considering spawning of males and females at 10−3 M 5-HT, these results indicate that selective 5-HT reuptake inhibitors stimulate spawning in Zebra mussel at concentrations lower than that of 5-HT. Further examinations have revealed that mianserin and cyproheptadine interfere with fluvoxamine-, fluoxetine-, and zimelidine-induced spawning [144] suggesting that antagonists of 5-HT<sup>2</sup> receptor block stimulatory function of selective 5-HT reuptake inhibitors in spawning. Inhibition of 5-HT reuptake may increase the synaptic 5-HT concentrations, which in turn activate postsynaptic 5-HT receptor to induce spawning. It is also possible that selective 5-HT reuptake inhibitors act as ligands at postsynaptic receptor rather than inhibition of SERT. Overall, these studies suggest that 5-HT transport plays a key role in reproduction; however, the mechanisms of action are largely unknown.

So far, histochemistry and immunohistochemistry methods have been employed to localize the 5-HT neurons and fibers, and spectrofluorometric and HPLC-EC methods have been used to identify 5-HT content in the nervous system and gonad of various bivalve species. Successful implication of various mammalian monoclonal or polyclonal antibodies indicates that 5-HT structure is highly conserved through evolution across the animal kingdom. However, mechanisms through which 5-HT acts on a biological system may differ. The present review shows that 5-HT content highly differs in the nervous system and gonad of bivalve species. The inter-species differences in 5-HT content might be related to capability of nervous system to synthesize 5-HT, differences in 5-HT inactivation or 5-HT transport from nervous system to the gonad. In the latter case, 5-HT content in the gonad may correspond to 5-HT concentration that requires to stimulate spawning. The present review shows that 5-HT concentration to induce spawning highly differs between sexes, and among species. It is worth to note that tissue sampling, extraction procedure, and analytical method affect the results of 5-HT content. In addition, 5-HT content undergoes seasonal variation and change following spawning.

#### **Conflict of interest**

The authors declare no conflicts of interest, financial or otherwise.

## **Acknowledgements**

knowledge to better understand 5-HT controls of feeding behaviors such as appetite and sati-

Mechanisms of 5-HT inactivation in the nervous system and peripheral organs of bivalves are poorly understood. It requires molecular identity, localization, and characterization of SERT and MOA. In this regard, several types of SERT and MOA inhibitors are available [80, 114, 141] that provide us with useful tools to elucidate molecular signaling that control 5-HT reuptake and metabolism. A few studies show that selective 5-HT reuptake inhibitors modulate 5-HT-induced spawning in bivalves. Fong [142] and Fong et al. [143, 144] reported spawning of Zebra mussel treated with selective 5-HT reuptake inhibitors (fluvoxamine, fluoxetine, zimelidine, and paroxetine). Both males and females are capable of releasing their gametes after treatment with fluvoxamine at 10−7 and 10−6 M, respectively. Following treatment with fluoxetine, 100% of males have spawned at 10−4 to 10−5 M, however spawning has induced in 50–60% of females at 10−5 M. Zimelidine induces spawning in 100 and 60–70% of males and females at 10−4 M. Paroxetine induces spawning in 50 and 20% of males and females at 10−6 and 10−5 M, respectively. Considering spawning of males and females at 10−3 M 5-HT, these results indicate that selective 5-HT reuptake inhibitors stimulate spawning in Zebra mussel at concentrations lower than that of 5-HT. Further examinations have revealed that mianserin and cyproheptadine interfere with fluvoxamine-, fluoxetine-, and

zimelidine-induced spawning [144] suggesting that antagonists of 5-HT<sup>2</sup>

The authors declare no conflicts of interest, financial or otherwise.

stimulatory function of selective 5-HT reuptake inhibitors in spawning. Inhibition of 5-HT reuptake may increase the synaptic 5-HT concentrations, which in turn activate postsynaptic 5-HT receptor to induce spawning. It is also possible that selective 5-HT reuptake inhibitors act as ligands at postsynaptic receptor rather than inhibition of SERT. Overall, these studies suggest that 5-HT transport plays a key role in reproduction; however, the mechanisms of

So far, histochemistry and immunohistochemistry methods have been employed to localize the 5-HT neurons and fibers, and spectrofluorometric and HPLC-EC methods have been used to identify 5-HT content in the nervous system and gonad of various bivalve species. Successful implication of various mammalian monoclonal or polyclonal antibodies indicates that 5-HT structure is highly conserved through evolution across the animal kingdom. However, mechanisms through which 5-HT acts on a biological system may differ. The present review shows that 5-HT content highly differs in the nervous system and gonad of bivalve species. The inter-species differences in 5-HT content might be related to capability of nervous system to synthesize 5-HT, differences in 5-HT inactivation or 5-HT transport from nervous system to the gonad. In the latter case, 5-HT content in the gonad may correspond to 5-HT concentration that requires to stimulate spawning. The present review shows that 5-HT concentration to induce spawning highly differs between sexes, and among species. It is worth to note that tissue sampling, extraction procedure, and analytical method affect the results of 5-HT content. In addition, 5-HT content undergoes seasonal variation and change following

ety, which have been demonstrated in mammals [140].

52 Serotonin - A Chemical Messenger Between All Types of Living Cells

action are largely unknown.

spawning.

**Conflict of interest**

This study was supported by Tohoku Ecosystem-Associated Marine Sciences (TEAMS) grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Japan, JSPS KAKENHI (16H04978), JSPS postdoctoral fellow (23-01404), and JAMBIO (23-02) to M.O.

## **Author details**

Sayyed Mohammad Hadi Alavi, Kazue Nagasawa, Keisuke G. Takahashi and Makoto Osada\*

\*Address all correspondence to: makoto.osada.a8@tohoku.ac.jp

Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai, Japan

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**Section 2**

## **Types**

## *4WD* **to Travel Inside the 5-HT1A Receptor World**

Wilma Quaglia, Carlo Cifani, Fabio Del Bello, Mario Giannella, Gianfabio Giorgioni, Maria Vittoria Micioni Di Bonaventura and Alessandro Piergentili

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69348

#### **Abstract**

5-HT1A receptor is one of the most important members of the numerous families of serotoninergic receptors. Though it was the first 5-HT receptor to be identified and cloned, the knowledge of its activation/transduction mechanisms, mediated effects, and connection with other systems is still uncompleted. For this reason, relevant is the study of the four Ws of the title: first of all "who" this receptor is, then "why" it continues to be a so attractive target after several years after its identification, then "where" is 5-HT1A receptor expressed within the body, and, finally, "what" effects this receptor can elicit under physiological and pathological conditions. Obviously, more and more potent, safe, and selective "drugs" might be discovered once the responses to these questions are given.

**Keywords:** 5-HT1A receptor, 5-HT1A transduction mechanisms, central nervous system diseases, 5-HT1A ligands, structure-activity relationship studies

## **1. Introduction**

The rational research of novel efficacious and safe drugs is mainly based on the knowledge of biological systems, whose dysfunctions cause several pathological conditions. Receptors and enzymes are the most common targets to which the so-called charmed bullets by Paul Ehrlich (1854–1915), Nobel Prize in Physiology and Medicine in 1908, should be addressed to mean the selectivity of interaction and, therefore, the reduced occurrence of unwanted side effects. Serotonin receptors (5-HTRs) are the most widespread targets of drugs because of the numerous

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Chemical structure of 5-HT.

biological effects of the endogenous ligand serotonin (5-HT; **Figure 1**) and the wide presence of different 5-HTR subtypes in both the central and peripheral nervous systems (CNS and PNS) [1].

5-HT is biosynthesized at the periphery into the gut by intestinal enterochromaffin cells and in the CNS in the raphe nucleus from the essential amino acid L-tryptophan. A 5-HT reuptake protein (SERT) is responsible for carrying the neurotransmitter from the synaptic cleft to its target nerve and acts as a regulator of 5-HT levels. In the CNS, SERT is a key target for various antidepressant drugs such as tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), and serotonin-norepinephrine reuptake inhibitors (SNRIs). 5-HT is mainly deaminated by monoamine oxidase A (MAO A) to the corresponding aldehyde in the liver. The physiological effects of 5-HT are mediated by several 5-HTRs, whose heterogeneity was hypothesized from pharmacological characterization in the 1950s. From radioligand experiments, the first evidences of 5-HT subtypes were reported in 1979 [2]. To date molecular cloning techniques, amino acid sequence determination, evaluation of its pharmacological properties, second messenger coupling, and signal transduction characterization have allowed the identification of at least seven subfamilies (5-HT1–7), some of which are further subdivided into different subtypes (**Figure 2**).

**Figure 2.** Classification of 5-HT1ARs.

While 5-HT3 Rs are cation-permeable ion channels, all the others are G-protein-coupled receptors (GPCRs) and are classified as rhodopsine-like receptors (class A). Among the 5-HTRs, the 5-HT1A subtype was the first to be cloned [3] and pharmacologically characterized, and it is one of the most studied. For this reason, it is often ironically called "old target" [4]. The human 5-HT1AR consists of 422 amino acid residues with a molecular weight of about 46,000 Da. Though its structure is still unknown, mutagenesis studies have allowed the identification of amino acid residues responsible for ligand binding and G-protein coupling [1].

## **2. Localization**

biological effects of the endogenous ligand serotonin (5-HT; **Figure 1**) and the wide presence of different 5-HTR subtypes in both the central and peripheral nervous systems (CNS and PNS) [1]. 5-HT is biosynthesized at the periphery into the gut by intestinal enterochromaffin cells and in the CNS in the raphe nucleus from the essential amino acid L-tryptophan. A 5-HT reuptake protein (SERT) is responsible for carrying the neurotransmitter from the synaptic cleft to its target nerve and acts as a regulator of 5-HT levels. In the CNS, SERT is a key target for various antidepressant drugs such as tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), and serotonin-norepinephrine reuptake inhibitors (SNRIs). 5-HT is mainly deaminated by monoamine oxidase A (MAO A) to the corresponding aldehyde in the liver. The physiological effects of 5-HT are mediated by several 5-HTRs, whose heterogeneity was hypothesized from pharmacological characterization in the 1950s. From radioligand experiments, the first evidences of 5-HT subtypes were reported in 1979 [2]. To date molecular cloning techniques, amino acid sequence determination, evaluation of its pharmacological properties, second messenger coupling, and signal transduction characterization have allowed the identification of at least seven subfamilies (5-HT1–7), some of which are further

subdivided into different subtypes (**Figure 2**).

**Figure 1.** Chemical structure of 5-HT.

68 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Figure 2.** Classification of 5-HT1ARs.

5-HT1ARs are widely expressed in the brain of mammals, including humans [5]. The main expressions are in limbic areas, such as the hippocampus, lateral septum, cortical brain regions, as well as dorsal and medial raphe nuclei (DRN and MRN) (**Figure 3**).

5-HT1ARs are located within the brain both pre- and postsynaptically. Presynaptic 5-HT1ARs are expressed in all 5-HT neurons (autoreceptors) and in a lot of non-5-HT neurons (heteroreceptors). The latter modulate the release of several neurotransmitters, including glutamate and dopamine, and hormones (adrenocorticotropin (ACHT), oxytocin, prolactin, growth hormone, β-endorphin). In the brainstem, presynaptic autoreceptors are expressed in serotonergic neurons in DRN and MRN, where their activation inhibits cell firing rate. These neurons send ascending 5-HT fibers to the forebrain attenuating 5-HT synthesis, turnover, and release in projection areas from axon terminals, working on a basis of a negative feedback. Presynaptic

**Figure 3.** Central localization of 5-HT1ARs (Adapted from CNSforum image bank, Lundbeck Institute "Distribution of 5-HT1A receptors" http://www.cnsforum.com/imagebank/item/hrl\_Rcpt\_sys\_SN1A\_dist/default.aspx).

5-HT1ARs expressed in DRN, through coupling to Gαi/o proteins, decrease rate of cell firing by the activation of inwardly rectifying potassium channels. Postsynaptic 5-HT1ARs are found at high density in limbic regions, such as the hippocampus and septum, and in the frontal and entorhinal cortices. Lower 5-HT1AR levels are observed in the amygdala. As in the case of presynaptic receptors, the activation of postsynaptic 5-HT1ARs generally decreases the firing rate of postsynaptic cells. Electrophysiological, pharmacological, and biochemical evidences have demonstrated that 5-HT1ARs are localized in primary afferent neurons [4]. They are also present in the gut, in the enteric nervous system, as well as in smooth muscle, where their activation inhibits relaxation or contraction.

## **3. Signal transduction pathways of 5-HT1ARs**

The primary transduction pathway of 5-HT1ARs is the inhibition of adenylate cyclase (AC). Nevertheless, various other pathways are coupled to this receptor depending on the target cell. Indeed, 5-HT1AR stimulation activates or inhibits different enzymes, channels, and kinases, as well as modulates the production of several second messengers (**Figure 4**) [6, 7].

Whatever is the activated second messenger, the signals initiated by the stimulation of 5-HT1ARs implicate the involvement of Gi/o protein. Moreover, a G-protein-independent pathway of 5-HT1AR coupling to a smooth inward current has also been suggested.

## **3.1. AC inhibition**

The activation of 5-HT1ARs inhibits AC and reduces the production of cAMP with a consequent decrease of protein kinase A (PKA) activity. The Gα<sup>i</sup> -induced inhibition of AC is coupled to 5-HT1A heteroreceptors, whereas the situation is still unclear for 5-HT1A autoreceptors. Indeed, some results reveal that 5-HT1AR partial agonists negatively regulate presynaptic AC activity in raphe nuclei. On the other hand, a lot of evidences highlight that 5-HT1AR agonists do not inhibit forskolin-stimulated AC activity in homogenates of the raphe region, suggesting that these autoreceptors do not couple to AC. 5-HT1AR agonists also reduce PKA activity in the hippocampus, determining increased protein phosphatase-1 activity and reduction of Calcium/calmodulin-dependent protein kinase II phosphorylation. This signaling effect is joined to cognitive deficits. Therefore, cognitive behaviors can be mediated by the inhibition of AC/PKA activity induced by 5-HT1ARs.

### **3.2. GIRK and Ca2+ channel opening**

Through coupling to Gαi/o proteins, 5-HT1ARs activate inwardly rectifying potassium channels (GIRKs) in the hippocampus and DRN. Such an action hyperpolarizes neurons and decreases firing. Moreover, Ca2+ entry is reduced by the inhibition of voltage-gated Ca2+ channel following 5-HT1AR activation.

### **3.3. ERK/MAPK pathway activation**

The stimulation of 5-HT1ARs induces the release of βγ-complex that participates in the activation of phosphatidylinositol-3 kinase (PI3K). It triggers the activation of extracellular signal-regulated

**Figure 4.** Main transduction pathways of 5-HT1ARs (Reprinted with permission from Ref. [6]).

5-HT1ARs expressed in DRN, through coupling to Gαi/o proteins, decrease rate of cell firing by the activation of inwardly rectifying potassium channels. Postsynaptic 5-HT1ARs are found at high density in limbic regions, such as the hippocampus and septum, and in the frontal and entorhinal cortices. Lower 5-HT1AR levels are observed in the amygdala. As in the case of presynaptic receptors, the activation of postsynaptic 5-HT1ARs generally decreases the firing rate of postsynaptic cells. Electrophysiological, pharmacological, and biochemical evidences have demonstrated that 5-HT1ARs are localized in primary afferent neurons [4]. They are also present in the gut, in the enteric nervous system, as well as in smooth muscle, where their activation

The primary transduction pathway of 5-HT1ARs is the inhibition of adenylate cyclase (AC). Nevertheless, various other pathways are coupled to this receptor depending on the target cell. Indeed, 5-HT1AR stimulation activates or inhibits different enzymes, channels, and kinases, as well as modulates the production of several second messengers (**Figure 4**) [6, 7].

Whatever is the activated second messenger, the signals initiated by the stimulation of 5-HT1ARs implicate the involvement of Gi/o protein. Moreover, a G-protein-independent path-

The activation of 5-HT1ARs inhibits AC and reduces the production of cAMP with a conse-

pled to 5-HT1A heteroreceptors, whereas the situation is still unclear for 5-HT1A autoreceptors. Indeed, some results reveal that 5-HT1AR partial agonists negatively regulate presynaptic AC activity in raphe nuclei. On the other hand, a lot of evidences highlight that 5-HT1AR agonists do not inhibit forskolin-stimulated AC activity in homogenates of the raphe region, suggesting that these autoreceptors do not couple to AC. 5-HT1AR agonists also reduce PKA activity in the hippocampus, determining increased protein phosphatase-1 activity and reduction of Calcium/calmodulin-dependent protein kinase II phosphorylation. This signaling effect is joined to cognitive deficits. Therefore, cognitive behaviors can be mediated by the inhibition

Through coupling to Gαi/o proteins, 5-HT1ARs activate inwardly rectifying potassium channels (GIRKs) in the hippocampus and DRN. Such an action hyperpolarizes neurons and decreases firing. Moreover, Ca2+ entry is reduced by the inhibition of voltage-gated Ca2+ channel following 5-HT1AR activation.

The stimulation of 5-HT1ARs induces the release of βγ-complex that participates in the activation of phosphatidylinositol-3 kinase (PI3K). It triggers the activation of extracellular signal-regulated


way of 5-HT1AR coupling to a smooth inward current has also been suggested.

inhibits relaxation or contraction.

**3.1. AC inhibition**

**3. Signal transduction pathways of 5-HT1ARs**

70 Serotonin - A Chemical Messenger Between All Types of Living Cells

quent decrease of protein kinase A (PKA) activity. The Gα<sup>i</sup>

of AC/PKA activity induced by 5-HT1ARs.

**3.2. GIRK and Ca2+ channel opening**

**3.3. ERK/MAPK pathway activation**

protein kinase (ERK) (or MAPK), implicated in cell proliferation and differentiation through two pathways involving Ras-Raf-MEK proteins. In addition, 5-HT1A-induced ERK activation in nonneuronal cells can be mediated by phosphatidylcholine-specific phospholipase C (PC-PLC) in a G-protein-dependent manner. In neuronal cells, the effects on ERK activity produced by 5-HT1ARs can be different. Indeed, in the hypothalamus a rapid but transient increase of ERK phosphorylation is observed, and this effect might be an intermediate step for the 5-HT1ARmediated increase of oxytocin, ACTH, and prolactin. In HN2-5 hippocampal-derived cell lines, 5-HT1AR activation favors ERK phosphorylation and activity. This effect does not occur in the primary culture of hippocampal or fetal rhombencephalic neurons. On the contrary, in the rat hippocampus, 5-HT1AR activation decreases ERK phosphorylation. Analogously it reduces MEK activity and ERK phosphorylation in differentiated raphe neurons. Different ERK-related effectors can be modulated by 5-HT1ARs: activation of the ribosomal S6 kinase (RSK), stimulation of nuclear factor κB (NF-κB), and inhibition of caspase 3. This pathway seems to be involved in neuroprotective mechanisms. ERK also activates cAMP response element binding (CREB), a transcription factor that plays fundamental roles in stress, anxiety, and depression. Finally, the activation of MAPK/ERK transduction pathway may inhibit apoptosis by phosphorylation of the proapoptotic protein Bad and by increasing the expression of antiapoptotic Bcl-2.

#### **3.4. PI3K and Akt pathway activation**

5-HT1AR stimulation can also regulate the activation of the PI3K/Akt signaling pathway through βγ-complex. The Akt protein kinase plays a key role in several cellular processes, such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. In the mammalian brain, the PI3K/Akt pathway is also implicated in synaptic plasticity, learning, and memory. Consequently, Akt dysfunction can be associated with metabolic diseases (e.g., diabetes and obesity), central disorders (e.g., depression, schizophrenia, and drug abuse), and the most frequent alterations observed in human cancer and tumor cells. Akt phosphorylates and inactivates the protein glycogen synthase kinase 3 (GSK3), whose inhibition produces antidepressant and antimanic effects. Active Akt also phosphorylates and inactivates Forkhead box O (FoxO) transcription factors, whose deficiency in mice develops antidepressive and anxiolytic behavioral phenotypes.

#### **3.5. Na+ /H+ exchanger activation**

Another complex pathway following 5-HT1AR stimulation and involving G(i2)α and/or G(i3)α induces Janus kinase 2 (Jak2) activation, which leads to tyrosine phosphorylation of calmodulin (CaM). The consequent increase of CaM binding to Na<sup>+</sup> /H<sup>+</sup> exchangers (NHEs) induces a conformational modification that activates NHEs, unmasking an obscured protonsensing and/or proton-transporting region. NHEs, expressed on the surface of all mammalian cells, regulate cell volume, intracellular pH, and transepithelial transport of Na<sup>+</sup> and acid-base equivalents.

### **3.6. NO production**

5-HT1ARs can also regulate the production of nitric oxide (NO) that plays an important role in the brain. In rat ventral prostate cells, 5-HT1ARs can stimulate NO synthase (NOS) activity, whereas in the adult rat hippocampus and in human neocortical slices, they inhibit NMDAinduced NO production. Therefore, the regulation of NO synthesis by 5-HT1ARs is complex and appears to be cell specific.

## **4. Biological interest of 5-HT1ARs**

5-HT1AR is one of the most important among the 5-HTRs because of its high affinity for 5-HT and involvement in nearly all 5-HT-mediated effects. The main behavioral and physiological functions mediated by this receptor are summarized in **Figure 5**.

**Figure 5.** Main behavioral and physiological functions mediated by 5-HT1ARs.

#### **4.1. Depression**

nuclear factor κB (NF-κB), and inhibition of caspase 3. This pathway seems to be involved in neuroprotective mechanisms. ERK also activates cAMP response element binding (CREB), a transcription factor that plays fundamental roles in stress, anxiety, and depression. Finally, the activation of MAPK/ERK transduction pathway may inhibit apoptosis by phosphorylation of

5-HT1AR stimulation can also regulate the activation of the PI3K/Akt signaling pathway through βγ-complex. The Akt protein kinase plays a key role in several cellular processes, such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. In the mammalian brain, the PI3K/Akt pathway is also implicated in synaptic plasticity, learning, and memory. Consequently, Akt dysfunction can be associated with metabolic diseases (e.g., diabetes and obesity), central disorders (e.g., depression, schizophrenia, and drug abuse), and the most frequent alterations observed in human cancer and tumor cells. Akt phosphorylates and inactivates the protein glycogen synthase kinase 3 (GSK3), whose inhibition produces antidepressant and antimanic effects. Active Akt also phosphorylates and inactivates Forkhead box O (FoxO) transcription factors, whose deficiency in mice develops

Another complex pathway following 5-HT1AR stimulation and involving G(i2)α and/or G(i3)α induces Janus kinase 2 (Jak2) activation, which leads to tyrosine phosphorylation of

induces a conformational modification that activates NHEs, unmasking an obscured protonsensing and/or proton-transporting region. NHEs, expressed on the surface of all mammalian

5-HT1ARs can also regulate the production of nitric oxide (NO) that plays an important role in the brain. In rat ventral prostate cells, 5-HT1ARs can stimulate NO synthase (NOS) activity, whereas in the adult rat hippocampus and in human neocortical slices, they inhibit NMDAinduced NO production. Therefore, the regulation of NO synthesis by 5-HT1ARs is complex

5-HT1AR is one of the most important among the 5-HTRs because of its high affinity for 5-HT and involvement in nearly all 5-HT-mediated effects. The main behavioral and physiological

/H<sup>+</sup>

exchangers (NHEs)

and acid-base

the proapoptotic protein Bad and by increasing the expression of antiapoptotic Bcl-2.

**3.4. PI3K and Akt pathway activation**

72 Serotonin - A Chemical Messenger Between All Types of Living Cells

**3.5. Na+**

equivalents.

**3.6. NO production**

and appears to be cell specific.

**4. Biological interest of 5-HT1ARs**

**/H+**

antidepressive and anxiolytic behavioral phenotypes.

calmodulin (CaM). The consequent increase of CaM binding to Na<sup>+</sup>

functions mediated by this receptor are summarized in **Figure 5**.

cells, regulate cell volume, intracellular pH, and transepithelial transport of Na<sup>+</sup>

 **exchanger activation**

The dysfunction of 5-HT1A autoreceptors has been proven to be associated with the major depressive disorders. This correlation is confirmed by the observation that significant antidepressant activity is elicited by 5-HT1AR agonists [4]. Though the mechanism responsible for their antidepressant action is still unclear, desensitization or downregulation of presynaptic 5-HT1ARs appears to be implicated in this pharmacological effect. Indeed, in DRN and MRN, prolonged treatment with 5-HT1AR agonists desensitizes presynaptic 5-HT1ARs inducing a reduction of autoreceptor-mediated inhibition of 5-HT release.

SSRIs represent the first-line treatment of depression. However, the inhibition of the reuptake of 5-HT increases 5-HT concentration in the synaptic cleft and simultaneously activates 5-HT1A autoreceptors, with a consequent suppression of 5-HT release from presynaptic terminals [8]. Therefore, only prolonged treatment with SSRIs allows the desensitization of 5-HT1A autoreceptors, leading to the recovery of neurotransmission in 5-HT neurons. Beneficial effects on depression are also produced by the combination of SSRIs with 5-HT1AR agonists or antagonists, leading to faster onset of antidepressant action and greater antidepressant efficacy. In particular, 5-HT1AR antagonists can improve the efficacy of SSRIs by blocking inhibitory 5-HT1A autoreceptors, while 5-HT1AR agonists exert antidepressant-like effect through the activation of postsynaptic 5-HT1ARs and/or faster desensitization of 5-HT1A autoreceptors. Finally, antidepressant-like effect can also be produced by 5-HT1A partial agonism combined with 5-HT reuptake inhibition [4].

## **4.2. Anxiety**

Several studies have been performed to demonstrate the possible role of 5-HT1ARs in anxiety [1]. Interestingly, mice with genetically inactivated 5-HT1AR gene develop an anxiety-like phenotype, probably resulting from impaired autoinhibitory control of midbrain 5-HT neurons. On the contrary, mice with overexpressed 5-HT1ARs display diminished anxiety when compared to wild-type animals. These findings support the crucial role of the stimulation of 5-HT1ARs in the control of anxiety-like behavior. Therefore, 5-HT1AR agonists and partial agonists have been developed as novel anxiolytic agents, devoid of dependence and side effect profile of other anxiolytics and antipsychotics.

### **4.3. Schizophrenia**

Several studies performed in postmortem schizophrenia patients report an overexpression of 5-HT1ARs in the prefrontal cortex, indicating that these receptors are not adequately stimulated by 5-HT [1]. Therefore, 5-HT1AR agonists might be useful to contrast this apparent deficit. Two mechanisms are advantageously activated by 5-HT1AR stimulation in the treatment of schizophrenia. The first one involves the attenuation of parkinsonian symptoms, such as catalepsy, caused by the antagonism at dopamine D<sup>2</sup> receptor (D2 R) produced by antipsychotics. Since atypical antipsychotic drugs, such as clozapine, quetiapine, and ziprasidone, also behave as potent 5-HT1AR agonists, it has been suggested that the reduced incidence of motor side effects observed with these drugs might be due to their inherent 5-HT1AR agonism. The second mechanism involves the ability of 5-HT1AR agonists to increase dopamine release in the prefrontal cortex, consequently reducing the negative symptoms of schizophrenia. Based on these observations, a novel approach in the treatment of schizophrenia concerns the development of novel atypical antipsychotic agents characterized by a mixed D<sup>2</sup> R antagonist/5- HT1AR agonist profile.

#### **4.4. Pain**

Full and partial 5-HT1AR agonists are beneficial in pain treatments, including efficacy in neuropathic pain models, arousing great interest as future therapeutic agents. In knockout mice, 5-HT1ARs have also been demonstrated to mediate an endogenous inhibitory control of nociception evoked by thermal noxious stimuli [4].

#### **4.5. Drug addiction**

A critical role in the effects of psychostimulants, including addiction, is played by 5-HT1ARs. Some psychostimulant drugs, including cocaine, amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA), increase not only dopamine but also 5-HT that can hyperactivate 5-HT1ARs. Interestingly, the contribution of pre- and postsynaptic 5-HT1ARs can be dissociated and frequently is responsible for opposite effects. In fact, 5-HT1A autoreceptors indirectly facilitate psychostimulant addiction-related behaviors by reducing 5-HT response in projection terminal areas, while postsynaptic 5-HT1ARs directly contrast the expression of various addiction-related behaviors [9]. Several studies have also demonstrated that 5-HT1AR agonists alleviate opioid-induced respiratory depression in rodent models. The mechanisms involved in this effect are still unclear. However, concomitant decreases in opioidinduced analgesia, as well as altered baseline ventilation and behavior, have also been observed.

#### **4.6. Dyskinesia**

In particular, 5-HT1AR antagonists can improve the efficacy of SSRIs by blocking inhibitory 5-HT1A autoreceptors, while 5-HT1AR agonists exert antidepressant-like effect through the activation of postsynaptic 5-HT1ARs and/or faster desensitization of 5-HT1A autoreceptors. Finally, antidepressant-like effect can also be produced by 5-HT1A partial agonism combined with

Several studies have been performed to demonstrate the possible role of 5-HT1ARs in anxiety [1]. Interestingly, mice with genetically inactivated 5-HT1AR gene develop an anxiety-like phenotype, probably resulting from impaired autoinhibitory control of midbrain 5-HT neurons. On the contrary, mice with overexpressed 5-HT1ARs display diminished anxiety when compared to wild-type animals. These findings support the crucial role of the stimulation of 5-HT1ARs in the control of anxiety-like behavior. Therefore, 5-HT1AR agonists and partial agonists have been developed as novel anxiolytic agents, devoid of dependence and side effect

Several studies performed in postmortem schizophrenia patients report an overexpression of 5-HT1ARs in the prefrontal cortex, indicating that these receptors are not adequately stimulated by 5-HT [1]. Therefore, 5-HT1AR agonists might be useful to contrast this apparent deficit. Two mechanisms are advantageously activated by 5-HT1AR stimulation in the treatment of schizophrenia. The first one involves the attenuation of parkinsonian symptoms, such as

ics. Since atypical antipsychotic drugs, such as clozapine, quetiapine, and ziprasidone, also behave as potent 5-HT1AR agonists, it has been suggested that the reduced incidence of motor side effects observed with these drugs might be due to their inherent 5-HT1AR agonism. The second mechanism involves the ability of 5-HT1AR agonists to increase dopamine release in the prefrontal cortex, consequently reducing the negative symptoms of schizophrenia. Based on these observations, a novel approach in the treatment of schizophrenia concerns the devel-

Full and partial 5-HT1AR agonists are beneficial in pain treatments, including efficacy in neuropathic pain models, arousing great interest as future therapeutic agents. In knockout mice, 5-HT1ARs have also been demonstrated to mediate an endogenous inhibitory control of noci-

A critical role in the effects of psychostimulants, including addiction, is played by 5-HT1ARs. Some psychostimulant drugs, including cocaine, amphetamine, methamphetamine,

opment of novel atypical antipsychotic agents characterized by a mixed D<sup>2</sup>

receptor (D2

R) produced by antipsychot-

R antagonist/5-

5-HT reuptake inhibition [4].

profile of other anxiolytics and antipsychotics.

74 Serotonin - A Chemical Messenger Between All Types of Living Cells

catalepsy, caused by the antagonism at dopamine D<sup>2</sup>

ception evoked by thermal noxious stimuli [4].

**4.2. Anxiety**

**4.3. Schizophrenia**

HT1AR agonist profile.

**4.5. Drug addiction**

**4.4. Pain**

5-HT1ARs are involved in the regulation of locomotor activity. In particular, the stimulation of 5-HT1ARs facilitates the establishment of locomotor sensitization [10]. Parkinsonian patients in therapy with L-3,4-dihydroxyphenylalanine (L-DOPA) may develop motor complications, such as dyskinesia. The development of this side effect involves several pathways, including an abnormal 5-HT-mediated neurotransmission [4]. It has been highlighted that parkinsonian animals chronically treated with L-DOPA have increased levels of 5-HT1ARs in the striatal matrix. Accordingly, treatment with 5-HT1AR agonists attenuates dyskinesia but, in some cases, also reduces the antiparkinsonian benefit of L-DOPA. Some evidences suggest that a lot of 5-HT1AR agonists are also endowed with D<sup>2</sup> R antagonism, which alleviates dyskinesia, though at the expense of worsening parkinsonism. The challenge is to obtain compounds able to selectively stimulate 5-HT1ARs in striatus and/or in middle layers of the cortex, avoiding the involvement of 5-HT1ARs in external cortical layers.

#### **4.7. Neuroprotection**

The activation of 5-HT1ARs exerts a neuroprotective effect in different animal models of ischemia, interfering with excitotoxic and apoptotic cell death processes in the postischemic brain [1]. Though the cellular mechanisms underlying such a neuroprotective effect are still unclear, the hyperpolarization of pyramidal neurons inhibits the glutamate-induced excitotoxicity consequent to cerebral ischemia. 5-HT1ARs may mediate brain protective mechanisms, by contrasting the effects of glutamatergic NMDA receptor overstimulation and the consequent NMDA-induced Ca2+ influx. Moreover, the inhibition of 5-HT1AR-induced cyclases might produce neuroprotective effects due to the reduction of adenylyl cyclase excess following reperfusion after ischemic attack. 5-HT1AR agonists can also be useful for the treatment of traumatic brain injury (TBI) [11].

#### **4.8. Memory**

Several experimental evidences highlight that the activation of postsynaptic 5-HT1ARs, attenuating the neuronal activity, impairs emotional memory. On the contrary, presynaptic 5-HT1AR activation reduces 5-HT release and exerts pro-cognitive effects. 5-HT1AR antagonism facilitates memory retention, probably by the activation of 5-HT7 Rs, and evidence is provided that 5-HT7 Rs can facilitate emotional memory upon reduced 5-HT1AR transmission [12]. Moreover, tonic and phasic 5-HT release can exert different and potentially opposite effects on emotional memory, depending on the states of 5-HT1ARs and 5-HT<sup>7</sup> Rs and their interaction. Consequently, individual differences due to genetic and/or epigenetic mechanisms play an essential role in the responsiveness to drug treatment [13].

#### **4.9. Sexual function**

5-HT1ARs and 5-HT2CRs produce two distinct and opposite effects on sexual function: the activation of 5-HT1ARs decreases ejaculatory latency and erection, directly promoting the sympathetic emission, while the activation of 5-HT2CRs increases them, directly favoring parasympathetic expulsion and erection [4]. Therefore, 5-HT1AR antagonists are under investigation for the treatment of primary premature ejaculation.

#### **4.10. Cardiovascular system**

Several studies have demonstrated that 5-HT1ARs in the medullary raphe mediate protective responses to stress [4]. Indeed, the activation of 5-HT1ARs induces bradycardia and blood pressure decrease, suggesting that 5-HT1ARs can reduce the sympathetic outflow. Moreover, 5-HT1AR agonists reduce the cutaneous vasoconstriction evoked by physical and psychological stressors. 5-HT1ARs located in limbic regions can also reduce stress-evoked cardiovascular responses. However, this action does not occur via a direct effect on brainstem cardiovascular neurons, but is consequent to the anxiolytic effect. Psychological stress, cold exposure, or fever might elicit cardiovascular responses also mediated by neurons within the dorsomedial hypothalamus. Therefore, 5-HT1AR agonists might be useful therapeutic agents to reduce the sympathetic responses occurring in some forms of hypertension and heart failure. The cardiovascular responses of 5-HT1AR agonists could also be useful to reduce side effects in the treatment of hyperphagia and obesity with noradrenaline (NA) uptake inhibitors. Such inhibitors are able to reduce food intake due to increased noradrenergic activity that also causes an increased cardiovascular activity. When 5-HT1AR agonists are combined with NA uptake inhibitors, side effects, such as hypertension and tachycardia, are mitigated. Postsynaptic 5-HT1AR activation may contribute to hypophagia efficacy. Moreover, presynaptic 5-HT1ARs may reduce food intake by inhibiting spontaneous noradrenergic cell firing.

#### **4.11. Urogenital system**

5-HT1ARs mediate effects in the lower urinary tract function [4]. Indeed, their stimulation activates the micturition reflex, inducing an increase in the frequency of isovolumic bladder contractions. Conversely, 5-HT1AR agonists elicit periodic external urethral sphincter relaxation, inducing an increase in micturition volume, a decrease in bladder capacity, and an increase in voiding efficiency.

#### **4.12. Pupillary dilation**

Pupillary response to 5-HT1AR agonists is species dependent [14]. Indeed, 5-HT1AR activation produces miosis in humans and rabbits and mydriasis in mice. In humans, 5-HT1ARs induce miosis solely by inhibiting sympathetic mechanisms. However, evidences suggest that the parasympathetic nerve is also involved. Indeed, the activation of central 5-HT1ARs induces NA release, which in turn reduces parasympathetic neuronal tone to the iris sphincter muscle by the stimulation of postsynaptic α<sup>2</sup> -adrenoceptors (α<sup>2</sup> -ARs) within the Edinger-Westphal nucleus.

#### **4.13. Cancer**

transmission [12]. Moreover, tonic and phasic 5-HT release can exert different and potentially opposite effects on emotional memory, depending on the states of 5-HT1ARs and

5-HT1ARs and 5-HT2CRs produce two distinct and opposite effects on sexual function: the activation of 5-HT1ARs decreases ejaculatory latency and erection, directly promoting the sympathetic emission, while the activation of 5-HT2CRs increases them, directly favoring parasympathetic expulsion and erection [4]. Therefore, 5-HT1AR antagonists are under inves-

Several studies have demonstrated that 5-HT1ARs in the medullary raphe mediate protective responses to stress [4]. Indeed, the activation of 5-HT1ARs induces bradycardia and blood pressure decrease, suggesting that 5-HT1ARs can reduce the sympathetic outflow. Moreover, 5-HT1AR agonists reduce the cutaneous vasoconstriction evoked by physical and psychological stressors. 5-HT1ARs located in limbic regions can also reduce stress-evoked cardiovascular responses. However, this action does not occur via a direct effect on brainstem cardiovascular neurons, but is consequent to the anxiolytic effect. Psychological stress, cold exposure, or fever might elicit cardiovascular responses also mediated by neurons within the dorsomedial hypothalamus. Therefore, 5-HT1AR agonists might be useful therapeutic agents to reduce the sympathetic responses occurring in some forms of hypertension and heart failure. The cardiovascular responses of 5-HT1AR agonists could also be useful to reduce side effects in the treatment of hyperphagia and obesity with noradrenaline (NA) uptake inhibitors. Such inhibitors are able to reduce food intake due to increased noradrenergic activity that also causes an increased cardiovascular activity. When 5-HT1AR agonists are combined with NA uptake inhibitors, side effects, such as hypertension and tachycardia, are mitigated. Postsynaptic 5-HT1AR activation may contribute to hypophagia efficacy. Moreover, presynaptic 5-HT1ARs may reduce food intake by inhibiting spontaneous noradrenergic cell firing.

5-HT1ARs mediate effects in the lower urinary tract function [4]. Indeed, their stimulation activates the micturition reflex, inducing an increase in the frequency of isovolumic bladder contractions. Conversely, 5-HT1AR agonists elicit periodic external urethral sphincter relaxation, inducing an increase in micturition volume, a decrease in bladder capacity, and an increase

Pupillary response to 5-HT1AR agonists is species dependent [14]. Indeed, 5-HT1AR activation produces miosis in humans and rabbits and mydriasis in mice. In humans, 5-HT1ARs induce

tigation for the treatment of primary premature ejaculation.

76 Serotonin - A Chemical Messenger Between All Types of Living Cells

Rs and their interaction. Consequently, individual differences due to genetic and/or epigenetic mechanisms play an essential role in the responsiveness to drug treatment [13].

5-HT<sup>7</sup>

**4.9. Sexual function**

**4.10. Cardiovascular system**

**4.11. Urogenital system**

in voiding efficiency.

**4.12. Pupillary dilation**

5-HT1ARs are known to be involved in the proliferation of human tumor cells, but their function still remains poorly understood [4]. 5-HT1AR antagonists inhibit the growth of different prostatic tumor cell lines, such as PC-3, DU-145, and LNCaP, as well as the proliferation of PC-3 xenografted subcutaneously in athymic nude mice. Multitarget ligands, acting as α1A/ α1D-AR and 5-HT1AR antagonists, in which a synergic effect occurs, have proved to be useful in the management of benign prostatic hyperplasia. 5-HT1ARs are also reported to be involved in the mitogenic effect of 5-HT in human small cell lung carcinoma cells.

## **5. Ligands**

Several structurally different ligands, such as aryloxyalkylamines, arylpiperazines, aminotetralins, indolyl-alkylamines, ergolines, and aporphines, are known to bind 5-HT1ARs [15]. Recently, new classes of ligands, including 2-imidazoline and 1,4-dioxane derivatives, have also shown high 5-HT1AR affinity. Due to the high homology among 5-HT1ARs and other receptor systems, in binding studies several molecules show nanomolar and subnanomolar affinity not only for 5-HT1ARs but also for other receptors (5-HT2ARs, 5-HT2CRs, 5-HT7 Rs, α<sup>1</sup> - and α <sup>2</sup> - ARs, as well as D<sup>1</sup> Rs and D<sup>2</sup> Rs).

#### **5.1. Aryloxyalkylamines**

The sequence analysis of the 5-HT1AR genomic clone indicates 43% amino acid homology with the β<sup>2</sup> -AR in the transmembrane domain. Therefore, some compounds show good affinity for both systems. The first examples of dualistic interaction are offered by pindolol (**1**) and propranolol (**2**) (**Figure 6**) [16].

In several studies, an Asn amino acid residue in the putative helix VII of 5-HT1ARs has been demonstrated to play a crucial role in the binding of aryloxypropanolamines. Indeed, for example, propranolol **2** shows significantly reduced affinity for human 5-HT1ARs, in which

**Figure 6.** Chemical structures of **1**–**3**.

the Asn386 is replaced by valine, while the affinity of the neurotransmitter 5-HT is hardly affected. It was initially hypothesized that the formation of two hydrogen bonds occurs between the oxypropanol moiety and the amide group of Asn386. Moreover, since the (*S*) enantiomer of propranolol is 13-fold more potent than the (*R*)-enantiomer at wild type (p*K*<sup>i</sup> 5-HT1AR = 6.8 and 5.7, respectively) and the enantioselectivity is significantly reduced (threefold) in Asn386Val mutant human 5-HT1ARs (p*K*<sup>i</sup> 5-HT1AR = 5.4 and 5.0, respectively), Asn386 proves to behave as a chiral discriminator. Moreover, the observation that the replacement of the hydroxyl substituent of **2** with a methoxy group does not affect the high affinity for the wild-type receptor suggests that one or both ether oxygen atoms of (*S*)-**3** may act as hydrogen bond acceptors. (*S*)-**3** (p*K*<sup>i</sup> 5-HT1AR = 6.8) also shows high affinity for the Asn386Val mutant receptor because of a favorable lipophilic contact of its methoxy group with Val386.

#### **5.2. Arylpiperazines**

Arylpiperazines are one of the most important classes of 5-HT1AR ligands from which a second generation of anxiolytics, including buspirone (**4**), the antipsychotics ziprasidone (**5**), perospirone (**6**), and aripiprazole (**7**), and several pharmacological tools originated (**Figure 7**) [8].

These ligands bind with high affinity to different GPCRs; the two multitarget drugs **5** and **6**, for example, acting as D<sup>2</sup> R antagonists and 5-HT1AR agonists, were marketed in 2001 and 2002, respectively, for the management of schizophrenia [4]. Compound **4** is the most known member of long-chain arylpiperazines (LCPAs) [17]. It was initially investigated as a putative antipsychotic agent devoid of the typical side effects of this class of drugs but was launched in the market as an anxiolytic in the USA in the 1980s. It behaves as a

**Figure 7.** Chemical structures of **4**–**8**.

potent but nonselective partial 5-HT1AR agonist and D<sup>2</sup> R antagonist. Since its launch, several *N*4-(2-pyrimidinyl)piperazines containing an *N*1-imidobutyl substituent have originated as the third generation of anxiolytic agents, including the partial agonist tandospirone (**8**) (**Figure 7**).

The general structure of arylpiperazines consists of a terminal fragment containing an amide, imide, alkyl, arylalkyl, heteroarylalkyl, or tetralin function linked through a flexible aliphatic chain of variable length to the *N*1-arylpiperazine moiety [8]. The search for new derivatives has been focused on the modification of one or more portions of such a pharmacophore. Some of the main changes are schematically reported in **Figure 8**.

#### *5.2.1. Modification of the aryl group*

the Asn386 is replaced by valine, while the affinity of the neurotransmitter 5-HT is hardly affected. It was initially hypothesized that the formation of two hydrogen bonds occurs between the oxypropanol moiety and the amide group of Asn386. Moreover, since the (*S*) enantiomer of propranolol is 13-fold more potent than the (*R*)-enantiomer at wild type (p*K*<sup>i</sup> 5-HT1AR = 6.8 and 5.7, respectively) and the enantioselectivity is significantly reduced (three-

proves to behave as a chiral discriminator. Moreover, the observation that the replacement of the hydroxyl substituent of **2** with a methoxy group does not affect the high affinity for the wild-type receptor suggests that one or both ether oxygen atoms of (*S*)-**3** may act as hydrogen

Arylpiperazines are one of the most important classes of 5-HT1AR ligands from which a second generation of anxiolytics, including buspirone (**4**), the antipsychotics ziprasidone (**5**), perospirone (**6**), and aripiprazole (**7**), and several pharmacological tools originated (**Figure 7**) [8].

These ligands bind with high affinity to different GPCRs; the two multitarget drugs **5** and

and 2002, respectively, for the management of schizophrenia [4]. Compound **4** is the most known member of long-chain arylpiperazines (LCPAs) [17]. It was initially investigated as a putative antipsychotic agent devoid of the typical side effects of this class of drugs but was launched in the market as an anxiolytic in the USA in the 1980s. It behaves as a

receptor because of a favorable lipophilic contact of its methoxy group with Val386.

5-HT1AR = 5.4 and 5.0, respectively), Asn386

5-HT1AR = 6.8) also shows high affinity for the Asn386Val mutant

R antagonists and 5-HT1AR agonists, were marketed in 2001

fold) in Asn386Val mutant human 5-HT1ARs (p*K*<sup>i</sup>

78 Serotonin - A Chemical Messenger Between All Types of Living Cells

bond acceptors. (*S*)-**3** (p*K*<sup>i</sup>

**6**, for example, acting as D<sup>2</sup>

**Figure 7.** Chemical structures of **4**–**8**.

**5.2. Arylpiperazines**

The replacement of the 2-pyrimidinyl moiety of **4** with a 2-methoxyphenyl group leads to the antidepressant BMY 8227 (**9**), from which BMY 7378 (**10**) originates by shortening its butyl

**Figure 8.** Pharmacophore of arylpiperazines.

to ethyl chain (**Figure 9**) [15]. Compounds **9** and **10** belong to a generation of postsynaptic 5-HT1AR antagonists, which also behave as low efficacy partial agonists [4].

The 2-methoxyphenyl group is also present in the WAY series, including WAY 100135 (**11**) and WAY 100635 (**12**) (**Figure 9**). These compounds, also called "silent" 5-HT1AR antagonists, behave as antagonists at both pre- and postsynaptic 5-HT1ARs. In the case of **11**, the (*S*) enantiomer is 28-fold more potent than its (*R*)-antipode.

The incorporation of the *o*-methoxy group into an annulated benzodioxane or benzofurane ring, affording two series of heterobicyclic arylpiperazines, is consistent with the maintenance of high 5-HT1AR affinity [15]. The benzodioxane fragment is present in the structure of flesinoxan (**13**) (**Figure 10**), a potent agonist at both pre- and postsynaptic 5-HT1ARs [15]. An example of benzofuran derivative showing high 5-HT1AR affinity is compound **14** (**Figure 10**).

Moderate to high affinity for 5-HT1ARs and SERT and low affinity for 5-HT2AR are recorded by ligands, whose four-carbon chain bears a quinoline moiety (**Figure 11**) [8].

**Figure 9.** Chemical structures of **9**–**12**.

**Figure 10.** Chemical structures of **13**–**14**.

**Figure 11.** General structure of quinoline derivatives.

#### *5.2.2. Modification of the piperazine ring*

to ethyl chain (**Figure 9**) [15]. Compounds **9** and **10** belong to a generation of postsynaptic

The 2-methoxyphenyl group is also present in the WAY series, including WAY 100135 (**11**) and WAY 100635 (**12**) (**Figure 9**). These compounds, also called "silent" 5-HT1AR antagonists, behave as antagonists at both pre- and postsynaptic 5-HT1ARs. In the case of **11**, the (*S*)-

The incorporation of the *o*-methoxy group into an annulated benzodioxane or benzofurane ring, affording two series of heterobicyclic arylpiperazines, is consistent with the maintenance of high 5-HT1AR affinity [15]. The benzodioxane fragment is present in the structure of flesinoxan (**13**) (**Figure 10**), a potent agonist at both pre- and postsynaptic 5-HT1ARs [15]. An example of benzofuran derivative showing high 5-HT1AR affinity is compound **14** (**Figure 10**). Moderate to high affinity for 5-HT1ARs and SERT and low affinity for 5-HT2AR are recorded by

5-HT1AR antagonists, which also behave as low efficacy partial agonists [4].

ligands, whose four-carbon chain bears a quinoline moiety (**Figure 11**) [8].

enantiomer is 28-fold more potent than its (*R*)-antipode.

80 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Figure 10.** Chemical structures of **13**–**14**.

**Figure 9.** Chemical structures of **9**–**12**.

**Figure 11.** General structure of quinoline derivatives.

*N*1-Arylpiperazine moiety plays an important role in the affinity for 5-HT1ARs. This template has been duplicated to successfully obtain selective homo- and heterobivalent ligands [18]. Indeed, compound **15** shows high affinity for 5-HT1ARs and selectivity over 5-HT7 Rs, whereas compound **16** selectively targets 5-HT7 Rs (p*K*<sup>i</sup> = 7.4) (**Figure 12**).

The piperazine ring can be replaced by a piperidine one. The most representative example is befiradol (**17**), a very potent and highly selective 5-HT1AR full agonist (**Figure 13**), that also shows efficacy in a rodent model of neuropathic, inflammatory, and surgical pain. It is endowed with potent analgesic and antiallodynic effects that are comparable to those of high doses of opioids. However, lower and fewer side effects are triggered, and little or no development of tolerance is manifested by **17**. In 2013, **17** was marketed by Neurolixis with indication for the treatment of L-DOPA-induced dyskinesia in Parkinson's disease [19]. The 3-chloro-4-fluorophenyl moiety of **17** can be bioisosterically replaced by both unsaturated and saturated lipophilic moieties [20]. Among the investigated compounds, the highly selective 5-HT1AR superagonist benzothiophene-3-carboxamide **18** almost exclusively recognizes 5-HT1ARs (**Figure 13**).

A series of 2*H*-pyrido[1,2-c]pyrimidine derivatives, bearing a piperidinyl-indole residue in their pharmacophore (**Figure 13**), shows very high-affinity values for both 5-HT1ARs and SERTs. Compound **19** is a representative example [21]. The presence of a tetrahydropyridinylindole moiety reduces binding to 5-HT1ARs, while a Cl substituent in R<sup>3</sup> reduces binding to both 5-HT1ARs and SERTs.

**Figure 13.** Chemical structures of **17**–**20**.

**Figure 12.** Chemical structures of **15** and **16**.

Finally, the presence of a 3β-aminotropane moiety instead of the piperazine or piperidine ring is unfavorable for the development of High affinity 5-HT1AR ligands (**Figure 14**) [22].

#### *5.2.3. Modification of the spacer*

In LCPAs, the four-carbon alkyl chain seems to be the most favorable for high 5-HT1AR affinity. Indeed, its shortening reduces affinity, according to the rank order of potency C-4 > C-2 > C-3 [4].

However, the butyl chain can be substituted by a propylthio bridge, as confirmed by the high 5-HT1AR affinity of compound **21** (**Figure 15**). The NH<sup>2</sup> function is responsible for its selectivity over α<sup>1</sup> -ARs (5-HT1AR/α<sup>1</sup> -AR = 55) [15].

The oxybutynin chain of aripiprazole (**7**) is also favorable for high 5-HT1AR affinity. Besides its main use in the treatment of schizophrenia and bipolar disorder, **7** is also employed as an add-on treatment in major depressive disorder, tic disorders, and irritability associated with autism. In addition, its systemic or local administration induces antinociceptive effects. Unlike other atypical antipsychotics approved by FDA (e.g., clozapine, olanzapine, quetiapine, ziprasidone, and risperidone), which are D<sup>2</sup> R antagonists, **7** behaves as a D<sup>2</sup> R and D<sup>3</sup> R partial agonist. Moreover, it shows partial agonism at 5-HT1ARs and, similarly to the other atypical antipsychotics, is an antagonist at 5-HT2ARs and 5-HT7 Rs as well as a partial agonist at 5-HT2CRs [23].

The presence of a hydroxyl group in the butyl chain is well tolerated. BMY 14802 (**22**) (**Figure 16**), for example, is a 5-HT1AR agonist that also attenuates dyskinesia produced by L-DOPA.

**Figure 14.** General structure of 3β-aminotropane derivatives.

A hydroxyalkyl chain also characterizes a series of molecules (**23**–**26**) (**Figure 17**), in which the combination of structural elements favoring the affinity for 5-HT1ARs (heterocyclic nucleus, hydroxyalkyl chain, and 4-substituted piperazine) was used to obtain ligands with high

**Figure 15.** Chemical structure of **21**.

5-HT1A affinity and selectivity over other 5-HT subtypes [24]. In particular, while compounds **23**–**25** show an outstanding 5-HT1AR affinity, compound **26** is selective for 5-HT2CRs (p*K*<sup>i</sup> = 8.3).

In a series of compounds prepared to discover mixed 5-HT/dopamine receptor agents as novel antipsychotics, amide **27** (**Figure 18**) emerges for its high affinity for D<sup>3</sup> Rs, 5-HT1ARs, and 5-HT2ARs. Its low affinity for D<sup>2</sup> Rs, 5-HT2CRs, and hERG channels reduces extrapyramidal side effects, risk of obesity under chronic treatment, and incidence of torsade des pointes, respectively [25]. The replacement of the ether/amide bridge with a sulfonamide function affords a series of quinoline or isoquinoline derivatives endowed with multireceptor 5-HT1AR/5-HT2AR/5-HT7 R/ D2 R/D<sup>3</sup> R profile and behaving as 5-HT1AR agonists, D<sup>2</sup> R partial agonists, and 5-HT2AR/5-HT7 R antagonists (**Figure 18**). They produce significant antidepressant activity in mice [26]. In particular, **28** also displays remarkable antipsychotic effects in MK-801-induced hyperlocomotor activity in mice.

The inclusion of the alkyl chain of LCAPs in a cyclohexyl ring leads to more conformationally constrained analogues (e.g., **29**) (**Figure 19**) [15]. Trans derivatives show 5-HT1AR affinity significantly higher than that of their corresponding cis isomers (e.g., trans **29** and cis **29**). The insertion of a hydroxyl substituent in the cyclohexyl moiety is also well tolerated (**30**). Interestingly, compared to flexible 4-carbon alkyl chain analogues, 1e,4e-disubstituted cyclohexane derivatives maintain very high 5-HT1AR affinity, but in some cases, the functional profile is modulated from partial agonism to antagonism [27].

**Figure 16.** Chemical structure of BMY 14802 (**22**).

Finally, the presence of a 3β-aminotropane moiety instead of the piperazine or piperidine ring

In LCPAs, the four-carbon alkyl chain seems to be the most favorable for high 5-HT1AR affinity. Indeed, its shortening reduces affinity, according to the rank order of potency C-4 > C-2 > C-3 [4]. However, the butyl chain can be substituted by a propylthio bridge, as confirmed by the high

The oxybutynin chain of aripiprazole (**7**) is also favorable for high 5-HT1AR affinity. Besides its main use in the treatment of schizophrenia and bipolar disorder, **7** is also employed as an add-on treatment in major depressive disorder, tic disorders, and irritability associated with autism. In addition, its systemic or local administration induces antinociceptive effects. Unlike other atypical antipsychotics approved by FDA (e.g., clozapine, olanzapine, quetiap-

partial agonist. Moreover, it shows partial agonism at 5-HT1ARs and, similarly to the other

The presence of a hydroxyl group in the butyl chain is well tolerated. BMY 14802 (**22**) (**Figure 16**),

A hydroxyalkyl chain also characterizes a series of molecules (**23**–**26**) (**Figure 17**), in which the combination of structural elements favoring the affinity for 5-HT1ARs (heterocyclic nucleus, hydroxyalkyl chain, and 4-substituted piperazine) was used to obtain ligands with high

for example, is a 5-HT1AR agonist that also attenuates dyskinesia produced by L-DOPA.

function is responsible for its selectiv-

R and D<sup>3</sup>

Rs as well as a partial agonist

R

R antagonists, **7** behaves as a D<sup>2</sup>

is unfavorable for the development of High affinity 5-HT1AR ligands (**Figure 14**) [22].

*5.2.3. Modification of the spacer*


ity over α<sup>1</sup>

at 5-HT2CRs [23].

5-HT1AR affinity of compound **21** (**Figure 15**). The NH<sup>2</sup>

82 Serotonin - A Chemical Messenger Between All Types of Living Cells

ine, ziprasidone, and risperidone), which are D<sup>2</sup>

**Figure 14.** General structure of 3β-aminotropane derivatives.

**Figure 15.** Chemical structure of **21**.

atypical antipsychotics, is an antagonist at 5-HT2ARs and 5-HT7


**Figure 17.** Chemical structures of **23**–**26**.

The alkyl chain can be partially included in aromatic functions, including pyrrole (RWJ 25730, **31**), phenyl (mazapertine, **32**), or benzimidazole (**33**) (**Figure 20**) [15]. The multireceptor affinity of **32** can be ascribed to its ability to adopt a variety of low-energy conformations. Indeed, constraining its 2-isopropoxyphenyl and piperazine moieties, affording compound **34**, significantly reduces affinities for α<sup>1</sup> -ARs and D<sup>2</sup> Rs, but not that for 5-HT1ARs.

The insertion of the 1,3-dioxolane nucleus in the chain is also well tolerated. Compound **35**, for example, is a potent partial agonist and shows moderate selectivity over α<sup>1</sup> -ARs (**Figure 21**) [28]. Substitutions at C-8 position of the 1,4-dioxaspiro[4, 5]decane moiety reduce 5-HT1AR/α<sup>1</sup> -AR selectivity ratio because of the significant decrease of binding affinity and intrinsic activity for 5-HT1ARs with respect to α<sup>1</sup> -ARs. The isosteric replacement of one (oxathiolane derivative **36**) and especially of two (dithiolane derivative **37**) oxygen atoms with sulfur atoms proves to be tolerated (**Figure 21**). The replacement of the piperazine ring with a more flexible basic chain affords compound **38**, which behaves as a potent and selective 5-HT1AR partial agonist endowed with neuroprotective activity in vitro and potent antinociceptive activity in an in vivo model [28]. A similar profile is shown by the unsubstituted analogue **39** characterized by good 5-HT1A/ α1 -AR selectivity (**Figure 21**).

Similar structure-activity relationships (SARs) can be observed when the spiro-cyclohexyl terminal fragment in both piperazine and open-chain series is replaced by a 2,2-diphenyl moiety.

**Figure 18.** Chemical structures of **27** and **28**.

**Figure 19.** Chemical structures of **29** and **30**.

**Figure 21.** Chemical structures of **35**–**39**.

**Figure 19.** Chemical structures of **29** and **30**.

affinities for α<sup>1</sup>

α1

5-HT1ARs with respect to α<sup>1</sup>


**Figure 18.** Chemical structures of **27** and **28**.


84 Serotonin - A Chemical Messenger Between All Types of Living Cells

The alkyl chain can be partially included in aromatic functions, including pyrrole (RWJ 25730, **31**), phenyl (mazapertine, **32**), or benzimidazole (**33**) (**Figure 20**) [15]. The multireceptor affinity of **32** can be ascribed to its ability to adopt a variety of low-energy conformations. Indeed, constraining its 2-isopropoxyphenyl and piperazine moieties, affording compound **34**, significantly reduces

The insertion of the 1,3-dioxolane nucleus in the chain is also well tolerated. Compound **35**, for

selectivity ratio because of the significant decrease of binding affinity and intrinsic activity for

and especially of two (dithiolane derivative **37**) oxygen atoms with sulfur atoms proves to be tolerated (**Figure 21**). The replacement of the piperazine ring with a more flexible basic chain affords compound **38**, which behaves as a potent and selective 5-HT1AR partial agonist endowed with neuroprotective activity in vitro and potent antinociceptive activity in an in vivo model [28]. A similar profile is shown by the unsubstituted analogue **39** characterized by good 5-HT1A/

Similar structure-activity relationships (SARs) can be observed when the spiro-cyclohexyl terminal fragment in both piperazine and open-chain series is replaced by a 2,2-diphenyl moiety.


Substitutions at C-8 position of the 1,4-dioxaspiro[4, 5]decane moiety reduce 5-HT1AR/α<sup>1</sup>



Rs, but not that for 5-HT1ARs.

example, is a potent partial agonist and shows moderate selectivity over α<sup>1</sup>

The replacement of the 1,3-dioxolane nucleus with other pentatomic rings bearing H-bond acceptor groups (tetrahydrofuran or cyclopentanone) or an H-bond acceptor and donor group (cyclopentanol) (**Figure 22**) causes an overall reduction of affinity at α<sup>1</sup> -ARs, while both potency and efficacy are increased at 5-HT1ARs.

#### *5.2.4. Modification of the terminal fragment*

The numerous structurally different terminal fragments, as already seen for ligands reported above, demonstrate that this moiety is less critical for 5-HT1AR interaction [8]. The dual SSRI and 5-HT1AR agonist vortioxetine (**40**), approved by FDA for the treatment of major depressive disorders in adult in 2013, even lacks this function (**Figure 23**).

The replacement of the azaspirodecanedione moiety of **9** with an *N*-phthalimido group affords the nonselective ligand **41** (**Figure 24**) [15]. Shortening the length of its butyl chain to three or two units significantly decreases the affinity. The presence of an isosteric sulfonyl function instead of a carbonyl group of the phthalimide moiety, as in ipsapirone (**42**), is compatible with

**Figure 22.** Bioisosteric replacement of oxygen atoms of 5-HT1AR 1,3-dioxolane ligands.

the maintenance of similar 5-HT1AR affinity and improved selectivity over α<sup>1</sup> -ARs (**Figure 24**) [15]. The replacement of the phthalimide moiety of **41** with an adamantyl amide group, leading to **43**, also increases the selectivity for 5-HT1ARs over α<sup>1</sup> -ARs (**Figure 24**) [15]. As in the case of the prototypical 5-HT1AR antagonist **12**, substituents can be present at amidic NH [15]. The replacement of the pyridine ring of **12** with a pyrimidine substituent leads to the similarly potent 5-HT1AR antagonist **44**. The isosteric inversion of the amide function and the presence of a phenyl group in the bridge, affording **45**, are tolerated (**Figure 24**). Considering both affinity and selectivity for 5-HT1ARs, among some 5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl derivatives obtained by inserting an alkyl chain of variable length (preferably a three-membered alkyl chain) in the α, β, or ω position, the best derivatives are **46** and **47** (**Figure 24**) [15].

Several molecules, bearing an isonicotinic moiety as the terminal fragment of LCAPs, show nanomolar and subnanomolar affinities for 5-HT1ARs, 5-HT2ARs, and 5-HT2CRs and moderate or no affinity for other relevant receptors (D<sup>1</sup> Rs, D<sup>2</sup> Rs, α<sup>1</sup> - and α<sup>2</sup> -ARs) [29]. In particular, derivative **48**, bearing a propyl chain as a spacer, shows the highest affinity for 5-HT1ARs and selectivity over dopaminergic, adrenergic, and other serotoninergic receptors (**Figure 25**). LCAPs bearing a 1,2,3,4-tetrahydroisoquinoline-3-carboxamide in the terminal fragment can show affinity for 5-HT1ARs and/or 5-HT7 Rs [30]. Indeed, while compounds **49** and **50**, with a methylthio substituent in the ortho-position show high

**Figure 23.** Chemical structure of vortioxetine (**40**).

**Figure 24.** Chemical structures of **41**–**47**.

**Figure 22.** Bioisosteric replacement of oxygen atoms of 5-HT1AR 1,3-dioxolane ligands.

ing to **43**, also increases the selectivity for 5-HT1ARs over α<sup>1</sup>

86 Serotonin - A Chemical Messenger Between All Types of Living Cells

moderate or no affinity for other relevant receptors (D<sup>1</sup>

the terminal fragment can show affinity for 5-HT1ARs and/or 5-HT7

the maintenance of similar 5-HT1AR affinity and improved selectivity over α<sup>1</sup>

chain) in the α, β, or ω position, the best derivatives are **46** and **47** (**Figure 24**) [15].

[15]. The replacement of the phthalimide moiety of **41** with an adamantyl amide group, lead-

case of the prototypical 5-HT1AR antagonist **12**, substituents can be present at amidic NH [15]. The replacement of the pyridine ring of **12** with a pyrimidine substituent leads to the similarly potent 5-HT1AR antagonist **44**. The isosteric inversion of the amide function and the presence of a phenyl group in the bridge, affording **45**, are tolerated (**Figure 24**). Considering both affinity and selectivity for 5-HT1ARs, among some 5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl derivatives obtained by inserting an alkyl chain of variable length (preferably a three-membered alkyl

Several molecules, bearing an isonicotinic moiety as the terminal fragment of LCAPs, show nanomolar and subnanomolar affinities for 5-HT1ARs, 5-HT2ARs, and 5-HT2CRs and

particular, derivative **48**, bearing a propyl chain as a spacer, shows the highest affinity for 5-HT1ARs and selectivity over dopaminergic, adrenergic, and other serotoninergic receptors (**Figure 25**). LCAPs bearing a 1,2,3,4-tetrahydroisoquinoline-3-carboxamide in

compounds **49** and **50**, with a methylthio substituent in the ortho-position show high

Rs, D<sup>2</sup>

Rs, α<sup>1</sup>




Rs [30]. Indeed, while


**Figure 23.** Chemical structure of vortioxetine (**40**).

5-HT1AR affinity, the replacement of the phenyl ring in the arylpiperazine moiety with a benzisoxazole system, affording, for example, **51** and **52**, significantly increases the affinity for 5-HT<sup>7</sup> R (p*K*<sup>i</sup> = 7.7 and 7.6, respectively) (**Figure 25**). The insertion of a spiro-cyclopentane or cyclohexane in position 3 of pyrrolidin-2,5-dione leads to a series of arylpiperazines, among which derivatives **53** and **54** with an ethylene spacer and a CF<sup>3</sup> substituent in meta position of the phenyl ring show both anticonvulsant activity and high 5-HT1AR and 5-HT2AR affinity (**Figure 25**) [31].

A β-tetralonohydantoin as terminal fragment characterizes a series of compounds, which show high 5-HT1AR affinity (p*K*<sup>i</sup> = 7.3–8.2) combined with moderate to high 5-HT2AR affinity (p*K*<sup>i</sup> = 6.7–7.3). Among them, compound **55** (**Figure 26**) is a postsynaptic 5-HT1AR antagonist and produces the characteristic effect of presynaptic 5-HT1AR agonists [32]. Moreover, it behaves as a 5-HT2AR antagonist. Due to its interesting 5-HT1A/5-HT2A functional profile, **55**, tested for its potential psychotropic activity, shows diazepam-like anxiolytic activity and behaves as a weak antidepressant.

Among new LNCPs with structural modifications in the terminal fragment, in the alkyl chain length and in the substituents of the piperazine fragment, the 2-ethoxy quinazolinone derivatives **56** and **57** are the most interesting ligands, showing high affinity for 5-HT1ARs and 5-HT<sup>7</sup> Rs (**Figure 26**) [33].

**Figure 25.** Chemical structures of **48**–**54**.

In a more recent work, the quinazolinone system has been replaced by 6-phenyl-4(3*H*)-pyrimidinone as a result of splitting bicyclic quinazolinone system [34]. The benzo-cracking strategy (compounds **58**–**62**) causes a decrease in affinity for both receptors. In functional assays, these derivatives behave as weak 5-HT1AR and 5-HT7 R antagonists (**Figure 26**).

1,2,4-Triazine-6(1*H*)-one derivatives also display dual affinity for 5-HT1ARs and 5-HT7 Rs. SAR studies have revealed that receptor affinity and selectivity depend on the nature of the substituent in position 3 of the triazinone fragment as well as on the substitution pattern of the phenylpiperazine moiety [35]. The best 5-HT1AR affinity values and selectivity over 5-HT7 Rs are displayed by compounds **63** and **64** (**Figure 26**).

The 3,5-dioxo-(2*H*,4*H*)-1,2,4-triazine-tethered arylpiperazines have been identified as agonists with high affinity for 5-HT1ARs. Several members of this series such as **65** show nanomolar affinity for 5-HT1ARs, high selectivity over α<sup>1</sup> -AR, and potent agonist activity (**Figure 26**) [36]. The 1,2,3-benzotriazin-4-one terminal fragment characterizes some 5-HT1AR antagonists prepared as potential antiproliferative agents in cancer cell lines [37]. These compounds are endowed with high 5-HT1AR affinity and moderate or no affinity for other receptors (5-HT2ARs, 5-HT2CRs, D<sup>1</sup> Rs, D<sup>2</sup> Rs, α<sup>1</sup> - and α<sup>2</sup> -ARs). In particular, derivative **66** shows picomolar affinity for 5-HT1ARs (**Figure 26**).

MP 3022 (**67**), the lead compound of a large series of 4-alkyl-1-(*o*-methoxyphenyl)-piperazines containing a benzotriazole terminal fragment, behaves as a potent pre- and postsynaptic 5-HT1AR antagonist, but it is not selective for 5-HT1ARs over α<sup>1</sup> -ARs (**Figure 26**) [15]. 4-Benzoyl-1,2,3-triazole derivatives (e.g., **68**), open-chain analogues of their benzotriazole bioisosteres, bind to 5-HT1ARs in a nanomolar range and are highly selective over 5-HT2ARs and 5-HT2CRs (**Figure 26**) [15].

Purine 2,6-dione core has also been used as a terminal fragment to combine the 5-HT1AR activity with the phosphodiesterase (PDE) inhibition [38]. Both effects might be advantageous in

**Figure 26.** Chemical structures of **55**–**68**.

In a more recent work, the quinazolinone system has been replaced by 6-phenyl-4(3*H*)-pyrimidinone as a result of splitting bicyclic quinazolinone system [34]. The benzo-cracking strategy (compounds **58**–**62**) causes a decrease in affinity for both receptors. In functional assays, these

studies have revealed that receptor affinity and selectivity depend on the nature of the substituent in position 3 of the triazinone fragment as well as on the substitution pattern of the phenylpiperazine moiety [35]. The best 5-HT1AR affinity values and selectivity over 5-HT7

The 3,5-dioxo-(2*H*,4*H*)-1,2,4-triazine-tethered arylpiperazines have been identified as agonists with high affinity for 5-HT1ARs. Several members of this series such as **65** show nanomo-

[36]. The 1,2,3-benzotriazin-4-one terminal fragment characterizes some 5-HT1AR antagonists prepared as potential antiproliferative agents in cancer cell lines [37]. These compounds are endowed with high 5-HT1AR affinity and moderate or no affinity for other receptors (5-HT2ARs,

MP 3022 (**67**), the lead compound of a large series of 4-alkyl-1-(*o*-methoxyphenyl)-piperazines containing a benzotriazole terminal fragment, behaves as a potent pre- and postsyn-

4-Benzoyl-1,2,3-triazole derivatives (e.g., **68**), open-chain analogues of their benzotriazole bioisosteres, bind to 5-HT1ARs in a nanomolar range and are highly selective over 5-HT2ARs and

Purine 2,6-dione core has also been used as a terminal fragment to combine the 5-HT1AR activity with the phosphodiesterase (PDE) inhibition [38]. Both effects might be advantageous in

1,2,4-Triazine-6(1*H*)-one derivatives also display dual affinity for 5-HT1ARs and 5-HT7

R antagonists (**Figure 26**).




Rs. SAR

Rs

derivatives behave as weak 5-HT1AR and 5-HT7

88 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Figure 25.** Chemical structures of **48**–**54**.

are displayed by compounds **63** and **64** (**Figure 26**).

lar affinity for 5-HT1ARs, high selectivity over α<sup>1</sup>

Rs, α<sup>1</sup>


aptic 5-HT1AR antagonist, but it is not selective for 5-HT1ARs over α<sup>1</sup>

5-HT2CRs, D<sup>1</sup>

Rs, D<sup>2</sup>

for 5-HT1ARs (**Figure 26**).

5-HT2CRs (**Figure 26**) [15].

the treatment of neuropsychiatric disorders. Among the compounds bearing this core, **69–72** show high affinity for 5-HT1ARs and, in the case of **69** and **70**, also for 5-HT7 R. At the same time, compounds **69–72** show a moderate to very low D<sup>2</sup> R affinity. From functional assays, **69**–**71** behave as 5-HT1AR antagonists, whereas **72** is an agonist (**Figure 27**) [38, 39]. The antidepressant activity of **69** and **70** at a dose of 1.25 mg/kg is similar to that of citalopram given at the same dose [38]. The annulation of the purine system at 7,8-positions with an imidazole moiety affords ligands with a wide spectrum of activities (high 5-HT1AR or 5-HT7 R affinity, mixed 5-HT1AR/5-HT7 R affinity, and additional affinity for D<sup>2</sup> R) [40]. The tested compounds are in the ranges defined by the "rule of five" (logP < 5), which indicates good intestinal permeability and metabolic stability. In preliminary pharmacological in vivo studies, the selected compound **73** behaves as a potential antidepressant in mice and, at the dose of 2.5 mg/kg, shows anxiolytic effect (**Figure 27**). Finally, purine 2,4,8-trione derivatives show affinity values lower than those of the corresponding purine 2,4-dione analogues (**Figure 27**) [41].

## *5.2.5. Main interactions of arylpiperazines with 5-HT1ARs*

Two main interactions prove to be important for the affinity of arylpiperazines for 5-HT1ARs: (a) an ionic bond between the protonated nitrogen atom of the piperazine ring and the carboxyl oxygen of the side chain of Asp3.32 and (b) an edge-to-face CH-π interaction between the aromatic ring and the Phe6.52 residue, which stabilizes the ligand binding. The basic pharmacophore of the 5-HT1AR is the same for agonists and antagonists and consists of an aromatic nucleus and a basic nitrogen atom, whose optimal distance is 5.2 Å, while the nitrogen lies at 0.2 Å above the plane defined by the reference ring (**Figure 28**) [4].

Due to the highly flexible linker (usually 2-4 methylene units), using different experimental and modeling techniques, various attempts have been conducted to determine the bioactive conformation of LCAPs [42]. Assuming that active conformations of LCAPs are closely related to those in solutions or in solid state, two-dimensional (2D) NMR and crystallographic methods were often applied. The 2D NMR studies indicated that compounds with tetramethylene spacer can adopt extended, bent, or folded conformations. On the other hand, analysis of Cambridge Structural Database showed that linear geometries predominated. Molecular

**Figure 27.** Chemical structures of **69**–**73** and general structure of purine 2,4,8-trione derivatives.

**Figure 28.** General structure of LCAPs and pharmacophoric model of 5-HT1AR (Adapted with permission from Ref. [4]. Copyright (2014) American Chemical Society).

modeling studies (conformational analysis, docking, dynamics), provided with structural investigations or conducted separately, also gave equivocal results suggesting the possibility of different bioactive conformations of LCAPs.

## **5.3. Aminotetralins**

**Figure 27.** Chemical structures of **69**–**73** and general structure of purine 2,4,8-trione derivatives.

mixed 5-HT1AR/5-HT7

R affinity, and additional affinity for D<sup>2</sup>

*5.2.5. Main interactions of arylpiperazines with 5-HT1ARs*

90 Serotonin - A Chemical Messenger Between All Types of Living Cells

are in the ranges defined by the "rule of five" (logP < 5), which indicates good intestinal permeability and metabolic stability. In preliminary pharmacological in vivo studies, the selected compound **73** behaves as a potential antidepressant in mice and, at the dose of 2.5 mg/kg, shows anxiolytic effect (**Figure 27**). Finally, purine 2,4,8-trione derivatives show affinity values lower than those of the corresponding purine 2,4-dione analogues (**Figure 27**) [41].

Two main interactions prove to be important for the affinity of arylpiperazines for 5-HT1ARs: (a) an ionic bond between the protonated nitrogen atom of the piperazine ring and the carboxyl oxygen of the side chain of Asp3.32 and (b) an edge-to-face CH-π interaction between the aromatic ring and the Phe6.52 residue, which stabilizes the ligand binding. The basic pharmacophore of the 5-HT1AR is the same for agonists and antagonists and consists of an aromatic nucleus and a basic nitrogen atom, whose optimal distance is 5.2 Å, while the nitrogen lies at 0.2 Å above the plane defined by the reference ring (**Figure 28**) [4]. Due to the highly flexible linker (usually 2-4 methylene units), using different experimental and modeling techniques, various attempts have been conducted to determine the bioactive conformation of LCAPs [42]. Assuming that active conformations of LCAPs are closely related to those in solutions or in solid state, two-dimensional (2D) NMR and crystallographic methods were often applied. The 2D NMR studies indicated that compounds with tetramethylene spacer can adopt extended, bent, or folded conformations. On the other hand, analysis of Cambridge Structural Database showed that linear geometries predominated. Molecular

R) [40]. The tested compounds

For a long time, 2-aminotetralin structure has been known to be pharmacologically important. Initially, aminotetralins were characterized by their sympathomimetic action, i.e., the induction of mydriasis, contraction of the uterus, changes in blood pressure, and respiration, as well as increased intestinal motility in in vivo experiments. During the late 1960s, the discovery of their activity at central dopamine receptor led to active synthesis programs all over the world. The 2-aminotetralin structure has proven to be a valuable scaffold not only for the development of 5-HTR ligands, but it also characterizes dopamine and adrenergic receptor ligands, as well as compounds interacting with melatonin receptors [15]. The main SARs of aminotetralins are summarized in **Figure 29**.

The position of the hydroxyl group in the aromatic ring of the tetralin scaffold is crucial to address ligands toward 5-HT or dopamine receptors. Indeed, 8-hydroxy-2-(*N*,*N*-di-*n*-propylamino)tetralin (8-OH-DPAT, **74**) (**Figure 30**) is a very potent and selective 5-HT receptor ligand, while its 5- and 7-hydroxy regioisomers (5- and 7-OH-DPAT) are potent dopamine receptor ligands. [<sup>3</sup> H]8-OH-DPAT is frequently used to label 5-HT1ARs. Both its enantiomers show high affinity for 5-HT1ARs. However, in functional experiments, the (*R*) enantiomer behaves as a full agonist while its antipode as a partial agonist.

Compounds obtained by replacing the 8-hydroxy substituent with 8-methoxy (8-MeO-DPAT, **75**), 8-acetyl (**76**), and 8-methoxycarbonyl (**77**) or 8-carboxamide (**78**) groups are about as potent as the parent compound, indicating that the proton of the 8-hydroxy group is not essential for drug-receptor interaction (**Figure 30**). A carboxylic group in the same position (**79**) is not favorable. Aryl and heteroaryl groups, such as phenyl, fluorophenyl, methoxyphenyl, acetylphenyl, 2-furyl, and benzylthio, are well tolerated. For most derivatives, the (*R*) enantiomers are more potent than the (*S*)-enantiomers. The introduction of a fluorine atom at position C-5 of **74**, affording **80**, slightly decreases 5-HT1AR affinity. In functional studies, the (*R*)-enantiomer behaves as a partial agonist, while the (*S*)-enantiomer is a pure antagonist at both pre- and postsynaptic receptors. An antagonist is also obtained by introducing a methyl group in 5-position of **74** (compound **81**) (**Figure 30**). The replacement of the *N*,*N*-di-*n*propyl groups of **74** or **75** with smaller or larger di-*n*-alkyl substituents results in a significant

**Figure 29.** Main SARs of aminotetralins.

**Figure 30.** Chemical structures of **74**–**87**.

decrease in affinity. The rank order of potency is *N*,*N*-dipropyl > *N*,*N*-diethyl > *N*,*N*-dibutyl > *N*,*N*-dimethyl group.

Compared to the *N*,*N*-dialkylated 8-MeO-DPAT (**75**), the monoalkylated N-propyl derivative **84** shows slightly lower affinity, whereas the non-substituted 8-methoxy-2-aminotetralin (**82**) is almost inactive (**Figure 30**). The piperidine analogue **83** (**Figure 30**) is 16–29-fold less active than the *N*-mono (**84**) or *N*,*N*-dipropyl derivative (**75**). Compounds with high-affinity values are obtained if the amino group is monosubstituted with relatively large substituents as a phenylalkyl moiety, with the 3-phenylpropyl-8-methoxy group being optimal (**85**). Even an extra *N*-methyl group (**86**) or bulky substituents such as an *N*-(phthalimidobutyl) group are also well tolerated (**87**).

The incorporation of the nitrogen atom in the tetralin nucleus furnishes the series of 1,2,3,4-tetrahydroisoquinoline (THIQ) derivatives, which bind to 5-HT1ARs and 5-HT2ARs. SAR studies performed on the THIQ class lead to the synthesis of 1-adamantoyloaminoalkyl derivatives endowed with high affinity for 5-HT1ARs (p*K*<sup>i</sup> = 7.3–8.3) and behaving as postsynaptic 5-HT1AR partial agonists (**Figure 31**).

Ring contraction (indamines) or ring expansion (benzocycloheptamines) of the cycloexyl ring of 2-aminotetralins decreases 5-HT1AR affinity. The replacement of the tetralin scaffold with the chroman nucleus does not influence affinity and selectivity.

Among the four enantiomers obtained by the introduction of a methyl group in position 1 of **75**, only (*S*,*R*)-**88** displays high affinity for 5-HT1ARs (**Figure 32**). In functional tests, it behaves as a mixed partial 5-HT1AR agonist/D<sup>2</sup> R antagonist.

The restriction of the conformation of **88** by the incorporation of the C-1 methyl and the C-2 nitrogen into an azetidine (**89**) or pyrrolidine (**90**) ring significantly enhances 5-HT1AR affinity (**Figure 32**). These more rigid four/six and five/six fused angular tricyclic 2-aminotetralins are *N*-substituted with either *n*-propyl or its bioequivalent 2-propenyl group. The cis racemates of both series are more potent than cis-**88**. The hydroxy derivatives display selective 5-HT1AR agonist activity, whereas the methoxy analogues show mixed 5-HT1AR agonist and dopamine antagonist activities. In general, the cis analogues are more potent than the corresponding trans analogues, and in the cis series, the (*S*,*R*)-enantiomers display higher potency (**Figure 32**). Nitrogen substitution with either an *n*-propyl or an allyl group leads to ligands with similar activities, whereas their replacement with a bulky α-methylbenzyl group produces a decrease in activity. The incorporation of the C-1 methyl and the C-2 nitrogen into a more flexible six-membered piperidine ring (**91**) is less favorable for 5-HT1AR affinity. In contrast to the pyrrolidine series, in these six/six fused angular tricyclic 2-aminotetralins, the trans enantiomers are more potent than the cis antipodes (**Figure 32**).

The introduction of a methyl group in position 3 of **75** is not favorable for high 5-HT1AR affinity. Consequently, the incorporation of the C-2 nitrogen and C-3 methyl into a five-membered

**Figure 31.** General structure of THIQ derivatives.

decrease in affinity. The rank order of potency is *N*,*N*-dipropyl > *N*,*N*-diethyl > *N*,*N*-dibutyl >

Compared to the *N*,*N*-dialkylated 8-MeO-DPAT (**75**), the monoalkylated N-propyl derivative **84** shows slightly lower affinity, whereas the non-substituted 8-methoxy-2-aminotetralin (**82**) is almost inactive (**Figure 30**). The piperidine analogue **83** (**Figure 30**) is 16–29-fold less active than the *N*-mono (**84**) or *N*,*N*-dipropyl derivative (**75**). Compounds with high-affinity values are obtained if the amino group is monosubstituted with relatively large substituents as a phenylalkyl moiety, with the 3-phenylpropyl-8-methoxy group being optimal (**85**). Even an extra *N*-methyl group (**86**) or bulky substituents such as an *N*-(phthalimidobutyl) group are

The incorporation of the nitrogen atom in the tetralin nucleus furnishes the series of 1,2,3,4-tetrahydroisoquinoline (THIQ) derivatives, which bind to 5-HT1ARs and 5-HT2ARs. SAR studies performed on the THIQ class lead to the synthesis of 1-adamantoyloaminoalkyl derivatives

Ring contraction (indamines) or ring expansion (benzocycloheptamines) of the cycloexyl ring of 2-aminotetralins decreases 5-HT1AR affinity. The replacement of the tetralin scaffold with

= 7.3–8.3) and behaving as postsynaptic 5-HT1AR

*N*,*N*-dimethyl group.

**Figure 30.** Chemical structures of **74**–**87**.

**Figure 29.** Main SARs of aminotetralins.

92 Serotonin - A Chemical Messenger Between All Types of Living Cells

also well tolerated (**87**).

partial agonists (**Figure 31**).

endowed with high affinity for 5-HT1ARs (p*K*<sup>i</sup>

the chroman nucleus does not influence affinity and selectivity.

**Figure 32.** Chemical structures of **88**–**91**.

pyrrolidine ring also leads to five/six fused linear tricyclic 2-aminotetralins, which are only moderately active.

A different six/six fused angular tricyclic of 2-aminotetralin is obtained by incorporating the 8-oxygen atom and C-7 into a six-membered ring, obtaining **92** and **93**, respectively. However, these modifications reduce affinity. The (*R*) configuration is more favorable than the (*S*) one (**Figure 33**).

A further decrease in affinity is shown by compounds bearing an annulated pyrrole ring in which the NH moiety is in the same position as the hydroxy group of **74**. On the contrary, the annulation in which the indole NH is in C-7 of the tetralin nucleus affords potent 5-HT1AR ligands (**94**) (**Figure 34**).

The introduction of a formyl group at C-1 of **94**, affording **95** (**Figure 34**), modulates the pharmacological profile from a mixed D<sup>2</sup> /5-HT1AR agonist to a selective 5-HT1AR agonist. The enantiomers of **95** are full agonists with affinities comparable to that of **74**. Both affinity and selectivity for 5-HT1ARs are improved by the substitution at C-1 of the pyrrole ring with a cyano group. In fact, the enantiomers of the 1-cyano derivative **96** are almost equipotent to the corresponding formyl derivative **95**, while 1-chloro (**97**) and 1-(1,1,1-trifluoroethyl) (**98**) substituents lead to less potent derivatives. The substitution at the C-2 of the pyrrole with a carboxamide (**99**) or cyano function (**100**) is also well tolerated, compound **100** being a potent 5-HT1AR agonist. In the C-1 and C-2 substituted series, the (*R*)-enantiomers display high and moderate affinity for 5-HT1ARs and D<sup>2</sup> Rs, respectively. The (*S*)-enantiomers are somewhat less potent but even more selective 5-HT1AR ligands. An unsubstituted indole-NH moiety is crucial for the interaction with 5-HT1ARs. Indeed, the *N*-methyl compounds are significantly less potent. Without loss in 5-HT1AR affinity, one of the propyl groups can be replaced by a variety of large substituents such as the glutarimide-butyl one (**101**–**103**) (**Figure 34**). In functional tests, most of the (*R*)-enantiomers behave as full agonists, whereas the corresponding (*S*)-enantiomers are partial agonists.

#### **5.4. Indolylalkylamines**

The prototype of this class of compounds is the endogenous ligand 5-HT (**Figure 1**), which behaves as a potent 5-HT1AR agonist (p*K*<sup>i</sup> = 8.4). The alkylation at α or β positions of tryptamine moiety, as well as the incorporation of its alkylamine side chain into a 4-substituted tetrahydropyridine ring, strongly decreases 5-HT1AR affinity [15]. The removal of the hydroxyl group at position C-5 also reduces 5-HT1AR affinity, the unsubstituted tryptamine analogue

**Figure 33.** Chemical structures of **92** and **93**.

**Figure 34.** Chemical structures of **94**–**103**.

pyrrolidine ring also leads to five/six fused linear tricyclic 2-aminotetralins, which are only

A different six/six fused angular tricyclic of 2-aminotetralin is obtained by incorporating the 8-oxygen atom and C-7 into a six-membered ring, obtaining **92** and **93**, respectively. However, these modifications reduce affinity. The (*R*) configuration is more favorable than the (*S*) one

A further decrease in affinity is shown by compounds bearing an annulated pyrrole ring in which the NH moiety is in the same position as the hydroxy group of **74**. On the contrary, the annulation in which the indole NH is in C-7 of the tetralin nucleus affords potent 5-HT1AR

The introduction of a formyl group at C-1 of **94**, affording **95** (**Figure 34**), modulates the

The enantiomers of **95** are full agonists with affinities comparable to that of **74**. Both affinity and selectivity for 5-HT1ARs are improved by the substitution at C-1 of the pyrrole ring with a cyano group. In fact, the enantiomers of the 1-cyano derivative **96** are almost equipotent to the corresponding formyl derivative **95**, while 1-chloro (**97**) and 1-(1,1,1-trifluoroethyl) (**98**) substituents lead to less potent derivatives. The substitution at the C-2 of the pyrrole with a carboxamide (**99**) or cyano function (**100**) is also well tolerated, compound **100** being a potent 5-HT1AR agonist. In the C-1 and C-2 substituted series, the

The (*S*)-enantiomers are somewhat less potent but even more selective 5-HT1AR ligands. An unsubstituted indole-NH moiety is crucial for the interaction with 5-HT1ARs. Indeed, the *N*-methyl compounds are significantly less potent. Without loss in 5-HT1AR affinity, one of the propyl groups can be replaced by a variety of large substituents such as the glutarimide-butyl one (**101**–**103**) (**Figure 34**). In functional tests, most of the (*R*)-enantiomers behave as full agonists, whereas the corresponding (*S*)-enantiomers are partial agonists.

The prototype of this class of compounds is the endogenous ligand 5-HT (**Figure 1**), which

amine moiety, as well as the incorporation of its alkylamine side chain into a 4-substituted tetrahydropyridine ring, strongly decreases 5-HT1AR affinity [15]. The removal of the hydroxyl group at position C-5 also reduces 5-HT1AR affinity, the unsubstituted tryptamine analogue

(*R*)-enantiomers display high and moderate affinity for 5-HT1ARs and D<sup>2</sup>

/5-HT1AR agonist to a selective 5-HT1AR agonist.

= 8.4). The alkylation at α or β positions of trypt-

Rs, respectively.

moderately active.

ligands (**94**) (**Figure 34**).

**5.4. Indolylalkylamines**

behaves as a potent 5-HT1AR agonist (p*K*<sup>i</sup>

**Figure 33.** Chemical structures of **92** and **93**.

pharmacological profile from a mixed D<sup>2</sup>

94 Serotonin - A Chemical Messenger Between All Types of Living Cells

(**Figure 33**).

being 30-fold less potent than 5-HT. However, the 5-hydroxyl group can be replaced by a 5-methoxy or 5-carboxamide function, leading to 5-MeOT (**104**) and 5-CT (**105**), respectively, which show high 5-HT1AR affinities (**Figure 35**).

The 4-substituted tetrahydropyridine analogue of **104** (RU 24969, **106**) and the *N*,*N*-di-*n*propyl analogue of **105** (DP-5-CT, **107**) also show high 5-HT1AR affinities and behave as potent and selective 5-HT1AR agonists (**Figure 35**). The incorporation of the side chain of **105** into a 3-substituted tetrahydropyridine, affording **108**, slightly decreases 5-HT1AR affinity, which is further reduced by the removal of the 5-carboxyamido function or its replacement with substituents such as a methoxy or cyano group. Linking the indolyl moiety to an *N*-substituted piperazine ring through a proper alkyl spacer (LCAPs) also proves to be compatible with high 5-HT1AR affinity and selectivity [43]. In particular, hydroxy, methoxy, or carboxamide groups in position 5 of the indole moiety yield ligands with high 5-HT1AR affinity. Such ligands tolerate several substituents in the piperazine ring. Though the optimal linker to connect the indolyl moiety to the *N*-substituted piperazine is the *n*-butyl chain, an *n*-propyl spacer is also suitable, as demonstrated by the good 5-HT1AR affinity showed by compounds **109** and **110** (**Figure 36**) [44].

A compound with an *n*-butyl chain is the potent and selective 5-HT1AR ligand **111** (**Figure 36**). Within this series of derivatives, the introduction of a residue in the para position of the phenyl ring reduces dopaminergic activity and, consequently, improves 5-HT1AR selectivity [45].

The indolylalkylamine moiety is also present in multitarget compounds simultaneously acting as SSRIs and 5-HT1AR antagonists and potentially useful for the treatment of depression. Among these, the benzoxazine derivative **112** shows high affinity for both 5-HT1ARs and SERTs (p*K*<sup>i</sup> SERT = 8.5), but no selectivity over α<sup>1</sup> -ARs. It behaves as a 5-HT1AR partial agonist [46]. On the contrary, the aryloxyalkylamine derivative **113** (p*K*<sup>i</sup> SERT = 9.3) behaves as a full 5-HT1AR antagonist (**Figure 37**) [47].

The hybridation between the chromane-based structure, present in 5-HT1AR antagonists, and the 3-indolyl-alkylamine moiety, embedded in numerous SSRIs, leads to compounds with mixed profiles. 5-Carboxamide-8-fluoro derivatives as well as 5-carboxamide-8-des-fluoro analogues with proper *N*-alkyl chains display good affinities for both 5-HT1ARs and 5-HT reuptake site [48]. In particular, **114** (**Figure 37**) behaves as a very potent 5-HT1AR antagonist and SSRI. The constrained amide conformation inherent in the lactam group results in less potent 5-HT1AR antagonist activity [49]. Another LCAP, obtained by combining 3-indolylalkylamine and arylpiperazine through a butyl chain (vilazodone, **115**), proves to be suitable for the interaction with both SERTs and 5-HT1ARs. Indeed **115**, showing subnanomolar 5-HT reuptake inhibitor activity and subnanomolar 5-HT1AR affinity, behaves as a 5-HT1AR agonist high selective over other GPCRs [43]. 5-Substituted *bis*-3-propylindole derivatives connected to *N*1 and *N*4 atoms of the piperazine ring also bind both SERTs and 5-HT1ARs, as suggested by compounds **116** and **117** (**Figure 37**), which show good affinities for both targets [50].

#### **5.5. Ergolines**

The tetracyclic ergoline skeleton is a common structural element contained in all ergot alkaloids. Such compounds are used in the treatment of several pathophysiological conditions, because of their wide spectrum of central and peripheral pharmacological activities. They can be considered as rigid analogues of both indolylalkylamines and catecholamines. Therefore, it is not surprising that they are able to nonselectively bind to adrenergic, dopaminergic, and serotoninergic receptors. Potent and selective 5-HT1AR ligands have been developed by combining the structural elements of the indolylethylamines and the 2-aminotetralins into a

**Figure 35.** Chemical structures of **104**–**108**.

**Figure 36.** Chemical structures of **109**–**111**.

**Figure 37.** Chemical structures of **112**–**117**.

The hybridation between the chromane-based structure, present in 5-HT1AR antagonists, and the 3-indolyl-alkylamine moiety, embedded in numerous SSRIs, leads to compounds with mixed profiles. 5-Carboxamide-8-fluoro derivatives as well as 5-carboxamide-8-des-fluoro analogues with proper *N*-alkyl chains display good affinities for both 5-HT1ARs and 5-HT reuptake site [48]. In particular, **114** (**Figure 37**) behaves as a very potent 5-HT1AR antagonist and SSRI. The constrained amide conformation inherent in the lactam group results in less potent 5-HT1AR antagonist activity [49]. Another LCAP, obtained by combining 3-indolylalkylamine and arylpiperazine through a butyl chain (vilazodone, **115**), proves to be suitable for the interaction with both SERTs and 5-HT1ARs. Indeed **115**, showing subnanomolar 5-HT reuptake inhibitor activity and subnanomolar 5-HT1AR affinity, behaves as a 5-HT1AR agonist high selective over other GPCRs [43]. 5-Substituted *bis*-3-propylindole derivatives connected to *N*1 and *N*4 atoms of the piperazine ring also bind both SERTs and 5-HT1ARs, as suggested by compounds **116** and **117** (**Figure 37**), which show good affinities for both targets [50].

96 Serotonin - A Chemical Messenger Between All Types of Living Cells

The tetracyclic ergoline skeleton is a common structural element contained in all ergot alkaloids. Such compounds are used in the treatment of several pathophysiological conditions, because of their wide spectrum of central and peripheral pharmacological activities. They can be considered as rigid analogues of both indolylalkylamines and catecholamines. Therefore, it is not surprising that they are able to nonselectively bind to adrenergic, dopaminergic, and serotoninergic receptors. Potent and selective 5-HT1AR ligands have been developed by combining the structural elements of the indolylethylamines and the 2-aminotetralins into a

**5.5. Ergolines**

**Figure 36.** Chemical structures of **109**–**111**.

**Figure 35.** Chemical structures of **104**–**108**.

partial ergoline skeleton [15]. Among the compounds belonging to this series, LY228729 (**118**; **Figure 38**) displays the highest affinity for 5-HT1ARs and good selectivity over a lot of other monoaminergic receptors. In functional assays, **118** behaves as a both pre- and postsynaptic 5-HT1AR agonists.

Though several tetracyclic ergolines, such as LSD (**119**), lisuride (**120**), or pergolide (**121**), show high affinities for 5-HT1ARs, they lack of selectivity over the other monoaminergic receptors. The improvement of the selectivity for 5-HT1ARs over 5-HT<sup>2</sup> Rs as well as D<sup>1</sup> Rs, D<sup>2</sup> Rs, and α-ARs can be obtained by introducing the bulky and metabolically stable *tert*-butyl group in the phenyl ring at C-13 of the ergoline skeleton. Some derivatives (**122–124**; **Figure 38**), bearing a heteroaryl substituent at C-9, display nM affinity for 5-HT1ARs and at least 100-fold selectivity over the other tested receptors. In contrast, the presence of a *tert-*butyl group at C-14 favors the selectivity for 5-HT<sup>2</sup> R.

Among the 5(10→9)*abeo*-ergoline derivatives, compound **125** displays good 5-HT1AR affinity and selectivity over 5-HT<sup>2</sup> Rs, D<sup>1</sup> Rs, D<sup>2</sup> Rs, and α-ARs. In this class of compounds, 5-HT1AR affinity is enhanced by the conversion of the 8β-hydroxymethyl group into a methyl group. Indeed, the transformation of **125** into the deoxy derivative **126** leads to appreciable increase of 5-HT1AR affinity. An improvement of 5-HT1AR selectivity can be obtained by the reduction of the 2,3-double bond of **126**, leading to the indolines **127** and **128** (**Figure 39**).

The stereochemistry at C-3 is very important for the 5-HT1AR profile. In particular, compound **128** displays an outstanding selectivity for 5-HT1ARs over 5-HT<sup>2</sup> Rs, D<sup>1</sup> Rs, D<sup>2</sup> Rs, and α<sup>1</sup> - and α<sup>2</sup> -ARs.

**Figure 39.** Chemical structures of **125**–**128**.

#### **5.6. Aporphines**

These compounds, whose prototype is (*R*)-apomorphine (**129**), have extensively been studied for their interaction with dopamine receptors in the CNS. In the effort to extend SAR studies of (*R*)-aporphines at dopamine receptors, (*R*)-(−)-10-methyl-11-hydroxyaporphine **130** (**Figure 40**), the 10-methyl substituted derivative of **129**, was surprisingly discovered [15] as a potent and selective 5-HT1AR agonist devoid of dopaminergic activity. The corresponding (*S*)-enantiomer behaves as an antagonist at postsynaptic 5-HT1ARs and is tenfold less potent than its antipode. Changes in steric bulk and/or electronic properties of the C10-substituent as compared to a C10-methyl group produce a decrease in 5-HT1AR affinity. For example, the substitution of the methyl at C-10 with an ethyl group (**131**) reduces the 5-HT1AR affinity of about 20-fold. Compound **132**, the *N*-desmethyl derivative of **130**, shows about 7-fold lower than 5-HT1AR affinity (**Figure 40**). However, such a modification mostly reduces the affinities for D1 Rs (62-fold) and D<sup>2</sup> Rs (>9.3-fold) and, consequently, improves 5-HT1AR selectivity. The removal of the substituent at position C-10 is compatible with 5-HT1AR interaction. In particular, among the C-11-monosubstituted aporphines, ethyl (**133**) and phenyl (**134**) derivatives show the highest affinities for 5-HT1ARs and good selectivity over both D<sup>1</sup> Rs and D<sup>2</sup> Rs (**Figure 40**).

Rigidifying (*R*)-aporphines derivatives by linking C-1 and C-11 into a fused pentacyclic or hexacyclic ring strongly reduces 5-HT1AR affinity. However, among the compounds within this series, the imino derivative **135** displays poor selectivity for 5-HT1ARs over both 5-HT7 Rs and D<sup>2</sup> Rs, whereas the regioisomer **136** is selective for 5-HT7 Rs.

#### **5.7. Imidazolines**

**5.6. Aporphines**

**Figure 38.** Chemical structures of **118**–**124**.

98 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Figure 39.** Chemical structures of **125**–**128**.

These compounds, whose prototype is (*R*)-apomorphine (**129**), have extensively been studied for their interaction with dopamine receptors in the CNS. In the effort to extend SAR studies of (*R*)-aporphines at dopamine receptors, (*R*)-(−)-10-methyl-11-hydroxyaporphine **130** (**Figure 40**), the 10-methyl substituted derivative of **129**, was surprisingly discovered [15] as a potent and selective 5-HT1AR agonist devoid of dopaminergic activity. The corresponding (*S*)-enantiomer behaves as an antagonist at postsynaptic 5-HT1ARs and is tenfold less potent than its antipode. Changes in steric bulk and/or electronic properties of the C10-substituent as compared to a C10-methyl group produce a decrease in 5-HT1AR affinity. For example, the substitution of the methyl at C-10 with an ethyl group (**131**) reduces the 5-HT1AR affinity of about 20-fold. Compound **132**, the *N*-desmethyl derivative of **130**, shows about 7-fold lower than 5-HT1AR affinity (**Figure 40**). However, such a modification mostly reduces the affinities The observation that the beneficial properties of the α2C-AR agonists and α2A-AR antagonists allyphenyline (**137**) and cyclomethyline (**138**) on morphine dependence proved to be associated to a significant antidepressant effect led to the hypothesis that ligands bearing the 2-substituted imidazoline nucleus as a structural motif can also be suitable to interact with 5-HT1ARs (**Figure 41**).

Experiments carried out in the presence of the 5-HT1AR antagonist WAY100135 confirmed that 5-HT1AR activation is involved in the observed antidepressant-like activity [51]. The investigation of a wide series of 2-substituted imidazolines linked to an aromatic moiety by

**Figure 40.** Chemical structures of **129**–**136**.

a biatomic bridge highlighted that a polar function (-O- or –NH- group) and a methyl group in the bridge as well as the suitable chirality and a proper steric hindrance in the aromatic area favor 5-HT1AR recognition and activation. In particular, (*S*)-naphthaline (**139**) shows the highest 5-HT1AR affinity within the series (**Figure 41**). In mice it displays antidepressantlike effect at a very low dose (0.01 mg/Kg) and proves to be more efficacious and potent than amitriptyline (15 mg/kg), a tricyclic antidepressant commonly used in human therapy [52].

#### **5.8. 1,4-Dioxanes**

The design and synthesis of 5-HT1AR ligands bearing the 1,4-dioxane nucleus were inspired by the observation that the potent α<sup>1</sup> -AR antagonist WB4101 (**140**) also shows high 5-HT1AR affinity [53]. In the effort to discriminate between 5-HT1AR and α<sup>1</sup> -ARs, the quite planar 1,4-benzodioxane structure of **140** was replaced by the less conformationally constrained 6-aryl-1,4-dioxane ring, maintaining the 2,6-dimethoxy substitution or removing one or both methoxy groups of the phenoxy terminal. The most interesting results are shown by the 6,6-diphenyl substituted compounds **141**–**143**, which display nanomolar 5-HT1AR affinities (**Figure 42**).

In particular, **143** behaves as a potent full 5-HT1AR agonist with a pD<sup>2</sup> value significantly higher than those of the reference compounds 5-HT and 8-OH-DPAT. This derivative also shows a good selectivity for 5-HT1ARs over α1A−, α1B−, and α1D−AR subtypes [54]. The stereogenic center in position 2 of the 1,4-dioxane nucleus appears to play a critical role in the

**Figure 42.** Chemical structures of **140**–**144**.

interaction with α<sup>1</sup> -AR and 5-HT1A R systems, a reversal enantioselectivity governing the 5-HT1AR or α<sup>1</sup> -AR recognition. Indeed, concerning 5-HT1ARs, the optimal affinity resides in the 2-(*S*) configuration, which, on the contrary, is less favorable for the interaction with α<sup>1</sup> - AR subtypes. This result is particularly interesting because, as the eutomers for 5-HT1ARs behave as distomers for α<sup>1</sup> -AR, the 5-HT1AR/α<sup>1</sup> -AR selectivity ratio significantly increases compared to the corresponding racemate [55].

A good selectivity for 5-HT1ARs over α<sup>1</sup> -ARs and dopamine D<sup>2</sup> -like receptors is also obtained by inserting a –OCH<sup>2</sup> OCH<sup>3</sup> group in 2-position of the phenoxy terminal (compound **144**; **Figure 42**). The pharmacological profile of **144** and docking studies suggest that 5-HT1ARs also accommodate substituents bulkier than the methoxy group. Instead, both α<sup>1</sup> -ARs and D<sup>2</sup> -like receptors have more stringent steric requirements being intolerant to the increase of steric bulk itself. Due to its 5-HT1AR activation, **144** significantly reduces anxiety-linked behaviors in mice [56].

## **6. Conclusion**

a biatomic bridge highlighted that a polar function (-O- or –NH- group) and a methyl group in the bridge as well as the suitable chirality and a proper steric hindrance in the aromatic area favor 5-HT1AR recognition and activation. In particular, (*S*)-naphthaline (**139**) shows the highest 5-HT1AR affinity within the series (**Figure 41**). In mice it displays antidepressantlike effect at a very low dose (0.01 mg/Kg) and proves to be more efficacious and potent than amitriptyline (15 mg/kg), a tricyclic antidepressant commonly used in human therapy [52].

The design and synthesis of 5-HT1AR ligands bearing the 1,4-dioxane nucleus were inspired

1,4-benzodioxane structure of **140** was replaced by the less conformationally constrained 6-aryl-1,4-dioxane ring, maintaining the 2,6-dimethoxy substitution or removing one or both methoxy groups of the phenoxy terminal. The most interesting results are shown by the 6,6-diphenyl substituted compounds **141**–**143**, which display nanomolar 5-HT1AR affinities

higher than those of the reference compounds 5-HT and 8-OH-DPAT. This derivative also shows a good selectivity for 5-HT1ARs over α1A−, α1B−, and α1D−AR subtypes [54]. The stereogenic center in position 2 of the 1,4-dioxane nucleus appears to play a critical role in the

affinity [53]. In the effort to discriminate between 5-HT1AR and α<sup>1</sup>

In particular, **143** behaves as a potent full 5-HT1AR agonist with a pD<sup>2</sup>



value significantly

**5.8. 1,4-Dioxanes**

(**Figure 42**).

by the observation that the potent α<sup>1</sup>

100 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Figure 41.** Chemical structures of **137**–**139**.

**Figure 42.** Chemical structures of **140**–**144**.

In summary, the currently main knowledges of the four-wheel drive (4WD: who, why, where, what, and drugs) vehicle by which to travel inside the 5-HT1AR world, have been presented. Such a travel, begun 30 years ago with the identification of 5-HT1AR coding gene, is far from the conclusion. Indeed, despite no X-ray structure is deposited to date, it is possible to answer quite exhaustively the question "who" this receptor is. However, the most intriguing question is "why" it continues to be a so attractive target several years after its identification. Several evidences are available about "where" 5-HT1AR is expressed throughout the body, at both central and peripheral levels. Between presynaptic (auto- and heteroreceptors) and postsynaptic receptors, are there differences which could allow us to target them selectively? Wider and wider is the field of "what" effects this receptor can elicit under physiological and pathological conditions directly or through the modulation of several other receptor systems or the stimulation of the secretion of various hormones. Well known is its involvement in anxiety, depression, epilepsy, mood disorders, learning, and memory. Consequently, growing is its importance in the treatment of such pathologies. Moreover, the interest for 5-HT1AR as an attractive target of drugs is increased by further physiologically governed functions, including feeding/ satiety, temperature regulation, sleep, pain perception, and sexual activity. The stimulation of 5-HT1ARs has been demonstrated to activate several different biochemical pathways and signals through both G-protein-dependent and G-protein-independent pathways. However, it cannot be ruled out that underlying mechanisms are far from being completely understood, making more and more complex the net of pathways through which the primary impulses unwind themselves. Finally, the discovery of "drugs" able to selectively activate or inhibit 5-HT1AR might help to better characterize such a receptor and the physiological functions in which it is involved. Despite the numerous published papers and synthesized and tested molecules, the results are not completely satisfactory yet. The reasons can be ascribed partly to the great similarity of the ligand recognition transmembrane region of 5-HT1ARs with other members of the family or other GPCRs, partly to bimodal effect of 5-HT1AR activation dependent on the neuroanatomical location of the receptors and the concentration of the ligand.

## **Author details**

Wilma Quaglia\*, Carlo Cifani, Fabio Del Bello, Mario Giannella, Gianfabio Giorgioni, Maria Vittoria Micioni Di Bonaventura and Alessandro Piergentili

\*Address all correspondence to: wilma.quaglia@unicam.it

School of Pharmacy, University of Camerino, Camerino, Italy

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## **Sculpting Cerebral Cortex with Serotonin in Rodent**

## **and Primate**

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108 Serotonin - A Chemical Messenger Between All Types of Living Cells

5-HT1A receptor over α<sup>1</sup>

Tania Vitalis and Catherine Verney

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69000

#### **Abstract**

The mammalian cerebral cortex is critical for sensory and motor integrations and, for higher-order cognitive functions. The construction of mammalian cortical circuits involves the coordinated interplay between cellular processes such as proliferation, migration and differentiation of neural and glial cell subtypes followed by accurate connectivity evolving in complexity in primates. Alteration in cortical development may induce the emergence of various pathological traits and behaviours. Among the large array of factors that regulate the assembly of cortical circuits, serotonin (5-HT) plays important role as a developmental signal that impacts on a broad diversity of cellular processes. 5-HT plays distinct roles during specific sensitive periods and is produced from various sources depending on the perinatal stage. Its roles are mediated by more than fourteen 5-HT receptors that are all G-protein coupled receptors except the ionotropic 5-HT type 3A receptor (5-HT3A) mediating rapid neuronal activation. Importantly, 5-HT metabolism and signalling are influenced by numerous epigenetic and genetic factors, including nutrition and gut microbiota, perinatal stress, infection and inflammation. In this review, we will recapitulate some evidences showing that dysregulation of 5-HT homeostasis and 5-HT3A signalling impairs distinct steps of cortical circuit formation leading to the predisposition of the onset of various psychiatric diseases.

**Keywords:** development, human, monoamine, plasticity, 5-HT3 receptor

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

The functions of the mammalian cerebral cortex are processed through the activation of multipartite neural networks composed of excitatory glutamatergic pyramidal neurons, local modulatory interneurons that release γ-aminobutyric acid (GABA), neuropeptides and vasoactive substances [1–5] and by 'glial cells' that do far more than just feeding neurones and scavenging debris [6, 7]. Developmental perturbations impacting the maturation of cortical circuits can trigger neuropsychiatric disorders [8–10]. Sensitive periods or windows of vulnerability have been demonstrated in various processes in particular for the rodent sensory systems as well as in the modulation of complex behaviours.

Mammalian cortical circuit formation is the result of a series of sequential events that take place mainly during embryonic and early post-natal development [11–14]. These events include the proliferation, migration and differentiation of neurons and 'glial cells' that are largely governed by genetic programs but are also sensitive to environmental factors. Such extrinsic signals are extremely diverse (including guidance cues, growth factors, cell adhesion molecules) and among them the monoamine serotonin (5-HT) has emerged as an important regulator of neural circuit formation [15, 16].

In mammals, cortical 5-HT arises from multiples sources depending on the developmental stage. At the onset of cortical development, 5-HT is of maternal and placental origin [17–19]. Later, by embryonic day 16 (E16 in mice) [15, 16, 20] and by gestational week 16 (GW16 in human) [13, 14], serotoninergic afferents invade the cerebral cortex and contribute to provide 5-HT locally. Not surprisingly, like in non-mammalian species, serotonin modulates neuronal proliferation, migration and differentiation. In addition, 5-HT is implicated in the emergence of many neuropsychiatric disorders, including mental retardation, autism, depression and anxiety [10, 15, 21–26]. Importantly, 5-HT signalling is influenced by numerous epigenetic and genetic factors, including nutrition and gut microbiota [27, 28], perinatal stress [29–31], infection and inflammation [32–35], 5-HT metabolism and storage [15, 36–38], pharmacological compounds such as selective serotonin reuptake inhibitors [38–40] and genetic alterations [41–44].

Our aim is to give a comprehensive overview on the possible roles of 5-HT receptor signalling and 5-HT homeostasis on the development of the cerebral cortex in rodent and primate with a specific emphasis on human. In this framework, we will highlight more particularly recent studies that have revealed new molecular targets of early-life 5-HT in the construction of cortical circuits; in particular, the ionotropic 5-HT type 3A receptor (5-HT3A). We will also review recent clinical studies suggesting that altered 5-HT homeostasis or signalling could participate in the emergence of human psychiatric disease, in particular of mood and anxiety disorders.

*In the following section, we will describe the general structure of the mammalian cerebral cortex focusing on rodent and then presenting the specificities observed in primate/human. Then we will describe the major steps of the development of the mammalian cerebral cortex that is governed by a series of sequential events including proliferation, migration and differentiation of neurons and glial cells. When numerous developmental similarities are observed very precociously in rodent versus primate, significant specificities arose later in development in primate especially in human*.

## **2. Structure and development of the mammalian cerebral cortex**

#### **2.1. Neuronal components and glial components**

**1. Introduction**

well as in the modulation of complex behaviours.

110 Serotonin - A Chemical Messenger Between All Types of Living Cells

regulator of neural circuit formation [15, 16].

The functions of the mammalian cerebral cortex are processed through the activation of multipartite neural networks composed of excitatory glutamatergic pyramidal neurons, local modulatory interneurons that release γ-aminobutyric acid (GABA), neuropeptides and vasoactive substances [1–5] and by 'glial cells' that do far more than just feeding neurones and scavenging debris [6, 7]. Developmental perturbations impacting the maturation of cortical circuits can trigger neuropsychiatric disorders [8–10]. Sensitive periods or windows of vulnerability have been demonstrated in various processes in particular for the rodent sensory systems as

Mammalian cortical circuit formation is the result of a series of sequential events that take place mainly during embryonic and early post-natal development [11–14]. These events include the proliferation, migration and differentiation of neurons and 'glial cells' that are largely governed by genetic programs but are also sensitive to environmental factors. Such extrinsic signals are extremely diverse (including guidance cues, growth factors, cell adhesion molecules) and among them the monoamine serotonin (5-HT) has emerged as an important

In mammals, cortical 5-HT arises from multiples sources depending on the developmental stage. At the onset of cortical development, 5-HT is of maternal and placental origin [17–19]. Later, by embryonic day 16 (E16 in mice) [15, 16, 20] and by gestational week 16 (GW16 in human) [13, 14], serotoninergic afferents invade the cerebral cortex and contribute to provide 5-HT locally. Not surprisingly, like in non-mammalian species, serotonin modulates neuronal proliferation, migration and differentiation. In addition, 5-HT is implicated in the emergence of many neuropsychiatric disorders, including mental retardation, autism, depression and anxiety [10, 15, 21–26]. Importantly, 5-HT signalling is influenced by numerous epigenetic and genetic factors, including nutrition and gut microbiota [27, 28], perinatal stress [29–31], infection and inflammation [32–35], 5-HT metabolism and storage [15, 36–38], pharmacological compounds

such as selective serotonin reuptake inhibitors [38–40] and genetic alterations [41–44].

*significant specificities arose later in development in primate especially in human*.

Our aim is to give a comprehensive overview on the possible roles of 5-HT receptor signalling and 5-HT homeostasis on the development of the cerebral cortex in rodent and primate with a specific emphasis on human. In this framework, we will highlight more particularly recent studies that have revealed new molecular targets of early-life 5-HT in the construction of cortical circuits; in particular, the ionotropic 5-HT type 3A receptor (5-HT3A). We will also review recent clinical studies suggesting that altered 5-HT homeostasis or signalling could participate in the emergence of human psychiatric disease, in particular of mood and anxiety disorders. *In the following section, we will describe the general structure of the mammalian cerebral cortex focusing on rodent and then presenting the specificities observed in primate/human. Then we will describe the major steps of the development of the mammalian cerebral cortex that is governed by a series of sequential events including proliferation, migration and differentiation of neurons and glial cells. When numerous developmental similarities are observed very precociously in rodent versus primate,* 

The mammalian cerebral cortex comprises of six lamina (layers), each containing specific combination of neurons and 'glial cells'. Cortical excitability is coordinated by the interplay of excitatory pyramidal neurons and inhibitory interneurons. Pyramidal cells, which make up the majority of all neurons in the adult cortex (80% in rodent cortex), are projection neurons that send axons to other areas inside or outside the cortex providing output excitatory drive by releasing glutamate [2]. Inhibitory neurons project locally, release the neurotransmitter GABA and refine cortical excitability. Although GABAergic interneurons are less abundant, they have crucial roles in the development and organization of cortical networks that underlie a wide range of cortical and mental functions [8, 45, 46]. They are extremely diverse, differing in shape, electrophysiological properties and in the combination of neuropeptides and calcium-binding proteins that they express in addition to GABA [1, 47]. To facilitate the description of GABAergic neurons, a consortium of experts has suggested using a unified nomenclature [4, 5]. Thus, one can distinguish four major and highly distinct classes of GABAergic neurons in the mammalian cerebral cortex (**Figure 1A**). First, fast-spiking interneurons expressing parvalbumin (PV) that gate incoming sensory information [48, 49]. Second, adapting Martinotti cells expressing somatostatin (SOM) that control dendritic information through local feedback inhibition [50]. Third, adapting bipolar interneurons expressing mainly the vasoactive intestinal peptide (VIP) and calretinin (CR) that preferentially target other interneurons and receive direct input from the thalamus [20, 51, 52]. Fourth, adapting neurogliaform interneurons expressing vasoactive substances, notably the neuropeptide Y (NPY) and/or nitric oxide (NO) that are responsible for the slow GABAergic inhibition of pyramidal cells and interneurons and vasomotion [53–56].

Although these different types of interneurons have been identified in the primate or human cerebral cortex, their diversity largely surpasses what is observed in rodent [12]. Interestingly, unique to human cerebral cortex, bipolar/von Economo neurons are present in layer V of the anterior cingulate and fronto-insular cortices expressing VMAT2 [57, 58]. Their possible involvement suggested in neuropsychiatric disorders needs to be further investigated [59]. In human and primate, the neuronal composition of the cerebral cortex is less homogeneous between areas with a higher level of arealisation than in rodent. Interestingly, the density of small interneurons appears very high in associative areas [60].

Besides neurons, mature 'glial cells' have been shown to exert roles that are extremely more complex than previously thought. Astrocytes are the largest glial population in the mammalian brain and are well-known to 'feed neurons' by transforming glucose into lactate that neurons can directly use as 'carburant', to scavenge debris and to regulate neural transmission and ionic homeostasis of the brain [61, 62]. Microglial cells play a role of sentinels of inflammatory state of the brain. In addition to these roles, astrocytes and microglial cells participate in regulating cell proliferation, neuronal migration and plasticity (for review, see Refs. [6, 61, 63]). Oligodendrocytes myelinate axons and increase their conduction velocity (they will not be further described in this chapter).

**Figure 1.** Structure of the rodent cerebral cortex and relation with serotoninergic afferents. A, The four main classes of interneurons (NG: neurogliaform, PV: parvalbumin+, VIP: vasoactive intestine peptide+, SOM: somatostatin+) and their relationship with a typical pyramidal glutamatergic neuron (adapted from [64]). B, Serotoninergic afferents arising from the median raphe (MnR) are thin, diffuse and display small varicosities. Serotoninergic afferents arising from the dorsal raphe (DR) are thick, beaded, preferentially located in superficial layer and make true synaptic contacts with small interneurons expressing VIP and with NG interneurons expressing the 5-HT receptor type 3A (3A). Adapted from [65]. 5-HT: serotonin.

#### **2.2. Development of the rodent cerebral cortex**

**Figure 1.** Structure of the rodent cerebral cortex and relation with serotoninergic afferents. A, The four main classes of interneurons (NG: neurogliaform, PV: parvalbumin+, VIP: vasoactive intestine peptide+, SOM: somatostatin+) and their relationship with a typical pyramidal glutamatergic neuron (adapted from [64]). B, Serotoninergic afferents arising from the median raphe (MnR) are thin, diffuse and display small varicosities. Serotoninergic afferents arising from the dorsal raphe (DR) are thick, beaded, preferentially located in superficial layer and make true synaptic contacts with small interneurons expressing VIP and with NG interneurons expressing the 5-HT receptor type 3A (3A). Adapted from [65].

5-HT: serotonin.

112 Serotonin - A Chemical Messenger Between All Types of Living Cells

The cerebral cortex develops from neuroepithelial germinal cells of the telencephalic pallium and subpallium that massively proliferate by E11-E12 in mice and GW5-6 in human, to form the cerebral vesicles [66]. At this stage, microglial cells—of extracerebral origin—have already started to invade the telencephalon (from E9.5 in rodent [67] and GW5 in human [63]) before blood vessels start to penetrate and ramify in the telencephalon [68]. They will both participate in regulating neurogenesis [69]. The first generated neurons, Cajal-Retzius (C-R) cells and subplate cells (SP; from E10 in mice, GW5-7 in human), constitute transient and heterogeneous populations of cells that originate from both pallial and subpallial territories and form the preplate (PP; Boulder Committee; [66, 70, 71]). SP and reelin-secreting C-R cells provide positioning cues and instructions to developing cortical neurons and afferents [71–74]. The cortical plate, is formed from E13-E17 in mice and GW7-20 in human by post-mitotic excitatory pyramidal neurons migrated along radial glial (RG) fibres in an inside out gradient of development from layer VIa to layer II [13]. At the beginning of cortical plate formation (E13-E14 in mice), pyramidal cells are generated from radial glial cells (RGC), whereas later (E15-E17 in mice), they mainly originate from intermediate progenitor cells (IPC) or basal progenitors deriving from RGC cells [75, 76] (**Figure 2**).

The primate/human cortical neurogenesis is far more complex than that of rodent involving more germinal zones and a larger number of cell types [77, 78]. In particular, beside the early RGC in the VZ, a novel class of radial cells, the outer RG (oRG), located in the outer subventricular zone (SVZ) could be responsible for the increasing number of excitatory neurons and the formation of gyration in primate. The second stage of human cortical development (GW18-20) corresponds to the genesis of the supragranular layers that likely expand from the oRG [14] (**Figure 2A**).

In rodent, the cortical GABAergic interneurons are generated outside the cortical VZ, in the subpallium: mainly in the medial ganglionic eminence (MGE) (E11-E14 in mice) and the caudal ganglionic eminence (CGE) (E14-E17 in mice) [11, 20, 52]. These regions are specified through a combination of distinct transcription factors and morphogenes that produce different classes of interneurons [80]. The ventral and the dorsal parts of the MGE expressing the homeobox transcription factor Lhx6 generate fast-spiking/PV+ and adapting/SOM+ interneurons [81–85]. The CGE, a region that expresses the transcription factor Gsh2, COUP-TFII but lacks the transcription factors Nkx2.1, Nkx6.2 and Lhx6 [80, 86, 87], generates VIP+, CR+, NPY+ and nNOS+ interneurons [20, 52, 85, 88]. Once produced, interneurons are targeted towards specific brain regions, including cortex, depending on the transcription factors and guidance cues they express [87, 89]. They initially follow parallel migratory streams, first in the IZ and MZ and later on along the SVZ, before they switch their migratory mode and incorporate into the developing CP through radial migration (see **Figure 2B**). In mice, cortical migration is almost completed by P4, and is followed by cortical expansion. However, during the first two post-natal weeks and decreasing with age the SVZ retains the capacity to produce CR+ interneurons contributing to the pools of GABAergic neurons mainly populating lower cortical layers and cingulate cortex [90–92]. These events are recapitulated in **Figure 3A** and **B**.

**Figure 2.** Early stages of development of the human (A) and mouse (B) cerebral cortex in relation with 5-HT afferents. A-B, Both in human and rodent intense proliferation of neuroepithelium and the formation of the preplate (PP) take place around (E10; GW5) and (E11-E12; GW6-7) respectively. By E13-E14 in mice and GW8-10 in human, PP is split by the migration of the first pyramidal neurons. Cajal-Retzius cells (C-R) will remain in the marginal zone (MZ) while subplate neurons (SP) will be positioned below the cortical plate (CP). In addition, in human around GW10, another source of progenitors arises: the outer radial glial (oRG) cells that do not maintain contacts with the apical surface. Monoaminergic axons and thalamocortical axons (TC) are already found in the MZ and in the intermediate zone (IZ) and, in the IZ respectively. By E15-E16 in mice most glutamatergic neurons are generated, 5-HT axons and TC run in the MZ and IZ and in the IZ respectively. By GW16 in human, SP occupy a large proportion of the cortical anlage and oRG are still producing a high amount of neurons. Interneurons migrating first tangentially to the pial surface and later radially to it, incorporating CP. C, Bars indicate the time at which different factors (maternal and environmental; 5-HT of placental origin, 5-HT produced by the embryo itself) could affect the development of the mouse embryo. A, is adapted from [20] and B is adapted from [13, 14, 79].

**Figure 3.** Presumptive genesis of cortical GABAergic neurons in the rodent and human/primate embryos and fetuses. (A and B) In rodent, PV+ and SOM+ interneurons (INs) are generated first from the medial ganglionic eminence (MGE) located in the anterior telencephalon. CR+, VIP+ and neurogliaform INs are generated mainly in the caudal GE (CGE) and in the lateral GE (LGE) located in the basal ganglia and to a lesser extent in the anterior entopeduncular area (AEP) and in the pre-optic area (POA). (C) In non-human primate and in human, the picture is less clear. However transcription factors expression suggest that the GE produce a large part of GABAergic neurons. By contrast to rodent brain numerous, INs may be generated in the cortical anlage. Panel C is adapted from Ref. [12]. CR: calretinin, NG: neurogliaform, PV: parvalbumin, SOM: somatostatin and VIP: vasoactive intestine peptide.

In non-human and human primate, the origin of the very heterogenous GABAergic interneurons is not so clear. Recently, studies have shown that in non-human primate, interneurons use a similar coding of transcription factors as in rodents and largely originate from the ganglionic eminences [93] (**Figure 3C**). However, a substantial proportion of them is likely to be generated in the pallium from the VZ and the SVZ [12, 94–96] (**Figure 3C**). Recently, migration of subclasses of human cortical interneurons has been reported to continue after birth [97].

#### **2.3. Specificities of the human and primate cerebral cortex**

**Figure 2.** Early stages of development of the human (A) and mouse (B) cerebral cortex in relation with 5-HT afferents. A-B, Both in human and rodent intense proliferation of neuroepithelium and the formation of the preplate (PP) take place around (E10; GW5) and (E11-E12; GW6-7) respectively. By E13-E14 in mice and GW8-10 in human, PP is split by the migration of the first pyramidal neurons. Cajal-Retzius cells (C-R) will remain in the marginal zone (MZ) while subplate neurons (SP) will be positioned below the cortical plate (CP). In addition, in human around GW10, another source of progenitors arises: the outer radial glial (oRG) cells that do not maintain contacts with the apical surface. Monoaminergic axons and thalamocortical axons (TC) are already found in the MZ and in the intermediate zone (IZ) and, in the IZ respectively. By E15-E16 in mice most glutamatergic neurons are generated, 5-HT axons and TC run in the MZ and IZ and in the IZ respectively. By GW16 in human, SP occupy a large proportion of the cortical anlage and oRG are still producing a high amount of neurons. Interneurons migrating first tangentially to the pial surface and later radially to it, incorporating CP. C, Bars indicate the time at which different factors (maternal and environmental; 5-HT of placental origin, 5-HT produced by the embryo itself) could affect the development of the mouse embryo. A, is adapted

from [20] and B is adapted from [13, 14, 79].

114 Serotonin - A Chemical Messenger Between All Types of Living Cells

As already mentioned, the first generated neurons, C-R and SP cells are located respectively in the presumptive Layer 1 and the SP zone of the human cortical anlage [66, 98, 99]. Specific to human, the SP zone is the largest transient compartment of the fetal neocortical anlage, about four times thicker than the cortical plate around midgestation [66, 100]. In humans and non-human primate, most SP neurons generated in the ventricular zone initially migrate radially, together with prospective layer VI neurons and secondarily get widespread into the expanding SP zone around midgestation [101]. Interestingly, at this stage, dispersion of SP cells in the extended SP zone is concomitant with the invasion of monoaminergic [102], thalamocortical and corticocortical axons in the cortical anlage [103]. SP zone begins slowly to disappear towards the end of gestation and during the early post-natal period. Finally, many subplate neurons survive postnatally and transform into interstitial neurons of the subcortical white matter of the adolescent and adult brain [104]. GABA+ interstitial neurons express CB and CR [105]. Subcortical interstitial neurons in the white matter, which have been associated with a variety of neurological and psychiatric disorders of infant and adults, need to be further investigated [105, 106]. Comparison of the rodent/human cortical development could be obtained by comparing **Figure 2A** with **B** and **Figure 3A** and **B** with **C**.

Microglial cells take part in normal establishment and maturation of neuronal circuitry during development [107]. In human, amoeboid microglial cells infiltrate the brain via the choroid plexus, the meninges and the ventricles around GW4,5, progressively colonize the cerebral wall from GW7 and became ramified [108, 109]. Passing through walls from GW10 on. Interestingly, amoeboid microglial cells cluster in a band at the limit of the CP/IZ-SP zone at GW9-13 where early synaptogenesis takes place in the cerebral anlage [110]. They also clustered in major axonal crossroads in the corpus callosum at GW16 and in the coronal radiata at GW19-24 [63]. Interestingly, this last fibres tract area is the target of white matter injury observed in inflammatory process of premature infant in cerebral palsy [111]. Similarly, a cluster of microglia/macrophages is detected in the cingulum bundle in the perinatal rat models of hypoxia and growth restriction developed by Verney and collaborators [112–114].

In mammals, the numerous cortical astrocytes are reported to be mainly generated not only from radial glial cells but also from other cell types that are not clearly elucidated such as progenitors in the SVZ [62]. Human astrocytes are far more complex in diversity and size, and the ratio of glia to neuron is higher when compared to rodent [115]. The protoplasmic and fibrous astrocytes appeared in waves in the cortical anlage [115], begin to differentiate around midgestation and co-expression between vimentin and GFAP is observed [116]. Functional

**Figure 4.** Presumptive comparative schedule for development of the cerebral cortex in rat and human.

astrocytes evolve in parallel with the maturation of the vascular endothelial cells involved in blood-brain barrier (BBB) formation [68, 117]. During development, monocarboxylates including lactate represent a major source of energy for the developing neurons [118]. The expression of monocarboxylate transporters such as MCT1 confirms the functionality of astrocytes in the energy trafficking occurring in the human visual cortex from GW19 [119].

Here, we provide a schematic drawing (**Figure 4**) comparing the schedule for the different key events occurring during the cortical development in human and in rat.

*Serotonin is provided to the developing mammalian cerebral cortex via many sources. Numerous studies, cited in the section below, have described this in rodent but only sparse data are available in primate especially in human*.

## **3. Sources of serotonin to the mammalian cortex**

#### **3.1. Serotonin synthesis and degradation**

subplate neurons survive postnatally and transform into interstitial neurons of the subcortical white matter of the adolescent and adult brain [104]. GABA+ interstitial neurons express CB and CR [105]. Subcortical interstitial neurons in the white matter, which have been associated with a variety of neurological and psychiatric disorders of infant and adults, need to be further investigated [105, 106]. Comparison of the rodent/human cortical development could be

Microglial cells take part in normal establishment and maturation of neuronal circuitry during development [107]. In human, amoeboid microglial cells infiltrate the brain via the choroid plexus, the meninges and the ventricles around GW4,5, progressively colonize the cerebral wall from GW7 and became ramified [108, 109]. Passing through walls from GW10 on. Interestingly, amoeboid microglial cells cluster in a band at the limit of the CP/IZ-SP zone at GW9-13 where early synaptogenesis takes place in the cerebral anlage [110]. They also clustered in major axonal crossroads in the corpus callosum at GW16 and in the coronal radiata at GW19-24 [63]. Interestingly, this last fibres tract area is the target of white matter injury observed in inflammatory process of premature infant in cerebral palsy [111]. Similarly, a cluster of microglia/macrophages is detected in the cingulum bundle in the perinatal rat models of hypoxia and growth restriction developed by Verney and collaborators [112–114].

In mammals, the numerous cortical astrocytes are reported to be mainly generated not only from radial glial cells but also from other cell types that are not clearly elucidated such as progenitors in the SVZ [62]. Human astrocytes are far more complex in diversity and size, and the ratio of glia to neuron is higher when compared to rodent [115]. The protoplasmic and fibrous astrocytes appeared in waves in the cortical anlage [115], begin to differentiate around midgestation and co-expression between vimentin and GFAP is observed [116]. Functional

**Figure 4.** Presumptive comparative schedule for development of the cerebral cortex in rat and human.

obtained by comparing **Figure 2A** with **B** and **Figure 3A** and **B** with **C**.

116 Serotonin - A Chemical Messenger Between All Types of Living Cells

Serotonin is synthesized from the essential amino acid tryptophan. In the blood stream, tryptophan is linked to serum albumin but a proportion that decreases with age is free to cross the BBB (10% at post-natal day 12 when BBB is thought fully functional [120]). Tryptophan is then transported, accumulated in 5-HT-producing cells and hydroxylated by the tryptophan hydroxylase enzymes (Tph). Tryptophan hydroxylase type 2 (Tph2) is expressed in serotoninergic neurons of the raphe nuclei and myenteric neurons [121, 122], while Tph1 is expressed in the pineal gland, in the placenta and in various peripheral tissues [18, 19, 122, 123]. 5-hydroxytryptophan is then further decarboxylated into 5-HT by the aromatic amino acid decarboxylase (AADC). The availability of tryptophan to synthesise 5-HT depends on the inflammatory status of the organism. In case of inflammation, indoleamine 2,3-dioxygenase (IDO) is generated, which can lead to 5-HT depletion in the organism [35].

5-HT is catabolized by monoamine oxidases A or B (MAOA or MAOB [124, 125]). MAOA has higher affinity for 5-HT than MAOB and is strongly co-expressed with MAOB between E12 to P7 in rodent serotoninergic neurons [126]. After P7, the expression of MAOB is largely predominant in 5-HT+ neurons [126]. MAOs are also expressed by many non-aminergic structures, in particular the placenta and in a subpopulation of VZ-SVZ cells ([126, 127] and our unpublished results) where they may regulate the amount of 5-HT locally. Interestingly, MAOs expression and protein synthesis are tightly regulated and have been shown to be sensitive to environmental factors such as inflammation and ischaemia-like conditions [34].

During embryonic development, the telencephalon receives 5-HT arising from multiple sources that are mainly of extra-embryonic or maternal origin at the beginning of gestation. Later, they progressively arise from different embryonic regions. Below, we will briefly recapitulate the sources of serotonin provided to the embryonic telencephalon in relation with cortical development.

## **3.2. Development of the serotoninergic neurons and projections**

In mammals, brainstem serotoninergic neurons are subdivided into 9 groups (B1–B9) forming a caudal and a rostral division. The rostral division (B5–B9; including the dorsal (B6, B7) and median raphe nuclei (B5, B8)) projects to the forebrain [65, 128, 129] (**Figure 1B**). Since these initial descriptions, recent mapping of 5-HT projections have been performed in mice revealing a higher level of refinement in the projections of raphe clusters towards specific targets [130]. Such level of analysis is lacking in primate and human.

In mice, the rostral division differentiates by E10-E11 (E12-E15 in rats); dorsal and median raphe send axons that reach the cortico-striatal junction by E14 in mice before entering the cortical anlage as two tangential streams, one above and the other below the CP [131, 132]. In the MZ, C-R cells and serotoninergic axons are in close apposition and make transient synaptic contacts [133, 134]. Below the CP, 5-HT afferents are mainly restricted to the IZ and the SP [131]. By E16-E17 in mice, thalamocortical axons (TCAs) invade the cortical anlage and are in close apposition with 5-HT axons running in the IZ. At the end of corticogenesis, 5-HT axons gradually arborize, sending numerous branches into the CP [131].

By P21, serotoninergic axons become evenly distributed in the different cortical territories showing their mature pattern of innervation [128]. Dorsal raphe axons are generally thin with pleiotropic varicosities that preferentially arborize in cortical layers IV and V. By contrast, median raphe axons show large spherical varicosities, form true chemical synapses, preferentially arborize in layer I and lower white matter, and contact interneurons containing VIP and cholecystokinin (CCK) [64, 65, 135] (**Figure 1**). Thus, 5-HT could be released along the entire axonal network through volume transmission or in synaptic clefts.

Anatomical studies have described the primate raphe nuclei and the serotonergic cortical innervation at mature stages [136–138], but only a few studies have reported their development. In Rhesus monkey, the genesis of raphe neurons was detected in the first quarter of gestation (E28-E45, birth: E165) [139] and 5HT+ fibres were reported in the entorhinal cortex at E70, similarly to tyrosine-hydroxylase+ catecholaminergic axons [140]. In human cortical anlage, one can suggest that the early afferents of serotoninergic axons as described for the catecholaminergic afferents may penetrate the cortical anlage around GW8 and invade the fetal cortex at midgestation in a mature-like pattern [102, 141]. In parallel, SERT expression in developing TCAs have been detected at GW10 in human cortical anlage [142]. Comparable expression has been described for the visual sensory system in the marmoset [143].

### **3.3. Other sources of serotonin**

The first demonstrations showing that 5-HT was influencing very early embryonic development were provided by pioneer groups showing that *ex vivo* application of 5-HT or alteration of 5-HT levels altered normal development of various embryonic structures before serotoninergic neurons have innervated these structures [144–149]. Several studies suggest that 5-HT derives from maternal or placental sources (see **Figure 5** that recapitulates those studies).

**3.2. Development of the serotoninergic neurons and projections**

118 Serotonin - A Chemical Messenger Between All Types of Living Cells

[130]. Such level of analysis is lacking in primate and human.

gradually arborize, sending numerous branches into the CP [131].

axonal network through volume transmission or in synaptic clefts.

the marmoset [143].

studies).

**3.3. Other sources of serotonin**

In mammals, brainstem serotoninergic neurons are subdivided into 9 groups (B1–B9) forming a caudal and a rostral division. The rostral division (B5–B9; including the dorsal (B6, B7) and median raphe nuclei (B5, B8)) projects to the forebrain [65, 128, 129] (**Figure 1B**). Since these initial descriptions, recent mapping of 5-HT projections have been performed in mice revealing a higher level of refinement in the projections of raphe clusters towards specific targets

In mice, the rostral division differentiates by E10-E11 (E12-E15 in rats); dorsal and median raphe send axons that reach the cortico-striatal junction by E14 in mice before entering the cortical anlage as two tangential streams, one above and the other below the CP [131, 132]. In the MZ, C-R cells and serotoninergic axons are in close apposition and make transient synaptic contacts [133, 134]. Below the CP, 5-HT afferents are mainly restricted to the IZ and the SP [131]. By E16-E17 in mice, thalamocortical axons (TCAs) invade the cortical anlage and are in close apposition with 5-HT axons running in the IZ. At the end of corticogenesis, 5-HT axons

By P21, serotoninergic axons become evenly distributed in the different cortical territories showing their mature pattern of innervation [128]. Dorsal raphe axons are generally thin with pleiotropic varicosities that preferentially arborize in cortical layers IV and V. By contrast, median raphe axons show large spherical varicosities, form true chemical synapses, preferentially arborize in layer I and lower white matter, and contact interneurons containing VIP and cholecystokinin (CCK) [64, 65, 135] (**Figure 1**). Thus, 5-HT could be released along the entire

Anatomical studies have described the primate raphe nuclei and the serotonergic cortical innervation at mature stages [136–138], but only a few studies have reported their development. In Rhesus monkey, the genesis of raphe neurons was detected in the first quarter of gestation (E28-E45, birth: E165) [139] and 5HT+ fibres were reported in the entorhinal cortex at E70, similarly to tyrosine-hydroxylase+ catecholaminergic axons [140]. In human cortical anlage, one can suggest that the early afferents of serotoninergic axons as described for the catecholaminergic afferents may penetrate the cortical anlage around GW8 and invade the fetal cortex at midgestation in a mature-like pattern [102, 141]. In parallel, SERT expression in developing TCAs have been detected at GW10 in human cortical anlage [142]. Comparable expression has been described for the visual sensory system in

The first demonstrations showing that 5-HT was influencing very early embryonic development were provided by pioneer groups showing that *ex vivo* application of 5-HT or alteration of 5-HT levels altered normal development of various embryonic structures before serotoninergic neurons have innervated these structures [144–149]. Several studies suggest that 5-HT derives from maternal or placental sources (see **Figure 5** that recapitulates those

**Figure 5.** Maternal, placental, genetic and pharmacological conditions determining the amount of serotonin supply to the developing telencephalon. Tryptophan is provided to the embryo but could also be converted into 5-HTP (5-hydroxytryptamine) or further into serotonin (5-HT) in the placenta via the expression of various metabolic enzymes expressed in the placenta. In addition, 5-HT from maternal sources could be taken up by the placenta that also expressed serotonin transporter (SERT). During early embryonic stages 5-HT could be delivered directly to the developing embryo. After E15-E16, when 5-HT axons of the hindbrain reach the cortex, 5-HT could act on various target cells (Cell) expressing selected arrays of 5-HT receptors. At this stage 5-HT could also be taken up and stored by thalamocortical afferents (TC) and released after specific stimulation. In addition 5-HTP is provided to the (tryptophan hydroxylase type 2) Tph2 and the (aromatic amino acid decarboxylase) AADC containing neurons that synthetize 5-HT. In this drawing adapted from [19], we have pointed in the large left arrow the maternal conditions that are best known to interfere with 5-HT availability to the embryo. We have also indicated that inhibitors of 5-HT uptake (SSRIs) that cross all barriers affect SERT function at all levels. Genetic polymorphisms or methylations mentioned in the text are indicated by a star. The major catabolic enzymes of 5-HT, monoamine oxidases are indicated (MAO). Tryptophan hydroxylase type 1; Tph1.

Several groups have suggested that, at early stages, 5-HT arises from maternal sources. Indeed, this was suggested when analysing the phenotype of embryos generated from Tph1+/− or Tph1+/+ mothers. Tph1−/− and Tph1+/− embryos obtained from crosses between heterozygous parents were indistinguishable from their wild-type littermates (the crown-rump length (CRL) was of 7.4– 7.5 mm). By contrast, 80–88.9% of Tph1−/− and Tph1+/− embryos born Tph1−/− mothers displayed low CRL values (5.8–7.4 mm). This suggests that the partial lack of maternal 5-HT provided to the embryo may be sufficient to explain some of the littermates phenotypes [18, 123].

Recently, the placenta (that is of embryonic origin) has been identified as an important source of 5-HT for the developing embryo. The placenta (syncytiothrophoblastic cells and sinusoidal throphoblastic giant cells) of the placenta contain Tph1, AADC and MAO [124, 125, 127], and convert tryptophan of maternal origin into 5-HT as soon as E10-E11 [150]. Homozygote knock-out embryos in which 5-HT neurons fail to fully differentiate or to produce normal amounts of 5-HT levels do not display severe cortical defects when gestating in heterozygous dams. Examples include mice lacking the transcription factors Lmx1b [151] or Pet-1 [152], in which all or 70–80% of 5-HT raphe neurons fail to develop, and mice lacking Tph2 [153, 154]. Further analysis revealed that Pet-1 knock-out embryos developing in heterozygous dams have normal 5-HT levels before the closure of the BBB (before E15 [68]). These studies suggest that 5-HT produced by the placenta may buffer maternal deficiency. However, the compensatory mechanisms remain to be clarified.

Outside the CNS, 5-HT is also produced in the periphery of the developing embryo: from the myenteric plexus (from E15-E16), from enterochromaffin cells of the lining lumen of the digestive tract (from E18), from neuroepithelial cells of the respiratory tracts, from the parafollicular cells of the thyroid and from pinealocytes (belonging to the CNS; from E12). 5-HT could also be taken up by SERT expressing cells and further delivered to a distant region. SERT is expressed in platelets and mast cells [155, 156] that become numerous around E12 in mice. These cells could cross the BBB, transit across blood vessels that start to invade the developing cortex by E10-E11 in mice [68]. Whether these structures and mechanisms provide substantial amount of 5-HT to the developing telencephalon remains to be clarified.

Transiently, sensory thalamic neurons express SERT (E15-P15 in mice) and the vesicular monoamine transporter type 2 (VMAT2) that are respectively responsible for the uptake and packaging of 5-HT into synaptic vesicles [37, 157, 158]. Sensory thalamic neurons do not contain MAOs [159] but are equipped to release 5-HT, possibly with other transmitters (e.g. glutamate), after specific stimulation (review in Ref. [15]). Interestingly, it has been suggested that thalamocortical axons (TCAs) could be implicated in the proliferation and migration of glutamatergic neurons [160, 161] in addition to their well-known role on axonal refinement (see below).

Tryptophan is provided to the embryo but could also be converted into 5-hydroxytryptamine (5-HTP) or further into serotonin (5-HT; violet) in the placenta via the expression of various metabolic enzymes expressed in the placenta. In addition, 5-HT from maternal sources could be taken up by the placenta that also expressed serotonin transporter (SERT). During early embryonic stages, 5-HT could be delivered directly to the developing embryo. After E15-E16, when 5-HT axons of the hindbrain reach the cortex, 5-HT could act on various target cells (Cell; maroon) expressing selected arrays of 5-HT receptors. At this stage, 5-HT could also be taken up and stored by thalamocortical afferents (TC) and released after specific stimulation. In addition, 5-HTP is provided to the tryptophan hydroxylase type 2 (Tph2) and the aromatic L-amino acid decarboxylase (AADC) containing neurons that synthesize 5-HT. In this drawing adapted from Ref. [19], we have pointed in orange the maternal conditions that are best known to interfere with 5-HT availability to the embryo. We have also indicated that inhibitors of 5-HT uptake (SSRIs) that cross all barriers affect SERT function at all levels. Genetic polymorphisms or methylations mentioned in the text are indicated by a star. The major catabolic enzymes of 5-HT, monoamine oxidases (MAO) are indicated.

*Serotonin receptor signalling has been shown to regulate various cellular events. However, the large spectrum of serotonin receptors still need to be investigated in cortical development in rodent and even more in primate*.

## **4. Serotonin receptors with specific attention to the 5-HT3A**

#### **4.1. Transducing pathways**

Several groups have suggested that, at early stages, 5-HT arises from maternal sources. Indeed, this was suggested when analysing the phenotype of embryos generated from Tph1+/− or Tph1+/+ mothers. Tph1−/− and Tph1+/− embryos obtained from crosses between heterozygous parents were indistinguishable from their wild-type littermates (the crown-rump length (CRL) was of 7.4– 7.5 mm). By contrast, 80–88.9% of Tph1−/− and Tph1+/− embryos born Tph1−/− mothers displayed low CRL values (5.8–7.4 mm). This suggests that the partial lack of maternal 5-HT provided to the embryo may be sufficient to explain some of the littermates phenotypes [18, 123].

Recently, the placenta (that is of embryonic origin) has been identified as an important source of 5-HT for the developing embryo. The placenta (syncytiothrophoblastic cells and sinusoidal throphoblastic giant cells) of the placenta contain Tph1, AADC and MAO [124, 125, 127], and convert tryptophan of maternal origin into 5-HT as soon as E10-E11 [150]. Homozygote knock-out embryos in which 5-HT neurons fail to fully differentiate or to produce normal amounts of 5-HT levels do not display severe cortical defects when gestating in heterozygous dams. Examples include mice lacking the transcription factors Lmx1b [151] or Pet-1 [152], in which all or 70–80% of 5-HT raphe neurons fail to develop, and mice lacking Tph2 [153, 154]. Further analysis revealed that Pet-1 knock-out embryos developing in heterozygous dams have normal 5-HT levels before the closure of the BBB (before E15 [68]). These studies suggest that 5-HT produced by the placenta may buffer maternal deficiency. However, the compensa-

Outside the CNS, 5-HT is also produced in the periphery of the developing embryo: from the myenteric plexus (from E15-E16), from enterochromaffin cells of the lining lumen of the digestive tract (from E18), from neuroepithelial cells of the respiratory tracts, from the parafollicular cells of the thyroid and from pinealocytes (belonging to the CNS; from E12). 5-HT could also be taken up by SERT expressing cells and further delivered to a distant region. SERT is expressed in platelets and mast cells [155, 156] that become numerous around E12 in mice. These cells could cross the BBB, transit across blood vessels that start to invade the developing cortex by E10-E11 in mice [68]. Whether these structures and mechanisms provide substantial amount of 5-HT to the developing telencephalon remains

Transiently, sensory thalamic neurons express SERT (E15-P15 in mice) and the vesicular monoamine transporter type 2 (VMAT2) that are respectively responsible for the uptake and packaging of 5-HT into synaptic vesicles [37, 157, 158]. Sensory thalamic neurons do not contain MAOs [159] but are equipped to release 5-HT, possibly with other transmitters (e.g. glutamate), after specific stimulation (review in Ref. [15]). Interestingly, it has been suggested that thalamocortical axons (TCAs) could be implicated in the proliferation and migration of glutamatergic neu-

Tryptophan is provided to the embryo but could also be converted into 5-hydroxytryptamine (5-HTP) or further into serotonin (5-HT; violet) in the placenta via the expression of various metabolic enzymes expressed in the placenta. In addition, 5-HT from maternal sources could be taken up by the placenta that also expressed serotonin transporter (SERT). During early embryonic stages, 5-HT could be delivered directly to the developing embryo. After E15-E16, when 5-HT axons of the hindbrain reach the cortex, 5-HT could act on various target cells (Cell; maroon) expressing selected arrays of 5-HT receptors. At this stage, 5-HT could also be

rons [160, 161] in addition to their well-known role on axonal refinement (see below).

tory mechanisms remain to be clarified.

120 Serotonin - A Chemical Messenger Between All Types of Living Cells

to be clarified.

At least fourteen genes encoding for 5-HT receptors have been identified and cloned in the mammalian brain [162–165]. In addition, isoform diversity, alternative splicing of some subtypes and RNA editing add to the diversity of serotoninergic receptors. With the exception of the 5-HT3 receptors, all 5-HT receptors are coupled to G-proteins. According to their second messenger coupling pathways, 5-HT receptors have been categorized into four groups. The 5-HT1 and 5-HT5 receptors are coupled to Gi/Go proteins and exert their inhibitory effects on adenylate cyclase, inhibiting cAMP formation. The 5-HT2 receptors are coupled to Gq proteins and stimulate phospholipase C to increase the hydrolysis of inositol phosphates and elevate intracellular Ca2+. The 5-HT4,6,7 receptors are coupled to Gs proteins and are positively linked to adenylate cyclase and increase cAMP formation. 5-HT3 receptors belong to a family of ligand-gated ion channel receptors that include nicotinic acetylcholine receptors, GABA<sup>A</sup> receptors and glycine receptors and are modulated by intracellular cyclic AMP [162]. The 5-HT3 receptors respond to neurotransmitter release via direct (through the 5-HT<sup>3</sup> receptor itself) or indirect activation of the voltage-gated Ca2+ channels and lead to Ca2+ entry into the cell [166]. 5-HT3 receptors are composed of five subunits, with the majority being homomers of 5-HT3A receptors. Heteromeric 5-HT3AB receptors have been observed in specific brain regions and display lower Ca2+ permeability than the homomeric 5-HT3A receptors [167–169].

#### **4.2. Expression patterns**

Despite the efforts of many laboratories and open databases, a complete description of the developmental expression pattern of 5-HT receptors in the cerebral cortex is still lacking in rodent and very few studies have been performed in primate. However, pictures are emerging in the rodent brain. For example, 5-HT1A,F are expressed in neocortical proliferative zones in E14.5 rodent brain [17] and the 5-HT2B are expressed in the proliferative zones of the human occipital cortex [129] and in all microglial cells [170, 171]. The 5-HT1A,B,D, 5-HT2A, 5-HT2C and 5-HT3A, are expressed in specific subpopulations of post-mitotic neurons [17, 88, 91, 167, 168, 172, 173], whereas the 5-HT6 is expressed in both migrating interneurons and pyramidal neurons [174, 175].

The dynamic expression pattern of the 5-HT3A receptor has been described in details recently in mice. In the developing cortex, 5-HT3A is expressed as early as E11-E12 in neurons expressing reelin (Cajal-Retzius cells) and/or GABA cells located in the PP [88, 173]. The 5-HT3A is expressed by newly post-mitotic GABAergic neurons located in the CGE and AEP/PO, where about 30% of cortical GABAergic neurons are generated ([52, 88]; see **Figure 3A** and **B**). Using homochronic in utero grafting in combination with a transgenic mouse line expressing GFP under the control of the 5-HT3A promoter (5-HT3A:GFP animals), we have shown that this expression was protracted in two large subpopulations of cortical GABAergic neurons: the multipolar interneurons expressing NPY displaying late spiking and accommodating properties and in VIP+ interneurons displaying adapting and bursting properties [52, 88, 176]. In addition, subpopulations of NO+ and reelin+ interneurons also express 5-HT3A ([52, 55]; **Figure 1A**). By post-natal stages and decreasing with age, 5-HT3A is also expressed by young neurons expressing doublecortin and/or calretinin generated in the SVZ and migrating towards the olfactory bulb (rostral medial stream) and various cortical and subcortical regions [90, 91]. In addition, we have reported that transient-amplifying precursors located in the white matter ventrally to the anterior cingulate cortex produced neurons destined to populate the anterior cingulate cortex and its vicinity [91].

*Serotonin homeostasis and signalling act as a sculptor of cortical circuitry. In this section, we will review the different steps of cortical assembly that have been shown to be modulated by serotonin*.

## **5. Impact of serotonin imbalance on cortical circuit assembly**

## **5.1. Serotonin and cell proliferation**

It has been postulated for some time that 5-HT regulates the proliferation of a wide variety of cell types including cortical neurons. Pharmacological studies inducing depletion of several monoamines triggered drawbacks due to the non-selectivity of the drugs used and they will not be discussed here.

Recently, transgenic models selectively targeting specific serotonin-related genes in different neuronal populations have started to provide more insights. For instance, mice deficient in Tph1 or Tph2 showed body weight reduction and delayed maturation of cortical layers [18, 153, 177]. Heterozygous embryos growing in null mutant Tph1−/− mice showed an average of 30% reduction in proliferating cells (BrdU+) in the VZ after a 2 h pulse of BrdU administration, an analog of thymidine that is incorporated during the S phase of the cell cycle [18]. Although these studies suggest that 5-HT from Tph1+ sources may regulate the proliferation of neuronal precursors, additional studies are needed to refine these observations.

Hyposerotonin-induced microcephaly could also be due to increased death of post-mitotic neurons or neuronal progenitors. Indeed, 5-HT2 stimulation promotes the survival of glutamatergic neurons *in vitro* with a maximal effect observed for stages E16 and E18 in rats [178], and 5-HT1A stimulation increases neuroprotection in models of ischaemia and protects neuronal cultures against serum withdrawal [179, 180]. Furthermore, activation of 5-HT2 reverts increased apoptosis observed in VMAT2:KO mice, in which dopamine, norepinephrine and 5-HT are depleted [181].

The analysis of mice lacking MAOA and B, which displays high 5-HT levels but normal dopamine and norepinephrine levels during development, revealed a specific reduction of symmetric divisions of intermediate precursors cells [76] in SVZ during late corticogenesis (E17.5) [182]. This unexpected alteration was reverted after pharmacological inhibition of 5-HT synthesis (with p-chlorophenylalanine; PCPA) between E14.5-E19.5. In addition, neurosphere formation was modulated by 5-HT in a dose-dependent manner *in vitro*, with proliferative effects observed for concentration ranging from 10 to 100 ng/ml and inhibitory effects observed for higher concentration (1000 ng/ml). In this study, these inhibitory effects were associated with decreased 5-HT1A labelling of neuronal precursors [182] previously known to trigger neurogenesis in adult dentate gyrus. Hence, 5-HT might modulate cortical density through its proliferation-inducing action on progenitors.

During early development, 5-HT could also promote gap junction coupling through 5-HT2 stimulation [183] that coordinates cell-cell assembly during cell cycle [184].

### **5.2. Serotonin and neuronal migration**

The dynamic expression pattern of the 5-HT3A receptor has been described in details recently in mice. In the developing cortex, 5-HT3A is expressed as early as E11-E12 in neurons expressing reelin (Cajal-Retzius cells) and/or GABA cells located in the PP [88, 173]. The 5-HT3A is expressed by newly post-mitotic GABAergic neurons located in the CGE and AEP/PO, where about 30% of cortical GABAergic neurons are generated ([52, 88]; see **Figure 3A** and **B**). Using homochronic in utero grafting in combination with a transgenic mouse line expressing GFP under the control of the 5-HT3A promoter (5-HT3A:GFP animals), we have shown that this expression was protracted in two large subpopulations of cortical GABAergic neurons: the multipolar interneurons expressing NPY displaying late spiking and accommodating properties and in VIP+ interneurons displaying adapting and bursting properties [52, 88, 176]. In addition, subpopulations of NO+ and reelin+ interneurons also express 5-HT3A ([52, 55]; **Figure 1A**). By post-natal stages and decreasing with age, 5-HT3A is also expressed by young neurons expressing doublecortin and/or calretinin generated in the SVZ and migrating towards the olfactory bulb (rostral medial stream) and various cortical and subcortical regions [90, 91]. In addition, we have reported that transient-amplifying precursors located in the white matter ventrally to the anterior cingulate cortex produced neurons destined to populate the anterior

*Serotonin homeostasis and signalling act as a sculptor of cortical circuitry. In this section, we will review the different steps of cortical assembly that have been shown to be modulated by serotonin*.

It has been postulated for some time that 5-HT regulates the proliferation of a wide variety of cell types including cortical neurons. Pharmacological studies inducing depletion of several monoamines triggered drawbacks due to the non-selectivity of the drugs used and they will

Recently, transgenic models selectively targeting specific serotonin-related genes in different neuronal populations have started to provide more insights. For instance, mice deficient in Tph1 or Tph2 showed body weight reduction and delayed maturation of cortical layers [18, 153, 177]. Heterozygous embryos growing in null mutant Tph1−/− mice showed an average of 30% reduction in proliferating cells (BrdU+) in the VZ after a 2 h pulse of BrdU administration, an analog of thymidine that is incorporated during the S phase of the cell cycle [18]. Although these studies suggest that 5-HT from Tph1+ sources may regulate the proliferation of neuronal precursors, additional studies are needed to refine these

Hyposerotonin-induced microcephaly could also be due to increased death of post-mitotic

matergic neurons *in vitro* with a maximal effect observed for stages E16 and E18 in rats [178], and 5-HT1A stimulation increases neuroprotection in models of ischaemia and protects neu-

ronal cultures against serum withdrawal [179, 180]. Furthermore, activation of 5-HT2

stimulation promotes the survival of gluta-

reverts

**5. Impact of serotonin imbalance on cortical circuit assembly**

cingulate cortex and its vicinity [91].

122 Serotonin - A Chemical Messenger Between All Types of Living Cells

**5.1. Serotonin and cell proliferation**

neurons or neuronal progenitors. Indeed, 5-HT2

not be discussed here.

observations.

In most phyla, 5-HT triggers motility of various cell types including vertebrate lymphocytes (chick, fish, rodent [185, 186]) and microglia towards the CNS [170]. In the mammalian cortex, a role for 5-HT in regulating the migration of cortical neurons has recently emerged. In this context, 5-HT produces opposite consequences depending on its concentration.

One of the first experiments to address this question was made *ex vivo* on cortical explants maintained in a serum-free medium and supplemented with low 5-HT concentration. The migration of glutamatergic neurons was examined and was found be faster in explants supplied with 5-HT suggesting that low 5-HT dosage may enhance the radial migration. Furthermore, decreasing 5-HT levels during development delayed or disrupted cortical migration, suggesting that 5-HT produces a positive drive on cortical migration [181]. In rats depleted in 5-HT by PCPA during the peak of migration (E12/E13 to E17 in rats), abnormal accumulation of GABAergic neurons below the subplate at E17 and a marked reduction of calretinin+ and CCK/VIP+ GABAergic neurons at adult stage were reported [187]. Interestingly, mice lacking Tph2 also display reductions of selective GABAergic populations in limbic structures [188]. 5-HT3A is protractedly expressed by 30% of GABAergic neurons leading to calcium entry into the cell (see above). Using electrophysiological recording and calcium imaging, it was recently shown that CGE-derived interneurons that expressed 5-HT3A increase their response to 5-HT3 activation while they migrate radially and integrate the cortical plate (late phase of migration; see **Figure 6A**). This activation leads to an increased growth cone activity and to a decrease resting-state of 5-HT3A+ interneurons. Further, using *in vivo* graft of 5-HT3A deficient interneurons into wild-type host, it was shown that this role was cell-autonomous. Interestingly, long-lasting alteration in the positioning of reelin+ cortical interneurons was reported. This suggests that 5-HT3A activation acts as a migratory signal for CGE-derived interneurons and alters definitively the positioning of their subpopulation [189]. A similar conclusion was suggested using SERT:KO animals that showed a specific increase in the migratory speed and positioning of VIP+ interneurons [92].

**Figure 6.** Modulation of cerebral circuit formation by 5-HT3A A, 5-HT3A (3A) is expressed by migrating interneurons generated in the caudal ganglionic eminence (CGE). Physiological concentration of serotonin (5-HT), induce an acceleration of the radial migration of 5-HT3A+ interneurons at E17. B, At early postnatal stage, Cajal-Retzius cells (C-R) that express 5-HT3A, respond to 5-HT application by releasing reelin that through the activation of the integrin signaling pathway induce pruning of apical dendrites of pyramidal neurons (Pyr). This figure is adapted from [200].

Although dynamic expression pattern of 5-HT receptors is lacking in developing primate and human cortex, a very recent study by the group of Alvarez-Bulla showed that in human, lateborn interneurons continue to migrate in the cingulate cortex even after birth. These interneurons expressed a combination of transcription factors and a substantial fraction of them expressed COUP-TFII or SP8 (22 or 28% respectively) that are mainly specific of 5-HT3A+ interneurons suggesting that 5-HT could also modulate the migration and positioning of these neurons in human [97]. Interestingly, in the primate cortex, it was shown that 5-HT3A is expressed by a subset of small GABA+, substance P+ or calbindin+ neurons and by medium-size CR+ neurons [190].

By contrast, 5-HT excess appears to have opposite role on migrating neurons. Using high dosage of 5-HT *ex vivo* on cortical slices, it has been shown that 5-HT induces a decrease in the migratory speed of non-GABAergic and GABAergic neurons [174]. High 5-HT levels induced a retraction of the leading processes of GABAergic neurons migrating into the intermediate zone and cortical plate. This effect was shown to be mediated, at least in part, by the 5-HT<sup>6</sup> receptor activating the cAMP-signalling pathway [191]. Such role was also reported for glutamatergic neurons (for review, see Ref. [175]).

#### **5.3. Serotonin and differentiation of cortical neurons and afferents**

Lauder and Krebs were the first to report that depletion of 5-HT delayed the cessation of cell division, a marker of cell differentiation [144, 192]. After these pioneering studies, numerous groups have shown that 5-HT can influence dendritic and axonal morphogenesis during cortical development.

#### *5.3.1. Serotonin and dendritic maturation of cortical neurons*

Although dynamic expression pattern of 5-HT receptors is lacking in developing primate and human cortex, a very recent study by the group of Alvarez-Bulla showed that in human, lateborn interneurons continue to migrate in the cingulate cortex even after birth. These interneurons expressed a combination of transcription factors and a substantial fraction of them expressed COUP-TFII or SP8 (22 or 28% respectively) that are mainly specific of 5-HT3A+ interneurons suggesting that 5-HT could also modulate the migration and positioning of these neurons in human [97]. Interestingly, in the primate cortex, it was shown that 5-HT3A is expressed by a subset of small GABA+, substance P+ or calbindin+ neurons and by medium-size CR+ neurons [190].

pathway induce pruning of apical dendrites of pyramidal neurons (Pyr). This figure is adapted from [200].

124 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Figure 6.** Modulation of cerebral circuit formation by 5-HT3A A, 5-HT3A (3A) is expressed by migrating interneurons generated in the caudal ganglionic eminence (CGE). Physiological concentration of serotonin (5-HT), induce an acceleration of the radial migration of 5-HT3A+ interneurons at E17. B, At early postnatal stage, Cajal-Retzius cells (C-R) that express 5-HT3A, respond to 5-HT application by releasing reelin that through the activation of the integrin signaling

By contrast, 5-HT excess appears to have opposite role on migrating neurons. Using high dosage of 5-HT *ex vivo* on cortical slices, it has been shown that 5-HT induces a decrease in the 5-HT was shown to regulate the physiology of C-R cells known to be key regulators of various aspects of cortical development including dendritic arborization. This role is largely mediated by the secretion of the glycoprotein, reelin [72, 74]. C-R cells receive serotoninergic projections with which they make transient synaptic contacts [134] and reelin secretion was shown to be regulated in part by the amount of brain 5-HT. Pharmacological perturbation of the serotoninergic system by 5-methoxytryptamine (a non-selective 5-HT receptor agonist) reduces reelin levels circulating in the blood flow at P0 [134], leading to the formation of abnormal microcolumns in the mice P7 presubicular cortex, a feature that is observed in autistic syndromes (ASDs). The activation of C-R cells was proposed to be modulated by 5-HT1A or by the 5-HT3A receptors, as they were both suspected to be expressed in the marginal zone during development [167, 193]. Interestingly, the 5-HT3A has been shown to be expressed by C-R cells (averaging 80% at P0) and the synaptic activation of 5-HT3A was shown to be sufficient to induce action-potential firing on C-R cells suggesting that 5-HT3A could play a role in dendritic development [173]. The contribution of the 5-HT3A was further analysed. The deletion or blockade of 5-HT3A receptors was shown to induce excessive arborization of layers II-III apical dendrites of pyramidal neurons. Application of the N-terminal region of reelin, that induces the activation of a signalling pathway that is independent from the classic ApoER2/VLDL-pathway, rescued the dendritic phenotype of cortical pyramidal neurons in 5-HT3A:KO cortical slices, whereas reelin blockade leads to an increased growth of apical dendrites ([173]; see **Figure 6B**). This study suggested that increased reelin secretion due to over-activation of the 5-HT3A receptor could induce a decreased growth of apical dendrites. Interestingly, fluoxetine (an inhibitor of 5-HT uptake, SSRI) administration from E8 to E18 decreased the dendritic basal and apical arbor complexity of layer II/III pyramidal neurons in the somatosensory cortex. Such a role is specific to a selective developmental period and SSRIs have opposite functions at mature stages [194]. Furthermore, the effects of SSRIs on developing dendrites were abolished when administered in the 5-HT3A:KO mice or after pharmacological blockade of the 5-HT3A receptor [173, 195]. Moreover, the fine tuning of 5-HT3A signalling has been shown to be responsible for the anxiety-like behaviours that are induced by prenatal fluoxetine treatment in wild type mice [196]. These results suggest that developmental excess of serotonin increases reelin secretion by overactivating 5-HT3A receptors expressed on C-R cells, consequently inhibiting dendritic growth of pyramidal neurons. Whether 5-HT3A+ interneurons participate in this process remains unclear.

Animals fed with low tryptophan diet [197, 198] display cortical pyramidal neurons with decreased dendritic complexity and spine density. Thus, 5-HT may regulate dendritic maturation and spine density through different types of 5-HT receptors that remain to be identified. In this respect, the 5-HT1A is strongly expressed in the developing cortical plate [17] and is known to be necessary for the dendritic maturation of CA1 pyramidal neurons [199]. The 5-HT6 receptor also appears as a good candidate for controlling neuritic and dendritic development due to its ability to engage signalling pathways (e.g. Fyn, mTOR and Cdk5) playing roles in these processes. *In vitro* studies strongly suggest a role of 5-HT6 on neuritic extension (for review see [175]). However, a clear view on the implication of the variety of 5-HT receptors expressed in the developing cortex remains to be elucidated.

### *5.3.2. Serotonin and axonal development within the cerebral cortex*

The first clear demonstration that serotonin acts on cellular processes involved in the formation of cortical circuits comes from works performed on the rodent barrel field in the somatosensory cortex (S1). The serendipitous generation of a mouse displaying deficiency in the gene encoding for MAOA was at the starting point of these discoveries. These studies showed that excessive 5-HT amounts (ninefold increase at P0) in the developing cortex induced an abnormal organization of thalamocortical afferents (TCAs) growing in the layer IV of the primary somatosensory cortex [36, 37]. These alterations were later interpreted as an abnormal refining of TC axons due to a specific rise of 5-HT occurring during a sensitive period (P0- P4: [201]). In addition, pharmacological normalization of 5-HT levels in MAOA:KO mice by P0-P4 PCPA-treatment was sufficient to revert to normal the organization of S1 in MAOA:KO mice [37]. Later, it was shown that genetic SERT deficiency affected S1 organization similarly in rodent. These alterations are not only structural but also impair whisker-mediated perception [10]. Hyper-activation of the 5-HT1B receptor, transiently expressed on TCAs during development, plays a key role in this process. Indeed, SERT:KO and MAOA:KO mice that are deficient in 5-HT1B receptors are rescued [202–205]. Interestingly, serotonin excess does not only impairs S1 organization, but also such a role could probably be generalized in other regions displaying transient 5-HT uptake [158] as this was shown for the visual system [202, 205, 206]. Moreover, such a role could also occur in primate cortex since SERT is transiently expressed in the visual sensory thalamic neurons, at least in the marmoset [143]. So far due to the difficulty to obtain human embryonic samples of late stages, clear sets of data are still lacking but numerous non-serotoninergic fibres, presumably TCAs, labelled by SERT have been detected at GW10 [142].

Surprisingly, perinatal 5-HT deficiency only induces a reduction of barrel field organization without altering its general organization [177, 207, 208]. Nevertheless, further studies need to be carried since early reduction of 5-HT during embryonic development induces the emergence of altered behaviour [153].

Other studies suggest a prenatal role for 5-HT in regulating initial TCAs pathfinding. TCAs express SERT, 5-HT1B and 5-HT1D receptors at a time when TCAs are navigating towards the pallium. Embryonic down-regulation of 5-HT1B/C receptors in TCAs using *in utero* electroporation leads to abnormal TCAs pathfinding [209]. Furthermore, it has been shown that 5-HT modifies the attractive versus repulsive responsiveness of TCAs to netrin-1 [209], an important guidance molecule for TCAs. Given these findings, it is thus likely that abnormal 5-HT levels could also affect these earlier stages of TCAs pathfinding and lead to abnormal long range of TCAs wiring [19, 150].

## **5.4. Serotonin and the regulation of astrocytes and microglial cell functions**

activating 5-HT3A receptors expressed on C-R cells, consequently inhibiting dendritic growth of pyramidal neurons. Whether 5-HT3A+ interneurons participate in this process remains unclear. Animals fed with low tryptophan diet [197, 198] display cortical pyramidal neurons with decreased dendritic complexity and spine density. Thus, 5-HT may regulate dendritic maturation and spine density through different types of 5-HT receptors that remain to be identified. In this respect, the 5-HT1A is strongly expressed in the developing cortical plate [17] and is known to be necessary for the dendritic maturation of CA1 pyramidal neurons [199]. The

 receptor also appears as a good candidate for controlling neuritic and dendritic development due to its ability to engage signalling pathways (e.g. Fyn, mTOR and Cdk5) playing

(for review see [175]). However, a clear view on the implication of the variety of 5-HT recep-

The first clear demonstration that serotonin acts on cellular processes involved in the formation of cortical circuits comes from works performed on the rodent barrel field in the somatosensory cortex (S1). The serendipitous generation of a mouse displaying deficiency in the gene encoding for MAOA was at the starting point of these discoveries. These studies showed that excessive 5-HT amounts (ninefold increase at P0) in the developing cortex induced an abnormal organization of thalamocortical afferents (TCAs) growing in the layer IV of the primary somatosensory cortex [36, 37]. These alterations were later interpreted as an abnormal refining of TC axons due to a specific rise of 5-HT occurring during a sensitive period (P0- P4: [201]). In addition, pharmacological normalization of 5-HT levels in MAOA:KO mice by P0-P4 PCPA-treatment was sufficient to revert to normal the organization of S1 in MAOA:KO mice [37]. Later, it was shown that genetic SERT deficiency affected S1 organization similarly in rodent. These alterations are not only structural but also impair whisker-mediated perception [10]. Hyper-activation of the 5-HT1B receptor, transiently expressed on TCAs during development, plays a key role in this process. Indeed, SERT:KO and MAOA:KO mice that are deficient in 5-HT1B receptors are rescued [202–205]. Interestingly, serotonin excess does not only impairs S1 organization, but also such a role could probably be generalized in other regions displaying transient 5-HT uptake [158] as this was shown for the visual system [202, 205, 206]. Moreover, such a role could also occur in primate cortex since SERT is transiently expressed in the visual sensory thalamic neurons, at least in the marmoset [143]. So far due to the difficulty to obtain human embryonic samples of late stages, clear sets of data are still lacking but numerous non-serotoninergic fibres, presumably TCAs, labelled by SERT have

Surprisingly, perinatal 5-HT deficiency only induces a reduction of barrel field organization without altering its general organization [177, 207, 208]. Nevertheless, further studies need to be carried since early reduction of 5-HT during embryonic development induces the emer-

Other studies suggest a prenatal role for 5-HT in regulating initial TCAs pathfinding. TCAs express SERT, 5-HT1B and 5-HT1D receptors at a time when TCAs are navigating towards the pallium. Embryonic down-regulation of 5-HT1B/C receptors in TCAs using *in utero* electropora-

on neuritic extension

roles in these processes. *In vitro* studies strongly suggest a role of 5-HT6

tors expressed in the developing cortex remains to be elucidated.

*5.3.2. Serotonin and axonal development within the cerebral cortex*

126 Serotonin - A Chemical Messenger Between All Types of Living Cells

5-HT6

been detected at GW10 [142].

gence of altered behaviour [153].

Astrocytes and microglial cells have been shown to be implicated in key processes—from neurogenesis to synaptogenesis—involved in cortical development (for review, see Ref. [61]). These cells bear several 5-HT receptors depending on their stage and state (resting or activated) making 5-HT an indirect actor of cortical development via the modulation of their functions [170]. Pioneer studies have shown that 5-HT1A and 5-HT2 are expressed by both immature and mature astrocytes in human and rodent cortex, and that 5-HT stimulates the release of several trophic factor produced by glial cells that promote neuritic extension and synaptogenesis of cortical and serotoninergic neurons such as S100β or BDNF. Conversely, lesions of the serotoninergic system were shown to increase GFAP and to decrease the release of several trophic factors [210, 211].

More recently, several groups have focused their attention on the implications of microglial cells that colonize the embryonic telencephalon at the very beginning of its formation in rodent and human (see above; [63, 212]). Through local phagocytic activities and the release of various molecules (such as interleukin-1beta or tumor necrosis factor-alpha), microglial cells have been shown to regulate neurogenesis, to participate in axonal and dendritic organizations and pruning [212–216]. From early stage of colonization, microglial cells have been shown to express, at least, the 5-HT2B receptor and at later stages or upon stimulation (such as inflammation), several other 5-HT receptors have been detected in rodent (5-HT1F,2A,2B,3B,5A and 5-HT7; [170]). The activation of these receptors has been shown to regulate their motility, their phagocytic properties and selective reshaping of axonal and dendritic arborizations. For instance, 5-HT2B has recently been shown to induce synaptic refinement of retinal projections to the thalamus since this process is impaired in mice lacking 5-HT2B selectively in microglial cells [171].

*During early development, the serotoninergic system is challenged by various genetic and epigenetic factors such as medications altering 5-HT transporter function, by nutrition and stress including ischaemia/hypoxia. In this section, we review how these factors may induce the emergence of various pathological disorders in primate and human*.

## **6. Serotonin imbalance and consequences in human pathology**

## **6.1. Serotonin imbalance and 5-HT3 receptor modulation in human pathology**

Developmental imbalance of 5-HT homeostasis or serotonin receptor signalling impacts various processes involved in the formation of cortical circuits and has consequences on the emergence of abnormal behaviour in rodent. Some similarities have been detected in primate and human but many aspects remain to be tested, in particular, the cellular processes implicated (conditioned by SERT or 5-HT receptors expressions) and the time windows of vulnerability.

In human, three major causes of 5-HT imbalance leading to psychiatric diseases have been clearly identified: abnormal metabolism of 5-HT, exposure of fetuses to SSRIs and genetic inheritance of SERT variants (these points of vulnerability have been indicated in **Figure 5**). Following the discovery of the lack of MAOA in Norrie disease [217], abnormal regulation of the enzymes implicated in 5-HT metabolism has been known for long to be associated with neuropsychiatric diseases (recently reviewed by Naoi et al. [218]). However, it is not known whether the alteration in prenatal or post-natal human life induces such illness. Pharmacological SSRIs treatment gave clearer answers. Indeed, SSRIs during pregnancy are still largely used among women ((2–13%) [219]); despite the high incidence of mood disorders in pregnant women (around 20% of pregnant women are affected) and the deleterious effect of maternal stress on fetal development. However, SSRIs crossing the placenta, are detectable in breast milk, reach the developing brain. Both, short-term (e.g. fetal cardiovascular malformations) and long-term drawbacks of the treatments have been revealed (see below). During gestation, SSRIs induced a reduction of blood flow in the middle cerebral artery at GW36 [220] and reduced fetal head growth [221]. SSRIs induce reduced motor movements and altered speech perception at 6–10 months of age, increased irritability, and persistent blunted pain reactivity [222, 223]. Children exposed to SSRIs during pregnancy have poor scores on psychomotor developmental scales [224] and higher risks to develop autism spectrum disorders [225]. The risk appeared higher when exposure to SSRIs occurred during the second trimester and with higher dosage of SSRIs, suggesting deleterious effects on early neural circuit formation. The third well-known cause of excessive 5-HT-signalling in human is of genetic origin. There are two variants of SERT alleles leading to different levels of SERT expression: the short form that induces decreased levels of SERT expression and SERT hypofunction [41] and the long form. Hypofunctional s-allele has been shown to increase the risk for a wide range of psychopathological traits. When combined with maternal anxiety during pregnancy, infants and children carrying the s-allele showed higher levels of negative emotionality compared to l-allele carriers [42] and increased scores of anxiety and depression [43, 226]. Interestingly, platelets that bear SERT (generally accepted to be identical to neuronal SERT), VMAT2 and 5-HT2 receptors have been suspected to play a role in the emergence of autistic disease in human. Dysregulation in platelets function has been largely used as a marker of autism, however clarifications need to emerge from further studies (for review, see Refs. [227, 228].

Although the consequences are subtle, they reveal that both genetic and environmental SERT deficiency impact human development and increase the risks of future psychiatric diseases [229, 230]. Overall, these findings point to the general conclusion that various clinical pathological traits, including autism, depression and anxiety-related phenotypes are associated to conditions of SERT deficiency during development. One should also consider that alteration of other genes may have synergistic effect on the emergence of those diseases or by contrast that bearing allelic variants of other genes could dampen the negative effects of SSRIs [231].

Rodent studies have revealed that the 5-HT3A regulates cellular events involved in cortical circuit formation (see above). Human genetic studies have recently explored more deeply the involvement of 5-HT3A polymorphisms and methylation in the emergence of various pathological traits and they now provide compelling evidence for such a role. In human genetic studies, it has been shown that a single-nucleotide polymorphism in 5-HT3A (SNP; rs1062613) was associated with bipolar disease [232]. Moreover, allelic variants or specific levels of methylation of the 5-HT3A have been shown to be tightly linked with alcohol-dependence, modulation of emotional networks and increase of depressive-related symptoms [233]. The emergence of depressive-like diseases was associated at the structural level with a decreased grey matter in the fronto-limbic region. Interestingly, 5-HT3A has been shown to interact with the brainderived neurotrophic factor, a key factor for circuit formation and consolidation [234, 235]. Thus, genetic polymorphism or methylation of 5-HT3A appears as a marker of susceptibility to develop a large panel of diseases.

human but many aspects remain to be tested, in particular, the cellular processes implicated (conditioned by SERT or 5-HT receptors expressions) and the time windows of vulnerability. In human, three major causes of 5-HT imbalance leading to psychiatric diseases have been clearly identified: abnormal metabolism of 5-HT, exposure of fetuses to SSRIs and genetic inheritance of SERT variants (these points of vulnerability have been indicated in **Figure 5**). Following the discovery of the lack of MAOA in Norrie disease [217], abnormal regulation of the enzymes implicated in 5-HT metabolism has been known for long to be associated with neuropsychiatric diseases (recently reviewed by Naoi et al. [218]). However, it is not known whether the alteration in prenatal or post-natal human life induces such illness. Pharmacological SSRIs treatment gave clearer answers. Indeed, SSRIs during pregnancy are still largely used among women ((2–13%) [219]); despite the high incidence of mood disorders in pregnant women (around 20% of pregnant women are affected) and the deleterious effect of maternal stress on fetal development. However, SSRIs crossing the placenta, are detectable in breast milk, reach the developing brain. Both, short-term (e.g. fetal cardiovascular malformations) and long-term drawbacks of the treatments have been revealed (see below). During gestation, SSRIs induced a reduction of blood flow in the middle cerebral artery at GW36 [220] and reduced fetal head growth [221]. SSRIs induce reduced motor movements and altered speech perception at 6–10 months of age, increased irritability, and persistent blunted pain reactivity [222, 223]. Children exposed to SSRIs during pregnancy have poor scores on psychomotor developmental scales [224] and higher risks to develop autism spectrum disorders [225]. The risk appeared higher when exposure to SSRIs occurred during the second trimester and with higher dosage of SSRIs, suggesting deleterious effects on early neural circuit formation. The third well-known cause of excessive 5-HT-signalling in human is of genetic origin. There are two variants of SERT alleles leading to different levels of SERT expression: the short form that induces decreased levels of SERT expression and SERT hypofunction [41] and the long form. Hypofunctional s-allele has been shown to increase the risk for a wide range of psychopathological traits. When combined with maternal anxiety during pregnancy, infants and children carrying the s-allele showed higher levels of negative emotionality compared to l-allele carriers [42] and increased scores of anxiety and depression [43, 226]. Interestingly, platelets that bear SERT (generally accepted

role in the emergence of autistic disease in human. Dysregulation in platelets function has been largely used as a marker of autism, however clarifications need to emerge from further studies

Although the consequences are subtle, they reveal that both genetic and environmental SERT deficiency impact human development and increase the risks of future psychiatric diseases [229, 230]. Overall, these findings point to the general conclusion that various clinical pathological traits, including autism, depression and anxiety-related phenotypes are associated to conditions of SERT deficiency during development. One should also consider that alteration of other genes may have synergistic effect on the emergence of those diseases or by contrast that bearing allelic variants of other genes could dampen the negative effects of SSRIs [231].

Rodent studies have revealed that the 5-HT3A regulates cellular events involved in cortical circuit formation (see above). Human genetic studies have recently explored more deeply the involvement of 5-HT3A polymorphisms and methylation in the emergence of various pathological

receptors have been suspected to play a

to be identical to neuronal SERT), VMAT2 and 5-HT2

128 Serotonin - A Chemical Messenger Between All Types of Living Cells

(for review, see Refs. [227, 228].

Together, this further confirms complex connections between early-life stress and the serotoninergic systems.

## **6.2. Linking serotoninergic system and neonatal inflammation/ischaemia with the emergence of neuropsychiatric diseases in children and adults**

Early-life inflammation modulates adulthood-inflammatory response [236]. In early brain injuries, activation of the immune system during fetal and neonatal life affects critical phases of brain development, with long-lasting consequences for neurological and mental health [237]. Neonatal stroke, systemic infection, or excitotoxicity/hypoxia-ischaemia (see **Figure 5**) induce perinatal insults activating the immune system and trigger peripheral and central responses that involve immune mediators (cytokines and chemokines), reactive oxygen species (ROS), reactive nitrosative species, excitotoxicity, mitochondrial impairment, and vascular integrity. In general, neonatal encephalopathy is of complex aetiology, encompassing several causal events, with strong evidence of fetal exposure to infection. The complex and multifactorial process of perinatal brain injury involves sensitization, whereby factors not severe enough by themselves to induce significant brain damage make the developing brain more susceptible to a second insult [238]. Substantial numbers of preclinical studies have demonstrated the sensitizing effects of gestational or neonatal systemic inflammation, gestational chronic mild maternal stress, and gestational hypoxia on perinatal excitotoxic or hypoxic-ischaemic lesions. Genetic factors have also been shown to influence the developing brain's response to sensitizing factors. Efforts to design therapies aimed to reduce the sensitizing effects of inflammation have been undertaken as neuroprotective agents, such as therapeutic hypothermia which have been performed mainly in models of pure hypoxia-ischaemia [238]. One of the main alterations following perinatal infection/inflammation is a persistent low-grade inflammation characterized by higher expression of inflammatory mediators and also microglial reactivity during adulthood [236]. Adult rodent exposed during early-life to LPS-enhanced expression of CD11b, IL-1β and IL-6 and also more activated microglia in the hippocampus, the striatum and substantia nigra/ventral tegmental area [239, 240]. This persistent low-grade inflammation sensitizes the brain to secondary injuries, which can lead to neurological disorders such as cerebral palsy, mood disorder, schizophrenia, or Parkinson disease [241].

Serotoninergic central system is vulnerable following a neonatal hypoxic-ischemic insult induced in a rat model [242] with a significant reduction in 5-HT levels, 5-HT transporter expression and 5-HT+ neurons is the dorsal raphe, 6 weeks after insult compared to control animals. Inhibition of neuroinflammation by Minocycline within the first week after injury is sufficient to prevent long-term neuroinflammation as well as serotonergic system damage still. The loss of dorsal raphe 5-HT+ neurons has been suspected to be induced by an alteration of one of their major target tissues: the prefrontal cortex [243].

## **7. Conclusion and perspectives**

Both genetic and environmental factors that influence serotonin signalling during specific sensitive periods of development impact specific cellular events involved in the development of cortical circuits. Such alterations depending on the cellular target and the time of occurrence could result in a predisposition to a large spectrum of cognitive or psychiatric illnesses including autism and depression.

## **Acknowledgements**

The work was supported by the INSERM. We warmly thank Pierre Gressens for his kind support, Stephane Peineau for kindly helping us with informatics and softwares and Zsolt Csaba for carefully reading and correcting our manuscript. T.V. thanks Hervé Langzam for fruitful discussions.

## **Author details**

Tania Vitalis\* and Catherine Verney

\*Address all correspondence to: tnvitalis@gmail.com

PROTECT U1141, French Institute of Health and Medical Research, University Paris Diderot, University Sorbonne Paris Cité, Paris, France

## **References**


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animals. Inhibition of neuroinflammation by Minocycline within the first week after injury is sufficient to prevent long-term neuroinflammation as well as serotonergic system damage still. The loss of dorsal raphe 5-HT+ neurons has been suspected to be induced by an altera-

Both genetic and environmental factors that influence serotonin signalling during specific sensitive periods of development impact specific cellular events involved in the development of cortical circuits. Such alterations depending on the cellular target and the time of occurrence could result in a predisposition to a large spectrum of cognitive or psychiatric illnesses

The work was supported by the INSERM. We warmly thank Pierre Gressens for his kind support, Stephane Peineau for kindly helping us with informatics and softwares and Zsolt Csaba for carefully reading and correcting our manuscript. T.V. thanks Hervé Langzam for fruitful

PROTECT U1141, French Institute of Health and Medical Research, University Paris Diderot,

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Tania Vitalis\* and Catherine Verney

\*Address all correspondence to: tnvitalis@gmail.com

University Sorbonne Paris Cité, Paris, France

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Cesar Soria-Fregozo, Maria Isabel Perez-Vega, Juan Francisco Rodríguez-Landa, León Jesús Germán-Ponciano, Rosa Isela García-Ríos and Armando Mora-Perez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68975

#### **Abstract**

Serotonin or 5-hydroxytryptamine (5-HT) is synthesized in both the brain and peripheral system, which exert their actions at a wide family of receptors classified as 5-HT1 to 5-HT7 . Pharmacological, behavioral, and clinical studies involve particularly to the 5-HT1A receptors (5-HT1A-R) - auto-receptors (presynaptic) and heteroreceptors (postsynaptic) - in the control of motivated behavior, and consequently in the physiopathology of affective disorders and in the action mechanism of antidepressant drugs. In this way, some research support that 5-HT1A-R participates in the delayed effect of different types of antidepressants, including selective serotonin reuptake inhibitors (SSRIs), and tricyclic drugs, principally. The therapeutic effect of serotonergic drugs as the SSRIs, starting with the binding to auto-receptors, which produces increases of 5-HT in the synaptic cleft as consequence of blockade of serotonin reuptake. While these molecular events occur initially, in the long-term are produced plastic changes at neuronal level, as well as down-regulation of the 5-HT1A-R, which is associated with the therapeutic effects of antidepressant drugs. The purpose of this chapter is to analyze and discuss the current information about of 5-HT1A-R-mediated signaling cascades, the intracellular signaling of 5-HT1A-R, in addition to their expression and pharmacology that are important to treatment of affective disorders symptoms.

**Keywords:** 5-HT1A receptors, affective disorders serotonin

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

5-Hydroxytryptamine (5-HT) regulates many important physiological processes, including body temperature, sleep, appetite, pain, motor activity, and affective disorders. One type of 5-HTergic functions is performed by the release of 5-HT into targeted areas and its action via at least 16 different pre-and postsynaptic 5-HT receptor (5-HTR) [1]. 5-HTRs are subdivided into seven groups—from 5-HT1 -R to 5-HT7 -R—according to their distribution, molecular structure, cell response, and function. Except for the 5-HT3 -Rs, which are ligand-gated ion channels, all other 5-HTR are G-protein-coupled receptors that influence different transduction pathways (**Table 1**). 5-HT1A-R auto-receptors located on the soma of 5-HTergic neurons are key components of the negative feedback loop that inhibits neuronal signaling and 5-HT release [2], while 5-HT1A-R heteroreceptors located on postsynaptic 5-HTergic and non-5-HTergic neurons [3, 4], particularly those in the limbic system, are involved in emotional states.

## **2. Distribution and ontogeny of the 5-HT1A-R**

The 5-HT can interact with different types of receptors, whose effect depends on the activation of different subtypes and location of these [1, 3] (**Table 1**). In this sense, the use of such techniques as ligand binding, immunohistochemistry, and hybridization *in situ* in the brains of the rat, mouse, cat, and human has reported significant levels of 5-HT1A-R in almost all regions of the brain [5–9]. A study performed in cats used positron emission tomography (PET) and 2′-methoxyphenyl-(N-2′-pyridinyl)-p-fluoro-benzamidoethyipiperazin marked with fluorine (MPPF [18F]) in combination with *in vitro* autoradiography with [<sup>3</sup> H] MPPF, 8-hydroxy-2-(din-propylamino)tetralin ([3H] 8-OH-DPAT) and [<sup>3</sup> H] paroxetine, to visualize the distribution of the 5-HT1A-R. These showed high levels of expression in the hippocampus, cingulate, septum, infralimbic cortex, and raphe nuclei, with low levels being detected in the cerebellum [9]. However, studies with PET using [11C] WAY-100635 reported some regional heterogeneity of the 5-HT1A-R in the human cerebellum [10]. The absence of 5-HT1A-R expression was observed in


**Table 1.** Classification and mechanism of the 5-HT receptor.

the cerebellar white matter, while the other regions displayed detectable levels of this receptor. On the other hand, studies of the cellular distribution of this receptor and its messenger ribonucleic acid (mRNA) have reported that approximately 60% of all glutamatergic cells express the transcript 5-HT1A-R, and about 25% of cells that express the enzyme glutamate decarboxylase (GAD) contain mRNA for 5-HT1A-R [5]. In addition, studies using immunohistochemistry, *in vitro* autoradiography with [<sup>3</sup> H] 8-OH-DPAT, and *in situ* hybridization have reported mRNA and protein expression for the 5-HT1A-R in the pyramidal neurons of layer 2 of the prefrontal, insular, and occipital cortex [9], but labeling with [<sup>3</sup>H] 8-OH-DPAT is only detected the layers 1 and 2 of the prefrontal and occipital cortex and in the pyramidal neurons of the cloister and the anterior olfactory nucleus. Neurons of the hippocampal CA1 region expressed the mRNA of the 5-HT1A-R, and [<sup>3</sup> H] 8-OH-DPAT labeling was observed in the stratum oriens and stratum radiatum. Low receptor expression was observed in CA3 pyramidal neurons, but the granule neurons in the dentate gyrus contained moderate concentrations of this receptor.

**1. Introduction**

states.

vided into seven groups—from 5-HT1

148 Serotonin - A Chemical Messenger Between All Types of Living Cells

5-Hydroxytryptamine (5-HT) regulates many important physiological processes, including body temperature, sleep, appetite, pain, motor activity, and affective disorders. One type of 5-HTergic functions is performed by the release of 5-HT into targeted areas and its action via at least 16 different pre-and postsynaptic 5-HT receptor (5-HTR) [1]. 5-HTRs are subdi-

ion channels, all other 5-HTR are G-protein-coupled receptors that influence different transduction pathways (**Table 1**). 5-HT1A-R auto-receptors located on the soma of 5-HTergic neurons are key components of the negative feedback loop that inhibits neuronal signaling and 5-HT release [2], while 5-HT1A-R heteroreceptors located on postsynaptic 5-HTergic and non-5-HTergic neurons [3, 4], particularly those in the limbic system, are involved in emotional

The 5-HT can interact with different types of receptors, whose effect depends on the activation of different subtypes and location of these [1, 3] (**Table 1**). In this sense, the use of such techniques as ligand binding, immunohistochemistry, and hybridization *in situ* in the brains of the rat, mouse, cat, and human has reported significant levels of 5-HT1A-R in almost all regions of the brain [5–9]. A study performed in cats used positron emission tomography (PET) and 2′-methoxyphenyl-(N-2′-pyridinyl)-p-fluoro-benzamidoethyipiperazin marked with fluorine

of the 5-HT1A-R. These showed high levels of expression in the hippocampus, cingulate, septum, infralimbic cortex, and raphe nuclei, with low levels being detected in the cerebellum [9]. However, studies with PET using [11C] WAY-100635 reported some regional heterogeneity of the 5-HT1A-R in the human cerebellum [10]. The absence of 5-HT1A-R expression was observed in

**Receptor family Subtype Mechanism Cellular response**

5-HT1 1A, 1B, 1D, 1E, 1F Adenylate cyclase Inhibitory 5-HT2 2A, 2B, 2C Phospholipase C Excitatory 5-HT3 3A, 3B, 3C Ligand-gated ion channel Excitatory 5-HT4 54 Adenylate cyclase Excitatory 5-HT5 5A, 5B Adenylate cyclase Inhibitory 5-HT6 56 Adenylate cyclase Excitatory 5-HT7 Adenylate cyclase Excitatory



H] MPPF, 8-hydroxy-2-(di-

H] paroxetine, to visualize the distribution


lar structure, cell response, and function. Except for the 5-HT3

**2. Distribution and ontogeny of the 5-HT1A-R**

(MPPF [18F]) in combination with *in vitro* autoradiography with [<sup>3</sup>

n-propylamino)tetralin ([3H] 8-OH-DPAT) and [<sup>3</sup>

**Table 1.** Classification and mechanism of the 5-HT receptor.

Turning now to the ontogeny of the 5-HT1A-R, immunohistochemistry has shown that almost all neurons of the hippocampus begin to express the 5-HT1A-R at the end of mitosis [11]. It is well known that at day 5 of postnatal age (P5), this receptor is expressed mainly in the cell bodies, while at day P10 it appears in the cell bodies and proximal apical dendrites. At the end of neuronal maturation (P21), a relatively scarce distribution is seen in the dendrites of the stratum radiatum and oriens of the hippocampus. During the early postnatal development of the hippocampus, glial cells that are positive to S100 (protein saturated ammonium sulfate soluble) and glial fibrillary acidic protein (GFAP) temporarily express the 5-HT1A-R and more than 90% of astrocytes that are positive to S100 in CA1, CA3, and the dentate gyrus also show moderate immunoreactivity to the 5-HT1A-R in P7, though this decreases sharply in P16. Although the specific distribution of the 5-HT1A-R has been studied in different brain regions, this does not ensure that receptor signaling activity will always be proportional to the levels of receptor expression. 5-HT1A-R signaling in neurons is important for functionality, and this intracellular effect is regulated by the coupling of second messengers.

## **3. Presynaptic and postsynaptic 5-HT1A-R and their signaling effects**

The main electrophysiological response to the activation of the 5-HT1A-R in neurons is mediated by the hyperpolarization of K+ channels [12, 13], which attenuates the propagation of action potentials, causing a consequent decrease in the release of the neurotransmitter. The hyperpolarizing effect is observed in both pre- and postsynaptic terminals; however, the desensitization profiles of those receptors and molecules activated in the pre- and postsynaptic terminals seem to differ. One of the mechanisms that cause desensitization of G-protein-coupled receptors is internalization, and studies have demonstrated the internalization (i.e., transfer of the plasmatic membrane in the cytoplasm) of the 5-HT1A auto-receptors in the dorsal raphe nucleus (DRN) of rats after acute treatment with the specific 8-OH-DPAT agonist to the 5-HT1A-R, or with recapture inhibitors of the 5-HT (selective inhibitors of serotonin reuptake, SSRIs). Although this phenomenon has not been observed in the hippocampus, we know that in this structure the 5-HT1A-Rs are located in the soma and dendrites of neurons (heteroreceptors) [14]. The SSRIs in the presynaptic terminals, in turn, increase the release of 5-HT, which binds to the 5-HT1A-R auto-receptors present in the soma of the raphe neurons, thus inhibiting neuronal firing. Subsequently, these auto-receptors are internalized, causing the end of 5-HT1A-R signaling in the presynaptic neurons, and again at onset of the 5-HT release of the rape neurons in the synapse with the dendritic terminals of the postsynaptic neurons. In the absence of the 5-HT1A auto-receptors, the 5HT released binds only to the postsynaptic 5-HT1A-R, thereby eliciting the anxiolytic effect of the SSRIs [15]. On the other hand, agonists to 5-HT1A-R, such as buspirone or flesinoxan, show an antidepressant effect, probably due to the desensitization of the 5-HT1A auto-receptors [16, 17]. Thus, the acute agonist treatment has its effect due to interaction with the auto-receptors present in the soma of the raphe neurons. The hyperpolarizing effect of the activation of this auto-receptor inhibits the release of 5-HT in the presynaptic terminal. It has been reported that under this treatment, the free or excess agonist can activate the postsynaptic (dendritic) 5-HT1A-R, resulting in the inhibition of postsynaptic neurons. Thus, an overstimulation of the receptor by an agonist causes desensitization and internalization of the 5-HT1A-R in raphe neurons, but not in postsynaptic neurons. The absence of 5-HT1A auto-receptors in the presynaptic raphe terminal facilitates neural firing by blocking inhibition by 5-HT, which is attached to the 5-HT1A-R in the postsynaptic neurons and causes the anxiolytic effect.

The activation of both pre- and postsynaptic 5-HT1A-R and their subsequent signaling seems to differ in at least one biochemical pathway. It has been shown that HN2-5 cells derived from neurons in the hippocampus, as well as in organotypic cultures of slices of the hippocampus, which are agonists to the 5-HT1A-R, stimulate the protein kinase pathway activated by mitogen (MAPK) [18]. However, in the raphe-derived cell line RN46A, activation of this receptor by agonists inhibits the basal activity of the MAPK pathway [19]. Nonetheless, it has been reported that activation of the 5-HT1A-R located both pre- and postsynaptically with the agonist inhibits intracellular cyclic adenosine monophosphate (cAMP) [20]. There are also reports that activation of 5-HT1A-R in a postsynaptic neuron-derived cell line and in non-neuronal cells promotes synthesis of phospholipase C (PLC), but this response has not been reported in presynaptic (serotonergic) or raphe-derived neurons [20, 21].

## **4. Aberrant 5-HT1A-R expression, anxiety and depression disorders**

In recent decades, such psychiatric disorders as anxiety (mainly generalized anxiety) and depression (mainly severe) have increased in prevalence and are now responsible for 3.12 and 6.86%, respectively, of years lived with disability (YLDs), according to estimates by the Global Burden of Diseases in 2015. Anxiety is a normal human emotion that allows us to respond to everyday stress situations, where the stressor—work, for example—can be identified. However, anxiety becomes a disorder when it no longer allows the individual to remain functional in her/his daily activities and when no trigger can be identified [22]. Both anxiety and depression have been attributed to a varied etiology that includes the person's social, economic, family, employment and academic condition, combined with the persistence of an inherent biological factor. In this sense, the findings of clinical and preclinical studies have identified a dysfunctionality of the serotonergic system associated with low availability of L-tryptophan (a precursor of 5-HT), low concentrations of 5-hydroxyindoleacetic acid (5-HIIA)—the main metabolite of 5-HT in the cerebrospinal fluid—a reduction in the synthesis, release, recapture, and metabolism of 5-HT, a decrease in the density of 5-HT1A-R pre- and postsynaptic, low neural activity in brain areas involved in regulating the emotions (such as the septum and prefrontal cortex), factors that increase the propensity (serotoninergic vulnerability) to suffer mood disorders like anxiety and depression. This is reinforced by the fact that serotonergic antidepressant treatments are prescribed to reverse these types of alterations [23–25]. In addition, functional brain imaging and postmortem studies of the limbic structures of depressed patients—which are responsible for integrating the emotions, and include the striatum, amygdala, and frontal cortex—have reported a low capacity for recapture 5-HT coupled with a decrease in the expression of 5-HT (5-HTT) transporters, which are responsible for recapturing the unused 5-HT in the 5-HT synapse and so regulate the magnitude and duration of serotoninergic neurotransmission [26]. Alterations of this kind in the 5-HTT have also been detected in patients with major depression using PET, which reveals a low capacity for 5-HT recapture in the thalamus, an area involved in controlling cortical excitability that contributes to establishing anxiety in patients so affected [27].

SSRIs in the presynaptic terminals, in turn, increase the release of 5-HT, which binds to the 5-HT1A-R auto-receptors present in the soma of the raphe neurons, thus inhibiting neuronal firing. Subsequently, these auto-receptors are internalized, causing the end of 5-HT1A-R signaling in the presynaptic neurons, and again at onset of the 5-HT release of the rape neurons in the synapse with the dendritic terminals of the postsynaptic neurons. In the absence of the 5-HT1A auto-receptors, the 5HT released binds only to the postsynaptic 5-HT1A-R, thereby eliciting the anxiolytic effect of the SSRIs [15]. On the other hand, agonists to 5-HT1A-R, such as buspirone or flesinoxan, show an antidepressant effect, probably due to the desensitization of the 5-HT1A auto-receptors [16, 17]. Thus, the acute agonist treatment has its effect due to interaction with the auto-receptors present in the soma of the raphe neurons. The hyperpolarizing effect of the activation of this auto-receptor inhibits the release of 5-HT in the presynaptic terminal. It has been reported that under this treatment, the free or excess agonist can activate the postsynaptic (dendritic) 5-HT1A-R, resulting in the inhibition of postsynaptic neurons. Thus, an overstimulation of the receptor by an agonist causes desensitization and internalization of the 5-HT1A-R in raphe neurons, but not in postsynaptic neurons. The absence of 5-HT1A auto-receptors in the presynaptic raphe terminal facilitates neural firing by blocking inhibition by 5-HT, which is

150 Serotonin - A Chemical Messenger Between All Types of Living Cells

attached to the 5-HT1A-R in the postsynaptic neurons and causes the anxiolytic effect.

**4. Aberrant 5-HT1A-R expression, anxiety and depression disorders**

In recent decades, such psychiatric disorders as anxiety (mainly generalized anxiety) and depression (mainly severe) have increased in prevalence and are now responsible for 3.12 and 6.86%, respectively, of years lived with disability (YLDs), according to estimates by the Global Burden of Diseases in 2015. Anxiety is a normal human emotion that allows us to respond to everyday stress situations, where the stressor—work, for example—can be identified. However, anxiety becomes a disorder when it no longer allows the individual to remain functional in her/his daily activities and when no trigger can be identified [22]. Both anxiety and depression have been attributed to a varied etiology that includes the person's social, economic, family, employment and academic condition, combined with the persistence of an inherent biological factor. In this sense, the findings of clinical and preclinical studies have identified a dysfunctionality of the serotonergic system associated with low availability

presynaptic (serotonergic) or raphe-derived neurons [20, 21].

The activation of both pre- and postsynaptic 5-HT1A-R and their subsequent signaling seems to differ in at least one biochemical pathway. It has been shown that HN2-5 cells derived from neurons in the hippocampus, as well as in organotypic cultures of slices of the hippocampus, which are agonists to the 5-HT1A-R, stimulate the protein kinase pathway activated by mitogen (MAPK) [18]. However, in the raphe-derived cell line RN46A, activation of this receptor by agonists inhibits the basal activity of the MAPK pathway [19]. Nonetheless, it has been reported that activation of the 5-HT1A-R located both pre- and postsynaptically with the agonist inhibits intracellular cyclic adenosine monophosphate (cAMP) [20]. There are also reports that activation of 5-HT1A-R in a postsynaptic neuron-derived cell line and in non-neuronal cells promotes synthesis of phospholipase C (PLC), but this response has not been reported in In addition, the involvement of deregulation of pre- and postsynaptic 5-HT1A-R in anxiety and depression is widely known, since it has been observed in patients with panic disorder by PET studies. There, reports indicate a reduction in the availability of both pre- and postsynaptic 5-HT1A-R in brain areas that regulate cognitive and emotional responses, such as the raphe, the orbitofrontal cortex, the temporal cortex, and the amygdala [28]. In support of this, preclinical studies have reported that knockout mice for 5-HT1A-R present an anxious phenotype that includes observations of such behaviors as a decrease of thigmotaxis (i.e., exploratory activity in central areas of an open field), increased fear in aversive environments, increased reactivity to stress, autonomic activation, and neuroendocrine alterations in models of experimental anxiety using the open-field, elevated-zero maze, and novel-object tests. However, an antidepressant-like effect has been observed in the tail suspension model of experimental depression, more markedly in females than in males. This is not associated with morphological abnormalities in brain tissues or changes in cell bodies or 5-HTergic fibers, nor is there evidence of changes in brain levels of 5-HT and 5-HIIA in the striatum, dorsal raphe, or frontal cortex [29, 30], though there is an increase in the turnover of 5-HT [31] and the firing of 5-HTergic neurons [32] in knockout mice to 5-HT1A-R. However, the possibility of such long-term changes cannot be discarded [33]. This situation can be interpreted as a disinhibition of 5-HTergic neuronal activity that increases the release of 5-HT in limbic areas, causing the establishment of anxiety through its interaction with other receptor subtypes, but without modifying levels of 5-HT or its metabolite, since the amount of stored 5-HT greatly exceeds the extracellular 5-HT content.

In support of this, differences in the function of the pre- and postsynaptic 5-HT1A-R in different brain areas seem to be decisive in establishing anxiety and depression, given that stimulation of the postsynaptic 5-HT1A-R in the dorsal hippocampus and amygdala produces anxiogenic effects, while anxiolytic effects are seen in areas such as the middle and dorsal raphe (where the 5-HT1A auto-receptors are located) [33–35]. In contrast, stimulation of the presynaptic receptors produces anxiolytic effects by suppressing 5-HTergic neuronal activity with the resulting decrease of 5-HT in axonal terminals of limbic areas [36]. These findings suggest that there are differences in the role played by pre- and postsynaptic 5-HT1A-R receptors in regulating emotions. This may be reflected in the fact that acute administration of antidepressants causes a reduction in neural activity due to the immediate stimulation of the 5-HT1A auto-receptors, while chronic antidepressant treatments cause desensitization and, consequently, the downregulation of the 5-HT1A auto-receptors, though with no changes in postsynaptic 5-HT1A-R. This leads to the recovery of 5-HTergic neuronal activity, which matches the long latency to the onset of the therapeutic effects of SSRIs antidepressants.

It is important to note that mice require proper 5-HTergic signaling through 5-HT1A-R stimulation of the prosencephalon during the early postnatal period as this produces lasting chemical and structural changes in the brain that are essential for effective response behaviors in the face of normal anxiety during adulthood [37]. Thus, clinically effective antidepressant or anti-anxiety treatments must stimulate the 5-HT1A auto-receptors with direct agonists (such as buspirone) or indirect agonists like fluoxetine to obtain therapeutic efficacy. This suggests that in both the developmental and adult stage efficient activation of the 5-HT1A auto-receptors can produce changes that decrease expressions of pathological anxiety.

Donaldson et al. [38] reported that a decrease in the 5-HT1A auto-receptors in the 21st postnatal leads to increased long-term anxiety levels but does not modify depressive behaviors. In this regard, lifelong abolition of the 5-HT1A auto-receptors suffices to increase anxiety behaviors in adult mice [39], though without necessarily affecting depressive-like behaviors in the forced swimming test [40]. Based on these results, it has been suggested that 5-HT1A auto-receptors are involved in establishing anxious and depressive phenotypes, while the heteroreceptor is implicated in the depressive phenotype observed in experimental tests of depression [40]. Moreover, Albert and François [41] suggest that a reduction in the activity of postsynaptic receptors is involved in anxiety and that an increase in the transcription of 5-HT1A auto-receptors is associated with both depression and resistance to chronic treatment with SSIR drugs [41]. Hence, the reduced expression of the auto-receptors with no modification of postsynaptic 5-HT1A-R expression is enough to produce depression-like behaviors in mice [42].

## **5. Therapeutic agents that function by regulating 5-HT1A-R signaling**

5-HT1A-R is involved in the pathology and treatment of mental disorders, such as anxiety and depression [23, 43, 44]. Several studies have suggested that the 5-HT1A-Rs are potential targets for these psychiatric disorders [45–49]. In this regard, agonists (total and partial) to the 5-HT1A-R have shown antidepressant and anxiolytic properties and have been employed as adjunct treatments to improve the therapeutic action of several antidepressant and anxiolytic drugs in several preclinical and clinical studies [50–53]. They offer a different pharmacological mechanism from that of the monoamine oxidase inhibitors (IMAO), tricyclic drugs, SSIRs, and other antidepressants.

Buspirone is perhaps the most widely studied partial 5-HT1A-R agonist. It belongs to the chemical class of the azapirones [54, 55] and has been used primarily due to its anxiolytic effects and absence of side effects such as sedation and dependence that are often associated with benzodiazepines [56]. It is also utilized to treat patients who are resistant to the SSRI<sup>S</sup> , due to its capacity to stimulate the release of catecholamines [57]. In this regard, a clinical trial carried out with ambulatory patients diagnosed with generalized anxiety disorder (GAD) found that after weeks 3 and 4, buspirone showed efficacy in relieving patients' symptoms with a therapeutic effect comparable to that of lorazepam. Also, after discontinuing this therapy, the individuals treated with buspirone showed no withdrawal symptoms, while those medicated with lorazepam saw their symptoms worsen in week 9 after ceasing treatment [58]. Similarly, buspirone (15 mg/day) prescribed for 4 weeks to ambulatory patients with GAD produced a significant reduction of anxiety symptoms compared to alprazolam. Moreover, the patients treated with buspirone experienced fewer adverse effects and symptoms of abstinence than those who received alprazolam [59]. The anxiolytic properties of buspirone have been confirmed in animal models. For example, in a study conducted with Swiss Albino mice that received buspirone at 2.5 and 5 mg/kg, i.p., the drug significantly increased the number of step-through by 46 and 61%, respectively [60]. This demonstrates that buspirone is effective in treating anxiety disorders without causing adverse effects or signs of benzodiazepine dependence.

activity with the resulting decrease of 5-HT in axonal terminals of limbic areas [36]. These findings suggest that there are differences in the role played by pre- and postsynaptic 5-HT1A-R receptors in regulating emotions. This may be reflected in the fact that acute administration of antidepressants causes a reduction in neural activity due to the immediate stimulation of the 5-HT1A auto-receptors, while chronic antidepressant treatments cause desensitization and, consequently, the downregulation of the 5-HT1A auto-receptors, though with no changes in postsynaptic 5-HT1A-R. This leads to the recovery of 5-HTergic neuronal activity, which matches the long latency to the onset of the therapeutic effects of

It is important to note that mice require proper 5-HTergic signaling through 5-HT1A-R stimulation of the prosencephalon during the early postnatal period as this produces lasting chemical and structural changes in the brain that are essential for effective response behaviors in the face of normal anxiety during adulthood [37]. Thus, clinically effective antidepressant or anti-anxiety treatments must stimulate the 5-HT1A auto-receptors with direct agonists (such as buspirone) or indirect agonists like fluoxetine to obtain therapeutic efficacy. This suggests that in both the developmental and adult stage efficient activation of the 5-HT1A auto-recep-

Donaldson et al. [38] reported that a decrease in the 5-HT1A auto-receptors in the 21st postnatal leads to increased long-term anxiety levels but does not modify depressive behaviors. In this regard, lifelong abolition of the 5-HT1A auto-receptors suffices to increase anxiety behaviors in adult mice [39], though without necessarily affecting depressive-like behaviors in the forced swimming test [40]. Based on these results, it has been suggested that 5-HT1A auto-receptors are involved in establishing anxious and depressive phenotypes, while the heteroreceptor is implicated in the depressive phenotype observed in experimental tests of depression [40]. Moreover, Albert and François [41] suggest that a reduction in the activity of postsynaptic receptors is involved in anxiety and that an increase in the transcription of 5-HT1A auto-receptors is associated with both depression and resistance to chronic treatment with SSIR drugs [41]. Hence, the reduced expression of the auto-receptors with no modification of postsynaptic 5-HT1A-R expression is enough to produce depression-like

**5. Therapeutic agents that function by regulating 5-HT1A-R signaling**

5-HT1A-R is involved in the pathology and treatment of mental disorders, such as anxiety and depression [23, 43, 44]. Several studies have suggested that the 5-HT1A-Rs are potential targets for these psychiatric disorders [45–49]. In this regard, agonists (total and partial) to the 5-HT1A-R have shown antidepressant and anxiolytic properties and have been employed as adjunct treatments to improve the therapeutic action of several antidepressant and anxiolytic drugs in several preclinical and clinical studies [50–53]. They offer a different pharmacological mechanism from that of the monoamine oxidase inhibitors (IMAO), tricyclic drugs, SSIRs,

tors can produce changes that decrease expressions of pathological anxiety.

SSRIs antidepressants.

152 Serotonin - A Chemical Messenger Between All Types of Living Cells

behaviors in mice [42].

and other antidepressants.

Gepirone is another component of the class of the azapirones that has shown antidepressant properties [61] due to its partial 5-HT1A-R antagonism, which improves 5-HTergic activity [62]. The structure of this azapirone is similar to that of buspirone, and it has similar anxiolytic properties that have been identified in clinical studies [63, 64]. But it also has antidepressant action. In a study of patients with major depressive disorder (DDM), prolonged-release gepirone (60–80 mg/day) administered for 3 weeks produced a significant reduction in total HAM-D17 scores (Hamilton Depression Scale) compared to a placebo group, thus improving the symptomatology of patients [65]. Similarly, gepirone (40–80 mg/day) prescribed for 8 weeks improved the sexual function of male patients diagnosed with DDM, in addition to its antidepressant action [66].

Tandospirone is a partial 5-HT1A-R agonist that has been shown to have antidepressant effects. In a study with male Sprague-Dawley rats, chronic treatment (28 days) with tandospirone at 10 mg/kg inhibited changes induced by psychosocial stress in the neurogenesis of the dorsal and ventral hippocampus, thus producing a type of antidepressant effect. It has been suggested that chronic administration of tandospirone desensitizes the 5-HT1A-R in the raphe. This decreases self-inhibition mediated by the somatodendritic receptor and, consequently, increases the firing rate and release of 5-HT [67].

Brexpiprazole is a second-generation antipsychotic that exerts partial antagonism to the 5-HT1A-R and D2. A study in adults diagnosed with DDM, but inadequate responses to antidepressants, showed that brexpiprazole as an adjunct therapy improved patients' symptoms. In that research, a series of drugs—escitalopram, fluoxetine, paroxetine, sertraline, duloxetine, and venlafaxine—all significantly improved scores on the Clinical Global Impressions Scale (CGI-I scale), Zung Self-Rating Depression Scale (SDS), and HAM-D17 scale when administered jointly with brexpiprazole (2 mg) for 6 weeks. Improvement was remarkable from the first week of treatment [68]. Finally, flesinoxan is a phenylpiperazine derivative initially developed as an antihypertensive [69]. This drug has total antagonism to 5-HT1A-R with high affinity [70]. Various studies have demonstrated its antidepressant properties, particularly in treatment-resistant DDM patients [71]. For example, in a double-blind, placebo-controlled and fixed-dose study of treatment-resistant DDM patients, flesinoxan (1.2 mg/day) administered for 6 weeks improved scores on the HAM-D17, Montgomery-Asberg Depression Rating Scale (MADRS) and CGI scales with improvement in subjects' mood. Nausea and dizziness were the most common side effects reported [72]. The therapeutic effects of flesinoxan have also been reported in animal models. In research with male Sprague-Dawley rats after olfactory bulbectomy, subjects were given flesinoxan (1 and 3 mg/ kg, s.c.) for 17 days. They presented reduced total immobility time on the forced swimming test [73]. This therapeutic action may be associated with the desensitization effect of the 5-HT1A-R in the nucleus of the dorsal raphe as an action mechanism [71].

The antidepressant activity of agonists to the 5-HT1A-R in presynaptic and postsynaptic neurons has been widely reported. Studies using the model of experimental learned helplessness in relation to depression have reported that stimulation of the 5-HT1A-R with 8-OH-DPAT at dosages of 0.03, 0.06, 0.125, 0.25, and 1 mg/kg i.p. for 5 days shows an antidepressant effect. To explore the role of the pre- and postsynaptic 5-HT1A-R, in that study, 8-OH-DPAT (0.1 and 1 µg/0.5 µl) was microinjected into the raphe and septum. While this showed an antidepressant effect when microinjected into the septum, no such effect was seen in the raphe of male rats [74]. This indicates that stimulation of the postsynaptic 5-HT1A receptors is responsible for establishing the antidepressant effect caused by 5-HT1A-R agonists when managed through a systemic pathway, since stimulation of the 5-HT1A somatodendritic auto-receptors in the raphe inhibits the release of 5-HT and the electrical activity of the raphe [75].

In recent years, administration of vilazodone has shown antidepressant [75, 76] and anxiolytic effects by eliminating physical and somatic symptoms in women with generalized anxiety disorder, after 8 weeks of treatment at daily doses of 20–40 mg [77, 78]. This effect is due to the action mechanism of this SSRI, which is a partial agonist of postsynaptic 5-HT1A receptors. In addition, it desensitizes 5-HT1A auto-receptors in the raphe more quickly than fluoxetine or paroxetine [79], is 30 times more powerful than serotonin transporter (SERT), and causes a larger increase of extracellular 5-HT in the ventral hippocampus and frontal cortex [80]. These facts justify the short latency to the appearance of therapeutic effects. Similar data have been reported in models of experimental anxiety using ultrasonic vocalizations. Observations suggest that vidazolam produces an anxiolytic effect that can be reversed by coadministration with an antagonist of the presynaptic 5-HT1A receptors such as WAY-100635.

This substance also produced an anxiolytic effect in the model of predator-induced stress at doses of 20–40 mg/kg and in the defensive burial model at doses of 10–40 mg/kg. However, no anxiolytic effect was seen in the elevated arms maze model [81]. Antidepressant effects at doses of 1 mg/kg were found in models of experimental depression based on the forced swimming and tail suspension tests [82].

## **6. Recent advances in the use of serotonin-norepinephrine reuptake inhibitors (SNRIs) for treating affective disorders**

scale when administered jointly with brexpiprazole (2 mg) for 6 weeks. Improvement was remarkable from the first week of treatment [68]. Finally, flesinoxan is a phenylpiperazine derivative initially developed as an antihypertensive [69]. This drug has total antagonism to 5-HT1A-R with high affinity [70]. Various studies have demonstrated its antidepressant properties, particularly in treatment-resistant DDM patients [71]. For example, in a double-blind, placebo-controlled and fixed-dose study of treatment-resistant DDM patients, flesinoxan (1.2 mg/day) administered for 6 weeks improved scores on the HAM-D17, Montgomery-Asberg Depression Rating Scale (MADRS) and CGI scales with improvement in subjects' mood. Nausea and dizziness were the most common side effects reported [72]. The therapeutic effects of flesinoxan have also been reported in animal models. In research with male Sprague-Dawley rats after olfactory bulbectomy, subjects were given flesinoxan (1 and 3 mg/ kg, s.c.) for 17 days. They presented reduced total immobility time on the forced swimming test [73]. This therapeutic action may be associated with the desensitization effect of the

The antidepressant activity of agonists to the 5-HT1A-R in presynaptic and postsynaptic neurons has been widely reported. Studies using the model of experimental learned helplessness in relation to depression have reported that stimulation of the 5-HT1A-R with 8-OH-DPAT at dosages of 0.03, 0.06, 0.125, 0.25, and 1 mg/kg i.p. for 5 days shows an antidepressant effect. To explore the role of the pre- and postsynaptic 5-HT1A-R, in that study, 8-OH-DPAT (0.1 and 1 µg/0.5 µl) was microinjected into the raphe and septum. While this showed an antidepressant effect when microinjected into the septum, no such effect was seen in the raphe of male rats [74]. This indicates that stimulation of the postsynaptic 5-HT1A receptors is responsible for establishing the antidepressant effect caused by 5-HT1A-R agonists when managed through a systemic pathway, since stimulation of the 5-HT1A somatodendritic auto-receptors in the raphe inhibits the release of 5-HT and the electrical activity

In recent years, administration of vilazodone has shown antidepressant [75, 76] and anxiolytic effects by eliminating physical and somatic symptoms in women with generalized anxiety disorder, after 8 weeks of treatment at daily doses of 20–40 mg [77, 78]. This effect is due to the action mechanism of this SSRI, which is a partial agonist of postsynaptic 5-HT1A receptors. In addition, it desensitizes 5-HT1A auto-receptors in the raphe more quickly than fluoxetine or paroxetine [79], is 30 times more powerful than serotonin transporter (SERT), and causes a larger increase of extracellular 5-HT in the ventral hippocampus and frontal cortex [80]. These facts justify the short latency to the appearance of therapeutic effects. Similar data have been reported in models of experimental anxiety using ultrasonic vocalizations. Observations suggest that vidazolam produces an anxiolytic effect that can be reversed by coadministration

This substance also produced an anxiolytic effect in the model of predator-induced stress at doses of 20–40 mg/kg and in the defensive burial model at doses of 10–40 mg/kg. However, no anxiolytic effect was seen in the elevated arms maze model [81]. Antidepressant effects at doses of 1 mg/kg were found in models of experimental depression based on the forced swim-

with an antagonist of the presynaptic 5-HT1A receptors such as WAY-100635.

5-HT1A-R in the nucleus of the dorsal raphe as an action mechanism [71].

154 Serotonin - A Chemical Messenger Between All Types of Living Cells

of the raphe [75].

ming and tail suspension tests [82].

Affective disorders are characterized by vigorousness in neurotransmission pathways at the cerebral level with reductions in serotonergic, noradrenergic, and dopaminergic concentrations, among other neurochemical and neuroanatomical changes. Consequently, therapeutic strategies designed to treat affective disorders include combinations of drugs and, in other cases, chemical compounds that act on one or more neurotransmission systems [83]. In this way, serotonin-norepinephrine reuptake inhibitors (SNRIs) have the capacity to block serotonin and noradrenalin reuptake in the brain, and so have been used successfully to treat such affective disorders as depression, emotional disorders like anxiety, and other illnesses related to the control of overweightness, fibromyalgia, peripheral diabetic neuropathic pain, and attention deficit-hyperactivity disorders, among others (**Table 2**).

The SNRIs were introduced into therapeutic use in the USA in 1993 under the name venlafaxine, a chemical compound included in a group of molecules named phenylethylamines, whose action mechanism principally involves the reuptake inhibition of serotonin and noradrenaline, though a lower degree of dopamine reuptake inhibition has also been reported. Through their dual action, these substances quickly increase concentrations of both neurotransmitters, apparently producing better therapeutic actions in major depression disorders than conventional antidepressant drugs that act upon only a single neurotransmission system. But SNRIs can produce side effects that include loss of appetite, reduced body weight and sleep, fatigue, headaches, nausea/vomiting, sexual dysfunction, and urinary retention, among others. To a lesser degree, they can also produce anxiety and high blood pressure. It is important to point out that some patients treated with SNRIs have increased suicidal thoughts, though this is still subject to controversy [84]. Despite their side effects, SNRIs are used frequently to control several depressive disorders due to their therapeutic efficacy.


Abbreviations: MDD, major depression disorder; BDD, bipolar depression disorder; AD, anxiety disorder; DPNP, diabetic peripheral neuropathy pain; ADHD, attention deficit hyperactivity disorder.

**Table 2.** Principal serotonin-norepinephrine reuptake inhibitors and their therapeutic uses.

Indeed, in some cases they work better than classic antidepressant drugs (e.g., SSRIs and tricyclic drugs) in certain groups of patients. For example, a clinical study of patients diagnosed with major depression disorder (aged 18–65) found remission of symptoms after 24 weeks of treatment with venlafaxine (initial dose of 75 mg/day, maximum dose of 225 mg/day) and milnacipran (50 mg twice a day), with a greater effect than that produced by 20 mg/day of the SSRI paroxetine [85]. However, in patients diagnosed with Alzheimer's and major depression disorders, the SSRIs sertraline and venlafaxine had a greater effect than the tricyclic antidepressant desipramine, all at doses of 150 mg/day during 12 weeks of treatment [86]. In a randomized, double-blind, parallel group study that evaluated the effect of long-term treatment (12 weeks) with venlafaxine in adult patients, there was a significant reduction of depressive symptoms compared to patients under the same conditions but treated with a lithium monotherapy [87]. Another SNRI used to treat major depression disorder is desvenlafaxine [88]. An integrated analysis of the efficacy of this drug found that treatment with 50 and 100 g/day reduced depression symptoms in patients diagnosed with major depression disorder compared to a placebo group [89].

Similarly, treatment with levomilnacipran (40–120 mg) in patients aged 18–80 diagnosed with some depression disorder, significantly reduced symptoms after 8–10 weeks of treatment [90]. These data show that the effect of SNRIs in treating major depression disorders depends on the characteristics of patients and the dosage schedule. One double-blind, controlled, randomized study compared two treatment schedules with venlafaxine: one fixed (75 mg/day) the other flexible (75–225 mg/day). It found that the fixed program gave a better response to this antidepressant treatment than the flexible approach [91]. Similarly, the use of SNRIs in young depressed patients (7–18) did not produce better therapeutic effects than a placebo treatment, though duloxetine has shown therapeutic potential in such patients [92]. A metaanalysis of the efficacy of venlafaxine, duloxetine, fluoxetine, and imipramine in children and adolescents found that SNRIs and tricyclic antidepressants do not seem to offer a significant advantage in treating major depression disorder in this population, as only fluoxetine produced an adequate therapeutic effect in those patients [93].

SNRIs are also often used to treat depressive symptoms associated with menopause. It is well known that in this biological phase, women are more susceptible and vulnerable to socioenvironmental factors that predispose them to develop emotional and affective disorders [94]. Menopausal women diagnosed with major depression disorders and vasomotor symptoms treated with duloxetine for 8 weeks experienced a reduction in their depressive and vasomotor symptoms, positive anxiolytic effects, and improved sleep quality, so it is believed that SNRIs may be an effective therapeutic option for treating mood and emotional disorders, as well as the more general symptoms associated with menopause [95]. In addition to its role as an effective treatment for major depression disorders associated with menopause, duloxetine is used to control other symptoms, such as hot flashes and anxiety [96]. Meanwhile, menopausal women treated with venlafaxine (75–300 mg/day) or fluoxetine (20–60 mg/day) felt a reduction in their depressive symptoms after 6 weeks of treatment, with no significant differences between these two antidepressants [97]. Administration of desvenlafaxine (50, 100, or 200 mg/day) to peri- and postmenopausal women also reduced depressive symptom compared to a placebo [98].

## **7. Mechanism of action of selective serotonin reuptake inhibitors (SSRIs) and affective disorders**

Indeed, in some cases they work better than classic antidepressant drugs (e.g., SSRIs and tricyclic drugs) in certain groups of patients. For example, a clinical study of patients diagnosed with major depression disorder (aged 18–65) found remission of symptoms after 24 weeks of treatment with venlafaxine (initial dose of 75 mg/day, maximum dose of 225 mg/day) and milnacipran (50 mg twice a day), with a greater effect than that produced by 20 mg/day of the SSRI paroxetine [85]. However, in patients diagnosed with Alzheimer's and major depression disorders, the SSRIs sertraline and venlafaxine had a greater effect than the tricyclic antidepressant desipramine, all at doses of 150 mg/day during 12 weeks of treatment [86]. In a randomized, double-blind, parallel group study that evaluated the effect of long-term treatment (12 weeks) with venlafaxine in adult patients, there was a significant reduction of depressive symptoms compared to patients under the same conditions but treated with a lithium monotherapy [87]. Another SNRI used to treat major depression disorder is desvenlafaxine [88]. An integrated analysis of the efficacy of this drug found that treatment with 50 and 100 g/day reduced depression symptoms in patients diagnosed with major depression

Similarly, treatment with levomilnacipran (40–120 mg) in patients aged 18–80 diagnosed with some depression disorder, significantly reduced symptoms after 8–10 weeks of treatment [90]. These data show that the effect of SNRIs in treating major depression disorders depends on the characteristics of patients and the dosage schedule. One double-blind, controlled, randomized study compared two treatment schedules with venlafaxine: one fixed (75 mg/day) the other flexible (75–225 mg/day). It found that the fixed program gave a better response to this antidepressant treatment than the flexible approach [91]. Similarly, the use of SNRIs in young depressed patients (7–18) did not produce better therapeutic effects than a placebo treatment, though duloxetine has shown therapeutic potential in such patients [92]. A metaanalysis of the efficacy of venlafaxine, duloxetine, fluoxetine, and imipramine in children and adolescents found that SNRIs and tricyclic antidepressants do not seem to offer a significant advantage in treating major depression disorder in this population, as only fluoxetine pro-

SNRIs are also often used to treat depressive symptoms associated with menopause. It is well known that in this biological phase, women are more susceptible and vulnerable to socioenvironmental factors that predispose them to develop emotional and affective disorders [94]. Menopausal women diagnosed with major depression disorders and vasomotor symptoms treated with duloxetine for 8 weeks experienced a reduction in their depressive and vasomotor symptoms, positive anxiolytic effects, and improved sleep quality, so it is believed that SNRIs may be an effective therapeutic option for treating mood and emotional disorders, as well as the more general symptoms associated with menopause [95]. In addition to its role as an effective treatment for major depression disorders associated with menopause, duloxetine is used to control other symptoms, such as hot flashes and anxiety [96]. Meanwhile, menopausal women treated with venlafaxine (75–300 mg/day) or fluoxetine (20–60 mg/day) felt a reduction in their depressive symptoms after 6 weeks of treatment, with no significant differences between these two antidepressants [97]. Administration of desvenlafaxine (50, 100, or 200 mg/day) to peri- and postmenopausal women also reduced depressive symptom com-

disorder compared to a placebo group [89].

156 Serotonin - A Chemical Messenger Between All Types of Living Cells

duced an adequate therapeutic effect in those patients [93].

pared to a placebo [98].

The action mechanism of SSRIs consists in inhibiting the 5-HT transporters (SERT) in the soma of raphe dorsal neurons (**Figure 1**). It has been shown that SSRIs, such as fluoxetine, that have an antidepressant effect possess a mechanism that inhibits SERT, thus increasing the availability of 5-HT in the synaptic cleft. This is accompanied by an increase in 5-HTergic neurotransmission associated with the establishment of the antidepressant effect [99]. This pharmacological effect is not immediate, suggesting that the 5-HT1A transporter blockade, *per se*, does not produce therapeutic effects during acute treatment, since in the first week of antidepressant therapy with SSRIs increases 5-HTergic neurotransmission due to the availability of 5-HT, which causes an overstimulation of the 5-HT1A auto-receptors, located in the cell body and dendrites of neurons in the raphe. Therefore, its neuronal activity, which is in charge of releasing 5-HT, is reduced in limbic areas, though we know that treatment with SSRI antidepressants requires 2–3 weeks to establish its therapeutic effect, because regulation of 5-HTergic neurotransmission in depressed patients requires the desensitization and subsequent internalization of the 5-HT1A auto-receptors of presynaptic neurons that eliminate the negative feedback on the raphe, thus increasing its neuronal activity and normalizing the release of 5-HT to the synaptic cleft that, finally, translates into an antidepressant effect.

The postsynaptic mechanism and cellular signaling of the 5-HT1A-R in relation to mood control are very complex. In this regard, it has been reported that some accompany the establishment of the therapeutic effect of SSRI antidepressants. One of the most important effects is the desensitization of the 5-HT1A auto-receptors. Normally in 5-HTergic neurotransmission, once the 5-HT is released into the synaptic cleft, it mainly has a three-point coupling. The

**Figure 1.** Mechanism of SSRIs: the 5-HT transporters (SERT) in the soma of raphe dorsal neurons; modified according to Garcia-Garcia et al. [40].

first is to the postsynaptic serotonergic receptors, mainly 5-HT1A. These receptors are coupled to the inhibition of protein G (Gi/o) and the consequent decrease in AMPc synthesis due to the inhibition of adenylate cyclase which, in conjunction with other second messengers, are responsible for activating the opening of ion channels, including Na<sup>+</sup> and K+ , for its input and output, respectively (**Figure 2**). This contributes to the hyperpolarization of the postsynaptic neurons so that they can go with the flow of neural inhibition. The second coupling is with the SERT, which are responsible for the reuptake of unused synapse 5-HT, which is returned to the presynaptic neuron through recycling, where it is stored for later release or to be metabolized to reset the synthesis of 5-HT. The energetic cost of its production is very high. The third coupling is with the 5-HT1A auto-receptors and, to a lesser extent, 5-HT1B and 5-HT1D. This causes inhibition of the opening of Ca2+ channels from the presynaptic neuron, which then inhibits the release of 5-HT into the synaptic cleft, thus regulating the intensity and duration of the nerve impulse from the presynaptic neuron (i.e., negative feedback or self-inhibition), mainly in neurons of the raphe, exerting the end the signaling of the presynaptic neurons and the resumption of the release of 5-HT neurons from the raphe to the postsynaptic neurons through the limbic areas [100]. In this context, chronic administration of SSRIs induces internalization of the 5-HT1A auto-receptors and the neurons of the raphe [101], since the increase in the availability of 5-HT in the cleft overstimulates those auto-receptors while also desensitizing and internalizing them. This process is associated with the phosphorylation of the carboxylic chain

**Figure 2.** Model of the transduction pathways that may be activated by the 5-HT1A-R; modified according to Polter and Li [100].

and the third intracellular loop of the receptor. The absence of 5-HT1A auto-receptors induces the binding of 5-HT only to postsynaptic 5-HT 1A receptors, which in turn triggers the antidepressant effect of SSRIs, though only after 2–3 weeks of treatment. However, this desensitization effect on the auto-receptors depends on the type of SSRIs administered, as it has not been observed when sertraline is administered chronically in humans [102].

## **8. Conclusion**

first is to the postsynaptic serotonergic receptors, mainly 5-HT1A. These receptors are coupled to the inhibition of protein G (Gi/o) and the consequent decrease in AMPc synthesis due to the inhibition of adenylate cyclase which, in conjunction with other second messengers, are

output, respectively (**Figure 2**). This contributes to the hyperpolarization of the postsynaptic neurons so that they can go with the flow of neural inhibition. The second coupling is with the SERT, which are responsible for the reuptake of unused synapse 5-HT, which is returned to the presynaptic neuron through recycling, where it is stored for later release or to be metabolized to reset the synthesis of 5-HT. The energetic cost of its production is very high. The third coupling is with the 5-HT1A auto-receptors and, to a lesser extent, 5-HT1B and 5-HT1D. This causes inhibition of the opening of Ca2+ channels from the presynaptic neuron, which then inhibits the release of 5-HT into the synaptic cleft, thus regulating the intensity and duration of the nerve impulse from the presynaptic neuron (i.e., negative feedback or self-inhibition), mainly in neurons of the raphe, exerting the end the signaling of the presynaptic neurons and the resumption of the release of 5-HT neurons from the raphe to the postsynaptic neurons through the limbic areas [100]. In this context, chronic administration of SSRIs induces internalization of the 5-HT1A auto-receptors and the neurons of the raphe [101], since the increase in the availability of 5-HT in the cleft overstimulates those auto-receptors while also desensitizing and internalizing them. This process is associated with the phosphorylation of the carboxylic chain

**Figure 2.** Model of the transduction pathways that may be activated by the 5-HT1A-R; modified according to Polter and

Li [100].

and K+

, for its input and

responsible for activating the opening of ion channels, including Na<sup>+</sup>

158 Serotonin - A Chemical Messenger Between All Types of Living Cells

Multiple antidepressant drugs are known to function through the 5-HT1A-R. New findings related to dysfunctions in the serotoninergic system, specifically in both pre- and postsynaptic 5-HT1A-R in the signaling pathways that modulate the 5-HT1A-R, demonstrate that 5HTergic alterations—whether in the expression or functionality associated with such disorders as anxiety and depression, and their subsequent association with alterations in signaling pathways that indirectly modulate and involve survival and neuronal development—can interfere with responses to antidepressant treatment. However, we require additional studies that accurately identify signaling mechanisms in different brain areas and differentiate their functions between the pre- and postsynaptic 5-HT1A-R present in intact animals and animals subjected to clinically effective antidepressant and anti-anxiety treatments. Since we know that differences in the distribution of receptors in the brain determine the physiological and behavioral functions, a better understanding of the underlying mechanisms associated with abnormal activity of the 5-HT1A-R will contribute to the search for novel therapeutic strategies that explore new ways of enhancing treatment of the most common psychiatric disorders around the world, including those of anxiety and depression, which severely impair the quality of life of individuals.

In general, the participation of the 5-HT1A-R in psychiatric disorders such as anxiety and depression has been widely explored in numerous clinical studies and animal models. All findings seem to indicate that including agonist components to the 5-HT1A-R in drug treatment of individuals with anxiety and depression is a promising option for improving the efficiency and implementation of the therapeutic effect of conventional drugs. It is important to emphasize that stimulation of the 5-HT1A-R activates indirect signaling mechanisms that have not yet been studied, so further research is necessary to explore possible alternative signaling mechanisms that accompany the establishment of the antidepressant effects mediated by 5-HT1A-R. Finally, in order to better understand the etiology of many disorders of brain development and advance in the elaboration of drugs that target 5-HT1A-R, it is important to study the profile of this receptor's activity in brain signaling during development.

In summary, there is ample clinical evidence to support the idea that SNRIs may be used to treat major depression disorder and other psychiatric disorders in certain groups of patients. However, the scarcity of controlled clinical studies and the wide age range of patients included in existing work, in addition to the scarce comparisons of the effects of SNRIs and classic antidepressant drugs (e.g., SSRIs and tricyclic antidepressants), raise the challenge of determining whether SNRIs produce greater, similar, or lower therapeutic effects than traditional therapeutic schedules. Nonetheless, the data currently available open doors for future research designed to explore new therapeutic options that will benefit patients with major depression disorders or other affective or emotional alterations.

## **Acknowledgements**

The writing of this chapter was made possible, in part, by funding from the *Programa de Apoyo a la Mejora de las Condiciones de Producción de los Miembros del SNI y SNCA* (PRO-SNI) 2017. The sixth author received financial support from Consejo Nacional de Ciencia y Tecnología (CONACyT) for postdoctoral studies at the University Center of Los Lagos, Universidad de Guadalajara (Laboratory of Biomedical Sciences/Histology). The fourth author received fellowship from CONACyT for postgraduate studies in Neuroethology Reg. 297560.

## **Author details**

Cesar Soria-Fregozo1 \*, Maria Isabel Perez-Vega<sup>1</sup> , Juan Francisco Rodríguez-Landa<sup>2</sup> , León Jesús Germán-Ponciano3 , Rosa Isela García-Ríos<sup>4</sup> and Armando Mora-Perez1

\*Address all correspondence to: csoria@culagos.udg.mx

1 Laboratory of Biomedical Sciences/Histology, University Center of Los Lagos, University of Guadalajara, Lagos de Moreno, Jalisco, Mexico

2 Laboratory of Neuropharmacology, Institute of Neuroethology, University Veracruzana, Xalapa, Veracruz, México

3 Postgraduate in Neuroethology, Institute of Neuroethology, Universidad Veracruzana, Xalapa, Veracruz, México

4 Department of Health Sciences, University Center of Tonalá, University of Guadalajara, México

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**Acknowledgements**

160 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Author details**

Cesar Soria-Fregozo1

Jesús Germán-Ponciano3

Xalapa, Veracruz, México

Xalapa, Veracruz, México

DOI: 10.1021/cr078224o

10.1016/S0091-3057(01)00746-8

México

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1 Laboratory of Biomedical Sciences/Histology, University Center of Los Lagos, University of

2 Laboratory of Neuropharmacology, Institute of Neuroethology, University Veracruzana,

3 Postgraduate in Neuroethology, Institute of Neuroethology, Universidad Veracruzana,

4 Department of Health Sciences, University Center of Tonalá, University of Guadalajara,

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**Section 3**

## **Metabolism**



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## **Application of 5-HT-SO4 in Biomarker Research**

## Raimond Lozda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69042

#### **Abstract**

A serotonin catabolite, serotonin O‐sulphate (5‐HT‐SO4 ), is hypothesised to accentuate the intensity of serotonin metabolism in the central nervous system (CNS). We hypothesised that serotonin O‐sulphate could be quantified in human plasma using modern liquid chromatography‐mass spectrometry. To test our hypothesis, we performed a critical lit‐ erature review and a three‐stage trial. First, a suitable liquid chromatography‐mass spec‐ trometry (LC‐MS/MS) method for detection of 5‐HT‐SO4 in human plasma samples was developed. Second, a pilot phase involving four healthy volunteers was executed. Finally, nine healthy volunteers were selected for the main study, where a basal plasma level of 5‐HT‐SO4 was measured before and after serotonergic stimulation of the central nervous system. One h after stimulation, six study subjects showed a decrease in 5‐HT‐SO<sup>4</sup> levels, while three subjects showed an increase. This was the first study in which naturally occur‐ ring 5‐HT‐SO<sup>4</sup> was detected by liquid chromatography–mass spectrometry (LC‐MS/MS) in the samples of human plasma obtained from healthy volunteers. The method devel‐ oped was specific to the measurement of 5‐HT‐SO<sup>4</sup> and opens up new possibilities to evaluate minor pathways or serotonin metabolism by minimally invasive methods.

**Keywords:** serotonin, serotonin O‐sulphate, biomarkers, depression

## **1. Introduction**

For several decades, it is noted that biomarkers are playing an increasingly important role in drug discovery and development from target identification and validation to clinical applica‐ tion, thereby making the overall process a more rational approach.

Indisputably, serotonin (5‐HT) plays a significant role in the course of depressive disorders, and majority of the drugs developed interferes with this pathway. Therefore, we decided to ascer‐ tain the metabolic processes to find perspective areas of research based on experience gathered so far. The most exploited laboratory biomarker methods investigating central nervous system

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(CNS) processes use the cerebrospinal fluid (CSF) as far as it is site specific and reveals local processes. Thus, measurement of indoleamine metabolites in the cerebrospinal fluid (CSF) remains an analytical evaluation method of drug efficacy during clinical trials. Nevertheless, due to patient's safety concerns and clinical convenience, a method employing less invasive approach is highly appreciated by health care professionals.

Thus, our objective was based on critical literature review approach and practical liquid chro‐ matography‐mass spectrometry (LC‐MS/MS) method implementation to investigate a possi‐ bility to use 5‐HT‐SO4 as a potential CNS‐specific serotonin metabolism biomarker based on a less invasive laboratory method suitable for clinical and pharmacological studies.

#### **2. Literature evidence related to 5‐HT‐SO4 appearance in animal and humans**

Sulphonate conjugation was first described in 1876 and has since been shown to be a signifi‐ cant pathway in the biotransformation of many neurotransmitters.

During a critical literature review, our special attention was drawn to the final phase of 5‐HT degradation by sulphotransferases (SULT) and the end product of biotransformation, 5‐HT‐ SO4 . We identified 66 papers and excluded 51 papers that did not contain data related to 5‐HT‐SO4 . In total, 15 papers were included in the final review with 10 analysed in terms of outcomes.

We found that during the last century, a sulphation of serotonin was described by Kishimoto and a final product of such biotransformation, serotonin‐O‐sulphate, was found [1]. Furthermore, during later years in animal experiments, it was approved that 5‐HT‐SO<sup>4</sup> is the final product of serotonin metabolism which is rapidly excreted from the organism [2, 3]. Later, a similar compound was found in human urine, cerebrospinal liquor and platelets [4–6]. During recent years, some research was done with marine molluscs determining 5‐HT‐ SO4 in their nervous system [7, 8]. Findings showed that the serotonin metabolite 5‐HT‐SO<sup>4</sup> forms from 5‐HT uptake and metabolism in central ganglia and other structures of nervous system but not in haemolymph itself.

**Table 1** summarises the evidence related to 5‐HT‐SO4 appearance in animals and humans.

As shown in **Table 1**, 5‐HT‐O‐SO4 was intensively investigated by G.M. Tyce during the 1980s and 1990s. Initially, a considerable amount of acid‐hydrolysable conjugates of dopamine, nor‐ epinephrine (NE) and 5‐HT were detected in lumbar CSF of normal individuals. The amounts of conjugated amines were small in comparison to the amounts of homovanillic acid and 5‐hydroxyindoleaceticacid [9]. In a further study performed with CSF from humans and ven‐ triculocisternal perfusion of African green monkeys found that the sulphates of NE, dopamine and 5‐HT are present in the CSF of laboratory animals and humans. All amines and metabo‐ lites were quantitated by using high‐performance liquid chromatography (HPLC) with elec‐ trochemical detection. The amounts of sulphated amines in human CSF always greatly exceed the amounts of the free amines [6]. This gave us a preliminary impression of 5‐HT‐SO<sup>4</sup> site


(CNS) processes use the cerebrospinal fluid (CSF) as far as it is site specific and reveals local processes. Thus, measurement of indoleamine metabolites in the cerebrospinal fluid (CSF) remains an analytical evaluation method of drug efficacy during clinical trials. Nevertheless, due to patient's safety concerns and clinical convenience, a method employing less invasive

Thus, our objective was based on critical literature review approach and practical liquid chro‐ matography‐mass spectrometry (LC‐MS/MS) method implementation to investigate a possi‐

Sulphonate conjugation was first described in 1876 and has since been shown to be a signifi‐

During a critical literature review, our special attention was drawn to the final phase of 5‐HT degradation by sulphotransferases (SULT) and the end product of biotransformation, 5‐HT‐

We found that during the last century, a sulphation of serotonin was described by Kishimoto and a final product of such biotransformation, serotonin‐O‐sulphate, was found [1]. Furthermore, during later years in animal experiments, it was approved that 5‐HT‐SO<sup>4</sup>

the final product of serotonin metabolism which is rapidly excreted from the organism [2, 3]. Later, a similar compound was found in human urine, cerebrospinal liquor and platelets [4–6]. During recent years, some research was done with marine molluscs determining 5‐HT‐

 in their nervous system [7, 8]. Findings showed that the serotonin metabolite 5‐HT‐SO<sup>4</sup> forms from 5‐HT uptake and metabolism in central ganglia and other structures of nervous

and 1990s. Initially, a considerable amount of acid‐hydrolysable conjugates of dopamine, nor‐ epinephrine (NE) and 5‐HT were detected in lumbar CSF of normal individuals. The amounts of conjugated amines were small in comparison to the amounts of homovanillic acid and 5‐hydroxyindoleaceticacid [9]. In a further study performed with CSF from humans and ven‐ triculocisternal perfusion of African green monkeys found that the sulphates of NE, dopamine and 5‐HT are present in the CSF of laboratory animals and humans. All amines and metabo‐ lites were quantitated by using high‐performance liquid chromatography (HPLC) with elec‐ trochemical detection. The amounts of sulphated amines in human CSF always greatly exceed the amounts of the free amines [6]. This gave us a preliminary impression of 5‐HT‐SO<sup>4</sup>

. We identified 66 papers and excluded 51 papers that did not contain data related to

. In total, 15 papers were included in the final review with 10 analysed in terms of

less invasive laboratory method suitable for clinical and pharmacological studies.

as a potential CNS‐specific serotonin metabolism biomarker based on a

 **appearance in animal and** 

appearance in animals and humans.

was intensively investigated by G.M. Tyce during the 1980s

is

site

approach is highly appreciated by health care professionals.

174 Serotonin - A Chemical Messenger Between All Types of Living Cells

**2. Literature evidence related to 5‐HT‐SO4**

cant pathway in the biotransformation of many neurotransmitters.

bility to use 5‐HT‐SO4

**humans**

SO4

SO4

system but not in haemolymph itself.

As shown in **Table 1**, 5‐HT‐O‐SO4

**Table 1** summarises the evidence related to 5‐HT‐SO4

5‐HT‐SO4

outcomes.

**Table 1.** Summary of the evidence found related to 5‐HT‐SO4 appearance in animals and humans. specificity. At the time of above mentioned studies, 5‐HT‐O‐SO<sup>4</sup> could not be detected in the plasma of untreated monkeys and the concentration of 5‐HT‐O‐SO4 in brain perfusates versus plasma increased after injection of 5‐HT sulphate. The ratio of amine sulphate in the brain versus amine sulphate in plasma was greater for 5‐HT‐O‐SO<sup>4</sup> than for DA‐O‐sulphate at 60 and 100 min after injection. Finally, it was concluded that although 5‐HT‐O‐SO<sup>4</sup> could not be detected in the plasma of monkeys or humans under normal conditions, the 5‐HT‐O‐SO4 in ventriculocisternal perfusates undoubtedly originates in the CNS [6].

This obstacle inspired us to analyse more recent studies selected during review.

Some research was conducted with marine molluscs determining 5‐HT‐O‐SO<sup>4</sup> in their ner‐ vous system [7, 8].

In one such research done in 2003, incubation of neuronal tissue of Aplysia revealed three novel 5‐HT catabolites. **Figure 1** summarises the metabolism of 5‐HT found in Aplysia central ganglia compared to human.

As seen from **Figure 1**, there is no difference of 5‐HT‐O‐SO<sup>4</sup> formation between humans and molluscs.

As shown in **Table 2**, the 5‐HT‐O‐SO4 can be detected in CNS and its formation though is site specific, and later animal studies confirm detection in periphery. Moreover, as far similar sulphotransfares exist in sea molluscs and mammals, an equal process should be theorised for humans [10]. Also, literature evidence exists that 5‐HT‐SO<sup>4</sup> was proposed to be measured in the animal urine or plasma which makes it to be relative simply detected by HPLC with various detectors [11].

**Figure 1.** Metabolites of 5‐HT in the marine mollusc *Aplysia californica* versus humans. The novel metabolites are shown for *Aplysia californica*. Only detailed 5‐HIAA metabolism is shown for humans.


**Table 2.** Summary of trials involving 5‐HT‐O‐SO<sup>4</sup> .

specificity. At the time of above mentioned studies, 5‐HT‐O‐SO<sup>4</sup>

176 Serotonin - A Chemical Messenger Between All Types of Living Cells

versus amine sulphate in plasma was greater for 5‐HT‐O‐SO<sup>4</sup>

As seen from **Figure 1**, there is no difference of 5‐HT‐O‐SO<sup>4</sup>

for humans [10]. Also, literature evidence exists that 5‐HT‐SO<sup>4</sup>

for *Aplysia californica*. Only detailed 5‐HIAA metabolism is shown for humans.

vous system [7, 8].

molluscs.

ganglia compared to human.

various detectors [11].

As shown in **Table 2**, the 5‐HT‐O‐SO4

plasma of untreated monkeys and the concentration of 5‐HT‐O‐SO4

ventriculocisternal perfusates undoubtedly originates in the CNS [6].

plasma increased after injection of 5‐HT sulphate. The ratio of amine sulphate in the brain

detected in the plasma of monkeys or humans under normal conditions, the 5‐HT‐O‐SO4

In one such research done in 2003, incubation of neuronal tissue of Aplysia revealed three novel 5‐HT catabolites. **Figure 1** summarises the metabolism of 5‐HT found in Aplysia central

site specific, and later animal studies confirm detection in periphery. Moreover, as far similar sulphotransfares exist in sea molluscs and mammals, an equal process should be theorised

in the animal urine or plasma which makes it to be relative simply detected by HPLC with

**Figure 1.** Metabolites of 5‐HT in the marine mollusc *Aplysia californica* versus humans. The novel metabolites are shown

and 100 min after injection. Finally, it was concluded that although 5‐HT‐O‐SO<sup>4</sup>

This obstacle inspired us to analyse more recent studies selected during review.

Some research was conducted with marine molluscs determining 5‐HT‐O‐SO<sup>4</sup>

could not be detected in the

than for DA‐O‐sulphate at 60

formation between humans and

was proposed to be measured

can be detected in CNS and its formation though is

in brain perfusates versus

could not be

in their ner‐

in

Summarising the literature review, there is evidence of similarities between human and ani‐ mal metabolic pathways, and as far as there is literature evidence of site‐specific 5‐HT‐SO<sup>4</sup> formation in animals, we can extrapolate the same to humans**.**

We noted that historically used methods assumed highly invasive approach of CSF sample collection from human subjects. Therefore, search for a minimally invasive method has signif‐ icant clinical benefit. Moreover, the latest 5‐HT‐SO<sup>4</sup> research revealed experience with LC‐MS application for such a purpose.

#### **3. The latest findings related to 5‐HT‐SO<sup>4</sup> appearance in humans**

The evidence in the scientific literature justifies the decision to employ detection of 5‐HT‐SO<sup>4</sup> in clinical practice. As far as there was no literature data particularly on 5‐HT‐SO<sup>4</sup> detection by LC‐MS/MS in the human plasma, we initiated a development of the chromatography method based on literature evidence related to detection of similar compounds such as indoleamines in the human plasma. The objective concerning analytical procedure was to demonstrate that it is suitable for its intended purpose—qualitative detection of 5‐HT‐SO4 in the human plasma.

Tandem mass spectrometric analysis (MS/MS) was made in a positive‐ion mode (ESI+). The electrospray ionisation of 5‐HT‐SO4 was weak. Thus, for the further quantitative analysis, a following ion transition was used: (257>>160) + (240>>160).

Specificity of the method was assessed visually by comparing multiple reaction monitor‐ ing (MRM) chromatograms of plasma sample spiked with serotonin O‐sulphate, samples of plasma and purified water. As seen in **Figure 2**, in the plasma‐based calibration stan‐ dard (A) and plasma (B), some 1.79–1.80 min retention time peaks can be observed [13]. In the plasma‐based calibration standard and "pure" plasma, some peaks with a retention time of 1.79–1.80 min were observed. The purified water samples treated similarly do not show such signals (C).This signal might be induced by native content of serotonin sul‐ phate found in plasma samples. Conclusion was reached because in the analytical solution made of 5% serum albumin, such a signal was not seen [13]. For the MRM chromatograms, the test solution of 5% serum albumin (buffered to pH = 7 in a phosphate buffer) was prepared.

The results obtained lead to conclusion that the method developed is specific to the com‐ pound of interest, 5‐HT‐SO4 .

The results obtained lead to the conclusion that the method developed is specific to the compound of interest, 5‐HT‐SO4 , in samples of human plasma. The linearity of detection was evaluated three times in different days by analysing calibration standard solutions of 5‐HT‐SO4 [13].

**Figure 2.** Chromatograms of 5‐HT‐SO<sup>4</sup> samples. (A) Plasma standard solution (containing 96 ng/mL of 5‐HT‐SO<sup>4</sup> ); (B) "pure" plasma sample and (C) water.

This method resulted in a linear relationship between concentration of the analyte (from 10 to 225 ng/mL) and a mass spectral signal of 5‐HT‐SO<sup>4</sup> with a calibration curve correlation coefficient of >0.98.

Specificity of the method was assessed visually by comparing multiple reaction monitor‐ ing (MRM) chromatograms of plasma sample spiked with serotonin O‐sulphate, samples of plasma and purified water. As seen in **Figure 2**, in the plasma‐based calibration stan‐ dard (A) and plasma (B), some 1.79–1.80 min retention time peaks can be observed [13]. In the plasma‐based calibration standard and "pure" plasma, some peaks with a retention time of 1.79–1.80 min were observed. The purified water samples treated similarly do not show such signals (C).This signal might be induced by native content of serotonin sul‐ phate found in plasma samples. Conclusion was reached because in the analytical solution made of 5% serum albumin, such a signal was not seen [13]. For the MRM chromatograms, the test solution of 5% serum albumin (buffered to pH = 7 in a phosphate buffer) was

The results obtained lead to conclusion that the method developed is specific to the com‐

The results obtained lead to the conclusion that the method developed is specific to the

was evaluated three times in different days by analysing calibration standard solutions of

, in samples of human plasma. The linearity of detection

samples. (A) Plasma standard solution (containing 96 ng/mL of 5‐HT‐SO<sup>4</sup>

); (B)

prepared.

5‐HT‐SO4

pound of interest, 5‐HT‐SO4

[13].

compound of interest, 5‐HT‐SO4

**Figure 2.** Chromatograms of 5‐HT‐SO<sup>4</sup>

"pure" plasma sample and (C) water.

.

178 Serotonin - A Chemical Messenger Between All Types of Living Cells

The optimal detection limit of 5‐HT‐SO4 in the plasma sample was determined to be 26.5 ng/mL. Four different concentrations of 5‐HT‐SO<sup>4</sup> were used for a recovery testing and the method gave correct 5‐HT‐SO<sup>4</sup> detection results, which were justified by the average level of recovery of the analyte at 116 ± 8%. The relatively high interval of recovery can be explained due to the matrix effect [13].

The intra‐laboratory accuracy of the method over a 3‐day period was characterised by a stan‐ dard deviation of ± 11.95% [13]. Taking into account above mentioned results, the method was concluded to be a suitable technique for measuring 5‐HT‐SO<sup>4</sup> in human blood samples.

The findings of method development phase led to the decision to perform the first‐in‐humans study in order to assess the clinical applicability of the LC‐MS/MS method developed, and the studies were designed to quantify intra‐individual results using a cohort of healthy subjects. The clinical study had a two‐stage design: a plot study and main Study.

The pilot study confirmed that the peaks with retention time 1.79–1.80 min are detected in the samples of plasma of healthy volunteers. These peaks corresponded to the signal of 5‐HT‐SO<sup>4</sup> . One study subject was exposed to oral intake of L‐5‐hydroxytryptophan (5‐HTP) containing food supplement to observe the influence of serotonergic stimulation to 5‐HT‐SO<sup>4</sup> level. The pilot study proved that the 5‐HT‐SO4 could be qualified in plasma samples obtained from healthy volunteers. The increase in 5‐HT‐SO4 level after serotonergic stimulation was observed. Unfortunately, all results were below the detection limit of the method and probably due to several matrix‐, method‐, or analyte‐specific reasons.

Our primary interest was to ascertain quantitative differences of basal 5‐HT‐SO<sup>4</sup> levels, the intra‐individual sensitivity of the quantitation as well as detection limit issues obtained in the pilot study on a larger number of subjects.

Thus, after measurement of the basal 5‐HT‐SO4 levels in nine subjects, all of them were exposed to serotonergic stimulation with a food supplement containing 100 mg of 5‐HTP and a second blood sample was analysed. In six study subjects, a decrease in 5‐HT‐SO<sup>4</sup> levels was observed 1 h after 5‐HTP ingestion. Three subjects, however, showed an increase in 5‐HT‐SO<sup>4</sup> 1 h after 5‐HTP ingestion. Out of nine study subjects and one pilot subject, an increase in 5‐HT‐SO<sup>4</sup> 1 h after 5‐HTP ingestion was observed in three women and one man, respectively. The others five men and one woman—showed decrease. A graphical chart of study results is shown in **Figure 3**.

The outcome of our studies is that we developed a liquid chromatography method, which is specific to the measurement of 5‐HT‐SO<sup>4</sup> in the samples of human plasma. It is the first time when 5‐HT‐SO<sup>4</sup> was detected in the plasma obtained from healthy volunteers [14]. The sen‐ sibility of the LC‐MS/MS method to detect intra‐individual changes of the compound in the healthy volunteers undergoing supplementation with 5‐HTP was observed, but the majority of results were below detection limit [13].

**Figure 3.** A graphical view of studies results.

## **4. Conclusions and discussion**

This chapter ascertains a possibility for **5‐HT‐SO**<sup>4</sup> to be employed as a potential serotonin metabolism biomarker based on a less invasive laboratory method. Based on critical literature review, **5‐HT‐SO**<sup>4</sup> is identified as a potential 5‐HT metabolism biomarker to be detected by a minimally invasive approach in human plasma. The novel LC‐MS/MS method, which is spe‐ cific to the measurement of **5‐HT‐SO**<sup>4</sup> in the human plasma, has been developed [13]. It was the first time when **5‐HT‐SO**<sup>4</sup> was detected in the plasma obtained from healthy volunteers [13]. However, the clinical applicability of the method was not justified as the majority of results were below detection limit of 26.5 ng/mL [13].

Concerns regarding the issue of whether 5‐HT‐SO<sup>4</sup> we found in the plasma has CNS ori‐ gin or not should be evaluated. It is known that L‐amino acid decarboxylase acts both in the periphery and in the CNS which can result with the ingested 5‐HTP being converted into serotonin in the periphery of the body too [15], but plasmatic serotonin mostly derived from peripheral tissues is primarily metabolised in the liver to 5‐hydroxyindole acetate and then excreted in the urine [16, 17]. Regarding 5‐HT locating in the gastrointestinal tract, it is known that once serotonin reuptake transporter (SERT) has brought serotonin into the epithelial cells, it is metabolised to 5‐HIAA by monoamine oxidase which is localised to all intestinal epithelial cells [18]. Alternatively, 5‐HT released into the lamina propria may enter the portal vein circulation and be detected either as free serotonin or within platelets (via the actions of SERT). As the liver processes the portal circulation, enzymes rapidly degrade the free 5‐HT. The monoamine oxidase degrades about one‐third to urine detectable 5‐HIAAn. The remaining two‐thirds of serotonin is degraded to the metabolite 5‐HT‐O‐glucuronide. It should be noted that 5‐HT taken up by platelets is protected from degradation in the liver and enters the general blood circulation [18]. Coincidentally, the only sites of 5‐HT sulpha‐ tion identified in humans are the CNS and enteric nervous systems [6, 19] while enteric nervous system 5‐HT‐SO4 was found only after induction of serotonin syndrome [19]. Some isoforms of SULT have been shown to have sulphate serotonin [20]. Also, findings in sea molluscs indicate that metabolism of serotonin with further formation of 5‐HT‐SO<sup>4</sup> depends upon the location of release. Thus, haemolymph 5‐HT‐ SO4 most probably originates from the nervous system [7, 8]. There is also no doubt about the entrance of ingested 5‐HTP into CNS [21]. Also, earlier studies concluded that under normal conditions, the 5‐HT‐O‐SO4 originates from CNS [6]. The most significant finding was that 5‐HT‐O‐SO<sup>4</sup> freely crosses blood‐CSF barrier, so physiological circumstances are not preventing the appearance of CNS‐originated 5‐HT‐O‐SO<sup>4</sup> in the venous blood circulation. Therefore, taking into account all aforementioned facts, we are more concerned that 5‐HT‐ SO<sup>4</sup> detected in the study [13] mimics serotonin metabolism in CNS. Future investigations are needed to justify this assumption.

The most disputable outcome of our research is the elevation or reduction of 5‐HT‐SO4 . The majority of volunteers from the study phase, six out of nine, had a drop of plasma sulphate concentration [12]. Although there is no data available regarding diurnal rhythmicity of serotonin sulphate levels in the human plasma, we are not able to confirm whether these changes are due to the direct influence of serotonergic stimulation. To investigate the possible link to the health status of study subjects with 5‐HT‐SO<sup>4</sup> level changes, we performed ques‐ tioning. Hamilton depression rating scale results revealed mild depression in four subjects. Unfortunately, we cannot make any clinical conclusion related to the correlation between symptoms and laboratory findings due to results below the detection limit, but the trend seems to be very intriguing.

**4. Conclusions and discussion**

**Figure 3.** A graphical view of studies results.

cific to the measurement of **5‐HT‐SO**<sup>4</sup>

the first time when **5‐HT‐SO**<sup>4</sup>

review, **5‐HT‐SO**<sup>4</sup>

This chapter ascertains a possibility for **5‐HT‐SO**<sup>4</sup>

180 Serotonin - A Chemical Messenger Between All Types of Living Cells

results were below detection limit of 26.5 ng/mL [13].

Concerns regarding the issue of whether 5‐HT‐SO<sup>4</sup>

metabolism biomarker based on a less invasive laboratory method. Based on critical literature

minimally invasive approach in human plasma. The novel LC‐MS/MS method, which is spe‐

[13]. However, the clinical applicability of the method was not justified as the majority of

gin or not should be evaluated. It is known that L‐amino acid decarboxylase acts both in the periphery and in the CNS which can result with the ingested 5‐HTP being converted into serotonin in the periphery of the body too [15], but plasmatic serotonin mostly derived from peripheral tissues is primarily metabolised in the liver to 5‐hydroxyindole acetate and then excreted in the urine [16, 17]. Regarding 5‐HT locating in the gastrointestinal tract, it is known that once serotonin reuptake transporter (SERT) has brought serotonin into the epithelial cells, it is metabolised to 5‐HIAA by monoamine oxidase which is localised to all intestinal epithelial cells [18]. Alternatively, 5‐HT released into the lamina propria may enter the portal vein circulation and be detected either as free serotonin or within platelets (via the actions of SERT). As the liver processes the portal circulation, enzymes rapidly degrade the

is identified as a potential 5‐HT metabolism biomarker to be detected by a

to be employed as a potential serotonin

we found in the plasma has CNS ori‐

in the human plasma, has been developed [13]. It was

was detected in the plasma obtained from healthy volunteers

Nevertheless, in the light of literature data, we tend to explain phenomena observed by sub‐ strate inhibition of SULT 1A3 [22, 23]. It could be concluded that under normal circumstances, quantity of serotonin synthesised and metabolised is kept under certain limits [20]. Data favouring this is evidence of paradoxical actions of the 5‐HTP on the activity of identified sero‐ tonergic neurons in a simple motor circuit. It was found that more serotonin did not lead to more potent swim motoraction, implying that serotonin synthesis must be kept withincertain limits for the circuit to function properly. Also, alteration of neurotransmitter synthesis can lead to grave consequencesfor the output of neuralnetworks [24]. Described mechanisms could be taken into account explaining our results. Thus, we hypothesise that a drop of 5‐HT‐SO<sup>4</sup> in plasma would be related to the overproduction of serotonin, leading to inhibition of SULT 1A3. Elevation of 5‐HT‐SO<sup>4</sup> was probably a sign of serotonin deficiency, but such an opinion also requires further investigation. The latter would correlate with the experiment made in sea molluscs when hungry animals had significantly higher levels of serotonin and 5‐HT‐SO<sup>4</sup> than their satiated partners [25]. It remains for future investigations to determine whether serotonin sulphate found in plasma has central nervous system origin and the reason for elevated or lowered 5‐HT‐SO<sup>4</sup> levels after serotonergic stimulation [13].

## **Author details**

Raimond Lozda

Address all correspondence to: ofiss@farma.lv

FMS Baltic Ltd, Latvia

## **References**


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[13] Lozda R, Purvinš I. Quantification of serotonin O‐sulfate by LC‐MS/MS method in plasma of healthy volunteers. Frontiers in Pharmacology. 2014;**5**:62

**Author details**

Raimond Lozda

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FMS Baltic Ltd, Latvia

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## **Energy Homeostasis by the Peripheral Serotonergic System**

Hitoshi Watanabe, Michael Rose, Yoshinori Kanayama, Hitoshi Shirakawa and Hisashi Aso

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68831

#### **Abstract**

Energy homeostasis is maintained by balancing energy intake and energy expenditure. In addition to the central nervous system, several hormones play a key role in energy homeostasis in the whole body. In particular, serotonin is regarded as one of the key regulators of energy homeostasis. Serotonin is unique in that it is able to act in both the brain as a neurotransmitter and the peripheral tissue as a gastrointestinal hormone. In the brain, serotonin is thought of as a pharmacological target for anti-obesity treatments because it greatly inhibits meal size and body weight gain. In contrast, serotonin in the periphery has not been targeted as a strategy for anti-obesity treatment, even though almost all of the serotonin produced in the body is produced in the peripheral tissue. Recently, the peripheral serotonergic signal has been shown to regulate glucose and lipid metabolism through autocrine and paracrine signals in energy homeostasis-related tissues, including the pancreatic β cell, liver, white adipose tissue, brown adipose tissue, and skeletal muscle. Thus, it is possible that the serotonergic system in the peripheral tissue is a new therapeutic target for metabolic disease, including obesity and diabetes. Here, we summarize the role of peripheral serotonin in the regulation of energy homeostasis.

**Keywords:** peripheral serotonin, energy homeostasis, obesity, pancreatic β cell, adipose tissue, skeletal muscle

## **1. Introduction**

Serotonin is a monoaminergic neurotransmitter that modulates central and peripheral functions. Serotonin has an association with food intake, sleep, anxiety, sexual behavior, and mood in the central nervous system, and about 2% of the body's serotonin is stored here. On the other hand, around 98% of the body's serotonin is found peripherally, where it functions

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

as a peripheral hormone. It affects vasoconstriction, intestinal motility, primary hemostasis, liver repair, and the control of the T-cell-mediated immune system [1–4].

The synthesis of serotonin from tryptophan begins with the enzyme tryptophan hydroxylase (TPH), which is also the rate-limiting enzyme in its biosynthesis. It is reported that TPH has two isoforms, TPH1 and TPH2 [5]. TPH1 mainly exists in the pineal gland, thymus, spleen, and enterochromaffin cells of the gastrointestinal tract. TPH2 is found only in neuronal cells, such as in the raphe nuclei of the brain stem. Moreover, serotonin is thought not to be able to pass the blood-brain barrier. Therefore, there are thought to be two independent serotonin systems in the body: one in the central nervous system and the other in the periphery.

Since serotonin has been shown to affect fat metabolism and feeding behavior, through independent molecular mechanisms in *Caenorhabditis elegans* [6], serotonin has therefore been suggested to contribute to energy homeostasis with independent modulation from the central nervous system. There are several peripheral tissue serotonin receptors (Htr's), and TPH1 has been shown to be expressed in peripheral tissues, which are related to energy metabolism of not only the gut but also pancreatic β cells and adipose tissue [7, 8]. The roles of serotonin in energy metabolism in these tissues have been further exposed after these discoveries. In the following section, the function of serotonin in peripheral tissues is summarized (**Figure 1**).

**Figure 1.** Role of serotonin in peripheral tissues related to energy homeostasis. Peripheral serotonin is mainly provided from the gut and regulates glucose and lipid metabolism through endocrine, autocrine, and paracrine matter. Gutderived serotonin suppresses glucose uptake and promotes gluconeogenesis through Htr2b. Serotonin regulates proliferation and insulin secretion in pancreatic β cell through several Htr's. Serotonin is also provided from adipocyte and may increase energy storage and adipogenesis in WAT and inhibit adaptive thermogenesis in BAT. BAT, brown adipose tissue; Htr, serotonin receptor; TPH, tryptophan hydroxylase; and WAT, white adipose tissue.

## **2. Role of serotonin in insulin secretion**

## **2.1. Insulin and glucose metabolism**

as a peripheral hormone. It affects vasoconstriction, intestinal motility, primary hemostasis,

The synthesis of serotonin from tryptophan begins with the enzyme tryptophan hydroxylase (TPH), which is also the rate-limiting enzyme in its biosynthesis. It is reported that TPH has two isoforms, TPH1 and TPH2 [5]. TPH1 mainly exists in the pineal gland, thymus, spleen, and enterochromaffin cells of the gastrointestinal tract. TPH2 is found only in neuronal cells, such as in the raphe nuclei of the brain stem. Moreover, serotonin is thought not to be able to pass the blood-brain barrier. Therefore, there are thought to be two independent serotonin

systems in the body: one in the central nervous system and the other in the periphery.

Since serotonin has been shown to affect fat metabolism and feeding behavior, through independent molecular mechanisms in *Caenorhabditis elegans* [6], serotonin has therefore been suggested to contribute to energy homeostasis with independent modulation from the central nervous system. There are several peripheral tissue serotonin receptors (Htr's), and TPH1 has been shown to be expressed in peripheral tissues, which are related to energy metabolism of not only the gut but also pancreatic β cells and adipose tissue [7, 8]. The roles of serotonin in energy metabolism in these tissues have been further exposed after these discoveries. In the following section, the function of serotonin in peripheral tissues is summarized (**Figure 1**).

**Figure 1.** Role of serotonin in peripheral tissues related to energy homeostasis. Peripheral serotonin is mainly provided from the gut and regulates glucose and lipid metabolism through endocrine, autocrine, and paracrine matter. Gutderived serotonin suppresses glucose uptake and promotes gluconeogenesis through Htr2b. Serotonin regulates proliferation and insulin secretion in pancreatic β cell through several Htr's. Serotonin is also provided from adipocyte and may increase energy storage and adipogenesis in WAT and inhibit adaptive thermogenesis in BAT. BAT, brown

adipose tissue; Htr, serotonin receptor; TPH, tryptophan hydroxylase; and WAT, white adipose tissue.

liver repair, and the control of the T-cell-mediated immune system [1–4].

186 Serotonin - A Chemical Messenger Between All Types of Living Cells

Insulin is secreted from pancreatic β cell and plays a key role in glucose homeostasis. Generally, insulin regulates plasma glucose level by suppression of gluconeogenesis in the liver and induces glucose uptake in the skeletal muscle and adipose tissue. Obesity induces insulin resistance in these tissues and glucose intolerance. To compensate for insulin dysfunction, the β cells increase their mass and secretion of insulin. The failure of compensation for this insulin resistance eventually results in type 2 diabetes.

Recently, serotonin has been implicated insulin secretion in the β cell. Pancreatic β cells express both Tph1 and Tph2 and synthesize serotonin [7, 9]. Indeed, mice lacking Tph1 (Tph1−/−) have impaired insulin secretion and are characterized as mildly diabetic [9]. Additionally, it has been reported that serotonin injection elevates plasma insulin levels [10, 11]. Therefore, serotonin might regulate insulin secretion from β cells by both local and systemic actions.

## **2.2. Insulin secretion by intracellular serotonin function**

Serotonin is synthesized and localized within β cell and co-released with insulin following stimulation by glucose [12, 13]. Tph1−/− mice have a normal pancreas mass and islet size. On the other hand, the level of serotonin in the pancreas is decreased by 90% compared with that of wild-type mice [9]. Insulin secretion in the Tph1−/− mice is suppressed, and high blood glucose and insulin resistance are observed. The adjustment of insulin secretion of this animal model has connection with GTPases (Rab3a and Rab27a) and serotonylation. The concentration of intracellular Ca2+ is raised by glucose stimulation that activates transglutaminase (TGase). The serotonylation of GTPases is promoted by TGase, and exocytosis of insulin granule is induced. TGase2−/− mice deteriorate β cell function as well as that of Tph1−/− mice [14]. Consequently, these data suggest that intracellular serotonin controls glucose-stimulated insulin secretion (GSIS) through modification of GTPase.

#### **2.3. Insulin secretion by extracellular serotonin function**

### *2.3.1. Insulin secretion by Htr3*

It has been reported that extracellular serotonin regulates insulin secretion through several Htr's. First of all, Htr3 is a ligand-gated cation channel [15]. Htr3 deletion mice have normal β-cell mass and amount of insulin. There is no change when Htr3 deletion mice are fed normal-fat diet, but impaired insulin secretion and glucose intolerance are shown when a high-fat diet (HFD) is fed. The islets derived from β-cell-specific Tph1 deletion mice show the same impaired insulin secretion as seen in Htr3 deletion mice, and these recover following serotonin treatment. However, the islets derived from Htr3 deletion mice do not recover. Thus, Htr3 is thought to be necessary in order to maintain normal GSIS from the β cell by serotonin.

#### *2.3.2. Insulin secretion by Htr2b*

In addition, other research has determined that Htr2b, G-protein-coupled receptor, also has an impact on GSIS [16]. In human and mouse islets, Htr2b is expressed in β cells but not in α cells. Htr2b knockdown depresses GSIS. Alpha-methylserotonin maleate salt (an Htr2 agonist) increases GSIS in wild-type INS-1 cells, but the effect of this drug does not show itself in Htr2b knockdown INS-1 cells.

## *2.3.3. Insulin secretion by Htr2c*

In contrast, Htr2c was reported to inhibit insulin secretion from pancreatic β cells in a mouse model of diabetes [17]. Htr2c expression is increased in pancreatic islets of db/db mice compared with that of lean mice. Treatment with an Htr2c antagonist increases insulin secretion from pancreatic islets isolated from db/db mice in a dose-dependent manner. This implies that Htr2c controls insulin secretion in diabetic subjects.

#### *2.3.4. Insulin secretion by serotonin transporter*

Furthermore, GSIS is obstructed by selective serotonin reuptake inhibitors (SSRIs) [18]. Shortterm treatment with SSRIs increases Ser/Thr phosphorylation of IRS-2 and inhibits IRS-2 functions and results in impaired GSIS from murine pancreatic islets. Long-term treatment with SSRIs induces ER stress and cellular apoptosis.

As a result, the former study shows that GSIS is adjusted through the Htr signal and the serotonin transporter, by both extracellular serotonin and intracellular serotonin.

#### *2.3.5. Role of serotonin in insulin function during pregnancy*

Pregnancy dramatically changes maternal metabolism. In order to maintain the flow of nutrition to fetus, insulin resistance in the mother increases, resulting in an increasing demand for insulin. In order to compensate for this, the mother enlarges the mass of the β cells and increases the secretion of insulin. This change in insulin secretion during gestation is intimately related to the synthesis of serotonin through lactogenic signaling [9]. Lactogenic signaling is increased during pregnancy (though prolactin and placental lactogen), which raises Tph1 expression in pancreatic β cells and enhances serotonin synthesis. Serotonin in islets regulates insulin function in a paracrine-autocrine fashion during pregnancy. The expression of Htr2b rises in β cells during the pregnancy period, and this returns to normal levels after the delivery of the young. Serotonin is increased during pregnancy, which raises β-cell proliferation and mass through the Htr2b signal. On the other hand, there has been shown to be an elevation in the expression of Htr1d in pancreatic β cells at the end of pregnancy and postpartum. After that, Htr1d restrains the proliferation of β cells.

In addition to the regulation of β-cell mass through Htr2b and Htr1d during pregnancy, insulin release is increased through Htr3 signaling as well in mice on a high-fat diet [19]. Because of the impaired insulin secretion, Htr3−/− mice demonstrate glucose intolerance during pregnancy. In conclusion, β-cell mass and function during pregnancy are controlled by serotonin through several Htr's.

## **3. Role of serotonin in the liver**

*2.3.2. Insulin secretion by Htr2b*

188 Serotonin - A Chemical Messenger Between All Types of Living Cells

knockdown INS-1 cells.

proliferation of β cells.

*2.3.3. Insulin secretion by Htr2c*

that Htr2c controls insulin secretion in diabetic subjects.

*2.3.5. Role of serotonin in insulin function during pregnancy*

*2.3.4. Insulin secretion by serotonin transporter*

SSRIs induces ER stress and cellular apoptosis.

In addition, other research has determined that Htr2b, G-protein-coupled receptor, also has an impact on GSIS [16]. In human and mouse islets, Htr2b is expressed in β cells but not in α cells. Htr2b knockdown depresses GSIS. Alpha-methylserotonin maleate salt (an Htr2 agonist) increases GSIS in wild-type INS-1 cells, but the effect of this drug does not show itself in Htr2b

In contrast, Htr2c was reported to inhibit insulin secretion from pancreatic β cells in a mouse model of diabetes [17]. Htr2c expression is increased in pancreatic islets of db/db mice compared with that of lean mice. Treatment with an Htr2c antagonist increases insulin secretion from pancreatic islets isolated from db/db mice in a dose-dependent manner. This implies

Furthermore, GSIS is obstructed by selective serotonin reuptake inhibitors (SSRIs) [18]. Shortterm treatment with SSRIs increases Ser/Thr phosphorylation of IRS-2 and inhibits IRS-2 functions and results in impaired GSIS from murine pancreatic islets. Long-term treatment with

As a result, the former study shows that GSIS is adjusted through the Htr signal and the sero-

Pregnancy dramatically changes maternal metabolism. In order to maintain the flow of nutrition to fetus, insulin resistance in the mother increases, resulting in an increasing demand for insulin. In order to compensate for this, the mother enlarges the mass of the β cells and increases the secretion of insulin. This change in insulin secretion during gestation is intimately related to the synthesis of serotonin through lactogenic signaling [9]. Lactogenic signaling is increased during pregnancy (though prolactin and placental lactogen), which raises Tph1 expression in pancreatic β cells and enhances serotonin synthesis. Serotonin in islets regulates insulin function in a paracrine-autocrine fashion during pregnancy. The expression of Htr2b rises in β cells during the pregnancy period, and this returns to normal levels after the delivery of the young. Serotonin is increased during pregnancy, which raises β-cell proliferation and mass through the Htr2b signal. On the other hand, there has been shown to be an elevation in the expression of Htr1d in pancreatic β cells at the end of pregnancy and postpartum. After that, Htr1d restrains the

In addition to the regulation of β-cell mass through Htr2b and Htr1d during pregnancy, insulin release is increased through Htr3 signaling as well in mice on a high-fat diet [19]. Because of the impaired insulin secretion, Htr3−/− mice demonstrate glucose intolerance during pregnancy.

tonin transporter, by both extracellular serotonin and intracellular serotonin.

## **3.1. The liver and glucose metabolism**

The liver has an important role in postprandial nutrient metabolism and in response to food deprivation. In particular, the liver maintains blood glucose levels through degradation of glycogen and gluconeogenesis in the fasted state and through glucose uptake in the fed state. It is known that diabetes is caused by an increase of gluconeogenesis and decline of glucose uptake in the liver. Moreover, the liver also controls the concentration of blood cholesterol and triglycerides. It is suggested that hormones such as insulin and glucagon mainly signal these liver functions.

Serotonin is a gastrointestinal hormone and is directly able to regulate the liver, as it is known that serotonin mediates liver regeneration [20]. Although there is still room for debate, several studies report that serotonin has a connection with glucose and lipid metabolism in the liver.

### **3.2. Serotonin and gluconeogenesis**

Sumara et al. revealed that gut-derived serotonin (GDS) increased gluconeogenesis in the liver through Htr2b [8]. Plasma glycerol, produced by adipose tissue and used for gluconeogenesis, is not increased in gut-specific Tph1 knockout mice during food deprivation, though that is increased in fasted wild-type mice. Additionally, the fat-specific Htr2b knockout mice do not also show an increase in plasma glycerol levels in these fasted mice. Mice lacking Tph1 in the gut demonstrate a reduction in hepatic glucose production during hyperinsulinemic-euglycemic clamps and a decrease in plasma glucose levels during pyruvate tolerance tests. Liver-specific Htr2b knockout mice also show similar phenotype as gut-specific Tph1 knockout mice in glucose metabolism. These data support the idea that serotonin signals play an important role in the control of gluconeogenesis in the liver through Htr2b signaling. Consequently, it is suggested that serotonin provides glycerol to the liver from the adipose tissue through Htr2b and thereby contributes to gluconeogenesis in the liver.

#### **3.3. Serotonin and glucose uptake in the liver**

Sumara et al. also report that hepatic glucose uptake decreases in liver-specific Htr2b deletion mice compared with wild-type mice. This is because serotonin is related to the degradation of glucose transporter 2 [8]. Nevertheless, not all studies agree. Injection of serotonin does not impact on the uptake of 2-deoxy-glucose in the liver of fasted mice [10]. Additionally, in an experiment using conscious dogs, portal vein injection of serotonin induced hepatic glucose uptake during a hyperinsulinemic-euglycemic clamp [21]. Agonists of Htr 1/2a reduce blood glucose and increase hepatic glycogen after oral glucose loading. The same study also reported that these agonists stimulate glycogen synthesis in freshly isolated hepatocytes. Furthermore, serotonin inhibits glycogen synthesis at micromolar concentrations but stimulates it at nanomolar concentrations in hepatocytes [22]. Thus, there are several reports on the control of hepatic glucose uptake by serotonin. It could be argued that the results vary according to the method used: genetic study or in vivo treatment study. Further progress in this field is expected.

#### **3.4. Serotonin and enterohepatic circulation of bile acids**

Bile acids are produced from the gallbladder and are deposited into the duodenum following feeding. They are associated with the absorption of nutrients and especially lipids. Nowadays, there is discussion about the role of bile acids with respect to glucose, lipid, and energy metabolism. It is suggested that activation of the farnesoid X receptor (FXR), a bile acid receptor, stimulates the liver concentrations of glycogen [23, 24]. In addition, hepatic triglyceride accumulation, very low-density lipoprotein (VLDL) secretion, and the elevation of serum triglyceride in mouse models of hypertriglyceridemia are impaired by bile acid cholic acid. In brown adipose tissue, administration of bile acids to mice raises energy consumption, preventing obesity and insulin resistance by inducing cAMP-dependent thyroid hormoneactivating enzyme type 2 iodothyronine deiodinase (D2) [25, 26].

In the enterohepatic circulation, bile acids are mainly reabsorbed from the ileum and return to the liver through the portal vein. The hepatocytes take up about 80% of this, and the remainder enters the general circulation. Serotonin is known to signal the enterohepatic circulation of bile acids. By stimulating the contraction of the smooth muscle in the gallbladder, serotonin induces the excretion of bile acids in a direct manner from the gallbladder into the duodenum [27, 28]. In addition to the excretion of bile acids from the gallbladder, serotonin is reported to enhance reabsorption of bile acids from the ileum and raise the level of plasma bile acids (**Figure 2**) [10]. Serotonin injection has been shown to cause an elevation of the expression of the apical sodium-dependent bile acid transporter (ASBT), which actively causes the reabsorption of bile acids from the lumen of the intestine into the body and decreases the content of bile acids in the feces. However, ASBT expression is negatively regulated by bile acids and FGF15 through the FXR-FGF15 signaling pathway. These data suggest the possibility that serotonin may increase ASBT expression through the FXR-FGF15-independent pathway.

## **3.5. Serotonin and lipid in the liver and the circulation**

It has been suggested that serotonin may affect the concentrations of lipid in the liver and blood [10]. In practical terms, plasma triglyceride, cholesterol, and nonesterified fatty acid concentrations are reduced following serotonin injection. The same report suggested that the level of the concentration of cholesterol in the liver was increased following the passage of 60 min after serotonin treatment. On the other hand, there was a reduction of the plasma concentration of cholesterol at the same time. These data show that the intake of the cholesterol by the liver from the blood through serotonin stimulation may cause a decrease in the plasma cholesterol concentration.

Besides this, Haub et al. have suggested that serotonin may raise the fat concentration of the liver [29]. Comparing lean control mice, there is an increase of duodenal Htr3a protein

**Figure 2.** Upregulation of bile acid turnover by peripheral serotonin. Peripheral serotonin induces excretion of bile acids from the gallbladder to the duodenum, reabsorption of bile acids in the ileum, and an elevation of the concentration of bile acids in the plasma of the portal vein (Ref. [1]). BAs, bile acids.

expression and plasma serotonin levels in ob/ob mice. The fat concentration, inflammation, and cell necrosis in the liver of ob/ob mice are all decreased following treatment with an Htr3 antagonist, this by means of reducing the elevated serotonin levels in the intestine. As a result, serotonin is indicated to regulate lipid metabolism functions in the liver, both directly and indirectly.

## **4. Role of serotonin in the adipose tissue**

### **4.1. Adipose tissue and energy metabolism**

Furthermore, serotonin inhibits glycogen synthesis at micromolar concentrations but stimulates it at nanomolar concentrations in hepatocytes [22]. Thus, there are several reports on the control of hepatic glucose uptake by serotonin. It could be argued that the results vary according to the method used: genetic study or in vivo treatment study. Further progress in

Bile acids are produced from the gallbladder and are deposited into the duodenum following feeding. They are associated with the absorption of nutrients and especially lipids. Nowadays, there is discussion about the role of bile acids with respect to glucose, lipid, and energy metabolism. It is suggested that activation of the farnesoid X receptor (FXR), a bile acid receptor, stimulates the liver concentrations of glycogen [23, 24]. In addition, hepatic triglyceride accumulation, very low-density lipoprotein (VLDL) secretion, and the elevation of serum triglyceride in mouse models of hypertriglyceridemia are impaired by bile acid cholic acid. In brown adipose tissue, administration of bile acids to mice raises energy consumption, preventing obesity and insulin resistance by inducing cAMP-dependent thyroid hormone-

In the enterohepatic circulation, bile acids are mainly reabsorbed from the ileum and return to the liver through the portal vein. The hepatocytes take up about 80% of this, and the remainder enters the general circulation. Serotonin is known to signal the enterohepatic circulation of bile acids. By stimulating the contraction of the smooth muscle in the gallbladder, serotonin induces the excretion of bile acids in a direct manner from the gallbladder into the duodenum [27, 28]. In addition to the excretion of bile acids from the gallbladder, serotonin is reported to enhance reabsorption of bile acids from the ileum and raise the level of plasma bile acids (**Figure 2**) [10]. Serotonin injection has been shown to cause an elevation of the expression of the apical sodium-dependent bile acid transporter (ASBT), which actively causes the reabsorption of bile acids from the lumen of the intestine into the body and decreases the content of bile acids in the feces. However, ASBT expression is negatively regulated by bile acids and FGF15 through the FXR-FGF15 signaling pathway. These data suggest the possibility that serotonin may increase ASBT expression through the FXR-FGF15-independent pathway.

It has been suggested that serotonin may affect the concentrations of lipid in the liver and blood [10]. In practical terms, plasma triglyceride, cholesterol, and nonesterified fatty acid concentrations are reduced following serotonin injection. The same report suggested that the level of the concentration of cholesterol in the liver was increased following the passage of 60 min after serotonin treatment. On the other hand, there was a reduction of the plasma concentration of cholesterol at the same time. These data show that the intake of the cholesterol by the liver from the blood through serotonin stimulation may cause a decrease in the plasma

Besides this, Haub et al. have suggested that serotonin may raise the fat concentration of the liver [29]. Comparing lean control mice, there is an increase of duodenal Htr3a protein

this field is expected.

**3.4. Serotonin and enterohepatic circulation of bile acids**

190 Serotonin - A Chemical Messenger Between All Types of Living Cells

activating enzyme type 2 iodothyronine deiodinase (D2) [25, 26].

**3.5. Serotonin and lipid in the liver and the circulation**

cholesterol concentration.

One of the features of the adipose tissue is to store a huge amount of energy. Adipose tissue can be roughly categorized into two types: white adipose tissue (WAT) and brown adipose tissue (BAT). In addition, more recently, the existence of beige adipose tissue has been noted. This is thought to be derived from WAT and has some of the features of BAT [30]. WAT functions as the main storage of energy in the body, in the form of triglycerides. WAT takes dietary absorbed glucose and lipids from the blood in the fed condition. On the other hand, in the fasted condition, WAT resolves stored triglyceride and releases free fatty acids and glycerol into the blood and supplies energy to the body [31]. The characteristic of BAT, on the other hand, is to have a small fat droplet and a large number of mitochondria, and it is considered as an important internal organ, because it produces heat for temperature homeostasis [30]. Indeed, these two adipose tissues are crucial for energy homeostasis and are intimately associated with the development of metabolic diseases such as obesity and type 2 diabetes.

## **4.2. Role of serotonin in white adipose tissue**

#### *4.2.1. Serotonin and adipocyte differentiation*

An expansion and growth in the number of individual adipocytes is a manifestation of obesity, and peripheral serotonin of adipose tissue origin is an autocrine element that is necessary for the adipocyte differentiation through the Htr2a and Htr2c receptors [32]. Serotonin production has been demonstrated in 3T3-L1 preadipocyte cells, and there was a gradual increase in the expression level of Tph1 protein and the concentration of serotonin after adipogenic induction in the same cells. Comparing wild-type 3T3-L1 preadipocyte cells, adipogenesis in 3T3-L1 preadipocyte cells was associated with a lack of Tph1 after treatment with differentiation-inducing agents. This phenotype in Tph1 mutant cells is recovered following treatment with serotonin. Furthermore, antagonists of Htr2a and Htr2c also inhibit the adipogenesis in 3T3-L1 preadipocyte cells. Additionally, it is suggested that serotonin metabolites operate as endogenous agonists for peroxisome proliferator-activated receptor gamma (PPARg) so that they control adipogenesis by means of directly binding to helix H12 of the PPARg binding site [33]. Consequently, these reports indicated that serotonin directly affects the differentiation from preadipocyte to adipocyte.

### *4.2.2. Regulation of energy homeostasis in white adipose tissue by serotonin*

There is a report that adipocyte-derived serotonin has the important role in energy homeostasis in the whole body [34]. Tph1 expression and serotonin concentrations were increased in epididymal and subcutaneous WAT in a diet-induced obesity mouse model. Intraperitoneal injection of the Tph1 inhibitor PCPA led to a reduction of weight gain and lower adiposity after a high-fat diet (HFD). Surprisingly, treatment of PCPA promotes beige adipogenesis in inguinal WAT by elevating UCP1 and DIO2 expression. Adipocyte-specific Tph1 KO also causes a reduction of bodyweight, an improvement of insulin resistance and beige adipogenesis in inguinal WAT. Therefore, adipocyte-derived serotonin is suggested to play important role both to induce adipogenesis and to maintain the feature and function of WAT.

#### **4.3. Regulation of energy homeostasis in brown adipose tissue by serotonin**

Serotonin is involved in energy homeostasis not only of WAT but also of BAT [35]. In a dietinduced obesity mouse model, Tph1 expression and tissue serotonin concentrations were increased in BAT as well as in WAT. Tph1-deficient mice on a high-fat diet (HFD) are prevented from becoming obese, as well as succumbing to insulin resistance and nonalcoholic fatty liver disease (NAFLD) while expressing energy generation by BAT and exhibiting energy expenditure in whole body. This BAT function in Tph1 KO mice elevated UCP1 dependently. These data are supported by Oh et al. [34]. Tph inhibitor PCPA and LP-533401 promoted the expression of UCP1 and DIO2 in BAT. Furthermore, it is indicated that Htr3 is involved in serotonergic signal to BAT. Tph1 KO mice demonstrate an increased expression of UCP1 and DIO2 of both WAT and BAT, whereas Htr3 KO mice show elevation of these thermogenic gene expressions solely of BAT. Thus, serotonin regulates BAT activation through Htr3. On the contrary, WAT of Htr3 KO mice did not show the beige adipogenesis in inguinal WAT which was observed in Tph1 KO mice. As a result, the serotonin function of WAT is connected to Htr's, for example, Htr2a which associated with adipogenesis, except for Htr3.

## **5. Role of serotonin in the skeletal muscle**

## **5.1. The skeletal muscle and energy metabolism**

as an important internal organ, because it produces heat for temperature homeostasis [30]. Indeed, these two adipose tissues are crucial for energy homeostasis and are intimately associated with the development of metabolic diseases such as obesity and type 2 diabetes.

An expansion and growth in the number of individual adipocytes is a manifestation of obesity, and peripheral serotonin of adipose tissue origin is an autocrine element that is necessary for the adipocyte differentiation through the Htr2a and Htr2c receptors [32]. Serotonin production has been demonstrated in 3T3-L1 preadipocyte cells, and there was a gradual increase in the expression level of Tph1 protein and the concentration of serotonin after adipogenic induction in the same cells. Comparing wild-type 3T3-L1 preadipocyte cells, adipogenesis in 3T3-L1 preadipocyte cells was associated with a lack of Tph1 after treatment with differentiation-inducing agents. This phenotype in Tph1 mutant cells is recovered following treatment with serotonin. Furthermore, antagonists of Htr2a and Htr2c also inhibit the adipogenesis in 3T3-L1 preadipocyte cells. Additionally, it is suggested that serotonin metabolites operate as endogenous agonists for peroxisome proliferator-activated receptor gamma (PPARg) so that they control adipogenesis by means of directly binding to helix H12 of the PPARg binding site [33]. Consequently, these reports indicated that serotonin directly affects the differentiation

There is a report that adipocyte-derived serotonin has the important role in energy homeostasis in the whole body [34]. Tph1 expression and serotonin concentrations were increased in epididymal and subcutaneous WAT in a diet-induced obesity mouse model. Intraperitoneal injection of the Tph1 inhibitor PCPA led to a reduction of weight gain and lower adiposity after a high-fat diet (HFD). Surprisingly, treatment of PCPA promotes beige adipogenesis in inguinal WAT by elevating UCP1 and DIO2 expression. Adipocyte-specific Tph1 KO also causes a reduction of bodyweight, an improvement of insulin resistance and beige adipogenesis in inguinal WAT. Therefore, adipocyte-derived serotonin is suggested to play important

Serotonin is involved in energy homeostasis not only of WAT but also of BAT [35]. In a dietinduced obesity mouse model, Tph1 expression and tissue serotonin concentrations were increased in BAT as well as in WAT. Tph1-deficient mice on a high-fat diet (HFD) are prevented from becoming obese, as well as succumbing to insulin resistance and nonalcoholic fatty liver disease (NAFLD) while expressing energy generation by BAT and exhibiting energy expenditure in whole body. This BAT function in Tph1 KO mice elevated UCP1 dependently. These data are supported by Oh et al. [34]. Tph inhibitor PCPA and LP-533401 promoted the expression of UCP1 and DIO2 in BAT. Furthermore, it is indicated that Htr3 is involved in

role both to induce adipogenesis and to maintain the feature and function of WAT.

**4.3. Regulation of energy homeostasis in brown adipose tissue by serotonin**

**4.2. Role of serotonin in white adipose tissue**

192 Serotonin - A Chemical Messenger Between All Types of Living Cells

*4.2.1. Serotonin and adipocyte differentiation*

from preadipocyte to adipocyte.

*4.2.2. Regulation of energy homeostasis in white adipose tissue by serotonin*

The skeletal muscle is an essential tissue in energy metabolism and glucose utilization, especially during exercise. Slow- and fast-type myosin heavy chain isoforms exist in normal mature muscle fibers. There is a high concentration of mitochondria in slow-type muscle fibers, and it produces energy by oxidative metabolism. On the other hand, glycolysis is utilized by fast-type muscle fibers as the chief adenosine triphosphate (ATP) source [36, 37]. Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1 a (PGC-1a) is confirmed as a nuclear receptor coactivator of PPARγ, and it is a principal physiological controller for slow-type muscle fiber specification [37, 38]. There is a significant impaired glucose tolerance in skeletal muscle-specific PGC-1α knockout mice [39], whereas humans with lower adiposities have a significantly higher percentage of slow-type muscle fibers than obese humans.

## **5.2. Effect of serotonin on glucose uptake and glycolysis in the skeletal muscle**

Some studies report that serotonin increases glucose uptake in the skeletal muscle. Serotonin promotes a fast stimulation in glucose uptake by 50% in both L6 myotubes and independent rat skeletal muscle mediated through the Htr2a receptor [40]. Apart from this, this serotonin function does not depend on the components that participate in the insulin signaling pathway. The other thesis insists that incubation with serotonin induced an increase in 2-deoxyglucose uptake in a concentration-dependent fashion by translocated GLUT4 to the cell membrane [41]. This GLUT4 translocation is thought to be caused by serotonylation of the small GTPase Rab 4.

In addition, serotonin signals 6-phosphofructo-1-kinase (PFK) through the Htr2a. This has been reported as the major rate-limiting enzyme of glycolysis and is related to the entire glycolytic pathway each other in the skeletal muscle [42]. Serotonin provokes PFK from the skeletal muscle via phospholipase C (PLC). The stimulation of PLC in the skeletal muscle promotes the recruitment of protein kinase C (PKC) and calmodulin and the activation of calmodulin kinase II, which connects with PFK upon serotonin action. Thus, serotonin may increase glucose uptake and glycolysis through Htr2a and intracellular serotonylation of Rab 4.

### **5.3. Effect of serotonin on skeletal muscle fiber type**

Obesity induced by feeding a high-fat diet is improved in Tph1 KO mice by increasing beige adipogenesis in WAT and thermogenic gene expressions in BAT. In contrast, Watanabe et al. report that long-term treatment of mice with peripheral serotonin interferes with weight gain, hyperglycemia, and insulin resistance and completely inhibited the enlargement of intra-abdominal adipocytes without having any impacts on food intake when on a high-fat diet, but not on a chow diet [43]. Amazingly, serotonin raises the percentage of slow muscle fibers and reduces the percentage of fast muscle fibers in serotonin-injected mice fed a high-fat diet (**Figure 3**). As a result, serotonin increases energy metabolism, O2 consumption, CO2 production, and the respiratory exchange ratio (RER). The function is caused by increase of PGC-1α expression in

**Figure 3.** Induction of the transformation of skeletal muscle fiber type into slow muscle fiber by serotonin. Serotonin increases the proportion of slow muscle fibers, which have a high concentration of mitochondria and produce energy by oxidative metabolism, in the soleus muscle from mice fed a high-fat diet (Ref. [43]). Serotonin may play an important role in the relief of obesity by accelerating energy consumption in the skeletal muscle. Con, control; G, gastrocnemius muscle; HFD, high-fat diet; NC, normal chow; S, soleus muscle; and Ser, serotonin.

the skeletal muscle. PGC-1α is a major regulator that induces mitochondrial biogenesis and a fiber switch to decelerate muscle fiber type in the skeletal muscle [37, 38]. The fact that PGC-1α mRNA has three isoforms, PGC-1α-a, PGC-1α-b, and PGC-1α-c, has been revealed recently [44]. There was an elevation of the expression of total PGC-1α in serotonin in the soleus muscle of mice on a high-fat diet, although following a significant increase of PGC-1α-b and PGC-1α-c expressions through the Htr2a and Htr7 signaling pathways. Previous reports suggest that an Htr2 agonist led to an increase of PGC-1α promoter activity, and it supports serotonin's PGC-1α expression promoter activity in the skeletal muscle [45].

that long-term treatment of mice with peripheral serotonin interferes with weight gain, hyperglycemia, and insulin resistance and completely inhibited the enlargement of intra-abdominal adipocytes without having any impacts on food intake when on a high-fat diet, but not on a chow diet [43]. Amazingly, serotonin raises the percentage of slow muscle fibers and reduces the percentage of fast muscle fibers in serotonin-injected mice fed a high-fat diet (**Figure 3**).

respiratory exchange ratio (RER). The function is caused by increase of PGC-1α expression in

**Figure 3.** Induction of the transformation of skeletal muscle fiber type into slow muscle fiber by serotonin. Serotonin increases the proportion of slow muscle fibers, which have a high concentration of mitochondria and produce energy by oxidative metabolism, in the soleus muscle from mice fed a high-fat diet (Ref. [43]). Serotonin may play an important role in the relief of obesity by accelerating energy consumption in the skeletal muscle. Con, control; G, gastrocnemius

muscle; HFD, high-fat diet; NC, normal chow; S, soleus muscle; and Ser, serotonin.

consumption, CO2

production, and the

As a result, serotonin increases energy metabolism, O2

194 Serotonin - A Chemical Messenger Between All Types of Living Cells

Serotonin in WAT and BAT has the possibility to reduce energy expenditure, whereas the skeletal muscle may transform skeletal muscle fiber type into slow muscle fiber and increase energy expenditure.

## **6. Will a tomato (with a high serotonin content) a day keep the doctor away?**

A variety of vegetables and fruits contain serotonin [46]. We have also reported that good plant sources of serotonin are the cherry tomato, tomato, kiwi, banana, and potato, using the HPLC-fluorescence detection method (**Table 1**) [47]. We have confirmed that the serum concentrations of serotonin increased in a dose- and time-dependent manner after oral administration and that a serotonin metabolite, 5-hydroxyindole-3-acetic acid, was detected in urine at higher concentrations in treated than in untreated mice [48]. The foods with a high serotonin content may represent excellent dietary sources of serotonin, and serotonin action may well offer new drug strategies for developing therapeutic drugs for the treatment of metabolic diseases such as hyperlipidemia, hypercholesterolemia, diabetes, and obesity. In the future, we may say that a tomato with a high serotonin content a day keeps the doctor away.


**Table 1.** Serotonin levels in common vegetables and fruits in Japan [47].

## **7. Conclusions**

Serotonin in the central nervous system has been studied as good strategy for dealing with obesity since the late twentieth century, because it affects behavior, especially food intake. On the other hand, despite the fact that peripheral tissue has almost all of the serotonin of the whole body, research into the function of serotonin in peripheral tissue has not significantly progressed. Since the beginning of the twenty-first century, the role of the peripheral serotonergic system in energy homeostasis has gradually been clarified and has been noticed as a new treatment target.

Peripheral serotonin is central to the control of energy homeostasis by means of stimulating several organs but especially pancreatic β cells, the liver, white adipose tissue, brown adipose tissue, and the skeletal muscle. These functions of peripheral serotonin are thought to operate through autocrine and paracrine means through at least the 14 Htr's or serotonin transporter.

It is considered that receptor-specific activation or inhibition is a better strategy for the development of drugs from this knowledge. Nevertheless, it has been reported that peripheral serotonin acts differently in different tissues, by functioning through different receptors in different cells. Thus, peripheral serotonin functions operate in a very complex manner when peripheral serotonin is considered as a therapeutic agent for the whole body. Indeed, there are still many points that need unraveling. For instance, serotonin in WAT and BAT regulates energy expenditure, while serotonin in the skeletal muscle increases glucose uptake and the proportion of slow muscle fibers and raises energy expenditure. This question may be resolved by using cell-specific deletion of Htr's and Tph1 mice. The solution of this question is expected to develop soon, because we anticipate that affecting energy homeostasis using the peripheral serotonergic system will eventually be a new treatment strategy for metabolic disease.

## **Acknowledgements**

This work was supported by a grant for Research Project on Development of Agricultural Products and Foods with Health-promoting benefits (NARO) from the Ministry of Agriculture, Forestry and Fisheries (to HA); Grant-in-Aid for challenging Exploratory Research (16K15021) from the Ministry of Education, Culture, Sports, Science and Technology (to HA); and the Japan Society for the Promotion of Science KAKENHI grants 16K00849 and 16J08117 (to HW).

## **Author details**

Hitoshi Watanabe1 , Michael Rose2 , Yoshinori Kanayama<sup>3</sup> , Hitoshi Shirakawa3 and Hisashi Aso3 \*

\*Address all correspondence to: asosan@bios.tohoku.ac.jp


## **References**

**7. Conclusions**

196 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Acknowledgements**

**Author details**

Hitoshi Watanabe1

\*

1 Kanazawa University, Japan

3 Tohoku University, Japan

2 Aberystwyth University, Wales

Hisashi Aso3

, Michael Rose2

\*Address all correspondence to: asosan@bios.tohoku.ac.jp

Serotonin in the central nervous system has been studied as good strategy for dealing with obesity since the late twentieth century, because it affects behavior, especially food intake. On the other hand, despite the fact that peripheral tissue has almost all of the serotonin of the whole body, research into the function of serotonin in peripheral tissue has not significantly progressed. Since the beginning of the twenty-first century, the role of the peripheral serotonergic system in energy homeostasis has gradually been clarified and has been noticed as a new treatment target. Peripheral serotonin is central to the control of energy homeostasis by means of stimulating several organs but especially pancreatic β cells, the liver, white adipose tissue, brown adipose tissue, and the skeletal muscle. These functions of peripheral serotonin are thought to operate through autocrine and paracrine means through at least the 14 Htr's or serotonin transporter. It is considered that receptor-specific activation or inhibition is a better strategy for the development of drugs from this knowledge. Nevertheless, it has been reported that peripheral serotonin acts differently in different tissues, by functioning through different receptors in different cells. Thus, peripheral serotonin functions operate in a very complex manner when peripheral serotonin is considered as a therapeutic agent for the whole body. Indeed, there are still many points that need unraveling. For instance, serotonin in WAT and BAT regulates energy expenditure, while serotonin in the skeletal muscle increases glucose uptake and the proportion of slow muscle fibers and raises energy expenditure. This question may be resolved by using cell-specific deletion of Htr's and Tph1 mice. The solution of this question is expected to develop soon, because we anticipate that affecting energy homeostasis using the peripheral

serotonergic system will eventually be a new treatment strategy for metabolic disease.

This work was supported by a grant for Research Project on Development of Agricultural Products and Foods with Health-promoting benefits (NARO) from the Ministry of Agriculture, Forestry and Fisheries (to HA); Grant-in-Aid for challenging Exploratory Research (16K15021) from the Ministry of Education, Culture, Sports, Science and Technology (to HA); and the Japan Society for the Promotion of Science KAKENHI grants 16K00849 and 16J08117 (to HW).

, Yoshinori Kanayama<sup>3</sup>

, Hitoshi Shirakawa3

and


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