Preface

The inhibition and excitation of neural networks form the basis of information transfer in the mammalian central nervous system. The key to most complex brain processes lies in the adequate balance between inhibitory and excitatory actions of amino acid neurotransmit‐ ters. The dominant inhibitory neurotransmitter is gamma-aminobutyric acid (GABA), while the principal excitatory neurotransmitter is glutamate. Furthermore, an increase or a de‐ crease in their activity is associated with a number of neurological and psychiatric diseases. The following seven chapters provide the reader with an overview of the latest research/ review data on GABA/glutamate system organization and function, receptor structure, sub‐ types and their ligands, as well as translational approaches and clinical implications.

The introductory chapter describes the basic functioning and the relevance of GABA/gluta‐ mate balance in the normal brain functioning, focusing on the role of their receptors. The second chapter, titled "Early Life Experience: Maternal Separation, Involvement of GABA and Glutamate Transporters," summarizes scientific data and opinion regarding maternal separation as a model of early life experience of postnatal stress, with focus on the involve‐ ment of GABA and glutamate transporters. The third chapter, "Notch Signaling in the As‐ troglial Phenotype: Relevance to Glutamatergic Transmission," addresses issues related to the role of notch signaling in radial glia, with emphasis on glial glutamate transporter regu‐ lation as a key element in the molecular mechanisms that support glutamatergic neurotrans‐ mission. "Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth in *Arabidopsis thaliana*" presents original data from a pharmacology-based functional study of ionotropic glutamate receptors (iGluRs) in plants, suggesting a correla‐ tion between the putative iGluR-like channel function and the modification of root growth and development in the Arabidopsis roots. This is followed by a very up-to-date review ti‐ tled "GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets," in which the authors address not only the role of both neurotransmitters during development but also the extra-neuronal glutamatergic and GABAergic signaling in pancreatic islets of Langerhans, and possible associations with type 1 diabetes mellitus. Further clinical implica‐ tions are discussed in the sixth chapter titled "Antagonists of Ionotropic Receptors for the Inhibitory Neurotransmitter GABA: Therapeutic Indications." The authors examine the an‐ tagonism of ionotropic GABA receptors, reflecting on the use of GABA receptor antagonists in the last 10 years and their possible therapeutic potential. Finally, the chapter "Clinical Ap‐ plications of MR Spectroscopy (MRS) in Neurosciences" delivers a detailed description of the methodology and the relevance of MRS as an important diagnostic and research tool in clinical neuroscience.

Therefore, the book offers readers a rich collection of data regarding current and future ap‐ plications of GABA and glutamate neurotransmission, including promising research strat‐ egies and potential clinical benefits.

#### **Janko Samardzic MD, PhD, Ass. Prof.**

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: GABA/Glutamate Balance: A Key**

**Introductory Chapter: GABA/Glutamate Balance: A Key** 

The basis of information transfer in the mammalian central nervous system (CNS) consists of excitation and inhibition of neuronal networks. The messengers responsible for propagating these excitatory and inhibitory actions are amino acid neurotransmitters [1]. The principal excitatory neurotransmitter is glutamate, while the principle inhibitory neurotransmitter is gamma-aminobutyric acid (GABA). Coordination between these two principal neurotransmitters ensures adequate rhythmic activity, which may involve either a single neuron or multiple neuronal groups, thus altering synaptic plasticity and ensuring a normal functioning of CNS [2]. As this spatiotemporal framework of different patterns in neural oscillations is essential for information processing throughout the brain [3], the deviations in normal activity of either system or their interactions are associated with a number of neurological and

The GABA/glutamate functional balance could be achieved by homeostatic control of presynaptic elements such as glutamate and GABA release, which could be the result of changes in their metabolism (synthesis or degradation involving various enzymes), compartmentation, and recycling (involving plasma transporters) and in the amounts of transmitters available for release from synaptic vesicles (involving vesicular transporters). However, it is generally considered that homeostatic plasticity mechanisms in the brain are mediated primarily by regulation of expression and function of glutamate and

> © 2016 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.

© 2018 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.

DOI: 10.5772/intechopen.74023

**for Normal Brain Functioning**

**for Normal Brain Functioning**

Jasna Jancic and Dubravka Svob Strac

Jasna Jancic and Dubravka Svob Strac

http://dx.doi.org/10.5772/intechopen.74023

**1. Introduction**

psychiatric diseases [4].

GABA receptors [5].

Janko Samardzic, Dragana Jadzic, Boris Hencic,

Janko Samardzic, Dragana Jadzic, Boris Hencic,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Institute of Pharmacology, Clinical Pharmacology and Toxicology Medical Faculty, University of Belgrade, Serbia

**Chapter 1**

**Provisional chapter**

### **Introductory Chapter: GABA/Glutamate Balance: A Key for Normal Brain Functioning Introductory Chapter: GABA/Glutamate Balance: A Key for Normal Brain Functioning**

DOI: 10.5772/intechopen.74023

Janko Samardzic, Dragana Jadzic, Boris Hencic, Janko Samardzic, Dragana Jadzic, Boris Hencic,

Jasna Jancic and Dubravka Svob Strac Jasna Jancic and Dubravka Svob Strac

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74023

### **1. Introduction**

Therefore, the book offers readers a rich collection of data regarding current and future ap‐ plications of GABA and glutamate neurotransmission, including promising research strat‐

**Janko Samardzic MD, PhD, Ass. Prof.**

Medical Faculty, University of Belgrade, Serbia

Institute of Pharmacology, Clinical Pharmacology and Toxicology

egies and potential clinical benefits.

VIII Preface

The basis of information transfer in the mammalian central nervous system (CNS) consists of excitation and inhibition of neuronal networks. The messengers responsible for propagating these excitatory and inhibitory actions are amino acid neurotransmitters [1]. The principal excitatory neurotransmitter is glutamate, while the principle inhibitory neurotransmitter is gamma-aminobutyric acid (GABA). Coordination between these two principal neurotransmitters ensures adequate rhythmic activity, which may involve either a single neuron or multiple neuronal groups, thus altering synaptic plasticity and ensuring a normal functioning of CNS [2]. As this spatiotemporal framework of different patterns in neural oscillations is essential for information processing throughout the brain [3], the deviations in normal activity of either system or their interactions are associated with a number of neurological and psychiatric diseases [4].

The GABA/glutamate functional balance could be achieved by homeostatic control of presynaptic elements such as glutamate and GABA release, which could be the result of changes in their metabolism (synthesis or degradation involving various enzymes), compartmentation, and recycling (involving plasma transporters) and in the amounts of transmitters available for release from synaptic vesicles (involving vesicular transporters). However, it is generally considered that homeostatic plasticity mechanisms in the brain are mediated primarily by regulation of expression and function of glutamate and GABA receptors [5].

© 2018 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.

© 2016 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. GABA and its receptors**

Every third chemical synapse in the brain uses neurotransmitter GABA as an integral part of the neurotransmission process. GABA mediates its effects via two types of receptors: ionotropic GABAA and metabotropic GABAB receptors [6]. Although a third type of GABA receptor with pharmacological specificities has been identified, the term GABAС has not received broad consensus among experts. Additionally, the International Union of Basic and Clinical Pharmacology (IUPHAR) has classified GABAC as a type of GABAA receptor [7].

On the other hand, GABAB metabotropic receptors, characterized by stereoselective ligand (−) baclofen, belong to the seven transmembrane G-protein-coupled receptor superfamily. They are pre- and postsynaptic G-protein-coupled receptors that negatively modulate adenylyl cyclase and inositol triphosphate synthesis. Heterodimeric structure as a result of GABAB1 and GABAB2 subunit assembly is necessary for appropriate GABAB receptor function. The extracellular domain of the GABAB1 subunit contains GABA-binding site, whereas GABAB2 subunit is important for the interaction with the G-proteins. GABAB receptor activation produces a cascade of signals that result in activation and/or inhibition of voltage-dependent calcium channels. GABAB receptor is located both centrally and peripherally, particularly in the thalamus, brain stem nuclei, and spinal cord. Depending on the localization of GABA<sup>B</sup> receptors, GABA-mediated inhibitory influences can be potentiated (postsynaptic receptors, presynaptic heteroreceptors on glutamatergic endings) or reduced (autoreceptors) [13, 14]. GABAB receptor function affects behavior, learning, and memory, and therefore their pharmacological targeting may be beneficial in various neuropsychiatric disorders [15, 16].

Introductory Chapter: GABA/Glutamate Balance: A Key for Normal Brain Functioning

http://dx.doi.org/10.5772/intechopen.74023

3

Glutamate, the most abundant neurotransmitter in vertebrates and precursor of GABA, is present in over 90% of all synaptic connections in the human brain and is essential for a wide variety of functions [17]. Over 20 types of mammalian glutamate receptors exist, generally classified into two main categories: voltage-sensitive (ionotropic) and ligand-sensitive (metabotropic) receptors. Ionotropic receptor channels are formed from various protein subunits assembled in heterotetrameric or homotetrameric receptors. The three types of ionotropic receptors are N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainic acid receptors. The discovery of these agonists, after which the receptors were originally named, led to the detection of other receptor agonists and antagonists [18]. The binding of glutamate to NMDA receptors (NMDARs) results in the opening of a nonselective cation channel. The opening and closing of the channel are primarily gated by ligand binding but are also voltage-dependent. Extracellular magnesium and zinc ions can bind to specific sites on the receptor, blocking the passage of other cations through the open ion channel. However, depolarization of the neuronal cell dislodges and repels these ions from the pore, therefore allowing a voltage-dependent influx of sodium and calcium ions and efflux of potassium ions [19]. The NMDA receptor is primarily a ligand-gated channel, but it does display weaker voltage-dependent modulation of the ligand-dependent gating. NMDA requires co-activation by two ligands: glutamate and either d-serine or glycine [20]. Furthermore, NMDA receptors are divided into subtypes, depending on their intracellular protein structure, NR1, NR2, and NR3. NR1 consists of eight different subunits originating from a single gene via alternative splicing. NR2 has four subunits (A–D), and NR3 has two subunits (A and B). NMDA receptors are highly expressed on both neurons and astrocytes [21]. NMDA signaling is crucial for learning, memory, recovery from injury, and brain plasticity. It is especially important for proper functioning of the hippocampus [22]. In pathological circumstances, overactivation of NMDA receptors can lead to excitotoxicity, involved in some neurodegen-

erative disorders such as Alzheimer's, Parkinson's, and Huntington's disease [23–25].

**3. Glutamate and its receptors**

GABAA receptors generally contain chloride ion channels but can, in varying degrees, also contain calcium, sodium, and potassium channels. GABAA receptors mediate the majority of GABA inhibitory actions in the CNS [4]. They are pentameric transmembrane receptors made up of 5 subunit proteins that form an ion channel selectively permeable to chloride anions. Although mainly localized on postsynaptic membranes, they can also be found extrasynaptically, especially GABAA receptors containing α4, α5, or α6 subunits [8]. Unexpectedly, GABAA receptors have also been found on glial cells, potentially providing adaptational support for adjacent neurons [9]. Activation of GABAA receptors leads to a change in the conformational state of associated ion channels, resulting in increased permeability to chloride ions. GABAergic mechanisms are also involved in metabolic processes [10], and a negative correlation between the intensity of GABAergic neurotransmission and metabolic processes in cerebral tissue has been established. So far, 19 subunits of GABAA receptors have been cloned and classified into several structurally related subfamilies (α 1–6, β 1–3, γ 1–3, δ, ε, θ, π, ρ 1–3). The most frequently found GABAA receptor composition is an aggregate composed of two α, two β, and one γ subunit [4]. Receptors that, in addition to two α and two β subunits, contain some other non-γ subunit are rare. Receptors composed only of α and β isoforms also exist. The subunit composition determines the functional and pharmacological properties of GABAA receptors. For example, α1 GABAA receptors mediate sedative and anticonvulsant actions, whereas the α2 subunit is responsible for anxiolytic action of benzodiazepines. Zolpidem, a commonly prescribed sedative for sleep initiation, has a high binding affinity for GABAA receptors containing the α1 subunit [11].

GABA action through GABAA receptors results in chloride channel opening and increased postsynaptic membrane permeability. In addition to the well-determined benzodiazepine binding site, at least 13 different and structurally specific sites on the GABAA receptors have been identified: (1) GABA and other agonist-binding sites, as well as competitive antagonists; (2) picrotoxin site near ion channel; (3) barbiturates binding site; (4) neuroactive steroids binding site; (5) ethanol binding site; (6) inhalation anesthetics stereoselective binding sites; (7) furosemide diuretic binding site; (8) Zn2+ ion binding site; (9) other divalent cation binding sites; (10) La3+ ions site; (11) sites for phosphorylation of specific protein kinases; (12) phospholipid-binding sites; and (13) sites involved in interaction of GABAA receptor and microtubules, which promote receptor grouping on postsynaptic membranes [12]. Modulators of GABAA receptor complex interact with these binding sites in three possible ways: positive allosteric modulators that potentiate chloride ion flux (agonists), negative modulators that reduce GABA-induced chloride ion flux (inverse agonists), and neutral allosteric modulators that competitively block the effects of these two types of agonists-antagonists.

On the other hand, GABAB metabotropic receptors, characterized by stereoselective ligand (−) baclofen, belong to the seven transmembrane G-protein-coupled receptor superfamily. They are pre- and postsynaptic G-protein-coupled receptors that negatively modulate adenylyl cyclase and inositol triphosphate synthesis. Heterodimeric structure as a result of GABAB1 and GABAB2 subunit assembly is necessary for appropriate GABAB receptor function. The extracellular domain of the GABAB1 subunit contains GABA-binding site, whereas GABAB2 subunit is important for the interaction with the G-proteins. GABAB receptor activation produces a cascade of signals that result in activation and/or inhibition of voltage-dependent calcium channels. GABAB receptor is located both centrally and peripherally, particularly in the thalamus, brain stem nuclei, and spinal cord. Depending on the localization of GABA<sup>B</sup> receptors, GABA-mediated inhibitory influences can be potentiated (postsynaptic receptors, presynaptic heteroreceptors on glutamatergic endings) or reduced (autoreceptors) [13, 14]. GABAB receptor function affects behavior, learning, and memory, and therefore their pharmacological targeting may be beneficial in various neuropsychiatric disorders [15, 16].

#### **3. Glutamate and its receptors**

**2. GABA and its receptors**

2 GABA And Glutamate - New Developments In Neurotransmission Research

GABAA receptors containing the α1 subunit [11].

Every third chemical synapse in the brain uses neurotransmitter GABA as an integral part of the neurotransmission process. GABA mediates its effects via two types of receptors: ionotropic GABAA and metabotropic GABAB receptors [6]. Although a third type of GABA receptor with pharmacological specificities has been identified, the term GABAС has not received broad consensus among experts. Additionally, the International Union of Basic and Clinical

GABAA receptors generally contain chloride ion channels but can, in varying degrees, also contain calcium, sodium, and potassium channels. GABAA receptors mediate the majority of GABA inhibitory actions in the CNS [4]. They are pentameric transmembrane receptors made up of 5 subunit proteins that form an ion channel selectively permeable to chloride anions. Although mainly localized on postsynaptic membranes, they can also be found extrasynaptically, especially GABAA receptors containing α4, α5, or α6 subunits [8]. Unexpectedly, GABAA receptors have also been found on glial cells, potentially providing adaptational support for adjacent neurons [9]. Activation of GABAA receptors leads to a change in the conformational state of associated ion channels, resulting in increased permeability to chloride ions. GABAergic mechanisms are also involved in metabolic processes [10], and a negative correlation between the intensity of GABAergic neurotransmission and metabolic processes in cerebral tissue has been established. So far, 19 subunits of GABAA receptors have been cloned and classified into several structurally related subfamilies (α 1–6, β 1–3, γ 1–3, δ, ε, θ, π, ρ 1–3). The most frequently found GABAA receptor composition is an aggregate composed of two α, two β, and one γ subunit [4]. Receptors that, in addition to two α and two β subunits, contain some other non-γ subunit are rare. Receptors composed only of α and β isoforms also exist. The subunit composition determines the functional and pharmacological properties of GABAA receptors. For example, α1 GABAA receptors mediate sedative and anticonvulsant actions, whereas the α2 subunit is responsible for anxiolytic action of benzodiazepines. Zolpidem, a commonly prescribed sedative for sleep initiation, has a high binding affinity for

GABA action through GABAA receptors results in chloride channel opening and increased postsynaptic membrane permeability. In addition to the well-determined benzodiazepine binding site, at least 13 different and structurally specific sites on the GABAA receptors have been identified: (1) GABA and other agonist-binding sites, as well as competitive antagonists; (2) picrotoxin site near ion channel; (3) barbiturates binding site; (4) neuroactive steroids binding site; (5) ethanol binding site; (6) inhalation anesthetics stereoselective binding sites; (7) furosemide diuretic binding site; (8) Zn2+ ion binding site; (9) other divalent cation binding sites; (10) La3+ ions site; (11) sites for phosphorylation of specific protein kinases; (12) phospholipid-binding sites; and (13) sites involved in interaction of GABAA receptor and microtubules, which promote receptor grouping on postsynaptic membranes [12]. Modulators of GABAA receptor complex interact with these binding sites in three possible ways: positive allosteric modulators that potentiate chloride ion flux (agonists), negative modulators that reduce GABA-induced chloride ion flux (inverse agonists), and neutral allosteric modulators

that competitively block the effects of these two types of agonists-antagonists.

Pharmacology (IUPHAR) has classified GABAC as a type of GABAA receptor [7].

Glutamate, the most abundant neurotransmitter in vertebrates and precursor of GABA, is present in over 90% of all synaptic connections in the human brain and is essential for a wide variety of functions [17]. Over 20 types of mammalian glutamate receptors exist, generally classified into two main categories: voltage-sensitive (ionotropic) and ligand-sensitive (metabotropic) receptors. Ionotropic receptor channels are formed from various protein subunits assembled in heterotetrameric or homotetrameric receptors. The three types of ionotropic receptors are N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainic acid receptors. The discovery of these agonists, after which the receptors were originally named, led to the detection of other receptor agonists and antagonists [18].

The binding of glutamate to NMDA receptors (NMDARs) results in the opening of a nonselective cation channel. The opening and closing of the channel are primarily gated by ligand binding but are also voltage-dependent. Extracellular magnesium and zinc ions can bind to specific sites on the receptor, blocking the passage of other cations through the open ion channel. However, depolarization of the neuronal cell dislodges and repels these ions from the pore, therefore allowing a voltage-dependent influx of sodium and calcium ions and efflux of potassium ions [19]. The NMDA receptor is primarily a ligand-gated channel, but it does display weaker voltage-dependent modulation of the ligand-dependent gating. NMDA requires co-activation by two ligands: glutamate and either d-serine or glycine [20]. Furthermore, NMDA receptors are divided into subtypes, depending on their intracellular protein structure, NR1, NR2, and NR3. NR1 consists of eight different subunits originating from a single gene via alternative splicing. NR2 has four subunits (A–D), and NR3 has two subunits (A and B). NMDA receptors are highly expressed on both neurons and astrocytes [21]. NMDA signaling is crucial for learning, memory, recovery from injury, and brain plasticity. It is especially important for proper functioning of the hippocampus [22]. In pathological circumstances, overactivation of NMDA receptors can lead to excitotoxicity, involved in some neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's disease [23–25].

AMPA receptors (AMPAR) are composed of four types of subunits, designated as GluA1, GluA2, GluA3, and GluA4 [26]. These receptors are heterotetrameric, containing GluA2 and either GluA1, GluA3, or GluA4 subunits in a "dimer of dimers" structure [27, 28]. Each AMPAR consists of four subunits which make up four binding sites to which an agonist (such as glutamate) can bind. The channel opens when two binding sites are simultaneously occupied, and the current increases as more binding sites become occupied [29]. Once opened, the channel may undergo rapid desensitization and current termination. Since AMPARs open and close quickly (1 ms), they are responsible for fast excitatory synaptic transmission in the CNS [30]. The GluA2 subunit regulates whether the AMPAR is permeable to calcium and other cations, such as sodium and potassium. If receptor does not contain a GluA2, the AMPAR will be permeable to calcium, sodium, and potassium. Both NMDA and AMPA ion channels are important for plasticity and synaptic transmission at many postsynaptic membranes.

number of neurological and psychiatric diseases. The GABAergic synapse is the site of action of several different classes of drugs that modulate inhibitory neurotransmission and are used in the pharmacotherapy of anxiety and sleep disorders, epilepsy, alcohol withdrawal, and induction and maintenance of anesthesia [34]. Moreover, glutamate dysfunction is also correlated with a wide range of nervous system disorders, such as Alzheimer's disease, and neuropsychiatric disorders, including schizophrenia, pain disorders, drug addiction, and traumatic brain and spinal cord injuries [35]. Given the importance of equilibrium of these two systems for neuronal excitability, synaptic plasticity, and cognitive functions such as learning and memory, as well as its involvement in the mood, feeding behavior, reproductive functions, pain sensitivity, aging, etc. [36], it is not surprising that the development of current and prospective pharmaceuticals, including anxiolytics, antidepressants, antipsychotics, antiepileptics, antidementia, and many other drugs, relies increasingly on GABA/glutamate balance.

Introductory Chapter: GABA/Glutamate Balance: A Key for Normal Brain Functioning

http://dx.doi.org/10.5772/intechopen.74023

5

Janko Samardzic's and Jasna Jancic's work has been supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant nos. 175076 and 175031). The authors declare that there are no competing interests regarding the publication

, Boris Hencic<sup>1</sup>

1 Institute of Pharmacology, Clinical Pharmacology and Toxicology, Medical Faculty,

2 Department of Biomedical Sciences, Neuropsychopharmacology Sect, University of

4 Laboratory for Molecular Neuropsychiatry, Division of Molecular Medicine, Rudjer

3 Clinic of Neurology and Psychiatry for Children and Youth, Medical Faculty, University of

[1] Petroff OA. GABA and glutamate in the human brain. The Neuroscientist. 2002;**8**(6):562-573 [2] Foster AC, Kemp JA. Glutamate- and GABA-based CNS therapeutics. Current Opinion

, Jasna Jancic<sup>3</sup>

and Dubravka Svob Strac<sup>4</sup>

**Acknowledgements**

of this chapter.

**Author details**

Janko Samardzic1

Cagliari, Cagliari, Italy

**References**

Belgrade, Belgrade, Serbia

Boskovic Institute, Zagreb, Croatia

in Pharmacology. 2006;**6**(1):7-17

\*, Dragana Jadzic<sup>2</sup>

University of Belgrade, Belgrade, Serbia

\*Address all correspondence to: jankomedico@yahoo.es

Kainate receptors (KAR) are heteromeric receptors assembled from four subunits, formerly referred to as GluR5, GluR6, GluR7, KA1, and KA2 but now named GluK1, GluK2, GluK3, GluK4, and GluK5, and grouped into low affinity (GluK1–3) and high affinity (GluK4–5) receptors. Each subunit has a large extracellular N-terminal domain, four helical transmembrane domains (M1–M4), and an intracellular C-terminal domain. GluK1–3 subunits can form both homomeric and heteromeric receptors, but GluK4 and GluK5 subunits can form only heteromeric functional ion channels together with GluK1–3 subunits. Despite their ion channel structure, KAR can also activate metabotropic signaling through noncanonical G-protein-coupled cascade. They are widely distributed in the brain and can be localized at pre-, post-, and/or extrasynaptic sites. Although KAR are less studied than AMPAR or NMDAR, it is not known that they are multifunctional neuronal modulators which play significant roles in health and disease [31].

Metabotropic glutamate receptors (mGluR) have a G-protein-linked receptor structure consisting of seven transmembrane domains with an extracellular N-terminal and an intracellular COOH terminal. When glutamate binds to a metabotropic receptor, it activates a postsynaptic intracellular G-protein, which eventually results in the opening of a membrane channel for signal transmission. Furthermore, G protein activation also triggers functional changes in the cytoplasm, resulting in gene expression and protein synthesis. For this reason, mGluR is generally considered slower acting channels than the ionotropic glutamate receptors. To date, three groups of mGluR exist. Group I receptors are coupled with phospholipase C, producing diacylglycerol and inositol triphosphate as second messengers. They are mainly expressed on the postsynaptic membrane. Group I receptors are involved in learning and memory, addiction, motor regulation, and Fragile X syndrome [32]. Groups II and III are negatively coupled to adenylyl cyclase. Impaired functioning of group II metabotropic receptors has been linked to anxiety, schizophrenia, and Alzheimer's disease. Group III metabotropic receptors also inhibit neurotransmitter release but are positioned presynaptically. They are found within the hippocampus and hypothalamus and may play a role in Parkinson's disease and anxiety disorders [33].

#### **4. Conclusion and clinical implications**

The adequate coordination of GABA and glutamate is essential to the normal functioning for the most complex brain processes. Decreased or increased GABA activity is associated with a number of neurological and psychiatric diseases. The GABAergic synapse is the site of action of several different classes of drugs that modulate inhibitory neurotransmission and are used in the pharmacotherapy of anxiety and sleep disorders, epilepsy, alcohol withdrawal, and induction and maintenance of anesthesia [34]. Moreover, glutamate dysfunction is also correlated with a wide range of nervous system disorders, such as Alzheimer's disease, and neuropsychiatric disorders, including schizophrenia, pain disorders, drug addiction, and traumatic brain and spinal cord injuries [35]. Given the importance of equilibrium of these two systems for neuronal excitability, synaptic plasticity, and cognitive functions such as learning and memory, as well as its involvement in the mood, feeding behavior, reproductive functions, pain sensitivity, aging, etc. [36], it is not surprising that the development of current and prospective pharmaceuticals, including anxiolytics, antidepressants, antipsychotics, antiepileptics, antidementia, and many other drugs, relies increasingly on GABA/glutamate balance.

#### **Acknowledgements**

AMPA receptors (AMPAR) are composed of four types of subunits, designated as GluA1, GluA2, GluA3, and GluA4 [26]. These receptors are heterotetrameric, containing GluA2 and either GluA1, GluA3, or GluA4 subunits in a "dimer of dimers" structure [27, 28]. Each AMPAR consists of four subunits which make up four binding sites to which an agonist (such as glutamate) can bind. The channel opens when two binding sites are simultaneously occupied, and the current increases as more binding sites become occupied [29]. Once opened, the channel may undergo rapid desensitization and current termination. Since AMPARs open and close quickly (1 ms), they are responsible for fast excitatory synaptic transmission in the CNS [30]. The GluA2 subunit regulates whether the AMPAR is permeable to calcium and other cations, such as sodium and potassium. If receptor does not contain a GluA2, the AMPAR will be permeable to calcium, sodium, and potassium. Both NMDA and AMPA ion channels are

4 GABA And Glutamate - New Developments In Neurotransmission Research

important for plasticity and synaptic transmission at many postsynaptic membranes.

neuronal modulators which play significant roles in health and disease [31].

thalamus and may play a role in Parkinson's disease and anxiety disorders [33].

The adequate coordination of GABA and glutamate is essential to the normal functioning for the most complex brain processes. Decreased or increased GABA activity is associated with a

**4. Conclusion and clinical implications**

Kainate receptors (KAR) are heteromeric receptors assembled from four subunits, formerly referred to as GluR5, GluR6, GluR7, KA1, and KA2 but now named GluK1, GluK2, GluK3, GluK4, and GluK5, and grouped into low affinity (GluK1–3) and high affinity (GluK4–5) receptors. Each subunit has a large extracellular N-terminal domain, four helical transmembrane domains (M1–M4), and an intracellular C-terminal domain. GluK1–3 subunits can form both homomeric and heteromeric receptors, but GluK4 and GluK5 subunits can form only heteromeric functional ion channels together with GluK1–3 subunits. Despite their ion channel structure, KAR can also activate metabotropic signaling through noncanonical G-protein-coupled cascade. They are widely distributed in the brain and can be localized at pre-, post-, and/or extrasynaptic sites. Although KAR are less studied than AMPAR or NMDAR, it is not known that they are multifunctional

Metabotropic glutamate receptors (mGluR) have a G-protein-linked receptor structure consisting of seven transmembrane domains with an extracellular N-terminal and an intracellular COOH terminal. When glutamate binds to a metabotropic receptor, it activates a postsynaptic intracellular G-protein, which eventually results in the opening of a membrane channel for signal transmission. Furthermore, G protein activation also triggers functional changes in the cytoplasm, resulting in gene expression and protein synthesis. For this reason, mGluR is generally considered slower acting channels than the ionotropic glutamate receptors. To date, three groups of mGluR exist. Group I receptors are coupled with phospholipase C, producing diacylglycerol and inositol triphosphate as second messengers. They are mainly expressed on the postsynaptic membrane. Group I receptors are involved in learning and memory, addiction, motor regulation, and Fragile X syndrome [32]. Groups II and III are negatively coupled to adenylyl cyclase. Impaired functioning of group II metabotropic receptors has been linked to anxiety, schizophrenia, and Alzheimer's disease. Group III metabotropic receptors also inhibit neurotransmitter release but are positioned presynaptically. They are found within the hippocampus and hypoJanko Samardzic's and Jasna Jancic's work has been supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant nos. 175076 and 175031). The authors declare that there are no competing interests regarding the publication of this chapter.

#### **Author details**

Janko Samardzic1 \*, Dragana Jadzic<sup>2</sup> , Boris Hencic<sup>1</sup> , Jasna Jancic<sup>3</sup> and Dubravka Svob Strac<sup>4</sup>

\*Address all correspondence to: jankomedico@yahoo.es

1 Institute of Pharmacology, Clinical Pharmacology and Toxicology, Medical Faculty, University of Belgrade, Belgrade, Serbia

2 Department of Biomedical Sciences, Neuropsychopharmacology Sect, University of Cagliari, Cagliari, Italy

3 Clinic of Neurology and Psychiatry for Children and Youth, Medical Faculty, University of Belgrade, Belgrade, Serbia

4 Laboratory for Molecular Neuropsychiatry, Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia

#### **References**


[3] Buzsaki G, Draguhn A. Neuronal oscillations in cortical networks. Science. 2004; **304**(5679):1926-1929

[20] Kleckner NW, Dingledine R. Requirement for glycine in activation of NMDA-receptors

Introductory Chapter: GABA/Glutamate Balance: A Key for Normal Brain Functioning

http://dx.doi.org/10.5772/intechopen.74023

7

[21] Lee MC, Ting KK, Adams S, Brew BJ, Chung R, Guillemin GJ. Characterisation of the expression of NMDA receptors in human astrocytes. PLoS One. 2010;**5**(11):e14123 [22] Barco A, Bailey CH, Kandel ER. Common molecular mechanisms in explicit and implicit

[23] Chen HS, Lipton SA. The chemical biology of clinically tolerated NMDA receptor antag-

[24] Lipton SA. Paradigm shift in neuroprotection by NMDA receptor blockade: Memantine

[25] Koch HJ, Szecsey A, Haen E. NMDA-antagonism (memantine): An alternative pharmacological therapeutic principle in Alzheimer's and vascular dementia. Current Pharma-

[26] Song I, Huganir RL. Regulation of AMPA receptors during synaptic plasticity. Trends in

[27] Mayer ML. Glutamate receptor ion channels. Current Opinion in Neurobiology. 2005;

[28] Greger IH, Ziff EB, Penn AC. Molecular determinants of AMPA receptor subunit assem-

[29] Rosenmund C, Stern-Bach Y, Stevens CF. The tetrameric structure of a glutamate recep-

[30] Platt SR. The role of glutamate in central nervous system health and disease—A review.

[31] Evans AJ, Gurung S, Henley JM, Nakamura Y, Wilkinson KA. Exciting times: New advances towards understanding the regulation and roles of kainate receptors. Neuro-

[32] Niswender CM, Conn PJ. Metabotropic glutamate receptors: Physiology, pharmacology, and disease. Annual Review of Pharmacology and Toxicology. 2010;**50**:295-322 [33] Swanson CJ, Bures M, Johnson MP, Linden AM, Monn JA, Schoepp DD. Metabotropic glutamate receptors as novel targets for anxiety and stress disorders. Nature Reviews.

[34] Samardzic J, Svob Strac D. Benzodiazepines and Anxiety disorders: From laboratory to clinic. In: Durbano F, editor. New Developments in Anxiety Disorders. Rijeka: InTech;

[35] Jadzic D. Role of the Bed Nucleus of Stria Terminalis (BNST) in addiction and depres-

[36] Car H, Wiśniewski K. Similarities and interactions between GABAergic and glutaminer-

sion: a microdialysis study [PhD thesis]. University of Cagliari; 2018

gic systems. Roczniki Akademii Medycznej w Białymstoku. 1998;**43**:5-26

expressed in Xenopus oocytes. Science. 1988;**241**(4867):835-837

memory. Journal of Neurochemistry. 2006;**97**(6):1520-1533

onists. Journal of Neurochemistry. 2006;**97**(6):1611-1626

ceutical Design. 2004;**10**(3):253-259

Neurosciences. 2002;**25**(11):578-588

bly. Trends in Neurosciences. 2007;**30**(8):407-416

tor channel. Science. 1998;**280**(5369):1596-1599

chemical Research. 2017 [Epub ahead of print]

Veterinary Journal. 2007;**173**(2):278-286

Drug Discovery. 2005;**4**(2):131-144

2016. pp. 23-45

**15**(3):282-288

and beyond. Nature Reviews. Drug Discovery. 2006;**5**(2):160-170


[20] Kleckner NW, Dingledine R. Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes. Science. 1988;**241**(4867):835-837

[3] Buzsaki G, Draguhn A. Neuronal oscillations in cortical networks. Science. 2004;

[4] Jacob TC, Moss SJ, Jurd R. GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nature Reviews. Neuroscience. 2008;**9**(5):331-343 [5] Erickson JD, De Gois S, Varoqui H, Schafer MK, Weihe E. Activity-dependent regulation of vesicular glutamate and GABA transporters: A means to scale quantal size.

[6] Chebib M, Johnston GA. The 'ABC' of GABA receptors: A brief review. Clinical and

[7] Olsen RW, Sieghart W. International Union of Pharmacology. LXX. Subtypes of gammaaminobutyric acid(A) receptors: Classification on the basis of subunit composition, phar-

[8] Fritschy JM, Brunig I. Formation and plasticity of GABAergic synapses: Physiological mechanisms and pathophysiological implications. Pharmacology & Therapeutics. 2003;

[9] Lin SC, Bergles DE. Synaptic signaling between neurons and glia. Glia. 2004;**47**(3):290-298 [10] Korpi ER, Grunder G, Luddens H. Drug interactions at GABA(A) receptors. Progress in

[11] Vlainić J, Jembrek MJ, Strac DS, Pericić D. The effects of zolpidem treatment and withdrawal on the in vitro expression of recombinant alpha1beta2gamma2s GABA(A) receptors expressed in HEK 293 cells. Naunyn-Schmiedeberg's Archives of Pharmacology.

[12] Chebib M, Johnston GA. GABA-activated ligand gated ion channels: Medicinal chemistry and molecular biology. Journal of Medicinal Chemistry. 2000;**43**(8):1427-1447 [13] Bettler B, Kaupmann K, Mosbacher J, Gassmann M. Molecular structure and physiological functions of GABA(B) receptors. Physiological Reviews. 2004;**84**(3):835-867

[14] Heaney CF, Kinney JW. Role of GABA(B) receptors in learning and memory and neuro-

[15] Cryan JF, Kaupman K. Don't worry 'B' happy!: A role for GABAB receptors in anxiety

[16] Bowery NG. GABAB receptor: A site of therapeutic benefit. Current Opinion in Pharma-

[17] Meldrum BS. Glutamate as a neurotransmitter in the brain: Review of physiology and

[18] Lesage A, Steckler T. Metabotropic glutamate mGlu1 receptor stimulation and blockade: Therapeutic opportunities in psychiatric illness. European Journal of Pharmacology.

[19] Paoletti P, Neyton J. NMDA receptor subunits: Function and pharmacology. Current

logical disorders. Neuroscience and Biobehavioral Reviews. 2016;**63**:1-28

and depression. Trends in Pharmacological Sciences. 2005;**26**(1):36-43

pathology. The Journal of Nutrition. 2000;**130**(4S):1007S-10015S

macology, and function. Update. Pharmacological Reviews. 2008;**60**(3):243-260

Neurochemistry International. 2006;**48**(6-7):643-649

6 GABA And Glutamate - New Developments In Neurotransmission Research

Experimental Pharmacology & Physiology. 1999;**26**(11):937-940

**304**(5679):1926-1929

**98**(3):299-323

2010;**382**(3):201-212

cology. 2006;**6**(1):37-43

2010;**639**(1-3):2-16

Opinion in Pharmacology. 2007;**7**(1):39-47

Neurobiology. 2002;**67**(2):113-159


**Chapter 2**

**Provisional chapter**

**Early Life Experience, Maternal Separation, and**

**Early Life Experience, Maternal Separation, and** 

Gabriela Beatriz Acosta

**Abstract**

Gabriela Beatriz Acosta

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70868

transporters, GABA, glutamate

ioral changes thus disturbing neuroplasticity [1, 2].

**1. Introduction**

**Involvement of GABA and Glutamate Transporters**

The physiological response initiates with activation of the hypothalamic-pituitaryadrenal axis, the autonomic nervous, and the immune systems. All actions promoted cellular adaptive changes in cells and tissues that protect the body and promote their survival. Diverse protocols of maternal separation (MS) in rodents presented alterations in central nervous system (CNS) such as learning disabilities, voluntary alcohol intake, and neurochemical changes. It is believed that the properties of these early life procedures are mediated by the high plasticity of the developing CNS. During critical development stage, brain regions, mainly those related to aggressive conditions, can have advancement abnormalities occasionally irreversible and thus adjust emotional processing when they grow to be adults. Early postnatal period and relationship between mother and infant are essential of normal stress response and emotional behavior. Probably, it involves the activation of intracellular signaling pathways, genome adaptations, adjusts in gene expression, and neural action. The objective of this article is to provide an overview of the current state of knowledge in the field focused on the maternal separation model, early life experience of postnatal stress, and the

involvement of γ-aminobutyric acid (GABA) and glutamate transporters.

**Keywords:** development plasticity, early maternal separation, acute and chronic stress,

Actually, it has been accepted that postnatal exposure to adverse events like stress can influence the offspring neurodevelopment, its neuroendocrine, and immune systems and induce behav-

**Involvement of GABA and Glutamate Transporters**

DOI: 10.5772/intechopen.70868

© 2016 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,

© 2018 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.

and reproduction in any medium, provided the original work is properly cited.

#### **Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters**

DOI: 10.5772/intechopen.70868

Gabriela Beatriz Acosta Gabriela Beatriz Acosta

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70868

#### **Abstract**

The physiological response initiates with activation of the hypothalamic-pituitaryadrenal axis, the autonomic nervous, and the immune systems. All actions promoted cellular adaptive changes in cells and tissues that protect the body and promote their survival. Diverse protocols of maternal separation (MS) in rodents presented alterations in central nervous system (CNS) such as learning disabilities, voluntary alcohol intake, and neurochemical changes. It is believed that the properties of these early life procedures are mediated by the high plasticity of the developing CNS. During critical development stage, brain regions, mainly those related to aggressive conditions, can have advancement abnormalities occasionally irreversible and thus adjust emotional processing when they grow to be adults. Early postnatal period and relationship between mother and infant are essential of normal stress response and emotional behavior. Probably, it involves the activation of intracellular signaling pathways, genome adaptations, adjusts in gene expression, and neural action. The objective of this article is to provide an overview of the current state of knowledge in the field focused on the maternal separation model, early life experience of postnatal stress, and the involvement of γ-aminobutyric acid (GABA) and glutamate transporters.

**Keywords:** development plasticity, early maternal separation, acute and chronic stress, transporters, GABA, glutamate

#### **1. Introduction**

Actually, it has been accepted that postnatal exposure to adverse events like stress can influence the offspring neurodevelopment, its neuroendocrine, and immune systems and induce behavioral changes thus disturbing neuroplasticity [1, 2].

and reproduction in any medium, provided the original work is properly cited.

© 2016 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, © 2018 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.

The central nervous system (CNS) maintains a degree of adaptive plasticity, which allows adjusting to certain situations and adapting innate designs from neuronal connections. There is an abundant bibliography where it is shown that the unfavorable measures in the early life of an individual present profound and persistent effects on the cerebral functions, being able to represent a risky influence for the future development of the psychopathology [4, 5]. Epidemiological studies have shown that postnatal stress or emotional trauma, especially when suffering from early life, is usually associated with an increased possibility of depression [6]. During the critical period of certain brain regions, mainly those related to adverse situations, such as the frontal cortex (CF), hippocampus (Hic), and amygdala, hypothalamic-pituitary-adrenal (HPA) axis can develop almost irreversible abnormalities and alter the response to stress throughout the life of the animal [6, 7]. A recent study of the consequences of maltreatment and stress during childhood at early ages has shown the effects of this experience on brain structures. These structural changes were associated with changes in levels of stress hormones and neurotransmitters, resulted in maltreatment and stress in childhood at early ages, a variety of disorders including depression, anxiety, aggression, impulsivity, hyperactivity, criminal tendency, or abuse of toxic substances [8–11]. With all these evidences, we can deduce that an excess of stress at early ages of the developing life restricts with the paused, progressive, and normal development of the brain [5, 12, 13].

**2. Early environmental experiences**

**3. Maternal separation**

logical and endocrine studies [31–33].

The brain in the early stages of development presents a high level of plasticity, facilitating both adaptive variations on behalf of opportunities, and malformations alterable vulnerability. At present, neuropsychiatric illnesses evident as complex combinations of cognitive, emotional, and behavioral discrepancies have their origins of development fixed primary in the initial placement of the functional impression of the brain [23]. Postnatal periods are critical for CNS development [24]. After birth, the brain continues to grow with its total volume doubling in the first year, measured by 15% rise in the second year [25]. In particular, this increase is explained by the development of neuronal connections in gray matter (synapses and dendrites), longrange axons, and myelination; all of these are necessary for the society of circulated functional systems; regressive development includes the pruning of the synapses and axons during the

Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters

http://dx.doi.org/10.5772/intechopen.70868

11

childhood period, permitting the restructuring of the primarily practical circuits [26].

long-term effects. In this review, we will focus in the models of MS.

vary in the frequency, the duration, and the age at which the MS occurred.

Animal models are useful tools that help us to understand how genetic vulnerability factors can modulate responses to early environmental factors and provide insights into behavioral and physiological mechanisms involved in the pathways through which early stress might produce

Adverse childhood experience is considered one of the main risk factors for the development of psychopathology. Maternal separation in rodents (rats or mice) is a well-known animal model of early stress to explore the neuroendocrine and behavioral properties of early difficulty. This paradigm discusses to the daily separation of puppies (usually rodents) from their mothers for a short period of time (1 hour) or prolonged (3–6 hours) during the first 1–3 weeks after birth. The paradigmatic MS puppies remain together as a litter [27]. This process is performed between birth and weaning for diverse periods of time and permits a set of experimental designs, which

Studies in animal models show the influence of life conditions during the postnatal period in the establishment of neurological factors that control behavior and response to stress [28]. Acute and chronic MS has both short and long-term effects on behavior and neuroendocrinal responses [29]. Different experimental protocols of MS in rodents have shown changes in CNS functioning: learning impediments, voluntary alcohol consumption, and behavioral variations [30]. This handling in animals is used for diverse experiments, for example, Studies in life sciences sometimes require repeated manipulation in rodents during the course of the experiment (handling). The main function of the handling is to minimize the stress associated with behavioral, pharmaco-

The early life of most mammals is expended in near contact with the mother and for the newborn. Early MS is a traumatic occurrence that, conditioning on the different situations, can form its behavioral and neurochemical phenotype in adulthood. Studies in rodents exhibited that a

The brain is vulnerable to early-life programming, and this can be manifested in childhood or adulthood as stress hyper-reactivity by deregulation of the HPA axis and increased susceptibility to affective disorders like anxiety, depression, and schizophrenia [14, 15]. Exposure to early stressful adverse life events may increase vulnerability to psychopathology in adult life. There are important memory disturbances in stress-related psychiatric disorders [16].

The term neuroplasticity refers to the potential of the brain to reorganize by creating new neural pathways to adapt, as it needs [2]. This phenomenon requires the stable modulation of gene expression, which is mediated at least in part by epigenetic processes such as DNA methylation and histone modifications. Both the genome and the epigenome cooperate interactively in the mature phenotype and determine the sensitivity to environmental factors and the subsequent risk of disease [17–19]. There is increasing evidence that environmental factors, particularly stressful events experienced early in life, increase the risk of developing a psychiatric illness and/or a behavioral disorder [3, 18, 19]. The experiences of chronic stress are a factor that mainly influences numerous neuropsychiatric diseases, since it often leads to maladaptive responses [20].

While the childhood adversity as a negative childhood experience associated with increased lifetime risk of poorer health and social outcomes have been described postnatal experiences. Several studies in psychiatry have shown a long-term negative effect on health and society such as depression [21], alcohol abuse, use of consumer drugs, family abuse, and other social practices that interact with these processes [22].

The aim of this article is to overview on the current state of knowledge in the field focusing an animal model of maternal separation (MS), early life experience of postnatal stress, and the involvement of γ-aminobutyric acid (GABA) and glutamate transporters.

#### **2. Early environmental experiences**

The central nervous system (CNS) maintains a degree of adaptive plasticity, which allows adjusting to certain situations and adapting innate designs from neuronal connections. There is an abundant bibliography where it is shown that the unfavorable measures in the early life of an individual present profound and persistent effects on the cerebral functions, being able to represent a risky influence for the future development of the psychopathology [4, 5]. Epidemiological studies have shown that postnatal stress or emotional trauma, especially when suffering from early life, is usually associated with an increased possibility of depression [6]. During the critical period of certain brain regions, mainly those related to adverse situations, such as the frontal cortex (CF), hippocampus (Hic), and amygdala, hypothalamic-pituitary-adrenal (HPA) axis can develop almost irreversible abnormalities and alter the response to stress throughout the life of the animal [6, 7]. A recent study of the consequences of maltreatment and stress during childhood at early ages has shown the effects of this experience on brain structures. These structural changes were associated with changes in levels of stress hormones and neurotransmitters, resulted in maltreatment and stress in childhood at early ages, a variety of disorders including depression, anxiety, aggression, impulsivity, hyperactivity, criminal tendency, or abuse of toxic substances [8–11]. With all these evidences, we can deduce that an excess of stress at early ages of the developing life restricts with the paused, progressive, and normal development of the brain

10 GABA And Glutamate - New Developments In Neurotransmission Research

The brain is vulnerable to early-life programming, and this can be manifested in childhood or adulthood as stress hyper-reactivity by deregulation of the HPA axis and increased susceptibility to affective disorders like anxiety, depression, and schizophrenia [14, 15]. Exposure to early stressful adverse life events may increase vulnerability to psychopathology in adult life. There are important memory disturbances in stress-related psychiatric

The term neuroplasticity refers to the potential of the brain to reorganize by creating new neural pathways to adapt, as it needs [2]. This phenomenon requires the stable modulation of gene expression, which is mediated at least in part by epigenetic processes such as DNA methylation and histone modifications. Both the genome and the epigenome cooperate interactively in the mature phenotype and determine the sensitivity to environmental factors and the subsequent risk of disease [17–19]. There is increasing evidence that environmental factors, particularly stressful events experienced early in life, increase the risk of developing a psychiatric illness and/or a behavioral disorder [3, 18, 19]. The experiences of chronic stress are a factor that mainly influences numerous neuropsychiatric diseases, since it often leads to maladaptive

While the childhood adversity as a negative childhood experience associated with increased lifetime risk of poorer health and social outcomes have been described postnatal experiences. Several studies in psychiatry have shown a long-term negative effect on health and society such as depression [21], alcohol abuse, use of consumer drugs, family abuse, and other social practices that inter-

The aim of this article is to overview on the current state of knowledge in the field focusing an animal model of maternal separation (MS), early life experience of postnatal stress, and the

involvement of γ-aminobutyric acid (GABA) and glutamate transporters.

[5, 12, 13].

disorders [16].

responses [20].

act with these processes [22].

The brain in the early stages of development presents a high level of plasticity, facilitating both adaptive variations on behalf of opportunities, and malformations alterable vulnerability. At present, neuropsychiatric illnesses evident as complex combinations of cognitive, emotional, and behavioral discrepancies have their origins of development fixed primary in the initial placement of the functional impression of the brain [23]. Postnatal periods are critical for CNS development [24]. After birth, the brain continues to grow with its total volume doubling in the first year, measured by 15% rise in the second year [25]. In particular, this increase is explained by the development of neuronal connections in gray matter (synapses and dendrites), longrange axons, and myelination; all of these are necessary for the society of circulated functional systems; regressive development includes the pruning of the synapses and axons during the childhood period, permitting the restructuring of the primarily practical circuits [26].

Animal models are useful tools that help us to understand how genetic vulnerability factors can modulate responses to early environmental factors and provide insights into behavioral and physiological mechanisms involved in the pathways through which early stress might produce long-term effects. In this review, we will focus in the models of MS.

#### **3. Maternal separation**

Adverse childhood experience is considered one of the main risk factors for the development of psychopathology. Maternal separation in rodents (rats or mice) is a well-known animal model of early stress to explore the neuroendocrine and behavioral properties of early difficulty. This paradigm discusses to the daily separation of puppies (usually rodents) from their mothers for a short period of time (1 hour) or prolonged (3–6 hours) during the first 1–3 weeks after birth. The paradigmatic MS puppies remain together as a litter [27]. This process is performed between birth and weaning for diverse periods of time and permits a set of experimental designs, which vary in the frequency, the duration, and the age at which the MS occurred.

Studies in animal models show the influence of life conditions during the postnatal period in the establishment of neurological factors that control behavior and response to stress [28]. Acute and chronic MS has both short and long-term effects on behavior and neuroendocrinal responses [29].

Different experimental protocols of MS in rodents have shown changes in CNS functioning: learning impediments, voluntary alcohol consumption, and behavioral variations [30]. This handling in animals is used for diverse experiments, for example, Studies in life sciences sometimes require repeated manipulation in rodents during the course of the experiment (handling). The main function of the handling is to minimize the stress associated with behavioral, pharmacological and endocrine studies [31–33].

The early life of most mammals is expended in near contact with the mother and for the newborn. Early MS is a traumatic occurrence that, conditioning on the different situations, can form its behavioral and neurochemical phenotype in adulthood. Studies in rodents exhibited that a very short separation cooled by a greater maternal care can completely affect the development of offspring. Nevertheless, prolonged MS origins stress. The significance of this stress and HPA axis hyper-reactivity is articulated in adulthood and continues throughout the life [34]. MS in rodents, particularly in rats, was used as a model for various psychotic conditions, especially depression and anxiety [30, 32]. The most popular MS technique of a daily separation of 3 hours from the second to the 12th postpartum day produces a model of high-construct depression and predictive validity. The results of studies of MS in rat lead to a discussion to its benefits for the neonates. This procedure might be contributed for the mental health of the offspring in adulthood [34].

GABA, and neuromodulators, such as neuropeptide Y, oxytocin, a gaseous molecule, and nitric oxide, have been implicated in the pathogenesis of stress-dependent disorders in early

Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters

http://dx.doi.org/10.5772/intechopen.70868

13

We will focus on the GABA and Glutamate neurotransmission, especially in their transporters.

γ-aminobutyric acid (GABA) is the chief inhibitory neurotransmitter of the adult mammalian CNS. During the early post-natal development, GABA acts as an excitatory neurotransmitter, serving as a neuronal and neurotrophic migration factor, separately from taking part in synaptogenesis. The GABAergic system has a large, complete molecular machine, by which it performs its actions, including enzymes for its synthesis and metabolism, membrane receptors, and transport proteins. Recent literature demonstrates that GABA transporters, as well as the GABAA receptor, are proteins of importance for the normal functioning and development of the central nervous system. All these molecules allow GABA to perform an essential function, both in the developing brain as in the adult brain. For this reason, the expression profile of the different subtypes of the molecules previously mentioned will be described in this article, in order to obtain a thorough knowledge of the molecular behavior of the GABAergic system

During the early postnatal development, GABA acts as an excitatory neurotransmitter, serving as a neuronal and neurotrophic migration factor, apart from taking part in synaptogenesis. The GABAergic system has a large, complete molecular machine, by which it performs its actions, including enzymes for its synthesis and metabolism, membrane receptors, and transport proteins. There is abundant literature showing that GABA transporters, as well as the GABA A receptor, are proteins of particular importance for the normal functioning and development of the central nervous system [46–49]. GABA-mediated inhibition exerts a powerful control over cortical neuronal activity, and GABA transporters (GATs) contribute to modulate the action of GABA [45]. Altered GATs activity and/or expression are likely to affect markedly cortical function, with their possible involvement in the pathophysiology of selected human disease.

The regulation of extracellular levels of GABA is essential for normal CNS development and functioning. The principal mechanism by which the levels of the neurotransmitter are regu-

transporter proteins called GABA transporters [50]. The others GAT-1, GAT-2, and GAT-3 have been identified from the GATs identified and cloned, although there is also a fourth isoform, BGT-1 (Betaine Carrier/GABA). Although all these transporter molecules have high affinity and selectivity for GABA, they present differential characteristics in pharmacology, localization, and functionality [13, 50, 51]. In fact, the levels of expression during postnatal CNS development vary markedly between the two transporters [13, 52]. There is another GAT, located intracellularly, which is the vesicular GABA transporter (VGAT), which plays a

primordial role in the normal development of the immature brain [52].


stages of life [43].

**5. γ-Aminobutyric acid (GABA)**

from conception to adulthood [44, 45].

**5.1. Transporters of GABA (GATs)**

lated is through Na+

### **4. Physiological mechanisms involved in early maternal separation: neurobiological responses to early life stress**

#### **4.1. HPA axis**

The hypothalamo-pituitary-adrenal axis is a key component of the stress reaction. Many studies have shown the impact of stress exposure during development on the HPA axis activity and on psychoemotional disorders during adulthood [19, 35].

Lifelong variations in HPA axis perform examined as a result of developmental complexities (maternal separation) demonstrate connections with psychiatric disorders containing schizophrenia depression, which may be characterized by irregularities in the activity of HPA axis and reaction to stress [4, 36, 37].

#### **4.2. Neurotrophins reaction**

Neurotrophins, also called neurotrophic factors, are a family of proteins that favor the survival of neurons. Family members include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT4/5) [38–40]. During development, limiting amounts of neurotrophins function as survival factors to ensure a match between the number of surviving neurons and the requirement for appropriate target innervation. In addition, they arrange cell destiny decisions, axon growth, dendrite pruning, the pattern of innervation, and the expression of proteins critical for normal neuronal role, such as neurotransmitters and ion channels. In the adult CNS, they control synaptic activity and plasticity, while ongoing to modulate neuronal survival [41].

#### **4.3. Neurochemical response**

Adverse early life practices can provoke neurochemical alterations that may underlie modifications in HPA axis reaction, emotionality, and cognition [42]. The impact of stress on brain function is known. Different substances are released in response to stress and can influence various neural circuits. The individual effects of functional neuronal mediators of stress (neurotransmitters, neuropeptides, and steroids) and plasticity are integrated. This causes the stress instruments to produce an orchestrated "symphony" that allows for adjusted responses to the various challenges [42]. Different neurotransmitters, such as NA, 5-HT, Glutamate, and GABA, and neuromodulators, such as neuropeptide Y, oxytocin, a gaseous molecule, and nitric oxide, have been implicated in the pathogenesis of stress-dependent disorders in early stages of life [43].

We will focus on the GABA and Glutamate neurotransmission, especially in their transporters.

### **5. γ-Aminobutyric acid (GABA)**

very short separation cooled by a greater maternal care can completely affect the development of offspring. Nevertheless, prolonged MS origins stress. The significance of this stress and HPA axis hyper-reactivity is articulated in adulthood and continues throughout the life [34]. MS in rodents, particularly in rats, was used as a model for various psychotic conditions, especially depression and anxiety [30, 32]. The most popular MS technique of a daily separation of 3 hours from the second to the 12th postpartum day produces a model of high-construct depression and predictive validity. The results of studies of MS in rat lead to a discussion to its benefits for the neonates. This

procedure might be contributed for the mental health of the offspring in adulthood [34].

**4. Physiological mechanisms involved in early maternal separation:** 

The hypothalamo-pituitary-adrenal axis is a key component of the stress reaction. Many studies have shown the impact of stress exposure during development on the HPA axis activity and on

Lifelong variations in HPA axis perform examined as a result of developmental complexities (maternal separation) demonstrate connections with psychiatric disorders containing schizophrenia depression, which may be characterized by irregularities in the activity of HPA axis

Neurotrophins, also called neurotrophic factors, are a family of proteins that favor the survival of neurons. Family members include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT4/5) [38–40]. During development, limiting amounts of neurotrophins function as survival factors to ensure a match between the number of surviving neurons and the requirement for appropriate target innervation. In addition, they arrange cell destiny decisions, axon growth, dendrite pruning, the pattern of innervation, and the expression of proteins critical for normal neuronal role, such as neurotransmitters and ion channels. In the adult CNS, they control synaptic activity and plasticity, while

Adverse early life practices can provoke neurochemical alterations that may underlie modifications in HPA axis reaction, emotionality, and cognition [42]. The impact of stress on brain function is known. Different substances are released in response to stress and can influence various neural circuits. The individual effects of functional neuronal mediators of stress (neurotransmitters, neuropeptides, and steroids) and plasticity are integrated. This causes the stress instruments to produce an orchestrated "symphony" that allows for adjusted responses to the various challenges [42]. Different neurotransmitters, such as NA, 5-HT, Glutamate, and

**neurobiological responses to early life stress**

12 GABA And Glutamate - New Developments In Neurotransmission Research

psychoemotional disorders during adulthood [19, 35].

and reaction to stress [4, 36, 37].

ongoing to modulate neuronal survival [41].

**4.2. Neurotrophins reaction**

**4.3. Neurochemical response**

**4.1. HPA axis**

γ-aminobutyric acid (GABA) is the chief inhibitory neurotransmitter of the adult mammalian CNS. During the early post-natal development, GABA acts as an excitatory neurotransmitter, serving as a neuronal and neurotrophic migration factor, separately from taking part in synaptogenesis. The GABAergic system has a large, complete molecular machine, by which it performs its actions, including enzymes for its synthesis and metabolism, membrane receptors, and transport proteins. Recent literature demonstrates that GABA transporters, as well as the GABAA receptor, are proteins of importance for the normal functioning and development of the central nervous system. All these molecules allow GABA to perform an essential function, both in the developing brain as in the adult brain. For this reason, the expression profile of the different subtypes of the molecules previously mentioned will be described in this article, in order to obtain a thorough knowledge of the molecular behavior of the GABAergic system from conception to adulthood [44, 45].

During the early postnatal development, GABA acts as an excitatory neurotransmitter, serving as a neuronal and neurotrophic migration factor, apart from taking part in synaptogenesis. The GABAergic system has a large, complete molecular machine, by which it performs its actions, including enzymes for its synthesis and metabolism, membrane receptors, and transport proteins. There is abundant literature showing that GABA transporters, as well as the GABA A receptor, are proteins of particular importance for the normal functioning and development of the central nervous system [46–49]. GABA-mediated inhibition exerts a powerful control over cortical neuronal activity, and GABA transporters (GATs) contribute to modulate the action of GABA [45]. Altered GATs activity and/or expression are likely to affect markedly cortical function, with their possible involvement in the pathophysiology of selected human disease.

#### **5.1. Transporters of GABA (GATs)**

The regulation of extracellular levels of GABA is essential for normal CNS development and functioning. The principal mechanism by which the levels of the neurotransmitter are regulated is through Na+ -dependent high-affinity uptake carried out by synaptic and glial located transporter proteins called GABA transporters [50]. The others GAT-1, GAT-2, and GAT-3 have been identified from the GATs identified and cloned, although there is also a fourth isoform, BGT-1 (Betaine Carrier/GABA). Although all these transporter molecules have high affinity and selectivity for GABA, they present differential characteristics in pharmacology, localization, and functionality [13, 50, 51]. In fact, the levels of expression during postnatal CNS development vary markedly between the two transporters [13, 52]. There is another GAT, located intracellularly, which is the vesicular GABA transporter (VGAT), which plays a primordial role in the normal development of the immature brain [52].

• **GAT-1**: This transporter is one of the most important isoforms of GATs. They are brain-specific proteins [53–56]. This transporter has the particularity of being found in both neurons and glial cells and is the main isoform of GATs in the mature brain [52].

The central nervous system maintains a degree of adaptive plasticity, which allows it to adjust to certain conditions and modify the innate patterns of neuronal connections [62]. These mediators exert a paradoxical damage-protection action. These variations can alter the functioning of the CNS, and consequently, the body's response to stress throughout life, as this treatment is done during the postnatal period, with the CNS in full development. A further support to this possibility came from the demonstration that prepulse inhibition disruption in maternally deprived rats occurs only after puberty [63, 64], with a temporal profile similar to the onset of schizophrenic symptomatology in patients, and was reversed by treatment with typical and atypical antipsychotic drugs [34], suggesting that the defects resulting from MS

Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters

http://dx.doi.org/10.5772/intechopen.70868

15

The identification of neurobiological substrates that are affected by early life adverse experience may have important diagnostic implications and could contribute to identify novel molecular targets for the development of more effective treatments of psychiatric disorders. Further studies are now warranted to elucidate the type or the timing of early life events that are associated with enhanced risk for depression or anxiety may be different from those

We will briefly describe glutamate as the main excitatory neurotransmitter in the brain. There are three families of ionotropic receptors with channels permeable to intrinsic cations: N-methyl-D-aspartate (NMDA), α- amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA), and Kainate. There are three groups of metabotropics, protein G-coupled glutamate receptors (mGluR) that modify neuronal and glial excitability through G-protein subunits acting on membrane ion channels and second messengers such as diacylglycerol and cAMP. Endogenous glutamate, by activating NMDA, AMPA or mGluR1 receptors, may contribute to acute brain damage following epilepsy, cerebral ischemia, or traumatic brain injury. It may also contribute to chronic neurodegeneration in disorders such as amyotrophic lateral sclerosis and Huntington's chorea. In animal models of cerebral ischemia and traumatic brain injury, NMDA and AMPA receptor antagonists protect against acute brain damage and delayed behavioral deficits. Other clinical conditions including epilepsy, amnesia, anxiety,

and psychosis may respond to drugs that act on glutamatergic transmission [67, 68].

A family of transporter proteins, excitatory amino acid transporters (EAAT), regulates extracellular concentration of Glu. Several lines of evidence suggest that increases of Glu in extracellular levels are involved in the stress response [68]. Astrocytes are the main protectors of neurons from excitotoxicity in the normal CNS, and this protection is conferred by clearance

The GluTs family is mediated by Na + dependent high affinity, this represents a critical factor in the Glu uptake and the regulation of homeostasis in the synaptic cleft [70]. Five

might be the consequence of an hyperactivity of the dopaminergic system [65].

relevant to schizophrenia [66].

**6.1. Transporters of glutamate (GluTs)**

of extracellular [69].

**6. Glutamate**


Odeon et al. [60] evaluated the effects of acute MS (AMS) and CMS (Chronic MS) + cold stress on the expression levels of GAT-1 in FC and Hic, whose appearance correlates with the concentration of corticosterone at different postnatal day from birth to young adulthood. In response to AMS + cold stress in FC, they demonstrated a decrease expression of GAT-1 at PD13. But in CMS, the levels of GAT-1 increased both at PD57 and PD63. At AMS in HIc, they observed an enhance in GAT-1 expression of either PD7 or PD13. Conversely, CMS decreased either PD57 or PD67 and increased at PD71 hippocampal levels expression of GAT-1. With respect to the levels of corticosterone, they observed an increase in all age groups studied in AMS. On the contrary, they showed a decrease in corticosterone levels in CMS. These authors concluded that a low responsiveness of the early postnatal period to stress, involvement of GABAergic system, suggesting that GATs may contribute to the deregulation of neuronal excitability that accompanies at neurobiological consequences of early stress. These dates obtained in this experimental condition serve as a starting point, elucidating the molecular mechanism of GAT regulation in GABA system throughout postnatal development.

In homogenates of FC and Hic acquired from either acute or chronic MS + cold stress, we found variations on the expression of GAT-1. GABA system plays a role in the pathophysiology of anxiety and mood disorders. The extracellular levels of GABA are regulated by specific high-affinity transporters, one of which, the plasma membrane GAT1, is considered the predominant neuronal transporter in the rodent brain [56, 61].

Although AMS might mimic a "dramatic" experience occurring at a precise developmental stage, the less dramatic repeated maternal separation can reproduce a more physiological situation [6].

The central nervous system maintains a degree of adaptive plasticity, which allows it to adjust to certain conditions and modify the innate patterns of neuronal connections [62]. These mediators exert a paradoxical damage-protection action. These variations can alter the functioning of the CNS, and consequently, the body's response to stress throughout life, as this treatment is done during the postnatal period, with the CNS in full development. A further support to this possibility came from the demonstration that prepulse inhibition disruption in maternally deprived rats occurs only after puberty [63, 64], with a temporal profile similar to the onset of schizophrenic symptomatology in patients, and was reversed by treatment with typical and atypical antipsychotic drugs [34], suggesting that the defects resulting from MS might be the consequence of an hyperactivity of the dopaminergic system [65].

The identification of neurobiological substrates that are affected by early life adverse experience may have important diagnostic implications and could contribute to identify novel molecular targets for the development of more effective treatments of psychiatric disorders. Further studies are now warranted to elucidate the type or the timing of early life events that are associated with enhanced risk for depression or anxiety may be different from those relevant to schizophrenia [66].

#### **6. Glutamate**

• **GAT-1**: This transporter is one of the most important isoforms of GATs. They are brain-specific proteins [53–56]. This transporter has the particularity of being found in both neurons and

• **GAT-2**: It is the least abundant isoform. Although low levels of GAT-2 have been detected in GABAergic neurons, this transporter is considered as extraparenchymal and its localization is limited to leptomeningeal and ependymal cells [52, 57]. In all postnatal stages, GAT-2 is detected in the arachnoid layer and in the arachnoid trabecula of the subarachnoid space. In some cases, expression of GAT-2 is observed across the entire diameter of the blood vessels supplying the cortex. The latter characteristic occurs mainly between postnatal day 0 and 5, and this transporter may be considered as the main source of pe-

• **GAT-3**: This isoform of the GABA transporter is among the most abundant along with GAT-1. GAT-3 is the predominant isoform during the early postnatal stages regulating neuronal excit-

• **VGAT**: The vesicular GABA transporter is essential for GABAergic neurotransmission to occur, as it introduces GABA into the presynaptic vesicles using a proton gradient. In this way, the GABA is stored to be released after the arrival of an action potential to the presynaptic

Odeon et al. [60] evaluated the effects of acute MS (AMS) and CMS (Chronic MS) + cold stress on the expression levels of GAT-1 in FC and Hic, whose appearance correlates with the concentration of corticosterone at different postnatal day from birth to young adulthood. In response to AMS + cold stress in FC, they demonstrated a decrease expression of GAT-1 at PD13. But in CMS, the levels of GAT-1 increased both at PD57 and PD63. At AMS in HIc, they observed an enhance in GAT-1 expression of either PD7 or PD13. Conversely, CMS decreased either PD57 or PD67 and increased at PD71 hippocampal levels expression of GAT-1. With respect to the levels of corticosterone, they observed an increase in all age groups studied in AMS. On the contrary, they showed a decrease in corticosterone levels in CMS. These authors concluded that a low responsiveness of the early postnatal period to stress, involvement of GABAergic system, suggesting that GATs may contribute to the deregulation of neuronal excitability that accompanies at neurobiological consequences of early stress. These dates obtained in this experimental condition serve as a starting point, elucidating the molecular mechanism of GAT

In homogenates of FC and Hic acquired from either acute or chronic MS + cold stress, we found variations on the expression of GAT-1. GABA system plays a role in the pathophysiology of anxiety and mood disorders. The extracellular levels of GABA are regulated by specific high-affinity transporters, one of which, the plasma membrane GAT1, is considered the pre-

Although AMS might mimic a "dramatic" experience occurring at a precise developmental stage, the less dramatic repeated maternal separation can reproduce a more physiological

ability at these times [52, 58] and has its unique location in astrocytes [52].

regulation in GABA system throughout postnatal development.

dominant neuronal transporter in the rodent brain [56, 61].

glial cells and is the main isoform of GATs in the mature brain [52].

14 GABA And Glutamate - New Developments In Neurotransmission Research

ripheral GABA [52].

terminal [59].

situation [6].

We will briefly describe glutamate as the main excitatory neurotransmitter in the brain. There are three families of ionotropic receptors with channels permeable to intrinsic cations: N-methyl-D-aspartate (NMDA), α- amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA), and Kainate. There are three groups of metabotropics, protein G-coupled glutamate receptors (mGluR) that modify neuronal and glial excitability through G-protein subunits acting on membrane ion channels and second messengers such as diacylglycerol and cAMP. Endogenous glutamate, by activating NMDA, AMPA or mGluR1 receptors, may contribute to acute brain damage following epilepsy, cerebral ischemia, or traumatic brain injury. It may also contribute to chronic neurodegeneration in disorders such as amyotrophic lateral sclerosis and Huntington's chorea. In animal models of cerebral ischemia and traumatic brain injury, NMDA and AMPA receptor antagonists protect against acute brain damage and delayed behavioral deficits. Other clinical conditions including epilepsy, amnesia, anxiety, and psychosis may respond to drugs that act on glutamatergic transmission [67, 68].

#### **6.1. Transporters of glutamate (GluTs)**

A family of transporter proteins, excitatory amino acid transporters (EAAT), regulates extracellular concentration of Glu. Several lines of evidence suggest that increases of Glu in extracellular levels are involved in the stress response [68]. Astrocytes are the main protectors of neurons from excitotoxicity in the normal CNS, and this protection is conferred by clearance of extracellular [69].

The GluTs family is mediated by Na + dependent high affinity, this represents a critical factor in the Glu uptake and the regulation of homeostasis in the synaptic cleft [70]. Five high-affinity GluTs were cloned into human and animal tissues and identified as a glutamate aspartate transporter (GLAST), excitatory amino acid carrier-1 (EAAT)-1, glial glutamate transporter-1 (GLT-1, EAAT-2); excitatory amino acid-carrier-1 (EAAC-1, EAAT-3), EAAT-4, and EAAT-5. Unlike other neurotransmitters, the action of Glu released into the synaptic cleft is terminated by uptake into neurons and surrounding glial cells via specific transporters. Within the nerve terminal, the glutamine released by glial cells and taken up by neurons is converted back to Glu [71]. Rapid removal of Glu from the extracellular space is required for the survival and normal function of neurons. Although GluTs are expressed by all CNS cell types, astrocytes are the cell type primarily responsible for Glu uptake [72]. Astrocytes express both GLT-1 and GLAST, while axon terminals in the neocortex only express GLT-1.

GluTs are neuronal and non-neuronal factors necessary for expression, maintenance, and transcriptional regulators of these proteins. The finding that RMS altered Glu regulation in the frontal cortex and hippocampus indicates a possible role for distorted glutamate regulation in the causal relationship between early life stress. Finally, I have some specific questions about this work: (1) Can early exposures with limited time produce lasting physiological changes? (2) Can these physiological changes lead to illness? (3) What factors could induce susceptibility to the adversity of normal development? Responses to these questions should influence

Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters

http://dx.doi.org/10.5772/intechopen.70868

17

the awareness of all social areas for the child's well-being and health throughout life.

The findings reviewed here explore some biological mechanisms that could explain the linkages between childhood negative experiences, possible diseases, and function of glutamate and GABA transporters. These results demonstrate efforts to improve quality of life throughout life. With the emergence of new tools, such as the biomarkers of early adversity, this will enable a new path of research with the close collaboration of physicians, health professionals, families, and communities on the basis of a deep understanding of the long term from early adversity.

This work was supported by UBACYT grants B019 from the University of Buenos Aires and PIP N° 114-2009-0100118 from National Scientific and Technologic Research Council (CONICET) to

The authors report no conflicts of interest. The authors alone are responsible for the content and

Institute of Pharmacological Research (ININFA), National Scientific and Technologic Research Council (CONICET), School of Pharmacy and Biochemistry, University of Buenos

**7. Conclusions**

**Acknowledgements**

**Conflict of interest**

writing of the paper.

**Author details**

Gabriela Beatriz Acosta

Aires, Buenos Aires, Argentina

Address all correspondence to: gacosta@ffyb.uba.ar

GBA. GBA is member of CONICET.

Previous studies have indicated that exposure to variable types of stressors during development produces persistent behavioral defects that are associated with hormonal, neurotransmitters, transporters, and functional changes, and resemble an array of psychopathological conditions.

Altered glutamate receptor (GluR) expression has been implicated in the pathogenesis of stressinduced disorders. Adrover et al. [73] have shown that glutamate neurotransmission might be impaired in the brain of prenatally stressed rats. They observed an increased uptake capacity for glutamate in the PFC of prenatal stress males, while no such changes were observed in the Hic. They concluded that prenatal stress produced long-term changes in the glutamatergic system, modulating the expression of glutamate transporters and altering synaptic transmission in the adult brain.

Odeon et al. [74] found that both ethanol intake and activity and protein expression of GluTs in certain areas of the rat brain are affected by repeated maternal separation (RMS). Also, they demonstrated that RMS increases glutamate uptake in frontal cortex and hippocampus, and RMS reduced both GLT-1 and EAAT-3 protein expression and increased GLAST protein levels.

Social loneliness has been used intensively as an animal model to study the consequences of social isolation during childhood on the brain and behavior. There is a crucial stage during which social isolation has very profound and sometimes irreversible effects.

Recent studies indicate that there are many aspects of alcohol and drug dependence that involve changes in glutamate transmission. Different investigations have reported that drugs of abuse, including alcohol and cocaine, modify GluTs [75, 76]. The effects of ethanol on glutamate transport may be mediated in part by the level of Ca2+/calmodulin kinase activity [77]. Similarly, Othman et al. [78] indicated that in rat cortical astrocytes *in vitro* ethanol affects [3 H]-Glutamate uptake by affecting protein kinase C (PKC) modulation of transporter activity.

Odeon et al. [74] observed changes following RMS in the glutamatergic system which could be an effect of glucocorticoid. It is known that this hormone may regulate GluT expression [79] and ethanol intake [80]. A significant increase in glutamate uptake is observed. However, protein levels of the major glial (GLT-1) and neuronal (EAAT-3) transporters declined. It should be noted that a third glutamate transporter, GLAST, was found in glia of the frontal cortex, and hippocampus was studied. This transporter exhibits increased levels of protein expression after treatment. This could be due to the decreased expression of the major glutamate uptake proteins and the probable excitotoxic consequence, which triggered a compensatory mechanism through the increase of GLAST.

GluTs are neuronal and non-neuronal factors necessary for expression, maintenance, and transcriptional regulators of these proteins. The finding that RMS altered Glu regulation in the frontal cortex and hippocampus indicates a possible role for distorted glutamate regulation in the causal relationship between early life stress. Finally, I have some specific questions about this work: (1) Can early exposures with limited time produce lasting physiological changes? (2) Can these physiological changes lead to illness? (3) What factors could induce susceptibility to the adversity of normal development? Responses to these questions should influence the awareness of all social areas for the child's well-being and health throughout life.

#### **7. Conclusions**

high-affinity GluTs were cloned into human and animal tissues and identified as a glutamate aspartate transporter (GLAST), excitatory amino acid carrier-1 (EAAT)-1, glial glutamate transporter-1 (GLT-1, EAAT-2); excitatory amino acid-carrier-1 (EAAC-1, EAAT-3), EAAT-4, and EAAT-5. Unlike other neurotransmitters, the action of Glu released into the synaptic cleft is terminated by uptake into neurons and surrounding glial cells via specific transporters. Within the nerve terminal, the glutamine released by glial cells and taken up by neurons is converted back to Glu [71]. Rapid removal of Glu from the extracellular space is required for the survival and normal function of neurons. Although GluTs are expressed by all CNS cell types, astrocytes are the cell type primarily responsible for Glu uptake [72]. Astrocytes express both GLT-1 and GLAST, while axon terminals in the neocortex only express GLT-1. Previous studies have indicated that exposure to variable types of stressors during development produces persistent behavioral defects that are associated with hormonal, neurotransmitters, transporters, and functional changes, and resemble an array of psychopathological conditions. Altered glutamate receptor (GluR) expression has been implicated in the pathogenesis of stressinduced disorders. Adrover et al. [73] have shown that glutamate neurotransmission might be impaired in the brain of prenatally stressed rats. They observed an increased uptake capacity for glutamate in the PFC of prenatal stress males, while no such changes were observed in the Hic. They concluded that prenatal stress produced long-term changes in the glutamatergic system, modulating the expression of glutamate transporters and altering synaptic transmission in the adult brain. Odeon et al. [74] found that both ethanol intake and activity and protein expression of GluTs in certain areas of the rat brain are affected by repeated maternal separation (RMS). Also, they demonstrated that RMS increases glutamate uptake in frontal cortex and hippocampus, and RMS reduced both GLT-1 and EAAT-3 protein expression and increased GLAST protein levels. Social loneliness has been used intensively as an animal model to study the consequences of social isolation during childhood on the brain and behavior. There is a crucial stage during

16 GABA And Glutamate - New Developments In Neurotransmission Research

which social isolation has very profound and sometimes irreversible effects.

tory mechanism through the increase of GLAST.

[3

Recent studies indicate that there are many aspects of alcohol and drug dependence that involve changes in glutamate transmission. Different investigations have reported that drugs of abuse, including alcohol and cocaine, modify GluTs [75, 76]. The effects of ethanol on glutamate transport may be mediated in part by the level of Ca2+/calmodulin kinase activity [77]. Similarly, Othman et al. [78] indicated that in rat cortical astrocytes *in vitro* ethanol affects

H]-Glutamate uptake by affecting protein kinase C (PKC) modulation of transporter activity. Odeon et al. [74] observed changes following RMS in the glutamatergic system which could be an effect of glucocorticoid. It is known that this hormone may regulate GluT expression [79] and ethanol intake [80]. A significant increase in glutamate uptake is observed. However, protein levels of the major glial (GLT-1) and neuronal (EAAT-3) transporters declined. It should be noted that a third glutamate transporter, GLAST, was found in glia of the frontal cortex, and hippocampus was studied. This transporter exhibits increased levels of protein expression after treatment. This could be due to the decreased expression of the major glutamate uptake proteins and the probable excitotoxic consequence, which triggered a compensaThe findings reviewed here explore some biological mechanisms that could explain the linkages between childhood negative experiences, possible diseases, and function of glutamate and GABA transporters. These results demonstrate efforts to improve quality of life throughout life. With the emergence of new tools, such as the biomarkers of early adversity, this will enable a new path of research with the close collaboration of physicians, health professionals, families, and communities on the basis of a deep understanding of the long term from early adversity.

#### **Acknowledgements**

This work was supported by UBACYT grants B019 from the University of Buenos Aires and PIP N° 114-2009-0100118 from National Scientific and Technologic Research Council (CONICET) to GBA. GBA is member of CONICET.

#### **Conflict of interest**

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

#### **Author details**

Gabriela Beatriz Acosta

Address all correspondence to: gacosta@ffyb.uba.ar

Institute of Pharmacological Research (ININFA), National Scientific and Technologic Research Council (CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

### **References**

[1] McEwen BS, Gianaros PJ. Stress-and allostasis-induced brain plasticity. Annual Review of Medicine. 2011;**62**:431-445

[15] Nestler EJ. From neurobiology to treatment: Progress against addiction. Nature Neuro-

Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters

http://dx.doi.org/10.5772/intechopen.70868

19

[16] Ehlert U, Gaab J, Heinrichs M. Psychoneuroendocrinological contributions to the etiology of depression, posttraumatic stress disorder, and stress-related bodily disorders: The role of the hypothalamus-pituitary-adrenal axis. Biological Psychology. 2001;**57**(1-3):141-152

[17] Gluckman PD, Hanson MA, Cooper C, Thornburg KL.Effect of in utero and early-life conditions on adult health and disease. The New England Journal of Medicine. 2008;**359**(1):61-73

[18] Zhang TY, Meaney MJ. Epigenetics and the environmental regulation of the genome and

[19] Heim C, Plotsky PM, Nemeroff CM. Importance of studying the contributions of early adverse experience to neurobiological findings in depression. Neuropsychopharmacology.

[20] Novais A, Monteiro S, Roque S, Correia-Neves M, Sousa N. How age, sex and genotype

[21] Bellis MA, Lowey H, Leckenby N, Hughes K, Harrison D. Adverse childhood experiences: Retrospective study to determine the impact on adult health behaviours and health

[22] Beckie TM.A systematic review of allostatic load, health, and health disparities. Biological

[23] Beardslee WR, Chien PL, Bell CC. Prevention of mental disorders, substance abuse, and problem behaviors: A developmental perspective. Psychiatric Services. 2011;**62**:247-254

[24] Ismail FY, Fatemi A, Johnston MV. Cerebral plasticity: Windows of opportunity in the developing brain. European Journal of Paediatric Neurology. 2017;**21**(1):23-48

[25] Knickmeyer RC, Gouttard S, Kang C, Evans D, Wilber K, Smith JK, Hamer RM, Lin W, Gerig G, Gilmore JH. A structural MRI study of human brain development from birth to 2

[26] Levitt P. Structural and functional maturation of the developing primate brain. The Journal

[27] Lehmann J, Feldon J. Long-term biobehavioral effects of maternal separation in the rat:

[28] Marco EM, Adriani W, Llorente R, Laviola G, Viveros MP. Detrimental psychophysiological effects of early maternal deprivation in adolescent and adult rodents: Altered responses to cannabinoid exposure. Neuroscience and Biobehavioral Reviews. 2009;**33**:498-507 [29] Anisman H, Zaharia MD, Meaney MJ, Merali Z. Do early life events permanently alter behavioral and hormonal response to stressors? Int J Dev Neurosc. 1998;**16**:149-167 [30] Roman E, Gustafsson L, Berg M, Nylander I. Behavioral profiles and stress induced corticosteroid secretion in male Wistar rats subjected to short and prolonged periods of

Consistent or confusing? Reviews in the Neurosciences. 2000;**11**(4):383-408

maternal separation. Hormones and Behavior. 2006;**50**:736-747

outcomes in a UK population. Journal of Public Health (Oxford). 2014;**36**:81-91

its function. Annual Review of Psychology. 2010;**61**:439-466

shape the stress response. Neurobiology of Stress. 2016;**6**:44-45

years. The Journal of Neuroscience. 2008;**28**:12176-12182

science. 2002;**5**(Suppl):1076-1079

Research for Nursing. 2012;**14**:311-346

of Pediatrics. 2003;**143**:S35-S45

2004;**29**:641-648


[15] Nestler EJ. From neurobiology to treatment: Progress against addiction. Nature Neuroscience. 2002;**5**(Suppl):1076-1079

**References**

of Medicine. 2011;**62**:431-445

18 GABA And Glutamate - New Developments In Neurotransmission Research

Pharmacology. 2013;**3**(2):121-125

endocrinology. 2014;**26**:707-723

2008;**42**(3):209-220

2013;**28**:196-206

2014;**283**:166-177

[1] McEwen BS, Gianaros PJ. Stress-and allostasis-induced brain plasticity. Annual Review

[2] Deppermann S, Storchak H, Fallgatter AJ, Ehlis A-C. Stress-induced neuroplasticity: (Mal) adaptation to adverse life events in patients with PTSD.A critical overview. Neuroscience.

[3] Odeon MM, Salatino AE, Acosta GB. Consequences of postnatal stress: Maternal separation in rats induces long-lasting changes on glutamate transporters. Clinical and Experimental

[4] Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB. The role of corticotropin-releasing factor in depression and anxiety disorders. The Journal of Endocrinology. 1999;**160**:1-12

[5] Smith SM, Vale WW. The role of the hypothalamic-pituitary-adrenal axis in neuroendo-

[6] Fumagalli F, Molteni R, Racagni G, Riva MA. Stress during development: Impact on neuroplasticity and relevance to psychopathology. Progress in Neurobiology. 2007;**1**(4):197-217

[7] Pryce CR, Rüedi-Bettschen D, Dettling AC, Weston A, Russig H, Ferger B, Feldon J. Longterm effects of early-life environmental manipulations in rodents and primates: Potential animal in depression research. Neuroscience and Biobehavioral Reviews. 2005;**29**:649-674

[8] Maccari S, Krugers HJ, Morley-Fletcher S, Szyf M, Brunton PJ. The consequences of earlylife adversity: Neurobiological: Behavioural and epigenetic adaptations. Journal of Neuro-

[9] Fletcher A, Bonell C, Hargreaves J. School effects on young people's drug use: A systematic review of intervention and observational studies. The Journal of Adolescent Health.

[10] Diz-Chaves Y, Astiz M, Bellini MJ, Garcia-Segura LM. Prenatal stress increases the expression of proinflammatory cytokines and exacerbates theinflammatory response to LPS in the hippocampal formation of adult male mice. Brain, Behavior, and Immunity.

[11] Berens AE, Jensen SKG, Nelson CA III. Biological embedding of childhood adversity: From physiological mechanisms to clinical implications. BMC Medicine. 2017;**15**:135-138

[12] Koenig JI, Kirkpatrick B, Lee P. Glucocorticoid hormones and early brain development

[13] Lupien SJ, McEwen BS, Gunnar MR, Heim C. Effects of stress throughout the lifespan on the brain, behaviour and cognition. Nature Reviews. Neuroscience. 2009;**10**(6):434-445

[14] Kofman O. The role of prenatal stress in the etiology of developmental behavioural dis-

in schizophrenia. Neuropsychopharmacology. 2002;**27**:309-318

orders. Neuroscience and Biobehavioral Reviews. 2002;**26**:457-470

crine responses to stress. Dialogues in Clinical Neuroscience. 2006;**8**:383-395


[31] O'Shea RD. Roles and regulation of glutamate transporters in the central nervous system. Clinical and Experimental Pharmacology & Physiology. 2002;**29**:1018-1023

[46] Ganguly K, Schinder AF, Wong ST, Poo M.GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell. 2001;**105**(4):521-532

Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters

http://dx.doi.org/10.5772/intechopen.70868

21

[47] Ben-Ari Y. Excitatory actions of GABA during development: The nature of the nurture.

[48] Tyzio R, Minlebaev M, Rheims S, Ivanov A, Jorquera I, Holmes GL, Zilberter Y, Ben-Ari Y, Khazipov R. Postnatal changes in somatic gamma-aminobutyric acid signalling in the rat

[49] Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, Fukuda A. Cl- uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1.

[50] McEwen BS. Sex, stress and the hippocampus: Allostasis, allostatic load and the aging

[51] McEwen BS. Physiology and neurobiology of stress and adaptation: Central role of the

[52] Minelli A, Barbaresi P, Conti F. Postnatal development of high-affinity plasma membrane GABA transporters GAT-2 and GAT-3 in the rat cerebral cortex. Brain Research. Develop-

[53] Yan XX, Cariaga WA, Ribak CE. Immunoreactivity for GABA plasma membrane transporter, GAT-1, in the developing rat cerebral cortex: Transient presence in the somata of neocortical and hippocampal neurons. Brain Research. Developmental Brain Research.

[54] Ikegaki N, Saito N, Hashima M, Tanaka C. Production of specific antibodies against GABA transporter subtypes (GAT-1, GAT-2, GAT-3) and their application to immunocytochemis-

[55] Liu QR, Lopez-Caarcuera B, Mandiyan S, Nelson H, Nelson N. Molecular characterization of four pharmacological distinct γ -aminobutyric acid transporters in the mouse brain.

[56] Radian R, Ottersen OP, Storm-Mathisen J, Castel M, Kanner BI. Immunocytochemical localization of the GABA transporter in rat brain. The Journal of Neuroscience. 1990;**10**(4):

[57] Conti F, Vitellaro-Zuccarello L, Barbaresi P, Minelli A, Brecha NCMelone M. Neuronal, glial, and epithelial localization of γ -aminobutyric acid transporter 2, a high-affinity γ aminobutyric acid plasma membrane transporter, in the cerebral cortex and neighboring

[58] Kinney GA. GAT-3 transporters regulate inhibition in the neocortex. Journal of Neuro-

[59] Minelli A, Alonso-Nanclares L, Edwards RH, De Felipe J, Conti F. Postnatal development of the vesicular GABA transporter in rat cerebral cortex. Neuroscience. 2003;**117**(2):

structures. The Journal of Comparative Neurology. 1999;**409**(3):482-494

hippocampus. The European Journal of Neuroscience. 2008;**27**(10):2515-2528

Nature Reviews. Neuroscience. 2002;(9):728-739

The Journal of Physiology. 2004;**557**(Pt 3):829-841

process. Neurobiology of Aging. 2002;**23**(5):921-939

brain. Physiological Reviews. 2007l;**87**(3):873-904

mental Brain Research. 2003;**142**(1):7-18

try. Molecular Brain Research. 1994;**26**:47-54

physiology. 2005;**94**(6):4533-4537

The Journal of Biological Chemistry. 1993;**268**(3):2106-2112

1997;**99**(1):1-19

1319-1330

337-346


[46] Ganguly K, Schinder AF, Wong ST, Poo M.GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell. 2001;**105**(4):521-532

[31] O'Shea RD. Roles and regulation of glutamate transporters in the central nervous system. Clinical and Experimental Pharmacology & Physiology. 2002;**29**:1018-1023

[32] Yehuda R, Antelman SM. Criteria for rationally evaluating animal models of posttrau-

[33] Tamaki K, Yamada K, Nakamichi N, Taniura H, Yoneda Y. Transient suppression of progenitor cell proliferation through NMDA receptors in hippocampal dentate gyrus of mice

[34] Kuhn CM, Pauk J, Schanberg SM. Endocrine responses to mother-infant separation in

[35] Vetulani J. Early maternal separation: A rodent model of depression and a prevailing human

[36] Jahng JW. An animal model of eating disorders associated with stressful experience in

[37] Lohof AM, Ip NY, Poo MM. Potentiation of developing neuromuscular synapses by the

[38] Corcoran C, Walker E, Huot R, Mittal V, Tessner K, Kestler L, Malaspina D. The stress cascade and schizophrenia: Etiology and onset. Schizophrenia Bulletin. 2003;**29**(4):671-692

[39] de Kloet ER, Joëls M, Holsboer F. Stress and the brain: From adaptation to disease. Nature

[40] Levine ES, Dreyfus CF, Black IB, Plummer MR. Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors. Proceedings of the National Academy of Sciences of the United States of America.

[41] Huang EJ, Reichardt LF. Neurotrophins: Roles in neuronal development and function.

[42] Giachino C, Canalia N, Capone F, Fasolo A, Alleva E, Riva MA, Cirulli F, Peretto P. Maternal deprivation and early handling affect density of calcium binding proteincontaining neurons in selected brain regions and emotional behavior in periadolescent

[43] Joëls M, Baram TZ. The neuro-symphony of stress. Nature Reviews. Neuroscience. 2009;

[44] Borden LA. GABA transporter heterogeneity: Pharmacology and cellular localization.

[45] Conti F, Minelli A, Melone M. GABA transporters in the mammalian cerebral cortex: Localization, development and pathological implications. Brain Research Brain Research

with traumatic stress experience. Journal of Neurochemistry. 2008;**105**:1642-1655

matic stress disorder. Biological Psychiatry. 1993;**33**:479-486

20 GABA And Glutamate - New Developments In Neurotransmission Research

developing rats. Developmental Psychobiology. 1990;**23**:395-410

condition. Pharmacological Reports. 2013;**65**:1451-1461

early life. Hormones and Behavior. 2011;**59**(2):213-220

Reviews. Neuroscience 2005; **6**(6):463-475

Annual Review of Neuroscience. 2001;**24**:677-736

Neurochemistry International. 1996;(4):335-356

rats. Neuroscience. 2007;**145**(2):568-578

1995;**92**:8074-8077

**10**(6):459-466

Review. 2004;**3**:196-212

neurotrophins NT-3 and BDNF. Nature. 1993;**363**:350-352


[60] Odeon MM, Salatino AE, Acosta GB. Consequences of early adverse life events on GABAergic neurons produce long term changes in brain function. Trends in Comparative Biochemistry & Physiology. 2011;**15**:67-72

[74] Odeon MM, Andreu M, Yamauchi L, Grosman M, Acosta GB. Chronic postnatal stress induces voluntary alcohol intake and modifies glutamate transporters in adolescent rats.

Early Life Experience, Maternal Separation, and Involvement of GABA and Glutamate Transporters

http://dx.doi.org/10.5772/intechopen.70868

23

[75] Chappell AM, Carter E, McCool BA, Weiner JL. Adolescent rearing conditions influence the relationship between initial anxiety-like behavior and ethanol drinking in male long Evans rats. Alcoholism, Clinical and Experimental Research. 2013;**37**(Suppl 1):E394-E403

[76] Rao PSS, Sari Y. Glutamate transporter 1: Target for the treatment of alcohol dependence.

[77] Smith TL, Navratilova E. Increased calcium/calmodulin protein kinase activity in astrocytes chronically exposed to ethanol: Influences on glutamate transport. Neuroscience Letters.

[78] Othman T, Sinclair CJ, Haughey N, Geiger JD, Parkinson FE. Ethanol alters glutamate but not adenosine uptake in rat astrocytes: Evidence for protein kinase C involvement. Neuro-

[80] Costin BN, Wolen AR, Fitting S, Shelton KL, Miles MF. Role of adrenal glucocorticoidsignaling in prefrontal cortex gene expression and acute behavioral responses to ethanol.

[79] Danbolt NC. Glutamate uptake. Progress in Neurobiology. 2001;**65**(1):1-105

Alcoholism, Clinical and Experimental Research. 2013;**37**(1):57-66

Stress. 2015;**18**(4):427-434

1999;**269**(3):145-148

Current Medicinal Chemistry. 2012;**19**:5148-5156

chemical Research. 2002;**27**(4):289-296


[74] Odeon MM, Andreu M, Yamauchi L, Grosman M, Acosta GB. Chronic postnatal stress induces voluntary alcohol intake and modifies glutamate transporters in adolescent rats. Stress. 2015;**18**(4):427-434

[60] Odeon MM, Salatino AE, Acosta GB. Consequences of early adverse life events on GABAergic neurons produce long term changes in brain function. Trends in Comparative

[61] Perego C, Bulbarelli A, Longhi R, Caimi M, Villa A, Caplan MJ, Pietrini G. Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized

[62] Levine S, Mody T. The long-term psychobiological consequences of intermittent postnatal separation in the squirrel monkey. Neuroscience and Biobehavioral Reviews.

[63] Ellenbroek BA, van den Kroonenberg PT, Cools AR. The effects of an early stressful life event on sensorimotor gating in adult rats. Schizophrenia Research 1998;**30**(3):251-260

[64] Lehmann J, Stöhr T, Schuller J, Domeney A, Heidbreder C, Feldon J. Long-term effects of repeated maternal separation on three different latent inhibition paradigms. Pharma-

[65] Ellenbroek BA, Cools AR. The long-term effects of maternal deprivation depend on the

[66] Komuro H, Rakic P. Modulation of neuronal migration by NMDA receptors. Science.

[67] LoTurco JJ, Blanton MG, Kriegstein AR. Initial expression and endogenous activation of NMDA channels in early neocortical development. The Journal of Neuroscience. 1991;

[68] Reznikov LR, Grillo CA, Piroli GG, Pasumarthi RK, Reagan LP, Fadel J. Acute stressmediated increases in extracellular glutamate levels in the rat amygdala: Differential effects of antidepressant treatment. The European Journal of Neuroscience. 2007;**25**(10):

[69] Matute C, Alberdi E, Ibarretxe G, Sánchez-Gómez MV. Excitotoxicity in glial cells. European

[70] Thomas CG, Tian H, Diamond JS. The relative roles of diffusion and uptake in clearing synaptically released glutamate change during early postnatal development. The Journal

[71] Kanai Y, Smith CP, Hediger MA. The elusive transporters with a high affinity for gluta-

[72] Anderson CM, Swanson RA. Astrocyte glutamate transport: Review of properties, regu-

[73] Adrover E, Pallarés ME, Baier CJ, Monteleone MC, Giuliani FA, Waagepetersen HS, Brocco MA, Cabrera R, Sonnewald U, Schousboe A, Antonelli MC. Glutamate neurotransmission is affected in prenatally stressed offspring. Neurochemistry International. 2005;**88**:73-87

epithelial cells. The Journal of Biological Chemistry. 1997;**272**(10):6584-6592

Biochemistry & Physiology. 2011;**15**:67-72

22 GABA And Glutamate - New Developments In Neurotransmission Research

cology, Biochemistry, and Behavior. 1998;**59**(4):873-882

Journal of Pharmacology. 2002;**447**(2-3):239-246

mate. Trends in Neurosciences. 1993;(9):365-370

lation, and physiological functions. Glia. 2000, 2000;**32**(1):1-14

of Neuroscience. 2011;**31**(12):4743-4754

genetic background. Neuropsychopharmacology. 2000;**23**(1):99-106

2003;**27**(1-2):83-89

1993;**260**:95-97

**11**:792-799

3109-3114


**Chapter 3**

**Provisional chapter**

**Notch Signaling in the Astroglial Phenotype: Relevance**

Glutamate (Glu), the major excitatory neurotransmitter, elicits its action through the activation of membrane receptors and transporters expressed in neurons and glial cells. Glial glutamate transporters, EAAT1 and EAAT2, remove this transmitter from the synaptic cleft preventing an excitotoxic insult. The Notch pathway is a signaling system involved in neuro- and gliogenesis. Radial glia (RG) generates neurons, oligodendrocytes, and astrocytes in a spatial and temporal pattern, in which Notch represses neurogenesis, maintaining the self-renewal potential of RG. Astrogenesis depends on several stimuli, Notch being a master regulator of the differentiation process. The cAMP-PKA-CREB signaling cascade cross talks with the Notch pathway, acting synergistically by reducing progenitor markers and inducing astrocytic differentiation. Notch1 mRNA is upregulated in a PKA/γ-secretase/NICD/CSL-dependent manner, suggesting a feedback loop to keep Notch active until astrocytic differentiation is complete. Glial differentiation is also modulated by PKC, which acts over NICD. In RG cells and astrocytes enwrapping glutamatergic synapses, EAAT1 transcriptional regulation is mediated by PKC, increasing Notch expression and its receptor intracellular traffic. It is clear that Notch represents an activity-dependent molecular key in RG cells that enable them to shape glutamatergic transmission through the expression of genes involved in glial/neuronal interactions.

**Notch Signaling in the Astroglial Phenotype: Relevance** 

DOI: 10.5772/intechopen.73318

© 2016 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,

© 2018 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.

and reproduction in any medium, provided the original work is properly cited.

Glutamate (Glu), the major excitatory neurotransmitter in the central nervous system (CNS), is a key player in higher brain functions such as learning and memory, and it is also

**Keywords:** glia cells, signal transduction, Notch, differentiation, protein kinase C

**to Glutamatergic Transmission**

**to Glutamatergic Transmission**

López-Bayghen Esther, Angulo-Rojo Carla,

López-Bayghen Esther, Angulo-Rojo Carla,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73318

Ramírez Leticia and Ortega Arturo

Hernández-Melchor Dinorah, Ramírez Leticia and

López-Bayghen Bruno, Hernández-Melchor Dinorah,

López-Bayghen Bruno,

Ortega Arturo

**Abstract**

**1. Introduction**

**Provisional chapter**

#### **Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission**

DOI: 10.5772/intechopen.73318

López-Bayghen Esther, Angulo-Rojo Carla, López-Bayghen Bruno, Hernández-Melchor Dinorah, Ramírez Leticia and Ortega Arturo López-Bayghen Esther, Angulo-Rojo Carla, López-Bayghen Bruno, Hernández-Melchor Dinorah, Ramírez Leticia and Ortega Arturo Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73318

#### **Abstract**

Glutamate (Glu), the major excitatory neurotransmitter, elicits its action through the activation of membrane receptors and transporters expressed in neurons and glial cells. Glial glutamate transporters, EAAT1 and EAAT2, remove this transmitter from the synaptic cleft preventing an excitotoxic insult. The Notch pathway is a signaling system involved in neuro- and gliogenesis. Radial glia (RG) generates neurons, oligodendrocytes, and astrocytes in a spatial and temporal pattern, in which Notch represses neurogenesis, maintaining the self-renewal potential of RG. Astrogenesis depends on several stimuli, Notch being a master regulator of the differentiation process. The cAMP-PKA-CREB signaling cascade cross talks with the Notch pathway, acting synergistically by reducing progenitor markers and inducing astrocytic differentiation. Notch1 mRNA is upregulated in a PKA/γ-secretase/NICD/CSL-dependent manner, suggesting a feedback loop to keep Notch active until astrocytic differentiation is complete. Glial differentiation is also modulated by PKC, which acts over NICD. In RG cells and astrocytes enwrapping glutamatergic synapses, EAAT1 transcriptional regulation is mediated by PKC, increasing Notch expression and its receptor intracellular traffic. It is clear that Notch represents an activity-dependent molecular key in RG cells that enable them to shape glutamatergic transmission through the expression of genes involved in glial/neuronal interactions.

**Keywords:** glia cells, signal transduction, Notch, differentiation, protein kinase C

#### **1. Introduction**

Glutamate (Glu), the major excitatory neurotransmitter in the central nervous system (CNS), is a key player in higher brain functions such as learning and memory, and it is also

© 2016 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. © 2018 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.

involved in cell differentiation and synaptogenesis. Glu exerts its function through specific receptors, according to which the signal transduction pathway mechanisms are classified into two major groups: ionotropic (iGluRs) and metabotropic receptors (mGluRs). iGluRs are ligand-gated ion channels subdivided upon pharmacological and electrophysiological properties into NMDA, AMPA, and KA receptors. mGluRs are G protein-coupled receptors subdivided in accordance with their amino acidic sequence and pharmacological properties into three subgroups, preferentially activated by quisqualate (Quis), t-ACPD, and L-AP4, respectively [1–4].

transporters particularly enriched in glial cells is responsible for the removal of this transmitter from the synaptic cleft [12]. These transporters, known as excitatory amino acid transporters (EAAT), are differentially expressed in neurons and astrocytes. EAAT3, 4, and 5 are mainly neuronal, whereas EAAT1 and EAAT2 are glial, although the latter one has also been found to be present in certain neuronal populations [18]. Once Glu has been taken up by glial cells, it is mostly converted to glutamine (Gln) by the glial-expressed Gln synthetase to be released in the vicinity of the presynaptic terminal, a process known as the Glu/Gln shuttle, in which an exquisite interplay between neurons and glial cells is fundamental for the proper function of glutamatergic transmission [12]. In this context, glutamatergic synapses are a perfect example

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

27

Notch signaling involves cell to cell communication and has a simple core. It initiates when the Notch receptor (Notch 1-4), present in the receiving-signal cell, binds its ligand (Jagged/Delta-like) present in the sending-signal cell. This binding promotes two sequential proteolytic cleavages on the Notch receptor: the first is mediated by the protease ADAM10/TACE (tumor necrosis factor α converting enzyme) to generate the membranetethered intermediated Notch extracellular truncation (NEXT). The second cleavage is mediated by the γ-Secretase enzyme on NEXT, to release the signal effector Notch intracellular domain (NICD) into the cytoplasm. NICD is translocated to the nucleus where it binds the transcription factor CSL (CBF1/RBPJκ in vertebrates, suppressor of hairless in *Drosophila*, Lag-1 in *C. elegans*) to activate what is known as the canonical Notch pathway. In the absence of NICD, CSL associates with the ubiquitous co-repressors (Co-R): SKIP, CtBP/Hairless, SMRT, CIR, FLH1C/KyoT2, SHARP/MINT and Gro/TLE proteins, and histone deacetylases (HDACs) to halt the transcription of Notch target genes. Once NICD binds CSL, allosteric changes may occur on CSL that facilitates displacement of transcriptional repressors. The transcriptional co-activator Mastermind (MAM) then recognizes the NICD/CSL interface, and this tri-protein complex recruits additional co-activators (Co-A) to promote transcription of target genes, as the astroglial markers shown in **Table 1**

The noncanonical Notch signaling pathway is CSL-independent and can as well be either ligand-dependent or independent. Nevertheless, one has to keep in mind that the NICD/ CSL complex is the major effector of Notch signaling. Several pieces of evidence have demonstrated that the Notch pathway may signal independently of CSL. It was first reported that Notch could signal via the RING-domain of E3 ubiquitin ligase Deltex1 (DTX1) [28]. It has also been shown that NEXT binds NICD on its ankyrin repeats [29], leading to its nuclear translocation. It has been documented as well that the NICD/DTX1 complex interacts with the transcriptional co-activator p300 inhibiting the transcriptional activation of the neural-specific transcription factor MASH1 [30]. As it will be described later, other genes important for astro-

of what has been lately known as a tripartite synapse [19].

glial differentiation are also targets of NICD/DTX (**Table 1**).

**3. Notch signaling**

[20–27].

Cerebellar Bergmann glia cells (BGC) are radial glia (RG) cells that are not differentiated into astrocytes after birth [5] and function as a neuronal reservoir [6, 7]. These cells extend processes through the molecular layer completely surrounding excitatory synapses between Purkinje cells and both parallel and climbing fibers. An exquisite and complex interplay between presynaptic-postsynaptic neurons and glia cells is fundamental for glutamatergic transmission. Glu recycling depends upon these interactions. Glu is removed from the synaptic cleft by a family of electrogenic sodium-dependent transporters expressed in neurons and glia cells [8]. Five subtypes of transporters named excitatory amino acid transporters 1-5 (EAAT1-5) have been characterized. The glial transporters EAAT-1 (GLAST) and EAAT-2 (GLT-1) account for more than 80% of the Glu uptake activity in the brain [9, 10]. Within BGC, EAAT-1/GLAST is the predominant transporter [11].

Once internalized, Glu is metabolized to Gln via Gln synthetase and released in the vicinity of the presynaptic neuron through sodium-dependent neutral amino acid transporter (SNAT) 3. Gln is then taken up by the presynaptic neuron through SNAT 2 and converted back to Glu by the enzyme glutaminase to be packed into synaptic vesicles completing the so-called Glu/ Gln shuttle (reviewed in [12]). It is this kind of glial/neuronal interactions that gave rise to what has been known in the last years as a tripartite synapse [13]. Evidence suggests that Glu transporters might also participate in the signaling transactions triggered by this excitatory amino acid. In fact, Glu regulates the uptake process in a receptor-independent manner [14]. More recently, it has also been reported that EAAT-1 is coupled to the Na<sup>+</sup> /K<sup>+</sup> ATPase [15, 16] and to the Gln transporter SNAT3 [17].

In this context, we reviewed in this contribution the role of Notch signaling in RG focusing in its role in EAAT-1/GLAST regulation as a key element in the molecular mechanisms that support the proven glia contribution to glutamatergic neurotransmission.

#### **2. Glutamatergic transmission: role of glial cells**

Glutamate (Glu) is the major excitatory neurotransmitter in the vertebrate brain. It elicits its action through the activation of specific membrane receptors and transporters expressed both in neurons and in glial cells. Extracellular glutamate levels have to be tightly regulated in order to prevent Glu receptors over-stimulation that has been shown to result in neuronal death, phenomena commonly known as excitotoxicity. A family of sodium-dependent Glu transporters particularly enriched in glial cells is responsible for the removal of this transmitter from the synaptic cleft [12]. These transporters, known as excitatory amino acid transporters (EAAT), are differentially expressed in neurons and astrocytes. EAAT3, 4, and 5 are mainly neuronal, whereas EAAT1 and EAAT2 are glial, although the latter one has also been found to be present in certain neuronal populations [18]. Once Glu has been taken up by glial cells, it is mostly converted to glutamine (Gln) by the glial-expressed Gln synthetase to be released in the vicinity of the presynaptic terminal, a process known as the Glu/Gln shuttle, in which an exquisite interplay between neurons and glial cells is fundamental for the proper function of glutamatergic transmission [12]. In this context, glutamatergic synapses are a perfect example of what has been lately known as a tripartite synapse [19].

#### **3. Notch signaling**

involved in cell differentiation and synaptogenesis. Glu exerts its function through specific receptors, according to which the signal transduction pathway mechanisms are classified into two major groups: ionotropic (iGluRs) and metabotropic receptors (mGluRs). iGluRs are ligand-gated ion channels subdivided upon pharmacological and electrophysiological properties into NMDA, AMPA, and KA receptors. mGluRs are G protein-coupled receptors subdivided in accordance with their amino acidic sequence and pharmacological properties into three subgroups, preferentially activated by quisqualate (Quis), t-ACPD, and L-AP4,

Cerebellar Bergmann glia cells (BGC) are radial glia (RG) cells that are not differentiated into astrocytes after birth [5] and function as a neuronal reservoir [6, 7]. These cells extend processes through the molecular layer completely surrounding excitatory synapses between Purkinje cells and both parallel and climbing fibers. An exquisite and complex interplay between presynaptic-postsynaptic neurons and glia cells is fundamental for glutamatergic transmission. Glu recycling depends upon these interactions. Glu is removed from the synaptic cleft by a family of electrogenic sodium-dependent transporters expressed in neurons and glia cells [8]. Five subtypes of transporters named excitatory amino acid transporters 1-5 (EAAT1-5) have been characterized. The glial transporters EAAT-1 (GLAST) and EAAT-2 (GLT-1) account for more than 80% of the Glu uptake activity in the brain [9, 10]. Within BGC,

Once internalized, Glu is metabolized to Gln via Gln synthetase and released in the vicinity of the presynaptic neuron through sodium-dependent neutral amino acid transporter (SNAT) 3. Gln is then taken up by the presynaptic neuron through SNAT 2 and converted back to Glu by the enzyme glutaminase to be packed into synaptic vesicles completing the so-called Glu/ Gln shuttle (reviewed in [12]). It is this kind of glial/neuronal interactions that gave rise to what has been known in the last years as a tripartite synapse [13]. Evidence suggests that Glu transporters might also participate in the signaling transactions triggered by this excitatory amino acid. In fact, Glu regulates the uptake process in a receptor-independent manner [14].

In this context, we reviewed in this contribution the role of Notch signaling in RG focusing in its role in EAAT-1/GLAST regulation as a key element in the molecular mechanisms that

Glutamate (Glu) is the major excitatory neurotransmitter in the vertebrate brain. It elicits its action through the activation of specific membrane receptors and transporters expressed both in neurons and in glial cells. Extracellular glutamate levels have to be tightly regulated in order to prevent Glu receptors over-stimulation that has been shown to result in neuronal death, phenomena commonly known as excitotoxicity. A family of sodium-dependent Glu

/K<sup>+</sup>

ATPase [15, 16]

More recently, it has also been reported that EAAT-1 is coupled to the Na<sup>+</sup>

support the proven glia contribution to glutamatergic neurotransmission.

**2. Glutamatergic transmission: role of glial cells**

respectively [1–4].

EAAT-1/GLAST is the predominant transporter [11].

26 GABA And Glutamate - New Developments In Neurotransmission Research

and to the Gln transporter SNAT3 [17].

Notch signaling involves cell to cell communication and has a simple core. It initiates when the Notch receptor (Notch 1-4), present in the receiving-signal cell, binds its ligand (Jagged/Delta-like) present in the sending-signal cell. This binding promotes two sequential proteolytic cleavages on the Notch receptor: the first is mediated by the protease ADAM10/TACE (tumor necrosis factor α converting enzyme) to generate the membranetethered intermediated Notch extracellular truncation (NEXT). The second cleavage is mediated by the γ-Secretase enzyme on NEXT, to release the signal effector Notch intracellular domain (NICD) into the cytoplasm. NICD is translocated to the nucleus where it binds the transcription factor CSL (CBF1/RBPJκ in vertebrates, suppressor of hairless in *Drosophila*, Lag-1 in *C. elegans*) to activate what is known as the canonical Notch pathway. In the absence of NICD, CSL associates with the ubiquitous co-repressors (Co-R): SKIP, CtBP/Hairless, SMRT, CIR, FLH1C/KyoT2, SHARP/MINT and Gro/TLE proteins, and histone deacetylases (HDACs) to halt the transcription of Notch target genes. Once NICD binds CSL, allosteric changes may occur on CSL that facilitates displacement of transcriptional repressors. The transcriptional co-activator Mastermind (MAM) then recognizes the NICD/CSL interface, and this tri-protein complex recruits additional co-activators (Co-A) to promote transcription of target genes, as the astroglial markers shown in **Table 1** [20–27].

The noncanonical Notch signaling pathway is CSL-independent and can as well be either ligand-dependent or independent. Nevertheless, one has to keep in mind that the NICD/ CSL complex is the major effector of Notch signaling. Several pieces of evidence have demonstrated that the Notch pathway may signal independently of CSL. It was first reported that Notch could signal via the RING-domain of E3 ubiquitin ligase Deltex1 (DTX1) [28]. It has also been shown that NEXT binds NICD on its ankyrin repeats [29], leading to its nuclear translocation. It has been documented as well that the NICD/DTX1 complex interacts with the transcriptional co-activator p300 inhibiting the transcriptional activation of the neural-specific transcription factor MASH1 [30]. As it will be described later, other genes important for astroglial differentiation are also targets of NICD/DTX (**Table 1**).


At the same time, Ngn1 becomes an astrogenesis inhibitor through the sequestration of p300/

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

29

In the cerebellum and in the immature RG, it has been demonstrated that Notch1 is activated by Jagged1 on newborn neuron progenitors; this interaction regulates the molecular and morphological differentiation of RG, through the transcriptional activation of BLBP and the erbB2 receptor tyrosine kinase. This effect is mediated by two downstream mechanisms, one that depends on RBPJκ (canonical activation) and the other depending on Deltex1 (DTX1) (noncanonical activation). In this manner, the induced erbB2 receptor interacts with its ligand neuregulin, present on neuronal progenitors, to facilitate cell migration through RG fibers (**Figure 1**) [32, 33].

After the neurogenesis period, at the end of embryonic development, most of the RG cells have lost their ventricular attachment and migrate toward the cortical plate by a process of somal translocation. In mammals, the majority of RG cells are transformed into astrocytes. During this period, astrocytic and oligodendrocytic intermediate precursors are also generated (aIPCs and oIPCs). Some studies suggest the presence of multipotent and bipotent pro-

Astrogenesis depends on several stimuli, being the Notch pathway a master regulator of the differentiation process. During the gliogenic phase, RG progenitors gain competence to generate astrocytes due to the activity of growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). This gain of competence allows them to respond to specific gliogenic signals acting at the extracellular level to activate astrocyte markers such as glial fibrillary acidic protein (GFAP), S100β, aquaporin 4, glutamate transporters (GLT-1, EACC1, and GLAST), and aldehyde dehydrogenase 1 family, member L1 (AldhL1) [35–39]. Before the astrocyte-marker promoters can respond to gliogenic signals, a chromatin epigenetic remodeling must occur. Notch canonical activation on RG induces expression of nuclear factor 1A (NFA1), an inhibitor of the DNA methyltransferase 1 (DNMT1). DNMT1 keeps

The extracellular signals are provided by neurotrophic cytokines such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1) secreted by newborn neurons. These cytokines activate heterodimeric cell surface receptors composed of two subunits named LIFRβ and gp130, which in turn activate to members of the JAK family of tyrosine kinases that result in the phosphorylation and nuclear translocation of signal transducer and activator of transcription (STAT) proteins. In RG, two of these proteins, STAT1 and STAT3, act on specific sites in the promoters of the astroglial genes GFAP and S100β to stimulate their transcription during the astrocyte differentiation process. Neural progenitors also respond to different neurotrophic factors from the bone morphogenetic proteins (BMP) family to generate astrocytes. In this case, BMP2 and BMP4 act on heterotrimeric receptors, which activate SMAD transcription factors. These, in turn, interact with activated STAT proteins to synergistically

stimulate transcription of glial-specific genes during astrocyte differentiation [42–47].

Another estrogen signal is the activation of the seven transmembrane domain G protein-coupled receptors by the pituitary adenylate cyclase-activating polypeptide (PACAP), triggering the differentiation of astrocytes by increasing intracellular cAMP and activating the cAMPdependent protein kinase (PKA), which translocates into the nucleus to phosphorylate and

genitors, and perhaps astrocyte-restricted progenitors in the neonatal SVZ [34].

STAT3 site of GFAP promoter methylated and inactive [40, 41].

CBP, a key inducer of astrocyte differentiation [29].

**Table 1.** Targets of Notch signaling pathway in central nervous system development.

#### **4. Notch pathway signaling in astroglial differentiation**

Notch pathway is a pivotal signaling system during neuro- and gliogenesis in the central nervous system (CNS) [24, 25]. Primary neural stem cells (NSC) are radial glia (RG) during development, characterized by the expression of astroglial markers such as the astrocytespecific glutamate/aspartate transporter (GLAST), the brain lipid-binding protein (BLBP), and tenascin C (TN-C) [31]. RG cells generate neurons, oligodendrocytes, and astrocytes in a characteristic spatial and temporal pattern [31]. In this context, the Notch pathway plays an essential role repressing neurogenesis and maintaining the self-renewal potential of RG.

On the neurogenic phase, RG divide asymmetrically for auto-renewal and generation of neurons or neuron-restricted intermediate progenitor cells (nIPCs, transit amplifying cells), which in turn populate the subventricular zone (SVZ) in the cortex. The newborn neurons migrate along parental RG fibers, even though RG are dividing [31]. The Notch pathway plays an important role during the neurogenic phase in several ways. In the cortex, recent findings suggest that Notch signaling among SVZ nIPCs and between nIPCs and RG is important in the regulation of progenitor proliferation and in the inhibition of precocious neuronal differentiation. RG receives Notch signaling to activate Hes1 and Hes5 transcription factors, which down-regulate pro-neuronal genes such as neurogenin 1 (Ngn1), Mash1, and Math. At the same time, Ngn1 becomes an astrogenesis inhibitor through the sequestration of p300/ CBP, a key inducer of astrocyte differentiation [29].

In the cerebellum and in the immature RG, it has been demonstrated that Notch1 is activated by Jagged1 on newborn neuron progenitors; this interaction regulates the molecular and morphological differentiation of RG, through the transcriptional activation of BLBP and the erbB2 receptor tyrosine kinase. This effect is mediated by two downstream mechanisms, one that depends on RBPJκ (canonical activation) and the other depending on Deltex1 (DTX1) (noncanonical activation). In this manner, the induced erbB2 receptor interacts with its ligand neuregulin, present on neuronal progenitors, to facilitate cell migration through RG fibers (**Figure 1**) [32, 33].

After the neurogenesis period, at the end of embryonic development, most of the RG cells have lost their ventricular attachment and migrate toward the cortical plate by a process of somal translocation. In mammals, the majority of RG cells are transformed into astrocytes. During this period, astrocytic and oligodendrocytic intermediate precursors are also generated (aIPCs and oIPCs). Some studies suggest the presence of multipotent and bipotent progenitors, and perhaps astrocyte-restricted progenitors in the neonatal SVZ [34].

Astrogenesis depends on several stimuli, being the Notch pathway a master regulator of the differentiation process. During the gliogenic phase, RG progenitors gain competence to generate astrocytes due to the activity of growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). This gain of competence allows them to respond to specific gliogenic signals acting at the extracellular level to activate astrocyte markers such as glial fibrillary acidic protein (GFAP), S100β, aquaporin 4, glutamate transporters (GLT-1, EACC1, and GLAST), and aldehyde dehydrogenase 1 family, member L1 (AldhL1) [35–39]. Before the astrocyte-marker promoters can respond to gliogenic signals, a chromatin epigenetic remodeling must occur. Notch canonical activation on RG induces expression of nuclear factor 1A (NFA1), an inhibitor of the DNA methyltransferase 1 (DNMT1). DNMT1 keeps STAT3 site of GFAP promoter methylated and inactive [40, 41].

**4. Notch pathway signaling in astroglial differentiation**

**Table 1.** Targets of Notch signaling pathway in central nervous system development.

Canonical pathway

RBPJκ-dependent)

Noncanonical (NICD/

RBPJκ-independent)

Glutamate aspartate transporter (GLAST)

28 GABA And Glutamate - New Developments In Neurotransmission Research

(Glial fibrillary acidic protein (GFAP)

Binding lipid-binding protein (BLBP)

β-catenin/Wnt signaling

(NICD/

Notch pathway is a pivotal signaling system during neuro- and gliogenesis in the central nervous system (CNS) [24, 25]. Primary neural stem cells (NSC) are radial glia (RG) during development, characterized by the expression of astroglial markers such as the astrocytespecific glutamate/aspartate transporter (GLAST), the brain lipid-binding protein (BLBP), and tenascin C (TN-C) [31]. RG cells generate neurons, oligodendrocytes, and astrocytes in a characteristic spatial and temporal pattern [31]. In this context, the Notch pathway plays an essential role repressing neurogenesis and maintaining the self-renewal potential of RG.

**Target Function Reference**

as Mash1, Math, and Neurogenin; which in turns regulate neural protein expression (p. ej.MAP2)

Principal protein (most abundant) forming an intermediate filament in mature astrocytes. Is important in radial glia cytoskeleton.

Hydrophobic protein member from the family FABP (Fatty acid–binding protein). Binds to ligands of nuclear receptors and participate regulating their

filaments in immature astrocytes and radial glia

Wnt/β-catenin signaling; Notch binds and titrates levels of the obligate Wnt-signaling component active

can induce Slug expression and subsequent neural crest

erbB2 Tyrosine kinase receptor [33] Slug Zinc-finger transcription factor that regulates neural crest formation and delamination

Glutamate transport This work

[72]

[50]

[32, 33]

[73]

[74]

[75, 76]

[74, 77]

Hes1/Hes5 Down-regulate pro-neural transcription factors,

transcriptional activity.

BMP4 Induce neural crest cells from the neural plate. Bmp4

Vimentin Most abundant protein forming intermediate

β-catenin.

On the neurogenic phase, RG divide asymmetrically for auto-renewal and generation of neurons or neuron-restricted intermediate progenitor cells (nIPCs, transit amplifying cells), which in turn populate the subventricular zone (SVZ) in the cortex. The newborn neurons migrate along parental RG fibers, even though RG are dividing [31]. The Notch pathway plays an important role during the neurogenic phase in several ways. In the cortex, recent findings suggest that Notch signaling among SVZ nIPCs and between nIPCs and RG is important in the regulation of progenitor proliferation and in the inhibition of precocious neuronal differentiation. RG receives Notch signaling to activate Hes1 and Hes5 transcription factors, which down-regulate pro-neuronal genes such as neurogenin 1 (Ngn1), Mash1, and Math. The extracellular signals are provided by neurotrophic cytokines such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1) secreted by newborn neurons. These cytokines activate heterodimeric cell surface receptors composed of two subunits named LIFRβ and gp130, which in turn activate to members of the JAK family of tyrosine kinases that result in the phosphorylation and nuclear translocation of signal transducer and activator of transcription (STAT) proteins. In RG, two of these proteins, STAT1 and STAT3, act on specific sites in the promoters of the astroglial genes GFAP and S100β to stimulate their transcription during the astrocyte differentiation process. Neural progenitors also respond to different neurotrophic factors from the bone morphogenetic proteins (BMP) family to generate astrocytes. In this case, BMP2 and BMP4 act on heterotrimeric receptors, which activate SMAD transcription factors. These, in turn, interact with activated STAT proteins to synergistically stimulate transcription of glial-specific genes during astrocyte differentiation [42–47].

Another estrogen signal is the activation of the seven transmembrane domain G protein-coupled receptors by the pituitary adenylate cyclase-activating polypeptide (PACAP), triggering the differentiation of astrocytes by increasing intracellular cAMP and activating the cAMPdependent protein kinase (PKA), which translocates into the nucleus to phosphorylate and

activate the cAMP-response element-binding protein (CREB) [42–48]. In a glial precursor-like model, C6 cells, cAMP-PKA-CREB activation leads to increase autocrine IL-6, which in turn activates STAT3, which induces GFAP promoter activation [49]. In the same model, the cAMP-PKA-CREB signaling cascade cross talks with the Notch pathway, and together they act synergistically to reduce the progenitor marker (Nestin) and to induce astrocytic differentiation; measured by induced astrocytic markers (GFAP, S100β, and GLAST) and glutamate uptake. In this context, Notch1 mRNA is up-regulated in a PKA/γ-secretase/NICD/CSL-dependent manner, suggesting the establishment of a feedback loop to keep Notch pathway active until astrocytic differentiation is complete [50]. It is not surprising that the Notch pathway interacts with other signaling cascades to complete astrocytic differentiation. A bioinformatic analysis of promoters for GFAP, S100β, GLAST, and Notch1, reveals specific sites for CREB, STAT3, and CSL transcription factors, suggesting that these three pathways, cAMP-PKA/JAK-STAT3/ Notch, cooperate to induce the transcription of astrocyte markers. Certainly, at this stage,

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

31

other crosstalk interactions cannot be discarded.

**protein kinase (PKC)**

**5. Glial differentiation and the Ca2+/diacylglycerol-dependent** 

subfamilies: classical, novel, and atypical isoforms described in **Table 2** [51–54].

astrocytic differentiation, while PKC β and γ induce astrocyte differentiation [51].

However, it has also been documented that undifferentiated C6 cells express the α, βΙΙ, γ, δ, ε, and ζ PKC isoforms and that long-term PKC inhibition after staurosporine treatment, which

As already mentioned, glial differentiation is modulated by extracellular signals, growth factors, hormones, cytokines, neurotrophins, and neurotransmitters that activate different signal pathways. PKC is one of the major mediators of these extracellular signals. The structure of this family of protein kinases contains a highly conserved catalytic domain and a regulatory domain (C1-C4 domains) responsible for its inactive conformation. Regulatory domains are separated by variable regions susceptible to proteolytic cleavage and essential for activation and conformational changes. The PKC family comprises 11 isoforms and is organized in three

PKC is highly expressed in the brain, with a significant role of this kinase in the function of neuronal and glial cells. The role of PKC in glial cells has been demonstrated in different reports, and PKC-activator PMA as well as the different PKC inhibitors, modify the cell morphology, proliferation, and differentiation [55–58]. Differential expression of PKC isoforms has been reported during neuronal development as four PKC isoforms are expressed in neuronal primary cultures of rat cerebellum. In contrast, only two isoforms, αPKC and βII PKC, are present in glial cultures [59, 60]. Brodie et al. reported that in undifferentiated C6 cells, the PKC isoforms θ, μ, ζ, and λ are present; however, the cAMP-dependent differentiated C6 cells expressed significantly lower levels of PKC α and PKC δ and higher levels of PKC γ, η, and θ. Concerning PKC and glial cell function, overexpression of the β and γ isoforms increases GFAP levels, as a response to exposure to the PKC activator phorbol 12-myristate 13-acetate (PMA) treatment. Glutamine synthetase (GS) levels increase with PKC γ overexpression and decrease with PKC δ. Therefore, it is plausible that PKC α and δ provide negative signals for

**Figure 1.** Notch pathway regulates astrocytic differentiation. (A) In timed cell genesis, a series of orchestrated events are activated to regulate cell differentiation; the Notch pathway is used as a signaling system during embryonic development and regulates different cell markers in astrocyte differentiation, such as Hes genes, GFAP and S100B. (nIPC/aIPC cell stands for neural or astrocytic intermediate precursor cell). (B) Neuroepithelial cells in early development proliferate by asymmetric cell division to generate more neuroepithelial cells (progenitor expansion phase). As brain development proceeds, neuroepithelial cells elongate to convert into radial glial (RG) cells and guide neuronal migration. Later, RG can divide asymmetrically to generate neuron, astrocyte, or oligodendrocyte intermediate progenitor cells.

activate the cAMP-response element-binding protein (CREB) [42–48]. In a glial precursor-like model, C6 cells, cAMP-PKA-CREB activation leads to increase autocrine IL-6, which in turn activates STAT3, which induces GFAP promoter activation [49]. In the same model, the cAMP-PKA-CREB signaling cascade cross talks with the Notch pathway, and together they act synergistically to reduce the progenitor marker (Nestin) and to induce astrocytic differentiation; measured by induced astrocytic markers (GFAP, S100β, and GLAST) and glutamate uptake. In this context, Notch1 mRNA is up-regulated in a PKA/γ-secretase/NICD/CSL-dependent manner, suggesting the establishment of a feedback loop to keep Notch pathway active until astrocytic differentiation is complete [50]. It is not surprising that the Notch pathway interacts with other signaling cascades to complete astrocytic differentiation. A bioinformatic analysis of promoters for GFAP, S100β, GLAST, and Notch1, reveals specific sites for CREB, STAT3, and CSL transcription factors, suggesting that these three pathways, cAMP-PKA/JAK-STAT3/ Notch, cooperate to induce the transcription of astrocyte markers. Certainly, at this stage, other crosstalk interactions cannot be discarded.

### **5. Glial differentiation and the Ca2+/diacylglycerol-dependent protein kinase (PKC)**

As already mentioned, glial differentiation is modulated by extracellular signals, growth factors, hormones, cytokines, neurotrophins, and neurotransmitters that activate different signal pathways. PKC is one of the major mediators of these extracellular signals. The structure of this family of protein kinases contains a highly conserved catalytic domain and a regulatory domain (C1-C4 domains) responsible for its inactive conformation. Regulatory domains are separated by variable regions susceptible to proteolytic cleavage and essential for activation and conformational changes. The PKC family comprises 11 isoforms and is organized in three subfamilies: classical, novel, and atypical isoforms described in **Table 2** [51–54].

PKC is highly expressed in the brain, with a significant role of this kinase in the function of neuronal and glial cells. The role of PKC in glial cells has been demonstrated in different reports, and PKC-activator PMA as well as the different PKC inhibitors, modify the cell morphology, proliferation, and differentiation [55–58]. Differential expression of PKC isoforms has been reported during neuronal development as four PKC isoforms are expressed in neuronal primary cultures of rat cerebellum. In contrast, only two isoforms, αPKC and βII PKC, are present in glial cultures [59, 60]. Brodie et al. reported that in undifferentiated C6 cells, the PKC isoforms θ, μ, ζ, and λ are present; however, the cAMP-dependent differentiated C6 cells expressed significantly lower levels of PKC α and PKC δ and higher levels of PKC γ, η, and θ.

Concerning PKC and glial cell function, overexpression of the β and γ isoforms increases GFAP levels, as a response to exposure to the PKC activator phorbol 12-myristate 13-acetate (PMA) treatment. Glutamine synthetase (GS) levels increase with PKC γ overexpression and decrease with PKC δ. Therefore, it is plausible that PKC α and δ provide negative signals for astrocytic differentiation, while PKC β and γ induce astrocyte differentiation [51].

**Figure 1.** Notch pathway regulates astrocytic differentiation. (A) In timed cell genesis, a series of orchestrated events are activated to regulate cell differentiation; the Notch pathway is used as a signaling system during embryonic development and regulates different cell markers in astrocyte differentiation, such as Hes genes, GFAP and S100B. (nIPC/aIPC cell stands for neural or astrocytic intermediate precursor cell). (B) Neuroepithelial cells in early development proliferate by asymmetric cell division to generate more neuroepithelial cells (progenitor expansion phase). As brain development proceeds, neuroepithelial cells elongate to convert into radial glial (RG) cells and guide neuronal migration. Later, RG

can divide asymmetrically to generate neuron, astrocyte, or oligodendrocyte intermediate progenitor cells.

30 GABA And Glutamate - New Developments In Neurotransmission Research

However, it has also been documented that undifferentiated C6 cells express the α, βΙΙ, γ, δ, ε, and ζ PKC isoforms and that long-term PKC inhibition after staurosporine treatment, which


In another example, PMA increased the expression of Notch1 in a PKCε-dependent manner in the context of astrocytic differentiation, and this is to say that in the course of PKCdependent astrocyte differentiation, an increase in Notch levels is found. In fact, serine 729 PKCε phosphorylation is as essential for the differentiation process. This data suggests that Notch1 is a plausible mediator of PKCε in astrocytic differentiation [68]. In the same line, Xu et al. reported that morphine-dependent astrocytic differentiation of neuronal progenitor cells (NPC) involves ERK via PKCε and TRBP phosphorylation that leads to miR-181a matu-

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

33

More recently, it has been demonstrated that atypical PKC isoforms participate in asymmetric cell division when glial differentiation starts. Sjoqvist et al. demonstrated that PKCζ regulates the Notch pathway by phosphorylation and regulation of Notch receptor traffic. When Notch signaling is active (after ligand stimulation or after expression of an activated membranetethered form of Notch), PKCζ enhances the production of NICD and shifts the localization of Notch from late endosomes to the nucleus, leading to an elevated Notch signaling. In contrast, when the Notch receptor is not activated, PKCζ interacts with the receptor to induce a shift in receptor distribution from the plasma membrane to intracellular vesicles [70]. In C6 glioma cells, increased cAMP levels promote astrocytic commitment with a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and its transcriptional activity [50]. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage as demonstrated by its sensitivity to PKA inhibitors. As expected, Notch cleavage and nuclear translocation result in the upregulation of the mRNA levels of one of its target genes, the transcription factor Hair, and enhancer of split 5. Moreover, glutamate uptake activity, expression of astrocytic markers (genes responsible for glial progenitor cell fate decision) such as the glial fibrillary acidic protein, the S100beta protein, and GLAST, are also enhanced in cAMP-exposed cells [50]. Interestingly, polychlorinated biphenyls (PCBs) disturb the cAMP-induced astrocytic differentiation of C6 cells via the PKC isoforms γ, β2, δ and ε [58]. Additionally, PMA promotes adult neurogenesis by inducing neural progenitor cell proliferation *in vitro* in NPCs obtained

To support a plausible role of a crosstalk between PKC and Notch pathways in embryonic glial differentiation, we used chick Bergman radial glia from cerebellum (BGC) at day 14 of embryonic development and stimulated PKC using TPA. In this system, it was observed that PKC activation increased NICD/RPBJκ-dependent transcription, measured by a reporter construct that senses directly the CSL activity (**Figure 2A**). This effect could be mediated by the classical PKC isoforms (α, β1, β2, and γ) and/or the novel isoforms (δ and ε), as it was observed when the specific inhibitor bisindolylmaleimide 1 (Bis1). The same effect was observed over a Hes1-responsive reporter (**Figure 2A**, right). Also, the MAPK/ERK pathway plays a role in PKC-mediated NICD/RPBJκ activation, as demonstrated when specific MAPK/ERK inhibitors (U0126, PD98059, and SB202190) were used in co-treatment with TPA (**Figure 2A**). In contrast, treatment of BGC with TPA, down-regulates astrocytic biomarkers such as GFAP, GS, GLAST, FABP7, and Notch1 mRNA levels, and keeps Nestin, a progenitor marker, up-regulated (**Figure 2B**). Our results suggest that the activation of PKC induces NICD/RBPJκ dependent transcriptional activation by a yet-to-be characterized mechanism, that perhaps

ration, thus regulating the expression of Prox1 and Notch1 [69].

from the SVZ of 7-day postnatal mice [71].

**Table 2.** PKC classification.

leads to differentiation, results in βII decrease, γ increase and ε translocation from the membrane to the cytosol [55]. Similar results were reported in the C6 cell differentiation process with dbcAMP [61]. It is clear that the molecular mechanisms triggered by glia differentiation agents are different, but that the various PKC isoforms are critically involved in the overall process.

In contrast, Watanabe et al. recently reported that overexpression of PKC βII synergistically enhanced differentiation in the presence of 1 nM of PACAP. These results indicate that the β isoform of PKC is important in PACAP-induced differentiation of mouse embryonic NSCs into astrocytes via the PAC1 receptor, resulting in activation of phospholipase C, followed by PKC activation. This latter observation was confirmed in NSCs. The cells were exposed to 2 nM PACAP, resulting in a transient increase in the βII isoform, that returned to basal levels by day 4, whereas the levels of PKC α increased linearly up to day 6 [62].

RG cells and astrocytes are involved in regulation of the brain microenvironment, and glutamate transporters control the extracellular levels of this neurotransmitter. Regulation of glutamate uptake involves several factors like neuronal interactions, glutamate, cAMP, and phorbol esters. GLAST is the major glutamate transporter expressed in RG cells. Interestingly, GLAST expression is regulated via PKC through the reduction of its protein and mRNA levels. Our work group demonstrated that *chglast* transcriptional regulation is mediated by PKC, especially the α and ε isoforms, which activate the AP-1 transcription factor [56, 63, 64].

### **6. Another Notch in the belt: PKC/Notch cross talk in glial differentiation**

The Notch signaling pathway plays an important role in the control of cell fate during developmental processes. Several reports have shown that Notch-induced signaling interacts with other signaling pathways, such as NF-κB, the mitogen-activated protein kinase (MAPK) pathway, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway [65–67]. Although there are only a few reports describing the crosstalk between Notch and PKC signaling during glial differentiation, some connections have been described so far. The most direct example concerns the direct PKC action over NICD: Kim et al. found that PKC δ down-regulates NICD transcriptional activity in a kinase-independent manner. The mechanism involves the inhibition of the nuclear localization of NICD, most possibly through a physical association between NICD and PKCδ causing the dissociation of NICD from target gene promoters like Hes5 [67].

In another example, PMA increased the expression of Notch1 in a PKCε-dependent manner in the context of astrocytic differentiation, and this is to say that in the course of PKCdependent astrocyte differentiation, an increase in Notch levels is found. In fact, serine 729 PKCε phosphorylation is as essential for the differentiation process. This data suggests that Notch1 is a plausible mediator of PKCε in astrocytic differentiation [68]. In the same line, Xu et al. reported that morphine-dependent astrocytic differentiation of neuronal progenitor cells (NPC) involves ERK via PKCε and TRBP phosphorylation that leads to miR-181a maturation, thus regulating the expression of Prox1 and Notch1 [69].

leads to differentiation, results in βII decrease, γ increase and ε translocation from the membrane to the cytosol [55]. Similar results were reported in the C6 cell differentiation process with dbcAMP [61]. It is clear that the molecular mechanisms triggered by glia differentiation agents are different, but that the various PKC isoforms are critically involved in the overall process. In contrast, Watanabe et al. recently reported that overexpression of PKC βII synergistically enhanced differentiation in the presence of 1 nM of PACAP. These results indicate that the β isoform of PKC is important in PACAP-induced differentiation of mouse embryonic NSCs into astrocytes via the PAC1 receptor, resulting in activation of phospholipase C, followed by PKC activation. This latter observation was confirmed in NSCs. The cells were exposed to 2 nM PACAP, resulting in a transient increase in the βII isoform, that returned to basal levels

RG cells and astrocytes are involved in regulation of the brain microenvironment, and glutamate transporters control the extracellular levels of this neurotransmitter. Regulation of glutamate uptake involves several factors like neuronal interactions, glutamate, cAMP, and phorbol esters. GLAST is the major glutamate transporter expressed in RG cells. Interestingly, GLAST expression is regulated via PKC through the reduction of its protein and mRNA levels. Our work group demonstrated that *chglast* transcriptional regulation is mediated by PKC, especially the α and ε isoforms, which activate the AP-1 transcription factor [56, 63, 64].

The Notch signaling pathway plays an important role in the control of cell fate during developmental processes. Several reports have shown that Notch-induced signaling interacts with other signaling pathways, such as NF-κB, the mitogen-activated protein kinase (MAPK) pathway, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway [65–67]. Although there are only a few reports describing the crosstalk between Notch and PKC signaling during glial differentiation, some connections have been described so far. The most direct example concerns the direct PKC action over NICD: Kim et al. found that PKC δ down-regulates NICD transcriptional activity in a kinase-independent manner. The mechanism involves the inhibition of the nuclear localization of NICD, most possibly through a physical association between NICD and PKCδ causing the dissociation of NICD from target

by day 4, whereas the levels of PKC α increased linearly up to day 6 [62].

**Isoforms Activity** Classical isoforms α, βΙ, βΙΙ, γ Dependent on DAG, PS, and Ca2+

32 GABA And Glutamate - New Developments In Neurotransmission Research

Novel PKC isoforms δ, ε, θ, η, μ Bind DAG, PS, and calcium-independent

Atypical PKC isoforms ι/λ, ξ Bind PIP3, calcium-independent and do not require DAG

Diacylglycerol (DAG), phosphatidylserine (PS), phosphatidylinositol 3-phosphate (PIP3) [53, 78].

**6. Another Notch in the belt: PKC/Notch cross talk in glial** 

**differentiation**

**Table 2.** PKC classification.

gene promoters like Hes5 [67].

More recently, it has been demonstrated that atypical PKC isoforms participate in asymmetric cell division when glial differentiation starts. Sjoqvist et al. demonstrated that PKCζ regulates the Notch pathway by phosphorylation and regulation of Notch receptor traffic. When Notch signaling is active (after ligand stimulation or after expression of an activated membranetethered form of Notch), PKCζ enhances the production of NICD and shifts the localization of Notch from late endosomes to the nucleus, leading to an elevated Notch signaling. In contrast, when the Notch receptor is not activated, PKCζ interacts with the receptor to induce a shift in receptor distribution from the plasma membrane to intracellular vesicles [70]. In C6 glioma cells, increased cAMP levels promote astrocytic commitment with a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and its transcriptional activity [50]. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage as demonstrated by its sensitivity to PKA inhibitors. As expected, Notch cleavage and nuclear translocation result in the upregulation of the mRNA levels of one of its target genes, the transcription factor Hair, and enhancer of split 5. Moreover, glutamate uptake activity, expression of astrocytic markers (genes responsible for glial progenitor cell fate decision) such as the glial fibrillary acidic protein, the S100beta protein, and GLAST, are also enhanced in cAMP-exposed cells [50]. Interestingly, polychlorinated biphenyls (PCBs) disturb the cAMP-induced astrocytic differentiation of C6 cells via the PKC isoforms γ, β2, δ and ε [58]. Additionally, PMA promotes adult neurogenesis by inducing neural progenitor cell proliferation *in vitro* in NPCs obtained from the SVZ of 7-day postnatal mice [71].

To support a plausible role of a crosstalk between PKC and Notch pathways in embryonic glial differentiation, we used chick Bergman radial glia from cerebellum (BGC) at day 14 of embryonic development and stimulated PKC using TPA. In this system, it was observed that PKC activation increased NICD/RPBJκ-dependent transcription, measured by a reporter construct that senses directly the CSL activity (**Figure 2A**). This effect could be mediated by the classical PKC isoforms (α, β1, β2, and γ) and/or the novel isoforms (δ and ε), as it was observed when the specific inhibitor bisindolylmaleimide 1 (Bis1). The same effect was observed over a Hes1-responsive reporter (**Figure 2A**, right). Also, the MAPK/ERK pathway plays a role in PKC-mediated NICD/RPBJκ activation, as demonstrated when specific MAPK/ERK inhibitors (U0126, PD98059, and SB202190) were used in co-treatment with TPA (**Figure 2A**). In contrast, treatment of BGC with TPA, down-regulates astrocytic biomarkers such as GFAP, GS, GLAST, FABP7, and Notch1 mRNA levels, and keeps Nestin, a progenitor marker, up-regulated (**Figure 2B**). Our results suggest that the activation of PKC induces NICD/RBPJκ dependent transcriptional activation by a yet-to-be characterized mechanism, that perhaps

is related to regulation of NICD routing and trafficking [67, 70]. The NICD/RBPJκ complex induces Hes1 expression, a well-known neurogenesis inhibitor. On the other hand, PKC activation blocks astrogenesis, perhaps modulating the access of NICD/RBPJκ to the astrocytic markers' promoters, like GFAP, which is dependent on Notch activation. In BGC, PKC activation regulates several genes that are closely related to glial function and induces radial glial phenotype as TPA down-regulates GLAST, GFAP, and GS and upregulates Nestin, PKCα, or

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

35

A plethora of pathological scenarios in the CNS are the result of neuronal degeneration. This cell loss needs to be compensated to keep the neural circuits working. In this context, neural stem cells can be differentiated into precise neuronal subtypes, but a common fact is that Notch signaling promotes astrocyte differentiation rather than neuronal differentiation. Therefore, Notch inhibition is an alternative therapeutic option in the clinical approach. Examples of the possible application of inhibiting Notch are presented

**Notch signaling role and possible therapeutic approach Ref**

[79]

[69]

[80] [81]

[82]

Pharmacological inhibition of Notch pathway selectively inhibited tumor growth. Conversely, activation of Notch signaling promotes cell proliferation and colony formation in the human GB cell line. Notch1 promotes invasive migratory properties of GB cells by stimulating β-catenin and NF-κB signaling and mediates GB cell proliferation and survival through the Akt-mammalian target of rapamycin (mTOR) signaling axis. Treatment with γ-secretase inhibitors reduces neurosphere growth, and inhibits xenograft tumor growth through decreased Akt and STAT3 phosphorylation. Combination of Notch inhibitor MRK003 and Akt inhibitor

MK-2206 effectively inhibited GB invasiveness.

Phosphorylation of TAR RNA-binding protein together with miR-181a maturation, as well as Dicer activity, is involved in morphine-induced astrocyte-preferential differentiation of

Activation of IL-6/gp130 and Notch signaling pathways in glial differentiation of HNPCs may cause problems in maintaining normal brain function and may contribute to AD pathology. Treatment with sAPP increased expression levels of GFAP in NT-2/D1 cells along with the generation of Notch intracellular domain (NICD) and expression of Hairy and enhancer of split 1 (Hes1), indicating that glial differentiation may aid in the development of novel therapeutic strategies for AD.

Neuron-derived Dll1 activates Notch signaling and is essential for brain endothelial cells' survival as wells as blood-brain barrier, selective substance crossing; physiology, pathology,

**6.1. Notch pathway in CNS: some aspects of clinical relevance**

**Targeted neural stem cells**

Glioblastoma cells

Hippocampal progenitor cells (HPC)

Human neural progenitor cells (HNPC)

Brain endothelial

cells

HPC.

and drug development

(GB)

Blockage of notch pathway in neural adult stem cells to promote neurogenesis

PKCε (PKC �2).

in the **Table 3**.

**Pathological context/ biological system**

(multiforme or grade IV

Glioblastoma

astrocytoma)

Lineage-specific differentiation of NPCs

Alzheimer's disease

Amyloid precursor protein (APP)

Blood-brain barrier, pathology

(AD)

**Figure 2.** PKC/MAPK signaling activates Notch pathway in cultured Bergmann glia cells (BGCs). (A) In order to determine the role of the Notch/PKC signaling pathway in BGCs, cells (primary culture) were transfected with the reporter plasmid 4xwtCBF1Luc (containing four repeated sequences for RBPJκ elements) or Hes1 promoter (a Notch effector gene) with Lipofectamine R 2000; after harvesting, the luciferase reporter activity was measured. 24 h post-transfection, the BGCs were treated with 100 nM TPA for other 24 h, where we showed that promoter activity increased five-fold in relation with nontreatment cells, and TPA induces the Notch signaling response. To analyze the signal pathway involved in Notch activation, BGCs were treated with 40 μM BisI (a PKC inhibitor), 50 μM U0126 (a MAPK1 inhibitor), PD (PD8059, a MAPK1 inhibitor), or SB20 (SB202190, a specific ERK inhibitor) 30 min prior to TPA, as indicated in the figure. Note that both PKC and MAPK inhibitors prevent Notch pathway activation. Results are presented as fold expression relative to nontreated cells. Data represent mean values ± SE (n = 3). Data were analyzed by a one-way ANOVA with a post hoc Dunn's test. \*\*\* p < 0.001; ### p < 0.003; #, p < 0.05. (B) qRT-PCR was performed to analyze expression of Notch pathway targets, and BGC cultures were treated or not with TPA (100 nM TPA) at different times. Total RNA was extracted to amplify GFAP, GLAST, vimentin, glutamine synthetase (astrocytic markers), and nestin (radial glial marker) with the KAPA SYBR FAST one-step qRT-PCR kit. Our data suggest that TPA downregulates GLAST, GFAP, and GS and upregulates nestin; this evidence indicates that the Notch pathway is important in the radial glial fate.

is related to regulation of NICD routing and trafficking [67, 70]. The NICD/RBPJκ complex induces Hes1 expression, a well-known neurogenesis inhibitor. On the other hand, PKC activation blocks astrogenesis, perhaps modulating the access of NICD/RBPJκ to the astrocytic markers' promoters, like GFAP, which is dependent on Notch activation. In BGC, PKC activation regulates several genes that are closely related to glial function and induces radial glial phenotype as TPA down-regulates GLAST, GFAP, and GS and upregulates Nestin, PKCα, or PKCε (PKC �2).

#### **6.1. Notch pathway in CNS: some aspects of clinical relevance**

A plethora of pathological scenarios in the CNS are the result of neuronal degeneration. This cell loss needs to be compensated to keep the neural circuits working. In this context, neural stem cells can be differentiated into precise neuronal subtypes, but a common fact is that Notch signaling promotes astrocyte differentiation rather than neuronal differentiation. Therefore, Notch inhibition is an alternative therapeutic option in the clinical approach. Examples of the possible application of inhibiting Notch are presented in the **Table 3**.


**Figure 2.** PKC/MAPK signaling activates Notch pathway in cultured Bergmann glia cells (BGCs). (A) In order to determine the role of the Notch/PKC signaling pathway in BGCs, cells (primary culture) were transfected with the reporter plasmid 4xwtCBF1Luc (containing four repeated sequences for RBPJκ elements) or Hes1 promoter (a Notch effector gene) with Lipofectamine R 2000; after harvesting, the luciferase reporter activity was measured. 24 h post-transfection, the BGCs were treated with 100 nM TPA for other 24 h, where we showed that promoter activity increased five-fold in relation with nontreatment cells, and TPA induces the Notch signaling response. To analyze the signal pathway involved in Notch activation, BGCs were treated with 40 μM BisI (a PKC inhibitor), 50 μM U0126 (a MAPK1 inhibitor), PD (PD8059, a MAPK1 inhibitor), or SB20 (SB202190, a specific ERK inhibitor) 30 min prior to TPA, as indicated in the figure. Note that both PKC and MAPK inhibitors prevent Notch pathway activation. Results are presented as fold expression relative to nontreated cells. Data represent mean values ± SE (n = 3). Data were analyzed by a one-way ANOVA with a post hoc Dunn's test. \*\*\* p < 0.001; ### p < 0.003; #, p < 0.05. (B) qRT-PCR was performed to analyze expression of Notch pathway targets, and BGC cultures were treated or not with TPA (100 nM TPA) at different times. Total RNA was extracted to amplify GFAP, GLAST, vimentin, glutamine synthetase (astrocytic markers), and nestin (radial glial marker) with the KAPA SYBR FAST one-step qRT-PCR kit. Our data suggest that TPA downregulates GLAST, GFAP, and GS and

upregulates nestin; this evidence indicates that the Notch pathway is important in the radial glial fate.

34 GABA And Glutamate - New Developments In Neurotransmission Research


**7. Conclusion**

phenotype.

**Acknowledgements**

**Conflict of interest**

**Abbreviations**

The authors declare that there are no conflicts of interest.

ADAM10/TACE Tumor necrosis factor-α converting enzyme

AldhL1 aldehyde dehydrogenase 1

bFGF Basic fibroblast growth factor

BGC Chick Bergmann Radial glia

BLBP Brain lipid-binding protein

CNS Central nervous system

CNTF Ciliary neurotrophic factor

BMP Bone morphogenetic proteins

cAMP Cyclic adenosine monophosphate

CIR Corepressor interacting with RBPJ 1

BisI Bisindolylmaleimide

Glia cells play an active role in glutamatergic transmission due to their compulsory intervention in the recycling of this excitatory neurotransmitter. The Notch signal transduction pathway is critically involved in the gene expression regulation of the major excitatory amino acid transporter expressed in early stages of astrocyte differentiation and in RG in the adult brain. Notch signaling involves the activation of diverse isoforms of PKC. Glial differentiation can be mediated by PKC and its isoforms, which act over NICD, increasing Notch expression, regulating several astrocytic markers related to glial function, and inducing the radial glial

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

37

The work in our labs is funded by Conacyt-Mexico 255087 and Soluciones para un México Verde, S.A. de C.V. granted to AO and Conacyt-PEI 212650 and 231793 granted to ELB.

**Table 3.** Notch signaling in cell therapy.

#### **7. Conclusion**

**Pathological context/ biological system**

Traumatic brain injury (TBI), inflammation and apoptosis and brain

Increased differentiation of neural progenitor cells when co-cultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin

edema

Experimental autoimmune encephalomyelitis (failure to repair demyelination) as Multiple Sclerosis model

Seizure as a serious complication of stroke

Familial and idiopathic Parkinson's disease (PD)

Dopamine release in the striatum, individual's susceptibility to neuropsychiatric disease

Length, polarity, and synaptogenesis

**Table 3.** Notch signaling in cell therapy.

(mice)

**Targeted neural stem cells**

36 GABA And Glutamate - New Developments In Neurotransmission Research

Cerebral cortices response

Neural progenitor

Oligodendrocyte precursor cells

Neurons in cortex and hippo-campus

Differentiated dopaminergic neurons

Spiral ganglion neurons (SGNs) vimentin.

repair.

development.

linked to *LRRK2.*

Neuronal cells RBP-J deficiency drastically reduced dopamine release in

dopamine responsiveness in the striatum.

exhibited significantly reduced dendritic length.

Notch signaling in neurodegenerative diseases and pathological glutamate mediated plasticity

cells

**Notch signaling role and possible therapeutic approach Ref**

[83]

[84]

[85, 86]

[87]

[88]

[89].

[90]

Inhibition of Notch signaling by crocin, an extract of saffron, has a neuroprotective effect against TBI, since in this type of injury an upregulation of Notch intracellular domain (NICD) and Hes1 mRNA levels is present, decreasing microglial activation and release of several pro-inflammatory cytokines.

Astrocytes negatively regulate neurogenesis through Notch pathway; endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on intermediate filament proteins GFAP and

Gamma-secretase inhibition of Notch signaling enhances tissue

Notch pathway inhibits oligodendrocytes differentiation and hampers their ability to produce myelin during CNS

In a global cerebral ischemia model (GCI), there is augmented excitatory synaptic neurotransmission by upregulating glutamate receptor subunits (GluN2A, GluA1) and cotransporter

NKCC1, but there is attenuated inhibitory synaptic neurotransmission by down-regulating amino butyric acid (GABA), and neuronal K-Cl cotransporter. Aberrant activation of Notch signaling is involved in poststroke seizures, as NICD 1 and 2 were upregulated in the cerebral cortex and hippocampus post-GCI. DAPT treatment normalized the homeostasis of excitatory and inhibitory synaptic neurotransmission.

Leucine-rich repeat kinase 2 (LRRK2) complex promotes recycling of Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through modulation of endosomal trafficking and negatively regulates Notch signaling through *cis*-inhibition by stabilizing Dll1/Dl, accelerating neural stem cell differentiation; alteration of Notch signaling in mature neurons is a component of PD etiology

the striatum and caused a subtle decrease in the number of dopaminergic neurons as Notch/RBP-J signaling regulates

DNER modulates length, polarity and synaptogenesis *via* the Notch signaling pathway. DNER was expressed in spiral ganglion neurons exhibiting significant polarity in early differentiation stages; DNER expression gradually decreased until polarity was lost on week 35. Silencing DNER expression altered the polarity of differentiated neurons and these cells

Glia cells play an active role in glutamatergic transmission due to their compulsory intervention in the recycling of this excitatory neurotransmitter. The Notch signal transduction pathway is critically involved in the gene expression regulation of the major excitatory amino acid transporter expressed in early stages of astrocyte differentiation and in RG in the adult brain. Notch signaling involves the activation of diverse isoforms of PKC. Glial differentiation can be mediated by PKC and its isoforms, which act over NICD, increasing Notch expression, regulating several astrocytic markers related to glial function, and inducing the radial glial phenotype.

#### **Acknowledgements**

The work in our labs is funded by Conacyt-Mexico 255087 and Soluciones para un México Verde, S.A. de C.V. granted to AO and Conacyt-PEI 212650 and 231793 granted to ELB.

#### **Conflict of interest**

The authors declare that there are no conflicts of interest.

#### **Abbreviations**



PACAP Pituitary adenylate cyclase-activating polypeptide

STAT3 Signal transducer and activator of transcription 3

, Angulo-Rojo Carla2

, Ramírez Leticia1

1 Departamento de Toxicología, Centro de Investigación de Estudios Avanzados del IPN,

[1] Gasic GP et al. Molecular neurobiology of glutamate receptors. Annual Review of

[2] Hollmann M et al. Cloned glutamate receptors. Annual Review of Neuroscience. 1994;**17**:

2 Unidad de Investigación, Facultad de Medicina, Universidad Autónoma de Sinaloa,

Physiology. 1992;**54**:507-536. DOI: 10.1146/annurev.ph.54.030192.002451

, López-Bayghen Bruno1

and Ortega Arturo1

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

39

,

\*

TPA 12-O-tetradecanoylphorbol 13-acetate

\*Address all correspondence to: arortega@cinvestav.mx

31-108. DOI: 10.1146/annurev.ne.17.030194.000335

PI3K Phosphatidylinositol 3-kinase

PKC Protein kinase C

RG Radial glia

TN-C Tenascin C

**Author details**

López-Bayghen Esther1

México City, Mexico

**References**

Culiacán, Sinaloa, Mexico

Hernández-Melchor Dinorah1

PKA cAMP-dependent protein kinase

PMA Phorbol 12-myristate 13-acetate

RBPJκ Recombining binding protein

SMRT Thyroid-hormone receptors

TRBP TAR RNA-binding protein

SVZ Subventricular Zone


#### **Author details**

CREB cAMP-response element-binding protein

38 GABA And Glutamate - New Developments In Neurotransmission Research

CT-1 Cardiotrophin-1

DTX1 Deltex1

DNMT1 DNA methyl transferase 1

EGF Epidermal growth factor

FABP7 Fatty acid–binding protein 7

GFAP Glial fibrillary acidic protein

GLT-1 Glutamate transporter 1

GS Glutamine synthetase

HDAC Histone deacetylases

MAM Mastermind

LIF Leukemia inhibitory factor

LIFRβ Leukemia inhibitory factor receptor β

MAP 2 Microtubule-associated protein 2

MAPK Mitogen-activated protein kinase

NEXT Notch extracellular truncation

NICD Notch intracellular domain

NIPC Intermediate progenitor cell

NFA1 Nuclear factor 1A

Ngn1 Neurogenin 1

MASH Mammalian achaete-scute homolog-1

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells

GLAST Glutamate aspartate transporter

EAATs Excitatory amino acid transporters

EACC1 Excitatory amino acid transporter 1

ERK Extracellular signal-regulated kinases

ErbB2 also known as HER2, Human epidermal growth factor receptor 2

López-Bayghen Esther1 , Angulo-Rojo Carla2 , López-Bayghen Bruno1 , Hernández-Melchor Dinorah1 , Ramírez Leticia1 and Ortega Arturo1 \*

\*Address all correspondence to: arortega@cinvestav.mx

1 Departamento de Toxicología, Centro de Investigación de Estudios Avanzados del IPN, México City, Mexico

2 Unidad de Investigación, Facultad de Medicina, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico

#### **References**


[3] Schoepp DD et al. Metabotropic glutamate receptors and neuronal degenerative disorders. Neurobiology of Aging. 1994;**15**(2):261-263

[20] Fortini ME. Notch and presenilin: A proteolytic mechanism emerges. Current Opinion

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

41

[21] Fortini ME. Gamma-secretase-mediated proteolysis in cell-surface-receptor signalling. Nature Reviews. Molecular Cell Biology. 2002;**3**(9):673-684. DOI: 10.1038/nrm910 [22] Fortini ME et al. The suppressor of hairless protein participates in notch receptor signal-

[23] Kopan R et al. The canonical notch signaling pathway: Unfolding the activation mecha-

[24] Bray SJ. Notch signalling: A simple pathway becomes complex. Nature Reviews. Mole-

[25] Chiba S. Concise review: Notch signaling in stem cell systems. Stem Cells. 2006;**24**(11):

[26] Selkoe D et al. Notch and presenilin: Regulated intramembrane proteolysis links development and degeneration. Annual Review of Neuroscience. 2003;**26**(1):565-597. DOI: 10.1146/

[27] Mumm JS et al. Notch signaling: From the outside in. Developmental Biology. 2000;

[28] Gorman MJ et al. A genetic analysis of Deltex and its interaction with the notch locus in

[29] Matsuno K et al. Deltex acts as a positive regulator of notch signaling through interac-

[30] Yamamoto N et al. Role of Deltex-1 as a transcriptional regulator downstream of the notch receptor. The Journal of Biological Chemistry. 2001;**276**(48):45031-45040. DOI:

[31] Kriegstein A et al. The glial nature of embryonic and adult neural stem cells. Annual Review of Neuroscience. 2009;**32**:149-184. DOI: 10.1146/annurev.neuro.051508.135600

[32] Patten BA et al. Sequential signaling through Notch1 and Erbb receptors mediates radial glia differentiation. The Journal of Neuroscience. 2003;**23**(14):6132-6140 23/14/6132 [pii]

[33] Patten BA et al. Notch1 signaling regulates radial glia differentiation through multiple transcriptional mechanisms. The Journal of Neuroscience. 2006;**26**(12):3102-3108. DOI:

[34] Aguirre A et al. Postnatal neurogenesis and gliogenesis in the olfactory bulb from Ng2-expressing progenitors of the subventricular zone. The Journal of Neuroscience.

[35] Spassky N et al. Adult ependymal cells are postmitotic and are derived from radial glial cells during embryogenesis. The Journal of Neuroscience. 2005;**25**(1):10-18. DOI:

2004;**24**(46):10530-10541. DOI: 10.1523/jneurosci.3572-04.2004

tions with the notch Ankyrin repeats. Development. 1995;**121**(8):2633-2644

in Cell Biology. 2001;**13**(5):627-634. DOI: 10.1016/S0955-0674(00)00261-1

ing. Cell. 1994;**79**(2):273-282. DOI: 10.1016/0092-8674(94)90196-1

nism. Cell. 2009;**137**(2):216-233. DOI: 10.1016/j.cell.2009.03.045

cular Cell Biology. 2006;**7**(9):678-689. DOI: 10.1038/nrm2009

2437-2447. DOI: 10.1634/stemcells.2005-0661

**228**(2):151-165. DOI: 10.1006/dbio.2000.9960

drosophila melanogaster. Genetics. 1992;**131**(1):99-112

annurev.neuro.26.041002.131334

10.1074/jbc.M105245200

10.1523/JNEUROSCI.4829-05.2006

10.1523/jneurosci.1108-04.2005


[20] Fortini ME. Notch and presenilin: A proteolytic mechanism emerges. Current Opinion in Cell Biology. 2001;**13**(5):627-634. DOI: 10.1016/S0955-0674(00)00261-1

[3] Schoepp DD et al. Metabotropic glutamate receptors and neuronal degenerative disor-

[4] Pin JP et al. The metabotropic glutamate receptors: Structure and functions. Neurophar-

[5] Cameron RS et al. Glial cell lineage in the cerebral cortex: A review and synthesis. Glia.

[6] Malatesta P et al. Neuronal or glial progeny: Regional differences in radial glia fate.

[7] Anthony TE et al. Radial glia serve as neuronal progenitors in all regions of the central

[9] Eulenburg V et al. Neurotransmitter transporters expressed in glial cells as regulators of synapse function. Brain Research Reviews. 2010;**63**(1-2):103-112. DOI: 10.1016/j.

[10] Swanson CJ et al. Metabotropic glutamate receptors as novel targets for anxiety and stress disorders. Nature Reviews. Drug Discovery. 2005;**4**(2):131-144. DOI: 10.1038/nrd1630 [11] Maragakis NJ et al. Glutamate transporter expression and function in human glial pro-

[12] Martinez-Lozada Z et al. Glutamatergic transmission: A matter of three. Neural Plasticity.

[13] Araque A et al. Glial modulation of synaptic transmission in culture. Glia. 2004;**47**(3):241-

[14] Gonzalez MI et al. Regulation of high-affinity glutamate uptake activity in Bergmann

[15] Gegelashvili M et al. Glutamate transporter Glast/Eaat1 directs cell surface expression of Fxyd2/gamma subunit of Na, K-Atpase in human fetal astrocytes. Neurochemistry

[16] Rose EM et al. Glutamate transporter coupling to Na,K-Atpase. The Journal of Neuro-

[17] Martinez-Lozada Z et al. Glast/Eaat1-induced glutamine release via Snat3 in Bergmann glial cells: Evidence of a functional and physical coupling. Journal of Neurochemistry.

[18] Danbolt NC et al. Neuronal vs glial glutamate uptake: Resolving the conundrum. Neuro-

[19] Flores-Mendez M et al. Glia plasma membrane transporters: Key players in Glutamatergic neurotransmission. Neurochemistry International. 2016;**98**:46-55. DOI: 10.1016/j.

[8] Danbolt NC. Glutamate uptake. Progress in Neurobiology. 2001;**65**(1):1-105

genitors. Glia. 2004;**45**(2):133-143. DOI: 10.1002/glia.10310

glia cells by glutamate. Brain Research. 2000;**866**(1-2):73-81

International. 2007;**50**(7-8):916-920. DOI: 10.1016/j.neuint.2006.12.015

science. 2009;**29**(25):8143-8155. DOI: 10.1523/JNEUROSCI.1081-09.2009

chemistry International. 2016;**98**:29-45. DOI: 10.1016/j.neuint.2016.05.009

ders. Neurobiology of Aging. 1994;**15**(2):261-263

40 GABA And Glutamate - New Developments In Neurotransmission Research

1991;**4**(2):124-137. DOI: 10.1002/glia.440040204

nervous system. Neuron. 2004;**41**(6):881-890

2015;**2015**:787396. DOI: 10.1155/2015/787396

2013;**125**(4):545-554. DOI: 10.1111/jnc.12211

macology. 1995;**34**(1):1-26

Neuron. 2003;**37**(5):751-764

brainresrev.2010.01.003

248. DOI: 10.1002/glia.20026

neuint.2016.04.004


[36] Wang DD et al. The astrocyte odyssey. Progress in Neurobiology. 2008;**86**(4):342-367. DOI: 10.1016/j.pneurobio.2008.09.015

[49] Takanaga H et al. Camp-induced astrocytic differentiation of C6 Glioma cells is mediated by Autocrine Interleukin-6. The Journal of Biological Chemistry. 2004;**279**(15):15441-

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

43

[50] Angulo-Rojo C et al. Involvement of the notch pathway in terminal astrocytic differentiation: Role of Pka. ASN Neuro. 2013;**5**(5):e00130. DOI: 10.1042/AN20130023

[51] Brodie C et al. Differential role of specific Pkc isoforms in the proliferation of glial cells and the expression of the astrocytic markers Gfap and glutamine Synthetase. Molecular

[52] Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. The

[53] Newton AC. Protein kinase C: Structure, function, and regulation. The Journal of Biolo-

[54] do Carmo A et al. Pkc signaling in glioblastoma. Cancer Biology & Therapy. 2013;**14**(4):

[55] Kronfeld I et al. Staurosporine induces astrocytic phenotypes and differential expression of specific Pkc isoforms in C6 glial cells. Journal of Neurochemistry. 1995;**65**(4):1505-

[56] Lopez-Bayghen E et al. Glutamate-dependent transcriptional regulation of Glast: Role of Pkc. Journal of Neurochemistry. 2004;**91**(1):200-209. DOI: 10.1111/j.1471-4159.2004.02706.x

[57] Moreton K et al. Protein expression of the Α, Γ, Δ and Ε subspecies of protein kinase C changes as C6 Glioma cells become contact inhibited and quiescent in the presence of serum. FEBS Letters. 1995;**372**(1):33-38 https://doi.org/10.1016/0014-5793(95)00941-2

[58] Adornetto A et al. Polychlorinated biphenyls impair Dibutyryl camp-induced astrocytic differentiation in rat C6 glial cell line. FEBS Open Bio. 2013;**3**:459-466. DOI: 10.1016/j.

[59] Masliah E et al. Differential involvement of protein kinase C Isozymes in Alzheimer's

[60] Moreton K et al. Changes in Pkc subspecies protein expression as C6 cells reach G(0) by contact inhibition in the presence of serum. Biochemical Society Transactions. 1995;

[61] S-i Y et al. Differential expression of rho family Gtp-binding proteins and protein kinase C Isozymes during C6 glial cell differentiation. Molecular Brain Research. 1997;**45**(1):

[62] Watanabe J et al. Pituitary adenylate cyclase-activating polypeptide-induced differentiation of embryonic neural stem cells into astrocytes is mediated via the Beta isoform of

disease. The Journal of Neuroscience. 1990;**10**(7):2113-2124

90-98. DOI: 10.1016/S0169-328X(96)00239-2

Brain Research. 1998;**56**(1):108-117. DOI: 10.1016/S0169-328X(98)00035-7

gical Chemistry. 1995;**270**(48):28495-28498. DOI: 10.1074/jbc.270.48.28495

15447. DOI: 10.1074/jbc.M311844200

FASEB Journal. 1995;**9**(7):484-496

287-294. Doi: 10.4161/cbt.23615

fob.2013.10.008

**23**(3):446S)

1514. DOI: 10.1046/j.1471-4159.1995.65041505.x


[49] Takanaga H et al. Camp-induced astrocytic differentiation of C6 Glioma cells is mediated by Autocrine Interleukin-6. The Journal of Biological Chemistry. 2004;**279**(15):15441- 15447. DOI: 10.1074/jbc.M311844200

[36] Wang DD et al. The astrocyte odyssey. Progress in Neurobiology. 2008;**86**(4):342-367.

[37] Liu X et al. Gfap-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes. Glia. 2006;**54**(5):394-

[38] Kimelberg HK. The problem of astrocyte identity. Neurochemistry International. 2004;

[39] Cahoy JD et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: A new resource for understanding brain development and function. The Journal of

[40] Wilczynska KM et al. Nuclear factor I isoforms regulate gene expression during the differentiation of human neural progenitors to astrocytes. Stem Cells. 2009;**27**(5):1173-1181.

[41] Cebolla B et al. Nuclear factor-I regulates glial fibrillary acidic protein gene expression in astrocytes differentiated from cortical precursor cells. Journal of Neurochemistry.

[42] Vallejo I et al. Pituitary adenylate cyclase-activating polypeptide induces astrocyte differentiation of precursor cells from developing cerebral cortex. Molecular and Cellular

[43] Jozwiak-Bebenista M et al. Effects of Pacap, Vip and related peptides on cyclic amp formation in rat neuronal and astrocyte cultures and cerebral cortical slices. Pharmacological

[44] Vallejo M. Pacap signaling to dream: A camp-dependent pathway that regulates cortical Astrogliogenesis. Molecular Neurobiology. 2009;**39**(2):90-100. DOI: 10.1007/s12035-

[45] Tatsuno I et al. Pituitary adenylate cyclase-activating polypeptide (Pacap) is a regulator of astrocytes: Pacap stimulates proliferation and production of interleukin 6 (Il-6), but not nerve growth factor (Ngf), in cultured rat astrocyte. Annals of the New York

Academy of Sciences. 1996;**805**:482-488. DOI: 10.1111/j.1749-6632.1996.tb17508.x

[46] Nakamachi T et al. Role of Pacap in neural stem/progenitor cell and astrocyte: From neural development to neural repair. Current Pharmaceutical Design. 2011;**17**(10):973-984.

[47] Nakamachi T et al. Pituitary adenylate cyclase-activating polypeptide (Pacap) type 1 receptor (Pac1r) co-localizes with activity-dependent Neuroprotective protein (Adnp) in the mouse brains. Regulatory Peptides. 2008;**145**(1):88-95. DOI: 10.1016/j.regpep.2007.09.025

[48] Sands WA et al. Regulating gene transcription in response to cyclic amp elevation. Cellu-

lar Signalling. 2008;**20**(3):460-466. DOI: 10.1016/j.cellsig.2007.10.005

Neuroscience. 2008;**28**(1):264-278. DOI: 10.1523/jneurosci.4178-07.2008

2006;**97**(4):1057-1070. DOI: 10.1111/j.1471-4159.2006.03804.x

Neurosciences. 2002;**21**(4):671-683. DOI: 10.1006/mcne.2002.1189

Reports. 2007;**59**(4):414-420. DOI: 10.1016/j.peptides.2007.04.007

DOI: 10.1016/j.pneurobio.2008.09.015

42 GABA And Glutamate - New Developments In Neurotransmission Research

**45**(2-3):191-202. DOI: 10.1016/j.neuint.2003.08.015

410. DOI: 10.1002/glia.20392

DOI: 10.1002/stem.35

009-8055-2

DOI: 10.2174/138161211795589346


protein kinase C. Journal of Neuroscience Research. 2006;**84**(8):1645-1655. DOI: 10.1002/ jnr.21065

[78] Kang JH et al. Protein kinase C (Pkc) isozyme-specific substrates and their design. Biotechnology Advances. 2012;**30**(6):1662-1672. DOI: 10.1016/j.biotechadv.2012.07.004

Notch Signaling in the Astroglial Phenotype: Relevance to Glutamatergic Transmission

http://dx.doi.org/10.5772/intechopen.73318

45

[79] Wu S et al. Targeting Glioma stem cells for therapy: Perspectives and challenges. Journal

[80] Sugaya K. Stem cell biology in the study of pathological conditions. Neurodegenerative

[81] Kwak Y-D et al. Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation. European Journal of Pharmacology. 2011;**650**(1):18-27.

[82] Yamamizu K et al. In vitro modeling of blood-brain barrier with human Ipsc-derived endothelial cells, pericytes, neurons, and astrocytes via notch signaling. Stem Cell

[83] Wang K et al. Neuroprotective effects of Crocin against traumatic brain injury in mice: Involvement of notch signaling pathway. Neuroscience Letters. 2015;**591**:53-58. DOI:

[84] Wilhelmsson U et al. Astrocytes negatively regulate neurogenesis through the Jagged1 mediated notch pathway. Stem Cells. 2012;**30**(10):2320-2329. DOI: 10.1002/stem.1196 [85] Jurynczyk M et al. Inhibition of notch signaling enhances tissue repair in an animal model of multiple sclerosis. Journal of Neuroimmunology. 2005;**170**(1-2):3-10. DOI:

[86] Jurynczyk M et al. Overcoming failure to repair demyelination in Eae: Gamma-secretase inhibition of notch signaling. Journal of the Neurological Sciences. 2008;**265**(1-2):5-11.

[87] Yang GS et al. Synergistic effect of mild hypothermia and the notch inhibitor Dapt against post stroke seizures. Biomedicine & Pharmacotherapy. 2017;**96**:675-684. DOI:

[88] Andersson ER et al. Therapeutic modulation of notch Signalling — Are we there yet? Nature Reviews Drug Discovery. 2014;**13**:357. DOI: 10.1038/nrd4252 https://www.nature.

[89] Toritsuka M et al. Regulation of striatal dopamine responsiveness by notch/Rbp-J signaling. Translational Psychiatry. 2017;**7**:e1049. DOI: 10.1038/tp.2017.21 https://www.nature.

[90] Du J et al. Dner modulates the length, polarity and synaptogenesis of spiral ganglion neurons via the notch signaling pathway. Molecular Medicine Reports. 2017;**17**:2357-2365.

of Cell Science and Therapy. 2015;**6**(207). DOI: 10.4172/2157-7013.1000207

Diseases. 2010;**7**(1-3):84-87. DOI: 10.1159/000285512

Reports. 2017;**8**(3):634-647. DOI: 10.1016/j.stemcr.2017.01.023

DOI: 10.1016/j.ejphar.2010.09.015

10.1016/j.neulet.2015.02.016

10.1016/j.jneuroim.2005.10.013

DOI: 10.1016/j.jns.2007.09.007

10.1016/j.biopha.2017.10.036

DOI: 10.3892/mmr.2017.8115

com/articles/nrd4252#supplementary-information

com/articles/tp201721#supplementary-information


[78] Kang JH et al. Protein kinase C (Pkc) isozyme-specific substrates and their design. Biotechnology Advances. 2012;**30**(6):1662-1672. DOI: 10.1016/j.biotechadv.2012.07.004

protein kinase C. Journal of Neuroscience Research. 2006;**84**(8):1645-1655. DOI: 10.1002/

[63] Bernabe A et al. Regulation of the Na+−dependent glutamate/aspartate transporter in rodent cerebellar astrocytes. Neurochemical Research. 2003;**28**(12):1843-1849 https://doi.

[64] Espinoza-Rojo M et al. Glast: Gene expression regulation by Phorbol esters. Neuroreport.

[65] Guo D et al. Notch-1 regulates Akt signaling pathway and the expression of cell cycle regulatory proteins Cyclin D1, Cdk2 and P21 in T-all cell lines. Leukemia Research. 2009;

[66] Fitzgerald K et al. Ras pathway signals are required for notch-mediated oncogenesis.

[67] Kim M et al. Protein kinase Cdelta negatively regulates Notch1-dependent transcription via a kinase-independent mechanism in vitro. Biochimica et Biophysica Acta.

[68] Steinhart R et al. Pkcepsilon induces astrocytic differentiation of multipotential neural

[69] Xu C et al. Morphine promotes astrocyte-preferential differentiation of mouse hippocampal progenitor cells via Pkcepsilon-dependent Erk activation and Trbp phosphory-

[70] Sjoqvist M et al. Pkczeta regulates notch receptor routing and activity in a notch signaling-dependent manner. Cell Research. 2014;**24**(4):433-450. DOI: 10.1038/cr.2014.34 [71] Guillem AM et al. Methylphenidate increases glutamate uptake in Bergmann glial cells. Neurochemical Research. 2015;**40**(11):2317-2324. DOI: 10.1007/s11064-015-1721-z [72] Ohtsuka T et al. Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation. The EMBO Journal. 1999;**18**(8):2196-2207. DOI: 10.1093/emboj/18.8.2196

[73] Lebkuechner I et al. Heterogeneity of notch signaling in astrocytes and the effects of Gfap and Vimentin deficiency. Journal of Neurochemistry. 2015;**135**(2):234-248. DOI:

[74] Endo Y et al. Bimodal functions of notch-mediated signaling are involved in neural crest formation during avian ectoderm development. Development. 2002;**129**(4):863-873 [75] Kwon C et al. Notch post-translationally regulates beta-catenin protein in stem and progenitor cells. Nature Cell Biology. 2011;**13**(10):1244-1251. DOI: 10.1038/ncb2313

[76] Ramain P et al. Novel notch alleles reveal a Deltex-dependent pathway repressing neural fate. Current Biology. 2001;**11**(22):1729-1738 https://doi.org/10.1016/S0960-9822

[77] Endo Y et al. Deltex/Dtx mediates notch signaling in regulation of Bmp4 expression in cranial neural crest formation during avian development. Development, Growth &

Differentiation. 2003;**45**(3):241-248. DOI: 10.1046/j.1524-4725.2003.693.x

jnr.21065

org/10.1023/A%3A1026171724963

44 GABA And Glutamate - New Developments In Neurotransmission Research

**33**(5):678-685. DOI: 10.1016/j.leukres.2008.10.026

Oncogene. 2000;**19**(37):4191-4198. DOI: 10.1038/sj.onc.1203766

precursor cells. Glia. 2007;**55**(2):224-232. DOI: 10.1002/glia.20454

lation. Stem Cells. 2015;**33**(9):2762-2772. DOI: 10.1002/stem.2055

2012;**1823**(2):387-397. DOI: 10.1016/j.bbamcr.2011.11.005

2000;**11**(12):2827-2832

10.1111/jnc.13213

(01)00562-0


**Chapter 4**

**Provisional chapter**

**Pharmacological Studies with Specific Agonist and**

Ionotropic glutamate receptors (iGluRs) are a group of proteins with a high degree of sequence homology. At least 20 type of putative ionotropic glutamate receptor (iGluR) like channels have been identified in *Arabidopsis thaliana*. To uncover the role of iGluRlike channels in plant root growth, we used a comprehensive set of compounds known to alter iGluR channels in the neurons. We found that Arabidopsis root system is highly sensitive to these compounds. iGluR competitive antagonists 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 6,7-dinitroquinoxaline-2,3-dione acted (DNQX) acts as a negative regulator of primary root and lateral root density. Continuous growth on antagonist also leads to impairment of root meristem size, which suggests that iGluR-like channels may play a role in meristem maintenance. However, application of iGluR agonists L-glutamate recovered Arabidopsis root growth. Taken together, these results suggest a correlation between the putative iGluR-like channel function and the alteration of root

**Pharmacological Studies with Specific Agonist and** 

DOI: 10.5772/intechopen.72121

© 2016 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,

© 2018 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.

and reproduction in any medium, provided the original work is properly cited.

A mixture of organic and inorganic materials that makes uppermost layer of the earth in which plants grow is known as Soil. The parent mineral rock derives inorganic materials and is found in the form of sand, silt and clay. However, organic materials come from dead and decayed parts of bacteria, fungi, algae, protozoa and soil animals such as nematodes, earthworms, beetles and termites. The inorganic nitrogen dissolved in soil is vital for nutritional requirements of plants, and it can be directly used in the synthesis of amino acids,

**Antagonist of Animal iGluR on Root Growth in**

**Antagonist of Animal iGluR on Root Growth in** 

*Arabidopsis thaliana*

*Arabidopsis thaliana*

Shashi Kant Singh and Ing-Feng Chang

Shashi Kant Singh and Ing-Feng Chang

http://dx.doi.org/10.5772/intechopen.72121

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

growth and development in the Arabidopsis roots.

**Keywords:** glutamate receptor, lateral root, Glu, calcium, DNQX, CNQX

Additional information is available at the end of the chapter

**Provisional chapter**

#### **Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth in** *Arabidopsis thaliana* **Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth in**  *Arabidopsis thaliana*

DOI: 10.5772/intechopen.72121

Shashi Kant Singh and Ing-Feng Chang Shashi Kant Singh and Ing-Feng Chang Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72121

#### **Abstract**

Ionotropic glutamate receptors (iGluRs) are a group of proteins with a high degree of sequence homology. At least 20 type of putative ionotropic glutamate receptor (iGluR) like channels have been identified in *Arabidopsis thaliana*. To uncover the role of iGluRlike channels in plant root growth, we used a comprehensive set of compounds known to alter iGluR channels in the neurons. We found that Arabidopsis root system is highly sensitive to these compounds. iGluR competitive antagonists 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 6,7-dinitroquinoxaline-2,3-dione acted (DNQX) acts as a negative regulator of primary root and lateral root density. Continuous growth on antagonist also leads to impairment of root meristem size, which suggests that iGluR-like channels may play a role in meristem maintenance. However, application of iGluR agonists L-glutamate recovered Arabidopsis root growth. Taken together, these results suggest a correlation between the putative iGluR-like channel function and the alteration of root growth and development in the Arabidopsis roots.

**Keywords:** glutamate receptor, lateral root, Glu, calcium, DNQX, CNQX

#### **1. Introduction**

A mixture of organic and inorganic materials that makes uppermost layer of the earth in which plants grow is known as Soil. The parent mineral rock derives inorganic materials and is found in the form of sand, silt and clay. However, organic materials come from dead and decayed parts of bacteria, fungi, algae, protozoa and soil animals such as nematodes, earthworms, beetles and termites. The inorganic nitrogen dissolved in soil is vital for nutritional requirements of plants, and it can be directly used in the synthesis of amino acids,

© 2016 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. © 2018 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.

peptides and proteins [1]. Plants absorb organic nitrogen from soil in the form of free amino acids [2, 3], which is derived mainly from decomposed organic matter and exudates produced by bacteria, fungus and living plants roots [4–9].

**2. Materials and methods**

**2.2. Chemicals**

for treatments.

22–23°C.

**2.3. Seed sterilization**

**2.4. Plant growth condition**

**2.5. Laser scanning and light microscopy**

a band-pass 500–550 nm filter.

**2.1. Plant materials and growth conditions**

quantified using ImageJ program (http://rsb.info.nih.gov/ij/).

*Arabidopsis thaliana* (Col-0) seedlings were used in different analyses on root development. All seed germination treatments were carried out at same half-strength Murashige and Skoog (MS) medium [39] at constant pH 5.8. The root elongation under various treatments was

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth…

http://dx.doi.org/10.5772/intechopen.72121

49

L-aspartic acid (Sigma, USA), L-glutamic acid, monosodium salt (Sigma, USA), N-Methyl-D-aspartate (NMDA; Sigma, USA), and 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid (AMPA; Sigma, USA) were dissolved in water, adjusted to pH 5.8 and filter sterilized. Both receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX; Sigma, USA) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris, USA) were dissolved in organic solvent Dimethyl sulfoxide (DMSO) (Sigma, USA). All treatments were used with variable concentrations as indicated in the figure legends. In order to study the role of Glu in auxin balance inside the root cells, we used a synthetic auxin Naphthaleneacetic acid (NAA, Sigma) and a polar auxin transport inhibitor NPA (1-N-Naphthylphthalamic acid, Sigma)

Prior to germination at growth media, seeds were first surface-sterilized in sodium hypochlorite in active chlorine. Sterilization was carried out in a hood cabinet, and aliquots of seeds

Arabidopsis seedlings for analysis were grown in sterile petri dishes using half strength MS medium and the plate was sealed using Micropore TM tape. After this, seeds were stratified in the dark at 4°C for 2–3 days to synchronize germination. Plates were then transferred to a growth chamber at illumination of 120–150 μmol/m2 s continuous light and at temperature

Confocal microscopy was performed using a Zeiss LSM510 META Confocal Imaging System (USA). To observe the apical root meristem through confocal microscopy, roots were counter-

were obtained by excitation with the Kr/Ar 488-nm laser line and emission was detected with

O. Images

stained in propidium iodide (PI, Sigma) (10 μM) for 2–3 min, rinsed, mounted in dH2

were placed in Eppendorf tubes and treated with active chlorine for 1–2 h.

Among the 20 common amino acids, the six amino acids (glutamic acid, glutamine, aspartic acid, asparagine, alanine and histidine) are mainly dominated in the soil, and they cover approximately 80% of the total soil amino acid pool [10–12].

An agonist is an inducing ligand that can bind to and induce channel-linked receptors. On the contrary, antagonist is a type of receptor ligand that can block the agonist-mediated responses. Since ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels, binding of L-glutamate (Glu) will open gates and increase ions conductance. However, both agonists and antagonists of iGluRs share structural similarity with glutamate and bind to iGluRs at the same site where Glu binds [13]. Interestingly, it has been observed that major amino acids (glutamate, glycine, alanine, serine, asparagine, and cysteine) present in the rhizosphere are strong agonist for iGlurRs [14].

Previous studies indicate that plant GLRs are functional, and involved in various functions, such as photosynthesis [15, 16], abiotic stress [17, 18], as C/N balance [19], plant-pathogen interaction [20, 21], root morphogenesis [22–24], pollen tube growth [25] and regulate cellular calcium homeostasis [14, 20, 26–29]. Among studies with various cell types in plants, it was found that Glu induces intracellular Ca2+ current. Glu-induced rise in the intracellular Ca2+ level can be inhibited by the use of iGluRs antagonists, which are quinoxalinediones, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) [18, 27, 30]. Therefore, it was proposed that glutamate receptors (GLRs) can contribute to the network of Ca2+ signaling pathways in plant cells [16]. *Atglr1.2* knock-out mutant plants displayed abnormalities in pollen growth [25]. Further, Analysis of *Arabidopsis* GLR mutant, *atglr3.6*, reveals a major role of the plant GLRs in the regulation of plant root development [24]. As a signaling molecule, glutamate is regarded to be the major neurotransmitter in the mammalian central nervous system. The application of exogenous Glu can also alter root phenotype [31, 32], indicating a role for GLR signaling in plants. Additionally, MEKK pathways can alter the glutamate sensitivity at the root tip suggesting for a glutamate signaling pathway in plants [33, 34].

These days pharmacology-based functional study of ionotropic glutamate receptors in plants has become very popular and useful approach [17, 18, 27, 32, 35–37]. We used comprehensive set of compounds that have been found to contain a strong ability to modulate the activity of mammalian iGluRs. In the present study, we introduced Glu to study the possible role of plant GLRs in root development. To minimize the chance of multiple effects of Glu, we also used artificial agonists (NMDA and AMPA) and competitive antagonists (DNQX and CNQX) to the glutamate binding site on receptors. In animals, these artificial agonists and antagonists are reported only for specific effects via their impact on iGluR activities [38]. In our pharmacological-based study, we investigate how glutamate and iGluRs antagonists directly affect plant root growth and development.

### **2. Materials and methods**

#### **2.1. Plant materials and growth conditions**

*Arabidopsis thaliana* (Col-0) seedlings were used in different analyses on root development. All seed germination treatments were carried out at same half-strength Murashige and Skoog (MS) medium [39] at constant pH 5.8. The root elongation under various treatments was quantified using ImageJ program (http://rsb.info.nih.gov/ij/).

#### **2.2. Chemicals**

peptides and proteins [1]. Plants absorb organic nitrogen from soil in the form of free amino acids [2, 3], which is derived mainly from decomposed organic matter and exudates pro-

Among the 20 common amino acids, the six amino acids (glutamic acid, glutamine, aspartic acid, asparagine, alanine and histidine) are mainly dominated in the soil, and they cover

An agonist is an inducing ligand that can bind to and induce channel-linked receptors. On the contrary, antagonist is a type of receptor ligand that can block the agonist-mediated responses. Since ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels, binding of L-glutamate (Glu) will open gates and increase ions conductance. However, both agonists and antagonists of iGluRs share structural similarity with glutamate and bind to iGluRs at the same site where Glu binds [13]. Interestingly, it has been observed that major amino acids (glutamate, glycine, alanine, serine, asparagine, and cysteine) present in the rhizosphere

Previous studies indicate that plant GLRs are functional, and involved in various functions, such as photosynthesis [15, 16], abiotic stress [17, 18], as C/N balance [19], plant-pathogen interaction [20, 21], root morphogenesis [22–24], pollen tube growth [25] and regulate cellular calcium homeostasis [14, 20, 26–29]. Among studies with various cell types in plants, it was found that Glu induces intracellular Ca2+ current. Glu-induced rise in the intracellular Ca2+ level can be inhibited by the use of iGluRs antagonists, which are quinoxalinediones, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) [18, 27, 30]. Therefore, it was proposed that glutamate receptors (GLRs) can contribute to the network of Ca2+ signaling pathways in plant cells [16]. *Atglr1.2* knock-out mutant plants displayed abnormalities in pollen growth [25]. Further, Analysis of *Arabidopsis* GLR mutant, *atglr3.6*, reveals a major role of the plant GLRs in the regulation of plant root development [24]. As a signaling molecule, glutamate is regarded to be the major neurotransmitter in the mammalian central nervous system. The application of exogenous Glu can also alter root phenotype [31, 32], indicating a role for GLR signaling in plants. Additionally, MEKK pathways can alter the glutamate sensitivity at the root tip suggesting for a glutamate signaling

These days pharmacology-based functional study of ionotropic glutamate receptors in plants has become very popular and useful approach [17, 18, 27, 32, 35–37]. We used comprehensive set of compounds that have been found to contain a strong ability to modulate the activity of mammalian iGluRs. In the present study, we introduced Glu to study the possible role of plant GLRs in root development. To minimize the chance of multiple effects of Glu, we also used artificial agonists (NMDA and AMPA) and competitive antagonists (DNQX and CNQX) to the glutamate binding site on receptors. In animals, these artificial agonists and antagonists are reported only for specific effects via their impact on iGluR activities [38]. In our pharmacological-based study, we investigate how glutamate and iGluRs antagonists directly affect

duced by bacteria, fungus and living plants roots [4–9].

48 GABA And Glutamate - New Developments In Neurotransmission Research

approximately 80% of the total soil amino acid pool [10–12].

are strong agonist for iGlurRs [14].

pathway in plants [33, 34].

plant root growth and development.

L-aspartic acid (Sigma, USA), L-glutamic acid, monosodium salt (Sigma, USA), N-Methyl-D-aspartate (NMDA; Sigma, USA), and 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid (AMPA; Sigma, USA) were dissolved in water, adjusted to pH 5.8 and filter sterilized. Both receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX; Sigma, USA) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris, USA) were dissolved in organic solvent Dimethyl sulfoxide (DMSO) (Sigma, USA). All treatments were used with variable concentrations as indicated in the figure legends. In order to study the role of Glu in auxin balance inside the root cells, we used a synthetic auxin Naphthaleneacetic acid (NAA, Sigma) and a polar auxin transport inhibitor NPA (1-N-Naphthylphthalamic acid, Sigma) for treatments.

#### **2.3. Seed sterilization**

Prior to germination at growth media, seeds were first surface-sterilized in sodium hypochlorite in active chlorine. Sterilization was carried out in a hood cabinet, and aliquots of seeds were placed in Eppendorf tubes and treated with active chlorine for 1–2 h.

#### **2.4. Plant growth condition**

Arabidopsis seedlings for analysis were grown in sterile petri dishes using half strength MS medium and the plate was sealed using Micropore TM tape. After this, seeds were stratified in the dark at 4°C for 2–3 days to synchronize germination. Plates were then transferred to a growth chamber at illumination of 120–150 μmol/m2 s continuous light and at temperature 22–23°C.

#### **2.5. Laser scanning and light microscopy**

Confocal microscopy was performed using a Zeiss LSM510 META Confocal Imaging System (USA). To observe the apical root meristem through confocal microscopy, roots were counterstained in propidium iodide (PI, Sigma) (10 μM) for 2–3 min, rinsed, mounted in dH2 O. Images were obtained by excitation with the Kr/Ar 488-nm laser line and emission was detected with a band-pass 500–550 nm filter.

#### **2.6. Statistical analysis**

Each experiment was repeated at least three times. Values are expressed as mean ± SD. The statistical significance was analyzed using Student's *t*-test analysis.

#### **3. Results**

#### **3.1. iGluR agonists and antagonists alter root growth in Arabidopsis**

We used a comprehensive set of compounds that have been found to modulate iGluRs. All treatments were performed with half strength of MS media [39] at a constant pH 5.8. The presence these compounds was observed to have a marked effect on root architecture of Arabidopsis. Both Glu and NMDA treatments had a stimulatory effect on primary root length (PRL) as well as lateral root density (LRD) in wild-type plants as compared to the non-treated plants. However, up to 10 days, AMPA showed a minor effect on root growth, but afterward, AMPA addition also nearly restored root growth of wild-type plants, making it visually indistinguishable from that of NMDA-treated plants (**Figure 1A** and **B**). These results indicated that glutamate receptor agonists likely interact with signaling pathways to control root growth in plants. Further, to test whether root growth was specific to natural iGluR ligands (Glu), we used another kind of neurotransmitter amino acid L-aspartate (Asp) [40]. Interestingly, after 12 days of growth, Asp treatment showed modest activity at inhibiting root growth and failed to increase lateral root formation when supplied at the same concentrations as Glu (**Figure 1A** and **B**). These results indicate that Glu and Asp have different activity in Arabidopsis root growth modulation and that the effects of Glu on root development are likely due to a specific effect of Glu rather than as a consequence of acidic behavior of amino acids.

To determine more closely the effects of plant iGluR-like receptor on the architecture of the Arabidopsis root system, wild-type Arabidopsis seedlings were germinated and grown on vertically oriented agar plates containing half strength MS medium supplemented with iGluR antagonists (DNQX and CNQX) alone or in combination of antagonists with Glu. As expected, our results show that both DNQX and CNQX drastically reduced root growth, and induced approximately similar kind of effects on root growth (**Figure 1C** and **D**). It was seen that the PRL approximately reduced by 64.65% and 69.24% and LRD by 76.1% and 76.55% (respectively for DNQX and CNQX treatment) (**Figure 1C** and **D**). To observe the effect of agonist and antagonist treatment together, we used naturally occurring agonist, Glu, to compete with DNQX and CNQX inhibitory actions [41]. It was observed that the external supplement of Glu (at 0.5 mM) successfully recovered the reduced root growth (both PRL and LRD) (**Figure 1C** and **D**). In summary, root growth was promoted by iGluR agonists, and use of iGluR antagonists (CNQX and DNQX) drastically reduced root growth and then, again subsequently recovered by addition of Glu suggesting molecular correlation. Since, these comprehensive set of compounds are called the great modulator of iGluRs in mammalian cells, our results suggested the involvement of Arabidopsis iGluR-like channel in root development.

**3.2. Short-root growth in antagonist treated wild-type roots is contributed by** 

Previously we concluded that the glutamate receptor signaling may be involved in regulatory mechanisms in the control of root growth, indicating an essential role for plant GLRs in root meristem maintenance Therefore we analyzed cell division and meristem size among wild-type and antagonist-treated wild-type roots at different growth duration (4 and 6 days). However, since treatments of both antagonists induced similar kind of inhibitory effect on root growth, and thus we selected only one antagonist (DNQX) for further studies.

**Figure 1.** Variable effects of animal iGluR effector compounds on *Arabidopsis thaliana* root development. Arabidopsis (wild-type, Col-0 ecotype) seedlings were germinated on half strength of MS medium (MS/2, [39]) supplemented with 1% of sucrose and agar. Immediately after germination, different treatments were done in MS/2 basal media adjusted to pH 5.8 with NaOH. Time course for agonist treatments response in days 2–14 of longitudinal primary root growth (A) and LR density (B) represented as LRs per centimeter primary root of Col-0 after incubation with 0.5 mM of each glutamate (Glu), NMDA, AMPA and aspartate (Asp) individually. Antagonist's treatments were done in MS/2 basal media but control seedlings (Mock) were grown with equal volume of solvent (DMSO) as in DNQX (1 mM) and CNQX (1 mM) treated seedlings. Comparison of root growth under antagonist given alone (1 mM) or together in the treatment of 0.5 mM Glu. Root length (C) and LR density (D) of 11-day-old Col-0 seedlings. Values represent the mean of 15–18 measurements in triplicate and error bars represent ±SD. The statistical analysis were performed by Student's *t*-test

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth…

http://dx.doi.org/10.5772/intechopen.72121

51

**reduced root meristem size**

(P < 0.005) indicated by asterisks.

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth… http://dx.doi.org/10.5772/intechopen.72121 51

**2.6. Statistical analysis**

**3. Results**

Each experiment was repeated at least three times. Values are expressed as mean ± SD. The

We used a comprehensive set of compounds that have been found to modulate iGluRs. All treatments were performed with half strength of MS media [39] at a constant pH 5.8. The presence these compounds was observed to have a marked effect on root architecture of Arabidopsis. Both Glu and NMDA treatments had a stimulatory effect on primary root length (PRL) as well as lateral root density (LRD) in wild-type plants as compared to the non-treated plants. However, up to 10 days, AMPA showed a minor effect on root growth, but afterward, AMPA addition also nearly restored root growth of wild-type plants, making it visually indistinguishable from that of NMDA-treated plants (**Figure 1A** and **B**). These results indicated that glutamate receptor agonists likely interact with signaling pathways to control root growth in plants. Further, to test whether root growth was specific to natural iGluR ligands (Glu), we used another kind of neurotransmitter amino acid L-aspartate (Asp) [40]. Interestingly, after 12 days of growth, Asp treatment showed modest activity at inhibiting root growth and failed to increase lateral root formation when supplied at the same concentrations as Glu (**Figure 1A** and **B**). These results indicate that Glu and Asp have different activity in Arabidopsis root growth modulation and that the effects of Glu on root development are likely due to a specific effect of Glu rather than

To determine more closely the effects of plant iGluR-like receptor on the architecture of the Arabidopsis root system, wild-type Arabidopsis seedlings were germinated and grown on vertically oriented agar plates containing half strength MS medium supplemented with iGluR antagonists (DNQX and CNQX) alone or in combination of antagonists with Glu. As expected, our results show that both DNQX and CNQX drastically reduced root growth, and induced approximately similar kind of effects on root growth (**Figure 1C** and **D**). It was seen that the PRL approximately reduced by 64.65% and 69.24% and LRD by 76.1% and 76.55% (respectively for DNQX and CNQX treatment) (**Figure 1C** and **D**). To observe the effect of agonist and antagonist treatment together, we used naturally occurring agonist, Glu, to compete with DNQX and CNQX inhibitory actions [41]. It was observed that the external supplement of Glu (at 0.5 mM) successfully recovered the reduced root growth (both PRL and LRD) (**Figure 1C** and **D**). In summary, root growth was promoted by iGluR agonists, and use of iGluR antagonists (CNQX and DNQX) drastically reduced root growth and then, again subsequently recovered by addition of Glu suggesting molecular correlation. Since, these comprehensive set of compounds are called the great modulator of iGluRs in mammalian cells, our results suggested the involvement of Arabidopsis iGluR-like channel in root development.

statistical significance was analyzed using Student's *t*-test analysis.

50 GABA And Glutamate - New Developments In Neurotransmission Research

**3.1. iGluR agonists and antagonists alter root growth in Arabidopsis**

as a consequence of acidic behavior of amino acids.

**Figure 1.** Variable effects of animal iGluR effector compounds on *Arabidopsis thaliana* root development. Arabidopsis (wild-type, Col-0 ecotype) seedlings were germinated on half strength of MS medium (MS/2, [39]) supplemented with 1% of sucrose and agar. Immediately after germination, different treatments were done in MS/2 basal media adjusted to pH 5.8 with NaOH. Time course for agonist treatments response in days 2–14 of longitudinal primary root growth (A) and LR density (B) represented as LRs per centimeter primary root of Col-0 after incubation with 0.5 mM of each glutamate (Glu), NMDA, AMPA and aspartate (Asp) individually. Antagonist's treatments were done in MS/2 basal media but control seedlings (Mock) were grown with equal volume of solvent (DMSO) as in DNQX (1 mM) and CNQX (1 mM) treated seedlings. Comparison of root growth under antagonist given alone (1 mM) or together in the treatment of 0.5 mM Glu. Root length (C) and LR density (D) of 11-day-old Col-0 seedlings. Values represent the mean of 15–18 measurements in triplicate and error bars represent ±SD. The statistical analysis were performed by Student's *t*-test (P < 0.005) indicated by asterisks.

#### **3.2. Short-root growth in antagonist treated wild-type roots is contributed by reduced root meristem size**

Previously we concluded that the glutamate receptor signaling may be involved in regulatory mechanisms in the control of root growth, indicating an essential role for plant GLRs in root meristem maintenance Therefore we analyzed cell division and meristem size among wild-type and antagonist-treated wild-type roots at different growth duration (4 and 6 days). However, since treatments of both antagonists induced similar kind of inhibitory effect on root growth, and thus we selected only one antagonist (DNQX) for further studies. Interestingly, we observed that antagonist-treated wild-type root illustrated a smaller meristem size compared to wild-type (**Figure 2A** and **B**). Simultaneously, the number of meristematic epidermal cells (in a single file) was also significantly reduced in both 4 and 6-day-old roots of DNQX-treated wild-type plants (**Figure 2C**). Reduced meristem-enriched tissues in DNQX treated roots showed a putative vital contribution of putative *AtGLR* signaling in Arabidopsis root development.

observed by Lugol staining of starch granule [43]. We found that DNQX treatment in wild-

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth…

There are many studies which showed that Arabidopsis AtGLRs engage in calcium homeostasis [27, 28, 30]. We investigated whether the induced root growth in Glu-treated seedlings was dependent on Ca2+. Various concentrations of EGTA (a Ca2+ chelator) was added to MS/2 supplemented with 0.5 mM Glu. At both concentrations of EGTA (0.5 and 1.0 mM), root elongation was drastically inhibited in wild-type seedlings. However, supplement of external Glu partially recovered root growth inhibited by low amount of EGTA (**Figure 4**). Collectively, these data suggest a role for Ca2+ in AtGLRs signaling to control root growth. Auxin has been recognized as a key regulator in root development [44, 45]. NPA is a drug that known for inhibition of polar auxin transport. An induction of cytosolic Ca2+ was observed after auxin application, indicating a strong correlations between Ca2+ and auxin signaling. Therefore, we investigated whether the higher root growth observed in the Glu-treated seedlings is linked to the auxin and calcium. To elucidate this, we investigated whether

are able to minimize the negative effect of NPA on Arabidopsis root growth.

**Figure 3.** Putative AtGLR regulates meristematic activity in primary-root apical meristem. Statistical significance in compared with wild-type were analyzed by Student's *t*-test (P < 0.005), indicated by asterisk. (A–B) Confocal images of 4-day-old PI-stained wild-type and DNQX treated wild-type roots. Columella cells have abnormal cell divisions in DNQX treated wild-type roots. Wild-type and DNQX treated wild-type roots in Lugol staining (C–D). Scale bar: 100 μm.

to NPA-treated wild-type seedling had restored

http://dx.doi.org/10.5772/intechopen.72121

53

type approximately abolished the starch grains from amyloplast (**Figure 3C** and **D**).

**3.3. Externally supplied Glu can rescue the EGTA-inhibited root phenotypes**

Glu and CaCl2

Interestingly, applications of Glu and CaCl2

the number of LR (**Figure 5A** and **B**).

QC surrounded with stem cells are pivotal in cell proliferation and meristem maintenance in root [42]. Thus we investigated the possibility of deformity in the stem cell niche which may result in impaired root growth after antagonist treatment. In confocal sections of propidium iodide (PI) stained roots (**Figure 3A** and **B**), we observed that in comparison to wild-type (four-celled QC), DNQX-treated wild-type roots were characterized by small dislocated columella cells with complicated-cellular-patterns. Altered columella root cap cells can also be

**Figure 2.** Putative AtGLR regulates meristematic activity in primary-root apical meristem. (A) Confocal microscopic images of PI-stained 4-day-old wild-type and DNQX treated wild-type root. The border of root apical meristem is indicated by arrows in PI-stained roots. The longitudinal distance between the quiescent center (QC, marked in lower arrow) and the first elongating cell is correspond to the root meristem length (B) and the number of meristematic epidermal cells in single file of cells in wild-type and DNQX treated wild-type root at various time points (4 and 6-dayold seedling) (C). Error bars represent SE (n > 15). Statistical significance in compared with wild-type were analyzed by Student's *t*-test (P < 0.005).

observed by Lugol staining of starch granule [43]. We found that DNQX treatment in wildtype approximately abolished the starch grains from amyloplast (**Figure 3C** and **D**).

#### **3.3. Externally supplied Glu can rescue the EGTA-inhibited root phenotypes**

Interestingly, we observed that antagonist-treated wild-type root illustrated a smaller meristem size compared to wild-type (**Figure 2A** and **B**). Simultaneously, the number of meristematic epidermal cells (in a single file) was also significantly reduced in both 4 and 6-day-old roots of DNQX-treated wild-type plants (**Figure 2C**). Reduced meristem-enriched tissues in DNQX treated roots showed a putative vital contribution of putative *AtGLR* signaling in

QC surrounded with stem cells are pivotal in cell proliferation and meristem maintenance in root [42]. Thus we investigated the possibility of deformity in the stem cell niche which may result in impaired root growth after antagonist treatment. In confocal sections of propidium iodide (PI) stained roots (**Figure 3A** and **B**), we observed that in comparison to wild-type (four-celled QC), DNQX-treated wild-type roots were characterized by small dislocated columella cells with complicated-cellular-patterns. Altered columella root cap cells can also be

**Figure 2.** Putative AtGLR regulates meristematic activity in primary-root apical meristem. (A) Confocal microscopic images of PI-stained 4-day-old wild-type and DNQX treated wild-type root. The border of root apical meristem is indicated by arrows in PI-stained roots. The longitudinal distance between the quiescent center (QC, marked in lower arrow) and the first elongating cell is correspond to the root meristem length (B) and the number of meristematic epidermal cells in single file of cells in wild-type and DNQX treated wild-type root at various time points (4 and 6-dayold seedling) (C). Error bars represent SE (n > 15). Statistical significance in compared with wild-type were analyzed by

Arabidopsis root development.

52 GABA And Glutamate - New Developments In Neurotransmission Research

Student's *t*-test (P < 0.005).

There are many studies which showed that Arabidopsis AtGLRs engage in calcium homeostasis [27, 28, 30]. We investigated whether the induced root growth in Glu-treated seedlings was dependent on Ca2+. Various concentrations of EGTA (a Ca2+ chelator) was added to MS/2 supplemented with 0.5 mM Glu. At both concentrations of EGTA (0.5 and 1.0 mM), root elongation was drastically inhibited in wild-type seedlings. However, supplement of external Glu partially recovered root growth inhibited by low amount of EGTA (**Figure 4**). Collectively, these data suggest a role for Ca2+ in AtGLRs signaling to control root growth.

Auxin has been recognized as a key regulator in root development [44, 45]. NPA is a drug that known for inhibition of polar auxin transport. An induction of cytosolic Ca2+ was observed after auxin application, indicating a strong correlations between Ca2+ and auxin signaling. Therefore, we investigated whether the higher root growth observed in the Glu-treated seedlings is linked to the auxin and calcium. To elucidate this, we investigated whether Glu and CaCl2 are able to minimize the negative effect of NPA on Arabidopsis root growth. Interestingly, applications of Glu and CaCl2 to NPA-treated wild-type seedling had restored the number of LR (**Figure 5A** and **B**).

**Figure 3.** Putative AtGLR regulates meristematic activity in primary-root apical meristem. Statistical significance in compared with wild-type were analyzed by Student's *t*-test (P < 0.005), indicated by asterisk. (A–B) Confocal images of 4-day-old PI-stained wild-type and DNQX treated wild-type roots. Columella cells have abnormal cell divisions in DNQX treated wild-type roots. Wild-type and DNQX treated wild-type roots in Lugol staining (C–D). Scale bar: 100 μm.

**4. Discussion**

**4.1. Effect of glutamate treatment on root growth**

acids, nucleic acids *etc*.) are derived from NH4

**4.2. Root meristematic activity**

Root plays pivotal role in plant life as it is crucial for nutrient and water absorption. In *Arabidopsis thaliana*, a total of 20 types of AtGLR subunits have been identified. They have significantly high sequence similarity with animal iGluR-like channels [46]. Probably due to phylogenetically conserved amino acid sequences, they may have a high potential for functional redundancy. Using specific drugs that alter the channel activities is a key to study the function of iGluR-like channels in Arabidopsis [21]. We used Glu, (a neurotransmitter), and other set of compounds known to agonize (activate) (NMDA and AMAP) and antagonize (deactivate) (DNQX and CNQX) the iGluR channels in mammalian cells. The use of broader set of drugs would allowed us to observe the specific effects related to Glu and iGluR-like channels in root cells. We observed that the application of these drugs potentially modulate the Arabidopsis root architectures indicating an importance for AtGLRs in root development. We observed that the application of iGluR agonists, Glu and NMDA were promoting root growth. In other studies it has also been reported that Glu could act as a root growth modifier [32, 47, 48]. Because Glu is an acidic amino acid which can cause low pH-induced toxicity (acidic), which could reduce root growth [31, 32, 48], we performed all experiments on constant pH range from 5.7 to 5.8 designed for plant tissue culture medium. Our result showing correlation with other evidences which have been proved that plants possess Glu-activated ion channels like iGluRs [30, 35, 36]. More specifically, NMDA-like iGluR receptors are also predicted in plants [27, 49]. Ammonium ion is a key form of inorganic nitrogen. Organic nitrogen compounds (amino

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth…

+

suggests signaling properties of Glu in animals may also develop in plant [53].

cial step in amino acid synthesis and nitrogen metabolism [51]. Glu is directly involved chlorophyll synthesis in developing leaves [52]. Although it cannot be ruled out that Glu metabolism plays an important part in plant nitrogen assimilation and its regulation, increasing evidence

The specificity of Glu to promote root development is individual. We used another kind of amino acid neurotransmitter, aspartate (Asp). Unlike Glu, it failed to induce root growth, showing Glu signaling in root development is highly specific [19]. Both DNQX and CNQX are the potent competitive AMPA/kainate glutamate receptor antagonists [38, 54]. We reported that iGluR antagonists have drastic effect on root growth. In animals they are known to block the ionotropic glutamate receptors very precisely [55]. Moreover, some studies in plants also have defined that animal iGluR antagonist are capable of changing the ion activity inside the cells and hence the phenotypes [16, 19, 21, 35, 56]. More interestingly, additional supply of Glu is able to counter the negative effect of each antagonist, suggesting a strong evidence of the existence of functional glutamate receptors in plant root development [17, 19, 20, 37, 56]. Similar evidence is also reported. Glu and Gly successfully revert back the effect of DNQX on Arabidopsis hypocotyl growth [27].

In Arabidopsis, root meristem develops from a stem-cell niche situated at the apical part of the root [57, 58]. Glutamate Receptor–Like protein (GLR3.1) has been described to be

[50]. The assimilation of NH4

+

http://dx.doi.org/10.5772/intechopen.72121

55

into Glu is the cru-

**Figure 4.** Ca2+-dependent growth phenotypes of EGTA treated wild-type root seedlings. Putative ligand Glu can overcome reduced root growth by low amount of EGTA. Root phenotype of the 10-day-old wild-type seedlings under the different treatments. Supplement of 0.5 mM Glu successfully recovered the primary root growth which was reduced by 0.5 mM and 1 mM EGTA. The data presented are averages of three biological replicates. Asterisks represent statistical difference analyzed with a Student's *t*-test; P < 0.005, n = 15.

**Figure 5.** Reduced root growth shown by NPA-treated wild-type roots can be rescued by the externally supplied Glu. Recovery of arrested root growth suggest the role of auxin. Application 1-N-Naphthylphthalamic acid (NPA) caused arrest of root growth. However, exogenous application of Glu and Ca2+ (0.5 mM) to NPA-treated root is successfully minimized the NPA effect (A and B). Asterisks represent statistical difference analyzed with a Student's *t*-test; P < 0.005, n = 15.

#### **4. Discussion**

**Figure 4.** Ca2+-dependent growth phenotypes of EGTA treated wild-type root seedlings. Putative ligand Glu can overcome reduced root growth by low amount of EGTA. Root phenotype of the 10-day-old wild-type seedlings under the different treatments. Supplement of 0.5 mM Glu successfully recovered the primary root growth which was reduced by 0.5 mM and 1 mM EGTA. The data presented are averages of three biological replicates. Asterisks represent statistical

**Figure 5.** Reduced root growth shown by NPA-treated wild-type roots can be rescued by the externally supplied Glu. Recovery of arrested root growth suggest the role of auxin. Application 1-N-Naphthylphthalamic acid (NPA) caused arrest of root growth. However, exogenous application of Glu and Ca2+ (0.5 mM) to NPA-treated root is successfully minimized the NPA effect (A and B). Asterisks represent statistical difference analyzed with a Student's *t*-test; P < 0.005,

difference analyzed with a Student's *t*-test; P < 0.005, n = 15.

54 GABA And Glutamate - New Developments In Neurotransmission Research

n = 15.

#### **4.1. Effect of glutamate treatment on root growth**

Root plays pivotal role in plant life as it is crucial for nutrient and water absorption. In *Arabidopsis thaliana*, a total of 20 types of AtGLR subunits have been identified. They have significantly high sequence similarity with animal iGluR-like channels [46]. Probably due to phylogenetically conserved amino acid sequences, they may have a high potential for functional redundancy. Using specific drugs that alter the channel activities is a key to study the function of iGluR-like channels in Arabidopsis [21]. We used Glu, (a neurotransmitter), and other set of compounds known to agonize (activate) (NMDA and AMAP) and antagonize (deactivate) (DNQX and CNQX) the iGluR channels in mammalian cells. The use of broader set of drugs would allowed us to observe the specific effects related to Glu and iGluR-like channels in root cells. We observed that the application of these drugs potentially modulate the Arabidopsis root architectures indicating an importance for AtGLRs in root development. We observed that the application of iGluR agonists, Glu and NMDA were promoting root growth. In other studies it has also been reported that Glu could act as a root growth modifier [32, 47, 48]. Because Glu is an acidic amino acid which can cause low pH-induced toxicity (acidic), which could reduce root growth [31, 32, 48], we performed all experiments on constant pH range from 5.7 to 5.8 designed for plant tissue culture medium. Our result showing correlation with other evidences which have been proved that plants possess Glu-activated ion channels like iGluRs [30, 35, 36]. More specifically, NMDA-like iGluR receptors are also predicted in plants [27, 49]. Ammonium ion is a key form of inorganic nitrogen. Organic nitrogen compounds (amino acids, nucleic acids *etc*.) are derived from NH4 + [50]. The assimilation of NH4 + into Glu is the crucial step in amino acid synthesis and nitrogen metabolism [51]. Glu is directly involved chlorophyll synthesis in developing leaves [52]. Although it cannot be ruled out that Glu metabolism plays an important part in plant nitrogen assimilation and its regulation, increasing evidence suggests signaling properties of Glu in animals may also develop in plant [53].

The specificity of Glu to promote root development is individual. We used another kind of amino acid neurotransmitter, aspartate (Asp). Unlike Glu, it failed to induce root growth, showing Glu signaling in root development is highly specific [19]. Both DNQX and CNQX are the potent competitive AMPA/kainate glutamate receptor antagonists [38, 54]. We reported that iGluR antagonists have drastic effect on root growth. In animals they are known to block the ionotropic glutamate receptors very precisely [55]. Moreover, some studies in plants also have defined that animal iGluR antagonist are capable of changing the ion activity inside the cells and hence the phenotypes [16, 19, 21, 35, 56]. More interestingly, additional supply of Glu is able to counter the negative effect of each antagonist, suggesting a strong evidence of the existence of functional glutamate receptors in plant root development [17, 19, 20, 37, 56]. Similar evidence is also reported. Glu and Gly successfully revert back the effect of DNQX on Arabidopsis hypocotyl growth [27].

#### **4.2. Root meristematic activity**

In Arabidopsis, root meristem develops from a stem-cell niche situated at the apical part of the root [57, 58]. Glutamate Receptor–Like protein (GLR3.1) has been described to be essential for meristematic activity in roots [22]. The roots grown by antagonist treatment significantly reduced meristematic cell number, and hence a contraction of meristem size was also observed. These observations certainly showed a correlation with less root growth under antagonist treatment [59]. The role of quiescent center (QC) is vital in the maintenance of root meristem [58, 60]. The majority of cells in the root meristem develop from stem cells which are derived from QC. In confocal microscopic analysis, antagonist treated-root showed a major change in QC organization which may resulted in less developed root meristem [61]. Numerous sedimented starch-filled amyloplasts in the root cap are distinguishing of columella cells [62]. In our study it was observed that columella cells of antagonist treated-roots possessed of defective amyloplasts in Lugol staining [63]. Therefore short root phenotype is highly consistence with defected organization of the root cap and QC [42, 64, 65].

versus an agar block with the calcium-chelating agent EGTA, suggesting that auxin transport is regulated by local [Ca2+]cyt levels [72]. NPA is a potent polar auxin transport inhibitor, which can highly reduce the lateral root emergence [75–77]. Supplement of Glu together with NPA (1-N-Naphthylphthalamic acid) (at 0.5 mM) showed approximately close root phenotype to the control seedlings. Addition of Glu in intact roots directly may induce Ca2+ which may lead to enhanced auxin transport and hence the suppressed negative effect of NPA Possibly application of Glu can enhance the auxin supply to other deserved root cells rather than showing

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth…

http://dx.doi.org/10.5772/intechopen.72121

57

In this study, we applied a comprehensive set of compounds to study how these compounds affect Arabidopsis root growth. Arabidopsis root system is highly sensitive to these compounds known to alter the iGluR channels. Both Glu and NMDA promote the primary root growth and lateral root density in Arabidopsis. On the other hand, iGluR antagonists drastically reduced root growth at both parameters. Exogenous application of Glu successfully rescued reduced root phenotype inhibited by EGTA. Moreover, root growth reduced by polar auxin transport inhibitor NPA, could be rescued by Glu and

. As for AtGLRs function, although the mechanisms are not yet clear, the results presented provide evidence in support of a role of AtGLRs in regulating Arabidopsis root

This work was supported by grants from Ministry of Science and Technology, Taiwan (MOST# 104-2311-B-002-034, MOST#106-2311-B-002-014, and MOST#106-2313-B-002-004). We thank TechComm (College of Life Science, NTU, Taiwan) for very helpful technical assistance.

3 Genome and Systems Biology Degree Program, National Taiwan University and Academia

and Ing-Feng Chang1,2,3\*

1 Institute of Plant Biology, National Taiwan University, Taipei, Taiwan

2 Department of Life Science, National Taiwan University, Taipei, Taiwan

\*Address all correspondence to: ifchang@ntu.edu.tw

competition with NPA blockage.

**5. Conclusion**

CaCl2

development.

**Acknowledgements**

**Author details**

Shashi Kant Singh1

Sinica, Taipei, Taiwan

#### **4.3. Glutamate and calcium in root growth**

The iGluR is known to be a Ca2+ permeable channel [66]. Many studies revealed that Arabidopsis AtGLR induces Ca2+ current upon activation by Glu [27, 30, 67]. We investigated whether the putative agonist and antagonist treatments alter the [Ca2+]cyt level in roots. EGTA is a well-known Ca2+-chelating agents [68]. In our study, application of EGTA shows a strong inhibition in root growth. Interestingly, however when Glu was introduced in same media, root growth was resumed. The presence of EGTA allows low availability of Ca2+ in free space. Animal cells and plant cells are similar in that they are both use endoplasmic reticulum (ER) as a calcium storage. Glutamate receptors are also reported to localized in ER [22, 69]. In animals, Glu-induced intracellular calcium levels through endoplasmic reticulum is reported [69]. However, application of Glu may lead to more activation of putative AtGLRs that allow more Ca2+ release to cytoplasm from endomembrane system which might play a role to recover the root growth. Calcium is key regulator of root growth [70, 71]. Previous report has also found that roots in EGTA containing media failed to grow toward gravity but it could be recovered by extra Ca2+ supply [72]. Furthermore, as we have discussed before that application of DNQX and CNQX reduced root apical meristem and hence also root growth, but application external Ca2+ could resume root growth. These results suggest a role of AtGLRs in Arabidopsis root development.

#### **4.4. Glutamate signaling and polar auxin transport in roots**

Expressions of *AtGLR* genes inside the root tissue give strong evidence that these receptors have vital role [46, 73]. Recent studies on chimeric and other plant iGLRs provided evidence for Ca2+ permeability across membranes. We have also found that the *glr3.6-1* mutant showed altered cytosolic calcium levels in root cells [24]. Calcium and auxin work together in many aspects of cellular processes. A similar effect has been observed in different studies in response to calcium-chelating agents. Dela Fuente and Leopold (1973) showed that basipetal transport of auxin is depressed by EDTA treatment and that subsequent addition of Ca2+ restores auxin transport in roots [74]. Root bend toward a calcium-containing agar block versus an agar block with the calcium-chelating agent EGTA, suggesting that auxin transport is regulated by local [Ca2+]cyt levels [72]. NPA is a potent polar auxin transport inhibitor, which can highly reduce the lateral root emergence [75–77]. Supplement of Glu together with NPA (1-N-Naphthylphthalamic acid) (at 0.5 mM) showed approximately close root phenotype to the control seedlings. Addition of Glu in intact roots directly may induce Ca2+ which may lead to enhanced auxin transport and hence the suppressed negative effect of NPA Possibly application of Glu can enhance the auxin supply to other deserved root cells rather than showing competition with NPA blockage.

#### **5. Conclusion**

essential for meristematic activity in roots [22]. The roots grown by antagonist treatment significantly reduced meristematic cell number, and hence a contraction of meristem size was also observed. These observations certainly showed a correlation with less root growth under antagonist treatment [59]. The role of quiescent center (QC) is vital in the maintenance of root meristem [58, 60]. The majority of cells in the root meristem develop from stem cells which are derived from QC. In confocal microscopic analysis, antagonist treated-root showed a major change in QC organization which may resulted in less developed root meristem [61]. Numerous sedimented starch-filled amyloplasts in the root cap are distinguishing of columella cells [62]. In our study it was observed that columella cells of antagonist treated-roots possessed of defective amyloplasts in Lugol staining [63]. Therefore short root phenotype is highly consistence with defected organization of the

The iGluR is known to be a Ca2+ permeable channel [66]. Many studies revealed that Arabidopsis AtGLR induces Ca2+ current upon activation by Glu [27, 30, 67]. We investigated whether the putative agonist and antagonist treatments alter the [Ca2+]cyt level in roots. EGTA is a well-known Ca2+-chelating agents [68]. In our study, application of EGTA shows a strong inhibition in root growth. Interestingly, however when Glu was introduced in same media, root growth was resumed. The presence of EGTA allows low availability of Ca2+ in free space. Animal cells and plant cells are similar in that they are both use endoplasmic reticulum (ER) as a calcium storage. Glutamate receptors are also reported to localized in ER [22, 69]. In animals, Glu-induced intracellular calcium levels through endoplasmic reticulum is reported [69]. However, application of Glu may lead to more activation of putative AtGLRs that allow more Ca2+ release to cytoplasm from endomembrane system which might play a role to recover the root growth. Calcium is key regulator of root growth [70, 71]. Previous report has also found that roots in EGTA containing media failed to grow toward gravity but it could be recovered by extra Ca2+ supply [72]. Furthermore, as we have discussed before that application of DNQX and CNQX reduced root apical meristem and hence also root growth, but application external Ca2+ could resume root growth.

These results suggest a role of AtGLRs in Arabidopsis root development.

Expressions of *AtGLR* genes inside the root tissue give strong evidence that these receptors have vital role [46, 73]. Recent studies on chimeric and other plant iGLRs provided evidence for Ca2+ permeability across membranes. We have also found that the *glr3.6-1* mutant showed altered cytosolic calcium levels in root cells [24]. Calcium and auxin work together in many aspects of cellular processes. A similar effect has been observed in different studies in response to calcium-chelating agents. Dela Fuente and Leopold (1973) showed that basipetal transport of auxin is depressed by EDTA treatment and that subsequent addition of Ca2+ restores auxin transport in roots [74]. Root bend toward a calcium-containing agar block

**4.4. Glutamate signaling and polar auxin transport in roots**

root cap and QC [42, 64, 65].

**4.3. Glutamate and calcium in root growth**

56 GABA And Glutamate - New Developments In Neurotransmission Research

In this study, we applied a comprehensive set of compounds to study how these compounds affect Arabidopsis root growth. Arabidopsis root system is highly sensitive to these compounds known to alter the iGluR channels. Both Glu and NMDA promote the primary root growth and lateral root density in Arabidopsis. On the other hand, iGluR antagonists drastically reduced root growth at both parameters. Exogenous application of Glu successfully rescued reduced root phenotype inhibited by EGTA. Moreover, root growth reduced by polar auxin transport inhibitor NPA, could be rescued by Glu and CaCl2 . As for AtGLRs function, although the mechanisms are not yet clear, the results presented provide evidence in support of a role of AtGLRs in regulating Arabidopsis root development.

#### **Acknowledgements**

This work was supported by grants from Ministry of Science and Technology, Taiwan (MOST# 104-2311-B-002-034, MOST#106-2311-B-002-014, and MOST#106-2313-B-002-004). We thank TechComm (College of Life Science, NTU, Taiwan) for very helpful technical assistance.

#### **Author details**

Shashi Kant Singh1 and Ing-Feng Chang1,2,3\*

\*Address all correspondence to: ifchang@ntu.edu.tw

1 Institute of Plant Biology, National Taiwan University, Taipei, Taiwan

2 Department of Life Science, National Taiwan University, Taipei, Taiwan

3 Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei, Taiwan

#### **References**

[1] Lipson D, Nasholm T. The unexpected versatility of plants: Organic nitrogen use and availability in terrestrial ecosystems. Oecologia. 2001;**128**:305-316

[14] Qi Z, Stephens NR, Spalding EP. Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile. Plant Physiology. 2006;**142**:963-971

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth…

http://dx.doi.org/10.5772/intechopen.72121

59

[15] Teardo E, Formentin E, Segalla A, Giacometti GM, Marin O, Zanetti M, Lo Schiavo F, Zoratti M, Szabo I. Dual localization of plant glutamate receptor AtGLR3.4 to plastids

[16] Teardo E, Segalla A, Formentin E, Zanetti M, Marin O, Giacometti GM, Lo Schiavo F, Zoratti M, Szabo I. Characterization of a plant glutamate receptor activity. Cellular

[17] Kang J, Mehta S, Turano FJ. The putative glutamate receptor 1.1 (AtGLR1.1) in *Arabidopsis thaliana* regulates abscisic acid biosynthesis and signaling to control development and

[18] Meyerhoff O, Muller K, Roelfsema MR, Latz A, Lacombe B, Hedrich R, Dietrich P, Becker D. AtGLR3.4, a glutamate receptor channel-like gene is sensitive to touch and cold.

[19] Kang J, Turano FJ. The putative glutamate receptor 1.1 (AtGLR1.1) functions as a regulator of carbon and nitrogen metabolism in *Arabidopsis thaliana*. Proceedings of the National Academy of Sciences of the United States of America. 2003;**100**:6872-6877

[20] Kang S, Kim HB, Lee H, Choi JY, Heu S, Oh CJ, Kwon SI, An CS. Overexpression in Arabidopsis of a plasma membrane-targeting glutamate receptor from small radish increases glutamate-mediated Ca2+ influx and delays fungal infection. Molecules and

[21] Kwaaitaal M, Huisman R, Maintz J, Reinstadler A, Panstruga R. Ionotropic glutamate receptor (iGluR)-like channels mediate MAMP-induced calcium influx in *Arabidopsis* 

[22] Li J, Zhu S, Song X, Shen Y, Chen H, Yu J, Yi K, Liu Y, Karplus VJ, Wu P, Deng XW. A rice glutamate receptor-like gene is critical for the division and survival of individual cells in

[23] Miller ND, Durham Brooks TL, Assadi AH, Spalding EP. Detection of a gravitropism phenotype in glutamate receptor-like 3.3 mutants of *Arabidopsis thaliana* using machine

[24] Singh SK, Chien CT, Chang IF. The Arabidopsis glutamate receptor-like gene GLR3.6 controls root development by repressing the Kip-related protein gene KRP4. Journal of

[25] Michard E, Lima PT, Borges F, Silva AC, Portes MT, Carvalho JE, Gilliham M, Liu LH, Obermeyer G, Feijo JA. Glutamate receptor-like genes form Ca2+ channels in pollen tubes

and plasma membrane. Biochimica et Biophysica Acta. 2011;**1807**:359-367

Physiology and Biochemistry. 2010;**26**:253-262

Planta. 2005;**222**:418-427

Cells. 2006;**21**:418-427

water loss. Plant & Cell Physiology. 2004;**45**:1380-1389

*thaliana*. The Biochemical Journal. 2011;**440**:355-365

the root apical meristem. Plant Cell. 2006;**18**:340-349

vision and computation. Genetics. 2010;**186**:585-593

and are regulated by pistil D-serine. Science. 2011;**332**:434-437

Experimental Botany. 2016;**67**:1853-1869


[14] Qi Z, Stephens NR, Spalding EP. Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile. Plant Physiology. 2006;**142**:963-971

**References**

[1] Lipson D, Nasholm T. The unexpected versatility of plants: Organic nitrogen use and

[2] Kielland K. Amino acid absorption by arctic plants: Implications for plant nutrition and

[3] Nordin A, Uggla C, Nasholm T. Nitrogen forms in bark, wood and foliage of nitrogen-

[4] Barzana G, Aroca R, Bienert GP, Chaumont F, Ruiz-Lozano JM. New insights into the regulation of aquaporins by the arbuscular mycorrhizal symbiosis in maize plants under drought stress and possible implications for plant performance. Molecular Plant-

[5] Butler JL, Williams MA, Bottomley PJ, Myrold DD. Microbial community dynamics associated with rhizosphere carbon flow. Applied and Environmental Microbiology.

[6] Dennis PG, Miller AJ, Hirsch PR. Are root exudates more important than other sources of rhizodeposits in structuring rhizosphere bacterial communities? FEMS Microbiology

[7] Ramos C, Licht TR, Sternberg C, Krogfelt KA, Molin S. Monitoring bacterial growth activity in biofilms from laboratory flow chambers, plant rhizosphere, and animal intes-

[8] Walder F, Brule D, Koegel S, Wiemken A, Boller T, Courty PE. Plant phosphorus acquisition in a common mycorrhizal network: Regulation of phosphate transporter genes of

[9] Weintraub MN, Scott-Denton LE, Schmidt SK, Monson RK. The effects of tree rhizodeposition on soil exoenzyme activity, dissolved organic carbon, and nutrient availability

[10] Bremner JM. The amino-acid composition of the protein material in soil. The Biochemical

[11] Martens DA, Loeffelmann KL. Soil amino acid composition quantified by acid hydrolysis and anion chromatography-pulsed amperometry. Journal of Agricultural and Food

[12] Nancy RW. Soil amino acid composition across a boreal forest successional sequence.

[13] Schwartz EA, Tachibana M. Electrophysiology of glutamate and sodium co-transport in a glial cell of the salamander retina. The Journal of Physiology. 1990;**426**:43-80

the Pht1 family in sorghum and flax. The New Phytologist. 2015;**205**:1632-1645

in a subalpine forest ecosystem. Oecologia. 2007;**154**:327-338

availability in terrestrial ecosystems. Oecologia. 2001;**128**:305-316

nitrogen cycling. Ecology. 1994;**75**:2373-2383

58 GABA And Glutamate - New Developments In Neurotransmission Research

Microbe Interactions. 2014;**27**:349-363

tine. Methods in Enzymology. 2001;**337**:21-42

2003;**69**:6793-6800

Ecology. 2010;**72**:313-327

Journal. 1950;**47**:538-542

Chemistry. 2003;**51**:6521-6529

Soil Biology & Biochemistry. 2009;**41**:1210-1220

fertilized *Pinus sylvestris*. Tree Physiology. 2001;**21**:59-64


[26] Cho D, Kim SA, Murata Y, Lee S, Jae SK, Nam HG, Kwak JM. De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca2+-programmed stomatal closure. The Plant Journal. 2009;**58**:437-449

[40] Cavallero A, Marte A, Fedele E. L-aspartate as an amino acid neurotransmitter: Mechanisms of the depolarization-induced release from cerebrocortical synaptosomes. Journal

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth…

http://dx.doi.org/10.5772/intechopen.72121

61

[41] Geoffroy M, Lambolez B, Audinat E, Hamon B, Crepel F, Rossier J, Kado RT. Reduction of desensitization of a glutamate ionotropic receptor by antagonists. Molecular

[42] van den Berg C, Willemsen V, Hendriks G, Weisbeek P, Scheres B. Short-range control of cell differentiation in the Arabidopsis root meristem. Nature. 1997;**390**:287-289

[43] Qin C, Yi KK, Wu P. Ammonium affects cell viability to inhibit root growth in

[44] Laskowski MJ, Williams ME, Nusbaum HC, Sussex IM. Formation of lateral root meri-

[45] Overvoorde P, Fukaki H, Beeckman T. Auxin control of root development. Cold Spring

[46] Chiu JC, Brenner ED, DeSalle R, Nitabach MN, Holmes TC, Coruzzi GM. Phylogenetic and expression analysis of the glutamate-receptor-like gene family in *Arabidopsis thali-*

[47] Walch-Liu P, Forde BG. Nitrate signalling mediated by the NRT1.1 nitrate transporter antagonises L-glutamate-induced changes in root architecture. The Plant Journal.

[48] Walch-Liu P, Liu LH, Remans T, Tester M, Forde BG. Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in *Arabidopsis thaliana*.

[49] Chiu J, DeSalle R, Lam HM, Meisel L, Coruzzi G. Molecular evolution of glutamate receptors: A primitive signaling mechanism that existed before plants and animals

[50] Storey R, Beevers L. Enzymology of glutamine metabolism related to senescence and seed development in the pea (*Pisum sativum* L.). Plant Physiology. 1978;**61**:494-500

[51] Suzuki A, Knaff DB. Glutamate synthase: Structural, mechanistic and regulatory properties, and role in the amino acid metabolism. Photosynthesis Research. 2005;**83**:191-217

[52] Yaronskaya E, Vershilovskaya I, Poers Y, Alawady AE, Averina N, Grimm B. Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings.

[53] Forde BG, Lea PJ. Glutamate in plants: Metabolism, regulation, and signalling. Journal

Arabidopsis. Journal of Zhejiang University. Science. B. 2011;**12**:477-484

stems is a two-stage process. Development. 1995;**121**:3303-3310

*ana*. Molecular Biology and Evolution. 2002;**19**:1066-1082

diverged. Molecular Biology and Evolution. 1999;**16**:826-838

Harbor Perspectives in Biology. 2010;**2**:a001537

Plant & Cell Physiology. 2006;**47**:1045-1057

of Experimental Botany. 2007;**58**:2339-2358

of Neurochemistry. 2009;**110**:924-934

Pharmacology. 1991;**39**:587-591

2008;**54**:820-828

Planta. 2006;**224**:700-709


[40] Cavallero A, Marte A, Fedele E. L-aspartate as an amino acid neurotransmitter: Mechanisms of the depolarization-induced release from cerebrocortical synaptosomes. Journal of Neurochemistry. 2009;**110**:924-934

[26] Cho D, Kim SA, Murata Y, Lee S, Jae SK, Nam HG, Kwak JM. De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca2+-programmed

[27] Dubos C, Huggins D, Grant GH, Knight MR, Campbell MM. A role for glycine in the

[28] Kim SA, Kwak JM, Jae SK, Wang MH, Nam HG. Overexpression of the AtGluR2 gene encoding an Arabidopsis homolog of mammalian glutamate receptors impairs calcium utilization and sensitivity to ionic stress in transgenic plants. Plant & Cell Physiology.

[29] Vincill ED, Bieck AM, Spalding EP. Ca(2+) conduction by an amino acid-gated ion chan-

[30] Dennison KL, Spalding EP. Glutamate-gated calcium fluxes in Arabidopsis. Plant

[31] Kim TH, Kim EC, Kim SW, Lee HS, Choi DW. Exogenous glutamate inhibits the root growth and increases the glutamine content in *Arabidopsis thaliana*. Journal of Plant

[32] Walch-Liu P, Forde BG. L-Glutamate as a novel modifier of root growth and branching:

[33] Forde BG. Glutamate signalling in roots. Journal of Experimental Botany. 2014;**65**:779-787

[34] Forde BG, Cutler SR, Zaman N, Krysan PJ. Glutamate signalling via a MEKK1 kinasedependent pathway induces changes in Arabidopsis root architecture. The Plant Journal.

[35] Brenner ED, Martinez-Barboza N, Clark AP, Liang QS, Stevenson DW, Coruzzi GM. Arabidopsis mutants resistant to S(+)-beta-methyl-alpha, beta-diaminopropionic acid, a

[36] Dubos C, Willment J, Huggins D, Grant GH, Campbell MM. Kanamycin reveals the role played by glutamate receptors in shaping plant resource allocation. The Plant Journal.

[37] Sivaguru M, Pike S, Gassmann W, Baskin TI. Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: Evidence that these responses are

[38] Alford S, Grillner S. CNQX and DNQX block non-NMDA synaptic transmission but not NMDA-evoked locomotion in lamprey spinal cord. Brain Research. 1990;**506**:297-302

[39] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco

mediated by a glutamate receptor. Plant & Cell Physiology. 2003;**44**:667-675

tissue cultures. Plant Physiology. 1962;**15**:473-497

cycad-derived glutamate receptor agonist. Plant Physiology. 2000;**124**:1615-1624

gating of plant NMDA-like receptors. The Plant Journal. 2003;**35**:800-810

nel related to glutamate receptors. Plant Physiology. 2012;**159**:40-46

What's the sensor? Plant Signaling & Behavior. 2007;**2**:284-286

stomatal closure. The Plant Journal. 2009;**58**:437-449

60 GABA And Glutamate - New Developments In Neurotransmission Research

2001;**42**:74-84

Physiology. 2000;**124**:1511-1514

Biology. 2010;**53**:45-51

2013;**75**:1-10

2005;**43**:348-355


[54] Lee SH, Govindaiah G, Cox CL. Selective excitatory actions of DNQX and CNQX in rat thalamic neurons. Journal of Neurophysiology. 2010;**103**:1728-1734

[69] Schapansky J, Morissette M, Odero G, Albensi B, Glazner G. Neuregulin beta1 enhances peak glutamate-induced intracellular calcium levels through endoplasmic reticulum calcium release in cultured hippocampal neurons. Canadian Journal of Physiology and

Pharmacological Studies with Specific Agonist and Antagonist of Animal iGluR on Root Growth…

http://dx.doi.org/10.5772/intechopen.72121

63

[70] Trewavas AJ, Malho R. Ca2+ signalling in plant cells: The big network! Current Opinion

[71] Xu J, Brearley CA, Lin WH, Wang Y, Ye R, Mueller-Roeber B, Xu ZH, Xue HW. A role of Arabidopsis inositol polyphosphate kinase, AtIPK2alpha, in pollen germination and

[72] Lee JS, Mulkey TJ, Evans ML. Reversible loss of gravitropic sensitivity in maize roots

[73] Roy SJ, Gilliham M, Berger B, Essah PA, Cheffings C, Miller AJ, Davenport RJ, Liu LH, Skynner MJ, Davies JM, Richardson P, Leigh RA, Tester M. Investigating glutamate receptor-like gene co-expression in *Arabidopsis thaliana*. Plant, Cell & Environment.

[74] Dela Fuente RK, Leopold AC. A role for calcium in auxin transport. Plant Physiology.

[75] Casimiro I, Marchant A, Bhalerao RP, Beeckman T, Dhooge S, Swarup R, Graham N, Inze D, Sandberg G, Casero PJ, Bennett M. Auxin transport promotes Arabidopsis lateral

[76] Muday GK, Haworth P. Tomato root growth, gravitropism, and lateral development: Correlation with auxin transport. Plant Physiology and Biochemistry. 1994;**32**:193-203

[77] Reed RC, Brady SR, Muday GK.Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis. Plant Physiology. 1998;**118**:1369-1378

after tip application of calcium chelators. Science. 1983;**220**:1375-1376

Pharmacology. 2009;**87**:883-891

in Plant Biology. 1998;**1**:428-433

2008;**31**:861-871

1973;**51**:845-847

root growth. Plant Physiology. 2005;**137**:94-103

root initiation. Plant Cell. 2001;**13**:843-852


[69] Schapansky J, Morissette M, Odero G, Albensi B, Glazner G. Neuregulin beta1 enhances peak glutamate-induced intracellular calcium levels through endoplasmic reticulum calcium release in cultured hippocampal neurons. Canadian Journal of Physiology and Pharmacology. 2009;**87**:883-891

[54] Lee SH, Govindaiah G, Cox CL. Selective excitatory actions of DNQX and CNQX in rat

[55] Bessho Y, Nakanishi S, Nawa H. Glutamate receptor agonists enhance the expression of BDNF mRNA in cultured cerebellar granule cells. Brain Research. Molecular Brain

[56] Lam HM, Chiu J, Hsieh MH, Meisel L, Oliveira IC, Shin M, Coruzzi G. Glutamate-

[57] Beemster GT, Baskin TI. Analysis of cell division and elongation underlying the developmental acceleration of root growth in *Arabidopsis thaliana*. Plant Physiology.

[58] Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, Scheres B. Cellular

[59] Gonzalez-Garcia MP, Vilarrasa-Blasi J, Zhiponova M, Divol F, Mora-Garcia S, Russinova E, Cano-Delgado AI. Brassinosteroids control meristem size by promoting cell cycle pro-

[60] Aida M, Beis D, Heidstra R, Willemsen V, Blilou I, Galinha C, Nussaume L, Noh YS, Amasino R, Scheres B. The PLETHORA genes mediate patterning of the Arabidopsis

[61] Wysocka-Diller JW, Helariutta Y, Fukaki H, Malamy JE, Benfey PN. Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and

[62] Moore R. A morphometric analysis of the redistribution of organelles in columella cells in primary roots of normal seedlings and agravitropic mutants of *Hordeum vulgare*.

[63] Stahl Y, Wink RH, Ingram GC, Simon R. A signaling module controlling the stem cell

[64] Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J, Heidstra R, Aida M, Palme K, Scheres B. The PIN auxin efflux facilitator network controls growth and patterning in

[65] Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold N, Weisbeek P, Scheres B. An auxin-dependent distal organizer of pattern and

[66] Swanson GT, Sakai R. Ligands for ionotropic glutamate receptors. Progress in Molecular

[67] Demidchik V, Essah PA, Tester M. Glutamate activates cation currents in the plasma

[68] Weisenberg RC. Microtubule formation in vitro in solutions containing low calcium con-

niche in Arabidopsis root meristems. Current Biology. 2009;**19**:909-914

organisation of the *Arabidopsis thaliana* root. Development. 1993;**119**:71-84

gression in Arabidopsis roots. Development. 2011;**138**:849-859

thalamic neurons. Journal of Neurophysiology. 2010;**103**:1728-1734

receptor genes in plants. Nature. 1998;**396**:125-126

62 GABA And Glutamate - New Developments In Neurotransmission Research

root stem cell niche. Cell. 2004;**119**:109-120

Journal of Experimental Botany. 1985;**36**:1275-1286

polarity in the Arabidopsis root. Cell. 1999;**99**:463-472

membrane of Arabidopsis root cells. Planta. 2004;**219**:167-175

shoot. Development. 2000;**127**:595-603

Arabidopsis roots. Nature. 2005;**433**:39-44

and Subcellular Biology. 2009;**46**:123-157

centrations. Science. 1972;**177**:1104-1105

Research. 1993;**18**:201-208

1998;**116**:1515-1526


**Chapter 5**

Provisional chapter

**GABA and Glutamate: Their Transmitter Role in the**

GABA and Glutamate: Their Transmitter Role in the CNS

DOI: 10.5772/intechopen.70958

Glutamate and gamma-aminobutyric acid (GABA) are the major neurotransmitters in the mammalian brain. Inhibitory GABA and excitatory glutamate work together to control many processes, including the brain's overall level of excitation. The contributions of GABA and glutamate in extra-neuronal signaling are by far less widely recognized. In this chapter, we first discuss the role of both neurotransmitters during development, emphasizing the importance of the shift from excitatory to inhibitory GABAergic neurotransmission. The second part summarizes the biosynthesis and role of GABA and glutamate in neurotransmission in the mature brain, and major neurological disorders associated with glutamate and GABA receptors and GABA release mechanisms. The final part focuses on extra-neuronal glutamatergic and GABAergic signaling in pancreatic islets of Langerhans, and possible associations with type 1 diabetes mellitus.

Keywords: glutamate, GABA, CNS, pancreatic islets, neurological disorders

Glutamate and gamma-aminobutyric acid (GABA) are the major neurotransmitters in the brain. Inhibitory GABA and excitatory glutamate work together to control many processes, including the brain's overall level of excitation. A balanced interaction is required to maintain the physiological homeostasis, while prolonged imbalance can lead to disease. Glutamatergic/ GABAergic imbalance can be found in autism spectrum disorders and anxiety disorders with elevated glutamatergic neurotransmission, while high levels of GABA produce more relaxation and even sedation. Neurotransmitter levels can be affected by external factors, for example, alcohol. Alcohol potentiates the sedentary effects of GABA, while inhibiting the excitatory aspects of glutamate, resulting in an overall increase in GABA/glutamate ratio. This leads to

> © 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 eproduction in any medium, provided the original work is properly cited.

© 2018 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 and Pancreatic Islets**

and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

Mario Manto

Mario Manto

Abstract

autoimmune diabetes

1. Introduction

Christiane S. Hampe, Hiroshi Mitoma and

Christiane S. Hampe, Hiroshi Mitoma and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets** GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

DOI: 10.5772/intechopen.70958

Christiane S. Hampe, Hiroshi Mitoma and Mario Manto Christiane S. Hampe, Hiroshi Mitoma and Mario Manto

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70958

#### Abstract

Glutamate and gamma-aminobutyric acid (GABA) are the major neurotransmitters in the mammalian brain. Inhibitory GABA and excitatory glutamate work together to control many processes, including the brain's overall level of excitation. The contributions of GABA and glutamate in extra-neuronal signaling are by far less widely recognized. In this chapter, we first discuss the role of both neurotransmitters during development, emphasizing the importance of the shift from excitatory to inhibitory GABAergic neurotransmission. The second part summarizes the biosynthesis and role of GABA and glutamate in neurotransmission in the mature brain, and major neurological disorders associated with glutamate and GABA receptors and GABA release mechanisms. The final part focuses on extra-neuronal glutamatergic and GABAergic signaling in pancreatic islets of Langerhans, and possible associations with type 1 diabetes mellitus.

Keywords: glutamate, GABA, CNS, pancreatic islets, neurological disorders autoimmune diabetes

#### 1. Introduction

Glutamate and gamma-aminobutyric acid (GABA) are the major neurotransmitters in the brain. Inhibitory GABA and excitatory glutamate work together to control many processes, including the brain's overall level of excitation. A balanced interaction is required to maintain the physiological homeostasis, while prolonged imbalance can lead to disease. Glutamatergic/ GABAergic imbalance can be found in autism spectrum disorders and anxiety disorders with elevated glutamatergic neurotransmission, while high levels of GABA produce more relaxation and even sedation. Neurotransmitter levels can be affected by external factors, for example, alcohol. Alcohol potentiates the sedentary effects of GABA, while inhibiting the excitatory aspects of glutamate, resulting in an overall increase in GABA/glutamate ratio. This leads to

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2018 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.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

sensations of relaxation and in later stages to loss of control with slurred speech, unsteady gait and loss of social anxiety. The GABA/glutamate balance can also be affected by autoimmunity and genetic disorders. The contributions of GABA and glutamate in extra-neuronal signaling are by far less recognized. We will discuss extra-neuronal GABAergic and glutamatergic signaling and its relevance in insulin secretion from the pancreatic islets of Langerhans.

The electrical activity of neurons is a guide for the genesis of neuronal connections. Indeed, neuronal activity exerts a key role in the development of inhibitory GABAergic synapses, interacting closely with genetic programs. Blocking neuronal activity in developing neurons decreases the density of inhibitory synapses, confirming an activity-dependent development [10, 11]. The expression of GABAergic plasticity is related to modifications in the quantity of neurotransmitter in individual vesicles. Migrating neurons express, already at an early stage, both GABA and glutamate receptors [1], but GABA receptors are likely to be established first [12]. Interestingly, tangentially migrating interneurons express AMPA but not GABA or NMDA receptors. Therefore, a modulation/targeting of neurons via selective activation of receptors can be achieved [13]. This has implications for understanding and treatment of migrating disorders

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

67

The building of brain networks is a highly complex task which requires organized sequential events, both spatially and from a timing standpoint. While an overdrive of GABAergic signaling slows the development, the overactivity of glutamatergic signaling causes excitotoxicity [1]. GABA receptors are the first to be active, even when synapses are still non-operant. This creates a shunting effect preventing excitotoxicity, since the Na<sup>+</sup> and Ca2+ spikes triggered by GABA require only a 30–40 mV driving force. Such a shunt is part of the synergistic interactions between GABA and glutamate. Thanks to these interactions, the neuronal networks in development generate primitive patterns of discharges, observed in vivo and in cultured networks, such as the giant depolarizing potentials (GDPs) which allow the building of functional units [1]. GDPs resemble interictal-like epileptiform discharges and provide synchronous Ca2+ oscillations also contributing to the development of networks. GDPs rely on the release of GABA, glutamate, and glycine at the onset of synaptogenesis [14]. The synchronized activity is one of the factors controlling the phenomenon of maturation [11]. Synchronization is also achieved, thanks to gap junctions, intrinsic voltage-dependent conductance [15], and non-

The capacity of developing nervous system to generate spontaneous activity in absence of external stimulation is a remarkable feature that has been observed in particular in the retina, the cerebral cortex, the hippocampus, the cerebellum, the hindbrain, and the spinal cord [14]. Recent works highlight that network bursts are driven by AMPA pathways in terms of coordination, whereas the shaping of the dynamics of spiking activities is regulated by

affecting the nervous system.

vesicular paracrine release of neurotransmitters [16].

NMDA- and GABA-associated currents [17].

3. Glutamate and GABA in the mature mammalian brain

3.1. Biosynthesis of glutamate and GABA: the glutamate/GABA-glutamine cycle

Glutamate and GABA do not cross the blood-brain barrier and must therefore be synthesized within the CNS. As neurons lack the enzyme pyruvate carboxylase and therefore cannot synthesize glutamate through the TCA cycle [18], they rely on astrocytes for the generation of glutamate. Astrocytes generate glutamate via de novo synthesis or by "recycling" glutamine from GABA and glutamate after reuptake. However, de novo synthesis makes up only ~15% of

#### 2. Glutamate and GABA during development

Both glutamatergic and GABAergic neurons are highly diversified in the central nervous system (CNS). More than half a century after the discovery of the effects of GABA, it is now established that in mature neurons, neuronal excitability is characterized by a balance between glutamatergic excitatory input and GABAergic inhibitory transmission. This balance is reached during development. However, the functions of GABAergic signaling are not restricted to a pure inhibitory mechanism at the synaptic level. This is a too simplistic view. For instance, GABA influences patterns and oscillations that are very relevant from the behavioral point of view [1]. GABA and glutamate expression are already widespread in the embryonic stage, and glutamate receptors are expressed in neurons even before glutamatergic synaptogenesis [1]. While glutamate receptor activities tune the developing GABAergic synapse [2], GABA is now considered the main excitatory transmitter during early development, acting not only at a synaptic and network level, but also on cell cycle and migration [1]. This excitatory function of GABA is caused by elevated neuronal intracellular chloride concentration at the early stages of development. The efflux of chloride mediated by GABA in immature neurons is excitatory, triggering sodium spikes and activating voltage-gated Ca2+ channels [1]. With time, a progressive reduction of chloride efflux occurs. This explains the shift from a depolarizing to a hyperpolarizing effect. Obata and colleagues were the first to suggest this developmentally regulated shift at the level of the spinal cord [3]. Neuronal chloride homeostasis is regulated by channels, exchangers and co-transporters. The developmental changes of sodium-potassiumchloride cotransporter 1 (NKCC1) (ensuring chloride uptake; higher expression in immature neurons) and potassium-chloride transporter 2 (KCC2) (principal chloride extruder; higher expression in mature neurons) are the masterpieces for the changes in chloride efflux associated with maturation. The developmental shift from local to large-scale network activity occurs in parallel with a gradual shift from electrical to chemical synaptic transmission [4].

It is noteworthy that in immature neurons activation of GABAA receptors leads to an increase in the intra-cellular concentration of Ca2+, as a consequence of the stimulation of voltage-gated Ca2+ channels, which exerts trophic effects on neuritic growth, migration and synaptogenesis. Blocking the GABAA receptor reduces the cytoplasmic concentrations of Ca2+ [5]. In addition, the activation of GABAB receptors depresses the GABAA receptor-mediated Ca2+ increase and therefore the GABAB pathway is likely supervising the entry of Ca2+ [6]. In granule cells of the cerebellum, the changes in the concentrations of Ca2+ outlast the exposure to GABA by several minutes [7]. The GABAA-activated Ca2+ influx regulates the expression of the chloride extruder KCC2 [8]. One example of the relevance of this physiological shift in the chloride gradient occurs during delivery when the maternal hormone oxytocin triggers labor [9].

The electrical activity of neurons is a guide for the genesis of neuronal connections. Indeed, neuronal activity exerts a key role in the development of inhibitory GABAergic synapses, interacting closely with genetic programs. Blocking neuronal activity in developing neurons decreases the density of inhibitory synapses, confirming an activity-dependent development [10, 11]. The expression of GABAergic plasticity is related to modifications in the quantity of neurotransmitter in individual vesicles. Migrating neurons express, already at an early stage, both GABA and glutamate receptors [1], but GABA receptors are likely to be established first [12]. Interestingly, tangentially migrating interneurons express AMPA but not GABA or NMDA receptors. Therefore, a modulation/targeting of neurons via selective activation of receptors can be achieved [13]. This has implications for understanding and treatment of migrating disorders affecting the nervous system.

sensations of relaxation and in later stages to loss of control with slurred speech, unsteady gait and loss of social anxiety. The GABA/glutamate balance can also be affected by autoimmunity and genetic disorders. The contributions of GABA and glutamate in extra-neuronal signaling are by far less recognized. We will discuss extra-neuronal GABAergic and glutamatergic signaling and its relevance in insulin secretion from the pancreatic islets of Langerhans.

Both glutamatergic and GABAergic neurons are highly diversified in the central nervous system (CNS). More than half a century after the discovery of the effects of GABA, it is now established that in mature neurons, neuronal excitability is characterized by a balance between glutamatergic excitatory input and GABAergic inhibitory transmission. This balance is reached during development. However, the functions of GABAergic signaling are not restricted to a pure inhibitory mechanism at the synaptic level. This is a too simplistic view. For instance, GABA influences patterns and oscillations that are very relevant from the behavioral point of view [1]. GABA and glutamate expression are already widespread in the embryonic stage, and glutamate receptors are expressed in neurons even before glutamatergic synaptogenesis [1]. While glutamate receptor activities tune the developing GABAergic synapse [2], GABA is now considered the main excitatory transmitter during early development, acting not only at a synaptic and network level, but also on cell cycle and migration [1]. This excitatory function of GABA is caused by elevated neuronal intracellular chloride concentration at the early stages of development. The efflux of chloride mediated by GABA in immature neurons is excitatory, triggering sodium spikes and activating voltage-gated Ca2+ channels [1]. With time, a progressive reduction of chloride efflux occurs. This explains the shift from a depolarizing to a hyperpolarizing effect. Obata and colleagues were the first to suggest this developmentally regulated shift at the level of the spinal cord [3]. Neuronal chloride homeostasis is regulated by channels, exchangers and co-transporters. The developmental changes of sodium-potassiumchloride cotransporter 1 (NKCC1) (ensuring chloride uptake; higher expression in immature neurons) and potassium-chloride transporter 2 (KCC2) (principal chloride extruder; higher expression in mature neurons) are the masterpieces for the changes in chloride efflux associated with maturation. The developmental shift from local to large-scale network activity occurs in

parallel with a gradual shift from electrical to chemical synaptic transmission [4].

occurs during delivery when the maternal hormone oxytocin triggers labor [9].

It is noteworthy that in immature neurons activation of GABAA receptors leads to an increase in the intra-cellular concentration of Ca2+, as a consequence of the stimulation of voltage-gated Ca2+ channels, which exerts trophic effects on neuritic growth, migration and synaptogenesis. Blocking the GABAA receptor reduces the cytoplasmic concentrations of Ca2+ [5]. In addition, the activation of GABAB receptors depresses the GABAA receptor-mediated Ca2+ increase and therefore the GABAB pathway is likely supervising the entry of Ca2+ [6]. In granule cells of the cerebellum, the changes in the concentrations of Ca2+ outlast the exposure to GABA by several minutes [7]. The GABAA-activated Ca2+ influx regulates the expression of the chloride extruder KCC2 [8]. One example of the relevance of this physiological shift in the chloride gradient

2. Glutamate and GABA during development

66 GABA And Glutamate - New Developments In Neurotransmission Research

The building of brain networks is a highly complex task which requires organized sequential events, both spatially and from a timing standpoint. While an overdrive of GABAergic signaling slows the development, the overactivity of glutamatergic signaling causes excitotoxicity [1]. GABA receptors are the first to be active, even when synapses are still non-operant. This creates a shunting effect preventing excitotoxicity, since the Na<sup>+</sup> and Ca2+ spikes triggered by GABA require only a 30–40 mV driving force. Such a shunt is part of the synergistic interactions between GABA and glutamate. Thanks to these interactions, the neuronal networks in development generate primitive patterns of discharges, observed in vivo and in cultured networks, such as the giant depolarizing potentials (GDPs) which allow the building of functional units [1]. GDPs resemble interictal-like epileptiform discharges and provide synchronous Ca2+ oscillations also contributing to the development of networks. GDPs rely on the release of GABA, glutamate, and glycine at the onset of synaptogenesis [14]. The synchronized activity is one of the factors controlling the phenomenon of maturation [11]. Synchronization is also achieved, thanks to gap junctions, intrinsic voltage-dependent conductance [15], and nonvesicular paracrine release of neurotransmitters [16].

The capacity of developing nervous system to generate spontaneous activity in absence of external stimulation is a remarkable feature that has been observed in particular in the retina, the cerebral cortex, the hippocampus, the cerebellum, the hindbrain, and the spinal cord [14]. Recent works highlight that network bursts are driven by AMPA pathways in terms of coordination, whereas the shaping of the dynamics of spiking activities is regulated by NMDA- and GABA-associated currents [17].

#### 3. Glutamate and GABA in the mature mammalian brain

#### 3.1. Biosynthesis of glutamate and GABA: the glutamate/GABA-glutamine cycle

Glutamate and GABA do not cross the blood-brain barrier and must therefore be synthesized within the CNS. As neurons lack the enzyme pyruvate carboxylase and therefore cannot synthesize glutamate through the TCA cycle [18], they rely on astrocytes for the generation of glutamate. Astrocytes generate glutamate via de novo synthesis or by "recycling" glutamine from GABA and glutamate after reuptake. However, de novo synthesis makes up only ~15% of astrocytic glutamate [19]. In this reaction, pyruvate is generated from glucose during glycolysis and enters the TCA after conversion to Acetyl CoA. The TCA product α-ketoglutarate can be converted to glutamate, which is converted to glutamine by glutamine synthetase, an enzyme that is predominately, if not exclusively, located in astrocytes [20]. Glutamine exits astrocytes via the bidirectional N system transporters, SNAT3 and SNAT5 [21], and enter neurons via the unidirectional system A transporters, SNAT1, SNAT2 [21], and SNAT7 [22]. There glutamine is converted back to glutamate by phosphate-activated glutaminase, an enzyme which is expressed preferentially in neurons [23]. GABAergic neurons require an additional step to convert glutamate to GABA through decarboxylation. After release from the neurons, GABA and glutamate reenter the astrocytes to be "recycled" to glutamine. A small portion of glutamate is oxidatively metabolized, thus making de novo synthesis of glutamate necessary to maintain adequate glutamate levels [24]. The continuous recycling of glutamate, GABA and glutamine between neurons and astrocytes is known as the glutamate/GABAglutamine cycle [25] (Figure 1).

after ligands that selectively bind to one receptor only: N-methyl-D-aspartate (NMDA), alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and Kainate, however all three are bound by glutamate [28]. NMDA receptors (NMDA-Rs) activate slower than AMPA and Kainate receptors. This delayed reaction is caused by the blockage of the cation pore of NMDA-Rs by external Mg2+ at resting membrane potential, which is removed upon depolarization of the neurons [29, 30]. Thus, non-NMDA-Rs activation is necessary to depolarize the neurons allowing NMDA-Rs activation. NMDA-Rs also close slower than non-NMDA-Rs, and

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

69

NMDA-Rs also allow influx of Ca2+ and have thereby a regulatory role in synaptic plasticity by connecting synaptic activity with Ca2+-mediated biochemical signaling [31]. Depending on the nature of the neuron's depolarization, NMDA-Rs can both strengthen synapses, through longterm potentiation (LTP) [32, 33], and weaken synapses, through long-term depression (LTD) [34]. For LTP, repetitive and strong depolarization of the neurons allows significant influx of Ca2+ into the cytoplasm and activation of protein kinases, including the calcium/calmodulindependent protein kinase II (CaMKII), which (a) phosphorylate and activate AMPA-Rs and (b) trigger the insertion of additional AMPA-Rs into the postsynaptic membrane [35]. This increases the postsynaptic neuron's responsiveness to glutamate leading to LTP. By strengthening the neighboring connections of similar activity patterns, the NMDA-R enforces the Hebbian postulate that 'cells that fire together, wire together' [36]. For LTD, weak depolarization by low frequency stimulation still activates NMDA-Rs, but promotes only a modest prolonged increase in Ca2+ levels. Protein phosphatases (protein phosphatase 1 and protein phosphatase calcineurin) have a much higher affinity for calcium/calmodulin compared to CaMKII and are activated at lower Ca2+ levels. Thus, under the above conditions protein phosphatases are activated [37], dephosphorylate AMPA-Rs, and induce the removal of AMPA-Rs from the postsynaptic membrane [38], thereby reducing the postsynaptic respon-

Metabotropic glutamate receptors (mGluRs) [39] are overall slower acting than iGluRs. In difference to iGluRs, mGluRs are not ion channels, but belong to a group of G-protein-coupled receptors. The associated G-protein consists of three subunits (α, β, and γ), of which the αsubunit is associated with GDP. Glutamate binding to the receptor induces a conformational change that allows the replacement of GDP with GTP and consequent dissociation of the three G-protein subunits. While the Gβ/γ-complex activates K<sup>+</sup> and Na+ channels, the α-subunit interacts with different enzymes [40]. Excitatory Gα-subunits (Gα<sup>q</sup> or Gα11) bind and activate phospholipase C-β (PLC-β), initiating a signaling cascade leading to the activation of protein kinase C (PKC) and the release of Ca2+ from the ER. Another excitatory Gα-subunit (Gαs) activates the membrane-associated adenylyl cyclase, which catalyzes the conversion of ATP to cAMP, leading to activation of protein kinase A (PKA). Inhibiting Gα-subunits (Gα<sup>i</sup> or Gαo) prevent activation of adenylyl cyclase and the activation of PKA [41]. The three mGluRs groups (I–III) differ in their alpha subunits. Generally, Group I mGluRs carry activating Gα<sup>s</sup> or Gα<sup>11</sup> subunits, are localized postsynaptically, and lead to cell depolarization and increased neuronal excitability. Groups II and III mGluRs carry inhibitory Gα<sup>i</sup> or Gα<sup>o</sup> subunits and are often localized on presynaptic terminals or preterminal axons, where they inhibit neurotrans-

therefore determine the duration of the synaptic current.

siveness to glutamate and leading to LTD.

mitter release [42].

#### 3.2. Glutamatergic neurotransmission

In glutamatergic neurons, glutamate is packaged into synaptic vesicles (SVs) by vesicular glutamate transporters (VGLUT1–3) [26]. The loaded SVs then dock near the release site, where they are primed into a state of competence for Ca2+-triggered fusion-pore opening. Once glutamate has been released, SVs can either fully collapse into the synaptic membrane, or close rapidly and undock ("kiss-and-run") [27].

Released glutamate is recognized by glutamate receptors (GluRs). Binding of glutamate changes the receptor's conformation and allows influx of extracellular Na+ and other cations, and an efflux of intracellular K<sup>+</sup> ions. GluRs fall into two major categories: ionotropic and metabotropic [28]. Ionotropic GluRs are tetrameric ligand-gated cation channels that induce depolarization of the postsynaptic membrane. The three types of ionotropic GluRs are named

Figure 1. Overview of the glutamate/GABA-glutamine cycle. For details see text.

after ligands that selectively bind to one receptor only: N-methyl-D-aspartate (NMDA), alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and Kainate, however all three are bound by glutamate [28]. NMDA receptors (NMDA-Rs) activate slower than AMPA and Kainate receptors. This delayed reaction is caused by the blockage of the cation pore of NMDA-Rs by external Mg2+ at resting membrane potential, which is removed upon depolarization of the neurons [29, 30]. Thus, non-NMDA-Rs activation is necessary to depolarize the neurons allowing NMDA-Rs activation. NMDA-Rs also close slower than non-NMDA-Rs, and therefore determine the duration of the synaptic current.

astrocytic glutamate [19]. In this reaction, pyruvate is generated from glucose during glycolysis and enters the TCA after conversion to Acetyl CoA. The TCA product α-ketoglutarate can be converted to glutamate, which is converted to glutamine by glutamine synthetase, an enzyme that is predominately, if not exclusively, located in astrocytes [20]. Glutamine exits astrocytes via the bidirectional N system transporters, SNAT3 and SNAT5 [21], and enter neurons via the unidirectional system A transporters, SNAT1, SNAT2 [21], and SNAT7 [22]. There glutamine is converted back to glutamate by phosphate-activated glutaminase, an enzyme which is expressed preferentially in neurons [23]. GABAergic neurons require an additional step to convert glutamate to GABA through decarboxylation. After release from the neurons, GABA and glutamate reenter the astrocytes to be "recycled" to glutamine. A small portion of glutamate is oxidatively metabolized, thus making de novo synthesis of glutamate necessary to maintain adequate glutamate levels [24]. The continuous recycling of glutamate, GABA and glutamine between neurons and astrocytes is known as the glutamate/GABA-

In glutamatergic neurons, glutamate is packaged into synaptic vesicles (SVs) by vesicular glutamate transporters (VGLUT1–3) [26]. The loaded SVs then dock near the release site, where they are primed into a state of competence for Ca2+-triggered fusion-pore opening. Once glutamate has been released, SVs can either fully collapse into the synaptic membrane, or close

Released glutamate is recognized by glutamate receptors (GluRs). Binding of glutamate changes the receptor's conformation and allows influx of extracellular Na+ and other cations, and an efflux of intracellular K<sup>+</sup> ions. GluRs fall into two major categories: ionotropic and metabotropic [28]. Ionotropic GluRs are tetrameric ligand-gated cation channels that induce depolarization of the postsynaptic membrane. The three types of ionotropic GluRs are named

glutamine cycle [25] (Figure 1).

3.2. Glutamatergic neurotransmission

68 GABA And Glutamate - New Developments In Neurotransmission Research

rapidly and undock ("kiss-and-run") [27].

Figure 1. Overview of the glutamate/GABA-glutamine cycle. For details see text.

NMDA-Rs also allow influx of Ca2+ and have thereby a regulatory role in synaptic plasticity by connecting synaptic activity with Ca2+-mediated biochemical signaling [31]. Depending on the nature of the neuron's depolarization, NMDA-Rs can both strengthen synapses, through longterm potentiation (LTP) [32, 33], and weaken synapses, through long-term depression (LTD) [34]. For LTP, repetitive and strong depolarization of the neurons allows significant influx of Ca2+ into the cytoplasm and activation of protein kinases, including the calcium/calmodulindependent protein kinase II (CaMKII), which (a) phosphorylate and activate AMPA-Rs and (b) trigger the insertion of additional AMPA-Rs into the postsynaptic membrane [35]. This increases the postsynaptic neuron's responsiveness to glutamate leading to LTP. By strengthening the neighboring connections of similar activity patterns, the NMDA-R enforces the Hebbian postulate that 'cells that fire together, wire together' [36]. For LTD, weak depolarization by low frequency stimulation still activates NMDA-Rs, but promotes only a modest prolonged increase in Ca2+ levels. Protein phosphatases (protein phosphatase 1 and protein phosphatase calcineurin) have a much higher affinity for calcium/calmodulin compared to CaMKII and are activated at lower Ca2+ levels. Thus, under the above conditions protein phosphatases are activated [37], dephosphorylate AMPA-Rs, and induce the removal of AMPA-Rs from the postsynaptic membrane [38], thereby reducing the postsynaptic responsiveness to glutamate and leading to LTD.

Metabotropic glutamate receptors (mGluRs) [39] are overall slower acting than iGluRs. In difference to iGluRs, mGluRs are not ion channels, but belong to a group of G-protein-coupled receptors. The associated G-protein consists of three subunits (α, β, and γ), of which the αsubunit is associated with GDP. Glutamate binding to the receptor induces a conformational change that allows the replacement of GDP with GTP and consequent dissociation of the three G-protein subunits. While the Gβ/γ-complex activates K<sup>+</sup> and Na+ channels, the α-subunit interacts with different enzymes [40]. Excitatory Gα-subunits (Gα<sup>q</sup> or Gα11) bind and activate phospholipase C-β (PLC-β), initiating a signaling cascade leading to the activation of protein kinase C (PKC) and the release of Ca2+ from the ER. Another excitatory Gα-subunit (Gαs) activates the membrane-associated adenylyl cyclase, which catalyzes the conversion of ATP to cAMP, leading to activation of protein kinase A (PKA). Inhibiting Gα-subunits (Gα<sup>i</sup> or Gαo) prevent activation of adenylyl cyclase and the activation of PKA [41]. The three mGluRs groups (I–III) differ in their alpha subunits. Generally, Group I mGluRs carry activating Gα<sup>s</sup> or Gα<sup>11</sup> subunits, are localized postsynaptically, and lead to cell depolarization and increased neuronal excitability. Groups II and III mGluRs carry inhibitory Gα<sup>i</sup> or Gα<sup>o</sup> subunits and are often localized on presynaptic terminals or preterminal axons, where they inhibit neurotransmitter release [42].

Released glutamate must be rapidly removed to avoid continuous stimulation and excitotoxicity [43, 44] (see also glutamate-induced excitotoxicity). Glutamate uptake is mediated via brain excitatory amino acid transporters (EAATs) on both pre- and postsynaptic neurons and on surrounding astrocytes [45]. Five subtypes of EAATs (EAAT1–5) have been cloned so far. A smaller portion of extracellular glutamate is reabsorbed by presynaptic or postsynaptic neurons.

Metabotropic G-protein coupled GABAB receptors are mostly located extrasynaptically and can be found both pre- and the postsynaptical. These heterodimers consist of GABAB1 and GABAB2 subunits. The GABAB1 subunit can be bound by GABA, while GABAB2 is coupled to the G-protein. Activation of GABAB receptors induces the dissociation of the subunits of the coupled G-protein. The Gβ/γ-subunit complex activates inwardly rectifying K<sup>+</sup> channels [57] and inhibits voltage-activated Ca2+ channels [58], resulting in hyperpolarization of the neuron and inhibition of neurotransmission [59]. The Gαi-subunit inhibits activation of adenylyl cyclase as described above for mGluR. Activation of presynaptical GABAB receptors impedes opening of voltage-activated Ca2+ channels and thereby reduces neurotransmitter release. Thus GABA can inhibit its own release through a negative feedback loop via GABAB receptors present on GABAergic axons [60]. Activation of postsynaptical GABAB receptors reduces

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

71

depolarization of the plasma membrane and thereby modulates excitatory signals.

terminals (GAT-1) and surrounding glial cells (GAT-3) [61].

release mechanisms

excitability [66].

3.4.2. Immune-mediated cerebellar ataxias

3.4.1. Limbic encephalitis

The reuptake of GABA is mediated by GABA transporter protein present in presynaptic nerve

The relation between anti-GluR antibodies (Abs) with limbic encephalitis has been investigated during the last two decades [62, 63]. Several autoantibodies against extracellular epitopes of GluR involved in synaptic transmission and plasticity, such as AMPA-Rs [64] and NMDA-Rs [65] are described. The affected patients develop complex neuropsychiatric symptoms, such as memory deficits, cognition impairment, psychosis, seizures, abnormal movements, or coma. These disorders affect mainly young women, though cases of men and children have been reported [62]. Some of these patients present with malignant tumors and the syndrome can be qualified as paraneoplastic [62], characterized by association of anti-NMDA-RAb in ovarian teratoma and anti-AMPA-RAb in lung small cell carcinoma. Paraneoplastic limbic encephalitis can be fatal, but is curable if treated at an early stage by surgical removal of the tumor and a combination of immunotherapeutic agents [62]. Mechanistically, autoantibodies directed against AMPA-Rs and NMDA-Rs decrease the numbers of the cell-surface receptors [63] and anti-AMPA-RAb may act as agonists and increase cell

Compared with autoimmune encephalitis affecting the limbic system, autoantibodies to GluRs, such as anti-mGluR1 Ab and anti-glutamate receptor delta2 (GluRδ2) Ab, are less frequently associated with immune-mediated cerebellar ataxias (IMCAs). Anti-mGluR1Ab was reported in two patients with malignant lymphoma and one patient with prostate adenocarcinoma [67, 68]. Anti-mGluR1Ab impairs the induction of LTD, which causes ataxia in mice [67]. Interestingly, IMCAs associated with anti-GluRδ2Ab are always preceded or accompanied by either infection or vaccination [58]. Polyclonal Abs toward the putative ligand-binding

3.4. Neurological diseases associated with glutamate and GABA receptors and GABA

Another mechanism involved in the extracellular glutamate regulation is the cystine/glutamate exchange transporter xc(). In contrast to the EAATs, xc() is involved in elevating extracellular glutamate concentrations. Here intracellular glutamate is exchanged for extracellular cystine. The stimulation of xc() modulates glutamate release from the presynaptic neurons [46]. xc() regulates glutamate homeostasis through the involvement of the presynaptic mGluR2/3. Moreover, a decrease of xc() expression can lead to a reduction in extrasynaptic glutamate level. This effect may cause a loss of glutamatergic tone on presynaptic mGluR2/3, which can lead to a marked increase in glutamate release from presynaptic glutamatergic neurons [47].

#### 3.3. GABAergic neurotransmission

For the synthesis of GABA, glutamate is decarboxylated to GABA by glutamate decarboxylase (GAD). GAD is expressed as two isoforms (GAD67 and GAD65) and can be found only in GABAergic neurons and in certain peripheral tissues, most prominently in the pancreatic islets [48]. GABA can also be taken up by presynaptic neurons after its release into the synapsis. However, this recycled GABA is mainly metabolized to generate ATP through the GABA shunt pathway [49], while newly synthesized GABA is preferentially taken up into SVs [49]. Transport of newly synthesized GABA into SVs is tightly linked to its synthesis, as GAD65 and the GABA transporter VGAT form a protein complex with chaperone protein HSC70, the vesicular cysteine string protein (CSP), and CaMKII [49]. In the absence of GAD65 from this complex, the active site of VGAT may be available to cytosolic GABA, and vesicular transport of GABA can be restored to a certain extent [49]. GAD65 has also a crucial role in the trafficking of GABAergic vesicles to presynaptic clusters [50]. Palmitoylation of cysteine residues located at the N-terminus of GAD65 is required for the transport of GAD65 to synaptic terminals [51], and decreased palmitoylation impairs GABAergic neurotransmission, as observed in Huntington's disease [52]. Similar to glutamate, GABA is released in a Ca2+ dependent manner upon depolarization of the presynaptic membrane.

GABA receptors present as ionotropic GABAA receptors that cause rapid inhibitory postsynaptic potentials, and metabotropic GABAB receptors that cause slow inhibitory postsynaptic potentials [53]. GABAA receptors are multi-subunit proteins [54] and consist of three major subunits (α, β, and γ). A total of five subunits are arranged around the central ion pore. The major receptor isoform consists of two α1, two β2, and one γ<sup>2</sup> subunits. GABA binding to GABAA receptors on the postsynaptic neuron induces rapid and transient opening of GABAA receptor Cl channels [55]. The subsequent influx of anions hyperpolarizes the membrane (phasic inhibition). GABA overspill can activate extrasynaptical GABAA receptors, inducing a more prolonged opening of the ion channel (tonic inhibition) [56].

Metabotropic G-protein coupled GABAB receptors are mostly located extrasynaptically and can be found both pre- and the postsynaptical. These heterodimers consist of GABAB1 and GABAB2 subunits. The GABAB1 subunit can be bound by GABA, while GABAB2 is coupled to the G-protein. Activation of GABAB receptors induces the dissociation of the subunits of the coupled G-protein. The Gβ/γ-subunit complex activates inwardly rectifying K<sup>+</sup> channels [57] and inhibits voltage-activated Ca2+ channels [58], resulting in hyperpolarization of the neuron and inhibition of neurotransmission [59]. The Gαi-subunit inhibits activation of adenylyl cyclase as described above for mGluR. Activation of presynaptical GABAB receptors impedes opening of voltage-activated Ca2+ channels and thereby reduces neurotransmitter release. Thus GABA can inhibit its own release through a negative feedback loop via GABAB receptors present on GABAergic axons [60]. Activation of postsynaptical GABAB receptors reduces depolarization of the plasma membrane and thereby modulates excitatory signals.

The reuptake of GABA is mediated by GABA transporter protein present in presynaptic nerve terminals (GAT-1) and surrounding glial cells (GAT-3) [61].

#### 3.4. Neurological diseases associated with glutamate and GABA receptors and GABA release mechanisms

#### 3.4.1. Limbic encephalitis

Released glutamate must be rapidly removed to avoid continuous stimulation and excitotoxicity [43, 44] (see also glutamate-induced excitotoxicity). Glutamate uptake is mediated via brain excitatory amino acid transporters (EAATs) on both pre- and postsynaptic neurons and on surrounding astrocytes [45]. Five subtypes of EAATs (EAAT1–5) have been cloned so far. A smaller portion of extracellular glutamate is reabsorbed by presynaptic or postsynaptic

70 GABA And Glutamate - New Developments In Neurotransmission Research

Another mechanism involved in the extracellular glutamate regulation is the cystine/glutamate exchange transporter xc(). In contrast to the EAATs, xc() is involved in elevating extracellular glutamate concentrations. Here intracellular glutamate is exchanged for extracellular cystine. The stimulation of xc() modulates glutamate release from the presynaptic neurons [46]. xc() regulates glutamate homeostasis through the involvement of the presynaptic mGluR2/3. Moreover, a decrease of xc() expression can lead to a reduction in extrasynaptic glutamate level. This effect may cause a loss of glutamatergic tone on presynaptic mGluR2/3, which can lead to a marked increase in glutamate release from presynaptic

For the synthesis of GABA, glutamate is decarboxylated to GABA by glutamate decarboxylase (GAD). GAD is expressed as two isoforms (GAD67 and GAD65) and can be found only in GABAergic neurons and in certain peripheral tissues, most prominently in the pancreatic islets [48]. GABA can also be taken up by presynaptic neurons after its release into the synapsis. However, this recycled GABA is mainly metabolized to generate ATP through the GABA shunt pathway [49], while newly synthesized GABA is preferentially taken up into SVs [49]. Transport of newly synthesized GABA into SVs is tightly linked to its synthesis, as GAD65 and the GABA transporter VGAT form a protein complex with chaperone protein HSC70, the vesicular cysteine string protein (CSP), and CaMKII [49]. In the absence of GAD65 from this complex, the active site of VGAT may be available to cytosolic GABA, and vesicular transport of GABA can be restored to a certain extent [49]. GAD65 has also a crucial role in the trafficking of GABAergic vesicles to presynaptic clusters [50]. Palmitoylation of cysteine residues located at the N-terminus of GAD65 is required for the transport of GAD65 to synaptic terminals [51], and decreased palmitoylation impairs GABAergic neurotransmission, as observed in Huntington's disease [52]. Similar to glutamate, GABA is released in a Ca2+-

GABA receptors present as ionotropic GABAA receptors that cause rapid inhibitory postsynaptic potentials, and metabotropic GABAB receptors that cause slow inhibitory postsynaptic potentials [53]. GABAA receptors are multi-subunit proteins [54] and consist of three major subunits (α, β, and γ). A total of five subunits are arranged around the central ion pore. The major receptor isoform consists of two α1, two β2, and one γ<sup>2</sup> subunits. GABA binding to GABAA receptors on the postsynaptic neuron induces rapid and transient opening of GABAA receptor Cl channels [55]. The subsequent influx of anions hyperpolarizes the membrane (phasic inhibition). GABA overspill can activate extrasynaptical GABAA receptors, inducing a

dependent manner upon depolarization of the presynaptic membrane.

more prolonged opening of the ion channel (tonic inhibition) [56].

neurons.

glutamatergic neurons [47].

3.3. GABAergic neurotransmission

The relation between anti-GluR antibodies (Abs) with limbic encephalitis has been investigated during the last two decades [62, 63]. Several autoantibodies against extracellular epitopes of GluR involved in synaptic transmission and plasticity, such as AMPA-Rs [64] and NMDA-Rs [65] are described. The affected patients develop complex neuropsychiatric symptoms, such as memory deficits, cognition impairment, psychosis, seizures, abnormal movements, or coma. These disorders affect mainly young women, though cases of men and children have been reported [62]. Some of these patients present with malignant tumors and the syndrome can be qualified as paraneoplastic [62], characterized by association of anti-NMDA-RAb in ovarian teratoma and anti-AMPA-RAb in lung small cell carcinoma. Paraneoplastic limbic encephalitis can be fatal, but is curable if treated at an early stage by surgical removal of the tumor and a combination of immunotherapeutic agents [62]. Mechanistically, autoantibodies directed against AMPA-Rs and NMDA-Rs decrease the numbers of the cell-surface receptors [63] and anti-AMPA-RAb may act as agonists and increase cell excitability [66].

#### 3.4.2. Immune-mediated cerebellar ataxias

Compared with autoimmune encephalitis affecting the limbic system, autoantibodies to GluRs, such as anti-mGluR1 Ab and anti-glutamate receptor delta2 (GluRδ2) Ab, are less frequently associated with immune-mediated cerebellar ataxias (IMCAs). Anti-mGluR1Ab was reported in two patients with malignant lymphoma and one patient with prostate adenocarcinoma [67, 68]. Anti-mGluR1Ab impairs the induction of LTD, which causes ataxia in mice [67]. Interestingly, IMCAs associated with anti-GluRδ2Ab are always preceded or accompanied by either infection or vaccination [58]. Polyclonal Abs toward the putative ligand-binding site of GluRδ2 are known to cause AMPA-R endocytosis and attenuate their synaptic transmission, resulting in the development of ataxia in mice [69].

CAs affect mostly women in their 50–60s and exhibit subacute or chronic CAs, which are sometimes associated with SPS or epilepsy [76]. Furthermore, the majority of patients also suffer

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

73

Based on the intracellular location of GAD65 (on the cytoplasmic side of SVs), the pathogenic role of anti-GAD65Ab in CA and SPS has been questioned. However, recent studies have shed new light on this issue. First, the pathogenic actions of anti-GAD65Ab have been clarified both in in vitro and in vivo preparations [77–82], for example, the Cerebrospinal fluid (CSF) of patients with SPS inhibited GABA synthesis [83]. Furthermore, SPS-like symptoms were reproduced in experimental rats and mice injected intrathecally or intraventricularly [84] or intracerebellarly [80] with IgGs obtained from the CSF of CA and SPS patients. Specifically, IgGs obtained from the CSF of patients with anti-GAD65Ab-associated CA depressed GABA release in cerebellar brain slices [77, 78, 82, 85], and their intracerebellar injection interfered with cerebellar control of the motor cortex and resulted in ataxic gait in rats [79]. These actions were reproduced by human anti-GAD65 monoclonal Ab b78, which binds to an epitope

Second, studies in animals have shown internalization of antibodies by cerebellar neurons [81, 86], demonstrating that anti-GAD65Ab can access their intracellular target. Together, these results indicate the possibility that anti-GAD65Ab may damage sufficient numbers of

The pathological effects of anti-GAD65Ab depend on epitope specificity. Specifically, the pathologic effects of anti-GAD65Abs on GABA synthesis and release were specific to anti-GAD65Ab b78—representing SPS- and CA-associated anti-GAD65Ab—and could not be reproduced by monoclonal anti-GAD65Ab b96.11, representing an epitope specificity associated with type 1 diabetes mellitus [80–82]. This epitope dependence might explain the differences in neurological phenotypes. Administration of CSF obtained from patients with SPS or CAs reproduced the clinical features of the corresponding disease in mice [80]. Thus anti-GAD65Ab in SPS that inhibit GABA synthesis, would attenuate inhibitory Purkinje cellmediated depression of the spino-cerebellar loop, resulting in increased muscle tone, whereas anti-GAD65Ab in CAs depress GABA release, resulting in disruption of Purkinje cell-mediated modulation of the cerebro-cerebellar loop to elicit disorganized movements [87]. An alternative explanation of the phenotypic differences is the involvement of other autoimmune-

In anti-GAD65Ab-associated CAs, anti-GAD65Ab impairs the association of GAD65 with the cytosolic side of SVs resulting in a decrease in vesicular GABA contents with low release probability [82]. Under normal conditions, the released GABA spills over to the neighboring excitatory synaptic terminals and inhibits presynaptic glutamate release through GABA receptors. However, this GABA-induced inhibition of glutamate release is disturbed in patients with CAs [77]. Taken together, anti-GAD65Ab elicit marked imbalance in neurotransmitters; including a decrease in GABA and an increase in glutamate. The imbalance between GABA and glutamate is accelerated following the involvement of microglia and astrocytes [87]. Microglia activated by excessive glutamate levels can secrete various cytokines, which facilitate glutamate release presumably through xc() on microglia, and suppress the uptake of glutamate

GABAergic neurons to result in the appearance of frank neurological symptoms [87].

similar to that recognized in SPS patients positive for anti-GAD65Ab [80–82].

from type 1 diabetes mellitus (T1D) [76].

mediated mechanisms.

#### 3.4.3. Autosomal recessive cerebellar degeneration

Autosomal recessive CAs (ARCAs) are affected by several gene mutations. One of these involves mutations in the GRM1 gene, which encodes mGluR1, known to play an important role in cerebellar differentiation [70]. Accordingly, the clinical features of this familial form of CAs appear already during childhood. The child shows global developmental delay, intellectual defects, severe CAs, and pyramidal signs. Brain imaging often shows progressive generalized cerebellar atrophy. Mutations affect a gene region critical for alternative splicing and the formation of the receptor structure.

#### 3.4.4. Glutamate-induced excitotoxicity

Deficits and mutations in GluR can also affect the level of extracellular glutamate with detrimental outcomes for neurotransmission and neuronal health. The level of extracellular glutamate is determined by three factors: (1) vesicular-released glutamate at the synapses, (2) nonvesicular-released glutamate from the system xc() (see above) [71], and (3) glutamate uptake by EAATs on astrocytes [72]. When glutamate release exceeds glutamate uptake, the excess glutamate activates a large number of postsynaptic NMDA-Rs, resulting in the induction of excitotoxic neuronal death by allowing excessive Ca2+ influx through the receptor-operated cation channels [73]. Excessive activation of NMDA-Rs and the associated Ca2+ influx result in stimulation of calpain I and nNOS [73]. This causes DNA damage and formation of ONOOd due to excess NO (nitrosative stress) and other free radicals. The combination of these two changes ultimately leads to mitochondrial dysfunction and cell death [73]. Recent studies reported the presence of high glutamate levels in the extracellular space in chronic degenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, and Huntington's disease [74]. Taken together, glutamate-induced excitotoxicity may be a common process for neuronal death throughout the CNS and accelerates the original pathological changes.

#### 3.4.5. Neurological diseases associated with deficits in GABA receptors

Anti-GABAB receptor Ab is associated with limbic encephalitis [75], which manifests clinically similar to anti-GluRAb-mediated limbic encephalitis. Some patients also develop CAs. About 50% of the patients have neoplastic diseases, especially small cell lung carcinoma [75]. Clinical studies indicate that surgical removal of the lung tumor and subsequent immunotherapy can be effective in the relief of the above neurological disorders, especially in the early stages of disease.

#### 3.4.6. Association of GAD65 dysfunction with neurological disorders

Anti-GAD65Abs are associated with stiff-person syndrome (SPS) and CAs [76]. Titers of anti-GAD65Ab are high (>1000 U/ml) in both conditions. SPS is characterized clinically by progressive rigidity and painful-muscle spasms in the axial and limb muscles. Electromyography (EMG) shows simultaneous activities of the agonist and antagonistic muscles. Anti-GAD65Ab-associated CAs affect mostly women in their 50–60s and exhibit subacute or chronic CAs, which are sometimes associated with SPS or epilepsy [76]. Furthermore, the majority of patients also suffer from type 1 diabetes mellitus (T1D) [76].

site of GluRδ2 are known to cause AMPA-R endocytosis and attenuate their synaptic trans-

Autosomal recessive CAs (ARCAs) are affected by several gene mutations. One of these involves mutations in the GRM1 gene, which encodes mGluR1, known to play an important role in cerebellar differentiation [70]. Accordingly, the clinical features of this familial form of CAs appear already during childhood. The child shows global developmental delay, intellectual defects, severe CAs, and pyramidal signs. Brain imaging often shows progressive generalized cerebellar atrophy. Mutations affect a gene region critical for alternative splicing and the

Deficits and mutations in GluR can also affect the level of extracellular glutamate with detrimental outcomes for neurotransmission and neuronal health. The level of extracellular glutamate is determined by three factors: (1) vesicular-released glutamate at the synapses, (2) nonvesicular-released glutamate from the system xc() (see above) [71], and (3) glutamate uptake by EAATs on astrocytes [72]. When glutamate release exceeds glutamate uptake, the excess glutamate activates a large number of postsynaptic NMDA-Rs, resulting in the induction of excitotoxic neuronal death by allowing excessive Ca2+ influx through the receptor-operated cation channels [73]. Excessive activation of NMDA-Rs and the associated Ca2+ influx result in stimulation of calpain I and nNOS [73]. This causes DNA damage and formation of ONOOd due to excess NO (nitrosative stress) and other free radicals. The combination of these two changes ultimately leads to mitochondrial dysfunction and cell death [73]. Recent studies reported the presence of high glutamate levels in the extracellular space in chronic degenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, and Huntington's disease [74]. Taken together, glutamate-induced excitotoxicity may be a common process for

neuronal death throughout the CNS and accelerates the original pathological changes.

Anti-GABAB receptor Ab is associated with limbic encephalitis [75], which manifests clinically similar to anti-GluRAb-mediated limbic encephalitis. Some patients also develop CAs. About 50% of the patients have neoplastic diseases, especially small cell lung carcinoma [75]. Clinical studies indicate that surgical removal of the lung tumor and subsequent immunotherapy can be effective in the relief of the above neurological disorders, especially in the early stages of

Anti-GAD65Abs are associated with stiff-person syndrome (SPS) and CAs [76]. Titers of anti-GAD65Ab are high (>1000 U/ml) in both conditions. SPS is characterized clinically by progressive rigidity and painful-muscle spasms in the axial and limb muscles. Electromyography (EMG) shows simultaneous activities of the agonist and antagonistic muscles. Anti-GAD65Ab-associated

3.4.5. Neurological diseases associated with deficits in GABA receptors

3.4.6. Association of GAD65 dysfunction with neurological disorders

mission, resulting in the development of ataxia in mice [69].

72 GABA And Glutamate - New Developments In Neurotransmission Research

3.4.3. Autosomal recessive cerebellar degeneration

formation of the receptor structure.

3.4.4. Glutamate-induced excitotoxicity

disease.

Based on the intracellular location of GAD65 (on the cytoplasmic side of SVs), the pathogenic role of anti-GAD65Ab in CA and SPS has been questioned. However, recent studies have shed new light on this issue. First, the pathogenic actions of anti-GAD65Ab have been clarified both in in vitro and in vivo preparations [77–82], for example, the Cerebrospinal fluid (CSF) of patients with SPS inhibited GABA synthesis [83]. Furthermore, SPS-like symptoms were reproduced in experimental rats and mice injected intrathecally or intraventricularly [84] or intracerebellarly [80] with IgGs obtained from the CSF of CA and SPS patients. Specifically, IgGs obtained from the CSF of patients with anti-GAD65Ab-associated CA depressed GABA release in cerebellar brain slices [77, 78, 82, 85], and their intracerebellar injection interfered with cerebellar control of the motor cortex and resulted in ataxic gait in rats [79]. These actions were reproduced by human anti-GAD65 monoclonal Ab b78, which binds to an epitope similar to that recognized in SPS patients positive for anti-GAD65Ab [80–82].

Second, studies in animals have shown internalization of antibodies by cerebellar neurons [81, 86], demonstrating that anti-GAD65Ab can access their intracellular target. Together, these results indicate the possibility that anti-GAD65Ab may damage sufficient numbers of GABAergic neurons to result in the appearance of frank neurological symptoms [87].

The pathological effects of anti-GAD65Ab depend on epitope specificity. Specifically, the pathologic effects of anti-GAD65Abs on GABA synthesis and release were specific to anti-GAD65Ab b78—representing SPS- and CA-associated anti-GAD65Ab—and could not be reproduced by monoclonal anti-GAD65Ab b96.11, representing an epitope specificity associated with type 1 diabetes mellitus [80–82]. This epitope dependence might explain the differences in neurological phenotypes. Administration of CSF obtained from patients with SPS or CAs reproduced the clinical features of the corresponding disease in mice [80]. Thus anti-GAD65Ab in SPS that inhibit GABA synthesis, would attenuate inhibitory Purkinje cellmediated depression of the spino-cerebellar loop, resulting in increased muscle tone, whereas anti-GAD65Ab in CAs depress GABA release, resulting in disruption of Purkinje cell-mediated modulation of the cerebro-cerebellar loop to elicit disorganized movements [87]. An alternative explanation of the phenotypic differences is the involvement of other autoimmunemediated mechanisms.

In anti-GAD65Ab-associated CAs, anti-GAD65Ab impairs the association of GAD65 with the cytosolic side of SVs resulting in a decrease in vesicular GABA contents with low release probability [82]. Under normal conditions, the released GABA spills over to the neighboring excitatory synaptic terminals and inhibits presynaptic glutamate release through GABA receptors. However, this GABA-induced inhibition of glutamate release is disturbed in patients with CAs [77]. Taken together, anti-GAD65Ab elicit marked imbalance in neurotransmitters; including a decrease in GABA and an increase in glutamate. The imbalance between GABA and glutamate is accelerated following the involvement of microglia and astrocytes [87]. Microglia activated by excessive glutamate levels can secrete various cytokines, which facilitate glutamate release presumably through xc() on microglia, and suppress the uptake of glutamate through EAATs on astrocytes [71, 88, 89]. Thus, the neuroinflammation-induced chain reactions accelerate the imbalance, leading to profound excitotoxicity. In agreement with this notion, the cerebellar neurons are completely lost in patients with advanced stage CAs [90].

[95]. Within the beta cells, glutamate is decarboxylated by GAD to yield GABA. GABA is packaged by the GABA transporter VGAT into small synaptic-like microvesicles (SLMVs) [96]. A smaller fraction of GABA is present in insulin-containing large dense core vesicles (LDCVs) [97, 98]. These vesicles also express VGAT, suggesting similar packaging mechanisms as for SLMVs [98]. GABA is present predominantly, if not exclusively, in the beta cells [96, 99, 100], and GABA and GAD expression levels in beta cells are similar to those in GABAergic

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

75

Both isoforms of GAD have been identified in pancreatic beta cells, GAD65 being the predominant isoform in rat and human beta cells, while mice beta cells only express GAD67 [48]. In human and rat beta cells GAD65 co-localizes with GABA at the SLMVs [101], while in murine pancreatic islets, GAD67 is firmly membrane anchored and efficiently transits to presynaptic clusters [102]. It remains unclear whether GAD is involved in SLMVs transport to the plasma

As outlined above, GABA is present in both SLMVs and insulin-containing LDCVs. The basal release of GABA from beta cells is relatively constant [101, 103], but can be modulated depending on the metabolic state of the cell [104]. The mechanisms involved in GABA release were first investigated in beta cell lines, where GABA secretion was shown to be dependent on the presence of extracellular Ca2+ [105], suggesting that GABA is released in response to an increase in cytosolic Ca2+ levels. A detection system for GABA release involving overexpressed GABAA receptors in dispersed rat islets allowed the sensitive detection of GABA release as fluctuations in current in whole-cell patch-clamped beta cells [106]. These studies confirmed that GABA release is dependent on the entry of extracellular Ca2+ through voltage-gated channels and not by membrane depolarization itself. The study further indicated that the

observed GABA release originated predominantly from SLMVs rather than LDCVs.

Both beta and alpha cells express GABAA and GABAB receptors [103]. Through the expression of GABA receptors on beta cells, GABA regulates its own secretion (autocrine regulation). GABAA receptor activation induces further GABA release (autocrine positive feedback loop) [97, 107]. However, GABA-mediated regulation of GABA release depends on the extracellular glucose concentration. At high glucose concentrations, GABA hyperpolarizes the membrane of isolated beta cells and beta cell lines [108]. This inhibitory effects appears be mediated via

Presence of GABA receptors on adjacent alpha cells enables a paracrine regulation of these cells [110]. Activation of GABAA receptors on alpha cells leads to hyperpolarization and suppression of glucagon and glutamate secretion [103, 111]. As glucagon and glutamate trigger insulin release from beta cells, GABAergic inhibition of glucagon and glutamate secretion indirectly downregulates insulin release from beta cells as a negative feedback regulation (Figure 2). Extracellular GABA is taken up by the plasma membrane GABA transporter GAT3

At low concentrations of glucose, alpha cells show high action potentials [103, 112], mediated by voltage-gated Na+ and Ca2+ channels [113]. This triggers the release of glucagon and glutamate. At high glucose concentrations, release of glucagon and glutamate is inhibited,

membrane, in analogy to GABAergic neurons (see also Section 3.3).

neurons [97].

GABAB receptors [106, 109].

expressed on both alpha and beta cells [98].

In conclusion, deficits in glutamate- and GABA-mediated synaptic mechanisms, upset the glutamate/GABA ratio. Notably, the level of glutamate is relatively high compared with that of GABA, caused by various etiologies, including glutamate release by damage-induced depolarization, exaggerated glutamate release through xc(), and attenuated uptake of glutamate through EAATs, or a decrease in GABA release with subsequent increase in glutamate release. The imbalance between glutamate and GABA can trigger excitotoxicity, one of several neuron death mechanisms.

#### 4. GABA/glutamate signaling pathways in pancreatic islets and implications in type 1 diabetes mellitus

While GABA and glutamate are best characterized for their role as neurotransmitter, they are also involved in extra-neuronal signaling. As a major building block in proteins synthesis, intracellular glutamate is abundantly present in the body. In contrast, GABA is present only in restricted non-neuronal tissues, including the pancreas [91]. Pancreatic islets are clusters of endocrine cells located in the exocrine pancreas and regulate blood glucose homeostasis. An islet typically contains insulin-releasing beta cells, glucagon-secreting alpha cells, somatostatincontaining delta cells, and pancreatic polypeptide-producing (PP) cells. The metabolic actions of insulin and glucagon are reviewed in great detail elsewhere [92]. Briefly, insulin is released at elevated blood glucose levels and acts as an anabolic hormone, causing cellular glucose uptake primarily in skeletal muscles, the liver, and fat tissue. Here glucose is converted to storable energy substrates including glycogen and triglycerides, respectively. At low blood glucose levels, glucagon is secreted from pancreatic alpha cells. Glucagon causes the liver to convert stored glycogen to glucose and induces lipolysis in fat tissue. Many regulatory mechanisms are in place to control the secretion of insulin and glucagon to maintain stable blood glucose levels. Within the islet, extracellular insulin inhibits glucagon secretion from alpha cells, while glucagon enhances both insulin and somatostatin secretion [93]. In recent years, GABA and glutamate have gained interest for their respective roles in the regulation of secretion of insulin and glucagon.

In the following sections, we will review GABAergic and glutamatergic signaling in the islets of Langerhans and possible implications for type 1 diabetes mellitus.

#### 4.1. Glutamate and GABA in pancreatic islets

Both glutamate and GABA are synthesized from glucose taken up by beta cells. The majority of glucose is metabolized to produce energy, however a portion is converted to glutamate. The initial step is catalyzed by pyruvate carboxylase present in the beta cells [94]. Thus, in contrast to GABAergic neurons, beta cells can synthesize glutamate on their own. In the alpha cells, glutamate is loaded into glucagon-containing secretory granules via VGLUT1 and VGLUT2 [95]. Within the beta cells, glutamate is decarboxylated by GAD to yield GABA. GABA is packaged by the GABA transporter VGAT into small synaptic-like microvesicles (SLMVs) [96]. A smaller fraction of GABA is present in insulin-containing large dense core vesicles (LDCVs) [97, 98]. These vesicles also express VGAT, suggesting similar packaging mechanisms as for SLMVs [98]. GABA is present predominantly, if not exclusively, in the beta cells [96, 99, 100], and GABA and GAD expression levels in beta cells are similar to those in GABAergic neurons [97].

through EAATs on astrocytes [71, 88, 89]. Thus, the neuroinflammation-induced chain reactions accelerate the imbalance, leading to profound excitotoxicity. In agreement with this notion, the cerebellar neurons are completely lost in patients with advanced stage CAs [90].

In conclusion, deficits in glutamate- and GABA-mediated synaptic mechanisms, upset the glutamate/GABA ratio. Notably, the level of glutamate is relatively high compared with that of GABA, caused by various etiologies, including glutamate release by damage-induced depolarization, exaggerated glutamate release through xc(), and attenuated uptake of glutamate through EAATs, or a decrease in GABA release with subsequent increase in glutamate release. The imbalance between glutamate and GABA can trigger excitotoxicity, one of several neuron

While GABA and glutamate are best characterized for their role as neurotransmitter, they are also involved in extra-neuronal signaling. As a major building block in proteins synthesis, intracellular glutamate is abundantly present in the body. In contrast, GABA is present only in restricted non-neuronal tissues, including the pancreas [91]. Pancreatic islets are clusters of endocrine cells located in the exocrine pancreas and regulate blood glucose homeostasis. An islet typically contains insulin-releasing beta cells, glucagon-secreting alpha cells, somatostatincontaining delta cells, and pancreatic polypeptide-producing (PP) cells. The metabolic actions of insulin and glucagon are reviewed in great detail elsewhere [92]. Briefly, insulin is released at elevated blood glucose levels and acts as an anabolic hormone, causing cellular glucose uptake primarily in skeletal muscles, the liver, and fat tissue. Here glucose is converted to storable energy substrates including glycogen and triglycerides, respectively. At low blood glucose levels, glucagon is secreted from pancreatic alpha cells. Glucagon causes the liver to convert stored glycogen to glucose and induces lipolysis in fat tissue. Many regulatory mechanisms are in place to control the secretion of insulin and glucagon to maintain stable blood glucose levels. Within the islet, extracellular insulin inhibits glucagon secretion from alpha cells, while glucagon enhances both insulin and somatostatin secretion [93]. In recent years, GABA and glutamate have gained interest for their respective roles in the regulation of secretion of insulin

In the following sections, we will review GABAergic and glutamatergic signaling in the islets

Both glutamate and GABA are synthesized from glucose taken up by beta cells. The majority of glucose is metabolized to produce energy, however a portion is converted to glutamate. The initial step is catalyzed by pyruvate carboxylase present in the beta cells [94]. Thus, in contrast to GABAergic neurons, beta cells can synthesize glutamate on their own. In the alpha cells, glutamate is loaded into glucagon-containing secretory granules via VGLUT1 and VGLUT2

of Langerhans and possible implications for type 1 diabetes mellitus.

4.1. Glutamate and GABA in pancreatic islets

4. GABA/glutamate signaling pathways in pancreatic islets and

implications in type 1 diabetes mellitus

74 GABA And Glutamate - New Developments In Neurotransmission Research

death mechanisms.

and glucagon.

Both isoforms of GAD have been identified in pancreatic beta cells, GAD65 being the predominant isoform in rat and human beta cells, while mice beta cells only express GAD67 [48]. In human and rat beta cells GAD65 co-localizes with GABA at the SLMVs [101], while in murine pancreatic islets, GAD67 is firmly membrane anchored and efficiently transits to presynaptic clusters [102]. It remains unclear whether GAD is involved in SLMVs transport to the plasma membrane, in analogy to GABAergic neurons (see also Section 3.3).

As outlined above, GABA is present in both SLMVs and insulin-containing LDCVs. The basal release of GABA from beta cells is relatively constant [101, 103], but can be modulated depending on the metabolic state of the cell [104]. The mechanisms involved in GABA release were first investigated in beta cell lines, where GABA secretion was shown to be dependent on the presence of extracellular Ca2+ [105], suggesting that GABA is released in response to an increase in cytosolic Ca2+ levels. A detection system for GABA release involving overexpressed GABAA receptors in dispersed rat islets allowed the sensitive detection of GABA release as fluctuations in current in whole-cell patch-clamped beta cells [106]. These studies confirmed that GABA release is dependent on the entry of extracellular Ca2+ through voltage-gated channels and not by membrane depolarization itself. The study further indicated that the observed GABA release originated predominantly from SLMVs rather than LDCVs.

Both beta and alpha cells express GABAA and GABAB receptors [103]. Through the expression of GABA receptors on beta cells, GABA regulates its own secretion (autocrine regulation). GABAA receptor activation induces further GABA release (autocrine positive feedback loop) [97, 107]. However, GABA-mediated regulation of GABA release depends on the extracellular glucose concentration. At high glucose concentrations, GABA hyperpolarizes the membrane of isolated beta cells and beta cell lines [108]. This inhibitory effects appears be mediated via GABAB receptors [106, 109].

Presence of GABA receptors on adjacent alpha cells enables a paracrine regulation of these cells [110]. Activation of GABAA receptors on alpha cells leads to hyperpolarization and suppression of glucagon and glutamate secretion [103, 111]. As glucagon and glutamate trigger insulin release from beta cells, GABAergic inhibition of glucagon and glutamate secretion indirectly downregulates insulin release from beta cells as a negative feedback regulation (Figure 2). Extracellular GABA is taken up by the plasma membrane GABA transporter GAT3 expressed on both alpha and beta cells [98].

At low concentrations of glucose, alpha cells show high action potentials [103, 112], mediated by voltage-gated Na+ and Ca2+ channels [113]. This triggers the release of glucagon and glutamate. At high glucose concentrations, release of glucagon and glutamate is inhibited,

thus serving as a regulatory factor to limit glucagon/glutamate release [106]. As in the GABAergic system, glutamate-mediated regulation of beta cells depends on the extracellular glucose concentration. The high-affinity NMDA-Rs on the beta cell are already saturated at physiological glutamate concentrations in the islet, and are mainly activated through depolarization of beta cells [121]. As islets are depolarized by external glucose, the NMDA-R-mediated repolarization of the beta cells is a negative feedback regulation of glucose-stimulated insulin secretion. Extracellular glutamate is taken up by EAAT1 and 2 expressed on alpha cells [122].

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

77

4.2. Proposed GABAergic and glutamatergic signaling in type 1 diabetes mellitus

generalized seizures [130].

Type 1 diabetes mellitus (T1D) is an autoimmune disease, characterized by the specific destruction of pancreatic beta cells. Exogenous administration of insulin is necessary to avoid hyperglycemia. Additionally, within 5 years of disease diagnosis, almost all patients with T1D lose their ability to generate an adequate glucagon response to hypoglycemia [123]. This loss has been attributed to the lack of intracellular regulation of beta- to alpha-cell signaling during hypoglycemia [124] and may account for the elevated plasma glucagon levels in diabetes patients, indicating alpha-cell hypersecretion [125, 126]. As observed for neurons, beta cells are sensitive to elevated extracellular glutamate levels and show signs of secretory defects and apoptosis at high glutamate levels [122]. This effect was not prevented by AMPA-R and Kainate-R antagonists and therefore unlikely caused by excitotoxicity. Instead, oxidative stress appears to be the underlying mechanism of glutamate-induced beta-cell death. As outlined in detail in the CNS portion of this chapter, the glutamate/cystine antiporter system xc() exchanges intracellular glutamate for extracellular cystine. Excess extracellular glutamate inhibits and/or reverts the activity of the antiporter, thus depleting the cells of cysteine, a building block of the antioxidant glutathione, possibly increasing the cells' vulnerability to oxidative stress [122]. Upregulation of EAAT1 expression on beta cells protects beta cells from glutamate-induced toxicity [122], indicating glutamate signaling as a potential therapeutic target. Notably, many effective antidiabetic drugs such as GLP-1, exenatide, and glitazones also show significant neuroprotective activity against glutamate-induced cytotoxicity in the brain [127, 128]. Moreover, topiramate, an anti-epileptic drug that provides neuroprotection by preventing glutamate toxicity, has antidiabetic and beta-cell cytoprotective effects [129] and long-lasting remission was observed in a T1D patient after treatment with topiramate for

GABA has an overall anti-inflammatory effect on the immune system [131]. GABAA receptors are expressed by T cells, B cells, and other mononuclear cells, and their activation suppresses lymphocyte proliferation [132, 133]. This GABA-mediated inhibition of T cell responses may provide the mechanism of GABA-associated protection of animal models for development of T1D [134]. GABA also promotes a shift from an inflammatory to an anti-inflammatory cytokine profile in vivo and in vitro [107]. Another aspect of GABA activity in regard to pancreatic beta cells has been only recently reported. Through activation of GABAB receptors, GABA significantly increases beta-cell viability [135] and replication [136]. In mouse models, GABA administration prevented and even reversed T1D [107]. One of the involved mechanisms may be GABA-mediated conversion of alpha cells to beta cells [137, 138]. While the details of the

Figure 2. Schematic outline of presumed GABAergic and glutamatergic signaling in pancreatic islets. At low glucose concentrations, beta cells release GABA in a glucose-independent manner [97, 108]. This tonic GABA release continuously activates GABAA receptors on beta cells and alpha cells. GABAA receptor activation leads to further GABA release from beta cells and suppresses release of glucagon and glutamate from alpha cells, thereby preventing insulin secretion. As extracellular glucose levels rise, the level of intracellular Ca2+ increases in beta cells, allowing release of insulin. Secreted insulin inhibits GABAA receptors on the beta cells, thereby decreasing the autocrine positive feedback release of GABA from beta cells and reducing GABA-mediated inhibition of alpha cells [140]. Moreover, under high glucose concentrations GABA inhibits its own release from beta cells (via GABAB receptor activation). Reduced extracellular GABA concentrations allow glucagon and glutamate secretion from alpha cells. Glutamate triggers glutamate and glucagon release via activation of AMPA-Rs and glucagon stimulates further insulin release from beta cells. Counter-regulation: eventually, the depolarization of beta cells activates NMDA-Rs present on beta cells, initiating the repolarization of beta cells and limiting insulin release. Reduced insulin levels lift the inhibition of GABAA receptors on beta cells and allow positive feedback regulation of GABA release. Elevated extracellular GABA levels can now activate GABAA receptors on alpha cells and inhibit glutamate and glucagon release. Of note, insulin secretion induces GABAA receptors phosphorylation and translocation to alpha cell plasma membrane [111]. This renders alpha cells more susceptible to GABA-mediated suppression of glucagon secretion, and ultimately limits insulin secretion from beta cells [111, 141].

although the mechanistic details of this regulation remain unclear. Paracrine GABA-mediated regulation (as described above) is suggested by the finding that isolated rat alpha cells no longer show reduced glucagon/glutamate release at elevated glucose concentrations [112], and the observation that in rat islets and purified alpha cells, GABA antagonists suppress glucagon secretion [114]. Insulin and GABA are suggested to serve as paracrine inhibitors of glutamate and glucagon release [101, 103, 115].

Once released, glutamate activates GluRs expressed on both alpha and beta cells. The cellspecific distribution of AMPA-R/Kainate receptors and NMDA-Rs remains debated, and earlier reports suggested that AMPA-Rs are expressed exclusively on alpha cells, while NMDA-Rs were reported to be specifically expressed on beta cells [116]. However, later reports suggest that AMPA-Rs are also expressed on mouse beta cells, and other studies suggest iGluRs expression only on alpha and not on beta cells [117]. mGluRs mGluR8, mGluR5 and mGluR2/3 are expressed by beta cells [109, 118], while mGluR4 protein is expressed on alpha cells [119]. Extracellular glutamate activates AMPA/kainate Rs present on alpha cells and triggers the corelease of glutamate and glucagon (positive feedback regulation) [117, 120, 121]. On the other hand, activation of mGluR inhibits glucagon/glutamate secretion from alpha cells [119] (negative feedback regulation). Few studies reported that activation of AMPA/Kainate-R and/or mGluR on beta cells triggers insulin secretion [118, 120], however subsequent studies could not confirm these results [117]. Still, through glucagon-mediated insulin release from beta cells, co-secreted glutamate indirectly stimulates insulin secretion from beta cells [121]. In addition, glutamate stimulates GABA release from SLMVs in beta cells independently of insulin release, thus serving as a regulatory factor to limit glucagon/glutamate release [106]. As in the GABAergic system, glutamate-mediated regulation of beta cells depends on the extracellular glucose concentration. The high-affinity NMDA-Rs on the beta cell are already saturated at physiological glutamate concentrations in the islet, and are mainly activated through depolarization of beta cells [121]. As islets are depolarized by external glucose, the NMDA-R-mediated repolarization of the beta cells is a negative feedback regulation of glucose-stimulated insulin secretion. Extracellular glutamate is taken up by EAAT1 and 2 expressed on alpha cells [122].

#### 4.2. Proposed GABAergic and glutamatergic signaling in type 1 diabetes mellitus

Type 1 diabetes mellitus (T1D) is an autoimmune disease, characterized by the specific destruction of pancreatic beta cells. Exogenous administration of insulin is necessary to avoid hyperglycemia. Additionally, within 5 years of disease diagnosis, almost all patients with T1D lose their ability to generate an adequate glucagon response to hypoglycemia [123]. This loss has been attributed to the lack of intracellular regulation of beta- to alpha-cell signaling during hypoglycemia [124] and may account for the elevated plasma glucagon levels in diabetes patients, indicating alpha-cell hypersecretion [125, 126]. As observed for neurons, beta cells are sensitive to elevated extracellular glutamate levels and show signs of secretory defects and apoptosis at high glutamate levels [122]. This effect was not prevented by AMPA-R and Kainate-R antagonists and therefore unlikely caused by excitotoxicity. Instead, oxidative stress appears to be the underlying mechanism of glutamate-induced beta-cell death. As outlined in detail in the CNS portion of this chapter, the glutamate/cystine antiporter system xc() exchanges intracellular glutamate for extracellular cystine. Excess extracellular glutamate inhibits and/or reverts the activity of the antiporter, thus depleting the cells of cysteine, a building block of the antioxidant glutathione, possibly increasing the cells' vulnerability to oxidative stress [122]. Upregulation of EAAT1 expression on beta cells protects beta cells from glutamate-induced toxicity [122], indicating glutamate signaling as a potential therapeutic target. Notably, many effective antidiabetic drugs such as GLP-1, exenatide, and glitazones also show significant neuroprotective activity against glutamate-induced cytotoxicity in the brain [127, 128]. Moreover, topiramate, an anti-epileptic drug that provides neuroprotection by preventing glutamate toxicity, has antidiabetic and beta-cell cytoprotective effects [129] and long-lasting remission was observed in a T1D patient after treatment with topiramate for generalized seizures [130].

although the mechanistic details of this regulation remain unclear. Paracrine GABA-mediated regulation (as described above) is suggested by the finding that isolated rat alpha cells no longer show reduced glucagon/glutamate release at elevated glucose concentrations [112], and the observation that in rat islets and purified alpha cells, GABA antagonists suppress glucagon secretion [114]. Insulin and GABA are suggested to serve as paracrine inhibitors of glutamate

of glucagon secretion, and ultimately limits insulin secretion from beta cells [111, 141].

76 GABA And Glutamate - New Developments In Neurotransmission Research

Figure 2. Schematic outline of presumed GABAergic and glutamatergic signaling in pancreatic islets. At low glucose concentrations, beta cells release GABA in a glucose-independent manner [97, 108]. This tonic GABA release continuously activates GABAA receptors on beta cells and alpha cells. GABAA receptor activation leads to further GABA release from beta cells and suppresses release of glucagon and glutamate from alpha cells, thereby preventing insulin secretion. As extracellular glucose levels rise, the level of intracellular Ca2+ increases in beta cells, allowing release of insulin. Secreted insulin inhibits GABAA receptors on the beta cells, thereby decreasing the autocrine positive feedback release of GABA from beta cells and reducing GABA-mediated inhibition of alpha cells [140]. Moreover, under high glucose concentrations GABA inhibits its own release from beta cells (via GABAB receptor activation). Reduced extracellular GABA concentrations allow glucagon and glutamate secretion from alpha cells. Glutamate triggers glutamate and glucagon release via activation of AMPA-Rs and glucagon stimulates further insulin release from beta cells. Counter-regulation: eventually, the depolarization of beta cells activates NMDA-Rs present on beta cells, initiating the repolarization of beta cells and limiting insulin release. Reduced insulin levels lift the inhibition of GABAA receptors on beta cells and allow positive feedback regulation of GABA release. Elevated extracellular GABA levels can now activate GABAA receptors on alpha cells and inhibit glutamate and glucagon release. Of note, insulin secretion induces GABAA receptors phosphorylation and translocation to alpha cell plasma membrane [111]. This renders alpha cells more susceptible to GABA-mediated suppression

Once released, glutamate activates GluRs expressed on both alpha and beta cells. The cellspecific distribution of AMPA-R/Kainate receptors and NMDA-Rs remains debated, and earlier reports suggested that AMPA-Rs are expressed exclusively on alpha cells, while NMDA-Rs were reported to be specifically expressed on beta cells [116]. However, later reports suggest that AMPA-Rs are also expressed on mouse beta cells, and other studies suggest iGluRs expression only on alpha and not on beta cells [117]. mGluRs mGluR8, mGluR5 and mGluR2/3 are expressed by beta cells [109, 118], while mGluR4 protein is expressed on alpha cells [119]. Extracellular glutamate activates AMPA/kainate Rs present on alpha cells and triggers the corelease of glutamate and glucagon (positive feedback regulation) [117, 120, 121]. On the other hand, activation of mGluR inhibits glucagon/glutamate secretion from alpha cells [119] (negative feedback regulation). Few studies reported that activation of AMPA/Kainate-R and/or mGluR on beta cells triggers insulin secretion [118, 120], however subsequent studies could not confirm these results [117]. Still, through glucagon-mediated insulin release from beta cells, co-secreted glutamate indirectly stimulates insulin secretion from beta cells [121]. In addition, glutamate stimulates GABA release from SLMVs in beta cells independently of insulin release,

and glucagon release [101, 103, 115].

GABA has an overall anti-inflammatory effect on the immune system [131]. GABAA receptors are expressed by T cells, B cells, and other mononuclear cells, and their activation suppresses lymphocyte proliferation [132, 133]. This GABA-mediated inhibition of T cell responses may provide the mechanism of GABA-associated protection of animal models for development of T1D [134]. GABA also promotes a shift from an inflammatory to an anti-inflammatory cytokine profile in vivo and in vitro [107]. Another aspect of GABA activity in regard to pancreatic beta cells has been only recently reported. Through activation of GABAB receptors, GABA significantly increases beta-cell viability [135] and replication [136]. In mouse models, GABA administration prevented and even reversed T1D [107]. One of the involved mechanisms may be GABA-mediated conversion of alpha cells to beta cells [137, 138]. While the details of the mechanisms involved need to be further investigated, these studies open the intriguing potential to use GABA treatment to re-generate beta cells in T1D [139].

[6] Obrietan K, van den Pol AN. GABAB receptor-mediated inhibition of GABAA receptor calcium elevations in developing hypothalamic neurons. Journal of Neurophysiology.

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

79

[7] Connor JA, Tseng HY, Hockberger PE. Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures. The

[8] Ganguly K, Schinder AF, Wong ST, Poo M. GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell. 2001;105:

[9] Tyzio R, Cossart R, Khalilov I, Minlebaev M, Hübner CA, Represa A, Ben-Ari Y, Khazipov R. Maternal oxytocin triggers a transient inhibitory switch in GABA signaling

[10] Hartman KN, Pal SK, Burrone J, Murthy VN. Activity-dependent regulation of inhibitory synaptic transmission in hippocampal neurons. Nature Neuroscience. 2006;9:642-649 [11] Spitzer NC. Electrical activity in early neuronal development. Nature. 2006;444:707-712 [12] Manent JB, Demarque M, Jorquera I, Pellegrino C, Ben-Ari Y, Aniksztejn L, Represa A. A noncanonical release of GABA and glutamate modulates neuronal migration. The

[13] Manent JB, Jorquera I, Ben-Ari Y, Aniksztejn L, Represa A. Glutamate acting on AMPA but not NMDA receptors modulates the migration of hippocampal interneurons. The

[14] Czarnecki A, Le Corronc H, Rigato C, Le Bras B, Couraud F, Scain AL, Allain AE, Mouffle C, Bullier E, Mangin JM, Branchereau P, Legendre P. Acetylcholine controls GABA-, glutamate-, and glycine-dependent giant depolarizing potentials that govern spontaneous motoneuron activity at the onset of synaptogenesis in the mouse embry-

[15] Allene C, Cossart R. Early NMDA receptor-driven waves of activity in the developing neocortex: Physiological or pathological network oscillations? The Journal of Physio-

[16] Scain AL, Le Corronc H, Allain AE, Muller E, Rigo JM, Meyrand P, Branchereau P, Legendre P. Glycine release from radial cells modulates the spontaneous activity and its propagation during early spinal cord development. The Journal of Neuroscience.

[17] Lonardoni D, Amin H, Di Marco S, Maccione A, Berdondini L, Nieus T. Recurrently connected and localized neuronal communities initiate coordinated spontaneous activ-

[18] Yu AC, Drejer J, Hertz L, Schousboe A. Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. Journal of Neurochemistry. 1983;41:1484-1487

ity in neuronal networks. PLoS Computational Biology. 2017;13:e1005672

onic spinal cord. The Journal of Neuroscience. 2014;34:6389-6404

in the fetal brain during delivery. Science. 2006;314:1788-1792

1998;79:1360-1370

521-532

Journal of Neuroscience. 1987;7:1384-1400

Journal of Neuroscience. 2005;25:4755-4765

Journal of Neuroscience. 2006;26:5901-5909

logy. 2010;588:83-91

2010;30:390-403

It remains unclear what may cause impaired GABAergic and glutamatergic signaling in the pancreatic islets. No mutations of receptors or other elements of the signaling mechanisms have been identified in T1D so far. GAD65Ab are present in the majority of patients with T1D and are regarded as a byproduct of the immune response without significant relevance for the disease progression. However, it is possible that in analogy to their role in neurological disorders, GAD65Ab are taken up by pancreatic beta cells and (a) uncouple the balanced regulation of insulin and glucagon secretion and (b) induce beta cell apoptosis through prolonged exposure to elevated extracellular glutamate levels. Further research is needed to determine whether GAD65Ab have a pathologic role in the development of T1D.

#### Author details

Christiane S. Hampe<sup>1</sup> \*, Hiroshi Mitoma<sup>2</sup> and Mario Manto3

\*Address all correspondence to: champe@u.washington.edu


#### References


[6] Obrietan K, van den Pol AN. GABAB receptor-mediated inhibition of GABAA receptor calcium elevations in developing hypothalamic neurons. Journal of Neurophysiology. 1998;79:1360-1370

mechanisms involved need to be further investigated, these studies open the intriguing poten-

It remains unclear what may cause impaired GABAergic and glutamatergic signaling in the pancreatic islets. No mutations of receptors or other elements of the signaling mechanisms have been identified in T1D so far. GAD65Ab are present in the majority of patients with T1D and are regarded as a byproduct of the immune response without significant relevance for the disease progression. However, it is possible that in analogy to their role in neurological disorders, GAD65Ab are taken up by pancreatic beta cells and (a) uncouple the balanced regulation of insulin and glucagon secretion and (b) induce beta cell apoptosis through prolonged exposure to elevated extracellular glutamate levels. Further research is needed to

determine whether GAD65Ab have a pathologic role in the development of T1D.

\*, Hiroshi Mitoma<sup>2</sup> and Mario Manto3

[1] Ben-Ari Y, Gaiarsa JL, Tyzio R, Khazipov R. GABA: A pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiological Reviews. 2007;

[2] Aamodt SM, Shi J, Colonnese MT, Veras W, Constantine-Paton M. Chronic NMDA exposure accelerates development of GABAergic inhibition in the superior colliculus.

[3] Obata K, Oide M, Tanaka H. Excitatory and inhibitory actions of GABA and glycine on

[4] Luhmann HJ, Sinning A, Yang JW, Reyes-Puerta V, Stüttgen MC, Kirischuk S, Kilb W. Spontaneous neuronal activity in developing neocortical networks: From single cells to

[5] Obrietan K, van den Pol AN. GABA neurotransmission in the hypothalamus: Developmental reversal from Ca2+ elevating to depressing. The Journal of Neuroscience. 1995;

embryonic chick spinal neurons in culture. Brain Research. 1978;144:179-184

\*Address all correspondence to: champe@u.washington.edu

Journal of Neurophysiology. 2000;83:1580-1591

large-scale interactions. Front Neural Circuits. 2016;10:40

1 University of Washington, Seattle, Washington, USA

2 Tokyo Medical University, Tokyo, Japan

3 ULB Erasme, Brussels, Belgium

tial to use GABA treatment to re-generate beta cells in T1D [139].

78 GABA And Glutamate - New Developments In Neurotransmission Research

Author details

References

87:1215-1284

15:5065-5077

Christiane S. Hampe<sup>1</sup>


[19] Hertz L. The glutamate-glutamine (GABA) cycle: Importance of late postnatal development and potential reciprocal interactions between biosynthesis and degradation. Front Endocrinol (Lausanne). 2013;4:59

[34] Collingridge GL, Kehl SJ, Loo R, McLennan H. Effects of kainic and other amino acids on synaptic excitation in rat hippocampal slices: 1. Extracellular analysis. Experimental

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

81

[35] Gnegy ME. Ca2+/calmodulin signaling in NMDA-induced synaptic plasticity. Critical

[37] Lisman J. A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory. Proceedings of the National Academy of Sciences of the United States of

[38] Carroll RC, Beattie EC, von Zastrow M, Malenka RC. Role of AMPA receptor endocy-

[39] Niswender CM, Conn PJ. Metabotropic glutamate receptors: Physiology, pharmacology, and disease. Annual Review of Pharmacology and Toxicology. 2010;50:295-322

[40] Leaney JL, Milligan G, Tinker A. The G protein alpha subunit has a key role in determining the specificity of coupling to, but not the activation of, G protein-gated inwardly

[41] Sunahara RK, Taussig R. Isoforms of mammalian adenylyl cyclase: Multiplicities of

[42] Shigemoto R, Kinoshita A, Wada E, Nomura S, Ohishi H, Takada M, Flor PJ, Neki A, Abe T, Nakanishi S, Mizuno N. Differential presynaptic localization of metabotropic glutamate receptor subtypes in the rat hippocampus. The Journal of Neuroscience.

[44] Choi DW. Calcium and excitotoxic neuronal injury. Annals of the New York Academy of

[45] Kanai Y, Stelzner M, Nussberger S, Khawaja S, Hebert SC, Smith CP, Hediger MA. The neuronal and epithelial human high affinity glutamate transporter. Insights into structure and mechanism of transport. The Journal of Biological Chemistry. 1994;269:20599-20606

[46] Pomierny-Chamioło L, Rup K, Pomierny B, Niedzielska E, Kalivas PW, Filip M. Metabotropic glutamatergic receptors and their ligands in drug addiction. Pharmacol-

[47] Moran MM, McFarland K, Melendez RI, Kalivas PW, Seamans JK. Cystine/glutamate exchange regulates metabotropic glutamate receptor presynaptic inhibition of excitatory transmission and vulnerability to cocaine seeking. The Journal of Neuroscience.

[48] Okada Y, Taniguchi H, Shimada C. High concentration of GABA and high glutamate decarboxylase activity in rat pancreatic islets and human insulinoma. Science. 1976;194:

[43] Danbolt NC. Glutamate uptake. Progress in Neurobiology. 2001;65:1-105

rectifying K(+) channels. The Journal of Biological Chemistry. 2000;275:921-929

tosis in synaptic plasticity. Nature Reviews. Neuroscience. 2001;2:315-324

[36] Hebb DO. The Organization of Behavior. New York: Wiley & Sons; 1949

Brain Research. 1983;52:170-178

America. 1989;86:9574-9578

1997;17:7503-7522

2005;25:6389-6393

620-622

Sciences. 1994;747:162-171

ogy & Therapeutics. 2014;142:281-305

Reviews in Neurobiology. 2000;14:91-129

signaling. Molecular Interventions. 2002;2:168-184


[19] Hertz L. The glutamate-glutamine (GABA) cycle: Importance of late postnatal development and potential reciprocal interactions between biosynthesis and degradation. Front

[20] Norenberg MD, Martinez-Hernandez A. Fine structural localization of glutamine syn-

[21] Chaudhry FA, Reimer RJ, Edwards RH. The glutamine commute: Take the N line and

[22] Hägglund MG, Sreedharan S, Nilsson VC, Shaik JH, Almkvist IM, Bäcklin S, Wrange O, Fredriksson R. Identification of SLC38A7 (SNAT7) protein as a glutamine transporter expressed in neurons. The Journal of Biological Chemistry. 2011;286:20500-

[23] Kvamme E, Torgner IA, Roberg B. Kinetics and localization of brain phosphate activated

[24] Hertz L, Peng L, Dienel GA. Energy metabolism in astrocytes: High rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis. Journal of

[25] Ottersen OP, Zhang N, Walberg F. Metabolic compartmentation of glutamate and glutamine: Morphological evidence obtained by quantitative immunocytochemistry in rat

[26] Omote H, Miyaji T, Juge N, Moriyama Y. Vesicular neurotransmitter transporter: Bioenergetics and regulation of glutamate transport. Biochemistry. 2011;50:5558-5565

[27] Rizzoli SO, Jahn R. Kiss-and-run, collapse and 'readily retrievable' vesicles. Traffic. 2007;

[28] Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R. Glutamate receptor ion channels: Structure, regulation,

[29] Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A. Magnesium gates glutamate-activated channels in mouse central neurones. Nature. 1984;307:462-465 [30] Mayer ML, Westbrook GL, Guthrie PB. Voltage-dependent block by Mg2+ of NMDA

[31] Pankratov Y, Lalo U. Calcium permeability of ligand-gated Ca2+ channels. European

[32] Collingridge GL, Kehl SJ, McLennan H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. The Journal of

[33] Volianskis A, France G, Jensen MS, Bortolotto ZA, Jane DE, Collingridge GL. Long-term potentiation and the role of N-methyl-D-aspartate receptors. Brain Research. 2015;1621:

thetase in astrocytes of rat brain. Brain Research. 1979;161:303-310

transfer to the A. The Journal of Cell Biology. 2002;157:349-355

glutaminase. Journal of Neuroscience Research. 2001;66:951-958

Cerebral Blood Flow and Metabolism. 2007;27:219-249

and function. Pharmacological Reviews. 2010;62:405-496

responses in spinal cord neurones. Nature. 1984;309:261-263

cerebellum. Neuroscience. 1992;46:519-534

Journal of Pharmacology. 2014;739:60-73

Physiology. 1983;334:33-46

5-16

Endocrinol (Lausanne). 2013;4:59

80 GABA And Glutamate - New Developments In Neurotransmission Research

20511

8:1137-1144


[49] Jin H, Wu H, Osterhaus G, Wei J, Davis K, Sha D, Floor E, Hsu CC, Kopke RD, JY W. Demonstration of functional coupling between gamma-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:4293-4298

Venkatesan A, Vincent A, Wandinger KP, Waters P, Dalmau J. A clinical approach to

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

83

[63] Dalmau J, Gleichman AJ, Hughes EG, Rossi JE, Peng X, Lai M, Dessain SK, Rosenfeld MR, Balice-Gordon R, Lynch DR. Anti-NMDA-receptor encephalitis: Case series and

[64] Rogers SW, Andrews PI, Gahring LC, Whisenand T, Cauley K, Crain B, Hughes TE, Heinemann SF, McNamara JO. Autoantibodies to glutamate receptor GluR3 in Rasmu-

[65] Dalmau J, Tüzün E, HY W, Masjuan J, Rossi JE, Voloschin A, Baehring JM, Shimazaki H, Koide R, King D, Mason W, Sansing LH, Dichter MA, Rosenfeld MR, Lynch DR. Paraneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian

[66] Twyman RE, Gahring LC, Spiess J, Rogers SW. Glutamate receptor antibodies activate a subset of receptors and reveal an agonist binding site. Neuron. 1995;14:755-762

[67] Sillevis Smitt P, Kinoshita A, De Leeuw B, Moll W, Coesmans M, Jaarsma D, Henzen-Logmans S, Vecht C, De Zeeuw C, Sekiyama N, Nakanishi S, Shigemoto R. Paraneoplastic cerebellar ataxia due to autoantibodies against a glutamate receptor. The New

[68] Jarius S, Wildemann B. 'Medusa-head ataxia': The expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 1: Anti-mGluR1, anti-Homer-3, anti-Sj/

[69] Hirai H, Launey T, Mikawa S, Torashima T, Yanagihara D, Kasaura T, Miyamoto A, Yuzaki M. New role of delta2-glutamate receptors in AMPA receptor trafficking and

[70] Guergueltcheva V, Azmanov DN, Angelicheva D, Smith KR, Chamova T, Florez L, Bynevelt M, Nguyen T, Cherninkova S, Bojinova V, Kaprelyan A, Angelova L, Morar B, Chandler D, Kaneva R, Bahlo M, Tournev I, Kalaydjieva L. Autosomal-recessive congenital cerebellar ataxia is caused by mutations in metabotropic glutamate receptor 1.

[71] Massie A, Boillée S, Hewett S, Knackstedt L, Lewerenz J. Main path and byways: Nonvesicular glutamate release by system xc() as an important modifier of glutamatergic

[72] Anderson CM, Swanson RA. Astrocyte glutamate transport: Review of properties, reg-

[73] Fujikawa DG. The role of excitotoxic programmed necrosis in acute brain injury. Com-

[74] Lewerenz J, Maher P. Chronic glutamate toxicity in neurodegenerative diseases-what is

ITPR1 and anti-CARP VIII. Journal of Neuroinflammation. 2015;12:166

cerebellar function. Nature Neuroscience. 2003;6:869-876

American Journal of Human Genetics. 2012;91:553-564

ulation, and physiological functions. Glia. 2000;32:1-14

the evidence? Frontiers in Neuroscience. 2015;9:469

neurotransmission. Journal of Neurochemistry. 2015;135:1062-1079

putational and Structural Biotechnology Journal. 2015;13:212-221

diagnosis of autoimmune encephalitis. Lancet Neurology. 2016;15:391-404

analysis of the effects of antibodies. Lancet Neurology. 2008;7:1091-1098

ssen's encephalitis. Science. 1994;265:648-651

teratoma. Annals of Neurology. 2007;61:25-36

England Journal of Medicine. 2000;342:21-27


Venkatesan A, Vincent A, Wandinger KP, Waters P, Dalmau J. A clinical approach to diagnosis of autoimmune encephalitis. Lancet Neurology. 2016;15:391-404

[63] Dalmau J, Gleichman AJ, Hughes EG, Rossi JE, Peng X, Lai M, Dessain SK, Rosenfeld MR, Balice-Gordon R, Lynch DR. Anti-NMDA-receptor encephalitis: Case series and analysis of the effects of antibodies. Lancet Neurology. 2008;7:1091-1098

[49] Jin H, Wu H, Osterhaus G, Wei J, Davis K, Sha D, Floor E, Hsu CC, Kopke RD, JY W. Demonstration of functional coupling between gamma-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proceedings of the National

[50] Tian N, Petersen C, Kash S, Baekkeskov S, Copenhagen D, Nicoll R. The role of the synthetic enzyme GAD65 in the control of neuronal gamma-aminobutyric acid release. Proceedings of the National Academy of Sciences of the United States of America.

[51] Baekkeskov S, Kanaani J. Palmitoylation cycles and regulation of protein function

[52] Rush DB, Leon RT, McCollum MH, Treu RW, Wei J. Palmitoylation and trafficking of GAD65 are impaired in a cellular model of Huntington's disease. The Biochemical

[53] Bormann J. The 'ABC' of GABA receptors. Trends in Pharmacological Sciences. 2000;

[54] Barnard EA, Skolnick P, Olsen RW, Mohler H, Sieghart W, Biggio G, Braestrup C, Bateson AN, Langer SZ. International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: Classification on the basis of subunit structure

[55] Sigel E, Steinmann ME. Structure, function, and modulation of GABA(A) receptors. The

[56] Brickley SG, Mody I. Extrasynaptic GABA(A) receptors: Their function in the CNS and

[57] Lüscher C, Slesinger PA. Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease. Nature Reviews. Neuroscience. 2010;11:

[58] Tedford HW, Zamponi GW. Direct G protein modulation of Cav2 calcium channels.

[59] Ong J, Kerr DI. Recent advances in GABAB receptors: From pharmacology to molecular

[60] Davies CH, Starkey SJ, Pozza MF, Collingridge GL. GABA autoreceptors regulate the

[61] Schousboe A. Pharmacological and functional characterization of astrocytic GABA

[62] Graus F, Titulaer MJ, Balu R, Benseler S, Bien CG, Cellucci T, Cortese I, Dale RC, Gelfand JM, Geschwind M, Glaser CA, Honnorat J, Höftberger R, Iizuka T, Irani SR, Lancaster E, Leypoldt F, Prüss H, Rae-Grant A, Reindl M, Rosenfeld MR, Rostásy K, Saiz A,

transport: A short review. Neurochemical Research. 2000;25:1241-1244

and receptor function. Pharmacological Reviews. 1998;50:291-313

Journal of Biological Chemistry. 2012;287:40224-40231

implications for disease. Neuron. 2012;73:23-34

Pharmacological Reviews. 2006;58:837-862

induction of LTP. Nature. 1991;349:609-611

biology. Acta Pharmacologica Sinica. 2000;21:111-123

Academy of Sciences of the United States of America. 2003;100:4293-4298

(review). Molecular Membrane Biology. 2009;26:42-54

82 GABA And Glutamate - New Developments In Neurotransmission Research

1999;96:12911-12916

Journal. 2012;442:39-48

21:16-19

301-315


[75] Höftberger R, Titulaer MJ, Sabater L, Dome B, Rózsás A, Hegedus B, Hoda MA, Laszlo V, Ankersmit HJ, Harms L, Boyero S, de Felipe A, Saiz A, Dalmau J, Graus F. Encephalitis and GABAB receptor antibodies: Novel findings in a new case series of 20 patients. Neurology. 2013;81:1500-1506

[86] Hill KE, Clawson SA, Rose JW, Carlson NG, Greenlee JE. Cerebellar Purkinje cells incorporate immunoglobulins and immunotoxins in vitro: Implications for human neurological disease and immunotherapeutics. Journal of Neuroinflammation. 2009;6:31 [87] Mitoma H, Manto M, Hampe CS: Pathogenic roles of glutamic acid decarboxylase 65 autoantibodies in cerebellar ataxias. Journal of Immunology Research 2017;2017:

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

85

[88] Wong WT, Wang M, Li W. Regulation of microglia by ionotropic glutamatergic and

[89] Olmos G, Lladó J. Tumor necrosis factor alpha: A link between neuroinflammation and

[90] Ishida K, Mitoma H, Wada Y, Oka T, Shibahara J, Saito Y, Murayama S, Mizusawa H. Selective loss of Purkinje cells in a patient with anti-glutamic acid decarboxylase antibodyassociated cerebellar ataxia. Journal of Neurology, Neurosurgery and Psychiatry. 2007;78:

[91] Erdö SL, Wolff JR. Gamma-aminobutyric acid outside the mammalian brain. Journal of

[92] Woods SC, Lutz TA, Geary N, Langhans W. Pancreatic signals controlling food intake; insulin, glucagon and amylin. Philosophical Transactions of the Royal Society of Lon-

[93] Gromada J, Franklin I, Wollheim CB. Alpha-cells of the endocrine pancreas: 35 years of

[94] MacDonald MJ. Influence of glucose on pyruvate carboxylase expression in pancreatic

[95] Hayashi M, Morimoto R, Yamamoto A, Moriyama Y. Expression and localization of vesicular glutamate transporters in pancreatic islets, upper gastrointestinal tract, and testis.

[96] Thomas-Reetz A, Hell JW, During MJ, Walch-Solimena C, Jahn R, De Camilli P. A g-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvewsicles of pancreatic ß cells. Proceedings of the National Academy of Sciences.

[97] Braun M, Ramracheya R, Bengtsson M, Clark A, Walker JN, Johnson PR, Rorsman P. Gamma-aminobutyric acid (GABA) is an autocrine excitatory transmitter in human

[98] Gammelsaeter R, Frøyland M, Aragón C, Danbolt NC, Fortin D, Storm-Mathisen J, Davanger S, Gundersen V. Glycine, GABA and their transporters in pancreatic islets of Langerhans: Evidence for a paracrine transmitter interplay. Journal of Cell Science.

research but the enigma remains. Endocrine Reviews. 2007;28:84-116

islets. Archives of Biochemistry and Biophysics. 1995;319:128-132

The Journal of Histochemistry and Cytochemistry. 2003;51:1375-1390

GABAergic neurotransmission. Neuron Glia Biology. 2011;7:41-46

excitotoxicity. Mediators of Inflammation. 2014;2014:861231

don. Series B, Biological Sciences. 2006;361:1219-1235

pancreatic beta-cells. Diabetes. 2010;59:1694-1701

2913297.

190-192

1993;90:5317-5321

2004;117:3749-3758

Neurochemistry. 1990;54:363-372


[86] Hill KE, Clawson SA, Rose JW, Carlson NG, Greenlee JE. Cerebellar Purkinje cells incorporate immunoglobulins and immunotoxins in vitro: Implications for human neurological disease and immunotherapeutics. Journal of Neuroinflammation. 2009;6:31

[75] Höftberger R, Titulaer MJ, Sabater L, Dome B, Rózsás A, Hegedus B, Hoda MA, Laszlo V, Ankersmit HJ, Harms L, Boyero S, de Felipe A, Saiz A, Dalmau J, Graus F. Encephalitis and GABAB receptor antibodies: Novel findings in a new case series of 20 patients.

[76] Honnorat J, Saiz A, Giometto B, Vincent A, Brieva L, de Andres C, Maestre J, Fabien N, Vighetto A, Casamitjana R, Thivolet C, Tavolato B, Antoine J, Trouillas P, Graus F. Cerebellar ataxia with anti-glutamic acid decarboxylase antibodies: Study of 14 patients.

[77] Mitoma H, Ishida K, Shizuka-Ikeda M, Mizusawa H. Dual impairment of GABAA- and GABAB-receptor-mediated synaptic responses by autoantibodies to glutamic acid

[78] Ishida K, Mitoma H, Song SY, Uchihara T, Inaba A, Eguchi S, Kobayashi T, Mizusawa H. Selective suppression of cerebellar GABAergic transmission by an autoantibody to

[79] Manto MU, Laute MA, Aguera M, Rogemond V, Pandolfo M, Honnorat J. Effects of antiglutamic acid decarboxylase antibodies associated with neurological diseases. Annals of

[80] Manto MU, Hampe CS, Rogemond V, Honnorat J. Respective implications of glutamate decarboxylase antibodies in stiff person syndrome and cerebellar ataxia. Orphanet Jour-

[81] Hampe CS, Petrosini L, De Bartolo P, Caporali P, Cutuli D, Laricchiuta D, Foti F, Radtke JR, Vidova V, Honnorat J, Manto M. Monoclonal antibodies to 65kDa glutamate decarboxylase induce epitope specific effects on motor and cognitive functions in rats. Orphanet

[82] Manto M, Honnorat J, Hampe CS, Guerra-Narbona R, Lopez-Ramos JC, Delgado-Garcia JM, Saitow F, Suzuki H, Yanagawa Y, Mizusawa H, Mitoma H. Disease-specific monoclonal antibodies targeting glutamate decarboxylase impair GABAergic neurotransmission and affect motor learning and behavioral functions. Frontiers in Behavioral

[83] Dinkel K, Meinck H, Jury KM, Karges W, Richter W. Inhibition of gamma-aminobutyric acid synthesis by glutamic acid decarboxylase autoantibodies in stiff-man syndrome.

[84] Hansen N, Grunewald B, Weishaupt A, Colaco MN, Toyka KV, Sommer C, Geis C. Human Stiff person syndrome IgG-containing high-titer anti-GAD65 autoantibodies

[85] Ishida K, Mitoma H, Mizusawa H. Reversibility of cerebellar GABAergic synapse impairment induced by anti-glutamic acid decarboxylase autoantibodies. Journal of the

induce motor dysfunction in rats. Experimental Neurology. 2013;239:202-209

decarboxylase. Journal of the Neurological Sciences. 2003;208:51-56

glutamic acid decarboxylase. Annals of Neurology. 1999;46:263-267

Neurology. 2013;81:1500-1506

Neurology. 2007;61:544-551

nal of Rare Diseases. 2011;6:3

Journal of Rare Diseases. 2013;8:82

Annals of Neurology. 1998;44:194-201

Neurological Sciences. 2008;271:186-190

Neuroscience. 2015;9:78

Archives of Neurology. 2001;58:225-230

84 GABA And Glutamate - New Developments In Neurotransmission Research


[99] Vincent ST, Hökfelt T, J-Y W, Eide RP, Morgan LM, Kimmel JR. Immunohistochemical studies of the GABA system in the pancreas. Neuroendocrinology. 1983;36:197-204

[112] Franklin I, Gromada J, Gjinovci A, Theander S, Wollheim CB. Beta-cell secretory products activate alpha-cell ATP-dependent potassium channels to inhibit glucagon release.

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

87

[113] Zhang Q, Ramracheya R, Lahmann C, Tarasov A, Bengtsson M, Braha O, Braun M, Brereton M, Collins S, Galvanovskis J, Gonzalez A, Groschner LN, Rorsman NJ, Salehi A, Travers ME, Walker JN, Gloyn AL, Gribble F, Johnson PR, Reimann F, Ashcroft FM, Rorsman P. Role of KATP channels in glucose-regulated glucagon secretion and

impaired counterregulation in type 2 diabetes. Cell Metabolism. 2013;18:871-882 [114] Wendt A, Birnir B, Buschard K, Gromada J, Salehi A, Sewing S, Rorsman P, Braun M. Glucose inhibition of glucagon secretion from rat alpha-cells is mediated by GABA

[115] Ostenson CG. Regulation of glucagon release: Effects of insulin on the pancreatic A2-cell

[116] Gonoi T, Mizuno N, Inagaki N, Kuromi H, Seino Y, Miyazaki J, Seino S. Functional neuronal ionotropic glutamate receptors are expressed in the non-neuronal cell line

[117] Cabrera O, Jacques-Silva MC, Speier S, Yang SN, Köhler M, Fachado A, Vieira E, Zierath JR, Kibbey R, Berman DM, Kenyon NS, Ricordi C, Caicedo A, Berggren PO. Glutamate is a positive autocrine signal for glucagon release. Cell Metabolism. 2008;7:545-554 [118] Storto M, Capobianco L, Battaglia G, Molinaro G, Gradini R, Riozzi B, Di Mambro A, Mitchell KJ, Bruno V, Vairetti MP, Rutter GA, Nicoletti F. Insulin secretion is controlled by mGlu5 metabotropic glutamate receptors. Molecular Pharmacology. 2006;69:1234-1241

[119] Uehara S, Muroyama A, Echigo N, Morimoto R, Otsuka M, Yatsushiro S, Moriyama Y. Metabotropic glutamate receptor type 4 is involved in autoinhibitory cascade for gluca-

[120] Bertrand G, Puech R, Loubatieres-Mariani MM, Bockaert J. Glutamate stimulates insulin secretion and improves glucose tolerance in rats. The American Journal of Physiology.

[121] Otter S, Lammert E. Exciting times for pancreatic islets: Glutamate signaling in endo-

[122] Di Cairano ES, Davalli AM, Perego L, Sala S, Sacchi VF, La Rosa S, Finzi G, Placidi C, Capella C, Conti P, Centonze VE, Casiraghi F, Bertuzzi F, Folli F, Perego C. The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced

[123] McCrimmon R. The mechanisms that underlie glucose sensing during hypoglycaemia

[124] Fukuda M, Tanaka A, Tahara Y, Ikegami H, Yamamoto Y, Kumahara Y, Shima K. Correlation between minimal secretory capacity of pancreatic beta-cells and stability of

crine cells. Trends in Endocrinology and Metabolism. 2016;27:177-188

beta-cell death. The Journal of Biological Chemistry. 2011;286:14007-14018

in diabetes. Diabetic Medicine. 2008;25:513-522

diabetic control. Diabetes. 1988;37:81-88

gon secretion by alpha-cells of islet of Langerhans. Diabetes. 2004;53:998-1006

released from neighboring beta-cells. Diabetes. 2004;53:1038-1045

MIN6. The Journal of Biological Chemistry. 1994;269:16989-16992

of the guinea pig. Diabetologia. 1979;17:325-330

Diabetes. 2005;54:1808-1815

1995;269:E551-E556


[112] Franklin I, Gromada J, Gjinovci A, Theander S, Wollheim CB. Beta-cell secretory products activate alpha-cell ATP-dependent potassium channels to inhibit glucagon release. Diabetes. 2005;54:1808-1815

[99] Vincent ST, Hökfelt T, J-Y W, Eide RP, Morgan LM, Kimmel JR. Immunohistochemical studies of the GABA system in the pancreas. Neuroendocrinology. 1983;36:197-204 [100] Garry DJ, Appel NM, Garry MG, Sorenson RL. Cellular and subcellular immunolocalization of L-glutamate decarboxylase in rat pancreatic islets. Journal of Histochemistry

[101] Reetz A, Solimena M, Matteoli M, Folli F, Takei K, De Camilli P. GABA and pancreatic β-cells: Co-localization of glutamic acid decarboxylase (GAD) and GABA with synapticlike microvesicles suggests their role in GABA storage and secretion. The EMBO Jour-

[102] Kanaani J, Lissin D, Kash SF, Baekkeskov S. The hydrophilic isoform of glutamate decarboxylase, GAD67, is targeted to membranes and nerve terminals independent of dimerization with the hydrophobic membrane-anchored isoform, GAD65. The Journal

[103] Rorsman P, Berggren PO, Bokvist K, Ericson H, Möhler H, Östensson CG, Smith PA. Glucose-inhibition of glucagon secretion involves activation of GABAA-receptor chlo-

[104] Winnock F, Ling Z, De Proft R, Dejonghe S, Schuit F, Gorus F, Pipeleers D. Correlation between GABA release from rat islet beta-cells and their metabolic state. American

[105] von Blankenfeld G, Turner J, Ahnert-Hilger G, John M, Enkvist MO, Stephenson F, Kettenmann H, Wiedenmann B: Expression of functional GABAA receptors in neuroen-

[106] Braun M, Wendt A, Birnir B, Broman J, Eliasson L, Galvanovskis J, Gromada J, Mulder H, Rorsman P. Regulated exocytosis of GABA-containing synaptic-like microvesicles in

[107] Soltani N, Qiu H, Aleksic M, Glinka Y, Zhao F, Liu R, Li Y, Zhang N, Chakrabarti R, Ng T, Jin T, Zhang H, Lu WY, Feng ZP, Prud'homme GJ, Wang Q. GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:11692-11697

[108] Smismans A, Schuit F, Pipeleers D. Nutrient regulation of gamma-aminobutyric acid

[109] Brice NL, Varadi A, Ashcroft SJ, Molnar E. Metabotropic glutamate and GABA(B) receptors contribute to the modulation of glucose-stimulated insulin secretion in pan-

[110] Sorenson RL, Garry DG, Brelje TC. Structural and functional considerations of GABA in

[111] Xu E, Kumar M, Zhang Y, Ju W, Obata T, Zhang N, Liu S, Wendt A, Deng S, Ebina Y, Wheeler MB, Braun M, Wang Q. Intra-islet insulin suppresses glucagon release via

islets of Langerhans. Beta-cells and nerves. Diabetes. 1991;40:1365-1374

GABA-GABAA receptor system. Cell Metabolism. 2006;3:47-58

Journal of Physiology. Endocrinology and Metabolism. 2002;282:E937-E942

pancreatic beta-cells. The Journal of General Physiology. 2004;123:191-204

docrine gastropancreatic cells. Pflügers Archiv 1995;430:381-388

release from islet beta cells. Diabetologia. 1997;40:1411-1415

creatic beta cells. Diabetologia. 2002;45:242-252

& Cytochemistry. 1988;36:573-580

86 GABA And Glutamate - New Developments In Neurotransmission Research

of Biological Chemistry. 1999;274:37200-37209

ride channels. Nature. 1989;341:233-236

nal. 1991;10:1275-1284


[125] Müller WA, Faloona GR, Unger RH. Hyperglucagonemia in diabetic ketoacidosis. Its prevalence and significance. American Journal of Medicine. 1973;54:52-57

Pattou F, Mansouri A, Collombat P. Long-term GABA administration induces alpha

GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets

http://dx.doi.org/10.5772/intechopen.70958

89

[139] Rutter GA. GABA signaling: A route to new pancreatic β cells. Cell Research. 2017;27:

[140] Bansal P, Wang S, Liu S, Xiang YY, WY L, Wang Q. GABA coordinates with insulin in regulating secretory function in pancreatic INS-1 β-cells. PLoS One. 2011;6:e26225 [141] Bailey SJ, Ravier MA, Rutter GA. Glucose-dependent regulation of gamma-aminobutyric acid (GABA A) receptor expression in mouse pancreatic islet alpha-cells. Diabetes. 2007;

cell-mediated beta-like cell neogenesis. Cell. 2017;168:73-85 e11

309-310

56:320-327


Pattou F, Mansouri A, Collombat P. Long-term GABA administration induces alpha cell-mediated beta-like cell neogenesis. Cell. 2017;168:73-85 e11

[139] Rutter GA. GABA signaling: A route to new pancreatic β cells. Cell Research. 2017;27: 309-310

[125] Müller WA, Faloona GR, Unger RH. Hyperglucagonemia in diabetic ketoacidosis. Its

[126] Reaven GM, Chen YD, Golay A, Swislocki AL, Jaspan JB. Documentation of hyperglucagonemia throughout the day in nonobese and obese patients with noninsulindependent diabetes mellitus. The Journal of Clinical Endocrinology and Metabolism.

[127] Perry T, Haughey NJ, Mattson MP, Egan JM, Greig NH. Protection and reversal of excitotoxic neuronal damage by glucagon-like peptide-1 and exendin-4. The Journal of

[128] Perry T, Greig NH. The glucagon-like peptides: A new genre in therapeutic targets for intervention in Alzheimer's disease. Journal of Alzheimer's Disease. 2002;4:487-496 [129] Rosenstock J, Hollander P, Gadde KM, Sun X, Strauss R, Leung A, Group O-S. A randomized, double-blind, placebo-controlled, multicenter study to assess the efficacy and safety of topiramate controlled release in the treatment of obese type 2 diabetic

[130] Davalli AM, Perego C, Folli FB, Bosi E. Long-lasting remission of type 1 diabetes following treatment with topiramate for generalized seizures. Acta Diabetologica. 2012;

[131] Wan Y, Wang Q, Prud'homme GJ. GABAergic system in the endocrine pancreas: A new target for diabetes treatment. Diabetes, Metabolic Syndrome and Obesity. 2015;8:79-87

[132] Alam S, Laughton DL, Walding A, Wolstenholme AJ. Human peripheral blood mononuclear cells express GABAA receptor subunits. Molecular Immunology. 2006;43:1432-1442

[133] Mendu SK, Bhandage A, Jin Z, Birnir B. Different subtypes of GABA-A receptors are expressed in human, mouse and rat T lymphocytes. PLoS One. 2012;7:e42959

[134] Tian J, Chau C, Hales TG, Kaufman DL. GABA(A) receptors mediate inhibition of T cell

[135] Ligon B, Yang J, Morin SB, Ruberti MF, Steer ML. Regulation of pancreatic islet cell survival and replication by gamma-aminobutyric acid. Diabetologia. 2007;50:764-773

[136] Tian J, Dang H, Chen Z, Guan A, Jin Y, Atkinson MA, Kaufman DL. gamma-Aminobutyric acid regulates both the survival and replication of human beta-cells. Diabetes. 2013;62:

[137] Thorel F, Népote V, Avril I, Kohno K, Desgraz R, Chera S, Herrera PL. Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss. Nature. 2010;464:

[138] Ben-Othman N, Vieira A, Courtney M, Record F, Gjernes E, Avolio F, Hadzic B, Druelle N, Napolitano T, Navarro-Sanz S, Silvano S, Al-Hasani K, Pfeifer A, Lacas-Gervais S, Leuckx G, Marroquí L, Thévenet J, Madsen OD, Eizirik DL, Heimberg H, Kerr-Conte J,

responses. Journal of Neuroimmunology. 1999;96:21-28

prevalence and significance. American Journal of Medicine. 1973;54:52-57

Pharmacology and Experimental Therapeutics. 2002;302:881-888

patients. Diabetes Care. 2007;30:1480-1486

88 GABA And Glutamate - New Developments In Neurotransmission Research

1987;64:106-110

49:75-79

3760-3765

1149-1154


**Chapter 6**

**Provisional chapter**

receptors. Many other structurally

**Antagonists of Ionotropic Receptors for the Inhibitory**

Agents that antagonize the action of GABA on ionotropic receptors are widely used to probe the function of this neurotransmitter. Three such agents are in common use: bicuculline, gabazine, and picrotoxinin. These three agents produce convulsions on systemic administration but act in significantly different ways. Bicuculline is a competitive antagonist of GABAA receptors. Gabazine is also a competitive antagonist of GABAA receptors, interacting with different residues on the receptors. Picrotoxinin is a noncompetitive antagonist acting on the chloride channel of GABAA and several other ionotropic CYS-

diverse agents are now known to act as GABA receptor antagonists, providing opportunities for the discovery of agents with selectivity for the myriad of ionotropic GABA receptors. TPMPA is a selective antagonist for GABAC receptors, which are insensitive to bicuculline. Like TPMPA, many antagonists of ionotropic GABA receptors are not convulsants, indicating that there is still much to be learnt about GABA function in the brain from the study of such agents and their possible therapeutic uses. The most recently discovered GABAA receptor nonconvulsive antagonist is S44819, which is subtype selective for α5-containing receptors, and is arousing much interest in relation to cognition.

**Keywords:** antagonists, GABA receptors, bicuculline, gabazine, picrotoxinin

**Antagonists of Ionotropic Receptors for the Inhibitory** 

DOI: 10.5772/intechopen.72678

© 2016 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,

© 2018 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.

and reproduction in any medium, provided the original work is properly cited.

"Advantages of an Antagonist" headed the Nature editorial in 1970 on the paper reporting the antagonist action of the convulsant alkaloid bicuculline on receptors for the neurotransmitter GABA in the cat spinal cord [1, 2]. The editorial predicted "With this tool it should now be possible to map fairly rapidly the distribution of GABA-inhibitory synapses in the CNS,

**Neurotransmitter GABA: Therapeutic Indications**

**Neurotransmitter GABA: Therapeutic Indications**

Tina Hinton and Graham A. R. Johnston

Tina Hinton and Graham A. R. Johnston

http://dx.doi.org/10.5772/intechopen.72678

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

loop receptors including glycine, GABAC, and 5-HT<sup>3</sup>

Additional information is available at the end of the chapter

#### **Antagonists of Ionotropic Receptors for the Inhibitory Neurotransmitter GABA: Therapeutic Indications Antagonists of Ionotropic Receptors for the Inhibitory Neurotransmitter GABA: Therapeutic Indications**

DOI: 10.5772/intechopen.72678

Tina Hinton and Graham A. R. Johnston Tina Hinton and Graham A. R. Johnston

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72678

#### **Abstract**

Agents that antagonize the action of GABA on ionotropic receptors are widely used to probe the function of this neurotransmitter. Three such agents are in common use: bicuculline, gabazine, and picrotoxinin. These three agents produce convulsions on systemic administration but act in significantly different ways. Bicuculline is a competitive antagonist of GABAA receptors. Gabazine is also a competitive antagonist of GABAA receptors, interacting with different residues on the receptors. Picrotoxinin is a noncompetitive antagonist acting on the chloride channel of GABAA and several other ionotropic CYSloop receptors including glycine, GABAC, and 5-HT<sup>3</sup> receptors. Many other structurally diverse agents are now known to act as GABA receptor antagonists, providing opportunities for the discovery of agents with selectivity for the myriad of ionotropic GABA receptors. TPMPA is a selective antagonist for GABAC receptors, which are insensitive to bicuculline. Like TPMPA, many antagonists of ionotropic GABA receptors are not convulsants, indicating that there is still much to be learnt about GABA function in the brain from the study of such agents and their possible therapeutic uses. The most recently discovered GABAA receptor nonconvulsive antagonist is S44819, which is subtype selective for α5-containing receptors, and is arousing much interest in relation to cognition.

**Keywords:** antagonists, GABA receptors, bicuculline, gabazine, picrotoxinin

#### **1. Introduction**

"Advantages of an Antagonist" headed the Nature editorial in 1970 on the paper reporting the antagonist action of the convulsant alkaloid bicuculline on receptors for the neurotransmitter GABA in the cat spinal cord [1, 2]. The editorial predicted "With this tool it should now be possible to map fairly rapidly the distribution of GABA-inhibitory synapses in the CNS,

and reproduction in any medium, provided the original work is properly cited.

© 2016 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, © 2018 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.

and to determine whether they are as numerous and widely distributed as the relatively high GABA content of the tissue would suggest." Indeed, interest in GABA antagonists continues today, with more than 120 publications per year containing the terms "bicuculline" and "GABA" since 1970 [3]. GABA-inhibitory synapses are widely distributed in the CNS with GABA being released by up to 40% of neurons in many brain regions [4]. Specific GABA receptor antagonists have been described as "essential tools of physiological and pharmacological elucidation of the different types of GABA receptor inhibition" [5].

site, i.e., the orthosteric site; negative allosteric modulation where the drug binds to a site distinct from the orthosteric site to reduce the affinity of the agonist; and noncompetitive antagonism where the drug binds to a site on the chloride channel to reduce chloride permeability or channel opening by GABA [13]. In this perspective, we consider examples of all three types

Antagonists of Ionotropic Receptors for the Inhibitory Neurotransmitter GABA: Therapeutic…

http://dx.doi.org/10.5772/intechopen.72678

93

The first reported GABA receptor antagonist was picrotoxin, a convulsant plant product, a combination of the Greek words "picros" (bitter) and "toxicon" (poison). It is a 50:50 mixture of picrotoxinin (**Figure 1**) and picrotin with picrotoxinin being the more active component as a GABA receptor antagonist. Early reports showed that picrotoxin antagonized the action of GABA at invertebrate inhibitory synapses and that it reduced presynaptic inhibition in the spinal cord [14]. In 1968, Davidoff and Aprison showed that picrotoxin antagonized the inhibitory

**2. Picrotoxin, a channel blocker of ionotropic GABA receptors**

of antagonism of the ionotropic GABA receptors.

**Figure 1.** GABAA receptor antagonists that are convulsants.

GABA receptors can be divided into two major types based on their mechanism of action dating from the studies by David Hill and Norman Bowery in 1981 on the binding of the GABA analog baclofen to rat brain membranes [6]. They described a receptor that "differs from the classical GABA site as it is unaffected by recognized GABA antagonists such as bicuculline." They went on to state "We propose to designate the classical site as the GABAA and the novel site as the GABAB receptor." We now know that GABAA receptors are ionotropic receptors and that GABAB receptors are metabotropic. This perspective on GABA receptor antagonists is limited to mammalian ionotropic receptors.

Ionotropic GABA receptors are ligand-gated ion channels, where binding of GABA necessitates a change in conformation, which leads to opening of the ion channel. The ion channel is permeable to chloride, and increased conductance of this anion stabilizes the membrane potential, thereby reducing excitatory depolarization of the postsynaptic membrane. On the other hand, metabotropic GABA receptors are G-protein-coupled receptors, where GABA binding activates a variety of second messengers that lead to closing of cation channels to prevent sodium and calcium entry and opening of potassium channels to permit potassium efflux. The net effect is a reduction in excitability of the pre- or postsynaptic cell.

Ionotropic GABA receptors are part of the CYS-loop group of receptors that include glycine and 5-HT<sup>3</sup> receptors. Ionotropic GABA receptors may be divided into two classes based on their sensitivity to antagonists. GABAA receptors may be antagonized selectively by bicuculline, while GABAC receptors are antagonized selectively by TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid) and are insensitive to bicuculline [7]. It turns out that these two classes differ in several other respects. While GABAC receptors are relatively simple homomeric pentameric receptors, GABAA receptors are complex heteromeric pentameric receptors consisting of a variety of protein subunits, resulting in different possible combinations and thus a myriad of receptor subtypes. The structural complexity of the GABAA receptors further supports a range of allosteric binding sites which are binding targets for endogenous and exogenous allosteric modulators of these receptors. For example, receptors containing α1 , α<sup>2</sup> , α3 , or α<sup>5</sup> subunits along with a γ2L subunit permit high-affinity benzodiazepine binding [8]. Barbiturates are known to bind to an allosteric site on all GABAA receptor subtypes [9]. As another example, receptors containing δ subunits are targets for endogenous neurosteroids and ethanol [10–12]. Development of subtype-selective agonists, antagonists, and modulators of the ionotropic GABA receptors is imperative to the provision of valuable experimental tools for elucidation of the distribution and various functions of these GABAA receptor subtypes.

Antagonism of ionotropic GABA receptors may result from three distinct mechanisms: competitive antagonism where the binding site for the drug may overlap with the GABA-binding site, i.e., the orthosteric site; negative allosteric modulation where the drug binds to a site distinct from the orthosteric site to reduce the affinity of the agonist; and noncompetitive antagonism where the drug binds to a site on the chloride channel to reduce chloride permeability or channel opening by GABA [13]. In this perspective, we consider examples of all three types of antagonism of the ionotropic GABA receptors.

#### **2. Picrotoxin, a channel blocker of ionotropic GABA receptors**

and to determine whether they are as numerous and widely distributed as the relatively high GABA content of the tissue would suggest." Indeed, interest in GABA antagonists continues today, with more than 120 publications per year containing the terms "bicuculline" and "GABA" since 1970 [3]. GABA-inhibitory synapses are widely distributed in the CNS with GABA being released by up to 40% of neurons in many brain regions [4]. Specific GABA receptor antagonists have been described as "essential tools of physiological and pharmaco-

GABA receptors can be divided into two major types based on their mechanism of action dating from the studies by David Hill and Norman Bowery in 1981 on the binding of the GABA analog baclofen to rat brain membranes [6]. They described a receptor that "differs from the classical GABA site as it is unaffected by recognized GABA antagonists such as bicuculline." They went on to state "We propose to designate the classical site as the GABAA and the novel site as the GABAB receptor." We now know that GABAA receptors are ionotropic receptors and that GABAB receptors are metabotropic. This perspective on GABA receptor antagonists

Ionotropic GABA receptors are ligand-gated ion channels, where binding of GABA necessitates a change in conformation, which leads to opening of the ion channel. The ion channel is permeable to chloride, and increased conductance of this anion stabilizes the membrane potential, thereby reducing excitatory depolarization of the postsynaptic membrane. On the other hand, metabotropic GABA receptors are G-protein-coupled receptors, where GABA binding activates a variety of second messengers that lead to closing of cation channels to prevent sodium and calcium entry and opening of potassium channels to permit potassium

Ionotropic GABA receptors are part of the CYS-loop group of receptors that include glycine

their sensitivity to antagonists. GABAA receptors may be antagonized selectively by bicuculline, while GABAC receptors are antagonized selectively by TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid) and are insensitive to bicuculline [7]. It turns out that these two classes differ in several other respects. While GABAC receptors are relatively simple homomeric pentameric receptors, GABAA receptors are complex heteromeric pentameric receptors consisting of a variety of protein subunits, resulting in different possible combinations and thus a myriad of receptor subtypes. The structural complexity of the GABAA receptors further supports a range of allosteric binding sites which are binding targets for endogenous and exogenous allosteric modulators of these receptors. For example, receptors containing α1

receptors. Ionotropic GABA receptors may be divided into two classes based on

subunits along with a γ2L subunit permit high-affinity benzodiazepine binding [8].

Barbiturates are known to bind to an allosteric site on all GABAA receptor subtypes [9]. As another example, receptors containing δ subunits are targets for endogenous neurosteroids and ethanol [10–12]. Development of subtype-selective agonists, antagonists, and modulators of the ionotropic GABA receptors is imperative to the provision of valuable experimental tools for elucidation of the distribution and various functions of these GABAA receptor subtypes. Antagonism of ionotropic GABA receptors may result from three distinct mechanisms: competitive antagonism where the binding site for the drug may overlap with the GABA-binding

, α<sup>2</sup> ,

efflux. The net effect is a reduction in excitability of the pre- or postsynaptic cell.

logical elucidation of the different types of GABA receptor inhibition" [5].

is limited to mammalian ionotropic receptors.

92 GABA And Glutamate - New Developments In Neurotransmission Research

and 5-HT<sup>3</sup>

α3 , or α<sup>5</sup> The first reported GABA receptor antagonist was picrotoxin, a convulsant plant product, a combination of the Greek words "picros" (bitter) and "toxicon" (poison). It is a 50:50 mixture of picrotoxinin (**Figure 1**) and picrotin with picrotoxinin being the more active component as a GABA receptor antagonist. Early reports showed that picrotoxin antagonized the action of GABA at invertebrate inhibitory synapses and that it reduced presynaptic inhibition in the spinal cord [14]. In 1968, Davidoff and Aprison showed that picrotoxin antagonized the inhibitory

**Figure 1.** GABAA receptor antagonists that are convulsants.

action of glycine on spinal neurones [15]. Curtis et al. [16] reported antagonist action against both glycine and GABA, but the results were inconsistent due to the lack of ionization using the microiontophoresis method of drug administration.

on systemic administration [27, 28]. The quaternary salts differ in their pharmacology to bicuculline itself in that they are much less selective. It is not always clear in publications whether the investigators use bicuculline or a quaternary salt [3]. The quaternary salts have significant actions on nicotinic receptors, calcium-activated potassium channels, and acetylcholinesterase [29–31]. Thus, while ensuring chemical stability of bicuculline, the quaternary salts may be less effective tools owing to their reduced binding specificity for GABAA receptors. Subject to these considerations, bicuculline and its quaternary salts continue to be used extensively as

Antagonists of Ionotropic Receptors for the Inhibitory Neurotransmitter GABA: Therapeutic…

http://dx.doi.org/10.5772/intechopen.72678

95

Extensive structure-activity studies have been carried out on bicuculline with little improvement on potency, selectivity, or stability [3]. Investigations of bicuculline analogs devoid of the phenyl ring fused to the lactone moiety have yielded positive allosteric modulators. These analogs do not bind to the orthosteric binding site on GABAA receptors. Instead, they bind to the high-affinity benzodiazepine site on GABAA receptor subtypes containing subunit combinations described above and show subtype selectivity that differs from that shown by benzodiazepines [32].

Gabazine (also known as SR 95531, **Figure 1**) resulted from a study of arylaminopyridazine analogs of GABA. It was found to be a relatively specific, potent, and competitive antagonist of GABAA receptors [34]. Although both are functionally competitive inhibitors, gabazine and bicuculline also interact with other residues on GABAA receptors [35, 36]. Neither gabazine nor bicuculline compete for the binding at the barbiturate or neurosteroid binding sites on GABAA receptors. It is suggested that both antagonists act "as allosteric inhibitors of channel opening for the GABAA receptor after binding to the GABA-binding site" [36]. Gabazine has little activity at GABAC receptors [37]. At binary β3δ recombinant GABAA receptors, gabazine antagonized GABA currents, whereas bicuculline activated these receptors [38]. Thus, while functioning as competitive antagonists for GABAA receptors, gabazine and bicuculline clearly

Structural analogs of gabazine have identified more potent agents [39]. Gabazine analogs incorporating photoactive groups, such as GZ-B1 (**Figure 1**), have been developed as photoactivated antagonists of GABAA receptors [40]. These antagonists provide dynamic tools for visualizing GABAA receptors, permitting a novel means of investigating receptor location,

GABAC receptors, also known as GABA-ρ and GABAA-ρ receptors, have distinctive distribution and pharmacological properties to GABAA receptors, making them particularly interesting [41].

**5. TPMPA and related compounds, competitive antagonists of** 

Bicuculline has been shown to improve special memory in the rat hippocampus [33].

**4. Gabazine, a competitive antagonist of GABAA receptors**

GABAA receptor antagonists in experimentation.

interact with different residues on GABAA receptors.

function, and trafficking [40].

**GABAC receptors**

These technical difficulties were eventually overcome using recombinant receptors with bath application of picrotoxin showing it to be a mixed/noncompetitive GABAA receptor antagonist [17]. But picrotoxin was also shown to be an antagonist of other CYS-loop receptors, including glycine, GABAC, and 5-HT<sup>3</sup> receptors [18]. Thus, the utility of picrotoxin (and also picrotoxinin) as an experimental tool is mitigated by its lack of selectivity for GABAA receptors. It also has no therapeutic potential owing to its potent convulsant effects.

Many terpenoids related to picrotoxinin are convulsants acting on ionotropic receptors for GABA and glycine [19]. Of particular interest is tutin that occurs in the berries and flowers of the indigenous New Zealand tutu plant, *Coriaria arborea*, and has been credited with convulsing a circus elephant that consumed the berries [20]. Toxic honey was also produced from bees that collected nectar from the flowers.

## **3. Bicuculline, a competitive antagonist of GABAA receptors**

The discovery of bicuculline as a selective antagonist of what became known as GABAA receptors arose out of a systematic study of convulsant alkaloids [3]. It was well known that the most widely understood convulsant alkaloid, strychnine, antagonized the inhibitory action of glycine without influencing the inhibitory action of GABA [21]. Indeed, most convulsant alkaloids turned out to be glycine receptor antagonists with the important exception of bicuculline (**Figure 1**), an alkaloid from *Dicentra cucullaria* [22].

While bicuculline is selective for GABAA receptors, having little effect on GABAB, GABAC, glycine, and 5-HT<sup>3</sup> receptors, its action is largely independent of GABAA subunit composition [17, 23]. Selectivity for GABAA receptors makes bicuculline a powerful experimental tool but without any therapeutic potential owing to the nonselective nature of binding to all GABA<sup>A</sup> receptor subtypes, causing profound convulsive effects. Bicuculline binds at the orthosteric site to stabilize the receptor in a closed state. It is three times the size of GABA and thus is able to bind to sites on the receptor that GABA cannot reach [3]. Bicuculline acts as a competitive antagonist in which it competitively inhibits GABA agonist binding to GABAA receptors, and GABA competitively inhibits bicuculline binding [24]. Single channel studies show that by competing with GABA for its binding site, bicuculline acts to reduce both chloride channel open time and opening frequency [25].

At physiological pH, bicuculline is slowly converted to bicucine, a much less active convulsant [26]. This transformation is slowly reversed at acidic pH. Thus, bicuculline solutions should always be freshly prepared in order to preserve maximum convulsant potency. Quaternary salts of bicuculline, such as bicuculline methiodide ("N-methyl bicuculline") or methochloride, are much more stable than bicuculline, are more water soluble, and are of similar potency as GABA receptor antagonists, but they do not cross the blood-brain barrier on systemic administration [27, 28]. The quaternary salts differ in their pharmacology to bicuculline itself in that they are much less selective. It is not always clear in publications whether the investigators use bicuculline or a quaternary salt [3]. The quaternary salts have significant actions on nicotinic receptors, calcium-activated potassium channels, and acetylcholinesterase [29–31]. Thus, while ensuring chemical stability of bicuculline, the quaternary salts may be less effective tools owing to their reduced binding specificity for GABAA receptors. Subject to these considerations, bicuculline and its quaternary salts continue to be used extensively as GABAA receptor antagonists in experimentation.

Extensive structure-activity studies have been carried out on bicuculline with little improvement on potency, selectivity, or stability [3]. Investigations of bicuculline analogs devoid of the phenyl ring fused to the lactone moiety have yielded positive allosteric modulators. These analogs do not bind to the orthosteric binding site on GABAA receptors. Instead, they bind to the high-affinity benzodiazepine site on GABAA receptor subtypes containing subunit combinations described above and show subtype selectivity that differs from that shown by benzodiazepines [32].

Bicuculline has been shown to improve special memory in the rat hippocampus [33].
