Neural Control in Unique Systems

### **Chapter 5**

## Sympathetic Innervation of the Mammalian Pineal Gland: Its Involvement in Ontogeny and Physiology, and in Pineal Dysfunction

*Martin Avila, Carlos L. Freites, Elena Vásquez, Juan B. Amiotti, Janina Borgonovo and Estela M. Muñoz*

#### **Abstract**

In mammals, the melatonin-producing pineal gland (PG) receives sympathetic innervation from the superior cervical ganglia (SCG). This chapter describes the role of this innervation on the PG's ontogeny and rhythmic function, along with consequences to physiology when this regulation is disrupted. The PG and the SCG are components of the circadian timing system (CTS). Therefore, the overall CTS is described, including its oscillatory basis, its synchronization to the light: dark (L:D) cycles, and the dissemination of timing cues to all cells throughout the body. Pineal cellular composition and heterogeneity, cell-cell interactions, and the molecular mechanisms involved in the circadian rhythm of melatonin (MEL), are discussed. The SCG's bilateral placement among surrounding anatomical landmarks, as well as their afferent and efferent connections, are described and illustrated. In addition, the SCG-related surgical models and the state-of-the art technology used to investigate the connection between SCG and PG are presented. Perspectives and gaps in our understanding are also discussed. We hope this chapter inspires readers to delve deeper into the field of the pineal gland and its main messenger, melatonin, as well as MEL's impact in health and disease, including as a remedial therapy.

**Keywords:** hormone, melatonin, pineal gland, pinealocyte, sympathetic innervation, superior cervical ganglia, norepinephrine, ontogeny, physiology, dysfunction

#### **1. Introduction**

In mammals, melatonin (MEL) is a circadian hormone that is released at high levels into the bloodstream and into the cerebrospinal fluid (CSF) at night, but then drops off to negligibly low levels throughout the daytime [1–3]. Almost all the body's cells

respond to this timing signal. MEL and other circadian cues orchestrate physiology in a rhythmic manner that impacts organ function, tissue healing and rejuvenation, and growth, as well as cognition, motivation, behavior, adaptation, and survival [4]. Taking melatonin supplements is growing in popularity, mainly to augment naturally produced MEL levels and as a sleep aide, but also for its powerful antioxidant, antiinflammatory, free-radical scavenging, and neuroprotective properties [5]. In mammals, circulating MEL is primarily synthesized by the pineal gland (PG), under direct control of sympathetic innervation stemming from the superior cervical ganglia (SCG) [6, 7]. The PG and the SCG are part of an endogenous circadian timing system (CTS) that synchronizes the whole organism to the environmental light: dark cycles (L:D; *Zeitgeber*). In this chapter, we present foundational and current knowledge about how the pineal gland is controlled by the sympathetic nervous system (SNS), with regard to its ontogeny and normal physiology, as well as under dysfunctional conditions. We hope this work inspires readers to seek a deeper understanding of the pineal gland and the functional role of its main messenger, melatonin, in both health and disease, as well as remedial therapy.

#### **2. The pineal gland's role in the mammalian circadian timing system**

The pineal gland (PG) is a highly vascularized neuroendocrine organ that rhythmically produces melatonin (MEL) [2]. The PG is located in the mid-line of the brain, attached to the roof of the third ventricle (III V) by a short stalk [8, 9]. The PG is positioned deep within the brain of humans, and more superficially in rodents. The mammalian PG is driven by a hierarchical series of oscillators from the photoneuroendocrine system (PNS) (**Figure 1**) [7, 12]. Furthermore, the nocturnal release of MEL by the PG provides downstream circadian synchronization to most cells throughout the body. All these elements taken together comprise the circadian timing system (CTS) [12]. The PNS transduces 24-hour light: dark (L:D) cycle information from the external environment into the circadian pattern of MEL synthesis and secretion. To do this, the multisynaptic PNS senses light using intrinsically photosensitive retinal ganglion cells (ipRGC) in the eye, in coordination with the retinal rods and cones. The ipRGC axons project into the GABAergic neurons in the suprachiasmatic nuclei (SCN) of the hypothalamus. SCN are considered to be the master circadian pacemaker, which synchronizes a complex and widely distributed network of peripheral clocks. Each of these oscillators has its own cell-autonomous circadian clock that is driven by interlocked transcriptional/translational feedback loops (TTFL) of core-clock genes (CG), that in turn regulate the expression of clock-controlled genes (CCG), and ultimately coordinate the timing of many biological processes throughout the body [12–15]. The CG family includes genes that encode either positive or negative regulators, such as CLOCK, BMAL, PERs (Period), and CRYs (Cryptochrome) proteins. During the light phase of the L:D cycle, glutamatergic ipRGC axons activate the SCN neurons. This inhibits the rest of the PNS, including the hypothalamic paraventricular nuclei (PVN), neurons of the intermediolateral columns (IMC) of the spinal cord (SC), and the superior cervical ganglia (SCG), and thus, prevents MEL synthesis and secretion by the PG. During the night phase, the SCN release their inhibition over the circuit, and SCG nerve ends release norepinephrine (NE) into the PG parenchyma [6]. This neurotransmitter binds to specific adrenergic receptors on the pinealocyte (Pc) plasma membrane and regulates key steps in the multienzymatic pathway that results in MEL synthesis.

*Sympathetic Innervation of the Mammalian Pineal Gland: Its Involvement in Ontogeny… DOI: http://dx.doi.org/10.5772/intechopen.112361*

#### **Figure 1.**

*A1-A3: The rodent photoneuroendocrine system and the circadian rhythm of melatonin. A1: BV: Blood vessels. ICN: Internal carotid nerves. IMC: Neurons of the intermediolateral columns of the spinal cord (SC). ipRGC: Intrinsically photosensitive retinal ganglion cells. MEL: Melatonin. NC: Nervi conarii. PG: Pineal gland. PVN: Paraventricular nuclei. RHT: Retinohypothalamic tracts. SCG: Superior cervical ganglia. SCN: Suprachiasmatic nuclei. A2: MEL-producing rat pinealocytes immunoreactive for serotonin (red; white arrowheads in the inset). White arrows: Interstitial cells negative for serotonin. DAPI: Nuclear marker 4*′*,6-diamidino-2-phenylindole (blue). Immunofluorescence and confocal microscopy; objective: 10X, scale bar: 150 μm (inset: 60X with 4X zoom, scale bar: 15 μm.). See Ibañez Rodriguez et al. [9] for further details about animal procedures and immunolabeling protocols. A3: Rat superior cervical ganglion and surrounding anatomical landmarks. CCA: Common carotid artery. ECA: External carotid artery. ECN: External carotid nerve. HN: Hypoglossal nerve. ICA: Internal carotid artery. IJV: Internal jugular vein. ST: Sympathetic trunk. VN: Vagus nerve. Modified from Savastano et al. [10], where further details about animal procedures can be found. The reproduction of this copyrighted material was authorized by Elsevier. B: Melatonin biosynthetic pathway. AADC: Aromatic L-amino acid decarboxylase. AANAT: Arylalkylamine N-acetyltransferase. ASMT: Acetylserotonin O-methyltransferase. CSF: Cerebrospinal fluid. TPH: Tryptophan hydroxylase. III V: Third ventricle. C: Adrenergic regulation of melatonin synthesis at night. α1B-ADR: α1B adrenergic receptor. AC: Adenylate cyclase. ATP: Adenosine triphosphate. β1-ADR: β1 adrenergic receptor. cAMP: Cyclic adenosine monophosphate. CRE: cAMP responsive element. G: G proteins. mRNA: Messenger ribonucleic acid. pCREB: Phosphorylated form of cAMP responsive element-binding protein (CREB). PKA: Protein kinase A. PLC: Phospholipase C. PKC: Protein kinase C. D1-D2. Transcriptionally distinguished cell types and subtypes within the adult rat pineal gland. D1: Distribution of the cell types profiled in Mays et al. [11] during the light (L) and dark (D) phases of the L:D cycle. VLMC: Vascular and leptomeningeal cells. D2: Crosstalk between α- and β-pinealocytes to produce nocturnal melatonin in a coordinated and efficient manner. See Mays et al. [11] for further details.*

#### **2.1 Cellular composition of the mature pineal gland**

Melatonin (MEL) is produced within the PG by its predominant cell population, the pinealocytes (Pc). One of the most modern classifications of rat Pc came with the application of single-cell RNA sequencing (scRNA-seq) (**Figure 1**) [11, 16]. This state-of-the-art technology provides gene expression profiles of isolated and individualized cells. Nowadays, it is accepted that at least two subtypes of pinealocytes, α-Pc and β-Pc, coexist and crosstalk in the rat PG, to produce MEL in a coordinated and efficient manner. β-pinealocytes are more abundant than α-Pc, but α-Pc are more effective in catalyzing the last step in the MEL biosynthetic pathway. The scRNA-seq analysis also discriminated interstitial cells. Among these transcriptionally distinguished non-pinealocyte cells are three astrocytes (α, β, and γ), two microglial subtypes (α and β), endothelial cells (EC), and vascular and leptomeningeal cells (VLMC). Several studies have shown that some non-pinealocyte cells modulate MEL production by pinealocytes, under both homeostatic and pathological conditions [17]. With respect to EC, they represent key elements within the PG because they form the inner lining of all blood vessels (BV) that make up its vast circulatory network, which are mainly fenestrated capillaries [8]. Therefore, the PG's blood vessels are more permeable and less selective than the tightly regulated blood-brain barrier (BBB) present in most of the central nervous system (CNS) [18]. The PG is included as one of the seven circumventricular organs (CVO) in the brain, and all of them have an incomplete barrier [8, 19]. This characteristic allows CVO to function as an intermediary pathway between the brain and the periphery, for bidirectional trafficking and interaction.

#### **2.2 Melatonin synthesis by pinealocytes**

Melatonin (MEL) is synthesized by pinealocytes (Pc) at night, via a multienzymatic pathway driven mainly by rhythmic neural inputs [2, 6, 7]. Circulating L-tryptophan (Trp) is an essential amino acid that acts as the biosynthetic precursor of the MEL molecule (**Figure 1**). Trp is hydroxylated and then decarboxylated enzymatically within the Pc cytoplasm. The product of these two reactions is serotonin or 5-hydroxytryptamine (5-HT). Serotonin is then converted into MEL by acetylation, followed by methylation [4]. The enzymes that catalyze the last two reactions, AANAT (Arylalkylamine N-acetyltransferase) and ASMT (Acetylserotonin O-methyltransferase), respectively, represent adjustable key points of the MELproducing pathway [2, 11, 20]. Sympathetic axons stemming from neurons located in both superior cervical ganglia (SCG), provide the norepinephrine (NE) neurotransmitter signal that is the main regulator of MEL production. NE impacts the MEL biosynthetic machinery at different levels, from gene expression to enzyme activities, among other target points (**Figure 1**) [6, 7].

#### **2.3 Sympathetic innervation of the mammalian pineal gland**

The mammalian PG receives a wide range of afferent nerve fibers and, therefore, it can be influenced by a plethora of neurotransmitters [6, 8]. Efferent projections from the PG have also been described, but only for some species and at particular ontogenetic stages [8]. Among the afferent innervations, sympathetic axons, originating from both the right and left SCG, are a fundamental regulatory element of PG rhythmicity in mammals (**Figure 2**) [2]. Classic transcriptomic and neurotranscriptomic

studies have shown that essentially all aspects of PG biology are subject to neural control. These aspects include thousands of genes associated with either MEL-related or MEL-unrelated functions, such as immune/inflammatory response and thyroid hormone signaling [11, 22–24].

#### *2.3.1 Superior cervical ganglia*

The SCG are the uppermost ganglia of the paravertebral sympathetic chain. They are well-defined structures with a variable number of neurons, which receive inputs from preganglionic fibers ascending in the sympathetic trunk (ST) (**Figure 1**) [10, 25, 26]. SCG neurons, mainly via the external and internal carotid nerves (ECN and ICN), establish a wide field of synapsis in the neck, face, and intracranial areas. The SCG not only innervate the pineal gland (PG), but also the hypophysis and median eminence, the thyroid and parathyroid glands, and the Muller's muscles (MM) that control the position of the upper eyelids (palpebral position). An important distinction is that the PG and the MM are innervated differently. Nerve fibers from both the right and left ICN innervate the PG bilaterally. Whereas for the MM, each MM is innervated unilaterally via efferent sympathetic axons present in the homolateral ICN. This innervation difference is used to evaluate the success of the SCG-related surgical procedures that are discussed in Section 2.3.3 (**Figure 2**). For all SCG targets and under tissue-specific stimuli (e.g., lights off for the PG), SCG-derived nerve terminals mainly release norepinephrine (NE) into the synaptic cleft and into the perivascular space. Additionally, other neuropeptides, such as the neuropeptide Y (NPY) in the PG, have been identified as sympathetic co-neurotransmitters [8]. The concentration of NE in the synaptic (or synaptic-like) gaps is affected by simple diffusion and uptake rates. NE uptake includes both its transport back into presynaptic nerve ends and its recapture by neighboring cells. NE re-uptake by sympathetic nerve terminals is crucial for stimulus termination, and for removal and deactivation of circulating stress-induced catecholamines. NE passes the message to the targets by stimulating specific adrenergic receptors on their cell membranes.

#### *2.3.2 Adrenergic reception in the mature pineal gland*

Pinealocytes (Pc) are the MEL-producing cells within the PG. As mentioned, Pc express adrenergic receptors on their cell membranes. These adrenoceptors bind and respond to the nocturnal NE released into the perivascular space from the sympathetic nerve ends (**Figure 1**). A recent scRNA-seq study confirmed the expression of two catecholamine receptor genes, *Adrb1* and *Adra1b*, in both α-Pc and β-Pc in the rat PG [11]. These genes encode β1 and α1B adrenergic receptors, respectively. Additionally, low levels of both transcripts were found in all the non-pinealocyte cells as well, with the exception that none were found in β-microglial cells. β1-ADR and α1B-ADR are seven-transmembrane (7TM) domain receptors that belong to the G protein-coupled receptor (GPCR) superfamily. The NE activation of these adrenoceptors triggers cooperative signaling pathways and several second messengers (e.g.: cyclic adenosine monophosphate, cAMP, and Ca2+) that impact the whole Pc, including its nucleus and transcriptome (e.g., NE induces the expression of the *Aanat* gene, which encodes the enzyme AANAT, via the phosphorylated form of cAMP responsive element-binding protein, pCREB) [6, 7, 27–29]. As soon as *de novo* MEL is synthesized, it is released immediately into the bloodstream and into the cerebrospinal fluid (CSF) [3, 30]. MEL is produced during the dark phase of the L:D cycle and it is used

#### **Figure 2**

*A1-A3: Surgical procedures related to the superior cervical ganglia and their impacts on pineal rhythmicity and palpebral position. A1: Bilateral sympathetic innervation of the pineal gland (PG) by both the right and left internal carotid nerves (ICN). This innervation provides nocturnal norepinephrine (NE), which drives the rhythmic synthesis of the hormone melatonin (MEL). ECN: External carotid nerves. SCG: Superior cervical ganglia. ST: Sympathetic trunks. A2: Reduction in the circulating MEL levels and homolateral blepharoptosis (\*) after unilateral disruption of the sympathetic innervation. (1) Decentralization, by removal of a segment of the afferent sympathetic trunk (STx; lesion of preganglionic axons with undamaged ganglion in situ). (2) Denervation, by removal of a portion of the ICN (ICNx; lesion of postganglionic axons with undamaged ganglion in situ). (3) Ganglionectomy, by complete excision of the ganglion (SCGx; ablation of neuronal cell bodies). A3: Disappearance of the MEL circadian rhythm after a bilateral procedure. Surgery efficiency can be confirmed by observing palpebral ptosis in both eyes (\*). The rat image also shows signs of chromodacryorrhoea (red tears). B1-B2: Norepinephrine re-uptake by nerve ends is abolished in the ganglionectomy model. B1: Norepinephrine (NE; light blue circles) released in the synaptic-like gaps, binds to specific adrenergic receptors (1) and diffuses outside the cleft (2). In addition, NE is transported back into the presynaptic nerve terminals in the healthy PG (3). NE re-uptake is crucial for stimulus termination, and for removal and deactivation of circulating stress-induced catecholamines (Orange circles: NE metabolites). α1B-ADR: α1B adrenergic receptor. β1-ADR: β1 adrenergic receptor. V: Vesicles. (?) Unknown. B2: NE re-uptake is abolished in the degenerating nerve terminals after SCGx. This is a difference with the STx procedure. C: Degeneration of the sympathetic nerve fibers within the pineal gland with age and after bilateral superior cervical ganglionectomy. Sections of rat pineal glands were immunolabeled for β III-tubulin, a marker of nerve fibers. SCGx: Bilateral ganglionectomy. SHAM: Bilateral sham surgery. Pineal glands from young (3 months) and aged (18 months) rats are shown. Immunofluorescence and confocal microscopy; objective: 10X, scale bar: 150 μm; objective: 40X, scale bar: 50 μm. See Ibañez Rodriguez et al. [9, 21] and Savastano et al. [10] for further details about animal procedures and immunolabeling protocols. The reproduction of the copyrighted rat images was authorized by Elsevier.*

*Sympathetic Innervation of the Mammalian Pineal Gland: Its Involvement in Ontogeny… DOI: http://dx.doi.org/10.5772/intechopen.112361*

to disseminate the nighttime circadian status to all cells of the body, via specific MT1 and MT2 MEL receptors on the target cell membranes [31]. Nocturnal MEL production subsides towards late night and is shutdown during the daytime, in response to both extra-Pc mechanisms (e.g.: NE diffusion and uptake) and intra-Pc mechanisms (e.g.: feedback inhibition) [2, 6, 7].

#### *2.3.3 SCG-related surgical procedures*

Different procedures have been extensively used to study the sympathetic innervation of the mammalian PG, including surgical and pharmacological interventions, and electric stimulation [10, 23, 26, 32, 33]. Right and left ICN ascend from each SCG via the internal carotid arteries (ICA) and extend further to form the *nervi conarii* (NC) (**Figure 1**). The NC penetrate the PG at its dorso-posterior border and then ramify throughout the whole organ. Sometimes, bundles of axons from the two NC become fused before entering the gland. The sympathetic nerve fibers that innervate the PG along its vasculature, arise from a small population of neurons (not all neurons) that are rostrally dispersed in each SCG. When SCG-related surgeries are performed to completely suppress PG rhythmicity, both SCG must be isolated and manipulated in order to shut down the neural NE source bilaterally (**Figure 2**). A complete and permanent disruption of the MEL rhythm is achieved only when the influence of both NC is fully and irreversibly disrupted. In circadian biology, SCGrelated surgeries are preferred to intracranial ones, such as a suprachiasmatic nuclei lesion (SCNx), due to the technical complications and the wider physiological impacts associated with these more invasive neurosurgeries. Because of the well-defined anatomy of the SCG and the surrounding structures, three types of surgical procedures can be executed to influence PG rhythmicity: (1) decentralization, by removal of a segment of the afferent sympathetic trunk (STx; lesion of preganglionic axons with undamaged ganglion *in situ*); (2) denervation, by removal of a portion of the ICN (ICNx; lesion of postganglionic axons with undamaged ganglion *in situ*), and (3) ganglionectomy, by complete excision of the ganglion (SCGx; ablation of neuronal cell bodies) (**Figure 2**). For those researchers who are interested in incorporating these procedures as routine techniques, there are straightforward protocols available in the literature, which provide step-by-step descriptions, illustrated with amazing images and detailed videos [10, 32, 33]. Each SCG-related surgery has advantages and disadvantages. For example, after a latency period following SCGx, the innervated target is seen to change through phases before stabilizing: (1) sympathetic stimulation due to the Wallerian degeneration of the sympathetic nerve terminals and a supraliminal release of neurotransmitter in the first few days following surgery (acute SCGx), and (2) sympathetic deprivation after the first post-surgery week (chronic SCGx). One complication of the SCGx is that it cannot prevent the influence of circulating stress-released catecholamines on the target tissue, because the local NE re-uptake mechanism is abolished in the degenerating sympathetic nerve ends (**Figure 2**). This obliges the users to exhaustively control animal housing conditions to eliminate any kind of stressor during the whole experimental time, even during animal euthanasia. On the contrary, STx preserves presynaptic re-uptake and the capacity to remove and deactivate circulating catecholamines. Additionally, SCGx induces microgliosis with damaging consequences over the PG parenchyma [21]. For these reasons, researchers prefer STx over SCGx. As mentioned, in the case of the PG, both SCG must be successfully manipulated to abolish the MEL circadian rhythm completely and permanently. This can be confirmed in a calm animal by bilateral blepharoptosis (palpebral

ptosis), a sign commonly considered to evaluate surgery effectiveness (**Figure 2**) [10]. This sign is used because both the PG and the MM are innervated by the ICN (see Section 2.3.1).

#### **2.4 Ontogeny of the mammalian pineal gland and its relationship with the sympathetic innervation**

The PG emerges as an evagination of the roof of the diencephalon, late in the embryonic (E) period (E14-E15 for rat) [8, 9]. The basis of pineal morphogenesis, and the dynamic and intricate network of transcription factors (TF) involved in the establishment and maintenance of the pineal phenotype, have been extensively characterized, as well as the consequences of certain gene mutations on these mechanisms [28, 34–39]. Cells that are positive for the essential ontogenetic TF Pax6 and the intermediate filament protein vimentin (Vim) are present in the pineal primordium. The Pax6+ /Vim+ precursor cells divide and go through an intrinsically and spatially programmed transformation, giving rise to pinealoblasts, which then mature perinatally to become pinealocytes. Astrocytes also derive from the Pax6+ /Vim+ precursors, but later than Pc. Beyond the well-characterized sympathetic regulation of PG rhythmicity in mammals, researchers have questioned what role sympathetic innervation may have on the definition and fate of the pinealocyte lineage. Disruption of the SCGderived innervation of the rat PG at 5 days after birth (P5; P: postnatal), by either STx or SCGx, did not substantially affect the establishment of the pineal-defining transcriptome (e.g., almost unaltered expression of the *Asmt* gene, which encodes the enzyme ASMT) [23]. As expected, both neonatal SCG-related surgeries did disrupt NE-dependent rhythms in the mature gland, including the circadian rhythm of melatonin (MEL). This suggests that functional sympathetic innervation might not be essential for pinealocyte definition, as it is for its circadian function. These results are consistent with previous classic reports about the ontogeny of adrenoceptors and the postnatal appearance of rhythms in adrenergic reception and signaling transduction, and in MEL-related enzymes [7, 40]. However, further comprehensive studies are necessary to confirm or not whether sympathetic and non-sympathetic innervations do indeed participate in the fine definition of the PG phenotype. This might include interventions earlier than P5, for example.

#### **2.5 Sympathetic dysfunction**

In general, an abnormal melatonin rhythm has been associated with a wide spectrum of human pathologies, including sleep disorders, obesity, diabetes, cancer, and genetic, trauma-induced, neurological, and neurodegenerative disorders [1, 4, 41–43]. Our modern life, with the use of artificial lighting, time-shifted work schedules, and travel jet lag, contributes to alterations in circulating MEL levels in humans [42, 44]. MEL production normally subsides as we age and is aggravated by the more prolonged life expectancy of current generations [45]. This directly affects sleep patterns and mental alertness, but it also has short-term and long-term impacts on overall health. Taking MEL supplements has become a popular remedial therapy when endogenous MEL production is deemed to be deficient or altered. However, basic questions regarding MEL consumption and optimal dosage have not yet been resolved. Nevertheless, MEL supplementation is being used mainly to improve sleep quality and to treat certain sleep disorders. Additionally, it is consumed to attenuate tissue damage due to the cytoprotective properties of MEL itself. Further studies

#### *Sympathetic Innervation of the Mammalian Pineal Gland: Its Involvement in Ontogeny… DOI: http://dx.doi.org/10.5772/intechopen.112361*

are needed to clarify the cellular and molecular mechanisms behind the altered MEL patterns for each of these pathological landscapes. In addition, the therapeutical potential of MEL and its analogs for a wide range of human pathologies needs further investigation [4, 5, 43]. As mentioned, the experimental disruption of the SCG-derived innervation, when it is executed bilaterally and irreversibly, shuts down NE-dependent pineal rhythmicity. In humans, there are several pathological conditions that may be accompanied by primary or secondary sympathetic alterations, that therefore may cause pineal dysfunction. For example, spinal cord injuries (SCI) can be a primary mechanism for sympathetic abnormality [46]. SCI at the upper thoracic segments or higher (cervical injury) may sever the descending axons from the hypothalamic PVN, which connect the SCN to the preganglionic neurons located in the IMC of the spinal cord (SC). As a result, these IMC neurons may not establish functional synapsis with SCG ganglionic cells, which would impair or abolish nocturnal MEL synthesis [47]. In fact, patients with certain upper SCI sometimes experience altered levels of circulating MEL, and disrupted sleep patterns and behaviors. Exogenous MEL has been used to ameliorate SCI consequences due to both MEL's chronobiotic and cytoprotective qualities [48]. On the other hand, a large number of studies has pointed to an age-related loss of the pineal function in both animals and humans (e.g.: elderly individuals, and preclinical and clinical patients with agingrelated pathologies, such as Alzheimer's disease and Parkinson's disease) [4, 43, 45, 49–51]. This loss has been linked to some or all the following features: anatomical abnormalities, reduced number of pinealocytes and variable numbers of glial cells, fibrosis, calcification, inflammation, altered CG expression and clock functionality, disconnection from the master circadian clock at the hypothalamic SCN, and impaired sympathetic regulation [49–54]. Sympathetic dysregulation may involve the loss of nerve terminals, as well as age-induced neuroaxonal dystrophy (NAD) of distal axons, altered denervation supersensitivity, and a decrease in adrenoceptor reception and responsiveness, among other mechanisms (**Figure 2**) [53, 55]. The sequence and progression of these structural and mechanistic alterations during aging and aging-associated pathologies have not yet been fully clarified. Acute and chronic MEL supplementation, in both early and late stages of these conditions, also warrants further investigation. The use of exogenous MEL supplements represents, however, a promising remedial therapy especially for those patients in preclinical phases due to both its chronobiotic and its cytoprotective properties [51, 56, 57].

#### **3. Conclusions**

In mammals, sympathetic innervation plays a key regulatory role in pineal biology and its circadian production of the hormone melatonin. A proper melatonin rhythm, with its classic level rise at night, requires an intact photoneuroendocrine system, that transduces light information from the external environment into the hormonal cue. To do this, a hierarchical series of oscillators, that includes the brain's master circadian clock, operates in a coordinated manner to assure that this information is communicated to the pineal gland through the right and left superior cervical ganglia. The well-characterized structural and functional features of this association have made the pineal gland one of the preferred models for understanding the role of sympathetic innervation in health and disease. A wide spectrum of human pathologies may be accompanied by pineal dysfunction. This may be related to different forms of sympathetic abnormalities. Further studies are necessary to delve deeper into the

cellular and molecular mechanisms responsible for altered melatonin rhythms in these pathological landscapes. In addition, further efforts are needed to elucidate whether melatonin supplementation is useful to prevent or to ameliorate the impacts of these conditions on health and life quality.

### **Acknowledgements**

We thank Raymond D. Astrue for editing the manuscript. This work was supported by grants from CONICET (Argentina; PUE 2017; http://www.conicet. gov.ar), and ANPCyT (Argentina; PICT 2017-499 and PICT 2021-314; http://www. agencia.mincyt.gob.ar).

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Martin Avila1†, Carlos L. Freites1†, Elena Vásquez1 , Juan B. Amiotti1 , Janina Borgonovo2 and Estela M. Muñoz1 \*

1 Institute of Histology and Embryology of Mendoza (IHEM), National University of Cuyo (UNCuyo), National Scientific and Technical Research Council (CONICET), Mendoza, Argentina

2 Laboratory of Experimental Ontogeny (LEO), Faculty of Medicine, Institute of Biomedical Sciences, Universidad de Chile, Santiago, Chile

\*Address all correspondence to: munoz.estela@fcm.uncu.edu.ar; emunoz@conicet-mendoza.gob.ar

† These authors contributed equally to this chapter.

© 2023 The Author(s). Licensee IntechOpen. 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.

*Sympathetic Innervation of the Mammalian Pineal Gland: Its Involvement in Ontogeny… DOI: http://dx.doi.org/10.5772/intechopen.112361*

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**Chapter 6**

## The Brain-Like Enteric Nervous System

*Flower M.J. Caycho Salazar, Deissy Herrera-Covarrubias, Genaro A. Coria-Ávila, Luis I. García-Hernández, María Rebeca Toledo-Cárdenas, María Elena Hernández-Aguilar and Jorge Manzo*

#### **Abstract**

Understanding the autonomic supply at the gastrointestinal tract is one of the significant challenges for science. Its complex network of neurons exists on a broad evolutionary scale, from Hydra to mammals, and in a higher number than those found in the vertebrate spinal cord. Inside the gastrointestinal tract, enteric neurons regulate several functions with intrinsic processes and communicate with the other complex known as the microbiome. Outside the gastrointestinal tract, the enteric neurons project to the brain stem and spinal cord via the gut–brain axis. Furthermore, this enteric system has close functional relationships with the immune system for a rapid response to unhealthy food. The present chapter focuses on the structure, function, and pathologies of the enteric nervous system.

**Keywords:** enteric nervous system, gastrointestinal, microbiome, brain–gut axis, second brain

#### **1. Introduction**

The enteric nervous system (ENS) is a complex network of neurons and glia that regulates the physiology of the gastrointestinal tract. It is the largest division of the autonomic nervous system and is responsible for controlling several functions, including motility, secretion, blood flow, and immune surveillance. The ENS spans the entire length of the gastrointestinal tract and comprises over 100 million neurons in humans, a higher number than those found in the spinal cord. It is considered a second brain because it carries out specific functions that do not depend on the central nervous system (CNS). From an evolutionary perspective, however, the ENS could be considered the first brain, as it evolved early in development in multicellular organisms to allow for efficient food processing and digestion. Although such a discussion is out of the scope of this chapter, it is worth saying that nutrients imposed an evolutionary pressure on all living species because it is a critical vital need; in animals, it was necessary to adapt a precise autonomic control, leading to the rise of a primitive system that became the ENS in contemporary species. Undoubtedly, this

later fact reveals that the ENS is the oldest region of the nervous system; the Hydra, for example, a 500 million years cnidarian [1], is the oldest known group with sensory neurons in the oral region to regulate feeding, and with clustered ganglion neurons at the hypostome-tentacle junction to trigger contraction burst pulses of the epithelium to allow movement and ingestion [2]. The organization of these neurons is of great significance since it persisted throughout evolution and is observed from Hydras up to humans [3].

The complex circuitry of the ENS allows for the organization of both local and long-distance reflexes. These reflexes start with sensory neurons that detect changes in the gut's environment, such as the presence of food, and then relay this information to the motor neurons that control gut function. At a glance, the ENS is an essential component of the autonomic nervous system, and its complex and sophisticated functions make it a critical player in regulating gastrointestinal function. Furthermore, its ability to function independently of the CNS, coupled with its communication with the brain, highlights the importance of this intricate neural network in maintaining overall health and well-being.

The autonomic control of the intestine was first described in 1847 by Robert Remak, who settled the basis for further descriptions by Georg Meissner (1852) and Leopold Auerbach (1862), establishing the initial studies about the ENS [4]. Now, data show that neurons and glia of the ENS have their embryological origin at the neural crest, and before and after arriving at the gastrointestinal tract, they differentiate into glia and different types of neurons [5]. A detailed analysis of cellular and molecular processes of ENS development is in an excellent review [6]. In brief, this system originates from neural crest cells (NCC) derived from the ectoderm at the neural tube. NCC delaminate, and during this epithelial-mesenchymal transition, other levels or axes of the neural tube arise, named cranial, cardiac, vagal, truncal, and sacral. Subsequently, they proliferate and migrate until colonize specific sites. In such a process, they differentiate into various types of cells to structure the tissues to make up the gastrointestinal tract [6, 7]. These tissues can be diverse: connective tissue, endocrine cells, glia, and enteric neurons. The process of cell differentiation is necessary for a functional ENS; hence it is gradual because markers for neuronal types appear and may continue to a particular postnatal stage [8]. Studies are increasing to determine which molecules are involved in cell differentiation, such as the transcription factors and signaling pathways. Many of these molecules are for neuronal differentiation, as the SRY-like high-mobility group (HMG)-box (Sox) family, Sox6, Sox10, Mash1 (now called Ascl1), Hand 2; and those known for glial differentiation, as the GDNF, Neurturin, and the signaling pathway Ret – Rearranged during transfection – Notch [9].

#### **2. Neurons and glia**

The neurons form at least two ganglionic nerve plexuses running along the submucose layer of the gastrointestinal tract, the inner and the outer submucose plexus, and even a third plexus observed in humans [10]. Also, they are called the myenteric or Auerbach's plexus, the submucous or Meissner's plexus, and the mucous plexus [11]. The first is a plexus that runs from the esophagus to the rectum, while the others are located mainly in the intestines, with some functions independent of the influence of the central nervous system [12]. Thus, the ENS is a specialized system with significant self-supporting processes.

#### *The Brain-Like Enteric Nervous System DOI: http://dx.doi.org/10.5772/intechopen.112571*

There are different kinds of neurons in the ENS. In the 19th century, the Russian neuroscientist Alexandre S. Dogiel described three types of neurons at the ENS for the first time. The current terminology recognizes them as Type I (one axon and short dendrites), Type II (one axon and long dendrites), and Type III neurons (one axon and long tapering and branching dendrites found in the guinea pig); all of them also recognized as multipolar neurons [13]. Then, Type IV was described by Stach in the 1980s as a radiate multidendritic uniaxonal neuron with branches between the myenteric and submucous plexus [14], and Type V referring neurons with long dendrites observed in pigs and humans [15, 16].

The Dogiel Type II neurons in the guinea pig are primary afferent neurons [17] also found in humans in the stomach, small intestine, and colon [13]. The Dogiel Type I neurons show specific subdivisions depending on the shape; stubby neurons have short and stubby dendrites, spiny neurons with short and thorny dendrites, and hairy neurons with short and thin dendrites [13].

ENS glial cells outnumber neurons, as occurs in the central nervous system. They are flat and stellate-shaped, extended over neurons and neuronal processes, with a similar arrangement between vertebrate species [18]. The structure and molecular characteristics of enteric glia suggest that they are astrocytes-like cells [19] subdivided into Type I or protoplasmic glia, and Type II or fibrous glia, an organization determined by the microenvironment [20]. Also, there is a description of a Type III glia showing long and branched processes, and the Type IV referring to that glia on nerve fibers in the muscle layer; notwithstanding, a new proposal is to name them according to their location, cells in the myenteric and submucosal plexuses referred as EGMP and EGSMP, and cells in the mucosa and musculature as EGMucosa and EGIM [21].

Although the classification of ENS neurons and glia is remarkable, as far as innovative methods become available, identifying and characterizing the structure of these cells will still be refined. The task will improve our understanding of gastrointestinal functions, diseases, and treatments.

#### **3. Microbiome**

The gastrointestinal microbiome (GM), previously known as the intestinal flora, is the world of microorganisms that live in the gut to support digestion and significantly impact health. It includes different bacterial taxonomic groups and their interrelations [22]; for example, 400+ bacterial species live just in the human colon [23]. No matter whether microbiomes also exist in the skin, mouth, or reproductive tract, GM is the most well-studied in humans. Thus, it is known that starting at birth, the GM is acquired from the mother during delivery and breastfeeding [24]. GM plays a significant role in breaking down food, extracting nutrients, and producing vitamins necessary for human health; consequently, it is modified during life depending on the environment of the subject [25]. Considering the complex ecosystem that several microbes species establish in the gut, the role they play for health, their physiology as a group that impacts health if modified, and the many functions that even affect behavior, the GM is considered an organ by itself [22, 26]. Such a statement is further consolidated due to the relationship between GM and ENS.

The afferent starting point is at the enteroendocrine cells within the gut. They are specialized cells that respond to ingested substances. Although not entirely known, they release hormones such as cholecystokinin to activate nerve pathways to the central nervous system [27]. Also, neurotransmitters such as the gamma-aminobutyric

acid (GABA) play a specific role in activating the afferent pathways [28]. Whatever the milieu chemicals, the activation of enteroendocrine cells, in turn, activates the two main types of ENS afferent neurons, the extrinsic and intrinsic afferents, which differ depending on the location of their cell bodies, outside (extrinsic) or inside (intrinsic) the ENS [29]. Notwithstanding, the exact mechanisms by which the GM activates the ENS are yet not completely understood, but it represents the first interface between intestinal content and ENS to activate the afferent pathways of the brain–gut axis [30].

#### **4. Brain–gut axis**

Gastrointestinal afferent neurons are Dogiel Type II cells representing about 20% of ENS neurons. The intrinsic complex is formed by the intrinsic primary afferent neurons (IPAN), interneurons, and motor neurons, which organize local circuits within the ENS to trigger CNS-independent reflexes that regulate several aspects of gut function [31]. The extrinsic neurons have cell bodies in the dorsal root ganglia and the complex jugular-nodose ganglia at the jugular foramen. Extrinsic fibers from the stomach and upper intestine run from the gut to the CNS via the vagus and splanchnic nerves, and those from the distal intestine run via pelvic nerves [32].

The jugular ganglion is the smallest afferent cluster of sensory neurons of the vagus nerve [33] and also has neurons with similar properties as small dorsal root ganglion neurons, suggesting a nociceptive role [34]. The afferents project to the brain stem, specifically to the nucleus of the solitary tract (NTS), area postrema, and the upper cervical dorsal horn [35]. The nodose ganglion and its neurons are organized in a viscerotopic position, i.e., located inside the ganglion, depending on the origin of the afferent information. Then, they project central fibers through the solitary tract that synapse on neurons of the NTS located in the medulla [36]. NTS is a complex nucleus with projections to different cortex areas and brain nuclei, such as the insular cortex, frontal cortex, or thalamus [37]. It is a region for inputs from several regions, such as the insular cortex, paraventricular nucleus, hypothalamus, and amygdala [38].

The cell bodies of splanchnic afferents neurons are in the dorsal roots ganglia of the thoracolumbar spinal cord. Such neurons activate ascendent fibers in the spinothalamic, spinoreticular, and dorsal column pathways that carry information about noxious stimuli to different parts of the CNS, where it is interpreted as pain or discomfort [39]. However, the information from splanchnic afferents has a less graded sensation in response to distention, implying more intense or unpleasant sensations than vagal and pelvic afferents [40].

Pelvic afferent neurons are the third input pathway critical in sending information from the gastrointestinal tract to the CNS. These neurons are subdivided into two types based on their firing pattern: tonic and phasic. Tonic afferents become active by colonic distention and mainly consist of unmyelinated C fibers, while phasic afferents discharge at the onset and cessation of distention and include myelinated A-delta fibers [41]. They enter the spinal cord through the lumbosacral dorsal root ganglia and activate different ascending tracts [42].

Activation of afferent pathways triggers the different reflexes of the gastrointestinal system. Intrinsic activity is represented by ascending and descending reflexes to increase the luminal content and initiate peristalsis [29]. Ascending reflexes are excitatory pathways that induce the peristaltic contraction of circular muscles, and

*The Brain-Like Enteric Nervous System DOI: http://dx.doi.org/10.5772/intechopen.112571*

descending reflexes were described as inhibitory [43]. However, specific inhibitory and excitatory neurons exist in ascending and descending pathways [44]. These reflexes initiate following the enteroendocrine cell's activation of the IPANs, then activate interneuron and motor neurons to produce the appropriate response [45]. IPANs are located in submucosal or myenteric areas, and they respectively trigger peristaltic and secretory reflexes or stretch contraction reflexes via cholinergic pathways [46].

The extrinsic activity allows reflexes to perform tasks involving neurons in the CNS. Neurons at the dorsal motor nucleus of the vagus and at the NTS activate efferent pathways via the vagal outflow to the ENS [37]. Such pathways allow a fine modulation of gastrointestinal functions, mainly in the upper gastrointestinal tract, although the vago-vagal reflexes also include esophagogastric, gastrograstric, and duodenogastric reflexes that still need more studies [47]. It is noteworthy that vagal reflexes are not a fixed response, as observed in spinal reflexes. Instead, they are modulated, and the response depends on the demand of the gastrointestinal tract [48].

#### **5. Immune system**

A healthy gastrointestinal system depends on the immune system, notwithstanding it also depends on the collaborative work that immunity maintains with the microbiota and the ENS. The gastrointestinal tract is the region with more concentration of immune cells, mainly macrophages. The microbiota stimulates both macrophages and ENS neurons to synthesize and release, respectively, the bone morphogenic protein 2 (BMP2) and the colony stimulator factor 1 (CSF1). ENS neurons have receptors for BMP2 and macrophages for CSF1, representing the complex crosstalk signal circuit that exists to control gut function [49, 50].

Macrophages represent a diverse group of guard cells for the custody of the surrounding environment aimed to prevent infections [51]. Those in the smooth muscle of the gastrointestinal tract are in close contact with ENS neurons and regulate synaptic functions that include control of neuropeptides and neurotransmitters, but also receive activation from the ENS for the neuroimmune responses [52]. The comparison between lamina propia macrophages located in the epithelium close to the lumen, and those muscularis macrophages, show that they have particular responses to support the specialized interaction of the ENS and the immune system [53]. In the event of a response, such as inflammation, both macrophages and the ENS become active to restore homeostasis. Such activation is observed in local circuits but also in central ones as the inflammatory reflex, in which central neurons in the NTS and motor neurons of the dorsal nucleus of the vagus nerve become active [54].

#### **6. Diseases**

Several disorders are linked to dysfunctions of the ENS. For example, the so-called enteric neuropathies arise from the loss, degeneration, or functional impairment of enteric neurons, which may be congenital disabilities during development induced by infectious agents or conditions such as diabetes and neurodegenerative diseases [55]. Furthermore, specific dysfunctions or damage to the submucosal plexus are linked to gastrointestinal disorders and other disorders.

#### **6.1 Irritable bowel syndrome (IBS)**

According to the Rome IV criteria, the symptoms of IBS are frequent abdominal pain associated with bloating or the rhythm of evacuations, such as constipation, diarrhea, or both. Also, IBS is subdivided according to the defecation pattern, those with diarrhea IBS-D, constipation IBS-C, a mixed subtype IBS-M, and even those not yet subtyped, known as IBS-U [56]. Diagnosis includes the frequency criterion, i.e., if the abdominal pain occurs once a week, for at least 3 months, and the onset of symptoms with a minimum of six months before diagnosis [57]. IBS is recognized by altered gastrointestinal motility, characterized by accelerated GI transit in response to enteric ganglionitis in severe cases, carbohydrate malabsorption, bacterial overpopulation [58], visceral hypersensitivity, mucosal permeability, and altered microbiota [59].

The etiology of IBS has not yet been fully clarified, but there is sufficient evidence to link the immune system interaction with the ENS in the syndrome's pathophysiology. Notably, inflammation in IBS is marked and considered a significant feature in diagnosis, including in patients with postinfectious IBS [60]. The immune mast cells in the submucosal plexus trigger multiple inflammatory responses and generate a neuroimmune response when interacting with enteric neurons. The signaling of mast cells to enteric neurons is via neurotransmitters and neurohormones such as histamine and tryptase, which exert an excitatory function on the submucosal plexus, and an increased density of such cells is correlated with visceral hypersensitivity [61]. Genes are also involved, some supporting neuronal functions associated with IBS [56].

#### **6.2 Hirschsprung disease (HSCR)**

HSCR is a primary enteric neuropathy and one of the most frequent gastrointestinal motility disorders, showing the absence of enteric ganglia mainly in the colon. The congenital absence of ganglia neurons at the submucosal and myenteric plexuses occurs following a failure in the migration process of cells from the enteric neural crest to the hindgut; thus, this disease is known as a neurochristopathy but also is known as congenital megacolon or intestinal aganglionosis [62, 63]. The absence of ganglia produces a reduced or no peristalsis at all, causing intestinal occlusion because of the cessation of the expelling of fecal material. HSCR has an incidence of 1/5000 newborns and is more prevalent in males, in a 4:1 ratio [62].

#### **6.3 Achalasia**

Achalasia is a rare disorder affecting the motility of the esophageal region, characterized by the loss of enteric neurons and inhibitory postganglionic neurons that produces the absence of peristalsis of the tubular esophagus and impaired relaxation of the lower esophageal sphincter, involved in the swallow reflex. Symptoms include dysphagia, heartburn, regurgitation, chest pain, and weight loss. There is no total clarity of the etiology, but evidence exists that it is associated with autoimmune processes to still unknown antigens [64]. Furthermore, similar manifestations are found in some cases, as those related to the Chagas disease [65, 66].

#### **6.4 Chronic constipation**

Constipation by itself is not a disease, but it is considered a widespread gastrointestinal disorder that turns out to be the primary symptom to diagnose a disease. During constipation, defecation is difficult, accompanied by pain and stiffness (Forootan 2018), which is more frequent in women [67]. It is classified into two types, primary and secondary. Primary refers to dysregulation of neuromuscular activity within the colon and rectum, sometimes called functional constipation, that includes irritable bowel syndrome, and slow-transit constipation, caused by dysfunction of the smooth muscle activity in the colonic region. The secondary is nonspecific because constipation can respond to multiple factors such as metabolic problems, intake of medications, diet, neurological disorders, or colon diseases [68].

#### **6.5 Autism spectrum disorder (ASD)**

ASD is a neurodevelopmental disorder characterized by two domains, social and communicative difficulties, and restricted and repetitive behaviors, that appear early in childhood. In addition to the whole manifestations, ASD is commonly accompanied by many comorbid conditions that include a significant prevalence of gastrointestinal alterations such as chronic gastrointestinal dysfunction (diarrhea, constipation, reflux, etc.), or food intolerance [69, 70]. Also, ASD children show physiological alterations such as increased intestinal permeability, microbiota modifications, and intestinal infection [71]. Furthermore, the upper and lower gastrointestinal tract can show mild to moderate inflammation [72].

More than 90% of ASD children have feeding problems with detrimental effects; one of the causes is the modification of the microbiome, suggesting that ASD behaviors could benefit from interventions to restore microbial balance [73]. Such modification, known as gut dysbiosis, has been investigated by studying the bacterial genus *Clostridium*, which contributes essential species to the human microbiome [74]. Data suggest an association of ASD with an increase in *Clostridium* and a decrease in other microbiome species [75]. Notwithstanding, dysbiosis of other species is also correlated to autism [76]. Beyond the microbiome, several changes occur in enteric neurons and enteric glia [77] that affect the appropriate communication to central structures [78], making the microbiota–gut–brain axis a pivotal center to study the underlying basis of autism.

#### **6.6 Alzheimer's disease (AD)**

AD is a neurodegenerative condition showing a progressive deterioration of higher brain functions, mainly memory, and is considered one of the most common dementias [79]. The striking tissue features are the presence of extracellular accumulations of the amyloid beta (Aβ) peptide, or amyloid plaques, and neurofibrillary tangles [80]. Unfortunately, the evidence to explain the correlation between ENS and AD remains scarce. However, AD patients suffer from gastrointestinal alterations [81], that Aβ is also accumulated in enteric neurons producing a number reduction and suggesting that ENS dysfunction is ligated to AD [82] and that gut dysbiosis could also be correlated to AD [83].

#### **6.7 Parkinson's disease (PD)**

PD is a neurodegenerative disease with a progressive reduction in the number of dopaminergic neurons in the substantia nigra pars compacta (SN), associated with abnormal cytoplasmic deposits mainly of alpha-synuclein, known as Lewy bodies [84, 85]. It is usually diagnosed by the motor characteristics of the patient, such as

progressive tremors, jaw rigidity, and bradykinesia [86]. Data indicate that Lewy bodies are also found in enteric neurons, correlated with gastrointestinal motility and constipation [87].

#### **6.8 Chagasic megacolon**

Chagas disease is caused after an infection by *Trypanosoma cruzi*. It is an endemic disease in South America, Central America, and Mexico. The acute symptoms often go unrecognized, but patients can develop many physiological alterations, including motor dysfunction of the gastrointestinal tract [88]. The mechanism of enteric neuronal lesions in the intestinal plexuses generates aperistalsis and megasyndromes. In the case of megacolon, motility problems are associated with colon enlargement and constipation, showing the widening of the luminal region and muscle hypertrophy. Such lesions occur because the infection causes immune reactions that progressively become cytotoxic, producing oxidative stress and a reduction in the number of neurons [89].

#### **7. Conclusions**

The ENS is a sophisticated nervous system by itself, with an elaborated organization immersed in the whole gastrointestinal tract, responsible for regulating gut physiology. Its million neurons include intrinsic and extrinsic neural pathways with a significant function independent from the central nervous system. This attribute insinuates that the ENS should be considered as a second brain. To sustain all functions around food processing, the complex network of neurons and glial cells and the relationships with the microbiome organize an exceptional bidirectional communication with the CNS and an intrinsic communication with its own afferents, interneurons, and motor neurons. These pathways are essential for maintaining a healthy gut and general homeostasis. Dysregulation of such networks produces a wide range of diseases. Thus, research on the ENS and its interaction with the CNS must grow to give new insights into the function and pathophysiology of the gastrointestinal tract to develop new and better therapeutic approaches.

#### **Acknowledgements**

This chapter was funded by Conacyt Fellowship 1036810 to FMJCS and by funds from the Cuerpos Académicos Neuroscience (UV-CA-28), and Neurochemistry (UV-CA-304) at the Brain Research Institute.

#### **Conflict of interest**

The authors declare no conflict of interest.

*The Brain-Like Enteric Nervous System DOI: http://dx.doi.org/10.5772/intechopen.112571*

#### **Author details**

Flower M.J. Caycho Salazar1,2, Deissy Herrera-Covarrubias2 , Genaro A. Coria-Ávila2 , Luis I. García-Hernández2 , María Rebeca Toledo-Cárdenas2 , María Elena Hernández-Aguilar2 and Jorge Manzo2 \*

1 PhD Program in Brain Research, University of Veracruzana, Xalapa, Veracruz, Mexico

2 Brain Research Institute, University of Veracruzana, Xalapa, Veracruz, Mexico

\*Address all correspondence to: jmanzo@uv.mx

© 2023 The Author(s). Licensee IntechOpen. 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.

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### Section 4
