Serotonin in the Nervous System: Few Neurons Regulating Many Functions

Abstract

Serotonin is synthesized from tryptophan in small groups of neurons within the central nervous system. These neurons, however, branch profusely and innervate all the nervous system, where, by releasing serotonin in different manners, they regulate a myriad of functions, including many behaviors. This chapter reviews the main functions of serotonin in the nervous system of invertebrates and vertebrates, showing that many of these have been conserved throughout evolution. It also summarizes the current knowledge about the mechanisms that control and regulate serotonin secretion from different compartments of the same neurons, evidencing their differences, which enable small numbers of neurons to display a wide variety of functions, including the regulation of our mood states.

1. Introduction

From the mid-nineteenth century, a substance in the blood serum was known to cause contraction of smooth muscle, regulating the “tone” of blood vessels. When this substance was isolated, it was called serotonin, for its first known function. Similarly, a substance that causes smooth muscle contraction in the digestive tract, secreted by enterochromaffin cells, was called “enteramine” [1, 2]. Both substances were later demonstrated to be the same molecule: 5-hydroxytryptamine (5-HT).

Serotonin, or 5-hydroxytryptamine is a monoamine that acts as a chemical messenger, both in and out of the nervous system of invertebrates and vertebrates. It is one of the most ancient molecules regulating cellular functions, since it is found from protozoans [3]. Serotonin regulates a wide variety of physiological functions, from those already mentioned on smooth muscle contraction to very complex ones, such as attention or social behavior. Serotonin is secreted by exocytosis from the cells that synthesize it and can act as a neurotransmitter, neuromodulator, or hormone.

Most of the serotonin in mammals is found in cells outside of the brain, such as platelets, mastocytes, or enterochromaffin cells. However, serotonin cannot go through the blood-brain barrier, and thus, cerebral serotonin must be synthesized in the brain. Within the nervous system, serotonin has multiple functions, acting as a neurotransmitter at synapses or as a neuromodulator both at synapses and at extrasynaptic sites. Its functions are notably well conserved throughout the phylogenetical scale, as are the basic characteristics of serotonergic systems, which are characterized by having small numbers of neurons that, nevertheless, act at multiple levels in the nervous system, regulating many different functions.

2. Metabolism and effects of serotonin in the nervous system

2.1 Synthesis and release

Serotonin is synthesized in the cytoplasm of neurons, from the amino acid L-tryptophan, which is captured by cells through a process of facilitated transport. The main source of tryptophan are the proteins obtained from the diet. The enzyme tryptophan hydroxylase transfers an oxygen atom to the fifth position of the ring, forming 5-hydroxytriptophan. For this, the enzyme requires molecular oxygen (O₂), as well as reduced pteridine as a cofactor. This reaction is the limiting step in serotonin synthesis. Since in the brain the concentration of tryptophan normally does not saturate the enzyme, serotonin synthesis is increased by increases in the ingest of this amino acid. The level of O₂ in the tissues also regulates the synthesis of serotonin. 5-hydroxytriptophan is immediately decarboxylated by the enzyme aromatic amino acid decarboxylase, to form 5-hydroxytryptamine or serotonin. In addition to the effects of serotonin, which will be discussed below, this molecule serves as a precursor for the hormone melatonin in the pineal gland and other tissues. Serotonin synthesis can be increased in situations that require its continuous release. For example, electrical stimulation of the serotonergic neurons increases serotonin synthesis in a frequency-dependent manner. This can occur without increasing the synthesis of tryptophan hydroxylase, probably by changing the kinetic properties of the enzyme by phosphorylation through calcium-dependent mechanisms [4].

Serotonin release occurs through exocytosis, since it is a charged molecule that cannot go through the plasma membrane. In addition, it is necessary to store serotonin in vesicles to protect it from degradation (see below). The rate of serotonin release depends on the firing frequency of the neurons that synthesize it. The mechanisms that regulate serotonin release will be described further below in this chapter.

2.2 Reuptake and degradation

The activity of serotonin is terminated principally by its reuptake in serotonergic neurons and in glial cells, through a membrane transporter. Reuptake is an active process, which depends on the temperature and of extracellular sodium and chloride. Energy is required to maintain the sodium gradient between the intra and extracellular media, which is indispensable for serotonin transport. Serotonin reuptake by serotonin transporter (SERT) may produce changes in the membrane potential, since it moves positively charged sodium ions into the cytoplasm, thus slightly depolarizing the neuron [5]. Many of the pharmacological treatments for depression are selective serotonin reuptake inhibitors (SSRIs).

Serotonin is degraded by the enzyme monoamine oxidase (MAO) that converts 5-HT to 5-hydroxindoleacetaldehyde, which in turn can be oxidized by a NAD⁺-dependent aldehyde dehydrogenase to form 5-hydroxindolacetic acid or reduced by a NADH-dependent aldehyde reductase to produce 5-hydroxy tryptophol, depending on the relative concentrations of NAD⁺ and NADH in the tissue. In the brain, the main metabolite of serotonin is 5-hydroxindolacetic acid.

2.3 Cellular effects of serotonin on the nervous system

Serotonin acts on seven groups of receptors, named 5-HT₁–5-HT₇, distinguished by their pharmacology and the intracellular pathways and responses they activate in the target cells. Each receptor type is differentially distributed in different areas of the nervous system and other tissues. All serotonin receptors, except the 5-HT₃ (which is an ionic channel), belong to the family of G-protein-coupled receptors, similar to rhodopsin, and activate intracellular signaling cascades. The effects that serotonin exerts on target neurons depend on the type of receptor activated. The G_i/G_o-coupled 5-HT₁ receptors generally mediate inhibitory effects on neuronal firing through an opening of inwardly rectifying potassium channels or a closing of voltage-gated calcium channels. The G_q/11-coupled 5-HT₂ family of receptors generally mediates slow excitatory effects through a decrease in the membrane permeability for potassium or an increase in the permeability for cations. The 5-HT₃ receptors, which are ligand-gated cation channels with structural homology to nicotinic receptors for acetylcholine, mediate fast excitatory effects of 5-HT. In invertebrates there seem to be ionotropic 5-HT receptors [6, 7, 8], which are channels for chloride (a negatively charged ion), unlike the vertebrate 5-HT₃ receptor, which allows the flux of positively charged ions.

The characteristics, signaling pathways, and distribution of each of the receptors for serotonin have been extensively reviewed elsewhere [9, 10, 11, 12, 13] and will not be further discussed here.

In this chapter, we provide an overview of the morphology and function of serotonergic neural systems, and show how, by secreting this substance in several different modes, a few neurons regulate a wide variety of functions in the nervous system.

3. The diverse functions of serotonin in the nervous system

The functions of serotonin have been well conserved along evolution, from invertebrates to humans. In addition to developmental, cardiovascular, gastrointestinal, and endocrine functions, serotonin has multiple functions in the nervous system, including the regulation of sensory perception and motor patterns (such as those that are activated during ingestion, locomotion, or respiration) [14, 15, 16], circadian rhythms, including sleep-wake cycles [17, 18], appetite, feeding, sexual behavior, mood, attention, cognition, and memory [19]. Importantly, serotonin regulates social behavior in invertebrates and vertebrates, and produces changes that span from aggression, associated with social dominance [20], to submission and depression. In humans, the alteration in serotonin metabolism is related to neuropsychiatric disorders, such as depression [21], schizophrenia [22, 23], and obsessive-compulsive [23, 24] or feeding disorders [25].

3.1 Serotonergic functions in invertebrates

The relatively small and simple nervous systems of invertebrates, with small numbers of neurons of big size and stereotyped localization, have been powerful model systems to study neural circuits in detail, from intracellular pathways to behavior. Serotonin regulates a wide variety of physiological functions in invertebrates, including sensory processing, locomotion, feeding, and social behavior. The accessibility of invertebrate nervous systems to perform intracellular recordings of identified neurons has enabled us to unveil complete circuits regulating these behaviors and the multiple ways in which serotonin modulates them.

3.1.1 Regulation of swimming, feeding, and learning in leeches

Serotonin released from Retzius neurons has a neuromodulatory effect on the block of conduction of action potentials that occurs at the branching points of the axons of mechanosensory neurons [26] that respond to touch (T), pressure (P), or noxious (N) stimuli in the skin of the leech [27]. In this way, serotonin modulates sensory perception of these animals.

Serotonin also regulates a wide variety of physiological functions in the leech, many of which are related to feeding behavior. Hungry leeches are usually located in shallow waters, and respond to water movements, possibly indicating the presence of prey, by swimming toward the source of the waves. Serotonergic neurons play a fundamental role in swim initiation. Swimming in these organisms is coordinated by a series of interneurons that, when activated, display an oscillatory activity that stimulates and inhibits, in a rhythmic and alternative manner, the motor neurons that excite and inhibit longitudinal ventral and dorsal muscles. Activation of this central pattern occurs progressively through the ganglion chain, giving rise to undulations of the body from head to tail [28]. The rhythmic activity of these neurons can be recorded in the segmental ganglia or the connective nerve and is maintained in isolated ganglion chains, which has facilitated the study of these circuits. Mechanosensory neurons excite several of the serotonergic neurons, including Retzius neurons, cells 21 and 61 (see Section 4.1 on the Structure of the serotonergic system in invertebrates below), through a polysynaptic pathway (i.e., with one or more interneurons connected in series to each other in between) [29]. Stimulation of cells 21 and/or 61 triggers the initiation of swimming episodes in ganglion chains [29], since these cells, in turn, excite interneurons that generate the central swimming pattern, receiving also feedback from them [30]. Stimulation of Retzius neurons or application of serotonin in the bath also produces swimming episodes [31], because serotonin modulates the activity of some of the oscillating interneurons that may initiate the rhythmic activity of the swimming circuit. For example, through modulating several sodium channels, serotonin changes the excitability (i.e., the likeliness to fire action potentials) of one of the interneurons that initiate the swimming cycle (called cell 204), decreasing the threshold for this neuron to trigger the activity of the circuit [32, 33]. Serotonin also modulates the activity of motor neurons that produce swimming, promoting their rhythmic alternated activation [34, 35]. Thus, serotonin seems to be the determinant factor for the activation of swimming in the leech. In fact, the probability of swimming in a leech is correlated with the concentration of serotonin in its blood [31] and in the CNS [36].

Locomotion is also regulated by serotonin in other animals. In larvae of the fruit fly Drosophila, increasing the level of 5-HT is able to decrease body wall contractions used for locomotion. In the larval stages, 5-HT is involved in turning behavior [37], whereas in adult flies, acute activation of 5-HT neurons disrupts normal locomotor activity [38].

Once a leech finds possible prey, it starts an exploration phase. During swimming and this exploratory phase, the activity of serotonergic neurons increases [39]. When the lips feel a warm surface, some of the serotonergic neurons are activated, firing in bursts at high frequencies, and serotonin activates ingestion, directly stimulating salivary glands to secrete saliva, the claws to bite, and the pharynx to generate peristaltic movements, which continue throughout the ingestion process. During ingestion, serotonin relaxes the body wall muscles by producing a hyperpolarization through the activation of chloride currents during the compound action potential in these muscles [40], and by inhibiting the production of excitatory synaptic potentials in the muscles through its action on central neurons [41]. In addition, Retzius neurons directly stimulate mucus secretion at the mucous glands in the skin [42], which is also a characteristic of the ingestion phase in the leech.

The distention of the body wall after feeding hyperpolarizes the serotonergic neurons and terminates ingestion, producing a satiety state. Satiated leeches have a lower amount of serotonin in the CNS and in blood than hungry leeches [43] and stay in deeper waters at the bottom of the ponds, swimming much less. Administration or elimination of serotonin cause alterations in feeding behavior, such as hyperphagia or anorexia, respectively [44].

Beyond feeding and swimming, serotonin modulates learning processes in the leech, such as facilitation, sensitization, and dishabituation, in the shortening reflex that occurs when touching the head of the animal [45, 46]. This modulation is exerted upon the excitability of a series of neurons that are essential for learning in these organisms, called S cells [47]. The activity of the S cell network all along the nervous system is modulated by serotonin [48].

3.1.2 Regulation of swimming and sensory learning in mollusks

Serotonin regulates swimming also in mollusks. In Clione limacine, the serotonergic system modulates the expression of different behaviors by producing different swim velocities [49]. This is achieved by independently regulating the contractility of the wings, the cycling frequency of the central pattern generator, recruiting motor neurons for swimming, and by stimulating the heart-exciting neuron, while inhibiting a circuit that competes with the swimming circuit.

In Aplysia californica, serotonin has been shown to regulate learning. This mollusk has a reflex that withdraws the gill and the siphon in response to a tactile stimulus in the tail. The strength and duration of this reflex can be enhanced by a noxious electrical stimulation applied to the tail, the head, or the body wall. This sensitization phenomenon, which can last for hours, is caused by (1) an increase in the excitability of sensory neurons and (2) a facilitation of transmitter release from these neurons to the motor neurons that produce the contraction of the mantle muscles [50]. Both of these mechanisms are activated by serotonin. Noxious stimulation of the tail activates serotonin release in the CNS, which can be detected by electrochemical methods, and lasts 30–40 seconds, peaking in the neuropil (i.e., the net of neuronal processes) surrounding the synapses between sensory and motor neurons in the tail [51]. Serotonin levels also increase in the hemolymph [52]. Serotonin acts on sensory neurons to activate the protein kinase A (PKA) and protein kinase C (PKC) intracellular pathways, which phosphorylate S-type potassium channels, to reduce the time they remain open [53]. In this way, the membrane excitability increases and the action potential is broadened [54, 55], allowing more calcium influx to the nerve terminals and therefore increasing transmitter release. PKA and PKC also increase transmitter release by enhancing the mobilization of synaptic vesicles to the active zone [56, 57, 58, 59, 60, 61], thus increasing the transmitter available for release. By increasing transmitter release, a stronger contraction is produced in the muscle that withdraws the siphon. The modulation of defensive reflex circuits by 5-HT also involves a variety of different cell types that respond differently to 5-HT. Serotonin blocks the synaptic enhancement in a polysynaptic pathway that inhibits the siphon-withdrawal reflex, thereby reducing potential for inhibition within the circuit. Serotonin also acts directly on motor neurons facilitating responses to glutamate mediated by AMPA-type receptors, thus possibly enhancing the response of motor neurons to sensory neuron activation (Reviewed by [62]).

3.1.3 Regulation of aggression associated with social behavior in arthropods

The modulation that serotonin exerts on behaviors associated with social hierarchy has been studied in detail in decapod crustaceans, such as lobsters and crayfish. When two individuals meet at the same space, at the beginning both of them show aggressive behavior, consisting of standing high on the tips of their walking legs, while bending them, and showing their claws up and forward. Both animals have aggressive encounters, which will determine which one will be dominant. The dominant individual then keeps a high posture with the limbs flexed, and walks throughout the space, while the other one tends to extend its limbs, to place the body low, near the bottom, and remains in the corners (when confined in a tank), or leaves the area (in the wild), avoiding the dominant individual. Serotonin, together with octopamine, is responsible for these opposite postures and behaviors. Serotonin injection to lobsters produces the dominant posture with flexed limbs and abdomen, while octopamine produces the submission behavior with the extension of the limbs [20, 63]. Serotonin activates a motor program for the flexion of the limbs, by increasing the firing frequency of the motor neurons that excite the flexor muscles and those that inhibit the extensor muscles while decreasing the firing frequency of the motor neurons that inhibit the flexor muscles and of those that excite the extensor muscles. This occurs by modifying the frequency of the synaptic inputs to the motor neurons, and not by acting directly upon them [64]. Octopamine produces the opposite effects [64]. Serotonin also acts directly on the muscles, by producing prolonged contractions, and on the neuromuscular junctions (i.e., the synapses between motor neurons and muscles), by increasing neurotransmitter release and therefore the strength of muscle contractions [65, 66, 67]. These effects are produced by serotonin released from two neurosecretory organs to the hemolymph [68]: (see Section 4.1 on the Structure of the serotonergic system in invertebrates) where it circulates as a neurohormone and regulates different targets, including the exoskeleton muscles and the heart.

The role of serotonin in aggressive behavior associated with social dominance has also been studied in the fruit fly Drosophila melanogaster. The multiple genetic manipulation methods developed in this organism have been powerful tools for studying a variety of functions. Recent tools to manipulate the biosynthesis and release of serotonin, the activity of serotonergic neurons, or the expression of 5-HT receptors, have revealed the role of serotonin in a variety of behaviors in these animals. As in lobsters, Drosophila males establish hierarchical relationships based on the results of aggressive encounters. These fights have several stages in which the animals show a number of characterized behaviors, including tapping on the opponent’s leg, lunging, holding, boxing, or tussling. The result of these encounters defines a dominant individual, who takes a food source, while the defeated individual retreats from the food surface [69, 70]. Increasing the level of serotonin in the fly’s nervous system, either by increasing 5-hydroxytryptophan in the diet or by constitutive over-expression of tryptophan hydroxylase in serotonergic neurons, increases aggressive behavior [71]. Similarly, when serotonergic neurons are selectively activated by expressing cationic channels that can be activated by temperature in these cells, aggression is expressed faster, fights become intensified and continue even after social dominance has been established. By contrast, the reduction of serotonergic neurotransmission by the selective expression of a temperature-sensitive form of dynamin, a protein involved in endocytosis, in serotonergic neurons, results in a reduction in male mid-intensity aggression and the development of fewer dominance relationships [72].

3.1.4 Regulation of circadian rhythms in invertebrates

The relationship between circadian rhythm and sleep-wake has been also studied in invertebrates. In Drosophila, modifying the serotonin system can break the link between sleep and the circadian rhythm. In fact, an increase in serotonin reinforces sleep, presumably via the 5-HT_1A receptor, while a decrease in serotonin by the deletion of tryptophan hydroxylase suppresses sleep at night [73]. In crustaceans, the regulation of circadian rhythms is operated in both the central brain and the eyestalk X-organ-sinus gland system, where serotonin and melatonin have been largely found [74]. In crayfish, serotonin concentration in tissue displays circadian fluctuations, linking the serotonin system with the pacemaker system involved in circadian rhythms [75].

3.2 Serotonergic functions in vertebrates

Serotonin regulates a wide variety of functions and behaviors also in vertebrates, many of which are conserved along the phylogenetic scale. However, in contrast to invertebrates, where the pathways and mechanisms through which serotonin acts are very well known, the precise pathways and mechanisms of serotonergic effects in vertebrates remain elusive, due to the complexity of the vertebrate nervous system.

3.2.1 Regulation of locomotion

As in invertebrates, central 5-HT is also a powerful neuromodulator of locomotor activities in vertebrates, including lamprey [76, 77, 78, 79], zebrafish [80, 81, 82, 83], Xenopus [84, 85], and rodents [86, 87, 88]. Serotonergic neurons terminate on specific target neurons with different types of serotonergic receptors in the spinal cord. Serotonergic neurons can initiate locomotor activity through actions on motoneurons and neurons of the locomotor central pattern generator (CPG) [89, 90]. In fact, the stimulation of serotonergic neurons projecting to the spinal cord, or application of 5-HT receptor agonists on ex vivo spinal cord from newborn rats, initiates and sustains episodes of so-called fictive locomotion [91, 92]. In rats and mice, serotonergic agonists promote the recovery of locomotor movements after spinal cord injury [89].

3.2.2 Regulation of feeding

As in invertebrates, serotonin regulates feeding in vertebrates. However, while in invertebrates serotonin promotes appetitive states, in vertebrates serotonin suppresses appetite [93]. In addition to the peripheral effects on metabolism through stimulating insulin and inhibiting glucagon secretion (reviewed in a different chapter), serotonin has important actions in the regulation of food intake at the central nervous system (CNS) level. Food ingestion is regulated in the hypothalamus, by two types of neurons in the arcuate nucleus: one that releases agouti-related peptide (AgRP) and neuropeptide Y (NPY) and one that releases alpha-melanocyte stimulating hormone (α-MSH), also called POMC neurons, because they express pro-opiomelanocortin. Both of these innervate neurons in the paraventricular nucleus, which express melanocortin MC4 receptors and, when activated, inhibit food ingestion. α-MSH is an agonist of melanocortin receptors, while AgRP/NPY is an antagonist of these receptors. In addition, NYP neurons release GABA onto POMC neurons, inhibiting them. Thus, activation of POMC neurons results in a decrease in food intake, while activation of NPY/AgRP neurons results in an increase in food intake. Serotonin produces decreases food intake, by depolarizing POMC neurons and by inhibiting NYP neurons. Depolarization of POMC neurons occurs through the activation of 5-HT_2C receptors, by inhibiting both GIRK and M potassium channels, which participate in the maintenance of the resting potential [94], and activating TRPC channels, thus decreasing their input resistance [95]. Hyperpolarization of NPY/AgRP neurons by activation of 5-HT_1B receptors decreases the release of AgRP/NPY onto paraventricular nucleus neurons and also decreases inhibitory synaptic input onto POMC neurons [96]. In consequence, inhibition of serotonin synthesis and release increases food intake and body weight in rats [97, 98]. Moreover, mutant mice that do not express serotonergic 5-HT_2C receptors show increased food intake and increased body weight and adipose tissue than wild-type mice [99, 100]. On the other hand, the treatment with fluoxetine [101], a selective serotonin reuptake inhibitor (SSRI), or with d-Fenfluramine [96], which inhibits serotonin reuptake and stimulates its secretion, decreases food intake in wild-type rodents, but not in mice lacking 5-HT_2C receptors [102]. Thus, serotonin in vertebrates decreases appetite and feeding. This is the main difference between the functions of serotonin in vertebrates and those in invertebrates, where serotonin stimulates feeding. The hypophagic effects of serotonin in humans have been related to feeding disorders, such as anorexia and bulimia [25].

3.2.3 Serotonergic regulation of circadian rhythms and sleep

In mammals, 5-HT is implicated in sleep-wake states. Studies using electrophysiological, neurochemical, genetic, and neuropharmacological approaches have shown that serotonin promotes wakefulness and inhibits rapid eye moment (REM) sleep. Indeed, mutant mice that do not express 5-HT_1A receptors exhibit greater amounts of REM sleep than their wild-type counterparts. Recordings from serotoninergic neurons in unanesthetized animals have shown that activity is highest during periods of waking arousal, reduced in quiet waking, reduced further in slow-wave sleep, and absent during REM sleep [103]. However, under certain circumstances, this neurotransmitter contributes to the increase in sleep propensity [104]. Thus, serotonergic activity may be accompanied by waking or sleep depending on the brain area and receptor type involved in the response and also on the concomitant agonism/antagonism of other neurotransmitter systems [105].

Serotonin also participates in the control of the circadian rhythms. This participation is supported by the fact that there is a significant projection of serotonergic neurons form the raphe nuclei to the suprachiasmatic nucleus, which is considered the master clock regulating circadian rhythms [106]. Also, one of the metabolites of serotonin is melatonin, a molecule that is known to regulate the sleep-wake cycle. In doves, serotonin levels in serum seem to be positively correlated with the circadian activity rhythm [107]. In mammals, it is well established that serotonin modulates the sensitivity of the circadian rhythm to light through the modulation of a presynaptic 5-HT_1B receptor on the retino-hypothalamic tract; the activation of this receptor attenuates photic input to the central nervous system, thereby reducing the phase response to light [106].

3.2.4 Regulation of aggression associated with social behavior in mammals

Aggressive behavior and the establishment of social dominance are also modulated by serotonin in vertebrates. In mammals, including humans, serotonin inhibits aggressive behavior by blocking the secretion of vasopressin and other transmitters [108, 109]. Serotonergic and vasopressinergic innervation of the hypothalamus can be altered in hamsters that are exposed to aggression during adolescence, resulting in altered aggressive behavior in the adult [108]. The activity of serotonergic neurons in the rat CNS determines the transition between normal and escalated types of aggression [110]. Mutant mice that do not express 5-HT_1B receptors show increased aggression, as well as auto administration of cocaine and ethanol [111].

Several lines of evidence point to low levels of serotonin in the cerebrospinal fluid and in specific areas of the brain in individuals with increased aggressive behavior, both in rodents [112, 113, 114, 115] and in humans [116, 117]. In contrast, higher levels of serotonin are related to adaptive social behaviors [118, 119]. In primates, individuals with a low serotonergic activity in the CNS [120], or treated with 5-HT antagonists [121], express impulsive, aggressive, and social isolation behaviors, and invariably become subordinates, while individuals treated with 5-HT agonists or 5-HT reuptake inhibitors show more affiliation behaviors and less aggressivity, and become dominants [121].

3.2.5 Participation of serotonin in mood, mental health, and other behaviors

Studying the neurobiological bases of mood disorders faces a number of difficulties, because the brain is not an approachable tissue in humans, and research on this topic has relied upon post-mortem studies, or neuroimaging of living subjects, both of which have great limitations. There are a number of animal models too, with concomitant challenges for their interpretation in terms of human mental health. However, several lines of evidence support that serotonin plays an important role in mood states, and is implicated in mental health disorders, such as depression, anxiety, or schizophrenia, among others [122].

Patients with depression and epilepsy have a deficit in serotonergic transmission [123]. Human clinical studies have employed a range of serotonin indexes, including the cerebrospinal fluid level of serotonin metabolites and the prolactin response to serotonin agonists. A positive correlation was found between low levels of serotonin metabolites in the cerebrospinal fluid and serious or high-intent suicidal acts [124, 125].

Serotonin transporter (SERT) is the main target of SSRI antidepressants. However, although the therapeutic effects induced by SSRI are initially triggered by blocking SERT, they rely on consequences of chronic exposure [126], including desensitization of somatodendritic 5-HT autoreceptors. One of the events that seem to mediate antidepressant effects is hippocampal neurogenesis, which is negatively regulated by stress and positively regulated by antidepressant treatment [127].

Exposure to chronic unpredictable stress has been found to induce depressive-like symptoms or behaviors, including passive behavioral coping and anhedonia in animal models, along with some other affective, cognitive behavioral symptoms that are also present in humans. In models of chronic unpredictable stress, it has been shown that serotonergic activity and neurotransmission, as well as autoreceptor sensitivity are altered [127, 128]. Along the same line, the pathway from the medial raphe nucleus to the hippocampus attenuates stress through facilitating transmission in that area [129].

It has been proposed that serotonergic pathways in mammals regulate anxiety. Activation of the ascending pathway of the dorsal raphe nucleus (DRN) facilitates defensive learned behaviors. On the other hand, activation of the pathway from the DRN to the periventricular area inhibits innate “fight or flight” reactions. It is thought that the disfunction of these pathways is related to generalized anxiety disorder and panic [130]. The pathway from the medial raphe nucleus to the hippocampus, on the other hand, attenuates stress through facilitating transmission in that area [129]. Mice lacking 5-HT_1A receptors show increased fear in several behavioral tests, suggesting that this receptor modulates some neural circuits related to fear [131]. In fact, 5-HT_1A agonists are used, in addition to benzodiazepines (which increase GABAergic activity), to treat generalized anxiety disorders [132]. Inhibition of the serotonin transporter can also reduce anxiety symptoms in obsessive-compulsive disorder [24].

Sexual behavior is also modulated by serotonin, possibly through the regulation of dopamine secretion in the hypothalamus. Serotonin inhibits some aspects of sexual behavior in both male and female rats; it increases the latency to copulation and ejaculation [133], as well as the refractory period between ejaculation and the next copulation in males, and decreases female receptivity (reviewed by Weiger WA [109]).

In vertebrates, serotonin also modulates pain. Descending pathways from the raphe nuclei to the spinal cord are activated upon peripheral injury and produce an inhibitory effect on pain perception [134]. It is interesting to note that some patients with pain disorders also present mood symptoms.

Serotonergic neurotransmission is altered in schizophrenia [135], and the treatment of this disease includes drugs that inhibit 5-HT₂ as well as dopaminergic receptors. Finally, some serotonergic 5-HT_1D agonists are successfully used in the treatment of migraine [136].

4. The serotonergic system: few neurons releasing serotonin in multiple ways

A striking characteristic of serotonergic systems is that they, in general, have small numbers of neurons in relation to the total neurons in the nervous system. This is conserved throughout the phylogenetic scale, from invertebrates to mammals. In the leech, for example, there are seven serotonergic neurons, out of 400 total neurons in each segmental ganglion. In rodents, there are around 9000 serotonergic out of 10¹² total neurons. In the human brain, the number of serotonergic neurons represents only one out of every million neurons [137]. Yet, serotonergic projections in vertebrates and invertebrates branch profusely and have complex innervations to virtually all areas of the central nervous system, and serotonin regulates a wide variety of functions, from the modulation of inputs at sensory systems to the performance of complex behaviors.

4.1 Structure of the serotonergic system in invertebrates

The large and identifiable neurons of invertebrates, in which nervous systems have small numbers of neurons compared to those in vertebrates, provide a unique accessibility to study neural circuits at the cellular level, allowing to trace the actions of serotonin throughout circuits and to study the functions of serotonin from the cellular level to behavior.

In invertebrates, including annelids, mollusks, and crustacea, where the nervous system is formed by a ventral chain of ganglia, the serotonergic system is comprised of a few neurons in each neural ganglion. In leeches, there are seven serotonergic neurons in each segmental ganglion [138]: the pair of Retzius neurons, which are the biggest neurons in the ganglion and the ones that contain most of the serotonin in this system, two pairs of lateral neurons (one in the dorsal side of the ganglion, also called cells 21, and one on the ventral side, also called cells 61), and an unpaired neuron in the medial posterior package. The three first ganglia have an additional pair, near the pair of Retzius neurons. Because of their large size, Retzius neurons have been used to study the detailed characteristics of serotonin secretion. Retzius neurons send axons toward the periphery, through lateral roots in each ganglion, while the rest of serotonergic neurons have their dendritic arbor restricted within the central nervous system (CNS). All these neurons are coupled among them by chemical excitatory synapses as well as by electrical synapses that allow the flux of current in both directions. In addition, they receive common (apparently cholinergic) synaptic inputs. Thus, all serotonergic neurons display somewhat synchronous electrical activity in bursts and form a “compartmental serotonergic system,” embedded in motor networks controlling feeding, escape swim/turn, and locomotor functions [49, 93]. Serotonergic neurons in these animals establish some synaptic contacts with other neurons in these networks and receive reciprocal innervation from the networks they innervate as well as external afferents. In addition, as will be explained below, these neurons contain large groups of serotonin-containing dense-core vesicles in the soma and at extrasynaptic sites of the axons.

In the lobster, there is at least one pair of serotonergic neurons in each ganglion. Two of these pairs, located in the fifth thoracic ganglion (T5) and the first abdominal ganglion (A1) are responsible for the secretion of serotonin in the neuropile and to the periphery at neurosecretory organs. These neurons are stimulated by the command interneurons that activate the flexor motor pattern of the legs (see Section 3.1 on Serotonergic functions in invertebrates), and serotonin then acts as an amplifier of the activation signal from the motor pattern, producing the effects already mentioned on motor neurons, muscles, and neuromuscular junctions [20]. On the other hand, when the motor pattern for the extension of the legs is activated, these serotonergic neurons are inhibited.

In the mollusk Clione limacine, there are 27 pairs of serotonergic neurons, 75% of which have been identified. The serotonergic system in this organism is compartmentalized, so that each subsystem can act independently or in synchrony with the others to produce variability in the speed of locomotion, thus modulating the expression of different behaviors. A cluster of podal 5-TH neurons increases the contractility of the wings without affecting their beating frequency or the activity of motoneurons. Two clusters of cerebral 5-HT neurons produce responses that increase the cycling frequency of the central pattern generator, recruit motoneurons for swimming, activate the podal 5-HT neurons, and excite the heart-exciting neuron. On the other hand, a pair of cerebral 5-HT neurons exerts a weak excitatory input to the swimming circuit and strongly inhibits neurons in another circuit, which competes with the swimming circuit [49].

4.2 Structure of the serotonergic system in vertebrates

In 1964, Dahlstrom and Fuxe found, using the Falck-Hillarp technique of histofluorescence, that most of the somata of serotonergic neurons are grouped in nine clusters localized at the midline of the brain stem, which previously had been designated as the raphe nuclei, based on cell body structural characteristics and organization, and they named B₁ through B₉ [139], although some serotonergic neurons are out of these nuclei and not all the neurons in the raphe nuclei are serotonergic. Therefore, unlike invertebrates, where serotonergic neurons are distributed throughout the nervous system in the different ganglia, vertebrates concentrate the serotonin neuron somata in a few nuclei in the brainstem. However, despite the restricted localization of the somata to this area, brainstem serotonin neurons send ascending projections that branch profusely and terminate in a defined and organized manner in cortical, limbic, midbrain, and hindbrain regions, as well as descending projections to the spinal cord, and thus the axons of serotonergic neurons innervate virtually all areas of the CNS (Figure 1). Groups B1 to B5, which are small and are in the most caudal raphe nuclei, send projections within the brainstem and toward the spinal cord, where they modulate the activity of motoneurons as well as synaptic transmission in the pain perception pathway. Group B7 (the largest group of serotonergic cells), which together with group B6 constitutes the dorsal raphe nucleus, B8, which corresponds to the medial or central superior nucleus, and B9, which is not considered one of the raphe nuclei, innervates all the forebrain, including the cortex. The two main ascending serotonergic pathways emerging from the midbrain raphe nuclei to the forebrain, named the dorsal periventricular path and the ventral tegmental radiations, converge in the caudal hypothalamus, where they join the medial forebrain bundle. Together with the projections of the locus coeruleus, they form part of the ascending reticular activator system, which regulates attention, motor control, and sleep-wake cycles, among other functions. Ascending projections from the raphe nuclei to forebrain structures are organized in a topographical manner. The dorsal and median raphe nuclei project to forebrain regions; the median raphe projects heavily to hippocampus, septum, and hypothalamus, whereas the dorsal raphe innervates the striatum, and both nuclei send overlapping projections to the neocortex. Within the dorsal and median raphe, cells are organized in particular zones or groups that send axons to specific areas of the brain that are related in function. Thus, different sets of serotonergic neurons seem to be specialized for certain functions, instead of a nonspecific general innervation of the CNS [4]. In the vertebrate serotonergic system, there is reciprocal connectivity between each of the raphe nuclei and the networks it innervates [103]. All brain regions express multiple serotonin receptors, with each receptor subtype showing a specific distribution [140].

Figure 1.

Structure of the serotonergic system in the central nervous system of vertebrates. The somata of all serotonergic neurons are located within nuclei in the brainstem. These neurons extend projections to the spinal cord, cerebellum, hypothalamus, hippocampus, and throughout the cerebral cortex, thereby innervating virtually all areas of the central nervous system.

Some of the serotonergic terminals establish specialized synaptic contacts with target neurons and release serotonin upon electrical activity. However, in most of the areas in the CNS, there are at least some sites where serotonin is released without evidence of synaptic specializations [141, 142]. The axons of the serotonergic neurons from the median raphe are thick and have big spherical varicosities that form well-defined synapses in the hippocampus [143, 144]. On the other hand, axons arising from the dorsal raphe are very thin and have small spherical fusiform varicosities. In these fibers, it is difficult to demonstrate defined synaptic connections [145]. Serotonergic axons innervating the ventral horns of the spinal cord [146, 147] or the substantia nigra reticulata [148] establish mostly synaptic contacts with well-defined target neurons; however, in dorsal horns of the spinal cord [149] and in the nucleus accumbens [150], nearly 60% of the 5-HT terminals do not form synapses. Recurrent axon collaterals ending in the dorsal raphe contain both synaptic and non-synaptic endings [151]. The dendrites of serotonergic neurons in the dorsal raphe nucleus also contain serotonin in small clear and large dense-core vesicles, densely packed in clusters [151, 152, 153], some of which are localized at defined synapses, but some others are not associated with synaptic structures and seem to be part of an extrasynaptic release machinery, as will be shown below.

5. Regulation of the firing rate in serotonergic neurons

Serotonergic neurons are classically thought to display a regular tonic firing at low frequencies (0.1–3 Hz), with pace-maker-like regularity [17], which is thought to maintain a tone of basal serotonin concentration in the nervous system, whereas phasic firing in bursts of higher firing rates (up to 17 Hz) [154, 155, 156] is associated with specific behaviors [157]. Serotonergic neurons in the raphe nuclei receive glutamatergic and GABAergic [158], as well as noradrenergic [159, 160] synaptic inputs that presumably contribute to the regulation of their firing frequency. In addition, serotonergic neurons of vertebrates and invertebrates connect with each other. In vertebrates, serotonergic neurons are connected through inhibitory dendro-dendritic synapses within a nucleus and by inhibitory afferents from other nuclei [93]; in invertebrates, 5-HT neurons connect through electrical synapses and through chemical synapses that are mostly excitatory, although many of the effects of serotonin are mediated by inhibitory receptors. Serotonergic neurons have 5-HT autoreceptors that participate in the regulation of their electrical activity [4, 161, 162, 163, 164, 165, 166]. In rodents, 5-HT_1A autoreceptors are localized at the somatodendritic compartment of serotonergic neurons in the raphe nuclei, whereas 5-HT_1B autoreceptors are localized at axon terminals [167, 168]. Activation of these autoreceptors activates potassium channels, producing an outward current and a robust membrane hyperpolarization [169], which decreases the firing rate of serotonergic neurons [170, 171]. On the other hand, 5-HT₂ autoreceptors positively regulate the activity of raphe serotonergic neurons.

In Retzius neurons of the leech an autoregulation mechanism has also been found, specifically at presynaptic terminals [172], where serotonin release activates autoreceptors of unidentified type, coupled to chloride channels, that decrease the input resistance of the terminals, and their excitability [173]. Through the presence of inhibitory autoreceptors, the subsequent release of serotonin is controlled by the previous activity and release history.

The serotonin transporter, which reuptakes this amine into serotonergic neurons, also plays an important role in the regulation of their firing, by regulating the extracellular levels of serotonin and thus the autoinhibitory effect of serotonin on these neurons [174, 175, 176].

6. Serotonin secretion in the nervous system

Serotonin is released from secretory vesicles, by exocytosis. It can be stored both in small (40–60 nm) clear synaptic vesicles and in large (90–120 nm) dense-core vesicles. Each vesicle type releases its contents from different sites of the neuron, producing responses on different targets and with different time courses. Small clear vesicles generally release their contents at the active zone of presynaptic terminals, producing fast and localized effects onto specific postsynaptic terminals. In contrast, large dense-core vesicles are released at extrasynaptic sites [177, 178, 179]. Since dense-core vesicles contain 17-times more serotonin than small clear vesicles [180], exocytosis from these large vesicles releases much more serotonin, which can diffuse in the extracellular fluid and reach distant targets to produce slow and long-lasting effects over vast areas of the nervous system, in what has been called “volume transmission” [181].

6.1 Synaptic secretion

Most of the current knowledge on serotonin secretion mechanisms has been obtained from studies in identified serotonergic neurons from the central nervous system of the leech. The “colossal” Retzius neurons, so-called after the nineteenth century anatomist who described them, are the largest neurons and contain most of the serotonin in the nervous system of this invertebrate. The large size of their soma (60–100 μm in diameter) and their stereotyped localization in the ganglia facilitate their identification under simple microscopes and their electrical recordings with intracellular electrodes. In addition, these neurons can be isolated and kept in culture, where they maintain their physiological characteristics and keep synthesizing and releasing serotonin [6, 182]. In addition, if placed in contact with an adequate target neuron, they can form synapses in culture [7], which are an ideal experimental preparation to study synaptic transmission, since the isolated neurons are isopotential (changes in membrane potential are not filtered out as happens in cells with long processes) and do not receive other inputs. In these synapses, serotonin is released from small clear vesicles, which cluster near the active zone [183]. Synaptic serotonin release is quantal [6, 184], i.e., it occurs in “packs” of constant size [185], corresponding to the contents of a synaptic vesicle; it depends on calcium and the presynaptic membrane potential [8]. Like neurotransmitter release at any synapse, serotonin release at presynaptic terminals occurs within milliseconds after each action potential, since synaptic vesicles are docked at the active zone, where calcium channels are clustered [186]. Calcium entry to the terminal in response to the arrival of an action potential produces the immediate fusion of these readily releasable vesicles, as shown in Figure 2. Moreover, since serotonin is released from the presynaptic terminal directly to the synaptic cleft, in close proximity to receptors in the postsynaptic terminal, its effect is very fast and localized. Because presynaptic serotonergic terminals have serotonin transporters that reuptake the transmitter, synaptic effects are also short-lived, lasting only hundreds of milliseconds. Upon repetitive stimulation, synaptic transmission displays plasticity phenomena, such as synaptic facilitation [187] or depression, depending on the stimulation frequency and the release probability. In addition, serotonin-containing large dense-core vesicles are present surrounding the synaptic terminals. These large vesicles release their contents upon repetitive activity and add to the synaptic responses produced by synaptic serotonin release. Synaptic serotonin release is also regulated by autoreceptors located in the presynaptic terminals, which are linked to potassium channels (in vertebrates) or to chloride channels (in invertebrates) that lead to hyperpolarization and decrease the excitability of the terminal (Figure 2).

Figure 2.

Serotonin release from presynaptic terminals. Synaptic secretory vesicles are organized near or at the active zone of the presynaptic terminal. Docked vesicles can fuse with the membrane in response to single action potentials upon influx of calcium (Ca²⁺) through voltage-gated N or P/Q types of calcium channels. Neurotransmitter released to the synaptic cleft, which is only a few nanometers wide, rapidly reaches the postsynaptic membrane and bind to its receptors. Serotonin also activates autoreceptors on the presynaptic membrane, which are coupled to potassium (K⁺)or chloride (Cl⁻) channels that hyperpolarize the membrane and decrease presynaptic excitability, thus immediately regulating subsequent release.

6.2 Extrasynaptic secretion

In addition to the classical mechanisms of release from presynaptic terminals, serotonin, like most neurotransmitters, is released from extrasynaptic sites of the neurons [188], including the soma, dendrites, and axonal shafts. As explained above (see Section 4.2 on the Structure of the serotonergic system), morphological evidence has shown that many of the serotonergic axonal varicosities in vertebrates contain the machinery necessary for serotonin release, but do not have postsynaptic counterparts [141, 142, 145, 149, 150, 151], suggesting that these sites display extrasynaptic secretion. In addition, evidence from microdialysis and cyclic voltammetry showed the presence of serotonin in the extracellular fluid in the CNS of vertebrates and invertebrates [189, 190, 191, 192, 193, 194, 195], in concentration that match the affinity of 5-HT receptors [189]. Moreover, there is extensive evidence for the existence of receptors and transporters for serotonin at extrasynaptic locations [141, 168, 175, 196, 197, 198].

Extrasynaptic secretion has characteristics that differ a lot from those of synaptic secretion and are more similar to hormone secretion by neuroendocrine cells. The first direct demonstration of extrasynaptic serotonin secretion was made in the soma of leech Retzius neurons [199]. In these neurons, in contrast to presynaptic terminals, where vesicles are docked at the presynaptic active zone, very near calcium channels, the soma has clusters of hundreds of large dense-core vesicles, forming different pools that rest at different distances from the plasma membrane. This difference in the localization of vesicles at rest produces large differences in the requirements for the activation of somatic secretion and in its time course. In contrast to synaptic secretion, which is activated within milliseconds by single action potentials, somatic secretion requires repetitive firing at high frequencies to be activated. Somatic secretion requires also the activation of L-type voltage-sensitive calcium channels [199] and depends on calcium-induced calcium release from intracellular calcium pools, such as the endoplasmic reticulum [200, 201], which amplifies and propagates the calcium signal in the cytoplasm and promotes the mobilization of vesicle clusters (Figure 3). This confers a long delay to the onset of somatic secretion after electrical activity, which contrasts with the fast release at synapses. Upon the fusion of the first vesicles that reach the membrane, released serotonin activates 5-HT₂ autoreceptors coupled to phospholipase C, which produces the second messenger IP3 that in turn releases calcium from the endoplasmic reticulum near the membrane. This calcium promotes the fusion of vesicles subsequently reaching the membrane, establishing a positive feedback cycle that sustains serotonin release for minutes after only a short burst of action potentials at high frequency, until all the vesicles mobilized by the first calcium signal release their contents [200]. The mechanism producing somatic serotonin secretion is illustrated in Figure 3.

Figure 3.

Schematic diagram of somatic secretion of serotonin (5-HT) from leech Retzius neurons. Clusters of dense-core vesicles distant from the plasma membrane are mobilized by motor proteins activated by ATP and by Ca²⁺ waves initiated by entry through L-type channels and propagated by calcium-induced calcium release. A 5-HT-activated feedback loop mediated by IP3-induced intracellular Ca²⁺ release sustains secretion in these cells. All of these mechanisms are also seen in vertebrates.

Somatic release of serotonin has also been shown by multi-photon microscopy in the raphe nuclei of mammals [202, 203]. Although the detailed mechanisms of somatic secretion in these neurons in vertebrates have not been elucidated, they seem to be similar to those described in invertebrates, and somatic release lasts also for minutes, reaching vast volumes in the nervous system [204].

In addition to somatic secretion, serotonergic neurons have been shown to display extrasynaptic secretion from the dendrites. Dendritic serotonin release can be activated by glutamatergic inputs, which open L-type calcium channels in the dendrites, independently of action potential firing [205].

The presence of clusters of secretory vesicles in different sites of serotonergic axons in the absence of postsynaptic counterparts, as described above, strongly suggests that extrasynaptic serotonin release takes place also from the axons. Extrasynaptic secretion of serotonin from the primary axon has been directly observed in leech Retzius neurons, where the requirements and characteristics of secretion are intermediate between those of release at presynaptic terminals and that of somatic secretion (Cercós and Trueta, in preparation). Extrasynaptic release from sites along the axons of serotonergic neurons could act in a more diffuse manner than serotonin released from presynaptic terminals, but produce effects still somewhat restricted in space, thus producing local neuromodulation at different levels of neural circuits.

In addition, in vertebrates and invertebrates, serotonin is secreted from neurosecretory organs to the circulatory system, through which it can reach peripheral organs and act as a neurohormone. Thus, serotonin can act in at least three ways: as a neurotransmitter at specialized synapses, a neuromodulator at extrasynaptic paracrine secretion sites, and a neurohormone in the general circulation.

Due to the differences in the localization of the vesicles that produce secretion in each neuronal compartment, the different modes of serotonin secretion in the nervous system have very different time courses. Synaptic release occurs upon each action potential, and is thus synchronized with electrical activity, and its effects are produced immediately, lasting only hundreds of milliseconds. In contrast, extrasynaptic release occurs with a long delay after electrical activity, and its effects are even more delayed, due to the diffusion of the transmitter in the extracellular space before reaching its targets. Extrasynaptic secretion also lasts for minutes after a short train of action potential [199, 200], and its effects may last for hours or even longer periods. Figure 4 schematizes the differences in the time courses of synaptic and extrasynaptic secretion and their effects on the nervous system.

Figure 4.

Differences in the time courses of synaptic and extrasynaptic release and their effects on the nervous system. In response to a burst of action potentials lasting 500 ms, release at synaptic terminals occurs synchronized with electrical activity and produces postsynaptic effects lasting a few hundred of milliseconds more. In contrast, extrasynaptic release begins with a delay of nearly a minute and lasts several minutes and its effects, mediated by diffusion, may last for hours or even longer.

Neurotransmitter release at synapses has immediate and localized effects that contribute to synaptic computation in neuronal networks. On the other hand, extrasynaptic release from the soma, axons, or dendrite is thought to produce slow and diffuse neuromodulatory effects on neuronal populations, changing the way in which the synaptic networks respond. These slow effects may be related to the regulation of mood, emotions, and social behavior, which have time courses that exceed by far the effects of synaptic transmission. However, there is no concrete evidence of the effects of extrasynaptic release for serotonin or other neurotransmitters in the nervous system. This is a fascinating subject for further research.

The different modes of serotonin release, from different compartments of the same neurons, enable these neurons to act as multifunctional cells. This may be the way in which these neurons, although reduced in number, are able to regulate such a wide diversity of functions in the nervous system.

7. Conclusions

Serotonin in the nervous system is synthesized by small numbers of neurons that release it by exocytosis from different compartments of their complex structure. The type of structure where serotonin is released defines the type of information processing that serotonin carries in each region. Presynaptic terminals release small amounts of serotonin, in synchrony with electrical activity, and with a strong association between the presynaptic neuron and its target, producing very fast, local, and short-lasting effects on specific postsynaptic targets that participate in immediate functions. On the other hand, extrasynaptic sites of these neurons, including the soma and the axons, slowly release massive amounts of serotonin that produce less localized and more dynamic interactions with the target neurons, whereby serotonin can reach distant and diverse targets by diffusion and produce slow and long-lasting neuromodulatory effects, such as those that characterize emotions and social behavior. By displaying different modes of neurotransmitter release, small numbers of serotonergic neurons can regulate a multiplicity of functions in the nervous system, which have been conserved throughout the phylogenetic scale.

Acknowledgments

We thank Ricardo Cruz Garduño for the elaboration of the figures for this chapter, and Silvia Rocío Mejía Mauríes for technical and administrative assistance.

This work has been funded by INPRFM internal resources. Dr. Nuria Segovia paid the publishing fee. We are thankful for her generosity.

Conflict of interest

The authors declare no conflict of interest.