Chapter 13:  Serotonin

The indolealkylamine 5-hydroxytryptamine, serotonin, was identified initially because of interest in its cardiovascular effects

It has been known since the mid-nineteenth century that after blood clots the resulting serum possesses a substance that constricts vascular smooth muscle so as to increase vascular tone. Around the turn of this century, platelets were identified as the source of this substance. In the late 1940s, Rapport and collaborators [1] isolated, purified and identified this “tonic” substance in “serum” (hence, serotonin) as the substituted indole 5-hydroxytryptamine (5-HT).

The structures of serotonin and related compounds are shown in Figure 13-1. The combination of the hydroxyl group in the 5 position of the indole nucleus and a primary amine nitrogen serving as a proton acceptor at physiological pH makes 5-HT a hydrophilic substance. As such, it does not pass the lipophilic blood—brain barrier readily. Thus, its discovery in brain indicated that 5-HT is synthesized in brain, where it might play an important role in brain function. The observation, at about the same time, that the psychedelic drug (+)lysergic acid diethylamide (LSD) antagonized a response produced by 5-HT further substantiated the idea that 5-HT might have important behavioral effects, even though the response was contraction of gastrointestinal smooth muscle. Subsequently, various theories arose linking abnormalities of 5-HT function to the development of a number of psychiatric disorders, particularly schizophrenia and depression. Psychotherapeutic drugs are now available that are effective in depression (see Chap. 52), anxiety disorders and schizophrenia; some of these drugs have potent, and in some cases selective, effects on serotonin neurons in brain.

Understanding the neuroanatomical organization of serotonergic cells in brain provides insight into the functions of this neurotransmitter

Serotonin-containing neuronal cell bodies are restricted to discrete clusters or groups of cells located along the midline of the brainstem. Their axons, however, innervate nearly every area of the CNS (Fig. 13-2). In 1964, Dahlstrom and Fuxe (discussed in [2]), using the Falck-Hillarp technique of histofluorescence, observed that the majority of serotonergic soma are found in cell body groups, which previously had been designated as the raphe nuclei. This earlier description of the raphe nuclei was based on cell body structural characteristics and organization. Dahlstrom and Fuxe described nine groups of serotonin-containing cell bodies, which they designated B₁ through B₉, and which correspond for the most part with the raphe nuclei (discussed in [2]) (Table 13-1). Some serotonergic neuronal cell bodies, however, are found outside the raphe nuclei, and not all of the cell bodies in the raphe nuclei are serotonergic. In most of the raphe nuclei, the majority of neurons are nonserotonergic. For example, the dorsal raphe contains the largest number of serotonergic neurons; however, only 40 to 50% of the cell bodies in the dorsal raphe are serotonergic (Fig. 13-3).

Since the late 1960s, a variety of techniques have been used to characterize the neuronal circuitry of serotonin cells in the CNS. The density of serotonergic innervation in the forebrain was underestimated initially because the original histofluorescence method was limited in sensitivity and did not permit the detection of many fine axons and terminals. Subsequent anatomical techniques, such as immunohistochemical localization of either 5-HT or tryptophan hydroxylase, an enzyme unique to the synthesis of 5-HT, in addition to retrograde and anterograde axonal transport studies, have allowed a more complete and accurate characterization of the serotonergic innervation of forebrain areas.

The largest group of serotonergic cells is B₇ (Fig. 13-2), which is continuous with a smaller group of serotonergic cells, B₆. Groups B₆ and B₇ often are considered together as the dorsal raphe nucleus, with B₆ being its caudal extension. Another prominent serotonergic cell body group is B₈, which corresponds to the median raphe nucleus, also termed the nucleus central superior. Group B₉, part of the ventrolateral tegmentum of the pons and midbrain, forms a lateral extension of the median raphe (Fig. 13-2) and, therefore, is not considered one of the midline raphe nuclei. Ascending serotonergic projections innervating the cerebral cortex and other regions of the forebrain arise primarily from the dorsal raphe, median raphe and B₉ cell group.

Two distinct ascending projections arise from the rostral serotonergic system. The two main ascending serotonergic pathways emerging from the midbrain raphe nuclei to the forebrain are the dorsal periventricular path and the ventral tegmental radiations. Both pathways converge in the caudal hypothalamus, where they join the medial forebrain bundle. Axons of both dopaminergic and noradrenergic neurons course anteriorly through the medial forebrain bundle as well (see Chap. 12).

Ascending projections from the raphe nuclei to forebrain structures are organized in a topographical manner. The dorsal and median raphe nuclei give rise to distinct projections to forebrain regions (Fig. 13-4). The median raphe projects heavily to hippocampus, septum and hypothalamus, whereas the striatum is innervated predominantly by the dorsal raphe. The dorsal and median raphe nuclei send overlapping neuronal 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 brain. For example, the frontal cortex receives heavy innervation from the rostral and lateral subregions of the dorsal raphe nucleus. Raphe neurons send collateral axons to areas of brain that are related in function, such as the amygdala and hippocampus or the substantia nigra and caudate putamen. The specific and highly organized innervation of forebrain structures by raphe neurons implies independent functions of sets of serotonergic neurons dependent on their origin and terminal projections, as opposed to a nonselective or general role for serotonin in the CNS.

Serotonergic axon terminals, labeled by uptake of [³H]5-HT or studied with immunohistochemical techniques, appear to exhibit morphological differences related to the raphe nucleus of origin (Fig. 13-4). Serotonergic axons from the median raphe nucleus, type M, look relatively coarse with large spherical varicosities. By contrast, axons from the dorsal raphe, type D, are very fine and typically have small, pleomorphic varicosities. Dorsal raphe axons appear to be more vulnerable to certain neurotoxic amphetamine derivatives, such as d-fenfluramine, 3,4-methylenedioxymethamphetamine (MDMA, commonly termed Ecstasy) or parachloroamphetamine (PCA). Median raphe axons appear to be more resistant to the neurotoxic effects of these drugs. Blockade of the serotonin transporter prevents the neurotoxic effects of these amphetamine derivatives, indicating that activity of this transporter is critical for the neurotoxic effects of these drugs.

As expected, serotonergic terminals make the usual specialized synaptic contacts with target neurons and release serotonin following nerve stimulation. In some areas of the mammalian CNS, there are sites where 5-HT is released and no evidence for synaptic specialization has been found. For example, it is difficult to demonstrate the synaptic contacts of the fine varicose fibers, whereas large terminals make well defined synapses with target cells. The percentage of 5-HT terminals associated with synaptic specializations appears to vary in particular brain regions. This may have important implications for the type of information processing in which 5-HT is involved in these brain areas. The appearance of specialized synaptic contacts suggests relatively stable and strong associations between a presynaptic neuron and its target. Conversely, the lack of synaptic specialization implies a dynamic, and perhaps less specific, interaction with target neurons. For example, neurotransmitter is released and then diffuses over distances as great as several hundred microns. In this case, 5-HT may act as a neuromodulator to adjust or tune ongoing synaptic activity.

The other raphe nuclei, B₁ to B₄, are situated more caudally in the midpons to caudal medulla and contain a smaller number of serotonergic cells. These cell body groups give rise to serotonergic axons that project within the brainstem and to the spinal cord (Fig. 13-2). The spinal cord receives a strong serotonergic innervation. Three principal descending pathways have been described: (i) from the raphe magnus nucleus (B₃) to laminae I and II of the dorsal horn, (ii) from the raphe obscurus nucleus (B₂, B) to lamina IX of the ventral horn and (iii) from the rostral ventrolateral medulla and the lateral paragigantocellular reticular nucleus (B₃) to the interomediolateral cell column. Projections from the caudal ventrolateral medulla (B₁) are not known at present [3].

Afferent connections to the raphe nuclei include those between the dorsal and median raphe nuclei, B₉, B₁ and B₃. Connections between the raphe nuclei have been described by retrograde tracing techniques using horseradish peroxidase and wheat germ agglutinin. Such innervation may have considerable physiological and/or pharmacological importance as serotonin released in the vicinity of serotonergic cell bodies regulates the firing of serotonergic neurons through the activation of somatodendritic autoreceptors. The raphe nuclei also receive input from other cell body groups in the brainstem, such as the substantia nigra and ventral tegmental area (dopamine), superior vestibular nucleus (acetylcholine), locus ceruleus (norepinephrine) and nucleus prepositus hypoglossi and nucleus of the solitary tract (epinephrine). Other afferents include neurons from the hypothalamus, thalamus and limbic forebrain structures (see [2–4] for a comprehensive review of serotonergic neuroanatomy).

The amino acid l-tryptophan serves as the precursor for the synthesis of 5-hydroxytryptamine

Not all cells that contain 5-HT synthesize it. For example, platelets do not synthesize 5-HT; rather, they accumulate 5-HT from plasma by an active-transport mechanism found on the platelet membrane. Certain brain cells do synthesize 5-HT. The synthesis and primary metabolic pathways of 5-HT are shown in Figure 13-5. The initial step in the synthesis of serotonin is the facilitated transport of the amino acid l-tryptophan from blood into brain. The primary source of tryptophan is dietary protein. Certain other neutral amino acids, such as phenylalanine, leucine and methionine, are transported into brain by the same carrier. The entry of tryptophan into brain is related not only to its concentration in blood but is also a function of its concentration in relation to the concentrations of other neutral amino acids. Consequently, lowering the dietary intake of tryptophan while raising intake of the amino acids that tryptophan competes with for transport into brain lowers the content of 5-HT in brain and changes certain behaviors associated with 5-HT function. This strategy for lowering the brain content of 5-HT has been used clinically to evaluate the importance of brain 5-HT in the mechanism of action of psychotherapeutic drugs [5].

Serotonergic neurons contain the enzyme l-tryptophan-5-monooxygenase (EC 1.14.16.4), more commonly termed tryptophan hydroxylase, which converts tryptophan to 5-hydroxytryptophan (5-HTP) (Fig. 13-5). This enzyme is synthesized in serotonergic cell bodies of the raphe nuclei and is found only in cells that synthesize 5-HT; its distribution in brain is similar to that of 5-HT itself. The enzyme requires both molecular oxygen and a reduced pteridine cofactor, such as l-erythro-tetrahydrobiopterin (BH₄), for activity. In the enzymatic reaction, one atom of oxygen is used to form 5-HTP and the other is reduced to water. The pteridine cofactor donates electrons, and the unstable quinonoid dihydrobiopterin that results is regenerated immediately to the tetrahydrobiopterin form by a NADPH-linked enzymatic reaction:

The K_m of partially purified tryptophan hydroxylase for tryptophan is approximately 30 to 60 μM, a concentration comparable to that of tryptophan in brain. If the concentration of tryptophan in serotonergic neurons is assumed to be comparable to that in whole brain, the enzyme would not be saturated with substrate and the formation of 5-HT in brain would be expected to rise as the brain concentration of tryptophan increases. This occurs specifically in response to raising the dietary intake of tryptophan. However, the relationship among tryptophan availability, total tissue 5-HT concentration and 5-HT release is not fully understood.

cDNAs encoding tryptophan hydroxylase from both brain and pineal gland have been cloned and sequenced. Some biochemical differences between the enzyme(s) obtained from brain and from pineal gland, such as molecular weight, substrate specificity and isoelectric point, have been reported previously. However, cDNAs isolated from both tissue sources appear to have identical nucleotide sequences, making it likely that tissue-specific differences in the properties of tryptophan hydroxylase result from differential post-translational processing. Tryptophan hydroxylase contains 444 amino acids, corresponding to a molecular weight of about 51,000, and is 50% homologous with tyrosine hydroxylase, the rate-limiting enzyme in catecholamine (CA) biosynthesis (see Chap. 12). The greatest homology resides in the central and C-terminus regions of these enzymes, making it likely that these areas contain the catalytic site. Substrate specificity may reside in those amino acids nearer the N-terminus.

The other enzyme involved in the synthesis of serotonin, aromatic l-amino acid decarboxylase (AADC) (EC 4.1.1.28), is a soluble pyridoxal-5′-phosphate-dependent enzyme which converts 5-HTP to 5-HT (Fig. 13-5). It has been demonstrated that administration of pyridoxine increases the rate of synthesis of 5-HT in monkey brain, as revealed using position emission tomography. This presumably reflects a regulatory effect of pyridoxine on AADC activity and raises the interesting issue of the use of pyridoxine supplementation in situations associated with 5-HT deficiency.

AADC is present not only in serotonergic neurons but also in catecholaminergic neurons, where it converts 3,4-dihydroxyphenylalanine (DOPA) to dopamine (see Chap. 12). However, different pH optima or concentrations of substrate or cofactor are required for optimal activity of the enzyme in brain homogenates when using either 5-HTP or DOPA as the substrate. cDNAs encoding AADC have been cloned from various species. The encoded protein contains 480 amino acids and has a molecular weight of 54,000 but it appears to exist as a dimer. Characterization of the protein expressed in cells transfected with the cDNA shows that it decarboxylates either DOPA or 5-HTP. Also, in situ hybridization of the mRNA for the enzyme revealed its presence both in serotonergic cells in the dorsal raphe nucleus and in catecholaminergic cells in brain regions containing catecholaminergic soma [6]. Taken together, these results support the idea that the enzymatic decarboxylation of both DOPA and 5-HTP is catalyzed by the same enzyme.

Because the decarboxylase enzyme is not saturated with 5-HTP under physiological conditions, that is, the concentration of 5-HTP is much less than the K_m of 10 μM, it is possible to raise the content of 5-HT in brain not only by increasing the dietary intake of tryptophan but also by raising the intake of 5-HTP. This procedure, though, results in the formation of 5-HT in cells that would not normally contain it, such as catecholaminergic neurons, because of the nonselective nature of AADC.

The initial hydroxylation of tryptophan, rather than the decarboxylation of 5-HTP, appears to be the rate-limiting step in serotonin synthesis. Evidence in support of this view includes the fact that 5-HTP is found only in trace amounts in brain, presumably because it is decarboxylated about as rapidly as it is formed. As might be expected if the hydroxylation reaction is rate-limiting, inhibition of this reaction results in a marked depletion of the content of 5-HT in brain. The enzyme inhibitor most widely used in experiments is parachlorophenylalanine (PCPA). In vivo, PCPA irreversibly inhibits tryptophan hydroxylase, presumably by incorporating itself into the enzyme to produce an inactive protein. This results in a long-lasting reduction of 5-HT levels. Recovery of enzyme activity and 5-HT biosynthesis requires the synthesis of new enzyme. Marked increases in levels of mRNA for tryptophan hydroxylase are found in the raphe nuclei 1 to 3 days after administration of PCPA [7].

The synthesis of 5-hydroxytryptamine can increase markedly under conditions requiring a continuous supply of the neurotransmitter

Plasticity is an important concept in neurobiology. In general, this refers to the ability of neuronal systems to conform to either short- or long-term demands placed upon their activity or function. One of the processes contributing to neuronal plasticity is the ability to increase the rate of neurotransmitter synthesis and release in response to increased neuronal activity. Serotonergic neurons have this capability; the synthesis of 5-HT from tryptophan is increased in a frequency-dependent manner in response to electrical stimulation of serotonergic soma [8]. The increase in synthesis results from the enhanced conversion of tryptophan to 5-HTP and has an absolute dependence on extracellular Ca²⁺. It is likely that the increased synthesis results in part from alterations in the kinetic properties of tryptophan hydroxylase, perhaps due to calcium-dependent phosphorylation of the enzyme. The enzyme can be phosphorylated directly by the action of calmodulin-dependent protein kinase II; an activator protein appears to be required for this interaction. In the presence of the activator, tryptophan hydroxylase also may be a substrate for cAMP-dependent protein kinase (PKA). The increased activity of tryptophan hydroxylase does not result from the removal of enzyme inhibition caused by either 5-HT or 5-HTP.

Short-term requirements for increases in the synthesis of 5-HT can be met by processes that change the kinetic properties of tryptophan hydroxylase, such as phosphorylation, without necessitating the synthesis of more molecules of tryptophan hydroxylase. By contrast, situations requiring long-term increases in the synthesis and release of 5-HT result in the synthesis of tryptophan hydroxylase protein. For example, partial but substantial destruction of >60% of central serotonergic neurons results in an increase in the synthesis of 5-HT in residual terminals. The increase in synthesis initially results from activation of existing tryptophan hydroxylase molecules, but the increased synthesis of 5-HT seen weeks after the lesion results from more tryptophan hydroxylase being present in the residual terminals. An increase in tryptophan hydroxylase mRNA has been reported in residual raphe serotonergic neurons after partial lesioning, consistent with the idea of an increase in the synthesis of tryptophan hydroxylase molecules in residual neurons.

As with other biogenic amine transmitters, 5-hydroxytryptamine is stored primarily in vesicles and released by an exocytotic mechanism

Peripheral sources of monoamine-containing cells have been utilized to study the properties of storage vesicles, such as chromaffin cells of the adrenal medulla for CAs and parafollicular cells of the thyroid gland for 5-HT. In some respects, the vesicles that store 5-HT resemble those that store CAs. For example, drugs such as reserpine and tetrabenazine, which inhibit the activity of the transporter localized to the vesicular membrane, deplete the brain content of 5-HT as well as of CAs. Storage of 5-HT in vesicles requires its active transport from the cytoplasm. The vesicular transporter uses the electrochemical gradient generated by a vesicular H⁺-ATPase to drive transport, such that a cytoplasmic amine is exchanged for a luminal proton; that is, uptake of 5-HT is coupled to efflux of H⁺ (Fig. 13-6).

Two synaptic vesicle transporters have been cloned, with the predicted amino acid sequence containing 12 transmembrane domains. The first transporter, cloned from chromaffin granules, has been termed vesicular membrane transporter₁ (VMAT₁). It contains 521 amino acids and a large loop between transmembrane domains 1 and 2, which faces the lumen of the vesicle, contains sites for glycosylation. Both the NH₂ and COOH termini face the cytoplasm. A second vesicular transporter (VMAT₂), cloned from rat brain, is 62% identical to VMAT₁. Human chromosome 8 contains the gene for VMAT₁ and chromosome 10 contains the gene for VMAT₂. These transporters show homology to a family of drug-resistant transporters in bacteria and yeast. All members of this family function as antiporters to remove toxic compounds from the cytoplasm. It has been suggested that this family, including VMAT₁ and VMAT₂, be termed T oxin-Extruding Antiporters (TEXANs) [9].

Although there are clear structural differences between the vesicular transporter and the Na⁺-dependent plasma membrane transporter for 5-HT (described below), there are also striking structural similarities. Nevertheless, drugs that inhibit the vesicular transporter generally do not block the plasma membrane transporter and vice versa. However, two drugs that have effects at both the vesicular transporter and the plasma membrane transporter are the anorectic agent fenfluramine and MDMA. Fenfluramine inhibits the vesicular transporter directly by competing for its substrate-binding site. It also dissipates the transmembrane pH gradient to inhibit further 5-HT uptake into the vesicle (Fig. 13-6). MDMA has these pharmacological effects as well. The effects of these drugs on the plasma membrane transporter are described below. The consequence of such pharmacological actions is stimulation of 5-HT release by a nonexocytotic mechanism.

Vesicles storing 5-HT exhibit some differences from those storing CAs. In contrast to CA-containing vesicles, there is virtually no ATP in serotonin vesicles. Also, serotonergic synaptic vesicles, but not chromaffin granules, contain a specific protein that binds 5-HT with high affinity. This serotonin-binding protein is present in serotonergic cells derived ontogenetically from the neuroectoderm [10]. It binds 5-HT with high affinity in the presence of Fe²⁺. There are three isoforms of serotonin-binding protein. The 45-kDa isoform appears to be packaged in secretory vesicles along with 5-HT, which probably accounts for the observation that newly taken up [³H]5-HT is rapidly complexed with this isoform in brain in situ. This isoform is secreted along with 5-HT by a calcium-dependent process.

There is considerable evidence that the release of 5-HT occurs by exocytosis, that is, by the discharge from the cell of the entire contents of individual storage vesicles. First, 5-HT is ionized sufficiently at physiological pH so that it does not cross plasma membranes by simple diffusion. Second, most intraneuronal 5-HT is contained in storage vesicles and other contents of the vesicle, including serotonin-binding protein (SPB), are released together with serotonin. By contrast, cytosolic proteins do not accompany electrical stimulation-elicited release of 5-HT. Third, the depolarization-induced release of 5-HT occurs by a calcium-dependent process; indeed, it appears that the influx of Ca²⁺ with or without membrane depolarization can increase the release of 5-HT. Ca²⁺ has been reported to stimulate the fusion of vesicular membranes with the plasma membrane.

The rate of serotonin release is dependent on the firing rate of serotonergic soma in the raphe nuclei. Numerous studies have revealed that an increase in raphe cell firing enhances the release of 5-HT in terminal fields. The opposite effect is observed when raphe cell firing decreases. This means that drugs that change the firing rate of serotonergic soma modify the release of serotonin as well. An important target for such drugs is the somatodendritic autoreceptor, which, as discussed later, is the 5-HT_1A receptor subtype (see Fig. 13-8). Administration of 5-HT_1A agonists, such as 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), into the dorsal raphe nucleus slows the rate of firing of serotonergic soma. Using the technique of in vivo microdialysis, application of 8-OH-DPAT in the dorsal raphe nucleus decreases the release of 5-HT in the striatum. Depending on the species, serotonergic autoreceptors in terminal fields appear to be either the 5-HT_1B or the 5-HT_1D subtype. Administration of agonists of these receptors into areas receiving serotonergic innervation decreases the synthesis and release of 5-HT measured in vitro or in situ, using the technique of microdialysis. However, in contrast to the activation of somatodendritic autoreceptors, such effects are not due to decreases in the firing rate of serotonergic soma.

The activity of 5-hydroxytryptamine in the synapse is terminated primarily by its re-uptake into serotonergic terminals

Synaptic effects of many amino acid and monoaminergic neurotransmitters, including 5-HT, are terminated by binding of these molecules to specific transporter proteins. The serotonin transporter (SERT) is located on serotonergic neurons. Evidence for this comes from studies showing that the selective lesioning of serotonergic neurons in brain markedly reduces both the high-affinity uptake of [³H]5-HT in areas of brain receiving serotonergic innervation and the specific binding of radioligands to the serotonin transporter. Activity of the SERT regulates the concentration of 5-HT in the synapse, thereby influencing synaptic transmission.

The uptake system for 5-HT is saturable and of high affinity, with a K_m value for 5-HT of approximately 0.2 to 0.5 μM. Uptake of 5-HT is an active process that is temperature-dependent and has an absolute requirement for external Na⁺ and C1⁻; it is inhibited by metabolic inhibitors as well as by inhibitors of Na/K ATPase activity. From these and other data, it has been inferred that the energy requirement for 5-HT uptake is not used directly to transport 5-HT but rather is necessary to maintain the gradient of Na⁺ across the plasma membrane, upon which 5-HT uptake is dependent. The current model of transport has one Na⁺, one C1⁻ and one protonated 5-HT binding to the transporter extracellularly prior to translocation to form a quaternary complex that subsequently undergoes a conformational change to release the neurotransmitter and the ions into the cytoplasm. The conformational change may involve the “opening” of a pore formed by some portion of the transmembrane domains of the SERT (see below). In the cytoplasm, K⁺ associates with the SERT to promote reorientation of the unloaded carrier for another transport cycle (Fig. 13-6).

The cloning, sequencing and expression of several transporter proteins, including that for 5-HT, has aided considerably in understanding structure/function relationships of transporter proteins [11,12]. The cDNA for the SERT isolated from rat brain predicts a protein containing 630 amino acids with a molecular weight of about 68,000. The SERT is encoded by a single gene on the long arm of chromosome 17. The mRNA for the SERT has been localized in brain exclusively to the serotonergic cells in the raphe nuclei. Of interest is the fact that mRNA for the SERT has not yet been detected in glia, even though primary cultures of astrocytes in vitro can take up 5-HT. The inability to detect message for the SERT in glia raises questions about the physiological relevance of the uptake of 5-HT by glia in vitro.

The putative structure of the SERT has 12 transmembrane domains (TMDs) with both the amino- and carboxy-termini being intracellular, and a large extracellular loop containing canonical glycosylation sites connecting TMDs 3 and 4 (Fig. 13-7). Glycosylation seems necessary for optimal stability of the transporter in the membrane but not for 5-HT transport or ligand binding. There are also six potential sites of phosphorylation by PKA and protein kinase C (PKC) on the human SERT. The predicted structure of the serotonin transporter is similar to that of other cloned neurotransmitter transporters and quite distinct, for example, from the seven-transmembrane domain structure of G protein-linked receptors. These transporters are considered members of the Na⁺ and C1⁻-dependent neurotransporter family, distinct from the vesicular transporter family described earlier. All members of this family are fragmented by multiple introns, raising the possibility of multiple transcripts by alternative RNA processing.

SERT exhibits about 50% absolute homology with the transporters for norepinephrine (NET) and dopamine (DAT), with the greatest homology being found in TMDs 1 and 2 and TMDs 4–8. The least conserved regions are the intracellular amino- and carboxy-terminal tails (Fig. 13-7). It has been proposed that the conserved regions may be involved in general transport functions and the least conserved regions in the unique attributes of each carrier, such as pharmacological specificity. However, studies in which the terminal regions were exchanged between the SERT and the NET revealed no alternation in substrate or antagonist selectivity. Thus, the NH₂ and COOH termini may not be important for ligand recognition.

Of considerable interest is whether the SERT is subject to physiological regulation. Such regulation could occur at the level of transcription or translation or by post-translational modifications, such as glycosylation or phosphorylation. The focus of much of this research, carried out in vitro using cells that naturally express the SERT or cells stably transfected with the SERT, has been on the role of second-messenger systems, particularly those activating protein kinases. It has been established that SERT gene expression is (i) influenced by cAMP-dependent pathways and (ii) rapidly regulated by changes in intracellular Ca²⁺, treatment with calmodulin inhibitors or activation of PKC as well as nitric oxide synthase (NOS)/cGMP pathways. Most of the kinetic alterations reflect changes in maximal transport capacity (V_max) rather than in apparent affinity (K_m). Activation of PKC causes a loss of SERT protein on the cell surface. It remains to be established, however, whether such processes contribute to modulation of SERT activity in vivo.

Although there is structural homology among the transporters for 5-HT, NE and DA, some drugs exhibit great selectivity at inhibiting the activity of just one of these proteins. For example, secondary amine tricyclic antidepressants, such as desipramine, are 25- to 150-fold more potent at inhibiting transport of NE than 5-HT. By contrast, some of the newer antidepressants, appropriately termed selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, sertraline and paroxetine, are 15 to 75 times more potent at inhibiting the uptake of 5-HT than the uptake of NE. However, there are also drugs, such as cocaine, that are nonselective inhibitors of all three transporters.

In addition to facilitating the removal of transmitters from the synapse, plasma membrane transporters function under certain circumstances to release transmitter by a non-Ca²⁺-dependent, that is, nonexocytotic, process. In contrast to exocytotic release, this release process is not modulated by presynaptic receptors. However, such transporter-mediated release is Na⁺-dependent and sensitive to transport inhibitors. The role of such transporter-mediated release in physiological circumstances is speculative. However, certain drugs elicit the release of 5-HT, at least in part, through such a mechanism. For example, both fenfluramine and MDMA act as substrates for the SERT and not only inhibit the transport of 5-HT into the cell but also facilitate its outward transport. The action of these drugs on 5-HT-storing vesicles raises cytoplasmic 5-HT and facilitates SERT-mediated efflux of 5-HT (Fig. 13-6). The release of 5-HT caused by drugs such as fenfluramine is prevented by inhibitors of the SERT.

The primary catabolic pathway for 5-hydroxytryptamine is oxidative deamination by the enzyme monoamine oxidase

Monoamine oxidase (MAO) (E.C.1.4.3.4.) converts serotonin to 5-hydroxyindoleacetaldehyde, and this product is oxidized by an NAD⁺-dependent aldehyde dehydrogenase to form 5-hydroxyindoleacetic acid (5-HIAA) (Fig. 13-5). The intermediate acetaldehyde also can be reduced by an NADH-dependent aldehyde reductase to form the alcohol 5-hydroxytryptophol. Whether oxidation or reduction takes place depends on the ratio of NAD⁺ to NADH in the tissue. In brain, 5-HIAA is the primary metabolite of serotonin.

There are at least two isoenzymes of MAO, referred to as MAO-A and MAO-B. These isoenzymes are integral flavoproteins of outer mitochondrial membranes in neurons, glia and other cells. Evidence for the existence of isoenzymes was based initially on differing substrate specificities and sensitivities to inhibitors of MAO. For example, both 5-HT and NE are metabolized preferentially by MAO-A. Selective inhibitors of each form of MAO exist: clorgyline or moclobemide for type A and deprenyl for type B. Definitive proof of the existence of these two forms of MAO comes from the cloning of cDNAs encoding subunits of MAO-A and MAO-B from human liver [13]. The deduced amino acid sequences of MAO-A and MAO-B show about 70% homology and have masses of 59.7 and 58.8 kDa, respectively. When each cDNA was cloned into an expression vector and transfected independently into a cell line, the activity of the proteins expressed resembled that of the endogenous enzymes from human brain, such that the expressed MAO-A preferred 5-HT as a substrate and was inhibited preferentially by clorgyline. From such data it was inferred that the functional differences between these two enzymes exist in their primary structures.

Several different techniques have been used to study the neuroanatomical localization of the two forms of MAO in brain. Originally, both histochemical and immunohistochemical techniques were used. More recently, in situ hybridization histochemistry has been used to demonstrate the location of the mRNAs for the two isoenzymes. The development of radioligands selective for each form has enabled their distribution to be revealed by quantitative autoradiography. In general, the results from the use of these different techniques are similar. It is of interest that there is more MAO-A than MAO-B throughout rat brain, whereas human brain contains more MAO-B than MAO-A. Serotonergic cell bodies contain predominantly MAO-B, for which 5-HT is not a preferred substrate. This has led to the hypothesis that MAO-B in serotonergic neurons prevents the cell from accumulating various other natural substrates, such as DA, that could interfere with the storage, release and uptake of 5-HT. Furthermore, treatment of rats with clorgyline, a selective inhibitor of MAO-A, raises the brain content of 5-HT and reduces the conversion of 5-HT to 5-HIAA in brain. Thus, 5-HT may well be oxidized preferentially by MAO-A in vivo, as it is in vitro, even though serotonergic neurons do not contain much of this form of the enzyme.

The use of transgenic mice permits the selective elimination, or “knockout,” of either MAO type [14]. In the brains of mice deficient in MAO-A, the content of 5-HT is elevated markedly for about 12 days after birth and then slowly declines, reaching values comparable to those in normal mice after about 7 months. In MAO-A-deficient mice, the selective inhibitor of MAO-B, deprenyl, had a greater effect on serotonin metabolism than it did in normal mice. Such observations indicate that, in the absence of MAO-A, MAO-B can metabolize 5-HT in vivo. However, mice lacking the MAO-B isoenzyme do not have elevated levels of 5-HT in brain. Of interest are the aggressive behaviors exhibited by mice deficient in MAO-A, consistent with a postulated role of serotonergic neurons in human aggressive behaviors.

Pharmacological and physiological studies have contributed to the definition of the many receptor subtypes for serotonin

The initial suggestion that there might be more than one type of receptor for serotonin came from experiments on the isolated guinea pig ileum, which demonstrated that only a portion of the contractile response to serotonin could be blocked by high concentrations of morphine, whereas the remainder of the response could be blocked by low concentrations of dibenzyline (phenoxybenzamine). Similarly, when maximally effective concentrations of dibenzyline were present, the remaining contractile response elicited by serotonin was blocked by low concentrations of morphine. It was speculated that there were two different receptors for 5-HT in the ileum: D receptors, which are blocked by dibenzyline, and M receptors, which are blocked by morphine. The D receptor was thought to be on the smooth muscle of the ileum, whereas the M receptor was considered to be on ganglia or nerves within the muscle.

In the 1970s, the development of radioligand-binding assays furthered our understanding of subtypes of receptors for serotonin. Initially, a number of radioligands, such as [³H]5-HT, [³H]LSD and [³H]spiperone, were used to label sites related to serotonin receptors. These radioligands originally were proposed to label two classes of serotonin receptor in brain. Binding sites that were labeled with high affinity by [³H]5-HT were designated 5-HT₁ receptors; binding sites labeled with high affinity by [³H]spiperone were termed 5-HT₂ receptors. Many subsequent experiments have shown that the D receptor and the 5-HT₂ receptor are pharmacologically indistinguishable.

The binding of [³H]5-HT to 5-HT₁ receptors was shown to be displaced by spiperone in a biphasic manner, suggesting that what was termed the 5-HT₁ receptor might be a heterogeneous population of receptors. The [³H]5-HT-binding site that showed high affinity for spiperone was termed the 5-HT_1A subtype, whereas the component of [³H]5-HT binding that showed low affinity for spiperone was called the 5-HT_1B subtype. A high density of binding sites for [³H]5-HT was found in the choroid plexus. These [³H]5-HT-binding sites were termed the 5-HT_1C subtype as they did not show the pharmacological characteristics used to classify the 5-HT_1A, 5-HT_1B or 5-HT₂ binding sites. Subsequently, a fourth binding site for [³H]5-HT was identified in bovine brain and called the 5-HT_1D receptor. The 5-HT_1D receptor was identified by pharmacological criteria only in brains of species devoid of the 5-HT_1B receptor, such as pig, cow, guinea pig and human.

Bradley and associates in 1986 proposed a classification scheme with three major types of receptors for serotonin, using pharmacological criteria and functional responses primarily in peripheral tissues [15]. The receptors were called “5-HT₁-like,” 5-HT₂ and 5-HT₃. The development of potent and selective antagonists of the 5-HT₂ receptor, such as ketanserin, facilitated the assignment of certain effects mediated by 5-HT to the 5-HT₂ receptor. The M receptor, originally described in guinea pig ileum, is pharmacologically distinct from all of the binding sites associated with the serotonin receptors just described. Bradley and associates renamed this the 5-HT₃ receptor. The development of potent selective antagonists and an agonist, 2-methyl-5-HT, provided useful tools for the pharmacological characterization of 5-HT₃ receptors.

Molecular biological techniques have led to the rapid discovery of additional serotonin-receptor subtypes and their properties

The first 5-HT receptor to be cloned was the 5-HT_1C receptor. Over the course of the next 5 years, the 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT₂ and 5-HT₃ receptors also were cloned. The 5-HT_1A, 5-HT_1B, 5-HT_1C, 5-HT_1D and 5-HT₂ receptors are single-subunit proteins that are members of the G protein receptor superfamily. This receptor family is characterized by the presence of seven transmembrane domains, an intracellular carboxy-terminus and an extracellular amino-terminus. It is the interaction of the receptor with the G protein that allows the receptor to modulate the activity of different effector systems, such as ion channels, phospholipase C and adenylyl cyclase. The transmembrane domains of G protein-coupled receptors are the most highly conserved regions of these proteins. The 5-HT₃ receptor differs from all other known subtypes of serotonin receptor in that it is a member of the ligand-gated ion channel superfamily. Members of this receptor superfamily consist of five subunits, each of which possesses four transmembrane segments and a large, extracellular N-terminal region.

The rapid discovery of additional subtypes of receptor for serotonin made it necessary to establish an unambiguous system of nomenclature. The current classification scheme takes into account not only operational criteria, such as drug-related characteristics, but also information about intracellular signal-transduction mechanisms and amino acid sequence of the receptor protein. For example, the 5-HT_1C receptor was reclassified as a 5-HT₂ receptor based on the sequence homology, similar pharmacological characteristics and effector coupling of the 5-HT₂ and 5-HT_1C receptors. The 5-HT₂ receptor was renamed 5-HT_2A and the 5-HT_1C receptor was renamed the 5-HT_2C [16]. The amino acid sequence of several new serotonin receptors has been reported. However, the classification of these receptors remains tentative due to limited knowledge of their operational and tranductional characteristics, which have only been described in transfected cell systems for these recombinant receptors. Because the functions mediated by these serotonin receptors in intact tissue are unknown, lowercase appellations are presently used [16].

The three serotonin receptor subfamilies, the 5-HT₁ family; the 5-HT₂ family; and the family that includes the 5-HT₄, 5-ht₆ and 5-HT₇ receptors, represent the three major classes of serotonin receptor that are members of the G protein-coupled receptor superfamily. As mentioned above, the 5-HT₃ receptor is a ligand-gated ion channel and is a separate subfamily. Although each serotonin receptor can be activated potently by serotonin, differences in signal-transduction mechanisms, neuroanatomical distribution and affinities for synthetic chemicals create opportunities for drug discovery and make each serotonin receptor subtype a potential therapeutic target.

The 5-HT₁ receptor family contains receptors that are negatively coupled to adenylyl cyclase and includes the 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-ht_1E and 5-ht_1F receptors. The 5-HT_1A receptor is coupled via G proteins to two distinct effector systems: (i) inhibition of adenylyl cyclase activity and (ii) the opening of K⁺ channels, which results in neuronal hyperpolarization. In terminal field areas of serotonergic innervation, such as the hippocampus, 5-HT_1A receptors are coupled to both effector systems (Table 13-2). However, in the dorsal raphe nucleus, 5-HT_1A receptors are coupled only to the opening of potassium channels.

The 5-HT_1B and 5-HT_1D receptor subtypes are also linked to inhibition of adenylyl cyclase activity (Table 13-2). Binding sites that have been defined pharmacologically as 5-HT_1B receptors have been characterized in the rat, mouse and hamster, whereas the 5-HT_1D receptor has been characterized using pharmacological criteria in species such as guinea pig, pig, cow and human. In the substantia nigra, where a high density of 5-HT_1B or 5-HT_1D receptors has been demonstrated by radioligand-binding studies, these serotonin receptors are linked to the inhibition of adenylyl cyclase through a G protein.

An issue raised by the use of molecular biological techniques for the study of neurotransmitter receptors is whether a receptor is a subtype or a species homolog, that is, an equivalent receptor in different species. For example, the 5-HT_1B and 5-HT_1D receptors originally were considered to be species variants of the same receptor because the pharmacological profiles of these two receptors are similar, although not identical; the distribution of these two receptors in brain is very similar; and both receptors are coupled to the inhibition of adenylyl cyclase. Although biochemical, pharmacological and functional data suggest that the 5-HT_1B receptor found in rats and mice and the 5-HT_1D receptor found in other species, including humans, are functionally equivalent species homologs, the story has been complicated somewhat by the discovery of two genes encoding the human 5-HT_1D receptor, 5-HT_1Dα and 5-HT_1Dβ [16].

Radioligand-binding studies currently do not allow the differentiation of 5-HT_1Dα and 5-HT_1Dβ receptors, and the binding profiles of these receptor subtypes match the previously described 5-HT_1D-binding site. Furthermore, a rat homolog of the human 5-HT_1Dα receptor has been isolated and shown to encode a receptor with a 5-HT_1D-binding site profile, suggesting that the 5-HT_1B and 5-HT_1D receptors may not be species homologs but distinct 5-HT receptor subtypes. There is still some debate as to whether a common appellation should be used to refer to the protein products of two distinct genes, the 5-HT_1Dα and the 5-HT_1Dβ receptor, and whether the human 5-HT_1Dβ receptor should be called the human 5-HT_1B receptor, even though it has a distinct pharmacological profile from that of the rat 5-HT_1B receptor. Because there are no compounds currently available to differentiate between the 5-HT_1Dα and the 5-HT_1Dβ receptors, we will refer to them in this chapter as 5-HT_1D. Furthermore, because of distinct pharmacological profiles of the 5-HT_1B receptor found in rat and the 5-HT_1D receptor found in other species, we will not refer to the 5-HT_1Dβ as the human 5-HT_1B receptor.

The 5-ht_1E receptor originally was identified in homogenates of human frontal cortex by radioligand-binding studies with [³H]5-HT in the presence of 5-carboxamidotryptamine (5-CT) to block 5-HT_1A and 5-HT_1D receptor sites. Because of the lack of specific radioligands for the 5-ht_1E receptor, the overall distribution in brain is unknown. With the cloning of the various subtypes of receptors for serotonin, knowledge of receptor sequences can be used to generate radioactive probes for mRNAs encoding individual serotonin receptor subtypes. Using in situ hybridization histochemistry, the localization of these mRNAs and, thus, the distribution of cells expressing the mRNAs for serotonin receptors can be established in brain. 5-ht_1E receptor mRNA has been found in the caudate putamen, parietal cortex and olfactory tubercle [17]. The function of the 5-ht_1E receptor in intact tissue is not known due to the lack of selective agonists or antagonists. In transfected cells, the 5ht_1E receptor is coupled to the inhibition of adenylyl cyclase activity. The 5-ht_1E receptor displays a higher degree of homology with the 5-HT_1D receptor (64%) than any other 5-HT₁ receptors [16].

The 5-HT_1F receptor was cloned and sequenced in 1993 and shares the greatest sequence homology with the 5-ht_1E receptor (61%). 5-ht_1F receptor mRNA is found in cortex, hippocampus, dentate gyrus, nucleus of the solitary tract, spinal cord, trigeminal ganglion neurons, uterus and mesentery. In transfected cells, the 5-ht_1F receptor is coupled to the inhibition of adenylyl cyclase [16]. Because selective agonists or antagonists for the 5-ht_1F receptor have not been available until very recently, little is known about the distribution or function of the 5-ht_1F receptor in brain. The selective agonist radioligand [³H]LY334370 has been used to demonstrate the presence of 5-ht_1F receptor sites in cortex, striatum, hippocampus and olfactory bulb [18]. Activation of 5-ht_1F receptors in vivo inhibits neurogenic dural inflammation and dural protein extravasation.

The 5-HT₂ receptor family stimulates phosphoinositide-specific phospholipase C (PI-PLC) and includes the 5-HT_2A, 5-HT_2B and 5-HT_2C (formerly the 5-HT_1C) receptors. 5-HT_2A receptor-mediated stimulation of phosphoinositide hydrolysis has been well characterized in cerebral cortex. 5-HT_2C receptor-mediated stimulation of inositol lipid hydrolysis has been studied in the choroid plexus (Table 13-2). Stimulation of phosphoinositide turnover by 5-HT in these tissues is not dependent on the activity of lipoxygenase or cyclooxygenase pathways, nor is it blocked by agents that inhibit neuronal firing, suggesting that coupling of the 5-HT_2A or 5-HT_2C receptor to the enzyme PI-PLC mediates the enhanced response (see Chap. 21). Activation of 5-HT_2A receptors also mediates neuronal depolarization, a result of the closing of potassium channels. The 5-HT_2A receptor was first cloned in the rat by homology with the rat 5-HT_2C receptor. The rat 5-HT_2A receptor is 49% homologous to the rat 5-HT_2C receptor.

Cloning of the 5-HT_2A receptor has been used to gain insight into a controversy over the nature of agonist binding to the 5-HT_2A receptor. The hallucinogenic amphetamine derivative [³H]2,5-Dimethoxy-4-bromoamphetamine (DOB), an agonist, binds to a small number of sites with properties very similar to those of the receptor labeled with the antagonist [³H]ketanserin. Agonists, though, have higher affinities for the receptor labeled with [³H]DOB than for that labeled with [³H]ketanserin. Some investigators have interpreted these and other data as evidence for the existence of a new subtype of 5-HT_2A receptor, whereas others have interpreted these data as indicative of agonist high-affinity and agonist low-affinity preferring states of the 5-HT_2A receptor. In experiments in which the cDNA encoding the 5-HT_2A receptor was transfected into clonal cells, binding sites for both the 5-HT_2A receptor antagonist [³H]ketanserin and the 5-HT₂ receptor agonist [³H]DOB were found. Furthermore, agonists had higher affinities for [³H]DOB binding than for [³H]ketanserin binding. Thus, a single gene produces a protein with both binding sites, substantiating the view that agonist and antagonist binding are to different states, rather than to two different subtypes, of the 5-HT_2A receptor.

Although the 5-HT_2B receptor is the most recently cloned of the 5-HT₂ receptor class, it was among the first of the serotonin receptors to be characterized using pharmacological criteria. The first report of the sensitivity of rat stomach fundus to serotonin was published by Vane in 1959. This receptor, whose activation results in the contraction of fundus smooth muscle, originally was placed in the 5-HT₁ receptor class by Bradley and associates [15] because of its sensitivity to serotonin and because responses mediated by it were not blocked by 5-HT₂ or 5-HT₃ receptor antagonists. It has been reclassified as a 5-HT₂ receptor because of its similar pharmacological profile to the 5-HT_2C receptor (Table 13-2). The recombinant receptor expressed in clonal cells is coupled to the stimulation of inositol lipid hydrolysis. However, in rat stomach fundus, the 5-HT_2B appears not to be coupled to phosphoinositide hydrolysis. 5-HT_2B receptor-mediated contraction of rat stomach fundus is dependent on the influx of calcium through voltage-sensitive channels, intracellular calcium release and activation of PKC [19]. The effector system to which this receptor is coupled in the CNS remains to be established. Using quantitative polymerase chain reaction (PCR), 5-HT_2B mRNA has been detected in the rat stomach fundus, intestine, kidney, heart, lung and dura mater but not in rat brain. In humans, 5-HT_2B receptor mRNA has been found peripherally and in cerebellum, cerebral cortex, amygdala, substantia nigra, caudate, thalamus, hypothalamus and retina [16].

The 5-HT₃ receptor is homomeric and belongs to the ligand-gated ion channel superfamily. As mentioned above, the 5-HT₃ receptor is a serotonin-gated cation channel that causes the rapid depolarization of neurons (Table 13-2). The depolarization mediated by 5-HT₃ receptors is caused by a transient inward current, specifically the opening of a channel for cations. A single subunit of the 5-HT₃ receptor, the 5-HT₃-A receptor subunit, has been cloned. An alternatively spiced variant, the 5-HT₃-As receptor subunit, has been identified in mouse, rat and human. The cloned receptor subunit exhibits sequence similarity to the α subunit of the nicotinic acetylcholine receptor and to the β₁ subunit of the GABA_A receptor. It is not known whether the native 5-HT₃ receptor is composed of this single subunit or several different subunits. Although single subunits of members of the ligand-gated ion channel receptor family can form functional homomeric receptors, they generally lack some of the properties of the native, multisubunit receptor. The cloned subunit of the 5-HT₃ receptor has been studied in Xenopus oocytes injected with mRNA encoding this receptor. Although the expressed 5-HT₃-A and 5-HT₃-As receptors are functional, they do not display all of the characteristics of native 5-HT₃ receptors. The 5-HT₃ receptor, like other members of the ligand-gated ion channel superfamily, appears to possess additional pharmacologically distinct recognition sites for alcohols and anesthetic agents, by which the function of this receptor can be allosterically modulated [20].

5-HT₄ , 5-ht₆ and 5-HT₇ receptors are included in a family of serotonin receptors coupled to the stimulation of adenylyl cyclase. The 5-HT₄ receptor originally was described in cultured murine collicular neurons as a serotonin receptor coupled to the stimulation of adenylyl cyclase activity, possessing pharmacological characteristics distinct from those of the 5-HT₁, 5-HT₂ or 5-HT₃ receptors. The 5-HT₄ receptor gene has been cloned from rat brain RNA by reverse transcriptase (RT)-PCR [21]. Two different cDNA clones, the long isoform, 5.5-kb 5-HT₄₁, and the short isoform, 4.5-kb 5-HT_4s, have been isolated and are most likely the result of alternative splicing of 5-HT₄ receptor mRNA.

The 5-ht₆ receptor is approximately 30% homologous to other serotonin receptors. When expressed in transfected cells, it shows high affinity for [¹²⁵I]LSD and [³H]5-HT. The pharmacology of this recombinant receptor is unique. Interestingly, this receptor has high affinity for various antipsychotic and antidepressant drugs, such as clozapine, amitriptyline, clomipramine, mianserin and ritanserin. The 5-ht₆ receptor stimulates adenylyl cyclase when expressed in some, but not all, cell systems. The function of the 5-ht₆ receptor in intact tissue has not been characterized due to the lack of selective agonists or antagonists. Expression of 5-ht₆ receptor mRNA has been detected in the striatum, nucleus accumbens, olfactory tubercle, hippocampus and cerebral cortex [16].

Two rat 5-HT₇ receptor clones, which differ only in the C terminus and presumably result from alternative mRNA splicing, have been identified. The 5-HT₇ receptor shows the highest amino acid sequence homology with the Drosophila 5-HT_1A receptor, 42%, and approximately 35% homology with all other serotonin receptors. To date, no selective agonists or antagonists have been described for the 5-HT₇ receptor. In transfected cells, the 5-HT₇ receptor stimulates adenylyl cyclase. 5-HT₇ receptors have been identified in human vascular smooth muscle cells and frontal cortical astrocytes in primary culture, where they are coupled to the stimulation of adenylyl cyclase.

The 5-ht_5A and 5-HT_5B receptors may constitute a new family of serotonin receptors since neither is coupled to adenylyl cyclase or PI-PLC; their effector systems are currently unknown. Both the 5-ht_5A and 5-HT_5B receptors were cloned by using degenerate oligonucleotides derived from TMDs III and VI of G protein-coupled serotonin receptors. Both genomic clones possess one intron in the middle of the third cytoplasmic loop. The receptor proteins are 77% identical to each other, whereas the homology to other serotonin receptors is low.

5-ht_5A receptor mRNA transcripts have been detected by in situ hybridization in the cerebral cortex, hippocampus, granule cells of the cerebellum, medial habenula, amygdala, septum, several thalamic nuclei and olfactory bulb of the rat and mouse. 5-HT_5B mRNA has been detected by in situ hybridization in the hippocampus, habenula and the dorsal raphe nucleus of rat and human [16].

Immunohistochemical studies with antibodies to the 5-ht_5A receptor have shown this receptor to be expressed predominantly by astrocytes, although some neurons in cortex were labeled as well. In transfected cells expressing the 5-ht_5A receptor, 5-HT does not stimulate the formation of cAMP as it does in wild-type cells. Furthermore, 5-HT inhibits forskolin-stimulated cAMP formation, an effect not seen in wild-type cells. Thus, the 5-ht_5A receptor appears to be coupled to the inhibition of adenylyl cyclase activity [22]. At the present time, the functional correlate and transductional properties are unknown for the 5-HT_5B receptor.

The many serotonin-receptor subtypes are differentiated by their localization in the central nervous system

5-HT_1A receptors are present in high density in the hippocampus, septum, amygdala, hypothalamus and neocortex (Table 13-2). Destruction of serotonergic neurons with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) does not reduce 5-HT_1A receptor number in forebrain areas, indicating that 5-HT_1A receptors are located postsynaptically in these brain regions. Many of these serotonergic terminal field areas are components of the limbic system, the pathway thought to be involved in the modulation of emotion. The presence of 5-HT_1A receptors in high density in the limbic system indicates that the reported effects of 5-HT or serotonergic drugs on emotional states could be mediated by 5-HT_1A receptors. The presence of 5-HT_1A receptors in the neocortex suggests that this receptor also may be involved in cognitive or integrative functions of the cortex. 5-HT_1A receptors are also present in high density in serotonergic cell body areas, in particular the dorsal and median raphe nuclei, where they function as somatodendritic autoreceptors, modulating the activity of serotonergic neurons. Activation of these autoreceptors causes a decrease in the rate of firing of serotonergic neurons and a reduction in the release of 5-HT from serotonergic terminals. Neurotoxin-induced destruction of serotonergic cell bodies dramatically reduces the number of 5-HT_1A receptors in these areas, consistent with their location on serotonergic soma.

The 5-HT_1B receptor in rats and mice and the 5-HT_1D receptor in bovine and human brain are located in high density in the basal ganglia, particularly in the globus pallidus and the substantia nigra (Table 13-2). Functional studies indicate that the 5-HT_1B and 5-HT_1D receptors are located on presynaptic terminals of serotonergic neurons and modulate the release of serotonin. Release of 5-HT from the dorsal raphe nucleus also appears to be under the control of 5-HT_1B/1D receptors, although it is unclear whether these receptors are located on serotonergic terminals or cell bodies. The 5-HT_1B and 5-HT_1D receptors also are located postsynaptically, where they may modulate the release of other neurotransmitters, such as acetylcholine (ACh) in the hippocampus and DA in the prefrontal cortex. The presence of these receptors in high density in the basal ganglia raises the interesting possibility that they may play a role in diseases of the brain which involve the basal ganglia, such as Parkinson's disease.

A high density of 5-HT_2A receptors is found in many cortical areas. These receptors are particularly concentrated in the frontal cortex. 5-HT_2A receptors also are found in high density in the claustrum, a region which is connected to the visual cortex; in parts of the limbic system; and in the basal ganglia and the olfactory nuclei (Table 13-2). 5-HT_2A receptors in the cortex are thought to be located postsynaptically on intrinsic cortical neurons as destruction of projections to the cortex does not reduce 5-HT_2A receptors. Because of the lack of selective agonists to differentiate between members of the 5-HT₂ receptor family, many of the functional and clinical correlates of the 5-HT_2A receptor may very well involve or be attributed to the 5-HT_2C receptor.

5-HT_2C receptors are present in high density in the choroid plexus. High-resolution autoradiography has shown that they are enriched on the epithelial cells of the choroid plexus. It has been proposed that 5-HT-induced activation of 5-HT_2C receptors could regulate the composition and volume of the cerebrospinal fluid. 5-HT_2C receptors also are found throughout the brain, particularly in areas of the limbic system, including the hypothalamus, hippocampus, septum, neocortex and regions associated with motor behavior, including the substantia nigra and globus pallidus. 5-HT_2C receptors are present in much lower concentrations in these areas than in the choroid plexus (Table 13-2). The lack of truly selective 5-HT_2C receptor agonists and antagonists has limited our knowledge about the functional role of these receptors in brain.

5-HT₃ receptors initially appeared to be confined to peripheral neurons, where they mediate depolarizing actions of 5-HT and modulate neurotransmitter release. 5-HT₃ receptors are found in high density in peripheral ganglia and nerves, including the superior cervical ganglion and vagus nerve, as well as in the substantia gelatinosa of the spinal cord. Their localization in spinal cord and medulla suggests that 5-HT could modulate nociceptive mechanisms via the 5-HT₃ receptor. 5-HT₃ receptors facilitate the release of substance P in the spinal cord [23]. The localization of 5-HT₃ receptor-binding sites in cortical and limbic areas of the brain is consistent with behavioral studies in animals which suggest that 5-HT₃ receptor antagonists may have potential anxiolytic, antidepressant and cognitive effects. 5-HT₃ receptors are located postsynaptically, where they modulate the release of neurotransmitters such as ACh or DA. 5-HT₃ receptors modulate the activity of dopaminergic neurons in the ventral tegmental area. In the cortex and hippocampus, the majority of neurons expressing 5-HT₃ receptor mRNA are GABAergic. The highest density of 5-HT₃ receptor sites in the brain is in the area postrema, the site of the chemoreceptor trigger zone (Table 13-2).

Studies of the 5-HT₄ receptor, originally characterized by measuring cAMP production in cultured mouse collicular neurons, have been hampered by the absence of a high-affinity radioligand. The synthesis and development of specific radioligands, [³H]GR 113808 and [¹²⁵I]SB 207710, have provided the necessary tools for the study and characterization of the 5-HT₄ receptor. 5-HT₄ receptor binding sites are localized with high densities in the striatum, substantia nigra and olfactory tubercle and have been reported in the hippocampus as well (Table 13-2). The 5-HT₄ receptor indirectly mediates the enhancement of striatal DA release by 5-HT, although 5-HT₄ receptors do not appear to be located on striatal DA terminals. In the alimentary tract, 5-HT₄ receptors are located on neurons, for example, the myenteric plexus of the ileum, smooth muscle cells and secretory cells, where they evoke secretions and the peristaltic reflex.

Although there is no known selective agonist or antagonist available for the 5-HT₇ receptor, the distinct pharmacological profile of 5-HT₇ receptor sites, that is, the potent agonism by the 5-HT₁ receptor agonist 5-CT and antagonism by methiothepin, clozapine and a variety of ergot compounds, has been used to delineate the function and distribution of this receptor in vivo. 5-HT₇ receptor-binding sites in the rat brain have been described using receptor autoradiography in layers 1–3 of the cortex, septum, thalamus, hypothalamus, amygdala and superior colliculus [24] (Table 13-2). In the periphery, the 5-HT₇ receptors mediate relaxation of vascular smooth muscle.

An atypical 5-HT receptor exists on the enteric neurons of the gut. This receptor has high affinity for [³H]5-HT and mediates a slow depolarization of particular myenteric neurons that is not blocked by selective 5-HT₃ antagonists. It has been termed the 5-HT_1P receptor as it has a high affinity for 5-HT and is found in the periphery. The available functional and radioligand-binding data confirm the orphan status of the 5-HT_1P receptor and emphasize the need to establish a rigorous basis for its positive identification [16].

Many serotonin-receptor subtypes do not appear to undergo compensatory regulatory changes

Classically, a decrease in exposure of a tissue to its endogenous transmitter leads to a supersensitive, or exaggerated, response to exogenous agonist, which may be accounted for by an increase in the density or upregulation of postsynaptic receptors for the transmitter. Conversely, increased exposure of a tissue to agonists overtime will result in a decreased responsiveness, or desensitization, to the agonist, which may be due, at least in part, to a decrease, or downregulation, in receptor density. Central β₁-noradrenergic and D2-dopaminergic receptors undergo such regulatory processes.

Chronic or repeated administration of antidepressant drugs, such as MAO inhibitors or inhibitors of serotonin uptake, or 5-HT_1A receptor agonists to laboratory rats results in a desensitization of behavioral and electrophysiological responses believed to be mediated by 5-HT_1A receptors. Lesioning serotonergic neurons results in increased behavioral and electrophysiological responses. However, these treatments do not result in changes in 5-HT_1A receptors as measured with binding assays. Some investigators have reported diminished 5-HT_1A receptor-mediated inhibition of adenylyl cylcase following repeated administration of some antidepressant drugs to rats. However, desensitization of second-messenger function has not been observed consistently after chronic antidepressant or agonist treatments.

Lesions of serotonergic neurons do not cause detectable changes in 5-HT_1B receptors in forebrain areas and have been reported to cause upregulation, downregulation or not to effect the density of 5-HT_1B receptors in substantia nigra. Interpretation of these reports may be complicated by the fact that the 5-HT_1B receptor is located both pre- and postsynaptically. Cells maintained in culture represent an alternative to in vivo systems. The 5-HT_1B receptor is found on an epithelial cell line from opossum kidney (OK cells). Exposure of OK cells to 5-HT results in a time- and dose-dependent decrease in the density of 5-HT_1B receptors and a desensitization of the 5-HT_1B receptor-mediated inhibition of forskolin-stimulated cAMP accumulation [25]. It seems, then, that the 5-HT_1B receptor can downregulate in response to prolonged exposure to an agonist.

5-HT_2A receptors do not respond to changes in agonist exposure in the classic manner. Specifically, no change in 5-HT_2A receptor density is observed after lesioning serotonergic neurons or after depletion of serotonin stores. 5-HT_2A receptor-mediated phosphoinositide hydrolysis is also unchanged after such treatments, suggesting that denervation supersensitivity does not occur. Thus, it appears that neither the 5-HT_2A receptor nor its second-messenger pathway is regulated by a decrease in neurotransmitter exposure. After administration of hallucinogenic 5-HT₂ receptor agonists or chronic administration of selective inhibitors of serotonin uptake, 5-HT_2A receptor-mediated inositol lipid hydrolysis becomes desensitized and 5-HT_2A receptors downregulate. Surprisingly, 5-HT_2A receptor desensitization and downregulation also occur following administration of drugs that are antagonists at 5-HT_2A receptors, such as ketanserin, the atypical antidepressant mianserin and atypical antipsychotic drugs. Given that agonist exposure causes desensitization of 5-HT₂ receptors, it has been proposed that the absence of supersensitivity after denervation may reflect low tonic activity at synapses innervating 5-HT₂ receptors [26].

Following the lesioning of serotonergic neurons with neurotoxin, 5-HT_2C receptor-mediated phosphoinositide hydrolysis in choroid plexus is increased, indicating that these receptors undergo denervation supersensitivity. However, radioligand-binding studies fail to show an increase in 5-HT_2C receptor number or in receptor upregulation. Paradoxically, chronic administration of the 5-HT₂ receptor antagonist mianserin to rats results in downregulation of the 5-HT_2C receptor. In a fibroblast cell line transfected with 5-HT_2C receptor cDNA, phosphorylation is increased by agonist treatment and accompanies agonist-mediated desensitization [27].

5-HT₃ receptors, located on neurons in the periphery and in the CNS, mediate fast, excitatory responses, that is, membrane depolarization to serotonin. Like many other receptors that are ligand-gated ion channels, the 5-HT₃ receptor exhibits rapid desensitization after sustained agonist exposure. In addition to preparations of peripheral neurons, cultured hippocampal cells and neuroblastoma cells have been used to study this phenomenon.

Studies of 5-HT₇ receptor regulation have been performed on rat frontal cortical astrocytes in primary culture. In these cells, 5-HT₇ receptors are coupled to the stimulation of adenylyl cyclase. Exposure of astrocytes in culture to the atypical antidepressant mianserin or to the tricyclic antidepressant amitriptyline for 3 days increased the stimulation of cAMP accumulation in response to 5-HT [28]. Whether such effects are relevant for the therapeutic effects of these drugs is a topic for future research.

Serotonin may set the tone of brain activity in relationship to the state of behavioral arousal/activity

Serotonin has been implicated in practically every type of behavior, such as appetitive, emotional, motor, cognitive and autonomic. However, from a physiological perspective, it is not clear whether 5-HT affects such behaviors specifically or more generally by coordinating the activity of the nervous system, particularly to set the tone of activity in conjunction with the amount of arousal.

The primary body of data that has contributed to the view that 5-HT has a general effect on behavior by modulating the tone of nervous system activity comes from studies of the firing rate of serotonergic soma in raphe nuclei [29]. Under quiet waking conditions, serotonergic neurons display a slow, clock-like activity of about 1 to 5 spikes/sec, which shows a gradual decline as the animal becomes drowsy and enters slow-wave sleep. A decrease in the regularity of firing accompanies this overall slowing of neuronal activity. During rapid eye movement (REM) sleep, the activity of these neurons becomes silent. In response to certain types of arousing stimuli, the firing rate of these serotonergic neurons increases. Not surprisingly, such data led to the idea that the activity of serotonergic neurons is related to the level of behavioral arousal/activity. Such data also have contributed to the idea that the activity of serotonergic neurons is associated with motor output since atonia of the major skeletal muscle groups occurs during REM sleep. Also oral—buccal motor activity, such as chewing, biting, licking or grooming, causes a marked increase in the firing rate of a subgroup of serotonergic soma that are also activated by somatosensory stimuli applied to the head, neck and face. However, exposing a cat to environmental stressors, such as a loud noise or seeing a dog, although producing strong sympathetic activation and typical behavioral responses, does not alter the firing rate of serotonergic neurons. Thus, the type of motor activity that activates serotonergic soma seems to be repetitive, like that mediated by central pattern generators. Furthermore, activation of serotonergic transmission inhibits information processing in afferent systems. From all such data, it has been suggested that the serotonergic neuronal system functions at the organismic level to integrate functions needed for behavioral output, that is, facilitation of motor output with suppression of activity in sensory systems irrelevant to the ongoing behavior.

Serotonin appears to be involved in a wide variety of physiological functions and behaviors, such as eating, sleep, circadian rhythmicity and neuroendocrine function

Perturbation of the 5-HT system by different types of drugs can elicit alterations in behaviors. Drugs affecting serotonergic neurons and their receptors are used to treat diseases such as depression, anxiety disorders and schizophrenia. In part because of this, 5-HT also has been speculated to be involved specifically in the regulation of all types of behaviors and physiological processes. The possible involvement of 5-HT in three areas, neuroendocrine function, circadian rhythms and feeding behavior, will be highlighted for illustrative purposes.

The hypothalamus secretes several releasing factors and release-inhibiting factors to control the secretion of hormones from the anterior pituitary gland. Serotonin is among the many neurotransmitters that participate in the hypothalamic control of pituitary secretion, particularly in the regulation of adrenocorticotropin (ACTH), prolactin and growth hormone secretion. A direct synaptic connection between serotonergic terminals and corticotropin-releasing hormone (CRH)-containing neurons in the paraventricular nucleus of the hypothalamus has been described. Precursors of 5-HT or drugs that enhance the effect of 5-HT increase CRH in portal blood and ACTH in plasma. In addition to effects at the hypothalamus, 5-HT may have direct effects on the anterior pituitary to stimulate the release of ACTH and at the level of the adrenal cortex to regulate release of corticosterone or cortisol. Actions of serotonin on 5-HT_1A, 5-HT₂, 5-HT₃ and 5-HT₄ receptors seem to be involved in these effects on the hypothalamic—pituitary—adrenal axis [30]. However, what role, if any, is played by serotonin in regulating stress-induced elevations of CRH or the circadian periodicity of the hypothalamic—pituitary—adrenal axis is unclear.

Measurement of these endocrine responses after administration of drugs that increase brain serotonin function provides one of the few methods currently available for assessing such function in humans. Precursors of 5-HT, releasing agents, reuptake inhibitors and receptor agonists and antagonists have been used to probe serotonergic function. For example, intravenous administration of the serotonin precursor l-tryptophan consistently increases plasma concentrations of prolactin and growth hormone but not of ACTH or cortisol. Fenfluramine causes a dose-dependent increase in plasma prolactin. When administered to humans, serotonin agonists that stimulate 5-HT_1A and 5-HT₂ receptors also increase plasma concentrations of ACTH, cortisol, prolactin and perhaps growth hormone. The neuroendocrine response in humans to such agents has been used clinically to assess the functioning of the central serotonergic system in patients with psychiatric disorders.

Serotonin also appears to be involved in the regulation of circadian rhythms. The suprachiasmatic nuclei (SCN) of the hypothalamus generate electrophysiological and metabolic cycles which repeat approximately every 24 hr. When isolated in vitro, the SCN continue to produce 24-hr rhythms in metabolism, vasopressin secretion and spontaneous electrical activity, indicating that circadian time-keeping functions or pacemaker activity are endogenous characteristics of the SCN. Ordinarily, this rhythm is synchronized or entrained to the environmental photoperiod, also about 24 hr. A serotonergic contribution to circadian rhythm regulation has been postulated because the SCN receive very dense serotonergic innervation from the midbrain raphe nuclei. In addition, there is a serotonergic innervation to the intergeniculate leaflet (IGL), an area of brain through which photic information indirectly accesses the SCN.

Serotonin appears to function as an inhibitory transmitter that modulates the effects of light on circadian rhythmicity. Direct application of 5-HT or receptor agonists to the SCN blocks light-induced phase shifts during the subjective night but causes phase advances during the subjective day. Such agents inhibit the excitatory effect of light, measured electrophysiologically, in either the SCN or the lateral geniculate complex. The nonselective 5-HT agonist quipazine resets or shifts the rhythm of spontaneous electrical activity of single cells recorded extracellularly in SCN isolated in brain slices.

Lesions of serotonergic neurons in laboratory animals have been reported by some investigators to disrupt locomotor rhythms or result in loss of the daily rhythm of corticosterone. In the hamster, the median raphe nucleus projects to the SCN, whereas the dorsal raphe nucleus innervates the IGL; furthermore, serotonergic innervation to the SCN, and not the IGL, is necessary for the photic entrainment of locomotor activity [31]. It appears, then, that the SCN circadian pacemaker, or clock, is modulated by stimulation of serotonergic receptors in the SCN and that serotonergic projections to the SCN may modulate the phase of the SCN in intact animals.

The possible involvement of 5-HT in feeding behavior has been an active area of research for many years. Pharmacological studies have contributed primarily to the idea that 5-HT has an inhibitory effect on feeding behavior. Drugs that either directly or indirectly activate postsynaptic 5-HT receptors decrease food consumption, whereas agents that inhibit serotonergic transmission increase food intake. Precisely how this occurs is controversial, with claims that 5-HT governs the selection of macronutrients in the diet, influences responses to the taste qualities of food or modulates gastric activity to reduce feeding. Perhaps the most comprehensive and enduring view is that enhanced serotonergic activity enhances satiety, particularly by increasing the rate of satiation and prolonging the state of satiety [32].

Fenfluramine, originally the racemate and more recently the d-isomer, has been the prototypical drug for studying serotonergic mechanisms in feeding behavior. As mentioned previously, fenfluramine elicits the release of 5-HT and inhibits its reuptake (Fig. 13-7). d-Fenfluramine has an active de-ethylated metabolite, d-norfenfluramine, that contributes to the appetite-suppressant effects of the parent compound. Fenfluramine decreases meal size, rate of eating and eating between meals. This probably is related to its ability in humans to decrease the sensation of hunger and to increase the feeling of “fullness.” Serotonin-reuptake inhibitors, such as fluoxetine and serotonin precursors, mimic these effects. The effects of fenfluramine on feeding behavior are blocked by the nonselective serotonin receptor antagonist metergoline.

Multiple mechanisms in brain appear to be responsible for the effects of serotonergic drugs on satiety; for example, postsynaptic 5-HT_1B receptors are involved in regulating the size of meals eaten, but 5-HT_2C receptors influence the rate of eating. The sites in brain where these drugs, and presumably 5-HT, cause such effects remain to be identified. The paraventricular nucleus (PVN) of the hypothalamus may be an important site, although there are data indicating that actions on the PVN may be sufficient, but not necessary, to reduce caloric intake. In addition to brain mechanisms, 5-HT may act through peripheral mechanisms to produce satiety.

The pharmacological effects produced by drugs such as fenfluramine on feeding behavior in animals have led to its use in the treatment of obesity in humans. In many double-blind, placebo-controlled trials, chronic administration of fenfluramine causes greater weight loss than placebo. Although not as extensively studied clinically, fluoxetine produces similar effects. Weight gain occurs when fenfluramine is stopped, which indicates that the weight loss was related to its administration [33]. Given all of the medical problems associated with obesity, anorectic agents are valuable tools to be used in association with other modalities, such as diet and exercise, in the treatment of the truly obese individual.

5-Hydroxytryptamine not only has important physiological effects of its own but also is the precursor of the hormone melatonin

The human pineal gland weighs about 150 mg and occupies the depression between the superior colliculi at the posterior border of the corpus callosum. Although there are physical connections between the pineal gland and brain, the pineal gland lies “outside” the blood—brain barrier (see Chap. 32) and is innervated primarily by sympathetic nerves arising from the superior cervical ganglia.

Extracts of the pineal gland were reported as early as 1917 to lighten frog skin in vitro; in the late 1950s, the pineal hormone, melatonin, which produces this effect was isolated and its chemical structure, 5-methoxy-N-acetyltryptamine, described (Fig. 13-5). Melatonin is synthesized from serotonin, and the pineal gland contains all of the enzymes necessary to synthesize serotonin from tryptophan as well as two additional enzymes required to convert serotonin to melatonin (Fig. 13-5). The rate-limiting enzyme, serotonin N-acetyltransferase (EC 2.31.87), or arlylalkylamine N-acetyltransferase (AANAT), converts serotonin to N-acetylserotonin; this product is converted to melatonin by the enzyme 5-hydroxyindole-O-methyltransferase (HIOMT), which uses S-adenosylmethionine as the methyl donor. The human AANAT gene has been cloned and has considerable sequence identity to the sheep and rat genes. The human gene is localized on chromosome 17. The gene product is a 23.2-kDa protein that contains putative phosphorylation sites. Such sites are likely to be involved in the cAMP-dependent regulation of enzyme activity.

A unique feature of pineal gland physiology is that the synthesis and secretion of melatonin is influenced markedly by the light—dark cycle, acting through a multisynaptic pathway that relays in the superior cervical ganglia of the sympathetic nervous system. During daylight, the synthesis and secretion of melatonin are reduced, as is impulse flow along the sympathetic nerves innervating the pineal gland. At the onset of darkness, there is activation of these nerves, and the increased release of NE from them activates β adrenoceptors on the pineal gland to increase the formation of cAMP, with activation of α₁ adrenoceptors further amplifying the response. This second messenger causes activation of AANAT so as to increase the synthesis of melatonin. The extent of the nighttime increase in AANAT activity is very species-dependent, being, for example, as much as 150-fold in rats but only 1.5-fold in sheep. What type of rhythm is exhibited by humans is not yet known, although mRNA for AANAT is abundant in the pineal gland of humans during the day, as it is in sheep, whereas the transcript is nearly undetectable during the day in the rat pineal gland.

Thus, the pineal gland functions as a neuroendocrine transducer. In mammals, photosensory information impinging on the retina influences the activity of its neuronal projections, which ultimately inhibits or stimulates the secretion of melatonin. A circadian rhythm of melatonin secretion persists in animals housed in continuous darkness. Thus, melatonin synthesis is turned on by an endogenous “clock,” probably located within the SCN of the hypothalamus, with the daily rhythm normally being entrained to the day—night, light—dark cycle [34].

The exact physiological and behavioral effects of melatonin in humans are unclear. Such effects primarily result from the actions of melatonin on the SCN to influence the timing of circadian rhythms. The effects of melatonin are mediated by its activation of specific receptors. Two mammalian receptors for melatonin have been cloned [35], both of which belong to the G protein-coupled receptor family. A third subtype has been cloned from chickens but not yet in mammals. The melatonin_1A receptor is expressed in the hypophyseal pars tuberalis (PT) and the SCN, presumed sites of the reproductive and circadian effects of melatonin. The human melatonin_1B receptor is 60% identical to, and exhibits similarity in, its pharmacological profile and second-messenger coupling to the melatonin_1A receptor. It is found most abundantly in retina and to a lesser extent in brain. It has not been detected in the PT or the SCN. It seems likely that the ability of melatonin to act in the retina to affect some light-dependent functions, such as photopigment disc shedding and phagocytosis, may be due to its activation of the melatonin_1B receptor.

Perhaps the strongest case can be made for melatonin playing a role in reproduction, particularly in seasonally breeding mammals such as hamsters or sheep, which time their reproductive cycles via changes in the photoperiod. Information on day length may be relayed to the hypothalamic—pituitary—gonadal axis by the pattern of melatonin production. Although the effects of melatonin on reproduction were believed to be solely antigonadotropic, melatonin has been shown to be capable of causing progonadotropic effects. The type of effect caused by melatonin is dependent on the time point in the photoperiod when it is administered, the length of the photoperiod, the species and the dose administered. In general, the effects of melatonin are most robust when given around the time of the light-to-dark transition.

Melatonin has potent sedative and hypnotic activity. This has been demonstrated in double-blind, placebo-controlled studies. The hypnotic effect of melatonin seems to be separable from its effects on circadian rhythms. Although far from being fully understood, the entraining effect of melatonin on biological rhythms has led to its being used by humans for disorders that may be related to disturbances of circadian rhythms. For example, it is used to alleviate symptoms of jet lag as well as alertness-related problems in shift workers. Much more research will be needed to establish its efficacy in such conditions. Unfortunately, claims of its usefulness for these types of problems far exceed any controlled clinical data demonstrating such effects. At a different level, but one receiving comparable attention in the popular press as those mentioned above, is the purported antiaging properties of melatonin due to its antioxidant properties. Based on its demonstrated antioxidant properties, seen both in vitro and in vivo, melatonin has been speculated to be part of the natural defense system of the body against the toxic effects caused by free radicals.