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.

Table 13-2

Serotonin Receptors Present in the Central Nervous System.

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.