Neuronal Serotonin Receptor and Transporter Pharmacology

The ancient biochemical 5-hydroxytryptamine—also known as 5-HT and serotonin—has been controlled through evolution to be used widely across the plant and animal kingdoms.

5-HT is used by mammals as a neurotransmitter within the peripheral and central nervous systems. It is also used as a local hormone by many other tissues, such as immune cells, the cardiovascular system and the gastrointestinal tract. Due to this multiplicity of function, 5-HT is implicated in a wide range of pathological and physiological processes. This vast number of roles has led to the development of a number of compounds of therapeutic value, such as numerous antiemetic, antipsychotic and antidepressant drugs.

5-HT has the ability to mediate an array of actions and this is partly due to the vast number of 5-HT receptor families and subtypes that have evolved¹. At present, 18 genes have been identified, which encode 14 discrete mammalian 5-HT receptor subtypes that are separated into seven families, all but one of which are part of the G-protein coupled receptor (GPCR) superfamily. The 5-HT₃ receptor is a Cys-loop ligand-gated ion channel (LGIC) that evolved independently of the GPCR 5-HT receptors together with other members of this superfamily (for example GABA_A receptor, nicotinic acetylcholine receptor, Zn²⁺-activated receptor and glycine receptor). Additional receptor heterogeneity is produced through RNA editing (the 5-HT_2C receptor), alternative splicing (for example 5-HT₃, 5-HT4 and 5-HT7 receptors), and the putative formation of heterodimers and homodimers (5-HT₄ and the β₂ adrenoceptor receptor)².

The 5-HT₁ Receptor Family

This family includes five individual gene products: 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-ht_1e, and 5-HT_1F receptors.

Previous, some of these receptors were believed to be species-specific homologs (for example, the 5-HT_1D receptor in humans and 5-HT_1B receptor in rats). However, the genes for each of these receptors are now known to exist in all mammalian species investigated to date. Each receptor is encoded by one, intron-less reading frame and they share significant sequence homology. All 5-HT₁ family receptors couple to G_i/o to inhibit adenylyl cyclase and lower cAMP levels, however, alternative signal transduction mechanisms have also been elucidated. While the molecular properties and gene structure of these receptors are analogous, discrete patterns of regional expression in the body and major cellular variations underlie divergent physiological traits. A number of these receptors are known to be autoreceptors that control the release of serotonin and the excitability of serotonin neurons³. However, these receptors are also expressed in nonserotonergic neurons, where they can have similar impacts on other neurotransmitters.

5-HT_1A Receptors

5-HT_1A receptors are widely distributed in the CNS, where they can also be found in the dendrites, soma, and in certain cases the axon hillock of neurons, and astrocytic cell body and processes. The 5-HT1A receptor is expressed by several nonserotonergic neurons (as heteroreceptors) and by all serotonin neurons (as autoreceptors). When the 5-HT_1A receptor is activated, its electrophysiological effect on neurons is usually inhibitory and acts by decreasing the neuronal firing rate.

Several highly selective ligands have been created, and they range from partial agonists to full agonists, inverse agonists, and antagonists. It is believed that 5-HT1A receptors are the therapeutic targets for a number of neuropsychiatric disorders such as schizophrenia, depression, and anxiety. Clinically used ligands of 5-HT_1A receptor include some of the atypical antipsychotics, which have have neutral antagonist or partial agonist activity, and buspirone, a partial agonist used for generalized anxiety disorder. 5-HT_1A receptor partial agonists are anxiolytic drugs that are clinically useful and could act on the autoreceptors to decrease serotonergic activity, while 5-HT_1A receptors in the hippocampus region have been implicated in the regulation of the hypothalamic-pituitary-adrenal axis and in the mechanism of antidepressant action (by promoting neurogenesis).

Serotonin syndrome, hyperphagia, and hypothermia are other physiological effects of activation of the CNS 5-HT_1A receptor¹. 5-HT_1A receptor knockout mice were found to have heightened anxiety and can display reduced depression-like features^4,5. Just like the other serotonin receptors, this could involve receptor actions during the early development of the brain and also during the processing of emotional experience in the adult.

Several highly selective ligands for 5-HT_1A receptors have been developed, but it must be noted that some of these share affinity for 5-HT₇ receptors (for example, 8-OH-DPAT) or for other 5-HT₁ subtypes or 5-HT2 receptors. Most often, WAY 100635 has been utilized as a highly selective 5-HT_1A receptor neutral antagonist, while selective agonists include S-14506 and Xaliproden.

5-HT_1B Receptors

Similar to 5-HT_1A receptors, 5-HT_1B receptors are also widely distributed in the CNS in nonserotonergic and serotonergic neurons. These receptors are mainly translocated to axon terminals: hence there is an anatomical discrepancy between the mature 5-HT_1B receptor protein and the localization of mRNA. The 5-HT_1B receptor was originally assumed to be the rat analog of 5-HT_1D receptors however, it is now evident that both types of receptors exist in all mammalian species inspected so far and that their regional distributions also vary⁶. β-adrenergic antagonists possess high affinity for 5-HT_1B receptors only in some species and not all.

5-HT_1B autoreceptors reduce 5-HT synthesis and release and improve reuptake through the serotonin transporter (SERT). In addition, 5-HT_1B heteroreceptors suppress the release of many different neurotransmitters, based on the types of neurons expressing them. When 5-HT_1B receptor agonists are systemically administrated, they lead to a number of behavioral effects such as decreased aggression, changes in brain reward mechanisms, and increased locomotion, while selective antagonists may have certain pro-cognitive potential^7,8. When these receptors are expressed in different and possibly competing sets of neurons this may have an impact on their utility as a clinical target, albeit many 5-HT_1B/D receptor agonists are known to be effective as anti-migraine treatments.

5-HT_1B receptor knockout mice have been widely tested and have a clear phenotype defined by increased aggression and a predisposition for addiction-like behaviors, in the majority of cases. However, the phenotype of these mice may rely on compensatory changes in the dopamine system during the course of development instead of being caused by reduced 5-HT_1B receptor signaling in adults.

Many moderately selective agonists, including the more brain-penetrant CP 94253 as well as CP 93129, have been developed, and antagonists like SB 224289 are generally used for identifying 5-HT_1B receptor-mediated responses.

5-HT_1D Receptors

Compared to 5-HT_1B receptors, 5-HT_1D receptors are expressed at more modest levels in the brain. However, the largest extent of expression appears to be in the raphe nuclei. Similarly, 5-HT_1D receptor binding sites exist at a lower level when compared to 5-HT_1B receptor binding sites in the majority of the brain regions⁹. Most of the evidence, utilizing 5-HT_1B receptor knockout mice as controls, denotes that the activity of terminal serotonin autoreceptors in the forebrain region is of the 5-HT_1B receptor type¹⁰. However, there may be somatodendritic 5-HT_1D autoreceptors that mediate the release of serotonin inside the raphe nuclei¹¹.

The problem for the majority of putative selective 5-HT_1D receptor ligands is that they have a high affinity for more than a single receptor, often either the 5-HT_1A or 5-HT_1B receptors, but usually not both. For example, ketanserin has about 100-fold higher affinity for human 5-HT1D when compared to 5-HT1B receptors, but it also has the highest affinity for 5-HT2A receptors. This enables drug combinations to attain conditions that are rationally selective for inhibition or activation of 5-HT_1D receptors.

5-ht_1e Receptors

The lower case letters in the 5-ht_1e receptors indicate that it has not been established to have meaningful physiological functions in vivo.

The existence of the 5-ht_1e receptor was initially hypothesized on the basis of radioligand binding studies utilizing brain homogenates, demonstrate a 5-HT₁-like receptor with low affinity for 5-carboxamidotryptamine. It is now evident that a number of binding sites could have contributed to this observation. For the 5-ht_1e receptor, the gene sequence has been cloned from a guinea pig brain genomic DNA and human placental library but has not been detected in mouse and rat¹².

Only a few pharmacological studies of this receptor have been performed in human tissue or in rodents, but despite this fact, it was identified by RT-PCR in numerous regions of guinea pig, and in the monkey and human brains through in-situ hybridization⁶. Moreover, highly selective ligands have not been developed, albeit several typical 5-HT₁ receptor antagonists and agonists exhibit modest affinity at these receptors in heterologous expression systems. A new technique for labeling 5-ht_1e binding sites in guinea pig was recently developed and this strategy may be useful for modeling human 5-ht_1e receptors¹³. The physiological importance of this receptor continues to be vague.

5-HT_1F Receptors

Detected in various species, the 5-HT_1F receptor has been cloned from mouse, guinea pig, rat, and human genomes. This receptor, similar to other members of the 5-HT₁ receptor family, suppresses adenylyl cyclase activity through a G_i-dependent mechanism. The 5-HT_1F receptor is expressed at modest levels in the CNS on both non-serotonergic and serotonergic cell bodies where it behaves as a heteroreceptor and autoreceptor, respectively.

Similar to 5-HT_1B receptors, 5-HT_1F receptors are expressed in vestibular nuclei and trigeminal ganglion neurons and have a high affinity for triptan drugs that are useful for treating migraine headache. The corresponding contribution of each of these two receptors to pain relief in migraine headaches has not been resolved yet. Less selective agonists that could bind multiple 5-HT₁ receptors (for example, 5-HT_1B/D/F subtypes, like the “triptans”) may alleviate migraine headaches through numerous mechanisms; hence, more selective drugs may have distinct side-effect profiles and clinical efficacy. For instance, the 5-HT_1F receptor agonist LY 334370 is relatively selective and has about 100-fold higher affinity for 5-HT_1F over 5-HT_1B receptors. Although active in animal models of anti-migraine activity, this compound appears to act on the trigeminal nucleus rather than through a vascular mechanism.¹⁴ No selective 5-HT_1F antagonists have been developed thus far.

The 5-HT₂ Receptor Family

5-HT_2A, 5-HT_2B, and 5-HT_2C receptors are the three members of the 5-HT₂ family. The pharmacological importance of these receptors is substantial owing to their complex pharmacological features and clinical significance. 5-HT₂ receptors were initially theorized based on a significant study performed by Picarelli and Gaddum in 1957¹⁵. With the help of a guinea pig ileum contraction bioassay, the duo identified two classes of “tryptamine” receptors (called “M” and “D”) corresponding to 5-HT₂ and 5-HT₃ receptors in present nomenclature.

Analogous sequence homology, overlapping pharmacology, and structural motifs are shared by the members of the 5-HT₂ receptor family, although highly selective ligands are available and significant ligand development has taken place¹⁶. Multiple signal transduction pathways, agonist-directed signaling, prominence of inverse agonists, and significant clinical roles in neuropsychiatric conditions some of the remarkable features of these receptors. The 5-HT₂ receptors—similar to 5-HT₁ receptors—have a seven-transmembrane domain motif but they have a tendency to conjugate with phospholipases C and A2. Moreover, the relative efficiency of coupling to these effectors differs based on the type of cell being studied.

5-HT_2A Receptors

Densely expressed in the forebrain, specifically the cortex, 5-HT_2A receptors are expressed in both pyramidal neurons and interneurons. Complex pharmacological patterns of activity are displayed by numerous ligands at 5-HT_2A receptors, spanning from full agonism to partial agonism, and also from neutral antagonism to inverse agonistic behavior.

It is believed that these different activities reflect ligand stabilization of various structural conformations, which have been successfully resolved by X-ray crystallography in certain cases. In addition, site-directed mutagenesis has been used for testing structure-activity models. Through the heterologous expression of 5-HT_2A receptors and molecular strategies, a large amount of biophysical data has been created. Concurrent analysis of numerous signal transduction mechanisms in cell culture systems has also demonstrated that the same ligand can have varying degrees of intrinsic activity for activating different second messenger systems by the same 5-HT_2A receptor population¹⁷. This additionally supports the concept of dynamic and complex structure-activity relationships for 5-HT_2C and 5-HT_2A receptors.

Compared to other 5-HT receptors, 5-HT has low affinity for the 5-HT_2A receptor, with a K_d being in the low micromolar range. Nevertheless, it can be debated that the active and high-affinity conformational state has about ten-fold higher affinity for the 5-HT_2A receptor. A number of agonists, including LSD and DOI, have a high affinity for 5-HT_2A receptors and others show selective potency for triggering one signal transduction pathway over another (for example, TCB-2). However, these compounds are not specifically selective because they also bind to other 5-HT₂ receptors.  Many well-characterized antagonists show high selectivity for 5-HT_2A receptors, including MDL 100907 and ketanserin. While several others have high affinity, their specificity is not completely described. There are certain 5-HT_2A receptor agonists that create psychotomimetic effects (LSD is the most well-known), and hence many antipsychotic medications are high-affinity 5-HT_2A receptor antagonists.

5-HT2_B Receptors

Although 5-HT_2B receptors are sporadically expressed in district subregions of CNS, they are densely expressed in heart, kidney, liver and the fundus of the stomach. This pattern of expression is different from 5-HT_2C and 5-HT_2A receptors, which are expressed at comparatively higher levels in the CNS. These receptors share affinity for many of the same kinds of drugs, but only a few highly selective 5-HT_2B receptor antagonists, including RS 127445, have been described. Although the physiological role of 5-HT_2B receptors is still vague, they have been implicated in anxiety, morphogenesis, and cardiac function. Despite the fact that 5-HT_2B receptors have analogous signal transduction coupling to other 5-HT₂ receptors in vitro, not much evidence has been generated for endogenous receptors.

5-HT_2C Receptors

5-HT_2C receptors are expressed at low levels beyond the brain, but they are robustly expressed across the CNS. Among 5-HT receptors, 5-HT_2C receptors are considered to be special because the mRNA transcript can be edited, causing slight changes in coding sequence that can have functionally pertinent influences on the mature receptor protein¹⁸. 5-HT_2C receptor knockout mice have been created which remarkably develop seizures, glucose intolerance, and mid-life obesity¹⁹.

The 5-HT_2C receptors’ pharmacology is akin to that of the other 5-HT₂ receptors; 5-HT_2C receptors show intricate interactions with agonist-directed signaling, signal transducing mechanisms, and inverse agonism by a few atypical antipsychotics. Data from animal models denotes that 5-HT_2C receptors may impact on addiction, appetite, anxiety, and antipsychotic drug actions. SB 242084 is considered a fairly selective 5-HT_2C receptor antagonist with anxiolytic activity. No highly selective 5-HT_2C agonists have been developed so far because those that were described also have affinity for other 5-HT₂ receptors. Lorcaserin shows some selectivity for the 5-HT_2C receptor, even though no readily available sources are there for this kind of molecule beyond custom synthesis²⁰.

The 5-HT₃ Receptor

The 5-HT₃ receptor is the sole 5-HT receptor that belongs to the Cys-loop ligand-gated ion channel (LGIC) family²¹. This receptor is believed to be a pentameric complex, which is consistent with other family members of the Cys-loop LGICs²². The receptor complex may consists of a mix of up to five varied subunits called 5-HT₃A-E, although only the 5-HT₃B and 5-HT₃A subunits have been comprehensively examined. The 5-HT₃ receptor complex—a non-selective cation channel (most permeable to K⁺, Na⁺, and Ca²⁺ ions)—facilitates rapid synaptic depolarizing neurotransmission in the brain and is liable to fast desensitization. Current attention is on the combination of subunits that form the functional channel in native tissue.

Within the recombinant systems, the expression of the 5-HT₃A subunit alone creates a functional receptor that shows several properties of native receptors. The limitation is that a relatively high conductance single-channel receptor cannot be generated by homomeric 5-HT₃A receptors, something that is obvious in certain populations of native neuronal receptors. Most importantly, co-expression of the 5-HT₃B and 5-HT₃A subunits leads to a heteromeric receptor that imitates the high conductance of certain populations of native receptors in a more faithful manner^23,24. Apart from 5-HT, the action of 5-HT₃ receptor is modulated allosterically through volatile alcohols and anesthetics^25,26,27. However, the actions of these compounds may rely on the receptor’s subunit composition.²⁸

The highest densities of 5-HT₃ receptors within the brain are within brainstem nuclei covering the chemoreceptor trigger zone—to be precise, the dorsal motor nucleus of the vagus nerve, nucleus tractus solitaries, and area postrema²⁹. Expression of the 5-HT₃ receptor is also evident in human forebrain regions such as the caudate-putamen, amygdala, and hippocampus³⁰. Importantly, expression within the extrapyramidal system (substantia nigra and caudate-putamen [striatum]) cannot be readily identified in other species (like rodents and/or non-human primates).

Within the 5-HT₃ receptor complex, the 5-HT binding site is formed by neighboring N-termini from adjacent subunits present in the pentameric complex. Through structural analysis, three peptide loops (labeled as A, B, and C) has been found to occur from the “principal” subunit, and an additional three peptide loops from the “complementary” subunit (D, E, and F) take part in ligand binding. Therefore, the initial report regarding the stoichiometry of the heteromeric 5-HT₃AB receptor (with B-A-B-B-A as subunit composition) created a great deal of debate about the potential to detect pharmacological compounds that would differentiate heteromeric 5-HT₃AB receptors from homomeric 5-HT₃A receptors. The former’s binding sites would emerge from A-A interfaces, while this structural interface was not present in heteromeric 5-HT₃AB receptors. However, the B-A-B-B-A stoichiometry has been questioned recently³¹.

Most of the compounds examined so far fail to differentiate between molecular isoforms of the 5-HT₃ receptor. Picrotoxin is a notable exception which shows weak (micromolar) affinity but excellent selectivity (about 100-fold for homomeric mouse 5-HT₃A against heteromeric mouse 5-HT₃AB receptors)³². This has been shown in functional recordings, and the molecular interaction could be a channel blockade of the 5-HT₃ receptor instead of competition at the 5-HT binding site.

While the search goes on for compounds that readily differentiate between 5-HT₃ receptor molecular isoforms, there are a vast number of ligands that exhibit high selectivity for the 5-HT₃ receptor against other neurotransmitter receptors. Apart from antagonists, high affinity and selective agonists are also available for the 5-HT₃ receptor, albeit these are likely to be partial agonists similar to methyl-5-HT—the non-selective exogenous agonist. mCPBG, PBG, and pumosetrag (DDP733) are a few examples of partial agonists, and SR 57227A is a classic example of an exogenous near full agonist.

The release of numerous neurotransmitters, including facilitation of GABA, dopamine, and 5-HT release, is modulated by the activation of the 5-HT₃ receptor, even though the receptor is not believed to be expressed by 5-HT neurons^34,35. On the other hand, the 5-HT₃ receptor has an inhibitory impact on acetylcholine release in the cortex^36,37, which could be mediated through GABAergic interneurons^37,38.

Several 5-HT₃ receptor ligands, such as palonosetron, tropisetron, granisetron, and ondansetron, have now been leveraged for therapeutic benefit. This is because of their potential to reduce the effects of vomiting and nausea ensuing from anticancer radiotherapy and chemotherapy, and also from post-operative emesis that is specifically evident after procedures involving the abdomen³⁹.

An additional therapeutic utility of the 5-HT₃ receptor ligands is related to the symptomatic relief from IBS—a well-known heterogeneous condition. Although this condition is not life-threatening, it does present a significant economic and health burden. Alosetron, selective and potent 5-HT₃ receptor antagonist, shows clear efficacy in decreasing the symptoms of IBS presenting with diarrhea (IBS-d). Rare occurrences of possibly fatal ischemic colitis had an impact on the marketing approval for this medication, resulting in its withdrawal. This side-effect was also recognized—again at a comparatively low incidence—in the aborted trials of cilansetron, which is another 5-HT₃ receptor antagonist. This indicates that this side effect is not an “off-target” phenomenon.

The comparatively high number of patients who received 5-HT₃ receptor antagonists to control emesis—without a single incidence of ischemic colitis—indicates that this side effect results from the combination of the IBS condition and 5-HT₃ receptor antagonism. Most importantly, patient pressure helped the reinstatement of alosetron, although with reduced availability. However, regulatory approval exists in Japan for the use of an extremely low dose of ramosetron—the selective 5-HT₃ receptor antagonist—with a maximum daily dose of 10 μg. This extremely low dose apparently lowers the occurrence of ischemic colitis by reducing the degree of blockade of the 5-HT₃ receptor, albeit the efficacy levels accomplished by these low doses are a debatable topic.

An alternative pharmacological approach targeting the 5-HT₃ receptor has been assessed for IBS presenting with constipation (IBS-c). In this case, the predicted prokinetic action of a 5-HT₃ receptor partial agonist, DDP733, was evaluated. Regrettably, the compound showed comparatively high levels of agonist activity (intrinsic activity) such that it led to emesis in certain patients (anticipated for 5-HT₃ receptor agonists with high intrinsic activity).

The potential efficacy of 5-HT₃ receptor antagonists to affect behaviors that are mediated through the forebrain (for instance, cognitive dysfunction, anxiety, and alcohol-induced reward) is yet to be completely understood. In fact, the initial potential of antagonists as a treatment for these effects did not translate in reliable clinical findings. One possible explanation for this is the major variations apparent in the regional and cellular expression of the 5-HT₃ receptor when a comparison was being made between humans and laboratory animals (New World primates and rodents). Fascinatingly, some effects of the 5-HT₃ receptor antagonists have been detected in humans without any previous detection in animal models, such as in chronic fatigue syndrome and fibromyalgia.

The 5-HT₄ Receptor

A functional 5-HT₄ receptor protein—consistent with other GPCRs—emerges from one gene. However, the emerging mRNA can be alternatively spliced within the region corresponding to the C-terminus, and the region corresponding to the extracellular association between the fourth and fifth transmembrane domain. This creates 10 isoforms—5-HT₄(n), 5-HT₄(a-g), 5-HT₄(hb), and 5-HT₄(i)—although even more may be identified in the future.

5-HT₄ receptor transcripts—except for the 5-HT₄(d) receptor isoform—are expressed in the brain. The role of the C-terminus is to enable subcellular localization and to communicate the activation of receptors, with little impact on the pharmacology of the orthosteric site. As a result, it is no surprise that 5-HT₄ receptor isoforms do not tend to vary pharmacologically, even though functional differences are obvious.

5-HT₄ receptor is highly expressed in the cardiovascular tissues, gut, and brain. mRNA and protein within the brain co-localize implying a post-synaptic location. The highest levels of expression are in the basal ganglia, including the globus pallidus, substantia nigra, nucleus accumbens, putamen, caudate nucleus, cortex, and hippocampus (CA1 and subiculum)⁴⁰.

The 5-HT₄ receptor is positively conjugated to adenylyl cyclase through G_s, with receptor activation leading to neuronal excitability, albeit coupling to ion channels is also apparent. Furthermore, excitatory 5-HT₄ receptors increase the release of several neurotransmitters including hippocampal 5-HT, nigral-striatal dopamine, and cortical acetylcholine.

It is thought that the 5-HT₄ receptor plays a role in memory and learning. The majority of studies have demonstrated that activation of 5-HT₄ receptors enhances performance in numerous experimental behavioral aspects of cognitive function⁴¹. The advantageous effects of the activation of 5-HT₄ receptors could be mediated by facilitating the release of acetylcholine in the cerebral cortex.

Another pertinent process associated with the 5-HT₄ receptor corresponds to the metabolism of amyloid precursor protein, or APP. 5-HT₄ receptors encourage the secretion of a neuroprotective peptide—sAPPα—that improves memory functions in behavioral paradigms, facilitates the growth of neurons, and also ablates the cellular toxicity related to extreme glutamatergic transmission that can lead to cognitive impairment⁴².

Furthermore, the 5-HT₄ receptor may also improve cognitive performance via depolarization of pyramidal cells within the hippocampus’ CA1 field, and thus encourage the induction of hippocampal long-term potentiation (LTP)—a cellular phenomenon considered to be ther neurophysiological basis of memory⁴³.

In addition, the 5-HT₄ receptor may possibly contribute to the generation of anxiety. 5-HT₄ receptor antagonists have been shown to display anxiolytic characteristics while the 5-HT₄ receptor knockout mouse displayed abnormal responses to stress, wherein stress-induced hypophagia was attenuated when compared to the wild-type strain^44,45. 5-HT₄ receptor antagonists—consistent with the dogma associating excessive 5-HT function with anxiety—reduce the release of hippocampal 5-HT.

There are several drug tools to activate or antagonize 5-HT₄ receptors: RS 100235, SB 204070, and GR 113808 are selective high-affinity antagonists, while BIMU8, ML 10302, and RS 67506 represent non-tryptamine selective agonists. 5-methoxytryptamine—the tryptamine derivative agonist—is also a powerful yet non-selective agonist of the 5-HT₄ receptor. Cisapride and other similar benzamide derivatives exhibit agonist actions. Cisapride was initially marketed as a gastro-prokinetic but was later withdrawn owing to cardiovascular side effects, which were consistent with the expression of 5-HT₄ receptors in atria. Tegaserod, an additional 5-HT₄ receptor agonist, created for IBS-c was withdrawn for analogous reasons.

The 5-ht₅ Receptors

Two gene products—5-ht_5a and 5-ht_5b receptors—make up the 5-ht₅ receptor subfamily. In spite of being identified almost two decades ago, they are the most poorly investigated 5-HT receptor subtypes. No conclusive evidence is available as to how they stimulate second messenger responses in native tissue, despite their structural classification as GPCRs. Due to the lack of clear physiological roles for these receptor subtypes, lower-case notation highlights their present status as purely gene products, conflicting with the upper-case notation of a receptor with recognized cellular functions in native tissues or cells.

Among the two subtypes, the 5-ht_5a receptor has triggered a great deal of interest, as the human 5-ht_5b receptor gene sequence could well be a pseudogene since it contains stop codons within its open reading frame. If the ensuing truncated protein was expressed, it would most probably lack functionality⁴⁶. However, the rodent 5-ht_5b receptor would seem to be capable of functional expression, albeit little proof has been established. Within the rat brain, 5-ht_5b receptor mRNA can be found in the hippocampus, entorhinal, habenula, piriform cortices, as well as the olfactory bulb⁴⁷.

The 5-ht_5a receptor, within heterologous expression systems, may suppress the activity of adenylyl cyclase, probably through G_i^48,49, however other investigations have failed to replicate this finding⁵⁰. Other transduction processes impacted by the 5-ht_5a receptor may include coupling to an inwardly rectifying potassium channel⁵⁰ or increased intracellular Ca²⁺ mobilization⁵¹. The lack of appropriate selective ligands has limited autoradiographic study of the 5-ht_5a receptor.

Expression of the 5-ht_5a receptor protein in the rat brain is linked with neurons and can be seen in the horizontal nucleus of the diagonal band, locus coeruleus, raphe nuclei, hypothalamus, amygdala, with more moderate immune-reactivity in the cerebral cortex (specifically entorhinal cortex), cerebellum, pons, ventral tegmental area, substantia nigra, lateral habenula, and hippocampus. In-situ hybridization utilizing human brain tissue has shown transcripts of the 5-ht_5a receptor in the cerebellum, cortex, amygdala, and hippocampus⁵². So far, no definitive role for native 5-ht_5a receptors has been discovered, but some studies have indicated putative functions. For example, 5-ht_5a receptor knockout mice exhibit improved exploratory behavior in response to a new environment⁴⁶. In addition, the 5-ht_5a receptor has been shown to play a role in the regulation of rodent circadian rhythm, although interpretation is complicated due to the limited number of pharmacological tools available to analyze this receptor.⁵³

SB 699551-A, the most promising compound, shows a 30-fold selectivity for the human 5-ht_5a receptor over other neuronal targets and other 5-HT receptor subtypes, apart from the serotonin transporter, which it impacts at just 10-fold higher concentrations⁵⁴. Regrettably, SB 699551-A shows inter-species difference in affinity for the 5-ht_5a receptor and exhibits comparatively low affinity for rodent 5-ht_5a receptors (pK = 6.3), which additionally restricts the utility of this compound to probe the function of the 5-ht_5a receptor via the common rodent paradigms.

The 5-HT₆ Receptor

The 5-HT₆ receptor is conjugated to G_s to stimulate adenylyl cyclase and displays moderate affinity for serotonin. This receptor is selectively and strongly expressed in CNS but contains species-specific patterns of expression with human and rat displaying strong expression in hippocampus and striatum. However, this expression is approximately 1/10^th of the expression level in mice and litter regional variation in different regions of the mouse brain⁵⁵.

While a 5-HT₆ receptor knockout mouse has been developed, the phenotypic relevance is not clear, considering the low expression levels of the 5-HT₆ receptor in wild-type mice as compared to human or rat⁵⁶. Compared to the mouse receptor, the human and rat 5-HT₆ receptors are more pharmacologically similar to each other. The 5-HT₆ receptor, detected in animal models, is a potential target for cognitive improvement and perhaps weight loss⁵⁷. The relatively selective 5-HT₆ receptor agonists are WAY 181,187, EMD 386088, and EMDT. Several selective antagonists, including Ro 4368554, SB 258585, and SB 399885, have also been developed. The less selective ligands are also likely to have high affinity for D₂ and 5-HT_2A receptors. Several clinically relevant antidepressant and antipsychotic drugs also share high affinity for this receptor along with their other targets⁵⁷.

The 5-HT₇ Receptor

5-HT₇ receptors also couple to G_s (triggering adenylyl cyclase) and are extensively distributed in the brain⁵⁸. A number of splice variants with varied patterns of distribution within the CNS region have been detected⁵⁹, although they do not display meaningful pharmacological distinctions in human isoforms⁶⁰.

A number of atypical antidepressant and antipsychotic drugs have sufficient affinity for this receptor and will occupy it at frequently used dosages. Certain ligands that were historically linked with other 5-HT receptors also couple to 5-HT₇ receptors, specifically those related to 5-HT₆, 5-HT_2A, and 5-HT_1A receptors. The agonist 8-OH-DPAT is a specific compound that should be considered: it is a full agonist and has just approximately 10-fold higher affinity for 5-HT_1A than 5-HT₇ receptors.

5-HT₇ receptors have been demonstrated to play a role in a wide range of physiological and behavioral processes, including vasodilation, circadian rhythmicity, and affective behavior. In the forced swim test, consistent with the pharmacological data, 5-HT₇ receptor knockout mice showed reduced immobility. This suggests that this receptor blockade can create antidepressant effects. A number of moderately selective agonists have been reported, including LP 12 and AS 19; SB 269970 and SB 258719 are highly selective antagonists of 5-HT₇ receptors.

The 5-HT transporter (SERT)

The 5-HT transporter (5-HTT or SERT) is a Na⁺/Cl^– dependent biogenic amine transporter, whose family comprises the noradrenaline (NET) and dopamine (DAT) transporters⁶¹. SERT is important for the functioning of the 5-HT system and limits the neurotransmission of the 5-HT by removing synaptic neurotransmitter via transport across the presynaptic membrane⁶². After the original sequencing of rat SERT⁶³, follow-up studies have suggested that the functional complex can exist as an oligomer^64,65.

Within the brain, SERT is found throughout 5-HT neurons, and therefore shows a distribution at the protein level that almost matches the areas receiving 5-HT neuron innervation. The protein provides a phenotypic marker for 5-HT neurons⁶⁶. Consistently, studies performed on in situ hybridization reveal that SERT transcript expression is linked with the cell bodies of 5-HT neurons⁶⁷. However, in the developing mouse brain, transporter expression occurs temporarily in glutamatergic thalamocortical afferents that are incapable of synthesizing 5-HT^68,69. Therefore, these neurons may sequester 5-HT, allowing the afferents to mediate serotonergic transmission during the course of brain development.

More than a single form of SERT protein seems to be present in vivo. Using a pair of selective antibodies, raised against varied epitopes within the N- and C-terminus, Shigematsu et al performed immunohistochemical studies on the mouse brain⁷⁰. The team noticed that immunoreactivity with the N-terminal antibody was not present in the CA3 field of the hippocampus, while the C-terminal antibody indicated the expression of SERT in this area. This suggests that variable N-terminal domains might be present in SERT, possibly through alternative splicing of exon 1. More molecular diversity seems to be evident in human immune cells, in which SERT may function to deliver 5-HT to other immune cells across the immunological synapse⁷¹.

The efficacy of an array of antidepressant drugs, specifically the selective serotonin reuptake inhibitors (SSRIs), has made it possible to describe the physiological roles of SERT in the brain. It is unquestionable that the transporter has an impact on depression, although its accurate function is still a debatable topic. More evidence arises from the genetic difference that takes place upstream of the SERT coding sequence: the 5-HTT gene-linked polymorphic region (5-HTTLPR). A range of repeated units integrated inside this sequence creates a promoter region that regulates the expression of SERT^72,73. Within this region, one common polymorphism is a 44 base pair deletion, indicated as a short form (S) for the gene, together with variations of a long form (LA and LG). The short form allele lowers the expression and function of SERT in relation to the long forms^72,74. However, it has been reported that LG may lead to SERT expression similar to the short form variant⁷⁵, and the short form allele may not be related to reduced SERT levels in the adult brain⁷⁶, further complicating the research field. Even if one short form allele is present in individuals, it predisposes them to depressive episodes.

In further support from in vivo imaging, expression of SERT is reduced within the brainstem⁷⁷, amygdala and midbrain⁷⁸ in patients suffering from depression. Several reports associate the expression of SERT with the brains of suicide victims, although this area continues to be a controversial topic⁷⁹. However, SERT knockout mice show behavioral abnormalities in relation to anxiety and depression^80,81.

Although SERT is a molecular therapeutic target, it is also a target for numerous drugs of abuse, such as cocaine and MDMA (ecstasy). For instance, MDMA blocks the reuptake of 5-HT and increases its release^82,83. While cocaine is mainly believed to act upon DAT, SERT interaction seems to contribute to cocaine’s rewarding actions⁸⁴.

Conclusion

The 5-HT receptor research field—right from the original discovery of serotonin in the mid-20^th century—continues to expand on both commercial and scientific levels. Over the last six decades, significant pharmacological and physiological processes involving 5-HT transporter and 5-HT receptors have been discovered. The successive discovery of the six classes of G-protein coupled 5-HT receptors (5-HT_1,2,4–7) and their subclasses, together with the ligand-gated ion channel 5-HT_3, has offered an interesting research platform that shows potential for upcoming drug discovery. 5-ht_1e and 5-ht₅ receptors are comparatively uncharacterized and the discovery of selective ligands for these receptors may help in interpreting their functions in vivo.

One major issue in this area has been the lack of adequately selective ligands to detect the relative contribution of various serotonin receptors to serotonin-mediated complex physiological and behavioral phenomena. As the latest pharmacological and molecular tools become available, targeting specific 5-HT receptors should result in the development of compounds of therapeutic value that will decrease the possibility for unwanted side effects. A new generation of serotonergic drugs that can be useful as antiemetic, antidepressant, precognitive, and antipsychotic treatments could be developed in the future. It can be expected that 5-HT receptor studies will continue to advance and yield interesting outcomes in the near future.

Special Mentions

This white paper was based on the original work of:

Nicholas M. Barnes: Cellular and Molecular Neuropharmacology Research Group, Section of Neuropharmacology and Neurobiology, Clincal and Experimental Medicine, The Medical School, University of Birmingham, UK. Email: [email protected]

John F. Neumaier: Department of Psychiatry, University of Washington, Seattle, USA. Email: [email protected]

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