The purpose of this study was to investigate the distribution of 5HT6 receptors in some

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The neurotransmitter 5-hydroxytryptamine (5HT), known as serotonin, is acknowledged, since long time ago, to be able to influence anxiety and depression, as well as various physiological functions such as sleep, feeding behavior, sexuality, mood and circadian rhythms. Hence, the most part of the research about the 5HT are focused on these fields. However, more attention has been recently directed to the relationship between 5HT neurotransmission and memory.

The serotonin receptors of type 6 (5HT6R) are recently identified. They are quiet different from all the other 5HT receptors, because they are characterized by a short third cytoplasmatic loop and a long C-terminal tail, and because they contain one intron, located in the middle of the third cytoplasmatic loop. After some initial controversies, the available findings are now apparently more congruent. Nevertheless, the discrepancies concerning, for example, the binding affinity, the effects of 5HT6 ligands on brain catecholamines and the behavioral syndromes mediated by them, still exist. Much interest on 5HT6 receptors was triggered by the evidence that some antipsychotics could bind to them. Subsequently, despite the lack of completed information on metabolic patterns of the various compounds, some of 5HT6 receptor ligands entered the clinical development as potential anti-dementia, antipsychotic and anti-obese drugs. Anyway, the available information on both the pharmacology of 5HT6 receptors is still quite inadequate.

The purpose of this study was to investigate the distribution of 5HT6 receptors in some

human brain areas obtained postmortem, by means of binding techniques, and to characterize

them pharmacologically where possible. In addition, autoradiography of brain sections were

performed, as well as immunohistochemistry and immunofluorescence for a better

localization of the receptors.

















AIM 36






[125I]SB-258585 BINDING STUDIES 40







[125I]SB-258585 BINDING STUDIES 47








Serotonin or 5-hydroxytryptamine (5HT) is a biogenic amine indole structured present in the central nervous system (CNS) and in the peripheral one (PNS). It was discovered in 1948 during experiments designed to identify a substance with vasoconstrictor activity, secreted during the process of blood clotting.

To highlight the biological action was called "serotonin." It was later also identified in the gastrointestinal tract at the level of the enterochromaffin cells of the intestinal mucosa. After the characterization of its chemical structure as 3-(β-aminoethyl)-5-hydroxytryptamine (Fig.1), the 5-HT was also identified in the nervous system as a mediator of humoral transmission (Gaddum, 1953). Subsequent studies established an important role as neurotransmitter and local hormone at the level of the peripheral vascular system and blood.

In evolutionary terms, serotonin is one of the oldest neurotransmitters thus underscoring the

high functional importance (Barnes et al., 1999). Serotonin is involved centrally in the

hypothalamic control of emotional reactions of the autonomic functions like sleep-wake cycle,

body temperature, sexual function, also regulates pain perception, acting as an inhibitor of the

afferent pathways to pain in the spinal cord and the feeling of hunger, which can be reduced

by drugs that increase levels of 5-HT in the brain. The 5HT system is one of the main targets

for the pharmacological treatment of several psychiatric disorders: 5HT activity is indeed

associated with mood disorders such as obsessive-compulsive disorders, panic attacks (Fekkes

et al., 1997) and depressive syndromes, effectively treated with drugs that block the re-uptake

of 5-HT (SSRIs) in the pre-synaptic axon terminal (Cowen et al., 1987).


Fig.1 Serotonin






The anatomical localization of 5HT pathway in the central nervous system was initially delineated in the rat brain by Dahlstroem and Fuxe (1964), who demonstrated that 5HT cell bodies are concentrated into the raphe nuclei of the brainstem. Later investigations have found a similar distribution of 5HT neurons in the human brain. (Azmitia et al, 1986; Baker et al, 1991a, 1991b; Hornung, 2003)

Nuclei of ascending serotonergic projections are mainly confined to the dorsal and median

raphe nuclei in the mesencephalon and rostral pons. These nuclei project to forebrain

structures though two major pathways: a lateral projection through the internal capsule,

innervating lateral cortical regions, and a medial projection through the medial forebrain

bundle to the hypothalamus, basal forebrain and amygdale. The medial pathway also innervate

medial cortical regions and hippocampus through fibers in the cingulum bundle. In addition

to the ascending 5HT pathway, a caudal group of neurons in the lower pons and medulla

sends descending serotonergic projections to the brainstem and spinal cord. (Azmitia et al,

1986; Hornung, 2003).


Although biochemical data of brain 5HT concentrations show a large variability in different investigations, the highest levels of 5HT are present in raphe nuclei, substantia nigra, striatum and hypothalamus of human brain; lower levels have been detected in cerebellum and cerebral cortex, where 5HT seems to be concentrated to sensory and limbic cortical regions.

The 5HT is present in high concentrations in specific areas of the midbrain, hippocampus, caudate nucleus and the floor of the fourth ventricle, where it acts as a neurotransmitter. The raphe nuclei cover a large number of serotonergic neurons whose axons show a wide distribution. Serotonergic neurons that project rostrally to areas of the cortex, hippocampus and limbic system, through the median forebrain bundle, are active during sleep, those that project caudally, receiving afferents from the gray matter, modulate sensitivity to pain.



Enterochromaffin cells (EC), which originate from the neural crest, contain about 90% of the total amount of 5-HT in the human body. A certain amount is also found in nerve cells in the myenteric plexus, where it seems to act as an excitatory neurotransmitter.

EC cells produce and secrete far more serotonin than either central or peripheral serotonergic neurons to reach the gastro-intestinal lumen and blood (Tamir et al. 1985). Overflowing serotonin from EC cells, which is taken up and concentrated in platelets, is virtually the sole source of blood serotonin.



At blood level, the 5HT is mainly contained within the platelets. Here, the serotonin can not

be synthesized since platelets have no nucleus and therefore the enzyme kit, but can be stored

through a system of active transport (SERT, the serotonin transporter). The 5HT is the

primary agent that promotes and maintains regular platelet aggregation (Vanhouette et al.,


1991), it is also a chemical intermediate in the inflammatory response and it regulates the immune response modulating the activity of natural killer cells and promoting the process of cellular cooperation macrophages- Natural Killer Cells (Hermodsson and Hellstrand, 1990); in particular 5HT defends lymphocyte Natural Killers from oxidation monocyte-induced.

Nevertheless the role of serotonin in the immune response remains unclear, but it seems that during this response the autonomic nervous system, innerving lymphoid areas


controls the release of 5HT. Literature studies show increased levels of 5-HT during the immune response in the prefrontal cortex (Gardier et al., 1994) and hippocampus (Linthorst et al., 1996).

Depending on the species, type of blood vessel and the condition of the layer of endothelial cells, exogenous serotonin can act as a vasoconstrictor or vasodilator substance; sub- contractile concentrations of 5HT also potentiate the action of contraction of other arterial constrictor agents as angiotensin II, norepinephrine and endotelina-1 (Yildiz et al., 1998;

Watts, 2000). Despite the concentration of circulating free 5HT in plasma is low, is sufficient to enhance the contraction, as practiced by other vasoconstrictors (Martin, 1994). The 5HT is mitogenic in smooth muscle cells and endothelial cells and, as in the event of contraction, may enhance the mitogenic effect of other hormones (Rapport et al., 1948; Mc Duffie et al., 2000).





The cardiomyocytes provide to the local resource of 5HT on the heart, the synthesis of

serotonin has been shown in a cardiomyocyte cell line (HL-1). At the central level the 5HT

inhibits the release of noradrenaline (NA) with a consequent reduction in heart rate

(Matsumoto et al., 1995). In contrast, the interaction of 5HT with receptors at level of

cardiomyocytes, causes a positive chronotropic effect (increase in heart rate), positive

ionotropic effect (increased force of contraction of the myocardium) and it results as mediator

of mitogenesis of cardio myocytes (Saxena et al., 1991; Brattelid et al., 2004).




The kidneys have the ability to synthesize serotonin, such as in the heart (Stier et al., 1985). In this district the 5HT has a mitogenic function of mesangial cells, increases renal perfusion (Moran et al., 1997), promotes the Na


retention and the removal of phosphates (Berndt et al., 2001).



The 5HT and its acid metabolite, the acid 5-hydroxy-3-indole-acetic acid (5-HIAA) were found in the adrenal gland in several species (Delarue et al., 1988; H. Lefebvre et al., 1998). At this level exogenous 5HT increases the release of adrenaline and noradrenaline, affecting the cardiovascular system and increasing its total peripheral resistance (Sugimoto et al., 1996).



The tryptophan derivative 5-HT is an important signaling molecule in the brain and periphery (Sodhi et al. 2004; McLean et al. 2007).

5HT was known to be synthesized from tryptophan early in its existence and the sequence of steps shown to be hydroxylation followed by decarboxylation. The precursor of 5HT is L- tryptophan, a neutral amino acid taken with the diet. Tryptophan is detected at the neuronal level by an active transport mechanism mediated by a transporter of neutral amino acids. The conversion of L-tryptophan to 5-hydroxy-tryptophan is mediated by tryptophan-5- hydroxylase, a specific enzyme in 5HT producing cells: tryptophan hydroxylase I (TpH-1) is a rate limiting enzyme present in enterochromaffin cells (EC), whereas enteric and central serotonergic neurons contain TpH-2 (Walther et al.2003).

Next is the decarboxylation of 5-hydroxy-tryptophan to serotonin by the action of a non-

specific decarboxylase that acts on many other substrates. The degradation of 5HT, however,


takes place mostly through oxidative deamination, catalyzed by monoamine oxidase (MAO) type A, with the formation of an aldehyde which is then oxidized by an enzyme 5-hydroxy acid dehydrogenase -indole-3-acetic acid (5-HIAA). Also in the cells of the pineal gland, 5HT is first converted to N-acetil-serotonin, and subsequently melatonin, with a reaction catalyzed by the enzyme hydroxy-indole-O-methyl transferase (HIOMT). Therefore melatonin is released into the circulation during the night, going to regulate biorhythms with a cyclical basis, such as sleep patterns and reproduction.

Serotonin exerts its action by binding to its receptors (5HT1 to 5HT7) present in both intrinsic and extrinsic primary afferent neurons. Serotonin in tissues metabolized mainly by enzyme monoamine oxidase. In the kidney and liver, monoamine oxidase and aldehyde dehydrogenase convert 5HT to 5-hydroxyindole acetic acid (5-HIAA), which is excreted in the urine. Approximately 2–10 mg of 5-HIAA is excreted daily by the normal adult as a result of metabolism of endogenous 5-HT. Serotonin present in the food, metabolized before entering the blood stream (Cote et al. 2003).



In the late 1950s, Gaddum and Picarelli demonstrated the presence of two different classes of 5HT receptors in the guinea pig ileum. The development of the radioligand technique in the 1970s made the classification underlying the currently used nomenclature possible.

The earlier subdivisions were based on pharmacological criteria, as proposed by Bradley et al.:

they defined three classes of receptors, 5HT1, 5HT2 and 5HT3 (Brandley et al., 1986;

Bockaert et al., 1990; Baxter et al., 1995; Kilpatrick et al., 1991), a fourth subtype, with low


sensitivity to 5HT receptor ligands, was later identified and was denoted 5HT4 (Dumuis et al, 1988).

To date, the number of recognized mammalian 5HT receptor subtypes in the CNS has more than doubled to 14, and these have been classified into seven receptor families (5HT1-7) on the basis of their structural, functional and to some extent pharmacological characteristics (Fig.2). Even this level of complexity will escalate given the existence in the brain of as yet unclassified novel 5HT binding sites, but particularly evidence that specific 5HT receptor subtypes can occur as multiple isoforms due to gene splicing or post-transcriptional RNA editing (Barnes et al, 1999). Moreover, many 5HT receptor subtypes have naturally occurring polymorphic variants, and these could be a major source of biological variation within the 5HT system.

Much new information about the CNS distribution and function of 5HT receptor subtypes has accrued as a result of the development, by the pharmaceutical industry, of novel compounds with high selectivity for individual 5HT receptor subtypes.

A striking feature of the 5HT receptors subtypes revealed by autoradiographic studies is that each has a highly distinct pattern of distribution in CNS, such that individual brain regions contain their own complement of 5HT receptor subtypes. All of the receptors are located post-synaptically where some are known to modulate ion flux to cause neuronal depolarization (5HT2A, 5HT2C, 5HT3, 5HT4) or hyper-polarization (5HT1A). Certain 5HT receptor subtypes are located on the 5HT neurons themselves where they serve as 5HT autoreceptors at the somato-dendritic or nerve terminal level; some receptors are also located on the nerve terminals of non-5HT neurons where they appear to function as heteroceptors, regulating neurotransmitter release.

The up to now identified serotonin receptors, except 5HT3, which is a ligand-gated ion

channel, belong to G-protein coupled receptors (GPCRs), and transduce the signal via the

interaction with different types of G-proteins (Fig.3). The stimulatory G-protein (Gs) is


coupled to 5HT4, 5HT6 and 5HT7 (Hoyer and Martin, 1997), whereas inhibitory G-protein (Gi) to 5HT1, 5HT5 (Hoyer and Schoeffter, 1991; Francken et al., 1998). Members of the 5HT1 receptor family are negatively coupled to adenylyl cyclase, 5HT2 receptors couple to the hydrolysis of inositol phosphates and 5HT4, 5HT6 and 5HT7 receptors are positively coupled to adenylyl cyclase (Hoyer et al, 2002).

It seems highly likely that 5HT receptor-mediated changes in gene expression have a significant role to play in the neuroadaptive processes that are though to be fundamental to the mechanism of psychotropic drug therapy and abuse.

Fig. 2: Dendrogram showing the evolutionary relationship between various human 5-HT receptor protein sequences. From Barnes et al, 1999


Fig. 3: 5HT receptor structure



Serotonin has long been known to influence anxiety and depression, as well as feeding behavior and circadian rhythms. Hence, the majority of 5HT research has focused on these fields. However, more attention has been recently directed to the relationship between 5HT neurotransmission and memory. Although several other 5HT receptors have been shown to alter memory functioning (Roth et al., 2004), the 5HT6 receptor is rather unusual among the 14 identified 5HT receptor subtypes since it appears to influence long-term memory more than anxiety (Mitchell, 2006).

The 5HT6 receptor is one of the most recently discovered serotonin receptors, first identified and sequenced in 1993 by 3 separate groups (Monsma et al., 1993; Plassat et al., 1993; Ruat et al., 1993).

It is 1 of the 3 serotonin metabotropic receptors, as 5HT4 and 5HT7, positively coupled to

the Gs protein, inducing cAMP production (Monsma et al., 1993; Plassat et al., 1993; Ruat et

al., 1993). They are quite different from all other 5HT receptors, as they are characterized by a


short third cytoplasmatic loop and a long C-terminal tail, and contain one intron located in the middle of the third cytoplasmatic loop.

The human and mouse 5HT6 receptors are glycoproteins of 440 amino acids, while in rats, the protein has 438 amino acids. The 5HT6R sequence contains a consensus sequence predictive of a N-linked glycosylation site in the putative N-terminal extracellular domain, and a number of consensus sequences indicating the presence of a number of phosphorylation sites in the presumed third cytoplasmic loop and the C-terminal (Barnes et al, 1999).

In comparison with the ontogenetic profiles of other 5HT receptors, 5HT6 receptor mRNA is expressed relatively early in brain development. It is at first expressed on embryonic day 12 (E12) in the developing rat brain, coinciding with the appearance of the first serotonergic neurons. 5HT6 mRNA decreases slightly at embryonic day 17, then increases until birth, and remains stable through young adulthood. 5HT6 message expression has been identified in the diencephalon on E12 and is expressed in the caudate by E17. Given its early and progressive expression throughout the brain and its ability to induce cAMP, 5HT6 receptors may be important for axonal growth and guidance (Grimaldi et al., 1998). Unfortunately, there is a very modest knowledge about 5HT6 receptors’ functional roles during development. In-depth developmental studies may also be relevant to 5HT6-related mechanisms of memory modulation. In adult animals, by means of immunohistochemical studies it was demonstrated high receptor expression in the striatum, nucleus accumbens, olfactory tubercle, and cortex, with moderate expression in the amygdale, hypothalamus, thalamus, cerebellum, and hippocampus (Gerard et al, 1997; Boess et al, 1998; Yoshioka et al, 1998). There appears to be negligible expression outside the CNS.

After some initial controversies, the available findings are now apparently more congruent.

Nevertheless, discrepancies still exist, such as those in binding affinity, effects of 5HT6 ligands


on brain catecholamines and the existence of a behavioral syndrome, made up by yawning, stretching and chewing, which may be mediated by them


Several 5HT6 drugs have been developed as potential enhancers of cognition and memory (Mitchell et al, 2005). However, compared with other extensively investigated 5HT receptors, such as the 5HT1A or 5HT2A, very little is understood about the regulation and function of the 5HT6, or how its manipulation improves long-term memory.



The human 5HT6 gene spans 4.1 kb and exhibits a similar genomic sequence to the rat and mouse homologues. Its homology is closest to the 5HT2 group of serotonin receptors, ranging between 33% and 40% in sequence homology. The gene for the human receptor maps to the chromosome region 1p35-p36 and has an open reading frame of 1320 bp (Kohen et al., 1996). The human 5HT6 receptor gene has 3 exons, which are separated by a 1.8-kb intron at bp position 714 and a second intron of 193 bp at position 873, corresponding to intracellular loop 3 and extracellular loop 3. There is a silent polymorphism at bp position 267 within a tyrosine codon, where a cytidine is substituted for a thymidine (T→C 267). Based on a number of genetic linkage studies, the distribution of C and T alleles appears to be more or less equal among the general population (Dubertret et al., 2004; Lane et al., 2004). Although this polymorphism does not affect the identity of the tyrosine codon, it has been further analyzed for biased distribution in several human diseases.

Because several atypical antipsychotics show high affinity for the 5HT6 receptor (Shinkai et

al., 1994), the first genetic studies of the T→C 267 polymorphism examined a possible

association in schizophrenia patients. A study published soon afterwards found several more

polymorphisms in the 5HT6 gene, none of which correlated with schizophrenia; however, one


of these was associated with a higher incidence of bipolar disorder (Vogt et al., 2000). Because different studies examined patients from different ethnic group (Shinkai et al., 1999; Tsai et al., 1999; Vogt et al., 2000), it is not surprising that contradicting polymorphism associations were reported (Mitchell et al, 2005).

This same confound of ethnic genetic variation is also apparent in most studies investigating the T→C 267 polymorphism for association with Alzheimer disease (Alvarez-Alvarez et al., 2003).

Polymorphisms in the sequence of the 5HT6 receptor gene thus may provide a genetic tool to advance our understanding of the differential responses of patients to antipsychotic medications (Branchek and Blackburn, 2000). However, previous studies regarding the association between the T→C 267 polymorphism of the 5HT6 receptor gene and response to clozapine (the prototype of atypical antipsychotics) have yielded conflicting findings (Yu et al., 1999; Masellis et al., 2001). Of note, clozapine is distinguished from all other newer atypical antipsychotics in pharmacological profiles (Meltzer, 1999).

In rodent studies, 5HT6 receptors also mediate anxiety response (Otano et al, 1999; Pouzet et al, 2002) and cognitive function (Branchek and Blackburn, 2000; Meneses, 2001).

Animal model studies have indicated that 5HT6 receptors may mediate positive schizophrenia symptoms, anxiety symptoms, and cognitive function (but not negative symptoms) (Otano et al., 1999; Branchek and Blackburn, 2000; Meneses, 2001; Pouzet et al., 2002).

Yu et al. (1999) hypothesized that an unidentified functional 5HT6 receptor polymorphism,

which confers response/non-response to antipsychotics, may be in linkage disequilibrium with

this T→C 267 variant. Vogt et al. (2000) performed a systematic mutation scan of the

complete coding region and splice junctions of the 5HT6 receptor gene and identified five

other base substitutions (G→T126, C→T873 + 30, A→C873 + 128, G→C1128, and

T→G1376) besides the T→C 267 polymorphism. None of the variants altered the amino acid

sequence of the receptor or was located in consensus sequences for splice sites (Vogt et al.,


2000). Interestingly, the T→C 267 polymorphism has also been indicated to be associated with bipolar affective disorder (Vogt et al., 2000), Alzheimer disease (Tsai et al., 1999), and Parkinson disease (Messina et al., 2002). Vogt et al. (2000) thus proposed that the T→C 267 polymorphism itself may be involved in mRNA processing or in the stability of the transcript although the existence of undetected variants cannot be completely excluded. Similarly, although the T→C102 substitution of the 5HT2A receptor gene does not alter amino acid sequence, recent evidence (Polesskaya and Sokolov, 2002) indicates that total levels of 5HT2A receptor mRNA and protein in normal individuals with the C/C genotype are lower than the ones in individuals with the T/T genotype (Lane et al., 2004).



The history of 5HT6 receptors started with the finding of a possible 5HT receptor in NCB-20 cells, a clonal cell line formed by a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain explant (MacDermont et al, 1979; Cossery et al, 1990).

However, the pharmacological characteristics of this receptor were ambiguous (Berry-Kravis, 1983). Conner and Mansour (1990) reported that LSD (Lysergic acid diethylamide) behaved as a potent partial agonist in the adenylate cyclase assay, but did not displace the bound [



in contrast, 8-OHDPAT, a 5HT1A/7 agonist, displaced the [


H]5HT binding, but had no effect on cAMP. When ascorbic acid was used to stabilize 5HT, no specific binding of [


H]5HT was detected. Therefore, the binding studies produced results that did not correspond to the potencies obtained with the 5HT-activated cyclase assay. In 1994, Usworth and Molinoff interpreted the previous data as characteristics of both the 5HT1C and 5-HT4 subtypes, and for these reasons, it could be not attributable to any known 5HT receptor.

These authors, thus, suggested that the neuroblastoma N18TG2 cells presented a 5HT

receptor subtype with 5HT6 pharmacological characteristics.


The results began to be definitively more consistent when the 5HT6 receptor was cloned in 1993 (Monsma, et al, 1993; Ruat, et al, 1993). The cloning was carried out with PCR amplification by using cDNA prepared from rat striatal mRNA (St-B17). The receptor was radiolabelled with [


I]LSD, [


H]LSD or [


H]5HT in COS-7 or HEK cells, whose binding sites were unaffected by the presence of guanine nucleotides, while suggesting, therefore, recognition of the low affinity, uncoupled state of the receptor.

A few years later, some frame shift errors were described in the previous gene sequence cloned previously, but the affinity values of different compounds for 5HT6 receptors seemed quite similar, with the exclusion of metergoline (K


= 400 nM in Kohen et al., 1996; K


= 30 nM in Monsma et al., 1993).

However, the affinity values of compounds for the 5HT6 receptors did not seem to be always congruent, for instance, the K


value of lisuride, ranged between 30 nM (Boess et al, 1997), and 1.3 nM (Grimaldi et al, 1998). What appears certain is that 5HT receptors in mice have different brain distribution and pharmacological characteristics, as compared with rats and humans (Setola et al, 2003). In fact, LSD, unlike rat and human 5HT6 receptors, behaves as a full agonist in mice (Sebben et al, 1994). In addition, no specific binding of the selective radioligand [


I]SB-258585 to 5HT6 receptors was observed in mouse brain (Hirst et al, 2003;

Bonasera et al, 2006), where also 5HT6 receptor mRNA levels were extremely low. Finally, the

ligand Ro04-6790, which binds to rat and human 5HT6 receptors, did not have effect in

cloned mouse 5HT6 receptors. However, a 4.2 kb band for 5HT6 receptor mRNA was

observed in whole mouse brain, similarly to that found in rats and humans, and 5HT6

receptor mRNA expression by RT-PCR was measured in prefrontal cortex, hypothalamus,

hippocampus and midbrain of mice (Bibancos et al, 2007). A non-functional variant of a

human 5HT6 receptor was found in humans, but not in mice or rats (Olsen et al, 1999).





In the rat brain, the highest levels of 5HT6 receptor mRNA are present in the striatum, olfactory tubercle, nucleus accumbens, hippocampus, cortex, cerebellum, hypothalamus, and amygdala (Monsma et al., 1993; Ruat et al., 1993;Ward et al., 1995). The highest density of mRNA within the hippocampus was found to be in the dentate gyrus, CA1, CA2 and CA3 regions (Ruat et al., 1993;Ward et al., 1995).

However, some discrepancies exist in literature, mainly due to different techniques of measurement 5HT6 mRNA and receptors. The mRNA was observed in the cerebellum, hippocampus and hypothalamus (Ruat et al, 1993; Ward et al, 1995) in the pituitary and in the spinal cord (Gerard et al, 1996), but at the same time negative findings are also available.

Similarly, some authors described it in the stomach (Ruat et al, 1993), in dorsal root ganglia (Gerard et al, 1996), but others did not (Pierce et al, 1996). The 5HT6 mRNA was also found in the immune system of rats (Stefuly et al, 2000) and in monkey blood mononuclear cells (Boess et al, 1998; Yang et al, 2006).

There are also some discrepancies between the localization of the 5HT6 receptor and its

mRNA (Marazziti et al, 2011). For example, intense 5HT6 receptor immunoautoradiographic

labelling was observed in the cerebellum, where only low to medium levels of 5HT6 receptors

mRNA were found. The lack of cerebellar [


I]SB-258585 binding despite the presence of

cerebellar 5HT6R mRNA and protein (Gérard et al., 1997) may reflect region-specific

perimortem influences (Barton et al., 1993; Harrison et al., 1995; Lewis, 2002), or regional

differences in the regulation of 5HT6R gene expression and receptor turnover. Interestingly,

antagonist binding of [


H]M100907 to 5HT2A receptors is also absent in the human

cerebellum (Hall et al., 2000), despite the presence of 5HT2A receptor mRNA, protein, and

agonist binding sites as determined with [


H]ketanserin (Eastwood et al., 2001).


Another mismatch is also present in the hippocampus, where a few 5HT6 receptors were described with high levels of 5HT6-mRNA.

Hence, there is a very good correlation between the distributions of 5HT6 receptor mRNA and protein. However, levels of both 5HT6 receptor mRNA and protein were found to be relatively low in mouse brain, and despite a high degree of receptor homology across the rat, human and mouse, important differences exist between the mouse 5HT6 receptor subtype and the rat and human homologues of this receptor (Hirst et al., 2003).

Thus, the 5HT6 receptor is not expressed at high levels in the basal ganglia of the mouse, unlike the human or rat, while in a number of other brain regions of these is expressed at a level much lower (Hirst et al., 2003). Furthermore, site directed mutagenesis experiments performed by Hirst and colleagues (2003) revealed that the binding pocket where antagonists and agonists bind is quite different in the mouse 5HT6 receptor compared with the agonist and antagonist docking sites present in the human or rat 5HT6 receptor subtypes.

In human tissues, mRNA for 5HT6 receptors was predominantly found in the caudate nucleus and nucleus accumbens, with lower concentrations in the hippocampus and amygdala, and very low levels in the thalamus, subthalamic nucleus and substantia nigra (Fig.4).

The human 5HT6 receptor was cloned by Kohen and Colleagues in 1996 and they found the

highest expression of mRNA to be in the caudate nucleus, and other less high value in the

hippocampus and amygdala. Low expression levels were found in the thalamus, subthalamic

nuclei and substantia nigra. East et al. (2002) found that the relative distribution of the

radiolabelled 5HT6 antagonist, SB-258585, in the striatum, hippocampus and cortex was

similar to that reported in the rat. In addition, the distribution was in agreement with 5HT6

receptor mRNA and determined by in situ hybridisation. No 5HT6-mRNA was detected in


the human spleen, stomach, small intestine, heart, liver, lung, skeletal muscle, kidney, pancreas, placenta, testis, prostate, and uterus (Hirst et al., 2003)

Together, these inconsistencies indicate that while rats are good surrogate species to predict the pharmacology of 5HT6 receptor ligands in humans, when we are evaluating data that have been generated using mice we have to use much more cautions (Heal et al, 2008).

It seems that the 5HT6 receptor is formed in somata and then moves to dendrites or axons.

Comparison between the localization between 5HT6R mRNA and [


I]SB-258585 signal (e.g., in the dentate gyrus) supports immunocytochemical observations indicating that the receptor has a somatodendritic localization (Gérard et al., 1997).

The 5HT6 receptors in the adult life are present on GABAergic cells, but not on serotonergic, cholinergic or dopaminergic neurons .

A supposed localization of 5HT6R on cholinergic neurons was discharged as a selective cholinergic lesion, induced by injection of the selective immunotoxin IgG Saporin, failed to alter the density of 5HT6 R mRNA or protein expression in the deafferentated frontal cortex (Marcos et al, 2006)

Lesioning studies have described that 5HT6 receptors are present within 5HT projection fields and not in serotonergic neurons of the raphe, indicating a probable postsynaptic role for these receptors (Gérard et al, 1996).

Similarly, when serotonergic neurones in the brains of rats were ablated with 5,7-

dihydroxytryptamine, this did not affect the levels of 5HT6 receptor mRNA in the

hippocampus, striatum and nucleus accumbens (Gérard et al., 1996), suggesting that it is not

present as an autoreceptor on serotonergic nerve terminals. Lesioning of the nigro-striatal

pathway with 6-hydroxydopamine also did not alter the level of the 5HT6 antagonist, [



SB258585, binding in any of the brain regions examined, suggesting that 5HT6 receptors are

not located on dopaminergic neurones (Roberts et al., 2002).


Neurones which express dendritic 5HT6 receptors have been shown to innervate glutamic acid decarboxylase-positive cells. Using a dual-labelling immunohistochemical technique, 5HT6 receptors were co-localized with GABAergic neurones in the hippocampus in greater than 20% of 5HT6 immunoreactive neurones (Fone et al., 2002).

It can be concluded that 5HT6 receptors in adults are heteroreceptors and not autoreceptors.

In addition, 5HT6 receptors appear in the developmental brain where their stimulation support embryonic interneurons migration (Riccio et al, 2009) and, according to Jackson et al., (1997), they work as autoreceptors.

Fig. 4: 5HT6-distribution





The 5HT6 receptor activity leads to activation of cAMP signaling pathway through adenylate cyclase stimulation. In fact, activity on adenylate cyclase confers the classical definition as agonist/antagonist upon 5HT6 receptors.

Recent studies have suggested that equally agonist and antagonist may have pro-cognitive activities, and so both activation and inhibition of this receptor could evoke similar responses.

The mechanism for this apparently paradoxical effect could be related to the existence of alternative biochemical pathways activated by 5HT6 receptors.

It coupling to Gs has been widely described, but coupling of 5HT6 receptors to other G

protein subunits (Gi/o or Gq) has also been reported by Dupuis and collaborators using a SPA/antibody-immunocapture technique (Dupuis et al, 2004). In addition, the coupling of 5HT6 R to Ca


signaling using a chimeric G-protein has been reported (Zhang et al, 2003).

On the contrary, it has also been described that the carboxyl-terminal region of 5HT6 receptors interacts with Fyn-tyrosine kinase, a member of the Src family of non-receptor protein-tyrosine kinase, and even more, 5HT6R activated the extracellular signal regulated kinase1/2 (ERK1/2) via Fyn-dependent pathway (Yun et al, 2007; Marcos et al, 2010).

Recently, it has been also described a physical interaction between 5HT6 receptor and the Jun activation domain-binding protein-1 (Jab-1) using different experimental approaches (Yun et al, 2010). Recent studies have shown functional coupling of the carboxyl terminus of the human 5HT6 receptor to Fyn-tyrosine kinase and it has been recorded a 5HT6 receptor- mediated depolarization involving K


channel opening, using the whole-cell patch-clamp technique in rat striatal cholinergic interneurones in brain slices (Bonsi et al, 2007), suggesting that this receptor may couple through multiple cellular signaling pathways.

Although in vitro studies show the coupling of the 5HT6 receptor to adenylyl cyclase, the

functional coupling of the human 5HT6 receptor to Fyn-tyrosine kinase, which is expressed in


neurons (Yun et al., 2007) could explain the differential affection of these two signal pathways by agonist and antagonist (Fone et al, 2008).

Other alternative mechanisms have been described, counting that agonists and antagonists could act on receptors located on distinct neuronal populations (Fone et al, 2008).

Even more, the 5HT6 receptor agonist LY-586713 has been found to increase expression of cortical and hippocampal BDNF (brain derived neurotrophic factor) which could underlie any pro-cognitive effect, but the cortical increase in BDNF was not antagonized by SB-271046 (De Foubert et al, 2007), consistent with a potential differential mechanism. However, 5HT6 receptor functionality is being revealed to be much more complex that initially defined. Based on the existing data different cellular pathways may be activated. The full characterization of the functional profile of 5HT6 receptor is still pending (Codony et al, 2011).

Fig. 5: Neurochemical and biochemical mechanisms mediating 5HT6 receptor functions. In addition to the activation of cAMP signalling pathways, 5HT6 receptors activate the extracellular signal regulated kinase1/2 (ERK1/2) via Fyn-dependent pathway.

AC: adenylate cyclase; ACH: acetylcholine; Glut: glutamate.

From Codony et al, 2011.




Regarding the biological activity of the compounds showing affinity for the 5HT6 receptors, it appears that the definition of such an activity depends on the biological system used. At least three different second messenger systems have been described for these receptors: one linked to the third intracellular loop and G protein (Romero et al, 2006), another linked to the terminal cytoplasmatic part of the receptor protein through Fyn-kynase activity (Marcos et al, 2009), and the third associated with K


channels (Bonsi et al, 2007).

Most pharmacological and behavioral studies have been conducted in rats. With regard to the rat and human receptor homologues, the K


of serotonin at the 5HT6 receptor is in the middle range of binding profiles as compared with other 5HT receptor subtypes. Reported values for serotonin affinity for 5HT6 range from 12.3 to 234.4nM, depending on the specific pharmacological assay and receptor preparation (Ruat et al., 1993; Zhukovskaya & Neumaier, 2000; Kohen et al., 2001).

5HT and LSD seem to interact differently on 5HT6 receptor, the latter having higher affinity but being capable of only partially activating rat and human receptors (Boess et al, 1997;

Dupuis et al, 2008). When the more selective 5HT6 receptor antagonists [


H]Ro63-0563 and



I]SB-258585 were used, the number of 5HT6R in human recombinant cloned cells ranged

from 1.6 pmol/mg with [


H]Ro63-0563 (Boess et al, 1998), to 6.1 pmol/mg with [



258585 (Hirst et al, 2000), in comparison with 2.8 pmol/mg with [


H]5HT (Boess et al, 1997)

and 3.9 pmol/mg with [


H]LSD (Hirst et al, 2000). These findings suggest that 5HT6 receptor

population is bound at different sites by radiolabeled compounds. Another important aspect is

the difference in their density between native tissues and recombinant cells, being the density

in the native tissues about 30 times lower than in cloned cells (Hirst et al, 2000). As the

definition of agonist/antagonist depends on receptor and G protein density, the use of cloned

cells for such definition may be misleading (Codony et al, 2011). More information on


compound selectivity is needed to clarify possible effects that do not seem mediated by 5HT6 receptors (Borsini et al, 2011).

Several labs have conducted mutation studies to dissect the functional domains of the mouse and human receptor homologues (Kohen et al., 2001; Purohit et al., 2003). When the serine residue (267) in the C-terminal region of the human receptor was substituted with a lysine, constitutive activity was detected. There was also a change in the Kd of clozapine, which made the atypical antipsychotic act as an agonist while previously it had an inverse agonist profile (Purohit et al., 2003). Interestingly, when the wild-type mouse 5HT6 receptor is transfected into cell cultures, it displays strong constitutive activity even when expressed at very low levels (Kohen et al., 2001). This basal constitutive activity is thought to be due to the BBXXB protein motif (B=basic peptides and X=nonbasic peptides) which confers constitutive activity to other serotonin receptors. Kohen et al. (2001) found that an amino acid substitution in the mouse 5HT6 receptor, via mutagenesis in the purported 5HT binding domain of the receptor, decreased basal activity to levels similar to those in human and rat homologues. Different sequences between rat and mouse 5HT6 homologues probably account for differences in the pharmacology of several 5HT6 ligands at these receptors. Indeed, this has recently been shown: the drug Ro 04-6790 has robust antagonist effects in rat 5HT6 receptors but negligible effects on mouse homologues (Lindner et al., 2003).

Because 5HT6 receptors have high homology to several other 5HT receptors, for many years, there were no selective agonists or antagonists, although there are several commonly known nonselective 5HT agonists, such as LSD, that strongly bind to 5HT6 receptors. However, several papers have been published on the chemistry of 5HT6 receptor ligands and on efficacy as agonists and antagonists by using adenylate cyclase as activity index (Marazziti et al, 2011).

To date, the selective 5HT6 agonists developed are few, ie EMDT, LY586713, and WAY-466,


WAY208466, WAY181187 (Glennon et al., 2000; De Foubert et al., 2004; Schechter et al., 2004), and only the last one has been more widely used.

With regard to the in vivo effects induced by 5HT6 receptor agonist, a lack of readily available selective 5HT6 agonists has limited research advances to date. In the 2008 was presented a new 5HT6 agonist, ST1936: it increased extracellular DA and NA, but not 5HT levels in medial prefrontal cortex and in the shell of nucleus accumbens in a dose-dependent manner (Borsini et al, 2008). Such data are in contrast with those obtained with another 5HT6 agonist, WAY181187, as showed by Schechter et al. (2008).

Pharmacokinetic differences across compounds may produce vastly different in vivo neurochemical and behavioral profiles.

Underlying some of these behavioral discrepancies are ambiguous results on some basic aspects of 5HT6 receptor pharmacology.

There are several 5HT6R antagonists with high affinity for cloned human 5HT6 receptors, ie SB271046 (K


=1.3nM; Bromidge et al, 1999), SB357134 (K


=0.3nM; Bromidge et al, 2001), SB399885 (K


=0.9nM; Hirst et al, 2006). However, not all of them can appreciably pass the blood-brain barrier (Sleight et al, 1998).

However, 5HT6 receptors are more complex than expected and their interaction with specific ligands might also depend on neuroanatomical region. In fact, there is a report (De Fourbet et al, 2007) that shows an up-regulation in Arc mRNA expression, caused by subcoutaneous administration of the purported agonist LY586713, and a blocking by the antagonist/inverse agonist SB271046 in hippocampus and parietal cortex but not in cingulated and orbital cortex.

Additionally, it remains to document whether the G proteins linked to 5HT6 receptors are

differently expressed in the different brain regions (Borsini et al, 2011).




The localization of 5HT6 in specific brain areas suggested that it might participate in the serotonergic control of motor function, mood-dependent behavior, depression, and cognition (Meneses, 2001; Rogers and Hagan, 2001; Woolley et al., 2001).

Furthermore, high binding affinities of 5HT6 to antipsychotic agents, such as chlorpromazine, amoxapine, clozapine and olanzapine, indicated that 5HT6 was related to the pathogenesis of psychiatric disorders. Their involvement in mental functions and high affinities against antipsychotic agents nominate 5HT6 as a potential target for the development of antidepressant and antipsychotic drugs (Choi et al, 2007).

Emphasis has been placed on understanding 5HT6 receptor effects on several groups of neurotransmitters and how these effects may translate into improved cognition during treatment of neurological disorders.

Although many enlightening discoveries have been made in 5HT6 genetics, pharmacology, and physiology, there are still conspicuous lacks in our knowledge, such as the role of 5HT6 receptors in development. Many research groups are in accordance over the pronounced influence of 5HT6 antagonists on memory consolidation, even if is not well-known the precise mechanism of enhanced consolidation.

Another untouched field of investigation is the possible synergistic interaction of 5HT6 receptors with other serotonin receptors that are also important for memory processes.

To date, no investigations into the role of 5HT6 receptors in sleep have been reported.

However, a role for these receptors in sleep and wake may be anticipated based on their association with brain regions known to be important in the regulation of these activities, such as the hypothalamus, thalamus, and striatum (Woolley et al, 2004; Gerard et al, 1996).

Literature evidences support the hypotheses that 5HT6 receptors may modulate GABAergic

and cholinergic neurotransmission (Woolley et al, 2004; Lieben et al, 2005, Schreiber et al,


2007), two neurotransmitter systems widely known to play roles in sleep-wake regulation. All These findings all together suggest that 5HT6 receptors could influence the regulation of sleep and wakefulness.





As can be inferred from the information presented above, 5HT6 interactions with other neurotransmitters are complex and not well understood. It has therefore been difficult to determine which interactions are responsible for 5HT6-mediated changes in behavior. The 5HT6 inhibition has been shown to raise extracellular acetylcholine levels in the cortex and hippocampus (Dawson et al., 2001; Riemer et al., 2003), yet there have been no studies showing that these increases are indeed responsible for enhanced memory consolidation.

However, acetylcholine release in hippocampal and cortical regions is known to be important

for memory acquisition and retention, and several groups have demonstrated that 5HT6

blockade overcomes scopolamine induced amnesia. In a 2004 paper, Foley proposed a

possible mechanism in which 5HT6 inhibition overcomes the memory deficit caused by

muscarinic blockade. The blockade of 5HT6 receptors disinhibits striatal GABAergic neurons,

thus allowing increased acetylcholine release in the cortex via indirect basal ganglia pathways

(Foley et al., 2004). As stated previously, the striatum robustly expresses 5HT6 receptors on

GABAergic medium spiny neurons. On the other hand, Woolley et al. (2004) proposed that

5HT6 blockade memory enhancement was due to the inhibition of tonic activity of receptors

located in GABAergic cells in the septum and the hippocampus, which releases acetylcholine

from the septum and the glutamate from hippocampal cells. This proposed model suggests

that the memory-enhancing component of 5HT6 inhibition may be due to both glutamatergic

and cholinergic transmission. It is interesting to note that the amnesic effects of cholinergic


blockade are reversed by several 5HT6 antagonists, but memory dysfunction due to MK-801, an NMDA inhibitor, is not overcome by SB 271076 (Woolley et al., 2004). The absolute necessity of either glutamate or acetylcholine for spatial memory in tests such as object recognition or water maze may be further revealed through micro dialysis or lesion studies. It is interesting to note that the above proposed mechanisms of memory enhancement do not involve the moderate 5HT6 receptor expression in the hippocampus, but rather through indirect activation of the hippocampus through other neurotransmitters. Determining the mechanism of 5HT6 memory enhancement is particularly important in light of possible clinical applications of 5HT6 drugs.

Several 5HT6 antagonists are being developed for Alzheimer disease (Kwon et al., 2004), a condition in which cholinergic neurons are selectively destroyed. If acetylcholine is the crucial neurotransmitter for 5HT6-mediated memory enhancement, then late-stage Alzheimer patients would not benefit from such drugs. Alternatively, there is also interest in using 5HT6 ligands for treatment of the cognitive deficits of schizophrenia, a disease that may stem from glutamatergic rather than cholinergic imbalances. The impact of 5HT6 blockade on emotional memory is less clear than the effects of 5HT6 ligands on memory tasks that involve spatial or reward-associated learning.

There is abundant 5HT6 expression in the striatum and hippocampus, regions important for

habituation and spatial learning (Gerard et al., 1997), and several studies have shown that

manipulation of 5HT6 activity in these areas produces physiological changes such as

neurotransmitter release. Yet, there have been no studies searching for similar changes in the

amygdale, where there is moderate 5HT6 expression. There is substantial evidence that stress

modulates memory consolidation, and a crucial region for such modulation is the amygdale

(Pelletier & Pare, 2004). Stress pathways may circumvent the memory-enhancing circuits

augmented by 5HT6 blockade. Clearly, more studies need to be conducted on the role of

5HT6 receptors in emotional learning.


There has been considerable interest in the enhanced memory consolidation mediated by 5HT6 receptor blockade; equally intriguing but less understood is the role of 5HT6 action in cognitive improvement from antipsychotics.

Atypical antipsychotics, such as clozapine and olanzapine, are particularly effective in enhancing cognitive function in schizophrenic patients. Some clinicians believe that atypical antipsychotics’ precognitive effects might stem from their ability to inhibit this receptor, although this has never been conclusively shown. On the other hand, it has also been suggested that 5HT6 antagonism is one of the reason because atypical antipsychotics do not produce the dopamine-mediated extra pyramidal side effects of older antipsychotics (Branchek & Blackburn, 2000). One proposed mechanism for the lack of such side effects in atypical antipsychotics is that 5HT6 inhibition attenuates striatal dopamine activity responsible for extrapyramidal syndrome, but there are no evidence.



Several atypical antipsychotics as clozapine and olanzapine exhibit strong affinity for 5HT6 receptors (Roth et al., 1994). Thus, several researchers have investigated the effects of chronic administration of antipsychotic medication on 5HT6 regulation and activity. When compared with traditional antipsychotics as haloperidol (which predominantly inhibits D2 dopamine receptors), clozapine significantly reduced 5HT6 mRNA in the hippocampus (Frederick &

Meador-Woodruff, 1999) and down-regulated 5HT6 receptors expressed in cultured cells (Zhukovskaya & Neumaier, 2000). The number of atypical antipsychotics with 5HT6

Schizophrenia symptoms are principally subdivided into two subtypes, positive and negative (Andreasen, 1985; Chang et al., 1990). The positive symptoms include delusions and hallucinations; the negative symptoms, blunted affect and social withdrawal. Typical antipsychotics, principally blocking dopamine D2

receptors, have limited effect on negative symptoms. Atypical antipsychotics, also called serotonin-dopamine antagonists, show advantages in both positive and negative symptoms (Leucht et al., 1999) and perhaps in schizophrenia’s mood symptoms (Tollefson et al., 1998; Lane et al., 1999, 2002b) and cognitive functions (Meltzer et al., 1999; Potkin et al., 2001; Sawa and Snyder, 2002). Serotonergic systems have thus been implicated in negative symptoms (Meltzer, 1999; Lane et al., 2002c).


antagonist activity continues to grow: rilapine, olanzapine, tiospirone, fluperlapine, clorotepine, and zotepine have all shown 5HT6 affinity.

Because the most effective antipsychotic medications have high affinities for several types of receptors, it is difficult to say whether this enhances efficacy or only produces side effects. As such, a major goal in schizophrenia research is parsing out the contributions of different receptors to therapeutic as well as unwanted effects.

There are several independent lines of evidence which link 5HT6 receptors to schizophrenia.

There is some evidence that either schizophrenia or antipsychotic drug treatment may alter 5HT6 receptor density; through these results is not clear if specific 5HT6 antagonists can be use as antipsychotic drugs and if 5HT6 blockade has a potent effect on some schizophrenia- like behaviors driven by dopamine. Altered cortical dopamine transmission has been proposed as a pathological characteristic of schizophrenia.

It is possible that 5HT6 receptor antagonism of atypical antipsychotics may improve some of the side effects of these drugs’ actions on other receptors. More research must be conducted in clinical settings in order to understand the contribution of 5HT6 inhibition to the therapeutic action of these drugs (Mitchell & Neumaier, 2005)





Significant reductions in 5HT6R density in cortical areas of Alzheimer Desease (AD) patients have been found, although reductions in the receptor density were unrelated to cognitive status before death (Garcia-Alloza et al, 2004).

This study has examined brains of Alzheimer patients for changes in 5HT6 receptor

expression, which may indicate a role in disease pathogenesis. 5HT6 receptor levels were

analyzed on postmortem Alzheimer and control brains by measuring binding of a radiolabeled

5HT6 antagonist, SB258585, detecting 56–58% reductions in 5HT6 receptor density in the


frontal and temporal cortex. The decreases in 5HT6 receptors may play a role in aggressive, anxious behaviors often seen in Alzheimer patients. It is not immediately clear how this data fits into the larger picture of how 5HT6 receptors in the aging brain regulate neurotransmission and behavior, making it an important focus of future investigations.

The available data supports the concept that 5HT6 antagonists increase glutamate and/or acetylcholine release and that these are likely mediators of enhanced memory consolidation (Mitchell & Neumaier, 2005). Literature findings indicate that the endogenous 5HT6 receptors are under tonic activation to suppress cholinergic neurotransmission. Because acetylcholine (ACh) was known to be directly involved in a range of cognitive and behavioural functions, including memory, anxiety, arousal, attention, and fatigue (Francis et al, 1999; Perry et al, 1999) a potential therapeutic role for 5HT6 receptor antagonists in cognitive and psychiatric diseases became apparent (Upton et al, 2008). As 5HT6R blockade induces ACh release, reductions in 5HT6R may represent an effort to restore ACh levels in a deteriorated cholinergic system. In addition, it has also been described that a dysregulation of 5HT6R activation by 5HT in the temporal cortex may be related to behavioral symptoms in AD (Garcia-Alloza et al, 2004; Marcos et al, 2008)

Little is known about the mechanism by which chronic administration of a 5HT6 antagonist improves memory consolidation. However Regan et al. (2003) have investigated one possible mechanism by examining markers of synaptic plasticity: it is possible that glutamate or ACh neurotransmission due to chronic 5HT6 antagonism primes neurons for synapse remodeling and growth during memory consolidation.

There is also evidence that the striatum may play an important role in 5HT6 modulation of

memory circuits.




Pharmacological manipulations identified an inverse relationship between the biogenic amine neurotransmitter serotonin and food intake. More specifically, a selective reduction in serotonin bioavailability was associated with hyperphagia and subsequent weight gain, whilst diminished food intake was induced by an increase in serotonin efficacy (Saller & Stricker, 1976; Fletcher & Paterson, 1989). Researchers further sought to clarify which of the 14 distinct serotonin receptors identified in vertebrates are critically involved in serotonin effects on ingestive behavior: the 5HT1


R, 5HT2


R and 5HT6R subtypes were shown to be the principal mediators through which serotonin exerts its anorectic effects in rodents, and as such, these receptors have been investigated as pharmacotherapeutic targets for the treatment of obesity (Garfield & Heisler, 2009).

5HT6R distribution within the CNS includes hypothalamic regions of immediate salience to a role in appetitive control, including the arcuate (ARC), paraventricular (PVH) and ventromedial (VMH) nuclei (Heal et al, 2008). Concordant with this expression profile, manipulation of 5HT6R signaling has been demonstrated to have potent effects on both food consumption and body weight. It is of note that it is the antagonism of this receptor that is generally associated with its antiobesity function (Garfield & Heisler 2009).

Initial experiments to characterize the function of 5HT6 receptors in the CNS did not yield any signal to indicate that modulating them would have a role to play in the regulation of food intake or body weight.

In 2000, Tecott and Brennan published a US patent covering the 5HT6 receptor knock-out mouse strain, and performed the first experiments to define the potential of the 5HT6 receptor as a viable target for the discovery and development of novel anti-obesity drugs.

Caldirola (2003) reported that the 5HT6 receptor knock-out mouse was resistant to dietary-

induced obesity when maintained on a high-fat diet. When overviewing these studies with


hindsight, it is probable that the early investigations did not observe any effect of 5HT6 antisense oligonucleotide injection on food intake or body weight because the degree of receptor ablation was relatively small, i.e. 25–30% (Bourson et al., 1995; Yoshioka et al., 1998;

Hamon et al., 1999).

In turn, this has led to a widespread view that 5HT6 receptor antagonists will evoke hypophagia and weight-loss only under conditions of very high receptor occupancy. Caldirola also presented the preliminary pharmacology results for a small molecule, 5-HT6 receptor antagonist, ie BVT5182 (5-HT6 K


=0.2 nM), showing that when given acutely this compound dose-dependently reduced the food intake of ob/ob mice by enhancing satiety, and when given repeatedly, BVT5182 produced a sustained reduction in food intake and weight-loss in DIO (diet-induced obese) mice; this weight-loss was shown to be accompanied by a reduction in visceral adiposity, and plasma leptin and insulin concentrations (Svartengren et al.,2003).

Importantly, in view of the report that the mouse 5HT6 receptor is substantially different from the homologues in either the rat or humans (Hirst et al., 2003), BVT5182 was also shown to produce a sustained reduction in food intake and body weight in DIO rats, together with decreases in visceral adiposity and plasma leptin concentrations (Svartengren et al.,2003).

The fact that the 5HT6 receptor is located almost exclusively within the CNS, has made it a popular target for drug discovery because of its perceived low liability for the induction of drug-mediated peripheral side effects. Whilst this hypothesis will undoubtedly hold true for a direct action of 5HT6 receptor agonists and antagonists on peripheral organ systems, it does not preclude peripheral side effects translated via a central action of these compounds.

It is well known that 5HT6 receptor antagonists improve several aspects of cognitive function

(Woolley et al., 2004; Mitchell & Neumaier, 2005), and consequently, these drugs could evoke

cognitive side-effects when used in the treatment of obesity. However, the actions of the


5HT6 receptor antagonists are pro-cognitive, and as such, their side effects are predicted to be neutral and perhaps even beneficial.

The only other potential CNS side-effects to have been revealed by preclinical research are related to depression and anxiety. Wesolowska and Nikiforuk (2007) reported that another 5HT6 receptor antagonist, SB 399885, had an antidepressant-like effect in both the tail suspension test and the forced-swim tests. In addition, these authors reported that SB 399885 also displayed anxiolytic-like activity in both the Vogel conflict drinking and the elevated plus- maze tests. On this basis, it is impossible to make any firm predictions, but once again, since 5HT6 receptor agonists and antagonists appear to produce antidepressant- and anxiolytic-like effects in animal models, it would seem unlikely, therefore, that these compounds will have a negative effect on mood when given to human subjects.





In line with the difficulty to interpret the afore mentioned evidences, the potential therapeutic effects also are still unclear.

Taking an overview of the data provided in literature, it is evident that the 5HT6 receptor has emerged as a highly interesting molecular target for drug development in a number of therapeutic indications.

5HT6 receptor compounds appear to act through multiple mechanisms (enhancing cortical

dopamine and cortical and hippocampal glutamate and acetylcholine release and may also

have longer-term neurotrophic actions) in normal adult and aged rats, restoring cognitive

deficits in a variety of behavioural paradigms which have translational relevance to the

dysfunction seen in schizophrenia and Alzheimer disease. This makes them a potential


candidate for adjunct therapy with antipsychotics and as an additive to cholinesterase inhibitors for the latter disorder. However, as agonists, partial agonists and full antagonists all appear to possess pro-cognitive (and anti-obesity) potential from pre-clinical studies, a key issue for pharmaceutical development is therefore which type of compound to pursue as clinical candidates (Fone, 2008).

Despite the lack of complete information on the metabolic pattern of the various compounds, some 5HT6 receptor ligands entered the clinical development as potential anti- dementia agents (Geldenhuys et al 2008), antipsychotics (Johnson et al 2008), and anti-obese drugs (Heal et al, 2008).

The 5HT6 receptors have also been involved in analgesic effects (Hedo et al, 2002;

Castaneda-Corral et al, 2009), in the mode of action of drugs of abuse (Kindlundh et al, 2006;

Ferguson et al, 2007; Pitsikas et al, 2009


Di Chiara et al,.2009; Van Gaalen et al, 2010), in sleep-wake regulation (Morairy et al, 2008), and in seizures (Routledge et al, 2000; Freitas et al, 2009).

Finally there is only limited evidence on the predictive validity of each preclinical cognitive

paradigm and scant knowledge of their relevance to individual domains of human cognitive





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