University of Groningen Augmentation of the neurochemical and behavioural effects of SSRIs Rea, Kieran

Augmentation of the neurochemical and behavioural effects of SSRIs Rea, Kieran

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Chapter 1

The Serotonergic System

Contents

1. Introduction

2. Neuroanatomy of serotonergic system 3. Synthesis and breakdown of 5-HT

4. Serotonergic modulation of the limbic system 5. Serotonin receptor subtypes

6. Signal transduction pathways

7. Clinical significance of 5-HT receptors 8. Effects of serotonin on behaviour

9. Receptors directly affecting 5-HT firing and release

1. Introduction

Serotonin (5-hydroxytryptamine; 5-HT) was discovered in 1948 by Rapport et al, as a potent vasotonic factor. As is the case with most neurotransmitters, it has a relatively simple structure but displays complex pharmacological properties. In 1957, Gaddum & Picarelli suggested that 5-HT interacted on two different receptors in isolated tissue; one receptor type specific for nervous tissue, and the other for smooth muscle.

Since then, the development of better pharmacological tools and techniques have helped gather a large amount of data on the serotonergic system.

Techniques such as histofluorescence (Dahlstrom & Fuxe, 1964), autoradiography using [³H] 5-HT (Aghajanian & Bloom, 1967; Calas et al, 1975; Descarries et al, 1975), and immunocytohistochemistry using anti-5HT antibodies (Steinbusch et al, 1978) as well as light and electron microscopy have allowed further study of this complex system. Numerous 5-HT markers, antibodies, and probes are now available for immunocytochemical and in-situ hybridisation studies of serotonergic neurons. To summarise, the serotonin system has been studied intensively, and determined to be comprised of 7 subfamilies with at least fourteen 5-HT receptor subtypes.

2. Neuroanatomy of Serotonergic System

Serotonin itself is found throughout the body, with only a relatively small portion present in the brain. It cannot cross the blood-brain barrier and must be synthesised in the brain. Between 60-75% of the brain’s serotonin is found within the nine main serotonergic nuclei (Pineyro & Blier, 1999).

Serotonergic Nuclei

During early prenatal development, two groups of serotonergic neurons are visible, a superior group at the boundary between the midbrain and pons, and a separate inferior group stretching from the caudal pons to the spinal cord (Wallace

& Lauder, 1983).

Figure 1. Serotonergic innervation of the brain. Neurons in the B1-3 groups, corresponding to the raphe magnus, raphe pallidus, and raphe obscurus nuclei in the medulla project to the lower brain stem and spinal cord. Neurons in the B4-9 groups including the raphe pontis, median raphe, and dorsal raphe nuclei, project forward to the rest of the brain.

2.1 Superior Raphe Nuclei (Rostrally located serotonergic nuclei)

The superior group of 5-HT neurons has been described as having two collections of neurons, rostral and caudal. The rostral collection gives rise to the caudal linear nucleus and most of the dorsal raphe nucleus. The caudal collection descends from the ependymal zone in two streams of cells that meet in the midline to form the superior central nucleus (median raphe nucleus and interfascicular portion of the dorsal raphe nucleus).

• Caudal Linear Nucleus (CLN)

The most rostral group is the CLN, which starts at the level of the red nucleus (Tork, 1990). The 5-HT neurons are located between the rootlets of the oculomotor nuclei and extend dorsally from the anterior edge of the interpeduncular nucleus to blend with the rostral dorsal raphe nucleus. The neurons are often situated rostral to the median raphe nucleus (MRN) and have

incorrectly been considered as part of the MRN. The projections from these regions extend to the thalamus and cortex.

• Dorsal Raphe Nucleus (DRN)

The DRN is divided into medial, lateral (the wings), and caudal components. The medial component can be further divided into a mediodorsal (superior) and an interfascicular component. The superior component is in the central gray, just below the cerebral aqueduct. The interfascicular component surrounds the MLF and is especially prominent between the fasciculi. These neurons blend with the caudal MRN. The lateral component (the wings) forms the larger division of the DRN and extends as far rostrally as the oculomotor nuclei. In the human, the lateral wings can be divided into a dorsal and ventral subdivision (Tork, 1990).

This is the largest of the brainstem serotonergic nuclei containing about 50% of 5- HT neurons in rat CNS (Wiklund & Bjorklund, 1980; Descarries et al, 1982), 40% in cat CNS (Wiklund et al, 1981), and 50-60% in the human CNS (Baker et al, 1990).

• Median Raphe Nucleus (MRN)

The MRN (previously B7) is composed of a paramedian and median cluster of cells lying below and caudal to the superior cerebellar decussation (SCD).

Scattered 5-HT cells of the MRN are seen ventrolateral to the MLF. These laterally situated cells lie in the nucleus pontis centralis oralis and form a ring around the central tegmental tract, one of the most primitive ascending pathways carrying reticulothalamic axons. The MRN is but one part of the larger superior central nucleus (SCN), which includes the interfascicular aspect of the DRN.

Laterally, the limits of the nucleus are poorly defined towards the reticular formation (Kohler & Steinbusch, 1982). The MRN forms the second largest cluster of 5-HT neurons in the mammalian CNS (Baker et al, 1990).

• Supralemniscal Nucleus (SLN)

This group (originally classified as B9) is located along the superior surface of the medial lemniscus, from the rostral border of the inferior olive to the level of the red nucleus. These cells are occasionally continuous with the cells of the MRN

and form the ventral border of the ring of scattered cells that surrounds the central tegmental tract in the pontine reticular formation. In rodents the supralemniscal cell cluster is predominantly mesencephalic, whereas in humans it is located entirely in the pons (Baker et al, 1990).

2.2 Inferior Raphe Nuclei (Caudally located serotonergic neurons)

These neurons display a different developmental pattern from the superior group of serotonergic neurons.

• Nucleus Raphe Obscrus (NRO)

This group (originally classified as B2) is a collection of large-medium multipolar neurons. They form a symmetrical paramedian cluster on either side of the midline. This dorsally situated nucleus extends from the caudal pons back into the cervical spinal cord. 5-HT neurons in the spinal cord lie ventral to the central canal and on the medial border of the ventral horn. The 5-HT neurons are commonly intermixed with the medial longitudinal fasciculus (MLF), the tectospinal tract (tst), and the dorsal aspect of the pyramidal decussation. The nucleus is denser caudally in the medulla; at the level of cranial nerve VI, it is less densely packed than either the ventrally situated NRM or the NRPa. These neurons were previously designated nucleus raphe ventricularis.

• Nucleus Raphe Pallidus (NRPa)

This group (originally designated as B1) is a group of medium sized multipolar 5- HT neurons in paramedian columns. The nucleus stretches from the cranial nerve XII to the anterior end of the inferior olive. The lateral aspects of the nucleus extend over the mediodorsal surface of the pyramidal tracts, while the main body of the nucleus lies between the pyramidal tracts. The cells appear to be contiguous with the NRM anteriorly and with the VLM laterally.

• Nucleus Raphe Magnus (NRM)

This collection of medium to large 5-HT neurons (originally classified as B3) extends from the rostral superior olive back to cranial nerve XII. This nucleus lies between NRPa and NRO, and at points the borders between these three nuclei are difficult to demarcate. Both the trapezoid body and the dorsal border of the medial lemniscus invade the nucleus. Occasionally, very large 5-HT neurons are seen more laterally in the boundary of the nucleus reticularis gigantocellularis.

• Ventral Lateral Medulla (VLM)

A large number of medium-sized, multipolar 5-HT neurons (originally part of B1/B3) are seen in the ventral lateral medulla. The nucleus extends from the Inferior olive to cranial nerve XII. The neurons are closely associated with the pyramidal tract, trapezoid body, and medial lemniscus. This nucleus overlaps with two important reticular nuclei; rostrally it forms the medial component of the reticular lateral paragigantocellularis nucleus, while caudally it forms the medial part of the inferior reticular nucleus. At its most ventral position, the neurons lie against the pial surface and are closely intertwined with the large blood vessels entering the medulla.

• Area Postrema

This is a large collection of very small 5-HT neurons that lie ventral to the fourth ventricle and are associated with the parabrachial area. The neurons are considered immature and have a bipolar or simple oval shape. (Dahlstrom &

Fuxe, 1964).

Serotonergic innervation of different brain areas

The serotonergic fibre pathways are extremely complex to describe, since they include aspects of all the main pathways in the brain. There are five routes into the forebrain and three routes into the spinal cord. These routes give rise to countless branches which follow other neuronal pathways, blood vessel ramifications, the ependymal lining of the ventricular system and even the pial surface (Azmitia et al, 1978).

The more caudal group of serotonergic neurons project mainly to the medulla and spinal cord. The more rostrally positioned neurons (median raphe nuclei, superior central nucleus and dorsal raphe nuclei) are thought to innervate ascending structures, while the remaining intermediate nuclei innervate both ascending and descending pathways.

The majority of innervation of the ascending structures is derived from the dorsal raphe nuclei and the median raphe nuclei (B7-B9), which innervate many structures including the cortex, limbic system, basal ganglia and hypothalamus. It is generally agreed that these terminal areas are ‘cross innervated’ by these nuclei.

Indeed, in the cortex, the 5-HT fibres stream across the superficial and deep layers to innervate all the cortical layers diffusely, and further extensive branching proceeds in the granular cell layers. There is also a large degree of co-localization of serotonergic and noradrenergic systems within the limbic system, which could account for some of the side effects observed in the treatment of certain conditions. Topographical differentiations do exist however, as it has been shown that the dorsal raphe nucleus preferentially innervates structures like the prefrontal cortex, lateral septum, amygdala, striatum and ventral hippocampus (Molliver, 1987), the median raphe nucleus innervation is most obvious in the hypothalamus, medial septum and dorsal hippocampus (McQuade & Sharp, 1997).

3. Synthesis and Breakdown of 5-HT

5-HT is an indoleamine transmitter, which is synthesised within the nerve ending from the amino acid L-tryptophan. Tryptophan is actively transported to the brain by an active transport system as the tryptophan found in the body is protein bound and as such is unable to penetrate the blood-brain barrier to be used in 5-HT synthesis. The levels of tryptophan in the brain are influenced not only by its plasma levels, but also by the concentrations of other amino acids which also compete for this uptake system. On entering the nerve terminal, tryptophan is hydroxylated by the enzyme tryptophan hydroxylase, which is the rate-limiting step in the synthesis of 5-HT. This enzyme requires tetrahydrobiopterin as a cofactor, which is located in serotonergic terminals (Yohrling et al, 2000).

Following the synthesis of 5-hydroxytryptophan, serotonin (5-hydroxytryptamine) is formed via a decarboxylation step by the enzyme DOPA decarboxylase. At this point the serotonin is compartmentalized into storage vesicles. The 5-HT will then be released upon the arrival of an action potential, and can then exert its neurochemical functions.

After release, 5-HT which is not metabolized is taken back into the neuron. This re-uptake mechanism is governed by a plasma membrane carrier protein, capable of transporting serotonin in both directions depending on the concentration gradient across the membrane. It has been hypothesized that there is a differential expression of this serotonin reuptake transporter protein in serotonergic neurons arising from dorsal and median raphe nuclei (Brown & Molliver, 2000). It has also been suggested that these reuptake receptors, as well as 5-HT1A, 5-HT1B and 5-HT2A receptors, are located outside the vicinity of the synapse and only become active in the presence of excess 5-HT release (Hensler, 2006). After reuptake, the 5-HT is then stored in vesicles or metabolized by monoamine oxidase (MAO) inside the neuron.

4. Serotonergic Modulation of the Limbic System

The limbic system is composed of cortical as well as subcortical structures, which are intimately connected. The major structures in the limbic system include the prefrontal cortex, cingulate cortex, entorhinal cortex, hippocampus, nucleus accumbens (ventral striatum), ventral pallidum, amygdala, and anterior hypothalamus (Swanson & Petrovich, 1998; Kandel et al, 2000; Heimer, 2003).

Connections between these different structures form complex circuits, which are organised in a strict topographical manner (van Groen et al, 2002; Heidbreder &

Groenwegen, 2003). The result is a global macrostructure for the mediation of motivation and emotion, and is the main target in the treatment of conditions such as anxiety and depression.

The serotonergic modulation of this system arises primarily from two distinct systems, the dorsal and median raphe nuclei. These systems differ in their electrophysiological characteristics, topographical organisation, and morphology as well as response to neurotoxins and therapeutic agents (Hensler, 2006). It is worth noting that not all neuronal cell bodies within the raphe nuclei are serotonergic (Descarries et al, 1982; Kohler & Steinbusch, 1982; Molliver, 1987;

Tork, 1990), and although serotonergic afferent connections between the dorsal and median raphe nuclei exist (Mosko et al, 1977; Descarries et al, 1982; Kapadia et al, 1985; Chazal & Ralston, 1987), there are also innervations from other brain areas including the substantia nigra and ventral tegmental area (dopamine), superior vestibular nucleus (acetylcholine), locus coeruleus (norepinephrine) as well as other afferents from areas such as hypothalamus, cortex, and limbic forebrain structures (Jacobs and Azmatia, 1992).

It has recently been reported that the median and dorsal raphe nuclei differ in their active and passive electrophysiological characteristics as well as their response to inhibition of somatodendritic 5-HT_1A autoreceptor activation (Kirby et al, 2003; Beck et al, 2004). It was shown that non-serotonergic neurons in the dorsal raphe nuclei were responsive to 5-HT_1A receptor agonists, while no response was seen in non-serotonergic neurons in the median raphe nuclei. This suggests that, in contrast to observations in the median raphe nuclei, 5-HT_1A receptors in the dorsal raphe nuclei act not only as somatodendritic autoreceptors,

but also as heteroreceptors on other neurotransmitter systems. This may be important in the future understanding of the role of these two separate serotonergic systems in the aetiology and treatment of disorders in the limbic system.

5. Serotonin Receptor Subtypes

The 5-HT receptors have been divided into 7 subfamilies by convention. These subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organisation, and secondary messenger coupling pathways (Hoyer et al, 2002). The 5-HT₁, 5-HT₂, 5-HT₄, 5-HT₅, 5-HT₆, and 5- HT₇ receptors are G-protein-coupled receptors (GPCRs), whereas the 5-HT₃ receptors are ligand gated ion channels.

The structure of the G-protein-coupled 5-HT receptors is similar to nearly all GPCRs, with the receptors being integral membrane proteins with 7 putative hydrophobic transmembrane domains connected by three intracellular loops and three extracellular loops. The amino terminal is oriented extracellularly while the carboxyl terminal is orientated towards the cytoplasm. The core proteins possess conserved or common sites for post translational modifications while the extracellular domains are typically glycosylated, with the conformation of the receptor being maintained by disulphide bridges.

Numerous agents have been used to investigate and characterize 5-HT receptors. Since the first cloning and sequencing of a gene encoding a 5-HT receptor subtype (Fargin et al, 1988), nucleic acid probes have been available to study the regional distribution and mRNA transcripts using Northern blotting, and more precisely their localization on brain sections using in-situ hybridisation and autoradiography (Chalmers & Watson, 1991; Miquel et al, 1991; Pompeiano et al, 1992). The in-situ hybridisation technique works complimentary to ligand autoradiography, as it allows the visualisation of transcripts at the somatic level, as well as the affinity at the binding sites. The comparison of the respective distributions is indicative of the compartmentalization of receptors in neurons:

superimposition indicates a somato-dendritic localization (i.e. 5-HT_1A receptors, Miquel et al, 1991), whereas mismatch suggests an axonal location (i.e. 5-HT_1B

receptors, Boschert et al, 1994). Similarly, if the genetic sequence is known, the corresponding amino acid sequence can be deduced, and selective antibodies can be developed to recognise their peptides or proteins, which can help determine if neurons are producing mRNA for the various 5-HT receptor subtypes.

However the characterizing and localization of 5-HT receptors is very difficult due to the lack of selectivity of various 5-HT agonists and antagonists.

No agent displays an absolute specificity for one population of 5-HT receptors. It has been shown that 5-HT₁ receptors have a high (nanomolar) affinity for 5-HT, compared to the 5-HT₂ receptors which bind with low (micromolar) affinity (Peroutka & Snyder, 1979; Barnes & Sharp, 1999).

5.1 5-HT

Receptors

There are 5 members of the 5-HT₁ receptor family, termed 5-HT_1A, 5-HT_1B, 5- HT_1D, 5-HT_1E, and 5-HT_1F. The 5-HT_1C receptor has been re-classified as a 5-HT₂ type receptor based on its similarity in structure and secondary messenger systems. 5-HT₁ receptors act primarily through G_i/o-proteins to the inhibition of adenylate cyclase (AC) and to a multitude of other signalling pathways and effectors. It has been shown that while the 5-HT_1A receptor is responsible for regulating the firing of a serotonergic neuron, the local release of 5-HT is regulated by 5-HT1B/1D. There are also other 5-HT1 receptors involved in the periphery but these receptors have been omitted.

5.1.1 5-HT1A receptor

The 5-HT1A receptor is the best characterized of the 5-HT1 receptor subtypes. Its characterization is largely due to the wide availability of many specific pharmacological tools and because its cDNA and gene were cloned and identified (Fargin et al, 1988) over a decade ago. Like all the 5-HT1 receptors, the 5-HT1A

receptor is characterized pharmacologically by its high affinity for 5-HT. It has a uniquely high affinity for second generation, arylpyperazine anxiolytic agents, such as busperidone, gepirone and ipsapirone. This receptor subtype also has high

affinity for 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Many structural derivatives of 8-OH-DPAT are also being developed and tested for their relative affinities for 5-HT1A autoreceptors.

The 5-HT1A receptor was one of the first GPCRs for which the cDNA and gene were cloned. It was determined that there is a 88% homology between the human and rat receptor genes at the nucleic acid level, however there was substantially less homology between the other 5-HT subtypes (5-HT_2A = 19%; 5- HT_2C = 18%; and 5-HT_1D = 43%) (Barnes & Sharp, 1999). The 5-HT_1A receptor is coupled to a broad range of secondary messengers including various enzymes, channels and kinases and indeed to regulate secondary messenger production.

This receptor has been reported to both inhibit and activate AC and phospholipase C (De Vivo & Maayani, 1990: Cadogan et al, 1994: Weiss et al, 1986: Fargin et al, 1991), and to stimulate nitric oxide synthase (NOS) and an NADPH oxidase-like enzyme (Raymond et al, 2001). It can activate K⁺ channels and high conductance anion channels, inhibit Ca²⁺ conductance, and regulate a number of channels and transporters. It has also been shown to activate protein kinase C (PKC), Src kinase, and mitogen-activated protein (MAP) kinases. The 5- HT1A receptor has also been shown to; inhibit or stimulate Ca²⁺ mobilization, activate or inhibit phosphatidyl inositol hydrolysis, and stimulate the production of reactive oxygen species such as H2O2 and superoxide radicles as well as arachidonic acid (AA). The signals, in almost every case, have been shown to be sensitive to pertussis toxin implicating Gi/o –proteins in the mechanism of action of 5-HT1A.

5-HT1A receptors are mainly localized in limbic structures: hippocampus, cortex, septum, amygdala, and in raphe and dorsal horn of the spinal cord (Marcinkiewicz et al, 1984; Pazos & Palacios, 1985; Verge et al, 1986; El Mestikawy et al, 1900; Kia et al, 1996). Serotonergic neurons in the raphe nucleus normally have a slow, rhythmic firing rate when the animals are awake, and the activity of these cells is mostly linked to motor activity (brainstem and spinal cord). Activation of the 5-HT_1A autoreceptor results in a powerful inhibition of the firing rate (Hamon et al, 1990) and thus affects the release of 5-HT from terminals in projection areas (Hjorth & Sharp, 1991). This effect is mediated through the resulting opening of K⁺ channels, which in turn leads to the depolarisation of the neuron. It has also been shown that 5-HT_1A receptors located post-synaptically are

involved in a long feedback loop of the serotonergic system. Post-synaptic 5-HT1A

heteroreceptors have been identified on hippocampal pyramidal and granular cells (Miquel et al, 1991; Pompeiano et al, 1992), and are believed to be involved in the manipulation of a number of other neurotransmitter systems, which in turn may mediate an effect on the serotonergic neuron.

5.1.2 5-HT_1B receptor

The 5-HT_1B receptor was initially recognised as a separate receptor from the 5- HT_1A receptor by its affinity for [³H] spiperone (Nelson et al, 1991; 1981). There has been considerable confusion over the identities of the 5-HT_1B and 5-HT_1D receptors, this is due to their differences and relative similarities in varying species, and most notably their similar distribution in particular brain areas.

Surprisingly the 5-HT_1B receptor was only found in rodents, while the 5-HT_1D was found in human, dog, and guinea-pig (Gerhardt & van Heerikhuizen, 1997). With the advent of 5-HT receptor cloning, it became apparent that genes encoding both 5-HT1B and 5-HT1D were present in rat as well as humans. The confusion in nomenclature were instigated by the fact that there is a 93% sequence homology between the human 5-HT1B/1Dβ receptor and the cloned rat 5-HT1B receptor (Voigt et al, 1991), yet there were significant pharmacological differences between them, and further confusion was added by the fact that there was a pharmacological resemblance between the 5-HT1B/1Dβ receptor and the cloned 5-HT1Dα receptor (Hamblin & Metcalf, 1991; Weinshank et al, 1992). It was later determined that there was a single amino acid difference, and so the human 5-HT1Dβ receptor was renamed the h 5-HT_1B receptor.

The 5-HT_1B receptor is widely expressed in brain tissue, probably in both presynaptic and postsynaptic locations (Buhlen et al, 1996). 5-HT_1B receptors were initially identified in rodent brain using radioligand binding techniques and shown to be densest in the substantia nigra, globus pallidus, and dorsal subiculum (Zifa et al, 1992). 5-HT_1B autoreceptors are present on 5-HT axons and terminals, and upon activation of these receptors release, as well as synthesis of 5-HT is directly inhibited (Engel et al, 1986; Maura et al, 1986; Crespi et al, 1990; Hoyer

& Middlemiss, 1989; Starke et al, 1989; Gothert, 1990). Post-synaptic heteroreceptors have also been shown on non-serotonergic terminals where it is

believed they are involved in the regulation of alternative neurotransmitter systems (Maura & Raiteri, 1986). Activation of the 5-HT1B receptor has been shown to inhibit AC when natively expressed in tissues such as the substantia nigra and rabbit mesenteric arteries amongst other tissues. This receptor is also capable of activating PLC in several cell types by a pertussis toxin-sensitive mechanism resulting in an increase of intracellular Ca²⁺ (Dickenson & Hill, 1998).

It has also been shown that 5-HT can stimulate PLD via endogenous 5-HT_1B receptors in rabbit mesenteric artery through a signalling pathway that requires extracellular Ca²⁺ and PKC activation but is independent of PLC activation. 5- HT_1B has also been linked with regulating extracellularly signal-regulated kinases (ERKs), and NOS.

A number of ligands have been tested for their selectivity of 5-HT_1B receptors; however no 5-HT_1B receptors have been developed. Two of the more selective agents are CP-93,129 and serotonin O-carboxymethylglycyltyrosinamide but these also bind to 5-HT1D receptors.

5.1.3 5-HT1D receptors

The 5-HT1D receptor was discovered by homology screening using the canine RDC4 gene. It has been difficult to establish the exact locations of 5-HT1D

receptors due to lack of specific ligands and apparently low levels of mRNA and receptor protein in the brain. The aforementioned problem of nomenclature and lack of presence of 5-HT1D in rat brain also hindered progress on this receptor.

Binding sites for the 5-HT1D receptors have been localized to substantia nigra, globus pallidus, and caudate, and in lower levels in the cortex and hippocampus putamen (Bruinvels et al, 1994). However it was noted that mRNA for the receptor was not found in the globus pallidus or substantia nigra indicating that 5- HT_1D receptors may be transported along axon terminals after their synthesis.

Many of the more recent agents for 5-HT_1D receptors are tryptamine derivatives or sumatriptan- related structures. Recently two 5-HT_1D receptor antagonists have been developed: GR127935 and GR55562, which display high selectivity and high affinity for 5-HT_1D receptors (Skingle et al, 1996).

The 5-HT_1D receptors are capable of inhibiting AC through pertussis sensitive G-proteins in certain cell types (C6 glioma and NIH 3T3 fibroblast

cells). It has also been noted that when expressed at high concentrations in CHO or Y1 adrenal cells, this receptor can weakly stimulate cAMP accumulation (Wurch et al, 1997). Similar to the other 5-HT1 receptors these receptors can regulate ion channels such as K⁺ and Ca²⁺ ions (Le Grand et al, 1998).

It has been reported that 5-HT_1D receptors are located on serotonergic cell bodies and are believed to locally inhibit 5-HT release in several brain regions (Gerhardt & van Heerikhuizen, 1997). 5-HT_1D autoreceptors are responsible for diminished 5-HT release in guinea pig mesencephalic raphe, hippocampus, and frontal cortical slices through a pathway involving G-proteins. However the respective roles of the 5-HT_1B and 5-HT_1D receptors in the inhibition of 5-HT remain unresolved in many brain areas. It has been reported that both receptor subtypes exist as monomers and homodimers when expressed alone and as monomers and heterodimers when co-expressed. Gene expression studies have shown that there are brain regions where the 5-HT_1B and 5-HT_1D receptors are co- localized and where heterodimerization may occur physiologically (Xie et al, 1999).

5.1.4 5-HT_1E Receptor

There are very few details linked to the signalling pathways of the 5-HT1E

receptor. It was first identified as a component of [³H] 5-HT binding in human cortical homogenates which was resistant to a cocktail of antagonists of 5-HT_1A, 5-HT_1B/1D, and 5-HT₂ receptors (Leonhardt et al, 1989). The low affinity of 5-CT and ergotamine for 5-HT_1E receptors allowed for their differentiation from 5-HT_1D receptors. The 5-HT_1E receptor has been shown to both inhibit and stimulate activation of AC in different cells. The 5-HT_1E receptor has not yet been demonstrated to stimulate PLC and/or PLA₂. The mRNA encoding cloned 5-HT_1E receptor was shown to be localized to cortical areas, caudate putamen and amygdala (Bruinvels et al, 1994).

5.1.5 5-HT1F Receptor

The 5-HT_1F receptor was originally termed 5-HT_1Eβ based on its pharmacological similarities with the 5-HT_1E receptor. It was first cloned from mouse and shows 70% homology to the 5-HT_1E receptor (Amlaiky et al, 1992). It is the newest of the 5-HT₁ receptors, and the mRNA for this receptor has been detected in dorsal raphe nucleus, hippocampus and cortex of the rat. This receptor has also been shown to be negatively linked to AC, while it has also been shown to couple to PI-PLC in a cell specific manner. In the guinea-pig brain, mRNA and binding sites for 5-HT_1F receptors were detected in the cortex, mamillary nuclei, thalamic nuclei and the oculomotor nucleus (Mengod et al, 1996).

5.2. 5-HT

Receptors

The 5-HT₂ receptor family consists of three receptor subtypes; 5-HT_2A, 5-HT_2B, and 5-HT_2C (Hoyer et al, 1994). All 5-HT₂ receptor subtypes couple to the PLC-β second messenger pathway in native tissues and heterologous cells (Peroutka et al, 1995). Similarly to the 5-HT₁ receptors, the 5-HT₂ receptors can also couple to other second messenger pathways in a cell specific manner. One main difference between the 5-HT₁, and 5-HT₂ receptors is that the 5-HT₂ receptors genes contain introns which can lead in some cases to a number of mRNA editing, or splice variants.

5.2.1 5-HT_2A receptor

5-HT2A receptor was originally identified as a [³H] spiperone binding site, with low affinity for 5-HT. It is located post-synaptically on GABA interneurons in the pyriform cortex and large pyramidal neurons located in layer 5 (Poblete et al, 1995). This receptor underlies many of the motor effects of 5-HT and is involved in the actions of the main hallucinogenic drugs. They are located on astroglial cells and can regulate energy availability by stimulating the breakdown of glycogen (Poblete et al, 1995). It was classified as a 5-HT receptor base on its

pharmacological similarities with other 5-HT receptors. The 5-HT2A receptor is widely distributed in the brain (cortex, caudate nucleus, olfactory tubercle, nucleus accumbens, and hippocampus), and exerts its effects predominantly through PLC-β resulting in an accumulation of inositol phosphates and an increase in intracellular Ca²⁺ (Pazos et al, 1985). These effects can further result in activation of L-type Ca²⁺ voltage channels and stimulation of PKC.

In specific cell types, the 5-HT_2A receptor is also capable of activating other signalling messenger systems through PLA₂ and PLD. The resulting consequences include the release of AA and receptor induced prostaglandins (Berg et al, 1996, 1998: Tournois et al, 1998). The 5-HT_2A can regulate cAMP accumulation in certain cells, and also activate ERK MAP kinases - extracellular signal-regulated mitogen activated protein kinases – the significance of which is yet uncertain in the brain (Watts, 1998).

Activation of 5-HT_2A receptors can increase Ca²⁺ levels by liberating intracellular Ca²⁺ stores and/or by activating Ca²⁺ channels, depending on the cell of interest (Jalonen et al, 1997: Watts, 1998). Small K+ channels can also become indirectly opened by this increase in Ca²⁺ (Jalonen et al, 1997).

The 5-HT2A receptor is not surprisingly linked with the utilisation of calmodulin, as calmodulin is a major signalling target of Ca²⁺ mobilization in a variety of pathways in the body. 5-HT2A is shown to require calmodulin and calmodulin/Ca²⁺ kinases to become upregulated. Berg et al, (1994) have shown that 5-HT2A receptor increases cAMP in A1A1 adrenal cells by the intermediary action of calmodulin.

There is a unique quality attributed to the 5-HT2A receptor – its ability to internalise. This receptor internalises in response to agonist and antagonist signals but to a different degree depending on whether it was an agonist or antagonist.

These effects may be significant in the “ligand-directed” activation of secondary messenger systems by the 5-HT_2A receptor. The recent development of the selective 5-HT_2A antagonist MDL 100907 (Johnson et al, 1996), has allowed further studies to determine the role of this receptor in binding studies, and the role of 5-HT_2A receptors in behaviour.

5.2.2 5-HT

receptor

There is not so much known about the exact function of the 5-HT2B receptor, mainly due to the lack of specific receptor ligands. 5-HT2B receptors are generally located peripherally in liver, kidney, pancreas and spleen, with some studies reporting their presence (although at relatively low amounts) in amygdala, septum, hypothalamus, and cerebellum (Kennett et al, 1996). Similarly to the 5- HT2A and 5-HT2C receptors, this receptor couples to PLC. The 5-HT2B receptor has been reported to stimulate Ca²⁺ mobilization in astrocytes derived from rat cerebral cortex, hippocampus, and brain stem (Sanden et al, 2000). This 5-HT2B

receptor has been linked to an increase in the accumulation of cAMP, and also to the activation of extracellular signal-regulated kinases in certain cell types. In peripheral cells, 5-HT2B has also been shown to increase NO production and to regulate ion channels and transport processes (Florian and Watts, 1998).

5.2.3 5-HT

receptor

The 5-HT2C receptor, originally called the 5-HT1C receptor, was first identified as a high affinity [³H] 5-HT-binding site in the mouse choroid plexus (Pazos et al, 1985). The cloning of mouse, rat, and human 5-HT_2C receptors led to the recognition that this receptor was much more closely related to the 5-HT2 family than the 5-HT₁ family. The gene for this receptor contains 3 introns and a non- functional splice variant has been identified (Canton et al, 1996: Xie et al, 1996).

To date no other splice variants have been identified but the 5-HT_2C receptor has a unique mechanism of generating multiple functional receptor variants through a process called mRNA editing (Burns et al, 1997). The 5-HT_2C receptor is expressed almost exclusively in brain. High levels of 5-HT_2C receptor expression have been detected by ligand autoradiography, immunocytohistochemistry, and in situ hybridization techniques. 5-HT_2C receptors are determined to be located in the choroid plexus, the raphe, the cortex, the nucleus accumbens, the amygdala, the hippocampus, the caudate nucleus, and the substantia nigra (Barnes & Sharp, 1999; Millan et al, 2005; Pazos et al, 1985: Abramowski et al, 1995: Serrats et al, 2003; 2005).

The 5-HT2C receptor has long been known as being capable of exerting its effects through PLC in the choroid plexus (Sanders-Bush et al, 1988) and other brain areas (Wolf and Schutz, 1997). After activating PLC through Gq-proteins (Price et al, 2001), there is an increase in inositol phosphate accumulation (Briddon et al, 1998). It has been shown that with different agonists of the 5-HT_2C receptor, there is preferential activation of either the PLA₂ or PLC (Berg et al, 1998). This can lead to a wide array of signalling pathways.

The 5-HT_2C receptor at physiological levels has not been reported to modulate cAMP levels, however at high density it has been reported that it can inhibit forskolin stimulated cAMP production.

The 5-HT_2C receptor can regulate K⁺ and Cl^- channels. This has been illustrated in mouse choroid plexus (Hung et al, 1993). 5-HT_2C receptors have been shown to inhibit GABA_A receptor channels by a Ca²⁺-dependent, phosphorylation-independent mechanism in Xenopus oocytes (Huidobro-Toro et al, 1996). Also shown in Xenopus oocytes is the fact that 5-HT2C receptors can stimulate Ca²⁺ release from intracellular stores resulting in the opening of Ca²⁺- gated Cl^- channels (DiMagno et al, 1996). However, further investigations involving the role of 5-HT2C receptors in modifying the activity of GABAA

receptors in vivo have not been performed.

While there are no selective agents for 5-HT2C receptor there are a few antagonists that display selectivity for 5-HT2C over the other 5-HT2 receptors. SB- 242084 and RS 102221 are two selective compounds which show a 100 fold preference for 5-HT2C over 5-HT2A or 5-HT2B receptors (Kennett et al, 1997;

Bonhaus et al, 1997). The 5-HT2C antagonist SB-200646A displays a 50 fold selectivity over 5-HT_2A but binds equally well at 5-HT_2B. Preclinical studies show that this compound may possess anxiolytic activity due to its 5-HT_2B/5-HT_2C antagonism (Kennett et al, 1995).

The 5-HT_2C receptor has a very unique mechanism of generating novel multiple functional variants through a process known as mRNA editing. The functional significance of the various edited forms of the receptor can be displayed in different concentrations depending on which area of the brain they are located in (Fitzgerald et al, 1999). It was also noted that the different splice variants displayed different abilities to bind various ligands, to mobilize intracellular Ca²⁺

and to stimulate accumulation of inositol phosphates (Niswender et al, 1998:

Fitzgerald et al, 1999).

5.3. 5-HT

Receptors

Among the characterized 5-HT receptors of the central nervous system, the 5-HT3

receptor subtype is the only one known to be a ligand-gated ion channel. Recently two receptor subunits (5-HT3A and 5-HT3B) have been cloned, and it has been shown that these subunits can form homomeric or heteromeric dimers (Morales &

Wang, 2002). In situ hybridization histochemistry and reverse transcriptase-PCR amplification were used to demonstrate that 5-HT_3A subunit transcripts are expressed in central and peripheral neurons. In contrast, 5-HT3B subunit transcripts are restricted to peripheral neurons.

Its early pharmacological characterization and mapping by radioligand binding autoradiography suggested that this receptor may, among other actions, regulate dopamine release in the nigro-striatal pathway and reduce alcohol consumption in experimental animals while antagonists of this receptor have been reported to treat anxiety disorders. Following the cloning of this receptor in 1991, direct cellular localization was made possible by in situ hybridization and immunohistochemical analysis. It has been shown that 5-HT₃ receptor-expressing neurons are mainly GABA containing cells in the rat neocortex, olfactory cortex, hippocampus, and amygdala.

Many indolealkylamines bind at 5-HT₃ receptors in a non-selective manner. Ergolines do not bind, or only with low affinity. 5-HT binds with only modest affinity. MDL 72222 was the first selective 5-HT3 antagonist. It is a structural modification of cocaine, and many other similar compounds are undergoing test to determine their relative selectivities (Aapro, 1991).

5.4. 5-HT

Receptors

The 5-HT4 receptor is coupled primarily to the activation of adenylate cyclase, and exert its effects through the action of PKA. The major functional effects of the 5-HT4 receptor are in the periphery, but 5-HT4 receptors binding sites and

mRNA were found in the olfactory system, striatum, cortex, habenular nuclei, septum, hippocampus, amygdala, dorsal hippocampus, substantia nigra, interpeduncular nucleus and superior colliculus (Waeber et al, 1994; Vilaro et al, 1996). The gene for the 5-HT4 receptor contains multiple introns which results in a number of functional splice variants, which differ only in the length of their intracellular carboxy terminal tails (Claeysen et al, 1999).

5-HT₄ receptors can regulate a variety of channels, such as L-type Ca²⁺

channels, Ca²⁺-activated K⁺ channels, but these effects are mainly mediated through the effects of PKA.

Currently there are a number of 5-HT₄ agonists and antagonists, as reviewed by (Eglen et al, 1995). The most potent and selective antagonists are benzoate esters such as SB 204070 and indole esters such as GR 113808, whereas the most potent agonists are the carbazimidamides.

5.5. 5-HT

Receptors

There is little known about the 5-HT₅ receptor. There are two types of 5-HT₅ receptor as has been determined from cloning studies; 5-HT_5A and 5-HT_5B (Rees et al, 1994). These receptors have high affinity for LSD and 5- carboxamidotryptamine, and both have been found in the brain, whereas only 5- HT_5B receptors have been identified in the periphery. Recently it was shown that 5-HT5A receptors are weakly detected on neurons in the cortex, and that their primary site of action is non-neuronal (Carson et al, 1995). Rat 5-HT_5A receptors are expressed in vivo and in vitro by astrocytes and have been shown to be negatively coupled to AC (Carson et al, 1995).

5.6. 5-HT

Receptors

This 5-HT receptor stimulates AC and has a high affinity for typical and atypical antipsychotics such as clozapine. The receptor is expressed in the caudate nucleus, the olfactory tubercle, the striatum, the hippocampus and the nucleus accumbens (Gerard et al, 1996: Grimaldi et al, 1998). Some receptors also expressed in the

periphery including the adrenal gland and the stomach. 5-HT6 receptors regulate cholinergic transmission in the brain implicating a function in learning and memory (Branchek & Blackburn, 2000). There are two introns in the gene for 5- HT6 receptor but to date only two truncated splice variants have been identified (Monsma et al, 1993: Olsen et al, 1999). It was the first 5-HT receptor shown to be coupled to the activation of AC.

5-HT binds at the 5-HT₆ receptor with moderate affinity, and one of the highest affinity agents is methiothepin. Other agents which bind with high affinity include 5-methoxytryptamine, clozapine and olanzapine.

5.7. 5-HT

Receptors

There are two introns in the gene for the 5-HT7 receptor, and at least four splice variants have been determined. It is highly expressed in the CNS especially in the hippocampus, hypothalamus, and the neocortex. It has been speculated to participate in the control of circadian rhythms because it is expressed in the suprachiasmatic nucleus (Lovenberg et al, 1993: Stowe & Barnes, 1998). The functional significance of the individual splice variants is till unclear, but all of them exert their effects through the activation of AC.

Many ligands bind to 5-HT₇ with low affinity and are used to label this receptor. Agents with k_i values of <10nM include 5-HT, 5-methoxytryptamine, LSD, methiothepin, and mesulergine. A number of antipsychotic and antidepressant agents bind the 5-HT₇ receptor with nanomolar or subnanomolar affinity.

6. Signal Transduction Pathways

Two major receptor-linked signal transduction pathways exist, and the numerous 5-HT receptors utilise one or other of them. They are either a multistep enzyme mediated pathway; or a direct regulation of ion channels. Both require a guanine nucleotide triphosphate (GTP)-binding protein (G protein) to link the receptor to the effector molecule.

The sequence of steps involved in enzyme-dependent biochemical signalling is: cell-surface receptor G protein effector enzyme activating a secondary messenger system which in turn activates further messengers such as protein kinase, or causes conformation changes to proteins within the cell e.g.

phosphoprotein. This multistep scheme applies to receptors linked to activation and inhibition of adenylate cyclase and activation of phospholipase C. These enzyme-dependent pathways lead to amplification of cellular signals. Each step involves proteins that exist in multiple forms. For example, G proteins are composed of α, β, and γ subunits, each of which exists in multiple isoforms. More than 15 α subunits and at least three β and γ subunits have been identified, leading to a tremendous diversity in G proteins. Multiple isozymes of both adenylate cyclase and phospholipase C have been found, and more than one isoform is involved in signal transduction. In addition, protein kinase A, protein kinase C, and calcium/calmodulin-dependent kinase exist as multiple isoforms. A final level of complexity, which may serve to integrate various signals, is protein phosphorylation. The protein substrates are numerous, and it is impossible to list them. Key proteins regulated by phosphorylation include neurotransmitter and growth factor receptors, G proteins, protein kinases, protein phosphatases, ion channels, neurotransmitter synthetic and metabolic enzymes, transport molecules, and DNA transcription factors.

Adenylate Cyclase (AC)

Activation of AC by the binding of a ligand to the receptor results in an increase of cyclic 3,5-adenosine monophosphate (cAMP). Proximal cellular events that result from an increase in (cAMP) include activation of protein kinase A, which, in turn, regulates the activity of cellular proteins by phosphorylation.

Phospholipase C (PLC)

(PLC), a membrane-bound enzyme, catalyzes the degradation of the inositol lipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), with the production of inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ mobilizes Ca²⁺ from an intracellular storage site by interacting with specific receptors. Ca²⁺ induces multiple responses in the cell, including activation of calcium/calmodulin-

dependent protein kinases, enzymes which phosphorylate/ dephosphorylate key protein substrates in the cell.

DAG activates protein kinase C (PKC) which is the enzyme responsible for regulating numerous processes of cell function. DAG is also hydrolysed by a specific lipase to release arachidonic acid with the subsequent formation of prostaglandins and prostacyclins. Thus, PLC activation induces diverse changes in the cell, leading to the regulation of many cellular processes.

Phospholipase A₂ (PLA₂)

In the early literature, it was found that 5-HT stimulates the activity PLA₂ in membranes of guinea-pig cerebral cortex. PLA₂ releases arachidonic acid, resulting in the production of arachidonic acid metabolites by lipoxygenase and cycloxygenase pathways with the formation of eicosanoids. Recently, it has been found that 5-HT stimulates the production of arachidonic acid in a number of brain tissues.

Cross-Talk of Signal Transduction Pathways

A given receptor may couple to more than one signal transduction pathway. The numerous and diverse possibilities of cross-talk are just beginning to be worked out. As well as the known effects of Gα subunits activating secondary messenger systems, recent evidence suggest that G-protein βγ subunits may also play a role in cross-talk between signalling pathways. Both adenylate cyclase and phospholipase-Cβ are activated by Gβγ in an isoform-specific manner. Gβγ

stimulation of PLC and adenylate cyclase requires high receptor occupancy and high expression levels. Thus the functional consequences of receptor–G-protein activation may vary from cell to cell, depending on both (a) the receptor and its level of expression and (b) the component of effector molecules within a given cell.

7. Clinical significance of 5-HT receptors

5-HT is known to be involved in numerous physiological and behavioural systems which explains the many 5-HT based drugs used as treatments in a variety of different clinical conditions. Even though alterations in 5-HT system function are observed in many of these clinical conditions, there is no direct evidence to suggest there is such a thing as a “serotonin disease”. At present there are many treatments directed at increasing or decreasing serotonin levels, as well as serotonergic transmission at their selected target cells, however there is considerable research yet to be performed to clarify the causative role of many of these clinical conditions.

Treatments are generally targeted towards the various 5-HT receptors, as altering the delicate balance of endogenous 5-HT would exert a more global response. As already mentioned there are a variety of selective or semi-selective agents for most of the 5-HT receptor subtypes, and with further research into the functions of each individual receptor, a more selective and effective agent can be developed.

5-HT1A receptors

5-HT1A ligands with agonist activity seem to possess antianxiety, antidepressant, antiaggressive and perhaps anticraving, anticataleptic, antiemetic, and neuroprotective properties (Jolas et al, 1995). These receptors are believed to be involved in impulsivity, alcoholism and sleep. However the main therapeutic potential of 5-HT1A receptors is in the treatment of anxiety and depression. The antianxiety actions of 5-HT1A (partial) agonists may involve primarily presynaptic somatodendritic 5-HT1A receptors (which results in a decrease in terminal 5-HT release) whereas the antidepressant action of 5-HT1A agents may primarily involve postsynaptic 5-HT_1A receptors (Jolas et al, 1995). Measurement of the density of 5-HT_1A receptors in frontal cortex of suicide victims reveals that non- violent suicide victims had a significantly B_max, compared with control and violent suicides (Matsubara et al, 1991). Other links to 5-HT_1A include alcoholism (Dillon et al, 1991), depression and anxiety (Rausch et al, 2001; Tollefson et al,

1993), sexual behaviour, appetite control, thermoregulation, and cardiovascular function (Saxena, 1995).

5-HT1B receptors

5-HT_1B receptors play a role in thermoregulation, respiration, appetite control, sexual behaviour, aggression and anxiety. Other studies have also implicated 5- HT_1B in sleep, sensorimotor inhibition, and locomotor activity (Monti et al, 1995:

Sipes et al, 1996).

Knockout studies have been particularly useful in elucidating the role of 5- HT_1B receptors. The absence of functional 5-HT_1B receptors in the homozygous mutants was confirmed by the reduction of [¹²⁵I]-cyanopindolol binding to brain sections. The animals exhibited no overt abnormalities in brain morphology or in the expression of related serotonin receptor subtypes. The absence of 5-HT_1B receptors did not appear to produce marked alterations in appearance or baseline behaviours of the mutant mice (Lucas & Hen., 1995).

Due to the proposed role of serotonin systems in aggression and locomotion, these behaviours were examined in 5-HT1B receptor knockout mice.

As for locomotion, baseline levels of aggression appeared normal in the mutants.

In the locomotion test the 5-HT1B knockout animals were insensitive to the hyperlocomotor effects of the 5-HT1A/1B receptor agonist RU24969, and marked differences were observed in the provocative "resident-intruder" aggression paradigm. Following isolation, resident mutant mice displayed hyperaggressive behaviour toward intruders, as evidenced by reduced attack latencies and increased frequencies of attack, relative to wild type animals. This result led to the suggestion that 5-HT_1B receptors contribute to the serotonergic regulation of aggression and to the actions of "serenics", a class of nonspecific 5-HT₁ receptor agonists with antiaggressive properties.

A potential role for 5-HT_1B receptors in the serotonergic modulation of alcohol intake was also supported in a recent study of 5-HT_1B receptor mutants (Crabbe et al, 1996). Mutant mice displayed elevated ethanol consumption in a two-bottle choice situation. In addition, they exhibited reductions in ethanol- induced ataxia and in the development of tolerance to ethanol, compared with wild-type mice. This study demonstrated that the phenomena of ethanol

sensitivity, tolerance, and drinking could be genetically dissected in this knockout model.

5-HT1D receptors

The clinical significance of 5-HT_1D receptors is largely unknown, but they are believed to be involved in anxiety, depression, and other neuropsychiatric disorders. It is also believed to play a role in the biochemical processes that lead to migraines. It has been postulated that the human 5-HT_1D receptor is also believed to be involved in neurogenic inflammation and vasoconstriction.

The clinical significance of 5-HT_1E and 5-HT_1F receptors is unknown at this time.

5-HT_1F receptors are believed to play some role in migraine (Adham et al, 1993), as they are expressed both in neural and vascular tissue in the CNS.

5-HT2A receptors

Many of the clinical actions of 5-HT2A receptors may actually involve 5-HT2C

receptors or a combination of the two. The specific clinical role of 5-HT2B is, as yet, unknown. 5-HT2A receptors play a role in appetite control, sleep and thermoregulation, and also play a role in cardiovascular function and muscle contraction (Saxena, 1995: Zifa et al, 1992). Various antipsychotic agents and antidepressants bind with relatively high affinity to this receptor implicating it in a beneficial neuropsychiatric role. The role of 5-HT2A has been reviewed in anxiety, and also in hallucinogenic potential (Fiorella et al, 1995: Schreiber et al, 1994).

5-HT_2C receptors

The activation of 5-HT_2C receptors are implicated in the anxiogenic, aversive, and endocrine actions of SSRIs as well as their involvements in sexual function, locomotor behaviour and sleep (Kennett et al, 1997; Olivier et al, 1998; Millan et al, 1999; Dekeyne et al, 2000). It has also been shown that the density and functional activity of 5-HT_2C receptors is increased in depressed patients and animal models of depression (Bos et al, 1997; Fone et al, 1998). As previously mentioned, there are no agents that display specificity for one population of 5-HT₂ receptors over another. Many experiments have been performed using 5-HT_2A/2C

or 5-HT2B/2C antagonists, but a more efficient way of determining the clinical implications of 5-HT2C receptors is by using knockout animals.

Through gene targeting procedures, a null mutation was introduced into the X-linked 5-HT2C receptor gene (Tecott et al, 1995). The absence of intact receptor protein in hemizygous mutant male mice was verified by the loss of 5- HT_2C receptor immunoreactivity and by the absence of functional 5-HT_2C receptors encoded by brain mRNA, as determined in a Xenopus oocyte expression assay. The mutant mice exhibited no overt abnormalities in appearance or in brain morphology.

Two major phenotypic abnormalities were reported in 5-HT_2C receptor mutants: epilepsy and obesity. Mutant mice were prone to occasional spontaneous episodes of tonic-clonic seizure activity. Furthermore, these animals displayed a markedly elevated sensitivity to the convulsant actions of the GABA_A receptor antagonist metrazol.

Mutant animals also exhibited an obesity syndrome, manifested by a 50%

increase in the deposition of white adipose tissue in young adult animals. The obesity was associated with elevated food intake and did not appear to result from metabolic alterations indicating that 5-HT2C receptors are normally involved in the serotonergic inhibition of appetite. Moreover, mutant mice were found to be resistant to the anorectic effects of mCPP, indicating that this drug reduces food intake through its action at 5-HT2C receptors. More generally, this demonstrates that receptor knockout models may be useful for determining the extent to which particular subtypes mediate the effects of nonspecific drugs.

5-HT₃ receptors

5-HT₃ receptors have been shown to be clinically effective for the treatment of chemotherapy-induced or radiation-induced nausea and vomiting (Gyermek et al, 1995), whereas they are ineffective against motion sickness and apomorphine- induced emesis). There are also indications that this receptor is involved with the mediation of pain and migraine. Preclinical studies suggest that 5-HT₃ antagonists may enhance memory, and may be beneficial in anxiety, depression, pain, and dementia treatment. It is believed that 5-HT₃ agonists display some anxiolytic potential (Rault et al, 1996).

5-HT4 receptors

Previously, a lack of selective agents for this receptor has hampered the investigation of its therapeutic potential. However, lately 5-HT4 agents are being examined both for central and peripheral effects. It has been suggested that 5-HT4

agonists may restore deficits in cognitive function and that 5-HT₄ antagonists may be useful as anxiolytics in. 5-HT₄ receptors may also be involved in memory and learning, as they are markedly decreased in Alzheimer’s patients (Eglen et al, 1995).

5-HT₅ receptors

The pharmacological function of 5-HT₅ receptors is currently unknown. It has been proposed that they are involved, based on their localization, in motor control, feeding, anxiety, depression, learning, memory consolidation, adaptive behaviour and brain development (Matthes et al, 1993: Rees et al, 1994).

5-HT6 receptors

The pharmacological function of 5-HT6 receptors is still unknown at this time.

Various anti-psychotics, and anti-depressants suggest a possible connection between this receptor and the therapeutic effects of the drug (Roth et al, 1994).

5-HT7 receptors

5-HT7 receptors have been shown to be involved in mood and learning, as well as endocrine and vegetative behaviours. It is also possibly linked to depression, sleep, and vasoactive properties.

8. Effects of Serotonin on Behaviour

Research over the past few decades has led to the development of specific theories regarding the function of the related neurotransmitter systems. 5-HT is believed to be involved in a variety of processes including cardiovascular and respiratory activity, sleep, aggression, sexual behaviour, nutrient intake, anxiety, depression,