Distribution of 5HT2C Receptors in the Basal Ganglia 18131 General Overview

The 5-HT2C receptor (termed 5-HTlC receptors before 1994; see Hoyer et al. 1994) belongs to the seven transmembrane G-protein-coupled receptor family. It is located exclusively within the central nervous system (CNS) with no messenger ribonucleic acid (mRNA) detected in heart, lungs, intestine, or kidney (Julius et al. 1988). Numerous studies have demonstrated a widespread and similar distribution of the 5-HT2C receptor subtype in rat, monkey, and human brains. The highest concentration is observed in choroid plexus and moderate levels have been reported in the different regions of the basal ganglia. Like the 5-HT axon terminals, the 5-HT2C receptors display a heterogeneous distribution in the basal ganglia (Mengod et al. 1990; Pompeiano et al. 1994). First of all, the mRNA has been observed in all regions except the GPe in rats, monkeys, and humans (Eberle-Wang et al. 1997; Lopez-Gimenez et al. 2001; Pasqualetti et al. 1999). In addition, rostral levels of the SNr are devoid of 5-HT2C receptor mRNA whereas the highest expression in this region has been reported in central and caudal parts, preferentially medially (Eberle-Wang et al. 1997). The highest concentration of mRNA has been found in the STN, SNc, SNr, VTA, and EPN with virtually all neurons in the latter region being labeled (Mengod et al. 1990; Eberle-Wang et al. 1997). In the striatum, 5-HT2C receptor mRNA shows a topographical gradient with higher levels medially and anteroventrally (Pompeiano et al. 1994; Eberle-Wang et al. 1997; Ward and Dorsa 1996). High levels are also seen within the nucleus accumbens (Wright et al. 1995).

Autoradiographic studies have reported moderate levels of 5-HT2C receptors in the basal ganglia. It should be noted, however, that the radioligands used in early studies, including [3H]lysergic acid diethylamide (LSD), [3H]5HT, [125I]LSD, or [125I]SCH 23982 are not selective for 5-HT2 receptors (Hoyer and Karpf 1988; Meibach et al. 1980; Young and Kuhar 1980). The radioligands used nowadays are [3H]mesulergine, and to a lesser extent, [3H]1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) that is also commonly used to label 5-HT2A receptors. Mesulergine is an ergot derivative with a high and preferential affinity for 5-HT2C receptors but also binds to 5-HT2A, 5-HT2B, 5-HT6, and 5-HT7 and D2 receptors. Furthermore, the cold ligands used to displace unwanted sites bound by [3H]mesu-lergine including 5-HT itself, mianserine (which displays equal affinity for 5-HT2A and 5-HT2C receptors), or spiperone (which displaces mesulergine from D2 and 5-HT2A receptors) are not selective. New ligands that are selective for 5-HT2C exist, but their use has been sporadic (Fox and Brotchie 2000a). Despite these limitations, ligand binding studies of 5-HT2C receptors within the basal ganglia show a high correlation with in situ hybridization for 5-HT2C mRNA in the SNr and GPi (high binding) and in the SNc, putamen, and caudate (low binding) (Mengod et al. 1990; Lopez-Gimenez et al. 2001; Wright et al. 1995; Hoyer et al. 1986; Pazos et al. 1987; Hoffman and Mezey 1989; Molineaux et al. 1989). Exceptions to a good match with mRNA distribution include the STN and the GPe. For example, Mengod et al. (1990)

have reported relatively low binding sites in the STN compared with the high levels of mRNA (Mengod et al. 1990). Furthermore, binding studies in rat and humans indicate equal levels of 5-HT2C receptors binding in the GPe and GPi, whereas GPe neurons do not express 5-HT2C mRNA (Eberle-Wang et al. 1997; Fox and Brotchie 2000a; Pazos et al. 1987; Pazos and Palacios 1985). This distribution of 5-HT2C receptors has been confirmed using immunohistochemistry (Clemett et al. 2000). These data suggest that 5-HT2C receptors in the basal ganglia are mostly somato-dentritic except in the GPe, where they are likely located presynaptically, on axon terminals of afferent neurons (Fig. 18.2).

In addition to the limited selectivity of the ligands, an important limitation of the radioligand binding studies to date is their inability to distinguish among various forms of the receptor. Indeed, the 5-HT2C receptor mRNA undergoes an editing process by RNA adenosine deaminases at various sites of the region encoding for the second intracellular loop of the receptor, which is critical for G-protein coupling (Burns et al.

Nucleus accumbens (5-HT2C) Dorsal Striatum ^

mRNA Binding sites Coupling Localization mRNA Binding sites

1 neurons, Ach

Medium-size spiny neurons, Ach

(>striosomal over matrix compartment),

Mediolateral, ^ ventrodorsal

Nucleus accumbens (5-HT2C) Dorsal Striatum ^

mRNA Binding sites Coupling Localization mRNA Binding sites

1 neurons, Ach

Medium-size spiny neurons, Ach

(>striosomal over matrix compartment),

Mediolateral, ^ ventrodorsal

Fig. 18.2 Distribution of 5-HT2C receptors mRNA and binding sites in the basal ganglia. In each region, the density of mRNA is presented first by ++, +, or 0, relative to their level of expression, while the relative density of binding sites is presented second. In some occasions, neuronal populations have been labeled to illustrate their ability to express the receptor. 5-HT neurons from the dorsal raphe nucleus (DRN) have been added. GPe and GPi external and internal globus pallidus, VP ventral pal-lidum, SNr and SNc substantia nigra pars reticulata and pars compacta, STN subthalamic nucleus, VTA ventral tegmental area. Filled arrows correspond to excitatory neurons; the other arrows correspond to inhibitory neurons. Dopaminergic neurons diffusely innervate all basal ganglia regions

STN

++, +

Fig. 18.2 Distribution of 5-HT2C receptors mRNA and binding sites in the basal ganglia. In each region, the density of mRNA is presented first by ++, +, or 0, relative to their level of expression, while the relative density of binding sites is presented second. In some occasions, neuronal populations have been labeled to illustrate their ability to express the receptor. 5-HT neurons from the dorsal raphe nucleus (DRN) have been added. GPe and GPi external and internal globus pallidus, VP ventral pal-lidum, SNr and SNc substantia nigra pars reticulata and pars compacta, STN subthalamic nucleus, VTA ventral tegmental area. Filled arrows correspond to excitatory neurons; the other arrows correspond to inhibitory neurons. Dopaminergic neurons diffusely innervate all basal ganglia regions

1997; Niswender et al. 1999; Berg et al. 2001). This process results in multiple 5-HT2C transcripts and isoforms that differ in their pharmacological properties. Mesulergine is not a pure neutral antagonist and displays inverse agonist properties. This implies that its binding is also dependent on the isoform considered. Therefore, other radioligands are needed to confirm the pattern of expression described earlier.

18.1.3.2 Cellular Types Expressing the 5-HT2C Receptors

Studies using in situ hybridization, chemical lesions of neuronal pathways, reverse transcription coupled to polymerase chain reaction (RT-PCR) and immuno-histochemistry have helped identify some of the cell types that express 5-HT2C receptors. In the striatum, most neurons expressing the receptor mRNA are medium-sized spiny neurons, the efferent GABA-ergic neurons innervating the GPe ("indirect" pathway), or the SNr/GPi ("direct" pathway). 5-HT2C receptor mRNA is similarly expressed in projection neurons forming both pathways because it colocalizes equally with enkephalin and substance P/dynorphin, respectively (Ward and Dorsa 1996). Recent RT-PCR studies have shown that the 5-HT2C mRNA is present in cholinergic interneurons. Interestingly, 5-HT2C mRNA showed a patchy distribution (Ward and Dorsa 1996) that could correspond to the patchy distribution of the 5-HT2C receptor binding of [3H]DOI reported by Waeber and Palacios (Waeber and Palacios 1994). This finding suggests a preferential localization in the striosomal versus the extrastriosomal matrix compartment. The strio-somes, or "patches," and the extrastriosomal "matrix" represent an anatomofunctional organization of the striatum, with the striosomes being low in acetylcholinesterase, rich in mu opioid receptors, and containing the cell bodies of the GABA-ergic neurons projecting to the SNc (Gerfen 1984, 1985; Graybiel 1991).

In the STN, the cells expressing 5-HT2C receptors are presumably the gluta-matergic neurons innervating the GPe, the GPi, and the SN. The terminals carrying the presynaptic 5-HT2C receptors in the GPe have not been identified; they could include afferents from striatal and/or STN neurons. The cells expressing 5-HT2C receptors in the GPi and SNr likely include the GABA-ergic efferent neurons that innervate the thalamus and superior colliculus. Importantly, two populations of GABA-ergic neurons can be distinguished in the SNr; some neurons express a high level of 5-HT2C mRNA, whereas others seem devoid of the mRNA (Eberle-Wang et al. 1997). It is interesting to note that some authors consider that the SNr is composed of a mixed population of neurons equivalent to GPe and GPi neurons; the heterogenous distribution of the 5-HT2C receptor mRNA may in fact identify these two populations since GPe neurons do not express the mRNA, whereas GPi neurons do. Furthermore, a rostrocaudal gradient of expression of the mRNA has been detected in this region (Eberle-Wang et al. 1997).

The cellular location of 5-HT2C receptors in the ventral mesencephalon (SNc/ VTA) may be more complex. In situ hybridization data suggest that, in the SNc, the receptor mRNA is selectively expressed by GABA-ergic neurons that are intermingled with DA neurons in this region. Indeed, 5-HT2C mRNA is colocalized with the mRNA for glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis, but not with tyrosine hydroxylase, the rate-limiting enzyme of DA biosynthesis in this region (Eberle-Wang et al. 1997). However, this apparently differential distribution may reflect large differences in levels of expression of the mRNA rather than a selective expression excluding the DA neurons. Indeed, more recent techniques of laser capture microdissection allow for the determination of mRNA expression in selected cell populations with quantitative polymerase chain reaction (Q-PCR), a technique that is much more sensitive than in situ hybridization histochemistry. With this approach, the 5HT2C mRNA can be detected in tyrosine hydroxylase positive neurons of the SNc (Mortazavi and Chesselet, unpublished observations). These data are compatible with results from a recent study reporting immunostaining with an antibody raised against 5-HT2C receptor in both GABA-ergic and DA neurons of the VTA (Bubar and Cunningham 2007). Even though such a colocalization appears to exist in the SN based on the PCR data, it may be limited because 5-HT2C binding sites tend to increase rather than decrease in the SNc after loss of nigrostriatal DA neurons both in rats and in humans (Fig. 18.2) (Fox and Brotchie 2000a; Radja et al. 1993).

18.1.3.3 Conclusions

5-HT2C receptors are heterogeneously expressed in the basal ganglia. A comparison between mRNA and binding sites suggests that this receptor is mostly a somatoden-dritic receptor, except in the GPe, where it may be located on axons. Numerous cell types express the receptor including GABA-ergic, glutamatergic and cholinergic neurons. The DA neuron may express very low levels of 5-HT2C receptors in the SNc but greater levels in the VTA. In general, the density of 5-HT2C receptors follows the density of the 5-HT innervation, the ventromedial parts of the basal ganglia being enriched in both. This pattern of expression of 5-HT2C receptors suggests that its functional influences may be stronger on associative and limbic circuits than on the sensorimotor pathways.

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