Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS
Introduction
The physiological effects of the neurotransmitter 5-hydroxytryptamine (5-HT) are mediated through activation of at least 14 different receptor proteins (Hoyer et al., 1994, Saxena, 1995) classified into seven subfamilies. Within the 5-HT2 family, the 5-HT2C (previously denoted 5-HT1C) receptor was first identified in porcine choroid plexus on the basis of its pharmacological characteristics (Pazos et al., 1984). On subsequent cloning the 5-HT2C receptor was found to be a 51,916 kDa molecular weight protein of 460 amino acids coupled to a G-protein (Julius et al., 1988). As with the structurally homologous 5-HT2A (Pritchett et al., 1988, Julius et al., 1990) and 5-HT2B receptors (Foguet et al., 1992, Kursar et al., 1992), 5-HT2C receptor activation stimulates phospholipase C activity and phosphatidyl inositol hydrolysis (Conn and Sanders-Bush, 1986). In situ hybridisation has shown that 5-HT2C receptor mRNA is abundantly expressed throughout the central nervous system of rat and man (Hoffman and Mezey, 1989, Molineaux et al., 1989, Mengod et al., 1990, Pompeiano et al., 1994). In order to establish the functional role of the 5-HT2C receptor, a precise anatomical localisation of this protein would be beneficial. Although 5-HT2C binding sites have been demonstrated by autoradiography using [3H]mesulergine (Pazos and Palacios, 1985, Hoyer et al., 1986, Mengod et al., 1990) resolution is poor due to the high non-specific binding associated with this radioligand. Although selective 5-HT2C antagonists have recently become available for functional studies (Bonhaus et al., 1997, Kennett et al., 1997), no selective high affinity ligand suitable for autoradiography is currently available to distinguish between the three 5-HT2 receptors (Baxter et al., 1995). To overcome this problem, we have used an immunological approach to specifically identify the 5-HT2C receptor distribution in the rat CNS.
Several groups have produced antisera against the 5-HT2C receptor protein (Abramowski and Staufenbiel, 1995, Backstrom et al., 1995, Sharma et al., 1997) but there is only one previous report on the immunohistochemical distribution in the rat brain (Abramowski et al., 1995). This study used an antiserum generated against the C-terminus of the mouse 5-HT2C protein and immunoautoradiography with a radiolabelled secondary antibody which enabled positive anatomical structures to be identified but, due to poor resolution, unlike the current technique gave no information on the cellular localisation.
Section snippets
Antibody production
A high titre polyclonal antiserum was produced against a 15 amino acid N-terminal portion of the rat 5-HT2C receptor protein (DGGRLFQFPDGVQNW, Washington Singer Laboratories, Exeter, UK, corresponding to amino acids 42–56 (Julius et al., 1988)), synthesised with two additional C-terminal residues (Y16 and C17). These additional amino acids enabled addition of radiolabel [125I] and coupling to Keyhole Limpet haemocyanin (KLH) to increase immunogenicity, respectively. This peptide sequence had no
Antibody specificity
The specificity of the 5-HT2C antibody was assessed using radioimmunoassay, Western blot analysis and immnuofluorescence with a h5-HT2C tranfected cell line. For radioimmunoassay, the cross-reactivity of the antiserum was tested with synthetic peptides derived from the N-terminal portions of the rat 5-HT2A and 5-HT2B receptors (Duxon et al., 1997) and the N-terminal 13 amino acids of the rat 5-HT2C receptor (a different sequence from that used for the current immunisation, see Sharma et al.,
Discussion
Seven major 5-HT2C receptor isoforms with brain region-specific distributions have been identified in the rat (Burns et al., 1997) and polymorphism occurs in man (Lappalainen et al., 1995). However, none of these variants contain alterations in the amino acid region used to generate the current polyclonal antiserum, which should therefore recognise all these variants. Differential splicing produces a transcript encoding a truncated 5-HT2C receptor protein (172 amino acid long and lacking either
Acknowledgements
We would like to thank John Humphrey (SmithKline Beecham Pharmaceuticals, Harlow) for the synthesis of the 5-HT2C peptide sequence. DAC was an MRC case student and both MSD (a BBSRC case student) and TP (Nottingham University half-funded studentship) were supported by SmithKline Beecham Pharmaceuticals who also provided the HEK293 cell lines.
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2021, Progress in Brain ResearchCitation Excerpt :Later studies confirmed that the choroid plexus in mammals express the highest quantities of 5-HT2C receptors, far beyond the other brain regions (Hoyer et al., 1986; Pazos and Palacios, 1985; Pazos et al., 1984). They are also expressed at different levels in various CNS regions including the basal ganglia, the cortex, the hippocampus, the septum, the amygdala, hypothalamic nuclei, habenula, the spinal cord to cite a few (Abramowski et al., 1995b; Clemett et al., 2000; Delicata et al., 2018; Mengod et al., 1990a; Pasqualetti et al., 1999; Pazos and Palacios, 1985). In the basal ganglia, their density is higher in the ventromedial striatum/Nac compared to the dorsolateral striatum, and elevated in the STN, the VTA, the SNc, the SNr, the GPe and the GPi (Bubar and Cunningham, 2007; Clemett et al., 2000; Molineaux et al., 1989; Shannak and Hornykiewicz, 1980; Wright et al., 1995).
- 1
Present address: Adis International Ltd, Chowley Oak Lane, Tattenhall, Chester CH3 9GA, UK.
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Present address: SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Harlow, Essex CM19 5AW, UK.
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Present address: Synaptic Pharmaceutical Corporation, 215 College Road, Paramus, NJ 07652-1431, USA.