Immunohistochemical evidence that 5-HT2C receptor agonist administration activates GABA neurones in the rat DRN

Josephine Raley, Franziska Denk, Laura Boothman & Trevor Sharp. University Department of Pharmacology, Mansfield Road, Oxford, OX1 3QT.

The firing of midbrain 5-HT neurones is controlled by 5-HT1A autoreceptors, but emerging evidence also indicates a role for postsynaptic 5-HT2 receptors. Thus, 5-HT2 receptor agonists inhibit the firing of 5-HT neurones in the dorsal raphe nucleus (DRN) in vivo (Boothman et al, 2003), and recent data show selective 5-HT2C agonists have this effect (Boothman et al, this meeting). Since 5-HT2C receptor activation evokes inhibitory postsynaptic currents in DRN 5-HT neurones via a GABAA receptor-mediated mechanism in vitro (Lui et al., 2000), it implies that 5-HT2C receptors may activate DRN GABA neurones, which then inhibit nearby 5-HT neurones. Here, Fos immunohistochemistry was used to investigate whether DRN neurones are activated by the 5-HT2C receptor agonist, WAY 161503 (Cryan et al. 2000), and whether these neurones are GABAergic. Male Sprague Dawley rats (250-270g, 6 per group) received i.p. injections of saline, WAY 161503 (1, 3 or 10 mg/kg) or mCPP (non-selective 5-HT 2C receptor agonist 5 mg/kg). In a separate study, rats received vehicle or SB 242084 (5-HT 2C receptor antagonist; 1 mg/kg) followed 30 min later by either saline or WAY 161503 (3 mg/kg). Two hr later rats killed by fixation-perfusion, and DRN sections were processed for FOS and glutamate decarboxylase immunoreactivity (Fos-ir and GAD-ir, respectively). Immunopositive cells were counted in a blind fashion by microscopic observation, and analysed statistically using one-way ANOVA with post-hoc tests. Double-labelling for GAD or Fos and the 5-HT2C receptor was also carried out. Compared to saline controls, WAY 161503 increased the number of Fos-ir DRN neurones at doses of 3 and 10 mg/kg (177±22 % and 171±13 %, respectively; p<0.001). mCPP (5 mg/kg) also had this effect (178±11 %, p<0.001). WAY 161503 increased the number of DRN neurones in which Fos-ir colocalised with GAD-ir (434±23 %, p<0.001) and this effect was significantly less in animals pre-treated with SB 242084 (p<0.001). Double-labelling studies revealed DRN cells in which 5-HT2C receptor-ir colocalised with GAD-ir or Fos-ir.

In summary, these findings indicate that administration of the 5-HT2C receptor agonist WAY 161503 increases the number of Fos-ir cells colocalised with GAD-ir neurones in the DRN and that this effect is partially blocked by the 5-HT2C receptor antagonist SB 242084. Furthermore, 5-HT2C receptor-ir was found colocalised with GAD-ir in DRN neurones. These findings are consistent with the hypothesis that 5-HT2C receptor agonist administration directly activates GABA neurones in the DRN, which in turn inhibit the firing of nearby 5-HT neurones (Boothman et al., this meeting).

Boothman L.J. et al. (2003) Br. J. Pharmacology 139: 998-1004.
Cryan J. F., et al. (2000) J. Pharmacol. Exp. Ther. 295: 1120-1126.
Lui R. et al. (2000) Brain Res. 873: 34-45.

Supported by Wellcome Trust Studentships (JR and FD) and a FP6 Integrated Network (NEWMOOD, LMSH-CT-2004-503474 ).