Evidence of a role for 5-HT2A receptors in the local regulation of dorsal raphe 5-HT cell firing in mice

L.J. Boothman, T. Renoir, J. Gingrich1, T. Sharp2, M. Hamon & L. Lanfumey. Neuropsychopharmacology, UMR 677 INSERM-UPMC, GHU Pitié-Salpétrière, 75013 Paris, France; 1Dept. Psychiatry, Columbia University , New York, NY10032, USA; 2University Dept. of Pharmacology, Mansfield Road, Oxford, OX1 3 QT, UK.

Recent studies indicate that in addition to 5-HT1A autoreceptors, 5-HT2A/2C receptors also regulate the firing of midbrain 5-hydroxytryptamine (5-HT) neurones. Thus, 5-HT2A/2C receptor agonists inhibit the firing of 5-HT neurones in the dorsal raphe nucleus (DRN) of anaesthetised rats (Boothman et al., 2003) . The mechanism underlying this effect may include local networks within the DRN (e.g. GABA neurones) as well as afferents from other brain regions (Boothman et al., 2005; Liu et al., 2000) . This study investigated the local effect of the 5-HT2 receptor agonist DOI on 5-HT cell firing in mice under in vitro conditions. The involvement of 5-HT2A receptors was tested using 5-HT2A receptor knock-out animals (Fiorica-Howells et al., 2002) .

Homozygous 5-HT2A receptor knock-out mice on a C57BL/6J background and their wild type littermates were used (2 months old, 20-25 g, both sexes). Brain slices (400 μm thick) containing the DRN were prepared (see Lanfumey et al., 1999) and superfused with oxygenated artificial cerebrospinal fluid (32-34˚C, 2-3 ml/min) containing the α 1-adrenoceptor agonist phenylephrine (3 μM). Extracellular recordings were made using glass microelectrodes (2 M NaCl, 12-15 MΩ). Cells were identified as 5-HT neurons according to established electrophysiological and pharmacological criteria (Lanfumey et al., 1999) . After 10-15 min stable recording, DOI ( (R)(−)−DOI hydrochloride) was applied via the perfusate for 3 min. Integrated cell firing rates were captured in 10 s intervals, and drug action evaluated by comparing the firing rate for the 2 min prior to application with that at peak effect. Data were analysed by 1- or 2-way ANOVA with appropriate post-hoc tests.

DOI (0.003 – 3 μM) caused a dose-related inhibition of firing in a sub-population (43%) of 5-HT cells in 5-HT2A receptor wild-type mice (P<0.0001, maximum effect: -78.8±5.1%, EC50~30 nM, n=18) . In the remaining cells in wild-type mice, DOI caused a small inhibitory effect which was not dose-related (maximum effect: -15.6±2.6%, n=24) . In comparison, DOI had no significant effect on 5-HT cell firing in 5-HT2A receptor knock-out mice (maximum effect: -5.6 ±4.3%, n=10). Comparison of both wild-type groups with knock-out indicated an effect of genotype (P<0.001 , P<0.05).

These data demonstrated that the 5-HT2 receptor agonist DOI inhibits the firing of a sub-population of DRN 5-HT neurones in mice. The presence of this effect in an in vitro DRN slice preparation is consistent with its mediation by local DRN-based mechanisms. In addition, the absence of an inhibitory effect of DOI in 5-HT2A receptor knock-out mice suggests the involvement of the 5-HT2A receptor subtype.

Boothman et al ., (2003). Br J Pharmacol, 139, 998-1004.
Boothman et al., (2005). Neuroreport, 16, 891-6.
Fiorica-Howells et al., (2002). Am J Physiol Gastrointest Liver Physiol, 282, G877-93.
Lanfumey et al. . (1999). Neuroreport , 10, 3369-74.
Liu et al., (2000). Brain Res, 873, 34-45.

Supported by the ‘Fondation Recherche Médicale - Accueil de chercheur étranger’ (LB) and EC 6 th Framework (NEWMOOD, LMSH-CT-20046503474) programmes.