In vitro autoradiography of serotonin 5-HT2A/2C receptor-activated G protein: Guanosine-5 '-(gamma-[S-35]thio)triphosphate binding in rat brain

Agonist activation of G protein-coupled receptors induces an increase in th e binding of guanosine 5'-(gamma-[S-35]thio)triphosphate ([S-35]GTP gamma S ); this increase in binding has been used as a tool to investigate receptor interaction with the heterotrimer guanine nucleotide-binding regulatory pr otein (G protein), The present study uses agonist-stimulated [S-35]GTP gamm a S binding to characterize serotonin 5-HMT2A/2C receptors in rat brain mem brane fractions and demonstrate the anatomical localization of the receptor s by in vitro autoradiography on slide-mounted sections. The stimulatory ef fect of the agonist [1-(2,5-dimethoxy-4-iodophenyl)]-2 aminopropane (DOI) i s compared to that of serotonin (5-HT). Autoradiography revealed a similar localization of DOI- and 5-HT-stimulated binding of [S-35]GTP gamma S in di stinct areas of prefrontal and parietal cortex, consistent with previously reported 5-HT2A receptor distribution. Specific binding was demonstrated in the frontal and parietal cortex, medial prefrontal, and cingular and orbit al-insular areas as well as in the hippocampal formation, septal areas, the nucleus accumbens, and the choroid plexus. MDL 100105, a specific 5-HT2A a ntagonist, and ketanserin, an antagonist of 5-HT2A/2C receptors, blocked DO I stimulation in all labeled areas, whereas 5-HT stimulation was only parti ally blocked (70-80%). A small but significant inhibition was observed with the specific antagonist of 5-HT2C/2B SB 206553, This autoradiographic tech nique provides a useful tool for measuring in situ changes in specific rece ptor-Gq protein coupling in anatomically discrete brain regions, under phys iological and pathological conditions. (C) 2000 Wiley-Liss, Inc.