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

Agonist activation of G protein‐coupled receptors induces an increase in the binding of guanosine 5′‐(γ‐[35S]thio)triphosphate ([35S]GTPγS); this increase in binding has been used as a tool to investigate receptor interaction with the heterotrimer guanine nucleotide‐binding regulatory protein (G protein). The present study uses agonist‐stimulated [35S]GTPγS binding to characterize serotonin 5‐HT2A/2C receptors in rat brain membrane fractions and demonstrate the anatomical localization of the receptors by in vitro autoradiography on slide‐mounted sections. The stimulatory effect of the agonist [1‐(2,5‐dimethoxy‐4‐iodophenyl)]‐2 aminopropane (DOI) is compared to that of serotonin (5‐HT). Autoradiography revealed a similar localization of DOI‐ and 5‐HT‐stimulated binding of [35S]GTPγS in distinct 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 orbital‐insular areas as well as in the hippocampal formation, septal areas, the nucleus accumbens, and the choroid plexus. MDL 100105, a specific 5‐HT2A antagonist, and ketanserin, an antagonist of 5‐HT2A/2C receptors, blocked DOI stimulation in all labeled areas, whereas 5‐HT stimulation was only partially blocked (70–80%). A small but significant inhibition was observed with the specific antagonist of 5‐HT2C/2B, SB 206553. This autoradiographic technique provides a useful tool for measuring in situ changes in specific receptor–Gq protein coupling in anatomically discrete brain regions, under physiological and pathological conditions. J. Neurosci. Res. 61:674–685, 2000. © 2000 Wiley‐Liss, Inc.