[gamma-S-35]GTP autoradiography allows region-specific detection of muscarinic receptor-dependent G-protein activation in the chick optic tectum

A recently introduced technique of [gamma-S-35]GTP autoradiography was used to localize and characterize muscarinic receptor-dependent activation of G-proteins in tissue sections of the chick optic tectum, a brain region with relatively high expression of G-protein-coupled receptors for the neurotransmitter acetylcholine. Within the highly stratified tectal structure, the bull; of muscarinic receptor-mediated [gamma-S-35]GTP signal was localized to the stratum griseum et fibrosum superficiale with considerably lower binding responses in other tectal layers. Quantitative comparison of [gamma-S-35]GTP binding responses in tectal sections and membranes revealed a close match between the two tissue preparations for the response elicited by the cholinergic agonist carbachol, its dose-dependent reversal with the non-selective muscarinic antagonist atropine, its similar to 100-fold sensitivity towards blockade with MI-type (pirenzepine) over M2-type (gallamine) muscarinic antagonists, as well as absolute requirement for micromolar concentrations of GDP (EC50 similar to 10 mu M) for the receptor-mediated [gamma-S-35]GTP response. The pharmacological profile is consistent with that of cm4, a recently cloned chicken homolog of the mammalian m4 muscarinic acetylcholine receptor. Moreover, the strict GDP-dependence of the binding response suggest activation of G(i/o), the inhibitory class of G-proteins. These data provide the first functional characterization of the chick tectal muscarinic receptors. A close match between [gamma-S-35]GTP responses in membranes and tissue sections strongly suggest that [gamma-S-35]GTP autoradiography offers great potential for studies on G-protein-mediated signaling, with particular use within anatomically restricted regions that are not readily approached with more conventional techniques. It is anticipated that [gamma-S-35]GTP autoradiography should greatly facilitate studies on signaling capacity of an individual receptor subtype whilst in its native cellular environment. (C) 1997 Elsevier Science B.V.