Differential effects on [35S]GTPγS binding using muscarinic agonists and antagonists in the gerbil brain

In this work, we studied the in vitro G-protein activation induced by muscarinic agonists using [35S]guanylyl-5′-O-(γ-thio)-triphosphate ([35S]GTPγS) autoradiographic methods to characterize the M2 and M4 muscarinic subtypes response. Thus, we describe a detailed characterization of the increases in [35S]GTPγS binding elicited by carbachol (Cch) and oxotremorine (OXO) (binding in the presence minus binding in the absence of agonist) throughout the gerbil brain (Meriones unguiculatus). For both agonists, the strongest stimulations were found in the superficial gray layer of the superior colliculus, the anteroventral and anteromedial thalamic nuclei, the anterior paraventricular thalamic nucleus, and the caudate-putamen. The comparative study using OXO and Cch suggested that OXO is able to detect differences in the response of structures enriched in M4 muscarinic receptors, showing a lower potency to stimulate these brain areas. Furthermore, using increasing concentrations of selective M2 (AF-DX 116) and M1/M4 (pirenzepine) antagonists to inhibit specific Cch- or OXO-induced [35S]GTPγS binding, significant differences were observed in M2-enriched structures but not in M4-enriched ones such as the caudate-putamen. These data indicate that appropriate muscarinic agonist stimulation, together with selective inhibition of this effect using functional autoradiography, can be used as a tool to unravel the M2- and M4-muscarinic subtype-mediated response.