Guanosine 5′‐(γ‐[35S]Thio)triphosphate Autoradiography Allows Selective Detection of Histamine H3 Receptor‐Dependent G Protein Activation in Rat Brain Tissue Sections

: Histamine elicits its biological effects via three distinct G protein‐coupled receptors, termed H1, H2, and H3. We have used guanosine 5′‐(γ‐[35S]thio)triphosphate (GTPγ[35S]) autoradiography to localize histamine receptor‐dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A1 receptor‐dependent GTPγ[35S] signal in several brain regions. All further incubations therefore contained 8‐cyclopentyl‐1,3‐dipropylxanthine (10 µM), a selective A1 receptor antagonist. Histamine elicited dose‐dependent increments in GTPγ[35S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine‐evoked binding response closely reflects the known distribution of H3 binding sites and was faithfully mimicked by Nα‐methylhistamine, (R)‐α‐methylhistamine, and immepip, three H3‐selective agonists. In all regions examined, the GTPγ[35S] signal was reversed with thioperamide and clobenpropit, two potent H3‐selective antagonists, whereas mepyramine, a specific H1 antagonist, and cimetidine, a prototypic H2 antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPγ[35S] autoradiography selectively detects H3 receptor‐dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[35S] autoradiography preferentially reveals responses to Gi/o‐coupled receptors, our data indicate that most, if not all, central H3 binding sites represent functional receptors coupling to Gi/o, the inhibitory class of G proteins. Besides allowing more detailed studies on H3 receptor signaling within anatomically restricted regions of the CNS, GTPγ[35S] autoradiography offers a novel approach for functional in vitro screening of H3 ligands.