DISTINCT KINETIC BINDING-PROPERTIES OF N-H-3!-METHYLSCOPOLAMINE AFFORD DIFFERENTIAL LABELING AND LOCALIZATION OF M1, M2, AND M3-MUSCARINIC-RECEPTOR SUBTYPES IN PRIMATE BRAIN

Three classes of muscarinic receptors in mammalian brain have been pos tulated on the basis of equilibrium and kinetic binding data. However, equilibrium binding assays alone have not permitted a clear demonstra tion of the localization of putative Ml, M2, and M3 receptor subtypes in the brain because of the overlapping affinities of virtually all mu scarinic antagonists. In the present study, the conditions for selecti ve occupancy of the Ml, M2, and M3 receptor subtypes in the brain of t he rhesus monkey were based on the distinct kinetic and equilibrium bi nding properties of N-H-3!-methylscopolamine (NMS) at cloned ml-m4 mu scarinic receptor subtypes expressed in A9L transfected cells. Quantit atitve autoradiography of the Ml, M2, and M3 muscarinic receptor subyt pes in the primate brain was performed according to the following stra tegy. The Ml (ml) receptor subtype was labeled directly with a non-sat urating concentration of H-3!-pirenzepine. The M2 (m2) subtype was la beled by incubations consisting of short, two minute pulses of H-3!-N MS after a preincubation with 0.3 muM pirenzepine to occlude ml, m3, a nd m4 sites. Selective occupancy of the M3 (m3) receptor (subtype) was achieved by pre-incubation with 0.5 nM unlabeled NMS to partially occ lude the ml, m2, and m4 sites, equilibrium with 0.5 nM H-3!-NMS, foll owed by a 60 minute tracer dissociation in the presence of 1 muM atrop ine.In vitro autoradiography demonstrated that the Ml receptor subtype was confined to forebrain structures. Ml receptors were prevalent thr oughout the cerebral cortical mantle, amygdala, hippocampus, and the s triatum. Low to background levels of the Ml receptor subtype were meas ured over the thalamus, hypothalamus, and brainstem. The M2 subtype wa s widely distributed with elevated densities of binding sites seen ove r all primary sensory cortical areas, and within discrete thalamic, hy pothalamic, and brainstem nuclei. The distribution of the M3 receptor subtype was largely coincident with the pattern of the Ml sites labele d by non-saturating concentrations of H-3!-pirenzepine with some nota ble exceptions. Within the cerebral cortical mantle, the M3 receptor e xhibited an elevated gradient over the orbitofrontal gyrus and the tem poral lobe. Within the striatum, the M3 subtype was elevated over the anterior and dorsal part of the caudate nucleus, while the Ml receptor s were most prevalent over the ventromedial sector. Selective labeling of M3 receptors was seen over the medial division of the globus palli dus and within the substantia nigra pars reticulata. In contrast to th e pattern of the Ml receptor subtype, M3 receptors were prevalent also over midline nuclei of the hypothalamus. These results demonstrate th at the distinct kinetic and equilibrium binding profiles of N-methylsc opolamine and pirenzepine for cloned muscarinic receptors provide a vi able ligand autoradiographic strategy for mapping the distribution of Ml, M2, and M3 receptors in brain. (C) 1993 Wiley-Liss, Inc.