Distinct kinetic binding propeties of N-[3H]-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 postulated on the basis of equilibrium and kinetic binding data. However, equilibrium binding assays alone have not permitted a clear demonstration of the localization of putative M1, M2, and M3 receptor subtypes in the brain because of the overlaping affinities of virtually all muscarinic antagonists. In the present study, the conditions for selective occupancy of the M1, M2, and M3 receptor subtypes in the brain of the rhesus monkey were based on the distinct kinetic and equilibrium binding properties of N-³H]-methylscopolamine (NMS) at cloned m1–m4 muscarinic receptor subtypes expressed in A9L transfected cells. Quantitative autoradiography of the M1, M2, and M3 muscarinic receptor subtypes in the primate brain was performed according to the following strategy. The M1 (m1) receptor subtype was labeled directly with a non-saturating concentration of ³H]-pirenzepine. The M2 (m2) subtype was labeled by incubations consisting of short, two minute pulses of ³H]-NMS after a preincubation with 0.3 μM pirenzepine to occlude m1, m3, and m4 sites. Selective occupancy of the M3 (m3) receptor (subtype) was achieved by pre-incubation with 0.5 nM unlabeled NMS to partially occlude the m1, m2, and m4 sites, equilibrium with 0.5 nM ³H]-NMS, followed by a 60 minute tracer dissociation in the presence of 1 μM atropine. In vitro autoradiography demonstrated that the M1 receptor subtype was confined to forebrain structures. M1 receptors were prevalent throughout the cerebral cortical mantle, amygdala, hippocampus, and the striatum. Low to background levels of the M1 receptor subtype were measured over the thalamus, hypothalamus, and brainstem. The M2 subtype was widely distributed with elevated densities of binding sites seen over all primary sensory cortical areas, and within discrete thalamic, hypothalamic, and brainstem nuclei. The distribution of the M3 receptor subtype was largely coincident with the pattern of the M1 sites labeled by non-saturating concentrations of ³H]-pirenzepine with some notable exceptions. Within the cerebral cortical mantle, the M3 receptor exhibited an elevated gradient over the orbitofrontal gyrus and the temporal lobe. Within the striatum, the M3 subtype was elevated over the anterior and dorsal part of the caudate nucleus, while the M1 receptors were most prevalent over the ventromedial sector. Selective labeling of M3 receptors was seen over the medial division of the globus pallidus and within the substantia nigra pars reticulata. In contrast to the pattern of the M1 receptor subtype, M3 receptors were prevalent also over midline nuclei of the hypothalamus. These results demonstrate that the distinct kinetic and equilibrium binding profiles of N-methylscopolamine and pirenzepine for cloned muscarinic receptors provide a viable ligand autoradiographic strategy for mapping the distribution of M1, M2, and M3 receptors in brain. © 1993 Wiley-Liss, Inc.