Autoradiography of (3)H-pirenzepine and (3)H-AFDX-384 in Mouse Brain Regions: Possible Insights into M(1), M(2), and M(4) Muscarinic Receptors Distribution

Autoradiography helps to determine the distribution and density of muscarinic receptor (MR) binding sites in the brain. However, it relies on the selectivity of radioligands toward their target. (3)H-Pirenzepine is commonly believed to label predominantly M(1)MR, (3)H-AFDX-384 is considered as M(2)MR selective ligand. Here we performed series of autoradiographies with (3)H-AFDX-384 (2 nM), and (3)H-pirenzepine (5 nM) in WT, M(1)KO, M(2)KO, and M(4)KO mice to address the ligand selectivity. Labeling with (3)H-pirenzepine using M(1)KO, M(2)KO, and M(4)KO brain sections showed the high selectivity toward M(1)MR. Selectivity of (3)H-AFDX-384 toward M(2)MR varies among brain regions and depends on individual MR subtype proportion. All binding sites in the medulla oblongata and pons, correspond to M(2)MR. In caudate putamen, nucleus accumbens and olfactory tubercle, 77.7, 74.2, and 74.6% of (3)H-AFDX-384 binding sites, respectively, are represented by M(4)MR and M(2)MR constitute only a minor portion. In cortex and hippocampus, (3)H-AFDX-384 labels almost similar amounts of M(2)MR and M(4)MR alongside significant amounts of non-M(2)/non-M(4)MR. In cortex, the proportion of (3)H-AFDX-384 binding sites attributable to M(2)MR can be increased by blocking M(4)MR with MT3 toxin without affecting non-M(4)MR. PD102807, which is considered as a highly selective M(4)MR antagonist failed to improve the discrimination of M(2)MR. Autoradiography with (3)H-QNB showed genotype specific loss of binding sites. In conclusion: while (3)H-pirenzepine showed the high selectivity toward M(1)MR, (3)H-AFDX-384 binding sites represent different populations of MR subtypes in a brain-region-specific manner. This finding has to be taken into account when interpreting the binding data.