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