Autoradiography of ³H-pirenzepine and ³H-AFDX-384 in Mouse Brain Regions: Possible Insights into M₁, M₂, and M₄ 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 M₁MR, ³H-AFDX-384 is considered as M₂MR selective ligand. Here we performed series of autoradiographies with ³H-AFDX-384 (2 nM), and ³H-pirenzepine (5 nM) in WT, M₁KO, M₂KO, and M₄KO mice to address the ligand selectivity. Labeling with ³H-pirenzepine using M₁KO, M₂KO, and M₄KO brain sections showed the high selectivity toward M₁MR. Selectivity of ³H-AFDX-384 toward M₂MR varies among brain regions and depends on individual MR subtype proportion. All binding sites in the medulla oblongata and pons, correspond to M₂MR. 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 M₄MR and M₂MR constitute only a minor portion. In cortex and hippocampus, ³H-AFDX-384 labels almost similar amounts of M₂MR and M₄MR alongside significant amounts of non-M₂/non-M₄MR. In cortex, the proportion of ³H-AFDX-384 binding sites attributable to M₂MR can be increased by blocking M₄MR with MT3 toxin without affecting non-M₄MR. PD102807, which is considered as a highly selective M₄MR antagonist failed to improve the discrimination of M₂MR. Autoradiography with ³H-QNB showed genotype specific loss of binding sites.

In conclusion: while ³H-pirenzepine showed the high selectivity toward M₁MR, 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.