The use of a modified [3H]4-DAMP radioligand binding assay with increased selectivity for muscarinic M3 receptor shows that cortical CHRM3 levels are not altered in mood disorders

•We have developed a modified, [3H]4-DAMP radioligand binding assay that is highly selective towards CHRM3.•We have shown that [3H]4-DAMP binding is not altered in bipolar disorder or major depressive disorders.•Using western blotting we have confirmed that CHRM3 protein levels are not altered in these mood disorders.•We have shown that CHRM3 mRNA levels are not altered in bipolar disorder or major depressive disorders suggesting CHRM3 is not involved in the pathophysiology of mood disorders.

[3H]4-DAMP is a radioligand that has been used to quantify levels of the muscarinic receptor CHRM3 protein in situ. However, in addition to high affinity binding to CHRM3, [3H]4-DAMP binds with low affinity to CHRM1 confounding the potential to discriminate between changes in these two muscarinic receptors. We have developed a [3H]4-DAMP binding assay, optimised for measuring CHRM3 protein levels in the cortex, with minimal selectivity towards CHRM1. The selectivity of our assay towards CHRM3 was confirmed using recombinant receptor-expressing, cell lysate preparations. [3H]4-DAMP binding levels were similar between wildtype and CHRM1 knockout mice, confirming that the amount of [3H]4-DAMP binding to CHRM1 was negligible. We used this assay to measure CHRM3 protein levels in the frontal pole, obtained post-mortem from subjects with bipolar disorder (n = 15), major depressive disorder (n = 15) and matched controls (n = 20) and showed that [3H]4-DAMP binding was not altered in either bipolar disorder or major depressive disorder. Western blotting confirmed that CHRM3 protein levels were unchanged in these subjects