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