The role of cholinergic muscarinic receptor 2 and 3 in mood disorders

© 2014 Dr. Won Je JeonVariation in cholinergic muscarinic receptor (CHRM) levels is thought to be involved in the pathophysiology of bipolar disorder (BPD) and major depressive disorder (MDD). Lower levels of CHRM2/CHRM4 protein in the human dorsolateral frontal cortex (Brodmann’s area (BA) 46) from subjects with BPD and MDD, and CHRM3 in the human orbitofrontal cortex (BA 10) from subjects with BPD have previously been reported by my laboratory. I sought to confirm those findings in BA 10 and 46 in a larger cohort from subjects with BPD (n = 15), MDD (n = 15) and controls (n = 20) as well as see whether these changes occur in other cortical regions by examining BA 17, 24 and 47, which are also thought to be affected in mood disorders. I investigated whether the binding densities of the CHRM selective radioligands [3H]4-DAMP, [3H]pirenzepine and [3H]AF-DX 384 are altered in mood disorders. Findings from the binding data were confirmed using Western blot and quantitative Real-Time PCR (qPCR), and oxotremorine M stimulated-[35S]GTPγS binding was used to see whether changes in protein levels affected the receptor activity. I also investigated whether muscarinic receptor levels, using [3H]AF-DX 384 and [3H]pirenzepine binding, are altered in the rat brain regions 24 hours after the last acute or chronic treatment with mood stabilisers or antidepressant drugs (n = 20 per group) as part of potential confounding factors. Previous protocols used to measure [3H]4-DAMP binding measured both CHRM3 and CHRM1. I adapted this protocol to make the [3H]4-DAMP binding assay more selective to CHRM3. Contrasting previous findings, there was no difference in levels of CHRM3 protein in any cortical region from subjects with BPD and MDD compared to controls. [3H]pirenzepine binding was lower in the outer layer of BA 17 and BA 24 in BPD and MDD, and in BA 47 from subjects with MDD, but not BPD. [3H]AF-DX 384 binding was lower in the outer layer of BA 17 and BA 24 from subjects with BPD and MDD, and in the inner layer of BA 17 and BA 46 from subjects with BPD, but not MDD, compared to controls. I subsequently examined levels of CHRM2 and CHRM3 protein using Western blotting. Two CHRM2-specific immunogenic bands were detected on the western blots. CHRM2 protein levels were significantly lower in the upper band (71 kDa), but not the lower band (67 kDa), in BA 24 in BPD only compared to controls. By contrast, there was no change in BA 46 from subjects with BPD and MDD. Moreover, levels of CHRM3 protein were not changed in BA 10 from subjects with BPD and MDD compared to control subjects. Similarly, the levels of CHRM3 mRNA using qPCR in BA 10 were also not altered. Levels of specific [35S]GTPγS binding stimulated by oxotremorine M were significantly lower in BA 24 from subjects with BPD, but not MDD, compared to controls. However, there was no change in oxotremorine M stimulated-[35S]GTPγS binding in BA 46. In neuropsychopharmacological studies, there were widespread regions-specific increases in levels of [3H]AF-DX 384 and [3H]pirenzepine binding in rats following chronic treatment lithium and sodium valproate. Chronic lithium administration resulted in a significant increase in the densities of [3H]AF-DX 384 binding in the cingulate, parietal cortices, caudate putamen, nucleus accumbens and olfactory tubercle, but not the frontal cortex. Interestingly, there were significantly higher levels of [3H]pirenzepine binding in all rat brain regions examined after treatment with lithium. Moreover, treatment with sodium valproate for 28 days caused an increase in levels of [3H]AF-DX 384 binding in the caudate putamen, nucleus accumbens and olfactory tubercle of rats and an increase in [3H]pirenzepine binding densities in the cingulate cortex, caudate putamen and nucleus accumbens. By contrast, acute and chronic antidepressant drugs, fluoxetine and imipramine, administration resulted in no change in the levels of [3H]AF-DX 384 binding in any brain region of rats. No change in [3H]pirenzepine binding was found in any rat brain region following acute and chronic administration of antidepressant drugs. These findings suggest that regionally specific decreases in the level of cortical muscarinic receptors are involved in the pathophysiology of mood disorders