Cholinergic regulation of the suprachiasmatic nucleus circadian rhythm via a muscarinic mechanism at night

In mammals, the suprachiasmatic nucleus (SCN) is responsible for the generation of most circadian rhythms and for their entrainment to environmental cues. Carbachol, an agonist of acetylcholine (ACh) has been shown to shift the phase of circadian rhythms in rodents when injected intracerebroventricularly. However, the site and receptor type mediating this action have been unknown. In the present experiments, we used the hypothalamic brain-slice technique to study the regulation of the SCN circadian rhythm of neuronal firing rate by cholinergic agonists and to identify the receptor subtypes involved. We found that the phase of the oscillation in SCN neuronal activity was reset by a 5 min treatment with a carbachol microdrop (1 μl, 100 μM), but only when applied during the subjective night, with the largest phase shift (+6 hr) elicited during the middle of the subjective night. This effect also was produced by ACh and two muscarinic receptor (mAChR) agonists, muscarine and McN-A-343 (M1-selective), but not by nicotine. Furthermore, the effect of carbachol was blocked by the mAChR antagonist atropine (0.1 μM), not by two nicotinic antagonists, dihydro-β-erythroidine (10 μM) and d-tubocurarine (10 μM). The M1- selective mAChR antagonist pirenzepine completely blocked the carbachol effect at 1 μM, whereas an M3-selective antagonist, 4,2-(4,4'- diacetoxydiphenylmethyl)pyridine, partially blocked the effect at the same concentration. These results demonstrate that carbachol acts directly on the SON to reset the phase of its firing rhythm during the subjective night via an M1-like mAChR.