Cholinergic regulation of circadian rhythms in the SCN: Characterization of signaling mechanisms and the role of intracellular calcium in the directionality of clock resetting

This study identifies the mechanisms by which cholinergic stimuli reset the circadian clock in the SCN and clarifies signaling components that are the key determinants of the divergent cholinergic and glutamatergic clock resetting. It provides strong evidence for the M₁ muscarinic acetylcholine receptor (M₁•R) as a key mediator of cholinerigic clock resetting by evaluating the expression of mRNA and protein of M ₁•Rs, radioligand analysis of [3H]-pirenzepine binding, and the requirement of InsP₃-induced Ca2+ release for the cholinergic resetting of the circadian rhythm in the middle of the subjective night. It identifies a significant role of heme oxygenase 2 in cholinergic clock resetting by showing that basal NO production generates cGMP tone that is permissive for cholinergic signaling, whereas HO-2/CO transmits muscarinic receptor activation to the cGMP-signaling pathway that modulates clock state. In light of the recently reported inhibitory interaction between HO-2 and beta-amyloid, a marker of Alzheimer's disease (AD), this study identifies HO-2/CO signaling as a potential defective component of cholinergic neurotransmission in the pathophysiology of AD, whose manifestations include disintegration of circadian timing.; This study shows that stimulus-specific intracellular Ca2+ (Ca2+_i) release controls the direction of clock resetting. Direct activation of the InsP₃-induced Ca2+ _i release (IICR) mimics cholinergic phase advance, while InsP₃ , but not ryanodine, receptor antagonists block it. In contrast, selective activation of CICR with ryanodine results in glutamate-like delay. Ca 2+ imaging using multiphoton microscopy revealed signal-specific differences in Ca2+ transients. Cholinergic-stimulated Ca2+ transients are localized to the microdomains in the cytoplasm, whereas glutamate-induced Ca2+ transients are predominantly uniform across the cytosol and nucleus. Thus, the direction of clock resetting is determined by differential Ca2+_i release that is likely to result in activation of spatially restricted Ca2+-dependent effector proteins.