Rhythmic clock gene expression in the spinal cord may underlie sympathetic nerve control of adrenal function

Abstract

The hypothalamic-pituitary-adrenal (HPA) axis is characterized by a circadian rhythm that entrains to the light-dark cycle via inputs from a central pacemaker in the suprachiasmatic nucleus (SCN). Similar molecular clock mechanisms have been identified in each component of the HPA axis including the adrenal cortex, which generates circadian rhythms in glucocorticoid secretion. However, the mechanisms by which the adrenal clock entrains to environmental signals have not been elucidated. Since corticosterone secretion in rodents is mediated in part by sympathetic nerve activity through the intermediolateral (IML) nucleus of the thoracic spinal cord, we tested the hypothesis that these neurons also possess an endogenous clock. Using immunohistochemistry, we detected expression of two clock gene proteins – Period 2 (PER2) and brain and muscle Arnt-like protein 1 (BMAL1) – in the dorsal horn, ventral horn, and intermediate gray within mouse thoracic and lumbar segments; double labeling for vesicular acetylcholine transporter showed cholinergic neurons immunoreactive for clock gene proteins in the IML and ventral horn. To examine whether spinal cord neurons possess clock gene rhythms, we used the reporter mPer2::Luciferase knockin mouse to monitor real-time gene expression in vitro. We found that explants of thoracic spinal segments exhibit rhythms in mPER2 bioluminescence with a 22.0 ± 0.3 h period and a peak phase of 14.8 ± 0.1 h circadian time (CT), which precedes the peak of the adrenal rhythm (CT~16). Our results demonstrate that spinal cord neurons, including those in the IML, express clock gene proteins and that explants of thoracic segments exhibit an endogenous rhythm in PER2 expression. These findings suggest that circadian expression of clock genes in the IML drives sympathetic nerve activity to synchronize adrenal glucocorticoid release with daily changes in the external environment.