Supplementary Material for: Rhythmic Release of Corticosterone Induces Circadian Clock Gene Expression in the Cerebellum

Neurons of the cerebellar cortex contain a circadian oscillator, with circadian expression of clock genes being controlled by the master clock of the suprachiasmatic nucleus (SCN). However, the signaling pathway connecting the SCN to the cerebellum is unknown. Glucocorticoids exhibit a prominent SCN-dependent circadian rhythm, and high levels of the glucocorticoid receptor have been reported in the cerebellar cortex; we therefore hypothesized that glucocorticoids may control the rhythmic expression of clock genes in the cerebellar cortex. We here applied a novel methodology by combining the electrolytic lesion of the SCN with implantation of a micropump programmed to release corticosterone in a circadian manner mimicking the endogenous hormone profile. By use of this approach, we were able to restore the corticosterone rhythm in SCN-lesioned male rats. Clock gene expression in the cerebellum was abolished in rats with a lesioned SCN, but exogenous corticosterone restored the daily rhythm in clock gene expression in the cerebellar cortex, as revealed by quantitative real-time PCR and radiochemical in situ hybridization for the detection of the core clock genes Per1, Per2, and Arntl. On the contrary, exogenous hormone did not restore circadian rhythms in body temperature and running activity. RNAscope in situ hybridization further revealed that the glucocorticoid receptor colocalizes with clock gene products in cells of the cerebellar cortex, suggesting that corticosterone exerts its actions by binding directly to receptors in neurons of the cerebellum. However, rhythmic clock gene expression in the cerebellum was also detectable in adrenalectomized rats, indicating that additional control mechanisms exist. These data show that the cerebellar circadian oscillator is influenced by SCN-dependent rhythmic release of corticosterone.