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 <i>Per1</i>, <i>Per2,</i> and <i>Arntl</i>. 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.

小脑皮层神经元携带昼夜节律振荡器，其时钟基因的昼夜表达受视交叉上核（suprachiasmatic nucleus, SCN）主时钟的调控。然而，连接视交叉上核与小脑的信号通路仍未阐明。糖皮质激素呈现显著的视交叉上核依赖性昼夜节律，且已有研究报道小脑皮层中糖皮质激素受体（glucocorticoid receptor）的高表达水平；基于此，我们提出假说：糖皮质激素或可调控小脑皮层时钟基因的节律性表达。本研究采用全新实验范式，将视交叉上核电解损毁术与微泵植入技术相结合，该微泵可按照模拟内源性激素谱的昼夜节律模式释放皮质酮（corticosterone）。利用该方法，我们成功恢复了视交叉上核损毁雄性大鼠体内的皮质酮节律。视交叉上核损毁大鼠的小脑时钟基因表达完全消失，但外源性皮质酮可恢复小脑皮层时钟基因的每日节律，该结论经由实时荧光定量PCR（quantitative real-time PCR）与针对核心时钟基因*Per1*、*Per2*及*Arntl*的放射化学原位杂交（radiochemical in situ hybridization）得以证实。与之相反，外源性激素无法恢复大鼠体温与自主活动的昼夜节律。RNAscope原位杂交技术进一步证实，糖皮质激素受体与时钟基因产物在小脑皮层细胞中共定位，提示皮质酮可通过直接结合小脑皮层神经元上的受体发挥调控作用。但在肾上腺切除大鼠的小脑中，仍可检测到时钟基因的节律性表达，表明小脑昼夜节律尚存在其他调控机制。上述实验结果表明，小脑昼夜节律振荡器受视交叉上核依赖性皮质酮节律性释放的调控。