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.小脑皮层神经元内存在昼夜节律振荡器，其时钟基因的昼夜表达受视交叉上核（suprachiasmatic nucleus, SCN）的主时钟调控。然而，连接视交叉上核与小脑的信号通路至今仍未明确。糖皮质激素呈现显著的视交叉上核依赖性昼夜节律，且已有研究证实小脑皮层中糖皮质激素受体表达水平较高；基于此，我们提出假说：糖皮质激素可调控小脑皮层时钟基因的节律性表达。本研究结合视交叉上核电解损毁术与微型泵植入技术，开发了一种全新实验方法：该微型泵可模拟内源性激素谱，以昼夜节律模式释放皮质酮。通过该策略，我们成功恢复了视交叉上核损毁雄性大鼠的皮质酮昼夜节律。实时定量聚合酶链反应（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结果显示：视交叉上核损毁大鼠的小脑皮层时钟基因表达节律消失，但外源性皮质酮可恢复其小脑皮层时钟基因的每日表达节律。与之相反，外源性皮质酮未能恢复大鼠的体温与自主跑步活动的昼夜节律。RNAscope原位杂交实验进一步揭示：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，这提示皮质酮通过直接结合小脑皮层神经元上的受体发挥调控作用。不过，在肾上腺切除（adrenalectomized）大鼠中仍可检测到小脑皮层的节律性时钟基因表达，这表明存在额外的调控机制。上述实验数据表明，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。