Calcium Signaling in Suprachiasmatic Nucleus Neurons

描述(由申请人提供)：昼夜节律紊乱会导致多种疾病，并损害精神和身体表现。生理和行为过程中的昼夜节律是由位于视交叉上核的分子钟产生的。这种时钟从光等信号中接收环境信息，随后产生定时信息，发送给有机体的其余部分。虽然对这一信息链中涉及的信号通路知之甚少，但有证据表明，钙离子可能在生物钟的输入和输出中作为信号分子发挥作用。但这种钙离子的来源尚不清楚。一种模型认为，从视黄素下丘脑束末端释放的谷氨酸激活了NMDA受体，后者产生一氧化氮，并从兰诺定敏感的储存区释放钙离子。然而，谷氨酸或一氧化氮对兰诺定敏感细胞的钙释放作用尚未被直接证实。激活NMDA受体可使SCN神经元胞核内钙离子浓度升高。核Ca~(2+)调控基因表达，可能包括时钟基因表达。这项工作的长期目标是表征SCN神经元的功能属性以及生物钟如何调节这些属性。这一建议将确定细胞质和细胞核中的钙离子作为昼夜节律输入和输出信号所扮演的角色。我们的中心假设是，细胞质和细胞核内钙离子浓度的变化是光调节生物钟的关键步骤。这项建议使用荧光成像技术、细胞培养和电生理记录技术的创新组合来研究昼夜节律不同时间段SCN神经元中钙离子的调节。这项研究将确定光信号通路的早期步骤。该方案的具体目的是：1)确定激活NMDA和AMPA受体所产生的胞浆钙浓度升高的机制和昼夜节律。2)确定NMDA受体激活引起核内钙离子浓度升高的机制和昼夜时相依赖性。3)确定PACAP在调节NMDA和AMPA受体激活引起的胞浆和胞核内钙离子浓度变化中的作用。4)探讨SCN神经元胞浆钙节律的峰值是否先于动作电位放电频率节律的峰值。总之，这些实验将识别光信号通路的早期步骤，从而有助于更好地理解昼夜节律的细胞基础。

DESCRIPTION (provided by applicant): Disturbances in circadian rhythms contribute to a variety of diseases and impair mental and physical performance. Circadian rhythms in physiological and behavioral processes are generated by a molecular clock located in the suprachiasmatic nucleus. This clock receives environmental information from cues such as light and subsequently creates timing information that is sent to the rest of the organism. While the signaling pathways involved in this chain of information are poorly understood, evidence suggests a possible role for Ca2+ as a signaling molecule for both input to and output from the circadian clock. But the source of this Ca2+ is unknown. One model proposes that glutamate released from terminals of the retinohypothalamic tract activates NMDA receptors, which generate nitric oxide and release Ca2+ from ryanodine-sensitive stores. However, the release of Ca2+ from ryanodine-sensitive stores by glutamate or nitric oxide has not been directly demonstrated. NMDA receptor activation increases the nuclear Ca2+ concentration of SCN neurons. Nuclear Ca2+ regulates gene expression, including possibly clock gene expression. The long-term goal of this work is to characterize the functional properties of SCN neurons and how the circadian clock regulates these properties. This proposal will determine the roles that cytoplasmic and nuclear Ca2+ play as circadian input and output signals. Our central hypothesis is that changes in cytoplasmic and nuclear Ca2+ concentration are a critical step in light's regulation of the circadian clock. This proposal uses an innovative combination of fluorescent imaging techniques, cell culture, and electrophysiological recording techniques to study the regulation of Ca2+ in SCN neurons during different portions of the circadian day. This research will identify the early steps in the light-signaling pathway. The Specific Aims of the proposal are: 1) Identify the mechanisms and circadian regulation of the increase of the cytoplasmic Ca2+ concentration produced by activating NMDA and AMPA receptors. 2) Determine the mechanisms and circadian phase dependence of the increase in the nuclear Ca2+ concentration produced by NMDA receptor activation. 3) Determine the role that PACAP plays in regulating changes in the cytoplasmic and nuclear Ca2+ concentration induced by NMDA and AMPA receptor activation. 4) Investigate whether the peak of the cytoplasmic Ca2+ rhythm precedes the peak of action potential firing frequency rhythm in SCN neurons. Together, these experiments will identify the early steps in the light-signaling pathway, thus contributing to a better understanding of the cellular basis of circadian rhythms.