Voltage-gated L-type calcium channels and circuit plasticity

钙离子对多种神经元功能是重要的，这些功能是通过钙通道进入细胞而实现的，钙通道是嵌入细胞膜的一族蛋白质。在钙通道家族中有一个亚类，称为L型钙通道。这些对于调节神经细胞的电兴奋性和释放神经递质尤其重要，这些神经递质是在反映记忆形成的电路功能中形成长期变化所必需的。鉴于L类钙通道的关键作用，在分子水平上了解这些通道是如何调节的，以调节其生物物理性质和位于质膜上的通道蛋白密度是至关重要的。这项设备应用将直接惠及NSERC资助的发现资助项目R.W.特纳博士和G.W.赞波尼博士，他们在一种名为海马体的皮质结构中研究L类型钙通道的不同方面。特纳博士的项目专注于调节通过L类通道的钙电流的大小的内部辅助蛋白(生物物理特性)，赞波尼博士的研究重点是调节通道进出细胞膜的分子步骤(通道密度)。这两个实验室目前都专注于记录体外分离的神经元或电路，并结合激光或LED对特定神经元中表达的光激活分子进行激发(光遗传学)。这项建议将介绍通过对记录和光遗传刺激的电极进行立体定位，在活体小鼠完整的海马区获得膜片钳记录的能力。它还将进一步允许使用无线系统来同时记录单位活动和光遗传刺激海马神经元或传入通路。通过这种方式，迄今为止进行的体外工作将被推进到解释活体和自由活动动物中L类型钙通道的功能，这些动物进行已知的依赖于海马体功能的行为测试。因此，这些研究将使我们的研究计划更上一层楼，使我们能够在活体动物中进行膜片钳记录和行为测试，大大提高我们的合作能力，在顶级期刊上发表竞争立场，并极大地扩大我们受训人员的实验机会。

Calcium ions are important for a variety of neuronal functions that are enabled by calcium entry into cells through calcium channels, a family of proteins embedded in the cell membrane. Among the family of calcium channels is a subclass called L-type calcium channels. These are particularly important for regulating the electrical excitability of nerve cells and for the release of neurotransmitters required to form long-term changes in circuit function reflecting memory formation. Given the pivotal roles for L-type calcium channels, it is critical to understand how these channels are modulated at the molecular level to regulate their biophysical properties and the density of channel protein positioned at the plasma membrane. This equipment application will directly benefit the NSERC funded Discovery grant programs of Drs. R.W. Turner and G.W. Zamponi who work on different aspects of L-type calcium channels in a cortical structure called the hippocampus. Dr. Turner’s program is focused on internal accessory proteins that regulate the magnitude of calcium current through L-type channels (biophysical properties), and Dr. Zamponi the molecular steps that regulate trafficking of channels to and from the membrane (channel density). Both labs currently focus their work on recordings from isolated neurons or circuits in vitro in conjunction with laser or LED-based excitation of light-activated molecules expressed in select neurons (optogenetics). This proposal will introduce the capability of obtaining patch clamp recordings in the intact hippocampus of mice in vivo through stereotaxic positioning of electrodes for recording and optogenetic stimulation. It will further allow the use of a wireless system to simultaneously record unit activity and optogenetically stimulate hippocampal neurons or the incoming pathways. In this way the in vitro work conducted to date will be advanced to interpreting the function of L-type calcium channels in live and freely moving animals performing behavioural tests known to depend on hippocampal function. These studies will thus elevate our research programs to the next level by enabling patch clamp recordings and behavioural testing in the live animal, substantially increasing our collaborative capabilities, competitive stance for publication in top journals, and greatly expand the experimental opportunities for our trainees.