Functions of a novel astrocyte calcium signal

描述（申请人提供）：星形胶质细胞遍布大脑，并发挥着良好的生理作用。星形胶质细胞的新作用包括向神经元传递信号和调节局部血流。星形胶质细胞的某些功能与胞质钙瞬变相关或受其调控，这是一种生理信号。在之前的资助周期中，我们开发并使用了一种称为Lck-GCaMP3的膜系系遗传编码钙指示剂，目的是测量星形胶质细胞钙瞬态。利用Lck-GCaMP3，我们偶然发现了TRPA1离子通道介导的跨膜通量在星形胶质细胞中产生的一种新的钙信号。此外，我们正在进行的实验表明，TRPA1介导的钙通量引起频繁和高度局部化的近膜钙信号，这不仅在单个星形胶质细胞内，而且在细胞培养的星形胶质细胞和神经元网络中，以及在急性脑切片中，对星形胶质细胞的静息钙水平有显著贡献。我们的初步数据还表明，TRPA1通道的药物阻断或基因缺失会降低中间神经元上的抑制性突触效应和锥体神经元上的Schaffer侧联突触的长期突触增强。我们有三个具体的目标，我们寻求进一步扩展这些发现，测试新的假设和评估星形胶质细胞TRPA1介导的钙信号的功能。在目的1中，我们将研究海马切片内星形胶质细胞的近膜钙信号。在Aim 2中，我们将采用多种方法系统地评估为什么阻断星形胶质细胞TRPA1通道或缓冲休息时星形胶质细胞钙水平会降低对中间神经元的抑制性突触效应，而不是海马辐射层（s.r）中的锥体神经元。在Aim 3中，我们将研究阻断TRPA1通道如何减少长期增强（LTP）。通过完成这些实验，我们将对星形胶质细胞钙信号的功能提供新的信息。这些信息将对我们的工作大有帮助

DESCRIPTION (provided by applicant): Astrocytes are found throughout the brain and play well documented physiological roles. Emerging roles for astrocytes include signaling to and from neurons and regulation of local blood flow. Certain astrocyte functions are correlated with or regulated by cytosolic calcium transients, which are a physiological signal. During the previous grant cycle, we developed and used a membrane tethered genetically encoded calcium indicator called Lck-GCaMP3 with the aim of measuring astrocyte calcium transients. Using Lck-GCaMP3 we made the serendipitous discovery of a novel calcium signal in astrocytes due to transmembrane fluxes mediated by TRPA1 ion channels. Moreover, our ongoing experiments show that TRPA1 mediated calcium fluxes give rise to frequent and highly localized near membrane calcium signals that contribute significantly to the resting calcium levels of astrocytes not only within a single astrocyte, but also in a network of astrocytes and neurons in cell cultures, as well as in acute brain slices. Our preliminary data also show that pharmacological block or genetic deletion of TRPA1 channels reduced inhibitory synapse efficacy onto interneurons and long term synaptic potentiation of Schaffer collateral synapses onto pyramidal neurons. We have three specific aims with which we seek to further extend these findings, test novel hypotheses and evaluate the function of astrocyte TRPA1 mediated calcium signals. In Aim 1 we will study near membrane calcium signals in astrocytes within hippocampal slices. In Aim 2 we will employ a variety of methods to systematically evaluate why blocking astrocyte TRPA1 channels or buffering astrocyte calcium levels below rest reduces inhibitory synapse efficacy onto interneurons, but not pyramidal neurons in the stratum radiatum (s.r.) of the hippocampus. In Aim 3 we will study how long-term potentiation (LTP) is reduced by blocking TRPA1 channels. By completing these experiments we will provide new information on the function of a novel astrocyte calcium signal. This information will contribute significantly to our understanding of astrocytes in neuronal networks and allow us and others to test novel hypotheses on the roles of near membrane Ca2+ signals in astrocyte-neuron signaling, and lay the foundations for determining if TRPA1 channels are valid drug targets in brain disorders that involve astrocytes.