Voltage imaging of astrocyte-neuron interactions

项目摘要 星形胶质细胞从细胞外空间清除兴奋性神经递质谷氨酸， 通过钠驱动的电压依赖性兴奋性氨基酸转运蛋白（EAAT）的活性。鲁棒谷氨酸盐 EAAT的摄取确保了谷氨酸信号传导的时间和空间保真度。有趣的是，我们最近 发现神经元活动迅速（在毫秒内），可逆地减缓了成年人对谷氨酸的吸收 大脑皮层这种减缓神经元NMDA反应，与细胞外 谷氨酸动力学，并且高度依赖于刺激的频率和持续时间。此外，本发明还 谷氨酸清除可以通过具有突触特异性的神经元活动来调节，即使在单个突触内也是如此。 星形胶质细胞我们相信这可能对神经传递、突触外受体 激活和突触可塑性。基于这一发现，我们假设神经元活动诱导 星形胶质细胞膜电位（Vm）的微区水平的变化，局部调节EAAT功能。GLT 1是 成年前脑中主要的星形胶质细胞EAAT大量表达，并确保谷氨酸在脑中的表达。 细胞外空间被EAAT结合迅速隔离。一旦绑定到EAAT， 谷氨酸进入星形胶质细胞是钠驱动和电压依赖的。正常情况下，星形胶质细胞 由于其对钾的高渗透性而超极化（-80 mV）。然而，神经元活动增加 细胞外钾、[K+]e和星形胶质细胞Vm对[K+]e的变化特别敏感。因此， 这是神经元活动可以通过使星形胶质细胞去极化来改变EAAT功能的原因。星形胶质细胞Vm的变化可能 在精细星形胶质细胞过程中尤其相关，其中EAAT集中，并且其中小的细胞内 与索马相比，体积可以放大Vm的变化。我们还将探索替代机制 包括通过增加[K+]e对EAAT的电压依赖性调节。测试我们的一个主要挑战是 然而，假设是由于低膜电阻，不能监测远端突起处的星形胶质细胞Vm 和工艺形态。克服这一挑战是重要的，因为星形胶质细胞远端过程是 突触相互作用位点和EAAT定位。为了检测星形胶质细胞Vm的远端变化，我们 开发了一种使用遗传编码电压指示器（GEVI）在星形胶质细胞过程中成像Vm的方法 显像利用星形胶质细胞和神经元电生理记录，星形胶质细胞Vm的光遗传学操纵， 和GEVI成像的星形胶质细胞膜电位，我们已经产生了初步的数据，支持我们的 假设EAAT功能可以通过星形胶质细胞Vm的活性诱导的变化来调节。

Project summary Astrocytes remove the excitatory neurotransmitter glutamate from the extracellular space following neuronal activity via sodium-driven, voltage-dependent excitatory amino acid transporters (EAATs). Robust glutamate uptake by EAATs ensures the temporal and spatial fidelity of glutamate signaling. Interestingly, we recently found that neuronal activity rapidly (within milliseconds) and reversibly slows glutamate uptake in the adult cerebral cortex. This slowing prolongs neuronal NMDA responses, consistent with prolonged extracellular glutamate dynamics, and is highly dependent on the frequency and duration of stimulation. Additionally, glutamate clearance can be modulated by neuronal activity with synapse specificity, even within a single astrocyte. We believe this may have important consequences on neurotransmission, extrasynaptic receptor activation, and synaptic plasticity. Based on this finding, we hypothesized that neuronal activity induces microdomain-level changes in astrocyte membrane potential (Vm) that locally modulate EAAT function. GLT1 is the predominant astrocytic EAAT in the adult forebrain, is abundantly expressed, and ensures that glutamate in the extracellular space is rapidly sequestered by EAAT binding. Once bound to EAATs, the transport of glutamate into the astrocyte is both sodium-driven and voltage-dependent. Under normal conditions, astrocytes are hyperpolarized (-80 mV) due to their high permeability to potassium. However, neuronal activity increases extracellular potassium, [K+]e, and astrocyte Vm is especially sensitive to [K+]e changes. Therefore, it stands to reason that neuronal activity can alter EAAT function by depolarizing astrocytes. Changes in astrocytic Vm may be especially relevant in fine astrocytic processes, where EAATs are concentrated, and where small intracellular volumes may amplify changes in Vm, as compared to soma. We will also explore alternative mechanisms including voltage-independent modulation of EAATs by increases in [K+]e. A major challenge to testing our hypothesis, however, is an inability to monitor astrocyte Vm at distal processes due to low membrane resistance and process morphology. Overcoming this challenge is important because astrocyte distal processes are the site of synaptic interaction and EAATs localization. In order to detect distal changes in astrocyte Vm, we developed an approach to image Vm in astrocyte processes using genetically-encoded voltage indicator (GEVI) imaging. Utilizing astrocyte and neuron electrophysiological recording, optogenetic manipulation of astrocyte Vm, and GEVI imaging of astrocyte membrane potential we have generated preliminary data that supports our hypothesis that EAAT function can be modulated by activity-induced changes in astrocyte Vm.