Voltage imaging of astrocyte-neuron interactions

项目总结 星形胶质细胞将兴奋性神经递质谷氨酸从神经元的胞外间隙中移除 通过钠驱动的、电压依赖的兴奋性氨基酸转运体(EAATs)的活性。健壮的谷氨酸 EAAT的摄取确保了谷氨酸信号在时间和空间上的保真度。有趣的是，我们最近 发现神经元活动迅速(在毫秒内)并可逆地减缓成人对谷氨酸的摄取 大脑皮层。这种减慢会延长神经元的NMDA反应，与延长细胞外的反应一致 谷氨酸动力学，并高度依赖于刺激的频率和持续时间。另外， 谷氨酸清除可以由具有突触特异性的神经元活动来调节，即使在单个 星形细胞。我们认为这可能会对神经传递、突触外受体产生重要影响 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 局部调节EAAT功能的星形胶质细胞膜电位(Vm)的微域水平变化。GLT1是 在成人前脑中占主导地位的星形细胞EAAT大量表达，并确保谷氨酸在 胞外空间被EAAT结合迅速隔离。一旦绑定到EAAT，运输 谷氨酸进入星形胶质细胞既是钠驱动的，也是电压依赖的。正常情况下，星形胶质细胞 由于对钾的高渗透性，它们是超极化的(-80 mV)。然而，神经元的活动增加 细胞外钾、[K]e和星形胶质细胞Vm对[K]e变化特别敏感。因此，它站在了 神经元活动可以通过去极化星形胶质细胞来改变EAAT功能的原因。星形细胞VM的变化可能 在细微的星形细胞突起中尤其相关，在星形细胞突起中，EAAT集中，细胞内较小 与SOMA相比，体积可能放大Vm的变化。我们还将探索替代机制。 包括通过增加[K]e对EAAT进行电压无关的调制。测试我们的 然而，假设是由于膜电阻低，无法监测星形胶质细胞远端突起的VM。 和过程形态。克服这一挑战很重要，因为星形胶质细胞的远端突起是 突触相互作用的部位和EAATs的定位。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)对星形胶质细胞突起中的VM进行成像的方法 成像。利用星形胶质细胞和神经元电生理记录，对星形胶质细胞VM进行光遗传操作， 和星形胶质细胞膜电位的GEVI成像，我们已经产生了支持我们的 假设EAAT功能可以通过活动诱导的星形胶质细胞Vm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.