Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114.
Department of Pediatrics and Neurology, Iowa Neuroscience Institute, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
J Neurosci. 2021 Jun 9;41(23):4957-4975. doi: 10.1523/JNEUROSCI.3175-20.2021. Epub 2021 Apr 26.
Developmental, cellular, and subcellular variations in the direction of neuronal Cl currents elicited by GABA receptor activation have been frequently reported. We found a corresponding variance in the GABA receptor reversal potential (E) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a similar heterogeneity in the cytoplasmic intracellular concentration of chloride ([Cl]) in individual dendrites. We determined [Cl] in the murine hippocampus and cerebral cortex of both sexes by (1) two-photon imaging of the Cl-sensitive, ratiometric fluorescent protein SuperClomeleon; (2) Fluorescence Lifetime IMaging (FLIM) of the Cl-sensitive fluorophore MEQ (6-methoxy--ethylquinolinium); and (3) electrophysiological measurements of E by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl] were highly correlated. [Cl] microdomains persisted after pharmacological inhibition of cation-chloride cotransporters, but were progressively modified after inhibiting the polymerization of the anionic biopolymer actin. These methods collectively demonstrated stable [Cl] microdomains in individual neurons and and the role of immobile anions in its stability. Our results highlight the existence of functionally significant neuronal Cl microdomains that modify the impact of GABAergic inputs. Microdomains of varying chloride concentrations in the neuronal cytoplasm are a predictable consequence of the inhomogeneous distribution of anionic polymers such as actin, tubulin, and nucleic acids. Here, we demonstrate the existence and stability of these microdomains, as well as the consequence for GABAergic synaptic signaling: each interneuron produces a postsynaptic GABA response with a unique reversal potential. In individual hippocampal pyramidal cells, the range of GABA reversal potentials evoked by stimulating different interneurons was >20 mV. Some interneurons generated postsynaptic responses in pyramidal cells that reversed at potentials beyond what would be considered purely inhibitory. Cytoplasmic chloride microdomains enable each pyramidal cell to maintain a compendium of unique postsynaptic responses to the activity of individual interneurons.
神经元 Cl 电流的方向在 GABA 受体激活后会发生发育、细胞和亚细胞变化，这一现象已被频繁报道。我们发现，源自单个中间神经元的 GABA 受体反转电位 (E) 在单个锥体神经元上的突触处存在相应的差异。这些发现表明，在单个树突的细胞质内氯离子 ([Cl]) 浓度中存在类似的异质性。我们通过以下方法确定了雌雄两性的小鼠海马体和大脑皮质中的 [Cl]：（1）利用 Cl 敏感型比率荧光蛋白 SuperClomeleon 的双光子成像；（2）利用 Cl 敏感型荧光团 MEQ（6-甲氧基-乙基喹啉鎓）的荧光寿命成像（FLIM）；（3）通过 GABA 和 RuBi-GABA 光解笼的压力施加进行电生理测量来测量 E。荧光和电生理对局部 [Cl] 的估计高度相关。在阳离子-Cl 共转运蛋白的药理学抑制后，[Cl]微区仍然存在，但在抑制阴离子生物聚合物肌动蛋白聚合后，[Cl]微区逐渐被修饰。这些方法共同证明了单个神经元中稳定的 [Cl] 微区的存在和不可移动阴离子在其稳定性中的作用。我们的结果突出了功能上重要的神经元 Cl 微区的存在，这些微区可以改变 GABA 能输入的影响。神经元细胞质中氯离子浓度不同的微区是阴离子聚合物（如肌动蛋白、微管蛋白和核酸）不均匀分布的可预测结果。在这里，我们证明了这些微区的存在和稳定性，以及它们对 GABA 能突触信号传递的影响：每个中间神经元产生具有独特反转电位的突触后 GABA 反应。在单个海马锥体细胞中，刺激不同中间神经元引起的 GABA 反转电位范围超过 20 mV。一些中间神经元在锥体细胞中产生反转电位超过纯抑制作用的突触后反应。细胞质氯离子微区使每个锥体细胞能够维持对单个中间神经元活动的独特突触后反应总集。
