Walch Erin, Bilas Alexander, Bebawy Valine, Lam Angelina, Murphy Thomas R, Sriram Sandhya, Fiacco Todd A
Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, CA, United States.
Center for Glial-Neuronal Interactions, University of California, Riverside, Riverside, CA, United States.
Front Cell Neurosci. 2022 Jul 22;16:930384. doi: 10.3389/fncel.2022.930384. eCollection 2022.
Rapid increases in cell volume reduce the size of the extracellular space (ECS) and are associated with elevated brain tissue excitability. We recently demonstrated that astrocytes, but not neurons, rapidly swell in elevated extracellular potassium ([K] ) up to 26 mM. However, effects of acute astrocyte volume fluctuations on neuronal excitability in [K] have been difficult to evaluate due to direct effects on neuronal membrane potential and generation of action potentials. Here we set out to isolate volume-specific effects occurring in [K] on CA1 pyramidal neurons in acute hippocampal slices by manipulating cell volume while recording neuronal glutamate currents in 10.5 mM [K] + tetrodotoxin (TTX) to prevent neuronal firing. Elevating [K] to 10.5 mM induced astrocyte swelling and produced significant increases in neuronal excitability in the form of mixed α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/N-methyl-D-aspartate (NMDA) receptor mEPSCs and NMDA receptor-dependent slow inward currents (SICs). Application of hyperosmolar artificial cerebrospinal fluid (ACSF) by addition of mannitol in the continued presence of 10.5 mM K forced shrinking of astrocytes and to a lesser extent neurons, which resisted swelling in [K] . Cell shrinking and dilation of the ECS significantly dampened neuronal excitability in 10.5 mM K. Subsequent removal of mannitol amplified effects on neuronal excitability and nearly doubled the volume increase in astrocytes, presumably due to continued glial uptake of K while mannitol was present. Slower, larger amplitude events mainly driven by NMDA receptors were abolished by mannitol-induced expansion of the ECS. Collectively, our findings suggest that cell volume regulation of the ECS in elevated [K] is driven predominantly by astrocytes, and that cell volume effects on neuronal excitability can be effectively isolated in elevated [K] conditions.
细胞体积的快速增加会减小细胞外间隙（ECS）的大小，并与脑组织兴奋性升高有关。我们最近证明，在细胞外钾浓度（[K⁺]）升高至26 mM时，星形胶质细胞而非神经元会迅速肿胀。然而，由于对神经元膜电位和动作电位产生的直接影响，急性星形胶质细胞体积波动对[K⁺]条件下神经元兴奋性的影响一直难以评估。在这里，我们通过在记录10.5 mM [K⁺] + 河豚毒素（TTX）中的神经元谷氨酸电流时操纵细胞体积，来分离[K⁺]条件下急性海马切片中CA1锥体神经元上发生的体积特异性效应，以防止神经元放电。将[K⁺]升高至10.5 mM会诱导星形胶质细胞肿胀，并以混合的α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）/N-甲基-D-天冬氨酸（NMDA）受体微小兴奋性突触后电流（mEPSCs）和NMDA受体依赖性缓慢内向电流（SICs）的形式显著增加神经元兴奋性。在持续存在10.5 mM K的情况下，通过添加甘露醇应用高渗人工脑脊液（ACSF）会迫使星形胶质细胞以及在较小程度上神经元发生收缩，神经元在[K⁺]条件下能抵抗肿胀。细胞收缩和ECS的扩张显著抑制了10.5 mM K条件下的神经元兴奋性。随后去除甘露醇会放大对神经元兴奋性的影响，并使星形胶质细胞的体积增加几乎翻倍，这可能是由于在存在甘露醇时胶质细胞持续摄取K⁺所致。主要由NMDA受体驱动的较慢、较大幅度的事件被甘露醇诱导的ECS扩张所消除。总的来说，我们的研究结果表明，[K⁺]升高时ECS的细胞体积调节主要由星形胶质细胞驱动，并且在[K⁺]升高的条件下，细胞体积对神经元兴奋性的影响可以有效地分离出来。
