Priel Avner, Dai Xiao-Qing, Chen Xing-Zhen, Scarinci Noelia, Cantero María Del Rocío, Cantiello Horacio F
The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.
Front Mol Neurosci. 2022 Aug 25;15:769725. doi: 10.3389/fnmol.2022.769725. eCollection 2022.
Dendritic spines (DS) are tiny protrusions implicated in excitatory postsynaptic responses in the CNS. To achieve their function, DS concentrate a high density of ion channels and dynamic actin networks in a tiny specialized compartment. However, to date there is no direct information on DS ionic conductances. Here, we used several experimental techniques to obtain direct electrical information from DS of the adult mouse hippocampus. First, we optimized a method to isolate DS from the dissected hippocampus. Second, we used the lipid bilayer membrane (BLM) reconstitution and patch clamping techniques and obtained heretofore unavailable electrical phenotypes on ion channels present in the DS membrane. Third, we also patch clamped DS directly in cultured adult mouse hippocampal neurons, to validate the electrical information observed with the isolated preparation. Electron microscopy and immunochemistry of PDS-95 and NMDA receptors and intrinsic actin networks confirmed the enrichment of the isolated DS preparation, showing open and closed DS, and multi-headed DS. The preparation was used to identify single channel activities and "whole-DS" electrical conductance. We identified NMDA and Ca-dependent intrinsic electrical activity in isolated DS and DS of cultured adult mouse hippocampal neurons. recordings in the presence of local NMDA, showed that individual DS intrinsic electrical activity often back-propagated to the dendrite from which it sprouted. The DS electrical oscillations were modulated by changes in actin cytoskeleton dynamics by addition of the F-actin disrupter agent, cytochalasin D, and exogenous actin-binding proteins. The data indicate that DS are elaborate excitable electrical devices, whose activity is a functional interplay between ion channels and the underlying actin networks. The data argue in favor of the active contribution of individual DS to the electrical activity of neurons at the level of both the membrane conductance and cytoskeletal signaling.
树突棘（DS）是中枢神经系统中与兴奋性突触后反应有关的微小突起。为实现其功能，树突棘在一个微小的特殊区域内集中了高密度的离子通道和动态肌动蛋白网络。然而，迄今为止，尚无关于树突棘离子电导的直接信息。在此，我们使用了几种实验技术，从成年小鼠海马体的树突棘中获取直接的电信息。首先，我们优化了一种从解剖的海马体中分离树突棘的方法。其次，我们使用脂质双分子层膜（BLM）重组和膜片钳技术，获得了迄今为止关于树突棘膜中存在的离子通道的电表型。第三，我们还直接对培养的成年小鼠海马神经元中的树突棘进行膜片钳记录，以验证从分离标本中观察到的电信息。对PSD-95和NMDA受体以及内在肌动蛋白网络的电子显微镜和免疫化学分析证实了分离的树突棘标本的富集，显示出开放和闭合的树突棘以及多头树突棘。该标本用于识别单通道活动和“整个树突棘”的电导。我们在分离的树突棘以及培养的成年小鼠海马神经元的树突棘中识别出了NMDA和钙依赖性内在电活动。在局部NMDA存在下的记录表明，单个树突棘的内在电活动通常会从其发出的树突反向传播。通过添加F-肌动蛋白破坏剂细胞松弛素D和外源性肌动蛋白结合蛋白，肌动蛋白细胞骨架动力学的变化调节了树突棘的电振荡。数据表明，树突棘是复杂的可兴奋电子装置，其活动是离子通道与潜在的肌动蛋白网络之间的功能相互作用。数据支持单个树突棘在膜电导和细胞骨架信号传导水平上对神经元电活动的积极贡献。
