Hidaka Soh, Akahori Yasushi, Kurosawa Yoshikazu
Department of Physiology, School of Medicine, Institute for Comprehensive Medical Sciences, Fujita Health University, Toyoake, Aichi 470-1192, Japan.
J Neurosci. 2004 Nov 17;24(46):10553-67. doi: 10.1523/JNEUROSCI.3319-04.2004.
Electrical synapses between alpha-type ganglion cells were detected using combined techniques of dual patch-clamp recordings, intracellular labeling, electron microscopy, and channel subunit connexin immunocytochemistry in the albino rat retina. After intracellular injection of Neurobiotin into alpha-cells of inner (ON-center) and outer (OFF-center) ramifying types, measurement of tracer coupling resulted in a preferentially homologous occurrence among cells of the same morphological type (n = 19 of 24). In high-voltage as well as conventional electron microscopic analysis, direct dendrodendritic gap junctions (average size, 0.86 mum long) were present in contact sites between tracer-coupled alpha-cells. In simultaneous dual whole-cell recordings from pairs of neighboring alpha-cells, these cells generated TTX-sensitive sustained spiking against extrinsic current injection, and bidirectional electrical synapses (maximum coupling coefficient, 0.32) with symmetrical junction conductance (average, 1.35 nS) were observed in pairs with cells of the same morphological type. Precise temporal synchronization of spike activity (average time delay, 2.7 msec) was detected when depolarizing currents were simultaneously injected into the pairs. To address whether physiologically identified electrical synapses constitute gap junctional connectivity between cell pairs, identified neuronal connexin36 immunoreactivity was undertaken in Lucifer yellow-labeled cell pairs after patch-clamp recordings. All alpha-cells expressed connexin36, and confocal laser-scanning imaging demonstrated that connexin36 is primarily located at dendritic crossings between electrically coupled cells (seven sites in a pair, on average). These results give conclusive evidence for electrical synapses via dendrodendritic gap junctions involving connexin36 in alpha retinal ganglion cells of the same physiological type.
利用双膜片钳记录、细胞内标记、电子显微镜和通道亚基连接蛋白免疫细胞化学等联合技术，在白化大鼠视网膜中检测到了α型神经节细胞之间的电突触。将神经生物素细胞内注射到内部分支型（ON中心）和外部分支型（OFF中心）的α细胞后，示踪剂耦合测量结果显示，在相同形态类型的细胞之间优先出现同源耦合（24对中有19对）。在高压以及传统电子显微镜分析中，示踪剂耦合的α细胞之间的接触部位存在直接的树突-树突间隙连接（平均长度为0.86μm）。在对相邻α细胞对进行同步双全细胞记录时，这些细胞在注入外部电流时产生对河豚毒素敏感的持续放电，并且在与相同形态类型的细胞对中观察到双向电突触（最大耦合系数为0.32），其连接电导对称（平均为1.35 nS）。当向细胞对同时注入去极化电流时，检测到了动作电位活动的精确时间同步（平均时间延迟为2.7毫秒）。为了确定生理上确定的电突触是否构成细胞对之间的间隙连接连通性，在膜片钳记录后，对荧光黄标记的细胞对进行了已鉴定的神经元连接蛋白36免疫反应性检测。所有α细胞均表达连接蛋白36，共聚焦激光扫描成像显示，连接蛋白36主要位于电耦合细胞之间的树突交叉处（平均每对有七个位点）。这些结果为同一生理类型的α视网膜神经节细胞中通过涉及连接蛋白36的树突-树突间隙连接形成电突触提供了确凿证据。
