Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, 16132, Genoa, Italy.
Department of Experimental Medicine, School of Medicine and Pharmacy, University of Genoa, 16132, Genoa, Italy.
Mol Neurobiol. 2019 Sep;56(9):6276-6292. doi: 10.1007/s12035-019-1506-5. Epub 2019 Feb 12.
Cultured hippocampal neurons represent the most widely used experimental substrate for in vitro electrophysiological studies. Nevertheless, in most cases, the nature of neuron under study is not identified as excitatory or inhibitory, or even worse, recorded neurons are considered as excitatory because of the paucity of GABAergic interneurons. Thus, the definition of reliable criteria able to guarantee an unequivocal identification of excitatory and inhibitory cultured hippocampal neurons is an unmet need. To reach this goal, we compared the electrophysiological properties and the localization and size of the axon initial segment (AIS) of cultured hippocampal neurons, taking advantage from GAD67-GFP knock-in mice, which expressing green fluorescent protein (GFP) in gamma-aminobutyric acid (GABA)-containing cells, allowed to unambiguously determine the precise nature of the neuron under study. Our results demonstrate that the passive electrophysiological properties, the localization and size of the AIS, and the shape and frequency of the action potential (AP) are not reliable to unequivocally identify neurons as excitatory or inhibitory. The only parameter, related to the shape of the single AP, showing minimal overlap between the sample-point distributions of the two neuronal subpopulations, was the AP half-width. However, the estimation of the AP failure ratio evoked by a short train of high-current steps applied at increasing frequency (40-140 Hz) resulted to be indisputably the safer and faster way to identify the excitatory or inhibitory nature of an unknown neuron. Our findings provide a precise framework for further electrophysiological investigations of in vitro hippocampal neurons.
培养的海马神经元是体外电生理研究中最常用的实验底物。然而，在大多数情况下，所研究的神经元的性质未被鉴定为兴奋性或抑制性，甚至更糟糕的是，由于 GABA 能中间神经元的缺乏，记录的神经元被认为是兴奋性的。因此，定义可靠的标准来保证兴奋性和抑制性培养海马神经元的明确鉴定是一个未满足的需求。为了实现这一目标，我们利用 GAD67-GFP 基因敲入小鼠（其在 GABA 能细胞中表达绿色荧光蛋白 [GFP]）比较了培养的海马神经元的电生理特性以及轴突起始段（AIS）的定位和大小，从而能够明确确定所研究神经元的精确性质。我们的结果表明，被动电生理特性、AIS 的定位和大小以及动作电位（AP）的形状和频率不能可靠地区分神经元是兴奋性的还是抑制性的。唯一与单个 AP 形状相关的参数是 AP 半宽度，两个神经元亚群的样本点分布之间的重叠最小。然而，通过在增加的频率（40-140 Hz）下施加短的高电流阶跃的刺激来估计 AP 失败比，结果证明是识别未知神经元的兴奋性或抑制性性质的更安全和更快的方法。我们的发现为体外海马神经元的进一步电生理研究提供了一个精确的框架。
