Spike-Related Electrophysiological Identification of Cultured Hippocampal Excitatory and Inhibitory Neurons
Spike-Related Electrophysiological Identification of Cultured Hippocampal Excitatory and Inhibitory Neurons
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DOI：
10.1007/s12035-019-1506-5
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发表时间：
2019-09-01
影响因子：
5.1
通讯作者：
Baldelli, Pietro
中科院分区：
文献类型：
--
作者：
Prestigio, Cosimo;Ferrante, Daniele;Baldelli, Pietro
培养的海马神经元代表了体外电生理研究中最广泛使用的实验基质。然而，在大多数情况下，所研究的神经元的性质不能被确定为兴奋性或抑制性，甚至更糟的是，记录的神经元被认为是兴奋性的，因为缺乏GABA能中间神经元。因此，能够保证明确鉴定兴奋性和抑制性培养海马神经元的可靠标准的定义是未满足的需求。为了达到这一目标，我们比较了培养的海马神经元的电生理特性和轴突起始段（AIS）的定位和大小，利用GAD 67-GFP基因敲入小鼠，其在含有γ-氨基丁酸（GABA）的细胞中表达绿色荧光蛋白（GFP），允许明确地确定所研究的神经元的精确性质。我们的研究结果表明，被动的电生理特性，AIS的定位和大小，以及动作电位（AP）的形状和频率是不可靠的，以明确识别神经元兴奋性或抑制性。唯一的参数，与单个AP的形状，显示两个神经元亚群的样本点分布之间的重叠最小，是AP半宽。然而，由以增加的频率（40-140 Hz）施加的短串高电流阶跃引起的AP故障率的估计无可争议地导致识别未知神经元的兴奋性或抑制性的更安全和更快的方式。我们的研究结果提供了一个精确的框架，为进一步的电生理研究在体外海马神经元。
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.