Caruso Department of Otolaryngology Head & Neck Surgery, Zilkha Neurogenetics Institute, Hearing and Communications Neuroscience Training Program, University of Southern California.
Caruso Department of Otolaryngology Head & Neck Surgery, Zilkha Neurogenetics Institute, Hearing and Communications Neuroscience Training Program, University of Southern California;
J Vis Exp. 2023 Apr 21(194). doi: 10.3791/64908.
The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.
内耳神经节神经元的形态紧凑，允许对离子通道和神经递质受体进行详细表征，这些通道和受体有助于该群体内细胞的多样性。本方案概述了成功解剖、分离和短期培养内耳双极神经元体细胞以进行膜片钳记录的必要步骤。提供了详细的前庭神经节神经元制备说明，并对用于培养螺旋神经节神经元所需的必要修改进行了说明。该方案包括在穿孔贴片配置中执行全细胞膜片钳记录的说明。示例结果说明了超极化激活环核苷酸门控 (HCN) 介导电流的电压钳记录，突出了穿孔贴片记录配置相对于更标准的破裂贴片配置的稳定性。这些方法的结合，即分离的体细胞加穿孔贴片钳记录，可以用于研究需要长期稳定记录和细胞内环境保存的细胞过程，例如通过 G 蛋白偶联受体进行信号转导。
