Gilin Naomi, Wattad Nadine, Tiroshi Lior, Goldberg Joshua A
Department of Medical Neurobiology, Institute of Medical Research Israel - Canada, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel.
Department of Neurobiology, Northwestern University, Evanston, IL, USA.
Bio Protoc. 2024 May 20;14(10):e4992. doi: 10.21769/BioProtoc.4992.
Understanding dendritic excitability is essential for a complete and precise characterization of neurons' input-output relationships. Theoretical and experimental work demonstrates that the electrotonic and nonlinear properties of dendrites can alter the amplitude (e.g., through amplification) and latency of synaptic inputs as viewed in the axosomatic region where spike timing is determined. The gold-standard technique to study dendritic excitability is using dual-patch recordings with a high-resistance electrode used to patch a piece of distal dendrite in addition to a somatic patch electrode. However, this approach is often impractical when distal dendrites are too fine to patch. Therefore, we developed a technique that utilizes the expression of Channelrhodopsin-2 (ChR2) to study dendritic excitability in acute brain slices through the combination of a somatic patch electrode and optogenetic activation. The protocol describes how to prepare acute slices from mice that express ChR2 in specific cell types, and how to use two modes of light stimulation: proximal (which activates the soma and proximal dendrites in a ~100 µm diameter surrounding the soma) with the use of a high-magnification objective and full-field stimulation through a low-magnification objective (which activates the entire somato-dendritic field of the neuron). We use this technique in conjunction with various stimulation protocols to estimate model-based spectral components of dendritic filtering and the impact of dendrites on phase response curves, peri-stimulus time histograms, and entrainment of pacemaking neurons. This technique provides a novel use of optogenetics to study intrinsic dendritic excitability through the use of standard patch-clamp slice physiology. Key features • A method for studying the effects of electrotonic and nonlinear dendritic properties on the sub- and suprathreshold responses of pacemaking neurons. • Combines somatic patch clamp or perforated patch recordings with optogenetic activation in acute brain slices to investigate dendritic linear transformation without patching the dendrite. • Oscillatory illumination at various frequencies estimates spectral properties of the dendrite using subthreshold voltage-clamp recordings and studies entrainment of pacemakers in current clamp recordings. • This protocol uses Poisson white noise illumination to estimate dendritic phase response curves and peri-stimulus time histograms.
了解树突兴奋性对于全面准确地表征神经元的输入-输出关系至关重要。理论和实验工作表明，树突的电紧张特性和非线性特性可以改变突触输入在轴突体区域（即确定动作电位发放时间的区域）所观察到的幅度（例如，通过放大）和潜伏期。研究树突兴奋性的金标准技术是采用双电极膜片钳记录，除了在胞体处使用膜片电极外，还使用高电阻电极膜片钳记录一段远端树突。然而，当远端树突太细而无法进行膜片钳记录时，这种方法通常不切实际。因此，我们开发了一种技术，通过结合胞体膜片钳电极和光遗传学激活，利用2型通道视紫红质（ChR2）的表达来研究急性脑片的树突兴奋性。本方案描述了如何从在特定细胞类型中表达ChR2的小鼠制备急性脑片，以及如何使用两种光刺激模式：近端刺激（使用高倍物镜激活胞体及其周围直径约100μm范围内的近端树突）和通过低倍物镜进行全场刺激（激活神经元的整个胞体-树突场）。我们将该技术与各种刺激方案结合使用，以估计基于模型的树突滤波频谱成分以及树突对相位响应曲线、刺激后时间直方图和起搏神经元同步化的影响。该技术通过使用标准的膜片钳脑片生理学方法，提供了一种利用光遗传学研究树突内在兴奋性的新方法。关键特性•一种研究电紧张和非线性树突特性对起搏神经元阈下和阈上反应影响的方法。•将急性脑片中的胞体膜片钳或穿孔膜片钳记录与光遗传学激活相结合，在不膜片钳记录树突的情况下研究树突线性转换。•使用亚阈值电压钳记录，通过不同频率的振荡光照估计树突的频谱特性，并在电流钳记录中研究起搏器的同步化。•本方案使用泊松白噪声光照来估计树突相位响应曲线和刺激后时间直方图。
