Van Hook Matthew J, Thoreson Wallace B
Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center.
J Vis Exp. 2013 Jun 1(76):50007. doi: 10.3791/50007.
One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells--the output cells of the retina. A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording. Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon--a specialized synaptic structure found in only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release--and how it contributes to the unique signaling properties of this first retinal synapse.
视网膜神经科学的核心任务之一是了解视网膜神经元的电路，以及这些连接如何塑造传递到大脑的信号。视网膜中的视杆和视锥光感受器能够检测光子，它们将光能转化为电信号，并将其传递给其他视网膜神经元，在那里信号被处理，然后通过视神经传递到大脑中的中枢靶点。对视网膜电路和视觉处理的早期重要见解来自于卡哈尔的组织学研究，以及后来对视网膜神经节细胞（视网膜的输出细胞）的脉冲活动进行的电生理记录。要详细了解视网膜中的视觉处理过程，需要了解从光感受器到视网膜神经节细胞这条通路中每一步的信号传递情况。然而，许多视网膜细胞类型深埋在组织中，因此相对难以进行电生理记录。通过使用垂直切片可以克服这一限制，在垂直切片中，每个视网膜层中的细胞都清晰可见，便于进行电生理记录。在这里，我们描述一种从虎纹钝口螈幼体视网膜制作垂直切片的方法。虽然这种制备方法最初是为使用尖锐微电极进行记录而开发的，但我们描述了一种从光感受器以及二级水平细胞和双极细胞进行双全细胞电压钳记录的方法，在该方法中，我们操纵光感受器的膜电位，同时记录水平细胞或双极细胞中的突触后反应。虎纹钝口螈的光感受器比哺乳动物的光感受器大得多，这使得它成为进行这种技术上具有挑战性的实验方法的理想样本。我们描述这些实验的目的是探究突触带的信号特性，突触带是一种仅在少数神经元中发现的特殊突触结构，包括视杆和视锥光感受器，它非常适合维持较高频率的持续性神经递质释放，以及它如何促成这个视网膜第一突触独特的信号特性。
