Use of SyCatCh for the restoration of light signals in the retina of mice with photoreceptor degeneration

视网膜是视觉系统处理光信号的第一阶段。高度专门化的感光神经元，视杆和视锥感光器，将光转换成神经信号，并将这些信号传输到接收后的视网膜网络。在这些信号被传输到大脑的高级视觉中心之前，视网膜对视觉信息进行了大量的处理和过滤。许多视网膜退行性疾病涉及光感受器的退化，经常导致完全失明。预防视力丧失甚至恢复视功能是视觉研究的重要目标。光遗传学是一种很有前途的恢复视力的策略，它涉及将编码光敏蛋白(如通道视紫红质)的基因引入光感受器下游的神经元。在光刺激下，这些蛋白质在神经元中产生电信号，而这些信号本质上不是光敏感的。RD1小鼠是一种广泛使用的早发性视网膜变性模型。此前研究表明，在这些小鼠的视网膜内细胞中表达通道视紫红质-2可以恢复视网膜和皮质水平的光敏性，证明了光遗传学在恢复视力方面的潜在应用。然而，刺激需要非常高的光强度，这最终可能会对视网膜造成光损伤。我们的目标是测试一种新的光遗传策略，以恢复光感受器退化后视网膜回路的光敏感性。这一策略是基于用光来特定地控制突触的活动，通过病毒将光激活的钙通道捕获引入双极细胞的突触终末。蓝光刺激应该导致钙离子的内流，并直接导致高度专门化的双极细胞带突触的囊泡融合，我们将使用突触活动的遗传编码报告结合多光子成像来测量这两者。我们将使用多电极阵列的电生理记录来测试光信号是否传递到神经节细胞以产生棘波。所有这些都将直接告诉我们，在双极细胞上的Catch表达如何很好地恢复了Rd1小鼠视网膜的突触传递。我们将通过测量视觉运动反应和成像视觉皮质中的神经活动来测试光信号是否传递到大脑的高级视觉中心。通过我们的方法，我们将有望恢复光感受器退化小鼠的视觉功能。我们认为，与以前使用的通道视紫红质相比，使用CATCH将需要显著降低激活双极细胞的光水平，从而消除对高强度光导致视网膜光损伤的担忧。

The retina is the first stage in the processing of light signals in the visual system. Highly specialized light-sensing neurons, the rod and cone photoreceptors, convert light into neural signals and transmit these to the post-receptoral retinal network. The retina performs considerable processing and filtering of the visual information before these signals are transmitted to the higher visual centers in the brain.Many retinal degenerative diseases involve the degeneration of photoreceptors and often lead to complete blindness. The prevention of vision loss or even the restoration of visual function are important goals in vision research. A promising strategy for the restoration of vision is optogenetics, which involves the introduction of genes encoding light-sensitive proteins (like channelrhodopsins) into neurons downstream of the photoreceptors. Upon light stimulation, these proteins generate electrical signals in neurons which are not intrinsically light-sensitive.The rd1 mouse is a widely used model of early onset retinal degeneration. It was previously demonstrated that expression of channelrhodopsin-2 in inner retinal cells of these mice can restore photosensitivity at the retinal and cortical level, proving the potential use of optogenetics in the restoration of vision. However, very high light intensities were needed for stimulation, which could ultimately cause photodamage to the retina. This might be overcome by using genetically engineered variants with altered properties.Our aim is to test a new optogenetic strategy for restoring light-sensitivity to the retinal circuitry following photoreceptor degeneration. This strategy is based on specifically controlling synaptic activity by light, by virally introducing the light-activated calcium channel CatCh into synaptic terminals of ON bipolar cells.Stimulation with blue light should lead to an influx of calcium ions and directly result in vesicle fusion at the highly specialized bipolar cell ribbon synapse, both of which we will measure using genetically encoded reporters of synaptic activity in combination with multiphoton imaging. We will use electrophysiological recordings with multi-electrode arrays to test whether the light signals are transmitted to the ganglion cells to generate spikes. All this will tell us directly how well CatCh expression in ON bipolar cells restores synaptic transmission in the retina of rd1 mice. We will test whether the light signals are transmitted to the higher visual centers in the brain by measuring optomotor responses and by imaging neural activity in the visual cortex.With our approach we will hopefully be able to restore visual function in mice with photoreceptor degeneration. We think that using CatCh will require significantly reduced light levels for activation of ON bipolar cells in comparison with previously used channelrhodopsins, thereby eliminating the concern that high intensity light induces photodamage in the retina.