Melanopsin-expressing retinal ganglion cells: novel genetic tools

描述（由申请人提供）：黑视蛋白（视蛋白4）是一种在投射到视交叉上核和其他大脑区域的视网膜神经节细胞的一小部分中发现的色素，涉及对环境光的非视觉反应，例如瞳孔光反射、生理学中的季节性适应、夜间褪黑激素释放的光抑制以及睡眠、警觉性和活动的调节。由于含有黑视蛋白的神经节细胞数量很少，并且分散在整个视网膜中，因此很难对其进行研究。为了解决这些限制，我们工程化了一种小鼠系，其中增强型绿色荧光蛋白（EGFP）在小鼠黑视蛋白启动子的控制下表达，采用BAC（细菌人工染色体）转基因。我们将在这些小鼠中进行两种类型的研究：（1）在整个安装视网膜中的EGFP阳性神经元的单细胞电生理记录，以表征其在发育过程中的功能特性，（2）急性分离的EGFP阳性神经元的电生理记录，以表征其内在特性。利用我们选择性靶向表达黑视蛋白的神经元的能力，我们还将工程化另外的小鼠系：Opn4tTA系，其中转录激活因子的表达将通过将转基因动物暴露于不同浓度的多西环素（Dox）来可逆地和定量地调节。Opn4 tTA系将用于允许破伤风毒素轻链的条件性表达，破伤风毒素轻链是抑制内向整流钾通道Kir2.1的突触释放或条件性过表达的分子，以降低表达黑视素的神经元的兴奋性。这些小鼠品系将为神经科学和视觉研究界提供重要的工具，以解决黑视素表达细胞中的信号转导机制，其生理特性及其在体内的作用。在脊椎动物眼睛的视网膜内，有光感受器，其捕获光以调节非视觉过程，例如昼夜节律和瞳孔的缩小和扩大。为了捕捉光线，这些特殊的细胞被称为内在感光视网膜神经节细胞，需要称为黑视素的色素，类似于视杆细胞和视锥细胞用于将光线转化为视觉的视蛋白。虽然视锥细胞和视杆细胞已经被广泛研究，但对表达黑视素的神经节细胞知之甚少，尽管它们的功能似乎更像无脊椎动物眼睛中发现的光感受器。这项研究将产生新的小鼠模型，将允许在体内和体外研究这些细胞。确定内在光敏神经节细胞如何工作可能有助于治疗睡眠障碍和季节性抑郁症等疾病。

DESCRIPTION (provided by applicant): Melanopsin (Opsin 4), a photopigment found in a small subset of retinal ganglion cells projecting to the suprachiasmatic nucleus and other brain areas, is implicated in nonvisual responses to environmental light such as the pupillary light reflex, seasonal adaptations in physiology, photic inhibition of nocturnal melatonin release, and modulation of sleep, alertness and activity. Because melanopsin containing ganglion cells are few in number and scattered throughout the retina, they are difficult to study. To address these limitations, we engineered a mouse line in which the Enhanced Green Fluorescent Protein (EGFP) is expressed under the control of the mouse melanopsin promoter employing BAC (bacterial artificial chromosome) transgenesis. We will perform two types of studies in these mice: (1) single cell electrophysiological recordings of EGFP positive neurons in whole mount retinas to characterize their functional properties during development, (2) electrophysiological recordings of acutely isolated EGFP positive neurons to characterize their intrinsic properties. Taking advantage of our ability to selectively target the melanopsin expressing neurons we will also engineer an additional mouse line: Opn4tTA line, in which expression of a transcriptional activator will be regulated both reversibly and quantitatively by exposing the transgenic animals to varying concentrations of doxycycline (Dox). The Opn4 tTA line will be used to allow the conditional expression of tetanus toxin light chain, a molecule that inhibits synaptic release or conditional overexpression of the inward rectifying potassium channel, Kir2.1, to diminish the excitability of melanopsin expressing neurons. These mouse lines will provide important tools to the neuroscience and vision research community to address the mechanisms of signal transduction in melanopsin expressing cells, their physiological properties, and their roles in vivo. Within the retina of the vertebrate eye there are photoreceptors that capture light to regulate non visual processes such as day night rhythms and the narrowing and widening of the pupil. To capture light, these special cells called intrinsically photoreceptive retinal ganglion cells, require pigments called melanopsins, similar to the opsins that the rod and cone cells use for turning light into vision. While cones and rods have been extensively studied, much less is known about the melanopsin expressing ganglion cells, although they appear to function more like the photoreceptors found in invertebrate eyes. This study will generate novel mouse models that will allow the study of these cells in vivo and in vitro. Determining how the intrinsically photosensitive ganglion cells work may allow the treatment of disorders such as sleep disorders and seasonal depression.