Genetic dissection of color-vision circuits

色觉回路的基因剖析

基本信息

批准号：
8941499
负责人：
Chi-Hon Lee
金额：
$ 151.62万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8941499
关键词：
Adult Animal Behavior Animals Behavior Behavioral Biological Assay Brain Cell physiology Choline O-Acetyltransferase Cholinergic Receptors Color Color Visions Detection Discrimination Dissection Drosophila genus Electron Microscope Electrons Flying body movement Functional Imaging Ganglia Gene Expression Profile Genetic Glutamate Transporter Glutamates Goals Human Immunohistochemistry Insecta Interneurons Label Learning Light Light Microscope Maps Mediating Memory Methods Microscopic Molecular Profiling Motion Muscarinic Acetylcholine Receptor Muscarinics Neurons Neurotransmitter Receptor Operant Conditioning Optic Lobe Output Pathway interactions Peripheral Photoreceptors Primates Process RNA Interference Retinal Ganglion Cells Retrieval Reverse Transcriptase Polymerase Chain Reaction Signal Transduction Stimulus Synapses Synaptic Receptors Training Transcript Ultraviolet Rays Vision Visual Visual Motion Visual system structure arm base cholinergic classical conditioning color processing detector fly functional restoration kainate knock-down mutant neural circuit novel object recognition preference promoter receptive field receptor expression reconstitution reconstruction relating to nervous system response single cell analysis transmission process visual information visual map

项目摘要

Color vision, which differentiates spectral compositions independent of brightness, provides animals, from insects to primates, great power for object recognition and memory registration and retrieval. Using a combination of genetic, histological, electrophysiological and behavioral approaches, we study how the visual system processes chromatic information to guide behaviors in Drosophila. Using color preference and color learning assays, we demonstrated that flies innately prefer short wavelengths of light but they can be trained to select specific wavelengths of light by Pavlovian conditioning, indicating that flies, like honeybee and human, have true color vision. Using both light and electron microscope, we mapped the synaptic circuits of the chromatic photoreceptors, R7s and R8s, and their synaptic target neurons in the medulla ganglion of the peripheral visual system. We focused on the amacrine neurons, which interconnect medulla columns and the medulla projection (Tm) neurons, which are thought to serve functions analogous to those of vertebrate retinal ganglion cells by processing and relaying photoreceptor information to higher visual center. We found that the chromatic photoreceptors, R7 (UV-sensing) and R8 (blue/green-sensing), provide inputs to a subset of first-order interneurons. The first-order interneurons Tm5a/b receive direct synaptic inputs from R7s while Tm9, Tm20 and Tm5c receive direct synaptic inputs from R8s. In addition, these Tm neurons receive indirect inputs from R1-6 via L3. These Tm neurons relay spectral information from the medulla to the higher visual center, the lobula. In addition to the direct pathways from photoreceptors to Tm neurons, the amacrine neuron Dm8 receives input from multiple R7s and provides input for Tm5a/b/c. To relate neural connectivity to functions, we assigned components of synaptic machinery to specific connections. To directly probe the usage of neurotransmitters and receptors which determine the polarity and dynamic of signal transmission, we developed a method to profile transcripts in single neurons. We used highly specific promoter-Gal4 constructs to label single types of neurons with GFP and to isolated these GFP-labeled neurons from adult fly brains and profiled their gene expression patterns by RT-PCR. Using this method, we determined that a large percentage of the first/second-order interneurons in the chromatic circuits express the vesicular glutamate transporter indicating that they are glutamatergic while the remaining chromatic circuits express choline acetyltransferase and therefore are likely cholinergic. This contrasts with the motion detection pathway, which is mostly cholinergic. By inactivating specific neurons and examining behavioral consequences, we previously found that the amacrine Dm8 neurons, which receive UV-sensing R7 photoreceptor inputs, are both required and sufficient for animals' innate spectral preference to UV light. RNAi-knock-down of vesicular glutamate transporter in the Dm8 neurons significantly reduced UV preference, suggesting that glutamatergic output of Dm8 is critical for its functions. While Dm8 provides inputs for three types of transmedulla neurons, Tm5a/b/c, inactivating Tm5c alone abolished UV preference, indicating that Tm5c is the key downstream targets for this behavior. Furthermore, RNAi-knockdown of Kainate-type iGluR in Tm5c, thus inhibiting its ability to receive glutamatergic Dm8 inputs, significantly reduced UV preference. Using a modified GRASP (GFP-reconstitution across synaptic partners) method, which allows single-cell analysis of bona fide synaptic connections, we demonstrated that Dm8 receives 16 R7 inputs and provides inputs for 1-2 Tm5c neurons in the center of Dm8's receptive field. Thus, the R7s->Dm8->Tm5c connections constitute a hard-wired pooling circuit for detecting dim UV light. To map the circuits that transform photoreceptor signals into color percepts, we developed a novel aversive operant conditioning assay for intensity-independent color discrimination. Single flying flies were magnetically tethered in an arena surrounded by blue/green LEDs and the flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies can be trained in this paradigm to avoid either blue or green while mutant lacking functional R7 and R8 photoreceptors can not, indicating that the color entrainment requires the function of the narrow-spectrum photoreceptors R7s and/or R8s. Genetically inactivating four classes of first-order interneurons, Tm5a/b/c and Tm20, abolishes color learning. However, inactivating subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. The apparent redundancy in learned color discrimination sharply contrasts with innate spectral preference, which is dominated by a single pathway. Using a similar strategy, we mapped the visual motion pathways in Drosophila. Previous studies revealed that two types of lobula plate neurons, T4 and T5, signal small-field direction-selective motion responses and are downstream of the first-order neurons, L1 and L2, or the ON- and OFF-channel neurons, respectively. Serial-EM reconstruction revealed that T5 receives direct synaptic inputs from four types of transmedulla neurons, Tm1, Tm2, Tm4, and Tm9. Our transcript profiling and immunohistochemistry revealed that these Tm neurons express choline acetyltransferase and therefore are likely cholinergic. Furthermore, we found that T5 expresses both nicotinic and muscarinic acetylcholine receptors. The Tm2 and Tm9 input synapses are spatially segregated on T5s dendritic arbor, thus providing candidate anatomical substrates for the two arms of elementary motion detector circuits. Based on the synaptic circuit and receptor expression profiles, we hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.

色觉能够区分与亮度无关的光谱成分，为从昆虫到灵长类动物提供了强大的物体识别、记忆登记和检索能力。我们结合遗传、组织学、电生理学和行为方法，研究果蝇的视觉系统如何处理色彩信息来指导行为。通过颜色偏好和颜色学习测定，我们证明苍蝇天生喜欢短波长的光，但它们可以通过巴甫洛夫条件训练来选择特定波长的光，这表明苍蝇像蜜蜂和人类一样，具有真正的色觉。使用光学和电子显微镜，我们绘制了周围视觉系统髓质神经节中彩色感光器 R7 和 R8 的突触回路及其突触目标神经元。我们关注的是无长突神经元，它连接髓质柱和髓质投射（Tm）神经元，它们被认为通过处理和将感光信息传递到更高的视觉中枢来提供类似于脊椎动物视网膜神经节细胞的功能。我们发现彩色感光器 R7（紫外线感应）和 R8（蓝色/绿色感应）为一级中间神经元的子集提供输入。一阶中间神经元 Tm5a/b 接收来自 R7 的直接突触输入，而 Tm9、Tm20 和 Tm5c 接收来自 R8 的直接突触输入。此外，这些 Tm 神经元通过 L3 接收来自 R1-6 的间接输入。这些 Tm 神经元将光谱信息从髓质传递到更高的视觉中心（小叶）。除了从光感受器到 Tm 神经元的直接通路外，无长突神经元 Dm8 还接收来自多个 R7 的输入，并为 Tm5a/b/c 提供输入。为了将神经连接与功能联系起来，我们将突触机制的组件分配给特定的连接。为了直接探测决定信号传输极性和动态的神经递质和受体的使用，我们开发了一种分析单个神经元转录本的方法。我们使用高度特异性的启动子-Gal4 构建体用 GFP 标记单一类型的神经元，并从成年果蝇大脑中分离这些 GFP 标记的神经元，并通过 RT-PCR 分析其基因表达模式。使用这种方法，我们确定彩色回路中很大一部分第一/第二级中间神经元表达囊泡谷氨酸转运蛋白，表明它们是谷氨酸能的，而其余的彩色回路表达胆碱乙酰转移酶，因此可能是胆碱能的。这与运动检测通路形成鲜明对比，运动检测通路主要是胆碱能的。通过灭活特定神经元并检查行为后果，我们之前发现接收紫外线感应 R7 光感受器输入的无长突 Dm8 神经元对于动物对紫外线的先天光谱偏好来说既是必需的也是充分的。 RNAi 敲低 Dm8 神经元中的囊泡谷氨酸转运蛋白显着降低了紫外线偏好，表明 Dm8 的谷氨酸输出对其功能至关重要。虽然 Dm8 为三种类型的跨髓神经元 Tm5a/b/c 提供输入，但单独灭活 Tm5c 会消除紫外线偏好，表明 Tm5c 是这种行为的关键下游目标。此外，Tm5c 中红藻氨酸型 iGluR 的 RNAi 敲低，从而抑制其接收谷氨酸能 Dm8 输入的能力，显着降低了紫外线偏好。使用改进的 GRASP（跨突触伙伴的 GFP 重建）方法，该方法允许对真正的突触连接进行单细胞分析，我们证明 Dm8 接收 16 个 R7 输入，并为 Dm8 感受野中心的 1-2 个 Tm5c 神经元提供输入。因此，R7s->Dm8->Tm5c 连接构成了用于检测暗淡 UV 光的硬连线池电路。为了绘制将光感受器信号转换为颜色感知的电路，我们开发了一种新颖的厌恶操作性条件反射分析，用于与强度无关的颜色辨别。单只飞行的果蝇被磁性拴在一个被蓝色/绿色 LED 包围的区域中，然后这些果蝇会被调节以区分等亮度的蓝色或绿色刺激。可以在该范例中训练野生型果蝇以避免蓝色或绿色，而缺乏功能性 R7 和 R8 光感受器的突变体则不能，这表明颜色夹带需要窄谱光感受器 R7s 和/或 R8s 的功能。通过基因灭活四类一级中间神经元（Tm5a/b/c 和 Tm20），可以消除颜色学习。然而，使这些神经元子集失活不足以阻止颜色学习，这表明真正的色觉是由多个冗余通路介导的。习得的颜色辨别中明显的冗余与先天的光谱偏好形成鲜明对比，先天的光谱偏好由单一途径主导。使用类似的策略，我们绘制了果蝇的视觉运动路径。先前的研究表明，两种类型的小叶板神经元 T4 和 T5 发出小场方向选择性运动响应信号，并且分别位于一级神经元 L1 和 L2 或 ON 通道神经元和 OFF 通道神经元的下游。串行电镜重建显示，T5 接收来自四种类型的跨髓神经元 Tm1、Tm2、Tm4 和 Tm9 的直接突触输入。我们的转录谱分析和免疫组织化学显示，这些 Tm 神经元表达胆碱乙酰转移酶，因此可能具有胆碱能。此外，我们发现 T5 表达烟碱和毒蕈碱乙酰胆碱受体。 Tm2 和 Tm9 输入突触在 T5 树突轴上空间隔离，从而为基本运动检测器电路的两个臂提供候选解剖基质。基于突触回路和受体表达谱，我们假设 T5 通过使用烟碱和毒蕈碱胆碱感受器整合多个胆碱能 Tm 输入来计算小场运动信号。