Pre- and Post-Synaptic Players in Target Specific mRGC Circuits

目标特定 mRGC 电路中的突触前和突触后参与者

• 批准号: 9052073
• 负责人: Yu Hsin Liu
• 金额: $ 4.68万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-16 至 2019-04-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9052073
• 关键词: Address; Affect; Area; Attention; Axon; Back; Behavior; Behavioral; Birds; Brain; Brain region; Cell Communication; Cell Nucleus; Cells; Cellular Morphology; Chronic; Circadian Rhythms; Cognitive deficits; Color; Data Set; Dendrites; Dependovirus; Depressed mood; Development; Diagnosis; Disease; Electrophysiology (science); Environment; Eye; Eye diseases; Fiber; Fluorescence-Activated Cell Sorting; Functional disorder; Future; Gene Expression; Genes; Genetic Transcription; Glycoproteins; Immunohistochemistry; Infection; Injection of therapeutic agent; Inner Plexiform Layer; Insulin; Jet Lag Syndrome; Label; Lead; Light; Location; Mammalian Cell; Maps; Mediating; Mental disorders; Metabolic Diseases; Metabolism; Molecular; Molecular Profiling; Mood Disorders; Moods; Morphology; Mus; Neurodegenerative Disorders; Pathway interactions; Patients; Phase; Physiological; Physiology; Play; Population; Presynaptic Terminals; Process; Proteins; Pupil light reflex; RNA; Rabies virus; Recombinants; Research; Retina; Retinal; Retinal Ganglion Cells; Role; Site; Sleep; Sorting - Cell Movement; Specificity; Stratification; Synapses; System; Target Populations; Techniques; Testing; Transgenic Mice; Unconscious State; Visual; base; cell type; effective therapy; experience; intravitreal injection; melanopsin; molecular marker; mouse model; neural circuit; neuronal circuitry; next generation sequencing; novel strategies; promoter; protein biomarkers; protein expression; public health relevance; rabies virus glycoprotein G; receptor; recombinase; research study; response; suprachiasmatic nucleus; targeted treatment; therapy development; transcriptome sequencing; visual process; visual processing

 DESCRIPTION (provided by applicant): A growing body of research is showing that chronic circadian rhythm disruption, as experienced by rotational shift workers and patients suffering from sleep phase disorders, neurodegenerative diseases, and psychiatric disorders, can lead to behavioral and physiological problems such as insulin insensitivity, depressed or agitated mood, impulse control issues, and cognitive deficits. To regulate circadian rhythms, light information reaches the brain via efferent projections of intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs) to the suprachiasmatic nucleus (SCN). In order to develop effective treatments for circadian rhythm disruption, it is imperative to understand and separate out the specific pathways through which light information reaches the SCN from other mRGC mediated effects, such as the pupillary light reflex which is mediated by mRGC projections to the olivary pretectal nucleus (OPN). This proposal aims to identify distinct retinal cell- mRGC-brain region circuits for different non-image forming (NIF) visual responses and molecular players that underlie those circuits. In order to identify mRGCs that specifically project to SCN o to OPN, TVA, an avian receptor not found in mammalian cells, will be expressed in the mRGCs of Opn4Cre (cre-recombinase driven by melanopsin promoter) mice via intravitreal injections of cre-dependent AAV. Stereotaxic injections of TVA dependent red glycoprotein-deleted rabies virus (EnvA-SADΔG-mCherry) into the SCN and blue glycoprotein- deleted rabies virus (EnvA-SADΔG-BFP) into the OPN will allow SADΔG to be taken up in mRGC axon terminals ending at the injection site. Glycoprotein deleted rabies virus is unable to spread transsynaptically but continues to replicate within the infected cells- effectively filling all processes with fluorescen protein and allowing for identification of mRGCs that specifically project to SCN (will express mCherry), those that specifically project to OPN (will express BFP), and subtypes of mRGCs. In a separate experiment, rabies virus glycoprotein (RV-G) will be targeted to mRGCs. Infection of RV-G-expressing mRGCs with SADΔG will allow the fluorescent rabies virus to spread one synapse in the retrograde direction. This will allow SCN or OPN specific mRGCs to label upstream retinal cells with the same colored SADΔG and may reveal exclusive retinal cell-mRGC-brain region di-synaptic circuits. Lastly, next generation sequencing will be applied to mRGC populations that are fluorescently labeled based on their central target projections to generate gene transcription profiles of target- specific mRGC populations. Comparisons between gene expression profiles of different target-defined mRGC populations will reveal genes that are preferentially expressed in mRGC populations that specifically project to SCN or OPN. All together, these experiments will identify brain region-specific mRGC projections and retinal cell inputs, uncover molecular players that underlie target-specific mRGC projections, and inform about potential targets for treating circadian rhythm disruption.

 描述（由适用提供）：越来越多的研究表明，慢性昼夜节律中断，旋转转移工人和患有睡眠期疾病，神经退行性疾病和精神疾病的患者所经历的，可以导致行为和身体问题，例如胰岛素性和胰岛素内抑制，抑制或搅动的情绪，情绪抑制或抗击，并施加了侵害性，并认识到了胰岛素内的疾病和抑制作用。为了调节昼夜节律，光信息通过有效地投射出表达黑素神经节细胞（MRGC）到肩部核核（SCN）的有效投射到大脑。为了开发有效的昼夜节律中断治疗方法，必须理解并分离光信息与其他MRGC介导的效果达到SCN的特定途径，例如由MRGC项目介导的瞳孔光反射，该途径是由MRGC项目介导的，该途径是由Olivary Protective Nucus（OPN）介导的。该建议旨在确定不同的非图像形成（NIF）视觉反应和分子参与者的不同视网膜细胞 - MRGC-脑区域电路，这些反应和分子参与者是这些电路的基础。为了确定专门向SCN O投射到OPN的MRGC，TVA（在哺乳动物细胞中找不到的鸟类受体TVA将通过OPN4CRE的MRGC（由黑色素蛋白启动子驱动的Cre-recombinase）在MRGC中表达，这是通过cre依赖依赖AAV的cretreal注射。对TVA依赖的红色糖蛋白缺失的狂犬病病毒（Enva-SADΔG-MCHERRY）的立体触及注射到SCN中，而蓝色糖蛋白骨蛋白缺失的狂犬病病毒（Enva-SADΔG-BFP）（ENVA-SADΔGBFP）将允许在MRGC Axon terminals在Endecection the Endecection的MRGC Axon终末中吸收SADΔG。糖蛋白删除的狂犬病病毒无法透射传播，但继续在受感染的细胞中复制，有效地用荧光蛋白填充所有过程，并允许识别特殊投射到SCN的MRGC（Will Express MCHERRY）（Will Express MCHERRY），专门向OPN投射了OPN（将表达OPN（将表达BFP）和MRGCSS和Supgcs的opn。在单独的实验中，狂犬病病毒糖蛋白（RV-G）将针对MRGC。用SADΔG感染表达RV-G的MRGC将使荧光狂犬病病毒在逆行方向上传播一个突触。这将允许SCN或OPN特定的MRGC标记具有相同有色SADΔG的视网膜细胞，并可能显示出独家残留的残留细胞MRGC-脑区域DI突触电路。最后，下一代测序将应用于MRGC种群，这些测序是根据其中心目标项目标记的荧光标记，以生成目标特异性MRGC种群的基因转录谱。不同靶标MRGC种群的基因表达谱之间的比较将揭示在MRGC种群中优选表达的基因，这些基因在MRGC种群中特异性表达，这些基因专门投射到SCN或OPN。这些实验将共同确定大脑区域特异性的MRGC项目和残留细胞输入，发现基于特定目标的MRGC项目的分子参与者，并告知治疗昼夜节律中断的潜在目标。