Cre, Dre Dual Lineage Model for Calcium Imaging and Optogenetic Manipulations

用于钙成像和光遗传学操作的 Cre、Dre 双谱系模型

基本信息

批准号：
8657497
负责人：
Petr Tvrdik
金额：
$ 17.77万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8657497
关键词：
Acute Address Affect Alzheimer&apos s Disease Animal Model Astrocytes Behavior Binding Sites Biocompatible Materials Biological Neural Networks Brain Breeding Calcium Cells Complement DNA Detection Development Disease Dyes Emerging Technologies Environment Enzymes Epilepsy Epileptogenesis Fluorescence Resonance Energy Transfer Fostering Fragile X Syndrome Funding Opportunities Future Gated Ion Channel Generations Genes Genetic Genetic Recombination Glial Fibrillary Acidic Protein Glues Goals Health Human Image Imaging technology Individual Inflammatory Injection of therapeutic agent Institutes Interdisciplinary Study Internal Ribosome Entry Site Investigation Knock-in Mouse Label Life Measurable Mental Retardation Microglia Mission Modeling Monitor Multiple Sclerosis Mus National Institute of Drug Abuse National Institute of Mental Health National Institute of Neurological Disorders and Stroke National Institute on Deafness and Other Communication Disorders Nervous system structure Neuroglia Neurons Neurosciences Neurotransmitter Receptor Obsessive-Compulsive Disorder Optics Other Genetics Outcome Parkinson Disease Performance Physiology Play Population Preparation Procedures Proteins Psyche structure Reporter Research Research Personnel Rett Syndrome Rhodopsin Role Scanning Schizophrenia Seizures Series Signal Transduction Site Slice Specificity Stimulus System Tamoxifen Techniques Technology Temporal Lobe Epilepsy Testing Transgenic Organisms Tyrosine United States National Institutes of Health Universities Utah Viral Work animal model development axonal pathfinding base calcium indicator cell type cofactor combinatorial cytokine design drug development drug testing homologous recombination in vivo mouse genome mouse model mutant nervous system disorder neural stimulation neuropathology novel strategies novel therapeutic intervention optogenetics public health relevance recombinase relating to nervous system research study response sensor synaptogenesis tool transmission process two-photon voltage

项目摘要

DESCRIPTION (provided by applicant): Glia play many critical yet poorly understood roles in normal brain physiology, as well as in a wide range of neuropathologies. Far from being neutral glue of the nervous system, glia are heterogeneous, active players in axonal pathfinding, synapse formation and signal transmission efficacy. Serious conditions arise when these functions become defective. Astrocytes, for example, have been implicated in neurodevelopmental diseases such as Rett syndrome, fragile X mental retardation and in epileptogenesis. Microglia are involved in the initiation and progression of neurological disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, as well as obsessive compulsive disorder and schizophrenia. Progress in understanding the causes of these diseases would be much faster if better tools were available for monitoring and manipulating glial cell activity. Intracellular calcium transients represent such convenient, measurable signals that strongly correlate with cellular activity. Here we propose to implement the latest generation of genetically encoded calcium indicators (GECI) into mouse reporters, using specific gene knock-in techniques. We will also explore the potential of using an alternative site-specific recombination system in the mouse, based on Dre, a tyrosine recombinase similar to Cre but possessing a distinct DNA specificity. Availability of this alternative genetic labeling technology will enable experiments investigating the relationship between two distinct cell populations, such as specific neurons and glia, without any confounding issues of dye loading efficiency, viral injections, etc., because all the required tools can be brought together by breeding. This technology will make it possible to stimulate, for example, a specific population of neurons with channel rhodopsin and monitor surrounding glia with calcium indicators, and vice versa. We will evaluate these experiments in acute brain slices, with ultrafast two-photon imaging technology in a raster scanning mode or using a powerful new approach termed targeted path scanning for rapid investigation of neural networks. The proposed tools will allow us to study reactive astrocytes in mouse models of temporal lobe epilepsy and to investigate Hoxb8 mutant microglia, which have been implicated in obsessive compulsive-like behavior in mice. Temporal lobe epilepsy is the most common type of epilepsy in humans. Only recently, astrocytes have been shown to play an important role in the disease progress. Dramatic changes occur in the expression of neurotransmitter receptors, voltage gated ion channels, inflammatory cytokines, and a variety of other proteins in response to seizure activity. Astrocytic networks will be studie in normal and epileptic brain slices, establishing an experimental framework for drug development and testing. Microglia arise from several developmental origins, including one in the Hoxb8 expression domain. Microglia are sensors of neuronal activity. We will be able to determine if Hoxb8 mutant microglia respond to neural stimuli correctly. Taken together, the proposed work will advance the set of tools available to address the underlying causes of glial and other disorders.

描述（由申请人提供）：神经胶质在正常的脑生理学以及广泛的神经病理学中起着许多关键但知之甚少的作用。神经胶质不是神经系统的中性胶水，而是异质的，有活跃的参与者，在轴突路径上，突触形成和信号传递功效。当这些功能变得有缺陷时，就会出现严重的条件。例如，星形胶质细胞与神经发育疾病有关，例如RETT综合征，脆弱的X智力低下和癫痫发生。小胶质细胞参与神经系统疾病的启动和进展包括阿尔茨海默氏病，帕金森氏病，多发性硬化症以及强迫症和精神分裂症。如果可以使用更好的工具来监测和操纵神经胶质细胞活性，那么了解这些疾病的原因的进展将更快。细胞内钙瞬变代表如此方便的可测量信号这与细胞活性密切相关。在这里，我们建议使用特定的基因敲入技术将最新一代的遗传编码钙指标（GECI）实施到小鼠记者中。我们还将探索在小鼠中使用替代位点特异性重组系统的潜力，基于DRE，DRE是一种类似于CRE的酪氨酸重组酶，但具有独特的DNA特异性。这种替代遗传标签技术的可用性将实现研究两个不同细胞群体（例如特定神经元和神经胶质）之间关系的实验，而没有任何混淆染料负荷效率，病毒注射等问题，因为所有必需的工具都可以通过育种来聚集在一起。这项技术将使刺激具有通道视紫红质的特定神经元的特定群体，并用钙指示器监测周围的神经胶质，反之亦然。我们将在急性脑切片中评估这些实验，并在栅格扫描模式下使用超快的两光量成像技术，或使用一种强大的新方法，称为靶向路径扫描，以快速研究神经网络。所提出的工具将使我们能够研究颞叶癫痫的小鼠模型中的反应性星形胶质细胞，并研究HOXB8突变小胶质细胞，这与小鼠中的强迫性行为有关。颞叶癫痫是人类最常见的癫痫类型。直到最近，星形胶质细胞才被证明在疾病进展中起着重要作用。在神经递质受体，电压门控通道，炎症细胞因子和多种其他蛋白质的表达中发生了巨大变化，这是对癫痫发作活性的响应。星形胶质细胞网络将在正常和癫痫脑切片中进行研究，从而建立一个用于药物开发和测试的实验框架。小胶质细胞来自几个发育起源，其中包括HOXB8表达域中的一个。小胶质细胞是神经元活性的传感器。我们将能够确定HOXB8突变小胶质细胞是否正确反应神经刺激。综上所述，拟议的工作将推进可用的工具集，以解决神经胶质和其他疾病的根本原因。