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

• 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
• 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' 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

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）：胶质细胞在正常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。胶质细胞并非神经系统的中性粘合剂，而是异质的，在轴突寻路、突触形成和信号传递功效中活跃的参与者。当这些功能出现缺陷时，就会出现严重的情况。例如，星形胶质细胞与神经发育疾病如Rett综合征、脆性X染色体智力低下和癫痫发生有关。小胶质细胞参与神经系统疾病的发生和发展 包括阿尔茨海默病、帕金森病、多发性硬化症以及强迫症和精神分裂症。如果有更好的工具来监测和操纵神经胶质细胞的活动，那么了解这些疾病的原因将会更快。细胞内钙瞬变代表了这样方便的，可测量的信号 与细胞活动密切相关。在这里，我们建议实施最新一代的遗传编码钙指标（GECI）到小鼠报告，使用特定的基因敲入技术。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，该系统基于Dre，一种类似于Cre但具有独特DNA特异性的酪氨酸重组酶。这种替代基因标记技术的可用性 将使实验能够研究两个不同的细胞群之间的关系，如特定的神经元和神经胶质，而没有任何混淆的问题，染料负载效率，病毒注射等，因为所有需要的工具都可以通过繁殖聚集在一起。例如，这项技术将使刺激特定的神经元群体成为可能，并用钙指示剂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，使用光栅扫描模式下的超快双光子成像技术或使用称为靶向路径扫描的强大新方法来快速研究神经网络。所提出的工具将使我们能够研究颞叶癫痫小鼠模型中的反应性星形胶质细胞，并研究Hoxb8突变型小胶质细胞，这些小胶质细胞与小鼠的强迫症样行为有关。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重要作用。神经递质受体、电压门控离子通道、炎性细胞因子和多种其他蛋白质的表达响应于癫痫发作活动而发生显著变化。星形胶质细胞网络将在正常和癫痫脑切片中进行研究，为药物开发和测试建立实验框架。小胶质细胞由几个发育起源产生，包括Hoxb8表达结构域中的一个。小胶质细胞是神经元活动的传感器。我们将能够确定Hoxb8突变型小胶质细胞是否对神经刺激做出正确反应。总之，拟议的工作将推进一套可用于解决神经胶质和其他疾病的根本原因的工具。