New approaches for chemical-genetic targeting of specific circuits and cell types in the mammalian brain

哺乳动物大脑中特定回路和细胞类型的化学遗传靶向新方法

基本信息

批准号：
10012597
负责人：
Anton Maximov
金额：
$ 266.69万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10012597
关键词：
Acute Adverse effects Affinity Alleles Animal Model Animals Antibiotics Behavior Behavioral Benchmarking Binding Blood - brain barrier anatomy Brain Brain Diseases Brain imaging CRISPR/Cas technology Cells Chemical Structure Color Communities Complex Cues DNA Sequence Data ERG gene Early Promoters Electron Microscopy Electrophysiology (science)Elements Environment Exocytosis Future Genetic Genetic Recombination Genetic Techniques Genomic DNA Genomics Goals Hour Image Kinetics Label Mammals Mediating Membrane Proteins Memory Methods Molecular Profiling Morphology Mus Neurons Neuropeptides Neurosciences Neurotransmitters Optics Output Pharmaceutical Preparations Pharmacology Physiological Population Property Proteins Publishing Recording of previous events Regulation Reporter Research Route SNAP receptor Sensory Side Structure Synapses System Tamoxifen Techniques Technology Tertiary Protein Structure Tetracycline Control Time Transgenic Mice Trimethoprim Viral Vector Work cDNA Expression cell type chemical genetics cognitive task experience experimental study genetic manipulation genome editing in vivo innovation interest mouse model nervous system disorder neural circuit novel strategies optical imaging optogenetics protein function recombinase reconstruction recruit relating to nervous system response sensory input side effect small molecule tool vesicle-associated membrane protein vesicular release viral DNA virtual

项目摘要

The goal of this research is to establish new robust methods for manipulation of specific circuits and genetically defined neuron types in brains of model organisms with small molecules. While several chemical-genetic techniques are already available, these techniques have drawbacks that limit their utility. Our recent work demonstrates that these obstacles can be overcome by using a strategy for acute control of function of proteins of interest (POI) containing destabilizing domains (DD) with the inexpensive commercially available drug, TMP. This compound efficiently crosses the blood-brain barrier, stabilizes DD-POIs with a rapid time course, and does not produce undesired side effects by itself. Since DD tags can be attached to virtually any protein, TMP-inducible stabilization is applicable to a broad spectrum of experimental paradigms. We propose to generate mouse alleles and viral vectors encoding DD-POI fusions suitable for applications ranging from ultra-structural imaging to assessment of complex behaviors. We will develop tools for TMP-dependent recombination of DNA and genome editing (Aim1), acute labeling of behaviorally relevant neuron populations with a reporter compatible with optical imaging and electron microscopy (Aim2), and cell-type-specific control of neurotransmitter secretion (Aim3). Our collaborative team will demonstrate the advantages of these techniques by combining genomics, whole brain imaging, serial electron microscopy, electrophysiology, optogenetics and behavior. New tools will be compared side-by-side against existing technologies, and then distributed to the neuroscience community. We anticipate that these efforts will significantly benefit future studies of the normal brain and mechanisms underlying neurological diseases.

这项研究的目标是建立新的稳健方法来操纵特定电路和基因用小分子定义了模型生物体大脑中的神经元类型。虽然有一些化学遗传虽然技术已经可用，但这些技术的缺点限制了它们的实用性。我们最近的工作表明可以通过使用精确控制蛋白质功能的策略来克服这些障碍含有不稳定结构域（DD）的感兴趣的（POI）与廉价的市售药物 TMP。该化合物可有效穿过血脑屏障，快速稳定 DD-POIs，并且本身不会产生不良副作用。由于 DD 标签几乎可以附着在任何蛋白质上，因此 TMP 诱导稳定性适用于广泛的实验范式。我们建议生成小鼠等位基因和编码 DD-POI 融合的病毒载体，适用于从超结构成像到复杂行为的评估。我们将开发用于 DNA 和基因组的 TMP 依赖性重组的工具编辑（Aim1），使用与光学兼容的报告器对行为相关的神经元群体进行急性标记成像和电子显微镜（Aim2），以及神经递质分泌的细胞类型特异性控制（Aim3）。我们的合作团队将通过结合基因组学、全脑技术来展示这些技术的优势成像、系列电子显微镜、电生理学、光遗传学和行为。将比较新工具与现有技术并列，然后分发给神经科学界。我们预计这些努力将极大地有益于未来对正常大脑及其机制的研究神经系统疾病。