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.

这项研究的目的是建立新的强大方法来操纵特定电路和遗传学具有小分子的模型生物的大脑中定义的神经元类型。而几种化学遗传学技术已经可用，这些技术具有限制其效用的缺点。我们最近的工作证明可以通过使用急性控制蛋白质功能的策略来克服这些障碍感兴趣的（POI），其中包含廉价的市售药物TMP的不稳定域（DD）。这种复合有效地越过血脑屏障，通过快速的时间过程稳定DD-Pois，并且确实可以不会单独产生不希望的副作用。由于DD标签几乎可以连接到任何蛋白质，TMP诱导稳定适用于广泛的实验范例。我们建议生成鼠标等位基因和编码DD-POI融合的病毒向量，适用于从超结构成像到评估复杂行为。我们将开发用于DNA和基因组TMP依赖性重组的工具编辑（AIM1），具有与光学兼容的记者的行为相关神经元种群的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）。我们的协作团队将通过结合基因组学，整个大脑来证明这些技术的优势成像，串行电子显微镜，电生理学，光遗传学和行为。将比较新工具并排反对现有技术，然后分发给神经科学界。我们期待这些努力将极大地有益于对正常大脑和基本机制的未来研究神经疾病。