Optogenetic Mapping of Adult Newborn Neuron Projections

成人新生儿神经元投影的光遗传学图谱

• 批准号: 8890528
• 负责人: Bret N Smith
• 金额: $ 22.54万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8890528
• 关键词: Abbreviations; Adult; Animal Model; Axon; Behavior assessment; Biological Neural Networks; Brain; Brain Injuries; Brain region; Cells; Dependovirus; Development; Diagnosis; Disease; Employee Strikes; Epilepsy; Epileptogenesis; Evaluation; Experimental Models; Frequencies; Generations; Hilar; Hippocampus (Brain); Injection of therapeutic agent; Injury; Intermediate Filament Proteins; Label; Laboratories; Light; Maps; Measures; Medical; Methodology; Methods; Mus; Neuronal Plasticity; Neurons; Newborn Infant; Outcome Measure; Output; Pharmaceutical Preparations; Population; Process; Property; Refractory; Reporting; Role; Seizures; Services; Severities; Signal Transduction; Slice; Stem cells; Stimulus; Stroke; Survivors; Synapses; Tamoxifen; Techniques; Temporal Lobe Epilepsy; Testing; Transgenic Organisms; Traumatic Brain Injury; United States; Work; adult neurogenesis; base; calretinin; cell preparation; controlled cortical impact; experience; fundamental research; gene therapy; granule cell; improved; in vivo; injured; mossy fiber; mouse model; nerve stem cell; nestin protein; neural circuit; neurogenesis; neurotransmission; newborn neuron; optogenetics; patch clamp; postsynaptic; prevent; public health relevance; recombinase; response; selective expression; therapeutic target; vector; voltage

 DESCRIPTION (provided by applicant): Over 2 million people in the United States have experienced unprovoked seizures or been diagnosed with epilepsy. In approximately 25% of cases, seizures are refractory to medical therapies. Inability to effectively treat epilepsy reflecs a lack of understanding of the basic mechanisms of this disorder. Up to 50% of traumatic brain injury survivors develop epilepsy. Posttraumatic epilepsy (PTE) is associated with alterations in hippocampal circuits including cell loss and reactive plasticity. In the mammalian brain, there is continual generation of new neurons in a few key brain regions throughout adulthood. This process, referred to as adult neurogenesis, represents a form of experience-dependent plasticity that is believed to support normal brain function. Brain insults including traumatic bran injury, seizures, and stroke are associated with increases in hippocampal adult neurogenesis, and abnormal integration of adult-born neurons within hippocampal circuitry may provide a substrate for hyperexcitable circuits that contribute to seizures. Epilepsy is associated with the emergence of adult-born dentate granule cells (DGCs) that display abnormal dendritic fields and axons that may project to unexpected targets. Adult-born DGCs are associated with spontaneous seizures in experimental epilepsy and blockade of adult neurogenesis reduces spontaneous seizure expression in an animal model of PTE. Despite these reports, inherent limitations of the techniques used have prevented the characterization of functional cellular connections formed by adult-born neurons with their synaptic targets. This proposal aims to develop a new technique to selectively label and stimulate newly-born neurons in the adult brain and then use this technique to assess functional outputs of hippocampal adult-born neurons in a mouse model of PTE. Work here aims to: 1) Selectively target expression of channelrhodopsin (ChR2) in adult-born progenitor cells based on their tamoxifen-inducible expression of nestin using Nestin-Cre mice. Nestin-Cre mice will be administered with a Cre-inducible adeno-associated virus (DIO-AAV) with double- floxed reverse cassettes containing channelrhodopsin (ChR2) and the fluorescent report mCherry (abbreviation: ChR2-mCherry; construct: pAAV-Ef1a-DIO-hChR2(H134R)-mCherry-WPRE-pA); and 2) Use blue-light stimulation parameters to activate adult-born neurons and drive signaling to their postsynaptic targets. Whole-cell patch-clamp recordings will be performed on DGCs in hippocampal slices from Nestin-Cre mice that have received injections of the ChR2-mCherry construct to describe the functional projections formed by adult-born neurons after brain injury. Improved understanding of how adult-born neurons incorporate into neural networks and signal during normal and PTE states will help define their relevance as therapeutic targets and will also provide new context for evaluation of clinically available drugs that have documented effects on adult-born cells.

 描述（由适用提供）：美国超过200万人经历了无端的癫痫发作或被诊断出患有癫痫病。在大约25％的病例中，癫痫发作是对医疗疗法的难治性。无法有效地治疗癫痫病，缺乏对这种疾病的基本机制的理解。高达50％的创伤性脑损伤存活发生癫痫。创伤后癫痫（PTE）与海马电路的改变有关，包括细胞损失和反应性可塑性。在哺乳动物的大脑中，整个成年期的一些关键大脑区域都有连续产生的新神经元。该过程被称为成人神经发生，代表了一种依赖经验的可塑性形式，据信支持正常的大脑功能。脑损伤包括创伤性麸皮损伤，癫痫发作和中风与海马成人神经发生的增加有关，以及海马电路中成年神经元的异常整合，可能为过度刺激的电路提供了有助于癫痫发作的过度兴奋的电路。癫痫与成年出生的牙齿颗粒细胞（DGC）的出现有关，这些颗粒细胞（DGC）显示出异常的树突状田地和轴突，可能会投射出意外的靶标。成人出生的DGC与实验性癫痫发作的癫痫发作有关，成人神经发生的盖鼠会降低PTE动物模型中的赞助性癫痫发作表达。尽管有这些报道，但所使用的技术的继承局限性阻止了成人出生神经元与其突触靶的功能性细胞连接的表征。该建议旨在开发一种新技术，以选择性地标记和刺激成人大脑中新生的神经元，然后使用该技术在PTE小鼠模型中评估海马成人出生神经元的功能输出。此处的工作目的是：1）基于使用Nestin-Cre小鼠的他莫昔芬诱导的表达，选择性地靶向成人祖细胞中通道旋转（CHR2）的表达。 Nestin-Cre小鼠将使用CRE诱导的腺相关病毒（Dio-AAV）和含有通道旋hodopsin（CHR2）的双flox反向盒和荧光报告MCHERRY（CHR2-MCHERRY：CHR2-MCHERRY; CHR2-MCHERRY; paav-ef1a-dio-dio-hchr2（H134R）; 2）使用蓝光刺激参数激活成人出生的神经元并向其突触后靶标发出信号。将对来自Nestin-Cre小鼠的海马切片中的DGC进行全细胞贴片钳记录，这些DGC已接受了CHR2-MCHERRY构建体的注射，以描述脑损伤后成人出生的神经元形成的功能性项目。对正常和PTE状态期间成年神经元如何纳入神经元的成年神经元如何纳入神经元和信号的了解将有助于定义其作为治疗靶标的相关性，还将为评估对成人出生细胞的临床可用药物的评估提供新的背景。