Optogenetic Mapping of Adult Newborn Neuron Projections

 描述(申请人提供)：在美国，超过200万人经历过无故癫痫发作或被诊断为癫痫。在大约25%的病例中，癫痫发作对药物治疗是无效的。无法有效地治疗癫痫反映了对这种疾病的基本机制缺乏了解。高达50%的创伤性脑损伤幸存者会患上癫痫。创伤后癫痫(PTE)与海马神经元的改变有关，包括细胞丢失和反应可塑性。在哺乳动物的大脑中，在整个成年期的几个关键脑区不断产生新的神经元。这一过程被称为成人神经发生，代表了一种依赖经验的可塑性，被认为支持正常的大脑功能。包括创伤性麸皮损伤、癫痫发作和中风在内的脑损伤与海马成年神经发生的增加有关，成年出生的神经元在海马回路中的异常整合可能为导致癫痫发作的过度兴奋回路提供了底物。癫痫与成人出生的齿状颗粒细胞(DGC)的出现有关，这些细胞显示异常的树突区和轴突，可能会投射到意想不到的靶点。在实验性癫痫中，成人出生的DGC与自发性癫痫发作有关，在PTE动物模型中，阻断成人神经发生减少了自发性癫痫发作的表达。尽管有这些报道，但所用技术的固有局限性阻碍了对成年出生的神经元与其突触目标形成的功能性细胞连接的表征。这项建议旨在开发一种新的技术来选择性地标记和刺激成年大脑中的新生神经元，然后使用这项技术来评估PTE小鼠模型中成年出生的海马神经元的功能输出。1)利用Nestin-Cre小鼠，基于他莫昔芬诱导的Nestin表达，选择性地靶向在成年出生的祖细胞中表达通道视紫红质(ChR2)。Nestin-CRE小鼠将被注射Cre诱导的腺相关病毒(DIO-AAV)，其中含有通道视紫红质(ChR2)和荧光报告mCherry(缩写：ChR2-mCherry；构建：pAAV-Ef1a-DIO-hChR2(H134R)-mCherry-WPRE-pa)；2)使用蓝光刺激参数激活成年出生的神经元，并向其突触后靶标发出信号。将对接受ChR2-mCherry构建的Nestin-CRE小鼠海马片中的DGC进行全细胞膜片钳记录，以描述成年出生的神经元在脑损伤后形成的功能投影。更好地理解成年出生的神经元如何在正常和PTE状态下合并到神经网络和信号中，将有助于确定它们作为治疗靶点的相关性，也将为评估已记录对成年出生细胞的影响的临床可用药物提供新的背景。

 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.