Effects of TrkB Activation on Abnormalities in Neocortical FS interneuron

据报道，小清蛋白 (PV) 和生长抑素 (SOM) 中间神经元的异常在许多 神经系统疾病，包括癫痫。改善有缺陷的中间神经元功能的疗法并不是 可用。中间神经元的结构发育和维持取决于提供的营养支持 通过脑源性神经营养因子 (BDNF) 激活 TrkB 受体。在底切 (UC) 模型中 致癫痫性新皮质损伤，选择性小分子部分激动剂慢性激活 TrkB-Rs （LM22A-4，下文“LM”）具有逆转抑制性结构和功能异常的长期作用 PV中间神经元末端，增强GABA释放并提高诱发癫痫样的阈值 活动和癫痫发作。为了确定这些影响是否适用于治疗或预防 具有不同癫痫发作原因的其他模型中的癫痫，例如遗传性癫痫、Dravet 综合征 (DS) 小鼠将用于一些实验。 PV 中间神经元膜钠通道减少 DS 小鼠中，导致抑制性递质 GABA 的释放减少，并产生自发性 以及高温引起的癫痫发作。将在 DS 和 UC 小鼠中使用多种实验方法 通过增加 SOM 神经末梢的 GABA 释放来确定 LM 是否可以长期治疗 和 PV 中间神经元，或诱导新的抑制性突触形成，将增强皮质网络的抑制 并抑制癫痫样放电：1）免疫细胞化学和共聚焦成像将用于评估 PV 和 SOM 突触前末梢的变化，包括 VGAT- 和 GAD65/67- 表达的变化 IR，和钙传感器蛋白突触结合蛋白2； 2) SOM/-和PV/gephyrin的密度分析接近 用于测试 TrkB 激活诱导的新抑制性突触形成的并置； 3）电生理学 从PV中间神经元到锥体神经元的抑制性突触传递的基本特性分析 体外切片检测 TrkB 激活对单一 IPSC 的影响、释放概率和传输失败； 3) 对 PV/CHR2 和 SOM/CHR2 小鼠的皮质切片进行激光扫描光刺激，以绘制 新皮质抑制回路中抑制连接的分布和强度； 4) 视频/脑电图监测 植入小鼠以评估 LM 治疗对自发性癫痫发作和高热诱发的效果 癫痫发作。这些实验的结果将提供有关由中间神经元引起的机制的信息 癫痫发展的异常和预防癫痫发生的潜在方法 增强中间神经元的营养支持。

Abnormalities in parvalbumin (PV) and somatostatin (SOM) interneurons are reported in a number of neurological disorders, including epilepsy. Therapy that improves function of defective interneurons is not available. Structural development and maintenance of interneurons is dependent on trophic support provided by brain derived neurotrophic factor (BDNF) activation of TrkB receptors. In the undercut (UC) model of epileptogenic neocortical injury, chronic activation of TrkB-Rs with a selective small molecule partial agonist (LM22A-4, “LM” below) has long-term effects to reverse structural and functional abnormalities in inhibitory terminals of PV interneurons, enhance GABA release and increase the threshold for evoking epileptiform activity and seizures. In order to determine whether these effects will be applicable to treatment or prevention of epilepsy in other models with different causes for seizures, such as genetic epilepsies, the Dravet syndrome (DS) mouse will be used in some experiments. Decreases in a membrane sodium channel in PV interneurons in DS mice causes decreased release of the inhibitory transmitter GABA, and development of spontaneous and high temperature-induced seizures. A variety of experimental approaches in DS and UC mice will be used to determine whether chronic treatment with LM, by increasing GABA release from nerve terminals of SOM and PV interneurons, or inducing new inhibitory synapse formation, will enhance inhibition in cortical networks and suppress epileptiform discharges: 1) immunocytochemistry and confocal imaging will be used to assess alterations in PV and SOM presynaptic terminals, including changes in expression of VGAT- and GAD65/67- IR, and the calcium sensor protein synaptotagmin 2; 2) analysis of density of SOM/- and PV/gephyrin close appositions to test for new inhibitory synapse formation induced by TrkB activation; 3) electrophysiological analysis of basic properties of inhibitory synaptic transmission from PV interneurons to pyramidal neurons of in vitro slices to detect effects of TrkB activation on unitary IPSCs, release probability and transmission failures; 3) laser scanning photostimulation of cortical slices from PV/CHR2 and SOM/CHR2 mice to map the distribution and strength of inhibitory connectivity in neocortical inhibitory circuits; and 4) video/EEG monitoring of implanted mice to assess effects of treatment with LM on spontaneous seizures and hyperthermia-induced seizures. Results of these experiments will provide information about mechanisms leading from interneuronal abnormalities to development of epilepsy and a potential approach to prophylaxis of epileptogenesis by enhancing trophic support of interneurons.