Molecular and Genetic Studies of TMEM16C Control of Thermoregulation and Neuronal Excitability

我们令人惊讶地发现，TMEM16C功能的丧失导致了大多数 视前区温度升高可使POA神经元的放电频率增加 下丘脑(POA)--处于体温调节指挥链顶端的温敏性神经元。至 TMEM16C参与体温调节和热性惊厥的测试，我们检测了TMEM16C 条件性基因敲除(CKO)小鼠，并发现携带TMEM16C的突变幼鼠从其中枢 神经元不能维持体温，容易发生高温诱发的癫痫。至 为了寻找温度敏感的POA神经元的分子标记，我们进行了68个单细胞RNAseq 脑片中记录的POA神经元受到温度变化的影响，以及 验证了温度敏感型POA神经元的分子和遗传标记。通过产生CKO小鼠 通过从温度敏感的POA神经元中去除TMEM16C，我们的目标是测试TMEM16C的功能 在温敏神经元、体温调节和高温诱发癫痫的规范中。 考虑到TMEM16C与发热性癫痫之间的联系，以及我们发现没有TMEM16C的啮齿动物幼崽 神经元TMEM16C易出现高温致痫，TMEM16C CKO小鼠提供动物 发热性癫痫的模型，影响2-8%的幼儿。因为复杂的发热性癫痫发作 与海马区硬化症相关的致痫病理，我们将检验以下假设 TMEM16C通过记录动作电位和突触电位调节海马神经元兴奋性 在TMEM16C去除的小鼠海马神经元中，通过在ALL中表达的Nestin-Cre 或通过靶向约50%的海马锥体神经元的Drd3-Cre。我们还将录制来自 TMEM16C CKO小鼠和同胞对照中温度敏感的POA神经元 调节温度敏感的POA神经元的活动。我们将进一步检验这一假设 来自中枢神经元的TMEM16C引起这些神经元钠激活钾电流的改变。 最后，我们将采用最近开发的分离绿色荧光蛋白方法，涉及使用质谱学来 识别特定神经元类型中与TMEM16C相关的蛋白质。

We made the surprise finding that loss of TMEM16C function led to the elimination of the majority of POA neurons that increase their firing rate with rising temperature of the preoptic area of the anterior hypothalamus (POA) – warm-sensitive neurons that are on top of a command chain for thermoregulation. To test for the involvement of TMEM16C in thermoregulation and febrile seizure, we examined TMEM16C conditional knockout (cKO) mice and found that mutant pups with TMEM16C removed from their central neurons cannot maintain their body temperature and are prone to develop hyperthermia-induced seizure. To look for molecular markers of temperature sensitive POA neurons, we conducted single-cell RNAseq of 68 POA neurons following recording from these neurons in brain slices subjected to temperature changes, and validated a molecular and genetic marker for temperature sensitive POA neurons. By generating cKO mice with TMEM16C removed from temperature sensitive POA neurons, we aim to test the function of TMEM16C in the specification of warm-sensitive neurons, thermoregulation, and hyperthermia-induced seizure. Given the GWAS association of TMEM16C with febrile seizure and our finding that rodent pups without neuronal TMEM16C are prone to exhibit hyperthermia-induced seizures, TMEM16C cKO mice provide animal models for febrile seizure, which affects 2-8% of young children. Because complex febrile seizures are associated with hippocampal sclerosis as the epileptogenic pathology, we will test the hypothesis that TMEM16C regulates hippocampal neuronal excitability by recording action potentials and synaptic potentials in hippocampal neurons from mice with TMEM16C removed either via nestin-Cre that is expressed in all neurons or via Drd3-Cre that targets ~50% of hippocampal pyramidal neurons. We will also record from temperature sensitive POA neurons in TMEM16C cKO mice and sibling controls to determine how TMEM16C modulates temperature sensitive POA neuronal activity. We will further test the hypothesis that removing TMEM16C from central neurons causes alteration of sodium-activated potassium current in these neurons. Finally, we will adopt the recently developed split GFP approach involving the use of mass spectrometry to identify proteins that are associated with TMEM16C in specific neuronal types.