Neuronal and non-neuronal excitatory transmission in the hypothalamus

下丘脑的神经元和非神经元兴奋性传递

• 批准号: RGPIN-2020-04269
• 负责人: Hirasawa, Michiru
• 金额: $ 3.06万
• 依托单位: Memorial University of Newfoundland
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716361
• 关键词: Neuronal; non; neuronal; excitatory; transmission

Chemical transmission is one of the most fundamental processes of the nervous system, which depends on the release, diffusion and clearance of neuroactive substances detected by various receptors. Particularly, neuron-to-neuron communication mediated by glutamate, the primary excitatory neurotransmitter in the CNS, is critical for information flow within neuronal networks. In addition, recent research has established that astrocytes can play multiple roles in glutamatergic transmission. Astrocytes sense, uptake and release glutamate and hence regulate the extracellular glutamate levels, which can underlie basal or tonic excitability in some neurons. The distinct time course of neuronal and astrocytic glutamatergic transmission suggests different mechanisms, regulatory processes and functional roles. However, these mechanisms remain obscure. Neuropeptide release from peptidergic neurons typically require prolonged firing activity, thus sustained elevation in excitability supported by astrocytes may have significant functional consequences. Melanin-concentrating hormone (MCH) neurons are peptidergic neurons in the hypothalamus critical for the control of energy balance, sleep-wake cycle and motivated behaviors. Our preliminary study indicates that MCH neurons represent a unique model to study neuronal and non-neuronal chemical transmission as they display several glutamatergic currents with distinct time courses: fast EPSC, slow EPSC and tonic inward current. Fast and slow EPSCs are triggered by synaptic stimulation while tonic currents are induced by glutamate transport inhibitors and are due to ambient glutamate that appear to be astrocytic in origin (gliotransmission). Furthermore, these currents are modulated/mediated by different glutamate transporters and receptors. Based on these results, we will test the hypothesis that depending on the source (presynaptic neuron or astrocyte), extracellular glutamate is regulated by distinct glutamate transporters and detected by different pools of receptors on MCH neurons. SHORT-TERM AIMS 1) Determine the mechanism of fast and slow EPSCs in MCH neurons 2) Identify the source of ambient glutamate and its mechanism of release 3) Determine the role of EAAC1 (neuronal glutamate transporter) and NMDA receptors in ambient glutamate action EPSCs and tonic currents will be recorded in mice brain slices using whole cell patch clamp, while extracellular glutamate levels will be assessed by genetically encoded glutamate sensors iGluSnFR expressed on MCH neurons. Subcellular localization of receptors and transporters will be investigated by electrophysiology and immunofluorescence microscopy combined with super-resolution radial fluctuation analysis. Proposed studies will be significant steps towards my long-term goal to establish a better understanding of neuronal and non-neuronal chemical transmission in the central nervous system.

化学传递是神经系统最基本的过程之一，它依赖于各种受体检测到的神经活性物质的释放、扩散和清除。特别是，由谷氨酸（CNS中的主要兴奋性神经递质）介导的神经元与神经元的通信对于神经元网络内的信息流至关重要。此外，最近的研究已经确定，星形胶质细胞可以发挥多种作用，在神经递质传递。星形胶质细胞感受、摄取和释放谷氨酸，从而调节细胞外谷氨酸水平，这可能是某些神经元基础或紧张兴奋性的基础。神经元和星形胶质细胞间质能传递的不同时间过程表明不同的机制、调节过程和功能作用。然而，这些机制仍然模糊不清。 从肽能神经元释放神经肽通常需要延长的放电活动，因此星形胶质细胞支持的兴奋性持续升高可能具有显著的功能后果。黑色素浓集激素（MCH）神经元是下丘脑中的肽能神经元，对能量平衡、睡眠-觉醒周期和动机行为的控制至关重要。我们的初步研究表明，MCH神经元代表了一个独特的模型来研究神经元和非神经元的化学传递，因为它们显示了几个具有不同时程的兴奋性电流：快EPSC，慢EPSC和紧张性内向电流。快和慢EPSC由突触刺激触发，而紧张性电流由谷氨酸转运抑制剂诱导，并且是由于似乎是星形胶质细胞起源的环境谷氨酸（胶质传递）。此外，这些电流由不同的谷氨酸转运蛋白和受体调节/介导。 基于这些结果，我们将测试的假设，根据不同的来源（突触前神经元或星形胶质细胞），细胞外谷氨酸调节不同的谷氨酸转运蛋白和检测MCH神经元上的不同池的受体。 短期目标 1)确定MCH神经元中快和慢EPSC的机制 2)确定环境谷氨酸的来源及其释放机制 3)确定EAAC 1（神经元谷氨酸转运蛋白）和NMDA受体在环境谷氨酸作用中的作用 将使用全细胞膜片钳在小鼠脑切片中记录EPSC和强直电流，而细胞外谷氨酸水平将通过MCH神经元上表达的遗传编码的谷氨酸传感器iGluSnFR来评估。受体和转运蛋白的亚细胞定位将通过电生理学和免疫荧光显微镜结合超分辨率径向波动分析进行研究。 建议的研究将是实现我的长期目标的重要步骤，以建立更好地了解中枢神经系统中的神经元和非神经元化学传递。