Neuronal and non-neuronal excitatory transmission in the hypothalamus

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，由谷氨酸介导的神经元间通信，中枢神经系统的主要兴奋性神经递质，对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在谷氨酸能传递中可以发挥多种作用。星形胶质细胞感知、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性或紧张性兴奋的基础。神经元和星形胶质细胞谷氨酸能传递的时间过程不同，提示其不同的机制、调控过程和功能作用。然而，这些机制仍然不清楚。多肽能神经元的神经肽释放通常需要长时间的放电活动，因此星形胶质细胞支持的兴奋性持续升高可能具有重要的功能后果。黑色素集中激素（Melanin-concentrating hormone， MCH）神经元是下丘脑中的肽能神经元，对控制能量平衡、睡眠-觉醒周期和动机行为至关重要。我们的初步研究表明，MCH神经元为研究神经元和非神经元化学传递提供了一种独特的模型，因为它们表现出几种具有不同时间进程的谷氨酸能电流：快速EPSC、慢速EPSC和滋补内向电流。快速和缓慢的EPSCs是由突触刺激触发的，而紧张电流是由谷氨酸运输抑制剂诱导的，并且是由于周围的谷氨酸在起源上似乎是星形细胞（胶质传递）。此外，这些电流由不同的谷氨酸转运体和受体调节/介导。基于这些结果，我们将验证不同来源（突触前神经元或星形胶质细胞）的细胞外谷氨酸由不同的谷氨酸转运体调节，并由MCH神经元上不同的受体池检测的假设。短期目的：1)确定快慢EPSCs在MCH神经元中的作用机制2)确定环境谷氨酸的来源及其释放机制3)利用全细胞膜片钳记录小鼠脑切片EPSCs和紧张电流，并通过MCH神经元上表达的遗传编码谷氨酸传感器iGluSnFR评估细胞外谷氨酸水平。受体和转运体的亚细胞定位将通过电生理和免疫荧光显微镜结合超分辨率径向波动分析来研究。我的长期目标是更好地理解中枢神经系统中的神经元和非神经元化学传递，拟议的研究将是实现这一目标的重要一步。

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.