Project 1 Determination of Vesicular Neurotransmitter

最近的证据表明，囊泡神经递质的内容是动态和自主调节的抑制性突触。囊泡不像以前认为的那样有一个静态的神经递质池。相反，囊泡的内容物在一分钟到一分钟的基础上变化，作为细胞质神经递质量的直接结果 可以打包。这反过来又取决于几个因素，包括摄取转运蛋白的活性，也对当地GABA合成可用的前体的量。由于抑制效果是突触互连神经元的净兴奋性的主要决定因素，GABA囊泡含量的活动依赖性减少在降低突触传递的基本货币，突触后受体水平方面处于关键地位。 通过释放单个量子来激活。鉴于GABA释放在调节脑功能中发挥的关键作用，我们计划表征调节囊泡GABA含量的关键因素。这将在集中于2个特定目标的实验中完成：AIM 1。确定神经胶质和神经元神经递质转运蛋白在调节 在高活性条件下抑制性突触囊泡含量。AIM 2.确定调节更多细胞募集的主要细胞间信使和细胞内中介体 在高活动期间，抑制性突触处的神经胶质神经递质再循环。 我们将研究GABA和谷氨酸神经胶质细胞和抑制性突触结和谷氨酸-谷氨酰胺循环的神经胶质细胞的摄取，因为这些过程影响囊泡GABA水平。这将通过记录突触后神经元中的IPSC监测GABA释放以及记录响应于突触刺激的神经胶质谷氨酸和GABA转运体电流来评估。我们将整合膜片钳记录技术、药理学、激光血管扩张技术， 神经递质和效应器，和分子生物学技术，以阐明机制调节囊泡GABA含量。康特中心的环境为我们提供了解决这些问题的独特机会。获得具有工程化神经胶质的转基因动物，靶向神经胶质的独特转染载体，MNI笼状GABA和谷氨酸适合于高分辨率2光子释放实验，并评估局部产生和调节 神经元过程中的蛋白质对于进行所提出的实验都是至关重要的。所有这些技术和资源都是由Coulter、Levitan、Haydon、Ellis-Davies和Eberwine实验室组成的Conte中心协作环境提供的。

Recent evidence has emerged that vesicular neurotransmitter content is dynamically and autonomously regulated at inhibitory synapses. Vesicles do not have a static pool of neurotransmitter, as previously believed. Instead, the content of vesicles varies on a minute to minute basis as a direct consequence of the amount of cytoplasmic neurotransmitter available to be packaged. This in turn depends on several factors, including the activity of uptake transporters and also on the amount of available precursors for local GABA synthesis. Since inhibitory efficacy is a prime determinant of the net excitability of synaptically interconnected neurons, activity-dependent reductions in GABA vesicle content is in a pivotal position to devalue the fundamental currency of synaptic transmission, the level of postsynaptic receptor activation by release of a single quanta. Given the critical role played by GABA release in regulating brain function, we plan to characterize the critical factors regulating vesicular GABA content. This is to be accomplished in experiments focused on 2 specific aims: AIM 1. Determine the relative importance of the glial and neuronal neurotransmitter transporters in regulating inhibitory synaptic vesicle content under high activity conditions. AIM 2. Identify the primary intercellular messengers and intracellutar intermediaries regulating recruitment of more robust glial neurotransmitter recycling at inhibitory synapses during periods of high activity. We will examine GABA and glutamate uptake by glia and inhibitory synaptic boutons and glutamate-glutamine cycling by glia, as these processes affect vesicular GABA levels. This will be assessed by monitoring GABA release through recordings of IPSCs in postsynaptic neurons, as well as recording glial glutamate and GABA transporter currents in response to synaptic stimulation. We will integrate patch clamp recording techniques, pharmacology, laser uncaging of neurotransmitters and effectors, and molecular biological techniques to elucidate mechanisms regulating vesicular GABA content. The Conte Center environment affords us a unique opportunity to address these questions. Access to transgenic animals with engineered glia, unique transfection vectors targeting glia, MNI caged GABA and glutamate amenable to high resolution 2 photon uncaging experiments, and assessment of local production and regulation of proteins in neuronal processes all are critical to conduct of the proposed experiments. All of these techniques and resources are provided by the collaborative Conte Center environment consisting of the Coulter, Levitan, Haydon, Ellis-Davies, and Eberwine laboratories.