Project 1 Determination of Vesicular Neurotransmitter Content at the Tripartite

最近出现的证据表明，囊泡神经递质的含量在抑制性突触处受到动态和自主的调节。小泡并不像以前认为的那样有一个静态的神经递质池。相反，作为细胞质神经递质数量的直接结果，小泡的含量在每分钟的基础上变化。 可供包装。这又取决于几个因素，包括摄取转运蛋白的活性，以及可用于局部GABA合成的前体的数量。由于抑制效应是突触相互连接神经元的净兴奋性的主要决定因素，GABA囊泡含量的活性依赖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释放在调节大脑功能中所起的关键作用，我们计划对调节囊泡GABA含量的关键因素进行表征。这将在集中于两个特定目标的实验中完成：目标1.确定神经胶质和神经递质转运体在调节 在高活性条件下抑制突触小泡的含量。目标2.确定调节More招募的主要细胞间信使和细胞内中介 在高活动期，抑制性突触的胶质神经递质的强健循环。 我们将检测神经胶质细胞对GABA和谷氨酸的摄取以及神经胶质细胞抑制突触启动子和谷氨酸-谷氨酰胺循环，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的IPSCs来监测GABA的释放，以及记录突触刺激下的胶质谷氨酸和GABA转运体电流来评估这一点。我们将结合膜片钳记录技术、药理学、激光去势治疗 神经递质和效应器，以及分子生物学技术，以阐明调节囊泡GABA含量的机制。孔特中心的环境为我们提供了一个独特的机会来解决这些问题。获得转基因动物的基因工程胶质细胞，独特的针对胶质细胞的载体，MNI笼式GABA和谷氨酸服从高分辨率双光子去除实验，以及对局部生产和调节的评估 神经元突起中的蛋白质对进行拟议的实验都是至关重要的。所有这些技术和资源都由Conte Center协作环境提供，该环境由Coulter、Levitan、Haydon、Ellis-Davies和Eberwin实验室组成。

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.