Functional dissection of dual acetylcholine/glutamate neurons

描述(申请人提供)：神经系统如何对信息进行编码、处理和传输，目前还远未完全了解。大多数神经元使用单一的神经递质向下游神经元传递信息。然而，现在被广泛接受的是，有一部分神经元利用两种不同的神经递质。然而，大多数关于双重神经递质使用的研究都集中在确定这种现象是真实存在的，而对潜在的功能目的相对较少关注。最近在这个实验室的实验中发现了果蝇幼虫神经系统中同时使用乙酰胆碱和谷氨酸的~100个中间神经元的亚群(~1%)。为了了解这些神经元的生物学作用，直接连接上游和下游神经元将使用本实验室最近开发的改进的突触特异性突触跨突触伙伴GFP重建(GGRST)方法来筛选7000株GAL4驱动程序中的幼虫表达亚集(419)，这些GAL4驱动程序是为精细神经电路图谱开发的。一旦确定了上下游神经元，它们是什么类型的神经元将根据神经解剖学进行识别，因为感觉神经元、运动神经元和中间神经元具有不同的形态。对上下游神经元类型的了解将把乙酰胆碱/谷氨酸双重神经递质神经元置于生物学背景中，从而提供对信息流的方向、它们携带的信息类型以及信息传输需求的洞察。为了了解乙酰胆碱/谷氨酸双神经递质神经元的功能用途，胆碱能和谷氨酸能传递将被单独或联合消除，特别是在这些神经元中，利用现有的突变，其后果将通过行为和功能钙成像进行分析。将分析幼虫对光、机械感应和温度的行为反应。由于这些不同的刺激各自激活不同的感觉和运动回路，这些行为分析将共同评估幼虫神经系统的相当大一部分。单个或双个沉默的Ach/vGlut双神经递质神经元将被光遗传兴奋，其下游神经元的激活将使用遗传编码的钙指示剂来评估。这些实验的结果将检验这一假设，即不同类型的信息，如刺激强度和刺激方向，或不同的传输模式，如快速兴奋和调制，是由乙酰胆碱和谷氨酸在同一神经元内携带的。

DESCRIPTION (provided by applicant): How information is encoded, processed, and transmitted by the nervous system is far from completely understood. Most neurons use a single neurotransmitter to transmit information to downstream neurons. However, it is now widely accepted that there is a subset of neurons that utilize two distinct neurotransmitters. Most studies on dual neurotransmitter usage have, however, focused on establishing that the phenomenon is real, with comparatively little attention given to the underlying functional purpose. Recent experiments in this laboratory have identified a subset of ~100 interneurons (~1% of total) in the Drosophila larval nervous system that use both acetylcholine and glutamate. To understand the biological role of these neurons, directly connecting upstream and downstream neurons will be identified using an improved synapse-specific GFP Reconstitution Across Synaptic Partners (GRASP) method recently developed in this laboratory to screen a larval-expressing subset (419) of a 7000 strain collection of GAL4 drivers developed for fine-scale neural circuit mapping. Once the upstream and downstream neurons have been identified, what types of neurons they are will be discerned based on neuroanatomy since sensory, motor, and interneurons have distinct morphologies. Knowledge of the types of neurons upstream and downstream will place the acetylcholine/glutamate dual neurotransmitter neurons in a biological context and thereby provide insights into the direction of information flow, the type of informatio they are carrying, and the demands of information transfer. To understand the functional purpose of the acetylcholine/glutamate dual neurotransmitter neurons, cholinergic and glutamatergic transmission will be eliminated, singly and in combination, specifically in these neurons using existing mutations and the consequences will be assayed behaviorally and by functional calcium imaging. Larval behavioral responses to light, mechanosensation, and temperature will be assayed. Since these different stimuli each activate distinct sensory and motor circuits, these behavioral assays will together assess a substantial portion of the larval nervous system. The single or dual silenced Ach/vGlut dual neurotransmitter neurons will be excited optogenetically and the activation of their downstream neurons will be assessed using genetically-encoded calcium indicators. The results of these experiments will test the hypothesis that distinct types of information, such as stimulus intensity and stimulus direction, or distinct modes of transmission, such as fast excitatory and modulatory, are being carried by acetylcholine and glutamate within the same neurons.