Functional dissection of dual acetylcholine/glutamate neurons

描述（由申请人提供）：信息是如何被神经系统编码、处理和传输的，我们还远未完全了解。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的，而对潜在的功能目的的关注相对较少。本实验室最近的实验发现，果蝇幼虫神经系统中约100个中间神经元（约占总数的1%）同时使用乙酰胆碱和谷氨酸。为了了解这些神经元的生物学作用，将使用该实验室最近开发的一种改进的突触特异性GFP跨突触伙伴重构（GRASP）方法来识别直接连接上游和下游神经元，以筛选用于精细神经回路映射的7000株GAL4驱动因子集合中的幼虫表达子集（419）。一旦确定了上游和下游神经元，就可以根据神经解剖学来区分它们是什么类型的神经元，因为感觉、运动和中间神经元具有不同的形态。了解上游和下游神经元的类型将把乙酰胆碱/谷氨酸双神经递质神经元置于生物学背景下，从而提供对信息流方向、它们携带的信息类型和信息传递需求的见解。为了了解乙酰胆碱/谷氨酸双神经递质神经元的功能目的，胆碱能和谷氨酸能的传递将被单独或联合消除，特别是在这些神经元中使用现有的突变，其后果将通过行为和功能钙成像进行分析。幼虫对光、机械感觉和温度的行为反应将被分析。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很大一部分。单或双沉默的Ach/vGlut双神经递质神经元将被光遗传学激活，其下游神经元的激活将使用遗传编码钙指标进行评估。这些实验的结果将验证一个假设，即不同类型的信息，如刺激强度和刺激方向，或不同的传递模式，如快速兴奋和调节，是由同一神经元内的乙酰胆碱和谷氨酸携带的。

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.