Vesicular monoamine transporter trafficking affects mode of neurotransmitter release and microcircuit function

摘要 单胺神经递质，如多巴胺和血清素，调节快速突触传递回路 调节许多复杂的行为，包括攻击性，睡眠，注意力和情绪。在精神病学中，许多 治疗剂将单胺系统靶向于介导和调节 单胺神经传递囊泡单胺转运蛋白（VMAT）负责装载所有 单胺神经递质进入突触囊泡（SV）和大致密核心囊泡（LDCV）， 分别介导突触和突触外释放。然而，每种类型的功能贡献 囊泡释放到回路的功能和行为是未知的。之前对果蝇的研究已经证明 胺释放的量和位点可以通过C-末端运输结构域的突变来改变， 果蝇VMAT（DVMAT）。在DVMAT无效遗传背景下，几种回路和行为的功能 受到干扰，但通过野生型和运输突变等位基因的转基因表达得以拯救。然而，在这方面， 有些行为是无法被贩卖突变体拯救的。导致DVMAT优先向 LDCV不能挽救产卵回路的功能。这表明，产卵电路是高度 对神经递质章鱼胺的突触和突触外释放之间的微妙平衡敏感 （OA）。我假设内源性DVMAT基因位点的运输突变将赋予电路 功能障碍，类似于基因拯救实验。为了进一步验证这一想法，我提议创造一种新的基因 使用CRISPR/Cas9改变内源基因中的运输信号的DVMAT运输模型。我 初步数据表明，破坏DVMAT C-末端表型的插入缺失突变， 实验这一新的遗传模型将有助于研究突变体对内源表达的影响 水平，促进了与遗传和分子工具的组合，用于电路分析，并代表了一种新的 用于基因筛选的平台，以寻找DVMAT功能的新型调节剂。我建议研究 果蝇产卵微回路中单胺释放的不同模式。我假设 胺释放模式的改变将导致肌肉收缩和节律活动的模式 在靶器官中的作用与野生型动物不同。这些研究将阐明新的机制 胺能信号传导和精神疾病新疗法的研究和开发的新途径 紊乱

Abstract Monoamine neurotransmitters, such as dopamine and serotonin, modulate fast synaptic transmission in circuits that mediate many complex behaviors including aggression, sleep, attention, and mood. In psychiatry, many therapeutics target monoamine systems at either the receptors or transporters that mediate and regulate monoamine neurotransmission. The vesicular monoamine transporter (VMAT) is responsible for loading all monoamine neurotransmitters into both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), which mediate synaptic and extrasynaptic release, respectively. However, the functional contribution of each type of vesicular release to circuit function and behavior is unknown. Previous studies in Drosophila have demonstrated that the amount and site of amine release can be altered by mutations in the C-terminal trafficking domain of Drosophila VMAT (DVMAT). In a DVMAT null genetic background, the function of several circuits and behaviors are perturbed, but are rescued by transgenic expression of wild-type and trafficking mutant alleles. However, some behaviors are not rescued by trafficking mutants. Mutations that cause DVMAT to preferentially traffic to LDCVs do not rescue function of the oviposition circuit. This suggests that the oviposition circuit is highly sensitive to the delicate balance between synaptic and extrasynaptic release of the neurotransmitter octopamine (OA). I hypothesize that trafficking mutations in the endogenous DVMAT gene locus will confer circuit dysfunction, resembling genetic rescue experiments. To further test this idea, I propose to create a new genetic model of DVMAT trafficking using CRISPR/Cas9 to alter trafficking signals in the endogenous gene. My preliminary data demonstrate that indel mutations that disrupt the DVMAT C-terminus phenocopy previous experiments. This novel genetic model will be useful to study the effects of mutants at endogenous expression levels, facilitates combinations with genetic and molecular tools for circuit analysis, and represents a new platform for genetic screens to find novel regulators of DVMAT function. I propose to study the contributions of the different modes of monoamine release in the Drosophila melanogaster oviposition microcircuit. I hypothesize that alteration of the mode of amine release will result in patterns of muscular contractions and rhythmic activity in target organs that will differ from wild-type animals. These proposed studies will elucidate novel mechanisms of aminergic signaling and new avenues for the research and development of new therapeutics for psychiatric disorders.