Mechanisms of specific (co-)transmission of distinct neuropeptides from a single neuron

神经系统由化学突触和电突触组成，它们定义了解剖学上的连接体。第三，神经调节剂或神经肽的“无线”网络存在，它调节神经元整体，甚至大脑系统，以采用不同的功能状态。特别是在紧凑的神经系统中，经济上需要使用很少的神经元，由有限数量的细胞执行多种功能。在这里，不同功能的神经元网络被覆盖在一个单一的解剖网络上，这些网络通过神经调节进行切换。当然，神经肽信号也用于高等动物，但它的意义往往才刚刚出现。我们分析了线虫中参与食物相关行为调控的神经元网络(Oranth等人，2018，Neuron 100：1414)。这个网络以中间神经元AVK为中心，使用神经肽来改变行为。AVK通过两个神经肽受体释放FLP-1神经肽来调节食物反应的各个方面。然而，AVK mRNA图谱显示，另一种神经肽NLP-49也同样大量表达。有趣的是，FLP-1和NLP-49神经肽对行为有不同的影响，正如我们在使用单个动物和群体行为跟踪的初步行为分析中所显示的那样，在各自的突变体中，在特定条件下(食物搜索行为，导致唤醒的机械刺激)。这些作用可能是通过不同靶细胞上不同的神经肽受体来实现的。此外，我们还表明，救援是通过在AVK中特异性地表达每个肽来实现的。我们发现FLP-1和NLP-49在AVK中表现出明显的定位和转运，即它们不被包装成相同的致密核心小泡(DCV)。因此，AVK神经元可以区别和特异地“处理”这两种神经肽。本项目旨在揭示1)这些神经肽的释放是否受到不同的调控，以及在什么情况下，2)这些神经肽的差异释放如何调节神经回路以引起不同的行为，以及3)这种差异释放是如何在分子水平上实现的，即使神经肽能够特定地包装到不同的DCV中的机制，以及使得含有不同神经肽的DCV能够特定地运输的机制。为此，我们将使用遗传学、光遗传学、荧光神经肽释放报告、钙离子成像、电子显微镜和行为分析，以及有限的RNAi屏幕来鉴定和表征单个神经元AVK释放FLP-1和NLP-49的差异调节所需的分子。阐明这一复杂的神经肽信号转导层将有助于从总体上理解神经系统的信息处理，并将为高等动物的类似分析提供蓝图，在高等动物中，小分子神经递质的共同传递已被识别，但这一概念尚未扩展到神经肽。

Nervous systems comprise chemical and electrical synapses, which define an anatomical connectome. A third, ‘wireless’ network of neuromodulators or neuropeptides exists, that modulate neuronal ensembles or even brain systems, to adopt distinct functional states. Particularly in compact nervous systems, that need to use few neurons economically, multiple functions are performed by a limited number of cells. Here, different functional neuronal networks are overlaid on a single anatomical network, which are switched by neuromodulation. Of course, neuropeptide signaling is also used in higher animals, yet its implications are often only emerging.We analyzed a neuronal network involved in the regulation of food-related behaviors in the nematode C. elegans (Oranth et al., 2018, Neuron 100: 1414). This network, centered around the interneuron AVK, uses neuropeptides to alter behavior. The AVKs release FLP-1 neuropeptides to regulate aspects of the food response via two neuropeptide receptors. However, the AVK mRNA profile shows that a second neuropeptide, NLP-49, is expressed equally abundantly. Interestingly, FLP-1 and NLP-49 neuropeptides have differential effects on behavior, as we show in preliminary behavioral analyses using single animal and population behavioral tracking, in the respective mutants, and in specific conditions (food search behavior, mechanical stimulation leading to arousal). These effects are likely mediated by different neuropeptide receptors on different target cells. Also, we show that rescue is achieved by expression of each peptide specifically in AVK. We found that FLP-1 and NLP-49 show distinct localization and trafficking in AVK, i.e., they are not packaged into the same dense core vesicles (DCVs). Thus, the AVK neuron can ‘handle’ these two neuropeptides distinctly and specifically. This project aims to reveal 1) whether release of these peptides is regulated differently, and in which context, 2) how the differential release of these neuropeptides modulates the neural circuits to evoke different behaviors, and 3) how this differential release is achieved at the molecular level, i.e. mechanisms enabling specific packaging of neuropeptides into different DCVs, as well as mechanisms enabling specific trafficking of DCVs containing different neuropeptides. To this end we will employ genetics, optogenetics, fluorescent neuropeptide release reporters, Ca2+ imaging, electron microscopy and behavioral analyses, as well as a limited RNAi screen to identify and characterize molecules required for the differential regulation of release of FLP-1 and NLP-49 from the single neuron AVK. Elucidation of this complex layer of neuropeptide signaling will contribute to understanding information processing by nervous systems in general, and will provide a blueprint for similar analyses in higher animals’, where co-transmission by small neurotransmitters has been recognized, but this concept has not yet been extended to neuropeptides.