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

神经系统包括化学和电突触，它们定义了解剖学上的连接体。第三种是神经调质或神经肽的“无线”网络，它调节神经元集合甚至大脑系统，以适应不同的功能状态。特别是在紧凑的神经系统中，需要经济地使用很少的神经元，多种功能由有限数量的细胞执行。在这里，不同的功能神经元网络覆盖在一个单一的解剖网络，这是由神经调节开关。当然，神经肽信号也被用于高等动物，但它的影响往往只是新兴的。秀丽隐杆线虫（Oranth等，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通过两种神经肽受体释放FLP-1神经肽来调节食物反应的各个方面。然而，AVK mRNA谱显示第二种神经肽NLP-49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示，在各自的突变体中，以及在特定条件下（食物搜索行为，机械刺激导致觉醒）。这些作用可能由不同靶细胞上的不同神经肽受体介导。此外，我们表明，救援是通过在AVK中特异性表达每种肽来实现的。我们发现FLP-1和NLP-49在AVK中显示出不同的定位和运输，即，它们不被包装在相同的致密核心囊泡（DCV）中。因此，AVK神经元可以“处理”这两种神经肽的区别和具体。该项目旨在揭示1）这些肽的释放是否受到不同的调节，以及在何种情况下，2）这些神经肽的差异释放如何调节神经回路以唤起不同的行为，以及3）这种差异释放如何在分子水平上实现，即能够将神经肽特异性包装到不同DCV中的机制，以及能够特异性运输含有不同神经肽的DCV的机制。为此，我们将采用遗传学，光遗传学，荧光神经肽释放报告，Ca 2+成像，电子显微镜和行为分析，以及有限的RNAi筛选，以确定和表征所需的分子的差异调节FLP-1和NLP-49从单个神经元AVK的释放。阐明神经肽信号传导的这一复杂层将有助于理解一般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，其中小神经递质的共传递已被认可，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.