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

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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.