Monoamines Differentially Modulate Neuropeptide Release from Distinct Sites Within A Single Neuron Pair

Monoamines and neuropeptides modulate behavior, but monoaminergic-peptidergic cross talk remains poorly understood. In C. elegans, serotonin and the adrenergic-like ligands, tyramine (TA) and octopamine (OA) require distinct subsets of neuropeptides in the two ASI sensory neurons to inhibit nociception. For example, the ASI neuropeptides required for TA inhibition, encoded by nlp-1, -14 and -18 are distinct from those required for OA inhibition, encoded by nlp-6, -7, and -9. In the present study, NLP-9, -14:: or -18::GFPs co-localized with the synaptic marker, mCHERRY::RAB-3 in the distal portion of the ASI axons, where synapses have been identified previously by electron microscopy. Neuropeptide and RAB-3 positive axonal puncta were also consistently observed extra-synaptically near ASI soma. ASI::NLP-14::GFP and ASI::NLP-9::mCHERRY co-localized to both synaptic and extra-synaptic puncta, but the GFP/mCHERRY ratios in extra-synaptic/synaptic puncta differed significantly, suggesting that TA- and OA-dependent neuropeptides may be differentially trafficked and/or localized. TA, but not OA, selectively increased the release of ASI neuropeptides encoded by nlp-14 or nlp-18 from either synaptic/perisynaptic regions of ASI axons or the ASI soma, respectively. In contrast, OA, but not TA, selectively increased the release of ASI neuropeptides encoded by nlp-9 asymmetrically, only from synaptic/perisynaptic regions of the right ASI axon. The predicted preprosequences of the three TA-dependent neuropeptide-encoding genes differed markedly from the three OA-dependent genes. However, these distinct preprosequences were not sufficient to confer monoamine-specificity, as the release of chimeric neuropeptides containing swapped pre-pro-sequences did not respond to the predicted monoamine. In contrast, the release of a truncated NLP-14::GFP containing only the first 95 amino acids and only five of a predicted 12 neuropeptides responded to TA but not OA, demonstrating that additional N-terminal sequence was required for monoamine-specificity. Collectively, our results demonstrate that TA and OA differently modulate the release of distinct subsets of neuropeptides from different subcellular sites within the ASIs and highlight the complexity of monoaminergic/peptidergic modulation, even animals with a relatively simple nervous system containing only 302 neurons.