Developing systems to optogenetically inhibit neuropeptide exocytosis

Neuropeptides are a diverse group of signaling molecules used by the nervous system as mediators of direct signaling or modulators of signaling pathways. Neuropeptides have conserved roles in driving homeostatic behaviours from peptidergic neurons, for which they have been most thoroughly described. But the roles of neuropeptides in modifying signaling are more ubiquitous. Neuropeptides are used throughout the nervous system, and often are co-transmitted alongside fast neurotransmitters to modify the strength of neuronal networks. Unlike neurotransmitters, neuropeptides are not spatially restricted to the presynapse and are secreted from the dendrites, soma and axon. From there these molecules can act as paracrine signaling molecules, affecting both connected and neighbouring cells, or can act as autocrine signaling molecules. To date, research is still unlocking the role in information processing that these co-transmitted neuromodulators perform, however there are no techniques available that allow spatially defined and inducible manipulation of neuropeptide secretion. Optogenetics provides an effective way to manipulate cellular function with high spatial and temporal resolution. Optogenetic ion channels, membrane pumps and signaling pathway manipulators have been used to modify the excitability of neurons, or to directly trigger exocytosis, but there exists no approaches that manipulate the release of different vesicles. The objective of this project is to develop an optogenetic tool to inhibit the secretion of neuropeptides, but not the release of neurotransmitters. The approach uses the ‘Inhibiting of Synapses with Chromophore Assisted Light Inactivation (CALI)’ tool, InSynC, modified to selectively target neuropeptide-containing dense core vesicles. InSynC uses the photosensitizing fluorescent protein miniSOG targeted to synaptic vesicles, where light-induced reactive oxygen species disrupts synaptic protein function. In this thesis, InSynC was modified to direct miniSOG to the synaptic vesicle lumen resulting in improved recovery rate of synaptic inhibition with no loss in efficacy. This suggests an increase in specificity with this approach. Next, dense core vesicles and neuropeptide secretion was targeted through fusion of miniSOG to neuropeptide processing enzymes packaged within dense core vesicles. Using two in vivo assays in the nematode C. elegans, blue light disrupted odour adaptation learning and carbon dioxide avoidance responses, both behaviours reported to be associated with neuropeptide release. These results were consistent with the expected phenotypes of neuropeptide suppression based on the recorded phenotypes of the appropriate neuropeptide/receptor mutant strains. The results suggest that neuropeptide secretion is inhibited while neurotransmitter release is minimally disrupted. For this thesis the tool has been named Peptide Secretion Inhibition with CALI (PepSI-CALI). PepSI-CALI is currently being assayed in mammalian culture systems to further validate its proposed mechanism and effect. This includes the direct measurement of neuropeptide and neurotransmitter release in primary culture neurons and high-resolution imaging to validate expression targeting. If successfully validated, PepSI-CALI will be a powerful optogenetic tool for uncovering the complicated roles of neuropeptides in complex nervous systems