Neuropeptides regulate many physiological functions, including neuromodulatory actions, gene transcription and emotional responses.1-3 They also have been implicated in neuroadaptation, influencing neuronal plasticity and memory formation. However, the neuropeptide-mediated pathways underpinning memory formation have not been elucidated.1,3,4 Complex expression patterns of their respective receptors in various brain regions, in combination with a lack of receptor-subtype selectivity, their long half-life in the cerebral spinal fluid and the resulting diffusion distances throughout the brain make precise in vivo studies difficult.5,6
To address these issues, we are developing molecular probes to simulate endogenous neuronal secretion for investigating signalling pathways, with a focus on the role of neuropeptides in emotional memory formation. Using organic chemistry, we synthesised a selection of photolabile protecting groups (photocages) that follow different cleavage mechanisms and wavelengths ranging from UV light to the visible spectrum (365-527 nm). We incorporated these photocages into the neuropeptides oxytocin (OT) and vasopressin (VP) using solid and solution phase synthesis strategies and characterised them pharmacologically at their respective G protein-coupled receptors (OTR, V1aR, V1bR) in cellular functional assays.
The photocaged neuropeptides had a >100-1000-fold reduced activity compared to the endogenous ligands, validating our design strategy. Removal of the photocages (uncaging) was carried out via irradiation with UV light and confirmed by LC-MS and confocal microscopy. Incubation of cells with the photocaged neuropeptides did not generate a cellular response, but uncaging with a laser at the probes’ specific uncaging wavelengths yielded the desired physiological response, including cellular contractions and neuropeptide-mediated receptor internalisation.