VISUALIZING IPUT: A NOVEL BIOORTHOGONAL PROBE REVEALS REDISTRIBUTION OF EXOGENOUS PUTRESCINE UNDER NEUROTOXIC STRESS IN HIPPOCAMPAL SLICES

This study aimed to track the regional, cellular, and subcellular distribution of exogenous putrescine in acute hippocampal slices using a novel clickable probe (iPUT) under neurotoxic stress conditions, including mimetic hypoxia and excitotoxicity.
Acute hippocampal slices from adult C57 mice were incubated with 500 µM iPUT and analyzed by confocal microscopy following fluorophore conjugation via a click chemistry reaction. Hypoxia was mimicked with 1 mM CoCl₂, and excitotoxicity was induced using 250 µM NMDA. Changes in fluorescence intensity, percentage of iPUT-positive astrocytes, and probe colocalization with cellular and subcellular markers were assessed across hippocampal subregions.
Under control conditions, iPUT localized to pyramidal neurons and astrocytes, showing extranuclear distribution in neurons, but not in astrocytes. iPUT accumulation was higher in CA1 than in CA2–3 under control conditions but decreased significantly in CA1 following CoCl₂ or NMDA exposure. Neurotoxic stress shifted the distribution towards the nucleus in CA1 neurons, while. no significant changes were observed in CA2-3. The percentage of probe-positive astrocytes remained stable across regions.
iPUT is a promising tool for visualizing putrescine dynamics under control and stress conditions , with potential utility in a range of experimental settings. The observed changes in putrescine distribution at the regional, cellular, and subcellular levels in the hippocampus may contribute to the differential sensitivity of hippocampal subregions to neurotoxic stress.