Abstract
Introduction
Synaptic dysfunction is an early and significant feature of aging, neurodegenerative disease, and radiation associated neurological defects. However, measuring functional changes at the level of individual synapses remains a challenge. Fluorescence-assisted single-synapse long-term potentiation (FASS-LTP) addresses this limitation by making use of both chemically induced long-term potentiation (cLTP) and flow cytometric detection of activity dependent synaptic markers. This study aimed to establish and optimize a reproducible FASS-LTP workflow for assessing synaptic plasticity in frozen brain tissue using spectral flow cytometry.
Methods
Synaptosomes were isolated from banked hippocampal samples that contained both non-irradiated control samples and samples from which animals were exposed to varying radiation conditions. To induce cLTP, the samples were exposed to glycine priming, followed by potassium chloride induced depolarization. Samples were immunolabeled for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit 1 (GluA1), a postsynaptic protein associated with synaptic potentiation, and neurexin-1β (Nrx1β), a presynaptic adhesion protein. Synaptosomal particles were identified using forward and side scatter characteristics with size reference beads to exclude debris. Fluorescence was used to quantify GluA1 positive, Nrx1β positive, and GluA1/Nrx1β double positive populations. Optimization focused on sample recovery, reagent preparation, stimulation conditions, immunolabeling, spectral unmixing, acquisition, and gating.
Results
Initial experiments produced limited Nrx1β fluorescence and no clear distinction between basal and stimulated samples. Following optimization, it was observed that non-irradiated hippocampal samples had an increase in GluA1/Nrx1β double positive synaptosomes following cLTP induction, whereas irradiated samples showed a reduced or absent response. Although this trend was not evaluated as a complete statistical comparison, it suggests that radiation exposure may alter activity-dependent synaptic potentiation and supports further investigation using the optimized FASS-LTP workflow.
Conclusion
Overall, an increase in GluA1/Nrx1β double positive synaptosomes following stimulation establishes a functional workflow for evaluating synaptic potentiation in stored hippocampal tissue and identify critical specifications affecting FASS-LTP reproducibility. Although a complete comparison of irradiated and control samples was not fulfilled, this optimization provides the foundation for successive investigation of radiation associated synaptic dysfunction. More broadly, the ability to examine functional synaptic changes in frozen tissue creates opportunities to expand to regional vulnerability, disease progression, and therapeutic response across existing animal and human tissue collections.