FROM HOURS TO DAYS: EXTENDING BRAIN SLICE VIABILITY

We established a preparation protocol for acute brain slices of mice aged P10 to two years, allowing whole-cell patch-clamp and Ca²⁺ imaging experiments on neurons that retain electrophysiological properties over a 36-hour period after slicing. Using only standard laboratory equipment, this markedly expands the viability of acute brain slices. We have optimized the composition of ACSFs for different phases of slice preparation to minimize oxidative stress and allow neurons to maintain their activity and energy dynamics. In addition, we have improved the design of the incubation chamber for slice storage, minimizing mechanical stress to the tissue when moving the chamber. We applied this protocol on neurons in the thalamic reticular nucleus (TRN), globus pallidus (GP), in layer 1 of the medial prefrontal cortex (L1 mPFC) and of the motor cortex (M1/M2). Typical electrophysiological properties of these neurons remained unchanged during 2 days after slicing. Using paired recordings to investigate electrical synaptic plasticity in L1 mPFC, we were able to find electrical coupling on the 3^rd day after slice preparation, which shows the robustness of our method. Cell viability over days was also confirmed by using a standard imaging assay. In summary, our protocol has the potential to a) reduce the number of animals used per study, b) allow obtaining healthy brain slices from adult animals of up to ~two years of age, c) allow prolonged experimental procedures, and d) reduce material cost and preparation effort for the experimenter.