Method for Prolonged Incubation of Brain Slices

Slices of neuronal tissue maintain a high degree of topographical and functional properties of neurons and glia and therefore are extensively used for measurements of neuronal activity at the molecular, cellular and network levels. However, the lifespan of slice preparations is narrow, averaging of 6-8 hours. Moreover, the average viability of brain slices varies according to animal age and region of interest, leading to the high variability and low reproducibility of recorded data. Previous techniques to increase the viability of brain slices focused on reducing cytotoxicity by chemical means, including alterations of the artificial cerebrospinal fluid (aCSF) composition to alleviate the direct damage of the slicing procedure or adding protective antioxidants to reduce cellular deterioration. In this protocol, we use a combination of hypothermia with firm control of the aCSF conditions in the recovery chamber (pH, temperature, and bacteria levels) to extend the slice viability significantly. Given the breadth of its usage, improving slice viability and longevity can considerably increase data reproducibility and reduce the cost, time, and number of animals used in neurophysiological studies.