The adult brain slice method we have described has been successfully implemented in a variety of experimental contexts for analysis of diverse brain regions and cell types. However, we would encourage adopters to view this method as a work in progress, and we believe there is still substantial room for systematic improvement. As a case in point, we have observed that mature adult brain slices experience high levels of oxidative stress due in large part to rapid depletion of cellular antioxidants including ascorbate and reduced glutathione (GSH). This can lead to lipid peroxidation, neuronal membrane rigidity, and tissue deterioration. There appears to be a nonuniform susceptibility to this form of oxidative damage, for example, CA1 and CA3 pyramidal neurons are particularly vulnerable, making patch clamp recording of these cells difficult in brain slices from adult and aging animals in spite of the protective recovery method.
The specific restoration of intracellular pools of neuronal GSH (e.g. supplementation with the cell-permeable GSH-ethyl ester) is highly effective at curbing deterioration and prolonging slice viability under these circumstances. Thus, we have been able to further improve the NMDG recovery method by devising strategies for stimulating de novo synthesis of glutathione during acute brain slice preparation and incubation. This is most readily accomplished by adding the inexpensive GSH precursor N-acetyl-L-cysteine (NAC, 5–12 mM) to the NMDG aCSF and HEPES holding aCSF formulas, but not the recording aCSF (seeNote 14 ). NAC is cell-permeable and has been shown to specifically increase neuronal glutathione levels in brain tissue (26 (link)). Within 1–2 hours of slice preparation we are able to observe notable improvements in the general appearance of neurons and in the ease of patch clamp recording, and the slices are able to be maintained in a healthy state for extended time periods.
Although these more advanced methods are not absolutely required for successful adult brain slice patch clamp recordings (as demonstrated by the specific application we have described in this chapter), we include this information in hopes of providing more options to extend the versatility of our method for particularly challenging applications. Glutathione restoration is highly effective at maintaining healthy brain slices but may not be appropriate in every experimental context, e.g. investigations focusing on oxidative stress in the aging brain. On the other hand, without implementing the NMDG protective recovery method together with glutathione restoration strategy, targeted patch clamp analysis in brain slices from very old animals is prohibitively challenging.
The specific restoration of intracellular pools of neuronal GSH (e.g. supplementation with the cell-permeable GSH-ethyl ester) is highly effective at curbing deterioration and prolonging slice viability under these circumstances. Thus, we have been able to further improve the NMDG recovery method by devising strategies for stimulating de novo synthesis of glutathione during acute brain slice preparation and incubation. This is most readily accomplished by adding the inexpensive GSH precursor N-acetyl-L-cysteine (NAC, 5–12 mM) to the NMDG aCSF and HEPES holding aCSF formulas, but not the recording aCSF (see
Although these more advanced methods are not absolutely required for successful adult brain slice patch clamp recordings (as demonstrated by the specific application we have described in this chapter), we include this information in hopes of providing more options to extend the versatility of our method for particularly challenging applications. Glutathione restoration is highly effective at maintaining healthy brain slices but may not be appropriate in every experimental context, e.g. investigations focusing on oxidative stress in the aging brain. On the other hand, without implementing the NMDG protective recovery method together with glutathione restoration strategy, targeted patch clamp analysis in brain slices from very old animals is prohibitively challenging.
