Extending the protective recovery method:
Glutathione depletion underlies rapid deterioration of adult brain slices
We have demonstrated that the protective recovery method is a versatile approach for both morphological and functional preservation of neurons in acute brain slices from adult and aging animals. This adult brain slice method can be adapted to access diverse neuronal populations across the entire lifespan of mice or rats, and effectively overcomes many limitations of existing methodologies. While this innovation represents an important step forward, more work is necessary to refine and extend the technique.
One limitation we have noted is the difficulty in sustaining preservation of pyramidal neurons in the hippocampal CA1 and CA3 regions in brain slices from adult and aging animals. Although the protective recovery method enables functional analysis of pyramidal neurons within the first few hours after slice preparation, the neurons still deteriorate relatively rapidly and the membranes become rigid and inaccessible for patch camp recording. Similar results were observed in other neuron subtypes such as cortical pyramidal neurons. We reasoned that this deterioration was mediated by excitotoxicity, perhaps owing to the high concentration of NMDA receptors in these neurons combined with the increased neuronal survival and higher synaptic activity with the new method. The membrane rigidity could reflect oxidative stress linked to NMDA receptor activation, such as lipid peroxidation.
One possible explanation for these events may be rapid depletion of endogenous antioxidants in acute brain slices. Ascorbate supplementation is beneficial for adult brain slices, as this antioxidant is readily taken up by neurons to replenish intracellular supply. In contrast, the powerful antioxidant glutathione (GSH) is unable to cross the neuronal membrane and does not have a notable impact on the health of adult brain slices when added to the aCSF. Importantly, the endogenous levels of GSH in neurons in the intact brain has been estimated in the low millimolar range. To address this issue we explored diverse strategies for restoring neuronal glutathione levels in adult brain slices. First, application of the cell-permeable glutathione precursor N-acetyl-L-cysteine (NAC, 12 mM) to adult brain slices during the slicing procedure and maintained throughout slice incubation was effective at preserving hippocampal pyramidal neuron morphology and functionality for up to 12 hours. This effect is likely due to on-line de-novo glutathione synthesis, as the beneficial effects were only fully apparent after one hour or more of slice incubation. Application of L-cysteine in place of NAC was not protective and instead was highly neurotoxic. Second, application of the cell-permeable version of glutathione (glutathione ethyl-ester, GSH-EE 1 mM) was even more effective than NAC at preserving hippocampal pyramidal neurons. This is presumably because glutathione supply is directly replenished and does not require de novo synthesis from precursors, thus providing rapid and sustained protection from oxidative stress following brain slice preparation. Thus, two separate treatments confirm the central role of glutathione depletion in the rapid deterioration of adult brain slices.