Impact of variation in tissue preparation methodology on the functional outcome of neocortical mouse brain slices

The in vitro cortical slice preparation is a widely used tool for electrophysiological investigation of brain neurophysiology. However, slice quality can be highly variable despite attempts to standardise practice. The purpose of this study was to determine the extent to which this variability is due to sensitivity to aspects of the preparation methodology. The study ultilised the no-magnesium seizure-like event (SLE) model and was in two parts. In the first, slice outcome was quantified following a standardised hypoxic-ischaemic insult applied to pre-prepared slices. The hypoxia-induced changes in SLE activity were used to characterise the effect of tissue damage on commonly measured electrophysiological variables. In the second, multivariate data associated with the slice preparation methodology was collected and related to tissue outcome, according to the same SLE characteristics. Hypoxia-ischaemia damage was reflected most strongly in a reduction in SLE amplitude, inter-event frequency and intra-event high frequency activity. The number of active locations was also markedly reduced. In the second part of the study, between 21% and 47% of outcome variation was attributable to variability in the methodological parameters tested. Most important were slice position from the tissue block, depth of slice submersion during recording and the experience level of the experimenter. The latter showed a paradoxical relationship between inexperience, heightened SLE amplitude and reduced spatial viability. While much of the variation in slice outcome remained unexplained, the factors shown to correlate with tissue viability will aid slice experimentalists in preparing tissue of optimal quality for electrophysiological investigation. In particular, using population event amplitude as the sole criterion of slice quality in the no-magnesium model is overly simplistic and must be viewed in the context of spatial activity.