Indeed, these studies focused mainly on the retention of maturityassociated electrophysiological properties possibly in response to comparative studies showing maturation of synaptic responses occur more rapidly in cultures prepared from older animals compared to younger animals. Interestingly, these techniques have not been attempted using mature mouse tissue, which would be particularly useful given the increasing availability of a range of transgenic mouse lines. Very few studies have attempted to culture whole brain slices from young adult mice, and in these circumstances the slices were mainly utilized as a substrate to culture dissociated cells. In the current study, we demonstrate an agedependant increase in cellular vulnerability when culturing whole brain slices in mice. However, we do show that mature slice cultures from P25 mice survive for 7 DIV, which are well suited for short-term studies and may be advantageous for experiments involving chronic stimulation or implanted recording electrodes for multi-site recording of electrical activity. Given cellular maturity and reduced morphological alterations during culture, P25 cultures could be an ideal extension of current acute brain slice techniques. It is important to note that although there are fewer morphological alterations in the cortex of more mature cultures, this needs to be further validated functionally using electrophysiological techniques. Additionally, there are constant improvements in markers of neuronal death/apoptosis/necrosis, which may prove useful in validating current results. In this study PI was utilized as a nuclei stain which would be indicative of membrane damage. Although this cell impermeant stain is by far the most commonly used fluorescent probe, it cannot be used to distinguish between necrosis and apoptosis. The results of AlamarBlue TM needs further qualification, particularly in P6 cultures, as increased signal may be indicative of cell proliferation over time and mask results on cell survival. Indeed, previous studies have shown increased cell proliferation in brain slice cultures. We observed a time-dependant increase in the expression of TWS119 synaptophysin in neonatal brain slice cultures over 14 DIV. This finding may be indicative of a gradual shift in these cultured brain slices to a mature phenotype. This is further supported by a significant and concomitant decrease in the expression of the growth-associated proteins GAP-43. There were no similar increases in synaptophysin expression in slices from mature animals, with the decrease in synaptophysin expression during culture temporally associated with the progressive death of cells over an extended culture period. Previous ultrastructural studies in rat organotypic hippocampal slice cultures have illustrated a significant increase in the density of synaptic contacts at 21 DIV double that observed at 7 DIV.