Nuclear shrinkage in live mouse hippocampal slices.

Brain slices are used extensively for biochemical, electrophysiological and molecular investigations. However, only the time frame for electrophysiological and biochemical investigations has as yet been defined. The goal of the present study was to investigate the time course of nuclear structure in live brain slices. Hippocampal slices (300 microm) were prepared from male CD1 mice (25-30 g), stained with Hoechst 33342 (10 microM), calcein-AM (2 microM) and ethidium homodimer (4 microM), and imaged with single- and dual-photon microscopy. The volume of CA1 pyramidal cell nuclei decreased from 759+/-229 microm3 in 40-50 microm depth 25 min after preparation to 453+/-169 microm3 (P<0.001) after 60 min, 315+/-112 microm3 (P<0.001) after 120 min and 128+/-71 microm3 (P<0.001) after 8 h. Similar results were obtained on a prolonged time scale in 70-80 microm depth and with an accelerated time scale in 20-30 microm depth. Live-dead staining showed that cell damage is progressing from the surface to deeper layers of the slices in a time-dependent fashion. We conclude that nuclei of CA1 hippocampal pyramidal cells show a time- and depth-dependent shrinkage converging 8 h after slice preparation to a volume of 90-130 microm; in any depth between 20 and 80 microm. The nucleus in the superficial 80 microm of each side appears dysfunctional even at times suitable for electrophysiological and biochemical experimentation in hippocampal slices. Molecular analysis of cell regulation in brain slices may, therefore, be time-dependently distorted by progressing cell death in at least half of the tissue under investigation.