Nuclear shrinkage in live mouse hippocampal slices

Area of Science:

• Neuroscience
• Cell Biology
• Microscopy

Background:

• Brain slices are crucial for neuroscience research, enabling biochemical, electrophysiological, and molecular studies.
• The temporal viability and structural integrity of brain slices, particularly nuclear structure, are not fully understood.
• Defining the time course of nuclear changes is essential for accurate interpretation of slice-based experiments.

Purpose of the Study:

• To investigate the time course of nuclear structure changes in live hippocampal brain slices.
• To assess the depth-dependent and time-dependent progression of cell damage in brain slices.
• To determine the implications of nuclear structural changes and cell death for molecular investigations.

Main Methods:

• Preparation of male CD1 mouse hippocampal slices (300 microm).
• Staining with Hoechst 33342, calcein-AM, and ethidium homodimer for nuclear and viability assessment.
• Imaging using single- and dual-photon microscopy to measure nuclear volume and cell integrity over time and depth.

Main Results:

• CA1 pyramidal cell nuclear volume significantly decreased over 8 hours post-preparation, from 759+/-229 microm3 to 128+/-71 microm3.
• Nuclear shrinkage was time- and depth-dependent, with significant changes observed within 60-120 minutes.
• Live-dead staining revealed progressive cell damage from superficial to deeper layers, indicating dysfunction in the superficial 80 microm within experimental timeframes.

Conclusions:

• Hippocampal pyramidal cell nuclei undergo significant, time- and depth-dependent shrinkage in brain slices.
• The superficial 80 microm of brain slices exhibit nuclear dysfunction and progressing cell death, even within typical experimental durations.
• Molecular analyses in brain slices may be compromised by time-dependent cell death, potentially distorting findings in over half the tissue.