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Effects of chronic depolarization on apoptosis of mutant leaner mice cerebellar granule cells in slice culture

Christine McCoy1, Bhupinder Bawa1, Louise C. Abbott1

1Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, 77843-4458 USA

ABSTRACT Christine picture

Objective— To determine the method of apoptosis in cerebellar granule cells in mutant leaner mice.

Animals— Eight 10-day-old wild type (C57BL/6) and eight age-matched leaner (C57BL/6:tgla/tgla ) mutant mice were used in this study.  Homozygous leaner carry a mutation in the pore-forming protein of P/Q-type calcium ion channels, causing decreased calcium ion current through P/Q-type channels, and exhibit cell death of approximately 50% of the cerebellar granule cells and Purkinje cells.

Procedure— Leaner and wild type 10-day-old mice were anesthetized with isoflurane and euthanized by exsanguination. The brains were surgically removed under sterile conditions and the cerebellum was cut into 350 µm coronal sections. The slices were cultured for 10 days in low (4mM) or increased (29 mM) potassium in the culture medium. The slices were fixed with 4% paraformaldehyde, then stained with Fluoro-Jade, an indicator of dying cells.

Results— In the low potassium group, wild type slices tended to exhibit more apoptotic cells per unit area than leaner slices.  This trend remained in the high potassium group.

Conclusions and Clinical Relevance—While these observations are still preliminary, the trend for more wild type cerebellar granule cells to die in culture compared to age-matched leaner cerebellar granule cells is intriguing.  This may mean that leaner cerebellar granule cells may be more resistant to stressful conditions compared to wild type cerebellar granule cells.  This is consistent with the observation that leaner mice live significantly longer (up to three years) than wild type mice, which live approximately two years.