Chronic mild acidosis specifically reduces functional expression of N-methyl-D-aspartate receptors and increases long-term survival in primary cultures of cerebellar granule cells.

Abstract Previous studies suggest that chronic depolarization by addition of 25 mM KCl or N -methyl- d -aspartate to primary cultures of cerebellar granule cells promotes expression of the N -methyl- d -aspartate subtype of glutamate receptor, as determined by electrophysiological responsiveness and susceptibility to excitotoxicity. Recent studies have demonstrated that acute mild acidosis reduces N -methyl- d -aspartate receptor channel activity by a non-competitive action of H + on an extracellular site of the receptor channel complex. Since the level of N -methyl- d -aspartate receptor expression in granule cell cultures is activity-dependent, we examined whether chronic mildly acidotic culture conditions would selectively diminish the level of N -methyl- d -aspartate responsiveness in granule cells, in effect producing a functional level of expression more comparable to that observed in vivo . To test this, cerebellar granule cells from eight-day neonatal rats were grown in an HCO 3 -buffered medium containing elevated K + (25 mM KCl) either under standard conditions (95% air/5% CO 2 , pH 7.4), or under chronic mildly acidotic conditions (90% air/10% CO 2 , estimated pH of 7.1). Glutamate receptor subtype expression was subsequently assessed using standard neurotoxicity assays, a quantitative immunoblotting assay for N -methyl- d -aspartate receptors and whole cell patch clamp recordings. Cells grown in the 10% CO 2 environment exhibited a significant reduction in susceptibility to l -glutamate neurotoxicity (at least 10-fold), but not kainate-induced neurotoxicity, relative to cells grown in 5% CO 2 . In both culture conditions, l -glutamate- and kainate-induced toxicity were mediated by activation of N -methyl- d -aspartate andnon- N -methyl- d -aspartate receptors, respectively, as determined by the sensitivity of agonist-induced toxicity to specific receptor antagonists. Using polyclonal antibodies generated against a peptide sequence recognizing five of eight splice variants in the common “R1” subunit of N -methyl- d -aspartate receptors, a 31% reduction in the amount of immunoreactive protein was observed in membrane preparations from cells grown in 10% CO 2 , relative to the amount detected in cells grown in 5% CO 2 . Moreover, perfusion of cells with glutamate (50 μM) in a nominally Mg 2+ -free solution containing glycine (2 μM) elicited N -methyl- d -aspartate antagonist-sensitive inward currents in proportionately fewer cells cultured in 10% CO 2 , relative to cells cultured in 5% CO 2 . Long-term survival was also significantly enhanced in cells exposed chronically to mild acidotic culture conditions, relative to cells grown under standard pH conditions (22 days, 10% CO 2 vs 16 days, 5% CO 2 ). Collectively, these results demonstrate that exposure of cerebellar granule cells to conditions of chronic mild acidosis reduces the number of N -methyl- d -aspartate receptors expressed in mature cultures. The N -methyl- d -aspartate responsiveness observed under such conditions may be more comparable to that observed in cerebellar granule cells in vivo following maturation.