Carol M. Beaman-Hall

Glia Modulate NMDA‐Mediated Signaling in Primary Cultures of Cerebellar Granule Cells

Abstract: Excessive activation of N‐methyl‐d‐aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR‐mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte‐rich and astrocyte‐poor cultures. In astrocyte‐poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte‐rich neuronal cultures demonstrated three significant differences, compared with astrocyte‐poor cultures: (a) Neuronal viability was increased; (b) glutamate‐mediated neurotoxicity was decreased, consistent with the presence of a sodium‐coupled glutamate transport system in astrocytes; and (c) NMDA‐ but not kainate‐mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT‐PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR‐mediated changes in intracellular Ca2+ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte‐rich and astrocyte‐poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA‐mediated toxicity in astrocyte‐poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR‐mediated toxicity.

Ca2+ and pH modulate alternative splicing of exon 5 in NMDA receptor subunit 1.

RT-PCR and intracellular Ca2+ measurements were used to identify factors that modulate alternative splicing of exon 5 in the NMDA receptor transcript encoding NR1, in cultured cerebellar granule neurons. Although cells grown in media containing 5 mM KCl demonstrate compromised survival, they show the predicted developmental transition from NR1a (-exon 5) to NR1b (+exon 5) mRNA expression. This transition was blocked under culture conditions that promote survival; inclusion or exclusion of exon 5 is a reversible process that is sensitive to alterations in Ca2+ and pH. We conclude that alternative splicing of NR1 pre-mRNA transcripts may be regulated by developmental cues that modulate the degree of glutamate receptor activation.

Transcriptional profiling of depolarization-dependent phenotypic alterations in primary cultures of developing granule neurons

Rat cerebellar granule neurons cultured in medium supplemented with elevated KCl are extensively used as a model to examine the coupling between neural activity and Ca(2+)-dependent gene expression. Elevated (25 mM) KCl is believed to mimic endogenous neural activity because it promotes depolarization and Ca(+2)-dependent survival and some aspects of maturation. By comparison, at least half of the granule neurons grown in standard medium containing 5 mM KCl undergo apoptosis beginning approximately 4 days in vitro. However, accumulating evidence suggests that chronic depolarization induces phenotypic abnormalities whereas growth in chemically defined medium containing 5 mM KCl more closely resembles the constitutive phenotype. To examine this, oligonucleotide microarrays and RT-PCR of selected mRNAs were used to compare transcription profiles of cultures grown in 5 mM and 25 mM KCl. In some cases, N-methyl-D-aspartate (NMDA) which, like elevated KCl, promotes long-term survival was also tested. Robust changes in several gene groups were observed and indicated that growth in elevated KCl: induces expression of mRNAs that are not normally observed; represses expression of mRNAs that should be present; maintains expression of mRNAs that are markers of immature neurons. Supplementation of the growth medium with NMDA instead of elevated KCl produces similar abnormalities. Altogether, these data indicate that growth in 5 mM KCl more closely mimics survival and maturation of granule neurons in vivo and should therefore be adopted in future studies.

Reduced blockade by extracellular Mg2+ is permissive to NMDA receptor activation in cerebellar granule neurons that model a migratory phenotype

J. Neurochem. (2010) 114, 191–202.