Stimulation of the N-methyl-D-aspartate receptor promotes the biochemical differentiation of cerebellar granule neurons and not astrocytes.

Cerebellar granule cells are believed to be glutamatergic, but, as they receive excitatory amino acidergic input from mossy fibers, they also possess N-methyl-D-aspartate (NMDA) receptors. The possible involvement of these NMDA receptors in the biochemical differentiation of cultured granule neurons was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 3 days (i.e. between 2 and 5 days in vitro), it elevated specific activity of glutaminase in the dose- and time-dependent manners. The half-maximal effect was obtained at about 10 microM NMDA, whereas the maximum concentration, which produced about a 2.7-fold increase in 5-day-old cultures, was about 50 microM NMDA. This increase in glutaminase was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca2+ ion channel blockers, MK 801 and Mg2+. The effect of NMDA was not related to the survival of the granule cells, as the experiments were carried out before the dependence on high K+ for the survival of granule cells develops in culture, and during the period of investigation none of the compounds used compromised the survival of these cells. The enhancement of glutaminase activity was due to an induction in enzyme protein, since it was completely blocked by cycloheximide and actinomycin D. In contrast to granule neurons, the treatment with NMDA had no significant effect on the activity of glutaminase and glutamine synthetase in cultured cerebellar astroglial cells. Our present results on glutaminase enzyme would indicate that an increase in the cellular concentration of free Ca2+ mediated through the NMDA induced increase in Ca2+ conductance, leads to long term changes in differentiating cerebellar granule neurons, and it is possible that this kind of physiological stimulation of granule cells is normally provided in vivo by the presynaptic glutamatergic mossy fibers.