Growth conditions differentially modulate the vulnerability of developing cerebellar granule cells to excitatory amino acids

Abstract

The survival of immature nerve cells in a cerebellar culture, predominantly excitatory granule cells, is known to be promoted by chronic exposure to high K + (> 20 mM) or glutamate (Glu) receptor agonists. These treatments are believed to mimic the in vivo effect of the incoming glutamatergic afferents, the mossy fibres. Here we report that with maturation the cells become vulnerable to excitatory amino acids (EAAs) and that the characteristics of EAA sensitivity are dependent on the environmental influences being either “trophic” (25 mM K + or 140 μM NMDA, K25 or K10 + NMDA) or “non-trophic” (10 mM K +, K10). Toxicity was assayed routenely at 9 days in vitro (DIV) after 24 h exposure to EAAs. Under all the tested conditions, the effect of Glu was mediated exclusively through NMDA receptors. However, the efficacy and potency of Glu were high in K25- and K10 + NMDA-grown cells compared with K10-grown cells. Growth conditions had the same influence on NMDA as on Glu-induced toxicity, but with the following special features: (1) in comparison with K25 cells, the potency of NMDA was significantly lower in K10 + NMDA cells. The K10 + NMDA cultures behaved as if they were completely insensitive to the NMDA which is present in their growth medium. (2) The K10-grown cells were not vulnerable to NMDA, unless the cell membrane was depolarised by shifting the cells into K25 medium. The efficacy of NMDA became then similar to that in K25 cultures, although the potency was about 7-fold less. Thus NMDA receptors can be activated by the depolarisation of K10 cells, implying the operation of Mg 2+ blockade of the channel at normal resting membrane potential. Although non-NMDA receptors did not seem to be involved in Glu toxicity, cells were vulnerable to kainate, which killed significantly more cells than Glu (about 80% vs 70%). This was partly due to the resistance of GABAergic interneurons present in the cultures to Glu- or NMDA-induced toxicity. In contrast to the effects of Glu or NMDA, KA vulnerability was lower in cells grown in K25 or K40 than K10 medium (rank order K10 > K25 > K40). Under our experimental conditions, cultured cells were resistant to AMPA, quisqualate and the selective metabotropic Glu receptor agonist 1 S,3 R-ACPD. Collectively, the observations indicated that EAA sensitivity of cultured cerebellar interneurons is significantly and differentially influenced by environmental factors, believed to mimic in vivo trophic influences on these cells.