Mogensen H S, Jørgensen O S
Department of Pharmacology, University of Copenhagen, Rigshospitalet, Denmark.
Neurochem Int. 1996 Nov;29(5):497-506. doi: 10.1016/0197-0186(96)00018-6.
We have previously reported that, unlike their rat counterparts, the survival of mouse cerebellar granule cells is independent of chronic stimulation whether owing to elevated K(+)-induced depolarization or NMDA (N-methyl-D-aspartate) receptor activation. One explanation could be that during the critical period mouse granule cells are very sensitive to NMDA receptor stimulation by endogenous glutamate released in the cultures. If so, this might be reflected by an increased expression of NMDA receptors or an increased response to their activation. We tested this hypothesis by measuring (a) the concentration of mRNA for the obligatory NMDA receptor subunit, NMDAR1, and (b) the glutamate/NMDA stimulated increase in cytosolic Ca(2+)-ion concentration in cultures at physiological or elevated K(+)-ion concentration. The expression of NMDAR1 mRNA was measured by competitive PCR of reversely transcribed mRNA and was normalized to that of the constitutively expressed H3.3 histone mRNA. The glutamate and NMDA stimulated increase in cytosolic Ca(2+)-ion concentration was measured using the fluorescent Ca(2+)-chelator Fluo3. In contrast to the hypothesis, we found NMDAR1 mRNA expression to be lower in mouse than in rat granule cells cultured for 4 days at physiological K(+)-ion concentration. However, the NMDA stimulated increase in cytosolic Ca(2+)-ion concentration did not differ in 4-day rat and mouse cultures. Although the glutamate-stimulated increase in cytosolic Ca(2+)-ion concentration in 2-day cultures was higher in mouse granule cells than in rat granule cells, the developmental profile of the glutamate-stimulated increase in cytosolic Ca(2+)-ion concentration was the same in both cases. In conclusion, we found no obvious evidence for increased NMDA receptor activity in mouse cerebellar granule cells cultured at physiological K(+)-ion concentration.
我们之前曾报道，与大鼠小脑颗粒细胞不同，小鼠小脑颗粒细胞的存活不依赖于慢性刺激，无论这种刺激是由高钾诱导的去极化还是NMDA（N-甲基-D-天冬氨酸）受体激活所引起。一种解释可能是，在关键时期，小鼠颗粒细胞对培养物中内源性谷氨酸释放所引起的NMDA受体刺激非常敏感。如果是这样，这可能会通过NMDA受体表达增加或对其激活的反应增强来体现。我们通过测量以下内容来检验这一假设：（a）必需的NMDA受体亚基NMDAR1的mRNA浓度，以及（b）在生理或高钾离子浓度下培养的细胞中，谷氨酸/NMDA刺激引起的胞质钙离子浓度升高。通过对逆转录mRNA进行竞争性PCR来测量NMDAR1 mRNA的表达，并将其标准化为组成型表达的H3.3组蛋白mRNA的表达。使用荧光钙螯合剂Fluo3测量谷氨酸和NMDA刺激引起的胞质钙离子浓度升高。与假设相反，我们发现，在生理钾离子浓度下培养4天的小鼠颗粒细胞中，NMDAR1 mRNA表达低于大鼠颗粒细胞。然而，在培养4天的大鼠和小鼠细胞中，NMDA刺激引起的胞质钙离子浓度升高并无差异。虽然在培养2天的细胞中，小鼠颗粒细胞中谷氨酸刺激引起的胞质钙离子浓度升高高于大鼠颗粒细胞，但在这两种情况下，谷氨酸刺激引起的胞质钙离子浓度升高的发育情况是相同的。总之，我们没有发现明显证据表明在生理钾离子浓度下培养的小鼠小脑颗粒细胞中NMDA受体活性增加。
