Garthwaite G, Hajós F, Garthwaite J
Neuroscience. 1986 Jun;18(2):437-47. doi: 10.1016/0306-4522(86)90164-8.
The ionic requirements for the neurotoxic effects of N-methyl-D-aspartate and kainate in incubated slices of developing rat cerebellum were studied using light and electron microscopy. Under normal conditions, 30 min exposure to 100 microM N-methyl-D-aspartate followed by a 90 min recovery period in agonist-free medium resulted in the necrosis of differentiating granule cells and deep nuclear neurons, while the corresponding effect of 100 microM kainate was the death of Golgi cells. Substitution of 96% of the Cl- in the medium with isethionate did not prevent the toxicity of either agonist. However, all the ordinarily vulnerable cells survived and exhibited normal ultrastructure if the slices were exposed to the excitants in a Ca2+-free medium and were subsequently allowed to recover in a Ca2+-containing solution. Prior to this recovery period, granule, Golgi and deep nuclear neurons exposed to N-methyl-D-aspartate were markedly swollen but their mitochondria were hypercontracted and there was no clumping of chromatin or obvious swelling of the rough endoplasmic reticulum or Golgi apparatus, in contrast to observations made on slices exposed to this agonist in normal medium. Substitution of all the Na+ in the medium with a mixture of choline (118 mM) and Tris (25 mM) itself caused necrosis of granule cells and deep nuclear neurons and an intense microvacuolation of Purkinje cells, due, in large part, to high amplitude mitochondrial swelling. A low (25 mM) Na+ medium was well tolerated under control conditions. This medium protected granule cells but not deep nuclear neurons from the toxicity of N-methyl-D-aspartate and failed to prevent kainate-induced death of Golgi cells. It is concluded that the acute neurotoxic effects of the two excitatory amino acid receptor agonists in the slices are dependent on extracellular Ca2+ and are independent of extracellular Cl-. Where apparent, the protective effect of reducing extracellular Na+ on the toxicity of N-methyl-D-aspartate is likely to reflect the involvement of this ion in the primary depolarizing mechanism.
利用光学显微镜和电子显微镜，研究了发育中大鼠小脑孵育切片中N-甲基-D-天冬氨酸和海人藻酸神经毒性作用的离子需求。在正常条件下，将切片暴露于100μM N-甲基-D-天冬氨酸30分钟，然后在无激动剂的培养基中恢复90分钟，导致分化中的颗粒细胞和深核神经元坏死，而100μM海人藻酸的相应作用是高尔基细胞死亡。用羟乙基磺酸替代培养基中96%的Cl-并不能防止任何一种激动剂的毒性作用。然而，如果切片在无Ca2+的培养基中暴露于兴奋性氨基酸，随后在含Ca2+的溶液中恢复，则所有通常易受损的细胞均存活并表现出正常的超微结构。在这个恢复期之前，暴露于N-甲基-D-天冬氨酸的颗粒细胞、高尔基细胞和深核神经元明显肿胀，但其线粒体过度收缩，染色质没有凝聚，粗面内质网或高尔基体也没有明显肿胀，这与在正常培养基中暴露于该激动剂的切片的观察结果相反。用胆碱（118 mM）和Tris（25 mM）的混合物替代培养基中所有的Na+本身会导致颗粒细胞和深核神经元坏死以及浦肯野细胞强烈的微空泡化，这在很大程度上是由于线粒体的高幅度肿胀所致。在对照条件下，低（25 mM）Na+培养基耐受性良好。这种培养基可保护颗粒细胞免受N-甲基-D-天冬氨酸的毒性作用，但不能保护深核神经元，也不能防止海人藻酸诱导的高尔基细胞死亡。结论是，两种兴奋性氨基酸受体激动剂在切片中的急性神经毒性作用依赖于细胞外Ca2+，且与细胞外Cl-无关。在明显的情况下，降低细胞外Na+对N-甲基-D-天冬氨酸毒性的保护作用可能反映了该离子参与了初级去极化机制。
