Johnson S M, Bywood P T
School of Medicine, Flinders University of South Australia, Adelaide 5001, South Australia, Australia.
Exp Neurol. 1998 Jun;151(2):229-36. doi: 10.1006/exnr.1998.6783.
We have recently established a rat substantia nigra (SN) slice preparation in which a sensitive index of excitatory amino acid (EAA) toxicity was degeneration of the dendritic arbor of catecholamine neurons labelled by immunostaining for tyrosine hydroxylase (TH). The present study examined the pharmacological characteristics of EAA-induced neurotoxicity. Rats were anesthetised by halothane inhalation and killed, the brain was rapidly removed, and 400-microm-thick SN slices cut in the horizontal plane on a vibratome. Slices were incubated in saline buffer at 35 degreesC for 15 min to 6 h in the presence or absence or absence of kainic acid (KA) or N-methyl-D-aspartate (NMDA) in concentrations ranging from 10 to 500 microM. The slices were then fixed and resectioned into 40-microm sections that were coplanar with the parent slice. Dopaminergic SN neurons were labeled using antibody to tyrosine hydroxylase (TH) coupled to diaminobenzidine. A feature of the immunostaining was that it labeled not only the cell body but also the prolific dendritic arborization of SN neurons. Dendritic damage was quantified by counting the proportion of neurons with intact dendrites after treatment with EAA. KA and NMDA caused loss of dendrites that was prevented by CNQX (20 microM) and MK-801 (20 microM), respectively, indicating that activation of either NMDA or non-NMDA receptors produces neurotoxicity. EAA-induced dendritic damage was observed within 2 h of treatment with a low concentration (10 microM) of KA and within 15 min if the concentration was increased to 500 microM. Thus the loss of dendrites occurs rapidly and precedes disintegration of the cell bodies. Furthermore, brief (15 min) exposure to EAA initiated damage in the dendrites which progressed after the EAA was removed from its receptor. The observations are consistent with the postulated role of EAAs in neurodegenerative diseases. Labeling the dendritic arbor provides a sensitive approach to investigating the cellular mechanisms of neurodegeneration of catecholamine neurons.
我们最近建立了一种大鼠黑质（SN）脑片制备方法，其中，通过对酪氨酸羟化酶（TH）进行免疫染色标记的儿茶酚胺能神经元树突状分支的退化，是兴奋性氨基酸（EAA）毒性的一个敏感指标。本研究检测了EAA诱导的神经毒性的药理学特征。通过吸入氟烷麻醉大鼠并将其处死，迅速取出大脑，在振动切片机上切成400微米厚的水平SN脑片。脑片在35℃的生理盐水缓冲液中孵育15分钟至6小时，存在或不存在浓度范围为10至500微摩尔的海藻酸（KA）或N-甲基-D-天冬氨酸（NMDA）。然后将脑片固定并切成与母脑片共面的40微米切片。使用与二氨基联苯胺偶联的酪氨酸羟化酶（TH）抗体标记多巴胺能SN神经元。免疫染色的一个特点是，它不仅标记了细胞体，还标记了SN神经元丰富的树突分支。通过计算EAA处理后树突完整的神经元比例来量化树突损伤。KA和NMDA分别导致树突丢失，而CNQX（20微摩尔）和MK-801（20微摩尔）可预防这种情况，这表明NMDA或非NMDA受体的激活都会产生神经毒性。用低浓度（10微摩尔）的KA处理2小时内即可观察到EAA诱导的树突损伤，如果浓度增加到500微摩尔，则在15分钟内即可观察到。因此，树突的丢失迅速发生，且先于细胞体的解体。此外，短暂（15分钟）暴露于EAA会引发树突损伤，在EAA从其受体上移除后这种损伤仍会继续发展。这些观察结果与EAA在神经退行性疾病中的假定作用一致。标记树突分支为研究儿茶酚胺能神经元神经退行性变的细胞机制提供了一种敏感的方法。
