CALRETININ-CONTAINING NEURONS AND EXCITOTOXIC INJURY

Calcretinin(CR)是一种钙结合蛋白， 主要发现于脑神经元的亚群中，如 黑质(SN)和腹侧被盖区(VTA)。 我们之前已经证明，一些SN/VTA多巴胺- 含细胞与CR共定位。进一步研究和探讨 完整的大鼠胚胎大脑显示共定位 从E16开始。我们观察到许多CR免疫反应(IR)。 酪氨酸羟化酶(TH)以外的神经元 组织培养中的免疫反应细胞，但我们还没有 培养的胚胎细胞(E14)中发现CR+TH共存 在长达12天的培养中，似乎有一些因素 是必需的，它存在于大脑中，但不是分离的 文化。我们还研究了兴奋性氨基酸的作用。 酸(EAA)对SN培养中CR和TH表达的影响。 CR和TH神经元在24小时内受到不同程度的影响 与红藻氨酸(KA)、NMDA或AMPA(100-500)孵育 总的来说，TH-IR神经元表现出更大的变化 与EAA治疗后的CR-IR神经元相比，两种类型的 细胞受影响的严重程度依次为：KA 大于AMPA大于NMDA。退化，退化 神经细胞和突起较少被观察到 在CR-IR细胞中，KA(47%)和NMDA(22%)与 TH-IR细胞(KA，57%；NMDA和AMPA，35%)。两人都幸存了下来 CR和TH细胞表现出更多的纤维分支， 但这些细胞的外观有所不同。而幸存的CR 细胞突起延伸到很长的距离，TH细胞 出现了越来越多的轮辐状突起 直接从细胞体中分离出来，其中许多已经肿胀 静脉曲张。长条分叉的戏剧性外观 EAA后的静脉曲张过程，尤其是KA 我们对新纤维是否发芽的关注 在地产代理监管局生效后的24小时内，或 CR的周转和/或轴浆流量增加 在控制下看不见的现有纤维 条件。定量CR的放射免疫分析显示 KA(42%)和AMPA(17%)后CR蛋白丢失，但没有 对NMDA处理的细胞的影响。一种裂解核糖核酸酶 保护实验显示显著丢失了对 KA治疗组CR和TH均为81%，TH为76%； AMPA治疗后较小的下降(CR：62%，TH：43%)和 NMDA处理的细胞(CR：58%TH：36%)。这些数据不会 明确地揭示了增加的 EAA后的神经丛，正如CR-IR显示的主要 蛋白质从细胞体进入挪威的运动 在一些细胞中萌发，而在两个细胞中 蛋白质和信使核糖核酸提示部分细胞的退化 细胞。这是否反映了两者之间的差异 SN致密(A9)或VTA(A10)细胞仍待显示。 这种区别以前是在以下情况下发现的 用SN神经毒素MPTP治疗猴子，它 摧毁了A9细胞，但没有破坏A10亚群。自.以来 CR在培养的TH细胞内共定位不是 我们不能从这些实验中得出结论 含铬的多巴胺细胞免受神经毒性的保护 由机管局造成的伤害。

Calcretinin (CR) is a calcium binding protein that is mostly found in subpopulations of brain neurons such as the substantia nigra (SN) and the ventral tegmental area (VTA). We have previously shown that some of the SN/VTA dopamine- containing cells colocalize with CR. A further study of intact rat embryo brains revealed that colocalization begins at E16. We observed many CR immunoreactive (IR) neurons in addition to tyrosine hydroxylase (TH) immunoreactive cells in tissue culture, but we have not found CR+TH colocalization in embryonic cells (E14) grown for up to 12 days in culture, It appears that some factor is required which is present in brain but not dissociated cultures. We also studied the effects of excitatory amino acids (EAA) on the expression of CR and TH in SN cultures. CR and TH neurons were differentially affected by 24 hour incubation with kainic acid (KA), NMDA or AMPA (100-500 muM)> In general, TH-IR neurons exhibited greater change than CR-IR neurons after EAA treatment, and both types of cells were affected in a decreasing order of severity: KA greater than AMPA greater than NMDA. Degeneration of neuronal cells and processes were less frequently observed in CR-IR cells after KA (47%) and NMDA (22%) compared with TH-IR cells (KA, 57%; NMDA and AMPA, 35%). Both surviving CR and TH cells exhibited increased fiber ramifications, but the cells' appearances differed. Whereas surviving CR cell processes extended for very long distances, TH cells had increased numbers of spoke-like processes emerging directly from cell bodies, many of which had swollen varicosities. The dramatic appearance of long ramifying varicose processes following EAA, especially KA, focuses our attention on whether sprouting of new fibers occurs within 24 h following the EAA, or on the possibility that there is an increased turnover and/or axoplasmic flow of CR to existing fibers that were not visible under control conditions. A radioimmunoassay to quantify CR revealed a loss of CR protein after KA (42%), and AMPA (17%), but no effect in NMDA-treated cells. A lysate ribonuclease protection assay revealed a significant loss of mRNA for both CR and TH in the KA-treated group (CR:81%, TH:76%) and a lesser decrease in the AMPA-treated (CR:62%, TH:43%) and NMDA-treated cells (CR:58% TH:36%). These data do not definitively reveal the significance of the increased plexus of fibers following EAA, as CR-IR suggests a major movement of protein from cell bodies into norway gnawer or sprouting in some cells while a reduction in both the protein and mRNA suggests degeneration of a subset of cells. Whether this is a reflection of differences between SN compacta (A9) or VTA (A10) cells remains to be shown. Such a distinction was previously uncovered following treatment in monkeys with the SN neurotoxin, MPTP, which destroyed A9 cells but not the A10 subpopulation. Since colocalization of CR within TH cells in culture was not revealed, we cannot conclude from these experiments that CR-containing dopamine cells are protected from neurotoxic injury by EAA.