Gundersen V, Chaudhry F A, Bjaalie J G, Fonnum F, Ottersen O P, Storm-Mathisen J
Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Blindern, N-0317 Oslo, Norway.
J Neurosci. 1998 Aug 15;18(16):6059-70. doi: 10.1523/JNEUROSCI.18-16-06059.1998.
To elucidate the role of aspartate as a signal molecule in the brain, its localization and those of related amino acids were examined by light and electron microscopic quantitative immunocytochemistry using antibodies specifically recognizing the aldehyde-fixed amino acids. Rat hippocampal slices were incubated at physiological and depolarizing [K+] before glutaraldehyde fixation. At normal [K+], aspartate-like and glutamate-like immunoreactivities were colocalized in nerve terminals forming asymmetrical synapses on spines in stratum radiatum of CA1 and the inner molecular layer of fascia dentata (i.e., excitatory afferents from CA3 and hilus, respectively). During K+ depolarization there was a loss of aspartate and glutamate from these terminals. Simultaneously the immunoreactivities strongly increased in glial cells. These changes were Ca2+-dependent and tetanus toxin-sensitive and did not comprise taurine-like immunoreactivity. Adding glutamine at CSF concentration prevented the loss of aspartate and glutamate and revealed an enhancement of aspartate in the terminals at moderate depolarization. In hippocampi from animals perfused with glutaraldehyde during insulin-induced hypoglycemia (to combine a strong aspartate signal with good ultrastructure) aspartate was colocalized with glutamate in excitatory terminals in stratum radiatum of CA1. The synaptic vesicle-to-cytoplasmic matrix ratios of immunogold particle density were similar for aspartate and glutamate, significantly higher than those observed for glutamine or taurine. Similar results were obtained in normoglycemic animals, although the nerve terminal contents of aspartate were lower. The results indicate that aspartate can be concentrated in synaptic vesicles and subject to sustained exocytotic release from the same nerve endings that contain and release glutamate.
为阐明天冬氨酸作为脑内信号分子的作用，我们使用特异性识别醛固定氨基酸的抗体，通过光镜和电镜定量免疫细胞化学方法检测了其定位以及相关氨基酸的定位。在戊二醛固定前，将大鼠海马切片置于生理浓度和去极化浓度的[K⁺]环境中孵育。在正常[K⁺]浓度下，天冬氨酸样和谷氨酸样免疫反应性共定位于在CA1辐射层的棘突上形成不对称突触的神经末梢以及齿状回内分子层（即分别来自CA3和海马回的兴奋性传入纤维）。在K⁺去极化过程中，这些末梢中天冬氨酸和谷氨酸减少。同时，神经胶质细胞中的免疫反应性强烈增加。这些变化依赖于Ca²⁺且对破伤风毒素敏感，不包括牛磺酸样免疫反应性。以脑脊液浓度添加谷氨酰胺可防止天冬氨酸和谷氨酸的减少，并显示在中度去极化时末梢中天冬氨酸增加。在胰岛素诱导低血糖期间用戊二醛灌注的动物海马中（为了将强烈的天冬氨酸信号与良好的超微结构相结合），天冬氨酸与谷氨酸在CA1辐射层的兴奋性末梢中共定位。天冬氨酸和谷氨酸的免疫金颗粒密度的突触小泡与细胞质基质比率相似，显著高于谷氨酰胺或牛磺酸的比率。在血糖正常的动物中也获得了类似结果，尽管天冬氨酸的神经末梢含量较低。结果表明，天冬氨酸可浓缩于突触小泡中，并可从含有和释放谷氨酸的相同神经末梢持续进行胞吐释放。
