Conti F, DeBiasi S, Minelli A, Rothstein J D, Melone M
Institute of Human Physiology, University of Ancona, Italy.
Cereb Cortex. 1998 Mar;8(2):108-16. doi: 10.1093/cercor/8.2.108.
High-affinity uptake of glutamate from the synaptic cleft plays a crucial role in regulating neuronal activity in physiological and pathological conditions. We have used affinity-purified specific polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of rabbit and rat EAAC1, a glutamate (Glu) transporter believed to be exclusively neuronal, to investigate its cellular and subcellular localization and whether it is expressed exclusively in glutamatergic cells of infragranular layers, as suggested by previous studies. Light microscopic studies revealed that EAAC1 immunoreactivity (ir) is localized to neurons and punctate elements in the neuropil. EAAC1-positive neurons were more numerous in layers II-III and V-VI, i.e. throughout all projection layers. Most EAAC1-positive neurons were pyramidal, although nonpyramidal cells were also observed. Some EAAC1-positive non-pyramidal neurons stained positively with an antiserum to GAD, thus demonstrating that EAAC1 is not confined to glutamatergic neurons. Non-neuronal EAAC1-positive cells were also observed in the white matter, and some of them stained positively with an antiserum to GFAP. Ultrastructural studies showed that EAAC1-ir was in neuronal cell bodies, dendrites and dendritic spines, but not in axon terminals, i.e. exclusively postsynaptic. Analysis of the type of axon terminals synapsing on EAAC1-ir profiles showed that 97% of them formed asymmetric contacts, thus indicating that EAAC1 is located at the very sites of excitatory amino acid release. Unexpectedly, EAAC1-ir was also found in a few astrocytic processes located in both the gray and the white matter. The localization of EAAC1 may explain the pathological symptoms that follow EAAC knockout (seizures and mild toxicity), as seizures could be due to the loss of EAAC1-mediated fine regulation of neuronal excitability at axodendritic and axospinous synapses, whereas the mild toxicity may be related to the functional inactivation of astrocytic EAAC1.
从突触间隙高亲和力摄取谷氨酸在生理和病理条件下调节神经元活动中起关键作用。我们使用针对与兔和大鼠EAAC1的C末端区域相对应的合成肽产生的亲和纯化特异性多克隆抗体，EAAC1是一种被认为仅存在于神经元中的谷氨酸（Glu）转运体，以研究其细胞和亚细胞定位，以及它是否如先前研究所暗示的那样仅在颗粒下层的谷氨酸能细胞中表达。光学显微镜研究表明，EAAC1免疫反应性（ir）定位于神经元和神经毡中的点状成分。EAAC1阳性神经元在II-III层和V-VI层中较多，即在所有投射层中都有。大多数EAAC1阳性神经元是锥体神经元，尽管也观察到了非锥体神经元。一些EAAC1阳性非锥体神经元用抗GAD抗血清染色呈阳性，因此表明EAAC1并不局限于谷氨酸能神经元。在白质中也观察到了非神经元EAAC1阳性细胞，其中一些用抗GFAP抗血清染色呈阳性。超微结构研究表明，EAAC1-ir存在于神经元细胞体、树突和树突棘中，但不存在于轴突终末，即仅存在于突触后。对与EAAC1-ir轮廓形成突触的轴突终末类型的分析表明，其中97%形成不对称接触，因此表明EAAC1位于兴奋性氨基酸释放的部位。出乎意料的是，在灰质和白质中的一些星形胶质细胞突起中也发现了EAAC1-ir。EAAC1的定位可能解释了EAAC基因敲除后出现的病理症状（癫痫发作和轻度毒性），因为癫痫发作可能是由于EAAC1介导的轴突-树突和轴突-棘突触处神经元兴奋性精细调节的丧失，而轻度毒性可能与星形胶质细胞EAAC1的功能失活有关。
