Aoki Chiye, Fujisawa Sho, Mahadomrongkul Veera, Shah Priti J, Nader Karim, Erisir Alev
Center for Neural Science, New York University, Rm 809, 4 Washington Pl., New York, NY 10003, USA.
Brain Res. 2003 Feb 14;963(1-2):139-49. doi: 10.1016/s0006-8993(02)03962-8.
Past in vitro studies have used immunofluorescence to show increased clustering of the NR1 subunits of NMDA receptors (NMDAR) following NMDAR blockade, indicating that NMDARs self-regulate trafficking to and from spines. However, since a substantial portion of spinous NMDAR subunits can reside at sites removed from plasma membranes, whether or not these immunofluorescent clusters are synaptic remains to be shown. Also, the NR2A/B subunits undergo activity-dependent switching at synapses, indicating that their subcellular distribution may be regulated differently from the NR1 subunits. We examined the issue of NMDAR autoregulation by determining whether in vivo NMDAR blockade enhances trafficking of the NR2A subunits toward spines and more specifically to postsynaptic densities (PSDs) of already mature synapses. Seven adult rats received unilateral intra-cortical infusion of the NMDAR antagonist, D-AP5 for 1/2-2 h and the inactive enantiomer or the solvent, alone, in the contralateral cortex. Using an electron microscope, approximately 5600 cortical spines originating from the two hemispheres of the seven adult animals were analyzed for the location of NR2A subunits. In six out of the seven cases analyzed, the D-AP5-treated neuropil exhibited increased immunolabeling at PSDs and a concomitantly great increase at non-synaptic sites within spines. NR2A subunits also increased presynaptically within 1/2 h but not after 1 h. These findings indicate that NR2A subunits in intact, adult cortical neurons are prompted to become trafficked into spines and axon terminals by NMDAR inactivity, yielding an increase of a readily available reserve pool and greater localization at both sides of synapses.
过去的体外研究利用免疫荧光技术显示，在NMDAR被阻断后，NMDA受体（NMDAR）的NR1亚基聚集增加，这表明NMDAR可对其在树突棘的内吞和外排进行自我调节。然而，由于相当一部分棘状NMDAR亚基可能位于远离质膜的位置，这些免疫荧光簇是否位于突触处仍有待证实。此外，NR2A/B亚基在突触处会发生活性依赖性转换，这表明它们的亚细胞分布可能与NR1亚基受到不同的调节。我们通过确定体内NMDAR阻断是否会增强NR2A亚基向树突棘的转运，更具体地说是向已成熟突触的突触后致密物（PSD）的转运，来研究NMDAR的自我调节问题。七只成年大鼠在单侧皮质内注射NMDAR拮抗剂D-AP5 1/2 - 2小时，对侧皮质则注射无活性对映体或溶剂。使用电子显微镜，对来自这七只成年动物两个半球的约5600个皮质树突棘进行分析，以确定NR2A亚基的位置。在分析的七个案例中的六个中，经D-AP5处理的神经毡在PSD处的免疫标记增加，同时在树突棘内的非突触部位也大幅增加。NR2A亚基在突触前也在1/2小时内增加，但在1小时后没有增加。这些发现表明，完整成年皮质神经元中的NR2A亚基会因NMDAR失活而被转运到树突棘和轴突终末，从而增加了易于利用的储备池，并在突触两侧有更多的定位。
