Rudolf G D, Cronin C A, Landwehrmeyer G B, Standaert D G, Penney J B, Young A B
Department of Neurology, Massachusetts General Hospital, Boston, USA.
Neuroscience. 1996 Jul;73(2):417-27. doi: 10.1016/0306-4522(96)00048-6.
The laminar distribution and cellular levels of expression of mRNAs encoding N-methyl-D-aspartate receptor subunits (NMDAR1, NMDAR2A-D and the alternatively spliced isoforms of NMDAR1) were examined in prefrontal cortex of rat by in situ hybridization using film and emulsion autoradiography. Film autoradiograms demonstrated a distinctive laminar distribution of hybridization signals for each of the probes recognizing NMDAR1, NMDAR2A, and NMDAR2B messenger RNA; hybridization with probes for NMDAR2C and NMDAR2D resulted in scattered signals without laminar organization. Grain counting disclosed that neurons in layer V displayed the highest and neurons in layer IV the lowest absolute number of grains for all probes examined. Correction for cell size demonstrated statistically significant differences in cellular labelling density of up to 50% across neurons in different cortical layers. The cellular density profiles across cortical laminae differed between probes. Hybridization with a probe recognizing all isoforms of NMDAR1 resulted in significantly lower densities of cellular labelling in neurons of layer IV than of layers II/III, V and VI. Cellular labelling densities following hybridization with probes recognizing alternatively spliced segments of NMDAR1 were examined. Densities were low in neurons of the upper cortical layers II/III and IV using probes for the messenger RNA encoding the amino terminal insert, NMDAR11XX and the second carboxy terminal deletion, NMDAR1XX1; hybridization with a probe for the messenger RNA encoding the first carboxy terminal deletion, NMDAR1X1X, resulted in low cellular signal densities in neurons of layers IV and VIb. NMDAR2A messenger RNA expression was of relatively uniform intensity in neurons of layers II-V but significantly lower in neurons of the inner part of layer VI. NMDAR2B expression was most dense in layer II neurons. These data indicate that neurons in different cortical laminae express distinct N-methyl-D-aspartate receptor subunit messenger RNA phenotypes. In addition, the observed differences in density of N-methyl-D-aspartate receptor subunit messenger RNA expression suggest that cortical laminae differ in the relative contribution of N-methyl-D-aspartate receptors to their excitatory responses.
运用胶片和乳胶放射自显影原位杂交技术，检测大鼠前额叶皮质中编码N-甲基-D-天冬氨酸受体亚基（NMDAR1、NMDAR2A-D以及NMDAR1的可变剪接异构体）的mRNA的层状分布及细胞表达水平。胶片放射自显影片显示，识别NMDAR1、NMDAR2A和NMDAR2B信使RNA的每种探针的杂交信号均呈现出独特的层状分布；与NMDAR2C和NMDAR2D探针杂交则产生无层状结构的散在信号。颗粒计数显示，对于所有检测的探针，V层神经元的颗粒绝对数量最高，IV层神经元的颗粒绝对数量最低。细胞大小校正表明，不同皮质层的神经元在细胞标记密度上存在高达50%的统计学显著差异。不同探针的皮质各层细胞密度分布情况有所不同。与识别NMDAR1所有异构体的探针杂交后，IV层神经元的细胞标记密度显著低于II/III层、V层和VI层。检测了与识别NMDAR1可变剪接片段的探针杂交后的细胞标记密度。使用编码氨基末端插入片段NMDAR11XX和第二个羧基末端缺失片段NMDAR1XX-1的信使RNA探针时，皮质上层II/III层和IV层神经元的密度较低；与编码第一个羧基末端缺失片段NMDAR1X1X的信使RNA探针杂交后，IV层和VIb层神经元的细胞信号密度较低。NMDAR2A信使RNA在II-V层神经元中的表达强度相对均匀，但在VI层内部神经元中的表达显著较低。NMDAR2B在II层神经元中的表达最为密集。这些数据表明，不同皮质层的神经元表达不同的N-甲基-D-天冬氨酸受体亚基信使RNA表型。此外，观察到的N-甲基-D-天冬氨酸受体亚基信使RNA表达密度差异表明，皮质各层在N-甲基-D-天冬氨酸受体对其兴奋性反应的相对贡献方面存在差异。
