大鼠听皮质NMDA受体来单位NR2BmRNA年龄—依赖性表达

应用原位杂交技术，研究了大鼠生后发育过程中，听皮质神经元NMDA受体亚单位NR2B mRNA年龄－依赖性的表达变化。特异性DIG标记寡核苷酸探针检测显示，NR2B亚单位mRNA阳性神经元数量从出生后即有高水平表达，之后，随着天龄增长逐渐递减，在出生后14d出现一过性表达高峰，14－21d时表达水平急剧降低（＞50．0％），21d后保持低水平表达至成年。研究结果为进一步在皮质水平上探讨出生后听觉功能发育可塑性的分子机制提供了重要资料。     

大鼠听皮质NMDA受体亚单位NR2BmRNA年龄-依赖性表达

应用原位杂交技术,研究了大鼠生后发育过程中,听皮质神经元NMDA受体亚单位NR2B mRNA年龄-依赖性的表达变化.特异性DIG标记寡核苷酸探针检测显示,NR2B亚单位mRNA阳性神经元数量从出生后即有高水平表达,之后,随着天龄增长逐渐递减,在出生后14 d出现一过性表达高峰,14～21 d时表达水平急剧降低(＞50.0%),21 d后保持低水平表达至成年.研究结果为进一步在皮质水平上探讨出生后听觉功能发育可塑性的分子机制提供了重要资料.     

大鼠生后发育中听皮层NR1 mRNA的表达

目的：研究大鼠生后发育过程中，听皮层神经元NMDA受体NR1 mRNA的表达。方法：采用特异性DIG标记的寡核苷酸探针，分别在动物出生后第7，14，2l，28，35，42，49，56天和成年，检测听皮层NR1 mRNA阳性神经元的分布。结果：NR1亚单位mRNA阳性神经元从出生后第7天即可检测到，之后，随着天龄增加，阳性神经元分布密度呈明显递增趋势。期间，从生后第7天到第14天阳性神经元密度增加最快，增加了33．80％。到生后第35天NR1 mRNA表达达到高峰，一直保持到成年。对称的两侧听皮层阳性神经元表达模式完全一致。结论：听皮层NR1 mRNA表达与动物年龄相关。研究结果为进一步在皮层水平上探讨生后听觉功能发育可塑性的分子机制提供了重要资料。     

大鼠生后发育中听皮层NR1 mRNA的表达

目的:研究大鼠生后发育过程中,听皮层神经元NMDA受体NR1 mRNA的表达.方法:采用特异性DIG标记的寡核苷酸探针,分别在动物出生后第7,14,21,28,35,42,49,56天和成年,检测听皮层NR1 mRNA阳性神经元的分布.结果:NR1亚单位mRNA阳性神经元从出生后第7天即可检测到,之后,随着天龄增加,阳性神经元分布密度呈明显递增趋势.期间,从生后第7天到第14天阳性神经元密度增加最快,增加了33.80%.到生后第35天NR1 mRNA表达达到高峰,一直保持到成年.对称的两侧听皮层阳性神经元表达模式完全一致.结论:听皮层NR1 mRNA表达与动物年龄相关.研究结果为进一步在皮层水平上探讨生后听觉功能发育可塑性的分子机制提供了重要资料.     

神经干细胞分化的神经元离子型谷氨酸受体NMDA亚单位的mRNA和蛋白表达

目的在体外研究由大鼠神经干细胞(NSCs)分化而来神经元细胞中离子型谷氨酸NMDA受体表达。方法分离培养孕14～16d胎鼠皮质和海马神经干细胞，对NSCs进行nestin和分化鉴定。通过RT—PCR、Western blot免疫印迹和免疫组化检测NSCs分化的神经元细胞中离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B的mRNA和蛋白表达。结果从孕14～16d胎鼠大脑中分离培养出NSCs，NSCs分化后的神经元可以表达离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B。结论由NSCs分化而来的神经元能表达离子型谷氨酸NMDA受体。     

大鼠听皮层GABAA受体亚单位mRNA年龄相关的表达变化

应用原位杂交技术,研究了大鼠出生后发育过程中,听皮层GABAA受体a2及β3亚单位mRNA年龄相关的表达变化。特异性DIG标记寡核苷酸探针检测显示,a2亚单位mRNA阳性神经元数量从出生后第1天开始逐渐增加,到第12d达到一个高峰,而后逐渐减少,出生后21d降至检测水平以下;β3亚单位mRNA阳性神经元数量从出生后第12d,一直保持较高表达水平,第12d后急剧降低,第21d后降至检测水平以下。研究结果为进一步在皮层水平上探讨出生后听觉功能发育可塑性的分子机制提供了重要资料。     

单耳气传导阻滞模型大鼠下丘脑N-甲基-D-门冬氨酸受体各亚基mRNA的表达

实验利用单耳外耳道皮下缝合的方法建立单侧气传导持续阻滞大鼠模型，观察环境变化对生后9，23，37 d的SD大鼠听觉中枢神经系统下丘脑NMDA受体NR1，NR2A，NR2B和NR2C mRNA基因表达的影响。PT-PCR结果显示，单耳缝合后缝耳对侧下丘脑NR1，NR2A，NR2B亚单位与缝耳同侧下丘脑NR1，NR2B依赖的听觉神经元发育临界期均在23 d附近，但同侧下丘脑NR2A亚单位依赖的听觉神经元发育的临界期的结束可能接近出生后37 d。结果证实了下丘脑NMDA受体亚基可受听觉环境调控的假设。     

大鼠螺旋神经节神经元r-氨基丁酸A受体及N-甲基-D-天门冬氨酸受体亚单位的表达

目的：研究大鼠耳蜗螺旋神经节神经元A型γ-氨基丁酸受体（Gamma-aminobutyric acid receptor GABAAR）和N—甲基—D—天门冬氨酸受体(N-methyl D-aspartat receptor,NMDAR)亚单位的表达及意义。 方法：原代培养大鼠螺旋神经节神经元，采用RT-PCR检测大鼠螺旋神经节神经元GABAAR和NMDAR亚单位的mRNA表达。 结果：大鼠螺旋神经节神经元mRNA表达GABAAR亚单位有α1-6,β1-3,γ1-3，且在α亚单位族中, α1，α3表达较高，差别无统计学意义（P>0.05）, α1,α3的表达量与其余亚单位表达量以及其余GABAAR亚单位之间表达量的差异有统计学意义（P<0.05），各亚单位表达量大小排列顺序是:α1/α3＞α6＞α5＞α4＞α2。β亚单位族中,β亚单位族中各亚单位表达量两两比较差异有统计学意义，表达量排列顺序β1＞β3＞β2（P<0.05）；γ亚单位族中各亚单位表达量两两比较差别有统计学意义（P<0.05），表达量排列顺序是γ2＞γ1＞γ3；NMDAR表达的亚单位有NR1,NR2A,NR2B,NR2C,NR2D,NR3A,NR3B，其中NR1亚单位的表达最高(P<0.05)。 结论：大鼠螺旋节神经元mRNA上表达GABAAR的主要亚单位α1-6,β1-3,γ1-3和NMDAR NR1,NR2-D,NR3A-B亚单位.     

无镁诱导惊厥后发育中神经元NMDA受体基因表达

目的　观察短暂无镁处理诱导惊厥后发育不同阶段的神经元NMDA受体亚单位基因的表达 ,以期进一步阐明惊厥对发育不同阶段的神经元所造成的影响。 方法　 以培养的发育中皮层神经元为研究对象 ,经短暂无镁处理诱导惊厥样放电 ,用real time定量RT PCR方法检测惊厥后NMDAR1,NMDAR2A ,NMDAR2BmRNA的表达。 结果　 ①培养 6 ,17d的神经元无镁处理后 6hNR1表达均较对照组增高 (P <0 .0 1) ,前者增高更明显 (P<0 .0 1)。②培养 6 ,17d的神经元无镁处理后 6hNR2A表达均较对照组增高 (P <0 .0 5 )。此后其表达较对照组降低 (P <0 .0 1) ,增高及降低的程度在两者间无明显差异 (P >0 .0 5 )。③培养 6 ,17d的神经元无镁处理后 6hNR2B表达均较对照组增高 (P <0 .0 1) ,后者增高更明显 (P <0 .0 5 )。此后其表达逐渐降低 ,在 6及 17d的神经元其表达存在差异。 结论　 短暂无镁处理诱导惊厥后在发育不同阶段的神经元NMDA受体亚单位基因表达不同     

脑缺血再灌注大鼠皮质NMDA受体亚单位NR2A/B蛋白的表达变化

目的 采用免疫细胞化学技术，探讨急性脑缺血再灌注不同时间段大鼠脑顶皮质NMDA受体亚单位NR2A及NR2B蛋白的表达变化。方法 雄性Wistar大鼠70只,随机分成正常对照组、假手术对照组、脑缺血再灌注对照组；以颈动脉引流法行全脑缺血7min再灌注造模，术后分6h、24h、72h3个时间段取脑．脑组织恒冷箱连续冠状切片,免疫细胞化学ABC反应，图像分析系统行顶皮质Ⅰ区V层免疫阳性面积检测。结果 （1）麻醉及假手术可导致顶皮质NR2A、NR2B蛋白表达短暂增多，24h内恢复正常；（2）缺血再灌注后6h前后形成表达高峰,24h恢复正常，随后表达急剧减少，持续至72h以后。结论（1）脑缺血再灌注可导致顶皮质神经元NR2A、NR2B蛋白表达变化，且表达存在明显的时间依赖性；（2）缺血再灌注早期皮质NR2A、NR2B蛋白高度表达可能是导致迟发性神经元丢失的重要原因。     

The effect of early auditory deprivation on the age-dependent expression pattern of NR2B mRNA in rat auditory cortex

NMDA receptors have been well shown to be involved in neuronal plasticity. In order to understand the role of NR2B subtype NMDA receptors in auditory function development, the present study investigated the effect of early auditory deprivation on the expression of NR2B mRNA in rat auditory cortex (AC) during postnatal development. For normal rats, the NR2B mRNA expression was highest at birth (postnatal day 1 [P1]) and declined rapidly to low level during adulthood. However, during the critical period of rat auditory development (two to three weeks after birth), there was a transient NR2B expression peak on postnatal day 21 (P21). For the auditory-deprived rats, the general declining trend of NR2B mRNA expression from birth to adult was similar to that observed in the normal group, whereas the expression level from P15 to P27 was significantly lower than normal and the transient peak on P21 disappeared. In both groups, the distribution pattern of NR2B mRNA-positive neurons was also examined in various layers and dorsal, medial and ventral subdistricts of AC. There is no significant effect on the spatial expression of the NR2B mRNA in the AC between normal and deprived group. Our results indicated that the early auditory deprivation decreased the expression levels of NR2B mRNA in AC during the critical period of rat auditory development, suggesting that NR2B plays an important role in the developmental plasticity of auditory function in rats.     

Early auditory enrichment with music enhances auditory discrimination learning and alters NR2B protein expression in rat auditory cortex

Previous studies have shown that the functional development of auditory system is substantially influenced by the structure of environmental acoustic inputs in early life. In our present study, we investigated the effects of early auditory enrichment with music on rat auditory discrimination learning. We found that early auditory enrichment with music from postnatal day (PND) 14 enhanced learning ability in auditory signal-detection task and in sound duration-discrimination task. In parallel, a significant increase was noted in NMDA receptor subunit NR2B protein expression in the auditory cortex. Furthermore, we found that auditory enrichment with music starting from PND 28 or 56 did not influence NR2B expression in the auditory cortex. No difference was found in the NR2B expression in the inferior colliculus (IC) between music-exposed and normal rats, regardless of when the auditory enrichment with music was initiated. Our findings suggest that early auditory enrichment with music influences NMDA-mediated neural plasticity, which results in enhanced auditory discrimination learning.     

Nicotine exposure during a postnatal critical period alters NR2A and NR2B mRNA expression in rat auditory forebrain

Chronic nicotine exposure (CNE) can alter brain development and is thought to produce deficits in auditory function. Previously, we found that CNE during the second postnatal week, but not before or after, increases the duration of excitatory postsynaptic potentials (EPSPs) mediated by N-methyl-D-aspartate receptors (NMDARs) in rat auditory cortex. It was proposed that a potential mechanism underlying increased EPSP duration could be over-stimulation of presynaptic nicotinic acetylcholine receptors, leading to prolonged glutamate release. Since glutamatergic activity regulates levels of postsynaptic NMDAR subunits, here we examine the effects of CNE on mRNA expression for the NR2A and NR2B subunits in auditory cortex and thalamus. Two days of CNE (postnatal days 8-9), produced no effects, but 5 days (postnatal days 8-12) enhanced cortical NR2A mRNA levels and reduced thalamic NR2B mRNA levels for up to 2 weeks. These effects are consistent with the hypothesis that CNE during a postnatal critical period disrupts auditory cortex development by over-stimulating glutamatergic synapses.     

Early auditory deprivation alters expression of NMDA receptor subunit NR1 mRNA in the rat auditory cortex

The expression of NMDA receptor NR1 subunit mRNA was studied in rat auditory cortex (AC) on different postnatal days using digoxigenin-labeled oligonucleotide probes. The results showed that NR1 expression increased from birth to postnatal day 35 (P35) and remained constant until P56. The most significant increases occurred between P7 and P14. Changes in NR1 mRNA expression in rats subjected to monaural hearing deprivation on P7, P21, P35, and P49 were examined on P56. Between P7 and P21, when the rat auditory system was still in a critical period of development, NR1 mRNA expression was lower in the contralateral AC, which received auditory signals from the plugged ear, than in the ipsilateral AC. However, no significant difference was observed between the rats deprived of hearing on P35 and those deprived of hearing on P42, the end of the critical period of auditory development. These results showed that monaural hearing deprivation during early postnatal development was associated with decreased NR1 mRNA expression in the contralateral AC and suggested the involvement of NR1 in auditory function during development. They also indicated that, during postnatal development, environmental factors changed the functional plasticity of neurons in the AC through NR1 receptor expression. Taken together, these findings provide a possible underlying mechanism for the development of postnatal auditory function.     

Perinatal exposure to PTU delays switching from NR2B to NR2A subunits of the NMDA receptor in the rat cerebellum

Certain kinds of developmental neurotoxicants are considered to act by affecting the levels of thyroid hormones, which are essential for the brain development of both humans and experimental animals. Hypothyroidism experimentally induced in rats with propylthiouracil (PTU) offers a useful animal model for developmental neurotoxicity. The purpose of the present study was to clarify developmental alterations in gene expression caused by PTU in this model, with the focus on eight genes implicated in neural network formation or synaptic functions, such as the brain-derived neurotrophic factor (BDNF) and NMDA receptors 2A/2B. First, we measured the developmental profile of gene expression in vehicle-dosed rat cerebellum by quantitative RT-PCR and then examined the effects of PTU on mRNA levels on postnatal day (PND) 22, when most of the cerebellar structures in mature animals are already formed. PTU induced up-regulation of NR2B mRNA and down-regulation of NR2A and BDNF mRNAs in the cerebellum on PND 22, but there were no changes in the other genes (growth associated protein-43, L1, neuronal cell adhesion molecule, synaptophysin, post synaptic density-95). Examination of the effects of PTU on maturation of NMDAR subunits (NR2A/NR2B) demonstrated changes in relative expression on PND 14, but not on PND 4, with recovery after maturation. The profile of NMDAR subunits in vehicle-dosed rats showed a shift from NR2B to NR2A during development. These results suggest PTU can delay this switching from NR2B to NR2A subunits in the maturation of NMDA receptors.     

Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats

Five N-methyl-D-aspartate (NMDA) receptor subunits have been identified thus far: NR1, NR2A, NR2B, NR2C, and NR2D. Here, we have analyzed the expression patterns of mRNAs for the NMDA receptor subunits in the developing and adult rats by in situ hybridization. The developmental changes of the expression patterns were most salient in the cerebellum. In the external granular layer, hybridization signals of mRNAs, for NR1, NR2A, NR2B, and NR2C appeared by postnatal day 3, but no NR2D mRNA was expressed at any developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stages examined. The signals for the NR2A mRNA appeared in Purkinje cells and granule cells during the second postnatal week. The signals for the NR2B mRNA in granule cells were seen transiently during the first 2 weeks after birth. The signals for NR2C mRNA appeared in granule cells and glial cells during the second postnatal week. The signals for NR2D mRNA appeared transiently in Purkinje cells during the first 8 postnatal days; in adult rats, these were seen in stellate and Golgi cells. In the cerebellar nuclei, mRNAs for NR1, NR2A, NR2B, and NR2D were more or less expressed on postnatal day 0, while expression signals for the NR2C mRNA were first detected in postnatal day 14. Thus, the most conspicuous changes of expression patterns were observed in the cerebellar cortex during the first 2 weeks after birth, when development and maturation of the cerebellum proceed most rapidly. © 1994 Wiley-Liss, Inc.     

NMDAR-2A subunit protein expression is reduced in the hippocampus of rats exposed to Pb2+ during development

Chronic exposure to lead (Pb2+) produces deficits of learning and memory in children and spatial learning deficits in developing rats. The N-methyl-D-aspartate receptor (NMDAR) has been identified as a principal target for Pb2+-induced neurotoxicity. Age-dependent changes in NMDAR subunit gene expression were observed in hippocampi of rats chronically exposed to Pb2+ during development [T.R. Guilarte, J.L. McGlothan, Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure, Brain Res. 790 (1998) 98-107]. These changes were present at blood Pb2+ levels ranging from 20-60 microg/dl. Littermates were used in the present study to determine whether the changes in gene expression were reflected in protein levels. NR1, NR2A, and NR2B subunit protein levels were measured in rat hippocampus and cortex at post-natal days (PND) 7, 14, 21, and 28 by Western blot and densitometric analysis. A treatment effect was apparent for NR2A subunit protein expression in the hippocampus (F1,28=10.224, p<0.01). NR2A subunit protein was reduced by 40%, 19%, and 27% from control levels in PND14, 21, and 28 Pb2+-exposed rats, respectively. Mean comparisons indicated that rats at PND14 exhibited the most significant reduction of NR2A (p<0.001). These data concur with our previous finding of reduced NR2A mRNA found in hippocampal pyramidal and granule cells of Pb2+-exposed rats. Pb2+ exposure during development had no effect on NR1 or NR2B subunit protein expression in the hippocampus at any age. No effect was observed on any subunit in the cortex at any age. The developmental profile of the NMDAR-2A subunit protein in the hippocampus is specifically changed by chronic exposure to Pb2+. These data suggest that composition of subunits comprising NMDAR may be altered in Pb2+-exposed rats.     

Changes in expression of NMDA receptor subunit mRNA by perinatal exposure to dioxin.

Since dioxin and related compounds are suspected of affecting permanently the brain function of offspring of human and experimental animals, effects of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of rat NMDA receptor NR2A and NR2B subunit mRNA were examined. The mRNA quantification by competitive RT-PCR clearly revealed that TCDD inhibited NR2B mRNA expression and enhanced NR2A mRNA expression in the neocortex and hippocampus on postnatal day (PND) 49, whereas these changes in mRNA expression were not found on PND 5. The results demonstrate for the first time that the perinatal exposure to TCDD can alter the molecular basis of brain of offspring in adulthood.