van den Pol A N, Kogelman L, Ghosh P, Liljelund P, Blackstone C
Section of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06510.
J Neurosci. 1994 Jun;14(6):3816-34. doi: 10.1523/JNEUROSCI.14-06-03816.1994.
The expression of the metabotropic glutamate receptor mGluR1 was studied with Northern and Western blot analysis, with immunocytochemistry, and with Ca2+ digital imaging in the developing rat hypothalamus. mGluR1 is coupled to a G protein and activation by glutamate and related agonists leads to intracellular phosphotidylinositol hydrolysis and Ca2+ mobilization. mGluR1 RNA could be detected in embryonic hypothalamus, and by the day of birth and prior to the primary period of synaptogenesis, both mGluR1 RNA and protein were strongly expressed. In parallel experiments with digital imaging of cultured hypothalamic cells, some embryonic day 18 hypothalamic neurons and many astrocytes after 3 d in vitro showed Ca2+ responses to quisqualate and t-ACPD, and to glutamate in the absence of extracellular Ca2+. A greater number of embryonic neurons responded to NMDA than to agonists of the metabotropic receptor. With increased development time in culture, the number of neurons that responded to metabotropic glutamate receptor agonists increased. In the adult hypothalamus, mGluR1-immunoreactive neurons were widespread, and particularly dense in the dorsomedial, lateral, and anterior hypothalamus/preoptic areas, and in the mammillary body. Strongly immunoreactive cells were interspersed among neurons with no immunoreactivity. In developing neurons a diffuse immunostaining appeared along dendrites and somata. With time, beginning in the first week after birth, strongly stained puncta appeared, possibly associated with synaptic specializations. These puncta were numerous on dendrites of some adult neurons, and were the most strongly stained regions of neurons. Neurons developing in vitro at low neuron densities showed a development of mGluR1 immunoreactivity similar to that of neurons in vivo, but with a delayed progression of immunostaining. We found no obvious staining of axons or of astrocytes. A strong expression of mGluR1 protein was found in the hypothalamus during the first 2 postnatal weeks; this expression was partially reduced in adults. In contrast, cerebellum showed no reduction in mGluR1 protein in adults. Together these data suggest a complex regulation of mGluR1 during development, with sufficient expression of functional receptors in the developing hypothalamus to modulate morphogenesis and synaptogenesis, and later to play a role in transduction of glutamate signals in the adult. Different regions of the brain showed dramatic differences in the way each expresses mGluR1 during development.
运用Northern印迹法、Western印迹分析、免疫细胞化学以及Ca²⁺数字成像技术，对发育中的大鼠下丘脑代谢型谷氨酸受体mGluR1的表达情况进行了研究。mGluR1与G蛋白偶联，谷氨酸及相关激动剂对其激活会导致细胞内磷脂酰肌醇水解和Ca²⁺动员。在胚胎下丘脑中可检测到mGluR1 RNA，在出生日及突触发生初期之前，mGluR1 RNA和蛋白质均强烈表达。在对培养的下丘脑细胞进行数字成像的平行实验中，一些胚胎第18天的下丘脑神经元以及体外培养3天后的许多星形胶质细胞，对quisqualate和t - ACPD以及在无细胞外Ca²⁺情况下的谷氨酸表现出Ca²⁺反应。对NMDA产生反应的胚胎神经元数量多于对代谢型受体激动剂产生反应的神经元。随着培养时间的增加，对代谢型谷氨酸受体激动剂产生反应的神经元数量增多。在成年下丘脑中，mGluR1免疫反应阳性神经元分布广泛，在背内侧、外侧、下丘脑前部/视前区以及乳头体中尤为密集。强免疫反应阳性细胞散布于无免疫反应的神经元之间。在发育中的神经元中，树突和胞体上出现弥漫性免疫染色。随着时间推移，从出生后的第一周开始，出现强染色的小点，可能与突触特化有关。这些小点在一些成年神经元的树突上很多，是神经元中染色最强的区域。在低神经元密度下体外培养的神经元，其mGluR1免疫反应性的发育与体内神经元相似，但免疫染色进程延迟。我们未发现轴突或星形胶质细胞有明显染色。出生后的前两周内，下丘脑中mGluR1蛋白强烈表达；在成年后这种表达部分降低。相比之下，成年小脑的mGluR1蛋白未减少。这些数据共同表明，mGluR1在发育过程中受到复杂调控，在发育中的下丘脑中功能性受体有足够表达以调节形态发生和突触发生，随后在成体中参与谷氨酸信号转导。大脑的不同区域在发育过程中表达mGluR1的方式存在显著差异。
