A cellular mechanism for the release of glutamate as a neural signal in hypothalamic peptidergic neurons

为了阐明内源性谷氨酸在下丘脑肽能神经元中的功能作用，本研究分析了表达囊泡谷氨酸转运体（VGLUT 2）的神经元的表型、VGLUT 2在神经垂体终末的超微结构定位以及VGLUT 2基因在室旁核（PVN）和视上核（SON）中表达的变化。原位杂交结合免疫组化显示VGLUT2基因表达的神经元含有加压素，催产素，食欲素或proopiomelanocortin的大鼠下丘脑，这表明存在的神经递质谷氨酸在这些肽能神经元。双标记免疫荧光染色显示VGLUT 2染色在大鼠神经垂体的含加压素的末端，但由于使用的抗VGLUT 2抗血清引起的高背景，这是无法观察到超微结构。BAS 5000和NIH图像的定量原位杂交分析表明，束缚应激小鼠PVN中促肾上腺皮质激素释放激素的基因表达显着上调，但VGLUT 2的基因表达未显着上调。在大鼠的渗透压挑战实验中，VGLUT2基因表达上调整个PVN和SON的缺水动物，而盐负荷（饮用2%NaCl）动物的表达上调，只有在特定的亚区，这些核加压素神经元主要位于。与对照动物（水分充足的大鼠）中的表达水平相比，由渗透刺激诱导的VGLUT2基因表达的这些变化具有统计学显著性。这些结果表明，VGLUT2的表达在转录水平上的调节，依赖于从神经垂体的激素分泌的状态，并建议自分泌或旁分泌调制内源性谷氨酸的肽释放在一些，但不是所有的，下丘脑的肽能神经元。

To clarify a functional role of endogenous glutamate in hypothalamic peptidergic neurons, the present study analyzed phenotypes of neurons which express a vesicular glutamate transporter (VGLUT2), the ultrastructural localization of VGLUT2 in neurohypophysial terminals and changes of VGLUT2 gene expression in the paraventricular (PVN) and supraoptic (SON) nuclei by stressful stimuli. In situ hybridization combined with immunohistochemistry revealed VGLUT2 gene expression in neurons containing vasopressin, oxytocin, orexin or proopiomelanocortin of the rat hypothalamus, suggesting the presence of neurotransmitter glutamate in these peptidergic neurons. Double-label immunofluorescence staining demonstrated VGLUT2-staining in vasopressin-containing terminals of the rat neurohypophysis but this was unable to observe ultrastructurally because of the high background caused by the anti-VGLUT2 antiserum used. Quantitative in situ hybridization analysis with BAS5000 and NIH image demonstrated that gene expression of corticotropin-releasing hormone but not VGLUT2 was significantly upregulated in the PVN of mice exposed to restraint stress. In experiments of osmotic challenges in rats, VGLUT2 gene expression was upregulated throughout the PVN and SON of water-deprivation animals, whereas the expression in salt-loading (drinking of 2% NaCl) animals was upregulated only in particular subregions of these nuclei where vasopressin neurons are predominantly located. These changes in VGLUT2 gene expression induced by osmotic stimulations were statistically significant as compared with the expression level in the control animal (euhydrated rats). These results show the regulation of VGLUT2 expression at the transcription level, dependent on the status of hormonal secretion from the neurohypophysis, and suggest autocrine or paracrine modulation by endogenous glutamate for the peptide release in some, but not all, peptidergic neurons of the hypothalamus.