GAD67-GFP基因敲入小鼠黑质网状部神经元GABA与氯离子转运体KCC2和NKCC1的共存

Colocalization of KCC2 or NKCC1 with GABA in the neurons within the substantia nigra pars reticulata of the GAD67-GFP knock-in mouse

为了观察谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠黑质网状部(SNr)内,表达GFP的GABA能神经元与一对功能相反的Cl-共转运体(K+-Cl-cotransporter2,KCC2;Na+-K+-Cl-cotransporter1,NKCC1)的共存情况,本研究分别运用原位分子杂交与免疫组织化学相结合以及GFP与KCC2或NKCC1免疫荧光染色相结合的双重标记方法,在光学显微镜和激光共聚焦显微镜下同时进行观察。结果显示:(1)SNr内95%以上的GFP阳性神经元同时表达KCC2 mRNA,而50%表达KCC2 mRNA的阳性神经元呈GFP阳性;(2)SNr内80%以上的GFP阳性神经元同时表达NKCC1 mRNA,约35%表达NKCC1 mRNA的阳性神经元呈GFP阳性;(3)SNr内90%以上的GFP阳性神经元同时表达KCC2,双标神经元约占KCC2阳性神经元的50.5%;(4)SNr内80.5%以上的GFP阳性神经元同时表达NKCC1,双标神经元约占NKCC1阳性神经元的42.5%。以上结果表明,SNr内表达GFP的GABA能神经元大部分与KCC2和NKCC1共存,提示氯离子共转运体可能对SNr内GABA能神经元起重要的调控作用。

To observe the eolocalization of KCC2 or NKCC1 with GFP-positive neurons within the substanti nigra pars reticulata （ SNr）, the glutamate decarboxylase 67-green fluorescence protein （GAD67-GFP） knock-in mice were used in the present study. Double-labeled technique was used by in situ hybridization combined with immunohistochemistry for GFP and double immunofluorescence histochemistry for GFP and KCC2 or NKCC1. The stained sections were observed under light microscope and a confocal laser-scanning microscope. The resuits showed that over 90% of GFP-positive neuronal cell bodies in the SNr showed hybridization signals for KCC2 mRNA, and that almost 50% of neuronal cell bodies with KCC2 mRNA signals were GFP-positive. At the same time, about 80% of GFP-positive neuronal cell bodies in the SNr showed hybridization signals for NKCC1 mRNA, and 35% of neuronal cell bodies with NKCC1 mRNA signals were GFP- positive. In addition, we found that about 50.5% of KCC2-positive neurons and 90.0% of GFP-positive neurons were KCC2/GFP doublelabeled neurons in the SNr. Similiarly, about 42.5% of NKCCl-positive neurons and 80.5% of GFP-positive neurons were NKCC1/GFP double-labeled neurons in the SNr. The present results indicate that majority of GABAergic neurons within the SNr of GAD67-GFP knockin mice, which are GFP-positive, are colocalizaed with KCC2 or NKCC1, suggesting an important role of the cation-chloride cotransporters in the regulation of GABAergic transmission in substantia nigra.