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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。
GFP-expressing GABAergic neurons were incubated with a pair of functionally opposite Cl-cotransporters (GAD67-GFP) in order to observe the GAD67-GFP knockdown in the substantia nigra pars reticulum (SNr) (K + -Cl-cotransporter2, KCC2; Na + -K + -Cl-cotransporter1, NKCC1), we used in situ hybridization with immunohistochemistry and in situ hybridization of GFP with KCC2 or NKCC1 immunofluorescence staining of dual The labeling method was observed simultaneously under a light microscope and a laser confocal microscope. The results showed that: (1) more than 95% of GFP positive neurons expressed KCC2 mRNA in SNr, while 50% of KCC2 mRNA positive neurons were GFP positive; (2) more than 80% GFP positive neurons were expressed in SNr NKCC1 mRNA and about 35% positive neurons expressing NKCC1 mRNA were GFP positive. (3) More than 90% of GFP positive neurons in SNr expressed KCC2 simultaneously, while double-labeled neurons accounted for 50.5% of KCC2 positive neurons. More than 80.5% of GFP-positive neurons in SNr expressed NKCC1 simultaneously, and double-labeled neurons accounted for 42.5% of NKCC1-positive neurons. The above results indicated that most of GABAergic neurons expressing GFP in SNr coexisted with KCC2 and NKCC1, suggesting that chloride ion co-transporter may play an important regulatory role on GABAergic neurons in SNr.