目的观察谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠三叉神经尾侧亚核(Vc)浅层内,表达GFP的GABA能神经元的分布及其与小白蛋白(PV)的共存。方法分别运用原位分子杂交与免疫组织化学相结合;GFP与神经元标记物——神经元核蛋白(NeuN)或PV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进行观察。结果1.Vc浅层内90%以上的GFP阳性神经元同时表达GAD67 mRNA,而几乎所有表达GAD67 mRNA的阳性神经元都呈GFP阳性;2.GFP阳性神经元主要分布于Vc的Ⅰ-Ⅱ层内,细胞较小,尤其在Ⅱ层内可见大量密集分布的GFP阳性细胞和突起。GFP阳性神经元分别占Ⅰ、Ⅱ层内NeuN阳性神经元总数的19.4%和24.3%;3.GFP/PV双标神经元主要分布于Vc的Ⅰ-Ⅱ层,这些双标神经元大约占PV阳性神经元的62.4%,占GFP阳性神经元的12.8%。结论在Vc表达GFP的GABA能神经元主要密集分布于与外周伤害性信息传递关系密切的板层内,且大部分PV样阳性神经元属于GABA能神经元。
Objective To observe the distribution of GABA-containing neurons revealed by GFP expression and the colocalization of the GFP with parvalbumin(PV) in the caudal spinal trigeminal nucleus(Vc), the glutamate decarboxylase 67- green fluorescence protein(GAI)67-GFP) knock-in mice were used in the present study. Methods Double-labeled techniques were used by in situ hybridization combined with immunohistochemistry for GFP and double immunofluorescence histochemistry for GFP and NeuN( neuronal nuclei protein, a neuronal marker) or PV. The stained sections were observed under light microscope and a confocal laser-scanning microscope. Results 1. Over 90% of GFP-positive neuronal cell bodies in the laminae Ⅰ and Ⅱ of the Vc showed hybridization signals for GAD67 mRNA, and that almost all neuronal cell bodies with GAD67 mRNA signals were GFP-positive; 2. GFP-positive neurons were mainly distributed in the laminae Ⅰ and Ⅱ of the Vc and the vast majority of them were small neurons. The considerable number of GFP-positive processes and somata were most densely observed in the lamina Ⅱ of the Vc. The proportion of GFP-positive neurons in the NeuN-labeled neurons of the Vc was about 19.4% and 24.3% in laminae Ⅰ and Ⅱ, respectively; 3. Double-labeled neurons for GFP/PV are mainly found in laminae Ⅰ and Ⅱ of the Vc. The proportion of GFP/PV double-labeled neurons was about 62.4% and 12.8% of total population of PV-and GFP-positive neurons in laminae Ⅰ - Ⅱ of the Vc, respectively. Conclusion GABAergic neurons are mainly distributed in the laminae Ⅰ and Ⅱ of the Vc which related closely to transmission of the nociceptive primary afferent information, and the majority of PV-positive neurons are GABAergic neurons.