利用GAD67-GFP基因敲入方法显示小鼠脊髓内表达GFP的GABA能神经元的分布(英文)

DISTRIBUTION OF GABA-CONTAINING NEURONS IN THE SPINAL CORD REVEALED BY GFP EXPRESSION IN THE GAD67-GFP KNOCK-IN MOUSE

γ-氨基丁酸 (GABA)是脊髓背角、前角内主要的抑制性神经递质。为了更好地观察脊髓背角内 GABA能神经元的形态和功能 ,本研究使用了两种谷氨酸脱羧酶 67-绿色荧光蛋白 (GAD67-GFP)基因敲入小鼠 ,并观察了敲入小鼠脊髓内的 GFP表达状况。用免疫荧光组织化学双标记方法显示脊髓内所有的 GF P阳性神经元基本上都呈 GAD67和 GABA阳性 ;GFP阳性神经元在脊髓背角的 ～ 层最为密集 ,背角深层内侧部及中央管周围呈中等密度分布 ,而在脊髓背角其它部位及前角则呈散在分布。脊髓内 GFP阳性神经元的分布与 GABA能神经元的分布一致。本文作者等还进一步在 GAD67-GFP敲入小鼠中观察了 GFP和神经元标志物神经元核蛋白 (Neu N )的共存状况。脊髓背角内 GFP阳性神经元分别占 、 和 层的 Neu N阳性神经元的 3 1.5 %、3 3 .3 %和 44 .7% ,与以往的 GABA免疫组化研究结果基本一致。本研究表明 GAD67-GFP基因敲入小鼠脊髓内的 GFP在GAD67启动子的调节下正确地表达于 GABA能神经元 ,该基因敲入小鼠可用于脊髓

aminobutyric acid (GABA) is the main inhibitory neurotransmitter in both dorsal and ventral horn of the spinal cord. In order to understand the morphology and function of GABAergic neurons in the spinal cord more precisely, two strains of glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice were used. The expression of GFP in the spinal cord of knock-in mice was observed. The results of the immunofluoroscent histochemical double-staining study showed that all GFP-positive neurons in the spinal cord were basically immunopositive for GAD67 and GABA. GFP-positive neurons were concentrated in laminae Ⅰ～Ⅲ of the spinal dorsal horn, presented in moderate amounts in the medial part of the deep dorsal horn and in the area around the central canal, and more sparsely distributed in the remainder of the dorsal horn and in the ventral horn. The distribution pattern of GFP expression neurons in the spinal cord was in consistent with that of GABAergic neurons. GAD67-GFP knock-in mice were further used to investigate the colocalization of GFP with a neuronal marker, neuronal nuclei protein (NeuN). The proportion of GFP-positive neurons among the NeuN-immunopositive neurons in the spinal cord was about 31.5%, 33.3% and 44.7% in laminae Ⅰ, Ⅱ and Ⅲ of the spinal dorsal horn, respectively, which was close to those obtained from GABA immunohistochemical studies. Our results indicated that the GFP expression in the spinal cord of the GAD67-GFP knock-in mouse was regulated properly in GAD67-positive, GABAergic neurons. The knock-in technique would be a useful tool for studying the morphology and physiological properties and developmental pattern of GABAergic neurons in the spinal cord.