In situ hybridization to localize mRNA encoding the neurotransmitter synthetic enzyme glutamate decarboxylase in mouse cerebellum.
In situ hybridization to localize mRNA encoding the neurotransmitter synthetic enzyme glutamate decarboxylase in mouse cerebellum.
复制标题
原位杂交定位小鼠小脑中编码神经递质合成酶谷氨酸脱羧酶的 mRNA。
DOI：
10.1073/pnas.83.16.6193
复制
发表时间：
1986
影响因子：
11.1
通讯作者：
Tobin,AJ
中科院分区：
文献类型：
--
作者：
Wuenschell,CW;Fisher,RS;Kaufman,DL;Tobin,AJ
谷氨酸脱羧酶（GAD; EC 4.1.1.15）负责神经递质γ-氨基丁酸（GABA）的合成。我们已经使用的cDNA序列编码GAD产生的单链RNA杂交探针GAD mRNA。该探针检测小鼠小脑切片中单个细胞中的GAD mRNA。用这种探针进行原位杂交的特异性取决于四个标准：标记细胞的分布与我们和其他人用GAD免疫组织化学获得的结果相匹配（浦肯野神经元、高尔基体II、星状神经元和篮状神经元被标记，而颗粒细胞和神经胶质没有被标记）;具有与GAD mRNA相同的序列的阴性对照探针没有特异性标记任何小脑细胞;预先用RNase处理切片消除了特异性标记;标记显示了核酸杂交体的典型解链行为。GAD mRNA的翻译显然仅限于神经元胞体，因为GAD mRNA在神经元胞体中可检测到，但在终末中不可检测到。此外，GABA作为神经递质的选择似乎是在转录水平上进行的，因为颗粒神经元不含可检测的GAD mRNA。GAD mRNA的水平在神经元类别之间以及在每个神经元类型内的细胞与细胞之间变化。
Glutamate decarboxylase (GAD; EC 4.1.1.15) is responsible for the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). We have used a cDNA sequence encoding GAD to produce a single-stranded RNA hybridization probe for GAD mRNA. This probe detects GAD mRNA in individual cells in sections of mouse cerebellum. The specificity of in situ hybridization with this probe rests on four criteria: the distribution of labeled cells matched the results we and others obtain with GAD immunohistochemistry (Purkinje, Golgi II, stellate, and basket neurons were labeled, whereas granule cells and glia were not); a negative control probe having a sequence identical to GAD mRNA did not specifically label any cerebellar cells; prior treatment of the sections with RNase abolished specific labeling; the labeling showed the melting behavior typical of nucleic acid hybrids. Translation of GAD mRNA is apparently restricted to neuronal cell bodies since GAD mRNA was detectable in neuronal perikarya but not in terminals. Also, the choice of GABA as a neurotransmitter appears to be made at the level of transcription since granule neurons did not contain detectable GAD mRNA. The level of GAD mRNA varied among the classes of neurons as well as from cell to cell within each neuron type.