Hendrickson A E, Tillakaratne N J, Mehra R D, Esclapez M, Erickson A, Vician L, Tobin A J
Department of Biological Structure, University of Washington, Seattle 98195.
J Comp Neurol. 1994 May 22;343(4):566-81. doi: 10.1002/cne.903430407.
Adult monkey primary visual cortex contains a diverse population of stellate neurons that utilize the neurotransmitter gamma aminobutyric acid (GABA). Two glutamic acid decarboxylase (GAD) enzymes that synthesize GABA, GAD65 and GAD67, were localized within these stellate neurons by in situ hybridization of 35S or digoxigenin (DIG) labeled riboprobes. Double labels were done by using 35S GAD67 riboprobe and GABA immunocytochemistry on the same section to verify that the neuronal population identified by immunocytochemistry was the same one studied in the in situ hybridization experiments. We find that GAD65 mRNA and GAD67 mRNA are widely distributed in the cortex, with four bands of heavily labeled neurons in upper layer 2, lower 3, 4C, and 6. GAD67 labeled neurons were more obvious in layer 4C beta, while GAD65 containing neurons were common in layer 1 and white matter. Northern blots and in situ hybridization on sections with both 35S and DIG riboprobes indicate that cortical neurons typically contain more GAD67 mRNA. Cell counts show that 18% of all cortical neurons contain GAD67 mRNA and 13% contain GAD65 mRNA, suggesting that a small population of GABA neurons might lack GAD65. Cell bodies that contain high amounts of GAD65 mRNA are prominent in layers deep 3, 4B, 4C alpha, and 6 and often are the largest cells in their respective layers. Double labels demonstrate that 96% of all GABA+ neurons contain GAD67 mRNA. Neurons heavily labeled for GABA tend to have smaller cell bodies and contain less GAD67 mRNA, while lightly labeled GABA neurons are larger and contain more GAD67 mRNA. These data indicate that most GABA neurons in monkey striate cortex contain both GAD enzymes. Although the differences in GABA content, cell size, laminar distribution, and GAD mRNA concentration suggest different requirements for GAD67 and GAD65 in cortical circuits, our experiments do not reveal what different roles these two enzymes subserve within GABAergic stellate neurons.
成年猴初级视皮层包含多种利用神经递质γ-氨基丁酸（GABA）的星状神经元。通过用35S或地高辛（DIG）标记的核糖探针进行原位杂交，将两种合成GABA的谷氨酸脱羧酶（GAD），即GAD65和GAD67，定位在这些星状神经元内。通过在同一切片上使用35S GAD67核糖探针和GABA免疫细胞化学进行双重标记，以验证免疫细胞化学鉴定的神经元群体与原位杂交实验中研究的是同一群体。我们发现GAD65 mRNA和GAD67 mRNA广泛分布于皮层，在第2层上部、第3层下部、第4C层和第6层有四条标记密集的神经元带。GAD67标记的神经元在第4Cβ层更明显，而含有GAD65的神经元在第1层和白质中常见。用35S和DIG核糖探针进行的Northern印迹和切片原位杂交表明，皮层神经元通常含有更多的GAD67 mRNA。细胞计数显示，所有皮层神经元中有18%含有GAD67 mRNA，13%含有GAD65 mRNA，这表明一小部分GABA能神经元可能缺乏GAD65。含有大量GAD65 mRNA的细胞体在第3层深部、第4B层、第4Cα层和第6层很突出，并且通常是各自层中最大的细胞。双重标记表明，所有GABA+神经元中有96%含有GAD67 mRNA。GABA标记密集的神经元往往细胞体较小，含有较少的GAD67 mRNA，而GABA标记较轻的神经元较大，含有较多的GAD67 mRNA。这些数据表明，猴纹状皮层中的大多数GABA能神经元都含有这两种GAD酶。尽管GABA含量、细胞大小、层状分布和GAD mRNA浓度的差异表明皮层回路中对GAD67和GAD65有不同需求，但我们的实验并未揭示这两种酶在GABA能星状神经元中发挥何种不同作用。
