Chaudhuri A, Matsubara J A, Cynader M S
Department of Ophthalmology, University of British Columbia, Vancouver, Canada.
Vis Neurosci. 1995 Jan-Feb;12(1):35-50. doi: 10.1017/s095252380000729x.
It is now well established that environmental signals mediated via neurotransmitters and hormones can induce responses in cells which involve a cascade of receptors, G proteins, and second messengers. These in turn can induce transcription factors which regulate long-term changes in gene expression. It has been proposed that the stimulus-transcription coupling properties of these DNA-binding proteins can be exploited to visualize activated neurons by way of immunostaining. We have used standard immunohistochemical techniques to detect the expression of one specific transcription factor, Zif268, in the visual cortex (area 17, V1) of vervet monkeys (Cercopithecus aethiops). Immunopositive neurons were present in large numbers throughout the visual cortex of the normal animal, being concentrated in layers 2/3 and 6 and at moderate levels in 4C beta and 5. To determine if Zif268 expression was affected by visual stimulation in the monkey, we restricted light input to one eye with the aim of revealing ocular-dominance columns in striate cortex. We found that short-term monocular deprivation induced either by enucleation, intravitreal TTX injection, or eyelid suturing resulted in dramatic changes in Zif268 levels, revealing vertically oriented columns of reduced Zif268 staining interdigitated with columns of normal expression. Furthermore, these columns were discernible after just 2 h of monocular blockade. A comparison of the ocular-dominance pattern obtained with Zif268 immunostaining and cytochrome oxidase histochemistry in long-term monocularly deprived animals showed a coincident reduction of both markers along columns that were precisely aligned in adjacent sections, indicating that Zif268 expression is restricted to cortical regions of high metabolic activity. Simultaneous immunostaining for Zif268 and the calcium-binding proteins calbindin and parvalbumin showed a negative correlation, suggesting that the Zif268 protein may be expressed selectively within excitatory neurons. A similar approach with immunostaining for neurofilament and microtubule-associated proteins (SMI-32 and MAP2) revealed pyramidal neurons which were regularly found to contain a Zif268-positive nucleus. Furthermore, confocal images of lucifer yellow filled neurons possessing Zif268-positive nuclei all showed pyramidal morphology. Taken together, these results point to activity-dependent expression of Zif268 within a subset of excitatory neurons.
现已充分证实，经由神经递质和激素介导的环境信号可在细胞中引发反应，这涉及一系列受体、G蛋白和第二信使。这些物质进而可诱导调节基因表达长期变化的转录因子。有人提出，这些DNA结合蛋白的刺激-转录偶联特性可用于通过免疫染色来可视化活化的神经元。我们使用标准免疫组织化学技术检测了绿猴（非洲长尾猴）视皮层（第17区，V1）中一种特定转录因子Zif268的表达。在正常动物的整个视皮层中存在大量免疫阳性神经元，集中在第2/3层和第6层，在4Cβ层和第5层中含量中等。为了确定Zif268的表达是否受猴子视觉刺激的影响，我们限制一只眼睛的光输入，目的是揭示纹状皮层中的眼优势柱。我们发现，通过摘除眼球、玻璃体内注射TTX或缝合眼睑诱导的短期单眼剥夺导致Zif268水平发生显著变化，显示出Zif268染色减少的垂直定向柱与正常表达的柱相互交错。此外，在单眼阻断仅2小时后就可辨别出这些柱。对长期单眼剥夺动物中用Zif268免疫染色和细胞色素氧化酶组织化学获得的眼优势模式进行比较，结果显示在相邻切片中精确对齐的柱上两种标记物同时减少，表明Zif268的表达局限于高代谢活性的皮层区域。对Zif268与钙结合蛋白钙结合蛋白和小白蛋白进行同时免疫染色显示呈负相关，这表明Zif268蛋白可能在兴奋性神经元中选择性表达。对神经丝和微管相关蛋白（SMI-32和MAP2）进行免疫染色的类似方法揭示了锥体细胞，这些细胞通常含有Zif268阳性核。此外，具有Zif268阳性核的荧光黄填充神经元的共聚焦图像均显示出锥体形态。综上所述，这些结果表明Zif268在一部分兴奋性神经元中存在活性依赖性表达。
