Interdisciplinary Institute of Neuroscience and Technology, Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China.
Center for Neuroscience, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, School of Medicine, Zhejiang University, Hangzhou, China.
Nat Protoc. 2018 Jan;13(1):118-133. doi: 10.1038/nprot.2017.134. Epub 2017 Dec 14.
Researchers in behavioral neuroscience have long sought imaging techniques that can identify and distinguish neural ensembles that are activated by sequentially applied stimuli at single-cell resolution across the whole brain. Taking advantage of the different kinetics of immediate-early genes' mRNA and protein expression, we addressed this problem by developing tyramide-amplified immunohistochemistry-fluorescence in situ hybridization (TAI-FISH), a dual-epoch neural-activity-dependent labeling protocol. Here we describe the step-by-step procedures for TAI-FISH on brain sections from mice that were sequentially stimulated by morphine (appetitive first stimulus) and foot shock (aversive second stimulus). We exemplify our approach by FISH-labeling the neural ensembles that were activated by the second stimulus for the mRNA expression of c-fos, a well-established marker of neural activation. We labeled neuronal ensembles activated by the first stimulus using fluorescence immunohistochemistry (IHC) for the c-fos protein. To further improve the temporal separation of the c-fos mRNA and protein signals, we provide instructions on how to enhance the protein signals using tyramide signal amplification (TSA). Compared with other dual-epoch labeling techniques, TAI-FISH provides better temporal separation of the activated neural ensembles and is better suited to investigation of whole-brain responses. TAI-FISH has been used to investigate neural activation patterns in response to appetitive and aversive stimuli, and we expect it to be more broadly utilized for visualizing brain responses to other types of stimuli, such as sensory stimuli or psychiatric drugs. From first stimulation to image analysis, TAI-FISH takes ∼9 d to complete.
行为神经科学的研究人员长期以来一直在寻找成像技术，可以在单细胞分辨率下识别和区分整个大脑中由顺序施加的刺激激活的神经集合。利用即时早期基因的 mRNA 和蛋白质表达的不同动力学，我们通过开发 tyramide 扩增免疫组织化学-荧光原位杂交（TAI-FISH），一种双时相神经活性依赖性标记方案来解决这个问题。在这里，我们描述了对连续用吗啡（食欲第一刺激）和足底电击（厌恶第二刺激）刺激的小鼠脑切片进行 TAI-FISH 的分步程序。我们通过 FISH 标记第二刺激激活的 c-fos 信使 RNA 表达的神经集合来说明我们的方法，c-fos 是神经激活的一个成熟标志物。我们使用荧光免疫组织化学（IHC）标记用于 c-fos 蛋白的第一个刺激激活的神经元集合。为了进一步提高 c-fos mRNA 和蛋白质信号的时间分离，我们提供了使用 tyramide 信号放大（TSA）增强蛋白质信号的说明。与其他双时相标记技术相比，TAI-FISH 提供了更好的激活神经集合的时间分离，更适合研究整个大脑的反应。TAI-FISH 已被用于研究对食欲和厌恶刺激的神经激活模式，我们期望它更广泛地用于可视化大脑对其他类型刺激的反应，例如感觉刺激或精神药物。从第一次刺激到图像分析，TAI-FISH 大约需要 9 天才能完成。
