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.

中文翻译：

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使用TAI-FISH可视化由多种刺激激活的神经集合体。

行为神经科学的研究人员长期以来一直在寻找能够识别和区分神经集合体的成像技术，这些神经集合体是通过在整个大脑中以单细胞分辨率依次施加刺激而激活的。利用即时早期基因mRNA和蛋白质表达的不同动力学，我们通过开发酪氨酰胺扩增的免疫组织化学-荧光原位杂交（TAI-FISH）（一种双历时神经活性依赖性标记方案）解决了这个问题。在这里，我们描述了在小鼠大脑切片上进行TAI-FISH的分步程序，这些小鼠依次被吗啡（竞争性第一刺激）和足部电击（平均第二刺激）刺激。我们通过FISH标记由第二种刺激激活的c-fos mRNA表达所激活的神经小体来举例说明我们的方法，公认的神经激活标记物。我们使用c-fos蛋白的荧光免疫组织化学（IHC）标记了由第一个刺激激活的神经元集合。为了进一步改善c-fos mRNA和蛋白质信号的时间分离，我们提供了有关如何使用酪酰胺信号放大（TSA）增强蛋白质信号的说明。与其他双历元标记技术相比，TAI-FISH可以更好地在时间上分离激活的神经集合，并且更适合于研究全脑反应。TAI-FISH已被用于研究对食性和厌恶性刺激作出反应的神经激活模式，我们希望它能被更广泛地用于可视化对其他类型刺激（例如感觉刺激或精神药物）的大脑反应。