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Use of TAI-FISH to visualize neural ensembles activated by multiple stimuli

Nature Protocols volume 13, pages 118133 (2018) | Download Citation

Abstract

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.

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Acknowledgements

We thank the Hu laboratory members for valuable discussions and advice. This study was supported by grants from the Ministry of Science and Technology of China (2011CBA00400, 2016YFA0501000), the National Natural Science Foundation of China (91432108, 31225010, and 81527901) and the Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB02030004) to H.H., and by a grant from the National Natural Science Foundation of China (81571335) to Q.H.

Author information

Author notes

    • Qi Zhang
    •  & Qiye He

    These authors contributed equally to this work.

Affiliations

  1. Interdisciplinary Institute of Neuroscience and Technology, Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China.

    • Qi Zhang
    • , Qiye He
    • , Jihua Wang
    • , Chaoying Fu
    •  & Hailan Hu
  2. Center for Neuroscience, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, School of Medicine, Zhejiang University, Hangzhou, China.

    • Qi Zhang
    • , Qiye He
    • , Jihua Wang
    • , Chaoying Fu
    •  & Hailan Hu
  3. Mental Health Center, School of Medicine, Zhejiang University, Hangzhou, China.

    • Hailan Hu

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Contributions

Q.Z. and H.H. designed the experimental strategy. Q.Z., J.W. and C.F. optimized experimental procedures. Q.H., Q.Z. and C.F. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Hailan Hu.

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DOI

https://doi.org/10.1038/nprot.2017.134

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