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Visualizing an emotional valence map in the limbic forebrain by TAI-FISH

Nature Neuroscience volume 17, pages 1552–1559 (2014)Cite this article

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Abstract

A fundamental problem in neuroscience is how emotional valences are represented in the brain. We know little about how appetitive and aversive systems interact and the extent to which information regarding these two opposite values segregate and converge. Here we used a new method, tyramide-amplified immunohistochemistry–fluorescence in situ hybridization, to simultaneously visualize the neural correlates of two stimuli of contrasting emotional valence across the limbic forebrain at single-cell resolution. We discovered characteristic patterns of interaction, segregated, convergent and intermingled, between the appetitive and aversive neural ensembles in mice. In nucleus accumbens, we identified a mosaic activation pattern by positive and negative emotional cues, and unraveled previously unappreciated functional heterogeneity in the D1- and D2-type medium-spiny neurons, which correspond to the Go and NoGo pathways. These results provide insights into the coding of emotional valence in the brain and act as a proof of principle of a powerful methodology for simultaneous functional mapping of two distinct behaviors.

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Figure 1: Use of TAI-FISH dual activity mapping to reveal the neural ensembles of appetitive and aversive emotional stimuli.
Figure 2: Segregated neural representations of morphine and foot shock in CEA.
Figure 3: Convergent neural representations of morphine and foot shock in PVN.
Figure 4: Intermingled neural representations of morphine and foot shock in NAcDMS.
Figure 5: Neural representations of morphine and foot shock in the whole field of NAc revealed by TAI-FISH.
Figure 6: Heterogeneity in D1- and D2-MSNs in the NAc in response to morphine and foot shock.
Figure 7: Representations of multiple positive and negative emotional cues in NAc.
Figure 8: An emotional valence map in the forebrain.

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Acknowledgements

We thank J. Feldman for critical review of the manuscript, S. Sarah, H. Kessels, A. Roe and M. Poo for comments on the manuscript, and S. Song, J. Huang, B. Lu and members of the Hu laboratory for stimulating discussions. This work was supported by the Chinese 973 Program (2011CBA00400), the Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB02030004), the One Hundred Talents Program and the Outstanding Youth Grant (to H.H.).

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  1. Jianbo Xiu and Qi Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, P.R. China

    Jianbo Xiu, Qi Zhang, Tao Zhou, Ting-ting Zhou, Yang Chen & Hailan Hu

  2. Graduate School of Chinese Academy of Sciences, University of Chinese Academy of sciences, Shanghai, P.R. China

    Jianbo Xiu, Qi Zhang, Tao Zhou & Ting-ting Zhou

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Contributions

J.X. designed the study and performed the TAI-FISH experiments. Q.Z. and Tao Zhou contributed to the TAI-FISH experiments. Ting-ting Zhou tested the time course for chocolate stimulation. Y.C. performed the statistical analysis. H.H. conceived the idea, designed the study and wrote the manuscript with input from J.X., Q.Z. and Tao Zhou.

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Correspondence to Hailan Hu.

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Integrated supplementary information

Supplementary Figure 1 Time course of c-fos mRNA and protein expression in CEA (a), PVN (b) and NAc (c) (related to Figs. 2–4)

The schematic was adapted from the mouse brain atlas (Paxinos and Franklin, 2001). Arrows indicate the time points chosen for the dual labeling experiment in Figures 2,3,4. TAI-FISH was used for the 4h and 6h time points in (c) as indicated by the circles. I-FISH was used for all other time points. n = 3 mice/group. Error bars represent s.e.m.

Supplementary Figure 2 Time course of c-fos mRNA and protein expression in basolateral amygdala (BLA) (a), medial prefrontal cortex (mPFC) (b) and dorsal raphe (DR) (c)

Note that no optimal time points were available for dual labeling analysis in these brain regions, due to persistent mRNA signals. All time points were examined by I-FISH. TAI-FISH was also examined for the 6h time points, but the mRNA signals were still present (data not shown). Error bars represent s.e.m.

Supplementary Figure 3 Representative images showing the double labeling (I-FISH for CEA and PVN, TAI-FISH for NAc) for c-fos protein (green channel) and mRNA (red channel) following single stimulation in CEA (a), PVN (b) and NAc (c) (related to Figs. 2–4)

0 min indicates the time point of animal sacrifice. Scale bars are 100 μm in a, 50 μm in b and c.

Supplementary Figure 4 MEA is activated by foot shock, but not morphine

a. Schematic illustrating the structure of MEA and its neighboring regions. Red box indicates the position of the image taken in (b). b. Representative images showing I-FISH double labeling for c-fos protein (green) and mRNA (red) in MEA following single stimulation of Mor or FS, and sequential stimulation of Mor-FS. Scale bars = 50 μm.

Supplementary Figure 5 BSTov is activated by morphine, but not foot shock

a.Schematic illustrating the structure of BSTov and its neighboring regions. Red box indicates the position of the image taken in (b). VP, ventral pallidum. b. Representative images showing the green (c-fos protein) and red (c-fos mRNA) channels of TAI-FISH double labeling in BSTov following single stimulation of Mor or FS, and sequential stimulation of Mor-FS. Scale bars = 50 μm.

Supplementary Figure 6 Partially convergent neural representations of morphine and foot shock in BSTfu

a. Schematic illustrating the structure of BSTfu and its neighboring regions. Red box indicates the position of the image taken in (b) and (c). b. Representative images showing the green (c-fos protein) and red (c-fos mRNA) channels of TAI-FISH double labeling in BSTfu following single stimulation of Mor or FS. c. Representative images showing the green (c-fos protein), red (c-fos mRNA) and merged channels of double labeling from five experiments: Sal-Ctx, Mor-Ctx, Sal-FS, Mor-Mor, and Mor-FS. Scale bars = 50 μm. d-e. Percentage of neurons expressing c-fos protein (d) and mRNA (e) in BSTfu in the six experimental conditions: Sal-Ctx, Mor-Ctx, Sal-FS, Mor-Mor, Mor-FS, and Mor-Coc. f. Percentage of overlap in the Mor-FS, Mor-Mor and Mor-Coc double labeling experiments. n = 3 mice/group. More than 250 neurons per mouse from 3 mice were counted for each group.Paired t-test adjusted by Benjamini-Hochberg procedure controlling the false discovery rate. *, p<0.05; **, p<0.01. Error bars represent s.e.m.

Supplementary Figure 7 Intermingled neural representations of morphine and foot shock in LSv

a. Schematic illustrating the structure of LSv and its neighboring regions. Red box indicates the position of the image taken in B and C. b. Representative images showing the green (c-fos protein) and red (c-fos mRNA) channels of TAI-FISH double labeling in LSv following single stimulation of Mor or FS. c. Representative images showing the green (c-fos protein), red (c-fos mRNA) and merged channels of double labeling from five experiments: Sal-Ctx, Mor-Ctx, Sal-FS, Mor-Mor, and Mor-FS. Scale bars = 50 μm. d-e Percentage of neurons expressing c-fos protein (d) and mRNA (e) in LSv in the six experimental conditions: Sal-Ctx, Mor-Ctx, Sal-FS, Mor-Mor, Mor-FS, and Mor-Coc. Note that the second morphine- and cocaine-induced mRNA signals were significantly reduced, presumably due to strong desensitization in this region. f. Percentage of overlap in the Mor-FS, Mor-Mor and Mor-Coc double labeling experiments. n = 3 mice/group. More than 250 neurons per mouse from 3 mice were counted for each group. Paired t-test adjusted by Benjamini-Hochberg procedure controlling the false discovery rate. *,p<0.05. Error bars represent s.e.m.

Supplementary Figure 8 Summary of neural representations for morphine and foot shock in different regions of limbic forebrain, as revealed by I-FISH and TAI-FISH in this study

Scale bars are 200 μm.

Supplementary Figure 9 Morphine did not alter the averseness of foot shock

a. Example trajectory plot of two representative mice, which received either saline or morphine 6 hours earlier, and were tested by the real-time place preference assay for 20 min. They received foot-shock every time they entered the shock chamber on the left. b. Quantification of the number of attempts mice tried to enter the shock chamber during the 20-min period, n = 4 mice each group, Mann-Whitney U test, n.s., not significant, P = 0.69. Error bars represent s.e.m.

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Xiu, J., Zhang, Q., Zhou, T. et al. Visualizing an emotional valence map in the limbic forebrain by TAI-FISH. Nat Neurosci 17, 1552–1559 (2014). https://doi.org/10.1038/nn.3813

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