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Visualizing mammalian brain area interactions by dual-axis two-photon calcium imaging

Nature Neuroscience volume 17, pages 18251829 (2014) | Download Citation

Abstract

Fluorescence Ca2+ imaging enables large-scale recordings of neural activity, but collective dynamics across mammalian brain regions are generally inaccessible within single fields of view. Here we introduce a two-photon microscope possessing two articulated arms that can simultaneously image two brain areas (0.38 mm2 each), either nearby or distal, using microendoscopes. Concurrent Ca2+ imaging of 100–300 neurons in primary visual cortex (V1) and lateromedial (LM) visual area in behaving mice revealed that the variability in LM neurons' visual responses was strongly dependent on that in V1, suggesting that fluctuations in sensory responses propagate through extended cortical networks.

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Acknowledgements

We thank K. Merkle for machining the various custom parts of the microscope, S. Sinha for help with coating miniature mirrors, and J. Li for help with histology and mouse husbandry and genotyping. We appreciate support from a Stanford Vision Translation Postdoctoral Fellowship (J.L.), a Swiss National Postdoctoral Fellowship (B.F.G.), graduate research fellowships from the US National Science Foundation, Stanford University and the Simons Foundation (L.J.K.) and research funding from a US National Institutes of Health (NIH) EUREKA grant (M.J.S.) and the NIH NDC for the Optical Control of Biological Function (J.L. and M.J.S.).

Author information

Affiliations

  1. James H. Clark Center for Biomedical Engineering & Sciences, Stanford University, Stanford, California, USA.

    • Jérôme Lecoq
    • , Joan Savall
    • , Dejan Vučinić
    • , Benjamin F Grewe
    • , Hyun Kim
    • , Jin Zhong Li
    • , Lacey J Kitch
    •  & Mark J Schnitzer
  2. Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.

    • Joan Savall
    •  & Mark J Schnitzer
  3. CNC Program, Stanford University, Stanford, California, USA.

    • Mark J Schnitzer

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Contributions

J.L., D.V. and M.J.S. initiated the project. J.L., J.S., D.V. and M.J.S. designed the microscope. J.L., B.F.G. and H.K. performed experiments. J.Z.L. prepared and titrated viral vectors. J.L. and L.J.K. performed analysis. J.L. and M.J.S. wrote the manuscript. All authors reviewed the manuscript. M.J.S. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jérôme Lecoq or Mark J Schnitzer.

Integrated supplementary information

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–6

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    Supplementary Methods Checklist

Videos

  1. 1.

    A head-restrained mouse behaving on a trackball views moving grating visual stimuli (left panel) during concurrent two-photon Ca2+-imaging in visual areas V1 (middle panel) and LM (right panel).

    Ca2+ signals are displayed as ΔF(x, y, t) /σ (x, y), where the normalization factor σ (x, y) provides an estimate of each pixel's baseline noise (Online Methods). The appearances of white arrows indicate periods of visual stimulation and the directions of grating movement on the video monitor. The increase in background fluorescence is due to neuropil activation and is not light leakage from the video monitor, which accounted for <0.2% of the photodetection currents. The V1 and LM panels are cropped from full size, and the scale bars are both 100 μm.

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DOI

https://doi.org/10.1038/nn.3867

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