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Anatomy and function of an excitatory network in the visual cortex

Nature volume 532, pages 370374 (21 April 2016) | Download Citation


Circuits in the cerebral cortex consist of thousands of neurons connected by millions of synapses. A precise understanding of these local networks requires relating circuit activity with the underlying network structure. For pyramidal cells in superficial mouse visual cortex (V1), a consensus is emerging that neurons with similar visual response properties excite each other1,2,3,4,5, but the anatomical basis of this recurrent synaptic network is unknown. Here we combined physiological imaging and large-scale electron microscopy to study an excitatory network in V1. We found that layer 2/3 neurons organized into subnetworks defined by anatomical connectivity, with more connections within than between groups. More specifically, we found that pyramidal neurons with similar orientation selectivity preferentially formed synapses with each other, despite the fact that axons and dendrites of all orientation selectivities pass near (<5 μm) each other with roughly equal probability. Therefore, we predict that mechanisms of functionally specific connectivity take place at the length scale of spines. Neurons with similar orientation tuning formed larger synapses, potentially enhancing the net effect of synaptic specificity. With the ability to study thousands of connections in a single circuit, functional connectomics is proving a powerful method to uncover the organizational logic of cortical networks.

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We thank R. Arora, E. Ashbolt, L. Bailey, L. Beaton, P. Chopra, A. Coda, M. Driesbach, R. Fang, M. Fisher, A. Giuliano, H. Godtfredsen, A. Henry, E. Holtzman, P. Hughes, K. Joines, J. Larson, S. Maillet, K. Moody, E. Nguyen, A. Orban, V. Osuiji, K. Robertson, D. Sandman, S. Schwartz, E. Sczerzenie, N. Thatra, L. Thomas, B. Titus, R. Torres for tracing and reconstruction; E. Raviola for discussions and advice; H. Kim for technical support at the beginning of the study; A. Wetzel for help with alignment and stitching; A. Cardona and S. Saalfeld for the CATMAID project being openly available; and N. da Costa for launching the EM annotation program at the Allen Institute for Brain Science, with help and support from B. Youngstrom, R. Young, C. Dang and J. Phillips. We also thank D. Brittain, W. Gray Roncal, L. Thomas, S. Yurgenson, H. Elliot, and the HMS Image and Data Analysis Core for programming; E. Benecchi and the HMS EM Core Facility for technical support; P.J. Manavalan, K. Lillaney, and R. Burns, for helping make the data freely available; and S. Druckmann, Y. Park, C. Priebe, and J. Vogelstein for discussions on statistical analyses. We are indebted to M. Andermann, S. Chatterjee, N. da Costa, L. Glickfeld, C. Harwell, D. Hildebrand, M. Histed, S. Mihalas, L. Ostroff, E. Raviola, and J. T. Vogelstein for critical reading of various versions of the manuscript. This work was supported by NIH grants to RCR (R01 EY10115 and R01 NS075436); through resources provided by the NRBSC (P41 RR06009) and MMBioS (P41 GM103712); the HMS Vision Core Grant (P30 EY12196); and the AIBS. We thank the Allen Institute founders, P. G. Allen and J. Allen, for their vision, encouragement, and support. WCL was also supported by the Bertarelli Program in Translational Neuroscience and Neuroengineering, Edward R. and Anne G. Lefler Center, and Stanley and Theodora Feldberg Fund, and the NIH (R21 NS085320). VB was also supported by Neuro-Electronics Research Flanders. The project described was partially supported by the NIH by the above named awards. Its content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author information


  1. Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Wei-Chung Allen Lee
    • , Vincent Bonin
    • , Michael Reed
    • , Brett J. Graham
    •  & R. Clay Reid
  2. Neuro-Electronics Research Flanders, a research initiative by imec, Vlaams Instituut voor Biotechnologie (VIB) and Katholieke Universiteit (KU) Leuven, 3001 Leuven, Belgium

    • Vincent Bonin
  3. Biomedical Applications Group, Pittsburgh Supercomputing Center, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA

    • Greg Hood
  4. Allen Institute for Brain Science, Seattle, Washington 98103, USA

    • Katie Glattfelder
    •  & R. Clay Reid


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W.-C.A.L. and V.B. performed the in vivo calcium imaging and analysed it. W.-C.A.L. processed the tissue for EM, sectioned the series, and aligned the block with the in vivo imaging. W.-C.A.L. and M.R. imaged it on the TEMCA. G.H. and W.-C.A.L. aligned the EM images into a volume. W.-C.A.L., M.R., K.G. annotated the EM dataset and W.-C.A.L. and K.G. supervised segmentation efforts. B.J.G. and W.-C.A.L. generated software for visualization and analysis. W.-C.A.L. performed quantitative analysis on the tracing. W.-C.A.L., V.B., and R.C.R. designed the experiment and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Wei-Chung Allen Lee or R. Clay Reid.

The aligned EM dataset will be publicly accessible at http://neurodata.io/lee16.

Extended data

Supplementary information

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  1. 1.

    Supplementary Data

    This file contains Supplementary Data 1-3.


  1. 1.

    In vivo fluorescence to EM correspondence

    Fluorescence in vivo two-photon microscopy data 3-D aligned with the EM volume. To visualize three imaging datasets (in vivo anatomy: fluorescence volume, in vivo physiology: fluorescence planes; and EM) first, the volume is flown-through, oriented with the EM sections (coronally, relative to the brain). Then the volume is rotated so that the perspective is above the surface and horizontal relative to the brain. Next, descending into the brain, note the correspondence of GCaMP3 (green) and functionally colored cell bodies with the nuclei in EM, the blood vessels (red) with clear vasculature in EM, and track apical dendrites in vivo (green radially oriented processes) in EM (large caliber cylindrical dendrites with spines).

  2. 2.

    EM volume overview

    The video shows a fly-through of the aligned EM series. Please see Author Information for directions to the publicly accessible high-resolution aligned dataset.

  3. 3.

    Higher resolution EM core

    The video shows fly-through of a cropped (16.4 x 16.4 µm) region traversing 400 of the aligned EM sections in the functionally imaged volume.

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