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Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex


Neurons in the cerebral cortex are organized into anatomical columns, with ensembles of cells arranged from the surface to the white matter. Within a column, neurons often share functional properties, such as selectivity for stimulus orientation; columns with distinct properties, such as different preferred orientations, tile the cortical surface in orderly patterns. This functional architecture was discovered with the relatively sparse sampling of microelectrode recordings. Optical imaging of membrane voltage or metabolic activity elucidated the overall geometry of functional maps, but is averaged over many cells (resolution >100 µm). Consequently, the purity of functional domains and the precision of the borders between them could not be resolved. Here, we labelled thousands of neurons of the visual cortex with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at single-cell resolution with two-photon microscopy up to a depth of 400 µm. In rat primary visual cortex, neurons had robust orientation selectivity but there was no discernible local structure; neighbouring neurons often responded to different orientations. In area 18 of cat visual cortex, functional maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two cells wide. These results indicate that cortical maps can be built with single-cell precision.

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We thank E. Takahashi for involvement in the first set of experiments; B. Sabatini, W. Regehr, R. Yuste and F. Engert for discussions and technical advice; S. Yurgenson for technical support and programming; A. Kerlin and J. Leong for programming; A. Vagodny for surgical assistance; and R. Yuste and J. Pezaris for comments on the manuscript. This work was supported by grants from the NEI and fellowships from the Uehara Foundation (K.O.), the Goldenson Fund (S.C.) and HHMI (Y.H.C.).Authors' contributions K.O. started this work and played the major role in the project; S.C., Y.H.C. and P.K. contributed equally to its completion.

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Competing interests

The authors declare that they have no competing financial interests.

Correspondence to R. Clay Reid.

Supplementary information

  1. Supplementary Figure 1

    The calcium indicator OGB-1 AM preferentially stains neurons rather than glia when pressure ejected directly into layer 2/3 of the visual cortex in vivo. (JPG 57 kb)

  2. Supplementary Figure 2

    Single condition ( F/F) maps in rat and cat visual cortex. (PDF 161 kb)

  3. Supplementary Figure 3

    Columnar organization of direction discontinuity maps in cat visual cortex. (JPG 55 kb)

  4. Supplementary Discussion

    This section addresses various technical issues concerning two-photon calcium imaging that we deemed too important to omit, but were of insufficient interest to the general readership to include in the main discussion. (PDF 89 kb)

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Further reading

Figure 1: Functional maps of selective responses in rat visual cortex with single-cell resolution.
Figure 2: Smoothly changing direction map in cat visual cortex.
Figure 3: Direction discontinuity in cat visual cortex.
Figure 4: Sharpness of direction discontinuity at multiple depths.
Figure 5: Correspondence of direction tuning obtained by calcium imaging and single-unit electrophysiology in cat visual cortex.
Figure 6: Three regimes of functional organization.


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