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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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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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  1. Mountcastle, V. B. Modality and topographic properties of single neurons of cat's somatic sensory cortex. J. Neurophysiol. 20, 408–434 (1957)

    Article  CAS  Google Scholar 

  2. Hubel, D. H. & Wiesel, T. N. Receptive fields, binocular interaction and functional architecture in the cat's visual cortex. J. Physiol. (Lond.) 160, 106–154 (1962)

    Article  CAS  Google Scholar 

  3. Grinvald, A., Anglister, L., Freeman, J. A., Hildesheim, R. & Manker, A. Real-time optical imaging of naturally evoked electrical activity in intact frog brain. Nature 308, 848–850 (1984)

    Article  ADS  CAS  Google Scholar 

  4. Blasdel, G. G. & Salama, G. Voltage-sensitive dyes reveal a modular organization in monkey striate cortex. Nature 321, 579–585 (1986)

    Article  ADS  CAS  Google Scholar 

  5. Grinvald, A., Lieke, E., Frostig, R. D., Gilbert, C. D. & Wiesel, T. N. Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature 324, 361–364 (1986)

    Article  ADS  CAS  Google Scholar 

  6. Bonhoeffer, T. & Grinvald, A. Iso-orientation domains in cat visual cortex are arranged in pinwheel-like patterns. Nature 353, 429–431 (1991)

    Article  ADS  CAS  Google Scholar 

  7. Shmuel, A. & Grinvald, A. Functional organization for direction of motion and its relationship to orientation maps in cat area 18. J. Neurosci. 16, 6945–6964 (1996)

    Article  CAS  Google Scholar 

  8. Weliky, M., Bosking, W. H. & Fitzpatrick, D. A systematic map of direction preference in primary visual cortex. Nature 379, 725–728 (1996)

    Article  ADS  CAS  Google Scholar 

  9. Maldonado, P. E., Godecke, I., Gray, C. M. & Bonhoeffer, T. Orientation selectivity in pinwheel centers in cat striate cortex. Science 276, 1551–1555 (1997)

    Article  CAS  Google Scholar 

  10. Parnavelas, J. G., Burne, R. A. & Lin, C. S. Receptive field properties of neurons in the visual cortex of the rat. Neurosci. Lett. 27, 291–296 (1981)

    Article  CAS  Google Scholar 

  11. Girman, S. V., Sauve, Y. & Lund, R. D. Receptive field properties of single neurons in rat primary visual cortex. J. Neurophysiol. 82, 301–311 (1999)

    Article  CAS  Google Scholar 

  12. Tsien, R. Y. Fluorescence measurement and photochemical manipulation of cytosolic free calcium. Trends Neurosci. 11, 419–424 (1988)

    Article  CAS  Google Scholar 

  13. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990)

    Article  ADS  CAS  Google Scholar 

  14. Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D. W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997)

    Article  ADS  CAS  Google Scholar 

  15. Waters, J., Larkum, M., Sakmann, B. & Helmchen, F. Supralinear Ca2+ influx into dendritic tufts of layer 2/3 neocortical pyramidal neurons in vitro and in vivo . J. Neurosci. 23, 8558–8567 (2003)

    Article  CAS  Google Scholar 

  16. Yuste, R. & Katz, L. C. Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters. Neuron 6, 333–344 (1991)

    Article  CAS  Google Scholar 

  17. Yuste, R., Peinado, A. & Katz, L. C. Neuronal domains in developing neocortex. Science 257, 665–669 (1992)

    Article  ADS  CAS  Google Scholar 

  18. Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl Acad. Sci. USA 100, 7319–7324 (2003)

    Article  ADS  CAS  Google Scholar 

  19. Wiesenfeld, Z. & Kornel, E. E. Receptive fields of single cells in the visual cortex of the hooded rat. Brain Res. 94, 401–412 (1975)

    Article  CAS  Google Scholar 

  20. Mao, B. Q., Hamzei-Sichani, F., Aronov, D., Froemke, R. C. & Yuste, R. Dynamics of spontaneous activity in neocortical slices. Neuron 32, 883–898 (2001)

    Article  CAS  Google Scholar 

  21. Ts'o, D. Y., Frostig, R. D., Lieke, E. E. & Grinvald, A. Functional organization of primate visual cortex revealed by high resolution optical imaging. Science 249, 417–420 (1990)

    Article  ADS  CAS  Google Scholar 

  22. Payne, B. R., Berman, N. & Murphy, E. H. Organization of direction preferences in cat visual cortex. Brain Res. 211, 445–450 (1981)

    Article  CAS  Google Scholar 

  23. Swindale, N. V., Matsubara, J. A. & Cynader, M. S. Surface organization of orientation and direction selectivity in cat area 18. J. Neurosci. 7, 1414–1427 (1987)

    Article  CAS  Google Scholar 

  24. Ohki, K., Matsuda, Y., Ajima, A., Kim, D. S. & Tanaka, S. Arrangement of orientation pinwheel centers around area 17/18 transition zone in cat visual cortex. Cereb. Cortex 10, 593–601 (2000)

    Article  CAS  Google Scholar 

  25. Gilbert, C. D. & Wiesel, T. N. Morphology and intracortical projections of functionally characterised neurones in the cat visual cortex. Nature 280, 120–125 (1979)

    Article  ADS  CAS  Google Scholar 

  26. Martin, K. A. & Whitteridge, D. The relationship of receptive field properties to the dendritic shape of neurones in the cat striate cortex. J. Physiol. (Lond.) 356, 291–302 (1984)

    Article  CAS  Google Scholar 

  27. Hirsch, J. A. Synaptic physiology and receptive field structure in the early visual pathway of the cat. Cereb. Cortex 13, 63–69 (2003)

    Article  Google Scholar 

  28. Adams, D. L. & Horton, J. C. Capricious expression of cortical columns in the primate brain. Nature Neurosci. 6, 113–114 (2003)

    Article  CAS  Google Scholar 

  29. Rose, D. & Blakemore, C. An analysis of orientation selectivity in the cat's visual cortex. Exp. Brain Res. 20, 1–17 (1974)

    Article  CAS  Google Scholar 

  30. Braitenberg, V. & Schuz, A. Anatomy of the Cortex: Statistics and Geometry (Springer, Berlin, 1991)

    Book  Google Scholar 

  31. Reid, R. C. & Alonso, J. M. Specificity of monosynaptic connections from thalamus to visual cortex. Nature 378, 281–284 (1995)

    Article  ADS  CAS  Google Scholar 

  32. Peters, A. & Yilmaz, E. Neuronal organization in area 17 of cat visual cortex. Cereb. Cortex 3, 49–68 (1993)

    Article  CAS  Google Scholar 

  33. Mountcastle, V. B. Perceptual Neuroscience: the Cerebral Cortex (Harvard University, Cambridge, 1998)

    Google Scholar 

  34. Pologruto, T. A., Sabatini, B. L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 〈〉 (2003)

    Article  Google Scholar 

  35. Kara, P. & Reid, R. C. Efficacy of retinal spikes in driving cortical responses. J. Neurosci. 23, 8547–8557 (2003)

    Article  CAS  Google Scholar 

  36. Berman, N. E., Wilkes, M. E. & Payne, B. R. Organization of orientation and direction selectivity in areas 17 and 18 of cat cerebral cortex. J. Neurophysiol. 58, 676–699 (1987)

    Article  CAS  Google Scholar 

  37. Kim, D. S., Matsuda, Y., Ohki, K., Ajima, A. & Tanaka, S. Geometrical and topological relationships between multiple functional maps in cat primary visual cortex. Neuroreport 10, 2515–2522 (1999)

    Article  CAS  Google Scholar 

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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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Correspondence to R. Clay Reid.

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Supplementary information

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)

Supplementary Figure 2

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

Supplementary Figure 3

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

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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Ohki, K., Chung, S., Ch'ng, Y. et al. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).

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