Figure 1. Experimental approach used to map in visual space the orientation and retinotopic organization of V1 inputs to single V2 orientation columns: example case MK373.

(A) Schematics of the experimental approach. Intrinsic signal optical imaging (OI) of a region encompassing V1 and V2 (red box) was performed in vivo to obtain functional maps of stimulus orientation and retinotopy, and injections of retrograde tracers were made into a single orientation column in V2. (B) In vivo image of the surface vasculature, obtained under green light illumination, used as reference to position pipettes for tracer injections to specific functional domains, as well as to register functional maps to confocal images of histological sections (see Extended Data Fig. 1). In this representative case, three different tracer injections were made; confocal images of these injection sites are shown superimposed to the surface vasculature inside the colored boxes, and at higher magnification in panel (F): CTB647 (purple), CTB488 (green), and CTB555 (red). The white contour here and in (C-E) delineates the V1/V2 border based on the functional maps. Scale bar: 1 mm and valid for B-E and H-J. (C, D) Difference orientation maps obtained by subtracting responses to two orthogonally-oriented gratings (45°–135° and 22.5° – 112.5°, respectively), as shown in the insets above the panels. The orientation maps in V2 show larger orientation domains than in V1, and a stripy organization, with regions having strong orientation responses corresponding to the thick (delineated by the cyan dotted contours) and pale stripes, and regions with weak or no orientation responses corresponding to the thin stripes (Tn; delineated by the white dotted contours). The outlines of the stripes shown on these maps are based on both the orientation and CO maps (see also Extended Data Fig. 1). Superimposed to the maps in (C-E) and (H-I) are manual plots of the locations of the V1 cells (green dots in C-D and H-I, black dots in E) labeled by the CTB488 injection, and the contour of the CTB488 injection site (green oval in V2). V1 label from the other tracer injections is shown in Figure 2 and Extended data Fig. 2. (E) V1 cells (black dots) labeled by the CTB488 injection (black oval) are shown superimposed to the composite orientation map. Other conventions are as in (C-D). (F) Confocal images of injection sites taken under 647 nm (left), 488 nm (middle), and 555 nm (right) light illumination. Injection sites in V2 were outlined manually as indicated by the colored ovals. Scale bar: 200μm. (G) Confocal images of V1 cells labeled by each respective tracer injection site taken under illumination with different light wavelengths. Scale bars: 100 μm. (H,I) Retinotopic maps generated by subtracting responses to 90° (H) or 0° (I) oriented gratings occupying complementary and adjacent strips (0.5° in width) of visual space (as shown in the insets above). This visual stimulation paradigm generates response stripes (manually delineated by white dashed contours) corresponding to the stimulated visual locations between the masks. The area encircled by the yellow contour is estimated to correspond to ~2.5° along an axis parallel to the vertical meridian (corresponding to the location of the V1/V2 border) and 1.5° along the orthogonal axis. (J) Visual cortex-to-visual space mapping grid generated within the retinotopic mask (area delimited by the yellow contour). This grid contains 125 points along the vertical meridian and 75 points along the orthogonal axis, which provides a resolution of 0.02° in both directions. CTB488-labeled V1 cells (green dots) are superimposed on the grid. For the purpose of the final analysis, the V1 cells that lay on vessels were discarded and are not shown here. (K) Visual field map of the retinotopic location and orientation preference of the CTB488-labeled V1 cells. The location of the vertical meridian is at 0° on the Horizontal Meridian axis and corresponds to the location of the V1/V2 border on the brain. As there is no landmark for the horizontal meridian representation on the brain, its location relative to the labeled cells could not be determined accurately, and as such it is not indicated on the vertical meridian axis. The labeled field is located at parafoveal eccentricities (3–7°).