Volume 26, Issue 13, Pages 1737-1743 (July 2016) Parallel Cortical Networks Formed by Modular Organization of Primary Motor Cortex Outputs  Adjia Hamadjida, Melvin Dea, Joan Deffeyes, Stephan Quessy, Numa Dancause  Current Biology  Volume 26, Issue 13, Pages 1737-1743 (July 2016) DOI: 10.1016/j.cub.2016.04.068 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Electrophysiological Mapping and Tracer Injections (A) Cartoon shows the approximate location of the craniotomy (gray square) on a cebus monkey’s brain and the color code used for movements evoked with ICMS in the motor areas or receptive fields defined with multi-unit recordings in the parietal cortex. M, medial; R, rostral. (B) Physiological mapping data collected in CB-7. Similar mapping was done in all three monkeys. Each small dot on the digital photograph of the cortex represents a microelectrode penetration site. The hand representations of PMv, PMd, SMA, and M1 are outlined in red. For SMA, the location of stimulation sites are approximated based on the distance from PMd and the depth of the electrode. In the parietal cortex, dotted lines show the location of the borders among areas 1, 2, and 5. Medial and lateral, they show the extent of the hand representation and its borders with the forearm and face representations, respectively. The large color dots in premotor areas and area 5 show the location of tracer injections (blue, FB in PMv; green, FE in PMd; red, FR in SMA; and orange, BDA in area 5). The location of each tracer was alternated in different monkeys (Table S1). AS, arcuate sulcus; CS, central sulcus; IPS, intraparietal sulcus; M1, primary motor cortex; PMd, dorsal premotor cortex; PMv, ventral premotor cortex; SMA, supplementary motor area; 1, area 1; 2, area 2; 5, area 5. Scale bar, 1 mm. (C) Photomicrographs show the injection sites of all four tracers injected into CB-7 in relation to the physiological maps (left column). Scale bars, 1 mm. The alignment of the flattened sections and physiological maps confirmed that each tracer was injected in the hand representation of the targeted area. The right column shows examples of cell bodies that the tracers injected into CB-7 labeled in M1. Scale bars, 100 μm. Current Biology 2016 26, 1737-1743DOI: (10.1016/j.cub.2016.04.068) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Reconstruction of the Ipsilateral Hemisphere and Labeled Cells for CB-7 Top left cartoon illustrates the location of the main areas present in the flattened sections on the brain. The main panel shows the anatomical reconstruction of the hemisphere and labeled cells in CB-7. The location and extent of the injection cores are indicated with large colored blobs (BDA, orange; FE, green; FR, red; and FB, blue) on the reconstruction. Each small dot represents a labeled cell, colored according to the same code. The hand representations in motor areas are outlined with dark red contours and the borders among area 1, area 2, and area 5 with black dashed lines. A pale gray dashed line shows the approximate location of the convexity of the medial wall (midline unfolded). Each tracer injection labeled many cells across the ipsilateral hemisphere and in M1. The general pattern of connections of M1 was similar to previous studies and consistent in all three monkeys (see Figures S1 and S2). AS, arcuate sulcus; CMA, cingulate motor area; CS, central sulcus; IPS, intraparietal sulcus; M1, primary motor cortex; PMd, dorsal premotor cortex; PMv, ventral premotor cortex; PO/IPC, posterior operculum/inferior parietal cortex; SMA, supplementary motor area; 1, area 1; 2, area 2; 5, area 5. Scale bar, 5 mm. Current Biology 2016 26, 1737-1743DOI: (10.1016/j.cub.2016.04.068) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Pattern of Labeled Cells within the Hand Representation of M1 Location of labeled cells in M1 following injections in PMv (A), PMd (B), SMA (C), and area 5 (D). The first three columns show data from CB-7, CB-5, and CB-4, respectively. For each tracer, the reconstruction shows the hand area of M1 in light gray, bordered on the right with the central sulcus (CS, black lines). Small colored dots are individually labeled cells and are color coded according to the area injected (PMv, orange; PMd, red; SMA, green; and area 5, blue). Overlaid on top of the hand area and labeled cells on each panel, an isodensity contour (dark gray line) shows the zone with the highest density of labeled cells for each injection (high-density zone; Supplemental Experimental Procedures). Scale bar, 5 mm. Bar graphs in the rightmost column show quantitative results. For each high-density zone, we calculated the percentage of labeled cells per millimeter2 projecting to PMv (orange bars), PMd (red bars), SMA (green bars), and area 5 (blue bars). We found the high-density zones systematically contained greater proportions of labeled cells projecting to one cortical area injected. In the high-density zone identified after PMv injections (A), the relative density of the labeled neurons projecting to PMv was significantly greater (9.7%/mm2 or 53.9% of the normalized labeling; p < 0.05), compared to only 4.1%/mm2 for PMd, 2.7%/mm2 for SMA, and 1.5%/mm2 for area 5. Similarly, the relative density of the labeled neurons projecting to PMd (9.7%/mm2 or 62.8%), SMA (13.61%/mm2 or 72.6%), and area 5 (15.40%/mm2 77.7%) was significantly greater (p < 0.05) in the high-density zone identified after PMd (B), SMA (C), and area 5 (D) injections, respectively. These results support that neurons projecting to the four injected areas were clustered in specific subregions of M1. Current Biology 2016 26, 1737-1743DOI: (10.1016/j.cub.2016.04.068) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Segregation of M1 Outputs to Different Areas of the Ipsilateral Hemisphere (A) Data from all three monkeys were warped to align anatomical and physiological landmarks and produce morphed images of the pattern of labeling across monkeys (Figure S3). The four panels show morphed images with zones of high and low intensities of labeling in auto-scaled color maps over a composite M1 hand area produced with all data from the three monkeys. After injections in PMv, the cluster of labeled cells was mainly in the RL part of M1. Although less dense, a cluster of labeled cells was found in the RM area after PMd injections. After injections in SMA and area 5, clusters of cells were found in the CM and CL areas, respectively. (B) Contour plot showing the location of three levels isodensity contours based on the pseudocolor images in (A) over the composite map of M1. They show that the regions with the highest density of labeling following injections in PMv, PMd, SMA, and area 5 are largely segregated in different regions of the hand representation. M, medial; R, rostral. Scale bar, 1 mm. (C) Summary diagram of the zones in the M1 hand representation sending the densest outputs to diverse cortical areas, based on the present dataset (green arrows) and the zone receiving the predominant projection from cortical areas (red arrows) [8]. Current Biology 2016 26, 1737-1743DOI: (10.1016/j.cub.2016.04.068) Copyright © 2016 Elsevier Ltd Terms and Conditions