Nature Neuroscience: doi: /nn.4332

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1 Nature Neuroscience: doi: /nn.4332

2 Supplementary Figure 1 Topography and termination patterns of cortico-striatal projections (a) Manual inspection of ~150 tracer-labeled cortico-striatal pathways revealed highly topographic projection patterns across the rostral-caudal extent of the CP. Tracer injections (column 1) were relatively small and confined within anatomically delineated cortical regions thereby producing discrete and specific labeling across the CP (columns 2-7). Injections in different cortical areas also produced a variety of termination patterns within the projection fields. Magnified images of the labeling at the intermediate CP is presented in the final column and demonstrates the different axonal terminal patterns. Some show dense terminal fields surrounded by diffuse labeling, others are arranged as horizontal or vertical stripes, and some are confined to the center parts of the CP while most others run along the periphery. (b) The large majority of the anterograde tracing data was confirmed with retrograde tracer injections placed in the CP. Injections of four different tracers infused into different regions within the CP retrogradely labeled different populations of cortical cells validating unique cortical projection sources to different parts of the CP. They also revealed the laminar organization of the CP projecting neurons. Nature Neuroscience: doi: /nn.4332

3 Nature Neuroscience: doi: /nn.4332

4 Supplementary Figure 2 Triple anterograde tracing and tracer-dependent labeling (a) Despite their potential confound of labeling fascicles (discussed in b below), AAV can be used to validate PHALtraced pathways and can be used in a triple anterograde tracing approach to showcase topography and interdigitation of terminations from different cortical sources. Injections in the MOs-fef/ACAd (AAV-RFP), PTLp rostral (AAV-GFP) and VISam (PHAL) validate the projections of these areas to their specific domains. They also show the topographic interdigitation of projections within the CPi.dm.d from MOs-fef/ACAd (red) and VISam (magenta) and between the CPi.dm.d and CPi.dm.dl. Insets show 20x confocal micrographs of boxed regions. (b) Comparison of labeled elements using PHAL versus viral tracers like AAV. Triple anterograde tracer injections of PHAL, AAV-GFP and AAV-RFP injections in the MOs upper limb demonstrate how the fascicles are intensely labeled by the AAV tracers, but not by the PHAL although a few PHAL-labeled axons can be detected within the fascicles. Hence, only PHAL-labeled pathways were used for reconstructions and computational analyses. (c) High resolution confocal imaging at 40x magnification shows the abundance of PHAL-labeled varicosities and boutons, both of which are indicative of synaptic contacts 54, suggesting that positive pixels are more likely indicative of connections rather than fibers of passage. Nature Neuroscience: doi: /nn.4332

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6 Supplementary Figure 3 Intensity, span and labeling density index values (a) Graphical representation of intensity and span as complementary measures to quantify density of CP labeling from individual cortical sources. Intensity is the sum of the labeled pixels across the CP and thereby indexes the concentration of labeling. Span is the count of cells containing labeling and therefore measures how diffuse the labeling is throughout the CP. Projections from the ACAd label a large majority of the 35 x 35 pixels within each cell of the CP (blue; intensity=4,046,768) and therefore show a higher intensity value than projections from the SS rostral, whose projections show far less labeling of pixels (red; intensity=161,560). On the other hand, the ACAd injection labels far fewer cells compared to projections from the SSs rostral and therefore has a much lower span value (1,453 compared to 2,406 for SSs rostral). The Labeling Density Index (LDI) summarizes these labeling properties in a single value, which is the ratio of the intensity to span. LDI values greater than 1.0 are designated as concentrated, with lesser LDI values defined as diffuse. (bd) Intensity, span and LDI values for each cortical area are presented for the rostral, intermediate and caudal CP. Nature Neuroscience: doi: /nn.4332

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8 Supplementary Figure 4 Table of communities and domains and their cortical constituents (a) Table showing all cortical areas that project to unique communities and domains within the intermediate CP. Cortical areas are color coded according to their projections to domains and are the same as their centroid colors (i.e., Fig. 5f). (bc) Table of cortical areas that project to unique communities and domains within the rostral and caudal CP. Colors for cortical areas are the same as those used for the intermediate CP to highlight the higher degree of projection integration from different cortical areas across the CP. Nature Neuroscience: doi: /nn.4332

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10 Supplementary Figure 5 Somatic sensorimotor cortical injections, projections and intracortical connections (a) Injection locations for all somatic sensorimotor cortical areas representing unique body subregions. Generally within each somatic sensorimotor cortical area, there was a caudomedial to rostrolateral representation of trunk, lower limb, upper limb, inner mouth and outer mouth. The literature available for somatotopic cortical maps largely support these findings, although this is the first report to identify all body subregions for all somatic sensorimotor cortical areas 61 (also see Woolsey et al., Res. Publ. Assoc. Res. Nerv. Ment. Dis.,1952; Hall & Lindholm, Exp. Brain Res., 1974; Welker, J. Comp. Neurol., 1976; Muakkassa & Strick, Brain Res., 1979; Donoghue & Wise, J. Comp. Neurol., 1982; Tanji & Kurata, J. Neurophysiol., 1982; Godschalk et al., Exp. Brain Res.,1984; Neafsey et al., Brain Res., 1986; Mitz & Wise, J. Neurosci., 1987). (b) Panels show retention of the CP somatotopy in more caudal parts of the CP (i.e., 0.28 mm from bregma). (c) The specificity of injections within the SSp-body subregions was validated by examining their thalamic and brainstem projections. Leftmost panels are schematic representations of the somatosensory thalamus (top) and brainstem (bottom) modified from 57 (also see Nord, J. Comp. Neurol., 1967). The thalamus schematic shows the approximate location of body representations within the ventral posteromedial (VPM) and ventral posterolateral (VPL) thalamic nuclei. Injections into the SSp-tr, ll, ul and m regions showed clear topographic projections to their corresponding VPM and VPL regions. Trunk representations were in dorsal regions, followed by lower limb which were lateral to upper limb representations, with orofacial representations in the most ventral and medial parvocellular regions (also see Emmers, J. Comp. Neurol., 1965). The somatosensory brainstem schematic shows the approximate location of the body representations in the spinal trigeminal, the cuneate and gracile nuclei. Consistent with these brainstem body representations, SSp-ll labeled the gracile nucleus, the SSp-ul the cuneate, and the SSp-m the dorsal parts of the spinal trigeminal (SPV) nucleus with SSp-m/o represented dorsal and lateral to the SSp-m/i regions 61 (also see Nord, J. Comp. Neurol., 1967; Kruger et al., J. Neurophysiol.,1961; Kruger & Michel, Exp. Neurol., 1962). The cortical location of the SSp-m/i and SSp-m/o also agree with data showing that SSp representations of the tongue are generally more rostral and lateral than those of the lips in rats 62 and monkeys 63. (d) Specificity of SSp-bfd injections was also validated by observing their topographic projections to the medial part of the posterior thalamus (POm), to the VPM and to approximately the ventral third of the spinal trigeminal, precisely where whisker representations reside 57, 58,64 (also see Nord, J. Comp. Neurol., 1967). (e) Panels showing strong intra-cortical connectivity among all somatic sensorimotor cortical regions for upper limb. Figure adapted from 1. (f, left panels) Mouth somatic sensorimotor region projections to the CPi.vl.v and vt were validated with a FG injection placed within these domains. FG retrogradely labeled cells within the MOp and SSp regions for mouth, but did not label cells within SSp and SSp-bfd regions for trunk. (f, right panels) CTb 647 injection in CP trunk region substantiated its inputs from somatic sensorimotor trunk regions. Cells were labeled in the trunk regions within the MOp, SSp and SSp-bfd. Nature Neuroscience: doi: /nn.4332

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12 Supplementary Figure 6 Cortical projections to dorsomedial (CPi.dm) and ventromedial (CPi.vm) CPi (a) A thalamic injection shows labeling of the CPi.vl.cvl substantiating the domain, but also validating the projection trend for this domain across the CP. Both rostral MOs (pole 2) and thalamic projections to the CPi.vl.cvl terminate in the lateral strip domain of the CPr and in the intermediate part of the CPc. (b) Centroids for the cortical areas that project to the central CP domains (denoted by *). Cortical areas that project to each center domain are listed next to the centroids. (c) Raw data showing cortical projections from the ORBvl and ORBl to the CPi center domain CPi.dm.cd and from the rostral MOs (pole 1) to the CPi.vm.cvm. (d) Raw data showcasing representative projections to the dorsomedial CPi domains. (e) Connectivity of the MOs-fef (ACAd/MOs region) compared to those of the VISam and from the somatomotor region for lower limb. Unlike the MOs ll, the MOs-fef is connected with other visual cortical areas like the VISam, VISp and RSPv. Similar to the VISam, the MOs-fef is also connected with visual thalamic nuclei like the lateral dorsal (LD) and projects to the CPi.dm.d, the domain of the CP that receives visual and spatial information from the VISam, VISal, ACAv and PTLp. The MOs ll on the other hand projects to the somatosensory lateral part of the CPi. (f) Topographically arranged projections from the VISp and VISpm to the CPi.dm.dm domain. The terminal field from the VISpm is typically in between the two patches of segregated terminals from the VISp. (g) Double anterograde tracers display the boundary between the CPi.dm.im and CPi.vm.vm domains. (h-j) Injections in different thalamic nuclei validate the CPi.dm.d, CPi.dm.dm and CPi.vl.v domains of the intermediate CP. (k) A CTb 647 injection in the CPi.dm.dl and CPi.dl.d (trunk region) retrogradely labels cells in the ACAd, MOp tr, SSp-tr and PTLp caudal lateral confirming their projections to these domains. Nature Neuroscience: doi: /nn.4332

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14 Supplementary Figure 7 Cortical projections to the rostral (CPr) and caudal (CPc) CP (a) Color-coded CP in the left panel shows the four CPr communities, which are also faithfully represented by the centroids (right panels). Centroid color assignment from the intermediate CP was maintained in the rostral and caudal CP to highlight the higher degree of cortical convergence that occurs at these CP divisions. This precluded identification of smaller domains within the CPr communities with the exception of the CPr.l, which was subdivided into two domains: the lateral strip (CPr.l.ls) and ventromedial (CPr.l.vm). All cortical areas projecting to each community are listed in a box next to the centroids. Boxes are color coded according to community structure. (b) Raw data demonstrating the domains of the CPr. (c) Thalamic injections validating the CPr.m and CPr.imd. (d) Raw images of representative cases for the dorsal (left) and intermediate (right) communities in the CPc. (e) Centroids representing dorsal (top; CPc.d), and intermediate (CPc.i) and ventral (CPc.v) (bottom) communities. The SSp-ll was grouped with the CPc.d communities by the algorithm, but its centroid (green, denoted with an *) fell in between the dorsal and intermediate communities. Based on its raw labeling and the centroid, the SSp-ll was grouped with cortical areas projecting to the CPc.i. All cortical areas projecting to each CPc domain are listed next to the centroids. Boxes are colored according to community assignment. Nature Neuroscience: doi: /nn.4332

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16 Supplementary Figure 8 MOs ul, MOp ul and ORBvl injections in zq175 and MAO A/B KO mice (a) Consistent placement of injections in layer V of MOs upper limb region in zq175 and WT subjects. Boxed regions are magnified and show individual neurons that absorbed the tracer at the center of the injection site. (b) Regression scatterplots showing a positive correlation between injection site size and label intensity in the CP for MOs upper limb, MOp upper limb and ORBvl injections as measured by Pearson s r. Injections in MOp upper limb showed a trend toward a significant correlation. [MOs ul: r 2 =0.1724, P=0.028; ORBvl: r 2 =0.3788, P=0.012; MOp ul: r 2 =0.1018, P=0.312]. (c) ORBvl injection captured with higher exposure setting to reveal labeling shows overexposed PHAL injection site. The same injection captured with lower exposure times reveals the number of neurons that absorbed the tracer for transport. (d) Boxplots showing that no group differences were detected in injection site size between zq175 and MAO A/B KO mice and their respective WT littermate controls. Boxes depict the range between the upper and lower quartiles, whiskers depict the maximum and minimum values, and the bar indicates the median value. [Injection site, zq175 or MAO Mean/SEM, WT Mean/SEM, P value, t value, degrees of freedom; MOs ul: 26.69/2.986, 21.60/2.640, 0.214, 1.278, 24; MOp ul: 36.00/7.452, 42.00/7.545, 0.585, , 9; ORBvl: 25.10/3.024, 26.83/5.043, 0.776, , 8]. * denotes significant difference (P<0.05) detected by two-tailed t test with Welch s correction. Nature Neuroscience: doi: /nn.4332

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18 Supplementary Figure 9 Cortico-striatal projections in zq175 and MAO A/B KO mice (a-b) Raw CP labeling resulting from injections placed in the MOs upper limb of zq175 mice and in the ORBvl of MAO A/B KO mice, respectively. (c) Boxplot of projections from MOs upper limb to contralateral CP in zq175 animals and their WT littermates. Boxes depict the range between the upper and lower quartiles, whiskers depict the maximum and minimum values, and the bar indicates the median value. [Domain, Mean/SEM zq175 group, Mean/SEM WT group, P value, t value, degrees of freedom: i.vl.imv (ul), 9437/1417, 16960/2595, 0.019, 2.543, 21; i.dl.imd (ll), 5284/927, 10070/1609, 0.017, 2.578, 22; i.dl.d (tr), 1801/376, 3104/538.6, 0.059, 1.984, 24; i.dm.d, 59.16/28.27, 125.6/97.46, 0.522, , 16; i.dm.dl, 316.1/123.4, 414.9/156.4, , , 25; i.dm.im, 25.83/8.460, 127.6/104.4, 0.347, 0.972, 14; i.dm.cd, 466.3/153.7, 631.3/182.8, 0.496, , 25; i.dm.dm, 29.55/21.95, 56.25/51.39, 0.639, , 18; i.vm.vm, 308.0/131.9, 607.6/178.5, 0.190, 1.350, 24; i.vm.v, 795.6/278.1, 2047/725.1, 0.126, 1.611, 17; i.vm.cvm, 811.7/233.2, 1609/345.2, 0.068, 1.914, 23; i.vl.v (m/i), 7706/1189, 12110/1844, 0.057, 2.007, 23; i.vl.vt (m/o), 5230/663, 10890/2551, 0.048, 2.149, 15; i.vl.cvl, 3625/566, 7937/1359, 0.009, 2.930, 18]. (d) Boxplot of projections from MOp upper limb to ipsilateral CP in zq175 and WT animals (zq175, n=6; WT, n=6). No significant differences in normalized signal intensity values were detected in ipsilateral or contralateral (data not shown) domains. [Ipsilateral: i.vl.imv (ul), 6384/1663, 4446/1353, 0.390, , 9; i.dl.imd (ll), 4111/1119, 2779/853.9, 0.369, , 9; i.dl.d (tr), 1453/629.2, 852.4/216.3, 0.401, , 6; i.dm.d, /0.3670, 17.85/15.35, 0.309, 1.132, 5; i.dm.dl, 449.9/304.9, 229.8/139.1, 0.536, , 6; i.dm.im, 1.424/1.352, 1.235/0.7757, 0.907, , 7; i.dm.cd, 80.18/31.34, 51.14/29.84, 0.519, , 9; i.dm.dm, /0.3299, /0.1159, , 1.236, 8; i.vm.v, 863.6,/332.0, 544.3/149.2, 0.414, , 6; i.vm.cvm, 646.7/228.7, 248.5/105.2, 0.158, 1.582, 7; i.vl.v (m/i), 3807/1336, 3188/785.4, 0.700, , 8; i.vl.vt (m/o), 3484/1156, 2894/680.2, 0.671, , 8; i.vl.cvl, 3703/1246, 1653/284.8, 0.170, 1.604, 5; Contralateral: i.vl.imv (ul), 4606/1480, 3967/1179, 0.743, , 9; i.dl.imd (ll), 3435/1328, 2743/591.0, 0.651, , 6; i.dl.d (tr), 1116/535.8, 725.9/153.7, 0.516, , 5; i.dm.d, 3.693/2.297, 7.712/3.790, 0.391, , 8; i.dm.dl, 128.7/87.71, 114.4/35.54, 0.885, , 6; i.dm.im, 1.270/0.7481, 4.077/2.045, 0.245, 1.289, 6; i.dm.cd, 78.44/42.89, 62.95/12.95, 0.743, , 5; i.dm.dm, /0.2096, 1.806/1.596, 0.396, , 5; i.vm.vm, 422.0/200.7, 301.3/73.54, 0.593, , 6; i.vm.v, 437.1/143.1, 561.0/122.0, 0.526, , 9; i.vm.cvm, 85.75/53.76, 60.22/12.83, 0.664, , 5; i.vl.v (m/i), 4533/1687, 4059/1589, 0.843, , 9; i.vl.vt (m/o), 3725/1384, 3715/1391, 0.996, , 9; i.vl.cvl, 1715/712.8, 1490/138.3, 0.769, , 5]. (e) Comparisons of MOp upper limb projection field span in ipsilateral and contralateral CP between zq175 and WT mice. [Ipsilateral: 4432/407, 4208/241, 0.648, , 8; Contralateral: 1017/31, 1044/39, 0.606, , 9]. (f) MOs upper limb and ORBvl projection field span in contralateral CP of zq175 (left) and MAO A/B KO (right) mice compared to their respective WT littermates. [MOs ul: 4843/302, 5187/296, 0.423, , 25; ORBvl: , 2314/370, 0.016, 3.142, 7]. * denotes significant difference (P<0.05) detected by two-tailed t test with Welch s correction. Intensity is measured as the total number of labeled pixels. Span is measured as the total number of labeled cells. Nature Neuroscience: doi: /nn.4332

Nature Neuroscience doi: /nn Supplementary Figure 1. Characterization of viral injections.

Nature Neuroscience doi: /nn Supplementary Figure 1. Characterization of viral injections. Supplementary Figure 1 Characterization of viral injections. (a) Dorsal view of a mouse brain (dashed white outline) after receiving a large, unilateral thalamic injection (~100 nl); demonstrating that