Extended Data Figure 1:. Single-neuron tracing protocol…
Extended Data Figure 1:. Single-neuron tracing protocol efficiently fills axons projecting to the ipsilateral striatum.
Extended Data Figure 1:. Single-neuron tracing protocol efficiently fills axons projecting to the ipsilateral striatum.We retrogradely labeled striatum projecting cells by stereotactically injecting cholera toxin subunit B conjugated with AlexaFluro594 or PRV-cre into the visual stri-atum of wild type mice or tdTomato reporter mice (Ai14, JAX), respectively (magenta). With visual guidance of two-photon microscopy, we electroporated single retrogradely labeled cells in V1 with a GFP expressing plasmid (cyan). (a) Coronal, maximum intensity projections of visual striatum. Scale bar = 1 mm. (b) Higher magnification view of the visual stratum. Scale bar = 0.2 mm. (c) Single channel images of the same axonal arbor as in (b). (d) Coronal maximum intensity projection containing V1. Scale bar = 1 mm. (e) Higher magnification view of V1. Scale bar = 0.2 mm. (f) Single channel images of V1. Scale bar = 0.2 mm. (g) Horizontal ARA-space projections of eight retrogradely labeled and electroportated cells. Cell ID numbers are indicated at the top right of each thumbnail. Scale bar = 1 mm. Note that one additional cell was retrogradely labeled and electroporated, which revealed its axonal projection to the striatum, but it is not shown because the brain was too distorted to allow accurate atlas registration.
Extended Data Figure 2:. Some axonal branches…
Extended Data Figure 2:. Some axonal branches terminate abruptly without arborizing, while other branches of…
Extended Data Figure 2:. Some axonal branches terminate abruptly without arborizing, while other branches of the same neuron arborise extensively within different target areas and appear to be completely filled.(a) Horizontal view of a representative cell in ARA space. The abrupt termination is labeled with a purple square. N=28 abruptly terminating cells. (b) The abrupt termination of the example cell shown as a maximum z-projection (left) and in the indi-vidual z-sections (right). (c) Two normal terminations of the same cell, shown as a maximum z-projection (left) and in two color-coded series of z-sections (right). (d) Distance of abrupt termi-nation from cell body vs. distance of farthest regular termination of the same cell. Dashed line indicates the unity line. (e) Pie charts illustrating the distribution of target numbers of all project-ing neurons without abrupt terminations (as shown in the main figures; left), of projecting cells with abrupt terminations (centre) and of all projecting cells (no abrupt terminations + abrupt ter-minations; right). (f) To test the effect of false negatives on our analyses, we simulated the ran-dom loss or gain of projections from the MAPseq dataset, while maintaining overall area projec-tion probabilities. N=553 neurons; 400 repeats. P-values based on a binomial test for all six pro-jection motifs determined as significantly over- or underrepresented in our dataset are plotted after removing (dropfraction < 1) or adding (dropfraction >1) connections. Mean (black line) and s.d. (shaded area) are indicated.
Extended Data Figure 3:. Thumbnails of traced…
Extended Data Figure 3:. Thumbnails of traced layer 2/3 V1 neurons, part 1.
Horizontal views…
Extended Data Figure 3:. Thumbnails of traced layer 2/3 V1 neurons, part 1.Horizontal views of the ARA space are shown, and cell ID numbers are indicated at the top right of each thumbnail. Scale bar = 1 mm.
Extended Data Figure 4:. Thumbnails of traced…
Extended Data Figure 4:. Thumbnails of traced layer 2/3 V1 neurons, part 2.
Horizontal views…
Extended Data Figure 4:. Thumbnails of traced layer 2/3 V1 neurons, part 2.Horizontal views of the ARA space are shown, and cell ID numbers are indicated at the top right of each thumbnail. Scale bar = 1 mm.
Extended Data Figure 5:. Individual neurons in…
Extended Data Figure 5:. Individual neurons in higher visual areas project to more than one…
Extended Data Figure 5:. Individual neurons in higher visual areas project to more than one target area.(a) Thumbnails of all traced neurons with cell bodies not in V1. Brain area identity is color-coded as in Figure 1. Cell identity is indicated at the top right of each thumbnail. Scale bar = 1 mm. (b) Histogram of the number of target areas per cell.
Extended Data Figure 6:. Density of axonal…
Extended Data Figure 6:. Density of axonal innervation by area and layer of V1 layer…
Extended Data Figure 6:. Density of axonal innervation by area and layer of V1 layer 2/3 projection neurons.(a) Total axon length plotted as a function of the number of targets inner-vated by every V1 projection neuron. (b) Axon length in area LM, PM or POR plotted as a func-tion of the total number of targets innervated by each neuron projecting to the respective area. (c–h) The axons of V1 neurons in target areas most densely innervate layers 2/3 and 5, with some density in layer 1, but less in layers 4 and 6, often recapitulating the laminar axonal profile within V1. Coronal views of each area are shown in ARA space (left) and axonal arbors of each neuron innervating the area are color coded. Scale bar = 200 m. A histogram of the laminar innervation is shown (right). Note that cells with abrupt terminations outside the shown area were included in this analysis. Areas depicted are (c) V1, (d) AL, (e) LI, (f) LM, (g) PM, (h) POR. White matter axons are not shown.
Extended Data Figure 7:. Conclusions from fluorescence-based…
Extended Data Figure 7:. Conclusions from fluorescence-based single neuron tracing data hold true if analysis…
Extended Data Figure 7:. Conclusions from fluorescence-based single neuron tracing data hold true if analysis is restricted to subset of target areas.(a) The projection patterns of re-constructed GFP-filled neurons when only the six target areas LI, LM, AL, PM, AM, and RL are considered. Projection strengths are normalized to the maximum projection of each neuron, and only neurons projecting to at least one target area are shown. (b) Pie chart showing the distribu-tion of target area numbers per projecting neuron. (c) Bar graph illustrating the fraction of all cells projecting to each target area. (d) The fraction dedicated input per area. (e) The number of times each binarized projection motif is observed. (f) The fraction of broadcasting cells as a function of the minimum projection strength (relative to the primary target) that each area needs to receive to be considered a target. (g) The fraction of broadcasting cells as a function of increasing buffer zones between areas within which axons are ignored, assuming a minimum projection of 1 mm of axon per target area. (h) The fraction of broadcasting cells as a function of the minimal amount of axon per area for it to be considered a target, assuming buffer zones of 100 μm width.
Extended Data Figure 8:. Location of cell…
Extended Data Figure 8:. Location of cell bodies in V1 as a function of their…
Extended Data Figure 8:. Location of cell bodies in V1 as a function of their projection targets.(a–l Horizontal views of ARA space are shown. The location of all traced V1 neurons are indicated as circles (cells with no abrupt terminations) or squares (cells with abrupt terminations). In every plot the cells projecting to the highlighted higher visual area are colored in solid blue. Target areas considered are (a) A, (b) AL, (c) AM, (d) ECT, (e) LI, (f) LM, (g) P, (h) PER, (i) PM, (j) POR, (k) RL, (l) TEA. (m-n) Quantification of cell body location in the rostro-caudal (m) and medio-lateral (n) direction. Dotted lines indicate expected number of cells based on a bootstrapping procedure, where we randomly selected neurons from the available positions to project to each area and repeated the process 10,000 times. P-values were derived from the boot-strapping probability distribution and are indicated for projection targets significantly deviating from this expectation (α=0.05). P-values below 10−4 are not exact and are therefore indicated as a range.
Extended Data Figure 9:. MAPseq dissection strategy.
Extended Data Figure 9:. MAPseq dissection strategy.
We identified the to-be-dissected high-er visual areas by…
Extended Data Figure 9:. MAPseq dissection strategy.We identified the to-be-dissected high-er visual areas by performing intrinsic imaging of visual cortex in response to stimuli at different positions in the contralateral visual field and mapping the resulting changes in intrinsic signals. (a) A representative retinotopic map, with responses to the two 25° visual stimuli pseudocolored in green and magenta (stimulus 1 position: 90° azimuth, 20° elevation; stimulus 2 position: 60° azimuth, 20° elevation). Based on this map, we fluorescently labelled retinotopically matched positions in the to-be-dissected cortical areas with a DiI stab (white circles). Putative borders between the higher visual areas are indicated in dashed lines for orientation. Scale bar = 1 mm. N=4 animals. (b) The MAPseq virus injection site is discernible in consecutive frozen 180 m thick coronal sections, using GFP fluorescence. Scale bar = 1 mm. (c) DiI injections targeted to matched retinotopic positions in six target areas identified by intrinsic signal imaging. DiI epifluorescence images of each 180 m thick slice are shown, and dissected areas are labeled. Scale bar = 1 mm.
Extended Data Figure 10:. Clustering of MAPseq…
Extended Data Figure 10:. Clustering of MAPseq data and data summary.
( a ) GAP…
Extended Data Figure 10:. Clustering of MAPseq data and data summary.(a) GAP and (b) Silhouette criteria for k-means clustering of the MAPseq neurons as a function of the number of clusters. Black arrow heads indicate chosen number of clusters (k=8). (c,d) Centroids for alterna-tive, near-optimal cluster number choices with (c) k=3 and (d) k=5. (e) Hierarchical clustering results of the MAPseq dataset using a cosine distance metric. Color intensity in (c,d,e) indicated projection strengths. (f,g) Summary of single-neurons projections from V1. (f) Cells targeting single higher visual areas (dedicated projection neurons) comprise the minority of layer 2/3 V1 projection neurons. Among the areas analysed by MAPseq, dedicated projection neurons pre-dominantly innervate cortical areas LM or PM. (g) Cells projecting to two or more areas (broad-casting projection neurons) are the dominant mode of information transfer from V1 to higher visual areas. In the six areas analysed by MAPseq, broadcasting neurons innervate combinations of target areas in a non-random manner, including those that are more or less abundant than ex-pected by chance. Line width indicates the absolute abundance of each projection type as ob-served in the MAPseq dataset.
Figure 1:. Brain-wide single-cell tracing reveals the…
Figure 1:. Brain-wide single-cell tracing reveals the diversity of axonal projection patterns of layer 2/3…
Figure 1:. Brain-wide single-cell tracing reveals the diversity of axonal projection patterns of layer 2/3 V1 neurons, with most cells projecting to more than one target area.(a) Three hypothetical modes of inter-areal information transfer from one area to its multiple targets. Neurons (arrows) could each project to a single area (top) or to several areas either randomly (middle) or in predefined projection patterns (bottom). (b) Maximum projection of a representative example GFP-filled neuron coronal view acquired by serial-section 2-photon microscopy. Auto-fluorescence from the red channel is used to show the brain’s ultrastructure (gray background). Scale bar = 600 μm. N = 71. (c-d) Higher magnification of the medial (c) and lateral (d) axonal arbor of the example cell. Scale bar = 300 μm. (e) Horizontal section through a sample brain (cyan) and Allen reference atlas (ARA; magenta) before (left) and after (right) rigid and non-rigid transformation of the brain to the atlas. (f) Coronal, sagittal and horizontal projections of the traced example cell overlaid in ARA space. Target cortical areas are coloured as indicated. Areas: A, anterior; AL: anterolateral; AM: anteromedial; LI: lateroitermediate; LM: lateral; P: posterior; PM: posteromedial; POR: postrhinal; RL: rostrolateral; TEA: temporal association; ECT: ectorhinal; PER: perirhinal. Scale bar = 1 mm. (g) Overlay of all traced single neurons (top left) and 11 example cells in Allen Reference Atlas (ARA) space; horizontal view (upper panel) and sagittal view (lower panel). Dashed outlines label non-visual target areas: AC: anterior cingulate cortex; STR, striatum; AMYG: amygdala. Note that these images are for illustration purposes only because a 2D projection cannot faithfully capture the true axonal arborisation pattern in 3D. Scale bar = 1 mm. (h) Pie chart illustrating the fraction of traced single neurons that project to at least one target area outside V1, where at least 1 mm of axonal innervation is required for an area to be considered a target. (i) Projection pattern of all GFP-filled V1 neurons targeted randomly (upper panel, n=31). The colour-code reflects the projection strengths of each neuron, determined as axon length per target area, normalized to the axon length in the target area receiving the densest innervation. Only brain areas that receive input form at least one neuron, as well as striatum, are shown. Areas: AUD: auditory cortex; ENT: entorhinal; HIPP: hippocampus; LA: lateral amygdala; RHIPP: retrohippocampal region; RS: retrosplenial. (j) The number of projection targets for every neuron that projects out of V1. (k) The proportion of cells targeting more than one area, when projection targets that receive projections weaker than the indicated projection strength are ignored. For each neuron, projection strengths are normalized to axon length in the target area receiving the densest innervation. (l) The fraction of neurons projecting to each of the 18 target areas of V1. (m) The fraction of neurons innervating a single target area (‘dedicated’ projection neurons) out of all neurons that innervate that area.
Figure 2:. MAPseq projection mapping reveals a…
Figure 2:. MAPseq projection mapping reveals a diversity of projection motifs.
( a ) Overview…
Figure 2:. MAPseq projection mapping reveals a diversity of projection motifs.(a) Overview of the MAPseq procedure. Six target areas were chosen for analysis: LI, LM, AL, PM, AM and RL. (b) Projection strength in the six target areas, as well as the olfactory bulb (OB) as a negative control, of 553 MAPseq-mapped neurons. Projection strengths per neuron are defined as the number of barcode copies per area, normalized to the efficiency of sequencing library generation and to the neuron’s maximum projection strength (n=4 mice). (c) Number of projection targets of V1 neurons when considering the six target areas only, based on the fluorescence-based axonal reconstructions (left) or the MAPseq data (right). (d) Distribution of cosine distances obtained by a bootstrapping procedure (1000 repeats) between MAPseq neurons (blue), fluorescence-based single neuron reconstructions and MAPseq neurons (orange), or random neurons (with projection strengths sampled from a uniform distribution) and MAPseq neurons (yellow). The distance distributions obtained from MAPseq neurons and fluorescence-based single-neuron reconstructions are statistically indistinguishable (Kolmogorov-Smirnov one-sided two sample test; p=0.94; α=0.05), whereas the distributions obtained from both MAPseq neurons or fluorescence-based reconstructed neurons are statistically different form the distribution obtained using random neurons (Kolmogorov-Smirnov two sample test; p<10−3; α=0.05). (e) Centroids and example cells for eight clusters obtained by k-means clustering of all MAPseq cells using a cosine distance metric. Target areas are coloured to indicate the projection strength of the plotted neuron. Projections strengths are normalize as in (b). (f) The probability of projecting to one area (Area A) given that the same neuron is projecting to another area (Area B) based on the MAPseq dataset.
Figure 3:. Over- and under-represented projection motifs…
Figure 3:. Over- and under-represented projection motifs of neurons in primary visual cortex.
( a…
Figure 3:. Over- and under-represented projection motifs of neurons in primary visual cortex.(a) The null hypothesis of independent projections to two target areas (left) and an example deviation (over-represented bifurcation) from the null hypothesis (right). (b) The observed and expected abundance of all possible bi-, tri- and quadfrucation motifs in the MAPseq dataset. Significantly over- or under-represented motifs, based on a binomial test with Bonferoni correction (see Methods), are indicated by black and grey arrowheads, respectively. N=553 neurons from 4 animals. (c) Statistical significance of over- and under-represented broadcasting motifs and associated effect sizes, based on a binomial test with Bonferoni correction (see Methods). N=553 neurons from 4 animals. (d-h) The projection strengths of the individual neurons (one per line) giving rise to the six under-represented (d,e) or over-represented (f-h) projection motifs. For each neuron, the projections strength in each target area is normalized to the neuron’s maximum projection strength. Lines of the same color represent neurons mapped in the same brain (n=4 mice).
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