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A mesoscale connectome of the mouse brain

Abstract

Comprehensive knowledge of the brain’s wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.

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Figure 1: Creation of the Connectivity Atlas.
Figure 2: Whole brain projection patterns from seven representative cortical regions.
Figure 3: Adult mouse brain connectivity matrix.
Figure 4: A computational model of inter-regional connection strengths.
Figure 5: Topography of cortico-striatal and cortico-thalamic projections.
Figure 6: A wiring diagram of connections between major cortical regions and thalamic nuclei.

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Acknowledgements

We wish to thank the Allen Mouse Brain Connectivity Atlas Advisory Council members, D. Anderson, E. M. Callaway, K. Svoboda, J. L. R. Rubenstein, C. B. Saper and M. P. Stryker for their insightful advice. We thank T. Ragan for providing invaluable support and advice in the development and customization of the TissueCyte 1000 systems. We are grateful for the technical support of the many staff members in the Allen Institute who are not part of the authorship of this paper. This work was funded by the Allen Institute for Brain Science. The authors wish to thank the Allen Institute founders, P. G. Allen and J. Allen, for their vision, encouragement and support.

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Contributions

H.Z., S.W.O., J.A.H. and L.N. contributed significantly to overall project design. S.W.O. and H.Z. did initial proof-of-principle studies. J.A.H., M.T.M., B.O., P.B., T.N.N., K.E.H. and S.A.S. conducted tracer injections and histological processing. B.W., C.R.S., E.N., A.H., P.W. and A.B. carried out imaging system establishment, maintenance, and imaging activities. L.N., C.L., L.K., W.W., Y.L., D.F. and H.P. conducted informatics data processing and online database development. A.M.H., K.M.J. and Q.W. conducted image quality control and data annotation. N.C., S.M. and C.K. performed computational modelling. H.Z., S.W.O., A.R.J., C.D., C.K., A.B., J.G.H., J.W.P. and M.J.H. performed managerial roles. H.Z., J.A.H., L.N., S.M., N.C., Q.W., S.W.O., C.R.G. and C.K. were main contributors to data analysis and manuscript writing, with input from other co-authors.

Corresponding author

Correspondence to Hongkui Zeng.

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The authors declare no competing financial interests.

Additional information

The Allen Mouse Brain Connectivity Atlas is accessible at (http://connectivity.brain-map.org). All AAV viral tracers are available at Penn Vector Core, and AAV viral vector DNA constructs have been deposited at the plasmid repository Addgene.

Extended data figures and tables

Extended Data Figure 1 AAV/BDA tracer comparison, using a primary motor cortex (MOp) injection as an example.

The cortical and subcortical projections from MOp injection are labelled similarly with the AAV tracer (green) and conventional tracer BDA (red). a, Injection sites of AAV and BDA are mostly overlapping (yellow), with a blue DAPI counterstain. b, Confocal image taken from the box in a shows BDA tracer uptake in individual neurons at the injection site. b’, The same box in a shows AAV infection of individual neurons. b”, Overlay of b and b’ shows the presence of both tracers in the same region and their colocalization in some neurons (yellow). c–f, Examples of cortical projections in the contralateral primary motor cortex, ipsilateral primary somatosensory cortex, agranular insular area (dorsal part), and perirhinal cortex labelled with red, green, or yellow. g–n, Examples of subcortical projections in the ipsilateral ventral posterolateral nucleus of the thalamus and posterior nucleus of the thalamus, superior colliculus, pontine grey, caudate putamen, zona incerta and subthalamic nucleus, midbrain reticular nucleus, parabrachial nucleus, and contralateral bed nucleus of the anterior commissure. Scale bars are 1,000 µm (a); 100 µm in (b, b’ and b’’); and 258 µm (cn). Approximately 18 brain regions were selected throughout the brain to represent broad anatomical areas and diverse cell types (3 cortical and 15 subcortical structures). AAV and BDA were co-injected into each selected brain region in wild-type mice using a sequential injection method developed to target virtually the same anatomical region. For most cases, the anatomical area(s) of tracer uptake are well matched. We found the long-range projections from all studied regions with both tracers. Their patterns were similar between the two tracers in mostly overlapped injection cases. There were more retrogradely labelled neurons with BDA than AAV, although a few retrograde neurons were observed in all studied regions with both tracers. BDA was clearly uptaken by passing fibres in some injections but AAV was not.

Extended Data Figure 2 Consistency among individual STP tomography image sets.

a, High-resolution images of 140 serial sections of a single brain are shown as an example (injection into the primary visual area). The injection site and major projection targets can be easily observed in this ‘contact-sheet’ view. b, Registration variability study. A set of ten 3D fiducial points of interest (POIs) were manually identified on the Nissl 3D reference space, the average template brain, and in 30 randomly selected individual image sets by three raters independently. The POIs were selected such that they span the brain and can be easily and repeatedly identified in 3D. Each POI (p) from each experiment was then projected into 3D reference space (p’) using the transform parameters computed by the Alignment module. Statistics were gathered on the target registration error between p’ and its ‘gold standard’ correspondence (computed as the mean of the labelling of 3 raters) in the Nissl (pNissl) and template (ptemplate) volumes. In summary, for all POIs in template space, the observed median variation in each direction are 28 µm for left-right, 35 µm for inferior-superior, and 49 µm for anterior-posterior. In Nissl space, these observed median variations are 42, 71 and 60 µm, respectively. The registration variations in different directions are shown here. For each POI, the green dot shows the position in template space (ptemplate) and the red dot shows the position in Nissl space (pNissl). The small yellow bar shows the median variation (among 30 image sets) away from ptemplate in each direction. POIs: AP: area postrema, midline; MM: medial mammillary nucleus, midline; cc1: corpus callosum, midline; cc2: corpus callosum, midline; acoL, acoR: anterior commisure, olfactory limb; arbL, arbR: arbor vitae; DGsgL, DGsgR: dentate gyrus, granule cell layer. c, Percent agreement between computationally assigned injection site voxels and manually assigned injection structures. Each histogram data point corresponds to a single injection, and 100% indicates that every injection site voxel was computationally assigned to a structure included on the manually annotated injection structures list. Voxels which were computationallyassigned to fibre tracts or ventricles are excluded from the computation. Neither fibre tracts nor ventricles were incorporated into the manual annotation process; their exclusion allows for a more commensurate comparison.

Extended Data Figure 3 Distribution of injection sites across the brain.

a, Locations of injection spheres within 12 major brain subdivisions are shown as a projection onto the mouse brain in sagittal views. n = 108 isocortex, 23 olfactory areas (OLF), 42 hippocampal formation (HPF), 8 cortical subplate (CTXsp), 38 striatum (STR), 9 pallidum (PAL), 57 thalamus (TH), 47 hypothalamus (HY), 50 midbrain (MB), 21 pons (P), 45 medulla (MY) and 21 cerebellum (CB). b, Frequency histogram for the injection site volumes of all 469 data sets is shown. c, The Allen Reference Ontology was collapsed into 295 non-overlapping, unique, anatomical structures for analyses, distributed across major brain subdivisions as shown (black bars). For most structures, a single injection was sufficient to infect the majority of neurons in that region. For larger structures (for example, primary motor cortex), multiple injections were made into several, spatially separate locations. The majority of these 295 regions have at least one injection targeted to that structure as either the primary or secondary injection site (white bars); only 18 structures are not covered at all (grey bars, for details see Supplementary Table 1). These missed structures (minimal to no infected cells in either the primary or secondary injection sites) were either very small (for example, nucleus y in the medulla), purposefully left out due to the presence of other injections under the same large parent structure (for example, four of the cerebellar cortex lobules), or technically challenging to reach via stereotaxic injection (for example, suprachiasmatic nucleus).

Extended Data Figure 4 Distribution of whole brain projections from different cortical source areas.

Pie charts show the percentage of total projection volume across all voxels outside of the injection site distributed in the 12 major brain subdivisions from both hemispheres. Each pie chart represents the average of 4 to 27 cortical injections grouped by the broad regions listed. A pie chart key of the volume distribution (number of voxels per structure/total number of voxels per brain) of these 12 subdivisions is at the bottom right for comparison. The largest projection signal from each cortical injection is found within isocortex (range of 45.4–69.8% of projection signals depending on source region, with an average of 59% for all cortical injections), although the isocortex accounts for only 30.2% of total brain volume. Differences in the subcortical distribution of relative signal strength between cortical sources were also observed. For example, within the striatum (light blue), the percentage of total signal is low from auditory, retrosplenial and visual areas (6.2%, 3.4% and 7.6%, respectively), but much greater from frontal, motor, cingulate and somatosensory areas (16.5%, 27.7%, 20.3% and 17.6%, respectively).

Extended Data Figure 5 Variability of brain-wide projection signal strength.

To examine animal-to-animal variability in projection patterns, 12 sources with two spatially overlapping injection experiments were identified from the full data set of tracer injections shown in Fig. 3. a, Rows show segmented projection volumes normalized to the injection volume (log10-transformed) in the 295 ipsilateral target regions for each of 2 individual overlapping tracer injections per source region indicated (above and below solid black line). The colour map is as shown in Fig. 3. b, Maximal density projections of whole brain signals from each of the two spatially overlapping injection experiments per source region visibly demonstrate consistency of brain-wide connections. Scatter plots of all ipsilateral and contralateral target structure values above a minimum threshold in both members of the pair (log10 = −3.5; non-blue values from a) show significant correlations between each pair of injections across a four orders of magnitude range of projection strengths. Values in the scatter plots are Pearson’s correlation coefficients (r). Note that in some cases (for example, PTLp) axon pathways appear to be labelled in only one of the pair. This could be due to random differences in the proportion of corticospinal projecting neurons labelled in a particular injection within the same source area. Signal in large annotated white matter tracts are computationally removed from the connectivity matrix, and thus not included in the scatter plots. c, Detected fluorescent signals from each of two injections into the same location of primary somatosensory cortex registered and overlaid with the average template brain (grey). Lower 2 rows, 2D raw images from each injection experiment at different anterior-posterior levels. The centres of these injection sites are in the far left panel and their major targets are in the right panels. See Supplementary Table 1 for the corresponding full name and acronym for each region.

Extended Data Figure 6 Cytoplasmic EGFP and synaptophysin-EGFP AAV tracer comparison, using primary motor cortex (MOp) injections as an example.

a–f, Two-photon images showing an example of the labelling obtained using the Phase I virus for whole-brain projection mapping, which consists of a human Synapsin I promoter driving expression of EGFP. g–l, To compare cytoplasmic labelling of projections and identification of terminal regions with a presynaptic reporter virus, we made a construct with the same hSynapsin promoter driving expression of a triple reporter cassette (AAV2/1.pSynI.nls-hrGFP-T2A-tagRFP-T2A-sypGFP.WPRE.bGH): a nuclear localization signal attached to humanized Renilla GFP (nls-hrGFP), a T2A sequence followed by cytoplasmic tagRFP, a second T2A sequence, and the synaptophysin-EGFP fusion (sypGFP). Owing to the two-photon imaging wavelength used (925 nm), the tagRFP (red) signal is weak to non-existent in these images. a, g, An image at the centre of the infected area after injection into the same region of MOp with the Phase I cytoplasmic viral tracer (a) and the nuclear and synaptic reporter viral tracer (g). Examples of viral labelling in the caudoputamen (b, h), somatosensory cortex (c, i), thalamus (d, j), pontine grey (e, k) and inferior olivary complex in the medulla (f, l) are shown for both tracers. The cytoplasmic viral tracer labels axons, revealing dense branching patterns in presumed terminal zones. The presynaptic reporter virus predominantly shows a punctate pattern of labelling consistent with presynaptic protein expression patterns, and indicative of terminal zones. Punctate or diffuse labelling was observed with the synaptic reporter virus in nearly all MOp target regions manually and computationally identified using the cytoplasmic reporter, including those with both large and small signals. Fluorescent signal originating in fibres outside of large white matter tracts are included in the signal quantification and matrix shown in Fig. 3, but signals from these large annotated fibre tracts are computationally removed. m–p, High-resolution images of terminal zones in the somatosensory cortex (m) and thalamus (o) identified using the cytoplasmic viral tracer show axon ramification and punctate structures consistent with bouton labelling, similar to the synaptic reporter in the corresponding regions (n, p). q–r’, To validate the presynaptic expression of the sypGFP fusion protein, sections including and adjacent to the thalamic region shown in j were collected after two-photon imaging and immunostained with antibodies against GFP (chicken polyclonal 1:500, Aves Labs, Inc. #GFP-1020) and synapsin I (rabbit polyclonal, 1:200, Millipore, #AB1543P) or GFP (rabbit polyclonal, Life Technologies, #A-11122) and SV2 (mouse monoclonal, DSHB, SV2-supernatant) presynaptic proteins. q, r, Confocal images at a single plane show punctate labelling indicative of presynaptic boutons for both sypGFP and synapsin or SV2. Many puncta were colocalized (yellow arrows show select examples in q’ and r’), although quantification was not reliable due to the very high density of presynaptic labelling by anti-Synapsin and anti-SV2.

Extended Data Figure 7 Distribution of log10-transformed normalized projection volumes from the entire matrix presented in Fig. 3.

The values (left-hand y axis, black bars) were number of target regions and derived from Supplementary Table 2. The entire range of the normalized projection volumes in this matrix was between log10 = −14 and log10 = 1.5, and it peaked between log10 = −3.5 and log10 = −3.0. A manual analysis of true positive and true negative signals from 20 randomly chosen injection experiments, representing the range of injection sizes, was used to estimate the false positive rate at different threshold levels, shown on the right-hand y axis (grey circles). True positive values predominantly fall within the range of log10 = −4 to 1.5. For example, at a threshold of log10 = −4, the false positive rate was 27%, dropping to 14.5% at log10 = −3.5. False positives were almost exclusively due to small segmentation artefacts in areas without actual fluorescently labelled axon fibres.

Extended Data Figure 8 Cortical domains identified by clustering analysis of projection patterns.

Eighty injections were used in the analysis for which the injection site is strictly within the isocortex and injection volume >0.07 mm3. a, Scatter plot of the voxel densities (excluding injection sites) of the whole brains from two nearby anterior cingulate injections (ACAd and ACAv) shows a strong correlation between the two (Spearman’s ρ = 0.82), whereas that of two distant injections (ACAd and SSp-m) shows little correlation (ρ = −0.03). b, Hierarchical clustering of the projection pattern based on Spearman’s rank correlation coefficient of voxel density over the entire brain. The pseudo-F statistics measures the coherence of clusters and is the ratio of mean sum of squares between groups to the mean sum of squares within group. Peaks in the pseudo-F statistics (for example, at n = 3, 8 and 21 clusters) are indicators of greater cluster separation. For n = 21 clusters, a systematic colour-code is given to each cluster to provide a visual guide to their cortical location (Fig. 5a), the numbers in parentheses indicate the number of injections in each group. c, Voxel densities from the 21 selected injections from Fig. 5a are overlaid as ‘dotograms’ at 8 coronal levels for the contralateral hemisphere.

Extended Data Figure 9 Topography of cortico-striatal and cortico-thalamic projections.

Average inter-group distance was used to quantify the degree of which inter-group spatial relationship within the cortex is preserved in target domains. a, Inter-group injection distances were obtained by computing the 3D Euclidean distances between injection site voxels of two experiments, one from each group, and then averaging over all injection pairs. For visualization, the distances are embedded into a 2D plane using multidimensional scaling to create a group-level injection flat-map. b, Inter-group projection distances were obtained by computing the 3D Euclidean distance between a pair of voxels in a target domain weighted by the product of voxel density of two injections, one from each group. The distances are then averaged over all voxel pairs in the target domain and injection pairs between groups. c, Inter-group projection distance matrices for each target domain visualized as false-coloured heatmaps. The black columns and rows in contralateral caudoputamen and thalamus are due to four missing structures. d, Inter-group projection distances are embedded into a 2D plane using multi-dimensional scaling to visualize the spatial relationship between groups. e, 3D tractography paths from decimated (every other indices in each dimension) voxels in both cortical hemispheres. Voxels belonging to the medial cortical groups have been omitted to reveal a reconstructed corpus callosum showing parallel crossings with a conserved spatial configuration. f, A top-down view of 3D tractography paths into the ipsilateral thalamus for all voxels excluding the RSP/VIS groups showing axonal projections passing through fibre tracts in the striatum, narrowing through the globus pallidus, before spreading throughout the thalamus.

Extended Data Figure 10 A matrix of major connections between functionally distinct cortical regions and thalamic nuclei, corresponding to Fig. 6.

Upper and lower panels show projections from cortex (source) to thalamus (target) or from thalamus (source) to cortex (target), respectively (ipsilateral projections only). The label ‘pc’ indicates that cortico-thalamic and thalamo-cortical projections in the gustatory/visceral pathway are between GU/VISC cortical areas and VPMpc/VPLpc nuclei. The number of pluses denotes relative connectivity strength and corresponds to the thickness of arrows in Fig. 6. All the connections described here were found in our data set. Connections labelled in red are previously known, whereas those labelled in black are not previously described in the rodent literature to our knowledge. There are also cases in which a connection was described in the literature but is excluded here because we could not find solid evidence in our data set to support it. All references that we have used to compare with our data are listed33,39,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127. Specifically, the cortico-thalamic system can be divided into six functional pathways: visual, somatosensory, auditory, motor, limbic and prefrontal. The visual pathway is composed of primary and associational visual cortical areas (VISp, VISam, VISal/l, and TEa) and thalamic nuclei LGd, LGv, and LP100,103,109, with LGd and VISp playing primary roles in processing incoming visual sensory information, visual associational areas involved in higher-order information processing and LP potentially modulating the function of all visual cortical areas (thus similar to the pulvinar in primates). LGv does not project back to cortex. Similarly, the somatosensory pathway is composed of primary and secondary somatosensory cortical areas (SSp and SSs) and thalamic nuclei VPM, VPL and PO48,74, with SSp and VPM/VPL playing primary roles in processing incoming somatosensory information, SSs in higher-order information processing and PO modulating the function of all somatosensory cortical areas. The gustatory and visceral pathway (involving GU/VISC cortical areas and VPMpc/VPLpc nuclei)55,108 and the auditory pathway (involving primary and secondary AUD areas and different MG nuclei)82,98 also have similar organizations, although our current data do not have sufficient resolution to resolve fine details. The motor pathway is composed of primary and secondary motor cortical areas (MOp and MOs) and the VAL nucleus92,94. The limbic pathway (which is closely integrated with the hippocampal formation system not discussed here) is composed of the retrosplenial (RSP) and anterior cingulate (ACA) cortical areas and thalamic nuclei AV, AD and LD107,114. The prefrontal pathway, which is considered to play major roles in cognitive and executive functions, is composed of the medial, orbital and lateral prefrontal cortical areas (including PL, ILA, ORB and AI) and many of the medial, midline, and intralaminar nuclei of the thalamus (including MD, VM, AM, PVT, CM, RH, RE and PF)111. The reticular nucleus (RT) is unique in that it is a relay nucleus for all these pathways, receiving collaterals from both cortico-thalamic and thalamo-cortical projections although itself only projecting within the thalamus. Between these pathways, we have observed cross-talks, mediated by specific associational cortical areas and thalamic nuclei that may be considered to play integrative functions. For example, the anterior cingulate cortex (ACA) appears to bridge the prefrontal and the limbic pathways, interconnecting extensively with both. The posterior parietal cortex (PTLp) and the LD nucleus may relay information between the visual and the limbic pathways. PTLp, while hardly receiving any inputs from the thalamus, projects strongly to both LP and LD. On the other hand, LD, while projecting quite exclusively to the limbic cortical areas, receives strong projections from all visual cortical areas. There is also extensive cross-talk between the motor pathway and the prefrontal pathway, with both MOp and MOs receiving strong inputs from VM and sending strong projections to MD, and additionally with MOs projecting widely into many medial, midline and intralaminar nuclei. Finally, the thalamic nuclei PO, VM, CM, RH and RE all send out widely distributed, albeit weak, projections to many cortical areas in different pathways, thus potentially capable of modulating activities in large cortical fields.

Supplementary information

Supplementary Information

This file contains a detailed description of the construction of the computational connectivity model and the network analyses based on this model, Supplementary Figures 1-5, Supplementary Tables 5-8 and Supplementary references. (PDF 901 kb)

Supplementary Table 1

The 295 non-overlapping, mid-level anatomical regions selected from the Allen Reference Atlas ontology that have tiling coverage of the entire brain space. These regions were used for both targeted stereotaxic injections and for connectivity data analysis. The first tab shows all 1204 non-fiber tract brain regions and fiber tracts delineated in the Allen Reference Atlas, in ontological order. The final selected 295 brain regions are marked as “1”. The second tab lists only the 295 selected regions, ordered by volumes (voxel counts). A histogram of the voxel counts for all 295 regions is also shown. The 18 regions that were not covered in the Phase I dataset are marked in the “Structure Hit with rAAV” column. The next two columns indicate whether a structure was infected as the primary site or secondary site of any injection experiment. The last column shows the structures that were included in the linear model matrix (figure 4a). (XLSX 87 kb)

Supplementary Table 2

Normalized projection strength values underlying the Connectivity Matrix in figure 3. Values from all 469 injections (in rows) and the 295 anatomical target regions on both ipsilateral and contralateral hemispheres (in columns) are shown here. Each value is the normalized projection volume (sum of segmented pixels across all voxels in one target region / sum of segmented pixels across voxels in injection volume). Also shown are the manually annotated primary and secondary anatomical structures contained within each injection site, as well as the volume of each injection site. (XLSX 2935 kb)

Supplementary Table 3

Quantitative projection strength values underlying the linear model based Connectivity Matrix in figure 4a. The 213 anatomical regions as both source regions (in rows) and target regions on both ipsilateral and contralateral hemispheres (in columns) are shown here. See Supplementary Table 1 for the corresponding full name and acronym of each region. (XLSX 1533 kb)

Supplementary Table 4

Cartesian distances between the centers of mass of all the interconnected source and target region pairs for the 213 anatomical regions used in the linear model based Connectivity Matrix in figure 4a. Names of source regions (in rows) are shown in Column A, and names for target regions on both ipsilateral and contralateral hemispheres (in columns) are shown in Row 1. See Supplementary Table 1 for the corresponding full name and acronym of each region. (XLSX 4395 kb)

Exemplar TissueCyte STP tomography image sets showing axonal projections throughout the brain

The image sets are from 5 anatomically distinct injection sites representing several major brain subdivisions: primary motor area (MOp), centromedial nucleus of the thalamus (CM), medial preoptic area of the hypothalamus (MPO), pontine central gray (PCG), and intermediate reticular nucleus of the medulla (IRN). (MOV 24167 kb)

Average template brain and its alignment with Allen Reference Atlas

Coronal image series of the average template brain at 10 µm X-Y and 25 µm Z sampling rate is shown first. Coronal image series of the alignment of the Allen Reference Atlas structures (at 27% opacity) onto the average template brain at the same sampling rate is shown next. (AVI 24297 kb)

Locations of all injection sites

Physical locations of all 469 injection sites are superimposed onto the coronal image series of the average template brain, to show the overall anatomical coverage of the Phase I experiments. Each injection site is shown as a group of 100 x 100 x 100 µm3 voxels contained within its injection site polygon, and in the ontology color of the primary anatomical structure it occupies. In some cases, viral infections along the injection tracts can be seen. (AVI 2423 kb)

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Oh, S., Harris, J., Ng, L. et al. A mesoscale connectome of the mouse brain. Nature 508, 207–214 (2014). https://doi.org/10.1038/nature13186

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