Cited by 2,612Functional Brain Connectivity StudiesAdvanced Neuroimaging Techniques and ApplicationsNeuroscience and Neuropharmacology Research

The DOI matches its Crossref record; 130 of the 130 references it cites were found in a registry or an index; none of the 42 sources its claims cite is checked for support yet.

A mesoscale connectome of the mouse brain

Open access

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.

Overview

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TL;DR

The paper presents the Allen Mouse Brain Connectivity Atlas, a brain-wide cellular-level mesoscale connectome of the adult mouse generated with EGFP-expressing AAV tracing and serial two-photon tomography. It reports a quantitative inter-areal connectivity matrix and analyses of network properties, including lognormal connection strengths, bilateral projections, and cortico-thalamic parallel pathways.

Key findings

  • Phase I includes 469 image sets covering 295 non-overlapping anatomical regions, with only 18 intended structures completely missed. Connectivity strengths span a greater than 10^5-fold range; ipsilateral projections are generally stronger than contralateral ones, with total normalized projection volumes at a 4.3:1 ratio. Strong connections are sparse: 63% ipsilateral and 51% contralateral targets exceed the minimal true-positive level of 10^-4, but only 21% ipsilateral and 9% contralateral targets exceed 10^-2.
  • A computational model inferred mutual connections among 213 regions. Sparsity upper bounds were 36% for the entire brain and 52% for cortico-cortical connections; lower bounds were 13% for the entire brain and 32% for cortico-cortical connections. Connection strengths negatively correlated with distance and were best fit by a lognormal distribution by AIC, although log-transformed strengths failed Shapiro-Wilk normality (ipsilateral P = 0.039; contralateral P = 0.023), with a two-component Gaussian mixture providing the best fit.
  • Network analyses found a mean clustering coefficient of 0.42 versus 0.12 expected by a random network with identical sparseness; random graphs matched for node degree distribution dropped to 0.16. Neither a small-world nor a scale-free model simultaneously fit both clustering and degree distributions. Across 12 duplicate injection sets, average correlation was 0.90.
  • In the cortico-thalamic analysis, 80 isocortical injections clustered into 21 groups, and the wiring diagram divided the cortico-thalamic system into six functional pathways: visual, somatosensory, auditory, motor, limbic, and prefrontal, with cross-talk mediated by associational cortical areas and integrative thalamic nuclei.

Methods

  • C57BL/6J male mice at P56 received unilateral stereotaxic iontophoretic injections of AAV serotype 1 expressing EGFP under the human synapsin I promoter. Phase I regional projection mapping used wild-type mice; Phase II Cre-dependent mapping is described but not reported here.
  • Brains were imaged with serial two-photon tomography at 0.35 μm x–y resolution, 100-μm z-sampling, 140 coronal sections, about 18.5 h per brain, and ~750 GB per brain. Image stacks were registered to a 3D Allen Reference Atlas via an iterative average template from 1,231 brains, segmented, and voxelized into 100 × 100 × 100 μm^3 voxels.
  • Projection strength was defined as total segmented target volume normalized by injection site volume. The raw connectivity matrix used 469 Phase I experiments across 295 structures; a computational model with regional homogeneity and projection additivity inferred inter-region connections among 213 regions, followed by graph-theoretic, clustering, and virtual tractography analyses.

Limitations

  • Stated limitations: Phase I lacks extensive redundancy; the AAV tracer may not be completely unbiased in labelling all neuronal types; the matrix contains false positives from tissue/imaging artefacts and injection tract contamination; cytoplasmic EGFP does not distinguish passing fibres from terminal zones; projection volume may underestimate dense projections; alignment between the average template brain and the Nissl-based Reference Atlas is imperfect; postsynaptic neuron identities are unknown; microscale synaptic details and gap junctions are missing; and the map is static structural connectivity, not functional connectivity.
  • Evident from the text: only the completed Phase I study is reported, so cell-type-specific Cre-driver connectivity mapping is not included in this dataset; the 100-μm z-sampling prevents real single-axon tractography, requiring virtual tractography instead.

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Integrity signals

Registry

Crossref · 10.1038/nature13186

Cites

130 of 130 references verified0 citation errors · 0 not checkable

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96 Own result · 27 Not checked yet · 25 Own interpretation · 18 Own method · 15 No basis given

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  • It may state two separate claims as one. 36
  • Probably not a substantial claim. 33
  • The quote only partly states this claim. 1
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  • The authors state this as an assumption.

    Regional homogeneity: projections between source X and target Y regions are homogeneously distributed, so that infection of a subarea of the region is representative of the entire region.

    “The first, regional homogeneity, assumes that projections between source X and target Y regions are homogeneously distributed, so that infection of a subarea of the region is representative of the entire region.”
  • The authors state this as an assumption.

    Projection additivity: the projection density of multiple source regions sum linearly to produce projection density in a target region.

    “The second assumption, projection additivity, assumes that the projection density of multiple source regions sum linearly to produce projection density in a target region.”
  • The paper relies on this without support.

    Much of the histological tract tracing data are qualitative, incomplete, variable, scattered and difficult to retrieve.

    “However, much of these data are qualitative, incomplete, variable, scattered and difficult to retrieve.”
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Citations

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Research works printed but not linked: 2

  1. Sporns, O. Networks of the Brain 412 (The MIT Press, 2011)
  2. Jones, E. G. The Thalamus (Cambridge Univ. Press, 2007)

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Metadata

Authors
Oh, S. W., Harris, J. A., Ng, L., Winslow, B., Cain, N., Mihalas, S., Wang, Q., Lau, C., Kuan, L., Henry, A. M., Mortrud, M. T., Ouellette, B., Nguyen, T. N., Sorensen, S. A., Slaughterbeck, C. R., Wakeman, W., Li, Y., Feng, D., Ho, A., Nicholas, E., Hirokawa, K. E., Bohn, P., Joines, K. M., Peng, H., Hawrylycz, M. J., Phillips, J. W., Hohmann, J. G., Wohnoutka, P., Gerfen, C. R., Koch, C., Bernard, A., Dang, C., Jones, A. R., & Zeng, H.
Affiliations
  • Allen Institute for Brain Science (US)
  • National Institutes of Health (US)
Corresponding author
Zeng, Hongkui
Venue
Nature
Published
2014-04
Volume
508
Issue
7495
Pages
207-214
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
OpenAlex
W1975389666
Cited by
2,612
References
130
Citations by year
2026: 176 · 2025: 233 · 2024: 274 · 2023: 254 · 2022: 238 · 2021: 247 · 2020: 250 · 2019: 229 · 2018: 187 · 2017: 153 · 2016: 176 · 2015: 132 · 2014: 61
FWCI
97.03
Type
journal article
Language
en
Open access
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • National Institutes of Health
  • National Institute of Mental Health (ZIAMH002497)
Topics
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Functional Brain Connectivity Studies
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Advanced Neuroimaging Techniques and Applications
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
whole-brain connectivity, axonal projections, Allen Mouse Brain Connectivity Atlas, brain network topology, human brain development, transcriptional atlas, BRAIN Initiative, neural circuits, neuroanatomy
MeSH terms
Animals, Atlases as Topic, Axons, Brain, Cerebral Cortex, Corpus Striatum, Male, Mice, Inbred C57BL, Models, Neurological, Thalamus, Mice, Neuroanatomical Tract-Tracing Techniques, Connectome

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