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A whole-brain monosynaptic input connectome to neuron classes in mouse visual cortex

Nature Neuroscience volume 26, pages 350–364 (2023)Cite this article

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Abstract

Identification of structural connections between neurons is a prerequisite to understanding brain function. Here we developed a pipeline to systematically map brain-wide monosynaptic input connections to genetically defined neuronal populations using an optimized rabies tracing system. We used mouse visual cortex as the exemplar system and revealed quantitative target-specific, layer-specific and cell-class-specific differences in its presynaptic connectomes. The retrograde connectivity indicates the presence of ventral and dorsal visual streams and further reveals topographically organized and continuously varying subnetworks mediated by different higher visual areas. The visual cortex hierarchy can be derived from intracortical feedforward and feedback pathways mediated by upper-layer and lower-layer input neurons. We also identify a new role for layer 6 neurons in mediating reciprocal interhemispheric connections. This study expands our knowledge of the visual system connectomes and demonstrates that the pipeline can be scaled up to dissect connectivity of different cell populations across the mouse brain.

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Fig. 1: Pipeline identifying monosynaptic inputs to specific neuronal populations in the visual cortex.
Fig. 2: Identification of monosynaptic inputs to Cre-labeled neuronal classes in different visual areas.
Fig. 3: Comparison of brain-wide inputs to neurons in the primary visual cortex and higher visual areas.
Fig. 4: Comparison of brain-wide input patterns to excitatory neuron subclasses in different layers of the primary visual cortex.
Fig. 5: Comparison of brain-wide input patterns to different interneuron subclasses in the primary visual cortex.
Fig. 6: Comparison of local inputs to excitatory neurons and inhibitory interneurons in different depths of the primary visual cortex.
Fig. 7: Relative hierarchical positions of the primary visual cortex and higher visual areas.

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Data availability

Plasmids for the generation of recombinant viruses will be deposited in Addgene. All anterograde tracing data (including high-resolution STPT images, and informatically processed axonal projections across brain structures) are available through the Allen Mouse Brain Connectivity Atlas portal (http://connectivity.brain-map.org/). A link for each anterograde tracing experiment is provided in Supplementary Tables 4 and 5. Original images for trans-synaptic rabies viral tracing will be or already are available through the Brain Image Library (https://www.brainimagelibrary.org/). A link for each trans-synaptic rabies tracing experiment that has been deposited in the Brain Image Library is provided in Supplementary Table 2. Normalized presynaptic input volumes as fractions of total inputs across the brain for all rabies virus tracing experiments and preliminary retrograde labeling data from initial informatic quantification are listed in Supplementary Table 3. Source data are provided with this paper.

Code availability

The Allen Mouse Brain CCFv3 ontology (http://atlas.brain-map.org/) was used to define brain regions. R software (version 3.6.0) was used for statistical tests and generation of graphs. Hierarchical clustering was conducted using the pvclust package (pvclust_2.2-0) in R (version 3.6.0). Numbers of starter cells in confocal images were quantified using the Cell Counter plugin in ImageJ (ImageJ 2.0.0-rc-43/1.53j).

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Acknowledgements

We are grateful to the Transgenic Colony Management, Neurosurgery & Behavior, Lab Animal Services, Molecular Genetics, Imaging, Histology, Technology and Project Management teams at the Allen Institute for technical and management support. We thank T. R. Reardon, A. J. Murray and I. Wickersham for providing cell lines and plasmids for the establishment of rabies virus production at the Allen Institute. This work was supported by the Allen Institute for Brain Science and by the National Institute of Mental Health of the NIH under award number U19MH114830 to H.Z. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH and its subsidiary institutes. We thank the Allen Institute founder, Paul G. Allen, for his vision, encouragement and support.

Author information

Author notes

  1. Karla E. Hirokawa, Kat North, Philip R. Nicovich & Julie A. Harris

    Present address: Cajal Neuroscience, Seattle, WA, USA

  2. Ali Cetin

    Present address: CNC Program, Stanford University, Palo Alto, CA, USA

Authors and Affiliations

  1. Allen Institute for Brain Science, Seattle, WA, USA

    Shenqin Yao, Quanxin Wang, Karla E. Hirokawa, Benjamin Ouellette, Ruweida Ahmed, Jasmin Bomben, Krissy Brouner, Linzy Casal, Shiella Caldejon, Andy Cho, Nadezhda I. Dotson, Tanya L. Daigle, Tom Egdorf, Rachel Enstrom, Amanda Gary, Emily Gelfand, Melissa Gorham, Fiona Griffin, Hong Gu, Nicole Hancock, Robert Howard, Leonard Kuan, Sophie Lambert, Eric Kenji Lee, Jennifer Luviano, Kyla Mace, Michelle Maxwell, Marty T. Mortrud, Maitham Naeemi, Chelsea Nayan, Nhan-Kiet Ngo, Thuyanh Nguyen, Kat North, Shea Ransford, Augustin Ruiz, Sam Seid, Jackie Swapp, Michael J. Taormina, Wayne Wakeman, Thomas Zhou, Philip R. Nicovich, Ali Williford, Lydia Potekhina, Medea McGraw, Lydia Ng, Peter A. Groblewski, Bosiljka Tasic, Stefan Mihalas, Julie A. Harris, Ali Cetin & Hongkui Zeng

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Contributions

H.Z., J.A.H., A. Cetin, S.M. and S.Y. contributed to overall project design. A. Cetin designed and orchestrated the viral tracing technology as well as viral production capability and established these with help from S.Y., T.Z. and M.T.M. S.Y., T.Z. and M.T.M. performed virus production. A. Cetin, S.Y., T.L.D. and B.O. conducted initial proof-of-principle studies. A.W. and P.A.G. supervised surgical procedures with contributions from B.O., C.N., K.M., S.L., A. Cho, L.C., K.N., N.H., E.G., J.L., R.A., R.H. and J.S. P.A.G. supervised ISI procedures with contributions from S.C., S.S., E.K.L., F.G. and T.N. M. McGraw supervised histological processing with contributions from T.E., J.B., M. Maxwell, H.G., A.G., K.B. and A.R. P.R.N. coordinated imaging procedures with contributions from R.E., M.G., S.R., L.P., N.I.D., N.-K.N. and M.J.T. L.N., L.K. and W.W. performed informatics data processing. K.E.H. and S.Y. coordinated workflow and carried out QC. M.N. contributed to the development of data visualization tools. Q.W., S.M., J.A.H., A. Cetin, S.Y. and H.Z. formulated data generation and analysis strategies. S.Y., A. Cetin, J.A.H., B.T. and H.Z. supervised the project. S.Y. analyzed data and prepared figures. S.Y. and H.Z. wrote the manuscript with inputs from all authors.

Corresponding authors

Correspondence to Shenqin Yao or Hongkui Zeng.

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Competing interests

J.A.H., K.E.H., P.R.N. and K.N. are currently employed by Cajal Neuroscience. The remaining authors declare no competing interests.

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Nature Neuroscience thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Comparison of different AAV helper viruses and rabies viruses for monosynaptic retrograde tracing.

(a–c) Comparison of spurious rabies infection from AAV helper viruses expressing wild-type TVA and mutant TVA66T. Tricistronic AAV helper viruses were constructed to conditionally express either the wild-type TVA or TVA66T, together with dTomato and RG (a). Cre-negative wild-type mice were sequentially injected with AAV helper viruses and EnvA-pseudotyped recombinant rabies viruses expressing EGFP. Each AAV helper virus/rabies virus pair was tested in two wild-type mice. Top-down view of whole brains (b) and observation of the injection sites under the confocal microscope (c) revealed fewer spurious rabies infection from AAV helper virus expressing TVA66T. (d–f) Comparison of monosynaptic retrograde tracing in VISp using SAD B19 strain of recombinant RV expressing EGFP (d, similar results observed in 6 independent experiments) or H2B-EGFP (e, similar results observed in 7 independent experiments) or CVS N2c strain of recombinant RV expressing H2B-EGFP (f, similar results observed in 303 experiments). Note that in H2B-EGFP expressing SAD B19 experiment there is still green fluorescence in the processes of infected and transmitted neurons (e), due to the very high-level transgene expression in this rabies strain, whereas in CVS N2c experiment H2B-EGFP is strictly contained within nuclei of infected and transmitted neurons (f). Scale bars, 100 µm in b, 1 mm in panels showing full brain sections in d–f, and 500 µm in panels showing selected brain areas in d–f.

Extended Data Fig. 2 Validation of the AAV helper virus and recombinant rabies used in the retrograde connectomic pipeline in wild-type mice and non-neuronal Cre lines.

(a) Sequential two-photon images of a Cre-negative wild-type mouse brain injected with the AAV helper virus and EnvA-pseudotyped CVS N2cΔG rabies virus expressing H2B-EGFP. Similar results observed in 3 independent experiments. (b) Absence of RV-labeled neurons except a few H2B-EGFP-expressing cells in the injection site. Virus injection was targeted to VISp and validation was conducted in two wild-type mice. Left and middle panels: corresponding 2D atlas plate of Allen CCFv3 and the section image from the outlining box in a showing the injection site. Right panel: Image magnified from the box in the middle panel. Applying the monosynaptic rabies tracing to wild-type mice led to only a few H2B-EGFP-labeled cells in the injection site, but no starter cells in the injection site and no H2B-GFP-labeled cells outside the injection site. This shows that our system does not have the issue of spurious local rabies virus uptake due to low-level expression from the AAV helper in the absence of Cre, or local infection by small quantities of non-pseudotyped, RG-coated RVdG virus particles that may be present in the EnvA-pseudotyped rabies virus preparation. We then confirmed that the trans-synaptic transfer of the recombinant rabies relies on the expression of rabies G from the AAV helper. A G-minus version of the AAV helper virus, which conditionally expresses TVA66T and dTomato after Cre-mediated recombination, was injected into Cre+ mice, followed by the injection of rabies virus three weeks later. We observed H2B-EGFP-labeled cells only at the injection site and nowhere else in the brain. This finding confirms that the presynaptic labeling is specific for the Cre+ starter cells expressing the tricistronic cassette and infected with the RV-H2B-GFP rabies virus. (c) Sequential two-photon images of rabies labeling in an astrocyte-specific Cre mouse brain injected with hSyn promoter-driven AAV helper virus and recombinant rabies virus into VISp. Similar results observed in 4 independent experiments. (d, e) Left and middle two panels: corresponding 2D atlas plates of Allen CCFv3 and the section images from the boxes in c. Right panels: Representative images magnified from the boxes in the middle panels reveal sparse labeling around the injection site (d) and in LGd (e). We tested the monosynaptic rabies tracing system in three non-neuronal Cre lines, Olig2-Cre, Tek-Cre, and Aldh1l1-CreERT2, which express Cre in oligodendrocytes, vascular endothelium, and astrocytes, respectively. Among all experiments using the non-neuronal Cre lines, with either the hSyn-driven AAV helper virus used in the pipeline or a similarly constructed CMV-driven helper virus, sporadic long-distance H2B-EGFP-labeled cells were found only in 50% of the injected Aldh1l1-CreERT2 mice. Our results show that the occasionally infected non-neuronal cells do not support the spread of rabies virus to neurons in local or distant areas.

Extended Data Fig. 3 Overview of experiments included in the final dataset, automatic input signal detection and characterization of inputs to cell classes defined by Cre lines in the visual cortex.

(a) Flow chart showing the injection methods, and numbers of experiments passing each QC step. QC1 excluded experiments with no rabies virus labeling or with tissue damage. QC2 excluded experiments with segmentation errors that prevent quantitative analysis or with targeting sites falling outside the visual cortex. One experiment targeting TEa was included in the final data set. For experiments guided by ISI, target validation was performed by overlaying injection polygons with sign maps derived from ISI, and overlaying injection centroids in the CCFv3. Inconsistency between ISI-assigned targets and CCFv3-derived targets were observed in 15 out of the total 136 ISI-guided experiments in the final data set. We assigned injection targets based on the overlaying of injection polygons with sign maps. (b–g) Relationship between per structure input signal volume measured by the informatics data pipeline and manual cell counts. Linear correlation between input signal volume and manually counted input cells was shown in various brain areas. In the six example structures from cortex, thalamus and cortical subplate, strong positive linear correlations were found between automatic measurement and manual counts (R2 in the 0.62–0.98 range). (h) Slopes from linear correlations between informatically measured input signals and manual cell counts in various brain areas. The numbers of independent experiments are as follows: CLA: n = 11; ORB: n = 10, ACA: n = 11, RSP: n = 19; LGd: n = 19; LP: n = 19. (i) Number of starter cells for experiments categorized in Cre lines. Box plots show median and interquartile range (IQR). Whiskers show the largest or smallest value no further than 1.5 × IQR from the hinge. The numbers of independent experiments are as follows: Emx1: n = 7, Sepw1: n = 19; Cux2: n = 29; Nr5a1: n = 25; Rbp4: n = 25; Tlx3: n = 26; A93-Tg1: n = 19; Ntsr1: n = 21; Ctfg: n = 19; Syt6: n = 1; Gad2: n = 6; Ndnf: n = 6; Vip: n = 24; Pvalb: n = 33; Sst: n = 40; Chat: n = 1; Htr3a: n = 1; Calb1: n = 1. (j) Distribution of numbers of starter cells across all experiments. (k) Relationship between numbers of starter cells and total inputs from the whole brain. (l) Fractions of total inputs from isocortex, thalamus and HPF to the mouse visual cortex. Dots represent the median values of input signals. (m-n) Fractions of total inputs from non-VIS isocortical areas (m) and thalamic areas (n). Brain areas are ordered according to their levels of input signals. Data are shown as mean ± s.e.m. A total of 303 independent experiments were included.

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Extended Data Fig. 4 Representative images of presynaptic inputs to the visual areas from anatomical structures outside of cortex and thalamus.

Coronal STPT images and their corresponding 2D atlas plates in Allen CCFv3 show labeled presynaptic neurons in OLF (a-a’), HPF (b-b’), CTXsp (c-c’), STR (d-c’), PAL (e-e’), HY (f-f’), MB (g-g’), pons (h-h’), and MY (i-i’). Claustrum (CLA) in CTXsp, diagonal band nucleus (NDB) in PAL, and lateral hypothalamic area (LHA) in HY each represent ~0.1% of whole brain inputs; globus pallidus, external segment (GPe) in PAL, basolateral amygdalar nucleus (BLA) in CTXsp, and zona incerta (ZI) in HY each account for ~0.01% of whole brain inputs; dorsal peduncular area (DP) in OLF, locus ceruleus (LC) in pons, and superior colliculus (SC) in MB each account for ~0.001% of whole brain inputs; areas in MY each account for ~0.0001% of whole brain inputs. Rare inputs in several structures of MY are also found in less than 10% of all experiments (Supplementary Table 3), which could be missed using other connectivity mapping techniques. Clustered inputs are found in NDB (fraction of whole brain inputs in NDB > 0 in 93% of all experiments) and substantia innominata (SI) (fraction of whole brain inputs in SI > 0 in 75% of all experiments) for putative cholinergic neurons, in LC (fraction of whole brain inputs in LC > 0 in 66% of all experiments) for putative noradrenergic neurons, and in DR (~0.01% of whole brain inputs, found in 63% of all experiments) for putative serotonergic neurons. The numbers of independent experiments with similar results are 121 in a, 61 in a’, 52 in a’, 254 in b, 165 in b’, 137 in b’, 278 in c, 187 in c’, 149 in c’, 236 in d, 187 in d’, 68 in d’, 226 in e, 110 in e’, 152 in e’, 275 in f, 224 in f’, 198 in f’, 214 in g, 187 in g’, 33 in g’, 231 in h, 193 in h’, 193 in h’, 53 in i, 52 in i’, and 30 in i’. Enlarged views of boxed areas are shown in the right-hand panels for each major brain structure. Arrows highlight the location of single labeled cells.

Extended Data Fig. 5 Connectivity matrices comparing the inputs to Rbp4 (a) and Sst (b) neurons in the visual areas from within the visual cortex, non-visual isocortical modules, and thalamus.

Gray indicates experiments not available. In the matrix, each row represents experiments with the same target area, and each cell shows the fraction of the total inputs in a given input structure measured from a single experiment or the average when n > 1.

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Extended Data Fig. 6 Brain-wide input patterns to excitatory neuron subclasses in different layers of the primary visual cortex.

(a, b) Overview of the layer selectivity and number of experiments of each transgenic Cre line (a) and the numbers of starter cells grouped by Cre lines (b) for the 48 experiments in VISp. (c) Laminar distribution of starter cells for each Cre line. For each transgenic line, different experiments are indicated by different colors. Box plots show median and interquartile range (IQR). Whiskers show the largest or smallest value no further than 1.5 × IQR from the hinge. The numbers of independent experiments are as follows: Sepw1: n = 3; Cux2: n = 13; Nr5a1: n = 10; Rbp4: n = 11; Tlx3: n = 12; A93-Tg1: n = 6; Ntsr1: n = 4; Ctfg: n = 3; Emx1: n = 2. (d) Representative 3D visualization of brain-wide inputs to neurons in different layers of VISp. (e) Brain-wide input patterns of major brain structures to different layer-specific excitatory neuron subclasses labeled by Cre lines.

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Extended Data Fig. 7 Comparison of subcortical inputs to excitatory neurons in different layers of VISp and brain-wide input patterns to excitatory neuron subclasses in VISl.

(a) Connectivity matrix showing presynaptic inputs from the ipsilateral and contralateral HPF, CTXsp, STR, and PAL to excitatory neurons in different layers of VISp. Each row of the matrix represents the mean per structure fraction of total input signals for experiments in each Cre line. Rows are organized based on layer-specific distribution of the starter cells. Brain regions are ordered by ontology order in the Allen CCFv3. (b) Connectivity matrix showing normalized projections from VISp to the brain regions shown in (a). Anterograde tracing experiments (Supplementary Table 4) from the Cre mouse lines used in (a) and C57BL/6J were included, and rows represent the mean per structure fraction of total projection signals for experiments in each mouse line. (c) Comparison of ENTl, ENTm, and CLA inputs to excitatory neurons in different layers of VISp. Box plots show median and interquartile range (IQR). Whiskers show the largest or smallest value no further than 1.5 × IQR from the hinge (same below). The numbers of independent experiments are as follows: L2/3: n = 16; L4: n = 10; L5: n = 29; L6: n = 7. (d) Connectivity matrix showing normalized inputs from the ipsilateral and contralateral isocortex, and ipsilateral thalamus to excitatory neurons in different layers of VISl. Each row of the matrix represents the mean per structure fraction of total input signals for experiments in each Cre line. Rows are organized based on layer-specific distribution of the starter cells. The cortical areas are ordered first by module membership (color coded) then by ontology order in the Allen CCFv3. The ten thalamic nuclei are ordered based on the strength of inputs, and are color coded by the thalamocortical projection classes (blue: core, green: intralaminar, brown: matrix-focal, and red: matrix-multiareal). (e) Connectivity matrix showing normalized axon projections from VISl to the ipsilateral and contralateral isocortex, and ipsilateral thalamus shown in (d). Anterograde tracing experiments (Supplementary Table 5) from the Cre mouse lines used in (d) and C57BL/6J were included, and rows represent the mean per structure fraction of total projection signals for experiments in each mouse line. (f) Comparison of inputs from ipsilateral and contralateral cortical areas and thalamic nuclei to excitatory neurons in different layers of VISl. The inset shows fraction of inputs from VISp to excitatory neurons in different layers of VISl. Data are shown as mean ± s.e.m. The numbers of independent experiments are as follows: L2/3: n = 5; L4: n = 4; L5: n = 11; L6: n = 8. (g, h) Representative STPT images showing laminar termination patterns of axon projections in VISl from higher-order association cortical areas and thalamic nuclei (g) and laminar distribution patterns of presynaptic input cells in the cortical areas that project to VISl (h). Ipsi, ipsilateral hemisphere. Contra, contralateral hemisphere. (i, j) Quantification of laminar distribution of inputs from ACAd (i, numbers of independent experiments: L2/3: n = 3; L5: n = 10; L6: n = 7) and ORBvl (j, numbers of independent experiments: L2/3: n = 2; L5: n = 11; L6: n = 7) to excitatory neurons in different layers of VISl. The fraction of layer inputs is calculated as the fraction of the total input signals in a given source area across layers. (k, l) Comparison of L5 and L6 preference for medial (k) or lateral (l) source cortical areas in the ipsilateral and contralateral hemispheres sending presynaptic inputs to VISl. The preference score for a given cortical area is calculated as (L5 input - L6 input)/(L5 input + L6 input). Each source cortical area was colored according to its preference score. Data are shown as mean ± s.e.m. The numbers of independent experiments included are as follows: ACAd (Ipsi): n = 35; ACAv (Ipsi): n = 26; RSPd (Ipsi): n = 40; RSPv (Ipsi): n = 43, RSPagl (Ipsi): n = 36, AUDp (Ipsi): n = 39, AUDv (Ipsi): n = 30; TEa (Ipsi): n = 42, ECT (Ipsi): n = 30, ACAd (Contra): n = 8; ACAv (Contra): n = 4; RSPd (Contra): n = 14; RSPv (Contra): n = 10, RSPagl (Contra): n = 13, AUDp (Contra): n = 17, AUDv (Contra): n = 13; TEa (Contra): n = 26, ECT (Contra): n = 14. Scale bars, 500 µm. We also find generally consistent input patterns to excitatory neurons in different layers of other HVAs as of VISp and VISl, though due to smaller number of experiments in each layer of each region (Fig. 2a) we do not provide quantitative analysis here.

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Extended Data Fig. 8 Comparison of contralateral cortical inputs to excitatory neuron subclasses in different layers of visual areas.

(a) Comparison between ipsilateral (left) and contralateral (right) brain-wide inputs to excitatory neuron subclasses with starter cells restricted to either L2/3, L4, L5 or L6 of visual areas. A total of 89 experiments with starter cells restricted in a single layer were identified, and the target areas included both VISp and HVAs. (b) Comparison between ipsilateral (top) and contralateral (bottom) isocortical inputs for each cortical module to excitatory neuron subclasses with starter cells restricted to either L2/3 (n = 20 numbers of independent experiments), L4 (n = 18 numbers of independent experiments), L5 (n = 25 numbers of independent experiments) or L6 (n = 27 numbers of independent experiments) of visual areas. (c) Representative top-down view of inputs to Ntsr1 and Ctgf Cre line-labeled L6 and L6b cell types in visual areas. (d) Laminar distribution of presynaptic inputs from the homotypic contralateral areas to Ntsr1 and Ctgf Cre line-labeled neurons in visual areas shown in (c). The fraction of layer inputs is calculated as the fraction of the total input signals in a given source area across layers. L1 is excluded from the analysis due to overall lack of signal in this layer. Box plots show median and interquartile range (IQR). Whiskers show the largest or smallest value no further than 1.5 × IQR from the hinge. The numbers of independent experiments are as follows: Ntsr1 in VISp: n = 4; Ctgf in VISp: n = 3; Ntsr1 in VISl: n = 4; Ctgf in VISl: n = 4; Ntsr1 in VISam: n = 2; Ctgf in VISam: n = 2; Ntsr1 in VISrl: n = 2; Ctgf in VISrl: n = 2; Ntsr1 in VISal: n = 2; Ctgf in VISal: n = 3.

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Extended Data Fig. 9 Analysis of brain-wide input patterns to interneuron subclasses in VISp, and comparison of local inputs to excitatory neurons in different layers of VISl.

(a) Summary of the numbers of starter cells for each interneuron subclass. Each dot represents one individual experiment. (b) Laminar distribution of starter cells for each interneuron subclass. For each transgenic line, different experiments are indicated by different colors. Box plots show median and interquartile range (IQR). Whiskers show the largest or smallest value no further than 1.5 × IQR from the hinge (same below). The number of independent experiments included are as follows: Gad2: n = 2; Ndnf: n = 2; Pvalb: n = 9; Sst: n = 17; Vip: n = 9. (c) Representative 3D visualization of brain-wide inputs to interneuron subclasses in VISp. (d) Overview of brain-wide inputs to different interneuron subclasses. (e-h) Layer-specific inputs of ipsilateral VISl to excitatory neurons in L2/3 (e, n = 5 independent experiments), L4 (f, n = 4 independent experiments), L5 (g, n = 11 independent experiments) and L6 (h, n = 8 independent experiments) of VISl and representative images of local VISl inputs. Starter cells are identified by the co-expression of dTomato from the AAV helper virus and nucleus-localized H2B-EGFP from the rabies virus.

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Extended Data Fig. 10 Comparison of laminar distribution of visual inputs to SSp-bfd-rll (a-b) and VISrl (c-d).

(a, c) Laminar distribution of inputs from various visual areas to SSp-bfd-rll (a, the numbers of independent experiments are: VISli and VISpm: n = 5, VISl and VISa: n = 12; VISp, VISal, and VISrl: n = 13; VISam: n = 7) and VISrl (c, the numbers of independent experiments are: VISli and VISpm: n = 20, VISl and VISa: n = 21; VISp: n = 23; VISal and VISrl: n = 22; VISam: n = 19). Box plots show median and interquartile range (IQR). Whiskers show the largest or smallest value no further than 1.5 × IQR from the hinge. (b, d) Representative images of inputs from VISp, VISpm and VISal to SSp-bfd-rll (b) and VISrl (d).

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Supplementary information

Reporting Summary

Supplementary Tables 1–5

Supplementary Table 1. Control experiments validating the improved monosynaptic rabies tracing system. Supplementary Table 2. Metadata and summary of starter cells for retrograde connectome experiments. Supplementary Table 3. Input connectivity matrix with normalized input strength per structure as a fraction of whole-brain total inputs and preliminary retrograde labeling data from initial informatic quantification. Supplementary Table 4. Summary of anterograde projection experiments from VISp and source areas projecting to VISp. Supplementary Table 5. Summary of anterograde projection experiments from VISl and source areas projecting to VISl.

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Yao, S., Wang, Q., Hirokawa, K.E. et al. A whole-brain monosynaptic input connectome to neuron classes in mouse visual cortex. Nat Neurosci 26, 350–364 (2023). https://doi.org/10.1038/s41593-022-01219-x

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