Abstract
Information processing in neocortical circuits requires integrating inputs over a wide range of spatial scales, from local microcircuits to long-range cortical and subcortical connections. We used rabies virus–based trans-synaptic tracing to analyze the laminar distribution of local and long-range inputs to pyramidal neurons in the mouse barrel cortex and medial prefrontal cortex (mPFC). In barrel cortex, we found substantial inputs from layer 3 (L3) to L6, prevalent translaminar inhibitory inputs, and long-range inputs to L2/3 or L5/6 preferentially from L2/3 or L5/6 of input cortical areas, respectively. These layer-specific input patterns were largely independent of NMDA receptor function in the recipient neurons. mPFC L5 received proportionally more long-range inputs and more local inhibitory inputs than barrel cortex L5. Our results provide new insight into the organization and development of neocortical networks and identify important differences in the circuit organization in sensory and association cortices.
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Acknowledgements
We thank K. Miyamichi for critical discussions while designing experiments, members of the Luo laboratory and K. Svoboda for helpful comments on the manuscript, P. Joshi and S. McConnell (Department of Biology, Stanford University) for Rbp4Cre and Ntsr1Cre mice, and C. Gerfen (Laboratory of Systems Neuroscience, National Institute of Mental Health) for SepW1Cre mice. L.L is an investigator of the Howard Hughes Medical Institute. This work was supported by US National Institutes of Health grants T32NS007280-27 and F32NS087860 (L.A.D.), F31DC013240 (D.S.B.) and R01-NS050835 (L.L.).
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L.A.D., D.S.B. and L.L. designed the experiments. L.A.D. and D.S.B. performed and analyzed layer-specific trans-synaptic tracing experiments. L.A.D. performed slice recording and CRACM experiments. K.D. performed and analyzed in situ hybridization experiments. L.A.D., D.S.B. and L.L wrote the manuscript.
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Supplementary Figure 1 Controls for rabies tracing
(a) Characterization of reagents and procedures. L2/3 neurons were electroporated in utero with pCAG-tdTomato and Cre-driver lines were crossed to Ai14 mice. Images show that tdTomato expression in barrel cortex was restricted to the intended layers at P21, and both Cre lines were active at P1. Individual barrels in L4 at P21 are outlined with dotted lines. (b) We observed 0 mCherry-positive or GFP-positive cells when tracing from wild-type mice that did not express Cre (n = 3 mice) using the trans-synaptic tracing experimental conditions. (c) Example image from a control tracing experiment when AAV carrying CAG-FLEx-G was omitted. Graphs show quantification of the number of red+green (yellow cells) divided by total green cells. (d) Tables list the lower limits (upper) and thicknesses (lower) of each layer for each set of experiments (IUEp-Cre, n = 6 mice; Rbp4Cre, n = 9 mice; Ntsr1Cre, n = 9 mice, layer markers, n = 6–8 sections from 2 mice). There were no significant differences between layer limits (Two-way ANOVA, F = 2.66, P = 0.07) or layer thicknesses (Two-way ANOVA, F = 1.39, P = 0.25). (e) Staining for layer markers Cux1 (L2-4) and Ctip2 (L5b/6) in barrel cortex. Scale bars represent 200 μm. Summary statistics presented as mean ± s.e.m. See Supplementary Table 6 for exact test results and P values.
Supplementary Figure 2 Characterization of Cre driver lines
(a) Characterization of SepW1Cre. SepW1Cre mice were injected with AAV carrying CAG-FLEx-TC66T-mCherry in barrel cortex at P25 and perfused at P40. Sections of barrel cortex were stained with antibodies against NeuN (n = 8 sections from 2 mice). (b-e) Characterization of Rbp4Cre. Brain sections from P40 Rbp4Cre;RosaAi14 mice were stained for NeuN or Ctip2. (b) NeuN staining in barrel cortex (n = 6 slices from 2 mice). (c) Ctip2 staining in barrel cortex (n = 12 slices from 2 mice). (d) NeuN staining in mPFC (n = 5 slices from 2 mice). (e) Ctip2 staining in mPFC (n = 5 slices from 2 mice). (f) Characterization of Ntsr1Cre. Barrel cortex sections from P40 Ntsr1Cre;RosaAi14 mice were stained for NeuN (n = 10 slices from 2 mice). (g) Quantification of marker colocalization. Summary statistics presented as mean ± s.e.m.
Supplementary Figure 3 Spatial analyses of Gad+ and Gad– synaptic inputs from barrel cortex to starter cells in L2/3, L5, and L6 along the M-L Axis
(a-f) Heat maps showing distribution of Gad– inputs (upper panels), Gad+ inputs (middle panels) and starter cells (SCs, lower panels) in each layer along the M-L axis (n = 3 mice each). Inputs to L2/3 (a), L5 (c), and L6 (e) scaled to highest fraction of Gad+ inputs. Inputs to L2/3 (b), L5 (d), and L6 (f) scaled to highest fraction of Gad– inputs. Colors represent fraction of barrel cortex inputs within the sections analyzed. Bin widths are 120 μm.
Supplementary Figure 4 Characterization of the EPSCs in control and GluN1-lacking neurons
(a) Example EPSCs recorded from a WT L6 neuron voltage-clamped at +40 mV while stimulating white matter. DNQX (blue trace) reduced the amplitude of the EPSC. (b) Example EPSCs recorded from a GluN1fl/Δ L6 neuron voltage-clamped at +40 mV while stimulating white matter. DNQX (blue trace) completely abolished the EPSC. (c) Quantification of fraction of EPSC remaining after DNQX wash-in (Control: 0.20 ± 0.07, n = 4 cells, from 2 mice; Grin1fl/Δ: 0.005 ± 0.001, n = 4 cells from 2 mice; P = 0.04, Student’s t-test). Summary statistics presented as mean s.e.m. See Supplementary Table 6 for exact test results and P values.
Supplementary Figure 5 Layer assignments for tracing from starter cells lacking GluN1 and comparison of long-range inputs to control and GluN1-lacking starter cells
(a) Layer limits were assigned based on the DAPI signal for each section. Table lists the lower limits of each layer for each set of experiments (IUEp-Cre;GluN1fl/Δ, n = 4 mice; Rbp4Cre;GluN1fl/Δ, n = 6 mice; Ntsr1Cre;GluN1fl/Δ, n = 7 mice). (b) Comparison of layer limits between control and GluN1fl/Δ animals. (c) Layer thicknesses derived from the limits based on DAPI signal within each section. Table lists the thickness of each layer for each set of experiments (IUEp-Cre;GluN1fl/Δ, n = 4 mice; Rbp4Cre;GluN1fl/Δ, n = 6 mice; Ntsr1Cre;GluN1fl/Δ, n = 7 mice). (d) Comparison of layer thickness between control and GluN1fl/Δ animals. (e) Laminar analysis of long-range inputs from to control (colors) and GluN1-lacking (black) starter cells in L5 from S1 (control: n = 9 mice; GluN1fl/Δ: n = 4 mice), S2 (control: n = 5 mice; GluN1fl/Δ: n = 5 mice; L6 comparison, P = 0.004), M1 (control: n = 7 mice; GluN1fl/Δ: n = 4 mice), and M2 (control: n = 5 mice; GluN1fl/Δ: n = 3 mice). (f) Laminar analysis of long-range inputs from to control (colors) and GluN1-lacking (black) starter cells in L6 from S1 (control: n = 9 mice; GluN1fl/Δ: n = 7 mice), S2 (control: n = 9 mice; GluN1fl/Δ: n = 7 mice; L6 comparison, P = 0.003), M1 (control: n = 8 mice; GluN1fl/Δ: n = 7 mice), and M2 (control: n = 8 mice; GluN1fl/Δ: n = 6 mice). * denotes significant P values from multiple t-tests with Holm-Sidak correction for multiple comparisons (α=0.05). Summary statistics presented as mean ± s.e.m. See Supplementary Table 6 for exact test results and P values.
Supplementary Figure 6 Controls for layer-specific tracing to mPFC
(a) Example images showing staining for layer markers Cux1 (L3) and Ctip2 (L5b/6). Scale bar represents 100 μm. (b) Summary of layer lower limits (upper) assigned based on DAPI or layer markers and layer thickness (lower) assigned based on DAPI or layer markers. (c) Example images showing rabies tracing in mPFC without CAG-FLEx-G. Green rabies-infected neurons have almost 100% overlap with red TVA66T-mCherry-positive neurons. (d) Results of control tracing experiments (n = 3 mice) without rabies glycoprotein, which mediates trans-synaptic spread. CAG-FLEx-TVA66T-mCherry was injected into Rbp4Cre animals in mPFC. Two weeks later, RVdG-EGFP was injected into the same location. Graphs show quantification of the sum of red+green (yellow) divided by the total green cells. (e) Comparison of layer distribution of total inputs to L5 of mPFC and barrel cortex, normalized by layer thickness for each respective area (mPFC: n = 4 mice; BC: n = 8 mice; L1: P = 6.31x10-6, L3: P = 0.0002). * denotes significant P values from multiple t-tests with Holm-Sidak correction for multiple comparisons (α=0.05). Summary statistics presented as mean ± s.e.m. See Supplementary Table 6 for exact test results and P values.
Supplementary Figure 7 Raw numbers of inputs and starter cells for tracing in Barrel Cortex and mPFC
Plots of raw numbers of inputs vs. starter cells (left) and convergence index (# inputs cells /# starter cells) (right) for barrel cortex (a) L2/3, (b) L5, and (c) L6, and (d) mPFC L5 tracing experiments. Summary statistics presented as mean±SEM.
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DeNardo, L., Berns, D., DeLoach, K. et al. Connectivity of mouse somatosensory and prefrontal cortex examined with trans-synaptic tracing. Nat Neurosci 18, 1687–1697 (2015). https://doi.org/10.1038/nn.4131
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DOI: https://doi.org/10.1038/nn.4131
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