Article Figures & Data
Figures
- Figure 1.
Imaging in multiple lamina in auditory cortex. A. Schematic of experimental setup. B, Image of loaded neurons in L4. Scale bar, 50 μm. C, Mean imaging depths for the two groups (L2/3 mean depth from pia, 190 ± 32 μm; L4 mean depth from pia, 348 ± 46 μm; average separation of imaging planes, 161 μm). D, Left, Injections of latex microspheres at L4 imaging depth confirm imaging in midcortical layers. Right, Expression of the layer 4-specific marker Scnn1a (Madisen et al., 2010; Viswanathan et al., 2012) indicates location of L4. We crossed a transgenic mouse expressing cre recombinase under the control of Scnn1a promoter [B6;C3-Tg(Scnn1a-cre)3Aibs/J; JAX strain 9613], with a mouse that expresses td Tomato in a cre-dependent manner [B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze; Jax strain 7909]. WM, White matter.
- Figure 2.
Sound-evoked fluorescence changes are larger in L4 neurons. A, Sound-evoked fluorescence traces (ΔF/F; mean ± SEM) from two neurons in L2/3 (black) and L4 (red) imaging planes. Horizontal colored bars indicate sound frequency and sound duration. Vertical scale bars are 5%. B, Comparison of average maximum ΔF/F of all responding neurons in pairs of imaging fields. L4 fields showed larger maximum ΔF/F values (p < 0.03, paired t test). C, Maximum ΔF/F response of all neurons in both layers; nL2/3 = 310 responsive neurons; nL4 = 341 responsive neurons. L4 neurons showed larger maximum ΔF/F (p < 10−23, Wilcoxon rank sum).
- Figure 3.
BF variability in L4 and L2/3. A, Fluorescence (ΔF/F) time course traces from three neighboring neurons in L2/3 (rows). Horizontal colored bars indicate sound frequency and duration (1 s). Arrowheads indicate BFs. B, Superimposed tuning curves of all responsive neurons in the imaged field. Filled circles indicate the BF for each neuron. C, Spatial map of BFs for all imaged neurons in the field. Color indicates BFs. White circles indicate unresponsive neurons (see Materials and Methods). D–F, Conventions are as in A–C. D, Fluorescent time course traces (ΔF/F) from three neighboring neurons in L4 (red). E, Frequency-tuning curves of all responsive neurons in the imaged field. F, Spatial map of BFs for all imaged neurons. Scale bar: C (for C, F) 25 μm.
- Figure 4.
BF variability in L4 and L2/3. A second example of paired imaging fields. Conventions are as in Figure 3. A, Superimposed tuning curves of all responsive neurons in L2/3. B, Spatial map of BFs for imaged neurons in the field. C, D, Frequency-tuning and spatial maps of BF for the corresponding L4 imaging field. Scale bars: 25 μm.
- Figure 5.
BF variability is larger in L2/3. A, BF variability (IQRBF) in each paired imaging field. Each set of connected dots represents a pair of imaging fields. L4 fields showed lower variability (medianL2/3, 0.47 ± 1.3 octaves; medianL4, 0.16 ± 0.56 octaves; p = 0.01, Wilcoxon sign rank). B, Cumulative distribution functions displaying local (<100 μm radius) BF variability around each neuron in L2/3 (black dashed line) and L4 (thick gray line). L4 neurons show lower variability (p < 10−6, Wilcoxon rank sum).
- Figure 6.
BF variability is due to spatial variability. A, Scatter plot of median BF of all responsive neurons in each paired imaging plane. The median BF is similar for each pair of imaging fields. B, Percentage of single-peaked neurons in each paired imaging field. Horizontal bars indicate the mean for each group. Mean values in each group were similar (L2/3, 59 ± 7% SEM; L4, 65 ± 7% SEM; p > 0.3, paired t test). C, Cumulative distribution functions displaying a measure of frequency-tuning curve bandwidth (BW75) for single-peaked L2/3 (black) and L4 (red; medianL2/3, 0.23 ± 0.18 IQR octaves; medianL4, 0.28 ± 0.18 IQR octaves; p < 0.001, Wilcoxon rank sum). D, BF variability (IQRBF) for only single-peak neurons in paired imaging fields. Each set of connected dots represents the IQRBF for all responding neurons within a pair of imaging fields. Horizontal bars indicate the medians of each group (medianL2/3, 0.31 ± 0.97 IQR octaves; medianL4, 0 ± 0.25 IQR octaves). BFs across entire L4 fields are less variable (p < 0.03, Wilcoxon sign rank). E, Cumulative distribution functions displaying local (<100 μm radius) BF variability of only single-peak neurons in L2/3 (black) or L4 (red). Local BF variability is less in L4 (medianL2/3, 0.25 ± 0.875 IQR octaves; medianL4, 0 ± 0.375 IQR octaves; p < 10−8, Wilcoxon rank sum). F, Distributions of ratios of peak frequencies of multipeak neurons in L2/3 (gray) and L4 (red) are similar. Arrow indicates secondary peak representing octave spacing.
- Figure 7.
Signal and noise correlations between neurons in L2/3 and L4 are different. A, Significant pairwise signal cross-correlations in L2/3 for an example imaging field. Light gray circles show correlations obtained from a randomized version of the data (see Materials and Methods; n = 91 pairs; p = 10−24, paired t test). B, Significant pairwise signal cross-correlations in L4 (n = 136 pairs; p = 10−59, paired t test). C, Comparison of distributions of signal correlation values in L2/3 (black) and L4 (red). Vertical lines indicate means of distributions (p < 10−9, unpaired t test). Inset, Scatter plots comparing SC values and SC-NC values. Each symbol represents an SC value (x-axis) or SC values corrected for the contributions of noise correlations (y-axis) for a pair of neurons in L2/3 (black) or L4 (red). D, E, Significant pairwise noise correlations in L2/3 and L4 in an example field (p = 10−39 and p = 10−23, respectively). F, Comparison of distributions of noise correlation values in L2/3 (black) and L4 (red). Vertical lines indicate means of distributions (p < 10−30, unpaired t test).
- Figure 8.
Signal and noise correlations between neurons in L2/3 and L4 are not always different. A, Significant pairwise signal cross-correlations in L2/3 for a second example field. Light gray circles show correlations obtained from a randomized version of the data (n = 78 pairs; p < 0.001, paired t test). B, Significant pairwise signal cross-correlations in L4 (n = 300 pairs; p = 10−67, paired t test). C, Comparison of distributions of signal correlation values in L2/3 (black) and L4 (red). Vertical lines indicate means of distributions (p < 10−7, unpaired t test). Inset, Scatter plots comparing SC values and SC-NC values. Each symbol represents an SC value (x-axis) or SC values corrected for the contributions of noise correlations (y-axis) for a pair of neurons in L2/3 (black) or L4 (red). D, E, Significant pairwise noise correlations in L2/3 and L4 in an example field (p < 0.001 and p < 10−11, respectively). F, Comparison of distributions of noise correlation values in L2/3 (black) and L4 (red). Vertical lines indicate means of distributions (p = 0.08, Wilcoxon rank sum).
- Figure 9.
Differences in signal and noise correlations in L2/3 and L4 neuron populations. A, B, Significant pairwise signal cross-correlations (SC) in the populations of L2/3 (black) and L4 (red). Light gray circles represent correlations obtained from a randomized version of the data; black or red circles indicate correlation values from the actual data. C, Distributions of SC values from L2/3 (black) and L4 (red) from all imaged fields (2968 L2/3 neuron pairs; 3988 L4 neuron pairs). Top, SC values. Bottom, SC values corrected for the contribution of noise correlations. Vertical lines indicate medians of distributions. p < 10−9 for SC; p < 10−26 for SC-NC (Wilcoxon rank sum test). D, Scatter plots comparing SC values and SC-NC values. Each symbol represents SC value (x-axis) or SC values corrected for the contributions of noise correlations (y-axis) for a pair of neurons in L2/3 (top) or L4 (bottom). E, F, Significant pairwise noise correlations in L2/3 and L4 layers. Light gray circles represent correlations obtained from a randomized version of the data; black or red circles indicate correlation values from the actual data. G, Comparison of distributions of noise correlation values in L2/3 (black) and L4 (red). Vertical lines indicate medians of distributions (p = 0.009, Wilcoxon rank sum).
- Figure 10.
Signal and noise correlations are correlated and decrease with distance. A, Noise and signal correlations for individual cell pairs are correlated. Data were normalized for comparison across animals and imaged regions by computing z scores (see Materials and Methods; slopeL2/3, 0.52; slopeL4, 0.45; p < 0.002). B, Display of cell densities in the two imaging planes (L2/3, black; L4, red). C, Signal correlations in L2/3 and L4 as function of cell-pair distance. Data were grouped into ∼10 μm bins, and all correlations values occurring within that distance increment were plotted. Data from the two groups (L2/3 and L4) were offset for clarity. Lines (black and red) represent the median values in each distance bin. D, Conventions are as in C. Noise correlations in L2/3 and L4 are shown as function of cell-pair distance.
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