Visual Deprivation during Mouse Critical Period Reorganizes Network-Level Functional Connectivity

Preprocessing of raw GCaMP6f signals. A, Flowchart illustrates the order and steps involved in data preprocessing. B, Exemplar FOV in grayscale. C, Preprocessed dF/F time course from the three ROIs illustrated in B. The average time course is shown in black and individual pixel's time courses are depicted in color. D, Exemplar outputs from each data preprocessing step shown in A. E, Distribution of dF/F measurements following log mean transformation, image smoothing, and global signal regression.

Preprocessed data are coregistered to a common atlas and bandpass filtered at 0.4–4 Hz. A, Exemplar images of the mouse dorsal cortex before coregistration and B, after coregistration using two anatomical landmarks: anterior midline suture and lambda. C, GCaMP6f power spectra of a 5 min run before (orange) and after (purple) 0.4–4 Hz bandpass filtering. D, Exemplar dF/F time course of pixels within ROI1 (shown in B) before (top) and after (bottom) 0.4–4 Hz bandpass filtering. The average time course is shown in black while each individual pixel's time course is shown in color. E, Example functional connectivity analysis between the selected ROIs shown in B. While ROI1 (red) and ROI2 (green) – both within the visual cortex – are moderately correlated, ROI1 and ROI3 (blue) – which are in different anatomical and functional regions – are moderately anticorrelated.

Assessment of signal to noise ratio of wide-field optical imaging system (WFOI) shows extremely low technical noise by comparing live to dead mouse. A, Distribution of pixels from a 5 min run on MD3 following image smoothing and global signal regression (GSR). The number on the top right of each panel indicates the histogram's full width at half maximum. B, Distribution of pixels from a 5 min run after euthanasia. The number on the top right of each panel indicates the histogram's full width at half maximum. C, Three ROIs are selected to illustrate the time course of spontaneous neural activity before (D) and after (E) 0.4–4 Hz bandpass filtering.

Monocular deprivation (MD) and calcium-based WFOI paradigm. A, Experimental timeline for optical imaging and lid suturing. For MD mice (n = 20;14 females and 6 males), the right eyelid was sutured after baseline imaging at P24. Sham-sutured mice (n = 15; 5 females and 10 males) were subjected to the same length of isoflurane anesthesia. Mice were subsequently imaged at MD1 (P25), MD2 (P26), and MD3 (P27). Mice were subsequently imaged at MD1 (P25), MD2 (P26), and MD3 (P27). Detailed data preprocessing steps are illustrated in Figures 1–4. B, Visualization of sampled seed centers overlaid with Paxinos atlas brain regions. C, Representative baseline interhemispheric homotopic connectivity map derived from sample 5 min run data, displaying Fisher z-transformed Pearson's correlation coefficients between each pixel in the left hemisphere and its corresponding pixel in the right hemisphere. Only the left hemisphere is displayed, as homotopic connectivity is symmetrical across the midline. D, Representative baseline left visual seeded rsFC map derived from 5 min run data. Black dot represents the Visual-a seed region, and colormap represents correlation to that seed of each pixel's time traces. E, rsFC matrix from the same 5 min run data displaying correlation values for each seed pair.

A 3 d critical period monocular deprivation (MD) decreases Δ frequency Ca²⁺ power in the visual cortex. A, Average MD calcium Δ power maps at each timepoint (n = 20). B, MD Δ calcium power plotted as the averaged percentage of each mouse's baseline values. C, Sham calcium Δ power plotted as the averaged percentage of each mouse's baseline values (n = 15). D, Mean difference maps comparing the changes in Δ calcium power from baseline between MD and sham mice that were shown in (B) and (C). E, Two sample t tests between each MD timepoint and baseline. Maps display pixels that had significant change in Δ calcium power. F, Δ calcium power in the visual cortex of MD and sham mice across time. # denotes significance on rmANOVA (MD mice, F_(3,57) = 9.50, p = 0.00035). G, Overlay of the significant cluster in the visual cortex with Paxinos atlas-based region outlines. Black dot indicates the visual seed for subsequent analysis. Error bars represent SEM here and in subsequent figures.

Critical period monocular deprivation (MD) alters homotopic functional connectivity patterns. A, Group averages of MD homotopic FC maps across time (n = 20). Exemplar traces of visual cortex activities in MD and sham mice are illustrated in Figures 8 and 9, respectively. B, MD homotopic FC plotted as the averaged change from each mouse's baseline values. C, Sham homotopic FC plotted as the averaged change from each mouse's baseline values (n = 15). D, Difference maps displaying the mean difference in the changes from baseline between MD and sham mice at each MD timepoint. E, Two-sample t tests compared the changes from baseline between MD and sham mice at each MD timepoint. F, Pearson correlations between homotopic contralateral Visual-a seeds (two-way ANOVA F_(3,99) = 3.73, p = 0.021 HF-corrected). † denotes significance on post hoc two-sample t tests comparing MD and sham mice at each timepoint (baseline, t₍₃₃₎ = 0.12, p = 0.90.; MD1, t₍₃₃₎ = −2.79, p = 0.0088; MD2, t₍₃₃₎ = −3.03, p = 0.0048; MD3, t₍₃₃₎ = −3.39, p = 0.0018). # denotes significance on rmANOVA (MD mice, F_(3,57) = 6.32, p = 0.0009; sham mice, F_(3,42) = 3.37, p = 0.027). * denotes significance on post hoc paired t tests comparing each MD timepoint to baseline values for MD mice (MD1 vs baseline, t₍₁₉₎ = 3.42, p = 0.0029; MD2 vs baseline, t₍₁₉₎ = 2.72, p = 0.014; MD3 vs baseline, t₍₁₉₎ = 2.51, p = 0.021). ¤ denotes significance on post hoc paired t tests comparing each MD timepoint to baseline values for sham mice (MD3 vs baseline, t₍₁₄₎ = 3.50, p = 0.0035). G, Pearson correlations between homotopic contralateral visual-b seeds (two-way ANOVA F_(3,99) = 3.88, p = 0.019 HF-corrected). † denotes significance on post hoc two-sample t tests comparing MD and sham mice at each timepoint (baseline, t₍₃₃₎ = −0.11, p = 0.92; MD1, t₍₃₃₎ = −2.71, p = 0.010; MD2, t₍₃₃₎ = −2.70, p = 0.011; MD3, t₍₃₃₎ = −3.37, p = 0.0020). # denotes significance on rmANOVA (MD mice, F_(3,57) = 4.64, p = 0.0057; sham mice, F_(3,42) = 4.66, p = 0.0067). *denotes significance on post hoc paired t tests comparing each MD timepoint to baseline of MD mice (MD1 vs baseline, t₍₁₉₎ = −2.75, p = 0.013; MD2 vs baseline, t₍₁₉₎ = −1.35, p = 0.19; MD3 vs baseline, t₍₁₉₎ = −2.24, p = 0.037). ¤ denotes significance on post hoc paired t tests comparing each MD timepoint to baseline values for sham mice (MD2 vs baseline, t₍₁₄₎ = 2.91, p = 0.011; MD3 vs baseline, (t14) = 4.99, p = 0.0002). *, ¤, † p < 0.05; **, ¤¤, †† p < 0.01; ¤¤¤, ††† p < 0.001 here and in subsequent figures.

Exemplar neural activity from the left and right visual cortex of an animal before and after monocular deprivation (MD) at baseline (A), MD1 (B), MD2 (C), and MD3 (D). ROIs are shown in the grayscale image on the right. The red trace depicts mean calcium activity of the left visual cortex, and the blue trace depicts mean calcium activity of the right visual cortex. Shading reflects the standard deviation.

Exemplar neural activity from the left and right visual cortex of a sham animal across the experiment timeline at baseline (A), day 1 (B), day 2 (C) and day 3 (D). ROIs are shown in the grayscale image on the right. The red trace depicts mean calcium activity of the left visual cortex, and the blue trace depicts mean calcium activity of the right visual cortex. Shading reflects the standard deviation.

Short-term monocular deprivation alters left visual-seeded rsFC patterns. A, MD average left (deprived) visual seed (represented by black dot)-based rsFC maps across time (n = 20). B, Pearson correlations of MD mice at each timepoint normalized to baseline values. C, Pearson correlations of sham mice at each MD timepoint normalized to baseline values. D, Mean difference maps illustrating the differences between normalized MD and sham mice shown in (B) and (C). (E) T-statistic maps showing significant clusters of pixels after two-sample t tests comparing normalized MD and sham mice at each timepoint, thresholded using a cluster-based technique (see Materials and Methods). F, Pearson correlations between left visual and parietal seeds (two-way ANOVA F_(3,99) = 3.73, p = 0.021 HF-corrected). † denotes significance on post hoc two-sample t tests comparing MD and sham mice at each timepoint (baseline, t₍₃₃₎ = 0.74, p = 0.47; MD1, t₍₃₃₎ = 2.25, p = 0.031; MD2, t₍₃₃₎ = 2.58, p = 0.015; MD3, t₍₃₃₎= 3.38, p = 0.0019). # denotes significance on rmANOVA (MD mice, F_(3,57) = 16.354, p < 0.0001). *denotes significance on post hoc paired t tests comparing each MD timepoint to baseline of MD mice (MD1 vs baseline, t₍₁₉₎ = 3.96, p = 8.4 × 10⁻⁴; MD2 vs baseline, t₍₁₉₎ = 4.9005, p = 9.9 × 10⁻⁵; MD3 vs baseline, t₍₁₉₎ = 5.86, p = 1.22 × 10⁻⁵). G, Pearson correlations between left visual and somatosensory-d seeds (two-way ANOVA, F_(3,99) = 3.92, p = 0.014 HF-corrected). † denotes significance on post hoc two-sample t tests comparing MD and sham mice at each timepoint (baseline, t₍₃₃₎ = 0.73, p = 0.47; MD1, t₍₃₃₎ = 2.42, p = 0.021 ; MD2, t₍₃₃₎ = 3.23, p = 0.0028 ; MD3, t₍₃₃₎ = 3.32, p = 0.0022). # denotes significance on rmANOVA (MD mice, F_(3,57) = 14.00, p < 0.0001). *denotes significance on post hoc paired t tests comparing each MD timepoint to baseline of MD mice (MD1 vs baseline, t₍₁₉₎ = 4.92, p = 9.4 × 10⁻⁴; MD2 vs baseline, t₍₁₉₎ = 4.63, p = 1.8 × 10⁻⁵; MD3 vs baseline, t₍₁₉₎ = 4.49, p = 2.5 × 10⁻⁵).

The nondeprived visual cortex exhibited decreased correlation to the deprived visual cortex. A, MD average right (nondeprived) visual seed (represented by black dot)-based rsFC maps across time (n = 20). B, Pearson correlations of MD mice at each timepoint normalized to baseline values. C, Pearson correlations of sham mice at each MD timepoint normalized to baseline values (n = 15). D, Mean difference maps illustrating the differences between normalized MD and sham mice shown in (B) and (C). E, T-statistic maps showing significant clusters of pixels after two-sample t tests comparing normalized MD and sham mice at each timepoint, thresholded using a cluster-based technique.

Gross body movement during head-fixed imaging is not significantly different between MD and sham mice. A, Exemplar frame of head-fixed behavioral recording (i) and optic flow vectors for movement quantification (ii–iv). B, Mean movement distributions of MD and sham mice following lid suturing or mock suturing MD n = 6; sham n = 6) . There was no significant influence of conditions (two-way ANOVA F_(1,20) = 0.30, p = 0.59), time (two-way ANOVA F_(2,20) = 2.4, p = 0.065), or their interaction (two-way ANOVA F_(2,20) = 1.0, p = 0.44) on movement distribution.

Global motion detection using global variance of temporal derivatives (GVTD). A, 100 s run represented as a matrix of dF/F measurements by time. Each row in the matrix represents a measurement signal and the column represents time. B, Temporal derivatives of the data shown in A. C, Squared values of the elements shown in B. D, GVTD time course represented as the RMS of the values shown in C. E, Mean GVTD scores of MD and sham mice across time (MD n = 20, sham n = 15). There was no significant effect of conditions (two-way F_(1,99) = 0.0059, p = 0.94), time (two-way F_(3,99) = 0.78, p = 0.48), or their interaction (two-way F_(3,99) = 0.31, p = 0.82) on mean GVTD scores. F, GVTD standard deviation values of MD and sham mice across time (MD n = 20), sham (n = 15). There was no significant influence of condition (two-way ANOVA F_(1,99) = 0.35, p = 0.56), time (two-way ANOVA F_(3,99) = 1.2, p = 0.33), or their interaction (two-way ANOVA F_(3,99) = 2.8, p = 0.051) on GVTD standard deviations.