Cortical Excitation:Inhibition Imbalance Causes Abnormal Brain Network Dynamics as Observed in Neurodevelopmental Disorders

Abstract

Abnormal brain development manifests itself at different spatial scales. However, whether abnormalities at the cellular level can be diagnosed from network activity measured with functional magnetic resonance imaging (fMRI) is largely unknown, yet of high clinical relevance. Here a putative mechanism reported in neurodevelopmental disorders, that is, excitation-to-inhibition ratio (E:I), was chemogenetically increased within cortical microcircuits of the mouse brain and measured via fMRI. Increased E:I caused a significant "reduction" of long-range connectivity, irrespective of whether excitatory neurons were facilitated or inhibitory Parvalbumin (PV) interneurons were suppressed. Training a classifier on fMRI signals, we were able to accurately classify cortical areas exhibiting increased E:I. This classifier was validated in an independent cohort of Fmr1y/- knockout mice, a model for autism with well-documented loss of parvalbumin neurons and chronic alterations of E:I. Our findings demonstrate a promising novel approach towards inferring microcircuit abnormalities from macroscopic fMRI measurements.

Keywords: DREADD; excitation: inhibition balance; fMRI; functional connectivity; hypoconnectivity.

Figure 1

Local changes in neural activity induced by activating hSyn-hM3Dq. (A) Neuronal firing rate before and after clozapine injection in the right SSp, indicating a steady increase in firing rate upon DREADD activation. (B) As A but for the left SSp (control area) indicating no change in the neuronal firing rate after clozapine injection. (C) Time-resolved firing rate in millisecond bins for the right (red) and left (black) SSp. A steady increase in firing rate occurs in the right SSp once clozapine is injected, while no change occurs in the left SSp. (D) Relative change of averaged multiunit activity recorded in the right and left SSp. (E), (F) Cerebral blood flow maps were collected every 15 min for wt-hSyn-hM3Dq and control mice (displayed in their native space), respectively. (G) Clozapine is injected 15 min after the start of the experiment. These 15 min are referred to as the baseline period (−15 to 0 min). The rest of the experiment is 45 min long and divided into three periods, that is, Post 1 (0–15 min), Post 2 (15–30 min), and Post 3 (30–45 min). For all analyses performed in this experiment (unless otherwise stated), Post data was expressed relative to baseline (Post 1/Post 2/Post 3—baseline) and is referred to as ∆1, ∆2, and ∆3, respectively. (H) Comparison of blood flow (mL of blood/100 g/min) between controls and wt-hSyn-hM3Dq mice over time (measured at the injection site). (I) Percentage change in blood flow over time between the controls (n = 7) and wt-hSyn-hM3Dq (n = 7) mice (measured at the injection site). Linear mixed models indicate a significant main groups × scan time effect (F_2,21 = 5.657; P = 0.04; asterisk indicates significant between-group independent samples post hoc t-test at Post 1 (t₁₂ = 3.23; P = 0.07), at Post 2 (t₁₂ = 2.66; P = 0.02), and at Post 3 (t₁₂ = 2.35; P = 0.04). (J) Percentage change in blood flow over time measured in left SSp. (K) Percentage change in blood flow over time measured in the left lateral striatum. (L) Average regional homogeneity (ReHo) change over time at the injection site. Repeated measures ANOVA (control [n = 13]; wt-hSyn-hM3Dq [n = 14]) showed a significant groups × scan time effect (F_1.93,75 = 11.8; P < 0.001; asterisk indicates significant between-group independent samples post hoc t-test at Post 1 (t₂₅ = −2.57; P = 0.01), at Post 2 (t₂₅ = −2.28; P = 0.03), and at Post 3 (t₂₅ = −2.1; P = 0.05). (M) Significant decrease in ReHo in wt-hSyn-hM3Dq mice versus controls for ∆1 depicted as a 3D image and in coronal slices (TFCE-corrected). The slice marked with a red rectangle shows the injectionsite.

Figure 2

Long-range connectivity changes induced by activating hSyn-hM3Dq. (A) Effect-size analysis (Cohen’s d) of functional connectivity is calculated for single edges (n = 844). Only cluster 1 shows a decrease in the effect size after clozapine injection. (B) Averaged effect size (Cohen’s d) over time for 20 edges of cluster 1 and 20 randomly selected edges of cluster 2. (C) Coronal brain slices depicting tracer data after unilateral injection in SSp (green, obtained from Allen Mouse Brain Connectivity Atlas, Injection ID: 114290938) and averaged voxel-wise Pearson’s correlation (Fisher’s z-transform) during the baseline period from a seed in the right SSp. (D) Pearson’s correlation between functional connectivity of the target region (right SSp) and tracer injection intensity obtained from images depicted in C. (E–G) Matrices represent averaged and z-transformed Pearson’s correlation of all control animals (lower triangular matrix) and all wt-hSyn-hM3Dq animals (upper triangular matrix) for the baseline period, Post 1 and Post 2, respectively. (H) The upper triangle of the matrix shows a reduction in FC in wt-hSyn-hM3Dq (n = 14) mice compared with controls (n = 13) during ∆1 (i.e., Post 1-baseline), while the lower triangle shows the same set of comparisons in ∆2 (i.e., Post 2-baseline). Color coding reflects randomized permutation testing, P < 0.05, uncorrected. Asterisks indicate significant differences that survived FDR correction for multiple comparisons. MO, somatomotor cortex; SSp-m, primary somatosensory cortex (mouth); SSs, supplementary somatosensory cortex; CP, caudoputamen; TEa, temporal association cortex; R, right; L, left. (I) Change over time of the mean brain-wide VMHC (symmetric connectivity) at the injection site. Repeated measures ANOVA (controls [n = 13]; wt-hSyn-hM3Dq [n = 14]) indicates significant groups × scan time effect (F_1.67,41.9 = 21.18; P < 0.001; asterisk indicates significant between-group independent samples post hoc t-test at Post 1 (t₂₄ = −6.01; P = 10⁻⁵), at Post 2 (t₂₄ = −5.93; P = 10⁻⁵), and at Post 3 (t₂₄ = −5.22; P = 10⁻⁵). (J) Significant decrease (TFCE-corrected) in symmetric connectivity depicted on 3D images and coronal slices for the ∆1 time point. The slice marked with a red rectangle is the injection slice.

Figure 3

Changes induced by activating CAMK-hM3Dq in wild-type mice. (A) Comparison of blood flow (mL of blood/100 g/min) over time between controls and wild-type mice injected with excitatory (hM3Di) DREADD on CAMKII promoter (measured at the injection site). We observe a significant repeated measures ANOVA groups × scan time effect (F(1.0, 20.0) = 7.3; P = 0.004; asterisk indicates significant between-group independent samples post hoc t-test at Post 1 (t_9.8 = 2.7; P = 0.02) and at Post 2 (t_9.4 = 2.1; P = 0.05) in blood flow in wt-CAMK-hM3Dq mice (n = 13) after clozapine injection (similar to wt-hSyn-hM3Dq from Fig. 2H). (B,C) Blood flow in the left SSp and left CP does not significantly differ between controls and wt-CAMK-hM3Dq mice. (D) Change over time of mean regional homogeneity at the injection site (right SSp). There is a significant decrease (repeated measures ANOVA significant groups × scan time effect: F(1.3, 25.2) = 7.74, P = 0.006; asterisk indicates significant between-group independent samples post hoc t-test at Post 1 [t₂₃ = −3.46; P = 0.002] and at Post 2 [t₂₃ = −3.49; P = 0.002]) in mean connectivity in wt-CAMK-hM3Dq (n = 13) mice as compared with controls (n = 13). (E) Change over time of mean symmetric connectivity at the injection site. There is a significant decrease (repeated measures ANOVA groups × scan time effect: F(1.16, 21.98) = 8.21, P = 0.007; asterisk indicates significant between-group independent samples post hoc t-test at Post 1 (t₂₄ = 3.85; P = 0.001) and at Post 2 [t₂₄ = 4.26; P = 10⁻⁵]) in mean connectivity in wt-CAMK-hM3Dq (n = 13) mice as compared with controls (n = 13). (F) Matrix represents statistically significant differences during ∆1 and ∆2 time period among three brain regions (MO, somatomotor cortex; SSp, somatosensory cortex; SSs, supplementary somatosensory cortex; R, right; L, left) between the controls and wt-CAMK-hM3Dq mice. The asterisk within ROIs indicates a statistically significant difference that survived correction of multiple comparisons.

Figure 4

Changes induced by activating hSyn-hM4Di in PVCre mice. (A) Neuronal firing rate at the right SSp DREADD injection site of the PVCre-hSyn-hM4Di mice before and after clozapine injection, indicating a steady increase in firing rate on DREADD activation. (B) Similar to figure A but for right caudoputamen, indicating no change in the neuronal firing rate after clozapine injection. (C) Comparison of blood flow (mL of blood/100 g/min) between controls (n = 7) and PVCre-hSyn-hM4Di (n = 13) mice over time (measured at the injection site). No significant differences observed between groups (repeated measures ANOVA: F_1.4,17.9 = 2.45; P = 0.1). (D) Normalized average regional homogeneity (ReHo) change over time at the injection site. Repeated measures ANOVA indicates significant groups × scan time effect: F_1,25 = 14.01; P = 0.001; asterisk indicates significant between-group independent samples post hoc t-test at ∆1 (t₃₀ = −4.17; P = 10⁻⁵) and at ∆2 (t₃₀ = −7.15; P = 10⁻⁶). (E) Normalized averaged symmetric connectivity over time at the injection site. Repeated measures ANOVA indicates significant groups × scan time effect: F_{1.2, 21.9} = 8.21; P = 0.007; asterisk indicates significant between-group independent samples post hoc t-test at ∆1 (t₂₉ = −3.52; P = 0.001) and at ∆2 (t₂₉ = −5.78; P = 10⁻⁵). (F) Seed-to-seed analysis indicates reduced FC (randomized permutation testing, P < 0.05, uncorrected) between controls (n = 13) and PVCre-hSyn-hM4Di (n = 19) mice. (G,H) Whole-brain connectome analysis shows a significant interhemispheric reduction between somatosensory cortices for ∆1 and ∆2 between PVCre-hSyn-hM4Di (n = 14) mice and controls (n = 13). Regions affected are as follows: SSp-m, primary somatosensory area, mouth; SSp-ul, primary somatosensory area, upper limb; SSs, supplementary somatosensory area; SSp-bdf, primary somatosensory area, barel field; SSp-n, primary somatosensory area, nose.

Figure 5

DREADD manipulations lead to characteristic changes in univariate BOLD dynamics. Classification of BOLD signal dynamics in three brain regions (the injected right SSp, left SSp, and VIS) across four conditions (wt-hSyn-hM3Dq, wt-CAMK-hM3Dq PVCre-hSyn-hM4Di, and control). A schematic of the approach is depicted in A–C: (A) BOLD dynamics are measured from each brain region as a univariate time series (a 15-min time series per time window and experiment), which was (B) converted to a set of properties (a “feature vector”) using hctsa. (C) For a given region and pair of classes, we used the features of each time series (relative to baseline) as the basis for classification, which was visualized using a low-dimensional principal component projection and quantified as the 10-fold cross-validated balanced classification accuracy (%). Classification results in each brain region at ∆1 are shown for (D) wt-hSyn-hM3Dq (n = 14) versus control (n = 13), (E) wt-CAMK-hM3Dq (n = 13) versus control (n = 13), and (F) PVCre-hSyn-hM4Di (n = 10) versus control (n = 13), revealing significant discriminability in the right SSp for wt-hSyn-hM3Dq and PVCre-hSyn-hM4Di versus control (permutation test, P < 0.05, annotated as “*”). There is a consistent trend of high discriminability in the injected region (right SSp), followed by the contralateral region (left SSp), and lowest discriminability in the control region (VIS). (G) Classification results in the right SSp at ∆1 are shown for wt-hSyn-hM3Dq versus wt-CAMK-hM4Di, wt-hSyn-hM3Dq versus PVCre-hSyn-hM4Di, and wt-CAMK-hM4Di versus PVCre-hSyn-hM4Di. High discriminability is only present for wt-hSyn-hM3Dq versus wt-CAMK-hM4Di (permutation test, P < 0.05, annotated as “*”). BOLD time series in right SSp are visualized in two-dimensional principal component spaces in (H–K) for the same three pairs of classes as in (D–G). Time series with similar properties are close in the space, revealing a visual depiction of the discriminability of wt-hSyn-hM3Dq, wt-CAMK-hM3Dq, and PVCre-hSyn-hM4Di relative to control (H–J), but a relative lack of discriminability in K. Shaded ellipses (∆1) have been added to guide the eye, and time series from each class are labeled for ∆1 and ∆2.

Figure 6

Fmr1^y/− exhibit similar local BOLD dynamics as DREADD-manipulated mice. (A) The classifier was trained on features of right SSp BOLD time series to distinguish PVCre-hSyn-hM4Di from control mice. (B) The trained classifier was used to predict the identity of cortical BOLD time series measured from Fmr1^y/− (knockout) and Fmr1^y/+ (control) mice, identifying predictions of PVCre-hSyn-hM4Di as Fmr1^y/−. Predictability in each brain region was assessed as balanced classification accuracy (%); P values were estimated using a permutation test. (C) Classification results are shown for the right hemisphere, highlighting regions with significant classification accuracy (P < 0.05, FDR-corrected) as bold, dark green, and marked with asterisks. (D) Scatter plot of balanced classification accuracy and PV cell density. (E) Balanced classification accuracy is plotted for each region in the right and left hemispheres, exhibiting a strong positive correlation, Spearman’s ρ = 0.69 (P = 3 × 10⁻⁴). (F) Balanced classification accuracy visualized on a 3D brain.