Gamma Entrainment Binds Higher-Order Brain Regions and Offers Neuroprotection

Abstract

Neuronal and synaptic loss is characteristic in many neurodegenerative diseases, such as frontotemporal dementia and Alzheimer's disease. Recently, we showed that inducing gamma oscillations with visual stimulation (gamma entrainment using sensory stimuli, or GENUS) reduced amyloid plaques and phosphorylated tau in multiple mouse models. Whether GENUS can affect neurodegeneration or cognitive performance remains unknown. Here, we demonstrate that GENUS can entrain gamma oscillations in the visual cortex, hippocampus, and prefrontal cortex in Tau P301S and CK-p25 mouse models of neurodegeneration. Tau P301S and CK-p25 mice subjected to chronic, daily GENUS from the early stages of neurodegeneration showed a preservation of neuronal and synaptic density across multiple brain areas and modified cognitive performance. Our transcriptomic and phosphoproteomic data suggest that chronic GENUS shifts neurons to a less degenerative state, improving synaptic function, enhancing neuroprotective factors, and reducing DNA damage in neurons while also reducing inflammatory response in microglia.

Figure 1. 40 Hz visual stimulation entrains gamma oscillations beyond visual cortex.

(A) Experimental protocol, and target regions for analyses is indicated. (B) Representative c-Fos (red) immunostaining images, and Hoechst labeling of cell nuclei (blue). Scale bar 50 μm. 40 Hz visual stimulation increased c-Fos expression (N = 4 mice/group. T-test; V1, t = 8.312, p = 0.0002; SS1, t = 4.071, p = 0.006; CA1, t = 5.33, p = 0.0018; DG, t = 2.56, p = 0.042; and CC, t = 4.771, p = 0.003). (C) Representative raw and filtered (30–50 Hz) LFP traces recorded concurrently in V1, SS1, CA1, and PFC with the LED delivering 40 Hz visual stimulation occluded (blue) and visible (red). (D) Representative spectra of LFPs recorded simultaneously from V1, SS1, CA1, and PFC. (E) Normalized group gamma power (see also, Figure S1C) (N = 7 mice. Wilcoxon-Ranksum test; V1, Z = 5.9, p < 0.0001; SS1, Z = 2.4, p = 0.018; CA1, Z = 3.4, p < 0.0001; and PFC, Z = 3.3, p < 0.0001). (F) Raster plots of single CA1 units (labeled in different colors) with concurrently recorded LFP (band-pass filtered for 30–50 Hz) from two representative mice. (G) Spike probability of all isolated CA1 units across 40 Hz phase. (H) Phase locking strength of neuronal spikes to local LFP analyzed by mean resultant length (N = 24 cells from 4 mice. Wilcoxon-Ranksum, Z = 2.5, p = 0.011). Mean firing rate of single CA1 units did not differ between occluded (2.0 ± 0.12 Hz) and visible 40 Hz stimulation (2.1 ± 0.13 Hz) (Z = 0.55, p = 0.58). (I) LFP coherence between pairs of recording sites, as indicated, quantified using WPLI (N = 7 mice; 40 Hz visual stimulation occluded (blue) and visible (red)). (J) Group changes in low gamma band (30–50 Hz) WPLI, related to Figure 1I (Wilcoxon-Ranksum; V1-CA1, p = 0.03; V1-SS1, p = 0.021; and V1-PFC, p = 0.014; n.s. = not significant).

Figure 2. Chronic 40 Hz stimulation reduces neurodegeneration in Tau P301S and CK-p25 mice.

(A) Representative LFP spectra from V1, CA1, and PFC from 8-month-old P301S (see Figure S2A–S2C). (B) Representative images for NeuN (red) from V1 and CA1 in No Stim or GENUS P301S, with Hoechst labeling of cell nuclei (blue) (Scale bar 50 μm). (C) Group data quantifying NeuN+ cells (N = 7–8 mice/group. Two-way ANOVA F (2, 76) = 21.8, p < 0.0001. Post-hoc test; WT control Vs No Stim P301S: V1, p = 0.0005; SS1, p = 0.051; CA1, p = 0.0006; CC, p = 0.0500; WT control Vs GENUS P301S: V1, p = 0.844; SS1, p = 0.573; CA1, p > 0.999; CC, p > 0.999; No Stim Vs GENUS P301S: V1, p = 0.016; SS1, p = 0.859; CA1, p = 0.0145; CC, p = 0.090). (D) Representative LFP spectra from V1, CA1, and PFC from CK-p25 mice after 6 weeks of p25 induction (see Figure S2J–S2L). (E) Bar graph of brain weight (N = 14–17 mice/groups. ANOVA F (2, 43) = 26.6, p < 0.0001. Post-hoc test; CK control Vs No Stim CK-p25: p < 0.0001; CK control Vs GENUS CK-p25: p = 0.001; No Stim Vs GENUS CK-p25: p = 0.005). (F) Representative IHC images with vGlut1 (green), GAD65 (red), and Hoechst (blue) of the lateral ventricles (outlined) at anterior-posterior (AP) −1.2 (top row) and −2.0 mm (bottom row) from bregma. (G) Fold change of size of lateral ventricles (N = 6–9 mice/group. Two-way RM-ANOVA, between groups F (2, 18) = 31.51, p < 0.0001. Post-hoc test; CK control Vs No Stim CK-p25: AP from bregma (mm): −1.2, p < 0.0001; −1.4, p = 0.042; −1.8, p = 0.033; −2.0, p < 0.0001, −2.5, p = 0.002; CK control Vs GENUS CK-p25: −1.2, p = 0.123; −1.4, p = 0.421; −1.8, p = 0.801; −2.0, p = 0.0137; −2.5, p = 0.393; No Stim Vs GENUS CK-p25: −1.2, p = 0.035; − 1.4, p = 0.523; −1.8, p = 0.194; −2.0, p < 0.0001; −2.5, p = 0.085). (H) Representative images for NeuN (red) in V1 and CA1, and Hoechst labeling of cell nuclei (blue) (Scale bar 50 μm). (I) Group data quantifying NeuN+ cells (N = 6–9 mice/group. Two-way ANOVA F (2, 72) = 31.38, p < 0.0001. Post-hoc test; CK control Vs No Stim CK-p25: V1, p < 0.0001; SS1, p = 0.001; CA1, p = 0.002; CC, p = 0.0007; CK control Vs GENUS CK-p25: V1, p > 0.99; SS1, p > 0.99; CA1, p = 0.765; CC, p > 0.999; No Stim CK-p25 Vs GENUS CK-p25: V1, p = 0.0001; SS1, p = 0.035; CA1, p = 0.106; CC, p = 0.026; n.s.= not significant).

Figure 3. Chronic GENUS modifies behavior in mouse models of neurodegeneration.

(A) Behavior schedule of CK-p25 with No Stim or GENUS during 6-weeks of p25 induction, with OF (day 40) test. (B) Representative occupancy heat maps from OF session. (C) Time spent in the center of OF (N= 15 mice/group. t = 0.467, p = 0.644). (D) Distance travelled in the OF test (t = 0.1179, p = 0.907). (E) Plasma corticosterone levels (t = 0.9314, p = 0.360). (F) MWM schedule of p25-induced CK-p25 with or without GENUS for 6-weeks. (G) Latency to find the platform in the training (Two-way RM-ANOVA, effect of days, F (5, 168) = 14.05, p < 0.001; effect between groups, F (1, 168) = 20.47, p < 0.001. D1, t = 1.768, p = 0.259; D2, t = 2.859, p = 0.039; D3, t = 0.682, p = 0.500; D4, t = 1.867, p = 0.259; D5, t = 3.089, p = 0.0269; D6, t = 1.687, p = 0.259). (H) Swimming velocity during MWM (Two-way RM-ANOVA, effect between groups, F (1, 168) = 0.05377, p = 0.816). (I) Number of platform crossings in the probe test (Mann-Whitney U = 53, p = 0.0100). (J) Time spent in the target quadrant (t = 2.754, p = 0.010). (K) Behavior schedule of P301S with No Stim or GENUS for 22-days, with OF (day 20) test. (L) Representative occupancy heat maps from OF session. (M) Time spent in the OF center (t = 0.8801, p = 0.388). (N) Distance travelled during OF (t = 0.3122, p = 0.757). (O) Plasma corticosterone levels following No Stim or GENUS (t = 0.9232, p = 0.364). (P) MWM schedule of P301S with or without GENUS for 22-days. (Q) Latency to the platform during training (Two-way RM-ANOVA, effect of days, F (4, 150) = 9.702, p < 0.0001; effect between groups, F (1, 150) = 7.096, p = 0.008. D1, t = 0.160, p = 0.873; D2, t = 0.488, p = 0.862; D3, t = 0.7342, p = 0.849; D4, t = 2.266, p = 0.03; D5, t = 2.465, p = 0.019). (R) Swimming velocity during training (Two-way RM-ANOVA, effect between groups F (1, 150) = 1.4, p = 0.238). (S) Number of platform crossings in the probe test (Mann-Whitney U = 83.5, p = 0.091). (T) Time in target quadrant in the probe test (t = 2.603, p = 0.014).

Figure 4. Chronic GENUS reduces inflammatory response in microglia.

(A) RNA-sequencing of FACS-isolated CD11b and CD45 double-positive microglia RNAs from CK control, No Stim CK-p25, and GENUS CK-p25. Volcano plots of DEGs (N = 4 mice/group). (B) Top 7 selected GO terms for biological processes associated with the DEGs. (C) Representative images of microglia stained with Iba1 (green) and Hoechst labeling of cell nuclei (blue). Scale bar, 100 μm. (D) Bar graph of number of Iba1+ cells (N = 6–7 mice/group. Two-way ANOVA F (2, 64) = 80.35, p < 0.0001. Post-hoc test; CK control Vs No Stim CK-p25: V1, p = 0.0001; SS1, p < 0.0001; CA1, p < 0.0001; CC, p < 0.0001; CK control Vs GENUS CK-p25: V1, p = 0.050; SS1, p = 0.049; CA1, p < 0.0001; CC, p = 0.0002; No Stim Vs GENUS CK-p25: V1, p = 0.046; SS1, p = 0.005; CA1, p = 0.0002. CC, p = 0.474). (E) Representative images of microglia (Iba1; green), CD40 (red), and Hoechst labeling of cell nuclei (blue). Arrowheads and arrow indicate bushy arborization and rod-shaped ramified processes, respectively (N = 6–7 mice/group. Scale bar 50 μm). (F) Distribution plot of volume of processes of microglia (excluding rod like microglia). Right: bar chart of microglia processes volume binned into four categories based on the distribution of CK controls: minimum-25% percentile, 25%-median, 75% percentile and upper 75% percentile-maximum (N = 73 microglia/group from 6–7 mice/group. Kruskal-Wallis test, H = 9.224, p = 0.009. Dunn’s multiple comparisons; CK control Vs No Stim CK-p25: p = 0.008; CK control Vs GENUS CK-p25: p = 0.908; No Stim Vs GENUS CK-p25: p = 0.150). (G) Distribution plot of minimum distance between microglia. Right: bar graph of microglia distance, binned as in Figure 4F (N = 68, 131 and 95 microglia from 9 CK control, 6 No Stim and 6 GENUS CK-p25 mice, respectively. Kruskal-Wallis test, H = 100.1, p < 0.0001. Dunn’s multiple comparisons; CK control Vs No Stim CK-p25: p < 0.0001; CK control Vs GENUS CK-p25: p = 0.1731; No Stim Vs GENUS CK-p25: p < 0.0001). (H) Bar graph of number of rod like microglia (N = 6–9 mice/group. ANOVA F (2, 18) = 24.05, p < 0.0001. Post-hoc test; CK control Vs No Stim CK-p25: p < 0.0001; CK control Vs GENUS CK-p25: p = 0.0100; No Stim Vs GENUS CK-p25: p = 0.0113). (I) Bar graph of CD40 signal intensity (ANOVA F (2, 16) = 36.84, p < 0.0001. N = 6–7 mice/group. Post-hoc test; CK control Vs No Stim CK-p25: p < 0.0001; CK control Vs GENUS CK-p25: p = 0.0003; No Stim Vs GENUS CK-p25: p = 0.01). (J) Representative C1q (red) and Iba1 (green) images (N = 6–7 mice/groups. Scale bar 50 μm (10 μm inset)). (K) Bar graph of C1q signal intensity (ANOVA F (2, 16) = 13.39, p = 0.0004). (L) Representative images of microglia (Iba1; green) and Hoechst labeling of cell nuclei (blue). Arrowhead indicates the complexity of microglia processes (scale bar, 50 μm). (M) Distribution plot of the volume of processes of microglia, and (right) based on WT controls binned as in Figure 4F (N = 7–8 mice/group; 58 microglia/group. Kruskal Wallis test H = 7.895, p = 0.0193. Dunn’s multiple comparisons; WT control Vs No Stim P301S: p = 0.210; WT control Vs GENUS P301: p > 0.99; No Stim Vs GENUS P301S: p = 0.0170). (N) Representative C1q (red) images (N = 7–8 mice/group. Scale bar 50 μm). (O) Bar graph of C1q signal intensity (ANOVA F (2, 19) = 6.887, p = 0.005). Post-hoc test in D, H, I, K, & O ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s. = not significant.

Figure 5. Chronic GENUS modifies synaptic function and reduces DNA damage in neurons.

(A) RNA-sequencing of FACS-isolated NeuN+ RNAs from CK control, No Stim CK-p25, and GENUS CK-p25. Heat-maps of the DEGs. Number of DEGs is indicated to the right (N = 4–5 mice/group). (B) GO terms associated with the DEGs. (C) P301S with No Stim or GENUS followed by NeuN+ RNA-seq. Heat-maps of the DEGs (N = 5–6 mice/group). (D) GO terms associated with the DEGs. (E) S/T phosphorylated proteins analysis in CK-p25 using LC-MS/MS. Venn diagram of overlap of total RNAs from neuron specific RNA-seq (Figure 5A) and total proteins from LC-MS/MS (N = 3–4 mice/group). (F) Volcano plot of differentially S/T phosphorylated proteins. (G) GO terms associated with the differentially S/T phosphorylated proteins. (H) S/T phosphorylated proteins analysis in P301S using LC-MS/MS. Overlap of total RNAs from neuron specific RNA-seq (Figure 5C) and total proteins from LC-MS/MS (N = 3–4 mice/group). (I) Volcano plot of differentially S/T phosphorylated proteins. (J) GO terms associated with the differentially S/T phosphorylated proteins. (K) Representative images of vGlut1 (green), synaptophysin (Syn; red), DNM1pS774 (green), γH2Ax (red), and Hoechst labeling of cell nuclei (blue) (N = 6–9 mice/group. Scale bar represents 10 μm, 10 μm, 5 μm and 50 μm for vGlut1, Syn, DNM1pS774, and γH2Ax, respectively). (L) Bar graph of vGlut1 puncta (ANOVA between groups effect F (2, 18) = 17.9, p < 0.0001. Post-hoc test; CK control Vs No Stim CK-p25: p < 0.0001; CK control Vs GENUS CK-p25: p = 0.577; No Stim Vs GENUS CK-p25: p < 0.0001). (M) Bar graph of synaptophysin puncta (ANOVA F (2, 18) = 7.693, p = 0.003. Post-hoc test; CK control Vs No Stim CK-p25: p = 0.003; CK control Vs GENUS CK-p25: p > 0.99; No Stim Vs GENUS CK-p25: p = 0.042). (N) Bar graph of vGlut1 puncta (ANOVA F (2, 19) = 6.681, p = 0.006. Post-hoc test; WT control Vs No Stim P301S: p = 0.02; WT control Vs GENUS P301S: p = 0.879; No Stim Vs GENUS P301S: p = 0.008). (O) Bar graph of synaptophysin puncta (Two-way ANOVA F (2, 19) = 5.099, p = 0.016. Post-hoc test; WT control Vs No Stim P301S: p = 0.034; WT control Vs GENUS P301S: p > 0.99; No Stim Vs GENUS P301S: p = 0.046). (P) Bar graph of DNM1pS774 expression (ANOVA F (2, 18) = 27.89, p < 0.0001. Post-hoc test; CK control Vs No Stim CK-p25: p < 0.0001; CK control Vs GENUS CK-p25: p = 0.0046; No Stim Vs GENUS CK-p25: p = 0.0102). (Q) Bar graph of DNM1pS774 expression (ANOVA F (2, 17) = 10.084, p = 0.0009. Post-hoc test; WT control Vs No Stim P301S: p = 0.0022; WT control Vs GENUS P301S: p = 0.9575; No Stim Vs GENUS P301S: p = 0.002). (R) Bar graph of γH2Ax+ cells. γH2Ax+ cells was not detectable in CK controls (N = 6 mice/group. t = 4.224, p = 0.001). (S) Bar graph of expression of γH2Ax (ANOVA F (2, 20) = 10.02, p = 0.0010. Post-hoc test; WT control Vs No Stim P301S: p = 0.0010; WT control Vs GENUS P301S: p = 0.341; No Stim Vs GENUS P301S: p = 0.019). (T) Immunoblots of γH2Ax and GAPDH.