Fig 1.
Multisensory gamma stimulation improves spatial working memory in Ts65Dn mice.
A) Experimental scheme. B) Novel Object Recognition (NOR) test. Quantification of recognition index during NOR. Average Novel object exploration time: ambient: 9.73 seconds, stimulation: 12.84 seconds. Average familiar object exploration time: ambient: 10.31 seconds, stimulation: 7.20 seconds. C) Novel Object Location (NOL) test. Quantification of recognition index during NOL. Average Novel location exploration time: ambient: 9.33 seconds, stimulation: 14.72 seconds. Average old location exploration time: ambient: 10.70 seconds, stimulation: 5.34 seconds. D) Spontaneous alteration based on Y-maze. Quantification of percent alteration and total number of entries. ambient = 12, stimulation = 12, unpaired t-test, two-tailed, *P<0.05, ****P<0.0001. Error bars indicate mean ± standard error mean.
Fig 2.
Molecular signatures underlying multisensory gamma stimulation-associated cognitive benefits in the hippocampus.
A) Experimental scheme for the single nuclei RNA-seq experiment in Ts65Dn mice B) Unbiased clustering of hippocampal snRNA-seq data for 15884 nuclei represented on a UMAP. Cell types are annotated based on the marker genes. Ex: Excitatory Neurons, Astro: Astrocytes, Oligo: Oligodendrocytes, In: Inhibitory Neurons, OPC: Oligodendrocyte precursor cells, Micro: Microglia. C) Heatmap showing number of differentially expressed genes in excitatory and inhibitory neurons in the hippocampal snRNA-seq data from stimulation vs ambient Ts65Dn mice. D) Gene ontology analyses for the upregulated and downregulated genes in hippocampal excitatory and inhibitory neurons in stimulation vs ambient Ts65Dn mice. Color represents statistical significance after multiple testing adjustments. E) Signed bar plot displaying significant co-expression changes of the identified 7 gene modules in hippocampal excitatory neurons in stimulation vs ambient TsD65n mice. Red color denotes increased and blue color decreased expression in stimulated mice. X-axis represents signed -log10 p value. F) Hub genes of modules Ex-3, Ex-6 and Ex-7. Genes that encode synaptic proteins are highlighted in green. G) Top gene ontology biological processes for modules Ex-3, Ex-6, and Ex-7. H) Quantification of mature synapses in granule cell molecular layer of dentate gyrus in stimulation and ambient Ts65Dn mice via co-localization of PSD95 and synaptophysin. Representative images of mouse brain slices stained with PSD95, synaptophysin and DAPI. Images were acquired using 63x objective. Triangles indicate colocalized puncta. Y-axis represents the absolute number of colocalized puncta. Scale bar = 10 microns. t-test, two-tailed, unpaired, *P<0.05. Error bars indicate mean ± sem. ambient = 6, stimulation = 7 mice. I) Hub genes overlapping with disease risk genes for Alzheimer´s disease. J) Quantification of Reln+ neurons after ambient or 40Hz stimulation. Representative confocal images of hippocampal CA3 area brain slices from Ts65Dn mice after staining for Reelin and NeuN with DAPI counterstain. Merged panel shows all three markers. 20x objective. Scale bar = 50 pixels. Bar plots show the mean proportion of Reln+ neurons between the ambient and stimulation groups. Error bars indicate mean ± sem. t-test, two-tailed, unpaired, **P<0.01. Number of mice = 5/group. K-L) Hypergeometric overlap of the genes in modules Ex-3, Ex-6, and Ex-7 with different sets of downregulated genes related to (K) aging and (L) Alzheimer’s disease (AD). Color represents fold enrichment. *FDR<0.05, **FDR<0.01, ***FDR<0.001.
Fig 3.
Multisensory gamma stimulation increases adult neurogenesis in Ts65Dn mice.
A) Top 10 ranked TFs for module Ex-7. Color represents -log10 enrichment score based on ChEA3 analysis. B) Mean TCF4 fluorescence intensity after treatment of mice with or without 40 Hz stimulation. Representative images of TS65Dn mouse brain slices from the hippocampal granule cell molecular layer of dentate gyrus area after staining for TCF4 (green) and DAPI (blue). Merged panel shows both markers. Scale bar = 15 µm. Bar plots show normalized mean intensity (% of ambient group) between the two groups. t-test, two-tailed, unpaired. *P<0.05. number of mice = 4/group. C) Sub clusters of immature granule cells represented on a UMAP D) Dot plots showing relative enrichment of marker genes across the three sub-clusters of immature granule cells (GC) E) UMAP plots separated by group reveal differential numbers of cluster 3 immature GC in stimulation group. F) Box plot shows increased proportion of Prox1+ immature GC cells in stimulation vs ambient Ts65Dn mice. Center line represents the median; lower and the upper lines represent the 25th and 75th percentiles, respectively; whiskers indicate the smallest and largest values in the 1.5x interquartile range. G-H) Increased cell proliferation in the dentate gyrus of stimulation vs ambient Ts65Dn mice tested via immunolabeling for G) Ki67 (ambient = 6 mice, stimulation = 4 mice); Scale bar = 10 µm. and H) EdU (ambient = 8 mice, stimulation = 8 mice); Scale bar = 50 microns. Error bar indicates mean ± standard error mean. Two tailed, unpaired t-test. *P<0.05, **P<0.01, ***P<0.001.