Article Figures & Data
Figures
- Figure 1.
Chemogenetic treatment modulates neural activity without causing inflammation or neurotoxicity. A, Schematics and experimental timeline of chemogenetic treatment of the mouse cortex. B, Representative confocal images and quantification of c-Fos immunohistochemistry in the cortex of mice receiving DREADD plus CNO treatment. Yellow arrows indicate cells expressing HA–hM3D (red; HA was used as a tag for detecting DREADDs). Nissl stain (blue) highlights neuronal cell bodies. n = 50 cells compiled from three animals in each ipsilateral and contralateral side groups. ***p < 0.001. C, Confocal image of NeuN and DAPI stains in the infected region. D, Relative proportion of the various infected cell types in layer IV–VI of the ipsilateral hemisphere. E, Mice that had received unilateral hM4D plus CNO treatment did not demonstrate differences in cell or neuron number, n = 6 mice. F, No difference in nuclear or soma size was detected between mice that had received hM4D plus CNO treatment and controls. n = 6 mice, 150 cells in each group. G, Image of Iba1 labeling showing no changes in the normal distribution or morphology of microglia in ipsilateral and contralateral sites after hM4D plus CNO treatment.
- Figure 2.
Inhibitory and excitatory DREADDs have opposing effects on fibrillar amyloid deposition. A–D, Confocal images of the ipsilateral and contralateral hemispheres in 5XFAD mice treated with hM3D (A, B) or hM4D (C, D). B and D show zoomed images of amyloid plaques (cyan; labeled by thioflavin S) and associated neuritic dystrophy (magenta; labeled by anti-Lamp1 antibody) in the infected cortical region and corresponding region in contralateral hemisphere (red and green dashed boxes). Yellow arrows point to small plaques as determined by the area of thioflavin S labeling. E, Quantification of plaque number (ipsilateral/contralateral ratios) in mice receiving different treatment combinations. F, Quantification of plaque number (ipsilateral/contralateral ratios) in infected cortical regions of hM4D-treated mice receiving CNO at varying frequencies. G, Quantification of plaque number (ipsilateral/contralateral ratios classified by plaque size) in infected cortical regions of 5XFAD mice. Last bar (with pattern) shows plaque number in ipsilateral and contralateral regions labeled before hM4D plus CNO treatment, demonstrating no plaque removal over the treatment interval. n = 6 mice for each group. H, Two months hM4D plus CNO treatment in 4-month-old PS/APP mice reduced amyloid deposition in the infected cortical regions. Neither CNO administration nor AAV-hM4D transfection alone produced significant changes in amyloid deposition. n = 3 mice for each group. I, Quantification of plaque number (ipsilateral/contralateral ratios classified by plaque size) in infected cortical regions of PS/APP mice. In all analyses (E–I), the number of plaques counted in each mouse were 150–300. Sample size (n) represents animal number. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 3.
Inhibitory DREADDs markedly reduce diffuse amyloid deposits. A, Confocal images of amyloid plaques, double labeled by anti-Aβ antibody (red) and thioflavin S (cyan), in the ipsilateral and contralateral hemispheres of 5XFAD mice treated with hM4D plus CNO. B, Quantification of plaque number using anti-Aβ immunohistochemistry (ipsilateral/contralateral ratios classified by plaque size) in infected cortical regions of 5XFAD mice. n = 6 mice, 150–300 plaques were counted in each animal. C, Examples of compact and diffuse plaques in 5XFAD mice. Images were maximal projections along z-axis covering the entire plaque. D, Quantification of compact and diffuse plaque number in cortical regions receiving hM4D plus CNO treatment and corresponding contralateral regions in 5XFAD mice. n = 6 mice. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 4.
hM4D treatment reduces total Aβ levels and the size of oligomeric Aβ halo around amyloid plaques. A, B, Quantification of Aβ40 and Aβ42 concentration by ELISA. n = 3 mice for each group. C, Confocal images of OC antibody staining of oligomeric amyloid (red) around fibrillar plaque cores (cyan) in ipsilateral and contralateral hemispheres of the same mouse. D, Quantification of the area of the OC+ amyloid halo showed reduced size in the ipsilateral treated hemisphere. n = 6 mice for each group, 50 plaques from each mouse were measured. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 5.
hM3D and hM4D chemogenetic treatment has opposite effects on the degree of synaptic pathology around amyloid plaques. A, Images of synaptic puncta (white; labeled by anti-PSD95 antibody) around amyloid plaques (cyan; labeled by thioflavin S). B, Quantification of synaptic density in mice that received hM4D treatment shows greater density of PSD95 puncta near amyloid plaques in the ipsilateral compared with contralateral hemispheres. C, Images of axonal dystrophy labeled with an antibody against Lamp1 (magenta) around amyloid plaques (cyan; labeled by thioflavin S) in ipsilateral and contralateral hemispheres of hM4D plus CNO treated mice. D, Quantification of axonal dystrophy area in mice that received hM4D or hM3D treatment. n = 6 mice in the hM4D group and 4 mice in the hM3D group. Fifty plaques were imaged in each animal for all the analyses. Statistical analyses were done using animal number as n. E, F, Statistical analyses of data in B and D using plaque number as n are consistent with analysis by animal number. G, Quantification of area of dystrophic neurites labeled using APP immunohistochemistry in mice that received hM4D plus CNO treatment. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 6.
DREADD treatment affects Aβ deposition in both presynaptic and postsynaptic regions. A, B, Thalamic LP nucleus, but not the adjacent posterior complex (PO) nucleus, contain axons of cortical neurons infected with inhibitory DREADDs (green). Neuronal cells were labeled with Nissl stain (red). Neuritic dystrophy around plaques were labeled by anti-Lamp1 immunostaining (magenta). C, hM4D and hM3D treatments showed opposite effects on amyloid deposition only in LP (innervated region) but not PO (adjacent uninnervated region). n = 4 mice in each group, 60–120 plaques were counted in each region. D, Quantification of c-Fos immunostaining in LP neurons. n = 3 mice, 50 neurons from each animal. E–G, CA1 stratum regions, but not the adjacent dentate gyrus (DG), contain the dendrites of AAV–hM4D-infected CA1 neurons. Chemogenetic manipulation reduced amyloid deposition in the somatodendritic field of CA1 neurons but not the adjacent DG region (upstream polysynaptic connection from CA1). n = 4 mice in each group, 20–50 plaques were counted in each region. **p < 0.01.
- Figure 7.
DREADDs likely modulate APP processing and Aβ secretion. A, Quantitative immunohistochemistry analyses for APP, NeuN, BACE1, NEP, and IDE proteins in DREADD-treated mice. APP was tested with two antibodies recognizing C- and N-terminal sequences. n = 50 cells from 3 mice for each group. B, Confocal images showing a greater degree of APP immunolabeling in the cell body of hM4D infected neurons (arrow; quantified in A). ***p < 0.001.
