Fig. 3

In situ illustration of neuron-secreted exosomes internalized into astrocytes. Representative confocal images (a) and quantification (b) of the number and size of CD63-GFP⁺ puncta in cortical slices of CaMKII-CreER⁺CD63-GFP^f/+ mice. Neurons were patched and filled with Alexa-568 dye Subpanels i: Alexa-568 dye filled neuron; ii: CD63-GFP fluorescence: iii: merge of Alexa-568 dye filled neuron and CD63-GFP; iv: magnified view of iii. A single dose of 4-OHT (10 mg/kg) was given to P15-16 mice. Scale bar: 50 μm (i–iii); 15 μm (enlarged view, iv); n = 24 images/4 mice; c Representative images of induced CD63-GFP⁺ puncta in proximal and distal sections to the injection site. Subpanels i, iii: CD63-GFP fluorescence; ii, iv: mCherry expression from AAV8-CaMKII-Cre_mCherry virus; The virus injection site and brain sections analyzed are illustrated in the diagram. Red star: distal sections; blue star: proximal sections; Scale bar: 40 μm; d Representative confocal images of the internalization of neuron-secreted exosomes (CD63-GFP⁺ puncta) into GFAP⁺ astrocytes in AAV8-CaMKII-Cre_mCherry virus injected CD63-GFP^f/+ mice. Subpanels i: CD63-GFP fluorescence; ii: GFAP immunostaining: iii: merge of CD63-GFP and GFAP staining; iv: magnified view of iii; Scale bar: 50 μm (i–iv); e Quantification of the size and number of CD63-GFP⁺ puncta that overlap with GFAP⁺ astrocytes. n = 24 images/4 mice; f Diagram of peripheral sciatic nerve injections of AAV9-CaMKII-Cre/FluoroGold to eaat2-tdT⁺CD63-GFP^f/+ mice. g Representative confocal and Imaris 3D images for astroglial localization of neuron-derived CD63-GFP⁺ exosomes. Subpanels i: tdT⁺ astrocyte; ii: CD63-GFP fluorescence; iii: merge of CD63-GFP and tdT fluorescence; iv: converted 3D astrocyte domain from (i); v: merge of CD63-GFP and converted 3D astrocyte domain; vi: the upper part of the transected image in (v) with 90 degree outward rotation; Scale bar: 10 μm; The dashed line indicates where the transection was. The upper part of the transected image turns 90 degree outward to show the representative CD63-GFP⁺ puncta (white arrows) inside tdT⁺ astrocytes. The data was presented in the box and whisker plot with defined elements, median (center line), upper and lower quartiles (bounds of box), and highest and lowest values (whiskers)