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Fig. 3 | Biological Procedures Online

Fig. 3

From: A 3D human co-culture to model neuron-astrocyte interactions in tauopathies

Fig. 3

Synapse formation and neuronal activity. Synapse formation (A-C) and calcium activity (D-F) was assessed in 4-week old 3D human neuron/astrocyte co-cultures A 12 µm-thick maximum intensity confocal image projection of axons, pre- and postsynapses in a 3D co-culture. Immunostaining was performed for axons (NF200, blue), pre- (SYP1, green) and postsynapses (PSD95, magenta). Single channels are shown in greyscale and zooms of the numbered boxed areas, indicated in the NF200 panel, are shown as merged. Intensity profiles of SYP1 (green) and PSD95 (magenta) were determined along the line segment indicated in merged zoom 3 to show co-localization of pre- and postsynaptic compartments. B Transmission electron micrograph of a pre- and postsynapse (pseudocolour in green and red, respectively). The boxed area indicated in the pseudocoloured image is shown as a greyscale image. C Transmission electron micrograph of an astrocyte (blue) in contact with a pre- and postsynaptic compartment (pseudocolour in green and red, respectively). Note that the astrocyte contains electron-dense glycogen granules. The boxed area indicated in the pseudocoloured image is shown as a greyscale image. A 4-week old 3D human neuron/astrocyte co-culture D, F was loaded with Fluo5-AM for calcium imaging and E subsequently labeled with β-3-tubulin and GFAP to assess the neuronal/astrocytic source of the calcium signal. D A 10-min maximum fluorescence intensity projection of Fluo5-AM. The heatmap corresponds to the calcium concentration. See Supplementary Video 2 for calcium signal over time. E After calcium imaging, immunofluorescent staining was performed for neurons (β-3-tubulin, green) and astrocytes (GFAP, magenta), and nuclei were visualized using DAPI (greyscale). F Calcium bursting in one cell indicated by the boxed area in D at three different time points (t = 16, 20 and 24 s) and the corresponding immunofluorescent image of the boxed area in E. The graph shows the Fluo5-AM fluorescence intensity of this cell (cell 2 in Supplementary Fig. 1A-C) over 1 min, with the lowest and highest value set to 0 and 1 respectively, and the blue box corresponds to the timeframe between t = 16 and 24 s for which the bursting activity is shown. Please note that no calcium signal was observed in the astrocyte. See Supplementary Fig. 1 for the neuronal/astrocytic annotation of calcium traces

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