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. 2017 Sep 20;11:275. doi: 10.3389/fncel.2017.00275

Figure 2.

Figure 2

Neurons and astrocytes both swell in hypoosmolar conditions. (A) Representative MIPs of a CA1 pyramidal neuron loaded with Alexa Fluor 488 dye via patch clamp, depicting soma volume at baseline (A1), followed by 5 min in 40% hACSF (A2), and a subsequent 5 min wash period in nACSF (A3). Swelling of the soma during hACSF application is readily apparent in (A2). (B1–B3) Time points in (A1–A3) are processed and binarized (see Figure 1) to allow measurement of soma area. Images are pseudocolored green for easier comparison with (A). (C) Each thresholded image in (B) is pseudocolored magenta and overlaid with green baseline image from (B) to reveal regions of soma expansion during 40% hACSF, which recover to near basal levels upon return to nACSF (magenta highlights, indicated by white arrows). Scale bars = 5 μm. (D) Quantification of neuronal soma volume as a percent change from baseline area in 17% and 40% hACSF conditions over a single 5-min application, followed by a 5 min wash period denoted with (5). Time points 0, 5 and (5) correspond to images in (A1–A3) respectively. (E) Representative thresholded images of an astrocyte loaded with Alexa Fluor 488 dextran (10,000 MW) at baseline (E1), after 5 min in 40% hACSF (E2), and after a 5 min wash in nACSF (E3). As in (A), images in (E2,E3) have been overlaid with the baseline image to illustrate changes in cell volume (white arrows). Scale bar = 5 μm. (F) Quantification of astrocyte soma volume as a percent change from baseline area in 17% and 40% hACSF conditions. *p < 0.05 and ***p < 0.001 for 17% vs. 40% at each time point in (D,F). All time points for both 17% and 40% hACSF were significantly elevated over baseline. (G) Expanded time course showing neuron and astrocyte volume changes over multiple re-applications of 40% or 17% hACSF. Neuron volume in control conditions (“no hACSF”, black line) is included for comparison. ***p < 0.001, **p < 0.01 (both cell types) and #p < 0.05 (astrocytes only), percent change vs. 0% (baseline). N = 7–9 cells per hACSF group and six cells for control.