Figure 4. Knockdown of aquaporin-4 in the prefrontal cortex shifts astrocyte phenotype and induces behavioral susceptibility to a sub-chronic stressor (7-days).

A) Adult male mice received infusions of either AAV5-scrambled-shRNA or AAV5-Aqp4-shRNA into the PFC. Following three weeks of recovery, animals were exposed to 7-days of sub-CUS or were left unstressed. All animals were subjected to behavioral testing (n = 11–16/group). In one cohort, mice received intravenous injections of tomato lectin, after which brains were processed for confocal microscopy (n = 6–8/group). In a separate cohort, astrocytes were isolated via FACS and astrocyte gene expression was analyzed (n = 7–8/group). B) Representative images of AAV5-Aqp4-ShRNA reporter (BFP, yellow) in the PFC. Astrocyte GFAP is shown in cyan, tomato lectin+ vessels are shown in magenta. Dotted line delineates barrier between the forceps minor and PFC. Left image: 10×, scale bar = 200 μm. Right image panel: 60×, scale bar = 20 μm. C) Normalized expression of Aqp4 in dissected PFC or SSCTX (S1J subregion). D) Total amount of distance traveled in the open field test (OFT). E) Time spent in the center during the OFT. F) Total amount of time spent exploring objects in the test phase of the temporal object recognition task (TOR). G) Discrimination index in the TOR. H) Time spent exploring both the novel and familiar arm in the T-maze task. I) Total number of alternations in the T-maze task. J) Proportion of spontaneous alternations in the T-maze task. K) Normalized astrocyte expression of marker genes (Gfap, Vim, S100b), synaptic interaction factors (Lamp1, Mertk, Megf10), and vascular interaction factors (Vegf, Bfgf, Aqp4, Angpt1, Agt) in the frontal cortex. Bars represent mean ± S.E.M. ^# p<0.05 main effect (no specific contrasts detected). * p<0.05 planned comparison indicated (Sidak’s test).