Fig. 6

Hypophyseal vascular permeability is regulated by oxytocin signaling (A) Graphical representation of the permeable hypophyseal capillary visualized in the Tg(l-fabp:DBP-EGFP) larvae. (A′) Scheme depicting the hypophyseal capillary permeability assay in which fluorescent recovery after photobleaching (FRAP) of the extravasated DBP-EGFP signal the Tg(l-fabp:DBP-EGFP) reporter was measured in a region of interest (ROI) within the hypophyseal capillary loop (red circle) compared to a control ROI outside of the loop. (B) A kymograph representing a line scan across time showing the bleached and unbleached ROIs on either side of the hypophyseal capillary. The bleaching time point is marked by an arrowhead. (C) Average FRAP recovery half-time T1/2 was decreased after osmotic challenge (OC) (p < 0.0001, paired t test, n = 8). (D) Kymographs representing FRAP traces of naive (unchallenged) larva displaying vehicle-treated control larva and larva treated with 25 μM of the Tgf-β receptor inhibitor (TgfβR in.) SB431542. The bleaching time point is marked by an arrowhead. Higher-intensity images of the FRAP kymographs before and after photobleaching are shown on the right (D′). (E) The basal T1/2 values of SB431542-treated larvae were significantly higher than their vehicle-treated siblings (p < 0.0001, Dunnett’s multiple comparison, TgfβR inh.− n = 7, TgfβR inh.+ n = 7). Larvae treated with SB431542 exhibit a decrease in T1/2 values after the OC (p = 0.0002, paired t test, n = 7), as do their vehicle-treated control larvae siblings (p = 0.0012, paired t test, n = 7). (F) Mean blood flow velocity values following OC were increased in larvae that were treated with the TgfβR inh. (p < 0.0001, paired t test, n = 7) as well as their vehicle-treated control siblings (p = 0.0007, paired t test, n = 7). (G) Representative kymographs of larvae exhibiting basal vs. reduced blood flow. (H) Application of 1 mg/mL of α-bungarotoxin for 30 s immobilizes larvae without affecting blood flow velocity (basal), whereas incubation with α-bungarotoxin for 150 s reduced mean blood flow velocity values (p < 0.0001, paired t test, n = 13). (I) Reduction in blood flow velocity did not affect capillary permeability as indicated by FRAP measurements of T1/2 values (p = 0.0854, paired t test, n = 13). (J) Hypophyseal capillary permeability was monitored at 20 min intervals before and after the application of Oxtr ant. A significant increase in T1/2 is seen after 40 and 60 min of antagonist treatment (p = 0.8090, basal vs. 20 min; p = 0.0004, basal vs. 40 min; and p < 0.0001, basal vs. 60 min; Dunnett’s multiple comparison, n = 7). (K) The increment in capillary permeability, which is induced by OC (p < 0.0001, paired t test, n = 7), is blocked in larvae that were treated for 60 min with the Oxtr ant. L-368,899, showing no change in T1/2 after OC (p = 0.6147, paired t test, n = 7). In accord with (J) above, basal T1/2 values of L-368,899-treated larvae were significantly higher than their vehicle-treated siblings (p < 0.0001, Dunnett’s multiple comparison, Oxtr ant.− n = 7, Oxtr ant.+ n = 7). (L) Optogenetic activation of OXT neurons was performed using oxt:Gal;UAS:CoChR-tdTomato transgenic larvae, as described in the legend of Figure 5, followed by FRAP-based permeability measurements. Blue light stimulation of CoChR+ larvae led to a decrease in T1/2 values (p = 0.0003, paired t test, n = 7). T1/2 values were unaffected by light in CoChR− larvae (p = 0.2559, paired t test, n = 7). Data are represented as mean ± SEM.