Fig. 1

Physiological osmotic challenge increases local hypophyseal blood flow (A) Representative image and scheme showing the hypophyseal neurovascular interface of the transgenic 6-day-old zebrafish larval Tg(oxt:EGFP;kdrl:mCherry). Oxytocin (OXT) axonal termini and blood vessels are labeled in green and red, respectively. (Right) Schema depicting the stereotypic anatomical structure of the hypophyseal artery (HyA), capillary (HyC), and vein (HyV), as well as the anterior mesencephalic central artery (AMCtA), which was used as a control. Arrows, which indicate the positions of line scans imaging through and across the vessel, were employed to acquire changes in capillary diameter and red blood cell (RBC) velocity traces. (B) Scheme showing the production of DBP-eGFP under the liver promoter, L-fabp, in the Tg(l-fabp:DBP-EGFP) transgenic line labeling the serum. (C) Snapshot of the hypophyseal capillary loop of the Tg(l-fabp:DBP-EGFP) transgenic larvae at 6 days post-fertilization (dpf). Dark shadows are created by the RBCs on a background of DBP-eGFP-labeled serum. (D and E) Transgenic Tg(oxt:EGFP) zebrafish larvae with labeled OXT neurons were incubated with hyperosmotic solution equivalent to 50% seawater (17.5 ppt) followed by in situ hybridization of the activity-dependent c-fos mRNA. Representative image of a single z plane (D) showing increased c-fos expression in OXT neurons following the osmotic challenge (OC), quantified (E) as the number of c-fos+ OXT neurons expressing cells (p < 0.0001, Mann-Whitney test, naive n = 9, OC = 10). Scale bar, 20 μm. (F–I) Representative blood flow traces in the HyC at basal conditions and following the OC (F), which led to increased mean RBC velocities in the HyC (G; p = 0.001, paired t test, n = 9) and higher variability of velocities measured by spread of individual velocities around the mean (H; p = 0.0039, Wilcoxon test, n = 9). Single-sided amplitude spectra of the velocities (I) indicate an increase in amplitudes for frequencies less than 2 Hz following the OC. (J–M) OC induced no changes to the blood flow traces (J), mean velocity (K; p = 0.2296, paired t test, n = 7), variability in velocities (L; p = 0.2669, paired t test, n = 7), or any changes in the spectra (M) in the AMCtA, which served as a control for the specific effect of the OC. Data are represented as mean ± SEM.