Hyperosmotic stimulus reversibly modifies the anatomical organization of SON vessels. Freshly dissected brains of rats normally hydrated (A-B); osmotically stimulated during 6 days (C-D) and rehydrated during 6 days following 6 days of osmotic stimulation (E-F) were fixed by immersion in the fixative and the content of blood vessels was labeled by an antibody against Rat IgG. A-F: Stack confocal images (10 μm-thicks) of sections double immunostained for Rat IgG and VP + OT. Insets show the morphology of VP or OT SON neurons (A, C, E), and the organization of Rat IgG-labeled vessels in the lateral hypothalamus (B, D, F). G: Quantitative evaluation of the vessels density within the SON and the lateral hypothalamus. For each section analyzed, a 100 point grid was centered on the core of the nucleus or on the overlying lateral hypothalamus (as shown in the schematic drawing), and the vessel density was estimated by scoring vessel contacts with the bars of the grid. The data are expressed as percent of the vessel density determined in the lateral hypothalamus overlying the SON of control rats. H: Quantitative evaluation of the total amount of SON vessels. For each section analyzed the SON surface was first determined by delineating the area containing the neuronal cell bodies labeled for OT or VP (H1 shows the SON area selected from the image C). The surface occupied by vessels was then quantified on binary images of the Rat-IgG-labeled structures within this SON area (H2 shows the binary image of labeled vessels selected from the image D). All the quantifications were performed on at least 4 sections containing the middle portions of the SON (i.e. the largest SON areas), for at least 3 rats per experiment. Note that the size of individual VP or OT immunostained neurons is highly increased in the SON of stimulated rat (inset C) as compared with control (inset A) or rehydrated rat (inset E). Double asterisk: P < 0.01, Mann-Whitney test, statistically different from the control SON. ct: control, normally hydrated; IgG: type G immunoglobulin; LH: lateral hypothalamus; OC: optic chiasma; OT: oxytocin; os 6d: osmotically stimulated during 6 days; reh 6d: rehydrated during 6 days following 6 days stimulation; SON: supraoptic nucleus; VP: vasopressin. Scale bars: A-C = 100 μm. Insets A, C, D = 25 μm.