The annual reproductive cycle in sheep may reflect a functional remodeling within the GnRH system. Specifically, changes in total synaptic input and association with the polysialylated form of neural cell adhesion molecule have been observed. Whether seasonal changes in a specific subset(s) of GnRH inputs occur or whether glial cells specifically play a role in this remodeling is not clear. We therefore examined GnRH neurons of breeding season (BS) and nonbreeding season (anestrus) ewes and tested the hypotheses that specific (i.e. gamma-aminobutyric acid, catecholamine, neuropeptide Y, or beta-endorphin) inputs to GnRH neurons change seasonally, and concomitant with any changes in neural inputs is a change in glial apposition. Using triple-label immunofluorescent visualization of GnRH, glial acidic fibrillary protein and neuromodulator/neural terminal markers combined with confocal microscopy and optical sectioning techniques, we confirmed that total numbers of neural inputs to GnRH neurons vary with season and demonstrated that specific inputs contribute to these overall changes. Specifically, neuropeptide Y and gamma-aminobutyric acid inputs to GnRH neurons increased during BS and beta-endorphin inputs were greater during either anestrus (GnRH somas) or BS (GnRH dendrites). Associated with the changes in GnRH inputs were seasonal changes in glial apposition, glial acidic fibrillary protein density, and the thickness of glial fibrils. These findings are interpreted to suggest an increase in net stimulatory inputs to GnRH neurons during the BS contributes to the seasonal changes in GnRH neurosecretion and that this increased innervation is perhaps stabilized by glial processes.
To determine whether profiles of decreasing concentration were generated among hepatocytes of the liver acinus during the transport of sulfobromophthalein sodium (BSP), rat livers were perfused with various concentrations of this dye (10 microM to 1 mM) in the presence and absence of albumin. After steady-state conditions for the biliary secretion of BSP had been attained, pieces of liver were rapidly frozen. Following the alkalinization of cryostat-cut sections, the relative concentration of BSP in hepatocytes of each zone and the effect of albumin on this localization were quantitated by microspectrophotometry. The results showed that BSP, perfused in the absence of albumin, was efficiently extracted by the liver (95% on a single pass), generating distinct profiles of decreasing cellular concentration from zone 1 to zone 3 at every concentration of BSP. However, the addition of albumin to the perfusate greatly reduced the extraction of BSP from the sinusoidal compartment and resulted in the abolition of the differences in BSP content between hepatocytes of zone 1 and zone 3. These results represent a direct demonstration that, as predicted by mathematical modeling, binding of BSP to albumin indeed results in a more homogeneous distribution of BSP within the liver acinus. A simple and direct microspectrophotometric method is therefore available to follow the changes in the relative concentration of BSP among the hepatocytes of the various acinar zones.
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