9 research outputs found
Expression of Vesicular Glutamate Transporter 2 (vGluT2) on Large Dense-Core Vesicles within GnRH Neuroterminals of Aging Female Rats
<div><p>The pulsatile release of GnRH is crucial for normal reproductive physiology across the life cycle, a process that is regulated by hypothalamic neurotransmitters. GnRH terminals co-express the vesicular glutamate transporter 2 (vGluT2) as a marker of a glutamatergic phenotype. The current study sought to elucidate the relationship between glutamate and GnRH nerve terminals in the median eminence—the site of GnRH release into the portal capillary vasculature. We also determined whether this co-expression may change during reproductive senescence, and if steroid hormones, which affect responsiveness of GnRH neurons to glutamate, may alter the co-expression pattern. Female Sprague-Dawley rats were ovariectomized at young adult, middle-aged and old ages (~4, 11, and 22 months, respectively) and treated four weeks later with sequential vehicle + vehicle (VEH + VEH), estradiol + vehicle (E<sub>2</sub> + VEH), or estradiol + progesterone (E<sub>2</sub>+P<sub>4</sub>). Rats were perfused 24 hours after the second hormone treatment. Confocal microscopy was used to determine colocalization of GnRH and vGluT2 immunofluorescence in the median eminence. Post-embedding immunogold labeling of GnRH and vGluT2, and a serial electron microscopy (EM) technique were used to determine the cellular interaction between GnRH terminals and glutamate signaling. Confocal analysis showed that GnRH and vGluT2 immunofluorescent puncta were extensively colocalized in the median eminence and that their density declined with age but was unaffected by short-term hormone treatment. EM results showed that vGluT2 immunoreactivity was extensively associated with large dense-core vesicles, suggesting a unique glutamatergic signaling pathway in GnRH terminals. Our results provide novel subcellular information about the intimate relationship between GnRH terminals and glutamate in the median eminence.</p></div
Confocal microscopic images show GnRH and vGluT2 co-localization in the lateral median eminence (ME).
<p>Images were scanned from a single plane of a representative middle-aged vehicle treated rat. (A) GnRH processes were labeled with FITC (green color) and are seen along the portal capillary region (Cap) in the caudal median eminence. (B) Texas Red signals (red color) representing vGluT2 were clearly seen in the pericapillary area with a similar pattern to that of GnRH. (C) A merged image showed colocalization of GnRH and vGluT2 fluorescent signal. (D) A region from panel C (framed) is shown at higher magnification. Some punctate structures were single labeled by vGluT2 (arrow head); however, considerable overlapping of GnRH and vGluT2 signals were seen in yellow (arrows). Scale bar (shown in panel C, applies to panels A-C) = 20 μm, scale bar D = 5 μm. Cap = portal capillaries,</p
Serial sections show subcellular distribution of vGluT2.
<p>Images were taken from a representative middle-aged vehicle treated female rat. (A) A low magnification electron microscopy image was pseudocolored to show neuroterminals with (light yellow) and without (orange) large dense-core vesicles, a convoluted basal lamina covered portal capillary area—typical in older rats (pink), and tanycytic elements (green). (B) A higher magnification image of panel A was shown. This section was used as a negative control, in which omitting primary vGluT2 antibody resulted in no immunogold labels in the terminal. Two neurosecretory terminals containing large dense-core vesicles in direct contact with the basal lamina (BL with arrow) of the portal capillary region were highlighted in light yellow, and surrounding neuronal elements without large secretory vesicles were highlighted in orange. Groups of small vesicles in neurosecretory terminals and surrounding neuronal elements were pseudocolored in blue. A cluster of small vesicles was distributed in the central portion of the lower left terminal. Another cluster of irregular shaped small vesicles was shown to the left. (C, D) Serial sections adjacent to panel B were used to label vGluT2 protein. Immunogold labels for vGluT2 were seen on large dense-core vesicles (arrow). Whether small vesicles (arrowhead) contained vGluT2 immunosignal was difficult to determine, even at higher magnification. (E, F) By examining adjacent sections, it could be seen that immunogold labels of vGluT2 were present in most dense-core vesicles. As indicated by double arrows, some large dense-core vesicles in image E showed no immunogold labels, while the same vesicle in the adjacent image F was immunolabeled. This is likely to be the case because the antibody reaction site on large dense-core vesicle (about 120 nm diameter) might only be captured in sections (each about 50 nm thick) in which antigen is exposed. BL (panel B) = basal lamina. Scale bar panel A = 2 μm, B-D = 500 nm, E-F = 200 nm.</p
Quantification of the density of GnRH, vGluT2 and co-expressed puncta in the pericapillary area of the median eminence of young, middle-aged, and old rats, treated with vehicle or hormones.
<p>(A) The number of GnRH puncta per 40 μm<sup>3</sup> were calculated and showed a decrease with age from the young to the old group (P < 0.05). (B) The density of vGluT2 puncta decreased with age from the young to the MA group (P < 0.05). (C) GnRH-vGluT2 colocalized puncta did not change significantly with age although a trend was found (P = 0.054). N = 8 rats per group.</p
3D reconstruction of electron microscopy image showed immunogold labels of vGluT2 associated with large dense-core vesicles in a neuroterminal of a representative middle-aged VEH treated rat.
<p>(A) Lower magnification electron microscopy image was taken from the portal capillary zone of the lateral median eminence. A vGluT2 immunolabeled neuroterminal (Term, pseudocolored in light orange) was separated from the portal capillary area (pseudocolored in pink) by tanycyte endfeet (Tan, pseudocolored in green). (B) An adjacent section showed the same vGluT2 immunopositive neuroterminal from panel A in higher magnification. The 10 nm immunogold labels for vGluT2 were seen on large dense-core vesicles (arrows). (C) The terminal shown in panel A and B was 3D reconstructed from 13 images taken from a serial section ribbon. Reconstruction showed this terminal was covered by tanycyte endfeet (green). The majority of the vesicles in this terminal were large dense-core vesicles (yellow). Immunogold beads labeled for vGluT2 were individually marked in black dots and showed extensive association to large dense-core vesicles. A large mitochondria (purple) was located in the center of the terminal. BL = basal lamina, Mit = mitochondria, Tan = Tanycyte, Term = Neuroterminal. Scale bar panel A = 100 μm, B = 500 nm, C = 200 nm.</p
Electron microscopy images show the subcellular distribution of immunoreactive vGluT2 in neural profile of a representative young VEH treated rat.
<p>Images were taken from the neuroprofile zone of the lateral median eminence, a region in which neurosecretory axons and GnRH dendrons are located. (A) Three puncta were framed and shown in higher magnification in panels B, C, and D. (B—D) The 10 nm immunogold labels for vGluT2 were seen on large dense-core vesicles (arrowheads). (E, F) Two representative adjacent sections from a series show consistent immunogold labeling of vGluT2 on large dense-core vesicles (white and black arrowheads). Glia = glial processes, Mit = mitochondria. Scale bar panel A = 2 μm, panel F (applies to B–F) = 200 nm.</p
Serum hormones and physiological parameters were measured to evaluate treatment outcomes.
<p>Serum estradiol levels were assayed in a subset of rats in each group (N = 3–7 per group). Other parameters were measured in all rats (N = 8 per group). (A) Serum estradiol levels at all ages were significantly elevated by E<sub>2</sub> in both E<sub>2</sub> + VEH (P < 0.0001) and E<sub>2</sub> + P<sub>4</sub> (P < 0.0001) groups, compared to VEH + VEH treatment. (B) Serum progesterone concentrations were significantly altered by hormone treatment (F = 3.91, P < 0.05) with the E<sub>2</sub> + P<sub>4</sub> group higher than VEH + VEH group. (C) The diameter of the uterine horn was significantly altered by hormone treatment (F = 114.9, P < 0.0001) with both E<sub>2</sub> +VEH (P < 0.001) and E<sub>2</sub> + P<sub>4</sub> (P < 0.001) higher than VEH + VEH group. (D) Pituitary weight index showed a main effect of age (F = 3.47, P < 0.05) and hormone (F = 4.67, P < 0.05) with E<sub>2</sub> +VEH higher than VEH + VEH group. (E) Body weight was significantly altered by age (F = 4.06, P < 0.05), with the young group lighter than old group (P < 0.05). There was a significant treatment effect on body weight (F = 3.235, P < 0.05), with significantly lower weight in the E<sub>2</sub> + P<sub>4</sub> group (p < 0.05), and a trend for lower weight in the E<sub>2</sub> + VEH group (p = 0.08) compared to VEH + VEH control group. (F) Statistical results were summarized to show main effects of age, treatment, and age x treatment interactions. n.s. = not significant. Abbreviations: MA: middle-aged, E<sub>2</sub>: estradiol; P<sub>4</sub>: progesterone; VEH: vehicle treatment.</p