Components of the basal lamina and dystrophin-dystroglycan complex in the neurointermediate lobe of rat pituitary gland: different localizations of beta-dystroglycan, dystrobrevins, alpha1-syntrophin, and aquaporin-4

Abstract

The so-called neurointermediate lobe is composed of the intermediate and neural lobes of the pituitary. The present immunohistochemical study investigated components of the basal lamina (laminin, agrin, and perlecan), the dystrophin-dystroglycan complex (dystrophin, beta-dystroglycan, alpha1-dystrobrevin, beta-dystrobrevin, utrophin, and alpha1-syntrophin), and the aquaporins (aquaporin-4 and -9). Glia markers (GFAP, S100, and glutamine synthetase) and components of connective tissue (collagen type I and fibronectin) were also labeled. In the neurohypophysis, immunostaining of basal lamina delineated meningeal invaginations. In these invaginations, vessels were seen to penetrate the organ without submerging into its parenchyma. On the parenchymal side of the invaginations, beta-dystroglycan was detected, whereas utrophin was detected in the walls of vessels. Immunostaining of alpha1-dystrobrevin and alpha1-syntrophin did not delineate the vessels. The cells of the intermediate lobe were fully immunoreactive to alpha1-dystrobrevin and alpha1-syntrophin, whereas components of the basal lamina delineated the contours of the cells. GFAP-immunoreactive processes surrounded them. Aquaporin-4 localized at the periphery of the neurohypophysis, mainly adjacent to the intermediate lobe but not along the vessels. It colocalized only partially with GFAP and not at all with alpha1-syntrophin. Aquaporin-9 was not detected. These results emphasize the possibility that the components of the dystrophin-dystroglycan complex localize differently and raise the question about the roles of dystrobrevins, alpha1-syntrophin, and aquaporin-4 in the functions of the intermediate and neural lobes, respectively.

Figure 1

Distribution of laminin and dystroglycan in the neurointermediate lobe. (A,B) General views of the neurointermediate lobe following laminin and β-dystroglycan immunostaining, respectively. Note the “empty” intermediate lobe and the border zone (arrowheads) between the neural and intermediate lobes. A part of the anterior lobe is also visible. Asterisk indicates the cleft remaining from Rathke's pouch. (C) At this magnification, the double contours of the “septa” (arrowheads) in the neural lobe are clearly visible. Laminin immunostaining. (D) β-Dystroglycan (arrows, red) localizes mainly on the outer “parenchymal” side of the basal laminae shown in C (arrowheads, green, laminin immunostaining), i.e., on the side of the neurohypophyseal cells and axons. Inset shows enlarged detail. The scattered immunoreactive, green punctae in the background are artifacts. (E–G) Utrophin immunostaining (red) visualizes vessels (arrowheads) inside the laminin “septa” (arrows, green). AL, anterior lobe; Dg, dystroglycan; IL, intermediate lobe; Lam, laminin; NL, neural lobe; Utr, utrophin. Bars: A,B = 100 μm; C–G = 50 μm; inset in D = 25 μm.

Figure 2

Basal lamina and dystroglycan–dystrophin complex in the neurohypophysis; relationship to cells. (A) Agrin (red) colocalizes (arrows, yellow) with laminin (green) along the “septa.” (B) Perlecan (red) colocalizes (arrows, yellow) with laminin (green) along the “septa” but also forms a finer mesh (arrowheads, red) within the holes of the laminin network. (C) S100 (green) and β-dystroglycan (red). S100-immunopositive cells (arrows) seem to form the β-dystroglycan layer. (D) Dystrophin (red) around S100-immunopositive cells (arrows, green). The scattered immunoreactive, green punctae in the background are artifacts. (E) Perlecan (arrowheads, red) mesh around S100-immunoreactive cells (arrows, green). Note the similar patterns in C and D. (F) Perlecan (here, green) mesh around glutamine synthetase-immunopositive cells (arrows, red). Note differences between the shapes of glutamine synthetase-positive cells (red) and those of S100-positive cells (green) in the insets. These two inset images are taken from different reactions. Agr, agrin; Lam, laminin; Per, perlecan; Gs, glutamine synthetase. Bars: A–F and insets in F = 50 μm; inset in C = 25 μm.

Figure 3

Relationship of axons to the basal lamina components and cells. (A) The double-walled septa (arrows) formed by basal lamina (here, immunoreaction to laminin, green) surround bundles of axons (arrowheads, neurofilament protein immunostaining, red). (B) S100 (green) and neurofilament (red) immunostaining. Note the close contact between pituicytes (arrows) and nerve fibers (arrowheads). Inset: nerve fibers (red) in the perlecan mesh shown in Figure 2B (perlecan is green here; this inset image is taken from another reaction). Lam, laminin; Nf, neurofilament protein. Bar = 50 μm.

Figure 4

Localization of dystrobrevins and syntrophin. (A) α1-Dystrobrevin immunostaining results in a massive labeling in the intermediate lobe. Inset shows increased detail. Cell borders are not visible; nuclei (arrows) were not labeled. No immunolabeling is visible in the neurohypophysis. Asterisk indicates the cleft remaining from Rathke's pouch. (B) α1-Syntrophin immunostaining has a similar pattern. Marks are like those in the previous panel. (C) α1-Dystrobrevin immunoreactivity (green) occurs in the intermediate lobe, whereas β-dystroglycan (red) is found mainly around the cell groups of the intermediate lobe (arrows) but also within them, between the cells (inset). The cuboidal epithelium (arrowheads), which lines the cleft remaining from Rathke's pouch (asterisk) is also labeled. (D) Immunostaining against β-dystrobrevin in neurohypophyseal cells (arrows). The scattered immunoreactive, green punctae in the background are artifacts. AL, anterior lobe; Db, dystrobrevin; Dg, dystroglycan; IL, intermediate lobe; NL, neural lobe. Bars: A–C = 100 μm; D and inset in A = 50 μm; inset in C = 65 μm.

Figure 5

Further investigations in the intermediate lobe show basal lamina and dystroglycan–dystrophin complex and their relationship to cells. (A) Utrophin immunostaining reveals scarce elements (double arrowheads) in the intermediate lobe. Note the faintly visible network (arrowheads) in the neurohypophysis. Asterisk indicates the cleft remaining from Rathke's pouch. (B) Dystrophin immunostaining, a fine network delineates cell contours (arrows = thin; double arrowheads = thicker parts). (C) S100-immunopositive cells (green, arrows point to nuclei unlabeled) in the intermediate lobe, separated by perlecan (double arrows, red). Arrowheads indicate the cuboidal epithelium. Note the faintly visible perlecan network (double arrowheads) in the neurohypophysis. (D) When double-labeling was applied, perlecan immunostaining resulted in a pattern identical with that of dystrophin (see B, same immunoreaction, different wavelengths). (E) The cuboidal epithelium (arrowheads) of the remnant of Rathke's pouch on its basal lamina (arrows). Laminin (green) and β-dystroglycan (red) immunostainings. (F) GFAP immunostaining within a cell group of the intermediate lobe: a radially oriented fiber pattern is visible (arrowheads) with endfeet on the surface (arrows). Some astrocyte-like cells (double arrows) in the neural lobe are also labeled. IL, intermediate lobe; Lam, laminin; NL, neural lobe; Per, perlecan; Utr, utrophin. Bars: A,F = 100 μm; B–E = 50 μm.

Figure 6

Aquaporin and glial structures. (A) Distribution of aquaporin-4. Note the thin labeling along the posterior surface (arrows) of the neural lobe, whereas a thick zone (double arrows) is labeled adjacent to the intermediate lobe. The cuboidal epithelium (arrowheads) of Rathke's pouch is also visible (inset a). Inset b shows enlarged detail of the zone (double arrows) behind the intermediate lobe. (B,C) Parts of the neural lobe adjacent to the intermediate lobe. Double labeling against GFAP (red) and aquaporin-4 (green). Note the astrocytes (arrowheads) and the confined areas of colocalization (arrows, yellow). (D) Neurohypophysis, border zone to the intermediate lobe. Double-labeling to β-dystroglycan (red) and aquaporin-4 (green) is shown. Here, astrocyte-like aquaporin-4-positive elements (arrows) are recognizable. (E) An area similar to that shown in the previous panel. Glutamine synthetase- (arrows, red) and aquaporin-4-immunoreactive cells (arrowheads, green). Partial colocalization is also visible (yellow). (F) The GFAP-immunoreactive radial fibers (red, see also Figure 5F) of the intermediate lobe colocalize (yellow, double arrows) with aquaporin-4 (green, arrows), at least in part. No colocalization is visible in the neural lobe. Arrowheads indicate cuboidal epithelium. (G) Aquaporin-4-immunopositive process-bearing cells (arrows, green) and the cuboidal epithelium (arrowheads, red, stained against glutamine synthetase) of the remnant of Rathke's pouch are shown. Note the endfeet-like structures (double arrows) on the surface of the intermediate lobe (see these structures also in F and Figure 5F). The scattered immunoreactive, red puncta in the background are artifacts. AQP4, aquaporin-4; Dg, dystroglycan; Gs, glutamine synthetase; IL, intermediate lobe; NL, neural lobe. Bars: A–C,F = 100 μm; D,E,G = 50 μm; insets in A = 25 μm.