Figure 1.
Representative images and size determination of the SDN-POA.
A1: anatomical localization of the SDN-POA as delineated by CB28 immunoreactivity. Cm = cerebrum; Ce = cerebellum; 3V = 3rd ventricle; OX = optic chiasm. A2: Graphic SDN-POA volumes (n = 6/sex/age, Mean±SE). #p<0.01 vs. weanling male. *p<0.001 vs. age-matched females. Panels B1, C1, D1, E1 illustrate triple-labeling images which include the bilateral preoptic area of the hypothalamus from male and female rats at postnatal day (PND) 21 and PND 110. Panels B2, C2, D2, E2 highlight the SDN-POA as was indicated in the white dotted area in panels B1, C1, D1, E1. B1&2: PND 21 male; C1&2: PND 21 female; D1&2: PND 110 male; E1&2: PND 110 female. Green fluorescence indicates calbindin D28K (CB28)-positive neurons, red fluorescence indicates NeuN-immunoreactivity, and blue (DAPI) fluorescence delineates cellular nuclei.
Figure 2.
Representative images showing nestin- or Ki67-immunoreactivity in the preoptic area of the hypothalamus.
Panel A displays the nestin-delineated subventricular zone (SVZ), which served as an internal positive control, while panel B shows the Ki67-labeled SVZ. Red fluorescence indicates nestin- or Ki67-immunoreactivity and blue (DAPI) fluorescence delineates cell nuclei. Insets in panels A and B are magnified images acquired at the locations indicated by the white dotted square. Panels C and E demonstrate the bilateral preoptic area of the hypothalamus labeled by nestin-immunoreactivity and DAPI from two PND 21 male animals. Images displayed under higher magnification in panels D and F were acquired at the locations indicated by the white dotted areas in Panels C and E. Arrows indicate non-vascular cells that expressed nestin. Nestin-immunoreactivity delineated the microvasculature well.
Figure 3.
Mapping the 3rd Ventricle Stem Cell Niche, weanling vs. adult.
Panel A displays the nestin-delineated subventricular zone (SVZ), which served as an internal positive control. Images in series B were acquired from a PND21 male rat and images in series C were taken from an adult male (PND110) rat, demonstrating the nestin-positive region/cells (red label) that extended into the parenchyma of the hypothalamus (∼0.1 mm or more from the ventricular wall) at the rostral end of the 3rd ventricle, namely the 3rd ventricle stem cell niche (3VSCN). Distance between the 2 adjacent brain slices is 90 µm. SVZ, subventricular zone; OC, optic chiazma 3V, the 3rd ventricle; blue labelings, DAPI-staining.
Figure 4.
Reconstruction of 3-dimensional images of the 3VSCN.
The sequential images demonstrated in Fig. 3 were employed to reconstruct 3-dimensional images of the 3VSCN using the Stereo Investigation software from MBF Bioscience. 3VSCN, the 3rd ventricular stem cell niche; PND, postnatal day; 3V, the 3rd ventricle.
Figure 5.
Representative images of the triple labeling of calbindin D28k (CB28), Ki67, and DAPI.
Four sets of sequential images are displayed defining the SDN-POA (CB28-delineated, green fluorescence) and proliferative cells (Ki67-labeled, red fluorescence) in the hypothalamic preoptic area. The blue (DAPI) fluorescence delineates cell nuclei. The four series of images are from a PND 21 male (A1–D1), PND 21 female (A2–D2), PND 110 male (A3–D3), and PND 110 female (A4–D4). A to C images are aligned in a direction from rostral to caudal with 90 µm between adjacent slices along the longitudinal axis. Images displayed under higher magnification in D were acquired at the locations indicated by the white dotted area in their corresponding series.
Figure 6.
Representative images showing the morphology of dividing cells.
A: This relatively low power image illustrates the CB28-immunoreactivity (green fluorescence) delineation of a unilateral adult male SDN-POA within which the red fluorescence (Ki67-immunoreactivity) is located. B, C and D demonstrate the cell at the telophase of cell division defined in A: B shows a cut view; C displays images with a sequential order at 1 µm intervals along the Z axis; and D highlights the 3-dimensional view with the stacked images.
Figure 7.
Ki67-positive cell counts in the SDN-POA (top) and hypothalamus (bottom).
In the SDN-POA area, the number of Ki67-positive cells was 3.4 times higher at weaning than at adulthood in male rats (p<0.01), whereas the number in females was not statistically significantly different between the weaning group and the adult group (p>0.05). In the hypothalamus, the Ki67-positive cell number was 3.5 and 3.3 times higher respectively, in PND 21 male and female rats (p<0.001 for both) compared to same-sex PND 110 rats. #suggests age difference.