Images and Movies:
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Images of research interests
These images highlight research interests within my laboratory. Below you'll find images and general descriptions of:
Seeing is believing...isn't it? We rely on confocal microscopy for our research, but confocal microscopy is only as good as the microscopist. To learn more about the pitfalls of confocal microscopy, check out this QuickTime movie. It depicts how poorly-performed confocal microscopy can give misleading results.
Figure legend: Simplified schematic of the mammalian cell cycle (center) surrounded by images depicting each developmental stage leading to SGZ neurogenesis. Cell cycle (center schematic): The length of the cell cycle in the SGZ of the young adult male rat is about 25 hours, with 9.5 hours consisting of the DNA synthesis, or S, phase, 4.5 hours consisting of G2 and mitosis (M), and the remainder consisting of G1 phase (Cameron and McKay, 2001). Systemic injection of BrdU or [3H]thymidine leads to incorporation of these mitotic markers into the DNA of cells in S Phase (note red in newly synthesized strand of DNA). This cell cycle and the surrounding images emphasize the continuum between the time points of proliferation or cytogenesis (left column), differentiation and migration (center column) and survival or neurogenesis (right column). Proliferation or cytogenesis (left column): Two hours after injection of BrdU, cells in the SGZ appear on the border of the granule cell layer (GCL), and are small and irregularly-shaped. BrdU-immunoreactive cells will often appear in dense clusters. A cluster is defined by any BrdU-immunoreactive cells that make contact. The top left panel is a light microscopic image depicting two BrdU-positive clusters (arrows). When examined at high magnification (X100) with continual scanning through the Z-plane of section, these clusters can be identified as containing eleven (top cluster) and three (bottom cluster) individual cells. The bottom panels depict confocal microscope images of a triple immunohistochemical stain of the SGZ from a rat given the mitotic marker BrdU 2 hours earlier. Cells are labeled for BrdU (green), the neuronal marker NeuN (red) and the glial marker GFAP (blue). Arrowheads indicate cells double-labeled with BrdU and GFAP, but not with NeuN. Merged images of the three labels (last panel) show that all BrdU-positive cells in the SGZ are GFAP-positive (blue-green cells) and NeuN-negative. Note that in contrast to the neuronal morphology of NeuN-labeled cells in the GCL, BrdU-labeled cells in the SGZ are small, clustered, and irregularly shaped. Differentiation, migration (center images): About four or more hours after injection of BrdU, some cells labeled with BrdU will have reached M phase and will divide. Some of these cells will reenter the cell cycle (or Go, not depicted); others will exit the cell cycle and begin to express markers of postmitotic, migrating, or differentiating cells. Two such markers are shown here. The center left panel is a confocal microscope image of the SGZ depicting staining for Hu (green) and nissl substance (red). Hu is a member of a family of RNA binding proteins shown to be expressed in neurons around the time of exit from the cell cycle (Marusich et al., 1994). The center right panel is a confocal microscope image of the SGZ depicting class III b-tubulin immunoreactivity (green). Class III b-tubulin is a cytoskeletal protein expressed in both postmitotic cells that may become neurons and in mature neurons. Hu and class III b-tubulin are markers of differentiating and migrating cells, but the fate of these maturing cells is considered malleable at least until the cells express markers of mature neurons, such as calbindin or NeuN. Survival or neurogenesis (right column): Two to four weeks after injection of BrdU, some BrdU-immunoreactive cells have migrated into the GCL and have achieved a neuronal morphology. The top right panel is a light microscopic image depicting four BrdU-positive cells (arrowheads). These four cells are round, about 10 mm in diameter, and solitary. Note that some surviving BrdU-immunoreactive cells are solid (black arrowhead) while others are speckled (blue arrowhead). Due to the depth of penetration of the BrdU antibody and narrow focal plane at this magnification, an additional speckled cell is out of focus (light blue arrowhead). The heterogeneous labeling reflects either time spent in S Phase or number of cycles of division, e.g. speckled cells were later in S-phase relative to solid cells when BrdU was injected, or speckled cells have gone through more cycles of division than solid cells (Miller and Nowakowski, 1988). The bottom panels depict confocal microscope images of a triple immunohistochemical stain of the subgranular zone from a rat given BrdU 4 weeks earlier. Cells are labeled for BrdU (green), the neuronal marker NeuN (red) and the glial marker GFAP (blue). Arrows indicate a cell double-labeled with BrdU and NeuN but not with GFAP. Merged images of the three labels (last panel) show that all BrdU-positive cells in the granule cell layer are NeuN-positive (yellow cell) and GFAP-negative. Note that in contrast to the neuronal morphology of BrdU-labeled cell in the granule cell layer, BrdU-labeled cells in the hilus are small and irregularly shaped. From (Eisch et al., 2000). All confocal images shown here were collected on a confocal microscope at X65 magnification using multitrack scanning. The images presented are taken from a 1 mm optical section through the Z plane of focus. GCL, granule cell layer; H, hilus.
Use of endogenous cell cycle markers to characterize dividing clusters in the hippocampal SGZ