Source Paper
Estrogen Stimulates a Transient Increase in the Number of New Neurons in the Dentate Gyrus of the Adult Female Rat
Patima Tanapat, Nicholas B. Hastings, Alison J. Reeves, Elizabeth Gould
Journal of Neuroscience • 1999
Estrous Cycle Analysis
Objective: To determine whether sex differences exist in hippocampal cell production during adulthood and whether ovarian hormones affect cell proliferation across estrous cycle stages
This is a Estrous Cycle Analysis protocol using rat as the model organism. The procedure involves 6 procedural steps, 2 equipment items, 1 materials. Extracted from a 1999 paper published in Journal of Neuroscience.
Model and subjects
rat • not specified • unknown • adult • not specified
Study window
~2 week study window
Core workflow
BrdU Administration • Estrous Cycle Stage Determination • Ovariectomy and Hormone Manipulation
Primary readouts
- Number of BrdU-labeled proliferating cells in dentate gyrus and subventricular zone
- Comparison of cell proliferation between males and females
- Cell proliferation rates across estrous cycle stages (proestrus, estrus, diestrus)
- Effects of ovariectomy and estrogen replacement on cell proliferation
Key equipment and reagents
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Protocol Steps
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BrdU Administration
Administer bromodeoxyuridine (BrdU) to label proliferating cells in adult rats
Note: BrdU is a thymidine analog that incorporates into DNA of dividing cells
View evidence from paper
“examined proliferating cells and their progeny in adult rats using the thymidine analog bromodeoxyuridine (BrdU)”
Estrous Cycle Stage Determination
Determine estrous cycle stage in female rats (proestrus, estrus, or diestrus)
Note: Proestrus is when estrogen levels are highest
View evidence from paper
“females produced more cells during proestrus (when estrogen levels are highest) compared with estrus and diestrus”
Ovariectomy and Hormone Manipulation
Perform ovariectomy on some female rats and administer estrogen replacement to examine effects on cell proliferation
Note: Ovariectomy diminished BrdU-labeled cells; effect was reversed by estrogen replacement
View evidence from paper
“ovariectomy diminished the number of BrdU-labeled cells, an effect reversed by estrogen replacement”
Immunohistochemical Staining
Perform immunohistochemistry using markers for neurons and glia to identify cell types
Note: Markers include Turned-On-After-Division 64 kDa (immature granule neurons) and calbindin (mature granule neurons)
View evidence from paper
“Many of these cells acquired neuronal characteristics, including the formation of dendrites and expression of Turned-On-After-Division 64 kDa, a marker of immature granule neurons, and the calcium-binding protein calbindin”
Stereological Analysis
Perform stereological analyses to quantify numbers of BrdU-labeled cells in dentate gyrus and subventricular zone
Note: Analysis conducted at different time points including 2 weeks after last BrdU injection
View evidence from paper
“Stereological analyses of the numbers of BrdU-labeled cells revealed that females produced more cells than males in the dentate gyrus but not in the subventricular zone”
Pyknotic Cell Examination
Examine numbers of pyknotic (degenerating) cells to assess cell death
Note: Many new cells in dentate gyrus eventually degenerate
View evidence from paper
“examination of the numbers of pyknotic cells and the numbers of BrdU-labeled cells at longer survival times revealed that many new cells in the dentate gyrus eventually degenerate”