Source Paper
Regulation of Gonadotropin-Releasing Hormone Secretion by Kisspeptin/Dynorphin/Neurokinin B Neurons in the Arcuate Nucleus of the Mouse
Victor M. Navarro, Michelle L. Gottsch, Charles Chavkin, Hiroaki Okamura, Donald K. Clifton et al.
Journal of Neuroscience • 2009
Knockout Mouse Analysis
Objective: Evaluate LH secretion changes after ovariectomy in dynorphin and KOR knockout mice compared to controls, and determine the physiological significance of dynorphin and neurokinin B coexpression in kisspeptin neurons
This is a Knockout Mouse Analysis protocol using mouse as the model organism. The procedure involves 5 procedural steps. Extracted from a 2009 paper published in Journal of Neuroscience.
Model and subjects
mouse • Not specified in provided text • female • Not specified in provided text • Not specified in provided text
Study window
Estimated timing pending
Core workflow
In situ hybridization analysis • Evaluate estradiol inhibition of gene expression • Confirm dynorphin and NKB inhibition of LH secretion
Primary readouts
- Coexpression of Kiss1, dynorphin, NKB, NK3, and KOR genes in arcuate nucleus neurons
- Estradiol-mediated inhibition of Dyn, NKB, KOR, and NK3 expression
- Inhibitory effects of dynorphin and NKB on LH secretion
- LH secretion levels after ovariectomy in knockout versus control mice
Key equipment and reagents
Verified items
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Protocol Steps
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In situ hybridization analysis
Perform in situ hybridization to identify coexpression of Kiss1, dynorphin (Dyn), neurokinin B (NKB), NK3 receptor, and KOR genes in arcuate nucleus neurons of female mice
Note: Analysis conducted on arcuate nucleus (Arc) tissue from female mice
View evidence from paper
“Using in situ hybridization, we found that Kiss1 neurons in the Arc of female mice not only express the Dyn and NKB genes but also the NKB receptor gene (NK3) and the Dyn receptor [the κ opioid receptor (KOR)] gene”
Evaluate estradiol inhibition of gene expression
Assess whether expression of Dyn, NKB, KOR, and NK3 in the arcuate nucleus are inhibited by estradiol
Note: Confirm that inhibition pattern matches that established for Kiss1
View evidence from paper
“We also found that expression of the Dyn, NKB, KOR, and NK3 in the Arc are inhibited by estradiol, as has been established for Kiss1”
Confirm dynorphin and NKB inhibition of LH secretion
Confirm that dynorphin and neurokinin B inhibit luteinizing hormone secretion
Note: Not specified in provided text
View evidence from paper
“confirmed that Dyn and NKB inhibit LH secretion”
Ovariectomy procedure
Perform ovariectomy on dynorphin knockout mice, KOR knockout mice, and control mice
Note: Surgical procedure to remove ovaries
View evidence from paper
“using Dyn and KOR knock-out mice, we found that long-term disruption of Dyn/KOR signaling compromises the rise of LH after ovariectomy”
Measure LH secretion post-ovariectomy
Measure luteinizing hormone secretion in dynorphin knockout, KOR knockout, and control mice following ovariectomy
Note: Compare LH rise between knockout and control groups
View evidence from paper
“we found that long-term disruption of Dyn/KOR signaling compromises the rise of LH after ovariectomy”