Intracerebroventricular Leptin Administration
Objective: To investigate whether leptin exerts effects on food intake in normal animals by administering leptin intracerebroventricularly and measuring changes in food consumption and hypothalamic neuropeptide expression
This is a Intracerebroventricular Leptin Administration protocol using rat as the model organism. The procedure involves 7 procedural steps, 2 equipment items, 2 materials. Extracted from a 1996 paper published in Journal of Clinical Investigation.
Model and subjects
rat • Long-Evans
Study window
~4 hour study window | ~80 hours hands-on
Core workflow
Leptin administration at onset of nocturnal feeding • Measure food intake at 1 hour • Measure food intake at 4 hours
Primary readouts
- Food intake at 1 hour post-injection
- Food intake at 4 hours post-injection
- Neuropeptide Y (NPY) mRNA expression levels in arcuate nucleus
- Corticotrophin releasing hormone (CRH) mRNA expression levels in paraventricular nucleus
Key equipment and reagents
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Protocol Steps
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Leptin administration at onset of nocturnal feeding
Administer leptin intracerebroventricularly to Long-Evans rats at the beginning of their nocturnal feeding period
Note: Dose: 3.5 micrograms icv
View evidence from paper
“Leptin administration (3.5 microg icv) at the onset of nocturnal feeding reduced food intake”
Measure food intake at 1 hour
Quantify food intake 1 hour after leptin administration and compare to vehicle-treated controls
Note: Leptin reduced food intake by 50% at this timepoint (P < 0.05)
View evidence from paper
“reduced food intake by 50% at 1 h and by 42% at 4 h, as compared with vehicle-treated controls”
Measure food intake at 4 hours
Quantify food intake 4 hours after leptin administration and compare to vehicle-treated controls
Note: Leptin reduced food intake by 42% at this timepoint (P < 0.05)
View evidence from paper
“reduced food intake by 50% at 1 h and by 42% at 4 h, as compared with vehicle-treated controls”
Conduct 40-hour fasting period
Fast rats for 40 hours prior to leptin injection to investigate neuropeptide expression changes
Note: Two injections of leptin (3.5 microg icv) administered during this fasting period
View evidence from paper
“Two injections of leptin (3.5 microg icv) during a 40 h fast significantly decreased levels of mRNA”
Perform in situ hybridization for neuropeptide Y (NPY)
Use in situ hybridization to measure mRNA expression levels for neuropeptide Y in the arcuate nucleus
Note: Leptin decreased NPY mRNA levels by 24% in arcuate nucleus (P < 0.05 vs. vehicle controls)
View evidence from paper
“significantly decreased levels of mRNA for neuropeptide Y (NPY, which stimulates food intake) in the arcuate nucleus (-24%)”
Perform in situ hybridization for corticotrophin releasing hormone (CRH)
Use in situ hybridization to measure mRNA expression levels for corticotrophin releasing hormone in the paraventricular nucleus
Note: Leptin increased CRH mRNA levels by 38% in paraventricular nucleus (P < 0.05 vs. vehicle controls)
View evidence from paper
“increased levels of mRNA for corticotrophin releasing hormone (CRH, an inhibitor of food intake) in the paraventricular nucleus (by 38%)”
Measure leptin receptor gene expression
Use in situ hybridization with probe complementary to mRNA for all leptin receptor splice variants to determine receptor distribution in rat brain
Note: Leptin receptor mRNA was densely concentrated in arcuate nucleus with lower levels in ventromedial and dorsomedial hypothalamic nuclei
View evidence from paper
“we measured leptin receptor gene expression in rat brain by ISH using a probe complementary to mRNA for all leptin receptor splice variants”