Difference between revisions of "GFA phospho-PDH and Taste Aversion"
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| − | Examine levels of | + | Examine levels of [[Phospho-Pyruvate Dehydrogenase (pPDH)]] by immunofluorescence in after intraoral conditioned taste aversion. |
Based on Yang et al. 2024 PMID 38266644 and report by Roitman lab at SSIB 2024. | Based on Yang et al. 2024 PMID 38266644 and report by Roitman lab at SSIB 2024. | ||
Employed SD-GFP-TH rats (Iacovitti et al 2014 PMID 25462571) from Taconic that express GFP under TH promoter with especially good expression in VTA. | Employed SD-GFP-TH rats (Iacovitti et al 2014 PMID 25462571) from Taconic that express GFP under TH promoter with especially good expression in VTA. | ||
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| + | For immunohistochemistry: | ||
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| + | * for pPDH: [https://www.cellsignal.com/products/primary-antibodies/phospho-pyruvate-dehydrogenase-a1-ser293-e4v9l-rabbit-mab/37115 Phospho-Pyruvate Dehydrogenase α1 (Ser293) (E4V9L) Rabbit mAb #37115] at 1:500 for immunofluoresence; BLUE secondary [https://www.jacksonimmuno.com/catalog/products/711-475-152 Jackson DyLight™ 405 AffiniPure™ Donkey Anti-Rabbit IgG (H+L) #711-475-152] [WHAT IS CONCENTRATION?] | ||
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| + | * for c-Fos: [https://www.scbt.com/p/c-fos-antibody-c-10 mouse anti-c-Fos antibody (C-10; Santa Cruz)] at a dilution of 1:1000. GET Antibody specification and secondary from Jason's work | ||
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{{Infobox experiment|align=right | {{Infobox experiment|align=right | ||
Latest revision as of 16:08, 4 February 2025
Examine levels of Phospho-Pyruvate Dehydrogenase (pPDH) by immunofluorescence in after intraoral conditioned taste aversion.
Based on Yang et al. 2024 PMID 38266644 and report by Roitman lab at SSIB 2024.
Employed SD-GFP-TH rats (Iacovitti et al 2014 PMID 25462571) from Taconic that express GFP under TH promoter with especially good expression in VTA.
For immunohistochemistry:
- for pPDH: Phospho-Pyruvate Dehydrogenase α1 (Ser293) (E4V9L) Rabbit mAb #37115 at 1:500 for immunofluoresence; BLUE secondary Jackson DyLight™ 405 AffiniPure™ Donkey Anti-Rabbit IgG (H+L) #711-475-152 [WHAT IS CONCENTRATION?]
- for c-Fos: mouse anti-c-Fos antibody (C-10; Santa Cruz) at a dilution of 1:1000. GET Antibody specification and secondary from Jason's work
| phospho-PDH and CTA | |
|---|---|
| Taste Aversion | |
| Expt Code | GFA |
| Start Date | 2024-12-3 |
| Investigators | Houpt, Cote, Trombley, Blakemore |
| Funding | Chair's Fund |
| Protocol | 202200000006 |
| Expt Type | CTA |
| Species | rat |
| Drugs | lithium |
| Data Link | https://houptlab.org/bartab/expt/?id=GFA |
Data
People
Jason Cote (breeding), Laura Blakemore and Paul Trombley (colony foundation and management)
CTA Conditioning
Agenda
Rats Arrive (December 3, 2024)
Rats (n=4 male, n=5 female, littermates, DOB: 7-???-2024) transferred from GFP-TH breeding protocol to individual cages.
Day 0: Surgery (December XXX, 2024)
Chronic Intraoral Catheters implanted under isoflurane anesthesia. All rats receive carprofen (XXX mg/kg) pre-operatively and 1 day post-op.
Days 1 - 3: Recovery (December XXXX - XXXX, 2024)
Weigh rats daily. Ad lib food and water. Carprofen (XXX mg/kg) on first day post-op.
Days 5-7: Habituation (December XXXX - XXXX, 2024)
Rats given intraoral infusion of 6 ml dH2O over 6 min to habituate to infusion procedure
Food removed on last day of habituation.
Noncontingent group: Half of rats injected with LiCl (0.15M, 12 ml/kg).
Day 8: Conditioning day 1 (December XXXX, 2024)
All rats given intraoral infusion of 6 ml sucrose 5% over 6 min.
Contingent group: Half of rats injected with LiCl (0.15M, 12 ml/kg).
Food returned overnight.
Day 9: Recovery Day (December XXXX, 2024)
Food removed.
Noncontingent group: Half of rats injected with LiCl (0.15M, 12 ml/kg).
Day 10: Conditioning day 2 (December XXXX, 2024)
All rats given intraoral infusion of 6 ml sucrose 5% over 6 min.
Contingent group: Half of rats injected with LiCl (0.15M, 12 ml/kg).
Food returned overnight.
Day 11: Recovery Day (December XXXX, 2024)
Food removed.
Noncontingent group: Half of rats injected with LiCl (0.15M, 12 ml/kg).
Day 12: Conditioning day 2 (December XXXX, 2024)
All rats given intraoral infusion of 6 ml sucrose 5% over 6 min.
Contingent group: Half of rats injected with LiCl (0.15M, 12 ml/kg).
Food returned overnight.
Day 13: Recovery Day (December XXXX, 2024)
Food removed.
Day 14: Expression Test (December XXXX, 2024)
All rats given intraoral infusion of 6 ml sucrose 5% over 6 min.
One hour after infusion, rats are overdosed with euthanasol and transcardially perfused. Brains postfixed for 1-2 hours, then stored in 0.1M PB.