Aug 30, 2024
Preparation of pharmacological agents
- Alexandra Nelson1,
- Allison Girasole1,
- Michael Ryan1
- 1UCSF
Protocol Citation: Alexandra Nelson, Allison Girasole, Michael Ryan 2024. Preparation of pharmacological agents . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1nbzlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 30, 2024
Last Modified: August 30, 2024
Protocol Integer ID: 106762
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinsons
Grant ID: ASAP-020529
NINDS
Grant ID: R01NS101354
Abstract
This is a description of how we prepare different drugs for use in live animals (in vivo) or in brain slices (ex vivo).
This protocol provides a description of the preparation, dilution, application and administration of pharmacological agents.
This protocol provides a description of the preparation, dilution, application and administration of pharmacological agents.
6-OHDA (Sigma Aldrich) for MFB dopamine depletions was prepared at 5 μg/μL in
normal saline solution.
Levodopa (Sigma Aldrich) was administered with benserazide (Sigma Aldrich) and prepared
in normal saline solution. Levodopa (5–10 mg/kg) and benserazide (2.5-5 mg/kg) were given via IP
injection 5–7 days per week over the course of the experiment.
4-hydroxytamoxifen (4-OHT, 50 mg/kg in Chen oil, IP) was prepared as previously
described (Guenthner et al, 2016; Girasole et al, 2018). To prepare a 20 mg/mL stock
in ethanol of 4-OHT, 4-OHT was added to 200 proof ethanol, vortexed, and placed
on a horizontal shaker at 37°C for 30 min or until the 4-OHT dissolved.
The stock solution was kept covered in foil to minimize light exposure. Next,
to prepare a 10 mg/mL working solution in oil, the 4-OHT/ethanol mixture
was combined with Chen Oil (a mixture of 4 parts sunflower
seed oil and 1 part castor oil) and placed into 1.5 mL Eppendorf
tubes. The tubes were vigorously mixed, wrapped in foil, and left on a nutator
for 45 min at room temperature, vortexed and shaken periodically. The
tubes were then placed in a speed-vac for 2-3 h to evaporate the ethanol.
If necessary, the final volume was adjusted with Chen Oil to 1 mL to reach
a final concentration of 10 mg/mL.
Picrotoxin (Sigma) was dissolved in warm water to prepare a 5 mM stock
solution. Picrotoxin stock which was subsequently diluted in ACSF for a final concentration of
50 μM in ex vivo (slice) electrophysiology experiments.
Tetrodotoxin (TTX, Abcam) was dissolved in water at a stock concentration of 1 mM and
added to ACSF for a final concentration of 1 μM in ex vivo (slice) electrophysiology experiments.
SKF 81297 (Tocris) was dissolved in water at a concentration of 1mM and added to
ACSF for a final concentration of 5 μM in ex vivo (slice) electrophysiology experiments.