Jan 09, 2024
Stock solutions of cocaine, isradipine, nomifensine, lidocaine, DHβE, CP8, L-741,626, and water-soluble cholesterol
- Katherine Brimblecombe1,2,3,
- Stephanie J Cragg1,2,3
- 1Department of Physiology, Anatomy and Genetics, University of Oxford, OX1 3PT, UK;
- 2Oxford Parkinson’s Disease Centre, University of Oxford, Oxford, United Kingdom;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Katherine Brimblecombe, Stephanie J Cragg 2024. Stock solutions of cocaine, isradipine, nomifensine, lidocaine, DHβE, CP8, L-741,626, and water-soluble cholesterol. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3bbbl3p/v1
Manuscript citation:
Brimblecombe KR, Connor-Robson N, Bataille CJR, Roberts BM, Gracie C, O'Connor B, Te Water Naude R, Karthik G, Russell AJ, Wade-Martins R, Cragg SJ. Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine co-varies with risk factors for Parkinson's. Eur J Neurosci. 2023 Nov 8. doi: 10.1111/ejn.16180. Epub ahead of print. PMID: 37941514.
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: December 20, 2023
Last Modified: January 09, 2024
Protocol Integer ID: 92560
Keywords: cocaine, isradipine, nomifensine, lidocaine, DHβE, CP8
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020370
Abstract
This protocol details the making of the following stock solutions used in Brimblecombe, K.R. et al. (2023):cocaine (inhibits the activity of dopamine transporters (DATs)), isradipine (inhibits L-type voltage-gated Ca2+ channels (LTCC)), nomifensine (inhibits monoamine re-uptake), lidocaine (blocks voltage-gated Na+- channels), dihydro-β-erythroidine (DHβE) (nicotinic acetlycholine receptor (nAChR) antagonist), 1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione (CP8) (a potent and highly selective CaV1.3 L-type calcium channel antagonist; see Protocol: Synthesis of 1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione (CP8)), L-741,626 (inhibits D2 receptors), and water-soluble cholesterol (DAT function in Snca-null mice is augmented, see Threlfell et al., 2021).
Guidelines
Drug concentrations were chosen in accordance with previous studies (Acevedo-Rodriguez et al., 2014; Brimblecombe et al., 2015).
Materials
Drugs:
- Dihydro-β-erythroidine (DHßE) (Mr 365.27, stock powder stored at room temperature)
- Isradipine (Mr 371.39, stock powder stored at 5°C)
- Cocaine (Torcis, Mr 339.82, stock powder stored at room temperature)
- Nomifensine (maleate salt) (Mr 354.4, stock powder stored at room temperature)
- Lidocaine (Mr 234.34, stock powder stored at room temperature)
- CP8 (Mr 334.8, stock powder stored at room temperature)
- L741-626 (Mr 340.85; stock powder stored at room temperature)
Controls:
- Water-soluble Cholesterol (Sigma Aldrich, #SKU C4951-30MG, stock powder stored at -20°C)
- Me-ß-cyclodextrin (Mr ~1320, stock powder stored at room temperature)
Solvents:
- Dimethylsulfoxide (DMSO)
- Ethanol
- 0.1M Hydrocloric Acid (HCl)
- dH2O
Cocaine (5 µM)
Cocaine (5 µM)
Make 50 mL aliquots of 10 mM stock solution:
- add 3.4 mg per 1 mL dH2O
- store at -20°C
Note
Controlled substance! Stored in locked cabinet. Keep log book up to date.
For each experiment, dilute 50 µL of 10 mM stock solution (one aliquot) in 100 mL of bicarbonate-buffered artificial cerebrospinal fluid (aCSF) solution for a final working concentration of 5 µM.
Isradipine (5 µM)
Isradipine (5 µM)
Make 50 µL aliquots of 10 mM stock solution:
- add 2.69 mL DMSO to 10 mg powder
- stored at -20°C
For each experiment, dilute 50 µL of 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 5 µM.
Nomifensine (10 µM)
Nomifensine (10 µM)
Make 100 µL aliquots of 10 mM stock solution:
- add 3.54 mg per 1 mL 0.1 HCl
- stored at -20°C
For each experiment, dilute 100 µL of 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 10 µM.
Lidocaine (100 µM)
Lidocaine (100 µM)
Make 100 µL aliquots of 100 mM stock solution:
- add 2.34 mg per 1 mL ethanol
- stored at -20°C
For each experiment, dilute 100 µL 100 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 100 µM.
Dihydro-β-erythroidine (DHβE)(1 µM)
Dihydro-β-erythroidine (DHβE)(1 µM)
Make 0.5 mL aliquots of 10 mM stock solution:
- add 2.8 mL dH2O to 10 mg powder
- store at -20°C
Make 100 µL aliquots of 1 mM working stock solution:
- add 4.5 mL dH2O to 0.5 mL 10 mM stock solution
- store at -20°C
For each experiment, dilute 100 µL of 1 mM working stock solution (one aliquot) in 100 mL of aCSF solution for a final working concentration of 1 µM.
CP8 (10 µM)
CP8 (10 µM)
Make 100 µL aliquots of 10 mM stock solution:
- add 3.35 mg per 1 mL DMSO
- store at -20°C
For each experiment, dilute 100 µL 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 100 µM.
L741-626 (1 µM)
L741-626 (1 µM)
Make 100 µL aliquots of 10 mM stock solution:
- add 2.93 mL DMSO to 10 mg powder
- store at -20°C
For each experiment, dilute 10 µL 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 1 µM.
Water-soluble Cholesterol (50 µg/mL)
Water-soluble Cholesterol (50 µg/mL)
Make 45 mg of cholesterol per gram:
- add 11.1 mg water-soluble cholesterol to aCSF
Note
Contains 1 mM Me-ß-cyclodextrin to make it water soluble. Therefore, control experiments are conducted in 1 mM Me-ß-cyclodextrin as a vehicle control.
Me-ß-cyclodextrin (1000 µM) - Control Solution
Me-ß-cyclodextrin (1000 µM) - Control Solution
Make 20 µL aliquots of 0.5 M stock solution:
- add 655 mg per 1 mL dH2O [Yes it really is this soluble!]
- store at 4°C
For each experiment dilute 20 µL 0.5M stock solution (one aliquot) in 10 mL aCSF for final working concentration of 1 mM.