TITLE: Stock solutions of cocaine, isradipine, nomifensine, lidocaine, DHβE, CP8, L-741,626, and water-soluble cholesterol
AUTHORS: Katherine Brimblecombe, Stephanie J Cragg

[DESCRIPTION]
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).

[STEPS]
SECTION: Cocaine (5 µM)
1. Make 50 mL aliquots of 10 mM stock solution:
add 3.4 mg per 1 mL dH2O
store at -20°C    

 

SECTION: Isradipine (5 µM)
3. Make 50 µL aliquots of 10 mM stock solution:
add 2.69 mL DMSO to 10 mg powder
stored at -20°C   

SECTION: Cocaine (5 µM)
2. 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.
SECTION: Isradipine (5 µM)
4. For each experiment, dilute 50 µL of 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 5 µM.
SECTION: Nomifensine (10 µM)
5. Make 100 µL aliquots of 10 mM stock solution:
add 3.54 mg per 1 mL 0.1 HCl
stored at -20°C 
SECTION: Nomifensine (10 µM)
6. For each experiment, dilute 100 µL of 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 10 µM.
SECTION: Lidocaine (100 µM)
7. Make 100 µL aliquots of 100 mM stock solution:
add 2.34 mg per 1 mL ethanol
stored at -20°C 

SECTION: Lidocaine (100 µM)
8. For each experiment, dilute 100 µL 100 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 100 µM.
SECTION: Dihydro-β-erythroidine (DHβE)(1 µM)
9. Make 0.5 mL aliquots of 10 mM stock solution:
add 2.8 mL dH2O to 10 mg powder
store at -20°C    
SECTION: Dihydro-β-erythroidine (DHβE)(1 µM)
10. 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   
SECTION: Dihydro-β-erythroidine (DHβE)(1 µM)
11. 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.
SECTION: CP8 (10 µM)
12. Make 100 µL aliquots of 10 mM stock solution:
add 3.35 mg per 1 mL DMSO
store at -20°C
SECTION: CP8 (10 µM)
13. For each experiment, dilute 100 µL 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 100 µM.
SECTION: L741-626 (1 µM)
14. Make 100 µL aliquots of 10 mM stock solution:
add 2.93 mL DMSO to 10 mg powder
store at -20°C
SECTION: Water-soluble Cholesterol (50 µg/mL)
16. Make 45 mg of cholesterol per gram:
add 11.1 mg water-soluble cholesterol to aCSF

 

SECTION: Me-ß-cyclodextrin (1000 µM) - Control Solution
17. 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
SECTION: L741-626 (1 µM)
15. For each experiment, dilute 10 µL 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 1 µM.
SECTION: Me-ß-cyclodextrin (1000 µM) - Control Solution
18. For each experiment dilute 20 µL 0.5M stock solution (one aliquot) in 10 mL aCSF for final working concentration of 1 mM.