I have been using quinpirole as D2 dopamine receptor agonist but I am wondering if the endogenous ligand Dopamine would activate the receptor in a similar way. I know that dopamine easily oxidizes, does anybody know how to bath apply it in brain slices recordings? Thank you
Thanks Shishu. How do you check the dopamine oxidation state? Thx
It helps immensely to make all your solutions fresh the day of the experiment.
Between making fresh solutions and including Na-ascorbate, we haven't had issues with oxidation
And you'll see oxidation as an increase in opacity in your solutions.
I have a slightly different question involving the same matter. On the experiment day I normally prepare a concentrated 50mM Dopamine solution. This solution is initially clear but becomes brown after about an hour. I can't get my head around whether the brown color means it has been oxidized and gone bad or if that's the normal color of Dopamine after it had become protonated. How much Ascorbic acid should I add to this Dopamine solution to guard it against oxidation?
we have used up to 1mM of Na-ascorbate. and obviously the process is temperature-dependent so cold storage helps too
One way to avoid the problem is to set up a Razel pump to deliver DA into the flowing aCSF. The aCSF normally flows at 2 ml/min. Using a 10cc syringe and a Razel set at low speed (20ul/min), you will dilute the DA by 100x. So if you want a bath concentration of 10 uM, you prepare a 1 mM syringe. The advantage to this approach is that you can keep the DA at low pH (2-3), preventing oxidation. The dilution into buffered aCSF means that the pH of the aCSF will not be affected.
As mentioned above (Alexander), using a pump to deliver DA to the bath is very convienent for avoiding oxidation (at least until the very last minute when it mixes in the recording chamber).
An additional step to prevent oxidation of DA (or 6-OHDA) is to bubble the stock with nitrogen to displace the oxygen. Also, use thick and short impermeable tubes to prevent oxygen entering the DA solution when it is sitting in the tube (prior to delivery).
Ascorbate will definitely help with oxidation but will also block T-type calcium channels ( Nelson MT, Joksovic PM, Su P, Kang H-W, Van Deusen A, Baumgart JP, et al. Molecular mechanisms of subtype-specific inhibition of neuronal T-type calcium channels by ascorbate. Journal of Neuroscience. Society for Neuroscience; 2007 Nov 14;27(46):12577–83.). It's probably better to use sodium metabisulfite and make the DA containing aCSF immediately prior to use.
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