Does anyone have any experience using cyclothiazide (CTZ, 100uM) plus kynurenate (KYN, 300uM) in patch clamp recordings on mouse or rat hippocampal slices? We are trying to use KYN block (competitive glutamate antagonist on APMA receptors) from evoked EPSCs at the CA1 synapse as an indirect way to measure glutamate's timecourse within the synapse.
I can isolate decent AMPA responses but the addition of CTZ is giving me some odd results. I first add 200uM APV and 10uM gabazine to isolate APMA responses (monopolar electrode, stimulating Schaffer collaterals in mice and rats aged P11-18).
In mice, I'm seeing APV, gabazine, CTZ, and KYN potentiate evoked EPSCs rather than inhibit/dampen them. To ensure our drugs worked, we performed two electrode voltage clamp experiments in oocytes expressing rat GluR1 and they worked just fine....CTZ potentiated due to relief of desensitization and CTZ plus KYN inhibited current to ~50% of CTZ plus saturating glutamate (100uM).
Currenty working on the same experimental set up in rat slices (CTZ then KYN inhibition) as we thought there may be a species difference. Most, if not all, experiments using these compounds are in rat, not mice.
Does anyone have any experience with these compounds, particularly when used together in slices? Any feedback would be greatly appreciated.
I have used CTZ together with Kyn (Chanda & Xu-Friedman, 2010). They pretty much work as advertised. I would recommend a somewhat higher concentration of Kyn, 1 or 2 mM. It prevents saturation by shielding receptors from activation, so the more shielded, the better. In fact, it shields receptors from desensitization too (see Wong, Graham, Billups, Forsythe 2003), so I don't know why one uses CTZ as well (though many do). See Sakaba and Neher for estimating glutamate from EPSCs. Also, I would recommend CPP, which blocks NMDA receptors at much lower concentrations, and more specifically than APV.
As for why your EPSCs potentiate after blocking, I am just guessing, but could that be a hippocampus thing? I seem to remember that recurrent excitation can get out of control in hippocampus when GABA is blocked. You may need to fiddle with Mg and Ca to keep the slice quiet (Vogt & Regehr 2001?).
One more thing: CTZ can enhance axon excitability. I am not clear why this happens. If you are stimulating multiple axons, then adding CTZ may increase the number that are activated, leading to a larger EPSC.
Matthew,
Thank you for your reply and providing some good papers to reference. I'm starting to think that the potentiation is due to me not allowing CTZ enough time to wash in and work before I add KYN. Do you recall the wash in time for CTZ in slices? I'm flowing at 2mL/min and I was seeing EPSCs increase even after 12 minutes. I'm going to do more experiments to estimate CTZ wash in time.
I also agree that my KYN concentration is probably too low. I'm thinking about going to 800uM.
Wash-in could depend on too many experiment-specific things. To verify wash-in, you can assess the peak EPSC and also EPSC tau of decay. When both look stable, analyze those.
To establish the appropriate concentration for kynurenate, I would recommend measuring a dose-response curve of paired-pulse ratio. We found 1 or 2 mM was necessary. DGG is even better, but higher concentration, and more expensive.
BTW, if I remember right, kynurenate is also an NMDA receptor antagonist, so APV may be unnecessary.
KYN is a low affinity nonselective antagonist. I suggest CPP for NMDA as Matthew has outlined and either NBQX or the noncompetitive antagonist GYKI-53655 (Derek Bowie's group uses 10 micromolar)
Linda and Matthew,
Thank you both for the responses. I used KYN at 1mM today and still got no block. If anything, my results are indicating that the presence of KYN decreases the amount of time it takes for CTZ to have its full affect and block desensitization. No idea why.
For our experiments, we want competition. I think we are going to try moving in another direction....using a low-affinity NMDA blocker to evaluate glutamate's timecourse within the synapse (Diamond 2001, J NeuroSci).
From my brief reading, NMDARs don't desensitize nearly as much as AMPARs. However, it definitely still happens. Are there any drugs to prevent NMDAR desensitization? Or is the desensitization of NMDARs negligible when recording synaptic events, i.e. time spent in the desensitized state?
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