A question about QX-314 in intracellular solution

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patchclamper
patchclamper's picture
A question about QX-314 in intracellular solution

Hi all,

First, thanks for all the folks here posting problems and anwering questions, I've learned a lot from here.

Recently I met a problem,

In my experiment, I stimulate schaffer collateral fibers and record evoked EPSCs in CA1 pyramidal neurons using whole-cell recording in mice hippocampus.

When I was using low stimulus strengh (~0.1mA), EPSCs looks normal, However, If I stimulate with a relatively high stimulus strength (~0.5mA), sometimes I will get a fast inward current spike at the peak of EPSC (with K-gluconate intracellular solution, see attached picture) and get prolonged large EPSCs (with Cs+ based intracellular solution, EPSC last for several hundred of miliseconds, the shape is definitely not normal), and I guess that's a result of losing voltage control over distal dentrites when the deporlarizing current was very big. So I put QX-314 in my intracellular solution to block Voltage-gated Na+ channels intracelluarly. After putting QX314, I saw much less prolonged EPSCs at first. However, after about a month, the prolonged EPSCs appear again...And I have almost no idea what's causing the "fat EPSCs" except worrying about the degradation of QX-314)

My questions are:
1, what concentration of QX-314 you guys put in intracellular solution when recording large EPSCs?(I am using 5mM)

2, QX-314 is said to be light sensitive. so I made the solution in dark condition and wrap my intracellular solution syringe with foil during recording) Since my recordings usually last for 1hour,  Is that necessary to cover the whole rig with some black cloth to avoid light?

3, On MSDS sheet of QX-314, it is said that the stock solution should be used within one month. So, should I  make intracellular solution with new stock of QX-314 every month? (I use to make a huge amount of intracelluar solution stock which is good for 3 months and put it in -80 freezer, have any of you tried store intracellular solution with QX-314 more than a month?)

Any other advices on handling QX-314 or getting better voltage control are welcome!!

Thanks!

Peter

pbm
pbm's picture
 Let me answer your questions

 Let me answer your questions serially:
1. 5 mM is fine. 
2. I've never had a problem with the light sensitivity, but then we usually record with the lights off in the rig rooms anyway. 
3. I always make a stock (about 5 ml at a shot) and store it in the -70, but it is aliquoted into 100 or 200 ul or 500 ul single-use tubes, and yes, it seems to work even after 6 months. The tubes never go through more than one thaw cycle.

Voltage clamp is not possible in an extended cell - there are multiple studies on this that come out every couple of years, but the latest  is:

1: Williams SR, Mitchell SJ. Direct measurement of somatic voltage clamp errors
in central neurons. Nat Neurosci. 2008 Jul;11(7):790-8. Epub 2008 Jun 15. PubMed
PMID: 18552844.

If you can do your experiment in current clamp, you'll be better off. However, it won't prevent engagement of active conductances in the dendrites.
 
To my eye, the fat epscs look like unclamped calcium spikes.

 hth...