1/ You might need to use a two electrodes voltage calmp (TEVC) amplifier if you are using sharp electrodes. The 200B from Axon is a patch clamp amplifier that will allow you to measure volatge (in current clamp mode) but like you said you don't want to mess up the Chloride concentration, then you can't. And in my opinion, even if you use perforated patch, because Chloride is a small and highly mobile ion, you are going to get chloride in your cell despite using the perforated technique.

2/ Regarding the the basic question of whether adding Glycine will depolarize or hyperpolarize your neuron? I think, you can answer that question by using the patch clamp method (using the amplificator 200B you have) either in perforated configuration or just using the regular patch clamp. When you are in Current Clamp Mode, I don't think you need to compensate for anything (If my memory serves e well, the system won't even allow you any compensation). You need to be in Volatge mode for the the compensation knobs to be active.

Aside from optics/imaging techniques, looks like perforated patch is the way to go - just don't use nystatin or amphoterecin B. Gramicidin perforated patch recording has been shown to not disturb intracellular chloride concentration. http://jp.physoc.org/content/484/Pt_1/77.full.pdf

Yip perforated patches with gramicidin are the way to go - although as you say this takes a little while to optimise and is quite a tricky technique. I've attached some notes on using gramicidin which I found made a huge difference - took me a while to discover what is important in getting a good perforated patch.

Thank you Joseph! However, I can't open your attached file. What format is it?

It's just a text file - but I'll paste the contents here, hope it helps.

**Making Gramicidin - New Technique**

Using gramicidin A from Sigma, 90% pure, 50845, 100mg

or Calbiochem, 96% pure, 368020, 25mg

-Take 1.5 ml ependorf

-Zero it on a scale (press tare)

make sure the scale is as accurate as possible by making sure the bubble is in the middle and the doors are closed while weighing

also don't lean on the table that the scale is positioned on, can also close doors to prevent drafts

-weigh out some gramicidin aim for about 0.5 to 0.9 mg

-however much you weigh out multiply by 1000 ie ,0.7*1000 = 700, then divide by 4 ie 700/4 = 175, and add this many microl of DMSO (to give 4mg/ml conc of gramicidin)

-mix on the rotamixer

-add 5 microl of this to 250 microl of internal to give a final desired concentration of 80 microg/ml (put the DMSO stock soln in the fridge)

-rotamix for about 40 secs

-sonicate for 5 secs

-do not let it get hot

-filter with a 0.45 microm filter

-use IMMEDIATELY!! the 1st 5 mins is optimum, that is why you should have everything else ready before you make the gramicidin, ie slice down puffer pipette in position etc

-do not use after 30 mins, rather make up new gramicidin from the DMSO stock in the fridge

-gramicidin loses potency maximally when it is in soln with the internal, not while it is dissolved in DMSO (something to do with H2O?)

-try keep divalent cations out of your internal

-generally make a new stock every one or 2 days

Thanks a lot! Didn't know that gramicidin loses potency that fast. In many papers, people wait 30-40 min for access resistance to fall below 20 megaohms before they do recordings. If that's the case, gramicidin will be useless by then.

Is it just gramicidin or other antibiotics as well?

Hey Cherry, no the others are correct and you'll likely find the same. Once you have your cell attached patch (with gramicidin) it can take up to 40 mins for the access resistance to fall (ie for the perforation to occur). The important thing though, which I was trying to write in my protocol, is that to keep this time to a minimum the gramicidin internal must be made fresh from the stock solution and used immediately. If you add the gramicidin stock to your internal solution and then wait around for an hour or two before patching, you will take much longer to get a perforation (if at all). It is worth mentioning though that there is quite a lot of variability in the quality and time to perforation. Good luck!

It just seems such a waste to make up 250 microl of internal with gramicidin but only use it once. The pipette can only take ~10 microl and the rest of the solution will be thrown away (since patching a cell requires 40min+). Is there a way around it?

I would think that sharp electrode recording is the way to go. Are you sure about the amplifier? Axon made an axoclamp 2b or an axopatch 200b. Which do you have?

The patch amp is not very good for current clamping.

Hi Cherry, sorry I missed your question from before. Yeah you're right, that's not ideal. You can keep the DMSO stock with gramicidin for a couple of days but I'm afraid I did end up wasting a lot of internal that contains gramicidin. Not really sure of a way around that other than perhaps using less internal when making up the gramicidin fresh each time ...

Hi Olivier, I have the 200B which I have been told that it is not good for sharp electrode current clamp. I have it for whole-cell current clamp though.

I don't know whether it is too late, but the easiest method - by far - and indisputable are cell attached recordings of single channels. First, you record NMDA channels, which give you the RMP of the cell. Second, you record GABAa channels, and you get Egaba in a totally non invasive way. And you know whether GABA is excitatory, depol, shunt or hyperpol.

We have used it recently; you can find details in Quilichini et al Neuron 2012 - you can download it with the suppl from Research Gate.

There are issues with gramicidin and/or patch, as the smaller the cell, the larger is the mistake (you can check a J Neurophysiol paper we made on that: authors Bernard Khazipov Tyzio and others.

Hope that helps.

Great idea, I've read about this method from Tyzio but never tried it - as you say sounds perfect for this situation (Chrisotophe will check your Neuron pub too) - Cherry perhaps you could use glycine instead of GABA in the internal if that's your focus? - although the result should be similar. Christophe would Cherry need to do dual patching on the same cell? One electrode with NMDA in the internal and another with GABA/ Glycine?

Yes, it sounds scary, but it is much much easier than you think.

It is just cell attached recordings.

I did the experiments myself, with two electrodes on the same soma (I like technical challenges!). But you don't need it (it was for the paper). Patch once, remove, and repatch.

There are two schools to remove the first pipette: super fast or super slow.

We do super slow, and the success of repatching the same cell (if the patch held during the first recording) is nearly 95%.

If you know how to do a good cell attached, it is super easy.

Ok great this sounds A LOT easier than gramicidin patching - getting cell attached recordings are quite routine. Will have to try it out myself at some point. Thanks so much for the advice.

That sounds exciting! My rig can't do dual recordings at the moment as I only have one headstage and the microscope is not movable (doesn't have a translator). Guess I can try re-patching it. If I don't succeed with that will try perforated patch with gramicidin then. Thanks so much for your help!

Question: The cell-attached single channel recordings require lower noise levels than whole-cell patch clamping right? At the moment my noise level is between 5-10pA. Wonder if that's too much and will mask the small single channel currents that I want to see. Also, I will be using external solution in the pipette with GABA/glycine dissolved in it right? Would the constant presence of neurotransmitters cause desensitization of the channels? I was thinking of doing an external puff of glycine using a picopritzer - then I can just record in the cell-attached mode as usual?

And also by assuming GABA/glycine only allows chloride ions to flow, a positive current is when chloride flows in and therefore is hyperpolarizing and vice versa for negative current?

Your easiest bet, if you have the ability to generate an evoked GABA-A IPSP, or record spontaneous GABA-A IPSP, is simmply to see where their reversal potential is found. You can do this with a sharp microelectrode. ...and yes, the 200b is an AxoPATCH. Not particularly goood for current clamp. My best advice is to ask Johanna ;-)