I'm sorry but why are you dissociating the neurons before patching?

If you think that gramicidin is giving you a trouble, why don't you try the whole cell mode instead of perforated patch? I have used the amphotericin B on isolated hair cells and had about 15-20 minutes to get set of whole-cell currents after gigaseal formation. And what I have noticed that you have to be pretty quick: within 1 minute I have seen an increase of tail currents, indicating that perforation of patch is already there,  even though I have prefilled the tip with antibiotic-free solution, which means that diffusion rate of antibiotic towards the tip is quite fast. In a lot of cases I even not able to form seal with membrane, because molecules are already leaving tip of electrode and bombarding membrane of cell, making holes in there. So, having said that, I'd risk to say that between back-filling the electrode with your antibiotic and forming the seal you have less than a minute before running into trouble. At least it's on my own experience. Again, I'd try 1st to DO NOT use antibiotic at all. And of course, you should balance the voltage on your electrode to 0 mV before you suck and break the membrane. If you are afraid the massive reciprocal diffusion of cell body components and electrode solution, find a proper recipe for it.

I've only access to a setup for dissociated cells, the other for slice is very often occupied. I think that the problem is the digestion or mechanical dissociation (too hard?!?!?), any suggestion for the enzyme?

I cannot use whole cell configuration due to the change in [Cl]i taking place during the first two postnatal weeks so whole cell will alter what I'm actually analyzing

From my own experience, gramicidin used at concentrations between 40 and 80µg/mL is not damaging granular neurons when recorded within the hippocampal slice and I can record from these neurons for more than one hour after getting the access. The delay to obtain a good access with gramicidin is about 20min.

To me, your problem is not related to gramicidin but mostly to the neuronal dissociation which probably makes the neurons very fragile.

Dear Isabelle,

Just out of curiosity: does what are you saying is applicable to Amphotericin as well, if you have used it, of course? I mean the time required to get antibiotic accessed to membrane?

Thank you

I confirm that the problem was linked to neuronal dissociation, I've tried to patch without gramicidin and neurons continued to die (they also die in whole cell configuration). The problem get worse as the mice age increase: till P7 I'm able to dissociate and record some neuron, then it becomes progressively harder. With younger mice won't be a problem, I'm considering to try perforated patch on slice for older ones (P10-15)

Dear Evgeny,

For my perforated PC experiments, I have never used amphotericin but only gramicidin and nystatin. With nystatin which belongs to the same polyene antibiotics family as amphotericin, the delay to get the access is a little bit shorter, more around 10 min. I've noticed that it also depends on the diameter of the pipette tip which seems quite logical. Some useful information regarding perforated PC technique can be found in this book: Patch-clamp analysis - Advanced techniques edited by Walz, Boulton and Baker.

Hope this helps

Thank you, Isabelle!

I have been adding the stock right near the eletrode tip, so that is probably why I had had if faster than you

HI Evgeny,

I was just reading your comments. Do you use patch pipettes without filament? 

Hi, Mr. Simone: I think Leon Jacobo has a good point. I cannot comment on the dissociation procedures, and I have not worked with this type of preparation before. For that, most everybody gave you good comments. In my experience with well stable cultured cell lines, two things will sometime cause the symptoms you describe (swelling and loss of membrane integrity, if I understood well). One is the osmolarity of the internal solution. We always measure and adjust internal and external osmolarity to be equal (between 310 and 330 max), or if anything, the external slightly higher than the internal. BUT EVAPORATION ALWAYS OCCUR. If you fill your electrode long before using them, or you filling needle dries out and is not purged, you may have been  using an internal solution with higher osmolarity. The second possibility is one of the most annoying things in electrophysiology: vibration. If you are using a floating table, check that it is properly floated. Also, that no element (tubing, wires) is applying mechanical stress on your electrodes or electrode holders, or transmitting vibration from the armrests. Sometimes, even a camera attached to your microscope might be producing a low hum or clicks that move, at the tiniest level, your preparation. Cheers.

Hi Brusco

I am also having trouble to get successful gramicidin perforated recording in the dissociated neurons. In my experiments, cells are either dying or become whole cell (appearance of the cell also changes as you mentioned).

Therefore, I am curious to know whether you get any solution?

Thanks !

My trouble are linked to dissociation protocol: with older mice (P13-14) I've been able to get vital neurons able to survive enough time to allow me recording EGaba without going in whole cell configuration. I digest PFC slices using Protease XIV 0.75 mg/mL for 15-16 minute then I proced to mechanical dissociation using Pasteur whose tip diameter has been reduced. I'm still trying to develop a succesfull protocol for younger mice PFC slices digestion: using Protease XIV neurons preserve a good structure and mirphology but when I'm trying to patch them they get dissolved. Gramicidin does not interphere with seal formation at 5ug/mL, must be prepared often (every 2h)