Bad luck with patching... any tips?

tomte tehgabriel at web.de
Mon Nov 29 08:09:05 EST 2004


BilZ0r <BilZ0r at TAKETHISOUThotmail.com> wrote in message news:<Xns95ADB653CED05BilZ0rhotmailcom at 202.20.93.13>...
> Hi,
> 
> So I've just started trying to patch cells and I'm not having much luck. 
> I'm using spike2v5 basically as an ossilocope, with a time base of about 
> 5 seconds, as I pass a 5mV step down the pipette (around 3MOhms K-
> gluconate). I manually drive into the CA1 pyramidal cell layer with 
> positive pressure on. I first see a very transient drop in the current 
> trace (I assume this is entering the slice, if I reverse the pressure 
> here I always get nothing). If I keep driving in a bit I usually don't 
> really see a drop in the current, but the current trace goes a bit wavey, 
> occilating up and down. If I drop the pressure here I get a 15% drop in 
> current, (maybe 30% of the time) and if I put on negative pressure the 
> "seal" forms more.
> 
> But I don't get zero current, Maybe down to 40-30% of original... I start 
> to see some capacatinces transients, but I can't get the current down to 
> nothing, or go whole cell.
 
> Basically
> 1) Is there any way to get a more definate signal when I approach a cell? 
> Or is it always subtle?


> 2) Do you thing I should try and increase my pipette resistance? 

I use pipettes with the same parameters (3MOhm, K-Gluc.) and they work
perfectly. But sure it may be one point you can check. Usually
increasing the pipette resistance (up to 5-6 MOhm, don't go higher)
will result in getting better seals but your access resistance gets
worse.

> 3) Any ideas on what I'm doing that makes it look live I've got a cell, 
> but yet I can't go whole cell (or even really on cell)?

You are using solitions of standard composition, i suppose. In most of
the cases when i had problems in getting good seals it turned out that
there was something wrong with the solutions i used. Even if you are
using a standard receipes check out pH (with regard to the temperature
you work at!) and osmolarity of your solutions (both, acsf and pipette
solution). Osmolarity of the pipette solution should be slightly - 10%
lower than acsf. These parameters are highly critical for obtaining
good seals.
I am working with acsf: 300 +- 5 mOsm, pH 7.45
                  pipette solution: 285 +- 5 mOsm, pH 7.3


Good luck!

Cheers,
Thomas



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