[Neuroscience] Re: problems with dissociated cells & Ca2+ current recording

Bill.Connelly via neur-sci%40net.bio.net (by connelly.bill from gmail.com)
Mon Apr 20 16:27:16 EST 2009


Hi Sibylle,

What is happening that is making you say that the cells aren't
surviving? Are you just loosing the seal?
You would assume that if the cells were dying because of some problem
with your solutions, they would be dead long before you put a patch
electrode on them.

I suppose my first suggestion is to try a different dissociation
techneque. There are a large number of them out there, from the Bill
Catterall school who use Na Isethionate for the dissociation and
bubble with pure oxygen (140 Na isethionate, 2 KCl, 4 MgCl2, 0.1
CaCl2, 23 glucose, 15 HEPES, pH 7.4  bubbled with
100% O2), to just a straight bicarbonate based aCSF with protease.

My other thought is maybe forget about dissociated cells. Try
nucleated patches. They're a lot easier than most people make it
sound. Especially from such young tissue. You can pull the nucleated
sphere out above the level of the slice, so you get excellent drug
access. You'll have a 10-15MOhm series resistance over a 500M-1GOhm
membrane, with 10pF capacitance, so you'll get excellent voltage
clamp.


On Apr 20, 8:30 pm, Sibylle Guggenmoos-Schreyer
<sibylle.guggenm... from uniklinik-freiburg.de> wrote:
> I am trying to record Ca2+ currents (whole-cell) from acutely  
> dissociated hippocampal CA1 neurons (7-10 day-old rats and mice).  
> However, the cells only survive a couple of minutes after having  
> established the patch. I wonder if this is a dissociating or a bath  
> solution problem, as I can't bubble the bath solution with carbogene  
> (it becomes too acidic). I tried recording Ca currents in acute slices  
> of very young rats before, and those currents were stable but very  
> difficult to influence with various substances which is why I think  
> the bath solution might be the cause of trouble.
> So, if anyone's got either a good bath solution recipe or a  
> dissociating method or any other idea - I'd be so grateful!
> Sibylle



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