patching dentate gyrus

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jack_prem
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patching dentate gyrus

Hello,
I have been patching Dentate gyrus pyramidal cells and do current clamp recording for the past couple of days. I use Multiclamp 700B, pClamp 10.2 and K-Gluconate as internal solution. Whenever I patch dentate gyrus pyramidal cells, I can get 1GOhm seal but as soon as I switch from voltage clamp to current clamp, the cells are depoalrised to -30 mV. Does anyone know how to fix it?

Thanks

James

The FFM
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Can you please give the

Can you please give the details of the ionic composition of your pipette solution and your extracellular solution?

Also I assume that you are current clamping at 0 pA?

jack_prem
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Thanks for geting back to me.

Thanks for geting back to me.

Yes, I current clamp at  0 pA. The composition of solutions are as follows:

Internal:
KGlu-130mM; KCl: 20 mM; HEPEP free acid: 10mM; EGTA: 0.2 mM; GTP-NA salt: 0.3; ATP-Mg salt: 4 mM

External (aCSF): NaCL: 124 mM, KCl: 3mM; NaHCO3:24 mM; NaH2PO4: 1.25 mM; MgSO4.7H2O: 1 mM, Glucose: 10 mM; Ca2+: 2mM

 
 

 
 

 
 

 
 

 
 

 
 

The FFM
The FFM's picture
 What does an I-V look like?

 What does an I-V look like?  Are the cells leaky (the seal goes bad quickly)?

 also do you double check that your aCSF is gassed correctly and maintaining the correct pH throughout the experiment?

It seems that he cells in the slice might just be sick after the prep.

jack_prem
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I have not done V-I yet. The

I have not done V-I yet. The seal goes bad within a minute after I patch.  I checked the osmomolarity and pH of aCSF and it stays almost constant throughout the experiment. The cells look good in DIC.