Perforated patch in brain slice prep

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JennaMo
JennaMo's picture
Perforated patch in brain slice prep

 Hi all,

I am going to start doing perforated patch in mouse brain slices to record action potential activity. I am a bit confused as to what the best perforating agent is. I am not specifically recording any chloride-dependent processes, although the action pottentials themselves could be affacted by GABA input. Do I need to use gramicidin? I am put off by the long wait for stable perforation.

My second issue is how you actually do this in a brain slice. It seems you can not use positive pressure on the pipette (because of the need for tip-filling of agent-freesolution) and I currently rely on this to get through the slice without debris buildup on the pipette and for cleaning the cell surface for seal formation. How can I get around this issue? 

Any info would be greatly appreciated!

Jenna.

fatchains
fatchains's picture
Chloride, like sodium,

Chloride, like sodium, potassium, and pretty much any ion, can affect various aspects of cellular physiology. However, due to the difficulty using gramacidin unless you are looking at Cl- currents or transporters directly, you may be able to use another perforating agent. The polyene antibiotics are not without their own drawbacks but Ive had much more success with them to look at modulation of AP properties.
If clearing debris is necessary, use a separate pipette. Either fix another manipulator or clear debris and then exchange the pipette. Alternatively, use more perforation-substance free solution at the tip and only apply positive pressure minimally. I always need to clear debris and it really hasnt reduced my yeild dramatically so I wouldnt worry too much if your tip is filled enough. That issue may be more of a problem for in vivo patching or in older animals.
Hope this helps.