acute hippocampal slices

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gsboris
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acute hippocampal slices

Hi there
If anybody could give me advice I would really appreciate it.
I am trying to patch pyramidal and granule cells in acute hippocampal slices. I get about 3-4GOhm seal quickly but it does not hold. It breaks away in <1min or so. If I apply -70mV, as suggested to make better seal, it breaks away even faster. In cultures I did not have this problem, it seals there up to 15GOhm and stays fro a long time. My bath solution is normal ACSF, pipette solution, 110 KGluconate, 10 KCl, 2mgMgATP, 10 EGTA, 10HEPES pH 7.2, Osm 290mOsm, R=6MOhm.
Thank you in advance
B

EGC
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Dissection and cutting is

Dissection and cutting is critical. What's your cutting solution? What's your ACSF's Osm? You can also try to wait a little bit longer before break the cell.

Shampa
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I was working on Cell culture

I was working on Cell culture recording few months back. I can refer you to a Prof at Shaheed Behesti Medical University, Tehran where Prof Janahmedi is working on very similar to your question. She is one os the experts in this field. Try to contact her at her webpage through internet. Best wishes.

gsboris
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Cutting solution is the same

Cutting solution is the same as ACSF but with elevated up to 3mM Mg. I will check ACSF osmolarity.

EGC
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Then try to reduce Ca to 0

Then try to reduce Ca to 0.5mM and increase Mg to 4 or more in cutting solution. Osm of ACSF shuold be higher than pipette solution.

gsboris
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Thanks a lot EGC

Thanks a lot EGC
I will try and post next week
B

gsboris
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Hi EGC:

Hi EGC:
Well, ACSF osmolarity is 310mOsm, pipette solution 280mOsm
I tryed what you suggested .5mM Ca & 4mM Mg. Same problem. It seals up to 3GOhm but as soon as I start holding pipette at -70mV seal is gone :( it is like repellent. For some reason glass pipette does not stick to the membrane good enough.
B

EGC
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It sounds to me the pipette

It sounds to me the pipette possibly. I use thin glass, which seems better than thick glass. Make sure the glass is very clean and you make pipette freshly. Dissection should be as fast as you can. Find papers doing similar experiment and check their methods carefully. Can you break the cell at 0 mV? 

gsboris
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Thanks EGC

Thanks EGC
To pull pipettes I use firepolished borosilicate glass OD 1.5mm ID 1.1mm.
I will try breaking in at 0mV

patchclamper
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Hi,

Hi,
Just wondering have you solved the problem yet? I am having a similar problem..Thanks for replying

hypa_dude
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This can happen if you don't

This can happen if you don't have vibration dampened properly, so I would check that. Make sure your table is floating and that cables hang freely so that they are not mechanically coupled to unisolated parts of your rig.  Also, make sure your slice is flat against the bottom of the chamber and anchored properly (I suggest using a warner harp).