We are working on Acute hippocampal slices for electrophysiological recordings using MEAs (Multichannel Systems) in rats.
We could not find any evoked or spontaneous firing of slices (350-400 microns) made with McIlwain tissue chopper either in young or adult rats.
Without spontaneous firing, can we proceed for further data streaming to analyse EPSP and population spikes???
We have a doubt, whether NMDG can be added directly to the conventional aCSF solution to get better response and prolonged viable slices.
Please advise and share your thoughts in this regard.
Thanking you,
Best Regards,
Grandhi V Ramalingayya
Dear Ramalingayya,
Is very interesting this research. Instead, answer now your ask. I prefer follow this work If do you accept me, of course!!!
Thank so much.
Best regards
Hi Grandhi,
I have no experience with MEA recordings, but to answer the question of whether you're having a slice health problem and not a technical problem with your MEA setup, you might consider using conventional stimulating electrodes and recording electrodes, and look to see if you're able to evoke potentials from Schaffer collateral stimulation in CA1, or by stimulation of dendritic field of DG. If you're having trouble with evoking potentials, even in young animals (3-4 weeks old), then most likely the slices are not in good health.
Another user of this forum appeared to be having problems with slice health, and so you might want to check that post (you can find it if you look through the questions I've answered previously - not many). There was also a helpful protocol, and solution recipes added from Brian Christie's lab.
As for NMDG, that is meant to replace Na+ in the buffer used for slicing only, and not typically included in the buffers you would incubate the slices in after completing slicing.
Another thing to consider while trying to improve your brain dissection/slicing, is to use a vibrating slicer if one is available. I have in the past used a McIlwain chopper to produce good quality slices from younger animals, but I think the general consensus is that a vibrating slicer is less damaging to the brain tissue. If the chopper is all you have access to, perhaps consult this protocol from Madison and Edson: Preparation of Hippocampal Brain Slices (2001) Current Protocols in Neuroscience, Unit 6.4.
Best of luck,
In addition to the previous excellent suggestions, such as using a vibrating slicer to cut 350 - 400-micron thick slices, I let the brain slices "rest" in a chamber for an hour or so. This chamber was filled with room temperature aCSF and had a bubbler at the bottom that gently percolated 95% O2/5% CO2 through the solution. It is simple to build one of your own. If interested, please send me a message.
Thank you all for your valuable suggestions.
We will try our level best to address the problems with the given advices.
Thanking you,
Best Regards,
Grandhi V Ramalingayya
From the point of my view important is very quick excision of brain from skull and short cooling of prepared hippocampus in ice-cold aCSF solution before cutting (cca 1 min). McIllwain tissue chopper is quite sufficient for cuting slices for electrophysiology, we use it also. Try to set it to such speed that you can gently take each slice separately with wet brush from the blade. I also argee with the most previous suggestions and I would added that temperature of aCSF solution is suitable up to 33-34 oC during the rest of slices and recordings in the measuring chamber. Good luck.
- Badges
- Science topic