I am fresh electrophysiologist in brain slices. I have worked only on extracellular signal detection and patch-clamp combination with voltage imaging in a dish before.
I have recently joined group that is working on neurons in preoptic area near hypocampus. Electrophysiologists working in this group left more then year ago and I am trying to repeat their experiments.
It took some time, but I stated to get relatively good mice brain slices. On other hand, my neurons are dead within 1hr. I am trying to patch more roudish, smooth membrane cells which do not have such visually exposed nucleos. For the first 10-20min neurons has nice spontaneous activity, but after 30min neurons become depolarized. If I keep cell at hyperpolarized stage, then I can evoke action potentials which amplitude overshoots 0mV.
Majority of cells has stable spontaneous activity if I keep cell hyperpolarized. Depolarized cells has resting potential about -35 to -25mV, all membrane parameters looks fine. Pipette offset a bit high (about 50mV), but its just because i am using LowCl intracellular solution with AgCl electrode inside.
I cleaned all set up few times, increased speed of brain preparation, checked pH and osmalarity (extracellular solution 300-305mOsm, intracellular solution calibrated just before experiment to have 11-15mOsm lower then extracellular).
Brain slices kept in my recording solution for 2-3hr looks relatively fine, cells do not shrink or expand. I have to admit that all cells looks a bit roundish after 1hr.
Based on observation cells looks intact and healthy, just these cells ''give up on life''. I am constantly giving carbogen to brain slices. Slicing and recording solutions made weekly. Digitized calibrated few weeks ago.
I will accept any suggestions.
Hi Julius! Welcome to the club :)
Does your slicing solution contain Na+? It can help to slice in mostly glucose or NMDG-based, cold solution, and then afterward let the slices recover in “standard” solution. So you avoid hyperactivity during the stressful cutting process. And it can be helpful to have ATP, GTP and glucose in your internal.
The offset sounds fine for low Cl solution.
And, do you mean by 1 h continuous recordings for one cell after break in? That already is a lot :) not every cell (type) will persist for that long!
Maybe something will be helpful, best of luck for your experiments!
Hi Julius,
As Victoria suggested, try the NMDG based ice cold aCSF for slicing, then allow to recover in standard aCSF. Also, an hour patch is pretty good.. however, you may need to consider changing tubings etc in case of contamination that may results in cell death
Also, are you working on mice/ rats or...? And what age mice/ rats (As you may need to take additional precautions for older mice/rats)..
What cells are you recording from
There used to be a website called Brainslicemethods.com (I just check that out, but could not find the brain slicing sections anymore.
Anyway, have a read of below paper
Article Acute Brain Slice Methods for Adult and Aging Animals: Appli...
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