We are trying perform patch clamp (whole cell) recordings over basal ganglia neurons (striatum and substantia nigra) from organotypic rat silices that have been cultured between one and two weeks. The issue is that, even when the aspect and morphology of the recorded neurons seem to be normal and healty, after achieving a good seal and acces to the cell the neurons did not present action potentials in response to depolarising current steps. Also, neuronal resistances tended to be higher than in normal BG neurons, in several cases over 1Gohm.
We are using ACSF of 300-310 mOsm and internal patch solution of 290 mOsm. The osmolarity of the culture medium was 280 mOsm and yesterday we have adjusted to 310 mOsm for the new group of slices.
Did you think that by adjusting de osmolarity of the medium will resolve the problem or what else can be done?
Hi Alejandra,
Interesting problem. It is hard to comment without some more data. Would you be able to post a picture of the morphology, and a picture of the voltage response to current traces? Also, the details of the growth medium would be good.
Generally speaking, neurons in culture, organotypic or dissociated, undergo a variety of alterations due the the huge changes that occur when slices/dissociated/removed from normal environment. However, I cant think of ever observing a loss of spiking, unless you had massive astrocyte proliferation (which the high input resistance and the normal morphology argue against).
The fact that you are observing an input resistance of 1GOhm, (i.e. 10x higher than normal for medium spines or SN neurons) shows that massive changes have taken place in the neurons. I strongly doubt changing the osmolarity of your aCSF is going to help in this regard. It seems to me that something terrible happened during the culture phase, but what, I don't know.
Dear Alejandra: Sometimes it is not easy to distinguish whole-cell from cell-attached current recordings, particularly small in neuronal size. As cell-attached mode was established, action currents which represent the configurations of action potentials would be measured.
Hi Alejandra,
I've never culture basal ganglia, but I've done nodose ganglia which is probably much different.
What is their baseline holding voltage in current clamp? A 1 gigaohm membrane resistance is indeed very high, as mentioned above it would be interesting to see the I-V curves for these neurons. First to see if channel kinetics are the same as slice BG neurons and second, to make sure you have good access to the cell.
Have you put on a control drug in voltage clamp and analyzed the holding current to make sure the cells are responding as normal? After two weeks of culture have the neurons grown interconnections with each other and can you see axonal growth? If they've developed mature interconnections you can also look at IPSC and EPSC to verify they're responding well and you have good access to the cell.
Unfortunately changing the osmolarity isn't going to impact your result at all.
Best of luck!
B
Thanks William, Sheng-Nan and Brandon for your kind replies. I was trying to have some new recordings from the new slices with the change in osmolarity but the history is the same.
William: I´m afraid to agree with you, something terrible is must be happening in some point of the culturing. I will post more information about the growing media and pictures of the current traces as soon as I can hopping to read your replies.
Sheng-Nan Wu: I agree with you, in some cases I’m not sure if have a good access to the cells, but even when I could be on cell attach mode, I don´t see action currents at all. In voltage clamp mode sometimes it looks like the cell it could be closed, but when I switch to current clamp I can see Taus of more than 50 ms then I suppose that I have access to the cytoplasm.
Brandon: Im using holding voltage of 80 or 70 mV and the membrane potentials are variable from -75 to -45. Surely I´ll reply soon with I-V curves to hear about the observations that you can give me. I´m not quite sure that the neurons are making new interconnections between each because the only ones that I have successfully record have just one week and the histology is not yet done, but as far as I have seen, I haven´t notice spontaneous synaptic currents. Also, I haven´t tried any drug, which one would you recommend.
Thank’s again for your help
hi Alejandra,
I've never worked with striatal cultures but keep in mind that if you if only culture striatum and SN you won't have any glutamatergic neuron population there. Usually there's extensive input to most striatal cells from cortex and thalamus and also from amygdala and hippocampus to more medial ventral regions. In your conditions striatal cells will develop without any glutamatergic input which will certainly alter their physiological maturation.
My guess is that this will change if you add cortical explants to your cultures. Good luck!
Rui
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