I’m having some problems with patch clamping iPS neurons as well as with embryonal mice neurons. They are anywhere from 20-40 days old and all have the same problem. Some of them have a smaller RMP (around -20mV), but even the ones with a more negative one ( around -40mV) have the same issue.
It is possible to seal them as well as to open them relatively easily, but then, when current-clamping, they show no action potentials, and, while voltage-clamping, there is no sodium current?
Of course that without sodium current, we can’t expect an action potential, but what could be causing this absence of sodium current? We’ve tried neurons with different ages, to exclude them being too immature or too old already. I’ve also thought about the RMP not being negative enough, thus inactivating the sodium channels, but I’m guessing that even at -40mV there should still be a significant portion of them on the resting conformation.
What do you think could the problem could be?
It is very common not to have action potentials nor sodium current probably you should use a external solution which significantly reduces or eliminates the outward K currents and also the Ca current and then look at the Na currents. If you are going to perform current clamp probably you have to had the cells for longer time in culture. But also depends on the isolation procedure,
Enrique Soto first of all, thank you very much for the suggestion. I’d like to ask you if you have any specific protocols for isolation and/or external solution that you could provide me with. The plan is to do current clamp as well as voltage clamp on the same neuron.
also, if we are blocking the outward K currents (responsible for the repolarization), how can we expect to see action potentials?
Yeah. iPS cells are very depolarized especially during early development. I would inject enough current to bring the membrane potential to -60 and stimulate with larger current pulse. If you have to inject a lot of current to attain -60 it’s a bad patch.
Michael Risner thank you for your feedback! I always use the holding command to bring the voltage down to around -60, and I play with it up and down but, unfortunately, still no action potentials or Na+ current at whichever voltage... how long do you suggest culturing before patching?
Have you measured your osmolarity between intracellular and extracellular solutions? Prior to recording I substitute Ames' medium for culture media .
- Badges
- Science topic
- Similar topics
- Cognitive Science
- Thinking