Dear Oleksandra,

It is impossible to give you an answer with the info you have provided above. Which cells are you trying to patch, Purkinje, granule? What is the resting membrane potential of your cells? What are you using as extracellular solution during recording? If you are using the solution you have mentioned above with 25 mM KCl, then the neurons will be very depolarised. Normally extracellular K should be around 3 mM. Can you stably voltage-clamp these cells? 

Best wishes, Refik

Dear Refik,

I keep neurons in 25mM KCl just during culturing!  For patching I have standard buffer compositiion (3mM K external 120mM K internal). I have a mixture of cerebellar cells in my prep. Resting membrane potential is -50-70 mV. And I want to do current-clamp now, so have not tried to voltage-clamp them. 

For hippocampal neurons I use picrotoxin and CNQX to get stable membrane potential and firing. Do you think it is helpful to use agonist in case of cerebellar neurons too?

Thanks in advance!!!

Dear Oleksandra,

I have previously recorded from cerebellar Purkinje cells in slices, and in human iPSC derived cerebellar neurons in organoids. For recording action potentials in current-clamp, I did not add any drugs, used just standard extracellular solution. In slices Purkinje cells often display typical complex spikes spontaneously. I think the resting potential of your cells are great, and therefore you should first voltage-clamp them and then switch to current-clamp once the cells are stabilized. If the cells are too depolarized you can apply some continuous negative voltage to slightly hyperpolarize them to around -60 mV; if they are too hyperpolarized (no spontaneous action potentials), then you can either inject continuous voltage/or apply steps/ramps to bring the membrane potential to firing theshold. In good and healthy cells you should not need to apply much voltage to the soma (close to the axon initial segment) to make the cells fire APs.

best wishes,

Refik

Hello

The problem with granule cell is its high resistance you can may be read the paper published by D'angelo several years ago concerning action potentials in granular cells. You can also try to do loss patch recording to see if the cell are spontaneously firing. If it is the case its mean that when you dialyse the cell you lost the firing properties .

Best wishes