Do cell-attached (CA) recordings sufficient for confirmation?
I recorded in CA mode with K-gluconate intracellular solution (as after I needed to record firing patterns) without adding any pharmacological agents. Some of my cells are self-firing in CA. So I am wondering, whether I can use this data to conclude on spontaneous activity? Or do I need any other tests?
Thank you in advance!
Dear Elina,
To identify spontaneous or self firing neurons, I do recommend the loose-seal or extracellular recording methods using 3-4 megaohm patch electrodes filled with the bath solution. With patch-clamp, it is less reliable to identify spontaneous activity compared to extracellular recording. This is particularly important when you have high levels of K in your pipette solution and blowing high levels of K onto the soma prior to sealing will induce or increase firing in neurons. You can check my following publications for details on whole-mount retinal ganglion cells (2008 Pflugers Arch paper), in brain slices (2013 paper), and in IPSC cells (2017 Frontiers paper).
Best wishes,
Refik
Article Semi-loose seal Neurobiotin electroporation for combined str...
Article Neurobiotin Electroporation for Combined Structural and Func...
Article Human iPSC-derived Cerebellar Neurons From a Patient With At...
In CA mode, it is necessary to check if you apply more positive charge into the cells and hence induce neuronal firing. Under such conditions, the firing behavior could be as a results of sustained current injection, rather than spontaneous.
Dear Sheng-Nan,
Thank you for your answer! Does that mean that I need to check whether in my recordings I have 0 pA baseline? And during recording I need to use not the Vclamp mode, but I=0?
Or do I need first read the Vm in I=0 mode and then during recording in Vclamp mode hold exactly this voltage, right?
I=0, the recordings would be change in membrane voltage. Under cell-attached voltage-clamp, it is better to hold the potential at about -70 mV with 0 pA in the baseline. The reason that the holding potential is set at ~70 mV is dependent on the bathing solution used and what the predicted intracellular K concentration would be.
Dear Elina,
To identify spontaneous or self firing neurons, I do recommend the loose-seal or extracellular recording methods using 3-4 megaohm patch electrodes filled with the bath solution. With patch-clamp, it is less reliable to identify spontaneous activity compared to extracellular recording. This is particularly important when you have high levels of K in your pipette solution and blowing high levels of K onto the soma prior to sealing will induce or increase firing in neurons. You can check my following publications for details on whole-mount retinal ganglion cells (2008 Pflugers Arch paper), in brain slices (2013 paper), and in IPSC cells (2017 Frontiers paper).
Best wishes,
Refik
Article Semi-loose seal Neurobiotin electroporation for combined str...
Article Neurobiotin Electroporation for Combined Structural and Func...
Article Human iPSC-derived Cerebellar Neurons From a Patient With At...
The firing might be due to synaptic activity. You can test this point by blocking synaptic transmission. This can be done by using post-synaptic receptors. Alternatively you can block transmitter release using low Ca, cobalt, etc.
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology