Hi there, all very good questions. Yes, you should consider carefully the composition of your internal solution, depending on your experimental needs. Cs based solutions are best for clamping small currents as Cs blocks K leak and allows better clamp of distal inputs. But they are harsh on neurons. K-gluc are milder and more physiological (?) but some reports suggests that they are not good for clamping currents. Also, in CC mode, these can drive cells into hyperactive mode. Check R. Nicoll, R. Malenka, G. Collingradge and others work for more details. As regards to the second part of your question, both the content and the osmolatiry as well as Ca2+ concentration in your internal are super-important, so please take close look into literature and make sure that you have all right. Otherwise, you are going to waste much time. Good luck with your entry into the patch clamp world - sv

Hello!

For voltage-clamp recordings the Cs-Methanesulfonate based solution is used in our lab.

127 CsMeS, 10 NaCl, 5 EGTA, 10 HEPES, 6 QX314, 4 ATP-Mg, and 0.3 GTP; pH adjusted to 7.25 with CsOH

It allows to record synaptic currents in pyramidal neurons in slices for prolonged period of time. As it contains the blockers of potassium and sodium channels, the input resistance in whole-cell configuration is rather high (about 250-400 MOhm). I would recommend to voltage-clamp the cell at -50 - -20 mV most of the time during the experiment. Prolonged recordings at more negative voltages decrease the cell viability during the recording. This solution can't be used for current-clamp recordings.

For current-clamp recordings the K-Gluconate based solution is used:

135 K-gluconate, 10 NaCl, 5 EGTA, 10 HEPES, 4 ATP-Mg, and 0.3 GTP (with pH adjusted to 7.25 with KOH)

It allows to record membrane voltage and action potentials in pyramidal neurons in slices. However it does not perform too well in voltage-clamp mode, as the input resistance in whole-cell configuration is quite low (60-180 MOhm depending on cell type). If you use it in voltage-clamp recordings I would recommend to clamp the cell at -90 - -60 mV most of the time during the experiment.

Both of the solutions are prepared in the same manner. First we dissolve all the components in water, except ATP and GTP. After that we rougly adjust pH. Then we rapidly add ATP and GTP, make a final adjustment of pH (ATP decreases the pH) and freeze the solution in 1 ml tubes. The osmolarity of the resulting solutions is about 300 mOsm (for better patching it should be a little lower than the osmolarity of the extracellular solution).

https://www.ncbi.nlm.nih.gov/pubmed/28912687

https://www.ncbi.nlm.nih.gov/pubmed/27790093

Good luck with your experiments.

Hi Darwin,

You can use Cs-gluconate internal soulution for your voltage clamped recordings as it will prevent interference by K currents. Adding QX314 helps reduce noise and improving seal. Make sure your osmolarity is at least 10 mOsm less than your extracellular solution so that your seal remains stable. You could use osmometer for that purpose. If you don't have access to one there are websites that will calculate your components in your solution to provide the osmolarity value. Try to weigh your chemicals as accurate as possible. Adjust pH only with CsOH.

You will get NMDA and AMPA current components when you clamp at -70mV. Holding at -80mV for AMPA currents would be okay provided you do all AMPA recordings at that voltage. You may not get any NMDA component at -80mV assuming you have Mg in your bath or ACSF. Additionally you can apply CPP to block NMDA currents completely and record just AMPA currents. However, you won't be able to record NMDA in that case. If you want only NMDA current, you can add NBQX and AMPA currents will be wiped off. But if you want both in one go, you can record AMPA first at -80mV followed by NMDA at +40mV and it should be acceptable. Double check some of John Lisman group papers. Start playing with it, make mistakes and you will be a pro! Good luck.