By convention, 5-8Mohm of 1-2 um glass microelectrode shall be used for patching the pyramidal neurons in the brain (please correct me if I were wrong). However, due to some adjustments to the hardwares, I changed my pulling protocol. Now, the glass microelectrodes in the bath has a resistance of around 8-9Mohms (mostly 8.2-8.5Mohms). I was wondering whether such bath resistance is acceptable.
Thank you very much in advance!
The quality of the recording, mostly related to series resistance, depends upon the pipette resistance. The thumb rule is that the best series resistance you can get is twice that of the pipette in the bath.
8-10 MOhm is acceptable. Provided that you keep your series resistance below 20 MOhm. For dendrites you need small pipette tips around 10 MOhm anyway.
Mid you polish the tip you have better whole cell recording.
By using smaller tips, you might get the membrane to reseal itself sometimes forcing you to re-establish the whole cell config. Yo might loose your seal by doing that or you might get a different access resistance. Since you are patching Pyramidal cells, there are relatively big cells (especially if you are in Layer V), 3 to 5 MOhms (standard CsCl based intracell media) is what I would recomend. Go back to your pulling protocol and tweak it to get bigger tips.
As it has already been mentioned, the pipette resistance will limit the series resistance you can achieve. The higher it gets, the more difficult will be series resistance if you need it, particular if you want to investigate fast responding and/or highly voltage-dependent channels. I would also rather try and find a puller protocol that gives you pipettes with 3-5MOhm. By the way , which puller do you use? for Sutter pullers, we have some protocols that work well with thick walled 1,5 mm glass
we also use a P97. From what you wrote it looks like you use a box filament, we use a trough filament but the parameters should work with both. Try this:
Heat: 0-5% less than RAMP value Pull: 30 Vel: 28 Time 250 (single line, 5-6 loops)
this works well with our Thick wall capillaries (1.5mmOD 0.87mmID) yielding 3-4MOhm pipettes. If resistance is still too high, decrease either Pull or Vel in increments of 3.
Hope that helps ....
Dear Ping
Usually the pipette resistance is determined according to the size (or better to say: according to the resistance) or recorded cells. Thus, if you are trying to record from pyramidal cells, it is better to change your puller program to achieve pipettes with lower resistance. In fact, learning and practicing how to adjust the resistance of pipette electrodes is an important skill for a patch clamp recorder.
If you are voltage-clamping, you may be in difficulty if the active conductance of the cell is ~50 nS (corresponding to 20 Mohm), since about half of your IR drop will be across the series resistance and since you will have difficulty in compensating for most of the series resistance. As others have pointed out, it's best to work with pipettes having as low a bath resistance as possible.
Hello,
of course, it depends on whether you want to do current or voltage clamping. However, I would consider any pipette bath resistance over 3 MOhm unacceptable since you will end up with an access resistance of more than 5 MOhm. I would aim at pipette resistances well below 2 MOhm.
Please consider the calculation of the maximum theoretical voltage error caused by the series resistance:
Voltage error = Rs*(1-Rs-compensation)*I
with I being the maximum current amplitude. If you put in your series resistance of at least 10 MOhm and a current of 10 nA, 80% compensated, you will end up with the following:
Voltage error=10^7 Ohm * 0.2 * 10^-8 A = 0.02V (!!!)
A theoretical voltage error of 20 mV is not acceptable! You should aim at something below 5 mV which brings you to series resistances of about 2.5 MOhm.
I would just add an extra consideration. The solutions you are using (both external and internal) will affect the resistance. So, e.g. given the same pipette wide tip with two different internal solutions, you will have two different resistances.
As stated by most of the people above, the higher your starting pipette resistance, the more problems you will have with series resistance during your recording.
A general rule of thumb is to use the largest diameter pipettes that you can to reliably get a GigaOhm seal on the membrane of your target cell/structure. Also be sure that this size pipette will allow you to maintain lasting stable recording. Typically 2-3 MOhm pipettes work well for most cells--including pyramidal neurons. But if you are trying to patch dendrites or extremely small cells, obviously you will need much smaller pipettes to be able to get a seal.
If your cell tolerate high chloride internals then you can improve a bit the series resistance with such an internal. Raising the bath temperature also lowers the Rs in our hands, perhaps because the glass expands. But you will need to use electronic series resistance compensation and if your pipette has a high Rs this may cause an oscillation in the voltage-clamp circuit and you may lose the recording. That is also why its good to start with a lower Rs pipette, if you can do so.
Everything said above about "nasty" effects of high pipette resistance is generally correct. But you should be pragmatic and tailor your technical requirements for pipette fabrication to your experimental needs. In the example given above by Martin Schink, if the magnitude of typical current under study is
Hi,
There is another practical consideration that we use to decide on the highest "acceptable" Rs ( and in extension, the pipette (bath) resistance)and works fairly well if voltage-gated calcium and sodium currents are involved: can you clamp your current of interest without Rs error? It has an objective measure: the slope of ICa or INa activation curve cannot exceed 1/Rs. If either one does, you are in trouble as it indicates the lack of voltage-clamp of you cell (Marty A, Neher E (1995) Tight-seal whole-cell recording. In: Single-channel recording (Sakmann B, Neher E, eds), pp 31–52. New York: Plenum.).
Once you establish your pipette parameters for your cells of interest using either one of these currents, in my opinion you are good to go, as potassium currents are generally slower to activate. With that being said, you always have to be realistic about what do you attempt to clamp/record and space clamp problems have to be taken seriously. Say, a small ICa that contributes to transmitter release hundreds of microns away CANNOT be realistically clamped from the soma, even if INa at the axon hillock looks OK. Similarly, clamping of small synaptic inputs to distant dendrites might be problematic.
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