Part of the answer may be the liquid junction potential, which tends to be quite substantial for electrode solutions intended to mimick the intracellular milieu (e.g. around -17 mV at 35°C in our case with K-gluconate-based solution). Some authors correct their RMP values for it, others don't. An excellent source on information and help with LJP is

http://web.med.unsw.edu.au/phbsoft/LJP_Calculator.htm

Furthermore, due to the high synaptic connectivity in organotypic cultures spontaneous activity, including miniature glutamatergic PSCs, is much higher than in acute slices, which may elevate the RMP somewhat (although certainly not by the amount you stated). A 2003 paper by De Simoni et al may be a good starting point for digging deeper into the comparison between hippocampal cultures and slices: http://jp.physoc.org/content/550/1/135.abstract

Harald is right. You should always correct for the Liquid junction potential which can be empirically calculated or better directly measured. Check E Neher literature for references on LJP corrections.

I agree with the LJP explanation and this is probably a major contribution to the error. Also though, consider your access resistance. If the solution exchange between the pipette and the cell is poor (high access resistance), the RMP will be affected.

I agree with Harald. it is likely that there is something about the culture conditions that account for the difference. Slice cultures are usually made form immature slices and maintained in artificial conditions. There may be a developmental issue with regard to the expression of ion channels contributing to RMP, or altered RMP resulting from enhanced synaptic transmission. Have you tried blocking glutamate receptors with kynurate or added TTX to block all impulse-driven transmitter release?

This is Neher's paper on measuring LJP's:

Neher E. (1992). Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 207:123-31. PMID:1528115

And here's a blog post with someone's step-by-step distillation of Neher's method:

http://junctionpotential.blogspot.com/2010/10/how-do-i-measure-liquid-junction.html

How long after breaking into the cell do you measure RMP? Sometimes the internal will change the RMP (especially if you have channel blockers in there). Switching to current-clamp immediately after breaking in will give you the "real" RMP.

you got to correct for LJP, some software such as pClamp provide an estimate of it. I also suggest to discard any recordings with a series resistance (i.e. tip+ access resistance) higher than 15MOhm.

I agree with the above that LJP needs to be corrected for which may bring you closer to your expected RMP. Also, it is true that if you want to measure the cell's actual RMP you must check immediately after breaking in. However, even then you are looking at a somewhat artificial RMP that is influenced by the ACSF and internal solutions used. Also, I think that R Alberto is referring to 15 MOhm rather than mOhm. This will also depend a lot on your experiment. We routinely use 6-8MOhm pipettes to record from NTS neurons so our series resistance tends to be slightly higher.

The theoretical LJP for the K-gluconate-based solution is only -7 mV. Even without the correction for the LJP the RMP of the CA1 neurons in acute slices or neurons in dissociated hippocampal cultures is around -62 mV. My opinion is that most likely the neurons in this particular organotypic slices are not very healthy. The RMP in my hands was stable for up to 4 hours.

Hello,

LJP can be directly measured also. I do prefer to do it in this way because give more accurate results and you can actually change your solutions in the way that you want, and just correct after. There is a paper from Erwin Neher in Methods in Enzymology (vol.207, 1992) which is very good in order to learn how to measure and what the junction potential means.

I do work in another kind of cells, but I can tell you, from my experience, that the seal need to be extremely good for to have reproducible measurements of RPM.

Good luck!

I agree with Harald, but I would tell you that we are all flawed in this respect! You can't actually record the rmp in whole-cell at all! This because the rmp is dependent on the composition of both the extracellular and intrecellular solutions. You are changing (note the active tense) the composition of the intracellular solution (cytosol) during the recording, as you dialyse electrode solution into the neuron. Therefore, you will never record the rmp, as the membrane potential changes as soon as you start to dialyse and the membrane potential you record when dialysis is complete is not the rmp!!

I agree with most of the above but perhaps lean towards what Neil M. and others above have said about dialysis of your intracellular solution as an explanation of the lower values as opposed to LJP. Another possibility might be that the h-current is active in your neurons and under your conditions, which would tend to depolarize the measured membrane potential. Gian Maria Maccaferri did publish a paper on this a few years ago (Maccaferri et al, J Neurophysiology 69:2129-2136, 1993). They measured rmps of >-55 mV and suggested that the h-curent is active at the rmp in these neurons. This contrasted with earlier by Halliwell and Adams (Brain Research 25071-92, 1982) that suggested the h-current did not contribute much to rmp. The degree to which this current contributes to rmp in neurons is an issue that is often debated.

Do you have very good gigaohm-seal. I think >10GΩ seal is very important.

I think that all the above comments as well as the experience of your's truly clearly show that it is NOT reliable to measure RMP in whole cell. You can only use it as a relative indication of the state of the cell i.e. seal. And yes - measure it fast after patch openning!

Can youg give me a protocol to record RMP?

Which patch-clamp amplifier are you using? In HEKA and Pulse software your RMP can be influenced by the clamp potential in initial voltage-clamp configuration. You have to disable the soft CC switch.

One main problem in RMP measurement is the relation of membrane resistance to seal resistance. If your seal-resistance is 1 GOhm and you membrane input resistance also about 1 GOhm then you will measure only 50% of the real MP because both are in series (not in parallel). I do not know the input resistance of your cells and how large your seal is. Small cells have a high input resistance big cell a lower. It is not a problem of series resistance because in voltage measurements there is almost no current flow through electrode and seal resistance. If you know Rm and Rseal you can calculate the "true" MP from you measurement and the relation of Rm/Rseal: Vm(true)=Vm(measured) x (Rm+Rseal)/Rm. The liquid junction potential will add to that problem. Summary: In many cases true membrane potentials of small cells cannot be measured with patch electrodes accurately.

Liquid junction potential is the first thing I will look at. There is software which can help you calculate how much junction potential you have by inputting all the ions from your intracellular and extracellular solution. I am using JPCalc, which is quite handy. Sameera proposed another possibility worthwhile to have a look. In the end of your current clamp experiment, you can simply slightly pull out your electrode (still in the bath), so you can check whether the membrane potential is zero or not.

Have you looked into measuring the RMP using cell-attached mode? This would get around all the problems highlighted above using WC.

As mentioned in all above answers, there are so many factors that will affect the number of RMP. I'd like to check if the input resistance is above 200megaohms and overshoot is >20mV and amplitude of action potentials is >80mV for those cells, if all the answers are yes, the recordings are acceptable no matter what RMP is. I'd also like to check the potential while the electrode was detached the cell after whole cell recording to see if there is big value. If it is big, please check again PH of internal solution. I'd also like to check if the threshold of action potential is near the -45mV, if it is no, that indicates the problems of offset and/or solution (internal and/or external).

Thank you everyone for the great responses. In answer to some of your questions...

I am switching to current clamp immediately after breaking into the cell to view RMP.

I have not been using any toxins, based on somewhat vague methods section in a publication that I am trying to follow.

I always have a decent giga ohm seal (>2 GOhm) but not usually as good as >10 GOhm.

The K concentration of my ACSF is very close to that of the publication I am trying to follow.

The slices are prepared from rat at P5 or P6 and used starting at P9 up to P14.

I am not very familiar with cell-attached mode. After recording RMP I would like to proceed to measuring sag amplitude following hyperpolarization and induce AP. How would this work with cell-attached?

I will be looking into all of your suggestions, particularly the LJP and comparing to acute slices. Thank you again.

the concentration of EGTA is also critical for the membrane potential. The membrane potential is more hyperpolarized in my whole-cell recordings with the high (2-10 mM) vs small (100 mkM) concentration of Ca buffers.

Check out Verheugen 1999 and Tyzio 2003 for the technique of recording RMP using cell attached.

Your certainly cannot measure sag in cell attached mode, however, you may be able to break in to WC using the same pipette and do your IC experiments after measuring RMP in cell attached if you use potassium as the equimolar ion (Verheugen 1999).

http://www.ncbi.nlm.nih.gov/pubmed/10087068 and http://www.ncbi.nlm.nih.gov/pubmed/12867526

Good luck! =)

A good check is to measure then membrane potential when you pull out of the cell at the end of recording and use this as the zero potential. You might find that it is above zero, and that your cells are more negative than you thought.

May be the liquid junction potential.

...also K+ in intracellular solution can affect on RMP.

The internal saline (high K+ in this case) blown onto the cell before the GOhm seal is formed can significantly depolarize the cell. I'd suggest that you wait 30 sec to a minute AFTER you've formed the seal and BEFORE you break in. See if that makes a difference. (I know that you're immediately switching to cc mode to check RMP; that's good.)

Who can give me a protocol to record RMP? Thank you. My mail is [email protected]

I also have similar experience before, although in my case, I forgot to correct the liquid junction

These are excellent suggestions and I would follow though to caution you that RMP measured by WC pipette should always be treated with some caution. This will depend on your internal solution and what additional conductances might be activated by intracellular perfusion. If it is really important to have an exact number for this, then measuring RMP with sharp micro-electrodes(20 Mohm and more see e.g. discussion in the Sutter user manual) using a good quality micro-electrode amplifier is the preferred way. Of course you can't clamp afterwards. But you could then use the values obtained to set your holding potential. Again this depends on the importance of having the real RMP for your analysis.

Best way to look at RMP is by using sharp electrode technique with (3M KCl or variations of KCl and Kgluconate)...if that is all you are measuring. so you impale the cell and measure quickly and also maybe in 5 min just to make sure its stabilized. whole cell allows leak of your internal solution into your cell over time whereas sharps are very small so very little may leak which wont affect your RMP. so really if your main point is to show differences in RMP sharps is the best technique. but for other recordings i would stay away.

There are two factors to think about in trying to record the resting membrane potential accurately:

1) Older generation patch clamps do not provide the most accurate current clamp, due to the electronic design, which incorporates less-than-ideal opamps wired up as current-to-voltage converters. The initial opamp needs to have the input offset currents trimmed to zero when the amplifier is already warmed up (takes about 20 minutes), but even if you have eliminated the input offset current at one point in time, the currents may change over time. An uncompenstated input offset current of only 1 pA, driven through a cell having a 10 GOhm input resistance leads to a spurious offset in the membrane potential of 10 mV. Newer generations of patch clamp use a better input stage, and different configuration for current clamp, so the error is much less.

2) Indeed, the liquid junction potential can be significant, as noted by others, and it is important to take it into account when reporting the resting membrane potential. An excellent source of information is Erwin Neher's chapter in Methods in Enzymology: Neher, E. (1992). CORRECTION FOR LIQUID JUNCTION POTENTIALS IN PATCH CLAMP EXPERIMENTS. Methods in Enzymology, 207, 123-131.

There are two factors to think about in trying to record the resting membrane potential accurately:

1) Older generation patch clamps do not provide the most accurate current clamp, due to the electronic design, which incorporates less-than-ideal opamps wired up as current-to-voltage converters. The initial opamp needs to have the input offset currents trimmed to zero when the amplifier is already warmed up (takes about 20 minutes), but even if you have eliminated the input offset current at one point in time, the currents may change over time. An input offset current of only 1 pA, driven through a cell having a 10 GOhm input resistance would lead to an error of 10 mV. Newer generations of patch clamp use a better input stage, and different configuration for current clamp, so the error is much less.

2) Indeed, the liquid junction potential can be significant, and it is important to take it into account when reporting the resting membrane potential. An excellent source of information is Erwin Neher's chapter in Methods in Enzymology: Neher, E. (1992). CORRECTION FOR LIQUID JUNCTION POTENTIALS IN PATCH CLAMP EXPERIMENTS. Methods in Enzymology, 207, 123-131.

I've patched pyramidal cells in hippocampal organotypic slice cultures with both KCl and K-gluconate internals and find the RMP to be between -60 and -70 mV (without liquid junction potential correction) so something slightly weird is going on. Are your slices healthy? bad batches of horse serum or b27 can sometimes cause trouble.

The potassium -gluconate-based solution may decrease the RMP or the junction potential. should be carefully see the internal solution composition.

Getting very good seal is very important, at least 10 GΩ.

Filter your bath/pipette solutions with 0.22um filter.

Polish the tip of pipette by microforge.

Lots of good information here. An apparently elevated resting potential (i.e. not hyperpolarized enough) can arise from 4 main sources: 1) Your internal solution has changed the equilibrium potential of ions participating in Erest, 2) the liquid junction potential (as discussed above) is causing an offset from 'real' Erest, and 3) stray offsets while using a voltage-clamp instrument for current clamp, and 4) excessive leak around your electrode/cell seal. 1-3 have been well-covered above. I'll just add to #3 - an AxoClamp is an excellent current clamp that can also be used for VC (or, for that matter, an Axon Multi-Clamp if you want a more modern instrument - the Multiclamp is a better voltage clamp than is the AxoClamp). For 4 - a good seal upon break-in is no guarantee that the seal remains good after break in. Deterioration of the seal can happen for a variety of reasons - bad mechanical break in, osmotic mismatch between internal and external solutions etc. You can measure your seal resistance after break in by injecting square waves of current and measuring the instantaneous voltage jumps (make sure your capacitance compensation is tuned up). Verifying Erests with sharp microelectrodes is also a good suggestion, but this is fairly difficult in organotypic slices due to their thinness.

Hi, you probably did most of this but you could try troubleshooting in the following way: (1) thoroughly check all arithmetic associated with solutions and stocks, discard & remake solutions and try again - or use a simpler recipe; (2) if using pClamp, use the LJP calculator to see what potential may need to be added; (3) to lessen the possibility of break-in causing leak, try perforated patch with amphotericin B or nystatin ; (4) stimulate the cells for action potentials or use voltage clamp to demonstrate known current(s) to be sure you the cells are behaving as expected...

Be cautious about using perforated patch. Amphotericin and Nystatin are commonly used for perforating the membrane, and they create pores that are somewhat selective, and which therefore will introduce a series potential, depending on the ionic composition of the internal and external solutions. Check out the original papers by Alain Marty in J. Gen. Physiol.

Yes, do examine literature for your particular preparation. Another key reference on PP is:-

Low access resistance perforated patch recordings using amphotericin B

James Rae, Kim Cooper, Peter Gates, Mitchell Watsky

Journal of Neuroscience Methods

Volume 37, Issue 1, March 1991, Pages 15–26

I have performed current clamp exp. with both acute and organotypic slices and didn't find any difference in RMP between both the preparations. However, i have faced such problem of persistent depolarized RMP for a while in acute preparation and it turned out to be due to unstable offset potential (LJP) between patch electrode and external solution. I think you should check for stability of the offset potential for a duration of 15-20 mins. Just dip your patch electrode in bath solution and monitor potential difference for a while. If unstable, then rechloride the silver wire as discussed on this forum earlier.

Another good overview of LJP and how to determine it within your system:

http://junctionpotential.blogspot.com/2010/10/how-do-i-measure-liquid-junction.html

Take a look at this: "http://www.sciencedirect.com/science/article/pii/S0165027006001221"

It is not easy but may help you to find out if the problem rises from the internal solution or LJP.

Some interesting answers, but the bottom line is you can't ever measure rmp properly, and certainly not with whole-cell.  As soon as you change the ionic composition of the cytosol, the rmp will change.  The only way is to do cell-attached patch and use the reversal of a potassium channel current to estimate rmp.

You may not be able to measure RMP, but you should be able to get close to your theoretical MP based on the ionic compositions of your solutions and the resulting LJP. In my experience, a depolarised membrane potential is indicative of an unhealthy cell selection, given that all other methods mentioned above have been rigorously adhered to. Try switching up the cells you are going for and go as deep as you possibly can.