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Measuring the rest potential of a membrane is relatively easy and straight forward: put one electrode inside the neuron, the other one (the "counter electrode") outside the neuron.

Because everything is at equilibrium (steady state), it's not important where exactly inside the neuron you place the first electrode, and even less important where outside the neuron you place the counter electrode: in the extracellular fluid next to the neuron or inside a container filled with extracellular fluid placed anywhere. One may even connect the other electrode to the ground (a lightning rod), if one does it consistently (over all measurements).

But now consider you want to measure the spread of depolarization along the membrane by measuring the membrane potential at two different locations of the membrane: You place one electrode underneath the membrane at point $p_1$ (and its counter electrode to the ground). And you place another electrode underneath the membrane at point $p_2$ (and its counter electrode to the ground).

Now it seems possibly important where exactly inside the neuron you place the two electrodes: the distance from the membrane might make a significant difference. (But maybe not.)

What probably would make a significant difference: Since the ionic compositions of the extracellular fluid in the vicinity of $p_1$ and $p_2$ will differ, it might be important to connect the counter electrodes not to the ground (or to some volume of normal extracellular fluid) but to corresponding points $p_1'$, $p_2'$ directly opposite to $p_1$, $p_2$ on the other side of the membrane.

I emphasized might be important because in practice it may be not important. That's what my question is for:

Is it OK, even for the measurement of the dynamics of depolarizations, to connect the counter electrodes to the ground (if only consistently). What would be the difference in the measured voltages, and can these be determinstically be cancelled out?

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It is extremely important where you put your electrode when measuring such delicate structures as dendrites (which is when you want to compare voltage changes between two parts of a neuron). That is why you put your electrode inside the dendrite. Each recording electrode has its own ground that goes inside the bath, i.e. outside the neural tissue. Alternatively, you can observe fluorescence changes of a calcium or voltage dependent indicator that you have introduced inside them.

https://www.janelia.org/sites/default/files/Labs/Spruston%20Lab/Stuart_Spruston_2015.pdf

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  • $\begingroup$ That's what I presumed: I put two electrodes inside the dendrite. But where to put their respective "counter electrodes"? Or do I use only two electrodes (both inside the dendrite), the second being the counter electrode of the first? But how to take measurements at three, four, many points on the membrane? $\endgroup$
    – Hans-Peter Stricker
    Commented Sep 15, 2017 at 13:48
  • $\begingroup$ The ground goes inside the bath, i.e. outside the tissue. I am not sure I've seen any paper with more than three recording electrodes, but of course I cannot exclude that it exists. A 'usual' setup is having one electrode in the soma and one or two more electrodes on the dendrite. Needless to say, these are very challenging recordings and very few labs in the world are able to perform them. That is why fluorescence imaging of activity is becoming more popular, despite its limitations. $\endgroup$
    – vkehayas
    Commented Sep 15, 2017 at 13:53
  • $\begingroup$ I've edited my answer to address your comment. $\endgroup$
    – vkehayas
    Commented Sep 15, 2017 at 14:00
  • $\begingroup$ Llinás in his "I of the vortex", p.9 evoked the following picture: "[The membrane voltage] may oscillate in a manner similar to the traveling, sinusoidal waves that we see as gentle ripples in calm water, and are weakly chaotic." What you say ("no paper with more than three recording electrodes") means - not surprisingly - that we will probably never see these ripples measured - but only simulated. (Do you know a reference for such a simulation?) $\endgroup$
    – Hans-Peter Stricker
    Commented Sep 15, 2017 at 14:36
  • $\begingroup$ (I already asked this here: biology.stackexchange.com/questions/65550/… - without explicitly mentioning membrane potentials, being another kind of "item"). $\endgroup$
    – Hans-Peter Stricker
    Commented Sep 15, 2017 at 14:37

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