IUBio

Properties of hippocampal neurons of rat embryons?

Matt Jones jonesmat at physiology.wisc.edu
Tue Jan 8 12:39:43 EST 2002


In article <a1f74j$qb76q$1 at ID-9504.news.dfncis.de> Edmund M¸ller,
edmund.mueller at freenet.de writes:
>Hi,
>
>> I'm not entirely sure I understand the question. Are you asking for
>> -recipes- for recording solutions for hippocampal embryonic
neurons?
>
>No, actually my problem is, that I had an intrcellular recording
solution
>with apparently had no Na+ in it. AFAIK the concentration of Na+
>intracellular is not high but surely higher than 0 in physiological
>condition. Afterwards I really wonder how the experiments worked
anyway, and
>how to calculate a nernst potential for Na+ without knowing the
>intracellular concentration of it. 



Aha. The old "why doesn't the sodium current reverse at plus infinity
if I don't include sodium in the pipette" question...

You are right  that internal sodium is "not high but surely higher
than 0 in physiological condition". I actually think it's likely that
sodium can get quite high, e.g., several millimolar at least,
depending on neuronal activity.

There are two seperable aspects to the question:
1) What is the internal sodium concentration under normal conditions
(i.e., -before- you start the recording and rupture the cell membrane)

2) Why does the cell behave as if it has some internal sodium when you
didn't put any in the pipette solution?

The second question is easier to answer: The internal concentration of
any ion is -never- excactly the same as the pipette concentration
because a) the cell has its own passive and active mechanisms for
trying to keep concentrations the way it likes them, and b) the most
ion channels through which reversals are measured are not purely
selective for a single ionic species. This is more obvious with
certain ions than others. For example, the measured reversal
potentials for potassium are often very close to the predicted Nernst
potential, whereas for chloride the measured reversal potential tends
to be dragged toward about -75 mV regardless of what's in the pipette
solution. The predicted chlordide reversal in my intracellular
solutions is usually around -90 to -120 mV, but the measured reversal
for the IPSC or GABA-activated currents is very reliably about -75 mV.
(This is not due to bad voltage-clamp, by the way, because the IPSC
has the same reversal potential as the current evoked by puffing GABA
directly at the soma).

You can't calculate the Nernst potential without knowing the internal
Na conc. However, you could try to estimate the internal concentration
if you measure the reversal potential. If you are studying a
voltage-gated sodium channels, this will not be easy to do in a
neuron. To get around the fact that current amplitude varies with
voltage due to channel  gating as well as due to ionic permeability,
you have to perform a "tail-current" experiment to properly measure
the open channel I-V. For a sodium current, this requires extremely
fast and accurate voltage-clamp, which really can't be obtained in a
neuron that has extensive dendritic and axonal processes.

If it's imperative that you measure the sodium channel reversal
potential, I suggest doing it either in acutely dissociated cells that
don't have a big space-clamp problem because they are small and round,
or in an outside-out patch.

>I wonder if it's possible, that on the
>way through the bath the pippette with the intracellular solution
gathered
>Na+ from the extracellular solution through the tip by diffusion that
>eventually I got something like a physiological Na+ concentration in
the
>pippette before sealing and going to whole cell mode. Otherwise if
there was
>no Na+ in the pipette then, the Na+ of the cell would have diffunded
away in
>the pipette, wouldn't it?

Although it's possible that the pipette got some sodium from the bath,
I think this would be a very small contribution. If you are using
positive pressure while in the bath, that should blow the external
solution away from the tip. And even if you're not, I would expect the
amount of sodium that got in to be pretty small, and to be diluted
almost to nothing by the huge excess volume of internal solution, as
you suggest above.

I think it's more likely that the cell simply has it's own mechanisms
for allowing sodium entry at rest, through non-specific cation leak
channels, pumps, etc. Most people -don't- put sodium in their internal
solution (I never do). The predicted Nernst potential in this case is
infinitely positive, which obviously doesn't happen.  So there must be
some sodium in there. It's either coming through leak channels,
through the leak in the pipette-to-membrane seal (for whole-cell, this
leak is often equivalent in conductance to many open channels), or
through sodium present in the other salts or water being used. Getting
absolutley pure water is almost impossible. People who study NMDA
chanels are acutely aware that even distilled deionized water contains
enough glycine and magnesium to create artifacts).


>My question is actually what
>would be the natural intracellular concentration of a living cell
>physiological extracellular solution. What are the nature made
recipes of
>these two solutions at let's say 25° C.

This is pretty hard to answer accurately. Think about it. How would
one go about determining the intracellular concentration of an ion
without disrupting the system somehow?  One could try sampling the
internal concentration with a whole-cell pipette, and then calculating
the original concentration from knowledge of the pipette volume, etc.
But that requires rupturing the membrane, which would alter the native
concentration  at least a little. One could try using
membrane-permeable ion-sensitive dyes, but that entails lots of
complicated issues about callibrating the dye for the internal
environment. Such calibrations are approximate at best. Or one could
try making an on-cell patch recording (without rupturing the
membrane), and measuring the reversal potential of channel currents in
the patch. This has two problems: a) you don't know the membrane
voltage accurately, and b) the channel might not be perfectly
selective for the ion you are interested in.

I don't know how people originally decided what internal solutions to
use to mimic the native state. Possibly by fractionating cells and
measuring the ion concentrations in various fractions, or something
like that. Would be interesting to know how this was originally done.


Cheers,

Matt




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