IUBio

Properties of hippocampal neurons of rat embryons?

Matt Jones jonesmat at physiology.wisc.edu
Thu Jan 10 12:18:25 EST 2002


In article <12to3u0ac5b4o01nr29br7pnvoe2ojrfm4 at 4ax.com> Edmund M¸ller,
edmund.mueller at freenet.de writes:
>Hi,
>
>>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
>
>Could you please give me some sources in literature for this, since I
cannot
>mention this posting in my work, I think :-)


Damn, I was afraid you would ask for that.  Unfortunately, I am not
sure this has been very well documented in the literature. In general,
chloride handling by neurons is not a particularly well-studied area,
although two authors you might look at are Kai Kaila and Kevin Staley.
 Both of them have worked on the issues of how chloride reversal
potentials can change dramatically under repeated synaptic
stimulation, partly due to redistribution of chloride concentrations.

Sorry I can't help with a more specific reference. I was simply
speaking from (unpublished) personal experience.







>
>>if you measure the reversal potential. If you are studying a
>
>Too late, no means and no time any more. Do you know a most likely Na
reversal
>potential I could simply assume for that kind of cells?


Well, I suppose that taking the -amplitude- of an action potential at
least gives a lower limit on the Na-reversal potential. so that would
mean at-least- +40 mV.  I think if you solved Nernst assuming 140 mM
Na_out and about 3-10 mM Na_in, you would probably be in the right
ballpark.


>
>>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. 
>
>That's right. Actually I couldn't believe this myself. But what do I
know? Maybe
>the force of brownian motion can overcome the flux in some way. I
have never
>seen a diffusion equation taking into account the presence of
material flow
>under pressure. So maybe with solution under pressure one can forget
about any
>diffusion equation. Correct me please if I'm wrong.


I'm sure that people have worked out diffusion equations with drift.
For sure there's a whole theory on diffusion with drift caused by an
electrical potential (I think Hille's book discusses this). 
Basically, if you make all the typical simplifying assumptions such as
uniform flow velocity at the pipette tip, etc, I think the argument
would look like this:

With -no- flow and -no- pipette potential, diffusion through the
pipette tip will be a simple (well, sort of simple, as diffusion
equations go) function of the tip size, the diffusion coefficient, and
the concentration difference across the tip. I can't solve this on the
fly, but I'm guessing that you would get some rate of flux into the
pipette tip, which corresponds to some average velocity of molecules
in that direction. This velocity would be in the vicinity of 1 micron
per milisecond probably. In the -presence- of flow, this diffusion
still occurs, but is opposed by the flow in the opposite direction.
Ergo, any flow greater than around 1 micron per millisecond would
effectively prevent accumulation of Na into the pipette. If you're
like me, your pipette flow under positive pressure is probably many
many microns per millisecond (you can guess how fast it is by watching
how fast junk at the bottom of the dish gets blown out ofthe way ->
pretty fast).

By the way, here are two  references for diffsion between pipettes and
cells. One using experimental measurements:
Pusch M, Neher E. Rates of diffusional exchange between small cells
and a measuring patch pipette. Pflugers Arch. 1988 Feb;411(2):204-11.

and another one theoretical:
Mathias RT, Cohen IS, Oliva C.
Limitations of the whole cell patch clamp technique in the control of
intracellular concentrations. Biophys J. 1990 Sep;58(3):759-70.


>>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. .... 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)
>
>That's what I thought, too. I supposed that the flow of Na out of the
cell into
>the pipette by diffusion would be compensated by an equally inflow
from leak
>channels and the seal into the cell. I could even imagine that most
of the
>external Na "tries" to get in at the seal where it is in direct
vicinity of the
>Na-hungry pipette tip which takes it away like a sort of vacuum
cleaner. In that
>case the original [Na] wouldn't perhaps even be touched very much.
But maybe
>these thoughts are too adventureous.
>
>Thnx
>Edmund


I think having the pipette in the cell, acting as a perfectly
absorbing sink, necessarily implies that the final cellular Na conc.
must be lower than what it would be if the pipette wasn't there. But
depending on how much leak there is into the cell, the pipette may not
lower the Na conc. very much.  The Na has to get into the pipette via
diffusion alone, and as i mentioned above that process would probably
occur reasonably slowly in the grand scheme of things. It has to
diffuse through a pipette tip hole that is only 1 or 2 microns wide. 
The cell, on the other hand, has hundreds and hundreds of square
microns of surface area, much of which contains channels that could at
least potentially act as a sodium source. So it could be that the
influx through the leak is quite able to compensate for the drain into
the pipette, and that a steady state is reached that's not very far
off from the original resting state, even if no sodium is present in
the pipette solution.


Cheers,

Matt




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