An electrophysiology quesiton

Matt Jones jonesmat at physiology.wisc.edu
Tue Feb 10 11:52:13 EST 2004


Xiaoshen Li <xli6 at gmu.edu> wrote in message news:<c08fgr$pm9 at portal.gmu.edu>...
> Hi,
> I have an electrophysiology question. I read somewhere "somatic EPSCs 
> under passive voltage clamp conditions...". What is "passive voltage 
> clamp"? I am always confused EPSC and EPSP. In voltage clamp condition, 
> are we measuring EPSCs?
> 
> Thank  you very much for your help.
> 
> Best Regards,
> Xiaoshen


Xiaoshen,

Think of Ohm's Law:

V = iR

In electrophysiology, we often refer to a "clamp" of some kind. This
means that we are using an amplifier to "clamp" one of the parameters
in Ohm's Law, so that it can't change. In Voltage Clamp, the amplifier
is maintaining the voltage, V, at some user-specified value, so that
regardless what happens to the cell (i.e., whatever channels may open
or close), the voltage will stay at the specified potential.

Now, suppose you are recording from a cell under voltage clamp (at -60
mV), and stimulate an excitatory synapse. This opens synaptic ion
channels that have a reversal potential near 0 mV (typical for AMPA,
NMDA and nicotinic receptors). Because the cell's voltage is at -60
mV, and not at the synaptic reversal potential, there is an electrical
force on ions that tends to drive them in one direction or another,
resulting in a current. This synaptic current is given by the
following equation:

Isyn = Gsyn * ( V - Esyn )

where Gsyn is the synaptic conductance (let's say 1 nanoSiemen), V is
the voltage (-60 mV) and Esyn is the synaptic reversal potential (0
mV). The difference between the cell's voltage and the synaptic
reversal potential is the "driving force" (it's not really a force,
but that's what everybody calls it). Solving this equation yields a
magnitude of -60 pA for the EPSC (excitatory postsynaptic CURRENT (not
"conductance", as another poster said)).

Isyn = 1x10^-9 S * (-60x10^-3 - 0) = -60 x10^-12 Amps = -60 pA

A negative current is a "downward" deflection on the oscilloscope, and
is called an "inward" current, because by convention a negative
current designates net positive charge flowing *toward* your
electrode. In this case net positive ions are flowing from outside the
cell to the inside, which is toward your electrode.

Ok, so -60 pA of current is going to flow into the cell. If you were
*not* in voltage clamp, this would depolarize the cell by adding
positive charge to the inside. However, you are in voltage clamp, so
the amplifier will try to prevent any change in membrane potential. It
does this by injecting an amount of current that exactly counteracts
the current flowing through the synapse (i.e., -60 pA). Again, the
minus sign means that the amplifier is causing positive charge to flow
toward the electrode, which in this case means from the cell into the
pipette. So the amplifier "steals" the same amount of charge that
flowed in through the synapse, returning the cell's interior to its
original charge state (and thus, its original voltage). It shows you
how much current it injected on the oscilloscope, and this is exactly
how much current flowed in through the synapse.

Voltage clamp is useful because it *clamps* the V in Ohm's Law. At the
same time, it *shows you* the current, i, in Ohm's Law. If you know
the voltage and the current simultaneously, you can solve Ohm's Law
for the resistance, R. A change in this resistance signifies the
opening or closing of some ion channels, and in fact, the reciprocal
of this resistance tells you the total conductance of the ion
channels, which is directly related to their open probability and
kinetics. So people use voltage clamp when they want to *clamp* the
voltage in order to study the opening and closing of ion channels.

Another thing that you can do with an amplifier is called "current
clamp". In current clamp, your amplifier is maintaining the amount of
current it injects at a user-specified value. Now, when you stimulate
the synapse, the amplifier *does not* compensate by injecting current,
it keeps the injected current fixed at whatever level you told it to.
Therefore, the synaptic current can now change the membrane potential,
and the result in the case above would be a depolarization (an EPSP,
excitatory postsynaptic potential). People use current clamp when they
want to study the *voltage changes*, including EPSPs, IPSPs and action
potentials, in response to some stimulus.

Finally, regarding the other terms you asked about: "somatic" and
"passive". Somatic means "at the soma", or "at the cell body". In
other words, the synapse is located right at the cell body, rather
than far away on a dendrite. The location of a synapse is very
important from a practical point of view in both voltage clamp and
current clamp experiments. Remember that in voltage clamp, the
amplifier has to inject current to compensate for the current flowing
through the synapse. If the synapse is far away on a dendrite, then
the current from the amplifier has to travel up the dendrite in order
to restore the potential at the location of the synapse. This is
problematic because the dendrite has a resistance to current flow, and
is also somewhat leaky, so not all of the injected current will
actually get to the synapse, and therefore the synapse will not be
completely "clamped". The potential at the synapse may actually
change. This means that a) you do not really know the V at the
synapse, and b)  the current your amplifier shows you is not exactly
the same current that flowed through the synapse. In this unclamped
situation, it is no longer possible to directly compute the
conductance of the synaptic channels, or to get a good picture of
their kinetics.

"Passive" refers to the cell acting as if it has no voltage-gated
channels. In electronics, passive components are resistors, capacitors
and inductors. They may store energy, but they cannot amplify a
signal. Nerve and muscle membranes, however, are "excitable", which
means that they *can* amplify small signals (e.g., when a small EPSP
crosses threshold, it triggers a huge action potential). So when they
say "under passive voltage clamp conditions", they mean that the
experiment is being performed so that the voltage-gated (i.e.,
amplifying) channels have either been blocked with drugs (e.g., TTX
and TEA), or that the EPSCs/EPSPs being triggered are so small that
they cannot cause these channels to turn on or off very much at all.

Passive *does not* necessarily mean that they are clamping the cell at
its resting potential, although that is often a good idea because
cells tend to be more passive near rest.


Cheers,

Matt



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