An electrophysiology quesiton

Xiaoshen Li xli6 at gmu.edu
Wed Feb 11 04:17:34 EST 2004


Matt,

Thank you for your wonderful reply. It clears my mind a lot.

Now I understand that, when somebody shows an action potential curve or 
a neuron is bursting, most of time that was obtained by current clamp 
experiment.(Voltage clamp holds the neuron at certain membrane potential 
below the AP threshold, therefore the neuron cannot go through 
Hodgkin-Huxley cycle).
For single channel studies, voltage-clamp is used.

To study synaptic transmission, I am still unclear why sometimes an 
electrophysiologist wants to measure EPSC and sometimes EPSP. (I read a 
paper which talks about EPSP data for a while, then EPSC data for a 
while. My eyes were searching "P" or "C" and my mind cannot follow the 
logic behind it).

Thank you very much for your help.


Sincerely,
Xiaoshen

By the way, you said that current clamp inject a fixed amount of current 
into the neuron and doesn't change it even when the channels are open 
and the current flows in. Originally I thought the amplifier circuit 
will adjust its battery to increase its injecting current so the 
resulted net current be the constant fixed value and this battery 
adjustment gives the reading of EPSP. Could you explain it a little 
more? Thank you again.

Matt Jones wrote:

> 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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