Neurophysiology – lecture 14

February 24, 2011

1 Review:
1. How are ECl and chloride concentrations determined?
2. Let us assume that Cl− concentration inside and outside the cell were in the beginning the same. But
the intracellular resting potential is negative and is maintained by K+ fluxes. So this intracellular neg-
ativity will drive Cl− ions out of the cell. This efflux of Cl− will increase the extracellular concentration
of Cl− and decrease its intracellular concentration. Therefore a concentration gradient for Cl− will
develop which will produce a small inward Cl− flux. The increase in extracellular Cl− and decrease in
intracellular Cl− will continue until the concentration gradient for Cl− is sufficient to produce as large
an inward flux of Cl− as the efflux of Cl− driven by the voltage gradient. At this time there will be no
net Cl− flux and an equilibrium will have been reached. The Vrest at this time (determined principally
by K+ fluxes) will then be equal to ECl since there is also no net flux of Cl− at this time. Thus ECl is
always very close to the resting potential of the cell and Cl− is said to be “passively distributed.”
3. The properties of action potentials were then outlined:
(a) They are All-or-None.
(b) A threshold depolarization is required to elicit an action potential.
(c) The peak of the action potential “overshoots.” 0 mV.
(d) For a short period of time after an action potential has been produced, it is impossible or simply
more difficult to produce a second action potential. This property is termed “refractoriness.”
4. But it was also demonstrated that during a long and strong depolarization a second and third action
potential is elicited and that these later action potentials are of smaller amplitude that the initial
action potential. Thus action potentials can be Some-or-None on occasion. Additionally, after a long
membrane hyperpolarization the transmembrane potential of the cell returns to resting potential but
often will produce an action potential during this return. In these “Anodal Break.” cases the threshold
for action potential production appears to be hyperpolarized relative to the resting potential, so a
depolarization is not always required to produce an action potential.
dVm
5. At the very peak of the action potential = 0 so one can analyze the ionic basis of the action
dt
potential peak using the Nernst or G-H-K equations since the assumptions on which these equations
are formulated are all met.
6. As a test of which ion permeates the cell membrane best at the peak of the action potential, the
concentration of Na+ in the seawater surrounding a squid axon was varied while the amplitude of
elicited action potentials was recorded. It was found that as the concentration of Na+ decreased the
amplitude of the elicited action potentials also decreased. These data when plotted on a graph of action

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 potential peak vs. log of the [Na+ ]o could be well fit with transmembrane potential values obtained
from the G-H-K Equation with
PK : PNa : PCl
1 : 25 : 0.45
Thus the overshoot of the action potential peak is attributable to a very high PNa causing Vm to
approach ENa .

2 What about other properties of action potentials - their thresh-

old, refractory periods, etc.?
1. Presently what we can explain about A.P.s is the resting potential from which they arise, the trans-
membrane potential at the very peak of the A.P. and the transmembrane potential at the very bottom
of the “afterhyperpolarization.”
2. The rest of the A.P. waveform is a mystery since we cannot employ the Nernst or G-H-K Equations
dVm
to potentials in these areas since does not equal 0 at other times during the A.P.
dt
3. Can we generate a better explanation of A.P. waveforms using the Electrical Membrane Model?
In trying to do this it must be noted that:
PNa must be changing since it is so different before the A.P. and at the A.P. peak .
gNa must be changing because it is proportional to PNa .
PK must be changing since it is different before the A.P. and during the afterhyperpolarization.
gK then must also be changing.
Vm is clearly changing.
Further to account for the rise and fall of the action potential INa must be increasing.
Likewise to account for the bottom of the afterhyperpolarization it must be that IK is increased.
Thus most of the parameters governing transmembrane potentials in the Electrical Membrane Model
seem to be changing.
This makes it impossible to solve any equations we might use like: INa = gNa (Vm − ENa ) since all the
terms but ENa are unknown and changing from rime to time during the A.P.
4. The solution to this problem has been to:
(a) Make one of the variables in the equation into a constant.
(b) Measure the values of another variable
(c) and then use these 2 known values to determine the value of the third variable.
5. Specifically,
(a) Vm is made a constant with the use of a Voltage Clamp.
(b) The current required to do this is measured.
Ii
(c) The conductance gi is calculated from gi =
V m − Ei

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3 The Voltage Clamp and Voltage Clamping
1. In Figure 9A two symbols for amplifiers are shown each with a + and a - sign inside the representative
symbol. The input at the + sign is called the “noninverting” input and indicates that voltage signals
presented to the amplifier at this contact point will be seen on the amplifiers output as having the
same polarity and form as the signal that went into the amplifier. (Figure 9A left) Signals presented
at the “inverting.”input will be seen at the output as having the opposite polarity from the signal
presented.(Figure 9A right)
2. The basic structure of a voltage clamp is portrayed in Figure 9B.
The amplifier portrayed to the right simply records the transmembrane potential, Vm , as other pream-
plifiers we have talked about do.
The output of this amplifier is fed to a second amplifier at the inverting input.
The voltage command from a stimulator or other controllable voltage producing device (the box labeled
“Command.”) is fed to noninverting input on this second amplifier.
Therefore the output of this second amplifier equals Vc − Vm .
The current generated at this output is proportional to (Vc − Vm ).
This current was fed into the squid axon on a thin wire electrode.
This current flows through the axon cytoplasm and then through the axon membrane.
The circuit in which the current flows is returned to the current source (command) through ground to
complete the circuit.
3. The basic idea is that the current supplied to the axon is proportional to (Vc − Vm ).
With this in mind let us consider 3 cases.
Case 1 The experimenter sets the Vc = Vm , then since (Vc − Vm ) = 0, no I will flow through the
voltage clamp and the transmembrane potential will stay at the voltage it was at.
Case 2 The experimenter sets the Vc = Vm − 10 mV, then (Vc − Vm 10 mV) = −10 mV and so the I
generated will be −I or inward I.
That is, the intracellular electrode will become negative relative to the intracellular potential
because it is removing + charge from within the axon. This means that + charge will be removed
from the membrane surface on the inside of the membrane leaving it more negatively charged.
Thus inside the axon becomes more negatively charged, i.e., it gets hyperpolarized. When it is
hyperpolarized by 10 mV, Vc = Vm and the capacitative current, Ic , will decrease to 0 and Vm will
stay very close to this more hyperpolarized voltage. However, the fact that the transmembrane
potential is now hyperpolarized relative to its previous resting potential means that the balance
of ionic currents that was present in the resting state will no longer be perfectly balanced. This
will result in a small sustained inward current across the membrane; this is called the “leakage
current,” IL .
The leakage current IL Rin = −10 mV.
The current record obtained from this voltage step will show a spike of inward current at the
onset of the voltage step which is the Ic charging the membrane by -10 mV followed by a small
sustained inward IL .
Case 3 The experimenter sets the Vc = Vm + 10 mV, then (Vc − Vm + 10 mV) = +10 mV and so
the I generated will be +I or outward I. That is, the intracellular electrode will become positive
relative to the intracellular potential because it is adding + charge to within the axon. This means

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 that + charge will be added to the membrane surface on the inside of the membrane. Thus the
inside of the axon becomes more depolarized. When it is depolarized by 10 mV, Vc = Vm and the
capacitative current, Ic , will decrease to 0 and Vm will stay very close to this more depolarized
voltage. However, the fact that the transmembrane potential is now depolarized relative to its
previous resting potential means that the balance of ionic currents that was present in the resting
state will no longer be perfectly balanced. This will result in a small sustained outward current
across the membrane; another example of a “leakage current,” IL .
This leakage current IL Rin = +10 mV.
The current record obtained from this voltage step will show a spike of outward current at the
onset of the voltage step which is the Ic charging the membrane by +10 mV followed by a small
sustained outward IL .
4. Note that the Ic and IL produced by the voltage step of -10mV should be of equal amplitude and
opposite direction to the Ic and IL produced by the voltage step of +10mV. Thus when these 2 current
records are summed together the resultant current should be 0pA.
5. Thus Ic and IL can be eliminated from a complex pattern of currents by summing the record obtained
with a particular voltage step with a current record produced by a voltage step of equal amplitude but
going in the opposite direction.
6. The ability to remove Ic and IL from the total current record will help in obtaining the final objective
of voltage clamping which is to determine the contribution of the various currents to the total current
record. That is, the Electric Membrane Model we presently have suggests that the total current through
the membrane, IT = Ic + IL + INa + IK , where each of these currents has a separate pathway in the
model. IL does not vary with time and so is similar to ICl but includes currents carried by other ions
as well.