Neurophysiology – lecture 26

April 19, 2011

1 Review:
Ca2+ is held at a low value — 0.2–0.7µM inside cells generally and in neurons in particular.
1. A.P. invading synaptic terminals where Ca2+ channels are concentrated can produce sufficient ICa to
cause the [Ca2+ ]i to increase 10 to 100 fold.
2. This increase in [Ca2+ ]i is necessary because the many enzymes that are sensitive to [Ca2+ ] only become
fully activated when [Ca2+ ]i reached a concentration between 10 and 400µM.
3. Note that this is a process in which electrical activity (A.P.s) are converted into biochemical processes.
Similarly G-protein activation is often an intermediary between electrical activity and the stimulation
of biochemical processes. In both cases the small electrical signal must be amplified in order to produce
a modification in biochemical processes.
4. Neural tissue uses 4 mechanisms to maintain a low concentration of Ca2+ intracellularly:
1) buffering of Ca2+ by intracellular proteins
2) sequestration of Ca2+ into mitochondria and endoplasmic reticulum
3) Na+ - Ca2+ exchanger in the plasma membrane and
4) Ca2+ ATPase in the plasma membrane.
5. Postsynaptic mechanisms
When a voltage clamp is used to measure current in a muscle fiber after a presynaptic axon terminating
on that muscle fiber is stimulated, only a brief (about 1-2ms long) pulse of inward current is recorded.
This pulse is called an “EPSC”, excitatory postsynaptic current.
When recording transmembrane potential without the use of a voltage clamp, stimulation of the same
axon produces a depolarization with a rapid rise time (1ms or so) but with a much slower decay of 10
to 50ms. This depolarization is called an “EPSP”, excitatory postsynaptic potential.
6. The sequence of events is that the inward EPSC is returned to the extracellular fluid by a depolarizing
outward capacitive current, thus producing the EPSP.

2 Determining the reversal potential (Vrev ) of the EPSC, Isyn

1. Referring to Figure 18A, we note that the scale to the left in the figure indicates 0nA when Vm =
-50mV. This is the cells resting potential when the net current flow across the cells membrane is 0. To
hyperpolarize the cell to -70mV the experimenter must pass current into the intracellular microelectrode

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 (thus removing + charge from the inside of the cell) and this removal causes + charge (current) to
flow into the cell. Therefore, there is a steady inward current shown throughout the -70mV trace. And
as greater and greater hyperpolarizations are required larger and larger inward currents are needed to
maintain those hyperpolarizations.
Conversely, to maintain a depolarization of the transmembrane potential a sustained outward current
must be applied.
Thus, all the current records in Figure 18A are offset from the 0nA value because of the steady currents
that must be passed to hold the Vm at some desired voltage.
2. When the axon is stimulated to produce an A.P. and release ACh , a brief (2 ms) inward current, Isyn ,
is seen when the transmembrane potential is held more negative than 0mV. This Isyn is outward when
the transmembrane potential is positive relative to 0mV.
3. I measured the amplitude of each of these 7 Isyn ’s and plotted those values against the transmembrane
potential at which the cells membrane was being held. This produced Figure 19A.
4. We note that the relationship between Isyn and Vm portrayed in this figure is linear. From our vast
mathematical background we remember that for a linear relation; y = ax + b, where a determines the
slope of the relation and b determines the intercept of the line with the Isyn = 0nA axis.
The line in Figure 19A fits the equation: Isyn = gsyn (Vm − Vrev ) where Vrev = the transmembrane
potential at which Isyn = 0nA.
Note that if I shift this equation around: Isyn = gsyn · Vm − gsyn · Vrev and consider Isyn to be y and
Vm to be x, then gsyn is equivalent to a and determines the slope of the line in the above graph while
gsyn · Vrev must be equivalent to b and determine the intercept of the line with the Isyn = 0nA axis.
5. If we add curare to the preparation (Figure 18B) we see that the slope (a) of the Isyn and Vm graph is
changed, as we would expect since curare blocks the postsynaptic receptors and hence would change
their collective conductance. On the other hand curare does not change the Isyn = 0nA axis intercept
and hence does not change Vrev .
6. To explain what currents contribute to Isyn , we initially assumed that all the ionic currents we know
about may be contributors, i.e., Isyn = INa + IK + ICl + ICa .
7. In Figure 18C we see that changing [Cl]o changes the slope and hence must change gsyn . On the other
hand changing [Cl]o does not change the Isyn = 0nA axis intercept and so must not change Vrev .
8. However, if ICl contributed to Isyn and ICl = gCl (Vm − ECl ), when we change [Cl]o we know that ECl
will change and hence ICl will change and therefore Isyn will change and specifically its Vrev should
change - but it didnt. Therefore, we conclude that ICl must not contribute to Isyn .
9. In Figure 18D we see that changing [Na+ ]o does not change the slope, a, of the graph and so does not
change gsyn . On the other hand changing [Na+ ]o does change the intercept of the line, which must
have occurred because ENa has changed which altered Vrev . Thus INa must contribute to Isyn .
10. In Figure 18E we see that changing [K+ ]o very slightly changes the slope of the line suggesting it does
not change gsyn much. But changing [K+ ]o does change the intercept, Vrev . So like INa , IK must
contribute to Isyn .
11. In summary, these tests for changes in Vrev indicate that Isyn = INa + IK .

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3 How much does INa and IK contribute to Isyn ?
1. At Vrev , Isyn = 0nA, so INa + IK = 0. And gNa (Vrev − ENa ) + gK (Vrev − EK ) = 0. This equation can
be rearranged to:
gNa EK − Vrev
= (1)
gK Vrev − ENa
gNa
2. So the ratio of gK can be calculated whenever the Vrev is known.
gNa
3. For example if ENa = +50 mV and EK = −90 mV and Vrev = 0mV, then gK = 1.8, i.e., the conduc-
tance for Na+ is higher than that for K+ .

4 Electrical models of synaptic inputs

1. The simple electrical model of a synapse will have 2 parallel pathways: one for INa and one for IK . In
each of these pathways there will be a fixed resistor, since each pathway allows only a limited flow of
current. The INa pathway will contain a battery equal to ENa , while the IK pathway will contain a
battery equal to EK .
2. As described above these pathways would always be present between the inside and the outside of the
cell, but of course synaptic inputs are not always active. To remedy this, we put a simple switch into
both pathways between the inside and outside of the cell. This switch is closed by acetylcholine and is
open when acetylcholine is not present.
3. Localization of acetylcholine receptors:
(a) Electrical stimulation of an axon going to a muscle fiber or ionophoretic application of ACh to
a muscle fiber elicits a sizeable EPSP only when the application is applied within fractions of a
millimeter of an actual NMJ not all along the muscle fiber. Thus ACh receptors appear to be
only found at the NMJ.
(b) In Figure 19B, ω-conotoxin which has been made fluorescent by attaching fluorescent antibodies
to it is located in the presynaptic terminals of the axons synapsing on muscle fibers (picture to
the left). Labeled α - Bungarotoxin which binds to ACh receptors, is shown in the center picture.
The two images are shown to neatly overlap in the right-hand picture.
Thus, the area on the muscle fiber membrane which has ACh receptors is strictly limited to the
area under the presynaptic terminal.
4. In Figure 18F we see that EPSPs generated by stimulation of the axon entering an NMJ are large
at the synapse, but decay in amplitude as one moves along the surface of the muscle fiber away from
the synapse. Four millimeters away from the synapse the EPSP is hardly visible. Since some muscle
fibers are 10 or more centimeters long, this EPSP decay would result in only a very poor mechanism
to excite muscle fibers to contract.
5. Instead, the EPSP are the NMJ, which can reach a depolarization of 50mV, acts by triggering an A.P.
in the electrically excitable membrane next to the synapse. This action potential is then propagated
in both directions along the muscle fiber in a manner similar to the way A.P.s are propagated along
axons. The propagated A.P. causes the influx of Ca2+ and the release of intracellular Ca2+ to produce
the muscle contraction.
6. As an exercise you might try to draw an Electrical Model of a synapse, as described above, surrounded
by electrically excitable muscle membrane.

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5 Neuromuscular Junction
Why is there no conflict between Na+ going into a muscle fiber through small open channels at a NMJ and
K+ coming out of that same pore?
1. In our formulation we wrote Isyn = INa + IK . This implies that INa and IK go through the same
channels and do not interfere with one another.
2. However, there are about 6 million ions/second that flow through an ACh receptor, you would think
there would be some conflict.
3. As Dillon pointed out, the trick is that it only takes an ion 10−13 seconds to traverse the cell membrane,
made possible by the fact that cell membranes are so thin.
4. Thus, (6 · 106 ions)(10−13 s · ion−1 ) = 6 · 10−7 s that the channel contains an ion.
The other way of saying this is 99.99994% of the time the channel is empty, and so poses no problem
for another ion to pass through.