EFFECT OF TEMPERATURE ON NERVOUS THRESHOLD
by
MICHAEL BINDER
SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF
SCIENCE
at the
MASSACHUSETTS INSTITUTE OF
TECHNOLOGY
June,
1975
Signature of Author...,..
Department of Biology,
Certified by...-,..
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A
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apei14,
1975
Supervisor
Thesis Supervisor
Accepted by...
Chairman, Departmental Committee
on Graduat,B Students
ARCHIVES
MAY 21 1975
1111A eF-*
Abstract
The effect of temperature on threshold bf excitation
is investigated in frog nerve by the methods of Newman
and Raymond (1971).
In a nerve depressed by past activity
threshold has a negative temperature coefficient.
When
temperature is changed very rapidly the corresponding
change in threshold is
The second,
seen to consist of two components,
slow component is
shown to bear resemblance
to the accumulation or decay of depression as defined by
Newman and Raymond (1971).
Table of Contents
Abstract
page 2.
List of Figures
page 4.
Acknowledgements
page 5.
Effect of Temperature on Threshold
Introduction
page 6.
Methods
page 15.
Results
page 22.
Discussion
page 33.
Conclusion
page 41.
Bibliography
page 42
List of Figures
Figure 1.. .....
Apparatus
page 16.
Figure 2.,,.....Stimulating Block
page 17.
Figure 3.......Adjusting the Electrode
page 20.
Figure 4.......Threshold vs Time in an
Undepressed axon
page 23.
Figure S.......Early Supernormal period
page 26,
Figure 6.......Accumulation and decay of
Depression
page 27.
Figure 7,,......Threshold vs Time in a
Depressed axon
page 29.
Figure 8.......Threshold Oscillation in
Slightly Depressed Axon
page 30.
Figure 9.......Threshold Oscillation in
Depressed Axon
page 31.
Figure 10. ..... Threshold vs Time in an
Undepressed Axon
page 39.
5
Acknowledgements
I must acknowledge a great personal debt to
Dr. J.Y. Lettvin for telling me how to be a scientist.
This work would not have been possible without the
assistance of America's batrachian masses and the staff
of R.L.E.
Lastly I dedicate this thesis to my advisor and
friend, Dr. S.A. Raymond, whose empathic tendencies have
caused him to suffer greatly during my many misguided
efforts.
-6-
I.
INTRODUCTION
Nerve physiologists have been so
concerned with mechanisms of transmission
that they have paid little attention to
events which persist longer than 100
milliseconds. Yet the most interesting
nervous impressions are those which last
Systefor seconds, minutes, lifetimes.
called
behavior,
matic modifications of
conditioning, have been produced...
Perhaps a comparison of persistent changes
in excitability in nerve centers of varying
degrees of organization may point the way
to understanding this most intriguing
property of nervous systems.
(Prosser 1952)
The mechanisms of nervous transmission mentioned
above are the fast nervous phenomena which directly give
rise to the action potential.
Neurophysiologists have
been very successful describing these fast processes
(Hodgkin and Huxley 1952, Fitzhugh 1969) in large measure
because the processes seem to persist when the nerve is
in a nonphysiological state.
The durability of the
nerve to conduct under adverse circumstances has allowed
investigators to reduce all manner of electrical artifact.
The study and theory of slow electrical processes
has not evolved as rapidly or successfully.
Because the
-7-
slow processes are closely related to the nerve's
metabolic
activity or to the nerve's intimate associ-
ation with other tissues, investigators have not been
able to reduce electrical artifact without introducing
severe physiological artifact.
A.
Slow Phenomena
Gotch and Burch (1898) were the first to notice
the slowly declining negative aftercurrent that follows
an action current, and Forbes and Thatcher (1920) found
that it might last for seconds in winter frogs.
With
frog nerve Boruttau and Frolich (1904) and Amberson
and Downing (1929) found respectively that condition of
the nerve and previous activity affected the aftercurrents.
Aftercurrents were measured in these studies across a
dead-live junction, and the 1920's were a time of debate
as to the propriety of making measurements this way.
In
particular there was confusion as to the very existence
of the positive afterpotential corresponding to the
positive aftercurrent (Woronzow 1924).
In 1930 Gasser
and Erlanger by differential application of drugs and
temperature changes to the nerve at the recording electrodes proved that the positive afterpotential following
-8-
a single impulse was an artifact of the dead-live junction.
By 1973 Gasser was able to publish a review of afterpotentials and it was perfectly clear that different
nerves, even different fiber types in the same nerve had
markedly different properties.
The positive afterpoten-
tial was reinstated as a phenomenon of type C fibers
following an impulse and of type A fibers following a
tetanus.
Following tetanus a positive afterpotential
might last for many minutes.
B.
Correlations
It was natural that physiologists should attempt
to correlate
afterpotentials with other measurable
signs of nervous activity.
In 1933 Hill showed that
heat production of the nerve occurred in two phases,
and that there was a temporal correspondence of these
two phases to the negative and positive afterpotentials.
Schmitt and Gasser (1933) poisoned a nerve with carbon
monoxide, reversed the action with bright light, and
concluded that the negative afterpotential is a sign of
metabolic activity.
This was in opposition to Levin's
(1929) conclusion that the negative potential was a sign
-9-
of accumulated catabolites and was reduced by metabolic
activity.
Shanes (1951) and Frankenhauser and Hodgkin
(1956) showed that accumulation of potassium played a
role in the production of afterpotentials.
reasonable to conclude from this history
It seems
that the
afterpotentials are the net result of several oscillating processes (Gasser 1937, Shanes 1958).
C.
Threshold as a Correlate of Afterpotential
By far the most striking correlate of afterpotentials
has been threshold of excitation.
In 1912 Adrian and
Lucas gave the first lucid description of the supernormal
phase of nervous excitability.
Gasser (1931) showed
that afternegativity and supernormal phase are both affected similarly in a variety of experimental circumstances.
Graham (1935) showed under what conditions a subnormal
phase of excitability might arise, and that these same
conditions generally accentuated the positive afterpotential.
Lorente de No (1947) gave the best description
to date of the complex interrelationship of membrane
potential to threshold.
All other membrane parameters
held equal, the excitability is a relatively simple
function of membrane potential; however, the membrane
-10-
potential usually varies as a function of some other
parameter of which excitability is also a function.
Despite the difficulties in relating membrane potential
to threshold Gasser and Grundfest (1936) felt that
rather than compromise the integrity of their preparation
by recording potential directly, they would infer the
potentials from a study of the threshold oscillations.
Recently Zucker (1973) used the same reasoning in his
determination of afterpotentials by measuring threshold
oscillation in crayfish motor-nerve terminals.
Threshold for Invadabilities' Sake
D.
Raymond (1969) and Bittner (1968) both offered interesting evidence of the role played by a branch point
in an axon.
Chung et al (1970) described a system
whereby an axonal arborization might function as a time
domain filter.
The arbor in this model does its filtering
at branch points which operate by means of thresholdmediated invasion.
Slow threshold oscillations are
crucial in this model if the time domain filter is to
operate over any significant period of time.
Newman and
Raymond (1971) through a novel measurement technique
obtained the clearest description to date of threshold
-11-
changes strong enough to mediate invasion of axons.
These changes were shown to last tens of minutes,
Curiosity about what happens at a point of low safety
factor when the temperature changes (as it might in any
poikilothermic animal) has motivated this investigation
of threshold.
E.
Acclimatization and Nervous Activity
Hering found in 1883 that the reflection of nerve
impulses back from the cut end of a frog nerve was
successful only in winter frogs.
Garten and Sulze (1913)
investigating the effects of temperature on nervous
activity found that previous acclimatization of the frog
before sacrifice would affect the results of the experiments.
Gasser & Erlanger (1930) found that the unusual
afterpotentials of the winter frogs reported by Forbes
and Thatcher (1920) could be normalized by holding the
frog at laboratory temperatures for a week before measurement.
Interestingly this property is not exclusive to
poikilothermic animals.
Chatfield et al (1948) found
a difference in the ability of excised nerves from hibernating and nonhibernating mammals to conduct at
extremes of temperature.
Chatfield et al (1953) investi-
-12-
gated the feet of the Herring Gull which in the winter
are often naturally at near freezing temperatures.
They
found that the excised peroneal nerve from a winter
gull had interesting properties:
at temperatures suit-
able for conduction in the proximal portion the distal
portion was heat blocked, and at temperatures suitable
for conduction into the distal portion the proximal
section was cold blocked.
If the gull was allowed to
acclimate to room temperature for a week before the
experiment the nerve would behave in a more uniform
manner.
It is not altogether surprising to find that a nervous system subject to temperature changes might have
evolved some mechanism to cope with these changes, and
this report will attempt to shed some further light
on the subject.
F.
Temperature and Nervous Activity
Despite contradictory results by Gasser (1931),
Tasaki and Fujita (1948), and others the literature
seems to have descended quite firmly on the notion that
action potential latency, duration, and peak amplitude
increase with decrease in temperature.
The notion is
-13-
well supported in both theory and practice (Hodgkin
and Katz 1949, Schoepfle and Erlanger 1941, Huxley
1959).
Maruhashi* was able to explain the results
of Tasaki and Fujita as an artifact of recording in
series with a substantial amount of axoplasmic resistivity which has a temperature coefficient of its own.
Tasaki and Spyropoulos
(1957) point out on theoretical
grounds that the membrane current during an action
potential is likely to be reduced by cold.
and Cole (1964) predict otherwise.
Fitzhugh
In any case it is
certain that the total net charge passed during an
action potential is increased by cold, and this is well
supported by nonelectrical measurement (Shanes 1954).
G.
Temperature and Excitability
Whether or not a zone of low safety factor will
block invasion depends upon at least these two factors:
the stimulus strength applied to the zone (by means
of the approaching action potential) and the threshold
of the membrane beyond the zone.
As mentioned above,
cold increases the stimulus but there is no conclusive
theory or data for the effect of temperature upon
threshold.
Both Tasaki and Spyropoulos (1957) and
as reported in Tasaki and Spyropoulos 1957
-14-
Fitzhugh (1966) emphasize that temperature-threshold
relations are dependent upon stimulus form, and draw
sharp distinctions between the expected temperature
influence on Q * and rheobase.
However, their predic-
tions are at variance with each other; Tasaki's theoretical emphasis is based upon cable constants, Fitzhugh's emphasis is upon the Hodgkin-Huxley rate
constants.
Sjoden and Mullins (1958), Guttman (1962,
1966), Tasaki and Fujita (1948), and Tasaki (1949)
have all investigated temperature-threshold relations.
The results do not fall into a pattern; e.g. Tasaki**
was plagued by an hysteresis phenomenon, and all of the
techniques are subject to serious criticims (e.g. the
sucrose gap technique causes hyperpolarization of the
membrane under test).
It is the intent of the author
to investigate the effects of temperature changes upon
threshold using primarily the methods of Newman and
Raymond (1971).
*minimum excitory charge
**discussed further on page 34
-15-
II.
METHODS
The frogs used in this research varied at the discretion of the supplier.
It is unfortunate that both
All frogs
northern and southern varieties were provided.
were kept for at least a week at 18-190 C before sacrifice.
The sciatic nerve was excised with great care to
prevent any damage to the epineurium, particularly in the
proximal portion which was to be stimulated.
The nerve
was mounted in the chamber as illustrated in Figures 1
and 2.
The proximal portion of the nerve was tied off with
ordinary cotton sewing thread, and the thread was pulled
taut until the nerve was just about to slip through the
vaseline seal.
The silver block was chloridized on its internal
surface and a silver-silver chloride electrode was mounted
upon a micrometer head for precise control of position.
The nerve was always positioned such that the stimulating
electrode intersected the nerve at least 2 1/2 centimeters
from the cut end.
The stimulus isolation unit provided
cathodal (with reference to the silver block)current pulses
(normally used in the range .05 - .05 milliamps) of duration
controlled by Dr. S. Raymond's* hunter circuit.
*described in Newman and Raymond (1971)
STOPPER
NERVE
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The silver block was mounted upon a Cambion Thermionic
Devices Peltier device.
A thermistor mounted inside the
silver block in conjunction with a temperature control
circuit was capable of holding the silver block at any
temperature between 12 and 250 C with fluctuations less
than 0.30 C.
When changing temperatures the system would
reach a new set point at a rate of 40 C per minute.
Both the silver block and the recording dish had
their own ringer supplies.
That ringer which perfused
the silver block first passed through a preheater (precooler)
also operated by the temperature control circuit.
Follow-
ing this step it was led past a glass pH electrode and
then into the silver block.
It emerged from the block and
was pumped back up to the aerating chamber from which it
would recirculate under the force of gravity.
The ringer was that used by Newman and Raymond (1971)
but it is reprinted here because of a typographical error
in their publication:
Salt
Millimolarity
NaCl
80.5
Salt
Millimolarity
Na 2 SO 4
0.66
1.99
Na 2 HPO 4
2.55
NaHCO 3
25.01
KH2PO 4
0.52
CaC1 2
1.70
C6 H 1 2 06
3.3
MgSO 4
1.17
KC1
-19-
The ringer was aerated by a humidified 5% CO 2 in
air mixture which held the pH at about 7.4.
The pH
never varied by more than .02 units during the course
of an experiment.
Although the nerve might remain func-
tional for up to 72 hours, all experiments were done
within 12 hours of pithing the frog.
After loading the nerve in place, and with the stimulating electrode advanced to within close proximity of
the nerve, the stimulator would be set to deliver a pulse
every 2.5 seconds.
A small group of fibers was teased
away from the nerve in the recording chamber and aspirated
into a suction electrode.
The fibers were cut or crushed
one by one until only one action potential remained upon
the monitoring oscilliscope.
In order to adjust the
position of the stimulating electrode the circuit was
set to hunt the threshold of this single unit.
If the
threshold plot was unsteady as in the left hand portion
of Figure 3 the electrode would be advanced very slightly
(corresponding to time "A" in Figure 3).
Moving the
electrode closer to the nerve would make the stimulus more
effective and thus the hunter would hunt a lower threshold.
The unsteadiness of the trace in the left hand portion of
Figure 3 is probably due to motion of the nerve as the
ringer flowed past.
After time "A" the electrode was
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probably in contact with the nerve.
The stimulus durations used in this report varied
from 50 to 250 microseconds.
This range falls upon the
hyperbolic portion of a frog strength-duration curve
(Verveen 1961).
Because a gross nerve is being stimulated it is
important to be sure that the threshold of the fiber that
is being recorded is not affected through ephaptic contact with its neighbors.
Pecher's (1939) demonstration
of the independence of two similar fibers in an axon
bundle can be invoked as long as resting threshold is
being measured.
To be certain that threshold oscillations,
such as the supernormal period, are not ephaptic artifacts
Newman and Raymond (1971) devised a control experiment
and showed the absence of ephaptic-like interaction.
-22-
III.
A.
RESULTS
Resting axon
Most axons are considered to be in their resting
state when they conduct impulses less often than once
per two seconds.
When in this rested state on axon
would maintain stability (as in figure 3 after time "A")
for several hours with less than 3/4 centimeter of
drift. This corresponds to a 5% variation in stimulus strength.
1.
Slow temperature changes
When changed slowly in the range 100 to 240,
temperature had no effect upon threshold in most fibers
The threshold of occasional fiberswould increase
tested.
0
as temperature was lowered below lab temperature (18 C),
however these fibers could be shown to be depressed* at
the temperatures below 180.
2.
Fast temperature changes
Figure 4 displays the result of an experiment
on a rested axon when the temperature was changed very
rapidly.
The threshold is measured two seconds after a
*depression as defined in Newman & Raymond (1971)
*depression as defined in Newman & Raymond (1971)
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conditioning impulse which is delivered to the nerve
every four seconds.
At time "A" the temperature was
dropped from 180 to 130 and at time "B" raised back to
180.
As in the experiment with slow temperature change
there was no net difference in threshold after changing
temperature, but the striking result of changing temperature rapidly was that threshold temporarily changed.
When the nerve was cooled(heated) rapidly the threshold
quickly (1 minute) decreased (increased) and then slowly
(5-15 minutes in figure 4) recovered its initial value.
There was some variation of this "recovery" among
different axons, and within the same axon as its age increased.
In particular, the "recovery" would often take
as long as 30 minutes, rarely exceeding 45 minutes.
In all axons the initial rapid deflection of threshold
was greater when temperature was raised than when lowered.
In those axons which became slighly depressed at lower
temperatures, the time course of threshold to a sudden
temperature drop was similar to the threshold change shown
in figure 4, with the exception that the "recovery" overshot its original position.
3.
Threshold Oscillation
Newman and Raymond (1971) showed that supernormality
-2 5-
in a rested axon lasted about a second.
The 180 curve
of figure 5 is in excellent agreement with their results.
Upon cooling, the relative refractory period and the
slightly decreased peak of supernormality were delayed.
This is in excellent agreement with the results of Gasser
and Erlanger
(1930) although their supernormality only
lasted about 200 milliseconds.
4.
Sudden
temperature change in the supernormal period
Figure 4 is a graph of threshold vs time
where the threshold is taken at a fixed delay of two
seconds after conditioning.
If the fixed delay was set
in the range 15-1000 milliseconds
(supernormality) figure
4 would remain a good representation of a sudden temperature
change;
however the initial and final values of threshold
would be as given in figure 5.
B.
Depressed axon
The initial
segment of figure 6 shows the threshold
of an axon measured 3 seconds after a single conditioning
pulse which is repeated every four seconds.
in its resting state.
At time "A"
was made a conditioning volley
The axon is
the conditioning pulse
(10 impulses at 30 impulses
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per second) and at time "B" the initial conditions were
reinstituted.
The total accumulation of depression
was quite variable from fiber to fiber, and in the
same fiber as the fiber aged.
The rates of accumulation
and decay were also quite variable, depression often
taking up to an hour to accumulate or decay.
Newman
and Raymond (1971) did not report such long time
periods.
1.
Temperature and depression
An experiment designed to show the effect of
temperature upon rate of accumulation (or decay) of
depression could not be done conclusively because the
experiment would have to last about 5 hours during
which time drift and ageing would cloud the results.
The amount of depression in a fiber was always decreased
with increase in temperature.
This is illustrated in
Figure 7 which is recorded in the same manner as figure
6, but at time "B" the temperature is suddenly dropped
from its initial value (17.50) to 14.50, and at time
"C" raised back to 17.50.
2.
Threshold Oscillatons
Figures 8 and 9 both show the time course of
threshold, measured at variable time after the last stimulus delivered in the conditioning volley (10 impulses
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-32-
at 30 impulses per second repeated every 5 seconds).
The fiber recorded from in figure 8 was slightly depressed by this proceedure and reached peak depression in
about 1.5 seconds.
The highly depressed fiber of figure
9 reached peak depression in about 2 seconds.
Newman
and Raymond found that in all depressed axons peak
depression was reached in 1-3 seconds,
3.
Sudden temperature change in supernormal or
depressed phase
If a threshold vs time graph (such as figure 7)
was plotted with any fixed delay of 20 milliseconds or more
after the conditioning volley, the threshold would undergo
a transition qualitatively similar to figure 7 but with
initial and final threshold values consistent with the
data plotted in a graph such as figure 8 or 9.
-33-
IV.
A.
DISCUSSION
Methods
1.
Circulation
Liesse (1938) and Parrack (1940) pointed out that
there are small differences in the excitation characteristics of nerves which are excised with respect to nerves
in situ, and that the reason for this is the lack of
natural circulation in the excised tissue.
Feng et al
(1950) and others have shown that brisk perfusion of a
nerve by ringers solution can, in many ways, negate the
need for a natural circulation.
The nerves used in this study are suspended in a
1/4 inch diameter canal (through the silver block) through
which ringers solution briskly flows.
The point made by
Liesse and Parrack is well taken, however the stability
of the threshold over long periods and the longevity of
the nerves used in this report are convincing evidence
that the nerves were in physiologically sound condition.
2. Ageing and deterioration
Ageing and deterioration of the nerve was manifest
through a tendency for the value of threshold to drift
_34-
slowly, especially when the nerve was depressed or heated
above room temperature.
When data such as figure 8 was recorded, at least
45 minutes elapsed after the first trace (180) before
the second trace (130) was taken.
The 45 minute time
interval was necessary to allow the threshold to stabilize at its new value.
After another 45 minutes a trace
was taken again under the initial conditions.
If there
was any significant difference between the two initial
(180) curves the data was not used.
Due to the increased
aging and deterioration of the nerve at warm temperatures,
it was most difficult to obtain data above 180.
B.
The Time Course
There has been no mention in the literature of any
threshold response to sudden temperature change similar
to the data presented in this report.
Tasaki (1949) has
reported a hysteresis associated with threshold measurements in his attempt to describe excitability in terms
of temperature.Because it is sometimes necessary to
wait 45 minutes between taking threshold measurements
at two different temperatures, waiting a lesser time
could give the illusion of a hysteresis.
-35 -
The time course that threshold follows after a
sudden change in temperature is not unique in the neurophysiological literature; an initial deflection followed
by a slow recovery has been reported for membrane potential and spike height under certain conditions.
Lorente de No (1947) reported that a frog nerve
in air if exposed to CO2 would quickly hyperpolarize
and than slowly depolarize.
Crescitelli (1957) reports
that in a nerve which has been sodium blocked, application
of sodium and potassium will quickly relieve the sodium block.
but that slowly a potassium block would be set up.
The action potential in this case would increase and
decrease in much the same manner as threshold in response
to sudden heating.
The mechanisms behind these processes
have not been elucidated although Shanes (1958) has
suggested that the action of CO2 causes a sudden decrease
in PNa followed by either slow movement of potassium out
of the cell or a slow decrease in PK.
The phenomena dis-
cussed in this section have been presented only because
their interesting temporal resemblence to some of the
phenomena presented in the results section.
C.
Speculations
What is to be presented in this section is pure
-36-
speculation.
It is useful in that it suggests meaning-
ful experiments that may be done to aid in the elucidation of a mechanism for the action of temperature upon
threshold.
1.
The fast threshold deflection
There is compelling theoretical evidence
(Fitzhughl966) that a sudden change in temperature
should cause a sudden change in threshold.
As Fitzhugh
points out it is extraordinarily difficult to actually
predict magnitude or direction of the effect in a real
system.
A speculative explanation for the quick threshold deflection is that increase (decrease) of temperature increases (decreases) the current through the
sodium pump which hyperpolarizes (depolarizes) the
membrane and therefore raises (lowers) the threshold.
2.
The "recovery" speculation
All other things being equal an exchange of
extracellular sodium with intracellular potassium will
raise threshold (assuming that the concentration of
extracellular potassium is not changed).
This is
because increasing the intracellular sodium concentration
-37-
will have an effect upon the sodium potential.
When
the potential is reduced there is less driving force
in the sodium activation process necessary for activation.
The concentration of intracellular sodium is increased by nervous activity and by membrane leakage.
It is reduced by the action of the sodium pump.
The
"recovery" that follows a temperature change is actually
a readjustment of the intracellular sodium concentration,
as is the accumulation and the decay of depression.
Newman and Raymond have shown clearly that the
accumulation of depression is related to how often the
nerve is stimulated.
After a sudden temperature change
it would not be surprising to find that the rate of
"recovery" is function of how often the axon is tested
for threshold.
Cold is known to reduce membrane leakage
and at the same time increase the ionic debt associated
with the conduction of each impulse; "recovery" would
than be hastened (hindered) after cooling (heating) by
testing for threshold.
The rate of recovery (after heating) is increased
by higher temperatures because the pumping capacity
of the nerve is increased with temperature.
This effect
would be opposed by the increased membrane leakage at
-38-
higher temperatures; however Shanes (1958) points out
that the cell may expend energy to exclude (as opposed
to transport) sodium, thereby effectively reducing its
leakage permeability.
The rate of "recovery" after cooling is increased
by lower temperatures, since the "recovery" is actually
an accumulating sodium debt.
This is seen in figure 10
where the temperature is lowered at times "A" and "B".
The experimental use of sodium pump inhibiters should
shed some light upon the soundness of these speculations.
D.
Invadability
A nerve fiber active at physiological frequencies
would normally be somewhat depressed.
If depression is
the result of a pumping debt, than surface to volume
considerations predict that smaller axons or axon branches
(which conduct impulses at lower frequencies in vivo)
would be depressed at lower frequencies.
It is expected
then, that increase of temperature will lower threshold
for most axons and their branches in vivo.
Since action
potential strength (as a stimulus to a zone of low safety
factor) is also reduced by increased temperature, it is
possible to speculate that the invadability of a zone of
low safety factor is not influenced by temperature.
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This could easily be tested through methods already
established by Bittner 1968 or Pomas 1972, although
neither of them has actually investigated the effects
of temperature in their studies of differential invadability of crustacean branch poiLh,
-41-
V.
Conclusion
In an axon conducting impulses at physiological
frequencies threshold has a negative temperature coefficient.
When the temperature of an axon was changed
suddenly, the corresponding threshold change was broken
into two components.
The second slow component bears
resemblance to accumulation or decay of depression.
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