- Email: [email protected]
The effect of temperature changes on the activity on the neurones of the snail Helix aspersa
Comp. Biochem. Physiol., 1962, Vol. 5, pp. 283 to 295. Pergamon Press Ltd., London. Printed in Great Britain
THE EFFECT OF TEMPERATURE CHANGES ON THE ACTIVITY OF THE NEURONES OF THE SNAIL HELIX ASPERSA G. A. K E R K U T and R. M. A. P. R I D G E Department of Physiology & Biochemistry, The University of Southampton, England
(Received 28 November 1961) A b s t r a c t - - 1 . This paper describes the effect of changing the temperature on
the spontaneous activity and resting potentials of the snail nerve cells. 2. Four types of response were noted : (a) An increase in temperature caused an increase in the frequency of the action potentials. A decrease in temperature caused a decrease in the frequency. (b) An increase in temperature caused a transient decrease in the frequency of the action potentials. This was followed by an increase in the frequency. Lowering the temperature brought about a similar transient; the frequency increased for a short time, then fell to a rate lower than the initial one.
(c) In a silent cell, i.e. one that did not show spontaneous activity, cooling brought about activity whilst warming stopped the activity. (d) Some cells did not show any response to warming or cooling. 3. The resting potential increased when the temperature was increased and decreased when the temperature was decreased. The Q10-~0oc varied from 1.04 to 1"96. 4. The resting potentials of many cells after a change in temperature tended to return to their initial value (adapted), even if the temperature was maintained at the new level. 5. The results are discussed in terms of two theories. (a) That the behaviour of individual cells changes with time. (b) That there are specific differences in the reactions of nerve cells. INTRODUCTION IN 1959, Burkhardt described the effect of temperature changes on the resting potential and activity of the crustacean stretch receptor. He found that lowering the temperature brought about a transient increase in the activity of the stretch receptor whilst warming brought about a transient decrease in activity. Warming increased the resting potential whilst cooling decreased the resting potential. On the other hand, at a steady temperature the rate of activity of the stretch receptor was almost independent of the temperature. There was a relationship between the effects of temperature on the resting potential, generator potential and the membrane threshold such that the rate of discharge of the sense organ remained constant. Van Gelder & Krnjevic (1961) have also reported on the manner in which the crustacean stretch receptor responded to a temperature change. 283
284
G. A. KERKUTAND R. M. A. P. RIDGE
Axelsson & Bfilbring (1961) studied the resting potential and the spontaneous activity of intestinal muscle and found that following a change from 23-27°C to 33-37°C there was a decrease in the spontaneous activity and an increase in the resting potential. A decrease in temperature from 33 to 30°C, or 30 to 27°C, caused an increase in the spontaneous activity and a decrease in the resting potential. These results were interpreted in terms of metabolic changes. Warming increased the rate of metabolism and slowed up the rate of spontaneous activity; this was correlated with changes in phosphorylase activity following a temperature change. In earlier reports (Kerkut & Taylor, 1956, 1958) it was shown that the activity from invertebrate central nervous systems responded to temperature changes in various ways. Following a temperature change the activity could increase, decrease or be unaffected. These results were obtained hy means of extracellular recording from complete ganglia, and it was thought mat the results might be easier to interpret if the experiments were repeated on individual nerve cells using intracellular electrodes. METHOD The experiments were carried out on large neurones in the suboesophageal ganglia of the snail Helix aspersa. An active snail was opened and the brain removed. The connective tissue covering the dorsal surface of the suboesophageal ganglia was carefully dissected away and the neurones were exposed. The preparation was then placed in a bath through which a Ringer solution (Cardot, 1921) of a specified temperature could be passed (Kerkut & Ridge, 1961). A 3 M~] KCl-filled glass microelectrode was inserted into an observed neurone and the potential recorded via a high impedance input stage. This then led to a d.c. power amplifier (Southern Instruments) and an Ediswan Pen Recorder, or Tektronix 502 oscilloscope fitted with a Shackman camera. The temperature was measured by means of a thermistor and amplifying system leading to a pen recorder. In those experiments where the d.c. changes were closely followed, the electrodes were tested for temperature sensitivity and any electrode showing a change greater than 0.5 mV/10°C was rejected. It was found that many electrodes were made temperature sensitive by the cytoplasm of the cell; the nerve cells in this way differ from muscle cells. RESULTS When an electrode is lowered into a cell, there is a gradual deformation of the membrane and the effects of this mechanical distortion may be initially seen (Fig. 1). This is followed by the electrode penetrating the membrane, and in the example shown in Fig. 1 the penetration caused a series of action potentials to be initiated in the cell. The frequency of these potentials decreased and reached a rate of one action potential every three or four seconds. This mechanical stimulation of the cell caused by the penetration of an electrode is not always seen. In most cases where the cell is spontaneously active, the activity can be detected
285
TEMPERATURECHANGESON NEURONES
when the electrode is close to but not actually touching the cell. This indicates that the spontaneous activity is not necessarily a result of stimulation due to the electrode. Some cells when penetrated do not show any spontaneous activity at all and this is further evidence that the activity is not necessarily caused by the electrode.
r
f
f
r
I0 i
sec t
FIG. 1. Penetration of a neurone by a micro-electrode. As the electrode pressed against the membrane the distortion caused a potential change in the electrode. On penetration a series of action potentials occurred ; then the frequency of action potentials decreased to a steady rate.
Activity
Temperature
5*C 15oC Time, sec -
-
5.50C
~
2L°C
Timet $ec
FIG. 2. Effect of temperature change on the frequency of the action potentials. There is a simple relationship between the temperature and rate; decreasing the temperature decreased the frequency, increasing the temperature increased the frequency.
(a) Effect of temperature changes on spontaneously active cells Many spontaneously active cells show a simple response to temperature changes (Fig. 2). An increase or decrease in temperature caused respectively an increase or decrease in activity. The unit shown in Fig. 2 had a steady rate of
286
G . A . KERKUTAND R. M. A. P. RIDGE
twenty-two action potentials every 10 sec at 15°C and four action potentials every 10 sec at 5°C. W h e n the unit was w a r m e d f r o m 5-5°C to 21°C the rate increased from five to thirty action potentials every 10 sec. In all the cells that responded in this way, there was no adaptation; the rate remained constant at the new temperature. T h e Q1o for the cell recorded in Fig. 2 was 3"3 over the temperature change 15-25°C.
! I{
HIt{I If!If !t !I !! i Continued
11.6 °
19"6°
./~
50mY
I
,
i
~
,
I 'L
I
~
1
, I,!
Continuation 11.6 °
20"0*
Time,
sec
FIG. 3. Effect of temperature change on the frequency of the action potentials. This example shows the anomalous transient. Cooling led to a transient increase in frequency of action potentials whilst warming led to a transient decrease in frequency. Other cells showed a more complex response to a t e m p e r a t u r e change. T h o u g h the final rate following an increase in t e m p e r a t u r e was higher than the initial rate, there was a transient decrease in rate immediately following the t e m p e r a t u r e change. A similar effect followed a decrease in temperature, the transient being an increase in the rate. T h e cell shown in Fig. 3 had a steady rate of one action potential every 2 sec. Cooling the cell f r o m 19-6 to 11.6°C brought about a transient increase in rate to one action potential every 1.3 sec, and a later steady rate of one every 3.8 sec. W a r m i n g f r o m 11.6 ° to 20°C brought about a slowing of the rate with no action potential for 15 sec. T h e rate increased to one every 2.4 sec.
287
TEMPERATURE CHANGES ON NEURONES
(b) Effect of temperature changes on inactive cells Some cells do not show any activity on penetration by the electrode. Fig. 4 shows a record from such a cell. Cooling from 20 ° to 10°C caused the cell to become active for 56 sec. The activity sho~ved adaptation and after the action potentials had stopped the "excitatory post synaptic potentials" were still noticeable.
~
_
~
[50 mV
IO°C
/ ~°°c
Id.U. ~
~
i
~
~
~ ~
~
~
~
~
~
~ J ~ J j j_l.uj_~
LLI L~ LLU
ILl
Time, sec
u
u
UJ I LU.IjOJ.LI
Lu
u
IJu
u
LI U •, L i u
u
LL| * I~U
LI U IJ~J
LI
FIG. 4. Cooling bringing about activity in a silent cell. W h e n the cell was cooled it
became active for 56 sec.
j[[!![c[[[f f 16.3oC
f 12"8°C
f 11.3oc
[ 8"7°C
Time, sec FIG. 5. Cooling bringing about activity in a silent cell. The cell activity rapidly adapted but could be elicited by further cooling. Figure 5, using a greater amplification, shows a similar pattern in an inactive cell which was stimulated to activity by cooling. The cell was cooled from 16.3 ° to 12.8°C and this brought about activity in the cell. The activity stopped, but cooling from 12.8 ° to 11.3°C brought about further activity as also did cooling from 11.3 ° to 8.7°C. In all cases, the cell adapted so that the activity slowed down and stopped. If the cell was warmed before the completion of adaptation, the activity was inhibited. Warming could only rarely bring about activity in a silent cell. Heating above 35°C often Caused activity, but this temperature is above the animal's physioIogical
288
G. A. KERKUTAND R. M. A. P. RIDGE
range. A case where warming within the physiological range caused activity to occur in the cell is shown in Fig. 6. (c) Resting potential changes with temperature A series of experiments was carried out using a higher amplification so that the detailed changes in the resting potentials following a temperature change could be examined. In all cases where a clear response was obtained, increasing the temperature led to an increase (hyperpolarization) of the resting potential,
( / (
5 9 " 3 mV
I
,
38'5mV
I1.3°C
IL '~ Time, it.
Lt2~.~i
.........
25.0oc
sec
i ~ i . i i t i [i iJ.LLIj.l~
tl
U t ~
1
[1LI
iJJ
IJ i I [I ljJjJJJJ...LlJ.Ltl
l Li I i I) t i I
Fic. 6. Warming bringing about activity in a silent cell. The cell was warmed and the resting potential increased to 41 mV, but then adapted to 38'5 mV and this depolarization brought about activity. whilst decreasing the temperature led to a decrease (depolarization) of the resting potential. Fig. 7(a) shows the resting potential of an inactive cell that was warmed. No adaptation of the resting potential to its original value was noted. Warming the cell from 13.0°C to 24.9°C increased the resting potential from 19.2 mV to 24.0 mV. The resting potential remained at the new level. Fig. 7(b) shows a similar cell but there was partial adaptation of the resting potential. Warming the cell from 10-7 ° to 22.0°C increased the resting potential from 34.5 mV to 39.2 mV. The resting potential of the cell at 22°C gradually fell to 37.5 mV. The Qlo values for warming and cooling between 10 ° and 20°C varied both for the same cell and between different cells. The range was from 1.04 to 1-96; the maximum changes being found in those cells that adapted. In those cells that were spontaneously active, the depolarization of the resting potential prior to an action potential ranged from 1 mV to 10 mV. Cooling an active cell brought about an increased rate in depolarization and this could elicit a transient increase in the frequency of the action potential. Cooling an inactive cell could bring about a depolarization sufficient to make the cell active. Figure 8(a) shows the relationship between resting potential and the frequency of action potentials following a temperature decrease. The average rate of activity for the 70 sec prior to the temperature change was one potential every 5 sec. The resting potential was 43 mV. The temperature was lowered from 20 ° to
289
TEMPERATURE CHANGES ON NEURONES
9°C. Immediately the frequency of the action potentials increased. The resting potential decreased to just under 41.5 mV, and then returned back to 42.5 mV, even though the temperature remained at 9°C. In a similar experiment where the temperature was increased from 9 ° to 20°C, the resting potential increased from 43 mV to 47 mV (Fig. 8(b)). The frequency of the action potentials decreased from one approximately every second to no activity for 50 sec. The activity then returned even though the resting potential was still at 47 mV. (a} I9.2 mV --.-.,-.-,.-.,,..~
--~- :- ~
_
24.0
.
13.0°C
24.9"C
~
i
i i | / | U L i U U Ll L| I ] U t i U
(b)
U U UU
Li LiLIU
|iU
UII
U LiU
UU
L} U I ]
L] U U ~
54.5 mV 39.2 rnV
37.5mV
10.7"C
\ ~
~
~
-
,,...
22.0"C
t
a Time.
J
t
~_LLLL~
sec
FIG. 7. Effect of warming on the resting potential. (a) Showing an increase in the resting potential, the new value being maintained. (b) Showing an increase in the resting potential, the value adapting back towards the initial level.
In many cases the resting potential, after responding to a temperature change, tended to adapt back to its former level. Fig. 6 shows an interesting example in which warming from 11-3° to 23°C brought about an increase in the resting potential from 39.3 mV to 41 mV. The resting potential slowly returned to a lower level till 32 see later it had reached 38 mV and the cell became active. This
290
G . A . KERKUT AND R. M. A. P. RIDGE
~
0
0
•
0 20
o
to
E
.E
2
c
3
43 o
I
g 42
c: 09
15 °C
~,
g
&•
"
es ing potential
g~ 41
<
-Temperature
tO
I
I
f0
I
20
]
30
I
40
50
sec
Time,
FIG. 8a.
48
47 > E
o
. . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.9
15 oC
4s
.E
20
/
Temperoture,~/
46 0-
o
Resting
potential
0.5 I~ 4 4
I 1.0
g
Action
A
j ~
43
Action
potential~
potential I0
1,5~t
42
I tO
20
I 40
30
Time,
I 50
I 60
sec
FIc. 8b.
FIG. 8. Effect of temperature changes on the resting potential and frequency of action potentials. (a) Cooling from 20 ° to 9°C led to a rapid increase in the frequency of the action potentials and a slow fall of the resting potential. (b) Warming from 9 ° to 20°C brought about a rise in the resting potential and a fall in the frequency of the action potentials. T h e action potentials returned even though the resting potential was at the higher level.
TEMPERATURE
CHANGES
291
ON NEURONE$
reaction was repeated many times. On each occasion there was a considerable lag between the temperature change and the onset of activity--for the specific cell shown in Fig. 6 the average lag over the nine experiments was 43 sec. (d) Effect of temperature change on the duration of the action potential The shape and parameters of the action potential vary considerably from one cell to another. Amplitudes ranging from 30 mV to 75 mV (average 53 mV) and durations from 4 msec to 500 msec (average 55 msec) have been found in ~00
9oi 8C > E
70 60
o
g
50
g. 4o30 20-l0 0
I
I
I
I
I
I
r
I
I
I
.5
i0
t ,5
20
2,5
30
35
4o
45
.50
Time,
55
m sec
FIa. 9. The effect of temperature on the duration of the action potentiM. Cooling led to an increase in the duration of the action potential. cells at room temperature. Cooling the cell brought about an increase in the duration of the action potential. This is seen in Fig. 9, where action potentials photographed from one cell at different temperatures are superimposed. The duration varied from 20 msec at 13.3°C to 45 msec at 3°C. This increase in the duration of the action potential agrees with that described by other workers: Tasaki & Fugita (1948), frog nerve fibres; Hodgkin & Katz (1949), Loligo axon; Coraboeuf & Weidmann (1954), Purkinje fibres; Boistel (1960), cockroach nerve; Loewenstein & Ishiko (1961), paccinian corpuscle-nerve. A decrease in temperature could also bring about a decrease in the amplitude of the action potential.
(e) Cells unaffected by temperature change A few cells showed no response to temperature changes. Five different cells showed a constant rate of spontaneous activity; the frequency being unaffected by temperature changes. In nine other cases the ceils were inactive and temperature changes did not bring about any activity, though mechanical stimulation of these ceils could bring them into activity.
292
G. A. KERKUTAND R. M. A. P. RIDGE
DISCUSSION Changes in the frequency of action potentials due to changes in the temperature of nerve cells have often been described. Some of these nerves are cold or warm receptors whose function is the conveying of information about temperature (e.g. cat cutaneous thermoreceptors, Hensel & Zottermann, 1951a, b, c; Hensel & Witt, 1959, 1960). Receptors with other primary functions can also convey information about temperature. Examples of these are the mechanoreceptors in the cat tongue (Hensel & Zotterman, 1951a); cutaneous touch receptors in the cat (Maruhashi et al., 1952; Douglas et al., 1959; Hunt & McIntyre, 1960); the mechanoreceptors of the facial pit of the rattlesnake Crotalus (Bullock & Diecke, 1956); and the crayfish stretch receptor (Burkhardt, 1959a, b). The effect of temperature changes on the blowfly contact chemoreceptors is reported by Hodgson (1956). Dethier & Arab (1958) later described specific temperature receptors in the tarsus. The behaviour of a nerve can vary with temperature in many ways. Thus some nerves increase their activity on warming, others on cooling. Inverse transients in nerve cells have been found in all the spontaneously active nerve preparations so far studied. The simplest explanation of this type of transient is that cooling the nerve reduces the resting potential; this depolarization leads to an increase in the frequency of the action potentials. Gradually the nerve adapts, physico-chemical systems controlling the membrane re-establish new equilibrium conditions at the low temperatures and the frequency of the action potentials decreases. Conversely, increasing the temperature increases the resting potential; this hyperpolarization causes a temporary slowing in the frequency of action potentials till once more a new equilibrium system is attained for the higher temperature. This raises the problem as to why certain nerve cells do not show these inverse transients but instead behave in a simple manner, the higher temperature leading to a direct increase in activity ? It is possible that this to some extent depends on the rate of change of temperature, but in most of our experiments the rate of temperature change was identical for cells that showed transients and those that did not. The relationship between the change in the resting potential and the frequency of the action potentials in an active neurone is probably not a simple one. Thus, as shown in Fig. 8a, the frequency of the action potentials changed before there was a detectable change of 0.25 mV in the resting potential. This supports the observation of Terzuolo & Bullock (1956) that the spontaneously active cell can be sensitive to very small potential changes. Nor have we found a simple relationship between the value of the resting potential, the depolarization potential and the frequency of the action potentials. An active cell may become silent when the resting potential is increased yet later it may become active at this new resting potential value with no difference in the value of the observed depolarization potential. One cannot fail to be impressed by the manner in which the resting potential of many cells returns back towards the original level in spite of a
TEMPERATURECHANGESON NEURONES
293
temperature change. Though it is possible that in a complex brain the surrounding cells may participate in the observed reactions, it seems more probable that the cell membrane itself has regulating properties. Certain cells during the course of an experiment changed their behaviour. Thus, often during experiments on single cells where the temperature responses were followed over 30 min to 1 hr, the cell initially showed a direct temperaturefrequency response with no transients. Gradually, as the preparation aged, the transients appeared. Later still the cell lost its spontaneous activity but could be Temperature
I
Cool
IIII IIII
I
Warm
I II I[ Iit I II It Ill
I IIIIIIII 1 I IIIIIIII I c
[lllllll IIIIIII
FIG. 10. Diagram to show the changes in behaviour of some neurones during the course of an experiment. (A) Initially cooling led to a decrease in the activity, warming to an increase. (B) Later cooling led to a transient increase in activity, warming to a transient decrease. (C) Later still, the cell lost its spontaneous activity. Cooling brought about activity which was stopped by warming.
brought into activity by cooling; activity being lost on warming. This last response is the exact opposite to the cells' initial behaviour. Initially cooling caused a decrease in activity, warming an increase in activity. Now cooling brings about an increase in activity whilst warming stops activity (Fig. 10). Though some of the cells under observation showed a diminution of the resting potential, we do not think that this is the explanation since other cells maintained their potential to the end of the experiment but still showed this change in behaviour. The present experimental results can be explained in terms of three different factors: 1. The change 'of resting potential following a change in temperature. In most cases this effect leads to the expected change of frequency of the action potentials.
294
G. A. KERKUTAND R. M. A. P. RIDGE
2. T h e adaptation of the resting potential to its initial level at constant temperature. This does not occur in all cases and does not completely explain the adaptation of the activity. 3. Change of the nerve threshold. T h e time relationship between this change in threshold and the resting potential change with temperature appears to be different in some cases. As yet we have no direct experimental evidence concerning the threshold changes with temperature, but such a system can explain the fact that there appears to be no absolute value of resting potential at which the cell will fire-off an action potential. T h e ability to adapt varies from one cell to another and this might be due to ageing and/or injury of the cell. On the other hand, we have not noted any correlation between adaptation and the occurrence of positive after potentials, the latter being an indication of ageing (Moore & Cole, 1959). T h o u g h a unifying concept can be developed to explain the different reactions of the nerve cells to temperature change in terms of various factors, it is possible that there are real and significant differences in the reactions of specific nerves. Such a situation appears to be true for the reactions of the Helix neurones to drugs (Kerkut & Walker, 1961), and though it is convenient to believe that the neurones will behave simply and homogeneously to temperature changes, it is equally possible that there are precise differences between the neurones and that they will behave in a non-uniform manner to temperature changes. Work is at present in progress on the nature and responses of known neurones to see if this throws further light on the subject.
REFERENCES AXELSSON J. ~ BI3LBRINGE. (1961) Metabolic factors affecting the electrical activity of intestinal smooth muscle. 07. Physiol. 156, 344-356. BOISTELJ. (1960) Caractdristiques fonctionnelles des fibres nerveuses et des rdcepteurs tactiles et olfactifs des Insectes. Librairie Arnette, Paris. BULLOCKT. H. & DmCKE F. J. P. (1956) Properties of an infra red receptor.07. Physiol. 134,
47-87. BURKHARDT D. (1959a) Effect of temperature on isolated stretch receptor organ of the crayfish. Science 129, 392-393. BURKH^RDT D. (1959b) Die erregungsvorg~inge sensibler Ganglienzellen in Abh~ingigkeit yon der Temperatur. Biol. Zbl. 78, 22-62. CARDOT H. (1921) Actions des solutions de Ringer hypotoniques sur le cceur isol6 d'Helix pomatia. C.R. Soc. Biol., Paris 85, 813-816. CORABOEUFE. & WEIDMANN S. (1954) Temperature effects upon the electrical activity of Purkinje fibres. Helv. Physiol. Acta 12, 32-41. DETHIERV. G. & ARABY. M. (1958) Effect of temperature on the contact chemoreceptors of the blowfly. 07. Ins. Physiol. 2, 153-161. DOUGLASW. W., RITCHIE J. M. & ST~UB R. W. (1959) Discharges in non myelinated (c) fibres in the cat saphenous nerve in response to changing the temperature of the skin. 07. Physiol. 146, 47-48P. VANGELDERN. & KRNJEVtCK. (1961) Sensitivity of crayfish stretch receptors to temperature changes. 07. Physiol. 156, 14-15P.
TEMPERATURE CHANGES ON NEURONES
295
HENSEL H., IGGO A. & WITT I. (1960) A quantitative study of sensitive cutaneous thermoreceptors with c afferent fibres. J . Physiol. 153, 113-126. HENSEL H. & WITT I. (1959) Spatial temperature gradient and thermoreceptor stimulation. `7. Physiol. 148, 180-187. HENSRL H. & ZOTTERMaN Y. (195 la) T h e response of mechanoreceptors to thermal stimulation..7. Physiol. 115, 16-24. HSNSRL H. & ZOTTEmV~N Y. (1951b) T h e response of the cold receptor to constant cooling. .4cta Physiol. Stand. 22, 97-105. HSNSRL H. & ZOTTEmMAN Y. (1951c) T h e persisting cold sensation. ,4cta Physiol. Seand. 22, 106-115. HODGKIN A. L. & KATZ B. (1949) T h e effect of temperature on the electrical activity of the giant axon of the squid..7. Physiol. 109, 240-249. HODGSON E. S. (1956) Temperature sensitivity of primary chemoreceptors of insects. Anat. Ree. 175, 560-561. HUNT C. C. & MClNTYRe A. K. (1960) Properties of cutaneous touch receptors in cat. .7. Physiol. 153, 88-98. K~mcUT G. A. & RIDGR R. M. A. P. (1961) T h e effect of temperature changes on the resting potential of crab, insect and frog muscle. Corap. Biochem. Physiol. 3, 64-70. KERKUT G. A. & TAYLOR B. J. R. (1956)Effect of temperature on the spontaneous activity from the isolated ganglia of the slug, cockroach and crayfish. Nature, Lond. 178, 426. K~RKUT G. A. & TAYLOR B. J. R. (1958) T h e effect of temperature changes on the activity of poikilotherms. Behaviour 13, 259-279. KEm~UT G. A. & WALKRR R. J. (1961) T h e effect of drugs on the neurones of the snail Helix aspersa. Corap. Biochem. Physiol. 3, 143-160. LORWENSTEIN W. R. & ISHIKO N. (1961) Effect of temperature on the generator and action potentials of a sense organ..7. Gen. Physiol. 45, 105-124. MARUHASHI J., MIzuOucm K. & TASAKI I. (1952) Action currents in single afferent nerve fibres elicited by stimulation of the skin of the toad and the cat..7. Physiol. 117, 129-151. MOORE J. W. & COLE K. S. (1959) Resting and action potentials of the squid giant axon in vivo. `7. Gen. Physiol. 43, 961-970. TASAKI I. & F'VGIXAM. (1948) Action currents of single nerve fibres as modified by temperature changes. J. Neurophysiol. 11, 311-315. TRRZUOLO C. A. & BULLOCKT . H. (1956) Measurement of imposed voltage gradient adequate to modulate neuronal firing. Proc. Nat. Acad. Sci., Wash. 42, 687-694.
THE EFFECT OF TEMPERATURE CHANGES ON THE ACTIVITY OF THE NEURONES OF THE SNAIL HELIX ASPERSA G. A. K E R K U T and R. M. A. P. R I D G E Department of Physiology & Biochemistry, The University of Southampton, England
(Received 28 November 1961) A b s t r a c t - - 1 . This paper describes the effect of changing the temperature on
the spontaneous activity and resting potentials of the snail nerve cells. 2. Four types of response were noted : (a) An increase in temperature caused an increase in the frequency of the action potentials. A decrease in temperature caused a decrease in the frequency. (b) An increase in temperature caused a transient decrease in the frequency of the action potentials. This was followed by an increase in the frequency. Lowering the temperature brought about a similar transient; the frequency increased for a short time, then fell to a rate lower than the initial one.
(c) In a silent cell, i.e. one that did not show spontaneous activity, cooling brought about activity whilst warming stopped the activity. (d) Some cells did not show any response to warming or cooling. 3. The resting potential increased when the temperature was increased and decreased when the temperature was decreased. The Q10-~0oc varied from 1.04 to 1"96. 4. The resting potentials of many cells after a change in temperature tended to return to their initial value (adapted), even if the temperature was maintained at the new level. 5. The results are discussed in terms of two theories. (a) That the behaviour of individual cells changes with time. (b) That there are specific differences in the reactions of nerve cells. INTRODUCTION IN 1959, Burkhardt described the effect of temperature changes on the resting potential and activity of the crustacean stretch receptor. He found that lowering the temperature brought about a transient increase in the activity of the stretch receptor whilst warming brought about a transient decrease in activity. Warming increased the resting potential whilst cooling decreased the resting potential. On the other hand, at a steady temperature the rate of activity of the stretch receptor was almost independent of the temperature. There was a relationship between the effects of temperature on the resting potential, generator potential and the membrane threshold such that the rate of discharge of the sense organ remained constant. Van Gelder & Krnjevic (1961) have also reported on the manner in which the crustacean stretch receptor responded to a temperature change. 283
284
G. A. KERKUTAND R. M. A. P. RIDGE
Axelsson & Bfilbring (1961) studied the resting potential and the spontaneous activity of intestinal muscle and found that following a change from 23-27°C to 33-37°C there was a decrease in the spontaneous activity and an increase in the resting potential. A decrease in temperature from 33 to 30°C, or 30 to 27°C, caused an increase in the spontaneous activity and a decrease in the resting potential. These results were interpreted in terms of metabolic changes. Warming increased the rate of metabolism and slowed up the rate of spontaneous activity; this was correlated with changes in phosphorylase activity following a temperature change. In earlier reports (Kerkut & Taylor, 1956, 1958) it was shown that the activity from invertebrate central nervous systems responded to temperature changes in various ways. Following a temperature change the activity could increase, decrease or be unaffected. These results were obtained hy means of extracellular recording from complete ganglia, and it was thought mat the results might be easier to interpret if the experiments were repeated on individual nerve cells using intracellular electrodes. METHOD The experiments were carried out on large neurones in the suboesophageal ganglia of the snail Helix aspersa. An active snail was opened and the brain removed. The connective tissue covering the dorsal surface of the suboesophageal ganglia was carefully dissected away and the neurones were exposed. The preparation was then placed in a bath through which a Ringer solution (Cardot, 1921) of a specified temperature could be passed (Kerkut & Ridge, 1961). A 3 M~] KCl-filled glass microelectrode was inserted into an observed neurone and the potential recorded via a high impedance input stage. This then led to a d.c. power amplifier (Southern Instruments) and an Ediswan Pen Recorder, or Tektronix 502 oscilloscope fitted with a Shackman camera. The temperature was measured by means of a thermistor and amplifying system leading to a pen recorder. In those experiments where the d.c. changes were closely followed, the electrodes were tested for temperature sensitivity and any electrode showing a change greater than 0.5 mV/10°C was rejected. It was found that many electrodes were made temperature sensitive by the cytoplasm of the cell; the nerve cells in this way differ from muscle cells. RESULTS When an electrode is lowered into a cell, there is a gradual deformation of the membrane and the effects of this mechanical distortion may be initially seen (Fig. 1). This is followed by the electrode penetrating the membrane, and in the example shown in Fig. 1 the penetration caused a series of action potentials to be initiated in the cell. The frequency of these potentials decreased and reached a rate of one action potential every three or four seconds. This mechanical stimulation of the cell caused by the penetration of an electrode is not always seen. In most cases where the cell is spontaneously active, the activity can be detected
285
TEMPERATURECHANGESON NEURONES
when the electrode is close to but not actually touching the cell. This indicates that the spontaneous activity is not necessarily a result of stimulation due to the electrode. Some cells when penetrated do not show any spontaneous activity at all and this is further evidence that the activity is not necessarily caused by the electrode.
r
f
f
r
I0 i
sec t
FIG. 1. Penetration of a neurone by a micro-electrode. As the electrode pressed against the membrane the distortion caused a potential change in the electrode. On penetration a series of action potentials occurred ; then the frequency of action potentials decreased to a steady rate.
Activity
Temperature
5*C 15oC Time, sec -
-
5.50C
~
2L°C
Timet $ec
FIG. 2. Effect of temperature change on the frequency of the action potentials. There is a simple relationship between the temperature and rate; decreasing the temperature decreased the frequency, increasing the temperature increased the frequency.
(a) Effect of temperature changes on spontaneously active cells Many spontaneously active cells show a simple response to temperature changes (Fig. 2). An increase or decrease in temperature caused respectively an increase or decrease in activity. The unit shown in Fig. 2 had a steady rate of
286
G . A . KERKUTAND R. M. A. P. RIDGE
twenty-two action potentials every 10 sec at 15°C and four action potentials every 10 sec at 5°C. W h e n the unit was w a r m e d f r o m 5-5°C to 21°C the rate increased from five to thirty action potentials every 10 sec. In all the cells that responded in this way, there was no adaptation; the rate remained constant at the new temperature. T h e Q1o for the cell recorded in Fig. 2 was 3"3 over the temperature change 15-25°C.
! I{
HIt{I If!If !t !I !! i Continued
11.6 °
19"6°
./~
50mY
I
,
i
~
,
I 'L
I
~
1
, I,!
Continuation 11.6 °
20"0*
Time,
sec
FIG. 3. Effect of temperature change on the frequency of the action potentials. This example shows the anomalous transient. Cooling led to a transient increase in frequency of action potentials whilst warming led to a transient decrease in frequency. Other cells showed a more complex response to a t e m p e r a t u r e change. T h o u g h the final rate following an increase in t e m p e r a t u r e was higher than the initial rate, there was a transient decrease in rate immediately following the t e m p e r a t u r e change. A similar effect followed a decrease in temperature, the transient being an increase in the rate. T h e cell shown in Fig. 3 had a steady rate of one action potential every 2 sec. Cooling the cell f r o m 19-6 to 11.6°C brought about a transient increase in rate to one action potential every 1.3 sec, and a later steady rate of one every 3.8 sec. W a r m i n g f r o m 11.6 ° to 20°C brought about a slowing of the rate with no action potential for 15 sec. T h e rate increased to one every 2.4 sec.
287
TEMPERATURE CHANGES ON NEURONES
(b) Effect of temperature changes on inactive cells Some cells do not show any activity on penetration by the electrode. Fig. 4 shows a record from such a cell. Cooling from 20 ° to 10°C caused the cell to become active for 56 sec. The activity sho~ved adaptation and after the action potentials had stopped the "excitatory post synaptic potentials" were still noticeable.
~
_
~
[50 mV
IO°C
/ ~°°c
Id.U. ~
~
i
~
~
~ ~
~
~
~
~
~
~ J ~ J j j_l.uj_~
LLI L~ LLU
ILl
Time, sec
u
u
UJ I LU.IjOJ.LI
Lu
u
IJu
u
LI U •, L i u
u
LL| * I~U
LI U IJ~J
LI
FIG. 4. Cooling bringing about activity in a silent cell. W h e n the cell was cooled it
became active for 56 sec.
j[[!![c[[[f f 16.3oC
f 12"8°C
f 11.3oc
[ 8"7°C
Time, sec FIG. 5. Cooling bringing about activity in a silent cell. The cell activity rapidly adapted but could be elicited by further cooling. Figure 5, using a greater amplification, shows a similar pattern in an inactive cell which was stimulated to activity by cooling. The cell was cooled from 16.3 ° to 12.8°C and this brought about activity in the cell. The activity stopped, but cooling from 12.8 ° to 11.3°C brought about further activity as also did cooling from 11.3 ° to 8.7°C. In all cases, the cell adapted so that the activity slowed down and stopped. If the cell was warmed before the completion of adaptation, the activity was inhibited. Warming could only rarely bring about activity in a silent cell. Heating above 35°C often Caused activity, but this temperature is above the animal's physioIogical
288
G. A. KERKUTAND R. M. A. P. RIDGE
range. A case where warming within the physiological range caused activity to occur in the cell is shown in Fig. 6. (c) Resting potential changes with temperature A series of experiments was carried out using a higher amplification so that the detailed changes in the resting potentials following a temperature change could be examined. In all cases where a clear response was obtained, increasing the temperature led to an increase (hyperpolarization) of the resting potential,
( / (
5 9 " 3 mV
I
,
38'5mV
I1.3°C
IL '~ Time, it.
Lt2~.~i
.........
25.0oc
sec
i ~ i . i i t i [i iJ.LLIj.l~
tl
U t ~
1
[1LI
iJJ
IJ i I [I ljJjJJJJ...LlJ.Ltl
l Li I i I) t i I
Fic. 6. Warming bringing about activity in a silent cell. The cell was warmed and the resting potential increased to 41 mV, but then adapted to 38'5 mV and this depolarization brought about activity. whilst decreasing the temperature led to a decrease (depolarization) of the resting potential. Fig. 7(a) shows the resting potential of an inactive cell that was warmed. No adaptation of the resting potential to its original value was noted. Warming the cell from 13.0°C to 24.9°C increased the resting potential from 19.2 mV to 24.0 mV. The resting potential remained at the new level. Fig. 7(b) shows a similar cell but there was partial adaptation of the resting potential. Warming the cell from 10-7 ° to 22.0°C increased the resting potential from 34.5 mV to 39.2 mV. The resting potential of the cell at 22°C gradually fell to 37.5 mV. The Qlo values for warming and cooling between 10 ° and 20°C varied both for the same cell and between different cells. The range was from 1.04 to 1-96; the maximum changes being found in those cells that adapted. In those cells that were spontaneously active, the depolarization of the resting potential prior to an action potential ranged from 1 mV to 10 mV. Cooling an active cell brought about an increased rate in depolarization and this could elicit a transient increase in the frequency of the action potential. Cooling an inactive cell could bring about a depolarization sufficient to make the cell active. Figure 8(a) shows the relationship between resting potential and the frequency of action potentials following a temperature decrease. The average rate of activity for the 70 sec prior to the temperature change was one potential every 5 sec. The resting potential was 43 mV. The temperature was lowered from 20 ° to
289
TEMPERATURE CHANGES ON NEURONES
9°C. Immediately the frequency of the action potentials increased. The resting potential decreased to just under 41.5 mV, and then returned back to 42.5 mV, even though the temperature remained at 9°C. In a similar experiment where the temperature was increased from 9 ° to 20°C, the resting potential increased from 43 mV to 47 mV (Fig. 8(b)). The frequency of the action potentials decreased from one approximately every second to no activity for 50 sec. The activity then returned even though the resting potential was still at 47 mV. (a} I9.2 mV --.-.,-.-,.-.,,..~
--~- :- ~
_
24.0
.
13.0°C
24.9"C
~
i
i i | / | U L i U U Ll L| I ] U t i U
(b)
U U UU
Li LiLIU
|iU
UII
U LiU
UU
L} U I ]
L] U U ~
54.5 mV 39.2 rnV
37.5mV
10.7"C
\ ~
~
~
-
,,...
22.0"C
t
a Time.
J
t
~_LLLL~
sec
FIG. 7. Effect of warming on the resting potential. (a) Showing an increase in the resting potential, the new value being maintained. (b) Showing an increase in the resting potential, the value adapting back towards the initial level.
In many cases the resting potential, after responding to a temperature change, tended to adapt back to its former level. Fig. 6 shows an interesting example in which warming from 11-3° to 23°C brought about an increase in the resting potential from 39.3 mV to 41 mV. The resting potential slowly returned to a lower level till 32 see later it had reached 38 mV and the cell became active. This
290
G . A . KERKUT AND R. M. A. P. RIDGE
~
0
0
•
0 20
o
to
E
.E
2
c
3
43 o
I
g 42
c: 09
15 °C
~,
g
&•
"
es ing potential
g~ 41
<
-Temperature
tO
I
I
f0
I
20
]
30
I
40
50
sec
Time,
FIG. 8a.
48
47 > E
o
. . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.9
15 oC
4s
.E
20
/
Temperoture,~/
46 0-
o
Resting
potential
0.5 I~ 4 4
I 1.0
g
Action
A
j ~
43
Action
potential~
potential I0
1,5~t
42
I tO
20
I 40
30
Time,
I 50
I 60
sec
FIc. 8b.
FIG. 8. Effect of temperature changes on the resting potential and frequency of action potentials. (a) Cooling from 20 ° to 9°C led to a rapid increase in the frequency of the action potentials and a slow fall of the resting potential. (b) Warming from 9 ° to 20°C brought about a rise in the resting potential and a fall in the frequency of the action potentials. T h e action potentials returned even though the resting potential was at the higher level.
TEMPERATURE
CHANGES
291
ON NEURONE$
reaction was repeated many times. On each occasion there was a considerable lag between the temperature change and the onset of activity--for the specific cell shown in Fig. 6 the average lag over the nine experiments was 43 sec. (d) Effect of temperature change on the duration of the action potential The shape and parameters of the action potential vary considerably from one cell to another. Amplitudes ranging from 30 mV to 75 mV (average 53 mV) and durations from 4 msec to 500 msec (average 55 msec) have been found in ~00
9oi 8C > E
70 60
o
g
50
g. 4o30 20-l0 0
I
I
I
I
I
I
r
I
I
I
.5
i0
t ,5
20
2,5
30
35
4o
45
.50
Time,
55
m sec
FIa. 9. The effect of temperature on the duration of the action potentiM. Cooling led to an increase in the duration of the action potential. cells at room temperature. Cooling the cell brought about an increase in the duration of the action potential. This is seen in Fig. 9, where action potentials photographed from one cell at different temperatures are superimposed. The duration varied from 20 msec at 13.3°C to 45 msec at 3°C. This increase in the duration of the action potential agrees with that described by other workers: Tasaki & Fugita (1948), frog nerve fibres; Hodgkin & Katz (1949), Loligo axon; Coraboeuf & Weidmann (1954), Purkinje fibres; Boistel (1960), cockroach nerve; Loewenstein & Ishiko (1961), paccinian corpuscle-nerve. A decrease in temperature could also bring about a decrease in the amplitude of the action potential.
(e) Cells unaffected by temperature change A few cells showed no response to temperature changes. Five different cells showed a constant rate of spontaneous activity; the frequency being unaffected by temperature changes. In nine other cases the ceils were inactive and temperature changes did not bring about any activity, though mechanical stimulation of these ceils could bring them into activity.
292
G. A. KERKUTAND R. M. A. P. RIDGE
DISCUSSION Changes in the frequency of action potentials due to changes in the temperature of nerve cells have often been described. Some of these nerves are cold or warm receptors whose function is the conveying of information about temperature (e.g. cat cutaneous thermoreceptors, Hensel & Zottermann, 1951a, b, c; Hensel & Witt, 1959, 1960). Receptors with other primary functions can also convey information about temperature. Examples of these are the mechanoreceptors in the cat tongue (Hensel & Zotterman, 1951a); cutaneous touch receptors in the cat (Maruhashi et al., 1952; Douglas et al., 1959; Hunt & McIntyre, 1960); the mechanoreceptors of the facial pit of the rattlesnake Crotalus (Bullock & Diecke, 1956); and the crayfish stretch receptor (Burkhardt, 1959a, b). The effect of temperature changes on the blowfly contact chemoreceptors is reported by Hodgson (1956). Dethier & Arab (1958) later described specific temperature receptors in the tarsus. The behaviour of a nerve can vary with temperature in many ways. Thus some nerves increase their activity on warming, others on cooling. Inverse transients in nerve cells have been found in all the spontaneously active nerve preparations so far studied. The simplest explanation of this type of transient is that cooling the nerve reduces the resting potential; this depolarization leads to an increase in the frequency of the action potentials. Gradually the nerve adapts, physico-chemical systems controlling the membrane re-establish new equilibrium conditions at the low temperatures and the frequency of the action potentials decreases. Conversely, increasing the temperature increases the resting potential; this hyperpolarization causes a temporary slowing in the frequency of action potentials till once more a new equilibrium system is attained for the higher temperature. This raises the problem as to why certain nerve cells do not show these inverse transients but instead behave in a simple manner, the higher temperature leading to a direct increase in activity ? It is possible that this to some extent depends on the rate of change of temperature, but in most of our experiments the rate of temperature change was identical for cells that showed transients and those that did not. The relationship between the change in the resting potential and the frequency of the action potentials in an active neurone is probably not a simple one. Thus, as shown in Fig. 8a, the frequency of the action potentials changed before there was a detectable change of 0.25 mV in the resting potential. This supports the observation of Terzuolo & Bullock (1956) that the spontaneously active cell can be sensitive to very small potential changes. Nor have we found a simple relationship between the value of the resting potential, the depolarization potential and the frequency of the action potentials. An active cell may become silent when the resting potential is increased yet later it may become active at this new resting potential value with no difference in the value of the observed depolarization potential. One cannot fail to be impressed by the manner in which the resting potential of many cells returns back towards the original level in spite of a
TEMPERATURECHANGESON NEURONES
293
temperature change. Though it is possible that in a complex brain the surrounding cells may participate in the observed reactions, it seems more probable that the cell membrane itself has regulating properties. Certain cells during the course of an experiment changed their behaviour. Thus, often during experiments on single cells where the temperature responses were followed over 30 min to 1 hr, the cell initially showed a direct temperaturefrequency response with no transients. Gradually, as the preparation aged, the transients appeared. Later still the cell lost its spontaneous activity but could be Temperature
I
Cool
IIII IIII
I
Warm
I II I[ Iit I II It Ill
I IIIIIIII 1 I IIIIIIII I c
[lllllll IIIIIII
FIG. 10. Diagram to show the changes in behaviour of some neurones during the course of an experiment. (A) Initially cooling led to a decrease in the activity, warming to an increase. (B) Later cooling led to a transient increase in activity, warming to a transient decrease. (C) Later still, the cell lost its spontaneous activity. Cooling brought about activity which was stopped by warming.
brought into activity by cooling; activity being lost on warming. This last response is the exact opposite to the cells' initial behaviour. Initially cooling caused a decrease in activity, warming an increase in activity. Now cooling brings about an increase in activity whilst warming stops activity (Fig. 10). Though some of the cells under observation showed a diminution of the resting potential, we do not think that this is the explanation since other cells maintained their potential to the end of the experiment but still showed this change in behaviour. The present experimental results can be explained in terms of three different factors: 1. The change 'of resting potential following a change in temperature. In most cases this effect leads to the expected change of frequency of the action potentials.
294
G. A. KERKUTAND R. M. A. P. RIDGE
2. T h e adaptation of the resting potential to its initial level at constant temperature. This does not occur in all cases and does not completely explain the adaptation of the activity. 3. Change of the nerve threshold. T h e time relationship between this change in threshold and the resting potential change with temperature appears to be different in some cases. As yet we have no direct experimental evidence concerning the threshold changes with temperature, but such a system can explain the fact that there appears to be no absolute value of resting potential at which the cell will fire-off an action potential. T h e ability to adapt varies from one cell to another and this might be due to ageing and/or injury of the cell. On the other hand, we have not noted any correlation between adaptation and the occurrence of positive after potentials, the latter being an indication of ageing (Moore & Cole, 1959). T h o u g h a unifying concept can be developed to explain the different reactions of the nerve cells to temperature change in terms of various factors, it is possible that there are real and significant differences in the reactions of specific nerves. Such a situation appears to be true for the reactions of the Helix neurones to drugs (Kerkut & Walker, 1961), and though it is convenient to believe that the neurones will behave simply and homogeneously to temperature changes, it is equally possible that there are precise differences between the neurones and that they will behave in a non-uniform manner to temperature changes. Work is at present in progress on the nature and responses of known neurones to see if this throws further light on the subject.
REFERENCES AXELSSON J. ~ BI3LBRINGE. (1961) Metabolic factors affecting the electrical activity of intestinal smooth muscle. 07. Physiol. 156, 344-356. BOISTELJ. (1960) Caractdristiques fonctionnelles des fibres nerveuses et des rdcepteurs tactiles et olfactifs des Insectes. Librairie Arnette, Paris. BULLOCKT. H. & DmCKE F. J. P. (1956) Properties of an infra red receptor.07. Physiol. 134,
47-87. BURKHARDT D. (1959a) Effect of temperature on isolated stretch receptor organ of the crayfish. Science 129, 392-393. BURKH^RDT D. (1959b) Die erregungsvorg~inge sensibler Ganglienzellen in Abh~ingigkeit yon der Temperatur. Biol. Zbl. 78, 22-62. CARDOT H. (1921) Actions des solutions de Ringer hypotoniques sur le cceur isol6 d'Helix pomatia. C.R. Soc. Biol., Paris 85, 813-816. CORABOEUFE. & WEIDMANN S. (1954) Temperature effects upon the electrical activity of Purkinje fibres. Helv. Physiol. Acta 12, 32-41. DETHIERV. G. & ARABY. M. (1958) Effect of temperature on the contact chemoreceptors of the blowfly. 07. Ins. Physiol. 2, 153-161. DOUGLASW. W., RITCHIE J. M. & ST~UB R. W. (1959) Discharges in non myelinated (c) fibres in the cat saphenous nerve in response to changing the temperature of the skin. 07. Physiol. 146, 47-48P. VANGELDERN. & KRNJEVtCK. (1961) Sensitivity of crayfish stretch receptors to temperature changes. 07. Physiol. 156, 14-15P.
TEMPERATURE CHANGES ON NEURONES
295
HENSEL H., IGGO A. & WITT I. (1960) A quantitative study of sensitive cutaneous thermoreceptors with c afferent fibres. J . Physiol. 153, 113-126. HENSEL H. & WITT I. (1959) Spatial temperature gradient and thermoreceptor stimulation. `7. Physiol. 148, 180-187. HENSRL H. & ZOTTERMaN Y. (195 la) T h e response of mechanoreceptors to thermal stimulation..7. Physiol. 115, 16-24. HSNSRL H. & ZOTTEmV~N Y. (1951b) T h e response of the cold receptor to constant cooling. .4cta Physiol. Stand. 22, 97-105. HSNSRL H. & ZOTTEmMAN Y. (1951c) T h e persisting cold sensation. ,4cta Physiol. Seand. 22, 106-115. HODGKIN A. L. & KATZ B. (1949) T h e effect of temperature on the electrical activity of the giant axon of the squid..7. Physiol. 109, 240-249. HODGSON E. S. (1956) Temperature sensitivity of primary chemoreceptors of insects. Anat. Ree. 175, 560-561. HUNT C. C. & MClNTYRe A. K. (1960) Properties of cutaneous touch receptors in cat. .7. Physiol. 153, 88-98. K~mcUT G. A. & RIDGR R. M. A. P. (1961) T h e effect of temperature changes on the resting potential of crab, insect and frog muscle. Corap. Biochem. Physiol. 3, 64-70. KERKUT G. A. & TAYLOR B. J. R. (1956)Effect of temperature on the spontaneous activity from the isolated ganglia of the slug, cockroach and crayfish. Nature, Lond. 178, 426. K~RKUT G. A. & TAYLOR B. J. R. (1958) T h e effect of temperature changes on the activity of poikilotherms. Behaviour 13, 259-279. KEm~UT G. A. & WALKRR R. J. (1961) T h e effect of drugs on the neurones of the snail Helix aspersa. Corap. Biochem. Physiol. 3, 143-160. LORWENSTEIN W. R. & ISHIKO N. (1961) Effect of temperature on the generator and action potentials of a sense organ..7. Gen. Physiol. 45, 105-124. MARUHASHI J., MIzuOucm K. & TASAKI I. (1952) Action currents in single afferent nerve fibres elicited by stimulation of the skin of the toad and the cat..7. Physiol. 117, 129-151. MOORE J. W. & COLE K. S. (1959) Resting and action potentials of the squid giant axon in vivo. `7. Gen. Physiol. 43, 961-970. TASAKI I. & F'VGIXAM. (1948) Action currents of single nerve fibres as modified by temperature changes. J. Neurophysiol. 11, 311-315. TRRZUOLO C. A. & BULLOCKT . H. (1956) Measurement of imposed voltage gradient adequate to modulate neuronal firing. Proc. Nat. Acad. Sci., Wash. 42, 687-694.