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Neuromuscular function at low temperatures in frogs from cold and warm climates
Comp. Biochem. Physiol., 1969, Vol. 28, pp. 915 to 921. Pergamon Press. Printed in Great Britain
NEUROMUSCULAR F U N C T I O N AT LOW TEMPERATURES IN FROGS FROM COLD AND WARM CLIMATES*t L. K E I T H
M I L L E R and P E T E R J. D E H L I N G E R : ~
Institute of Arctic Biology, University of Alaska, College, Alaska 99701 (Received 20 )tune 1968)
Abstract--1. Nerve-muscle function was studied at a variety of temperatures in the most northerly American frog, Rana sylvatica, and in a southern (westcentral Mexico) population of Rana pipiens. 2. Maximal twitch tension in isolated gastrocnemius muscles increased with decreasing temperature in R. sylvatica but decreased with decreasing temperature in R. pipiens. 3. R. sylvatica muscles remained excitable to sciatic nerve stimulation until the freezing point ( - 3 to - 5°C) was reached. Muscles from R. pipiens became inexcitable to either nerve or direct stimulation at about 0°C. 4. No temperature-related differences in nerve function were seen between the two species. INTRODUCTION IN MUCH of the cold boreal region of N o r t h America k is common during spring and summer to encounter a small resident amphibian, the wood frog, R a n a sylvatica. This species ranges considerably farther north than any other amphibian in the western hemisphere (Wright & Wright, 1949), and one must suspect that it possesses certain characteristics that enable it to live in an environment apparently hostile to other amphibians. During late summer it is possible to recognize at least three major sizes of adult frogs, and Bellis (1961) states that adults may survive for at least 3 years. T h i s means that in northern Canada and Alaska the adult frogs must overwinter in an environment where air temperatures not uncommonly reach - 50°C. T h e survival of the wood frog in northern latitudes probably depends upon a n u m b e r of factors. Moore (1949) has emphasized the importance of embryonic development and behavior in limiting the distribution of frogs. Herreid & Kinney (1967) have applied Moore's criteria in a study of the Alaskan wood frog. T h e y conclude that the Alaskan population is not noticeably different from more southern wood frog populations with respect to larval temperature tolerance and rate of development. * This study was supported by Grant No. GM-10402 from the National Institute of Health. t Publication No. 83, Institute of Arctic Biology. ++Present address: Biophysics Laboratory, Stanford University, Palo Alto, California. 915
916
L. KEITH MILLER AND PETER J. DEHLINGER
T h e ability to p e r f o r m co-ordinated m o v e m e n t s at low t e m p e r a t u r e would appear to be an important factor in the survival of a poikilotherm. I n interior Alaska, cold spells during the mating period in late April or M a y m a y result in air temperatures well below freezing, with light ice forming on smaller ponds. Even in m i d - s u m m e r cold periods m a y occur during which ambient temperatures approach freezing. I n the present paper we have examined the performance of peripheral nerve and nerve-muscle preparations of Rana sylvatica at different temperatures. For comparative purposes similar preparations from a southern (Mexico) population of the ubiquitous Rana pipiens were studied over the same t e m p e r a t u r e range. MATERIALS AND M E T H O D S Adult Rana sylvatica were collected in open fields in the vicinity of Fairbanks, Alaska (latitude 65°N.). The Rana pipiens were collected near Guasave or Guamuchil, Sinaloa, Mexico (latitude 28°N.), and were supplied by College Biological Supply (Escondido, California). Total body length ofR. sylvatica was A a. 5 cm; R.pipiens was 7-9 cm in length. In the laboratory frogs were maintained at room temperature (about 23 °C) for periods of up to 1 week in open aquaria. They were fed a light diet of mealworms. The sciatic nerve-gastrocnemius unit was removed from decapitated frogs and placed on a Plexiglas mount in which were embedded platinum-iridium electrodes. Multiple, alternate positive-negative electrodes provided nearly simultaneous direct muscle stimulation. Paired platinum electrodes were used for nerve stimulation and recording. Isometric twitch tension was measured with a Sanborn 100-g transducer and recorded photographically from oscilloscope display. Nerve and muscle action potentials were also displayed on the oscilloscope. The mounted preparation was suspended in a 1300-ml cooling chamber containing a frog Ringer solution (NaC1, 6"5 g/l; KC1, 0"12 g/l; Caclz, 0"14 g/l; NaHCOa, 0"2 g/l). Chamber temperature was controlled by circulating methanol through the chamber jacket. A 1-1. Dewar flask served as a reservoir to raise or lower the level of saline. The preparation was immersed in saline except during actual stimulation and recording. Temperatures were changed in 5°C steps between 25 and 0°C, after which the temperature was gradually lowered until the preparation froze or became inexcitable. At each temperature level nerves were tested for excitability (electrical threshold at 0"l-msec pulse duration), conduction velocity and absolute refractory period. Only the fast fiber component was used for these determinations. RESULTS I n tests of combined nerve-muscle function striking differences in maximal twitch tension were seen between R. sylvatica and R. pipiens at low temperatures. R. sylvatica muscles developed m o r e tension at 0 than at 25°C, the increase amounting to 150-200 per cent of the 25°C value (Fig. 1A). In contrast, twitch amplitudes in R. pipiens declined as t e m p e r a t u r e decreased, and the muscles became inexcitable at about 0°C (Fig. 1B). R. sylvatica muscles did not become inexcitable until they actually froze. Freezing occurred in muscles of both species at approximately - 3°C. Direct muscle stimulation did not greatly alter or improve twitch responses at any t e m p e r a t u r e in either species. T h e stimulus voltage required to elicit a
NEUROMUSCULARFUNCTIONAT LOW TEMPERATURESIN FROGS
917
maximal twitch response was considerably greater for direct muscle stimulation than for stimulation via nerves. As would be expected, the electrical activity of the muscles showed changes with t e m p e r a t u r e in keeping with the changes in twitch amplitude (Fig. 1A, B, middle sweep in each frame). Muscle action potential amplitude increased with decreasing
tA Rana sylvat[ca
(Alosko)
m
m IB Rono pipiens
(Mexico)
m
FIG. 1. A. R. sylvatica sciatic nerve action potentials (small arrow, upper trace), gastrocnemius muscle action potential (middle trace) and twitch tension (lower trace) at various temperatures. B. R. pipiens nerve and muscle action potentials and twitch tension at various temperatures; same sequence as for A.
918
L . K E I T H M I L L E R AND PETER
J.
DEHLINGER
temperature in R. sylvatica, but showed a steady decline with decreasing temperature in R. pipiens. Analysis of nerve electrical characteristics failed to point up any important temperature-dependent differences between the Alaska R. sylvatica and the Mexico R. pipiens (Figs. 2, 3, 4). In nerves of both species action potentials of considerable magnitude could be produced by electrical stimulation just prior to freezing at I. 5 r
o
pip/ens
•
$ylvat/CO
1
L
I0
o
.3
-5
5 15 25 TEMPERATURE (%)
FIG. 2. Sciatic nerve excitability in R. sylvatica and R. pipiens at different temperatures. 40o e
30-
o
o
g
2o
-
•
o
E
10--
o
°
• I
-5
o pipiens * sy/vaftco I [
5 15 25 TEMPERATURE (°C)
FIG. 3. Sciatic nerve c o n d u c t i o n velocity at different temperatures in R. sylvatica and R. pipiens. BO
o plptens sylvotico •
2O
.= EIO
0 -5
I
?
"
¢
5 15 25 TEMPERATURE (°C)
FIG. 4. Sciatic nerve absolute refractory period at different temperatures in R. sylvatica and R. pipiens.
NEUROMUSCULAR FUNCTION AT LOW TEMPERATURES I N FROGS
919
2 to - 5°C. The Q10 of nerve conduction velocity was about 2.0 in both frogs at mid-temperature range. Temperature coefficients of excitability and absolute refractory period were close to those found in most vertebrate nerves. -
DISCUSSION The continuous decrease in twitch tension with decreasing temperature seen in R. pipiens muscle is a feature unusual enough to warrant special comment. A number of papers dealing with the basic physiology of muscle function have noted an increase in maximal twitch tension with a decrease in temperature (see Hill, 1951). The majority of these studies have been done on English frogs, species usually unspecified but assumed to be R. temporaria. England has a temperate, cool winter climate, so the frogs found there are much closer to R. sylvatica than to our Mexico R. pipiens in their degree of cold exposure. As far as we can determine, R. pipiens muscles used in the present study are unique in showing a decrease in twitch tension at temperatures below 20°C, and we suggest that this characteristic is due to the muscles being accustomed, if not adapted, to operation at higher temperature. We cannot make a quantitative comparison between the twitch tension data from R. sylvatica and previous results obtained with English frogs, so it is not possible to say if R. sylvatica muscles might operate better at low temperatures than the English species. Other evidence exists for temperature adaptation in amphibian muscle. Ushakov & Zander (1961) compared heat resistance and muscle excitability in lake frogs (R. ridibunda) from cold and warm habitats. Strength-duration curves for sartorius muscle from "warm" frogs showed that a constant excitability level to stimuli of long duration was maintained at higher temperatures and that rheobase increased more slowly with increasing temperature. As shown by Brattstrom & Lawrence (1962), auran amphibians can acclimate to temperature changes in a rather short time. Using the critical thermal maximum (point at which locomotory activity becomes disorganized--CTM) as a criterion of acclimation, Brattstrom & Lawrence showed that R. pipiens had a C T M near 31.4°C when acclimated to 5°C, but the C T M increased to about 34.5°C within 48 hr after transferring the frogs to 23°C. Since all of our frogs were kept at room temperature for at least 24 hr, and most were at room temperature for several days before testing, the differences in muscle function between our cold and warm climate frogs must be relatively long lasting, if not permanent. It is not possible to say if the C T M changes observed by Brattstrom & Lawrence involved changes only in muscle, only in nerve, or a combination of the two, but from our data it is most likely that the changes primarily involved muscle. In view of the numerous examples of temperature adaptation in mammalian nerves it is surprising to find no apparent temperature differences in nerves of Alaska R. sylvatica and Mexico R. pipiens. However, this finding agrees with previous work reported in the extensive literature on temperature relations in frog nerve. With one exception, all reports of action potential extinction temperatures
920
L . KEITH MILLER AND PETER J. DEHLINGER
indicate that frog nerves conduct until they freeze, usually at - 5 to - 7°C. Sjodin & Mullins (1958) stated that their frog sciatic nerves (species unnamed) did not conduct below 2°C. For warm acclimatized R. pipiens the limiting factor in low temperature activity must then be muscle or neuromuscular junction rather than nerve. In our experiments little increase in twitch tension occurred when the muscles were stimulated directly instead of via nerve, indicating that the neuromuscular junction is not the limiting factor in neuromuscular function in the cold. Some property or properties of the muscle itself must limit low temperature activity in R. pipiens. R. sylvatica muscles frozen at - 3 to - 5 ° C recovered their excitability and contractility, though not always to a pre-freezing level, if they did not remain frozen much more than 30 rain. This finding agrees with the classic studies of Moran (1929), who states that frog (presumably English) sartorius muscles lose irritability after being frozen for 0.5 hr at - 3.5°C. It seems safe to assume that the wood frog, even in interior Alaska, does not survive freezing. Because of dormancy behavior it is probable that a variety of frogs found in cooler parts of the world are exposed to temperatures similar to those encountered by R. sylvatica in Alaska, i.e. the Alaskan frogs must overwinter in situations where their tissue temperatures never reach freezing. Such situations obtain in both of the postulated overwintering sites: pond bottoms and forest floor. Pond mud covered by water would be unfrozen, through near 0°C. Figure 5 shows O
-I.0~ / ~ D
..,
~-2~o-
/,
-3.O--
-4,.0
---e
.
,
~. '~
~n~
,e
/ ".._"-: / p.
/
l
I~'X I.~". i(
Li
""I / "I •" ~ i l ,i / 1 : I
/
/7p4o cm
I/I
Ill
I'l U/ I~I I.:.1
~l Ill
"X
!
i qX. %. I I "~
Y~'~
~ ~ ~ ~ ~, ~ t,.~.
~
30cm
V '., v
V r.,
/
; I
i
~20cm
lelOcm
/l~ l ~. ill lil~. ~ • HI UI ~'t NI I,I I" ~ U/ I':.1 ~"..I Ill
I"1
~'...t I/I
15 20 25
5 I0
15 ~0 ~5
5
DEC 1966
dAN
1967
FEB 1967
I0 15
FIG. 5. G r o u n d level (0 cm) and subsoil temperatures (to 40-cra depth) d u r i n g
winter, near Fairbanks, Alaska.
N E U R O M U S C U L A R F U N C T I O N AT L O W TEMPERATURES I N F R O G S
921
a typical midwinter temperature profile of the ground in a field near Fairbanks. It is very unlikely that the frogs could burrow to the 50-60-cm depth that would have to be reached to be completely safe from freezing. We therefore feel that our results point to the use of pond bottoms as overwintering sites. If there is any adaptive advantage in the low temperature neuromuscular function in the northern wood frog it probably derives from the fact that this amphibian encounters low temperatures more frequently or for greater periods of time than southern frogs and, perhaps most important, it seldom or never encounters the high temperatures commonly experienced by southern frogs. Amphibians living in the warmest climates should provide excellent material for additional neuromuscular studies concerned with temperature acclimatization or adaptation. REFERENCES
BELLISE. m. (1961) Growth of the wood frog, Rana sylvatica. Copeia 74-77. BRATTSTROM B. H. & LAWRENCEP. (1962) The rate of thermal acclimation in anuran amphibians. Physiol Zool. 35, 148-156. HERREIDC. F., II & KINNEYS. (1967) Temperature and development of the wood frog, Rana sylvatica, in Alaska. Ecology 48, 579-590. HILL A. V. (1951) The influence of temperature on the tension developed in an isometric twitch. Proc. Roy. Soc. (Lond.) B 138, 349-354. MOORE J. A. (1949) Patterns of Evolution in the Genus Rana, in Genetics, Paleontology, and Evolution (Edited by JEPSF_NG. L., MAYa E. & SIMPSONG. G.), pp. 315-338. Princeton University Press, New Jersey. MORAN T. (1929) Critical temperature of freezing--living muscle. Proc. Roy Soc. (Lond.), B 105, 177-197. SJODIN R. A. & MULLINS L. J. (1958) The action potential of frog nerve as affected by temperature, narcosis, and stimulation frequency..7, cell. comp. Physiol. 51,425-436. USHAKOVB. P. & ZANDERN. V. (1961) Adaptation of muscle fibers of lake frogs in warm habitat to the temperature factor. Biofi~ika 6, 322-327. WRIGHT A. H. & WRIGHT A. A. (1949) Handbook of Frogs and Toads of the United States andCanada, pp. 540-544. Comstoek, Ithaca, New York.
NEUROMUSCULAR F U N C T I O N AT LOW TEMPERATURES IN FROGS FROM COLD AND WARM CLIMATES*t L. K E I T H
M I L L E R and P E T E R J. D E H L I N G E R : ~
Institute of Arctic Biology, University of Alaska, College, Alaska 99701 (Received 20 )tune 1968)
Abstract--1. Nerve-muscle function was studied at a variety of temperatures in the most northerly American frog, Rana sylvatica, and in a southern (westcentral Mexico) population of Rana pipiens. 2. Maximal twitch tension in isolated gastrocnemius muscles increased with decreasing temperature in R. sylvatica but decreased with decreasing temperature in R. pipiens. 3. R. sylvatica muscles remained excitable to sciatic nerve stimulation until the freezing point ( - 3 to - 5°C) was reached. Muscles from R. pipiens became inexcitable to either nerve or direct stimulation at about 0°C. 4. No temperature-related differences in nerve function were seen between the two species. INTRODUCTION IN MUCH of the cold boreal region of N o r t h America k is common during spring and summer to encounter a small resident amphibian, the wood frog, R a n a sylvatica. This species ranges considerably farther north than any other amphibian in the western hemisphere (Wright & Wright, 1949), and one must suspect that it possesses certain characteristics that enable it to live in an environment apparently hostile to other amphibians. During late summer it is possible to recognize at least three major sizes of adult frogs, and Bellis (1961) states that adults may survive for at least 3 years. T h i s means that in northern Canada and Alaska the adult frogs must overwinter in an environment where air temperatures not uncommonly reach - 50°C. T h e survival of the wood frog in northern latitudes probably depends upon a n u m b e r of factors. Moore (1949) has emphasized the importance of embryonic development and behavior in limiting the distribution of frogs. Herreid & Kinney (1967) have applied Moore's criteria in a study of the Alaskan wood frog. T h e y conclude that the Alaskan population is not noticeably different from more southern wood frog populations with respect to larval temperature tolerance and rate of development. * This study was supported by Grant No. GM-10402 from the National Institute of Health. t Publication No. 83, Institute of Arctic Biology. ++Present address: Biophysics Laboratory, Stanford University, Palo Alto, California. 915
916
L. KEITH MILLER AND PETER J. DEHLINGER
T h e ability to p e r f o r m co-ordinated m o v e m e n t s at low t e m p e r a t u r e would appear to be an important factor in the survival of a poikilotherm. I n interior Alaska, cold spells during the mating period in late April or M a y m a y result in air temperatures well below freezing, with light ice forming on smaller ponds. Even in m i d - s u m m e r cold periods m a y occur during which ambient temperatures approach freezing. I n the present paper we have examined the performance of peripheral nerve and nerve-muscle preparations of Rana sylvatica at different temperatures. For comparative purposes similar preparations from a southern (Mexico) population of the ubiquitous Rana pipiens were studied over the same t e m p e r a t u r e range. MATERIALS AND M E T H O D S Adult Rana sylvatica were collected in open fields in the vicinity of Fairbanks, Alaska (latitude 65°N.). The Rana pipiens were collected near Guasave or Guamuchil, Sinaloa, Mexico (latitude 28°N.), and were supplied by College Biological Supply (Escondido, California). Total body length ofR. sylvatica was A a. 5 cm; R.pipiens was 7-9 cm in length. In the laboratory frogs were maintained at room temperature (about 23 °C) for periods of up to 1 week in open aquaria. They were fed a light diet of mealworms. The sciatic nerve-gastrocnemius unit was removed from decapitated frogs and placed on a Plexiglas mount in which were embedded platinum-iridium electrodes. Multiple, alternate positive-negative electrodes provided nearly simultaneous direct muscle stimulation. Paired platinum electrodes were used for nerve stimulation and recording. Isometric twitch tension was measured with a Sanborn 100-g transducer and recorded photographically from oscilloscope display. Nerve and muscle action potentials were also displayed on the oscilloscope. The mounted preparation was suspended in a 1300-ml cooling chamber containing a frog Ringer solution (NaC1, 6"5 g/l; KC1, 0"12 g/l; Caclz, 0"14 g/l; NaHCOa, 0"2 g/l). Chamber temperature was controlled by circulating methanol through the chamber jacket. A 1-1. Dewar flask served as a reservoir to raise or lower the level of saline. The preparation was immersed in saline except during actual stimulation and recording. Temperatures were changed in 5°C steps between 25 and 0°C, after which the temperature was gradually lowered until the preparation froze or became inexcitable. At each temperature level nerves were tested for excitability (electrical threshold at 0"l-msec pulse duration), conduction velocity and absolute refractory period. Only the fast fiber component was used for these determinations. RESULTS I n tests of combined nerve-muscle function striking differences in maximal twitch tension were seen between R. sylvatica and R. pipiens at low temperatures. R. sylvatica muscles developed m o r e tension at 0 than at 25°C, the increase amounting to 150-200 per cent of the 25°C value (Fig. 1A). In contrast, twitch amplitudes in R. pipiens declined as t e m p e r a t u r e decreased, and the muscles became inexcitable at about 0°C (Fig. 1B). R. sylvatica muscles did not become inexcitable until they actually froze. Freezing occurred in muscles of both species at approximately - 3°C. Direct muscle stimulation did not greatly alter or improve twitch responses at any t e m p e r a t u r e in either species. T h e stimulus voltage required to elicit a
NEUROMUSCULARFUNCTIONAT LOW TEMPERATURESIN FROGS
917
maximal twitch response was considerably greater for direct muscle stimulation than for stimulation via nerves. As would be expected, the electrical activity of the muscles showed changes with t e m p e r a t u r e in keeping with the changes in twitch amplitude (Fig. 1A, B, middle sweep in each frame). Muscle action potential amplitude increased with decreasing
tA Rana sylvat[ca
(Alosko)
m
m IB Rono pipiens
(Mexico)
m
FIG. 1. A. R. sylvatica sciatic nerve action potentials (small arrow, upper trace), gastrocnemius muscle action potential (middle trace) and twitch tension (lower trace) at various temperatures. B. R. pipiens nerve and muscle action potentials and twitch tension at various temperatures; same sequence as for A.
918
L . K E I T H M I L L E R AND PETER
J.
DEHLINGER
temperature in R. sylvatica, but showed a steady decline with decreasing temperature in R. pipiens. Analysis of nerve electrical characteristics failed to point up any important temperature-dependent differences between the Alaska R. sylvatica and the Mexico R. pipiens (Figs. 2, 3, 4). In nerves of both species action potentials of considerable magnitude could be produced by electrical stimulation just prior to freezing at I. 5 r
o
pip/ens
•
$ylvat/CO
1
L
I0
o
.3
-5
5 15 25 TEMPERATURE (%)
FIG. 2. Sciatic nerve excitability in R. sylvatica and R. pipiens at different temperatures. 40o e
30-
o
o
g
2o
-
•
o
E
10--
o
°
• I
-5
o pipiens * sy/vaftco I [
5 15 25 TEMPERATURE (°C)
FIG. 3. Sciatic nerve c o n d u c t i o n velocity at different temperatures in R. sylvatica and R. pipiens. BO
o plptens sylvotico •
2O
.= EIO
0 -5
I
?
"
¢
5 15 25 TEMPERATURE (°C)
FIG. 4. Sciatic nerve absolute refractory period at different temperatures in R. sylvatica and R. pipiens.
NEUROMUSCULAR FUNCTION AT LOW TEMPERATURES I N FROGS
919
2 to - 5°C. The Q10 of nerve conduction velocity was about 2.0 in both frogs at mid-temperature range. Temperature coefficients of excitability and absolute refractory period were close to those found in most vertebrate nerves. -
DISCUSSION The continuous decrease in twitch tension with decreasing temperature seen in R. pipiens muscle is a feature unusual enough to warrant special comment. A number of papers dealing with the basic physiology of muscle function have noted an increase in maximal twitch tension with a decrease in temperature (see Hill, 1951). The majority of these studies have been done on English frogs, species usually unspecified but assumed to be R. temporaria. England has a temperate, cool winter climate, so the frogs found there are much closer to R. sylvatica than to our Mexico R. pipiens in their degree of cold exposure. As far as we can determine, R. pipiens muscles used in the present study are unique in showing a decrease in twitch tension at temperatures below 20°C, and we suggest that this characteristic is due to the muscles being accustomed, if not adapted, to operation at higher temperature. We cannot make a quantitative comparison between the twitch tension data from R. sylvatica and previous results obtained with English frogs, so it is not possible to say if R. sylvatica muscles might operate better at low temperatures than the English species. Other evidence exists for temperature adaptation in amphibian muscle. Ushakov & Zander (1961) compared heat resistance and muscle excitability in lake frogs (R. ridibunda) from cold and warm habitats. Strength-duration curves for sartorius muscle from "warm" frogs showed that a constant excitability level to stimuli of long duration was maintained at higher temperatures and that rheobase increased more slowly with increasing temperature. As shown by Brattstrom & Lawrence (1962), auran amphibians can acclimate to temperature changes in a rather short time. Using the critical thermal maximum (point at which locomotory activity becomes disorganized--CTM) as a criterion of acclimation, Brattstrom & Lawrence showed that R. pipiens had a C T M near 31.4°C when acclimated to 5°C, but the C T M increased to about 34.5°C within 48 hr after transferring the frogs to 23°C. Since all of our frogs were kept at room temperature for at least 24 hr, and most were at room temperature for several days before testing, the differences in muscle function between our cold and warm climate frogs must be relatively long lasting, if not permanent. It is not possible to say if the C T M changes observed by Brattstrom & Lawrence involved changes only in muscle, only in nerve, or a combination of the two, but from our data it is most likely that the changes primarily involved muscle. In view of the numerous examples of temperature adaptation in mammalian nerves it is surprising to find no apparent temperature differences in nerves of Alaska R. sylvatica and Mexico R. pipiens. However, this finding agrees with previous work reported in the extensive literature on temperature relations in frog nerve. With one exception, all reports of action potential extinction temperatures
920
L . KEITH MILLER AND PETER J. DEHLINGER
indicate that frog nerves conduct until they freeze, usually at - 5 to - 7°C. Sjodin & Mullins (1958) stated that their frog sciatic nerves (species unnamed) did not conduct below 2°C. For warm acclimatized R. pipiens the limiting factor in low temperature activity must then be muscle or neuromuscular junction rather than nerve. In our experiments little increase in twitch tension occurred when the muscles were stimulated directly instead of via nerve, indicating that the neuromuscular junction is not the limiting factor in neuromuscular function in the cold. Some property or properties of the muscle itself must limit low temperature activity in R. pipiens. R. sylvatica muscles frozen at - 3 to - 5 ° C recovered their excitability and contractility, though not always to a pre-freezing level, if they did not remain frozen much more than 30 rain. This finding agrees with the classic studies of Moran (1929), who states that frog (presumably English) sartorius muscles lose irritability after being frozen for 0.5 hr at - 3.5°C. It seems safe to assume that the wood frog, even in interior Alaska, does not survive freezing. Because of dormancy behavior it is probable that a variety of frogs found in cooler parts of the world are exposed to temperatures similar to those encountered by R. sylvatica in Alaska, i.e. the Alaskan frogs must overwinter in situations where their tissue temperatures never reach freezing. Such situations obtain in both of the postulated overwintering sites: pond bottoms and forest floor. Pond mud covered by water would be unfrozen, through near 0°C. Figure 5 shows O
-I.0~ / ~ D
..,
~-2~o-
/,
-3.O--
-4,.0
---e
.
,
~. '~
~n~
,e
/ ".._"-: / p.
/
l
I~'X I.~". i(
Li
""I / "I •" ~ i l ,i / 1 : I
/
/7p4o cm
I/I
Ill
I'l U/ I~I I.:.1
~l Ill
"X
!
i qX. %. I I "~
Y~'~
~ ~ ~ ~ ~, ~ t,.~.
~
30cm
V '., v
V r.,
/
; I
i
~20cm
lelOcm
/l~ l ~. ill lil~. ~ • HI UI ~'t NI I,I I" ~ U/ I':.1 ~"..I Ill
I"1
~'...t I/I
15 20 25
5 I0
15 ~0 ~5
5
DEC 1966
dAN
1967
FEB 1967
I0 15
FIG. 5. G r o u n d level (0 cm) and subsoil temperatures (to 40-cra depth) d u r i n g
winter, near Fairbanks, Alaska.
N E U R O M U S C U L A R F U N C T I O N AT L O W TEMPERATURES I N F R O G S
921
a typical midwinter temperature profile of the ground in a field near Fairbanks. It is very unlikely that the frogs could burrow to the 50-60-cm depth that would have to be reached to be completely safe from freezing. We therefore feel that our results point to the use of pond bottoms as overwintering sites. If there is any adaptive advantage in the low temperature neuromuscular function in the northern wood frog it probably derives from the fact that this amphibian encounters low temperatures more frequently or for greater periods of time than southern frogs and, perhaps most important, it seldom or never encounters the high temperatures commonly experienced by southern frogs. Amphibians living in the warmest climates should provide excellent material for additional neuromuscular studies concerned with temperature acclimatization or adaptation. REFERENCES
BELLISE. m. (1961) Growth of the wood frog, Rana sylvatica. Copeia 74-77. BRATTSTROM B. H. & LAWRENCEP. (1962) The rate of thermal acclimation in anuran amphibians. Physiol Zool. 35, 148-156. HERREIDC. F., II & KINNEYS. (1967) Temperature and development of the wood frog, Rana sylvatica, in Alaska. Ecology 48, 579-590. HILL A. V. (1951) The influence of temperature on the tension developed in an isometric twitch. Proc. Roy. Soc. (Lond.) B 138, 349-354. MOORE J. A. (1949) Patterns of Evolution in the Genus Rana, in Genetics, Paleontology, and Evolution (Edited by JEPSF_NG. L., MAYa E. & SIMPSONG. G.), pp. 315-338. Princeton University Press, New Jersey. MORAN T. (1929) Critical temperature of freezing--living muscle. Proc. Roy Soc. (Lond.), B 105, 177-197. SJODIN R. A. & MULLINS L. J. (1958) The action potential of frog nerve as affected by temperature, narcosis, and stimulation frequency..7, cell. comp. Physiol. 51,425-436. USHAKOVB. P. & ZANDERN. V. (1961) Adaptation of muscle fibers of lake frogs in warm habitat to the temperature factor. Biofi~ika 6, 322-327. WRIGHT A. H. & WRIGHT A. A. (1949) Handbook of Frogs and Toads of the United States andCanada, pp. 540-544. Comstoek, Ithaca, New York.