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Tonic immobility and shivering in birds: Evolutionary implications
Physiology & Behavior, Vol. 27, pp. 475-480. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.
Tonic Immobility and Shivering in Birds: Evolutionary Implications ESA HOHTOLA
University o f Oulu, Department o f Zoology Zoophysiological Laboratory SF-90100 Oulu 10, Finland R e c e i v e d 13 A p r i l 1981 HOHTOLA, E. Tonic immobility and shivering in birds: Evolutionary implications. PHYSIOL. BEHAV. 27(3) 475-480, 1981 .--The influence of ambient temperature and lighting on the duration of tonic immobility (TI) in birds was studied in conjunction with simultaneous shivering measurements. Contrary to what has been found in mammals, low ambient temperature did not disrupt TI in pigeons and house sparrows: the TI durations were equal at +2°C and +25°C. Electromyographic recordings in pigeons showed that after an initial suppression, shivering soon reappeared and reached normal levels during TI at +2°C. When the duration of TI was prolonged (indicating slower recovery of postural activity) by experimental manipulation (darkness), shivering likewise reappeared more slowly. The ability of birds to maintain shivering during TI, and the congruent changes in the duration of TI and the intensity of shivering suggest that shivering has neuromuscular and evolutionary connections to the postural activity of skeletal muscles also in birds. The difference between birds and mammals in reference to the effects of low ambient temperature on TI may be related to a differential recruitment of muscles for shivering thermogenesis. Tonic immobility
Columba livia
Shivering thermogenesis
Postural activity
Endothermy
Birds
Passer domesticus
S H I V E R I N G is a major mechanism for regulatory heat production in endotherms [4, 7, 31]. It is characterized by a generalized muscle tension, linearly related to input from specific cold sensors. Usually, shivering is best detected as an increased electromyographic (EMG) activity of skeletal muscles, but upon severe cooling, overt tremors are also demonstrable [10]. The exclusive occurrence of shivering in mammals and birds provides an interesting question about its evolutionary origins. To explain this phenomenon, other physiological traits specific to birds and mammals must be sought. A salient feature in the general behavior of mammals and birds is their limb-supported stance that is maintained for long periods of time. This is in marked contrast with the sprawled posture of reptiles. The inevitable neuromuscular adjustments needed for this postural change led Heath [8] to suggest that shivering most probably developed from the posturai activity of skeletal muscles, as it provides a mechanism for maintaining prolonged muscle tension (see also [21]). This hypothesis is not readily amenable to experimental tests, and so far only circumstantial evidence is available. Recently, however, studies on tonic immobility (TI) have unexpectedly shed light on the hypothetical relationship between shivering and postural activity. TI (also: animal hypnosis) is a naturally occurring antipredator response that can be elicited also experimentally by a brief physical restraint (for references, see [17]). TI provides a unique opportunity to study the relation of shivering to postural activity: It is
characterized by a profound inhibition of gross motor activity [5], while at least part of the postural mechanisms remain functional, resulting often in exaggerated postures and visible tremors of the extremities [17,19]. The gradual recovery of postural activity eventually leads to self-righting which terminates TI. Studies on TI [29,30] have now shown that in adult mammals, external cooling disrupts TI. The authors conclude that TI is disrupted because shivering , stimulated by low temperature, enhances postural activity and induces the righting reflex. These studies, and the previous observation [12] that shivering does not terminate TI in pigeons and house sparrows, prompted a more detailed study on the relationship between postural activity and shivering in birds. The experimental approach to this problem was to study the effects of low ambient temperature on TI and, by using EMGtechniques, to characterize the shivering response during TI. EXPERIMENT 1 Studies on rats and rabbits [29,30] have shown that external cooling potentiates TI during the early postnatal period, but disrupts it in fully-grown animals. In an earlier study with birds [12], it was observed that there were no difficulties in inducing TI in pigeons and house sparrows at low ambient temperatures. As a first step towards elucidating potential differences between the immobility reactions of mammals and birds, the first experiment was designed to study the
C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/090475-06502.00/0
476
HOHTOLA
influence of low ambient temperature on the duration of TI in birds.
500['~--'
METHOD
PIGEON
~ 400 i
HOUSE
PIGEON
SPARROW
HOUSE SPARROW
g 3oo
Animals Adult pigeons (Columba livia, mass 270-350 g), captured in Southern Finland and house sparrows (Passer domesticus, mass 27-29 g), captured near the laboratory were used. The birds were housed in metal wire cages in an animal room maintained at +24°C with a 12:12-hour light/dark regime. All birds were experimentally naive and were kept in the animal room for at least 5 weeks before experiments. They had free access to food and water at all time except during the actual test procedures.
Procedure and Equipment The duration of TI was measured at +2°C and +25°C in both species. Before experiments, birds of each species were randomly divided into two groups, which were then tested on a Latin square basis: Each birds was tested at either of the two temperatures on day 1, and at the other temperature on the following day at the same time. All experiments were done 3--8 hours after lights-on (6 a.m.). The experiments were performed in a dark-walled box placed in a thermostatically controlled cold-room. The bottom of the box was covered with a layer of paper sheets to avoid direct thermal contact with the floor during TI. To mask disturbing ambient sounds, a steady background noise was produced by feeding a 30-Hz sinusoidal signal from an audio generator into a horizontally mounted loudspeaker and by placing small glass beads on the loudspeaker diaphragm. Before an experiment, each bird had a 20-minute adaptation time to the test temperature in a small side compartment of the experimental box. TI was then induced by restraining the bird manually in a supine position for 15 seconds. Maximally 3 inductions were needed for any bird. The duration of TI was measured as the time elapsed from the cessation of restrainment to the point of self-righting. The TI of pigeons was monitored remotely using a video-system (Philips LDH25 camera and Finnvideo VM 12FI monitor), while the TI of house sparrows was observed directly by placing the lid of the experimental box so that the bird could not see the experimenter. RESULTS
Figure 1 presents the mean durations of TI in pigeons and house sparrows as a function of ambient temperature and trial number. Clearly, ambient temperature has no influence on TI duration in either species. In agreement with previous reports (see [17]), a habituation of the responses was observed: The duration of TI during the second trial was significantly shorter in both species (pigeon, p <0.001; house sparrow, p=0.05; Wilcox's signed-ranks test for paired values). The mean number of inductions needed to elicit TI showed no variation with respect to the experimental conditions, being always 1.7-1.8 in pigeons and 1.%2.1 in house sparrows. EXPERIMENT 2 Whishaw et al. [29,30] postulate that the inhibition (short duration) of TI in mammals at low ambient temperatures is related to the development of shivering thermogenesis. They
200)
'-'
i
, ,°2r +25
+2
+25
+2 °C
AMBIENT TEMPERATURE
1.
2 TRIAL
1.
2.
NUMBER
FIG. 1. The durations (mean ~+ SEM) of tonic immobility (TI) as a function of ambient temperature and trial number (Latin square procedure) in pigeons and house sparrows. N = 10 and 6, respectively.
assert that in adult animals external cooling stimulates shivering which disrupts TI by driving a set of postural adjustments that cause the animal to take an upright posture. In newborn animals, postural activity is not enhanced due to the immaturity of the shivering response. Unfortunately, no EMG-recordings were made in these experiments. The second experiment of the present study was thus designed to measure and characterize, by EMG-techniques, the shivering response of the pigeon during TI. METHOD
Animals Adult pigeons, obtained and maintained as in Experiment 1, were used.
Procedure and equipment TI was induced at +2°C as in Experiment 1. Shivering was measured as described previously [13]. Briefly, three monopolar stainless steel electrodes in a fixed close arrangement were inserted into the pectoral muscle. The EMG obtained was amplified, filtered (10-500 Hz), and monitored with a Tektronix 502A oscilloscope. Samples of EMG were also stored on magnetic tape for further analysis. For a quantitative evaluation, the EMG was rectified and averaged using an RC-integrator. The output of the integrator was plotted by a potentiometric recorder (Rikadenki DP6, Tohshin). A two-channel EMG-processing system constructed in our laboratory was used for these procedures. RESULTS
Figure 2 shows an authentic and representative recording of shivering during a TI-experiment in a pigeon. During induction, and for some time thereafter, shivering is suppressed, but then gradually reappears, reaching preinduction levels in 3 minutes. A previous study [12] showed that the EMG-activity of the pectoral muscle during TI is a genuine shivering response, as it was not elicited at +25°C. It also showed that although body temperature decreases during TI (see also [18]), it is regulated at a stable level, indicating an umimpaired control of temperature regulation in general.
TONIC I M M O B I L I T Y A N D S H I V E R I N G IN BIRDS
477
TI
Q lOOseN
| OL
0
2
4
6
8
MINUTES FIG. 2. A representative electromyographic recording of shivering in a pigeon during a tonic immobility (TI) experiment. The horizontal bar depicts the duration of TI, while the dotted line shows the induction period.
Although some shivering preceded self-righting in most cases, the termination of TI was not correlated to the level of shivering reached. If TI terminated at an early stage, shivering was immediately restored, often with a rebound (see Fig. 3). Furthermore, in each bird, shivering reappeared in a highly specific and repeatable fashion. It was also observed that the occurrence of tremors in the extremities does not correlate with the presence of shivering : Tremors were also observed at +25°C, where no shivering occurs, and at +2°C the intensity of shivering in the pectoral muscle was often reduced during bouts of leg tremors. In summary, this experiment shows that although an upright posture seems to facilitate the recovery of shivering, pigeons are quite capable of shivering also during TI. Furthermore, shivering per se does not induce the termination of TI. EXPERIMENT 3 The two previous experiments show that in the pigeon, the appearance of shivering during TI does not necessitate the ultimate postural action, an upright posture. A relationship between postural activity and shivering should thus be demonstrable by correlating the duration of TI to the intensity of the simultaneous shivering response. Because of the highly individual patterns of shivering, a direct comparison between the duration of TI and the intensity of shivering was considered inadequate for this. To reliably elucidate the relationship between shivering and postural activity in the pigeon, a method for manipulating the duration of TI in individual pigeons was needed, so that potential changes could be evaluated by using each bird as its own control. Casual experimentation showed that TI was prolonged by darkness (see also [20]). In the third experiment, then, this technique was used to manipulate TI. By using the duration of TI as a measure of the rate at which postural activity was
resumed and by simultaneously measuring the intensity of shivering, a comparison between these variables was feasible. METHOD Animals
The 10 pigeons used in Experiment 1 were again used. The care of animals was the same as previously. The interval between Experiments 1 and 3 was 5 weeks. Procedure and Equipment
The pigeons were randomly divided into two groups which were tested on a Latin square basis: The duration of TI was measured for each bird at +2°C in a light (approximately 100 lux) and in a dark (approximately 0.1 lux) environment. The induction procedure was identical to that of Experiment 1, except that for the test in the dark, lighting was reduced to 0.1 lux 5 minutes before induction. The monitoring of TI was direct in these experiments. The 0.1-1ux lighting permitted the experimenter to detect the silhouette of the bird against the box floor and so judge the duration of TI. In addition to the duration of TI, the intensity of shivering was measured in each experiment. The recording was done as in Experiment 2. To obtain a measure of the general intensity of shivering during TI, the output from the RC-circuit was further integrated by digitizing the analog signal manually at 30-sec intervals. Identical lengths of recordings, corresponding to the duration of the shorter of the two TIepidsodes, were used for each bird. The numerical integral was then used to calculate the mean intensity of shivering for each bird in light and dark conditions. To account for interindividual variation in TI duration, relative values were also calculated by giving the intensity of shivering in light a value of 100 for each bird.
478
HOHTOLA L D
100
D
~
L9 Z tr LU
L
50
"1O3
0
5
lb
5
MINUTES
10
1
MINUTES
FIG. 3. Representative recordings of shivering (integrated EMG) in two pigeons during tonic immobility (TI) in a light (L) and in a dark (D) environment. The horizontal bars depict the durations of TI.
TABLE 1 RESULTS
Figure 3 presents two examples of the effects of environmental lighting on TI duration and the concurrent course of shivering in pigeons. In both cases the duration of TI is prolonged and the intensity of shivering reduced. Table 1 is a summary of the results. It shows that these reciprocal changes are highly significant: There is a two-fold prolongation of TI duration and a corresponding two-fold reduction in the intensity of shivering in the dark. GENERAL DISCUSSION The present results show that external cooling has no influence on the duration of TI in birds. This is, as such, in contrast with mammals, in which TI is disrupted by cooling in adult animals [29,30]. This dissimilarity may be related to a difference between birds and mammals in the recruitment of various muscle groups for shivering thermogenesis. In birds, the massive non-postural (i.e. not participating in the maintenance of actual stance) pectoral muscle is by far the most important site for thermogenesis in cold [23,28], while in mammals the postural musculature of the limbs and neck region have a more important role [ 10]. It is probable that the incipient shivering in these muscles also drives the neuromuscular systems used in the maintenance of stance, which facilitates self-righting and thus terminates TI in mammals. Due to the major role of pectoral muscle in shivering, this does not occur in birds, although an upright posture seems to accelerate the reappearance of shivering. The most important point for the following discussion is, however, that despite these differences, both mammals and birds are able to initiate shivering during TI. What is, then, the relation of shivering and postural activity? An inevitable premise for the maintenance of postural and other sustained motor activity, and also shivering, by birds and mammals is their increased capacity and scope for aerobic metabolism, especially in muscles [ 1,2]. These properties correlate with the profound differences in the general
E F F E C T S O F E N V I R O N M E N T A L L I G H T I N G ON T H E DURATION O F T O N I C IMMOBILITY (TI) A N D T H E S I M U L T A N E O U S INTENSITY O F S H I V E R I N G IN T H E PIGEON AT +2°C
Intensity of shivering Conditions
Duration* of TI (sec)
p~V
Light Dark
149 + 55 360 _+ 104
32.0 _+ 12.7 14.4 _+ 4.9
= 0.001
<0.0005
p$
Relativet 100 55 _+ 11.2
*Durations are expressed as means + SEM (N= 10). tRelative values were calculated by giving the intensity of shivering in light a value of 100 for each pigeon. 5;The p-values refer to a Wilcoxon's signed-ranks test for paired values.
behavior and physiology of these two groups compared to reptiles. More specifically, shivering occurs only in those animal groups (i.e. mammals, birds) in which the postural tone of muscles is maintained mainly by a summation of twitches produced by oxidative twitch-type muscle fibers (see [8]). In lower vertebrates, posture is maintained by slow and graded contractures of the tonic fibers [11,32]. Maintenance of posture by a continuous and smooth summation of twitching motor units is necessary for the rapid adjustments of muscle tone needed in a limb-supported posture, let alone flying. An important point is that this mechanism also consumes much more energy and consequently, produces more heat than the tonic mechanism. Thus the change in postural control that occured early in the evolution of birds and mammals was preadaptive for shivering.
TONIC IMMOBILITY AND SHIVERING IN BIRDS Birds and mammals have evolved from separate reptilian ancestors. As an alternative to convergent evolution, it might be possible that reptiles as a group already showed indications of a twitch-type muscular tone. This has, in fact, been shown to be true for some modern reptiles [32]. However, not until the emergence of the highly dynamic mammals and birds did the selection pressure for this type of muscular tone become significant. These facts, as such, do not confirm that shivering actually developed from postural activity. However, there is no reason to postulate a de n o v o mechanism for shivering thermogenesis, because most thermoregulatory effectors have evolved from functions that are of non-thermoregulatory origin [21]. A reasonable hypothesis is, then, that shivering evolved as a modification of postural activity in mammals and birds. The present study provides two lines of evidence for birds in favor of this hypothesis. First, birds are able to produce heat by shivering during TI, a condition where all gross motor activity is efficiently suppressed [5], but where postural activity, often in exaggerated forms, is still present [19]. Second, an inverse relationship was found between the duration of TI and the intensity of shivering when TI was experimentally manipulated. It was shown that when postural activity develops more slowly (resulting in a prolonged duration of TI), the rate of increase in the intensity of shivering also decreases. This congruency strongly suggests a common neuromuscular basis for shivering and postural activity. Several reports in the literature provide further evidence for a relationship between postural tone and shivering, although this aspect is not usually explicitly stated in these studies. (1) Shivering and postural activity are similarly influenced by changes in the state of wakefulness both in mammals and birds (for a review, see [9]). Both decrease in magnitude during slow-wave sleep and disappear totally during paradoxical sleep. It should be noted that mammals and birds are the only animals that have a well-defined slowwave sleep that enables precise temperature regulation at a lower energy consumption level. In fact, it is possible that slow-wave sleep evolved to compensate for the increased energy consumption in endotherms that resulted from a higher basal metabolism to which the sustained postural activity significantly contributed [3]. (2) The mechanical tremor frequency in muscles induced by postural activity and shivering are similar [16, 24, 25]. Although the results available are mainly from human studies, they suggest that similar neuromuscular circuits are recruited for shivering and postural tone. (3) Certain brain lesions with 6-OHDA in
479 rats produce akinetic states where only postural mechanisms remain functional, while exploratory motor systems, including eating, are suppressed. SchaUert et al. [22] have reported that these animals attain the same cataleptic posture seen in intact but hypothermic rats that are shivering vigorously. The authors conlcude that in addition to postural systems, the capability for shivering thermogenesis is also spared by this brain lesion. This suggests that these mechanisms are related anatomically in the CNS. We have also found [14] that pigeons with a hypothalamic 6-OHDA-lesion are capable of shivering vigorously although their feeding behavior is impaired. An interesting neuropharmacological relationship must also be mentioned: Several monoamines, notably serotonin (5-HT), have independently been shown to be involved in the control of both TI and heat production. Regarding Experiment 3, it is noteworthy that 5-HT is known to increase the duration of TI [27] and to inhibit shivering in most cases [6]. These observatons suggest still another parallelism between postural control and shivering. Among vertebrates, incubating pythons also use their muscles for thermogenesis [15,26]. As they have no postural activity comparable to that of mammals and birds, this might be claimed to invalidate the hypothesis about the relationship between shivering and postural activity. However, pythons produce heat by "spasmodic contractions" of body musculature that can be counted visually, and they do so only during brooding. Furthermore, heat production ceases to increase below +26°C ambient [15]. Thus, this reaction is a behaviorally mediated response for thermogenesis that bears no true resemblance to shivering. In fact, it is probably the result of another line of preadaptation for muscular thermogenesis that pythons achieved with their special predation strategies and large size. In summary, the present results together with those compilated from the literature afford many connections between postural mechanisms and shivering in birds and mammals. Although the evidence is not conclusive, the most parsimonious hypothesis is that shivering developed directly from the postural activity of skeletal muscles. Differences in the physiology of birds and mammals have resulted in fine adjustments of these mechanisms, which can be experimentally demonstrated. Tonic immobility is an effective tool for elucidating such differences, and may be a useful model for thermoregulatory studies in general. ACKNOWLEDGEMENTS This work was supported by the Emil Aaltonen Foundation.
REFERENCES 1. Bennett, A. F. Activity metabolism of the lower vertebrates. A Rev. Physiol. 40: 447--469, 1978. 2. Bennett, A. F. and J. A. Ruben. Endothermy and activity in vertebrates. Science 206: 649-654, 1979. 3. Berger, R. J. Bioenergetic functions of sleep and activity rhythms and their possible relevance to aging. Fed. Proc. 34: 97-102, 1975. 4. Calder, W. A. and J. R. King. Thermal and caloric relations of birds. In: Avian Biology, Vol. 4, edited by D. S. Farner and J. R. King. New York: Academic Press, 1974, pp. 259-413. 5. Carli, G., M. Coltelli and M. Sabourin. Effects of animal hypnosis on the performance and the extinction of an avoidance response. Brain Res. 66: 365-366, 1974.
6. Clark, W. G. and Y. L. Clark. Changes in body temperature after administration of adrenergic and serotonergic agents and related drugs including antidepressants. Neurosci. Biobehav. Rev. 4: 281-375, 1980. 7. Hammel, H. T. Regulation of internal body temperature. A Rev. Physiol. 30: 641-710, 1968. 8. Heath, J. E. The origins of thermoregulation. In: Evolution and Environment, edited by E. T. Drake. New Haven: Yale University Press, 1968, pp. 259-278. 9. HeUer, H. C. and S. F. Glotzbach. Thermoregulation during sleep and hibernation. In: Environmental Physiology H, Vol. 15, edited by D. Robertshaw. Baltimore: University Park Press, 1977, pp. 147-188.
480 10. Hemingway, A. Shivering. Physiol. Rev. 43: 397-422, 1963. 11. Hess, A. Vertebrate slow muscle fibers. Physiol. Rev. 50: 40-61, 1970. 12. Hohtola, E. Shivering during tonic immobility in birds. In: Contributions to Thermal Physiology, edited by Z. Zelrnyi and M. Sz~kely. Budapest: Akad~mial Kiad6 1980, pp. 447-449. 13. Hohtola, E., H. Rintam~iki and R. Hissa. Shivering and ptiloerection as complementary cold defense responses in the pigeon during sleep and wakefulness. J. comp. Physiol. 136B: 77-81, 1980. 14. Hohtola, E. and R. Hissa. Shivering and body temperature in the pigeon after depletion of hypothalamic noradrenaline. Comp. Biochem. Physiol. 1981, in press. 15. Hutchison, V. H., H. G. Dowling and A. Vinegar. Thermoregulation in a brooding female python, Python molurus bivittatus. Science 151: 695-696, 1966. 16. Lippoid, O. C. J. Oscillation in the stretch reflex arc and the origin of the rhythmical, 8-12 c/s component of physiological tremor. J. Physiol., Lond. 206: 35%382, 1970. 17. Maser, J. D. and G. G. Gallup. Tonic immobility and related phenomena: A partially annotated, tricentennial bibliography, 1636--1976. Psychol. Rec. 27: 177-217, 1977. 18. Nash, R. F., G. G. Gallup and D. A. Czech. Psychophysiological correlates of tonic immobility in the domestic chicken (Gallus gallus). Physiol. Behav. 17: 413-418, 1976. 19. Ratner, S. C. Comparative aspects of hypnosis. In: Handbook of Clinical and Experimental Hypnosis, edited by J. E. Gordon. New York: Macmillan, 1967, pp. 550-587. 20. Rovee, C. K., L. W. Kaufman and G. H. Collier. Components of predation defense behavior in chickens: Evidence for endogeffous rhytmicity. Physiol. Behav. 19: 663--671, 1977. 21. Satinoff, E. Neural organization and evolution of thermal regulation in mammals. Science 201: 16-22, 1978.
HOHTOLA 22. Schallert, T., I. Q. Whishaw, M. de Ryck and P. Teitelbaum. The postures of catecholamine-depletion catalepsy: Their possible adaptive value in thermoregulation. Physiol. Behav. 21: 817-820, 1978. 23. Steen, J. and P. S. Enger. Muscular heat production in pigeons during exposure to cold. Am. J. Physiol. 191: 157-158, 1957. 24. Stiles, R. N. Mechanical and neural feedback factors in postural hand tremor of normal subjects. J. Neurophysiol. 44: 40-59, 1980. 25. Stuart, D., K. Ott, K. Ishikawa and E. Eldred. The rhythm of shivering. Am. J. phys. Med. 45: 61-104, 1966. 26. Vinegar, A., V. H. Hutchison and H. G. Dowling. Metabolism, energetics, and thermoregulation during brooding of snakes of the genus Python (Reptilia, Boidae). Zoologica, N. Y. 55: 1%48, 1970. 27. Wallnau, L. B. and G. G. Gallup. A serotonergic, midbrainraphe model of tonic immobility. Biobehav. Rev. 1." 35-43, 1977. 28. West, G. C. Shivering and heat production in wild birds. Physiol. Zool. 38:111-120, 1965. 29. Whishaw, I. Q., K. P. Flannigan and R. H. Barnsley. Development of tonic immobility in the rabbit: Relation to body temperature. Devl Psychobiol. 12: 595-605, 1979. 30. Whishaw, I. Q., T. Schallert and B. Kolb. The thermal control of immobility in developing infant rats: Is the neocortex involved? Physiol. Behav. 23: 757-762, 1979. 31. Whittow, G. C. Comparative Physiology of Thermoregulation. Vols. 1-3. New York: Academic Press, 1970-1973. 32. Zhukov, Ye. K. Evolution of physiological mechanism of tonus in the vertebrates. In: Essays on Physiological Evolution, edited by T. M. Turpayev. London: Pergamon Press, 1965, pp. 33% 349.
Tonic Immobility and Shivering in Birds: Evolutionary Implications ESA HOHTOLA
University o f Oulu, Department o f Zoology Zoophysiological Laboratory SF-90100 Oulu 10, Finland R e c e i v e d 13 A p r i l 1981 HOHTOLA, E. Tonic immobility and shivering in birds: Evolutionary implications. PHYSIOL. BEHAV. 27(3) 475-480, 1981 .--The influence of ambient temperature and lighting on the duration of tonic immobility (TI) in birds was studied in conjunction with simultaneous shivering measurements. Contrary to what has been found in mammals, low ambient temperature did not disrupt TI in pigeons and house sparrows: the TI durations were equal at +2°C and +25°C. Electromyographic recordings in pigeons showed that after an initial suppression, shivering soon reappeared and reached normal levels during TI at +2°C. When the duration of TI was prolonged (indicating slower recovery of postural activity) by experimental manipulation (darkness), shivering likewise reappeared more slowly. The ability of birds to maintain shivering during TI, and the congruent changes in the duration of TI and the intensity of shivering suggest that shivering has neuromuscular and evolutionary connections to the postural activity of skeletal muscles also in birds. The difference between birds and mammals in reference to the effects of low ambient temperature on TI may be related to a differential recruitment of muscles for shivering thermogenesis. Tonic immobility
Columba livia
Shivering thermogenesis
Postural activity
Endothermy
Birds
Passer domesticus
S H I V E R I N G is a major mechanism for regulatory heat production in endotherms [4, 7, 31]. It is characterized by a generalized muscle tension, linearly related to input from specific cold sensors. Usually, shivering is best detected as an increased electromyographic (EMG) activity of skeletal muscles, but upon severe cooling, overt tremors are also demonstrable [10]. The exclusive occurrence of shivering in mammals and birds provides an interesting question about its evolutionary origins. To explain this phenomenon, other physiological traits specific to birds and mammals must be sought. A salient feature in the general behavior of mammals and birds is their limb-supported stance that is maintained for long periods of time. This is in marked contrast with the sprawled posture of reptiles. The inevitable neuromuscular adjustments needed for this postural change led Heath [8] to suggest that shivering most probably developed from the posturai activity of skeletal muscles, as it provides a mechanism for maintaining prolonged muscle tension (see also [21]). This hypothesis is not readily amenable to experimental tests, and so far only circumstantial evidence is available. Recently, however, studies on tonic immobility (TI) have unexpectedly shed light on the hypothetical relationship between shivering and postural activity. TI (also: animal hypnosis) is a naturally occurring antipredator response that can be elicited also experimentally by a brief physical restraint (for references, see [17]). TI provides a unique opportunity to study the relation of shivering to postural activity: It is
characterized by a profound inhibition of gross motor activity [5], while at least part of the postural mechanisms remain functional, resulting often in exaggerated postures and visible tremors of the extremities [17,19]. The gradual recovery of postural activity eventually leads to self-righting which terminates TI. Studies on TI [29,30] have now shown that in adult mammals, external cooling disrupts TI. The authors conclude that TI is disrupted because shivering , stimulated by low temperature, enhances postural activity and induces the righting reflex. These studies, and the previous observation [12] that shivering does not terminate TI in pigeons and house sparrows, prompted a more detailed study on the relationship between postural activity and shivering in birds. The experimental approach to this problem was to study the effects of low ambient temperature on TI and, by using EMGtechniques, to characterize the shivering response during TI. EXPERIMENT 1 Studies on rats and rabbits [29,30] have shown that external cooling potentiates TI during the early postnatal period, but disrupts it in fully-grown animals. In an earlier study with birds [12], it was observed that there were no difficulties in inducing TI in pigeons and house sparrows at low ambient temperatures. As a first step towards elucidating potential differences between the immobility reactions of mammals and birds, the first experiment was designed to study the
C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/090475-06502.00/0
476
HOHTOLA
influence of low ambient temperature on the duration of TI in birds.
500['~--'
METHOD
PIGEON
~ 400 i
HOUSE
PIGEON
SPARROW
HOUSE SPARROW
g 3oo
Animals Adult pigeons (Columba livia, mass 270-350 g), captured in Southern Finland and house sparrows (Passer domesticus, mass 27-29 g), captured near the laboratory were used. The birds were housed in metal wire cages in an animal room maintained at +24°C with a 12:12-hour light/dark regime. All birds were experimentally naive and were kept in the animal room for at least 5 weeks before experiments. They had free access to food and water at all time except during the actual test procedures.
Procedure and Equipment The duration of TI was measured at +2°C and +25°C in both species. Before experiments, birds of each species were randomly divided into two groups, which were then tested on a Latin square basis: Each birds was tested at either of the two temperatures on day 1, and at the other temperature on the following day at the same time. All experiments were done 3--8 hours after lights-on (6 a.m.). The experiments were performed in a dark-walled box placed in a thermostatically controlled cold-room. The bottom of the box was covered with a layer of paper sheets to avoid direct thermal contact with the floor during TI. To mask disturbing ambient sounds, a steady background noise was produced by feeding a 30-Hz sinusoidal signal from an audio generator into a horizontally mounted loudspeaker and by placing small glass beads on the loudspeaker diaphragm. Before an experiment, each bird had a 20-minute adaptation time to the test temperature in a small side compartment of the experimental box. TI was then induced by restraining the bird manually in a supine position for 15 seconds. Maximally 3 inductions were needed for any bird. The duration of TI was measured as the time elapsed from the cessation of restrainment to the point of self-righting. The TI of pigeons was monitored remotely using a video-system (Philips LDH25 camera and Finnvideo VM 12FI monitor), while the TI of house sparrows was observed directly by placing the lid of the experimental box so that the bird could not see the experimenter. RESULTS
Figure 1 presents the mean durations of TI in pigeons and house sparrows as a function of ambient temperature and trial number. Clearly, ambient temperature has no influence on TI duration in either species. In agreement with previous reports (see [17]), a habituation of the responses was observed: The duration of TI during the second trial was significantly shorter in both species (pigeon, p <0.001; house sparrow, p=0.05; Wilcox's signed-ranks test for paired values). The mean number of inductions needed to elicit TI showed no variation with respect to the experimental conditions, being always 1.7-1.8 in pigeons and 1.%2.1 in house sparrows. EXPERIMENT 2 Whishaw et al. [29,30] postulate that the inhibition (short duration) of TI in mammals at low ambient temperatures is related to the development of shivering thermogenesis. They
200)
'-'
i
, ,°2r +25
+2
+25
+2 °C
AMBIENT TEMPERATURE
1.
2 TRIAL
1.
2.
NUMBER
FIG. 1. The durations (mean ~+ SEM) of tonic immobility (TI) as a function of ambient temperature and trial number (Latin square procedure) in pigeons and house sparrows. N = 10 and 6, respectively.
assert that in adult animals external cooling stimulates shivering which disrupts TI by driving a set of postural adjustments that cause the animal to take an upright posture. In newborn animals, postural activity is not enhanced due to the immaturity of the shivering response. Unfortunately, no EMG-recordings were made in these experiments. The second experiment of the present study was thus designed to measure and characterize, by EMG-techniques, the shivering response of the pigeon during TI. METHOD
Animals Adult pigeons, obtained and maintained as in Experiment 1, were used.
Procedure and equipment TI was induced at +2°C as in Experiment 1. Shivering was measured as described previously [13]. Briefly, three monopolar stainless steel electrodes in a fixed close arrangement were inserted into the pectoral muscle. The EMG obtained was amplified, filtered (10-500 Hz), and monitored with a Tektronix 502A oscilloscope. Samples of EMG were also stored on magnetic tape for further analysis. For a quantitative evaluation, the EMG was rectified and averaged using an RC-integrator. The output of the integrator was plotted by a potentiometric recorder (Rikadenki DP6, Tohshin). A two-channel EMG-processing system constructed in our laboratory was used for these procedures. RESULTS
Figure 2 shows an authentic and representative recording of shivering during a TI-experiment in a pigeon. During induction, and for some time thereafter, shivering is suppressed, but then gradually reappears, reaching preinduction levels in 3 minutes. A previous study [12] showed that the EMG-activity of the pectoral muscle during TI is a genuine shivering response, as it was not elicited at +25°C. It also showed that although body temperature decreases during TI (see also [18]), it is regulated at a stable level, indicating an umimpaired control of temperature regulation in general.
TONIC I M M O B I L I T Y A N D S H I V E R I N G IN BIRDS
477
TI
Q lOOseN
| OL
0
2
4
6
8
MINUTES FIG. 2. A representative electromyographic recording of shivering in a pigeon during a tonic immobility (TI) experiment. The horizontal bar depicts the duration of TI, while the dotted line shows the induction period.
Although some shivering preceded self-righting in most cases, the termination of TI was not correlated to the level of shivering reached. If TI terminated at an early stage, shivering was immediately restored, often with a rebound (see Fig. 3). Furthermore, in each bird, shivering reappeared in a highly specific and repeatable fashion. It was also observed that the occurrence of tremors in the extremities does not correlate with the presence of shivering : Tremors were also observed at +25°C, where no shivering occurs, and at +2°C the intensity of shivering in the pectoral muscle was often reduced during bouts of leg tremors. In summary, this experiment shows that although an upright posture seems to facilitate the recovery of shivering, pigeons are quite capable of shivering also during TI. Furthermore, shivering per se does not induce the termination of TI. EXPERIMENT 3 The two previous experiments show that in the pigeon, the appearance of shivering during TI does not necessitate the ultimate postural action, an upright posture. A relationship between postural activity and shivering should thus be demonstrable by correlating the duration of TI to the intensity of the simultaneous shivering response. Because of the highly individual patterns of shivering, a direct comparison between the duration of TI and the intensity of shivering was considered inadequate for this. To reliably elucidate the relationship between shivering and postural activity in the pigeon, a method for manipulating the duration of TI in individual pigeons was needed, so that potential changes could be evaluated by using each bird as its own control. Casual experimentation showed that TI was prolonged by darkness (see also [20]). In the third experiment, then, this technique was used to manipulate TI. By using the duration of TI as a measure of the rate at which postural activity was
resumed and by simultaneously measuring the intensity of shivering, a comparison between these variables was feasible. METHOD Animals
The 10 pigeons used in Experiment 1 were again used. The care of animals was the same as previously. The interval between Experiments 1 and 3 was 5 weeks. Procedure and Equipment
The pigeons were randomly divided into two groups which were tested on a Latin square basis: The duration of TI was measured for each bird at +2°C in a light (approximately 100 lux) and in a dark (approximately 0.1 lux) environment. The induction procedure was identical to that of Experiment 1, except that for the test in the dark, lighting was reduced to 0.1 lux 5 minutes before induction. The monitoring of TI was direct in these experiments. The 0.1-1ux lighting permitted the experimenter to detect the silhouette of the bird against the box floor and so judge the duration of TI. In addition to the duration of TI, the intensity of shivering was measured in each experiment. The recording was done as in Experiment 2. To obtain a measure of the general intensity of shivering during TI, the output from the RC-circuit was further integrated by digitizing the analog signal manually at 30-sec intervals. Identical lengths of recordings, corresponding to the duration of the shorter of the two TIepidsodes, were used for each bird. The numerical integral was then used to calculate the mean intensity of shivering for each bird in light and dark conditions. To account for interindividual variation in TI duration, relative values were also calculated by giving the intensity of shivering in light a value of 100 for each bird.
478
HOHTOLA L D
100
D
~
L9 Z tr LU
L
50
"1O3
0
5
lb
5
MINUTES
10
1
MINUTES
FIG. 3. Representative recordings of shivering (integrated EMG) in two pigeons during tonic immobility (TI) in a light (L) and in a dark (D) environment. The horizontal bars depict the durations of TI.
TABLE 1 RESULTS
Figure 3 presents two examples of the effects of environmental lighting on TI duration and the concurrent course of shivering in pigeons. In both cases the duration of TI is prolonged and the intensity of shivering reduced. Table 1 is a summary of the results. It shows that these reciprocal changes are highly significant: There is a two-fold prolongation of TI duration and a corresponding two-fold reduction in the intensity of shivering in the dark. GENERAL DISCUSSION The present results show that external cooling has no influence on the duration of TI in birds. This is, as such, in contrast with mammals, in which TI is disrupted by cooling in adult animals [29,30]. This dissimilarity may be related to a difference between birds and mammals in the recruitment of various muscle groups for shivering thermogenesis. In birds, the massive non-postural (i.e. not participating in the maintenance of actual stance) pectoral muscle is by far the most important site for thermogenesis in cold [23,28], while in mammals the postural musculature of the limbs and neck region have a more important role [ 10]. It is probable that the incipient shivering in these muscles also drives the neuromuscular systems used in the maintenance of stance, which facilitates self-righting and thus terminates TI in mammals. Due to the major role of pectoral muscle in shivering, this does not occur in birds, although an upright posture seems to accelerate the reappearance of shivering. The most important point for the following discussion is, however, that despite these differences, both mammals and birds are able to initiate shivering during TI. What is, then, the relation of shivering and postural activity? An inevitable premise for the maintenance of postural and other sustained motor activity, and also shivering, by birds and mammals is their increased capacity and scope for aerobic metabolism, especially in muscles [ 1,2]. These properties correlate with the profound differences in the general
E F F E C T S O F E N V I R O N M E N T A L L I G H T I N G ON T H E DURATION O F T O N I C IMMOBILITY (TI) A N D T H E S I M U L T A N E O U S INTENSITY O F S H I V E R I N G IN T H E PIGEON AT +2°C
Intensity of shivering Conditions
Duration* of TI (sec)
p~V
Light Dark
149 + 55 360 _+ 104
32.0 _+ 12.7 14.4 _+ 4.9
= 0.001
<0.0005
p$
Relativet 100 55 _+ 11.2
*Durations are expressed as means + SEM (N= 10). tRelative values were calculated by giving the intensity of shivering in light a value of 100 for each pigeon. 5;The p-values refer to a Wilcoxon's signed-ranks test for paired values.
behavior and physiology of these two groups compared to reptiles. More specifically, shivering occurs only in those animal groups (i.e. mammals, birds) in which the postural tone of muscles is maintained mainly by a summation of twitches produced by oxidative twitch-type muscle fibers (see [8]). In lower vertebrates, posture is maintained by slow and graded contractures of the tonic fibers [11,32]. Maintenance of posture by a continuous and smooth summation of twitching motor units is necessary for the rapid adjustments of muscle tone needed in a limb-supported posture, let alone flying. An important point is that this mechanism also consumes much more energy and consequently, produces more heat than the tonic mechanism. Thus the change in postural control that occured early in the evolution of birds and mammals was preadaptive for shivering.
TONIC IMMOBILITY AND SHIVERING IN BIRDS Birds and mammals have evolved from separate reptilian ancestors. As an alternative to convergent evolution, it might be possible that reptiles as a group already showed indications of a twitch-type muscular tone. This has, in fact, been shown to be true for some modern reptiles [32]. However, not until the emergence of the highly dynamic mammals and birds did the selection pressure for this type of muscular tone become significant. These facts, as such, do not confirm that shivering actually developed from postural activity. However, there is no reason to postulate a de n o v o mechanism for shivering thermogenesis, because most thermoregulatory effectors have evolved from functions that are of non-thermoregulatory origin [21]. A reasonable hypothesis is, then, that shivering evolved as a modification of postural activity in mammals and birds. The present study provides two lines of evidence for birds in favor of this hypothesis. First, birds are able to produce heat by shivering during TI, a condition where all gross motor activity is efficiently suppressed [5], but where postural activity, often in exaggerated forms, is still present [19]. Second, an inverse relationship was found between the duration of TI and the intensity of shivering when TI was experimentally manipulated. It was shown that when postural activity develops more slowly (resulting in a prolonged duration of TI), the rate of increase in the intensity of shivering also decreases. This congruency strongly suggests a common neuromuscular basis for shivering and postural activity. Several reports in the literature provide further evidence for a relationship between postural tone and shivering, although this aspect is not usually explicitly stated in these studies. (1) Shivering and postural activity are similarly influenced by changes in the state of wakefulness both in mammals and birds (for a review, see [9]). Both decrease in magnitude during slow-wave sleep and disappear totally during paradoxical sleep. It should be noted that mammals and birds are the only animals that have a well-defined slowwave sleep that enables precise temperature regulation at a lower energy consumption level. In fact, it is possible that slow-wave sleep evolved to compensate for the increased energy consumption in endotherms that resulted from a higher basal metabolism to which the sustained postural activity significantly contributed [3]. (2) The mechanical tremor frequency in muscles induced by postural activity and shivering are similar [16, 24, 25]. Although the results available are mainly from human studies, they suggest that similar neuromuscular circuits are recruited for shivering and postural tone. (3) Certain brain lesions with 6-OHDA in
479 rats produce akinetic states where only postural mechanisms remain functional, while exploratory motor systems, including eating, are suppressed. SchaUert et al. [22] have reported that these animals attain the same cataleptic posture seen in intact but hypothermic rats that are shivering vigorously. The authors conlcude that in addition to postural systems, the capability for shivering thermogenesis is also spared by this brain lesion. This suggests that these mechanisms are related anatomically in the CNS. We have also found [14] that pigeons with a hypothalamic 6-OHDA-lesion are capable of shivering vigorously although their feeding behavior is impaired. An interesting neuropharmacological relationship must also be mentioned: Several monoamines, notably serotonin (5-HT), have independently been shown to be involved in the control of both TI and heat production. Regarding Experiment 3, it is noteworthy that 5-HT is known to increase the duration of TI [27] and to inhibit shivering in most cases [6]. These observatons suggest still another parallelism between postural control and shivering. Among vertebrates, incubating pythons also use their muscles for thermogenesis [15,26]. As they have no postural activity comparable to that of mammals and birds, this might be claimed to invalidate the hypothesis about the relationship between shivering and postural activity. However, pythons produce heat by "spasmodic contractions" of body musculature that can be counted visually, and they do so only during brooding. Furthermore, heat production ceases to increase below +26°C ambient [15]. Thus, this reaction is a behaviorally mediated response for thermogenesis that bears no true resemblance to shivering. In fact, it is probably the result of another line of preadaptation for muscular thermogenesis that pythons achieved with their special predation strategies and large size. In summary, the present results together with those compilated from the literature afford many connections between postural mechanisms and shivering in birds and mammals. Although the evidence is not conclusive, the most parsimonious hypothesis is that shivering developed directly from the postural activity of skeletal muscles. Differences in the physiology of birds and mammals have resulted in fine adjustments of these mechanisms, which can be experimentally demonstrated. Tonic immobility is an effective tool for elucidating such differences, and may be a useful model for thermoregulatory studies in general. ACKNOWLEDGEMENTS This work was supported by the Emil Aaltonen Foundation.
REFERENCES 1. Bennett, A. F. Activity metabolism of the lower vertebrates. A Rev. Physiol. 40: 447--469, 1978. 2. Bennett, A. F. and J. A. Ruben. Endothermy and activity in vertebrates. Science 206: 649-654, 1979. 3. Berger, R. J. Bioenergetic functions of sleep and activity rhythms and their possible relevance to aging. Fed. Proc. 34: 97-102, 1975. 4. Calder, W. A. and J. R. King. Thermal and caloric relations of birds. In: Avian Biology, Vol. 4, edited by D. S. Farner and J. R. King. New York: Academic Press, 1974, pp. 259-413. 5. Carli, G., M. Coltelli and M. Sabourin. Effects of animal hypnosis on the performance and the extinction of an avoidance response. Brain Res. 66: 365-366, 1974.
6. Clark, W. G. and Y. L. Clark. Changes in body temperature after administration of adrenergic and serotonergic agents and related drugs including antidepressants. Neurosci. Biobehav. Rev. 4: 281-375, 1980. 7. Hammel, H. T. Regulation of internal body temperature. A Rev. Physiol. 30: 641-710, 1968. 8. Heath, J. E. The origins of thermoregulation. In: Evolution and Environment, edited by E. T. Drake. New Haven: Yale University Press, 1968, pp. 259-278. 9. HeUer, H. C. and S. F. Glotzbach. Thermoregulation during sleep and hibernation. In: Environmental Physiology H, Vol. 15, edited by D. Robertshaw. Baltimore: University Park Press, 1977, pp. 147-188.
480 10. Hemingway, A. Shivering. Physiol. Rev. 43: 397-422, 1963. 11. Hess, A. Vertebrate slow muscle fibers. Physiol. Rev. 50: 40-61, 1970. 12. Hohtola, E. Shivering during tonic immobility in birds. In: Contributions to Thermal Physiology, edited by Z. Zelrnyi and M. Sz~kely. Budapest: Akad~mial Kiad6 1980, pp. 447-449. 13. Hohtola, E., H. Rintam~iki and R. Hissa. Shivering and ptiloerection as complementary cold defense responses in the pigeon during sleep and wakefulness. J. comp. Physiol. 136B: 77-81, 1980. 14. Hohtola, E. and R. Hissa. Shivering and body temperature in the pigeon after depletion of hypothalamic noradrenaline. Comp. Biochem. Physiol. 1981, in press. 15. Hutchison, V. H., H. G. Dowling and A. Vinegar. Thermoregulation in a brooding female python, Python molurus bivittatus. Science 151: 695-696, 1966. 16. Lippoid, O. C. J. Oscillation in the stretch reflex arc and the origin of the rhythmical, 8-12 c/s component of physiological tremor. J. Physiol., Lond. 206: 35%382, 1970. 17. Maser, J. D. and G. G. Gallup. Tonic immobility and related phenomena: A partially annotated, tricentennial bibliography, 1636--1976. Psychol. Rec. 27: 177-217, 1977. 18. Nash, R. F., G. G. Gallup and D. A. Czech. Psychophysiological correlates of tonic immobility in the domestic chicken (Gallus gallus). Physiol. Behav. 17: 413-418, 1976. 19. Ratner, S. C. Comparative aspects of hypnosis. In: Handbook of Clinical and Experimental Hypnosis, edited by J. E. Gordon. New York: Macmillan, 1967, pp. 550-587. 20. Rovee, C. K., L. W. Kaufman and G. H. Collier. Components of predation defense behavior in chickens: Evidence for endogeffous rhytmicity. Physiol. Behav. 19: 663--671, 1977. 21. Satinoff, E. Neural organization and evolution of thermal regulation in mammals. Science 201: 16-22, 1978.
HOHTOLA 22. Schallert, T., I. Q. Whishaw, M. de Ryck and P. Teitelbaum. The postures of catecholamine-depletion catalepsy: Their possible adaptive value in thermoregulation. Physiol. Behav. 21: 817-820, 1978. 23. Steen, J. and P. S. Enger. Muscular heat production in pigeons during exposure to cold. Am. J. Physiol. 191: 157-158, 1957. 24. Stiles, R. N. Mechanical and neural feedback factors in postural hand tremor of normal subjects. J. Neurophysiol. 44: 40-59, 1980. 25. Stuart, D., K. Ott, K. Ishikawa and E. Eldred. The rhythm of shivering. Am. J. phys. Med. 45: 61-104, 1966. 26. Vinegar, A., V. H. Hutchison and H. G. Dowling. Metabolism, energetics, and thermoregulation during brooding of snakes of the genus Python (Reptilia, Boidae). Zoologica, N. Y. 55: 1%48, 1970. 27. Wallnau, L. B. and G. G. Gallup. A serotonergic, midbrainraphe model of tonic immobility. Biobehav. Rev. 1." 35-43, 1977. 28. West, G. C. Shivering and heat production in wild birds. Physiol. Zool. 38:111-120, 1965. 29. Whishaw, I. Q., K. P. Flannigan and R. H. Barnsley. Development of tonic immobility in the rabbit: Relation to body temperature. Devl Psychobiol. 12: 595-605, 1979. 30. Whishaw, I. Q., T. Schallert and B. Kolb. The thermal control of immobility in developing infant rats: Is the neocortex involved? Physiol. Behav. 23: 757-762, 1979. 31. Whittow, G. C. Comparative Physiology of Thermoregulation. Vols. 1-3. New York: Academic Press, 1970-1973. 32. Zhukov, Ye. K. Evolution of physiological mechanism of tonus in the vertebrates. In: Essays on Physiological Evolution, edited by T. M. Turpayev. London: Pergamon Press, 1965, pp. 33% 349.