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Entry into hibernation in M. Flaviventris: Sleep and behavioral thermoregulation
Physiology & Behavior, Vol. 27, pp. 989--993.Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.
Entry into Hibernation in M. Flaviventris: Sleep and Behavioral Thermoregulation V I R G I N I A M. M I L L E R A N D F R A N K
E. S O U T H
School o f Life and Health Sciences, University o f Delaware, N e w a r k , D E 19711 R e c e i v e d 19 M a r c h 1981 MILLER, V. M. AND F. E. SOUTH. Entry into hibernation in M. flaviventris: Sleep and behavioral thermoregulation. PHYSIOL. BEHAV. 27(6) 989--993, 1981.--Hibernators of the genus Marmota (wt=3-5 kg) differ from smaller hibernators (wt< 1 kg) in thermoregulatory characteristics during entry into hibernation. They might be expected to differ also with respect to the distributions of activity, awake and sleep states during entry. Marmots implanted stereotaxically with electrodes to record electroencephalograms and brain temperature (TBr) were monitored remotely by a polygraph as well as video transmission as they entered hibernation. During entry, awake (A), slow wave sleep (SWS), and paradoxical sleep (PS) states could be identified. Activity which included nest building, grooming, and shivering slowed entry and became progressively more stereotyped as Ta~ decreased. All animals exhibited at least one PS bout between Tsr=33-32°C. SWS as percent of total sleep increased (80 to 92%) as Tar decreased from 35-25°C. This increase represented a decrease in number and increase in duration of SWS episodes. The length of individual SWS and PS episodes of the marmot did not differ from those reported in Citellus. These data suggest that entry into hibernation is a more complex phenomenon than merely an extension of slow wave sleep. Similar changes in arousal state distribution occur in hibernators of different sizes. Marmot M. flaviventris Hibernation Hibernation entry Arousal states Slow wave sleep Paradoxical sleep
I N T E R M I T T E N T periods of apparent activity have been observed repeatedly in animals entering hibernation [2, 8, 13, 14, 16, 19]. Although the thermoregulatory significance of these activity bouts remains to be determined, it has been suggested [14] that they slow or " b r a k e " too rapid a drop in brain temperature (Tar). However, this also could be due to relatively nonspecific movements or shivering by the animals within their nest. A review of the literature suggests that behavior patterns during entry are specific and relatively stereotyped among a variety of hibernators (Citellus spp., E. europeanus, P. Iongimembris, M. flaviventris, and M. monax). The complexity of these behavior patterns which include grooming [2, 14, 16], nest rearranging [2, 13, 14], curling [2, 13, 14, 16], and shivering [13, 14, 19] have led us [13] to postulate that the net effect of these activities increased the heat capacity of the animal's nest allowing for more precise regulation of TR~ during hibernation. Although these bouts of movement are indeed striking, animals entering hibernation do spend the majority of entry time in a quiescent state. Electrophysiological observations from hibernating mammals have led to the hypothesis that hibernation is entered into from a sleep state [5, 6, 10, 11, 15, 17, 19]. Specifically, in Citellus spp. [19], slow wave sleep [SWS] as a percent of total sleep time (TST) increased from the 80% of normothermia to 90% during entry into hibernation. Increased amounts of SWS were concomitant with decreased amounts of paradoxical sleep (PS) [10,19]. However, similar observations in a larger hibernator, M. flaviventris [11], at a brain temperature (Tar) of 33°C suggest that the amount of SWS during entry does not alter from the 80% of TST seen during normothermia. The hibernators of the genus Marmota (adult w t = 3 - 5 kg) represent smaller
Thermoregulation
Sleep
surface-to-volume ratios when compared to hibernators of weight < 1 kg. The thermoregulatory characteristics of Marmota during entry into hibernation therefore might differ from other hibernators. This difference also might be reflected in the relative distribution during entry of the awake and sleep states. The observations of this report are the first in which undisturbed behavioral activity, electroencephalogram and Tar were monitored continuously during entry into hibernation in Marmota. Thus they provide complete data of the entry phenomena for comparison among hibernators of widely differing weights. METHOD Male and female yellow bellied marmots (Marmota flaviventris) weighing between 3-5 kg were implanted stereotaxically with electrodes to monitor cortical electroencephalogram (EEG), dorsal hippocampal theta activity and the electrooculogram (EOG). Surgical anesthesia was obtained with sodium pentobarbital (40 mg/kg intraperitoneally). E E G electrodes were stainless steel machine screws with recording wires soldered to the heads. Electrodes were placed bilaterally over the frontal and occipital cortices. The hippocampal electrode (In Vivo Metric NEX-200, l0 mm) was placed anteriorly from earbar reference 5.6 mm, laterally 6.2 mm and 5.2 mm below brain surface. A 0.010 mm stainless steel wire stripped of insulation and formed into a flat coil was placed on the orbit of the eye as the EOG electrode. A 22 ga stainless steel tube with one end closed and coated with Epoxylite electrode coat was placed in the internal capsule. Insertion of a thermocouple (36 ga copper-
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.--0031-9384/81/120989-05502.00/0
990
M I L L E R A N D SOUTH
AWAKE
TB R = 3 5
moving
° C
q uiet
30 s ec EMG
2oo.v I EEG
\
~\
400 u VI EOG
f
IOOpV I
FIG. 1. Electrophysiological tracing of the awake state observed during entry into hibernation. During movement, EMG activity is high and EOG spikes appear. During quiet awake, the frequency of EOG spikes and EMG activity is reduced while low voltage fast activity dominates the EEG. As slow wave sleep begins, spindles appear in the EEG.
constantan) into this sleeve provided a sensitive record of brain temperature. All electrodes were soldered to a DE-95 Cinch plug and secured to the skull with dental acrylic. Animals were treated postoperatively with 100,000 units of Procaine Penicillin G intramuscularly. Two weeks were allowed before animals were placed in the hibernaculum. Prior to surgery and during the recovery period, animals were housed individually at an ambient temperature (Ta) of 20___I°C, light:dark cycle 12:12 hrs with food and water provided ad lib. After recovery from surgery, recording cables were attached to the exposed pedestal plugs on the animal's head. At this time, electromyographic (EMG) pins, designed after South et al. [12], were inserted into the skin in the nucal region for monitoring heart rate, movement, and shivering. Animals were then placed in an environmental chamber (Ta=5°C) in individual cages. Recording cables were attached to a slipring assembly so that the animal was free to move about the cage. Food, water and cotton nesting material were provided ad lib. Red incandescent lights were on continuously, allowing for behavior to be monitored by remote video. EEG, EMG, and EOG were monitored through 7P5A Grass AC preamplifiers. THr was referenced to an Omega thermocouple reference junction box and recorded through a Grass 7P1 DC preamplifier. Animals were left undisturbed except for routine feeding and watering. Hibernation usually proceeded within 5 weeks time. At the conclusion of the hibernating season, animals were overdosed with sodium pentobarbital (50 mg/kg intraperitoneally) and were perfused via the carotid arteries with 0.9% saline followed by 10% Formalin. The brains were removed, embedded in paraffin, and cut into 40 micron sections for confirmation of electrode placement. Polygraph records for the entry periods were scored at 0.5 min intervals. Variability existed in the lability of TBr among
animals and between bouts of hibernation. Therefore, initiation of entry into hibernation was defined for this study as a decrease in TBr from the maximum TBr measured or 35°C with no subsequent i n c r e a s e ~ TBr of over an hour duration. Standard criteria [7, 9, 11, 18, 19] were used to define arousal states. Two independent scorings were made of each record and found not to differ significantly. Intermittent periods (0.5-2.5 min duration) of low voltage fast E E G activity and spindles were scored individually as awake and sleep, respectively, whereas other have scored these periods collectively as awake [18], drowsy [16], or sleep [19]. RESULTS Nine entries into hibernation were observed in 7 animals between the months of November and March. No correlation between the initiation of entry to time of day could be made. During the initial phase of entry, TBr = 35-25°C, all arousal states could be identified (Figs. 1 and 2). The awake phase (A) was characterized by low voltage fast activity (~12 cycles/sec) in the EEG, theta activity (4-10 cycles/sec) from the hippocampus (not shown) during movement, and high background EMG activity. EOG spikes occurred during movement but were absent during quiescent awake periods and slow wave sleep (Fig. 1). Sleep could be identified (Fig. 2) as slow wave sleep (SWS) and paradoxical sleep (PS). During SWS, high voltage slow wave activity (0--5 cycles/sec) dominated the E E G trace. EMG activity and heart rate were low and no spikes were seen on the EOG. Frequently, intermittent periods of A and SWS (each state <2.5 min) were observed. These alternating A and SWS sequences preceded a prolonged SWS bout of 6.2___ 1 min (mean_+SE) duration in 29 of 34 instances. As shown in Fig. 2, PS always proceeded
ENTRY INTO H I B E R N A T I O N IN THE MARMOT
991
SWS
PS
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..............................................................
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FIG. 2. Electrophysiological tracing of the sleep state observed during entry into hibernation. SWS=slow wave sleep. PS=paradoxical sleep. See text for further description.
from SWS. During PS, low voltage fast activity was seen in the E E G trace, theta activity emanated from the hippocampus (not shown), heart rate was irregular, background EMG was low with periodic jerking movements, and EOG record reflected the characteristic rapid eye movement. The above criteria for arousal states could not be used satisfactorily below TBr=25°C. However, alternating periods of slow and fast frequency EEG were observed as TBr continued to decline during the remainder of the entry. Periodic alterations in EEG activity were also observed during deep hibernation. Any correlation between the initiation of entry and a given arousal state was difficult to ascertain. Prior to entry, TBr among animals varied greatly. Three animals showed cycles of decreases followed by increases in TBr. In two other animals TBr plateaued at 33°C before Tar continued to decrease to values of deep hibernation. SWS during the initial stages of entry, however, was always associated with a decline in Tar. Periods of movement and activity could be identified throughout the entire entry. The distribution of activity during two typical successive entries by one animal is illustrated in Fig. 3. The overall rate of Tar decline and the behaviors observed were similar in both instances. However, the distribution of the behaviors during the TBr decline differed. In general, behavior patterns during the initial phase of entry were complex and involved extensive nest building, circling, and shivering. These became increasingly stereotyped as Tar decreased: raising of the head, rocking from side to side, curling into the hibernation position. Relatively complex behaviors were still observed at Tar as low as 24°C. Since conventional arousal states could be identified only during the initial phase of entry (TBr>~25°C), the relative contributions of activity, A, SWS and PS during this stage of entry were analyzed. The total recording time (TRT) aver-
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HOURS FIG. 3. Brain temperature decline and distribution of activity during two entries into hibernation observed from a single animal.
aged 324_+3%7 min (mean_-+SE) for the 9 entries observed. The A state occupied 27.4±2.75% of this T R T . This represented 22.8-.+4.5 episodes of 4.2-+0.7 min duration per animal. Total activity for all animals represented 32.5±4.2% of the A state. The amount of activity was linearly related to TRT. The relationship was defined by the equation Y' = 0.17x - 24.25 (r=0.90)
992
MILLER AND SOUTH
TABLE 1 COMPARISONOF EPISODE LENGTH BETWEENCITELLUS AND MARMOTA Citellus spp. [19]
A SWS PS
80
M. flaviventris
1.7 ± 0.8 (min)*
12.3 ± 2.3t N =204 13.7 _+ 1.1 N=272 3.5 ± 0.3 N= 94
11.4 _+ 5.4 3.0 ± 0.9
IIII
I00
kz i,i co i,i a.
60
40
20
*Values represent Mean - SD. +Values represent Mean _+ SE. I
I
35-30 where Y' is the total activity time (min) displayed by an animal and x is the TRT for that animal. In 31 of 43 instances, activity occurred during an A episode which followed SWS; in the remaining 12 instances, the activity followed an episode of PS. The distribution of the activity was such that few animals were observed to engage in activity at the extremes of the Tar range, 35° and 25°C (2 of 7 and 3 of 9, respectively). However, all animals (9 of 9) engaged in at least one activity bout of 6.9_+2.3 min duration (N= 14) between Tar 32° and 30°C. Total sleep time ( T S T = T R T - A ) was composed of 85.1-+1.3% SWS. This represented 30.2-+5.0 episodes per animal with each episode averaging 6.5-+0.5 min length. Animals averaged 10.4-+ 1.4 episodes of PS of 3.5-+0.3 min duration between Tsr 35-25°C. PS occurred most frequently early in the entry with 8 of 9 animals (p<0.02 Wilcoxon matched-pairs signed-ranks test) engaged in PS at both TB~of 33° and 32°C. No correlation could be made between the number or duration of PS bouts with the amount of activity or TRT. When the average lengths of A, SWS, and PS episodes are compared to those values reported for CiteUus ([19]; see Table 1, column 1) several differences are suggested. The length of SWS episodes were less and the length of A episodes were longer in the marmot than in the ground squirrel. However, these apparent differences could be due to the way in which the lengths of A and SWS bouts were calculated. If the periods of intermittent A and SWS are considered as SWS (as in the Citellus study [19]), then the lengths of SWS are remarkably similar between species (Table 1). The
A TRT
I
I
I
I
30-25 ~5-3o 30-25 35-30 3o-25 x I00
SWS TST
x I00
AC
x
FIG. 4. Percent, A, SWS and activity (AC) during each 5°C drop in Tar during the initial phase of entry. Horizontal bar represents mean; boxes represent standard error; vertical line represents range; N=9, ***o<0.005.
difference between the length of the A episodes is increased. The length of the PS episodes did not differ between species. The distribution of PS and activity above TBr of 30°C in the marmot suggested that the character of the entry gradually changes as TBr decreases. The initial phase of entry was further subdivided to Tsr = 35~<30°C and TBr=30>25°C. TRT did not differ between the two temperature ranges: 198.1-+36.7 min at Tsr 35-30°C and 125.9-+10.22 min at TBr 30-25°C. T-tests were used to test significant differences between the contribution of each arousal state in each temperature range (Fig. 4). Awake state as percent of TRT did not differ over the two temperature ranges (25.32-+3.63% vs 27.72-+6.30%). Activity as a percent of A tended to decline as Ts~ declined, but because of the large variability among animals the decline was not significant statistically. SWS as a percent of TST did increase significantly as TB~ declined (80.81---2.24% vs 92.43-+2.16% p<0.005). This increase in SWS occurred at the expense of PS and represented a decrease in the number of SWS episodes with an increase in duration of each episode (Table 2). The number of A and PS episodes also decreased as T,r declined. However, no change in episode length of A and PS was observed.
A 35~<30 16.6 _+ 3.6* N =9 3.8 -+ 0.6 N = 147 0.5-50
*Values represent Mean _+ SE. tp<0.02.
SWS 30>25 6.3 ± 1.4t N=9 5.9 ± 1.9 N =57 0.5-100
(*C)
A
TABLE 2 NUMBER AND DURATIONOF AROUSALEPISODESDURINGENTRY
Tsr (°C) No. of episodes/ animal Mean duration of episodes (min) Range (min)
TBR
I00
35~<30 21.0 _+ 4.3 N=9 5.7 _+ 0.5 N= 189 0.5-31.5
30>25 9.2 -+ 1.3t N=9 8.6 _+ 1.4t N=83 0.5-68
PS 35~<30 7.1 _+ 0.9 N=9 3.8 _+ 0.4 N=64 0.5-14
30>25 3.3 ± 1.0t N=9 2.7 _+ 0.4 N=30 0.5-7.5
E N T R Y INTO H I B E R N A T I O N IN T H E MARMOT
993
DISCUSSION Entry into hibernation in the marmot is a complex phenomenon in which all arousal states are represented. These data do not support, as originally suggested by South et al. [11], the hypothesis that hibernation is entered into as an extension of SWS because, (1) the initial drop in Tar could occur during A, (2) the last identifiable arousal state in some cases was A accompanied by complex behavior patterns. These behavior patterns were similar to those observed in the ground squirrel (Citellus beecheyi, [14]), hedgehog (Erinaceus europeanus, [16]) and pocket mouse (Perognathus longimernbris, [2]). The activity periods of the marmot also acted to slow the rate of entry. However, such " b r a k i n g " well may be no more than fortuitous since many of the behaviors involved purposeful building and rearranging of the nest rather than shivering in a curled position. A pattern does emerge that would be consistent with the hypothesis that the activity not only defends against the cold as Tar drops but alters also the total thermal insulation of the animal plus its nest. This would allow for more precise regulation of Tar during hibernation. A further test of this hypothesis would be to observe the behavior patterns of animals with no nesting material and with materials of widely differing heat conductances. The stereotypic nature of the activity as Tar decreased reflects the gradual cold inactivation of the central nervous system at reduced temperature; however, "absolute activity" [13] how has a profound effect on Tar because of the limited sources of body heat. The mean episode lengths of arousal states during the initial phase of entry were remarkable similar in the small and large hibernator (Table 1). The greatest difference was the length of awake periods. The criticism could be put forth that differences in bout lengths are masked by the way in which the intermittent periods of A and SWS are considered. Other rodent species [16,18] have been reported to exhibit alternating periods of A and SWS, however, the significance of their frequency and distribution remains to be determined. Additional differences in episode lengths between species
may emerge if the episodes were measured over 5°C temperature increments. The characteristics of entry in the marmot do change within each 5°C increment in TBr. AS has been reported in the ground squirrel [19], the percent of PS decreased and SWS increased as Tar declined. During SWS, hypothalamic thermosensitivity is less than during A [4,9] but greater than during PS [4]. If this same relationship exists in Marmota, then progressive increases in the amount of SWS would render the system capable of continuous regulation of body temperature (as has been shown, [3,5]) at reduced sensitivity. In addition to the thermoregulatory advantage of increased amounts of SWS during entry, the animals are also more responsive to external stimuli than during PS [7]. Reduction in PS during entry is compatible with the hypothesis that as the overall danger (ecological and predatory factors) increases PS decreases [1]. Animals entering hibernation are more vulnerable to predation and environmental problems (ground water, change in freeze line, etc.) than normothermic animals because response time is increased by lowered body temperature. The relationship between PS and activity is not clear. In the hedgehog [16] the activity periods occurred at predictable intervals and always followed PS. No such patterns were seen in the marmot. Although conventional criteria for arousal states could not be used with confidence below TBr = 25°C, consistent and periodic alterations in E E G waveform could be identified down to the achievement of actual deep hibernation (ca. 7°C). What these alterations mean in terms of animal responsiveness and thermoregulatory sensitivity during hibernation remains to be defined. ACKNOWLEDGMENT The authors wish to acknowledge the technical assistance of W. C. Hartner and H. K. Jacobs.
REFERENCES
1. Allison, T. and D. V. Cicchetti. Sleep in mammals: Ecological and constitutional correlates. Science 194: 732-734, 1976. 2. Bartholomew, G. A. and T. J. Cade. Temperature regulation, hibernation and aestivation in the little pocket mouse, Perognathus longimembris. J. Mammal. 38: 60-72, 1957. 3. Florant, G. L. and H. C. Heller. CNS regulation of body temperature in euthermic and hibernating marmots (Marmota flaviventris). Am. J. Physiol. 232: R203-208, 1977. 4. Glotzbach, S. F. and H. C. Heller. Central nervous regulation of body temperature during sleep. Science 194: 537-539, 1976. 5. Heller, H. C., G. W. Colliver and J. Beard. Thermoregulation during entrance into hibernation. Pfliigers Arch. 369: 55-59, 1977. 6. Heller, H. C. and S. F. Glotzbach. Thermoregulation during sleep and hibernation. In: International Review of Physiology: Environmental Physiology II, Vol. 15, edited by D. Robertshaw. Baltimore: University Park Press, 1977, pp. 147188. 7. Jouvet, M. Neurophysiology of the states of sleep. Physiol. Rev. 47: 117-177, 1967. 8. Lyman, C. P. Oxygen consumption, body temperature and heart rate of woodchucks entering hibernation. Am. J. Physiol. 194: 83-91, 1958. 9. Miller, V. M. and F. E. South. Spinal cord, hypothalamic and air temperature interaction with arousal states in the marmot. Am. J. Physiol. 236: RI07-RII6, 1979. 10. Satinoff, E, Hibernation and the central nervous system. In: Progress in Physiological Psychology, Vol. 3, edited by E. Stellar and J. M. Sprague. New York: Academic Press, 1970, pp. 201-236.
11. South F. E., J. E. Breazile, H. D. Dellmann and A. D. Epperly. Sleep, hibernation and hypothermia. In: Depressed Metabolism, edited by X. J. Musacchia and J. F. Saunders. New York: American Elsevier, 1969, pp. 277-312. 12. South, F. E., W. C. Hartner and R. H. Luecke. Responses to peroptic temperature manipulation in the awake and hibernating Marmot. Am. J. Physiol. 229: 150--160, 1975. 13. South, F. E., V. M. Miller and W. C. Hartner. Neuronal models of temperature regulation in euthermic and hibernating mammals: an alternative model for hibernation. In: Strategies in Cold, edited by L. C. H. Wang and J. W. Hudson. New York: Academic Press, 1978, pp. 187-224. 14. Strumwasser, F. Thermoregulatory brain and behavioral mechanisms during entrance into hibernation in the squirrel, Citellus beecheyi. Am. J. Physiol. 196: 15-22, 1959. 15. Suomalainen, P. Hibernation and sleep. In: The Nature oJ Sleep, edited by G. E. W. Wolstenhohne and M. O'Connor. London: J. H. Churchill, 1961, pp. 307-316. 16. Toutain, P. L. and Y. Ruckebush. Arousal as cyclic phenomena during sleep and hibernation in hedgehogs (Erinaceus europeanus). Experientia 31: 312-314, 1975. 17. Twente, J. W. and J. A. Twente. Regulation of hibernating periods by temperature. Proc. natn Acad. Sci. U.S.A. 54: 1058--1061, 1965. 18. VanTwyver, H. Sleep patterns of five rodent species. Physiol. Behav. 4: 901-905, 1969. 19. Walker, J. M., S. F. Glotzbach, R. J. Berger and H. C. Heller. Sleep and hibernation in ground squirrels (Citellus spp.) electrophysiological observations. Am. J. Physiol. 233: R213-R221, 1977.
Entry into Hibernation in M. Flaviventris: Sleep and Behavioral Thermoregulation V I R G I N I A M. M I L L E R A N D F R A N K
E. S O U T H
School o f Life and Health Sciences, University o f Delaware, N e w a r k , D E 19711 R e c e i v e d 19 M a r c h 1981 MILLER, V. M. AND F. E. SOUTH. Entry into hibernation in M. flaviventris: Sleep and behavioral thermoregulation. PHYSIOL. BEHAV. 27(6) 989--993, 1981.--Hibernators of the genus Marmota (wt=3-5 kg) differ from smaller hibernators (wt< 1 kg) in thermoregulatory characteristics during entry into hibernation. They might be expected to differ also with respect to the distributions of activity, awake and sleep states during entry. Marmots implanted stereotaxically with electrodes to record electroencephalograms and brain temperature (TBr) were monitored remotely by a polygraph as well as video transmission as they entered hibernation. During entry, awake (A), slow wave sleep (SWS), and paradoxical sleep (PS) states could be identified. Activity which included nest building, grooming, and shivering slowed entry and became progressively more stereotyped as Ta~ decreased. All animals exhibited at least one PS bout between Tsr=33-32°C. SWS as percent of total sleep increased (80 to 92%) as Tar decreased from 35-25°C. This increase represented a decrease in number and increase in duration of SWS episodes. The length of individual SWS and PS episodes of the marmot did not differ from those reported in Citellus. These data suggest that entry into hibernation is a more complex phenomenon than merely an extension of slow wave sleep. Similar changes in arousal state distribution occur in hibernators of different sizes. Marmot M. flaviventris Hibernation Hibernation entry Arousal states Slow wave sleep Paradoxical sleep
I N T E R M I T T E N T periods of apparent activity have been observed repeatedly in animals entering hibernation [2, 8, 13, 14, 16, 19]. Although the thermoregulatory significance of these activity bouts remains to be determined, it has been suggested [14] that they slow or " b r a k e " too rapid a drop in brain temperature (Tar). However, this also could be due to relatively nonspecific movements or shivering by the animals within their nest. A review of the literature suggests that behavior patterns during entry are specific and relatively stereotyped among a variety of hibernators (Citellus spp., E. europeanus, P. Iongimembris, M. flaviventris, and M. monax). The complexity of these behavior patterns which include grooming [2, 14, 16], nest rearranging [2, 13, 14], curling [2, 13, 14, 16], and shivering [13, 14, 19] have led us [13] to postulate that the net effect of these activities increased the heat capacity of the animal's nest allowing for more precise regulation of TR~ during hibernation. Although these bouts of movement are indeed striking, animals entering hibernation do spend the majority of entry time in a quiescent state. Electrophysiological observations from hibernating mammals have led to the hypothesis that hibernation is entered into from a sleep state [5, 6, 10, 11, 15, 17, 19]. Specifically, in Citellus spp. [19], slow wave sleep [SWS] as a percent of total sleep time (TST) increased from the 80% of normothermia to 90% during entry into hibernation. Increased amounts of SWS were concomitant with decreased amounts of paradoxical sleep (PS) [10,19]. However, similar observations in a larger hibernator, M. flaviventris [11], at a brain temperature (Tar) of 33°C suggest that the amount of SWS during entry does not alter from the 80% of TST seen during normothermia. The hibernators of the genus Marmota (adult w t = 3 - 5 kg) represent smaller
Thermoregulation
Sleep
surface-to-volume ratios when compared to hibernators of weight < 1 kg. The thermoregulatory characteristics of Marmota during entry into hibernation therefore might differ from other hibernators. This difference also might be reflected in the relative distribution during entry of the awake and sleep states. The observations of this report are the first in which undisturbed behavioral activity, electroencephalogram and Tar were monitored continuously during entry into hibernation in Marmota. Thus they provide complete data of the entry phenomena for comparison among hibernators of widely differing weights. METHOD Male and female yellow bellied marmots (Marmota flaviventris) weighing between 3-5 kg were implanted stereotaxically with electrodes to monitor cortical electroencephalogram (EEG), dorsal hippocampal theta activity and the electrooculogram (EOG). Surgical anesthesia was obtained with sodium pentobarbital (40 mg/kg intraperitoneally). E E G electrodes were stainless steel machine screws with recording wires soldered to the heads. Electrodes were placed bilaterally over the frontal and occipital cortices. The hippocampal electrode (In Vivo Metric NEX-200, l0 mm) was placed anteriorly from earbar reference 5.6 mm, laterally 6.2 mm and 5.2 mm below brain surface. A 0.010 mm stainless steel wire stripped of insulation and formed into a flat coil was placed on the orbit of the eye as the EOG electrode. A 22 ga stainless steel tube with one end closed and coated with Epoxylite electrode coat was placed in the internal capsule. Insertion of a thermocouple (36 ga copper-
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.--0031-9384/81/120989-05502.00/0
990
M I L L E R A N D SOUTH
AWAKE
TB R = 3 5
moving
° C
q uiet
30 s ec EMG
2oo.v I EEG
\
~\
400 u VI EOG
f
IOOpV I
FIG. 1. Electrophysiological tracing of the awake state observed during entry into hibernation. During movement, EMG activity is high and EOG spikes appear. During quiet awake, the frequency of EOG spikes and EMG activity is reduced while low voltage fast activity dominates the EEG. As slow wave sleep begins, spindles appear in the EEG.
constantan) into this sleeve provided a sensitive record of brain temperature. All electrodes were soldered to a DE-95 Cinch plug and secured to the skull with dental acrylic. Animals were treated postoperatively with 100,000 units of Procaine Penicillin G intramuscularly. Two weeks were allowed before animals were placed in the hibernaculum. Prior to surgery and during the recovery period, animals were housed individually at an ambient temperature (Ta) of 20___I°C, light:dark cycle 12:12 hrs with food and water provided ad lib. After recovery from surgery, recording cables were attached to the exposed pedestal plugs on the animal's head. At this time, electromyographic (EMG) pins, designed after South et al. [12], were inserted into the skin in the nucal region for monitoring heart rate, movement, and shivering. Animals were then placed in an environmental chamber (Ta=5°C) in individual cages. Recording cables were attached to a slipring assembly so that the animal was free to move about the cage. Food, water and cotton nesting material were provided ad lib. Red incandescent lights were on continuously, allowing for behavior to be monitored by remote video. EEG, EMG, and EOG were monitored through 7P5A Grass AC preamplifiers. THr was referenced to an Omega thermocouple reference junction box and recorded through a Grass 7P1 DC preamplifier. Animals were left undisturbed except for routine feeding and watering. Hibernation usually proceeded within 5 weeks time. At the conclusion of the hibernating season, animals were overdosed with sodium pentobarbital (50 mg/kg intraperitoneally) and were perfused via the carotid arteries with 0.9% saline followed by 10% Formalin. The brains were removed, embedded in paraffin, and cut into 40 micron sections for confirmation of electrode placement. Polygraph records for the entry periods were scored at 0.5 min intervals. Variability existed in the lability of TBr among
animals and between bouts of hibernation. Therefore, initiation of entry into hibernation was defined for this study as a decrease in TBr from the maximum TBr measured or 35°C with no subsequent i n c r e a s e ~ TBr of over an hour duration. Standard criteria [7, 9, 11, 18, 19] were used to define arousal states. Two independent scorings were made of each record and found not to differ significantly. Intermittent periods (0.5-2.5 min duration) of low voltage fast E E G activity and spindles were scored individually as awake and sleep, respectively, whereas other have scored these periods collectively as awake [18], drowsy [16], or sleep [19]. RESULTS Nine entries into hibernation were observed in 7 animals between the months of November and March. No correlation between the initiation of entry to time of day could be made. During the initial phase of entry, TBr = 35-25°C, all arousal states could be identified (Figs. 1 and 2). The awake phase (A) was characterized by low voltage fast activity (~12 cycles/sec) in the EEG, theta activity (4-10 cycles/sec) from the hippocampus (not shown) during movement, and high background EMG activity. EOG spikes occurred during movement but were absent during quiescent awake periods and slow wave sleep (Fig. 1). Sleep could be identified (Fig. 2) as slow wave sleep (SWS) and paradoxical sleep (PS). During SWS, high voltage slow wave activity (0--5 cycles/sec) dominated the E E G trace. EMG activity and heart rate were low and no spikes were seen on the EOG. Frequently, intermittent periods of A and SWS (each state <2.5 min) were observed. These alternating A and SWS sequences preceded a prolonged SWS bout of 6.2___ 1 min (mean_+SE) duration in 29 of 34 instances. As shown in Fig. 2, PS always proceeded
ENTRY INTO H I B E R N A T I O N IN THE MARMOT
991
SWS
PS
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FIG. 2. Electrophysiological tracing of the sleep state observed during entry into hibernation. SWS=slow wave sleep. PS=paradoxical sleep. See text for further description.
from SWS. During PS, low voltage fast activity was seen in the E E G trace, theta activity emanated from the hippocampus (not shown), heart rate was irregular, background EMG was low with periodic jerking movements, and EOG record reflected the characteristic rapid eye movement. The above criteria for arousal states could not be used satisfactorily below TBr=25°C. However, alternating periods of slow and fast frequency EEG were observed as TBr continued to decline during the remainder of the entry. Periodic alterations in EEG activity were also observed during deep hibernation. Any correlation between the initiation of entry and a given arousal state was difficult to ascertain. Prior to entry, TBr among animals varied greatly. Three animals showed cycles of decreases followed by increases in TBr. In two other animals TBr plateaued at 33°C before Tar continued to decrease to values of deep hibernation. SWS during the initial stages of entry, however, was always associated with a decline in Tar. Periods of movement and activity could be identified throughout the entire entry. The distribution of activity during two typical successive entries by one animal is illustrated in Fig. 3. The overall rate of Tar decline and the behaviors observed were similar in both instances. However, the distribution of the behaviors during the TBr decline differed. In general, behavior patterns during the initial phase of entry were complex and involved extensive nest building, circling, and shivering. These became increasingly stereotyped as Tar decreased: raising of the head, rocking from side to side, curling into the hibernation position. Relatively complex behaviors were still observed at Tar as low as 24°C. Since conventional arousal states could be identified only during the initial phase of entry (TBr>~25°C), the relative contributions of activity, A, SWS and PS during this stage of entry were analyzed. The total recording time (TRT) aver-
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HOURS FIG. 3. Brain temperature decline and distribution of activity during two entries into hibernation observed from a single animal.
aged 324_+3%7 min (mean_-+SE) for the 9 entries observed. The A state occupied 27.4±2.75% of this T R T . This represented 22.8-.+4.5 episodes of 4.2-+0.7 min duration per animal. Total activity for all animals represented 32.5±4.2% of the A state. The amount of activity was linearly related to TRT. The relationship was defined by the equation Y' = 0.17x - 24.25 (r=0.90)
992
MILLER AND SOUTH
TABLE 1 COMPARISONOF EPISODE LENGTH BETWEENCITELLUS AND MARMOTA Citellus spp. [19]
A SWS PS
80
M. flaviventris
1.7 ± 0.8 (min)*
12.3 ± 2.3t N =204 13.7 _+ 1.1 N=272 3.5 ± 0.3 N= 94
11.4 _+ 5.4 3.0 ± 0.9
IIII
I00
kz i,i co i,i a.
60
40
20
*Values represent Mean - SD. +Values represent Mean _+ SE. I
I
35-30 where Y' is the total activity time (min) displayed by an animal and x is the TRT for that animal. In 31 of 43 instances, activity occurred during an A episode which followed SWS; in the remaining 12 instances, the activity followed an episode of PS. The distribution of the activity was such that few animals were observed to engage in activity at the extremes of the Tar range, 35° and 25°C (2 of 7 and 3 of 9, respectively). However, all animals (9 of 9) engaged in at least one activity bout of 6.9_+2.3 min duration (N= 14) between Tar 32° and 30°C. Total sleep time ( T S T = T R T - A ) was composed of 85.1-+1.3% SWS. This represented 30.2-+5.0 episodes per animal with each episode averaging 6.5-+0.5 min length. Animals averaged 10.4-+ 1.4 episodes of PS of 3.5-+0.3 min duration between Tsr 35-25°C. PS occurred most frequently early in the entry with 8 of 9 animals (p<0.02 Wilcoxon matched-pairs signed-ranks test) engaged in PS at both TB~of 33° and 32°C. No correlation could be made between the number or duration of PS bouts with the amount of activity or TRT. When the average lengths of A, SWS, and PS episodes are compared to those values reported for CiteUus ([19]; see Table 1, column 1) several differences are suggested. The length of SWS episodes were less and the length of A episodes were longer in the marmot than in the ground squirrel. However, these apparent differences could be due to the way in which the lengths of A and SWS bouts were calculated. If the periods of intermittent A and SWS are considered as SWS (as in the Citellus study [19]), then the lengths of SWS are remarkably similar between species (Table 1). The
A TRT
I
I
I
I
30-25 ~5-3o 30-25 35-30 3o-25 x I00
SWS TST
x I00
AC
x
FIG. 4. Percent, A, SWS and activity (AC) during each 5°C drop in Tar during the initial phase of entry. Horizontal bar represents mean; boxes represent standard error; vertical line represents range; N=9, ***o<0.005.
difference between the length of the A episodes is increased. The length of the PS episodes did not differ between species. The distribution of PS and activity above TBr of 30°C in the marmot suggested that the character of the entry gradually changes as TBr decreases. The initial phase of entry was further subdivided to Tsr = 35~<30°C and TBr=30>25°C. TRT did not differ between the two temperature ranges: 198.1-+36.7 min at Tsr 35-30°C and 125.9-+10.22 min at TBr 30-25°C. T-tests were used to test significant differences between the contribution of each arousal state in each temperature range (Fig. 4). Awake state as percent of TRT did not differ over the two temperature ranges (25.32-+3.63% vs 27.72-+6.30%). Activity as a percent of A tended to decline as Ts~ declined, but because of the large variability among animals the decline was not significant statistically. SWS as a percent of TST did increase significantly as TB~ declined (80.81---2.24% vs 92.43-+2.16% p<0.005). This increase in SWS occurred at the expense of PS and represented a decrease in the number of SWS episodes with an increase in duration of each episode (Table 2). The number of A and PS episodes also decreased as T,r declined. However, no change in episode length of A and PS was observed.
A 35~<30 16.6 _+ 3.6* N =9 3.8 -+ 0.6 N = 147 0.5-50
*Values represent Mean _+ SE. tp<0.02.
SWS 30>25 6.3 ± 1.4t N=9 5.9 ± 1.9 N =57 0.5-100
(*C)
A
TABLE 2 NUMBER AND DURATIONOF AROUSALEPISODESDURINGENTRY
Tsr (°C) No. of episodes/ animal Mean duration of episodes (min) Range (min)
TBR
I00
35~<30 21.0 _+ 4.3 N=9 5.7 _+ 0.5 N= 189 0.5-31.5
30>25 9.2 -+ 1.3t N=9 8.6 _+ 1.4t N=83 0.5-68
PS 35~<30 7.1 _+ 0.9 N=9 3.8 _+ 0.4 N=64 0.5-14
30>25 3.3 ± 1.0t N=9 2.7 _+ 0.4 N=30 0.5-7.5
E N T R Y INTO H I B E R N A T I O N IN T H E MARMOT
993
DISCUSSION Entry into hibernation in the marmot is a complex phenomenon in which all arousal states are represented. These data do not support, as originally suggested by South et al. [11], the hypothesis that hibernation is entered into as an extension of SWS because, (1) the initial drop in Tar could occur during A, (2) the last identifiable arousal state in some cases was A accompanied by complex behavior patterns. These behavior patterns were similar to those observed in the ground squirrel (Citellus beecheyi, [14]), hedgehog (Erinaceus europeanus, [16]) and pocket mouse (Perognathus longimernbris, [2]). The activity periods of the marmot also acted to slow the rate of entry. However, such " b r a k i n g " well may be no more than fortuitous since many of the behaviors involved purposeful building and rearranging of the nest rather than shivering in a curled position. A pattern does emerge that would be consistent with the hypothesis that the activity not only defends against the cold as Tar drops but alters also the total thermal insulation of the animal plus its nest. This would allow for more precise regulation of Tar during hibernation. A further test of this hypothesis would be to observe the behavior patterns of animals with no nesting material and with materials of widely differing heat conductances. The stereotypic nature of the activity as Tar decreased reflects the gradual cold inactivation of the central nervous system at reduced temperature; however, "absolute activity" [13] how has a profound effect on Tar because of the limited sources of body heat. The mean episode lengths of arousal states during the initial phase of entry were remarkable similar in the small and large hibernator (Table 1). The greatest difference was the length of awake periods. The criticism could be put forth that differences in bout lengths are masked by the way in which the intermittent periods of A and SWS are considered. Other rodent species [16,18] have been reported to exhibit alternating periods of A and SWS, however, the significance of their frequency and distribution remains to be determined. Additional differences in episode lengths between species
may emerge if the episodes were measured over 5°C temperature increments. The characteristics of entry in the marmot do change within each 5°C increment in TBr. AS has been reported in the ground squirrel [19], the percent of PS decreased and SWS increased as Tar declined. During SWS, hypothalamic thermosensitivity is less than during A [4,9] but greater than during PS [4]. If this same relationship exists in Marmota, then progressive increases in the amount of SWS would render the system capable of continuous regulation of body temperature (as has been shown, [3,5]) at reduced sensitivity. In addition to the thermoregulatory advantage of increased amounts of SWS during entry, the animals are also more responsive to external stimuli than during PS [7]. Reduction in PS during entry is compatible with the hypothesis that as the overall danger (ecological and predatory factors) increases PS decreases [1]. Animals entering hibernation are more vulnerable to predation and environmental problems (ground water, change in freeze line, etc.) than normothermic animals because response time is increased by lowered body temperature. The relationship between PS and activity is not clear. In the hedgehog [16] the activity periods occurred at predictable intervals and always followed PS. No such patterns were seen in the marmot. Although conventional criteria for arousal states could not be used with confidence below TBr = 25°C, consistent and periodic alterations in E E G waveform could be identified down to the achievement of actual deep hibernation (ca. 7°C). What these alterations mean in terms of animal responsiveness and thermoregulatory sensitivity during hibernation remains to be defined. ACKNOWLEDGMENT The authors wish to acknowledge the technical assistance of W. C. Hartner and H. K. Jacobs.
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1. Allison, T. and D. V. Cicchetti. Sleep in mammals: Ecological and constitutional correlates. Science 194: 732-734, 1976. 2. Bartholomew, G. A. and T. J. Cade. Temperature regulation, hibernation and aestivation in the little pocket mouse, Perognathus longimembris. J. Mammal. 38: 60-72, 1957. 3. Florant, G. L. and H. C. Heller. CNS regulation of body temperature in euthermic and hibernating marmots (Marmota flaviventris). Am. J. Physiol. 232: R203-208, 1977. 4. Glotzbach, S. F. and H. C. Heller. Central nervous regulation of body temperature during sleep. Science 194: 537-539, 1976. 5. Heller, H. C., G. W. Colliver and J. Beard. Thermoregulation during entrance into hibernation. Pfliigers Arch. 369: 55-59, 1977. 6. Heller, H. C. and S. F. Glotzbach. Thermoregulation during sleep and hibernation. In: International Review of Physiology: Environmental Physiology II, Vol. 15, edited by D. Robertshaw. Baltimore: University Park Press, 1977, pp. 147188. 7. Jouvet, M. Neurophysiology of the states of sleep. Physiol. Rev. 47: 117-177, 1967. 8. Lyman, C. P. Oxygen consumption, body temperature and heart rate of woodchucks entering hibernation. Am. J. Physiol. 194: 83-91, 1958. 9. Miller, V. M. and F. E. South. Spinal cord, hypothalamic and air temperature interaction with arousal states in the marmot. Am. J. Physiol. 236: RI07-RII6, 1979. 10. Satinoff, E, Hibernation and the central nervous system. In: Progress in Physiological Psychology, Vol. 3, edited by E. Stellar and J. M. Sprague. New York: Academic Press, 1970, pp. 201-236.
11. South F. E., J. E. Breazile, H. D. Dellmann and A. D. Epperly. Sleep, hibernation and hypothermia. In: Depressed Metabolism, edited by X. J. Musacchia and J. F. Saunders. New York: American Elsevier, 1969, pp. 277-312. 12. South, F. E., W. C. Hartner and R. H. Luecke. Responses to peroptic temperature manipulation in the awake and hibernating Marmot. Am. J. Physiol. 229: 150--160, 1975. 13. South, F. E., V. M. Miller and W. C. Hartner. Neuronal models of temperature regulation in euthermic and hibernating mammals: an alternative model for hibernation. In: Strategies in Cold, edited by L. C. H. Wang and J. W. Hudson. New York: Academic Press, 1978, pp. 187-224. 14. Strumwasser, F. Thermoregulatory brain and behavioral mechanisms during entrance into hibernation in the squirrel, Citellus beecheyi. Am. J. Physiol. 196: 15-22, 1959. 15. Suomalainen, P. Hibernation and sleep. In: The Nature oJ Sleep, edited by G. E. W. Wolstenhohne and M. O'Connor. London: J. H. Churchill, 1961, pp. 307-316. 16. Toutain, P. L. and Y. Ruckebush. Arousal as cyclic phenomena during sleep and hibernation in hedgehogs (Erinaceus europeanus). Experientia 31: 312-314, 1975. 17. Twente, J. W. and J. A. Twente. Regulation of hibernating periods by temperature. Proc. natn Acad. Sci. U.S.A. 54: 1058--1061, 1965. 18. VanTwyver, H. Sleep patterns of five rodent species. Physiol. Behav. 4: 901-905, 1969. 19. Walker, J. M., S. F. Glotzbach, R. J. Berger and H. C. Heller. Sleep and hibernation in ground squirrels (Citellus spp.) electrophysiological observations. Am. J. Physiol. 233: R213-R221, 1977.