Progressive irritability of hibernating Citellus lateralis

Comp. Biochcm. Physiol., 1968, VoL 25, pp. 467 to 474. Pergamon Press. Printed in Great Britain

PROGRESSIVE IRRITABILITY OF HIBERNATING C I T E L L US L A T E R A L I S * JOHN W. T W E N T E and JANET A. T W E N T E Department of Zoology and Space Sciences Research Center, University of Missouri, Columbia

(Received 17 October 1967) Abstracts1. Intraperitoneal Lnjection of 0.5 ml isotonic saline to hibernating

Citellus lateralis does not evoke premature arousal ff administered during the first 50 per cent of the expected hibernating period. After that time, the number arousing in response to injection increases as the hibernating period progresses. 2. The greater the volume injected the earlier premature arousal is evoked. Hypo- or hypertonic saline (0"5 ml) evokes arousal earlier than 0-5 ml isotonic saline. 3. Electrocardiographic patterns after sound and injection stimuli and at the initiation of spontaneous arousal indicate that evoked and spontaneous arousal are similar, centrally mediated phenomena. INTRODUCTION DURATIONS of hibernating periods of golden-mantled ground squirrels (Citellus lateralis) are a linear function of body temperature over a range of 2-25°C. An Arrhenius plot of the frequency of spontaneous arousal is also linear suggesting that the duration of the hibernating period may be limited by a single, regulating process (Twente & Twente, 1965a). If animals are undisturbed, hibernating periods are relatively constant in duration for an animal at a temperature level. However, premature arousals may be evoked at any time during the hibernating period providing that a strong enough stimulus is given. As the hibernating period progresses, these animals become increasingly sensitive to mild, external stimuli and the frequency of evoked, premature arousals increases as the normal termination of the hibernating period is approached. We have cruddy shown that sound stimuli evoke premature arousals with increasing frequency after 50 per cent of the expected hibernating period has elapsed (Twente & Twente, 1965b) and have designated this phenomenon as "progressive irritability" (Twente & Twente, 1966). Changes in irritability during hibernating periods of mammals were first reported on the basis of field observations of vespertilionid bats (Twente, 1955). Kristoffersson & Soivio (1964a) indicated that hedgehogs (Erinaeeus europeaus) did not arouse in response to pricking with injection needles just after entry into hibernation, but that similar treatment shortly before the time of the estimated spontaneous arousal always evoked arousal. * This work was supported in part by USPHS grant AM-11320. 467

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AX~ JANET A. T w ~ s

T h e following experiments were designed to m e a s u r e progressive irritability in a m o r e substantial fashion; c o n c u r r e n t l y it was d e t e r m i n e d at w h a t intervals of the h i b e r n a t i n g period injections could be administered w i t h o u t evoking p r e m a t u r e arousal. METHODS The average durations of hibernating periods of adult Citellus lateralis of both sexes were determined through continuous monitoring from permanently implanted, Tefloncoated, iron--constantan thermocouples tied to a lower rib (Twente & Twente, 1965b). Temperature records were printed at 6-rain intervals on time-printed chart paper by Leeds and Northrup Speedomax G 16-point recording potentiometers calibrated from 0 to 50°C. The duration of the hibernating period was determined as the time elapsed, in hours, after the body temperature reached 2°C above the microenvironmental temperature during induction until the initial temperature increase indicative of arousal. All animals exhibited the winter pattern of hibernating behavior which is characterized by hibernating periods of relatively constant duration for each animal at any given temperature level (Twente & Twente, 1967). A single hibernating period of an undisturbed ground squirrel seldom varies more than _+10 per cent from the average duration for that animal. Variation between animals, however, is great; the average duration of hibernating periods of some animals may be twice as long as that for others at the same temperature level (Twente & Twente, 1965a). We have equated hibernating periods of ground squirrels with different durations by expressing each animal's average duration as constituting 100 per cent. Fractions thereof, expressed as percentages, are considered to be physiologically comparable and comparisons between animals have been made after specific intervals of the elapsed hibernating period. The dose-response curve derived from injections of different concentrations of epinephrine administered after specific intervals of the hibernating period indicates that this assumption is valid with respect to progressive irritability (Twente & Twente, 1968). Animals were caged individually and maintained, undisturbed, at 4, 5 and 6°C. Sunflower seeds and cut oranges were present at all times. After different percentages of the expected hibernating periods had elapsed, approximately 2 ml cold tincture of benzalkonium chloride was applied to tl~e side prior to lateral, intraperitoneal injection with No. 26, 9 rnm, hypodermic needles. The animals were not handled. Evoked arousal or continued hibernation after injection indicated whether or not the animals were responsive to injection. Arousals after injections of saline were apparent from temperature records. If arousals were not associated with any known, external stimulus, they were considered to be spontaneous. All solutions injected were approximately 5°C except for the 37°C series.

Single injection studies A. The average durations of hibernating periods of fifty-five ground squirrels were established on the basis of four or more hibernating periods for each animal. The experiment was designed so that one of us knew at what percentage interval of the hibernating period the injection was to be given. The other then blindly administered 0"5 ml cold, pyrogen-free 0"9% saline. If arousal was not evoked, the ground squirrel was allowed to complete its normal hibernating period. B. The hibernating periods of seventeen additional animals were established as above and were subjected to sham injections wherein the peritoneal cavity was only pierced with the hypodermic needle.

Multiple-injection studies The average durations of hibernating periods of eleven additional ground squirrels were established on the basis of three or more hibernating periods for each animal. Series

PROGRESSIVE I R R I T A B I L I T Y O F H I B m I N A T I N O CITEI.,Z,U,..q ~TE_RMLZS

469

of injections were initiated early in the hibernating period and administered at intervals ranging from 15 to 48 hr. Five animah were used for the study of the effect of the volume injected and six were used for the study of the effects of solution temperature and tonicity.

Electrocardiograms Records were obtained of twenty-eight hibernating periods of sixteen animals. Recordings from standard Limb Lead II (right fore leg, left hind leg) needle electrode placements were made using a recording osciUograph (Grass Model 7). Segments selected from these recordings representing the following are presented: Comparisons of heart rates prior to, the initiation of, and immediately foUowing (1) spontaneous arousal; (2) arousal evoked by noise; (3) arousal evoked by injection; (4) noise stimulus which did not evoke arousal; (5) injection which did not evoke arousal. RESULTS

Single-injection studies A. Arousals were evident from temperature records within 18 min after injection; no partial arousals occurred. Figure 1 shows that injections of 0.5 ml isotonic (0"9%) saline did not evoke arousal if administered before 50 per cent of the expected hibernating period had elapsed. After 50 per cent, the number of evoked arousals in response to injection increased as the hibernating period progressed, the highest percentage of animals arousing at the 90-99 per cent level. '~

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DURATION OF HIBERNATIONEXPRESSEDAS PERCENTOF EXPECTEDTOTAL

FIc. 1. Per cent evoked arousal/per cent no arousal in response to single injections of 0"5 ml isotonic saline. The number of animals used is shown in the histograms.

B. Arousals evoked by sham injections occurred later in the hibernating period than those evoked by 0-5 ml isotonic saline (Table 1).

Multiple-injection studies Figures 2 and 3 demonstrate: a. The greater the volume injected, the earlier in the hibernating period the evoked arousal occurred. One ml isotonic saline initiated arousal earlier than 0.5 ml; 0.5 ml evoked arousal earlier than 0.25 ml; 0.25 ml caused arousal earlier than sham injections.

470

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A_~D JANETA.

TABLE 1--Nu~mEm

o F G R O U N D SQUIRRELS S H O W I N G E V O K E D A R O U S A L / N O E V O K E D A R O U S A L IN R R S P O N S E T O S H A M I/~IJECTIONS O V E R T H E R A N G E O F T H E E X P E C £ ~ o H I B E R N A T I N G PERIOD

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50-59%

60-69%

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Fro. 2. Effects of different volumes of isotonic saline and sham injections administered at 15-48-hr intervals. The top of each histogram represents the average duration of the hibernating period for each ground squirrel expressed as 100 per cent of the expected hibernating period at the temperature level indicated. Arrows around the top of each histogram represent the durations of hibernating periods upon which the average is based. Animals are indicated by their laboratory

designations (0-739, etc.). b. Arousals were evoked earlier in the hibernating period with 0.5 ml hypertonic (1.8%) saline than with 0.5 ml hypotonic (0.45%) saline which, in turn, tended to cause arousal earlier than 0"5 ml isotonic saline.

471

PROGRESSIVE I R R I T A B I L I T Y OF H I B m ~ I A T I N G CITELLUS L A T E R A L I S

c. No greater stimulus to arousal was constituted by 0.5 m137°C isotonic saline

than by 0.5 ml 5°C isotonic saline. d. Some animals accumulated 3-4 ml of the various solutions apparently without affecting arousal patterns (Fig. 3, 0-85 and 0-667). Considerable variation existed between animals regarding the sensitivity to injection. 250

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Fxo. 3. Effects of different concentrations of 0"5 ml saline injected at 15--48-hr intervals: All solutions were 5°C except for the 37°C series. See legend of Fig. 2 for explanation.

Electrocardiograms Figures 4 and 5 show that electrocardiographic patterns of spontaneous arousals and premature arousals evoked by sound and injection stimuli were similar. The initial electrocardiographic response after stimuli that did not culminate in arousal appears to be no different than that of complete arousal (Fig. 6). DISCUSSION Progressive irritability is characteristic of golden-mantled ground squirrels that are exhibiting the winter pattern of hibernating behavior. It is suggested that a correlation exists between the time-temperature relationship of the duration of the hibernating period (Twente & Twente, 1965a) and a changing physiological state

472

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AND J ~ z

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throughout the hibernating period which results in progressive irritability and culminates in spontaneous arousal, providing the animal is undisturbed. Since these ground squirrels behave similarly in response to sound and injection stimuli in regard to evoked arousals, it appears that the central nervous system is involved in changes in irritability. On the basis of the similarity of electrocardiographic patterns at the initiation of arousal in response to sound or injection stimuli and the pattern of spontaneous arousal, it would seem that the mechanisms of spontaneous arousal and induced arousal are identical in nature. While the nervous input is obviously different, the nervous output and response appear to be the same. T h e immediate increase in heart rate after injection or sound stimuli, as well as the electrocardiographic pattern at the initiation of spontaneous arousal, implicates the sympathetic nervous system as a controlling mechanism of the arousal process itself, as indicated by Lyman & O'Brien (1963, 1964). The data suggest, however, that the initiation of arousal is under central nervous system control. It does not seem unreasonable that progressive irritability during the hibernating period and the culminating, internally triggered, spontaneous arousal are the result of the same metabolic phenomenon and its effect upon the central nervous system. The nature of this change is suggested in that different concentrations of epinephrine administered early in the hibernating period produce a graded response. A large dosage (50/~g) evokes arousal at the beginning of the hibernating period (0-5 per cent), whereas 2.5 #g does not evoke arousal until approximately 30 per cent of the hibernating period has elapsed (Twente & Twente, 1968). We have determined that our earlier interpretation of premature arousals in response to gradually elevated environmental temperatures as a measure of progressive irritability (Twente & Twente, 1965b) was probably erroneous. Integration of the time the animals spent at each temperature level before the initiation of arousal showed that they were probably passively responding to the time-temperature relationship. T h e arousals were not, therefore, premature. Kristoffersson & Soivio (1964b) reported that hibernating hedgehogs subjected to elevated environmental temperatures were induced to arouse in successive order depending upon the length of time the animals had been in hibernation. They concluded that hibernation became shallower as the end of the hibernating period is approached. It is not unlikely that their animals were similarly responding to the time-temperature relationship. These data relating to injections indicate that pharmacological agents may be intraperitoneally administered in 0.5 ml isotonic saline without inducing premature arousal by the vehicle or injection. For such studies it is necessary that the animal exhibit the winter pattern of hibernating behavior and that the injection be given before 50 per cent of the expected hibernating period has clasped. The temperature of the solution is not critical nor is, for most individuals, the tonicity within the ranges studied. Ground squirrels that exhibit the adaptive autumn or spring patterns of hibernating behavior or show changed hibernating patterns resulting from handling

FIG. 4.

(Caption

overleaf.)

. . . . . . .

FIG. 5.

(Caption

overleaf.)

FIG. 6. FIGS. 4, 5 and 6. Electrocardiographic patterns of hibernating ground squirrels 2 min before and 8 min after stimuli (arrows). A and B = spontaneous initiation of complete arousal; C = noise-evoked initiation of complete arousal; D = injection-evoked initiation of complete arousal; E = noise stimulus, no arousal, no change in body temperature (animal in the last stage of induction); F = injection stimulus, no arousal, no change in body temperature. The heart rate and body temperature are plotted 10 min before and 30 min after the stimulus. The segment represented by the electrocardiograms is indicated beneath each plot.

PROGRESSIVE IRRITABILITY OF HIBERNATING CITELLUS LATERMLIS

473

during periods of activity between hibernating periods (Twente & Twente, 1967) are much more susceptible to stimuli than animals exhibiting the winter pattern of hibernating behavior. Injections of saline administered during these phases, even when given within a few hours after the animal has finished induction, always evoke partial or complete arousal, the latter being more frequent. On the basis of the findings reported here, it would appear that the published results of the arousing effects of substances intraperitoneally injected into hibernating mammals should be critically re-evaluated. SUMMARY Hibernating golden-mantled ground squirrels (Citellus lateralis), exhibiting the winter pattern of hibernating periods of relatively constant durations, became increasingly sensitive to external stimuli as hibernating periods progressed. Progressive irritability, as determined by evoked arousal in response to intraperitoneal injection of saline, was demonstrated. Studies of the laboratory population indicate: I. Premature arousal is not evoked by 0.5 ml isotonic saline if administered before 50 per cent of the expected hibernating period has elapsed. 2. After 50 per cent of the hibernating period has elapsed, the number of animals arousing in response to injection increases, the greatest percentage arousing at the 90-99 per cent level. 3. Animals are less sensitive to sham injections than to 0.5 ml isotonic saline. Studies of series of injections during the same hibernating period indicate: I. T h e greater the volume injected, the earlier in the hibernating period evoked arousal occurs. 2. Arousals are evoked earlier with 0.5 ml hypertonic saline than with 0.5 ml hypotonic saline which, in turn, evokes arousal earlier than 0.5 ml isotonic saline. 3. No greater stimulus to arousal is constituted by 0.5 ml 37°C isotonic saline than by 0.5 ml 5°C isotonic saline. It was suggested that a correlation exists between the time-temperature relationship of the duration of the hibernating period and progressive irritability. Because an increase in heart rate is immediately evident after noise or injection stimuli, it was thought that a centrally initiated and mediated sympathetic discharge was responsible for evoked arousal. T h e similarity of evoked arousal and spontaneous arousal led to the speculation that while the nervous input which triggers the two types of arousal is different, the nervous output and response is the same. AcknowledgementmWe are indebted to Frank E. South for the use of the recording oscillograph. REFERENCES

KRISTOFFERSSONR. & SoIvIO A. (1964a) Hibernation of the hedgehog (Erinaceus europaeus L.). The periodicity of hibernation of undisturbed animals during the winter in a constant ambient temperature. Ann..4cad. Sci. fenn. A, IV, 80, 5-22.

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JOHN W. ~

AND JANETA. T w m ~

I~xsro~,~SSON R. & SoIwo A. (1964b) Hibernation in the hedgehog (Erinac~ europacus L.). Changes of respiratory pattern, heart rate and body temperature in response to gradually decreasing or increasing ambient temperature. Ann. Acad. Sc/. fenn. A, IV, 82, 3-17. LYMAN C. P. & O ' B s n ~ R. C. (1963) Autonomic control of circulation during the hibernating cycle in ground squirrels..~. Physiol. 168, 477-499. L ~ C. P. & O'Bmm¢ R. C. (1964) The effect of some autonomic drugs on Citellus tr/decemh'neatus during the hibernating cycle. Ann. Acad. Sci. fenn. A IV, 71, 314-323. Tw~crs J. W. (1955) Some aspects of habitat selection and other behavior of caverndwelling bats. Ecology 36, 706-732. J. W. & TwJ~T~ J. A. (1965a) Regulation of hibernating periods by temperature. Proc. nat. Acad. Sci. 54, 1058-1061. J. W. & TWSN~ J. A. (1965b) Effects of core temperature upon duration of hibernation of Citellus lateralis. •. appl. Physiol. 20, 411-416. J. W. & Twmcrs J. A. (1966) Progressive irritability of hibernating CiteUus lateralis as determined by injection of physiological saline. Am. Zool. 6, 326. J. W. & TWeN~ J. A. (1967) Seasonal variation in the hibernating behavior of Citellus lateralis. In Proc. 3rd int. ,Syrup. Natur. Mammalian Hibernation (Edited by FIsmm K. C., DAWE A. W., LYdiAN C. P., SCHtNeAUM E. & SOUTH F. E.) Vol. I I I , pp. 47-63. Oliver & Boyd, Edinburgh. TWRNTEJ. W. & T w i T s J. A. (1968) Effects of epinephrine upon progressive irritability of hibernating Citellus lateralis. Comp. Biochem. Physiol. 25, 475-483.