Effects of epinephrine upon progressive irritability of hibernating Citellus lateralis

Comp. Biochem. Physiol., 1968, Vol. 25, pp. 475 to 483. PergamonPress. Printed in Great Britain

EFFECTS OF EPINEPHRINE UPON PROGRESSIVE IRRITABILITY OF HIBERNATING CITELLU8 LATERALIS* 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)

Abstract--1. Intraperitoneal injections of epinephrine to hibernating Citellus latera[is show that progressively smaller dosages are required to evoke arousal

as the hibernating period progresses. Fifty/zg cause arousal at the beginning of the hibernating period whereas 2"5/zg do not evoke arousal until approximately 30 per cent of the hibernating period has elapsed. The dose-response relationship is linear. 2. Dosages which do not evoke complete arousal result in partial arousal. 3. Dosages up to 20/*g of norepinephrine cause neither calorigenesis nor premature arousal. INTRODUCTION A CORRELATIONappears to exist between the time-temperature relationship of the duration of hibernating periods of golden-mantled ground squirrels (CiteUu, lateralls) (Twente & Twente, 1965a) and a changing state of irritability in response to external stimuli as the hibernating period progresses (Twente & Twente, 1965b, 1968). We have designated this phenomenon as "progressive irritability" (Twente & Twente, 1966, 1968). Hibernating C. lateralis intraperitoneally injected with 0.5 ml isotonic saline are not evoked to arouse if the injection is administered before 50 per cent of the expected hibernating period has elapsed. After 50 per cent the percentage of animals that arouses in response to injection stimulus increases as the hibernating period approaches the terminal stage (Twente & Twente, 1968). Animals at this time, if left undisturbed, appear to arouse spontaneously, apparently as the result of an internally triggered, arousal mechanism. Because an increase in heart rate is immediately evident after sound or injection stimuli, it was thought that a centrally initiated and mediated sympathetic discharge was responsible for evoked arousal (Twente & Twente, 1968). A seemingly identical pattern, apparently independent of external stimuli, characterizes the beginning of spontaneous arousal. This similarity between evoked and spontaneous arousal led us to postulate that while the nervous input which triggers arousal may be different, the nervous output and response are probably the same (Twente & Twente, 1968). * This work was supported in part by USPHS grant AM-11320. 475

476

JOHN W. ~

A~roJANETA. Tw-~,~TE

Evidence indicating sympathetic control of the arousal process itself has been presented (Lyman & O'Brien, 1963, 1964), but the triggering mechanism has yet to be elucidated. Epinephrine was originally administered to determine whether substances injected intraperitoneally entered the circulation. Seemingly, contradictory results relating to the dosage effects upon arousal patterns led us to the experiments reported. METHODS Citellus lateralis were caged singly and maintained, undisturbed, at 4, 5, and 6°C.

Average durations of hibernating periods of thirty-one animals exhibiting the winter pattern of hibernating periods of relatively constant duration (Twente & Twente, 1967a) were calculated on the basis of four or more hibernating periods for each ground squirrel. These averages were characteristically variable ranging from 140 to 238 hr. Teflon-coated, iron-constantan, permanently implanted thermocouples (Twente & Twente, 1965b) yielded records of body temperature. Records were printed at 6-rain intervals by Leeds and Northrup Speedomax G 16-point recording potentiometers calibrated from 0 to 50°C. Electrocardiograms were obtained with a recording oscillograph (Grass Model 7) using standard Limb Lead II (right fore leg, left hind leg) needle electrode placements. Since injections after 50 per cent of the expected hibernating period may evoke premature arousal, injections were performed only during the first 40 per cent of the hibernating period. Epinephrine was administered in 0"5 ml, 0.9%, pyrogen-free saline and intraperitoneally injected with No. 26, 9 mm needles. Response to injection was determined through recordings of body temperature which indicated the time of response and whether partial or complete arousal occurred. Animals were weighed only in the autumn prior to being placed in the cold rooms and again in late winter or spring after removal from the cold rooms. This procedure was necessary because animals handled during hibernating periods are frequently evoked to amuse prematurely; animals handled during periods of activity between hibernating periods exhibit changed patterns of hibernating behavior (Twente & Twe~te, 1967a). None were weighed prior to injection and the dosages were administered on a per animal basis. Extrapolated weights of the thirty-one animals at the time of injection averaged 202 g. A partial arousal was defined as any elevation of body temperature which is maintained for several hours and does not culminate in the rapid rise to at least 36°C. The latter is characteristic of complete arousal. RESULTS Figure 1 shows that complete arousals were evoked earlier in the hibernating period with the higher dosages. Note in Fig. 1 that a boundary can be seen which separates partial and complete arousals and describes a linear dose-response relationship over a wide range of dosages. All arousals shown in Fig. 1, however, were evoked considerably before 50 per cent of the hibernating period had elapsed. Whether or not complete arousal would be evoked by any dosage could be predicted within several hours by referring to the dose-response line (Fig. 1). This predictability was consistent despite the variations between animals in estimated weight and average duration of hibernating periods. Injections of saline never resulted in partial arousals when administered to animals exhibiting the winter pattern of hibernating behavior. Except for the 2.5/~g/animal dosage which elicited no response in four of fifteen animals injected

~CTS

OF EPINEPHRINE U P O N PROGRESSIVE IRRITABILITY

477

(Fig. 2), partial arousals of 0.5-11.0°C above the hibernating temperature resulted in response to all of the injections of epinephrine that did not evoke complete arousal. These temporary increases in body temperature appear to be related to dosage and are considered to be peripherally induced. An average increase of 0.9°C (0.3- 1.8°C) was shown by the eleven animals that exhibited partial arousals in

SINGLE INJECTIONS

3o

4

~t

COMPLETE AROUSAL • NO COMPLETE AROUSAL

20

, •4~

SERIES INJECTIONS TO O - - 7 2 1 d COMPLETE AROUSAL • NO COMPLETE AROUSAL

iv

,c i l l •

,,i, 4 1

~

4

Lu

,L

2.5 " • • •

ee I 10

I 20

I 30

I 40

,I 50

DURATION OF HIBERNATION EXPRESSED AS PERCENTOF EXPECTEDTOTAL

FIG. 1. Dose-response relationships to injections of epinephrine. Symbols with arrows represent complete arousals. Symbols without arrows represent partial arousals or no response. Thirty-one animals were used for single-injection studies. Data obtained from a single injection (10 pg) to No. 0-721 and series of injections during each of two hibernating periods of 5 pg and 2"5 pg to the same animal are indicated by triangles. response to the 2.5/~g/animal dosage. An average increase of 4.8°C (0"6-11.0°C) was recorded from the thirteen animals receiving the 10 pg/animal dosage (Fig. 2). No correlation between the degree of elevation of body temperature during partial arousal and the elapsed fraction of the hibernating period when injections were administered could be discerned. The characteristic, electrocardiographic pattern after a control injection of saline which did not evoke arousal and a subsequent injection of 5 pg/animal dosage of epinephrine which evoked a partial arousal (Fig. 3) shows that the increase in heart rate after saline was transitory, but after epinephrine a gradual, prolonged increase occurred. The increase in body temperature was not evident, ,6

478

JOHN W. ~

~

JANgr A.

however, until approximately 50 min after injection. These data suggest that a quantity of epinephrine reaches the circulation shortly after injection, but the calorigenic effect requires either greater concentration or time for mediation. The EPINEPHRINE u°

-

ua u~

~

<

~

--4

~1-

~

O

~

10-

a

= 30/Jg

•

=20~g

'~

= 10 pg

~

= 2.5 Pg

~

I

,~ •

5-

~ ~' ~

"

-r

0

I

I

10o'/o

|

~

20=/o

30%

DURATION OF HIBERNATION EXPRESSED AS PERCENT OF EXPECTED TOTAL PARTIAL AROUSALS OCCUR LEFT, FULL AROUSALS OCCUR TO RIGHT OF EPINEPHRINE DOSAGE AS INDICATED. FzG. 2.

Change in body temperature during pe_rtid arousal in response to

different dosages of epinephrine administered at diiferent elapsed percentages of the expected hibernating periods. Symbols with arrows at the bottom of the plot represent the point on the dose-response curve after which cornp]ete arousals are always evoked (see Fig. 1).

latter seems to be more likely because the 50 and 100 pg/animal dosages required as long a time to produce calorigenic effects as the 5/~g/animal dosage (Fig. 4). Evoked arousals of animals after control injections of saline occurred after 50 per cent of the expected hibernating period had elapsed and were apparent within 18 min. Increases in body temperature indicative of partial or complete arousal after epinephrine were not initiated until 30-120 min after injection (Fig. 4). Characteristic effects of different concentrations of epinephrine as illustrated by arousal patterns of one animal during four successive hibernating periods (Fig. 5) indicate that the dose-response relationship applies even when injections are administered in series. The pattern of progressive irritability, therefore, was not altered by previous injections during the same hibernating period. The progressive increase in the height of the partial arousal curves after successive injections during the same hibernating period (Fig. 5), however, appears to be the result of residual epinephrine from the previous injections. The lack of

FIG. 3. Continuous heart rate and body temperature records are shown for 670 min. The animal was recorded at the termination of induction into hibernation. Segments represented by electrocardiograms prior to and after injections (arrows) of saline and 5 pg epinephrine are indicated beneath each plot.

EFFECTS OF EPINEPHRINE UPON PROGRESSIVEIRRITABILITY

479

response to the third injection during the third hibernating period (Fig. 5) was attributed to injection into the gut or mesenteric fat. This, coupled with the absence of partial arousals in response to saline, indicates that partial arousals are SALINE *

EPINEPHRINE

= 0.5 mi

.o.. :

o

= 100~g

•

=

a

= 30 . g

•

=20#g

50 ~g

~,

= 10 #g

•

= 5 #g

I~"

= 2.5 , g

.o

°

°

0 e..

•>r~

O •

A

-r U

3O ELASIPEDTIME BETWEEN INJECTION AND DETECTABLE RESPONSE (minutes]

FIG. 4. T i m e elapsed between injection and initial temperature rise indicative of partial and complete arousals in response to injections of saline and different dosages of epinephrine. The three groups of complete arousals in response to saline represent initial elevations in body temperature (1) before 6 rain; (2) 6-12 rain; and (3) 12-18 rain after injection, respectively.

peripherally, rather than centrally, induced. We consider that partial arousals may be the result of direct or indirect stimulation of the brown adipose tissue. Except for the series experiments, the animals injected with epinephrine which did not arouse completely were allowed to complete their hibernating periods without further disturbance. Injections of epinephrine that did not evoke complete arousal did not change the duration of the hibernating period. It appears that concentrations of circulating epinephrine which do not evoke complete arousal do not alter the normal pattern of progressive irritability and spontaneous arousal. Five additional animals were selected for a preliminary study of the effects of norepinephrine upon progressive irritability. Results of 10 and 20 pg[animal

480

JoHN W. Twmcrs AND J,~,,'BT A. TWBN~

dosages administered in 0.5 ml isotonic saline were essentiaUy negative (Table 1); no partial or premature arousals were evoked. Significantly, these relatively large dosages did not affect the pattern of irritability even after 40 per cent of the expected hibernating period had elapsed. Series of injections during the same

_

10 •

I

10/

EPINEPHRINE INJECTION TO O--721 cf

50

E

10~

2.<

5e

10o

2.5#g

o

2.Swg

2.5~g

2.Sxg

2.

+

5o

2.5~g

2.Sxg

$o 0

i 0

I 10

i

I 20

i

I 30

I

I ,Lo

i

I 50

i

I 6o

i

I 70

i

2,iJ

I 80

~

I 90

D U R A T I O N O F HIBERNATION IN HOURS

I O%

I

I

I

I

I

I

I

D U R A T I O N O F HIBERNATION EXPRESSED AS PERCENT OF EXPECTED TOTAL

FIG. 5. Effects of injections of epinephrine administered to the same animal during four consecutive hibernating periods. Body and environmental temperatures were 4°C. TABLE 1--INACTIONS

OF NOREPINEPHRINE TO HIBERNATING GROUND SQUIRRELS

(BODY AND ENVIRONMENTAL TEMPERATURES 5 ° C ) W H I C H EVOKED NO CHANGES I N BODY TEMPERATURE

Animal No.

Dosage (pg)

0-601 ~ 0-234 ~ 0--418 c~ 0-16 ~ 0--476 ~

10 10 10 20 20

% of elapsed hibernating period at time of injection 46 12 11 20 8

37 23

48* 35

49

62*

21

29

41

53"

* Injections administered in series during the same hibernating period. hibernating period similarly caused no calorigenesis. Increases in heart rate after norepinephrine, however, showed the same time lag and were apparently identical to those induced by epinephrine. The absence of calorigenesis in response to

I

40~

EFFECTS OF EPINEPHRINE U P O N PROGRESSIVE IRRITABILITY

481

norepinephrine supports a logical contention that mechanisms of cold adaptation are not applicable to hibernating ground squirrels. DISCUSSION 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, have been considered to be physiologically comparable (Twente & Twente, 1967b, 1968). The predicted results relating to evoked arousal in response to epinephrine were consistent despite the variation of the average durations of the hibernating periods of the animals used. These data lend confidence to the normalizing procedure employed. It seems unlikely that the glycolytic effects of epinephrine are involved in the shifted progressive irritability curve. Concentrations of glucose in the blood are no different at the beginning than at the end of the normal, hibernating period (Twente & Twente, 1967b). Drask6czy & Lyman (1967) have shown that the turnover of epinephrine and norepinephrine from tritium-labeled Dopa ceases in the brain of C. tridecemlineatus at the beginning of induction into hibernation. They concluded that possibly the complete lack of activity of adrenergic neurons of the brain is the cause of induction into hibernation. Weil-Malherbe et al. (1961) elegantly demonstrated that epinephrine cannot effectively cross the blood-brain barrier of cats except in the region of the hypothalamus. Although the hypothalamus has not yet been identified as the regulator of the duration of hibernating periods and arousal from hibernation, its thermoregulatory nature (Hardy, 1961) and role in sleep and wakefulness (Herrfindez Pe6n, 1965) would seem to indicate involvement. Rothballer (1959) postulated central, adrenoceptive mechanisms, possibly within the hypothalamus, which are more sensitive to epinephrine than to norepinephrine. The duration of each normal hibernating period appears to be limited by the development of a progressive irritability in the central nervous system which culminates in the triggering of spontaneous arousal. Our data indicate that the progressive irritability curve is shifted towards the beginning of the hibernating period by epinephrine but not by norepinephrine. Since the duration of the hibernating period is regulated by temperature and may be the result of a single, metabolic process (Twente & Twente, 1965a), it does not seem unreasonable that spontaneous arousal may be initiated by the accumulation of epinephrine, probably at the level of the hypothalamus. SUMMARY Injections to hibernating Citellus laterah's exhibking the winter pattern of hibernating behavior indicated the following: 1. The progressive irritability curve was shifted towards the beginning of the hibernating period by epinephrine. A linear dose-response relationship was

482

JOHN W. TW~Tn ANDJANETA. TWITS

demonstrated; animals were evoked to arouse progressively earlier with increasing dosages. Dosages necessary to elicit arousal were predictable after specific percentages of the expected hibernating periods had elapsed. 2. Animals with different durations of hibernating periods behaved similarly in respect to the time of the predicted, evoked arousal in response to epinephrine. The normalizing process of equating average durations of hibernating periods as constituting 100 per cent and considering fractions thereof as being physiologically comparable was therefore considered valid. 3. Animals that did not arouse completely in response to epinephrine usually showed partial arousals. The height of the partial arousal curves was a function of dosage and thought to be peripherally induced. 4. Complete arousals were initiated within 18 rain after injections of saline; 30120 min were required before the initiation of complete arousal with epinephrine. 5. Injections of epinephrine that did not evoke complete arousal had no effect upon the duration of the hibernating period. 6. Arousals were not evoked earlier in the hibernating period than predicted as a result of the administration of series of injections during the same hibernating period. The pattern of progressive irritability was therefore unaffected. 7. Simultaneous recordings of body temperature and electrocardiograms indicated that the heart rate increased immediately and then subsided after a control injection of saline; a subsequent injection of epinephrine caused a gradual and prolonged increase in heart rate. Calorigenesis, however, was delayed. 8. Neither calorigenesis nor irritability resulted from 10- and 20-/~g dosages of norepinephrine administered singly and in series. Arousals were not evoked even after 40 per cent of the expected hibernating period had elapsed. It was thought that spontaneous arousal may be initiated by the accumulation of epinephrine, probably at the level of the hypothalamus. Achnowledgement~We are indebted to Frank E. South for the use of the recording

oscillograph. REFERENCES DI~.SK6CZYP. R. & LYM.~ C. P. (1967) Turnover of catecholamines in active and hibernating ground squirrels. ~. Pharmac. exp. Ther. 155, 101-111. t-D,maY J. D. (1961) Physiology of temperature regulation. Physiol. Rev. 41, 521-606. HSRNg~DEZPS6N R. (1965) Central neuro-humoral transmission in sleep and wakefulness. In Sleep Mechanisms, Progress in Brain Research (Edited by AmmT K., BALLYC. & SCHAD~J. P.) Vol. 18, pp. 96-117. Elsevier, Amsterdam. LYMANC. P. & O'BRmN R. C. (1963) Autonomic control of circulation during the hibernating cycle in ground squirrels. ~. Physiol. 168, 477-499. LYMAN C. P. & O'BI~mN R. C. (1964) The effect of some autonomic drugs on Citellus tridecendineatus during the hibernating cycle. Ann. Acad. Sd. fenn. A, IV, 71, 314-323. ROTH~ALLeaA. B. (1959) The effects of catecholamines on the central nervous system. Pharmac. Rev. 11, 494-547. TW~TE J. W. & Tw-mcrB J. A. (1965a) Regulation of hibernating periods by temperature. Proc. nat. Acad. Sci. 54, 1058-1061.

~ ¥ ~ C T S OF EPINEPHRINE UPON PROGRESSIVE IRRITABILITY

483

J. W. & ~ J. A. (1965b) Effects of core temperature upon duration of hibernation of Citellus lateralis. 3. appl. Physiol. 20, 411-416. Tw~rrR J. W. & ~ J. A. (1966) Progressive irritability of hibernating Citellus lateralis as determined by injection of physiological saline. Am. Zool. 6, 326. J. W. & ~ J. A. (1967a) Seasonal variation in the hibernating behavior of CiteUus lateralis. In Mammalian Hibernation (Edited by FXSH~ K. C., DAWE A. W., L Y M ~ C. P., SCHI3NBAUME. & SOUTH F. E.) Vol. III, pp. 47-63. Oliver & Boyd, Edinburgh. J. W. & TW]~TE J. A. (1967b) Concentrations of v-glucose in the blood of CiteUus lateralis after known intervals of hibernating periods..7. Mature. 48, 381-386. Tw]~TB J. W. & TW]~TB J. A. (1968) Progressive irritability of hibernating Citellus lateralis. Comp. Biochem. Physiol. 25, 467-474. WEIL-MALH]3~E H., WHXTBY L. G. & AXELROD J. (1961) The blood-brain barrier for catecholamines in different regions of the brain. In Regional Neurochonistry (Edited by S. S. & ELKESJ.) pp. 284-292. Pergamon Press, Oxford.