Palmer, Dennis L., and M. L. Riedesel. L976.

Responses of whole-animal and isolated heart of ground squirrels' Cilgfrye lateralis, to melatonin. C_omp. Biochem. Physiol. 53C: 69-72.
Canp. Bio.heDt. Physirt.. 1976, Vol. 53C.
t1t1.

69 to 12. Pergamott Press

Prited in

Gredt Btitaitl

RESPONSES OF WHOLE-ANIMAL AND ISOLATED HEARTS OF GROUND SQUIRRELS, CITELLUS LATERAT,IS

TO MELATONIN1
DrNNrs L.

Parwn aNl M. L. Rrprpsel

Department of Biology, University of New Mexico, Albuquerque, 87131, U.S.A.

(Receir:ed 14 August 1975) 1. The pineal hormone, melatonin, has been demonstrated to influence hibernation in the gronnd squilrel, C. lateralis. Daily subcutaneous iniections of a commercial preparation of melatonin resulted in an increase in the incidence and duration of hibernation. 2. Isolated hearts from hibernating and non-hibernating ground squirrels had a depressed duration of electrical aotivity when melatonin was present in the bathing solution.

Abstract

INTRODUCTION

IN pnrpanarroN for the hibernating state, the mammalian hibernator becomes increasingly obese during the late summer months with the deposition of both
the Chatfleld, 1955). The pineal hormone, melatonin (5-methovaries and testes before hibernation (Lyman

to a reduction in the number of impulses sent out from the pacemaker (Dawe & Morrison, 1955). This
infers that the decrease in rate is attributable to biochemical effects on cardiac tissue or pacemaker. Therefore, the objective of the second portion of this study was to determine if melatonin had a direct effect on the heart.

brown and white fat. There is also atrophy

ol

&

oxy N-acetyltryptamine), has been demonstrated to stimulate the growth of brown adipose tissrre within the Djungarian hamster (Heldmaier & Hoffman, 1914).It also has been described to produce antigonadal effects within rodents (Wurtman et al., 1963). Spafford & Pengelley (1971) reported an influence of serotonin, the metabolic precursor to melatonin, on hibernating golden-mantied ground squirrels,
Citellus lateralis. Their results demonstrate that whole-

MATERIALS AND METHODS:

brain serotonin levels decrease during the entrance

into hibelnation and that inhibition of serotonin synthesis disrupted hibernation.

The metabolic conversion of serotonin into melatonin is a light dependent mechanism with the highest activity of the melatonin forming enzyme coming in darkness (Ax1erod, 1974). Within C. lateralis, Pengel1ey (1969) observed that light does play a role in the period of hibernation. In view of these reports, the purpose of this study was to determine if melatonin had an influence on hibernation within the goldenmantled ground squirrel. Our results from administering a commercial preparation of melatonin into C. lateralis indicate that melatonin does have an influence on hibernation. These results in turn posed the question of what was the physiological role of melatonin within the ground
squirrel.

The golden-mantled ground squirrels, C. Iateralis, werc trapped in the area of Fenton Lake, New Mexico. Animals were caged individually with food and water ad lib. The body weights of the animals selected for the study ranged from 200 Io 324 g. The hibernaculum temperature was 5 + 2"C with a daily 10 L and 14 D photoperiod. Animals were provided with burlap as a nesting material. Melatonin was obtained from Sigma Biochemicals, St. Louis, Missouri and Nutritional Biochemicals, Cleveland,

Ohio. Each commercial preparation of melatonin

was assayed for its relative biological effectiveness by observing

the degree of compensatory ovarian hypertrophy within

lab mice. Crystalline melatonin was stored frozen until the soiutions were to be prepared. Fresh solutions were prepared weekly and refrigerated, 5"C. Melatonin, either 12'5 or 25 0 mg, was dissolved in 0'3 ml absolute ethyl alcohol and diluted to l0 m1 with 0'9?4 NaCl. The study of the response of whole animals to melatonin consisted of two continuous phases which involved four groups of nine animals per group. In the first phase the animals of groups A and B were administered injections of physiological saline. The experimental animals of groups C and D were given injections equalling 100 and
500 pg of melatonin per day respectively. The second phase was a reversal ofthe first phase in which the ground squirrels of groups A and B were administered dosages of 100 and 500 pg of melatonin per day respectively and those

A dramatic slowing ol the heart rate is one of the first physiological changes identified with the onset of hibernation (Dawe & Morrison, 1955). It is

believed that the hibernating heart is devoid of influences of vagal tone (Dawe & Landau, 1960) but

that the slow rate in hibernation is due primarily

I Taken in part from a thesis submitted in partial fu1fiilment of the M.S. degree. Universitl' of New Mexico.
69

animals of groups D and C received injections of physiological saline. The ground squirrels were observed twice daily (7 00 a.m. & 5 00 p.m.) at which time skin temperature measurements were made with a copper constantan thermocouple probe and a Leeds Northrup Potentiometer. Each animal was administered two subcutaneous injections daily iust after the observations for hibernation. Each phase continued for 10 days.

70

DrNNrs L.

Pelwn aNo M. L. Rrror:sr

Table 2. Duration of hibernation period (24 hr). p values represent differences in the length of the hibernation period for individual animals in the two phases
6roup
SaI ine

Skin temperature was the criterion for determining whether a ground squirrel was hibernating or active. Animals with skin temperature below 10"C were considered to be hibernating. Hibernating animals disturbed regularly require several days to become habituated to regular handling and injections (Pengelley & Fisher, 1967). Once habitllated most animals do not arouse following injections, however, bouts of hibernation less than 24 consecutive hours were not included in the statistical analvsis. The data on the frequency and duration ol the hibernation periods were submitted to statistical analysis in accordance with methods discussed by Simpson et al. (1960). The experimental animals in the study of isolated hearts consisted of two groups: summer-active and winter-hibernating ground squirrels. The animals were sacrificed by decapitation and the heart was immediately excised and bathed in two consecutive washes of cotd safine preparation (7 + 2'C) for 3 min. The heart was then placed
one on an auricle and one on a ventricle. Experimental hearts had melatonin added to the fina1 saline bath (46 x l0 4 M). Electrical activity was recorded continuously on a Narco DMP 44 Physiograph. Total

Inj ections
3.70L
0.84
11
0

Me1

atonin Inj ect ions

4.13 0,71
11

0.01

2.50 .29 1t

3.25
0.44
11

0.02

2.94 0.22
15

3.60 0.33
15
3 0

0.01

2.44 0.15
15

.93 .36
1.5

0. 001

into a final saline bath with two graphite eleclrodes arranged to make contact with the surface of the heart,

1.

x
N

s.E.

a significant increase in the duration of hibernation

when injected with melatonin. The results of this experiment indicate that melatonin did have an influence on hibernation within the golden-mantled ground squirrel, C. lateralis.

lated hearts continued to show activity were recorded. After t hr of having no activity the hearts were considered
cal11 stimulate them.

number of electrical pulses and the length of time the iso-

to be terminated and no attempt was made to mechani-

SULTS

tion containing melatonin

of isolated hearts to melatonin. From the two groups of experimental animals, i.e. summer-active and winter-hibernating ground squirrels, isolated hearts bathed in a standard saline soluResponse

(4.6

Response of ground squirrels

to cold and melatonin

In the first phase, two groups of ground squirrels were administered physiological saline injections
while the other two groups received 100 and 500 pg of melatonin respectively as a daily dosage. In the second phase the dosage given to the groups was
reversed, i.e. the two groups of ground squirrels which

had initially received the saline injections were given melatonin injections and those two groups which had been administered the melatonin in the first phase were given the saline injections. The two phases were continuous with the duration of each phase being 10
days.

resulted in a lower total number of pulses recorded and a shorter duration of electricai activity than the control isolated hearts (Figs. i & 2). Within the summer-active animals the mean values for the total number of pulses recorded were 576 for the control animals and 253 for the experimental ground squirrels. Totai duration of organized activity ranged from 122 to 188 min for the controls and 80 88 min for the isolated hearts bathed in the saline preparation containing melatonin. Winter-hibernating ground squirrels ranged from 738 to 1335 in the total number
HIBERNATING JANUARY 196

x 10-aM)

always

The incidence of hibernation bouts (Table 1) increased in all groups when melatonin was injected daily. Whereas there was no difference in the mean duration of hibernation (Table 2), each animal had
Table
6raup

N:4 e

btqalls

l.

Average number ofhibernations observed per day

Sa1

& U G)
H

o o
UJ

ine Inj ections

Melatonin 6.9 0.70

9

lnl ectlons
0.01

+.al
9

o_

a u l

0.s0

0.37
9

6.3
0.77
9

0.001

o t. ul
co

l.!

f,
0.05

0-70

I
9

!!'1 .7
9
'J.2

z
J

.02
Z

5.5 0.69

p
0. 001

.
9

0.94

1. f

coNTRq_

S,E.

rcLAloilrL 48xld4m

@arTRoL tLAroiltt{ 4.6xlo-4 n

Fig. 1. Total number ofpulses recorded for isolated hearts

Responses of whole-animal and isolated hearts of ground squirrels

HIEERI\IATING c. /otffi//s JANUARY

7l

ACTIVE

JUNE 1974
(h

s?s,l,t{"llTf*

trl

z
=
F F

strated this not to be the case for isolated guinea pig and rat hearts periused with melatonin at a concentration of 10 a M. Studies conducted to date within our laboratory on perfused ground squirrel hearts confirm their report of no response. The

ola response to melatonin by periused hearts whereas non-perfused hearts do respond, presumably indicates melatonin is most effective in a deteriorating
absence heart.

?
o L!
N

z
CD

E.

o b

When the data obtained for two groups were compared both the duration of electrical activity and the total number ol' pulses recorded were higher within the winter-hibernating animals than in the summeractive animals. These data support the hypothesis

z o

:) o

Fig. 2. Duration of pulses recorded for isolated hearts

of pulses recorded for the controls and 370 538 for the experimental animals. The mean values for the duration of organized activity were 197 min for the control ground squirrels and 96 for the experimental
groups.
DISCUSSION

The pineal has been reported to participate in the regulation of seasonal cycles (Reiter, 1973). In mammals, melatonin is produced in the pineai gland and

(Dawe & Landau, 1960) that the mammalian hibernating heart has undergone some physiological transition(s) which enables it to have a longer survival time than the heart from the active animal. It is interesting to speculate as to the relationship between melatonin and the hibernation "trigger" described by Dawe & Spurrier (1974). The action ol melatonin in this seasonal hibernator, C- lateralis, is very different from the action of the blood borne trigger. Both compounds change the activity of isolated hearts but the trigger increased whereas melatonin decreased the number of spontaneous heartbeats. Melatonin may either be responsible for changing the receptiveness of target tissue to the trigger or associated with the seasonal changes in the amount of trigger which is present in the blood. Most certainly there needs to be additional information before it is possible to relate these two systems within any
reasonable doubt.

the activity of

hydroxyindole-O-methyltansferase
step

(HIOMT), the enzyme responsible for the last

in melatonin formation, has been reported to be light dependent with high activity in darkness and 1ow activity in light (Wurtman et al., 1968). Environmentai lighting messages reach the pineal via the retina of
the mammalian eye (Axlerod,1974). Pengelley (1969) noted light does play a role in the period of hibernation in C. lateralis as evidenced by experiments involving enucleation. Hoffman and his colleagues (1965) reported that in the golden hamster there is an increase of brown adipose tissue (BAT) weight following exposure to short photoperiods. There was a drastic increase of BAT mass in hamsters treated with melatonin which indicates regulation of the growth of BAT involves the pineal and increased BAT is not simply a consequence ol cold exposure (Heldmaier & Hoffman,
1974\.

The data collected in these whole-animal and isolated heart experiments support the hypothesis that melatonin has a role in the preparation for and maintenance of hibernation within the golden-mantled ground squirrel, C. lateralis.
REFERENCES

Axr.rnoo J. (1974) The Pineal Gland: A Neurochemical Transducer. Science. N.I 184. 1341-1348. Brrncnas J., D.q.Cosrn F. & SpEcron S. (1967) Acute pharmacology of melatonin. Nature, Lond. 214,919 920. Dl,ws A. R.& LlNor,u B. R. (1960) The hibernating mammalian heart. Am. Heart J. 59, 78-89. D,qwE A. R. & MonnrsoN P. R. (1955) Characteristics of
the hibernating hearl. Ant. Heart J. 49,361*389. & Spunnnn W. A. (1974) Summer hibernation of infant (six-week old) l3Jined ground squirrels. Citellus tridecemlineatus. Cryobiology. l1(1), 33 43. HsroMAlen G. & HoFpnr.tr K. (1974) Melatonin stimulates growth of brown adipose tissue. l{atare, Lond. 247,
D,q.wr A. R.
t1/ 11<

of

These previous reports along with the results presented in this study support the concept that melatonin may be involved in the onset and maintenance

HoFFMAN R. A., Hpsren R. J.

&

TowNs C. (1965) Effects

Waterhouse.

of light and temperature on the endocrine system of the

hibernation. The physiological mechanisms by which this pineal indoleamine could influence hibernation are unknown. Our results from the isolated heart experiment indicate the heart may be an important melatonin target tissue in the mammalian hibernator. Because many
compounds which prolong the effects of hypnotics are vasoactive agents, Barchas et al., (1967) speculated that melatonin would alter the inotropic and chronotropic actions ol isolated hearts. Their resuits demon-

golden hamster, Mesocricetus auratus

Comp. Biochenl Pltysiol.15, 525 533.

& Cnerpnro P. O. (1955) Physiology of hibernation in mammals. Physiol. Reu. 35, 403 425. PsNcuLrrv R. T. (1969) Influence of light on hibernation in the Mojave Ground Squirrel, Citellus mohauensis. In P hy siolo qical Sys/enrs in S e miar id E nuir onmen t s. (Ediled by Horr C. C. & RrroEssr- M.L.), pp. 11 16. University of New Mexico Press, New Mexico.
LvlreN C. P.
ground squirrels, Citellus lateralis, to be conditioned to stimuli while in htbernation. J. Mammal. 49, 56I-564.

72

DsNNrs

L.

Pllwn rNo M. L. RrElEsEi.

den-mantled ground squirrel, Citellus lateralis. Comp. Biochem. Physiol. 38A, 239 249. Wr;nrurN R. J.. Axrrnoo J. & Cnu E. W. (1963) Melatonin, a pineal substance effect on the rat ovary. Science, r{.}a r4l. 217 218. Wunru,rN R. J., Axrrnoo J. & Krr-rv D. E. (1968) Ifte Pineal. Academic Press^ New York.

RElrr:n R. J. (1973) Edocrine aspects ol the mammalian pineal gland. A. Reu. Physiol. 35, 305 328. StrrapsoN G., Ror, A. & LtwoNrrN R. (1960) Quantitatirje Zoology. Harcourt, Brace World, New York. Spllrono D. C. & PlNcErLpy E. T. (1971) The influcnce of the neurohumor serotonin on hibernation in the gol-