The effect of pinealectomy on non-shivering thermogenesis and hibernation of the wyoming ground squirrel, Spermophilus elegans

J. therm. Biol. Vol. 8, No. 4, pp. 321-326, 1983

0306-4565/83 $3.00 + 0.00 Copyright ~ 1983 Pergamon Press Ltd

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THE EFFECT OF PINEALECTOMY ON NON-SHIVERING THERMOGENESIS AND HIBERNATION OF THE WYOMING GROUND SQUIRREL, SPERMOPHILUS ELEGANS H. J. HARLOWand L. M. WALTERS Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, U.S.A.

(Received 11 October 1982; revised 22 March 1983; accepted 18 May 1983) AImraet--1. Pineal gland removal (PX) performed on Wyoming ground squirrels (Spermophilus elegans) during the summer significantly lowered their non-shivering thermogenic (NST) response and impaired their ability to hibernate the subsequent winter. 2. This impairment was not observed in previous studies on squirrels PX'd later in the season. A period of responsiveness for involvement of the pineal gland in NST and hibernation is, therefore, presented. 3. Environmental signals translated by the pineal gland which influence the NST response do not appear to be thermal cues but are more than likely photic information. 4. Reduced impairment of the NST response was observed in squirrels PX'd for 3 yr compared to the 1st PX'd group. However, this may simply be a result of the 3-yr PX'd group being tested later in the hibernation cycle.

Key Word Index--Brown adipose tissue; hibernation; pineal; melatonin; non-shivering thermogenesis; photoperiod; Spermophilus elegans.

INTRODUCTION The pineal gland receives neural signals from the eyes and responds to photic information by increasing the production and release of its primary hormone melatonin during scotophase. As a result of the pineal's and melatonin's ability to modify circannual events such as reproduction (Reiter, 1978; Reiter et aL, 1981), the pineal gland has been implicated as being involved in the hibernation cycle (Reiter, 1981). Previous studies have suggested that pineal gland removal (PX) impairs hibernation (Smit-Vis, 1972) while exogenous melatonin enhances the duration of hibernation by small mammals (Palmer and Riedesel, 1976). These data, however, have met with some opposition. For example, Sinnamon and Pivorun (1981a), unlike Palmer and Riedesel (1976), have been unable to induce consistent changes in the duration of hibernation with exogenouslyadministered melatonin. As a partial explanation of this contradiction, Phillips and Harlow (1982) have found that PX appears to demonstrate a delayed effect on the ability of squirrels to hibernate. When both Wyoming ground squirrels, (Spermophilus eiegans, formerly S. richardsoni) and golden-mantled ground squirrels (S. lateralis) were PX'd in late September no impairment of the hibernation cycle was observed during the subsequent winter by either species (Harlow et al., 1980). But, when PX and sham-PX groups of golden-mantled ground squirrels were maintained under simulated natural photoperiod and temperature through the following winter the PX group exhibited an impaired ability to hibernate (Phillips and Harlow, 1982). A period of influence by the pineal gland during which it participates in programming the animal to its cir-

cannual cycle is, therefore, hypothesized. After this period has been passed the gland's involvement may not be needed for the remainder of that annual cycle. One possible influence of the pineal gland on the hibernation process may be via its stimulatory effect on brown adipose tissue (BAT) deposition in the late summer and early autumn and the consequent non-shivering thermogenic (NST) response during repeated arousals from hibernation. It is generally believed that BAT plays a major role in NST heat production of hibernators, especially during the arousal stage (Jansky, 1973; Wang 1978; Abbotts and Wang, 1980). The animal perceives shortened daylength as summer progresses and this, in turn, stimulates BAT synthesis and deposition in preparation for hibernation. Photoperiod and cold both affect the development of BAT; however, seasonal changes in NST have been observed in animals maintained under short photoperiod but constant thermoneutral temperatures (Heldmaier and Steinlechner, 1981). Short photoperiod, therefore, appears to be a major stimulus for the hypertrophy of BAT in small hibernating or torporous mammals (Lynch, 1973; Heldmaier and Steinlechner, 1981; Hoffman et al., 1965; Joel, 1965). As an important correlate, chronic administration of melatonin (simulating shortened daylength) can induce hypertrophy of BAT both in the Djungarian hamster (Phodopus sungorus) (Heldmaier and Hoffman, 1974) and the 13-lined ground squirrel (Spermophilus tridecemlineatus) (Sinnamon and Pivorun, 1981b). The pineal gland is, therefore, linked to the circannual events of hibernation through melatonin's influence on BAT synthesis in preparation for repeated arousals during the heterothermic phases of hibernation.

321

322

H . J . HARLOW and L. M. WALTERS

This study is designed to establish more precisely the period of activity of the pineal gland in the hibernation process by comparing various hibernation parameters of a late-fall PX'd group of squirrels (previous data) to a summer PX'd group and long-term PX'd (3 yr) group. The other purposes are to: (1) test the hypothesis that PX may hamper the NST response and thereby contribute to an impaired ability of the animal to hibernate; and (2) to determine if the pineal gland is involved in the mechanism of a cold-induced increase in NST capacity.

MATERIALS AND METHODS

Adult Wyoming ground squirrels, Spermophilus elegans, were live-trapped during September of 1979 and August of 1981 in Albany Co., Wyoming (42°00"N, 105°20'W), at approx. 2220m elevation. All squirrels were individually housed in 20 x 25 x 50 cm cages with a wooden nesting platform and bedding material and maintained under natural photoperiod at 23°C. Laboratory rat chow (LabBlox, Wayne Labs), sunflower seeds and water were available ad libitum. Male and female squirrels were randomly divided into 3 groups: intact (I), sham-pinealectomized (sham-PX) and pinealectomized (PX). Pinealectomy was performed within 2 weeks after capture by removing a bone disc (4.0mm dia) overlying the pineal gland (Hoffman and Reiter, 1965), then hooked jeweller's forceps were inserted through a small hole in the meninges at the confluence of the cerebral blood sinuses and the pineal gland and the pineal stalk removed intact. The hole was covered with sterile bone wax and the incision was closed by sutures (Harlow et aL, 1980). Squirrels were considered to be PX'd only if the entire gland was removed, as determined by microscopic examination of the gland suspended in ethanol. Intact animals from 1979 served as a control group as well as sham animals in the 1981 group. Sham-PX were radical in that the cerebral membranes were ruptured and the pineal gland was touched but not removed. Surgeries were completed by 30 September 1979 in the 3-yr PX group and the animals maintained under natural photoperiod with a 7°C winter and 23°C summer temperature regime until the 1982 test period. Surgery was completed on the lst-year PX group by 13 August 1982. Experiment 1: the effect of summer P X on the following winter hibernation patterns Squirrels PX'd and sham PX'd in early August of 1982 were placed in the cold (7°C) with low-intensity lighting (50 Ix) that was periodically altered in duration to simulate the natural photoperiod duration. Periods of torpor and normotherrny were determined by direct observation (see Pengelley and Fisher, 1961), August through to November. All conditions in the present experiment were made to duplicate those conditions in the previous 1979 study on squirrels PX'd in late September and monitored through the subsequent winter (Harlow et al., 1980).

Experiment 2." the effect of sumrner P)( on the/o/lowing winter N S T response In December, all squirrels were induced to arouse by continuous disturbance as individual squirrels were removed for testing their NST response. Each squirrel was placed in a 15 x 15 x 35cm wire cage which was inserted into a SEC-A gradient layer calorimeter at 20°C for determining the animal's dry heat production. Air, previously scrubbed of water, was pushed through the closed calorimeter housing the animal and then through the sensor of an EG&G Dew Point hygrometer for evaporative water loss (EWL) determination. Ultimately, the exiting air sample was passed through a Beckman F-3 O, analyser for O2-consumption determination (I)'o3. These simultaneous measurements were then converted into the common units of calories from: (1) direct measurements in watts from the calorimeter: (2) conversion of ml of EWL to calories from the latent heat of evaporation; and (3) caloric equivalence of consumed 02 with an assumed RQ of 0.7. Calories of heat storage was assumed to be the difference between the caloric equivalence of O, consumption and the sum of calories from heat loss (HL) and water loss. After the animal was equilibrated to the chamber it was rapidly removed, injected intramuscularly (i.m.) with 0.08mgkg -~ isoproterenol (Sigma) in saline and replaced in the calorimeter for monitoring of the above stated parameters for the NST response. Injection of saline produced no significant change in I;'o2, EWL or HL compared to the NST response elicited by isoproterenol. Injections of 0.3mgkg -~ norepinephrine (NE, Sigma) in saline were administered i.m. at least 2 weeks subsequent to isoproterenol testing in order to compare the NST response and side effects induced by both adrenergic agents. The doses for both drugs were established from dose-response curves generated in preliminary studies. Experiment 3: the effect of long-term P X (3 yr) on the N S T response and the involvement of the pineal gland in coM-induced B A T deposition The 3-yr PX and intact groups were maintained under natural photoperiod and room temperature (23°C) until November when animals were individually removed and the NST response measured as previously described. At the end of December, these groups were then placed under simulated low-lux photoperiod and cold temperatures until March when the NST response of the individuals was again tested. Statistical analysis A one-way ANOVA followed by a Student-Newman-Keuls test for interaction was used to distinguish significant differences between the means of the test groups. RESULTS

During the first several months (AugustNovember) of exposure to low-lux light and cold prior to measuring the NST response: 40°o of the Intact and 50~o of the Sham-PX squirrels consistently

323

Pinealectomy on NST and hibccnation Table I. Percent,~f' PX, intact"and sham sqMl'rds sutgi~lly manip~ated in the summer which successfully hibernated during the subsequent period. August-November 1982 Surgical groups PX Intact Sham-PX (, = 10) (n = 10) (n = 12) Hibernation success (%) 0 40 50

NOREPINEPHRI

NE

20JO

A 0

20]SO

0

<]

2o.5o

Z" 20. 70

2o.~o

o

2o

,o

6o

,oo

,;o TIME

,'~o

,,o

,so

(rain)

Fig. 1. A representative profile of O2 uptake by squirrels comparing the effects of isoproterenol and NE, inj~ed i.m.

hibernated while no PX animals were ever observed in a torporous state (Table 1)• NE and isoproterenol both produced a substantial NST response at the doses established from the dose-response curves. While NE initiated a response significantly greater than isoproterenol (330 vs 210%), the latter drug has a tendency to bind longer to receptors and produce a more extended response (Lefkowitz, 1978; Flaim et al., 1977) (Fig. 1). NE, however, was accompanied many times by the adverse side effect of local tissue destruction and bruising. Isoproterenol initiated a characteristic response of elevated HL and EWL by all squirrels tested. When also considering the calories associated with increased stored body heat during the response, these three factors combine to produce the calorigenic equivalent associated with the rise in 02 consumption seen during NST (Fig. 2). In the group of 1982 trapped squirrels both HL and EWL were lower in the PX group than in the sham-PX or intact groups, but not to the 0.05 significance level. However, when the calorigenic equivalents of these two parameters are considered along with the body temperature increase, as represented by the elevated Vo:, there is a significantly lower peak I?o: and overall calorigenic response by the PX group (Table 2). In the 3-yr group, again the PX squirrels exhibited a lower HL, EWL and f'o2 increase in response to isoproterenol even though all parameters were not significant to the 0.05 level. When placed in the cold, both the PX and intact groups exhibited a significantly enhanced NST response. Again, the PX group demonstrated a consistently lower response

than the intact group but not to the 0.05 significance level (Table 3). DISCUSSION

Pengelley et al• (1972) have argued that the CNS of hibernators may respond to various stimuli only during specific time "windows"• Thus, if the pineal gland participates in programming the animal prior to pinealectomy, the gland's activity may not be mandatory for the remainder of that annual cycle. This appears to be the case for the reproductive cycle of birds (Gwinner and Dittami, 1979) and the horse

,[

Vo,

20

40

60

80

I00

120

140

160

I00

200

TI ME (rain)

Fig. 2. A representative profile of the NST response (cal g-~ h -j) depicting the relationship between EWL, HL, Oz consumption and consequent heat storage.

324

H.J. HARLOWand L. M. WALTERS Table 2. NST response to i.m. injections of 0.08 mg kg- ' isoproterenol (02 consumption, HL and EWL) of intact, sham and PX squirrels surgically manipulated in the summer and tested in the subsequent winter, 1982 NST response

Group

Peak I:o2 (cm3g-'h-') ~

ANST from ~>o, (calg-th-t) a

ACalories from HL (calg-lh -1)

ACalories from EWL (calg-Ih -t)

Intact ( n = 9 ) Sham-PX (n = 12) PX (n =8)

3.32(+0.75) 3.54(+0.74) 2.18(+_0.39)

10.83(+2.77) 10.96(+_2.40) 6.53(+_1.38)

6.11(+2.09) 5.29(+2.15) 4.69(+_1.30)

1.66(+_0.73) 1.18(+_0.59) 1.11 (+_0.40)

'PX group significantly different from sham-PX and intact groups (P < 0.05). Values in parentheses represent +_ I SD.

(Sharp et al., 1979). Further support for this hypothesis, with regard to circannual events of hibernation, is presented by Phillips and Harlow (1982). They observed that while golden-mantled ground squirrels PX'd in the autumn had a normal winter hibernation cycle the first winter, they had an impaired heterothermic phase the subsequent winter. Also, in that same study, the Wyoming ground squirrels PX'd in late autumn failed to show an impaired hibernation ability during the first winter (Harlow et al., 1980). But Wyoming ground squirrels in the present study PX'd earlier in the cycle (early August) did exhibitan impairment of their ability to hibernate under the same conditions as in the initial study. A period of influence by the pineal gland on the physiological mechanisms of hibernation is, therefore, suggested to occur during the late summer (through August) thereby influencing circannual events leading up to the preparation for hibernation. While not specifically delineating the boundaries of a "window" of activity, this study establishes more precisely (within several weeks) the time of year at which the pineal gland no longer has an influence on the hibernation cycle. BAT is an important component of the total fat reserve which is established in preparation for the winter heterothermic phase of hibernation. Since Wyoming ground squirrels seem to put on a majority of their fat stores during late summer and early autumn, as measured via the NST response (Abbotts and Wang, 1980), PX during the summer may eliminate a critical signal for hormonal control of BAT deposition and consequently the animal's ability to hibernate effectively. Indeed, Heldmaier and Hoffman (1974) and Sinnamon and Pivorun (1981b) both found that subcutaneous (s.c.) meiatonin beeswax implants initiated a significant increase in BAT weight in animals kept on both long- and short-day

photoperiods. In addition, Glass and Lynch (1981) found that pinealectomy diminished BAT reserves in the white-footed mouse. Squirrels that have more BAT should be able to tolerate longer and/or more frequent bouts of hypothermia. Lynch et al. (1980), using intraperitoneal beeswax implants, demonstrated that melatonin-treated mice exhibited a reversal in the effects of pinealectomy by increasing their number of torpor bouts. And Palmer and Riedesel (1976) also observed an increase in the number and duration of hibernation bouts of the golden-mantled ground squirrel treated for 10 days with 2 daily s.c. injections of meiatonin. Because of the close involvement of the pineal gland and melatonin in BAT deposition and because of the large thermogenic contribution of BAT to the NST response (Jansky, 1973; Wang, 1978; Abbots and Wang, 1980), it is felt that an impairment of the NST response by pinealectomy may well be the result of altered BAT metabolism. Indeed, other mechanisms contributing to the NST response, i.e. muscle thermogenesis (Himms-Hagen, 1978; Horwitz, 1978), may also be influenced by the pineal gland. However, because of the relatively minor contribution of these mechanisms to the NST response in comparison to BAT metabolism (Foster and Frydman, 1978), we feel that an alteration in the NST response is at least in part a consequence of altered BAT thermogenesis. Indeed, pinealectomy does appear to diminish the NST response of Wyoming ground squirrels. This diminished response may be a result of altered synthesis or activity of BAT caused by eliminating a critical signal to this tissue in the late summer period of influence by the pineal gland. The question, therefore, arises as to why the differen~ in NST between intact squirrels and squirrels PX'd 3 yr previously was not as marked as the differences between the Ist-yr PX and intact groups.

Table 3. NST response to i.m. injections of 0.08 mg kg- ~(02 consumption, HL and EWL) of intact and PX squirrels surgically nmnipulated in the autman of 1979 and tested in tim winter, 1982 NST response

Group W a r m exposed Intact (n = 7) PX (n = 9)

Peak I7o2 (crn~g-' h-'y

2.15(-4-0.61) 1.94(±0.41)

ANST from P'o2 (calg-I h-l)•

ACalories from HL (calg-t h -l)

ACalories from EWL (calg-' h-')

"6.78(:I:0.93) 6,03(±1.19)

5.21(±0.86) 4.78 (± 1.19)

1.13(+0.47 0.83(+0.36)

Cold exposed 1.54(+0.78 2.82 (+ 0.27) 9.79 (+-2.00) 5.68 (+ 0.30) Intact (n = 6) I.II (_0.51) 2.68(__.0.37) 8.76(+ 1.39) 5.08(+ 1.01) PX (n = 6) ~Cold-exposed groups significantlydifferentfrom warm-exposed groups (P < 0.05). Values in parentheses represent+- I SD.

Pinealectomy on NST and hibernation It is well known that the duration O f t o r p o r for S:: elegans is maximal in about February and is then

progressively shortened throughout the remainder of winter (Harlow et al., 1980). It is possible that a diminished difference between the treatment groups in the cold-exposed long-term-study animals is a result of their being tested later in the hibernation cycle than the lst-yr treated animals. More specifically, if BAT stores were lower in March than in December, the influence of PX on the NST response may be more difficult to note. In addition to the previously-discussed influences of photoperiod on BAT, it is well established that cold temperatures also stimulate hypertrophy of BAT (Roberts and Smith, 1967; Leblanc and Villemaire, 1970). While this study reaffirms the pineal gland's involvement in BAT desposition through its response to photic information, it suggests no influence of thermal stimuli on this gland to promote BAT deposition. For, even though cold enhanced the NST response in squirrels, the enhancement was identical in both the PX and intact groups, suggesting that another pathway may be involved in influencing BAT metabolism along with the pineal path. The central neural mechanism whereby the pineal gland and melatonin may initiate BAT synthesis and deposition is not precisely known. Preliminary studies (Gem et al., personal communication) using receptor-binding assay techniques detected no melatonin receptors on BAT and also observations by Glass and Lynch (1982), that central implants in the hypothalamus stimulate BAT deposition, suggest that the pineal gland elicits its response on BAT via a central neural pathway. Melatonin may be acting directly on certain areas in the hypothalamus which, by way of efferent projections to the BAT, or by way of sympathetic pathways to other peripheral endocrine glands, subsequently elicit changes in the fattyacid metabolism of BAT. In addition, the involvement of the adrenal medulla, its release of NE and resulting increase in circulating insulin may be of importance in glucose-mediated fatty-acid synthesis by BAT. In summary, this study has demonstrated that the pineal gland may be influencing the hibernation cycle during a period of activity, temporally functional in the late summer. This influence by the pineal may be acting to promote BAT synthesis and is not affected by the cold. However, this is not to suggest that the pineal gland and its hormone melatonin do not interact with other processes that influence NST and hibernation. On the contrary they may be acting in concert with alternate mechanisms such as thyroxine regulation (Phillips and Harlow, 1982) or even directly on hypothalamic thermoregulatory centres that eventually influence other endocrine functions. Acknowledgements--We would like to thank J. A. Phillips

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