Antagonism of brain opioid peptide action reduces hibernation bout duration

Brain Research, 328 (1985) 2111-205 Elsevier

201

BRE 10557

Research Reports

Antagonism of Brain Opioid Peptide Action Reduces Hibernation Bout Duration ALEXANDER L. BECKMAN and CARMEN LI,ADOS-ECKMAN A !l?ed 1. duPont Institute. Research Departrnent, Wilmington, D E 19899 ( U. S. A. )

(Accepted May 29th, 19841 Key words." naloxone - - hibernation - - antagonism of endogenous opioids - - ground squirrels

The effect of continuous intracerebroventricular (i.c.v.) administration of naloxonc on the duration of indMdual bouts of hibernation was investigated in the golden-mantled ground squirrel (Citellus lateralis). Following entrance into hibernation, naloxone xvas continuously administered by an osmotic minipump at a rate of 1 ul/h through a chronically implanted unilateral i.c.v cannula guktc. Naloxone (1, 5 and 7.5 ,ug/ul) produced a dose-dependent reduction in hibernation bout duration ranging lrom I to 4 days (13.9-62.3% of expected bout duration). These data suggest that selected endogenous opioid system neurons may contribute to the CNS maintenance ot the hibcrnating state and, consequently, to the overall conservation of energy in this species.

INTRODUCTION The wide distribution of endogenous opioid-containing neurons within the central nervous system (CNS) 22,23 suggests that endogenous opioid peptides may be involved in the CNS control of numerous behavioral and physiological variables. In view of the substantial representation of enkephalin- and fl-endorphin-containing neurons in the limbic system22, 2~, it is not surprising that components of energy balance (temperature regulation, food intake, behavioral activity) are sensitive to the action of these compounds. In general, intracerebral application of opioid peptides produces dose-selected bidirectional effects on body t e m p e r a t u r e (low doses, increase: higher doses, d e c r e a s e p ,5.J~,24,2~ and complex effects on the level of behavioral activity~,7,s,22,23 E n d o g e n o u s opioids increase~9.z0,> and their antagonists decrease<~.~2,e~ food intake. These findings lend themselves to the hypothesis, p r o p o s e d earlier 1~, that if a widespread and sustained neuronal release of opioid peptides exerts a coherent effect on energy balance, it is to

promote energy conservation. Hibernation is a dramatic example of a m a m m a lian adaptive specialization for energy conservation and therefore should be an important model in which to test this hypothesis. Recent work L~has shown that intracerebral levels of enkephalins in ground squirrels are, in fact, elevated during hibernation, Accordingly, the experiments reported here were designed to determine if continuous suppression of endogenous opioid efficacy during hibernation would shorten the hibernation bout. We report that, indeed, continuous intracerebroventricular (i.c.v.) infusion of low doses of the opioid antagonist naloxone produces a d o s e - d e p e n d e n t decrease in the duration of hibernation. MATERIALS AND METHODS G o l d e n - m a n t l e d ground squirrels ((Titellus alis, n = 16), trapped near Redding, California, maintained in our animal colony (illumination, bination of natural and artificial light; ambient

laterwere comtem-

Correspondence: A. L. Beckman, Research Department, Allred I. duPont Institute, P.O. Box 269, Wilmington, DE 19~9t) LI.S.A.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. ( Biomedical Division )

202 perature, 20-22 °C) in individual cages. Food (Purina rat chow; sunflower seeds) and water were available ad libitum, and each animal was given a generous amount of cotton from which they constructed fully enclosed nests. Prior to being transferred to a walk-in cold room (dim light/dark: 12 h/12 h; ambient temperature 4 °C) in late fall, 16 male and female animals were anesthetized (Nembutal, 75 mg/kg, i.p.) and stereotaxically implanted aseptically with a unilateral cannula guide tube (21-gauge stainless steel) that was fixed to the skull with dental acrylic and stainless steel (size 0-80) anchor screws. The tip of the cannula guide was positioned just above the lateral ventricle. at atlas t0 coordinates AP 7.1, L 1.8, and H 9.5. Following a post-surgical recovery period of at least 1 week, the animals were placed in the cold room where they remained until the end of the hibernation season. Following an animal's entrance into hibernation, the top of its cotton nest was parted and folded down sufficiently to facilitate twice daily observations of respiratory movement, posture and level of activity. Sawdust was sprinkled atop the animal to provide an indicator of gross postural changes and/or behavioral activity in the interval between observations. The duration of each bout was measured by the number of days in which characteristic overt features of deep hibernation (respiratory rate approximately one breath/min; body curled into a ball with head tucked beneath body; absence of movement) were maintained. Continuous infusion of naloxone hydrochloride (1, 5 and 7.5 pg/lul, dissolved in sterile 0.9% NaC1) or 0.9% NaC1 was accomplished by an osmotic minipump (Alza Model 2001) immersed in a saline bath at 38 °C (to maintain a pumping rate of 1 pl/h) and connected via a length of PE 20 polyethylene tubing (ex-

posed to ambient air temperature oi 5 ':~(i) to an injection cannula (26-gauge, stainless steel). On day 1 of the bout the animal was gently m~d briefly repositioned in its nest to facilitate insertion of the injection cannula into the cannula guide, bringing the tip into the ventricular lumen, 1 mm beneath the guide tube. The animal was not handled or otherwise disturbed for the remainder of the hibernation bout. Following completion of the experiments, the animals were anesthetized with Nembutal (75 mg/kg, i.p.) and perfused through the heart with 0.9% NaCI followed by 10% formalin. The brain was removed from the calvarium and immersed for I week in 10f~k formalin. Following this, serial frozen sections were cut (80 pm) in the coronal plane, mounted on glass slides and stained with thionin for histological verilication of cannula placement. Statistical evaluation of the data was carried out using repeated measures analysis of variance and Duncan's multiple comparisons test. RESULTS In order to minimize the influence of the natural variation in hibernation bout duration that occurs as a function of elapsed time in the hibernation season, the experiments reported here (n = 32) were performed during the same month (February) in two consecutive years. Comparisons of the mean length of the pre-infusion (baseline) hibernation bout immediately preceding the test bout showed no statistical differences (P > 0.05) across control (0,9% NaCI) and naloxone (1, 5 and 7.5 pg/pl) test conditions. The mean length of these pre-infusion bouts ranged from 6.7 to 7.2 days (Table I). Chronic i.c.v, infusion of naloxone (1 ul/h) reduced the number of days per hibernation bout below this baseline in a dose-dependent manner, No other

TABLE I Mean duration (days + S.E.M.) of hibernation bouts immediately previous to, during, and immediately after continuous i.c.v, infusion with 0.9% NaCIor naloxone. Numbers in parentheses, number of experiments/number of animals. Test condition

Pre-infusion Infusion Post-infusion

0.9% NaCl (7/5) ling (7/7)

51~g (8/8)

7.5 pg (8/7~

7.0 _+0.2 6.7 + 0.4 7.0 4- 0.4

7.2 _+0.4 3.8 ± 0.2 5.0 _+0.8

7.0 4- 0.6 2.6 ± 0.3 4.7 ± ().~

6.7 _+ 0.4 5.7 + 0.3 5.7 ± 0.7

203 100

z O kO

8O

~

6o

m 0

40

re

2O

1

5 NALOXONE,

7.5

ug/hr.

Fig. 1. Percent reduction in hibernation bout duration produced by i.c.v, infusion of naloxone. Percent reduction of naloxone-infused bout calculated with respect to the duration of the immediately preceeding bout. Slope of the line calculated by linear regression analysis: y = 7.8 × + 5.5; r = 0.997. Bars indicate S.E.M. overt changes (e.g., posture, increased movements) in hibernation characteristics were noted. As summarized in Table I, the mean bout length (+ S.E.M.) during control infusion of 0.9% NaC1 was 6.7 + 0.4 days whereas the mean values (_+ S.E.M.) during naloxone infusion were 5.7 + 0.3 days at 1 ~tg/~l/h, 3.8 + 0.2 days at 5 ~g/~l/h, and only 2.6 + 0.3 days at 7.5 pg/jA/h. These values yielded a decrease in mean bout length of 0.3 days during control infusion of 0.9% NaC1 (not significant, P > 0.5) and 1.0 days during infusion of naloxone at 1 ~g/h. The decrease in bout length produced by naloxone at 1 pg/h was not statistically different from that produced by 0.9% NaC1 (P > 0.05). However, the decreases in mean bout length produced by naloxone infusion at 5 j~g/h (3.4 days) and 7.5 pg/h (4.4 days) were significantly greater than control (P < 0.01, both comparisons). Expressed as a percentage of the pre-infusion bout, i.c.v, administration of naloxone reduced the length of hibernation (mean _+ S.E.M.) 13.9 + 4.2, 47.7 + 1.9, and 62.3 + 2.4% at doses of 1, 5 and 7.5 ~tg/j~l/h, respectively (Fig. 1). Infusion of~0.9% NaCI reduced the length of the bout by 4.1 + 5.3%. 1.c.v. infusions of naloxone or 0.9% NaC1 were discontinued when arousal from hibernation began. During and following arousal, the animals displayed normal behavior. Those animals that received 0.9% NaC1 or naloxone at 1 and 5 ~g/h remained euthermic for the normal period of time (24 h or less) prior to re-entering hibernation. Of the 7 animals that re-

ceived naloxone at 7.5/~g/h, 4 remained euthermic for longer (2-6 days) than the usual periods prior to re-entering hibernation. As shown in Table I, the mean duration of the first post-infusion bout following 0.9% NaCI or naloxone at 5 ktg/h and 7.5 ~g/h showed recovery toward the pre-infusion baseline values. This trend continued in subsequent contiguous bouts (not shown). The mean duration of the post-infusion bout following 1 ~g/h of naloxone was the same as that during infusion, but recovery toward baseline duration was evident by the second post-infusion bout (not shown). The recovery of post-infusion bout lengths to pre-infusion baseline levels was confirmed by analysis of variance which showed no statistically significant differences (P > 0.05) between the series of contiguous pre- and postinfusion bouts in each test condition. Two animals were tested at a naloxone concentration of 10~g/pl/h. At this high dose, one animal died following arousal from hibernation in the 6th day of the bout (pre-infusion bout length, 10 days; 40% reduction). The second animal aroused after hibernating 5 days (pre-infusion bout length, 9 days; 44.4c~ reduction), displaying convulsions and morphine abstinence-like signs in the arousal and post-arousal periods. This animal then displayed irregular hibernation cycles, consisting of multi-day euthermic periods interposed between bouts lasting 1-3 days. Histological examination following completion of the experiments revealed penetration of the ventricular lumen beneath the cannula guide tract in all animals. The guide tracts were located at anterior-posterior coordinates 6.8=7.7 and lateral coordinates 1.6-1.8 in the ground squirrel atlas of Joseph et al. 10. DISCUSSION Our results show that, in C. lateralis, i.c.v, infusion of low doses (1 and 5 pg//A) of naloxone decrease the length of hibernation bouts in a dose-dependent manner. Hibernating ground squirrels receiving naloxone displayed no other overt differences from animals hibernating in the absence of naloxone infusion. Similarly, the arousal from hibernation following naloxone administration was normal, as was the postarousal euthermic phase between hibernation bouts. These data therefore offer evidence to support the hypothesis~6 that activity of endogenous opioid-re-

204 leasing neurons contributes to CNS maintenance of the state of hibernation, a powerful adaptive specialization for energy conservation. The precise anatomical localization of these neurons cannot be determined from our methodology, although it is safe to assume that active structures must be within the range of diffusion from the ventricular system. Previous work on the CNS control of hibernation2,11 make it likely that such structures include components of the limbic system. As noted in the Introduction, whole-brain content of Met- and Leu-enkephalin is elevated in the hibernating ground squirrel (C. suslicus) 13. Our present results, combined with our earlier finding l that hibernating ground squirrels (C. lateralis) fail to develop physical dependence on morphine, indicate that the elevated brain content of opioid peptides during hibernation is associated with functionally significant levels of activity in the endogenous opioid system. Other attempts to study the effects of opioid antagonist administration on physiological parameters in hibernation have been reported, with results that are compatable with those we report here. Naloxone (subcutaneous injection of low doses) appeared to influence the heart rate of hibernating hamsters, producing either increases or biphasic changes Iv, However, methodological considerations involving the use of subcutaneous injections (i.e., potentially arousing stimulation produced by needle penetration) suggest caution in the interpretation of these data because increases in heart rate are a sensitive in-

REFERENCES 1 Beckman, A. L., Llados-Eckman, C., Stanton, T. L. and Adler, M. W., Physical dependence on morphine fails to develop during the hibernating state, Science, 212 (1981) 1527-1529. 2 Beckman, A. L. and Stanton, T. L., Properties of the CNS during hibernation. In A. L. Beckman (Ed,), The Neural Basis of Behavior, Spectrum, New York, 1982, pp. 19-45. 3 Bloom, F., Segal, D., Ling, N. and Guillemin, R., Endorphins: profound behavioral effects in rats suggest new etiological factors in mental illness, Science, 194 (1976) 630-632. 4 Ferri, S., Arrigo Reina, R., Santagostino, A., Scoto, G. M. and Spadaro, C., Effects of met-enkephalin on body temperature of normal and morphine-tolerant rats, Pharmacology, 58 (1978) 277-281. 5 Geller, E. B., Hawk, C., Tallarida, R. and Adler, M, W., Postulated thermoregulatory roles for different opiate receptors in rats, Life Sci., 31 (1982) 2241-2244.

dex of the response of hibernating animals to peripheral disturbancel5 In another paper 14. multiple (4/day) daily injections of high doses of naltrexone (25 mg/kg, i.p. ) were reported to produce in some animals a decline in the incidence of sleep or hibernation at only one (midnight) of 4 daily observation periods (06.00. 12.00. 18.00 and 24.00 h I. The emphemeral nature of this effect, coupled with the use of a high dose of naltrexone, make this study difficult to interpret with confidence. Taken together, the results ol the present study and those noted above support the hypothesis thai widespread release of opioid peptides in the CNS promotes energy conservation. Whereas studies of opioid agonist or antagonist actions on feeding, temperature regulation, or behavioral activity have demonstrated opioid system influence on individual energy balance responses, the work we report here indicates that this influence extends to l he broader control of energy conserving states. ACKNOWLEDGEMENTS This study was supported by N I D A Grant DA02254 and the Alfred I. duPont Institute. The authors wish to thank E n d o Laboratories for their generous gift of naloxone hydrochioride. Nimrah Sartin for excellent technical assistance. Dr. Frederick Steier for statistical evaluation of the data. and Susan Keenan for typing the manuscript.

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