HORMONES
AND
BEHAVIOR
26, 394-405 (1992)
Testosterone and Opioids Interact to Regulate Feeding in a Male Migratory Songbird PIERRE DEVICHE’ Institute of Arctic Biology,
University
of Alaska Fairbanks,
Fairbanks,
Alaska 99775
A male migratory songbird (dark-eyed junco, Junco hyemalis) was used as a model for studies on the influence of testosterone (T) on feeding, and on interactive effects on this behavior between T and the opioid antagonist naloxone hydrochloride (Nal). Administered chronically to birds exposed to nonstimulating photoperiods, T increased food intake by 30-58% without altering the body mass, the fat index, or the standard metabolic rate. An intramuscular injection of Nal decreased feeding temporarily in a dose-related manner. T-treated juncos exhibited a decreased sensitivity to the anorexic influence of Nal administration, demonstrating that T interacts with opioids to control food consumption. Neuroendocrine mechanisms that potentially account for this interaction are discussed. 0 1992 Academic
Press. Inc.
Accumulating evidence indicates that opioid mechanisms participate in the control of avian feeding behavior. Food intake is decreased in a doserelated manner by the opioid antagonist naloxone (Nal) given to pigeons (Columba livia; Cooper and Turkish, 1981; Deviche and Schepers, 1984a; Deviche and Wohland, 1984a), chicks (Gallus domesticus, McCormack and Denbow, 1987), and dark-eyed juncos (Junco hyemalis, Deviche, 1992). In pigeons, the anorexic influence of opioid antagonists is behaviorally specific and stereoselective (Deviche and Schepers, 1984a; Deviche and Wohland, 1984b). Nal decreasesfeeding when given centrally at doses that are ineffective when administered peripherally (Deviche, Melmer, and Schepers, 1984), and an intracerebroventricular injection of opioid agonists in some cases enhances feeding (Deviche and Schepers, 1984b; McCormack and Denbow, 1988, 1989). These results support the hypothesis that central opioid mechanisms regulate food intake in birds, as is the case in mammals (Gosnell, 1987; Morley, 1987; Morley, Levine, Gosnell, Mitchell, Krahn, and Nizielski, 1985). In mammals, gonadal steroids also control feeding (Gentry and Wade,
1976; Gray and Greenwood, 1982; Morley, Levine, Grace, Kneip, and ’ To whom correspondence should be addressed. 394 0018-506x/92 $4.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form resewed.
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Gosnell, 1984; Wade and Gray, 1979), and they modulate the influence of opioids on this behavior. Specifically, Morley et al. (1984) reported that the sensitivity of female rats to the anorexic influence of Na! is increased by ovariectomy, whereas estrogen administration to ovariectomized females attenuates their sensitivity to the antagonist. The neuroendocrine mechanisms that account for these observations are not yet identified, but possibly involve effects of gonadal steroids on endogenous opioids (Almeida, Nikolarakis, and Herz, 1987; Tang, 1991; Lapchak, 1991; Wardlaw, 1986) and/or on opioid receptors (Hammer, 1985; Weiland and Wise, 1990; Wilkinson, Brawer, and Wilkinson, 1985). Furthermore, interactions between gonadal steroids and opioids in effects on food intake in male vertebrates have not yet been reported. To address this question, we studied interactions on feeding between testosterone (T) and Nal in male juncos, a highly seasonal, migratory passerine songbird. Our results show that T administration stimulates food consumption and decreases the sensitivity of feeding to the anorexic influence of Nal. These observations constitute the first indication that androgens interact with opioids to regulate feeding in male vertebrates. METHODS Birds
Immature male dark-eyed juncos were caught in July and August 1990 on the campus of the University of Alaska Fairbanks. In the laboratory, they were exposed to artificial, progressively decreasing photoperiods to approximate natural conditions. When the photoperiod reached 12 hr, it was held constantly at that level (lights on at 8:00 AM). During the experiments, birds were kept in individual cages, in visual isolation from each other. Unless otherwise specified, pelleted food (Purina finch chow) and tap water were continuously available. T Treatment
On February 21, 1991 (Day 0, DO), birds were randomly divided into two groups. Birds belonging to one group (T birds; N = 10) received two subcutaneous, l-cm-long Silastic capsules (Dow Corning, Midland, MI; i.d. 1.45 mm; o.d. 1.93 mm) filled with crystalline T (Sigma Chemical Co., St. Louis, MO), to produce tonically high circulating concentrations of the steroid (Ketterson, Nolan, Wolf, Ziegenfus, Dufty, Ball and Johnsen, 1991). The remaining birds (C group; N = 10) received two empty capsules of identical size. Before implantation, capsules were weighed to the nearest 0.1 mg, then incubated overnight in a 0.9% NaCl solution (saline) at 40°C. Birds were sacrificed after 50 days of treatment (D50). at which time capsules were removed, dehydrated at 45°C for 48 hr, and weighed.
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PIERRE DEVICHE
Nal Treatment
Between D33 and D47, each bird received an intramuscular injection of Nal hydrochloride (Endo Pharmaceuticals, NJ) at four different doses (0, 5, 10, and 20 mg/kg body mass; vehicle: 0.1 ml saline/injection). Injections were given in a random order, after 5 hr of food deprivation, and between 1:45 and 2:45 PM. At least 4 days separated successive injections to a same individual. Food Intake
To determine the influence of T administration on ad fibitum food consumption, we measured the 24-hr food intake of all birds on D’s 7, 11, 15, 25, and 50. For this, birds received a preweighed amount of food at 2:00 PM, and the amount left in the containers after 24 hr was measured to the nearest 0.1 g. The shape of the containers prevented significant food spillage. To study the influence on feeding of Nal treatment, birds received a preweighed amount of food 15 min after a Nal injection, and the amount left in the containers was measured to the nearest 0.05 g after 1, 2, 4, and 24 hr. Morphology;
The body mass (BM) and the diameter of the cloaca1 protuberance (CP, an androgen-sensitive secondary sex characteristic, Schwab1and Farner, 1989) of all males were measured on D’s 0, 3, 7, 11, 15, 25, 35, and 50. On these dates, we also recorded the amount of subcutaneous fat contained in the furcular space (fat index, ranging from 0 (no visible fat) to 5 (furcula bulging with fat), Silverin, Viebke, and Westin, 1989). In juncos, the fat index constitutes a reliable indicator of the total amount of extractable body lipids (Deviche, unpublished data; Rogers, 1991). Standard Metabolic Rate
The standard metabolic rate of all birds was determined between D19 and D22 by measuring oxygen consumption, using an open-circuit, automatic respiratory system (Morrison, 1951). During the measurements, birds were kept in the dark and at a stable ambient temperature (24.5 ? 0.4”C, within the genus thermoneutral zone, Weathers and Sullivan, 1989). Measurements were made after 4 hr of food deprivation, during the active phase of the daily cycle. Results are expressed in milliliters of O2 consumed/g BM/hr. Statistical Analyses
To determine the influence of Nal treatment on feeding and to study interactions between an injection of the antagonist and T administration,
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data were analyzed using a three-factor cross-nested experimental design (Neter, Wasserman, and Kutner, 1985, p. 1025), using PROC GLM (SAS Institute, 1985), with testosterone and Nal as fixed factors and individuais as a random factor nested within the T factor. A preliminary examination of the data revealed that assumptions of normality and homoscedasticity were upheld. Data obtained for the 24-hr ad libitum food consumption, the BM, the CP diameter, and the metabolic rate were analyzed using two-sample Student’s tests. Fat index data were analyzed using Mann-Whitney U tests (Siegel, 1956). Two-tailed probabilities 0.05. See legend of Fig. 2 for additional information.
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DISCUSSION This study demonstrates that the administration of Nal to male juncos decreases their food intake, and it shows that the anorexic influence of the antagonist is dose related and temporary, not lasting for more than 1 hr after an injection. The data are consistent with previous research on this and other avian species (see introduction), and they suggest that opioids regulate food intake in birds in general. Our results provide the first evidence that opioids and T interact to regulate feeding in male vertebrates. Specifically, T juncos exhibited a lower sensitivity than C birds to the anorexic influence of a Nal injection. This observation parallels results obtained on female rats, whose feeding response to Nal treatment is attenuated by estrogen administration (Morley et al., 1984). In birds, central opioid mechanisms participate in the regulation of food intake (see introduction), and hypothalamic regions including the lateral and the ventromedial hypothalamus (LHy, VMH) control this behavior (Denbow. 1985; Foreman, Lea, and Buntin, 1990; Kuenzel, 1982a,b; Robinzon and Katz, 1980). The avian LHy and VMH contain opioidbinding sites and immunoreactive opioid peptides (Blahser and Dubois, 1980; de Lanerolle, Elde, Sparber, and Frick, 1981; Deviche and Cotter, 1991; Reiner, Brauth, Kitt, and Quirion, 1989). In mammals, these regions mediate the influence of opioids on feeding (Grandison and Guidotti, 1977; Tepperman, Hirst, and Gowdey, 1981). Thus, the avian LHy and VMH may be sites where opioids act to influence food consumption. Cells located in the avian LHy and VMH possess androgen-binding sites (Arnold, Nottebohm, and Pfaff, 1976; Watson and Adkins-Regan, 1989), and hypothalamic regions have been implicated in the control of feeding by T (Yokoyama, 1976). Further, opioid peptide-containing hypothalamic cells concentrate gonadal steroids in rats (Morrell, McGinty, and Pfaff, 1985; Akesson and Micevych. 1991; Olster and Blaustein, 1990). Hence, hypothalamic areas may mediate T-opioid behavioral interactions. Several mechanisms may account for gonadal steroid-induced alterations of the sensitivity of feeding to opioids. One such mechanism may consist in steroid-induced alterations of the release of opioid peptic& into brain areas that control food intake. In rats, castration reduces the release of opioid peptides by hypothalamic tissues, whereas in viva or ipl vitro T administration in some circumstances stimulates this release (Almeida et al., 1987; Nakano. Suda, Sumitomo, Tozawa, and Demura, 1991). Thus, elevated circulating concentrations of T possibly increase the proportion of opioid receptors that are occupied by endogenous ligands. As a result, the number of these receptors available for exogenous ligands (e.g., Nal) binding may be reduced, causing a decreased behavioral response to these ligands.
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In female rats, estrogen and progesterone influence hypothalamic opioid receptors (Weiland and Wise, 1990; Wilkinson et al., 1985). In male rats, T decreased the density of opioid receptors purified from whole brain preparations in some (Hahn and Fishman, 1979, 1985), but not in other (Cicero, O’Connor, and Bell, 1987; Diez and Roberts, 1982) studies. In addition, Clark, Ball, and Hughes (1984) reported that hypothalamic opioid receptors of rats are not regulated by androgens. These observations do not preclude effects of androgens on opioid receptors located in specific brain regions (preoptic area: Hammer, 1988). However, they do not support the hypothesis that, in males, T modifies the sensitivity of feeding to Nal by directly altering the density of central opioid receptors that regulate this behavior. In T juncos, the size of the cloaca1 protuberance (an androgen-dependent sexual characteristic) reached a maximum value (5.5 mm) approximating that measured in free-living males at the peak of the breeding season (7.5 mm; Deviche, unpublished data). In a recent study on the same species (Ketterson et al., 1991), administration of T-filled Silastic capsules of identical length but of smaller diameter than those used in this study induced chronically high but physiological circulating concentrations of the steroid. Thus, the amount of T released by the Silastic capsules (average: 92.3 pg/day) used in this investigation probably resulted in physiological or close to physiological circulating concentrations of the steroid. In the present study, the stimulating influence of T treatment on feeding did not concur with effects on the body mass, the fat index, or the standard metabolic rate of the birds. One explanation for these observations may be that T administration produced behavioral and/or metabolic changes that enhanced overall energy expenditure. In birds, T treatment stimulates presumably energy-consuming activities such as locomotion, vocalizations, and aggressive and exploratory behavior (Wada, 1981, 1982; Watson, 1970; Wingfield and Ramenofsky, 1985). The role played by such modifications in the control of energy balance in natural conditions is currently unclear, however, since the total daily energy expenditure of free-living male yellow-eyed juncos (Bunco phaeonotus) remained relatively constant through the breeding season (Weathers and Sullivan, 1989), despite the fact that their plasma concentrations of T probably underwent marked seasonal changes. Ketterson et al. (1991) recently reported that treating free-living male juncos with T decreases their body mass and their fat reserves when the hormone is given in early spring, at which time these males are heavy (23 g) and normally possesslarge fat reserves. In contrast, and similar to the present study, T administration did not influence the weight of juncos treated with the steroid during the breeding season, when their body mass is relatively low (21 g; i.d.). These observations suggest that the effects
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of T on body mass and possibly on energy expenditure vary depending on the initial physiological condition of the birds. Additional studies are required to confirm this hypothesis and to elucidate the mechanisms involved. ACKNOWLEDGMENTS It is a pleasure to thank Dr. Kent Schwaegerle for help with the statistical analyses. The work was supported by a UAF Faculty Small Grant to the author.
REFERENCES Akesson, T. R., and Micevych, P. E. (1991). Endogenous opioid-immunoreactive neurons of the ventromedial hypothalamic nucleus concentrate estrogen in male and female rats. J. Neurosci. Rex 28, 359-366. Almeida, 0. F. X., Nikolarakis, K. E., and Herz, A. (1987). Significance of testosterone in regulating hypothalamic content and in vitro release of P-endorphin and dynorphin. J. Neurochem.
49, 742-747.
Arnold, A. P.. Nottebohm, F.. and Pfaff, D. W. (1976). Hormone concentrating cells in vocal control and other areas of the brain of the Zebra finch (Poephila guttata). J. Comp. Neurol. 165, 487-512. Blahser, S., and Dubois, M. P. (1980). Immunocytochemical demonstration of metenkephalin in the central nervous system of the domestic fowl. Cell Tissue Rex 213, 5368. Cicero, T. J., O’Connor, L. H., and Bell, R. D. (1987). Reevaluation of the effects of castration on naloxone-sensitive opiate receptors in the male rat brain. Neuroendocrinology 46, 176-184. Clark, C. R., Ball, A. J., and Hughes, J. (1984). Hypothalamic opiate receptors (mu, kappa, delta) are refractory to testosterone. Sot. Neurosci. Abrf. 1, 478. Cooper, S. J., and Turkish, S. (1981). Food and water intake in the nondeprived pigeon after morphine or naloxone administration. Neuropharmacology 20, 1053-1058. de Lanerolle, N. C., Elde, R. P., Sparber, S. B., and Frick, M. (1981). Distribution of methionine-enkephalin immunoreactivity in the chick brain: An immunohistochemical study. J. Comp. Neural. 199, 513-533. Denbow, D. M. (1985). Food intake control in birds. Neurosci. Biobehav. Rev. 9, 223232. Deviche, P. (1992). Regulation of food intake in a migratory songbird (Bunco hyemalis): Participation of endorphinergic mechanisms. Ornis Stand., in press. Deviche, P., and Cotter, P. (1991). Hypothalamic localization of kappa, delta, and mu opioid receptors in the songbird brain. Sot. Neurosci. A&. 1, 596. Deviche, P., and Schepers, G. (1984a). Naloxone treatment attenuates food but not water intake in domestic pigeons. Psychopharmacology 82, 122-126. Deviche, P., and Schepers, G. (1984b). Intracerebroventricular injection of ostrich p-endorphin to satiated pigeons produces hyperphagia but not hyperdipsia. Peptides 5,691694.
Deviche, P., and Wohland, A. (1984a). Behavioural characterization of the anorexic effect of naloxone in domestic pigeons. IRCS Med. Sci. 12, 427-428. Deviche, P., and Wohland, A. (1984b). Opiate antagonists stereoselectively attenuate the consumption of food but not of water by pigeons. Pharmacol. Biochem. Behav. 21, 507-512. Deviche, P., Melmer, G., and Schepers, G. (1984). Evidence that naloxone attenuates the
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ON FEEDING
403
consumption of food by domestic pigeons through a central influence. Nenrophamrncology 23, 1173-1178. Diez, J. A.. and Roberts, D. L. (1982). Evidence contradicting the notion that gonadal hormones regulate brain opiate receptors. Biochem. Biophys. Res. Commun. 108,13131319. Foreman, K. ‘I’., Lea, R. W., and Buntin, J. D. (1990). Changes in feeding activity. plasma luteinizing hormone, and testes weight in ring doves following hypothalamic injections of prolactin. .I. Neuroendocrinol. 2, 667-673. Gentry, R. T., and Wade, G. N. (1976). Androgenic control of food intake and body weight in male rats. J. Comp. Physiol. Psychol. 90, 18-25. Gosnell, B. A. (1987). Central structures involved in opioid-induced feeding. Fed. Proc. 46, 163-167. Grandison. L., and Guidotti, A. (1977). Stimulation of food intake by muscimol and beta endorphin. Neuropharmacology 16, 533-536. Gray. J. M., and Greenwood, M. R. C. (1982). Time course of effects of ovarian hormones on food intake and metabolism. Am. J. Physiol. 243, E407-412. Hahn, E. F., and Fishman, J. (1979). Changes in rat brain opiate receptor content upon castration and testosterone replacement. Biochem. Biophys. Rex Commun. 90, 819823. Hahn. E. F., and Fishman, .I. (1985). Castration affects male rat brain opiate receptor content. Neuroendocrinology 41, 60-63. Hammer, R. P. (1985). The sex hormone-dependent development of opiate receptors in the rat medial preoptic area. Brain Res. 360, 65-74. Hammer, R. P. (1988). Opiate receptor ontogeny in the rat medial preoptic area is androgendependent. Neuroendocrinology 48, 336-341. Ketterson, E. D., Nolan, V., Jr., Wolf, L., Ziegenfus, C., Dufty, A. M., Ball, G. F., and Johnsen, T. S. (1991). Testosterone and avian life histories: The effect of experimentally elevated testosterone on corticosterone and body mass in dark-eyed juncos. Harm. Behnv. 25, 489-503. Kuenzel, W. J. (1982a). Transient aphagia produced following bilateral destruction of the lateral hypothalamic area and quinto-frontal tract of chicks. Physiol. Behav. 28, 237244. Kuenzel, W. J. (1982b). Central neural structures affecting food intake in birds: The lateral and ventral hypothalamic areas. In C. G. Scanes, M. A. Ottinger. A. D. Kenny, J. Balthazart, J. Cronshaw, and I. Chester Jones. (Eds.). Aspects of Asian Endocrkology, pp. 211-216. Texas Tech. Univ. Lapchak, P. A. (1991). Effect of estradiol treatment on &endorphin content and release in the female rat hypothalamus. Brain Res. 554, 198-202. McCormack, J. F., and Denbow, D. M. (1987). The effects of opioid antagonists on ingestive behavior in the domestic fowl. Pharmacol. Biochem. Behav. 27, 25-33. McCormack, J. F., and Denbow, D. M. (1988). Feeding. drinking and temperature responses to intracerebroventricular P-endorphin in the domestic fowl. Peptides 9, 709-715. McCormack, J. F.. and Denbow, D. M. (1989). Ingestive responses to mu and delta opioid receptor agonists in the domestic fowl. Br. Poultry Sci. 30, 327-340. Morley, J. E. (1987). Neuropeptide regulation of appetite and weight. Endocr. Rev. 8,256287. Morley, J. E.. and Levine, A. S. (1985). Neuropeptides and appetite regulation, Med. 1. Aust. 142, Sll-S13. Morley, J. E., Levine, A. S., Gosnell, B. A., Mitchell, J. E., Krahn, D. D., and Nizielski. S. E. (1985). Peptides and feeding. Pepfides 6(2), 181-192. Morley, J. E., Levine, A. S.. Grace, M., Kneip, J., and Gosnell. B. A. (1984). The effect
404
PIERRE
DEVICHE
of ovariectomy, estradiol and progesterone on opioid modulation of feeding. Physio/. Behav. 33, 237-241. Morrell, J. I., McGinty, J. F., and Pfaff, D. W. (198.5). A subset of P-endorphin- or dynorphin-containing neurons in the medial basal hypothalamus accumulates estradiol. Neuroendocrinolog~~ MorrisOn,
41, 41 J-426.
P. R. (1951). An automatic manometric respirometer. Rev. Sci. Instru. 22, 264-
267. Nakano, Y., Suda, T., Sumitomo, T., Tozawa, F., and Demura, H. (1991). Effects of sex steroids on /3-endorphin release from rat hypothalamus in virro. Bruin Res. 553, I-3. Neter, J., Wasserman, W., and Kutner, M. H. (1985). Applied Linear Statistical Models. Irwin, Homewood, IL. Olster, D. H., and Blaustein, J. D. (1990). Immunocytochemical colocalization of progestin receptors and /3-endorphin or enkephalin in the hypothalamus of female guinea pigs. J. Neurobiol.
21, 768-780.
Reiner, A., Brauth, S. E., Kitt, C. A., and Quirion, R. (1989). Distribution of mu, delta, and kappa opiate receptor types in the forebrain and midbrain of pigeons. J. Comp. Neural.
280, 359-382.
Robinzon, B., and Katz, Y. (1980). The effects of hypothalamic and mesencephalic lesions on food and water intakes, adiposity and some endocrine criteria, in the red-winged blackbird (Agelaius phoeniceus). Physiol. Behav. 24, 347-356. Rogers, C. M. (1991). An evaluation of the method of estimating body fat in birds by quantifying visible subcutaneous fat. J. Field Omithot. 62, 349-356. SAS Institute, Inc. (1985). SAS User’s Guide: Statistics. SAS Institute Inc., NC. Schwabl, H., and Farner, D. S. (1989). Endocrine and environmental control of vernal migration in male white-crowned sparrows, Zonotrichia leucophrys gambelii. Phpsiol. 2001. 62, l-10. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Silverin, B., Viebke, P. A., and Westin, J. (1989). Hormonal correlates of migration and territorial behavior in juvenile willow tits during autumn. Gen. Comp. Endocrinol. 75, 148-156. Tang, F. (1991). Endocrine control of hypothalamic and pituitary met-enkephalin and pendorphin contents. Neuroendocrinology 53, 68-76. Tepperman, F. S., Hirst, M., and Gowdey, C. W. (1981). Hypothalamic injection of morphine: Feeding and temperature responses. Life Sci. 28, 2459-2467. Wada, M. (1981). Effects of photostimulation, castration, and testosterone replacement on daily patterns of calling and locomotor activity in Japanese quail. Harm. Behav. 15, 270-281. Wada, M. (1982). Effects of sex steroids on calling, locomotor activity, and sexual behavior in castrated male Japanese quail. Horm. Behav. 16, 147-157. Wade, G. N., and Gray, J. M. (1979). Gonadal effects on food intake and adiposity: A metabolic hypothesis. Physiol. Behav. 22, 583-593. Wardlaw, S. L. (1986). Regulation of P-endorphin, corticotropin-like intermediate lobe peptide, and cY-melanotropin-stimulating hormone in the hypothalamus by testosterone. Endocrinology
119, 19-24.
Watson, A. (1970). Territorial and reproductive behavior of red grouse. J. Reprod. Fe&. Suppl. 11, 3-14.
Watson, J. T., and Adkins-Regan, E. (1989). Neuroanatomical localization of sex steroidconcentrating cells in the Japanese quail (Coturnix japonica): Autoradiography with [3HJtestosterone, [3H]estradiol, and [3H]dihydrotestosterone. Neuroendocrinobgy 49, 51-64.
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Weathers. W. W., and Sullivan, K. A. (1989). Juvenile foraging proficiency, parental effort. and avian reproductive success. Ecol. Monogr. 59, 223-246. Weiland, N. G., and Wise, P. M. (1990). Estrogen and progesterone regulate opiate receptor densities in multiple brain regions. Endocrinology 126, 804-808. Wilkinson, M.. Brawer, J. R., and Wilkinson, D. A. (1985). Gonadal steroid-induced modification of opiate binding sites in anterior hypothalamus of female rats. Biol. Reprod. 32, 501-506. Wingfield, J. C., and Ramenofsky. M. (1985). Testosterone and aggressive behaviour during the reproductive cycle of male birds. In R. Gilles and J. Balthazart (Eds.), Neurobiology pp. 92-104. Springer-Verlag, Berlin. Yokoyama, K. (1976). Hypothalamic and hormonal control of the photoperiodically induced vernal functions in the white-crowned sparrow, ZonotricRia leucophrys gambelii. Ce!i Tissue Res. 174, 391-416.
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Testosterone and Opioids Interact to Regulate Feeding in a Male Migratory Songbird PIERRE DEVICHE’ Institute of Arctic Biology,
University
of Alaska Fairbanks,
Fairbanks,
Alaska 99775
A male migratory songbird (dark-eyed junco, Junco hyemalis) was used as a model for studies on the influence of testosterone (T) on feeding, and on interactive effects on this behavior between T and the opioid antagonist naloxone hydrochloride (Nal). Administered chronically to birds exposed to nonstimulating photoperiods, T increased food intake by 30-58% without altering the body mass, the fat index, or the standard metabolic rate. An intramuscular injection of Nal decreased feeding temporarily in a dose-related manner. T-treated juncos exhibited a decreased sensitivity to the anorexic influence of Nal administration, demonstrating that T interacts with opioids to control food consumption. Neuroendocrine mechanisms that potentially account for this interaction are discussed. 0 1992 Academic
Press. Inc.
Accumulating evidence indicates that opioid mechanisms participate in the control of avian feeding behavior. Food intake is decreased in a doserelated manner by the opioid antagonist naloxone (Nal) given to pigeons (Columba livia; Cooper and Turkish, 1981; Deviche and Schepers, 1984a; Deviche and Wohland, 1984a), chicks (Gallus domesticus, McCormack and Denbow, 1987), and dark-eyed juncos (Junco hyemalis, Deviche, 1992). In pigeons, the anorexic influence of opioid antagonists is behaviorally specific and stereoselective (Deviche and Schepers, 1984a; Deviche and Wohland, 1984b). Nal decreasesfeeding when given centrally at doses that are ineffective when administered peripherally (Deviche, Melmer, and Schepers, 1984), and an intracerebroventricular injection of opioid agonists in some cases enhances feeding (Deviche and Schepers, 1984b; McCormack and Denbow, 1988, 1989). These results support the hypothesis that central opioid mechanisms regulate food intake in birds, as is the case in mammals (Gosnell, 1987; Morley, 1987; Morley, Levine, Gosnell, Mitchell, Krahn, and Nizielski, 1985). In mammals, gonadal steroids also control feeding (Gentry and Wade,
1976; Gray and Greenwood, 1982; Morley, Levine, Grace, Kneip, and ’ To whom correspondence should be addressed. 394 0018-506x/92 $4.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form resewed.
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395
Gosnell, 1984; Wade and Gray, 1979), and they modulate the influence of opioids on this behavior. Specifically, Morley et al. (1984) reported that the sensitivity of female rats to the anorexic influence of Na! is increased by ovariectomy, whereas estrogen administration to ovariectomized females attenuates their sensitivity to the antagonist. The neuroendocrine mechanisms that account for these observations are not yet identified, but possibly involve effects of gonadal steroids on endogenous opioids (Almeida, Nikolarakis, and Herz, 1987; Tang, 1991; Lapchak, 1991; Wardlaw, 1986) and/or on opioid receptors (Hammer, 1985; Weiland and Wise, 1990; Wilkinson, Brawer, and Wilkinson, 1985). Furthermore, interactions between gonadal steroids and opioids in effects on food intake in male vertebrates have not yet been reported. To address this question, we studied interactions on feeding between testosterone (T) and Nal in male juncos, a highly seasonal, migratory passerine songbird. Our results show that T administration stimulates food consumption and decreases the sensitivity of feeding to the anorexic influence of Nal. These observations constitute the first indication that androgens interact with opioids to regulate feeding in male vertebrates. METHODS Birds
Immature male dark-eyed juncos were caught in July and August 1990 on the campus of the University of Alaska Fairbanks. In the laboratory, they were exposed to artificial, progressively decreasing photoperiods to approximate natural conditions. When the photoperiod reached 12 hr, it was held constantly at that level (lights on at 8:00 AM). During the experiments, birds were kept in individual cages, in visual isolation from each other. Unless otherwise specified, pelleted food (Purina finch chow) and tap water were continuously available. T Treatment
On February 21, 1991 (Day 0, DO), birds were randomly divided into two groups. Birds belonging to one group (T birds; N = 10) received two subcutaneous, l-cm-long Silastic capsules (Dow Corning, Midland, MI; i.d. 1.45 mm; o.d. 1.93 mm) filled with crystalline T (Sigma Chemical Co., St. Louis, MO), to produce tonically high circulating concentrations of the steroid (Ketterson, Nolan, Wolf, Ziegenfus, Dufty, Ball and Johnsen, 1991). The remaining birds (C group; N = 10) received two empty capsules of identical size. Before implantation, capsules were weighed to the nearest 0.1 mg, then incubated overnight in a 0.9% NaCl solution (saline) at 40°C. Birds were sacrificed after 50 days of treatment (D50). at which time capsules were removed, dehydrated at 45°C for 48 hr, and weighed.
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Nal Treatment
Between D33 and D47, each bird received an intramuscular injection of Nal hydrochloride (Endo Pharmaceuticals, NJ) at four different doses (0, 5, 10, and 20 mg/kg body mass; vehicle: 0.1 ml saline/injection). Injections were given in a random order, after 5 hr of food deprivation, and between 1:45 and 2:45 PM. At least 4 days separated successive injections to a same individual. Food Intake
To determine the influence of T administration on ad fibitum food consumption, we measured the 24-hr food intake of all birds on D’s 7, 11, 15, 25, and 50. For this, birds received a preweighed amount of food at 2:00 PM, and the amount left in the containers after 24 hr was measured to the nearest 0.1 g. The shape of the containers prevented significant food spillage. To study the influence on feeding of Nal treatment, birds received a preweighed amount of food 15 min after a Nal injection, and the amount left in the containers was measured to the nearest 0.05 g after 1, 2, 4, and 24 hr. Morphology;
The body mass (BM) and the diameter of the cloaca1 protuberance (CP, an androgen-sensitive secondary sex characteristic, Schwab1and Farner, 1989) of all males were measured on D’s 0, 3, 7, 11, 15, 25, 35, and 50. On these dates, we also recorded the amount of subcutaneous fat contained in the furcular space (fat index, ranging from 0 (no visible fat) to 5 (furcula bulging with fat), Silverin, Viebke, and Westin, 1989). In juncos, the fat index constitutes a reliable indicator of the total amount of extractable body lipids (Deviche, unpublished data; Rogers, 1991). Standard Metabolic Rate
The standard metabolic rate of all birds was determined between D19 and D22 by measuring oxygen consumption, using an open-circuit, automatic respiratory system (Morrison, 1951). During the measurements, birds were kept in the dark and at a stable ambient temperature (24.5 ? 0.4”C, within the genus thermoneutral zone, Weathers and Sullivan, 1989). Measurements were made after 4 hr of food deprivation, during the active phase of the daily cycle. Results are expressed in milliliters of O2 consumed/g BM/hr. Statistical Analyses
To determine the influence of Nal treatment on feeding and to study interactions between an injection of the antagonist and T administration,
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data were analyzed using a three-factor cross-nested experimental design (Neter, Wasserman, and Kutner, 1985, p. 1025), using PROC GLM (SAS Institute, 1985), with testosterone and Nal as fixed factors and individuais as a random factor nested within the T factor. A preliminary examination of the data revealed that assumptions of normality and homoscedasticity were upheld. Data obtained for the 24-hr ad libitum food consumption, the BM, the CP diameter, and the metabolic rate were analyzed using two-sample Student’s tests. Fat index data were analyzed using Mann-Whitney U tests (Siegel, 1956). Two-tailed probabilities 0.05. See legend of Fig. 2 for additional information.
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DISCUSSION This study demonstrates that the administration of Nal to male juncos decreases their food intake, and it shows that the anorexic influence of the antagonist is dose related and temporary, not lasting for more than 1 hr after an injection. The data are consistent with previous research on this and other avian species (see introduction), and they suggest that opioids regulate food intake in birds in general. Our results provide the first evidence that opioids and T interact to regulate feeding in male vertebrates. Specifically, T juncos exhibited a lower sensitivity than C birds to the anorexic influence of a Nal injection. This observation parallels results obtained on female rats, whose feeding response to Nal treatment is attenuated by estrogen administration (Morley et al., 1984). In birds, central opioid mechanisms participate in the regulation of food intake (see introduction), and hypothalamic regions including the lateral and the ventromedial hypothalamus (LHy, VMH) control this behavior (Denbow. 1985; Foreman, Lea, and Buntin, 1990; Kuenzel, 1982a,b; Robinzon and Katz, 1980). The avian LHy and VMH contain opioidbinding sites and immunoreactive opioid peptides (Blahser and Dubois, 1980; de Lanerolle, Elde, Sparber, and Frick, 1981; Deviche and Cotter, 1991; Reiner, Brauth, Kitt, and Quirion, 1989). In mammals, these regions mediate the influence of opioids on feeding (Grandison and Guidotti, 1977; Tepperman, Hirst, and Gowdey, 1981). Thus, the avian LHy and VMH may be sites where opioids act to influence food consumption. Cells located in the avian LHy and VMH possess androgen-binding sites (Arnold, Nottebohm, and Pfaff, 1976; Watson and Adkins-Regan, 1989), and hypothalamic regions have been implicated in the control of feeding by T (Yokoyama, 1976). Further, opioid peptide-containing hypothalamic cells concentrate gonadal steroids in rats (Morrell, McGinty, and Pfaff, 1985; Akesson and Micevych. 1991; Olster and Blaustein, 1990). Hence, hypothalamic areas may mediate T-opioid behavioral interactions. Several mechanisms may account for gonadal steroid-induced alterations of the sensitivity of feeding to opioids. One such mechanism may consist in steroid-induced alterations of the release of opioid peptic& into brain areas that control food intake. In rats, castration reduces the release of opioid peptides by hypothalamic tissues, whereas in viva or ipl vitro T administration in some circumstances stimulates this release (Almeida et al., 1987; Nakano. Suda, Sumitomo, Tozawa, and Demura, 1991). Thus, elevated circulating concentrations of T possibly increase the proportion of opioid receptors that are occupied by endogenous ligands. As a result, the number of these receptors available for exogenous ligands (e.g., Nal) binding may be reduced, causing a decreased behavioral response to these ligands.
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In female rats, estrogen and progesterone influence hypothalamic opioid receptors (Weiland and Wise, 1990; Wilkinson et al., 1985). In male rats, T decreased the density of opioid receptors purified from whole brain preparations in some (Hahn and Fishman, 1979, 1985), but not in other (Cicero, O’Connor, and Bell, 1987; Diez and Roberts, 1982) studies. In addition, Clark, Ball, and Hughes (1984) reported that hypothalamic opioid receptors of rats are not regulated by androgens. These observations do not preclude effects of androgens on opioid receptors located in specific brain regions (preoptic area: Hammer, 1988). However, they do not support the hypothesis that, in males, T modifies the sensitivity of feeding to Nal by directly altering the density of central opioid receptors that regulate this behavior. In T juncos, the size of the cloaca1 protuberance (an androgen-dependent sexual characteristic) reached a maximum value (5.5 mm) approximating that measured in free-living males at the peak of the breeding season (7.5 mm; Deviche, unpublished data). In a recent study on the same species (Ketterson et al., 1991), administration of T-filled Silastic capsules of identical length but of smaller diameter than those used in this study induced chronically high but physiological circulating concentrations of the steroid. Thus, the amount of T released by the Silastic capsules (average: 92.3 pg/day) used in this investigation probably resulted in physiological or close to physiological circulating concentrations of the steroid. In the present study, the stimulating influence of T treatment on feeding did not concur with effects on the body mass, the fat index, or the standard metabolic rate of the birds. One explanation for these observations may be that T administration produced behavioral and/or metabolic changes that enhanced overall energy expenditure. In birds, T treatment stimulates presumably energy-consuming activities such as locomotion, vocalizations, and aggressive and exploratory behavior (Wada, 1981, 1982; Watson, 1970; Wingfield and Ramenofsky, 1985). The role played by such modifications in the control of energy balance in natural conditions is currently unclear, however, since the total daily energy expenditure of free-living male yellow-eyed juncos (Bunco phaeonotus) remained relatively constant through the breeding season (Weathers and Sullivan, 1989), despite the fact that their plasma concentrations of T probably underwent marked seasonal changes. Ketterson et al. (1991) recently reported that treating free-living male juncos with T decreases their body mass and their fat reserves when the hormone is given in early spring, at which time these males are heavy (23 g) and normally possesslarge fat reserves. In contrast, and similar to the present study, T administration did not influence the weight of juncos treated with the steroid during the breeding season, when their body mass is relatively low (21 g; i.d.). These observations suggest that the effects
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of T on body mass and possibly on energy expenditure vary depending on the initial physiological condition of the birds. Additional studies are required to confirm this hypothesis and to elucidate the mechanisms involved. ACKNOWLEDGMENTS It is a pleasure to thank Dr. Kent Schwaegerle for help with the statistical analyses. The work was supported by a UAF Faculty Small Grant to the author.
REFERENCES Akesson, T. R., and Micevych, P. E. (1991). Endogenous opioid-immunoreactive neurons of the ventromedial hypothalamic nucleus concentrate estrogen in male and female rats. J. Neurosci. Rex 28, 359-366. Almeida, 0. F. X., Nikolarakis, K. E., and Herz, A. (1987). Significance of testosterone in regulating hypothalamic content and in vitro release of P-endorphin and dynorphin. J. Neurochem.
49, 742-747.
Arnold, A. P.. Nottebohm, F.. and Pfaff, D. W. (1976). Hormone concentrating cells in vocal control and other areas of the brain of the Zebra finch (Poephila guttata). J. Comp. Neurol. 165, 487-512. Blahser, S., and Dubois, M. P. (1980). Immunocytochemical demonstration of metenkephalin in the central nervous system of the domestic fowl. Cell Tissue Rex 213, 5368. Cicero, T. J., O’Connor, L. H., and Bell, R. D. (1987). Reevaluation of the effects of castration on naloxone-sensitive opiate receptors in the male rat brain. Neuroendocrinology 46, 176-184. Clark, C. R., Ball, A. J., and Hughes, J. (1984). Hypothalamic opiate receptors (mu, kappa, delta) are refractory to testosterone. Sot. Neurosci. Abrf. 1, 478. Cooper, S. J., and Turkish, S. (1981). Food and water intake in the nondeprived pigeon after morphine or naloxone administration. Neuropharmacology 20, 1053-1058. de Lanerolle, N. C., Elde, R. P., Sparber, S. B., and Frick, M. (1981). Distribution of methionine-enkephalin immunoreactivity in the chick brain: An immunohistochemical study. J. Comp. Neural. 199, 513-533. Denbow, D. M. (1985). Food intake control in birds. Neurosci. Biobehav. Rev. 9, 223232. Deviche, P. (1992). Regulation of food intake in a migratory songbird (Bunco hyemalis): Participation of endorphinergic mechanisms. Ornis Stand., in press. Deviche, P., and Cotter, P. (1991). Hypothalamic localization of kappa, delta, and mu opioid receptors in the songbird brain. Sot. Neurosci. A&. 1, 596. Deviche, P., and Schepers, G. (1984a). Naloxone treatment attenuates food but not water intake in domestic pigeons. Psychopharmacology 82, 122-126. Deviche, P., and Schepers, G. (1984b). Intracerebroventricular injection of ostrich p-endorphin to satiated pigeons produces hyperphagia but not hyperdipsia. Peptides 5,691694.
Deviche, P., and Wohland, A. (1984a). Behavioural characterization of the anorexic effect of naloxone in domestic pigeons. IRCS Med. Sci. 12, 427-428. Deviche, P., and Wohland, A. (1984b). Opiate antagonists stereoselectively attenuate the consumption of food but not of water by pigeons. Pharmacol. Biochem. Behav. 21, 507-512. Deviche, P., Melmer, G., and Schepers, G. (1984). Evidence that naloxone attenuates the
T-OPIOID INTERACTIONS
ON FEEDING
403
consumption of food by domestic pigeons through a central influence. Nenrophamrncology 23, 1173-1178. Diez, J. A.. and Roberts, D. L. (1982). Evidence contradicting the notion that gonadal hormones regulate brain opiate receptors. Biochem. Biophys. Res. Commun. 108,13131319. Foreman, K. ‘I’., Lea, R. W., and Buntin, J. D. (1990). Changes in feeding activity. plasma luteinizing hormone, and testes weight in ring doves following hypothalamic injections of prolactin. .I. Neuroendocrinol. 2, 667-673. Gentry, R. T., and Wade, G. N. (1976). Androgenic control of food intake and body weight in male rats. J. Comp. Physiol. Psychol. 90, 18-25. Gosnell, B. A. (1987). Central structures involved in opioid-induced feeding. Fed. Proc. 46, 163-167. Grandison. L., and Guidotti, A. (1977). Stimulation of food intake by muscimol and beta endorphin. Neuropharmacology 16, 533-536. Gray. J. M., and Greenwood, M. R. C. (1982). Time course of effects of ovarian hormones on food intake and metabolism. Am. J. Physiol. 243, E407-412. Hahn, E. F., and Fishman, J. (1979). Changes in rat brain opiate receptor content upon castration and testosterone replacement. Biochem. Biophys. Rex Commun. 90, 819823. Hahn. E. F., and Fishman, .I. (1985). Castration affects male rat brain opiate receptor content. Neuroendocrinology 41, 60-63. Hammer, R. P. (1985). The sex hormone-dependent development of opiate receptors in the rat medial preoptic area. Brain Res. 360, 65-74. Hammer, R. P. (1988). Opiate receptor ontogeny in the rat medial preoptic area is androgendependent. Neuroendocrinology 48, 336-341. Ketterson, E. D., Nolan, V., Jr., Wolf, L., Ziegenfus, C., Dufty, A. M., Ball, G. F., and Johnsen, T. S. (1991). Testosterone and avian life histories: The effect of experimentally elevated testosterone on corticosterone and body mass in dark-eyed juncos. Harm. Behnv. 25, 489-503. Kuenzel, W. J. (1982a). Transient aphagia produced following bilateral destruction of the lateral hypothalamic area and quinto-frontal tract of chicks. Physiol. Behav. 28, 237244. Kuenzel, W. J. (1982b). Central neural structures affecting food intake in birds: The lateral and ventral hypothalamic areas. In C. G. Scanes, M. A. Ottinger. A. D. Kenny, J. Balthazart, J. Cronshaw, and I. Chester Jones. (Eds.). Aspects of Asian Endocrkology, pp. 211-216. Texas Tech. Univ. Lapchak, P. A. (1991). Effect of estradiol treatment on &endorphin content and release in the female rat hypothalamus. Brain Res. 554, 198-202. McCormack, J. F., and Denbow, D. M. (1987). The effects of opioid antagonists on ingestive behavior in the domestic fowl. Pharmacol. Biochem. Behav. 27, 25-33. McCormack, J. F., and Denbow, D. M. (1988). Feeding. drinking and temperature responses to intracerebroventricular P-endorphin in the domestic fowl. Peptides 9, 709-715. McCormack, J. F.. and Denbow, D. M. (1989). Ingestive responses to mu and delta opioid receptor agonists in the domestic fowl. Br. Poultry Sci. 30, 327-340. Morley, J. E. (1987). Neuropeptide regulation of appetite and weight. Endocr. Rev. 8,256287. Morley, J. E.. and Levine, A. S. (1985). Neuropeptides and appetite regulation, Med. 1. Aust. 142, Sll-S13. Morley, J. E., Levine, A. S., Gosnell, B. A., Mitchell, J. E., Krahn, D. D., and Nizielski. S. E. (1985). Peptides and feeding. Pepfides 6(2), 181-192. Morley, J. E., Levine, A. S.. Grace, M., Kneip, J., and Gosnell. B. A. (1984). The effect
404
PIERRE
DEVICHE
of ovariectomy, estradiol and progesterone on opioid modulation of feeding. Physio/. Behav. 33, 237-241. Morrell, J. I., McGinty, J. F., and Pfaff, D. W. (198.5). A subset of P-endorphin- or dynorphin-containing neurons in the medial basal hypothalamus accumulates estradiol. Neuroendocrinolog~~ MorrisOn,
41, 41 J-426.
P. R. (1951). An automatic manometric respirometer. Rev. Sci. Instru. 22, 264-
267. Nakano, Y., Suda, T., Sumitomo, T., Tozawa, F., and Demura, H. (1991). Effects of sex steroids on /3-endorphin release from rat hypothalamus in virro. Bruin Res. 553, I-3. Neter, J., Wasserman, W., and Kutner, M. H. (1985). Applied Linear Statistical Models. Irwin, Homewood, IL. Olster, D. H., and Blaustein, J. D. (1990). Immunocytochemical colocalization of progestin receptors and /3-endorphin or enkephalin in the hypothalamus of female guinea pigs. J. Neurobiol.
21, 768-780.
Reiner, A., Brauth, S. E., Kitt, C. A., and Quirion, R. (1989). Distribution of mu, delta, and kappa opiate receptor types in the forebrain and midbrain of pigeons. J. Comp. Neural.
280, 359-382.
Robinzon, B., and Katz, Y. (1980). The effects of hypothalamic and mesencephalic lesions on food and water intakes, adiposity and some endocrine criteria, in the red-winged blackbird (Agelaius phoeniceus). Physiol. Behav. 24, 347-356. Rogers, C. M. (1991). An evaluation of the method of estimating body fat in birds by quantifying visible subcutaneous fat. J. Field Omithot. 62, 349-356. SAS Institute, Inc. (1985). SAS User’s Guide: Statistics. SAS Institute Inc., NC. Schwabl, H., and Farner, D. S. (1989). Endocrine and environmental control of vernal migration in male white-crowned sparrows, Zonotrichia leucophrys gambelii. Phpsiol. 2001. 62, l-10. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Silverin, B., Viebke, P. A., and Westin, J. (1989). Hormonal correlates of migration and territorial behavior in juvenile willow tits during autumn. Gen. Comp. Endocrinol. 75, 148-156. Tang, F. (1991). Endocrine control of hypothalamic and pituitary met-enkephalin and pendorphin contents. Neuroendocrinology 53, 68-76. Tepperman, F. S., Hirst, M., and Gowdey, C. W. (1981). Hypothalamic injection of morphine: Feeding and temperature responses. Life Sci. 28, 2459-2467. Wada, M. (1981). Effects of photostimulation, castration, and testosterone replacement on daily patterns of calling and locomotor activity in Japanese quail. Harm. Behav. 15, 270-281. Wada, M. (1982). Effects of sex steroids on calling, locomotor activity, and sexual behavior in castrated male Japanese quail. Horm. Behav. 16, 147-157. Wade, G. N., and Gray, J. M. (1979). Gonadal effects on food intake and adiposity: A metabolic hypothesis. Physiol. Behav. 22, 583-593. Wardlaw, S. L. (1986). Regulation of P-endorphin, corticotropin-like intermediate lobe peptide, and cY-melanotropin-stimulating hormone in the hypothalamus by testosterone. Endocrinology
119, 19-24.
Watson, A. (1970). Territorial and reproductive behavior of red grouse. J. Reprod. Fe&. Suppl. 11, 3-14.
Watson, J. T., and Adkins-Regan, E. (1989). Neuroanatomical localization of sex steroidconcentrating cells in the Japanese quail (Coturnix japonica): Autoradiography with [3HJtestosterone, [3H]estradiol, and [3H]dihydrotestosterone. Neuroendocrinobgy 49, 51-64.
T-OPIOID INTERACTIONS
ON FEEDING
405
Weathers. W. W., and Sullivan, K. A. (1989). Juvenile foraging proficiency, parental effort. and avian reproductive success. Ecol. Monogr. 59, 223-246. Weiland, N. G., and Wise, P. M. (1990). Estrogen and progesterone regulate opiate receptor densities in multiple brain regions. Endocrinology 126, 804-808. Wilkinson, M.. Brawer, J. R., and Wilkinson, D. A. (1985). Gonadal steroid-induced modification of opiate binding sites in anterior hypothalamus of female rats. Biol. Reprod. 32, 501-506. Wingfield, J. C., and Ramenofsky. M. (1985). Testosterone and aggressive behaviour during the reproductive cycle of male birds. In R. Gilles and J. Balthazart (Eds.), Neurobiology pp. 92-104. Springer-Verlag, Berlin. Yokoyama, K. (1976). Hypothalamic and hormonal control of the photoperiodically induced vernal functions in the white-crowned sparrow, ZonotricRia leucophrys gambelii. Ce!i Tissue Res. 174, 391-416.
