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Site-Specific Opioid Receptor Blockade Allows Prepubertal Guinea Pigs to Display Progesterone-Facilitated Lordosis
Hormones and Behavior 33, 115–124 (1998) Article No. HB981442
Site-Specific Opioid Receptor Blockade Allows Prepubertal Guinea Pigs to Display Progesterone-Facilitated Lordosis Deborah H. Olster1 Department of Psychology, University of California, Santa Barbara, California 93106 Received November 24, 1997; revised January 26, 1998; accepted February 27, 1998
Ovariectomized (OVX) juvenile guinea pigs (;3 weeks old) rarely display steroid-induced sexual receptivity. Systemic administration of the opioid receptor antagonist naloxone enhances the display of progesteronefacilitated lordosis in prepubertal females, suggesting that endogenous opioids tonically inhibit the expression of sexual receptivity at this age. This study was designed to ascertain the neural site(s) at which naloxone injection would stimulate lordosis in juvenile guinea pigs. Hartley guinea pigs were OVX at 10 –11 days of age and 2– 6 days later implanted with bilateral cannulae aimed at the medial preoptic area/anterior hypothalamus (MPOA/ AH), ventrolateral hypothalamus/ventromedial hypothalamus (VLH/VMH), or mesencephalic central gray (MCG). At 21–23 days of age, following administration of estradiol benzoate (10 mg) and progesterone (0.5 mg), naloxone (100 ng/side) or 0.9% saline was injected through the cannulae and the guinea pigs were tested for the display of lordosis. The MPOA/AH was the only site at which application of naloxone reliably elicited lordosis (87% positive response vs 12% for saline). Few females (<17%) displayed lordosis following injections of naloxone or saline into the VLH/VMH or MCG. A second experiment demonstrated that the stimulation of lordosis following MPOA/AH naloxone application was prevented by prior injection of the opioid agonist morphine (500 ng/side) at the same site. These data support the hypothesis that endogenous opioids acting in the MPOA/ AH, but not the VLH/VMH or MCG, tonically inhibit the display of progesterone-facilitated lordosis in prepubertal guinea pigs. © 1998 Academic Press
Sexual receptivity in adult rats and guinea pigs in response to sensory stimulation occurs only if the ovarian steroid hormone milieu is appropriate. Ovariectomized (OVX) females of these species do not dis1 To whom correspondence and reprint requests should be addressed. E-mail: [email protected].
0018-506X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
play lordosis, but replacement with estradiol alone or with subthreshold doses of estradiol followed by progesterone reinstates reproductive behaviors (Pfaff, Schwartz-Giblin, McCarthy, and Kow, 1994). Neonatal female rats (4 – 6 days of age) also display a lordosis response that is facilitated by ovarian steroid hormones (Williams and Blaustein, 1988). Newborn guinea pigs, which are highly precocious, exhibit the lordosis posture in response to anogenital licking by their dams, but the behavior is not facilitated by ovarian steroid hormones and disappears within 8 h after birth (Beach, 1966; Goy, Phoenix, and Meidinger, 1967). The ability to display steroid-induced sexual receptivity does not develop until 3–9 weeks of age in this species (Wilson and Young, 1941; Beach, 1966; Goy et al., 1967; Ryer and Feder, 1984a). In this laboratory, 3-week-old OVX Hartley guinea pigs rarely display progesterone-facilitated lordosis; by 40 –50 days of age, adult-typical behavioral responses are observed (Olster and Blaustein, 1989a,b). One approach to studying the mechanism(s) underlying the marked behavioral hyporesponsiveness to ovarian steroid hormones in immature guinea pigs focuses on putative deficiencies in components of the neural systems known to be involved in the display of steroidinduced sexual receptivity in adults. Among the deficiencies reported in immature compared to adult females are lower concentrations of hypothalamic estrogen receptors and estradiol-induced progestin receptors (Ryer and Feder, 1984b; Olster and Blaustein, 1989b, 1991; Olster, 1994b) and the inability to exhibit lordosis in response to a-noradrenergic receptor stimulation (Olster, 1998), a treatment which is effective in adults (Nock and Feder, 1979). A second (not mutually exclusive) thesis posits that the display of steroid-induced lordosis in prepubertal females is under tonic, inhibitory control. Evidence supporting this theory in115
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cludes reports that bilateral lesions of the medial preoptic area (MPOA) result in adult-typical lordosis responses in 3-week-old guinea pigs (Olster, 1995; Olster and Paulson, 1997) and the observation that injection of the opioid receptor antagonist naloxone allows juvenile females to display progesterone-facilitated lordosis (Olster, 1994a). The second observation is consistent with the hypothesis that endogenous opioids tonically suppress the display of lordosis in prepubertal guinea pigs. Notably, whereas injection of an exogenous opioid (i.e., morphine) suppresses the exhibition of lordosis in mature guinea pigs, administration of naloxone does not enhance the display of sexual receptivity in OVX adults pretreated with subthreshold doses of estradiol and progesterone (Nock and Cicero, 1991). Thus, adult guinea pigs are sensitive to the inhibitory effects of exogenous opioids on sexual receptivity, but tonic suppression of steroid-induced lordosis by endogenous opioid action is absent. This experiment was designed to ascertain the neural site(s) at which endogenous opioids act to inhibit the display of progesterone-facilitated lordosis in prepubertal guinea pigs. Since the site at which opioids suppress lordosis in adult guinea pigs is not known, three candidate sites were chosen based on data obtained in adult female rats: the medial preoptic area/ anterior hypothalamus (MPOA/AH), ventrolateral hypothalamus/ventromedial hypothalamus (VLH/ VMH), and mesencephalic central gray (MCG). Local application of endogenous or exogenous opioids (bendorphin or morphine, respectively) at these sites suppresses steroid-induced lordosis in adult rats, and infusion of naloxone to the MPOA and MCG stimulates the display of lordosis in OVX rats receiving subthreshold doses of estradiol and progesterone (Sirinathsinghji, 1984, 1986; Vathy, Van der Plas, Vincent, and Etgen, 1991). These inhibitory actions of opioids on progesterone-facilitated lordosis in rats appear to be mediated by m receptors (Wiesner and Moss, 1986; Pfaus and Pfaff, 1992). Selective m receptor binding has been found in MCG and MPOA in rats, but is nearly absent in the VMH (Mansour, Khachaturian, Lewis, Akil, and Watson, 1987; Hammer, 1990). A precise mapping of this receptor subtype in guinea pig brain has not been reported, but m receptor binding has been documented in homogenates of hypothalamus and midbrain of this species (Robson, Gillian, and Kosterlitz, 1985; Lahti, Mickelson, Jodelis, and McCall, 1989). m receptor mRNA has also been extracted from the MPOA and mediobasal hypothalamus of guinea pigs (Ro¨nnekleiv, Bosch, Cunningham, Wagner, Grandy, and Kelly, 1996). Furthermore, b-endorphin fibers are heavily concentrated in the
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preoptic region and scattered throughout the hypothalamus in adult guinea pigs (Thornton, Kelly, Loose, and Ro¨nnekleiv, 1994).
METHODS Animals Hartley guinea pigs were purchased from Charles River Laboratories (Wilmington, MA) and arrived at the University of California, Santa Barbara vivarium at 8 –9 days of age. They were housed in pairs in plastic tubs until brain cannula implantation, after which time they were housed individually. The animal room was maintained on a 12:12 photocycle with lights on from 0700 to 1900 PST. Food (5025 Guinea Pig Diet, PMI Feeds, Inc., St. Louis, MO) and water were available ad libitum. Surgery At 10 –11 days of age, all females were OVX under anesthesia induced by Metofane (methoxyflurane) and Innovar-Vet (0.4 mg droperidol plus 8.0 mg fentanyl im; both drugs purchased from Pittman-Moore, Washington Crossing, NJ). Two to six days later, the guinea pigs were anesthetized with a combination of chloral hydrate and sodium pentobarbital (72 and 15 mg/kg ip, respectively) for stereotaxic implantation of bilateral guide cannulae aimed at the MPOA/AH, VLH/VMH (26 gauge), or MCG (22 gauge). All cannulae were purchased from Plastics One, Inc. (Roanoke, VA). The stereotaxic coordinates were as follows (with the animal’s head in the flat-skull position, determined empirically): MPOA/AH: anterior/posterior, Bregma; medial/lateral, midline 6 0.5 mm; dorsal/ventral, dura 28.5 or 9.0 mm (Experiment 1) or dura 24.5 mm (Experiment 2, shorter guide cannulae used); VLH/VMH: anterior/posterior, Bregma 21.0 or 1.4 mm; medial/lateral, midline 6 1.0 mm; dorsal/ ventral, dura 210.0 mm; MCG (dorsal portion): anterior/posterior, Bregma 27.0 mm; medial/lateral, midline 60.6 mm; dorsal/ventral, dura 26.0 or 6.5 mm. Stylets (cut to the same lengths as the guide cannulae) were placed in the cannulae to maintain patency until intracerebral injections were performed. Experiment 1 Between 18 and 20 days of age, all females received single injections of estradiol benzoate (EB, 10 mg sc, in sesame oil, at 1600 –1700 h) followed 40 h later by
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progesterone (0.5 mg sc, in propylene glycol; both steroids purchased from Sigma Chemical Co., St. Louis, MO). Immediately before the progesterone injection and at hourly intervals for 4 h thereafter, females were tested for the display of lordosis by manual palpation in their home cages (Young, Dempsey, Hagquist, and Boling, 1937). Females displaying lordosis for 2 s or more during these first four hourly tests were dropped from the remainder of the experiment (N 5 2). Four and a half to five hours after injection of progesterone, while the animals were gently restrained by the investigator, the stylets were removed and replaced with injection cannulae (28 or 33 gauge) that extended 1.0 mm beyond the tips of the guide cannulae. Each female then received either naloxone (100 ng/0.5 ml/side; naloxone HCl, pH 7.0, Sigma) or the 0.9% saline vehicle (0.5 ml/side, pH 7.0), injected with a Hamilton syringe attached via PE-50 tubing to the injection cannulae. The injection cannulae were left in place for 1 min to facilitate diffusion of the fluid around the injection site and to prevent reflux. Beginning 15 min after the intracerebral injections, all animals were tested for the display of lordosis at 15-min intervals for 2.5 h and then at 30-min intervals until all females ceased displaying lordosis (as defined by the absence of the behavior on three consecutive 30-min tests). One hour after that (i.e., 3– 4 h after the first set of naloxone/saline injections), the treatment groups were reversed, i.e., those females previously receiving naloxone received saline and vice versa, and the testing protocol was repeated. Behavioral testing was performed with the investigator blind to the treatment groups. A positive lordosis response after intracerebral injection was defined as the display of lordosis for a minimum of 2 s on two consecutive 15-min tests. This criterion was used in anticipation of a relatively short-duration, stimulatory effect of naloxone on sexual receptivity, as has been observed previously following systemic administration of the compound (Olster, 1994a). In addition to the percentage of positive responders following the various treatments, the latency to lordosis (relative to the intracerebral injection), maximum lordosis duration (maximum length of time the animal held the posture on any given test), and heat duration were recorded (in positive responders). Experiment 2 After a positive response to the injection of naloxone into the MPOA/AH was observed in Experiment 1, a second experiment was performed to demonstrate
blockade of the naloxone effect by injection of the opioid agonist morphine into the same site. OVX females received the same hormone regimen as described above (10 mg EB at 18 –20 days of age, followed 40 h later by 0.5 mg progesterone) and were tested for the display of lordosis immediately before and at hourly intervals for 4 h after progesterone administration. After verification that none of the guinea pigs was sexually receptive, morphine (500 ng/0.5 ml/side, morphine sulfate, pH 7.0, Sigma) or the 0.9% saline vehicle (0.5 ml/side) was injected 4.5–5 h after progesterone administration, through injection cannulae that extended 5 mm beyond the tips of the more dorsally placed (compared to Experiment 1) MPOA/AH guide cannulae. Twenty to thirty minutes later, all of the morphine-treated guinea pigs received an injection of naloxone (100 ng/0.5 ml/side, pH 7.0); the animals which had received saline were given injections of naloxone or a second saline injection to the same site. This protocol yielded three treatment groups: saline– saline, saline–naloxone, morphine–naloxone. One round of behavioral testing was performed as described above, beginning 15 min after the second intracerebral injection.
Histology After the behavioral testing was completed, all females were transcardially perfused with 0.9% saline followed by 10% buffered formalin. Brains were stored for several days in 20% sucrose/10% buffered formalin and then sectioned at 40 mm, mounted, and Nissl stained with cresyl violet. The histology was examined without knowledge of the behavioral data. An injection was considered correctly placed if the tips of the injection cannulae were located bilaterally anywhere in the target region. Data from animals whose cannulae missed the intended targets were excluded from the subsequent statistical analysis. In Experiment 1, for each brain site tested, the frequency of positive responses following intracerebral injections of naloxone or saline was compared by the binomial test (Siegel and Castellan, 1988). In Experiment 2, the percentages of females displaying lordosis following sequential injections of morphine and naloxone or saline and naloxone compared to animals receiving two saline injections were compared by Fisher’s Exact Probability test. In all cases P , 0.05 was the criterion for statistical significance. The latency to display lordosis, maximum lordosis duration, and heat duration data in drug- vs vehicle-treated groups could not be analyzed statistically due to the small number of positive responders in the
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groups receiving intracerebral injections of saline or morphine plus naloxone.
RESULTS The locations of the correctly placed injection sites for the three targets (MPOA/AH, VLH/VMH, and MCG) from Experiment 1 are illustrated in Fig. 1. The injections aimed at the MPOA/AH also reached the medial preoptic nucleus and extended rostrally into the nucleus of the diagonal band of Broca (Fig. 1A). The VLH/VMH injection sites were concentrated along the lateral edge of the VMH or in the VLH (Fig. 1B), a steroid hormone receptor-containing region in the guinea pig hypothalamus that includes the ventrolateral nucleus and its dorsal surround, extending up to the fornix (Blaustein, King, Toft, and Turcotte, 1988). The MCG injections were in the dorsal portion of this area (Fig. 1C). Data from 21 guinea pigs were excluded from the analysis due to misplaced injections. These ‘‘miss– hits’’ were usually due to injections that were below the base of the brain or too rostral (for the MPOA/AH and VLH/VMH targets) or too dorsal (for the MCG target). Of these animals with misplaced injections, none (of three) targeted for the MPOA/AH, one (of nine) targeted for the VLH/ VMH, and two (of nine) targeted for the MCG showed lordosis following injections of naloxone. None responded to saline administration. The percentages of females displaying positive lordosis responses following correctly placed injections of 0.9% saline or naloxone (100 ng/side, bilaterally) are shown in Fig. 2. The MPOA/AH was the only site at which naloxone administration reliably induced the display of lordosis: 87% of females displayed lordosis following naloxone injections to this site, compared to a 12% response rate to the saline injections. The order of injections did not influence the response to naloxone: 4/4 animals receiving naloxone first displayed lordosis vs 3/4 which received the saline first. The characteristics of the lordosis display in the positive responders with MPOA/AH cannulae are shown in Table 1. In contrast to the positive results in MPOA/AH-targeted animals, injection of naloxone or saline into the VLH/VMH or MCG rarely induced the display of lordosis. Fewer than 17% of females (i.e., not more than one animal in any group) responded (Fig. 2). Having found that naloxone injections into the MPOA/AH enhance the display of progesterone-facilitated lordosis, Experiment 2 was designed to demonstrate the specificity of an opioid effect, by attempting to prevent the stimulatory effects of naloxone administra-
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tion to the MPOA/AH by prior injection of the opioid agonist morphine into the same region. The correctly placed injection sites and behavioral data from animals in this second experiment are illustrated in Figs. 3 and 4, respectively. The injection sites for the animals in this study were a bit more rostrally located than those in Experiment 1 (compare Figs. 3 and 1A), but still ranged from the nucleus of the diagonal band of Broca caudally to the AH. Replicating the results of Experiment 1, injections of saline followed by naloxone into the MPOA/AH induced the display of lordosis in a significantly higher percentage of females than two injections of saline. Furthermore, injection of morphine (500 ng/side, bilaterally) into the MPOA/AH 20–30 min prior to naloxone administration at the same site prevented the facilitatory effect of naloxone on lordosis, with animals in this group responding no differently from those receiving two saline injections (i.e., ,15% of females displaying lordosis). The characteristics of the lordosis response stimulated by naloxone in this second experiment are shown in Table 1. These could not be compared statistically to the results obtained in Experiment 1 due to the differences in experimental protocol, but positive responses to naloxone in Experiment 2 tended to be stronger (shorter latency, longer maximum lordosis duration). Data from 11 animals were discarded from Experiment 2 because the injections were below the base of the brain or asymmetrical, with one injection going into the third ventricle and the other into the lateral POA. Interestingly, three (of five) of the animals which received sequential injections of saline and naloxone into the third ventricle and lateral hypothalamus also showed lordosis, possibly due to diffusion of the drug from the ventricular system to the MPOA/AH.
DISCUSSION These data extend the previous observation that systemic administration of the opioid receptor antagonist naloxone induces the display of progesteronefacilitated lordosis in otherwise steroid-hyporesponsive, juvenile guinea pigs, by demonstrating that the MPOA/AH is one site at which opioid receptor blockade is effective in this regard. The characteristics of the lordosis response (i.e., latency, maximum lordosis duration, heat duration) following injection of 100 ng of the drug bilaterally into the MPOA/AH were very similar to those observed following the optimal dose of systemically administered naloxone (2 mg/kg SC; Olster, 1994a), but in both cases, not as robust as observed in adult females responding to these doses of EB and progesterone (Olster and Blaustein, 1989a,b).
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FIG. 1. Injection sites from correctly placed cannulae aimed at the medial preoptic area/anterior hypothalamus (A, N 5 8), ventrolateral/ ventromedial hypothalamus (B, N 5 6) and mesencephalic central gray (C, N 5 7) in Experiment 1. AC, anterior commissure; AH, anterior hypothalamus; Aq, cerebral aqueduct; ARC, arcuate nucleus; CC, corpus callosum; CPu, caudate putamen; CS, superior colliculus; f, fornix; IP, interpeduncular nucleus; LS, lateral septum; MCG, mesencephalic central gray; MPN, medial preoptic nucleus; MPOA, medial preoptic area; oc, optic chiasm; ot, optic tract; PON, pons; PVN, paraventricular nucleus; SN, substantia nigra; VLH, ventrolateral hypothalamus; VMH, ventromedial hypothalamus.
This short-duration effect of naloxone is probably due to its 30- to 40-min half-life (as measured in the circulation and brain of rats; Ngai, Berkowitz, Yang, Hemp-
stead, and Spector, 1976; these data are not available for guinea pigs). Furthermore, the stimulatory effect of naloxone on sexual receptivity appears to be opioid
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FIG. 2. Percentage of steroid-treated, OVX females displaying lordosis following bilateral injections of naloxone (100 ng/0.5 ml/side) or vehicle (0.5 ml 0.9% saline/side into the medial preoptic area/anterior hypothalamus (MPOA/AH, N 5 8), ventrolateral hypothalamus/ ventromedial hypothalamus (VLH/VMH, N 5 6) or mesencephalic central gray (MCG, N 5 7). *P , 0.05 vs response to saline.
specific, as its action was eliminated by prior injection of morphine at the same site. The results of this study suggest that the exhibition of steroid-induced lordosis in prepubertal females is under tonic inhibitory con-
TABLE 1 Characteristics of the Lordosis Response Following Injections into the Medial Preoptic Area/Anterior Hypothalamusa
Treatment Experiment 1 Naloxone Saline Experiment 2 Saline/naloxone Saline/saline Morphine/naloxone
Latency to lordosis (min)
Maximum lordosis duration (s)
Heat duration (min)
70.7 6 10.2 60
5.8 6 0.7 5
57.8 6 10.0 45
32.1 6 8.3 45 30
10.0 6 3.7 7 22
57.8 6 14.7 90 60
a Positive responders only (N 5 7/8 for naloxone trial and N 5 1/8 for saline trial in Experiment 1; N 5 7/10 for saline/naloxone group, N 5 1/9 for saline/saline group, and N 5 1/11 for morphine/naloxone group in Experiment 2).
trol by endogenous opioids acting in the MPOA/AH. In contrast, naloxone injections into two other candidate sites, i.e., the VLH/VMH and MCG, were ineffective. All three are sites at which injections of morphine (VMH) or b-endorphin (MPOA, MCG) suppress the display of steroid-induced lordosis in adult rats (Sirinathsinghji, 1984, 1986; Vathy et al., 1991). It is possible that higher doses of naloxone injected into the VLH/VMH or MCG would have been effective. The 100-ng dose of naloxone (per side) is optimal, when administered into the MPOA or MCG, for stimulating lordosis in OVX adult rats receiving subthreshold replacement doses of estradiol and progesterone (Sirinathsinghji, 1984, 1986). Furthermore, the optimal systemic dose for stimulation of lordosis in adult rats and juvenile guinea pigs is the same (2 mg/kg SC; Saito, Aoki, Hokao, Amao, Wakafuji, Sugiyama, and Takahashi, 1992; Olster, 1994a). Naloxone and morphine are bound with high affinity and preferentially by m opioid receptors in guinea pig brain (Paterson, Robson, and Kosterlitz, 1983; Loew, Toll, Lawson, Frenking, and Polgar, 1989). Although this experiment could not ascertain the opioid
Opioid Blockade and Lordosis
FIG. 3. Correctly placed injection sites of cannulae aimed at the medial preoptic area/anterior hypothalamus (MPOA/AH) in Experiment 2 (N 5 30). DBB, nucleus of the diagonal band of Broca; MS, medial septum; other abbreviations defined in legend to Fig. 1.
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receptor subtype involved in the sexual behavioral response to these drugs, the possibility of it being mediated by m receptors is tenable, as m receptor mRNA has been isolated from guinea pig POA (Ro¨nnekleiv et al., 1996). Furthermore, the POA and rostral hypothalamus have also been shown to receive substantial b-endorphin innervation in adult guinea pigs (Thornton et al., 1994). Although the source of these fibers has not been determined in this species, b-endorphin neurons originating in the arcuate nucleus innervate the MPOA in rats (Wilcox, Roberts, Chronwall, Bishop, and O’Donohue, 1986). In both species, b-endorphin neurons in the arcuate nucleus contain ovarian steroid hormone receptors (Fox, Harlan, Shivers, and Pfaff, 1990; Olster and Blaustein, 1990) and this population appears to be steroid responsive in guinea pigs (Thornton et al., 1994). The notion that puberty may result from the removal of an overriding, inhibitory system stems from early work demonstrating acceleration of puberty by specific brain lesions (Donovan and van der Werff den Bosch, 1956). A decrease in responsiveness to the inhibitory actions of opioid peptides on luteinizing hormone secretion is characteristic of sexual maturation in rats (Bhanot and Wilkinson, 1983). In fact, chronic opioid receptor blockade by naloxone treatment accelerates puberty (vaginal opening, first estrus, and ovulation) in rats and hamsters (Sirinathsinghji, Motta, and Martini, 1985; Donham, Sarafidis, and Stetson, 1986). The results of the current guinea pig study and those obtained by other investigators in adults (Nock and Cicero, 1991) are consistent with the notion that the behavioral component of sexual maturation in this species may entail abatement of inhibitory opiatergic tone. Exactly how this is achieved— e.g. reduction in opioid release or responsiveness or activity downstream from opioid action—remains to be determined. The mechanism underlying opioid inhibition over the display of steroid-induced lordosis in guinea pigs has not been elucidated. In steroid-treated, OVX rats, systemic morphine administration inhibits the release of norepinephrine in the VMH (as measured by in vivo microdialysis) that usually occurs coincident with the onset of mating, and morphine applied directly to this brain region suppresses the display of steroid-induced lordosis (Vathy et al., 1991). These data suggest that morphine acts directly in the VMH to inhibit norepinephrine release, the latter being a critical event for the onset of sexual receptivity (Etgen, Ungar, and Petitti, 1992). In guinea pigs, too, there is strong evidence that noradrenergic activity may mediate the stimulatory effects of ovarian steroids on lordosis (Nock and Feder, 1979). However, it is unlikely that tonic, endog-
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FIG. 4. Percentage of steroid-treated, OVX females displaying lordosis following sequential, bilateral injections of the saline vehicle (Sal–Sal; 0.5 ml/side/injection, N 5 9), saline and naloxone (Sal–Nalox; 0.5 ml saline/side 1 100 ng naloxone/side, N 5 10), or morphine and naloxone (Mor–Nalox; 500 ng morphine/side 1 100 ng naloxone/side, N 5 11) into the medial preoptic area/anterior hypothalamus. *P , 0.05 vs. Sal-Sal group.
enous opioid inhibition of norepinephrine release in the VMH is the mechanism underlying the behavioral hyporesponsiveness of juvenile guinea pigs to estradiol and progesterone. For instance, the current study demonstrates that opioid receptor blockade in the VLH/VMH does not enhance the display of steroidinduced lordosis in prepubertal females. Although one could posit that endogenous opioids released in the MPOA could be preventing lordosis by inhibiting norepinephrine release in the same region, this idea is inconsistent with data showing that noradrenergic receptor stimulation in the mediobasal hypothalamus, and not the MPOA, stimulates sexual receptivity in adult guinea pigs (Malik, Morrell, and Feder, 1993). Furthermore, the suppressive effect of systemic morphine injection on the display of sexual receptivity in adults is not blocked by administration of the a-noradrenergic receptor agonist, clonidine (Nock and Ci-
cero, 1991). Finally, Hartley guinea pigs do not become responsive to the lordosis-enhancing actions of noradrenergic stimulation until after 48 days of age (Olster, 1998). Therefore, even if naloxone were antagonizing opioid inhibition over norepinephrine release elsewhere in the brain, animals at this age (approximately 3 weeks) presumably would be unresponsive to noradrenergic stimulation of lordosis. The data from this experiment fit well with another observation from this laboratory, i.e., that destruction of the MPOA/AH (by electrolytic lesion) allows prepubertal guinea pigs to display adult-typical lordosis responses to estradiol and progesterone (Olster, 1995; Olster and Paulson, 1997). Perhaps MPOA/AH lesions are behaviorally effective because this manipulation removes this region’s inhibitory, opiatergic influence over the display of sexual receptivity at this age. Both observations are consistent with the hypoth-
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esis that the exhibition of adult-typical, behavioral responses to ovarian steroid hormones is under tonic, inhibitory control in prepubertal guinea pigs. The mechanism by which this suppressive influence diminishes as animals mature will be the subject of future study.
ACKNOWLEDGMENTS The author thanks Gavin Magnuson, Adam Fox, and Stephanie Richardson for their technical assistance. This work was supported by NIH HD 28636.
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kappa opioid receptors in the rat forebrain and midbrain. J. Neurosci. 7, 2445–2464. Ngai, S. H., Berkowitz, B. A., Yang, J. C., Hempstead, J., and Spector, S. (1976). Pharmacokinetics of naloxone in rats and in man: Basis for its potency and short duration of action. Anesthesiology 44, 398 – 401. Nock, B., and Cicero, T. J. (1991). Morphine suppresses the ovarian steroid hormone-dependent lordosis response of female guinea pigs: Reversal by naloxone but not clonidine. Horm. Behav. 25, 29 –37. Nock, B., and Feder, H. H. (1979). Noradrenergic transmission and female sexual behavior of guinea pigs. Brain Res. 166, 369 –380. Olster, D. H. (1994a). Opiate receptor blockade enhances the display of progesterone-facilitated lordosis in juvenile female guinea pigs. Horm. Behav. 28, 84 –95. Olster, D. H. (1994b). Hypothalamic estrogen receptor-immunoreactivity in prepubertal vs adult female guinea pigs. J. Neuroendocrinol. 6, 617– 625. Olster, D. H. (1995). Progesterone-facilitated lordosis in medial preoptic area-lesioned, juvenile guinea pigs. Horm. Behav. 29, 519 –530. Olster, D. H. (1998). Reproductive behavioral responsiveness to noradrenergic stimulation in developing guinea pigs. Pharmacol. Biochem. Behav., 59, 551–556. Olster, D. H., and Blaustein, J. D. (1989a). Development of steroidinduced lordosis in female guinea pigs: Effects of different estradiol and progesterone treatments, clonidine, and early weaning. Horm. Behav. 23, 118 –129. Olster, D. H., and Blaustein, J. D. (1989b). Development of progesterone-facilitated lordosis in female guinea pigs: Relationship to neural estrogen and progestin receptors. Brain Res. 484, 168 –176. Olster, D. H., and Blaustein, J. D. (1990). Immunocytochemical colocalization of progestin receptors and b-endorphin or enkephalin in the hypothalamus of female guinea pigs. J. Neurobiol. 21, 768 –780. Olster, D. H., and Blaustein, J. D. (1991). Development of estradiolinduced progestin receptor immunoreactivity in the hypothalamus of female guinea pigs. J. Neurobiol. 22, 195–203. Olster, D. H., and Paulson, K. C. (1997). Effects of medial preoptic area and septal lesions on puberty in female guinea pigs. Biol. Reprod. 56, 731–738. Paterson, S. J., Robson, L. E., and Kosterlitz, H. W. (1983). Classification of opioid receptors. Br. Med. Bull. 39, 31–36. Pfaff, D. W., Schwartz-Giblin, S., McCarthy, M. M., and Kow, L.-M. (1994). Cellular and molecular mechanisms of female reproductive behaviors. In E. Knobil and J. D. Neill (Eds.), The Physiology of Reproduction, 2nd ed., Vol. 2, pp. 107–220. Raven Press, New York. Pfaus, J. G., and Pfaff, D. W. (1992). m-, d- and k-opioid receptor agonists selectively modulate sexual behaviors in the female rat: Differential dependence on progesterone. Horm. Behav. 26, 457– 473. Robson, L. E., Gillian, M. G. C., and Kosterlitz, H. W. (1985). Species differences in the concentrations and distributions of opioid binding sites. Eur. J. Pharmacol. 112, 65–71. Ro¨nnekleiv, O. K., Bosch, M. A., Cunningham, M. J., Wagner, E. J., Grandy, D. K., and Kelly, M. J. (1996). Downregulation of m-opioid receptor mRNA in the mediobasal hypothalamus of the female guinea pig following morphine treatment. Neurosci. Lett. 216, 129 –132. Ryer, H. I., and Feder, H. H. (1984a). Development of steroid– receptor systems in guinea pig brain. I. Cytoplasmic estrogen receptors. Dev. Brain Res. 13, 9 –14. Ryer, H. I., and Feder, H. H. (1984b). Development of steroid– receptor systems in guinea pig brain. II. Cytoplasmic progestin receptors. Dev. Brain Res. 13, 15–21.
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Saito, T. R., Aoki, S., Hokao, R., Amao, H., Wakafuji, Y., Sugiyama, M., and Takahashi, K. W. (1992). Effects of naloxone and naltrexone on receptive and proceptive behaviors in female rats. Exp. Anim. 41, 75–77. Siegel, S., and Castellan, N. J., Jr. (1988) Nonparametric Statistics for the Behavioral Sciences, 2nd ed., pp. 1–399. McGraw-Hill, New York. Sirinathsinghji, D. J. S. (1984). Modulation of lordosis behavior of female rats by naloxone, b-endorphin and its antiserum in the mesencephalic central gray: Possible mediation via GnRH. Neuroendocrinology 39, 222–230. Sirinathsinghji, D. J. S. (1986). Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, b-endorphin/ corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area. Brain Res. 375, 49 –56. Sirinathsinghji, D. J. S., Motta, M., and Martini, L. (1985). Induction of precocious puberty in the female rat after chronic naloxone administration during the neonatal period: The opiate ‘brake’ on prepubertal gonadotrophin secretion. J. Endocrinol. 104, 299 –307. Thornton, J. E., Loose, M. D., Kelly, M. J., and Ro¨nnekleiv, O. K. (1994). Effects of estrogen on the number of neurons expressing
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b-endorphin in the medial basal hypothalamus of the female guinea pig. J. Comp. Neurol. 341, 68 –77. Vathy, I., Van der Plas, J., Vincent, P. A., and Etgen, A. M. (1991). Intracranial dialysis and microinfusion studies suggest that morphine may act in the ventromedial hypothalamus to inhibit female rat sexual behavior. Horm. Behav. 25, 354 –366. Wiesner, J. B., and Moss, R. L. (1986). Behavioral specificity of b-endorphin suppression of sexual behavior: Differential receptor antagonism. Pharmacol. Biochem. Behav. 24, 1235–1239. Wilcox, J. N., Roberts, J. L., Chronwall, B. M., Bishop, J. F., and O’Donohue, T. (1986). Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections. J. Neurosci. Res. 16, 89 –96. Williams, C. L., and Blaustein, J. D. (1988). Steroids induce hypothalamic progestin receptors and facilitate female sexual behavior in neonatal rats. Brain Res. 449, 403– 407. Wilson, J. G., and Young, W. C. (1941). Sensitivity to estrogen studied by means of experimentally induced mating responses in the female guinea pig and rat. Endocrinology 29, 779 –783. Young, W. C., Dempsey, E. W., Hagquist, C. W., and Boling, J. L. (1937). The determination of heat in the guinea pig. J. Lab. Clin. Med. 23, 300 –303.
Site-Specific Opioid Receptor Blockade Allows Prepubertal Guinea Pigs to Display Progesterone-Facilitated Lordosis Deborah H. Olster1 Department of Psychology, University of California, Santa Barbara, California 93106 Received November 24, 1997; revised January 26, 1998; accepted February 27, 1998
Ovariectomized (OVX) juvenile guinea pigs (;3 weeks old) rarely display steroid-induced sexual receptivity. Systemic administration of the opioid receptor antagonist naloxone enhances the display of progesteronefacilitated lordosis in prepubertal females, suggesting that endogenous opioids tonically inhibit the expression of sexual receptivity at this age. This study was designed to ascertain the neural site(s) at which naloxone injection would stimulate lordosis in juvenile guinea pigs. Hartley guinea pigs were OVX at 10 –11 days of age and 2– 6 days later implanted with bilateral cannulae aimed at the medial preoptic area/anterior hypothalamus (MPOA/ AH), ventrolateral hypothalamus/ventromedial hypothalamus (VLH/VMH), or mesencephalic central gray (MCG). At 21–23 days of age, following administration of estradiol benzoate (10 mg) and progesterone (0.5 mg), naloxone (100 ng/side) or 0.9% saline was injected through the cannulae and the guinea pigs were tested for the display of lordosis. The MPOA/AH was the only site at which application of naloxone reliably elicited lordosis (87% positive response vs 12% for saline). Few females (<17%) displayed lordosis following injections of naloxone or saline into the VLH/VMH or MCG. A second experiment demonstrated that the stimulation of lordosis following MPOA/AH naloxone application was prevented by prior injection of the opioid agonist morphine (500 ng/side) at the same site. These data support the hypothesis that endogenous opioids acting in the MPOA/ AH, but not the VLH/VMH or MCG, tonically inhibit the display of progesterone-facilitated lordosis in prepubertal guinea pigs. © 1998 Academic Press
Sexual receptivity in adult rats and guinea pigs in response to sensory stimulation occurs only if the ovarian steroid hormone milieu is appropriate. Ovariectomized (OVX) females of these species do not dis1 To whom correspondence and reprint requests should be addressed. E-mail: [email protected].
0018-506X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
play lordosis, but replacement with estradiol alone or with subthreshold doses of estradiol followed by progesterone reinstates reproductive behaviors (Pfaff, Schwartz-Giblin, McCarthy, and Kow, 1994). Neonatal female rats (4 – 6 days of age) also display a lordosis response that is facilitated by ovarian steroid hormones (Williams and Blaustein, 1988). Newborn guinea pigs, which are highly precocious, exhibit the lordosis posture in response to anogenital licking by their dams, but the behavior is not facilitated by ovarian steroid hormones and disappears within 8 h after birth (Beach, 1966; Goy, Phoenix, and Meidinger, 1967). The ability to display steroid-induced sexual receptivity does not develop until 3–9 weeks of age in this species (Wilson and Young, 1941; Beach, 1966; Goy et al., 1967; Ryer and Feder, 1984a). In this laboratory, 3-week-old OVX Hartley guinea pigs rarely display progesterone-facilitated lordosis; by 40 –50 days of age, adult-typical behavioral responses are observed (Olster and Blaustein, 1989a,b). One approach to studying the mechanism(s) underlying the marked behavioral hyporesponsiveness to ovarian steroid hormones in immature guinea pigs focuses on putative deficiencies in components of the neural systems known to be involved in the display of steroidinduced sexual receptivity in adults. Among the deficiencies reported in immature compared to adult females are lower concentrations of hypothalamic estrogen receptors and estradiol-induced progestin receptors (Ryer and Feder, 1984b; Olster and Blaustein, 1989b, 1991; Olster, 1994b) and the inability to exhibit lordosis in response to a-noradrenergic receptor stimulation (Olster, 1998), a treatment which is effective in adults (Nock and Feder, 1979). A second (not mutually exclusive) thesis posits that the display of steroid-induced lordosis in prepubertal females is under tonic, inhibitory control. Evidence supporting this theory in115
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cludes reports that bilateral lesions of the medial preoptic area (MPOA) result in adult-typical lordosis responses in 3-week-old guinea pigs (Olster, 1995; Olster and Paulson, 1997) and the observation that injection of the opioid receptor antagonist naloxone allows juvenile females to display progesterone-facilitated lordosis (Olster, 1994a). The second observation is consistent with the hypothesis that endogenous opioids tonically suppress the display of lordosis in prepubertal guinea pigs. Notably, whereas injection of an exogenous opioid (i.e., morphine) suppresses the exhibition of lordosis in mature guinea pigs, administration of naloxone does not enhance the display of sexual receptivity in OVX adults pretreated with subthreshold doses of estradiol and progesterone (Nock and Cicero, 1991). Thus, adult guinea pigs are sensitive to the inhibitory effects of exogenous opioids on sexual receptivity, but tonic suppression of steroid-induced lordosis by endogenous opioid action is absent. This experiment was designed to ascertain the neural site(s) at which endogenous opioids act to inhibit the display of progesterone-facilitated lordosis in prepubertal guinea pigs. Since the site at which opioids suppress lordosis in adult guinea pigs is not known, three candidate sites were chosen based on data obtained in adult female rats: the medial preoptic area/ anterior hypothalamus (MPOA/AH), ventrolateral hypothalamus/ventromedial hypothalamus (VLH/ VMH), and mesencephalic central gray (MCG). Local application of endogenous or exogenous opioids (bendorphin or morphine, respectively) at these sites suppresses steroid-induced lordosis in adult rats, and infusion of naloxone to the MPOA and MCG stimulates the display of lordosis in OVX rats receiving subthreshold doses of estradiol and progesterone (Sirinathsinghji, 1984, 1986; Vathy, Van der Plas, Vincent, and Etgen, 1991). These inhibitory actions of opioids on progesterone-facilitated lordosis in rats appear to be mediated by m receptors (Wiesner and Moss, 1986; Pfaus and Pfaff, 1992). Selective m receptor binding has been found in MCG and MPOA in rats, but is nearly absent in the VMH (Mansour, Khachaturian, Lewis, Akil, and Watson, 1987; Hammer, 1990). A precise mapping of this receptor subtype in guinea pig brain has not been reported, but m receptor binding has been documented in homogenates of hypothalamus and midbrain of this species (Robson, Gillian, and Kosterlitz, 1985; Lahti, Mickelson, Jodelis, and McCall, 1989). m receptor mRNA has also been extracted from the MPOA and mediobasal hypothalamus of guinea pigs (Ro¨nnekleiv, Bosch, Cunningham, Wagner, Grandy, and Kelly, 1996). Furthermore, b-endorphin fibers are heavily concentrated in the
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preoptic region and scattered throughout the hypothalamus in adult guinea pigs (Thornton, Kelly, Loose, and Ro¨nnekleiv, 1994).
METHODS Animals Hartley guinea pigs were purchased from Charles River Laboratories (Wilmington, MA) and arrived at the University of California, Santa Barbara vivarium at 8 –9 days of age. They were housed in pairs in plastic tubs until brain cannula implantation, after which time they were housed individually. The animal room was maintained on a 12:12 photocycle with lights on from 0700 to 1900 PST. Food (5025 Guinea Pig Diet, PMI Feeds, Inc., St. Louis, MO) and water were available ad libitum. Surgery At 10 –11 days of age, all females were OVX under anesthesia induced by Metofane (methoxyflurane) and Innovar-Vet (0.4 mg droperidol plus 8.0 mg fentanyl im; both drugs purchased from Pittman-Moore, Washington Crossing, NJ). Two to six days later, the guinea pigs were anesthetized with a combination of chloral hydrate and sodium pentobarbital (72 and 15 mg/kg ip, respectively) for stereotaxic implantation of bilateral guide cannulae aimed at the MPOA/AH, VLH/VMH (26 gauge), or MCG (22 gauge). All cannulae were purchased from Plastics One, Inc. (Roanoke, VA). The stereotaxic coordinates were as follows (with the animal’s head in the flat-skull position, determined empirically): MPOA/AH: anterior/posterior, Bregma; medial/lateral, midline 6 0.5 mm; dorsal/ventral, dura 28.5 or 9.0 mm (Experiment 1) or dura 24.5 mm (Experiment 2, shorter guide cannulae used); VLH/VMH: anterior/posterior, Bregma 21.0 or 1.4 mm; medial/lateral, midline 6 1.0 mm; dorsal/ ventral, dura 210.0 mm; MCG (dorsal portion): anterior/posterior, Bregma 27.0 mm; medial/lateral, midline 60.6 mm; dorsal/ventral, dura 26.0 or 6.5 mm. Stylets (cut to the same lengths as the guide cannulae) were placed in the cannulae to maintain patency until intracerebral injections were performed. Experiment 1 Between 18 and 20 days of age, all females received single injections of estradiol benzoate (EB, 10 mg sc, in sesame oil, at 1600 –1700 h) followed 40 h later by
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progesterone (0.5 mg sc, in propylene glycol; both steroids purchased from Sigma Chemical Co., St. Louis, MO). Immediately before the progesterone injection and at hourly intervals for 4 h thereafter, females were tested for the display of lordosis by manual palpation in their home cages (Young, Dempsey, Hagquist, and Boling, 1937). Females displaying lordosis for 2 s or more during these first four hourly tests were dropped from the remainder of the experiment (N 5 2). Four and a half to five hours after injection of progesterone, while the animals were gently restrained by the investigator, the stylets were removed and replaced with injection cannulae (28 or 33 gauge) that extended 1.0 mm beyond the tips of the guide cannulae. Each female then received either naloxone (100 ng/0.5 ml/side; naloxone HCl, pH 7.0, Sigma) or the 0.9% saline vehicle (0.5 ml/side, pH 7.0), injected with a Hamilton syringe attached via PE-50 tubing to the injection cannulae. The injection cannulae were left in place for 1 min to facilitate diffusion of the fluid around the injection site and to prevent reflux. Beginning 15 min after the intracerebral injections, all animals were tested for the display of lordosis at 15-min intervals for 2.5 h and then at 30-min intervals until all females ceased displaying lordosis (as defined by the absence of the behavior on three consecutive 30-min tests). One hour after that (i.e., 3– 4 h after the first set of naloxone/saline injections), the treatment groups were reversed, i.e., those females previously receiving naloxone received saline and vice versa, and the testing protocol was repeated. Behavioral testing was performed with the investigator blind to the treatment groups. A positive lordosis response after intracerebral injection was defined as the display of lordosis for a minimum of 2 s on two consecutive 15-min tests. This criterion was used in anticipation of a relatively short-duration, stimulatory effect of naloxone on sexual receptivity, as has been observed previously following systemic administration of the compound (Olster, 1994a). In addition to the percentage of positive responders following the various treatments, the latency to lordosis (relative to the intracerebral injection), maximum lordosis duration (maximum length of time the animal held the posture on any given test), and heat duration were recorded (in positive responders). Experiment 2 After a positive response to the injection of naloxone into the MPOA/AH was observed in Experiment 1, a second experiment was performed to demonstrate
blockade of the naloxone effect by injection of the opioid agonist morphine into the same site. OVX females received the same hormone regimen as described above (10 mg EB at 18 –20 days of age, followed 40 h later by 0.5 mg progesterone) and were tested for the display of lordosis immediately before and at hourly intervals for 4 h after progesterone administration. After verification that none of the guinea pigs was sexually receptive, morphine (500 ng/0.5 ml/side, morphine sulfate, pH 7.0, Sigma) or the 0.9% saline vehicle (0.5 ml/side) was injected 4.5–5 h after progesterone administration, through injection cannulae that extended 5 mm beyond the tips of the more dorsally placed (compared to Experiment 1) MPOA/AH guide cannulae. Twenty to thirty minutes later, all of the morphine-treated guinea pigs received an injection of naloxone (100 ng/0.5 ml/side, pH 7.0); the animals which had received saline were given injections of naloxone or a second saline injection to the same site. This protocol yielded three treatment groups: saline– saline, saline–naloxone, morphine–naloxone. One round of behavioral testing was performed as described above, beginning 15 min after the second intracerebral injection.
Histology After the behavioral testing was completed, all females were transcardially perfused with 0.9% saline followed by 10% buffered formalin. Brains were stored for several days in 20% sucrose/10% buffered formalin and then sectioned at 40 mm, mounted, and Nissl stained with cresyl violet. The histology was examined without knowledge of the behavioral data. An injection was considered correctly placed if the tips of the injection cannulae were located bilaterally anywhere in the target region. Data from animals whose cannulae missed the intended targets were excluded from the subsequent statistical analysis. In Experiment 1, for each brain site tested, the frequency of positive responses following intracerebral injections of naloxone or saline was compared by the binomial test (Siegel and Castellan, 1988). In Experiment 2, the percentages of females displaying lordosis following sequential injections of morphine and naloxone or saline and naloxone compared to animals receiving two saline injections were compared by Fisher’s Exact Probability test. In all cases P , 0.05 was the criterion for statistical significance. The latency to display lordosis, maximum lordosis duration, and heat duration data in drug- vs vehicle-treated groups could not be analyzed statistically due to the small number of positive responders in the
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groups receiving intracerebral injections of saline or morphine plus naloxone.
RESULTS The locations of the correctly placed injection sites for the three targets (MPOA/AH, VLH/VMH, and MCG) from Experiment 1 are illustrated in Fig. 1. The injections aimed at the MPOA/AH also reached the medial preoptic nucleus and extended rostrally into the nucleus of the diagonal band of Broca (Fig. 1A). The VLH/VMH injection sites were concentrated along the lateral edge of the VMH or in the VLH (Fig. 1B), a steroid hormone receptor-containing region in the guinea pig hypothalamus that includes the ventrolateral nucleus and its dorsal surround, extending up to the fornix (Blaustein, King, Toft, and Turcotte, 1988). The MCG injections were in the dorsal portion of this area (Fig. 1C). Data from 21 guinea pigs were excluded from the analysis due to misplaced injections. These ‘‘miss– hits’’ were usually due to injections that were below the base of the brain or too rostral (for the MPOA/AH and VLH/VMH targets) or too dorsal (for the MCG target). Of these animals with misplaced injections, none (of three) targeted for the MPOA/AH, one (of nine) targeted for the VLH/ VMH, and two (of nine) targeted for the MCG showed lordosis following injections of naloxone. None responded to saline administration. The percentages of females displaying positive lordosis responses following correctly placed injections of 0.9% saline or naloxone (100 ng/side, bilaterally) are shown in Fig. 2. The MPOA/AH was the only site at which naloxone administration reliably induced the display of lordosis: 87% of females displayed lordosis following naloxone injections to this site, compared to a 12% response rate to the saline injections. The order of injections did not influence the response to naloxone: 4/4 animals receiving naloxone first displayed lordosis vs 3/4 which received the saline first. The characteristics of the lordosis display in the positive responders with MPOA/AH cannulae are shown in Table 1. In contrast to the positive results in MPOA/AH-targeted animals, injection of naloxone or saline into the VLH/VMH or MCG rarely induced the display of lordosis. Fewer than 17% of females (i.e., not more than one animal in any group) responded (Fig. 2). Having found that naloxone injections into the MPOA/AH enhance the display of progesterone-facilitated lordosis, Experiment 2 was designed to demonstrate the specificity of an opioid effect, by attempting to prevent the stimulatory effects of naloxone administra-
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tion to the MPOA/AH by prior injection of the opioid agonist morphine into the same region. The correctly placed injection sites and behavioral data from animals in this second experiment are illustrated in Figs. 3 and 4, respectively. The injection sites for the animals in this study were a bit more rostrally located than those in Experiment 1 (compare Figs. 3 and 1A), but still ranged from the nucleus of the diagonal band of Broca caudally to the AH. Replicating the results of Experiment 1, injections of saline followed by naloxone into the MPOA/AH induced the display of lordosis in a significantly higher percentage of females than two injections of saline. Furthermore, injection of morphine (500 ng/side, bilaterally) into the MPOA/AH 20–30 min prior to naloxone administration at the same site prevented the facilitatory effect of naloxone on lordosis, with animals in this group responding no differently from those receiving two saline injections (i.e., ,15% of females displaying lordosis). The characteristics of the lordosis response stimulated by naloxone in this second experiment are shown in Table 1. These could not be compared statistically to the results obtained in Experiment 1 due to the differences in experimental protocol, but positive responses to naloxone in Experiment 2 tended to be stronger (shorter latency, longer maximum lordosis duration). Data from 11 animals were discarded from Experiment 2 because the injections were below the base of the brain or asymmetrical, with one injection going into the third ventricle and the other into the lateral POA. Interestingly, three (of five) of the animals which received sequential injections of saline and naloxone into the third ventricle and lateral hypothalamus also showed lordosis, possibly due to diffusion of the drug from the ventricular system to the MPOA/AH.
DISCUSSION These data extend the previous observation that systemic administration of the opioid receptor antagonist naloxone induces the display of progesteronefacilitated lordosis in otherwise steroid-hyporesponsive, juvenile guinea pigs, by demonstrating that the MPOA/AH is one site at which opioid receptor blockade is effective in this regard. The characteristics of the lordosis response (i.e., latency, maximum lordosis duration, heat duration) following injection of 100 ng of the drug bilaterally into the MPOA/AH were very similar to those observed following the optimal dose of systemically administered naloxone (2 mg/kg SC; Olster, 1994a), but in both cases, not as robust as observed in adult females responding to these doses of EB and progesterone (Olster and Blaustein, 1989a,b).
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FIG. 1. Injection sites from correctly placed cannulae aimed at the medial preoptic area/anterior hypothalamus (A, N 5 8), ventrolateral/ ventromedial hypothalamus (B, N 5 6) and mesencephalic central gray (C, N 5 7) in Experiment 1. AC, anterior commissure; AH, anterior hypothalamus; Aq, cerebral aqueduct; ARC, arcuate nucleus; CC, corpus callosum; CPu, caudate putamen; CS, superior colliculus; f, fornix; IP, interpeduncular nucleus; LS, lateral septum; MCG, mesencephalic central gray; MPN, medial preoptic nucleus; MPOA, medial preoptic area; oc, optic chiasm; ot, optic tract; PON, pons; PVN, paraventricular nucleus; SN, substantia nigra; VLH, ventrolateral hypothalamus; VMH, ventromedial hypothalamus.
This short-duration effect of naloxone is probably due to its 30- to 40-min half-life (as measured in the circulation and brain of rats; Ngai, Berkowitz, Yang, Hemp-
stead, and Spector, 1976; these data are not available for guinea pigs). Furthermore, the stimulatory effect of naloxone on sexual receptivity appears to be opioid
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FIG. 2. Percentage of steroid-treated, OVX females displaying lordosis following bilateral injections of naloxone (100 ng/0.5 ml/side) or vehicle (0.5 ml 0.9% saline/side into the medial preoptic area/anterior hypothalamus (MPOA/AH, N 5 8), ventrolateral hypothalamus/ ventromedial hypothalamus (VLH/VMH, N 5 6) or mesencephalic central gray (MCG, N 5 7). *P , 0.05 vs response to saline.
specific, as its action was eliminated by prior injection of morphine at the same site. The results of this study suggest that the exhibition of steroid-induced lordosis in prepubertal females is under tonic inhibitory con-
TABLE 1 Characteristics of the Lordosis Response Following Injections into the Medial Preoptic Area/Anterior Hypothalamusa
Treatment Experiment 1 Naloxone Saline Experiment 2 Saline/naloxone Saline/saline Morphine/naloxone
Latency to lordosis (min)
Maximum lordosis duration (s)
Heat duration (min)
70.7 6 10.2 60
5.8 6 0.7 5
57.8 6 10.0 45
32.1 6 8.3 45 30
10.0 6 3.7 7 22
57.8 6 14.7 90 60
a Positive responders only (N 5 7/8 for naloxone trial and N 5 1/8 for saline trial in Experiment 1; N 5 7/10 for saline/naloxone group, N 5 1/9 for saline/saline group, and N 5 1/11 for morphine/naloxone group in Experiment 2).
trol by endogenous opioids acting in the MPOA/AH. In contrast, naloxone injections into two other candidate sites, i.e., the VLH/VMH and MCG, were ineffective. All three are sites at which injections of morphine (VMH) or b-endorphin (MPOA, MCG) suppress the display of steroid-induced lordosis in adult rats (Sirinathsinghji, 1984, 1986; Vathy et al., 1991). It is possible that higher doses of naloxone injected into the VLH/VMH or MCG would have been effective. The 100-ng dose of naloxone (per side) is optimal, when administered into the MPOA or MCG, for stimulating lordosis in OVX adult rats receiving subthreshold replacement doses of estradiol and progesterone (Sirinathsinghji, 1984, 1986). Furthermore, the optimal systemic dose for stimulation of lordosis in adult rats and juvenile guinea pigs is the same (2 mg/kg SC; Saito, Aoki, Hokao, Amao, Wakafuji, Sugiyama, and Takahashi, 1992; Olster, 1994a). Naloxone and morphine are bound with high affinity and preferentially by m opioid receptors in guinea pig brain (Paterson, Robson, and Kosterlitz, 1983; Loew, Toll, Lawson, Frenking, and Polgar, 1989). Although this experiment could not ascertain the opioid
Opioid Blockade and Lordosis
FIG. 3. Correctly placed injection sites of cannulae aimed at the medial preoptic area/anterior hypothalamus (MPOA/AH) in Experiment 2 (N 5 30). DBB, nucleus of the diagonal band of Broca; MS, medial septum; other abbreviations defined in legend to Fig. 1.
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receptor subtype involved in the sexual behavioral response to these drugs, the possibility of it being mediated by m receptors is tenable, as m receptor mRNA has been isolated from guinea pig POA (Ro¨nnekleiv et al., 1996). Furthermore, the POA and rostral hypothalamus have also been shown to receive substantial b-endorphin innervation in adult guinea pigs (Thornton et al., 1994). Although the source of these fibers has not been determined in this species, b-endorphin neurons originating in the arcuate nucleus innervate the MPOA in rats (Wilcox, Roberts, Chronwall, Bishop, and O’Donohue, 1986). In both species, b-endorphin neurons in the arcuate nucleus contain ovarian steroid hormone receptors (Fox, Harlan, Shivers, and Pfaff, 1990; Olster and Blaustein, 1990) and this population appears to be steroid responsive in guinea pigs (Thornton et al., 1994). The notion that puberty may result from the removal of an overriding, inhibitory system stems from early work demonstrating acceleration of puberty by specific brain lesions (Donovan and van der Werff den Bosch, 1956). A decrease in responsiveness to the inhibitory actions of opioid peptides on luteinizing hormone secretion is characteristic of sexual maturation in rats (Bhanot and Wilkinson, 1983). In fact, chronic opioid receptor blockade by naloxone treatment accelerates puberty (vaginal opening, first estrus, and ovulation) in rats and hamsters (Sirinathsinghji, Motta, and Martini, 1985; Donham, Sarafidis, and Stetson, 1986). The results of the current guinea pig study and those obtained by other investigators in adults (Nock and Cicero, 1991) are consistent with the notion that the behavioral component of sexual maturation in this species may entail abatement of inhibitory opiatergic tone. Exactly how this is achieved— e.g. reduction in opioid release or responsiveness or activity downstream from opioid action—remains to be determined. The mechanism underlying opioid inhibition over the display of steroid-induced lordosis in guinea pigs has not been elucidated. In steroid-treated, OVX rats, systemic morphine administration inhibits the release of norepinephrine in the VMH (as measured by in vivo microdialysis) that usually occurs coincident with the onset of mating, and morphine applied directly to this brain region suppresses the display of steroid-induced lordosis (Vathy et al., 1991). These data suggest that morphine acts directly in the VMH to inhibit norepinephrine release, the latter being a critical event for the onset of sexual receptivity (Etgen, Ungar, and Petitti, 1992). In guinea pigs, too, there is strong evidence that noradrenergic activity may mediate the stimulatory effects of ovarian steroids on lordosis (Nock and Feder, 1979). However, it is unlikely that tonic, endog-
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FIG. 4. Percentage of steroid-treated, OVX females displaying lordosis following sequential, bilateral injections of the saline vehicle (Sal–Sal; 0.5 ml/side/injection, N 5 9), saline and naloxone (Sal–Nalox; 0.5 ml saline/side 1 100 ng naloxone/side, N 5 10), or morphine and naloxone (Mor–Nalox; 500 ng morphine/side 1 100 ng naloxone/side, N 5 11) into the medial preoptic area/anterior hypothalamus. *P , 0.05 vs. Sal-Sal group.
enous opioid inhibition of norepinephrine release in the VMH is the mechanism underlying the behavioral hyporesponsiveness of juvenile guinea pigs to estradiol and progesterone. For instance, the current study demonstrates that opioid receptor blockade in the VLH/VMH does not enhance the display of steroidinduced lordosis in prepubertal females. Although one could posit that endogenous opioids released in the MPOA could be preventing lordosis by inhibiting norepinephrine release in the same region, this idea is inconsistent with data showing that noradrenergic receptor stimulation in the mediobasal hypothalamus, and not the MPOA, stimulates sexual receptivity in adult guinea pigs (Malik, Morrell, and Feder, 1993). Furthermore, the suppressive effect of systemic morphine injection on the display of sexual receptivity in adults is not blocked by administration of the a-noradrenergic receptor agonist, clonidine (Nock and Ci-
cero, 1991). Finally, Hartley guinea pigs do not become responsive to the lordosis-enhancing actions of noradrenergic stimulation until after 48 days of age (Olster, 1998). Therefore, even if naloxone were antagonizing opioid inhibition over norepinephrine release elsewhere in the brain, animals at this age (approximately 3 weeks) presumably would be unresponsive to noradrenergic stimulation of lordosis. The data from this experiment fit well with another observation from this laboratory, i.e., that destruction of the MPOA/AH (by electrolytic lesion) allows prepubertal guinea pigs to display adult-typical lordosis responses to estradiol and progesterone (Olster, 1995; Olster and Paulson, 1997). Perhaps MPOA/AH lesions are behaviorally effective because this manipulation removes this region’s inhibitory, opiatergic influence over the display of sexual receptivity at this age. Both observations are consistent with the hypoth-
Opioid Blockade and Lordosis
esis that the exhibition of adult-typical, behavioral responses to ovarian steroid hormones is under tonic, inhibitory control in prepubertal guinea pigs. The mechanism by which this suppressive influence diminishes as animals mature will be the subject of future study.
ACKNOWLEDGMENTS The author thanks Gavin Magnuson, Adam Fox, and Stephanie Richardson for their technical assistance. This work was supported by NIH HD 28636.
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