Preoptic Area Infusions of Morphine Disrupt—and Naloxone Restores—Parental-Like Behavior in Juvenile Rats

Brain Research Bulletin, Vol. 44, No. 2, pp. 183–191, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 1 .00

PII S0361-9230(97)00111-1

Preoptic Area Infusions of Morphine Disrupt—and Naloxone Restores—Parental-Like Behavior in Juvenile Rats JACQUELINE WELLMAN, DAVID CARR, AMANDA GRAHAM, HENDREE JONES, J. LEIGH HUMM, MICHAEL RUSCIO, BLASE BILLACK AND CRAIG H. KINSLEY1 Department of Psychology, University of Richmond, Richmond, VA 23173, USA [Received 22 October 1996; Revised 17 March 1997; Accepted 22 April 1997] ABSTRACT: As in the adult lactating female, opioids disrupt (and naloxone restores), parental behavior in juvenile rats (;25 days of age). Because the preoptic area regulates the display of parental behavior in lactating females, we examined its parental behavior role in the juvenile rat. At 21 days of age, juvenile rats were implanted with bilateral cannulae aimed at the preoptic area using a modified Kopf stereotaxic and extrapolating from a developing-rat brain atlas [58], and divided into two groups: initiation and maintenance. On day 25, the Initiation group received bilateral infusions of either morphine (0.50 mg), saline (0.25 ml), or morphine plus naloxone (0.25 mg). Thirty minutes later, they were exposed to three 1– 6-day-old pups; the maintenance group was exposed to pups until they displayed 2 consecutive days of parental behavior, then infused. Morphine disrupted parental behavior in both the Initiation and Maintenance groups, and naloxone restored the behavior to control/ saline levels. Parental behavior in the juvenile animal of both sexes, therefore, is under opioid regulation that parallels the adult female. © 1997 Elsevier Science Inc.

parous females the latency to respond to pups approximates 4 – 6 days and in males, 6 – 8 days [52,53,55]. Laboratory and field research has shown that the juveniles of many species perform various parental-like behaviors including food provision, defense, and retrieval of young [7]. Parental behavior is subject to disruption and interference [2]. Bridges and Grimm [3] demonstrated an inhibitory influence of morphine on parental behavior in studies using pregnancy-terminated females (who normally display a rapid onset of the behavior). When the effect was blocked with the opiate antagonist naloxone the females’ parental behavior was completely restored. Grimm and Bridges [19] replicated and extended these data, showing additional disruptions in parental behavior following morphine treatment. And, Rubin and Bridges [54] demonstrated central mediation of the opioid effect by specific administration of morphine sulfate. When pregnancy-terminated females were infused with morphine into the POA, 100% of the females failed to engage in the behavior and, when naloxone was infused concurrent with the morphine, the effect was reversed. Therefore, opiates, in particular mu-receptor ligands (cf., [33]) disrupt parental behavior in a very selective, naloxone-reversible fashion. The medial preoptic area (mPOA) regulates parental behavior in females [12,42– 47]. For instance, lesions of the mPOA disrupt many components of parental behavior in the rat, including retrieving, nest building, nursing behaviors, and other pup-directed responses [20,42,47]. The mPOA contains a high concentration of estrogen- and progesterone-binding neurons, and may be the primary central site wherein gonadal steroids such as progesterone and estradiol influence the behavior [12,51] in the adult. Given the behavioral similarities between the parental responsiveness of lactating females and juvenile animals, would morphine decrease parental behavior in the prepubertal, and obviously, nonlactating animal? Juvenile male and female animals (25 days of age) treated systemically with morphine (5.0 mg/kg) and saline failed to respond to neonates over a 10-day period [29]. Morphine and naloxone (0.5 mg/kg) or saline-treated juveniles responded in just a few days, latencies that are comparable to nontreated juveniles. In a second experiment, juveniles that had been sensitized to foster neonates and then treated with morphine and saline failed to

KEY WORDS: Preoptic area infusions, Morphine, Naloxone.

INTRODUCTION Parental care is not limited to the reproductively experienced. Prepubertal rats will exhibit parental behavior toward pups in the absence of the conditions of pregnancy and/or parturition, similar to sensitized/concaveated virgin females (i.e., those exposed to neonates; [36,52]). Behaviors elicited from these sensitized juveniles include nest building, retrieval and grouping of pups, licking of the anogenital region to stimulate urination and defecation, lying close to or on top of the pups, and the adoption of a crouching posture [35–37]. Juvenile rats (20 –30 days of age) of both sexes show a rapid onset (2–3 day latency) of full parental behavior (FPB), which includes the retrieval and grouping of, and crouching over, pups [5,8,18,36,37]. (Although licking of pups is another integral part of parental behavior we do not include it as a variable in the present study.) In these particular behavioral aspects, therefore, juvenile rats resemble adult postpartum females. The rapid onset of parental behavior at this age stands in marked contrast to the adult expression of the behavior, where in nulli1

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respond to pups with FPB, compared to juveniles treated with morphine and naloxone or saline. These data [29] suggest that the onset and maintenance of parental behavior in juvenile animals appear to be regulated by opiates. The current work examines if central administration of the opiate morphine directly into the POA disrupts parental behavior in juvenile rats. Furthermore, will blockade of morphine’s effects with the narcotic antagonist naloxone restore the behavior within juvenile rats? MATERIALS AND METHODS Animals Female Sprague–Dawley rats originally purchased from Charles River Laboratories, Inc. were timed-mated with stud males. Following positive sperm identification in the vaginal lavage, the females were isolated until parturition. At 20 days of age (day of birth 5 day 0), offspring of both sexes were housed individually in 20 3 45 3 25 polypropylene cages, the floors of which were covered with pine shavings. Food (Purina rat chow) and water were available ad lib and all animals were housed in light (on from 0500 –1900 h) and temperature (21–24°C)-controlled testing rooms. Animals used in this study in were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Richmond and those prepared by the Committee on Care and Use of Laboratory Animal Resources, National Research Council [DHHS publication No. (NIH) 85-23, Revised, 1985, 1996]. Stereotaxic Surgery At 21 days of age, each animal was outfitted with bilateral, double-barreled cannulae assemblies (Plastics One, Roanoke, VA) aimed at the preoptic area (POA) consisting of 23-gauge outer guide cannulae and 28-gauge inserts. Animals were anesthetized with sodium pentobarbital and guide cannulae were implanted using standard stereotaxic procedures and a Kopf stereotaxic instrument modified for the juvenile skull by blunting the ear bars. Implant coordinates were chosen according to the developmental atlas of Sherwood and Timiras [58]. Because the animals did not start testing until 25 days of age, and could conceivably be tested until day 36, 15 days following stereotaxic surgery, implant coordinates were chosen, accounting for predicted maturation, by averaging the anterior–posterior, medial–lateral, and ventral– dorsal measurements of rats 21 and 39 days of age (the next age for which coordinates were provided in the atlas). Using the auditory meatus as the zero or reference point, the extrapolated POA coordinates we used for rats at surgery (21 days of age) was 6.5 mm anterior– posterior, 0.8 mm medial–lateral, and 3.6 mm ventral– dorsal. Guide cannulae were cut to 6.2 mm in length and lowered into the brain through two small holes drilled into the skull with a dental drill. The implant was secured to the skull with dental cement and the skin sutured around it. Dummy insert cannulae cut to just the same length as the guide cannulae were placed in the guide cannulae between testing periods. Removable infusion cannulae were cut 1 mm longer than the guide cannulae to reach the specific brain sites. Drug Administration Drugs were dissolved in sterile saline and a total volume of 0.5 ml of the solution was infused bilaterally (0.25 m 3 2/side) through the infusion cannulae at a rate of 1.0 ml/min. using an automatic syringe infusion pump (Harvard Instruments). The infusion cannula was left in place for 30 s following the infusion to allow for adequate dispersion and to prevent backflow.

Procedure Experiment 1—initiation. All testing began at 0800 h at 25 days of age. In this first phase of the study, the juvenile males and females were administered one of three regimens (n 5 6/group): Morphine (0.50 mg/0.25 ml saline) 1 saline (MOR 1 SAL); morphine 1 naloxone (0.25 mg/0.25 ml saline; MOR 1 NAL); or saline (0.25 ml) 1 saline (0.25 ml; SAL 1 SAL). One dosage of drug or control substance (0.25 ml) was administered, the infusion cannula was removed, and the second dosage (0.25 ml) was then administered; the process was repeated on the contralateral side. Thus, each hemisphere received 0.50 ml, for a total volume per brain of 1.0 ml. To initiate testing, 30-min after drug/saline infusion, three 1- to 6-day-old freshly-suckled pups obtained from donor mothers were proffered to the subjects (into a corner opposite the location of the subject) and their behavior was scored (by observers blind to the treatment) continuously for 15 min and then spot-checked again at 30, 45, and 60 min. The behaviors scored (and hereafter designated as FPB) consisted of retrieving, grouping, and crouching over young during the 60-min test session. Animals were scored as fully parental if they retrieved the three test young, grouped them, and crouched over them within the 60-min test period for 2 consecutive testing days. The latency of the animal (in days) to exhibit a parental response was based on the first day of two consecutive test sessions on which FPB was observed. (For example, if an animal displayed FPB on day 3 and then again on day 4, a latency of 3 days was assigned.) Each animal was exposed to rat pups continuously for the duration of testing (10 days). On each day at the time of testing, the position of the juvenile and the pups that had remained in the cage overnight was recorded. Regular testing occurred 30 min after this pretest. Any animal not responding for the duration of the experiment was assigned a latency of 10 days. Experiment 2—maintenance. Juvenile male and female rats (n 5 6/group) were exposed to three freshly suckled 1- to 6-dayold neonates for a period of days until they displayed 2 consecutive days of FPB as described above. On the day following the second day of FPB, the three different groups were treated with one of the same three regimens as in the initiation phase (Experiment 1): MOR 1 SAL, MOR 1 NAL or SAL 1 SAL. Thirty minutes later, animals were exposed to three 1- to 6-day-old neonates and behavior was observed and scored for 60 min, continuously for the initial 15 min, and at 15-min intervals thereafter. Histological Analysis Animals were killed for verification of cannulae placement sites no later than 38 days of age. All animals were killed with an overdose of sodium pentobarbital and perfused intracardially with saline followed by 10% formalin. The brain was frozen-sectioned at 80-mm intervals through the area of the cannulae and stained with thionin. Cannula placement for each animal was examined for its proximity to/above the POA. Data were discarded for any animal in which cannula tracks were not in the vicinity of the POA (6 100 m), and analyses were done only on verified animals. Statistical Analysis To determine the overall significant differences in the latencies to the onset of FPB among the three groups in Experiment 1, the Kruskal–Wallis H-statistic was used. The Mann–Whitney U-statistic for small samples was used to analyze the differences when comparing the latencies between any two groups. To analyze Experiment 2, the percentage of animals displaying FPB after treatment was calculated and the Fishers’ exact probability test was used.

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FIG. 1. Median latency (in days) to display two consecutive days of full parental behavior (FPB) for morphine plus saline (MOR 1 SAL), saline-alone (SAL 1 SAL), and morphine plus naloxone (MOR 1 NAL) males and females. In males, latency to display FPB was significantly longer for MOR 1 SAL compared to both SAL 1 SAL (p , 0.032) and MOR 1 NAL (p , 0.004). In females, latency to display FPB was significantly longer for MOR 1 SAL compared to SAL 1 SAL (p , 0.05). Note: Data in Experiment 1 were analyzed using the Kruskal–Wallis test followed by the Mann–Whitney-U. The significant differences reported in this figure are from the Mann–Whitney-U test. See the text and Table 1 for additional significant differences in individual components of FPB.

RESULTS Experiment 1—Initiation Figure 1 displays the median latencies for the three treatment groups, morphine 1 saline (MOR 1 SAL), morphine 1 naloxone (MOR 1 NAL), or saline 1 saline (SAL 1 SAL) to exhibit full parental behavior in Experiment 1, Initiation. It is evident that morphine had a significant effect on the parental behavior displayed by juvenile rats. For the males, the overall Kruskal–Wallis revealed a significant effect of morphine treatment on the latency to display two consecutive days of FPB (H 5 7.94, p , 0.05). Further, latencies to display one day of FPB (H 5 12.03, p , 0.01), to retrieve one (H 5 7.80, p , 0.05), two (H 5 8.54, p , 0.05), and three pups (H 5 8.17, p , 0.05), to group (H 5 11.94, p , 0.01), and to crouch over pups (H 5 9.77, p , 0.01) were significantly extended by morphine treatment in juvenile males. The follow-up with the Mann–Whitney U showed that, in males, latencies to display two consecutive days of FPB were

significantly longer in the MOR 1 SAL group vs. the SAL 1 SAL group (U 5 6.0, p , 0.032) (see Fig. 1), as were latencies to exhibit one day of FPB (U 5 0.0, p , 0.001), to retrieve one (U 5 2.0, p , 0.004), two (U 5 2.0, p , 0.004), and three pups (U 5 3.5, p , 0.01) (see Table 1). Further, latencies to group pups (U 5 0.0, p , 0.001), and to crouch over the pups (U 5 1.0, p , 0.002) were significantly longer for the MOR 1 SAL group relative to the SAL 1 SAL group. Moreover, the MOR 1 SAL group required significantly longer than the MOR 1 NAL group to display 2 consecutive days of FPB (U 5 0.0, p , 0.004) (see Fig. 1), to display 1 day of FPB (U 5 0.0, p , 0.004), to retrieve one (U 5 5.0, p , 0.021), two (U 5 7.5, p , 0.057), and three pups (U 5 3.0, p , 0.008), to group pups (U 5 0.0, p , 0.001), and to crouch over the pups (U 5 8.0, p , 0.066) (see Table 1). There were significant differences between the MOR 1 NAL and the SAL 1 SAL groups for latencies to retrieve two pups (U 5 6.5, p , 0.04), and the latency to crouch over pups (U 5 4.5, p , 0.017), demonstrating that naloxone was mostly capable of antagonizing

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WELLMAN ET AL. TABLE 1 MEDIAN LATENCIES IN DAYS FOR JUVENILE MALE AND FEMALE RATS TO EXHIBIT FULL PARENTAL BEHAVIOR (FPB) AND ITS VARIOUS COMPONENTS FOR EXPERIMENT 1, INITIATION Juvenile Males

1 day FPB Retrieve 1 pup Retrieve 2 pups Retrieve 3 pups Group Crouch N’s

Juvenile Females

MOR 1 SAL

MOR 1 NAL

SAL 1 SAL

MOR 1 SAL

MOR 1 NAL

SAL 1 SAL

10a†b† 7a*b† 7b†a‡ 10a†b† 10a†b† 8a‡b† 6

4 2 3b* 4 4 3b* 6

2 1 1c 1 2 1c 6

10a*b* 6a†b† 6a†b* 10a†b* 10a†b* 6a†b† 5

4 3 3 3 3 1 6

4 2 4 4 4 2 5

Overall data were analyzed with the Kruskal–Wallis H statistic, with individual comparisons performed with the Mann–Whitney U. * p , 0.05; † at least p , 0.02; ‡ trend, p , 0.06. a Significantly different from MOR 1 NAL; b significantly different from SAL 1 SAL; c significant sex difference, same treatment group, p , 0.05. See comparisons in text for specific p-values.

the disruptive effects of morphine on the parental behavior displayed by juveniles, though morphine effects were not completely antagonized. For the juvenile females in Experiment 1, sample size for both the MOR 1 SAL and SAL 1 SAL groups was only five each, instead of the intended six; unfortunately, data were discarded following the histological analysis because cannulae placement could not be accurately determined. Consequently, some of the probability values are affected (see below). The Kruskal–Wallis revealed, however, that morphine treatment in females was similarly disruptive on the latencies to retrieve one (H 5 8.11, p , 0.05), two (H 5 8.42, p , 0.05), and three pups (H 5 7.71, p , 0.05), to group pups (H 5 8.22, p , 0.05), and to crouch over pups (H 5 9.12, p , 0.05). Individual comparisons within the female groups showed that the MOR 1 SAL group took significantly longer than the SAL 1 SAL group to display 2 consecutive days of FPB (U 5 3.0, p , 0.041) (see Fig. 1), to display 1 day of FPB (U 5 2.5, p , 0.012), to retrieve one (U 5 2.0, p , 0.009), two (U 5 2.5, p , 0.012), and three pups (U 5 3.5, p , 0.021), to group pups (U 5 2.5, p , 0.012), and to crouch over pups (U 5 0.0, p , 0.041) (see Table 1). Furthermore, MOR 1 SAL females took significantly longer than MOR 1 NAL females to display 1 day FPB (U 5 4.5, p , 0.017), to retrieve one (U 5 0.5, p , 0.003), two (U 5 0.5, p , 0.003), and three pups (U 5 0.5, p , 0.003), to group (U 5 0.5, p , 0.003), and to crouch over the pups (U 5 1.0, p , 0.004) (see Table 1). There was a trend for MOR 1 SAL females to be different in latency to display 2 consecutive days of FPB (U 5 7.5, p , 0.106). There were no significant differences between MOR 1 NAL animals and SAL 1 SAL animals. There were few individual sex differences in behavior. The Mann–Whitney U statistic revealed, however, significant differences between male and female SAL 1 SAL groups for retrieving two pups (U 5 4.5, p , 0.017) and for crouching over pups (U 5 5.5, p , 0.027) (see Table 1). There were no significant differences in any behaviors between male and female MOR 1 SAL or MOR 1 NAL groups. Experiment 2—Maintenance Experiment 1 demonstrated that morphine disrupted, and naloxone could reverse, the initiation of parental behavior displayed by male and female juvenile rats. Experiment 2 sought to examine

how morphine would affect the established behavior in sensitized juvenile males and females (Fig. 2, Table 2). For males, following 2 consecutive days of FPB in the absence of treatment, significantly fewer of the MOR 1 SAL group displayed FPB on the day of injection compared to either the MOR 1 NAL or SAL 1 SAL groups, according to the Fishers Exact test, (MOR 1 SAL vs. MOR 1 NAL, p 5 0.001; vs. SAL 1 SAL, p 5 0.001). There were no significant differences between MOR 1 NAL and SAL 1 SAL males. Additional analyses revealed that significantly fewer MOR 1 SAL than MOR 1 NAL males retrieved one (p 5 0.001), two (p 5 0.001), and three pups (p 5 0.001), grouped (p 5 0.001), and crouched-over pups (p 5 0.001) following treatment. For MOR 1 SAL vs. SAL 1 SAL, there were significant differences for retrieving one (p 5 0.001), two (p 5 0.001), and three pups (p 5 0.001), grouping pups (p 5 0.001), and crouching over pups (p 5 0.001). There were no significant differences between MOR 1 NAL and SAL 1 SAL. For females, sample size for all three groups was five instead of six due to misplacement of cannula location observed during histological analysis (see Fig. 3 for example of cannula placement in representative animal). Significantly fewer MOR 1 SAL treated females displayed FPB compared to both MOR 1 NAL and SAL 1 SAL animals (MOR 1 SAL vs. MOR 1 NAL, p 5 0.024; vs. SAL 1 SAL, p 5 0.004). There were no significant differences between MOR 1 NAL and SAL 1 SAL females. Significantly fewer females from the MOR 1 SAL group than from the MOR 1 NAL group retrieved one (p 5 0.024), two (p 5 0.024), and three pups (p 5 0.024), grouped (p 5 0.024), or crouched-over pups (p 5 0.024). For MOR 1 SAL vs. SAL 1 SAL, there were significant differences in retrieving one (p 5 0.004), two (p 5 0.004), and three pups (p 5 0.004), grouping pups (p 5 0.004), and crouching over pups (p 5 0.004). There were no significant differences between MOR 1 NAL and SAL 1 SAL. There were no significant sex differences observed in Experiment 2. DISCUSSION Previous research suggests that opioids depress parental responsiveness. The current work demonstrates that parental behavior in the juvenile animal is regulated, in part, by the POA in much

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187

FIG. 2. Percentage of previously parental males and females that responded with full parental behavior (FPB) following morphine plus saline (MOR 1 SAL), saline-alone (SAL 1 SAL), and morphine plus naloxone (MOR 1 NAL) treatment. In males, a significantly smaller percentage of MOR 1 SAL displayed FPB compared to both SAL 1 SAL (p , 0.001) and MOR 1 NAL (p , 0.001). In females, a significantly smaller percentage of MOR 1 SAL displayed FPB compared to both SAL 1 SAL (p , 0.004) and MOR 1 NAL (p , 0.024). Note: data in Experiment 2 were analyzed using the Fisher’s Exact Probability test. See the text and Table 2 for additional significant differences in individual components of FPB.

the same manner as the behavior in adulthood. Administration of morphine into the POA of the juvenile rat disrupted parental behavior and naloxone, a narcotic antagonist, restored the behavior in both males and females. These effects were observed during the initial exposure to neonates (Experiment 1, Initiation), as well as in juveniles that had been sensitized to young and in which parental behavior had been established (Experiment 2, Maintenance). Furthermore, morphine seems to affect full parental behavior (2 consecutive days in which the juveniles retrieved, grouped and crouched-over three pups within 60 min) as well as separate components (including the retrieval of one to three pups). In both experiments, when the juveniles were treated with MOR 1 SAL, the animals made little or no attempt to be parental. The disruption in behavior appeared specific as the MOR 1 SAL animals were active and approached the pups, made contact by either cursorily prodding or sniffing them, or ignored the neonates and spent most of the time either eating or jumping about the cage. In no case was the morphine observed to have nonspecific activity effects, result-

ing in a loss of motor capabilities (as high doses are known to do; [25]). For females that received MOR 1 SAL in Experiment 1, the latency to display full parental behavior for 2 consecutive days was significantly longer than those receiving saline alone (SAL 1 SAL) but was not significantly different from those receiving morphine and naloxone (MOR 1 NAL), due most likely to small sample size within each group (two animals in Experiment 1 and three in Experiment 2 had to be dropped because of misplaced cannula sites observed with the follow-up histology). In Experiment 2, the FPB of females treated with MOR 1 SAL was significantly impaired relative to both SAL 1 SAL- and MOR 1 NAL-treated females. Together, the data support the view that opioids regulate parental-like behavior in juvenile males and females [29] as they do maternal behavior in adult female rats [3,19]. As previously discussed, within juveniles there is a reversal of the sex difference that normally accompanies pup-induced parental behavior in adults; juvenile males respond more quickly to foster

188

WELLMAN ET AL. TABLE 2 PERCENTAGES OF JUVENILE MALE AND FEMALE RATS THAT EXHIBITED THE VARIOUS COMPONENTS OF FULL PARENTAL BEHAVIOR (FPB) FOR EXPERIMENT 2, MAINTENANCE Juvenile Males

Retrieve 1 pup Retrieve 2 pups Retrieve 3 pups Group Crouch N’s

Juvenile Females

MOR 1 SAL

MOR 1 NAL

SAL 1 SAL

MOR 1 SAL

MOR 1 NAL

SAL 1 SAL

0a†b† 0a†b† 0a†b† 0a†b† 0a†b† 6

100 100 100 100 100 6

100 100 100 100 100 6

0a*b† 0a*b† 0a*b† 0a*b† 0a*b† 5

80 80 80 80 80 5

100 100 100 100 100 5

Data analyzed using the Fisher’s exact probability test. * p , 0.024; † at least p , 0.004. a Significantly different from MOR 1 NAL. b significantly different from SAL 1 SAL.

young that do juvenile females [5,18,26]. In the present experiment, however, there were few significant sex differences in the display of parental behavior. (We only examined differences between the SAL 1 SAL males and females because of possible treatment effects with the other drug groups.) In the first experiment, there was a significant sex difference between the SAL 1 SAL groups only in the latency to retrieve two pups and to crouch over the pups. There were no significant sex differences in Experiment 2. Once again, small group numbers, or the stress associated with the extra handling associated with being infused with the solutions, may have influenced the lack of gender difference observed (though, we should point out, trends in Experiment 1 are in the expected direction). Recent work by Bridges and his colleagues [64] demonstrates that long-term treatment of juveniles (5–9 days prior to exposure to neonates) with naltrexone, a potent opiate antagonist, interfers with the onset of parental behavior. The authors interpret their data

as evidence for opioid facilitation of parental behavior during the juvenile “window,” and that previous work in the area [29] may have used too-high dosages of morphine that masked a positive regulation of the behavior. We posit an alternative explanation. For instance, other research supports the view that the endogenous opioid system is still maturing in juveniles [21]. Furthermore, chronic treatment with naltrexone is capable of stimulating luteinizing hormone (LH) release, especially during sexual maturation [1]. High levels of LH could stimulate gonadal hormone release, which is associated with the decline of parental responsiveness [23,24]. Therefore, what Zais et al. [64] may have done is simply accelerate sexual maturation and a fully functioning pituitary– gonadal axis in the juvenile animal, thereby hastening the close of the “window” of parental responsiveness as the animals approach sexual maturity. Though this possibility merits further study, Stern [61] reported that sexual maturation, per se, may not be involved. If endogenous opioids do inhibit parental-like behavior, an

FIG. 3. Photomicrograph of Nissl-stained (thionin) coronal section, taken from representative juvenile brain to exemplify location of cannula placements. The open expanse is the track left by the implanted guide cannula. POA: preoptic area; 3V: third ventricle (actually, off to the left, approximately 500 m); Ac: anterior commissure. Scale bar 5 300 m.

MORPHINE, POA, AND PARENTAL BEHAVIOR IN JUVENILE RATS interesting question becomes on what particular areas of the brain involved in the display of parental behavior, do opioids act? The data from the present experiment demonstrate that the POA regulates the display of parental behavior in juveniles, as in the adult female and male [42– 47,54]. That the POA is part of a larger neural circuitry important for parental behavior has been provided by studies using knife cuts, which showed that lateral efferent projections are critical for maternal behavior [15,39,40,42,44,63]. To date, few studies have examined the neural sites that regulate parental behavior in the juvenile animal, but evidence is mounting that the POA is critical for the juvenile-period display of the behavior (M. Kalinichev and J. S. Rosenblatt, personal communication). The sense of smell may be as important to juveniles in the display of parental behavior as it is for the adult. Olfactory input reaches the mPOA via the medial amygdala and bed nucleus of the stria terminalis (BNST) and is probably the most important afferent input to the mPOA [44,59]. Fleming, Vaccarino, and Luebke [14] hypothesized that the medial amygdala may be a part of the neural circuit underlying olfactory inhibition of parental behavior in nulliparous rats. Lesions of the BNST, and hence, disruption of olfactory input to mPOA, facilitated parental behavior in virgin rats [14]. Thus, olfactory input may inhibit parental behavior by depressing the activity of the mPOA. Morphine, which disrupts parental behavior in adult females [3], apparently works through altering the olfactory information available to the animal: it renders the odors of pups aversive to the test animal [27,28], thereby preventing the display of parental behavior. Perhaps similar mechanisms are at work in the juvenile. When neurons receive extracellular and intracellular signals, a class of genes referred to as immediate early genes (IEGs), is expressed, which code for proteins that serve as transcriptional factors, either activating or repressing other target genes. c-fos, located within the nucleus, is an immediate early gene that codes for the protein Fos [41,57] and serves as a marker for individual cells that become active under certain conditions; its detection, therefore, provides a literal picture of the neural circuits that are involved in a behavior as a result of particular forms of stimulation. Numan and Numan [46] found that sensitized virgin females had more fos-labeled cells in the lateral preoptic area and in the BNST than nonparentally responding females. Recently, Stafisso– Sandoz et al. [60] demonstrated that morphine administration, in a dose that disrupts maternal behavior in lactating females, significantly attenuates c-fos expression in the mPOA. Treatment with a maternal behavior-restoring dose of naloxone was able to increase c-fos expression in morphine-treated females [60]. Further, in animals in which the gene for fosB was lacking (so-called “knockouts”), parental behavior was virtually absent, as was fos-activity in the POA [6]. In the juvenile rat, therefore, the mapping of neural circuits with fos-labeling could provide further information on the larger neural circuitry within which the POA operates to influence parental behavior at this age. Comparisons between the juvenile and the adult may yield an interesting set of data regarding maturation and development of parental behavior-regulating systems. As discussed, opiates inhibit parental behavior in adult rats [3,19,25] and in juvenile animals [29]. These studies, as well as the current one, support the view that parental behavior is under an inhibitory influence by opiates. Neurons expressing three different opioid precursors, pro-enkephalin, pro-dynorphin, and proopiomelanocortin (POMC) are distributed throughout the CNS, binding to one of three major classes of opioid receptors, m, d, k, (also widely distributed throughout the central and peripheral nervous systems; [31]). The activation of these receptors usually results in an inhibition of activity [56]. The m-receptor has high affinity for morphine-like drugs and for several endogenous opioid peptides

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including b-endorphin, which is cleaved from POMC [31]. b-Endorphin producing neurons have widespread projections to several brain regions including the POA [13,22,38], an area that is rich in m-opiate receptors [11,34]. Other areas of the brain thought to be important for parental behavior also contain m-opiate receptors including certain amygdaloid nuclei, the periaqueductal gray, several raphe nuclei, and the ventral tegmental area [31]. It may be endorphin, then, acting at the level of the medial POA, that regulates parental behavior by binding to m-opioid receptor sites [32,33]. b-Endorphin activity in the juvenile is very labile. Preliminary data gathered by Kinsley and his colleagues suggest that in juveniles, the level of b-endorphin in the POA varies, with the females having greater levels compared to males, and the levels increase in the POA in both sexes between the ages of 20 to 36 (Kinsley, Graham, Billack, and Bridges, unpublished observations). Thus, endogenous opioids may play a developmental role in certain regions of the brain, particularly the POA, in the expression of parental behavior. Also important for the promotion of parental responsiveness in juvenile male and female rats is the prepubertal hormonal contribution as examined by Kinsley and Bridges [26]. They reported that juvenile males have significantly higher levels of prolactin than females and, when treated with bromocriptine, a dopamine agonist, the males’ parental behavior was significantly disrupted. Given prolactin’s facilitatory role in parental behavior in the adult [4], an association between the levels of prolactin and responsiveness to pups at this age is clear. There exists the possibility that the behavior we (and others) have observed and refer to as parental may, in fact, be an expression of either play behavior or affiliation [7–9,48 –50]. Indeed, juveniles placed in the presence of neonates engage in a set of behaviors that, to an observer, looks for all the world to be play-like. For example, when exposed to pups, the nontreated juvenile may engage in the following sequence of behaviors: the juvenile approaches the pups and investigates them with its snout. It will sometimes move away, coming back again and again to gently nudge and prod the squirming young. The juvenile then begins to rapidly jump about the cage, moving rapidly to-and-fro as it attempts to engage the pups. Together with these apparent play behaviors, the juvenile will pick up a pup and carry it about the cage, coming to rest at its nest site. [Because the pups represent a much greater load to the juvenile relative to the adult (the pups weigh about 5–10% of the juvenile, compared to about 1.5–3% of the adult), the juvenile must work harder and the behavior is not as “clean” or organized as the adult’s.] There it deposits the pup, and will return to repeat the process until all the pups are in the nest. It may continue to bound around the cage for a while longer, but will eventually (within our 60-min test period) attempt to crouch over the pups. Again, due to the greater relative size, the juvenile is hard pressed to crouch over all the pups simultaneously. Collectively, then, the behaviors that are characteristic of the juvenile when it is exposed to pups appear primarily, though not exclusively, parental. A large body of evidence exists that the POA is involved in the regulation of parental responsiveness in adults of a diverse number of species [10,30,40]. Why, however, should juvenile animals be so parentally responsive? In other words, what is the adaptive value—if any— of such behavior? First, these organisms, just as their adult counterparts, have a genetic investment to protect: by ensuring the reproductive success of their siblings, they ensure their own genetic fitness. Second, when a female rat has overlapping litters (and is, at one point, simultaneously pregnant and lactating), benefits accrue to the helpers from the first, upon the birth of the second, litter. For example, in exchange for providing

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care to their siblings, these animals benefit from their mother’s protection, and their own nursing period may be extended [16,17]. Third, by slightly delaying their own independence, these juveniles may increase their foraging efficiency, thus maximizing their breeding preparedness once sexual maturity has been achieved. Last, early parental experience may provide these animals with the opportunity to learn parenting skills that can be used later in adulthood [18,62]. Whatever the reason(s), the display of parental behavior by juveniles remains an interesting and thought-provoking behavior to observe for insights into the larger role of parental care in the rat and other species, including humans. ACKNOWLEDGEMENTS

The authors gratefully acknowledge the fine and insightful comments provided by Drs. Kenneth Blick, Robert Bridges, and Frederick Kozub. We would like to thank the National Science Foundation (USE-9250537), the Keck Foundation, the George L. Suhor Foundation, and the University of Richmond Faculty, Graduate and Undergraduate Research Committees for their very generous support during the conduct of this project.

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