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A modified method of applying ZnSO4 to the olfactory mucosa is described. Treated rats experienced severe nasal congestion that cleared within 24 h; more persistent morbidity did not occur. Nonpregnant females observed with male intruders 24 h following ZnSO4 showed no alterations in behavior other than a reduction in anogenital sniffing, indicating that they were not hypoactive or irritable. In other experiments, lactating females were observed in a hole-board apparatus; 2 days posttreatment anosmia was confirmed in 80% of bilaterally ZnSO4-treated females by the absence of preference for pup odors. After bilateral but not unilateral ZnSO4 treatment, initially activity scores and nose pokes were equivalent in all groups, but later they both were lower than in controls, probably due to a more rapid habituation to the novel apparatus. We conclude that intranasal ZnSO4 by small-volume spray is a useful experimental tool.

We examined the effects in postpartum female rats (Rattus norvegicus) of olfactory deafferentation by intranasal ZnSO4 on maternal aggression and retention of aggression 24 hr after pups are removed. When females were tested with pups, effects of deafferentation depended on postpartum interval; anosmia failed to reduce aggression on Lactation Day 3 (L3), but anosmia was associated with moderate reductions on L8-L12. We propose that postpartum aggression is stimulated initially by hormonal factors associated with late pregnancy but later is supported by exteroceptive stimulation. After removal of pups, although aggression scores were lower across treatment conditions, anosmic females had shorter latencies to attack and tended to elicit more submissive behavior from intruders. Data suggest that odor cues from conspecifics normally inhibit aggression by females, but this inhibition is reduced during lactation.

Two experiments explored the effects on subsequent maternal aggression of interspersing 5-24 days between pregnancy termination and pup exposure to elicit maternal behavior. Pregnancy-terminated females displaying maternal behavior were compared to nonpregnant females also stimulated by pups to behave maternally and to pregnancy-terminated groups that were not exposed to pups. Pregnancies were terminated by hysterectomy-ovariectomy on Gestation Day 19 (G19), with pup exposure commencing after 6 days (Expt. 1), and by Cesarean section + ovariectomy (OVX), after delays of 5, 10, or 24 days (Expt. 2). In each study pregnancy-terminated, maternal groups were more aggressive than both nonpregnant, maternal groups and pregnancy-terminated, nonmaternal groups regardless of the imposed delays. In Expt. 2, postsurgical interval also was a significant factor, suggesting that surgical stress may elevate maternal aggression for several days. Prolonged treatment of nonpregnant OVX females (Expt. 3) with estrogen and progesterone elevated maternal aggression when pup exposure commenced 2 but not 7 days after termination of hormone treatment (Expt. 3). Therefore the physiological changes initiated during pregnancy that support maternal aggression persist for at least 4 weeks and may involve factors in addition to ovarian hormones that stimulate maternal behavior and maternal aggression. An additional unexpected finding was that nonpregnant (OVX) females had significantly higher Aggression scores after initiating maternal behavior.

Bilateral peripeduncular (PPN) lesions made on the seventh postpartum day (L7) with either radiofrequency (RF) current or N-methyl-d,l-aspartic acid (NMDA)/phosphate buffered saline (PBS) reduced maternal aggression (MA) and partially inhibited lactation without producing significant deficits in other items of maternal behavior (MB). RF-PPN lesions did not interfere with prolactin secretion, which suggests that there was deficient oxytocinergic activity. The deficit in MA was not due to interruption of afferent suckling input to the PPN: either thelectomizing females (day L6) or producing bilateral knifecuts in the mesencephalon (placed caudal to the level of the PPN; Day L7) had no effect on MA, but both procedures impaired lactation. Deficits in MA produced by RF-PPN lesions developed gradually between Days L4 and L7; lesions made either prepartum or on Day L1 did not impair MA or MB. Deficits in lactation first appeared after RF-PPN lesions on Day L1.

A 16-day treatment of nonpregnant, ovariectomized rats using 5-mm Silastic implants of estradiol (E), daily injections of 4 mg of progesterone (P), and terminal injections of 5 micrograms/kg of estradiol benzoate (EB) to provide a pregnancy-like pattern of hormone exposure, stimulates (a) home-cage aggression toward unfamiliar intruder rats, (b) short-latency maternal behavior when the females are exposed continuously to pups, and (c) maternal aggression after maternal care has been initiated. Preliminary experiments examined the persistence of stimulation of aggression by the 16-day treatment in the absence of exposure to pups eliciting maternal care, and whether an abbreviated, 1-week treatment stimulates aggression equally. Subsequent experiments examined the importance of the elements of the treatment (E implants, P injections, EB injection), and whether prolonging exposure to P or E would alter its behavioral effects. The full 16-day E/P/EB treatment stimulated higher levels of home-cage and maternal aggression, and shorter maternal behavior latencies than abbreviated and partial treatments. E in combination with P or EB significantly raised home-cage aggression, whereas P alone was without effect. Administering P for 2 additional days attenuated reductions in maternal behavior latencies by E/P/EB, but did not reduce home-cage or maternal aggressiveness. Continuous exposure to E throughout testing did not affect any dependent variable. Comparing these findings to earlier data and reports suggests that hormone exposure for 2 weeks or more, and provision of P levels approaching those of pregnancy are important to the effects of the E/P/EB treatment on aggression.

In a previous study, high nuclear estrogen receptor concentrations in the preoptic area (POA) were found on Day 16 of pregnancy to prime females to respond to a subsequent low dose of estradiol benzoate (EB) after hysterectomy-ovariectomy by exhibiting maternal behavior in 48 hr. Receptor concentrations in the POA were found to be higher than those in the hypothalamus (HYP). The present study investigated when nuclear estrogen receptors increase during pregnancy in POA and when the difference in receptor concentrations between POA and HYP occurs. An attempt was made to reproduce these pregnancy changes with a 16-day treatment of estrogen and progesterone in ovariectomized (OVX), nulliparous rats. In Experiment 1, we measured cytosol and nuclear estrogen receptor concentrations in the POA and HYP of female rats during pregnancy. Nuclear receptor concentrations in the POA increased beginning on Day 10, increased again on Day 16, and continued at this high level for the remainder of pregnancy. Nuclear estrogen receptor concentrations in the HYP remained at a lower level throughout most of pregnancy until Day 22 when they increased significantly. In Experiment 2, we tested the maternal behavior and measured estrogen receptor concentrations in OVX, steroid-primed, nulliparous rats after hysterectomy (H) and EB treatment. While 90% of estradiol (E) + progesterone (P)-primed females displayed short-latency maternal behavior 48 hr after H and EB treatment, 46% of E + vehicle (V)-treated controls were maternal. At 0 hr (prior to H and EB treatment), there was a significantly larger nuclear receptor accumulation in the POA but significantly attenuated receptor binding in the HYP. P treatment significantly affected cytosol and nuclear estrogen receptor dynamics. Differences in nuclear estrogen receptor concentrations were shown to be based on the number of available binding sites and not to changes in receptor affinity for estradiol.

Two experiments were undertaken to examine the stimulation of home-cage and/or maternal aggressiveness by a hormonal treatment stimulating short-latency maternal behavior. Nonpregnant ovariectomized rats were treated with a 16-day regimen providing pregnancy levels of estrogen (E, 5-mm Silastic capsule) and progesterone (P, daily injection of 4 mg) followed by E and P withdrawal, with or without a terminal injection of estradiol benzoate (EB, 5 micrograms/kg). In Experiment 1, hormonally treated and control females were exposed continuously to pups and tested for aggression toward male intruders on the fifth day of pup exposure. Females receiving E/P/Oil and E/P/EB were highly aggressive whether or not they had yet shown maternal behavior, whereas vehicle-treated females were nonaggressive. In Experiment 2, hypophysectomized (HYPX) and Sham-HYPX females received either E/P/EB or a control treatment and were tested with male intruders (a) immediately preceding and (b) on the fifth day of continuous pup exposure. HYPX and Sham-HYPX females treated with E/P/EB were almost equally aggressive both preceding and following pup exposure (during which they initiated maternal care), whereas HYPX and Sham-HYPX vehicle-treated females were nonaggressive at both tests. In contrast, maternal behavior latencies were reduced by E/P/EB only among Sham-HYPX females. The results establish that an E/P/EB-treatment which elicits short-latency maternal responses also increases aggressiveness toward intruders. Pituitary products, although involved in the mediation of maternal responsiveness, do not contribute significantly to the stimulation of female aggressiveness by ovarian hormones.

Previous hormonal studies have identified the hormonal and stimulus factors mediating the initiation of maternal behavior but have failed to reduce hormone-induced latencies of nonpregnant females to less than 1-2 days of continuous pup exposure. For the purpose of testing whether this delay is due to an olfactory-vomeronasal-mediated aversive reaction to pups like that found in untreated virgins, estrogen-injected hysterectomized-ovariectomized (HO-EB) nonpregnant females were subjected to olfactory-vomeronasal deafferentation. Median latencies were reduced to 0 and 1.5 hr compared with 72 hr for HO-EB nondeafferented females, which indicates that normally after HO-EB treatment, tendencies to initially avoid pup contact remain strong. Next, the hypothesis was explored that experiences during late pregnancy and/or parturition interact with hormonal priming to modify pup avoidance. Nonpregnant HO-EB females that had been exposed to pregnant-parturient females for 2 wk were tested under conditions simulating parturition. A high percentage rapidly initiated maternal behavior, but conditions during testing proved more important than prior exposure to pregnancy/parturition. The contributions of three stimulus/situation factors were analyzed: pup age--newborn versus 3-8 day; method of pup presentation--four at once versus staggered introduction; and time of day--lights versus dark phase. Hormonally treated but not sham-treated females initiated maternal behavior most rapidly when first exposed to one newborn in the nest during the light phase (76% within 1.5 hr). Prepartum caesarean-delivered females, however, responded maternally to four 3-8-day-old pups outside the nest, which indicates that additional factors operate at parturition.

In mice, tactile stimulation of the nipples appears to be critical for the onset of postpartum maternal aggression. Surgical removal of the nipples (thelectomy) blocks aggression if performed prior to parturition. In rats, indirect evidence suggests a similar role for nipple stimulation in maternal aggression. Two experiments were undertaken to determine whether thelectomy prior to mating reduces pregnancy-induced and/or postpartum aggression in this species. In the first, thelectomized and sham-thelectomized females were subjected to home cage tests (pups, if any, present) with unfamiliar male intruders on Gestation Days 18 and 21 and Lactation Days 3 and 5. Additional groups of thelectomized females were tested one time only on either Lactation Day 5 or 12. Thelectomized and control females were equally aggressive; postpartum, nearly all females in both groups attacked. Experiment 2 used females that were hysterectomized-ovariectomized (HO) on Gestation Day 16. Such females are not aggressive prior to initiating maternal behavior, but become highly aggressive (over 80% attacking) after commencing maternal care. Females again were thelectomized or sham-thelectomized prior to mating. On Day 16 HO was performed, and 48 hr later continuous exposure to pups was begun. After the females had displayed maternal behavior for 1.5-2 days, intruder tests were conducted. All females attacked at least once, with no differences between treatment groups. Thus thelectomy does not reduce maternal aggression in the rat. This finding, however, does not preclude a role for tactile ventral stimulation in mediating maternal aggression.

The responses of unweaned juveniles, 18 to 27 days of age, in brief tests with pups younger than 10 days were observed during initial exposure and at 4-6-day intervals. They showed no aversive responses and actively sought contact with the pups until 24 days when contact-seeking declined sharply. Young exposed to pups before 24 days continued to seek contact with them after 24 days showing an effect of the prior experience. Juveniles given a choice between young pups and a warm bowl chose the pups; given a choice between young pups and age mates, they chose the young pups until day 23, but chose the age mates after then. When housed continuously with young pups at 22 days of age, 5 of 9 retrieved and grouped pups with a latency of 1 day. Our results indicate that timidity or fear of novelty develops in rats in this situation about Day 24 and thereafter is a factor inhibiting the expression of positive social behavior toward pups.

This experiment addressed the hypothesis that aggressiveness toward conspecifics is stimulated by hormonal factors known to mediate the onset of maternal care. Subjects included both pregnant and virgin females. Sixteen-day pregnant rats were hysterectomized (H), hysterectomized-ovariectomized and injected with estrogen (HO-EB), or subjected to sham procedures. Nonpregnant females were HO-EB or sham operated. The females were sensitized by continuous exposure to pups and were judged to have initiated maternal care when all pups were retrieved and grouped, Aggressiveness was observed during 5-min intruder tests using unfamiliar males, administered (a) 10 min prior to the introduction of test pups, (b) following the first 3 hr of pup exposure, and (c) after females had initiated maternal care. The results revealed that treatments known to reduce sensitization latencies also increased aggressiveness even prior to exposure to pups. Aggressiveness was displayed before sensitization only in groups having elevated estrogen levels. After initiating maternal behavior, pregnant and pregnancy-terminated females increased further in aggressiveness whereas nonpregnant females did not. Pregnancy-terminated, HO-Oil females became aggressive (only) after initiating maternal behavior, indicating that factors other than estrogen also influence the onset of maternal aggression.

This article reviews the current state of our knowledge about the hormonal basis of maternal behavior in the rat. Considered are the ovarian hormones estrogen and progesterone, the pituitary hormones beta-endorphin and prolactin, and the hormone oxytocin, secreted by several hypothalamic nuclei and associated brain regions. The hormones of pregnancy, estrogen and progesterone, prime the female to respond to a terminal rise in estrogen that stimulates a high level of maternal responsiveness even before parturition begins. Studies on the role of prolactin, using hypophysectomy, prolactin release blockers and anterior pituitary and prolactin replacement, indicate that prolactin is required for the ovarian hormones to be effective in stimulating maternal behavior. During the latter half of pregnancy, placental lactogen may displace prolactin in this role. Although prolactin serves as a chronic stimulus for maternal behavior, it also may act over a short period. Oxytocin stimulates maternal behavior in a specific strain of rat, but not in other strains, and only when administered introcerebroventricularly (ICV) in estrogen-primed females. The decline in the high brain levels of beta-endorphin around parturition has been proposed as a requirement for the onset of maternal behavior; morphine blocks the onset of maternal behavior and disrupts ongoing maternal behavior and maternal aggression in lactating females. However, blocking beta-endorphin action at parturition interferes with pup cleaning and eating of the placenta as well.

Sensitized virgins and postpartum lactating mothers, both exhibiting maternal behavior, were given donor litters that increased in age by 1 day, for 28 days, starting at the onset of maternal behavior. Each day females were tested for maternal behavior with pups 4-8 days old: Maternal care (i.e., nursing/crouching, retrieving, nest building and licking) and maternal withdrawal, rejection, and prevention of nursing were recorded. After the ninth day, females were also tested with the progressively older pups from 10 to 28 days of age with which they were living. Virgins and lactating mothers showed generally similar patterns of maternal care although some differences were found, and they declined in maternal behavior toward the older pups in a similar manner. Maternal behavior did not decline in tests with younger pups. The results are interpreted as supporting the hypothesis that the decline as well as the maintenance of maternal behavior postpartum is nonhormonally mediated.

To test the hypothesis that the onset of maternal behavior is stimulated by estrogen, we examined the effects of medial preoptic area (MPOA) or ventromedial hypothalamus (VMH) implants of the antiestrogen 4-hydroxytamoxifen (OH-TAM) on pre- and postpartum maternal behavior and on postpartum estrus in rats. On day 20 of pregnancy, animals were implanted bilaterally with OH-TAM or cholesterol cannulae into MPOA or VMH. Unilateral cannulae of OH-TAM were also placed into MPOA. Females were tested with newborn pups for the onset of immediate retrieval, prepartum, at noon on day 21, at midnight of the day 21, and 1 day following parturition (which occurred on day 22). On the evening of parturition, implanted animals were tested with stimulus males for the occurrence of postpartum estrus. In order to examine the influence of estrogen on maternal behavior in the absence of parturitional experience, antiestrogen-implanted animals were delivered surgically (cesarean section) and were observed for the display of maternal behavior at various times after surgery. At noon of day 21, only a few animals in any group retrieved pups. However, 12 h later, females that received bilateral OH-TAM implants into MPOA remained nonresponsive, while over 80% animals in other groups retrieved and gathered pups. The antiestrogen did not disrupt the display of postpartum maternal behavior in those females that were allowed to undergo normal parturition, but it significantly reduced the number of cesarean-delivered animals showing maternal behavior. Bilateral implants of OH-TAM into VMH, but not into MPOA, effectively blocked postpartum estrus. The MPOA implants of OH-TAM resulted in a significant reduction in cytoplasmic estrogen receptor levels and an increase in nuclear estrogen receptor concentrations. The results of these experiments lend support to the current hypothesis that estrogen acts most effectively in the MPOA to stimulate maternal behavior and in the VMH to facilitate sexual receptivity.

A previous study showed that intranasal zinc sulfate (ZnSO4) treatment shortens the latencies for the onset of maternal behavior in nonpregnant rats. The aim of this study was to see whether after recovery from the effects of ZnSO4 treatment, the latencies for maternal behavior increased. In Experiment 1 nonpregnant females were intranasally infused with ZnSO4 or air or were left untreated and exposed to pups starting 48 hr later. Olfactory discrimination tests using chocolate bits were done simultaneously. The ZnSO4 treatment resulted in short latency maternal behavior (1.1 days) compared with latencies of control females (3.8 days), but neither group showed any loss of olfactory discrimination. Three weeks later- ZnSO4-treated females showed increased latencies for all but retrieving, while control groups showed decreased latencies for all maternal behaviors. In Experiment 2, intranasal ZnSO4- and air-treated females were given olfactory discrimination tests under food deprivation, using chocolate bits and guinea pigs pellets. Choice of guinea pig pellets was more severely affected than choice of chocolate bits, but recovery of the discriminations was complete in 4-5 days. The effects of ZnSO4 on maternal behavior and on olfactory discrimination in nonpregnant females were discussed.

Since endogenous opiate mechanisms are activated during parturition, the present study examined in rats the effects of opiate antagonism on maternal care during and shortly after parturition. Endogenous opiate mechanisms were blocked in late pregnant rats by (1) naltrexone pellet implants (Experiment 1); (2) acute naloxone injections of 10 mg/kg (Experiment 2) or 0.1 mg/kg (Experiment 7); or (3) induction of opiate tolerance (Experiment 3). All methods resulted in a significant decrease in placentophagia and/or in cleaning pups of umbilical cords and birth fluids (Experiment 6). Other aspects of maternal care appeared relatively unaffected and 24 hr pup survival rats were lowered only by induction of morphine tolerance (probably via its effects on the young). In nonpregnant females, naloxone produced a small but significant decrease in placentophagia (Experiment 4) whereas morphine-tolerant nonpregnant females consumed placentas as readily as controls (Experiment 5). Thus the inhibition of placentophagia produced by opiate antagonism may be specific to conditions associated with parturition. These findings suggest that endogenous opiates support placenta eating and pup cleaning during and immediately after birth. Mediation may be via opiate effects on ingestive behavior, and/or via a reduction in the stress of parturition which otherwise can interfere with the female's ability to perform these tasks.

Changes in maternal responsiveness during late pregnancy were traced by exposing females to foster pups (15-30-min tests) under conditions favoring the rapid initiation of maternal behavior. Groups were tested when nonpregnant or on Days 17, 20, 21, or 22 of pregnancy (Day 22 = parturition). Nulliparous females were compared with primiparous and with "experienced breeders." Nest defense was observed by introducing unfamiliar males (2-min tests) on Day 22. Results focused attention on three periods: (a) Days 17-20, when maternal responsiveness was lower than in the nonpregnant condition, (b) Day 21, when maternal responsiveness returned to or rose above nonpregnant levels, and (c) on Day 22, the 3.5 hr prior to delivery, during which 90% of females almost immediately retrieved, gathered, and tended foster pups and during which 92% attacked the unfamiliar intruders. (Attacks were rare earlier.) Maternally experienced females were more responsive to pups than nulliparous females when nonpregnant and throughout late pregnancy, but both groups were equally likely to show prepartum aggression.

Male and female rats that were nonhandled or that were handled from weaning, and that had intact or impaired olfaction (intranasal zinc sulfate), were sensitized through continuous pup exposure commencing at 30, 45, 60, or 90 days of age. Nonhandled males and females were alike in latencies to become maternal at Day 30 but thereafter latencies of females became shorter and latencies of males lengthened; by 90 days males had markedly longer latencies than females and only 1/3 became maternal. Handling facilitated sensitization at 30 days among males and females but only among males at 45 and 60 days. Intranasal zinc sulfate reduced latencies of both males and females at all ages tested, but appeared most effective after 45 days of age. Gender differences in latencies persisted in adult animals even after combined treatments. Results indicate that both timidity and olfaction inhibit the onset of maternal responses to pups in virgin males and females, but they differ in relative importance by age and gender.