Comparison of the "nursing" and other parental behaviors of nulliparous and lactating female rats

Virgin female rats display maternal behaviors after continuous exposure to pups (sensitization) that are in some respects similar to those of postpartum females. We herein provide a detailed comparison of the "nursing" and other parental behaviors of maternally sensitized virgin females and postpartum lactating dams. Ovariectomized and intact virgin females were exposed to pups until displaying maternal behavior. On the females' fourth day of maternal responsiveness, the pups were removed for 3 h and then returned, and subject-litter interactions were observed for 45 min. Behavior of maternal virgins was compared with that of lactating dams observed on day 4 postpartum interacting with either suckling pups or pups unable to suckle due to perioral anesthesia. Ovariectomy had no effect on behavior of virgins. Retrieval and licking of pups were deficient in virgins compared with lactating dams. Suckled dams showed prolonged kyphosis (upright crouched nursing), whereas nonsuckled dams displayed little kyphosis but rather were often in a hunched position over pups. Some aspects of quiescent "nursing" behaviors of virgins were surprisingly similar to those of suckled dams, including the latency to and duration of quiescence. Nonsuckling pup stimulation elicited more kyphosis in virgins than in lactating dams, which was still much less than in suckled dams. Virgins also "nursed" pups in hunched and prone postures. Differences between sensitized and postpartum females in their maternal behaviors likely reflect differences in motivation as well as sensory inputs they receive from pups. In particular, sensory regulation of "nursing" behaviors is influenced by reproductive state because nonsuckling pups elicit different postural responses in sensitized and lactating mothers.     

Hormonal factors influence the onset of maternal aggression in laboratory rats

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.     

The effects of adrenalectomy and corticosterone replacement on induction of maternal behavior in the virgin female rat

Maternal behavior of the sensitized virgin rat is affected by approach-avoidance systems as well as by hypothalamic-pituitary-adrenal (HPA) axis, which is also activated during stress. The present experiments investigated the effects of adrenalectomy and of varying corticosterone concentrations on the onset and expression of maternal behavior in sensitized virgin rats. In the first experiment, latency to onset of maternal behavior and time spent licking once maternal were positively related to endogenous levels of corticosterone. However, few rats showed licking. In the second experiment, virgin rats were adrenalectomized or given sham surgeries before being sensitized and being given 0, 25, 100, 300, or 500 microg/mL of corticosterone in their drinking water. In the third experiment, virgin rats were adrenalectomized or given sham surgeries and given either control or corticosterone time-release pellets after being sensitized. Maternal behavior was then tested. Adrenalectomy increased licking in the second experiment and time over pups in the third experiment. Corticosterone replacement reduced licking in the second experiment and both licking and time over pups in the third experiment. In conclusion, exogenous corticosterone had an inhibitory effect on the expression of maternal behavior in the sensitized virgin rat, unlike the facilitatory effect previously found in the postpartum rat.     

Impacts of ceftriaxone exposure during pregnancy on maternal gut and placental microbiota and its influence on maternal and offspring immunity in mice

This study aimed to investigate the association between microbiota found in the maternal gut and placenta, and whether ceftriaxone exposure during pregnancy could alter these microbiota, and consequently affect the immunity of the mothers and their offspring. The microbiota in the feces and placenta of the dams were comprehensively analyzed using16S rRNA sequencing. Furthermore, viable bacteria in the placentas and blood of pups were also isolated by plate cultivation then taxonomically identified in detail by clone sequencing. Serum cytokines collected from dams and pups were quantitatively profiled using Luminex. The spleen organ index of dams was significantly lower and the offspring serum interleukin-6 levels were significantly higher in ceftriaxone-treated mice compared with the control group. The maternal fecal microbiota community was drastically altered in ceftriaxone-treated mice with significantly decreased diversity, depletion of Bacteroidetes and the blooming of Tenericutes. However, the placenta microbiota was dominated by Proteobacteria especially characteristically by Ralstonia, which was distinct from the maternal gut microbiota, regardless of whether ceftriaxone treatment or not. Viable bacteria have been found in placenta and blood cultures. These results indicated that ceftriaxone exposure in pregnancy could dramatically alter maternal intestinal microbiota, which affected the immunity of the mothers and their offspring at least partly, characteristically by enhanced pro-inflammatory responses. This study also indicated that the placenta might harbor its own microbes and the microbes were distinct from maternal gut microbiota, which may not be affected by oral administration of ceftriaxone during pregnancy.     

Effects of prolactin deficiency during the early postnatal period on the development of maternal behavior in female rats: Mother's milk makes the difference

During early life, prolactin (PRL) ingested by the pups through the milk participates in the development of neuroendocrine, immunological and reproductive systems. The present study tested whether a deficiency in PRL in the dam's milk during early lactation affected the offspring in terms of the maternal responsiveness in the sensitization paradigm and behavioral response to a novel environment in the offspring. Thus, lactating rats were injected (sc) on postnatal days (PND) 2–5 with bromocriptine (125 μg/day), bromocriptine + ovine PRL (125 μg + 300 μg/day), or vehicle. As juveniles (at PND 24) or adults (PND 90–100), one female from each litter was exposed to 5 foster pups continuously for 8 days and their maternal responsiveness was recorded. Female offspring were also tested in an open field arena. Adult, but not juvenile, female offspring of bromocriptine-treated mothers showed an increased latency to become maternal, in comparison to latencies displayed by the offspring of control mothers. Furthermore, the proportion of adult, but not juvenile, offspring of bromocriptine-treated mothers that became maternal was lower than that showed by the offspring of vehicle-treated mothers. In comparison to female offspring of vehicle-treated mothers, female offspring of bromocriptine-treated mothers spent less time hovering over the pups (as juvenile females), body licking (as both juvenile and adult females), and in close proximity to pups (as adult females) during the maternal behavior test. Simultaneous administration of ovine PRL and bromocriptine reversed almost all the negative effects of bromocriptine. These data suggest that maternally-derived PRL participates during the early postnatal period in the development of neural systems that underlie the control of maternal behavior.     

Adrenal inhibition of maternal behavior in virgin female rats.

The role of the adrenal gland in suppressing the onset of maternal behavior in virgin female rats was investigated. Virgin female rats were either ovariectomized, adrenalectomized, or subjected to the combined operation and tested for the induction of maternal behavior by being exposed to young pups. Females that were both ovariectomized and adrenalectomized 4 weeks prior to testing exhibited significantly shorter latencies to the onset of maternal behavior than that of females subjected to either ovariectomy or adrenalectomy alone. Replacement of either estrogen or progesterone in the group that was both adrenalectomized and ovariectomized resulted in a prolongation of the average latency to become maternal. The results indicated that both estrogen and progesterone can act to inhibit the onset of maternal behavior and that the adrenal gland may act to suppress the onset of such behavior by supplying an extra-ovarian source of endogenous progesterone and estrogen.     

Postpartum,hormonal, and nonhormonal induction of maternal behavior in rats: Effects on T-maze retrieval of pups

It is known that the home-cage maternal behavior of rats which become maternal after daily pup exposure (sensitization) is almost indistinguishable from that of lactating mothers, but that sensitized and lactating rats can be distinguished by their pup-retrieval performance in a T-maze extension of the home cage. The present study explored this difference further. Postpartum mothers which could not suckle due to prior nipple removal (thelectomy) retrieved as well, if not better, than intact controls in the T-maze. Hormonal induction of maternal behavior (in ? 3 days) was carried out by hysterectomy-ovariectomy plus 100 μg/kg estradiol benzoate; the performance of these females was similar to that of the postpartum groups. In contrast, only a small percentage of the sensitized mothers retrieved in the T-maze, whether the latency to onset of their maternal behavior was long (4–10 days) or short (? 3 days). Thus, hormonal factors associated with pregnancy and/or parturition, but not suckling stimulation, may facilitate T-maze retrieval of pups. The possible ethological significance of the T-maze test as a measure of maternal responsiveness is discussed.     

Patterns of maternal behavior of rats genetically selected for opposite coping styles

Maternal behavior of Koltushi High- and Low-avoidance (KHA and KLA) rats strains was assessed over the prewealing period (days 6-21). Ten litters of each strain were observed during the light phase of the light/dark cycle. In a series of experiments, rat pups were taken from the maternal nest and placed into the opposite corner of the cage. The following parameters of the maternal behavior were recorded: the latency of the first contact with the pups, pup licking, latency of carrying/retrieval of the first pup back to the nest, time of returning to the nest of the whole litter, and mother's spontaneous behavior (grooming and locomotion time) over the course of 10 min of observation. KLA mothers stayed with their pups and took care of them more frequently than KHA mothers during the light phase of the circadian cycle. Time of self-grooming was significantly higher in KHA rats. The highest levels of self-grooming of mothers was registered on the first day of testing. The latency of the first coming to pups after their removal from the nest was lower in KHA rats, but they needed more time to returned all pups to the nest. The experimental evidence suggests that the KHA but not KLA rats with the active coping style and higher stress reactivity display disorders in maternal behavior in a novel situation.     

The effects of adrenalectomy and corticosterone replacement on maternal memory in postpartum rats

Hormones associated with parturition prime rats to behave maternally, although hormonal changes are not necessary for these behaviors to occur. Experience with pups after birth enhances maternal responsiveness after a period of isolation, creating a maternal memory. The purpose of this study was to determine the role of corticosterone in the formation of maternal memory. Adrenalectomy or sham surgeries were performed in late gestation with corticosterone or vehicle pellets being given to adrenalectomized rats. Pups were removed immediately following parturition, and half of the rats received 4 h of pup experience, while the other half received only brief pup experience associated with parturition. Ten days following pup experience, foster pups were given to all rats. Latency to become maternal and maternal behaviors on the first 2 days of re-exposure and the first two maternal days were recorded. Among adrenalectomized rats given corticosterone, 4-h experience with pups decreased maternal latency when compared to brief experience with pups. This maternal experience effect was not found in comparisons between adrenalectomized rats not given corticosterone. In addition, corticosterone decreased latencies regardless of pup experience. Corticosterone also increased maternal behavior upon initial exposure to foster pups. In conclusion, corticosterone enhanced maternal memory and initial maternal behavior in postpartum rats.     

Serum prolactin concentrations and the initiation of maternal behavior in the rat

Maternal behavior and serum prolactin were measured in pregnant and virgin female rats. Pregnant rats were either ovariectomized or shamovariectomized on Day 17 of pregnancy, while virgin females were ovariectomized at the same age. Two days after surgery nests were rated and the three treatment groups were tested for responsiveness to rat pups. Both pregnant treatment groups built superior nests compared to the virgin group and also responded more frequently to rat pups within a 1 hr test period than the virgin controls. In addition, significantly more ovariectomized pregnant subjects responded to pups than did intact pregnant females. Serum prolactin levels did not differ among the three treatments nor did exposure to pups affect serum prolactin levels. In each treatment group serum prolactin was less than 15 ng/ml, well below the 139.7 ng/ml mean found on Day 23 of pregnancy. These data suggest that high levels of serum prolactin during late pregnancy are not essential for the initiation of maternal behavior in the rat.     

Corticotropin-releasing hormone inhibits maternal behavior and induces pup-killing

Behavioral responses to stressors and the effects of stressors on maternal behavior change with mothering experience. Corticotropin-releasing hormone (CRH) is released by stressors and produces stress-like behavioral effects. We tested the effects of ICV infusion of ovine CRH (0.5-4 ug) on pup-directed behaviors in ovariectomized, ovarian steroid-treated virgin rats that were either naive to pups or that had three days of mothering experience. CRH inhibited maternal behavior in naive and experienced rats in a dose-related manner. The magnitude and duration of inhibition, especially at the 1 ug dose, were less in rats with mothering experience. Higher doses of CRH (1 - 4 ug) significantly increased pup-killing in rats that were naive to pups. In contrast, CRH produced no pup-killing in rats with mothering experience.     

Effects of repeated pup exposure on behavioral,neural, and adrenocortical responses to pups in male California mice (Peromyscus californicus)

In biparental mammals, the factors facilitating the onset of male parental behavior are not well understood. While hormonal changes in fathers may play a role, prior experience with pups has also been implicated. We evaluated effects of prior exposure to pups on paternal responsiveness in the biparental California mouse (Peromyscus californicus). We analyzed behavioral, neural, and corticosterone responses to pups in adult virgin males that were interacting with a pup for the first time, adult virgin males that had been exposed to pups 3 times for 20 min each in the previous week, and new fathers. Control groups of virgins were similarly tested with a novel object (marble). Previous exposure to pups decreased virgins' latency to approach pups and initiate paternal care, and increased time spent in paternal care. Responses to pups did not differ between virgins with repeated exposure to pups and new fathers. In contrast, repeated exposure to a marble had no effects. Neither basal corticosterone levels nor corticosterone levels following acute pup or marble exposure differed among groups. Finally, Fos expression in the medial preoptic area, ventral and dorsal bed nucleus of the stria terminalis was higher following exposure to a pup than to a marble. Fos expression was not, however, affected by previous exposure to these stimuli. These results suggest that previous experience with pups can facilitate the onset of parental behavior in male California mice, similar to findings in female rodents, and that this effect is not associated with a general reduction in neophobia.     

Acute effects of ethanol on brain,plasma and adrenal monoamine concentrations in virgin and pregnant rats and their fetuses

The dose-response relationship in brain, plasma, and adrenal monoamine changes after acute oral ethanol administration (1, 2, 4 g/kg body wt) was studied in virgin rats to determine whether the response to the highest dose differed in 21-day pregnant animals, and to assess the potential consequences of ethanol on the neurotransmitter systems of their fetuses. Blood ethanol and acetaldehyde concentrations in blood increased progressively with the ethanol dose in virgin rats, and values in pregnant animals were very similar. Ethanol concentration in fetal blood and amniotic fluid did not differ from that in mother's blood whereas fetal acetaldehyde concentrations were negligible. In a dose-related manner, ethanol decreased brain DA, DOPAC and 5HT concentrations did not affect those of NA and 5HIAA, or adrenal A and NA concentrations, whereas it enhanced plasma NA levels. Basal levels of monoamines and their changes after ethanol intake did not differ in pregnant and virgin rats. Monoamine and metabolite concentrations were much lower in fetal than in maternal brains whereas plasma and adrenal catecholamine concentrations were very similar and maternal ethanol intake did not modify these fetal parameters in the fetus. Results are in agreement with the known similar metabolic response to ethanol in fed pregnant and virgin rats. The lack of fetal monoamine response to maternal ethanol intake may be a consequence of the incapacity of fetal liver to form acetaldehyde and the ability of the placenta to oxidize maternal acetaldehyde which protects the fetus from maternal alcohol intake at late gestation.     

An olfactory basis for maternal discrimination of sex of offspring in rats (rattus norvegicus)

Lactating Long-Evans rats (Rattus norvegicus) were presented with three-day-old male and female pups that were either untreated, coated with collodion in the anogenital region or on the neck, or perfumed; or with female pups odorized with either male or female pup urine. As measured by related t-tests of maternal anogenital licking, male and female pups were discriminated in the untreated condition, but not when masking stimuli were present. Female pups treated with male urine elicited more licking than female pups treated with female urine. Overall, maternal licking was decreased by olfactory masking agents and increased by pup urine. It was concluded that olfactory stimuli from pups both stimulate maternal licking and serve as a basis for discriminating male and female offspring.     

Effect of preoptic region implants of dilute estradiol on the maternal behavior of ovariectomized, nulliparous rats

Estrogen implanted directly into the medial preoptic region of pregnant Charles River Sprague-Dawley rats hysterectomized and ovariectomized on Day 16 of gestation mimics the effects of systemic estrogen treatment at this time by reducing the latency to respond to foster pups with maternal behavior (Numan, Rosenblatt, and Komisaruk, 1977). The present report describes the pup-directed responses of ovariectomized, nulliparous Zivic-Miller Sprague-Dawley rats that received bilateral medial preoptic implants of either cholesterol (n = 11) or estradiol diluted 1:10 with cholesterol (n = 11). Two days after treatment these animals were housed with three foster pups: their responsivity to pups and quality of nests built were then assessed, at first hourly and then daily. Rats receiving intracranial estradiol required significantly shorter exposures to pups than did cholesterol-treated animals before initiating carrying and grouping of 3 dispersed pups in a maternal nest during a 15-min test. On other measures, however, the groups did not differ (e.g., proportion grouping pups overnight, time required to complete retrieval of pups to the nest, time required to rebuild a disrupted nest). Animals treated with cholesterol and animals with estradiol implants did not differ in uterine weight at the time of sacrifice, suggesting that estrogen did not leak, even from this well-vascularized implant site, into the circulation. Thus, as in the pregnant animal, the facilitating effects of estrogen on maternal behavior can be mediated through the medial preoptic region; however, the effects were evident only when a test requiring retrieval of several pups within an arbitrarily short interval was given.     

Antagonism of V1b receptors promotes maternal motivation to retrieve pups in the MPOA and impairs pup-directed behavior during maternal defense in the mpBNST of lactating rats

Recent studies using V1b receptor (V1bR) knockout mice or central pharmacological manipulations in lactating rats highlighted the influence of this receptor for maternal behavior. However, its role in specific brain sites known to be important for maternal behavior has not been investigated to date. In the present study, we reveal that V1bR mRNA (qPCR) and protein levels (Western blot) within either the medial preoptic area (MPOA) or the medial-posterior part of the bed nucleus of the stria terminalis (mpBNST) did not differ between virgin and lactating rats. Furthermore, we characterized the effects of V1bR blockade via bilateral injections of the receptor subtype-specific antagonist SSR149415 within the MPOA or the mpBNST on maternal behavior (maternal care under non-stress and stress conditions, maternal motivation to retrieve pups in a novel environment, maternal aggression) and anxiety-related behavior in lactating rats. Blocking V1bR within the MPOA increased pup retrieval, whereas within the mpBNST it decreased pup-directed behavior, specifically licking/grooming the pups, during the maternal defense test. In addition, immediately after termination of the maternal defense test, V1bR antagonism in both brain regions reduced nursing, particularly arched back nursing. Anxiety-related behavior was not affected by V1bR antagonism in either brain region. In conclusion our data indicate that V1bR antagonism significantly modulates different aspects of maternal behavior in a brain region-dependent manner.     

Different uptake and handling of oleoyl-estrone by fetuses and neonatal rats.

To determine whether oleoyl-estrone can be transferred from mothers to their offspring either during pregnancy or lactation, a gavage of tracer dose of (3)H-Oleoyl-estrone was given to 21-day pregnant rats and to lactating rats on day 15 after delivery. In pregnant rats, the label was found in maternal blood as well as in liver and fetal serum, the latter showing the highest specific activity observed. In lactating rats, oleoyl-estrone label was found both in the mammary gland and maternal serum; in the pups, label was found in their stomach contents (i.e., clotted milk) and serum. The results suggest that the placenta effectively blocks the passage of oleoyl-estrone to the fetuses probably because of its high esterase activity. On the other hand, oleoyl-estrone is easily transferred from dams to pups, as a component of milk.     

Maternal condition reduces fear behaviors but not the endocrine response to an emotional threat in virgin female rats

Lactating dams and maternal virgin females are less fearful in behavioral tests compared with non-maternal animals, suggesting that maternal condition per se reduces the negative value of threatening stimuli. In addition, lactating females exhibit a diminished hypothalamic-pituitary-adrenal response to potential environmental threats. Can the maternal condition, independently of the endocrine profile of lactation, promote a reduction in the behavioral as well as in the endocrine response to an emotional stressor? To answer this question, anxiety-related and fear behaviors as well as the levels of corticosterone were evaluated in response to a bright-lit open field-loud noise model in maternal and non-maternal non-ovariectomized virgin females and lactating dams in the presence of the pups. Maternal animals, both lactating and virgin, presented an increased exploration of the bright-lit open field and a significant reduction of fear behaviors, indicated by the decreased flight and immobility responses to the subsequent activation of a loud noise, in comparison to non-maternal virgins. Interestingly, maternal virgin females, as non-maternal rats, showed high corticosterone plasma levels, in contrast to the lower endocrine response exhibited by lactating dams when confronted to this threat. Present results suggest that maternal condition allows females to take risks when caring for their young, a behavioral strategy that is independent of the reduced hypothalamic-pituitary-adrenal axis response characteristic of lactation. This evidence points towards a clear dissociation in the mechanisms regulating behavioral and endocrine responses to emotional stressors during motherhood.     

Effect of prenatal androgen treatment on maternal behavior in the female rat

The perinatal critical period when androgen suppresses the capacity of virgin female rats to display maternal behavior in response to pups in adulthood was studied. A single direct injection of a large dose of testosterone propionate (TP) to the fetuses on Days 19 or 21 of pregnancy, but not during the neonatal period, significantly suppressed maternal responses in females. Percentages of females with anovulatory ovaries were largest in groups treated with TP within 2 days after birth. It is suggested that the androgen-sensitive period of the maternal mediating systems in the female rat exists prenatally, whereas the critical period of the systems regulating the cyclic release of ovulatory hormone is in the neonatal period.