Fear response of rat pups to a non-aversive social stimulus: Evidence for the involvement of memory processes.

Learning processes in rats during early development are importantly mediated by the mother, which represents the primary source of environmental information. This study aimed to determine whether aversive early experiences can induce the expression of pups' fear responses toward a non-aversive stimulus as a consequence of a memory process. First, we determined pups' fear responses toward an anesthetized female after being exposed to this stimulus or an empty cage together with their mothers from Postnatal Day (PNDs) 1 to 4. Second, we evaluated if the administration of the protein synthesis inhibitor cycloheximide (CHX; 0.2 mg/kg, subcutaneously (sc).) disrupted the reconsolidation processes and abolished the fear response on PND 9. Only female pups previously exposed to the female intruder expressed fear responses toward an anesthetized female on PND 8. CHX administration to female pups immediately after exposure to an anesthetized female on PND 8 suppressed fear responses on PND 9, indicating that the fear expression was the result of a memory process, probably mediated by the mother. These findings demonstrated that early experiences can shape responses to social stimuli in a sex-dependent manner and emphasize the critical role of the mother in influencing fear learning in a social context.

Sexual behaviour of the female rat during late adolescence: effect of chronic cocaine treatment.

Sexual behaviour is highly motivated and female rats begin to express it during adolescence. The circuitries implicated in the control of motivated behaviours continue to mature during adolescence and seem more sensitive to the effects of psychostimulants such as cocaine. However, a putative differential effect of this drug on the sexual behaviour of females according to age has not yet been studied. Therefore, we compared the motivational value of a male and the expression of sexual behaviour of late-adolescent and adult female rats after chronic treatment with a vehicle or 15.0 mg/kg cocaine. The strong incentive value of a male rat, in a male versus female preference test, for adolescent and adult female rats, was not affected by cocaine. During sexual interaction, adolescents were as sexually receptive as were adults; however, they expressed more runaways and social investigation. Cocaine treatment did not modify the expression of sexual behaviour in either group, but increased social investigation in adolescent rats. These results indicate that late-adolescent pro-oestrus females are highly sexually motivated and might express behaviours typical of this life period during sexual interaction. Moreover, although chronic cocaine treatment seemed to affect more adolescents, it did not alter the sexual motivation or behaviour of females.

Neuroanatomical and neurochemical basis of parenting: Dynamic coordination of motivational, affective and cognitive processes.

This article is part of a Special Issue "Parental Care". Becoming a parent is arguably the most profound transforming experience in life. It is also inherently very emotionally and physically demanding, such that the reciprocal interaction with the young changes the brain and behavior of the parents. In this review, we examine the neurobiological mechanisms of parenting primarily discussing recent research findings in rodents and primates, especially humans. We argue that it is essential to consider parenting within a conceptual framework that recognizes the dynamics of the reciprocal mother-young relationship, including both the complexity and neuroplasticity of its underlying mechanisms. Converging research suggests that the concerted activity of a distributed network of subcortical and cortical brain structures regulates different key aspects of parenting, including the sensory analysis of infant stimuli as well as motivational, affective and cognitive processes. The interplay among these processes depends on the action of various neurotransmitters and hormones that modulate the timely and coordinated execution of caregiving responses of the maternal circuitry exquisitely attuned to the young's affect, needs and developmental stage. We conclude with a summary and a set of questions that may guide future research.

Social aversive stimuli presented to the mother produce the precocious expression of fear in rat pups.

During the stress hypo-responsive period, rat pups do not display fear responses toward adult males, yet they exhibit distress behavior in isolation. Since the mother modulates her offspring's affective development, we hypothesized that by altering the mother's behavior, a prolonged stressful situation would modify the ontogeny of the fear responses and distress behaviors in pups. Therefore, we repeatedly exposed the mother-litter dyad to different socially stressful stimuli and subsequently evaluated in 8-day-old pups their fear responses toward an anesthetized male, as well as their distress behavior in isolation. Our results show that repeated exposure to unfamiliar males and females, which altered maternal behavior by eliciting aggression in the mother, was associated with the precocious fear responses in pups, though without altering their distress behavior in isolation. We propose that the mother, as the principal mediator of environmental influences, provokes the precocious expression of fear in pups through alterations in her maternal behavior.

Hypocretins, sleep, and maternal behavior.

The postpartum period is a demanding time during which mothers experience numerous physiological adaptations that enable them to care for their offspring while maintaining their wellbeing. Hypocretins, also known as orexins, are neuropeptides synthesized by hypothalamic neurons that play a fundamental role in several functions, including the promotion of wakefulness and motivated behaviors, such as maternal care. In this regard, several findings suggest that the activity of the hypocretinergic system increases in the early postpartum period and begins to decline as weaning approaches. In particular, hypocretins within the medial preoptic area, a crucial region during this period, modulate both maternal behavior and sleep. Although further studies are necessary to obtain a comprehensive understanding of the role of hypocretins in lactating females, current research suggests that this system participates in promoting active components of maternal behavior and regulating wakefulness and sleep adjustments during the postpartum period, potentially leading to increased wakefulness during this stage. These adaptive adjustments enable the mother to cope with the continuously changing demands of the pups.

Is sleep critical for lactation in rat?

Sleep deprivation is a feature shared by most studied mammals at some point during the postpartum period. Unlike the rabbit, the pig, or the human mother, sleep has been claimed as an essential state for milk ejection in mother rats, where sleep deprivation using gentle handling (GH) prevents milk ejection and pup weight gain. Though sleep deprivation is a stressful situation itself, most common methodologies used in laboratory animals, including GH, usually involve aversive stimulus to prevent sleep, adding further stress to the animal. Deep brain electrical stimulation (DBES) of the brainstem reticular formation is a less common technique used to prevent sleep, and while this methodology may also carry unwanted effects, it avoids stressful conditions. In the present study, we examined the relationship between sleep and nursing, and how different sleep deprivation methodologies impact nursing and lactation. For this purpose, we carried out two sets of experiments. First, we correlated sleep and waking states with different nursing parameters of lactating rats under undisturbed conditions. Second, we slept deprived another group of mother rats using two different techniques: GH and DBES. Our main findings show that sleeping time was positively correlated with the time devote to nurse the pups, but not either with milk ejection or pup weight gain. When mother rats were sleep deprived, maternal behavior was fragmented using both methods, but was substantially more disrupted when using GH. Additionally, lactating dams were capable of ejecting milk and their pups gained weight despite of being sleep deprived using both techniques, but these parameters were significantly reduced using GH compared to control values, while DBES did not differ from control group. Overall, these results suggest that sleep and nursing are behaviorally compatible, but in disagreement with previous findings, we concluded that sleep is not necessary for milk ejection. These observations have critical implications for using the rat as a model to explore sleep loss during the postpartum period.

The Integration of the Maternal Care with Sleep During the Postpartum Period.

Our entire life occurs in a constant alternation between wakefulness and sleep. The impossibility of living without sleep implies that any behavior must adapt to the need for sleep, and maternal behavior does not escape from this determination. Additionally, maternal behavior in mammals is a highly motivated behavior, essential for the survival of the offspring. Thus, the mother has to adapt her physiology of sleep to the constant demands of the pups, where each species will have different strategies to merge these two physiological needs. However, all studied female mammals will experience sleep disturbances at some point of the postpartum period.

Electrophysiological characterization of medial preoptic neurons in lactating rats and its modulation by hypocretin-1.

The medial preoptic area (mPOA) undergoes through neuroanatomical changes across the postpartum period, during which its neurons play a critical role in the regulation of maternal behavior. In addition, this area is also crucial for sleep-wake regulation. We have previously shown that hypocretins (HCRT) within the mPOA facilitate active maternal behaviors in postpartum rats, while the blockade of endogenous HCRT in this area promotes nursing and sleep. To explore the mechanisms behind these HCRT actions, we aimed to evaluate the effects of juxta-cellular HCRT-1 administration on mPOA neurons in urethane-anesthetized postpartum and virgin female rats. We recorded mPOA single units and the electroencephalogram (EEG) and applied HCRT-1 juxta-cellular by pressure pulses. Our main results show that the electrophysiological characteristics of the mPOA neurons and their relationship with the EEG of postpartum rats did not differ from virgin rats. Additionally, neurons that respond to HCRT-1 had a slower firing rate than those that did not. In addition, administration of HCRT increased the activity in one group of neurons while decreasing it in another, both in postpartum and virgin rats. This study suggests that the mechanisms by which HCRT modulate functions controlled by the mPOA involve different cell populations.

Local administration of bicuculline into the ventrolateral and medial preoptic nuclei modifies sleep and maternal behavior in lactating rats.

The preoptic area (POA) is a brain structure classically involved in a wide variety of animal behavior including sleep and maternal care. In the current study, we evaluate the specific effect of disinhibition of two specific regions of the POA, the medial POA nucleus (mPOA) and the ventrolateral POA area (VLPO) on sleep and maternal behavior in lactating rats. For this purpose, mother rats on postpartum day 1 (PPD1) were implanted for polysomnographic recordings and with bilateral cannulae either in the mPOA or in the VLPO. The rats were tested for sleep and maternal behavior on PPD4-8 after the infusion of the GABA-A antagonist, bicuculline (0, 10 or 30 ng/0.2 µl/side). Infusion of bicuculline into the mPOA augmented retrieving and nest building behaviors and reduced both nursing and milk ejections but had almost no effect on sleep. When bicuculine was microinjected into the VLPO, the rats significantly increase the number of retrievings and mouthings and reduced the nursing time without changes in milk ejections, which was associated with an increase in wakefulness and a reduction in light sleep. Our results show that disinhibition of the mPOA, a key area in the control of maternal behavior, increased active maternal behaviors and reduced nursing without affecting wakefulness or sleep time. In contrast, the enhancement of some active maternal behaviors when the drug was infused into the VLPO, a sleep-promoting area, with a concomitant increase in wakefulness suggests that mother rats devote this additional waking time in the active maternal care of the pups. We hypothesize that maternal behavior changes after bicuculine microinjection into the VLPO are caused by a reduction in the sleep drive, rather than a direct effect on maternal behavior.

Role of Hypocretin in the Medial Preoptic Area in the Regulation of Sleep, Maternal Behavior and Body Temperature of Lactating Rats.

Hypocretins (HCRT), also known as orexins, includes two neuroexcitatory peptides, HCRT-1 and HCRT-2 (orexin A y B, respectively), synthesized by neurons located in the postero-lateral hypothalamus, whose projections and receptors are widely distributed throughout the brain, including the medial preoptic area (mPOA). HCRT have been associated with a wide range of physiological functions including sleep-wake cycle, maternal behavior and body temperature, all regulated by the mPOA. Previously, we showed that HCRT in the mPOA facilitates certain active maternal behaviors, while the blockade of HCRT-R1 increases the time spent in nursing. As mother rats mainly sleep while they nurse, we hypothesize that HCRT in the mPOA of lactating rats reduce sleep and nursing, while intra-mPOA administration of a dual orexin receptor antagonist (DORA) would cause the opposite effect. Therefore, the aim of this study was to determine the role of HCRT within the mPOA, in the regulation and integration of the sleep-wake cycle, maternal behavior and body temperature of lactating rats. For that purpose, we assessed the sleep-wake states, maternal behavior and body temperature of lactating rats following microinjections of HCRT-1 (100 and 200 µM) and DORA (5 mM) into the mPOA. As expected, our data show that HCRT-1 in mPOA promote wakefulness and a slightly increase in body temperature, whereas DORA increases both NREM and REM sleep together with an increment of nursing and milk ejection. Taken together, our results strongly suggest that the endogenous reduction of HCRT within the mPOA contribute to the promotion of sleep, milk ejection and nursing behavior in lactating rats.

Effects of litter-overlapping on emotionality, stress response, and reproductive functions in male and female rats.

In rats, mating at postpartum estrus and delayed dispersal of the young would result in the overlapping of two different-age litters. As a consequence, newborn pups' early experience will include not only that acquired during the interaction with the mother and age-matched littermates, but also with older siblings. As early-life experience modulates rodents' brain function, behavior and reproduction, we aimed to assess how changes in the early environment provoked by the overlapping of litters would affect emotionality, stress response and reproductive functions of male and female pups during adulthood. Results showed that both male and female overlapped reared pups exhibited a reduced behavioral inhibition in the open field test during adulthood. In addition, overlapped reared adult females, but not males, showed a blunted corticosterone response to an acute stressor during diestrus and a reduction in sexual behavior. In summary, natural changes in early experience provoked by the overlapping of litters, long-term modulate affective and reproductive behaviors, and the endocrine stress response in a sex dimorphic manner.

Cocaine treatment before pregnancy differentially affects the anxiety and brain glucose metabolism of lactating rats if performed during adulthood or adolescence.

Cocaine exposure disrupts the maternal behavior of lactating rats, yet it is less known whether it alters the affective changes that accompany motherhood. As the long-term action of cocaine on anxiety varies according to the developmental stage of the individuals, this study aimed to compare the effect of a chronic treatment with cocaine to adult and adolescent non-pregnant females on their anxiety-like behavior and basal brain metabolic activity during lactation. Thus, adult and adolescent virgin rats were exposed to cocaine (0.0 or 15.0 mg/kg ip) during 10 days and were mated four days later. Anxiety behavior was evaluated on postpartum days 3-4 in the elevated plus maze test, and the basal brain glucose metabolism was determined on postpartum days 7-9 by means of [18F] fluorodeoxyglucose positron emission tomography. Cocaine treatment during adulthood increased the anxiety-like behavior of lactating females whereas its administration during adolescence decreased it. Also, the basal glucose metabolism of the medial prefrontal cortex differed between lactating females treated with cocaine during adulthood and adolescence. These differential effects of cocaine, according to the age at which the drug was administered, support the idea that the adolescent and adult brains have a distinct susceptibility to this drug, which leads to divergent long-term changes in the neural circuits that regulate anxiety during lactation.

The reproductive stage and experience of sexually receptive mothers alter their preference for pups or males.

Female rats show postpartum estrus, a unique stage in their reproductive cycle in which they are able to display maternal and sexual responses at the same time. To assess the relative value of pups or males for sexually receptive mothers with different hormonal profiles and reproductive experiences, we employed a 3-point star maze with 3 choice compartments containing: pups, a sexually active male, or no stimulus (neutral). Cycling maternal and nonmaternal females in late proestrus, independently of their previous reproductive experience, strongly preferred the male to the pups, although most postpartum estrous dams did not exhibit preference for the male. The majority of the postpartum primiparous females did not prefer the litter's chamber either, but a previous reproductive experience strongly determined their preference for the pups. These results suggest that the hormonal changes of the proestrus, in contrast to those of the postpartum estrus, promote a strong preference for the male that is not diminished by the maternal condition. Conversely, the endocrine changes of the postpartum facilitate the effect of previous reproductive experience in strengthening the incentive value of the pups.

Overlapping litters in rats: effects on maternal behavior and offspring emotionality.

Female rats have a fertile postpartum estrus, which can result in a simultaneous gestation and lactation and later in the overlapping of two different-age litters. These different physiological and contextual situations may affect the maternal behavior of lactating rats and provoke long-lasting changes in the affective behavior of the litter. Therefore, we aimed to assess the effect of pregnancy and of litter overlapping on the maternal behavior of lactating rats and to describe the maternal- and anxiety-like behaviors of the juveniles that remained in contact with their younger siblings. Results showed that pregnant lactating rats spent more time outside the nest and less time nursing than non-pregnant mothers. On the other side, mothers with overlapping litters licked less the newborn pups than mothers with single litters. These deficits in maternal licking received by neonates were overridden by the juveniles' licking behavior to their younger siblings. Adult male and diestrous female rats reared with younger siblings showed a reduced anxiety-like behavior as compared to age-weaning matched animals without this experience. Thus, natural changes in the reproductive conditions and in the early experience, affect the maternal behavior and long-term modulate affective behavior of the individuals.

Incentive value of newborn pups relative to juveniles for mother rats raising overlapping litters.

In rats, successful mating during the postpartum estrus results in the temporal overlapping of successive litters within the maternal nest. Mothers with two overlapping-litters (OLM) simultaneously take care of neonate and juvenile pups; however, they mostly direct their attention to the neonates. We hypothesized that these differences reflect an adaptation to the specific characteristics and needs of the two litters and not a lack of interest in the juveniles. To test this hypothesis, we assessed the relative incentive value of newborns and juveniles for OLM in a preference test and compared it with that exhibited by mothers in early (EPM) and late (LPM) postpartum, which were raising only newborns or only juveniles, respectively. Results showed that OLM spent similar time in the newborns and juveniles compartments and did not prefer the newborns as did the EPM, however, similarly to them, OLM made more attempts to get access to the newborns than the juveniles. On the other hand, OLM and LPM did not exhibit a clear preference between the stimuli. These results indicate that both neonates and juveniles have incentive value for OLM, although these mothers invest more effort in the newborns. These results point out to a unique behavioral profile of OLM, which shows similarities with EPM and LPM on different behavioral measures. They also support the idea that motivational processes underlying maternal behavior are complex and dynamic, adapting the response of the mother to pups' needs and the context.

Dopaminergic activity mediates pups' over male preference of postpartum estrous rats.

Pups have greater incentive value than males for rats during the postpartum estrus (PPE); a period when females are both maternally and sexually motivated. Mesolimbic dopaminergic system has been proposed as a general motivational circuit; however in the literature it has been more related to the control of the motivational aspects of maternal than sexual motivation of females. Therefore, we aimed to assess the effect of antagonizing dopaminergic neurotransmission of PPE females on their preference for pups over a male. To achieve this objective we tested PPE rats in a Y-maze with three-choice chambers (one containing eight pups, the other a male and the last one no stimulus) after the systemic administration of the dopaminergic antagonist haloperidol (0.0; 0.025 or 0.05 mg/kg). Furthermore, to determine if this dopaminergic antagonist differentially affects maternal and sexual motivations when pups and male are not competing, we evaluated the effect of haloperidol in the preference of females for pups vs. a non-receptive female and for a male vs. a non-receptive female. In the preference test for pups vs. male, both doses of haloperidol decreased the time that females spent in pups' chamber while increased the time that they spent in male's chamber, resulting in a lack of preference between both incentives. Besides, haloperidol reduced the effort -attempts to get access to the stimuli- made by the females to obtain the pups. Conversely, 0.05 mg/kg of haloperidol did not affect the preference for both incentives when they were confronted to a non-receptive female. Together, these results indicate that the dopaminergic activity mediates pups' preference over male during the PPE and point toward a more relevant role of this system in females' behavioral output when incentives are competing.

Microinjection of the dopamine D2-receptor antagonist Raclopride into the medial preoptic area reduces REM sleep in lactating rats.

The medial preoptic area (mPOA) is a brain structure classically related to both non-REM (NREM) sleep and maternal behavior. Although the dopaminergic system is known to play a role in the control of the states of sleep and wakefulness, its effects within the mPOA on sleep are still not clear. Microinjection of the dopamine D2 receptor antagonist Raclopride into the mPOA has been shown to promote nursing postures in lactating dams with no effects on active maternal behavior. We hypothesized that the facilitation of nursing postures may be also associated with the promotion of NREM sleep. In order to test the hypothesis, Raclopride was microinjected into the mPOA and maternal behavior and sleep were assessed in lactating rats. The changes observed included a reduction of the latency to start nursing and an increase of the time to reunite the entire litter. Contrary to our hypothesis, NREM sleep was not affected by Raclopride. On the other hand, REM sleep and its transitional stage from NREM sleep, were significantly reduced by this pharmacological agent. These data suggest that dopamine D2 receptors within the mPOA are involved in the transition from NREM to REM sleep.

Ovarian steroids counteract serotonergic drugs actions in an animal model of obsessive-compulsive disorder.

Recently, we reported the existence of differences according to the reproductive stage of female rats in a pharmacologically induced animal model of obsessive-compulsive disorder. Therefore, the aim of the present study was to examine the role of endogenous and exogenous ovarian steroids in the induction of perseverative responses in a T-maze by the 5-HT1A agonist 8-OH-DPAT (1.0 and 2.0 mg/kg, SC) and in the preventive action of the selective serotonin reuptake inhibitor, fluoxetine (10.0 mg/kg, three times, SC). The results showed that the perseverant action of 8-OH-DPAT as well as the prevention of such perseverance by fluoxetine were reduced during the estrous-metestrous phases of the female reproductive cycle, and by the exogenous ovarian steroids administration to ovariectomized animals. Data are discussed from the standpoint of the action of ovarian steroids on the serotonergic system and on the putative influence of these hormones on the physiopathology and treatment of this disorder in women.

Demanding pups improve maternal behavioral impairments in sensitized and haloperidol-treated lactating female rats.

The impairments in the maternal behavior of ovariectomized sensitized females, relative to lactating dams, resemble those deficits found in lactating females after treatment with the D1/D2 DA receptor antagonist haloperidol, which interferes with maternal motivation. Therefore, it could be speculated that these behavioral deficits found in sensitized females and haloperidol-treated dams are due to a reduced motivation to interact with pups. In support of this hypothesis, we have found that both sensitized and haloperidol-treated lactating females exhibited remarkably similar impairments in the expression of all active maternal behaviors relative to lactating dams. Furthermore, these deficits were overridden when they were allowed to interact with 12h-isolated pups (demanding pups). Interestingly, lactating dams also improved their maternal behavior in the presence of demanding pups, and clearly chose demanding more than non-demanding pups in a preference paradigm. These data support the idea that the behavioral deficits of sensitized and haloperidol-treated lactating females are due to a reduced behavioral activation in response to the incentive cues from pups compared to lactating dams, and not because of a motor inability to express maternal behavior. These findings ultimately suggest that pups modulate the activity of DA system involved in the regulation of maternal behavior.

Hypocretinergic system in the medial preoptic area promotes maternal behavior in lactating rats.

Hypocretin-1 and 2 (HCRT-1 and HCRT-2, respectively) are neuropeptides synthesized by neurons located in the postero-lateral hypothalamus, whose projections are widely distributed throughout the brain. The hypocretinergic (HCRTergic) system has been associated with the generation and maintenance of wakefulness, as well as with the promotion of motivated behaviors. In lactating rats, intra-cerebroventricular HCRT-1 administration stimulates maternal behavior, whilst lactation per se increases the expression of HCRT type 1 receptor (HCRT-R1). Due to the fact that HCRTergic receptors are expressed in the medial preoptic area (mPOA), a region critically involved in maternal behavior, we hypothesize that HCRT-1 promotes maternal behavior acting on this region. In order to evaluate this hypothesis, we assessed the maternal behavior of lactating rats following microinjections of HCRT-1 (10 or 100µM) and the selective HCRT-R1 antagonist SB-334867 (250µM) into the mPOA, during the first and second postpartum weeks. While intra-mPOA microinjections of HCRT-1 (100µM) increased corporal pup licking during the second postpartum week, the blockade of HCRT-R1 significantly decreased active components of maternal behavior, such as retrievals, corporal and ano-genital lickings, and increased the time spent in nursing postures in both postpartum periods. We conclude that HCRTergic system in the mPOA may stimulate maternal behavior, suggesting that endogenous HCRT-1 is necessary for the natural display of this behavior.