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.

Increase in sexual motivation throughout adolescence in the cycling female rat.

Sexual behavior in the female rat is a highly motivated behavior first displayed during adolescence, a developmental period when neural circuits underlying motivation are not mature. This study characterizes the natural development of sexual motivation and behavior of female rats. We compared the incentive value of the male for mid-adolescent (PNDs:39-43), late adolescent (PNDs:49-53), and adult (PNDs:90-115) cycling females, using a male-female preference task and an ultrasonic vocalization emission test following exposure to a male or female stimulus animal. Furthermore, display of sexual and social behaviors during an interaction with a male or a nonreceptive female was assessed. Mid-adolescent rats exhibited a reduced preference for the male than adults and performed less attempts to access the male. Unlike late adolescent and adult females, mid-adolescent rats did not increase their ultrasonic vocalization emission after interacting with a male relative to a female. Although most of the sexual behavior did not differ between groups, mid-adolescent females showed lower lordosis magnitude and higher levels of play and social investigation during a sexual interaction, giving rise to a unique behavioral profile. Present results indicate that the sexual behavior repertoire is fully displayed by mid-adolescence, but sexual motivation is low and increases into late adolescence.

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.

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.

What can challenging reproductive contexts tell us about the rat's maternal behavior?

Maternal behavior in mammals encompasses a complex repertoire of activities that ensure the survival of the offspring and shape their neural and behavioral development. The laboratory rat has been employed as a classic model for investigating maternal behavior, and recently with the use of advanced techniques, the knowledge of its neural basis has been expanded significantly. However, the standard laboratory testing conditions in which rats take care of a single litter impose constraints on the study of maternal flexibility. Interestingly, the reproductive characteristics of this species, including the existence of a fertile postpartum estrus, allow us to study maternal behavior in more complex and ethologically relevant contexts, even in laboratory settings. Here we review how maternal and sexual motivations interact during the postpartum estrus, shaping the behavioral response of females according to the presence of the pups and males. Next, we describe how impregnation during the postpartum estrus creates a new reproductive context in which mothers simultaneously care for two successive litters, adapting their responses to different behavioral and physiological demands of pups. These findings illustrate the behavioral adaptability of maternal rats to pups' needs and the presence of other reinforcers, as well as its dependence on the context. In our view, future perspectives in the field, by incorporating the use of cutting-edge techniques, should analyze maternal flexibility and its neural substrates in models that incorporate complex and challenging contexts. This approach would allow a more comprehensive understanding of brain circuits involved in the adaptive and flexible nature of parenting.

Central Alteration in Peripheral Neuropathy of Trembler-J Mice: Hippocampal pmp22 Expression and Behavioral Profile in Anxiety Tests.

Charcot-Marie-Tooth (CMT) type 1 disease is the most common human hereditary demyelinating neuropathy. Mutations in pmp22 cause about 70% of all CMT1. Trembler-J (TrJ/+) mice are an animal model of CMT1E, having the same spontaneous pmp22 mutation that is found in humans. We compared the behavior profile of TrJ/+ and +/+ (wild-type) in open-field and elevated-plus-maze anxiety tests. In these tests, TrJ/+ showed an exclusive head shake movement, a lower frequency of rearing, but a greater frequency of grooming. In elevated-plus-maze, TrJ/+ defecate more frequently, performed fewer total entries, and have fewer entries to closed arms. These hippocampus-associated behaviors in TrJ/+ are consistent with increased anxiety levels. The expression of pmp22 and soluble PMP22 were evaluated in E17-hippocampal neurons and adult hippocampus by in situ hybridization and successive immunohistochemistry. Likewise, the expression of pmp22 was confirmed by RT-qPCR in the entire isolated hippocampi of both genotypes. Moreover, the presence of aggregated PMP22 was evidenced in unmasked granular hippocampal adult neurons and shows genotypic differences. We showed for the first time a behavior profile trait associated with anxiety and a differential expression of pmp22/PMP22 in hippocampal neurons of TrJ/+ and +/+ mice, demonstrating the involvement at the central level in an animal model of peripheral neuropathy (CMT1E).

Reorganization of perineuronal nets in the medial Preoptic Area during the reproductive cycle in female rats.

Perineuronal nets (PNNs) are aggregations of extracellular matrix associated with specific neuronal populations in the central nervous system, suggested to play key roles in neural development, synaptogenesis and experience-dependent synaptic plasticity. Pregnancy and lactation are characterized by a dramatic increase in neuroplasticity. However, dynamic changes in the extracellular matrix associated with maternal circuits have been mostly overlooked. We analyzed the structure of PNNs in an essential nucleus of the maternal circuit, the medial preoptic area (mPOA), during the reproductive cycle of rats, using the Wisteria floribunda (WFA) label. PNNs associated to neurons in the mPOA start to assemble halfway through gestation and become highly organized prior to parturition, fading through the postpartum period. This high expression of PNNs during pregnancy appears to be mediated by the influence of estrogen, progesterone and prolactin, since a hormonal simulated-gestation treatment induced the expression of PNNs in ovariectomized females. We found that PNNs associated neurons in the mPOA express estrogen receptor α and progesterone receptors, supporting a putative role of reproductive hormones in the signaling mechanisms that trigger the assembly of PNNs in the mPOA. This is the first report of PNNs presence and remodeling in mPOA during adulthood induced by physiological variables.

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.

Raising overlapping litters: Differential activation of rat maternal neural circuitry after interacting with newborn or juvenile pups.

The maternal behaviour of a rat dynamically changes during the postpartum period, adjusting to the characteristics and physiological needs of the pups. This adaptation has been attributed to functional modifications in the maternal circuitry. Maternal behaviour can also flexibly adapt according to different litter compositions. Thus, mothers with two overlapping litters can concurrently take care of neonate and juvenile pups, mostly directing their attention to the newborns. We hypothesised that the maternal circuitry of these mothers would show a differential activation pattern after interacting with pups depending on the developmental stage of their offspring. Thus, we evaluated the activation of several areas of the maternal circuitry in mothers of overlapping litters, using c-Fos immunoreactivity as a marker of neuronal activation, after interacting with newborns or juveniles. The results showed that mothers with overlapping litters display different behavioural responses towards their newborn and their juvenile pups. Interestingly, these behavioural displays co-occurred with specific patterns of activation of the maternal neural circuitry. Thus, a similar expression of c-Fos was observed in some key brain areas of mothers that interacted with newborns or juveniles, such as the medial preoptic area and the nucleus accumbens, whereas a differential activation was quantified in the ventral region of the bed nucleus of the stria terminalis, the infralimbic and prelimbic subregions of the medial prefrontal cortex and the basolateral and medial nuclei of the amygdala. We posit that the specific profile of activation of the neural circuitry controlling maternal behaviour in mothers with overlapping litters enables dams to respond adequately to the newborn and the juvenile 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.

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.

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.

Previous and recent maternal experiences modulate pups' incentive value relative to a male without affecting maternal behavior in postpartum estrous rats.

This study extends the behavioral analysis of the postpartum estrus (PPE) which represents a unique period in the female rat's lifetime when maternal and sexual motivations co-exist. The aim of this study was to explore how previous and recent maternal experiences influence the maternal responses to pups when confronted with a male in a preference test or when they are presented independently in the home cage. To achieve this objective, we firstly compared the maternal behavior in the home cage and the preference for pups or a male in a Y-maze of primiparous and multiparous females approximately twelve hours after delivery. No differences were observed in the active and passive components of the maternal behavior of primiparous and multiparous rats; however second-time mothers made more efforts to gain access to the pups and tended to spend more time with them in the Y-maze than maternally inexperienced dams. In a second experiment, we assessed the influence of recent maternal experience with pups on PPE females' behavior by comparing pups vs. male preference and maternal behavior of females that had experienced continuous or limited (approximately two hours) interaction with their litters after parturition was completed. PPE rats subjected to reduced interaction with their pups preferred the male, while females continuously exposed to pups chose them over the male. This change in females' preference was not accompanied by significant alterations of maternal performance in the home cage, although anogenital licking tended to decrease in females with limited mother-litter interaction. Together, the results of these experiments indicate that previous and recent maternal experiences influence the motivational responses of PPE females, and that these effects are more evident when both motivations compete.

Compulsive-like behaviour according to the sex and the reproductive stage of female rats.

The aim of the present study was to explore putative differences in the responses assessed in an animal model of obsessive-compulsive disorder (OCD) according to the sex and the reproductive cycle of female rats. The model consists of the induction of perseveration (repetitive choices of the same arm in a T-maze) by 8-OH-DPAT (1.0mg/kg). Males and females (pooled in all stages of their oestrous cycle) persevered after 8-OH-DPAT administration and no differences were observed between groups. During the oestrous cycle, this 5-HT(1A) agonist induced perseveration in metoestrus, dioestrus and prooestrus and reduced levels of this behaviour in oestrus. 8-OH-DPAT provoked perseveration in mid-gestation, an effect that was reduced in late-gestation and blocked during lactation. Reproductive cycle changes in the induced perseveration are discussed from the standpoint of the ovarian steroids' action on the serotoninergic system and on the bases of the variations in stress responsiveness along the reproductive cycle of the female. Present results validate the use of females in this model of OCD and could be relevant for studying the role of reproductive hormones in the pathophysiology of this disorder.

Maternal and affective behaviors of lactating rats reared in overlapping litters.

Postpartum mating in rats gives rise to complex family units consisting of the mother and two overlapping litters. As a consequence, newborn pups of the second litter, since the moment they are born, acquire experience not only from interaction with the mother and age-matched littermates but also from interaction with older siblings. Newborn pups reared in overlapping litters (OLs) receive a different pattern of maternal stimulation compared to those reared in single litters (SL: one litter of same aged pups), as the mothers reduce some maternal behavior components and juvenile pups from the first litter develop maternal behavior. Since there is strong evidence showing that variations in maternal behavior are transmitted throughout generations, we hypothesized that the altered pattern of maternal stimulation received by OL reared females would modify their behavior during motherhood. To test this hypothesis maternal behavior, maternal aggression and experimental anxiety of dams reared under OL and SL conditions during the first postpartum week were compared. No differences were found between the groups in their maternal behavior and aggression. This result may be explained by the maternal behavior of the juveniles that could compensate for the deficits in the caregiving behaviors received by OL litters. However, a subtle temporal reorganization of the licking behavior was found in OL reared mothers, together with an increased anxiety-related behavior in the plus maze test. These results suggest dissociation in the effects provoked by early environmental alterations on different behavioral systems, and more importantly, that independently of their early family composition, both groups can cope effectively with the changing demands of the pups.

Flexibility and adaptation of the neural substrate that supports maternal behavior in mammals.

Maternal behavior is species-specific and expressed under different physiological conditions, and contexts. It is the result of neural processes that support different forms (e.g. postpartum, cycling sensitized and spontaneous maternal behavior) and modalities of mother-offspring interaction (e.g. maternal interaction with altricial/precocious young; selective/non-selective bond). To understand how the brain adapts to and regulates maternal behavior in different species, and physiological and social conditions we propose new neural models to explain different forms of maternal expression (e.g. sensitized and spontaneous maternal behavior) and the behavioral changes that occur across the postpartum period. We emphasize the changing role of the medial preoptic area in the neural circuitry that supports maternal behavior and the cortical regulation and adjustment of ongoing behavioral performance. Finally, we discuss how our accumulated knowledge about the psychobiology of mothering in animal models supports the validity of animal studies to guide our understanding of human mothering and to improve human welfare and health.

New theoretical and experimental approaches on maternal motivation in mammals.

Maternal behavior is expressed in different modalities, physiological conditions, and contexts. It is the result of a highly motivated brain, that allows the female to flexibily adapt her caring activities to different situations and social demands. To understand how mothers coordinate maternal and other motivated behaviors we discuss the limitations of current theoretical approaches to study maternal motivation (e.g. distinction between appetitive and consummatory behaviors), and propose a different approach (i.e. motorically active vs. passive motivations) and a distinction between maternal motivated state and maternal motivated behaviors. We review the evidence supporting dopamine mediation of maternal motivation and describe how different phases of the dopaminergic response - basal, tonic, and phasic release in the nucleus accumbens - relate to increased salience, invigorating behavior, and behavioral switching. The existing and new experimental paradigms to investigate maternal motivation, and its coexpression and coordination with other social or non-social motivations are also analyzed. An example of how specificity of motivational systems (e.g. maternal and sexual behavior at postpartum estrus) could be processed at the neural level is also provided. This revision offers new theoretical and experimental approaches to address the fundamental question of how mothers flexibly adapt and coordinate the different components of maternal behavior with other motivated behaviors, also critical for the survival of the species.

Different chemical fractions of fetal fluids account for their attractiveness at parturition and their repulsiveness during late-gestation in the ewe.

We have investigated whether the chemical components of fetal fluids (FFs), which elicit repulsion in late gestating ewes, are also those responsible for the attractiveness of fetal fluids at parturition. An aqueous fraction of FFs (A1), obtained after extraction with hexane, was tested for repulsion in late-pregnant ewes and for attraction at parturition. We also investigated if the repulsive and attractive characteristics of this A1 fraction were maintained after an additional extraction with dichloromethane (DCM, CH(2)Cl(2)) that produced two more fractions (aqueous/high polarity: A2 and dichloromethane/medium polarity: DCM). Thus, late-pregnant ewes were tested for repulsion of aqueous extracts of FFs (A1, A2 and DCM fractions) in a two-choice test of food preference, whereas parturient ewes were tested for attraction toward these same fractions in a two-choice test of licking warm spongy cloths. The A1 fraction was repulsive to late-pregnant ewes and attractive to parturient females. In contrast, neither the A2 nor the DCM fractions were repulsive to late-pregnant ewes, whereas both fractions were attractive to parturient ones. The discordance between the repulsive and attractive properties of the A2 and DCM fractions suggests that the attractiveness of FFs for parturient ewes and its repulsiveness for females outside the peripartum period depend on mixtures of substances that are at least partially different. Some compounds with high and medium polarity in the A2 and DCM fractions would act synergistically to generate the repulsiveness of FFs, whereas both high and medium polarity compounds can evoke attraction independently of each other.