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Infant maltreatment increases vulnerability to physical and mental disorders, yet specific mechanisms embedded within this complex infant experience that induce this vulnerability remain elusive. To define critical features of maltreatment-induced vulnerability, rat pups were reared from postnatal day 8 (PN8) with a maltreating mother, which produced amygdala and hippocampal deficits and decreased social behavior at PN13. Next, we deconstructed the maltreatment experience to reveal sufficient and necessary conditions to induce this phenotype. Social behavior and amygdala deficits (volume, neurogenesis, c-Fos, local field potential) required combined chronic high corticosterone and maternal presence (not maternal behavior). Hippocampal deficits were induced by chronic high corticosterone regardless of social context. Causation was shown by blocking corticosterone during maltreatment and suppressing amygdala activity during social behavior testing. These results highlight (1) that early life maltreatment initiates multiple pathways to pathology, each with distinct causal mechanisms and outcomes, and (2) the importance of social presence on brain development.

The dense expression of glucocorticoid receptors (GR) within the amygdala, medial prefrontal cortex (mPFC) and paraventricular nucleus of hypothalamus (PVN) mediates many aspects of emotional and stress regulation. Importantly, both prenatal alcohol exposure (PAE) and adolescent stress are known to induce emotional and stress dysregulation. Little is known, however, about how PAE and/or adolescent stress may alter the expression of GR in the amygdala, mPFC, and PVN. To fill this gap, we exposed PAE and control adolescent male and female rats to chronic mild stress (CMS) and assessed GR mRNA expression in the amygdala, mPFC, and PVN immediately following stress or in adulthood. We found that the effects of PAE on GR expression were more prevalent in the amygdala, while effects of adolescent stress on GR expression were more prevalent in the mPFC. Moreover, PAE effects in the amygdala were more pronounced during adolescence and adolescent stress effects in the mPFC were more pronounced in adulthood. GR expression in the PVN was affected by both PAE and adolescent stress. Finally, PAE and/or adolescent stress effects were distinct between males and females. Together, these results suggest that PAE and adolescent CMS induce dynamic alterations in GR expression in the amygdala, mPFC, and PVN, which manifest differently depending on the brain area, age, and sex of the animal. Additionally, these data indicate that PAE-induced hyperresponsiveness to stress and increased vulnerability to mental health problems may be mediated by different neural mechanisms depending on the sex and age of the animal.

Additional file 1 Table S1. Cytokine Assay Lower Limits of Detection. Table S2. Component Loadings.

Social interaction deficits seen in psychiatric disorders emerge in early-life and are most closely linked to aberrant neural circuit organization and function. Due to technical limitations, we have had little understanding of the typical ontogeny of social behavior neural circuits or how early caregiving adversity impacts infant neurobiology to perturb lifelong social interaction. Using a suite of invasive procedures in awake, behaving young infant rats, including optogenetics, microdialysis and microinfusions, we charted the gradual increase in social behavior deficits following adversity-rearing and dissected circuits controlling this process. Persistently elevated dopamine in basolateral amygdala (BLA) was necessary and sufficient in initiating social behavior pathology, as demonstrated by manipulation of amygdala dopamine during adversity and during expression of social behavior deficits with the mother and peers. Taken together, these data highlight mesolimbic dopamine circuit organization and function as a potential therapeutic target in understanding behavioral deficits associated with psychiatric disorders.

New findings What is the central question of this study? Early-life adversity is associated with increased risk for obesity and metabolic dysfunction. However, it is unclear whether obesity and metabolic dysfunction result from coping strategies to deal with adversity-related emotional dysregulation, a direct programming of systems regulating metabolic function, or a combination of both. What is the main finding and its importance? Early-life adversity increases vulnerability to later-life obesity and metabolic dysfunction, indicating that genetics and adult lifestyle are not the only determinants of obesity and related metabolic dysfunction. Moreover, consumption of cafeteria diet exacerbated metabolic dysfunction associated with early-life adversity, suggesting that poor dietary choices might have a bigger impact in the context of early-life adversity. Abstract Early-life adversity has become recognized as an important factor contributing to adult obesity and associated metabolic dysfunction. However, it is unclear whether obesity and metabolic dysfunction associated with early-life adversity result from coping strategies to deal with adversity-related emotional dysregulation, a direct programming of systems regulating metabolic function, or a combination. Interestingly, both early-life adversity and later-life dietary choices affect immune function, favouring pro-inflammatory mechanisms that are associated with obesity-related metabolic dysfunction. To investigate the unique and/or interactive effects of early-life adversity and later-life dietary choices for increased vulnerability to obesity and metabolic dysfunction, and specifically the role of the immune system in this vulnerability, we combined a naturalistic rat model of early-life scarcity-adversity with a rat model of obesity, the cafeteria diet. Our results indicate that early-life adversity alone induces insulin resistance, reduces pancreatic insulin secretion, plasma concentrations of triglycerides and cholesterol, and increases fasting glucose and tumour necrosis factor-α plasma concentrations. Importantly, animals exposed to adverse rearing were more vulnerable to metabolic dysregulation associated with the cafeteria diet, given that they consumed more energy, showed more severe hepatic steatosis and increased concentrations of the pro-inflammatory cytokine interleukin-1β than normally reared animals fed the cafeteria diet. Together, our results suggest that early-life adversity negatively programmes physiological systems that regulate metabolic function and increases vulnerability to obesity and metabolic dysfunction in adulthood. These results highlight the intrinsic relationship between the quality of the early postnatal environment and later-life dietary choices on adult health outcomes.

The immediate and long-term effects of exposure to early life stress (ELS) have been documented in humans and animal models. Even relatively brief periods of stress during the first 10 days of life in rodents can impact later behavioral regulation and the vulnerability to develop adult pathologies, in particular an impairment of cognitive functions and neurogenesis, but also modified social, emotional, and conditioned fear responses. The development of preclinical models of ELS exposure allows the examination of mechanisms and testing of therapeutic approaches that are not possible in humans. Here, we describe limited bedding and nesting (LBN) procedures, with models that produce altered maternal behavior ranging from fragmentation of care to maltreatment of infants. The purpose of this paper is to discuss important issues related to the implementation of this chronic ELS procedure and to describe some of the most prominent endpoints and consequences, focusing on areas of convergence between laboratories. Effects on the hypothalamic-pituitary adrenal (HPA) axis, gut axis and metabolism are presented in addition to changes in cognitive and emotional functions. Interestingly, recent data have suggested a strong sex difference in some of the reported consequences of the LBN paradigm, with females being more resilient in general than males. As both the chronic and intermittent variants of the LBN procedure have profound consequences on the offspring with minimal external intervention from the investigator, this model is advantageous ecologically and has a large translational potential. In addition to the direct effect of ELS on neurodevelopmental outcomes, exposure to adverse early environments can also have intergenerational impacts on mental health and function in subsequent generation offspring. Thus, advancing our understanding of the effect of ELS on brain and behavioral development is of critical concern for the health and wellbeing of both the current population, and for generations to come.

Prenatal alcohol exposure (PAE) and early-life adversity (ELA) both negatively impact social neurobehavioral development, including social recognition memory. Importantly, while individuals with PAE are more likely to experience ELA, relatively few studies have assessed the interaction of these two early insults on adolescent social behavior development. Here, we combine animal models of PAE and ELA to investigate both their unique and interactive effects on social neurobehavioral function in early and late adolescent male and female rats. Behavioral testing was followed by assessment of hypothalamic expression of oxytocin (OT) and vasopressin (AVP), key neuropeptides in the regulation of social behavior. Our results indicate that PAE and ELA have unique sex- and age-specific effects on social recognition memory and OT/AVP expression, with more pronounced neurobehavioral changes observed in males than in females in both early and late adolescence. Specifically, ELA impaired social recognition in early adolescent females regardless of prenatal treatment, while males showed deficits in both early and late adolescence in response to unique and interactive effects of PAE and ELA. Neurobiological data suggest that these perinatal insults differentially impact the OT and AVP systems in a sexually dimorphic manner, such that the OT system appears to be particularly sensitive to PAE in males while the AVP system appears to be more vulnerable to ELA in females. Taken together, our data provide novel insight into how the early postnatal environment may mediate outcomes of PAE as well as the power of animal models to interrogate the relationship between these pre- and postnatal insults.

Cytokines and chemokines are potent modulators of brain development and as such, dysregulation of the maternal immune system can result in deviations in the fetal cytokine balance, altering the course of typical brain development, and putting the individual on a "pathway to pathology". In the current study, we used a multi-variate approach to evaluate networks of interacting cytokines and investigated whether alterations in the maternal immune milieu could be linked to alcohol-related and alcohol-independent child neurodevelopmental delay. This was achieved through the measurement of 40 cytokines/chemokines from maternal blood samples collected during the second and third trimesters of pregnancy. Importantly, during the second trimester we identified network enrichment in levels of cytokines including IFN-ɣ, IL-10, TNF-β, TNF-α, and CRP associated with offspring neurodevelopmental delay. However, as elevations in levels of these cytokines have previously been reported in a wide range of neurodevelopmental disorders including autism spectrum disorder and schizophrenia, we suggest that this cytokine profile is likely not disorder specific, but rather may be an indicator of neurodevelopmental delay in general. By contrast, distinct clusters of activated/inhibited cytokines were identified based on maternal alcohol consumption and child neurodevelopmental outcome. Specifically, cytokines including IL-15, IL-10, MDC, and members of the VEGF sub-family were highest in alcohol-consuming mothers of children with neurodevelopmental delay and were identified in both network analyses and examination of individual cytokines, whereas a differential and unique cytokine profile was identified in the case of alcohol-independent child neurodevelopmental delay. We propose that the current findings could provide a critical step towards the development of early biomarkers and possibly interventions for alcohol-related neurodevelopmental delay. Importantly, the current approach could be informative for understanding mechanisms linking maternal immune system dysfunction and adverse child outcomes in a range of other neurodevelopmental disorders.

Children and adults prenatally exposed to alcohol show higher rates of mental health problems than unexposed individuals, with depression and anxiety being among the more commonly encountered disorders. Previous studies in rats showed that prenatal alcohol exposure (PAE) can indeed increase depressive- and anxiety-like behavior in adulthood; however, depression and anxiety are often observed in the context of stress and/or a dysregulated stress response system (the hypothalamic-pituitary-adrenal [HPA] axis). PAE can dysregulate the HPA axis, resulting in hyperresponsivity to stress. In turn, this may predispose individuals prenatally exposed to alcohol to the adverse effects of stress compared to unexposed individuals. We have shown previously that PAE animals may be more sensitive to the effects of chronic stress on behavior, showing increased anxiety- and depressive-like behavior following chronic unpredictable stress (CUS) exposure. Here, we investigated the independent and interactive effects of PAE and adult CUS on anxiety-like behavior and receptor systems (corticotropin-releasing hormone receptor type 1 [CRHR1], mineralocorticoid receptor [MR], and glucocorticoid receptor [GR]), and underlying stress and emotional regulation, and whether exposure to CUS differentially results in immediate or delayed effects. Adult male and female offspring from PAE, pair-fed (PF), and ad libitum-fed control (C) dams were exposed to either 10 days of CUS or left undisturbed. Behavioral testing began 1 or 14 days post-CUS, and brains were collected following testing. Anxiety-like behaviors were evaluated using the open field, elevated plus maze and dark-light emergence tests. CRHR1, MR, and GR mRNA expression were assessed in the medial prefrontal cortex (mPFC), amygdala, and hippocampal formation, brain areas key to both stress and emotional regulation. We found that PAE differentially increased anxiety-like behavior and altered GR mRNA in males and females compared to their control counterparts. Furthermore, depending on the timing of testing, CUS unmasked alterations in GR and CRHR1 mRNA expression in the mPFC and amygdala in PAE males, and MR mRNA in the hippocampal formation in PAE females compared to their C counterparts. Overall, the changes observed in these receptor systems may underlie the increase in anxiety-like behavior following PAE and CUS exposure in adulthood. That CUS differentially affected brain and behavioral outcome of PAE and C animals, and did so in a sexually-dimorphic manner, has important implications for understanding the etiology of psychopathology in individuals prenatally exposed to alcohol.

Many functions of the hippocampus are affected by prenatal alcohol exposure (PAE). In particular, dysregulation of the stress response is especially important because individuals with PAE carry increased risks for exposure to stressful environments throughout life. Little is known, though, about how adolescent stress in the context of PAE-related stress system dysregulation may further alter hippocampal development. Here, we investigate the short- and long-term effects of adolescent chronic mild stress (CMS) on mRNA expression of stress-related mineralocorticoid (MR), glucocorticoid (GR), and type 1 CRH (CRHR1) receptors in the dorsal and ventral hippocampal formation of PAE and control rats. Our results indicate that PAE affects the expression of stress-related receptors in the hippocampus; however, PAE effects were more prominent during adolescence, as MR and CRHR1 mRNA expression were altered in both male and female PAE animals, with GR mRNA expression alterations observed only in PAE female. In adulthood, the effects of PAE were restricted to alterations in CRHR1 mRNA expression in females, while there were no effects in males. In contrast, the effects of adolescent CMS were more pronounced in adulthood, long after stress exposure termination. Importantly, PAE animals were less responsive to adolescent CMS, with effects only on CRHR1 in PAE animals compared to the altered MR, GR, and CRHR1 mRNA expression observed in controls. Together, our results show that PAE and adolescent CMS induce dynamic alterations in the expression of stress-related receptors in the hippocampal formation that manifest differently depending on the age and sex of the animal.

The contribution of the early postnatal environment to the pervasive effects of prenatal alcohol exposure (PAE) is poorly understood. Moreover, PAE often carries increased risk of exposure to adversity/stress during early life. Dysregulation of immune function may play a role in how pre- and/or postnatal adversity/stress alters brain development. Here, we combine two animal models to examine whether PAE differentially increases vulnerability to immune dysregulation in response to early-life adversity. PAE and control litters were exposed to either limited bedding (postnatal day [PN] 8-12) to model early-life adversity or normal bedding, and maternal behavior and pup vocalizations were recorded. Peripheral (serum) and central (amygdala) immune (cytokines and C-reactive protein - CRP) responses of PAE animals to early-life adversity were evaluated at PN12. Insufficient bedding increased negative maternal behavior in both groups. Early-life adversity increased vocalization in all animals; however, PAE pups vocalized less than controls. Early-life adversity reduced serum TNF-α, KC/GRO, and IL-10 levels in control but not PAE animals. PAE increased serum CRP, and levels were even higher in pups exposed to adversity. Finally, PAE reduced KC/GRO and increased IL-10 levels in the amygdala. Our results indicate that PAE alters immune system development and both behavioral and immune responses to early-life adversity, which could have subsequent consequences for brain development and later life health.

Altricial infants, such as humans and rats, must form an attachment to their caregiver for survival, even if the caregiver is abusive. A unique pathway for learning appears to support this attachment learning, which provides robust and rapid learning regardless of the quality of care received. This abuse-related attachment is associated with diminished cognitive and emotional regulation from the caregiver and vulnerability to later-life neurobehavioral and psychiatric disorders.

Caregiver-associated cues, including those learned in abusive attachment, provide a sense of safety and security to the child. Here, we explore how cues associated with abusive attachment, such as maternal odor, can modify the enduring neurobehavioral effects of early-life abuse. Two early-life abuse models were used: a naturalistic paradigm, where rat pups were reared by an abusive mother; and a more controlled paradigm, where pups underwent peppermint odor-shock conditioning that produces an artificial maternal odor through engagement of the attachment circuit. Animals were tested for maternal odor preference in infancy, forced swim test (FST), social behavior, and sexual motivation in adulthood—in the presence or absence of maternal odors (natural or peppermint). Amygdala odor-evoked local field potentials (LFPs) via wireless electrodes were also examined in response to the maternal odors in adulthood. Both early-life abuse models induced preference for the maternal odors in infancy. In adulthood, these early-life abuse models produced FST deficits and decreased social behavior, but did not change sexual motivation. Presentation of the maternal odors rescued FST and social behavior deficits induced by early-life abuse and enhanced sexual motivation in all animals. In addition, amygdala LFPs from both abuse animal models showed unique activation within the gamma frequency (70–90 Hz) bands in response to the specific maternal odor present during early-life abuse. These results suggest that attachment-related cues learned during infancy have a profound ability to rescue neurobehavioral dysregulation caused by early-life abuse. Paradoxically, abuse-associated cues seem to acquire powerful and enduring antidepressive properties and alter amygdala modulation.

As one of the first rodent models designed to investigate the effects of early-life experiences, the neonatal handling paradigm has helped us better understand how subtle changes in the infant environment can powerfully drive neurodevelopment of the immature brain in typical or atypical trajectories. Here, we review data from more than 50 years demonstrating the compelling effects of neonatal handling on behavior, physiology, and neural function across the lifespan. Moreover, we present data that challenge the classical view of neonatal handling as an animal model that results only in positive/beneficial outcomes. Indeed, the overall goal of this review is to offer the suggestion that the effects of early-life experiences-including neonatal handling-are nuanced rather than unidirectional. Both beneficial and negative outcomes may occur, depending on the parameters of testing, sex of the subject, and neurobehavioral system analyzed.

A major component of perception is hedonic valence: perceiving stimuli as pleasant or unpleasant. Here, we used early olfactory experiences that shape odor preferences and aversions to explore developmental plasticity in circuits mediating odor hedonics. We used 2-deoxyglucose autoradiographic mapping of neural activity to identify circuits differentially activated by biologically relevant preferred and avoided odors across rat development. We then further probed this system by increasing or decreasing hedonic value. Using both region of interest and functional connectivity analyses, we identified regions within primary olfactory, amygdala/hippocampal, and prefrontal cortical networks that were activated differentially by maternal and male odors. Although some activated regions remained stable across development (postnatal days 7–23), there was a developmental emergence of others that resulted in an age-dependent elaboration of hedonic-response-specific circuitry despite stable behavioral responses (approach/avoidance) to the odors across age. Hedonic responses to these biologically important odors were modified through diet suppression of the maternal odor and co-rearing with a male. This allowed assessment of hedonic circuits in isolation of the specific odor quality and/or intensity. Early experience significantly modified odor-evoked circuitry in an age-dependent manner. For example, co-rearing with a male, which induced pup attraction to male odor, reduced activity in amygdala regions normally activated by the unfamiliar avoided male odor, making this region more consistent with maternal odor. Understanding the development of odor hedonics, particularly within the context of altered early life experience, provides insight into the development of sensory processes, food preferences, and the formation of social affiliations, among other behaviors. SIGNIFICANCE STATEMENT Odor hedonic valence controls approach–avoidance behaviors, but also modulates ongoing behaviors ranging from food preferences and social affiliation with the caregiver to avoidance of predator odors. Experiences can shape hedonic valence. This study explored brain circuitry involved in odor hedonic encoding throughout development using maternal and predator odors and assessed the effects of early life experience on odor hedonic encoding by increasing/decreasing the hedonic value of these odors. Understanding the role of changing brain circuitry during development and its impact on behavioral function is critical for understanding sensory processing across development. These data converge with exciting literature on the brain9s hedonic network and highlight the significant role of early life experience in shaping the neural networks of highly biologically relevant stimuli.

Abuse during early life, especially from the caregiver, increases vulnerability to develop later-life psychopathologies such as depression. Although signs of depression are typically not expressed until later life, signs of dysfunctional social behavior have been found earlier. How infant abuse alters the trajectory of brain development to produce pathways to pathology is not completely understood. Here we address this question using two different but complementary rat models of early-life abuse from postnatal day 8 (P8) to P12: a naturalistic paradigm, where the mother is provided with insufficient bedding for nest building; and a more controlled paradigm, where infants undergo olfactory classical conditioning. Amygdala neural assessment (c-Fos), as well as social behavior and forced swim tests were performed at preweaning (P20) and adolescence (P45). Our results show that both models of early-life abuse induce deficits in social behavior, even during the preweaning period; however, depressive-like behaviors were observed only during adolescence. Adolescent depressive-like behavior corresponds with an increase in amygdala neural activity in response to forced swim test. A causal relationship between the amygdala and depressive-like behavior was suggested through amygdala temporary deactivation (muscimol infusions), which rescued the depressive-like behavior in the forced swim test. Our results indicate that social behavior deficits in infancy could serve as an early marker for later psychopathology. Moreover, the implication of the amygdala in the ontogeny of depressive-like behaviors in infant abused animals is an important step toward understanding the underlying mechanisms of later-life mental disease associated with early-life abuse.

Neonatal handling is an experimental procedure used to understand how early-life adversity can negatively affect neurobehavioral development and place animals on a pathway to pathology. Decreased preference for the maternal odor during infancy is one of many behavioral deficits induced by neonatal handling. Here, we hypothesize that deficits in maternal odor preference may interfere with partner preference in the adult. To test this hypothesis, we assessed infant maternal odor preference and adult partner preference in different reproductive stages in both male and female rats that received neonatal handling. Our results indicate that only neonatally handled females present deficits in maternal odor preference during infancy, but both male and females present deficits in adult partner preference. However, sexual experience was effective in rescuing partner preference deficits in males. These results indicate that, considering infant and adult social interactions, females are more susceptible to the effects of neonatal handling than males.

Both odor-preference and odor-aversion learning occur in perinatal pups before the maturation of brain structures that support this learning in adults. To characterize the development of odor learning, we compared three learning paradigms: (1) odor-LiCl (0.3M; 1% body weight, ip) and (2) odor-1.2-mA shock (hindlimb, 1sec)—both of which consistently produce odor-aversion learning throughout life and (3) odor-0.5-mA shock, which produces an odor preference in early life but an odor avoidance as pups mature. Pups were trained at postnatal day (PN) 7–8, 12–13, or 23–24, using odor-LiCl and two odor-shock conditioning paradigms of odor-0.5-mA shock and odor-1.2-mA shock. Here we show that in the youngest pups (PN7–8), odor-preference learning was associated with activity in the anterior piriform (olfactory) cortex, while odor-aversion learning was associated with activity in the posterior piriform cortex. At PN12–13, when all conditioning paradigms produced an odor aversion, the odor-0.5-mA shock, odor-1.2-mA shock, and odor-LiCl all continued producing learning-associated changes in the posterior piriform cortex. However, only odor-0.5-mA shock induced learning-associated changes within the basolateral amygdala. At weaning (PN23–24), all learning paradigms produced learning-associated changes in the posterior piriform cortex and basolateral amygdala complex. These results suggest at least two basic principles of the development of the neurobiology of learning: (1) Learning that appears similar throughout development can be supported by neural systems showing very robust developmental changes, and (2) the emergence of amygdala function depends on the learning protocol and reinforcement condition being assessed.

Chronic alcohol consumption negatively affects health, and has additional consequences if consumption occurs during pregnancy as prenatal alcohol exposure adversely affects offspring development. While much is known on the effects of prenatal alcohol exposure in offspring less is known about effects of alcohol in dams. Here, we examine whether chronic alcohol consumption during gestation alters maternal behavior, hippocampal neurogenesis and HPA axis activity in late postpartum female rats compared with nulliparous females. Rats were assigned to alcohol, pair-fed or ad libitum control treatment groups for 21 days (for pregnant rats, this occurred gestation days 1 – 21). Maternal behavior was assessed throughout the postpartum period. Twenty-one days after alcohol exposure, we assessed doublecortin (DCX) (an endogenous protein expressed in immature neurons) expression in the dorsal and ventral hippocampus and HPA axis activity. Alcohol consumption during pregnancy reduced nursing and increased self-directed and negative behaviors, but spared licking and grooming behavior. Alcohol consumption increased corticosterone and adrenal mass only in nulliparous females. Surprisingly, alcohol consumption did not alter DCX-expressing cell density. However, postpartum females had fewer DCX-expressing cells (and of these cells more immature proliferating cells but fewer postmitotic cells) than nulliparous females. Collectively, these data suggest that alcohol consumption during pregnancy disrupts maternal care without affecting HPA function or neurogenesis in dams. Conversely, alcohol altered HPA function in nulliparous females only, suggesting that reproductive experience buffers the long-term effects of alcohol on the HPA axis.

Neonatal handling induces anovulatory estrous cycles and decreases sexual receptivity in female rats. The synchronous secretion of hormones from the gonads (estradiol (E2) and progesterone (P)), pituitary (luteinizing (LH) and follicle-stimulating (FSH) hormones) and hypothalamus (LH-releasing hormone (LHRH)) are essential for the reproductive functions in female rats. The present study aimed to describe the plasma levels of E2 and P throughout the estrous cycle and LH, FSH and prolactin (PRL) in the afternoon of the proestrus, and the LHRH content in the medial preoptic area (MPOA), median eminence (ME) and medial septal area (MSA) in the proestrus, in the neonatal handled rats. Wistar pup rats were handled for 1 min during the first 10 days after delivery (neonatal handled group) or left undisturbed (nonhandled group). When they reached adulthood, blood samples were collected through a jugular cannula and the MPOA, ME and MSA were microdissected. Plasma levels of the hormones and the content of LHRH were determined by RIA. The number of oocytes counted in the morning of the estrus day in the handled rats was significantly lower than in the nonhandled ones. Neonatal handling reduces E2 levels only on the proestrus day while P levels decreased in metestrus and estrus. Handled females also showed reduced plasma levels of LH, FSH and PRL in the afternoon of the proestrus. The LHRH content in the MPOA was significantly higher than in the nonhandled group. The reduced secretion of E2, LH, FSH and LHRH on the proestrus day may explain the anovulatory estrous cycle in neonatal handled rats. The reduced secretion of PRL in the proestrus may be related to the decreased sexual receptiveness in handled females. In conclusion, early-life environmental stimulation can induce long-lasting effects on the hypothalamus-pituitary-gonad axis.

Caregiver maltreatment induces vulnerability to later-life psychopathology. Clinical and preclinical evidence suggest changes in prefrontal and limbic circuitry underlie this susceptibility. We examined this question using a rat model of maternal maltreatment and methods translated from humans, resting-state functional magnetic resonance imaging (R-fMRI). Rat pups were reared by mothers provided with insufficient or abundant bedding for nest building from postnatal (PN) days 8 to 12 and underwent behavioral assessments of affect-related behaviors (forced swim, sucrose preference and social interaction) in adolescence (PN45) and early adulthood (PN60). R-fMRI sessions were conducted under light anesthesia at both ages. Offspring reared with insufficient bedding (that is, maltreated) displayed enduring negative affective behaviors. Amygdala-prefrontal cortex (PFC) functional connectivity increased significantly from adolescence to adulthood in controls, but not in maltreated animals. We computed the fractional amplitude of low-frequency fluctuations (fALFF), an index of intrinsic brain activity, and found that fALFF in medial prefrontal cortex and anterior cingulate cortex (MPFC/ACC) increased significantly with age in controls but remained unchanged in maltreated animals during adolescence and adulthood. We used a seed-based analysis to explore changes in functional connectivity between this region and the whole brain. Compared with controls, maltreated animals demonstrated reduced functional connectivity between MPFC/ACC and left caudate/putamen across both ages. Functional connectivity between MPFC/ACC and right caudate/putamen showed a group by age interaction: decreased in controls but increased in maltreated animals. These data suggest that maltreatment induces vulnerability to psychopathology and is associated with differential developmental trajectories of prefrontal and subcortical circuits underlying affect regulation.

During the postnatal period, the nervous system is modified and shaped by experience, in order to adjust it to the particular environment in which the animal will live. This plasticity, one of the most remarkable characteristics of the nervous system, promotes adaptive changes, but it also makes brain more vulnerable to insults. This chapter will focus on the effects of interventions during the postnatal development in animal models of neonatal handling (usually up to 15 min of handling) and maternal separation (usually at least for 3 h). Sex-specific changes and effects of prepubertal stress such as social isolation later on in life were also considered. These interventions during development induce long-lasting traces in the pups' nervous system, which will be reflected in changes in neuroendocrine functions, including the hypothalamus-pituitary-adrenal and hypothalamus-pituitary-gonadal axes; anxiety and cognitive performance; and feeding, sexual, and social behavior. These enduring changes may be adaptive or maladaptive, depending on the environment in which the animal will live. The challenge researchers facing now is to determine how to reverse the deleterious effects that may result from early-life stress exposure.

During the postnatal period, the nervous system is modified and shaped by experience, in order to adjust it to the particular environment in which the animal will live. This plasticity, one of the most remarkable characteristics of the nervous system, promotes adaptive changes, but it also makes brain more vulnerable to insults. This chapter will focus on the effects of interventions during the postnatal development in animal models of neonatal handling (usually up to 15 min of handling) and maternal separation (usually at least for 3 h). Sex-specific changes and effects of prepubertal stress such as social isolation later on in life were also considered. These interventions during development induce long-lasting traces in the pups’ nervous system, which will be reflected in changes in neuroendocrine functions, including the hypothalamus–pituitary–adrenal and hypothalamus–pituitary–gonadal axes; anxiety and cognitive performance; and feeding, sexual, and social behavior. These enduring changes may be adaptive or maladaptive, depending on the environment in which the animal will live. The challenge researchers facing now is to determine how to reverse the deleterious effects that may result from early-life stress exposure.

Individuals exposed to alcohol during gestation show higher rates of psychopathologies. The hyperresponsivity to stress induced by prenatal alcohol exposure may be related to this increased rate of psychopathologies, especially because this population is more likely to be exposed to stressful environments throughout life. However, alcohol-induced changes in the overlapping neurocircuitries that underlie stress and the expression of psychopathologies are not fully understood. Here, we performed a comprehensive analysis of the neural activity within central areas known to play key roles in both emotional and stress regulation. Adult male and female offspring from prenatal alcohol exposure (PAE), pair-fed, and ad libitum-fed control conditions were exposed to chronic mild stress (CMS). Following CMS, the neural activity (c-fos mRNA) of the amygdala, ventral hippocampal formation, medial prefrontal cortex (mPFC), and paraventricular nucleus of hypothalamus (PVN) was assessed in response to an acute stress (elevated plus maze). Our results demonstrate that, overall, PAE decreased neural activity within the amygdala and hippocampal formation in males and increased neural activity within the amygdala and mPFC in females. CMS reduced neural activity within the mPFC and PVN in PAE males, but reduced activity in all areas analyzed in control males. By contrast, CMS reduced neural activity in the mPFC in PAE females and had no effects in control females. Furthermore, the constrained principal component analysis revealed that these patterns of neural activity resulted in differential activation of functional neural networks in males compared to females, indicating sexually dimorphic effects of PAE and CMS. Importantly, the altered networks of brain activation in PAE animals may underlie the hyperresponsivity to stress and increased psychopathologies observed among individuals prenatally exposed to alcohol.

As one of the first rodent models designed to investigate the effects of early-life experiences, the neonatal handling paradigm has helped us better understand how subtle changes in the infant environment can powerfully drive neurodevelopment of the immature brain in typical or atypical trajectories. Here, we review data from more than 50 years demonstrating the compelling effects of neonatal handling on behavior, physiology, and neural function across the lifespan. Moreover, we present data that challenge the classical view of neonatal handling as an animal model that results only in positive/beneficial outcomes. Indeed, the overall goal of this review is to offer the suggestion that the effects of early-life experiences—including neonatal handling—are nuanced rather than unidirectional. Both beneficial and negative outcomes may occur, depending on the parameters of testing, sex of the subject, and neurobehavioral system analyzed.

Attachment to an abusive caregiver has wide phylogenetic representation, suggesting that animal models are useful in understanding the neural basis underlying this phenomenon and subsequent behavioral outcomes. We previously developed a rat model, in which we use classical conditioning to parallel learning processes evoked during secure attachment (odor-stroke, with stroke mimicking tactile stimulation from the caregiver) or attachment despite adversity (odor-shock, with shock mimicking maltreatment). Here we extend this model to mice. We conditioned infant mice (postnatal day (PN) 7–9 or 13–14) with presentations of peppermint odor and either stroking or shock. We used 14C 2-deoxyglucose (2-DG) to assess olfactory bulb and amygdala metabolic changes following learning. PN7-9 mice learned to prefer an odor following either odor-stroke or shock conditioning, whereas odor-shock conditioning at PN13-14 resulted in aversion/fear learning. 2-DG data indicated enhanced bulbar activity in PN7-9 preference learning, whereas significant amygdala activity was present following aversion learning at PN13-14. Overall, the mouse results parallel behavioral and neural results in the rat model of attachment, and provide the foundation for the use of transgenic and knockout models to assess the impact of both genetic (biological vulnerabilities) and environmental factors (abusive) on attachment-related behaviors and behavioral development.

BACKGROUND Hypertension is a major public health epidemic that is highly associated with sexual dysfunction in both men and women. Despite its high prevalence, clinical and animal literature on the underlying mechanisms of sexual dysfunction in hypertensive women is remarkably limited. METHODS Using a well-established rodent model of renovascular hypertension-the 2-kidney, 1-clip (2K1C) Goldblatt model-we investigated possible reproductive deficits in female rats. We evaluated several aspects of reproductive function in hypertensive female rats: estrous cycle, sexual behavior, ovulation, and plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol at proestrus afternoon. RESULTS Clipping of the left renal artery resulted in dramatic elevations in systolic blood pressure and heart rate. Renovascular hypertension was associated with a delay for reestablishing estrous cyclicity (50% of 2K1C rats failed to resume cycling by 15 days after surgery). In rats that resumed cycling, 2K1C female rats showed a decrease in sexual behavior, evidenced by a decreased lordosis quotient and a reduction in ovulation, as demonstrated by a decreased number of oocytes. Moreover, plasma levels of LH on the proestrus afternoon were reduced in hypertensive female rats, but no changes in estradiol or FSH were observed. CONCLUSIONS Our results demonstrate that renovascular hypertension induces an overall decrease in reproductive function in female rats. Most important, our results indicate that the animal model of renovascular hypertension could be used as a relevant tool to understand better the pathophysiological mechanisms involved in the reproductive deficits in women with renovascular hypertension.

Obesity is an epidemic disease most commonly caused by a combination of increased energy intake and lack of physical activity. The cholinergic system has been shown to be involved in the regulation of food intake and energy expenditure. Moreover, physical exercise promotes a reduction of fat pads and body mass by increasing energy expenditure, but also influences the cholinergic system. The aim of this study is to evaluate the interaction between physical exercise (swimming) and central cholinergic activity in rats treated with monosodium glutamate (MSG, a model for obesity) during infancy. Our results show that MSG treatment is able to induce obesity in male and female rats. Specifically, MSG-treated rats presented a reduced body mass and nasoanal length, and increased perigonadal and retroperitoneal fat pads in relation to the body mass. Physical exercise was able to reduce body mass in both male and female rats, but did not change the fat pads in MSG-treated rats. Increased food intake was only seen in MSG-treated females submitted to exercise. Cholinergic activity was increased in the cortex of MSG-treated females and physical exercise was able to reduce this activity. Thalamic cholinergic activity was higher in sedentary MSG-treated females and exercised MSG-treated males. Hypothalamic cholinergic activity was higher in male and female MSG-treated rats, and was not reduced by exercise in the 2 sexes. Taken together, these results show that MSG treatment and physical exercise have different effects in the cholinergic activity of males and females.

International audience; Maltreatment from the caregiver induces vulnerability to later life psychopathologies, yet attraction and comfort is sometimes provided by cues associated with early life maltreatment. We used a rat model of early life maltreatment with odor-0.5 mA shock conditioning to produce depressive-like behaviors and questioned whether stimuli associated with maltreatment would restore emotional neurobehavioral function to control levels. Pups received daily novel odor-0.5 mA shock conditioning from postnatal day 8 to 12. This procedure produces a new maternal odor that controls pups' attachment behaviors. In adulthood, either with or without the infant odor, animals received a Forced Swim Test, Sucrose Preference Test or assessment of amygdala and olfactory system functioning using field potential signal evoked by olfactory bulb paired-pulse electrical stimulation. Following neonatal odor-shock pairings, but not unpaired controls, adults without the odor present showed increased depression-like behavior in the Forced Swim Test and Sucrose Preference Test and a deficit in paired-pulse inhibition in amygdala and piriform (olfactory) cortex. All effects were brought to control levels when the infant conditioned odor was presented during behavioral and neural tests. The ability of cues associated with early life maltreatment to normalize behavior and amygdala activity suggests these cues provide adaptive value in adulthood.

Infant rats must learn to identify their mother’s diet-dependent odor. Once learned, maternal odor controls pups’ approach to the mother, their social behavior and nipple attachment. Here we present a review of the research from four different laboratories, which suggests that neural and behavioral responses to the natural maternal odor and neonatal learned odors are similar. Together, these data indicate that pups have a unique learning circuit relying on the olfactory bulb for neural plasticity and on the hyperfunctioning noradrenergic locus coeruleus flooding the olfactory bulb with norepinephrine to support the neural changes. Another important factor making this system unique is the inability of the amygdala to become incorporated into the infant learning circuit. Thus, infant rats appear to be primed in early life to learn odors that will evoke approach responses supporting attachment to the caregiver.

Altricial infants, such as humans and rats, must form an attachment to their caregiver for survival. A unique pathway for learning appears to support this attachment learning, which provides robust and rapid learning.

Background Both abused and well cared for infants show attachment to their caregivers, although the quality of that attachment differs. Moreover, the infant's attachment to the abusive caregiver is associated with compromised mental health, especially under stress. In an attempt to better understand how abuse by the caregiver can compromise mental health, we explore the neural basis of attachment in both typical and abusive environments using infant rats, which form attachments to the mother through learning her odor. Here, we hypothesize that the neural circuitry for infant attachment differs based on the quality of the attachment, which can be uncovered during stressful situations. Methods We used infant rats to compare infant attachment social behaviors and supporting neurobiology using natural maternal odor, as well as two odor-learning attachment paradigms: odor-stroke (mimics typical attachment) and odor-.5 mA shock conditioning (mimics abusive attachment). Next, to uncover differences in behavior and brain, these pups were injected with systemic corticosterone. Finally, pups were reared with an abusive mother to determine ecological relevance. Results Our results suggest that the natural and learned attachment odors indistinguishably control social behavior in infancy (approach to the odor and interactions with the mother). However, with corticosterone injection, pups with an abusive attachment show disrupted infant social behavior with the mother and engagement of the amygdala. Conclusions This animal model of attachment accommodates both abusive and typical attachment and suggests that pups' social behavior and underlying neural circuitry may provide clues to understanding attachment in children with various conditions of care.

The hippocampus is a part of the limbic system and is important for the formation of associative memories, such as acquiring information about the context (e.g., the place where an experience occurred) during emotional learning (e.g., fear conditioning). Here, we assess whether the hippocampus is responsible for pups' newly emerging context learning. In all experiments, postnatal day (PN) 21 and PN24 rat pups received 10 pairings of odor-0.5 mA shock or control unpaired odor-shock, odor only, or shock only. Some pups were used for context, cue or odor avoidance tests, while the remaining pups were used for c-Fos immunohistochemistry to assess hippocampal activity during acquisition. Our results show that cue and odor avoidance learning were similar at both ages, while contextual fear learning and learning-associated hippocampal (CA1, CA3, and dentate gyrus) activity (c-Fos) only occurred in PN24 paired pups. To assess a causal relationship between the hippocampus and context conditioning, we infused muscimol into the hippocampus, which blocked acquisition of context fear learning in the PN24 pups. Muscimol or vehicle infusions did not affect cue learning or aversion to the odor at PN21 or PN24. The results suggest that the newly emerging contextual learning exhibited by PN24 pups is supported by the hippocampus.

Stress might influence the reproductive behavior in females, and central angiotensin II (Ang II) is a peptide that plays a role in stress response and in the modulation of sexual behavior. The medial amygdala (MeA), an important structure that regulates this behavior, is strongly involved in stress response. The aim of the present study was to evaluate the effect of acute restraint stress on the night of proestrus on sexual receptivity in female rats and the participation of Ang II and MeA in this effect. Adult female Wistar rats with regular estrous cycles were utilized. The acute stress protocol utilized was the restraint stress for 15 min on the night of proestrus. The participation of Ang II was evaluated by injecting Ang II and Ang II receptor antagonists (losartan and PD12319) into the MeA. The lordosis quotient was recorded. The stress or the microinjection of Ang II into the MeA significantly reduced sexual behavior. The blockade of AT(1) or AT(2) receptors in the MeA prevented the effect of stress and the effect of Ang II microinjection into this nucleus on sexual receptivity. We concluded that acute restraint stress on the night of proestrus reduces sexual behavior in rats, and this effect is mediated by both AT(1) and AT(2) receptors in the MeA.

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic-pituitary-adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age.

Early-life environmental events that disrupt the mother-pup relationship may induce profound long-lasting changes on several behavioral and neuroendocrine systems. The neonatal handling procedure, which involves repeated brief maternal separations followed by experimental manipulations, reduces sexual behavior and induces anovulatory estrous cycles in female rats. On the afternoon of proestrus, neonatally handled females show a reduced surge of luteinizing hormone (LH) and an increased content of gonadotropin-releasing hormone in the medial preoptic area (MPOA). In order to detect the possible causes for the reduced ovulation and sexual behavior, the present study aimed to analyze the effects of neonatal handling on noradrenaline (NA) and nitric oxide (NO) levels in the MPOA on the afternoon of proestrus. Neonatal handling reduced MHPG (NA metabolite) levels and MHPG/NA ratio in the MPOA, indicating decreased NAergic activity. Additionally, neonatal handling decreased NO levels, as measured by the metabolites (NOx), nitrite and nitrate in the same period. We may conclude that the neonatal handling procedure decreased activity of the NAergic and NOergic systems in the MPOA during proestrus, which is involved in the control of LH and FSH secretion, and this may possibly explain the anovulatory estrous cycles and reduced sexual behavior of the neonatally handled female rats.