Neonatal maternal separation in male rats increases intestinal permeability and affects behavior after chronic social stress

Prolonged maternal separation in rats has several effects on health and behavior. Here we investigated how maternal separation might interact with social stress in adulthood on behavior and gastrointenstinal permeability. The effects of either daily 180 min long term pup-dam separation (LMS) during the stress hyporesponsive period or daily 10 min brief maternal separation (BMS) on behavior, corticosterone and intestinal permeability were investigated, compared to a non-handling (NH) condition in male offspring. The animals from each separation condition were then randomly assigned to adult stress and control conditions, where the stress condition was exposure to 14 days of social instability (CSI). Sucrose preference, elevated plus maze behavior and corticosterone were measured. Colitis was experimentally induced by dextran sulfate sodium for 7 days, followed by measurement of intestinal permeability using the (51)CrEDTA method. Granulocyte marker protein was measured in feces and colons were examined histologically for inflammation. Prior to the social stress, the LMS offspring showed elevated corticosterone levels, lower elevated plus maze activity and less fluid consumption. After social stress, corticosterone levels were suppressed in LMS animals and again they showed less fluid consumption. LMS animals had significantly higher intestinal permeability, but only when also exposed to the social stress in adulthood. The current results support a two-hit model, whereby early life events interact with adult life events in altering animals' vulnerability.     

Neonatal maternal separation in male rats increases intestinal permeability and affects behavior after chronic social stress

Prolonged maternal separation in rats has several effects on health and behavior. Here we investigated how maternal separation might interact with social stress in adulthood on behavior and gastrointenstinal permeability. The effects of either daily 180 min long term pup-dam separation (LMS) during the stress hyporesponsive period or daily 10 min brief maternal separation (BMS) on behavior, corticosterone and intestinal permeability were investigated, compared to a non-handling (NH) condition in male offspring. The animals from each separation condition were then randomly assigned to adult stress and control conditions, where the stress condition was exposure to 14 days of social instability (CSI). Sucrose preference, elevated plus maze behavior and corticosterone were measured. Colitis was experimentally induced by dextran sulfate sodium for 7 days, followed by measurement of intestinal permeability using the ⁵¹CrEDTA method. Granulocyte marker protein was measured in feces and colons were examined histologically for inflammation. Prior to the social stress, the LMS offspring showed elevated corticosterone levels, lower elevated plus maze activity and less fluid consumption. After social stress, corticosterone levels were suppressed in LMS animals and again they showed less fluid consumption. LMS animals had significantly higher intestinal permeability, but only when also exposed to the social stress in adulthood. The current results support a two-hit model, whereby early life events interact with adult life events in altering animals' vulnerability.     

Sexually dimorphic behavioral effects of maternal separation in anorexic rats

Exposure to negative events during the neonatal period is one of the leading factors contributing to the development of psychiatric disorders, including anorexia nervosa. In this study, we investigated the effects of maternal separation (MS) on the development of anorexia in rodents using the mild-stress form of the activity-based anorexia (ABA) model (2 hr of free access to a running wheel and a 1-hr feeding test) in both male and female rats. We assessed anxiety-like and locomotor behavior and hyperactivity with the open field and elevated plus maze tests. Our results showed that ABA rats of both sexes displayed hyperactive behavior associated with reduced anxiety-like behavior when compared to controls. However, a sexually dimorphic effect of MS emerged in anorexic rats: while the females exposed to MS + ABA were hyperactive with diminished anxiety-related behaviors compared to females of the ABA group, MS in males attenuated or did not alter the effects of the ABA protocol. In conclusion, our data reveal that the synergistic effects of MS and ABA on physical activity and anxiety-like behavior act in opposite directions in the two sexes.     

The effects of postnatal maternal separation on stress responsivity and experimentally induced colitis in adult rats

In this study, we investigated the effects of three neonatal conditions on adult corticosterone (CORT) levels, acoustic startle responses (ASRs), and vulnerability to colitis induced by dextran sulfate sodium (DSS) and how these early manipulations might interact with a brief stress exposure in adulthood on the same measures. Infant animals were subjected daily to either 180-min maternal separation [prolonged maternal separation (LMS)], 10-min maternal separation [brief maternal separation (BMS)], or nonhandling (NH) conditions during postnatal days 1-14. As adults, half of the animals were exposed to a series of 10 uncontrollable foot shocks. Animals were tested for CORT levels prior to and 10 days following shock/nonshock procedures before being tested for ASRs. Finally, all animals were exposed to 4% DSS in their drinking water for 6 days. LMS animals showed enhanced vulnerability to DSS-induced colitis when previously exposed to shock and enhanced stress reactivity responses as shown by elevated startle and CORT levels. Among the nonshocked animals, NH animals showed most colonic damage. Taken together, the results support previous findings suggesting that BMS has a protective effect on adult stress exposure. Additionally, BMS protects the animals from chemically induced colitis. The NH condition has clearly an effect on sensitizing mucosal response to DSS exposure.     

Maternally separated rats show deficits in maternal care in adulthood

Although there is considerable research on the phenomenology, neuroendocrinology, neuroanatomy, and sensory control of maternal behavior, little is known about the influences of early postnatal and postweaning experiences on the development of maternal behavior. The purpose of this study was to assess how early life separation from the mother rat affects development of the offspring's juvenile and adult maternal behavior. From postnatal Days 1 to 17, 3 female rats within each litter were separated (SEP) from the mother and the rest of the litter for 5 hr daily while 3 of their sisters were not maternally separated (NSEP). On postnatal Day 21, all subjects were weaned and randomly assigned to one of three juvenile conditions. One female from both SEP and NSEP groups was either isolated (I), given a social conspecific (S), or given 1- to 4-day-old pups (P) for 5 consecutive days. Maternal behavior of SEP and NSEP animals was assessed and recorded on each of the 5 days. Once all animals reached adulthood, they were mated, gave birth, and were assessed for their maternal behavior. We found that the effects of maternal separation on juvenile maternal-like behaviors were minimal. On the other hand, maternal separation reduced adult maternal licking and crouching over pups. In addition, there was a significant interaction between postnatal and juvenile experience on maternal crouching in maternal animals. These results are discussed in terms of the variety of possible behavioral, endocrine, and neurochemical mechanisms that mediate the effects of early life experiences on adult maternal behavior.     

Adolescent female rats are more resistant than males to the effects of early stress on prefrontal cortex and impulsive behavior

We tested the hypothesis that adolescent Sprague–Dawley females may be more resistant than males to display impulsive behavior and lower prefrontal cortex thickness after mother‐infant separation (MS). Starting at postnatal day 2 (P2), the MS group was separated 6 hr/day and the early handled (EH) group 15 min/day for 10 days, and another group was standard facility reared (SFR). Subjects were examined for novel open‐field activity (P28), light–dark apparatus (P29), familiar open‐field (P30) and frontal cortical thickness. This protocol resulted in impulsive behavior in MS rats relative to EH and SFR, but this effect was less pronounced in females than males. MS affected the two sexes differently in terms of decreased prefrontal cortex dorsoventral thickness, with this effect being significant in males but not females. Neuroanatomical and behavioral documentation that adolescent females are more resistant than males to ADHD‐like effects of maternal separation have not been previously reported. © 2009 Wiley Periodicals, Inc. Dev Psychobiol 51: 277–288, 2009     

Effects of neonatal handling and maternal separation on rough-and-tumble play in the rat

The extent to which brief daily handling and longer periods of separation from the mother during the first 2 weeks of life can affect play behavior in juvenile rats was assessed. Rat pups were separated from the mother for either 15 min daily (handling) or for 3 hr daily (maternal separation), and play was observed as juveniles. Overall levels of playfulness were not affected by either manipulation, although certain aspects of playful responsiveness were affected in males, but not females. In particular, the pattern of responsiveness to playful contacts was feminized in both handled and separated male rats. Activity in a novel open field at 15 days of age was increased in both males and females from the separated group, but not in the handled animals, as were the number of rears exhibited during the play bouts. These data suggest that early rearing experiences can have subtle gender-dependent effects on some aspects of play in juvenile rats and that the underlying mechanism(s) responsible for these effects may differ from those associated with other effects reported for handling and maternal separation.     

Sex, but not repeated maternal separation during the first postnatal week, influences novel object exploration and amphetamine sensitivity

Sensation seeking and early life stress are both risk factors for developing substance use disorders. Neural adaptations resulting from early life stress may mediate individual differences in novelty responsiveness, and, in turn, contribute to drug abuse vulnerability. Animal models also demonstrate associations between novelty responsiveness or early life stress and increased sensitivity to psychostimulants. We investigated whether repeated maternal separation affects responses to novelty during adolescence and to amphetamine during adulthood, and whether maternal separation alters the relationship between these behavioral variables. Rat pups underwent separation (180 min/day) or control procedures (15 min/day) on postnatal days (PND) 2-8. Novel object exploration and amphetamine response were tested at PND 38 and 60, respectively. Adolescent males were less active in a novel environment and approached novel objects more frequently than females, but adult females showed greater amphetamine-induced locomotion. Maternal separation did not affect novelty responsiveness or amphetamine sensitivity. Locomotor activity in an inescapable, novel environment during adolescence predicted amphetamine-induced locomotor activity during adulthood in maternally separated rats, but not in controls. The results of this study suggest that adolescent responses to novelty may be particularly predictive of future substance abuse among survivors of early life trauma. Furthermore, sex differences in novelty and amphetamine responsiveness may complicate the relationship between these behavioral variables.     

Long lasting alteration in REM sleep of female rats submitted to long maternal separation

Early adverse experiences represent risk factors for the development of anxiety and mood disorders. Maternal separation can induce biobehavioral alterations in male rodents similar to those seen in depressed humans, such as hyperresponsiveness to stress and sleep disturbances. Nonetheless, no study has yet explored the effects of early life events on the relationship between stress and sleep in female rats. Whole litters of Wistar rats were submitted to brief- or long maternal separations (15 [BMS] or 180 min/day [LMS], from postnatal days 2-14) or kept undisturbed with their mothers (CTL). When adults, female rats were sleep-recorded for 22 h before (baseline) and after a 1 h exposure to cold stress (post-stress). Additional subsets of animals were sacrificed before, 1 or 3 h after the stressor for plasma corticosterone determination. No differences in baseline sleep were observed among the groups. Female rats submitted to LMS exhibited a significant increase of REM sleep on the night following a 1 h exposure to cold stress, whereas the sleep of BMS rats was barely altered by stress. All groups exhibited similar basal and stress-induced corticosterone levels. The present results are compared to a previous study performed in male rats, and corroborate that manipulations applied during infancy modify the expression of stress-induced sleep rebound.     

Impact of mothers’ experience and early-life stress on aggression and cognition in adult male mice

The postnatal period is important for brain development and behavioral programming. Here, we hypothesized that females’ stressful experience early in life can lead to disruption of mother–offspring interactions with their own progeny. The objective of this study was to assess the effects of mothers’ stressful experience, early-life stress, or both on the behavior of adult male mice. In this study, female mice were allowed to raise their pups either without exposure to stress (normal rearing conditions, NC) or with exposure to maternal separation (3 hr/day, maternal separation, MS). Adult F1 female mice who had experienced MS (stressed mothers, SM) or had been reared normally (undisturbed mothers, UM) were used for generating F2 offspring, which was then exposed (or not exposed) to early-life stress. We assessed anxiety-like behavior, exploratory activity, locomotor activity, aggression, and cognition in four groups of adult F2 males (UM+NC, UM+MS, SM+NC, and SM+MS). We found that SM+MS males become more aggressive if agonistic contact is long enough; these results point to a change in their social coping strategy. Moreover, these aggressive males tended to show better long-term spatial memory. Overall, our findings suggest that mothers’ early-life experience may have important implications for the adult behavior of their offspring.     

Sexually dimorphic effects of postnatal treatment on the development of activity‐based anorexia in adolescent and adult rats

Hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis is a marked feature of anorexia nervosa. Using a modified version of the activity‐based animal model of anorexia nervosa, we examine whether factors known to affect HPA axis activity influence the development of activity‐based anorexia (ABA). Male and female rats were subjected to maternal separation or handling procedures during the first two postnatal weeks and tested in a mild version of the ABA paradigm, comprised of 2‐hr daily running wheel access followed by 1‐hr food access, either in adolescence or adulthood. Compared to handled females, maternally separated females demonstrated greater increases in wheel running and a more pronounced running‐induced suppression of food intake during adolescence, but not in adulthood. In contrast, it was only in adulthood that wheel running produced more prolonged anorexic effects in maternally separated than in handled males. These findings highlight the interplay between early postnatal treatment, sex of the animal, and developmental age on running, food intake, and rate of body weight loss in a mild version of the ABA paradigm. © 2009 Wiley Periodicals, Inc. Dev Psychobiol 51: 679–695, 2009.     

Both long and brief maternal separation produces persistent changes in tissue levels of brain monoamines in middle-aged female rats

Adverse experiences early in life are associated with an increased incidence of later psychopathology including depression. Based on evidence that dysfunction of central monoaminergic systems is involved in the pathophysiology of depression, we hypothesize that early adversity could negatively affect these systems. To test this we have investigated the effects of maternal separation, which has been suggested to model early-life stress and the development of a depression-like syndrome in the rat, on brain monoaminergic systems. Since depression is more common in women and the risk of developing this disorder appears to increase with age, we have studied such effects in middle-aged female rats. Rat pups were separated for 180 min (long maternal separation; LMS) or 15 min (brief maternal separation; BMS, often referred to as neonatal handling) twice daily for 2 weeks postpartum. An animal facility-reared (AFR) group was also included. At 15 months of age tissue levels of monoamines and their metabolites in several different brain regions were analyzed. In the LMS females tissue levels of both 5-HT and 5-hydroxyindole acetic acid (5-HIAA) were significantly increased in the dorsal raphe nucleus, and 5-HIAA and homovanillic acid levels were also elevated in the nucleus accumbens as compared with AFR and BMS rats. In the cingulate cortex both LMS and BMS decreased noradrenaline (NA) levels, although this effect was more pronounced in the LMS rats. On the other hand, BMS decreased 5-HT, 5-HIAA, dopamine (DA) as well as NA levels in the amygdala and produced an increase in DA levels in response to acute stress in the hypothalamus, an effect not seen in AFR rats. Our results demonstrate that LMS produced persistent alterations in both serotonergic, noradrenergic and dopaminergic systems in brain regions that have been suggested to be implicated in the pathophysiology of depression. In addition, BMS affected brain monoaminergic levels mainly in the amygdala.     

Adverse experience during early life and adulthood interact to elevate tph2 mRNA expression in serotonergic neurons within the dorsal raphe nucleus

Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of brain serotonergic systems. These effects could result from genetic influences, adverse early life experiences (ELE), or acute stressful life events, all of which can alter serotonergic neurotransmission and have been implicated in determining vulnerability to neuropsychiatric disorders. To evaluate the effects of ELE, adverse experiences during adulthood, and potential interactions between these factors on neuronal tryptophan hydroxylase 2 (tph2) mRNA expression, we investigated in rats the effects of maternal separation (MS)(separation from the dam for 180 min/day from postnatal day 2–14; MS180, a model of vulnerability to a depression-like syndrome), neonatal handling (separation from the dam for 15 min/day from postnatal day 2–14; MS15, a model of decreased stress sensitivity), or normal animal facility rearing (AFR) control conditions, with or without subsequent exposure to adult social defeat, on tph2 mRNA expression in the dorsal raphe nucleus (DR). Among rats exposed to social defeat, MS180 rats had increased tph2 mRNA expression in the DR, while MS15 rats had decreased tph2 mRNA expression compared to AFR rats. Social defeat increased tph2 mRNA expression, but only in MS180 rats and only in the “lateral wings” of the DR, a subdivision of the DR that is part of a sympathomotor command center. Overall, these data demonstrate that ELE and stressful experience during adulthood interact to determine tph2 mRNA expression. These changes in tph2 mRNA expression represent a potential mechanism through which adverse ELEs and stressful life experiences during adulthood may interact to increase vulnerability to stress-related psychiatric disease.     

Depressive behaviors and decreased expression of serotonin reuptake transporter in rats that experienced neonatal maternal separation

This study was conducted to examine the pathophysiologic mechanisms of long-term adverse effects by neonatal maternal separation on neurobehaviors of the offspring. Sprague-Dawley pups were separated from dam daily for 180min during the first 2 weeks of life (MS) or undisturbed (NH), and subjected to behavioral sessions for ambulatory activity, forced swim, and elevated plus maze tests at 2 months of age. Serotonin reuptake transporter (5-HTT) mRNA levels in the raphe nucleus and the contents of serotonin (5-HT) and its metabolite 5-hydroxyindol acetic acid in the raphe and the hippocampus were examined as well. Ambulatory counts decreased and immobility duration in swim test increased in MS rats compared with NH rats. MS rats spent more time in the closed arms, less time in the open arms, of elevated plus maze, compared to NH rats. The hippocampal contents of 5-HT and the raphe expression of 5-HTT mRNA were decreased in MS rats compared with NH rats. These results suggest that neonatal maternal separation may result in the development of depression- and/or anxiety-like behaviors in later life, in which the long-term alterations in 5-HTergic neurotransmission may take a role.     

Experimental model of unpredictable maternal stress and diabetes risk of offspring: An immunohistochemical study

The effect of stress on the occurrence of diabetes mellitus (DM) is commonly reported in recent studies. Maternal stress may have a negative effect on the later life of offspring. However, most studies only investigated long-term intrauterine stress on behavioral, emotional, psychological, and immunological disorders of offspring. The relationship between maternal stress and DM occurrence in the later life period of offspring is not known. This rat model study aimed to evaluate the susceptibility of offspring to DM after exposure to intrauterine stress. The purpose of this study is to examine serum glucose levels of mothers and offspring exposed to maternal stress and to evaluate pancreatic tissues pathologically and immunohistochemically. Twelve, Wistar Albino female rats were equally divided into two groups: controls and maternal stress groups. Normal routine conditions were applied to the control group without any stress. The pregnant rats in the maternal stress group were exposed to chronic unpredictable stressors throughout the 21-day gestation. One female and one male offspring and mothers from each term delivery were randomly selected and euthanatized at the 35th day. During the necropsy, blood and pancreatic tissue samples were collected from both mothers and pups. High serum glucose levels from mothers and offspring in the maternal stress group and the control group were compared. Additionally, histopathological examinations assessed the increased cell degeneration in mother rats and offspring. Immunohistochemical examinations revealed decreased insulin, amylin, and insulin receptor expressions and slightly increased glucagon expression in Langerhans islet cells in the maternal stress group. These results indicated that maternal stress may be a predisposing factor for DM in both mothers and offspring in their later life periods.     

Effect of maternal separation on feeding behavior of rats in later life

Effects of maternal separation on feeding behavior, particularly on rebound hyperphagia, in adult rats were examined. Time-restricted scheduled feeding (2 h per day for 6 days), was given at the age of 3, 6, 9 or 12 weeks in rats that were maternal separated from postnatal days (PD) 1–21 and control rats. Following the time-restricted scheduled feeding, rats were fed freely for 24 h (rebound hyperphagia). Body weight, daily normal food consumption and food consumption during time-restricted scheduled feeding and rebound hyperphagia were measured. Body weight of 3-week-old maternally separated rats were less than those of control rats. There was no significant difference in normal daily food consumption. Food consumption during rebound hyperphagia was significantly increased in 6- to 9-week-old female maternally separated rats, but there was no difference observed in males. Postnatal maternal separation enhanced rebound hyperphagia of female rats in later life. These results indicate that postnatal maternal separation made rats more vulnerable to the development of abnormal feeding behavior in response to food restriction in later life.     

Ethanol-induced effects on opioid peptides in adult male Wistar rats are dependent on early environmental factors

The vulnerability to develop alcoholism is dependent on both genetic and environmental factors. The neurobiological mechanisms underlying these factors are not fully understood but individual divergence in the endogenous opioid peptide system may contribute. We have previously reported that early-life experiences can affect endogenous opioids and also adult voluntary ethanol intake. In the present study, this line of research was continued and the effects of long-term voluntary ethanol drinking on the opioid system are described in animals reared in different environmental settings. Rat pups were subjected to 15 min (MS15) or 360 min (MS360) of daily maternal separation during postnatal days 1-21. At 10 weeks of age, male rats were exposed to voluntary ethanol drinking in a four-bottle paradigm with 5%, 10% and 20% ethanol solution in addition to water for 2 months. Age-matched controls received water during the same period. Immunoreactive (ir) Met-enkephalin-Arg6Phe7 (MEAP) and dynorphin B (DYNB) peptide levels were thereafter measured in the pituitary gland and several brain areas. In water-drinking animals, lower ir MEAP levels were observed in the MS360 rats in the hypothalamus, medial prefrontal cortex, striatum and the periaqueductal gray, whereas no differences were seen in ir DYNB levels. Long-term ethanol drinking induced lower ir MEAP levels in MS15 rats in the medial prefrontal cortex and the periaqueductal gray, whereas higher levels were detected in MS360 rats in the hypothalamus, striatum and the substantia nigra. Chronic voluntary drinking affected ir DYNB levels in the pituitary gland, hypothalamus and the substantia nigra, with minor differences between MS15 and MS360. In conclusion, manipulation of the early environment caused changes in the opioid system and a subsequent altered response to ethanol. The altered sensitivity of the opioid peptides to ethanol may contribute to the previously reported differences in ethanol intake between MS15 and MS360 rats.     

Neonatal handling and the maternal odor preference in rat pups: Involvement of monoamines and cyclic AMP response element-binding protein pathway in the olfactory bulb

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.     

Maternal separation alters maternal care, but has minor effects on behavior and brain opioid peptides in adult offspring

The aim of the study was to investigate the effect of repeated maternal separation (MS; 4 hr per day) during postnatal Days 1 to 15 on emotionality and voluntary ethanol intake in adult male and female Wistar rat offspring relative to controls exposed to a brief (5-min) daily handling procedure. Brain immunoreactive opioid peptide levels and plasma levels of corticosterone also were measured. There were mainly no alterations in any of the tested behaviors (i.e., fleeing and freezing responses, exploratory behavior, spontaneous and amphetamine-induced locomotor activity and competitive behavior), ethanol intake, or immunoreactive opioid peptide levels in MS offspring, either in males or females, compared to their respective controls nor were there any differences in plasma corticosterone between groups. In addition, the dams' retrieval behavior of the pups also was studied, showing that MS dams spent more time in the nest with the pups after the 4-hr separation period compared to control dams. With respect to the used protocol of the MS procedure in the present study, our results do not provide support for the suggestion that this procedure is a relevant model for studying development of psychopathology and vulnerability to drug abuse.