Prolonged Maternal Separation Research Articles

Brief versus long maternal separation in lactating rats: Consequences on maternal behavior, emotionality, and brain oxytocin receptor binding.

Maternal separation is a widely used animal model to study early life adversity in offspring. However, only a few studies have focused on the impact of disrupting the maternal bond from the mother's perspective. Such studies reveal alterations in behavior, whereas the underlying neuroendocrine mechanisms remain largely unknown. In this study, we compared the consequences of daily brief maternal separation (BMS; 15 min) versus long maternal separation (LMS; 180 min) during the first week postpartum with respect to behavioral and neuroendocrine changes in lactating Sprague-Dawley dams. Mothers were tested for their maternal care before and after separation, maternal motivation to retrieve pups, as well as anxiety-related and stress-coping behaviors. In addition, we analyzed their basal plasma corticosterone levels and oxytocin receptor binding in selected brain regions of the limbic system and maternal network. LMS dams showed higher levels of behavioral alterations compared to BMS and non-maternally separated (NMS) dams, including increased licking and grooming of the pups and decreased maternal motivation. Anxiety-related behavior was not affected by either separation paradigm, whereas passive stress-coping behavior tended to increase in the LMS group. Plasma corticosterone concentrations were not different between groups. Oxytocin receptor binding was higher in the medial preoptic area and tended to be higher in the prelimbic cortex of LMS dams, only. Our results demonstrate that especially daily prolonged maternal separation impacts on the mothers' behavior and oxytocin system, which suggests that enhanced oxytocin receptor binding could be a compensatory mechanism for potentially decreased central oxytocin release due to limited pup contact.

Prolonged Maternal Separation Reduces Anxiety State and Increases Compulsive Burying Activity in the Offspring of BALB/c Mice.

The elevated plus maze (EPM) and the marble burying (MB) tests are common behavioral tests used for behavioral phenotyping in mouse models for neurodevelopmental disorders. However, the behavioral effects of maternal separation (MS), a standard paradigm for early life stress in animals, in both the EPM and MB tests remain incompletely known. This study aimed to investigate the behavioral effects of prolonged MS in the offspring of mice using the EPM and MB tests. Male BALB/c mice were isolated from their mothers for 4 h each day during the first 30 days after birth. On day 50 postnatal, groups of separated and non-separated mice (n = 18/each group) were subjected to the EPM and MB tests for comparative behavioral evaluations. In addition, the locomotor activity of mice was evaluated using the actophotometer test. The findings of the EPM test revealed that separated mice exhibited anxiolytic-like behaviors, as evidenced by a significant increase in the latency to closed arms and the time spent in the open arms compared with non-separated mice. Separated mice also showed compulsive burying activity in the MB test, as determined by a significant increase in the number of buried marbles. The results of the actophotometer test did not show any significant change in locomotor activity. Prolonged MS caused the adult offspring of mice to exhibit a decrease in anxiety state and increased compulsive burying activity, which were not associated with a change in locomotor activity. Further investigations with validated tests are needed to support these findings.

Childhood separation from parents with cognitive and psychopathological outcomes in adolescence.

To examine the effects of childhood parent-child separation with varying duration and form on later cognitive performance and psychopathological problems over a 6-year period, we use data from the China Family Panel Study (CFPS), which is an ongoing, prospective nationally representative study across 25 provinces in China. Of the 4033 children aged 4-10 years followed in 2012, 3522 children with complete data on separation from fathers and mothers were included. In 2018, 2560 participants were followed and interviewed to assess cognitive performance, behavioral problems as well as depressive symptoms at age 10-16 years. A total of 3522 children (mean [SD] age at baseline, 6.9 [2.0] years) were included. About 17.9% and 13.3% had separated from father and mother over 11 months or more in the year prior to the survey. Prolonged separation from parents was associated with lower scores in vocabulary (maternal: adjusted β=-1.64, P=0.002; paternal: adjusted β=-1.58, P<0.001) and mathematics performance (maternal: adjusted β=-1.10, P=0.009; paternal: adjusted β=-0.94, P=0.004) over a 6-year period. Prolonged maternal separation was associated with 0.34-point higher internalizing behaviors (P<0.001) and 0.21-point higher externalizing behaviors (P=0.009), as well as 0.89-point higher depressive symptoms (P=0.018), while prolonged paternal separation was associated with 0.23-point higher internalizing behaviors (P=0.005) and 0.76-point higher depressive symptoms (P=0.011). The finding demonstrates substantial negative impacts of childhood paternal and maternal separations on cognitive and mental well-being during adolescence, which might be dependent on the "stress-dose" (duration of separation) and urban-rural residence. RESEARCH HIGHLIGHTS: Prolonged parent-child separation is independently associated with cognitive impairment and psychopathological problems during adolescence. The adverse effects of childhood separation on cognition and psychopathology varied by separation duration and separation forms. Age- and residence-specific associations between parent-child separation with cognition and psychopathology are observed.

Early life stress influences basal ganglia dopamine receptors and novel object recognition of adolescent and adult rats.

Environmental stimuli in early life are recognized to affect brain development and behavior. Mother-pup interaction constitutes a determinant stimulus during this critical period. It is known that the dopaminergic system undergoes significant reorganization during adolescence and that dopamine receptors are involved in recognition memory. Based on the above, we examined the effects of brief and prolonged maternal separation during the neonatal period (15 or 180 min daily) on basal ganglia dopamine receptors and on the behavior in the novel object recognition task of adolescent and adult male rats. Using the NOR task, we observed that the discrimination index (DI) was decreased in rats with brief maternal separations independent of age. Using receptor autoradiography, we observed that brief maternal separation induced decreases in D1, D2 and D4 receptor binding levels in adult basal ganglia nuclei, while prolonged maternal separation induced increases in D1 receptor binding levels in caudate - putamen (CPu) of adolescent rats. With immunoblotting experiments, we found decreases in D1 and increases in D2 total protein levels in CPu of adult rats with prolonged maternal separations. Α positive correlation was observed between DI and D1 binding levels in CPu, internal globus pallidus and substantia nigra, and D2 binding levels in nucleus accumbens core in adult rats, using the Pearson correlation coefficient. Our results indicate that the long-lasting effects of neonatal mother-offspring separation on dopamine receptors depend on the duration of maternal separation and age and that this early life experience impairs recognition memory in adolescent and adult rats. Furthermore, the present results suggest that modulation of striatal dopamine receptors might underlie the reduced recognition memory of adult rats with brief neonatal maternal separations.

Alcohol Deprivation Differentially Changes Alcohol Intake in Female and Male Rats Depending on Early-Life Stressful Experience.

Experiencing early-life adverse events has enduring effects on individual vulnerability to alcohol abuse and the development of addiction-related behaviors. In rodents, it can be studied using maternal separation (MS) stress. Studies have shown that, depending on the protocol used, MS can affect the mother and pups' behavior and are associated with behavioral alterations later in adulthood, associated with both positive or negative outcomes. However, it is not fully elucidated how MS affects relapse-like behaviors when experienced by female or male individuals. Therefore, the aim of our study was to evaluate the effects of brief and prolonged MS on the alcohol deprivation effect (ADE) in female and male rats. Female and male Wistar rats were exposed to brief (15 min/day) or prolonged (180 min/day) MS from postnatal day (PND) 2 to 10. Later, during adulthood (PND 70), animals were submitted to an ADE protocol. Brief MS exposure prevented the ADE in both females and males, while prolonged MS exposure also prevented the ADE in female rats. Moreover, the ADE was more robust in females when compared to males. In conclusion, we showed that male and female rats are differentially affected by alcohol deprivation periods depending on their early-life experiences.

Prolonged Maternal Separation Induces the Depression-Like Behavior Susceptibility to Chronic Unpredictable Mild Stress Exposure in Mice.

Early life stress is an important determinant for developing depression later in life. It is reported that maternal separation (MS) could trigger stress sensitivity in adulthood when exposed to stress again. However, it could also result in resilience to stress-induced depression. The conclusions are contradictory. To address this issue, C57BL/6N newborn pups were exposed to either daily short MS (MS for 15 min per day; MS15) or prolonged MS (MS for 180 min per day; MS180) from the first day postpartum (PD1) to PD21. Adult mice were then subjected to chronic unpredictable mild stress (CUMS) exposure from PD64 to PD105. The behavior tests such as the forced swimming test (FST), tail suspension test (TST), and open-field test were performed once a week during this time. Besides, the hippocampal neurosteroids, serum stress hormones, and hippocampal monoamine neurotransmitters were measured at PD106. We found that mice in the MS180 group displayed the reduced struggling time and the increased latency to immobility in both FST and TST. However, there was no significant difference in the MS15 group. The levels of hippocampal neurosteroids (progesterone and allopregnanolone) were decreased, and the serum levels of corticosterone, corticotropin-releasing hormone, and adrenocorticotropic hormone were overexpressed in the MS180 group. Besides, the expressions of monoamine neurotransmitters such as 5-hydroxytryptamine and 5-hydroxy indole acetic acid significantly decreased in the MS180 group, but not in the MS15 group. All findings revealed that prolonged MS, rather than short MS, could increase the susceptibility to depression-like behavior when reexposed to stress in adulthood. However, future studies are warranted to identify the underlying neuromolecular mechanism of the MS experience on the susceptibility to adult stress reexposure.

Proteome profiling of different rat brain regions reveals the modulatory effect of prolonged maternal separation on proteins involved in cell death-related processes

BackgroundEarly-life stress in the form of maternal separation can be associated with alterations in offspring neurodevelopment and brain functioning. Here, we aimed to investigate the potential impact of prolonged maternal separation on proteomic profiling of prefrontal cortex, hippocampus and cerebellum of juvenile and young adult rats. A special attention was devoted to proteins involved in the process of cell death and redox state maintenance.MethodsLong-Evans pups were separated from their mothers for 3 h daily over the first 3 weeks of life (during days 2–21 of age). Brain tissue samples collected from juvenile (22-day-old) and young adult (90-day-old) rats were used for label-free quantitative (LFQ) proteomic analysis. In parallel, selected oxidative stress markers and apoptosis-related proteins were assessed biochemically and by Western blot, respectively.ResultsIn total, 5526 proteins were detected in our proteomic analysis of rat brain tissue. Approximately one tenth of them (586 proteins) represented those involved in cell death processes or regulation of oxidative stress balance. Prolonged maternal separation caused changes in less than half of these proteins (271). The observed alterations in protein expression levels were age-, sex- and brain region-dependent. Interestingly, the proteins detected by mass spectrometry that are known to be involved in the maintenance of redox state were not markedly altered. Accordingly, we did not observe any significant differences between selected oxidative stress markers, such as the levels of hydrogen peroxide, reduced glutathione, protein carbonylation and lipid peroxidation in brain samples from rats that underwent maternal separation and from the corresponding controls. On the other hand, a number of changes were found in cell death-associated proteins, mainly in those involved in the apoptotic and autophagic pathways. However, there were no detectable alterations in the levels of cleaved products of caspases or Bcl-2 family members. Taken together, these data indicate that the apoptotic and autophagic cell death pathways were not activated by maternal separation either in adolescent or young adult rats.ConclusionProlonged maternal separation can distinctly modulate expression profiles of proteins associated with cell death pathways in prefrontal cortex, hippocampus and cerebellum of juvenile rats and the consequences of early-life stress may last into adulthood and likely participate in variations in stress reactivity.

Prolonged maternal separation alters neurogenesis and synaptogenesis in postnatal dentate gyrus of mice.

As a common model for adverse early experience and depression, maternal separation (MS) is always used to investigate the psychological disease. Despite extensive and strong evidence verified the depression-like state induced by MS, little is known about the specific mechanism of MS. Therefore, the present study aimed to investigate the neurobiology mechanism of the MS-induced depression-like state. To verify the depression-like behaviors of offspring induced by MS, a series of behavioral tests were performed. Then, in vivo electroporation and three-dimensional reconstruction, combining with immunohistochemistry and BrdU labeling, were mainly used to explore the neurogenesis and synaptogenesis in postnatal dentate gyrus. Prolonged MS indeed induced the depression-like behaviors of offspring in adulthood. Surprisingly, learning and memory were enhanced by prolonged MS. Further investigation indicated that prolonged MS inhibited the proliferation of neural stem cells, impaired the survival, and altered the fate decision of newborn cells, whereas the total length and terminal tips of dendrite, and the spine density, especially thin spine, were significantly increased in prolonged MS mice. Our results elucidated that prolonged MS induced the depression-like state by impairing postnatal neurogenesis of dentate gyrus. Importantly, our results emphasized that prolonged MS increased the spine density, especially thin spine, by increasing the total length and number of terminal tips of dendrite, thereby enhancing learning and memory.

Effects of BDNF Signaling on Anxiety-Related Behavior and Spatial Memory of Adolescent Rats in Different Length of Maternal Separation.

As an adverse form of early-life stress (ELS), maternal separation (MS) can interfere with the development of cognition and behaviors of adolescent rodents. Brain-derived neurotrophic factor (BDNF) is involved in the regulation of brain development and function, but the molecular mechanisms by which BDNF regulates brain function and behavior in MS with different stressor strengths remain unclear. This descriptive study characterized the levels of BDNF in the prefrontal cortex (PFC) and plasma corticosterone (CORT) from the offspring of rats exposed to early handling (EH, 15-min separation per day) and prolonged MS (PMS, 180-min separation per day), during postnatal days (PND) 1‑21. The behavioral and biochemical analyses were performed during adolescence (PND 42‑56). PMS resulted in reduced weight and decreased locomotor activity in the open field test and Y-maze task compared to control (CON) group, with EH showing an intermediate phenotype. BDNF protein levels in the PFC were lower in PMS compared to EH and further reduced in CON male rats. Plasma CORT levels were higher in PMS compared to CON with EH again showing intermediate levels. Neither PMS or EH affected spatial learning in the Y-maze task. These findings indicate that longer periods of maternal separation are necessary to increase anxiety-like behavior, elevate CORT levels, and further suppress BDNF levels in the PFC, providing a possible mechanism to explain why more severe forms of ELS lead to more significant psychiatric and medical consequences later in life.

Sex-Specific Effects of Early Life Stress on Brain Mitochondrial Function, Monoamine Levels and Neuroinflammation.

Sex differences have been reported in the susceptibility to early life stress and its neurobiological correlates in humans and experimental animals. However, most of the current research with animal models of early stress has been performed mainly in males. In the present study, prolonged maternal separation (MS) paradigm was applied as an animal model to resemble the effects of adverse early experiences in male and female rats. Regional brain mitochondrial function, monoaminergic activity, and neuroinflammation were evaluated as adults. Mitochondrial energy metabolism was greatly decreased in MS females as compared with MS males in the prefrontal cortex, dorsal hippocampus, and the nucleus accumbens shell. In addition, MS males had lower serotonin levels and increased serotonin turnover in the prefrontal cortex and the hippocampus. However, MS females showed increased dopamine turnover in the prefrontal cortex and increased norepinephrine turnover in the striatum, but decreased dopamine turnover in the hippocampus. Sex differences were also found for pro-inflammatory cytokine levels, with increased levels of TNF-α and IL-6 in the prefrontal cortex and hippocampus of MS males, and increased IL-6 levels in the striatum of MS females. These results evidence the complex sex- and brain region-specific long-term consequences of early life stress.

Asymmetry of turning behavior in rats is modulated by early life stress

Atypical leftward behavioral asymmetries have been associated with early life stress and psychopathologies in humans and animals. Maternal separation (MS) is a frequently used model to investigate early life stress and psychopathologies but has not yet been studied in terms of asymmetries. This study aims to investigate whether prolonged MS induces atypical leftward asymmetries in the turning behavior of rats. MS was performed from postnatal days 2–20 followed by a second stressor from postnatal days 21−40. Asymmetry of turning behavior was then examined in the elevated plus-maze test upon weaning (juveniles and dams) or adolescence. The number of left and right turns was calculated per animal using the deep learning software package DeepLabCut enabling markerless pose estimation. Then, a lateralization quotient (LQ) was determined for each animal allowing to investigate the strength as well as the preferred side of asymmetry. LQ analysis revealed a significant leftward asymmetry in the prolonged stress group. Moreover, analyzing the number of turns revealed significantly more left than right turns in total in this group. Control animals showed no asymmetries in turning behavior. These results indicate that prolonged stress during the early postnatal days led to atypical leftward turning behavior. The stress-induced atypical asymmetry might be a mediator of early life stress and the development of psychiatric disorders.

Maternal separation changes maternal care, anxiety-like behaviour and expression of paraventricular oxytocin and corticotrophin-releasing factor immunoreactivity in lactating rats.

The early postnatal period is a time of tremendous change for the dam and her offspring. During this time, environmental insults such as repeated stress exposure can have detrimental effects. In research that has focused on the effect of postnatal stress exposure on the dams, conflicting changes in maternal care and anxiety-like behaviour have been reported. Additionally, changes to hypothalamic neuropeptides that are crucially involved in the transition to motherhood and stress regulation, namely oxytocin and corticotrophin-releasing factor (CRF), have not been examined. Accordingly, the present study aimed to determine (i) whether repeated postpartum stress increases engagement in maternal care behaviours and anxiety-like behaviour and (ii) whether these behavioural changes correspond with changes to CRF- or oxytocin-immunoreactive (-IR) cells in the paraventricular nucleus (PVN) of the hypothalamus. A non-lactating group was also included to control for the effects of lactation on anxiety and the hypothalamic neuroendocrine system. Following the birth of their litters, Long-Evans dams were separated from their pups from postnatal day (PND) 1 to PND21 for either 15minutes (maternal separation [MS]15) or 6hours (MS360). Maternal behaviours were recorded for 30minutes on select PNDs following the separation. On PND22, dams were exposed to the elevated plus maze, brains were collected, and immunofluorescence analysis of PVN oxytocin- and CRF-IR cells was conducted. Our findings demonstrate that prolonged maternal separation altered typical maternal behaviours and reduced anxiety relative to MS15 dams. At the cellular level, oxytocin-IR cells in the caudal PVN were reduced in MS360 dams to a level similar to that in non-lactating controls, and PVN CRF-IR cells were reduced relative to both MS15 and non-lactating controls. Taken together, these data reveal the behavioural and neuronal changes that occur in the mother dam following repeated postnatal stress exposure.

Behavioral Profiling in Early Adolescence and Early Adulthood of Male Wistar Rats After Short and Prolonged Maternal Separation.

Early-life stress and its possible correlations to genes, environment, and later health outcomes can only be studied retrospectively in humans. Animal models enable the exploration of such connections with prospective, well-controlled study designs. However, with the recent awareness of replicability issues in preclinical research, the reproducibility of results from animal models has been highlighted. The present study aims to reproduce the behavioral effects of maternal separation (MS) previously observed in the multivariate concentric square fieldTM (MCSF) test. A second objective was to replicate the adolescent behavioral profiles previously described in the MCSF test. Male rats, subjected to short or prolonged MS or standard rearing, were subjected to behavioral testing in early adolescence and early adulthood. As seen in previous studies, the behavioral effects of MS in the MCSF were small at both tested time points. When tested in early adolescence, the animals exhibited a similar behavioral profile as previously seen, and the finding of adolescent behavioral types was also reproduced. The distribution of animals into the behavioral types was different than in the initial study, but in a manner consistent with developmental theories, as the current cohort was younger than the previous. Notably, the Shelter seeker behavioral type persisted through development, while the Explorer type did not. The lack of basal behavioral effect after MS is in line with the literature on this MS paradigm; the working hypothesis is that the prolonged MS gives rise to a phenotype predisposed to negative health outcomes but which is not apparent without additional provocation.

Environmental enrichment effects after early stress on behavior and functional brain networks in adult rats.

Early life stress is associated with long-term and pervasive adverse effects on neuroendocrine development, affecting normal cognitive and emotional development. Experimental manipulations like environmental enrichment (EE) may potentially reverse the effects of early life stress induced by maternal separation (MS) paradigm in rodents. However, the functional brain networks involved in the effects of EE after prolonged exposure to MS have not yet been investigated. In order to evaluate possible changes in brain functional connectivity induced by EE after MS, quantitative cytochrome c oxidase (CCO) histochemistry was applied to determine regional brain oxidative metabolism in adult male rats. Unexpectedly, results show that prolonged MS during the entire weaning period did not cause any detrimental effects on spatial learning and memory, including depressive-like behavior evaluated in the forced-swim test, and decreased anxiety-like behavior. However, EE seemed to alter anxiety- and depression-like behaviors in both control and MS groups, but improved spatial memory in the latter groups. Analysis of brain CCO activity showed significantly lower metabolic capacity in most brain regions selected in EE groups probably associated with chronic stress, but no effects of MS on brain metabolic capacity. In addition, principal component analysis of CCO activity revealed increased large-scale functional brain connectivity comprising at least three main networks affected by EE in both MS and control groups. Moreover, EE induced a pattern of functional brain connectivity associated with stress and anxiety-like behavior as compared with non-enriched groups. In conclusion, EE had differential effects on cognition and emotional behavior irrespective of exposure to MS. In view of the remarkable effects of EE on brain function and behavior, implementation of rodent housing conditions should be optimized by evaluating the balance between scientific validity and animal welfare.

Sex-dependent impact of different degrees of maternal separation experience on OFT behavioral performances after adult chronic unpredictable mild stress exposure in rats

Early-life social-environmental factors are important for normal development, and different degrees of early-life stress experience have different impacts on adult behaviors and stress responsiveness. The aim of present study was to investigate the long-term effects of different degrees of maternal separation (MS) on male and female rats and subsequent responsiveness to chronic unpredictable mild stress (CUMS) exposure in adults. Sprague-Dawley (SD) newborn pups were exposed to either 15 min/day of MS (MS15), 360 min/day of MS (MS360) or no separation (NS) during postnatal day (PND)4-PND10. At PND56, behavioral tasks, including sucrose preference test (SPT), forced swimming test (FST) and open field test (OFT), were used to explore depressive and anxiety-like behaviors. Then the rats received a series of CUMS for 28 days, behavioral tasks were recorded after CUMS. Prior to CUMS, the behavioral performances in male and female rats were consistent, MS360 led to increased immobile time in FST and decreased activity in OFT, while MS15 rats exhibited behavioral performances similar to NS group. After CUMS, sexual dimorphism was observed in the OFT behavioral responses to adult stress re-exposure, but no differences in FST were observed. CUMS male rats with MS360 experiences showed the worst behavioral performances in OFT compared to those of the other male rats groups, while CUMS female rats without MS experience showed the worst behavioral performances in OFT compared to those of the other female rats groups. Both CUMS male and female rats with MS15 experiences showed better trend in OFT performances than those of CUMS rats with MS360 experience and without MS experiences. These results suggest that brief MS experiences increase the OFT behavioral resilience of rats to adult stress re-exposure, and prolonged MS promotes OFT behavioral resilience of female rats to adult stress re-exposure, while increases vulnerability of male rats to adult stress re-exposure.

The Long-Term Effects of Early Postnatal Stress on Cognitive Abilities and Expression of Genes of the Glutamatergic System in Mice

Stressing events in the early period of life affect neuronal plasticity and cognitive functions in adulthood. A key role in the mechanisms of formation of memory and attention is played by the glutamatergic system. However, there has been virtually no systematic study on the effect of early postnatal stress on the expression of glutamatergic system genes in various regions of the brain in mice. In this study, we used two types of early postnatal stress: prolonged separation of pups from mothers (for 3 hours per day) and shortterm separation (15 minutes per day) during the first 2 weeks of life. We used an object recognition test to evaluate attention and memory to assess cognitive abilities in adults. We found that prolonged maternal separation reduced the ability to recognize a novel object and also disrupted motor and exploratory activities in adult animals, while short-term separation did not affect the studied parameters. We assessed the expression of the major genes of the glutamatergic system (AMPA receptor subunits Gria1, Gria2; NMDA subunits Grin1, Grin2a, and Grin2b; metabotropic receptor subunits Grm1, Grm2, and Grm3; glutamate transporters Vglut2, Eaat2, and Rab4a) in the frontal cortex, hippocampus, and hypothalamus. In the group with prolonged maternal separation, we found a decrease in the expression of Grin2b in the hypothalamus in comparison with the control, which led to a decrease in the mRNA ratio of this subunit to Grin2a mRNA, and possibly to a change in the ratio of these subunits in the NMDA receptor. In spite of the revealed cognitive impairments, we did not find significant changes in the expression of genes in the frontal cortex and hippocampus. Shortterm daily separation from mothers did not lead to changes in cognitive abilities and expression of genes of the glutamatergic system in mice. Thus, our results show that prolonged maternal separation may lead to a redistribution of the receptor subunits in the hypothalamus, which can modify the activity of the HPAA and determine the response to stress in these mice.

The impact of early-life stress on the expression of HPA-associated genes in the adult murine brain

Abstract Early life is an important period for the development of the nervous system and for the programming of behavioural phenotypes in adulthood. In our study, two types of early-life stress were used: prolonged separation of pups from their mothers (for 3 h/day, maternal separation (MS)) and brief separation (for 15 min/day, handling (HD)). We analysed the effects of early-life stress on behaviour and the expression of HPA-associated genes in the hypothalamus, hippocampus, and frontal cortex of male mice. Adult mice in the MS group demonstrated reduced locomotor activity and deficiencies in spatial long-term memory, while the HD showed no significant changes. Additionally, early-life MS resulted in reduced hippocampal Crhr1 mRNA, increased MR/GR mRNA in the hippocampus and hypothalamus. Both groups, HD and MS, showed increased Avp mRNA in the hypothalamus. Thus, prolonged maternal separation but not brief leads to adverse behavioural changes and influences the expression of HPA-associated genes in a brain region-specific manner.

Few long-term consequences after prolonged maternal separation in female Wistar rats.

Environmental factors during the early-life period are known to have long-term consequences for the adult phenotype. An intimate interplay between genes and environment shape the individual and may affect vulnerability for psychopathology in a sex-dependent manner. A rodent maternal separation model was here used to study the long-term effects of different early-life rearing conditions on adult behavior, HPA axis activity and long-term voluntary alcohol intake in female rats. Litters were subjected to 15 min (MS15) or 360 min (MS360) of daily maternal separation during postnatal day 1–21. In adulthood, the behavioral profiles were investigated using the multivariate concentric square field™ (MCSF) test or examined for HPA axis reactivity by cat-odor exposure with subsequent characterization of voluntary alcohol intake and associated changes in HPA axis activity. Adult female MS360 offspring showed mostly no, or only minor, effects on behavior, HPA axis reactivity and long-term alcohol intake relative to MS15. Instead, more pronounced effects were found dependent on changes in the natural hormonal cycle or by the choice of animal supplier. However, changes were revealed in corticosterone load after long-term alcohol access, as females subjected to MS360 had higher concentrations of fecal corticosterone. The present findings are in line with and expand on previous studies on the long-term effects of maternal separation in female rats with regard to behavior, HPA axis activity and voluntary alcohol intake. It can also be a window into further studies detailing how early-life experiences interact with other risk and protective factors to impact the adult phenotype and how possible sex differences play a role.

Prolonged maternal separation attenuates BDNF-ERK signaling correlated with spine formation in the hippocampus during early brain development.

Maternal separation (MS) is known to affect hippocampal function such as learning and memory, yet the molecular mechanism remains unknown. We hypothesized that these impairments are attributed to abnormities of neural circuit formation by MS, and focused on brain-derived neurotrophic factor (BDNF) as key factor because BDNF signaling has an essential role in synapse formation during early brain development. Using rat offspring exposed to MS for 6h/day during postnatal days (PD) 2-20, we estimated BDNF signaling in the hippocampus during brain development. Our results show that MS attenuated BDNF expression and activation of extracellular signal-regulated kinase (ERK) around PD 7. Moreover, plasticity-related immediate early genes, which are transcriptionally regulated by BDNF-ERK signaling, were also reduced by MS around PD 7. Interestingly, detailed analysis revealed that MS particularly reduced expression of BDNF gene and immediate early genes in the cornu ammonis 1 (CA1) of hippocampus at PD 7. Considering that BDNF-ERK signaling is involved in spine formation, we next evaluated spine formation in the hippocampus during the weaning period. Our results show that MS particularly reduced mature spine density in proximal apical dendrites of CA1 pyramidal neurons at PD 21. These results suggest that MS could attenuate BDNF-ERK signaling during primary synaptogenesis with a region-specific manner, which is likely to lead to decreased spine formation and maturation observed in the hippocampal CA1 region. It is speculated that this incomplete spine formation during early brain development has an influence on learning capabilities throughout adulthood.

Altered corticosterone levels and social play behavior after prolonged maternal separation in adolescent male but not female Wistar rats

Early-life socio-environmental factors are crucial for normal developmental processes; adverse experiences early in life can therefore lead to detrimental effects in several physiological systems. The aim of this study was to examine short-term effects of early adverse experiences in a maternal separation (MS) rodent model. In this study two separation conditions were used: daily 15- (MS15) or 360-min (MS360) separation of the litter from the dam during postnatal day 1–21. In early adolescence, male and female offspring were subjected to a single-isolation procedure with analysis of corticosterone levels prior to and after isolation. In addition, social play behavior was assessed during mid-adolescence. There was a clear difference between male and female offspring in both tests performed. There was no difference in corticosterone levels between the female MS groups, whereas MS360 males showed higher baseline and recovery corticosterone levels than MS15 males. The amount of pinning, a specific social play behavior, was affected by rearing with MS360 males having a higher frequency than MS15 males, while there was no difference between the female MS groups. The observation that males but not females are affected by MS360 has previously been reported for adult animals, and herein we show that this difference is present already in adolescence. Changes in corticosterone levels and social behavior following early-life adversity have been associated with adult behavioral alterations, and our results confirm that these changes emerge already within adolescence.