Single Maternal Separation Research Articles

Repeated and single maternal separation specifically alter microglial morphology in the prefrontal cortex and neurogenesis in the hippocampus of 15-day-old male mice.

Early-life adversity impairs neuronal plasticity of the developing brain. In rodents, brain maturation processes, including neuro- and synaptogenesis, myelination, microglial maturation, and hypothalamic-pituitary-adrenal (HPA) axis development continue in the postnatal period. In our study, two models of early-life stress were used: repeated maternal separation (MS) from postnatal day (PND) 2 to PND14 for 3 h daily and single maternal deprivation (MD) on PND9 for 24 h. Effects of each type of early-life stress on neuron density, neurogenesis, microglial morphology, and HPA axis programming were studied in 15-day-old male mice. Neither early-life stress paradigm affected the expression of stress-related genes (Crh, Avp, Crhr1, Crhr2, Nr3c1, and Nr3c2) and the serum level of corticosterone on PND15. Immunohistochemical analysis was performed on slices of the hippocampus and prefrontal cortex (PFC) with antibodies against a marker of mature neurons (NeuN), of microglia (Iba1), proliferating cells (Ki67), and immature neurons (DCX). We found higher density of ameboid microglia and intermediate microglia in the PFC in groups MS and MD, respectively, than in a control group. In both stressed groups, a higher number of Ki67-positive cells was noted in the dentate gyrus. Thus, in mice, the process of transformation of ameboid microglia into ramified ones as well as a neurogenesis reduction take place during the second postnatal week, whereas early-life stress can disturb these processes in a stress- and region-dependent manner.

Low Fos expression in newly generated neurons of the main and accessory olfactory bulb following single maternal separation

The main and accessory olfactory bulbs (MOB and AOB) are unique in that they produce new neurons throughout adulthood. Despite the recent knowledge about the involvement of postnatally generated cells in several aspects of olfaction, the functional role of these neurons is still not sufficiently understood. The function of newly generated olfactory bulb neurons is primarily investigated in relation to activities related to smell. Stress-induced activation of new olfactory neurons has not yet been studied. Thus, our work was aimed to investigate whether a stressful event, such as maternal separation (MS) can induce Fos expression in postnatally-born neurons in the MOB and AOB. Rat pups were exposed to single maternal separation (SMS) for 2 h at the postnatal days: P7, P14, and P21. Quantification of immunohistochemically labeled Fos + cells revealed that exposure to SMS in different age stages during the first postnatal month stimulates activity in cells of individual MOB/AOB layers in an age-dependent manner. In order to find out whether newly generated cells in the MOB/AOB could express Fos protein as a response to SMS, newborn rats were administrated with the marker of proliferation, bromodeoxyuridine (BrdU) at P0, and three weeks later (at P21) colocalization of Fos and BrdU in the neurons of the MOB and AOB was assessed. Quantitative analysis of BrdU/Fos double-labeled cells showed that Fos is expressed only in a small number of postnatally generated cells within the MOB/AOB. Our results indicate that postnatally generated MOB/AOB neurons are less sensitive to stress caused by MS than preexisting ones. LAY SUMMARY Our results showed that single maternal separation (SMS) is a stressful event that in age-dependent manner stimulates cellular activity in the main and accessory olfactory bulb (AOB) – the structures dedicated to odor information processing. The low level of Fos expression in newborn neurons of the main and accessory bulb indicates that postnatally generated cells are less sensitive to neonatal stress than preexisting neurons.

Relationship between motor function and ultrasonic vocalizations induced by maternal separation in rat pups.

When rat pups are isolated from their mothers, they emit ultrasonic vocalizations (USVs). Although previous studies have reported that USVs are related to anxiety, others have reported that they are related to simple, nonemotional factors, such as physiological reactions to coldness. In this study, we examined the influence of three maternal separations on rat pups. The number of USVs during 5 min of USV test under maternal separation, latency in the righting reflex as motor function, and body temperature were recorded twice (the first and second tests) before and after the pups were put in various environments for 10 min. The environments were no maternal separation (Control: CON), maternal separation with littermates (LMS), and single maternal separation with a heater (SMS). In the second test, the SMS pups had fewer USVs, a lower body temperature, and a more rapid righting reflex than the CON and LMS pups. In addition, there was no strong correlation between USVs and righting reflex. As a result, pups undergoing 10 min of SMS while being kept warm by the heater showed rapid righting reflex. Thus, by a single maternal separation, the number of USVs decreased but the decrease was unrelated to decrease in motor function.

Subsequent maternal separation exacerbates neurobehavioral abnormalities in rats neonatally exposed to sevoflurane anesthesia.

Several recent studies suggest that in the human population, a routine, short anesthetic in otherwise healthy infants is void of neurodevelopmental insult. On the other hand, many human retrospective epidemiological studies report evidence of cognitive abnormalities in children after testing those who had different anesthesia-requiring procedures in early childhood. We tested in a rat model whether post-anesthesia stressful environmental factors can contribute to developmental abnormalities that were initiated by a relatively short exposure to sevoflurane, the most widely used anesthetic in pediatric anesthesia, whose polyvalent actions include enhancement of gamma-aminobutyric acid type A receptor (GABAAR) activity. Postnatal day 6 (P6) male Sprague-Dawley rats were anesthetized with sevoflurane for 60min. To simulate subsequent stress, the animals were subjected to a single maternal separation for 180min at P10. To study the role of GABAAR-mediated depolarization, subgroups of P6 rats received a single injection of the Na+-K+-2Cl- (NKCC1) inhibitor, bumetanide, prior to initiation of anesthesia with sevoflurane. Rats that were exposed to sevoflurane had decreased hypothalamic K+-2Cl- (KCC2) mRNA level (F(2,13)=3.839, P=0.049), increased NKCC1/KCC2 mRNA ratio (F(2,13)=5.043, P=0.024) and increased corticotropin-releasing hormone (CRH) mRNA level (F(2,12)=9.450, P=0.003) at P10, the age at which maternal separation was imposed. Adult rats, neonatally exposed to a combination of sevoflurane and maternal separation, exhibited increases in the escape latencies greater than animals exposed to sevoflurane only (P=0.012), and only rats in the sevoflurane plus maternal separation group spent significantly less time in the target quadrant during the Morris water maze test (F(4,55)=4.856, P=0.002). Bumetanide ameliorated abnormalities induced by sevoflurane and a combination of sevoflurane plus maternal separation. Neonatal exposure to sevoflurane may sensitize to stressors later in life, and post-exposure stress may exacerbate neurodevelopmental abnormalities even after a relatively short exposure to sevoflurane in rodents. The NKCC1 downregulation prior to exposure to the anesthetic may be therapeutic.

Role of environmental stressors in determining the developmental outcome of neonatal anesthesia

BackgroundThe majority of studies evaluating neurocognition in humans who had procedures under anesthesia early in life found long-term deficits even though the typical anesthesia duration normalized to the human life span is much shorter than that shown to induce developmental abnormalities in rodents. Therefore, we studied whether subsequent environmental stressors contribute to deficiencies programmed by a brief neonatal etomidate exposure. MethodsPostnatal days (P) 4, 5, or 6, Sprague-Dawley rats, pretreated with vehicle or the Na+-K+-2Cl− (NKCC1) inhibitor, bumetanide, received two injections of etomidate resulting in anesthesia for 2h. To simulate stress after anesthesia, the animals were exposed to a single maternal separation for 3h at P10. 3–7days after exposure to etomidate the rats had increased hypothalamic NKCC1 mRNA and corticotropin releasing hormone (CRH) mRNA and decreased K+-2Cl− (KCC2) mRNA levels with greater changes in males. In rats neonatally exposed to both etomidate and maternal separation, these abnormalities persisted into adulthood. These animals also exhibited extended corticosterone responses to restraint stress with increases in total plasma corticosterone more robust in males, as well as behavioral abnormalities. Pretreatment with the NKCC1 inhibitor ameliorated most of these effects. ConclusionsPost-anesthesia stressors may exacerbate/unmask neurodevelopmental abnormalities even after a relatively short anesthetic with etomidate, leading to dysregulated stress response systems and neurobehavioral deficiencies in adulthood. Amelioration by bumetanide suggests a mechanistic role for etomidate-enhanced gamma-aminobutyric acid type A receptor-mediated depolarization in initiating long-lasting alterations in gene expression that are further potentiated by subsequent maternal separation.

Effects of early life adverse experiences on the brain: implications from maternal separation models in rodents.

During postnatal development, adverse early life experiences affect the formation of neuronal networks and exert long-lasting effects on neural function. Many studies have shown that daily repeated maternal separation (MS), an animal model of early life stress, can regulate the hypothalamic-pituitary-adrenal axis (HPA axis) and affect subsequent brain function and behavior during adulthood. However, the molecular basis of the long-lasting effects of early life stress on brain function has not been fully elucidated. In this mini review, we present various cases of MS in rodents and illustrate the alterations in HPA axis activity by focusing on corticosterone (CORT). We then show a characterization of the brain regions affected by various patterns of MS, including repeated MS and single time MS at various stages before weaning, by investigating c-Fos expression. These CORT and c-Fos studies suggest that repeated early life stress may affect neuronal function in region- and temporal-specific manners, indicating a critical period for habituation to early life stress. Next, we introduce how early life stress can impact behavior, namely by inducing depression, anxiety or eating disorders, and alterations in gene expression in adult mice subjected to MS.

Interactions Between Age, Stress and Insulin on Cognition: Implications for Alzheimer's Disease

There is much interest in understanding the mechanisms responsible for interactions among stress, aging, memory and Alzheimer's disease. Glucocorticoid secretion associated with early life stress may contribute to the variability of the aging process and to the development of neuro- and psychopathologies. Maternal separation (MS), a model of early life stress in which rats experience 3 h of daily separation from the dam during the first 3 weeks of life, was used to study the interactions between stress and aging. Young (3 months) MS rats showed an altered hypothalamic-pituitary-adrenal (HPA) axis reactivity, depressive-like behavior in the Porsolt swimming test and cognitive impairments in the Morris water maze and new object recognition test that persisted in aged (18 months) rats. Levels of insulin receptor, phosphorylated insulin receptor and markers of downstream signaling pathways (pAkt, pGSK3 beta, pTau, and pERK1 levels) were significantly decreased in aged rats. There was a significant decrease in pERK2 and in the plasticity marker ARC in MS aged rats compared with single MS or aged rats. It is interesting to note that there was a significant increase in the C99 : C83 ratio, A beta levels, and BACE1 levels the hippocampus of MS aged rats, suggesting that in aged rats subjected to early life stress, there was an increase in the amyloidogenic processing of amyloid precursor protein (APP). These results are integrated in a tentative mechanism through which aging interplay with stress to influence cognition as the basis of Alzheimer disease (AD). The present results may provide the proof-of-concept for the use of glucocorticoid-/insulin-related drugs in the treatment of AD.

The impact of maternal separation on adult mouse behaviour and on the total neuron number in the mouse hippocampus

The maternal separation paradigm has been applied to C57BL/6J mice as an animal developmental model for understanding structural deficits leading to abnormal behaviour. A maternal separation (MS) model was used on postnatal day (PND) 9, where the pups were removed from their mother for 24 h (MS24). When the pups were 10 weeks old, the level of anxiety and fear was measured with two behavioural tests; an open field test and an elevated plus maze test. The Barnes platform maze was used to test spatial learning, and memory by using acquisition trials followed by reverse trial sessions. The MS24 mice spent more time in the open arms of the elevated plus maze compared to controls, but no other treatment differences were found in the emotional behavioural tests. However, in the reverse trial for the Barnes maze test there was a significant difference in the frequency of visits to the old goal, the number of errors made by the MS24 mice compared to controls and in total distance moved. The mice were subsequently sacrificed and the total number of neurons estimated in the hippocampus using the optical fractionator. We found a significant loss of neurons in the dentate gyrus in MS mice compared to controls. Apparently a single maternal separation can impact the number of neurons in mouse hippocampus either by a decrease of neurogenesis or as an increase in neuron apoptosis. This study is the first to assess the result of maternal separation combining behaviour and stereology.

Effect of a single maternal separation at different pup ages on the corticosterone stress response in adult and aged rats

Postnatal days (PNDs) 4–14 constitute the stress hyporesponsive period (SHRP) of the rat's pituitary–adrenal axis. The impact of manipulation of the pup–dam relationship during the SHRP on neuroendocrine and behavioural function has been the subject of considerable investigation. A single period of 24-h separation of the litter from the dam (maternal separation, MS) during the SHRP increases pup pituitary–adrenal activity and attenuates the SHRP. The MS manipulation also allows for the age-specific analysis of the chronic effects of early-life stress. Here we report on the effects of MS performed at the beginning of (PND 4), or about midway into (PND 9), or after (PND 18) the SHRP, on basal and stress-related blood corticosterone (CORT) titers in mature (month 5) and old (month 20) adult males. MS at PND 4, 9, or 18 did not affect basal CORT plasma titers. MS at each of these ontogenetic stages led to a similar and significant increase in the CORT response to restraint in adults but not in old adults. Therefore, whereas MS exerts a chronic impact on stress-related pituitary–adrenal activity in adult male rats, the effect of this postnatal experience does not depend upon the ontogenetic/SHRP status of the pup, and nor does it persist into senescence.

Long-term effects of a single 24-hour maternal separation on three different latent inhibition paradigms

Basing animal models for psychiatric diseases on purely environmental manipulations would be a great asset in biological research, such as in the screening of antipsychotic drugs, since they preclude possible interference from pharmacologically induced deficits. Recent investigations have yielded evidence that a single 24-h maternal separation (MS) in rats during the stress hyporesponsive period (SHRP) alters hypothalamic-adrenal-pituitary (HPA) axis response in adult rats and produces long-term behavioral effects, such as deficits in sensorimotor gating (prepulse inhibition) and selective attention (latent inhibition [LI]), which have been related to schizophrenia. Since there are implications that the age of the pups at the time of separation may be a crucial parameter for the neuroendocrine and behavioral effects on adult animals, we investigated the effects of a single 24-h MS on postnatal day (PND) 4, 9, or 18 in unrelated adult male and female Wistar rats on three paradigms of LI: conditioned taste aversion (CTA), active avoidance (AA), and conditioned emotional response (CER). Whereas there was no effect of MS on CER, MS on PND 9 enhanced LI in the CTA paradigm, and MS on PND 18 disrupted LI in the AA paradigm in males only. Within subjects separated on PND 4, males displayed a severe learning deficit in the AA paradigm, and consequently LI was not observed. The results suggest that a single 24-h MS, irrespective of the developmental stage of the pups when separated, does not provide a robust animal model for adult disruption of selective attention similar to that observed in schizophrenia.

Natural Cytotoxicity toward K562 Cells by Macaque Lymphocytes from Infancy through Puberty: Effects of Early Social Challenge

The consequences of a single maternal separation experience followed by reunion at 6–7 months of age was studied in socially housed pigtail (Macaca nemestrina) and bonnet (Macaca radiata) macaques. At 15 months, these subjects were removed from their natal group and placed in same species social groups, consisting of other separated and matched control subjects. Some subjects were followed until they reached an average age of 4 years while remaining in this group. Blood samples were drawn to permit assessment of the ability of peripheral blood lymphocytes to lyse,in vitro,K562 cells. Maternal separation failed to affect lysis of K562 cells acutely, although lysis by matched control subjects appeared to be transiently reduced 2 h after removal of the adult female. A longer-term influence was noted such that lysis of targets in previously separated subjects was greater than that in matched controls. Lysis rose over time regardless of species or experimental condition. A striking internal consistency in the lysis was also noted. Lysis was highly intercorrelated (r’s > .60,p≤ .005) within subjects across time during baseline, separation, and reunion phases of the acute social challenge. In addition, there was a tendency for this correlation to hold over the longitudinal phases. Lysis of K562 targets by macaque lymphocytes would appear to possess trait-like stability; however, the range of lysis may be modified by early experiences.