The transgenerational transmission of childhood adversity: behavioral, cellular, and epigenetic correlates.

The view that the functional maturation of the brain is the result of an environmentally driven adaptation of genetically preprogrammed neuronal networks is an important current concept in developmental neuroscience and psychology. This hypothesis proposes that early traumatic experiences or early life stress (ELS) as a negative environmental experience provide a major risk factor for the development of dysfunctional brain circuits and as a consequence for the emergence of behavioral dysfunctions and mental disorders in later life periods. This view is supported by an increasing number of clinical as well as experimental animal studies revealing that early life traumas can induce functional 'scars' in the brain, especially in brain circuits, which are essential for emotional control, learning, and memory functions. Such gene × environment interactions are modulated by specific epigenetic mechanisms, which are suggested to be the key factors of transgenerational epigenetic inheritance. Indeed, there is increasing evidence for inter- and transgenerational cycles of environmentally driven neuronal and behavioral adaptations mediated by epigenetic mechanisms. Finally, recent concepts postulate that, dependent on type, time point, and duration of ELS exposure, also positive functional adaptations may occur in the relevant brain pathways, leading to better stress coping and resilience against adversities later in life.

Infant cognitive training preshapes learning-relevant prefrontal circuits for adult learning: learning-induced tagging of dendritic spines.

Work in various animal models has demonstrated that cognitive training in infancy has a greater effect on adult cognitive performance than pretraining in adulthood. Since the underlying synaptic mechanisms are unclear, the aim of this study was to test the working hypothesis that associative training "preshapes" synaptic circuits in the developing infant brain and thereby improves learning in adulthood. Using a two-way active avoidance (TWA) paradigm, we found that avoidance training during infancy, even though the infant rats were not capable to learn a successful avoidance strategy, improves avoidance learning in adulthood. On the neuroanatomical level we show here for the first time that infant TWA training in the ventromedial prefrontal cortex suppresses developmental spine formation. In contrast in the lateral orbitofrontal cortex, developmental spine pruning is suppressed, possibly by "tagging" activated synapses, which thereby are protected from being eliminated. Moreover, we demonstrate that infant TWA training alters learning-induced synaptic plasticity in the adult brain. The synaptic and dendritic changes correlate with specific behavioral parameters. Taken together, these results support the working hypothesis that infant cognitive training interferes with developmental reorganization and maturation of dendritic spines and thereby "optimizes" prefrontal neuronal circuits for adult learning.

Paternal deprivation alters region- and age-specific interneuron expression patterns in the biparental rodent, Octodon degus.

The impact of paternal care on the postnatal development of inhibitory neuronal subpopulations in prefrontal and limbic brain regions was studied in the rodent Octodon degus. Comparing offspring from biparental families with animals raised by a single mother revealed region-specific deprivation-induced changes in the density of PARV- and CaBP-D28k expressing cells. Some deprivation-induced changes were only seen at P21: elevated CaBP-D28k-positive neurons in the orbitofrontal cortex, CA1, CA3, and dentate gyrus (DG) and elevated PARV-positive neurons in the lateral orbitofrontal, prelimbic/infralimbic (PL/IL), DG and CA1, nucleus accumbens, and amygdala. Some deprivation-induced changes were obvious in both age groups: increased CaBP-D28k-positive neurons in the nucleus accumbens shell and increased PARV-positive neurons in the ventral orbitofrontal. Some deprivation-induced changes were only seen in adulthood: increased CaBP-D28k-positive neurons in the amygdala and decreased PARV-positive neurons in the PL/IL and in CA3. In CA1, PARV-positive neurons were increased at P21 and decreased in adulthood. The functional significance of the deprivation-induced changes in PARV-positive neurons, which are involved in gamma oscillations and thereby affect information processing and which appear to be key players for critical period plasticity in sensory cortex development, as well as the behavioral implications remain to be further elucidated.

Infant avoidance training alters cellular activation patterns in prefronto-limbic circuits during adult avoidance learning: II. Cellular imaging of neurons expressing the activity-regulated cytoskeleton-associated protein (Arc/Arg3.1).

Positive and negative feedback learning is essential to optimize behavioral performance. We used the two-way active avoidance (TWA) task as an experimental paradigm for negative feedback learning with the aim to test the hypothesis that neuronal ensembles activate the activity-regulated cytoskeletal (Arc/Arg3.1) protein during different phases of avoidance learning and during retrieval. A variety of studies in humans and other animals revealed that the ability of aversive feedback learning emerges postnatally. Our previous findings demonstrated that rats, which as infants are not capable to learn an active avoidance strategy, show improved avoidance learning as adults. Based on these findings, we further tested the hypothesis that specific neuronal ensembles are "tagged" during infant TWA training and then reactivated during adult re-exposure to the same learning task. Using cellular imaging by immunocytochemical detection of Arc/Arg3.1, we observed that, compared to the untrained control group, (1) only in the dentate gyrus the density of Arc/Arg3.1-expressing neurons was elevated during the acquisition phase of TWA learning, and (2) this increase in Arc/Arg3.1-expressing neurons was not specific for the TWA learning task. With respect to the effects of infant TWA training we found that compared to the naïve non-pretrained group (a) the infant pretraining group displayed a higher density of Arc/Arg3.1-expressing neurons in the anterior cingulate cortex during acquisition on training day 1, and (b) the infant pretraining group displayed elevated density of Arc/Arg3.1-expressing neurons in the dentate gyrus during retrieval on test day 5. Correlation analysis for the acquisition phase revealed for the ACd that the animals which showed the highest number of avoidances and the fastest escape latencies displayed the highest density of Arc/Arg3.1-expressing neurons. Taken together, we are the first to use the synaptic plasticity protein Arc/Arg3.1 to label neuronal ensembles which are involved in different phases of active avoidance learning and whose activity patterns are changing in response to previous learning experience during infancy. Our results indicate (1) that, despite the inability to learn an active avoidance response in infancy, lasting memory traces are formed encoding the subtasks that are learned in infancy (e.g., the association of the CS and UCS, escape strategy), which are encoded in the infant brain by neuronal ensembles, which alter their synaptic connectivity via activation of specific synaptic plasticity proteins such as Arc/Arg3.1 and Egr1, and (2) that during adult training these memories can be retrieved by reactivating these neuronal ensembles and their synaptic circuits and thereby accelerate learning.

Infant avoidance training alters cellular activation patterns in prefronto-limbic circuits during adult avoidance learning: I. Cellular imaging of neurons expressing the synaptic plasticity early growth response protein 1 (Egr1).

Both positive feedback learning and negative feedback learning are essential for adapting and optimizing behavioral performance. There is increasing evidence in humans and animals that the ability of negative feedback learning emerges postnatally. Our work in rats, using a two-way active avoidance task (TWA) as an experimental paradigm for negative feedback learning, revealed that medial and lateral prefrontal regions of infant rats undergo dramatic synaptic reorganization during avoidance training, resulting in improved avoidance learning in adulthood. The aim of this study was to identify changes of cellular activation patterns during the course of training and in relation to infant pretraining. We applied a quantitative cellular imaging technique using the immunocytochemical detection of the activity marker early growth response protein 1 (Egr1) as a candidate contributing to learning-induced synaptic plasticity. We found region-specific cellular activity patterns, which indicate that during the acquisition phase, Egr1 expression is specifically elevated in cellular ensembles of the orbitofrontal, dorsal anterior cingulate and hippocampal CA1 region. During memory retrieval Egr1 expression is elevated in cellular ensembles of the dentate gyrus. Moreover, we, for the first time, show here that TWA training during infancy alters adult learning- and memory-related patterns of Egr1 expression in these brain regions. It is tempting to speculate that during infant learning, specific Egr1-expressing cellular ensembles are "tagged" representing long-term memory formation, and that these cell ensembles may be reactivated during adult learning.

Stress in utero alters neonatal stress-induced regulation of the synaptic plasticity proteins Arc and Egr1 in a sex-specific manner.

The present study in juvenile rats investigated a "two-hit model" to test the impact of prenatal stress exposure ("first hit") on the regulation of the synaptic plasticity immediate early genes Arc and Egr1 in response to a second neonatal stressor ("second hit") in a sex-specific manner. Three stress-exposed animal groups were compared at the age of 21 days in relation to unstressed controls (CON): preS animals were exposed to various unpredictable stressors during the last gestational trimester; postS animals were exposed to 45 min restraint stress at postnatal day 21, pre/postS animals were exposed to a combination of pre- and postnatal stress as described for the two previous groups. The postS and pre/postS groups were killed 2 h after exposure to the postnatal stressor, males and females were separately analyzed. In line with our hypothesis we detected sex-specific stress sensitivity for both analyzed proteins. Males did not show any significant changes in Arc expression irrespective of the stress condition. In contrast, females, which had been pre-exposed to prenatal stress, displayed an "amplified" Arc upregulation in response to postnatal stress (pre/postS group) compared to unstressed controls, which may reflect a "sensitization" effect of prenatal stress. For Egr1, the females did not show any stress-induced regulation irrespective of the stress condition, whereas in males, which were pre-exposed to prenatal stress, we observed a "protective" effect of prenatal stress on postnatal stress-induced downregulation of Egr1 expression (pre/postS group), which may indicate that prenatal stress exposure may induce "resilience".

Transgenerational sex-specific impact of preconception stress on the development of dendritic spines and dendritic length in the medial prefrontal cortex.

Perinatal adverse experience programs social and emotional behavioral traits and is a major risk factor for the development of behavioral and psychiatric disorders. Little information is available on how adversity to the mother prior to her first pregnancy (preconception stress, PCS) may affect brain structural development, which may underlie behavioral dysfunction in the offspring. Moreover, little is known about possible sex-dependent consequences of PCS in the offspring. This study examined spine number/density and dendritic length/complexity of layer II/III pyramidal neurons in the anterior cingulate (ACd), prelimbic/infralimbic (PL/IL) and orbitofrontal cortex (OFC) of male and female rats born to mothers exposed to unpredictable variable stress at different time points prior to reproduction. Our main findings are that in line with our hypothesis adversity to the mother before her pregnancy results in highly complex changes in neuronal morphology in the medial prefrontal, but not in the orbitofrontal cortical regions of her future offspring that persist into adulthood. Moreover, our study revealed that (1) in the PCS2 group (offspring of dams mated two weeks after stress) spine numbers and dendritic length and complexity were increased in response to PCS in the ACd and PL/IL, (2) these regional effects depended on the temporal proximity of adversity and conception, (3) in the ACd of the PCS2 group only males and the left hemispheres were affected. We speculate that these transgenerational brain structural changes are mediated by stress-induced epigenetic (re)programming of future gene activity in the oocyte.

Separation-induced receptor changes in the hippocampus and amygdala of Octodon degus: influence of maternal vocalizations.

Relatively little is known about the basic mechanisms that play a role in the vulnerability of the developing brain toward adverse environmental influences. Our study in the South American rodent Octodon degus revealed that repeated brief separation from the parents and exposure to an unfamiliar environment induces in the hippocampal formation of male and female pups an upregulation of D1 and 5-HT1A receptor density in the stratum radiatum and stratum lacunosum moleculare of the CA1 region. In the CA3 region, only the 5-HT1A receptors were upregulated; no changes were observed for D1 receptors in this region. GABA(A) receptor density in the hippocampus and amygdala was downregulated (nonsignificant trend) after parental separation. The acoustic presence of the mother during parental separation suppressed the D1 and 5-HT1A receptor upregulation in some regions of the hippocampus; no such suppressing influence was observed for the GABA(A) receptors. In the basomedial amygdala, the maternal calls enhanced the separation-induced 5-HT1A receptor upregulation in the male pups, whereas in the female pups the separation-induced receptor densities were not only suppressed by the maternal call but further downregulated, compared with the control group. These results demonstrate that early adverse emotional experience alters aminergic function within the hippocampus and amygdala and that the mother's voice, a powerful emotional signal, can modulate these effects in the developing limbic system.

Paternal deprivation affects the functional maturation of corticotropin-releasing hormone (CRH)- and calbindin-D28k-expressing neurons in the bed nucleus of the stria terminalis (BNST) of the biparental Octodon degus.

While the critical role of maternal care on the development of brain and behavior of the offspring has been extensively studied, our knowledge about the importance of paternal care for brain development of his offspring is still comparatively scarce. The aim of this study in the biparental caviomorph rodent Octodon degus was to analyze the impact of paternal care on the development of corticotropin-releasing hormone (CRH)-expressing neurons in the bed nucleus of the stria terminalis (BNST) and hypothalamic paraventricular nucleus (PVN). Both brain areas are key players in neuronal circuits that regulate hypothalamic-pituitary-adrenal axis (HPA) activity. At the age of postnatal day (PND) 21, we found that paternal deprivation resulted in a decreased density of CRH-containing neurons in the medial, but not in the lateral BNST, whereas no changes were observed in the PVN. These deprivation-induced changes were still prominent in adulthood. At PND 21, the density of Ca-binding protein calbindin D28K (CaBP-D28K)-expressing neurons was specifically increased in the medial, but not lateral BNST of father-deprived animals. In contrast, adult father-deprived animals show significantly decreased density of CaBP-D28K-expressing neurons in the lateral, but not medial BNST. Taken together, these results may have important implications for our understanding of the experience-driven development of neural circuits that regulate HPA activity mediating acute responses to stress and chronic anxiety.

Paternal deprivation alters the development of catecholaminergic innervation in the prefrontal cortex and related limbic brain regions.

The impact of paternal care on the development of catecholaminergic fiber innervations in the prefrontal cortex, nucleus accumbens, hippocampus and the amygdala was quantitatively investigated in the biparental Octodon degus. Two age (juvenile, adult) and rearing groups: (1) degus reared without father and (2) degus raised by both parents were compared. Juvenile father-deprived animals showed significantly elevated densities of TH-immunoreactive fibers in all analyzed regions, except in the orbitofrontal cortex, as compared to biparentally reared animals. This difference between the two rearing groups was still evident in adulthood in the prelimbic and infralimbic cortices and in the hippocampal formation. Interestingly, the elevated TH fiber density in both nucleus accumbens subregions was reversed in adulthood, i.e. adult father-deprived animals showed strongly reduced TH fiber densities as compared to biparentally reared animals. We show here that paternal care plays a critical role in the functional maturation of catecholaminergic innervation patterns in prefrontal and limbic brain circuits.

Repeated neonatal separation stress alters the composition of neurochemically characterized interneuron subpopulations in the rodent dentate gyrus and basolateral amygdala.

Emotional experience during early life has been shown to interfere with the development of excitatory synaptic networks in the prefrontal cortex, hippocampus, and the amygdala of rodents and primates. The aim of the present study was to investigate a developmental "homoeostatic synaptic plasticity" hypothesis and to test whether stress-induced changes of excitatory synaptic composition are counterbalanced by parallel changes of inhibitory synaptic networks. The impact of repeated early separation stress on the development of two GABAergic neuronal subpopulations was quantitatively analyzed in the brain of the semiprecocial rodent Octodon degus. Assuming that PARV- and CaBP-D28k-expression are negatively correlated to the level of inhibitory activity, the previously described reduced density of excitatory spine synapses in the dentate gyrus of stressed animals appears to be "amplified" by elevated GABAergic inhibition, reflected by reduced PARV- (down to 85%) and CaBP-D28k-immunoreactivity (down to 74%). In opposite direction, the previously observed elevated excitatory spine density in the CA1 region of stressed animals appears to be amplified by reduced inhibition, reflected by elevated CaPB-D28k-immunoreactivity (up to 149%). In the (baso)lateral amygdala, the previously described reduction of excitatory spine synapses appears to be "compensated" by reduced inhibitory activity, reflected by dramatically elevated PARV- (up to 395%) and CaPB-D28k-immunoreactivity (up to 327%). No significant differences were found in the central nucleus of the amygdala, the piriform, and somatosensory cortices and in the hypothalamic paraventricular nucleus. Thus during stress-evoked neuronal and synaptic reorganization, a homeostatic balance between excitation and inhibition is not maintained in all regions of the juvenile brain.

Stress-induced synaptic changes in the rat anterior cingulate cortex are dependent on endocrine developmental time windows.

Fetal and neonatal brain development is characterized by developmental time windows during which brain regions or neuron types are specifically sensitive to environmental influences. Previous studies on cortical development have revealed evidence for the hypothesis that the extent and the direction of experience-induced neuronal and synaptic changes correlate with time windows of endocrine development. To further test this hypothesis we exposed rats to neonatal separation stress during different phases of endocrine maturation, i.e. prior, during and after the stress hyporesponsive period (SHRP) of the hypothalamic-pituitary-adrenal (HPA) axis. We show here that only stress during the SHRP resulted in significantly decreased (-29%) spines densities on the basal dendrites of pyramidal cells in layer V of the anterior cingulate cortex (ACd), whereas stress during the other two tested time windows had no effect on these parameters. Dendritic length remained unaffected by stress exposure at any of the tested time windows. These results reveal specific developmental time window for synaptic wiring within the deeper layers of the anterior cingulate cortex, which seem not to be mediated by hormonally induced mechanisms.

Long-term consequences of early experience on adult avoidance learning in female rats: role of the dopaminergic system.

Following our hypothesis that juvenile emotional and/or cognitive experience should affect learning performance at preweaning age as well as adulthood, the present study in female Wistar rats aimed to examine the impact of (i) avoidance training at preweaning age, (ii) exposure to repeated maternal separation, (iii) the combination of both, and (iv) the blockade of dopaminergic neurotransmission on adult two-way active avoidance learning in rats. We found that preweaning, i.e. three week old, rats were less capable of avoidance learning compared to adults. Our main findings revealed that preweaning avoidance training alone improved avoidance learning in adulthood. Furthermore, maternal separation alone also improved avoidance learning in preweaning and in adult rats, but this effect of maternal separation did not add up to the beneficial effect of preweaning avoidance training on adult learning. In addition, the pharmacological blockade of dopamine receptors during preweaning avoidance training via systemic application of haloperidol impaired preweaning avoidance performance in a dose-dependent manner. Testing the haloperidol-treated preweaning presumed "non-learners" as adults revealed that they still showed improved learning as adults. Taken together, our results strongly support the hypothesis that emotional as well as cognitive experience at preweaning age leaves an enduring "memory trace," which can facilitate learning in adulthood. Our pharmaco-behavioral studies suggest that unlike the adult brain, preweaning learning and memory formation is less dependent on dopaminergic mechanisms, which raises the intriguing question of possible alternative pathways.

Influence of parental deprivation on the behavioral development in Octodon degus: modulation by maternal vocalizations.

Repeated separation from the family during very early stages of life is a stressful emotional experience which induces a variety of neuronal and synaptic changes in limbic cortical areas that may be related to behavioral alterations. First, we investigated whether repeated parental separation and handling, without separation from the family, leads to altered spontaneous exploratory behavior in a novel environment (open field test) in 8-day-old Octodon degus. Second, we tested whether the parentally deprived and handled animals display different stimulus-evoked exploratory behaviors in a modified open field version, in which a positive emotional stimulus, the maternal call, was presented. In the open field test a significant influence of previous emotional experience was found for the parameters of running, rearing, and vocalization. Parentally deprived degus displayed increased horizontal (running) and vertical (rearing) motoric activities, but decreased vocalization, compared to normal and handled controls. The presentation of maternal vocalizations significantly modified running, vocalization, and grooming activities, which in the case of running activity was dependent on previous emotional experience. Both deprivation-induced locomotor hyperactivity together with the reduced behavioral response towards a familiar acoustic emotional signal are similar to behavioral disturbances observed in human attachment disorders.

Juvenile emotional experience alters synaptic composition in the rodent cortex, hippocampus, and lateral amygdala.

A quantitative anatomical study in the rodent anterior cingulate and somatosensory cortex, hippocampus, and lateral amygdala revealed region-, cell-, and dendrite-specific changes of spine densities in 3-week-old Octodon degus after repeated parental separation. In parentally separated animals significantly higher spine densities were found on the apical and basal dendrites of the cingulate cortex (up to 143% on apical and 138% on basal dendrite). Branching order analysis revealed that this effect is seen on all segments of the apical dendrite, whereas on the basal dendrites significantly higher spine densities were seen only on the outer branches (third to fifth dendritic segments). Increased spine densities were also observed on the hippocampal CA1 pyramidal neurons (up to 109% on the distal apical segments and up to 106% on the basal segment) compared with the control group. In contrast, significantly reduced spine densities were observed on the granule cell dendrites in the dentate gyrus (down to 92%) and on the apical dendrites in the medial nucleus of the amygdala (down to 95%). No significant changes of spine densities were seen in the somatosensory cortex (except for an increase in the proximal apical segments) and in the lateral nucleus of the dorsal amygdala (except for an increase in the proximal basal dendritic segments). These results demonstrate that repeated stressful emotional experience alters the balance of presumably excitatory synaptic inputs of pyramidal neurons in the limbic system. Such experience-induced modulations of limbic circuits may determine psychosocial and cognitive capacities during later life.