Pre-reproductive stress in adolescent female rats alters maternal care and DNA methylation patterns across generations.

Stress during development affects maternal behavior and offspring phenotypes. Stress in adolescence is particularly consequential on brain development and maturation, and is implicated in several psychiatric disorders. We previously showed that pre-reproductive stress (PRS) in female adolescent rats affects behavior and corticotropin releasing hormone receptor 1 (CRHR1) expression in first- (F1) and second- (F2) generation offspring. We further showed that offspring phenotypes are partially reversed by post-stress treatment with fluoxetine (FLX) or the CRHR1 antagonist NBI27914 (NBI). Epigenetic processes, such as DNA methylation, are implicated in the stress response and interact with maternal care quality across generations. Here, we asked whether PRS and FLX or NBI exposure would affect maternal care and global DNA methylation in the brains of exposed dams and their adult F1 and paternally-derived F2 offspring. We found that PRS decreased self-care while increasing pup-care behaviors. PRS also increased DNA methylation in the amygdala of dams and their F1 male offspring, but decreased it in F2 females. Drug treatment had no effect on maternal care, but affected DNA methylation patterns in F0 and F1 generations. Furthermore, PRS altered the expression of DNA methylating enzymes in brain, blood and oocytes. Finally, maternal care variables differentially predicted methylation levels in PRS and control offspring. Thus, the effects of adolescent stress are long-lasting and impact methylation levels across three generations. Combined with our findings of epigenetic changes in PRS-exposed oocytes, the present data imply that biological changes and social mechanisms act in concert to influence adult offspring phenotypes.

Beyond NMDA Receptors: Homeostasis at the Glutamate Tripartite Synapse and Its Contributions to Cognitive Dysfunction in Schizophrenia.

Cognitive deficits are core symptoms of schizophrenia but remain poorly addressed by dopamine-based antipsychotic medications. Glutamate abnormalities are implicated in schizophrenia-related cognitive deficits. While the role of the NMDA receptor has been extensively studied, less attention was given to other components that control glutamate homeostasis. Glutamate dynamics at the tripartite synapse include presynaptic and postsynaptic components and are tightly regulated by neuron-astrocyte crosstalk. Here, we delineate the role of glutamate homeostasis at the tripartite synapse in schizophrenia-related cognitive dysfunction. We focus on cognitive domains that can be readily measured in humans and rodents, i.e., working memory, recognition memory, cognitive flexibility, and response inhibition. We describe tasks used to measure cognitive function in these domains in humans and rodents, and the relevance of glutamate alterations in these domains. Next, we delve into glutamate tripartite synaptic components and summarize findings that implicate the relevance of these components to specific cognitive domains. These collective findings indicate that neuron-astrocyte crosstalk at the tripartite synapse is essential for cognition, and that pre- and postsynaptic components play a critical role in maintaining glutamate homeostasis and cognitive well-being. The contribution of these components to cognitive function should be considered in order to better understand the role played by glutamate signaling in cognition and develop efficient pharmacological treatment avenues for schizophrenia treatment-resistant symptoms.

FAAH Inhibition Restores Early Life Stress-Induced Alterations in PFC microRNAs Associated with Depressive-Like Behavior in Male and Female Rats.

Early life stress (ELS) increases predisposition to depression. We compared the effects of treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597, and the selective serotonin reuptake inhibitor paroxetine, on ELS-induced depressive-like behavior and the expression of microRNAs (miRs) associated with depression in the medial prefrontal cortex (mPFC), hippocampal CA1 area, lateral habenula and dorsal raphe in rats. We also examined the mRNA expression of serotonergic (htr1a and slc6a4) and endocannabinoid (cnr1, cnr2 and faah) targets in the mPFC following ELS and pharmacological treatment. Adult males and females exposed to the 'Limited Bedding and Nesting' ELS paradigm demonstrated a depressive-like phenotype and late-adolescence URB597 treatment, but not paroxetine, reversed this phenotype. In the mPFC, ELS downregulated miR-16 in males and miR-135a in females and URB597 treatment restored this effect. In ELS females, the increase in cnr2 and decrease in faah mRNAs in the mPFC were reversed by URB597 treatment. We show for the first time that URB597 reversed ELS-induced mPFC downregulation in specific miRs and stress-related behaviors, suggesting a novel mechanism for the beneficial effects of FAAH inhibition. The differential effects of ELS and URB597 on males and females highlight the importance of developing sex-specific treatment approaches.

A-to-I RNA editing in the rat brain is age-dependent, region-specific and sensitive to environmental stress across generations.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is an epigenetic modification catalyzed by adenosine deaminases acting on RNA (ADARs), and is especially prevalent in the brain. We used the highly accurate microfluidics-based multiplex PCR sequencing (mmPCR-seq) technique to assess the effects of development and environmental stress on A-to-I editing at 146 pre-selected, conserved sites in the rat prefrontal cortex and amygdala. Furthermore, we asked whether changes in editing can be observed in offspring of stress-exposed rats. In parallel, we assessed changes in ADARs expression levels. RESULTS: In agreement with previous studies, we found editing to be generally higher in adult compared to neonatal rat brain. At birth, editing was generally lower in prefrontal cortex than in amygdala. Stress affected editing at the serotonin receptor 2c (Htr2c), and editing at this site was significantly altered in offspring of rats exposed to prereproductive stress across two generations. Stress-induced changes in Htr2c editing measured with mmPCR-seq were comparable to changes measured with Sanger and Illumina sequencing. Developmental and stress-induced changes in Adar and Adarb1 mRNA expression were observed but did not correlate with editing changes. CONCLUSIONS: Our findings indicate that mmPCR-seq can accurately detect A-to-I RNA editing in rat brain samples, and confirm previous accounts of a developmental increase in RNA editing rates. Our findings also point to stress in adolescence as an environmental factor that alters RNA editing patterns several generations forward, joining a growing body of literature describing the transgenerational effects of stress.

Acquisition of conditioned fear is followed by region-specific changes in RNA editing of glutamate receptors.

Adenosine (A) to inosine (I) RNA editing is a post-transcriptional modification process that can affect synaptic function. Transcripts encoding the kainate GRIK1 and AMPA GluA2 glutamate receptor subunits undergo editing that leads to a glycine/arginine (Q/R) exchange and reduced Ca(2+) permeability. We hypothesized that editing at these sites could be experience-dependent, temporally dynamic and region-specific. We trained C57/Bl6 mice in trace and contextual fear conditioning protocols, and examined editing levels at GRIK1 and GluA2 Q/R sites in the amygdala (CeA) and hippocampus (CA1 and CA3), at two time points after training. We also examined experience-dependent changes in the expression of RNA editing enzymes and editing targets. Animals trained in the trace fear conditioning protocol exhibited a transient increase in unedited GRIK1 RNA in the amygdala, and their learning efficiency correlated with unedited RNA levels in CA1. In line with previous reports, GluA2 RNA editing levels were nearly 100%. Additionally, we observed experience-dependent changes in mRNA expression of the RNA editing enzymes ADAR2 and ADAR1 in amygdala and hippocampus, and a learning-dependent increase in the alternatively spliced inactive form of ADAR2 in the amygdala. Since unedited transcripts code for Ca(2+)-permeable receptor subunits, these findings suggest that RNA editing at Q/R sites of glutamate receptors plays an important role in experience-dependent synaptic modification processes.

nNOS in Erbb4-positive neurons regulates GABAergic transmission in mouse hippocampus.

Neuronal nitric oxide synthase (nNOS, gene name Nos1) orchestrates the synthesis of nitric oxide (NO) within neurons, pivotal for diverse neural processes encompassing synaptic transmission, plasticity, neuronal excitability, learning, memory, and neurogenesis. Despite its significance, the precise regulation of nNOS activity across distinct neuronal types remains incompletely understood. Erb-b2 receptor tyrosine kinase 4 (ErbB4), selectively expressed in GABAergic interneurons and activated by its ligand neuregulin 1 (NRG1), modulates GABA release in the brain. Our investigation reveals the presence of nNOS in a subset of GABAergic interneurons expressing ErbB4. Notably, NRG1 activates nNOS via ErbB4 and its downstream phosphatidylinositol 3-kinase (PI3K), critical for NRG1-induced GABA release. Genetic removal of nNos from Erbb4-positive neurons impairs GABAergic transmission, partially rescued by the NO donor sodium nitroprusside (SNP). Intriguingly, the genetic deletion of nNos from Erbb4-positive neurons induces schizophrenia-relevant behavioral deficits, including hyperactivity, impaired sensorimotor gating, and deficient working memory and social interaction. These deficits are ameliorated by the atypical antipsychotic clozapine. This study underscores the role and regulation of nNOS within a specific subset of GABAergic interneurons, offering insights into the pathophysiological mechanisms of schizophrenia, given the association of Nrg1, Erbb4, Pi3k, and Nos1 genes with this mental disorder.

Synergistic, long-term effects of glutamate dehydrogenase 1 deficiency and mild stress on cognitive function and mPFC gene and miRNA expression.

Glutamate abnormalities in the medial prefrontal cortex (mPFC) are associated with cognitive deficits. We previously showed that homozygous deletion of CNS glutamate dehydrogenase 1 (Glud1), a metabolic enzyme critical for glutamate metabolism, leads to schizophrenia-like behavioral abnormalities and increased mPFC glutamate; mice heterozygous for CNS Glud1 deletion (C-Glud1+/- mice) showed no cognitive or molecular abnormalities. Here, we examined the protracted behavioral and molecular effects of mild injection stress on C-Glud1+/- mice. We found spatial and reversal learning deficits, as well as large-scale mPFC transcriptional changes in pathways associated with glutamate and GABA signaling, in stress-exposed C-Glud1+/- mice, but not in their stress-naïve or C-Glud1+/+ littermates. These effects were observed several weeks following stress exposure, and the expression levels of specific glutamatergic and GABAergic genes differentiated between high and low reversal learning performance. An increase in miR203-5p expression immediately following stress may provide a translational regulatory mechanism to account for the delayed effect of stress exposure on cognitive function. Our findings show that chronic glutamate abnormalities interact with acute stress to induce cognitive deficits, and resonate with gene x environment theories of schizophrenia. Stress-exposed C-Glud1+/- mice may model a schizophrenia high-risk population, which is uniquely sensitive to stress-related 'trigger' events.

Synaptic underpinnings of altered hippocampal function in glutaminase-deficient mice during maturation.

Glutaminase-deficient mice (GLS1 hets), with reduced glutamate recycling, have a focal reduction in hippocampal activity, mainly in CA1, and manifest behavioral and neurochemical phenotypes suggestive of schizophrenia resilience. To address the basis for the hippocampal hypoactivity, we examined synaptic plastic mechanisms and glutamate receptor expression. Although baseline synaptic strength was unaffected in Schaffer collateral inputs to CA1, we found that long-term potentiation was attenuated. In wild-type (WT) mice, GLS1 gene expression was highest in the hippocampus and cortex, where it was reduced by about 50% in GLS1 hets. In other brain regions with lower WT GLS1 gene expression, there were no genotypic reductions. In adult GLS1 hets, NMDA receptor NR1 subunit gene expression was reduced, but not AMPA receptor GluR1 subunit gene expression. In contrast, juvenile GLS1 hets showed no reductions in NR1 gene expression. In concert with this, adult GLS1 hets showed a deficit in hippocampal-dependent contextual fear conditioning, whereas juvenile GLS1 hets did not. These alterations in glutamatergic synaptic function may partly explain the hippocampal hypoactivity seen in the GLS1 hets. The maturity-onset reduction in NR1 gene expression and in contextual learning supports the premise that glutaminase inhibition in adulthood should prove therapeutic in schizophrenia.

Anandamide Hydrolysis Inhibition Reverses the Long-Term Behavioral and Gene Expression Alterations Induced by MK-801 in Male Rats: Differential CB1 and CB2 Receptor-Mediated Effects.

NMDA receptor blockade in rodents is commonly used to induce schizophrenia-like behavioral abnormalities, including cognitive deficits and social dysfunction. Aberrant glutamate and GABA transmission, particularly in adolescence, is implicated in these behavioral abnormalities. The endocannabinoid system modulates glutamate and GABA transmission, but the impact of endocannabinoid modulation on cognitive and social dysfunction is unclear. Here, we asked whether late-adolescence administration of the anandamide hydrolysis inhibitor URB597 can reverse behavioral deficits induced by early-adolescence administration of the NMDA receptor blocker MK-801. In parallel, we assessed the impact of MK-801 and URB597 on mRNA expression of glutamate and GABA markers. We found that URB597 prevented MK-801-induced novel object recognition deficits and social interaction abnormalities in adult rats, and reversed glutamate and GABA aberrations in the prelimbic PFC. URB597-mediated reversal of MK-801-induced social interaction deficits was mediated by the CB1 receptor, whereas the reversal of cognitive deficits was mediated by the CB2 receptor. This was paralleled by the reversal of CB1 and CB2 receptor expression abnormalities in the basolateral amygdala and prelimbic PFC, respectively. Together, our findings show that interfering with NMDA receptor function in early adolescence has a lasting impact on phenotypes resembling the negative symptoms and cognitive deficits of schizophrenia and on glutamate and GABA marker expression in the PFC. Prevention of behavioral and molecular abnormalities by late-adolescence URB597 via CB1 and CB2 receptors suggests that endocannabinoid stimulation may have therapeutic potential in addressing treatment-resistant symptoms.

Pre-reproductive stress in adolescent female rats alters oocyte microRNA expression and offspring phenotypes: pharmacological interventions and putative mechanisms.

Pre-reproductive stress (PRS) to adolescent female rats alters anxiogenic behavior in first (F1)- and second-generation (F2) offspring and increases mRNA expression of corticotropin-releasing factor receptor type 1 (Crhr1) in oocytes and in neonate offspring brain. Here, we ask whether the expression of Crhr1 and Crhr1-targeting microRNA is altered in brain, blood, and oocytes of exposed females and in the brain of their neonate and adult F1 and F2 offspring. In addition, we inquire whether maternal post-stress drug treatment reverses PRS-induced abnormalities in offspring. We find that PRS induces a selective increase in Crhr1-targeting mir-34a and mir-34c in blood and oocytes, while non-Crhr1 microRNA molecules remain unaltered. PRS induces similar microRNA changes in prefrontal cortex of F1 and F2 neonates. In adult animals, cortical Crhr1, but not mir-34, expression is affected by both maternal and direct stress exposure. Post-PRS fluoxetine (FLX) treatment increases pup mortality, and both FLX and the Crhr1 antagonist NBI 27914 reverse some of the effects of PRS and also have independent effects on F1 behavior and gene expression. PRS also alters behavior as well as gene and miRNA expression patterns in paternally derived F2 offspring, producing effects that are different from those previously found in maternally derived F2 offspring. These findings extend current knowledge on inter- and trans-generational transfer of stress effects, point to microRNA changes in stress-exposed oocytes as a potential mechanism, and highlight the consequences of post-stress pharmacological interventions in adolescence.

Non-canonical glutamine transamination sustains efferocytosis by coupling redox buffering to oxidative phosphorylation.

Macrophages rely on tightly integrated metabolic rewiring to clear dying neighboring cells by efferocytosis during homeostasis and disease. Here we reveal that glutaminase-1-mediated glutaminolysis is critical to promote apoptotic cell clearance by macrophages during homeostasis in mice. In addition, impaired macrophage glutaminolysis exacerbates atherosclerosis, a condition during which, efficient apoptotic cell debris clearance is critical to limit disease progression. Glutaminase-1 expression strongly correlates with atherosclerotic plaque necrosis in patients with cardiovascular diseases. High-throughput transcriptional and metabolic profiling reveals that macrophage efferocytic capacity relies on a non-canonical transaminase pathway, independent from the traditional requirement of glutamate dehydrogenase to fuel ɑ-ketoglutarate-dependent immunometabolism. This pathway is necessary to meet the unique requirements of efferocytosis for cellular detoxification and high-energy cytoskeletal rearrangements. Thus, we uncover a role for non-canonical glutamine metabolism for efficient clearance of dying cells and maintenance of tissue homeostasis during health and disease in mouse and humans.

Glutamate dehydrogenase deficiency disrupts glutamate homeostasis in hippocampus and prefrontal cortex and impairs recognition memory.

Glutamate Dehydrogenase 1 (GDH), encoded by the Glud1 gene in rodents, is a mitochondrial enzyme critical for maintaining glutamate homeostasis at the tripartite synapse. Our previous studies indicate that the hippocampus may be particularly vulnerable to GDH deficiency in central nervous system (CNS). Here, we first asked whether mice with a homozygous deletion of Glud1 in CNS (CNS-Glud1 -/- mice) express different levels of glutamate in hippocampus, and found elevated glutamate as well as glutamine in dorsal and ventral hippocampus, and increased glutamine in medial prefrontal cortex (mPFC). l-serine and d-serine, which contribute to glutamate homeostasis and NMDA receptor function, are increased in ventral but not dorsal hippocampus, and in mPFC. Protein expression levels of the GABA synthesis enzyme glutamate decarboxylase (GAD) GAD67 were decreased in the ventral hippocampus as well. Behavioral analysis revealed deficits in visual, spatial and social novelty recognition abilities, which require intact hippocampal-prefrontal cortex circuitry. Finally, hippocampus-dependent contextual fear retrieval was deficient in CNS-Glud1 -/- mice, and c-Fos expression (indicative of neuronal activation) in the CA1 pyramidal layer was reduced immediately following this task. These data point to hippocampal subregion-dependent disruption in glutamate homeostasis and excitatory/inhibitory balance, and to behavioral deficits that support a decline in hippocampal-prefrontal cortex connectivity. Together with our previous data, these findings also point to different patterns of basal and activity-induced hippocampal abnormalities in these mice. In sum, GDH contributes to healthy hippocampal and PFC function; disturbed GDH function is relevant to several psychiatric and neurological disorders.

Experience-induced transgenerational (re-)programming of neuronal structure and functions: Impact of stress prior and during pregnancy.

This review focuses on the inter- and transgenerational effects of stress experience prior to and during gestation. We provide an overview of findings from studies in humans as well as in animal models on brain structural and physiological functions and on the development of cognitive and executive functions. We also discuss the concept of stress-induced (re-)programming in more detail by highlighting epigenetic mechanisms and particularly those affecting the development of monoaminergic transmitter systems, which constitute the brains reward system. As the majority of studies have focused on male individuals we will emphasize sex-specific differences in stress vulnerability and resilience. Finally, we offer some perspectives on the development of protective and therapeutic interventions in cognitive and emotional disturbances resulting from pre-conception and prenatal stress.

Glutamate Dehydrogenase-Deficient Mice Display Schizophrenia-Like Behavioral Abnormalities and CA1-Specific Hippocampal Dysfunction.

Brain imaging has revealed that the CA1 subregion of the hippocampus is hyperactive in prodromal and diagnosed patients with schizophrenia (SCZ), and that glutamate is a driver of this hyperactivity. Strikingly, mice deficient in the glutamate synthetic enzyme glutaminase have CA1 hypoactivity and a SCZ-resilience profile, implicating glutamate-metabolizing enzymes. To address this further, we examined mice with a brain-wide deficit in the glutamate-metabolizing enzyme glutamate dehydrogenase (GDH), encoded by Glud1, which should lead to glutamate excess due to reduced glutamate metabolism in astrocytes. We found that Glud1-deficient mice have behavioral abnormalities in the 3 SCZ symptom domains, with increased baseline and amphetamine-induced hyperlocomotion as a positive symptom proxy, nest building and social preference as a negative symptom proxy, and reversal/extradimensional set shifting in the water T-maze and contextual fear conditioning as a cognitive symptom proxy. Neuroimaging of cerebral blood volume revealed hippocampal hyperactivity in CA1, which was associated with volume reduction. Parameters of hippocampal synaptic function revealed excess glutamate release and an elevated excitatory/inhibitory balance in CA1. Finally, in a direct clinical correlation using imaging-guided microarray, we found a significant SCZ-associated postmortem reduction in GLUD1 expression in CA1. These findings advance GLUD1 deficiency as a driver of excess hippocampal excitatory transmission and SCZ symptoms, and identify GDH as a target for glutamate modulation pharmacotherapy for SCZ. More broadly, these findings point to the likely involvement of alterations in glutamate metabolism in the pathophysiology of SCZ.

Pre-reproductive stress and fluoxetine treatment in rats affect offspring A-to-I RNA editing, gene expression and social behavior.

Adenosine to inosine RNA editing is an epigenetic process that entails site-specific modifications in double-stranded RNA molecules, catalyzed by adenosine deaminases acting on RNA (ADARs). Using the multiplex microfluidic PCR and deep sequencing technique, we recently showed that exposing adolescent female rats to chronic unpredictable stress before reproduction affects editing in the prefrontal cortex and amygdala of their newborn offspring, particularly at the serotonin receptor 5-HT2c (encoded by Htr2c). Here, we used the same technique to determine whether post-stress, pre-reproductive maternal treatment with fluoxetine (5 mg/kg, 7 days) reverses the effects of stress on editing. We also examined the mRNA expression of ADAR enzymes in these regions, and asked whether social behavior in adult offspring would be altered by maternal exposure to stress and/or fluoxetine. Maternal treatment with fluoxetine altered Htr2c editing in offspring amygdala at birth, enhanced the expression of Htr2c mRNA and RNA editing enzymes in the prefrontal cortex, and reversed the effects of pre-reproductive stress on Htr2c editing in this region. Furthermore, maternal fluoxetine treatment enhanced differences in editing of glutamate receptors between offspring of control and stress-exposed rats, and led to enhanced social preference in adult offspring. Our findings indicate that pre-gestational fluoxetine treatment affects patterns of RNA editing and editing enzyme expression in neonatal offspring brain in a region-specific manner, in interaction with pre-reproductive stress. Overall, these findings imply that fluoxetine treatment affects serotonergic signaling in offspring brain even when treatment is discontinued before gestation, and its effects may depend upon prior exposure to stress.

Differential effects of social isolation in adolescent and adult mice on behavior and cortical gene expression.

Intact function of the medial prefrontal cortex (mPFC) function relies on proper development of excitatory and inhibitory neuronal populations and on integral myelination processes. Social isolation (SI) affects behavior and brain circuitry in adulthood, but previous rodent studies typically induced prolonged (post-weaning) exposure and failed to directly compare between the effects of SI in adolescent and adulthood. Here, we assessed the impact of a 3-week SI period, starting in mid-adolescence (around the onset of puberty) or adulthood, on a wide range of behaviors in adult male mice. Additionally, we asked whether adolescent SI would differentially affect the expression of excitatory and inhibitory neuronal markers and myelin-related genes in mPFC. Our findings indicate that mid-adolescent or adult SI increase anxiogenic behavior and locomotor activity. However, SI in adolescence uniquely affects the response to the psychotomimetic drug amphetamine, social and novelty exploration and performance in reversal and attentional set shifting tasks. Furthermore, adolescent but not adult SI increased the expression of glutamate markers in the adult mPFC. Our results imply that adolescent social deprivation is detrimental for normal development and may be particularly relevant to the investigation of developmental psychopathology.

Dopamine neuron dependent behaviors mediated by glutamate cotransmission.

Dopamine neurons in the ventral tegmental area use glutamate as a cotransmitter. To elucidate the behavioral role of the cotransmission, we targeted the glutamate-recycling enzyme glutaminase (gene Gls1). In mice with a dopamine transporter (Slc6a3)-driven conditional heterozygous (cHET) reduction of Gls1 in their dopamine neurons, dopamine neuron survival and transmission were unaffected, while glutamate cotransmission at phasic firing frequencies was reduced, enabling a selective focus on the cotransmission. The mice showed normal emotional and motor behaviors, and an unaffected response to acute amphetamine. Strikingly, amphetamine sensitization was reduced and latent inhibition potentiated. These behavioral effects, also seen in global GLS1 HETs with a schizophrenia resilience phenotype, were not seen in mice with an Emx1-driven forebrain reduction affecting most brain glutamatergic neurons. Thus, a reduction in dopamine neuron glutamate cotransmission appears to mediate significant components of the GLS1 HET schizophrenia resilience phenotype, and glutamate cotransmission appears to be important in attribution of motivational salience.

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.

Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex.

Neuronal epigenomes, including chromosomal loopings moving distal cis-regulatory elements into proximity of target genes, could serve as molecular proxy linking present-day-behaviour to past exposures. However, longitudinal assessment of chromatin state is challenging, because conventional chromosome conformation capture assays essentially provide single snapshots at a given time point, thus reflecting genome organization at the time of brain harvest and therefore are non-informative about the past. Here we introduce 'NeuroDam' to assess epigenome status retrospectively. Short-term expression of the bacterial DNA adenine methyltransferase Dam, tethered to the Gad1 gene promoter in mouse prefrontal cortex neurons, results in stable G(methyl)ATC tags at Gad1-bound chromosomal contacts. We show by NeuroDam that mice with defective cognition 4 months after pharmacological NMDA receptor blockade already were affected by disrupted chromosomal conformations shortly after drug exposure. Retrospective profiling of neuronal epigenomes is likely to illuminate epigenetic determinants of normal and diseased brain development in longitudinal context.