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BackgroundEarly-life adversity (ELA) is associated with increased risk for mood disorders including depression and substance use disorders. These are characterized by impaired reward-related behaviors, suggesting compromised operations of reward-related brain circuits. However, the brain regions engaged by ELA that mediate these enduring consequences of ELA remain largely unknown. In an animal model of ELA, we have identified aberrant reward-seeking behaviors, a discovery that provides a framework for assessing the underlying circuits.MethodsEmploying TRAP2 male and female mice, in which neurons activated within a defined timeframe are permanently tagged, we compared ELA and control-reared mice, assessing the quantity and distribution of ELA-related neuronal activation. After validating the TRAP2 results using native cFos labeling, we defined the molecular identity of this population of activated neurons.ResultsWe uniquely demonstrate that the TRAP2 system is feasible and efficacious in neonatal mice. Surprisingly, the paraventricular nucleus of the thalamus (PVT) is robustly and almost exclusively activated by ELA and is the only region distinguishing ELA from typical rearing. Remarkably, a large proportion of ELA-activated PVT neurons express CRFR1, the receptor for the stress-related peptide, corticotropin-releasing hormone (CRH), but these neurons do not express CRH itself.ConclusionsWe show here that the PVT, an important component of reward circuits which is known to encode remote, emotionally salient experiences to influence future motivated behaviors, encodes adverse experiences as remote as those occurring during the early postnatal period and is thus poised to contribute to the enduring deficits in reward-related behaviors consequent to ELA.

Disrupted operations of the reward circuit are thought to underlie major emotional disorders including depression and drug abuse1–3. These disorders commonly arise following early life stress4,5; however, how stress early in life enduringly impacts reward circuit functions to promote disease remains unclear. Here, we discover and characterize a novel stress-sensitive reward-circuit projection connecting the basolateral amygdala (BLA) and nucleus accumbens (NAc) that co-expresses GABA and the stress-reactive neuropeptide corticotropin-releasing hormone (CRH). We then identify a crucial role for this projection in executing the disrupted reward behaviors provoked by early-life adversity (ELA): Chemogenetic and optogenetic stimulations of the CRHGABA BLA→NAc projection in typically reared mice suppressed several reward seeking behaviors, recapitulating deficits resulting from ELA and demonstrating a key contribution of this pathway in the normal operations of the reward circuit. Next, inhibition of the CRHGABA BLA→NAc projection in adult mice that experienced ELA restored typical reward behaviors in these mice, and, in contrast, had little effect in typically reared mice, indicating a selective ELA-induced maladaptive plasticity of this reward-circuit projection. We discover a novel, stress-sensitive, reward inhibiting projection from the BLA→NAc with unique molecular features, which may provide targets for intervention in disabling mental illnesses.

Febrile status epilepticus (FSE) is an important risk factor for temporal lobe epilepsy and early identification is vital. In a rat model of FSE, we identified an acute novel MRI signal in the basolateral amygdala (BLA) at 2 hours post FSE that predicted epilepsy in adulthood. This signal remains incompletely understood and hypothesized that it might derive from changes to vascular topology. Experimental FSE was induced in rat pups and compared to normothermic littermate controls. We examined cerebral vascular topology at 2 hours, using a novel vessel painting and analysis protocol. Blood vessel density of the cortical vasculature was significantly reduced in FSE rats, and this effect was lateralized, as reported for the MRI signal. The middle cerebral artery (MCA) exhibited abnormal topology in FSE pups but not in controls. In the BLA, significant vessel junction reductions and decreased vessel diameter were observed, together with a strong trend for reduced vessel length. In summary, FSE results in acute vascular topological changes in the cortex and BLA that may underlie the acute MRI signal that predicts progression to future epilepsy. The altered vasculature may be amenable to intervention treatments to potentially reduce the probability of progression to epilepsy following FSE.

Mental and cognitive health, as well as vulnerability to neuropsychiatric disorders, involve the interplay of genes with the environment, particularly during sensitive developmental periods. Early-life stress / adversity (ELA) promotes vulnerabilities to stress-related affective disorders, yet it is unknown how a transient ELA dictates life-long neuroendocrine and behavioral reactions to stress. The population of hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons that regulate stress-responses is a promising candidate to mediate the enduring influences of ELA on stress-related behavioral and hormonal responses via enduring transcriptional and epigenetic mechanisms. Capitalizing on a well-characterized model of ELA, we examined here the ELA-induced changes in gene expression profiles of stress-sensitive CRH-neurons in the hypothalamic paraventricular nucleus (PVN) of male mice. Given the known heterogeneity of these neuronal populations, we employed single-cell RNA sequencing (RNA-seq) approaches. The use of single-cell transcriptomics identified distinct CRH-expressing neuronal populations characterized by both their gene expression repertoire and their neurotransmitter profiles. Expression changes provoked by ELA clustered around genes involved in neuronal differentiation, synapse formation, altered energy metabolism and the cellular responses to stress and injury. Notably, the ELA-induced transcriptional changes took place primarily in subpopulations of glutamatergic CRH cells. Finally, ELA-induced transcriptional reprogramming of hypothalamic CRH-expressing neurons heralded significant, enduring disruptions of both hormonal and behavioral responses to stress throughout life.

Early life adversity (ELA) comprises a wide variety of negative experiences during early life and has been linked to cognitive impairments, reduced experiences of pleasure (anhedonia), and other long-term consequences implying that ELA impacts the reward circuitry. In this study, we focused on the projections from the dorsal raphe (DR) to the ventral tegmental area (VTA) and on to the nucleus accumbens (NAcc), an important pathway within the reward circuit. We hypothesized that ELA alters connectivity within the DR-VTA- NAcc pathway, manifested behaviorally as anhedonia in adulthood. We used the limited bedding and nesting model to induce ELA in mice and measured reward-related behaviors in adulthood using the three-chamber social interaction and sucrose preference tests. High resolution ex vivo diffusion tensor imaging (DTI) was acquired and processed for regional DTI metrics, including tractography to assess circuit organization. We found brain-wide changes in radial diffusivity (RD) and altered connectivity of the reward circuit in the ELA group. DR-VTA-NAcc circuit tractography and axial diffusivity (AD) along this tract exhibited dispersed organization where AD was increased in the VTA segment. Behaviorally, ELA elicited an anhedonic phenotype in adulthood with decreased direct social approach and time spent with peer but no overt differences in sucrose preference test. Our findings suggest that reward circuits, assessed using DTI, are altered following ELA and that these changes may drive enduring reward deficits.

The developmental origins of stress-related mental illnesses are well-established, and early-life stress/adversity (ELA) is an important risk factor. However, it is unclear how ELA impacts the maturation of salient brain circuits, provoking enduring vulnerability to stress and stress-related disorders. Here we find that ELA increases the number and function of excitatory synapses onto stress-sensitive hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons, and implicate disrupted synapse pruning by microglia as a key mechanism. Microglial process dynamics on live imaging, and engulfment of synaptic elements by microglia, were both attenuated in ELA mice, associated with deficient signaling of the microglial phagocytic receptor Mer. Accordingly, selective chemogenetic activation of ELA microglia increased microglial process dynamics and reduced excitatory synapse density to control levels. Selective early-life microglial activation also mitigated the adrenal hypertrophy and prolonged stress responses in adult ELA mice, establishing microglial actions during development as powerful contributors to experience-dependent sculpting of stress-related brain circuits.

Spatial memory, the aspect of memory involving encoding and retrieval of information regarding one's environment and spatial orientation, is a complex biological function incorporating multiple neuronal networks. Hippocampus-dependent spatial memory is not innate and emerges during development in both humans and rodents. In children, nonhippocampal dependent egocentric (self-to-object) memory develops before hippocampal-dependent allocentric (object-to-object) memory. The onset of allocentric spatial memory abilities in children around 22 mo of age occurs at an age-equivalent time in rodents when spatially tuned grid and place cells arise from patterned activity propagating through the entorhinal–hippocampal circuit. Neuronal activity, often driven by specific sensory signals, is critical for the normal maturation of brain circuits This patterned activity fine-tunes synaptic connectivity of the network and drives the emergence of specific firing necessary for spatial memory. Whereas normal activity patterns are required for circuit maturation, aberrant neuronal activity during development can have major adverse consequences, disrupting the development of spatial memory. Seizures during infancy, involving massive bursts of synchronized network activity, result in impaired spatial memory when animals are tested as adolescents or adults. This impaired spatial memory is accompanied by alterations in spatial and temporal coding of place cells. The molecular mechanisms by which early-life seizures lead to disruptions at the cellular and network levels are now becoming better understood, and provide a target for intervention, potentially leading to improved cognitive outcome in individuals experiencing early-life seizures.

The origins and neural bases of the current opioid addiction epidemic are unclear. Genetics plays a major role in addiction vulnerability, but cannot account for the recent exponential rise in opioid abuse, so environmental factors must contribute. Individuals with history of early-life adversity (ELA) are disproportionately prone to opioid addiction, yet whether ELA interacts with factors such as increased access to opioids to directly influence brain development and function and cause opioid addiction vulnerability is unknown. We simulated ELA in female rats and this led to a striking opioid addiction-like phenotype. This was characterized by resistance to extinction, increased relapse-like behavior, and, as in addicted humans, major increases in opioid economic demand. By contrast, seeking of a less salient natural reward was unaffected by ELA, whereas demand for highly palatable treats was augmented. These discoveries provide novel insights into the origins and nature of reward circuit malfunction that may set the stage for addiction.

In humans, early-life adversity (ELA) is associated with impairments in learning and memory that may emerge later in life. In rodent models, ELA directly impacts hippocampal neuron structure and connectivity with progressive deficits in long-term potentiation and spatial memory function. Previous work has demonstrated that augmented release and actions of the stress-activated neuropeptide, CRH, contribute to the deleterious effects of ELA on hippocampal structure and memory-function. Early-life adversity increases CRH production and levels, and blocking CRH receptor type 1 (CRHR1) within the hippocampus immediately following adversity prevented the memory and LTP problems caused by ELA. Here we queried if blocking CRHR1 during adulthood ameliorates the adverse impact of ELA on memory in middle age. Blocking CRHR1 for a week in two month old male rats prevented ELA-induced deficits in object recognition memory that emerge during middle age. The intervention failed to mitigate the reduction of spatial memory at 4 and 8 months, but restored hippocampus-dependent location memory in ELA-experiencing rats during middle age (12 months of age).Notably, neither ELA nor blocking CRHR1 influenced anxiety- or depression-related behaviors These findings suggest a sensitive period during which interventions can fully prevent long-lasting effects of ELA, yet indicate that interventions later in life offer significant benefits.

The amygdaloid complex, including the basolateral nucleus (BLA) contributes crucially to emotional and cognitive brain functions, and is thus a major target of research in both humans and rodents. However, delineating structural amygdala plasticity in both normal and disease-related contexts using neuroimaging has been hampered by the difficulty of unequivocally identifying the boundaries of the BLA. This challenge is a result of poor contrast between BLA and the surrounding gray matter, including other amygdala nuclei. Here we describe a novel DTI approach to enhance contrast, enabling optimal identification of BLA in rodent brain from MR images. We employed this methodology together with a slice-shifting approach to measure BLA volume. We then validated the results by direct comparison to both histological and cellular-identity (parvalbumin)-based conventional techniques for defining BLA in the same brains used for MRI. We also confirmed the BLA region using DTI based tractography. The novel approach used here enables accurate and reliable delineation of BLA. Because this nucleus is involved in, and is changed by, developmental, degenerative and adaptive processes, the instruments provided here should be highly useful to a broad range of neuroimaging studies. Finally, the principles used here are readily applicable to numerous brain regions and across species.Summary StatementUse of MRI directionally encoded diffusion tensor imaging (DTI) can delineate the basolateral amygdala (BLA) and volumes derived from DTI were found to match those obtained using histological methods. Our approach can be used to identify the BLA.

Repression of target genes by the transcriptional repressor neuronal restrictive silencing factor (NRSF)/repressor element 1 silencing transcription factor (REST) contributes to enduring plasticity in the developing brain. However, the cofactor(s) interacting with NRSF to enable target gene repressor are not well understood, and may vary among neuronal populations and brain regions as well as with different contexts. Here we employed the novel designer drug mS-11 to block the interactions of the cofactor Sin3 with NRSF. We tested if NRSF-Sin3 interaction is required for repression of NRSF target genes in developing hypothalamus after activity-dependent modulation of NRSF function. In the hypothalamus in vitro, blocking glutamatergic neurotransmission robustly increased NRSF binding to the target gene Crh, resulting in its repression. Blocking the binding of NRSF to the chromatin with decoy NRSE-oligodeoxynucleotides abrogated this repression. In contrast, mS-11 at several concentrations did not impede Crh repression. NRSF-mediated repression may underlie disease processes such as the onset of epilepsy. Therefore, identifying small-molecule antagonists of NRSF is crucial for the development of disease-preventing or modifying interventions.

BackgroundMaternal care during early-life plays a crucial role in the sculpting of the mammalian brain. Augmented maternal care during the first postnatal week promotes life-long stress resilience and improved memory compared with the outcome of routine rearing conditions. Recent evidence suggests that this potent phenotypic change commences with altered synaptic connectivity of stress sensitive hypothalamic neurons. However, the epigenomic basis of the long-lived consequences is not well understood.MethodsHere, we employed whole-genome bisulfite sequencing (WGBS), RNA-sequencing (RNA-seq), and a multiplex microRNA (miRNA) assay to examine the effects of augmented maternal care on DNA cytosine methylation, gene expression, and miRNA expression.ResultsA significant decrease in global DNA methylation was observed in offspring hypothalamus following a week of augmented maternal care, corresponding to differential methylation and expression of thousands of genes. Differentially methylated and expressed genes were enriched for functions in neurotransmission, neurodevelopment, protein synthesis, and oxidative phosphorylation, as well as known stress response genes. Twenty prioritized genes with three lines of evidence (methylation, expression, and altered miRNA target) were identified as highly relevant to the stress resiliency phenotype.ConclusionsThis combined unbiased approach enabled the discovery of novel genes and gene pathways that advance our understanding of the central epigenomic mechanisms underlying the profound effects of maternal care on the developing brain.

Epilepsy is a common brain disorder characterized by the occurrence of spontaneous seizures. These bursts of synchronous firing arise from abnormalities of neuronal networks. Excitability of individual neurons and neuronal networks is largely governed by ion channels and, indeed, abnormalities of a number of ion channels resulting from mutations or aberrant expression and trafficking underlie several types of epilepsy. Here, we focus on the hyperpolarization-activated cyclic nucleotide-gated ion (HCN) channels that conduct Ih current. This conductance plays complex and diverse roles in the regulation of neuronal and network excitability. We describe the normal function of HCN channels and discuss how aberrant expression, assembly, trafficking, and posttranslational modifications contribute to experimental and human epilepsy.