Experience-dependent neuroplasticity of the developing hypothalamus: integrative epigenomic approaches.

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 programming commences with altered synaptic connectivity of stress sensitive hypothalamic neurons. However, the epigenomic basis of the long-lived consequences is not well understood. Here, 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. A total of 9,439 differentially methylated regions (DMRs) associated with augmented maternal care were identified in male offspring hypothalamus, as well as a modest but significant decrease in global DNA methylation. 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 were identified as highly relevant to the stress resiliency phenotype. This combined unbiased approach enabled the discovery of novel genes and gene pathways that advance our understanding of the epigenomic mechanisms underlying the effects of maternal care on the developing brain.

Diversity of Reporter Expression Patterns in Transgenic Mouse Lines Targeting Corticotropin-Releasing Hormone-Expressing Neurons.

Transgenic mice, including lines targeting corticotropin-releasing factor (CRF or CRH), have been extensively employed to study stress neurobiology. These powerful tools are poised to revolutionize our understanding of the localization and connectivity of CRH-expressing neurons, and the crucial roles of CRH in normal and pathological conditions. Accurate interpretation of studies using cell type-specific transgenic mice vitally depends on congruence between expression of the endogenous peptide and reporter. If reporter expression does not faithfully reproduce native gene expression, then effects of manipulating unintentionally targeted cells may be misattributed. Here, we studied CRH and reporter expression patterns in 3 adult transgenic mice: Crh-IRES-Cre;Ai14 (tdTomato mouse), Crfp3.0CreGFP, and Crh-GFP BAC. We employed the CRH antiserum generated by Vale after validating its specificity using CRH-null mice. We focused the analyses on stress-salient regions, including hypothalamus, amygdala, bed nucleus of the stria terminalis, and hippocampus. Expression patterns of endogenous CRH were consistent among wild-type and transgenic mice. In tdTomato mice, most CRH-expressing neurons coexpressed the reporter, yet the reporter identified a few non-CRH-expressing pyramidal-like cells in hippocampal CA1 and CA3. In Crfp3.0CreGFP mice, coexpression of CRH and the reporter was found in central amygdala and, less commonly, in other evaluated regions. In Crh-GFP BAC mice, the large majority of neurons expressed either CRH or reporter, with little overlap. These data highlight significant diversity in concordant expression of reporter and endogenous CRH among 3 available transgenic mice. These findings should be instrumental in interpreting important scientific findings emerging from the use of these potent neurobiological tools.

Differential contribution of CBP:CREB binding to corticotropin-releasing hormone expression in the infant and adult hypothalamus.

Corticotropin-releasing hormone (CRH) contributes crucially to the regulation of central and peripheral responses to stress. Because of the importance of a finely tuned stress system, CRH expression is tightly regulated in an organ- and brain region-specific manner. Thus, in the hypothalamus, CRH is constitutively expressed and this expression is further enhanced by stress; however, the underlying regulatory mechanisms are not fully understood. The regulatory region of the crh gene contains several elements, including the cyclic-AMP response element (CRE), and the role of the CRE interaction with the cyclic-AMP response element binding protein (CREB) in CRH expression has been a focus of intensive research. Notably, whereas thousands of genes contain a CRE, the functional regulation of gene expression by the CRE:CREB system is limited to ∼100 genes, and likely requires additional proteins. Here, we investigated the role of a member of the CREB complex, CREB binding protein (CBP), in basal and stress-induced CRH expression during development and in the adult. Using mice with a deficient CREB-binding site on CBP, we found that CBP:CREB interaction is necessary for normal basal CRH expression at the mRNA and protein level in the nine-day-old mouse, prior to onset of functional regulation of hypothalamic CRH expression by glucocorticoids. This interaction, which functions directly on crh or indirectly via regulation of other genes, was no longer required for maintenance of basal CRH expression levels in the adult. However, CBP:CREB binding contributed to stress-induced CRH expression in the adult, enabling rapid CRH synthesis in hypothalamus. CBP:CREB binding deficiency did not disrupt basal corticosterone plasma levels or acute stress-evoked corticosterone release. Because dysregulation of CRH expression occurs in stress-related disorders including depression, a full understanding of the complex regulation of this gene is important in both health and disease.

Postnatal expression pattern of HCN channel isoforms in thalamic neurons: relationship to maturation of thalamocortical oscillations.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (I(h)). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and I(h) properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in I(h) density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the I(h) activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on I(h) conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I(h) density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I(h) properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

A novel mouse model for acute and long-lasting consequences of early life stress.

Chronic early-life stress (ES) exerts profound acute and long-lasting effects on the hypothalamic-pituitary-adrenal system, with relevance to cognitive function and affective disorders. Our ability to determine the molecular mechanisms underlying these effects should benefit greatly from appropriate mouse models because these would enable use of powerful transgenic methods. Therefore, we have characterized a mouse model of chronic ES, which was provoked in mouse pups by abnormal, fragmented interactions with the dam. Dam-pup interaction was disrupted by limiting the nesting and bedding material in the cages, a manipulation that affected this parameter in a dose-dependent manner. At the end of their week-long rearing in the limited-nesting cages, mouse pups were stressed, as apparent from elevated basal plasma corticosterone levels. In addition, steady-state mRNA levels of CRH in the hypothalamic paraventricular nucleus of ES-experiencing pups were reduced, without significant change in mRNA levels of arginine vasopressin. Rearing mouse pups in this stress-provoking cage environment resulted in enduring effects: basal plasma corticosterone levels were still increased, and CRH mRNA levels in paraventricular nucleus remained reduced in adult ES mice, compared with those of controls. In addition, hippocampus-dependent learning and memory functions were impaired in 4- to 8-month-old ES mice. In summary, this novel, robust model of chronic early life stress in the mouse results in acute and enduring neuroendocrine and cognitive abnormalities. This model should facilitate the examination of the specific genes and molecules involved in the generation of this stress as well as in its consequences.

Functional stabilization of weakened thalamic pacemaker channel regulation in rat absence epilepsy.

Aberrant function of pacemaker currents (Ih), carried by hyperpolarization-activated cation non-selective (HCN) channels, affects neuronal excitability and accompanies epilepsy, but its distinct roles in epileptogenesis and chronic epilepsy are unclear. We probed Ih function and subunit composition during both pre- and chronically epileptic stages in thalamocortical (TC) neurones of the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). Voltage gating of Ih was unaltered in mature somatosensory TC cells, both in vivo and in vitro. However, the enhancement of Ih by phasic, near-physiological, cAMP pulses was diminished by approximately 40% and the half-maximal cAMP concentration increased by approximately 5-fold. This decreased responsiveness of Ih to its major cellular modulator preceded epilepsy onset in GAERS, persisted throughout the chronic state, and was accompanied by an enhanced expression of the cAMP-insensitive HCN1 channel mRNA (> 50%), without changes in the mRNA levels of HCN2 and HCN4. To assess for alterations in TC cell excitability, we monitored the slow up-regulation of Ih that is induced by Ca2+-triggered cAMP synthesis and important for terminating in vitro synchronized oscillations. Remarkably, repetitive rebound Ca2+ spikes evoked normal slow Ih up-regulation in mature GAERS neurones; that sufficed to attenuate spontaneous rhythmic burst discharges. These adaptive mechanisms occurred upstream of cAMP turnover and involved enhanced intracellular Ca2+ accumulation upon repetitive low-threshold Ca2+ discharges. Therefore, HCN channels appear to play a dual role in epilepsy. Weakened cAMP binding to HCN channels precedes, and likely promotes, epileptogenesis in GAERS, whereas compensatory mechanisms stabilizing Ih function contribute to the termination of spike-and-wave discharges in chronic epilepsy.

Hippocampal neurogenesis is not enhanced by lifelong reduction of glucocorticoid levels.

Neurogenesis of dentate gyrus granule cells is generally considered to be negatively regulated by glucocorticoids. We tested the hypothesis that exposure to low plasma corticosteroid levels starting in the early postnatal period enhances granule cell proliferation rate during adulthood. Rat pups were adrenalectomized (ADX) on postnatal day 10 and were then "clamped" throughout life at low corticosterone levels via oral supplementation. Neurogenesis was determined using BrdU immunochemistry at 3 and 12 months in clamped rats as compared with age-matched, sham-operated controls. Rate of neurogenesis did not differ between the groups at either 3 or 12 months. It was significantly lower in 12-month-old compared with 3-month-old rats, despite the presence of an age-dependent increase of plasma corticosterone only in the sham-ADX rats. Granule cell layer volume, granule cell density, and granule cell degeneration (determined using apoptotic markers) were indistinguishable in the two groups, further supporting the comparable rate of neurogenesis under differing chronic glucocorticoid levels. In addition, whereas acute deprivation of plasma glucocorticoids (adrenalectomy) in adult rats evoked a burst of granule cell neurogenesis, complete elimination of these hormones (by stopping hormone supplementation) in adult, early-life ADX/clamped rats did not. These data do not support a simple inverse relationship between chronic plasma glucocorticoid levels and granule cell neurogenesis. Specifically, chronic modulation of glucocorticoid levels commencing early in life evokes additional, adaptive, and compensatory mechanisms that contribute to the regulation of granule cell proliferation.

Developmental febrile seizures modulate hippocampal gene expression of hyperpolarization-activated channels in an isoform- and cell-specific manner.

Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (I(H)). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.