The nucleus reuniens: a key node in the neurocircuitry of stress and depression.

The hippocampus and prefrontal cortex (PFC) are connected in a reciprocal manner: whereas the hippocampus projects directly to the PFC, a polysynaptic pathway that passes through the nucleus reuniens (RE) of the thalamus relays inputs from the PFC to the hippocampus. The present study demonstrates that lesioning and/or inactivation of the RE reduces coherence in the PFC-hippocampal pathway, provokes an antidepressant-like behavioral response in the forced swim test and prevents, but does not ameliorate, anhedonia in the chronic mild stress (CMS) model of depression. Additionally, RE lesioning before CMS abrogates the well-known neuromorphological and endocrine correlates of CMS. In summary, this work highlights the importance of the reciprocal connectivity between the hippocampus and PFC in the establishment of stress-induced brain pathology and suggests a role for the RE in promoting resilience to depressive illness.

Mechanisms of initiation and reversal of drug-seeking behavior induced by prenatal exposure to glucocorticoids.

Stress and exposure to glucocorticoids (GC) during early life render individuals vulnerable to brain disorders by inducing structural and chemical alterations in specific neural substrates. Here we show that adult rats that had been exposed to in utero GCs (iuGC) display increased preference for opiates and ethanol, and are more responsive to the psychostimulatory actions of morphine. These animals presented prominent changes in the nucleus accumbens (NAcc), a key component of the mesolimbic reward circuitry; specifically, cell numbers and dopamine (DA) levels were significantly reduced, whereas DA receptor 2 (Drd2) mRNA expression levels were markedly upregulated in the NAcc. Interestingly, repeated morphine exposure significantly downregulated Drd2 expression in iuGC-exposed animals, in parallel with increased DNA methylation of the Drd2 gene. Administration of a therapeutic dose of L-dopa reverted the hypodopaminergic state in the NAcc of iuGC animals, normalized Drd2 expression and prevented morphine-induced hypermethylation of the Drd2 promoter. In addition, L-dopa treatment promoted dendritic and synaptic plasticity in the NAcc and, importantly, reversed drug-seeking behavior. These results reveal a new mechanism through which drug-seeking behaviors may emerge and suggest that a brief and simple pharmacological intervention can restrain these behaviors in vulnerable individuals.

Sex Hormone Depletion Augments Glucocorticoid Induction of Tau Hyperphosphorylation in Male Rat Brain.

Steroid hormones secreted by the gonads (sex steroids) and adrenal glands (glucocorticoids, GC) are known to influence brain structure and function. While levels of sex steroids wane in late adulthood, corticosteroid levels tend to rise in many individuals due to age-related impairments in their feedback on central mechanisms regulating adrenal function. These fluctuations in sex and adrenal steroid secretion may be relevant to age-related neurodegenerative disorders such as Alzheimer's disease (AD) in which hyperphosphorylation of Tau protein is a key pathological event. We here report that both, long-term GC deprivation (by adrenalectomy) and exogenous GC administration with natural or synthetic glucocorticoid receptor ligands (corticosterone and dexamethasone, respectively) induce Tau hyperphosphorylation in the hippocampus and frontocortical regions at epitopes associated with disruption of cytoskeletal and synaptic function. Interestingly, we observed that the changes in Tau induced by manipulation of the GC milieu of male rats were exacerbated by testosterone depletion (by orchiectomy). While this finding supports previous suggestions of a neuroprotective role of male sex hormones, this is the first study to address interactions between adrenal and sex steroids on Tau hyperphosphorylation and accumulation that are known to endanger neuronal function and plasticity. These results are particularly important for understanding the mechanisms that can precipitate AD because, besides being modulated by age, GC are elevated by stress, a phenomenon now established as a trigger of deficits in neural plasticity and survival, cognitive behaviour and AD-like Tau pathology.

The amyloidogenic potential and behavioral correlates of stress.

Observations of elevated basal cortisol levels in Alzheimer's disease (AD) patients prompted the hypothesis that stress and glucocorticoids (GC) may contribute to the development and/or maintenance of AD. Consistent with that hypothesis, we show that stress and GC provoke misprocessing of amyloid precursor peptide in the rat hippocampus and prefrontal cortex, resulting in increased levels of the peptide C-terminal fragment 99 (C99), whose further proteolytic cleavage results in the generation of amyloid-beta (Abeta). We also show that exogenous Abeta can reproduce the effects of stress and GC on C99 production and that a history of stress strikingly potentiates the C99-inducing effects of Abeta and GC. Previous work has indicated a role for Abeta in disruption of synaptic function and cognitive behaviors, and AD patients reportedly show signs of heightened anxiety. Here, behavioral analysis revealed that like stress and GC, Abeta administration causes spatial memory deficits that are exacerbated by stress and GC; additionally, Abeta, stress and GC induced a state of hyperanxiety. Given that the intrinsic properties of C99 and Abeta include neuroendangerment and behavioral impairment, our findings suggest a causal role for stress and GC in the etiopathogenesis of AD, and demonstrate that stressful life events and GC therapy can have a cumulative impact on the course of AD development and progression.

The mood-improving actions of antidepressants do not depend on neurogenesis but are associated with neuronal remodeling.

The mechanisms underlying the initiation/onset of, and the recovery from, depression are still largely unknown; views that neurogenesis in the hippocampus may be important for the pathogenesis and amelioration of depressive symptoms have gained currency over the years although the original evidence has been challenged. In this study, an unpredictable chronic mild stress protocol was used to induce a depressive-like phenotype in rats. In the last 2 weeks of stress exposure, animals were treated with the antidepressants fluoxetine, imipramine, CP 156,526 or SSR 1494515, alone or combined with methylazoxymethanol, a cytostatic agent used to arrest neurogenesis. We found that antidepressants retain their therapeutic efficacy in reducing both measured indices of depression-like behavior (learned helplessness and anhedonia), even when neurogenesis is blocked. Instead, our experiments suggest re-establishment of neuronal plasticity (dendritic remodeling and synaptic contacts) in the hippocampus and prefrontal cortex, rather than neurogenesis, as the basis for the restoration of behavioral homeostasis by antidepressants.

Melatonin: a coordinating signal for mammalian reproduction?

There is a daily rhythm in the production of the pineal hormone melatonin in all mammalian species. Production is stimulated by darkness and inhibited by light. This provides a signal reflecting the changing environmental lighting cycle. In seasonally breeding mammals that use changes in the photoperiod to time their reproductive cycles, temporal signals to the reproductive system are controlled by the daily rhythm in melatonin production.

Neuroplasticity-related correlates of environmental enrichment combined with physical activity differ between the sexes.

Environmental enrichment (EE), comprising positive physical (exercise) and cognitive stimuli, influences neuronal structure and usually improves brain function. The promise of EE as a preventative strategy against neuropsychiatric disease is especially high during early postnatal development when the brain is still amenable to reorganization. Despite the fact that male and female brains differ in terms of connectivity and function that may reflect early life experiences, knowledge of the neural substrates and mechanisms by which such changes arise remains limited. This study compared the impact of EE combined with physical activity on neuroplasticity and its functional consequences in adult male and female rats; EE was provided during the first 3 months of life and our analysis focused on the hippocampus, an area implicated in cognitive behavior as well as the neuroendocrine response to stress. Both male and female rats reared in EE displayed better object recognition memory than their control counterparts. Interestingly, sex differences were revealed in the effects of EE on time spent exploring the objects during this test. Independently of sex, EE increased hippocampal turnover rates of dopamine and serotonin and reduced expression of 5-HT1A receptors; in addition, EE upregulated expression of synaptophysin, a presynaptic protein, in the hippocampus. As compared to their respective controls, EE-exposed males exhibited parallel increases in phosphorylated Tau and the GluN2B receptor, whereas females responded to EE with reduced hippocampal levels of glutamate and GluN2B. Together, these observations provide further evidence on the differential effects of EE on markers of hippocampal neuroplasticity in males and females.

Stress and glucocorticoid footprints in the brain-the path from depression to Alzheimer's disease.

Increasingly, stress is recognized as a trigger of depressive episodes and recent evidence suggests a causal role of stress in the onset and progression of Alzheimer's disease (AD) pathology. Besides aging, sex is an important determinant of prevalence rates for both AD and mood disorders. In light of a recent meta-analysis indicating that depressed subjects have a higher likelihood of developing AD, a key message in this article will be that both depression and AD are stress-related disorders and may represent a continuum that should receive more attention in future neurobiological studies. Accordingly, this review considers some of the cellular mechanisms that may be involved in regulating this transition threshold. In addition, it highlights the importance of addressing the question of how aging and sex interplay with stress to influence mood and cognition, with a bias towards consideration of neuroplastic events in particular brain regions, as the basis of AD and depressive disorders.

Dissociation of the morphological correlates of stress-induced anxiety and fear.

Chronic stress is a powerful modulator of emotional behaviour. Previous studies have shown that distinct neuronal pathways modulate different emotional behaviours: while the amygdala plays a key role in fear-conditioned-to-cue stimuli, the bed nucleus of stria terminalis (BNST) is implicated in anxiety behaviour and responses to contextual stimuli. In addition, the BNST is directly involved in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis. In the present study, we assessed anxiety (measured in the elevated-plus maze and acoustic startle apparatus) and fear-conditioned responses to light stimuli in rats that had been exposed to either chronic unpredictable stress or corticosterone for 28 days; thereafter, stereological estimates of the BNST and amygdaloid complex were performed, followed by three-dimensional morphometric dendritic analysis. Results show that chronic stress induces hyperanxiety without influencing fear conditioning or locomotion and exploratory activity. Stress-induced hyperanxiety was correlated with increased volumes of the BNST but not of the amygdala. Dendritic remodelling was found to make a significant contribution to the stress-induced increase in BNST volume, primarily due to changes in the anteromedial area of the BNST, an area strongly implicated in emotional behaviour and in the neuroendocrine control of the stress response. Importantly, all of the effects of stress were recapitulated by exogenous corticosterone. In conclusion, this study shows that chronic stress impacts on BNST structure and function; its findings pertain to the modulation of emotional behaviour and the maladaptive response to stress.

Lithium blocks stress-induced changes in depressive-like behavior and hippocampal cell fate: the role of glycogen-synthase-kinase-3beta.

Mood disorders are the most common psychiatric disorders. Although the mechanisms implicated in the genesis of mood disorders are still unclear, stress is known to predispose to depression, and recently, studies have related hippocampal neurogenesis and apoptosis to depression. In the present study we first examined the balance between cell birth-death in the hippocampus and subventricular zone (SVZ) of pre-pubertal and adult rats subjected to chronic-mild-stress (CMS). CMS led to increased corticosterone secretion and induced depressive-like symptoms (assessed in the forced-swimming test); these endocrine and behavioral effects were paralleled by decreased hippocampal, but not SVZ, cell proliferation/differentiation and by increased apoptotic rate. In order to determine if lithium, a known mood stabilizer with antidepressant properties, could prevent the stress-induced events, we analyzed the same parameters in a group of rats treated with lithium during the stress exposure period (CMS+Li) and observed that the hormonal, behavioral and cell turnover effects of CMS were abrogated in these animals. Subsequently, to search for possible pathways through which CMS and lithium influence behavior, cell fate and synaptic plasticity, we analyzed the expression of glycogen-synthase-kinase-3beta (GSK-3beta), as well as some of its downstream targets (B-cell-CLL/lymphoma2-associated athanonege (BAG-1) and synapsin-I). CMS increased GSK-3beta and decreased synapsin-I and BAG-1 expression in the hippocampus. Interestingly, co-administration of lithium precluded the CMS-induced effects in GSK-3beta, synapsin-I and BAG-1 expression. Our observation that specific inhibition of this kinase with AR-A014418 blocked the effects of CMS in depressive-like behavior and in BAG-1 and synapsin-I expression confirmed the involvement of the GSK-3beta pathway in stress-induced effects. In summary, these results reveal that lithium, by regulating the activity of GSK-3beta, prevents the deleterious effects of stress on behavior and cellular functions.

Lactation as a model for naturally reversible hypercorticalism plasticity in the mechanisms governing hypothalamo-pituitary- adrenocortical activity in rats.

Steady state levels of hypothalamic expression of the genes encoding corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), arginine vasopressin (AVP), and oxytocin (OT) were studied in rats to investigate the mechanisms underlying the transitions between hypercorticalism during lactation and normocorticalism upon weaning. During lactation, CRH mRNA levels and blood titers of adrenocorticotropin (ACTH) were found to be significantly reduced, although POMC mRNA levels in the anterior pituitary were not significantly different from those found in cycling virgin (control) rats; during all phases of lactation, an inverse relationship was observed between the blood levels of ACTH and corticosterone (CORT). Plasma prolactin (PRL) concentrations were elevated approximately 30-fold during lactation. Whereas steady state levels of OT mRNA were markedly increased throughout lactation, those of AVP mRNA were only transiently (initially) elevated, and the blood levels of these hormones were not significantly altered in lactating as compared with cycling virgin and postlactating rats. CRH and POMC gene expression and blood levels of ACTH, CORT, and PRL were normalized within 1-3 d of removal of suckling pups. The temporal relationships between the biosynthetic profiles of the various peptide hormones and the patterns of ACTH and CORT secretion during the two physiological states suggest that lactation-associated hypercorticalism does not merely result from increased ACTH secretion; although still not well substantiated at this time, the evidence points to contributory roles of PRL, OT, and AVP in the hypercorticalismic state found during lactation.

Gender differences in ethanol preference and ingestion in rats. The role of the gonadal steroid environment.

An ethanol oral self administration paradigm showed the existence of gender differences in alcohol preference in rats: whereas males and females initiated alcohol drinking at similar rates, females maintained their preference for ethanol over a longer duration. Neonatal estrogenization of females, which effectively confers a male phenotype on a genetically female brain, resulted in patterns of drinking that were similar to those displayed by intact male rats, indicating that gender differences in alcohol drinking patterns may be, at least partially, accounted for by sexual differentiation of the brain. To test whether gonadal steroids also exert activational effects on ethanol-seeking behavior, we also examined the effects of gonadectomy alone, or in combination with gonadal steroid replacement therapy. Castration did not significantly alter ethanol consumption in males, although treatment of castrated rats with dihydrotestosterone resulted in a significant inhibition of this parameter. As compared with the situation in intact female rats, ethanol ingestion was significantly reduced in ovariectomized female rats receiving estradiol (E2) and in ovariectomized female rats receiving combined E2 and progesterone replacement therapy. However, neither ovariectomy nor progesterone replacement in ovariectomized rats resulted in ethanol drinking patterns that were different compared to those observed in intact female controls. Thus, dihydrotestosterone and E2, respectively, appear to exert modulatory influences on the male and female rats' preference for ethanol, but further investigations are necessary to determine to what extent these effects result from activational actions on the brain.

Neonatal treatment of rats with the neuroactive steroid tetrahydrodeoxycorticosterone (THDOC) abolishes the behavioral and neuroendocrine consequences of adverse early life events.

Stressful experience during early brain development has been shown to produce profound alterations in several mechanisms of adaptation, while several signs of behavioral and neuroendocrine impairment resulting from neonatal exposure to stress resemble symptoms of dysregulation associated with major depression. This study demonstrates that when applied concomitantly with the stressful challenge, the steroid GABA(A) receptor agonist 3,21-dihydropregnan-20-one (tetrahydrodeoxycorticosterone, THDOC) can attenuate the behavioral and neuroendocrine consequences of repeated maternal separation during early life, e.g., increased anxiety, an exaggerated adrenocortical secretory response to stress, impaired responsiveness to glucocorticoid feedback, and altered transcription of the genes encoding corticotropin-releasing hormone (CRH) in the hypothalamus and glucocorticoid receptors in the hippocampus. These data indicate that neuroactive steroid derivatives with GABA-agonistic properties may exert persisting stress-protective effects in the developing brain, and may form the basis for therapeutic agents which have the potential to prevent mental disorders resulting from adverse experience during neonatal life.

Neurodevelopment milestone abnormalities in rats exposed to stress in early life.

Manipulation of the corticosteroid milieu by interfering with the mother-newborn relationship has received much attention because of its potential bearing on psychopathology later in life. In the present study, infant rats that were deprived of maternal contact between the 2nd and the 15th postnatal days (MS2-15) for 6 h/day were subjected to a systematic assessment of neurodevelopmental milestones between postnatal days 2 and 21. The analyses included measurements of physical growth and maturation and evaluation of neurological reflexes. Although some somatic milestones (e.g. eye opening) were anticipated, MS2-15 animals showed retardation in the acquisition of postural reflex, air righting and surface righting reflexes, and in the wire suspension test; the latter two abnormalities were only found in males. A gender effect was also observed in negative geotaxis, with retardation being observed in females but not males. To better understand the delay of neurological maturation in MS2-15 rats, we determined the levels of various monoamines in different regions of the brain stem, including the vestibular area, the substantia nigra, ventral tegmental area and dorsal raphe nuclei. In the vestibular region of MS2-15 rats the levels of 5-HT were reduced, while 5-HT turnover was increased. There was also a significant increase of the 5-HT turnover in MS2-15 animals in the raphe nuclei, mainly due to increased 5-hydroxyindoleacetic acid (5-HIAA) levels, and an increase of 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the ventral tegmental area (VTA) of stressed females. No significant differences were found in the immunohistochemical sections for tyrosine and tryptophan hydroxylase in these regions of the brain stem. In conclusion, the present results show that postnatal stress induces signs of neurological pathology that may contribute to the genesis of behavioral abnormalities later in life.

Sumoylation and proteasomal activity determine the transactivation properties of the mineralocorticoid receptor.

MR is a hormone-activated transcription factor that carries a strong synergy inhibitory function at its N-terminus. Using this region as bait in a yeast two-hybrid screening, we isolated major components of the sumoylation pathway, including the SUMO-1-conjugating enzyme Ubc9, and SUMO-1 itself. We found that MR interacts with both, Ubc9 and SUMO-1 in mammalian cells, and that the receptor is sumoylated at four acceptor sites which are clustered within its AF-1 domain. We observed that MR can be poly-ubiquitinated and that proteasome activity is essential for MR-activated transcription. Disruption of the SUMO-1 attachment sites abolished MR sumoylation but interfered with neither the poly-ubiquitination of the receptor nor its transactivation potential on MMTV. However, the hormone-activated mutant displayed enhanced synergistic potential on a compound promoter and delayed mobility in the nucleus. FRAP analysis further showed that proteasome inhibition immobilizes a subpopulation of unliganded MR receptors in the nucleus, a phenomenon that is significantly attenuated in the presence of aldosterone. Interestingly, the ability of the hormone to counteract the immobilizing effect of MG132 requires the sumoylation-competent form of MR. Moreover, increasing exogenously SUMO-1 cellular levels resulted in a selective, dose-dependent inhibition of the activity of the sumoylation-deficient MR. This effect was observed only on a synergy-competent promoter, revealing a mode for negative regulation of synergy that might involve sumoylation of factors different from MR. The data suggest that the overall transcriptional activity of MR can be modulated by its sumoylation potential as well as the sumoylation level of MR-interacting proteins, and requires the continuous function of the proteasome.

Antidepressant responsiveness in adulthood is permanently impaired after neonatal destruction of the neurogenic pool.

The dynamic turnover of hippocampal neurons is implicated in the regulation of cognitive and affective behavior. Extending our previous demonstration that administration of dexamethasone (ND) to neonatal rats depletes the resident population of neural precursor cells (NPC) and restrains the size of the neurogenic regions, we now show that the adverse effects of ND persist into adulthood. Specifically, ND impairs repletion of the neurogenic pool and neurogenesis; ND also compromises cognitive performance, the ability to actively adapt to an acute stressor and, the efficacy of glucocorticoid (GC) negative feedback. Interestingly, although ND depletes the neurogenic pool, it does not permanently abolish the proliferative machinery of the residual NPC population; however, ND increases the susceptibility of hippocampal granule neurons to apoptosis. Although the antidepressant fluoxetine (FLX) reverses the latter phenomenon, it does not replenish the NPC pool. Treatment of ND-treated adult rats with FLX also improves GC negative feedback, albeit without rescuing the deleterious effects of ND on behavior. In summary, ND leads to protracted disruption of mental functions, some of which are resistant to antidepressant interventions. We conclude that manipulation of the NPC pool during early life may jeopardize the therapeutic potential of antidepressants in adulthood.

Soluble beta-amyloid1-40 induces NMDA-dependent degradation of postsynaptic density-95 at glutamatergic synapses.

Amyloid-beta (Abeta) has been implicated in memory loss and disruption of synaptic plasticity observed in early-stage Alzheimer's disease. Recently, it has been shown that soluble Abeta oligomers target synapses in cultured rat hippocampal neurons, suggesting a direct role of Abeta in the regulation of synaptic structure and function. Postsynaptic density-95 (PSD-95) is a postsynaptic scaffolding protein that plays a critical role in synaptic plasticity and the stabilization of AMPA (AMPARs) and NMDA (NMDARs) receptors at synapses. Here, we show that exposure of cultured cortical neurons to soluble oligomers of Abeta(1-40) reduces PSD-95 protein levels in a dose- and time-dependent manner and that the Abeta1(1-40)-dependent decrease in PSD-95 requires NMDAR activity. We also show that the decrease in PSD-95 requires cyclin-dependent kinase 5 activity and involves the proteasome pathway. Immunostaining analysis of cortical cultured neurons revealed that Abeta treatment induces concomitant decreases in PSD-95 at synapses and in the surface expression of the AMPAR glutamate receptor subunit 2. Together, these data suggest a novel pathway by which Abeta triggers synaptic dysfunction, namely, by altering the molecular composition of glutamatergic synapses.

A hitchhiker's guide to behavioral analysis in laboratory rodents.

Genes and environment are both essential and interdependent determinants of behavioral responses. Behavioral genetics focuses on the role of genes on behavior. In this article, we aim to provide a succinct, but comprehensive, overview of the different means through which behavioral analysis may be performed in rodents. We give general recommendations for planning and performing behavioral experiments in rats and mice, followed by brief descriptions of experimental paradigms most commonly used for the analysis of reflexes, sensory function, motor function and exploratory, social, emotional and cognitive behavior. We end with a discussion of some of the shortcomings of current concepts of genetic determinism and argue that the genetic basis of behavior should be analyzed in the context of environmental factors.

Mismatch between anxiety status and morphometric parameters in the amygdala and bed nucleus of the stria terminalis.

Aging is associated with behavioral changes, including increased anxiety. In this study we confirmed a hyperanxious status in aged animals, measured in the elevated-plus maze and in the acoustic startle. Subsequently, we searched for age-related changes in the volume and cell numbers in the amygdala or in the bed nucleus of the stria terminalis, but failed to detect gross structural changes in these two brain areas, both implicated in emotionality.

Corticosteroid status influences the volume of the rat cingulate cortex - a magnetic resonance imaging study.

Imbalances in the corticosteroid milieu result in reductions in hippocampal volume in humans and experimental rodents. The functional correlates of these changes include deficits in cognitive performance and regulation of the hypothalamic-pituitary-adrenal axis. Since other limbic structures which are intricately connected with the hippocampal formation, also play an important role in behavioural and neuroendocrine functions, we here used magnetic resonance imaging (MRI) to analyse how two of these areas, the anterior cingulate and retrosplenial cortex, respond to chronic alterations of adrenocortical status: hypocortisolism (induced by adrenalectomy, ADX), normocortisolism (ADX with low-dose corticosterone replacement), and hypercortisolism (ADX with high-dose dexamethasone supplementation). Hypercortisolism was associated with a significant reduction in the volume (absolute and normalized) of the left anterior cingulate gyrus as measured by MRI and confirmed using classical histological methods; a similar trend was observed in the right anterior cingulate region. In contrast, hypercortisolism did not influence the volume of the adjacent retrosplenial cortex. The volumes of the anterior cingulate gyrus and retrosplenial cortex were unaffected by the absence of adrenocortical hormones. These findings are the first to suggest that corticosteroid influences on the structure of the limbic system extend beyond the hippocampal formation, i.e., to fronto-limbic areas also.