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The brain is the central organ of stress and adaptation to social and physical stressors because it determines what is threatening, stores memories, and regulates the physiological as well as behavioral responses to stressors that may be damaging or protective1. The physi- ological responses that produce adaptation through ‘allostasis’ include not only the hypothalamic-pituitary-adrenal (HPA) axis and the auto- nomic nervous system, but also their nonlinear interactions with the metabolic system and the pro- and anti-inflammatory components of the immune defense system1,2. Exposure to multiple stressors and the dysregulation of the nonlinear interactions (such as failure to turning on or off responses efficiently) lead to wear and tear on the body and brain that is termed allostatic load and overload1,3.

Allostasis is the active process of adapting to stressors via mediators such as cortisol and the autonomic, metabolic and immune system that act together in a nonlinear fashion to maintain homeostasis2. Allostatic load refers to the cumulative effect of multiple stressors as well as the dysregulation of the nonlinear network of allostasis (for example, production of cortisol, adrenalin or inflammation in response to a challenge). Allostatic overload refers to the cumulative pathophysiology that can result from this dysregulation and excess stress. Allostasis, and allostatic load and overload, are more precise biological concepts than ‘stress’ to describe adaptation and mal- adaptation to ‘stressors’, and they include the physiological effects of health-promoting and health-damaging behaviors as well as stressful experiences1,2. Health behaviors (such as smoking, alcohol, poor diet, or lack of sleep), resulting from the experience of stress, also have a role and contribute to allostatic load and overload1,3.

‘Stress’ can be divided into ‘good stress’, ‘tolerable stress’ and ‘toxic stress’4. Early life stress can alter neural architecture to increase adverse reactions to stressors, leading to toxic stress4. ‘Biological embedding’4,5 of these effects during critical or sensitive periods of early development has lasting effects through the life course6,7. Among the most important early life experiences are those that involve abuse and neglect, on the one hand, versus the establishment of strong, positive attachment of child to caregiver; these alter the ability of the individual to engage in cooperative social experiences or to feel excluded and hostile to the social environment later in life8.

The brain is a target of stressful experiences, and glucocorticoids, along with excitatory amino acid neurotransmitters, alter neuro- nal architecture by causing dendritic retraction or expansion and decreased or increased synapse density, depending on the brain region, along with inhibition of dentate gyrus neurogenesis9–11. Many intra- and intercellular mediators and processes are involved in changing the brain during stress and recovery from stressful experiences12,13 (Box 1).

This Review provides an overview of the mechanisms and media- tors through which stressors alter brain structure and function. It does so by focusing primarily on three brain regions, the hippocampus, amygdala and prefrontal cortex (PFC), although in full recognition of the fact that stress has widespread effects throughout the brain. This Review also emphasizes the complex nonlinear interactions between different stress mediators that are central to the concept of allostasis and allostatic load and overload3, in which nonlinearity applies not only to systemic hormones but also to intra- and extra- cellular mediators in the brain. Because of this, the many changes caused by stress often result in an inverted-U-shaped dose-response relationship, as represented in Figure 1.

Mechanisms underlying stress effects on the brain Stressors alter gene expression through multiple mechanisms, includ- ing direct effects of glucocorticoids on gene transcription as well as the activation of epigenetic mechanisms in which histone modifica- tions and methylation and hydroxymethylation of CpG residues in

1Laboratory of Neuroendocrinology, The Rockefeller University, New York, New York, USA. 2Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada. 3Department of Psychology, University of Massachusetts Boston, Boston, Massachusetts, USA. 4Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, Washington, USA. Correspondence should be addressed to B.S.M. ([email protected]).

Received 19 April; accepted 8 July; published online 25 September 2015; doi:10.1038/nn.4086

Mechanisms of stress in the brain Bruce S McEwen1, Nicole P Bowles1, Jason D Gray1, Matthew N Hill2, Richard G Hunter3, Ilia N Karatsoreos4 & Carla Nasca1

The brain is the central organ involved in perceiving and adapting to social and physical stressors via multiple interacting mediators, from the cell surface to the cytoskeleton to epigenetic regulation and nongenomic mechanisms. A key result of stress is structural remodeling of neural architecture, which may be a sign of successful adaptation, whereas persistence of these changes when stress ends indicates failed resilience. Excitatory amino acids and glucocorticoids have key roles in these processes, along with a growing list of extra- and intracellular mediators that includes endocannabinoids and brain-derived neurotrophic factor (BDNF). The result is a continually changing pattern of gene expression mediated by epigenetic mechanisms involving histone modifications and CpG methylation and hydroxymethylation as well as by the activity of retrotransposons that may alter genomic stability. Elucidation of the underlying mechanisms of plasticity and vulnerability of the brain provides a basis for understanding the efficacy of interventions for anxiety and depressive disorders as well as age-related cognitive decline.

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DNA have roles leading to repression and activation of genetic fac- tors, including retrotransposons14,15. Glucocorticoids are not the sole mediators of these effects, in which excitatory amino acids and many other cellular mediators also play important parts (Box 1). These mediators span influences from extracellular adhesion molecules to cytoskeletal elements and at least one nuclear pore complex protein.

In addition to their critical role in complex behavior and cognition, the hippocampus, amygdala and PFC are important in regulating the autonomic and HPA stress response, and they are the main focus of this review (Box 2).

Stress effects on gene expression in an ever-changing brain. As the first extra-hypothalamic brain structure recognized to have receptors

for adrenal steroids16, the hippocampus has been an important gate- way to understanding the effects of glucocorticoids and stress on gene expression in the brain. Recent technological advances have allowed high-throughput analysis of gene expression changes in response to stress17. For example, microarray analysis of whole hippocampus after acute stress, chronic stress and stress recovery in mice revealed that acute and chronic stress modulate a core set of genes, but that numer- ous changes are exclusive to each condition, highlighting how duration and intensity of stress alters reactivity18. Furthermore, corticosterone injections do not produce the same expression profile as acute stress, suggesting that in vivo stressors activate a diverse set of pathways independent of glucocorticoid receptor (GR) activation18 (Fig. 2). Finally, characterization of expression profiles after extended recov-

ery from 21 d of chronic stress showed that, despite a normalization of anxiety-related behaviors, recovery does not represent a return to the stress-naive baseline but rather a new state in which reactivity to a novel stres- sor produces a unique expression profile18. Studies in rats confirm that gene expres- sion profiles can change significantly from the immediate end of stress to 24 h later19 and that chronic stress can alter the tran- scriptional response to an acute corticos- terone injection in dentate gyrus20 (Fig. 2). Together, these studies demonstrate that a history of stress exposure can have a last- ing impact on future stress reactivity and hippocampal function.

Box 1 Examples of molecules that are necessary or permissive for remodeling

Brain-derived neurotrophic factor (BDNF)93,94

• Facilitator of plasticity or growth • Overexpression occludes effects of chronic stress • Haploinsufficiency prevents stress-induced plasticity Tissue plasminogen activator (tPA)73,74

• Secreted signaling molecule and protease • Required for stress-induced spine loss in hippocampus and medial amygdala • Required for acute stress-induced increase in anxiety; secretion activated by CRF • In amygdala, regulates tPA release Corticotropin-releasing factor (CRF)74,135

• Secreted in hippocampus by interneurons • Downregulates thin spines via RhoA signaling Lipocalin-2 • Secreted protein of previously unknown function77,78

• Induced by acute stress • Downregulates mushroom spines • Knockout increases neuronal excitability and anxiety Endocannabinoids136–138

• Induced via glucocorticoids • Regulate emotionality and HPA habituation and shutoff • CB1 receptor knockout increases anxiety and basolateral amygdala dendrite length and causes stress-like retraction of prefrontal cortical dendrites, likely through the regulation of glutamatergic transmission • Fatty acid aminohydrolase (FAAH) is a key regulator of endocannabinoid action

Synaptic functions: enhancement of • Synaptic transmission • Long-term potentiation • Learning for self-preservation

Synaptic functions: suppression of • Synaptic transmission • Long-term potentiation • Learning for less important things

Adaptive plasticity: • Suppression of neurogenesis • Mediates dendritic remodeling

Loss of resilience: • Neurochemical distortion • Impaired remodeling and lack of recovery from stress

Damage potentiation: • Mediates excitotoxicity in seizures, stroke and head trauma

Brain aging: • Extrasynaptic glutamate • Free radicals and inflammation

Adrenal steroids and excitatory amino acids modulate both limbs of inverted U

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intervention required

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permanent damage External intervention needed

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Figure 1 Effects of acute and chronic stress, mediated in part by glutamate and glucocorticoids as well as other molecules described in the text and in Box 1. These effects follow an inverted U-shaped curve in dose and time. The timeline shows how acute and chronic stress and aging interact with the intensity and duration of stressor.

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Many of the genes altered after glucocorticoid and chronic stress exposure in the hippocampus are known epigenetic regulators21, providing one possible mechanism underlying the persistent altera- tions in the expression response beyond the end of stress exposure. The continually changing pattern of gene expression is consistent with the finding that, although stress-induced dendritic retraction in PFC neurons appears to be reversible in terms of dendritic length and branching, the recovered neurons are different, in that dendrites that regrow after recovery from stress are more proximal to the cell body than those that retracted22.

Epigenetic mediation via post-translational histone modifica- tions. Stress has a clear impact on many types of molecular epige- netic mechanisms, from histone modifications to DNA methylation and hydroxymethylation and expression of noncoding RNA14,23–25 (Fig. 3). For instance, social defeat stress in rodents causes changes in both histone methylation and acetylation26. Acute and chronic stress promote histone modifications leading to repression or activation of genes related to memory and other processes. Studies of memory acquired in the forced swim test and Morris water maze uncovered a novel, rapid mechanism: glucocorticoids, via GRs, facilitate signaling of the ERK-MAPK pathway to the downstream nuclear kinases MSK1 and Elk-1 in dentate gyrus granule neurons; and activation of this path- way results in phosphorylation of serine 10 (S10) acetylation of lysine 14 (K14) of histone H3 (H3S10p-K14ac), leading to the induction of the immediate-early gene products c-Fos and Egr-1 (ref. 27).

Unlike that of other immediate-early gene products, FosB and its splice variant ∆FosB increases and remain elevated in the nucleus accumbens (NAc) after social defeat stress and is deficient in those animals that show depressive-like behavior, as well as in human patients with depression postmortem. Moreover, increased FosB/∆FosB expres- sion in NAc protects animals from the deleterious effects of chronic stress28. Epigenetically, the FosB promoter is enriched for dimethylation of H3 lysine 9 (H3K9me2) in the NAc of humans with depression rela- tive to that of controls without depression, implicating this repressive epigenetic modification in the repression of FosB. Moreover, in mice

zinc finger protein (ZFP)-induced enrichment of H3K9me2 at FosB in NAc not only was sufficient to reduce FosB/∆FosB expression, but also induced depression- and anxiety-like behaviors after social stress28.

A current practical application of this approach is the investiga- tion of rapidly acting antidepressants28,29 that act, at least in part, via epigenetic mechanisms, as does electroconvulsive therapy26,30. An epigenetic mechanism connects excitatory amino acid function with neural remodeling and stress-related behavior in one genetic and one stress-induced rodent model of anxiety- and depressive-like behavior in which downregulation of the presynaptic inhibitor of neuronal gluta- mate release, the mGlu2 receptors, in hippocampus is a key biomar- ker29. In that connection, drugs that modify glutamate overflow, such as ketamine, acetyl-l-carnitine and riluzole, have been show to exert rapid antidepressant-like effects in animal models29,31 and in humans32.

The novel antidepressant candidate acetyl-l-carnitine (LAC) appears to act inside and outside the nucleus to exert fast antidepres- sant responses: LAC corrects mGlu2 deficits by increasing acetylation of histone H3 lysine 27 (H3K27) bound to Grm2 promoter gene as well as acetylation of the NF-κB p65 subunit29. Using the same animal models, 14 d of treatment with the tricyclic antidepressant clomi- pramine were needed to promote antidepressant responses, which disappeared when the treatment was stopped, whereas antidepressant effects of LAC were still evident after 2 weeks of drug withdrawal. The persistent effects of LAC highlight the involvement of stable molecular adaptations that are reflected at the level of histone modi- fications in controlling mGlu2 transcription in hippocampus.

Transposons and retrotransposons. Acute restraint stress also has repressive epigenetic effects in the hippocampus and most promi- nently in the dentate gyrus via trimethylation of H3K27 and H3K9. The latter is associated with repression of a number of retrotrans- posable elements (RTE) and reduction of the coding and noncod- ing RNA normally produced by the repressed DNA, so far only in hippocampus30,33. This repression is lost with repeated stress, suggesting that those RTEs may impair genomic stability under conditions of chronic stress15.

Box 2 Overview of stress effects on the hippocampus, amygdala and prefrontal cortex

Three regions of the brain shown in the illustration have important roles in behavior and cognitive function as well as in regulating the autonomic and HPA stress response and are the main focus of this review. Glucocorticoid and mineralocorticoid receptors were first recognized in the hippocampal formation16, showing that adrenal steroids affect the brain in more ways than through the hypothalamus, which is now known to include effects on spatial and episodic memory and mood regulation. In the hippocampus, stress and glucocorticoids were first shown to cause dendritic shrinkage and loss of spines. The rediscovery of neurogenesis in the dentate gyrus11 galvanized widespread interest in the functional role of neuronal replacement in the adult brain. It was in the hippocampus that the role of excitatory amino acids in stress effects was first recognized9. Effects of acute and chronic stress on the amygdala differ from those in the hippocampus. Acute traumatic stressors cause increased spine density on basolateral amygdala neurons, and chronic stress leads to the expansion of basolateral amygdala dendrites139. Yet the medial amygdala shows a chronic stress–induced loss of spines75. These alterations are implicated in increased anxiety and in PTSD-like behaviors67,139. Within the prefrontal cortex, chronic stress causes medial PFC neurons to develop debranching and shrinkage of dendrites that is associated with cognitive rigidity, while orbitofrontal cortical neurons expand dendrites that may be related to increased vigilance122,140. The PFC under stress has provided important clues to age-related loss of resilience and impaired memory as well as to the effects of circadian disruption and extinction of fear memory141. These three brain regions have contributed to our knowledge of cellular and molecular mechanisms and cellular processes that are described in this Review, revealing brain regional specializations as well as common mediators and mechanisms and the complex interactions among the mediators.

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Figure 2 Gene expression changes in hippocampus in response to stress and glucocorticoid challenge depend on the prior stress history of the subject. Hippocampal microarray data reveals stress-induced changes in gene expression. (a) Dark colors represent the number of genes with significantly increased expression and pastels represent those with significantly decreased expression as identified by pairwise comparisons of each stress group with age-matched controls. FST, forced swim test; Cort, glucocorticoid injection; CRS, chronic restraint stress; Rec, recovery from CRS. (b) Proportional Venn diagram illustrating the genes with expression significantly altered by the acute stress, chronic stress and glucocorticoid (Cort) injection conditions and their overlap. The numbers of genes unique to each comparison whose expression was increased or decreased are listed next to arrows indicating the direction of change. (c) Venn diagram of genes whose expression was altered by each FST condition reveals a core of 95 genes that were always changed by this stressor. The large number of unique gene expression changes in each condition shows that the response to FST is altered by the stress history of the group, with the vast majority of changes occurring when an animal is exposed to a novel stressor immediately after a chronic stress exposure, as also shown in a. (d) Scatter plot of normalized expression values for each microarray probe comparing CRS (x axis) with recovery from CRS (y axis). For the majority of genes, expression is increased by CRS, but decreased after recovery; however, some probes show expression that is increased by CRS and remains elevated after recovery or that is suppressed by CRS and remains low in recovery. Highlighted probes are those that reached significance when compared with age-matched controls (blue, CRS; red, recovery from CRS; gray, not significant). Several examples of the highlighted genes are listed below the scatter plot by color designation and quadrant. For example, blue points in the lower left quadrant represent genes, such as Nrg3 and Scn1b, whose expression is significantly changed by CRS as compared with that in unstressed controls and is also decreased after recovery from CRS. By contrast, red points in the upper right quadrant represent genes, such as Cdk2 and Gria2, whose expression remains significantly different from that of controls after recovery from CRS and is also increased immediately following CRS. (e) Venn diagram illustrating that the genes whose expression was significantly different from that of controls after recovery from CRS are mostly distinct from those whose expression was significantly altered by CRS. Reprinted from ref. 18 with permission from Macmillan Publishers Ltd. (f) Pie charts of Gene Ontology (GO) terms that are over-represented among the 576 genes that were differentially expressed upon GC challenge in naive as compared to chronically stressed rats. The differentially expressed genes were divided into groups that responded to GCs in both controls and CRS animals (center) or only in controls (left) or in CRS animals (right). The pie charts represent the GO terms that were overrepresented in the three groups of GC-responsive genes and show that after CRS, GC challenge gives rise to a different gene signature than is seen in control animals. Reprinted from ref. 20 with permission.

Retrotransposons constitute a tenfold larger fraction of mammalian genomes than protein coding genes and appear to be unusually active in brain and steroidogenic tissues15. Consequently, they have recently attracted increasing attention from neuroscientists, who have shown that they contribute to neural diversity, cell fate and development as well as brain disease15,34–36. In addition to transposons’ mobility, they also seem to contribute the largest fraction of functional elements to what might be referred to as ‘the RNA genome’37–39. This other genome is composed of genes for noncoding RNAs that are being found to govern a growing number of cellular processes, including develop- ment, cell differentiation, chromosome imprinting and the regulation of the epigenetic machinery40,41; thus RTE-derived RNAs represent a substantial store of both genetic and epigenetic information.

Barbara McClintock, who discovered transposons over 60 years ago, noted that they were important contributors to an organism’s ability to deal with environmental stress42,43, and this insight appears to hold true with regard to the neurobiology of stress, though as with many other aspects of stress, transposons are likely to have both adaptive34 and possibly deleterious effects given that their dysregulation has been observed in both humans with post-traumatic stress disorder (PTSD) and animal models of stress disorders44–46. Brain transposons there- fore appear to represent a significant new frontier for stress research.

Role of excitatory amino acids. Excitatory amino acids, particularly glutamate, play key roles in structural as well as functional changes in the brain (Fig. 4). Initial studies of restraint stress, in which chronic stress causes shrinkage of apical dendrites of hippocampal CA3 neu- rons, showed that acute restraint stress elevates extracellular glutamate levels through a process that is absent in adrenalectomized animals, suggesting a role for the adrenal cortex47. Indeed, corticosterone acts directly via membrane-associated mineralocorticoid receptors (MRs) and GRs to cause glutamate release29,48,49. Importantly, blocking NMDA receptors and interfering with excitatory stimulation of ion channels blocks stress-induced dendritic remodeling within the hip- pocampus, an effect similar to that of blockade of adrenal steroid synthesis50,51. Similarly, stress-induced NMDA-dependent dendritic remodeling has been reported in medial PFC neurons52. Excess gluta- matergic activity, without adequate reuptake in the aftermath of trauma from seizures, ischemia and head trauma, leads to permanent neuronal loss by a process that is exacerbated by glucocorticoids53. These relationships can be summarized in an inverted-U-shaped dose- and time-response curve (Fig. 1).

In that connection, the shrinkage of apical dendrites as a result of stress in CA3 pyramidal neurons can be thought of as a protective mech- anism against permanent damage and neuron loss that is caused by the

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metastable dentate gyrus–CA3 feedforward and feedback circuitry that is the basis of its function54 and yet makes it vulnerable to seizure-induced damage55. This is well illustrated in hibernation, a state of low energy supply to the brain that is accompanied by rapidly reversible (within hours) shrinkage of CA3 apical dendrites in the hippocampus56,57. This hypothesis is further substantiated by studies in which removal of poly- sialic acid residues from neural cell adhesion molecule (NCAM) leads to marked increases in dendritic length of CA3 neurons and increased vul- nerability to excitotoxic damage, supporting the notion that shorter den- drites reduce the vulnerability of CA3 neurons to overstimulation58.

Role of glucocorticoids via multiple intracellular sites and mecha- nisms. Glucocorticoids produce both genomic and nongenomic effects in the brain through multiple sites and pathways. In addition, glucocorticoids have biphasic effects in which the timing and the level of GR expression are critical29,59. Glucocorticoid actions via genomic mechanisms involve both direct interactions with glucocorticoid response elements (GRE) and indirect actions via tethering to other transcription factors60. Glucocorticoids can directly stimulate release of excitatory amino acids via membrane-associated receptors, and they can indirectly regulate both glutamate and GABA release through induction of local synthesis of endocannabinoids61 (see below).

In addition, glucocorticoids can also translocate GRs, along with the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), to mitochondria where they together promote Ca2+ sequestration and regulate mitochondrial oxidation, free radical formation and membrane potential, three independent measures of mitochondrial function. Bcl-2 is able to inhibit the formation of Bax-containing pores on the mitochondrial outer membrane and reduce the release of calcium and cytochrome c from the mitochondria. Importantly, these three glucocorticoid effects show an inverted U–shaped dose-response curve and are biphasic, and high glucocorticoid levels cause a failure of this mechanism over 72 h, leading to increased free-radical formation62 (Fig. 5).

Just as location of GR action is an essential consideration, the level of expression of GRs is also very important. Genetically induced overexpression of GR in forebrain leads to increased lability of mood-related behaviors, yet also confers greater responsiveness to antidepressant drugs63, whereas genetic knockdown of GR has the opposite effect64. Epigenetic regulation of GR activity also has significant functional implications, as increased CpG methylation within the GR promoter is associated with a suboptimal HPA stress response and with poor maternal care in rodents and early life abuse in human suicide victims65,66.

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Figure 3 Molecular epigenetic modifications. Among the molecular mechanisms that fall under the epigenetic rubric are covalent modifications of the histone proteins that package and control access to the DNA, which include acetylation, methylation and phosphorylation as well as a growing number of more exotic modifications. The DNA itself may be methylated or hydroxymethylated at cytosine residues. A suite of noncoding RNA species such as microRNA (miRNA), piwi-interacting RNA (piRNA) and long noncoding RNA (lncRNA) also act to convey epigenetic information and to coordinate interactions between DNA and the transcriptional and chromatin modification machinery. Many of these mechanisms seem to have evolved in part from, or as a consequence of the presence of, transposable elements in eukaryotic genomes. Adapted from ref. 24 with permission of Elsevier.

Figure 4 Glucocorticoids are released from the adrenal glands. Basal release varies in a diurnal pattern, and release increases severalfold after exposure to a stressor. Glucocorticoids can bind, with different affinities, to glucocorticoid and mineralocorticoid receptors, which are expressed throughout the brain and seem to exist in both membrane-bound form and nuclear form. Adrenal steroids can have both rapid and delayed effects. The effects can result from nongenomic mechanisms (mediated by membrane receptors, see the figure), indirect genomic mechanisms (mediated by membrane receptors and second messengers, see the figure) and genomic mechanisms (mediated by cytoplasmic receptors that move to the nucleus and act as transcription factors; see figure), as seems now to be the case for all steroid hormones. Although mineralocorticoid and glucocorticoid receptors seem to mediate many of these effects, other membrane-associated receptors, including G protein–coupled receptors, may also be involved in some of these actions. In addition, activated glucocorticoid receptors can translocate to mitochondria and enhance their calcium buffering capacity. Glucocorticoids rapidly induce glutamate release in the hippocampus through a mechanism that is absent when the mineralocorticoid receptor is deleted and that may involve a membrane-associated form of the mineralocorticoid receptor. An indirect way by which glucocorticoids can influence neurotransmission (glutamatergic, as well as GABAergic, cholinergic, noradrenergic and serotonergic) is through cross-talk with the endocannabinoid system. They rapidly stimulate endocannabinoid production in the brain, whereupon endocannabinoids bind to cannabinoid receptor 1 (CB1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) and inhibit neurotransmitter release (see the figure). Although a G protein–coupled receptor is implicated in endocannabinoid production, there is also evidence for a mechanism blocked by Ru486—a selective antagonist of the classical cytoplasmic glucocorticoid receptor—in the rapid actions of glucocorticoids in prefrontal cortex. Modified from ref. 32 with permission from Macmillan Publishers Ltd.

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Figure 5 Biphasic effect of glucocorticoids (Cort) in regulating mitochondrial function. (a) Cort readily penetrate the cell membrane and interact with cytoplasmic glucocorticoid receptors (GRs), causing a dose-dependent increase in GR translocation into cell nuclei. Con, vehicle control. (b) Translocation of GRs into mitochondria as a complex with the anti-apoptotic protein Bcl-2, where they upregulate mitochondrial calcium levels, membrane potential and oxidation; this is stabilized at a 100 nM dose of Cort (b) and decreases with time at the high, 1 µM Cort dose (c), where, after a 3-d treatment, high Cort leads to decreased abundance of GR and Bcl-2 in mitochondria. (d,e) Cort modulates membrane potential, measured by Janus-1 (JC-1) staining, in a dose- and time-dependent manner. Time course of JC-1 staining after Cort treatment shows sustained potential at 100 nM dose and loss of potential at 1 µM dose (d); dose-dependent curve for JC-1 staining after Cort treatment shows that both low and high Cort maintain potential at 24 h, but high Cort causes failure of membrane potential at 72 h (e). This regulation of mitochondrial function by Cort parallels neuroprotection: that is, treatment with low doses of Cort has a neuroprotective effect, whereas high Cort enhances kainic acid–induced toxicity of cortical neurons62,133, consistent with the “glucocorticoid endangerment” hypothesis134. Error bars, s.e.m.; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Modified from ref. 62 with permission from Macmillan Publishers Ltd.

The actions of glucocorticoids are biphasic, as illustrated above for mitochondria62, and their timing is important. For example, in several animal models of traumatic stress–induced PTSD-like delayed anxiety and traumatic stress–induced spine synapse formation in basolateral amygdala (BLA), a timed elevation of glucocorticoids, before the induction of stress, prevents development of the anxiety and synapse formation67. Data on human PTSD supports a protective role for ade- quate glucocorticoid levels at time of traumatic stress68,69. Yet repeated high-dose glucocorticoid treatment mimics chronic stress and induces dendritic lengthening in BLA70, a result that emphasizes the differ- ences between acute and chronic elevations of glucocorticoids.

Regarding MRs, which exert both genomic actions and nongenomic actions to stimulate glutamate release48, mice that spontaneously show increased anxiety have elevated expression of hippocampal MR, which mediate a stress-induced suppression of mGlu2 expression and increased levels of anxiety- and depression-like behavior71. Importantly, blocking MR receptors and interfering with glucocor- ticoid stimulation of glutamate activity blocks stress-induced mood abnormalities. The nature of the experiences of the animals that develop higher MR is not known, but it may involve epigenetic influ- ences early in life, such as maternal care and stressors in the neo- natal nesting environment72. The epigenetic allostasis model points to developmental origins of individual differences in the responses to stress and implies that unknown early life epigenetic influences program each individual to different trajectories of behavioral and physiological responses to later stressful life events (Fig. 6).

Involvement of secreted signaling molecules. In addition to glu- cocorticoids, secreted signaling molecules have important roles in the remodeling of neural tissue during stress (Box 1). Corticotropin- releasing factor (CRF), which is better known for its role in governing the secretion of adrenocorticotropic hormone (ACTH) and glucocor- ticoids, plays a key part in stress-induced dendritic remodeling in the CA1 region of the hippocampus73,74. Findings over the past decade have also implicated new players in the regulation of dendritic remod- eling. For instance, tissue plasminogen activator (tPA), a secreted

signaling molecule as well as protease, is implicated in stress-induced dendritic remodeling and spine loss in medial amygdala as well as in the CA1 hippocampus. Specifically, tPA-knockout mice fail to show chronic stress impairment of memory and spine reduction in CA1 (refs. 73,75). Linking these two factors together, there is evidence that in the amygdala tPA release is stimulated by CRF76. Similarly, lipocalin-2 is a novel modulator of spine plasticity with different effects in amygdala and hippocampus77,78. Acute stress increases lipocalin-2 levels, and lipocalin-2 downregulates mushroom spines and generally inhibits actin motility in hippocampus. Remarkably, dele- tion of lipocalin-2 increases neuronal excitability and anxiety, and, in amygdala, the absence of lipocalin-2 increases the basal number of spines and prevents a stress-induced increase in spine density.

Endocannabinoids are another class of signaling molecules that importantly regulate multiple aspects of the stress response. In addition to contributing to the termination79 of the acute response to stress, as well as habituation to repeated stress80, endocannabinoids also seem to be important for the regulation of structural plasticity under conditions of repeated stress (Box 1). For example, cannabi- noid 1 (CB1) receptor–deficient mice exhibit reductions in prefron- tal cortical dendritic length and complexity, while having enhanced and more complex dendritic arbors within the BLA, both of which effects parallel the effects of chronic stress81,82. More importantly, chronic stress and corticosterone treatment are both known to impair endocannabinoid signaling at multiple levels, through both a down- regulation of the CB1 receptor83 and a reduction in the levels of the endocannabinoid anandamide that is mediated by an increase in its hydrolysis by the enzyme fatty acid amide hydrolase (FAAH)84,85.

Given the parallels between genetic deletion of the CB1 receptor and the ability of chronic stress to impair endocannabinoid signaling, it is interesting to note that elevation of anandamide via CB1 receptor signaling, through genetic or pharmacological impairment of FAAH, retards the ability of chronic stress to produce dendritic hypertrophy in the BLA as well as concomitant changes in emotional behavior85–88,89. Collectively, these data indicate that endocannabinoid signaling buff- ers against many of the effects of stress and seems to be important for

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Figure 6 Individual differences in naive C57Bl6 mice in anxiety-related behavior reveal animals more sensitive to stress-induced downregulation of hippocampal mGlu2 expression, a biomarker of depressive-like behavior and antidepressant response. (a–c) The use of the light-dark test as a screening method (a) allows identification of clusters of animals with a different baseline anxiety profile (b) along with differences in mineralocorticoid receptor gene (MR; Nr3c2) transcript levels in hippocampus (c). Error bars, s.e.m.; **P ≤ 0.01, ***P ≤ 0.001. (d) The susceptible (HS) mice, which are characterized by higher baseline MR transcript levels, show reduced hippocampal mGlu2 (Grm2) transcript expression associated with exacerbation of anxious and of depressive-like behaviors after acute and chronic stress, respectively. Conversely, individuals with lower baseline MR transcript levels (low susceptible, LS) cope better with stress and show adaptation in mGlu2 receptor expression in hippocampus. Ctrl, unstressed sex and age-matched control animals; ARS, acute restraint stress. (e) The epigenetic allostasis model points to the developmental origins of these individual differences, suggesting that as-yet-unknown epigenetic influences early in life may lead to alterations in hippocampal MR transcript levels71. (f) Representative mechanisms of action of acetyl-l-carnitine (LAC): work in our and other laboratories has shown that a decrease in mGlu2 receptors either following stress exposure or occurring in a genetic animal model of depression is rapidly corrected by 3 d of intraperitoneal administration of the novel antidepressant candidate LAC via acetylation of either the H3K27 bound to the Grm2 promoter, which encodes mGlu2 receptors, or the NF-κB p65 subunit30. A, acetyl; M, methyl; P, phosphate; MR (Nr3c2), MGI 99459; mGlu2 (Grm2), MGI 1351339.

limiting the effects of chronic stress on structural plasticity within these identified limbic circuits. At a mechanistic level, this is likely to be due to the ability of CB1 receptor signaling to gate glutamatergic release, as it has been demonstrated that CB1 receptor–deficient mice show greater changes in glutamatergic signaling and excitotoxicity within the PFC following chronic stress90. Moreover, in a pattern similar to the protective effects of CB1 receptor activation identified within the amygdala, administration of a CB1 receptor agonist during repeated stress can reduce the increase in glutamatergic signaling, the induction of pro-inflammatory cytokines and lipid peroxidation within the PFC90. Thus, the release of endocannabinoids during stress may temper changes in structural plasticity by limiting the magnitude of glutamate release in response to stress; and under conditions of chronic stress, when this system becomes compromised, the loss of this endogenous buffer facilitates excess glutamate release and the ensuing changes in dendritic morphology. Linking this model with the factors previously described, it is interesting to note that in addi- tion to promoting tPA release, CRF has also been found to induce anandamide hydrolysis by FAAH91, suggesting that CRF could act as an orchestrator of multiple signaling molecules, all of which converge in structural changes within the brain following chronic stress.

Recent studies have suggested that blood-based biomarkers may be able to predict aspects of brain signaling associated with trauma-related effects in both males and females, specifically with respect to conver- gence onto GR signaling pathways. After a predator-scent-stress (PSS) exposure, male and female rats were classified into vulnerable (“PTSD- like”) and resilient (minimally affected) phenotypes on the basis of their performance on a variety of behavioral measures92. Genome-wide expression profiling in blood, amygdala and hippocampus indicated that glucocorticoid signaling was the only convergent pathway asso- ciated with individual differences in susceptibility. Moreover, corti- costerone treatment 1 h after PSS exposure prevented anxiety and

hyperarousal 7 d later in both sexes, consistent with prior findings in the same as well as in another PTSD animal model67,68, confirming the involvement of the GR in sequelae of traumatic stress.

Roles of brain-derived neurotrophic factor (BDNF). BDNF plays an important role in dendritic remodeling in both hippocampus and BLA (Box 1). BDNF-overexpressing mice show increases dendritic length in both CA3 and BLA, which occludes the effects of chronic stress in decreasing dendritic branching in CA3 and increasing it in BLA93. On the other hand, in WT animals, chronic stress causes a downregulation of BDNF in CA3 hippocampus and an upregulation of BDNF in the BLA. Although the increase in BLA persists beyond 21 d after the stress, the effect in CA3 normalizes94. These intriguing timing issues become even more interesting when one considers that after a single acute stress, BDNF expression in the BLA rises and stays elevated for 10 d, while that in CA3 shows only a transient increase94. This increase in the BLA is associated with both increased anxiety and increased density of spines in BLA neurons85. The mechanism of these effects on BDNF remain enigmatic, but they are not entirely mediated by glucocorticoid actions as corticosterone levels increase after both acute and chronic stress and remained elevated after chronic, but not acute, stress. Thus, it is clear that BDNF-mediated signaling is involved in the structural effects of stress, but that the direction and nature of signaling is region specific and stress specific and is influenced by epigenetic modifications89 along with post-translational modifications94,95. The epigenetic mechanisms controlling BDNF expression are influenced by maternal separation early in life, which, in turn, leads to changes in BDNF expression and epigenetic regulation via histone acetylation and methylation over the life course, with consequences for anxiety-like behaviors96.

Cellular processes in remodeling of neural architecture. The neu- ronal surface, cytoskeleton and nuclear envelope are each implicated

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in the mechanisms of stress-induced retraction and expansion of den- drites and synapse turnover. The polysialylated form of neural cell adhesion molecule (PSA-NCAM) is expressed in the CA3 and DG regions of the hippocampus and is believed to denote the capacity for adaptive structural plasticity in many parts of the CNS97–99. Repeated stress causes retraction of CA3 hippocampal dendrites accompanied by a modest increase in PSA-NCAM expression, possibly as the result of glucocorticoid mediation100. Using endoneuraminidase N (EndoN) to remove PSA from NCAM, Sandi reported impairment of consolidation of contextual fear conditioning101. Using the same treatment, we observed considerable expansion of the dendritic tree in both CA3 and CA1 and a marked increase in excitotoxicity and damage to CA3 neurons; repeated stress still caused some dendrite retraction after PSA removal58. Thus, although PSA-NCAM is a facilitator of plasticity, the PSA moiety appears to also limit the extent of dendritic growth and yet is not necessary for dendritic retraction under stress.

Two other classes of cell adhesion molecules are reported to change with chronic stress, with behavioral consequences. Neuroligins (NLGNs) are important for proper synaptic formation and function- ing and are critical regulators of the balance between neural excitation and inhibition (E/I), and chronic restraint stress reduces hippocampal NLGN-2 levels, in association with reduced sociability and increased aggression102,103. This occurred along with a reduction of NLGN-2 expression throughout the hippocampus, detectable in different layers of the CA1, CA3 and DG subfields. Intrahippocampal administration of neurolide-2, which interferes with the interaction between NLGN-2 and neurexin, led to reduced sociability and increased aggression, thus mimicking effects of chronic stress102.

Chronic restraint stress also increases activity of matrix metallopro- teinase-9 (MMP-9) in the CA1. MMP-9 carries out proteolytic process- ing of another cell adhesion molecule, nectin-3. Chronic stress reduced nectin-3 in the perisynaptic CA1, but not in the CA3, with conse- quences for social exploration and social recognition and for a CA1- dependent cognitive task. Implicated in this is a stress-related increase in extracellular glutamate and NMDA receptor mediation of MMP-9 (ref. 104). These findings are reminiscent of the CA1-specific effects of tPA in mediating stress effects on spine density in CA1 (ref. 73).

Actin polymerization plays a key role in filopodial extension and spine synapse formation as well as in plasticity within the synapse itself105, and cytoskeletal remodeling is an important factor in the effects of stress and other environmental manipulations. Hibernation in European hamsters and ground squirrels results in rapid retraction of dendrites of CA3 pyramidal neurons, and an equally rapid expan- sion occurs when hibernation torpor is reversed56,57. The retraction

of dendrites is accompanied by increases in a soluble phosphor- ylated form of tau that may indicate disruption of the cytoskeleton, which permits the dendrite shortening and possible protection from excitotoxicity; at the same time, PSA-NCAM expression is lost during hibernation torpor, reducing the capacity for plasticity106. This model highlights the important role that tau plays in normal cytoskeletal function, a fact that should be emphasized when attempt- ing to understand its role in pathology107.

Even though dendrite retraction and regrowth would appear to involve a reversible depolymerization and repolymerization of the cytoskeleton, there are other processes that point to the importance of nuclear factors. A recent example is the unexpected role of a cell nuclear pore complex protein, NUP-62, in stress-induced dendritic remodeling in the CA3 region of the hippocampus108. First identified as the product of a gene whose expression was downregulated in the prefrontal cortex of depressed patients109, NUP-62 was also found to be reduced in response to chronic stress in CA3 neurons of rodents108. Importantly, the levels of other nuclear pore complex genes were unchanged with chronic stress, supporting the specificity of its role in stress remodeling. Subsequent in vitro studies confirmed that the downregulation of NUP-62 is associated with dendritic retraction and that this effect is regulated at the molecular level by NUP-62 phosphor- ylation at a PYK2 site which results in its retention in the cytoplasm108. A role of NUP-62 in maintaining chromatin structure for transcription is suggested as well as in nucleocytoplasmic transport108.

Stress: not always what one thinks it is Just as stress is not a unitary phenomenon at the cell and circuit level, neither is it one at the level of the whole organism. As noted in the introduction, a key aspect of stress effects on the brain and body is the nonlinear interaction of multiple mediators of stress and adapta- tion that is part of the concept of allostasis1, which refers to the active process of maintaining homeostasis through the output of hormones and ANS activity along with immune and metabolic system media- tors and the mediators in the brain that are the main focus of this review. When one mediator system changes, the others adjust, and the resulting output can be distorted, as in chronic inflammation or a flat cortisol diurnal rhythm caused by sleep deprivation or depres- sion2. Moreover, the actions of any one mediator may depend on the actions of other mediators. For example, glucocorticoids and excitatory amino acids are both involved in stress-induced suppression of neurogenesis, which has been found not only in rodents but also in tree shrews and rhesus monkeys110–112. Yet, glucocorticoid levels alone do not predict the direction of neurogenesis, as shown by studies of male sexual behavior, which results in increased neurogenesis but also high glucocorticoid levels; in this scenario oxytocin appears to play an important role, emphasizing the importance of understanding the interaction of these distinct signaling molecules113,114.

In seeking to understand where and how stress affects neural cir- cuits, it has become evident that when these mediators act is also an important consideration. In most vertebrate species, plasma glucocor- ticoids rise just before the active phase. This rhythm is largely driven by changes in the amplitude and frequency of the ultradian secretion of glucocorticoids115. Indeed, the natural ultradian fluctuations of gluco- corticoids mediate turnover of a subset of synapses in cerebral cortex; and inhibiting the fluctuations with a minimal dose of dexamethasone impairs spine turnover116. Moreover, these diurnal changes in spine formation and removal are important for motor learning117.

In addition to ultradian pulses, circadian (or diurnal) rhythms are a crucial factor that impact the stress response. Rhythmic HPA function seems to be necessary for the normal initiation and

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termination of the stress response of ACTH, cortisol and other medi- ators118. Epidemiologically, disrupted sleep and circadian rhythms lead to increased risk for development of psychiatric, cardiovascular or other physiological syndromes in shift workers or populations undergoing chronic circadian disruption119. Housing mice in a light- dark cycle of 20 h (10 h light/10 h dark), rather than standard 24-h cycles, to drive circadian disruption results in metabolic signs of allostatic load120, with increased weight, adiposity and leptin levels, as well as an imbalance between insulin and plasma glucose suggest- ing a pre-diabetic state121. The metabolic changes are accompanied by changes in PFC cellular morphology, mirroring those observed in chronic stress122, with circadian-disrupted animals having shrunken and less complex apical dendritic trees of cells in layer II/III of the medial PFC123 (Fig. 7). In addition, circadian-disrupted mice show altered responses to endotoxin challenge with lipopolysaccharide124, highlighting the similarities between chronic circadian disrup- tion and chronic stress. The mechanisms by which these systems interact is not yet fully understood, but they do not appear to be driven simply by elevation of glucocorticoids.

Intriguingly, glucocorticoids are able to regulate the expression of circadian clock genes in several brain regions125 as well as in liver126. As such, disruption of normal oscillatory profiles of glucocorticoids could lead to desynchronized activity between different brain regions as well as peripheral organ systems. This dissonance is thought to contribute to several pathologies that are similar to the effects of chronic stress, including obesity and metabolic syndrome119. Thus circadian disruption is both a ‘stressor’, in that it increases allostatic load or overload, and a risk factor for other stressful experiences, emphasizing the importance of timing in glucocorticoid actions through the brain and body.

Conclusions and future directions The response of the brain to stressors is a complex process involv- ing multiple interacting mediators that utilizes both genomic and nongenomic mechanisms, from the cell surface to the cytoskeleton to epigenetic regulation via the cell nucleus. Resilience in the face of stress is a key aspect of a healthy brain, even though gene expression shows a brain that continually changes with experience127. Therefore, recovery from stress-induced changes in neural architecture after stress is not a ‘reversal’ but a form of neuroplastic adaptation that also may be impaired in mood disorders and reduced with aging (Fig. 1). Resilience may be thought of as an active process that implies ongoing adaptive plasticity without external intervention128.

On the other hand, resilience is decreased and vulnerability is increased by adverse childhood experiences that lead to ‘biological embedding’ of trajectories of response to stressful life events4 through- out the life course6, which contribute disproportionately to allostatic overload in the form of physical and mental health disorders over the life course7. Evidence from CpG methylation of DNA indicates the embedded influence of early adversity66. From the original definition of epigenetics129 as the emergence of characteristics of each individual of each species, not evident from prior stages of development, inter- ventions to counteract adverse childhood experiences cannot ‘roll back the clock’ but rather may be able change the trajectory of brain and body development in a more positive direction.

Can the effects of stress on the brain be treated even though there are no ‘magic bullets’ like penicillin for stress-related disorders6? For psychiatric illnesses such as depression and anxiety disorders, including PTSD, it is necessary to complement and even replace exist- ing drugs and adopt strategies that center around the use of targeted behavioral therapies along with treatments, including pharmaceutical

agents, that open up ‘windows of plasticity’ in the brain and facilitate the efficacy of the behavioral interventions5,130,131. To that extent, meeting the demands imposed by stressful experiences through vari- ous coping resources can lead to growth, adaptation and learning to promote resilience and improved mental health128,132.

ACkNowlEDgmENTS The McEwen laboratory acknowledges research support from US National Institutes of Health (R01 MH41256), and the Hope for Depression Research Network and the American Foundation for Suicide Prevention to C.N. and National Research Service Award F32 MH102065 to J.D.G.

ComPETINg FINANCIAl INTERESTS The authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.

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