Crystallin Mu in Medial Amygdala Mediates the Effect of Social Experience on Cocaine Seeking in Males but Not in Females.

BACKGROUND: Social experiences influence susceptibility to substance use disorder. The adolescent period is associated with the development of social reward and is exceptionally sensitive to disruptions to reward-associated behaviors by social experiences. Social isolation (SI) during adolescence alters anxiety- and reward-related behaviors in adult males, but little is known about females. The medial amygdala (meA) is a likely candidate for the modulation of social influence on drug reward because it regulates social reward, develops during adolescence, and is sensitive to social stress. However, little is known regarding how the meA responds to drugs of abuse. METHODS: We used adolescent SI coupled with RNA sequencing to better understand the molecular mechanisms underlying meA regulation of social influence on reward. RESULTS: We show that SI in adolescence, a well-established preclinical model for addiction susceptibility, enhances preference for cocaine in male but not in female mice and alters cocaine-induced protein and transcriptional profiles within the adult meA particularly in males. To determine whether transcriptional mechanisms within the meA are important for these behavioral effects, we manipulated Crym expression, a sex-specific key driver gene identified through differential gene expression and coexpression network analyses, specifically in meA neurons. Overexpression of Crym, but not another key driver that did not meet our sex-specific criteria, recapitulated the behavioral and transcriptional effects of adolescent SI. CONCLUSIONS: These results show that the meA is essential for modulating the sex-specific effects of social experience on drug reward and establish Crym as a critical mediator of sex-specific behavioral and transcriptional plasticity.

Hippocampal BDNF regulates a shift from flexible, goal-directed to habit memory system function following cocaine abstinence.

The transition from recreational drug use to addiction involves pathological learning processes that support a persistent shift from flexible, goal-directed to habit behavioral control. Here, we examined the molecular mechanisms supporting altered function in hippocampal (HPC) and dorsolateral striatum (DLS) memory systems following abstinence from repeated cocaine. After 3 weeks of cocaine abstinence (experimenter- or self-administered), we tested new behavioral learning in male rats using a dual-solution maze task, which provides an unbiased approach to assess HPC- versus DLS-dependent learning strategies. Dorsal hippocampus (dHPC) and DLS brain tissues were collected after memory testing to identify transcriptional adaptations associated with cocaine-induced shifts in behavioral learning. Our results demonstrate that following prolonged cocaine abstinence rats show a bias toward the use of an inflexible, habit memory system (DLS) in lieu of a more flexible, easily updated memory system involving the HPC. This memory system bias was associated with upregulation and downregulation of brain-derived neurotrophic factor (BDNF) gene expression and transcriptionally permissive histone acetylation (acetylated histone H3, AcH3) in the DLS and dHPC, respectively. Using viral-mediated gene transfer, we overexpressed BDNF in the dHPC during cocaine abstinence and new maze learning. This manipulation restored HPC-dependent behavioral control. These findings provide a system-level understanding of altered plasticity and behavioral learning following cocaine abstinence and inform mechanisms mediating the organization of learning and memory more broadly.

Genome-wide transcriptional profiling of central amygdala and orbitofrontal cortex during incubation of methamphetamine craving.

Methamphetamine (Meth) seeking progressively increases after withdrawal (incubation of Meth craving), but the transcriptional mechanisms that contribute to this incubation are unknown. Here we used RNA-sequencing to analyze transcriptional profiles associated with incubation of Meth craving in central amygdala (CeA) and orbitofrontal cortex (OFC), two brain areas previously implicated in relapse to drug seeking. We trained rats to self-administer either saline (control condition) or Meth (10 days; 9 h/day, 0.1 mg/kg/infusion). Next, we collected brain tissue from CeA and OFC on withdrawal day 2 (when Meth seeking is low and non-incubated) and on day 35 (when Meth seeking is high and incubated), for subsequent RNA-sequencing. In CeA, we identified 10-fold more differentially expressed genes (DEGs) on withdrawal day 35 than day 2. These genes were enriched for several biological processes, including protein ubiquitination and histone methylation. In OFC, we identified much fewer expression changes than in CeA, with more DEGs on withdrawal day 2 than on day 35. There was a significant overlap between upregulated genes on withdrawal day 2 and downregulated genes on withdrawal day 35 in OFC. Our analyses highlight the CeA as a key region of transcriptional regulation associated with incubation of Meth seeking. In contrast, transcriptional regulation in OFC may contribute to Meth seeking during early withdrawal. Overall, these findings provide a unique resource of gene expression data for future studies examining transcriptional mechanisms in CeA that mediate Meth seeking after prolonged withdrawal.

Habits Are Negatively Regulated by Histone Deacetylase 3 in the Dorsal Striatum.

BACKGROUND: Optimal behavior and decision making result from a balance of control between two strategies, one cognitive/goal-directed and one habitual. These systems are known to rely on the anatomically distinct dorsomedial and dorsolateral striatum, respectively. However, the transcriptional regulatory mechanisms required to learn and transition between these strategies are unknown. Here we examined the role of one chromatin-based transcriptional regulator, histone modification via histone deacetylases (HDACs), in this process. METHODS: We combined procedures that diagnose behavioral strategy in rats with pharmacological and viral-mediated HDAC manipulations, chromatin immunoprecipitation, and messenger RNA quantification. RESULTS: The results indicate that dorsal striatal HDAC3 activity constrains habit formation. Systemic HDAC inhibition following instrumental (lever press → reward) conditioning increased histone acetylation throughout the dorsal striatum and accelerated habitual control of behavior. HDAC3 was removed from the promoters of key learning-related genes in the dorsal striatum as habits formed with overtraining and with posttraining HDAC inhibition. Decreasing HDAC3 function, either by selective pharmacological inhibition or by expression of dominant-negative mutated HDAC3, in either the dorsolateral striatum or the dorsomedial striatum accelerated habit formation, while HDAC3 overexpression in either region prevented habit. CONCLUSIONS: These results challenge the strict dissociation between dorsomedial striatum and dorsolateral striatum function in goal-directed versus habitual behavioral control and identify dorsostriatal HDAC3 as a critical molecular directive of the transition to habit. Because this transition is disrupted in many neurodegenerative and psychiatric diseases, these data suggest a potential molecular mechanism for the negative behavioral symptoms of these conditions and a target for therapeutic intervention.

Bidirectional Synaptic Structural Plasticity after Chronic Cocaine Administration Occurs through Rap1 Small GTPase Signaling.

Dendritic spines are the sites of most excitatory synapses in the CNS, and opposing alterations in the synaptic structure of medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a primary brain reward region, are seen at early versus late time points after cocaine administration. Here we investigate the time-dependent molecular and biochemical processes that regulate this bidirectional synaptic structural plasticity of NAc MSNs and associated changes in cocaine reward in response to chronic cocaine exposure. Our findings reveal key roles for the bidirectional synaptic expression of the Rap1b small GTPase and an associated local synaptic protein translation network in this process. The transcriptional mechanisms and pathway-specific inputs to NAc that regulate Rap1b expression are also characterized. Collectively, these findings provide a precise mechanism by which nuclear to synaptic interactions induce "metaplasticity" in NAc MSNs, and we reveal the specific effects of this plasticity on reward behavior in a brain circuit-specific manner.

Experience-Dependent Induction of Hippocampal ΔFosB Controls Learning.

The hippocampus (HPC) is known to play an important role in learning, a process dependent on synaptic plasticity; however, the molecular mechanisms underlying this are poorly understood. ΔFosB is a transcription factor that is induced throughout the brain by chronic exposure to drugs, stress, and variety of other stimuli and regulates synaptic plasticity and behavior in other brain regions, including the nucleus accumbens. We show here that ΔFosB is also induced in HPC CA1 and DG subfields by spatial learning and novel environmental exposure. The goal of the current study was to examine the role of ΔFosB in hippocampal-dependent learning and memory and the structural plasticity of HPC synapses. Using viral-mediated gene transfer to silence ΔFosB transcriptional activity by expressing ΔJunD (a negative modulator of ΔFosB transcriptional function) or to overexpress ΔFosB, we demonstrate that HPC ΔFosB regulates learning and memory. Specifically, ΔJunD expression in HPC impaired learning and memory on a battery of hippocampal-dependent tasks in mice. Similarly, general ΔFosB overexpression also impaired learning. ΔJunD expression in HPC did not affect anxiety or natural reward, but ΔFosB overexpression induced anxiogenic behaviors, suggesting that ΔFosB may mediate attentional gating in addition to learning. Finally, we found that overexpression of ΔFosB increases immature dendritic spines on CA1 pyramidal cells, whereas ΔJunD reduced the number of immature and mature spine types, indicating that ΔFosB may exert its behavioral effects through modulation of HPC synaptic function. Together, these results suggest collectively that ΔFosB plays a significant role in HPC cellular morphology and HPC-dependent learning and memory. SIGNIFICANCE STATEMENT: Consolidation of our explicit memories occurs within the hippocampus, and it is in this brain region that the molecular and cellular processes of learning have been most closely studied. We know that connections between hippocampal neurons are formed, eliminated, enhanced, and weakened during learning, and we know that some stages of this process involve alterations in the transcription of specific genes. However, the specific transcription factors involved in this process are not fully understood. Here, we demonstrate that the transcription factor ΔFosB is induced in the hippocampus by learning, regulates the shape of hippocampal synapses, and is required for memory formation, opening up a host of new possibilities for hippocampal transcriptional regulation.

ACF chromatin-remodeling complex mediates stress-induced depressive-like behavior.

Improved treatment for major depressive disorder (MDD) remains elusive because of the limited understanding of its underlying biological mechanisms. It is likely that stress-induced maladaptive transcriptional regulation in limbic neural circuits contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF (ATP-utilizing chromatin assembly and remodeling factor) ATP-dependent chromatin-remodeling complex, occurring in the nucleus accumbens of stress-susceptible mice and depressed humans, is necessary for stress-induced depressive-like behaviors. We found that altered ACF binding after chronic stress was correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning were associated with repressed expression of genes implicated in susceptibility to stress. Together, our findings identify the ACF chromatin-remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress-related behaviors.

PSMC5, a 19S Proteasomal ATPase, Regulates Cocaine Action in the Nucleus Accumbens.

ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain's reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5-also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex-as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity.

Histone Deacetylases as Potential Targets for Cocaine Addiction.

Drug-induced changes in gene expression likely contribute to long-lasting structural and functional alterations in the brain's reward circuitry and the persistence of addiction. Modulation of chromatin structure through covalent histone modifications has emerged as an important regulator of gene transcription in brain and increasing evidence suggests that misregulation of histone acetylation contributes to the establishment and maintenance of aberrant neuronal gene programs and behaviors associated with cocaine or amphetamine exposure. In this review, we summarize evidence supporting a role for histone acetylation in psychostimulant-induced plasticity and discuss findings from preclinical studies investigating histone deacetylase (HDAC) action and the use of small-molecule HDAC inhibitors (HDACis) to correct drug-mediated transcriptional dysregulation.

Incubation of methamphetamine craving is associated with selective increases in expression of Bdnf and trkb, glutamate receptors, and epigenetic enzymes in cue-activated fos-expressing dorsal striatal neurons.

Cue-induced methamphetamine seeking progressively increases after withdrawal (incubation of methamphetamine craving), but the underlying mechanisms are largely unknown. We determined whether this incubation is associated with alterations in candidate genes in dorsal striatum (DS), a brain area implicated in cue- and context-induced drug relapse. We first measured mRNA expression of 24 candidate genes in whole DS extracts after short (2 d) or prolonged (1 month) withdrawal in rats following extended-access methamphetamine or saline (control condition) self-administration (9 h/d, 10 d). We found minimal changes. Next, using fluorescence-activated cell sorting, we compared gene expression in Fos-positive dorsal striatal neurons, which were activated during "incubated" cue-induced drug-seeking tests after prolonged withdrawal, with nonactivated Fos-negative neurons. We found significant increases in mRNA expression of immediate early genes (Arc, Egr1), Bdnf and its receptor (Trkb), glutamate receptor subunits (Gria1, Gria3, Grm1), and epigenetic enzymes (Hdac3, Hdac4, Hdac5, GLP, Dnmt3a, Kdm1a) in the Fos-positive neurons only. Using RNAscope to determine striatal subregion and cell-type specificity of the activated neurons, we measured colabeling of Fos with Drd1 and Drd2 in three DS subregions. Fos expression was neither subregion nor cell-type specific (52.5 and 39.2% of Fos expression colabeled with Drd1 and Drd2, respectively). Finally, we found that DS injections of SCH23390 (C17H18ClNO), a D1-family receptor antagonist known to block cue-induced Fos induction, decreased incubated cue-induced methamphetamine seeking after prolonged withdrawal. Results demonstrate a critical role of DS in incubation of methamphetamine craving and that this incubation is associated with selective gene-expression alterations in cue-activated D1- and D2-expressing DS neurons.

Morphine and cocaine increase serum- and glucocorticoid-inducible kinase 1 activity in the ventral tegmental area.

Drugs of abuse modulate the function and activity of the mesolimbic dopamine circuit. To identify novel mediators of drug-induced neuroadaptations in the ventral tegmental area (VTA), we performed RNA sequencing analysis on VTA samples from mice administered repeated saline, morphine, or cocaine injections. One gene that was similarly up-regulated by both drugs was serum- and glucocorticoid-inducible kinase 1 (SGK1). SGK1 activity, as measured by phosphorylation of its substrate N-myc downstream regulated gene (NDRG), was also increased robustly by chronic drug treatment. Increased NDRG phosphorylation was evident 1 but not 24 h after the last drug injection. SGK1 phosphorylation itself was similarly modulated. To determine the role of increased SGK1 activity on drug-related behaviors, we over-expressed constitutively active (CA) SGK1 in the VTA. SGK1-CA expression reduced locomotor sensitization elicited by repeated cocaine, but surprisingly had the opposite effect and promoted locomotor sensitization to morphine, without affecting the initial locomotor responses to either drug. SGK1-CA expression did not significantly affect morphine or cocaine conditioned place preference, although there was a trend toward increased conditioned place preference with both drugs. Further characterizing the role of this kinase in drug-induced changes in VTA may lead to improved understanding of neuroadaptations critical to drug dependence and addiction. We find that repeated, but not acute, morphine or cocaine administration induces an increase in serum- and glucocorticoid-inducible kinase (SGK1) gene expression and activity in the ventral tegmental area (VTA). This increase in SGK1 activity may play a role in drug-dependent behaviors and suggests a novel signaling cascade for potential intervention in drug dependence and addiction.

Implementation of influenza-like illness sentinel surveillance in Togo.

BACKGROUND: The emergence of avian influenza A/H5N1 in 2003 as well as the pandemic influenza A (H1N1) pdm09 highlighted the need to establish influenza sentinel surveillance in Togo. The Ministry of Health decided to introduce Influenza to the list of diseases with epidemic potential. By April 2010, Togo was actively involved in influenza surveillance. This study aims to describe the implementation of ILI surveillance and results obtained from April 2010 to December 2012. METHODS: Two sites were selected based on their accessibility and affordability to patients, their adequate specimen storage capacity and transportation system. Patients with ILI presenting at sentinel sites were enrolled by trained medical staff based on the World Health Organization (WHO) case definitions. Oropharyngeal and nasopharyngeal samples were collected and they were tested at the National Influenza Reference Laboratory using a U.S. Centers for Disease Control and Prevention (CDC) validated real time RT-PCR protocol. Laboratory results and epidemiological data were reported weekly and shared with all sentinel sites, Ministry of Health, Division of Epidemiology, WHO and CDC/NAMRU-3. RESULTS: From April 2010 to December 2012, a total of 955 samples were collected with 52% of the study population aged between 0 and 4 years. Of the 955 samples, 236 (24.7%) tested positive for influenza viruses; with 136 (14.2%) positive for influenza A and 100 (10.5%) positive for influenza B. The highest influenza positive percentage (30%) was observed in 5-14 years old and patients aged 0-4 and >60 years had the lowest percentage (20%). Clinical symptoms such as cough and rhinorrhea were associated more with ILI patients who were positive for influenza type A than influenza type B. Influenza viruses circulated throughout the year with the positivity rate peaking around the months of January, May and again in October; corresponding respectively to the dry-dusty harmattan season and the long and then the short raining season. The pandemic A (H1N1) pdm09 was the predominantly circulating strain in 2010 while influenza B was the predominantly circulating strain in 2011. The seasonal A/H3N2 was observed throughout 2012 year. CONCLUSIONS: This study provides information on influenza epidemiology in the capital city of Togo.

G9a influences neuronal subtype specification in striatum.

Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we found that G9a repression by cocaine occurred in both Drd1-expressing (striatonigral) and Drd2-expressing (striatopallidal) medium spiny neurons. Conditional knockout and overexpression of G9a within these distinct cell types, however, revealed divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicated that such developmental deletion of G9a selectively in Drd2 neurons resulted in the unsilencing of transcriptional programs normally specific to striatonigral neurons and in the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral 'switching' phenotype in mice indicates a new role for G9a in contributing to neuronal subtype identity and suggests a critical function for cell type-specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli.

National inventory of core capabilities for pandemic influenza preparedness and response: results from 36 countries with reviews in 2008 and 2010.

BACKGROUND: Re-emergence in 2003 of human cases of avian H5N1 and the resultant spread of the disease highlighted the need to improve the capacity of countries to detect and contain novel viruses. To assess development in this capacity, the Centers for Disease Control and Prevention (CDC) produced a tool for assessing a country's capability in 12 critical areas related to pandemic preparedness, including monitoring and identifying novel influenza viruses. OBJECTIVES: Capabilities the CDC tool assesses range from how well a country has planned and is prepared for an outbreak to how prepared a country is to respond when a pandemic occurs. Included in this assessment tool are questions to determine whether a country has a detailed preparedness plan and the laboratory capacity to identify various strains of influenza quickly and accurately. METHODS: The tool was used first in 2008 when 40 countries in collaboration with CDC calculated baseline scores and used a second time in 2010 by 36 of the original 40 countries to determine whether they had improved their preparedness. Using basic mathematical comparison and statistical analyses, we compared data at the aggregate capability level as well as at the indicator and country levels. Additionally, we examined the comments of respondents to the assessment questionnaire for reasons (positive and negative) that would explain changes in scores from 2008 to 2010. RESULTS: Analysis of results of two assessments in 36 countries shows statistically significant improvement in all 12 capabilities on an aggregate level and 47 of 50 indicators.

ΔFosB induction in prefrontal cortex by antipsychotic drugs is associated with negative behavioral outcomes.

ΔFosB, a FosB gene product, is induced in the prefrontal cortex (PFC) by repeated exposure to several stimuli including antipsychotic drugs such as haloperidol. However, the functional consequences of increased ΔFosB expression following antipsychotic treatment have not been explored. Here, we assessed whether ΔFosB induction by haloperidol mediates the positive or negative consequences or clinical-related actions of antipsychotic treatment. We show that individuals with schizophrenia who were medicated with antipsychotic drugs at their time of death display increased ΔFosB levels in the PFC, an effect that is replicated in rats treated chronically with haloperidol. In contrast, individuals with schizophrenia who were medication-free did not exhibit this effect. Viral-mediated overexpression of ΔFosB in the PFC of rodents induced cognitive deficits as measured by inhibitory avoidance, increased startle responses in prepulse inhibition tasks, and increased MK-801-induced anxiety-like behaviors. Together, these results suggest that ΔFosB induction in the PFC by antipsychotic treatment contributes to the deleterious effects of these drugs and not to their therapeutic actions.

Class I HDAC inhibition blocks cocaine-induced plasticity by targeted changes in histone methylation.

Induction of histone acetylation in the nucleus accumbens (NAc), a key brain reward region, promotes cocaine-induced alterations in gene expression. Histone deacetylases (HDACs) tightly regulate the acetylation of histone tails, but little is known about the functional specificity of different HDAC isoforms in the development and maintenance of cocaine-induced plasticity, and previous studies of HDAC inhibitors report conflicting effects on cocaine-elicited behavioral adaptations. Here we demonstrate that specific and prolonged blockade of HDAC1 in NAc of mice increased global levels of histone acetylation, but also induced repressive histone methylation and antagonized cocaine-induced changes in behavior, an effect mediated in part through a chromatin-mediated suppression of GABAA receptor subunit expression and inhibitory tone on NAc neurons. Our findings suggest a new mechanism by which prolonged and selective HDAC inhibition can alter behavioral and molecular adaptations to cocaine and inform the development of therapeutics for cocaine addiction.

Epigenetic regulation of RAC1 induces synaptic remodeling in stress disorders and depression.

Depression induces structural and functional synaptic plasticity in brain reward circuits, although the mechanisms promoting these changes and their relevance to behavioral outcomes are unknown. Transcriptional profiling of the nucleus accumbens (NAc) for Rho GTPase-related genes, which are known regulators of synaptic structure, revealed a sustained reduction in RAS-related C3 botulinum toxin substrate 1 (Rac1) expression after chronic social defeat stress. This was associated with a repressive chromatin state surrounding the proximal promoter of Rac1. Inhibition of class 1 histone deacetylases (HDACs) with MS-275 rescued both the decrease in Rac1 transcription after social defeat stress and depression-related behavior, such as social avoidance. We found a similar repressive chromatin state surrounding the RAC1 promoter in the NAc of subjects with depression, which corresponded with reduced RAC1 transcription. Viral-mediated reduction of Rac1 expression or inhibition of Rac1 activity in the NAc increases social defeat-induced social avoidance and anhedonia in mice. Chronic social defeat stress induces the formation of stubby excitatory spines through a Rac1-dependent mechanism involving the redistribution of synaptic cofilin, an actin-severing protein downstream of Rac1. Overexpression of constitutively active Rac1 in the NAc of mice after chronic social defeat stress reverses depression-related behaviors and prunes stubby spines. Taken together, our data identify epigenetic regulation of RAC1 in the NAc as a disease mechanism in depression and reveal a functional role for Rac1 in rodents in regulating stress-related behaviors.

Neurobiological sequelae of witnessing stressful events in adult mice.

BACKGROUND: It is well known that exposure to severe stress increases the risk for developing mood disorders. However, most chronic stress models in rodents involve at least some form of physically experiencing traumatic events. METHODS: This study assessed the effects of a novel social stress paradigm that is insulated from the effects of physical stress. Specifically, adult male C57BL/6J mice were exposed to either emotional (ES) or physical stress (PS) for 10 minutes per day for 10 days. The ES mice were exposed to the social defeat of a PS mouse by a larger, more aggressive CD-1 mouse from the safety of an adjacent compartment. RESULTS: Like PS mice, ES mice exhibited a range of depression- and anxiety-like behaviors both 24 hours and 1 month after the stress. Increased levels of serum corticosterone, part of the stress response, accompanied these behavioral deficits. Based on previous work that implicated gene expression changes in the ventral tegmental area (a key brain reward region) in the PS phenotype, we compared genome-wide mRNA expression patterns in this brain region of ES and PS mice using RNA-seq. We found significant overlap between these conditions, which suggests several potential gene targets for mediating the behavioral abnormalities observed. CONCLUSIONS: These findings demonstrate that witnessing traumatic events is a potent stress in adult male mice capable of inducing long-lasting neurobiological perturbations.