Chronic alcohol-induced long-lasting working memory deficits are associated with altered histone H3K9 dimethylation in the prefrontal cortex.

Introduction: Epigenetic modifications have emerged as key contributors to the enduring behavioral, molecular and epigenetic neuroadaptations during withdrawal from chronic alcohol exposure. The present study investigated the long-term consequences of chronic alcohol exposure on spatial working memory (WM) and associated changes of transcriptionally repressive histone H3 lysine 9 dimethylation (H3K9me2) in the prefrontal cortex (PFC). Methods: Male C57BL/6 mice were allowed free access to either 12% (v/v) ethanol for 5 months followed by a 3-week abstinence period or water. Spatial WM was assessed through the spontaneous alternation T-maze test. Alcoholic and water mice received daily injections of GABAB agonist baclofen or saline during alcohol fading and early withdrawal. Global levels of histone modifications were determined by immunohistochemistry. Results: Withdrawal mice displayed WM impairments along with reduced prefrontal H3K9me2 levels, compared to water-drinking mice. The withdrawal-induced decrease of H3K9me2 occurred concomitantly with increased level of permissive H3K9 acetylation (H3K9ac) in the PFC. Baclofen treatment rescued withdrawal-related WM deficits and fully restored prefrontal H3K9me2 and H3K9ac. Alcohol withdrawal induced brain region-specific changes of H3K9me2 and H3K9ac after testing, with significant decreases of both histone marks in the dorsal hippocampus and no changes in the amygdala and dorsal striatum. Furthermore, the magnitude of H3K9me2 in the PFC, but not the hippocampus, significantly and positively correlated with individual WM performances. No correlation was observed between H3K9ac and behavioral performance. Results also indicate that pre-testing intraperitoneal injection of UNC0642, a selective inhibitor of histone methyltransferase G9a responsible for H3K9me2, led to WM impairments in water-drinking and withdrawal-baclofen mice. Collectively, our results demonstrate that alcohol withdrawal induced brain-region specific alterations of H3K9me2 and H3K9ac, an effect that persisted for at least three weeks after cessation of chronic alcohol intake. Conclusion: The findings suggest a role for long-lasting decreased H3K9me2 specifically in the PFC in the persistent WM impairments related to alcohol withdrawal.

Acute pre-learning stress selectively impairs hippocampus-dependent fear memory consolidation: Behavioral and molecular evidence.

Despite compelling evidence that stress or stress-related hormones influence fear memory consolidation processes, the understanding of molecular mechanisms underlying the effects of stress is still fragmentary. The release of corticosterone in response to pre-learning stress exposure has been demonstrated to modulate positively or negatively memory encoding and/or consolidation according to many variables such as stress intensity, the emotional valence of the learned material or the interval between stressful episode and learning experience. Here, we report that contextual but not cued fear memory consolidation was selectively impaired in male mice exposed to a 50 min-period of restraint stress just before the unpaired fear conditioning session. In addition to behavioral impairment, acute stress down-regulated activated/phosphorylated ERK1/2 (pERK1/2) in dorsal hippocampal area CA1 in mice sacrificed 60 min and 9 h after unpaired conditioning. In lateral amygdala, although acute stress by itself increased the level of pERK1/2 it nevertheless blocked the peak of pERK1/2 that was normally observed 15 min after unpaired conditioning. To examine whether stress-induced corticosterone overflow was responsible of these detrimental effects, the corticosterone synthesis inhibitor, metyrapone, was administered 30 min before stress exposure. Metyrapone abrogated the stress-induced contextual fear memory deficits but did not alleviate the effects of stress on pERK1/2 and its downstream target phosphorylated CREB (pCREB) in hippocampus CA1 and lateral amygdala. Collectively, our observations suggest that consolidation of hippocampus-dependent memory and the associated signaling pathway are particularly sensitive to stress. However, behavioral normalization by preventive metyrapone treatment was not accompanied by renormalization of the canonical signaling pathway. A new avenue would be to consider surrogate mechanisms involving proper metyrapone influence on both nongenomic and genomic actions of glucocorticoid receptors.

The histone H3 lysine 9 methyltransferase G9a/GLP complex activity is required for long-term consolidation of spatial memory in mice.

The G9a/G9a-like protein (GLP) histone lysine dimethyltransferase complex and downstream histone H3 lysine 9 dimethylation (H3K9me2) repressive mark have recently emerged as key transcriptional regulators of gene expression programs necessary for long-term memory (LTM) formation in the dorsal hippocampus. However, the role for hippocampal G9a/GLP complex in mediating the consolidation of spatial LTM remains largely unknown. Using a water maze competition task in which both dorsal hippocampus-dependent spatial and striatum-dependent cue navigation strategies are effective to solve the maze, we found that pharmacological inhibition of G9a/GLP activity immediately after learning disrupts long-term consolidation of previously learned spatial information in male mice, hence producing cue bias on the competition test performed 24 h later. Importantly, the inhibition of hippocampal G9a/GLP did not disrupt short-term memory retention. Immunohistochemical analyses revealed increases in global levels of permissive histone H3K9 acetylation in the dorsal hippocampus and dorsal striatum at 1 h post-training, which persisted up to 24 h in the hippocampus. Conversely, H3K9me2 levels were either unchanged in the dorsal hippocampus or transiently decreased at 15 min post-training in the dorsal striatum. Finally, the inhibition of G9a/GLP activity further increased global levels of H3K9 acetylation while decreasing H3K9me2 in the hippocampus at 1 h post-training. However, both marks returned to vehicle control levels at 24 h. Together, these findings support the possibility that G9a/GLP in the dorsal hippocampus is required for the transcriptional switch from short-term to long-term spatial memory formation.

Targeting the Glucocorticoid Receptors During Alcohol Withdrawal to Reduce Protracted Neurocognitive Disorders.

Persistent regional glucocorticoid (GC) dysregulation in alcohol-withdrawn subjects emerges as a key factor responsible for protracted molecular and neural alterations associated with long-term cognitive dysfunction. Regional brain concentrations of corticosterone vary independently from plasma concentrations in alcohol-withdrawn subjects, which may account for the treatment of alcohol withdrawal-induced persistent pathology. Thus, from a pharmacological point of view, a main issue remains to determine the relative efficacy of compounds targeting the GC receptors to attenuate or suppress the long-lasting persistence of brain regional GC dysfunctions in abstinent alcoholics, as well as persistent changes of neural plasticity. Data from animal research show that acting directly on GC receptors during the withdrawal period, via selective antagonists, can significantly counteract the development and persistence of cognitive and neural plasticity disorders during protracted abstinence. A critical remaining issue is to better assess the relative long-term efficacy of GC antagonists and other compounds targeting the corticotropic axis activity such as gamma-aminobutyric acid A (GABAA) and GABAB agonists. Indeed, benzodiazepines (acting indirectly on GABAA receptors) and baclofen (agonist of the GABAB receptor) are the compounds most widely used to reduce alcohol dependence. Clinical and preclinical data suggest that baclofen exerts an effective and more powerful counteracting action on such persistent cognitive and endocrine dysfunctions as compared to diazepam, even though its potential negative effects on memory processes, particularly at high doses, should be better taken into account.

Sustained corticosterone rise in the prefrontal cortex is a key factor for chronic stress-induced working memory deficits in mice.

Exposure to prolonged, unpredictable stress leads to glucocorticoids-mediated long-lasting neuroendocrine abnormalities associated with emotional and cognitive impairments. Excessive levels of serum glucocorticoids (cortisol in humans, corticosterone in rodents) contribute notably to deficits in working memory (WM), a task which heavily relies on functional interactions between the medial prefrontal cortex (PFC) and the dorsal hippocampus (dHPC). However, it is unknown whether stress-induced increases in plasma corticosterone mirror corticosterone levels in specific brain regions critical for WM. After a 6 week-UCMS exposure, C57BL/6 J male mice exhibited increased anxiety- and depressive-like behaviors when measured one week later and displayed WM impairments timely associated with increased plasma corticosterone response. In chronically stressed mice, basal phosphorylated/activated CREB (pCREB) was markedly increased in the PFC and the CA1 area of the dHPC and WM testing did not elicit any further increase in pCREB in the two regions. Using microdialysis samples from freely-moving mice, we found that WM testing co-occurred with a rapid and sustained increase in corticosterone response in the PFC while there was a late, non-significant rise of corticosterone in the dHPC. The results also show that non-stressed mice injected with corticosterone (2 mg/kg i.p.) before WM testing displayed behavioral and molecular alterations similar to those observed in stressed animals while a pre-WM testing metyrapone injection (35 mg/kg i.p.), a corticosterone synthesis inhibitor, prevented the effects of UCMS exposure. Overall, the abnormal regional increase of corticosterone concentrations mainly in the PFC emerges as a key factor of enduring WM dysfunctions in UCMS-treated animals.

Protocols to Study Declarative Memory Formation in Mice and Humans:Optogenetics and Translational Behavioral Approaches.

Declarative memory formation depends on the hippocampus and declines in aging. Two functions of the hippocampus, temporal binding and relational organization (Rawlins and Tsaltas, 1983; Eichenbaum et al., 1992 ; Cohen et al., 1997 ), are known to decline in aging (Leal and Yassa, 2015). However, in the literature distinct procedures have been used to study these two functions. Here, we describe the experimental procedures used to investigate how these two processes are related in the formation of declarative memory and how they are compromised in aging ( Sellami et al., 2017 ). First, we studied temporal binding using a one-trial learning procedure: trace fear conditioning. It is classical Pavlovian conditioning requiring temporal binding since a brief temporal gap separates the conditioned stimulus (CS) and unconditioned stimulus (US) presentations. We combined the trace fear condition procedure with an optogenetic approach, and we showed that the temporal binding relies on dorsal (d)CA1 activity over temporal gaps. Then, we studied the interaction between temporal binding and relational organization in declarative memory formation using a two-phase radial-maze task in mice and its virtual analog in humans. The behavioral procedure comprises an initial learning phase where subjects learned the constant rewarding /no rewarding valence of each arm, followed by a test phase where the reward contingencies among the arms remained unchanged but where the arms were recombined to assess flexibility, a cardinal property of declarative memory. We demonstrated that dCA1-dependent temporal binding is necessary for the development of a relational organization of memories that allows flexible declarative memory expression. Furthermore, in aging, the degradation of declarative memory is due to a reduction of temporal binding capacity that prevents relation organization.

Changes in striatal activity and functional connectivity in a mouse model of Huntington's disease.

Hereditary Huntington's disease (HD) is associated with progressive motor, cognitive and psychiatric symptoms. A primary consequence of the HD mutation is the preferential loss of medium spiny projection cells with relative sparing of local interneurons in the striatum. In addition, among GABAergic striatal projection cells, indirect pathway cells expressing D2 dopamine receptors are lost earlier than direct pathway cells expressing D1 receptors. To test in vivo the functional integrity of direct and indirect pathways as well as interneurons in the striatum of male R6/1 transgenic mice, we assessed their c-Fos expression levels induced by a striatal-dependent cognitive task and compared them with age-matched wild-type littermates. We found a significant increase of c-Fos+ nuclei in the dorsomedial striatum, and this only at 2 months, when our HD mouse model is still pre-motor symptomatic, the increase disappearing with symptom manifestation. Contrary to our expectation, the indirect pathway projection neurons did not undergo any severer changes of c-Fos expression regardless of age in R6/1 mice. We also found a decreased activation of interneurons that express parvalbumin in the dorsomedial striatum at both presymptomatic and symptomatic ages. Finally, analysis of c-Fos expression in extended brain regions involved in the cognitive learning used in our study, demonstrates, throughout ages studied, changes in the functional connectivity between regions in the transgenic mice. Further analysis of the cellular and molecular changes underlying the transient striatal hyperactivity in the HD mice may help to understand the mechanisms involved in the disease onset.

Temporal binding function of dorsal CA1 is critical for declarative memory formation.

Temporal binding, the process that enables association between discontiguous stimuli in memory, and relational organization, a process that enables the flexibility of declarative memories, are both hippocampus-dependent and decline in aging. However, how these two processes are related in supporting declarative memory formation and how they are compromised in age-related memory loss remain hypothetical. We here identify a causal link between these two features of declarative memory: Temporal binding is a necessary condition for the relational organization of discontiguous events. We demonstrate that the formation of a relational memory is limited by the capability of temporal binding, which depends on dorsal (d)CA1 activity over time intervals and diminishes in aging. Conversely, relational representation is successful even in aged individuals when the demand on temporal binding is minimized, showing that relational/declarative memory per se is not impaired in aging. Thus, bridging temporal intervals by dCA1 activity is a critical foundation of relational representation, and a deterioration of this mechanism is responsible for the age-associated memory impairment.

Alcohol withdrawal induces long-lasting spatial working memory impairments: relationship with changes in corticosterone response in the prefrontal cortex.

This study intends to determine whether long-lasting glucocorticoids (GCs) dysregulation in the prefrontal cortex (PFC) or the dorsal hippocampus (dHPC) play a causal role in the maintenance of working memory (WM) deficits observed after alcohol withdrawal. Here, we report that C57/BL6 male mice submitted to 6 months alcohol consumption (12 percent v/v) followed by 1 (1W) or 6 weeks (6W) withdrawal periods exhibit WM deficits in a spatial alternation task and an exaggerated corticosterone rise during and after memory testing in the PFC but not the dHPC. In contrast, emotional reactivity evaluated in a plus-maze is altered only in the 1W group. No behavioral alterations are observed in mice still drinking alcohol. To determine the causal role of corticosterone in the withdrawal-associated long-lasting WM deficits, we further show that a single intraperitoneal injection injection of metyrapone (an inhibitor of corticosterone synthesis) 30 minutes before testing, prevents withdrawal-associated WM deficits and reestablishes PFC activity, as assessed by increased phosphorylated C-AMP Response Element-binding protein (CREB) immunoreactivity in withdrawn mice. Finally, we show that intra-PFC blockade of mineralocorticoid receptors by infusion of spironolactone and, to a lesser extent, of GCs receptors by injection of mifepristone reverses the WM deficits induced by withdrawal whereas the same injections into the dHPC do not. Overall, our study evidences that long-lasting GCs dysfunction selectively in the PFC is responsible for the emergence and maintenance of WM impairments after withdrawal and that blocking prefrontal mineralocorticoid receptors receptors restores WM in withdrawn animals.

Behavioral Neuroadaptation to Alcohol: From Glucocorticoids to Histone Acetylation.

A prime mechanism that contributes to the development and maintenance of alcoholism is the dysregulation of the hypothalamic-pituitary-adrenal axis activity and the release of glucocorticoids (cortisol in humans and primates, corticosterone in rodents) from the adrenal glands. In the brain, sustained, local elevation of glucocorticoid concentration even long after cessation of chronic alcohol consumption compromises functional integrity of a circuit, including the prefrontal cortex (PFC), the hippocampus (HPC), and the amygdala (AMG). These structures are implicated in learning and memory processes as well as in orchestrating neuroadaptive responses to stress and anxiety responses. Thus, potentiation of anxiety-related neuroadaptation by alcohol is characterized by an abnormally AMG hyperactivity coupled with a hypofunction of the PFC and the HPC. This review describes research on molecular and epigenetic mechanisms by which alcohol causes distinct region-specific adaptive changes in gene expression patterns and ultimately leads to a variety of cognitive and behavioral impairments on prefrontal- and hippocampal-based tasks. Alcohol-induced neuroadaptations involve the dysregulation of numerous signaling cascades, leading to long-term changes in transcriptional profiles of genes, through the actions of transcription factors such as [cAMP response element-binding protein (CREB)] and chromatin remodeling due to posttranslational modifications of histone proteins. We describe the role of prefrontal-HPC-AMG circuit in mediating the effects of acute and chronic alcohol on learning and memory, and region-specific molecular and epigenetic mechanisms involved in this process. This review first discusses the importance of brain region-specific dysregulation of glucocorticoid concentration in the development of alcohol dependence and describes how persistently increased glucocorticoid levels in PFC may be involved in mediating working memory impairments and neuroadaptive changes during withdrawal from chronic alcohol intake. It then highlights the role of cAMP-PKA-CREB signaling cascade and histone acetylation within the PFC and limbic structures in alcohol-induced anxiety and behavioral impairments, and how an understanding of functional alterations of these pathways might lead to better treatments for neuropsychiatric disorders.