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The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA’s 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU ‘on the ground’ in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Understanding compulsive drinking behavior is key to improving outcomes in the treatment of addiction. In the present study, we investigated compulsive-like drinking in alcohol-addicted rats using the alcohol deprivation effect (ADE) model of relapse behavior, which involves repeated deprivation and reintroduction phases; the latter approximate relapse.High-resolution longitudinal drinking and locomotor data were measured while rats (n = 30) underwent a four-bottle (water, 5%, 10%, 20% alcohol v/v) free-choice ADE paradigm. Alcohol bottles were adulterated with the bitter compound quinine during a reintroduction phase to test for compulsive behavior. We characterized how drinking and locomotor behavior during ADE + quinine differed from a regular ADE and how, at the individual level, behavioral parameters extracted from the regular ADE related to compulsive-like drinking. Associations of drinking with locomotor activity were also examined.In the ADE with quinine, we observed reduced consumption of alcohol and a shift to preference for stronger alcohol. Quinine acted by decreasing both the access size and frequency of drinking of 5% alcohol while increasing the frequency of consumption of 20% alcohol. Preference for higher alcohol concentrations prior to the quinine challenge was associated with greater compulsive-like drinking behavior; higher baseline consumption of 20% alcohol correlated with more drinking of quinine-adulterated solutions while high frequency and amount of 5% alcohol consumption at baseline were correlated with being more strongly affected by quinine. Associations between locomotor activity and drinking behavior were observed at the hourly level. These associations reflected changing preferences across experimental phases.Drinking patterns, and specifically solution preference, may offer insights into the presentation of compulsive-like drinking. The findings provide a preclinical basis for observations from epidemiological studies that link higher risk and burden of alcohol-related disease to stronger alcohol concentrations and encourage further translational studies to better understand the underlying mechanisms.

The potential of psychedelics to persistently treat substance use disorders is known since the 1960s. However, the biological mechanisms responsible for their therapeutic effects have not yet been fully elucidated. While it is known that serotonergic hallucinogens induce changes in gene expression and neuroplasticity, particularly in prefrontal regions, theories on how specifically this counteracts the alterations that occur in neuronal circuitry throughout the course of addiction are largely unknown. This narrative mini-review endeavors to synthesize well-established knowledge from addiction research with findings and theories regarding the neurobiological effects of psychedelics to give an overview of the potential mechanisms that underlie the treatment of substance use disorders with classical hallucinogenic compounds and point out gaps in the current understanding.

There is increasing evidence for a daily rhythm of µ-opioid receptor (MOR) efficacy and the development of alcohol dependence. Previous studies show that β-arrestin 2 (bArr2) has an impact on alcohol intake, at least partially mediated via modulation of MOR signaling, which in turn mediates the alcohol rewarding effects. Considering the interplay of circadian rhythms on MOR and alcohol dependence, we aimed to investigate bArr2 in alcohol dependence at different time points of the day/light cycle on the level of bArr2 mRNA (in situ hybridization), MOR availability (receptor autoradiography), and MOR signaling (Damgo-stimulated G-protein coupling) in the nucleus accumbens of alcohol-dependent and non-dependent Wistar rats. Using a microarray data set we found that bArr2, but not bArr1, shows a diurnal transcription pattern in the accumbens of naïve rats with higher expression levels during the active cycle. In 3-week abstinent rats, bArr2 is up-regulated in the accumbens at the beginning of the active cycle (ZT15), whereas no differences were found at the beginning of the inactive cycle (ZT3) compared with controls. This effect was accompanied by a specific down-regulation of MOR binding in the active cycle. Additionally, we detect a higher receptor coupling during the inactive cycle compared with the active cycle in alcohol-dependent animals. Together, we report daily rhythmicity for bArr2 expression linked to an inverse pattern of MOR, suggesting an involvement for bArr2 on circadian regulation of G-protein coupled receptors in alcohol dependence. The presented data may have implications for the development of novel bArr2-related treatment targets for alcoholism. (Figure presented.).

In Alzheimer disease, Tau pathology is thought to propagate from cell to cell throughout interconnected brain areas. However, the forms of Tau released into the brain interstitial fluid (ISF) in vivo during the development of Tauopathy and their pathological relevance remain unclear. Combining in vivo microdialysis and biochemical analysis, we find that in Tau transgenic mice, human Tau (hTau) present in brain ISF is truncated and comprises at least 10 distinct fragments spanning the entire Tau protein. The fragmentation pattern is similar across different Tau transgenic models, pathological stages and brain areas. ISF hTau concentration decreases during Tauopathy progression, while its phosphorylation increases. ISF from mice with established Tauopathy induces Tau aggregation in HEK293-Tau biosensor cells. Notably, immunodepletion of ISF phosphorylated Tau, but not Tau fragments, significantly reduces its ability to seed Tau aggregation and only a fraction of Tau, separated by ultracentrifugation, is seeding competent. These results indicate that ISF seeding competence is driven by a small subset of Tau, which potentially contribute to the propagation of Tau pathology.HighlightsIn interstitial fluid (ISF) of transgenic mice, Tau comprises >10 distinct fragmentsISF Tau decreases with Tauopathy progression, while its phosphorylation increasesOnly ISF from mice with established Tauopathy is seeding competent in vitroRemoval of phospho-Tau reduces ISF seeding competenceISF seeding competence is driven by less soluble, aggregated and phosphorylated TauGraphical abstract

Biological factors are known to influence disease trajectories and treatment effectiveness in alcohol addiction. One important biological factor influencing disease dynamics in alcohol use disorder is sex, as preclinical and clinical evidence suggests that gender influences disease trajectories in subjects with alcohol use disorder. Another critical factor is age at first intoxicating drink, which has been identified as a risk factor for later alcohol binging. Preclinical research allows prospective monitoring of rodents throughout the lifespan, providing very detailed information that cannot be acquired in humans. Lifetime monitoring in rodents can be conducted under highly controlled conditions, during which one can systematically introduce multiple biological and environmental factors that impact behaviors of interest.Here, we used the alcohol deprivation effect (ADE) rat model of alcohol addiction in a computerized drinkometer system to study disease dynamics and transition points of addictive behavior in cohorts of adolescent vs. adult as well as male vs. female rats in direct comparison. Early age of onset of drinking (postnatal day 40) in male rats had surprisingly little impact on the development of drinking behavior and compulsivity (quinine taste adulteration) when compared to rats that started drinking during early adulthood (postnatal day 72). However, female rats showed a stronger resistance to quinine taste adulteration after a history of ADEs, indicative of more pronounced compulsive behavior in females as compared to male rats. Specifically, female rats exhibit during quinine taste adulteration a higher frequency of drinking events and a larger access size, especially from the 20% alcohol solution, resulting in fast intoxication despite taste aversion in a relapse-like situation. Our results suggest that drinking patterns, and specifically solution preference as well as access size, provide a valuable indication into determining compulsive drinking. These findings provide a better understanding of sex and age factors involved in the development of drinking behavior that are important in preclinical development of models of addiction, drug development and exploration options for new treatments.

Clinical translation from bench to bedside often remains challenging even despite promising preclinical evidence. Among many drivers like biological complexity or poorly understood disease pathology, preclinical evidence often lacks desired robustness. Reasons include low sample sizes, selective reporting, publication bias, and consequently inflated effect sizes. In this context, there is growing consensus that confirmatory multicenter studies -by weeding out false positives- represent an important step in strengthening and generating preclinical evidence before moving on to clinical research. However, there is little guidance on what such a preclinical confirmatory study entails and when it should be conducted in the research trajectory. To close this gap, we organized a workshop to bring together statisticians, clinicians, preclinical scientists, and meta-researcher to discuss and develop recommendations that are solution-oriented and feasible for practitioners. Herein, we summarize and review current approaches and outline strategies that provide decision-critical guidance on when to start and subsequently how to plan a confirmatory study. We define a set of minimum criteria and strategies to strengthen validity before engaging in a confirmatory preclinical trial, including sample size considerations that take the inherent uncertainty of initial (exploratory) studies into account. Beyond this specific guidance, we highlight knowledge gaps that require further research and discuss the role of confirmatory studies in translational biomedical research. In conclusion, this workshop report highlights the need for close interaction and open and honest debate between statisticians, preclinical scientists, meta-researchers (that conduct research on research), and clinicians already at an early stage of a given preclinical research trajectory.

BACKGROUND Accumulating clinical evidence suggests that women with prior exposure to adverse childhood experiences are more susceptible to heavy drinking and other health-related behaviors. Yet, preclinical studies investigating sex-dependent effects of adolescent adverse social experiences (ASEs) on later alcohol-seeking behavior are lacking. This is mainly due to the unavailability of valid animal models and a shortage of studies that compare effects in males and females. Therefore, we sought to investigate the sex-dependent effects of ASE on adult alcohol-seeking behavior, locomotion, and reward sensitivity in male and female rats. METHODS We recently developed a rat model for childhood/adolescent peer rejection that allows us to study the long-term consequences of ASEs. Adolescent Wistar rats were reared from postnatal day (pd) 21 to pd 50 either within a group of Fischer 344 rats (ASE) or within a group of Wistar rats (control). Wistar rats housed with Fischer 344 rats do not reciprocate social play in adolescence. This reduced play across adolescence mimics peer rejection and results in chronic dysregulation of social and pain-related behaviors. We tested adult male and female rats in the reinstatement paradigm for cue-induced alcohol-seeking behavior, circadian locomotor activity, and sucrose consumption long after the termination of the peer rejection condition. RESULTS Peer rejection induced persistent sex-dependent changes in alcohol cue-induced reinstatement. Females showed an increased reinstatement effect while peer-rejected males demonstrated a decrease. Sex differences were observed in locomotor activity or reward sensitivity to sucrose. CONCLUSIONS Peer rejection has long-lasting sex-dependent consequences on alcohol-seeking behavior without affecting locomotion or sweet reward sensitivity. Our results suggest that peer-rejected female rats represent a vulnerable population in which to study relapse-like behaviors that are similar to clinical findings, while males seem to buffer the peer rejection effect and demonstrate resilience to later life alcohol-seeking behaviors, as measured by the reinstatement effect. Finally, we provide a novel approach to investigate the molecular and neurobiological underpinnings of ASEs on alcohol and other drug-seeking behaviors.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term ‘the Three Rs’, which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years — not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA’s 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as ‘on the ground’ facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Alcohol abuse is a leading risk factor for the public health burden worldwide. Approved pharmacotherapies have demonstrated limited effectiveness over the last few decades in treating alcohol use disorders (AUD). New therapeutic approaches are therefore urgently needed. Historical and recent clinical trials using psychedelics in conjunction with psychotherapy demonstrated encouraging results in reducing heavy drinking in AUD patients, with psilocybin being the most promising candidate. While psychedelics are known to induce changes in gene expression and neuroplasticity, we still lack crucial information about how this specifically counteracts the alterations that occur in neuronal circuits throughout the course of addiction. This review synthesizes well-established knowledge from addiction research about pathophysiological mechanisms related to the metabotropic glutamate receptor 2 (mGlu2), with findings and theories on how mGlu2 connects to the major signaling pathways induced by psychedelics via serotonin 2A receptors (2AR). We provide literature evidence that mGlu2 and 2AR are able to regulate each other’s downstream signaling pathways, either through monovalent crosstalk or through the formation of a 2AR-mGlu2 heteromer, and highlight epigenetic mechanisms by which 2ARs can modulate mGlu2 expression. Lastly, we discuss how these pathways might be targeted therapeutically to restore mGlu2 function in AUD patients, thereby reducing the propensity to relapse.

Description, Alcohol-induced mGluR2 deficits are restored by psilocybin, resulting in a rescue of pathological behaviors in alcoholism., Alcohol-dependent patients commonly show impairments in executive functions that facilitate craving and can lead to relapse. However, the molecular mechanisms leading to executive dysfunction in alcoholism are poorly understood, and new effective pharmacological treatments are desired. Here, using a bidirectional neuromodulation approach, we demonstrate a causal link between reduced prefrontal mGluR2 function and both impaired executive control and alcohol craving. A neuron-specific prefrontal mGluR2 knockdown in rats generated a phenotype of reduced cognitive flexibility and excessive alcohol seeking. Conversely, virally restoring prefrontal mGluR2 levels in alcohol-dependent rats rescued these pathological behaviors. In the search for a pharmacological intervention with high translational potential, psilocybin was capable of restoring mGluR2 expression and reducing relapse behavior. Last, we propose a FDG-PET biomarker strategy to identify mGluR2 treatment-responsive individuals. In conclusion, we identified a common molecular pathological mechanism for both executive dysfunction and alcohol craving and provided a personalized mGluR2 mechanism-based intervention strategy for medication development for alcoholism.

BackgroundAccumulating clinical evidence suggests women with prior exposure to adverse childhood experiences are more susceptible to alcohol relapse and other health-related issues. Yet, preclinical studies investigating sex-dependent effects of adolescent adverse social experiences (ASEs) on later alcohol-seeking behavior are lacking. This is mainly due to a lack of valid animal models and a shortage of studies comparing sexes. Therefore, we sought to investigate the sex-dependent effects of ASE on adult alcohol-seeking behavior, locomotion and reward sensitivity in both male and female rats.MethodsWe recently developed a rat model for adolescent peer-rejection which allows us to study the long-term consequences of ASEs. Peer-rejection interferes with adolescent rats‘ability to engage in adequate and reciprocal play behaviors that result in persistent dysregulation of social and pain-related behavior. Adolescent Wistar rats were reared from postnatal day (pd) 21 to pd 50 either within a group of Fischer 344 rats (ASE) or with Wistar rats (control). Adult male and female rats were tested in the reinstatement paradigm for cue-induced alcohol-seeking behavior, circadian locomotor activity, and sucrose consumption in adulthood long-after the termination of the peer-rejection condition.ResultsPeer-rejection induced persistent sex-dependent changes to cue-induced reinstatement. Females showed an increased reinstatement effect while peer-rejected males demonstrated a decrease. No differences were observed in circadian locomotor activity or reward sensitivity to sucrose.ConclusionsPeer-rejection has lasting sex-dependent consequences on alcohol-seeking behavior without affecting locomotion or sweet reward sensitivity. Our results suggest that peer-rejected female rats represent a vulnerable population to study relapse-like behaviors similar to clinical findings. While males seem to buffer the peer-rejection effect and demonstrate resilience to later-life alcohol-seeking behaviors, measured by the reinstatement effect. Finally, we provide a novel approach to investigate the molecular and neurobiological underpinnings of ASEs on alcohol and other drug-seeking behaviors.

Anthrax lethal toxin (LT) is a protease virulence factor produced by Bacillus anthracis that is required for its pathogenicity. LT treatment causes a rapid degradation of c-Jun protein that follows inactivation of the MEK1/2-Erk1/2 signaling pathway. Here we identify COP1 as the ubiquitin E3 ligase that is essential for LT-induced c-Jun degradation. COP1 knockdown using siRNA prevents degradation of c-Jun, ETV4, and ETV5 in cells treated with either LT or the MEK1/2 inhibitor, U0126. Immunofluorescence staining reveals that COP1 preferentially localizes to the nuclear envelope, but it is released from the nuclear envelope into the nucleoplasm following Erk1/2 inactivation. At baseline, COP1 attaches to the nuclear envelope via interaction with translocated promoter region (TPR), a component of the nuclear pore complex. Disruption of this COP1–TPR interaction, through Erk1/2 inactivation or TPR knockdown, leads to rapid COP1 release from the nuclear envelope into the nucleoplasm where it degrades COP1 substrates. COP1-mediated degradation of c-Jun protein, combined with LT-mediated blockade of the JNK1/2 signaling pathway, inhibits cellular proliferation. This effect on proliferation is reversed by COP1 knockdown and ectopic expression of an LT-resistant MKK7-4 fusion protein. Taken together, this study reveals that the nuclear envelope acts as a reservoir, maintaining COP1 poised for action. Upon Erk1/2 inactivation, COP1 is rapidly released from the nuclear envelope, promoting the degradation of its nuclear substrates, including c-Jun, a critical transcription factor that promotes cellular proliferation. This regulation allows mammalian cells to respond rapidly to changes in extracellular cues and mediates pathogenic mechanisms in disease states.

For most psychiatric disorders, including alcohol use disorder (AUD), approved pharmacological treatments are limited in their effectiveness, and new drugs that can easily be translated into the clinic are needed. Currently, great hope lies in the potential of psychedelics to effectively treat AUD. The primary hypothesis is that a single session of psychedelic-guided psychotherapy can restore normal brain function in AUD individuals and thereby reduce the risk of relapse in the long run. Here we applied three different treatment schedules with psilocybin/LSD in order to investigate relapse-like drinking in the alcohol deprivation effect (ADE) model. In contrast to the primary hypothesis, psychedelics had no long-lasting effects on the ADE in male and female rats, neither when administered in a high dosage regime that is comparable to the one used in clinical studies, nor in a chronic microdosing scheme. Only sub-chronic treatment with psilocybin produced a short-lasting anti-relapse effect. However, it is not a translatable treatment option to give psychedelics sub-chronically for relapse prevention. In conclusion, our results in the ADE model do not support the hypothesis that microdosing or high doses of psychedelic reduce relapse behavior. This conclusion has to be confirmed by applying other animal models of AUD. It could also well be that animal models of AUD might be unable to fully capture the therapeutic potential of psychedelic drugs and that only future large-scale clinical trials will be able to demonstrate the efficacy of psychedelics as a new treatment option for AUD.

Despite its limited immediate reinforcement value, alcohol has a potent ability to induce neuroadaptations that promote its incentive salience, escalation of voluntary alcohol intake and aversion resistant alcohol seeking. A constellation of these traits, collectively called “post-dependent”, emerges following brain exposure to repeated cycles of intoxication and withdrawal. The medial prefrontal cortex (mPFC) and its subdivisions exert top-down regulation of approach and avoidance behaviors, including those that lead to alcohol intake. Here, we review an emerging literature which indicates that a reprogramming of mPFC function occurs with prolonged exposure of the brain to cycles of alcohol intoxication and withdrawal. This reprogramming results in molecular dysregulations that contribute to the post-dependent syndrome. Convergent evidence has identified neuroadaptations resulting in altered glutamatergic and BDNF-mediated signaling, and for these pathways, direct evidence for a mechanistic role has been obtained. Additional evidence points to a dysregulation of pathways involving calcium homeostasis and neurotransmitter release. Recent findings indicate that global DNA hypermethylation is a key factor in reprograming the mPFC genome after a history of dependence. As one of the results of this epigenetic remodeling, several histone modifying epigenetic enzymes are repressed. Among these, PR-domain zinc-finger protein 2 (PRDM2), a methyltransferase that selectively mono-methylates histone H3 at lysine 9 (H3K9) has been functionally validated to drive several of the molecular and behavioral long-term consequences of alcohol dependence. Information processing within the mPFC involves formation of dynamic neuronal networks, or functional ensembles that are shaped by transcriptional responses. The epigenetic dysregulations identified by our molecular studies are likely to alter this dynamic processing in multiple ways. In summary, epigenetic molecular switches in the mPFC appear to be turned on as alcoholism develops. Strategies to reverse these processes may offer targets for disease modifying treatments.

Epigenetic enzymes oversee long‐term changes in gene expression by integrating genetic and environmental cues. While there are hundreds of enzymes that control histone and DNA modifications, their potential roles in substance abuse and alcohol dependence remain underexplored. A few recent studies have suggested that epigenetic processes could underlie transcriptomic and behavioral hallmarks of alcohol addiction. In the present study, we sought to identify epigenetic enzymes in the brain that are dysregulated during protracted abstinence as a consequence of chronic and intermittent alcohol exposure. Through quantitative mRNA expression analysis of over 100 epigenetic enzymes, we identified 11 that are significantly altered in alcohol‐dependent rats compared with controls. Follow‐up studies of one of these enzymes, the histone demethylase KDM6B, showed that this enzyme exhibits region‐specific dysregulation in the prefrontal cortex and nucleus accumbens of alcohol‐dependent rats. KDM6B was also upregulated in the human alcoholic brain. Upregulation of KDM6B protein in alcohol‐dependent rats was accompanied by a decrease of trimethylation levels at histone H3, lysine 27 (H3K27me3), consistent with the known demethylase specificity of KDM6B. Subsequent epigenetic (chromatin immunoprecipitation [ChIP]–sequencing) analysis showed that alcohol‐induced changes in H3K27me3 were significantly enriched at genes in the IL‐6 signaling pathway, consistent with the well‐characterized role of KDM6B in modulation of inflammatory responses. Knockdown of KDM6B in cultured microglial cells diminished IL‐6 induction in response to an inflammatory stimulus. Our findings implicate a novel KDM6B‐mediated epigenetic signaling pathway integrated with inflammatory signaling pathways that are known to underlie the development of alcohol addiction., Through quantitative mRNA expression analysis of over 100 epigenetic enzymes, we identified 11 that are dysregulated in the brain of alcohol dependent rats. The histone 3, lysine 27 (H3K27me3) demethylase KDM6B, was found to have region‐specific dysregulation in the prefrontal cortex and nucleus accumbens and also in human alcoholic brain tissues. A ChIP‐sequencing analysis showed that alcohol‐induced changes in H3K27me3 were enriched at genes in the IL‐6 signaling pathway, thereby implicating a novel KDM6B‐mediated epigenetic signaling pathway integrated with inflammatory signaling pathways that underlie alcohol addiction.

Preclinical studies revealed contribution of N-methyl-D-aspartate receptors (NMDARs) to a variety of neuropsychiatric diseases including alcoholism, but development of NMDAR antagonists for therapeutic use has been a challenge, in part due to severe side effects. One of the key intracellular events resulting from stimulation of NMDAR is activation of calpains—calcium-dependent cysteine proteases. Here we studied whether inhibition of calpains would produce therapeutic-like effects of NMDAR antagonists but without their NMDAR-mediated side-effect profile. The calpain inhibitor A-705253 (3–10 mg/kg) was tested in a model of cue-induced reinstatement of alcohol-seeking behavior in post-dependent Wistar rats and in an alcohol deprivation effect (ADE) model in long-term alcohol drinking Wistar rats, two behavioral models for alcohol-seeking and relapse, respectively. We also tested the effect of A-705253 on the saccharine deprivation effect (SDE) as a selectivity measure. Acute treatment with A-705253 dose-dependently reduced cue-induced reinstatement of alcohol-seeking behavior. Repeated administration of A-705253 caused significant reductions of relapse-like excessive alcohol intake during the post-abstinence drinking days, an effect that persisted during two more successive drug-free drinking weeks, which was selective for the ADE as the SDE was unaffected. However, A-705253 did not produce psychostimulant, cognition impairing (delayed-matching-to-position), or psychotomimetic effects (specifically, phencyclidine discriminative stimulus effects). Taken together, these results demonstrate the involvement of calpains in alcohol-seeking and relapse and present a rationale for a novel pharmacological intervention that may reduce craving and relapse with minimal side effects in alcohol-dependent patients.

‘Omics' techniques are widely used to identify novel mechanisms underlying brain function and pathology. Here we applied a novel metabolomics approach to further ascertain the role of frontostriatal brain regions for the expression of addiction-like behaviors in rat models of alcoholism. Rats were made alcohol dependent via chronic intermittent alcohol vapor exposure. Following a 3-week abstinence period, rats had continuous access to alcohol in a two-bottle, free-choice paradigm for 7 weeks. Nontargeted flow injection time-of-flight mass spectrometry was used to assess global metabolic profiles of two cortical (prelimbic and infralimbic) and two striatal (accumbens core and shell) brain regions. Alcohol consumption produces pronounced global effects on neurometabolomic profiles leading to a clear separation of metabolic phenotypes between treatment groups, particularly. Further comparisons of regional tissue levels of various metabolites, most notably dopamine and Met-enkephalin, allow the extrapolation of alcohol consumption history. Finally, a high-drinking metabolic fingerprint was identified indicating a distinct alteration of central energy metabolism in the accumbens shell of excessively drinking rats that could indicate a so far unrecognized pathophysiological mechanism in alcohol addiction. In conclusion, global metabolic profiling from distinct brain regions by mass spectrometry identifies profiles reflective of an animal's drinking history and provides a versatile tool to further investigate pathophysiological mechanisms in alcohol dependence.

Rational development of novel therapeutic strategies for alcoholism requires understanding of its underlying neurobiology and pathophysiology. Obtaining this knowledge largely relies on animal studies. Thus, choosing the appropriate animal model is one of the most critical steps in pre-clinical medication development. Among the range of animal models that have been used to investigate excessive alcohol consumption in rodents, the postdependent model stands out. It was specifically developed to test the role of negative affect as a key driving force in a perpetuating addiction cycle for alcoholism. Here, we will describe our approach to make rats dependent via chronic intermittent exposure to alcohol, discuss the validity of this model, and compare it with other commonly used animal models of alcoholism. We will summarize evidence that postdependent rats fulfill several criteria of a 'Diagnostic and Statistical Manual of Mental Disorders IV/V-like' diagnostic system. Importantly, these animals show long-lasting excessive consumption of and increased motivation for alcohol, and evidence for loss of control over alcohol intake. Our conclusion that postdependent rats are an excellent model for medication development for alcoholism is underscored by a summary of more than two dozen pharmacological tests aimed at reversing these abnormal alcohol responses. We will end with open questions on the use of this model. In the tradition of the Sanchis-Segura and Spanagel review, we provide comic strips that illustrate the postdependent procedure and relevant phenotypes in this review.

A key deficit in alcohol dependence is disrupted prefrontal function leading to excessive alcohol seeking, but the molecular events underlying the emergence of addictive responses remain unknown. Here we show by convergent transcriptome analysis that the pyramidal neurons of the infralimbic cortex are particularly vulnerable for the long-term effects of chronic intermittent ethanol intoxication. These neurons exhibit a pronounced deficit in metabotropic glutamate receptor subtype 2 (mGluR2). Also, alcohol-dependent rats do not respond to mGluR2/3agonist treatment with reducing extracellular glutamate levels in the nucleus accumbens. Together these data imply a loss of autoreceptor feedback control. Alcohol-dependent rats show escalation of ethanol seeking, which was abolished by restoring mGluR2expression in the infralimbic cortex via viral-mediated gene transfer. Human anterior cingulate cortex from alcoholic patients shows a significant reduction in mGluR2transcripts compared to control subjects, suggesting that mGluR2loss in the rodent and human corticoaccumbal neurocircuitry may be a major consequence of alcohol dependence and a key pathophysiological mechanism mediating increased propensity to relapse. Normalization of mGluR2function within this brain circuit may be of therapeutic value.

A major hypothesis in addiction research is that alcohol induces neuroadaptations in the mesolimbic dopamine (DA) system and that these neuroadaptations represent a key neurochemical event in compulsive drug use and relapse. Whether these neuroadaptations lead to a hypo- or hyperdopaminergic state during abstinence is a long-standing, unresolved debate among addiction researchers. The answer is of critical importance for understanding the neurobiological mechanism of addictive behavior. Here we set out to study systematically the neuroadaptive changes in the DA system during the addiction cycle in alcohol-dependent patients and rats. In postmortem brain samples from human alcoholics we found a strong down-regulation of the D1 receptor- and DA transporter (DAT)-binding sites, but D2-like receptor binding was unaffected. To gain insight into the time course of these neuroadaptations, we compared the human data with that from alcohol-dependent rats at several time points during abstinence. We found a dynamic regulation of D1 and DAT during 3 wk of abstinence. After the third week the rat data mirrored our human data. This time point was characterized by elevated extracellular DA levels, lack of synaptic response to D1 stimulation, and augmented motor activity. Further functional evidence is given by a genetic rat model for hyperdopaminergia that resembles a phenocopy of alcohol-dependent rats during protracted abstinence. In summary, we provide a new dynamic model of abstinence-related changes in the striatal DA system; in this model a hyperdopaminergic state during protracted abstinence is associated with vulnerability for relapse.

Long-term changes in brain gene expression have been identified in alcohol dependence, but underlying mechanisms remain unknown. Here, we examined the potential role of microRNAs (miRNAs) for persistent gene expression changes in the rat medial prefrontal cortex (mPFC) after a history of alcohol dependence. Two-bottle free-choice alcohol consumption increased following 7-week exposure to intermittent alcohol intoxication. A bioinformatic approach using microarray analysis, quantitative PCR (qPCR), bioinformatic analysis and microRNA-messenger RNA (mRNA) integrative analysis identified expression patterns indicative of a disruption in synaptic processes and neuroplasticity. About 41 rat miRNAs and 165 mRNAs in the mPFC were significantly altered after chronic alcohol exposure. A subset of the miRNAs and mRNAs was confirmed by qPCR. Gene ontology categories of differential expression pointed to functional processes commonly associated with neurotransmission, neuroadaptation and synaptic plasticity. microRNA-mRNA expression pairing identified 33 miRNAs putatively targeting 89 mRNAs suggesting transcriptional networks involved in axonal guidance and neurotransmitter signaling. Our results demonstrate a significant shift in microRNA expression patterns in the mPFC following a history of dependence. Owing to their global regulation of multiple downstream target transcripts, miRNAs may have a pivotal role in the reorganization of synaptic connections and long-term neuroadaptations in alcohol dependence. MicroRNA-mediated alterations of transcriptional networks may be involved in disrupted prefrontal control over alcohol drinking observed in alcoholic patients.

Loss of control over drinking is a key deficit in alcoholism causally associated with malfunction of the medial prefrontal cortex (mPFC), but underlying molecular and cellular mechanisms remain unclear. Cue-induced reinstatement of alcohol seeking activates a subset of mPFC neurons in rats, identified by their common expression of the activity marker cFos and comprised of both principal and interneurons. Here, we used cFos-lacZ and pCAG-lacZ transgenic rats for activity-dependent or nonselective inactivation of neurons, respectively, which by their lacZ encoded β-galactosidase activity convert the inactive prodrug Daun02 into the neurotoxin daunorubicin. We report that activity-dependent ablation of a neuronal ensemble in the infralimbic but not the prelimbic subregion induced excessive alcohol seeking. The targeted neuronal ensemble was specific for the cue-induced response because stress-induced reinstatement was not affected in these animals. Importantly, nonselective inactivation of infralimbic neurons, using pCAG-lacZ rats, was without functional consequence on the cue-induced reinstatement task. Thus, inhibitory control over alcohol seeking is exerted by distinct functional ensembles within the infralimbic cortex rather than by a general inhibitory tone of this region on the behavioral output. This indicates a high level of functional compartmentation within the rat mPFC whereat many functional ensembles could coexist and interact within the same subregion. SIGNIFICANCE STATEMENT [Hebb's (1949)][1] idea of memories as being represented in local neuronal networks is supported by identification of transiently stable activity patterns within subgroups of neurons. However, it is difficult to link individual networks to specific memory tasks, for example a learned behavior. By a novel approach of activity-dependent ablation, here we identify a specific neuronal ensemble located in the infralimbic subregion of the medial prefrontal cortex that controls a seeking response for alcohol in rats. Our data demonstrate that functional output depends on specific neuronal ensembles within a given brain region rather than on the global activity of that region, which raises important questions about the interpretation of numerous earlier experiments using site-directed silencing or stimulation for elucidating brain function. [1]: #ref-23

Rational development of novel therapeutic strategies for alcoholism requires understanding of its underlying neurobiology and pathophysiology. Obtaining this knowledge largely relies on animal studies. Thus, choosing the appropriate animal model is one of the most critical steps in pre-clinical medication development. Among the range of animal models that have been used to investigate excessive alcohol consumption in rodents, the postdependent model stands out. It was specifically developed to test the role of negative affect as a key driving force in a perpetuating addiction cycle for alcoholism. Here, we will describe our approach to make rats dependent via chronic intermittent exposure to alcohol, discuss the validity of this model, and compare it with other commonly used animal models of alcoholism. We will summarize evidence that postdependent rats fulfill several criteria of a 'Diagnostic and Statistical Manual of Mental Disorders IV/V-like' diagnostic system. Importantly, these animals show long-lasting excessive consumption of and increased motivation for alcohol, and evidence for loss of control over alcohol intake. Our conclusion that postdependent rats are an excellent model for medication development for alcoholism is underscored by a summary of more than two dozen pharmacological tests aimed at reversing these abnormal alcohol responses. We will end with open questions on the use of this model. In the tradition of the Sanchis-Segura and Spanagel review, we provide comic strips that illustrate the postdependent procedure and relevant phenotypes in this review.

Preclinical studies revealed contribution of N-methyl-D-aspartate receptors (NMDAR) to a variety of neuropsychiatric diseases including alcoholism but development of NMDAR antagonists for therapeutic use has been a challenge in part due to severe side effects. One of the key intracellular events resulting from stimulation of NMDAR is activation of …

The neurobiological mechanisms underlying craving and relapse into alcohol drinking are poorly understood. Human and animal studies implicate the prefrontal cortex in drug and alcohol addiction. So far the involvement of the infralimbic (IL) and prelimbic (PL) cortices, as parts of the medial prefrontal cortex in rats, has been shown …

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine β-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences.