Alcohol metabolism contributes to brain histone acetylation.

Emerging evidence suggests that epigenetic regulation is dependent on metabolic state, and implicates specific metabolic factors in neural functions that drive behaviour1. In neurons, acetylation of histones relies on the metabolite acetyl-CoA, which is produced from acetate by chromatin-bound acetyl-CoA synthetase 2 (ACSS2)2. Notably, the breakdown of alcohol in the liver leads to a rapid increase in levels of blood acetate3, and alcohol is therefore a major source of acetate in the body. Histone acetylation in neurons may thus be under the influence of acetate that is derived from alcohol4, with potential effects on alcohol-induced gene expression in the brain, and on behaviour5. Here, using in vivo stable-isotope labelling in mice, we show that the metabolism of alcohol contributes to rapid acetylation of histones in the brain, and that this occurs in part through the direct deposition of acetyl groups that are derived from alcohol onto histones in an ACSS2-dependent manner. A similar direct deposition was observed when mice were injected with heavy-labelled acetate in vivo. In a pregnant mouse, exposure to labelled alcohol resulted in the incorporation of labelled acetyl groups into gestating fetal brains. In isolated primary hippocampal neurons ex vivo, extracellular acetate induced transcriptional programs related to learning and memory, which were sensitive to ACSS2 inhibition. We show that alcohol-related associative learning requires ACSS2 in vivo. These findings suggest that there is a direct link between alcohol metabolism and gene regulation, through the ACSS2-dependent acetylation of histones in the brain.

VGF function in depression and antidepressant efficacy.

Brain-derived neurotrophic factor (BDNF) is a critical effector of depression-like behaviors and antidepressant responses. Here, we show that VGF (non-acronymic), which is robustly regulated by BDNF/TrkB signaling, is downregulated in hippocampus (male/female) and upregulated in nucleus accumbens (NAc) (male) in depressed human subjects and in mice subjected to chronic social defeat stress (CSDS). Adeno-associated virus (AAV)-Cre-mediated Vgf ablation in floxed VGF mice, in dorsal hippocampus (dHc) or NAc, led to pro-depressant or antidepressant behaviors, respectively, while dHc- or NAc-AAV-VGF overexpression induced opposite outcomes. Mice with reduced VGF levels in the germ line (Vgf+/-) or in dHc (AAV-Cre-injected floxed mice) showed increased susceptibility to CSDS and impaired responses to ketamine treatment in the forced swim test. Floxed mice with conditional pan-neuronal (Synapsin-Cre) but not those with forebrain (αCaMKII-Cre) Vgf ablation displayed increased susceptibility to subthreshold social defeat stress, suggesting that neuronal VGF, expressed in part in inhibitory interneurons, regulates depression-like behavior. Acute antibody-mediated sequestration of VGF-derived C-terminal peptides AQEE-30 and TLQP-62 in dHc induced pro-depressant effects. Conversely, dHc TLQP-62 infusion had rapid antidepressant efficacy, which was reduced in BDNF floxed mice injected in dHc with AAV-Cre, and in NBQX- and rapamycin-pretreated wild-type mice, these compounds blocking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mammalian target of rapamycin (mTOR) signaling, respectively. VGF is therefore a critical modulator of depression-like behaviors in dHc and NAc. In hippocampus, the antidepressant response to ketamine is associated with rapid VGF translation, is impaired by reduced VGF expression, and as previously reported, requires coincident, rapid BDNF translation and release.

The dendritic spine morphogenic effects of repeated cocaine use occur through the regulation of serum response factor signaling.

The nucleus accumbens (NAc) is a primary brain reward region composed predominantly of medium spiny neurons (MSNs). In response to early withdrawal from repeated cocaine administration, de novo dendritic spine formation occurs in NAc MSNs. Much evidence indicates that this new spine formation facilitates the rewarding properties of cocaine. Early withdrawal from repeated cocaine also produces dramatic alterations in the transcriptome of NAc MSNs, but how such alterations influence cocaine's effects on dendritic spine formation remain unclear. Studies in non-neuronal cells indicate that actin cytoskeletal regulatory pathways in nuclei have a direct role in the regulation of gene transcription in part by controlling the access of co-activators to their transcription factor partners. In particular, actin state dictates the interaction between the serum response factor (SRF) transcription factor and one of its principal co-activators, MAL. Here we show that cocaine induces alterations in nuclear F-actin signaling pathways in the NAc with associated changes in the nuclear subcellular localization of SRF and MAL. Using in vivo optogenetics, the brain region-specific inputs to the NAc that mediate these nuclear changes are investigated. Finally, we demonstrate that regulated SRF expression, in turn, is critical for the effects of cocaine on dendritic spine formation and for cocaine-mediated behavioral sensitization. Collectively, these findings reveal a mechanism by which nuclear-based changes influence the structure of NAc MSNs in response to cocaine.

Genes and addiction.

Drug addiction, like all psychiatric disorders, is defined solely in behavioural terms. For example, addiction can be considered a loss of control over drug-taking, or compulsive drug-seeking and -taking despite horrendous consequences. Abnormal behaviours are a consequence of aberrant brain function, which means that it is a tangible goal to identify the biological underpinnings of addiction. The genetic basis of addiction encompasses two broad areas of enquiry. One of these is the identification of genetic variation in humans that partly determines susceptibility to addiction. The other is the use of animal models to investigate the role of specific genes in mediating the development of addiction. Whereas recent advances in this latter effort are heartening, a major challenge remains: to understand how the many genes implicated in rodent models interact to yield as complex a phenotype as addiction.

Overexpression of DeltaFosB transcription factor(s) increases bone formation and inhibits adipogenesis.

Members of the AP-1 family of transcription factors participate in the regulation of bone cell proliferation and differentiation. We report here a potent AP-1-related regulator of osteoblast function: DeltaFosB, a naturally occurring truncated form of FosB that arises from alternative splicing of the fosB transcript and is expressed in osteoblasts. Overexpression of DeltaFosB in transgenic mice leads to increased bone formation throughout the skeleton and a continuous post-developmental increase in bone mass, leading to osteosclerosis. In contrast, DeltaFosB inhibits adipogenesis both in vivo and in vitro, and downregulates the expression of early markers of adipocyte differentiation. Because osteoblasts and adipocytes are thought to share a common precursor, it is concluded that DeltaFosB transcriptionally regulates osteoblastogenesis, possibly at the expense of adipogenesis.

Phosphorylation of DeltaFosB mediates its stability in vivo.

The transcription factor, DeltaFosB, accumulates in a region-specific manner in brain in response to many types of chronic stimulation due to the unusual stability of the protein. The phosphorylation of Ser27 in DeltaFosB has been shown to promote this stability in vitro. We show here that this phosphorylation reaction is also important for DeltaFosB's stability in the brain in vivo and for the unique behavioral plasticity mediated by this transcription factor.

Molecular and cellular basis of addiction.

Drug addiction results from adaptations in specific brain neurons caused by repeated exposure to a drug of abuse. These adaptations combine to produce the complex behaviors that define an addicted state. Progress is being made in identifying such time-dependent, drug-induced adaptations and relating them to specific behavioral features of addiction. Current research needs to understand the types of adaptations that underlie the particularly long-lived aspects of addiction, such as drug craving and relapse, and to identify specific genes that contribute to individual differences in vulnerability to addiction. Understanding the molecular and cellular basis of addictive states will lead to major changes in how addiction is viewed and ultimately treated.

Neuronal phosphoproteins: physiological and clinical implications.

The presence of a great variety of neuron-specific phosphoproteins in nervous tissue supports the view that protein phosphorylation plays many roles in neuronal function. The physiological significance of several of these phosphoproteins has already been established. Some neuronal phosphoproteins have been detected throughout the entire nervous system, whereas the distribution of others is limited to one or a few neuronal cell types. These various neuron-specific phosphoproteins are proving of value in the study of the physiology, anatomy, developmental biology, and pathophysiology of the nervous system.

Corticosterone increases the amount of protein 1, a neuron-specific phosphoprotein, in rat hippocampus.

Corticosterone increased the amount of the neuron-specific phosphoprotein protein 1 in the hippocampus, a brain region rich in corticosterone receptors, but not in several brain regions that contain relatively few corticosterone receptors.

Opposite modulation of cocaine-seeking behavior by D1- and D2-like dopamine receptor agonists.

Activation of the mesolimbic dopamine system is known to trigger relapse in animal models of cocaine-seeking behavior. We found that this "priming" effect was selectively induced by D2-like, and not by D1-like, dopamine receptor agonists in rats. Moreover, D1-like receptor agonists prevented cocaine-seeking behavior induced by cocaine itself, whereas D2-like receptor agonists enhanced this behavior. These results demonstrate an important dissociation between D1- and D2-like receptor processes in cocaine-seeking behavior and support further evaluation of D1-like receptor agonists as a possible pharmacotherapy for cocaine addiction.

Regulation of cocaine reward by CREB.

Cocaine regulates the transcription factor CREB (adenosine 3', 5'-monophosphate response element binding protein) in rat nucleus accumbens, a brain region that is important for addiction. Overexpression of CREB in this region decreases the rewarding effects of cocaine and makes low doses of the drug aversive. Conversely, overexpression of a dominant-negative mutant CREB increases the rewarding effects of cocaine. Altered transcription of dynorphin likely contributes to these effects: Its expression is increased by overexpression of CREB and decreased by overexpression of mutant CREB. Moreover, blockade of kappa opioid receptors (on which dynorphin acts) antagonizes the negative effect of CREB on cocaine reward. These results identify an intracellular cascade-culminating in gene expression-through which exposure to cocaine modifies subsequent responsiveness to the drug.

Sensitization to morphine induced by viral-mediated gene transfer.

Repeated administration of morphine sensitizes animals to the stimulant and rewarding properties of the drug. It also selectively increases expression of GluR1 (an AMPA glutamate receptor subunit) in the ventral tegmental area, a midbrain region implicated in morphine action. By viral-mediated gene transfer, a causal relation is shown between these behavioral and biochemical adaptations: Morphine's stimulant and rewarding properties are intensified after microinjections of a viral vector expressing GluR1 into the ventral tegmental area. These results confirm the importance of AMPA receptors in morphine action and demonstrate specific locomotor and motivational adaptations resulting from altered expression of a single localized gene product.

DARPP-32: regulator of the efficacy of dopaminergic neurotransmission.

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.

Molecular mechanisms of psychostimulant-induced structural plasticity.

Drug addiction is characterized by persistent behavioral and cellular plasticity throughout the brain's reward regions. Among the many neuroadaptations that occur following repeated drug administration are alterations in cell morphology including changes in dendritic spines. While this phenomenon has been well documented, the underlying molecular mechanisms are poorly understood. Here, within the context of drug abuse, we review and integrate several of the established pathways known to regulate synaptic remodeling, and discuss the contributions of neurotrophic and dopamine signaling in mediating this structural plasticity. Finally, we discuss how such upstream mechanisms could regulate actin dynamics, the common endpoint involved in structural remodeling in neurons.

Role for GDNF in biochemical and behavioral adaptations to drugs of abuse.

The present study examined a role for GDNF in adaptations to drugs of abuse. Infusion of GDNF into the ventral tegmental area (VTA), a dopaminergic brain region important for addiction, blocks certain biochemical adaptations to chronic cocaine or morphine as well as the rewarding effects of cocaine. Conversely, responses to cocaine are enhanced in rats by intra-VTA infusion of an anti-GDNF antibody and in mice heterozygous for a null mutation in the GDNF gene. Chronic morphine or cocaine exposure decreases levels of phosphoRet, the protein kinase that mediates GDNF signaling, in the VTA. Together, these results suggest a feedback loop, whereby drugs of abuse decrease signaling through endogenous GDNF pathways in the VTA, which then increases the behavioral sensitivity to subsequent drug exposure.

Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments.

Following chronic cocaine treatment, we have found a long-lasting increase in AP-1 binding in the rat nucleus accumbens and striatum, two important targets of the behavioral effects of cocaine. This increase develops gradually over several days and remains at 50% of maximal levels 7 days after the last cocaine exposure. Supershift experiments, along with one- and two-dimensional Western blots, indicate that this chronic AP-1 complex contains at least four Fos-related antigens (FRAs), some of which display delta FosB-like immunoreactivity, that are induced selectively by chronic, but not acute, cocaine treatment. The same chronic FRAs were also induced by several different types of chronic treatments in a region-specific manner in the brain. Thus, the chronic FRAs and associated chronic AP-1 complex could mediate some of the long-term changes in gene expression unique to the chronic-treated state as opposed to the acute-treated and normal states.

Correlates of aldosterone-induced increases in Cai2+ and Isc suggest that Cai2+ is the second messenger for stimulation of apical membrane conductance.

Studies were performed on monolayers of cultured A6 cells, grown on permeable filters, to determine the second messenger system involved in the aldosterone-induced increase in electrogenic sodium transport. Addition of aldosterone (1 microM) to the solution bathing the basal surface of cells caused both an increase in Isc and threefold transient rise in intracellular calcium Cai2+ after a delay of approximately 60 min. Because both events were inhibited by actinomycin D and cyclohexamide, they appeared to require transcriptional and translational processes. Addition of BAPTA to the bathing media to chelate Cai2+ reduced Isc and the delayed Cai2+ transient; 50 microM BAPTA inhibited Isc and the rise in Cai2+ by greater than 80%. Further studies suggested that the action of aldosterone to increase Isc may be dependent on a calcium/calmodulin-dependent protein kinase, because W-7 and trifluoperazine reduced the aldosterone-induced Isc in a dose-dependent manner. Taken together, these observations suggest that calcium is a second messenger for the action of aldosterone on sodium transport, and suggest, for the first time, that agonists which bind to intracellular receptors can utilize, via delayed processes dependent on de novo transcription and translation, intracellular second messenger systems to regulate target cell function.

Dopamine-receptor stimulation: biobehavioral and biochemical consequences.

The MPTP monkey is a well-characterized animal model of parkinsonism and provides an exceptional tool for the study of dyskinesias induced by dopamine-like agents. Several such agents have been tested during the past 15 years, and it has been found that the duration of action of these compounds is the most reliable variable with which to predict their dyskinesiogenic profile. It is proposed that L-dopa-induced dyskinesias represent a form of pathological learning caused by chronic pulsatile (nonphysiological) stimulation of dopamine receptors, which activates a cascade of molecular and biochemical events. These events include defective regulation of Fos proteins that belong to the deltaFosB family, increased expression of neuropeptides, and defective GABA- and glutamate-mediated neurotransmission in the output structures of the basal ganglia.

Molecular mechanisms of opiate and cocaine addiction.

Chronic administration of opiates or cocaine has been shown to alter the activity or expression of diverse types of cellular proteins in specific target neurons within the central nervous system. Prominent examples include signaling proteins, such as receptors, G proteins, second-messenger synthetic enzymes, and protein kinases. It is now increasingly possible to relate particular molecular adaptations to specific behavioral actions of drugs of abuse in animal models of addiction. In addition, recent work has focused on a role for transcription factors, and the associated alterations in gene expression, in mediating part of this long-lasting, drug-induced molecular and behavioral plasticity.