One of the major challenges of cocaine addiction is the high rate of relapse to drug use after periods of withdrawal. During the first few weeks of withdrawal, cue-induced cocaine craving intensifies, or "incubates," and persists over extended periods of time. Although several brain regions and molecular mechanisms were found to be involved in this process, the underlying epigenetic mechanisms are still unknown. Herein, we used a rat model of incubation of cocaine craving, in which rats were trained to self-administer cocaine (0.75 mg/kg, 6 h/d, 10 d), and cue-induced cocaine-seeking was examined in an extinction test after 1 or 30 d of withdrawal. We show that the withdrawal periods, as well as cue-induced cocaine seeking, are associated with broad, time-dependent enhancement of DNA methylation alterations in the nucleus accumbens (NAc). These gene methylation alterations were partly negatively correlated with gene expression changes. Furthermore, intra-NAc injections of a DNA methyltransferase inhibitor (RG108, 100 M) abolished cue-induced cocaine seeking on day 30, an effect that persisted 1 month, whereas the methyl donor S-adenosylmethionine (500 M) had an opposite effect on cocaine seeking. We then targeted two proteins whose genes were demethylated by RG108-estrogen receptor 1 (ESR1) and cyclin-dependent kinase 5 (CDK5). Treatment with an intra-NAc injection of the ESR1 agonist propyl pyrazole triol (10 nM) or the CDK5 inhibitor roscovitine (28 M) on day 30 of withdrawal significantly decreased cue-induced cocaine seeking. These results demonstrate a role for NAc DNA methylation, and downstream targets of DNA demethylation, in incubation of cocaine craving.

The lateral habenula (LHb) is critical for modulation of negative reinforcement, punishment and aversive responses. In light of the success of deep-brain-stimulation (DBS) in the treatment of neurological disorders, we explored the use of LHb DBS as a method of intervention in cocaine selfadministration, extinction, and reinstatement in rats. An electrode was implanted into the LHb and rats were trained to self-administer cocaine (21 days; 0.25-1 mg/kg) until they achieved at least three days of stable performance (as measured by daily recordings of active lever presses in selfadministration cages). Thereafter, rats received DBS in the presence or absence of cocaine. DBS reduced cocaine seeking behavior during both self-administration and extinction training. DBS also attenuated the rats' lever presses following cocaine reinstatement (5-20 mg/kg) in comparison to sham-operated rats. These results were also controlled by the assessment of physical performance as measured by water self-administration and an open field test, and by evaluation of depressive-like manifestations as measured by the swim and two-bottles-choice tests. In contrast, LHb lesioned rats demonstrated increased cocaine seeking behavior as demonstrated by a delayed extinction response. In the ventral tegmental area, cocaine self-administration elevated glutamatergic receptor subunits NR1 and GluR1 and scaffolding protein PSD95, but not GABA A β, protein levels. Following DBS treatment, levels of these subunits returned to control values. We postulate that the effect of both LHb modulation and LHb DBS on cocaine reinforcement may be via attenuation of the cocaineinduced increase in glutaminergic input to the VTA.

The lateral habenula (LHb) plays a role in prediction of negative reinforcement, punishment and aversive responses. In the current study, we examined the role that the LHb plays in the regulation of negative reward responses and aversion. First, we tested the effect of intervention in LHb activity on sucrose reinforcing behavior. An electrode was implanted into the LHb and rats were trained to self-administer sucrose (20%; 16 days) until at least three days of stable performance were achieved (as represented by the number of active lever presses in self-administration cages). Rats subsequently received deep brain stimulation (DBS) of the LHb, which significantly reduced sucrose self-administration levels. In contrast, lesion of the LHb increased sucrose-seeking behavior, as demonstrated by a delayed extinction response to substitution of sucrose with water. Furthermore, in a modified non-rewarding conditioned-place-preference paradigm, DBS of the LHb led to aversion to the context associated with stimulation of this brain region.We postulate that electrical stimulation of the LHb attenuates positive reward-associated reinforcement by natural substances.

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Long known for its anti-nociceptive effects, the opioid beta-endorphin is also reported to have rewarding and reinforcing properties and to be involved in stress response. In this manuscript we summarize the present neurobiological and behavioral evidence regarding the role of beta-endorphin in stress-related psychiatric disorders, depression and PTSD. There is existing data that support the importance of beta-endorphin neurotransmission in mediating depression. As for PTSD, however, the data is thus far circumstantial. The studies described herein used diverse techniques, such as biochemical measurements of beta-endorphin in various brain sites and behavioral monitoring, in two animal models of depression and PTSD. We suggest that the pathways for stress-related psychiatric disorders, depression and PTSD, converge to a common pathway in which beta-endorphin is a modulating element of distress. This may occur via its interaction with the mesolimbic monoaminergic system and also by its interesting effects on learning and memory. The possible involvement of beta-endorphin in the process of stress-related psychiatric disorders, depression and PTSD, is discussed.

Cue-induced cocaine craving intensifies, or 'incubates', during the first few weeks of abstinence and persists over extended periods of time. One important factor implicated in cocaine addiction is the endogenous opioid β-endorphin. In the present study, we examined the possible involvement of β-endorphin in the incubation of cocaine craving. Rats were trained to self-administer cocaine (0.75 mg/kg, 10 days, 6 h/day), followed by either a 1-day or a 30-day period of forced abstinence. Subsequent testing for cue-induced cocaine-seeking behavior (without cocaine reinforcement) was performed. Rats exposed to the drug-associated cue on day 1 of forced abstinence demonstrated minimal cue-induced cocaine-seeking behavior concurrently with a significant increase in β-endorphin release in the nucleus accumbens (NAc). Conversely, exposure to the cue on day 30 increased cocaine seeking, while β-endorphin levels remained unchanged. Intra-NAc infusion of an anti-β-endorphin antibody (4 μg) on day 1 increased cue-induced cocaine seeking, whereas infusion of a synthetic β-endorphin peptide (100 ng) on day 30 significantly decreased cue response. Both intra-NAc infusions of the δ opioid receptor antagonist naltrindole (1 μg) on day 1 and naltrindole together with β-endorphin on day 30 increased cue-induced cocaine-seeking behavior. Intra-NAc infusion of the μ opioid receptor antagonist CTAP (30 ng and 3 μg) had no behavioral effect. Altogether, these results demonstrate a novel role for β-endorphin and the δ opioid receptor in the development of the incubation of cocaine craving.

Anhedonia and lack of motivation are core symptoms of depression. In contrast, hyper-motivation and euphoria characterize intoxicated states. In order to explore the relationship between these two behavioral states we examined cocaine self-administration tasks in an animal model of depression [Flinders Sensitive Line (FSL) rats]. We found that FSL rats exhibit sub-sensitivity in their cocaine-seeking behavior, which was normalized following a chronic treatment with the antidepressant desipramine. However, when the cocaine dosage was increased, FSL rats demonstrated a similar cocaine-seeking behavior to that of controls. In light of dopamine's central role in modulating cocaine reinforcement, we examined dopaminergic neurotransmission in the nucleus accumbens, a brain region implicated in the rewarding and hedonic effects of substances of misuse. FSL rats exhibited low but dose-dependent increases in extracellular levels of dopamine in the nucleus accumbens after acute intravenous cocaine injection. Furthermore, by using the dopamine transporter blocker GBR-12909 we were able to demonstrate that the low extracellular dopamine levels, observed in FSL rats, were a consequence of low dopamine release in the nucleus accumbens, as opposed to the possibility of increased uptake. Treatment of FSL rats with the antidepressant desipramine raised cocaine- and GBR-12909-induced dopamine release to the level of controls. This treatment also resulted in increased cocaine-seeking behavior.

Homocysteine is a thiol-containing amino acid synthesized in mammals ⁄ humans as part of the normal metabolism of the essential amino acid methionine. Studies conducted over the past three decades have shown that high levels of homocysteine in the plasma (hyperhomocysteinemia, i.e. > 15 lmolAEL ) constitute a risk factor for cardiovascular diseases and stroke [1]. Elevated homocysteine is also a risk factor for several neurodegenerative disorders, such as dementia [2], Alzheimer's disease [3], and Parkinson's disease [4]. As elevated homocysteine is associated with an increasing number of pathologies, the regulation of homocysteine levels is of clinical importance.Several factors contribute to elevated homocysteine levels: (a) genetic disorders stemming from mutations in the enzymes involved in homocysteine remethylation to methionine (e.g. 5,10-methylenetetrahydrofolate reductase) [ Ammonium trichloro(dioxoethylene-o,o¢)tellurate (AS101) is an organotellurium compound with pleiotropic functions that has been associated with antitumoral, immunomodulatory and antineurodegenerative activities. Tellurium compounds with a +4 oxidation state, such as AS101, react uniquely with thiols, forming disulfide molecules. In light of this, we tested whether AS101 can react with the amino acid homocysteine both in vitro and in vivo. AS101 conferred protection against homocysteine-induced apoptosis of HL-60 cells. The protective mechanism of AS101 against homocysteine toxicity was directly mediated by its chemical reactivity, whereby AS101 reacted with homocysteine to form homocystine, the less toxic disulfide form of homocysteine. Moreover, AS101 was shown here to reduce the levels of total homocysteine in an in vivo model of hyperhomocysteinemia. As a result, AS101 also prevented sperm cells from undergoing homocysteine-induced DNA fragmentation. Taken together, our results suggest that the organotellurium compound AS101 may be of clinical value in reducing total circulatory homocysteine levels.Abbreviations AS101, ammonium trichloro(dioxoethylene-o,o¢)tellurate; ddw, double-deionized water; DEVD, Ac-benzyloxycarbonyl aspartyl glutamylvalylaspartic acid; DFI, DNA fragmentation index; FACS, fluorescence-activated cell sorter; Nbs 2 , 5,5¢-dithiobis(2-nitrobenzoic acid); PI, propidium iodide; pNA, p-nitroaniline; RP, reaction product; SCSA, sperm chromatin structure assay.