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 alcoholdependent 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.alcoholism | translational studies | dopamine release | in silico analysis | postmortem brain tissue A bout 10% of the total burden of disease in developed countries is caused by alcohol use alone (1). A large proportion of alcohol-related disability results from alcohol addiction. The condition affects more than 12% of the United States population at some point in their lives and is one of the most prevalent psychiatric disorders in Europe (2, 3). The relapsing course of alcoholism is associated with compulsive drinking, loss of control over intake, and emergence of a negative emotional state during abstinence (4). Afflicted individuals go through repeated cycles of alcohol intoxication and withdrawal leading to persistent alterations in brain activity that are hypothesized to drive relapse and compulsive alcohol use even long after detoxification (5).Seminal studies in experimental animals established that alcohol's rewarding properties are associated with increased dopamine (DA) in regions such as the nucleus accumbens (Acb) (6), whereas withdrawal after chronic alcohol use decreases DA neurotransmission (7). In humans, the binding of a DA receptor ligand, typically one for the D2-like receptor subgroup, i.e., [11 C] raclopride, can be monitored by PET. Displacement of the radioligand provides an indirect measure of DA release and has been used to demonstrate alcohol-evoked DA release in...

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Blockade of the μ-opioid receptor (MOR) by naltrexone reduces relapse risk in a subpopulation of alcohol-dependent patients. Previous positron-emission-tomography (PET) studies using the MOR ligand [C]carfentanil have found increased MOR availability in abstinent alcoholics, which may reflect either increased MOR expression or lower endogenous ligand concentration. To differentiate between both effects, we investigated two cohorts of alcoholic subjects using either post-mortem or clinical PET analysis. Post-mortem brain tissue of alcohol-dependent subjects and controls (N=43/group) was quantitatively analyzed for MOR ([H]DAMGO)-binding sites and OPRM1 mRNA in striatal regions. [C]carfentanil PET was performed in detoxified, medication free alcohol-dependent patients (N=38), followed by a randomized controlled study of naltrexone versus placebo and follow-up for 1 year (clinical trial number: NCT00317031). Because the functional OPRM1 variant rs1799971:A>G affects the ligand binding, allele carrier status was considered in the analyses. MOR-binding sites were reduced by 23-51% in post-mortem striatal tissue of alcoholics. In the PET study, a significant interaction of OPRM1 genotype, binding potential (BP) for [C]carfentanil in the ventral striatum, and relapse risk was found. Particularly in G-allele carriers, lower striatal BP was associated with a higher relapse risk. Interestingly, this effect was more pronounced in the naltrexone treatment group. Reduced MOR is interpreted as a neuroadaptation to an alcohol-induced release of endogenous ligands in patients with severe alcoholism. Low MOR availability may explain the ineffectiveness of naltrexone treatment in this subpopulation. Finally, low MOR-binding sites are proposed as a molecular marker for a negative disease course.

It has previously been shown that the inhibition of L-type calcium channels (LTCCs) decreases alcohol consumption, although the contribution of the central LTCC subtypes Cav1.2 and Cav1.3 remains unknown. Here, we determined changes in Cav1.2 (Cacna1c) and Cav1.3 (Cacna1d) mRNA and protein expression in alcohol-dependent rats during protracted abstinence and naive controls using in situ hybridization and western blot analysis. Functional validation was obtained by electrophysiological recordings of calcium currents in dissociated hippocampal pyramidal neurons. We then measured alcohol self-administration and cue-induced reinstatement of alcohol seeking in dependent and nondependent rats after intracerebroventricular (i.c.v.) injection of the LTCC antagonist verapamil, as well as in mice with an inducible knockout (KO) of Cav1.2 in Ca/calmodulin-dependent protein kinase IIα (CaMKIIα)-expressing neurons. Our results show that Cacna1c mRNA concentration was increased in the amygdala and hippocampus of alcohol-dependent rats after 21 days of abstinence, with no changes in Cacna1d mRNA. This was associated with increased Cav1.2 protein concentration and L-type calcium current amplitudes. Further analysis of Cacna1c mRNA in the CA1, basolateral amygdala (BLA), and central amygdala (CeA) revealed a dynamic regulation over time during the development of alcohol dependence. The inhibition of central LTCCs via i.c.v. administration of verapamil prevented cue-induced reinstatement of alcohol seeking in alcohol-dependent rats. Further studies in conditional Cav1.2-KO mice showed a lack of dependence-induced increase of alcohol-seeking behavior. Together, our data indicate that central Cav1.2 channels, rather than Cav1.3, mediate alcohol-seeking behavior. This finding may be of interest for the development of new antirelapse medications.

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Rationale The rewarding effects of alcohol have been attributed to interactions between opioid and dopaminergic system within the mesolimbic reward pathway. We have previously shown that ablation of β-arrestin 2 (Arrb2), a crucial regulator of μ-opioid receptor function, attenuates alcohol-induced hyperlocomotion and c-fos activation in the nucleus accumbens. Objectives Here, we further investigated the role of Arrb2 in modulating alcohol-induced dopamine (DA) release and conditioned place preference (CPP). We also assessed the functional importance of Arrb2 for μ-opioid receptor surface expression and signaling following an acute alcohol challenge. Methods Alcohol-evoked (0.375, 0.75 and 1.5 g/kg intraperitoneally, i.p.) DA release was measured by in vivo microdialysis in the shell of nucleus accumbens. Reward was assessed by the CPP paradigm. Receptor function was assessed by μ-receptor binding and [35S]GTP-γ-S autoradiography. Results In Arrb2 knockout mice accumbal DA levels reach maximum response at a lower dose compared to wild-type (wt) animals. In line with these results, Arrb2 knockout mice display increased CPP for alcohol as compared to wt mice. Finally, Arrb2 mutant mice display increased μ-opioid receptor signaling in the ventral and dorsal striatum and amygdala in response to a low dose of alcohol, indicating impaired desensitization mechanisms in these mice. Conclusions Our results show that Arrb2 modulates the response to low doses of alcohol on various levels including μ-opioid receptor signaling, DA release, and reward. They also reveal a clear dissociation between the effects of Arrb2 on psychomotor and reward behaviors.

Background:The veterinary antibiotic Sulfamethazine (SMZ) contaminates soils via manure applications. Like other soil contaminants (herbicides, fungicides, and nematicides), it has to be degraded. The main challenge is that SMZ biodegradation with bacteria is impeded, since SMZ is a bacteriostatic antibiotic, designed to block microbes in their growth. Results:In this study, we enriched the indigenous soil microbial community (including the single strain Microbacterium sp. C448, adapted to SMZ degradation) from a Canadian soil and we present a suitable approach, for soil remediation by inoculating a German soil with this microbial community established on carrier particles, at environmentally relevant concentrations of 1 mg kg. When compared with the isolated SMZ-degrading strain (also obtained from Canada), the microbial community outperformed the mineralization rates of the isolated strain in soil. The negligible soil native SMZ mineralization was successfully increased to 44 and 57 % within 46 days, by the microbial community. The sustainability of this increased SMZ mineralization capacity was proven by the rapid mineralization of a second application of 14 C-SMZ 112 days after the first. Conclusions:The pronounced SMZ mineralization and the high amount of non-extractable 14 C-residues (NER) in the inoculated soil indicate that the NER are mainly of biogenic origin (metabolically fixed 14 C). Therefore, the applied inoculation approach decreased the risk of persistent non-extractable SMZ residues. Together with our former studies, this specific soil inoculation approach was tested for three substances with different physico-chemical properties, indicating that this soil bioremediation technique might also be used for other substances.