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Glutamatergic neurotransmission plays a critical role in addictive behaviors, and recent evidence indicates that genetic or pharmacological inactivation of the type 5 metabotropic glutamate receptor (mGluR5) reduces the self-administration of cocaine, nicotine, and alcohol. Because mGluR5 is coupled to activation of protein kinase C (PKC), and targeted deletion of the epsilon isoform (PKCepsilon) in mice reduces ethanol self-administration, we investigated whether there is a functional link between mGluR5 and PKCepsilon. Here, we show that acute administration of the mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine to mice increases phosphorylation of PKCepsilon in its activation loop (T566) as well as in its C-terminal region (S729). Increases in phospho-PKCepsilon are dependent not only on mGluR5 stimulation but also on phosphatidylinositol-3 kinase (PI3K). In addition, the selective mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP) reduced basal levels of phosphorylation of PKCepsilon at S729. We also show that MPEP dose dependently reduced ethanol consumption in wild-type but not in PKCepsilon-null mice, suggesting that PKCepsilon is an important signaling target for modulation of ethanol consumption by mGluR5 antagonists. Radioligand binding experiments using [(3)H]MPEP revealed that these genotypic differences in response to MPEP were not a result of altered mGluR5 levels or binding in PKCepsilon-null mice. Our data indicate that mGluR5 is coupled to PKCepsilon via a PI3K-dependent pathway and that PKCepsilon is required for the ability of the mGluR5 antagonist MPEP to reduce ethanol consumption.

The present experiment tested the effects of intraaccumbens injections of dopamine (DA) agonists and antagonists on operant responding reinforced by sucrose and sucrose/ethanol solutions. The mixed DA agonist d-amphetamine (20.0 micrograms/microliters) significantly reduced responding reinforced by a low concentration sucrose solution (2% w/v) by 48% and 38% compared to no injection and sham control values, respectively. The addition of ethanol (10%) to a low concentration sucrose solution (3%) presented as the reinforcer changed the response pattern from a continuous moderate response rate, over a 30 min session, to an initial high response rate that terminated after approximately 10 min. With sucrose/ethanol reinforcement, d-amphetamine slowed the initial high response rate but extended responding throughout the 30 min sessions. However, no significant changes were observed in number of responses per session. When 75% sucrose (w/v) was presented as the reinforcer, d-amphetamine did not change the total number of responses/session, but response patterns were again altered from high initial rates with early offset to slow steady rates that continued for the duration of sessions. The D2 DA antagonist raclopride (0.1-5.0 micrograms/microliters) resulted in a dose-dependent decrease in responding reinforced by 75% sucrose. The baseline patterns, response totals, and effects of the DA antagonists resemble our previously reported findings with 10% ethanol (v/v) reinforcement. These data support the conclusion that mesolimbic DA activity may be a common mechanism in ethanol reinforced behavior and behavior reinforced by other substances, but suggest that the nature of behavioral change may depend upon the reinforcer.

It appears clear that ethanol reinforcement, like that of many abused drugs, utilizes the mesolimbic DA pathways. From the data presented on microinjection of DA agonists and antagonists, it would seem that only part of the regulatory process controlling ethanol drinking is directly involved with this pathway. Once drinking has begun, the DA antagonist raclopride results in a rapid termination of drinking. This appears to be a blocking effect of what may be conditioned reinforcement resulting from prior ethanol reinforcement initiation procedures. Microinjection of the DA agonists d-amphetamine and quinpirole prolonged drinking, with little signs of normal termination apparent in the 30-min session in many animals. This appeared to be the result of interference with normal termination processes. While it remains to be demonstrated that oral ethanol consumption results in the release of DA in the nucleus accumbens, evidence from prior work and the present studies support a role for the mesolimbic DA system in ethanol reinforcement.

The positive reinforcing effects of alcohol (ethanol) drive repetitive use and contribute to alcohol use disorder (AUD). Ethanol alters the expression of glutamate AMPA receptor (AMPAR) subunits in reward-related brain regions, but the extent to which this effect regulates ethanol's reinforcing properties is unclear.This study investigates whether ethanol self-administration changes AMPAR subunit expression and synaptic activity in the nucleus accumbens core (AcbC) to regulate ethanol's reinforcing effects in male C57BL/6 J mice.Sucrose-sweetened ethanol self-administration (0.81 g/kg/day) increased AMPAR GluA2 protein expression in the AcbC, without effect on GluA1, compared to sucrose-only controls. Infusion of myristoylated Pep2m in the AcbC, which blocks GluA2 binding to N-ethylmaleimide-sensitive fusion protein (NSF) and reduces GluA2-containing AMPAR activity, reduced ethanol-reinforced responding without affecting sucrose-only self-administration or motor activity. Antagonizing GluA2-lacking AMPARs, through AcbC infusion of NASPM, had no effect on ethanol self-administration. AcbC neurons receiving projections from the basolateral amygdala (BLA) showed increased sEPSC area under the curve (a measurement of charge transfer) and slower decay kinetics in ethanol self-administering mice as compared to sucrose. Optogenetic activation of these neurons revealed an ethanol-enhanced AMPA/NMDA ratio and significantly reduced paired-pulse ratio, suggesting elevated GluA2 contributions specifically within the BLA➔AcbC pathway.Ethanol use upregulates GluA2 protein expression in the AcbC and AMPAR synaptic activity in AcbC neurons receiving BLA projections and enhances synaptic plasticity directly within the BLA➔AcbC circuit. GluA2-containing AMPAR activity in the AcbC regulates the positive reinforcing effects of ethanol through an NSF-dependent mechanism, highlighting a potential therapeutic target in AUD.

Rats initiated to self-administer 10% ethanol (v/v) in an operant situation using the sucrose-substitution technique received bilateral n. accumbens or caudate nucleus microinjections of d-amphetamine (4, 10, and 20 micrograms/brain), quinpirole (4 micrograms/brain), and/or raclopride (0.1, 0.5, and 1.0 micrograms/brain). Only microinjections into the n. accumbens produced changes in rate and pattern of responding. With d-amphetamine, an increase in total responding and a slowing of initial response rate was seen, whereas with raclopride administration a dose-related decrease in total responding was observed with no alteration in momentary response rates. Drug-dependent behavioral rate and pattern differences suggest that DA activity in the n. accumbens influences ethanol reinforced behavior.

The effects of microinjections in the ventral tegmental area (VTA) of the dopamine D2/D3 agonist quinpirole on ethanol‐ and sucrosereinforced responding were tested. Two groups of Long‐Evans rats were trained to lever press on a fixed‐ratio 4 schedule with 10% ethanol (v/v) (n= 8) and 75% sucrose (w/v) (n= 10) presented as the reinforcer. Weekly bilateral injections of quinpirole were tested in the ethanol group (0.0, 0.001, 0.01, 0.1, and 1.0 μg/μl) and sucrose group (0.0, 0.01, 0.1, 1.0, and 10.0 μg/μl). Quinpirole dose dependently decreased ethanol‐ and sucrose‐reinforced responding, but the dose‐effect curve for sucrose was shifted two orders of magnitude to the right of the ethanol curve. Temporal response patterns during control sessions for both reinforcers were characterized by initial high rates that terminated after approximately 10 min. VTA injections of quinpirole dose dependently delayed the onset of the first response and the initial high rate period, and resulted in an early termination of responding following onset. These data support the hypothesis that dopamine activity in the VTA is involved in the regulation of ethanol‐reinforced responding in a manner similar to that of other reinforcers.

The actions of ethanol on extracellular dopamine levels in the n. accumbens were examined in both anesthetized and unanesthetized rats using either in vivo voltammetry or microdialysis. In the voltammetry studies, ethanol was microinjected directly into the accumbens. For the microdialysis studies, the ethanol was injected systemically. The voltammetry studies failed to find any direct effect of local ethanol on extracellular dopamine levels. However, exposure to high ethanol concentrations directly injected into the n. accumbens showed the rise rate and the return to baseline rate to a n. accumbens KCl-stimulated dopamine release. In the microdialysis studies, increased levels of extracellular dopamine in the n. accumbens were found in unanesthetized rats, similar to those reported in the literature. However, in the anesthetized rats, the extracellular dopamine levels were not increased, even with similar local ethanol levels measured in the dialysate. Taken together, the data suggest that the actions of ethanol to increase extracellular dopamine levels in the n. accumbens are most likely not an effect of ethanol at the level of the accumbens but rather an action which increases neural activity within the mesoaccumbens pathway, perhaps via actions at the ventral tegmental area.

Adaptive changes in gene expression are thought to contribute to dependence, addiction and other behavioral responses to chronic ethanol abuse. DNA array studies provide a nonbiased detection of networks of gene expression changes, allowing insight into functional consequences and mechanisms of such molecular responses. We used oligonucleotide arrays to study nearly 6000 genes in human SH-SY5Y neuroblastoma cells exposed to chronic ethanol. A set of 42 genes had consistently increased or decreased mRNA abundance after 3 days of ethanol treatment. Groups of genes related to norepinephrine production, glutathione metabolism, and protection against apoptosis were identified. Genes involved in catecholamine metabolism are of special interest because of the role of this pathway in mediating ethanol withdrawal symptoms (physical dependence). Ethanol treatment elevated dopamine beta-hydroxylase (DBH, EC 1.14.17.1) mRNA and protein levels and increased releasable norepinephrine in SH-SY5Y cultures. Acute ethanol also increased DBH mRNA levels in mouse adrenal gland, suggesting in vivo functional consequences for ethanol regulation of DBH. In SH-SY5Y cells, ethanol also decreased mRNA and secreted protein levels for monocyte chemotactic protein 1, an effect that could contribute to the protective role of moderate ethanol consumption in atherosclerotic vascular disease. Finally, we identified a subset of genes similarly regulated by both ethanol and dibutyryl-cAMP treatment in SH-SY5Y cells. This suggests that ethanol and cAMP signaling share mechanistic features in regulating a subset of ethanol-responsive genes. Our findings offer new insights regarding possible molecular mechanisms underlying behavioral responses or medical consequences of ethanol consumption and alcoholism.

The development and progression of alcohol use disorder (AUD) are widely viewed as maladaptive neuroplasticity. The transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory protein γ8 (TARP γ-8) is a molecular mechanism of neuroplasticity that has not been evaluated in AUD or other addictions.To address this gap in knowledge, we evaluated the mechanistic role of TARP γ-8 bound AMPAR activity in the basolateral amygdala (BLA) and ventral hippocampus (vHPC) in the positive reinforcing effects of alcohol, which drive repetitive alcohol use throughout the course of AUD, in male C57BL/6 J mice. These brain regions were selected because they exhibit high levels of TARP γ-8 expression and send glutamate projections to the nucleus accumbens (NAc), which is a key nucleus in the brain reward pathway.Site-specific pharmacological inhibition of AMPARs bound to TARP γ-8 in the BLA via bilateral infusion of the selective negative modulator JNJ-55511118 (0-2 µg/µl/side) significantly decreased operant alcohol self-administration with no effect on sucrose self-administration in behavior-matched controls. Temporal analysis showed that reductions in alcohol-reinforced response rate occurred > 25 min after the onset of responding, consistent with a blunting of the positive reinforcing effects of alcohol in the absence of nonspecific behavioral effects. In contrast, inhibition of TARP γ-8 bound AMPARs in the vHPC selectively decreased sucrose self-administration with no effect on alcohol.This study reveals a novel brain region-specific role of TARP γ-8 bound AMPARs as a molecular mechanism of the positive reinforcing effects of alcohol and non-drug rewards.