In vivo evaluation of a microtubule PET ligand, [11C]MPC-6827, in mice following chronic alcohol consumption.

BACKGROUND: Excessive alcohol consumption is a global health burden and requires a better understanding of its neurobiology. A lower density of brain microtubules is found in alcohol-related human brain disease postmortem and in rodent models of chronic alcohol consumption. Here, we report in vivo imaging studies of microtubules in brain using our recently reported Positron Emission Tomography (PET) tracer, [11C]MPC-6827, in chronic alcohol-consuming adult male C57BL/6 J mice and control mice. METHODS: In vivo PET imaging studies of [11C]MPC-6827 (3.7 ± 0.8 MBq) were performed in two groups of adult male mice: (1) water-consuming control mice (n = 4) and (2) mice that consumed 20% alcohol (w/v) for 4 months using the intermittent 2-bottle choice procedure that has been shown to lead to signs of alcohol dependence. Dynamic 63 min PET images were acquired using a microPET Inveon system (Siemens, Germany). PET images were reconstructed using the 3D-OSEM algorithm and analyzed using VivoQuant version 4 (Invicro, MA). Tracer uptake in ROIs that included whole brain, prefrontal cortex (PFC), liver and heart was measured and plotted as %ID/g over time (0-63 min) to generate time-activity curves (TACs). RESULTS: In general, a trend for lower binding of [11C]MPC-6827 in the whole brain and PFC of mice in the chronic alcohol group was found compared with control group. No group difference in radiotracer binding was found in the peripheral organs such as liver and heart. CONCLUSIONS: This pilot study indicates a trend of loss of microtubule binding in whole brain and prefrontal cortex of chronic alcohol administered mice brain compared to control mice, but no loss in heart or liver. These results indicate the potential of [11C]MPC-6827 as a PET ligand for further in vivo imaging investigations of AUD in human.

Negative allosteric modulation of metabotropic glutamate receptor 5 attenuates alcohol self-administration in baboons.

Many of the behavioral symptoms that define alcohol use disorder (AUD) are thought to be mediated by amplified glutamatergic activity. As a result, previous preclinical studies have investigated glutamate receptor inhibition as a potential pharmacotherapy for AUD, particularly the metabotropic glutamate receptor 5 (mGlu5). In rodents, mGlu5 negative allosteric modulators (NAMs) have been shown to decrease alcohol self-administration. However, their effect on non-human primates has not previously been explored. To bridge this gap, the effects of mGlu5 NAM pretreatment on sweetened alcohol (8% w/v in diluted KoolAid) self-administration in female baboons were evaluated. Two different mGlu5 NAMs were tested: 1) 3-2((-Methyl-4-thiazolyl) ethynyl) pyridine (MTEP) which was administered at a dose of 2 mg/kg IM; and 2) auglurant (N-(5-fluoropyridin-2-yl)-6-methyl-4-(pyrimidin-5-yloxy)picolinamide), a newly developed NAM, which was tested under two different routes (0.001, 0.01, 0.03, 0.1 mg/kg IM and 0.1, 0.3, 1.0 mg/kg PO). MTEP decreased both fixed ratio and progressive ratio responding for sweetened alcohol. Auglurant, administered IM, decreased alcohol self-administration at doses that did not affect self-administration of an alcohol-free sweet liquid reward (0.01 to 0.1 mg/kg). Oral administration of auglurant was not effective in decreasing alcohol self-administration. Our results extend positive findings from rodent studies on mGlu5 regulation of alcohol drinking to female baboons and further strengthen the rationale for targeting mGlu5 in clinical trials for AUD.

Alcohol reduces the activity of somatostatin interneurons in the mouse prefrontal cortex: A neural basis for its disinhibitory effect?

The prefrontal cortex (PFC) is involved in executive ("top-down") control of behavior and its function is especially susceptible to the effects of alcohol, leading to behavioral disinhibition that is associated with alterations in decision making, response inhibition, social anxiety and working memory. The circuitry of the PFC involves a complex interplay between pyramidal neurons (PNs) and several subclasses of inhibitory interneurons (INs), including somatostatin (SST)-expressing INs. Using in vivo calcium imaging, we showed that alcohol dose-dependently altered network activity in layers 2/3 of the prelimbic subregion of the mouse PFC. Low doses of alcohol (1 g/kg, intraperitoneal, i.p.) caused moderate activation of SST INs and weak inhibition of PNs. At moderate to high doses, alcohol (2-3 g/kg) strongly inhibited the activity of SST INs in vivo, and this effect may result in disinhibition, as the activity of a subpopulation of PNs was simultaneously enhanced. In contrast, recordings in brain slices using ex vivo electrophysiology revealed no direct effect of alcohol on the excitability of either SST INs or PNs over a range of concentrations (20 and 50 mM) consistent with the blood alcohol levels reached in the in vivo experiments. This dose-dependent effect of alcohol on SST INs in vivo may reveal a neural basis for the disinhibitory effect of alcohol in the PFC mediated by other neurons within or external to the PFC circuitry.

Constitutive Genetic Deletion of Hcn1 Increases Alcohol Preference during Adolescence.

The hyperpolarization-activated cyclic nucleotide-gated channel (HCN), which underlies the hyperpolarization-activated cation current (Ih), has diverse roles in regulating neuronal excitability across cell types and brain regions. Recently, HCN channels have been implicated in preclinical models of substance abuse including alcohol. In the prefrontal cortex of rodents, HCN expression and Ih magnitude are developmentally regulated during adolescence and may be vulnerable to alcohol's effects. In mice, binge alcohol consumption during the adolescent period results in a sustained reduction in Ih that coincides with increased alcohol consumption in adulthood, yet the direct role HCN channels have on alcohol consumption are unknown. Here, we show that the genetic deletion of Hcn1 causes an increase in alcohol preference on intermittent 2-bottle choice task in homozygous null (HCN1-/-) male mice compared to wild-type littermates without affecting saccharine or quinine preference. The targeted viral deletion of HCN1 in pyramidal neurons of the medial prefrontal cortex resulted in a gradual loss of Hcn1 expression and a reduction in Ih magnitude during adolescence, however, this did not significantly affect alcohol consumption or preference. We conclude that while HCN1 regulates alcohol preference, the genetic deletion of Hcn1 in the medial prefrontal cortex does not appear to be the locus for this effect.

Photoactivatable Cre recombinase 3.0 for in vivo mouse applications.

Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.

Kappa-opioid receptors, dynorphin, and cocaine addiction: a positron emission tomography study.

Animal studies indicate that the kappa-opioid receptor/dynorphin system plays an important role in cocaine binges and stress-induced relapse. Our goal was to investigate changes in kappa-opioid receptor (KOR) availability in the human brain using positron emission tomography (PET), before and after a cocaine binge. We also investigated the correlation between KOR and stress-induced cocaine self-administration. PET imaging was performed with the KOR selective agonist [11C]GR103545. Subjects with cocaine-use disorder (CUD) underwent PET scans and performed two types of cocaine self-administration sessions in the laboratory as follows: (1) choice sessions following a cold pressor test, to induce stress, and (2) binge dosing of cocaine. This allowed us investigate the following: (1) the association between KOR binding and a laboratory model of stress-induced relapse and (2) the change in KOR binding following a 3-day cocaine binge, which is thought to represent a change in endogenous dynorphin. A group of matched healthy controls was included to investigate between group differences in KOR availability. A significant association between [11C]GR103545 binding and cocaine self-administration was seen: greater KOR availability was associated with more choices for cocaine. In addition, the 3-day cocaine binge significantly reduced [11C]GR103545 binding by 18% in the striatum and 14% across brain regions. No difference in [11C]GR103545 binding was found between the CUD subjects and matched controls. In the context of previous studies, these findings add to the growing evidence that pharmacotherapies targeting the KOR have the potential to significantly impact treatment development for cocaine-use disorder.

Alcohol Consumption during Adolescence in a Mouse Model of Binge Drinking Alters the Intrinsic Excitability and Function of the Prefrontal Cortex through a Reduction in the Hyperpolarization-Activated Cation Current.

Periodic episodes of excessive alcohol consumption ("binge drinking") occur frequently among adolescents, and early binge drinking is associated with an increased risk of alcohol use disorders later in life. The PFC undergoes significant development during adolescence and hence may be especially susceptible to the effects of binge drinking. In humans and in animal models, adolescent alcohol exposure is known to alter PFC neuronal activity and produce deficits in PFC-dependent behaviors, such as decision making, response inhibition, and working memory. Using a voluntary intermittent access to alcohol (IA EtOH) procedure in male mice, we demonstrate that binge-level alcohol consumption during adolescence leads to altered drinking patterns and working memory deficits in young adulthood, two outcomes that suggest medial PFC dysfunction. We recorded from pyramidal neurons (PNs) in the prelimbic subregion of the medial PFC in slices obtained from mice that had IA EtOH and found that they display altered excitability, including a hyperpolarization of the resting membrane potential and reductions in the hyperpolarization-activated cation current (Ih) and in intrinsic persistent activity (a mode of neuronal firing that is dependent on Ih). Many of these effects on intrinsic excitability were sustained following abstinence and observed in mice that showed working memory deficits. In addition, we found that resting membrane potential and the Ih-dependent voltage "sag" in prelimbic PFC PNs are developmentally regulated during adolescence, suggesting that adolescent alcohol exposure may compromise PFC function by arresting the normal developmental trajectory of PN intrinsic excitability.SIGNIFICANCE STATEMENT Binge alcohol drinking during adolescence has negative consequences for the function of the developing PFC. Using a mouse model of voluntary binge drinking during adolescence, we found that this behavior leads to working memory deficits and altered drinking behavior in adulthood. In addition, we found that adolescent drinking is associated with specific changes to the intrinsic excitability of pyramidal neurons in the PFC, reducing the ability of these neurons to generate intrinsic persistent activity, a phenomenon thought to be important for working memory. These findings may help explain why human adolescent binge drinkers show performance deficits on tasks mediated by the PFC.

Cue-induced reinstatement of alcohol-seeking behavior is associated with increased CaMKII T286 phosphorylation in the reward pathway of mice.

Cue-induced reinstatement of alcohol-seeking is a hallmark behavioral pathology of addiction. Evidence suggests that reinstatement (e.g., relapse), may be regulated by cell signaling systems that underlie neuroplasticity. A variety of plasticity events require activation of calcium calmodulin-dependent protein kinase II (CaMKII) in components of the reward pathway, such as the nucleus accumbens and amygdala. We sought to determine if cue-induced reinstatement of alcohol-seeking behavior is associated with changes in the activation state (e.g., phosphorylation) of CaMKII-T286. Male C57BL/6J mice (n=14) were trained to lever press on a fixed-ratio-4 schedule of sweetened alcohol (2% sucrose+9% EtOH) reinforcement. After 14-d of extinction (no cues or reinforcers), mice underwent a response-contingent reinstatement (n=7) vs. an additional day of extinction (n=7). Brains were removed immediately after the test and processed for evaluation of pCaMKII-T286 immunoreactivity (IR). Number of pCaMKII-T286 positive cells/mm2 was quantified from coronal brain sections using Bioquant Image Analysis software. Mice emitted significantly more responses on the alcohol vs. the inactive lever throughout the baseline phase with average alcohol intake of 1.1±0.03g/kg/1-h. During extinction, responses on the alcohol lever decreased to inactive lever levels by day 7. Significant cue-induced reinstatement of alcohol-seeking was observed during a single test with no effects on the inactive lever. Reinstatement was associated with increased pCaMKII-T286 IR specifically in amygdala (LA and BLA), nucleus accumbens (AcbSh), lateral septum, mediodorsal thalamus, and piriform cortex as compared to extinction control. Brain regions showing no change included the dorsal striatum, medial septum, cingulate cortex, habenula, paraventricular thalamus, and ventral hypothalamus. These results show response contingent cue-induced reinstatement of alcohol-seeking behavior is associated with selective increases in pCaMKII-T286 in specific reward- and memory-related brain regions of male C57BL/6J mice. Primary molecular mechanisms of associative learning and memory may regulate relapse in alcohol addiction.

Glycine receptor α3 and α2 subunits mediate tonic and exogenous agonist-induced currents in forebrain.

Neuronal inhibition can occur via synaptic mechanisms or through tonic activation of extrasynaptic receptors. In spinal cord, glycine mediates synaptic inhibition through the activation of heteromeric glycine receptors (GlyRs) composed primarily of α1 and ß subunits. Inhibitory GlyRs are also found throughout the brain, where GlyR α2 and α3 subunit expression exceeds that of α1, particularly in forebrain structures, and coassembly of these α subunits with the ß subunit appears to occur to a lesser extent than in spinal cord. Here, we analyzed GlyR currents in several regions of the adolescent mouse forebrain (striatum, prefrontal cortex, hippocampus, amygdala, and bed nucleus of the stria terminalis). Our results show ubiquitous expression of GlyRs that mediate large-amplitude currents in response to exogenously applied glycine in these forebrain structures. Additionally, tonic inward currents were also detected, but only in the striatum, hippocampus, and prefrontal cortex (PFC). These tonic currents were sensitive to both strychnine and picrotoxin, indicating that they are mediated by extrasynaptic homomeric GlyRs. Recordings from mice deficient in the GlyR α3 subunit (Glra3-/-) revealed a lack of tonic GlyR currents in the striatum and the PFC. In Glra2-/Y animals, GlyR tonic currents were preserved; however, the amplitudes of current responses to exogenous glycine were significantly reduced. We conclude that functional α2 and α3 GlyRs are present in various regions of the forebrain and that α3 GlyRs specifically participate in tonic inhibition in the striatum and PFC. Our findings suggest roles for glycine in regulating neuronal excitability in the forebrain.

Effects of acute alcohol on excitability in the CNS.

Alcohol has many effects on brain function and hence on human behavior, ranging from anxiolytic and mild disinhibitory effects, sedation and motor incoordination, amnesia, emesis, hypnosis and eventually unconsciousness. In recent years a variety of studies have shown that acute and chronic exposure to alcohol can modulate ion channels that regulate excitability. Modulation of intrinsic excitability provides another way in which alcohol can influence neuronal network activity, in addition to its actions on synaptic inputs. In this review, we review "low dose" effects [between 2 and 20 mM EtOH], and "medium dose"; effects [between 20 and 50 mM], by considering in turn each of the many networks and brain regions affected by alcohol, and thereby attempt to integrate in vitro physiological studies in specific brain regions (e.g. amygdala, ventral tegmental area, prefrontal cortex, thalamus, cerebellum etc.) within the context of alcohol's behavioral actions in vivo (e.g. anxiolysis, euphoria, sedation, motor incoordination). This article is part of the Special Issue entitled "Alcoholism".

Voluntary adolescent drinking enhances excitation by low levels of alcohol in a subset of dopaminergic neurons in the ventral tegmental area.

Enhanced dopamine (DA) neurotransmission from the ventral tegmental area (VTA) to the ventral striatum is thought to drive drug self-administration and mediate positive reinforcement. We examined neuronal firing rates in slices of mouse midbrain following adolescent binge-like alcohol drinking and find that prior alcohol experience greatly enhanced the sensitivity to excitation by ethanol itself (10-50 mM) in a subset of ventral midbrain DA neurons located in the medial VTA. This enhanced response after drinking was not associated with alterations of firing rate or other measures of intrinsic excitability. In addition, the phenomenon appears to be specific to adolescent drinking, as mice that established a drinking preference only after the onset of adulthood showed no change in alcohol sensitivity. Here we demonstrate not only that drinking during adolescence induces enhanced alcohol sensitivity, but also that this DA neuronal response occurs over a range of alcohol concentrations associated with social drinking in humans.

Brain Stimulation in Addiction.

Localized stimulation of the human brain to treat neuropsychiatric disorders has been in place for over 20 years. Although these methods have been used to a greater extent for mood and movement disorders, recent work has explored brain stimulation methods as potential treatments for addiction. The rationale behind stimulation therapy in addiction involves reestablishing normal brain function in target regions in an effort to dampen addictive behaviors. In this review, we present the rationale and studies investigating brain stimulation in addiction, including transcranial magnetic stimulation, transcranial direct current stimulation, and deep brain stimulation. Overall, these studies indicate that brain stimulation has an acute effect on craving for drugs and alcohol, but few studies have investigated the effect of brain stimulation on actual drug and alcohol use or relapse. Stimulation therapies may achieve their effect through direct or indirect modulation of brain regions involved in addiction, either acutely or through plastic changes in neuronal transmission. Although these mechanisms are not well understood, further identification of the underlying neurobiology of addiction and rigorous evaluation of brain stimulation methods has the potential for unlocking an effective, long-term treatment of addiction.

Moderate Alcohol Drinking and the Amygdala Proteome: Identification and Validation of Calcium/Calmodulin Dependent Kinase II and AMPA Receptor Activity as Novel Molecular Mechanisms of the Positive Reinforcing Effects of Alcohol.

BACKGROUND: Despite worldwide consumption of moderate amounts of alcohol, the neural mechanisms that mediate the transition from use to abuse are not fully understood. METHODS: Here, we conducted a high-throughput screen of the amygdala proteome in mice after moderate alcohol drinking (n = 12/group) followed by behavioral studies (n = 6-8/group) to uncover novel molecular mechanisms of the positive reinforcing properties of alcohol that strongly influence the development of addiction. RESULTS: Two-dimensional difference in-gel electrophoresis with matrix assisted laser desorption ionization tandem time-of-flight identified 29 differentially expressed proteins in the amygdala of nondependent C57BL/6J mice following 24 days of alcohol drinking. Alcohol-sensitive proteins included calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) and a network of functionally linked proteins that regulate neural plasticity and glutamate-mediated synaptic activity. Accordingly, alcohol drinking increased α-amino-3-hydroxy-5-methyl-4-isooxazole receptor (AMPAR) in central amygdala (CeA) and phosphorylation of AMPAR GluA1 subunit at a CaMKII locus (GluA1-Ser831) in CeA and lateral amygdala. Further, CaMKIIα-Thr286 and GluA1-Ser831 phosphorylation was increased in CeA and lateral amygdala of mice that lever-pressed for alcohol versus the nondrug reinforcer sucrose. Mechanistic studies showed that targeted pharmacologic inhibition of amygdala CaMKII or AMPAR activity specifically inhibited the positive reinforcing properties of alcohol but not sucrose. CONCLUSIONS: Moderate alcohol drinking increases the activity and function of plasticity-linked protein networks in the amygdala that regulate the positive reinforcing effects of the drug. Given the prominence of positive reinforcement in the etiology of addiction, we propose that alcohol-induced adaptations in CaMKIIα and AMPAR signaling in the amygdala may serve as a molecular gateway from use to abuse.

Operant ethanol self-administration increases extracellular-signal regulated protein kinase (ERK) phosphorylation in reward-related brain regions: selective regulation of positive reinforcement in the prefrontal cortex of C57BL/6J mice.

RATIONALE: Extracellular-signal regulated protein kinase (ERK1/2) is activated by ethanol in reward-related brain regions. Accordingly, systemic inhibition of ERK1/2 potentiates ethanol reinforcement. However, the brain region(s) that mediate this effect are unknown. OBJECTIVE: This study aims to pharmacologically inhibit ERK1/2 in the medial prefrontal cortex (PFC), nucleus accumbens (NAC), and amygdala (AMY) prior to ethanol or sucrose self-administration, and evaluate effects of operant ethanol self-administration on ERK1/2 phosphorylation (pERK1/2). METHODS: Male C57BL/6J mice were trained to lever press on a fixed-ratio-4 schedule of 9% ethanol + 2% sucrose (ethanol) or 2% sucrose (sucrose) reinforcement. Mice were sacrificed immediately after the 30th self-administration session and pERK1/2 immunoreactivity was quantified in targeted brain regions. Additional groups of mice were injected with SL 327 (0-1.7 µg/side) in PFC, NAC, or AMY prior to self-administration. RESULTS: pERK1/2 immunoreactivity was significantly increased by operant ethanol (g/kg = 1.21 g/kg; BAC = 54.9 mg/dl) in the PFC, NAC (core and shell), and AMY (central nucleus) as compared to sucrose. Microinjection of SL 327 (1.7 µg) into the PFC selectively increased ethanol self-administration. Intra-NAC injection of SL 327 had no effect on ethanol- but suppressed sucrose-reinforced responding. Intra-AMY microinjection of SL 327 had no effect on either ethanol- or sucrose-reinforced responding. Locomotor activity was unaffected under all conditions. CONCLUSIONS: Operant ethanol self-administration increases pERK1/2 activation in the PFC, NAC, and AMY. However, ERK1/2 activity only in the PFC mechanistically regulates ethanol self-administration. These data suggest that ethanol-induced activation of ERK1/2 in the PFC is a critical pharmacological effect that mediates the reinforcing properties of the drug.

Upregulation of dopamine D2 receptors in the nucleus accumbens indirect pathway increases locomotion but does not reduce alcohol consumption.

Brain imaging studies performed in humans have associated low striatal dopamine release and D2R binding with alcohol dependence. Conversely, high striatal D2R binding has been observed in unaffected members of alcoholic families suggesting that high D2R function may protect against alcohol dependence. A possible protective role of increased D2R levels in the striatum is further supported by preclinical studies in non-human primates and rodents. Here, we determined whether there is a causal relationship between D2R levels and alcohol intake. To this end, we upregulated D2R expression levels in the nucleus accumbens of the adult mouse, but selectively restricted the upregulation to the indirect striatal output pathway, which endogenously expresses D2Rs. After overexpression was established, mice were tested in two models of free-choice alcohol drinking: the continuous and intermittent access two-bottle choice models. As anticipated, we found that D2R upregulation leads to hyperactivity in the open field. Contrary to our expectation, D2R upregulation did not reduce alcohol intake during continuous or intermittent access or when alcohol drinking was tested in the context of aversive outcomes. These data argue against a protective role of accumbal indirect pathway D2Rs in alcohol consumption but emphasize their importance in promoting locomotor activity.

Strychnine-sensitive glycine receptors on pyramidal neurons in layers II/III of the mouse prefrontal cortex are tonically activated.

Processing of signals within the cerebral cortex requires integration of synaptic inputs and a coordination between excitatory and inhibitory neurotransmission. In addition to the classic form of synaptic inhibition, another important mechanism that can regulate neuronal excitability is tonic inhibition via sustained activation of receptors by ambient levels of inhibitory neurotransmitter, usually GABA. The purpose of this study was to determine whether this occurs in layer II/III pyramidal neurons (PNs) in the prelimbic region of the mouse medial prefrontal cortex (mPFC). We found that these neurons respond to exogenous GABA and to the α4δ-containing GABAA receptor (GABA(A)R)-selective agonist gaboxadol, consistent with the presence of extrasynaptic GABA(A)R populations. Spontaneous and miniature synaptic currents were blocked by the GABA(A)R antagonist gabazine and had fast decay kinetics, consistent with typical synaptic GABA(A)Rs. Very few layer II/III neurons showed a baseline current shift in response to gabazine, but almost all showed a current shift (15-25 pA) in response to picrotoxin. In addition to being a noncompetitive antagonist at GABA(A)Rs, picrotoxin also blocks homomeric glycine receptors (GlyRs). Application of the GlyR antagonist strychnine caused a modest but consistent shift (∼15 pA) in membrane current, without affecting spontaneous synaptic events, consistent with the tonic activation of GlyRs. Further investigation showed that these neurons respond in a concentration-dependent manner to glycine and taurine. Inhibition of glycine transporter 1 (GlyT1) with sarcosine resulted in an inward current and an increase of the strychnine-sensitive current. Our data demonstrate the existence of functional GlyRs in layer II/III of the mPFC and a role for these receptors in tonic inhibition that can have an important influence on mPFC excitability and signal processing.

Interoceptive effects of alcohol require mGlu5 receptor activity in the nucleus accumbens.

The interoceptive effects of alcohol are major determinants of addiction liability. Metabotropic glutamate (mGlu) receptors are widely expressed in striatal circuits known to modulate drug-seeking. Given that the interoceptive effects of drugs can be important determinants of abuse liability, we hypothesized that striatal mGlu receptors modulate the interoceptive effects of alcohol. Using drug discrimination learning, rats were trained to discriminate alcohol (1 g/kg, i.g.) versus water. We found that systemic antagonism of metabotropic glutamate subtype 5 (mGlu5) receptors [10 mg/kg 2-methyl-6-(phenylethynyl)pyridine (MPEP) and 3 mg/kg 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine], but not mGlu1 receptors ([0.3-3 mg/kg JNJ16259685) (3,4-dihydro-2H-pyrano[2,3]beta-quinolin-7-yl)(cis-4-methoxycyclohexyl) methanone)], inhibited the discriminative stimulus effects of alcohol. Furthermore, mGlu5 receptor antagonism (10 mg/kg MPEP) significantly inhibited neuronal activity in the nucleus accumbens core as levels of the transcription factor c-Fos were significantly reduced. Accordingly, targeted inhibition of mGlu5 receptors (20 microg of MPEP) in the nucleus accumbens core blunted the discriminative stimulus effects of alcohol (1 g/kg). Anatomical specificity was confirmed by the lack of effect of inhibition of mGlu5 receptors (10-30 microg of MPEP) in the dorsomedial caudate-putamen and the similar cytological expression patterns and relative density of mGlu5 receptors between the brain regions. Functional involvement of intra-accumbens mGlu5 receptors was confirmed as activation of mGlu5 receptors [10 microg of (RS)-2-amino-2-(2-chloro-5-hydroxyphenyl)acetic acid sodium salt] enhanced the discriminative stimulus effects of a low alcohol dose (0.5 g/kg), and mGlu5 receptor inhibition (20 microg of MPEP) prevented the agonist-induced enhancement. These results show that mGlu5 receptor activity in the nucleus accumbens is required for the expression of the interoceptive effects of alcohol.

Nonselective suppression of operant ethanol and sucrose self-administration by the mGluR7 positive allosteric modulator AMN082.

Emerging evidence indicates that specific metabotropic glutamate receptors (mGluRs) modulate ethanol self-administration. In general, inhibition of glutamate transmission through blockade of postsynaptic mGluRs, or activation of presynaptic mGluRs, inhibits ethanol self-administration. The goal of this preclinical study was to further characterize mGluR regulation of ethanol self-administration by examining effects of AMN082, an allosteric positive modulator of presynaptic mGluR7 activity. Separate groups of C57BL/6J male mice were trained to self-administer ethanol or sucrose on a fixed-ratio 4 schedule of reinforcement during 1 h sessions. On test days, mice were pretreated with AMN082 (0, 1.0, 3.0, 5.6, or 10 mg/kg) 30 min prior to self-administration sessions. Functional specificity and activity was examined by testing the effects of AMN082 (0-10 mg/kg) on open-field locomotor activity and HPA axis function as measured by plasma corticosterone levels. AMN082 (10 mg/kg) produced a significant reduction in ethanol and sucrose reinforced responding, and inhibited locomotor activity. Plasma corticosterone levels were significantly increased following AMN082 (5.6 and 10 mg/kg) suggesting a dose-dependent dissociation between the behavioral and hormonal effects of the compound. These data suggest that activation of mGluR7 by AMNO82 produces nonspecific reductions in motivated behavior that are associated with negative effects on motor activity.

Cue-induced reinstatement of alcohol-seeking behavior is associated with increased ERK1/2 phosphorylation in specific limbic brain regions: blockade by the mGluR5 antagonist MPEP.

Relapse to alcohol use after periods of abstinence is a hallmark behavioral pathology of alcoholism and a major clinical problem. Emerging evidence indicates that metabotropic glutamate receptor 5 (mGluR5) antagonists attenuate relapse to alcohol-seeking behavior but the molecular mechanisms of this potential therapeutic effect remain unexplored. The extracellular signal-regulated kinase (ERK1/2) pathway is downstream of mGluR5 and has been implicated in addiction. We sought to determine if cue-induced reinstatement of alcohol-seeking behavior, and its reduction by an mGluR5 antagonist, is associated with changes in ERK1/2 activation in reward-related limbic brain regions. Selectively-bred alcohol-preferring (P) rats were trained to lever press on a concurrent schedule of alcohol (15% v/v) vs. water reinforcement. Following 9 days of extinction, rats were given an additional extinction trial or injected with the mGluR5 antagonist MPEP (0, 1, 3, or 10mg/kg) and tested for cue-induced reinstatement. Brains were removed 90-min later from the rats in the extinction and MPEP (0 or 10mg/kg) conditions for analysis of p-ERK1/2, total ERK1/2, and p-ERK5 immunoreactivity (IR). Cue-induced reinstatement of alcohol-seeking behavior was associated with a three to five-fold increase in p-ERK1/2 IR in the basolateral amygdala and nucleus accumbens shell. MPEP administration blocked both the relapse-like behavior and increase in p-ERK1/2 IR. p-ERK1/2 IR in the central amygdala and NAcb core was dissociated with the relapse-like behavior and the pharmacological effect of mGluR5 blockade. No changes in total ERK or p-ERK5 were observed. These results suggest that exposure to cues previously associated with alcohol self-administration is sufficient to produce concomitant increases in relapse-like behavior and ERK1/2 activation in specific limbic brain regions. Pharmacological compounds, such as mGluR5 antagonists, that reduce cue-induced ERK1/2 activation may be useful for treatment of relapse in alcoholics that is triggered by exposure to environmental events.