Adaptor protein complex 2 in the orbitofrontal cortex predicts alcohol use disorder.

Alcohol use disorder (AUD) is a life-threatening disease characterized by compulsive drinking, cognitive deficits, and social impairment that continue despite negative consequences. The inability of individuals with AUD to regulate drinking may involve functional deficits in cortical areas that normally balance actions that have aspects of both reward and risk. Among these, the orbitofrontal cortex (OFC) is critically involved in goal-directed behavior and is thought to maintain a representation of reward value that guides decision making. In the present study, we analyzed post-mortem OFC brain samples collected from age- and sex-matched control subjects and those with AUD using proteomics, bioinformatics, machine learning, and reverse genetics approaches. Of the 4,500+ total unique proteins identified in the proteomics screen, there were 47 proteins that differed significantly by sex that were enriched in processes regulating extracellular matrix and axonal structure. Gene ontology enrichment analysis revealed that proteins differentially expressed in AUD cases were involved in synaptic and mitochondrial function, as well as transmembrane transporter activity. Alcohol-sensitive OFC proteins also mapped to abnormal social behaviors and social interactions. Machine learning analysis of the post-mortem OFC proteome revealed dysregulation of presynaptic (e.g., AP2A1) and mitochondrial proteins that predicted the occurrence and severity of AUD. Using a reverse genetics approach to validate a target protein, we found that prefrontal Ap2a1 expression significantly correlated with voluntary alcohol drinking in male and female genetically diverse mouse strains. Moreover, recombinant inbred strains that inherited the C57BL/6J allele at the Ap2a1 interval consumed higher amounts of alcohol than those that inherited the DBA/2J allele. Together, these findings highlight the impact of excessive alcohol consumption on the human OFC proteome and identify important cross-species cortical mechanisms and proteins that control drinking in individuals with AUD.

The ethanol inhibition of basolateral amygdala neuron spiking is mediated by a γ-aminobutyric acid type A-mediated tonic current.

BACKGROUND: The basolateral nucleus of the amygdala (BLA) plays an important role in the development of fear and anxiety-related behaviors. The BLA receives inputs from all sensory stimuli. After processing those stimuli, BLA neurons signal neurons within the central amygdala and other brain regions, including the ventral and dorsal striatum and frontal cortex. Studies suggest that the BLA is involved in drug dependence and in the reinforcing actions of ethanol. For example, acute exposure to ethanol reduces anxiety, while withdrawal from chronic ethanol exposure alters BLA synaptic transmission, which increases anxiety, a common underlying cause of relapse. Exposure to and withdrawal from chronic alcohol also disrupts many brain areas that connect with the BLA. Despite these important findings, the acute actions of alcohol on the intrinsic excitability of BLA neurons have not been fully characterized. METHODS: Brain slices containing the BLA were prepared from adult C57BL/6J male mice. Whole-cell and sharp electrode electrophysiological recordings were performed to characterize the effects of acute ethanol on BLA neuronal and astrocyte function, respectively. RESULTS: Ethanol inhibited action potential (AP) firing of BLA neurons but had no effect on BLA astrocyte resting membrane potential. The ethanol-induced inhibition of firing was concentration-dependent (11 to 66 mM) and accompanied by a reduction in the input resistance and an increase in the rheobase of BLA neurons. The inhibitory effect of ethanol was suppressed by picrotoxin, which blocks both γ-aminobutyric acid type A (GABAA ) and glycine receptors, but not by the selective glycine receptor antagonist strychnine, which suggests an involvement of GABAA receptors. Ethanol did not affect spontaneous inhibitory postsynaptic currents suggesting that the inhibition of BLA neuronal excitability by ethanol was not due to an increase in GABAA -mediated synaptic transmission. However, acute ethanol enhanced the amplitude of the holding current of BLA neurons, an effect that was prevented by picrotoxin, which by itself reduced the holding current. CONCLUSIONS: These results suggest that BLA neurons express a GABA-mediated tonic current that is enhanced by acute ethanol, which leads to reduced excitability of BLA neurons.

The Abused Inhalant Toluene Impairs Medial Prefrontal Cortex Activity and Risk/Reward Decision-Making during a Probabilistic Discounting Task.

Inhalant (e.g., toluene) misuse is linked to behavioral and cognitive deficits in humans, yet preclinical studies of the effect of inhalants on higher-order cognition are limited. We addressed this gap in the literature by examining the effect of toluene vapor exposure on risk/reward decision-making in male and female Sprague-Dawley rats using a probabilistic discounting task. In this task, rodents chose a risky/large reward or a safe/small reward, with the odds of risky reinforcement descending or ascending throughout the test session. We observed a dose-dependent, sex-independent deficit in behavioral flexibility during probabilistic discounting caused by acute toluene exposure. Rats exposed to toluene vapor during adolescence and tested as adults performed comparably to air-treated controls and were susceptible to the effects of an acute toluene challenge. These behavioral flexibility deficits observed suggests dysfunctional medial prefrontal cortex (mPFC) activity. To address this hypothesis, we virally expressed the genetically encoded calcium sensor GCaMP6f in glutamatergic mPFC neurons and monitored calcium transients in real-time using in vivo fiber photometry. mPFC activity peaked before either lever press during free-choice trials in toluene- and air-treated animals. During forced-choice trials, GCaMP6f transients shifted from pre-risky to pre-safe choice, an effect mitigated by acute toluene exposure. mPFC activity decreased during rewarded trials, with larger decreases following risky/large wins compared with safe/small wins. Toluene-treated animals also had decreased mPFC activity during rewarded trials, but there was no distinction between risky/large wins and safe/small wins. These results provide physiological evidence for mPFC-dependent behavioral deficits caused by toluene.SIGNIFICANCE STATEMENT Inhalants (e.g., toluene) are an understudied class of drugs of abuse that cause devastating behavioral and cognitive deficits in humans. Understanding the neurobiological interactions of toluene vapor using animal models is important for developing effective treatment strategies for inhalant addicts. Here we find that toluene vapor reduces behavioral flexibility in rodents making risk/reward-based decisions. The medial prefrontal cortex (mPFC) drives behavioral flexibility during this type of decision-making and we show that toluene reduces the ability of mPFC neurons to track optimal choices as reward probabilities change. Toluene also reduces these neurons' ability to distinguish between small and large rewards. A combination of these factors likely leads to the impaired performance in probabilistic discounting following acute toluene exposure.

Knock-in Mice Expressing an Ethanol-Resistant GluN2A NMDA Receptor Subunit Show Altered Responses to Ethanol.

BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are glutamate-activated, heterotetrameric ligand-gated ion channels critically important in virtually all aspects of glutamatergic signaling. Ethanol (EtOH) inhibition of NMDARs is thought to mediate specific actions of EtOH during acute and chronic exposure. Studies from our laboratory, and others, identified EtOH-sensitive sites within specific transmembrane (TM) domains involved in channel gating as well as those in subdomains of extracellular and intracellular regions of GluN1 and GluN2 subunits that affect channel function. In this study, we characterize for the first time the physiological and behavioral effects of EtOH on knock-in mice expressing a GluN2A subunit that shows reduced sensitivity to EtOH. METHODS: A battery of tests evaluating locomotion, anxiety, sedation, motor coordination, and voluntary alcohol intake were performed in wild-type mice and those expressing the GluN2A A825W knock-in mutation. Whole-cell patch-clamp electrophysiological recordings were used to confirm reduced EtOH sensitivity of NMDAR-mediated currents in 2 separate brain regions (mPFC and the cerebellum) where the GluN2A subunit is known to contribute to NMDAR-mediated responses. RESULTS: Male and female mice homozygous for the GluN2A(A825W) knock-in mutation showed reduced EtOH inhibition of NMDAR-mediated synaptic currents in mPFC and cerebellar neurons as compared to their wild-type counterparts. GluN2A(A825W) male but not female mice were less sensitive to the sedative and motor-incoordinating effects of EtOH and showed a rightward shift in locomotor-stimulating effects of EtOH. There was no effect of the mutation on EtOH-induced anxiolysis or voluntary EtOH consumption in either male or female mice. CONCLUSIONS: These findings show that expression of EtOH-resistant GluN2A NMDARs results in selective and sex-specific changes in the behavioral sensitivity to EtOH.

Sex-dependent differences in ethanol inhibition of mouse lateral orbitofrontal cortex neurons.

Biological differences between males and females likely influence responses to alcohol and the propensity to engage in excessive drinking. In both humans and rodents, females escalate alcohol use and develop addiction-like behaviors faster than males, while males exhibit more severe withdrawal symptoms during abstinence. The mechanisms underlying these differences are not yet known but may reflect fundamental differences in the ethanol sensitivity of neurons in reward and control areas of the brain. To address this question, we recorded current-evoked spiking of lateral orbitofrontal cortex (lOFC) neurons in male and female C57BL/6J mice following acute and chronic exposure to ethanol. Ethanol (11-66 mM) reduced firing of lOFC neurons but produced less inhibition in neurons from female mice. As previously reported for male mice, the glycine receptor blocker strychnine blocked ethanol inhibition of spiking of lOFC neurons from female mice and prevented the ethanol-induced increase in tonic current. Following chronic intermittent ethanol (CIE) exposure, current-evoked spiking of lOFC neurons was significantly enhanced with a greater effect observed in males. After CIE treatment, acute ethanol had no effect on spiking in neurons from male mice, while it produced a slight but significant decrease in firing in females. Finally, like male mice, the inhibitory effect of the glycine transport inhibitor sarcosine was blunted in CIE-exposed female mice. Together, these results suggest that while lOFC neurons in male and female mice are similarly affected by ethanol, there are significant differences in sensitivity that may contribute to differences in alcohol actions between males and females.

Chemogenetic Excitation of Accumbens-Projecting Infralimbic Cortical Neurons Blocks Toluene-Induced Conditioned Place Preference.

Abuse rates for inhalants among adolescents continue to be high, yet preclinical models for studying mechanisms underlying inhalant abuse remain limited. Our laboratory has previously shown that, in male rats, an acute binge-like exposure to toluene vapor that mimics human solvent abuse modifies the intrinsic excitability of mPFC pyramidal neurons projecting to the NAc. These changes showed region (infralimbic; IL vs prelimbic; PRL), layer (shallow; 2/3 vs deep; 5/6), target (core vs shell), and age (adolescent vs adult) dependent differences (Wayman and Woodward, 2017). To expand these findings using reward-based models that may better mimic human drug abuse, we used whole-cell electrophysiology and drug receptors exclusively activated by designer drugs to examine changes in neuronal function and behavior in rats showing a conditioned place preference (CPP) to toluene. Repeated pairings of adolescent rats to binge concentrations of toluene vapor previously shown to enhance dopamine release in reward-sensitive areas of the brain produced CPP that persisted for 7 but not 30 d. Toluene-induced CPP was associated with increased excitability of IL5/6 mPFC neurons projecting to the core of the NAc and reduced excitability of those projecting to the NAc shell. No changes in PRL-NAc-projecting neurons were found in toluene-CPP rats. Chemogenetic reversal of the toluene-induced decrease in IL5/6-NAc shell neurons blocked the expression of toluene-induced CPP while manipulating IL5/6-NAc core neuron activity had no effect. These data reveal that alterations in selective mPFC-NAc pathways are required for expression of toluene-induced CPP.SIGNIFICANCE STATEMENT Disturbed physiology of pyramidal neurons projecting from the mPFC to the NAc has been shown to have different roles in drug-seeking behaviors for a number of drugs (e.g., methamphetamine, cocaine, ecstasy, alcohol, heroin). Here, we report that rats repeatedly exposed to the volatile organic solvent toluene, a member of the class of abused inhalants often used for intoxicating purposes by adolescents, induces a preference for the drug-paired environment that is accompanied by altered physiology of a specific population of NAc-projecting mPFC neurons. Chemogenetic correction of this deficit before testing prevented expression of drug preference. Overall, these findings highlight the importance of corticolimbic circuitry in mediating the rewarding properties of abused inhalants.

Prefrontal Cortex KCa2 Channels Regulate mGlu5-Dependent Plasticity and Extinction of Alcohol-Seeking Behavior.

Identifying novel treatments that facilitate extinction learning could enhance cue-exposure therapy and reduce high relapse rates in alcoholics. Activation of mGlu5 receptors in the infralimbic prefrontal cortex (IL-PFC) facilitates learning during extinction of cue-conditioned alcohol-seeking behavior. Small-conductance calcium-activated potassium (KCa2) channels have also been implicated in extinction learning of fear memories, and mGlu5 receptor activation can reduce KCa2 channel function. Using a combination of electrophysiological, pharmacological, and behavioral approaches, this study examined KCa2 channels as a novel target to facilitate extinction of alcohol-seeking behavior in rats. This study also explored related neuronal and synaptic mechanisms within the IL-PFC that underlie mGlu5-dependent enhancement of extinction learning. Using whole-cell patch-clamp electrophysiology, activation of mGlu5 in ex vivo slices significantly reduced KCa2 channel currents in layer V IL-PFC pyramidal neurons, confirming functional downregulation of KCa2 channel activity by mGlu5 receptors. Additionally, positive modulation of KCa2 channels prevented mGlu5 receptor-dependent facilitation of long-term potentiation in the IL-PFC. Systemic and intra-IL-PFC treatment with apamin (KCa2 channel allosteric inhibitor) significantly enhanced extinction of alcohol-seeking behavior across multiple extinction sessions, an effect that persisted for 3 weeks, but was not observed after apamin microinfusions into the prelimbic PFC. Positive modulation of IL-PFC KCa2 channels significantly attenuated mGlu5-dependent facilitation of alcohol cue-conditioned extinction learning. These data suggest that mGlu5-dependent facilitation of extinction learning and synaptic plasticity in the IL-PFC involves functional inhibition of KCa2 channels. Moreover, these findings demonstrate that KCa2 channels are a novel target to facilitate long-lasting extinction of alcohol-seeking behavior.SIGNIFICANCE STATEMENT Alcohol use disorder is a chronic relapsing disorder that is associated with compulsive alcohol-seeking behavior. One of the main causes of alcohol relapse is the craving caused by environmental cues that are associated with alcohol. These cues are formed by normal learning and memory principles, and the understanding of the brain mechanisms that help form these associations can lead to the development of drugs and/or behavior therapies that reduce the impact that these cues have on relapse in alcoholics.

Orbitofrontal Neuroadaptations and Cross-Species Synaptic Biomarkers in Heavy-Drinking Macaques.

Cognitive impairments, uncontrolled drinking, and neuropathological cortical changes characterize alcohol use disorder. Dysfunction of the orbitofrontal cortex (OFC), a critical cortical subregion that controls learning, decision-making, and prediction of reward outcomes, contributes to executive cognitive function deficits in alcoholic individuals. Electrophysiological and quantitative synaptomics techniques were used to test the hypothesis that heavy drinking produces neuroadaptations in the macaque OFC. Integrative bioinformatics and reverse genetic approaches were used to identify and validate synaptic proteins with novel links to heavy drinking in BXD mice. In drinking monkeys, evoked firing of OFC pyramidal neurons was reduced, whereas the amplitude and frequency of postsynaptic currents were enhanced compared with controls. Bath application of alcohol reduced evoked firing in neurons from control monkeys, but not drinking monkeys. Profiling of the OFC synaptome identified alcohol-sensitive proteins that control glutamate release (e.g., SV2A, synaptogyrin-1) and postsynaptic signaling (e.g., GluA1, PRRT2) with no changes in synaptic GABAergic proteins. Western blot analysis confirmed the increase in GluA1 expression in drinking monkeys. An exploratory analysis of the OFC synaptome found cross-species genetic links to alcohol intake in discrete proteins (e.g., C2CD2L, DIRAS2) that discriminated between low- and heavy-drinking monkeys. Validation studies revealed that BXD mouse strains with the D allele at the C2cd2l interval drank less alcohol than B allele strains. Thus, by profiling of the OFC synaptome, we identified changes in proteins controlling glutamate release and postsynaptic signaling and discovered several proteins related to heavy drinking that have potential as novel targets for treating alcohol use disorder.SIGNIFICANCE STATEMENT Clinical research identified cognitive deficits in alcoholic individuals as a risk factor for relapse, and alcoholic individuals display deficits on cognitive tasks that are dependent upon the orbitofrontal cortex (OFC). To identify neurobiological mechanisms that underpin OFC dysfunction, this study used electrophysiology and integrative synaptomics in a translational nonhuman primate model of heavy alcohol consumption. We found adaptations in synaptic proteins that control glutamatergic signaling in chronically drinking monkeys. Our functional genomic exploratory analyses identified proteins with genetic links to alcohol and cocaine intake across mice, monkeys, and humans. Future work is necessary to determine whether targeting these novel targets reduces excessive and harmful levels of alcohol drinking.

Loss of Ethanol Inhibition of N-Methyl-D-Aspartate Receptor-Mediated Currents and Plasticity of Cerebellar Synapses in Mice Expressing the GluN1(F639A) Subunit.

BACKGROUND: Glutamatergic N-methyl-d-aspartate receptors (NMDARs) are well known for their sensitivity to ethanol (EtOH) inhibition. However, the specific manner in which EtOH inhibits channel activity and how such inhibition affects neurotransmission, and ultimately behavior, remains unclear. Replacement of phenylalanine 639 with alanine (F639A) in the GluN1 subunit reduces EtOH inhibition of recombinant NMDARs. Mice expressing this subunit show reduced EtOH-induced anxiolysis, blunted locomotor stimulation following low-dose EtOH administration, and faster recovery of motor function after moderate doses of EtOH, suggesting that cerebellar dysfunction may contribute to some of these behaviors. In the mature mouse cerebellum, NMDARs at the cerebellar climbing fiber (CF) to Purkinje cell (PC) synapse are inhibited by low concentrations of EtOH and the long-term depression (LTD) of parallel fiber (PF)-mediated currents induced by concurrent activation of PFs and CFs (PF-LTD) requires activation of EtOH-sensitive NMDARs. In this study, we examined cerebellar NMDA responses and NMDA-mediated synaptic plasticity in wild-type (WT) and GluN1(F639A) mice. METHODS: Patch-clamp electrophysiological recordings were performed in acute cerebellar slices from adult WT and GluN1(F639A) mice. NMDAR-mediated currents at the CF-PC synapse and NMDAR-dependent PF-LTD induction were compared for genotype-dependent differences. RESULTS: Stimulation of CFs evoked robust NMDA-mediated excitatory postsynaptic currents (EPSCs) in PCs that were similar in amplitude and kinetics between WT and GluN1(F639A) mice. NMDA-mediated CF-PC EPSCs in WT mice were significantly inhibited by EtOH (50 mM) while those in mutant mice were unaffected. Concurrent stimulation of CF and PF inputs induced synaptic depression of PF-PC EPSCs in both WT and mutant mice, and this depression was blocked by the NMDA antagonist DL-APV. The synaptic depression of PF-PC EPSCs in WT mice was also blocked by a low concentration of EtOH (10 mM) that had no effect on plasticity in GluN1(F639A) mice. CONCLUSIONS: These results demonstrate that inhibition of cerebellar NMDARs may be a key mechanism by which EtOH affects cerebellar-dependent behaviors.

Chronic Alcohol, Intrinsic Excitability, and Potassium Channels: Neuroadaptations and Drinking Behavior.

Neural mechanisms underlying alcohol use disorder remain elusive, and this lack of understanding has slowed the development of efficacious treatment strategies for reducing relapse rates and prolonging abstinence. While synaptic adaptations produced by chronic alcohol exposure have been extensively characterized in a variety of brain regions, changes in intrinsic excitability of critical projection neurons are understudied. Accumulating evidence suggests that prolonged alcohol drinking and alcohol dependence produce plasticity of intrinsic excitability as measured by changes in evoked action potential firing and after-hyperpolarization amplitude. In this chapter, we describe functional changes in cell firing of projection neurons after long-term alcohol exposure that occur across species and in multiple brain regions. Adaptations in calcium-activated (KCa2), voltage-dependent (KV7), and G protein-coupled inwardly rectifying (Kir3 or GIRK) potassium channels that regulate the evoked firing and after-hyperpolarization parallel functional changes in intrinsic excitability induced by chronic alcohol. Moreover, there are strong genetic links between alcohol-related behaviors and genes encoding KCa2, KV7, and GIRK channels, and pharmacologically targeting these channels reduces alcohol consumption and alcohol-related behaviors. Together, these studies demonstrate that chronic alcohol drinking produces adaptations in KCa2, KV7, and GIRK channels leading to impaired regulation of the after-hyperpolarization and aberrant cell firing. Correcting the deficit in the after-hyperpolarization with positive modulators of KCa2 and KV7 channels and altering the GIRK channel binding pocket to block the access of alcohol represent a potentially highly effective pharmacological approach that can restore changes in intrinsic excitability and reduce alcohol consumption in affected individuals.

Correction to: Chronic Alcohol, Intrinsic Excitability, and Potassium Channels: Neuroadaptations and Drinking Behavior.

In section 8.1 on the 10th line in first paragraph the reference citation Mateos-Aparicio et al. 2014 is incorrect.

Altered NMDA receptor function in primary cultures of hippocampal neurons from mice lacking the Homer2 gene.

N-Methyl-D-Aspartate (NMDA) receptors are inhibited during acute exposure to ethanol and are involved in changes in neuronal plasticity following repeated ethanol exposure. The postsynaptic scaffolding protein Homer2 can regulate the cell surface expression of NMDA receptors in vivo, and mice with a null mutation of the Homer2 gene exhibit an alcohol-avoiding and -intolerant phenotype that is accompanied by a lack of ethanol-induced glutamate sensitization. Thus, Homer2 deletion may perturb the function or acute ethanol sensitivity of the NMDA receptor. In this study, the function and ethanol sensitivity of glutamate receptors in cultured hippocampal neurons from wild-type (WT) and Homer2 knock-out (KO) mice were examined at 7 and 14 days in vitro (DIV) using standard whole-cell voltage-clamp electrophysiology. As compared with wild-type controls, NMDA receptor current density was reduced in cultured hippocampal neurons from Homer2 KO mice at 14 DIV, but not at 7 DIV. There were no genotype-dependent changes in whole-cell capacitance or in currents evoked by kainic acid. The GluN2B-selective antagonist ifenprodil inhibited NMDA-evoked currents to a similar extent in both wild-type and Homer2 KO neurons and inhibition was greater at 7 versus 14 DIV. NMDA receptor currents from both WT and KO mice were inhibited by ethanol (10-100 mM) and the degree of inhibition did not differ as a function of genotype. In conclusion, NMDA receptor function, but not ethanol sensitivity, is reduced in hippocampal neurons lacking the Homer2 gene.

Phenotype-dependent inhibition of glutamatergic transmission on nucleus accumbens medium spiny neurons by the abused inhalant toluene.

Abused inhalants are voluntarily inhaled at high concentrations to produce intoxicating effects. Results from animal studies show that the abused inhalant toluene triggers behaviors, such as self-administration and conditioned place preference, which are commonly associated with addictive drugs. However, little is known about how toluene affects neurons within the nucleus accumbens (NAc), a brain region within the basal ganglia that mediates goal-directed behaviors and is implicated in the development and maintenance of addictive behaviors. Here we report that toluene inhibits a component of the after-hyperpolarization potential, and dose-dependently inhibits N-methyl-D-aspartate (NMDA)-mediated currents in rat NAc medium spiny neurons (MSN). Moreover, using the multivariate statistical technique, partial least squares discriminative analysis to analyze electrophysiological measures from rat NAc MSNs, we show that toluene induces a persistent depression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated currents in one subtype of NAc MSNs, and that the electrophysiological features of MSN neurons predicts their sensitivity to toluene. The CB1 receptor antagonist AM281 blocked the toluene-induced long-term depression of AMPA currents, indicating that this process is dependent on endocannabinoid signaling. The neuronal identity of recorded cells was examined using dual histochemistry and shows that toluene-sensitive NAc neurons are dopamine D2 MSNs that express preproenkephalin mRNA. Overall, the results from these studies indicate that physiological characteristics obtained from NAc MSNs during whole-cell patch-clamp recordings reliably predict neuronal phenotype, and that the abused inhalant toluene differentially depresses excitatory neurotransmission in NAc neuronal subtypes.

Cysteine substitution of transmembrane domain amino acids alters the ethanol inhibition of GluN1/GluN2A N-methyl-D-aspartate receptors.

N-Methyl-d-aspartate receptors (NMDARs) are inhibited by behaviorally relevant concentrations of ethanol, and residues within transmembrane (TM) domains of NMDARs, including TM3 GluN1 phenylalanine 639 (F639), regulate this sensitivity. In the present study, we used cysteine (C) mutagenesis to determine whether there are additional residues within nearby TM domains that regulate ethanol inhibition on NMDARs. GluN1(F639C)/GluN2A receptors were less inhibited by ethanol than wild-type receptors, and inhibition was restored to wild-type levels following treatment with ethanol-like methanethiosulfonate reagents. Molecular modeling identified six residues in the GluN1 TM1 domain (valine V566; serine S569) and the GluN2A TM4 domain (methionine, M817; V820, F821, and leucine, L824) that were in close vicinity to the TM3 F639 residue, and these were individually mutated to cysteine and tested for ethanol inhibition and receptor function. The F639C-induced decrease in ethanol inhibition was blunted by coexpression of GluN1 TM1 mutants V566C and S569C, and statistically significant interactions were observed for ethanol inhibition among V566C, F639C, and GluN2A TM4 mutants V820C and F821C and S569C, F639C, and GluN2A TM4 mutants F821C and L824C. Ethanol inhibition was also reduced when either GluN1 TM1 mutant V566C or S569C was combined with GluN2A V820C, suggesting a novel TM1:TM4 intrasubunit site of action for ethanol. Cysteines substituted at TM3 and TM4 sites previously suggested to interact with ethanol had less dramatic effects on ethanol inhibition. Overall, the results from these studies suggest that interactions among TM1, TM3, and TM4 amino acids in NMDARs are important determinants of ethanol action at these receptors.

Medial prefrontal cortex inversely regulates toluene-induced changes in markers of synaptic plasticity of mesolimbic dopamine neurons.

Toluene is a volatile solvent that is intentionally inhaled by children, adolescents, and adults for its intoxicating effects. Although voluntary use of toluene suggests that it possesses rewarding properties and abuse potential, it is unknown whether toluene alters excitatory synaptic transmission in reward-sensitive dopamine neurons like other drugs of abuse. Here, using a combination of retrograde labeling and slice electrophysiology, we show that a brief in vivo exposure of rats to a behaviorally relevant concentration of toluene vapor enhances glutamatergic synaptic strength of dopamine (DA) neurons projecting to nucleus accumbens core and medial shell neurons. This effect persisted for up to 3 d in mesoaccumbens core DA neurons and for at least 21 d in those projecting to the medial shell. In contrast, toluene vapor exposure had no effect on synaptic strength of DA neurons that project to the medial prefrontal cortex (mPFC). Furthermore, infusion of GABAergic modulators into the mPFC before vapor exposure to pharmacologically manipulate output, inhibited, or potentiated toluene's action on mesoaccumbens DA neurons. Together, the results of these studies indicate that toluene induces a target-selective increase in mesolimbic DA neuron synaptic transmission and strongly implicates the mPFC as an important regulator of drug-induced plasticity of mesolimbic dopamine neurons.

Toluene alters the intrinsic excitability and excitatory synaptic transmission of basolateral amygdala neurons.

Introduction: Inhalant abuse is an important health issue especially among children and adolescents who often encounter these agents in the home. Research into the neurobiological targets of inhalants has lagged behind that of other drugs such as alcohol and psychostimulants. However, studies from our lab and others have begun to reveal how inhalants such as the organic solvent toluene affect neurons in key addiction related areas of the brain including the ventral tegmental area, nucleus accumbens and medial prefrontal cortex. In the present study, we extend these findings and examine the effect of toluene on electrophysiological responses of pyramidal neurons in the basolateral amygdala BLA, a region important for generating emotional and reward based information needed to guide future behavior. Methods: Whole-cell patch-clamp electrophysiology recordings of BLA pyramidal neurons in rat brain slices were used to assess toluene effects on intrinsic excitability and excitatory glutamatergic synaptic transmission. Results: Acute application of 3 mM but not 0.3 mM toluene produced a small but significant (~20%) increase in current-evoked action potential (AP) firing that reversed following washout of the toluene containing solution. The change in firing during exposure to 3 mM toluene was accompanied by selective changes in AP parameters including reduced latency to first spike, increased AP rise time and decay and a reduction in the fast after-hyperpolization. To examine whether toluene also affects excitatory synaptic signaling, we expressed channelrhodopsin-2 in medial prefrontal cortex neurons and elicited synaptic currents in BLA neurons via light pulses. Toluene (3 mM) reduced light-evoked AMPA-mediated synaptic currents while a lower concentration (0.3 mM) had no effect. The toluene-induced reduction in AMPA-mediated BLA synaptic currents was prevented by the cannabinoid receptor-1 antagonist AM281. Discussion: These findings are the first to demonstrate effects of acute toluene on BLA pyramidal neurons and add to existing findings showing that abused inhalants such as toluene have significant effects on neurons in brain regions involved in natural and drug induced reward.

Deletion of the N-terminal domain alters the ethanol inhibition of N-methyl-D-aspartate receptors in a subunit-dependent manner.

BACKGROUND: Ethanol (EtOH) inhibition of N-methyl-d-aspartate (NMDA) receptors is poorly understood due in part to the organizational complexity of the receptor that provides ample locations for sites of action. Among these, the N-terminal domain (NTD) of NMDA receptor subunits contains binding sites for a variety of modulatory agents including zinc, protons, and GluN2B selective antagonists such as ifenprodil or Ro-25-6981. EtOH inhibition of neuronal NMDA receptors expressed in some brain areas has been reported to be occluded by the presence of ifenprodil or similar compounds suggesting that the NTD may be important in regulating the EtOH sensitivity of NMDA receptors. METHODS: Wild-type GluN1 and GluN2 subunits and those in which the coding sequence for the NTD was deleted were expressed in HEK293 cells. Whole-cell voltage-clamp recording was used to assess EtOH inhibition of wild-type and mutant receptors lacking the NTD. RESULTS: As compared to wild-type GluN1/GluN2A receptors, EtOH inhibition was slightly greater in cells expressing GluN2A subunits lacking the NTD. In contrast, GluN2B N-terminal deletion mutants showed normal EtOH inhibition while those lacking the NTD in both GluN1 and GluN2B subunits had decreased EtOH inhibition as compared to wild-type receptors. NTD lacking GluN2B receptors were insensitive to ifenprodil but retained normal sensitivity to EtOH. CONCLUSIONS: These findings indicate that the NTD modestly influences the EtOH sensitivity of NMDA receptors in a subunit-dependent manner. They also show that ifenprodil's actions on GluN2B-containing receptors can be dissociated from those of EtOH. These results suggest that while the NTD is not a primary site of action for EtOH on NMDA receptors, it likely affects sensitivity via actions on intrinsic channel properties.

The Effects of the Inhalant Toluene on Cognitive Function and Behavioral Flexibility: A Review of Recent Findings.

Substance use disorder (SUD) is characterized, in part, by lack of control over drug seeking and taking. The prefrontal cortex (PFC) is highly involved in control of behavior and deficits in PFC structure and function have been demonstrated in clinical and preclinical studies of SUD. Of the various classes of drugs associated with the development of SUD, inhalants are among the least studied despite their widespread use among adolescents and children. In this work, we review what is currently known regarding the sites and mechanisms of action of inhalants with a focus on the volatile solvent toluene that is contained in a wide variety of legal and easily obtained products. We then describe how inhalants including toluene affect various behaviors with an emphasis on those associated with PFC function and how chronic use of inhalants alters brain structure and neuronal signaling. Findings from these studies highlight advances made in recent years that have expanded our understanding of the effects of inhalants on brain structure and reinforce the need for continued work in this field.

A brief exposure to toluene vapor alters the intrinsic excitability of D2 medium spiny neurons in the rat ventral striatum.

Although volatile organic solvents such as toluene are used for commercial and industrial uses, they are often voluntarily inhaled for their intoxicating and euphoric effects. Research into the effects of inhalants such as toluene on brain function have revealed actions on a variety of ligand-gated and voltage-activated ion channels involved in regulating neuronal excitability. Previous work from this laboratory has also shown that brief exposures to toluene vapor induce changes in the intrinsic excitability and synaptic transmission of neurons within the medial prefrontal cortex and ventral tegmental area that vary depending on projection target. In the present study, we recorded current-evoked spiking of medium spiny neurons (MSNs) in the nucleus accumbens (NAc) core and shell in adolescent rats exposed to an intoxicating concentration of toluene vapor. Compared to air controls, firing of NAc core MSNs in Sprague-Dawley rats was not altered 24 h after exposure to 10,500 ppm toluene vapor while spiking of NAc shell MSNs was enhanced at low current steps but reduced at higher current steps. When the rheobase current was used to putatively identify MSN subtypes, both "D1-like" and "D2-like" MSNs within the NAc shell but not core showed toluene-induced changes in firing. As toluene may itself have altered the rheobase resulting in misclassification of neuron subtype, we conducted additional studies using adolescent D2-Cre rats infused with a Cre-dependent mCherry reporter virus. Following toluene vapor exposure, spiking of NAc shell D2+ MSNs was enhanced at low current steps but inhibited at higher currents as compared to air controls while there were no differences in the firing of NAc shell D2- MSNs. The toluene-induced change in NAc D2+ shell MSN firing was accompanied by alterations in membrane resistance, rheobase, action potential rise time and height with no changes noted in D2- MSNs. Overall, these data add to a growing literature showing that brief exposures to intoxicating concentrations of toluene vapor causes selective alterations in the excitability of neurons within the addiction neurocircuitry that vary depending on sub-region, cell-type and projection target.

Alcohol dependence and the ventral hippocampal influence on alcohol drinking in male mice.

Examining neural circuits underlying persistent, heavy drinking provides insight into the neurobiological mechanisms driving alcohol use disorder. Facilitated by its connectivity with other parts of the brain such as the nucleus accumbens (NAc), the ventral hippocampus (vHC) supports many behaviors, including those related to reward seeking and addiction. These studies used a well-established mouse model of alcohol (ethanol) dependence. After surgery to infuse DREADD-expressing viruses (hM4Di, hM3Dq, or mCherry-only) into the vHC and position guide cannula above the NAc, male C57BL/6J mice were treated in the CIE drinking model that involved repeated cycles of chronic intermittent alcohol (CIE) vapor or air (CTL) exposure alternating with weekly test drinking cycles in which mice were offered alcohol (15% v/v) 2 h/day. Additionally, smaller groups of mice were evaluated for either cFos expression or glutamate release using microdialysis procedures. In CIE mice expressing inhibitory (hM4Di) DREADDs in the vHC, drinking increased as expected, but CNO (3 mg/kg intraperitoneally [i.p.]) given 30 min before testing did not alter alcohol intake. However, in CTL mice expressing hM4Di, CNO significantly increased alcohol drinking (∼30%; p < 0.05) to levels similar to the CIE mice. The vHC-NAc pathway was targeted by infusing CNO into the NAc (3 or 10 µM/side) 30 min before testing. CNO activation of the pathway in mice expressing excitatory (hM3Dq) DREADDs selectively reduced consumption in CIE mice back to CTL levels (∼35-45%; p < 0.05) without affecting CTL alcohol intake. Lastly, activating the vHC-NAc pathway increased cFos expression and evoked significant glutamate release from the vHC terminals in the NAc. These data indicate that reduced activity of the vHC increases alcohol consumption and that targeted, increased activity of the vHC-NAc pathway attenuates excessive drinking associated with alcohol dependence. Thus, these findings indicate that the vHC and its glutamatergic projections to the NAc are involved in excessive alcohol drinking.