Inhibitory and excitatory alcohol-seeking cues distinct roles in behavior, neurochemistry, and mesolimbic pathway in alcohol preferring (P) rats.

Cues associated with alcohol use can readily enhance self-reported cravings for alcohol, which increases the likelihood of reusing alcohol. Understanding the neuronal mechanisms involved in alcohol-seeking behavior is important for developing strategies to treat alcohol use disorder. In all experiments, adult female alcohol-preferring (P) rats were exposed to three conditioned odor cues; CS+ associated with EtOH self-administration, CS- associated with the absence of EtOH (extinction training), and a CS0, a neutral stimulus. The data indicated that presentation of an excitatory conditioned cue (CS+) can enhance EtOH- seeking while the CS- can inhibit EtOH-seeking under multiple test conditions. Presentation of the CS+ activates a subpopulation of dopamine neurons within the interfascicular nucleus of the posterior ventral tegmental area (posterior VTA) and basolateral amygdala (BLA). Pharmacological inactivation of the BLA with GABA agonists inhibits the ability of the CS+ to enhance EtOH-seeking but does not alter context-induced EtOH-seeking or the ability of the CS- to inhibit EtOH-seeking. Presentation of the conditioned odor cues in a non-drug-paired environment indicated that presentation of the CS+ increased dopamine levels in the BLA. In contrast, presentation of the CS- decreased both glutamate and dopamine levels in the BLA. Further analysis revealed that presentation of a CS+ EtOH-associated conditioned cue activates GABA interneurons but not glutamate projection neurons. Overall, the data indicate that excitatory and inhibitory conditioned cues can contrarily alter EtOH-seeking behaviors and that different neurocircuitries are mediating these distinct cues in critical brain regions. Pharmacotherapeutics for craving should inhibit the CS+ and enhance the CS- neurocircuits.

The Rewarding and Anxiolytic Properties of Ethanol within the Central Nucleus of the Amygdala: Mediated by Genetic Background and Nociceptin.

In humans, alcohol is consumed for its rewarding and anxiolytic effects. The central nucleus of the amygdala (CeA) is considered a neuronal nexus that regulates fear, anxiety, and drug self-administration. Manipulations of the CeA alter ethanol (EtOH) consumption under numerous EtOH self-administration models. The experiments determined whether EtOH is reinforcing/anxiolytic within the CeA, whether selective breeding for high alcohol consumption alters the rewarding properties of EtOH in the CeA, and whether the reinforcing/anxiolytic effects of EtOH in the CeA are mediated by the neuropeptides corticotropin-releasing factor (CRF) and nociceptin. The reinforcing properties of EtOH were determined by having male Wistar and Taconic alcohol-preferring (tP) rats self-administer EtOH directly into the CeA. The expression of anxiety-like behaviors was assessed through multiple behavioral models (social interaction, acoustic startle, and open field). Coadministration of EtOH and a CRF1 antagonist (NBI35965) or nociceptin on self-administration into the CeA and anxiety-like behaviors was determined. EtOH was self-administered directly into the lateral CeA, and tP rats self-administered a lower concentration of EtOH than Wistar rats. EtOH microinjected into the lateral CeA reduced the expression of anxiety-like behaviors, indicating an anxiolytic effect. Coadministration of NBI35965 failed to alter the rewarding/anxiolytic properties of EtOH in the CeA. In contrast, coadministration of the nociceptin enhanced both EtOH reward and anxiolysis in the CeA. Overall, the data indicate that the lateral CeA is a key anatomic location that mediates the rewarding and anxiolytic effects of EtOH, and local nociceptin receptors, but not local CRF1 receptors, are involved in these behaviors. SIGNIFICANCE STATEMENT: Alcohol is consumed for the stimulatory, rewarding, and anxiolytic properties of the drug of abuse. The current data are the first to establish that alcohol is reinforcing and anxiolytic within the lateral central nucleus of the amygdala (CeA) and that the nociceptin system regulates these effects of alcohol within the CeA.

Co-administration of ethanol and nicotine heightens sensitivity to ethanol reward within the nucleus accumbens (NAc) shell and increasing NAc shell BDNF is sufficient to enhance ethanol reward in naïve Wistar rats.

Alcohol use disorder most commonly presents as a polydrug disorder where greater than 85% are estimated to smoke. EtOH and nicotine (NIC) co-abuse or exposure results in unique neuroadaptations that are linked to behaviors that promote drug use. The current experiments aimed to identify neuroadaptations within the mesolimbic pathway produced by concurrent EtOH and NIC exposure. The experiments used four overall groups of male Wistar rats consisting of vehicle, EtOH or NIC alone, and EtOH+NIC. Drug exposure through direct infusion into the posterior ventral tegmental area (pVTA) stimulated release of glutamate and dopamine in the nucleus accumbens (NAc) shell, which was quantified through high-performance liquid chromatography. Additionally, brain-derived neurotrophic factor (BDNF) protein levels were measured via enzyme-linked immunosorbent assay (ELISA). A second experiment investigated the effects of drug pretreatment within the pVTA on the reinforcing properties of EtOH within the NAc shell through intracranial self-administration (ICSA). The concluding experiment evaluated the effect of NAc shell pretreatment with BDNF on EtOH reward utilizing ICSA within that region. The data indicated that only EtOH+NIC administration into the pVTA simultaneously increased glutamate, dopamine, and BDNF in the NAc shell. Moreover, only pVTA pretreatment with EtOH+NIC enhanced the reinforcing properties of EtOH in the NAc shell. BDNF pretreatment in the NAc shell was also sufficient to enhance the reinforcing properties of EtOH in the NAc shell. The collected data suggest that concurrent EtOH+NIC exposure results in a distinct neurochemical response and neuroadaptations within the mesolimbic pathway that alter EtOH reward.

Peri-adolescent alcohol consumption increases sensitivity and dopaminergic response to nicotine during adulthood in female alcohol-preferring (P) rats: Alterations to α7 nicotinic acetylcholine receptor expression.

Adolescent alcohol drinking has been linked to increased risk for drug abuse during adulthood. Nicotine microinjected directly into the posterior ventral tegmental area (pVTA) stimulates dopamine (DA) release in the nucleus accumbens (NAc) shell. The α7 nicotinic acetylcholine receptor (nAChR) is a potent regulator of dopaminergic activity in the pVTA. The current experiments examined the effects of peri-adolescent ethanol (EtOH) drinking on the ability of intra-pVTA nicotine to stimulate DA release during adulthood and alterations in α7 nAChR expression within the pVTA. Alcohol-preferring (P) female rats consumed EtOH and/or water during adolescence (post-natal day [PND] 30-60) or adulthood (PND 90-120). Thirty days following removal of EtOH, subjects received microinjections of 1 µM, 10 µM, or 50 µM nicotine into the pVTA concurrently with microdialysis for extracellular DA in the NAc shell. Brains were harvested from an additional cohort after PND 90 for quantification of α7 nAChR within the pVTA. The results indicated that only adolescent EtOH consumption produced a leftward and upward shift in the dose response curve for nicotine to stimulate DA release in the NAc shell. Investigation of α7 nAChR expression within the pVTA revealed a significant increase in animals that consumed EtOH during adolescence compared to naïve animals. The data suggests that peri-adolescent EtOH consumption produced cross-sensitization to the effects of nicotine during adulthood. The interaction between adolescent EtOH consumption and inflated adult risk for drug dependency could be predicated, at least in part, upon alterations in α7 nAChR expression within the mesolimbic reward pathway.

Adolescent Intermittent Ethanol Increases the Sensitivity to the Reinforcing Properties of Ethanol and the Expression of Select Cholinergic and Dopaminergic Genes within the Posterior Ventral Tegmental Area.

BACKGROUND: Although not legally allowed to consume alcohol, adolescents account for 11% of all alcohol use in the United States and approximately 90% of adolescent intake is in the form of an alcohol binge. The adolescent intermittent ethanol (AIE) model developed by the NADIA consortium produces binge-like EtOH exposure episodes. The current experiment examined the effects of AIE on the reinforcing properties of EtOH and genetic expression of cholinergic and dopaminergic factors within the posterior ventral tegmental area (pVTA) in Wistar male and female rats and in male alcohol-preferring (P) rats. METHODS: Rats were exposed to the AIE or water during adolescence, and all testing occurred during adulthood. Wistar control and AIE rats were randomly assigned to groups that self-administered 0 to 200 mg% EtOH. Male P rats self-administered 0 to 100 mg%. RESULTS: The data indicated that exposure to AIE in both Wistar male and female rats (and male P rats) resulted in a significant leftward shift in dose-response curve for EtOH self-administration into the pVTA. TaqMan array indicated that AIE exposure had divergent effects on the expression of nicotinic receptors (increased a7, reduction in a4 and a5). There were also sex-specific effects of AIE on gene expression; male only reduction in D3 receptors. CONCLUSION: Binge-like EtOH exposure during adolescence enhances the sensitivity to the reinforcing properties of EtOH during adulthood which could be part of biological sequelae that are the basis for the deleterious effects of adolescent alcohol consumption on the rate of alcoholism during adulthood.

Conditioned stimuli affect ethanol-seeking by female alcohol-preferring (P) rats: the role of repeated-deprivations, cue-pretreatment, and cue-temporal intervals.

RATIONALE: Evidence indicates that drug-paired stimuli can evoke drug-craving leading to drug-seeking and repeated relapse periods can influence drug-seeking behaviors. OBJECTIVES: The present study examined (1) the effect of an interaction between repeated deprivation cycles and excitatory conditioning stimuli (CS+) on ethanol (EtOH)-seeking; (2) the effects of EtOH-paired cue-exposure in a non-drug-paired environment on subsequent conditioning in a drug-paired environment; and (3) the temporal effects of conditioned cues on subsequent EtOH-seeking. METHODS: Adult female alcohol-preferring (P) rats were exposed to three conditioned odor cues; CS+ associated with EtOH self-administration, CS- associated with the absence of EtOH (extinction training), and a neutral stimulus (CS0) presented in a neutral non-drug-paired environment. The rats underwent four deprivation cycles or were non-deprived, following extinction they were maintained in a home cage for an EtOH-free period, and then exposed to no cue, CS+, CS-, or CS0 to assess the effect of the conditioned cues on EtOH-seeking behavior. RESULTS: Repeated deprivations enhanced and prolonged the duration of CS+ effects on EtOH-seeking. Presentation of the CS- in a non-drug-paired environment blocked the ability of a CS+ to enhance EtOH-seeking in a drug-paired environment. Presentation of the CS+ or CS- in a non-drug-paired environment 2 or 4 h earlier significantly altered EtOH-seeking. CONCLUSION: Results indicated an interaction between repeated deprivation cycles and CS+ resulted in a potentiation of CS+ evoked EtOH-seeking. In addition, a CS- may have therapeutic potential by providing prophylactic protection against relapse behavior in the presence of cues in the drug-using environment.

Therapeutic challenges for concurrent ethanol and nicotine consumption: naltrexone and varenicline fail to alter simultaneous ethanol and nicotine intake by female alcohol-preferring (P) rats.

RATIONALE AND OBJECTIVES: Simultaneous alcohol and nicotine consumption occurs in the majority of individuals with alcohol use disorder (AUD) and nicotine dependence. Varenicline (Var) is used to assist in the cessation of nicotine use, while naltrexone (Nal) is the standard treatment for AUD. Despite evidence that ethanol (EtOH) and nicotine (NIC) co-use produces unique neuroadaptations, preclinical research has focused on the effects of pharmacotherapeutics on a single reinforcer. The current experiments examined the effects of Var and Nal on EtOH, NIC, or EtOH+NIC intake. METHODS: Animals were randomly assigned to one of four drinking conditions of 24-h access to a three-bottle choice paradigm, one of which always contained water. Drinking conditions were water only, 0.07 and 0.14 mg/mL NIC (NIC only), 15% and 30% EtOH (EtOH only), or 15% and 30% EtOH with 0.14 mg/mL NIC (EtOH+NIC). The effects of Var (0, 1, or 2 mg/kg) or Nal (0, 1, or 10 mg/kg) injections on maintenance and relapse consumption were determined during four consecutive days. RESULTS: Var reduced maintenance and relapse NIC intake but had no effect on EtOH or EtOH+NIC drinking. Conversely, Nal reduced EtOH maintenance and relapse drinking, but had no effect on NIC or EtOH+NIC drinking. DISCUSSION: The results indicate the standard pharmacological treatments for nicotine dependence and AUD were effective at reducing consumption of the targeted reinforcer but neither reduced EtOH+NIC co-use/abuse. These findings suggest that co-abuse may promote unique neuroadaptations that require models of polysubstance abuse to develop pharmacotherapeutics to treat AUD and nicotine dependence.

Selective breeding for high alcohol consumption and response to nicotine: locomotor activity, dopaminergic in the mesolimbic system, and innate genetic differences in male and female alcohol-preferring, non-preferring, and replicate lines of high-alcohol drinking and low-alcohol drinking rats.

RATIONALE: There is evidence for a common genetic link between alcohol and nicotine dependence. Rodents selectively bred for high alcohol consumption/responsivity are also more likely to self-administer nicotine than controls. OBJECTIVES: The experiments examined the response to systemic nicotine, the effects of nicotine within the drug reward pathway, and innate expression of nicotine-related genes in a brain region regulating drug reward/self-administration in multiple lines of rats selectively bred for high and low alcohol consumption. METHODS: The experiments examined the effects of systemic administration of nicotine on locomotor activity, the effects of nicotine administered directly into the (posterior ventral tegmental area; pVTA) on dopamine (DA) release in the nucleus accumbens shell (AcbSh), and innate mRNA levels of acetylcholine receptor genes in the pVTA were determined in 6 selectively bred high/low alcohol consuming and Wistar rat lines. RESULTS: The high alcohol-consuming rat lines had greater nicotine-induced locomotor activity compared to low alcohol-consuming rat lines. Microinjections of nicotine into the pVTA resulted in DA release in the AcbSh with the dose response curves for high alcohol-consuming rats shifted leftward and upward. Genetic analysis of the pVTA indicated P rats expressed higher levels of α2 and ß4. CONCLUSION: Selective breeding for high alcohol preference resulted in a genetically divergent behavioral and neurobiological sensitivity to nicotine. The observed behavioral and neurochemical differences between the rat lines would predict an increased likelihood of nicotine reinforcement. The data support the hypothesis of a common genetic basis for drug addiction and identifies potential receptor targets.

Parameters of Context-Induced Ethanol (EtOH)-Seeking in Alcohol-Preferring (P) Rats: Temporal Analysis, Effects of Repeated Deprivation, and EtOH Priming Injections.

BACKGROUND: Drug-paired environments can act as stimuli that elicit drug craving. In humans, drug craving is influenced by the amount of time abstinent, number of past periods of abstinence, and inadvertent exposure to the previously abused drug. The current experiments were designed to determine the effects of (i) the duration of abstinence on expression of ethanol (EtOH)-seeking; (ii) EtOH priming following a short and long abstinence period; and (iii) repeated deprivation cycles on relapse drinking and EtOH-seeking. METHODS: Rats were allowed to self-administer 15% EtOH, processed through extinction training, maintained in a home cage for a designated EtOH-free period, and then reintroduced to the operant context in the absence of EtOH. The experiments examined the effects of: (i) various home-cage duration periods (1 to 8 weeks), (ii) priming injections of EtOH in the Pavlovian spontaneous recovery (PSR; 14 days after extinction) and reinstatement of responding (RoR; 1 day after extinction) models, and (iii) exposure to repeated cycles of EtOH access-deprivation on relapse drinking and EtOH-seeking behavior. RESULTS: Highest expression of EtOH-seeking was observed following 6 weeks of home-cage maintenance. Priming injections of EtOH were more efficacious at stimulating/enhancing EtOH-seeking in the PSR than RoR model. Exposure to repeated cycles of EtOH deprivation and access enhanced and prolonged relapse drinking and the expression of EtOH-seeking (318 ± 22 responses), which was not observed in rats given equivalent consistent exposure to EtOH (66 ± 11 responses). CONCLUSIONS: Overall, the data indicated that the PSR model has ecological validity; factors that enhance EtOH craving in humans enhance the expression of EtOH-seeking in the PSR test. The data also detail factors that need to be examined to determine the biological basis of EtOH-seeking (e.g., neuroadaptations that occur during the incubation period and following repeated cycles of EtOH drinking and abstinence).

Peripheral Administration of Ethanol Results in a Correlated Increase in Dopamine and Serotonin Within the Posterior Ventral Tegmental Area.

AIMS: Two critical neurotransmitter systems regulating ethanol (EtOH) reward are serotonin (5-HT) and dopamine (DA). Within the posterior ventral tegmental area (pVTA), 5-HT receptors have been shown to regulate DA neuronal activity. Increased pVTA neuronal activity has been linked to drug reinforcement. The current experiment sought to determine the effect of EtOH on 5-HT and DA levels within the pVTA. METHODS: Wistar rats were implanted with cannula aimed at the pVTA. Neurochemical levels were determined using standard microdialysis procedures with concentric probes. Rats were randomly assigned to one of the five groups (n = 41; 7-9 per group) that were treated with 0-3.0 g/kg EtOH (intraperitoneally). RESULTS: Ethanol produced increased extracellular DA levels in the pVTA that resembled an inverted U-shape dose-response curve with peak levels (~200% of baseline) at the 2.25 g/kg dose. The increase in DA levels was observed for an extended period of time (~100 minutes). The effects of EtOH on extracellular 5-HT levels in the pVTA also resembled an inverted U-shape dose-response curve. However, increased 5-HT levels were only observed during the initial post-injection sample. The increases in extracellular DA and 5-HT levels were significantly correlated. CONCLUSION: The data indicate intraperitoneal EtOH administration stimulated the release of both 5-HT and DA within the pVTA, the levels of which were significantly correlated. Overall, the current findings suggest that the ability of EtOH to stimulate DA activity within the mesolimbic system may be modulated by increases in 5-HT release within the pVTA. SHORT SUMMARY: Two critical neurotransmitter systems regulating ethanol reward are serotonin and dopamine. The current experiment determined that intraperitoneal ethanol administration increased serotonin and dopamine levels within the pVTA (levels were significantly correlated). The current findings suggest the ability of EtOH to stimulate serotonin and dopamine activity within the mesolimbic system.

Oral Conditioned Cues Can Enhance or Inhibit Ethanol (EtOH)-Seeking and EtOH-Relapse Drinking by Alcohol-Preferring (P) Rats.

BACKGROUND: Conditioned cues can elicit drug-seeking in both humans and rodents. The majority of preclinical research has employed excitatory conditioned cues (stimuli present throughout the availability of a reinforcer), but oral consumption of alcohol is similar to a conditional stimuli (presence of stimuli is paired with the delivery of the reinforcer) approach. The current experiments attempted to determine the effects of conditional stimuli (both excitatory and inhibitory) on the expression of context-induced ethanol (EtOH)-seeking. METHODS: Alcohol-preferring (P) rats self-administered EtOH and water in standard 2-lever operant chambers. A flavor was added to the EtOH solution (CS+) during the EtOH self-administration sessions. After 10 weeks, rats underwent extinction training (7 sessions), followed by a 2-week home cage period. Another flavor was present during extinction (CS-). Rats were exposed to a third flavor in a non-drug-paired environment (CS(0)). EtOH-seeking was assessed in the presence of no cue, CS+, CS-, or CS(0) in the dipper previously associated with EtOH self-administration (no EtOH available). Rats were maintained a week in their home cage before being returned to the operant chambers with access to EtOH (flavored with no cue, CS+, CS-, or CS(0)). RESULTS: The results indicated that the presence of the CS+ enhanced EtOH-seeking, while the presence of the CS- suppressed EtOH-seeking. Similarly, adding the CS- flavor to 15% EtOH reduced responding for EtOH while the CS+ enhanced responding for EtOH during relapse testing. CONCLUSIONS: Overall, the data indicate that conditional stimuli are effective at altering both EtOH-seeking behavior and EtOH-relapse drinking.

Co-administration of ethanol and nicotine: the enduring alterations in the rewarding properties of nicotine and glutamate activity within the mesocorticolimbic system of female alcohol-preferring (P) rats.

RATIONALE: The co-abuse of ethanol (EtOH) and nicotine (NIC) increases the likelihood that an individual will relapse to drug use while attempting to maintain abstinence. There is limited research examining the consequences of long-term EtOH and NIC co-abuse. OBJECTIVES: The current experiments determined the enduring effects of chronic EtOH, NIC, or EtOH + NIC intake on the reinforcing properties of NIC and glutamate (GLU) activity within the mesocorticolimbic (MCL) system. METHODS: Alcohol-preferring (P) rats self-administered EtOH, Sacc + NIC, or EtOH + NIC combined for 10 weeks. The reinforcing properties of 0.1-3.0 µM NIC within the nucleus accumbens shell (AcbSh) were assessed following a 2-3-week drug-free period using intracranial self-administration (ICSA) procedures. The effects of EtOH, Sacc, Sacc + NIC, or EtOH + NIC intake on extracellular levels and clearance of glutamate (GLU) in the medial prefrontal cortex (mPFC) were also determined. RESULTS: Binge intake of EtOH (96-100 mg%) and NIC (21-27 mg/mL) were attained. All groups of P rats self-infused 3.0 µM NIC directly into the AcbSh, whereas only animals in the EtOH + NIC co-abuse group self-infused the 0.3 and 1.0 µM NIC concentrations. Additionally, self-administration of EtOH + NIC, but not EtOH, Sacc or Sacc + NIC, resulted in enduring increases in basal extracellular GLU levels in the mPFC. CONCLUSIONS: Overall, the co-abuse of EtOH + NIC produced enduring neuronal alterations within the MCL which enhanced the rewarding properties of NIC in the AcbSh and elevated extracellular GLU levels within the mPFC.

Stimulation of adult neural stem cells with a novel glycolipid biosurfactant.

Glycolipids are amphipathic molecules which are highly expressed on cell membranes in skin and brain where they mediate several key cellular processes. Neural stem cells are defined as undifferentiated, proliferative, multipotential cells with extensive self-renewal and are responsive to brain injury. Di-rhamnolipid: α-L-rhamnopyranosyl-(1-2)α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid, also referred to as di-rhamnolipid BAC-3, is a glycolipid isolated from the bacteria Pseudomonas aeruginosa. In the previous studies, di-rhamnolipid enhanced dermal tissue healing and regeneration. The present study provides the first assessment of di-rhamnolipid, and glycolipid biosurfactants in general, on the nervous system. Treatment of neural stem cells isolated from the lateral ventricle of adult mice and cultured in defined media containing growth factors at 0.5 and 1 µg/ml of di-rhamnolipid increased the number of neurospheres (2.7- and 2.8-fold, respectively) compared to controls and this effect remained even after passaging in the absence of di-rhamnolipid. In addition, neural stem cells treated with di-rhamnolipid at 50 and 100 µg/ml in defined media supplemented with fetal calf serum and without growth factors exhibited increased cell viability, indicating an interaction between di-rhamnolipid and serum components in the regulation of neural stem cells and neuroprogenitors. Intracerebroventricular administration of di-rhamnolipid at 300 and 120 ng/day increased the number of neurospheres (1.3- and 1.63-fold, respectively) that could be derived from the anterior lateral ventricles of adult mice. These results indicate that di-rhamnolipid stimulates proliferation of neural stem cells and increases their endogenous pools which may have therapeutic potential in managing neurodegenerative or neuropsychiatric disorders and promoting nervous tissue regeneration following injury.