Publications

Previously, it was shown that ethanol-dependent animals display increased sensitivity to the general opioid receptor antagonist nalmefene compared to naltrexone. It was hypothesized that the dissociable effects of the two antagonists were attributable to a κ-opioid receptor mechanism. Nucleus accumbens dynorphin is upregulated following chronic ethanol exposure and such neuroadaptations could contribute to nalmefene's increased potency in ethanol-dependent animals. To test this hypothesis, male Wistar rats were trained to self-administer ethanol using an operant conditioning procedure. Animals were then implanted with bilateral intra-accumbens shell guide cannulae and assigned to either a chronic intermittent ethanol vapor-exposure condition (to induce dependence) or an air-exposed control group. Following a one-month exposure period, nalmefene, nor-binaltorphimine (nor-BNI; selective for κ-opioid receptors) or a combination of the selective opioid receptor antagonists CTOP and naltrindole (selective for the μ- and δ-opioid receptors, respectively) were site-specifically infused into the nucleus accumbens shell prior to ethanol self-administration sessions during acute withdrawal. Nalmefene and CTOP/naltrindole dose-dependently reduced ethanol self-administration in nondependent and dependent animals, whereas nor-BNI selectively attenuated ethanol self-administration in ethanol-dependent animals without affecting the self-administration of nondependent animals. Further analysis indentified that intra-accumbens shell nalmefene was more potent in ethanol-dependent animals and that the increased potency was attributable to a κ-opioid receptor mechanism. These data support the concept that dysregulation of DYN/κ-opioid receptor systems contributes to the excessive self-administration observed in dependent animals and suggest that pharmacotherapeutics for ethanol dependence that target κ-opioid receptors, in addition to μ- and δ-opioid receptors, are preferable to those that target μ- and δ-opioid receptor mechanisms alone.

Repeated cycles of ethanol intoxication and withdrawal associated with dependence induce neuroadaptations in a variety of brain systems. Withdrawal-induced negative emotional states can be ameliorated by ethanol consumption; a learned process termed negative reinforcement. Accordingly, a dependence-induced phenotype is escalated ethanol self-administration. Matrix metalloproteinases (MMPs) are proteolytic enzymes which degrade the extracellular matrix to allow for synaptic reorganization and plasticity. To test the hypothesis that an intact MMP system is required for animals to learn about the negative reinforcing effects of ethanol and display escalated self-administration during acute withdrawal when ethanol-dependent, male Wistar rats were trained to self-administer ethanol and then assigned to either acute or chronic MMP inhibition treatment groups. The chronic treatment group received intracerebroventricular (ICV) infusions of the broad spectrum MMP inhibitor FN-439 or artificial cerebrospinal fluid (aCSF) via osmotic minipumps during a 1 month ethanol dependence induction period and subsequent post-dependence induction self-administration sessions that occurred during acute withdrawal. The acute treatment group only received ICV FN-439 or aCSF on the day of self-administration sessions following dependence induction during acute withdrawal. The results showed that inhibition of MMPs attenuated escalated ethanol self-administration following chronic and acute exposure conditions. Furthermore, once learning (i.e., plasticity) had occurred, MMP inhibition had no impact on escalated response patterns and animals previously subjected to MMP inhibition that did not escalate evidenced normal escalations in operant ethanol self-administration once FN-439 treatments were terminated. Thus, the present data identified that an intact MMP system is required for the escalated responding that occurs during acute withdrawal in dependent animals and implicate such escalation as a learned response.

Altered dynorphin opioid peptide systems contribute to increased ethanol self-administration during withdrawal following chronic alcohol exposure. We previously identified that the κ-opioid receptor antagonist nor-binaltorphimine (nor-BNI) selectively reduced ethanol self-administration in dependent animals. The purpose of this study was twofold: (1) determine whether peripherally administered nor-BNI could reduce dependence-induced ethanol self-administration and (2) confirm the selective κ-opioid effects of nor-BNI by administering it 24 hours prior to ethanol self-administration sessions occurring during acute withdrawal. Nor-BNI decreased ethanol self-administration in ethanol-dependent animals, with no effect in nondependent animals. Thus, the κ-opioid/dynorphin system is a viable pharmacotherapeutic target for the treatment of alcoholism.

Depressive symptoms in alcohol-dependent individuals are well-recognized and clinically relevant phenomena. The etiology has not been elucidated although it is clear that the depressive symptoms may be alcohol independent or alcohol induced. To contribute to the understanding of the neurobiology of chronic ethanol use, we investigated the effects of chronic intermittent ethanol vapor exposure on behaviors in the forced swim test (FST) and neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) levels in specific brain regions. Adult male Wistar rats were subjected to intermittent ethanol vapor (14 h on/10 h off) or air exposure for 2 weeks and were then tested at three time points corresponding to acute withdrawal (8-12 h into withdrawal) and protracted withdrawal (30 and 60 days of withdrawal) in the FST. The behaviors that were measured in the five-min FST consisted of latency to immobility, swim time, immobility time, and climbing time. The FST results showed that the vapor-exposed animals displayed depressive-like behaviors; for instance, decreased latency to immobility in acute withdrawal and decreased latency to immobility, decreased swim time and increased immobility time in protracted withdrawal, with differences between air- and vapor-exposed animals becoming more pronounced over the 60-day withdrawal period. NPY levels in the frontal cortex of the vapor-exposed animals were decreased compared with the control animals, and CRF levels in the amygdala were correlated with increased immobility time. Thus, extended ethanol vapor exposure produced long-lasting changes in FST behavior and NPY levels in the brain.

Knowledge of blood alcohol levels (BALs) that are achieved following ethanol administration is critical for contemporary efforts to develop animal models of alcoholism. Adolescent and adult male Wistar rats were administered varying doses of ethanol (0.75, 1.5 and 3.0 g/kg) via gavage or intraperitoneal injection and BALs were measured over a two hour period. The results showed that adolescent animals had lower BALs across all time points in comparison to adults following administration of 0.75 g/kg ethanol and that 1 h after administration of 1.5 g/kg ethanol, adolescent animals showed an enhanced rate of elimination. The highest dose of ethanol (3.0 g/kg) produced comparable BALs for both adolescents and adults during the two-hour sampling period; however, the BALs for both ages were lower following administration of ethanol by gavage at this dose. Furthermore, an order effects analysis highlights that depending on the route of administration, initial dose size can influence the BALs produced by lower doses of ethanol. The current data identify the importance of measuring the level of alcohol in the blood to confirm that target BALs are achieved for adolescents and equivalent BALs are being reached for both adolescent and adult animals when such comparisons are made.

Previous studies have reported that noradrenergic antagonists alleviate some of the symptoms of opiate withdrawal and dependence. Clinical studies also have shown that modification of the noradrenergic system may help protect patients from relapse. The present study tested the hypothesis that a dysregulated noradrenergic system has motivational significance in heroin self-administration of dependent rats. Prazosin, an alpha1-adrenergic antagonist (0.5, 1.0, 1.5 and 2.0 mg/kg, i.p.), was administered to adult male Wistar rats with a history of limited (1 h/day; short access) or extended (12 h/day; long access) access to intravenous heroin self-administration. Prazosin dose-dependently reduced heroin self-administration in long-access rats but not short-access rats, with 2 mg/kg of systemic prazosin significantly decreasing 1 h and 2 h heroin intake. Prazosin also reversed some changes in meal pattern associated with extended heroin access, including the taking of smaller and briefer meals (at 3 h), while also increasing total food intake and slowing the eating rate within meals (both 3 h and 12 h). Thus, prazosin appears to stimulate food intake in extended access rats by restoring meals to the normal size and duration. The data suggest that the alpha1 adrenergic system may contribute to mechanisms that promote dependence in rats with extended access.

Early onset of alcohol consumption increases the risk for the development of dependence. Whether adolescent consumption of other highly palatable solutions may also affect alcohol drinking in adulthood is not known. The purpose of this study was to determine the effects of adolescent consumption of four solutions: water, sucrose, sucrose-milk and milk on ethanol drinking in adult rats. Rats had limited access to one of the four solutions from day PND 29 to PND 51 and were subsequently trained to consume ethanol (E) using a sucrose (S) fade-out procedure. Adolescent consumption of sucrose and sucrose-milk solutions increased intake of 2.5% E when it was combined with 10% S but it had no effect on the drinking of 10% E alone. Adolescent consumption of milk and sucrose-milk significantly decreased the intake of 10% E when it was combined with 10% S, and milk significantly reduced 10% E consumption alone and when it was combined with 5% S. Adolescent exposure to the sucrose-milk and sucrose solutions was also found to increase sucrose and sucrose-milk consumption. Our findings suggest adolescent exposure to sucrose increases, whereas, exposure to milk reduces ethanol consumption in adult rats. Our results may provide a new theoretical approach to the early prevention of alcoholism.

Exposure to alcohol during adolescence is predictive of adult alcohol abuse and dependence. The present experiment was designed to measure the impact of appetitive motivational engagement with ethanol during adolescence on adult ethanol consumption. To this end, a group of adolescent male Wistar rats was allowed to traverse an operant runway to obtain access to a sweetened 10% ethanol solution (wt/vol) over 18 sessions. An additional yoked-control group was allowed access to an identical solution; however, exposure to the solution was contingent on the experimental groups entry into the goal box of the runway. Once the adolescent exposure sessions were completed, the animals were allowed to mature into adults and then tested for differences in ethanol consumption during 30-min 2-bottle limited access sessions. Following 14 ethanol consumption sessions during adulthood, a naltrexone dose-response challenge (0-0.4 mg/kg) was initiated for both groups. The results of the experiment showed that the animals allowed to traverse the runway during adolescence displayed increased ethanol consumption during adulthood when compared to the yoked-control group. In addition, both groups showed dose-dependent attenuation of ethanol consumption by naltrexone. Thus, appetitive motivational experience during adolescence can impact adult ethanol consummatory behavior, a process that appears to involve common reinforcement-related neural substrates. This model should prove useful in delineating appetitive motivation-related factors that contribute to excessive ethanol consumption.

The purpose of this study was to test the hypothesis that activation of the dynorphin/kappa (kappa)-opioid system has a role in the increased consumption of ethanol in dependent animals. The effects of three opioid receptor antagonists with different effects on opioid receptors, naltrexone, nalmefene, and nor-binaltorphimine (nor-BNI), were compared in their ability to decrease ethanol self-administration in nondependent and ethanol-dependent male Wistar rats. Nalmefene and naltrexone are both opioid receptor ligands with comparable molecular weights and pharmacokinetic profiles, but differing specificity for the three opioid receptor subtypes at low doses, while nor-BNI is a selective kappa-opioid receptor antagonist. Dependence was induced in half the animals by subjecting them to a 4-week intermittent vapor exposure period in which animals were exposed to ethanol vapor for 14 h per day. Subsequent to dependence induction, nalmefene, naltrexone, and nor-BNI were tested for their ability to modulate self-administration of ethanol in vapor-exposed and control rats. The results indicated that both nalmefene and naltrexone induced a significant dose-dependent decrease in the number of lever presses for ethanol in both groups of animals. However, in ethanol-dependent animals, nalmefene was significantly more effective in suppressing ethanol intake than naltrexone. Nor-BNI selectively attenuated ethanol-dependent self-administration while leaving nondependent ethanol self-administration intact. Because naltrexone is primarily selective for the mu-opioid receptor, and nalmefene is primarily selective for the mu- and kappa-opioid receptor subtypes, the fact that nalmefene demonstrates more suppression in dependent animals suggests that opioid systems distinct from the mu-regulated portion may be involved in the increased drinking seen during withdrawal in dependent animals. The results with nor-BNI confirm that kappa-opioid receptor antagonism selectively decreases dependence-induced ethanol self-administration, which supports the hypothesis that dynorphin/kappa-opioid systems are dysregulated in dependence and contribute to the increased drinking seen during acute withdrawal in dependent rats.

Age-related developmental differences in sensitivity to the acute effects of alcohol may play an important role in the development of alcoholism. The present study was designed to evaluate the acute effects of alcohol on cortical electroencephalogram (EEG) in adolescent (P36) and adult (P78) Wistar rats. Five minutes of EEG was recorded after administration of 0, 0.75 or 1.5 g/kg alcohol. The righting reflex was performed to measure the sedative effects of alcohol (3.5 g/kg) and total sleeping time for each rat. Our results showed that alcohol (1.5 g/kg) increased power in the 1-2 Hz band and decreased the power in the 32-50 Hz band in the parietal cortical region of adolescent rats. Alcohol (1.5 g/kg) also increased stability of the EEG power in the slow-wave frequency bands (2-4 Hz, 4-6 Hz, and 6-8 Hz) of adolescent rats. In the frontal cortex of adult rats, but not in adolescent rats, alcohol (1.5 or 0.75 g/kg) decreased the power in the 16-32 Hz frequency band. Alcohol (1.5 g/kg) differentially increased power in a multiple of slow-wave frequency bands (2-4 Hz and 4-6 Hz) in the parietal cortex of adult rats as compared to adolescent rats. Adolescent rats were shown significantly shorter sleeping time and higher blood alcohol levels after regaining reflex than adult rats. Our results provide additional evidence of age-related differences in the effects of acute alcohol on cortical EEG, sedation and tolerance.