Publications

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.

Investigations into the physiological mechanisms of sleep control require an animal psychomotor vigilance task (PVT) with fast response times (<300 ms). Rats provide a good PVT model since whisker stimulation produces a rapid and robust cortical evoked response, and animals can be trained to lick following stimulation. Our prior experiments used deprivation-based approaches to maximize motivation for operant conditioned responses. However, deprivation can influence physiological and neurobehavioral effects. In order to maintain motivation without water deprivation, we conditioned rats for immobilization and head restraint, then trained them to lick for a 10% sucrose solution in response to whisker stimulation. After approximately 8 training sessions, animals produced greater than 80% correct hits to the stimulus. Over the course of training, reaction times became faster and correct hits increased. Performance in the PVT was examined after 3, 6 and 12 h of sleep deprivation achieved by gentle handling. A significant decrease in percent correct hits occurred following 6 and 12 h of sleep deprivation and reaction times increased significantly following 12 h of sleep deprivation. While behaviorally the animals appeared to be awake, we observed significant increases in EEG delta power prior to misses. The rat PVT with fast response times allows investigation of sleep deprivation effects, time-on-task and pharmacological agents. Fast response times also allow closer parallel studies to ongoing human protocols.

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.

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.

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.

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.

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.

In adolescence, high levels of drinking over short episodes (binge drinking) is commonly seen in a proportion of the population. Because adolescence is an important neurodevelopmental period, the effects of binge drinking on brain and behavior has become a significant health concern. However, robust animal models of binge drinking in rats are still being developed and therefore further efforts are needed to optimize paradigms for inducing maximal self-administration of alcohol. In the present experiment, 1-h limited-access self-administration sessions were instituted to model excessive drinking behavior in adolescent and adult Wistar rats. In addition to age, the involvement of sex and phase within the light/dark cycle (i.e., drinking in the light or dark) on sweetened 5% ethanol intake were also evaluated over 14 limited-access sessions using a between-groups design. The results of the experiment showed that over 14 limited-access sessions, sweetened ethanol intake (g/kg) was significantly higher for adolescents compared to adults. Females were also found to drink more sweetened ethanol as compared to males. Additionally, drinking in the light produced a robust increase in sweetened ethanol intake (g/kg) in adolescents, as compared to adults during the light phase and as compared to both adolescent and adult rats drinking in the dark. Furthermore, the increase in ethanol consumption observed in adolescents drinking during the light phase was dissociable from sweetened solution intake patterns. These results identify that age, sex, and time of day all significantly influence consumption of sweetened ethanol in Wistar rats. Knowledge of these parameters should be useful for future experiments attempting to evaluate the effects of self-administered ethanol exposure in adult and adolescent rats.