Dysregulated kappa-opioid receptors in the medial prefrontal cortex contribute to working memory deficits in alcohol dependence.

Impaired working memory is one symptom contributing to compromised executive function in alcohol use disorder (AUD). Dysregulation of cortical dynorphin (DYN) and κ-opioid receptors (KORs) has been implicated in alcohol dependence-induced impairment in executive function. The present experiments test the hypothesis that dysregulated medial prefrontal cortex (mPFC) KORs contribute to impaired working memory in alcohol dependence. Alcohol dependence was induced in male Wistar rats via 4 months of intermittent ethanol vapor exposure prior to training/testing in an mPFC-dependent working memory task (delayed nonmatching-to-sample task; DNMST). mPFC KOR function in alcohol-naïve rats was compared with that of alcohol-dependent and nondependent rats using a DYN A-stimulated [35S ]GTPγS coupling assay. A functional role for mPFC KORs in the regulation of working memory was assessed via intra-mPFC infusions of a KOR agonist prior to assessment in the DNMST, and the contribution of mPFC KORs to compromised working memory in dependence was assessed via mPFC infusions of the KOR antagonist norbinaltorphimine (nor-BNI). In alcohol-dependent rats, impaired performance in the DNMST confirmed compromised working memory. Furthermore, DYN A-stimulated mPFC KOR function was pathologically increased in alcohol-dependent rats compared with nondependent and alcohol-naïve rats. Additionally, mPFC KOR involvement in working memory was functionally confirmed by intra-mPFC KOR agonist-induced deficits in DNMST performance. Importantly, alcohol dependence-induced impairment in the DNMST was ameliorated by intra-mPFC KOR antagonism. Regulation of working memory by mPFC KORs and alcohol dependence-induced dysregulation of mPFC KOR function identify a novel therapeutic target to treat AUD-related symptoms of working memory impairment.

The role of matrix metalloproteinase-9 in negative reinforcement learning and plasticity in alcohol dependence.

A role for matrix metalloproteinases (MMPs) in plasticity-dependent learning has been established. MMPs degrade the extracellular matrix (ECM) when synaptic reorganization is warranted. Previously, we showed that escalation of alcohol self-administration is a learned plasticity-dependent process that requires an intact MMP system. To identify the MMP subtypes within specific brain regions that are associated with plasticity underlying the negative reinforcing effects of alcohol (as measured by escalated alcohol self-administration) during acute withdrawal in alcohol dependence, male Wistar rats were trained to self-administer alcohol in an operant paradigm, subjected to one month of intermittent alcohol vapor exposure to induce alcohol dependence and then allowed to self-administer alcohol during repeated acute withdrawal self-administration sessions. Subsequently, rat brains were extracted after initial or stable escalated alcohol self-administration phases of acute withdrawal and analyzed by immunoblot to detect MMP-2, -3, and -9 levels in the anterior cingulate cortex (ACC), bed nucleus of the stria terminalis, central amygdala (CeA), hippocampus, and nucleus accumbens (NAc). The results showed that MMP-9 expression in the CeA and NAc of alcohol-dependent rats was increased, however, MMP-9 expression in the ACC was decreased during negative reinforcement learning. Subsequently, the importance of plasticity mediated by MMP-9 in escalated alcohol self-administration during acute withdrawal was functionally assessed through site-specific intra-CeA MMP-9 inhibition during repeated acute withdrawal self-administration sessions. MMP-9 inhibition prevented acute withdrawal-induced escalation of alcohol self-administration in a manner that was not confounded by locomotor effects or a permanent inability to learn about the negative reinforcing effects of alcohol.

Inflammatory Pain Promotes Increased Opioid Self-Administration: Role of Dysregulated Ventral Tegmental Area µ Opioid Receptors.

Pain management in opioid abusers engenders ethical and practical difficulties for clinicians, often resulting in pain mismanagement. Although chronic opioid administration may alter pain states, the presence of pain itself may alter the propensity to self-administer opioids, and previous history of drug abuse comorbid with chronic pain promotes higher rates of opioid misuse. Here, we tested the hypothesis that inflammatory pain leads to increased heroin self-administration resulting from altered mu opioid receptor (MOR) regulation of mesolimbic dopamine (DA) transmission. To this end, the complete Freund's adjuvant (CFA) model of inflammation was used to assess the neurochemical and functional changes induced by inflammatory pain on MOR-mediated mesolimbic DA transmission and on rat intravenous heroin self-administration under fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement. In the presence of inflammatory pain, heroin intake under an FR schedule was increased for high, but attenuated for low, heroin doses with concomitant alterations in mesolimbic MOR function suggested by DA microdialysis. Consistent with the reduction in low dose FR heroin self-administration, inflammatory pain reduced motivation for a low dose of heroin, as measured by responding under a PR schedule of reinforcement, an effect dissociable from high heroin dose PR responding. Together, these results identify a connection between inflammatory pain and loss of MOR function in the mesolimbic dopaminergic pathway that increases intake of high doses of heroin. These findings suggest that pain-induced loss of MOR function in the mesolimbic pathway may promote opioid dose escalation and contribute to opioid abuse-associated phenotypes. SIGNIFICANCE STATEMENT: This study provides critical new insights that show that inflammatory pain alters heroin intake through a desensitization of MORs located within the VTA. These findings expand our knowledge of the interactions between inflammatory pain and opioid abuse liability, and should help to facilitate the development of novel and safer opioid-based strategies for treating chronic pain.

Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

Recapitulating phenotypes of alcohol dependence via overexpression of Oprk1 in the ventral tegmental area of non-dependent TH::Cre rats.

The dynorphin (DYN)/kappa-opioid receptor (KOR) system is involved in dysphoria and negative emotional states. Dysregulation of KOR function promotes maladaptive behavioral regulation during withdrawal associated with alcohol dependence. Mesolimbic dopaminergic (DA) projections from the ventral tegmental area (VTA) innervate the extended amygdala circuitry and presynaptic KORs attenuate DA in these regions leading to an excessive alcohol consumption and negative affective-like behavior, whereas mesocortical KOR-regulated DA projections have been implicated in executive function and decision-making. Thus, the neuroadaptations occurring in DYN/KOR systems are important aspects to consider for the development of personalized therapeutic solutions. Herein, we study the contribution of the VTA DA neuron Oprk1 (KOR gene) in excessive alcohol consumption, negative emotional state, and executive function. To do so, Oprk1 mRNA expression and KOR function were characterized to confirm alcohol dependence-induced dysregulation in the VTA. Then, a transgenic Cre-Lox rat model (male and female TH::Cre rats) was used to allow for conditional and inducible overexpression of Oprk1 in VTA DA neurons. The effect of this overexpression was evaluated on operant alcohol self-administration, negative emotional states, and executive function. We found that VTA Oprk1 overexpression recapitulates some phenotypes of alcohol dependence including escalated alcohol self-administration and depressive-like behavior. However, working memory performance was not impacted following VTA Oprk1 overexpression in TH::Cre rats. This supports the hypothesis that dysregulated KOR signaling within the mesolimbic DA system is an important contributor to symptoms of alcohol dependence and shows that understanding Oprk1-mediated contributions to alcohol use disorder (AUD) should be an important future goal.

Plasticity associated with escalated operant ethanol self-administration during acute withdrawal in ethanol-dependent rats requires intact matrix metalloproteinase systems.

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.

Systemic κ-opioid receptor antagonism by nor-binaltorphimine reduces dependence-induced excessive alcohol self-administration in rats.

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.

Age-related differences in the blood alcohol levels of Wistar rats.

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.

The alpha1 adrenergic receptor antagonist prazosin reduces heroin self-administration in rats with extended access to heroin administration.

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.

Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid System.

Negative affective states affect quality of life for patients suffering from pain. These maladaptive emotional states can lead to involuntary opioid overdose and many neuropsychiatric comorbidities. Uncovering the mechanisms responsible for pain-induced negative affect is critical in addressing these comorbid outcomes. The nucleus accumbens (NAc) shell, which integrates the aversive and rewarding valence of stimuli, exhibits plastic adaptations in the presence of pain. In discrete regions of the NAc, activation of the kappa opioid receptor (KOR) decreases the reinforcing properties of rewards and induces aversive behaviors. Using complementary techniques, we report that in vivo recruitment of NAc shell dynorphin neurons, acting through KOR, is necessary and sufficient to drive pain-induced negative affect. Taken together, our results provide evidence that pain-induced adaptations in the kappa opioid system within the NAc shell represent a functional target for therapeutic intervention that could circumvent pain-induced affective disorders. VIDEO ABSTRACT.

Pharmacological evidence for a motivational role of kappa-opioid systems in ethanol dependence.

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.

Differential effects of acute alcohol on EEG and sedative responses in adolescent and adult Wistar 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.

Electrophysiological effects of dizocilpine (MK-801) in adult rats exposed to ethanol during adolescence.

BACKGROUND: Despite evidence showing persistent changes in N-methyl-D-aspartate (NMDA)-receptor function following ethanol (EtOH) exposure, the contribution of NMDA systems to the long-term neurophysiological consequences of adolescent EtOH exposure is unclear. The aims of this study were the following: (1) to determine whether adolescent EtOH exposure produces neurophysiological changes after a prolonged withdrawal period in adult rats and (2) to assess protracted alterations in neurophysiological responses to the NMDA antagonist MK-801 in adult rats exposed to EtOH during adolescence. METHODS: Adolescent male Wistar rats were exposed to EtOH vapor for 12 h/d for 5 weeks. The effects of MK-801 (0.0 to 0.1 mg/kg, intraperitoneally) on the electroencephalogram (EEG) and auditory event-related potentials (ERPs) were assessed after 8 weeks of abstinence from EtOH. RESULTS: Experiments in aim 1 revealed that adolescent EtOH exposure reduced EEG variability in the frontal cortex in the 4 to 6 Hz band but had no effect on cortical and hippocampal EEG power and ERPs. Experiments in aim 2 showed that MK-801 significantly reduced EEG power in the parietal cortex (4 to 6 Hz, 6 to 8 Hz, 8 to 16 Hz, 16 to 32 Hz) and hippocampus (16 to 32 Hz) and EEG variability in the parietal cortex (6 to 8 Hz, 16 to 32 Hz) following adolescent EtOH exposure. MK-801 produced a significant decrease in hippocampal EEG variability (4 to 6 Hz, 8 to 16 Hz, 16 to 32 Hz) in control, but not in EtOH-exposed rats. MK-801 reduced frontal P1 ERP amplitude and latency in response to the rare tone in EtOH-exposed rats compared to controls. In contrast, MK-801 significantly reduced P3 ERP amplitude and latency in control, but not in EtOH-exposed rats. CONCLUSIONS: The effects of MK-801 on hippocampal EEG variability and P3 ERP amplitude and latency are significantly attenuated after a prolonged withdrawal period following adolescent EtOH exposure. However, the inhibitory effects of MK-801 on cortical and hippocampal EEG power were enhanced in rats exposed to EtOH during adolescence. Taken together, these data suggest protracted changes in NMDA systems following adolescent EtOH exposure.

Dissociable effects of ethanol consumption during the light and dark phase in adolescent and adult Wistar rats.

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.

alpha1-noradrenergic receptor antagonism blocks dependence-induced increases in responding for ethanol.

The purpose of this study was to test the hypothesis that blockade of alpha1-adrenergic receptors may suppress the excessive ethanol consumption associated with acute withdrawal in ethanol-dependent rats. Following the acquisition and stabilization of operant ethanol self-administration in male Wistar rats, 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 14h/day. Subsequent to dependence induction, the effect of alpha1-noradrenergic receptor antagonist prazosin (0.0, 0.25, 0.5, 1, 1.5, and 2.0mg/kg IP) was tested on operant responding for ethanol in vapor-exposed and control rats during acute withdrawal. In ethanol-dependent animals, prazosin significantly suppressed responding at the 1.5 and 2.0mg/kg doses, whereas only the 2.0mg/kg dose was effective in nondependent animals, identifying an increase in the sensitivity to prazosin in dependent animals. Conversely, at the lowest dose tested (0.25mg/kg), prazosin increased responding in nondependent animals, which is consistent with the effect of anxiolytics on ethanol self-administration in nondependent animals. None of the doses tested reliably affected concurrent water self-administration. These results suggest the involvement of the noradrenergic system in the excessive alcohol drinking seen during acute withdrawal in ethanol-dependent rats.

Maladaptive behavioral regulation in alcohol dependence: Role of kappa-opioid receptors in the bed nucleus of the stria terminalis.

There is an important emerging role for the endogenous opioid dynorphin (DYN) and the kappa-opioid receptor (KOR) in the treatment of alcohol dependence. Evidence suggests that the DYN/KOR system in the bed nucleus of the stria terminalis (BNST) contributes to maladaptive behavioral regulation during withdrawal in alcohol dependence. The current experiments were designed to assess dysregulation of the BNST DYN/KOR system by evaluating alcohol dependence-induced changes in DYN/KOR gene expression (Pdyn and Oprk1, respectively), and the sensitivity of alcohol self-administration, negative affective-like behavior and physiological withdrawal to intra-BNST KOR antagonism during acute withdrawal. Wistar rats trained to self-administer alcohol, or not trained, were subjected to an alcohol dependence induction procedure (14 h alcohol vapor/10 h air) or air-exposure. BNST micropunches from air- and vapor-exposed animals were analyzed using RT-qPCR to quantify dependence-induced changes in Pdyn and Oprk1 mRNA expression. In addition, vapor- and air-exposed groups received an intra-BNST infusion of a KOR antagonist or vehicle prior to measurement of alcohol self-administration. A separate cohort of vapor-exposed rats was assessed for physiological withdrawal and negative affective-like behavior signs following intra-BNST KOR antagonism. During acute withdrawal, following alcohol dependence induction, there was an upregulation in Oprk1 mRNA expression in alcohol self-administering animals, but not non-alcohol self-administering animals, that confirmed dysregulation of the KOR/DYN system within the BNST. Furthermore, intra-BNST KOR antagonism attenuated escalated alcohol self-administration and negative affective-like behavior during acute withdrawal without reliably impacting physiological symptoms of withdrawal. The results confirm KOR system dysregulation in the BNST in alcohol dependence, illustrating the therapeutic potential of targeting the KOR to treat alcohol dependence.

Intracerebroventricular ethanol-induced conditioned place preferences are prevented by fluphenazine infusions into the nucleus accumbens of rats.

The rewarding properties of centrally administered ethanol (EtOH) were examined using a conditioned place preference (CPP) test. Male rats subjected to bilateral intracerebroventricular (icv) infusions of EtOH (0-240 nmol) produced a dose-dependent preference for the drug-paired environment that was potentiated by concurrent intravenous (iv) administration of heroin (0.025 mg/kg). The role of mesolimbic dopamine (DA) pathways in the development of EtOH reward was then examined by challenging EtOH-treated rats with bilateral intra-accumbens shell applications of a DA receptor antagonist. Fluphenazine (10 or 50 microg/side), infused immediately prior to daily place conditioning trials, was found to reliably attenuate the development of CPPs produced by icv EtOH administration. When fluphenazine was administered into the nucleus accumbens shell prior to the final test trial only (i.e., in already conditioned rats), intra-accumbens shell DA receptor blockade was found to prevent the expression of CPPs produced by icv EtOH. In summary, rats form reliable learned preferences for EtOH-paired locations (CPPs) that are potentiated by iv heroin and whose acquisition and expression rely on intact DA functionality within the nucleus accumbens.

Increased alcohol drinking in isolate-housed alcohol-preferring rats.

Alcoholism is a complex disorder influenced by interactions between genetic and environmental risk factors. This study examined the influence of isolate housing on ethanol intake in alcohol-preferring (P) and non-alcohol-preferring (NP) rats. Rats were isolate-housed or pair-housed for 8 weeks when between 45 and 96 days old. Ethanol drinking was assessed using a 24-hr preference test (10% ethanol vs. water) and 20-min limited access tests. A behavioral test battery was used to assess anxiety-like, depressive-like, acoustic startle, and motor behavior. Isolate housing increased home cage drinking in both lines and increased limited access drinking selectively in P rats. Isolation also reduced swim test immobility and prepulse inhibition in P rats and increased locomotor stereotypies in NP rats. Taken together, these data demonstrate that LinexEnvironment interactions influence the effects of isolation. Furthermore, isolation selectively increased ethanol intake in high drinking P rats. This effect was not correlated with changes in other behaviors. Selective enhancement of limited access ethanol drinking in P rats may represent a model whereby genetic liability to excessive drinking is enhanced by specific environmental exposures.

Dissociating Motivational From Physiological Withdrawal in Alcohol Dependence: Role of Central Amygdala κ-Opioid Receptors.

Chronic intermittent alcohol vapor exposure leads to increased dynorphin (DYN) A-like peptide expression and heightened kappa-opioid receptor (KOR) signaling in the central nucleus of the amygdala (CeA) and these neuroadaptive responses differentiate alcohol-dependent from non-dependent phenotypes. Important for therapeutic development efforts is understanding the nature of the stimulus that drives dependence-like phenotypes such as escalated alcohol self-administration. Accordingly, the present study examined the impact of intra-CeA KOR antagonism on escalated operant alcohol self-administration and physiological withdrawal symptoms during acute withdrawal and protracted abstinence in rats previously exposed to chronic intermittent alcohol vapor. Following operant training, rats were implanted with intra-CeA guide cannula and exposed to long-term intermittent alcohol vapor exposure that resulted in escalated alcohol self-administration and elevated physiological withdrawal signs during acute withdrawal. Animals received intra-CeA infusions of the KOR antagonist nor-binaltorphimine (nor-BNI; 0, 2, 4, or 6 µg) prior to operant alcohol self-administration sessions and physiological withdrawal assessment during acute withdrawal and protracted abstinence. The results indicated that site-specific KOR antagonism in the CeA ameliorated escalated alcohol self-administration during both acute withdrawal and protracted abstinence test sessions, whereas KOR antagonism had no effect on physiological withdrawal scores at either time point. These results dissociate escalated alcohol self-administration from physiological withdrawal symptoms in relation to KOR signaling in the CeA and help clarify the nature of the stimulus that drives escalated alcohol self-administration during acute withdrawal and protracted abstinence.

Species differences in the effects of the κ-opioid receptor antagonist zyklophin.

We have shown that dysregulation of the dynorphin/kappa-opioid receptor (DYN/KOR) system contributes to escalated alcohol self-administration in alcohol dependence and that KOR antagonists with extended durations of action selectively reduce escalated alcohol consumption in alcohol-dependent animals. As KOR antagonism has gained widespread attention as a potential therapeutic target to treat alcoholism and multiple neuropsychiatric disorders, we tested the effect of zyklophin (a short-acting KOR antagonist) on escalated alcohol self-administration in rats made alcohol-dependent using intermittent alcohol vapor exposure. Following dependence induction, zyklophin was infused centrally prior to alcohol self-administration sessions and locomotor activity tests during acute withdrawal. Zyklophin did not impact alcohol self-administration or locomotor activity in either exposure condition. To investigate the neurobiological basis of this atypical effect for a KOR antagonist, we utilized a κ-, µ-, and δ-opioid receptor agonist-stimulated GTPyS coupling assay to examine the opioid receptor specificity of zyklophin in the rat brain and mouse brain. In rats, zyklophin did not affect U50488-, DAMGO-, or DADLE-stimulated GTPyS coupling, whereas the prototypical KOR antagonist nor-binaltorphimine (norBNI) attenuated U50488-induced stimulation in the rat brain tissue at concentrations that did not impact µ- and δ-receptor function. To reconcile the discrepancy between the present rat data and published mouse data, comparable GTPyS assays were conducted using mouse brain tissue; zyklophin effects were consistent with KOR antagonism in mice. Moreover, at higher concentrations, zyklophin exhibited agonist properties in rat and mouse brains. These results identify species differences in zyklophin efficacy that, given the rising interest in the development of short-duration KOR antagonists, should provide valuable information for therapeutic development efforts.