Neither Amphetamine nor Sub-Anesthetic Ketamine Treatment during Adolescence Impairs Devaluation in Rats Tested during Adulthood.

BACKGROUND: Much of the existing animal literature on the devaluation task suggests that prior repeated exposure to drugs of abuse during adulthood can impair goal-directed action, but the literature on human drug users is mixed. Also, the initiation of drug use often occurs during adolescence, but examinations of the effects of drug exposure during adolescence on behavior in the devaluation task are lacking. METHODS: We examined whether repeated exposure during adolescence to amphetamine (3 mg/kg injections every-other day from post-natal day 27-45) or ketamine (twice daily 30 mg/kg injections from post-natal day 35-44) would impair behavior in a devaluation test when tested drug-free in adulthood. Rats were trained to press a left lever with a steady cue-light above it for one reinforcer and a right lever with a flashing cue-light above it for a different reinforcer. We tested whether any impairments in goal-directed action could be overcome by compensation between strategies by giving rats information based on lever-location and cue-lights during the test that was either congruent (allowing compensation) or incongruent (preventing compensation between strategies) with the configurations during training. RESULTS: Our results provided no evidence for impairment of goal-directed action during adulthood after adolescent amphetamine or ketamine exposure. CONCLUSIONS: We discuss possible reasons for this discrepancy with the prior literature, including (1) the age of exposure and (2) the pattern in the previous literature that most previous demonstrations of drug exposure impairing devaluation in laboratory animals may be attributed to either drug-associated cues present in the testing environment and/or accelerated habit learning in tasks that predispose laboratory animals towards habit formation with extended training (with training procedures that should resist the formation of habits in the current experiment). However, additional research is needed to examine the effects of these factors, as well a potential role for the particular doses and washout periods to determine the cause of our finding of no devaluation impairment after drug exposure.

A novel multichoice touchscreen paradigm for assessing cognitive flexibility in mice.

Cognitive flexibility refers to various processes which enable behaviors to be modified on the basis of a change in the contingencies between stimuli or responses and their associated outcomes. Reversal learning is a form of cognitive flexibility which measures the ability to adjust responding based on a switch in the stimulus-outcome contingencies of, typically two, perceptually distinct stimuli. Reversal tasks have provided valuable insight into the neural basis of cognitive flexibility, implicating brain regions including the lateral orbitofrontal cortex (lOFC) and dorsomedial prefrontal cortex (dmPFC). However, with two-stimulus reversal, it is difficult to determine whether response errors are due excessive perseveration, deficient learning, or other problems with updating. To address this limitation, we developed a mouse three-choice touchscreen-based visual reversal task, in which the contingencies of two stimuli were switched on reversal but a third, simultaneously presented, stimulus was never reinforced. We found that, in male C57BL/6J mice, responding at the previously rewarded stimulus predominated over the newly and never-reinforced stimuli during early reversal. Next, we showed that acute pharmacological inhibition of lOFC, but not dmPFC, impaired early reversal performance, relative to noninactivated controls. Interestingly, however, lOFC inactivation deficits were characterized by increased choice of the never-reinforced stimulus and a decrease in (perseverative-like) responding at the previously rewarded stimulus. These effects are inconsistent with the historical notion of lOFC mediating response inhibition and closer to recent views of the lOFC's role in response/outcome tracking. Overall, these findings provide initial support the utility of this novel paradigm for studying cognitive flexibility and its underlying neural substrates.

Relationship of low doses of alcohol voluntarily consumed during adolescence and early adulthood with subsequent behavioral flexibility.

Previous alcohol use is associated with impaired decision-making and impulsivity in humans, but the relationship between alcohol use and decision-making/impulsivity is unclear. In two experiments, we determined whether chronic intermittent access to alcohol during adolescence and early adulthood would alter or be correlated with performance in a go/no-go reversal task, a devaluation task, or operant extinction. Rats received 6 weeks of chronic intermittent access to 20% alcohol or water from postnatal day 26 to 66 and then behavioral testing was initiated 1.5-2.5 weeks later. We found no evidence that voluntary alcohol consumption altered behavior in either task. However, we found that rats that consumed more alcohol made fewer commission errors in reversal learning compared with rats that drank less. There was no relationship between alcohol consumption and reversal learning omission errors. Alcohol consumption was not correlated with the magnitude of the devaluation effect, but rats that consumed more alcohol showed faster extinction during the devaluation test. Our results suggest that the relationships between behavioral flexibility and alcohol consumption may represent individual differences. Future work will determine the neurobiological and genetic bases of these behavioral differences.

Subchronic anesthetic ketamine injections in rats impair choice reversal learning, but have no effect on reinforcer devaluation.

Previous exposure to a variety of drugs of abuse has been shown to cause long-term impairments in reversal learning and reinforcer devaluation tasks. However, there is mixed evidence in the literature for a long-term effect of ketamine exposure on reversal learning and the long-term effect of ketamine exposure on devaluation is not known. We determined whether repeated injections of an anesthetic dose of ketamine would lead to impairments in choice reversal learning after discrimination learning or impairments in reinforcer devaluation. In two experiments, rats received three injections once-daily of ketamine (100 mg/kg, intraperitoneally) or saline and then began behavioral training 19 days later so that the key reversal learning and devaluation tests would occur about 1 month after the final ketamine injection. This ketamine exposure regimen did not impair learning in our discrimination task, but led to an increase in perseverative errors in reversal learning. However, the same ketamine exposure regimen (or injections of a lower 50 mg/kg dose) had no effect on behavior in the devaluation task. The behavioral patterns observed suggest possible neural mechanisms for the effects of ketamine, but future neurobiological investigations will be needed to isolate these mechanisms.

Chronic alcohol produces neuroadaptations to prime dorsal striatal learning.

Drug addictions including alcoholism are characterized by degradation of executive control over behavior and increased compulsive drug seeking. These profound behavioral changes are hypothesized to involve a shift in the regulation of behavior from prefrontal cortex to dorsal striatum (DLS). Studies in rodents have shown that ethanol disrupts cognitive processes mediated by the prefrontal cortex, but the potential effects of chronic ethanol on DLS-mediated cognition and learning are much less well understood. Here, we first examined the effects of chronic EtOH on DLS neuronal morphology, synaptic plasticity, and endocannabinoid-CB1R signaling. We next tested for ethanol-induced changes in striatal-related learning and DLS in vivo single-unit activity during learning. Mice exposed to chronic intermittent ethanol (CIE) vapor exhibited expansion of dendritic material in DLS neurons. Following CIE, DLS endocannabinoid CB1 receptor signaling was down-regulated, and CB1 receptor-dependent long-term depression at DLS synapses was absent. CIE mice showed facilitation of DLS-dependent pairwise visual discrimination and reversal learning, relative to air-exposed controls. CIE mice were also quicker to extinguish a stimulus-reward instrumental response and faster to reduce Pavlovian approach behavior under an omission schedule. In vivo single-unit recording during learning revealed that CIE mice had augmented DLS neuronal activity during correct responses. Collectively, these findings support a model in which chronic ethanol causes neuroadaptations in the DLS that prime for greater DLS control over learning. The shift to striatal dominance over behavior may be a critical step in the progression of alcoholism.

Blockade of CB1 receptors prevents retention of extinction but does not increase low preincubated conditioned fear in the fear incubation procedure.

We recently developed a procedure to study fear incubation, in which rats given 100 tone-shock pairings over 10 days show low fear 2 days after conditioned fear training and high fear after 30 days. Notably, fear 2 days after 10 sessions of fear conditioning is lower than fear seen 2 days after a single session of fear conditioning, suggesting that fear is suppressed. Here, we investigate the potential role of CB1 receptor activation by endocannabinoids in this fear suppression. We subjected rats to 10 days of fear conditioning and then administered systemic injections of the CB1 receptor antagonist SR141716 before a conditioned fear test was conducted 2 days later under extinction conditions. A second test was conducted without any injections on the following day (3 days after training) to examine retention of fear extinction. SR141716 injections did not increase fear expression 2 days after extended fear conditioning or affect within-session extinction; however, it impaired retention of between-session fear extinction in the day 3 test. These data suggest that CB1 receptor activation does not suppress fear soon after extended fear conditioning in the fear incubation task. The data also add to the existing literature on the role of CB1 receptors in extinction of conditioned fear.

Impairments in expression of devaluation in a Pavlovian goal-tracking task, but not a free operant devaluation task, after fentanyl exposure in female rats.

In laboratory animals, there are numerous demonstrations that past exposure to drugs of abuse can lead to devaluation impairments weeks after the final drug exposure, with the majority of these demonstrations examining effects of exposure to psychostimulants. There has been minimal investigation into whether prior exposure to opiates can lead to devaluation impairments. Here, we first trained female rats that two separate cuelights predicted two different foods and measured Pavlovian goal-tracking responses (Experiment 1) or trained female rats to press two levers to earn two different foods and measured this operant response (Experiment 2). In both experiments, we subsequently gave the rats injections of fentanyl twice daily for 6 days, and then tested rats for conditioned responses after satiation on one of the foods 48-h after the final injection. We found that rats were impaired in the expression of devaluation in the Pavlovian task after fentanyl exposure, but were unimpaired in the expression of devaluation in the operant task. The pattern of results is most consistent with an impairment in lateral orbitofrontal cortex function, but additional research is needed to determine the neurobiological cause of this pattern of results.

Endogenous GDNF in ventral tegmental area and nucleus accumbens does not play a role in the incubation of heroin craving.

Glial cell line-derived neurotrophic factor (GDNF) activity in ventral tegmental area (VTA) mediates the time-dependent increases in cue-induced cocaine-seeking after withdrawal (incubation of cocaine craving). Here, we studied the generality of these findings to incubation of heroin craving. Rats were trained to self-administer heroin for 10 days (6 hours/day; 0.075 mg/kg/infusion; infusions were paired with a tone-light cue) and tested for cue-induced heroin-seeking in extinction tests after 1, 11 or 30 withdrawal days. Cue-induced heroin seeking was higher after 11 or 30 days than after 1 day (incubation of heroin craving), and the time-dependent increases in extinction responding were associated with time-dependent changes in GDNF mRNA expression in VTA and nucleus accumbens. Additionally, acute accumbens (but not VTA) GDNF injections (12.5 µg/side) administered 1-3 hours after the last heroin self-administration training session enhanced the time-dependent increases in extinction responding after withdrawal. However, the time-dependent increases in extinction responding after withdrawal were not associated with changes in GDNF protein expression in VTA and accumbens. Additionally, interfering with endogenous GDNF function by chronic delivery of anti-GDNF monoclonal neutralizing antibodies (600 ng/side/day) into VTA or accumbens had no effect on the time-dependent increases in extinction responding. In summary, heroin self-administration and withdrawal regulate VTA and accumbens GDNF mRNA expression in a time-dependent manner, and exogenous GDNF administration into accumbens but not VTA potentiates cue-induced heroin seeking. However, based on the GDNF protein expression and the anti-GDNF monoclonal neutralizing antibodies manipulation data, we conclude that neither accumbens nor VTA endogenous GDNF mediates the incubation of heroin craving.

Pre-training naltrexone increases conditioned fear learning independent of adolescent alcohol consumption history.

Our previous research has shown a relationship between low voluntary alcohol consumption and high conditioned fear in male Long Evans rats. Here, we examined whether differences in the endogenous opioid systems might be responsible for these differences. Rats received 6 weeks of chronic intermittent to 20% alcohol (v/v) or water-only from PND 26-66. Based on their consumption during the last 2 weeks of alcohol access, the alcohol-access rats were divided into high drinking (>2.5 g/kg/24-h) or low drinking (<2 g/kg/24-h). Rats were then given injections of the preferential mu opioid receptor antagonist naltrexone (1 mg/kg, s.c.) or the selective kappa opioid receptor antagonist LY2456302 (10 mg/kg, s.c.) prior to fear conditioning and were then tested for conditioned fear 2 days later. Pre-training naltrexone increased conditioned suppression of lever-pressing during training and testing, with no differences between high versus low alcohol drinkers or between water-only versus alcohol access groups (averaged across drinking levels). There was no effect of LY2456302 on conditioned fear in any comparison. We also found no differences between high and low alcohol drinkers and no reliable effect of prior alcohol access (averaged across drinking levels) on conditioned fear. Our experiment replicates and extends previous demonstrations that a preferential MOR antagonist can increase fear learning using conditioned suppression of lever-pressing as a fear measure. However, additional research is needed to determine the cause of the differences in conditioned fear that we previously observed (as they were not observed in the current experiments).

Extended operant training increases infralimbic and prelimbic cortex Fos regardless of fear conditioning experience.

Extended fear training can lead to initially low fear expression that grows over time, termed fear incubation. Conversely, a single fear conditioning session typically results in high fear initially that is sustained over time. Fear expression decreases across extended training, suggesting that a fear extinction-like process might be responsible for low fear observed soon after training. Because of the prominent role medial prefrontal cortex (mPFC) plays in fear conditioning and extinction, we decided to examine Fos expression resulting from a cued fear retrieval test to gain insight into possible mechanisms involved in extended training fear incubation. Male Long-Evans rats received 1 or 10 days of tone-shock pairings or tone-only exposure (while lever-pressing for food). Two days after the end of fear training, rats received a cued fear test, with perfusions timed to visualize Fos expression during test. As expected, the limited fear conditioning group exhibited higher fear in the test than any of the other groups (as measured with conditioned suppression of lever-pressing). Interestingly, we found that extended training animals (whether they received tone-shock pairings or tone-only exposure) expressed higher levels of Fos in both prelimbic and infralimbic cortices than limited training animals. There was no association between fear expression and mPFC Fos expression. These results suggest we may have visualized Fos expression related to operant overtraining rather than conditioned fear related processes. Further research is needed to determine the neurobiological basis of extended training fear incubation and to determine processes represented by the pattern of Fos expression we observed.

Dose-dependent effects of alcohol injections on omission-contingency learning have an inverted-U pattern.

Previous examinations of the long-term effects of alcohol exposure on omission-contingency learning have produced mixed results across different age or sex groups, with evidence for faster learning or no effect. However, none of these experiments made comparisons using the same exposure-dose across the age/sex groups. Here, we exposed rats to 6 weeks of alcohol injections (3 days/week, 1.75 or 3.5 g/kg/24-h, i.p. broken up into 2 injections/day) in adolescent/early adult males or females (PND27-66) or adult males (PND62-101). We then tested the rats in autoshaping and omission-contingency tasks. In contrast to our hypotheses, the low 1.75-g/kg/24-h dose led to slower omission learning and the higher 3.5-g/kg/24-h dose had no effect. There were no age- or sex-differences in omission learning. Additionally, during autoshaping training, rats exposed in adolescence/early adulthood had a faster shift to sign-tracking in their sign-tracking/goal-tracking ratios than rats exposed in adulthood, with no consistent effect of alcohol exposure or sex-differences. Our results suggest complex effects of alcohol on the neural substrates of omission-contingency learning at different doses, which will require future investigation.

Pre-training inactivation of basolateral amygdala and mediodorsal thalamus, but not orbitofrontal cortex or prelimbic cortex, impairs devaluation in a multiple-response/multiple-reinforcer cued operant task.

Reinforcer devaluation is a task often used to model flexible goal-directed behavior. Here, we inactivated basolateral amygdala (BLA), orbitofrontal cortex (OFC), mediodorsal thalamus (MD) (Exp. 1) and prelimbic cortex (PL) (Exp. 3) in rats during multiple-response/multiple-reinforcer operant training with levers available to earn reinforcers during cued trials. After two training days with each lever-food relationship, a reinforcer was devalued through selective satiety and devaluation was assessed in a choice test with the brain areas non-inactivated. The control and OFC and PL inactivation groups exhibited a devaluation effect, but the BLA or MD groups did not. Since the OFC is proposed to be required in devaluation tasks when a discrete cue signals an outcome and PL is proposed to be required when responses based on lever spatial-location guide behavior, we ran new rats through a cue-switching experiment (Exp. 2) to determine the strategy rats use to complete our task (attending to the discrete light cue or spatial lever location). Both groups (cue switched and cue normal) showed a devaluation effect based on the lever spatial location, suggesting that rats rely on the spatial lever location to guide behavior. Future studies will determine whether OFC and PL can compensate for each other to show intact devaluation when the functioning of one of them is impaired.

Voluntary alcohol access during adolescence/early adulthood, but not during adulthood, causes faster omission contingency learning.

In omission contingency training, rodents learn to suppress their natural tendency to approach or touch a reward-predictive cue (termed "autoshaping" or "sign-tracking" responses) if the approach/touching responses lead to the omission of the reward. Previous research has shown that high levels of alcohol exposure (through alcohol vapor exposure) or adolescent alcohol consumption (with some versions of the omission contingency task) can lead to faster omission contingency learning. However, the alcohol exposure procedures and/or omission contingency task parameters differed between these different demonstrations. It was unclear whether the same voluntary alcohol consumption procedures during adolescence/early adulthood and/or adulthood would lead to faster omission contingency learning in one or both age groups. Here, rats received 6 weeks of chronic intermittent access to 20% alcohol or water from PND 26-66 (adolescence/early adulthood in Exp. 1) or PND 68-108 (adulthood in Exp. 2) and began behavioral training (autoshaping training followed by omission contingency training) several days later. We found no evidence that alcohol access at either age altered the number of trials with a sign-tracking response on the levers during autoshaping training. However, alcohol access during adolescence/early adulthood, but not during adulthood, led to faster learning to withhold responding on the lever under omission contingencies during the subsequent phase. We also found no evidence that the level of alcohol consumption was correlated with sign-tracking behavior in the autoshaping phase or with the suppression of lever-pressing during the omission contingency phase. Our results suggest that adolescent/early adult rats have increased vulnerability, compared with adults, to some long-term behavioral effects of voluntary alcohol consumption.

Individual differences in voluntary alcohol consumption are associated with conditioned fear in the fear incubation model.

Previous research in male Long Evans rats has shown a relationship between low voluntary alcohol consumption and high conditioned fear after a single training session. Here, we determined whether chronic intermittent access (CIA) to alcohol during adolescence/early adulthood or during adulthood would alter or be associated with auditory-cued conditioned fear levels using an extended training fear incubation procedure. This training procedure leads to low fear soon after training that grows over one month. Rats received 6 weeks of CIA to 20% alcohol or water from PND 26-66. Ten or eleven days later, the rats began behavioral testing that included 10 sessions of tone-shock pairings. Rats then received 4 weeks of CIA exposure during the 1-month fear incubation period and were tested for conditioned fear 6 days after the end of alcohol access. We found no evidence that voluntary alcohol consumption during adolescence/early adulthood or adulthood altered fear expression. However, we found that rats that consumed more alcohol during early adulthood (PND 54-66) had lower fear than low-consumption rats on day 1 of conditioned fear training and in the day 2 and 1-month tests. This extends associations we previously found between individual differences in alcohol consumption and conditioned fear to a different fear conditioning procedure. Combined with our previous data that show that the rate of instrumental extinction is associated with both alcohol consumption and conditioned fear, these data provide further support for the generality and reliability of a pair of phenotypes that encompass a wide variety of learning traits.

A limited role for mediodorsal thalamus in devaluation tasks.

Six experiments were performed to determine the role of mediodorsal thalamus (MD) in the devaluation task, varying the type of contingencies (Pavlovian or operant), the number of reinforcers (one vs. two), and the order of experiments (in naïve or experimentally experienced rats). MD-lesioned rats were impaired in devaluation performance when switched between Pavlovian and operant devaluation tasks, but not when switched from one Pavlovian devaluation task to another Pavlovian devaluation task. MD lesions caused no devaluation impairment in a multiple-reinforcer Pavlovian devaluation task. These results suggest that MD lesions impair performance in devaluation tasks as a result of an inability to switch the form of associations made from one type of outcome-encoding association to another. This is in accord with previous literature suggesting that MD is needed for strategy set shifting. The results further suggest that MD is a necessary part of devaluation circuits only in cases in which previous associations need to be suppressed in order for new associations to be learned and control behavior, and otherwise the devaluation circuit does not require MD.

Individual differences in conditioned fear are associated with levels of adolescent/early adult alcohol consumption and instrumental extinction.

Previous research has shown a relationship between alcohol exposure and conditioned fear, but the nature of this relationship remains unclear. We determined whether chronic intermittent access to alcohol during adolescence and early adulthood would alter or be associated with the level of conditioned fear to an auditory cue in male Long Evans rats. Rats received 6 weeks of chronic intermittent access to 20% alcohol or water from PND 26-66 and began behavioral testing 10 days later. We found no evidence that voluntary alcohol consumption altered fear. However, we found that rats that consumed more alcohol had lower fear, as measured with conditioned suppression of lever-pressing and conditioned freezing to an auditory cue. We have previously shown that higher levels of alcohol consumption are correlated with faster instrumental extinction learning. Therefore, we determined whether instrumental extinction would be directly associated with conditioned fear in rats never given alcohol access. As predicted, we found that rats that exhibited faster instrumental extinction also exhibited lower conditioned fear, as measured with conditioned suppression of lever-pressing and conditioned freezing. Our results suggest that at least part of the relationship between alcohol consumption levels and fear learning differences may be due to pre-existing individual differences. In addition, our finding that conditioned fear and instrumental extinction abilities (both separately associated with alcohol consumption levels) are associated with each other suggests that alcohol consumption levels may be a marker that can distinguish two separate phenotypes that encompass a wide variety of learning traits.

Operant over-responding is more sensitive than reversal learning for revealing behavioral changes after withdrawal from alcohol consumption.

In humans, prior alcohol use is linked with impulsivity and impaired decision-making, but the nature of this relationship is unclear. In a previous study in rats, we found that prior alcohol access led to over-responding in go/no-go discrimination training, but had no effect on discrimination learning. It was unclear whether this over-responding effect would occur in a reversal learning task, or whether prior alcohol would impair reversal learning in our task. In the present experiments, we determined whether six weeks of chronic intermittent alcohol access would induce over-responding or impair reversal learning in our task. Our task allowed for multiple responses/trial with limited reinforcement, so over-responding could be assessed. In Exp. 1, we gave three days of discrimination training prior to access to 20% alcohol or water, then reversed task contingencies starting 4 days after the end of alcohol access. In Exp. 2, we gave either three or six days of discrimination training prior to the same alcohol access and reversal learning procedures to determine if the original training length would affect alcohol's behavioral effects. We found no reversal learning deficits in either experiment. Across both experiments, we found that the Alcohol group exhibited over-responding to the active lever, but this effect was smaller than in our previous discrimination experiments. Our data suggest that there are behavioral changes after voluntary alcohol access that can be missed by some discrimination/reversal learning assessments, and our over-responding task can detect these transient changes. However, over-responding is more pronounced in discrimination than reversal learning.

Dorsolateral Striatum Engagement Interferes with Early Discrimination Learning.

In current models, learning the relationship between environmental stimuli and the outcomes of actions involves both stimulus-driven and goal-directed systems, mediated in part by the DLS and DMS, respectively. However, though these models emphasize the importance of the DLS in governing actions after extensive experience has accumulated, there is growing evidence of DLS engagement from the onset of training. Here, we used in vivo photosilencing to reveal that DLS recruitment interferes with early touchscreen discrimination learning. We also show that the direct output pathway of the DLS is preferentially recruited and causally involved in early learning and find that silencing the normal contribution of the DLS produces plasticity-related alterations in a PL-DMS circuit. These data provide further evidence suggesting that the DLS is recruited in the construction of stimulus-elicited actions that ultimately automate behavior and liberate cognitive resources for other demands, but with a cost to performance at the outset of learning.

Prior alcohol consumption does not impair go/no-go discrimination learning, but causes over-responding on go trials, in rats.

Prior alcohol use is associated with impaired response inhibition in humans, including in laboratory go/no-go discrimination tasks. In two experiments, we determined whether chronic intermittent access to alcohol would alter go/no-go discrimination learning. Rats received 4-6 weeks of chronic intermittent access to 20% alcohol (alone or accompanied by saline or 1.5g/kg alcohol injections) or water. Rats then began discrimination training 4-5days after the end of the alcohol access. Each lever was available for 40s with one lever intermittently reinforced ("active lever") and the other lever non-reinforced ("inactive lever"). The rats given access to alcohol without concurrent alcohol injections drank ∼10g/kg/24-h on average during the last three weeks of alcohol access. The groups given alcohol injections (Alcohol+Injection groups) exhibited suppressed drinking, but the Alcohol+Injection groups exhibited higher blood alcohol spikes than all other alcohol groups (195 vs. 85-90mg/dl, respectively). We found no evidence for impaired go/no-go discrimination learning in either experiment. However, the alcohol access groups with moderate-to-high average alcohol consumption (>3g/kg/24-h) exhibited over-responding to the active lever compared to the water-only groups. One group given alcohol injections (Alcohol+Injection group) that exhibited very low voluntary drinking (<1g/kg/24-h) did not exhibit the over-responding effect, suggesting that the total 24-h alcohol dose matters more than short-lived blood alcohol spikes. Our findings are in accord with previous research showing that repeated alcohol withdrawal causes over-responding for responses that lead to limited reinforcement. Future work will determine the psychological and neurobiological basis of this behavioral change.