Acute stress increases voluntary consumption of alcohol in undergraduates.

AIMS: The primary aim of this study was to assess whether an acute stressor directly increases alcohol intake among undergraduates. A secondary aim was to examine whether individual differences in state anxiety predict alcohol intake. METHOD: Following random assignment, undergraduate students (n = 75; 47% males; mean age = 20.1 ± 2.8) completed the Trier Social Stress Test or no-stress protocol, and then engaged in a 30-min free-drinking session (alcohol, placebo, or non-alcoholic beverage). The State-Trait Anxiety Inventory was completed upon arrival, post-stressor, and after drinking. RESULTS: Planned comparisons demonstrated that psychosocial stress increased voluntary intake of alcohol, but not placebo or non-alcoholic beverages. In linear regression analyses, individual differences in anxiety did not predict voluntary alcohol consumption. CONCLUSION: A proximal relationship exists between acute stress and single-session alcohol intake in undergraduates, which may explain the relationship between life stressors and increased drinking in this group. These findings demonstrate that stress management is an important target for reducing heavy episodic drinking on university campuses.

Prolonged morphine treatment alters δ opioid receptor post-internalization trafficking.

BACKGROUND AND PURPOSE: The δ opioid receptor (DOP receptor) undergoes internalization both constitutively and in response to agonists. Previous work has shown that DOP receptors traffic from intracellular compartments to neuronal cell membranes following prolonged morphine treatment. Here, we examined the effects of prolonged morphine treatment on the post-internalization trafficking of DOP receptors. EXPERIMENTAL APPROACH: Using primary cultures of dorsal root ganglia neurons, we measured the co-localization of endogenous DOP receptors with post-endocytic compartments following both prolonged and acute agonist treatments. KEY RESULTS: A departure from the constitutive trafficking pathway was observed following acute DOP receptor agonist-induced internalization by deltorphin II. That is, the DOP receptor underwent distinct agonist-induced post-endocytic sorting. Following prolonged morphine treatment, constitutive DOP receptor trafficking was augmented. SNC80 following prolonged morphine treatment also caused non-constitutive DOP receptor agonist-induced post-endocytic sorting. The µ opioid receptor (MOP receptor) agonist DAMGO induced DOP receptor internalization and trafficking following prolonged morphine treatment. Finally, all of the alterations to DOP receptor trafficking induced by both DOP and MOP receptor agonists were inhibited or absent when those agonists were co-administered with a DOP receptor antagonist, SDM-25N. CONCLUSIONS AND IMPLICATIONS: The results support the hypothesis that prolonged morphine treatment induces the formation of MOP-DOP receptor interactions and subsequent augmentation of the available cell surface DOP receptors, at least some of which are in the form of a MOP/DOP receptor species. The pharmacology and trafficking of this species appear to be unique compared to those of its individual constituents. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Alcohol intoxication alters cognitive skills mediated by frontal and temporal brain regions.

Alcohol intoxication affects frontal and temporal brain areas and may functionally impair cognitive processes mediated by these regions. This study examined this hypothesis by testing the effects of alcohol on sustained attention, impulsivity, and verbal memory. Sober and placebo control groups were used to distinguish pharmacological from expectancy effects of alcohol. One hundred nine university students were assigned to an alcohol (low, medium, or high dose), placebo or sober group. Moderate and high doses of alcohol impaired all cognitive measures. A gender effect was revealed in that alcohol impaired sustained attention in males, but not females. Both sustained attention and verbal memory exhibited a U-shaped pattern, in that the medium-dose alcohol group showed the greatest impairment. This study adds to knowledge about the effects of alcohol intoxication on frontally- and temporally-mediated cognitive function. These findings have specific relevance for heavy-drinking undergraduate populations, particularly in light of the fact that repeated alcohol administration produces persistent changes in brain neurocircuitry.

neuroendocrinological responses to alcohol intoxication in healthy males: relationship with impulsivity, drinking behavior, and subjective effects.

Ambiguous biochemical and subjective responses to alcohol may relate to preexisting individual differences in alcohol expectations, experience, or impulsivity. This study examined cortisol and alpha-amylase responses to alcohol and their association with trait impulsivity, alcohol expectancy, and subjective reports of alcohol's effects. Eighty-seven males assigned to an alcohol, sober, or placebo group provided biochemical and self-report measures. Both cortisol and alpha-amylase increased following alcohol administration. Impulsivity correlated with cortisol changes, and the greatest rise in cortisol correlated with high stimulating effects in the alcohol group. These findings emphasize the importance of individual differences in alcohol responses and support a relationship between hormonal responses and alcohol use.

Ultra-low-dose naloxone suppresses opioid tolerance, dependence and associated changes in mu opioid receptor-G protein coupling and Gbetagamma signaling.

Opiates produce analgesia by activating mu opioid receptor-linked inhibitory G protein signaling cascades and related ion channel interactions that suppress cellular activities by hyperpolarization. After chronic opiate exposure, an excitatory effect emerges contributing to analgesic tolerance and opioid-induced hyperalgesia. Ultra-low-dose opioid antagonist co-treatment blocks the excitatory effects of opiates in vitro, as well as opioid analgesic tolerance and dependence, as was demonstrated here with ultra-low-dose naloxone combined with morphine. While the molecular mechanism for the excitatory effects of opiates is unclear, a switch in the G protein coupling profile of the mu opioid receptor and adenylyl cyclase activation by Gbetagamma have both been suggested. Using CNS regions from rats chronically treated with vehicle, morphine, morphine+ultra-low-dose naloxone or ultra-low-dose naloxone alone, we examined whether altered mu opioid receptor coupling to G proteins or adenylyl cyclase activation by Gbetagamma occurs after chronic opioid treatment. In morphine-naïve rats, mu opioid receptors coupled to Go in striatum and to both Gi and Go in periaqueductal gray and spinal cord. Although chronic morphine decreased Gi/o coupling by mu opioid receptors, a pronounced coupling to Gs emerged coincident with a Gbetagamma interaction with adenylyl cyclase types II and IV. Co-treatment with ultra-low-dose naloxone attenuated both the chronic morphine-induced Gs coupling and the Gbetagamma signaling to adenylyl cyclase, while increasing Gi/o coupling toward or beyond vehicle control levels. These findings provide a molecular mechanism underpinning opioid tolerance and dependence and their attenuation by ultra-low-dose opioid antagonists.

Chronic prenatal ethanol exposure impairs conditioned responding and enhances GABA release in the hippocampus of the adult guinea pig.

In this study, we assessed the effects of chronic prenatal ethanol exposure (CPEE) on spatial navigation in the water maze, conditioned responding using food-reinforced lever pressing, and amino acid neurotransmitter release from the hippocampus of the adult guinea pig. Pregnant guinea pigs were treated with ethanol (3 g/kg of maternal body weight/day), isocaloric-sucrose/pair-feeding, or water throughout gestation. Adult offspring were trained in two-lever operant chambers to respond for sucrose pellets, with one lever designated as the reward lever. There were no group differences in response acquisition or lever discrimination on a fixed-ratio 1 (FR-1) schedule. During extinction sessions, CPEE offspring maintained higher levels of responding on the previously reinforced lever, suggesting that CPEE increases perseveration and/or impairs response inhibition but does not affect operant responding for an appetitive reinforcer or the ability to discriminate rewarding from nonrewarding stimuli. In contrast, there was no effect of CPEE on performance in the water maze in the maternal ethanol regimen used in this study. CPEE did not alter electrically evoked glutamate or GABA release from hippocampal brain slices. However, when slices were tested after delivery of a tetanizing stimulation (five 5-s trains at 100 Hz), post-tetanic potentiation of electrically stimulated GABA release was greater in hippocampal slices obtained from CPEE offspring, whereas post-tetanic potentiation of electrically stimulated glutamate release was unaffected. These data suggest that conditioned learning is a sensitive behavioral measure of CPEE-induced brain injury. Increased activity-dependent potentiation of GABA release in the hippocampus may contribute to alterations in synaptic plasticity observed in CPEE offspring.

Integrated contributions of basal forebrain and thalamus to neocortical activation elicited by pedunculopontine tegmental stimulation in urethane-anesthetized rats.

Efferents from the pedunculopontine tegmentum (PPTg) exert widespread control over neocortical electrocorticographic (ECoG) activity and aid in maintaining high-frequency ECoG activation during waking and rapid eye movement sleep. The mechanisms and subcortical routes that allow the PPTg to influence cortical activity remain controversial. We examined the relative contributions of the thalamus and basal forebrain in ECoG activation elicited by PPTg stimulation in urethane-anesthetized rats. Stimulation (100 Hz, 2 s) of the PPTg suppressed large-amplitude, low-frequency oscillations, replacing them with high-frequency beta-gamma activity. Systemic administration of the anti-muscarinic drug scopolamine (1 mg/kg, i.p.) abolished activation elicited by PPTg stimulation, suggestive of an essential role of acetylcholine in this effect. Local infusions of lidocaine (1 microl, 1%) into the region of the cholinergic basal forebrain complex produced a strong reduction in activation elicited by PPTg stimulation. Lidocaine infusions into the reticular thalamic nucleus had no effect, but infusions into central thalamus produced a small attenuation of PPTg-evoked cortical activation. Combined basal forebrain-central thalamic infusions (1 microl/site) produced roughly additive effects, leading to a greater loss of activation than single-site infusions. These results indicate that, under the present experimental conditions, high-frequency cortical ECoG activation elicited by the PPTg involves relays in both the basal forebrain and central thalamus, with a predominant role of the basal forebrain. After concurrent central thalamic-basal forebrain inactivation, the forebrain can maintain only limited, short-lasting activation in response to PPTg stimulation. The additivity of infusion effects suggests that, rather than participating in one serial system, basal forebrain and central thalamus constitute parallel activating pathways. These findings aid in resolving previous controversies regarding the role of thalamus and basal forebrain in activation by emphasizing the importance of multiple, large-scale networks between brainstem and cortex in regulating the activation state of the mammalian neocortex.

Cocaine-induced anxiety: alleviation by diazepam, but not buspirone, dimenhydrinate or diphenhydramine.

Clinical reports and animal experiments indicate that both cocaine administration and cocaine withdrawal increase anxiety. We investigated the ability of a number of putative anxiolytic agents to alleviate these anxiety states using the elevated plus-maze. Rats in the cocaine condition received either saline or cocaine (20 mg/kg) 40 min prior to testing; those in the withdrawal condition were tested 48 h following a chronic treatment regime (saline or cocaine 20 mg/kg per day for 14 days). Prior to testing, animals received a benzodiazepine (1.0 or 2.0 mg/kg diazepam), a serotonergic agonist (0.5 or 1.0 mg/kg buspirone), an antihistamine (50 mg/kg dimenhydrinate or 27 mg/kg diphenhydramine) or a saline injection. All drugs were administered intraperitoneally. Cocaine administration and cocaine withdrawal reduced the percentage time spent on and the number of entries into the open arms. Diazepam dose-dependently alleviated cocaine withdrawal-induced anxiety and non-significantly attenuated cocaine-induced anxiety. Buspirone, dimenhydrinate and diphenhydramine did not consistently alleviate the anxiety caused by either cocaine pre-treatment regime; in the saline conditions, however, each of these treatments was anxiogenic. In summary, benzodiazepines alleviated cocaine-induced anxiety, while future research on the ability of serotonergic and antihistaminergic drugs to alleviate these anxiety states is warranted.

Selective deficits in attentional performance on the 5-choice serial reaction time task following pedunculopontine tegmental nucleus lesions.

Sustained attention requires the integrity of basal forebrain cholinergic systems. The pedunculopontine tegmental nucleus (PPTg) has direct and indirect connections (via the thalamus) with the basal forebrain, suggesting that the PPTg may also play an important role in attentional processes. We examined this hypothesis by testing the effects of PPTg lesions in rats on performance in the 5-choice serial reaction time test. Bilateral lesions reduced accuracy, increased errors of omission, and increased the latency to correct responses. The deficits were more severe when neuronal damage was bilateral and concentrated in the posterior PPTg. Attentional demands of the task were increased by decreasing the stimulus duration, the stimulus brightness, or the inter-trial interval, and by introducing random bursts of white noise. These challenges impaired performance of all animals, but the magnitude of deficit was increased in the lesioned group. Conversely, lesion-induced deficits were partially alleviated when the attentional demands of the task were reduced. This pattern of results suggests that PPTg lesions produce a global deficit in attention, rather than a specific impairment in one process. The PPTg may control attentional processes through its direct projections to the forebrain cholinergic system or, indirectly, through activation of thalamocortical projections.

Cocaine seeking by rats is a goal-directed action.

In two experiments rats were trained to self-administer intravenous cocaine on chained schedules using different responses in the initial (drug-seeking) and terminal (drug-taking) links. In both between- (Experiment 1) and within-subject designs (Experiment 2), the drug-taking response was then either extinguished or reinforced in the absence of the opportunity to perform the seeking response. In a subsequent extinction test with the seeking manipulanda alone, the rate of drug seeking was reduced after the prior extinction of the associated taking response. An additional group trained with a sucrose reinforcer showed a comparable devaluation effect. These findings demonstrate that seeking responses for cocaine and food rewards are mediated by a representation of the contingency between seeking responses and the opportunity to take the reward.

Pimozide, like extinction, devalues stimuli associated with sucrose taking.

Conditioned stimuli (CS) can be devalued by exposure to those stimuli in the absence of primary reward. We tested the hypothesis that dopamine (DA) mediates the control of behavior by conditioned appetitive stimuli. Long-Evans rats were trained to respond for sucrose under a heterogeneous chain schedule in which seeking responses (lever press) turned on a houselight [variable interval (VI)-120 s]; taking responses (wheel turn or chain pull) in the presence of the houselight were reinforced [fixed ratio (FR)-1] by a sucrose pellet. When responding on this schedule was stable, the levers were retracted and subjects had access to the sucrose-taking manipulandum only. Sucrose-taking responses were either extinguished or reinforced under the influence of the DA antagonist, pimozide. Control groups were also reinforced for sucrose-taking responses but received no injection or a vehicle injection prior to each session. Responses of extinction and pimozide-treated groups declined over sessions. Sucrose-seeking responses were measured in a later test when subjects had no access to the sucrose-taking manipulandum or to the reinforcer. Both extinction and pimozide manipulations reduced seeking responses, relative to the respective control groups. Pimozide injections in the home cage had no effect. These data support the idea that DA mediates the conditioned reinforcing properties provided by access to the taking link of the chain.

Cocaine-seeking by rats: regulation, reinforcement and activation.

RATIONALE: In animal models of drug self-administration, response rates often decrease with dose suggesting that a regulative process may mask the reinforcing effects of the drug. OBJECTIVE: The purpose of the present experiments was to dissociate the role of regulative and reinforcement processes in intravenous cocaine self-administration by rats using a paradigm that explicitly distinguishes between drug-seeking and drug-taking. METHODS: Rats were trained to respond for intravenous cocaine (0.25 mg/infusion) under a heterogeneous chain (tandem FR1 RI 30 s) FR1 schedule of reinforcement using different levers in the first (seeking) and second (taking) links of the chain. After 10 days of training, rats were switched to one of three doses of cocaine (0.08, 0.25, or 0.5 mg/infusion) and self-administration patterns were recorded for a further ten sessions in experiment 1. In experiment 2, a time-out (TO) period (0, 4, or 12 min) was imposed between successive cycles of the chain schedule. Finally, the effect of allowing animals to perform a drug-taking response on subsequent drug-seeking was assessed in experiment 3. RESULTS: Having verified that seeking responses for a conventional reinforcer (sucrose) were sensitive to changes in reward magnitude, experiment 1 demonstrated that the number of self-administered infusions was inversely related to dose whereas the latency to initiate drug-seeking increased with dose. Variations in the cocaine dose had no reliable effect on the number of drug seeking response per cycle of the chain schedule. The effect of dose on the latency to initiate drug-seeking was reversed in experiment 2 with increasing TO periods. Moreover, at the longest TO period, drug-seeking responses per cycle increased and the latency to initiate drug seeking decreased with dose. Experiment 3 showed that the latency to drug-seek for the low dose was reduced dramatically when the first drug-seeking response was preceded by a drug-taking response, even when this response did not produce a drug infusion. CONCLUSIONS: The overall pattern of results suggests that drug-seeking and drug-taking are controlled by three interacting processes: a regulative process depresses drug-seeking in the short-term; behavioral activation enhances drug-seeking and is sustained over longer intervals by higher drug doses; the reinforcing effect of cocaine increases with dose once the satiety producing effects of the drug dissipate.

Pedunculopontine tegmental nucleus lesions impair stimulus--reward learning in autoshaping and conditioned reinforcement paradigms.

The role of the pedunculopontine tegmental nucleus (PPTg) in stimulus-reward learning was assessed by testing the effects of PPTg lesions on performance in visual autoshaping and conditioned reinforcement (CRf) paradigms. Rats with PPTg lesions were unable to learn an association between a conditioned stimulus (CS) and a primary reward in either paradigm. In the autoshaping experiment, PPTg-lesioned rats approached the CS+ and CS- with equal frequency, and the latencies to respond to the two stimuli did not differ. PPTg lesions also disrupted discriminated approaches to an appetitive CS in the CRf paradigm and completely abolished the acquisition of responding with CRf. These data are discussed in the context of a possible cognitive function of the PPTg, particularly in terms of lesion-induced disruptions of attentional processes that are mediated by the thalamus.

Lesions of the pedunculopontine tegmental nucleus increase sucrose consumption but do not affect discrimination or contrast effects.

Pedunculopontine tegmental nucleus (PPTg) lesions block place preferences to drugs or food only when animals are nondeprived. PPTg lesions also disrupt operant responding, but lesioned rats cannot discriminate active from inactive levers. It is not clear, therefore, whether PPTg lesions block reward or disrupt the ability to differentiate changes in reward magnitude. These hypotheses were tested by measuring sucrose consumption, choice, and contrast effects after PPTg lesions. Both sham and lesioned rats consumed greater amounts of a sucrose solution as the concentration and level of deprivation were increased. Given a choice between 2 solutions, all rats consumed more of the higher concentration. Both groups exhibited contrast effects when the concentration was shifted from 32% to 4% within a session. Somewhat surprisingly, lesions increased sucrose intake when rats were food-restricted. These results suggest that PPTg lesions do not disrupt primary motivation or the ability to evaluate and respond to changes in reward strength.

Associative processes in addiction and reward. The role of amygdala-ventral striatal subsystems.

Only recently have the functional implications of the organization of the ventral striatum, amygdala, and related limbic-cortical structures, and their neuroanatomical interactions begun to be clarified. Processes of activation and reward have long been associated with the NAcc and its dopamine innervation, but the precise relationships between these constructs have remained elusive. We have sought to enrich our understanding of the special role of the ventral striatum in coordinating the contribution of different functional subsystems to confer flexibility, as well as coherence and vigor, to goal-directed behavior, through different forms of associative learning. Such appetitive behavior comprises many subcomponents, some of which we have isolated in these experiments to reveal that, not surprisingly, the mechanisms by which an animal sequences responding to reach a goal are complex. The data reveal how the different components, pavlovian approach (or sign-tracking), conditioned reinforcement (whereby pavlovian stimuli control goal-directed action), and also more general response-invigorating processes (often called "activation," "stress," or "drive") may be integrated within the ventral striatum through convergent interactions of the amygdala, other limbic cortical structures, and the mesolimbic dopamine system to produce coherent behavior. The position is probably not far different when considering aversively motivated behavior. Although it may be necessary to employ simplified, even abstract, paradigms for isolating these mechanisms, their concerted action can readily be appreciated in an adaptive, functional setting, such as the responding by rats for intravenous cocaine under a second-order schedule of reinforcement. Here, the interactions of primary reinforcement, psychomotor activation, pavlovian conditioning, and the control that drug cues exert over the integrated drug-seeking response can be seen to operate both serially and concurrently. The power of our analytic techniques for understanding complex motivated behavior has been evident for some time. However, the crucial point is that we are now able to map these components with increasing certainty onto discrete amygdaloid, and other limbic cortical-ventral striatal subsystems. The neural dissection of these mechanisms also serves an important theoretical purpose in helping to validate the various hypothetical constructs and further developing theory. Major challenges remain, not the least of which is an understanding of the operation of the ventral striatum together with its dopaminergic innervation and its interactions with the basolateral amygdala, hippocampal formation, and prefrontal cortex at a more mechanistic, neuronal level.

Dissociation in effects of lesions of the nucleus accumbens core and shell on appetitive pavlovian approach behavior and the potentiation of conditioned reinforcement and locomotor activity by D-amphetamine.

Dopamine release within the nucleus accumbens (NAcc) has been associated with both the rewarding and locomotor-stimulant effects of abused drugs. The functions of the NAcc core and shell were investigated in mediating amphetamine-potentiated conditioned reinforcement and locomotion. Rats were initially trained to associate a neutral stimulus (Pavlovian CS) with food reinforcement (US). After excitotoxic lesions that selectively destroyed either the NAcc core or shell, animals underwent additional CS-US training sessions and then were tested for the acquisition of a new instrumental response that produced the CS acting as a conditioned reinforcer (CR). Animals were infused intra-NAcc with D-amphetamine (0, 1, 3, 10, or 20 microg) before each session. Shell lesions affected neither Pavlovian nor instrumental conditioning but completely abolished the potentiative effect of intra-NAcc amphetamine on responding with CR. Core-lesioned animals were impaired during the Pavlovian retraining sessions but showed no deficit in the acquisition of responding with CR. However, the selectivity in stimulant-induced potentiation of the CR lever was reduced, as intra-NAcc amphetamine infusions dose-dependently increased responding on both the CR lever and a nonreinforced (control) lever. Shell lesions produced hypoactivity and attenuated amphetamine-induced activity. In contrast, core lesions resulted in hyperactivity and enhanced the locomotor-stimulating effect of amphetamine. These results indicate a functional dissociation of subregions of the NAcc; the shell is a critical site for stimulant effects underlying the enhancement of responding with CR and locomotion after intra-NAcc injections of amphetamine, whereas the core is implicated in mechanisms underlying the expression of CS-US associations.

Basal forebrain cholinergic lesions enhance conditioned approach responses to stimuli predictive of food.

This study examined the effects of lesions to different neuronal populations within the basal forebrain on reward-related learning. Rats received bilateral alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or quinolinate lesions that preferentially destroy the cholinergic nucleus basalis magnocellularis (NBM) or noncholinergic ventral pallidal neurons, respectively. Both lesions enhanced conditioned approach responses to stimuli predictive of food but did not increase the locomotor stimulating effect of d-amphetamine. Although both lesions disrupted the discriminative control over behavior by a conditioned stimulus, they did not impair the subsequent acquisition of instrumental responding with conditioned reinforcement (CR). Indeed, both lesions were associated with an increased responding with CR following intra-accumbens infusions of d-amphetamine (0, 1, 3, 10, and 20 microg). Quinolinate lesions also increased responses on an inactive control lever. Neither lesion altered consummatory responses to food or sucrose. Results suggest that NBM lesions may disrupt the balance between cortical and subcortical dopamine levels, and/or produce a deficit in attentional mechanisms that is manifested as increased responding to specific stimuli.

Effects of pedunculopontine tegmental nucleus lesions on responding for intravenous heroin under different schedules of reinforcement.

The pedunculopontine tegmental nucleus (PPTg) is believed to play important roles in reward and learning. We examined the effect of PPTg lesions (0.5 microl of 0.1 M NMDA injected bilaterally over 10 min) on the learning of an operant response for opiate reward. In 14 adult male Long-Evans rats, bilateral lesions of the PPTg disrupted the acquisition of responding for intravenous heroin (0.1 mg/kg infused at a rate of 0.25 ml/28 sec) on a fixed ratio-1 (FR-1) schedule of reinforcement. The 12 remaining lesioned animals increased their heroin intake over the acquisition sessions but did not reach the response levels of sham-lesioned animals on the 15th and final session. The sham- and PPTg-lesioned animals that learned the FR-1 task exhibited similar patterns of responding during extinction and reacquisition sessions. When tested on a progressive ratio (PR) schedule of reinforcement, however, PPTg-lesioned animals had lower break points than sham-lesioned animals. Asymmetric lesions, which destroyed the majority of the nucleus in one hemisphere only, did not produce any behavioral deficits. Rats that were lesioned after training also did not show deficits in responding under either FR or PR schedules. These findings suggest that PPTg lesions reduce the rewarding effect of opiates but do not disrupt the ability either to learn an operant response or the response requirements of a PR schedule.

The development of a conditioned place preference to morphine: effects of lesions of various CNS sites.

This study examined the neural substrates underlying the development of a conditioned place preference (CPP) to morphine (2 mg/kg x 3 pairings) by testing whether lesions of 7 different neural sites block a morphine-induced CPP. Lesions of the pedunculopontine tegmental nucleus (PPTg), the periaqueductal gray (PAG), or the fornix reduced the preference for a morphine-paired compartment. When they were retested following morphine administration, fornix- or PAG-lesioned animals exhibited a CPP indicating that lesions did not block morphine-induced reward or the ability to associate this effect with salient environmental cues. PPTg-lesioned animals did not express a CPP during state-dependent testing, suggesting that the lesions may attenuate the rewarding effect of the drug. Lesions of the mesolimbic dopamine system, the ventral pallidum, the lateral nucleus of the amygdala, or the caudate putamen had no effect on a morphine-induced CPP.