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.

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.

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.

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.

Differential effects of ventral striatal lesions on the conditioned place preference induced by morphine or amphetamine.

The present experiment examined the role of the ventral striatum in the rewarding effect of morphine and amphetamine by testing whether lesions of cell bodies within this region disrupt the development of a conditioned place preference to either drug. Bilateral, N-methyl-D-aspartate- or kainic acid-induced lesions of the ventral striatum block a conditioned place preference to amphetamine (1.5 mg/kg x 3 pairings) but not to morphine (2 mg/kg x 3 pairings). Because both lesions spared anterior portions of the ventral striatum, we examined the effect of larger or more selective ventral striatal lesions on a conditioned place preference induced by morphine. Destruction of the entire ventral striatum reduced, but did not eliminate, a conditioned place preference to morphine, whereas selective lesions of the anterior ventral striatum were ineffective. These results indicate that the ventral striatum is not critically involved in morphine's rewarding effect and support the suggestion that the rewarding effects of opiates and stimulants do not involve identical neural substrates.

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.

Effects of pedunculopontine tegmental nucleus lesions on morphine-induced conditioned place preference and analgesia in the formalin test.

The development of a conditioned place preference to morphine (2 mg/kg; three pairings) and the analgesic effect of morphine (0, 4 or 8 mg/kg) in the formalin test were studied in rats with sham or neurotoxin lesions of the pedunculopontine tegmental nucleus. Lesions were induced by bilateral infusions of N-methyl-D-aspartate (0.5 microliter of 0.1 M solution) or vehicle over 10 min. No anti-seizure medication was administered in the first experiment, whereas animals in the second experiment were injected with diazepam (1 mg/kg) immediately after surgery. In Experiment 1, behaviour in the conditioned place preference and formalin tests was assessed in separate groups of lesioned and control rats. In Experiment 2, the same animals received both sets of tests. In both experiments lesions of the pedunculopontine tegmental nucleus blocked the development of a conditioned place preference to morphine, but had no effect on the behavioural response to formalin, or on its inhibition by morphine. Examination of cholinergic-stained cells found no correlation between the magnitude of behavioural effects and the number of acetylcholine cells destroyed by the lesions. These results confirm that the pedunculopontine tegmental nucleus mediates the development of a morphine-induced conditioned place preference, but not the analgesic effect of morphine.

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.

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

Experiment 1 examined whether microinjections of morphine (1 microg in 0.5 microl over 1 min x 2 pairings) into 13 different CNS sites produced a conditioned place preference (CPP). Injections into the lateral ventricles (LV), ventral tegmental area (VTA), or periaqueductal gray (PAG) produced a CPP; injections 1 mm dorsal to the PAG or VTA, or into the caudate putamen, medial frontal cortex, hippocampus, lateral nucleus of the amygdala, lateral hypothalamus, pedunculopontine tegmental nucleus, posterior hypothalamus, ventral palladium, or nucleus accumbens septi (core or shell) did not. In Experiment 2, morphine 0.2 microg produced a CPP when injected into the VTA but not in the PAG, while 5.0 microg was effective in both sites. The CPP induced by systemic morphine (4 mg/kg x 1 pairing) was blocked by naloxone methiodide (NM) injected (2 nmol in 0.5 microl) into the VTA. PAG injections of 2 nmol reduced, and 5 nmol NM eliminated, the CPP. The results confirm that morphine injections into the VTA or the PAG are rewarding, that blockade of opioid receptors in either site disrupts a morphine-induced CPP, and that the VTA is more sensitive to both effects.

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.

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.

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.

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.

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.

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.

Differential effects of atropine, procaine and dopamine in the rat ventral tegmentum on lateral hypothalamic rewarding brain stimulation.

Microinjections of the muscarinic antagonist, atropine, of dopamine, or of the local anesthetic, procaine, in the ventral tegmentum elevated frequency thresholds for lateral hypothalamic self-stimulation. The largest and most robust effects were observed following atropine (30 or 60 micrograms) microinjections. The most sensitive sites for the atropine effect were near dopamine cells. In order to determine if the effects of atropine can be reversed by pretreatment with a cholinergic agonist, carbachol (1-3 micrograms) was microinjected 15 min prior to atropine. Carbachol pretreatment attenuated the frequency threshold elevation of atropine by 47-95%. Since atropine is a local anesthetic, the effects of procaine on self-stimulation thresholds were tested as well. Procaine (100 or 250 micrograms) in ventral tegmentum elevated frequency thresholds by much less than atropine. Therefore, while atropine attenuates reward primarily through blockade of muscarinic receptors, the local anesthetic effect of atropine may enhance the threshold elevation. Dopamine (1-10 micrograms) also elevated frequency thresholds, but when dopamine injections were repeated daily, the threshold elevations were attenuated. This attenuation contrasted with the robust effects of atropine, and may reflect the development of autoreceptor subsensitivity. Hence, both dopaminergic and muscarinic receptors in ventral tegmentum are involved in lateral hypothalamic brain stimulation reward.

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.

Lesions of the pedunculopontine tegmental nucleus block drug-induced reinforcement but not amphetamine-induced locomotion.

It has been proposed that the positive reinforcing and motor stimulating effects of drugs involve the activation of a common neural substrate. Reinforcing effects of food, drugs and brain stimulation are blocked by lesions of the pedunculopontine tegmental nucleus (PPTg), which is a component of the mesencephalic locomotor region. This has suggested that the PPTg may be involved in both positive reinforcement and forward locomotion. In four separate experiments, rats were prepared with NMDA (0.5 microliters of 0.1 M solution) or sham lesions of the PPTg. Animals in the first two experiments were tested for the development of a conditioned place preference (CPP) to morphine (2 mg/kg x 3 pairings) or amphetamine (1.5 mg/kg x 3 pairings). Ten days later, spontaneous motor activity (SMA) was assessed in these animals following a subcutaneous injection of saline or amphetamine (1.5 mg/kg). In two further experiments, drug-naive lesioned and control animals were tested for SMA only (saline or 1.5 mg/kg amphetamine in Experiment 3, and saline, 0.5 mg/kg, or 3 mg/kg amphetamine in Experiment 4). Lesions of the PPTg blocked the development of a CPP to both morphine and amphetamine. In contrast, lesions had no effect on saline or amphetamine-stimulated SMA. The PPTg, therefore, appears to be involved in the reinforcing effects of amphetamine and morphine, but is not necessary for the expression of amphetamine-induced activity.

Lesions of the pedunculopontine tegmental nucleus abolish catalepsy and locomotor depression induced by morphine.

The cataleptic and locomotor depressant effects of morphine are mediated by a series of neural structures, all of which project to the pedunculopontine tegmental nucleus (PPTg). To test the idea that the PPTg is also involved in mediating morphine's effect on these behaviours, we examined catalepsy and spontaneous motor activity following bilateral lesions of the PPTg (0.5 microliters of 0.1 M N-methyl-D-aspartate infused over 10 min). We also examined the effect of PPTg lesions on motor functioning by observing sensorimotor responses, limb use, muscle tone and locomotion. PPTg lesions completely abolished the catalepsy and decreased activity that normally follows morphine administration. In addition lesioned animals exhibited increased muscle tone, and impairments in limb use and righting reflexes. Although the deficits were subtle, these results confirm that damage to the PPTg is associated with motor abnormalities.