Kelly J. Clemens

Renewal of extinguished cocaine-seeking.

Rats were trained to self-administer cocaine in a distinctive context (context A). They were then extinguished in a second context (context B) prior to test for cocaine-seeking in the original training context, context A (group ABA), context B (group ABB) or no test (group AB0). Group ABA showed renewal of extinguished cocaine-seeking associated with c-Fos induction in basolateral amygdala, lateral hypothalamus, and infralimbic prefrontal cortex. Groups ABA and ABB showed test-associated c-Fos induction in prelimbic prefrontal cortex, nucleus accumbens (core, shell, rostral pole), striatum, lateral amygdala, perifornical hypothalamus, and ventral tegmental area. Double immunofluorescence revealed that renewal-associated c-Fos was expressed in orexin-negative lateral hypothalamic neurons whereas test-associated c-Fos was expressed in orexin-positive perifornical hypothalamic neurons. Retrograde tracing from lateral hypothalamus with cholera toxin revealed only sparse dual-labeled neurons in basolateral amygdala and infralimbic prefrontal cortex, suggesting that these regions contribute to renewal of cocaine-seeking independently of their projections to lateral hypothalamus. Retrograde tracing from the ventral tegmental area suggested that hypothalamic contributions to cocaine-seeking are likewise independent of projections to the midbrain. These results suggest that renewal of cocaine-seeking depends critically on basolateral amygdala, lateral hypothalamus, and infralimbic prefrontal cortex. Whereas basolateral amygdala and lateral hypothalamus contributions may be common to renewal of extinguished cocaine-, alcohol-, and sucrose-seeking, infralimbic prefrontal cortex contributions appear unique to renewal of cocaine-seeking and may reflect the habitual nature of relapse to cocaine.

Increased anxiety 3 months after brief exposure to MDMA ("Ecstasy") in rats: association with altered 5-HT transporter and receptor density.

Male Wistar rats were treated with 3,4-methylenedioxymethamphetamine (MDMA, ‘Ecstasy’) using either a high dose (4 × 5 mg/kg over 4 h) or low dose (1 × 5 mg/kg over 4 h) regimen on each of 2 consecutive days. After 10 weeks, rats were tested in the social interaction and emergence tests of anxiety. Rats previously given either of the MDMA dose regimens were significantly more anxious on both tests. After behavioral testing, and 3 months after the MDMA treatment, the rats were killed and their brains examined. Rats given the high-, but not the low-, dose MDMA treatment regimen exhibited significant loss of 5-hydroxytryptamine (5-HT) and 5-HIAA in the amygdala, hippocampus, striatum, and cortex. Quantitative autoradiography showed loss of SERT binding in cortical, hippocampal, thalamic, and hypothalamic sites with the high-dose MDMA regime, while low-dose MDMA only produced significant loss in the medial hypothalamus. Neither high- nor low-dose MDMA affected 5HT1A receptor density. High-dose MDMA increased 5HT1B receptor density in the nucleus accumbens and lateral septum but decreased binding in the globus pallidus, insular cortex and medial thalamus. Low-dose MDMA decreased 5HT1B receptor density in the hippocampus, globus pallidus, and medial thalamus. High-dose MDMA caused dramatic decreases in cortical, striatal, thalamic, and hypothalamic 5HT2A/2C receptor density, while low-dose MDMA tended to produce similar effects but only significantly in the piriform cortex. These data suggest that even brief, relatively low-dose MDMA exposure can produce significant, long-term changes in 5-HT receptor and transporter function and associated emotional behavior. Interestingly, long-term 5-HT depletion may not be necessary to produce lasting effects on anxiety-like behavior after low-dose MDMA.

Paraventricular thalamus mediates context-induced reinstatement (renewal) of extinguished reward seeking

Paraventricular thalamus (PvTh) is uniquely placed to contribute to reinstatement of drug and reward seeking. It projects extensively to regions implicated in reinstatement including accumbens shell (AcbSh), prefrontal cortex (PFC) and basolateral amygdala (BLA), and receives afferents from other regions important for reinstatement such as lateral hypothalamus. We used complementary neuroanatomical and functional approaches to study the role of PvTh in context‐induced reinstatement (renewal) of extinguished reward‐seeking. Rats were trained to respond for a reward in context A, extinguished in context B and tested in context A or B. We applied the neuronal tracer cholera toxin B subunit (CTb) to AcbSh and examined retrograde‐labelled neurons, c‐Fos immunoreactivity (IR) and dual c‐Fos/CTb labelled neurons in PvTh and other AcbSh afferents. In PvTh there was c‐Fos IR in CTb‐positive neurons associated with renewal showing activation of a PvTh–AcbSh pathway during renewal. In PFC there was little c‐Fos IR in CTb‐positive or negative neurons associated with renewal. In BLA, two distinct patterns of activation and retrograde labelling were observed. In rostral BLA there was significant c‐Fos IR in CTb‐negative neurons associated with renewal. In caudal BLA there was significant c‐Fos IR in CTb‐positive neurons associated with being tested in either the extinction (ABB) or training (ABA) context. We then studied the functional role of PvTh in renewal. Excitotoxic lesions of PvTh prevented renewal. These lesions had no effect on the acquisition of reward seeking. These results show that PvTh mediates context‐induced reinstatement and that this renewal is associated with recruitment of a PvTh–AcbSh pathway.

The addition of five minor tobacco alkaloids increases nicotine-induced hyperactivity, sensitization and intravenous self-administration in rats

Several minor tobacco alkaloids have been found to exhibit properties pharmacologically relevant to the addictive profile of tobacco; however, little is known of their effects on a behavioural model of drug addiction. In this study we compared the locomotor and reinforcing effects of intravenous nicotine (30 microg/kg per infusion) vs. a cocktail of nicotine plus five minor alkaloids found in tobacco smoke (anabasine, nornicotine, anatabine, cotinine and myosmine). Rats were initially tested for their locomotor response to nicotine or nicotine plus the minor alkaloids with six intravenous injections over 1 h. We then assessed the spontaneous acquisition of intravenous self-administration with nicotine or nicotine plus the minor alkaloids, under a fixed-ratio 1 schedule followed by responding on a fixed-ratio 5 schedule, progressive-ratio schedule and a single within-session ascending dose-response test. The activity test was repeated following the progressive-ratio phase to assess locomotor sensitization. A second group of rats were then tested on the locomotor procedure to better clarify the role of each individual minor alkaloid in nicotine-induced locomotor activity. Compared to nicotine alone, addition of the minor tobacco alkaloids increased locomotor activity and increased locomotor sensitization following self-administration. During fixed-ratio 5, progressive ratio and the dose-response test, rats receiving nicotine plus the minor alkaloids responded significantly more than those receiving nicotine alone. Testing of each minor alkaloid in the second experiment indicated that anatabine, cotinine and myosmine individually increased nicotine-induced locomotor activity. These results suggest that the minor tobacco alkaloids, particularly anatabine, cotinine and myosmine, may increase the motivation for nicotine and thus facilitate smoking behaviour.

CHRONIC FLUOXETINE TREATMENT PARTLY ATTENUATES THE LONG-TERM ANXIETY AND DEPRESSIVE SYMPTOMS INDUCED BY MDMA (ECSTASY) IN RATS

Use of the drug 3,4-methylenedioxymethamphetamine (MDMA, ‘Ecstasy’) can have long-term adverse effects on emotion in both humans and laboratory animals. The present study examined whether chronic treatment with the antidepressant drug fluoxetine could reverse such effects. Male Wistar rats were briefly exposed to MDMA (4 × 5 mg/kg over 4 h) or vehicle on 2 consecutive days. Approximately 9–12 weeks later, half of the rats received a dose of approximately 6 mg/kg/day fluoxetine in their drinking water for a 5-week period. Fluoxetine administration reduced fluid intake and body weight in MDMA and vehicle pretreated rats. After several weeks of fluoxetine treatment, rats were assessed on the social interaction test, the emergence test of anxiety and the forced swim model of depression. MDMA pretreated rats showed reduced social interaction, increased anxiety on the emergence test, and increased immobility and decreased active responses in the forced swim test. Fluoxetine treatment reversed MDMA-induced anxiety in the emergence test and depressive-like effects in the forced swim test, yet exhibited no effects on the social interaction test. MDMA pretreated rats had decreased 5-HT and 5-HIAA levels in limbic and cortical regions, and decreased density of serotonin transporter sites in the cortex. Fluoxetine treatment did not greatly affect 5-HT levels in MDMA pretreated rats, but significantly decreased 5-HIAA levels in all brain sites examined. Postmortem blood serum levels of fluoxetine and norfluoxetine did not differ in MDMA and vehicle pretreated rats. These results indicate that fluoxetine may provide a treatment option for some of the deleterious long-term effects resulting from MDMA exposure.

High levels of intravenous mephedrone (4-methylmethcathinone) self-administration in rats: Neural consequences and comparison with methamphetamine

Mephedrone (MMC) is a relatively new recreational drug that has rapidly increased in popularity in recent years. This study explored the characteristics of intravenous MMC self-administration in the rat, with methamphetamine (METH) used as a comparator drug. Male Sprague-Dawley rats were trained to nose poke for intravenous MMC or METH in daily 2 h sessions over a 10 d acquisition period. Dose-response functions were then established under fixed- and progressive-ratio (FR and PR) schedules over three subsequent weeks of testing. Brains were analyzed ex vivo for striatal serotonin (5-HT) and dopamine (DA) levels and metabolites, while autoradiography assessed changes in the regional density of 5-HT and serotonin transporter (SERT) and DA transporter (DAT) and induction of the inflammation marker translocator protein (TSPO). Results showed that MMC was readily and vigorously self-administered via the intravenous route. Under a FR1 schedule, peak responding for MMC was obtained at 0.1 mg/kg/infusion, versus 0.01 mg/kg/infusion for METH. Break points under a PR schedule peaked at 1 mg/kg/infusion MMC versus 0.3 mg/kg/infusion for METH. Final intakes of MMC were 31.3 mg/kg/d compared to 4 mg/kg/d for METH. Rats self-administering MMC, but not METH, gained weight at a slower rate than control rats. METH, but not MMC, self-administration elevated TSPO receptor density in the nucleus accumbens and hippocampus, while MMC, but not METH, self-administration decreased striatal 5-hydroxyindolacetic acid (5-HIAA) concentrations. In summary, MMC supported high levels of self-administration, matching or exceeding those previously reported with other drugs of abuse.

The effects of response operandum and prior food training on intravenous nicotine self-administration in rats

RationaleNicotine intravenous self-administration (IVSA) in rats has been conducted using a variety of methodological procedures with equally variable results.ObjectivesHere, we addressed the importance of the type of response operandum and prior instrumental training with a natural reinforcer on nicotine IVSA and reinstatement.MethodsRats were tested for spontaneous acquisition of IVSA using either nose poke (NP) or lever press (LVR) operandum. A dose-response test was then conducted, followed by extinction and cue- and nicotine-induced reinstatement.ResultsThe use of the NP operandum resulted in markedly higher levels of IVSA across acquisition and across dose-response testing compared with the LVR group. Whereas both groups reinstated following a nicotine prime, only the LVR group demonstrated cue-induced reinstatement. As a positive control, the experiment was repeated with cocaine as the reinforcer: equivalent levels of IVSA were observed across all tests, irrespective of operandum. When rats self-administering nicotine received instrumental training with a sucrose reinforcer prior to IVSA, a facilitated acquisition of IVSA was observed in both LVR and NP groups to a similar extent (the effect of operandum remained), but had little effect on responding thereafter. During reinstatement testing, both groups now displayed cue- and nicotine-induced reinstatement, but this was also evident in saline control animals that had never received nicotine.ConclusionsThese results suggest that, unlike cocaine, an increased physical response requirement can decrease nicotine intake. It also indicates that operandum and prior sucrose training may influence the role that visual cues play in nicotine dependence.

Modeling Nicotine Addiction in Rats

Among the human population, 15% of drug users develop a pathological drug addiction. This figure increases substantially with nicotine, whereby more than 30% of those who try smoking develop a nicotine addiction. Drug addiction is characterized by compulsive drug-seeking and drug-taking behaviors (craving), and loss of control over intake despite impairment in health, social, and occupational functions. This behavior can be accurately modeled in the rat using an intravenous self-administration (IVSA) paradigm. Initial attempts at establishing nicotine self-administration had been problematic, yet in recent times increasingly reliable models of nicotine self-administration have been developed. The present article reviews different characteristics of the nicotine IVSA model that has been developed to examine nicotine reinforcing and motivational properties in rats.

Behavioral and Neural Substrates of Habit Formation in Rats Intravenously Self-Administering Nicotine

Tobacco addiction involves a transition from occasional, voluntary smoking towards habitual behavior that becomes increasingly resistant to quitting. The development of smoking habits may reflect a loss of behavioral control that can be modeled in rats. This study investigated the behavioral and neural substrates of habit formation in rats exposed to either brief (10 days) or extended (47 days) intravenous (IV) nicotine self-administration training. Following training, the first cohort of rats were exposed to a nicotine devaluation treatment, which involved repeated pairings of IV nicotine with lithium injection. They were then tested for sensitivity of responding to nicotine devaluation under extinction and reinstatement conditions. The second cohort of rats received equivalent self-administration training followed by processing of brain tissue for c-Fos immunohistochemistry. After brief training, devaluation suppressed nicotine-seeking during tests of extinction and reinstatement, confirming that responding is initially sensitive to current nicotine value, and therefore, goal directed. In contrast, after extended training, devaluation had no effect on extinction or reinstatement of responding, indicating that responding had become habitual. Complementary neuroanalysis revealed that extended nicotine self-administration was associated with increased c-Fos expression in brain regions implicated in habitual control of reward seeking, including activation of the dorsolateral striatum and substantia nigra pars compacta. These findings provide evidence of direct devaluation of an IV drug reward, that nicotine self-administration is initially goal-directed but becomes habitual with extended training, and that this behavioral transition involves activation of brain areas associated with the nigrostriatal system.

Reduced alcohol drinking in adult rats exposed to sucrose during adolescence

Intake of sweet-alcoholic drinks during adolescence is believed to favor alcohol abuse and dependence in adulthood. This study examined the influence of early exposure to ethanol with or without sucrose on the consumption of sweet or alcoholic solutions in adulthood. Adolescent rats (from post-natal day 30-46) were given continuous free access to tap water and either 5% sucrose, 5% ethanol or mixed 5% sucrose-5% ethanol. The control group was given access to water only. Upon reaching adulthood (post-natal day 60), rats were tested for saccharin (sweet), quinine (bitter) and ethanol consumption using a two-bottle free-choice paradigm. The results indicated that pre-exposure to ethanol did not alter the intake of sweet or ethanol solutions in adulthood. However, rats exposed to sucrose during adolescence showed a decreased consumption of both sweet and ethanol solutions. Because alcohol has a sweet taste component, an additional group of rats, pre-exposed to either 5% sucrose or water during adolescence, was tested for intravenous ethanol self-administration (preventing oral sensory stimulation) and in a new model of simultaneous access to oral saccharin and intravenous ethanol that results in higher total ethanol intake. Relative to controls, sucrose-exposed rats showed reduced operant self-administration of saccharin, yet no differences were found for intravenous ethanol self-administration. Altogether, these findings indicate that sucrose exposure during adolescence persistently affected the perception of sweet taste reward and thereby alcohols acceptance in adulthood.