A synthetic small-molecule Isoxazole-9 protects against methamphetamine relapse.

Adult neurogenesis in the dentate gyrus (DG) is strongly influenced by drug-taking behavior and may have a role in the etiology of drug-seeking behavior. However, mechanistic studies on the relationship of neurogenesis on drug seeking are limited. Outbred Wistar rats experienced extended access methamphetamine self-administration and individual differences in drug taking defined animals with higher preferred and lower preferred levels of drug intake. Forced abstinence from higher preferred levels of drug taking enhanced neurogenesis and neuronal activation of granule cell neurons (GCNs) in the DG and produced compulsive-like drug reinstatement. Systemic treatment with the drug Isoxazole-9 (a synthetic small molecule known to modulate neurogenesis in the adult rodent brain) during abstinence blocked compulsive-like context-driven methamphetamine reinstatement. Isoxazole-9 modulated neurogenesis, neuronal activation and structural plasticity of GCNs, and expression of synaptic proteins associated with learning and memory in the DG. These findings identify a subset of newly born GCNs within the DG that could directly contribute to drug-seeking behavior. Taken together, these results support a direct role for the importance of adult neurogenesis during abstinence in compulsive-like drug reinstatement.

DARPP-32 knockout mice exhibit impaired reversal learning in a discriminated operant task.

The current study was conducted to examine the performance of mice with a targeted deletion of the gene for DARPP-32 in a discriminated operant task using food reinforcement. DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission. Initially, wild-type and DARPP-32 knockout mice were trained to nose-poke for food on a continuous reinforcement schedule. The minimum response requirement was increased every 5 days until the animals were responding on an FR-15 schedule of reinforcement. At the completion of extensive operant training, reversal learning was assessed. Wild-type and DARPP-32 knockout mice exhibited equivalent performance during acquisition of this task, with both groups increasing operant responding as the schedule of reinforcement was raised. However, significant differences in discrimination learning were observed during the reversal phase, with DARPP-32 knockout mice requiring significantly more trials to reach criterion than wild-type controls. These results provide evidence for a functional role of DARPP-32 in the mediation of processes underlying learning and memory.

mu-Opioid receptor knockout mice do not self-administer alcohol.

Opioid peptides long have been hypothesized to play a role in ethanol reinforcement. Neuropharmacological studies have shown that opioid receptor antagonists decrease ethanol self-administration in rodents and prevent relapse in humans. However, the exact mechanism for such powerful effects has remained elusive. The availability of mu-opioid receptor knockout mice has made possible the direct examination of the role of the mu-opioid receptor in mediating ethanol self-administration. In the present experiments, both nosepoke and lever operant ethanol self-administration and several tests of two bottle-choice ethanol drinking were studied in these genetically engineered mice. In no case did knockout mice show evidence of ethanol self-administration, and, in fact, these mice showed evidence of an aversion to ethanol under several experimental conditions. These data provide new evidence for a critical role for mu-opioid receptors in ethanol self-administration assessed with a variety of behavioral paradigms and new insights into the neuropharmacological basis for ethanol reinforcement.

Excessive ethanol drinking following a history of dependence: animal model of allostasis.

Alcohol withdrawal symptoms, particularly negative emotional states, can persist for months following the removal of alcohol. These protracted withdrawal symptoms have been implicated as an important trigger of relapse to excessive drinking in alcoholics and may represent a long lasting shift in affective tone as a result of chronic alcohol exposure. It was shown previously that ethanol-dependent rats increased their operant responding for ethanol when tested during the first 12 hr after withdrawal. The purpose of the present experiments was to determine the persistence of this finding by examining operant oral ethanol self-administration in rats with a history of physical dependence upon ethanol, detoxified and then allowed a two week period of protracted abstinence. The results of these experiments indicate that operant responding for ethanol was enhanced during protracted abstinence by 30-100% and remained elevated for 4-8 weeks post acute withdrawal. These results have important implications for understanding the characteristics and mechanisms underlying vulnerability to relapse.

Behavioral effects of central administration of the novel CRF antagonist astressin in rats.

Astressin, a novel corticotropin releasing factor (CRF) antagonist, has been found to be particularly potent at inhibiting the hypothalamo-pituitary-adrenal axis. The aim of the present study was to determine the effects in rats of astressin in attenuating the anxiogenic-like response produced by social stress and intracerebroventricular (ICV) CRF administration on the elevated plus-maze, and ICV CRF-induced locomotor activation in the rat. Astressin significantly reversed the anxiogenic-like response induced by both social stress and ICV rat/humanCRF (r/hCRF) on the elevated plus-maze, but failed to block the effects of r/hCRF-induced locomotor activity in a familiar environment. When these results were compared to previous studies performed with the same paradigms using other CRF antagonists, astressin showed effects similar to those of D-PheCRF(12-41) on plus-maze performance. However, contrary to alpha-helicalCRF(9-41) and D-PheCRF(12-41), astressin had no effect on CRF-induced locomotor activity. These results suggest that astressin may have a unique anti-CRF profile compared to previously tested antagonists.

Central administration of an opiate antagonist decreases oral ethanol self-administration in rats.

BACKGROUND: Opioid peptides have been implicated in various behavioral actions of alcohol, including its reinforcing effects; however, the role of specific brain sites for these actions remains to be explored. The present study examined the effects of intracerebroventricular (ICV) or intracerebral injections of an opiate antagonist (methylnaloxonium) on ethanol self-administration. The nucleus accumbens and amygdala were selected as intracerebral sites because these regions have been implicated in the reinforcing effects of drugs of abuse. METHODS: Male Wistar rats were trained in a limited-access paradigm (30 min/day) to respond for ethanol (10% w/v) or water in a two-lever free-choice condition using a saccharin fading procedure. After the establishment of stable baseline responding for ethanol, animals were implanted stereotaxically with a guide cannula above the lateral ventricle or with bilateral guide cannulae either above the nucleus accumbens or amygdala. After postoperative recovery of stable baseline responding, the rats were tested 15 min after ICV or intracerebral microinjections of methylnaloxonium (0-2000 ng). RESULTS: Injections of methylnaloxonium into the amygdala significantly reduced responding for ethanol at doses of 250-500 ng. Injections of methylnaloxonium into the nucleus accumbens significantly reduced responding for ethanol at doses of 500-1000 ng, whereas higher doses were needed ICV. CONCLUSIONS: These results provide evidence that opioid receptors located in the amygdala and nucleus accumbens may be involved in the regulation of ethanol self-administration.

Operant self-administration of sweetened versus unsweetened ethanol: effects on blood alcohol levels.

BACKGROUND: Sweeteners are often added to ethanol solutions to increase ethanol intake. However, literature on studies that use human subjects and laboratory animals suggests that sucrose, other sugars, and carbohydrate-rich foods alter ethanol absorption and metabolism, which leads to lower blood alcohol levels (BAL) relative to ethanol absorbed alone. This experiment was designed to test whether the addition of the nutritive sweetener sucrose, or the nonnutritive sweetener saccharin, to a 10% ethanol solution, self-administered in an oral operant paradigm, affected BAL in rats relative to self-administration of an unsweetened 10% ethanol solution. METHODS: All rats were trained to lever press for ethanol by use of a saccharin fading procedure. Half of the rats then received 30-min sessions in which ethanol + 2% sucrose and water were available and were alternated daily with sessions in which ethanol + 0.2% saccharin and water were available. The other half of the rats went on to receive daily sessions of unsweetened ethanol and water. BAL were taken after these standard daily sessions as well as after a 1-week period of alcohol deprivation (to enhance ethanol intake). RESULTS: Rats responded for more ethanol + sucrose than unsweetened ethanol, but had lower BAL per gram ethanol consumed in both the baseline test and alcohol deprivation effect test. No effect of saccharin on BAL was detected. An additional experiment that examined the effects of four concentrations of both sucrose and saccharin on self-administration of ethanol and BAL showed that, whereas rats consumed more ethanol + sucrose than ethanol + saccharin, BAL were significantly lower per gram ethanol consumed in the sucrose group. CONCLUSIONS: These results confirm previous reports and suggest that the addition of sucrose to an ethanol solution can result in lower BAL relative to unsweetened ethanol in an oral operant self-administration paradigm.

Strain distribution of mice in discriminated Y-maze avoidance learning: genetic and procedural differences.

The current study was conducted to characterize discriminated avoidance learning in mice by using a Y-maze task. In Experiment 1, the task parameters were manipulated, including the amount of time spent in the start arm, the amount of time to make the avoidance response, and the intertrial interval (ITI) using C57 x SJL F1 hybrid mice. Avoidance performance was significantly improved with longer times to avoid the shock and longer ITIs. In Experiment 2, mice from 4 inbred strains (BALB/cByJ, DBA/2J, C57BL/6J, and SJL/J), an F1 hybrid (C57 x SJL), and 1 outbred strain (CD1) were tested with various ITIs. Strain differences were observed in avoidance learning, with BALB, DBA, C57 x SJL and CD1 mice showing significantly better avoidance learning than C57 mice, which were better than SJL mice. These data demonstrate that Y-maze performance is significantly influenced by the genetic background of the mouse and the parameters of the task.

Differential expression of response-disruptive and somatic indices of opiate withdrawal during the initiation and development of opiate dependence.

The current study examined the conditions that are necessary and sufficient for the initiation and progression of acute morphine dependence using two indices of opiate withdrawal: suppression of operant response rates and a somatic withdrawal rating scale. Separate groups of rats were pretreated with morphine (5 mg/kg, s.c.) a total of three times at intervals of 24 h, 1, 3, or 6 weeks. Rats received a single dose of naloxone 4 h after each morphine pretreatment. Naloxone-induced suppression of operant responding (0.33 mg/kg, s.c.) was significantly potentiated with repeated exposure to morphine even at the 6-week inter-treatment interval (ITI). At 24-h, 1-week and 3-week ITIs, rats treated with naloxone only after the third and final morphine pretreatment showed similar suppression of operant responding following naloxone to rats treated with naloxone after all three morphine pretreatments. However, at the 6-week ITI, the response-disruptive effects of naloxone administered for the first time after the third morphine pretreatment were no greater than the effects of naloxone administered after a single morphine pretreatment. In contrast to results seen with suppression of operant responding as the withdrawal index, potentiation of somatic signs of withdrawal was observed only at the 24-h ITI. These results indicate that a neuroadaptive state resembling opiate dependence can be initiated after just one injection of morphine, and that the response-disruptive effects of naloxone appear to be a particularly sensitive index of the initiation and progression of acute opiate dependence.

Evidence for behavioral sensitization to cocaine in preweanling rat pups.

While chronic intermittent administration of stimulants often induces behavioral sensitization in adulthood, stimulant sensitization has rarely been reported prior to weaning [around postnatal day (P) 21]. Consistent pairing of drug administration with the test context often facilitates sensitization in adults, yet young animals have been typically returned to the home cage immediately post-injection. To determine whether promoting context-dependent sensitization might facilitate expression of sensitization in preweanlings, Sprague-Dawley rats were injected daily from P14 to P20 with 0, 5, 15, or 30 mg/kg cocaine HCl and placed for 30 min in either the experimental chamber or home cage. On P21 (test day), subjects were challenged with either 15 mg/kg cocaine or saline prior to placement in the experimental chamber. Significant sensitization of cocaine-induced stereotyped head movements was evident in animals given 15 or 30 mg/kg chronically in the experimental chamber, but not when these same doses were given in the home cage. Less consistent evidence for cocaine-induced sensitization was seen when examining locomotion, although trends for sensitization of this behavior were seen in animals chronically injected in either the test chamber or home cage. Thus, preweanlings can exhibit cocaine sensitization, particularly in terms of stereotypy, when tested shortly after the chronic exposure period, with expression of this sensitization being facilitated by pairing the chronic injections with the test context.

Substance dependence as a compulsive behavior.

A compulsion to take a drug combined with a loss of control in limiting intake is the defining feature of substance dependence or addiction, and is the conceptual framework for the criteria of substance dependence or addiction outlined by the World Health Organization and the American Psychiatric Association. However, defining exactly what constitutes loss of control and compulsive drug taking at the level of animal models is a daunting task, and it is clear that no validated animal model exists for the whole syndrome of addiction. The present discussion redefines loss of control as a narrowing of the behavioral repertoire toward drug-seeking behavior and suggests that there are many sources of reinforcement that contribute to this behavioral focus on drug seeking. Evidence is presented demonstrating separate animal models for many of these sources of reinforcement as well as for most of the criteria for substance dependence. Evidence is also presented showing that the brain neurochemical systems involved in processing drug reward are altered by chronic drug exposure to contribute additional sources of reinforcement. Challenges for the future involve not only elucidation of the neurobiological substrates of the different behavioral components of addiction, but better animal models of these components with which to effect such studies.

Neurocircuitry targets in ethanol reward and dependence.

Alcoholism is a complex behavioral disorder characterized by excessive consumption of ethanol, a narrowing of the behavioral repertoire toward excessive consumption, the development of tolerance and dependence, and impairment in social and occupational functioning. Animal models of the complete syndrome of alcoholism are difficult if not impossible to achieve, but validated animal models exist for many of the different components of the syndrome. Recent work has begun to define the neurocircuits responsible for the two major sources of reinforcement key to animal models of excessive ethanol intake: positive and negative reinforcement. Ethanol appears to interact with ethanol-sensitive elements within neuronal membranes that convey the specificity of neurochemical action. Ethanol reinforcement appears to be mediated by an activation of GABA-A receptors, release of opioid peptides, release of dopamine, inhibition of glutamate receptors, and interaction with serotonin systems. These neurocircuits may be altered by chronic ethanol administration as reflected by opposite effects during acute ethanol withdrawal and by the recruitment of other neurotransmitter systems such as the stress neuropeptide corticotropin-releasing factor. Future challenges will include a focus on understanding how these neuroadaptive changes convey vulnerability to relapse in animals with a history of ethanol dependence.

Chronic acamprosate eliminates the alcohol deprivation effect while having limited effects on baseline responding for ethanol in rats.

Acamprosate (calcium-acetyl homotaurinate) is a relatively new compound developed for the treatment of alcoholism and has been shown to be effective in attenuating relapse in human alcoholics. In the current study, the effects of this drug were further examined using an animal model of oral ethanol self-administration in a limited access paradigm. Male Wistar rats were trained to respond for ethanol (10% w/v) or water in a two-lever free-choice operant condition. Acute administration of acamprosate (400 mg/kg) reduced ethanol consumption and increased responding for water. Chronic administration of lower daily doses of acamprosate (100 and 200 mg/kg) blocked the increased ethanol consumption typically observed in rats after an imposed abstinence period. This effect of acamprosate was selective for ethanol, as responding for water was unaffected at any dose tested. These results with rats suggest a model by which to explore the mechanisms for anti-relapse effects of acamprosate.

Increased ethanol self-administration after a period of imposed ethanol deprivation in rats trained in a limited access paradigm.

A predominant feature in human alcohol abuse is the reported desire or "craving" to consume ethanol along with frequent episodes of drinking after periods of abstinence. These and other factors may be responsible for relapse to uncontrolled ethanol drinking. When relapse occurs after a period of abstinence, ethanol drinking has been shown to be temporarily increased. Two aspects of drug dependence could contribute to these increases. One may be the development of a need state; the other may involve changes in the perception of the positive reinforcing effects of ethanol when reinforcer access is limited. To investigate this phenomenon further, the present study was conducted to examine in nondependent rats the effect of forced time-off on oral ethanol self-administration in a limited access paradigm (30 min/day). Male Wistar rats were trained to respond for ethanol (10% w/v) or water in a two-lever, free-choice condition using a saccharin fading procedure. After the establishment of stable baseline responding for ethanol, various ethanol deprivation periods (3, 5, 7, 14, or 28 days) were imposed, during which no ethanol was available. Responding for ethanol increased as a function of the duration of the deprivation period when compared with baseline levels. This increase was temporary and returned to baseline levels within 2 to 3 days. Given that the shortest time-off period was 5 days and the rats showed no signs of withdrawal, this transient increase in ethanol responding does not seem to be related to the manifestation of dependence and withdrawal, and may be related to changes in ethanol's reinforcement properties. These results with rats may provide a useful tool to elucidate mechanisms underlying human alcohol seeking behavior and relapse.

Differences in the liability to self-administer intravenous cocaine between C57BL/6 x SJL and BALB/cByJ mice.

Application of animal models of psychostimulant abuse for experimentation in mice is becoming increasingly important for studying the contribution of genetic differences, as well as the roles of selected (targeted) genes, in specific behaviors. The purpose of this study was to investigate strain differences in cocaine self-administration behavior between C57BL/6 x SJL hybrid mice and BALB/cByJ mice. These two strains were chosen because BALB/cByJ mice have a well-developed behavioral pharmacological profile, and hybrid strains on a C57BL/6 background are commonly used for generating transgenic expressing and knockout mutant mice. C57BL/6 x SJL mice dose-dependently acquired cocaine self-administration (1.0 mg/kg/injection but not 0.25 mg/kg/injection) by responding selectively in the active nose-poke hole and maintaining stable levels of daily drug intake; they also exhibited a characteristic inverted-U-shaped cocaine dose-effect function. BALB/cByJ mice failed to acquire cocaine self-administration at either dose under the same test conditions. The strain differences observed in self-administration did not seem to be attributed to other behavioral differences because the two strains exhibited similar amounts of spontaneous nose-poking in the absence of reinforcers, and BALB/cByJ mice responded more than C57BL/6 x SJL mice in a food-reinforced nose-poke operant task. Importantly, the dose-effect function for the motor stimulating effects of cocaine (3.8-30 mg/kg intraperitoneally) suggests enhanced sensitivity but reduced efficacy of cocaine in stimulating motor activity in BALB/cByJ mice relative to the C57BL/6 x SJL hybrid mice. These results indicate that the decreased liability of BALB/cByJ mice to acquire cocaine self-administration is not the result of differences in spontaneous activity or performance, but may reflect different sensitivities to the reinforcing, or rate-disrupting, properties of cocaine. The data support an influence of genetic background in the liability to self-administer cocaine. Thus, a hypothesis is proposed that the decreased liability of BALB/cByJ mice to acquire cocaine self-administration is related to differences in brain monoamine systems linked to the high "emotionality" profile of BALB/c mice in novel or fearful situations, including perhaps cocaine administration.

The effects of cocaine on operant responding for food in several strains of mice.

The availability of numerous genetically homogenous mouse strains permits the analysis of genetic influences on behavior and also behavioral sensitivity (responsivity) to drugs of abuse. The current study was conducted to characterize discriminated operant responding for food in four inbred strains (Balb/cByJ, DBA/2J, C57BL/6J, SJL/J), an F1 Hybrid (C57BL/6xSJL), and one outbred strain (CD1) of mouse. The effect of cocaine on this operant behavior was also examined. Initially, all animals were trained to nosepoke for food on a continuous reinforcement schedule. The minimum response requirement for reinforcement was increased every 5 days until the animals were responding on an FR-15 schedule of reinforcement. All strains increased operant responding as the schedule of reinforcement was raised. However, significant differences in response rate and discrimination learning were observed among the various strains of mice. Cocaine administration reduced operant responding for food in Balb/cByJ, C57BL/6J, C57BL/6xSJL/J and CD1 mice at a dose of 15.0 mg/kg, whereas higher doses were required in DBA/2J mice (30.0 mg/kg) and SJL/J mice (56.0 mg/kg). These results suggest that operant performance and the effect of cocaine on this behavior is differentially influenced by genetic make-up.

Transgenic models to assess the pathogenic actions of cytokines in the central nervous system.

In order to better understand the actions of proinflammatory cytokines in the mammalian CNS, a transgenic approach was employed in which the expression of IL-6, IL-3 or TNF-alpha was targeted to astrocytes in the intact CNS of mice. Transgenic mice exhibited distinct chronic-progressive neurological disorders with neurodegeneration and cognitive decline due to IL-6 expression, macrophage/microglial-mediated primary demyelination with motor impairment due to IL-3 expression and lymphocytic meningoencephalomyelitis with paralysis induced by TNF-alpha expression. Thus, expression of specific cytokines alone in the intact CNS results in unique neuropathological alterations and functional impairments, thereby directly implicating these mediators in the pathogenesis of CNS disease.

Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain.

Inflammation with expression of interleukin 6 (IL-6) in the brain occurs in many neurodegenerative disorders. To better understand the role of IL-6 in such disorders, we examined performance in a learning task in conjunction with molecular and cellular neuropathology in transgenic mice that express IL-6 chronically from astrocytes in the brain. Transgenic mice exhibited dose- and age-related deficits in avoidance learning that closely corresponded with specific progressive neuropathological changes. These results establish a link between the central nervous system expression of IL-6, inflammatory neurodegeneration, and a learning impairment in transgenic mice. They suggest a critical role for a proinflammatory cytokine in the cognitive deficits and associated neuroinflammatory changes that have been documented in neurodegenerative diseases such as Alzheimer disease and AIDS.

Opiate withdrawal signs precipitated by naloxone following a single exposure to morphine: potentiation with a second morphine exposure.

Recent studies in humans with no prior history of opiate abuse indicated that naloxone-precipitated signs of opiate withdrawal could be observed after a single exposure to morphine, and that the severity of withdrawal was enhanced following a second morphine exposure 24 h later. The current study was conducted to establish a paradigm in rodents that resembled these conditions described in humans. To that end, naloxone-precipitated (0.03-3.0 mg/kg) suppression of operant response rates and somatic signs of withdrawal following single or repeated treatments with morphine (5.0 mg/kg) were assessed in previously opiate-naive rats. In one group of rats, naloxone was administered 4 h after both the first and second morphine pretreatment, while in a separate group of rats naloxone was administered 4 h after the second morphine pretreatment only. A single morphine pretreatment significantly increased naloxone's potency to suppress operant response rates, and resulted in the precipitation by naloxone of certain somatic signs of withdrawal. The effects of naloxone on both dependent measures (operant response rates and somatic signs) were potentiated following a second morphine pretreatment, regardless of whether naloxone was administered following both morphine exposures or only following the second morphine exposure. Thus, repeated morphine administration appears to be the critical factor underlying the progressive increase in antagonist potency, whereas prior experience with naloxone is not a necessary factor. The results provide additional support for the hypothesis that the development of dependence on opiates is a progressive phenomenon that may begin with a single dosing.