Start-of-day oculomotor screening demonstrates the effects of fatigue and rest during a total immersion training program.

BACKGROUND: Investigating changes in sleep and fatigue metrics during intensive surgical and trauma skills training, this study explored the dynamic association between oculomotor metrics and fatigue. Specifically, alterations in these relations over extended stress exposure, the influence of time of day, and the impact of fatigue exposure on sleep metrics were examined. METHODS: Thirty-nine military medical students participated in 6 days of immersion, hyper-realistic, and high-stress experiential casualty training. Participants completed surveys assessing the state of sleepiness with oculomotor tests performed each morning and evening, analyzing eye movement and pupillary change to characterize fatigue. Participants wore Fitbit TM devices to measure overall time asleep and time in each sleep stage during the training. RESULTS: Fitbit data showed increased average minutes in rapid eye movement, deep sleep, and less time in light sleep from day 1 to day 4. The microsaccade peak velocity-to-displacement ratio exhibited a morning decrease but not in afternoon sessions, indicating repeated but temporary effects of accumulated fatigue. There were no findings regarding pupil reactivity to illumination changes. CONCLUSION: This study describes characteristics of fatigue measured by rapid and individually calibrated oculomotor tests. It demonstrates oculomotor relationships to fatigue in start-of-day testing, providing a direction for timing for optimal fatigue testing. These data suggest that improved sleep could signal resilience to fatigue during afternoon testing. Further investigation with more participants and longer duration is warranted. A deeper understanding of the interrelationships between training, sleep, and fatigue could improve surgical and military fitness.

Compulsive alcohol seeking and relapse: Central role of conditioning factors associated with alleviation of withdrawal states by alcohol.

BACKGROUND AND PURPOSE: Learned associations between environmental stimuli and drugs of abuse represent a major factor in the chronically relapsing nature of drug addiction. In drug dependent subjects these associations must be presumed to include associations linked to reversal of adverse withdrawal states by drug use-"withdrawal-associated learning" (WDL). However, their significance in drug seeking has received little experimental scrutiny. EXPERIMENTAL APPROACH: Using alcohol as a drug of abuse, the behavioural consequences of WDL were investigated in animal models of relapse and compulsive drug seeking by comparing the effects of WD L-associated stimuli versus stimuli associated with alcohol without WDL experience in nondependent and post-dependent rats. Brain sites activated by exposure to the respective stimuli were identified by c-fos immunohistochemistry. KEY RESULTS: (1) WDL-associated stimuli elicited significant alcohol seeking. In rats with WDL experience, stimuli associated with alcohol in the nondependent state no longer elicited robust alcohol seeking. (2) Responding elicited by WDL-associated stimuli, but not stimuli conditioned to alcohol in the nondependent state, was resistant to footshock punishment and increased response effort requirements for presentation of WDL-related stimuli. (3) Stimuli conditioned to alcohol in rats with a dependence but not WDL history did not sustain punished responding or tolerance of increased effort. (4) The central nucleus of the amygdala was identified as a site selectively responsive to WDL stimulus exposure. CONCLUSION AND IMPLICATIONS: Environmental stimuli associated with reversal of adverse withdrawal states by alcohol elicit compulsive-like alcohol seeking and establish WDL as a major, not well-recognized factor, in relapse vulnerability.

Sensitization to the motor stimulant effects of 3,4-methylenedioxypyrovalerone (MDPV) and cross-sensitization to methamphetamine in rats.

BACKGROUND: In recent years, there has been a dramatic increase in abuse of the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV), often in combination with other illicit stimulants. PURPOSE: We sought to determine if repeated exposure to MDPV would produce sensitization to the motor stimulant effects of the drug, and whether cross-sensitization would develop with the stimulant effects of methamphetamine (METH). STUDY DESIGN: Male Sprague-Dawley rats were administered MDPV (1 or 5 mg/kg) or saline once daily for 5 days at 24 hour intervals, or were administered MDPV (1 mg/kg) or saline once daily for 5 days at 48 hour intervals. For cross-sensitization experiments, rats were administered METH (1 mg/kg) or MDPV (1 or 5 mg/kg) once daily for 5 days at 48 hour intervals, and following a 5 day incubation period, were given an acute challenge injection of either MDPV (0.5 mg/kg) or METH (0.5 mg/kg), respectively. RESULTS: Rats repeatedly administered MDPV (1 mg/kg) every 48 hours, but not every 24 hours, demonstrated increased motor activity when given either a subsequent challenge of MDPV (0.5 mg/kg i.p.) or METH (0.5 mg/kg), indicating the development of behavioral sensitization and cross-sensitization, respectively. Moreover, rats repeatedly administered METH (1 mg/kg) every 48 hours did not exhibit cross-sensitization to the motor stimulating effects of a subsequent challenge with MDPV (0.5 mg/kg). CONCLUSION: These results suggest that specific patterns of MDPV administration may lead to lasting changes in behavioral responses to subsequent METH exposure.

Region-specific effects of isoflurane anesthesia on Fos immunoreactivity in response to intravenous cocaine challenge in rats with a history of repeated cocaine administration.

We have previously shown that acute intravenous (i.v.) administration of cocaine increases Fos immunoreactivity in rats under isoflurane anesthesia. Given that Fos expression is a marker of neural activation, the results suggested that isoflurane is appropriate for imaging cocaine effects under anesthesia. However, most imaging research in this area utilizes subjects with a history of repeated cocaine exposure and this drug history may interact with anesthetic use differently from acute cocaine exposure. Thus, this study further examined Fos expression under isoflurane in rats with a history of repeated i.v. cocaine administration. Rats received daily injections of either saline or cocaine (2mg/kg, i.v.) across 7 consecutive days, followed by 5 days of no drug exposure. On the test day, rats were either nonanesthetized or anesthetized under isoflurane and were given an acute challenge of cocaine (2mg/kg, i.v.). Additional saline-exposed controls received a saline challenge. Ninety min after the drug challenge, the rats were perfused under isoflurane anesthesia and their brains were processed for Fos protein immunohistochemistry. We found that challenge injections of cocaine following a regimen of repeated cocaine exposure resulted in Fos expression in the prefrontal cortex and striatum roughly equivalent to that found in rats who had received the cocaine challenge after a history of vehicle injections. Additionally, isoflurane anesthesia resulted in a heterogeneous attenuation of cocaine-induced Fos expression, with the most robust effect in the orbital cortex but no effect in the nucleus accumbens core (NAcC). These results indicate that cocaine-induced Fos is preserved in the NAcC under isoflurane, suggesting that isoflurane can be used in imaging studies involving cocaine effects in this region.

The development of acamprosate as a treatment against alcohol relapse.

INTRODUCTION: Globally, alcohol abuse and dependence are significant contributors to chronic disease and injury and are responsible for nearly 4% of all deaths annually. Acamprosate (Campral), one of only three pharmacological treatments approved for the treatment of alcohol dependence, has shown mixed efficacy in clinical trials in maintaining abstinence of detoxified alcoholics since studies began in the 1980s. Yielding inconsistent results, these studies have prompted skepticism. AREAS COVERED: Herein, the authors review the preclinical studies which have assessed the efficacy of acamprosate in various animal models of alcohol dependence and discuss the disparate findings from the major clinical trials. Moreover, the authors discuss the major limitations of these preclinical and clinical studies and offer explanations for the often-contradictory findings. The article also looks at the importance of the calcium moiety that accompanies the salt form of acamprosate and its relevance to its activity. EXPERT OPINION: The recent discovery that large doses of calcium largely duplicate the effects of acamprosate in animal models has introduced a serious challenge to the widely held functional association between this drug and the glutamate neurotransmission system. Future research on acamprosate or newer pharmacotherapeutics should consider assessing plasma and/or brain levels of calcium as a correlate or mediating factor in anti-relapse efficacy. Further, preclinical research on acamprosate has thus far lacked animal models of chemical dependence on alcohol, and the testing of rodents with histories of alcohol intoxication and withdrawal is suggested.

Potent rewarding and reinforcing effects of the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV).

Reports of abuse and toxic effects of synthetic cathinones, frequently sold as 'bath salts' or 'legal highs', have increased dramatically in recent years. One of the most widely used synthetic cathinones is 3,4-methylenedioxypyrovalerone (MDPV). The current study evaluated the abuse potential of MDPV by assessing its ability to support intravenous self-administration and to lower thresholds for intracranial self-stimulation (ICSS) in rats. In the first experiment, the rats were trained to intravenously self-administer MDPV in daily 2-hour sessions for 10 days at doses of 0.05, 0.1 or 0.2 mg/kg per infusion. The rats were then allowed to self-administer MDPV under a progressive ratio (PR) schedule of reinforcement. Next, the rats self-administered MDPV for an additional 10 days under short access (ShA; 2 hours/day) or long access (LgA; 6 hours/day) conditions to assess escalation of intake. A separate group of rats underwent the same procedures, with the exception of self-administering methamphetamine (0.05 mg/kg per infusion) instead of MDPV. In the second experiment, the effects of MDPV on ICSS thresholds following acute administration (0.1, 0.5, 1 and 2 mg/kg, i.p.) were assessed. MDPV maintained self-administration across all doses tested. A positive relationship between MDPV dose and breakpoints for reinforcement under PR conditions was observed. LgA conditions led to escalation of drug intake at 0.1 and 0.2 mg/kg doses, and rats self-administering methamphetamine showed similar patterns of escalation. Finally, MDPV significantly lowered ICSS thresholds at all doses tested. Together, these findings indicate that MDPV has reinforcing properties and activates brain reward circuitry, suggesting a potential for abuse and addiction in humans.

Positive or negative allosteric modulation of metabotropic glutamate receptor 5 (mGluR5) does not alter expression of behavioral sensitization to methamphetamine.

We investigated the role of metabotropic glutamate receptor type 5 (mGluR5) in methamphetamine-induced behavioral sensitization. The mGluR5 positive allosteric modulator (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) and negative allosteric modulator fenobam were tested in separate experiments. Sprague-Dawley rats were repeatedly injected with 1 mg/kg methamphetamine or saline, and then given a locomotor challenge test using a dose of 0.5 mg/kg methamphetamine. Prior to the challenge test session, rats were injected with CDPPB, fenobam, or a vehicle.  Doses from previous studies showed reduced drug-conditioned behavior; however in this study neither CDPPB nor fenobam pretreatment resulted in an altered expression of behavioral sensitization, indicating a lack of mGluR5 involvement in sensitized methamphetamine-induced locomotion. Additionally, the high dose (30 mg/kg) of fenobam resulted in decreased methamphetamine-induced locomotion in rats regardless of drug exposure history, which suggests evidence of nonspecific behavioral inhibition.

Attenuation of methamphetamine seeking by the mGluR2/3 agonist LY379268 in rats with histories of restricted and escalated self-administration.

Recent findings implicate group II metabotropic glutamate receptors (mGluR(2/3)) in the reinforcing effects of psychostimulants and have identified these receptors as potential treatment targets for drug addiction. Here, we investigated the effects of mGluR(2/3) stimulation on cue- and drug-primed reinstatement in rats with different histories of methamphetamine (METH) self-administration training, under two conditions: 16 daily sessions of short access (90 min/day, ShA), or 8 daily sessions of short access followed by 8 sessions of long access (6 h/day, LgA). Following self-administration and subsequent extinction training, rats were pretreated with the selective mGluR(2/3) agonist LY379268 (variable dose, 0-3 mg/kg), exposed to METH-paired cues or a priming injection of METH (1 mg/kg), and tested for reinstatement of METH-seeking behavior. LgA rats self-administered greater amounts of METH during the second half of training, but when pretreated with vehicle, ShA and LgA rats showed cue- and drug-primed reinstatement at equivalent response rates. However, LgA rats demonstrated greater sensitivity to mGluR(2/3) stimulation with attenuated responding during cue-induced reinstatement after 0.3 mg/kg and higher doses of LY379268, whereas ShA rats decreased cue-induced reinstatement behavior following 1.0 mg/kg and 3.0 mg/kg LY379268. Additionally, both LgA and ShA rats exhibited decreased METH-primed reinstatement behavior following 0.3 mg/kg and higher doses of LY379268. A separate group of control rats was trained to self-administer sucrose pellets, and demonstrated attenuated cue-induced sucrose-seeking behavior following 1.0 and 3.0 mg/kg LY379268. Together, the results indicate that LY379268 has differential attenuating effects on cue-induced reinstatement behavior in rats with different histories of METH intake. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Attenuation of reinstatement of methamphetamine-, sucrose-, and food-seeking behavior in rats by fenobam, a metabotropic glutamate receptor 5 negative allosteric modulator.

RATIONALE: Methamphetamine (METH) is a highly potent and addictive psychostimulant with severe detrimental effects to the health of users. Currently, METH addiction is treated with a combination of cognitive and behavioral therapies, but these traditional approaches suffer from high relapse rates. Furthermore, there are currently no pharmacological treatment interventions approved by the FDA specifically for the treatment of METH addiction. OBJECTIVES: Metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators (NAMs) have shown promise in significantly attenuating drug self-administration and drug-seeking in reinstatement paradigms. However, studies assessing the potential efficacy of mGluR5 NAMs that have been tested in human subjects are lacking. The current study sought to assess the effect of the mGluR5 NAM fenobam on METH-seeking behavior. METHODS: Rats were trained to self-administer METH (0.05 mg/kg i.v.), and following extinction, tested for effects of fenobam (5, 10, or 15 mg/kg intraperitoneal) on cue- and drug-induced reinstatement of METH-seeking. To determine if fenobam also alters reinstatement of seeking of natural reinforcers, separate groups of rats were trained to self-administer sucrose or food pellets and were tested for the effects of fenobam on cue-induced reinstatement of sucrose- and food-seeking. RESULTS: Fenobam attenuated drug- and cue-induced reinstatement of METH-seeking behavior at doses of 10 and 15 mg/kg. Fenobam also attenuated cue-induced reinstatement of sucrose- and food-seeking at all doses tested. CONCLUSIONS: The mGluR5 NAM fenobam attenuates the reinstatement of METH-seeking behavior, but these effects may be due to nonspecific suppression of general appetitive behaviors.

Positive Allosteric Modulation of mGluR5 Accelerates Extinction Learning but Not Relearning Following Methamphetamine Self-Administration.

Recent studies have implicated glutamate neurotransmission as an important substrate for the extinction of conditioned behaviors, including responding for drug reinforcement. Positive allosteric modulation of the type-5 metabotropic glutamate receptor (mGluR5) in particular has emerged as a treatment strategy for the enhancement of extinction of drug-motivated behaviors. Here, we investigated the effects of the mGluR5 positive allosteric modulator CDPPB, a compound known for its cognitive enhancing effects in rodents, on extinction learning in rats with different histories of methamphetamine (METH) training. Rats were trained to self-administer METH under two conditions: 16 daily sessions of short access (90 min/day, ShA), or eight daily sessions of short access followed by eight sessions of long access (6 h/day, LgA). Control rats self-administered sucrose pellets in daily 30 min sessions. Next, rats were administered vehicle or 30 mg/kg CDPPB prior to seven consecutive daily extinction sessions, subjected to additional extinction sessions to re-establish a post-treatment baseline, and then tested for reinstatement of behavior in the presence of METH- or sucrose-paired cues. Rats were then subjected to a second series of extinction sessions, preceded by vehicle or 30 mg/kg CDPPB, and an additional test for cue-triggered reinstatement. CDPPB treatment resulted in a more rapid extinction of responding on the active lever, especially in the early sessions of the first extinction sequence. However, treatment effects were minimal during subsequent cue reinstatement tests and non-existent during the second series of extinction sessions. Rats with histories of ShA, LgA, and sucrose training expressed similar behavioral sensitivities to CDPPB, with LgA rats demonstrating a modestly higher treatment effect. Positive allosteric modulation of mGluR5 may therefore have some beneficial effects on efforts to facilitate extinction learning and reduce methamphetamine seeking.

Novel cues reinstate cocaine-seeking behavior and induce Fos protein expression as effectively as conditioned cues.

Cue reinstatement of extinguished cocaine-seeking behavior is a widely used model of cue-elicited craving in abstinent human addicts. This study examined Fos protein expression in response to cocaine cues or to novel cues as a control for activation produced by test novelty. Rats were trained to self-administer cocaine paired with either a light or a tone cue, or received yoked saline and cue presentations, and then underwent daily extinction training. They were then tested for reinstatement of extinguished cocaine-seeking behavior elicited by response-contingent presentations of either the cocaine-paired cue or a novel cue (that is, tone for those trained with a light or vice versa). Surprisingly, conditioned and novel cues both reinstated responding and increased Fos similarly in most brain regions. Exceptions included the anterior cingulate, which was sensitive to test cue modality in saline controls and the dorsomedial caudate-putamen, where Fos was correlated with responding in the novel, but not conditioned, cue groups. In subsequent experiments, we observed a similar pattern of reinstatement in rats trained and tested for sucrose-seeking behavior, whereas rats trained and tested with the cues only reinstated to a novel, and not a familiar, light or tone. The results suggest that novel cues reinstate responding to a similar extent as conditioned cues regardless of whether animals have a reinforcement history with cocaine or sucrose, and that both types of cues activate similar brain circuits. Several explanations as to why converging processes may drive drug and novel cue reinforcement and seeking behavior are discussed.

Enhanced sensitivity to attenuation of conditioned reinstatement by the mGluR 2/3 agonist LY379268 and increased functional activity of mGluR 2/3 in rats with a history of ethanol dependence.

Recent findings implicate group II metabotropic glutamate receptors (mGluR(2/3)) in the reinforcing and dependence-inducing actions of ethanol and identify these receptors as treatment targets for alcoholism. Here, we investigated the effects of mGLuR(2/3) activation on conditioned reinstatement in rats with different ethanol-dependence histories and examined dependence-associated changes in the functional activity of mGluR(2/3). Following ethanol self-administration training and conditioning procedures, rats were made ethanol dependent, using ethanol vapor inhalation, under three conditions: a single intoxication and withdrawal episode (SW), repeated cycles of intoxication and withdrawal (RW), or no intoxication (CTRL). At 1 week after removal from ethanol vapor, self-administration resumed until stable baseline performance was reached, followed by extinction of operant responding and reinstatement tests. Post-withdrawal self-administration was increased in the RW group, but all groups showed conditioned reinstatement. The mGluR(2/3) agonist LY379268 dose -dependently reduced reinstatement in all groups, but was more effective at low doses in the SW and RW groups. The highest dose of LY379268 tested reduced spontaneous locomotor activity and operant responding maintained by a non-drug reinforcer, without differences among groups. The heightened sensitivity to the effects of LY379268 in rats with an ethanol-dependence history was therefore specific to behavior motivated by ethanol-related stimuli. Both the SW and RW groups showed elevated [(35)S]GTPγS binding in the central nucleus of the amygdala (CeA) and bed nucleus of stria terminalis (BNST), relative to the CTRL group. The findings implicate changes in mGluR(2/3) functional activity as a factor in ethanol dependence and support treatment target potential of mGlu(2/3) receptors for craving and relapse prevention.

Investigating Methamphetamine Craving Using the Extinction-Reinstatement Model in the Rat.

Like all other drugs of abuse, the primary therapeutic objective for treating methamphetamine addiction research is the maintenance of abstinence and prevention of relapse to habitual drug-taking. Compounds with the potential to prevent relapse are often investigated in rats that are trained to self-administer intravenous methamphetamine, subjected to extinction training where responding is no longer reinforced, and then given tests for reinstatement of drug-seeking behavior triggered by methamphetamine injections or re-exposure to drug-paired cues. Experimental compounds are administered to the animals prior to the reinstatement tests to evaluate their potential for attenuating or preventing drug-seeking behavior. This article describes the common procedures of the extinction-reinstatement model in studies of this type, and identifies areas of discrepancy. This is followed by a comprehensive overview of the currently published anti-reinstatement effects of pharmacological compounds, classified by the most relevant neurological systems associated with these compounds. The article concludes with a brief discussion of how the study of anti-reinstatement effects can be expanded to further verify existing positive results or to find novel neurobiological targets.

c-Fos expression associated with reinstatement of cocaine-seeking behavior by response-contingent conditioned cues.

The capability of cocaine cues to generate craving in cocaine-dependent humans, even after extended abstinence, is modeled in rats using cue reinstatement of extinguished cocaine-seeking behavior. We investigated neural activity associated with incentive motivational effects of cocaine cues using c-fos mRNA and Fos protein expression as markers. Unlike preceding studies, we used response-contingent presentation of discrete cues to elicit cocaine seeking. Rats were first trained to press a lever, resulting in cocaine reinforcement and light and tone cues. Rats then underwent extinction training, during which lever presses decreased. On the test day, rats either received response-contingent cocaine cues or received no cues. The cues reinstated extinguished cocaine-seeking behavior on the test day. In general, cue-elicited c-fos mRNA and protein expression were similar and both were enhanced in the prefrontal cortex, ventral tegmental area (VTA), dorsal striatum, and nucleus accumbens. Cues elicited more widespread Fos protein expression relative to our previous research in which cues were presented noncontingently without prior extinction training, including increases in the VTA, substantia nigra, ventral subiculum, and lateral entorhinal cortex. We also observed a correlation between cocaine-seeking behavior and Fos in the agranular insula (AgI) and basolateral amygdala (BLA). The findings suggest that connections between BLA and AgI play a role in cue-elicited incentive motivation for cocaine and that reinstatement of cocaine seeking by response-contingent cues activates a similar corticolimbic circuit as that observed with other modes of cue presentation; however, activation of midbrain and ventral hippocampal regions may be unique to reinstatement by response-contingent cues.

Cocaine-induced Fos expression is detectable in the frontal cortex and striatum of rats under isoflurane but not alpha-chloralose anesthesia: implications for FMRI.

The ability of intravenous cocaine to induce Fos protein expression in anesthetized rats was tested. Two anesthetic regimens commonly used for in vivo FMRI of animals, i.v. alpha-chloralose and gaseous isoflurane, were studied in separate cohorts. The first experiment included three groups that received the following treatments: saline i.v. and no anesthetic; 2 mg/kg cocaine i.v. and no anesthetic; and 2mg/kg cocaine i.v. under 36 mg/kg/h alpha-chloralose anesthesia. The second experiment had a factorial design of four groups that were either nonanesthetized or isoflurane-treated and were either given saline or cocaine (2 mg/kg, i.v.). Anesthetized rats were maintained for 2 h under 2.5-3.5% isoflurane anesthesia, while nonanesthetized rats were kept in an alternative environment for the same time period. Rats were given 2 mg/kg cocaine or saline i.v., 30 min into the test session. Rats were perfused and their brains were processed for Fos immunohistochemistry 90 min after the i.v. treatment. In both experiments, the frontal cortex and striatum of the cocaine-treated nonanesthetized rats expressed Fos in greater amounts than the saline-treated nonanesthetized rats, as expected. The alpha-chloralose treatment prevented cocaine-induced Fos expression across all eight subregions of the striatum and frontal cortex that were examined. In contrast, isoflurane only partially attenuated Fos expression in the orbital and Cg2 subregions of frontal cortex. These results suggest a strong advantage for using isoflurane, as opposed to alpha-chloralose, when studying anesthetized rats for in vivo effects of psychostimulants.

Neural responses to acute cocaine administration in the human brain detected by fMRI.

An improved functional MRI (fMRI) method for the reduction of susceptibility artifacts has been utilized to measure blood oxygen level-dependent (BOLD) responses to acute cocaine administration in the human brain of cocaine users. Intravenous administration of cocaine (20 mg/70 kg) activated mesolimbic and mesocortical dopaminergic projection regions and showed temporal positive or negative BOLD responses. These results obtained from human cocaine users supported the involvement of the dopaminergic pathway in cocaine addiction from animal models. In addition, the cocaine administration also induced activations in the hierarchical brain networks in the anterior prefrontal cortex (aPFC) of the Brodmann area 10 (BA10) and orbitofrontal cortex (OFC). It is suggested that the dopaminergic pathways and the hierarchical brain networks may participate in mediating cocaine reward processes, associative learning, motivation, and memory in cocaine addiction in the human brain.