Research Findings - Basic Behavioral Research
Cue-Induced Craving in Smokers: Real-time Monitoring in Naturalistic Settings
Laboratory investigations have documented self-reported craving when individuals are exposed to stimuli previously paired with drugs. Cue-induced cravings have been the target of extinction-based therapies but these treatment approaches show limited effectiveness in a laboratory setting. NIDA supported researchers are working to develop cues that better resemble those addicts encounter in the real world (for examples, personalized or social stimuli). Another approach has been to deliver drug-related cues in a real world setting using remote technology. Drs. Stephen Tiffany and Matthew Warthen recently completed a study in heavy smokers, wherein cues were provided via hand-held personal digital assistants (PDAs). Using a newly developed "CREMA" procedure, the investigators couple cue reactivity (CR) with ecological momentary assessment (EMA) to both deliver cues and record real-time subject reactions (such as mood rating, cravings, and latency to smoke). Parallel studies were conducted with the same subjects in a laboratory setting, comparing cue presentation of smoking-related cues versus non-smoking cues. Cues were both visual – photographs – and imagery in the form of written scripts (for subjects to read and then actively imagine). Over an 8-day study period, subjects recorded responses for 24 h/day, including cigarettes smoked and reactions to cue presentations on a subscale from the Questionnaire of Smoking Urges and mood questions. Subjects were prompted for CREMA sessions four times per day during a 12 h window that was set by the participant. Results reveal completion of 29.07 (average) of 32 CREMA trials in 43 smokers. Post-cue craving ratings were significantly greater following smoking stimuli than non-smoking stimuli, (as has been reliably demonstrated in previous laboratory studies). Craving ratings were also significantly higher following imagery than following photographs. Similar data were derived from analogous laboratory trials given before and after the CREMA procedure. Overall this study supports the viability of using experimental CR procedures in naturalistic environments that allow data collection for context, social and individual subject variables. Future studies might target these variables in interventions that can be delivered along with experimental data collection in real time. Tiffany ST, Warthen MW. Evaluation of cue reactivity in the natural environment of smokers using ecological momentary assessment. Exper Clin Psychopharmacol. 2009;17(2):70-77.
Naltrexone Attenuates the Incentive Motivational Effects of Nicotine-Associated Cues
While the direct reinforcing effect of nicotine (NIC) contributes to human smoking, environmental stimuli paired with this drug effect and smoking behavior are potent motivators for continued use and relapse. Animal models can mimic these relapse effects by demonstrating that NIC-associated cues presented after extinction prompt drug-seeking behavior in an i.v. self-adminstration paradigm. This, and many other studies, suggest that NIC associated cues acquire incentive motivational properties. It is of interest to determine the neurobiological mechanisms through which these incentive cues activate behavior. Endogenous opioid systems have been implicated in the conditioned incentive effects of environmental stimuli associated with a number of drugs of abuse. Conditioned place preference with NIC is blocked in preproenkephalin knock-out mice and in those with reduced expression of opioid receptors in the ventral tegmental area – the location of dopamine cells important in central reinforcing effects of drugs of abuse. In fact, endogenous opioid systems modulate central dopaminergic activity. However, inconsistent effects have been reported with the opiate antagonist, naltrexone, in smoking cessation efforts, and pre-treatment with this antagonist is without effect on i.v. NIC self-administration in animal studies. Recently Dr. Xiu Liu and his colleagues at the University of Pittsburgh assessed naltrexone effects on (1) NIC's direct reinforcing properties, and (2) incentive motivational effects of cues paired with NIC. In this study, rats self-administered i.v. NIC with paired presentation of a tone+light (conditioned stimuli) over 30 sessions. For one group (cue-induced reinstatement) operant responding was then extinguished by placing rats in the experimental chambers without NIC-associated stimuli, and delivering saline instead of NIC for each bar press. Then, reinstatement tests were conducted wherein the conditioned stimuli were re-introduced and operant responses elicited by these stimuli were measured, again in the absence of NIC delivery. Half of these animals were pretreated with naltrexone before reinstatement, and half received saline instead. For the second group (cue-maintained lever pressing), responding during extinction resulted in the presentation of conditioned stimuli (also with saline infusions instead of NIC). Thus, for this group, the conditioned stimuli reinforced operant responding during extinction. Animals in this second group were also divided into those receiving pretreatment with naltrexone during extinction, and controls who received saline. Additional control groups were tested to examine the effects of acute and chronic naltrexone on operant responding for i.v. NIC. Group comparisons revealed that naltrexone dose dependently blocked cue-induced reinstatement in group one, and suppressed cue-maintained lever responding during extinction in group two. By contrast, the same doses of naltrexone that blocked cue-induced reinstatement and cue-maintained operant responding were without effect on operant responding for NIC, whether naltrexone was given acutely or chronically for seven days. Taken together, these findings suggest that endogenous opioid systems may have an important role in the incentive motivational properties of NIC-associated cues and that these systems may be a target for interventions directed at associative influences on smoking behavior. Xiu L, Palmatier MI, Caggiula AR, Sved AF, Donny EC, Gharib M, Booth S. Naltrexone attenuation of conditioned but not primary reinforcement of nicotine in rats. Psychopharmacol. 2009; 202:589-598.
Different Neurobiological Substrates for Aversive Versus Reinforcing Effects of Cocaine
Cocaine has strong reinforcing properties and also induces anxiogenic effects that can be measured in an animal behavioral assay. In Dr. Aaron Ettenberg's runway procedure, rats run along a straight alley (in a non-drugged state) to receive an i.v. cocaine infusion in the goal box at the end. In this paradigm, "retreats" away from the goal box provide a quantitative measure of the aversive properties of the drug. Thus, the procedure provides a model of the approach-avoidance conflict that typifies human drug seeking. Retreats have also been measured in rats trained to run for intracerebroventricular cocaine infusions, and Dr. Ettenberg is interested in determining the neurobiological circuits responsible for the drug's aversive properties. While prior research has indicated importance of the mesocorticolimbic dopamine system (including the prefrontal cortex, PFC) in cocaine's rewarding effects, it is not known if aversive properties arise from this, or a different, brain system. In a recent study, rats were trained to run for bilateral cocaine infusion into the medial PFC. Training began with an acclimation phase with the goal box closed. On the following 15 daily trials rats were placed in the start box and a guillotine door to the goal box was opened. Movement through the runway was recorded and bilateral cocaine infusions, (three doses or vehicle, administered to separate groups), were delivered upon entry into the goal area. Rats were removed 5-min after cocaine delivery. Dependent measures included run times and retreats, providing behavioral assessments of reinforcing and aversive drug effects, respectively. The investigator found that all doses of cocaine were associated with faster running speeds in the apparatus, as has previously been observed with i.v. cocaine delivery. However, unlike previous studies with this procedure, mPFC cocaine infusions were not associated with retreats. Since these behaviors are measured prior to drug delivery (in the goal box), they are believed to index the animal's memory for the drug effects – positive or negative. The findings indicate that while PFC is an important substrate for positive effects, aversive properties of this drug arise from neurotransmitter activation in some other part of the central dopamine system. Guzman D, Moscarello JM, Ettenberg A. The effects of medial prefrontal cortex infusions of cocaine in a runway model of drug self-administration: Evidence of reinforcing but not anxiogenic actions. Eur J Pharmacol. 2009; 605:117-122.
Drug-Conditioned Place Preference in Human Subjects
Animal behavioral models that mimic human drug abuse include those that directly measure reinforcing properties of drugs (such as i.v. self-administration) and those that measure a drug's ability to impart reinforcing properties to stimuli associated with the drug's effects. The latter can be assessed using conditioned place preference procedures (CPP) where rats are placed in a particular environment, repeatedly after drug administration, so that the drug-induced subjective experience becomes associated with discrete stimuli in that environment. Subsequently, on tests of CPP expression, animals will spend more time on the drug-associated side of a test chamber, than a side previously paired with only saline, suggesting these drug- related-stimuli elicit approach behavior by virtue of their acquired motivational properties. Investigators infer that the drug induces a positive subjective state, much like those reported when human subjects are queried for their subjective ratings of drug effects in a laboratory. However, there has yet to be an empirical demonstration of CPP in human subjects. Drs. Emma Childs and Harriet de Wit undertook a study to establish amphetamine induced CPP with 20 mg oral d-amphetamine in healthy, adult volunteers. The study involved two pairings with drug and a test room, and two pairings of placebo with a different test room. Each pairing lasted four hours, during which time subjective drug effects (Profile of Mood States, the Addiction Research Centre Inventory – ARCI, and Drug Effects Questionnaire) were measured. A separate control group experienced drug and placebo in both rooms (unpaired condition). During the test for CPP, rather than assess subject's choice of room, subjects completed a room preference questionnaire while in a new setting. Results showed a significantly higher rating for the drug-associated room in the paired condition (i.e., amphetamine-paired-room only), suggesting establishment of a CPP. Subsequent regression analysis revealed that room liking negatively correlated with POMS anxiety; and that overall, POMS anxiety, "drug liking", the hallucinogen-like scale of the ARCI, and positive mood, accounted for 43% of the variance in room liking. Thus, there was an orderly relationship between the CPP and subjective effects of d-amphetamine; thus, both negative subjective effects and drug liking contributed to the establishment of the CPP to amphetamine. This study validates a common procedure used to study drug effects in animal models and supports the underlying assumption that CPP expression can be related to subjective effects. Childs E, de Wit H. Amphetamine-induced place preference in humans. Biol Psychiat. 2009; 65:900-904.
New Mouse Self-Administration Procedure for Genetics Studies in Nicotine Addiction
Supported in part by a NIDA grant to Dr. Rafael Maldonado, in Barcelona, Spain, a new procedure has been developed to establish i.v. nicotine (NIC) self-administration in C57BL/6 mice. The investigators note that nicotine intake is difficult to establish in laboratory animals and that previously there has only been one demonstration of nicotine relapse using laboratory models. Human smokers relapse during abstinence for a number of difference reasons, and these paths to relapse can be modeled in animal 'reinstatement' paradigms. In these paradigms, animals are extinguished from i.v. self-administration and then, later, given a drug-associated cue, with a priming dose of the drug, or with a stressor. Under all three conditions, animals show a reinstatement of drug seeking behavior, resembling the human condition of relapse. In the Maldonado study, mice make nose-poke responses for i.v. NIC (0.03mg/kg/infusion), paired with pump noise and cue lights. After stable responding was established, extinction was conducted without cues, and saline was delivered instead of NIC. During reinstatement, nose pokes were measured in response to cue presentation, 0.18 mg/kg s.c. NIC, or a mild foot shock stressor, in different groups of subjects. Cue induced reinstatement was robust, and observed in 90% of the animals, whereas only 30% reinstated following the priming dose of NIC. Stress with mild shock induced drug-seeking in 50% of the animals. These results appear to mimic the powerful effects of conditioned stimuli in human relapse to smoking, even after prolonged abstinence. Martin-Garcia E, Barbano MF, Galeote L, Maldonado R. New operant model of nicotine-seeking behaviour in mice. Int J Neuropsychopharm. 2009;12:343-356. Moreover, establishment of this procedure allowed the researchers to investigate the genetic basis of NIC reinforcement using a mouse model. Morphine and opioid peptides derived from pre-proenkephalin (which bind to mu opioid receptors) have been demonstrated to participate in NIC reward and in NIC withdrawal symptoms. However, the dynorpyhin/kappa-opioid receptor system is also implicated in the reinforcing effects of several drugs of abuse, and activating this system decreases drug reward. Dr. Maldonado and colleagues trained wild-type, and prodynorpyhin knock-out mice, for NIC induced conditioned place preference, and for i.v. self-administration of NIC. They also measured NIC induced locomotion, antinociception and precipitated withdrawal. Knock-out mice were no different on any measures, with the exception of i.v. NIC self-administration: knock-out mice self-administered doses that were sub-threshold for wild-type animals (5.2 ug/kg/infusion) and would not take higher doses that were readily self-infused by the wild type group (21.3 to 85.5 ug/kg/infusion), indicating a shift to the left in the dose-response curve. This is the first study to reveal opioid peptide modulation of nicotine intake, possibly via the mediation of NIC's aversive effects, and suggests that NIC's subjective properties can be separated neurochemically from psychomotor stimulation, associative effects, and ability to induce physical dependence and antinociception. Galeote L, Berrendero F, Bura SA, Zimmer A, Maldonado R. Prodynorphin gene disruption increases the sensitivity to nicotine self-administration in mice. Int J Neuropsychopharm. 2009; 12:615-625.
Genetic Differences Play a Role in Sensitivity to Delayed Rewards
Emerging evidence suggests a genetic component to addiction, which is characterized by heightened impulsivity. Comparisons of multiple inbred mouse strains reveal significant differences in delay discounting (DD, a measure of impulsivity). However, findings from mouse and rat models do not always correspond, and although there have been several studies of DD comparing two inbred rat strains, calculation of strain differences requires more than two lines. NIDA grantees Drs. Wilhelm and Mitchell compared 6 rat strains (Brown Norway [BN], Copenhagen [C], Fischer [F], Lewis [L], Noble [N] and Wistar Furth [WF]) that, with the exception that L rats were derived from Wistar stock over 50 years ago, are genetically unrelated. Although primarily interested in potential differences in DD behavior, they also measured sucrose preferences, sucrose consumption, and locomotor activity. Using 10% sucrose solution as a reinforcer, rats received training in a 3-lever paradigm where responding on the middle lever "activated" the outer levers for a period of time. During the activated period, responding on one of the outer levers resulted in an adjusting sucrose reinforcer. Responding on the other outer lever delivered a standard (150ul) sucrose reinforcer. The volume of the adjusted reinforcer started at 75% sucrose and increased or decreased by 10%, depending on whether the rat chose the standard, or the adjusting lever, on the previous trial, respectively. During a DD session, responding on the standard lever resulted in delivery of 150ul following a delay of 0, 2, 4, 8 or 16 seconds. This delay didn't change within a session, but did vary between sessions. With all rat strains, the "indifference points" – that is, the point at which rats select from each end lever with roughly equal frequency, decreased as time to reward increased. Strain differences were most apparent at the longer delays, with N rats having significantly higher indifference points as compared to 4 of the 5 other strains. This suggests that N rats were more adverse to delay of reinforcement, and also suggests a heritability component in DD behavior. After the DD procedure, rats were given two-bottle sucrose preference tests to evaluate potential differences in baseline preferences that may affect interpretation of the DD data. For example, if a lower preference for sucrose were detected, there may appear to be an increase in impulsivity that may actually be the result of a faster devaluation of sucrose reward. During 50 minute test sessions, rats were given access to two bottles offering water or varied percents of sucrose. Significant strain differences were seen in the choice of water versus 10% sucrose, with N rats selecting more sucrose than all but the C strain. C rats also drank significantly more sucrose than F and WF rats. The researchers concluded that heredity also influences sucrose preference. Given this finding, results from the DD task cannot be attributed to differential reward preferences (or reward sensitivity) in the N rat strain. Next, animals were tested for locomotor activity in two 30-min test sessions. WF and L rats were significantly less active than most other rat strains, however activity was not correlated with DD or sucrose preference. Taken together, these data suggest that there is a heritability component in aversion to delayed reinforcement. This may have significant implications for disorders that are characterized by heightened aversion to delayed rewards. Understanding the role of specific genes and gene combinations may provide viable targets for the development of pharmacotherapies for disorders characterized by heightened impulsivity including drug addiction. Wilhelm CJ, Mitchell SH. Strain differences in delay discounting using inbred rats. Genes Brain Behav. 2009; 8:426-434.
Increased Sensitivity to Methamphetamine Following Prenatal Exposure to Lead
Children who live in inner cities have increased risk of exposure to lead above the "safe" range of less than 10ug/dl. Even when blood lead levels are within this so-called "safe" range, exposure to lead is known to be associated with neurobehavioral impairment and disturbed cognitive function. Additionally, lead exposure may also be associated with an enhanced vulnerability for drug abuse. Given a rise in methamphetamine (METH) use among people in low socioeconomic status, NIDA investigator Paul Wellman and colleagues were interested in the effects of prenatal lead exposure on response to METH. In these studies they employed an animal model of behavioral sensitization, which reveals the enhanced behavioral activation over repeated METH administration (as compared to initial, acute response). Female rats were administered either lead-free vehicle or 16 mg lead for 30 days prior to breeding. Once pregnant, they continued to receive either vehicle or lead treatment until the pups were weaned. After weaning, offspring were given no experimental treatment until post-natal day 70, when they were assigned to one of four test groups: no lead/10 days of systemic vehicle, no lead/10 days systemic METH (1 mg/kg/day), lead/10 days of systemic vehicle, or lead/10 days systemic METH (1 mg/kg/day). On days 1-10, animals were placed in locomotor activity (LMA) chambers, and LMA was monitored 15 minutes prior to treatment and for 45 min. post-treatment. On days 11-14, a dose-effect relationship was established in all animals given daily systemic injections of 0, 1, 2 and 4 mg/kg METH. For the first two exposures to METH (days 1 and 2), there were no differences in METH-induced LMA when comparing lead versus no lead exposure. However, with repeated METH treatment, animals exposed to lead showed significantly more LMA--an effect that lasted until day 6 when response levels of the two groups converged. Animals receiving vehicle treatments showed no increase in LMA. Dose-response testing indicated that METH produced an inverted U-shaped curve, with 1 and 2 mg/kg producing peak elevations in LMA. Prenatal treatment did not affect this dose-response relationship. Blood samples at breeding, postnatal day 2, and postnatal day 21, verified lead exposure of the dams and the pups. However, by the end of the experiment, there were no differences in the blood levels of lead in the exposed versus the non-exposed rats, indicating that the lead had been cleared from the blood. Therefore, increased sensitization of lead-exposed animals given METH challenges was due to residual effects of the lead and not a direct interaction between lead and METH. These results indicate increased behavioral responsiveness to METH if animals are exposed to lead during the prenatal and weaning period. These findings may have significant public health implications, as lead also affects cognitive development. Since lead affects both behavioral response to chronic psychostimulant administration (results of the present study) and impairs cognition, children exposed to lead during development may be particularly vulnerable for problems of drug abuse. Clifford PS, Hart N, Thompson J, Buckman S, Wellman PJ, Bratton GR, Nation JR. Prenatal lead exposure enhances meth-amphetamine sensitization in rats. Pharm Biochem Behav. 2009;93:165-169.
Blockade of Hypocretin Neurons in the Insula Decreases Nicotine Reinforcement
Damage to the insula from stroke has been associated with spontaneous tobacco cessation and reductions in urge to smoke, and imaging studies in smokers have shown that abstinence-induced cigarette craving is associated with activation of this cortical region. These findings suggest a role for the insula in nicotine reinforcement. In a recent study, NIDA grantees Paul Kenny, Jonathan Hollander and colleagues studied hypocretin (Hcrt) peptides (also known as orexins) in the insula, as these peptides have been implicated as potential regulators of reward and motivation. To investigate hypocretin's role in nicotine reinforcement, they first trained rats to self-administer intravenous nicotine. Rats were then pretreated with the Hcrt-1 receptor antagonist, SB-334867, and nicotine self-administration was assessed. Responding to receive nicotine decreased following Hcrt-1 blockade. This was due to a nonspecific response suppression, as responding for food was not similarly reduced. They next tested animals in an ICSS (intra-cranial self-stimulation) paradigm in which an animal lever-presses to receive rewarding electrical stimulation. Typically, nicotine lowers the current threshold that maintains self-stimulation, indicating reward enhancement. Administra-tion of the Hcrt-1 antagonist attenuated reward-enhancing effects of nicotine, supporting a role for Hcrt-1 receptors nicotine's rewarding properties. Next the investigators infused SB-334867 directly into the insular cortex. Infusion here, but not the somatosensory cortex control region, reduced responding for nicotine. Taken together, these data support a significant role for insular hypocretin receptors in nicotine reward and suggest that Hcrt-1 receptors may be a viable new target for tobacco cessation pharmacotherapies. Hollander JA, Lu Q, Cameron MD, Kamenecka TM, Kenny PJ. Insular hypocretin transmission regulates nicotine reward. PNAS. 2008;105(49):19480-19485.
Anxiety and Dysphoria During Spontaneous Withdrawal from Acute Morphine Exposure
Negative motivational states induced by drug withdrawal contribute to the pattern of addiction with drugs that induce physical dependence, such as the opiates. Withdrawal from chronic exposure to heroin or morphine includes symptoms of both anxiety and depression. Such negative emotional states can be induced by acute drug exposure and researchers have studied precipitated withdrawal after single drug treatments to study somatic and affective components of withdrawal from drugs like the opiates. The influence of withdrawal states induced by each individual opiate experience, over the course of morphine or heroin addiction, has not been studied. In animal behavioral models, anxiety can be indexed validly and reliably by elevated acoustic startle reflex, whereas conditioned place aversion is used to assess dysphoria or anhedonia associated with depression. The present study investigated the effects of spontaneous and naloxone precipitated withdrawal from one or two morphine injections using acoustic startle and place aversion. Results indicated that anxiety-like behavior (startle potentiation) emerges spontaneously after a single exposure to morphine and seems to be related to a decrease in opiate receptor occupancy. Moreover, it shares a pharmacological profile with naloxone precipitated opiate withdrawal, in that both can be blocked by anxiolytic drugs such as the benzodiazepines. They also found that during spontaneous withdrawal, startle potentiation develops before the rewarding effects of morphine have subsided (i.e., during the post-drug interval when conditioned place preference can be established). In contrast to these spontaneous withdrawal effects, naloxone potentiated startle and conditioned place aversion develop concurrently. Thus, increased anxiety-like behavior develops independently from decreased reward system activity. Furthermore, anxiogenic and dysphoric manifestations of opiate withdrawal may be mediated by distinct neural mechanisms, which may be progressively engaged during withdrawal after individual acute exposures to morphine during addiction. In conclusion, negative emotional states accompany the earliest stages of drug exposure, are likely a recurrent feature of intermittent drug use in humans, and thus may contribute significantly to the development of addiction. Rothwell PE, Thomas MJ, Gewirtz JC. Distinct profiles of anxiety and dysphoria during spontaneous withdrawal from acute morphine exposure. Neuropsychopharmacol. published online, June 10, 2009.
Learning Deficits Associated with Adolescent Cocaine in the Rat
Since previous epidemiological and preclinical research have suggested that the consequences of cocaine consumption may be different depending upon age of the individual, the present study investigated the effects of self-administered and passively administered cocaine, on odor-reward learning in adult rats exposed to cocaine either during adolescence or adulthood. Odor-reward learning was chosen as a task mediated by orbitofrontal cortex. Over 18 training days, rats were exposed to cocaine during adolescence or adulthood and tested during adulthood. The exposure conditions used a three group yoked design, with one-third self-administering cocaine, one-third yoked and receiving cocaine passively and one-third yoked and receiving saline. Following drug exposure, all rats were returned to their home cages for an 18 day drug free period. Then they were trained in two spatial maze-learning tasks. In the first task (easy) they learned to find food in four distinctive-odor arms of the maze. In the second task (difficult), they learned to find food in four distinctive-odor arms of the maze, but the maze had eight arms, four of which had the previous odors and now had to be avoided (so-called win-shift task). Results indicated that cocaine self-administration behavior was similar in adolescent and adult rats. However, despite similarities in the amount of cocaine consumed in adult and adolescent rats, subsequent learning was affected differentially after the drug-free period. On the difficult test, adolescent and adult rats exposed to cocaine both showed learning deficits compared to saline controls, but adolescents were somewhat more impaired, which was evident on the trials to criterion measure. Rats self-administering cocaine were also more impaired than those receiving cocaine passively, indicating that the observed deficits were not due exclusively to the pharmacological effects of cocaine, but may be related also to the expectancy and/or controllability of cocaine delivery. These findings support a view that similar intakes of cocaine can have difference consequences when drug use begins during adolescence vs. adulthood. Harvey RC, Dembro KA, Rajagopalan K, Mutebi MM, Kantak KM. Effects of self-administered cocaine in adolescent and adult male rats on orbitofrontal cortex-related neurocognitive functioning. Psychopharmacol, published online, June 10, 2009.
Coexpression of Alpha 2-Adrenergic and Opioid Receptors on Primary Afferent Nociceptive Fibers Which May Represent An Anatomical Substrate for Analgesic Synergy
Agonists acting at alpha 2-adrenergic and opioid receptors (alpha 2ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although this interaction is well established, its mechanism remains poorly understood. In the present study, NIDA-grantee Dr. George Wilcox and colleagues (University of Minnesota) used immunohistochemistry to investigate the spatial relationship between alpha 2ARs and ORs in the rat spinal cord. They observed extensive colocalization between alpha 2A-adrenergic and delta-opioid receptors (DOR) on substance P (SP)-immunoreactive (-ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. Furthermore, coincubation of isolated synaptosomes with alpha 2AR and DOR agonists resulted in a synergistic increase in the inhibition of potassium-stimulated neuropeptide release. These findings suggest that coexpression of 2AAR-DOR on primary afferent nociceptive fibers represent an anatomical substrate for analgesic synergy. Riedl MS, Schnell SA, Overland AC, Chabot-Dore AJ, Taylor AM, Ribeiro-Da-Silva A, Elde RP, Wilcox GL, Stone LS. Coexpression of alpha 2A-adrenergic and delta-opioid receptors in substance P-containing terminals in rat dorsal horn. J Comp Neurol. 2009;513:385–398.
Contribution of CD14 for Glial Mediated Neuropathic Pain
The CNS toll-like receptor 4 (TLR4) plays a key role in the development of behavioral hypersensitivity in the neuropathic pain spinal nerve L5 transection (L5Tx) model in rats. The TLR4 is a well-known receptor for lipopolysaccharide (LPS) in innate immune responses. NIDA-grantee Dr. Joyce Deleo and colleagues (Dartmouth Medical School) investigated the role of CD14, an accessory molecule in the LPS–TLR4 signaling pathway, in the development of L5Tx-induced neuropathic pain. CD14 knockout mice displayed significantly decreased behavioral sensitivity as early as day 1 post-L5Tx, indicating a nociceptive role of CD14. By flow cytometric analyses, they found elevated microglial surface CD14 expression in the ipsilateral lumbar spinal cord 3 days post-L5Tx, as well as substantial increases in microglial size. Further, intrathecal injection of soluble CD14 induced significantly greater mechanical hypersensitivity in wild type (C3H/HeN) mice compared with TLR4-deficient (C3H/HeJ) mice. Together, these data demonstrate that CD14 plays a role in TLR4-dependent nerve injury–induced neuropathic pain. Cao L, Tanga FY and DeLeo JA. The contribu-ting role of CD14 in toll-like receptor 4 dependent neuropathic pain. Neurosci. 2009; 158:896–903.
The Glutamate Transport Protein GLT1 is a Potential New Target for Treating Cocaine Addiction
A variety of studies in animal models indicate that increased glutamate transmission in several brain regions is a key neuroadaptation that promotes relapse to cocaine-seeking behavior. Therapeutic agents that can decrease glutamate transmission are therefore possible candidates for treatment of cocaine addiction. In this study, Dr. George Rebec and his colleagues tested the hypothesis that decreasing glutamate transmission by upregulation of a glutamate transporter would attenuate cocaine relapse. They targeted GLT1, which is expressed on astrocytes and is responsible for taking up most extracellular glutamate released by synaptic activation. Rats were trained to self-administer cocaine in sessions where lever pressing delivered intravenous cocaine paired with presentation of a combined light and tone cue. After learning to self-administer, the rats went through five days of extinction training, where the lever was present, but pressing it delivered neither cocaine nor the cue. At the end of each extinction session, they received an injection of 50, 100, or 200 mg/kg ceftriaxone, a β-lactam antibiotic, believed to increase GLT1 expression. Control animals received injections of saline vehicle. By the end of these five sessions, none of the rats was pressing the lever previously associated with cocaine more than a few times. On the sixth day, they were tested for reinstatement of cocaine-seeking with presentation of the cue. During reinstatement testing, lever presses delivered the cue, but not cocaine and the number of lever presses was used to assess reinstatement of cocaine-seeking behavior. Animals that had received saline or the lowest does of ceftriaxone pressed the lever significantly more than those who had received either of the two higher doses, indicating that ceftriaxone treatment at these doses significantly attenuated relapse behavior. Ceftriaxone at the highest dose had no effect on reinstatement of food seeking in similar experiments in which animals were trained to respond for food, indicating that the effect was specific for cocaine and not a general enhancement of extinction. Western blot analysis showed that GLT1 protein levels were significantly increased in both the prefrontal cortex and nucleus accumbens of rats treated with the highest dose of ceftriaxone compared to those treated with saline or the 50 mg/kg dose (brains of animals treated with 100mg/kg were not used in this analysis). The results implicate GLT1 as a potential target for treatment of cocaine addiction, and may be an attractive target for translational research because ceftriaxone is an approved drug commonly used to treat postoperative infections. Sari Y, Smith KD, Ali PK, Rebec GV. Upregulation of GLT1 attenuates cue-induced reinstatement of cocaine-seeking behavior in rats. J Neurosci. 2009; Jul 22;29(29):9239-9243.
Individual Rats with Higher Addiction Liability Show More Persistent Neuroadaptations in Ventral Tegmental Dopamine Neurons Following Cocaine Self-Administration
Studies in animal models have shown that addictive drugs produce neuroadaptations in dopamine neurons of the ventral tegmental area (VTA) that are critical for certain features of drug addiction. This study asked whether individual differences in these neuroadaptive responses are correlated with naturally occurring individual differences in drug addiction liability. To study this question, the investigators took advantage of high-responder (HR) and low-responder (LR) rats, which are distinguished on the basis of how much they move around in a novel environment. These spontaneous differences have been associated with increased addiction liability for HR over LR rats in several models of addiction. They first trained HR and LR rats to self-administer cocaine, using a dosing regimen that equalized drug intake across individuals. They then recorded the activity of VTA neurons at various periods of withdrawal. Withdrawal from cocaine self-administration increased VTA dopamine cell firing and bursting in all animals. However, these changes in firing rates and patterns were more persistent in HR than in LR rats. In the LR rats, elevated firing rats persisted for only one day, and then declined to baseline levels, whereas in the HR rats, the increase in firing was apparent at both withdrawal days 1 and 3 and then declined to baseline by withdrawal day 10. These results demonstrate individual differences in the duration of drug-induced neuroadaptations in dopamine neurons of the VTA. More persistent elevation of dopamine cell activity and reduced capacity to return to baseline levels may be an important factor contributing to the development of addiction in "at-risk" individuals. McCutcheon JE, White FJ, Marinelli M. Individual Differences in Dopamine Cell Neuroadaptations Following Cocaine Self-Administration. Biol Psychiatry. 2009; Jun 16. [E-pub ahead of print].
Prenatal Exposure to Cocaine Increases Dopamine Release Induced by Chronic Exposure to Cocaine in Adulthood
Studies in rodents have shown that prenatal exposure to cocaine can increase the rewarding potency of cocaine on operant or instrumental learning, e.g., in drug self-administration paradigms. Conversely, the potency of cocaine on Pavlovian learning seen in conditioned place preference, appears to be decreased in adult rodents after exposure in utero. The investigators current research focuses on cellular mechanisms underlying the adaptation of mesolimbic brain reward circuitry in response to adult drug exposure, and understanding how these mechanisms differ between developmentally exposed and non-exposed offspring, in a model of prenatal cocaine exposure. Previously, they have demonstrated increased cocaine sensitivity in operant tasks, and decreased sensitivity in classical Pavlovian tasks, upon cocaine challenge. Here, they studied the effects of prenatal cocaine exposure on dopamine (DA) levels in the nucleus accumbens (NAcc) of mice subjected to chronic non-contingent cocaine administration in adulthood using in vivo microdialysis. The mice were injected with cocaine every other day for a total of seven doses, withdrawn for 21 days, and then challenged with an additional injection. Microdialysis samples obtained after the first, seventh challenge doses showed increasing cocaine-stimulated DA release in the nucleus accumbens, which was significantly enhanced after prenatal cocaine exposure. Interestingly, the same dosing regimen that produces reduced levels of locomotor sensitization in prenatally exposed animals produced augmented DA release at days 7 and 21, compared to non-exposed controls. This sensitized DA release may therefore reflect the enhanced rewarding effects of cocaine in prenatally exposed animals rather than locomotor stimulating effects. The results show that early developmental cocaine exposure can alter adapta-tion of brain reward systems to chronic psychostimulant exposure in adulthood. Malanga CJ, Ren JQ, Guerriero RM, Kosofsky BE. Augmentation of cocaine-sensitized dopamine release in the nucleus accumbens of adult mice following prenatal cocaine exposure. Dev Neurosci. 2009;31(1-2):76-89.
Emotional Arousal in Cocaine Exposed Toddlers: Prediction of Behavior Problems
A recent study by Dr. Linda Mayes and colleagues at Yale University School of Medicine examined the relationship between prenatal cocaine exposure and agitated emotional arousal, self-regulation, and references to caregivers during a toy-wait task. The study involved 225 2½ year-old toddlers (including 129 Prenatally Cocaine and Other Drug Exposed [PCE], 30 Non Cocaine but other drug Exposed [NCE], 66 Non Drug Exposed [NDE]). Children's behaviors in the toy-wait task were coded for emotional arousal and regulation behaviors. The findings revealed a non-significant trend for PCE toddlers to exhibit greater agitated emotional arousal than NCE and NDE toddlers. In addition, PCE boys made significantly more references to their caregivers during the task than NDE boys; however, PCE boys did not differ from NCE suggesting that the effect was not cocaine-specific, but rather related to other drug exposures. Among girls, there were no differences in reference to caregivers as a function of drug exposure status. References to caregivers during the task included such behaviors as looking to, approaching, reaching and making physical contact, behaviors interpreted by the authors as reflecting "an attempt to regulate emotion by drawing in the caregiver." Results also suggested that higher agitated arousal at age 2½ years was related to greater decreases in externalizing behaviors through age 5½ years, but did not predict change changes in internalizing symptoms. Neither of these outcomes was affected by drug exposure status. Drug exposure status also did not predict externalizing or internalizing problems over time. In sum, the results of this study suggest a possible link between drug exposure and emotional arousal and emotional regulation. This finding is important because of possible implications for risk for or protection from later psychopathology. Outcomes showing no relationship between drug exposure status and externalizing and internalizing problems over time can be viewed as an encouraging finding regarding drug-exposed children given the historical concerns about possible adverse consequences of prenatal exposure to drugs of abuse. Chaplin TM, Fahy T, Sinha R, Mayes LC. Emotional arousal in cocaine exposed toddlers: Prediction of behavior problems. Neurotoxicol Teratol. 2009; May 21 [E-pub ahead of print].
The Changing Role of the Medial Preoptic Area in the Regulation of Maternal Behavior Across the Postpartum Period
Maternal behavior in rats undergoes considerable plasticity in parallel to the developmental stage of the pups, resulting in distinct patterns of maternal behavior and care at different postpartum time points. For example, Dr. Morrell and her colleagues have shown that rat mothers prefer their pups over cocaine through the first 8 days postpartum, but their preference begins to switch to a preference for drug at PPD10. In the current study, they investi-gated a neural substrate that may be involved in this change in preference. The medial preoptic area (mPOA) of the hypothalamus is one critical neural substrate underlying the onset and early expression of maternal behavior in rats, but little is known about its specific functional role in the evolving expression of maternal behavior across the postpartum period. To study this question, the investigators used a reversible local neural inactivation method to examine the role of the mPOA in the regulation of maternal behavior throughout the postpartum period. This approach avoids the compensatory plasticity in CNS that occurs after permanent lesions and allows repeated testing of the same individuals. Early (PPD7–8) and late (PPD13–14) postpartum maternal behavior was evaluated in female rats following infusions of bupivacaine or vehicle into the mPOA or into control areas. Consistent with the study's hypotheses, mPOA inactivation severely, but transiently, disrupted early postpartum maternal behavior, whereas infusion of vehicle or inactivation of adjacent control sites was without effect. Later in the postpartum period, however, transient mPOA inactivation facilitated the expression of maternal behaviors, in clear contrast with the lower level of expression of these behaviors characteristic of this later period. The results demonstrate that the mPOA is differentially engaged throughout the entire postpartum period in orchestrating appropriate maternal responses with the developmental stage of the pups. Further, the results suggest that the mPOA may be part of the neural circuit involved in the changing preference for cocaine over maternal care shown in the earlier experiments. Pereira, M, Morrell, JI. The changing role of the medial preoptic area in the regulation of maternal behavior across the postpartum period: Facilitation followed by inhibition. Behav Brain Res. 2009; June 21. [E-pub ahead of print].
Sex and Ovarian Hormones Influence Vulnerability and Motivation for Nicotine During Adolescence in Rats
In animal models of nicotine self-administration in adult rats, several sex differences have been observed. For example, a greater percentage of females acquire self-administration under low dose conditions and females show higher progressive ratio responding, suggesting greater motivation to obtain nicotine. In human laboratory studies, subjective responses to nicotine have been shown to vary with the menstrual cycle and to be affected by exogenous delivery of the ovarian hormone progesterone. Research has not examined whether these nicotine effects observed in adults occur also in adolescents. Thus, in the present study, Dr. Wendy Lynch of the University of Virginia sought to determine whether sex differences in nicotine self-administration are also seen in adolescent rats and if this difference is modulated by ovarian hormones. Nicotine self-administration training began on postnatal day 30 with either 5 or 10 microg/kg/infusion followed by testing on a progressive ratio schedule. Dr. Lynch found that under the lower nicotine dose, a greater percentage of females than males acquired self-administration. Early in adolescence, there were no sex differences in progressive ratio responding, but differences emerged in late adolescence in that females had higher break points; thus, they were willing to make more operant responses to obtain drug. Progressive ratio responding in females also was correlated with estrogen and progesterone levels, varying negatively with progesterone and positively with the ratio of estradiol to progesterone. Overall, these data indicate that sex differences in nicotine self-administration reported adult rats have their origin in adolescence and, as in humans, nicotine's effects are modulated by ovarian hormones. Lynch WJ. Sex and ovarian hormones influence vulnerability and motivation for nicotine during adolescence in rats. Pharmacol Biochem Behav. 2009; Jul 17. [E-pub ahead of print].
Housing Conditions Affect Conditioned Place Preference and Dopamingeric Markers in Adolescent Male Rats
Dr. Sari Izenwasser and colleagues at the University of Miami Miller School of Medicine assessed whether cocaine reward during early adolescence, in male rats, is affected by social and environmental housing conditions. She also examined the relation between cocaine reward outcomes and dopaminergic proteins that mediate drug reward. Social conditions were manipulated by housing rats either alone or with two or three conspecifics per cage. Environmental conditions were manipulated by housing rats either with toys (enrichment) or without toys (impoverishment). These conditions, which began on postnatal day (PND) 23, constituted six experimental conditions: socially isolated rats housed alone, either impoverished with no toys (II) or enriched with toys (IE); and social rats housed two/cage either with no toys (SI2) or with toys (SE2), or three/cage with (SE3) or without (SI3) toys. Cocaine reward was determined through use of the cocaine conditioned place preference (CPP) procedure conducted on PND 43-47 using either 5 or 10 mg/kg cocaine. Results indicated that CPP was established in the II rats (isolated, no toys) under both doses of cocaine. With additional cage mates or toys, CPP was decreased. Rats housed three/cage, either with (SE3) or without (SI3) toys, did not exhibit CPP with either cocaine dose. Thus, both social housing and toys diminished CPP, and housing with two cage mates prevented CPP. Further, there were correlations between these housing conditions and markers of dopaminergic transmission. In the nucleus accumbens increased dopamine transporter (DAT) protein was observed in rats housed three/cage with toys (SE3) compared to those housed alone without toys (II). Administration of cocaine also increased tyrosine hydroxylase and DAT in the II rats, but not in the SE3 rats. Zakharova E, Miller J, Unterwald E, Wade D, Izenwasser S. Social and physical environment alter cocaine conditioned place preference and dopaminergic markers in adolescent male rats. Neurosci. 2009; Jul 3. [E-pub ahead of print].
Hedonic Sensitivity in Adolescent and Adult Rats: Taste Reactivity and Voluntary Sucrose Consumption
In her 2000 article, "The adolescent brain and age-related manifestations," Dr. Linda Spear at Binghamton University suggested that adolescents may have a partial anhedonia, that leads them to seek out natural and drug rewards as compensation for this attenuation in hedonic sensitivity. She and colleague Carrie Wilmouth recently tested this hypothesis by assessing hedonic reactions to sucrose using a well-established taste-reactivity (TR) test, which measures oral-facial appetitive and aversive reactions to tastants. Contrary to their prediction, results suggested greater hedonic sensitivity in adolescents. They found that appetitive taste responses to 10% sucrose solution were nearly three times higher in adolescent than adult rats. Further evidence for greater hedonic sensitivity in adolescents was seen the concentration-effect curves, as well as more pronounced paw licking in adolescents in response to a 34% sucrose solution. In response to quinine, adolescents exhibited fewer negative oral-facial responses. In a two-bottle taste test, adolescents consumed more sucrose than adults; however, adolescents and adults equally preferred sucrose over water. The authors conclude that these results do not support the hypothesis that adolescents exhibit an age-related, partial anhedonia. Wilmouth CE, Spear LP. Hedonic sensitivity in adolescent and adult rats: Taste reactivity and voluntary sucrose consumption. Pharmacol Biochem Behav. 2009 Jun;92(4):566-573.
Nicotine and Social Interaction Interact to Enhance Reward in an Adolescent Rodent Model
NIDA grantee Dr. Janet Neisewander has been using the conditioned place preference paradigm (CPP) to study drug, and social interaction, rewards in adolescent rats. She has demonstrated that cocaine, and time with "play partners" independently can induce a CPP during adolescence. She also reported that doses of both, which are sub-threshold for inducing CPP alone, can summate to induce CPP when conditioned together. This suggests that inherently rewarding properties of social interaction might increase cocaine's reinforcing value, or that cocaine may enhance the reward value of social activities. In the new study she established nicotine (nic) CPP in adolescent rats, using either the s.c or i.v. route of administration. Using a biased CPP design, where drug is paired with the initially non-preferred side of a two-sided test box, she exposed rats to two times per day pairings – once per day when nic was paired with the non-preferred side, and once per day where vehicle was paired with the preferred side. Different groups were conditioned with .01, .03 or .06 i.v. nic, and 0.1, 0.3 or 0.6 s.c nic, in 10-min sessions over four days. In studies where social interaction was paired with nic during conditioning sessions, only two 10-min conditioning sessions were conducted, to test sub-threshold doses of reward. Social conditions included isolation versus conditioning with one playmate. In the final study, the investigator sought to determine if social and drug variables interact in an additive, or synergistic, manner. In this study, she eliminated the associative strength of nic alone, as a contributing factor to CPP, by pairing nic with both sides of the apparatus (thus negating the conditioned rewarding effects of the drug). Social reward continued to be paired with only the conditioning side, and groups receiving nic (both sides) + social reward were compared with groups receiving nic (paired side only) + social reward. Results of the studies revealed dose-orderly CPP with both routes of nic administration. She also replicated the original CPP finding that was established with social interaction, in that rats conditioned with a playmate showed greater preference for the paired side than isolates. Also similar to the results with cocaine, rats conditioned with a drug+social interaction combination (0.1 s.c. nic + playmate) exhibited significantly more time on the paired side during the test for CPP than those conditioned with nic or playmate alone. (This interaction could be demonstrated only with the s.c. route of administration). The final study replicated this finding, even though the nic was paired with both sides, suggesting nic enhancement of the playmate CPP. These findings suggest that nic administration, via smoking in adolescents, may enhance the rewarding effects of social interaction during a developmental period when these interactions are highly valued and influential. This interaction may contribute to the development and maintenance of smoking in teens. Thiel KJ, Sanabria F, Neisewander JL. Synergistic interaction between nicotine and social rewards in adolescent male rats. Psychopharm. 2009; 204:391-402.
Varenicline Both Mimics and Blocks Nicotine's Discriminative Stimulus Effects
Varenicline is an alpha4beta2 (a4b2) partial agonist that acts as an agonist with partial efficacy, but also acts as a functional competitive antagonist because occupying the receptor makes it unavailable for full agonist binding. Varenicline is the newest pharmacotherapy for smoking cessation available in the United States, but little is known about its ability to attenuate behavioral effects of nicotine in animal models outside of the self-administration model. NIDA grantee Mark LeSage and colleagues therefore used drug discrimination methodology to investigate varenicline's partial agonist effects. Rats trained to discriminate nicotine from saline were challenged with varenicline or cytisine (an older a4b2 partial agonist used as control). While varenicline partially generalized to nicotine (up to 63% responding on nicotine-paired lever), cytisine only marginally generalized (max of 23% responding on nicotine-paired lever). Similarly, when given prior to nicotine, varenicline significantly attenuated responding on the nicotine-paired lever while cytisine only slightly reduced responding on the nicotine-paired lever. Given these data, varenicline's behavioral profile in drug discrimination is consistent with its label as a partial agonist. Furthermore, drug discrimination appears to be a more sensitive paradigm for differentiating between a4b2 partial agonists. LeSage MG, Shelley D, Ross JT, Carroll FI, Corrigall WA. Effects of the nicotine receptor partial agonists varenicline and cytisine on the discriminative stimulus properties of nicotine in rats. Pharm Biochem Behav. 2009; 91:461-467.