Research Findings - Intramural Research

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Cannabinoid CB1 Receptor and Serotonin 3 Receptor Subunit A (5-HT3A) are Co-expressed in GABA Neurons in the Rat Telencephalon

Among all described serotonin (5-HT) receptors in mammals, the type three (5-HT3) is the only ligand-gated ion channel receptor for serotonin. By using double in situ hybridization histochemistry, we found co-expression of the functional 5-HT3A subunit of the 5-HT3 receptor and the central CB1 cannabinoid receptor in neurons of the rat telencephalon. Double-labeled 5-HT3A/CB1 neurons were found in the anterior olfactory nucleus, superficial and deep layers of the cortex, hippocampal formation (hippocampus, dentate gyrus, subiculum, and entorhinal cortex) and amygdala. Analysis of the proportion of neurons co-expressing 5-HT3A and CB1 receptors in the cortex and amygdala showed that, depending on the brain region, 37-53% of all neurons expressing the 5-HT3A subunit also expressed CB1 transcripts; 16-72% of the total population of neurons expressing CB1 mRNA co-expressed the 5-HT3A subunit. By using a combination of double in situ hybridization and immunohistochemistry, we demonstrated that 5-HT3A/CB1-expressing neurons contained the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). These results imply that in distinct regions of the telencephalon, GABA neurons that react to cannabinoids may also be responsive to serotonin through 5-HT3 receptors. Cellular coexistence of 5-HT3A and CB1 transcripts in interneurons of the cortex, hippocampal formation, and amygdala suggest possible interactions between the cannabinoid and serotonergic systems at the level of GABA neurotransmission in brain areas involved in cognition, memory, and emotion. Morales, M., Wang, S.D., Diaz-Ruiz, O., and Jho, D.H. Journal of Comparative Neurology, 468, pp. 205-216, 2004.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Cocaine Inhibits Cromakalim-Activated K(+) Currents in Follicle-Enclosed Xenopus Oocytes The effect of cocaine on K(+) currents activated by the K(ATP) channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3-500 microM reversibly inhibits cromakalim-induced K(+) currents. The IC(50) value for cocaine was 96 microM. Inhibition of the cromakalim-activated K(+) current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca(2+) chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K(+) currents, suggesting that Ca(2+)-activated second messenger pathways are not involved in the actions of cocaine. Outward K(+) currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC(50) and slope values for the activation of K(+) currents by cromakalim were 184+/-19 microM and 1.14 in the absence of cocaine as compared to 191+/-23 microM and 1.03 in the presence of cocaine (300 microM). Cocaine also blocked K(+) currents mediated through C-terminally deleted form of Kir6.2 (KirDeltaC26) in the absence of sulfonylurea receptor with an IC(50) value of 87 microM, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 microM) did not affect the binding characteristics of the K(ATP) ligand, [(3)H]glibenclamide. These results demonstrate that cromakalim-activated K(+) currents in follicular cells of Xenopus oocytes are modulated by cocaine. Oz, M., Zakharova, I., Dinc, M., and Shippenberg, T. Naunyn Schmiedebergs Archives of Pharmacology, 369, pp. 252-259, 2004.

Molecular Neuropsychiatry Section, Molecular Neuropsychiatry Research Branch

Chronic Methamphetamine Increases Fighting in Mice A propensity for violent behaviors to develop in chronic methamphetamine (METH) abusers has been noted. The idea that increased aggressiveness might result from chronic METH administration was tested in mice after chronic (long-term intermittent, 8 weeks) or single exposures to the drug. A single injection of METH (6 mg/kg) did not augment fighting. In contrast, chronic METH administration significantly increased the number of animals that initiated bite attacks. This regimen also shortened the latency before the first attack. Latency before the first attack was shorter at 20 h after the METH injection than at 15 min after injection. Locomotor activity was not different at 20 h after METH injection, indicating that increased fighting was not secondary to METH-induced hyperactivity. METH-induced increases in fighting were not related to the duration of persistent sniffing after the initial encounter with an intruder since the duration of this behavior was significantly increased at 15 min after METH but not at 20 h post drug. These results indicate that repeated injections of METH can increase fighting behaviors and also alter social interactions in mice. Thus, intermittent administration of METH might be useful as a pharmacological model to study the biochemical and molecular bases of aggressiveness. Sokolov, B.P., Schindler, C.W. and Cadet, J.L. Pharmacology Biochemistry and Behavior, 77, pp. 319-326, 2004.

Methamphetamine Induces Neuronal Apoptosis via Cross-talks Between Endoplasmic Reticulum and Mitochondria-dependent Death Cascades Methamphetamine (METH) is an illicit drug that causes neurodegenerative effects in humans. In rodents, METH induces apoptosis of striatal glutamic acid decarboxylase (GAD) -containing neurons. This paper provides evidence that METH-induced cell death occurs consequent to interactions of ER stress and mitochondrial death pathways. Specifically, injections of METH are followed by an almost immediate activation of proteases calpain and caspase-12, events consistent with drug-induced ER stress. Involvement of ER stress was further supported by observations of increases in the expression of GRP78/BiP and CHOP. Participation of the mitochondrial pathway was demonstrated by the transition of AIF, smac/DIABLO, and cytochrome c from mitochondrial into cytoplasmic fractions. These changes occur before the apoptosome-associated pro-caspase-9 cleavage. Effector caspases-3 and -6, but not -7, were cleaved with the initial time of caspase-3 activation occurring before caspase 9 cleavage; this suggests possible earlier cleavage of caspase-3 by caspase-12. These events preceded proteolysis of the caspase substrates DFF-45, lamin A, and PARP in nuclear fractions. These findings indicate that METH causes neuronal apoptosis in part via cross-talks between ER- and mitochondria-generated processes, which cause activation of both caspase-dependent and -independent pathways. Jayanthi, S., Deng, X., Noailles, P.A., Ladenheim, B. and Cadet, J.L. FASEB Journal, 18, pp. 238-251, 2004.

Molecular Neurobiology Branch

Estimating the Burden of Complex Genetics in Brain Disorders Few data estimate the impact of complex genetics in neuropsychiatric illness, making it likely that this impact could be underappreciated. Investigators provide estimates of the impact of complex genetics in neuropsychiatric disorders in the United States, based on estimates of disease costs to US society, disease heritability, and mendelian contributions to disease. Costs were estimated from literature sources and Lewin-National Foundation for Brain Research estimates updated for population growth and consumer price index inflation. Heritability estimates came from available twin data. Estimates of mendelian contributions came from the Online Mendelian Inheritance in Man database and our perspectives. Brain and nervous system disorders may cost the United States as much as $1.2 trillion annually, and affect many millions of Americans each year. Twin data suggest that more than 40% of the societal burden of brain disorders is likely to be genetically mediated. Most of this disease burden arises from complex multigene genetics as well as from environmental influences. The large sizes of these complex genetic burdens should encourage careful molecular and clinical work to link disease vulnerability allelic variants with the pathogenesis, nosologic characteristics, prevention, diagnostics, and therapeutics of brain disorders. Uhl, G.R. and Grow, R.W. Archives of General Psychiatry, 61, pp. 223-229, 2004.

Psychobiology Section, Medications Discovery Research Branch

What is Represented by Vertical Shifts in Self-administration Dose-response Curves? Several recent papers have made suggested mechanisms for changes in the drug self-administration dose-effect curve. In particular, a substantial amount of theory construction surrounds the observations of vertical shifts in dose-effect curves. In this paper, IRP investigators make suggestions regarding ways in which these changes can be conceptualized so that they become subject to objective rational study. Importantly it should be appreciated that there are many factors that contribute to the shape of the curve, including the potential for tolerance to various effects of the drug being self administered. Potential mechanisms thought to underlie changes in the self administration of cocaine are discussed from a perspective based on clinical findings. These findings suggest some problems resulting from the unfettered extrapolation of some preclinical theories, providing a focus for refinements in theory. Katz, J.L. and Higgins, S.T. Psychopharmacology, 171, pp. 360-361, 2004.

Medicinal Chemistry Section, Medications Discovery Research Branch

Synthesis and Monoamine Transporter Binding of 2-(Diarylmethoxymethyl)-3_-aryltropane Derivatives A series of 3_-aryl tropane analogues wherein the 2-position was substituted with various diarylmethoxyalkyl groups was synthesized and evaluated for binding at the dopamine (DAT), serotonin (SERT), norepinephrine transporter (NET) and muscarinic (M1) receptors. The 2_-analogues generally demonstrated high to moderate binding affinities (Ki = 34 -112 nM) at the DAT with good selectivity over SERT, NET and M1 receptors. Alternatively, the 2_-isomers were 10-fold less potent at the DAT with poor selectivity over SERT. These Structure-Activity Relationship (SAR) studies provide further evidence for the varied binding requirements of structurally diverse tropane-based ligands and support future studies to elucidate DAT binding requirements in relation to cocaine-like behavioral endpoints. Xu, L., Kulkarni, S. S., Izenwasser, S., Katz, J. L., Kopajtic, T., Lomenzo, S. A., Newman, A. H., and Trudell, M. L. Journal of Medicinal Chemistry, 47, pp. 1676-1682, 2004

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

A Single Infusion of BDNF into the Ventral Tegmental Area Induces Long-lasting Potentiation of Cocaine-seeking After Withdrawal Cocaine addiction in humans is associated with long-term propensity to relapse. Using a rat relapse model, IRP scientists found that cocaine-seeking induced by exposure to cocaine-associated cues progressively increases after withdrawal. This progressive increase is associated with increases in brain-derived nerve growth-factor (BDNF) levels within the mesolimbic dopamine system. Based on these findings, we studied whether BDNF infusions into the ventral tegmental area (VTA), the cell body region of mesolimbic dopamine neurons, would potentiate cocaine-seeking after withdrawal. Rats were trained to self-administer cocaine for 10 days and cocaine-seeking was measured in extinction tests 3, 10 or 30 d after withdrawal. During testing, rats were exposed to contextual cues that had predicted cocaine availability during training and lever-presses resulted in contingent presentations of a discrete tone-light cue that was previously temporally paired with cocaine infusions. BDNF (0-0.75 µg/site) or nerve growth factor (NGF, 0-0.75 µg/site) was infused into the VTA 1-2 h after the last self-administration session. To examine the role of the mitogen-activated protein kinase (MAPK) pathway in BDNF effects, U0126 (1 µg/site), a MEK inhibitor, was used. A single intra-VTA infusion of BDNF, but not NGF, induced long-lasting enhancement of cocaine-seeking for up to 30 days, an effect reversed by U0126. In contrast, neither BDNF infusions into the substantia nigra, nor acute intra-VTA BDNF infusions 2 h prior to testing on day 3 of withdrawal, were effective. These data suggest that BDNF-mediated neuroadaptations in mesolimbic areas are involved in the persistent cocaine-seeking induced by exposure to drug cues after withdrawal. Lu, L., Dempsey, J., Liu, S., Bossert, J. and Shaham, Y. The Journal of Neuroscience, 24, pp. 1604-1611, 2004

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Exposure to D-9-Tetrahydrocannabinol (THC) Increases Subsequent Heroin Taking but not Heroin's Reinforcing Efficacy: A Self-Administration Study in Rats One concern about the widespread use of cannabis is that exposure to its active ingredient, Delta-9-tetrahydrocannabinol (THC), might increase future reinforcing effects of other abused drugs such as heroin. In this study, IRP scientists investigated the effects of pre-exposure to THC on subsequent intravenous self-administration of heroin by Sprague-Dawley rats. We studied (1) acquisition of heroin self-administration behavior using a continuous-reinforcement (fixed-ratio (FR) 1) schedule, (2) heroin dose-response relationships using a FR1/variable-dose schedule, and (3) reinforcing efficacy of heroin using a progressive-ratio schedule. The number of rats pre-exposed to THC that subsequently learned to self-administer 50_g/kg injections of heroin within 10 daily sessions did not differ from vehicle-pretreated controls. In contrast, rats pre-exposed to THC subsequently self-administered significantly more heroin injections per session and showed significantly shorter post-injection pauses over a range of heroin doses (12.5-100 microg/kg/injection) using the variable-dose schedule. Interestingly, the maximum effort rats would exert to receive an injection of the different doses of heroin under the progressive-ratio schedule was not altered by THC pre-exposure. In other rats, we varied the 'price' of heroin (responses required/dose), by manipulating FR response requirements at different doses of heroin across sessions, to calculate demand and response output curves. Again, consumption was significantly higher in the THC-treated rats at the lowest prices of heroin (FR1/100_g/kg and FR1/50_g/kg) but there were no differences in the reinforcing efficacy of heroin between THC- and vehicle-pretreated rats. These results demonstrate that pre-exposure to THC alters some pharmacological effects of heroin that determine frequency of heroin taking, but offer no support for the hypothesis that pre-exposure to THC alters heroin's efficacy as a reinforcer. Solinas, M., Panlilio, L.V. and Goldberg, S.R. Neuropsychopharmacology DOI:10.1038/sj.npp.1300431

Sigma1 Receptor Upregulation after Chronic Methamphetamine Self-Administration in Rats: A Study with Yoked Controls Sigma-1 receptors (Sig-1R) are implicated in behavioral sensitization, conditioned place preference, and cellular restructuring induced by psychostimulants. IRP investigators previously reported that rats that actively self-administered methamphetamine for 5 weeks and were then withdrawn from methamphetamine for 24 h showed downregulation of dopamine D2 autoreceptors (approximately 30%) in the midbrain and this was not seen in rats that passively received injections of methamphetamine or saline at the same time (yoked controls). Involvement of Sig-1R in the self-administration of psychostimulants, however, has never been reported. This study examined neuroadaptive changes in Sig-1R in the brains of rats self-administering methamphetamine. Three groups of rats were tested simultaneously 5 days per week, for 5 weeks (25 daily sessions). Two groups served as yoked controls and passively received an injection of either 0.1 mg/kg methamphetamine or saline (not contingent on responding) each time a response-contingent injection of 0.1 mg/kg methamphetamine was actively self-administered by the first group of rats. Protein and mRNA levels of Sig-1R were then measured by Western and Northern blottings, respectively. Results showed a marked upregulation of Sig-1R proteins (50%) in the midbrain and altered levels of Sig-1R mRNA in the frontal cortex and hippocampus of rats that learned to actively self-administer methamphetamine, but not in yoked methamphetamine- or saline-control rats. The authors conclude that neuroadaptive increases in Sig-1R seen in this study may contribute to the reinforcing effects of methamphetamine. This upregulation of Sig-1R may be mediated by increased protein kinase A activity due to downregulation of dopamine D2 autoreceptors. Stefanski, R., Justinova, Z., Hayashi, T., Takebayashi, M., Goldberg, S.R. and Su, T-S. Psychopharmacology DOI 10.1007/s00213-004-1779-9

The Opioid Antagonist Naltrexone Reduces the Reinforcing Effects of D9-Tetrahydro- cannabinol (THC) in Squirrel Monkeys Experimental evidence from animal studies suggests reciprocal functional interactions between endogenous brain cannabinoid and opioid systems. There is recent evidence for a role of the opioid system in the modulation of the reinforcing effects of synthetic cannabinoid CB1 receptor agonists in rodents. Since _9-tetrahydrocannabinol (THC), the natural psychoactive ingredient in marijuana, is actively and persistently self-administered by squirrel monkeys, this provides an opportunity to directly study involvement of opioid systems in the reinforcing effects of THC in non-human primates. The objective of this study was to assess the effects of naltrexone, an opioid antagonist, on THC self-administration behavior in squirrel monkeys. Monkeys pressed a lever for intravenous injections of THC under a 10-response, fixed-ratio (FR) schedule with a 60-s time-out after each injection. Effects of pre-session treatment with naltrexone (0.03-0.3 mg/kg intramuscularly, 15 min before session) for 5 consecutive days on self-administration of different doses of THC (2 to 8 µg/kg/injection) were studied. Self-administration responding for THC was significantly reduced by pretreatment with 0.1 mg/kg of naltrexone for five consecutive daily sessions. Naltrexone pretreatment had no significant effect on cocaine self-administration responding under identical conditions. Self-administration behavior under a fixed-ratio schedule of intravenous THC injection was markedly reduced by daily pre-session treatment with naltrexone, but remained above saline self-administration levels. These findings demonstrate for the first time the modulation of the reinforcing effects of THC by an opioid antagonist in a non-human primate model of marijuana abuse. Justinova, Z., Tanda, G., Munzar, P. and Goldberg, S. R. Psychopharmacology DOI: 10.1007/s00213-003-1693-6

Neuropsychopharmacology Section, Behavioral Neuroscience Research Branch

Cocaine Exposure Increases the Expression of a Newly-Identified Brain Protein in Brain Areas Relevant to Addiction IRP scientists have previously described a family of brain-specific catecholamine-regulated proteins, which bind the addiction-relevant neurotransmitter dopamine in brain areas that are involved in the brain changes that accompany drug addiction. Now, they have identified a new member of that family by molecular cloning - a 40 kDa catecholamine-regulated protein termed CRP40. CRP40 is dopamine-inducible and has properties similar to previously-identified members of the family. The effects of acute and chronic cocaine treatment on CRP40 in brain areas relevant to addiction have now been studied in laboratory animals. Acute and chronic cocaine treatment significantly increased CRP40 expression in the nucleus accumbens and neostriatum, whereas chronic cocaine treatment increased CRP40 expression in the nucleus accumbens only. No effects were seen in prefrontal cortex or medulla. Pretreatment with anisomycin, a protein synthesis inhibitor, blocked cocaine-induced expression of CRP40, suggesting that cocaine's effects on CRP40 involves protein synthesis. Cocaine treatment did not affect levels of other brain proteins studied. These findings suggest that CRP40 is associated with high extracellular dopamine levels, and could play a neuroprotective role against cocaine-induced oxidative stress. Sharan, N., Chong, V.Z., Nair, V.D., Mishra, R.K., Hayes, R.J. and Gardner, E.L. Synapse, 47, pp. 33-44, 2003.

Dopamine D3 Receptor Antagonists as Potential Anti-Addiction, Anti-Craving and Anti-Relapse Medications for the Treatment of Addiction IRP scientists have previously found that acute blockade of the dopamine D3 receptor in the rat brain (which is neuroanatomically restricted to the mesolimbic dopamine system, implicated in drug-induced reward and drug-seeking behavior) dose-dependently attenuates cocaine-enhanced brain-stimulation reward, acquisition of cocaine-induced conditioned cue preference, expression of cocaine-induced conditioned cue preference, acquisition of heroin-induced conditioned cue preference, expression of heroin-induced conditioned cue preference, and cocaine-triggered relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their intravenous cocaine-taking behavior. Now, these researchers have found that SB277011A, a high-potency high-selectivity dopamine D3 receptor antagonist inhibits intravenous cocaine self-administration if the work-demand to receive intravenous cocaine is increased from low to moderate, or if the unit-dose of intravenous cocaine received is lowered from 1.0 mg/kg to 0.75 mg/kg, 0.5 mg/kg, 0.25 mg/kg, or 0.125 mg/kg. Also, the break-point for intravenous cocaine self-administration under progressive-ratio reinforcement is lowered by SB277011A, suggesting that dopamine D3 receptor antagonism lowers cocaine's reward value. These findings suggest that dopamine D3 receptor antagonists are worthy of further investigation as potential anti-addiction, anti-craving, and anti-relapse medications for the treatment of drug abuse. Gilbert, J., Xi, Z.-X., Campos, A.C., Ashby, C.R. Jr., Heidbreder, C.A. and Gardner, E.L. Poster, 2003. Abstract 422.10. Society for Neuroscience Annual Meeting, New Orleans, LA, November 8-12, 2003.

Dopamine D3 Receptor Antagonists as Potential Anti-Addiction, Anti-Craving and Anti-Relapse Medications for the Treatment of Addiction IRP scientists have found that SB277011A, a high-potency high-selectivity dopamine D3 receptor antagonist dose-dependently inhibits nicotine-enhanced brain-stimulation reward. As drug-enhanced brain reward is believed to be a neural substrate for addiction, these findings suggest that dopamine D3 receptor antagonists are worthy of further investigation as potential anti-addiction, anti-craving, and anti-relapse medications for the treatment of drug abuse. These findings also suggest a specific utility for dopamine D3 antagonists - to assist cigarette smokers in breaking their nicotine dependence and to quit smoking. Campos, A.C., Xi, Z.-X., Gilbert, J., Ashby, C.R. Jr., Heidbreder, C.A. and Gardner, E.L. Poster, 2003. Abstract 322.8. Society for Neuroscience Annual Meeting, New Orleans, LA, November 8-12, 2003.

Dopamine D3 Receptor Antagonists as Potential Anti-Addiction, Anti-Craving and Anti-Relapse Medications for the Treatment of Addiction IRP scientists have previously found that blockade of dopamine D3 receptors in the rat brain by the high-potency high-selectivity dopamine D3 receptor antagonist SB277011A dose-dependently attenuates cocaine-enhanced brain-stimulation reward and cocaine-triggered relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their intravenous cocaine-taking behavior. Now, these researchers have found that NGB2904, another high-potency high-selectivity dopamine D3 receptor antagonist, likewise dose-dependently attenuates cocaine-enhanced brain-stimulation reward and cocaine-triggered relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their intravenous cocaine-taking behavior. These confirmatory findings with a new D3 receptor antagonist suggest that dopamine D3 receptor antagonists are worthy of further investigation as potential anti-addiction, anti-craving, and anti-relapse medications for the treatment of drug abuse. Xi, Z.-X., Gilbert, J., Campos, A.C., Ashby, C.R. Jr., Gardner, E.L. and Newman, A.H. Poster, 2003. Abstract 422.9. Society for Neuroscience Annual Meeting, New Orleans, LA, November 8-12, 2003.

The Basolateral Amygdaloid Nucleus in the Brain Mediates the Modulation of Brain-Reward by Drug-Associated Environmental Cues Environmental cues (sights, smells, sounds) that have been previously associated with drug-taking or alcohol-taking are powerful triggers that provoke relapse to drug-seeking and drug-taking behavior. IRP scientists have previously reported that the ability of such environmental cues to trigger relapse depends upon the intact functioning of the basolateral amygdaloid nucleus in the brain. IRP scientists have also previously shown that such environmental cues alter brain-stimulation reward, and that electrical stimulation of the amygdaloid nucleus in the brain alters brain-reward functions. Now, these researchers have discovered that the ability of drug-associated environmental cues to alter brain-reward functions depends upon the intact functioning of the basolateral amygdaloid nucleus in the brain. Thus, the basolateral amygdala is necessary for cues associated with previous drug or alcohol exposure to modulate reward functions within the classically-described reward circuitry of the brain. These findings have implications for understanding the brain substrates that underlie the motivation to engage in drug-taking behavior, and may help to elucidate the brain mechanisms underlying drug craving. Hayes, R.J. and Gardner, E.L. Poster, 2003. Abstract 421.6. Society for Neuroscience Annual Meeting, New Orleans, LA, November 8-12, 2003.

Chemistry and Drug Metabolism Section, Clinical Pharmacology & Therapeutics Research Branch

Positive Chemical ionization Gas Chromatography-Mass Spectrometry Method Development IRP scientists have developed a new method for the simultaneous analysis of tetrahydrocannabinol, the primary psychoactive component of cannabis, and two of its metabolites. This method has been applied to the analysis of cannabinoids in plasma following controlled oral administration. The oral route of administration is important not only as a route for cannabis abuse, but also for hemp products containing active cannabinoids and for therapeutic use of Dronabinol for AIDS wasting disease and as an anti-emetic. Plasma concentrations following the oral route are very low compared to those following smoked cannabis. Gustafson, R.A., Moolchan, E.T., Barnes, A., Levine, B., and Huestis, M.A. Journal of Chromatography B, 798, pp. 145-154, 2003.

Oral Fluid Testing for Drug Impaired Driving Drug impaired driving is a major safety issue in the United States and internationally. IRP scientists have contributed important information on the disposition of cocaine, methamphetamine and opiates and metabolites into oral fluid, a new alternative matrix for the detection of drug use, following controlled drug administration. These data help to provide a science-based rationale for setting policy, identifying drugged drivers, and identifying individuals for treatment based interventions. These data are useful for interpreting the results of oral fluid drug tests. Kacinko, S.L., Barnes, A.J., Kim, I., Moolchan, E.T., Wilson, L., Cooper, G.A., Reid, C., Baldwin, D., Hand, C.W. and Huestis, M.A. Forensic Science International, 141, pp. 41-48, 2004.

In Utero Drug Exposure In utero exposure to cocaine and heroin poses significant health and behavioral development risks to the unborn fetus. IRP investigators are conducting several clinical protocols directed at improving monitoring of drug use by the pregnant addict during gestation and in improving the detection of drug exposure at birth. These data are essential for determining the relationship between the magnitude, frequency and timing of drug use and maternal and neonatal outcomes. It is not yet clear to what degree each of these factors contributes to adverse outcomes. In addition, these data are essential for differentiating drug from non-drug exposed infants in research addressing physiological and developmental outcomes of in utero drug exposure. Dams, R., Huestis, M.A., Lambert, W.E. and Murphy, C.M. Journal of the American Society for Mass Spectrometry, 14, pp. 1290-1294, 2003.

Nicotine Serves as an Effective Reinforcer of Intravenous Drug-Taking Behavior in Human Cigarette Smokers Although numerous studies have documented that nicotine can function as an effective reinforcer of intravenous self-administration behavior in animals, it has not been clearly shown to maintain intravenous self-administration behavior above vehicle placebo levels in humans. Here, IRP scientists compared the reinforcing effectiveness of nicotine versus saline placebo in human research volunteers responding under fixed-ratio (FR) schedules of intravenous drug self-administration while systematically increasing response requirements. Eight male cigarette smokers resided in an inpatient research unit. During 3-hr sessions, intravenous injections of nicotine and saline were available concurrently and were contingent on responding (pulling a lever). Nicotine dose (0.75, 1.5, 3.0 mg/injection), time-out (TO) value after each injection (1 to 20 min) and FR response requirement (10 to 1600) were varied in different subjects over consecutive sessions. Number of nicotine injections/session significantly decreased as dose/injection increased and the number of self-administered nicotine injections was significantly greater than the number of self-administered saline injections across conditions. When FR value was progressively increased over sessions, response rates for nicotine, but not saline, injections increased, with maximal rates at the highest FR values. Rates of responding and injections/session were markedly and significantly higher for nicotine than for saline at FR values of 200 and above. Subjects rated effects of nicotine as both significantly more positive and more negative than saline placebo, with positive ratings significantly higher than negative ratings. Nicotine functioned as a prototypic drug of abuse, serving as an effective reinforcer of intravenous drug-taking behavior in human cigarette smokers. Subjects adjusted their responding to response requirements in a way that maintained relatively constant levels of nicotine injections per session. Harvey, D.M., Yasar, S., Heishman, S.J., Panlilio, L.V., Henningfield, J. E. and Goldberg, S.R. Psychopharmacology DOI: 10.1007/s00213-004-1818-6.

Treatment of Adolescent Tobacco Dependence Over 1,300 adolescent smokers were screened and 156 patients enrolled. This first study is now completed showing both the feasibility and practicality of an outpatient clinical laboratory for highly-dependent smokers who are motivated to quit. Of the 50 completers (age 15.4±1.5 years, 64% female, 66% Caucasian, 53% at least one psychiatric diagnosis, 41% current marijuana use), biochemically-confirmed (exhaled carbon monoxide less than 6 ppm) continuous and point prevalence abstinence rates at 3 months were 24% and 44% respectively (10% and 19% of 120 randomized); mean decrease in self-reported cigarette consumption was 14.4±8.3 CPD (85%) across groups for completers at 3 months. Findings demonstrated that adolescent smokers randomized to the nicotine patch, compared to those receiving placebo, have significantly higher prolonged abstinence (quit) rates (18% vs. 2.5% Chi square p= 0.028) (OR 8.36 CI 0.95- 73.1). In this first study of the gum in teenage smokers, compliance rates were suboptimal indicating the need for higher instructional support for use of this treatment modality. Overall, the study showed reduced cigarette consumption but not smoke exposure indicating compensatory smoking among adolescents who did not quit. While the effect of behavioral intervention in our study might have exceeded that of NRT, cessation rates for this highly dependent sample of comorbid adolescents are somewhat encouraging. Several analyses (e.g., adequacy of nicotine and cotinine concentrations) are pending. Moolchan, E.T., Robinson, M.L., Schroeder, J.R., Ernst, M., Heishman, S.J., Pickworth, W.B., Cone, E.J., Cadet, J.L. and Henningfield, J.E. Oral presentation at the 10th Annual Meeting of the Society for Research on Nicotine and Tobacco, Scottsdale, AZ 2/10-15, 2004.