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Director's Report to the National Advisory Council on Drug Abuse - September, 2003

Research Findings - Basic Research

Effect of Buproprion on Nicotine Self-Adminstration in Rats

This study investigated the effects of bupropion, which inhibits monoamine uptake and nicotine acetylcholine receptor (AChR) function, on nicotine self-administration in rats. The effects of bupropion were compared with methamphetamine and apomorphine, which do not affect nAChR function. The specificity of bupropion's effects on nicotine self-administration, sucrose-maintained responding and amphetamine self-administration were also investigated. Bupropion produced a biphasic dose-response pattern at both doses (0.01 and 0.02 mg/kg/inf) of nicotine. Low doses of bupropion increased nicotine self-administration while high doses decreased self-administration. A similar pattern of responding was produced with methamphetamine challenges. Apomorphine had no effect at low doses, but reduced nicotine self-administration at high doses. The low dose of bupropion appeared to be selective, as it did not alter responding for sucrose and amphetamine. The high dose of bupropion, however, appeared to be non-specific, since it also dose-dependently decreased responding for sucrose and amphetamine. The investigators conclude that the increase in nicotine self-administration produced by low doses of bupropion is likely due to inhibition of dopamine and norepinephrine transporters combined with inhibition of nAChRs. Rauhut, A.S., Neugebauer, N., Dwoskin, L and Bardo, M.T. Effect of Bupropion on Nicotine Self-administration in Rats. Psychopharmacology (online publishing: June2003).

Silent Antagonist of Anandamide Receptor

The cannabinoid analog abnormal cannabidiol [abn-cbd; (-)-4-(3-3,4-trans-p-menthadien-[1,8]-yl)-olivetol] does not bind to CB1 or CB2 receptors, yet it acts as a full agonist in relaxing rat isolated mesenteric artery segments. Vasorelaxation by abn-cbd is endothelium-dependent, pertussis toxin-sensitive, and is inhibited by the BKCa channel inhibitor charybdotoxin, but not by the nitric-oxide synthase inhibitor N_-nitro-L-arginine methyl ester or by the vanilloid VR1 receptor antagonist capsazepine. The cannabidiol analog O-1918 does not bind to CB1 or CB2 receptors and does not cause vasorelaxation at concentrations up to 30 _M, but it does cause concentration-dependent (1-30_M) inhibition of the vasorelaxant effects of abn-cbd and anandamide. In anesthetized mice, O-1918 dose-dependently inhibits the hypotensive effect of abn-cbd but not the hypotensive effect of the CB1 receptor agonist (-)-11-OH-_9-tetrahydrocannabinol dimethylheptyl. In human umbilical vein endothelial cells, abn-cbd induces phosphorylation of p42/44 mitogen activated protein kinase and protein kinase B/Akt, which is inhibited by O-1918, by pertussis toxin or by phosphatidylinositol 3 (PI3) kinase inhibitors. These findings indicate that abn-cbd is a selective agonist and that O-1918 is a selective, silent antagonist of an endothelial "anandamide receptor," which is distinct from CB1 or CB2 receptors and is coupled through Gi/Go to the PI3 kinase/Akt signaling pathway. Offertáler, L., Mo, F.M., B‡tkai, S., Liu, J, Begg, M., Razdan R.K., Martin, B.R., Bukoski, R.D. and Kunos, G. Selective Ligands and Cellular Effectors of a G Protein-Coupled Endothelial Cannabinoid Receptor. Molecular Pharmacology, 63, pp. 699-705, 2003.

FAAH Inhibition

The available extracellular concentration of the endogenous cannabinoid anandamide is regulated, in part, by uptake into neurons and glia where it is degraded by the fatty acid amide hydrolase (FAAH), among other enzymes. Inhibitors of FAAH can be considered as indirect cannabinoid receptor agonists, but without the common side effects of traditional agonists such as THC. Thus, FAAH inhibitors are useful pharmacological tools and have potential as pharmaceutical agents. Previously investigated classes of FAAH inhibitors include: palmitylsulfonyl fluoride, organophosphorous pesticides, the anesthetic propofol, alpha-keto esters and amides, and fatty acid sulfonyl fluorides. In a collaborative study with the University of Urbino, Dr. Daniele Piomelli has recently reported on the preparation, modeling, and pharmacological testing of a new class of FAAH inhibitor, which are alkylcarbamic acid aryl esters. Several of these compounds displayed nanomolar or low micromolar IC50 inhibitory values toward FAAH activity, without affecting acetylcholinesterase or butylcholinesterase activity, and without appreciable binding to the CB1/CB2 receptors. In general, ester or urea analogs of these compounds were not effective as inhibitors, and replacement of oxygen with sulfur (resulting in a thiocarbamate) was also not successful. The use of molecular modeling has suggested that carbamates with a meta-substituted aryl group possess a curvature which somewhat resembles the "bent" or "U-shape" adopted by fatty acids such as arachidonic acid in binding to different proteins. To date, these carbamates exhibit little in-vivo cannabimimetic effects (such as catalepsy, reduced body temperature, or stimulation of feeding), but they do show potential as anxiolytic agents in rodent models. Tarzia, G., Duranti, A., Tontini, A., Piersanti, G., Mor, M., Rivara, S., Plazzi, P., Park, C., Kathuria, S., and Piomelli, D. Journal of Medicinal Chemistry, 46, pp. 2352-2360, 2003.

Methamphetamine Neurotoxicity and Neurokinin Receptors

Methamphetamine (METH), a long lasting derivative of d-amphetamine, is an addictive substance and is known to cause extensive dopaminergic (DA) neural degeneration in the central nervous system. As METH also augments striatal Substance P (SP) levels, a neuropeptide intimately involved in postsynaptic actions of DA in striatum region of brain, Dr. Jing Yu and associates at Hunter College CUNY speculated that SP plays a role in METH-induced DA toxicity and neural damage in the striatum. To test this hypothesis, they designed studies to assess the effects of concurrent administration of METH and selective non-peptide neurokinin-1 (NK-1) receptor antagonists on several markers of DA terminal toxicity in the mouse striatum. Their studies demonstrate for the first time that pharmacological blockade of NK-1 receptors is protective against neurochemical toxicity of striatal DA terminals induced by METH. Their results also provide evidence that tachykinins, particularly SP, acting through NK-1 receptors, play a crucial role in the pathogenesis of nigrostriatal DA terminal degeneration induced by METH. This finding could lead to novel therapeutic strategies to offset drug addictions as well as in the treatment of a number of other disorders such as, Parkinson's and Hunting's diseases. Yu, J., Cadet, J.L. and Angulo, J.A. Neurokinin-1 (NK-1) Receptor Antagonists Abrogate Methamphetamine-induced Striatal Dopaminergic Neurotoxicity in the Murine Brain. Journal of Neurochemistry, 83, pp. 613-622, 2002.

Gene Therapy Increases the Efficacy of Morphine Analgesia

Li-Yen Mae Huang of the University of Texas Medical Branch and her colleagues have been investigating the use of gene therapy to make pain treatments more effective. Specifically, she injected adeno-associated viral (AAV) vectors encoding mu-opioid receptor cDNA into the dorsal root ganglia (DRG) or sciatic nerves of rats. This resulted in a substantial long-term increase in the number of mu-opioid receptors identified in DRG neurons, many of which relay pain signals. In rats with this treatment, morphine was much more effective at producing analgesia, and there was a decrease in the tolerance typically seen with repeated morphine administration. This treatment might provide a novel way to make opioid treatments more effective, allowing smaller doses to be used, and decreasing the incidence of side effects. Yanping, Y.X., Gu, Y., Li, G., and Huang, L.M. Efficiencies of Transgene Expression in Nociceptive Neurons Through Different Routes of Delivery of Adeno-Associated Viral Vectors, Human Gene Therapy, 14, pp. 897-906, 2003.

Targeting the Purinergic Neuromodulator -- A Potential Therapeutic Strategy to Reduce Withdrawal from Opiates

While neuromodulators are known for their roles in normal brain function, much less is documented about their function in the addicted brain. Studies have suggested that the neuromodulator adenosine is involved in drug addiction and withdrawal. Recently, Dr. Williams' work suggests that chronic exposure to morphine may alter adenosine metabolism in the nucleus accumbens. His lab examined the effects of chronic morphine treatment (subcutaneous implantation of morphine pellets in rats for one week) on the ability of adenosine to inhibit excitatory postsynaptic currents in the medium spiny neurons of the nucleus accumbens. Although the treatment did not alter the level of adenosine-mediated tonic inhibition of excitatory synapses in the nucleus accumbens, it did induce a leftward shift in the adenosine dose-response curve, indicating an increase in the sensitivity of synaptic currents to exogenously applied adenosine. This shift was not due to a change in adenosine receptors or their effectors, because chronic morphine treatment had no effect on the dose-response relationship of a non-metabolized adenosine receptor agonist. When adenosine transport was blocked, it eliminated the shift of the adenosine dose-response curve induced by chronic morphine. These data suggest that the increase in the sensitivity of nucleus accumbens synapses to the inhibitory effects of adenosine may be due to a decrease in adenosine transport. The identification of these changes in the adenosine system after chronic drug exposure may help identify new therapeutic strategies aimed at reducing the symptoms of withdrawal from opiates. Brundege, J.M. and Williams, J.T. Increase in Adenosine Sensitivity in the Nucleus Accumbens following Chronic Morphine Treatment. Journal of Neurophysiology, 87, pp. 1369-1375, 2002.

Counter-Regulation of Serotonin Release by GABAergic and Glutamatergic Inputs is Modulated by Morphine

Dysregulation of serotonin (5-HT) levels in the nucleus accumbens (NAc) plays a role in opiate addiction and withdrawal symptoms. Dr. Tao's work previously showed that release of 5-HT is regulated in a counter-balanced manner by glutamatergic and GABAergic inputs to 5-HT neurons. Activation of glutamate receptors enhances 5-HT release while activation of the GABA receptors does the opposite. This counter-regulation is, in turn, modulated by opioids. For example, in the dorsal raphe nucleus (DRN) -- whose 5-HT neurons are afferent to the NAc -- release of 5-HT is tonically induced by endogenous opioid peptide via delta receptors and enhanced by exogenous morphine via activation of both mu and delta receptors. These effects are mediated by opioid receptors on both GABAergic and glutamatergic inputs to 5-HT neurons in the DRN, as release is increased after blockade of GABAA receptors and decreased after blockade of glutamate receptors. However, local infusion of morphine into the DRN inhibits both glutamate and GABA release, which suggests that the overall impact of opioids on 5-HT release depends on the balance of GABAergic and glutamatergic afferents. In this study, the investigators used microdialysis to characterize the effects of systemic morphine on 5-HT release in the rat. Their first aim was to determine whether opioids alter 5-HT release in the NAc locally (by modulating DRN afferents) or, alternatively, by effects on 5-HT neurons within the DRN. They found that the morphine-induced increase of 5-HT was blocked or attenuated when antagonists to either opioid, GABAA, GABAB, or glutamate receptors were locally infused into the DRN, but not when infused into the NAc. Thus the site of regulation appears to be only within the DRN. A second aim was to further characterize the GABA/glutamate counter regulation of 5-HT release. Their results indicate that morphine inhibits GABA release more than glutamate release, thus the net effect of morphine is to increase 5-HT efflux. Further studies are needed to determine how the counter-regulation system is affected when brain homeostasis is altered by chronic exposure to drugs of abuse or stress. Tao, R. and Auerbach, S.B. GABAergic and Glutamatergic Afferents in the Dorsal Raphe Nucleus Mediate Morphine-induced Increase in Serotonin Efflux in the Rat CNS. Journal of Pharmacology and Experimental Therapeutics, 303, pp. 704-710, 2002.

Ofloxacin as a Reference Marker in Hair of Various Colors

Diana Wilkins and her colleagues, proposed that the administration of a reliable marker substance to human subjects may enhance the ability to identify drug use and treatment compliance in drug treatment programs. The goal of the study was to determine if an oral dose of the antibiotic, Ofloxacin (OFLX) could be used as a marker substance to establish reference points with respect to time in hair of various colors. Male and female subjects (n=32) between 18 and 40 years of age received 800 mg of OFLX as a divided oral dose on a single day. Subjects were restricted from cutting their hair or performing chemical treatments. Hair was collected (by cutting) before, and at weeks 4, 5, 6, and 7 after OFLX administration. Subjects were classified as having black (n=5), brown (n=13), blonde (n=8), or red (n=6) hair. Hair was segmented into 3.0 cm segments prior to chemical digestion, extraction, and analysis by HPLC. At 7 weeks, the OFLX concentrations in the first 3.0 cm of hair closest to the scalp were as follows: 30.6±8.5 (black), 6.0±1.8 (brown), 3.5±1.6 (blonde), and 1.4±0.3 (red). A similar pattern was found in hair collected at weeks 4-6. Quantitative eumelanin (EUM) hair concentrations for each subject were also determined by HPLC. A strong relationship between OFLX concentration at 7 weeks and EUM was noted. For six subjects, the authors determined the intra-subject variability of OFLX incorporation into individual hair strands. Four strands from each subject were cut into 2-mm segments and analyzed. OFLX appeared in segments 1-10 at week 5 (the first centimeter of hair). OFLX appeared in segments 2-20 at week 7 (first and second centimeter of hair). The maximum OFLX concentration (the "band" of drug) and location was then determined for each strand. The maximum OFLX concentration was measured in segments 2-5 at week 5 for all subjects (within the first centimeter of hair length). The maximum OFLX concentration was measured in segments 3-8 at week 7 (within the first and second centimeter of hair). This was consistent with a growth rate of less than 1.0 cm/month, although considerable inter-subject variability was found. No significant axial diffusion of OFLX along the hair shaft beyond the first 3.0 cm of hair was noted. Despite a strong effect of hair color, these data suggest that OFLX may be a suitable marker substance for hair, allowing subjects to serve as their own "controls." Wilkins, D.G., Mizuno, A., Borges, C.R., Slawson, M.H., and Rollins, D.E. Ofloxacin as a Reference Marker in Hair of Various Colors, Journal of Analytical Toxicology, 27, pp. 130-136, 2003.

Navigated Laser Capture Microdissection as an Alternative to Direct Histological Staining for Proteomic Analysis of Brain Samples

Advances in biology often arise as a result of advances in technology. A relatively new technology is laser capture microdissection (LCM). This technique uses a laser to dissect individual cells or cell clusters such as a brain nucleus from tissue slices. In this study, the researchers were interested in analyzing the protein from the microdissected sample using the proteomic technologies, two-dimensional electrophoresis (2-DE) and mass spectrometry. It had previously been demonstrated that histological stains used to visualize cells can interfere with 2-DE analysis of proteins. Therefore, in these studies, the researchers set out to examine an approach called "navigated LCM" as an alternative to LCM of stained tissue. The navigated LCM uses an adjacent, stained tissue slice to navigate dissection of the unstained tissue slice. Navigated LCM requires that the tissue slice to be dissected be fixed with 70% ethanol and cleared with xylene. Three major questions had to be answered. When compared to manually dissected, unstained, unfixed tissue: Does navigated LCM affect total protein recovery? Does navigated LCM affect 2-DE protein spot resolution? Does navigated LCM affect protein identification or protein coverage by mass spectrometry? Examining brain tissue slices, the researchers found no significant difference between manually dissected, unstained, unfixed tissue and the navigated LCM, ethanol-fixed tissue in terms of protein recovery, 2-DE resolution or protein identification and coverage by mass spectrometry. This demonstrates that the ethanol fixation had no significant effect on protein analysis and that navigated LCM could be used for proteomics analysis of brain nuclei regardless of size, shape or location. This approach could be used to perform proteomics analysis of changes in protein expression in specific brain regions associated with disease or pharmacological treatment. Mouledous, L., Hunt, S., Harcourt, R., Harry, J., Williams, K.L., and Gutstein, H.B. Proteomics, 3, pp. 610-615, 2003.

Naltrexone Response in Alcohol-dependent Patients is Associated with a Functional Polymorphism of the m-Opioid Receptor Gene

Naltrexone, which primarily blocks the endogenous m-opioid receptor, is prescribed to treat alcohol dependence and narcotic addiction. Two specific polymorphisms in exon 1 of the mu-opioid receptor gene have been shown to have potential functional relevance. Therefore, David Oslin and colleagues examined the hypothesis that there may be an association between these polymorphisms and treatment responses to naltrexone. A total of 82 patients who were randomized to naltrexone and 59 who were randomized to placebo were genotyped for the two variants (Asn40Asp and Ala6Val). The association between genotype and drinking outcomes was measured over 12 weeks of treatment. Subjects with one or two copies of the Asp40 allele who were treated with naltrexone had significantly lower rates of relapse (p=0.04) and a longer time to return to heavy drinking (p=0.04) than those homozygous for the Asn40 allele. Abstinence rates and relapse rates did not differ between the two genotyped groups among those assigned to placebo. Unfortunately, the number of subjects in the study was too small to examine the effects of the Ala6Val variant. However, the findings for the Asn40Asp variant suggest that genotyping of the m-opioid receptor may be useful for identifying patients who are most likely to respond to naltrexone. Oslin, D.W., Berrettini, W., Kranzler, H.R., Pettinati, H., Gelernter, J., Volpicelli, J.R., and O'Brien C.P. A Functional Polymorphism of the m-Opioid Receptor Gene is Associated with Naltrexone Response in Alcohol-Dependent Patients. Neuropsychopharmacology, 28, pp. 1546-1552, 2003.

A Developmental Role for Nicotinic Acetylcholine Receptors (NAChr) in Modulation of Passive Avoidance Behavior

Chronic nicotine exposure exerts multiple effects on the developing brain that likely contribute to subsequent impaired function as adults. High affinity nicotinic cholinergic receptors are expressed normally throughout the brain and are thought to be involved in the development and function of synaptic activity. Because of the extensive distribution of these receptors, however, efforts to determine a definitive role for nAChRs and/or the underlying anatomical regions have been problematic. Dr. Marina Picciotto and her colleagues at Yale studied passive avoidance behavior, a test of fear associated learning, in transgenic mice with inducible expression of the ß2 subunit of the AChRs in corticothalamic projection neurons. They found that nicotine-induced changes in neurotransmission through AChRs during development were associated with impaired passive avoidance learning as adults. King, S., Marks, M., Grady, S., Calderone, B., Koren, A., Muhkin, A., Collins, A., and Picciotto, M. Conditional Expression in Corticothalamic Efferents Reveals a Developmental Role for Nicotinic Acetylcholine Receptors in Modulation of Passive Avoidance Behavior. Journal of Neuroscience, 23(9), pp. 3837-3843, 2003.

Repeated Nicotine Administration Enhances Cocaine-Seeking in Rats

Drugs with high abuse liability such as psychostimulants, opioids, nicotine or alcohol are likely to be co-administered, and the use of one drug may trigger craving and relapse for another drug. Dr. Greg Mark and his colleagues at the Oregon Health and Science University School of Medicine studied the effects of acute and chronic nicotine treatment on the self-administration of cocaine in rats. Acute, subcutaneous injection of nicotine 3 minutes before access to cocaine resulted in an inverted U-shaped dose-response function (low nicotine doses increased and high doses decreased the number of cocaine infusions). Repeated nicotine administration with the high dose before each daily session significantly increased the number of cocaine infusions by the 8th day of nicotine exposure. In addition, in sessions where responding no longer led to cocaine infusions, i.e. extinction, injections of nicotine caused a reinstatement of the previously reinforced responding. Dr. Mark concluded that nicotine facilitates cocaine reinforcement, can contribute to the transition from moderate drug taking to an escalation of drug intake, and may trigger relapse to drug taking. Bechtholt, A.J. and Mark, G.P. Enhancements of Cocaine-Seeking Behavior by Repeated Nicotine Exposure in Rats. Psychopharmacology, 162, pp. 178-185, 2002.

A Conditional Deletion of the NR1 Subunit of the NMDA Receptor in Adult Spinal Cord Dorsal Horn Reduces NMDA Currents and Injury-induced Pain

In order to determine the importance of the NMDA receptor (NMDAR) in pain hypersensitivity after injury, Dr. Charles Insturrisi and his research team at the Weill Medical College of Cornell University selectively deleted the NMDAR1 (NR1) subunit in the lumbar spinal cord of adult mice by the localized injection of an adenoassociated virus expressing Cre recombinase into floxed NR1 mice. NR1 subunit mRNA and dendritic protein were reduced by 80% in the area of the virus injection, and NMDA currents, but not AMPA currents, were reduced 86-88% in lamina II neurons. The spatially-restricted NR1 knockdown did not alter heat or cold paw-withdrawal latencies, mechanical threshold, or motor function. However, injury-induced pain produced by intraplantar formalin is reduced by 70%. These results demonstrate conclusively that the postsynaptic NR1 receptor subunit in the lumbar dorsal horn of the spinal cord is required for central sensitization, the central facilitation of pain transmission produced by peripheral injury. South, S.M., Kohno, T., Kaspar, B.K., Hegarty, D., Vissel, B., Drake, C.T., Ohata, M., Jenab, S., Sailer, A.W., Malkmus, S., Masuyama, T., Horner, P., Bogulavsky, J., Gage, F.H., Yaksh, T.L., Woolf, C.J., Heinemann, S.F., and Inturrisi, C.E. A Conditional Deletion of the NR1 Subunit of the NMDA Receptor in Adult Spinal Cord Dorsal Horn Reduces NMDA Currents and Injury-induced Pain. Journal of Neuroscience, 23, pp. 5031-5040, 2003.

Endogenous RGS Protein Action Modulates Mu-Opioid Signaling through G_o: Effects on Adenyly cyclase, Extracellular Signal-Regulated Kinases, and Intracellular Calcium Pathways

RGS (regulators of G protein signaling) proteins are GTPase-activating proteins (GAP) for the G_ subunits of heterotrimeric G proteins and act to regulate signaling by rapidly cycling G protein. RGS proteins may integrate receptors and signaling pathways by physical or kinetic scaffolding mechanisms. To determine whether this results in enhancement and/or selectivity of agonist signaling, Dr. John Traynor and his research team at the University of Michigan have prepared C6 cells stably expressing the mu-opioid receptor and either pertussis toxin-insensitive or RGS- and pertussis toxin-insensitive G_o. They have compared the activation of G protein, inhibition of adenylyl cyclase, stimulation of intracellular calcium release, and activation of the ERK1/2 MAPK pathway between cells expressing mutant G_o that is either RGS-insensitive or RGS-sensitive. The mu-receptor agonist DAMGO and partial agonist morphine were much more potent and/or had an increased maximal effect in inhibiting adenylyl cyclase and in activating MAPK in cells expressing RGS-insensitive G_o. In contrast, mu-opioid agonist increases in intracellular calcium were less affected. The results are consistent with the hypothesis that the GAP activity of RGS proteins provides a control that limits agonist action through effector pathways and may contribute to selectivity of activation of intracellular signaling pathways. Clark, M.J., Harrison, C., Zhong, H., Neubig, R.R., and Traynor, J.R. Endogenous RGS Protein Action Modulates Mu-Opioid Signaling through G_o: Effects on Adenylyl Cyclase, Extracellular Signal-Regulated Kinases, and Intracellular Calcium Pathways. Journal of Biological Chemistry, 278, pp. 9418-9425, 2003. Epub Jan 10, 2003.

Differential Mechanisms of Morphine Antinociceptive Tolerance Revealed in Arrestin-2 Knock-Out Mice

Morphine induces antinociception by activating µ opioid receptors (µORs) in spinal and supraspinal regions of the CNS. Arrestin-2 (arr2), a G-protein-coupled receptor-regulating protein, regulates the µOR in vivo. Dr. Laura Bohn and her colleagues at the Duke University Medical Center previously showed that mice lacking arr2 experience enhanced morphine-induced analgesia and do not become tolerant to morphine as determined in the hot-plate test, a paradigm that primarily assesses supraspinal pain responsiveness. To determine the general applicability of the arr2-µOR interaction in other neuronal systems, they tested arr2 knockout (arr2-KO) mice using the warm water tail-immersion paradigm, which primarily assesses spinal reflexes to painful thermal stimuli. In this test, the arr2-KO mice had greater basal nociceptive thresholds and markedly enhanced sensitivity to morphine. Interestingly, however, after a delay onset, they do ultimately develop morphine tolerance, although to a lesser degree than the wild-type (WT) controls. In the arr2-KO but not WT mice, morphine tolerance could be completely reversed with a low dose of the classical protein kinase C (PKC) inhibitor chelerythrine. These findings provide in vivo evidence that the µOR is differentially regulated in diverse regions of the CNS. Furthermore, although arr2 appears to be the most prominent and proximal determinant of µOR desensitization and morphine tolerance, in the absence of this mechanism, the contributions of a PKC-dependent regulatory system become readily apparent. Bohn, L.M., Lefkowitz, R.J., and Caron, M.G. Differential Mechanisms of Morphine Antinociceptive Tolerance Revealed in Arrestin-2 Knock-Out Mice. J Neurosci., 22(23), pp. 10494-10500, December 1, 2002.

The Cystine-Glutamate Exchanger: A Neurobiological Mechanism Underlying Cocaine Relapse

Repeated cocaine abuse can lead to cocaine addiction, which is often characterized as a compulsive pattern of continued drug taking despite potential harm. For many years NIDA supported researchers have studied this phenomenon in animals using the drug self-administration model in which animals are trained to press a response lever to receive an intravenous injection of cocaine. With this procedure, the animal continues to escalate its drug taking over several days. In extinction conditions, when the drug is no longer provided for lever responding, drug-seeking behavior declines. However, self-administration can be reinstated by administering a priming injection of the drug itself, exposing the animal to environmental cues associated with drug taking, or exposing the animal to a stressor. Because many of these same factors have often been reported to lead to drug abuse relapse, this reinstatement procedure is thought to model drug abuse relapse in human addicts. Through the reinstatement model much has been learned about the changes in brain function (i.e., neuroadaptations) and neural substrates that are thought to be involved in drug-seeking behavior and relapse to drug use. Although it has been known for quite some time that dopamine release in the nucleus accumbens (NAc) is associated with the reinforcing effects of cocaine, it has become evident that this is not the only neurotransmitter or substrate involved in drug addiction or relapse. In particular, increased glutamate neurotransmission in the NAc has been shown to mediate drug-seeking behavior, and withdrawal from cocaine reduces glutamate levels in the NAc. Further detailed investigation of this neurotransmitter and it role in cocaine relapse has been extensively studied by Dr. Peter Kalivas and his colleagues at Medical University of South Carolina. Their recent research has shown that reduction in glutamate brought about by repeated administration of cocaine results from modulation of the cystine/glutamate exchanger, which is found throughout the brain. The exchanger normally helps maintain appropriate glutamate levels in the brain. As cystine is brought into the neuronal cells for glutathion synthesis, glutamate is taken out to help maintain glutamate neurotransmission and glutamatergic tone among neurons in the brain. Through a series of experiments, the investigators concluded that cocaine administration cause a long lasting reduction in glutamate by reducing the activity of the cystine/glutamate exchanger. Cystine administered directly to the NAc restored this exchange and returned the reduced amount of extracellular glutamate caused by cocaine to normal levels. Similarly, administrating N-acetylcysteine systemically, a cystine pro-drug, elevated glutamate in the brains of rats that had been repeatedly administered cocaine and were experiencing cocaine withdrawal. Furthermore, and most importantly, when N-acetylcysteine was administered to rats after withdrawal from repeated cocaine administration, this cystine pro-drug successfully blocked cocaine-induced reinstatement (relapse). These results indicate that repeated abuse of cocaine and cocaine withdrawal compromises glutamatergic tone in the brain and this effect is mediated through the cystine/glutamate exchanger. How cocaine affects the exchanger to reduce glutamate is not clear and will need further investigation. This research, however, does identify the cystine/glutamate exchanger as a neurobiological mechanism that is affected by repeated cocaine administration and possibly linked to drug abuse relapse. Further investigation of this neurobiological mechanism and cocaine-induced neuroadaptation could lead to exciting new leads for pharmacotherapies for treating cocaine addiction and relapse. Baker, D.A., McFarland K., Russell, L.W., Shen, H., Tang,X.C., Toda, S. and Kalivas, P.W. Neuroadaptations in the Cystine-Glutamate Exchange Underlie Cocaine Relapse. Nature Neurosci., 6, pp. 743-749, 2003.

Methylphenidate (Ritalin") and MDMA ("Ecstasy") Adolescent Exposure in Mice: Long-Lasting Consequences on Cocaine-induced Reward and Psychomotor Stimulation in Adulthood

The consequences of methylphenidate and MDMA abuse during adolescence on vulnerability to subsequent drug abuse and relapse in adulthood are unknown. To investigate this question, researchers administered methylphenidate, MDMA or saline to adolescent (26 day old) mice for 7 days. After one month (62 days old; when mice are considered 'adults'), the three groups of mice received cocaine injections for 4 or 5 days to determine their sensitivity to the rewarding and psychomotor stimulating effects of cocaine in adulthood. The rewarding effect of cocaine was determined in the conditioned place preference paradigm, and cocaine-induced psychomotor stimulation was determined by measuring locomotion. They found that cocaine induced the same magnitude of place preference in the control and MDMA groups, suggesting that exposure to MDMA did not alter the acquisition of the rewarding effect of cocaine. The methylphenidate group developed lower place preference, suggesting that exposure to methylphenidate diminished the acquisition of the rewarding effect of cocaine. In the locomotor activity studies, the methylphenidate group showed enhanced sensitivity to cocaine compared to the saline and MDMA groups. To investigate their response to challenge cocaine injection following cocaine withdrawal (a measure for drug relapse), mice remained drug free for 14 days and were re-tested in response to a low dose of cocaine. This low dose of cocaine induced very high place preference response as well as pronounced locomotor stimulation in the methylphenidate and MDMA pre-exposed groups, but not in the saline group. These findings demonstrate that (1) adolescent exposure to methylphenidate or MDMA ultimately elicits similar consequences in response to cocaine during adulthood and (2) the sensitized or heightened response to cocaine is expressed primarily after withdrawal from the initial exposure to cocaine. Thus, although the initial responses to cocaine of the drug pre-exposed mice were not always different from those of the saline pretreated mice, the methylphenidate and MDMA pre-exposed subjects ultimately developed significantly higher sensitivity to both the rewarding and psychomotor stimulating effects of cocaine after the withdrawal period from cocaine. The implications of these findings for human adolescent exposure to methylphenidate and MDMA should be cautiously regarded. However, these results suggest that although methylphenidate and MDMA pre-exposed subjects may not be more prone to initiate drug use, they may exhibit greater vulnerability than unexposed subjects to drug relapse after drug use has been initiated. Achat-Mendes, C., Anderson, K.L., and Itzhak, Y. Methylphenidate and MDMA Adolescent Exposure in Mice: Long-Lasting Consequences on Cocaine-induced Reward and Psychomotor Stimulation in Adulthood. Neuropharmacology, 45, pp. 106-115, 2003.

Mu-Opioid Receptors Mediate Immunosuppression Induced by Chronic Restraint Stress

Psychological stress is associated with immunosuppression in both humans and animals. Although it was well established that psychological stressors stimulate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, resulting in the release of various hormones and neurotransmitters, the mechanisms underlying these phenomena are poorly understood. In this study, the investigators used mice with a genetic knockout of the mu-opioid receptor knockout (MORKO) to examine whether the mu-opioid receptor mediates the immunosuppression induced by restraint stress. Their results showed that wild-type (WT) mice subjected to chronic 12-h daily restraint stress for 2 days exhibited a significant decrease in splenocyte number with a substantial increase in apoptosis and CD95 (Fas/APO-1) expression of splenocytes. The effects were essentially abolished in MORKO mice. Furthermore, inhibition of splenic lymphocyte proliferation, IL-2, and IFN-gamma production induced by restraint stress in WT mice was also significantly abolished in MORKO mice. Interestingly, both stressed WT and MORKO mice showed a significant elevation in plasma corticosterone and pituitary proopiomelanocortin mRNA expression, although the increase was significantly lower in MORKO mice. Adrenalectomy did not reverse restraint stress-induced immunosuppression in WT mice. These data clearly established that the mu-opioid receptor is involved in restraint stress-induced immune alterations via a mechanism of apoptotic cell death, and that the effect is not mediated exclusively through the glucocorticoid pathway. Wang, J.H., Charboneau, R., Barke, R.A., Loh, H.H., and Roy, S. Mu-Opioid Receptor Mediates Chronic Restraint Stress-Induced Lymphocyte Apoptosis. Journal of Immunology, 169, pp. 3630-3636, 2002.

Stress-Induced Suppression of the Immune System after Withdrawal from Chronic Cocaine

Recent evidence suggests that withdrawal from cocaine shares similarities to the stress response. In this study, the investigators examined whether withdrawal from chronic cocaine produces immune system alterations and whether the hypothalamic-pituitary-adrenal axis is involved. Male Sprague-Dawley rats received daily cocaine injections (10 mg/kg i.p.) for or saline for 7 days, followed by 2 h, 1, 2, 4, 6, and 14 days of withdrawal. Proliferation responses of peripheral blood lymphocytes to concanavalin A were significantly suppressed and persisted for up to 6 days during withdrawal from chronic cocaine. Flow cytometric analysis revealed no significant differences in the immunophenotype of blood lymphocytic populations of T cells, B cells, or monocytes at 2 or 6 days of withdrawal from cocaine. Consistent with the suppression in cellular immunity observed in the in vitro response, the in vivo delayed-type hypersensitivity response was also significantly decreased in cocaine withdrawing animals. Plasma corticosterone levels were significantly elevated after cessation of cocaine but returned to basal values by 2 days of withdrawal. The suppressive effects of cocaine withdrawal were no longer observed in either adrenalectomized animals or those treated with the glucocorticoid receptor antagonist mifepristone (RU486), when administered during the first 2 days of withdrawal. These data argue that repeated exposure to cocaine followed by withdrawal leads to an activation of the neuroendocrine stress response, which alters cellular immunity during the initial withdrawal phase and may contribute to an increased susceptibility to infection. Avila, A.H., Morgan, C.A., and Bayer, B.M. Stress-Induced Suppression of the Immune System after Withdrawal from Chronic Cocaine. Journal of Pharmacology and Experimental Therapeutics, 305, pp. 290-297, 2003.

Dual Infection Processes and Opiates

An aspect of disease that is often overlooked are problems associated with multiple infections. However, a recent paper describes experiments examining the effects of opiates on the growth of two disease viruses in culture. Infection of injection drug users (IDUs) with the human T-cell leukemia viruses (HTLVs) or HIV is considerably higher than in the non-IDU population. Also, coinfection with HIV-1 and HTLV type 1 (HTLV-1) occurs more frequently. There is little or no information on the effects of opiates (i.e., morphine) on HTLV infection alone or on coinfection of HTLV-1-infected cells with HIV-1. Therefore, the authors analyzed the in vitro effects of morphine on HIV or HTLV infection alone as well as on dual infection with both viruses. Morphine decreased the in vitro levels of interferon-gamma (IFN-gamma) and IL-2 during single infections, and this effect was reversed by the addition of the opioid antagonist naloxone. In contrast, treatment with morphine resulted in a 31% and 36% increase in IFN-gamma and IL-2 levels, respectively, during dual infection. In addition, naloxone had an apparent additive effect on the morphine-associated enhancement of IFN-gamma and IL-2 expression in the dual-infection model. Despite the high levels of IFN-gamma expression, the viability of the coinfected cells in the presence of morphine was maintained. Importantly, morphine treatment was associated with augmented viral reverse transcription activity in dually infected cultures, apparently to the benefit of HTLV-1. If a similar putative morphine-induced advantage for HTLV-1 production also occurs during in vivo coinfection, opiates such as morphine could contribute to the observed increased rate of HIV-1/HTLV-1 infection in the IDU population in a more direct fashion than was previously believed. Nyland, S.B., Cao, C.H., Bai, Y., Loughran, T.P., and Ugen, K.E. Modulation of Infection and Type 1 Cytokine Expression Parameters by Morphine during in vitro Coinfection with Human T-cell Leukemia Virus Type I and HIV-1. Journal of Acquired Immune Deficiency Syndromes, 32, pp. 406-416, 2003.

A Contingent Payment Model of Smoking Cessation

This study characterized nicotine withdrawal during a 5-day period. Smokers were assigned to one of two contingent monetary groups or a control group. Subjects provided CO samples 3 times/day (morning, afternoon, evening), with completion of subjective questionnaires following the evening sampling. Subjects in the contingent groups had significantly lower CO and salivary cotinine levels, regardless of amount paid. Subjects in the contingent groups reported increased withdrawal symptoms, including increased anxiety and restlessness, hunger, and desire to smoke. The investigators concluded that contingent payment procedures may provide an effective method for studying nicotine withdrawal in smokers. Heil, S.H., Tidey, J.W., Holmes, H.W., Badger, G.J. and Higgins, S.T. A Contigent Payment Model of Smoking Cessation: Effects on Abstinence and Withdrawal. Nicotine and Tobacco Research, 5(2), pp. 205-213, 2003.


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