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National Institute on Drug Abuse

Director's Report to the National Advisory Council on Drug Abuse

September, 2000

Research Findings

Basic Research

Separate Neural Circuits in the Nucleus Accumbens Encode Information About Cocaine Versus Natural Reward

Considerable experimental evidence has implicated the nucleus accumbens as a key brain structure mediating information about the reward value of drugs and other reinforcing stimuli such as food, water, and sexual behaviors. Do drugs activate the same neural circuits regardless of the type of reward, or do separate circuits encode information about different types of rewards? Dr. Regina Carelli and her associates at the University of North Carolina recorded neuronal activity in the n. accumbens of rats as they pressed a lever for either of two natural reinforcers, food and water, or for either food or water paired with intravenous self-administered cocaine. In the rats responding for food or water only, many neurons were phasically active during various phases of the task. Most of the responding neurons showed a similar activity pattern for each of the two natural reinforcers. In contrast, in the n. accumbens of the rats rewarded with water and cocaine, very few of the phasically active neurons responded in a similar way to the two reinforcers, i.e. most cells responded selectively to either water or cocaine, but not both. Similar results were obtained from rats trained with food and cocaine. These findings indicate that in the well-trained animal, cocaine activates a neural circuit in the n. accumbens that is largely separate from the circuit that processes information about food and water reward. These results are consistent with studies showing that selective lesions or pharmacological manipulation of the mesolimbic system can alter cocaine self-administration but will leave operant responding for natural reinforcers relatively unaltered. These results underscore the possibility that pharmacotherapies for cocaine addiction can be developed that leave natural reward systems and consummatory behaviors intact. Carelli, R.M., James, S.G., and Crumling A.J. Evidence that Separate Neural Circuits in the Nucleus Accumbens Encode Cocaine Versus "Natural" (Water and Food) Reward. Journal of Neuroscience, 20, pp. 4255-4266, 2000.

Morphine-6beta-Glucuronide Antagonist 3-O-Methylnaltrexone Antagonized Heroin and Morphine Self-Administration in Rats

In mice, 3-O-methylnaltrexone, a morphine-6beta-glucuronide antagonist, blocks the analgesic actions of morphine-6beta-glucuronide and heroin at doses that are inactive against morphine. Dr. Pasternak and his colleagues found a similar selectivity in rats. 3-O-methylnaltrexone antagonized the analgesic actions of 6-acetylmorphine in Sprague-Dawley rats and heroin in Wistar rats at doses that were inactive against morphine. Inclusion of a fixed dose of 3-O-methylnaltrexone significantly shifted the analgesic dose-response curves for 6-aceylmorphine and heroin without altering the morphine dose-response curves. In a self-administration model, 3-O-methylnaltrexone treatment significantly increased both heroin and morphine intake during the first hour, suggestive of an antagonist effect. This effect at doses of 3-O-methylnaltrexone that were inactive against morphine analgesia implied a role for the morphine-6beta-glucuronide opioid receptor in the reinforcing properties of heroin and morphine. Walker, J.R., King, M., Izzo, E., Koob, G.F., and Pasternak, G. Antagonism of Heroin and Morphine Self-Administration in Rats by the Morphine-6beta-Glucuronide Antagonist 3-O-Methylnaltrexone. Eur. J. Pharmacology, 383(2), pp. 115-9, 1999.

Local Administration of Opioid Agonists Attenuates Capsaicin-Induced Thermal Hyperalgesia

Dr. Woods and his research team at the University of Michigan have conducted work characterizing capsaicin-induced thermal hyperalgesia in rats and evaluating the hypothesis that local administration of either mu or kappa opioid agonists (fentanyl and U50,488, respectively) can attenuate capsaicin-induced nociception. Capsaicin dose-dependently produced thermal hyperalgesia in the rat tail-withdrawal assay. Co-administration of either fentanyl or U50,488 with capsaicin attenuated capsaicin-induced hyperalgesia in a dose-dependent manner. This local antinociception was antagonized by small doses of an opioid antagonist, quadazocine, applied to the tail. However, the locally effective doses of quadazocine, when applied to the back near the scapular region did not antagonize either fentanyl or U50,488. It is therefore concluded that activation of peripheral mu or kappa opioid receptors, in this experimental pain model, can attenuate capsaicin-induced thermal hyperalgesia. These data also support the notion that peripheral antinociception can be achieved by the local administration of analgesics into the injured tissue without producing central side effects. Ko, M.C.H., Tuchman, J.E., Johnson, M.D., Wiesenauer, K. and Woods, J.H. Local Administration of Mu or Kappa Opioid Agonists Attenuates Capsaicin-Induced Thermal Hyperalgesia via Peripheral Opioid Receptors in Rats. Psychopharmacology (Berl), 148(2), pp. 180-185, 2000.

Regulating Cellular Receptors

The delta-opioid receptor is a G-protein coupled receptor that is implicated in the development of morphine tolerance and is found in brain regions associated with reward, motivation, and response to abused drugs. University of California, San Francisco researcher Dr. Mark von Zastrow and his student, Patricia Tsao, are studying the regulation of the delta-opioid receptor and other G-protein coupled receptors on the cellular level. In a recent article, they demonstrated that, when expressed in tissue culture cells, the delta-opioid receptor is down regulated by a novel membrane trafficking mechanism in response to even a brief application of agonist. After agonist-induced endocytosis, the delta-opioid receptor is sorted into a non-recycling pathway, leading to protein degradation. Native delta-opioid receptors expressed in brain tissue exhibit significant agonist-induced down regulation, suggesting that this new membrane trafficking pathway has physiological relevance and may mediate, in part, the long-term actions of agonist drugs. Tsao, P.I. and von Zastrow, M. Type-Specific Sorting of G Protein-Coupled Receptors After Endocytosis. J. Biol. Chem., 275, pp. 11130-11140, 2000.

Functional Imaging of Cocaine-Induced Brain Activation in Rats

Cocaine exerts a complex set of effects on the central nervous system, in part through stimulation of dopaminergic neurotransmission within brain cortical and subcortical limbic structures. To elucidate the anatomic and temporal patterns of regional brain activation following cocaine administration in rats, Dr. Barry Kosofsky and his colleagues at Massachusetts General Hospital performed functional MRI (fMRI). During the course of brain activation in rats that had never been exposed to drugs, cocaine produced anatomically distinct regional differences. Using a selective antagonist, the researchers demonstrated that the D1 dopamine receptor plays an important role in mediating the cocaine effects seen by fMRI. These data affirm the relevance of the rat model system in the study of the cellular and molecular bases of cocaine-induced brain activation in drug addicts. Marota, J.J.A., Mandeville, J.B., et al. Cocaine Activation Discriminates Dopaminergic Projections by Temporal Response: An fMRI Study in Rat. NeuroImage, 11, pp. 13-23, 2000.

Neuronal Nicotine Acetylcholine Receptor Populations

In a recent report by Drs. Allan Collins and Michael Marks, a subpopulation of nicotine acetylcholine receptors in mouse brain has been better defined by the use of the ligands epibatidine and a neurotoxin known as alpha-conotoxin MII. Alpha-conotoxin MII, a naturally occurring ligand isolated from the snail, was labeled with iodine-125 for autoradiographic and binding experiments, and used in competition with tritiated and unlabeled cytisine, nicotine, alpha-bungarotoxin, and epibatidine. The distribution of labeled alpha-conotoxin MII was highest in the mouse brain regions of the geniculate nucleus, olivary pretectal nucleus, and in the zonal layer of the superior colliculus. As an antagonist, alpha-conotoxin MII was a weak inhibitor of nicotine and alpha-bungarotoxin, generally believed to bind at alpha4beta2 or alpha7 receptor populations, respectively. The distribution of tritiated epibatidine was determined in various mouse brain regions, many of which are believed to correspond to cytisine-sensitive alpha4beta2 populations. There are, however, other regions to which epibatidine binds even in the presence of excess cytisine (i.e., cytisine-resistant locations) and these could be divided into those that were sensitive and those that were relatively insensitive to alpha-conotoxin MII. Since both of these latter regions were found to contain alpha3 messenger RNA, the tentative assignments for alpha-conotoxin MII binding regions have been proposed as alpha3beta2 and alpha3beta4 subunits of the neuronal acetylcholine receptors. Whiteaeker, P., McIntosh, J.M., Luo, S., Collins, A.C., Marks, M.J., Molecular Pharmacology, 57, pp. 913-925, 2000.

In Utero Cocaine Exposure Affects the Respiratory System

Dr. Immanuela Moss and her associates recently reported on the role of endogenous opioid systems, specifically met-enkephalin, in the attenuation of respiration by repeated prenatal cocaine exposure. These investigations were designed to delineate the contribution of met-enkephalin, an endogenous opioid peptide, in the attenuation of cocaine-induced respiratory and arousal responses in six- to seven-day-old and in 20- to 21-day-old piglets. In both age groups, prenatal exposure to cocaine increased met-enkephalin immunoreactivity in the respiratory- and arousal-related medullary regions. These findings support the investigators' hypothesis of a modulatory role of met-enkephalin in the normal development of respiratory control and an involvement of this peptide in the attenuation of respiration by repeated prenatal cocaine exposure. Liu, J-K., Laferriere, A. and Moss, I.R. Brain Research Bulletin. 51, pp. 419-424, 2000.

Candidates for the Genetic Basis for Individual Differences in Sensitivity to Cocaine-Induced Seizures

Quantitative trait loci (QTL) studies are used to determine the number and chromosomal location of genes involved in complex behaviors that are influenced by more than one gene. Seizures are a well-known but poorly understood consequence of human cocaine abuse, particularly with respect to individual differences in susceptibility. In rodent models, sensitivity to cocaine seizures is strongly influenced by genotype. For example, several studies have reported significant differences between the C57BL/6 (B6) and DBA/2 (D2) inbred mouse strains in their sensitivity to cocaine-induced seizures. These findings prompted Dr. Belknap and his colleagues to undertake a QTL study of cocaine-induced seizures in two populations derived from the C59BL/6 and DBA/2 mouse strains. Three QTLs emerged as significant (P<0.00005): one for clonic seizures on chromosome 9 (distal), and two for tonic seizures on chromosomes 14 (proximal to mid) and 15 (distal). Two additional QTLs emerged as suggestive (P<0.0015), both associated with clonic seizures on chromosomes 9 (proximal) and 15 (distal). Both QTLs on chromosome 9 were sex-specific, with much larger effects on the phenotype seen in females than in males. The results suggest that different types of seizures have both common and distinct underlying mechanisms, and that there are a variety of factors that may account for individual differences in seizure sensitivity. Some of the QTLs were located in regions of the chromosomes that have been identified as QTLs for other seizure types such as a mouse model of epilepsy. Some of these chromosomal regions are also known to contain nervous system-relevant genes such as those for the dopamine D2 receptor, the serotonin 1B receptor, various glutamate receptors, and ion channel genes. These candidate genes can now be used as the basis of studies on the physiology of cocaine-induced seizures. Hain, H.S., Crabbe J.C., Bergeson, S.E., Belknap, J.K. Cocaine-Induced Seizure Thresholds: Quantitative Trait Loci Detection and Mapping in Two Populations Derived from the C59BL/6 and DBA/2 Mouse Strains. Journal of Pharmacology and Experimental Therapeutics, 293, pp. 180-187, 2000.

Prenatal Exposure to Cocaine Can Affect the Outcome of Serotonin Receptor Stimulation in the Brains of the Offspring When They Are Tested As Adults

Dopamine normally inhibits the activity of cholinergic neurons in the striatum, and this inhibition is an important aspect of the control of motor activity. However, the activity of the dopamine-containing neurons is regulated by serotonergic activity at the 5HT3 receptor site, the only serotonin receptor of the 17 identified thus far that is an ion channel. The application of a 5HT3 agonist normally limits the release of acetylcholine in a dose-dependent manner. However, prenatal exposure to cocaine alters this relationship in female, but not in male rats. Denise Jackson and her colleagues treated rat mothers with 20 mg/kg of cocaine or saline solution (the control) twice daily from embryonic days 15 to 21 (day of birth). The rats were tested as adults (postnatal days 80-120). Both males and females (at two times in the estrous cycle) that had been treated with saline in utero showed the expected inhibition of cholinergic activity after graded doses of a 5HT3 agonist were applied to the neurons; however, females in diestrus, a relatively quiescent period during the rodent estrous cycle, were much less sensitive to the lower doses of the agonist. This is important because it demonstrates a functional difference in the brains of females given cocaine prenatally; a difference that is apparent in adulthood. The data also suggest that estrogen may modify the response, as animals in the phase of the cycle when estrogen is high did not display this deficit. This result suggests that persistent modification of the dopaminergic system has occurred as the cholinergic output is altered, and suggests that other parts of the mesocorticolimbic dopamine system may be functionally altered, possibly including regions that control motor activity and the perception of reward. Bolanos, C.A., Trksak, G.H., Glatt, S.J. and Jackson, D. Synapse, 36, pp. 1-11, 2000.

Two Recent Articles Demonstrate That Opioids/Cannabinoids Are Important in Regulating the Basic Comunicators (Cytokines and Chemokines) of Immune Function

This basic link of these endogenous 'drug' systems with basic elements of immunity are important in both the understanding of basic immune function as well as in understanding disease (e.g. HIV infection) modulation by abused drugs.

1) Regulation of Cytokine and Chemokine Receptors by Opioids
Endogenous and exogenous kappa-opioid agonists have been widely reported to modulate the immune response. Published results of L. Zhang et. al. show that the superantigen-induced proliferative response of thymocytes is inhibited by the selective kappa-opioid agonist (U50,488H). Previous work has established that the kappa-opioid receptor is widely expressed within the thymus; however, little is known about the role of the kappa-opioid receptor in the function of thymocytes. Zhang et. al. measured detectable levels of the cytokines IL-2, IL-4, IL-5, IL-13, and IFN-gamma, and the chemokines, lymphotactin and RANTES, in stimulated thymocyte cultures; however, addition of U50,488H did not alter the expression of these cytokines. Examination of cytokine receptor expression by these thymocytes revealed a significant inhibition in the expression of the transcript For the IL-7 receptor alpha-chain (IL-7R alpha), and these results were confirmed by flow cytometry. Surprisingly, the expression of several other cytokine receptor chains including the common gamma-chain, IL-2R beta, or the IL-2R alpha, IL-4R alpha, and IL-15R alpha chains, was not altered. In contrast to these results, a significant elevation in the expression of the chemokine receptor CCR2 was observed in U50,488H-treated cultures. These results suggest that the kappa-opioid receptor may function to promote cellular migration at the expense of the sensitivity to the growth-promoting/maturation activity of IL-7. Zhang, L and Rogers, T.J. Kappa-Opioid Regulation of Thymocyte IL-7 Receptor and C-C Chemokine Receptor 2 Expression. J. Immunology, 164, pp. 5088-5093, 2000.

2) Regulation of Cytokine Systems by Cannabinoids
Regulation of the activator protein-1 (AP-1) complex is very intricate because it involves phosphorylation state, protein-protein, and protein-DNA interactions. In studies by B.L. Faubert et. al., the regulation of AP-1 activity, with emphasis on c-fos and c-jun regulation, was investigated using cannabinol (CBN) in primary mouse splenocytes In vitroo. Cannabinoid compounds exhibit immunosuppressive actions that are putatively mediated through Gi-protein coupled receptors that negatively regulate adenylate cyclase. However, recent studies suggest that cannabinoids modulate other signaling cascades. Indeed, CBN inhibited binding to AP-1-containing sites from the interleukin-2 promoter. This inhibition of binding was, in part, due to decreased nuclear expression of c-fos and c-jun. B.L. Faubert et. al. further determined that the effects of CBN were due to posttranslational modifications of these phosphoproteins and showed that CBN inhibited the activation of ERK MAP kinases. Thus, cannabinoid-induced immunosuppression involves disruption of the ERK signaling cascade. Faubert, B.L. and Kaminski, N.E. AP-1 Activity is Negatively Regulated by Cannabinol Through Inhibition of its Protein Components, c-fos and c-jun, J. Leukocyte Biology, 67, pp. 259-266, 2000.

Cannabinoid Effects on Antigen Processing by Macrophages

Another basic study of immune system processing focuses on how cannabinoids modulate the presentation of antigens to lymphocytes by macrophages. This is an important step in the attack on microorganisms by the immune system. Delta(9)-tetrahydrocannabinol (THC) causes an antigen-dependent defect in the ability of macrophages to activate helper T cells, and this drug-induced impairment is mediated through the peripheral CB2 receptor. Various requirements for the processing of the antigen, lysozyme, were examined by C.B. Hartmann et. al. to determine where along the pathway THC exerts its influence. A THC-exposed macrophage hybridoma inefficiently stimulated interleukin-2 secretion by a helper T cell hybridoma in response to native lysozyme and its reduced form, suggesting that disulfide bond reduction was unaffected. Cell surface expression of major histocompatibility complex class II molecules was normal on THC-exposed macrophages. The drug-exposed macrophages also competently presented a lysozyme peptide to the T cells, indicating that the class II molecules were functional. The proteolytic activity of two thiol cathepsins was unaltered, but aspartyl cathepsin D activity was significantly increased in THC-exposed macrophages. Thus, selective up-regulation of aspartyl cathepsin activity accompanied the deficiency in lysozyme processing and may contribute, at least in part, to the antigen-dependent processing defect in THC-exposed macrophages. Hartmann, C.B., Harrison, M.T., Cabral, G.A., McCoy, K.L. Delta(9)-Tetrahydrocannabinol Selectively Increases Aspartyl Cathepsin D Proteolytic Activity and Impairs Lysozyme Processing by Macrophages. International J. Immunopharmacology, 22, pp. 373-381, 2000.

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