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Director's Report to the National Advisory Council on Drug Abuse
February, 2002

Research Findings

Basic Research

Separate Neural Pathways Mediate the Rewarding Effects of Cocaine and Reinstatement of Cocaine-Taking Behavior

Although the acute pharmacological effects of psychostimulants is mediated by changes in dopamine transmission, it is becoming increasingly apparent that addiction is associated with long-term changes in the cortical and allocortical circuitry. The mesolimbic dopamine system, originating in the VTA, projects to the nucleus accumbens, the amygdala, the prefrontal cortex, and the ventral pallidum. However, this system can be divided into two sub-circuits, the limbic circuit comprised of the ventral prefrontal cortex, the shell of the accumbens, the medial ventral pallidum, amygdala, and the VTA and the other primarily a motor circuit, comprised of the dorsal prefrontal cortex, the core of the accumbens, the dorsolateral ventral pallidum, and the substantia nigra. Most research suggests that the limbic circuit is intimately involved with the rewarding effects of abused drugs. By microinjecting GABA-A and GABA-B agonists to limit the activity of dopamine within specific nuclei of the motor sub-circuit, researchers in South Carolina were able to block the cocaine-primed reinstatement of responding for cocaine in animals in whom drug-seeking behaviors had been extinguished. Although dopamine projections from the VTA project to accumbens core, the ventral pallidum, and the dorsal prefrontal cortex, only the blockade of dopamine receptors in the dorsal prefrontal cortex antagonized the cocaine-induced reinstatement; furthermore, microinjection of dopamine in the same area elicited a reinstatement in drug-primed responding. These data showing that brain nuclei subserving motor functions, with limited limbic involvement, are important for inducing reinstatement of drug-taking behavior in drug-experienced subjects may be part of the substrate underlying the compulsive behavior associated with drug addiction. McFarland, K. and Kalivas, P.W., J. Neuroscience, 21, pp. 8655-8663, 2001.

Regulation of the Vesicular Monoamine Transporter-2

The plasmalemmal dopamine (DA) transporter (DAT) is a principal site of action for cocaine. Dr. Annette Fleckenstein of University of Utah and her research team reported the novel finding that in addition to inhibiting DAT function, cocaine administration rapidly alters vesicular DA transport. Specifically, cocaine treatment abruptly and reversibly increased both the Vmax of DA uptake and the Bmax of vesicular monoamine transporter-2 (VMAT-2) ligand (dihydrotetrabenazine) binding, as assessed ex vivo in purified rat striatal synaptic vesicles. Selective inhibitors of the DAT (amfonelic acid and GBR12935), but not the plasmalemmal serotonin transporter (fluoxetine), also increased vesicular DA uptake. Moreover, DA depletion resulting from administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine had cocaine-like effects. Conversely, administration of the DA-releasing agent methamphetamine rapidly decreased vesicular uptake. Taken together, these data demonstrate for the first time ex vivo that cocaine treatment rapidly alters vesicular monoamine transport, and suggest that alterations in cytoplasmic DA concentrations contribute to stimulant-induced changes in vesicular DA uptake. Hence, VMAT-2 may be an important target for developing strategies to treat not only cocaine addiction but also other disorders involving alterations in neuronal DA disposition, including Parkinson's disease. Brown, J.M., Hanson, G.R., and Fleckenstein, A.E. Regulation of the Vesicular Monoamine Transporter-2: A Novel Mechanism for Cocaine and Other Psychostimulants. J Pharmacol Exp Ther, 296(3), pp. 762-767, 2001.

New Family of Receptors that Binds Amphetamine, MDMA and LSD Cloned

The roles of the transporters and receptors of the biogenic amines, especially dopamine and serotonin, are well appreciated with regard to their importance in mediating the effects of psychostimulants such as cocaine and the amphetamines. However, antagonism of these proteins has often led to mixed results in the search for a medication that would limit their psychoactive effects. Researchers in Portland, OR, have recently cloned a trace amine receptor. These receptors bind to the major meta-O-methyl metabolites of the biogenic amines and trace amines such as tyramine, tryptamine, and _-_phenylethylamine, but not the classical neurotransmitters such as dopamine or serotonin, and then activate specific G-protein coupled receptors with nanomolar potency in pre- and post-synaptic membranes of target neurons. Importantly, these receptors also bind amphetamine, MDMA (“ecstasy”) and LSD with high affinity, suggesting that the action of these widely used drugs of abuse may be mediated in part by these novel receptors. The trace amine receptor may be a new target for the development of anti-stimulant medications. Bunzow, J.R., Sonders, M.S., Arttamangkul, S., Harrison, L.M., Zhang, G., Quigley, D.I., Darland, T., Suchland, K.L., Pasumamula, S., Kennedy, J.L., Olson, S.B., Magenis, R.E., Amara, S.G. and Grandy, D.K. Molecular Pharmacology, 60, pp. 1181-1188, 2001.

Endogenous Nicotinic Cholinergic Activity Regulates Dopamine Release in the Striatum

Dr. John Dani of Baylor College of Medicine and his co-workers Drs. Fu-Ming Zhou and Yong Liang recently showed that the neurotransmitter acetylcholine is important for controlling the release of dopamine in the target. Dopamine has long been known to be important for movement and in the learning process that enables us to adapt to our environment. It is now also widely regarded as playing a critical role in the rewarding properties of addictive drugs such as cocaine, amphetamines, and nicotine. Dr. Dani and his colleagues found that nicotinic receptors, responding to the neurotransmitter acetylcholine, control the release of dopamine in the target regions of the forebrain responsible for many of these behaviors. These findings have important implications for addiction in general, and for nicotine dependence in particular. In addition, his findings may also be important for other disorders that involve dopamine, such as Parkinson’s disease and schizophrenia. Zhou, F.M., Liang, Y., and Dani, J.A. Endogenous Nicotinic Cholinergic Activity Regulates Dopamine Release in the Striatum. Nature Neuroscience, 4, 1224–1229, 2001.

Marijuana and Lung Cell Death Pathways

In a recent paper, NIDA supported researchers demonstrate that the delta-9-THC contained in marijuana smoke disrupts elements of the apoptotic pathway, thereby shifting the balance between apoptotic (preprogrammed) and necrotic (gross tissue damage) cell death. They noted that exposure to whole marijuana and tobacco smoke blocked the induction of caspase-3 (a key regulatory enzyme in the apoptotic process) in A549 lung tumor cells. In contrast, gas-phase smoke, which generates high levels of intracellular reactive oxygen species, had no effect on caspase-3 activity. Exposure to marijuana or tobacco smoke is known to be toxic to respiratory epithelium. However, the researchers also observed that the balance between apoptotic and necrotic cell death may play a key role in determining host response to injury. These findings are important because they demonstrate that the observed shift in the two cell death pathways may very well affect both the carcinogenic and immunologic consequences of marijuana smoke exposure. Sarafian, T.A., Tashkin, D.P. and Roth, M.D. Marijuana Smoke and Delta-9 Tetrahydrocannabinol Promote Necrotic Cell Death But Inhibit Fas-Mediated Apoptosis. Toxicology and Applied Pharmacology, 174, pp. 264-272, 2001.

Endocannabinoid Synthesis

The endocannabinoids are a family of endogenous lipids, which include N-acylethanolamines such as anandamide and palmitoylethanolamine, and 2-monoacylglycerols such as 2-arachidonylglycerol (2-AG). Anandamide and 2-AG bind to both the CB1 and CB2 cannabinoid receptors, while palmitoylethanolamine (pamitylethanolamide) does not. Endocannabinoids are produced from cell membrane lipid precursors via several enzyme-catalyzed biosynthetic pathways. The enzymes are stimulated by an increase in cellular calcium ion concentration, which can occur during neuronal injury or insult. This has suggested a "neuroprotective" role for endocannabinoids. Some examples of this include increased anandamide by direct NMDA injection (NMDA is released naturally during brain injury) into the brain, increased anandamide levels found in post-mortem brain, elevation of both anandamide and 2-AG levels in endotoxic shock, and formation of 2-AG in animal models of head injury. It has been proposed that endocannabinoids may play a role as “reverse” or feedback signaling molecules, in which increases in postsynaptic neuronal calcium levels first trigger synthesis and release of endocannabinoids. These then reach (by an unknown mechanism) and activate presynaptic CB1 receptors. This leads to an inhibition of calcium channels, and a decrease in neurotransmitter release. This phenomenon is observed in-vitro by the reduction of GABA release from hippocampal neurons following depolarizing suppression of synaptic current, and in Purkinje cells, where elevation of postsynaptic calcium levels suppresses both excitatory and inhibitory inputs. The phenomenon is absent in knockout mice lacking the CB1 receptor. The production of endocannabinoids might be due to the stimulation of electrical potentials in the cells, or to receptor activation, or both. Recently, Dr. Daniele Piomelli and colleagues found that receptor activation is a requirement for endocannabinoid formation in cortical neurons. The researchers found that the coactivation of NMDA receptors (using NMDA) and cholinergic receptors (using carbachol) stimulates the production of the endogenous cannabinoids palmitylethanolamide and oleylethanolamide. They found that this process is inhibited by intracellular calcium chelation, and by the cholinergic antagonist atropine. Activation of NMDA receptors alone (by glutamate or NMDA) caused a substantial increase in 2-AG formation, which was blocked by application of an external cellular calcium chelator, which reduces the concentration of extracellular calcium available to activate the NMDA receptors. Anandamide formation required activation by both glutamate and carbachol, but in this case antagonism was found with the alpha 7 nicotinic acetylcholine antagonist methyllycaconitine. Anandamide formation was also blocked by an intracellular chelation of calcium ions. This work suggests that endocannabinoid synthesis may be a response modulating the effects of the neurotransmitters glutamate and/or acetylcholine in primary rat cortical neurons, and that the type of endocannabinoid released depends on the type of receptor activated. Stella, N. and Piomelli, D. European Journal of Pharmacology, 425, pp. 189-196, 2001.

Cannabinoids Prevent Formation of Synapses between Hippocampal Neurons in Culture

Marijuana (cannabis) is well known to impair the formation of memories in humans and animal models. However, the mechanisms by which the active constituents of marijuana, principally _9-tetrahydrocannabinol (_9-THC), bring about this memory impairment are not known. Drs. Daniel Kim and Stanley Thayer, University of Minnesota Medical School, grew rat hippocampal neurons in culture, and induced synapse formation between these neurons in culture by elevating cAMP with an agent called forskolin. The forskolin-induced formation of new synapses was prevented by the cannabinoids Win55212-2, _9-THC, and anandamide (an endogenous cannabinoid). The cannabinoids did not prevent the formation of synapses induced directly by a cAMP analog, indicating that they act by inhibiting the formation of cAMP. A cannabinoid CB1 receptor antagonist blocked the cannabinoid action, indicating that this action is CB1 receptor-mediated. These researchers concluded that preventing the formation of new synapses might contribute to the impairment of memory produced by cannabinoids. Kim, D., and Thayer, S.A. Cannabinoids Inhibit the Formation of New Synapses between Hippocampal Neurons in Culture. J. Neurosci., 21:RC146, pp. 1-5, 2001.

Interaction of Co-expressed Mu- and Delta-Opioid Receptors

Mu- and delta-opioid agonists interact in a synergistic manner to produce analgesia in several animal models. Additionally, receptor binding studies using membranes derived from brain tissue indicate that interactions between mu- and delta-opioid receptors might be responsible for the observation of multiple opioid receptor subtypes. To examine potential interactions between mu- and delta-opioid receptors, Dr. Paul Prather and his colleagues at University of Arkansas for Medical Sciences examined receptor binding and functional characteristics of mu-, delta-, or both mu- and delta-opioid receptors stably transfected in rat pituitary GH3 cells [GH3MOR, GH3DOR, and GH3MORDOR, respectively]. Saturation and competition binding experiments revealed that co-expression of mu- and delta-opioid receptors resulted in the appearance of multiple affinity states for mu- but not delta-opioid receptors. Additionally, coadministration of selective mu- and delta-opioid agonists in GH3MORDOR cells resulted in a synergistic competition with [3H][D-Pen2,5]enkephalin (DPDPE) for delta-opioid receptors. Finally, when equally effective concentrations of [D-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO) and two different delta-opioid agonists (DPDPE or 2-methyl-4a-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aa-octahydroquinolino-[2,3,3-g]-isoquinoline; TAN67) were coadministered in GH3MORDOR cells, a synergistic inhibition of adenylyl cyclase activity was observed. These results strongly suggest that cotransfection of mu- and delta-opioid receptors alters the binding and functional characteristics of the receptors. Therefore, they propose that the simultaneous exposure of GH3MORDOR cells to selective mu- and delta-opioid agonists produces an interaction between receptors resulting in enhanced receptor binding. This effect is translated into an augmented ability of these agonists to inhibit adenylyl cyclase activity. Similar interactions occurring in neurons that express both mu- and delta-opioid receptors could explain observations of multiple opioid receptor subtypes in receptor binding studies and the synergistic interaction of mu- and delta-opioids in analgesic assays. Martin, N.A., and Prather, P.L. Interaction of Co-Expressed Mu- and Delta-Opioid Receptors in Transfected Rat Pituitary GH3 cells. Mol. Pharmacol., 59(4), pp. 74-83, 2001.

Identification of a New Class of Molecules Involved in Pain Transmission

Dr. J. Michael Walker of Brown University and his colleagues have identified the existence of lipoamino acids in mammals. Lipoamino acids are conjugates of lipids and amino acids, and have previously been found to have biological activity in bacteria. Dr. Walker and his colleagues have found three examples of one class of lipoamino acid, the arachidonyl amino acids, in bovine brain. One of these, N-arachidonylglycine (NAGly), was also present in rat brain and other tissues. While the biological role of NAGly needs clarification, it was found that NAGly inhibited tonic inflammation pain in the rat. These data suggest a novel class of molecules that might be involved in pain processing, and may ultimately lead to novel pain treatments. Huang, L.-Y. et al., Identification of a New Class of Molecules, the Arachidonyl Amino Acids, and Characterization if One Member That Inhibits Pain, The Journal of Biological Chemistry, 276 (46), pp. 42639-42644, 2001.

Candidate Molecules Involved in Mechanisms of Chronic Cocaine-Mediated Changes are Identified Using Microarray Technology

The molecular composition of a cell is altered when receptors on that cell come in contact with molecules that the receptor recognizes and responds to. The response will often take the form of a change in the expression of various genes and production of proteins. In this study, the investigators used microarray technology to identify molecules involved in chronic cocaine-mediated changes in cells of the nucleus accumbens. They studied these cells in a non-human primate model, the cynomolgus macaque. Using a microarray with a limited number of genes, Freeman and associates identified 18 genes with significant changes in expression. The protein production for 8 of the 18 genes was examined to determine whether the mRNA levels measured with the microarray were consistent with levels of corresponding protein produced. They confirmed that, in their model system, chronic cocaine use (with increasing doses over the period of one year) lead to a statistically significant increase in the following proteins in the cells of the nucleus accumbens: protein kinase A _ _ catalytic subunit, cell adhesion tyrosine kinase __ (PYK2), mitogen activated protein kinase 1 (MEK1), and __-catenin. In the case of PKA_ _ catalytic subunit and MEK1, these results correlated with cocaine-induced changes in gene expression observed in other model systems. All four proteins are members of a common regulatory pathway that affects downstream molecules that have implications for drug addiction. Freeman, W.M., Nader, M.A., Nader, S.H., Robertson, D.J., Gioia, L., Mitchell, S.M., Daunais, J.B., Porrino, L.J., Friedman, D.P., and Vrana, K.E. Chronic Cocaine-Mediated Changes in Non-Human Primate Nucleus Accumbens Gene Expression. J. Neurochem., 77, pp. 542-549, 2001.

Rats Exposed to Methylphenidate during Development Respond Differently to Cocaine as Adults

Previous research had suggested that children with attention-deficit hyperactivity disorder who are treated with Ritalin (methylphenidate) are less likely to become substance abusers later in life than similar children who do not receive such treatment (Biederman et al, 1999). NIDA-supported researchers in Boston recently reported that the administration of methylphenidate to pre-adolescent rats results in behavioral and molecular adaptations that persist into adulthood and that the same treatment in adult rats results in different patterns of behavior and molecular adaptations. Rats were given a clinically relevant dose of methylphenidate or vehicle daily from postnatal days 20-35 or from days 50-65 and were studied 25 days later. Cocaine reward was assessed using a conditioned place preference procedure at a low and a high dose of cocaine. Rats treated in the earlier developmental period with vehicle showed the expected preference for cocaine in this test whereas those treated with the methylphenidate failed to establish a place preference to cocaine, suggesting that cocaine was less rewarding or aversive in these animals. Animals treated and tested as adults consistently demonstrated a preference for cocaine. Numerous neuroadaptations have been described in adult rats after treatment with cocaine, including increases in CREB (cAMP response element binding protein) and changes in NMDA receptors (GluR1, GluR2, NMDAR1). When the brains of the animals treated as juveniles were examined, CREB had increased as much as in animals treated as adults. However, there was no corresponding increase in GluR2, as there was in the animals treated as adults. These results show that neurobiological adaptations persist after treatment with methylphenidate and that the adaptive responses are different, depending on the age at which treatment occurred. Furthermore, they may represent neurobiological substrates that mediate the rewarding and aversive properties of stimulants. These findings suggest that the neurobiological impact of methylphenidate depends critically on the developmental stage during which it is administered. Andersen, S.L., Aravanitogiannis, A., Pliakas, A.M., LeBlanc, C. and Carlezon, Jr., W.A., Altered Responsiveness to Cocaine in Rats Exposed to Methylphenidate during Development. Nature Neuroscience, 5, pp. 13-14, 2002.

Studies of Opiate-Systems Regulation of Immune Function

Previously, it has been observed that opiates potently inhibit chemotaxis in phagocytic type cells such as macrophages. Thus, opiates diminish the capacity of these cells to reach and engulf foreign bodies in humans. This study explores this feature of other non-phagocytic immune cells and elaborates on the mechanism of cross sensitivity of opiates and chemotaxic peptides involved in this process. Opioids are known to suppress a number of elements of the immune response, including antimicrobial resistance, antibody production, and delayed-type hypersensitivity. Phagocytic cells may be particularly susceptible to opioid administration, since reduced production of the cytokines IL-1, IL-6 and TNF-alpha, monocyte-mediated phagocytosis, and both neutrophil and monocyte chemotaxis have all been well established. Earlier studies have shown that both mu- and delta -opioid agonists induce a chemotactic response in monocytes and neutrophils. In addition, mu- and delta -opioid administration inhibited the chemotactic response of these cell populations to a number of chemokines through a process of heterologous desensitization. Authors report here that mu-, delta-, and kappa-opioid agonists also induce a chemotactic response in T lymphocytes. Using the human T-cell line Jurkat, they confirmed previous observations that pre-incubation with met-enkephalin (MetEnk), an endogenous opioid agonist, prevents the subsequent chemotactic response to the chemokine RANTES. On the other hand, treatment with MetEnk does not alter the response to the chemokine SDF-1 alpha. Moreover, they found that pretreatment with RANTES prevented a subsequent response of monocytes to the mu-opioid agonist DAMGO. These results suggest that activation of members of the opioid and chemokine receptor families leads to downregulation of each other's leukocyte migratory activities. Rogers, T.J., Steele, A.D., Howard, O.M.Z., and Oppenheim, J.J. Bidirectional Heterologous Desensitization of Opioid and Chemokine Receptors. Neuroimmunomodulation: Annals NY Acad. Sci., 917, pp. 19-28, 2000.

There is considerable controversy about whether opioids modulate immunity centrally or in the periphery. Opioid receptors are present in lower quantities on T-cells or macrophages in comparison to the neural cells but the opiates have a higher affinity for immune cells. This paper clarifies the role of these systems in immunomodulation. Administration of morphine to rats was found to decrease the proliferative potential of blood lymphocytes by 60-80% and concurrently elevate circulating levels of the cytokine, interleukin-6 (IL-6), 2- to 4-fold. Both parameters were similarly altered upon the central administration of morphine and were blocked upon pretreatment of animals with the opioid receptor antagonist, naltrexone. These results suggest that the activation of central opioid receptors is involved in morphine-induced inhibition of lymphocyte proliferation as well as increases in circulating levels of IL-6. Studies addressing the potential peripheral mechanisms demonstrated that intact ganglionic transmission was required for both effects of morphine. Although the suppression by morphine of lymphocyte proliferation appeared to be largely independent of stimulation of the hypothalamic-pituitary-adrenal axis, the elevation of IL-6 was completely abolished in adrenalectomized animals. Collectively, these results suggest that central opioid receptor activation results in changes in different immune parameters that can be mediated through distinct peripheral mechanisms. Houghtling, R.A., Mellon, R.D., Tan, R.J., and Bayer, B.M. Acute Effects of Morphine on Blood Lymphocyte Proliferation and Plasma IL-6 Levels. Annals NY Acad. Sci., 917, pp. 771-777, 2000.

Cholecystokinin (CCK) Gene Polymorphism is Significantly More Prevalent in Two Independent Samples of Smokers

Two independent samples, one from Dr. Comings at the City of Hope, selected from women seeking treatment for obesity, and the other from parents of twins studied by Drs. Iacono, McGue and colleagues at the University of Minnesota were assessed for prevalence of the polymorphic SNP, C-45T, in relation to smoking history. The allele was 4 times more prevalent in smokers compared to never smokers of the obesity sample and nearly twice as prevalent in those diagnosed with nicotine dependence in the second sample. Even though CCK is considered a satiety gene, the polymorphism was not associated with obesity. It is concluded that there may be a role for the CCK gene to be a risk factor for
smoking. Comings, D.E., Wu, S., Gonzalez, N., Iacono, W.G., McGue, M., Peters, W.W., and MacMurray, J.P., Mol Genet Metab, 73, pp. 349-353, 2001.

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