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National Institute on Drug Abuse

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

September, 1997

Research Findings

Basic Research

Neurochemical Basis of Marijuana Withdrawal

George Koob and co-workers at Scripps Research Institute in La Jolla, California recently report a link between withdrawal from marijuana, behavior that appears to be like an anxiety state, and a peptide called corticotrophin-releasing factor (CRF). When the researchers measured CRF levels in the amygdala of THC-withdrawn rats, there was a doubling to tripling of the peptide which paralleled the anxiety and stress levels of the rats. Science 276, pp. 2050-2054, 1997.

Methamphetamine and Neurotoxicity

Dr. Charles Vorhees and his co-workers recently reported that in adult rats, exposure to methamphetamine (MA) produces localized depletion of glutamate-positive neurons and astrogliosis in somatosensory cortex. In developing animals, MA had no effect on glutamate-positive neurons suggesting that the mechanism underlying glutamatergic neurotoxicity is not acquired before postnatal age of 40 days. Other studies by these investigators show that administration of alpha-phenyl-N-tert-butylnitrone, a free radical spin trapping compound, attenuated MA-induced dopamine depletion in the adult rat caudate-putamen without altering MA-induced hyperthermia. These results support a role for free radicals in the generation of MA-induced dopaminergic neurotoxicity. Pu, C., Broening, H.W., and Vorhees, C. Synapse. 23, pp. 328-334, 1996; Cappon, G.D., Broening, H.W., Pu, C., Morford, L. and Vorhees, C. Synapse, 24, pp. 173-181, 1996.

Methylenedioxyamphetamines (MDA) and Neurotoxicity

Recent research reports that a putative metabolite of MDA, 2,5-bis-(glutathiol-S-yl)-alpha-methyldopamine, reproduced the neurobehavioral effects of the parent amphetamine. In addition, it produced changes in serotonergic system which appeared to be selective for nerve terminal fields as the levels of 5-HT were unaffected in the regions of the cell bodies. Since this metabolite caused long-term depletion in 5-HT without adversely affecting the dopaminergic system, it mimics the selectivity of MDA/MDMA. In contrast, administration of other metabolites of MDA produced only behavioral changes in animals that were similar to MDA. Thus, these results suggest a possible role of quinone-thioethers in the neurobehavioral and neurotoxicological effects of MDA/MDMA. Miller, R.T., Lau, S.S., and Monks, T.J., European J. Pharmacology, 323, pp. 173-180, 1997.

Orphanin FQ(OFQ)/Nociceptin(N), Antinociceptive Mechanisms and Receptor Subtypes

Orphanin FQ/Nociceptin, is a heptadecapeptide and a closely related to the cloned opioid receptor family . Orphanin produces hyperalgesia in mice when administered intra-ventricularly. Dr. Gavril W. Pasternak and colleagues of Sloan Kettering Institute for Cancer Research have been characterizing the pharmacology of OFQ. They found, in addition to hyperalgesia, which is observed soon after administration of OFQ, OFQ also produces a delayed analgesia response. Unlike OFQ-induced hyperalgesia, OFQ-induced analgesia is readily reversed by the opioid antagonist naloxone, implying an opioid mechanism of action. In view of the very poor affinity of OFQ for all the known traditional opioid receptors and the low affinity of opioids for the 125I[Tyr14]OFQ binding site, OFQ-induced analgesia is probably mediated through a novel OFQ receptor subtype. In contrast to supraspinal administration, spinal administration of OFQ elicits a rapidly appearing, naltrexone-reversible, dose-dependent analgesia in the tailflick assay without any indication of hyperalgesia. Two OFQ/N (1-7) and OFQ/N (1-11), are active, but far weaker. Blockade of sigma receptors with haloperidol enhances the analgesic potency of spinal OFQ/N, OFQ (1-7) and OFQ (1-11), but not as dramatically as supraspinal OFQ. Antisense probes targeting the second and third coding exons, but not the first exon, of the cloned mouse OFQ/N receptor (KOR-3) partially block OFQ/N analgesia. It appears that while spinal analgesia is mediated through an OFQ/N receptor, it is not clear that this is the same receptor which has been cloned. Future work is needed to define the receptor mechanisms mediating these analgesia actions. King, M.A., Rossi, G.C., Chang, A.H., Williams, L. and Pasternak, G.W. Spinal Analgesic Activity of Orphanin FQ/Nociceptin and its Fragments. Neurosci. Lett., 223, pp. 113 116, 1997; Rossi, G.C., Leventhal, L. and Pasternak, G.W., Naloxone-Sensitive Orphanin FQ/Nociceptin Analgesia in Mice. Eur. J. Pharmacol. 311, pp. R7-R8, 1996.

The PCP-Induced "Olney Lesion" Is Reproduced by Carbachol + Kainic Acid

Antagonists of the NMDA glutamate receptor, such as phencyclidine (PCP), ketamine, and dizocilpine, injure (produce vacuoles in) pyramidal neurons of the posterior cingulate/ retrosplenial (PC/RS) cortex when administered systemically to adult rats. This is sometimes termed the "Olney lesion," after the NIDA grantee, John W. Olney, M.D., its discoverer. He and other investigators hypothesize that this action may underlie the psychotomimetic effect of this class of drugs. This neurotoxic action of NMDA antagonists is hypothesized by Dr. Olney to be mediated by a complex disinhibition mechanism in which NMDA antagonists abolish GABAergic inhibition, resulting in excessive release of acetylcholine at muscarinic receptors on PC/RS cortex neurons. However, microinjection of carbachol (muscarinic agonist) into PC/RS cortex did not reproduce the Olney lesion. Because other evidence suggests that the NMDA antagonist disinhibition mechanism also releases excessive glutamate at non-NMDA receptors on PC/RS neurons, they microinjected a combination of carbachol and kainic acid (non-NMDA glutamate agonist) in very low doses into the PC/RS cortex. The combination reproduced the Olney lesion. Kainic acid alone did not reproduce the lesion. Thus, simultaneous hyperactivation of both muscarinic and non-NMDA glutamate receptors on PC/RS neurons is required for this lesion. Farber, et al., Society for Neuroscience Abstract, 1997.

Inhibition of Glutamate Transport in Synaptosomes by Dopamine Oxidation and Reactive Oxygen Species (ROS)

Dopamine can form reactive oxygen species (ROS) and other reactive metabolites that can modify proteins and other cellular constituents. Dr. Teresa G. Hastings of the University of Pittsburgh and her research team tested the effect of dopamine oxidation products, other generators of ROS, and a sulfhydryl modifier on the function of glutamate transporter proteins. They also compared any effects with those on the dopamine transporter, a protein whose function has previously been shown to be inhibited by dopamine oxidation. Preincubation with the generators of ROS, ascorbate or xanthine plus xanthine oxidase inhibited the uptake of [3H]glutamate into rat striatal synaptosomes (-54 and -74%, respectively). The sulfhydryl-modifying agent N-ethylmaleimide also led to a dose-dependent inhibition of [3H]glutamate uptake. Preincubation with dopamine (100 M) under oxidizing conditions inhibited [3H]glutamate uptake by 25%. Exposure of synaptosomes to increasing amount of dopamine quinone by enzymatically oxidizing dopamine with tyrosinase further inhibited [3H]glutamate uptake, an effect prevented by the addition of gultathione. The effects of free radical generators and dopamine oxidation on [3H]glutamate uptake were similar to the effects on [3H]dopamine uptake. These findings suggest that ROS and dopamine oxidation products can modify glutamate transport function, which may have implications for neurodegenerative processes such as ischemia, methamphetamine-induced toxicity, and Parkinson's disease. Berman, S.B. and Hastings, T.G. Inhibition of Glutamate Transport in Synaptosomes by Dopamine Oxidation and Reactive Oxygen Species. J. Neurochem. 69 (3), pp. 1185-1195, 1997.

Moving from the Orphanin FQ Receptor to an Opioid Receptor Using Four Point Mutations

In spite of the high homology at both ligand and receptor level there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. The opioid peptides show either relatively low affinity or no affinity towards the Orphanin FQ receptor. Conversely, Orphanin FQ has no affinity towards any of the opioid receptors. To investigate the molecular basis of such discrimination Dr. Huda Akil and her coworkers discovered that by mutating as few as four amino acids, they can produce a receptor which recognizes prodynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild type receptor. This suggests that the Orphanin FQ receptor has developed features which specifically exclude the opioids, and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

In a follow-up study, Dr. Akil and her coworkers aimed at shifting the binding profile of the Orphanin FQ receptor towards the opioid receptors, as a means of better understanding the critical characteristics of the opiate binding pocket. After two rounds of mutagenesis, several Orphanin FQ receptor mutants could be labeled with [3H]naltrindole and showed greatly increased affinities toward the opiate antagonists naltrexone, nBNI and (-)bremazocine. Furthermore, these Orphanin FQ receptor mutants exhibited stereospecificity similar to that of opioid receptors. In addition, the binding of several opioid alkaloids to an Orphanin FQ mutant and to the delta receptor showed similar affinity-shift profiles in the presence and the absence of GTP and high salt concentration, suggesting that their agonist/antagonist nature was preserved. These results indicate that several residues in the Orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists in the benzomorphan family. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a common binding pocket for opiate alkaloids. These findings may also be helpful to medicinal chemists in designing ligands for the Orphanin FQ receptor based on the structure of the opiate alkaloids. J. Biol. Chem., 271, 32016, December, 1996.

Carboxypeptidase E Activity Is Deficient in Mice with the Fat Mutation

Carboxypeptidase E (CPE) is involved in the biosynthesis of many peptide hormones and neurotransmitters. In collaboration with Dr. Leiter of the Jackson Laboratory, Dr. Lloyd D. Fricker of Albert Einstein College of Medicine was fortunate to find a mouse that has a normally occurring point mutation within CPE. This mutation (Cpefat/Cpefat), which completely eliminates CPE activity, causes the mice to be sterile and obese. Male mice, but not female mice, show markedly elevated serum glucose levels. These defects are due to the absence of CPE activity, presumably to a folding defect that causes the mutant CPE to be degraded in the endoplasmic reticulum. Peptide processing is abnormal in these mice, but not completely eliminated. This raised the possibility that a second carboxypeptidase is involved in peptide processing, and that this second enzyme can partially compensate for the defective CPE in the Cpefat/Cpefat mice. Dr. Fricker and his coworkers recently found a novel enzyme, carboxypeptidase D (CPD), that has CPE-like enzymatic properties. Recently they have discovered that in bovine and rat tissues CPD has a broad distribution. This is consistent with a role for this enzyme in the processing of numerous peptides, which would explain the ability of the Cpefat/Cpefat mice to produce small amounts of peptides.

Dr. Fricker and his coworkers also found that the opiate peptide precursor, prodynorphin, is not correctly processed in the Cpefat/Cpefat mouse brain. The processing of prodynorphin by the endopeptidases (the step that precedes CPE) is decreased in the Cpefat/Cpefat mice, suggesting that the CPE defect somehow feeds back on the previous step. CPE could play a role in the sorting of peptide precursors into the regulated secretory pathway, and the absence of CPE in the Cpefat/Cpefat mice causes missorting which then alters the processing. J. Biol. Chem., 271, 30619, November, 1996.

Placenta and Neurotransporter

Dr. Ganapathy and his research team has recently published reports that demonstrate that tyrosine phosphorylation is an essential component in the signaling pathways participating in the regulation of the human serotonin transporter gene expression. Regulation of the transporter gene expression also appeared to be modulated by a neuro-protective agent, aurin tricarboxylic acid (ATA) and epidermal growth factor (EGF) as both agents increased the transporter activity in JAR human placental choriocarcinoma cells. However, ATA appeared to elicit this, at least in part, by activating the EFG receptor through tyrosine phosphorylation. Other findings demonstrated that interleukin-1b turned on the human serotonin transporter gene expression by activating the transcription factor NF-kB via the mitogen-activated protein kinase signaling pathways. Although the physiologic role of the serotonin transporter in normal human placenta remains to be established, these data suggest a possibility that the placental serotonin transporter may be involved in the maintenance of serotonin levels in the intervillous space and thereby optimize utero placental blood flow. A defective clearance of serotonin from the maternal circulation due to dysfunctional serotonin expression in the placenta may play a role in the pathogenesis of intra-uterine growth retardation. Prasad, P.D. et al., European J Pharmacology, 325, pp. 85-92, 1997; Kekuda, R. et al., J Neurochemistry, 68, pp. 1443-1450, 1997.

Morphine and Melanocortin-4 Expression

Opiate dependence and tolerance is antagonized by melanocortin peptide but the receptors activated by these peptides has not been fully characterized. Duman and his colleagues at Yale University report the cloning and characterization of the full length rat melanocortin-4 receptor (MC4-R). This 332 amino acid long receptor shares a 95% amino acid sequence identity to the human MCR-4 receptor. The MCR-4 transcripts are most abundant in the septum and nucleus while only being moderately expressed in the periaquaductal gray, hypothalamus, neostriatum, ventral tegmentum, and olfactory bulb. Little expression is found in the cerebellum, substantia nigra, frontal cortex, and hippocampus. Chronic administration of morphine for five days was associated with a time-dependent down regulation of the MC4-R mRNA expression in the striatum and periaqueductal grey. Morphine treatment also resulted in time-dependent reduction in expression of the MC4R in the nucleus accumbens and the olfactory tubercle that was faster, occurring over 1 to 3 days. Other areas expressing the MC4-R were not affected by chronic morphine. Based on previous studies showing that melanocortins block tolerance and dependence as well as the results of the current study, Duman suggests that the down regulation of MC4-R by morphine may play a significant role in neuroadaptations to opiates and other drugs of abuse. Alvaro, J.D., Tatro, J.B., Quillan, J.M., Fogliano, M., Eisenhard, M., Lerner, M.R., Nestler, E.J., and Duman, R.S. Morphine Down-regulates Melanocortin-4 Receptor Expression in Brain Regions that Mediate Opiate Addiction. Molecular Pharmacology, 50, pp. 583-591, 1996.

Ligand Recognition by Monoamine Transporters

Transfection of cells with cDNA libraries and selection for survival in the presence of MPP+, a neurotoxin that destroys dopaminergic and other aminergic cells led to the discovery of two vesicular monoamine transporters, VMAT 1 and VMAT2. Cells overexpressing either VMAT survive because these transporters sequester the neurotoxin MPP+ in vesicles and prevent MPP+ uptake into mitochondria where MPP+ would be converted into cytotoxic compounds. VMAT1 expression is restricted to adrenal chromaffin cells while VMAT2 is expressed monoaminergic neurons in the CNS, neurons in sympathetic ganglia, mast cells, and histamine containing cells of the gut. VMAT2 has a higher affinity for serotonin, histamine and tetrabenazine than VMAT1. The differences in affinity between the two transporters appears to be conferred by differences in the transmembrane domains 5-8 (TMD 5-8) and transmembrane 9-12 (TMD 9-12). In this study Finn and Edwards determined how specific mutations in TMD 9-12 converted the activity of VMAT2 to VMAT1. Substitution of tyrosine at amino acid position 434 with phenylalanine and the substitution of asparagine for aspartate at amino acid position 461 reduced the affinity for tetrabenazine, histamine, and serotonin without affecting the recognition of dopamine. The replacement of lysine at amino acid 446 with glutamine reduced the affinity of VMAT2 for tetrabenzanine and serotonin and not histamine whereas substitution of tyrosine for phenylalanine at amino acid 464 reduced serotonin affinity but not tetrabenazine, providing evidence of specificity. These observations suggest that tyrosine-434, Lysine-446, and Aspartate-461 account for the preferential recognition of serotonin over dopamine in VMAT2. Finn III, J.P., and Edwards, R.H. Individual Residues Contribute to Multiple Differences in Ligand Recognition between Vesicular Monoamine Transporters 1 and 2. Journal of Biological Chemistry, 272, pp. 16301-16307, 1997.

Dopaminergic Mechanisms in Mediating Euphoria

The feeling of well being produced by natural rewarding stimuli and drugs of abuse appears to be mediated by a common mechanism. Opiates, cocaine, amphetamine, marijuana, caffeine, nicotine, and alcohol as well as food and sex all stimulate the release of dopamine from mesolimbic dopamine neurons. These mesolimbic dopamine neurons originate in the midbrain's ventral tegmental region and extend processes called axons to a region in the basal forebrain known as the nucleus accumbens. It is here in the nucleus accumbens that dopamine is released into the synapse. The release of dopamine in the nucleus accumbens inhibits both inhibitory and excitatory synaptic transmission by activating presynaptic D1 receptors. In this study Nicola and Malenka report that inhibition of excitatory and inhibitory synaptic transmission by dopamine and amphetamine occurs by two distinct mechanisms. Nicola and Malenka suggest that dopamine depresses the release of GABA, an inhibitory neurotransmitter by reducing calcium influx into the presynaptic terminal while excitatory synaptic transmission is reduced by a mechanism that is independent of calcium influx. Elucidation of the mechanisms by which dopamine inhibits both excitatory and inhibitory synaptic transmission in the nucleus accumbens may lead to better pharmacological interventions and manipulations of the reward mechanism involved in addiction. Nicola, S.M. and Malenka, R.C. Dopamine Depresses Excitatory and Inhibitory Synaptic Transmission by Distinct Mechanisms in the Nucleus Accumbens. Journal of Neuroscience. 17, pp. 5697-5710, 1997.

SPECT Imaging of Dopamine Transporter

A recent report has described the preparation and in-vivo binding of a 99mTc-labeled tropane which crosses the blood-brain barrier in monkeys, and selectively labels the dopamine transporter, as visualized by SPECT imaging. The compound represents one of a series of materials containing a tropane skeleton linked by a carbon chain to a lipophilic chelator which will chelate rhenium; the latter can be displaced by technetium for imaging studies. Meltzer, P.C., Madras, B. et al, J. Medicinal Chemistry, 40, pp. 1835-1844, 1997.

Pharmacokinetic Model for Nicotine Tolerance

The parameters of a pharmacokinetic model for nicotine tolerance in humans have been recently published. The study was based on a computer-controlled infusion of nicotine to reach blood levels of 25 ng/mL, with determination of blood pressure, epinephrine level, heart rate, energy expenditure (metabolic rate) and free fatty acids. The "half-life" of tolerance reported for the first three measurements was approximately 70 minutes, with a value of 15 minutes for tolerance shown by increase in metabolic rate, and no tolerance shown for the release of fatty acids. It was suggested that there may be differences in the mechanisms of tolerance, i.e., sympathetic neural response in the case of the first three parameters, and receptor subtype desensitization in the case of metabolic rate. Benowitz, N., Verotta, D., and Fattinger, K. J. Pharmacology and Experimental Therapeutics, 281, pp. 1238-1246, 1997.

Signal Effects of a Novel Environment in the Forebrain

Novelty seeking is a trait which has been associated with drug taking behavior. In order to further explore the role of catecholamine containing neurons in the rewarding properties of novel stimuli, voltammetric recordings with electrochemically modified carbon-fiber electrodes were obtained from specific regions of the forebrain in rats given free choice access to a novel environment. Entry into novelty increased the catechol signal in the medial prefrontal cortex and shell of nucleus accumbens by more than 100% but had no consistent effect in either neostriatum or accumbal core. In both medial prefrontal cortex and accumbal shell, the novelty-induced increase in catecholaminergic activity was detectable only during initial entry into the novel compartment and did not reappear when animals returned to the familiar environment. These results support increasing evidence for a functional distinction between accumbal core and shell with the latter having been linked to brain reward mechanisms. The results also indicate that novelty activates, albeit very transiently, some of the same neurochemical systems believed to play a critical role in the reinforcing effects of certain drugs of abuse. Rebec, G.V., Grabner, C.P., Johnson, M., Pierce, R.C., and Bardo, M.T. Transient Increases in Catecholamine Activity in Medial Prefrontal Cortex and Nucleus Accumbens Shell During Novelty. Neuroscience, In press.

Review of Mesolimbic Dopamine System in Drug Reward

This paper summarizes recent work that examines the neuropharmacologic mechanisms by which drugs impinge on the mesolimbic dopamine system. Whereas dopamine plays a critical role in this circuit, other neurotransmitters (e.g., serotonin, acetylcholine, glutamate, GABA) are involved. In addition, other structures (e.g., ventral pallidus, amygdala, hippocampus and pedunculopontine) interact with the mesolimbic system to play a role in drug reward. Finally, the activation of this reward circuitry is achieved differently for different drugs of abuse. Multiple lines of research are summarized in this review article. Bardo, M.T. Neuropharmacological Mechanisms of Drug Reward: Beyond Dopamine in the Nucleus Accumbens. Neurobiology, In press.

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