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National Institute on Drug Abuse

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

February, 2000

Research Findings

Basic Research

A Brain Protein Is Involved in Switching On Cocaine Addiction

Chronic exposure to cocaine causes the delta-FosB transcription factor to be expressed persistently in the nucleus accumbens. Thus, researchers hypothesized that delta-FosB may mediate some of the long-lived increases in sensitivity to the stimulant and rewarding effects of cocaine. Dr. Eric Nestler of Yale University and his colleagues tested whether delta-FosB expression actually increased the responsiveness to cocaine by generating genetically altered mice that produced large quantities of delta-FosB in the nucleus accumbens. The animals exhibited increased responsiveness to the rewarding and locomotor-activating effects of cocaine. The researchers then showed that delta-FosB increases the expression of a glutamate receptor, GluR2, in the nucleus accumbens. Indeed, overexpression of GluR2 expression in the nucleus accumbens was sufficient to account for the enhanced sensitivity to cocaine's rewarding effects seen in the mice with augmented levels of delta-FosB. The authors concluded that delta-FosB, by altering gene expression, enhances sensitivity to cocaine and may thereby contribute to cocaine addiction. This research offers insight into the nature of the switch from use to addiction, by demonstrating that sustained expression of delta-FosB is involved in the increased sensitivity to cocaine that may be related to addiction. Kelz, M.B., Chen, J., Carlezon, W.A. Jr., Whisler, K., Gilden, L., Beckmann, A.M., Steffen, C., Zhang, Y.J., Marotti, L., Self, D.W., Tkatch, T., Baranauskas, G., Surmeier, D.J., Neve, R.L., Duman, R.S., Picciotto, M.R., Nestler, E.J. Expression of the Transcription Factor Delta FosB in the Brain Controls Sensitivity to Cocaine. Nature, 401(6750), pp. 272-276, 1999.

Methamphetamine May Kill Cortical Neurons via Excessive Sensory Stimulation

Dr. John F. Marshall found that a group of pyramidal neurons located in the somatosensory cortex of the rat are killed by moderate, repeated doses of methamphetamine, in addition to the well-known injury to dopamine- and serotonin-containing terminals elsewhere in the brain. He hypothesizes that the cortical neurons die from a combination of hyperthermia and intense, prolonged excitation resulting from the rats' repetitive head and whisker movements during exposure to methamphetamine. His first experiments determined that the cortical neurons that die following a neurotoxic regimen of methamphetamine are located within the histochemically identified whisker "barrels" of the rat. The methamphetamine treatment induced Fluoro-Jade (fluorescent dye that specifically marks dying cells) positive cortical neurons only in cytochrome oxidase-stained "barrels" (discrete units containing about 2500 neurons arranged in a cylindrical array; each barrel contains the sensory representation of an individual whisker) of the somatosensory cortex. He reasons that some of the affected neurons are corticostriatal glutamatergic neurons, which, as they begin to degenerate, release excessive glutamate in the striatum, which contributes to damaging dopamine terminals in that structure. His most recent experiments demonstrated that removal of one set (left or right) of whiskers leads to reduced numbers of degenerating cortical neurons within the contralateral somatosensory cortex (consistent with the crossed projections of the whiskers). This provides evidence that the degeneration of somatosensory cortex neurons after methamphetamine depends at least in part on the stimuli arising from body movement. O'Dell, S.J. and Marshall, J.F. Effects of Vibrissae Removal on Methamphetamine-Induced Damage to Neurons in Rat Somatosensory Cortex. Society for Neuroscience, 25, Abstract 836.17, 1999.

Differential Effects of Neuropeptide Y and the Mu-Agonist DAMGO on 'Palatability' vs. 'Energy'

A variety of studies suggest that Neuropeptide Y (NPY) is an important regulator of energy metabolism. In contrast, the opioid peptides appear to influence the rewarding aspects of feeding. In this study, Dr. Levine and his research team stimulated feeding by injecting NPY or the mu-opioid agonist DAMGO into the paraventricular nucleus of rats. Following injection, rats were given free access to laboratory chow and a ten percent sucrose solution. Animals injected with saline derived ten percent of their kilocalories from the chow and 90 percent from the sucrose solution. Those rats injected with NPY derived 48 percent of their energy from chow and 52 percent from the sucrose solution. The DAMGO-injected rats derived only 15 percent of their kilocalories from chow and the remainder from the sucrose solution. Thus, while NPY and DAMGO both stimulated energy intake compared to saline controls, the effect on intake of a palatable dilute energy solution versus a bland laboratory chow was different. The results of this study reinforce the notion that NPY has a major effect on energy needs, whereas opioids influence the rewarding characteristics of foods. Giraudo, S.Q., Grace, M.K., Billington, C.J., Levine, A.S. Differential Effects of Neuropeptide Y and the Mu-Agonist DAMGO on 'Palatability' vs. 'Energy'. Brain Research, 834(1-2), pp. 160-163, 1999.

Tolerance to Morphine-Induced Antinociception is Decreased by Chronic Sucrose or Polycose Intake

Chronic intake of palatable fluids alters morphine-induced antinociception. Two experiments were conducted to evaluate how long-term access to palatable fluids alters the development of tolerance to morphine-induced antinociception. In the first experiment, adult male Long-Evans rats were given one of the following to drink for a three weeks period: either a 0.15 percent saccharin solution, a 32 percent sucrose solution, a 32 percent Polycose solution, or chow and water alone (control). Half of the animals in each dietary condition were pre-exposed to 7.5 mg/kg morphine while the other half received saline. After injection, all rats were given a tail flick (TF) test. To determine whether tolerance developed, a cumulative dose paradigm was employed 1 week after the initial morphine injection, and this was repeated at weekly intervals for 3 weeks. Antinociception was significantly lower in rats pre-exposed to morphine. Although all rats displayed decreased antinociception relative to the first morphine injection, rats that drank saccharin showed greater reductions in morphine-induced antinociception relative to rats that drank sucrose or Polycose. A second experiment was conducted to determine whether the initial pairing of the TF with morphine pre-exposure produced differences in the development of opioid tolerance. All conditions and procedures were identical to those used in the first experiment except that the initial morphine and saline injections were not followed by TF. As in the first experiment, rats that drank saccharin showed less antinociception than rats that drank sucrose or Polycose. The present results suggest that long-term intake of palatable nutritive solutions curbs tolerance to morphine-induced antinociception, whereas long-term intake of a nonnutritive, sweet saccharin solution does not. D'Anci, K.E. Tolerance to Morphine-Induced Antinociception is Decreased by Chronic Sucrose or Polycose Intake. Pharmacology. Biochem. Behav., 63(1), pp. 1-11, 1999.

Cannabinoid Receptor Selectivity

The cannabimimetic ligand WIN 55212-2 is an aminoalkylindole that has been variously reported to have a 10-20 fold higher affinity for the peripheral CB2 receptor than for the CNS CB1 receptor. A recent report by NIDA grantees Reggio and Song has provided new insight into the molecular basis for the differences in selectivity. It had previously been shown that lysine 192 in the third transmembrane helix of the CB1 receptor was necessary to maintain binding of the "classical" cannabinoid structure HU-210 and that of the endogenous ligand anandamide. However, mutating the lysine to alanine did not change the binding of WIN 55212-2. Further, the carbonyl oxygen in the latter is not necessary for binding, and the morpholino ring can be replaced by an alkyl group without loss of binding. The researchers have modeled the interaction of WIN 55212-2 in its predominant s-trans conformation, and obtained the minimum interaction between the ligand and the transmembrane helices 3-5 of the CB1 and CB2 receptors. For the CB1 receptor, they have found an interaction between the naphthyl ring of the ligand and phenylalanine 189 in helix three, as well as with tryptophan 279 of helix five. There is also an interaction between the aromatic indole ring of the ligand and phenylalanine 200 in helix three. For the CB2 receptor, in addition to these three interactions, there is a fourth interaction between the indole ring and phenylalanine 197 in helix five. By expressing mutated receptors (changing phenylalanine 192 to valine in CB2 and making the corresponding valine to phenylalanine in CB1), the authors have shown a decrease in affinity of the ligand toward CB2 by 14 fold, and a corresponding increase in affinity toward the mutated CB1. The single amino acid mutation did not affect the binding of HU-210, the ligand CP-55940, or that of anandamide for either receptor. Inhibition of cyclic AMP accumulation was used as a functional assay measurement. The single point mutation in CB2 produced an increase in the concentration needed to yield a 50 percent inhibition of cAMP accumulation, consistent with a corresponding decrease in the binding affinity. The selectivity of WIN 55212-2 for CB2 over CB1 was attributed in part to the presence of phenylalanine in transmembrane helix five. Song, Z.H., Slowey, C.A., Hurst, D.P., and Reggio, P.H., Molecular Pharmacology, 56(4), pp. 834-840, 1999.

Opiates, Nitric Oxide, Lungs and Blood Vessels

Dr. George Stefano and his co-workers, at SUNY Stony Brook, demonstrated that the endothelia from the human blood vessels express both delta and mu opioid receptors and the exposure of these cells to opioid peptides antagonize the morphine-stimulated release of nitric oxide in a dose-dependent manner. These findings suggest that opioid peptides and opiate alkaloids regulate endothelial function in an antagonistic manner, thereby influencing the micro-vascular environment. Stefano, G.B., Salzet, M., Hughes, T.K,, Bilfinger, T.V. Delta(2) Opioid Receptor Subtype on Human Vascular Endothelium Uncouples Morphine Stimulated Nitric Oxide Release. International Journal of Cardiology, Supplement 1, 64, pp. S43-51, 1998.

In another study, Dr. Stefano and his international colleagues reported activation of the mu3 opiate receptor by opiate alkaloids in tumor cells coupled with a rapid and substantial release of nitric oxide. From these findings the research suggests that "endogenous opiates, through their release of nitric oxide, may play a role in cancer progression." Fimiani, C., Arcuri, E., et al. Mu3 Opiate Receptor Expression in Lung and Lung Carcinoma: Ligand Binding and Coupling to Nitric Oxide Release. Cancer Letters, 146, pp. 45-51, 1999.

Fruit Flies Offer Insight Into Genes Involved In Response To Cocaine

In response to exposure to volatilized freebase cocaine, fruit flies (Drosophila) perform a set of behaviors similar to those observed in vertebrate animals, including grooming, proboscis extension, and unusual circling. Flies can show sensitization, increased severity of response, after even a single exposure to cocaine. Sensitization has been linked to the addictive process in humans and may be associated with craving. NIDA grantee Dr. Jay Hirsh of the University of Virginia and colleagues have demonstrated a connection between the biological clock and cocaine sensitization in fruit flies. Flies missing several genes that play a critical role in the insects' internal biological clock did not become sensitized to cocaine. Besides enabling the potential development of drugs to treat cocaine addiction, this research holds out the prospect that the "clock" genes, which are involved in setting and maintaining the body's internal clock, play other roles in the body and brain, such as affecting vulnerability to addiction. This research emphasizes the usefulness of flies as a model to unravel the complex genetics of drug abuse behavior. Andretic, R., Chaney, S., and Hirsh, J. Requirement of Circadian Genes for Cocaine Sensitization in Drosophila. Science, 285(5430), pp. 1066-1068, 1999.

EPH Molecules Mediate the Development of Reward Circuits

Dr. Renping Zhou and his colleagues at Rutgers University have found that interactions between the receptor EphB1 and the ligand ephrin B2 contribute to the establishment of distinct mesolimbic (VTA to nucleus accumbens) and mesostriatal (substantia nigra to caudate-putamen) pathways. They showed that this receptor ligand pair plays an important role in assuring that dopamine neurons from the ventral tegmental area connect with neurons in the nucleus accumbens and that dopamine neurons from the substantia nigra connect with neurons in the caudate-putamen or dorsal striatum. These molecules belong to the same family of tyrosine kinases (enzymes that phosphorylate a tyrosine amino acid in proteins) that control the development of the visual system and hippocampus. The ephrin B2 ligand is anchored to the membrane that enables it to guide the formation of nerve pathways. Receptor and ligand appear together in different brain regions. When paired, their interaction appears to inhibit neurite outgrowth and to even induce cell loss, perhaps preventing substantia nigra neurons from connecting with nucleus accumbens and prefrontal cortex neurons. Injections of cocaine and amphetamine induce the formation of this receptor-ligand pair in adult mice, suggesting the pair may play a role in the plasticity of adult dopamine nerve cells. This work is important because understanding how nerve pathways form aids in understanding their function and their relationships to each other. Revealing the molecular markers for developmental plasticity may lead to new insights into both the mechanisms of adult learning and the mechanisms of drug induced neuronal adaptation. Environmental influences during development may alter the neural circuitry of the brain and lead to increased susceptibility to drug addiction. Yue, Y., Widmer, D.A., Halladay, A.K., Cerretti, D.P., Wagner, G.C., Dreyer, J.L., Zhou, R. Specification of Distinct Dopaminergic Neural Pathways: Roles of the Eph Family Receptor EphB1 and Ligand Ephrin-B2. J. Neurosci., 19(6), pp. 2090-2101, 1999.

Serine Racemase: A Glial Enzyme Synthezing D-serine Regulates Glutamate-N-methyl-d-aspartate (NMDA) Neurotransmission

Ischemic brain injury or stroke can be a consequence of addiction to nicotine and cocaine. Much of the injury resulting from ischemia in the brain is thought to be mediated by the activation of N-methyl-d-aspartate (NMDA) receptors through the massive release of glutamate. Activation of the NMDA receptor requires glutamate and coactivation at a "glycine" binding site on the NMDA receptor. D-serine is up to three times more potent than glycine as a coactivator of the NMDA receptor, and is released by glutamate from astrocytic processes that ensheath the synapse. Extracellular levels of endogenous D-serine are twice as high as glycine in the striatum, and D-serine is present at concentrations equal to glycine in the prefrontal cortex. This evidence suggests that D-serine acts as a neuromodulator on neurotransmitter in the brain. The discovery of D-serine as a modulator of synaptic transmission is surprising since D amino acids are prominent in bacteria but not in mammals. Until now the presence of D-amino acids in mammals has been attributed to dietary origin or intestinal bacteria. To show a physiological role for D-serine and that the enzyme is produced endogenously, the enzyme converting L-serine to D-serine must be identified. In the November 9, 1999 issue of PNAS Dr. Solomon Snyder and his group report the cloning of a serine racemase that converts L-serine to D-serine. The enzyme was cloned from rat brain using the partial amino acid sequence from a purified preparation of enzyme that converts L-serine to D-serine. The serine racemace enzyme was localized to astrocytes where D-serine is produced, which is consistent with the idea that cloned racemase is the major enzyme required for D-serine production. The cloning of this enzyme will permit scientists to generate large quantities of purified enzyme needed to develop drugs that block and activate serine racimase as well as characterize the role of serine racemace in NMDA neurotransmission. The development of selective inhibitors may provide treatment for stroke and neurodegenerative diseases that are mediated by glutamate excitotoxicity. Wolosker, H., Blackshaw, S., Snyder, S.H, Serine Racemase: A Glial Enzyme Synthesizing D-Serine to Regulate Glutamate-N-Methyl-D-Aspartate Neurotransmission. Proc. Natl. Acad. Sci., 96(23), pp. 13409-13414, 1999.

Knockout of the Nicotinic Receptor Subunit Alpha3: Possible Mouse Model for Rare Human Genetic Disease

The nicotinic acetylcholine receptor (nAchR) gene family consists of eight alpha subunits (a2-a9) and three beta subunits (Beta1-Beta3). These subunits can combine with one another in many different combinations to form diverse classes of nicotinic receptors in different regions of the nervous system and within the same nerve cell type. The alpha3 receptor subunit is expressed in autonomic ganglion that controls functions such as heart rate, urination, pupil size, and is expressed in brain regions such as pontine-mescencephalic micturation center. To test the physiological role that the alpha3 receptor plays, scientists at Columbia University led by Dr. Lorna Role and scientists at Baylor College of Medicine led by Dr. James Patrick and Dr. Arthur Beaudet generated a transgenic mouse lacking the alpha3 receptor subunit. Mice lacking the alpha3 receptor subunit have a growth deficiency, urinary retention, bladder stones, develop urinary tract infections, and widely dilated pupils. Drs. Role, Patrick and Beaudet suggest that urinary tract infections and urinary retention are caused by the absence of alpha3 containing nAchR in the parasympathetic intramural ganglia and may be caused by the lack of expression in the pontine-mescepahlic micturation. Drs. Role, Patrick and Beaudet also suggest that the failure of the pupils to constrict in response to bright light is caused by the loss of the alpha3 subunit in the superior cervical ganglion that mediates pupilary contraction to light. The phenotype of the mice lacking the alpha3 receptor subunit is similar to the rare human genetic disease known as megacystis-microcolon-intestinal hypoperstalsis syndrome (MMIHS) that is an autosomal recessive condition. Xu, W., Gelber, S., Orr-Urtreger, A., Armstrong, D., Lewis, R.A., Ou, C.N., Patrick, J., Role, L., De Biasi, M., Beaudet, A.L. Megacystis, Mydriasis, and Ion Channel Defect in Mice Lacking the alpha3 Neuronal Nicotinic Acetylcholine Receptor. Proc. Natl. Acad. Sci., 96(10), pp. 5746-51, 1999.

Retention of Supraspinal Delta-like Analgesia and Loss of Morphine Tolerance in Delta Opioid Receptor (DOR-1) Knock-out Mice

The delta opioid receptor (DOR-1) has been proposed to mediate several physiological functions including analgesia, tolerance, and reproduction. Pharmacological studies have suggested at least two DOR-1 subtypes. The delta 1(d1) receptor subtype is preferentially activated by the agonist [D-Pen2, D-Pen5] enkephalin (DPDPE) and antagonized by [D-Ala2, Leu5, Cys6] enkephalin (DALCE), while the delta 2(d2) receptor is preferentially activated by [D-Ala2, D-Glu4] deltorphin (deltorphin-2) and antagonized by naltrindole-5'-isothiocyanate (5'-NTII). This classification is further supported by analgesic, adenylyl cyclase and antisense approaches. NIDA grantee John E. Pintar of Robert Wood Johnson Medical School and his coworkers have recently discovered that disruption of the mouse delta opioid receptor by targeting exon 2 of the DOR-1 gene eliminates essentially all 3H-DPDPE and 3H-[D-Ala2, D-Glu4] deltorphin binding in the homozygous DOR-1 mutant mice, demonstrating that this locus encodes both the pharmacologically-defined d1 and d2 receptors. Disruption of the DOR-1 gene markedly reduced spinal delta analgesia, shifting the dose-response curve of DPDPE significantly to the right. Supraspinally, peptide delta agonists retained analgesic potency in thermal nociceptive assays that was only partially antagonized by naltrindole. Retained DPDPE analgesia was also demonstrated in the formalin test, while the nonpeptide delta agonist BW373U69 exhibited markedly enhanced activity in DOR-1 mutant mice as compared to wild-type mice. Together, these findings suggest the presence of a second delta analgesic system mediated through a different opioid receptor. Finally, DOR-1 knockout mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for the DOR-1 receptor in this process. Zhu, Y., King, M.A., Schuller, A.G.P., Nitsche, J.F., Reid, M., Elde, R.P., Unterwald, E., Pasternak, G.W. and Pintar, J.E. Neuron, 24, pp. 243-252, 1999.

Role of Medial Prefrontal Cortex in Behavioral Sensitization

Behavioral sensitization to psychomotor stimulants in animal models can reveal neuroadaptations that are associated with the development of addiction to these drugs. Two such neuroadaptations in the mesoaccumbens dopamine (DA) system are DA autoreceptor subsensitivity in the ventral tegmental area (VTA) and DA D1 supersensitivity in the nucleus accumbens (NAc). Both behavioral sensitization to cocaine and these cellular correlates in the VTA and NAc were prevented by blockers of either the NMDA or the AMPA type of glutamate receptors. Similarly, lesions of the medial prefrontal cortex prevented behavioral and cellular manifestations of sensitization. These results indicate that excitatory, glutamatergic feedback connections from the medial prefrontal cortex are not only necessary for the induction of behavioral sensitization, but also are either directly or indirectly responsible for the neuroadaptations within the VTA and NAc that accompany the behavioral change. These results add to a growing body of evidence that implicates the medial prefrontal cortex in both the causes and consequences of drug addiction. Li, Y., Hu, X.T., Berney, T.G., Vartanian, A.J., Stine, C.D., Wolf, M.E., and White, F.J. Both Glutamate Receptor Antagonists and Prefrontal Cortex Lesions Prevent Induction of Cocaine Sensitization and Associated Neuroadaptations. Synapse, 34, pp. 169-180, 1999.

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