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

Research Findings - Basic Research

Working Memory and the Endocannabinoid System

The endocannabinoid system has recently been proposed to modulate a variety of physiological processes, including those that underlie cognition. In the past year, NIDA researchers conducted studies to further elucidate the function of this system in learning and memory using mice lacking the endocannabinoid receptor (CB1). Findings from these studies showed that mice lacking the CB1 receptor exhibited significant deficits in a reversal task. Moreover, the deficit persisted despite being repeatedly shown the new task solution. These investigators also report that administration of three different cannabinoid agonists, delta-9-THC, WIN 55,212-2 and methanandamide disrupted performance in wild-type mice. Furthermore, these cannabinoid-agonist disruptive effects found in wild-type mice were blocked by the cannabinoid antagonist SR 141716A. These findings provide strong evidence that cannabinoids disrupt working memory through a CB1 receptor mechanism of action and suggest that the endocannabinoid system may have a role in facilitating extinction and/or forgetting processes. Varvel, S.A. and Lichtman, A.H. Evaluation of CB1 Receptors Knockout Mice in Morris Water Maze. Journal of Pharmacology and Experimental Therapeutics, 301(3), pp. 915-924, 2002.

Structural Adaptations in a Membrane Enzyme that Terminates Endocannabinoid Signaling

Endocannabinoids are naturally occurring compounds that bind to receptors in the brain and in peripheral tissues that are also the target of delta-9-tetrahydrocannabinol (THC), the active ingredient in marijuana. Researchers have been studying the brain's endocannabinoid system to learn more about its function. So far, endocannabinoids have been shown to modulate pain, cognition, feeding, and locomotor activity. In an article in the November 29, 2002, issue of Science, NIDA grantee, Dr. Benjamin Cravatt and his colleagues describe the crystal structure of an enzyme (FAAH) that terminates the activity of endocannabinoids. The structure of FAAH complexed with an arachidonyl inhibitor reveals how a set of discrete structural alterations allows this enzyme, in contrast to soluble hydrolases of the same family, to integrate into cell membranes and establish direct access to the bilayer from its active site. The intimate relationship between the active site of FAAH and cell membranes revealed by the enzyme's structure raises the possibility that fatty acid amides need not be transported through aqueous cellular compartments in order to proceed from site of action to site of degradation. As a consequence, proper endocannabinoid tone may rely on both the expression levels of FAAH and its localization relative to CB receptor systems in vivo. This knowledge should facilitate the design of novel targeted compounds that could be used to alter the activity of endocannabinoids, with the potential for use in treating pain, a variety of related nervous system disorders, and possibly marijuana addiction. Bracey, M.H., Hanson, M.A., Masuda, K.R., Stevens, R.C., and Cravatt, B.F. Structural Adaptations in a Membrane Enzyme that Terminates Endocannabinoid Signaling. Science, 298, pp. 1793-1796, 2002.

Bupropion and Nicotine-Like Discriminative Stimulus

Bupropion, an antidepressant approved in 1997 for smoking cessation, shares discriminative stimulus effects with cocaine and methamphetamine. The discriminative stimulus effects of these drugs, in turn, overlap with those of nicotine. Studies conducted by NIDA-supported researchers investigated the overlap in discriminative stimulus effects of bupropion and nicotine in a rodent model. These investigators found that bupropion substituted for nicotine. That is, the effects of bupropion were similar to nicotine using this procedure. However, bupropion effects were not blocked by a nicotine antagonist, mecamylamine, suggesting that bupropion may be producing its nicotine-like discriminative stimulus effects through neurobiological mechanisms different from nicotine itself. Given bupropion's shared pharmacology with dopamine transporter inhibitors and nicotine's effects on dopamine, these effects may be produced in part through bupropion's actions on dopaminergic neurotransmission. Wiley, J.E., LaVechia, K.L., Martin, B.R. and Damaj, M.I. Nicotine-like Discriminative Stimulus Effects of Bupropion in Rats. Experimental and Clinical Pharmacology, 10(2), pp. 129-135, 2002.

Opiate-Induced Analgesia and Role of Steroids

In a recent paper, Dr. Theodore Cicero and his associates at Washington University School of Medicine, St. Louis, determined the role of either the organizational or activational sex steroids in mediating gender differences observed in morphine-induced antinociception in the rat. To examine the organizational aspects, male pups were castrated early in life at postnatal days 1 and 2; females were masculinized by large doses of testosterone early in life on postnatal days 1 and 2. Older adult male and female rats were also castrated over a period of 2 months to examine the role of the acute activational effects of the opiates in the already sexually differentiated adult rat brain. The results showed that the gender differences in opiate analgesia were still evident in castrated older adult male and female rats. On the other hand, in male rats castrated early in life at postnatal days 1 and 2, the sensitivity to morphine analgesic effects was similar to females and, in fact, was almost identical to that observed in untreated females. Conversely, in female rats, masculinized by large doses of testosterone early in prenatal life, the morphine dose-response curve shifted to the left, yielding a dose-response curve similar to the dose-effect function seen in normal males. These results suggest that the sex differences that have been observed in morphine induced analgesia are due to the organizational effects of sex steroids in the early development of the rat brain, rather than to their acute activational effects occurring later in adulthood. These findings could be important in determining further gender differences and drug abuse liability. Cicero, T.J., Nock, B., O'Connor, L., and Meyer, E.R. Role of Steroids in Sex Differences in Morphine-induced Analgesia: Activational and Organizational Effects. Journal of Pharmacology and Experimental Therapeutics, 300, pp. 695-701, 2002.

Neuronal Nicotinic Receptor Ligands

There is interest in developing new nicotinic ligands in addition to those currently available, in part because of the diversity of nicotinic receptors in the brain. Nicotine receptors comprise various combinations of alpha and beta peptides, suggesting that ligands selective for these subtypes might have differing pharmacology. Additionally, there is an interest in attempting to separate the toxic side effects of nicotine or related nicotine compounds from their more positive potential medicinal uses in areas such as analgesia, depression, smoking cessation, and neuroimaging. Past efforts to modify the basic structure of nicotine have included the substitution of various groups on the pyridine ring (positions five and six), ring-opened analogs such as 3-aminomethyl or 3-aminoethylpyridines, or the introduction of other heterocyclic systems, such as the imidazole or isoxazole ring rather than the pyrrolidine ring of the parent nicotine. Drs. Linda Dwoskin and Peter Crooks have observed the structural similarity between d-tubocurarine (a dicationic competitive antagonist blocking cholinergic receptors) and bis dicationic nicotinic ligands separated by alkyl chains of varying length. This represents an extension of earlier work indicating that the addition of an alkyl group (such as octyl) to the pyridine nitrogen produced a nicotinic antagonist, capable of inhibiting nicotine binding (alpha4beta2) and blocking the nicotine-induced DA release from rat striatal slices. These researchers have now extended this work to the development of bis-azaaromatic quaternary ammonium analogues, in which two nicotine molecules (or two quinoline analogs) are present, positively charged on the pyridine nitrogen, and separated by an alkyl chain of varying length. The significant finding reported was that some of these structures exhibited considerable receptor selectivity for nicotine receptor subtypes. For example. a ten-carbon chain produced alpha4beta2 receptor antagonism alone in the bis-nicotine series, while a twelve-carbon chain in the bis-quinoline series produced alpha7 antagonism.. Ayers, J.T., Dwoskin, L.P., Deaciuc, A.G., Grinevich, V.P., Zhu, J., and Crooks, P.A. Bis-Azaaromatic Quarternary Ammonium Analogues: Ligands for alpha4beta2 and alpha7 Subtypes of Neuronal Nicotinic Receptors. Bioorganic & Medicinal Chemistry Letters, 12, pp. 3067-3071, 2002.

MDMA ("Ecstasy") Produces Severe Dopaminergic Neurotoxicity in Primates After a Single Dose Regimen

The prevailing view is that MDMA is a selective serotonin neurotoxin, sparing noradrenergic terminals, and sparing dopaminergic terminals except at very high doses. Five squirrel monkeys were given 2 mg/kg subcutaneously 3 times, at 3-hour intervals, to model human MDMA use in a rave setting. Subjects received this regimen only once. One monkey was acutely symptomatic after two doses, and another died of hyperthermia. The other three, tested 2 weeks later, evidenced severe dopaminergic neurotoxicity and less serotonergic neurotoxicity. The investigators then repeated the experiment in 5 baboons (im route of administration). One died of hyperthermia, and one was acutely symptomatic. The neurotoxicity in the baboons was the same as in the monkeys. Noradrenergic axons and terminals were unaffected in both species. There was no evidence that MDMA induced parkinsonism, but the MDMA-treated monkeys were more sensitive to alpha-methyl-para-tyrosine (AMPT) -induced motor dysfunction. AMPT, a tyrosine hydroxylase inhibitor, gradually lowers dopamine levels. Earlier studies in primates generally involved administration of higher MDMA doses (5 or 10 mg/kg) twice daily, morning and evening, for 4 days, which engendered more severe toxicity toward brain serotonin neurons, with no long-term effects on brain dopamine neurons. One explanatory factor for dopaminergic neurotoxicity may be the result of closely spaced doses of MDMA resulting in a changed metabolic profile and nonlinear pharmacokinetics. Ricaurte, G.A., Yuan, J., Hatzidimitriou, G., Cord, B.J., and McCann, U.D. Severe Dopaminergic Neurotoxicity in Primates after a Common Recreational Dose Regimen of MDMA ("Ecstasy"). Science, 297(5590), pp. 2260-2263, 2002.

Methamphetamine-Induced Degeneration of Dopaminergic Neurons Involves Autophagy

Methamphetamine damages dopamine and serotonin-releasing neurons in primates and rodents when administered at levels equivalent to those abused by humans. The mechanism by which this drug induces neural damage, however, is not clear. An unusual aspect of the damage is that methamphetamine damages the axons and dendrites of neurons, but does not kill the neurons. The present work indicates that a novel cellular pathway, "neuronal macroautophagy," is responsible for this unusual form of degeneration. The authors showed that methamphetamine releases dopamine from synaptic vesicles, where it is normally stored and protected from breakdown, into the neuronal cytosol. Methamphetamine also increases dopamine synthesis. The excess dopamine in the cytosol oxidizes to produce highly reactive compounds that destroy cytosolic proteins by binding to the cysteine molecules that are common constituents of these proteins. This promotes the formation of "macroautophagic granules" in the dendrites and axons, which are visually striking, large, double layered organelles adapted for "swallowing" cellular constituents, and are mostly known for being induced in the liver during fasting or starvation. The granules, in an apparent attempt to save the neuron from death during periods of oxidative stress, consume the local axonal and dendritic consitutents and transport them to the cell body, thus destroying the neurites but saving the cell. Larsen, K. E., Fon, E.A., Hastings,T.G., Edwards, R.H., and Sulzer, D. Methamphetamine-induced Degeneration of Dopaminergic Neurons Involves Autophagy and Upregulation of Dopamine Synthesis. Journal of Neuroscience, 22(20), pp. 8951-60, 2002.

Pain Treatment Device Approved by FDA and is in Commercial Production

NIDA SBIR grantee Steven Michelson (Cyclotec Advanced Medical Technologies, Inc.) and his colleagues have developed and tested the CT1 pain control stimulator. The CT1 uses transcutaneous electrical nerve stimulation (TENS) technology, a technology that has been found over the past 30 years to be very effective in attenuating certain types of pain. The CT1 is a sophisticated yet simple device that delivers TENS directly to the areas in pain, without cumbersome wires or complicated electrode placements. This device has minimal side effects, and offers an effective alternative to drugs in the treatment of some types of pain. The device has recently received FDA approval as a pain treatment, and has gone into commercial production. A second generation TENS device (CT2) is now also being developed by Cyclotec with funding from a NIDA STTR grant.

Crystal Structures of Dipeptides Containing the Dmt-Tic Pharmacophore

Despite enormous progress in understanding the opioid systems of the brain, the mechanism of action of opioids is still not completely known. Neither the three-dimensional structures of the opioid receptors nor their ligand binding sites have been determined. There is need to design new opioid compounds to understand the opioid system as well as to provide leads for new therapeutic compounds that have fewer side effects than the known existing compounds. An essential goal in the development of new opioid compounds is the formation of agonists and antagonists with a high degree of selectivity for either one specific opioid receptor subtype or a combination of characteristics that permit the ligand to interact with high affinity at two distinct receptors while eliciting opposite effects as a bi- or heterofunctional molecule. Toward this end, NIDA-supported investigators determined the crystal structures of three analogues of the potent delta-opioid receptor antagonist H-Dmt-Tic-OH (2',6'-dimethyl-L-tyrosine-L-1,2,3,4-tetrahydroisoquinoline-3-carboxylate), N,N(CH3)2-Dmt-Tic-OH (1), H-Dmt-Tic-NH-1-adamantane (2), and N,N (CH3)2-Dmt-Tic-NH-1-adamantane (3) were determined by X-ray single crystal analysis. Crystals of 1 were grown by slow evaporation, while those of 2 and 3 were grown by vapor diffusion. Compounds 1 and 3 crystallized in the monoclinic space group P2 1, and 2 crystallized in the tetragonal space group P43. Common backbone atom superimpositions of structures derived from x-ray diffraction studies resulted in root-mean-square (rms) deviations of 0.2-0.5 'A, while all atom superimpositions gave higher rms deviations from 0.8 to 1.2 'A. Intramolecular distances between the aromatic ring centers of Dmt and Tic were 5.1 'A in 1, 6.3 'A in 2, and 6.5 'A in 3. The orientation of the C-terminal substituent 1-adamantane in 2 and 3 was affected by differences in the psi torsion angles and strong hydrogen bonds with adjacent molecules. Despite the high delta-opioid receptor affinity exhibited by each analogue (ki < 0.3 nM), high mu-receptor affinity (ki < 1 nM) was manifested only with the bulky C-terminal 1-adamantane analogues 2 and 3. Furthermore, the bioactivity of both 2 and 3 exhibited mu-agonism, while 3 also had potent delta-antagonist activity. These data demonstrate that a C-terminal hydrophobic group is an important determinant for eliciting mu-agonism, whereas N-methylation maintained delta-antagonism. Furthermore, the structural results support the hypothesis that expanded dimensions between aromatic nuclei is important for acquiring mu-agonism. Bryant, S.D., George, C., Flippen-Anderson, J.L., Deschamps, J.R., Salvadori, S., Balboni, G., Guerrini, R., and Lazarus, L.H. Crystal Structures of Dipeptides Containing the Dmt-Tic Pharmacophore. Journal of Medicinal Chemistry, 45, pp. 5506-5513, 2002.

Increasing Brain Serotonin Activity Attenuates the Reinforcing and Neurochemical Effects of Cocaine in Monkeys

NIDA grantee Leonard Howell and his colleagues at the Yerkes Regional Primate Research Center in Georgia evaluated the ability of serotonergic (5-HT) treatments to modulate the reinforcing and neurochemical effects of cocaine in nonhuman primates. Alaproclate, a 5-HT uptake inhibitor, and quipazine, a 5-HT direct agonist, were examined first and found to decrease cocaine self-administration in a manner that was not due to nonspecific effects. The neurochemical bases of these effects were examined subsequently by means of in vivo microdialysis studies in awake monkeys. Pretreatment with either compound at doses that reduced self-administration of cocaine also attenuated cocaine induced elevation of brain dopamine. Although the interactions of the serotonergic and dopaminergic systems in manifestation of cocaine's behavioral and reinforcing effects are likely complex, these findings nonetheless suggest an important avenue for developing new medications to treat cocaine abuse. Czoty, P.W., Ginsberg, B.C., and Howell, L.L. Serotonergic Attenuation of the Reinforcing and Neurochemical Effects of Cocaine in Squirrel Monkeys. Journal of Pharmacology and Experimental Therapeutics, 300, pp. 831-837, 2002.

Chronic Morphine Exposure Induces a Spinal Dynorphin-Dependent Enhancement of Excitatory Transmitter Release From Primary Afferent Fibers

Paradoxical opioid-induced pain has been demonstrated repeatedly in humans and animals. The mechanisms of such pain are unknown but may relate to opioid-induced activation of descending pain facilitatory systems and enhanced expression and proprioceptive actions of spinal dynorphin. Dr. Frank Porreca and his research team at the University of Arizona tested the possibility that these opioid-induced central changes might mediate increased excitability to the spinal cord. Tactile and thermal hypersensitivity was observed at 7, but not 1, days after subcutaneous morphine pellet implantation; placebo pellets produced no effects. Basal and capsaicin-evoked release of calcitonin gene-related peptide (CGRP) was measured in spinal tissues taken from naive rats or rats on post-pellet days 1 and 7. The content and evoked release of CGRP were significantly increased in tissues from morphine-exposed rats at 7, but not 1, days after implantation. Morphine increased spinal dynorphin content on day 7 in rats with sham bilateral lesions of the dorsolateral funiculus (DLF) but not in rats with DLF lesions. Pharmacological application of dynorphin A(2-13), a non-opioid fragment, to tissues from naive rats enhanced the evoked release of CGRP. Enhanced evoked release of CGRP from morphine-pelleted rats was blocked by dynorphin antiserum or by previous lesions of the DLF. Sustained morphine induces plasticity in both primary afferents and spinal cord, including increased CGRP and dynorphin content. Morphine-induced elevation of spinal dynorphin content depends on descending influences and enhances stimulated CGRP release. Enhanced transmitter release may allow increased stimulus-evoked spinal excitation, which is likely to be critical for opioid-induced paradoxical pain. Such pain may manifest behaviorally as antinociceptive tolerance. Gardell, L.R., Wang, R., Burgess, S.E., Ossipov, M.H., Vanderah, T.W., Malan, T.P., Jr., Lai, J., and Porreca, F. Sustained Morphine Exposure Induces a Spinal Dynorphin-dependent Enhancement of Excitatory Transmitter Release from Primary Afferent Fibers. Journal of Neuroscience, 22(15), pp. 6747-6755, 2002.

Neuronal Apoptosis Associated with Morphine Tolerance: Evidence for an Opioid-Induced Neurotoxic Mechanism

Tolerance to the analgesic effect of an opioid is a pharmacological phenomenon that occurs after its prolonged administration. Activation of the NMDA receptor (NMDAR) has been implicated in the cellular mechanisms of opioid tolerance. However, activation of NMDARs can lead to neurotoxicity under many circumstances. Dr. Mao at the Pain Center and his colleagues in the Neural Plasticity Research Group, at the Massachusetts General Hospital, Harvard Medical School, demonstrated that spinal neuronal apoptosis (cell death) occurred in rats made tolerant to morphine administered through intrathecal boluses or continuous infusion. The apoptotic cells were predominantly located in the superficial spinal cord dorsal horn, and most apoptotic cells also expressed glutamic acid decarboxylase, a key enzyme for the synthesis of the inhibitory neurotransmitter GABA. Consistently, increased nociceptive sensitivity to heat stimulation was observed in these same rats. Spinal glutamatergic activity modulated morphine-induced neuronal apoptosis, because pharmacological perturbation of the spinal glutamate transporter activity or coadministration of morphine with the NMDAR antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate, affected both morphine tolerance and neuronal apoptosis. At the intracellular level, prolonged morphine administration resulted in an upregulation of the proapoptotic caspase-3 and Bax proteins, and a downregulation of the anti-apoptotic Bcl-2 protein in the spinal cord dorsal horn. Furthermore, co-administration of morphine with N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (a pan-caspase inhibitor) or acetyl-aspartyl-glutamyl-valyl-aspart-1-aldehyde (a relatively selective caspase-3 inhibitor) blocked morphine-induced neuronal apoptosis. Blockade of the spinal caspase-like activity also partially prevented morphine tolerance and the associated increase in nociceptive sensitivity. These results indicate an opioid-induced neurotoxic consequence regulated by the NMDAR-caspase pathway, a mechanism that may have clinical implications in opioid therapy and substance abuse. Mao, J., Sung, B., Ji, R-R. and Lim, L. Neuronal Apoptosis Associated with Morphine Tolerance: Evidence for an Opioid-Induced Neurotoxic Mechanism. Journal of Neuroscience, 22(17), pp. 7650-7661, 2002.

A Common Receptor and More Defined Pathway is Revealed for Stopping Neuronal Growth in its Tracks

Plasticity is the process by which the proper formation and breaking of connections among neurons occurs. Understanding neuronal plasticity is a goal for many behavioral neuroscientists because this process is fundamental to the acquisition of many types of memory and addiction. Plastic changes produced by drugs of abuse are likely to use the same mechanism as those employed during development and learning. A great deal of insight into the mechanism of plasticity has been obtained by studying the regulation of outgrowth and retraction of both axons (the cable-like structure that sends electrical impulses to end of the neuron) and dendrites (a cable-like structure involved in receiving and integrating the signals from neighboring neurons). Nogo-A, oligodendrocyte-myelin glycoprotein (OMgp), and myelin-associate glycoprotein (MAG) are three protein signals that are now known to inhibit neuronal outgrowth through a common pathway. Previous work has suggested that Nogo-A and MAG exert their actions through the Nogo receptor (NgR). In a 2002 Nature paper, Dr. Zhigang He and his colleagues demonstrate that OMgp also acts through the NgR. In a subsequent report in Nature, this same group showed that NgR elicits its inhibitory response indirectly, via the transmembrane protein p75. Since NgR is a GPI-linked receptor, it is not physically protruding into the cytoplasm. As a result, its connection to the cytoplasm had always been a mystery. Using either cultured cerebellar granule neurons or dorsal root ganglia neurons, the He group showed that p75 both physically interacts with NgR and was likely the transducer of the inhibitory signal. Both of these findings provide real insight into how axonal outgrowth is regulated. Given that this same NgR mechanism is used by neurons to direct plasticity in the regions of the brain associated with substance abuse (the cortex, amygdala and others), this information should be very useful in our ability to understand, and potentially treat, addiction and other psychiatric disorders. In addition, this discovery has major implications for the development of small chemical ligands for the treatment of spinal cord injury. Wang, K.C., Koprivica, V., Kim, J.A., Sivasankaran, R., Guo, Y., Neve, R.L., and He, Z. Oligodendrocyte-myelin Glycoprotein is a Nogo Receptor Ligand that Inhibits Neurite Outgrowth. Nature, 417(6892), pp. 941-944, 2002; Wang, K.C., Kim, J.A., Sivasankaran, R., Segal, R., and He, Z. P75 Interacts with the Nogo Receptor as a Co-receptor for Nogo, MAG and OMgp. Nature, 420(6911), pp. 74-78, 2002.

Amphetamine Enhanced VTA Neuron Burst Firing and Phasic Release of DA May Account for Individuals' Tendency for Self-administration of Psychostimulants

Dr. John Williams and his colleagues found an adaptive change in the alpha- adrenergic receptors mediated membrane excitability of VTA dopamine neurons to psychostimulants. This was likely accompanied by a change in the pattern of dopamine release, which may account for individuals' tendency to self-administer psychostimulants. VTA dopamine neurons receive inputs from glutamatergic and noradrenergic neurons. Glutamatergic afferents activate both ionotropic and metabotropic glutamate receptors of the VTA dopamine neurons. Activation of the ionotropic glutamate receptor depolarized the DA neurons. Activation of the metabotropic glutamate receptors (mGlu) opens calcium-sensitive K+ channels and causes cell membrane hyperpolarization. The net result is a burst firing pattern of the DA neurons -- the ionotropic glutamate activation initiates neuron firing, which is followed by a transient pause due to the metabotropic glutamate hyperpolarization. The consequence is a phasic release of dopamine. The mGlu receptor-mediated hyperpolarization is attenuated and therefore the excitability of the VTA neuron is increased after activation of alpha adrenergic receptors, and dopamine receptors, similar to observations seen with the psychostimulants such as amphetamine or cocaine. The disinhibitory action of psychostimulants is mediated by the alpha-1 adrenergic receptor as their effects are reversed after application of the alpha-1 adrenergic receptor antagonist. The kinetics and attenuation of the magnitude of the membrane hyperpolarization would extend the depolarization of the DA neurons, curtail their burst activity and increase the phasic release of dopamine. The present results shed light on how the psychostimulant amphetamine acts at the cellular level. In addition, this research is implicating an alternative mechanism for how amphetamine might exert its effects on dopamine neurons and dopamine-dependent behavior. Paladini, C.A., Fiorillo, C.D., Morikawa, H., and Williams, J.T. Amphetamine Selectively Blocks Inhibitory Glutamate Transmission in Dopamine Neurons. Nature Neuroscience, 4(3), pp. 275-281, 2002.

Involvement of DARPP-32 Phosphorylation in the Stimulant Action of Caffeine

Dr. Lindskog and colleagues have elucidated some of the intracellular mechanisms that are involved in the stimulatory effects of caffeine on motor activity in mice. It had been previously reported by others that the psychostimulant effects of caffeine are due to blockade of adenosine A2A receptors. In the studies reported here, the investigators used A2A receptor agonist and antagonist reagents and mice lacking the DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of relative molecular mass 32,000) phosphoprotein to determine whether or not the motor activity of caffeine is regulated through DARPP-32. DARPP-32 is known for its role as a mediating molecule between slow and fast synaptic transmission. The activity of DARPP-32 is determined in part by the state of phosphorylation of various amino acids along the DARPP-32 molecule itself. What researchers found in this study is that caffeine, like adenosine A2A receptor antagonist, causes an increase in the phosphorylation of DARPP-32 at amino acid threonine 75. Phosphorylation at this particular residue makes DARPP-32 an inhibitor of a kinase known as PKA. These researchers expect that the caffeine-induced increase would lower PKA activity and provide a positive feedback amplification mechanism for shutting down adenosine A2A receptor-stimulated PKA signaling pathways. In this study they were able to demonstrate the involvement of DARPP-32 and its phosphorylation/dephosphorylation in the stimulant action of caffeine. Lindskog, M., Svenningsson, P., Pozzi, L., Kim, Y., Fienberg, A.A., Bibb, J.A., Fredholm, B.B., Nairn, A.C., Greengard, P., and Fisone, G. Involvement of DARPP-32 Phosphorylation in the Stimulant Action of Caffeine. Nature, 418(6899), pp. 774-778, 2002.

Elevated Expression of 5-HT1B Receptors in Nucleus Accumbens Efferents Sensitizes Animals to Cocaine

Although the effects of psychostimulants on brain dopamine systems are well recognized, the direct actions of cocaine on serotonin systems also appear to be important to its addictive properties. For example, serotonin actions at 5-HT1B receptors in the ventral tegmental area (VTA) modulate cocaine-induced dopamine release in the nucleus accumbens (NAcc) and alter the rewarding and stimulant properties of cocaine. However, the mechanisms of these effects have been unclear, because several neuron types in VTA express 5-HT1B receptors. One possibility is that 5-HT1B receptors on the terminals of GABAergic projections from NAcc to VTA inhibit local GABA release, thereby disinhibiting VTA neurons. Dr. Carlezon's lab tested this hypothesis directly by using viral-mediated gene transfer to overexpress 5-HT1B receptors in NAcc projections to VTA. A viral vector containing either epitope hemagglutinin-tagged 5-HT1B and green fluorescent protein (HA1B-GFP) cassettes or green fluorescent protein cassette alone (GFP-only) was injected into the NAcc shell, which sends projections to the VTA. HA1B-GFP injection induced elevated expression of 5-HT1B receptors in neuronal fibers in VTA and increased cocaine-induced locomotor hyperactivity without affecting baseline locomotion. Overexpression of 5-HT1B receptors also shifted the dose-response curve for cocaine-conditioned place preference to the left, indicating alterations in the rewarding effects of cocaine. Thus, increased expression of 5-HT1B receptors in NAcc efferents, probably in the terminals of medium spiny neurons projecting to the VTA, may contribute to psychomotor sensitization and offer an important target for regulating the addictive effects of cocaine. Neumaier, J.F., Vincow, E.S., Arvanitogiannis, A., Wise, R.A., and Carlezon, W.A., Jr. Elevated Expression of 5-HT1B Receptors in Nucleus Accumbens Efferents Sensitizes Animals to Cocaine. Journal of Neuroscience, 22, pp. 10856-10863, 2002.

Extinction-Induced Upregulation in AMPA Receptors Reduces Cocaine-Seeking Behavior

Preventing relapse is the greatest challenge to successful treatment of drug addiction. Furthermore, the risk of relapse may be present at almost anytime during the period of abstinence, from a few days to a few years, and is especially pronounced during stressful situations. Relapse may be reduced, however, through extinction training, a procedure wherein drug-seeking behavior is no longer reinforced by drug administration. In a recent report, Dr. David Self and his colleagues demonstrate that extinction training during withdrawal from cocaine self-administration in rats produces increases in GluR1 and GluR2/3 subunits of AMPA glutamate receptors in the shell of the nucleus accumbens (NAcc), a brain region critical for cocaine reward. Following the acquisition of cocaine self-administration, three groups of rats were withdrawn from cocaine for one week under different conditions. The extinction group (EXT) was returned to the self-administration environment, but lever presses no longer delivered cocaine; the home cage group (HC) remained undisturbed in the home cage during this time; the levers retracted group (LR) was returned to the self-administration environment, but the levers were unavailable. Thus, only Group EXT had the opportunity to learn the futility of lever responding and, as expected, this group ceased responding compared to the HC and LR groups. The new finding is that extinction training increased the amount of GluR1 and GluR2/3 subunits in the NAcc shell and the GluR1 increase was directly related to the level of extinction achieved, suggesting that GluR1 might promote extinction of cocaine seeking. Dr. Self and his colleagues confirmed this hypothesis by demonstrating that viral-mediated over- expression of these receptor subunits in the NAcc facilitated cocaine-seeking but not sucrose-seeking responses. Moreover, another experiment demonstrated that the facilitatory effect of GluR on extinction is due to reduced motivation to seek cocaine, not to the enhancement of extinction learning. A final experiment demonstrated that a single extinction session, when conducted during GluR over-expression, attenuated stress-induced (but not cue- or drug prime-induced) relapse to cocaine seeking behavior, even after GluR levels returned to baseline levels. In summary, these results indicate that extinction-induced changes in glutamate receptors may facilitate control over cocaine seeking by restoring glutamatergic tone in the NAcc, and, thereby, reduce the propensity for relapse under stressful conditions after prolonged abstinence. The results also suggest that behavioral approaches have the potential to reverse or ameliorate the harmful consequences of drug use. Sutton, M.A., Schmidt, E.F., Choi, K.H., Schad, C.A., Whisler, L., Simmons, D., Karanian, D.A., Monteggia, L.M., Neve, R.L., and Self, D.S. Extinction-induced Upregulation in AMPA Receptors Reduces Cocaine-seeking Behaviour. Nature, 421, pp. 70-75, 2002.


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