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NIDA Home > Publications > Director's Reports > September, 2008 Index    

Director's Report to the National Advisory Council on Drug Abuse - September, 2008

Research Findings - Basic Neuroscience Research

Voluntary Oral

Nicotine Intake in Mice Down-Regulates Glur2 But Not Depression-Like Behaviors Dr. Marina Picciotto and her colleagues at Yale University have continued their studies on the adaptive changes in brain and behavior that accompany repeated exposure to nicotine. Some of these adaptive changes are thought to be mediated by glutamate receptors (GluR) and cAMP response element-binding protein (CREB) in the nucleus accumbens. Dr. Picciotto studied the effects of nicotine exposure via the drinking water on nicotine preference, locomotor activity, and anxiety and depression-like behaviors in inbred mice. She and her colleagues reported few behavioral changes following extended nicotine exposure, but clear down-regulation of GluR2 in the mesolimbic system. When given a choice between nicotine and control solutions, mice showed a significant preference for nicotine. Dr. Picciotto interpreted these findings to indicate that voluntary nicotine drinking induces nicotine preference in mice with accompanying down regulation of GluR, but that differences are not sufficient to explain preference for nicotine. Vieryra-Reyes, P. Picciotto, M., and Mineur, Y. Voluntary Oral Nicotine Intake in Mice Down-regulates GluR2 but Does Not Modulate Depression-like Behaviors. Neuroscience Letters, 434, pp. 18-22, 2008.

Improved Synthesis of the ORL Antagonist, 1-[(3R, 4R)-1-Cyclooctylmethyl-3-ethoxycarbonyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397)

Four subtypes of opioid receptors have been identified based upon structural homology. These include the classical mu, delta, and kappa receptors (MOP, DOP, and KOP, respectively) and the more recently identified opioid-receptor-like-1 (ORL-1), which is termed NOP. The endogenous ligand for NOP is a heptapeptide named as nociceptin by one group of investigators and orphanin FQ (N/OFQ) by another group. Several studies have shown that NOP may be involved in pain regulation, drug dependence, anxiety and stress, depression, learning and memory, motor activity, epilepsy/seizures, cardiovascular effects, urinary incontinence, cough and immunoregulation. A number of ORL-1 (NOP) receptor agonists and antagonists have been synthesized and used to characterize the pharmacological effects of the NOP system. The first and most studied small molecule antagonist is 1-[(3R, 4R)-1-Cyclooctylmethyl-3-ethoxycarbonyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397). The synthesis of this molecule has been reported previously. In two reports the optical resolution of the racemate of J-113397 was achieved by chromatography with a chiral column, and in another report a new synthesis of (+)- and (-)- J-113397 was reported. In the present study the authors have avoided the expense of large scale chiral column and provided an alternate practical synthesis of (+/-)-J113397 by separating the racemic compound into individual (+) - and (-)-isomers using a chiral auxiliary. Carroll, F.I. and Brieaddy, L.E. Improved Synthesis of the ORL Antagonist 1-[(3R,4R)-1-Cyclooctylmethyl-3-ethoxycarbonyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397). Synthetic Communication, 38, pp. 1926-1930, 2008.

Microchip Capillary Electrophoresis

Members of the mammalian family of proteins known as RGS proteins (regulators of G-protein signaling) serve as GTPase-activating proteins, since they can bind to G proteins, reduce the normal lifetime of the GTP-G complex by hydrolysis, and effectively "turn off" the associated G protein-coupled receptor (GPCR) signaling pathway. RGS proteins have become a target for the design of RGS inhibitors, in order to potentiate the effects of GPCRs activated by agonist drugs. Recently, Dr. Richard Neubig and his colleagues at the University of Michigan reported on the development and validation of a micro-fabricated channel electrophoresis device for the determination of GTP hydrolysis and enzyme kinetics, which promises to improve the capability of high throughput screening. The completed device was subjected to validation for intra- and inter-channel deviation. The microchip could demonstrate the acceleration of G GTPase activity by RGs (based on the increase in product BGDP), as well as a reduction in BGDP in the presence of an RGS4 inhibitor, such as the YJ34 peptide, or the small molecule inhibitor CCG-4986. The screening of RGS inhibitors could be done at the rate of 4300 samples/hour for one determination each, if a library of inhibitors were to be tested. Finally, the microchip could also be used to obtain a complete dose response curve, in this case, one that provides a determination of the EC50 for inhibition of basal BGTP hydrolysis by formation of a GTPgammaS-BGTP complex. Pei, J. Dishinger, D.L. Roman, C. Rungwanitcha, R.R. Neubig and Kennedy, R.T. Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes using RGS-G Protein Interactions as a Model System. Analytical Chemistry, 80(13), pp. 5225-5231, 2008.

CD4+ T Lymphocytes Contribute To Spinal Nerve Transection-Induced Neuropathic Pain

Neuropathic pain is initiated or caused by a primary lesion or dysfunction in the nervous system. Current treatments for neuropathic pain are not effective and are often associated with various side effects such as physical dependence, hyperalgesia, tolerance, and sedation. Understanding the underlying mechanisms of neuropathic pain may help to develop novel, non-addictive analgesic agents. This study investigated the role of infiltrating T lymphocytes in the etiology of persistent pain by using a murine spinal nerve L5 transection (L5Tx) neuropathic pain model. T lymphocyte-deficient mice showed no evident mechanical hypersensitivity after day 3 of L5Tx compared to wild-type mice. Employing a Fluorescence Activated Cell Sorter (FACS) technique, these investigators observed significant leukocytic infiltration (CD45(hi)) into the lumbar spinal cord that peaked at day 7 post L5Tx. These infiltrating leukocytes contained predominantly CD4(+), but not CD8(+) T lymphocytes. B lymphocytes, natural killer cells, and macrophages were not detected at day 7 post L5Tx. No differences in the activation of peripheral CD4(+) T lymphocytes were detected in either the spleen or lumbar lymph nodes between L5Tx and sham surgery groups. Furthermore, CD4 KO mice displayed significantly decreased mechanical hypersensitivity after day 7 of L5Tx; adoptive transfer of CD4(+) leukocytes reversed this effect. Decreased immunoreactivity of glial fibrillary acidic protein observed in CD4 KO mice post L5Tx indicated possible T lymphocyte-glial interactions. These results suggest a contributing role of spinal cord-infiltrating CD4(+) T lymphocytes versus peripheral CD4(+) T lymphocytes in the maintenance of nerve injury-induced neuropathic pain. Approaches to reduce CD4+ T lymphocyte infiltration may reduce CD4+ T lymphocyte-mediated CNS responses after nerve injury and thus may be useful in chronic neuropathic pain management. Cao, L. and DeLeo, J.A. CNS-infiltrating CD4+ T Lymphocytes Contribute to Murine Spinal Nerve Transection-induced Neuropathic Pain. European Journal of Immunology, 38(2), pp. 448-458, 2008.

The Endogenous Cannabinoid System Modulates Nicotine Reward and Dependence

A growing body of evidence suggests that the endogenous cannabinoid system modulates the addictive properties of nicotine, the main component of tobacco that produces rewarding effects. In this study, complementary transgenic and pharmacological approaches were used to test the hypothesis that the endocannabinoid system modulates nicotine reward and dependence. An acute injection of nicotine elicited normal analgesic and hypothermic effects in knockout (KO) mice lacking the cannabinoid-1 receptors (CB1) and mice treated with the CB1 antagonist Rimonabant. However, disruption of CB1 receptor signaling blocked nicotine reward, as assessed in the conditioned place preference (CPP) paradigm. Conversely, genetic deletion or pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for catabolism of the endocannabinoid anandamide, enhanced the expression of nicotine CPP. Although the expression of spontaneous nicotine withdrawal was unaffected in CB1 KO mice, acute administration of Rimonabant ameliorated somatic withdrawal signs in wild type mice. Increasing endogenous levels of anandamide through genetic or pharmacological approaches exacerbated the physical somatic signs of spontaneous nicotine withdrawal in a milder withdrawal model. Moreover, FAAH-compromised mice displayed increased conditioned place aversion in a mecamylamine-precipitated model of nicotine withdrawal. These findings suggested that endocannabinoids play a role in the rewarding properties of nicotine, as well as in nicotine dependence liability. Specifically, increasing endogenous cannabinoid levels magnifies, whereas disrupting CB1 receptor signaling attenuates, nicotine reward and withdrawal. Collectively, these results support the hypothesis that cannabinoid receptor antagonists may offer therapeutic advantages to treat tobacco dependence. Merritt, L.L. Martin, B.R., Walters, C., Lichtman, A.H., and Damaj, M.I. The Endogenous Cannabinoid System Modulates Nicotine Reward and Dependence. Journal of Pharmacology and Experimental Therapeutics, May 2008, E-pub ahead of print.

Lithium Protects against Phencyclidine (PCP) Neurotoxicity

PCP, ketamine, and other N-methyl-D-aspartate (NMDA) receptor antagonists are known to produce neuroapoptosis when administered during early postnatal development in rats. Early treatment with NMDA antagonists (equivalent to exposure in humans in the prenatal third trimester) results in schizophrenia-like behaviors when the rats reach early adolescence and young adulthood. Lithium is clinically used to treat schizoaffective and bipolar disorders and has recently been shown to have neuroprotective properties. The present study used corticostriatal slices taken from postnatal day-2 rat pups to investigate the possible protective effect of lithium and the role of the phosphatidylinositol-3 kinase (PI-3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in PCP-induced neuroapoptosis. It is known that PI-3K/Akt generally promotes cell survival by phosphorylating (and thus inhibiting) pro-apoptotic proteins such as caspase-9 and glycogen synthase kinase-3 (GSK-3). They found that lithium pretreatment dose-dependently reduced PCP-induced caspase-3 activation and DNA fragmentation in layer II-IV of the cortex. PCP elicited time-dependent inhibition of the ERK and PI-3K/Akt pathways, as indicated by dephosphorylation of ERK1/2 and Akt. GSK-3 was also dephosphorylated at serine 9 and thus activated by PCP. Lithium prevented PCP-induced inhibition of the two pathways and activation of GSK-3. Further, blocking either the PI-3K/Akt or ERK pathway abolished the protective effect of lithium, and inhibiting GSK-3 activity mimicked the protective effect of lithium. No crosstalk between the two pathways was found, and specific GSK-3 inhibition did not prevent PCP-induced dephosphorylation of Akt and ERK. These data indicate that the protective effect of lithium against PCP-induced neuroapoptosis is mediated through independent stimulation of the PI-3K/Akt and ERK pathways and suppression of GSK-3 activity. Xia, Y., Wang, C.Z., Liu, J., Anastasio, N.C., and Johnson, K.M. Lithium Protection of Phencyclidine-induced Neurotoxicity in Developing Brain: the Role of PI-3K/Akt and MEK/ERK Signaling Pathways. Journal of Pharmacology and Experimental Therapeutics, 2008 Jun 10 [E-pub ahead of print].

Glutamate Release in the Nucleus Accumbens Core Is Necessary for Heroin Seeking

Although critical roles for glutamatergic neuroplasticity (i.e., the ability of glutamatergic synapses to change long-term as circumstances dictate) have been well described after exposure to the stimulants cocaine and amphetamine, roles for glutamate in opiate addiction are less established. However, very recent findings indicate that glutamatergic systems plasticity does indeed play critical roles in opiate addiction. Peter Kalivas' group showed, in rats trained to self-administer heroin and then extinguished, that reinstatement by either noncontingent heroin or cue (models of relapse) was accompanied by increased extracellular glutamate in the nucleus accumbens (NAc) core (NAcore). As has been shown for reinstatement of cocaine self-administration, the increase in glutamate during heroin-induced reinstatement was abolished by inhibiting glutamatergic afferents from the prelimbic cortex to the NAcore with the use of GABA agonists. Both cocaine and heroin reinstatement were blocked by inhibiting AMPA-type glutamate receptors in the NAcore. This new finding complements that of the Shaham lab at the NIDA IRP showing that activation of inhibitory mGluR2/3 autoreceptors in the NAcore or shell, which reduces glutamate release, prevents cue-induced reinstatement of heroin seeking (Bossert et al. 2006). LaLumiere, R.T. and Kalivas, P.W. Glutamate Release in the Nucleus Accumbens Core Is Necessary for Heroin Seeking. J. Neurosci., 28, pp. 3170-3177, 2008.

Agonist-Directed Signaling of the Serotonin-2A Receptor Depends on -Arrestin-2 Interactions In Vivo

Visual and auditory hallucinations accompany certain neuropsychiatric disorders, such as schizophrenia, and they also can be induced by the use or abuse of certain drugs. The serotonin 2A receptors (5-HT2ARs) are molecular targets for drug-induced hallucinations. However, the cellular mechanisms by which the 5-HT2AR mediates these effects are not well understood. Drugs acting at the 5-HT2AR can trigger diverse signaling pathways that may be directed by the chemical properties of the drug. -arrestins are intracellular proteins that bind to these receptors and are a point where such divergences in ligand-directed functional signaling could occur. Dr. Laura Bohn at the Ohio State University College of Medicine compares the endogenous agonist, serotonin, to a synthetic 5-HT2AR hallucinogenic agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), in mice lacking -arrestin-2, as well as in cells lacking -arrestins. In mice, they find that serotonin induces a head twitch response by a -arrestin-2-dependent mechanism. However, DOI invokes the behavior independent of -arrestin-2. The two structurally distinct agonists elicit different signal transduction and trafficking patterns upon activation of 5-HT2AR, which hinge on the presence of -arrestins. Their study suggests that the 5-HT2AR--arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, and this could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia. Schmid, C.L., Raehal, K.M., and Bohn, L.M. Agonist-directed Signaling of the Serotonin-2A receptor Depends on Beta-arrestin-2 Interactions In Vivo. Proceedings of the National Academy of Sciences USA, 105(3), pp. 1079-1084, 2008.

Morphine-Induced Receptor Endocytosis in a Novel Knockin Mouse Reduces Tolerance and Dependence

Opioid drugs, such as morphine, are among the most effective analgesics available, but their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R), which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an ''OFF'' switch by uncoupling receptors from G protein. Second, endocytosis functions as an ''ON'' switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the OFF and ON function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, Dr. Whistler and her group at the Ernest Gallo Clinic and Research Center generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence. Kim, J.A., Bartlett, S., He, L., Nielsen, C.K., Chang, A.M., Kharazia, V., Waldhoer, M., Ou, C.J., Taylor, S., Ferwerda, M., Cado, D., and Whistler, J.L. Morphine-induced Receptor Endocytosis in a Novel Knockin Mouse Reduces Tolerance and Dependence. Current Biology, 18(2), pp. 129-135, 2008.

Anomalous Dopamine Release Associated with a Human Dopamine Transporter Coding Variant

Dopamine (DA) signaling at synapses is tightly coordinated through opposing mechanisms of vesicular fusion-mediated DA release and transporter-mediated DA clearance. Altered brain DA signaling is thought to underlie multiple brain disorders, including drug addiction, Parkinson's disease, and a number of psychiatric syndromes. Dr. Blakely and his group identified a pedigree containing two male children diagnosed with ADHD who share a rare human DA transporter (DAT; SLC6A3) coding variant, Ala559Val. Although hDAT Ala559Val supports normal DAT protein and cell surface expression, as well as normal DA uptake, the variant exhibits anomalous DA efflux from DA-loaded cells. That is, they appear to reverse transport at the DAT. Additionally, they also show that hDAT Ala599Val exhibits increased sensitivity to intracellular Na(+), but not intracellular DA, and displays exaggerated DA efflux at depolarized potentials. Remarkably, the two most common ADHD medications, amphetamine and methylphenidate, both block hDAT Ala559Val-mediated DA efflux, whereas these drugs have opposite actions at wild-type hDAT. Their findings reveal that DA efflux, typically associated with amphetamine-like psychostimulants, can be produced through a heritable change in hDAT structure. Because multiple gene products are known to coordinate to support amphetamine-mediated DA efflux, the properties of hDAT Ala559Val may have broader significance in identifying a new mechanism through which DA signaling disorders arise. Additionally, they suggest that block of inappropriate neurotransmitter efflux may be an unsuspected mechanism supporting the therapeutic actions of existing transporter-directed medications. Mazei-Robison, M.S., Bowton, E., Holy, M., Schmudermaier, M., Freissmuth, M., Sitte, H.H., Galli, A. and Blakely, R.D., Journal of Neuroscience, 28(28), pp. 7040-7046, 2008.

There is Metaplastic Control of the Endocannabinoid System At Inhibitory Synapses In Hippocampus

Endocannabinoids (eCBs) are important signaling molecules that modulate synaptic plasticity. This ability to induce additional modulation (plasticity) of neuronal plasticity responses has been termed metaplasticity. eCB system (eCB synthesis, release, transport) activation can occur either in response to increased intracellular Ca2+ concentrations, or through activation of G-protein coupled receptors including metabotropic glutamate receptors (mGluRs). This paper reports the identification of a novel calcium-dependent mechanism that regulates mGluR-dependent eCB system activation in the hippocampus. In this mechanism, a transient rise in intracellular Ca2+ concentration that does not activate the eCB system primes cells to release eCBs with a subsequent (not concurrent) activation of mGluRs. Conversely, calcium-dependent release of eCBs can be enhanced by prior mGluR activation. These results show that eCB system activation is also subject to metaplasticity, adding another layer of complexity to the regulation of neuronal activity within the hippocampus. Edwards, D.A., Zhang, L., and Alger, B.A. Metaplastic Control of the Endocannabinoid System at Inhibitory Synapses in Hippocampus. Proceedings of the National Academy of Sciences of the United States, 105, pp. 8142-8147, 2008.

Increased Impulsivity during Withdrawal from Cocaine Self-Administration: Role for DFosB in the Orbitofrontal Cortex

Impulsivity is associated with the development and maintenance of addiction in which low prefrontal cortical activity is observed. It is not clear whether impulsivity is an antecedent or a consequence of addiction or both. Furthermore, most studies on impulsivity in cocaine addicts are done when addicts are abstinent and thus may reflect a consequence of withdrawal. Animal models using a 5-choice serial reaction time test (5CSRT) that measures impulsivity and cognitive performance permit scientists to tease out these possibilities. Eric Nestler and his colleagues examined the effect of cocaine self-administration on performance of the 5CSRT during acquisition and withdrawal from cocaine self-administration and determined whether the FosB induction in the orbital frontal cortex (OFC) would alter drug induced affects on impulse control. FosB is induced in many parts of the brain, including the OFC by chronic self-administration of cocaine. Rats self-administering cocaine developed tolerance to the initial errors of omissions and premature responses on the 5CSRT produced by cocaine. In contrast, rats during periods of withdrawal from cocaine showed increased premature responding. Over expression of FosB in the OFC attenuated errors of premature responses during acute self-administration of cocaine but dramatically increased the number of premature responses during cocaine withdrawal. Over expression of JunB (a dominant-negative antagonist of FosB) blocked the development of tolerance to the errors of omission and premature responses. These results suggest that FosB induction by cocaine plays a role in mediating the deficits in impulse control during withdrawal from chronic cocaine self-administration and acts a compensatory mechanism to produce tolerance to the errors produced by acute cocaine. One question remains is whether FosB mimics low prefrontal activity observed in cocaine addicts. While the paper suggests that chronic cocaine impairs impulse control in the withdrawn state, the paper does not address the question of how chronic self-administration of cocaine might affect animals displaying traits of high and low impulsivity as measured by 5CSRT. Thus, the cognitive deficits observed could still be both an antecedent and a consequence of chronic cocaine self-administration. Winstanley, C.A., LaPlant, Q., Theobald, D.E., Green, T.A., Bachtell, R.K., Perrotti, L.I., DiLeone, R.J., Russo, S.J., Garth, W.J., Self, D.W., and Nestler, E.J. Journal of Neuroscience, 27(39), pp. 10497-10507, 2008; Winstanley C.A., Bachtell, R.K., Theobald, D.E.H., Laali, S., Green, T.A., Kumar, A., Chakravarty, S., Self, D.W. and Nestler, E.J. Increased Impulsivity during Withdrawal from Cocaine Self-Administration: Role for FosB in the Orbitofrontal Cortex. Cerebral Cortex., 2008 Jun 6. [E-pub ahead of print]

EphB and EphrinB Are Charged for the Initiation of Synaptogenesis

During synaptogenesis the postsynaptic neuron sends out dendrites which form filopodia to explore and contact the presynaptic axon terminals. If a contact is made and recognized as partner, the cell adhesion triggers synaptogenesis. Until recently, how dendritic filopodial motility is linked to cell-cell interactions and the identity of molecules that regulate the filopodial motility and initiate synaptogenesis were not known. A team led by NIDA researcher Mathew Dalva, at University of Pennsylvania, reports that their new findings provide evidence for EphB-ephrinB, a receptor-ligand cell adhesion molecule pair, functioning as a unifying molecular mechanism that is able to control filopodia motility, cell-cell interactions, and induce synapse differentiation. Eph receptors are transmembrane signaling molecules and are the largest known family of receptor tyrosine kinases in the human genome. They are divided into A and B subclasses based on affinity for their membrane-associated ligands, ephrinA and ephrinB. A number of lines of evidence suggest that EphB receptors are a key regulator of synapse development and spine formation. In the present work, EphB knock-out mice cortical neurons showed the specific loss of synaptic specializations in dendritic protrusions, but not along dendritic shafts, demonstrating that EphBs selectively regulate the formation of many dendritic spine synapses. Expression of EphB2 in neurons that lack endogenous EphB1-3 was sufficient to rescue the phenotype, indicating that EphBs function in cell-autonomously. Moreover, immunostaining in sections from cortex of these animals revealed a ~40% decrease in the number of excitatory synapses. EphB tyrosine kinase activity can trigger intracellular signaling cascades that lead to actin remodeling and, as such, can influence movement and retraction of filopodia. Downstream signaling pathways, through Rho, Cdc42, and PAK, that link activation of EphB receptors to changes in spine morphology are elucidated. Strikingly, the combination of PAK and kinase inactive EphB2 rescues synaptogenesis, but deletion of the ephrin-binding domain from EphB2 precludes rescue, indicating that both motility and trans-cellular interactions are required for synaptic initiation. Thus, EphB-ephrin signaling and cell-cell interactions coordinate both the motility of dendritic filopodia exploration and synaptogenesis. Kayser, M.S., Nolt, M.K., and Dalva, M.B. EphB Receptors Couple Dendritic Filopodia Motility to Synapse Formation. Neuron 59, pp. 56-69, 2008.


Cellular membrane and membrane remodeling are involved in every aspect of cell survival and function, including receptor-ligand signaling, metabolite transport, as well as drug uptake and degradation. However, a structural description of membrane associated macromolecules and their roles in regulating membrane structures are largely missing. The BAR (Bin, amphiphysin, Rvs) domain superfamily proteins have recently been found to be recruited from cytoplasm to the membrane, and trigger the formation of membrane extensions, invaginations, tubular organelles, transport intermediates and endocytic vesicles. To understand how BAR proteins play their roles, NIDA researcher Vinzenz Unger examined the structure and role of one subfamily of BAR protein, the F-BAR, using electron cryomicroscopy. He reports that, during membrane tubule formation, the self segregated F-BAR proteins self assemble into a helical coat on the membrane, providing scaffolding for the folding of the membrane. The F-BAR modules readily bind to the originally flat membranes and generate membrane curvature de novo. By visualizing and following the F-BAR scaffolding, he observed the domain binds to the flat membrane via a surface other than the concave face, and therefore F-BAR helical coats are more than just curvature sensors or stabilizers. There is also no obvious need to invoke membrane curvature-mediated attractive forces since the dimers interact directly and extensively with each other. At the same time, the structural determinants of tubule formation serve to spatially segregate F-BAR activity from other membrane-binding domains. This work is an important first step toward a structural exploration of membrane remodeling during cell endocytosis. Frost, A., Perera, R., Rous A., Spasov, K., Destaing, O., Egelman, E.H., De Camilli, P., and Unger, V.M. Structural Basis of Membrane Invagination by F-BAR Domains. Cell, 132, pp. 807-817, 2008.

Differences between Dorsal and Ventral Striatum in Drd1a Dopamine Receptor Coupling of Dopamine- and cAMP-Regulated Phosphoprotein-32 to Activation of Extracellular Signal-Regulated Kinase

The striatum of the brain plays a role in processing context-and reward-related information to shape learning and behavior. Dopaminergic signals carried from the substantia nigra to the striatum modulate synaptic plasticity of glutaminergi c corticostriatal neurons. Dysregulation of the above process has been implicated in psychostimulant addiction and movement disorders. Sensitization of dopamine D1 receptor (Drd1a) responses occurs in reaction to both psychostimulants and loss of dopamine input to the striatum, as seen in models of Parkinson's disease (PD). This sensitization is influenced by phosphorylation of extracellular signaling-regulated kinase 1/2 (ERK 1/2), whose activity, in turn, is amplified by dopamine receptor protein phosphatase inhibitor [dopamine- and cAMP-related phosphoprotein 32 (DARPP-32)]. Psychostimulant and Drd1a agonist induced Drd1a sensitization coincides with a large increase of phsphorylated ERK1/2 (pERK 1/2) within neurons in the ventral striatum and nucleus accumbens (NAc), but not within neurons of the dorsal striatum. In a dopamine-depleted model of PD, however, there is a significant amount of ERK 1/2 activation within the dorsal striatum. Dr. Paul Worley and colleagues determined that the specific mechanism of Drd1a sensitization associated with PD is not dependent on DARPP-32-induced amplification of ERK 1/2. In this study, the investigators demonstrate an identical marked increase of pERK 1/2 within dopamine-depleted dorsal striatum in both wild-type and DARPP-32 knock-out mice. In addition, the investigators assessed the potential role of DARPP-32 activation of ERK 1/2 L-DOPA-associated dyskinesias. No appreciable difference was observed in the number of pERK 1/2 immunoreactive neurons between wild-type or DARPP-32 KO mice treated with either Drd1a agonist or L-DOPA. These results indicate that the mechanism of DARPP-32 regulated activation of ERK 1/2 is confined to the ventral striatum and NAc. In addition, the above mechanism of dopamine sensitization is not evident in a dopamine-depleted model of PD. Gerfen, C.R., Paletzki, R., and Worley, P. Differences between Dorsal and Ventral Striatum in Drd1a Dopamine Receptor Coupling of Dopamine- and cAMP-Regulated Phosphoprotein-32 to Activation of Extracellular Signal-Regulated Kinase. Journal of Neuroscience, 28(28) pp. 7113-7120, 2008.

Binding Sites For Cocaine and Dopamine In the Dopamine Transporter Overlap

The dopamine transporter (DAT) regulates dopaminergic signaling via reuptake of dopamine from the synaptic cleft. Cocaine binds to DAT with high affinity and inhibits dopamine reuptake, which results in a large increase in extracellular dopamine levels. Previous studies have attempted to elucidate the cocaine binding site within the DAT; however, without an accurate molecular model of DAT, identifying these sites has been difficult. Recently, Loland et al. constructed molecular models for DAT binding of cocaine and the cocaine analog CFT ((-)-2-carbomethoxy-3-(4-fluorophenyl)tropane) based on a high-resolution crystallized structure of the bacterial transporter LeuT, a homolog within the neurotransmitter/Na+ symporter (NSS) family. The investigators report that cocaine and its analogs bind to a site located deep between the transmembrane domains (TMD) 1, 3, 6 and 8 of DAT, which overlaps extensively with the binding sites of dopamine. The model indicates that dopamine and the cocaine analog CFT both interact with the same protein side chains within DAT. For example, asparagine residue 79 within TMD1 interacts with the amine group of dopamine and CFT. Furthermore, both CFT and dopamine interact with hydrophobic and aliphatic residues located within TMD 1, 3, and 6. The above results were confirmed via mutation of residues that interacted with dopamine and cocaine/CFT. The docking model was further validated by introducing structural limitations at sites located extracellularly to cocaine/CFT in order to trap the radiolabeled ligand within its binding pocket. This study presents a complete and experimentally verified model of cocaine and dopamine binding sites within DAT and demonstrates that these sites extensively overlap. As a result, these findings may render the creation of a competitive inhibitor of cocaine that does not disrupt dopamine uptake unfeasible. Beuming, T., Kniazeff, J., Bergmann, M.L., Shi, L., Gracia, L., Raniszewska, K., Newman, A.H., Javitch, J.A., Weinstein, H., Gether, U., and Loland, C.J. The Binding Sites for Cocaine and Dopamine in the Dopamine Transporter Overlap. Nature Neuroscience, 11(7), pp. 780-789, 2008.

Multiple Actions of Spinophilin Regulate Mu Opioid Receptor Function

Spinophilin is a ubiquitous, neuronal dendritic-spine enriched protein that interacts with and modulates the activity of several elements within the G-protein-coupled receptor (GPCR) signaling network. Recent studies have demonstrated that spinophilin modulates alpha-2-adrenergic signaling pathways, which are very similar to those involved with mu-opioid receptor (MOR) responses. In this study, Dr. Venetia Zachariou and colleagues evaluate the effects of spinophilin on the acute and chronic actions of opiates. Spinophilin indirectly promotes MOR internalization and recycling and regulates signal transduction events that are induced after MOR activation. Over expression of spinophilin within PC12 cells results in fast internalization of MOR within 30 minutes of exposure to morphine. Conversely, under control conditions exposure to morphine leads to delayed MOR endocytosis. Previous studies have demonstrated that MOR internalization is positively correlated with increased analgesic tolerance and opiate dependence. Thus, spinophilin modulates responses to chronic opiate exposure by preventing the development of tolerance and dependence. Deletion of the spinophilin gene results in an amplification of the consequences of repeated morphine exposure. When compared to normal wild-type mice, spinophilin knock out (KO) mice display decreased sensitivity to low morphine doses as determined by the 52*C hot plate test. In addition, spinophilin KO mice develop analgesic tolerance to morphine twice as quickly as wild-type mice (2 daily 20 mg/kg i.p. injections of morphine vs. 4 injections). Furthermore, mutant spinophilin mice show withdrawal symptoms that are two times as intense as compared to the wild-type controls. These findings describe the role of spinophilin in regulating MOR signaling pathways and the behavioral responses of opiate exposure. Spinophilin may serve as a target to aid in the investigation and development of opiate drugs that display effective analgesic actions while also limiting the adverse behavioral consequences of opiate exposure. Charlton, J.J., Allen, P.B., Psifogeorgou, K., Chakravarty, S., Gomes, I., Neve, R.L., Devi, L.A., Greengard, P., Nestler, E.J., and Zachariou, V. Multiple Actions of Spinophilin Regulate Mu Opioid Receptor Function. Neuron, 58(2), pp. 238-247, 2008.

Rewarding Stimuli Regulate Chromatin Via A Protein Phosphatase Cascade

Drugs of abuse and natural rewards such as food enhance extracellular dopamine levels in the nucleus accumbens and other brain structures leading to reinforcement learning. The precise mechanisms by which these rewards lead to long term synaptic changes are of great scientific interest. A current paradigm is that dopamine D1 receptor activation leads to phosphorylation of the signaling molecule DARPP-32. This in turn leads to inhibition of protein phosphatase PP1 leading to increased phosphorylation of channels and other cytoplasmic molecules critical for synaptic plasticity in neurons. Although these cytoplasmic functions of DARPP-32 are understood, drug and food rewards can cause DARPP-32 to accumulate rapidly in the cell nucleus. The nuclear functions of DARPP-32 have not been well characterized. In this paper, Dr. Greengard and colleagues have found that phosphorylation of DARPP-32 specifically on serine 97 (S97) controls nuclear localization of DARPP-32. Phosphorylation of DARPP-32 S97 by the casein kinase 2 leads to cytoplasmic localization of DARPP-32, while dephosphorylation of S97 by protein phosphatase 2A leads to nuclear localization of DARPP-32. To test the functional consequences of this exquisite regulation of DARPP-32 localization, a team of researchers mutated serine 97 to alanine (S97A) such that DARPP-32 could no longer be phosphorylated at this position and was in large part localized to the nucleus. The S97A mutation had no effect on the initial responses of the animals to cocaine. However, in response to a second cocaine injection, S97A mice had reduced locomotor sensitivity to cocaine and rewarding effects of cocaine were not observed. S97A animals also had decreased motivation for food rewards. What does DARPP-32 do in the nucleus? DARPP-32 is known to inhibit the PP1 protein phosphatase and phosphorylation of the chromatin structural protein histone H3 on serine 10 (S10) which is known to be crucial for memory formation. Dr. Greengard and colleagues found that cocaine could induce phosphorylation of histone H3 S10 but this effect was blocked by the DARPP-32 S97A mutant as well as by a T34A mutation. This data reveals that DARPP-32 phosphorylation on threonine 34 is required to inhibit protein phosphatase PP1 function and allow phosphorylation of histone H3 S10. There is an additional layer of regulatory complexity through modulation of DARPP-32 levels in the nucleus. In this work Dr. Greengard and colleagues have linked DARPP-32 to the regulation of chromatin in the nucleus. This regulation presumably can lead to long term changes in gene expression in these neurons which ultimately are required for processes such as memory formation and the long term rewarding effects of drugs of abuse. A deeper understanding of the precise molecular steps in this process may lead to the identification of new therapeutic targets and agents to treat addiction. Stipanovich, A., Valjent, E., Matamales, M., Nishi, A., Ahn, J.H., Maroteaux, M., Bertran-Gonzalez, J., Brami-Cherrier, K., Enslen, H., Corbille, A.G., Filhol, O., Nairn, A.C., Greengard, P., Herve, D., Girault, J.A. A Phosphatase Cascade by Which Rewarding Stimuli Control Nucleosomal Response. Nature, 453, pp. 879-885, 2008.


Research Findings

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