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Director's Report to the National Advisory Council on Drug Abuse - September, 2005

Research Findings - Basic Neuroscience Research

Stress-Induced Analgesia is Mediated by Endogenous Cannabinioids

Stress produces analgesia in a variety of paradigms, and opioids have long been understood to play a critical role in this process. However, NIDA grantees Andrea Hohmann, and Daniele Piomelli and their colleagues report that endogenous cannabinoids also play an important role in producing stress-induced analgesia, that is independent from the role of opioids. They found in rats that blockade of cannabinoid CB1 receptors in the periaqueductal gray (PAG) of the midbrain prevents non-opioid stress-induced analgesia. Further, increasing CB1 agonist availability in the PAG enhanced stress-induced analgesia in a CB1-receptor dependent manner. Their results indicate that cannabiniod release within the PAG mediates opioid-independent stress-induced analgesia. Hohmann, A.G., Suplita, R.L., Bolton, N.M., Neely, M.H., Fegley, D., Mangieri, R., Krey, J.F., Walker, J.M., Holmes, P.V., Crystal, J.D., Duranti, A., Tontini, A., Mor, M., Tarzia, G. and Piomelli, D. An Endocannabinoid Mechanism for Stress-induced Analgesia, Nature, 435(7045), pp. 1108-1112, 2005.

Anandamide Deactivation

The second step in the biological processing of the endogenous cannabinoid known as anandamide, following its uptake by neurons and glial cells, is hydrolysis by fatty-acid amide hydrolase (FAAH), an intracellular membrane-bound enzyme, whose crystal structure was determined in 2002. The action of this particular enzyme is believed to predominate among available lipases and amidases, since mice lacking the FAAH gene demonstrate a rate of brain hydrolytic activity about 1/100 that of wild-type mice. There is considerable research interest in studying the pharmacological and behavioral effects in animals lacking the FAAH gene, which include enhanced anandamide concentration in the brain, along with immobility, analgesia, catalepsy, and hypothermia. These latter properties are considered to be driven by CB1 receptor activation, since they are greatly reduced by prior administration of the CB1 inverse agonist SR 141716A. Recently, a pharmacological means of reducing the activity of FAAH has been studied by Dr. Danielle Piomelli and Dr. Georgio Tarzia and associates, namely, the introduction of a class of O-arylcarbamate inhibitors of FAAH activity. One of these compounds, URB 597, showed a potent IC50 inhibitory value of 5 nM toward FAAH enzymatic activity, which is supported by modeling data indicating a good fit of URB 597 into the binding cavity of FAAH. In wild-type rat brain membranes, URB 597 produced a time-dependent inhibition of tritiated anandamide hydrolysis, as well as a time-dependent increase in brain levels of anandamide, oleoylethanolamide, and palmitoylethanolamide. When the FAAH was absent, as in the FAAH null mouse, prior administration of URB 597 produced no significant increase in anandamide hydrolysis or anandamide brain levels. It also did not increase the hypothermia in FAAH null mice, produced by anandamide, and did not affect feeding behavior (satiety) in FAAH null mice, a process normally regulated by oleoylethanolamide. In the mouse intestine, the absence or presence of FAAH was not affected by URB 597 in terms of anandamide hydrolysis, but anandamide levels did not increase in the FAAH null mouse, suggesting that other enzymes may serve to regulate anandamide in the intestine, besides FAAH. The overall conclusion of this work is that synthetic inhibitors such as URB 597 can be shown to pharmacologically inhibit the action of FAAH, which is an enzyme of prime importance in the CNS for deactivating anandamide. Fegley, D., Gaetani, S., Duranti, A., Tontini, A., Mor, M., Tarzia, G. and Piomelli, D. JPET, 313(1), pp. 352-358, 2005.

Opioid Actions on Neuroimmune Processes

Chemokines (the main site of HIV action in cells) and their receptors have been implicated in the pathogenesis of neuroAIDS. NIDA supported investigators have observed that morphine regulates the expression of several key chemokine systems. Some are down regulated while others are upregulated. This indicates reciprocal actions of morphine on neuroimmune systems and may be very important in demonstrating how opiates and other drugs influence the course and action of HIV infections and neurotoxicity. Specifically, the effects of morphine on the gene expression of beta chemokines and their receptors by primary normal human astrocytes (NHA) were studied. The results show that NHA treated with morphine showed significant down regulation of the gene expression of beta chemokines, MCP-1, and MIP-1 beta, while reciprocally upregulating the expression of their specific receptors, CCR2b, CCR3, and CCR5 as detected by real-time quantitative PCR. These morphine-induced effects on NHA cells were reversed by the opioid mu receptor antagonist, naloxone. Further results indicate that morphine-induced effects are mediated via the modulation of MAPK and CREB signaling pathways. These results support the hypothesis that opiates act as co-factors in the neuropathogenesis of HIV infection. Mahajan, S.D., Schwartz, S.A., Aalinkeel, R.V., Chawda, R.P., Sykes, D.E. and Nair, M.P.N. Morphine Modulates Chemokine Gene Regulation in Normal Human Astrocytes. Clinical Immunology, 115, pp. 323-332, 2005.

The Conformation, Location, and Dynamic Properties of the Endocannabinoid Ligand Anandamide in a Membrane Bilayer

The endogenous cannabinoid ligand anandamide is biosynthesized from membrane phospholipid precursors and is believed to reach its sites of action on the CB1 and CB2 receptors through fast lateral diffusion within the cell membrane. To gain a better insight on the stereochemical features of its association with the cell membrane and its interaction with the cannabinoid receptors, the conformation, location, and dynamic properties were studied in a dipalmitoyl-phosphatidylcholine multilamellar model membrane bilayer system. By exploiting the bilayer lattice as an internal three-dimensional reference grid, the conformation and location of anandamide were determined by measuring selected inter- and intra-molecular distances between strategically introduced isotopic labels using rotational echo double resonance (REDOR) NMR method. A molecular model was proposed to represent the structural features of the anandamide/lipid system and was subsequently used in calculating the multi-spin dephasing curves. Results demonstrate that anandamide adopts an extended conformation within the membrane with its headgroup at the level of the phospholipid polar group and its terminal methyl group near the bilayer center. Parallel static 2H-NMR experiments further confirmed these findings and provided evidence that anandamide has dynamic properties similar to those of the membrane phospholipids and produces no perturbation to the bilayer. The results are congruent with a hypothesis that anandamide approaches its binding site by laterally diffusing within one membrane leaflet in an extended conformation and interacts with a hydrophobic groove formed by helices 3 and 6 of CB1 while its terminal carbon is closely positioned to a key cysteine residue in helix 6 leading to receptor activation. Tian, X., Guo, J., Yao, F., Yang, D.P. and Makriyannis, A. The Conformation, Location, and Dynamic Properties of the Endocannabinoid Ligand Anandamide in a Membrane Bilayer. Journal of Biological Chemistry, 280, pp. 29788-29795, 2005.

Pharmacological Properties of JDTic: A Novel _-Opioid Receptor Antagonist

NIDA investigators, in a recent study, have described the pharmacological properties of a novel kappa opioid receptor antagonist, (3R)-7-Hydroxy-N-{(1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]
methyl}-2-methylpropyl}-1,2,3,4-tetrahydro-3-isoquinoline-carboxamide (JDTic), JDTic is the first potent _-selective opioid receptor antagonist, not derived from an opiate class of compounds. In the mouse tail-flick test, JDTic blocked antinociceptive (analgesic) activity for up to two weeks. When JDTic was administered either before the selective KOP (_)-opioid receptor agonist, enadoline, it blocked the analgesic properties of enalodine. These and other results reprted by this research team suggest that JDTic is a potent long- and orally acting selective _-opioid antagonist. Carroll, I., Thomas, J.B., Dykstra, L.A., Granger, A.L., Allen, R.M., Howard, J.L., Pollard, G.T., Aceto, M.D. and Harris, L.S. Pharmacological Properties of JDTic: A Novel _-opioid Receptor Antagonist. European Journal of Pharmacology, 501, pp. 111-119, 2004.

Flavonoid Glycosides and Cannabinoids from the Pollen of Cannabis sativa L

Chemical investigation of the pollen grain collected from male plants of Cannabis sativa L. resulted in the isolation for the first time of two flavanol glycosides from the methanol extract, and the identification of 16 cannabinoids in the hexane extract. The two glycosides were identified as kaempferol 3-O-sophoroside and quercetin 3-o-sophoroside by spectroscopic methods including high field two-dimensional NMR experiments. The characterization of each cannabinoid was performed by GC-FID and GC-MS analyses and by comparison with both available reference cannbinoids and reported data. The identified cannabinoids were delta-9-tetrahydrocannabinol, cannabidivarin, cannabicitran, delta-9-tetrahydrocannabivarin, cannabicyclol, cannabidiol, cannabichromene, delta-9-tetrahydrocannabiorcol, cannabigerol, cannabinol, dihydrocannabinol, cannabielsoin, 6a,7-10a-trihydroxytetrahydrocannabinol, 9,10-epoxycannabitriol, 10-O-ethylcannabitriol, and 7,8-dehydro-10-O-ethylcannabitriol. These results demonstrate a diverse and large number of cannabinoids are found in Cannabis sativa. Research is needed to further characterize these newly discovered cannabinoids. Ross, S.A., ElSohly, M.A., Sultana, G.N.N., Mehmedic, Z., Hossain, C.F. and Chandra, S. Flavonoid Glycosides and Cannabinoids from the Pollen of Cannabis sativa L. Phytochemical Analysis, 16(1), pp. 45-48, 2005.

Cocaine and Development

Cocaine use during pregnancy is associated with neurobehavioral problems in school-aged children that implicate alterations in attentional processes, potentially due to impairments in the noradrenergic system. In a recent study, NIDA supported researchers report a direct, disruptive effect of cocaine on noradrenergic neurons that may provide a neurobiological basis for changes in attentional function observed, in clinical and pre-clinical investigations, in offspring exposed to cocaine in utero. In this study, rats were administered cocaine in a physiologically relevant dose during critical phases of gestation. The locus coeruleus, a brain region, was analyzed for neurite outgrowth characteristics. Results showed that cocaine inhibited locus coeruleus neurite outgrowth and development and female offspring appeared most vulnerable to such effects. Snow, D.S., Carman, H.M., Smith, J.D., Booze, R.M., Welch, M.A. and Mactutus, C.F. Cocaine-induced Inhibition of Process Outgrowth in Locus Coeruleus Neurons: Role of Gestational Exposure Period and Offspring Sex. International Journal of Developmental Neuroscience, 22(5), pp. 297-308, 2004.

THC Systems and Pain

Pain, a critical component of host defense, is one hallmark of the inflammatory response. These investigators hypothesized that pain might be exacerbated by proinflammatory chemokines. To test this hypothesis, CCR1 was cotransfected into human embryonic kidney (HEK)293 cells together with transient receptor potential vanilloid 1 (TRPV1), a cation channel required for certain types of thermal hyperalgesia. In these cells, capsaicin and anandamide induced Ca2 influx mediated by TRPV1. When CCR1:TRPV1 HEK293 cells were pretreated with proinflammatory chemokine C CL3, the sensitivity of TRPV1, as indicated by the Ca2 influx, was increased 3-fold. RT-PCR analysis showed that a spectrum of chemokine and cytokine receptors is expressed in rat dorsal root ganglia (DRG). Immunohistochemical staining of DRG showed that CCR1 is coexpressed with TRPV1 in >85% of small-diameter neurons. CCR1 on DRG neurons was functional, as demonstrated by CCL3-induced Ca2 ion influx and PKC activation. Pretreatment with CCL3 enhanced the response of DRG neurons to capsaicin or anandamide. This sensitization was inhibited by pertussis toxin, U73122, or chelerythrine chloride, inhibitors of Gi-protein, phospholipase C, and protein kinase C, respectively. Intraplantar injection of mice with CCL3 decreased their hot-plate response latency. That a proinflammatory chemokine, by interacting with its receptor on small-diameter neurons, sensitizes TRPV1 reveals a previously undescribed mechanism of receptor cross-sensitization that may contribute to hyperalgesia during inflammation. Zhang, N., Inan, S., Cowan, A., Sun, R., Wang, J.M., Rogers, T.J., Caterina, M., Oppenheim, J.J. A Proinflammatory Chemokine, CCL3, Sensitizes the Heat- and Capsaicingated Ion Channel TRPV1, Proceedings of the National Academy of Sciences, 102(19), p. 7050, 2005.

Antidepressants Attenuate Dopamine Signals and Enhance Serotonin Signals Due to the Reuptake, Accumulation and Co-Release of Serotonin and Dopamine at Dopamine Terminals

The brain's striatum receives and is rich in dopamine and serotonin neurotransmitters. After neurotransmitter release, neurotransmitter transporters take up, at their respective nerve terminals, dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT). DA is taken up by its transporters (DATs), which are expressed at the highest density in the striatum. There are far fewer serotonin transporters (SERTs) in the striatum. Although DA nerve terminals do not express SERT, they have some affinity for serotonin. Serotonin and DA terminals are often located next to each other, but they do not normally overlap, and this anatomy provides a physical basis for the interaction of these two neurotransmitters. Recent work by Dr. Dani and his group showed, that after treatment with an anti-depressant, which inhibits SERT, DATs take up the serotonin and store it along with DA in the dopaminergic terminals. Co-release of these two neurotransmitters then occurs upon stimulation of these DA neurons. As SERT is inhibited by anti-depressants, higher concentrations of 5-HT enter into DA terminals via the extremely dense striatal DAT-mediated reuptake. As a result of this and other mechanisms there is a prolonged enhancement of the serotonin and a reduced DA signal. A consistent, parallel recording of small spontaneous signals of both 5-HT and DA indicated a co-release of these two transmitters. In addition, application of fluoxetine, an anti-depressant and DAT inhibitor, blocked the reuptake, accumulation and co-release of 5-HT in DA terminals, suggesting these were DAT-mediated events. These investigators plan to extend these studies and determine if DAT function is altered by pre-exposure to its blocking drugs. Zhou, F.M., Liang, Y., Salas, R., Zhang, L., De Biasi, M. and Dani, J.A. Corelease of Dopamine and Serotonin from Striatal Dopamine Terminals, Neuron, 46(1), pp. 65-74, 2005.

Genes Tell About Nicotine Associated Reward, Tolerance and Sensitization

Although eleven neuronal acetylcholine (ACh) receptor subunits with distinct functional properties and pharmacological characteristics have been identified in humans, until recently the identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence was unknown. Dr. Henry A. Lester's lab engineered mutant mice that genetically over-express alpha4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering alpha 4 receptor subunits hypersensitive to nicotine. With this genetic "knock-in" model, selective activation of alpha 4 nicotinic ACh receptors with ultra low doses of agonist recapitulated nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of alpha 4 receptors would be sufficient for nicotine-induced reward, tolerance, and sensitization. Genetic studies have revealed the existence of polymorphisms in genes encoding neuronal AChR alpha4 and beta2 subunits. Dr. Lester's report points to a coordinated study of polymorphisms in nAChR genes, smoking behavior, and functional characterization of mutated receptors that will shed light on our understanding of the roles of polymorphisms, and the role of the alpha 4 AChR subunit in the susceptibility to addiction. Tapper, A., McKinney, S., Nashmi, R., Schwarz, J., Deshpande, P., Labarca, C., Whiteaker, P., Collins, A. and Lester, H. Nicotine Action on _4* Receptors: Sufficient for Reward, Tolerance and Sensitization, Science, 306, pp. 1029-1032, 2004.

Hallucinogens of Three Types Increase Extracellular Glutamate in the Prefrontal Cortex

In this in vivo study, the indoleamine hallucinogen LSD increased glutamate efflux in rat prefrontal cortex (PFC). The dose of LSD was identical to that used in drug discrimination stimulus training, and produced maximal LSD-appropriate responding in rats. The effects of LSD were abolished by the selective 5-HT2A antagonist M100907. The 5-HT2A/C agonist DOM, which is a phenethylamine hallucinogen, also increased glutamate efflux in rat PFC at the relevant dose. It was previously reported that phencyclidine (PCP) and ketamine also increase glutamate efflux in the PFC, though these compounds act as antagonists at the NMDA receptor. Thus, the present study provides evidence, in support of the hypothesis first presented by Aghajanian and Marek on electrophysiological grounds, that an enhanced release of glutamate is a mechanism common to indoleamine, phenethylamine, and glutamatergic hallucinogens. Muschamp, J.W., Regina, M.J., Hull, E.M., Winter, J.C. and Rabin, R.A. Lysergic Acid Diethylamide and [-]-2,5-Dimethoxy-4-methylamphetamine Increase Extracellular Glutamate in Rat Prefrontal Cortex. Brain Research, 1023, pp. 134-140, 2004.

Methamphetamine Damage to Dopamine Nerve Endings May be Due, in Part, to

Microglia are the primary immune defense cells in the brain. They safeguard and support neuronal functions. Nevertheless, excessive microglial activation can cause microglia to harm neurons. Donald M. Kuhn's group recently demonstrated that microglia, activated by methamphetamine (meth), may contribute to meth's characteristic neurotoxicity to dopamine-containing neuron terminals. They found, in mice, that: (1) Meth activates microglia in a dose-related manner and along a time course that is coincident with damage to dopamine (DA) nerve endings, (2) Low ambient temperature prevents both the neurotoxicity and the microglial activation; (3) The microglial response to meth and the damage to DA nerve endings both occur in the striatum; (4) Drugs that are not characteristically neurotoxic (e.g., cocaine) do not mimic the effect of methamphetamine on microglia; (5) Numerous genes linked to microglia are activated within hours of meth administration, suggesting that microglial activation occurs early in the meth toxic cascade. Thomas, D.M., Francescutti-Verbeem, D.M., Liu, X. and Kuhn, D.M. Identification of Differentially Regulated Transcripts in Mouse Striatum following Methamphetamine Treatment - an Oligonucleotide Microarray Approach. Journal of Neurochemistry, 88, pp. 380-393, 2004. It was separately reported that other DA neurotoxic drugs in mice (e.g., MDMA) also produce the microglial activation. Thomas, D.M., Dowgiert, J., Geddes, T.J., Francescutti-Verbeem, D., Liu, X. and Kuhn, D.M. Microglial Activation Is a Pharmacologically Specific Marker for the Neurotoxic Amphetamines. Neuroscience Letters, 367, pp. 349-354, 2004. Kuhn and his colleagues also demonstrated that a low dosage meth regimen that makes mice tolerant to meth neurotoxicity attenuates meth-induced microglial activation. Thomas, D.M. and Kuhn, D.M. Attenuated Microglial Activation Mediates Tolerance to the Neurotoxic Effects of Methamphetamine. Journal of Neurochemistry, 92, pp. 790-797, 2005. Finally, since microglia are known to produce many of the reactive chemicals (e.g., nitric oxide, superoxide, cytokines) that mediate the neurotoxicity of the amphetamines, their activation could represent an early and essential event in the neurotoxic cascade associated with high-dose amphetamine intoxication. Thomas, D.M., Walker, P.D., Benjamins, J.A., Geddes, T.J. and Kuhn, D.M. Methamphetamine Neurotoxicity in Dopamine Nerve Endings of the Striatum Is Associated with Microglial Activation. Journal of Pharmacology and Experimental Therapeutics, 311, pp. 1-7, 2004.

Separate Dopamine Transients In The Nucleus Accumbens Are Associated With Conditioning and With the Pharmacological Effects of Cocaine

Cocaine acts as a reinforcer through its pharmacological effects on central monoaminergic systems and its administration results in the accumulation of dopamine in the nucleus accumbens. Carelli and Wightman, using fast-scan cyclic voltammetry to rapidly monitor changing dopamine signals in animals that self-administer cocaine and in those that receive it non-contingently, show that two separate dopamine signals occur. Consistent, time-locked dopamine transients occurred about 1.5 sec after each self-administered dose of cocaine, and this signal was associated with the drug-associated cues. No pharmacological effect of cocaine was observed within 10 seconds of its administration in animals given the drug non-contingently. Instead, the pharmacological effects of cocaine were observed about 40 seconds after cocaine delivery in both groups and dopamine remained elevated for at least 5 minutes afterward. The data show that this pharmacological action of cocaine occurs for an extended period of time following either contingent or non-contingent administration of the drug and that this signal is distinct from those dopamine transients that are time-locked to each lever-press in self-administering animals. Stuber, G.D., Roitman, M.F., Phillips, P.E.M., Carelli, R.M. and Wightman, R.M. Rapid Dopamine Signaling in the Nucleus Accumbens During Contingent and Noncontingent Cocaine Administration, Neuropsychopharmacology, 30, pp. 853-863, 2005.

Morphine Side Effects Are Dramatically Attenuated in Beta-Arrestin-2

Morphine is a potent analgesic, yet, like most opioid narcotics, its unwanted side effects such as constipation and respiratory suppression limit its clinical utility. Pharmacological approaches taken to preserve the analgesic properties, while eliminating the untoward side effects, have met with very limited success. Dr. Laura Bohn and her research team provide evidence that altering mu opioid receptor regulation may provide a novel approach to discriminate morphine's beneficial and deleterious effects in vivo. They have previously reported that mice lacking the G protein-coupled receptor regulatory protein, beta-arrestin-2 have enhanced and prolonged morphine analgesia with very little morphine tolerance. In a recent report they examine whether the side-effects of morphine treatment are also augmented in this animal model. Surprisingly, the genetic disruption of opioid receptor regulation, while enhancing and prolonging analgesia, dramatically attenuates the respiratory suppression and acute constipation caused by morphine. Raehal, K.M., Walker, J.K. and Bohn, L.M. Morphine Side Effects in {beta}-Arrestin-2 Knockout Mice, Journal of Pharmacology and Experimental Therapeutics (epub), 2005.

Dynorphin-Induced Allodynia is Prevented By a Spatial Knockout of NMDA Receptors in the Lumbar Spinal Cord Dorsal Horn (SCDH)

A single intrathecal (IT) injection of dynorphin A (1-17) (DYN) produces allodynia (i.e., pain from stimuli that are not normally painful) in mice that is blocked by an NMDA receptor antagonist. To confirm and extend this observation, Dr. Charles Inturrisi and his colleagues used a spatial-temporal knockout (KO) of the NR1 subunits of the NMDA receptor (NR1 KO). Mechanical allodynia (von Frey), cold allodynia and thermal hyperalgesia were measured prior to and 2 to 5 days after IT DYN. DYN produced mechanical allodynia but not thermal hyperalgesia or cold allodynia in both the Control and the NR1 KO. However, while the allodynia was bilateral in the Controls, it was observed only with the contralateral paw in the NR1 KO mice. Thus, a spatial KO of the NMDA receptor, confined to one side of the SCDH, provided protection on that side from DYN-induced allodynia. These results demonstrate conclusively that postsynaptic NMDA receptors, at the level of the SCDH, are required for development of the mechanical allodynia induced by DYN. They may also offer some insight into the mechanism by which endogenous DYN mediates the allodynia that occurs following injury. South, S.M., Ohata, M., Hegarty, D., Xu, Q. and Inturrisi, C.E. Dynorphin-induced Allodynia is Prevented by a Spatial Knockout of NMDA Receptors in the Lumbar Spinal Cord Dorsal Horn, International Narcotics Research Conference, July 2005.

The Ror Receptor Tyrosine Kinase CAM-1 Is Required for ACR-16-Mediated

Communication among neurons in the nervous system is achieved by a neuron releasing a chemical substance or neurotransmitter that is detected by a neighboring neuron through a receptor. One of the major neurotransmitters in the nervous system is acetylcholine. After acetylcholine is released by a neuron, the acetylcholine signal is detected by either nicotinic or muscarinic receptors in neighboring neurons. The rewarding and addictive properties of tobacco are mediated initially by nicotine acting through nicotinic acetylcholine receptors in the central nervous system. The similarities of the neuromuscular junction of the worm, C. elegans to the vertebrate nicotinic cholinergic synapse together with the wonderful genetic tools available in c. elegans provide a powerful approach to understand the regulation of cholinergic synapses. Francis et al., identified two types of cholinergic receptors at the C. elegans neuromuscular junction by screening an RNAi library in which worms are placed into different wells and exposed to different type of RNAi. Each different RNAi type in the RNAi library selectively decreases the expression of a unique gene when taken up by the worm. The two types of cholinergic receptors that were identified are a levamisole sensitive receptor and nicotinic receptor, acr-16, which encodes a nicotinic AChR subunit homologous to the vertebrate _7 subunit. Worms lacking the acr-16 gene are uncoordinated and lack the fast synaptic current that is normally observed when nicotine is applied to the worm neuromuscular junction. Because worms lacking the receptor tyrosine kinase CAM-1 also show uncoordinated locomotion, Dr. Francis and his collegues tested the hypothesis that CAM-1 regulates nicotinic cholinergic transmission at the C. elegans neuromuscular junction. Dr. Francis and his colleagues show that the nicotinic receptors encoded by acr-16 are mislocalized at the neuromuscular junction and the ACR-16-dependent currents are greatly diminished. The effect of CAM-1 is selective because the response to levamisole and GABA were unaffected. In addition, the localization of vesicles and presynaptic proteins involved in the release of acetylcholine are altered in CAM-1 mutants. Dr. Francis suggests that CAM-1 acts to regulate or stabilize post-synaptic ACR-16 receptors and pre-release sites. Dr. Francis also suggest that CAM-1 action are not mediated by the kinase activity in the molecule but by other portion of the molecule because mutants lacking tyrosine kinase activity have little effect while worms completely lacking CAM-1 show the mutant phenotype. Francis, M.M., Evans, S.P., Jensen, M., Madsen, D.M., Mancuso, J., Norman, K.R. and Maricq, A.V., The Ror Receptor Tyrosine Kinase CAM-1 Is Required for ACR-16-Mediated Synaptic Transmission at the C. elegans Neuromuscular Junction, Neuron, 46(4), pp. 581-594, 2005.

Rapid Upregulation of Alpha7 Nicotinic Acetylcholine Receptors by Tyrosine Dephosphorylation

Although the tyrosine kinase activity of CAM-1 does not appear to regulate the C. elegans acr16, a nicotinic receptor homologous to the vertebrate alpha7 nicotinic receptor (see above), a recent report by Cho and his colleagues suggests that a tyrosine phosphatase, an enzyme that removes phosphate bonds from proteins regulates the expression of the rat alpha 7 nicotinic receptor. In oocytes expressing alpha 7 receptors, the size of currents elicited by nicotine was enhanced in the presence of genistein, an inhibitor of tyrosine kinase and decrease by pervandate, an inhibitor of tyrosine phosphatase activity. Genistein was found not to increase the size of the alpha7 receptor current by altering the time that the receptor-gated channel remains open nor alter the phosphorylation state of the alpha 7 nicotine receptor. Instead, the increased size of the currents elicited by nicotine in the presence of genistein appears to be the consequence of increased insertion of alpha 7. Increased expression of the alpha 7 nicotinic receptor was shown to be increased following genistein treatment by immunobloting and alpha-bungarotoxin receptor binding. Tyrosine kinase inhibition also increased the expression of alpha7 nicotine receptors in the rat hippocampus suggesting that the increase observed is not an artifact of the Xenopus oocyte systems. The increased expression of the alpha 7 receptor by tyrosine kinase inhibition appears to be mediated by increased exocytosis of vesicles containing the alpha 7 receptor and not decreased endocytosis (turn over) of the alpha7 nicotinic receptor. A dominant negative mutant of dynamin that blocks endocytosis did not block the increased expression of the alpha7 nicotine receptor in oocytes but did block increased expression of the cystic fibrosis transmembrane conductance regulator as had previously been shown. Botulinum toxin that inhibits SNARE dependent exocytosis abrogated increased expression of the the alpha7 receptor. These results suggest that regulators of tyrosine phosphorylation may mediate the changes in number of nicotine receptors observed in animals treated with chronic nicotine. Cho, C.H., Song, W., Leitzell, K., Teo, E., Meleth, A.D., Quick, M.W. and Lester, R.A. Rapid Upregulation of alpha7 Nicotinic Acetylcholine Receptors by Tyrosine Dephosphorylation, Journal of Neuroscience, 25(14), pp. 3712-3723, 2005.

Proteome Analysis of Liver Cells Expressing a Full-Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver From

Approximately 2 percent of the population in the United States is infected with hepatitis C, a blood borne virus. One of the leading causes of hepatitis C infection in the United States is intravenous drug abuse. 85% of those infected with hepatitis C will develop hepatic inflammation and fibrosis, cirrhosis, and hepatocellular carcinoma. Until recently, gene expression profiling that examines the expression of all the mRNAs in liver cells has been used to examine the host response and has been used to predict the clinical outcome of hepatitis C infection. One of the difficulties of this approach is that mRNA expression does not always correspond to the pattern of expression of proteins in the same cells. To overcome this problem, Dr. Michael Katze at the University of Washington in collaboration with Dr. Richard Smith at Pacific Northwest Laboratory have developed highly sensitive methods to assay all the proteins, the proteome, of a liver cell line, Huh-7.5 in the presence or absence of a replicating full length length HCV genome. In their analysis they observed changes in the level of more than 4,200 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry. Dr. Katze and his colleagues were able to extend this analysis to liver biopsy material from HCV-infected patients. Dr. Katze and his colleagues identified 1500 proteins from less than 2 microgram of liver biopsy tissue. Analysis of both the huh-7 cells in the presence of the full HCV genome and liver biopsies of livers infected with HCV suggests that HCV alters lipid metabolism that may contribute to a state of oxidative stress in infected cells. The ability to analyze the proteome from limited amounts of biopsied liver samples is a technological breakthrough that permits evaluation of clinical significance of changes in protein expression levels associated with HCV infection. Jacobs, J.M., Diamond, D.L., Chan, E.Y., Gritsenko, M.A., Qian, W., Stastna, M., Baas, T., Camp, D.G., Carithers, R.L. Jr., Smith, R.D. and Katze, M.G. Proteome Analysis of Liver Cells Expressing a Full-length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-infected Patients, Journal of Virology, 79(12), pp. 7558-7569, 2005.

A Mouse Model For Study of Systemic HIV-1 Infection, Antiviral Immune Responses, and Neuroinvasiveness

HIV readily infects humans and some non-human primates such as chimpanzees but species specific barriers prevent infection in other animals such as mice. A mouse model of HIV infection would have great utility because mice are cheaper than chimpanzees to maintain, the immune system is well characterized, and the mouse is a genetically tractable model system. Attempts so far to genetically engineer transgenic mice expressing key proteins involved in HIV infection such as CD4, CCR4, CXCR4, cyclin T1 have failed to produce a mouse host susceptible to HIV infection. Dr. Volsky and his colleagues have adopted a different strategy in which the HIV virus envelope is altered instead of altering the host to produce a mouse model of HIV infection. The gp80 envelope from ecotropic murine leukemia virus, a retrovirus that infects only rodents was used to replace coding region of gp120 in HIV-1 to produce a productive infection in mouse lymphocytes (white cells) but not in human lymphocytes. Dr. Volsky and his colleagues made two chimeric viruses, one based on a backbone of clade B NL4-3 (EcoHIV) and EcoNDK on a backbone of clade D NDK. Dr. Volsky and his colleagues then showed that intravenous inoculation of p24 EcoHIV, the genetically modified virus, infected nervous tissue, macrophages, spleen cells, and lymphocytes. The EcoHIV virus infection was replication competent because co-culturing of infected spleen cells with uninfected spleen cells resulted in the uninfected spleen cells becoming infected. In response to infection with the EcoHIV virus the mice mount a humoral response by producing antibodies against tat and gag, two HIV proteins. Although Dr. Volsky and his colleagues did not observe an overt disease in the mice infected with the chimeric virus, mice infected with EcoNDK showed expression of the chimeric virus in the brain associated with increased host expression of IL-1, MCP-1, and STAT-1. Increased expression of IL-1, MCP-1, and STAT-1 are associated with HIV dementia. The successful development of chimeric mouse HIV virus model of HIV infection provides an extremely useful model to study HIV pathogenesis, and a model to develop vaccines and other therapeutics. The model also provides a means for investigator to analyze the interaction of drugs of abuse and HIV infection. Potash, M.J., Chao, W., Bentsman, G., Paris, N., Saini, M., Nitkiewicz, J., Belem, P., Sharer, L., Brooks, A.I. and Volsky, D.J. A Mouse Model for Study of Systemic HIV-1 Infection, Antiviral Immune Responses, and Neuroinvasiveness, Proceedings of the National Academy of Sciences, 102(10), pp. 3760-3765, 2005.

Shank Expression is Sufficient to Induce Functional Dendritic Spine Synapses in Aspiny Neurons

In the mammalian nervous system most excitatory inputs are received by glutamate receptors located on dendritic spines at post-synaptic densities. The post synaptic density is composed of glutamate receptors, PSD-95, shank, homer, guanylate associated kinase, and cystoskeletal protein. Shank proteins couple glutamate receptors to the cytoskeleton and intracellular signaling molecules. A key question is whether shank plays a role in the formation of dendritic spines and the maturation of excitatory synapses. This is an important question for drug abuse research because drugs of abuse alter the number of dendritic spines. Dr. Worley and his colleagues have now shown that shank is both necessary and sufficient to induce spine formation. Expression of RNAi against shank expression in hippocampal neurons reduces the number of dendritic spines. In aspiny cerebellar granule cells transfection of shank into these cells induces dendritic spines. To determine the function of different domains in the shank3 protein, Worley and his colleague deleted various portions of the shank gene and then transfected the mutant shank gene into aspiny cerebellar granule cells. Deletion of the PSD and homer domains in the shank gene prevented the shank protein from being targeted to dendrites, a necessary condition for forming dendritic spines. Worley and his colleagues also report that the ankyrin repeats in the SH3 region of shank and the cortactin binding site are required for spine maturation. The ankyrin repeats in the SH3 region of shank appears to regulate only spine enlargement while the cortacting-site regulates head enlargement and retraction of spines. The formation of spines induced by transfection of shank is associated with the recruitment of glutamate receptors and the formation of functional synapses. Glutamate receptor blockers reduced the number of spines induced by shank suggesting that the release of glutamate from presynaptic terminals is required for the formation of spines induced by shank. The results reported by Dr. Worley and colleagues suggest that shank3 and glutamate are essential for the maturation and formation of synapses. Roussignol, G., Ango, F., Stefano, R., Tu, J.C., Sala, C., Worley, P.F., Bockaert, J.L. and Fagni, L. Shank Expression is Sufficient to Induce Functional Dendritic Spine Synapses in Aspiny Neurons, Journal of Neuroscience, 25(14), pp. 3560 -3570, 2005.

Regulation of Dopaminergic Transmission and Cocaine Reward By The Clock Gene

Molecular mechanisms regulating circadian rhythms regulate responses to cocaine. Previous work conducted by Jay Hirsh and his colleagues in fruit flies has shown that mutations in Period, Clock, Cycle, and Doubletime genes, regulating circadian rhythms, also alters cocaine's sensitizing effects. Subsequent work by Abarca and his colleagues showed that mutant mice lacking the period 1 gene do not sensitize to repeated administration of cocaine whereas mutant mice lacking the period-2 gene become hyper-responsive to repeated cocaine injection. Similarly, knockout mice lacking the period 1 gene showed a complete lack of cocaine reward while mice lacking the period 2 gene showed enhanced cocaine reward. In the June 28 issue of the Proceeding of the National Academy Science, Dr. Eric Nestler's laboratory led by Dr. Colleen McClung in collaboration with Dr. Joseph Takashi and Dr. Frank White now establish the role of the circadian rhythm gene, Clock, in regulating cocaine reward behavior. Clock mutant mice show increased activity to novel environments and have elevated activity throughout the dark/light cycle, especially at the beginning of the light cycle and the beginning of the dark cycle. Increased locomotor activity was associated with increased firing of dopamine neurons in the brain's ventral tegmental area (VTA) in clock mutant mice as compared to wild type mice. Although sensitization to cocaine is unaffected in the clock mutants, the clock mutants show increased place preference conditioning to lower doses of cocaine than wild type mice. This suggests that cocaine is more rewarding in clock mutant mice. The clock gene is expressed in dopamine neurons in the VTA that mediates the rewarding effects of cocaine. Because the clock gene is a transcription factor, the Nestler lab examined whether the clock mutant mice affected the expression of other genes. Clock mutants showed increased expression of tyrosine hydroxylase the rate-limiting enzyme for dopamine biosynthesis, and reduced expression of the beta-1 subunit of the GABAA receptor was observed. Decreased expression of the beta-1 subunit of the GABAA receptor which has previously been shown to inhibit the excitability of dopamine neurons may mediate the increased firing of dopamine neurons. Increased firing of dopamine VTA neurons in the clock mutant is also associated with decreased expression of a voltage-gated potassium channel. At the same time, the increase in the NR1 subunit of the AMPA receptor in VTA neurons of the clock mutants were observed by Dr. McClung and her colleagues. Previous work has suggested that upregulation of the NR1 subunit of the AMPA receptor increases the rewarding properties of cocaine. In conclusion, this work not only shows the role of circadian rhythm genes in regulating the effects of cocaine but also shows the power of fruit fly genetics to identify new pathways that mediate the effects of drugs of abuse in vertebrates. McClung, C.A., Sidiropoulou, K., Vitaterna, M., Takahashi, J.S., White, F.J., Cooper, D.C. and Nestler, E.J. Regulation of Dopaminergic Transmission and Cocaine Reward by the Clock Gene, Proceedings of the National Academy of Sciences, 102(26), pp. 9377-9381, 2005.

S-Nitrosylated GAPDH Initiates Apoptotic Cell Death by Nuclear Translocation Following Siah1 Binding

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its role in metabolizing glucose. More recently, work has shown that translocation of GAPDH to the nucleus is associated with cell death and cytotoxicity (degree to which a substance is poisonous to cells). Hara and his colleague now report the signaling mechanism by which GAPDH translocates to the nucleus and causes cell death. Hara and his colleague show that nitrosylation of GAPDH by nitric oxide synthase, an enzyme activated by stress, cause GAPDH to bind Siah1 (Sevenless in absentia homologue1), a ubiquitin E3 ligase and translocate to the nucleus. The binding of nitrosylated GAPDH to Siah1 decreases the rate of degredation of Siah1 in the nucleus, thereby increasing the amount and activity of Siah1 in the cell nucleus. The stabilization of Siah1 by GAPDH and increased activity cause increased ubiquination of nuclear proteins leading to increased degradation of nuclear proteins and cell death. To test the role of GAPDH in cell death, a macrophage cell lines was depleted of GAPDH using RNAi and stimulated with lipopolysacharide (LPS) that activates nitric oxide synthase. Depletion of GAPDH prevented cell death induced by LPS. Furthermore, Hara and his colleague's further show that depletion of GAPDH or Siah1 using RNAi prevents glutamate mediated cell death in cerebellar granule cells. Inhibitors of GAPDH nitrosylation by inhibitors of MAOB such as deprenyl not only have significant implications for the treatment of neurodegenerative diseases but also for treating the neurotoxic effects of MDMA and methamphetamine. Hara, M.R., Agrawal, N., Kim, S.F., Cascio, M.B., Fujimuro, M., Ozeki, Y., Takahashi, M., Cheah, J.H., Tankou, S.K., Hester, L.D., Ferris, C.D., Hayward, S.D., Snyder, S.H. and Sawa, A. S-nitrosylated GAPDH Initiates Apoptotic Cell Death by Nuclear Translocation Following Siah1 Binding, Nature Cell Biology, 7, pp. 665-674, 2005.

Regulation of Drug Reward by Cyclic-Amp Response Element-Binding Protein: Evidence For Two Functionally Distinct Subregions of the Ventral Tegmental Area

The cyclic-AMP response element (CRE) is present in the promoter region of cAMP-responsive genes. When the CRE binding protein (CREB) is activated by phosphorylation, it binds to the CRE and increases expression of those genes. Many groups have shown that CREB is activated in several brain regions following exposure to drugs of abuse. In this study, Olson et al., show that morphine upregulates CREB in the ventral tegmental area (VTA), a very important brain structure for drug abuse. More specifically, they went on to show that active CREB expressed in two different regions of the VTA produces opposite effects on drug reward. This is unusual since the VTA has traditionally been treated as a single structure. They found that these two sub-regions of the VTA contained differential proportions of two types of neurons, those that produce Gamma-Aminobutryic Acid (GABA) and those that produce dopamine. These differential cell populations may account for opposing responses they observed in the two regions. Specifically, they found that in the sub-region containing predominantly dopamine cells, the induced CREB expression made a threshold dose of cocaine or morphine aversive, while they were rewarding when expressed in the sub-region containing fewer dopamine cells and more GABA cells. They further confirmed that the dopamine cells are projecting to the nucleus accumbens shell, which has been shown by others previously. These findings suggest that studies on addiction involving the VTA may need to consider sub-regions of the VTA since this study clearly shows differential responses to morphine as it relates to CREB activity. Olson, V.G., Zabetian, C.P., Bolanos, C.A., Edwards, S., Barrot, M., Eisch, A.J., Hughes, T., Self, D.W., Neve, R.L., and Nestler, E.J. Regulation of Drug Reward by cAMP Response Element-binding Protein: Evidence for Two Functionally Distinct Subregions of the Ventral Tegmental Area, Journal of Neuroscience, 25(23), pp. 5553-5562, 2005.

-Opioid Receptor and CREB Activation are Required for Nicotine Reward

Understanding the molecular and genetic mechanisms that underlie the addictive properties of nicotine is important for creating successful treatments for smoking cessation, but these mechanisms are currently poorly delineated. Dr. Blendy and her colleagues have recently found that exposure to an environment previously associated with rewarding properties of nicotine results in an increase of CREB phosphorylation similar to that seen following nicotine administration, and this response is absent in mu opioid receptor knock-out (MOR-/-) mice. Administration of a single dose of the mu opioid antagonist, naloxone, blocks both the conditioned molecular response (CREB phosphorylation) and the conditioned behavioral response (nicotine reward) in a place preference paradigm. In related experiments, this group also found that repeated nicotine administration results in increased expression of the mu opioid receptors. In mice with a mutated CREB gene, however, expression of the mu receptor is abrogated as well as the rewarding properties of nicotine. These data suggest that activation of both the endogenous opioid system and CREB are critical for the expression of conditioned nicotine reward. This effect may also be seen in other substances of abuse, but has yet to be examined. Walters, C.J., Cleck, J.N., Kuo, Y-C. and Blendy, J.A. Mu-opioid Receptor and CREB Activation are Required for Nicotine Reward, Neuron, 46, pp. 933-943, 2005.

Remote Control of Behavior Through Genetically Targeted Photostimulation of Neurons

Dr. Miesenbock and his colleagues have recently published the first use of genetically encoded phototriggers for defining the neuronal substrates of specific behaviors in invertebrates. The ionotropic purinoreceptor P2X2, not normally expressed in the fly, was ectopically expressed in circumscribed groups of neurons in the Drosophila CNS so that broad illumination of flies evoked action potentials only in genetically designated target cells. Illumination of "caged" agonists of the P2X2 receptor causes the agonist to be released and bind to the receptor, thereby acting as a phototrigger. Flies harboring the phototriggers in different sets of neurons responded to laser light with behaviors specific to the sites of phototrigger expression. Photostimulation of neurons in the giant fiber system elicited the characteristic escape behaviors of jumping, wing beating, and flight; photostimulation of dopaminergic neurons caused changes in locomotor activity and locomotor patterns. These responses reflected the direct optical activation of central neuronal targets rather than from confounding visual input. These experiments demonstrate that encodable phototriggers provide non-invasive control interfaces for studying the connectivity and dynamics of neural circuits, for assigning behavioral content to neurons and their activity patterns, and, potentially, for restoring information corrupted by injury or disease. The capacity to remote-control genetically delineated sets of neuronal targets promises to open many new possibilities for the analysis of neural circuits and the search for the cellular substrates of behavior. The strategy developed by Dr. Meisenbock and his colleagues for two systems of neurons and their associated behaviors, i.e., the Giant Fiber system and escape movements and the dopaminergic system and locomotion, can be extended immediately to screens of existing collections of enhancer trap lines (or mosaic offspring in which expression of the phototrigger is restricted to smaller subsets of neurons) and other behaviors. Examples include searches for the neuronal signals guiding different forms of movement, courtship, mating, aggression, feeding, grooming, learning, and sleep and wakefulness, as well as the neural symbols representing reward and punishment, expectation, and categories of generalization. Lima, S.Q. and Miesenbock, G. Remote Control of Behavior Through Genetically Targeted Photostimulation of Neurons, Cell, 121, pp. 141-152, 2005.


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