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NIDA Home > Publications > Director's Reports > February, 2009 Index    

Director's Report to the National Advisory Council on Drug Abuse - February, 2009

Research Findings - Basic Neuroscience Research

AMPA Glutamate Receptors Mediate Time Dependent Cue-Induced Craving for Cocaine

Increased time dependent craving for cocaine is frequently seen among cocaine addicts following abstinence and may account for relapse after periods of abstinence. This can be modeled in rats. Rats are trained for 10 days to nose poke to self-administer cocaine when a light cue is presented. 1 or 45 days are allowed to pass before placing the rat back into the chamber where the rat had learned to self-administer cocaine in the presence of the light cue. After the 1 or 45 period of abstinence the light is presented but the rat is no longer rewarded for nose poking with a cocaine injection, a process called extinction. Conrad and colleagues report that rats after 45 days of abstinence made more nose pokes in the presence of the cue as compared to rats after 1 day of abstinence even though no cocaine was delivered showing a time-dependent increase in cue-induced cocaine seeking. Conrad and colleagues then examined whether glutamate synaptic transmission is altered in the nucleus accumbens, a subcortical structure involved in reward that receives input from dopamine neurons and neurons in prefrontal cortex and limbic regions of the brain. Glutamate can act on three classes of receptors: NMDA, AMPA, and metabotropic receptors. The NMDA and AMPA receptors excite the neuron upon sensing the release of glutamate from an adjacent neuron by allowing ions to pass through while metabotropic receptors change neuronal excitability by activating enzymes. Conrad and colleagues report that number of GluR1 and GluR3 receptors, but not GluR2 AMPA receptors are increased in the shell and core of the nucleus accumbens. Naspm, a blocker of GluR1 and GluR3 receptors, inhibited the enhanced cue-induced cocaine seeking after prolonged withdrawal from cocaine withdrawal after injections into the nucleus accumbens on the test withdrawal days. These results suggest that prolonged withdrawal from cocaine produces increases in the number of GluR1 and GluR3 receptors leading to time dependent increased responding for cocaine associated cues and may be responsible for cocaine craving in human addicts. Conrad, K.L., Tseng, K.Y., Uejima, J.L., Reimers, J.M., Heng, L.J., Shaham, Y., Marinelli, M., and Wolf, M.E. Formation of Accumbens GluR2-lacking AMPA Receptors Mediates Incubation of Cocaine Craving. Nature, 454(7200), pp. 118-121, 2008.

Phosphorylation Dependent Trafficking of GluR2 Receptors in the Nucleus Accumbens Play a Key Role in Drug-Induced Reinstatement of Drug Seeking Behavior

Previous work suggests that AMPA glutamategic transmission plays a key role in reinstatement of drug seeking behavior when drug seeking is primed by a drug such as cocaine. In this paradigm animals are trained to press a lever for cocaine. The behavior is then extinguished by no longer administering cocaine after a lever press. A week later, the rats are given an injection of cocaine which primes the animal to press the lever again. Injections of AMPA receptor agonists reinstate drug seeking behavior after it has been extinguished and AMPA antagonists injected into the nucleus accumbens block cocaine seeking behavior following a priming injection of cocaine. In the October 22, 2008 issue of the Journal of Neuroscience Dr. Chris Pierce and his colleagues report that the injection of the AMPA receptor antagonist CNQX into the nucleus accumbens shell or core, injected just before a priming dose of cocaine, attenuated cocaine seeking behavior. Dr. Pierce and his colleagues then showed that reinstatement of cocaine seeking behavior by a priming dose of cocaine is mediated by phosphorylation of the GluR2 receptor at serine 880 in the nucleus accumbens shell but not core. The effect on phosphorylation of the GluR2 receptor is specific because phosphorylation of the receptor is not observed for animals receiving a priming dose that were not receiving contingent cocaine. Phosphorylation of GluR2 at serine 880 is required for association of GluR2 to Pick1 and subsequent internalization of the receptor. To test whether phosphorylation of the GluR2 receptor is causally involved in drug reinstatement, Dr. Pierce and his colleagues injected Pep2-EVKI, a peptide that blocks GluR2 trafficking by preventing the phosphorylation of GluR2 and subsequent association of GluR2 with the gene, PICK1, into the nucleus accumbens an hour before the priming dose of cocaine. The Pep2-EVKI but not a control peptide, Pep2-SVKE, attenuated drug induced reinstatement of cocaine seeking behavior. These results suggest that the phosphorylation of GluR2 receptor and its internalization promote drug seeking behavior. Famous, K.R., Kumaresan, V., Sadri-Vakili, G., Schmidt, H.D., Mierke, D.F., Cha, J.H., and Pierce, R.C. Phosphorylation-Dependent Trafficking of GluR2-Containing AMPA Receptors in the Nucleus Accumbens Plays a Critical Role in the Reinstatement of Cocaine Seeking J. Neurosci. 28(43), pp. 11061-11070, 2008.

The Nicotinic Acetylcholine Receptor β2 Subunit (CHRNB2) Gene is Implicated in Smoking Cessation: A Genetic Marker for Bupropion Treatment And Relapse Risk?

It is well-documented that there is substantial variability among individuals in response to bupropion treatment for tobacco dependence. David Conti and his colleagues performed a systems-based candidate gene study of 1295 single nucleotide polymorphisms (SNPs) in 58 genes within the neuronal nicotinic receptor and dopamine systems to investigate their role in smoking cessation in a bupropion placebo-controlled randomized clinical trial. In global tests of main effects and treatment interactions, a SNP (rs2072661) in the 3' UTR region of the β2 nicotinic acetylcholine receptor subunit (CHRNB2) has an impact on abstinence rates at the end of treatment (adjusted P= 0.01) and after a 6-month follow-up period (adjusted P = 0.0002). Independent of treatment at 6-month follow-up, individuals carrying the minor allele have substantially decreased the odds of quitting (OR = 0.31; 95% CI 0.18-0.55). Effect of estimates indicate that the treatment is more effective for individuals with the wild-type (OR = 2.14, 95% CI 1.20-3.81) compared with individuals carrying the minor allele (OR = 0.83, 95% CI 0.32-2.19), although this difference is only suggestive (P = 0.10). Furthermore, this SNP demonstrated a role in the time to relapse (P = 0.0002) and an impact on withdrawal symptoms at target quit date (TQD) (P = 0.0009). Overall, while the results indicate strong evidence for CHRNB2 in ability to quit smoking, these results require replication in an independent sample. Conti, D.V., Lee, W., Li, D., Liu, J., Van Den Berg, D., Thomas, P.D., Bergen, A.W., Swan, G.E., Tyndale, R.F., Benowitz, N.L., and Lerman, C. for the Pharmacogenetics of Nicotine Addiction and Treatment Consortium. Nicotinic Acetylcholine Receptor β2 Subunit Gene Implicated in a Systems-based Candidate Gene Study of Smoking Cessation. Hum. Mol. Genetics, 17, pp. 2834-2848, 2008.

Dopamine, Norepinephrine and Serotonin Modulate Monoamine Transporter Function via TAAR1

The trace amine-associated receptor 1 (TAAR1) is expressed in brain monoaminergic systems and activated by dopamine, norepinephrine and serotonin. This study used transfected HEK293 cells and brain synaptosomes to evaluate the interaction of these monoamines with TAAR1 and with monoamine autoreceptors to explore their modulatory effects on monoamine transporters. The researchers demonstrated that TAAR1 signaling was attenuated by monoamine autoreceptors following exposure to dopamine, norepinephrine or serotonin. In transfected cells, TAAR1 in response to dopamine, norepinephrine or serotonin significantly inhibited uptake and promoted efflux of dopamine, [norepinephrine or serotonin, respectively, whereas the monoamine autoreceptors, D2s, alpha (2A) and 5-HT(1B), enhanced the uptake function under the same condition. In brain synaptosomes, dopamine, norepinephrine or serotonin significantly altered the uptake and efflux of dopamine, norepinephrine or serotonin, respectively, when the monoamine autoreceptors were blocked. By comparing the effects of dopamine, norepinephrine and serotonin in non human primate and wild-type mouse synaptosomes to their effects in TAAR1 knockout mouse synaptosomes, they deduced that TAAR1 activity inhibited uptake and promoted efflux by monoamine transporters and that monoamine autoreceptors exerted opposite effects. These data provide the first evidence that common biogenic amines modulate monoamine transporter function via both TAAR1 and monoamine autoreceptors, which may balance their activity. In this regard, the data reveal that TAAR1 functions as a monoamine autoreceptor in monoaminergic neurons. Xie, Z., Westmoreland, S.V., and Miller, G.M. Modulation of Monoamine Transporters by Common Biogenic Amines via Trace Amine-Associated Receptor 1 and Monoamine Autoreceptors in Human Embryonic Kidney 293 Cells and Brain Synaptosomes. J. Pharm. Exp. Ther., 325, pp. 629-640, 2008.

Cocaine Preferentially Enhances Firing and Evokes Release of Dopamine in the Shell of the Nucleus Accumbens

In this study, characterization of electrophysiological transients (firing of neurons) was accomplished by pharmacologically blocking dopamine autoreceptors, one of the regulatory elements of neurotransmission. Comparisons of real-time measurements using in vivo voltammetry after cocaine administration and systemic manipulation of dopamine autoreceptors provided reliable resolution between increases in the frequency of dopamine release events and the concentration of dopamine released. Wightman and his colleagues showed that the preferential enhancement of dopamine transmission within the NAc shell that is evoked by cocaine is attributable to a greater number of phasic release events originating from midbrain dopaminergic neuron activity. This subregion difference was abolished by autoreceptor blockade before cocaine administration. Finally, cocaine administration in the absence of autoregulation resulted in a synergistic increase in [DA], describing one mechanism by which cocaine induces burst firing from dopaminergic neurons. This study provides novel characterization of the distinct processes that encompass extracellular dopamine transmission, shows the first evidence that cocaine directly increases dopamine release events in a temporally and regionally specific manner, and demonstrates the significance of autoregulation in cocaine-evoked dopamine transmission. Aragona, B.J., Cleaveland, N.A., Stuber, G.D., Day, J.J., Carelli, R.M., and Wightman, R.M. Preferential Enhancement of Dopamine Transmission within the Nucleus Accumbens Shell by Cocaine Is Attributable to a Direct Increase in Phasic Dopamine Release Events. J. Neuroscience, 28, pp. 8821-8831, 2008.

β-Catenin is Required for Memory Consolidation

Recent work by Magaschuk and Ressler suggests that β-catenin, a molecule that governs some of the structural changes in synaptic morphology at excitatory synapses, has a role in long-term memory formation. β-catenin is highly expressed in the adult mouse amygdala and is dynamically regulated at both the transcriptional and post-translational levels with fear learning. Pharmacological stabilization of β-catenin with LiCl resulted in enhanced learning, whereas genetic deletion of its gene, Ctnnb1, in the amygdala resulted in deficient learning. By studying the effects of Ctnnb1 deletion in adult mice, these researchers have identified a role for β-catenin in learning and memory that is distinct from its role in development. Their data suggest that β-catenin is required for the consolidation, but not the acquisition, of fear memory. However, once memory consolidation has occurred, β-catenin is no longer needed to express the memory. During this consolidation period, the interaction between β-catenin and cadherin, a cell adhesion protein, is dynamically regulated, suggesting that β-catenin is involved in the structural conversion of short-term labile to long-term stable memory traces and that this effect was specific to the basal lateral amygdala. This mechanism may also prove to be important in memory consolidation processes associated with drug addiction. Magaschak, K.A. and Ressler, K.J. β-catenin is Required for Memory Consolidation. Nature Neurosci., 11, pp. 1319-1326, 2008.

Pharmacotherapies Targeted at Alpha 6 Nicotinic Acetylcholine Receptors May Be Useful in Treating Human Disorders Involving Changes in Dopamine

Nicotinic acetylcholine receptors (nAChRs) modulate dopaminergic transmission within the nervous system. One subtype of nAChR, the alpha6-containing (alpha6*) nAChR, is selectively expressed in dopamine neurons. Previous studies using gene knockouts (alpha6 knockout mice) or pharmacological inhibition of alpha6 nAChRs have identified behavioral and physiological responses that are regulated by alpha6 nAChR function. This recent study takes these previous results further by generating and studying mice with gain-of-function alpha6* nAChRs that amplify rather than inhibit cholinergic control of dopaminergic transmission. These gain-of-function mice show increased dopamine neuron excitability and dopamine release. And intriguingly, these mice display behavioral phenotypes consistent with increased dopaminergic transmission. Alpha6 gain-of-function mice exhibited locomotor hyperactivity in their home cage and failed to habituate to a novel environment. Selective activation of alpha6* nAChRs with low doses of nicotine, thereby stimulating dopamine but not GABA neurons, exaggerated these phenotypes and produced a hyper-dopaminergic state in vivo. Experiments with additional nicotinic drugs showed that altering agonist efficacy at alpha6* receptors provided fine tuning of dopamine release and locomotor responses. Thus, alpha6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms in midbrain or striatum, provide a method for treating neural disorders that result from aberrant dopaminergic transmission. Drenan, R.M., Grady, S.R., Whiteaker, P., McClure-Begley, T., McKinney, S., Miwa, J.M., Bupp, S., Heintz, N., McIntosh, J.M., Bencherif, M., Marks, M.J., and Lester, H.A. In Vivo Activation of Midbrain Dopamine Neurons Via Sensitized, High-affinity alpha 6 Nicotinic Acetylcholine Receptors. Neuron. 60(1), pp. 123-136, 2008.

Tolerance to Short-Term Repeated Morphine Administration is Associated with Increased, As Well As Decreased, Potency of Opioid Agonists

Tolerance to the pain-relieving effects of opiates limits their clinical use. Morphine tolerance is associated with desensitization of mu-opioid receptors. However, one problem with the desensitization hypothesis is that acute morphine does not readily desensitize mu-opioid receptors. Thus, studies of opioid tolerance have been typically performed after prolonged, continuous opioid pretreatment. These studies have routinely shown that continuous opioid pretreatment decreases opioid activation of G-protein-mediated inwardly rectifying potassium (GIRK) currents, subsequently altering neuronal activity within the periaqueductal gray region that then contributes to the expression of antinociceptive tolerance. The paper cited here sought to determine whether similar changes occurred with short-term repeated morphine exposure. Using this exposure paradigm, it was found that short-term repeated exposure to opioids produced a biphasic effect on GIRK currents in morphine tolerant rats. Unexpectedly, opioid activation of GIRK currents was initially potentiated in morphine compared to saline pretreated rats. These currents were inhibited by the mu-opioid receptor antagonist beta-funaltrexamine suggesting that short-term repeated morphine administration enhances agonist stimulation of mu-opioid receptor coupling to G-proteins. Longer application of opioids produced mu-opioid receptor desensitization consistent with results obtained with continuous opioid exposure. However, peak GIRK currents from short-term repeated exposure and tolerant rats desensitized more than currents from saline pretreated rats. These data indicate that antinociceptive tolerance due to short-term repeated opioid exposure may be triggered by enhanced agonist potency followed by an increased desensitization of mu-opioid receptors. Thus, this paper may have uncovered a novel action of opioids at mu-opioid receptors. Ingram, S.L., Macey, T.A., Fossum, E.N., and Morgan, M.M. Tolerance to Repeated Morphine Administration is Associated with Increased Potency of Opioid Agonists. Neuropsychopharmacology, 33(10), pp. 2494-504, 2008.

Striatal Misregulation of Cdk5 Alters Locomotor Responses to Cocaine, Motor Learning, and Dendritic Morphology

Cyclin dependent kinase 5 (Cdk5), an enzyme expressed primarily in neural cells, regulates dopamine (DA) neurotransmission and striatal neuron excitability and may play a role in mediating synaptic plasticity and learning. In the striatum, medium spiny neurons (MSNs) receive midbrain dopaminergic and cortical glutamergic inputs which influence reward and motor learning pathways. Recently, James Bibb and colleagues investigated the effects of Cdk5 misregulation on striatal-mediated behavior and learning. Cdk5 regulation of DA signaling alters behavioral and biochemical responses to stimulants such as cocaine. Cleavage of the Cdk5 cofactor p35 to form a Cdk5/p25 complex results in abnormal Cdk5 activity and, in turn, neurotoxicity and neurodegeneration. In this study, the investigators generated a transgenic mouse in which p25 could be selectively overexpressed. Using this mouse strain, Bibb and colleagues demonstrated that overexpression of p25 and the resultant misregulation of Cdk5 leads to reduced locomotor sensitization to cocaine over a five day exposure period. In addition, these mice displayed decreased motor coordination, which further indicates that p25-mediated Cdk5 misregulation results in abnormal DA- and striatal-dependent motor behavior. Histological evaluation of p25 overexpression in striatum revealed elevated amounts of glial fibrillary acidic protein (GFAP) and altered cellular morphology, including abnormally shaped cell bodies and decreased dendritic spine density. GFAP is a marker of activated astrocytes and astrogliosis, which accompanies excitotoxic neuronal cell loss. However, in the case of p25 overexpression-dependent Cdk5 misregulation within the striatum, MSNs remained alive, although with significant loss of function. The above findings show that abnormal Cdk5 regulation results in impaired function of this brain region without the neurodegeneration and neuronal cell loss normally associated with p25 overexpression and, as a result, may prove useful for investigation of non-neurodegenerative neurological disorders. Meyer, D.A., Richer, E., Benkovic, S.A., Hayashi, K., Kansy, J.W., Hale, C.F., Moy, L.Y., Kim, Y., O'Callaghan, J.P., Tsai, L.H., Greengard, P., Nairn, A.C., Cowan, C.W., Miller, D.B., Antich, P., and Bibb, J.A. Striatal Dysregulation of Cdk5 Alters Locomotor Responses to Cocaine, Motor Learning, and Dendritic Morphology. Proc. Natl. Acad. Sci. U S A., 105, pp. 18561-18566. Epub, 2008.

Cdk5 Regulates Neurogenesis in Adult Hippocampus

Many substances of abuse affect the dopaminergic neural circuits by inducing specific gene expression. One of the genes substantially affected by substances of abuse, such as cocaine and nicotine, is Cdk5, as it is immediately downstream of several upregulated transcription factors, and is significantly upregulated. During brain development, Cdk5 plays a role in neuronal differentiation and migration, but its role in the adult brain, is not clear. A group of NIDA researchers at University of Texas Southwestern, led by Dr. Amelia Eisch, report that Cdk5 has an essential role in the survival of adult-generated neurons in hippocampus. These researchers assessed the role of Cdk5 in the generation of dentate gyrus (DG) granule cell neurons in adult mice. They demonstrate that Cre recombinase- mediated conditional knockout (KO) of Cdk5 from stem cells and their progeny in the DG subgranular zone (SGZ) prevented maturation of new neurons. In addition, selective KO of Cdk5 from mature neurons throughout the hippocampus reduced the number of immature neurons. Furthermore, Cdk5 gene deletion specifically from DG granule neurons via viral-mediated gene transfer also resulted in fewer immature neurons. In each case, the total number of proliferating cells was unaffected, indicating that Cdk5 is necessary for progression of adult-generated neurons to maturity. In addition, they found that the role for Cdk5 in neurogenesis was activating-cofactor specific, as p35 KO but not p39 KO mice also had fewer immature neurons. These findings suggest that abnormal regulation of Cdk5, induced by substances of abuse, may result in abnormal neurogenesis in hippocampus, with consequences in learning and memory. Lagace, D.C., Benavides, D.R., Kansy, J.W., Mapelli, M., Greengard, P., Bibb, J.A., and Eisch, A.J. Cdk5 is Essential for Adult Hippocampal Neurogenesis. PNAS 105, pp. 18567-18571, 2008.

Cocaine Binge Alters Cellular Signaling Through Akt-GSK3

The mechanism of cocaine addiction, initiated through blocking dopamine transporters in the dopaminergic neural circuits in the brain, is well known. However, the cellular signaling pathways inside the cell membrane upon cocaine exposure and challenge are not well understood. Ikeda et al (2006), showed, that Akt, also called Protein kinase B, is involved in the cellular mechanisms of addiction. Akt negatively regulates GSK3 activity. Typically, following dopamine receptor activation, a time-dependent decrease in phosphorylated Akt in striatum is observed which leads to increased GSK3 activity (by decreasing phosphorylated GSK3) and subsequent increase in gene transcription. Whether cocaine initiates the same signaling pathway, and where such signaling events occur in the dopaminergic circuits are not known. Dr. Ellen Unterwald, at Temple University, reports that cocaine induces a spatially restricted pattern of phosphorylation of Akt and GSK3, wherein the nucleus accumbens and amygdala but not caudate putamen and hippocampus are affected. Regulation of these signaling molecules is also dependent on the length of cocaine exposure. In their study, rats were injected with cocaine (15 mg/kg) or saline (1 mL/kg) in a binge-pattern (three injections at 1 h intervals beginning at 9 AM) for 1, 3, or 14 days. After the last injection, brains were dissected, and the phosphorylation level of Akt and GSK3 in amygdala, nucleus accumbens, caudate putamen, and hippocampus were decided in protein assay. The team found that phosphorylation of Akt on the threonine-308 (Thr308) residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 was significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The researchers suggest that these findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure. Since GSK3 is downstream of the gene Wnt and catenin which are involved in axonal growth, dendritic remodeling and synaptic regulation, this study may also suggest roles for Wnt signaling pathway in substance abuse. Perrine, S.A., Miller, J.S., and Unterwald, E.M. Cocaine Regulates Protein Kinase B and Glycogen Synthase kinase-3 Activity in Selective Regions of Rat Brain. J. Neurochem. 107, pp. 570-577, 2008.

Translational Profiling Approach for the Analysis of CNS Cell Types

In the November 14th issue of Cell Dr. Paul Greengard, in collaboration with Dr. Nat Heintz, report the development of a method to identify cell types in the nervous system based on a molecular phenotype. This method, called, translating ribosome affinity purification (TRAP), overcomes the problems associated with isolating the highly heterogenous and highly intermixed cell types in the nervous system. Dissocation of neuron induces changes in gene express resulting from mechanical stress and the properties of neurons may change when not in contact with their local environment. To overcome this problem the Greengard and Heintz laboratory have invented a rapid isolation strategy for isolation of polysomal RNA from genetically targeted cell types. In this method an enhanced green fluorescent protein is fused to the N terminus of the large-subunit ribosomal protein L10a (EGFP-L10a). This protein is incorporated into the polysomes where mRNA is translated into protein. An EGFP-L10a antibody attached to magnetic beads is used to isolate and purify the polysomes and the message is then profiled on a silicon chip or sequenced. This method eliminates the expression profiling of RNAs that are not translated into cells. The reporter, EGFP-L10a, is genetically targeted to neurons by inserting the reporter into a large piece of mouse genomic DNA in a bacterial artificial chromosome. The elements inside the BAC that control expression of the gene direct EGFP-L10a to be expressed in specific cell types. To profile striatalnigal cells and striatalpallidal cells, two different populations in the striatum, EGP-L10a was inserted into a Drd1a and Drd2 receptor BACs, respectively. Striatalnigral and striatalpallidal neurons could be distinguished from one another on the basis of expression with translation of Eya1, Isl1, Gng2, and Crym in striatonigral neurons and Gpr6, Lhx8, Gpr88, Trpc4, and Tpm2 in striatopallidal neurons. Mice were then chronically treated with cocaine and gene expression profiling was performed on striatopallidal (Drd2) and striatalnigral cells (Drd1). The GABA signaling pathway (Gabrb3, Gabra1, Cacnb4, and Gabra4) was observed to be significantly increased in striatalnigral cells but not in striatalpallidal neurons. This change in the expression of these GABA receptors was associated with an increase in the frequency of small-amplitude GABAergic mIPSCs in striatalnigral neurons but not in striatalpallidal neurons. To show that the approach can be generalized to other cell types in the nervous system, the Greengard and Heintz labs used the translating ribosome affinity purification (TRAP) technique to profile 24 CNS populations. They report the identification of thousands of cell specific mRNAs not detected by whole tissue gene expression profiling experiments. Gene expression obtained from these cells using TRAP matched the expression of known genes using other methods. Hierarchial clustering, a statistical method, suggests that gene expression of cortical projection neurons are more similar than cortical interneurons, motorneurons, and Purkinje cells. Similarly, gene expression of astroglial from different regions of the brain show greater similarity in pattern of gene expression than other types of glia. This suggests that cells with similar functions share similar gene expression patterns. They also show that much of the diversity of cell types is marked by cell surface proteins. This method provides a means by which cells in the nervous system can be defined by a molecular phenotype. This method provides the basis by which investigators can induce human induced pleuripotent stem (iPS) cells derived from fibroblast to become defined cell types in the nervous system. These neuronal derived human iPS cells can be used for studies of human cell in culture or used for restoring function in the nervous system. Furthermore, the effects of mutations on the expression of genes in single cells can now be studied by crossing the TRAP transgene into the genetic background of the mutant. Doyle, J.P., Dougherty, J.D., Heiman, M., Schmidt, E.F., Stevens, T.R,, Ma, G., Bupp, S., Shrestha, P., Shah, R.D., Doughty, M.L., Gong, S., Greengard, P., and Heintz, N. Application of a Translational Profiling Approach for the Comparative Analysis of CNS Cell Types. Cell. 135(4), pp. 749-762, 2008; Heiman, M., Schaefer, A., Gong, S., Peterson, J.D., Day, M., Ramsey, K.E., Suarez-Farinas, M., Schwarz, C., Stephan, D.A., Surmeier, D.J., Greengard, P., and Heintz, N. A Translational Profiling Approach for the Molecular Characterization of CNS Cell Types. Cell, 135(4), pp. 738-748, 2008.

A Radiolabeled Alpha7 nAChR Antagonist Probe

Occurrence of the alpha7 subtype of nicotinic acetylcholine receptors has been documented in brain hippocampus and cortex, and further reported in keratinocytes, lymphocytes, macrophage, astrocytes, microglia, and retinal neurons. Besides the use of alpha7 subunit-detecting antibodies, the most commonly used probes are the peptide alpha-bungarotoxin (alpha-Bgt) and the non-peptide methyllycaconitine (MLA). These antagonists have some limitations: MLA also binds to alpha6-containing subtpyes, while alpha-Bgt also binds to other non-alpha7 subtypes (alpha1, alpha 8, and alpha 9/10), and displays a rather slow rate of dissociation from the receptors, which makes it difficult to determine equilibrium binding. Previous work by Drs. Paul Whiteaker and J. Michael McIntosh has produced two modified c. arenatus conotoxin peptide antagonists, with high selectivity for the alpha7 nAChR subtype. These researchers now report that one of these conotoxins has been modified at the single histidine residue with one or two iodine atoms (either I-127 or I-125), and the association/dissociation kinetics, saturation binding, and autoradiographic properties examined. In mouse hippocampal membranes, the monoiodo and diiodo nonradioactive ligands (I-127) had inhibition of I-125 alpha-Bgt greater than that of their parent peptide (low nM Ki values), and their radioactive counterparts produced association (specific binding) with the hippocampal membranes which was complete in about 120-180 minutes, and with reversible dissociation of this binding complete in 180 minutes at 22 degrees Celcius. The binding density (Bmax at saturation) of both the mono-iodo-125 and di-iodo-125 ligands was approximately equal to that of iodo-125 alpha-Bgt. Autoradiographic results for both iodinated ligands showed labeling of wild-type mouse brain in the hippocampus, amygdala, and cortex regions, with only background labeling in the alpha 7 null mouse brain. In terms of stability, the monoiodinatedq I-125 derivative displayed only slight decay over a twelve week period, based on its value of Kd, Bmax, and signal/noise ratio. Both iodinated derivatives showed a selectivity of more than 100-fold for alpha7 compared to alpha6beta2* and alpha9alpha10 subtypes, using a 1 nM concentration of iodinated peptide, displacing appropriate tritiated standards in various membranes. Whiteaker, P., Marks, M.J., Christensen, S., Dowell, C., Collins, A.C., and McIntosh, J.M. Synthesis and Characterization of I-125-alpha-Conotoxin ArIB[V11L;V16A], a Selective alpha7 Nicotinic Acetylcholine Receptor Antagonist. The Journal of Pharmacology and Experimental Therapeutics, 325(3), pp. 910-919, 2008.

Isolation and Characterization of New Cannabis Constituents

Cannabis sativa L., one of the oldest plants known in medicine, is the most widely used illicit drug in the world today. A total of almost 500 natural constituents have been isolated and/or identified from Cannabis, with 9-THC as the main biologically active component. The availability of high potency marijuana on the illicit market with unprecedented 9-THC concentrations (>20% by dry weight) has led researchers to discover new constituents from Cannabis. This publication reports the isolation and structure elucidation of six new metabolites, (+/-)-6,7-trans-epoxy-cannabigerolic acid, (+/-)-6,7-cis-epoxycannabigerolic acid, (+/-)-6,7-cis-epoxycannabigerol, (+/-)-6,7-trans-epoxy-cannabigerol, 5'-methyl-4-phenylbiphenyl-2,2',6-triol, and 7-methoxycannabispirone along with seven known compounds namely, cannabigerolic acid, 5'-methoxycannabigerolic acid, cannabispirone, ss-cannabispiranol, dehydrocannabifuran, cannflavin B, and cannabigerol. Their antimicrobial, as well as the antileishmanial activities, were also investigated. Radawan, M.M., Ross, S.R., Slade, D., Ahmed, S.A., Zulfiqar, F., and ElSohly, M.A. Isolation and Characteri-zation of New Cannabis Constituents from a High Potency Variety. Planta Med.,74(3), pp. 267-272, 2008.

Selective Blockade of 2-Arachidonoylglycerol Hydrolysis Produces Cannabinoid Behavioral Effects

2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate the cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis; for anandamide this process is mediated by fatty acid amide hydrolase (FAAH), and for 2-AG is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors have been shown to generate a subset of the behavioral effects observed with CB1 agonists, which suggests a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Dr. Cravatt and his colleagues describe a potent and selective inhibitor of MAGL (JZL184) that raises brain 2-AG by eight-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia and hypomotility. These findings indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and suggest overlapping and distinctive functions for 2-AG and anandamide in vivo. Long, J.Z., Li, W., Booker, L., Burston, J.J., Kinsey, S.G., Schlosburg, J.E., Pavon, F.J., Serrano, A.M., Selley, D.E., Parsons, L.H., Lichtman, A.H., and Cravatt, B.F. Selective Blockade of 2-arachidonoylglycerol Hydrolysis Produces Cannabinoid Behavioral Effects. Nature Chem. Bio., Published online 23 November 2008.

Nicotinic Acetylcholine Receptors Contribute to Neural Processing of Sensory Stimuli and Sensory-Cognitive Behaviors

Dr. Raju Metherate and his colleagues at the University of California, Irvine, have continued their studies on the role of nicotinic acetylcholine receptors (nAChRs) in modulating tone-evoked responses in auditory cortex. In this study they sought to test the prediction that nicotinic enhancement of sensory physiology had a direct relation to behavioral performance. They trained adult rats in an auditory-cued, active avoidance task and classified their performance as good, intermediate, or poor. Next, they anesthetized the animals and recorded tone-evoked local field potentials in layer 4 of the auditory cortex, before and after a test dose of nicotine or saline. The investigators found that nicotine enhanced the amplitude and decreased the threshold of responses of neurons to the smallest sound intensities in rats classified as good performers, but not in rats considered as intermediate or poor performers. Interestingly, nicotine had the opposite effects on responses to spectrally distant stimuli, in effect showing that cortical receptive fields became more selective to the appropriate stimuli, but once more only in the good performers. Nicotine did reduce onset latencies in all three groups, which the investigators interpreted as evidence that the drug did attain appropriate dose levels. These findings do not answer the questions of whether or not poor learning is associated with nonfunctional AChRs, whether higher doses of nicotine would be effective in poor performers, or whether robust learning results from or produces improved nAChR expression. These findings do, however, suggest that nicotine-enhanced receptive field selectivity in auditory cortex may contribute to improved cognitive function by enhanced responses to relevant stimuli and suppression of distracters. Liang, K. Poytress, B. Weinberger, N. and Metherate, R. Nicotinic Modulation of Tone-Evoked Responses in Auditory Cortex Reflects the Strength of Prior Auditory Learning. Neurobiology of Learning and Memory, 90, pp. 138-146, 2008.


Research Findings

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