Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Transcriptional Changes Common to Human Cocaine, Cannabis and Phencyclidine Abuse A major goal of drug abuse research is to identify and understand drug-induced changes in brain function that are common to many or all drugs of abuse. As these may underlie drug dependence and addiction, the purpose of the present study was to examine if different drugs of abuse effect changes in gene expression that converge in common molecular pathways. Microarray analysis was employed to assay brain gene expression in postmortem anterior prefrontal cortex (aPFC) from 42 human cocaine, cannabis and/or phencyclidine abuse cases and 30 control cases, which were characterized by toxicology and drug abuse history. Common transcriptional changes were demonstrated for a majority of drug abuse cases (N = 34), representing a number of consistently changed functional classes: Calmodulin-related transcripts (CALM1, CALM2, CAMK2B) were decreased, while transcripts related to cholesterol biosynthesis and trafficking (FDFT1, APOL2, SCARB1), and Golgi/endoplasmic reticulum (ER) functions (SEMA3B, GCC1) were all increased. Quantitative PCR validated decreases in calmodulin 2 (CALM2) mRNA and increases in apolipoprotein L, 2 (APOL2) and semaphorin 3B (SEMA3B) mRNA for individual cases. A comparison between control cases with and without cardiovascular disease and elevated body mass index indicated that these changes were not due to general cellular and metabolic stress, but appeared specific to the use of drugs. Therefore, humans who abused cocaine, cannabis and/or phencyclidine share a decrease in transcription of calmodulin-related genes and increased transcription related to lipid/cholesterol and Golgi/ER function. These changes represent common molecular features of drug abuse, which may underlie changes in synaptic function and plasticity that could have important ramifications for decision-making capabilities in drug abusers. Lehrmann, E., Colantuoni, C., Deep-Soboslay, A., Becker, K.G., Lowe, R., Huestis, M.A., Hyde, T.M., Kleinman, J.E., and Freed. W.J. PLoS ONE, 1:e, pp. 114, 2006.

Cellular Pathobiology Unit, Development and Plasticity Section, Cellular Neurobiology Research Branch

PKA Activation Downregulates whereas ERK Activation Upregulates Sigma-1 Receptors in B-104 Cells: Implication for Neuroplasticity The sigma-1 receptor (Sig-1R) can bind psychostimulants and was shown to be upregulated in the brain of methamphetamine self-administering rats. Upregulation of Sig-1Rs has been implicated in neuroplasticity. However, the mechanism(s) whereby Sig-1Rs are upregulated by psychostimulants is unknown. Here, IRP investigators employed a neuroblastoma cell line B-104, devoid of dopamine receptors and transporter, and examined the effects of psychostimulants as well as cAMP on the expression of Sig-1Rs in this cell line, with a specific goal to identify signal transduction pathway(s) that may regulate Sig-1R expression. Chronic treatments of B-104 cells with physiological concentrations of cocaine or methamphetamine failed to alter the expression of Sig-1Rs. Dibutyryl cAMP (dB-cAMP), when used at 0.5 mM, caused a downregulation of Sig-1Rs that could be blocked by a protein kinase A (PKA) inhibitor H-89. However, dB-cAMP, when used at 2 mM, caused an upregulation of Sig-1Rs that was insensitive to the H-89 blockade but was partially blocked by an extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK) inhibitor PD98059. Further, 2 mM of dB-cAMP induced an ERK phosphorylation lasting at least 90 min, at which time the phosphorylation caused by 0.5 mM of dB-cAMP had already diminished. PD98059, applied 90 min after addition of 2 mM of dB-cAMP, attenuated the Sig-1R upregulation. These results indicate that cAMP is bimodal in regulating Sig-1R expression: a downregulation via PKA and an upregulation via ERK. Results also suggest that psychostimulants may manipulate the cAMP-PKA-Sig-1R and/or the cAMP-ERK-Sig-1R pathways to achieve a neuroplasticity that favors addictive behaviors. Cormaci, G., Mori, T., Hayashi, T., and Su, T.P. The Journal of Pharmacology and Experimental Therapeutics, 320(1), pp. 202-210, 2007.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Parkinson's Disease Model Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson's disease (PD), a common age-associated neurodegenerative disease characterized by intraneuronal inclusions (Lewy bodies) and progressive degeneration of the nigrostriatal dopamine (DA) system. It has recently been demonstrated that midbrain DA neurons of PD patients and elderly humans contain high levels of somatic mtDNA mutations, which may impair respiratory chain function. However, clinical studies have not established whether the respiratory chain deficiency is a primary abnormality leading to inclusion formation and DA neuron death, or whether generalized metabolic abnormalities within the degenerating DA neurons cause secondary damage to mitochondria. IRP scientists have used a reverse genetic approach to investigate this question and created conditional knockout mice (termed MitoPark mice), with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. The knockout mice have reduced mtDNA expression and respiratory chain deficiency in midbrain DA neurons, which, in turn, leads to a parkinsonism phenotype with adult onset of slowly progressive impairment of motor function accompanied by formation of intraneuronal inclusions and dopamine nerve cell death. Confocal and electron microscopy show that the inclusions contain both mitochondrial protein and membrane components. These experiments demonstrate that respiratory chain dysfunction in DA neurons may be of pathophysiological importance in PD. Ekstrand, M., Terzioglu, M., Galter, D., Zhu, S., Hofstetter, C., Lindqvist, E., Thams, S., Bergstrand, A., Hansson, F.S., Trifunovic, A., Hoffer, B., Cullheim, S., Mohammed, A.H., Olson, L., and Larsson, N.G. PNAS, 104, pp. 1325-1330, 2007.

Glutamatergic Neurons are Present in the Rat Ventral Tegmental Area The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or gamma-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, IRP researchers have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry the authors investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. They found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro-caudal and medio-lateral aspects of the VTA, with the highest concentration detected in rostro-medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely co-expressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on the results described here, the authors concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non-DAergic and non-GABAergic. Yamaguchi, T., Sheen, W., and Morales, M. European Journal of Neuroscience, 25(1), pp. 106-118, 2007.

Alterations in Prodynorphin, Proenkephalin, and GAD67 mRNA Levels in the Aged Human Putamen: Correlation with Parkinson's Disease A real-time quantitative PCR approach was used to quantify mRNA levels corresponding to the neuropeptides enkephalin, dynorphin, and the 67-kDa isoform of glutamic acid decarboxylase (GAD67) in the human putamen from young and aged individuals as well as from aged patients affected by Parkinson's disease (PD). cDNA-specific primers were designed to amplify GAD67, proenkephalin (pENK), prodynorphin (pDYN), and the housekeeping genes glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and guanine nucleotide binding protein, beta-peptide 2-like I (GNB2LI). GAPDH and GNB2LI mRNA levels were similarly expressed among the groups and were therefore used as endogenous reference genes. Normalized data showed that mRNA levels for both pENK and pDYN were reduced in the putamen of aged controls and aged individuals affected by PD, compared with young controls. In addition, the authors showed that GAD67 mRNA levels did not change during aging and PD. Further analyses showed no differences in mRNA levels, for pENK, pDYN, or GAD67 mRNA, between PD patients and aged matched controls. These findings contrast with animal models of parkinsonism, for which expression of pDYN, pENK, and GAD67 mRNA has been reported to change after striatal dopamine denervation. Compensatory mechanisms and regional differences within the human putamen as well as the severity index of the disease, clinical diagnosis, and response to pharmacological therapy are possible reasons for these results. The present study suggests that alteration of neuropeptide pathways in the human putamen may be involved in the functional deterioration of parts of the extrapyramidal system during aging. Backman, C.M., Shan, L., Zhang, Y., Hoffer, B.J., and Tomac A.C. Journal of Neuroscience Research, 85(4), pp. 798-804, 2007.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Opposing Actions of Chronic Delta9-Tetrahydrocannabinol and Cannabinoid Antagonists on Hippocampal Long-Term Potentiation Memory deficits produced by marijuana arise partly via interaction of the psychoactive component, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), with cannabinoid receptors in the hippocampus. Although cannabinoids acutely reduce glutamate release and block hippocampal long-term potentiation (LTP), a potential substrate for learning and memory, the consequences of prolonged exposure to Delta(9)-THC for hippocampal function are poorly understood. Rats were injected with Delta(9)-THC (10 mg/kg, i.p., q.d.) for 1, 3, or 7 d, and electrophysiological recordings were performed in hippocampal slices 1d after the final injection. At this time, Delta(9)-THC was undetectable in hippocampus using liquid chromatography-mass spectrometry (LC-MS). Hippocampal LTP generated using high-frequency (HFS) or theta burst stimulation was not observed in brain slices from the 7-d Delta(9)-THC-treated animals. Delta(9)-THC also blocked HFS-LTP after 3 d, but not 1 d of treatment. The complete blockade of LTP persisted for 3 d after the last Delta(9)-THC injection, and full reversal of the LTP deficit was not observed up to 14 d following Delta(9)-THC withdrawal. The cannabinoid antagonist AM251 (2 mg/kg), administered before each Delta(9)-THC injection prevented the blockade of LTP, and 7-d treatment with AM251 alone significantly increased the level of LTP. Chronic Delta(9)-THC also produced tolerance to the inhibition of synaptic GABA, but not glutamate release by the agonist WIN55,212-2. These data define consequences of repeated Delta(9)-THC exposure for synaptic plasticity in the hippocampus that may help explain memory impairments in humans following chronic marijuana use. Hoffman, A.F., Oz, M., Yang, R., Lichtman, A.H., and Lupica, C.R.. Learning Memory, 14(1), pp. 63-74, 2007.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Sulfation, the Up-and-Coming Post-Translational Modification: Its Role and Mechanism in Protein-Protein Interaction Tyrosine sulfation is a post-translational modification entailing covalent attachment of sulfate to tyrosine residues. It takes place in the trans-Golgi, is necessary for the bioactivity of some proteins, and improves their ability to interact with other proteins. In the present work, IRP scientists show that a protein containing a sulfated tyrosine with a delocalized negative charge forms a salt bridge with another protein if it has two or more adjacent arginine residues containing positive delocalized charges. These noncovalent complexes are so stable that, when submitted to collision induced dissociation, the peptides forming the complex dissociate. Just one covalent bond fragments, the covalent bond between the tyrosine oxygen and the SO3 sulfur, and is represented by the appearance of a new peak (basic peptide + SO3), suggesting that in some instances covalent bonds will break down before the noncovalent bonds between the arginine guanidinium and SO3 dissociate. The data implies that the dissociation pathway is preferred; however, fragmentation between tyrosine and the sulfate residue is a major pathway. Woods, A.S., Wang, H.Y., and Jackson, S.N. Journal of Proteome Research, 6(3), pp. 1176-1182, 2007.

In Situ Structural Characterization of Glycerophospholipids and Sulfatides in Brain Tissue Using MALDI-MS/MS Lipids are major structural components of biomembranes. Negatively charged species such as phosphatidylinositol, phosphatidylserine, sulfatides, and the zwitterionic phosphatidylethanolamines are major components of the cytoplasmic surface of the cellular membrane lipid bilayer and play a key role in several receptors signaling functions. Lipids are not just involved in metabolic and neurological diseases; negatively charged lipids in particular play crucial roles in physiological events such as signal transduction, receptors, and enzymatic activation, as well as storage and release of therapeutic drugs and toxic chemicals in the body. Due to the importance of their role in signaling, the field of lipidomics has rapidly expanded in recent years. In the present study, direct probing of tissue slices with negative ion mode matrix assisted laser desorption/ionization mass spectrometry was employed to profile the distribution of lipids in the brain. In total, 32 lipid species consisting of phosphatidylethanolamines, phosphatidylglycerol, phosphatidylinositols, phosphatidylserines, and sulfatides were assigned. To confirm the structure of lipid species, MALDI-MS/MS analysis was conducted. Product-ion spectra obtained in negative ion mode allow for the assignment of the head groups and the fatty acid chains for the lipid species. Jackson, S.N., Wang, H.Y., and Woods, A.S. Journal of the American Society for Mass Spectrometry, 18(1), pp. 17-26, 2007.

Abeta Peptides as one of the Crucial Volume Transmission Signals in the Trophic Units and their Interactions with Homocysteine: Physiological Implications and Relevance for Alzheimer's Disease Amyloid peptides (Abeta) can operate as volume transmission (VT) signals since they are continuously released from cells of the central nervous system and diffuse in the extra-cellular space of the brain. They have both regulatory and trophic functions on cellular networks. In agreement with Abeta regulatory actions on glial-neuronal networks, the present paper reports new findings demonstrating that intrastriatal injections of Abeta peptides reduce striatal tyrosine hydroxylase, increase striatal GFAP immunoreactivities and lower pain threshold in experimental rats. Furthermore, it has been demonstrated that exogenous homocysteine (Hcy) binds Abeta(1-40) favoring its beta-sheet conformation both in vitro and in vivo and hence the formation of beta-fibrils and development of neurotoxicity. Thus, the hypothesis is discussed that Abeta peptides represent crucial VT-signals in the brain and their action is altered by dysmetabolic signals such as high Hcy extra-cellular levels, known to be an important risk factor for Alzheimer's disease. Agnati, L.F., Genedani, S., Leo, G., Forni, A., Woods, A.S., Filaferro, M., Franco, R., and Fuxe, K. Journal of Neural Transmission, 114(1), pp. 21-31, 2007.

Direct MALDI-MS Analysis of Cardiolipin from Rat Organs Sections Cardiolipins (CL) are mitochondria specific lipids. They play a critical role in ATP synthesis mediated by oxidative phosphorylation. Abnormal CL distribution is associated with several disease states. MALDI-MS and MALDI-MS/MS were used to demonstrate in situ analysis and characterization of CL from tissue sections of organs containing high concentrations of mitochondria. Once the experimental parameters were established, a survey of CL distribution in heart, liver, kidney, leg muscle, and testis was undertaken. The major CL specie in the heart muscle, leg muscle, liver, and kidney is the (18:2)(4) CL, while liver and kidney also contain a minor specie, (18:2)(3)/(18:1) CL. The major CL specie in testis is the (16:0)(4) CL. The CL species distribution in various organs appeared to be in agreement with prior reports. Overall, proper matrix selection, tissue section handling, instrument tuning, and the inclusion of cesium ion in matrix ensured successful in situ MALDI-MS and MALDI-MS/MS analysis of CL. Upon modification and standardization, this method could be streamlined for rapid pathological diagnosis with short turnaround time in clinical settings. Wang, H.Y., Jackson, S.N., and Woods, A.S. Journal of the American Society for Mass Spectrometry, 18(3), pp. 567-577, 2007.

Medicinal Chemistry Section, Medications Discovery Research Branch

Novel Metabotropic Glutamate Receptor Subtype 5 Antagonists The metabotropic glutamate receptor subtype 5 (mGluR5) has been implicated in drug abuse and other neuropsychiatric disorders. The lead compounds for this receptor subtype, MPEP and MTEP, have provided important tools with which to study the role these receptors play in the central nervous system, but have limited application as potential medications. In this report, IRP researchers design and synthesize a novel series of heterobicyclic amides that provide novel structural templates with sub-micromolar binding affinities at the mGluR5. In the functional assay measuring the hydrolysis of phosphoinositide at mGluR5 in CHO cells, the lead compound showed antagonist activity (IC50=0.26+/-0.05 micromolar). Hence this template provides a new lead for further structure-activity relationship investigation and these compounds may serve as molecular tools with which to further study the mGluR5. Kulkarni, S. S., Newman A. H. Bioorganic Medicinal Chemistry Letters, 17, pp. 2074-2079, 2007.

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Salvinorin A: Allosteric Interactions at the Mu-Opioid Receptor Salvinorin A is a hallucinogenic kappa opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. IRP researchers' first published studies indicated that Salvinorin A weakly inhibited mu receptor binding and subsequent experiments revealed that Salvinorin A partially inhibited mu receptor binding. Authors therefore hypothesized that Salvinorin A allosterically modulates mu receptor binding. To test this hypothesis, IRP researchers used CHO cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [3H]DAMGO (0.5, 2.0 and 8.0 nM) binding with EMAX values of 78.6%, 72.1% and 45.7%, respectively and EC50 values of 955, 1124 and 4527 nM, respectively. Salvinorin A also partially inhibited [3H]diprenorphine (0.02, 0.1 and 0.5 nM) binding with EMAX values of 86.2%, 64%, and 33.6%, respectively and EC50 values of 1231, 866, 3078 nM, respectively. Saturation binding studies with [3H]DAMGO showed that Salvinorin A (10 and 30 uM) decreased the mu receptor Bmax and increased the Kd in a dose-dependent non-linear manner. Saturation binding studies with [3H]diprenorphine showed that Salvinorin A (10 and 40 uM) decreased the mu receptor Bmax and increased the Kd in a dose-dependent non-linear manner. Similar findings were observed in rat brain with [3H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [3H]DAMGO and [3H]diprenorphine binding to mu receptors. Additionally, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated [35S]-GTP-g-S binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the mu opioid receptor. Rothman, R.B., Murphy, D.L., Xu H., Godin J.A., Dersch C.M., Partilla J.S., Tidgewell K. Schmidt M., Prisinzano T.E. J Pharmacol Exp Ther 320, pp. 801-810, 2007.

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

The Midbrain Raphe Nuclei Mediate Primary Reinforcement via GABA(A) Receptors Because rats learn to lever-press for brief electrical stimulation of the median and dorsal raphe nuclei (MRN and DRN, respectively), these brain sites have long been implicated in reward processes. However, it is not clear whether the MRN and DRN integrate reward-related signals or merely contain fibers of passage involved in reward processes. To shed light on this issue, the present study employed chemicals that selectively modulate neurotransmission, in particular the GABA(A) receptor agonist muscimol. Rats quickly learned to lever-press for muscimol infusions (50 and 100 microm) into the MRN or DRN. Muscimol was not self-administered when cannulae were placed just outside these nuclei. The reinforcing effects of muscimol appeared to be greater when the drug was administered into the MRN than into the DRN, as demonstrated by higher infusion rates and better response discrimination. These observations are consistent with the additional finding that muscimol administration into the MRN, but not the DRN, induced conditioned place preference. The reinforcing effects of muscimol administration into the MRN were blocked by coadministration of the GABA(A) antagonist picrotoxin (100 microm) and by pretreatment with the dopamine receptor antagonist SCH 23390 (0.025 mg/kg, i.p.). The present results suggest that median and dorsal raphe neurons presumably inhibited by muscimol via GABA(A) receptors are involved in integration of primary reinforcement, and that median raphe neurons exert tonic inhibition over dopamine-dependent reward circuitry. The midbrain raphe nuclei may be involved in a variety of reward-related phenomena including drug addiction. Liu, Z.H. and Ikemoto, S. European Journal of Neuroscience, 25, pp. 735-743, 2007.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

High Reinforcing Efficacy of Nicotine in Non-human Primates Although tobacco appears highly addictive in humans, there has been persistent controversy about the ability of its psychoactive ingredient nicotine to induce self-administration behavior in laboratory animals, bringing into question nicotine's role in reinforcing tobacco smoking. Because of ethical difficulties in inducing nicotine dependence in naive human subjects, IRP scientists explored reinforcing effects of nicotine in experimentally-naive non-human primates given access to nicotine for periods of time up to two years. Five squirrel monkeys with no experimental history were allowed to intravenously self-administer nicotine by pressing one of two levers. The number of presses on the active lever needed to obtain each injection was fixed (fixed-ratio schedule) or increased progressively with successive injections during the session (progressive-ratio schedule), allowing evaluation of both reinforcing and motivational effects of nicotine under conditions of increasing response cost. Over time, a progressive shift toward high rates of responding on the active lever, but not the inactive lever, developed. The monkeys' behavior was clearly directed toward nicotine self-administration, rather than presentation of environmental stimuli associated with nicotine injection. Both schedules of reinforcement revealed a high motivation to self-administer nicotine, with monkeys continuing to press the lever when up to 600 lever-presses were needed for each injection of nicotine. Thus, nicotine, by itself, in the absence of behavioral or drug-exposure history, is a robust and highly effective reinforcer of drug-taking behavior in a non-human primate model predictive of human behavior. This supports the use of nicotinic ligands for the treatment of smokers, and this novel preclinical model offers opportunities to test future medications for the treatment of nicotine dependence. Le Foll, B., Wertheim, C. and Goldberg, S. R. Plos One, 2, pp. e230, 2007.

Differential Glutamate-dependent and Glutamate-independent Adenosine A(1) Receptor-mediated Modulation of Dopamine Release in Different Striatal Compartments Adenosine and dopamine are two important modulators of glutamatergic neurotransmission in the striatum. However, conflicting reports exist about the role of adenosine and adenosine receptors in the modulation of striatal dopamine release. It has been previously suggested that adenosine A(1) receptors localized in glutamatergic nerve terminals indirectly modulate dopamine release, by their ability to modulate glutamate release. In the present study, using in vivo microdialysis, IRP researchers provide evidence for the existence of a significant glutamate-independent tonic modulation of dopamine release in most of the analyzed striatal compartments. In the dorsal, but not in the ventral, part of the shell of the nucleus accumbens (NAc), blockade of A(1) receptors by local perfusion with the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine or by systemic administration of the non-selective adenosine antagonist caffeine induced a glutamate-dependent release of dopamine. On the contrary, A(1) receptor blockade induced a glutamate-independent dopamine release in the core of the NAc and the nucleus caudate-putamen. Furthermore, using immunocytochemical and functional studies in rat striatal synaptosomes, the authors demonstrate that a fraction of striatal dopaminergic terminals contains adenosine A(1) receptors, which directly inhibit dopamine release independently of glutamatergic transmission. Borycz, J., Pereira, M. F., Melani, A., Rodrigues, R. J., Kofalvi, A., Panlilio, L., Pedata, F., Goldberg, S. R., Cunha, R. A. and Ferre, S. Journal of Neurochemistry, January 24, 2007, Epub ahead of print, PMID 17254024.

The Endogenous Cannabinoid Anandamide Produces THC-like Discriminative and Neurochemical Effects that are Enhanced by Inhibition of Fatty Acid Amide Hydrolase (FAAH) but Not by Inhibition of Anandamide Transport Anandamide is an endogenous ligand for brain cannabinoid CB1 receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here IRP scientists used a drug-discrimination procedure to test the hypothesis that anandamide, given intravenously (i.v.) or intraperitoneally, would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. The authors also used an in-vivo microdialysis procedure to investigate effects of anandamide, given i.v. or intraperitoneally, on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methanandamide (AM-356), a metabolically stable anandamide analog, produced clear dose-related THC-like discriminative effects, but anandamide produced THC-like discriminative effects only at a high 10 mg/kg dose that almost eliminated lever-press responding. URB-597, an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for anandamide's metabolic inactivation, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of anandamide, with 3 mg/kg anandamide completely substituting for the THC training dose. URB-597 also potentiated anandamide's ability to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of methanandamide and anandamide after URB-597 were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the anandamide transport inhibitors AM-404 and UCM-707 did not potentiate anandamide's THC-like discriminative effects or its dopamine-elevating effects. Thus, anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting brain area specificity for FAAH vs. transport/FAAH inactivation of anandamide. Solinas, M., Tanda, G., Justinova, Z., Wertheim, C.E., Yasar, S., Piomelli, D., Vadivel, S.K., Makriyannis, A. and Goldberg, S.R. Journal of Pharmacology and Experimental Therapeutics, January 8, 2007, Epub ahead of print, PMID 17210800.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

Long-term Upregulation of Protein Kinase A and Adenylate Cyclase Levels in Human Smokers Repeated injections of cocaine and morphine in laboratory rats cause a variety of molecular neuroadaptations in the cAMP signaling pathway in nucleus accumbens and ventral tegmental area. Here IRP investigators report similar neuroadaptations in postmortem tissue from the brains of human smokers and former smokers. Activity levels of two major components of cAMP signaling, cAMP-dependent protein kinase A (PKA) and adenylate cyclase, were abnormally elevated in nucleus accumbens of smokers and in ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of the catalytic subunit of PKA were correspondingly higher in the ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of other candidate neuroadaptations, including glutamate receptor subunits, tyrosine hydroxylase, and other protein kinases, were within normal range. These findings extend our understanding of addiction-related neuroadaptations of cAMP signaling to tobacco smoking in human subjects and suggest that smoking-induced brain neuroadaptations can persist for significant periods in former smokers. Hope, B.T., Nagarkar, D., Leonard, S., and Wise, R.A. Journal of Neuroscience, 27, pp. 1964-1972, 2007.

Tolerance to Opiate Reward: Role of Midbrain IRS2-Akt Pathway Addicts report that opiate drugs lose their rewarding effects over time, but the molecular mechanisms underlying this effect are unknown. A study now reports that tolerance to morphine reward in rats is due to downregulation of IRS2-Akt signaling in the ventral tegmental area (VTA), the cell body region of the mesolimbic dopamine reward system. Harvey, B.K., Hope, B.T., and Shaham, Y. Nature Neuroscience, 10, pp. 9-10, 2007.

Role of ERK in Cocaine Addiction Cocaine addiction is characterized by compulsive drug-taking behavior and high rates of relapse. According to recent theories, this addiction is due to drug-induced adaptations in the cellular mechanisms that underlie normal learning and memory. Such mechanisms involve signaling by extracellular signal-regulated kinase (ERK). As IRP scientists review here, evidence from rodent studies also implicates ERK in cocaine psychomotor sensitization, cocaine reward, consolidation and reconsolidation of memories for cocaine cues, and time-dependent increases in cocaine seeking after withdrawal (incubation of cocaine craving). The role of ERK in these behaviors involves long-term stable alterations in synaptic plasticity that result from repeated cocaine exposure, and also rapidly induced alterations in synaptic transmission events that acutely control cocaine-seeking behaviors. Pharmacological manipulations that decrease the extent to which cocaine and cocaine cues induce ERK activity might therefore be considered as potential treatments for cocaine addiction. Lu, L., Koya, E., Zhai, H., Hope, B.T., and Shaham, Y. Trends in Neurosciences, 29, pp. 695-703, 2006.

Cocaine-induced Locomotor Activity and Fos Expression in Nucleus Accumbens are Sensitized for 6 Months After Repeated Cocaine Administration Outside the Home Cage Induction of the immediate early gene protein product Fos has been used extensively to assess neural activation in the striatum after repeated cocaine administration to rats in their home cages but rarely after repeated administration outside the home cage, which produces more robust locomotor sensitization. In the present study, IRP investigators found cocaine-induced Fos expression in nucleus accumbens, but not caudate-putamen, was enhanced 1 and 6 months after repeated drug administration in locomotor activity chambers. Double-labeling of Fos protein and enkephalin mRNA indicated that Fox expression in nucleus accumbens was enhanced in enkephalin-positive, but not enkephalin-negative, medium spiny neurons. In contrast, cocaine-induced Fos expression was absent altogether in nucleus accumbens and unaltered in caudate-putamen 1 month after repeated cocaine administration in the home cage. As cocaine-induced locomotor activity was also enhanced 1 and 6 months after repeated cocaine administration in locomotor activity chambers, the authors wanted to confirm that neuronal activity in nucleus accumbens mediates cocaine-induced locomotor activity using our particular treatment regimen. Bilateral infusions of the GABA agonists baclofen and muscimol (1 microg/side) into nucleus accumbens of sensitized rats blocked cocaine-induced Fos expression and locomotor activity. Thus, while neuronal activity in both D1- and D2-type neurons in nucleus accumbens can mediate acute cocaine-induced locomotor activity, the enhanced activation of enkephalinergic D2-type neurons suggests that these latter neurons mediate the enhancement of cocaine-induced locomotor activity for up to 6 months after repeated drug administration outside the home cage. Hope, B.T., Simmons, D.E., Mitchell, T.B., Kreuter, J.D., and Mattson, B.J. European Journal of Neuroscience, 24, pp. 867-875, 2006.

Systemic and Central Amygdala Injections of the mGluR(2/3) Agonist LY379268 Attenuate the Expression of Incubation of Cocaine Craving IRP investigators and others reported time-dependent increases in cue-induced cocaine seeking after withdrawal, suggesting that craving incubates over time. Recently, the authors found that central amygdala extracellular signal-regulated kinases (ERK) and glutamate are involved in this incubation. Here, they further explored the role of central amygdala glutamate in the incubation of cocaine craving by determining the effect of systemic or central amygdala injections of the mGluR(2/3) agonist LY379268 (which decreases glutamate release) on cue-induced cocaine seeking during early and late withdrawal. Rats were trained to self-administer cocaine for 10 days (6 hours/day); infusions were paired with a tone-light cue. Cocaine seeking and craving after systemic or central amygdala injections of LY379268 were then assessed in extinction tests in the presence of the cocaine-associated cues during early (day 3) or late (day 21) withdrawal. Systemic (1.5 or 3 mg/kg) or central amygdala (.5 or 1.0 mug/side) injections of LY379268 attenuated enhanced extinction responding on day 21 but had no effect on lower extinction responding on day 3. Results confirm the authors previous findings on the role of central amygdala glutamate in the incubation of cocaine craving and together with previous reports suggest that mGluR(2/3) agonists should be considered in the treatment of drug relapse. Lu, L., Uejima, J., Gray, S., Bossert, J., and Shaham, Y. Biological Psychiatry, 61, pp. 591-598, 2007.

The Anxiogenic Drug Yohimbine Reinstates Palatable Food Seeking in a Rat Relapse Model: a Role of CRF1 Receptors The major problem in treating excessive eating is high rates of relapse to maladaptive eating habits during diet treatments; this relapse is often induced by stress or anxiety states. Preclinical studies have not explored this clinical problem. Here, IRP scientists adapted a reinstatement model (commonly used to study relapse to abused drugs) to examine the role of stress and anxiety in relapse to palatable food seeking during dieting. Rats were placed on restricted diet (75 - 80% of daily standard food) and for 12 intermittent training days (9 h/day, every other day) lever-pressed for palatable food pellets (25% fat, 48% carbohydrate) under a fixed ratio 1 (20-s timeout) reinforcement schedule. Subsequently, the rats were given 10 daily extinction sessions during which lever presses were not reinforced, and were then injected with yohimbine (an alpha-2 adrenoceptor antagonist that induces stress and anxiety in humans and non-humans) or given a single food pellet to assess reinstatement of food seeking. The rats rapidly learned to lever press for the palatable pellets and across the training days the ratio timeout nonreinforced lever presses to reinforced lever presses progressively increased more than three-fold, suggesting the development of compulsive eating behavior. After extinction, yohimbine injections and pellet priming reliably reinstated food seeking. The corticotropin-releasing factor1 (CRF1) receptor antagonist antalarmin attenuated the reinstatement induced by yohimbine, but not pellet priming. Antalarmin also reversed yohimbine's anxiogenic effects in the social interaction test. These data suggest that CRF is involved in stress-induced relapse to palatable food seeking, and that CRF1 antagonists should be considered for the treatment of maladaptive eating habits. Ghitza, U.E., Gray, S.M., Epstein, D.H., Rice, K.C., and Shaham, Y. Neuropsychopharmacology, 31, pp. 2188-2196, 2006.

Activation of Group II Metabotropic Glutamate Receptors in the Nucleus Accumbens Shell Attenuates Context-induced Relapse to Heroin Seeking Using a rat relapse model, IRP investigators previously reported that re-exposing rats to a drug-associated context, following extinction of operant responding in a different context, reinstates heroin seeking. In an initial pharmacological characterization, the authors found that the mGluR2/3 agonist LY379268, which acts centrally to reduce evoked glutamate release, attenuates context-induced reinstatement of heroin seeking when injected systemically or into the ventral tegmental area, the cell body region of the mesolimbic dopamine system. Here, the authors tested whether injections of LY379268 into the nucleus accumbens (NAc), a terminal region of the mesolimbic dopamine system, would also attenuate context-induced reinstatement of heroin seeking. Rats were trained to self-administer heroin; drug infusions were paired with a discrete tone-light cue. Subsequently, lever pressing was extinguished in the presence of the discrete cue in a context that differed from the drug self-administration context in terms of visual, auditory, tactile, and circadian cues. After extinction of responding, LY379268 was injected to different groups of rats into the NAc core or shell or into the caudate-putamen, a terminal region of the nigrastriatal dopamine system. Injections of LY379268 into the NAc shell (0.3 or 1.0 microg) dose-dependently attenuated context-induced reinstatement of heroin seeking. Injections of 1.0 microg of LY379268 into the NAc core had no effect, while a higher dose (3.0 microg) decreased this reinstatement. Injections of LY379268 (3.0 microg) 1.5 mm dorsal from the NAc core into the caudate-putamen were ineffective. Results suggest an important role of glutamate transmission in the NAc shell in context-induced reinstatement of heroin seeking. Bossert, J.M., Gray, S.M., Lu, L., and Shaham, Y. Neuropsychopharmacology, 31, pp. 2197-2209, 2006.

Nicotine Psychopharmacology Unit, Treatment Section, Clinical Pharmacology and Therapeutics Research Branch

Effect of Tobacco Deprivation on the Attentional Blink in Rapid Serial Visual Presentation When two targets are imbedded in rapid serial visual presentation (RSVP), identification of the second target (T2) is impaired if it occurs within 500 ms of the first target (T1). This attentional blink (AB) is thought to involve interference of resources in processing T1 and T2. The deleterious effect of tobacco deprivation on attention has been documented, but no studies have examined the AB. Nonsmokers (n = 30), 12-hr tobacco-deprived smokers (n = 30), and nondeprived smokers (n = 30) were randomly assigned to perform the RSVP with one of three stimulus-duration conditions (96, 113, or 130 ms). Participants completed 48 RSVP trials. Each trial consisted of 16 individually presented words (T1, T2, and 14 distractors), and T2 lagged T1 at serial positions 1 to 8. Participants verbalized T1 and T2 in order immediately after each trial. Identification of T2 (for correct T1 trials) was impaired at early vs. late lag positions, which was especially pronounced in the most difficult (96 ms) condition. There was no evidence for group differences on the AB; however, deprived smokers were worse identifying T1 in the 113-ms condition. These results suggest that the AB is influenced by stimulus duration, but not by 12 hr of tobacco deprivation. Heinz, A., Waters, A.J., Taylor, R.C., Myers, C.S., Moolchan, E.T., and Heishman, S.J. Human Psychopharmacology: Clinical and Experimental, 22, pp. 89-96, 2007.