Research Findings - Intramural Research

Biomedical Informatics Section, Administrative Management Branch

A Clinical Recruiting Management System for Complex Multi-Site Clinical Trials Using Qualification Decision Support Systems
A clinical recruiting management system implementing a qualification decision support systems was developed to increase the efficiency of screening and evaluation of participants during a recruiting process whereby recruiting for various protocols are conducted at multiple sites by different groups with process interdependencies. This system is seamlessly integrated into the authors' enterprise-scale Human Research Information System (HuRIS), encompassing research participants' electronic health records (EHR), with real-time access to the clinical trial data. Vahabzadeh, M., Lin, J.-L., Mezghanni, M., Contoreggi, C., and Leff, M. Proc. AMIA Annual Symposium on Biomedical and Health Informatics: From Foundations to Applications to Policy (AMIA 2007), pp. 1141, 2007.

Office of the Scientific Director

EEG Absolute Power during Extended Cocaine Abstinence
Cocaine causes acute changes in the human EEG, such as increased beta activity, but few studies have evaluated longitudinal changes in EEG during extended cocaine abstinence. Such changes may be relevant for assessing the neurophysiological effects of cocaine use and risk of relapse. This study evaluated EEG absolute power in 6 frequency bands in 20 adult chronic cocaine users during 3 months of monitored abstinence on a closed clinical research ward. The first EEG was recorded 0-7 weeks after their last cocaine use. Compared to non-drug using, healthy controls, the first EEG in the 8 cocaine users who had last used within 2 weeks showed decreased absolute power in most frequency bands in the left frontal brain region. During extended cocaine abstinence, absolute power among all 20 subjects increased in the beta1 frequency band in the left temporal region and in the delta frequency band in the right temporal region. These findings suggest that chronic cocaine use is associated with EEG changes that may reflect persisting brain electrophysiological abnormalities during cocaine abstinence. Levin K.H., Herning R.I., Better W.E., Epstein DH, Cadet J-L., and Gorelick D.A. Journal of Addiction Medicine, 1, pp. 139-144, 2007.

Neural Protection and Regeneration Section, Molecular Neuropsychiatry Branch

Tropism and Toxicity of Adeno-associated Viral Vector Serotypes 1, 2, 5, 6, 7, 8, and 9 in Rat Neurons and Glia in vitro
Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1 and 5 through 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using cell viability assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity. Howard, D.B., Powers, K., Wang, Y., and Harvey, B.K. Virology November 20, 2007 [Epub ahead of print].

Development and Plasticity Section, Cellular Neurobiology Research Branch

An Immortalized Rat Ventral Mesencephalic Cell Line, RTC4, is Protective in a Rodent Model of Stroke
One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, IRP researchers have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here the authors describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100beta, but not the neuronal markers NeuN, MAP2, or beta-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, the authors examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15-20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, the authors generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke. Harvey, B.K., Chen, J., Schoen, C.J., Lee, C.T., Howard, D.B., Dillon-Carter, O., Coggiano, M., Freed, W.J., Wang, Y., Hoffer, B.J., and Sanchez, J.F. Cell Transplantation, 16(5), pp. 483-491, 2007.

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

Sigma-1 Receptor Chaperones at the ER-Mitochondrion Interface Regulate Ca(2+) Signaling and Cell Survival
Communication between the endoplasmic reticulum (ER) and mitochondrion is important for bioenergetics and cellular survival. The ER supplies Ca(2+) directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). IRP scientists found here that the ER protein sigma-1 receptor (Sig-1R), which is implicated in neuroprotection, carcinogenesis, and neuroplasticity, is a Ca(2+)-sensitive and ligand-operated receptor chaperone at MAM. Normally, Sig-1Rs form a complex at MAM with another chaperone, BiP. Upon ER Ca(2+) depletion or via ligand stimulation, Sig-1Rs dissociate from BiP, leading to a prolonged Ca(2+) signaling into mitochondria via IP3Rs. Sig-1Rs can translocate under chronic ER stress. Increasing Sig-1Rs in cells counteracts ER stress response, whereas decreasing them enhances apoptosis. These results reveal that the orchestrated ER chaperone machinery at MAM, by sensing ER Ca(2+) concentrations, regulates ER-mitochondrial interorganellar Ca(2+) signaling and cell survival. Hayashi, T. and Su, T.P. Cell, 131(3), pp. 596-610, 2007.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

A Snapshot of Tissue Glycerolipids
The lipid membrane is the portal to the cell and its first line of defense against the outside world. Its plasticity, diversity and powers of accommodation in a myriad of environments, mirrored by the varied make up of the cells it protects, are unparalleled. Glycerophospholipids are one of its major components. In cell membranes the extracellular layer is mainly made up of positively charged glycolipids, while the intracellular one's main components are negatively charged. Advances in mass spectrometry have allowed the direct probing of tissues, and thus a direct approach to probing membranes make up was developed. Until recently most studies have focused on proteins. An overview of the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) for the direct analysis of phospholipids in various tissues is presented. Molecular ions corresponding to phosphatidlycholines, sphingomyelin, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols and sulfatides were mapped. Woods, A.S., Wang, H.Y., and Jackson, S.N. Current Pharmaceutical Design, 13(32), pp. 3344-3356, 2007.

A Stargardt Disease-3 Mutation in the Mouse Elovl4 Gene Causes Retinal Deficiency of C32-C36 Acyl Phosphatidlycholines
Stargardt disease-3 (STGD3) is a juvenile dominant macular degeneration caused by mutations in elongase of very long chain fatty acid-4. All identified mutations produce a truncated protein which lacks a motif for protein retention in endoplasmic reticulum, the site of fatty acid synthesis. In these studies of Stgd3-knockin mice carrying a human pathogenic mutation, IRP investigators examined two potential pathogenic mechanisms: truncated protein-induced cellular stress and lipid product deficiency. Analysis of mutant retinas detected no cellular stress but demonstrated selective deficiency of C32-C36 acyl phosphatidylcholines. The authors conclude that this deficit leads to the human STGD3 pathology. McMahon, A., Jackson, S.N., Woods, A.S., and Kedzierski, W. FEBS Letters, 581(28), pp. 5459-5463, 2007.

Adenosine Receptor Heteromers and their Integrative Role in Striatal Function
By analyzing the functional role of adenosine receptor heteromers, IRP scientists review a series of new concepts that should modify our classical views of neurotransmission in the central nervous system (CNS). Neurotransmitter receptors cannot be considered as single functional units anymore. Heteromerization of neurotransmitter receptors confers functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Some of these characteristics can be used as a "biochemical fingerprint" to identify neurotransmitter receptor heteromers in the CNS. This is exemplified by changes in binding characteristics that are dependent on coactivation of the receptor units of different adenosine receptor heteromers. Neurotransmitter receptor heteromers can act as "processors" of computations that modulate cell signaling, sometimes critically involved in the control of pre- and postsynaptic neurotransmission. For instance, the adenosine A1-A2A receptor heteromer acts as a concentration-dependent switch that controls striatal glutamatergic neurotransmission. Neurotransmitter receptor heteromers play a particularly important integrative role in the "local module" (the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit), where they act as processors mediating computations that convey information from diverse volume-transmitted signals. For instance, the adenosine A2A-dopamine D2 receptor heteromers work as integrators of two different neurotransmitters in the striatal spine module. Ferre, S., Ciruela, F., Quiroz, C., Lujan, R., Popoli, P., Cunha, R.A., Agnati, L.F., Fuxe, K., Woods, A.S., Lluis, C., and Franco, R. Scientific World Journal, 7, pp. 74-85, 2007.

Functional Relevance of Neurotransmitter Receptor Heteromers in the Central Nervous System
The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their functional significance is being revealed. Heteromerization of neurotransmitter receptors produces functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Neurotransmitter receptor heteromers can function as processors of computations that modulate cell signaling. Thus, the quantitative or qualitative aspects of the signaling generated by stimulation of any of the individual receptor units in the heteromer are different from those obtained during coactivation. Furthermore, recent studies demonstrate that some neurotransmitter receptor heteromers can exert an effect as processors of computations that directly modulate both pre- and postsynaptic neurotransmission. This is illustrated by the analysis of striatal receptor heteromers that control striatal glutamatergic neurotransmission. Ferre, S., Ciruela, F., Woods, A.S., Lluis, C., and Franco, R. Trends in Neuroscience, 30(9), pp. 440-446, 2007.

Electrophysiology Unit, Cellular Neurophysiology Section

The Endocannabinoid Anandamide Inhibits the Function of Alpha4beta2 Nicotinic Acetylcholine Receptors
The effects of the endocannabinoid anandamide (arachidonylethanolamide, AEA) on the function of alpha4beta2 nicotinic acetylcholine receptors (nAChR) stably expressed in SH-EP1 cells were investigated using the whole-cell patch-clamp technique. In the concentration range of 200 nM to 2 microM, AEA significantly reduced the maximal amplitudes and increased the desensitization of acetylcholine (ACh)-induced currents. The effects of AEA could be neither replicated by the exogenous cannabinoid Delta(9)-tetrahydrocannabinol (1 microM) nor reversed by the selective CB1 receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (SR-141716A) (1 microM). The actions of AEA were apparent when applied extracellularly but not during intracellular dialysis. Furthermore, the effects of AEA ACh currents were not altered by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The onset and washout of the AEA effects required several minutes (10-30 min), but the latter was significantly decreased in the presence of lipid-free bovine serum albumin (BSA). Moreover, BSA alone increased peak ACh current amplitudes and diminished desensitization rates in naive cells, suggesting a tonic modulation of alpha4beta2 nAChR function by an endogenous AEA-like lipid. Further analysis of AEA effects on alpha4beta2 nAChR-mediated currents, using a two-stage desensitization model, indicated that the first forward rate constant leading to desensitization, k(1), increased nearly 30-fold as a linear function of the AEA concentration. In contrast, the observation that the other three rate constants were unaltered by AEA suggested that AEA raised the energy of the activated state. These results indicate that AEA directly inhibits the function of alpha4beta2 nAChRs in a CB1 receptor-independent manner. Spivak, C.E., Lupica, C.R., and Oz, M. Molecular Pharmacology, 72(4), pp. 1024-1032, 2007.

Medicinal Chemistry Section, Medications Discovery Research Branch

Novel Dopamine D3 Receptor Ligands: Potential Substance Abuse Therapeutic Agents
Dopamine D3 receptor antagonists and partial agonists have been shown to modulate drug-seeking effects induced by cocaine and other abused substances. IRP scientists have recently discovered a potent and selective dopamine D3 receptor antagonist, PG01037, and related analogues that are currently being evaluated in animal models of drug addiction. In these studies, a discrepancy between in vitro binding affinity, in vivo occupancy and behavioral potency has been observed. The purpose of this study was to examine 1) modifications of the 2-pyridylphenyl moiety of PG01037 and 2) incorporate hydroxyl, acetyl and cyclopropyl substitutions on the butyl amide linking-chain systematically coupled with 2-fluorenylamide or 2-pyridylphenyl amide and 2-methoxy or 2,3-dichloro-substituted phenylpiperazines to measure impact on binding affinity, D2/D3 selectivity, lipophilicity and function. In general, these modifications were well tolerated at the human dopamine D3 (hD3) receptor (Ki=1-5 nM) as measured in competition binding assays. Several analogues showed >100-fold selectivity for dopamine D3 over D2 and D4 receptors. In addition, while all the derivatives with an olefinic linker were antagonists, in quinpirole-stimulated mitogenesis at hD3 receptors, several of the hydroxy-butyl-linked analogues showed partial agonist activity. Finally, several structural modifications reduced lipophilicities while retaining the desired binding profile. These compounds will provide novel tools with which to further elucidate the role of dopamine D3 receptors in drug abuse, in vivo, and may serve as leads for therapeutic agents for the treatment of addiction. Grundt, P., Prevatt, K.M., Cao, J., Taylor, J., Floresca, C.Z., Choi, J.-K., Jenkins, B.G., Luedtke, R.R., and Newman, A.H. Journal of Medicinal Chemistry, 50, pp. 4135-4146, 2007.

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Chronic Fenfluramine Administration Increases Plasma Serotonin (5-HT) to Non-Toxic Levels
Large elevations in blood serotonin (5-HT) can produce valvular heart disease in humans and laboratory animals. Accordingly, one prevailing hypothesis (i.e., the "5-HT hypothesis") suggests 5-HT transporter substrates like fenfluramine increase the risk for valvular heart disease by elevating plasma 5-HT, secondary to the release of 5-HT from platelets. The main purpose of this study was to determine if chronic administration of fenfluramine increases plasma 5-HT to concentrations that are associated with the development of valvular heart disease. To the best of the study authors' knowledge, this is the first study to address this issue using an in vivo microdialysis method that measures plasma 5-HT in non-hypoxic rats. They examined the effects of chronic (+/-)-fenfluramine and fluoxetine on plasma levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in blood samples from conscious catheterized rats. Plasma indoles were measured by HPLC-ECD in dialysates of whole blood. Baseline plasma 5-HT was < 1.0 nM. Chronic fenfluramine (14-day minipump infusion) produced small increases in baseline plasma 5-HT (~2-to-4-fold), while chronic fluoxetine had no effect. Chronic fenfluramine and fluoxetine markedly decreased whole blood 5-HT, and reduced the ability of acute fenfluramine to evoke 5-HT release. Elevations in baseline plasma 5-HT produced by chronic fenfluramine are far below M levels necessary to produce valvular heart disease. Furthermore, chronic fenfluramine reduces the ability of acute fenfluramine to increase plasma 5-HT, suggesting the "5-HT hypothesis" can not explain the increased risk of valvular heart disease in patients treated with fenfluramine. Zolkowska, D., Baumann, M.H., and Rothman, R.B. J.Pharmacol.Exp.Ther. Nov. 21, 2007 (e-pub ahead of print).

Dopamine Transport Inhibitors Based on GBR12909 and Benztropine as Potential Medications to Treat Cocaine Addiction
The discovery and development of medications to treat addiction and notably, cocaine addiction, have been frustrated by both the complexity of the disorder and the lack of target validation in human subjects. The dopamine transporter has historically been a primary target for cocaine abuse medication development, but addictive liability and other confounds of such inhibitors of dopamine uptake have limited clinical evaluation and validation. Herein IRP researchers describe efforts to develop analogues of the dopamine uptake inhibitors GBR 12909 and benztropine that show promising profiles in animal models of cocaine abuse that contrast to that of cocaine. Their unique pharmacological profiles have provided important insights into the reinforcing actions of cocaine and the authors propose that clinical investigation of novel dopamine uptake inhibitors will facilitate the discovery of cocaine-abuse medications. Rothman, R.B., Baumann, M.H., Prisinzano, T.E., and Newman, A.H. Biochemical Pharmacology August 9, 2007 (e-pub ahead of print).

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

A Role for Conditioned Ventral Tegmental Glutamate Release in Cocaine Seeking
Initiation of cocaine self-administration in rats was associated with release of glutamate in the ventral tegmental area (VTA). The glutamate release was transient, despite continued cocaine intake. Similar glutamate release was seen in rats earning, for the first time, unexpected saline rather than expected cocaine. VTA glutamate release was not seen in similarly trained rats earning saline instead of cocaine for the 13th time. VTA glutamate release was also seen in similarly trained rats that received yoked rather than earned cocaine injections on test day. VTA glutamate release was not seen in a group of rats that had never earned cocaine but had received yoked injections during the training period. Glutamate release was also not seen in a group of rats that received yoked injections but had no previous experience with cocaine. VTA GABA levels did not fluctuate during any aspect of cocaine seeking. Blockade of VTA glutamate receptors appeared to attenuate the rewarding effects of intravenous cocaine injections and blocked almost completely the conditioned responding normally seen during extinction trials. These findings indicate that VTA glutamate release is a conditioned response dependent on an associative process and is not a simple consequence of previous cocaine exposure. The findings implicate glutamate as at least one of the sources of VTA signals from reward-associated environmental stimuli. You, Z.B., Wang, B., Zitzman, D., Azari, S., and Wise, R.A. Journal of Neuroscience, 27, pp. 10546-10555, 2007.

Dopamine Reward Circuitry: Two Projection Systems from the Ventral Midbrain to the Nucleus Accumbens-Olfactory Tubercle Complex
Anatomical and functional refinements of the mesolimbic dopamine system of the rat are discussed. Present experiments suggest that dopaminergic neurons localized in the posteromedial ventral tegmental area (VTA) and central linear nucleus raphe selectively project to the ventromedial striatum (medial olfactory tubercle and medial nucleus accumbens shell), whereas the anteromedial VTA has few if any projections to the ventral striatum, and the lateral VTA largely projects to the ventrolateral striatum (accumbens core, lateral shell and lateral tubercle). These findings complement the recent behavioral findings that cocaine and amphetamine are more rewarding when administered into the ventromedial striatum than into the ventrolateral striatum. Drugs such as nicotine and opiates are more rewarding when administered into the posterior VTA or the central linear nucleus than into the anterior VTA. A review of the literature suggests that (1) the midbrain has corresponding zones for the accumbens core and medial shell; (2) the striatal portion of the olfactory tubercle is a ventral extension of the nucleus accumbens shell; and (3) a model of two dopamine projection systems from the ventral midbrain to the ventral striatum is useful for understanding reward function. The medial projection system is important in the regulation of arousal characterized by affect and drive and plays a different role in goal-directed learning than the lateral projection system, as described in the variation-selection hypothesis of striatal functional organization. Ikemoto, S. Brain Research Reviews, 56, pp. 27-78, 2007.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

Differential Effects of Blockade of Dopamine D1-family Receptors in Nucleus Accumbens Core or Shell on Reinstatement of Heroin-Seeking Induced by Contextual and Discrete Cues
In humans, exposure to environmental contexts previously associated with heroin intake can provoke drug relapse, but the neuronal mechanisms mediating this relapse are unknown. Using a drug relapse model, IRP scientists found previously that re-exposing rats to heroin-associated contexts, after extinction of drug-reinforced responding in different contexts, reinstates heroin seeking. This effect is attenuated by inhibition of glutamate transmission in the ventral tegmental area and medial accumbens shell, components of the mesolimbic dopamine system. Here, these investigators explored the role of dopamine of the accumbens in context-induced reinstatement by using the D1-family receptor antagonist SCH 23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride]. Rats were trained to self-administer heroin for 12 d; drug infusions were paired with a discrete tone-light cue. Subsequently, the heroin-reinforced 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. When tested in the original drug self-administration context, systemic and medial or lateral accumbens shell SCH 23390 injections attenuated context-induced reinstatement of heroin seeking, whereas accumbens core SCH 23390 injections were ineffective. In contrast, core but not lateral or medial shell SCH 23390 injections attenuated discrete-cue-induced reinstatement in a non-drug context after extinction of lever presses without this cue. Results indicate that activation of medial and lateral accumbens shell D1-family dopamine receptors mediate context-induced reinstatement of heroin seeking and provide the first demonstration for a role of lateral shell dopamine in conditioned drug effects. Results also demonstrate novel dissociable roles of accumbens core and shell in context- versus discrete-cue-induced reinstatement of heroin seeking. Bossert, J.M., Poles, G.C., Wihbey, K.A., Koya, E. and Shaham, Y. Journal of Neuroscience, 27, pp. 12655-12663, 2007.

Peptide YY3-36 Decreases Reinstatement of High-fat Food Seeking During Dieting in a Rat Relapse Model
A major problem in treating obesity is high rates of relapse to maladaptive food-taking habits during dieting. This relapse is often provoked by acute re-exposure to palatable food, food-associated cues, or stress. IRP researchers used a reinstatement model, commonly used to study relapse to abused drugs, to explore the effect of peptide YY3-36 (PYY3-36) on reinstatement of high-fat (35%, 45 mg pellets) food seeking induced by acute exposure to the pellets (pellet priming), a cue previously associated with pellet delivery (pellet cue), or yohimbine (2 mg/kg, a pharmacological stressor). Rats were placed on a restricted diet (16 g of chow per day) and lever-pressed for the pellets for 9-12 sessions (6 h/d, every 48 h); pellet delivery was paired with a tone-light cue. They were then given 10-20 extinction sessions wherein lever presses were not reinforced with the pellets and subsequently tested for reinstatement of food seeking. Systemic PYY3-36 injections (100-200 micro g/kg) decreased pellet priming- and pellet cue-induced reinstatement of food seeking but not yohimbine-induced reinstatement. Arcuate nucleus (Arc) injections of PYY3-36 (0.4 microg per side) decreased pellet priming-induced reinstatement. The attenuation of pellet priming-induced reinstatement by systemic PYY3-36 was reversed by systemic (2 mg/kg) but not Arc (0.5 microg per side) injections of the Y2 receptor antagonist BIIE0246. Arc PYY3-36 injections did not decrease pellet cue-induced reinstatement. Finally, systemic PYY3-36 injections had minimal effects on ongoing food self-administration or heroin priming- or heroin cue-induced reinstatement of heroin seeking. These data identify an effect of systemic PYY3-36 on relapse to food seeking that is independent of Y2 receptor activation in Arc and suggest that PYY3-36 should be considered for the treatment of relapse to maladaptive food-taking habits during dieting. Ghitza, U.E., Nair, S.G., Golden, S.A., Gray, S.M., Uejima, J.L., Bossert, J.M., and Shaham, Y. Journal of Neuroscience, 27, pp. 11522-11532, 2007.

Repeated Amphetamine Administration Outside the Home Cage Enhances Drug-induced Fos Expression in Rat Nucleus Accumbens
Induction of the immediate early gene protein product Fos has been used extensively to assess neural activation in the striatum after repeated amphetamine administration to rats in their home cages. However, this technique has not been used to examine striatal activation after repeated administration outside the home cage, an environment where repeated drug administration produces more robust psychomotor sensitization. IRP scientists determined the dose-response relationship for amphetamine-induced psychomotor activity and Fos expression in nucleus accumbens and caudate-putamen 1 week after repeated administration of amphetamine or saline in locomotor activity chambers. Repeated administration of amphetamine enhanced amphetamine-induced locomotor activity and stereotypy and Fos expression in nucleus accumbens, but not in caudate-putamen. In comparison, levels of Fos expression induced by 1mg/kg amphetamine were not altered in nucleus accumbens or caudate-putamen by repeated amphetamine administration in the home cage. Double-labeling of Fos protein and enkephalin mRNA indicates that Fos is expressed in approximately equal numbers of enkephalin-negative and enkephalin-positive neurons in nucleus accumbens and caudate-putamen following injections outside the home cage. Furthermore, repeated amphetamine administration increased drug-induced Fos expression in enkephalin-positive, but not enkephalin-negative, neurons in nucleus accumbens. The authors conclude that repeated amphetamine administration outside the home cage recruits the activation of enkephalin-containing nucleus accumbens neurons during sensitized amphetamine-induced psychomotor activity. Mattson, B.J., Crombag, H.S., Mitchell, T., Simmons, D.E., Kreuter, J.D., Morales, M. and Hope, B.T. Behavioural Brain Research, 185, pp. 88-98, 2007.

In Vivo Electrophysiology Unit, Behavioral Neuroscience Research Branch

I.V. Cocaine Induces Rapid, Transient Excitation of Striatal Neurons via its Action on Peripheral Neural Elements: Single-cell, Iontophoretic Study in Awake and Anesthetized Rats
Cocaine's (COC) direct interaction with the dopamine (DA) transporter is usually considered the most important action underlying the psychomotor stimulant and reinforcing effects of this drug. However, some physiological, behavioral and psycho-emotional effects of COC are very rapid and brief and they remain intact during DA receptor blockade, suggesting possible involvement of peripheral non-DA neural mechanisms. To assess this issue, single-unit recording with microiontophoresis was used to examine changes in impulse activity of dorsal and ventral striatal neurons to i.v. COC (0.25-0.5 mg/kg) in the same rats under two conditions: awake with DA receptor blockade and anesthetized with urethane. In the awake preparation approximately 70% striatal neurons showed rapid and transient (latency approximately 6 s, duration approximately 15 s) COC-induced excitations. These effects were stronger in ventral than dorsal striatum. During anesthesia, these phasic effects were fully blocked and COC slowly decreased neuronal discharge rate. Cocaine-methiodide (COC-M), a derivative that cannot cross the blood-brain barrier, also caused phasic excitations in the awake, but not anesthetized condition. In contrast to regular COC, COC-M had no tonic effect on discharge rate in either preparation. Most striatal neurons that were phasically excited by both COC forms also showed short-latency excitations during tail-touch and tail-pinch in the awake preparation, an effect strongly attenuated during anesthesia. Finally, most striatal neurons that in awake conditions were phasically excited by somato-sensory stimuli and COC salts were also excited by iontophoretic glutamate (GLU). Although striatal neurons were sensitive to GLU in both preparations, the response magnitude at the same GLU current was higher in awake than anesthetized conditions. These data suggest that in awake animals, i.v. COC, like somato-sensory stimuli, transiently excites striatal neurons via its action on peripheral neural elements and rapid neural transmission. While the nature of these neuronal elements needs to be clarified using other analytical techniques, they might involve voltage-gated K(+) and Na(+) channels, which have a high affinity for COC and are located on terminals of visceral sensory nerves that densely innervate peripheral vessels. Therefore, along with direct action on specific brain substrates, central excitatory effects of COC may occur via indirect action, involving afferents of visceral sensory nerves and rapid neural transmission. By providing a rapid sensory signal and triggering transient neural activation, such a peripherally triggered action might play a crucial role in the sensory effects of COC, thus contributing to learning and development of drug-taking behavior. Kiyatkin, E.A. and Brown, P.L. Neuroscience, 148, pp. 978-995, 2007.

Brain Edema and Breakdown of the Blood-brain Barrier during Methamphetamine Intoxication: Critical Role of Brain Hyperthermia
To clarify the role of brain temperature in permeability of the blood-brain barrier (BBB), rats were injected with methamphetamine (METH 9 mg/kg) at normal (23 degrees C) and warm (29 degrees C) environmental conditions and internal temperatures were monitored both centrally (nucleus accumbens, NAcc) and peripherally (skin and nonlocomotor muscle). Once NAcc temperatures peaked or reached 41.5 degrees C (a level suggesting possible lethality), animals were administered Evans blue dye (protein tracer that does not normally cross the BBB), rapidly anaesthetized, perfused and had their brains removed. All METH-treated animals showed brain and body hyperthermia associated with relative skin hypothermia, suggesting metabolic activation coupled with peripheral vasoconstriction. While METH-induced NAcc temperature elevation varied from 37.60 to 42.46 degrees C (or 1.2-5.1 degrees C above baseline), it was stronger at 29 degrees C (+4.13 degrees C) than 23 degrees C (+2.31 degrees C). Relative to control, METH-treated animals had significantly higher brain levels of water, Na(+), K(+) and Cl(-), suggesting brain edema, and intense immunostaining for albumin, indicating breakdown of the BBB. METH-treated animals also showed strong immunoreactivity for glial fibrillary acidic protein (GFAP), possibly suggesting acute abnormality or damage of astrocytes. METH-induced changes in brain water, albumin and GFAP correlated linearly with NAcc temperature (r = 0.93, 0.98 and 0.98, respectively), suggesting a key role of brain hyperthermia in BBB permeability, development of brain edema and subsequent functional and structural neural abnormalities. Therefore, along with a direct destructive action on neural cells and functions, brain hyperthermia, via breakdown of the BBB, may be crucial for both decompensation of brain functions and cell injury following acute METH intoxication, possibly contributing to neurodegeneration resulting from chronic drug use. Kiyatkin, E.A., Brown, P.L., and Sharma, H.S. European Journal of Neuroscience, 26, pp. 1242-1253, 2007.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Endocannabinoid System Involvement in Brain Reward Processes Related to Drug Abuse
Cannabis is the most commonly abused illegal drug in the world and its main psychoactive ingredient, delta-9-tetrahydrocannabinol (THC), produces rewarding effects in humans and non-human primates. Over the last several decades, an endogenous system comprised of cannabinoid receptors, endogenous ligands for these receptors and enzymes responsible for the synthesis and degradation of these endogenous cannabinoid ligands has been discovered and partly characterized. Experimental findings strongly suggest a major involvement of the endocannabinoid system in general brain reward functions and drug abuse. First, natural and synthetic cannabinoids and endocannabinoids can produce rewarding effects in humans and laboratory animals. Second, activation or blockade of the endogenous cannabinoid system has been shown to modulate the rewarding effects of non-cannabinoid psychoactive drugs. Third, most abused drugs alter brain levels of endocannabinoids in the brain. In addition to reward functions, the endocannabinoid cannabinoid system appears to be involved in the ability of drugs and drug-related cues to reinstate drug-seeking behavior in animal models of relapse. Altogether, evidence points to the endocannabinoid system as a promising target for the development of medications for the treatment of drug abuse. Solinas, M., Yasar, S. and Goldberg, S.R. Pharmacological Research, 56, pp. 393-405, 2007.

A Stimulus-control Account of Regulated Drug Intake in Rats
Patterns of drug self-administration are often highly regular, with a consistent pause after each self-injection. This pausing might occur because the animal has learned that additional injections are not reinforcing once the drug effect has reached a certain level, possibly due to the reinforcement system reaching full capacity. Thus, interoceptive effects of the drug might function as a discriminative stimulus, signaling when additional drug will be reinforcing and when it will not. This hypothetical stimulus control aspect of drug self-administration was emulated using a schedule of food reinforcement. Rats' nose-poke responses produced food only when a cue light was present. No drug was administered at any time. However, the state of the light stimulus was determined by calculating what the whole-body drug level would have been if each response in the session had produced a drug injection. The light was only presented while this virtual drug level was below a specific threshold. A range of doses of cocaine and remifentanil were emulated using parameters based on previous self-administration experiments. Response patterns were highly regular, dose-dependent, and remarkably similar to actual drug self-administration. This similarity suggests that the emulation schedule may provide a reasonable model of the contingencies inherent in drug reinforcement. Thus, these results support a stimulus control account of regulated drug intake in which rats learn to discriminate when the level of drug effect has fallen to a point where another self-injection will be reinforcing. Panlilio L.V., Thorndike, E.B. and Schindler, C.W. Psychopharmacology (Berl), October 24, 2007 (e-pub ahead of print).

Differential Involvement of Dopamine Receptors in Conditioned Suppression Induced by Cocaine
Cocaine-paired stimuli can suppress food-reinforced operant behavior in rats, providing an animal model of conditioned drug effects. To study the neuropharmacological basis of this phenomenon, IRP researchers examined the effects of various dopamine receptor antagonists on the acquisition and expression of cocaine-induced conditioned suppression in rats. Superimposed on an ongoing baseline of food-reinforced operant responding, a stimulus was paired with response-independent cocaine (3.0 mg/kg, i.v.) during each of 8 training sessions. To study acquisition, independent groups of rats were given saline, the dopamine D(1)-like receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390) (0.001-0.03 mg/kg, i.p.), or the dopamine D(2)-like receptor antagonist eticlopride (0.001-0.03 mg/kg, i.p.) prior to each training session. To study expression, independent groups of rats were trained first, then given saline, SCH 23390, eticlopride, or N-[4-(4-(2-methoxyphenyl) piperazinyl)butyl]-2-naphthamide (BP 897) (a dopamine D(3) partial receptor agonist; 0.1-1.0 mg/kg, i.p.) before test sessions in which the stimulus was presented without cocaine. Pre-treatment with either SCH 23390 or eticlopride during acquisition reduced the direct suppressant effects of cocaine, but conditioning was blocked only in rats that were treated with SCH 23390 during acquisition training. Expression of conditioning was attenuated only by eticlopride. Thus, dopamine at least partially mediates both the acquisition and expression of cocaine-induced conditioned suppression, with activation of dopamine D(1)- and D(2)-like receptors underlying these respective processes. Grakalic, I., Panlilio, L.V., Thorndike, E.B., and Schindler, C.W. European Journal of Pharmacology, 573, pp. 116-123, 2007.

Self-administration of Drugs in Animals and Humans as a Model and an Investigative Tool
The aim of this research was to review briefly the methods, assumptions, models, accomplishments, drawbacks and future directions of research using drug self-administration in animals and humans. The use of drug self-administration to study addiction is based on the assumption that drugs reinforce the behavior that results in their delivery. A wide range of drug self-administration techniques have been developed to model specific aspects of addiction. These techniques are highly amenable to being combined with a wide variety of neuroscience techniques. The identification of drug use as behavior that is reinforced by drugs has contributed greatly to the understanding and treatment of addiction. As part of a program of pre-clinical research that also involves screening with a variety of simpler behavioral techniques, drug self-administration procedures can provide an important last step in testing potential treatments for addiction. There is currently a concerted effort to develop self-administration procedures that model the extreme nature of the behavior engendered by addiction. As advances continue to be made in neuroscience techniques, self-administration should continue to provide a means of applying these techniques within a sophisticated and valid model of human drug addiction. Panlilio, L. and Goldberg, S.R. Addiction, 102, pp. 1863-1870, 2007.

Adenosine A1-A2A Receptor Heteromers: New Targets for Caffeine in the Brain
The contribution of blockade of adenosine A1 and A2A receptor to the psychostimulant effects of caffeine is still a matter of debate. When analyzing motor activity in rats, acutely administered caffeine shows a profile of a non-selective adenosine receptor antagonist, although with preferential A1 receptor antagonism. On the other hand, tolerance to the effects of A1 receptor blockade seems to be mostly responsible for the tolerance to the motor-activating effects of caffeine, while the residual motor-activating effects of caffeine in tolerant individuals seem to involve A2A receptor blockade. These behavioral studies correlate with in vivo microdialysis experiments that suggest that A1 receptor-mediated modulation of striatal glutamate release is involved in the psychostimulant effects of caffeine. Experiments in transfected cells demonstrate the ability of A1 receptors to heteromerize with A2A receptors and the A1-A2A receptor heteromer can be biochemically identified in the striatum, in striatal glutamatergic terminals. The striatal A1-A2A receptor heteromer provides a "concentration-dependent switch" mechanism by which low and high concentrations of synaptic adenosine produce the opposite effects on glutamate release. The analysis of the function of A1-A2A receptor heteromers during chronic treatment with caffeine gives new clues about the well-known phenomenon of tolerance to the psychostimulant effects of caffeine. Ferre, S., Ciruela, F., Borycz, J., Solinas, M., Quarta, D., Antoniou, K., Quiroz, C., Justinova, Z., Lluis, C., Franco, R. and Goldberg, S.R. Front Bioscience, 13, pp. 2391-2399, 2008.

The Endogenous Cannabinoid Anandamide has Effects on Motivation and Anxiety that are Revealed by Fatty Acid Amide Hydrolase (FAAH) Inhibition
Converging evidence suggests that the endocannabinoid system is an important constituent of neuronal substrates involved in brain reward processes and emotional responses to stress. Here, IRP researchers evaluated motivational effects of intravenously administered anandamide, an endogenous ligand for cannabinoid CB1-receptors, in Sprague-Dawley rats, using a place-conditioning procedure in which drugs abused by humans generally produce conditioned place preferences (reward). Anandamide (0.03-3mg/kg intravenous) produced neither conditioned place preferences nor aversions. However, when rats were pre-treated with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester; 0.3mg/kg intraperitoneal), which blocks anandamide's metabolic degradation, anandamide produced dose-related conditioned place aversions. In contrast, URB597 alone showed no motivational effects. Like URB597 plus anandamide, the synthetic CB1-receptor ligand WIN 55,212-2 (50-300mug/kg, intravenous) produced dose-related conditioned place aversions. When anxiety-related effects of anandamide and URB597 were evaluated in a light/dark box, both a low anandamide dose (0.3mg/kg) and URB597 (0.1 and 0.3mg/kg) produced anxiolytic effects when given alone, but produced anxiogenic effects when combined. A higher dose of anandamide (3mg/kg) produced anxiogenic effects and depressed locomotor activity when given alone and these effects were potentiated after URB597 treatment. Finally, anxiogenic effects of anandamide plus URB597 and development of place aversions with URB597 plus anandamide were prevented by the CB1-receptor antagonist AM251 (3mg/kg intraperitoneal). Thus, additive interactions between the effects of anandamide on brain reward processes and on anxiety may account for its aversive effects when intravenously administered during FAAH inhibition with URB597. Scherma, M., Medalie, J., Fratta, W., Vadivel, S. K., Makriyannis, A., Piomelli, D., Mikics, E., Haller, J., Yasar, S., Tanda, G. and Goldberg, S. R. Neuropharmacology, August 19, 2007 (e-pub ahead of print).

Effects of Kappa Opioid Agonists Alone and in Combination with Cocaine on Heart Rate and Blood Pressure in Conscious Squirrel Monkeys
As kappa agonists have been proposed as treatments for cocaine abuse, the cardiovascular effects of the kappa opioid receptor agonists ethylketocyclazocine (EKC) and enadoline were investigated in conscious squirrel monkeys. Both EKC and enadoline increased heart rate with little effect on blood pressure. This effect appeared to be specific for kappa receptors as the mu opioid agonist morphine did not mimic the effects of the kappa agonists. The opioid antagonist naltrexone, at a dose of 1.0 mg/kg, blocked the effect of EKC. An action at both central and peripheral receptors may be responsible for the heart rate increase following kappa agonist treatment. The ganglionic blocker chlorisondamine partially antagonized the effect of EKC on heart rate, suggesting central involvement, while the peripherally-acting agonist ICI 204,448 ((+/-)-1-[2,3- (Dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) also increased heart rate, supporting a peripheral site of action. When given in combination with cocaine, EKC produced effects that were sub-additive, suggesting that the kappa agonists may be used safely as cocaine abuse treatments. Schindler, C.W., Graczyk, Z., Gilman, J.P., Negus, S.S., Bergman, J., Mello, N.K. and Goldberg, S.R. European Journal Pharmacology, 576, pp. 107-113, 2007.

Validation of an Extracerebral Reference Region Approach for the Quantification of Brain Nicotinic Acetylcholine Receptors in Squirrel Monkeys with PET and 2-18F-flour-A-85380
The aim of the present study was to explore the applicability of an extracerebral reference region for the quantification of cerebral receptors with PET. Male squirrel monkeys underwent quantitative PET studies of cerebral nicotinic acetylcholine receptors (nAChRs) with 2-(18)F-fluoro-A-85380 (2-FA). Data from dynamic PET scans were analyzed with various compartment- and non-compartment-based models, including a simplified reference tissue model (SRTM). Nondisplaceable volume-of-distribution (VDnd) values were determined in regions of interest after the blockade of 2-FA-specific binding by nicotine infusion. Binding potential values, estimated with the cerebellum and muscle as reference regions, were compared and the reproducibility of measurements was determined. One- and 2-tissue-compartment modeling and linear graphic analysis provided similar total volume-of-distribution (VD(T)) values for each studied region. VD(T) values were high in the thalamus, intermediate in the cortex and midbrain, and low in the cerebellum and muscle, consistent with the distribution pattern of nAChR containing alpha(4) and beta(2) receptor subunits (alpha(4)beta(2)*). The administration of nicotine at 2 mg/kg/d via an osmotic pump resulted in a nearly complete saturation of 2-FA-specific binding and led to very small changes in volumes of distribution in the cerebellum and muscle (-9% +/- 4% [mean +/- SEM] and 0% +/- 6%, respectively), suggesting limited specific binding of the radioligand in these areas. VD(T) measured in muscle in 15 monkeys was reasonably constant (3.0 +/- 0.2, with a coefficient of variation of 8%). VDnd in studied brain regions exceeded VD(T) in muscles by a factor of 1.3. With this factor and with muscle as a reference region, BP* values calculated for studied brain regions with the SRTM were in good agreement with those obtained with the cerebellum as a reference region. Significant correlations were observed between BP* values estimated with these 2 approaches. The reproducibilities of BP* measurements obtained with the 2 methods were comparable, with coefficients of variation of less than 11% and 13% for the thalamus and the cortex, respectively. These results suggest that the accurate quantification of nAChRs can be performed with 2-FA and a reference region outside the brain, providing a novel approach for the quantification of brain receptors when no suitable cerebral reference region is available. LeFoll, B., Chefer, S.I., Kimes, A.S., Shumway, D., Goldberg, S.R., Stein, E.A. and Mukhin, A.G. Journal Nuclear Medicine, 48, pp. 1492-1500, 2007.

G-protein-coupled Receptor Heteromers: Function and Ligand Pharmacology
Almost all existing models for G-protein-coupled receptors (GPCRs) are based on the occurrence of monomers. Recent studies show that many GPCRs are dimers. Therefore for some receptors dimers and not monomers are the main species interacting with hormones/neurotransmitters/drugs. There are reasons for equivocal interpretations of the data fitting to receptor dimers assuming they are monomers. Fitting data using a dimer-based model gives not only the equilibrium dissociation constants for high and low affinity binding to receptor dimers but also a 'cooperativity index' that reflects the molecular communication between monomers within the dimer. The dimer cooperativity index (D(C)) is a valuable tool that enables to interpret and quantify, for instance, the effect of allosteric regulators. For different receptors heteromerization confers a specific functional property for the receptor heteromer that can be considered as a 'dimer fingerprint'. The occurrence of heteromers with different pharmacological and signalling properties opens a complete new field to search for novel drug targets useful to combat a variety of diseases and potentially with fewer side effects. Antagonists, which are quite common marketed drugs targeting GPCRs, display variable affinities when a given receptor is expressed with different heteromeric partners. This fact should be taken into account in the development of new drugs. Franco, R., Casado, V., Cortes, A., Mallol, J., Ciruela, F., Ferre, S., Lluis, C. and Canela, E.I. British Journal Pharmacology, November 26, 2007 (e-pub ahead of print).

Reduction of Cocaine Seeking by a Food-based Inhibitor in Rats
Environmental stimuli can exert a powerful influence over drug seeking and taking. For example, previous experiments found that combining multiple drug-related stimuli tripled drug seeking and doubled drug intake (L.V. Panlilio, S.J. Weiss, & C.W. Schindler, 1996, 2000), whereas a signal for the absence of cocaine (i.e., a drug-related inhibitor) dramatically reduced cocaine seeking in rats by over 90% (D.N. Kearns, S.J. Weiss, C.W. Schindler, & L.V. Panlilio, 2005). In the present experiment, a signal for the absence of food created through the A+/AB- conditioned inhibition paradigm also suppressed responding for cocaine by approximately 90%. Symmetrically, a signal for the absence of cocaine (i.e., a cocaine-based inhibitor) suppressed food seeking to a similar degree. These findings, consistent with the appetitive-aversive interaction theory of motivation, suggest that using inhibitors based on non-drug appetitive reinforcers might be a practical method of reducing drug seeking in human drug abusers and should be seriously considered for clinical test and application. Weiss, S.J., Kearns, D.N., Christensen, C.J., Huntsberry, M.E., Schindler, C.W. and Panlilio, L.V. Experimental Clinical Psychopharmacology, 15, pp. 359-367, 2007.

Adenosine Receptor Heteromers and their Integrative Role in Striatal Function
By analyzing the functional role of adenosine receptor heteromers, IRP scientists review a series of new concepts that should modify our classical views of neurotransmission in the central nervous system (CNS). Neurotransmitter receptors cannot be considered as single functional units anymore. Heteromerization of neurotransmitter receptors confers functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Some of these characteristics can be used as a "biochemical fingerprint" to identify neurotransmitter receptor heteromers in the CNS. This is exemplified by changes in binding characteristics that are dependent on coactivation of the receptor units of different adenosine receptor heteromers. Neurotransmitter receptor heteromers can act as "processors" of computations that modulate cell signaling, sometimes critically involved in the control of pre- and postsynaptic neurotransmission. For instance, the adenosine A1-A2A receptor heteromer acts as a concentration-dependent switch that controls striatal glutamatergic neurotransmission. Neurotransmitter receptor heteromers play a particularly important integrative role in the "local module" (the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit), where they act as processors mediating computations that convey information from diverse volume-transmitted signals. For instance, the adenosine A2A-dopamine D2 receptor heteromers work as integrators of two different neurotransmitters in the striatal spine module. Ferre, S., Ciruela, F., Quiroz, C., Lujan, R., Popoli, P. Cunha, R. A., Agnati, L.F., Fuxe, K., Woods, A.S., Lluis, C. and Franco, R. Scientific World Journal, 2, pp. 74-85, 2007.

Basic Concepts in G-protein-coupled Receptor Homo- and Heterodimerization
Until recently, heptahelical G-protein-coupled receptors (GPCRs) were considered to be expressed as monomers on the cell surface of neuronal and non-neuronal cells. It is now becoming evident that this view must be overtly changed since these receptors can form homodimers, heterodimers, and higher-order oligomers on the plasma membrane. Here IRP researchers discuss some of the basics and some new concepts of receptor homo- and heteromerization. Dimers-oligomers modify pharmacology, trafficking, and signaling of receptors. First of all, GPCR dimers must be considered as the main molecules that are targeted by neurotransmitters or by drugs. Thus, binding data must be fitted to dimer-based models. In these models, it is considered that the conformational changes transmitted within the dimer molecule lead to cooperativity. Cooperativity must be taken into account in the binding of agonists-antagonists-drugs and also in the binding of the so-called allosteric modulators. Cooperativity results from the intramolecular cross-talk in the homodimer. As an intramolecular cross-talk in the heterodimer, the binding of one neurotransmitter to one receptor often affects the binding of the second neurotransmitter to the partner receptor. Coactivation of the two receptors in a heterodimer can change completely the signaling pathway triggered by the neurotransmitter as well as the trafficking of the receptors. Heterodimer-specific drugs or dual drugs able to activate the two receptors in the heterodimer simultaneously emerge as novel and promising drugs for a variety of central nervous system (CNS) therapeutic applications. Franco, R., Casado, V., Cortes, A., Ferrada, C., Mallol, J., Woods, A., Lluis, C., Canela, E.I. and Ferre, S. Scientific World Journal, 7, pp. 48-57, 2007.

Old and New Ways to Calculate the Affinity of Agonists and Antagonists Interacting with G-protein-coupled Monomeric and Dimeric Receptors: The Receptor-dimer Cooperativity Index
Almost all existing models that explain heptahelical G-protein-coupled receptor (GPCR) operation are based on the occurrence of monomeric receptor species. However, an increasing number of studies show that many G-protein-coupled heptahelical membrane receptors (HMR) are expressed in the plasma membrane as dimers. IRP investigators here review the approaches for fitting ligand binding data that are based on the existence of receptor monomers and also the new ones based on the existence of receptor dimers. The reasons for equivocal interpretations of the fitting of data to receptor dimers, assuming they are monomers, are also discussed. A recently devised model for receptor dimers provides a new approach for fitting data that eventually gives more accurate and physiological relevant parameters. Fitting data using the new procedure gives not only the equilibrium dissociation constants for high- and low-affinity binding to receptor dimers but also a "cooperativity index" that reflects the molecular communication within the dimer. A comprehensive way to fit binding data from saturation isotherms and from competition assays to a dimer receptor model is reported and compared with the traditional way of fitting data. The new procedure can be applied to any receptor forming dimers; from receptor tyrosine kinases to intracellular receptors (e.g., estrogen receptor) and in general for ligand binding to proteins forming dimers. Cadaso, V., Cortes, A., Ciruela, F., Mallol, J., Ferre, S., Lluis, C., Canela, E.I. and Franco, R. Pharmacological Therapeutics, 116, pp. 343-354, 2007.

Functional Relevance of Neurotransmitter Receptor Heteromers in the Central Nervous System
The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their functional significance is being revealed. Heteromerization of neurotransmitter receptors produces functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Neurotransmitter receptor heteromers can function as processors of computations that modulate cell signaling. Thus, the quantitative or qualitative aspects of the signaling generated by stimulation of any of the individual receptor units in the heteromer are different from those obtained during coactivation. Furthermore, recent studies demonstrate that some neurotransmitter receptor heteromers can exert an effect as processors of computations that directly modulate both pre- and postsynaptic neurotransmission. This is illustrated by the analysis of striatal receptor heteromers that control striatal glutamatergic neurotransmission. Ferre, S., Ciruela, F., Woods, A.S., Lluis, C. and Franco, R. Trends Neuroscience, 30, pp. 440-446, 2007.

Molecular Neuropsychiatry Research Branch

Interactions of HIV and Methamphetamine: Cellular and Molecular Mechanisms of Toxicity Potentiation
Methamphetamine (METH) is a highly addictive psychostimulant drug, whose abuse has reached epidemic proportions worldwide. METH use is disproportionally represented among populations at high risks for developing HIV infection or who are already infected with the virus. Psychostimulant abuse has been reported to exacerbate the cognitive deficits and neurodegenerative abnormalities observed in HIV-positive patients. Thus, the purpose of the present paper is to review the clinical and basic observations that METH potentiates the adverse effects of HIV infection. An additional purpose is to provide a synthesis of the cellular and molecular mechanisms that might be responsible for the increased toxicity observed in co-morbid patients. The reviewed data indicate that METH and HIV proteins, including gp120, gp41, Tat, Vpr and Nef, converge on various caspase-dependent death pathways to cause neuronal apoptosis. The role of reactive microgliosis in METH- and in HIV-induced toxicity is also discussed. Cadet, J.L. and Krasnova, I.N. Neurotoxicology Research 12(3), pp. 181-204, 2007.

A Marked Increase in Cocaine-related Deaths in the State of Florida: Precursor to an Epidemic?
The history of cocaine misuse includes a destructive epidemic during the 1980s. While recent surveys suggest cocaine use is stable or decreasing, IRP scientists have observed increasing trends of cocaine-related death through analysis of medical examiner data collected by the Florida Department of Law Enforcement (FDLE). Florida's per capita cocaine-related death rates nearly doubled from 2001 to 2005. Electronic collection of data such as that collected by the FDLE nationally and in real-time would greatly advance understanding of drug-use patterns and consequences. For example, results from Florida suggest that high school and college students, and members of higher socioeconomic status, appear to be at increased risk of cocaine abuse. Public health interventions are necessary to prevent another full-fledged epidemic. Goldberger, B., Grahan, N., Nelson, S., Cadet, J.L., and Gold, M. J. of Addict..Diseases 26(3), pp. 113-116, 2007.

Transcriptional Responses to Reinforcing Effects of Cocaine in the Rat Hippocampus and Cortex
The psychostimulant effects of cocaine are thought to result from its ability to block dopamine (DA) uptake and increase DA levels in ventral striatum. In addition, cocaine causes biochemical changes in the brain areas involved in learning and memory, including hippocampus and cortex, whose role in drug reinforcement is now being actively investigated. Thus, IRP researchers studied molecular events in the hippocampus and frontal cortex of rats treated with cocaine conditioned place preference (CPP) paradigm. After exposure to cocaine conditioning (cocaine paired), cocaine alone (cocaine non-paired) or saline rats were tested for place conditioning. Cocaine (10 mg/kg) caused increases in time spent in the drug-paired compartment. By using microarray analyses, the authors examined gene expression in the hippocampi and frontal cortices of cocaine-paired rats, cocaine non-paired and saline-treated controls. Their study revealed that 214 transcripts were differentially regulated in the hippocampi of cocaine-paired rats. These include genes that play roles in protein phosphorylation, RNA processing and protein synthesis, ubiquitin-dependent protein degradation and cytoskeleton organization. In contrast, 39 genes were differently expressed in the frontal cortex. These data support the possibility that molecular changes in the hippocampus might participate in the formation and maintenance of memory patterns induced by cocaine in the brain. Differences in the transcriptional responses in the hippocampus and cortex suggest the primary importance of the hippocampus for recent memory processing associated with cocaine-induced CPP. Krasnova, I.N., Li, S.M., Wood, W.H., McCoy, M.T., Prabhu, V.V., Becker, K.G., Katz, J.L. and Cadet, J.L. Genes Brain Behavior July 19, 2007.

Sex-dependent Metabolic, Neuroendocrine, and Cognitive Responses to Dietary Energy Restriction and Excess
Females and males typically play different roles in survival of the species and would be expected to respond differently to food scarcity or excess. To elucidate the physiological basis of sex differences in responses to energy intake, IRP scientists maintained groups of male and female rats for 6 months on diets with usual, reduced [20% and 40% caloric restriction (CR), and intermittent fasting (IF)], or elevated (high-fat/high-glucose) energy levels and measured multiple physiological variables related to reproduction, energy metabolism, and behavior. In response to 40% CR, females became emaciated, ceased cycling, underwent endocrine masculinization, exhibited a heightened stress response, increased their spontaneous activity, improved their learning and memory, and 44 maintained elevated levels of circulating brain-derived neurotrophic factor. In contrast, males on 40% CR maintained a higher body weight than the 40% CR females and did not change their activity levels as significantly as the 40% CR females. Additionally, there was no significant change in the cognitive ability of the males on the 40% CR diet. Males and females exhibited similar responses of circulating lipids (cholesterols/triglycerides) and energy-regulating hormones (insulin, leptin, adiponectin, ghrelin) to energy restriction, with the changes being quantitatively greater in males. The high-fat/high-glucose diet had no significant effects on most variables measured but adversely affected the reproductive cycle in females. Heightened cognition and motor activity, combined with reproductive shutdown, in females may maximize the probability of their survival during periods of energy scarcity and may be an evolutionary basis for the vulnerability of women to anorexia nervosa. Martin, B., Pearson, M., Kebejian Golden, E., Keselman, A., Bender, M., Carlson, O., Egan, J., Ladenheim, B., Cadet, J.L., Becker, K.G., Wood, W., Duffy, K., Vinayakumar, P., Muudsley, S. and Mattson, M.P. Endocrinology 148(9), pp. 4318-4333, 2007.