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NIDA Home > Publications > Director's Reports > February, 2010 Index    

Director's Report to the National Advisory Council on Drug Abuse - February, 2010

Research Findings - Intramural Research

Biomedical Informatics Section, Administrative Management Branch

A Usability Study of Transactional Electronic Diary: Results From Expert Evaluation To Participants Use
This usability study suggests that the Transactional Electronic Diary can be efficiently used by polydrug abusing individuals to record their behaviors and moods in real time in their day-to-day environments. Lin JL, Vahabzadeh M, Mezghanni M, Epstein DH, Preston KL. A usability study of transactional electronic diary: results from expert evaluation to participants use. AMIA Annu Symp Proc. 2009: 937.

Clinical Pharmacology and Therapeutics

Cocaine Craving and Use During Daily Life
Craving is often assumed to cause ongoing drug use and relapse, but its relationship to drug use in daily life has not been documented in a rigorous, prospective manner. In a prospective, longitudinal, cohort design, 112 cocaine-abusing individuals on methadone maintenance rated their craving and mood at random times (two to five times daily prompted by electronic diaries) as they went about their everyday activities. They also initiated an electronic-diary entry each time they used cocaine. Cocaine use was further monitored by thrice-weekly urine testing. The authors found that during periods of urine-verified cocaine use, ratings of cocaine craving increased across the day and were higher than during periods of urine-verified abstinence. During the 5 hours prior to individual episodes of cocaine use, ratings of craving significantly increased. These patterns were not seen in ratings of heroin craving or mood. Thus, at both weekly and hourly time scales, cocaine craving is tightly coupled to cocaine use in users' normal environments. These findings provide previously unavailable support for a relationship that has been seriously questioned in some theoretical accounts. Preston KL, Vahabzadeh, M, Schmittner J, et al. Psychopharmacology 2009;207:291-301.

Office of the Scientific Director

Signal-Averaged Electrocardiogram In Physically Healthy, Recently Abstinent Chronic Cocaine Users
Cocaine use is associated with cardiac arrhythmias, but predicting who is at risk is difficult. Signal-averaged electrocardiography (SA-ECG), unlike standard ECG, can detect markers of ventricular late potentials (VLP), which may be a precursor to malignant ventricular arrhythmias. IRP scientists evaluated SA-ECG parameters in 60 medically screened, physically healthy, recently abstinent, chronic cocaine users and in 54 non-drug-using controls. SA-ECGs were done periodically for up to 12 weeks of monitored abstinence in 25 of the cocaine users. Cocaine users differed significantly from controls in only one of three SA-ECG parameters considered markers of VLPs. The proportion of subjects with abnormal SA-ECG parameters did not differ significantly between male cocaine users and male controls. There were no significant changes over time in either the mean values or proportion of subjects with abnormal values for any SA-ECG parameter. There were significant gender differences among controls, but not among cocaine users. These findings suggest that chronic cocaine use is not associated with a higher prevalence of abnormal SA-ECG parameters in physically healthy users. Kanneganti P, Copersino ML, Nelson RA, et al. Signal-averaged electrocardiogram in physically healthy, recently abstinent chronic cocaine users. Journal of Addiction Medicine. 2009;3(3):128-133.

Interest In Marijuana Treatment Programs Among Teenage Smokers and Nonsmokers
Many adolescents smoke marijuana, but little is known about adolescents' interest in marijuana treatment programs. IRP scientists evaluated this question by telephone interview in a convenience sample of 575 adolescents (13-17 years old) responding to advertisements for tobacco research studies at the NIDA IRP. 81% of respondents endorsed the need for marijuana treatment programs for adolescents. These adolescents were younger and less likely to smoke tobacco, smoke marijuana, or use alcohol than those not endorsing such a need. Among the 192 marijuana smokers, the 58.8% who endorsed the need for marijuana treatment programs took their first puff of marijuana at a younger age than those who did not endorse the need. Those who were willing to participate in a marijuana treatment program were more likely African-American and took their first marijuana puff at a younger age than those not interested in treatment. These findings suggest that a majority of adolescent marijuana smokers endorse the need for and are willing to attend marijuana treatment programs. Sheer AJ, Gorelick DA, Collins CC, et al. Interest in marijuana treatment programs among teenage smokers and nonsmokers. Journal of Substance Abuse Treatment. 2009; 37(4):421-425.

Behavioral Neuroscience Research Branch

A Ventral Tegmental CRF-Glutamate-Dopamine Interaction In Addiction
Stress-induced reinstatement of cocaine-seeking is blocked by antagonists for the stress-related neurohormone corticotropin-releasing factor (CRF). One site of this action is the ventral tegmental area (VTA), where mild footshock stress causes CRF release, glutamate release, and dopaminergic activation in cocaine-experienced but not cocaine-naive animals. Infusion of CRF into VTA has similar effects to footshock in cocaine-experienced animals but fails to cause significant VTA glutamate release or dopaminergic activation in cocaine-naive animals. The reinstatement, glutamate release, and dopamine release are prevented by VTA infusions of CRF-receptor 2 (CRF-R2) but not CRF-R1 antagonists. Reinstatement is triggered by some but not all CRF-R2 agonists and some but not all CRF-R1 agonists; the common denominator of the effective agonists is that they bind to the CRF-binding protein (CRF-BP), which appears to be essential for the behavioral and VTA effects of stress and CRF in cocaine-experienced animals. In situ hybridization reveals mRNA for CRF-R1 and CRF-BP but not CRF-R2 in a subset of VTA dopamine neurons. Electron microscopy reveals primarily asymmetric synapses between a subset of VTA terminals containing glutamate and CRF and a subset of VTA dopaminergic neurons and primarily symmetric synapses between a subset of CRF terminals that do not contain glutamate and a subset of GABAergic neurons in VTA. Thus, a complex and not yet fully understood interaction of CRF, glutamate, and the mesocorticolimbic dopamine system is established by experience with cocaine, and this alteration appears to contribute importantly to the transition from casual to compulsive cocaine-seeking. Wise RA, Morales M. A ventral tegmental CRF-glutamate-dopamine interaction in addiction. Brain Res. 2009 [Epub ahead of print]

Roles For Nigrostriatal--Not Just Mesocorticolimbic--Dopamine In Reward and Addiction
Forebrain dopamine circuitry has traditionally been studied by two largely independent specialist groups: students of Parkinson's disease who study the nigrostriatal dopamine system that originates in the substantia nigra (SN), and students of motivation and addiction who study the role of the mesolimbic and mesocortical dopamine systems that originate in the ventral tegmental area (VTA). The anatomical evidence for independent nigrostriatal and mesolimbic dopamine systems has, however, long been obsolete. There is now compelling evidence that both nominal "systems" participate in reward function and addiction. Electrical stimulation of both SN and VTA is rewarding, blockade of glutamatergic or cholinergic input to either SN or VTA attenuates the habit-forming effects of intravenous cocaine, and dopamine in both nigrostriatal and mesocorticolimbic terminal fields participates in the defining property of rewarding events: the reinforcement of memory consolidation. Thus the similarities between nigrostriatal and mesolimbic dopamine systems can be as important as their differences. Wise RA. Roles for nigrostriatal--not just mesocorticolimbic--dopamine in reward and addiction. Trends Neurosci. 2009; 32(10):517-524.

Control of Within-Binge Cocaine-Seeking By Dopamine and Glutamate In the Core of Nucleus Accumbens
Dopamine and glutamate are thought to interact in the ventral striatum and to play important roles there in the cocaine-seeking of cocaine-experienced animals. IRP scientists sought to determine the relative roles of the two transmitters in the two major zones of the nucleus accumbens (NAS), the core and shell subregions. They assessed the effects of dopamine and glutamate receptor blockade in the core and shell on intravenous cocaine self-administration in rats. Trained animals were allowed to self-administer cocaine for an initial hour, and then D1-type or D2-type dopamine receptor blockers or NMDA-type or AMPA-type glutamate receptor blockers were infused by reverse microdialysis into one of the two regions for an additional 3 h of testing. The D1-type antagonist SCH23390 and the D2-type antagonist raclopride each increased cocaine intake whereas the AMPA-type antagonist CNQX decreased responding when infused into the core. SCH23390 increased cocaine intake less strongly when infused into the shell, while raclopride and CNQX were each ineffective when infused into the shell. The NMDA-antagonist CPP failed to affect cocaine self-administration when infused into either site. These findings implicate the core of NAS in the maintenance of established cocaine self-administration in trained animals, despite the fact that the reinforcement of responding in untrained animals appears to results from cocaine actions in the olfactory tubercle and medial shell and not the core of accumbens. Suto N, Ecke LE, Wise RA. Control of within-binge cocaine-seeking by dopamine and glutamate in the core of nucleus accumbens. Psychopharmacology (Berl). 2009;205(3):431-439.

Reinstatement Of Cocaine Seeking By Hypocretin (Orexin) in the Ventral Tegmental Area: Independence From the Local Corticotropin-Releasing Factor Network
Hypocretin (Hcrt), an arousal- and feeding-associated peptide, is expressed in lateral hypothalamic neurons that project to the rewardŃassociated dopaminergic neurons of the ventral tegmental area (VTA). Intra-VTA Hcrt reinstates morphine-conditioned place preferences, and intracerebroventricular and intra-VTA corticotropin-releasing factor (CRF) reinstate cocaine seeking. Each is presumed to act, at least in part, through actions local to the VTA. Here, IRP investigators examined the possibility that VTA perfusion of Hcrt reinstates cocaine seeking and, if so, whether it does so through the VTA mechanism that is implicated in reinstatement by CRF. Rats were trained to lever-press for intravenous cocaine (2 weeks) and then underwent extinction training (saline substituted for cocaine: 3 weeks). Reinstatement behavior was tested and VTA dialysates were collected and assayed for glutamate or dopamine following footshock or perfusion of Hcrt or CRF, with or without Hcrt or CRF antagonists, into the VTA. Ventral tegmental area perfusion of Hcrt-1 or footshock stress reinstated cocaine seeking and caused release of VTA glutamate and dopamine. The effects of Hcrt-1 were blocked by a selective Hcrt-1 antagonist, but not a CRF antagonist, and were not mimicked by Hcrt-2. The Hcrt-1 antagonist did not block CRF-dependent footshock-induced reinstatement or glutamate or dopamine release. The behavioral and neurochemical effects of Hcrt-1 were attenuated but not blocked by kynurenic acid, an ionotropic glutamate antagonist that blocks footshock-induced reinstatement and glutamate release. While Hcrt and CRF are known to interact in some area of the brain, in the VTA proper they appear to have largely independent actions on the mesolimbic dopamine mechanisms of cocaine seeking. Wang B, You ZB, Wise RA. Reinstatement of cocaine seeking by hypocretin (orexin) in the ventral tegmental area: independence from the local corticotropin-releasing factor network. Biol Psychiatry. 2009 15;65(10):857-862.

Rapid EEG Desynchronization and EMG Activation Induced By Intravenous Cocaine In Freely Moving Rats: A Peripheral, Non-Dopamine Neural Triggering
Many important physiological, behavioral and psychoemotional effects of intravenous (iv) cocaine (COC) are too fast and transient when compared to pharmacokinetic predictions, suggesting a possible involvement of peripheral neural mechanisms in their triggering. In the present study, IRP researchers examined changes in cortical electroencephalogram (EEG) and neck electromyogram (EMG) induced in freely moving rats by iv COC administration at low, reinforcing doses (0.25-1.0 mg/kg) and compared them with those induced by an auditory stimulus and iv COC methiodide which cannot cross the blood-brain barrier. The authors found that COC induces rapid, strong, and prolonged EEG desynchronization, associated with decrease in alpha and increase in beta and gamma activities, and EMG activation that both begin within 2-6 s following the start of a 10-s injection; immediate components of this effect were dose-independent. The rapid COC-induced changes in EEG and EMG resembled those induced by an auditory stimulus; the latter effects had shorter onset latencies and durations and were fully blocked during urethane anesthesia. Although urethane anesthesia completely blocked COC-induced EMG activation and rapid components of EEG response, COC still induced EEG desynchronization that was much weaker, greatly delayed (~60 s), and associated with tonic decreases in delta and increases in alpha, beta and gamma activities. Peripherally acting COC methiodide fully mimicked rapid EEG and EMG effects of regular COC, but the effects at an equimolar dose were less prolonged than those with regular COC. These data suggest that in awake animals iv COC, like somato-sensory stimuli, induces cortical activation and a subsequent motor response via its action on peripheral neural elements and involving rapid neural transmission. By providing a rapid neural signal and triggering transient neural activation, such an action might play a crucial role in the sensory effects of COC, thus contributing to the learning and development of drug-taking behavior. Kiyatkin EA, Smirnov MS. Rapid EEG desynchronization and EMG activation induced by intravenous cocaine in freely moving rats: a peripheral, non-dopamine neural triggering. Am J Physiol Regul Integr Comp Physiol. 2009 [Epub ahead of print]

Phasic and Tonic Fluctuations In Brain, Muscle, and Skin Temperatures During Motivated Drinking Behavior In Rats: Physiological Correlates of Motivation and Reward
Since brain metabolism is accompanied by heat production, measurement of brain temperature offers a method for assessing global alterations in metabolic neural activity. This approach, high-resolution (5-s bin) temperature recording from the nucleus accumbens (NAcc), temporal muscle, and facial skin, was used to study motivated drinking behavior in rats. Experienced animals were presented with a cup containing 5-ml of Coca-Cola(R) (Coke) beverage that resulted, within certain latencies, in initiation of a continuous chain of licking until all liquid was fully consumed. While cup presentation induced rapid, gradual NAcc temperature increase peaking at the start of drinking, temperatures slowly decreased during Coke consumption, but phasically increased again in the post-consumption period when rats were hyperactive, showing multiple interactions with an empty cup. Muscle temperatures followed a similar pattern, but the changes were weaker and delayed compared to those in the brain. Skin temperature rapidly dropped after cup presentation, steadily maintained at low levels during consumption, and slowly restored during the post-consumption period. Substitution of the expected Coke with either sugar-free Diet Coke(R) or water resulted in numerous drinking attempts but ultimately no consumption. During these tests, locomotor activation was much greater and more prolonged, brain and muscle temperatures increased monophasically, and their elevation was significantly greater than that with regular Coke tests. Food deprivation decreased drinking latencies, did not change the pattern of temperature fluctuations during Coke consumption, but temperature elevations were greater than in controls. These data suggest sustained neural activation triggered by appetitive stimuli and associated with activational (seeking) aspects of appetitive motivated behavior. This seeking-related activation is rapidly ceased following consumption, suggesting this change as a neural correlate of reward. In contrast, inability to obtain an expected reward maintains neural activation and seeking behavior, resulting in larger deviations in physiological parameters. Smirnov MS, Kiyatkin EA. Phasic and tonic fluctuations in brain, muscle, and skin temperatures during motivated drinking behavior in rats: Physiological correlates of motivation and reward. Brain Res. 2009 Nov 22. [Epub ahead of print]

Acute Methamphetamine Intoxication Brain Hyperthermia, Blood-Brain Barrier, Brain Edema, and Morphological Cell Abnormalities
Methamphetamine (METH) is a powerful and often abused stimulant with potent addictive and neurotoxic properties. While it is generally assumed that multiple chemical substances released in the brain following METH-induced metabolic activation (or oxidative stress) are primary factors underlying damage of neural cells, in this work IRP researchers present data suggesting a role of brain hyperthermia and associated leakage of the blood-brain barrier (BBB) in acute METH-induced toxicity. First, they show that METH induces a dose-dependent brain and body hyperthermia, which is strongly potentiated by associated physiological activation and in warm environments that prevent proper heat dissipation to the external environment. Second, they demonstrate that acute METH intoxication induces robust, widespread but structure-specific leakage of the BBB, acute glial activation, and increased water content (edema), which are related to drug-induced brain hyperthermia. Third, they document widespread morphological abnormalities of brain cells, including neurons, glia, epithelial, and endothelial cells developing rapidly during acute METH intoxication. These structural abnormalities are tightly related to the extent of brain hyperthermia, leakage of the BBB, and brain edema. While it is unclear whether these rapidly developed morphological abnormalities are reversible, this study demonstrates that METH induces multiple functional and structural perturbations in the brain, determining its acute toxicity and possibly contributing to neurotoxicity. Kiyatkin EA, Sharma HS. Acute methamphetamine intoxication brain hyperthermia, blood-brain barrier, brain edema, and morphological cell abnormalities. Int Rev Neurobiol. 2009;88:65-100.

Cocaine Action on Peripheral, Non-Monoamine Neural Substrates as a Trigger of Electroencephalographic Desynchronization and Electromyographic Activation Following I.V. Administration In Freely Moving Rats
Many important physiological, behavioral and subjective effects of i.v. cocaine (COC) are exceptionally rapid and transient, suggesting a possible involvement of peripheral neural substrates in their triggering. In the present study, IRP scientists used high-speed electroencephalographic (EEG) and electromyographic (EMG) recordings (4-s resolution) in freely moving rats to characterize the central electrophysiological effects of i.v. COC at low doses within a self-administration range (0.25-1.0 mg/kg). They found that COC induces rapid, strong, and prolonged desynchronization of cortical EEG (decrease in alpha and increase in beta and gamma activity) and activation of the neck EMG that begin within 2-6 s following the start of a 10-s injection; immediate components of both effects were dose-independent. The rapid effects of COC were mimicked by i.v. COC methiodide (COC-MET), a derivative that cannot cross the blood-brain barrier. At equimolar doses (0.33-1.33 mg/kg), COC-MET had equally fast and strong effects on EEG and EMG total powers, decreasing alpha and increasing beta and gamma activities. Rapid EEG desynchronization and EMG activation was also induced by i.v. procaine, a structurally similar, short-acting local anesthetic with virtually no effects on monoamine uptake; at equipotential doses (1.25-5.0 mg/kg), these effects were weaker and shorter in duration than those of COC. Surprisingly, i.v. saline injection delivered during slow-wave sleep (but not during quiet wakefulness) also induced a transient EEG desynchronization but without changes in EMG and motor activity; these effects were significantly weaker and much shorter than those induced by all tested drugs. These data suggest that in awake animals, i.v. COC induces rapid cortical activation and a subsequent motor response via its action on peripheral non-monoamine neural elements, involving neural transmission via visceral sensory pathways. By providing a rapid neural signal and triggering neural activation, such an action might play a crucial role in the sensory effects of COC, thus contributing to the learning and development of drug-taking behavior. Smirnov MS, Kiyatkin EA. Cocaine action on peripheral, non-monoamine neural substrates as a trigger of electroencephalographic desynchronization and electromyographic activation following i.v. administration in freely moving rats. Neuroscience. 2009 Oct 25. [Epub ahead of print]

Permeability of the Blood-Brain Barrier Depends on Brain Temperature
Increased permeability of the blood-brain barrier (BBB) has been reported in different conditions accompanied by hyperthermia, but the role of brain temperature per se in modulating brain barrier functions has not been directly examined. To delineate the contribution of this factor, IRP researchers examined albumin immunoreactivity in several brain structures (cortex, hippocampus, thalamus and hypothalamus) of pentobarbital-anesthetized rats (50 mg/kg i.p.), which were passively warmed to different levels of brain temperature (32-42 degrees C). Similar brain structures were also examined for the expression of glial fibrillary acidic protein (GFAP), an index of astrocytic activation, water and ion content, and morphological cell abnormalities. Data were compared with those obtained from drug-free awake rats with normal brain temperatures (36-37 degrees C). The numbers of albumin- and GFAP-positive cells strongly correlate with brain temperature, gradually increasing from approximately 38.5 degrees C and plateauing at 41-42 degrees C. Brains maintained at hyperthermia also showed larger content of brain water and Na(+), K(+) and Cl(-) as well as structural abnormalities of brain cells, all suggesting acute brain edema. The latter alterations were seen at approximately 39 degrees C, gradually progressed with temperature increase, and peaked at maximum hyperthermia. Temperature-dependent changes in albumin immunoreactivity tightly correlated with GFAP immunoreactivity, brain water, and numbers of abnormal cells; they were found in each tested area, but showed some structural specificity. Notably, a mild BBB leakage, selective glial activation, and specific cellular abnormalities were also found in the hypothalamus and piriform cortex during extreme hypothermia (32-33 degrees C); in contrast to hyperthermia these changes were associated with decreased levels of brain water, Na(+) and K(+), suggesting acute brain dehydration. Therefore, brain temperature per se is an important factor in regulating BBB permeability, alterations in brain water homeostasis, and subsequent structural abnormalities of brain cells. Kiyatkin EA, Sharma HS. Permeability of the blood-brain barrier depends on brain temperature. Neuroscience. 2009;161(3):926-939.

Neurobiology of Relapse Section

Role of Ventral Tegmental Area GDNF In Incubation of Cocaine Craving
Ventral tegmental area (VTA) brain-derived neurotrophic factor (BDNF) contributes to the time-dependent increases in cue-induced cocaine-seeking after withdrawal (incubation of cocaine craving). Here, IRP investigators studied the role in incubation of cocaine craving of glial cell-line-derived neurotrophic factor (GDNF) that, like BDNF, supports the survival and function of midbrain dopamine neurons. They first trained rats to self-administer intravenous cocaine for 10 d (6 h/d, cocaine injections were paired with a tone-light cue). They then manipulated VTA GDNF function and assessed cue-induced cocaine-seeking in extinction tests after withdrawal from cocaine. VTA injections of an adeno-associated virus (AAV) vector containing rat GDNF cDNA (5x108 viral genomes) on withdrawal day 1 increased cue-induced cocaine-seeking on withdrawal days 11 and 31; this effect was not observed after VTA injections of an AAV viral vector containing red fluorescent protein (RFP). Additionally, VTA, but not substantia nigra, GDNF injections (1.25 or 12.5 µg/side) immediately after the last cocaine self-administration session increased cue-induced drug-seeking on withdrawal days 3 and 10; this effect was reversed by VTA injections of U0126, which inhibits the activity of extracellular signal-regulated kinases (ERK). Finally, interfering with VTA GDNF function by chronic delivery of anti-GDNF monoclonal neutralizing antibodies via minipumps (600 ng/side/d) during withdrawal days 1-14 prevented the time-dependent increases in cue-induced cocaine-seeking on withdrawal days 11 and 31. Results indicate that during the first weeks of withdrawal from cocaine self-administration GDNF-dependent neuroadaptations in midbrain VTA neurons play an important role in the development of incubation of cocaine craving. Lu L, Wang X, Wu P, et al. Role of ventral tegmental area GDNF in incubation of cocaine craving. Biological Psychiatry 2009;66:137-145.

Preclinical Pharmacology Section

Regulation of Sigma-1 Receptors and Endoplasmic Reticulum Chaperones in the Brain of Methamphetamine Self-Administering Rats
Sigma-1 receptors are endoplasmic reticulum (ER) chaperones that are implicated in the neuroplasticity associated with psychostimulant abuse. IRP scientists immunocytochemically examined the distribution of sigma-1 receptors in the brain of drug naive rats, then examined the dynamics of sigma-1 receptors and other ER chaperones in specific brain subregions of rats that self-administered methamphetamine, received methamphetamine passively, or received only saline injections. Sigma-1 receptors were found to be expressed in moderate to high levels in the olfactory bulb, striatum, nucleus accumbens shell, olfactory tubercle, amygdala, hippocampus, red nucleus, ventral tegmental area, substantia nigra, and locus ceruleus. Methamphetamine, whether self-administered or passively received, significantly elevated ER chaperones including the sigma-1 receptor, BiP, and calreticulin in the ventral tegmental area and substantia nigra. In the olfactory bulb, however, only the sigma-1 receptor chaperone was increased, and this increase occurred only in rats that actively self-administered methamphetamine. Consistent with an increase in sigma-1 receptors, ERK was found to be activated and PKA attenuated in the olfactory bulb of methamphetamine self-administering rats. Sigma-1 receptors in the olfactory bulb were found to be co-localized with dopamine D1 receptors. These results indicate that methamphet-amine induces ER stress in the ventral tegmental area and substantia nigra in rats whether the drug is received actively or passively. However, the changes seen only in rats that actively self-administered methamphetamine suggest that D1 and sigma-1 receptors in the olfactory bulb might play an important role in the motivational conditioning/learning aspects of methamphetamine self-administration in the rat. Hayashi T, Justinova Z, Hayashi E, et al. Journal of Pharmacology and Experimental Therapeutics, 2010;332:1-10.

Environmental Enrichment Reduces Cocaine Seeking and Reinstatement Induced By Cues and Stress But Not By Cocaine
Whereas earlier studies have focused on the preventive effects of enriched environments (EE) in drug addiction, in a recent study IRP researchers suggested that EE can also have 'curative' effects. In fact, they found that cocaine addiction-related behaviors can be eliminated by housing cocaine-treated mice in EE during periods of forced abstinence. However, those results were obtained with two simple models of addiction, conditioned place preference (CPP), and behavioral sensitization. In this study, the authors used intravenous drug self-administration procedures in rats to further investigate the beneficial effects of EE on cocaine addiction in a reinstatement model of relapse. Singly housed rats learned to self-administer cocaine during 10 consecutive daily sessions (0.6 mg/injection, 6 h/day). They were then housed three per cage in either standard environments (SE) or EE and were kept abstinent in the animal facility until testing for extinction and reinstatement. They found that 30 days of EE significantly and consistently reduced cocaine seeking during a 6-h extinction session. In addition, EE significantly reduced cue- and stress-induced reinstatement. Surprisingly, given our earlier results in mice with CPP, EE did not reduce cocaine-induced reinstatement regardless of the level of exposure to cocaine and the duration of the period of abstinence and exposure to EE. Altogether, these results support the hypothesis that EE can reduce cocaine-induced craving and highlight the importance of positive life conditions in facilitating abstinence and preventing relapse to cocaine addiction.Chauvet C, Lardeux V, Goldberg SR, Jaber M, Solinas M. Neuropsychopharma-cology. 2009, 34(13):2767-2778.

Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition Through Activation of PPAR-Alpha Nuclear Receptors
Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB(1)-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain. Using a passive-avoidance task in rats, IRP scientists found that memory acquisition was enhanced by the FAAH inhibitor URB597 or by the PPAR-alpha agonist WY14643, and these enhancements were blocked by the PPAR-alpha antagonist MK886. These findings demonstrate novel mechanisms for memory enhancement by activation of PPAR-alpha, either directly by administering a PPAR-alpha agonist or indirectly by administering a FAAH inhibitor. Mazzola C, Medalie J, Scherma M, et al. Learning and Memory. 2009;16(5):332-337.

Effects of Nicotine In Experimental Animals and Humans: An Update on Addictive Properties
Tobacco use through cigarette smoking is the leading preventable cause of death in the developed world. Nicotine, a psychoactive component of tobacco, appears to play a major role in tobacco dependence, but the reinforcing effects of nicotine have often been difficult to demonstrate directly in controlled studies with laboratory animals or human subjects. Here IRP researchers review results obtained with drug self-administration, conditioned place preference, subjective reports of nicotine effects and nicotine discrimination indicate that nicotine can function as an effective reinforcer of drug-seeking and drug-taking behavior both in experimental animals and humans under appropriate conditions. Interruption of chronic nicotine exposure produces ratings of drug withdrawal and withdrawal symptoms that may contribute to relapse. Difficulties encountered in demonstrating reinforcing effects of nicotine under some conditions, relative to other drugs of abuse, may be due to weaker primary reinforcing effects of nicotine, to aversive effects produced by nicotine, or to a more critical contribution of environmental stimuli to the maintenance of drug-seeking and drug-taking behavior with nicotine than with other drugs of abuse. Several recent reports suggest that other chemical substances inhaled along with nicotine in tobacco smoke may play a role in sustaining smoking behavior. However, conflicting results have been obtained with mice and rats and these findings have not yet been validated in nonhuman primates or human subjects. Taken together, these findings suggest that nicotine acts as a typical drug of abuse in experimental animals and humans in appropriate situations. Le Foll B, Goldberg SR. Handbook of Experimental Pharmacology. 2009;192:335-367.

Anandamide-Induced Behavioral Disruption Through A Vanilloid-Dependent Mechanism In Rats
Endocannabinoids are involved in a variety of behavioral and physiological processes that are just beginning to be understood. In the five-choice serial reaction-time task, exogenous cannabinoids have been found to alter attention, but endocannabinoids such as anandamide have not been studied. IRP scientists used this task to evaluate the effects of anandamide in rats. Since anandamide is a ligand for not only cannabinoid receptors but also transient receptor potential vanilloid 1 (TRPV1) receptors, and as recently suggested, peroxisome proliferator-activated nuclear receptor-alpha (PPARalpha), they also determined whether anandamide's effects in this task were mediated by each of these receptors. Whenever one of five holes was illuminated for 2 s, a food pellet was delivered if a response occurred in that hole during the light or within 2 s after the light. Anandamide increased omission errors and decreased responding during inter-trial intervals. These effects were blocked by the TRPV1 antagonist capsazepine, but not by the cannabinoid-receptor antagonist rimonabant or the PPARalpha antagonist MK886. Testing with open-field activity and food-consumption procedures in the same rats suggested that the disruption of operant responding observed in the attention task was not due to motor depression, anxiety, decreased appetite, or an inability to find and consume food pellets. The vanilloid-dependent behavioral disruption induced by anandamide was specific to the operant attention task. These effects of anandamide resemble effects of systemically administered dopamine antagonists and might reflect changes in vanilloid-mediated dopamine transmission. Panlilio LV, Mazzola C, Medalie J, et al. Psychopharmacology 2009;203(3):529-538.

Looking For the Role of Cannabinoid Receptor Heteromers In Striatal Function
The introduction of two concepts, "local module" and "receptor heteromer", facilitates the understanding of the role of interactions between different neurotransmitters in the brain. In artificial cell systems, cannabinoid CB(1) receptors form receptor heteromers with dopamine D2, adenosine A2A and mu opioid receptors. There is indirect but compelling evidence for the existence of the same CB1 receptor heteromers in striatal local modules centered in the dendritic spines of striatal GABAergic efferent neurons, particularly at a postsynaptic location. Their analysis provides new clues for the role of endocannabinoids in striatal function, which cannot only be considered as retrograde signals that inhibit neurotransmitter release. Recent studies using a new method to detect heteromerization of more than two proteins, which consists of sequential BRET-FRET (SRET) analysis, has demonstrated that CB1, D2 and A2A receptors can form heterotrimers in transfected cells. It is likely that functional CB1-A2A-D2 receptor heteromers can be found where they are highly co-expressed, in the dendritic spines of GABAergic enkephalinergic neurons. The functional properties of these multiple receptor heteromers and their role in striatal function need to be determined. Ferré S, Goldberg SR, Lluis C, Franco R. Neuropharmacology 2009;56 Suppl 1:226-234.

Effect of Rate of Delivery of Intravenous Cocaine on Self-Administration In Rats
Many studies of drug self-administration in primates have shown that faster infusions of a drug are more reinforcing than slower infusions. Similar effects have not been shown in rats. IRP researchers assessed the influence of delivery rate by allowing rats to choose between the same doses of intravenous cocaine delivered over two different infusion speeds. Rats were trained in chambers containing two nose-poke response devices. In Experiment 1, responses in one nose-poke delivered 0.3 mg/kg/injection of cocaine over 10 s, and responses in the other delivered the same dose over 100 s. In Experiment 2, the same procedure was used, but with 1.0 mg/kg/injection dose delivered over 1.7 versus 100 s. During acquisition, most rats preferred the faster infusion. When the delivery rates associated with the nose pokes were reversed, rats trained with 0.3 mg/kg/injection failed to switch nose-poke preference, but half the rats trained with 1.0 mg/kg/injection did switch. In Experiment 3, the choice was between 1 mg/kg cocaine delivered over 1.7 s and no reinforcement. Here, rats quickly learned to respond in the nose-poke associated with cocaine and quickly switched their choice during reversal. In Experiment 4, two groups of rats were allowed to choose between food delivered with a delay of 1 versus 5 s or 1 versus 10 s, respectively. Rats preferred the shorter delay during initial training. In reversal, some rats in the 1 vs 5 s group failed to reverse, while all the rats in the 1 vs 10 s group reversed. These results show that faster infusions of cocaine are clearly more reinforcing during acquisition, but delivery rate may not be as important to the maintenance of self-administration once it has been established. The results with food suggest that these findings represent general principles of behavior and are not unique to drug self-administration. Schindler CW, Panlilio LV, Thorndike EB. Pharmacology Biochemistry and Behavior 2009;93(4):375-381.

Effects of Orbitofrontal Cortex Lesions on Cocaine Self-Administration
Previous research has implicated limbic and prefrontal cortical areas in the control of drug-seeking behavior. The present study examined the effects of orbitofrontal-cortex (OFC) lesions on acquisition, dose-dependence, within-session patterning, and reinstatement of cocaine self-administration. Rats received OFC or sham lesions before or after acquisition (0.3 mg/kg/injection, paired with a visual stimulus), then were tested with a range of doses (0, 0.03, 0.1, 0.3 and 1). Compared to controls, rats lesioned before acquisition acquired the behavior sooner, responded more at low doses, and responded more on the first day of extinction. Rats that were lesioned after acquisition showed an even larger increase in responding (approximately 250%) at the lowest dose, and they also showed increased timeout responding and drug "loading" at low doses. Pre-acquisition lesions were tested and found to have no effect on cocaine-induced reinstatement. In parallel experiments examining effects of pre-acquisition OFC lesions on food-reinforced responding, lesions did not alter acquisition, maintenance, or reinstatement, but accelerated the course of extinction. The increased cocaine self-administration seen in OFC-lesioned rats did not resemble the dysregulated drug intake observed in long-access models of addiction but might be due to impaired response inhibition or impaired tracking of the reward value of drug-related cues. Grakalic I, Panlilio LV, Quiroz C, Schindler CW. Neuroscience 2010 Jan 20;165(2):313-324.

Interactions Between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors
The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, IRP scientists provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents. Navarro G, Aymerich MS, Marcellino D, et al. J Biol Chem. 2009;284(41):28058-28068.

GPCR Homomers and Heteromers: A Better Choice As Targets For Drug Development Than GPCR Monomers?
G protein-coupled receptors (GPCR) are targeted by many therapeutic drugs marketed to fight against a variety of diseases. Selection of novel lead compounds are based on pharmacological parameters obtained assuming that GPCR are monomers. However, many GPCR are expressed as dimers/oligomers. Therefore, drug development may consider GPCR as homo- and hetero-oligomers. A two-state dimer receptor model is now available to understand GPCR operation and to interpret data obtained from drugs interacting with dimers, and even from mixtures of monomers and dimers. Heteromers are distinct entities and therefore a given drug is expected to have different affinities and different efficacies depending on the heteromer. All these concepts would lead to broaden the therapeutic potential of drugs targeting GPCRs, including receptor heteromer-selective drugs with a lower incidence of side effects, or to identify novel pharmacological profiles using cell models expressing receptor heteromers. Casad— V, Cortés A, Mallol J, et al. Pharmacol. Ther. 2009;124(2):248-257.

Key Modulatory Role of Presynaptic Adenosine A2A Receptors In Cortical Neurotransmission To the Striatal Direct Pathway
Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, IRP researchers demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders. Quiroz C, Luj‡n R, Uchigashima M, et al. Scientific World Journal. 2009;9:1321-1344.

Calcium-Mediated Modulation of the Quaternary Structure and Function of Adenosine A(2A)-Dopamine D(2) Receptor Heteromers
The adenosine A(2A)-dopamine D(2) receptor heteromer is one of the most studied receptor heteromers. It has important implications for basal ganglia function and pathology. Recent studies using Bioluminescence and Sequential Resonance Energy Transfer techniques shed light on the role of Ca(2+) in the modulation of the quaternary structure of the A(2A)-D(2) receptor heteromer, which was found to depend on the binding of calmodulin (CaM) to the carboxy-terminus of the A(2A) receptor in the A(2A)-D(2) receptor heteromer. Importantly, the changes in quaternary structure correlate with changes in function. A Ca(2+)/CaM-dependent modulation of MAPK signaling upon agonist treatment could be observed in cells expressing A(2A)-D(2) receptor heteromers. These studies provide a first example of a Ca(2+)-mediated modulation of the quaternary structure and function of a receptor heteromer. Ferré S, Woods AS, Navarro G, Aymerich M, Llu’s C, Franco R. Curr. Opin. Pharmacol. 2009 Nov 5. [Epub ahead of print]

Chemical Biology Research Branch

Synthesis, Radiosynthesis and In Vivo Evaluation Of [123I]-4-(2-(Bis(4-Fluorophenyl) Methoxy) Ethyl)-1-(4-Iodobenzyl)Piperidine as a Selective Tracer For Imaging the Dopamine Transporter
Dopamine transporter (DAT) neuroimaging is a useful tool in Parkinson's disease diagnosis, staging and follow-up providing information on the integrity of the dopaminergic neurotransmitter system in vivo. 4-(2-(Bis(4-fluorophenyl)methoxy)ethyl)-1-(4-iodobenzyl)piperidine (7) has nanomolar affinity for DAT and better selectivity over the other monoamine transporters compared with the existing SPECT radioligands for DAT. The aim of this study was to synthesize and evaluate [123I]-7 as an in vivo tracer for DAT.The tributylstannyl precursor was synthesized with an overall yield of 25%. [123I]-7 was synthesized by electrophilic destannylation with a yield of 40±10%. Radiochemical purity appeared to be >98%, whereas specific activity was at least 667 GBq/µmol. Biodistribution studies in mice showed brain uptake of 0.96±0.53%ID/g at 30 s post injection (p.i.) and 0.26±0.02%ID/g at 3 h p.i. High blood activity was observed at all time points. Pretreatment with Cyclosporin A raised brain uptake indicating that [123I]-7 is transported by P-glycoprotein (P-gp) pumps. In rats, regional brain distribution of [123I]-7 was not in agreement with DAT distribution. These results indicate that [123I]-7 is not suitable for mapping DAT in vivo but could be a useful tracer for the P-gp transporter. De Bruyne S, Boos TL, Wyffels L, et al. J Labelled Comp Radiopharm. 2009;52(8):304-311.

[76Br]BMK-I-152, A Non-Peptide Analogue For PET Imaging of Corticotropin-Releasing Hormone Type 1 Receptor (CRHR1)
The study of corticotropin-releasing hormone is of significant interest in mental health. IRP investigators have developed a radiobromination procedure for the preparation of [76Br]BMK-I-152, a high-affinity corticotropin-releasing hormone type 1 receptor antagonist. The radiobromination procedure resulted in the formation of two radiobrominated products from the same trialkyltin precursor. Utilizing the results of several reaction conditions and the chromatographic and mass spectral data obtained from Waters Acquity and Q-TOF, the authors determined that both 3-bromo and 4-bromo isomers could be obtained. The authentic sample of the 3-bromo isomer was prepared to confirm the identity of a previously unknown radioactive side product; affinity assays revealed that the 4-bromo isomer had 70 times higher affinity than that of the 3-bromo compound. By manipulation of reaction conditions, the individual products could be selected. Under no-carrier-added conditions at room temperature in aqueous acetonitrile, the major radioactive product (>80%) was identified as the 3-[76Br]bromo-4-tributylstannyl analogue of BMK-I-152. The 4-[76Br]bromo isomer accounted for less than 1% of the total activity. The 3-[76Br]bromo BMK-I-152 could be obtained by treating this intermediate with trifluoroacetic acid to effect removal of the trialkyltin. If the radiobromination was conducted after first evaporating the water from the aqueous ammonium hydroxide solution of [76Br]bromide, the desired 4-[76Br]bromo isomer was obtained with a 58% radiochemical yield. Lang L, Ma Y, Kim BM, et al. J Labelled Comp Radiopharm. 2009;52(9):394-400.

Evidence That Opioids May Have Toll-Like Receptor 4 and MD-2 Effects
Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-(TLR4) was examined using in vitro, in vivo, and in silico techniques.Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling. Hutchinson MR, Zhang Y, Shridhar M, et al. Brain Behav Immun. 2010;24(1): 83-85. Epub 2009 Aug 10.

Evidence For a Role of Heat Shock Protein-90 In Toll Like Receptor 4 Mediated Pain Enhancement In Rats
Spinal cord microglial toll-like receptor 4 (TLR4) has been implicated in enhancing neuropathic pain and opposing morphine analgesia. The present study was initiated to explore TLR4-mediated pain modulation by intrathecal lipopolysaccharide, a classic TLR4 agonist. However, IRP researchers'initial study revealed that intrathecal lipopolysaccharide failed to induce low-threshold mechanical allodynia in naive rats, suggestive that TLR4 agonism may be insufficient to enhance pain. These studies explore the possibility that a second signal is required; namely, heat shock protein-90 (HSP90). This candidate was chosen for study given its known importance as a regulator of TLR4 signaling. A combination of in vitro TLR4 cell signaling and in vivo behavioral studies of pain modulation suggest that TLR4-enhancement of neuropathic pain and TLR4-suppression of morphine analgesia each likely require HSP90 as a cofactor for the effects observed. In vitro studies revealed that dimethyl sulfoxide (DMSO) enhances HSP90 release, suggestive that this may be a means by which DMSO enhances TLR4 signaling. While 2 and 100 microg lipopolysaccharide intrathecally did not induce mechanical allodynia across the time course tested, co-administration of 1 microg lipopolysaccharide with a drug that enhances HSP90-mediated TLR4 signaling now induced robust allodynia. In support of this allodynia being mediated via a TLR4/HSP90 pathway, it was prevented or reversed by intrathecal co-administration of a HSP90 inhibitor, a TLR4 inhibitor, a microglia/monocyte activation inhibitor (as monocyte-derived cells are the predominant cell type expressing TLR4), and interleukin-1 receptor antagonist (as this proinflammatory cytokine is a downstream consequence of TLR4 activation). Together, these results suggest for the first time that TLR4 activation is necessary but not sufficient to induce spinally mediated pain enhancement. Rather, the data suggest that TLR4-dependent pain phenomena may require contributions by multiple components of the TLR4 receptor complex. Hutchinson MR, Ramos KM, Loram LC, et al. Neuroscience. 2009 Dec 29;164(4):1821-32. Epub 2009 Sep 27.

Stress Induced Potentiation Of Cocaine Reward Is Dependent On CRF1 Receptor and CREB
Both clinical and preclinical research have shown that stress can potentiate drug use; however, the underlying mechanisms of this interaction are unknown. Previously, IRP scientists have shown that a single exposure to forced swim (FS) reinstates extinguished conditioned place preference (CPP) to cocaine and that cAMP response element binding protein (CREB) is necessary for this response. CREB can be activated by corticotropin releasing factor (CRF) receptor type 1 (CRFR1) binding, which mediates neuroendocrine and behavioral responses to stress as well as to drugs of abuse. The present experiments investigate whether changes in cocaine reward elicited by previous exposure to stress are mediated by CREB and/or CRFR1. Chronic exposure to FS in advance of conditioning enhances cocaine CPP in wild-type mice, but this is blocked in CREB-deficient mice. In addition, pretreatment with the CRFR1 antagonist, antalarmin, before FS exposure blocks this stress-induced enhancement of cocaine CPP. Furthermore, FS-induced increase in phosphorylated CREB (pCREB), specifically in the lateral septum (LS) and nucleus accumbens (NAc) is also blocked by antalarmin. Taken together, these studies suggest that both CREB and CRFR1 activation are necessary for stress-induced potentiation of drug reward. Kreibich AS, Cleck JN, Rice KC, et al. Neuropsychopharm. 2009;34(12):2609-2617.

Discriminative Stimulus (R)-(+)-{Alpha}-(2,3-Dimethoxyphenyl)-1-[2-(4-Fluorophenyl)Ethyl]-4-Pipidinemethanol (MDL100907) In Rats
Very little is known about constitutive activity in vivo. This study examined whether constitutive activity and inverse agonism contribute to discriminative stimulus effects of drugs acting at serotonin (5-HT)(2A) receptors. Rats were trained to discriminate between saline and either 0.56 mg/kg 5-HT(2) receptor agonist 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), 1.0 mg/kg 5-HT(2A) receptor antagonist ketanserin, or 0.1 mg/kg purported 5-HT(2A) receptor inverse agonist (R)-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-pipidinemethanol (MDL100907). Discriminative control was established with each drug after 33 to 35 sessions. MDL100907 and ketanserin did not occasion DOM lever responding but attenuated the discriminative stimulus effects of DOM. DOM did not occasion responding on the drug-associated lever in rats discriminating MDL100907 or ketanserin, but attenuated the discriminative stimulus effects of both drugs. Ketanserin and ritanserin occasioned MDL100907-lever responding, whereas rats discriminating ketanserin responded only partially on the drug-associated lever after receiving MDL100907, ritanserin, or the alpha(1)-adrenergic antagonist prazosin. Combining prazosin with MDL100907 or ritanserin resulted in near-complete ketanserin-lever responding, indicating that the ketanserin stimulus involves both 5-HT(2A) and alpha(1)-adrenergic receptors. Administration of p-chlorophenylalanine methyl ester, then fenfluramine, significantly decreased cortical 5-HT, enhanced sensitivity to the discriminative stimulus effects of DOM, and occasioned partial MDL100907-lever responding. Collectively, these results show that DOM and MDL100907 discriminative stimulus effects are mediated by 5-HT(2A) receptors and that ketanserin discriminative stimulus effects involve both 5-HT(2A) and alpha(1)-adrenergic receptors. Results in 5-HT-depleted rats further suggest that the discriminative stimulus effects of MDL100907 might involve antagonism of endogenous 5-HT and/or inverse agonism at 5-HT(2A) receptors. Li JX, Unzeitig A, Javors MA, Rice KC, Koek W, France CP. J Pharmacol Exp Ther. 2009 Nov;331(2):671-679. Epub 2009 Aug 17.

The "Toll" Of Opioid-Induced Glial Activation: Improving the Clinical Efficacy of Opioids By Targeting Glia
Glial activation participates in the mediation of pain including neuropathic pain, due to release of neuroexcitatory, proinflammatory products. Glial activation is now known to occur in response to opioids as well. Opioid-induced glial activation opposes opioid analgesia and enhances opioid tolerance, dependence, reward and respiratory depression. Such effects can occur, not via classical opioid receptors, but rather via non-stereoselective activation of toll-like receptor 4 (TLR4), a recently recognized key glial receptor participating in neuropathic pain as well. This discovery identifies a means for separating the beneficial actions of opioids (opioid receptor mediated) from the unwanted side-effects (TLR4/glial mediated) by pharmacologically targeting TLR4. Such a drug should be a stand-alone therapeutic for treating neuropathic pain as well. Excitingly, with newly-established clinical trials of two glial modulators for treating neuropathic pain and improving the utility of opioids, translation from rats-to-humans now begins with the promise of improved clinical pain control.Watkins LR, Hutchinson MR, et al. Trends Pharmacol.Sci.2009;30(11):581-591.

CRF System Recruitment Mediates Dark Side of Compulsive Eating
Dieting to control body weight involves cycles of deprivation from palatable food that can promote compulsive eating. The present study shows that rats withdrawn from intermittent access to palatable food exhibit overeating of palatable food upon renewed access and an affective withdrawal-like state characterized by corticotropin-releasing factor-1 (CRF(1)) receptor antagonist-reversible behaviors, including hypophagia, motivational deficits to obtain less palatable food, and anxiogenic-like behavior. Withdrawal was accompanied by increased CRF expression and CRF(1) electrophysiological responsiveness in the central nucleus of the amygdala. IRP investigators propose that recruitment of anti-reward extrahypothalamic CRF-CRF(1) systems during withdrawal from palatable food, analogous to abstinence from abused drugs, may promote compulsive selection of palatable food, undereating of healthier alternatives, and a negative emotional state when intake of palatable food is prevented. Cottone P, Sabino V, Roberto M, et al. PNAS USA. 2009 Nov 24;106(47):20016-20020.

Probes For Narcotic Receptor Mediated Phenomena. 40. N-Substituted Cis-4a-Ethyl-1,2,3,4,4a ,9a-Hexahydrobenzofuro[2,3-C]Pyridin-8-Ols
A series of N-substituted rac-cis-4a-ethyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-8-ols have been prepared using a simple synthetic route previously designed for synthesis of related cis-2-methyl-4a-alkyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ols. The new phenolic compounds, where the aromatic hydroxy moiety is situated ortho to the oxygen atom in the oxide-bridged ring, do not interact as well as the pyridin-6-ols with opioid receptors. The N-para-fluorophenethyl derivative had the highest μ-opioid receptor affinity of the examined compounds (Ki = 0.35 μM). Iyer MR, Lee YS, Deschamps JR, et al. Bioorg. Med. Chem. Epub 2009 Nov 18.

Effects of Dose and Route of Administration on Pharmacokinetics of (±)-3,4-Methylenedioxy-methamphetamine (MDMA) in the Rat
Based on animal data, there is speculation that (±)-3,4-methylenedioxymethamphetamine (MDMA) is neurotoxic to humans. Extrapolation of MDMA findings from animals to humans requires assessment of pharmacokinetics in various species, and low-dose administration data from rats are lacking. Here IRP scientists examine MDMA pharmacokinetics in rats given low (2 mg/kg) and high (10 mg/kg) doses of racemic MDMA via i.p., s.c. and p.o. routes. Repeated blood specimens were collected from venous catheters, and plasma was assayed for MDMA and its metabolites, 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphe-tamine (MDA), by gas chromatography mass spectrometry. After 2 mg/kg, maximum MDMA concentrations (Cmax) were ~200 ng/mL for i.p. and s.c. routes, but less for the p.o. route. MDMA plasma half-lives were < 1 h for low-dose groups, while HMMA and MDA half-lives were > 2 h. After 10 mg/kg, MDMA areas-under-the-curve (AUC) were 21-fold (i.p.), 10-fold (s.c.) and 36-fold (p.o.) greater than those at 2 mg/kg. By contrast, HMMA AUC values in high-dose groups were < 3-fold above those at 2 mg/kg. Several new findings emerge from this report of low-dose MDMA pharmacokinetics in rats. First, 2 mg/kg MDMA in rats can produce MDMA Cmax similar to those in humans, perhaps explaining why both species discriminate 1.5 mg/kg MDMA in laboratory paradigms. Second, these data provide additional support for non-linear kinetics of MDMA in rats, and analogous to humans, this phenomenon appears to involve impaired drug metabolism. Finally, given key similarities between MDMA pharmacokinetics in rats and humans, data from rats may be clinically-relevant when appropriate dosing conditions are employed. Baumann MH, Zolkowska D, Kim I, Scheidweiler KB, et al. Effects of dose and route of administration on pharmacokinetics of (±)-3,4-methylenedioxymethamphetamine (MDMA) in the rat. Drug Metab Dispos. 2009;37:2163-2170.

Identification of a Novel "Almost Neutral" Mu-Opioid Receptor Antagonist In CHO Cells Expressing the Cloned Human Mu-Opioid Receptor
The basal (constitutive) activity of G protein-coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The mu-opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. IRP scientists therefore sought to identify novel MOR inverse agonists and novel neutral MOR antagonists in both untreated and agonist-treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR-CHO cells) were incubated for 20 h with medium (control) or 10 muM (2S,4aR,6aR,7R,9S, 10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10 b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin, HERK). HERK treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [(35)S]-GTP-gamma-S assays were conducted using established methods. The authors screened 21 MOR "antagonists" using membranes prepared from HERK-treated hMOR-CHO cells. All antagonists, including CTAP and 6beta-naltrexol, were inverse agonists. However, LTC-274 ((-)-3-cyclopropylmethyl-2,3,4,4alpha, 5,6,7,7alpha-octahydro-1H-benzofuro[ 3,2-e]isoquinolin-9-ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [(35)S]-GTP-gamma-S binding. Other efforts in this study identified KC-2-009 ((+)-3-((1R,5S)-2-((Z)-3-phenylallyl)-2-azabicyclo[3.3.1]nonan-5-yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK-treated cells, KC-2-009 had the highest efficacy as an inverse agonist. In summary, the authors identified a novel and selective MOR inverse agonist (KC-2-009) and a novel MOR antagonist (LTC-274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC-274 is a promising lead compound for developing a true MOR neutral antagonist. Sally EJ, Xu H, Dersch CM, et al. Identification of a novel "almost neutral" mu-opioid receptor antagonist in CHO cells expressing the cloned human mu-opioid receptor. 2009 Synapse 64:280-288.

Cellular Neurobiology Research Branch

Development and Plasticity Section, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Human Embryonic Stem Cells Which Express Hrgfp In the Undifferentiated State and During Dopaminergic Differentiation
Human embryonic stem cells (hESCs) which express a reporter gene consistently during all phases of differentiation would be valuable for basic research on cell transplantation. In this study, IRP investigators describe karyotypically-abnormal variant hESCs, BGO1V2-EFG, which express hrGFP driven by the EF1 promoter. BGO1V2-EFG cells were analyzed by using immunocytochemistry, single cell-based confocal image, and in vitro differentiation, including dopaminergic differentiation. Undifferentiated BGO1V2-EFG cells expressed pluripotent ESC markers and retained the ability to differentiate into cell types of all three germ layers. BGO1V2-EFG cells maintained stable and robust hrGFP expression in vitro in the undifferentiated state and during differentiation. The EF1 promoter retained activity during dopaminergic differentiation, as 76% of tyrosine hydroxlase (TH)-positive cells co-expressed hrGFP by confocal analysis. Treated with sodium butyrate (0.02 mM to 2.0 mM), an inhibitor of histone deacetylase (HDAC), during differentiation did not affect hrGFP expression, although TH expression was reduced by higher concentrations of sodium butyrate. BGO1V2-EFG cells maintain stable and robust hrGFP expression in the undifferentiated state and during neural differentiation. Especially, the EF1 promoter was effective in driving hrGFP expression during dopaminergic differentiation. BGO1V2-EFG cells may be useful for transplantation studies in Parkinson disease animal models. Chen J, Tsai SY, Vazin T, Coggiano M, Freed WJ. Human embryonic stem cells which express hrGFP in the undifferentiated state and during dopaminergic differentiation. Restor Neurol Neurosci. 2009;27(4):359-370.

A Novel Combination of Factors, Termed SPIE, Which Promotes Dopaminergic Neuron Differentiation From Human Embryonic Stem Cells
Stromal-Derived Inducing Activity (SDIA) is one of the most efficient methods of generating dopaminergic (DA) neurons from embryonic stem cells (ESC). DA neuron induction can be achieved by co-culturing ESC with the mouse stromal cell lines PA6 or MS5. The molecular nature of this effect, which has been termed "SDIA" is so far unknown. Recently, IRP researchers found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC). In the present study, the authors compared PA6 cells to various cell lines lacking the SDIA effect, and employed genome expression analysis to identify differentially-expressed signaling molecules. Among the factors highly expressed by PA6 cells, and known to be associated with CNS development, were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these four factors, the combination of which was termed SPIE, were applied to hESC, they induced differentiation to TH-positive neurons in vitro. RT-PCR and western blot analysis confirmed the expression of midbrain specific markers, including engrailed 1, Nurr1, Pitx3, and dopamine transporter (DAT) in cultures influenced by these four molecules. Electrophysiological recordings showed that treatment of hESC with SPIE induced differentiation of neurons that were capable of generating action potentials and forming functional synaptic connections. The combination of SDF-1, PTN, IGF2, and EFNB1 mimics the DA phenotype-inducing property of SDIA and was sufficient to promote differentiation of hESC to functional midbrain DA neurons. These findings provide a method for differentiating hESC to form DA neurons, without a requirement for the use of animal-derived cell lines or products. Vazin T, Becker KG, Chen J, et al. A novel combination of factors, termed SPIE, which promotes dopaminergic neuron differentiation from human embryonic stem cells. PLoS One. 2009 Aug 12;4(8):e6606.

Electrophysiology Research Section, Cellular Neurobiology Research Branch

Nogo Receptor 1 Regulates Formation Of Lasting Memories
Formation of lasting memories is believed to rely on structural alterations at the synaptic level. IRP scientists had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. They now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction. Karlén A, Karlsson TE, Mattsson A, et al. Nogo receptor 1 regulates formation of lasting memories. Proc Natl Acad Sci USA. 2009 Nov 13. [Epub ahead of print].

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Expression Pattern of NuIP Gene In Adult Mouse Brain
IRP investigators previously reported the identification of the Nurr1 interacting protein (NuIP) that was demonstrated to modulate the transcriptional activity of Nurr1, the orphan nuclear receptor required for midbrain dopaminergic neuron differentiation. NuIP was also cloned by others and referred to as a small G protein signaling modulators. The open reading frame of NuIP predicts a protein with an N-terminal RUN domain (RPIP8, UNC-14, and NESCA) and a C-terminal TBC domain (Tre-2, Bub2, and Cdc16) both of which are found in proteins of the GTPase activating protein (GAP) family, involved in the GTPase signaling pathway. To characterize the NuIP gene product, the authors developed a polyclonal antibody. Since NuIP gene is expressed most abundantly in adult and the level of expression during development is below the detection limit of immunohistochemistry, we now report the expression pattern of NuIP in adult mouse brain compared with the expression pattern of Nurr1 protein. Many regions co-expressed Nurr1 and NuIP including cortex, hippocampus, substantia nigra, and the cerebellum. However, there are also regions that exclusively express NuIP such as striatum, septum, globus pallidus, and the reticular thalamic nucleus. The authors also find that NuIP protein expresses mainly in NeuN-positive (neuronal nuclei) neurons but can be detected in GFAP-positive (glial fibrillary acidic protein) glial cells in hippocampus. Interestingly, NuIP is expressed in high levels in midbrain dopaminergic neurons including ventral tegmental area (VTA) and substantia nigra (SN) dopaminergic neurons but is not expressed or expressed in low levels in other dopaminergic neurons such as olfactory bulb and hypothalamus. Overall, the expression pattern of NuIP in adult mouse brain suggests that it may be involved in motor activity control in basal ganglia as well as higher central nervous system (CNS) functions such as cognition and memory in cortex and hippocampus. Luo Y, Sarabi SA, Backman C, Shan L, Hoffer B, Federoff H. Expression pattern of NuIP gene in adult mouse brain. Brain Res. 2009;1302:42-53.

Selective Deletion Of PTEN In Dopamine Neurons Leads To Trophic Effects and Adaptation of Striatal Medium Spiny Projecting Neurons
The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. PTEN deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate PTEN in dopamine (DA) neurons (PTEN KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in PTEN KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinson's disease, and other neurodegenerative disorders. Diaz-Ruiz O, Zapata A, Shan L, Zhang Y, Tomac AC, Malik N, de la Cruz F, BŠckman CM. Selective deletion of PTEN in dopamine neurons leads to trophic effects and adaptation of striatal medium spiny projecting neurons. PLoS One. 2009;4(9):e7027.

MAM: More Than Just A Housekeeper
The physical association between the endoplasmic reticulum (ER) and mitochondria, known as the mitochondria-associated ER membrane (MAM), plays important roles in various cellular functions including the highly efficient transmission of Ca2+ from the ER to mitochondria to stimulate the production of ATP from oxidative metabolism and, conversely, may enable the metabolically energized mitochondria to regulate the ER Ca2+ homeostasis. These two major functions affect each other. Recent studies have shed light on the molecular chaperones (e.g., calnexin, calreticulin, ERp44, ERp57, grp75, the sigma-1 receptor) at the interface that regulate the association between the two organelles. The MAM thus integrates signal transduction with energy metabolism to regulate the communication and functional interactions between the ER and mitochondrion. Hayashi T, Rizzuto R, Hajnoczky G, Su TP. Trends in Cell Biology. 2009;19:81-88.

When the Endogenous Hallucinogenic Trace Amine N,N-Dimethyltryptamine Meets the Sigma-1 Receptor
N,N-dimethyltryptamine (DMT) is a hallucinogen found endogenously in human brain that is commonly recognized to target the 5-hydroxytryptamine 2A receptor or the trace amine-associated receptor to exert its psychedelic effect. DMT has been recently shown to bind sigma-1 receptors, which are ligand-regulated molecular chaperones whose function includes inhibiting various voltage-sensitive ion channels. Thus, it is possible that the psychedelic action of DMT might be mediated in part through sigma-1 receptors. Here, IRP scientists present a hypothetical signaling scheme that might be triggered by the binding of DMT to sigma-1 receptors. Specifically, DMT might cause the translocation of sigma-1 receptors from the endoplasmic reticulum to the proximity directly apposing plasma membrane to chaperone the receptors or ion channels on the plasma membrane. Su TP, Hayashi T, Vaupel DB. Science-Signaling. 2009;2:12, online publication.

Delta Opioid Peptide DADLE Causes Cell Cycle Arrest and Differentiation In A CNS Neuronal Progenitor Cell Line
Opioids have been demonstrated to play an important role in CNS development by affecting proliferation and differentiation in various types of neural cells. This study examined the effect of a stable delta opioid peptide [D-Ala(2),D-Leu(5)]-enkephalin (DADLE) on proliferation and differentiation in an AF5 CNS neural progenitor cell line derived from rat mesencephalic cells. DADLE (1 pM, 0.1 nM, or 10 nM) caused a significant growth inhibition on AF5 cells. The opioid antagonist naltrexone at 0.1 nM also caused growth inhibition in the same cells. When DADLE and naltrexone were both added to the AF5 cells, the resultant growth inhibition was apparently additive. DADLE alone or DADLE in combination with naltrexone did not cause apoptosis as evidenced by negative TUNEL staining. The cell-cycle progression analysis indicated that both DADLE (0.1 nM) and naltrexone (0.1 nM) caused an arrest of AF5 cell cycle progression at the G1 checkpoint. Neuronal marker indicated that DADLE- or naltrexone-treated AF5 cells tend to differentiate more when compared to controls. Results demonstrate the non-opioid action of both DADLE and naltrexone on cell cycle arrest and differentiation in a CNS neural progenitor cell line. Results also suggest some potential utilization of DADLE and/or naltrexone in stem cell research. Tsai SY, Lee CT, Hayashi T, Freed WJ, Su TP. Synapse. 2010;64:267-273.

Hibernation-Like State Induced By An Opioid Peptide Protects Against Experimental Stroke
Delta opioid peptide [D-ala2,D-leU5]enkephalin (DADLE) induces hibernation in summer ground squirrels and enhances preservation and survival of isolated or transplanted lungs and hearts. The present study investigated the protective effect of DADLE in the CNS by using a rat model of stroke. Adult Sprague-Dawley rats were pretreated with DADLE (4 mg/kg every 2 hr X 4 injections, i.p.) or saline prior to unilateral occlusion of the middle cerebral artery (MCA). Daily behavioral tests revealed that ischemic animals treated with DADLE did not show any significant behavioral dysfunctions compared to saline-treated ischemic animals. Opioid antagonists only transiently inhibited the protective effect of DADLE indicating the participation of non-opioid mechanisms in DADLE neuroprotection. Histological examination using triphenyltetrazolium chloride revealed that brains from ischemic animals treated with DADLE, either alone or with adjuvant opioid blockers, exhibited almost completely intact striata. In contrast, brains from ischemic animals that received saline showed significant infarction in the lateral striatum. Analyses of apoptotic cell death revealed a significant increase in the p-53 mRNA expression in the striatum of ischemic animals that received saline, while those that received DADLE exhibited near normal striatal p-53 expression. This protective effect was accompanied by significant increments in protein levels of glial cell line-derived neurotrophic factor in the striatum of DADLE-treated ischemic animals. These results indicate that DADLE protected against necrotic and apoptotic cell death processes associated with ischemia-reperfusion injury. The present study demonstrates that delta opioids are crucially involved in stroke suggesting that the opioid system is important in the study of brain injury and protection. Borlongan CV, Hayashi T, Oeltgen PR, Su TP, Wang Y. BMC Biology. 2009;7:31, online publication.

Sigma-1 Receptors Regulate Hippocampal Dendritic Spine Formation Via A Free Radical-Sensitive Mechanism Involving Rac.GTP Pathway
Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER)-resident proteins known to be involved in learning and memory. Dendritic spines in hippocampal neurons play important roles in neuroplasticity and learning and memory. This study tested if Sig-1Rs might regulate denritic spine formation in hippocampal neurons and examined potential mechanisms therein. In rat hippocampal primary neurons the knockdown of Sig-1Rs by siRNAs causes a deficit in the formation of dendritic spines that is unrelated to ER Ca2+ signaling or apoptosis but correlates with the mitochondrial permeability transition and cytochrome c release, followed by caspase-3 activation, Tiam1 cleavage, and a reduction in Rac1įGTP. Sig-1R-knockdown neurons contain higher levels of free radicals when compared to control neurons. The activation of superoxide dismutase or the application of a hydroxyl free radical scavenger N-acetyl cysteine (NAC) to the Sig-1R-knockdown neurons rescues dendritic spines and mitochondria from deficits caused by the Sig-1R siRNA. Further, the caspase-3 resistant TIAM1 construct C1199DN, a stable guanine exchange factor able to constitutively activate Rac1 in the form of Rac1įGTP, also rescues the siRNA-caused dendritic spine deficits. These results implicate Sig-1Rs as endogenous regulators in hippopcampal dendritic spine formation and suggest a free radical-sensitive ER-mitochondrion-Rac1įGTP pathway in the regulation of dendritic spine formation in the hippocampus. The results also suggest the importance of free radicals in the learning and unlearning aspects of neurobiology and provide a potential mechanistic explanation on why an established mucolytic agent NAC might have a therapeutic potential as an antiamnesic agent. Tsai Y, Hayashi T, Harvey B, et al. PNAS USA. 2009; Dec 11; Epubmed ahead of print.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Theoretical Considerations on the Topological Organization of Receptor Mosaics
The concept of Receptor Mosaic (RM) is discussed; hence the integrative functions of the assemblage of G-protein coupled receptors physically interacting in the plane of the plasma membrane. The main focus is on a hetero-trimer of G-protein coupled receptors, namely the A2A-D2-CB1 receptor trimer. A bioinformatics analysis was carried out on the amino acid sequence of these receptors to indicate domains possibly involved in the receptor-receptor interactions. Such a bioinformatic analysis was also carried out on the RM formed by mGLU R5, D2 and A2A. The importance of topology, i.e., of the reciprocal localization of the three interacting receptors in the plan of the membrane for the RM integrative functions is underlined. However, it is also pointed out that this fundamental aspect still awaits techniques capable of an appropriate investigation. Finally, it is discussed how RM topology can give hints for a structural definition of the concept of hub receptor. Thus, just as in any network, the receptor operating as a hub is the one that in the molecular network formed by the receptors has the highest number of inputs. Agnati LF, Fuxe K, Woods AS, Genedani S, Guidolin D. Theoretical considerations on the topological organization of receptor mosaics. Curr Protein Pept Sci. 2009 Sep 15. [Epub ahead of print].

Calcium-Mediated Modulation of the Quaternary Structure and Function of Adenosine A(2A)-Dopamine D(2) Receptor Heteromers
The adenosine A(2A)-dopamine D(2) receptor heteromer is one of the most studied receptor heteromers. It has important implications for basal ganglia function and pathology. Recent studies using bioluminescence and sequential resonance energy transfer techniques shed light on the role of Ca(2+) in the modulation of the quaternary structure of the A(2A)-D(2) receptor heteromer, which was found to depend on the binding of calmodulin (CaM) to the carboxy-terminus of the A(2A) receptor in the A(2A)-D(2) receptor heteromer. Importantly, the changes in quaternary structure correlate with changes in function. A Ca(2+)/CaM-dependent modulation of MAPK signaling upon agonist treatment could be observed in cells expressing A(2A)-D(2) receptor heteromers. These studies provide a first example of a Ca(2+)-mediated modulation of the quaternary structure and function of a receptor heteromer. Ferré S, Woods AS, Navarro G, Aymerich M, Llu’s C, Franco R. Calcium-mediated modulation of the quaternary structure and function of adenosine A(2A)-dopamine D(2) receptor heteromers. Curr Opin Pharmacol. 2009 Nov 5. [Epub ahead of print]

Ammonium Sulfate and MALDI In-Source Decay: A Winning Combination For Sequencing Peptides
In previous papers, IRP investigators highlighted the role of ammonium sulfate in increasing peptide fragmentation by in-source decay (ISD). The current work systematically investigated effects of matrix assisted laser desorption ionization (MALDI) extraction delay, peptide amino acid composition, matrix, and ammonium sulfate concentration on peptide ISD fragmentation. The data confirmed that ammonium sulfate increased peptides signal-to-noise ratio as well as their in-source fragmentation, resulting in complete sequence coverage regardless of the amino acid composition. This method is easy, inexpensive, and generates the peptides sequence instantly. Delvolve A, Woods AS. Ammonium sulfate and MALDI in-source decay: A winning combination for sequencing peptides. Anal Chem. 2009 Oct 30. [Epub ahead of print]

Molecular Neurobiology Research Branch

Neural Protection and Regeneration Section

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Reduces Ischemic Brain Injury and Promotes Behavioral Recovery In Rats
Mesencephalic astrocyte-derived neurotrophic factor (MANF), also known as Arginine-rich, Mutated in Early Stage of Tumors (ARMET), is a secreted protein which reduces endoplasmic reticulum (ER) stress. Previous studies have shown that MANF mRNA expression and protein levels are increased in the cerebral cortex after brain ischemia, a condition which induces ER stress. The function of MANF during brain ischemia is still not known. The purpose of this study was to examine the protective effect of MANF after ischemic brain injury. Recombinant human MANF was administrated locally to the cerebral cortex before a 60-min middle cerebral artery occlusion (MCAo) in adult rats. Triphenyltetrazolium chloride (TTC) staining indicated that pretreatment with MANF significantly reduced the volume of infarction at two days after MCAo. MANF also attenuated TUNEL labeling, a marker of cell necrosis/apoptosis, in the ischemic cortex. Animals receiving MANF pretreatment demonstrated a decrease in body asymmetry and neurological score as well as an increase in locomotor activity after MCAo. Taken together, these data suggest that MANF has neuroprotective effects against cerebral ischemia, possibly through the inhibition of cell necrosis/apoptosis in cerebral cortex. Airavaara M, Shen H, Kuo CC, Peranen J, Saarma M, Hoffer BJ, Wang Y. Mesencephalic astrocyte-derived neurotrophic factor (MANF) reduces ischemic brain injury and promotes behavioral recovery in rats. J Comp Neurol 2009;515:116-124.

Diadenosine Tetraphosphate Reduces Toxicity Caused By High-Dose Methamphetamine Administration
Diadenosine tetraphosphate (AP4A), two adenosine moieties bridged by four phosphates, is an endogenous purinergic ligand found in brain. Previous studies have shown that AP4A reduced neurodegeneration caused by the dopaminergic neurotoxin 6-hydroxydopamine in rat striatum and substantia nigra. The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA) -mediated toxicity. Primary neuronal cultures were prepared from rat embryonic (E14- E15) ventral mesencephalic tissue. Cultures treated with 2 mM MA exhibited decreased tyrosine hydroxylase (TH) immunoreactivity and increased cleaved caspase-3 immunoreactivity and TUNEL labeling. All these changes were lessened by pretreatment with AP4A. The protective effect of AP4A was also found in vivo. Adult Sprague-Dawley rats were injected with AP4A (25 µg/ 20 µl) or vehicle intracerebroventricularly followed by 4 doses of MA (5 or 10 mg/ kg), given subcutaneously every two hours. Administration of MA reduced locomotor activity one day after injection, which was significantly antagonized by the pretreatment with AP4A. Using immunohistochemical analysis, TH fiber density at the substantia nigra pars reticulata was found reduced while cleaved caspase-3 immunoreactivity in striatum was increased after MA treatment; these responses were also significantly antagonized by AP4A. Taken together, our data show that AP4A has protective effects against MA-mediated toxicity both in vitro and in vivo. The mechanism of action involves suppression of MA -induced apoptosis. Harvey BK, Chou J, Shen H, Hoffer BJ, Wang Y. Diadenosine tetraphosphate reduces toxicity caused by high-dose methamphetamine administration. Neurotoxicology 2009; 30:436-444.

GLP-1 Receptor Stimulation Preserve Primary Cortical and Dopaminergic Neurons In Cellular and Rodent Models Of Stroke and Parkinsonism
Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic peptide secreted from the gastrointestinal tract in response to food intake. It enhances pancreatic islet beta-cell proliferation and glucose-dependent insulin secretion, and lowers blood glucose and food intake in patients with type 2 diabetes mellitus (T2DM). A long-acting GLP-1 receptor (GLP-1R) agonist, exendin-4 (Ex-4), is the first of this new class of antihypergly-cemia drugs approved to treat T2DM. GLP-1Rs are coupled to the cAMP second messenger pathway and, along with pancreatic cells, are expressed within the nervous system of rodents and humans, where receptor activation elicits neurotrophic actions. IRP scientists detected GLP-1R mRNA expression in both cultured embryonic primary cerebral cortical and ventral mesencephalic (dopaminergic) neurons. These cells are vulnerable to hypoxia- and 6-hydroxydopamine-induced cell death, respectively. They found that GLP-1 and Ex-4 conferred protection in these cells, but not in cells from Glp1r knockout (-/-) mice. Administration of Ex-4 reduced brain damage and improved functional outcome in a transient middle cerebral artery occlusion stroke model. Ex-4 treatment also protected dopaminergic neurons against degeneration, preserved dopamine levels, and improved motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). These findings demonstrate that Ex-4 can protect neurons against metabolic and oxidative insults, and they provide preclinical support for the therapeutic potential for Ex-4 in the treatment of stroke and PD. Li Y, Perry T, Kindy MS, et al. GLP-1 receptor stimulation preserve primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism. PNAS USA 2009;106:1285-1290.

Delayed Treatment With A P53 Inhibitor Enhances Recovery In Stroke Brain
Cerebral ischemia can activate endogenous reparative processes, such as proliferation of endogenous neural progenitor cells (NPCs) in the subventricular zone (SVZ). Most of these new cells die shortly after injury. The purpose of this study was to examine a novel strategy for treatment of stroke at one week after injury by enhancing the survival of ischemia-induced endogenous NPCs in SVZ. Adult rats were subjected to a 90-min middle cerebral artery occlusion (MCAo). A p53 inhibitor pifithrin-alpha (PFT-a) was administered to stroke rats from days 6 to 9 after MCAo. Locomotor behavior was measured using an activity chamber. Proliferation, survival, migration, and differentiation of endogenous NPCs were examined using qRT-PCR, TUNEL, and immunohistochemistry. IRP researchers found that PFT-a enhanced functional recovery as assessed by a significant increase in multiple behavioral measurements. Delayed PFT-a treatment had no effect on the cell death processes in the lesioned cortical region. However, it enhanced the survival of SVZ progenitor cells and promoted their proliferation and migration. PFT-a inhibited the expression of a p53-dependent pro-apoptotic gene, termed PUMA (p53-upregulated modulator of apoptosis), within the SVZ of stroke animals. The enhancement of survival/proliferation of NPCs was further found in SVZ neurospheres in tissue culture. PFT-a dose-dependently increased the number and size of new neurosphere formation. In conclusion, our data suggest that delayed treatment with a p53 inhibitor PFT-a is able to modify stroke-induced endogenous neurogenesis and improve the functional recovery in stroke animals. Luo Y, Kuo CC, Shen H, Chou J, Greig NH, Hoffer BJ, Wang Y. Delayed treatment with a p53 inhibitor enhances recovery in stroke brain. Ann Neurol 2009;65:520-530.

9-Cis-Retinoic Acid Reduces Ischemic Brain Injury In Rodents Via Bone Morphogenetic Protein
Retinoic acid (RA), a biologically active derivative of vitamin A, has protective effects against damage caused by H2O2 or oxygen-glucose deprivation in mesangial and PC12 cells. In cultured human osteosarcoma cells, RA enhances the expression of bone morphogenetic protein -7 (BMP7), a trophic factor that reduces ischemia- or neurotoxin -mediated neurodegeneration in vivo. The purpose of this study is to examine whether RA reduces ischemic brain injury through a BMP7 mechanism. The authors found that intracerebroventricular administration of 9-cis-retinoic acid (9cRA) enhanced BMP7 mRNA expression, detected by RTPCR, in rat cerebral cortex at 24 hours after injection. Rats were also subjected to transient focal ischemia induced by ligation of the middle cerebral artery (MCA) at one day after 9cRA injection. Pretreatment with 9cRA increased locomotor activity and attenuated neurological deficits 2 days after MCA ligation. 9cRA also reduced cerebral infarction and TUNEL labeling. These protective responses were antagonized by BMP antagonist noggin given at one day after 9cRA injection. Taken together, these data suggest that 9cRA has protective effects against ischemia -induced injury and these effects involve BMPs. Shen H, Luo Y, Kuo CC, Deng X, et al. 9-Cis-Retinoic acid reduces ischemic brain injury in rodents via bone morphogenetic protein . J Neurosci Res 2009; 87:545-555.

Astaxanthin Reduces Ischemic Brain Injury In Adult Rats
Astaxanthin (ATX) is a dietary carotenoid of crustaceans and fish that contributes to their coloration. Dietary ATX is important for development and survival of salmonids and crustaceans and has been shown to reduce cardiac ischemic injury in rodents. The purpose of this study was to examine whether ATX can protect against ischemic injury in the mammalian brain. Adult rats were injected intracerebroventricularly with ATX or vehicle prior to a 60-min middle cerebral artery occlusion (MCAo). Treatment with ATX, compared to vehicle, increased locomotor activity in stroke rats and reduced cerebral infarction at 2 days after MCAo. To evaluate the protective mechanism(s) of ATX against stroke, brain tissues were assayed for free radical damage, apoptosis and excitoxicity. ATX antagonized ischemia -mediated loss of aconitase activity and reduced glutamate release, lipid peroxidation, translocation of cytochrome C and TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as body temperature, brain temperature, cerebral blood flow, blood gases, blood pressure, and pH. Collectively, these data suggest that ATX can reduce ischemia-related injury in brain through the inhibition of oxidative stress, reduction of glutamate release, and anti-apoptosis. ATX may be clinically useful for patients vulnerable or prone to ischemic events. Shen H, Kuo CC, Chou J, et al. Astaxanthin reduces ischemic brain injury in adult rats. FASEB J 2009;23:1958-1968.

Severity of Controlled Cortical Impact Traumatic Brain Injury In Rats and Mice Dictates Degree Of Behavioral Deficits
The clinical presentation of traumatic brain injury (TBI) involves either mild, moderate, or severe injury to the head resulting in long-term and even permanent disability. The recapitulation of this clinical scenario in animal models should allow examination of the pathophysiology of the trauma and its treatment. To date, only a few studies have demonstrated TBI animal models encompassing the three levels of trauma severity. Thus, in the present study IRP scientists characterized in mice and rats both brain histopathologic and behavioral alterations across a range of injury magnitudes arising from mild, moderate, and severe TBI produced by controlled cortical impact injury technique. Here, they replicated the previously observed TBI severity-dependent brain damage as revealed by 2,3,5-triphenyltetrazolium chloride staining (severe > moderate > mild) in rats, but also extended this pattern of histopathologic changes in mice. Moreover, they showed severity-dependent abnormalities in locomotor and cognitive behaviors in TBI-exposed rats and mice. Taken together, these results support the use of rodent models of TBI as a sensitive platform for investigations of the injury-induced neurostructural and behavioral deficits, which should serve as key outcome parameters for testing experimental therapeutics. Yu S, Kaneko Y, Bae E, et al. Severity of controlled cortical impact traumatic brain injury in rats and mice dictates degree of behavioral deficits. Brain Res 2009;1287:157-163.

Psychobiology Section, Medications Discovery Research Branch

Reinforcing Effects Of ∑-Receptor Agonists In Rats Trained To Self-Administer Cocaine
Sigma receptor (σR) antagonists have been reported to block certain effects of psychostimulant drugs IRP researchers found that these same σR antagonists did not affect cocaine self-administration. Interestingly, these drugs block conditioned place preference induced by cocaine indicating that the two procedures used to assess abuse liability of drugs tap different aspects of the activity of drugs of abuse. In contrast to the inactivity of σR antagonists against cocaine self administration, σR agonists were effective reinforcers in subjects trained to self administer cocaine. In addition, σR agonists, like dopamine uptake inhibitors, potentiated the reinforcing effects of cocaine in the self-administration procedure. The σR antagonists antagonized the self administration of σR agonists, despite their inactivity against cocaine. Response rates maintained by maximally effective doses of σR agonists were selectively decreased by σR antagonists (effective doses did not alter response rates maintained by food reinforcement). Although σR antagonists block some cocaine-induced effects, the lack of effect on cocaine self-administration suggests that the primary reinforcing effects of cocaine do not involve direct effects at σRs. However, the self-administration of σR agonists in cocaine-trained subjects, potentiation of cocaine self-administration by σR-agonists suggest enhanced abuse-related effects resulting from concomitant dopaminergically-mediated actions and σR-mediated actions of the drugs. Hiranita T, Soto PL, Tanda G, Katz JL. Reinforcing effects of σ-receptor agonists in rats trained to self-administer cocaine. JPET Fast Forward. DOI:10.1124/jpet.109.159236.


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