Research Findings - Intramural Research
Cellular Neurobiology Research Branch
Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch
Interactions Between Calmodulin, Adenosine A2A and Dopamine D2 Receptors
The Ca2+-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors (GPCRs), including the dopamine D2 receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D2 receptor, within an Arg-rich epitope that is also involved in the binding to Gi/o proteins and to the adenosine A2A receptor, with the formation of A2A-D2 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 A2A receptor. By using BRET and sequential resonance energy transfer (SRET) techniques, evidence was obtained for CaM-A2A-D2 receptor oligomerization. BRET competition experiments indicated that, in the A2A-D2 receptor heteromer, CaM binds preferentially to a proximal C-terminus epitope of the A2A receptor. Furthermore, Ca2+ was found to induce conformational changes in the CaM-A2A-D2 receptor oligomer and to selectively modulate A2A and D2 receptor-mediated MAPK signaling in the A2A-D2 receptor heteromer. These results may have implications for basal ganglia disorders since A2A-D2 receptor heteromers are being considered as a target for anti-parkinsonian agents. Navarro G, Aymerich MS, Marcellino D, Cortes A, Casado V, Mallol J, Canela EI, Agnati LF, Woods AS, Fuxe K, Lluis C, Lanciego JL, Ferre S, Franco R. Interactions between calmodulin, adenosine A2A and dopamine D2 receptors. J Biol Chem. 2009 Jul 24. [E-pub ahead of print].
Neuroimaging Research Branch
Association of Nicotine Addiction and Nicotine's Actions with Separate Cingulate Cortex Functional Circuits
Understanding the mechanisms underlying nicotine addiction to develop more effective treatment is a public health priority. Research consistently shows that nicotine transiently improves multiple cognitive functions. However, using nicotine replacement to treat nicotine addiction yields generally inconsistent results. Although this dichotomy is well known, the reasons are unclear. Imaging studies showed that nicotine challenges almost always involves the cingulate cortex, suggesting that this locus may be a key region associated with nicotine addiction and its treatment. IRP researchers thus sought to identify cingulate functional circuits that are associated with the severity of nicotine addiction and study how nicotine affects them by means of region-specific resting-state functional magnetic resonance imaging. Clearly separated pathways that correlated with nicotine addiction vs. nicotine's action were found. The severity of nicotine addiction was associated with the strength of dorsal anterior cingulate cortex (dACC)–striatal circuits, which were not modified by nicotine patch administration. In contrast, short-term nicotine administration enhanced cingulate-neocortical functional connectivity patterns, which may play a role in nicotine's cognition-enhancing properties. These data suggest that resting-state dACC-striatum functional connectivity may serve as a circuit-level biomarker for nicotine addiction, and that the development of new therapeutic agents aiming to enhance the dACC-striatum functional pathways may be effective for nicotine addiction treatment. Hong Le, Gu H, Yang Y, Ross TJ, Salmeron BJ, Buchholz B, Thaker GK, Stein EA. Association of nicotine addiction and nicotine's actions with separate cingulate cortex functional circuits. Arch Gen Psych. 2009;66:431-441.
Baseline Expression of a4b2* Nicotinic Acetylcholine Receptors Predicts Motivation to Self-Administer Nicotine
Marked interindividual differences in vulnerability to nicotine dependence exist, but factors underlying such differences are not well understood. The midbrain α4Β2* subtype of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediation of the reinforcing effects of nicotine responsible for dependence. However, no study has been performed evaluating the impact of interindividual differences in midbrain nAChR levels on motivation to self-administer nicotine. As such, baseline levels of α4Β2* nAChRs were measured using 2-[18F]fluoro-A-85380 (2-FA) and positron emission tomography (PET) in five squirrel monkeys. Motivation to self-administer nicotine (number of lever presses) was subsequently measured using a progressive-ratio (PR) schedule of reinforcement. Greater motivation to self-administer nicotine was associated with lower levels of midbrain nAChRs. These results suggest that level of expression of nAChRs is a contributing factor in the development of nicotine dependence. Similarly, it has been previously shown that low levels of dopamine D2 receptors (DRD2) are associated with a higher preference for psychostimulant use in humans and nonhuman primates. Together, results from these PET studies of dopaminergic and nicotinic cholinergic transmission suggest that an inverse relationship between the availability of receptors that mediate reinforcement and the motivation to take drugs exists across different neuro-transmitter systems. Le Foll B, Chefer SI, Kimes AS, Shumway D, Stein EA, Mukhin AG, Goldberg SR. Baseline expression of a4b2* nicotinic acetylcholine receptors predicts motivation to self-administer nicotine. Biological Psychiatry. 2009;65:714-716.
Early Life Stress Induces Long-term Morphological Changes in Primate Brain
Traumatic experiences in early childhood are associated with increased risk of developing stress-related disorders, which are linked to structural brain abnormalities. However, it is unclear whether these volumetric brain changes are present before disease onset or reflect the consequences of disease progression. As such, IRP scientists sought to identify structural abnormalities in the nonhuman primate brain that may predict increased risk of stress-related neuropsychiatric disorders in human beings. Rhesus monkeys were divided into 2 groups at birth: a group raised with their mothers and other juvenile and adult animals (mother reared) and a group raised with 3 age-matched monkeys only (peer reared) for the first 6 months of life. Anatomical brain images were acquired in juvenile male and female rhesus monkeys using magnetic resonance imaging. Volumetric measures of the anterior cingulate cortex, medial prefrontal cortex, hippocampus, corpus callosum, and cerebellar vermis were compared between mother-reared (n = 15) and peer-reared animals (n = 13). Compared with mother-reared monkeys, the authors found an enlarged vermis, dorsomedial prefrontal cortex, and dorsal anterior cingulate cortex in peer-reared monkeys without any apparent differences in the corpus callosum and hippocampus. These changes may be a structural phenotype for increased risk of stress-related neuropsychiatric disorders in human beings. Spinelli S, Chefer SI, Suomi SJ, Higley D, Barr CS, Stein EA. Early life stress induces long-term morphological changes in primate brain. Arch Gen Psych. 2009;66:658-665.
Patients with Schizophrenia Have A Reduced Neural Response to Both Unpredictable and Predictable Primary Reinforcers
One prevalent theory of learning states that dopamine neurons signal mismatches between expected and actual outcomes, called temporal difference errors (TDEs). Evidence indicates that dopamine system dysfunction is involved in negative symptoms of schizophrenia (SZ), including avolition and anhedonia. As such, IRP investigators predicted that brain responses to TDEs in dopamine midbrain nuclei and target areas would be abnormal in SZ. A total of 18 clinically stable patients with chronic SZ and 18 controls participated in an fMRI study, which used a passive conditioning task. In the task, the delivery of a small amount of juice followed a light stimulus by exactly 6 s on approximately 75% of 78 total trials, and was further delayed by 4–7 s on the remaining trials. The delayed juice delivery was designed to elicit the two types of TDE signals, associated with the recognition that a reward was omitted at the expected time, and delivered at an unexpected time. Main effects of TDE valence and group differences in the positive–negative TDE contrast (unexpected juice deliveries–juice omissions) were assessed through whole-brain and regions of interest (ROI) analyses. Main effects of TDE valence were observed for the entire sample in the midbrain, left putamen, left cerebellum, and primary gustatory cortex, bilaterally. Whole-brain analyses revealed group differences in the positive–negative TDE contrast in the right putamen and left precentral gyrus, whereas ROI analyses revealed additional group differences in the midbrain, insula, and parietal operculum, on the right, the putamen and cerebellum, on the left, and the frontal operculum, bilaterally. Further, these group differences were generally driven by attenuated responses in patients to positive TDEs (unexpected juice deliveries), whereas responses to negative TDEs (unexpected juice omissions) were largely intact. Patients also showed reductions in responses to juice deliveries on standard trials, and more blunted reinforcer responses in the left putamen corresponded to higher ratings of avolition. These results provide evidence that SZ patients show abnormal brain responses associated with the processing of a primary reinforcer, which may be a source of motivational deficits. Waltz JA, Schweitzer JB, Gold JM, Kurup PK, Ross TJ, Salmeron BJ, Rose EJ, McClure SM, Stein EA. Patients with Schizophrenia have a reduced neural response to both unpredictable and predictable primary reinforcers. Neuropsychopharmacology. 2009;34:1567-1577.
Mapping Functional Connectivity Based On Synchronized CMRO2 Fluctuations During the Resting State
Synchronized low-frequency fluctuations in the resting state functional MRI (fMRI) signal have been suggested to be associated with functional connectivity in brain networks. However, the underlying mechanism of this connectivity is still poorly understood. Synchronized fluctuations could either originate from hemodynamic oscillations or represent true neuronal signaling. To better interpret the resting signal, in the current work, IRP researchers examined spontaneous fluctuations at the level of cerebral metabolic rate of oxygenation (CMRO2), an index reflecting regional oxygen consumption and metabolism, and thus less sensitive to vascular dynamics. The CMRO2 signal was obtained based on a biophysical model with data acquired from simultaneous blood oxygenation level dependent (BOLD) and perfusion signals. CMRO2-based functional connectivity maps were generated in three brain networks: visual, default-mode, and hippocampus. Experiments were performed on twelve healthy participants during 'resting state' and as a comparison, with a visual task. CMRO2 signals in each of the above mentioned brain networks showed significant correlations. Functional connectivity maps from the CMRO2 signal are, in general, similar to those from BOLD and perfusion. In addition, the authors demonstrated that the three parameters (M, α and Β) in the biophysical model for calculating CMRO2 have negligible effects on the determination of the CMRO2-based connectivity strength. This study provides evidence that the spontaneous fluctuations in fMRI at rest likely originate from dynamic changes of cerebral metabolism reflecting neuronal activity. Wu CW, Gu H, Lu H, Stein EA, Chen J-H, Yang Y. Mapping functional connectivity based on synchronized CMRO2 fluctuations during the resting state. NeuroImage. 2009;45:694-701.
A Single High Dose of Methamphetamine Increases Cocaine Self Administration by Depletion of Striatal Dopamine in Rats
Psychostimulant addicts often take high doses of drugs, and high doses of psychostimulants such as methamphetamine (METH) are neurotoxic to striatal dopamine (DA) terminals. Yet, the effects of high doses of METH on drug-seeking and drug-taking behavior have not been examined. In the present study, IRP scientists found that single high doses of METH in rats (10–20 mg/kg) dose-dependently increased cocaine self-administration under fixed-ratio 2 (FR2) reinforcement conditions, while higher doses (40 mg/kgx1 or 10 mg/kg/2 hx4) caused high mortality among rats maintained on daily cocaine self-administration. The increased cocaine self-administration appeared to be a compensatory response to reduced cocaine reward after METH, because the same doses of METH caused a dose-dependent reduction both in "break-point" levels for cocaine self-administration under progressive-ratio reinforcement and in nucleus accumbens DA response to acute cocaine. Further, METH (10–20 mg/kg) produced large DA release (4000%–6000% over baseline), followed by a significant reduction in striatal DA and 3,4-dihydroxyphenylacetic acid (DOPAC) contents, but without significant changes in striatal DA transporter levels. These findings suggest that the present high doses of METH caused striatal DA depletion or hypofunction without severe damage in DA terminals, which may contribute to the increased cocaine-taking behavior observed in the present study. Provided that the present doses of METH may mimic METH overdose incidents in humans, the present findings suggest that METH-induced DA depletion or neurotoxicity may lead to an increase in subsequent drug-taking and drug-seeking behavior. Xi Z-X, Kleitz H, Deng X, Ladenheim B, Peng X-Q, Li X, Gardner EL, Stein EA, Cadet JL A single high dose of methamphetamine increases cocaine self administration by depletion of striatal dopamine in rats. Neuroscience. 2009;161:392-402.
Clinical Psychopharmacology Section, Chemical Biology Research Branch
The Impact of Early Environmental Rearing Condition on the Discriminative Stimulus Effects and Fos Expression Induced by Cocaine in Adult Male and Female Rats
A number of environmental manipulations, including maternal separation (MS), have been shown to alter behavioral responses to drugs of abuse. This study assessed if MS affected the stimulus and Fos-inducing effects of cocaine. In experiment 1, male and female Sprague-Dawley rats were exposed to brief maternal separations (BMS), long maternal separations (LMS), or animal facility rearing (AFR) and then trained as adults to discriminate cocaine (10 mg/kg, intraperitoneally) from saline. Following training, generalization tests to novel doses of cocaine and other dopaminergic compounds were performed. Assessments of variations in training dose pretreatment times were also made. In experiment 2, male and female rats exposed to MS conditions were administered cocaine or saline for 14 days, and Fos expression in the mesolimbic system was measured. In males, BMS retarded the acquisition of the cocaine discrimination. Generalization to novel doses of cocaine did not differ among rearing conditions, but the training dose cue lasted longer in LMS. Distinct generalization and ED(50) profiles were found between male rearing conditions for all dopamine compounds. While BMS females had higher cocaine ED(50) estimates, no other differences were found in females. LMS males and females, as well as AFR females, had significant increases in Fos expression after cocaine in a region-specific manner. No differences were found with other rearing groups. Early environmental variables altered the stimulus effects (in a sex-dependent manner) as well as the neuronal responsiveness to cocaine, which may be mediated by the dopamine system. Kohut SJ, Roma PG, Davis CM, Zernig G, Saria A, Dominguez JM, Rice KC, Riley AL. The impact of early environmental rearing condition on the discriminative stimulus effects and Fos expression induced by cocaine in adult male and female rats. Psychopharmacology (Berl). 2009 Apr;203(2): 383-397. E-pub 2008 Oct 25.
D2-like Agonist-Induced PE and Yawning are Differentially Mediated by the D3 (Induction) and D2 Receptors (Inhibition)
Dopamine D2-like agonists induce penile erection (PE) and yawning in a variety of species, effects that have recently been suggested to be specifically mediated by the D4 and D3 receptors, respectively. The current studies were aimed at characterizing a series of D2, D3, and D4 agonists with respect to their capacity to induce PE and yawning in the rat, as well as the pro-erectile effects of apomorphine in wild-type and D4 receptor (R) knock-out (KO) mice. All D3 agonists induced dose-dependent increases in PE and yawning over a similar range of doses, whereas significant increases in PE or yawning were not observed with any of the D4 agonists. Likewise, D2, D3, and D4 antagonists were assessed for their capacity to alter apomorphine- and pramipexole-induced PE and yawning. The D3 antagonist, PG01037, inhibited the induction of PE and yawning, whereas the D2 antagonist, L-741,626, reversed the inhibition of PE and yawning observed at higher doses. The D4 antagonist, L-745,870, did not alter apomorphine- or pramipexole-induced PE or yawning. A role for the D3 receptor was further supported as apomorphine was equipotent at inducing PE in wild-type and D4R KO mice, effects that were inhibited by the D3 antagonist, PG01037, in both wild-type and D4R KO mice. Together, these studies provide strong support that D2-like agonist-induced PE and yawning are differentially mediated by the D3 (induction) and D2 receptors (inhibition). These studies fail to support a role for the D4 receptor in the regulation of PE or yawning by D2-like agonists. Collins GT, Truccone A, Haji-Abdi F, Newman AH, Grundt P, Rice K, Husbands SM, Greedy BM, Enguehard-Gueiffier C, Gueiffier A, Chen J, Wang S, Katz JL, Grandy DK, Sunahara RK, Woods JH. Proerectile effects of dopamine D2-like agonists are mediated by the D3 receptor in rats and mice. J Pharm Exp Ther. 2009 Apr;329(1):210-217. E-pub 2009 Jan 9.
The Sigma-Receptor Antagonist BD-1063 Decreases Ethanol Intake and Reinforcement in Animal Models of Excessive Drinking
Sigma-Receptors (SigRs) have been implicated in behavioral and appetitive effects of psychostimulants and may also modulate the motivating properties of ethanol. This study tested the hypothesis that SigRs modulate ethanol reinforcement and contribute to excessive ethanol intake. The effects of subcutaneous treatment with the potent, selective Sig-1R antagonist BD-1063 on operant ethanol self-administration were studied in two models of excessive drinking-Sardinian alcohol-preferring (sP) rats and acutely withdrawn ethanol-dependent Wistar rats-and compared to ethanol self administration in nondependent Wistar controls. To assess the specificity of action, the effects of BD-1063 on self-administration of an equally reinforcing saccharin solution were determined in Wistar and sP rats. Gene expression of Sig-1R in reward-related brain areas implicated in ethanol reinforcement was compared between ethanol-naēve sP and Wistar rats and withdrawn ethanol-dependent Wistar rats. BD-1063 dose dependently reduced ethanol self-administration in sP rats (3.3-11 mg/kg) and withdrawn, dependent Wistar rats (4-11 mg/kg) at doses that did not modify mean ethanol self-administration in nondependent Wistar controls. BD-1063 did not reduce concurrent water self-administration and did not comparably suppress saccharin self administration, suggesting selectivity of action. BD-1063 also reduced the breakpoints of sP rats to work for ethanol under a progressive-ratio reinforcement schedule. Ethanol naive sP rats and 24-h withdrawn, dependent Wistar rats showed reduced Sig-1R mRNA expression in the nucleus accumbens. The results suggest that SigR systems may contribute to innate or ethanol-induced increases in susceptibility to self-administer high ethanol levels, identifying a potential neuroadaptive mechanism contributing to excessive drinking and a therapeutic target for alcohol abuse and dependence. Sabino V, Cottone P, Zhao Y, Iyer MR, Steardo L Jr., Steardo L, Rice KC, Conti B, Koob GF, Zorrilla EP. The sigma-receptor antagonist BD-1063 decreases ethanol intake and reinforcement in animal models of excessive drinking. Neuropsychopharmacology. 2009 May;34(6):1482-1493. E-pub 2008 Oct 22.
Discriminative Stimulus Effects of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane in Rhesus Monkeys: Antagonism and Apparent pA2 Analyses
Discriminative stimulus effects of the serotonin (5-HT) receptor agonist 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) have been studied in rats and, more recently, in rhesus monkeys. This study examined DOM, 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7), and dipropyltryptamine hydrochloride (DPT) alone and in combination with three antagonists, MDL100907 [(+/-)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]], ketanserin [3-[2-[4-(4-fluorobenzoyl) piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione], and ritanserin [6-[2-[4-[bis(4-fluorophenyl) methylidene]piperidin-1-yl]ethyl]-7-methyl-[1,3]thiazolo[2,3-b]pyrimidin-5-one], to identify the 5-HT receptor subtype(s) that mediates the discriminative stimulus effects of these 5-HT receptor agonists. Four adult rhesus monkeys discriminated between 0.32 mg/kg s.c. DOM and vehicle while responding under a fixed ratio 5 schedule of stimulus shock termination. DOM, 2C-T-7, and DPT dose-dependently increased responding on the DOM-associated lever. MDL100907 (0.001-0.01 mg/kg), ketanserin (0.01-0.1 mg/kg), and ritanserin (0.01-0.1 mg/kg) each shifted the dose-response curves of DOM, 2C-T-7, and DPT rightward in a parallel manner. Schild analysis of each drug combination was consistent with a simple, competitive, and reversible interaction. Similar apparent affinity (pA(2)) values were obtained for MDL100907 in combination with DOM (8.61), 2C-T-7 (8.58), or DPT (8.50), for ketanserin with DOM (7.67), 2C-T-7 (7.75), or DPT (7.71), and for ritanserin with DOM (7.65), 2C-T-7 (7.75), or DPT (7.65). Potency of antagonists in this study was correlated with binding affinity at 5-HT(2A) receptors and not at 5-HT(2C) or alpha(1) adrenergic receptors. This study used Schild analysis to examine receptor mechanisms mediating the discriminative stimulus effects of hallucinogenic drugs acting at 5-HT receptors; results provide quantitative evidence for the predominant, if not exclusive, role of 5-HT(2A) receptors in the discriminative stimulus effects of DOM, 2C-T-7, and DPT in rhesus monkeys. Li JX, Rice KC, France CP. Discriminative stimulus effects of 1- (2,5-dimethoxy-4-methylphenyl)-2-aminopropane in rhesus monkeys: antagonism and apparent pA2 analyses. J Pharmacol Exp Ther. 2009 Mar;328(3):976-981. E-pub 2008 Dec 19.
Blockade of the Serotonin 5-HT2A Receptor Suppresses Cue-Evoked Reinstatement of Cocaine-Seeking Behavior in a Rat Self-Administration Model
The serotonin 5-HT2A receptor (5-HT-sub(2A)R) may play a role in reinstatement of drug-seeking. This study investigated the ability of a selective 5-HT-sub(2A)R antagonist to suppress reinstatement evoked by exposure to cues conditioned to cocaine self administration. Cocaine self-administration (0.75 mg/kg/0.1 mL/6 s infusion; FR 4) was trained in naēve, free-fed rats to allow interpretation of results independent from changes related to food deprivation stress. Pretreatment with the selective 5-HTsub(2A)R antagonist M100907 (volinanserin) failed to reduce rates of operant responding for cocaine infusions. On the other hand, M100907 (0.001-0.8 mg/kg ip) significantly suppressed the cue-induced reinstatement of cocaine-seeking behavior following extinction; effective M100907 doses did not alter operant responding for cues previously associated with sucrose self-administration. Importantly, a greater magnitude of active lever presses on the initial extinction session (high extinction responders) predicted the maximal susceptibility to M100907-induced suppression of cue-evoked reinstatement. The findings indicate that blockade of the 5-HT-sub(2A)R attenuates the incentive-motivational effects of cocaine-paired cues, particularly in high extinction responders, and suggests that M100907 may afford a therapeutic advance in suppression of cue-evoked craving and/or relapse. Nic Dhonnchadha BA, Fox RG, Stutz SJ, Rice KC, Cunningham KA. Blockade of the serotonin 5-HT2A receptor suppresses cue-evoked reinstatement of cocaine-seeking behavior in a rat self-administration model. Behav Neurosci. 2009 Apr;123(2):382-396.
Protracted Withdrawal from Alcohol and Drugs of Abuse Impairs Long term Potentiation of Intrinsic Excitability in the Juxtacapsular Bed Nucleus of the Stria Terminalis
The juxta-capsular bed nucleus of the stria terminalis (jcBNST) is activated in response to basolateral amygdala (BLA) inputs through the stria terminalis and projects back to the anterior BLA and to the central nucleus of the amygdala. Here IRP scientists show a form of long term potentiation of the intrinsic excitability (LTP-IE) of jcBNST neurons in response to high-frequency stimulation of the stria terminalis. This LTP-IE, which was characterized by a decrease in the firing threshold and increased temporal fidelity of firing, was impaired during protracted withdrawal from self-administration of alcohol, cocaine, and heroin. Such impairment was graded and was more pronounced in rats that self administered amounts of the drugs sufficient to maintain dependence. Dysregulation of the corticotropin-releasing factor (CRF) system has been implicated in manifestation of protracted withdrawal from dependent drug use. Administration of the selective corticotropin-releasing factor receptor 1 (CRF(1)) antagonist R121919 [2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylamino-pyrazolo[1,5-a]pyrimidine)], but not of the CRF(2) antagonist astressin(2)-B, normalized jcBNST LTP-IE in animals with a history of alcohol dependence; repeated, but not acute, administration of CRF itself produced a decreased jcBNST LTP-IE. Thus, changes in the intrinsic properties of jcBNST neurons mediated by chronic activation of the CRF system may contribute to the persistent emotional dysregulation associated with protracted withdrawal. Francesconi W, Berton F, Repunte-Canonigo V, Hagihara K, Thurbon, D, LeKic D, Specio SE, Greenwell TN, Chen SA, Rice KC, Richardson HN, O'Dell LE, Zorrilla EP, Morales M, Koob GF, SannaPP. Protracted withdrawal from alcohol and drugs of abuse impairs long term potentiation of intrinsic excitability in the juxtacapsular bed nucleus of the stria terminalis. J Neurosci. 2009 Apr 29;29(17):5389-5401.
Location, Structure, and Dynamics of the Synthetic Cannabinoid Ligand CP-55,940 in Lipid Bilayers
The widely used hydrophobic cannabinoid ligand CP-55,940 partitions with high efficiency into biomembranes. IRP researchers studied the location, orientation, and dynamics of CP-55,940 in POPC bilayers by solid-state NMR. Chemical-shift perturbation of POPC protons from the aromatic ring-current effect, as well as 1H NMR cross-relaxation rates,
locate the hydroxyphenyl ring of the ligand near the lipid glycerol, carbonyls, and upper acyl-chain methylenes. Order parameters of the hydroxyphenyl ring determined by the 1H-13C DIPSHIFT experiment indicate that the bond between the hydroxyphenyl and hydroxyl-cyclohexyl rings is oriented perpendicular to the bilayer normal. 2H NMR order parameters of the nonyl tail are very low, indicating that the hydrophobic chain maintains a high level of conformational flexibility in the membrane. Lateral diffusion rates of CP-55,940 and POPC were measured by 1H magic-angle spinning NMR with pulsed magnetic field gradients. The rate of CP-55,940 diffusion is comparable to the rate of lipid diffusion. The magnitude of cross-relaxation and diffusion rates suggests that associations between CP-55,940 and lipids are with lifetimes of a fraction of a microsecond. With its flexible hydrophobic tail, CP-55,940 may efficiently approach the binding site of the cannabinoid receptor from the lipid-water interface by lateral diffusion. Kimura T, Cheng K, Rice KC, Gawrisch K. Location, structure, and dynamics of the synthetic cannabinoid ligand CP-55,940 in lipid bilayers. Biophys J. 2009 Jun 17;96(12): 4916-4924.
Probes for Narcotic Receptor Mediated Phenomena
Enantiomers of N-substituted benzofuro [2,3-c]pyridin-6-ols have been synthesized, and the subnanomolar affinity and potent agonist activity of the known racemic N-phenethyl substituted benzofuro[2,3-c]pyridin-6-ol can now be ascribed to the 4aS,9aR enantiomer. The energy-minimized structures suggest that the active enantiomer bears a greater three-dimensional resemblance to morphine than to an ostensibly structurally similar oxide-bridged phenylmorphan. Structural features of the conformers of Nsubstituted benzofuro[2,3-c]pyridin-6-ols were compared to provide the rationale for their binding affinity. Zhang Y, Lee YS, Rothman RB, Dersch CM, Deschamps JR, Jacobson AE, Rice KC. Probes for Narcotic Receptor Mediated Phenomena. 39. (1) Enantiomeric NSubstituted Benzofuro[2,3-c]pyridin-6-ols: Synthesis and Topological Relationship to Oxide-Bridged Phenylmorphans (2). J. Med Chem. 2009 Jul 24. [E-pub ahead of print].
Serotonergic Hyperinnervation and Effective Serotonin Blockade in an FGF Receptor Developmental Model of Psychosis
The role of fibroblast growth factor receptors (FGFR) in normal brain development has been well-documented in transgenic and knock-out mouse models. Changes in FGF and its receptors have also been observed in schizophrenia and related developmental disorders. The current study examines a transgenic th(tk-)/th(tk-) mouse model with FGF receptor signaling disruption targeted to dopamine (DA) neurons, resulting in
neurodevelopmental, anatomical, and biochemical alterations similar to those observed in human schizophrenia. IRP researchers show in th(tk-)/th(tk-) mice that hypoplastic development of DA systems induces serotonergic hyperinnervation of midbrain DA nuclei, demonstrating the co-developmental relationship between DA and 5-HT systems. Behaviorally, th(tk-)/th(tk-) mice displayed impaired sensory gaiting and reduced social interactions correctable by atypical antipsychotics (AAPD) and a specific 5-HT2A antagonist, M100907. The adult onset of neurochemical and behavioral deficits was consistent with the postpubertal time course of psychotic symptoms in schizophrenia and related disorders. The spectrum of abnormalities observed in th(tk-)/th(tk-) mice and the ability of AAPD to correct the behavioral deficits consistent with human psychosis suggests that midbrain 5-HT2A-controlling systems are important loci of therapeutic action. These results may provide further insight into the complex multineurotransmitter etiology of neurodevelopmental diseases such autism, bipolar disorder, Asperger's Syndrome and schizophrenia. Klejbor I, Kucinski A, Wersinger SR, Corso T, Spodnik JH, Dziewiatkowski J, Morys J, Hesse RA, Rice KC, Miletich R, Stachowiak EK, Stachowiak MK. Serotonergic hyperinnervation and effective serotonin blockade in an FGF receptor developmental model of psychosis. Schizophr Res. 2009 Jun 29. [E-pub ahead of print].
Differential Involvement of the Norepinephrine, Serotonin and Dopamine Reuptake
Transporter Proteins in Cocaine-Induced Taste Aversion Despite the impact of cocaine's aversive effects on its abuse potential, the neurochemical basis of these aversive effects remains poorly understood. By blocking the reuptake of the monoamine neurotransmitters dopamine (DA), norepinephrine (NE) and serotonin (5-HT) into the presynaptic terminal, cocaine acts as a potent indirect agonist of each of these systems. The following studies attempted to assess the extent of monoaminergic mediation of cocaine's aversive effects using conditioned taste aversion (CTA) learning [Garcia J, Kimeldorf DJ, Koelling RA. Conditioned aversion to saccharin resulting from exposure to gamma radiation. Science 1955;122:157-158.]. Specifically, Experiment 1 assessed the ability of selective monoamine transporter inhibitors, e.g., DAT (vanoxerine), NET (nisoxetine) and SERT (fluoxetine), to induce taste aversions (relative to cocaine). Only the NET inhibitor approximated the aversive strength of cocaine. Experiment 2 compared the effects of pretreatment of each of these transport inhibitors on the development of a cocaine-induced CTA. Pretreatment with nisoxetine and fluoxetine both attenuated cocaine-induced aversions in a manner comparable to that produced by cocaine itself. The DAT inhibitor was without effect. Combined, the results of these investigations indicate little or no involvement of dopaminergic systems in cocaine's aversive effects while NE appears to contribute most substantially, with a possible modulatory involvement by serotonin. Jones JD, Hall FS, Uhl GR, Rice K, Riley AL. Differential Involvement of the Norepinephrine, Serotonin and Dopamine Reuptake Transporter Proteins in Cocaine-Induced Taste Aversion. Pharm Biochem Behav. 2009 Jul;93(1):75-81. E-pub 2009 Apr 17.
Opiate-Agonist Induced Taste Aversion Learning in the Fischer 344 and Lewis Inbred Rat Strains: Evidence for Differential Mu Opioid Receptor Activation
The Fischer 344 (F344) and Lewis (LEW) inbred rat strains react differently to morphine in a number of behavioral and physiological preparations, including the acquisition of aversions induced by this compound. The present experiment tested the ability of various compounds with relative selectivity at kappa, delta and mu receptor subtypes to assess the relative roles of these subtypes in mediating the differential aversive effects of morphine in the two strains. In the assessment of the role of the kappa receptor in morphine-induced aversions, animals in both strains were given access to saccharin followed by varying doses of the kappa agonist (-)-U50,488H (0.0, 0.28, 0.90 and 1.60 mg/kg). Although (-)-U50,488H induced aversions in both strains, no strain differences emerged. A separate subset of subjects was trained with the selective delta opioid agonist, SNC80 (0.0, 5.6, 10.0 and 18.0 mg/kg), and again although SNC80 induced aversions, there were no strain differences. Finally, a third subset of subjects was trained with heroin (0.0, 3.2, 5.6 and 10.0 mg/kg), a compound with activity at all three opiate receptor subtypes. Although heroin induced aversions in both strains, the aversions were significantly greater in the F344 strain, suggesting that differential activation of the mu opioid receptor likely mediates the reported strain differences in morphine-induced aversion learning. These data were discussed in terms of strain differences in opioid system functioning and the implications of such differences for other morphine-induced behavioral effects reported in F344 and LEW rats. Davis CM, Rice KC, Riley AL. Opiate-agonist induced taste aversion learning in the Fischer 344 and Lewis inbred rat strains: Evidence for differential mu opioid receptor activation. Pharmacol Biochem Behav. 2009 Jun 7. [E-pub ahead of print].
Nicotine Psychopharmacology Section, Clinical Pharmacology and Therapeutics Branch
Reliability and Validity of a Short Form of the Marijuana Craving Questionnaire
The Marijuana Craving Questionnaire (MCQ) is a valid and reliable, 47-item self-report instrument that assesses marijuana craving along four dimensions: compulsivity, emotionality, expectancy, and purposefulness. For use in research and clinical settings, IRP investigators constructed a 12-item version of the MCQ by selecting three items from each of the four factors that exhibited the greatest within-factor internal consistency (Cronbach's alpha coefficient). Adult marijuana users (n = 490), who had made at least one serious attempt to quit marijuana use but were not seeking treatment, completed the MCQ-Short Form (MCQ-SF) in a single session. Confirmatory factor analysis of the MCQ-SF indicated good fit with the 4-factor MCQ model, and the coefficient of congruence indicated moderate similarity in factor patterns and loadings between the MCQ and MCQ-SF. Homogeneity (unidimensionality and internal consistency) of MCQ-SF factors was also consistent with reliability values obtained in the initial validation of the MCQ. Findings of psychometric fidelity indicate that the MCQ-SF is a reliable and valid measure of the same multidimensional aspects of marijuana craving as the MCQ in marijuana users not seeking treatment. Heishman SJ, Evans RJ, Singleton EG, Levin KH, Copersino ML, Gorelick DA. Reliability and validity of a short form of the Marijuana Craving Questionnaire. Drug Alcohol Depend. 2009;102:35-40.
Psychobiology Section, Medications Discovery Research Branch
Assessment of Reinforcing Effects of Benztropine Analogs and their Effects on Cocaine Self-Administration in Rats: Comparisons with Monoamine Uptake Inhibitors
Benztropine (BZT) analogs inhibit dopamine uptake but are less effective than cocaine in producing behavioral effects predicting abuse liability. IRP scientists compared reinforcing effects of intravenous BZT analogs with those of standard monoamine uptake inhibitors and the effects of their oral pretreatment on cocaine self-administration. Rats self-administered cocaine [0.032-1.0 mg/kg/injection (inj)] with maximal rates maintained by 0.32 mg/kg/inj cocaine or methylphenidate, when substituted for cocaine. The N-methyl BZT analog, AHN 1-055 was also self administered, although only at a single dose and to less of an extent than cocaine. Neither the N-allyl (AHN 2-005) nor N-butyl (JHW 007) BZT analogs were self administered. Similarly, neither nisoxetine nor citalopram were self administered. Presession treatment with methylphenidate shifted the cocaine self-administration dose-effect curve leftward, i.e. potentiated cocaine. Effects of nisoxetine and citalopram pretreatments were not significant. An intermediate dose of AHN 1-055 (32 mg/kg, p.o.) increased self administration of low cocaine doses and decreased self administration of higher cocaine doses. A higher dose of AHN 1-055 completely eliminated cocaine self administration. Both AHN 2-005 and JHW 007 dose-dependently (10-32 mg/kg) decreased cocaine self-administration, shifting its dose-effect curve down. Decreases in cocaine self administration responding occurred at doses of BZT analogs that did not affect responding maintained by food reinforcement. These findings further support the low abuse liability of BZT analogs and their potential development as medications for cocaine abuse. Hiranita T, Soto PL, Newman AH, Katz JL. Assessment of reinforcing effects of benztropine analogs and their effects on cocaine self-administration in rats: Comparisons with monoamine uptake inhibitors. Journal of Pharmacology and Experimental Therapeutics. 2009 May; 329(2):677-686.
Medicinal Chemistry Section, Medications Discovery Research Branch
High Affinity and Enantioselective D3 Receptor Antagonists
In the present report, the D3 receptor pharmacophore is modified in the 2,3-diCl- and 2-OCH3-phenyl piperazine class of compounds with the goal to improve D3 receptor affinity and selectivity. This extension of structure-activity relationships (SAR) has resulted in the identification of the first enantioselective D3 antagonists (R- and S-PG 648) to be reported, wherein enantioselectivity is more pronounced at D3 than at D2, and that a binding region on the second extracellular loop (E2) may play a role in both enantioselectivity and D3 receptor selectivity. Moreover, IRP scientists have discovered some of the most D3-selective compounds reported to date that show high affinity (Ki =1 nM) for D3 and ~400-fold selectivity over the D2 receptor subtype. Several of these analogues showed exquisite selectivity for D3 receptors over >60 other receptors further underscoring their value as in vivo research tools. These lead compounds also have appropriate physical characteristics for in vivo exploration and therefore will be useful in determining how intrinsic activity at D3 receptors tested in vitro is related to behaviors in animal models of addiction and other neuropsychiatric disorders. Newman AH, Grundt P, Cyriac GC, Deschamps JR, Taylor M, Kumar R, Ho D, Luedtke RR. N-(4-(4-(2,3-Dichloro- or 2-methoxyphenyl) piperazin-1-yl)-butyl)-heterobiarylcarboxamides with functionalized linking chains as high affinity and enantioselective D3 receptor antagonists. J Med Chem. 2009;52:2559-2570.
Novel mGluR5 Antagonists
The metabotropic glutamate receptor subtype 5 (mGluR5) has been implicated in anxiety, depression, pain, mental retardation and addiction. The potent and selective noncompetitive mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) has been a critically important tool used to further elucidate the role of mGluR5 in these CNS disorders. In an effort to provide novel and structurally diverse selective mGluR5 antagonists, IRP investigators previously described a set of analogues with moderate activity wherein the alkyne bond was replaced with an amide group. In the present report, extended series of both amide and alkyne-based ligands were synthesized. MGluR5 binding and functional data were obtained that identified 1) several novel alkynes with higher affinity than MPEP at mGluR5 but 2) most structural variations to the amide template were not well tolerated, although a few potent amides were discovered. Several of these novel analogues show drug-like physical properties (e.g. cLogP range= 2-5) that support their use for in vivo investigation into the role of mGluR5 in CNS disorders. Kulkarni SS, Zou M-F, Cao J, Deschamps JR, Rodriguez A, Conn PJ, Newman AH. Structure Activity Relationships Comparing N-(6-methylpyridin-yl)-substituted Aryl Amides to 2-Methyl-6-(substituted-arylethynyl) pyridines or 2-Methyl-4-(substituted-aryl ethynyl)thiazoles as Novel mGluR5 antagonists. J Med Chem. 2009;52:3563-3575.
Visualization of Dopamine Transporter Trafficking in Live Neurons by Use of Fluorescent Cocaine Analogues
The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, still little is known about the cellular distribution and trafficking of natively expressed DAT. Here IRP researchers use novel fluorescently tagged cocaine analogues to visualize DAT and DAT trafficking in cultured live midbrain dopaminergic neurons. The fluorescent tags were extended from the tropane N- position of 2Β-carbomethoxy-3Β-(3,4-dichlorophenyl)tropane using an ethylamino-linker. The rhodamine, Oregon Green or Cy3 labeled ligands had high binding affinity for DAT and enabled specific labeling of DAT in live neurons and visualization by confocal imaging. In the dopaminergic neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions and varicosities along these extensions. FRAP (fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction <~30%). DAT was constitutively internalized into vesicular structures likely representing intracellular transporter pools. The internalization was blocked by lentiviral-mediated expression of dominant-negative dynamin and internalized DAT displayed partial co-localization with the early endosomal marker EGFP-Rab5 and with the transferring receptor. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC. Eriksen J, Rasmussen SGF, Vaegter CB, Cha JH, Zou M-F, Newman AH, Gether U. Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogues. J Neurosci. 2009;29(21):6794-6808.