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

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

Research Findings - Intramural Research

Office of the Scientific Director

Brain Mu-Opioid Receptor Binding Predicts Treatment Outcome In Cocaine-Abusing Outpatients
Cocaine users not seeking treatment have increased regional brain mu-opioid receptor (mOR) binding that correlates with cocaine craving and tendency to relapse, but this relationship had not been evaluated in cocaine-abusing outpatients in treatment. In collaboration with the Treatment Section, Clinical Pharmacology and Therapeutics Branch, and the Johns Hopkins PET Center, IRP researchers determined whether regional brain mOR binding before treatment correlates with outcome, and compared it with standard clinical predictors of outcome, in 25 individuals seeking outpatient treatment for cocaine abuse or dependence (DSM-IV). Patients received up to 12 weeks of cognitive-behavioral therapy and cocaine abstinence reinforcement, whereby each cocaine-free urine was reinforced with vouchers redeemable for goods. Regional brain mOR binding was measured before treatment using positron emission tomography with [11C]-carfentanil (a selective mOR agonist). Elevated mOR binding in the medial frontal and middle frontal gyri before treatment correlated with greater cocaine use (higher proportion of cocaine-positive urine samples) during treatment. Elevated mOR binding in the anterior cingulate, medial frontal, middle frontal, middle temporal, and sublobar insular gyri correlated with shorter duration of cocaine abstinence during treatment. Regional mOR binding contributed significant predictive power for treatment outcome beyond that of standard clinical variables such as baseline drug and alcohol use. These findings show that elevated mOR binding in brain regions associated with reward sensitivity is a significant independent predictor of treatment outcome in cocaine-abusing outpatients. They suggest a key role for the brain endogenous opioid system in cocaine addiction and a possible role for PET scanning in predicting cocaine-abuse treatment response and improving allocation of treatment resources. Ghitza UE, Preston KL, Epstein DH, Kuwabara H, Endres CJ, Bencherif B, Boyd SJ, Copersino ML, Frost JJ, and Gorelick DA. Brain mu-opioid receptor binding predicts treatment outcome in cocaine-abusing outpatients. Biological Psychiatry. 2010 published online.

Cannabis Withdrawal Symptoms In Non-Treatment-Seeking Adult Cannabis Smokers
Cannabis withdrawal symptoms are reported in the medical literature, but a cannabis withdrawal syndrome is not recognized in DSM-IV because of doubts about its clinical significance. IRP scientists assessed the phenomenon of cannabis withdrawal and its relationship to relapse in a convenience sample of 469 nontreatment-seeking adults who had made a quit attempt without formal treatment while not in a controlled environment. Subjects completed a 176-item Marijuana Quit Questionnaire collecting information on sociodemographic characteristics, cannabis use history, and their "most difficult" cannabis quit attempt. 42.4% of subjects had experienced a lifetime withdrawal syndrome, of whom 70.4% reported using cannabis in response to withdrawal. During the index quit attempt, 95.5% of subjects reported 1 individual withdrawal symptom. Number of withdrawal symptoms was significantly associated with greater frequency and amount of cannabis use, but symptoms occurred even in those using less than weekly. Symptoms were usually of at least moderate intensity and often prompted actions to relieve them. Alcohol (41.5%) and tobacco (48.2%) were used more often than cannabis (33.3%) for this purpose. There was little change during withdrawal in use of other legal or illegal substances. These findings show that cannabis withdrawal is a common syndrome among adults not seeking treatment and that withdrawal can serve as a negative reinforcer for relapse to cannabis use (as well as other substance use). Therefore, cannabis withdrawal symptoms do have clinical significance and should be a target of treatment efforts. Levin KH, Copersino ML, Heishman SJ, Liu F, Kelly DL, Boggs DL, and Gorelick DA. Cannabis withdrawal symptoms in non-treatment-seeking adult cannabis smokers. Drug and Alcohol Dependence. 2010 published online.

Clinical Pharmacology and Therapeutics

Ecological Momentary Assessment Study
The effects of an intervention cannot be understood without precise knowledge of the baseline behavior on which the intervention is superimposed. For misusers of illicit drugs, patterns of daily activities and moods have not been studied in a way that is amenable to statistical aggregation. The objective of the study was to compare hour-by-hour daily activities in cocaine-dependent outpatients during urine-verified periods of use and abstinence. In a cohort design, a volunteer sample of 112 methadone-maintained cocaine- and heroin-abusing outpatients provided ecological momentary assessment (EMA) data on handheld computers for 10,781 person-days. Drug use was monitored in three-times weekly urine drug screens. EMA responses to questions about current location, activities, companions, moods, and recent exposure to putative drug-use triggers were compared across periods of use and abstinence (defined as at least three consecutive cocaine-positive or cocaine-negative drug screens, respectively) using SAS Proc Glimmix (for binary outcomes) and Proc Mixed (for continuous outcomes). Periods of cocaine use were associated with idle, solitary, affectively negative afternoons, but, unexpectedly, were also associated with a greater likelihood of early-morning or late-evening work. The whole-day concomitants of cocaine use were often distinct from the acute predecessors of use seen in prior analyses from the same sample. Several measures of negative mood increased during abstinence. Weeks of cocaine use and abstinence in outpatients are associated with distinct patterns of mood and behavior; the detailed hourly data reported here should help inform treatment interventions aimed at changing daily activities. The findings also argue against the contention that cocaine abstinence symptoms decrease monotonically from the day of cessation. Epstein DH, Preston KL. Daily life hour by hour, with and without cocaine: an ecological momentary assessment study. Psychopharmacology, 2010; 211: 223-232. Epub 2010 June 9.

Nicotine Psychopharmacology Section/Clinical Pharmacology and Therapeutics Branch

Validity of the 12-item French version of the Tobacco Craving Questionnaire
The French version (FTCQ) of the Tobacco Craving Questionnaire (TCQ) is a valid and reliable 47-item self-report instrument that assesses tobacco craving in four factors: emotionality, expectancy, compulsivity, and purposefulness. For use in research and clinical settings, IRP researchers constructed a 12-item version of the FTCQ (FTCQ-12). The FTCQ-12 was administered to treatment-seeking French smokers (n=310) enrolled in the Adjustment of DOses of NIcotine in Smoking Cessation (ADONIS) trial. The authors conducted confirmatory factor analysis (CFA) and examined congruence in factor loadings between the FTCQ and FTCQ-12 to determine the validity and reliability of the FTCQ-12. Measures of tobacco craving, withdrawal, smoking patterns, and smoking history were included to explore the concurrent validity of the FTCQ-12. The authors used craving scores to distinguish participants who were highly dependent on nicotine from those less dependent on nicotine. CFA indicated perfect fit for a 4-factor model, with congruence coefficients indicating moderate similarity in factor patterns and loadings between the FTCQ and FTCQ-12. Individual factors of the FTCQ-12 correlated positively with smoking history and withdrawal variables. Participants who were highly dependent on nicotine were nearly six times more likely to score >5 on the general factor (maximum: 7) than those less dependent on nicotine. Findings suggest the FTCQ-12 measures the same four factors as the FTCQ, TCQ, and TCQ-12, and these four constructs have unique properties. The FTCQ-12 yields valid and reliable indices of tobacco craving, and has potential clinical utility for rapid assessment of tobacco craving in smokers seeking treatment. Berlin I, Singleton EG, Heishman SJ. Validity of the 12-item French version of the Tobacco Craving Questionnaire in treatment-seeking smokers. Nicotine Tob Res. 2010;12: 500-507.

Meta-Analysis of the Acute Effects of Nicotine and Smoking On Human Performance
Empirical studies indicate that nicotine enhances some aspects of attention and cognition, suggesting a role in the maintenance of tobacco dependence. The purpose of this review was to update the literature since our previous review (Heishman et al. 1994) and to determine which aspects of human performance were most sensitive to the effects of nicotine and smoking. The authors conducted a meta-analysis on the outcome measures of 41 double-blind, placebo-controlled laboratory studies published from 1994 to 2008. In all studies, nicotine was administered and performance was assessed in healthy adult nonsmokers or smokers who were not tobacco deprived or minimally deprived ( 2 h). There were sufficient effect size data to conduct meta-analyses on nine performance domains, including motor abilities, alerting and orienting attention, and episodic and working memory. The authors found significant positive effects of nicotine or smoking on six domains: fine motor, alerting attention-accuracy and response time (RT), orienting attention-RT, short-term episodic memory-accuracy, and working memory-RT (effect size range = 0.16 to 0.44). The signficant effects of nicotine on motor abilities, attention, and memory likely represent true performance enhancement because they are not confounded by withdrawal relief. The beneficial cognitive effects of nicotine have implications for initiation of smoking and maintenance of tobacco dependence. Heishman SJ, Kleykamp BA, Singleton EG. Meta-analysis of the acute effects of nicotine and smoking on human performance. Psychopharmacology. 2010; 210: 453-469.

Comparison of Reactivity to Smoking Cues and Smoking Imagery
Increases in self-reported craving and changes in autonomic functioning are reliably elicited when smokers are exposed to tobacco-related stimuli compared with neutral stimuli. However, few studies have reported the time course of cue-elicited craving or have directly compared the effectiveness of smoking cues versus imagery to evoke a craving response. In addition to these two issues, IRP scientists investigated the influence of tobacco deprivation and sex on craving, mood, and autonomic responses. Sixty cigarette smokers (30 men, 30 women) were tested in two counterbalanced sessions, one after overnight tobacco deprivation and one during ad libitum smoking. At each session, participants were exposed to four randomized experimental trials: smoking imagery, neutral imagery, smoking cues, and neutral cues. Tobacco craving and mood were assessed repeatedly and physiological measures were recorded continuously for 30 min after imagery or cue exposure. Compared with neutral trials, smoking cues and smoking imagery reliably increased tobacco craving, negative mood, heart rate, and blood pressure and decreased positive mood ratings. Changes were observed immediately after cue and imagery presentation and remained unchanged for 30 min. Responding was greater in the nondeprived condition, and cues elicited more robust responding than imagery for most measures. Women responded more robustly to smoking cues only in the nondeprived condition, whereas imagery evoked greater responses in men during both conditions. These findings provide new data on the time course, magnitude, and tobacco deprivation effects on elicited craving. Sex differences were dependent on stimulus type and deprivation condition. Heishman SJ, Lee DC, Taylor RC, Singleton EG. Prolonged duration of craving, mood, and autonomic responses elicited by cues and imagery in smokers: effects of tobacco deprivation and sex. Exp Clin Psychopharmacol. 2010; 18: 245-256.

Chemical Biology Research Branch, Drug Design and Synthesis Section

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 mu-opioid receptor affinity of the examined compounds (K(i)=0.35 microM). Iyer MR, Lee YS, Deschamps JR, Rothman RB, Dersch CM, Jacobson AE, Rice KC. Bioorg Med Chem. 2010 Jan 1;18(1): 91-99. Epub 2009 Nov 18.

Identification of a Novel "Almost Neutral" Micro-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 investigators 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 microM (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl) dodecahydro-6a,10b-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, Hsin L-W, Chang L-T, Prisinzano TE, Simpson D, Giuvelis D, Rice KC, Jacobson, AE, Bilsky EJ, Rothman RB. Synapse. 2010 Apr;64(4): 280-288.

Evidence That Tricyclic Small Molecules May Possess Toll-Like Receptor and Myeloid Differentiation Protein 2 Activity
Opioids have been discovered to have Toll-like receptor (TLR) activity, beyond actions at classical opioid receptors. This raises the question whether other pharmacotherapies for pain control may also possess TLR activity, contributing to or opposing their clinical effects. IRP scientists document that tricyclics can alter TLR4 and TLR2 signaling. In silico simulations revealed that several tricyclics docked to the same binding pocket on the TLR accessory protein, myeloid differentiation protein 2 (MD-2), as do opioids. Eight tricyclics were tested for effects on TLR4 signaling in HEK293 cells over-expressing human TLR4. Six exhibited mild (desipramine), moderate (mianserin, cyclobenzaprine, imiprimine, ketotifen) or strong (amitriptyline) TLR4 inhibition, and no TLR4 activation. In contrast, carbamazepine and oxcarbazepine exhibited mild and strong TLR4 activation, respectively, and no TLR4 inhibition. Amitriptyline but not carbamazepine also significantly inhibited TLR2 signaling in a comparable cell line. Live imaging of TLR4 activation in RAW264.7 cells and TLR4-dependent interleukin-1 release from BV-2 microglia revealed that amitriptyline blocked TLR4 signaling. Lastly, tricyclics with no (carbamazepine), moderate (cyclobenzeprine), and strong (amitriptyline) TLR4 inhibition were tested intrathecally (rats) and amitriptyline tested systemically in wildtype and knockout mice (TLR4 or MyD88). While tricyclics had no effect on basal pain responsivity, they potentiated morphine analgesia in rank-order with their potency as TLR4 inhibitors. This occurred in a TLR4/MyD88-dependent manner as no potentiation of morphine analgesia by amitriptyline occurred in these knockout mice. This suggests that TLR2 and TLR4 inhibition, possibly by interactions with MD2, contributes to effects of tricyclics in vivo. These studies provide converging lines of evidence that several tricyclics or their active metabolites may exert their biological actions, in part, via modulation of TLR4 and TLR2 signaling and suggest that inhibition of TLR4 and TLR2 signaling may potentially contribute to the efficacy of tricyclics in treating chronic pain and enhancing the analgesic efficacy of opioids. 2010 IBRO. Hutchinson MR, Loram LC, Zhang Y, Shridhar M, Rezvani N, Berkelhammer D, Phipps S, Foster PS, Landgraf K, Falke JJ, Rice KC, Maier SF, Yin H, Watkins LR. Neuroscience. 2010 Jun 30;168(2): 551-563. Epub 2010 Apr 8.

Reversal of Pancreatitis-Induced Pain By an Orally Available, Small Molecule Interleukin-6 Receptor Antagonist
Pancreatic pain resulting from chronic inflammation of the pancreas is often intractable and clinically difficult to manage with available analgesics reflecting the need for more effective therapies. The mechanisms underlying pancreatitis pain are not well understood. Here, the possibility that interleukin-6 (IL-6) may promote pancreatitis pain was investigated with TB-2-081 (3-O-formyl-20R,21-epoxyresibufogenin, EBRF), a small molecule IL-6 receptor antagonist that was semi-synthetically derived from natural sources. The potential activity and mechanism of TB-2-081 were investigated following the induction of persistent pancreatitis using dibutyltin dichloride (DBTC) in rats. TB-2-081 displaces the binding of IL-6 to the human recombinant soluble IL-6 receptor with apparent high affinity and inhibits IL-6 mediated cell growth. Systemic or oral, but not intrathecal, administration of TB-2-081 reversed DBTC-induced abdominal hypersensitivity in a dose- and time-dependent manner. IL-6 levels were significantly up-regulated in the dorsal root ganglia (DRG) of rats with pancreatitis on day 6 after DBTC injection. IL-6-enhanced capsaicin-evoked release of calcitonin gene-related peptide from cultured DRG neurons was blocked by TB-2-081. These data demonstrate that TB-2-081 acts as a systemically available and orally active small molecule IL-6 receptor antagonist. TB-2-081 effectively reduces pancreatitis-induced pain through peripheral mechanisms that are likely due to (a) increased expression of IL-6 in the DRG and (b) IL-6-mediated sensitization of nociceptive neurons. The activity of TB-2-081 implicates an important role for IL-6 in sustaining pancreatitis pain. Strategies targeting IL-6 actions through small molecule antagonists may offer novel approaches to improve the therapy of chronic pancreatitis and other chronic pain states. Vardanyan M, Melemedjian OK, Price TJ, Ossipov MH, Lai J, Roberts E, Boos TL, Deschamps JR, Jacobson AE, Rice KC, Porreca F. Pain. 2010 Jul 2. [Epub ahead of print].

Evidence for Noncompetitive Modulation of Substrate-Induced Serotonin Release
Prior work indicated that serotonin transporter (SERT) inhibitors competitively inhibit substrate-induced [3H]5-HT release, producing rightward shifts in the substrate-dose response curve and increasing the EC50 value without altering the Emax. IRP researchers hypothesized that this finding would not generalize across a number of SERT inhibitors and substrates, and that the functional dissociation constant (Ke) of a given SERT inhibitor would not be the same for all tested substrates. To test this hypothesis, the authors utilized a well-characterized [3H]5-HT release assay that measures the ability of a SERT substrate to release preloaded [3H]5-HT from rat brain synaptosomes. Dose-response curves were generated for six substrates (PAL-287 [naphthyliso propylamine], (+)-fenfluramine, (+)-norfenfluramine, mCPP [meta-chlorophenylpiperazine], ()-MDMA, 5-HT) in the absence and presence of a fixed concentration of three SERT inhibitors (indatraline, BW723C86, EG-1-149 [4-(2-(benzhydryloxy)ethyl)-1-(4-bromobenzyl)piperidine oxalate]). Consistent with simple competitive inhibition, all SERT inhibitors increased the EC50 value of all substrates. However, in many cases a SERT inhibitor decreased the Emax value as well, indicating that in the presence of the SERT inhibitor the substrate became a partial releaser. Moreover, the Ke values of a given SERT inhibitor differed among the six SERT substrates, indicating that each inhibitor/substrate combination had a unique interaction with the transporter. Viewed collectively, these findings suggest that it may be possible to design SERT inhibitors that differentially regulate SERT function. Rothman RB, Baumann MH, Blough BE, Jacobson AE, Rice KC, Partilla JS. Synapse. 2010 April 23. [Epub ahead of print].

Antinociceptive Interactions Between Mu Opioid Receptor Agonists and the Serotonin Uptake Inhibitor Clomipramine In Rhesus Monkeys: Role Of Mu Agonist Efficacy
Mu-opioid agonists are effective analgesics but have undesirable effects such as sedation and abuse liability that limit their clinical effectiveness. Serotonergic systems also modulate nociception, and serotonin uptake inhibitors may be useful as adjuncts to enhance analgesic effects and/or attenuate undesirable effects of mu agonists. This study examined effects of the serotonin uptake inhibitor clomipramine on behavioral effects produced in rhesus monkeys by mu agonists with varying efficacy at mu receptors (nalbuphine< morphine< methadone). Clomipramine and each mu agonist were studied alone and in fixed-proportion mixtures in assays of schedule-controlled responding, thermal nociception and capsaicin-induced thermal allodynia. In the assay of schedule-controlled responding, all mu agonists dose-dependently decreased response rates. Clomipramine was inactive alone and did not alter effects of mu agonists. In the assay of thermal nociception, all mu agonists produced dose-dependent antinociception. Clomipramine was inactive alone but produced a proportion-dependent enhancement of the antinociceptive effects of nalbuphine>morphine> methadone. In the assay of capsaicin-induced allodynia, nalbuphine produced dose-dependent anti-allodynia. Clomipramine alone was inactive, but as in the assay of thermal nociception, it produced a proportion-dependent enhancement in the effects of nalbuphine. These findings suggest that serotonin uptake inhibitors can selectively enhance the antinociceptive effects of mu agonists in nonhuman primates. These effects of serotonin uptake inhibitors may be dependent on the proportion of the serotonin uptake inhibitor and the efficacy of the mu agonist. The greatest enhancement was observed with intermediate proportions of clomipramine in combination with the low-efficacy mu agonist nalbuphine. Banks ML, Rice KC, Negus SS. J. Pharmacol. Exp. Ther. 2010 July 30. [Epub ahead of print].

In Vivo Evaluation Of [123I]-4-(2-(Bis(4-Fluorophenyl)Methoxy)Ethyl)-1-(4- Iodobenzyl) Piperidine, An Iodinated SPECT Tracer For Imaging The P-Gp Transporter
P-glyco protein (P-gp) is an energy-dependent transporter that contributes to the efflux of a wide range of xenobiotics at the blood-brain barrier playing a role in drug-resistance or therapy failure. In this study, IRP scientists evaluated [(123)I]-4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(4-iodobenzyl) piperidine ([(123)I]-FMIP) as a novel single photon emission computed tomography (SPECT) tracer for imaging P-gp at the brain in vivo. The tissue distribution and brain uptake as well as the metabolic profile of [(123)I]-FMIP in wild-type and mdr1a (-/-) mice after pretreatment with physiological saline or cyclosporin A (CsA) (50 mg/kg) was investigated. The influence of increasing doses CsA on brain uptake of [(123)I]-FMIP was explored. microSPECT images of mice brain after injection of 11.1 MBq [(123)I]-FMIP were obtained for different treatment strategies thereby using the Milabs U-SPECT-II. Modulation of P-gp with CsA (50 mg/kg) as well as mdr1a gene depletion resulted in significant increase in cerebral uptake of [(123)I]-FMIP with only minor effect on blood activity. [(123)I]-FMIP is relatively stable in vivo with >80% intact [(123)I]-FMIP in brain at 60 min p.i. in the different treatment regiments. A dose-dependent sigmoidal increase in brain uptake of [(123)I]-FMIP with increasing doses of CsA was observed. In vivo region of interest-based SPECT measurements correlated well with the observations of the biodistribution studies. These findings indicate that [(123)I]-FMIP can be applied to assess the efficacy of newly developed P-gp modulators. It is also suggested that [(123)I]-FMIP is a promising SPECT tracer for imaging P-gp at the blood-brain barrier. De Bruyne S, Wyffels L, Boos TL, Staelens S, Deleye S, Rice KC, De Vos F. Nucl Med Biol. 2010 May;37(4): 469-477. Epub 2010 Apr 7.

GABAB Receptor Positive Modulators: Enhancement of GABAB Receptor Agonist Effects In Vivo
In vivo effects of GABA(B) receptor positive modulators suggest them to have therapeutic potential to treat CNS disorders such as anxiety, depression, and drug abuse. Although these effects are generally thought to be mediated by positive modulation of GABA(B) receptors, such modulation has been examined primarily in vitro. The present study was aimed at further examining the in vivo positive modulatory properties of the GABA(B) receptor positive modulators, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethylpropyl) phenol (CGP7930) and (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF). Both compounds enhanced loss of righting induced by baclofen in mice. However, CGP7930 was less effective and rac-BHFF was less potent to enhance loss of righting induced by GHB, which, like baclofen, has GABA(B) receptor agonist properties. In contrast with baclofen- and GHB-induced loss of righting, the hypothermic effects of baclofen and GHB were not enhanced by rac-BHFF, and were enhanced by CGP7930 only at doses that produced hypothermia when given alone. CGP7930-induced hypothermia was not attenuated by the GABA(B) receptor antagonist CGP35348, at doses that blocked baclofen-induced hypothermia, and was not increased by the NOS inhibitor L-NAME, at doses that increased the hypothermic effects of baclofen and GHB. The results are evidence that CGP7930 and rac-BHFF act in vivo as positive modulators at GABA(B) receptors mediating loss of righting, but not at GABA(B) receptors mediating hypothermia. Conceivably, CGP7930, but not rac-BHFF, acts as an allosteric agonist at these latter receptors. Taken together, the results are further evidence of pharmacologically distinct GABA(B) receptor subtypes, possibly allowing for a more selective therapeutic interference with the GABA(B) system. Koek W, France CP, Cheng K, Rice KC. J Pharmacol Exp Ther. 2010 Jul 13. [Epub ahead of print].

Cellular Neurobiology Research Branch

Development and Plasticity Section, Cellular Neurobiology Research Branch

Cocaine Causes Deficits In Radial Migration and Alters the Distribution of Glutamate and GABA Neurons In The Developing Rat Cerebral Cortex
Prenatal cocaine exposure induces cytoarchitectural changes in the embryonic neocortex; however, the biological mechanisms and type of cortical neurons involved in these changes are not known. Previously IRP scientists found that neural progenitor proliferation in the neocortical ventricular zone (VZ) is inhibited by cocaine; here they examine the changes in cortical neurogenesis and migration of glutamate and GABA neurons induced by prenatal cocaine exposure. Pregnant rats received 20 mg/kg of cocaine intraperitoneally twice at an interval of 12 hr during three periods of neocortical neurogenesis. Neocortical area and distribution of developing neurons were examined by counting Tuj1+, glutamate+ or GABA+ cells in different areas of the cerebral cortex. Cocaine decreased neocortical area by reducing the size of the Tuj1+ layer, but only when administered during early periods of neocortical neurogenesis. The number of glutamatergic neurons was increased in the VZ, but was decreased in the outer cortical laminae. Although the number of GABA+ neurons in the VZ of both the neocortex and ganglionic eminences was unchanged, GABA+ cells decreased in all other neocortical laminae. Tangential migration of GABA+ cells was also disrupted by cocaine. These findings suggest that in utero cocaine exposure disturbs radial migration of neocortical neurons, possibly due to decreased radial glia guiding support through enhanced differentiation of neocortical VZ progenitors. Cocaine interrupts radial migration of both glutamatergic and GABAergic neurons within the neocortex, in addition to the tangential migration of GABAergic neurons from the subcortical telecephalon. This may result in abnormal neocortical cytoarchitecture and concomitant adverse functional effects. Lee CT, Chen J, Worden, LT, Freed WJ. Cocaine causes deficits in radial migration and alters the distribution of glutamate and GABA neurons in the developing rat cerebral cortex. Synapse. 2010 May 4. [Epub ahead of print].

Electrophysiology Research Section, Cellular Neurobiology Research Branch

Afferent-Specific AMPA Receptor Subunit Composition and Regulation of Synaptic Plasticity In Midbrain Dopamine Neurons By Abused Drugs
Ventral tegmental area (VTA) dopamine (DA) neurons play a pivotal role in processing reward-related information and are involved in drug addiction and mental illness in humans. Information is conveyed to the VTA in large part by glutamatergic afferents that arise in various brain nuclei, including the pedunculopontine nucleus (PPN). Using a unique rat brain slice preparation, IRP investigators found that PPN stimulation activates afferents targeting GluR2-containing AMPA receptors (AMPAR) on VTA DA neurons, and these afferents did not exhibit long-term depression (LTD). In contrast, activation of glutamate afferents onto the same DA neurons via stimulation within the VTA evoked EPSCs mediated by GluR2-lacking AMPARs that demonstrated LTD or EPSCs mediated by GluR2-containing AMPA receptors that did not express LTD. Twenty-four hours after single cocaine injections to rats, GluR2-lacking AMPARs were increased at both PPN and local VTA projections, and this permitted LTD expression in both pathways. Single injections with the main psychoactive ingredient of marijuana, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), increased GluR2-lacking AMPA receptors and permitted LTD in only the PPN pathway, and these effects were prevented by in vivo pretreatment with the cannabinoid CB1 receptor antagonist AM251. These results demonstrate that cocaine more globally increases GluR2-lacking AMPA receptors at all glutamate synapses on VTA dopamine neurons, whereas Delta(9)-THC selectively increased GluR2-lacking AMPA receptors at subcortical PPN synapses. This suggests that different abused drugs may exert influence over distinct sets of glutamatergic afferents to VTA DA neurons which may be associated with different reinforcing or addictive properties of these drugs. Good CH, Lupica CR. Afferent-specific AMPA receptor subunit composition and regulation of synaptic plasticity in midbrain dopamine neurons by abused drugs. J Neurosci. 2010;30(23): 7900-7909.

Delta9-Tetrahydrocannabinol Is A Full Agonist At CB1 Receptors On GABA Neuron Axon Terminals In the Hippocampus
Marijuana impairs learning and memory through actions of its psychoactive constituent, delta-9-tetrahydrocannabinol (Delta(9)-THC), in the hippocampus, through activation of cannabinoid CB1 receptors (CB1R). CB1Rs are found on glutamate and GABA neuron axon terminals in the hippocampus where they control neurotransmitter release. Previous studies suggest that Delta(9)-THC is a partial agonist of CB1Rs on glutamate axon terminals in the hippocampus, whereas its effects on GABA terminals have not been described. Using whole-cell electrophysiology in brain slices from C57BL6/J mice, IRP scientists examined Delta(9)-THC effects on synaptic GABA IPSCs and postsynaptic GABA currents elicited by laser-induced photo-uncaging (photolysis) of alpha-carboxy-2-nitrobenzyl (CNB) caged GABA. Despite robust inhibition of synaptic IPSCs in wildtype mice by the full synthetic agonist WIN55,212-2, using a Tween-80 and DMSO vehicle, Delta(9)-THC had no effects on IPSCs in this, or in a low concentration of another vehicle, randomly-methylated beta-cyclodextrin (RAMEB, 0.023%). However, IPSCs were inhibited by Delta(9)-THC in 0.1% RAMEB, but not in neurons from CB1R knockout mice. Whereas Delta(9)-THC did not affect photolysis-evoked GABA currents, these responses were prolonged by a GABA uptake inhibitor. Concentration-response curves revealed that the maximal effects of Delta(9)-THC and WIN55,212-2 were similar, indicating that Delta(9)-THC is a full agonist at CB1Rs on GABA axon terminals. These results suggest that Delta(9)-THC inhibits GABA release, but does not directly alter GABA(A) receptors or GABA uptake in the hippocampus. Furthermore, full agonist effects of Delta(9)-THC on IPSCs likely result from a much higher expression of CB1Rs on GABA versus glutamate axon terminals in the hippocampus. Laaris N, Good CH, Lupica CR. Delta9-tetrahydrocannabinol is a full agonist at CB1 receptors on GABA neuron axon terminals in the hippocampus. Neuropharmacology. 2010; 59(1-2): 121-127.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Interactions Between Intracellular Domains As Key Determinants of the Quaternary Structure and Function of Receptor Heteromers
G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In the present study, using Resonance Energy Transfer (RET) techniques in experiments with mutated receptors, IRP researchers provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A2A, cannabinoid CB1 and dopamine D2 receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A2A, CB1 and D2) was found to include two evolutionary conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB1 receptor with A2A and D2 receptors are fundamental for the correct formation of the quaternary structure needed for the function (mitogen-activated protein kinase, MAPK, signaling) of the A2A-CB1-D2 receptor heteromers. Analysis of MAPK signaling in striatal slices of CB1 receptor KO mice and wild-type littermates supported the existence of A1-CB1-D2 receptor heteromer in the brain. These findings allowed the authors to propose the first molecular model of the quaternary structure of a receptor heteromultimer. Navarro G, Ferre S, Cordomi A, Moreno E, Mallol J, Casado V, Cortes A, Hoffmann H, Ortiz J, Canela EI, Lluis C, Pardo L, Franco R, Woods AS. Interactions between intracellular domains as key determinants of the quaternary structure and function of receptor heteromers. J Biol Chem 2010. Jun 18. [Epub ahead of print].

Neuroimaging Research Branch

A Genetically Modulated, Intrinsic Cingulated Circuit Supports Human Nicotine Addiction
Genome-wide searches have yielded genetic variants tied to human nicotine dependence. One such variant, called Asp398Asn, in a gene on human chromosome 15 that encodes an alpha 5 subunit of the nicotinic acetylcholine receptor has been repeatedly linked to smoking. Hong et al recently identified a brain circuit that reflects nicotine addiction. However, the relationship between dependence, neural circuitry and genetic predisposition are poorly understood. Elliot Hong et al. used a resting state functional magnetic resonance imaging technique on smokers and non-smokers to compare the strength of a circuit linking the dorsal anterior cingulate-ventral striatum-extended amygdala, brain regions where the gene is active and that have been implicated in addiction. The authors found that Asp398Asn was linked to a greater drop in the circuit's strength in smokers than in non-smokers, and that the circuit strength predicts the severity of the dependence among smokers. The authors report that the circuit is also impaired in people with mental illnesses, who are known to be at a higher risk of smoking, irrespective of the gene variant, suggesting that the circuit might be a common link underpinning nicotine dependence in smokers and people with mental illnesses. The findings could be used to develop an assay of the circuit's function in animal models of human nicotine dependence, possibly leading to novel treatment targets for smoking cessation, according to the authors. Hong LE, Hodgkinson CA, Yang Y, Sampath H, Ross TJ, Buchholz B, Salmeron BJ, Srivastava V, Thaker GK, Goldman D, Stein EA. A genetically modulated, intrinsic cingulated circuit supports human nicotine addiction. Proceedings of the National Academy of Science USA 107(30): 13509-13514, 2010.

Medications Discovery Research Branch, Medicinal Chemistry Section

Dopamine D3 Receptor Partial Agonist CJB090 and Antagonist PG01037 Decrease Progressive Ratio Responding For Methamphetamine In Rats With Extended-Access
The development of novel compounds acting selectively at dopamine D3 receptors has opened new possibilities to explore the role of these receptors in animal models of psychostimulant dependence. Here IRP scientists investigated whether the dopamine D3 partial agonist CJB090 (1-10 mg/kg, i.v) and the D3 antagonist PG01037 (8-32 mg/kg, s.c.,) modified methamphetamine (0.05 mg/kg/injection) intravenous self-administration under fixed- (FR) and progressive- (PR) ratio schedules in rats allowed limited (short access, ShA; 1h sessions 3 days/week) or extended access (long access, LgA; 6h sessions 6 days/week). Under a FR1 schedule, the highest dose of the D3 partial agonist CJB090 selectively reduced methamphetamine self-administration in LgA but not in ShA rats, whereas the full D3 antagonist PG01037 produced no effect in any group. Under a PR schedule of reinforcement, the D3 partial agonist CJB090 reduced the maximum number of responses performed ("breakpoint") for methamphetamine in LgA rats at the doses of 5 and 10 mg/kg and also it produced a significant reduction in the ShA group at the highest dose. However, the D3 full antagonist PG01037 only reduced PR methamphetamine self-administration in LgA rats at the highest dose of 32 mg/kg with no effect in the ShA group. The results suggest that rats might be more sensitive to pharmacological modulation of dopamine D3 receptors following extended access to methamphetamine self-administration, opening the possibility that D3 receptors play a role in excessive methamphetamine intake. Orio L, Wee S, Newman AH, Pulvirenti L, Koob GF. The dopamine D3 receptor partial agonist CJB090 and antagonist PG01037 decrease progressive ratio responding for methamphetamine in rats with extended-access. Addict Biol. 2010; 15(3): 312-323.

Dopamine D3 and D2 Receptor Mechanisms in the Abuse-Related Behavioral Effects of Cocaine
Dopamine (DA) D3 and D2 receptor mechanisms are implicated in cocaine's abuse-related behavioral effects, but the relative contribution of the two receptor subtypes is only partially characterized. This study investigated the role of D3 and D2 subtype mechanisms by determining the degree to which the D3-prefering antagonist PG01037 [N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide HCl] and the D2-prefering antagonist L-741,626 [3-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl-1H-indole] attenuated several behavioral effects of cocaine in squirrel monkeys. Quantitative observational studies established doses of each antagonist that did not produce untoward effects, which was used in subsequent comparisons. Additionally, the capacities of the D3-preferring agonist PD128907 [(R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol)] and the D2-preferring agonist sumanirole to reproduce cocaine's discriminative stimulus (DS) and priming effects were compared. In monkeys trained to discriminate cocaine from vehicle, both DA antagonists attenuated and both DA agonists partially reproduced cocaine's DS effects. PG01037 also attenuated the cocaine-like DS effects of PD128907, but not sumanirole, whereas L-741,626 attenuated the cocaine-like DS effects of both agonists. In self-administration studies, L-741,626 non-selectively reduced cocaine- and food-maintained responding, whereas PG01037 was ineffective against either reinforcer. In studies involving reinstatement of extinguished cocaine-seeking, both antagonists attenuated cocaine-induced reinstatement of responding, and both agonists induced reinstatement of cocaine-seeking. L-741,626 also attenuated sumanirole- but not PD128907-induced reinstatement of responding, whereas PG01037 was ineffective against either DA agonist. The results are consistent with a role for D3 and D2 receptor mechanisms in cocaine's DS effects and cocaine-induced reinstatement of drug-seeking, but provide no evidence for a major role of D3 receptors in the direct reinforcing effects of cocaine. Achat-Mendes C, Grundt P, Cao J, Platt DM, Newman AH, Spealman RD. Dopamine D3 and D2 Receptor Mechanisms in the Abuse-Related Behavioral Effects of Cocaine: Studies with Preferential Antagonists and Agonists in Squirrel Monkeys. J Pharmacol Exp Ther e-pub May 21, 2010.

Structure-Activity Relationships at the Metabotropic Glutamate Receptor Subtype 5
The metabotropic glutamate receptor subtype 5 (mGluR5) has been implicated in numerous neuropsychiatric disorders including addiction. IRP researchers hypothesized that the rigid diaryl alkyne template, derived from the potent and selective noncompetitive mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), can serve to guide the design of novel quinoline analogues and pharmacophore optimization has resulted in potent mGluR5 noncompetitive antagonists (EC50 range 60-100 nM) in the quinoline series. This study demonstrates that the structure-activity relationships derived from MPEP analogues can be used to direct the further investigation of novel quinoline analogues. In vivo evaluation of the lead compounds herein will provide valuable direction toward future drug design. Zhang P, Zou M-F, Rodriguez AL, Conn PJ, Newman AH. Structure-Activity Relationships in a Novel Series of 7-Substituted-Aryl Quinolines and 5-Substituted-Aryl Benzothiazoles at the Metabotropic Glutamate Receptor Subtype 5. Bioorg Med Chem. 2010; 18(9): 3026-3035.

Postendocytic Sorting of Constitutively Internalized Dopamine Transporter in Dopaminergic Neurons
The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for psychostimulants such as cocaine and amphetamine. DAT is known to undergo marked constitutive endocytosis but little is known about the fate and sorting of the endocytosed transporter. To study sorting in heterologous cells lines IRP scientists fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular N-terminal antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary colocalization with the late endosomal marker Rab7, less with the 'short loop' recycling marker Rab4 and little colocalization with the marker of 'long loop' recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the 2-adrenergic receptor, colocalizing primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue, JHC 1-64, exhibited the same co-localization pattern as TacDAT both in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. The authors conclude that independent of cell type, constitutively internalized DAT is sorted in an ubiqutination-independent manner to late endosomes/lysosomes and in part to a Rab4 positive 'short loop' recycling pathway. Ericksen J, Yoshimoto WBE, Jorgensen TN, Cha JH, Newman AH, Gether U. Postendocytic sorting of Constitutively Internalized Dopamine Transporter in Cell Lines and Dopaminergic Neurons. J Biol Chem. e-pub June 15, 2010.

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Neural and Cardiac Toxicities Associated With 3,4-Methylenedioxymethamphetamine (MDMA)
(+/-)-3,4-Methylenedioxymethamphetamine (MDMA) is a commonly abused illicit drug which affects multiple organ systems. In animals, high-dose administration of MDMA produces deficits in serotonin (5-HT) neurons (e.g., depletion of forebrain 5-HT) that have been viewed as neurotoxicity. Recent data implicate MDMA in the development of valvular heart disease (VHD). The present paper reviews several issues related to MDMA-associated neural and cardiac toxicities. The hypothesis of MDMA neurotoxicity in rats is evaluated in terms of the effects of MDMA on monoamine neurons, the use of scaling methods to extrapolate MDMA doses across species, and functional consequences of MDMA exposure. A potential treatment regimen (l-5-hydroxytryptophan plus carbidopa) for MDMA-associated neural deficits is discussed. The pathogenesis of MDMA-associated VHD is reviewed with specific reference to the role of valvular 5-HT(2B) receptors. The authors conclude that pharmacological effects of MDMA occur at the same doses in rats and humans. High doses of MDMA that produce 5-HT depletions in rats are associated with tolerance and impaired 5-HT release. Doses of MDMA that fail to deplete 5-HT in rats can cause persistent behavioral dysfunction, suggesting even moderate doses may pose risks. Finally, the MDMA metabolite, 3,4-methylenedioxyamphetamine (MDA), is a potent 5-HT(2B) agonist which could contribute to the increased risk of VHD observed in heavy MDMA users. Baumann MH, Rothman RB. Neural and cardiac toxicities associated with 3,4-methylenedioxymethamphetamine (MDMA). Int Rev Neurobiol 2009; 88: 257-296.

Synthetic Studies of Neoclerodane Diterpenes From Salvia Divinorum: Role of the Furan In Affinity for Opioid Receptors
Further synthetic modification of the furan ring of salvinorin A (1), the major active component of Salvia divinorum, has resulted in novel neoclerodane diterpenes with opioid receptor affinity and activity. A computational study has predicted 1 to be a reproductive toxicant in mammals and is suggestive that use of 1 may be associated with adverse effects. IRP scientists report in this study that piperidine 21 and thiomorpholine 23 have been identified as selective partial agonists at kappa opioid receptors. This indicates that additional structural modifications of 1 may provide ligands with good selectivity for opioid receptors but with reduced potential for toxicity. Simpson DS, Lovell KM, Lozama A, Han N, Day VW, Dersch CM, Rothman RB, Prisinzano TE. Synthetic studies of neoclerodane diterpenes from Salvia divinorum: role of the furan in affinity for opioid receptors. Organic & Biomolecular Chemistry 2009;7(18): 3748-3756.

Serotonergic Drugs and Valvular Heart Disease
The serotonin (5-HT) releasers (+/-)-fenfluramine and (+)-fenfluramine were withdrawn from clinical use owing to increased risk of valvular heart disease. One prevailing hypothesis (i.e., the '5-HT hypothesis') suggests that fenfluramine-induced increases in plasma 5-HT underlie the disease. Here, IRP researchers critically evaluate the possible mechanisms responsible for fenfluramine-associated valve disease. Findings from in vitro and in vivo experiments performed in the authors' laboratory are reviewed. The data are integrated with existing literature to address the validity of the 5-HT hypothesis and suggest alternative explanations. The overwhelming majority of evidence refutes the 5-HT hypothesis. A more likely cause of fenfluramine-induced valvulopathy is activation of 5-HT(2B) receptors on heart valves by the metabolite norfenfluramine. Future serotonergic medications should be designed to lack 5-HT(2B) agonist activity. Rothman RB, Baumann MH. Serotonergic drugs and valvular heart disease. Expert Opin Drug Safety 2009; 8(3): 317-329.


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