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NIDA Home > Publications > Director's Reports > May, 2009 Index    

Director's Report to the National Advisory Council on Drug Abuse - May, 2009

Research Findings - Intramural Research

Biomedical Informatics Section, Administrative Management Branch

Automation in an Addiction Treatment Research Clinic: Computerized Contingency Management, Ecological Momentary Assessment, and a Protocol Workflow System
IRP researchers have automated several major functions of our outpatient treatment research clinic for studies in drug abuse and dependence to enhance efficient operations, adherence to protocol and better communications among research staff. IRP researchers describe three such specialized applications within an integrated platform: the Automated Contingency Management (ACM) system for delivery of behavioral interventions, the Transactional Electronic Diary (TED) system for management of behavioral assessments, and the Protocol Workflow System (PWS) for computerized workflow automation and guidance of each participant's daily clinic activities. ACM and TED have each permitted us to conduct research that was not previously possible. In addition, the time to data analysis at the end of each study is substantially shorter. With the implementation of the PWS, the authors have been able to manage a research clinic with an 80-patient capacity having an annual average of 18,000 patient-visits and 7,300 urine collections with a research staff of five. Finally, automated data management has considerably enhanced the authors' ability to monitor and summarize participant-safety data for research oversight. Vahabzadeh M, Lin JL, Mezghanni M, Epstein DH, Preston KL. Automation in an addiction treatment research clinic: Computerised contingency management, ecological momentary assessment and a protocol workflow system. Drug Alcohol Rev. 2009;28(1):3-11.

Office of the Scientific Director

EEG and Cerebral Blood Flow Velocity Abnormalities in Chronic Cocaine Users
EEG and cerebral blood flow abnormalities occur in chronic cocaine users, but the relationship between these two phenomena is not well studied. IRP scientists evaluated this issue in 99 adult, chronic, heavy cocaine users tested within 5 days of admission to a closed research unit and 42 healthy, non-drug-using, age-matched controls. Absolute power in 6 frequency bands of resting EEG was compared with blood flow velocity in the middle cerebral artery as measured by transcranial Doppler sonography (which uses focused, high-frequency sound waves). The cocaine users had decreased blood flow velocity during cardiac diastole (relaxation), increased pulsatility index (a measure of small blood vessel resistance), and decreased high-frequency EEG power in posterior brain regions. The greater the amount of recent cocaine use, the greater the pulsatility index and the lower the low-frequency EEG power. Only high-frequency EEG power was significantly correlated with pulsatility index. These findings suggest that EEG and cerebral blood flow velocity changes during early cocaine abstinence reflect related physiological processes. Copersino ML, Herning RI, Better W, Cadet J-L, Gorelick DA. EEG and cerebral blood flow velocity abnormalities in chronic cocaine users. Clin EEG Neurosci. 2009;40(1):39-42.

Cellular Pathobiology Section, Cellular Neurobiology Research Branch

When the Endogenous Hallucinogenic Trace Amine N,N-dimethyltryptamine Meets the sigma-1 Receptor
N,N-dimethyltryptamine (DMT) is a hallucinogen found endogenously in human brain that is commonly recognized to target the 5-hydroxytryptamine 2A receptor or the trace amine-associated receptor to exert its psychedelic effect. DMT has been recently shown to bind sigma-1 receptors, which are ligand-regulated molecular chaperones whose function includes inhibiting various voltage-sensitive ion channels. Thus, it is possible that the psychedelic action of DMT might be mediated in part through sigma-1 receptors. Here, IRP researchers present a hypothetical signaling scheme that might be triggered by the binding of DMT to sigma-1 receptors. Su TP, Hayashi T, Vaupel DB. When the endogenous hallucinogenic trace amine N,N-dimethyltryptamine meets the sigma-1 receptor. Sci Signal. 2009;2(61):pe12.

MAM: More Than Just a Housekeeper
The physical association between the endoplasmic reticulum (ER) and mitochondria, which is known as the mitochondria-associated ER membrane (MAM), has important roles in various cellular 'housekeeping' functions including the non-vesicular transports of phospholipids. It has recently become clear that the MAM also enables highly efficient transmission of Ca(2+) from the ER to mitochondria to stimulate oxidative metabolism and, conversely, might enable the metabolically energized mitochondria to regulate the ER Ca(2+) homeostasis. Recent studies have shed light on molecular chaperones such as calnexin, calreticulin, ERp44, ERp57, grp75 and the sigma-1 receptor at the MAM, which regulate the association between the two organelles. The MAM thus integrates signal transduction with metabolic pathways to regulate the communication and functional interactions between the ER and mitochondrion. Hayashi T, Rizzuto R, Hajnoczky G, Su TP. MAM: more than just a housekeeper. Trends Cell Biol. 2009;19(2):81-8.

Electrophysiology Section, Cellular Neurobiology Research Branch

Properties of Distinct Ventral Tegmental Area Synapses Activated Via Pedunculopontine or Ventral Tegmental
Anatomical studies indicate that synaptic inputs from many cortical and subcortical structures converge on neurons of the ventral tegmental area (VTA). Although in vitro electrophysiological studies have examined synaptic inputs to dopamine (DA) and non-DA neurons in the VTA, they have largely relied upon local electrical stimulation to activate these synapses. This provides little information regarding the distinct properties of synapses originating from different brain areas. Using whole-cell recordings in parasagittal rat brain slices that preserved subcortical axons from the pedunculopontine nucleus (PPN) to the VTA, IRP scientists compared these synapses with those activated by intra-VTA stimulation. PPN-evoked currents demonstrated longer latencies than intra-VTA-evoked currents, and both VTA and PPN responses were mediated by GABA(A) and AMPA receptors. However, unlike VTA-evoked currents, PPN currents were exclusively mediated by glutamate in 25-40% of the VTA neurons. Consistent with a cholinergic projection from the PPN to the VTA, nicotinic acetylcholine receptors (nAChR) were activated by endogenous acetylcholine released during PPN, but not VTA, stimulation. This was seen as a reduction of PPN-evoked, and not VTA-evoked, synaptic currents by the alpha7-nAChR antagonist methyllycaconitine (MLA) and the agonist nicotine. The beta2-nAChR subunit antagonist dihydro-beta-erythroidine had no effect on VTA- or PPN-evoked synaptic currents. The effects of MLA on PPN-evoked currents were unchanged by the GABA(A) receptor blocker picrotoxin, indicating that alpha7-nAChRs presynaptically modulated glutamate and not GABA release. These differences in physiological and pharmacological properties demonstrate that ascending PPN and presumed descending inputs to VTA utilize distinct mechanisms to differentially modulate neuronal activity and encode cortical and subcortical information. Good CH, Lupica CR. Properties of distinct ventral tegmental area synapses activated via pedunculopontine or ventral tegmental. The Journal of Physiology. 2009;587(Pt 6):1233-47.

Neurophysiology Section, Cellular Neurobiology Research Branch

Generalized Tetracycline Induced Cre Recombinase Expression
Through the ROSA26 Locus of Recombinant Mice Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here IRP investigators describe the generation of a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5 kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. The mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development. BŠckman CM, Zhang Y, Malik N, Shan L, Hoffer BJ, Westphal H, Tomac AC. Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice. J Neurosci Methods. 2009;176(1):16-23.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Direct Profiling of Tissue Lipids by MALDI-TOFMS
Advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have allowed for the direct analysis of biological molecules from tissue. Although most of the early studies of direct tissue profiling by MALDI-TOFMS have focused on proteins and peptides, analysis of lipids has increased dramatically in recent years. This review gives an overview of the factors to consider when analyzing lipids directly from tissue and some recent examples of the use of MALDI-TOFMS for the direct profiling of lipids in tissue. Jackson SN, Woods AS. Direct profiling of tissue lipids by MALDI-TOFMS. J Chromatogr B Analyt Technol Biomed Life Sci. 2008; Nov 28. [Epub ahead of print].

Integrated Signaling in Heterodimers and Receptor Mosaics of Different Types of GPCRs of the Forebrain: Relevance for Schizophrenia
Receptor-receptor interactions within receptor heterodimers and receptor mosaics formed by different types of GPCRs represent an important integrative mechanism for signaling in brain networks at the level of the plasma membrane. The malfunction of special heterodimers and receptor mosaics in the ventral striatum containing D(2) receptors and 5-HT(2A) receptors in cortical networks may contribute to disturbances of key pathways involving ventral striato-pallidal GABA neurons and mediodorsal thalamic prefrontal glutamate neurons that may lead to the development of schizophrenia. The ventral striatum transmits emotional information to the cerebral cortex through a D(2) regulated accumbal-ventral pallidal-mediodorsal-prefrontal circuit which is of special interest to schizophrenia in view of the reduced number of glutamate mediodorsal-prefrontal projections associated with this disease. This circuit is especially vulnerable to D(2) receptor activity in the nucleus accumbens, since it produces a reduction in the prefrontal glutamate drive from the mediodorsal nucleus. The following D(2) receptor containing heterodimers/receptor mosaics are of special interest to schizophrenia: A(2A)-D(2), mGluR5-D(2), CB(1)-D(2), NTS(1)-D(2) and D(2)-D(3) and are discussed in this review. Fuxe K, Marcellino D, Woods AS, Giuseppina L, Antonelli T, Ferraro L, Tanganelli S, Agnati LF. Integrated signaling in heterodimers and receptor mosaics of different types of GPCRs of the forebrain: relevance for schizophrenia. J Neural Transm. 2009; Jan 21. [Epub ahead of print].

Building A New Conceptual Framework for Receptor Heteromers
Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, IRP scientists recommend clear definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. The authors also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development. FerrŽ S, Baler R, Bouvier M, Caron MG, Devi LA, Durroux T, Fuxe K, George SR, Javitch JA, Lohse MJ, Mackie K, Milligan G, Pfleger KD, Pin JP, Volkow ND, Waldhoer M, Woods AS, Franco R. Building new conceptual framework for receptor heteromers. Nat Chem Biol. 2009;5(3):131-4.

Anatomy and Cell Biology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Pedunculopontine and Laterodorsal Tegmental Nuclei Contain Distinct Populations of Cholinergic, Glutamatergic and GABAergic Neurons in the Rat
The pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) provide cholinergic afferents to several brain areas. This cholinergic complex has been suggested to play a role in sleep, waking, motor function, learning and reward. To have a better understanding of the neurochemical organization of the PPTg/LDTg IRP researchers characterized the phenotype of PPTg/LDTg neurons by determining in these cells the expression of transcripts encoding choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or the vesicular glutamate transporters (vGluT1, vGluT2 and vGluT3). Within the PPTg/LDTg complex the authors found neurons expressing ChAT, vGluT2 or GAD transcripts, these neuronal phenotypes were intermingled, but not homogeneously distributed within the PPTg or LDTg. Previous studies suggested the presence of either glutamate or gamma-aminobutyric acid (GABA) immunolabeling in a large number of PPTg/LDTg cholinergic neurons, leading to the widespread notion that PPTg/LDTg cholinergic neurons co-release acetylcholine together with either glutamate or GABA. To assess the glutamatergic or GABAergic nature of the PPTg/LDTg cholinergic neurons, the authors combined in situ hybridization (to detect vGluT2 or GAD transcripts) and immunohistochemistry (to detect ChAT), and found that over 95% of all PPTg/LDTg cholinergic neurons lack transcripts encoding either vGluT2 mRNA or GAD mRNA. As the vast majority of PPTg/LDTg cholinergic neurons lack transcripts encoding essential proteins for the vesicular transport of glutamate or for the synthesis of GABA, co-release of acetylcholine with either glutamate or GABA is unlikely to be a major factor in the interactions between acetylcholine, glutamate and GABA at the postsynaptic site. Wang HL, Morales M. Pedunculopontine and laterodorsal tegmental nuclei contain distinct populations of cholinergic, glutamatergic and GABAergic neurons in the rat. Eur J Neurosci. 2009;29(2):340-58.

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Studies of the Biogenic Amine Transporters 13. Identification of "Agonist" and "Antagonist" Allosteric Modulators of Amphetamine-Induced Dopamine Release
Recent studies identified novel allosteric modulators of the dopamine transporter (DAT). N-(diphenylmethyl)-2-phenyl-4-quinazolinamine (SoRI-9804), N-(2,2-diphenylethyl)-2-phenyl-4-quinazolinamine (SoRI-20040), and N-(3,3-diphenylpropyl)-2-phenyl-4-quinazolinamine (SoRI-20041) partially inhibited [(125)I]RTI-55 binding, slowed the dissociation rate of [(125)I]RTI-55 from the DAT, and partially inhibited [(3)H]dopamine uptake. In the present study IRP scientists report that SoRI-9804 and SoRI-20040, at doses that do not alter release, partially inhibited D-amphetamine-induced DAT-mediated release of [(3)H]MPP(+) or [(3)H]dopamine from striatal synaptosomes ("DAT-mediated DA release") in a dose-dependent manner. SoRI-20041, which does not alter DAT-mediated DA release measured with [(3)H]DA, reversed the effect of SoRI-20040. SoRI-20040 and SoRI-9804 also partially inhibited DAT-mediated DA release induced by DA or (+/-)-3,4-methylenedioxyamphetamine, demonstrating that the observed partial inhibition is not specific for a particular DAT substrate. SoRI-9804 and SoRI-20040 did not attenuate D-amphetamine-induced release of [(3)H]5-HT from serotonergic, or [(3)H]MPP(+) from noradrenergic, nerve terminals. Kinetic experiments demonstrated that SoRI-9804, in contrast to cocaine, slowed D-amphetamine-induced release of [(3)H]MPP(+) from dopaminergic nerve terminals without altering the apparent rate constants. The two major findings of this paper are: 1) the identification of both "agonist" (SoRI-9804, SoRI-20040) and "antagonist" (SoRI-20041) allosteric modulators of D-amphetamine-induced DAT-mediated DA release, and 2) [(3)H]DA uptake and D-amphetamine-induced DAT-mediated efflux can be separately modulated. Such agents may have therapeutic potential for the treatment of stimulant addiction, parkinson's disease and other psychiatric disorders. Rothman RB, Dersch CM, Ananthan S, Partilla JS. J Pharmacol Exp Ther. 2009; (Epub ahead of print)

Evidence for the Involvement of Dopamine Transporters in Behavioral Stimulant Effects of Modafinil
Modafinil is prescribed for numerous medical conditions, but the drug's mechanism of action is unclear. Here IRP investigstors examined the interaction of modafinil with receptors and transporters in vitro, and compared pharmacological effects of the drug to those produced by indirect dopamine (DA) agonists, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) and (+)-methamphetamine (METH). Modafinil was screened at various receptors and transporters using binding assays. Transporter-mediated uptake and release were examined in rat brain synaptosomes. Effects of modafinil on motor activity and neurochemistry were determined in rats undergoing in vivo microdialysis in n. accumbens. Of the receptors and transporters assayed, modafinil displayed measurable potency only at DA transporters (DAT), inhibiting [(3)H]DA uptake with an IC50 of 4.0 microM. Accordingly, modafinil pretreatment (10 microM) antagonized METH-induced release of the DAT substrate [(3)H]1-methyl-4-phenylpyridinium. Intravenous modafinil (20 & 60 mg/kg) produced dose-dependent increases in motor activity and extracellular DA, without affecting serotonin (5-HT). Analogous results were observed for GBR12909 (1 & 3 mg/kg), whereas METH (0.3 & 1 mg/kg) increased DA and 5-HT. Locomotor effects of all drugs were positively correlated with dialysate DA (P<0.001). Interestingly, modafinil pretreatment reduced METH-induced ambulation and DA release. These data show that modafinil interacts with DAT sites in rat brain, a property shared with agonist medications under investigation for treating cocaine dependence. Non-dopaminergic mechanisms may also contribute to the pharmacology of modafinil. Finally, the results suggest that modafinil should be tested as an adjunct for treating METH addiction. Zolkowska D, Jain R, Rothman RB, Partilla JS, Roth BL, Setola V, Prisinzano TE, Baumann MH. J Pharmacol Exp Ther. 2009; (Epub ahead of print).

Selective Suppression of Cocaine- versus Food-Maintained Responding by Monoamine Releasers in Rhesus Monkeys: Benzylpiperazine, (+)Phenmetrazine, and 4- Benzylpiperidine
Monoamine releasers constitute one class of drugs currently under investigation as potential agonist medications for the treatment of cocaine dependence. The efficacy and safety of monoamine releasers as candidate medications may be influenced in part by their relative potency to release dopamine and serotonin, and we reported previously that releasers with approximately 30-fold selectivity for dopamine vs. serotonin release may be especially promising. The present study examined the effects of the releasers benzylpiperazine, (+)phenmetrazine and 4-benzylpiperidine, which have 20-48-fold selectivity in vitro for releasing dopamine vs. serotonin. In an assay of cocaine discrimination, rhesus monkeys were trained to discriminate 0.4 mg/kg cocaine i.m. from saline in a two-key, food-reinforced procedure. Each of the releasers produced a dose- and time-dependent substitution for cocaine. 4-Benzylpiperidine had the most rapid onset and shortest duration of action. Phenmetrazine and benzylpiperazine had slower onsets and longer durations of action. In an assay of cocaine self-administration, rhesus monkeys were trained to respond for cocaine injections and food pellets under a second- order FR2(VR16:S) schedule. Treatment for 7 days with each of the releasers produced a dose-dependent and selective reduction in self-administration of cocaine (0.01 mg/kg/inj). The most selective effects were produced by phenmetrazine. Phenmetrazine also produced a downward shift in the cocaine self-administration dose effect curve, virtually eliminating responding maintained by a 30-fold range of cocaine doses (0.0032-0.1 mg/kg/inj) while having only small and transient effects on food-maintained responding. These findings support the potential utility of dopamine-selective releasers as candidate treatments for cocaine dependence. Negus SS, Baumann MH, Rothman RB, Mello NK, Blough BE. J Pharm Exp Ther. 2009; (Epub ahead of print).

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

The Future of Endocannabinoid-Oriented Clinical Research After CB(1) Antagonists
Great interest has been shown by the medical community and the public in the cannabinoid CB(1) receptor antagonists, such as rimonabant, for treatment of obesity, metabolic syndrome, and possibly drug addiction. This novel class of drug has therapeutic potential for other disorders, as the endocannabinoid system is involved in various health conditions. However, rimonabant, the first clinically available member of this class of drugs, has been linked to increased risk of anxiety, depression, and suicidality. Due to those risks, the European Medicines Agency called for its withdrawal from the market in October, 2008. Shortly after this decision, several pharmaceutical companies (Sanofi-aventis, Merck, Pfizer, Solvay) announced that they would stop further clinical research on this class of drug. Here, IRP scientists provide an overview of those events and make several suggestions for continuing such clinical research, while safeguarding the safety of patients and clinical trial subjects. Le Foll B, Gorelick DA, Goldberg SR. The future of endocannabinoid-oriented clinical research after CB(1) antagonists. Psychopharmacology. 2009;Mar 20; Epub ahead of print.

The Endocannabinoid System: A New Molecular Target for the Treatment of Tobacco Addiction
Tobacco addiction is one of the leading preventable causes of mortality in the world and nicotine appears to be the main critical psychoactive component in establishing and maintaining tobacco dependence. Several lines of evidence suggest that the rewarding effects of nicotine, which underlie its abuse potential, can be modulated by manipulating the endocannabinoid system. For example, pharmacological blockade or genetic deletion of cannabinoid CB(1) receptors reduces or eliminates many behavioral and neurochemical effects of nicotine that are related to its addictive potential. This review will focus on the recently published literature about the role of the endocannabinoid system in nicotine addiction and on the endocannabinoid system as a novel molecular target for the discovery of medications for tobacco dependence. Scherma M, Fadda P, Le Foll B, Forget B, Fratta W, Goldberg SR, Tanda G. The endocannabinoid system: a new molecular target for the treatment of tobacco addiction. CNS Neurol Disord Drug Targets. 2008;7(5):468-81.

Endogenous Fatty Acid Ethanolamides Suppress Nicotine-induced Activation of Mesolimbic Dopamine Neurons Through Nuclear Receptors
Nicotine stimulates the activity of mesolimbic dopamine neurons, which is believed to mediate the rewarding and addictive properties of tobacco use. Accumulating evidence suggests that the endocannabinoid system might play a major role in neuronal mechanisms underlying the rewarding properties of drugs of abuse, including nicotine. Here, IRP scientists investigated the modulation of nicotine effects by the endocannabinoid system on dopamine neurons in the ventral tegmental area with electrophysiological techniques in vivo and in vitro. They discovered that pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme that catabolizes fatty acid ethanolamides, among which the endocannabinoid anandamide (AEA) is the best known, suppressed nicotine-induced excitation of dopamine cells. Importantly, this effect was mimicked by the administration of the FAAH substrates oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), but not methanandamide, the hydrolysis resistant analog of AEA. OEA and PEA are naturally occurring lipid signaling molecules structurally related to AEA, but devoid of affinity for cannabinoid receptors. They blocked the effects of nicotine by activation of the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), a nuclear receptor transcription factor involved in several aspects of lipid metabolism and energy balance. Activation of PPAR-alpha triggered a nongenomic stimulation of tyrosine kinases, which might lead to phosphorylation and negative regulation of neuronal nicotinic acetylcholine receptors. These data indicate for the first time that the anorexic lipids OEA and PEA possess neuromodulatory properties as endogenous ligands of PPAR-alpha in the brain and provide a potential new target for the treatment of nicotine addiction. Melis M, Pillolla G, Luchicchi A, Muntoni AL, Yasar S, Goldberg SR, Pistis M. Endogenous fatty acid ethanolamides suppress nicotine-induced activation of mesolimbic dopamine neurons through nuclear receptors. J Neurosci. 2008;28(51):13985-94.

Interactions Between Environmental Aversiveness and the Anxiolytic Effects of Enhanced Cannabinoid Signaling by FAAH Inhibition in Rats
Since the discovery of endogenous cannabinoid signaling, the number of studies exploring its role in health and disease has increased exponentially. Fatty acid amide hydrolase (FAAH), the enzyme responsible for degradation of the endocannabinoid anandamide, has emerged as a promising target for anxiety-related disorders. FAAH inhibitors (e.g., URB597) increase brain levels of anandamide and induce anxiolytic-like effects in rodents. Recent findings, however, questioned the efficacy of URB597 as an anxiolytic. IRP scientists tested here the hypothesis that conflicting findings are due to variations in the stressfulness of experimental conditions employed in various studies. They found that URB597 (0.1-0.3 mg/kg) did not produce anxiolytic effects when the aversiveness of testing procedures was minimized by handling rats daily before experimentation, by habituating them to the experimental room, or by employing low illumination during testing. In contrast, URB597 had robust anxiolytic effects when the aversiveness of the testing environment was increased by eliminating habituation to the experimental room or by employing bright lighting conditions. Unlike URB597, the benzodiazepine chlordiazepoxide (5 mg/kg) had anxiolytic effects under all testing conditions. The anxiolytic effects of URB597 were abolished by the cannabinoid CB1-receptor antagonist AM251, showing that they were mediated by CB1 receptors. Close inspection of experimental conditions employed in earlier reports suggests that conflicting findings with URB597 can be explained by different testing conditions, such as those manipulated in the present study. These findings show that FAAH inhibition does not affect anxiety under mildly stressful circumstances but protects against the anxiogenic effects of aversive stimuli. Haller J, Barna I, Barsvari B, Gyimesi Pelczer K, Yasar S, Panlilio LV, Goldberg S. Interactions between environmental aversiveness and the anxiolytic effects of enhanced cannabinoid signaling by FAAH inhibition in rats. Psychopharmacology. 2009;Mar 4; Epub ahead of print.

Building a New Conceptual Framework for Receptor Heteromers
Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, IRP researchers recommend clear definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. They also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development. FerrŽ S, Baler R, Bouvier M, Caron MG, Devi LA, Durroux T, Fuxe K, George SR, Javitch JA, Lohse MJ, Mackie K, Milligan G, Pfleger KD, Pin JP, Volkow ND, Waldhoer M, Woods AS, Franco R. Building a new conceptual framework for receptor heteromers. Nat Chem Biol. 2009;5(3):131-4.

GDNF Control of the Glutamatergic Cortico-Striatal Pathway Requires Tonic Activation of Adenosine A Receptors
Glial cell line-derived neurotrophic factor (GDNF) affords neuroprotection in Parkinson's disease in accordance with its ability to bolster nigrostriatal innervation. IRP scientists previously found that GDNF facilitates dopamine release in a manner dependent on adenosine A(2A) receptor activation. As motor dysfunction also involves modifications of striatal glutamatergic innervation, they now tested if GDNF and its receptor system, Ret (rearranged during transfection) and GDNF family receptor alpha1 controlled the cortico-striatal glutamatergic pathway in an A(2A) receptor-dependent manner. GDNF (10 ng/mL) enhanced (by approximately 13%) glutamate release from rat striatal nerve endings, an effect potentiated (up to approximately 30%) by the A(2A) receptor agonist CGS 21680 (10 nM) and prevented by the A(2A) receptor antagonist, SCH 58261 (50 nM). Triple immunocytochemical studies revealed that Ret and GDNF family receptor alpha1 were located in 50% of rat striatal glutamatergic terminals (immunopositive for vesicular glutamate transporters-1/2), where they were found to be co-located with A(2A) receptors. Activation of the glutamatergic system upon in vivo electrical stimulation of the rat cortico-striatal input induced striatal Ret phosphorylation that was prevented by pre-treatment with the A(2A) receptor antagonist, MSX-3 (3 mg/kg). The results provide the first functional and morphological evidence that GDNF controls cortico-striatal glutamatergic pathways in a manner largely dependent on the co-activation of adenosine A(2A) receptors. Gomes CA, Sim›es PF, Canas PM, Quiroz C, Sebasti‹o AM, FerrŽ S, Cunha RA, Ribeiro JA. GDNF control of the glutamatergic cortico-striatal pathway requires tonic activation of adenosine A receptors. J Neurochem. 2009;108(5):1208-19.

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

Age at Menarche and Weight Concerns in Adolescent Smokers
Many girls adopt dieting and other practices (i.e. cigarette smoking) to control weight during puberty. This analysis explored the relationship between age at menarche and onset of daily smoking, and whether this relationship was influenced by weight concerns among treatment seeking female adolescents. The sample consisted of 71 participants enrolled in a smoking cessation trial (age 15.2±1.3 years; 74.7% European American, baseline BMI 24.7±5.4, age at menarche 11.7±1.3 years, Fagerstršm Test for Nicotine Dependence score 7.0±1.2). Over 60% of participants reported weight concerns at baseline, based on responses to the Eating Disorders module from the Diagnostic Interview for Children and Adolescents. Linear regression analyses revealed a significant association between age at menarche and age of onset of daily smoking (β=0.18±0.09, p=0.038). Having weight concerns did not modify the relationships between age at menarche and smoking trajectory/severity or abstinence. Findings support previous research showing that early maturation represents a risk factor for substance use. Further study in larger samples that include non-treatment-seeking adolescent female smokers is warranted. Jaszyna-Gasior M, Schroeder JR, Thorner ED, Heishman SJ, Collins CC, Lo S, Moolchan ET. Addictive Behaviors. 2009;34:92-5.

Behavioral Neuroscience Research Branch

Rapid Morphological Brain Abnormalities During Acute Methamphetamine Intoxication in the Rat: An Experimental Study Using Light and Electron Microscopy
This study describes morphological abnormalities of brain cells during acute methamphetamine (METH) intoxication in the rat and demonstrates the role of hyperthermia, disruption of the blood-brain barrier (BBB) and edema in their development. Rats with chronically implanted brain, muscle and skin temperature probes and an intravenous (i.v.) catheter were exposed to METH (9 mg/kg) at standard (23 ˇC) and warm (29 ˇC) ambient temperatures, allowing for the observation of hyperthermia ranging from mild to pathological (38-42 ˇC). When brain temperature peaked or reached a level suggestive of possible lethality (>41.5 ˇC), rats were injected with Evans blue (EB), rapidly anesthetized, perfused, and their brains were taken for further analyses. Four brain areas (cortex, hippocampus, thalamus and hypothalamus) were analyzed for EB extravasation, water and electrolyte (Na+, K+, Cl-) contents, immunostained for albumin and glial fibrillary acidic protein (GFAP), and examined for neuronal, glial and axonal alterations using standard light and electron microscopy. These examinations revealed profound abnormalities in neuronal, glial, and endothelial cells, which were stronger with METH administered at 29 ˇC than 23 ˇC and tightly correlated with brain and body hyperthermia. These changes had some structural specificity, but in each structure they tightly correlated with increases in EB levels, the numbers of albumin-positive cells, and water and ion contents, suggesting leakage of the BBB, acutely developing brain edema, and serious shifts in brain ion homeostasis as leading factors underlying brain abnormalities. While most of these acute structural and functional abnormalities appear to be reversible, they could trigger subsequent cellular alterations in the brain and accelerate neurodegeneration—the most dangerous complication of chronic amphetamine-like drug abuse. Sharma, HS, Kiyatkin, EA. Rapid morphological brain abnormalities during acute methamphetamine intoxication in the rat: An experimental study using light and electron microscopy. Journal of Chemical Neuroanatomy. 2009;37:18-32.

Reinstatement of Cocaine Seeking by Hypocretin (Orexin) in the Ventral Tegmental Area: Independence from the Local Corticotropin-Releasing Factor Network
Hypocretin (Hcrt), an arousal- and feeding-associated peptide, is expressed in lateral hypothalamic neurons that project to the ventral tegmental area (VTA). Intra-VTA Hcrt reinstates morphine-conditioned place preferences, and intracerebroventricular and intra-VTA corticotropin-releasing factor (CRF) reinstate cocaine seeking. Each is presumed to act, at least in part, through actions local to the VTA. Here, IRP scientists examined the possibility that VTA perfusion of Hcrt reinstates cocaine seeking and, if so, whether it does so through the VTA mechanism that is implicated in reinstatement by CRF. Rats were trained to lever-press for intravenous cocaine (2 weeks) and then underwent extinction training (saline substituted for cocaine: 3 weeks). Reinstatement behavior was tested and VTA dialysates were collected and assayed for glutamate or dopamine following footshock or perfusion of Hcrt or CRF, with or without Hcrt or CRF antagonists, into the VTA. Ventral tegmental area perfusion of Hcrt-1 or footshock stress reinstated cocaine seeking and caused release of VTA glutamate and dopamine. The effects of Hcrt-1 were blocked by a selective Hcrt-1 antagonist, but not a CRF antagonist, and were not mimicked by Hcrt-2. The Hcrt-1 antagonist did not block CRF-dependent footshock-induced reinstatement or glutamate or dopamine release. The behavioral and neurochemical effects of Hcrt-1 were attenuated but not blocked by kynurenic acid, an ionotropic glutamate antagonist that blocks footshock-induced reinstatement and glutamate release. While Hcrt and CRF are known to interact in some area of the brain, in the VTA proper they appear to have largely independent actions on the mesolimbic dopamine mechanisms of cocaine seeking. Wang B, You ZB, Wise RA. Reinstatement of cocaine seeking by hypocretin (orexin) in the ventral tegmental area: independence from the local corticotroping-releasing factor network. Biological Psychiatry. 2009; Feb 27 [E-pub ahead of print].

Long-Lasting Incubation of Conditioned Fear in Rats
In 1937, Diven (1) reported that human fear responses to cues previously paired with shock progressively increase or incubate over 24 hours. Since then, fear incubation has been demonstrated in both humans and nonhumans. However, the difficulty of demonstrating long-lasting fear incubation in rodents has hampered the study of the underlying mechanisms of this incubation. Here, IRP investigators describe a rat procedure where fear reliably incubates over time. They trained food-restricted rats to lever-press for food pellets in daily 90-min sessions. They then gave each rat one-hundred 30-s tones co-terminating with a 0.5-s, 0.5 mA footshock over 10 days (10 pairings per day). Groups of rats (n=10-15) were then given 4 presentations of the tone (the fear cue) 2, 15, 31 or 61 days after fear conditioning training and were assessed for conditioned suppression of lever-pressing. The authors found that conditioned fear responses were significantly higher 31 and 61 days after fear training than after 2 or 15 days. In control experiments, the authors showed that extensive tone-shock pairing is necessary for the emergence of fear incubation, and that it is unlikely that non-associative factors contribute to this incubation. The authors describe a procedure for generating reliable and long-lasting conditioned fear incubation. This procedure can be used to study mechanisms of fear incubation, and may provide a model for studying the mechanisms of delayed-onset posttraumatic stress disorder that occur in a sub-population of people previously exposed to chronic stressors. Pickens CL, Golden SA, Adams-Deutsch T, Nair SG, Shaham Y. Long-lasting incubation of conditioned fear in rats. Biological Psychiatry. 2009; Jan 22 Epub ahead of print].

Ventral mPFC Neuronal Activity Plays an Important Role in the Incubation of Cocaine Craving
Cue-induced drug-seeking in rodents progressively increases after withdrawal from cocaine, suggesting that cue-induced cocaine craving incubates over time. Here, IRP scientists explored the role of the medial prefrontal cortex (mPFC, a brain area previously implicated in cue-induced cocaine seeking) in this incubation. They trained rats to self-administer cocaine for 10 d (6 h/d, infusions were paired with a tone-light cue), and then assessed after 1 or 30 withdrawal days the effect of exposure to cocaine cues on lever presses in extinction tests. They found that cue-induced cocaine-seeking in the extinction tests was higher after 30 withdrawal days than after 1 day. The time-dependent increases in extinction responding were associated with large (ventral mPFC) or modest (dorsal mPFC) increases in ERK phosphorylation (a measure of ERK activity and an index of neuronal activation). After 30 withdrawal days, ventral but not dorsal injections of muscimol+baclofen (GABAa+GABAb receptor agonists that inhibit neuronal activity) decreased extinction responding. After 1 withdrawal day, ventral but not dorsal mPFC injections of bicuculline+saclofen (GABAa+GABAb receptor antagonists that increase neuronal activity) strongly increased extinction responding. Finally, muscimol+baclofen had minimal effect on extinction responding after 1 day, and in cocaine-experienced rats, ventral mPFC injections of muscimol+baclofen or bicuculline+saclofen had no effect on lever presses for an oral sucrose solution. The present results indicate that ventral mPFC neuronal activity plays an important role in the incubation of cocaine craving. Koya E, Uejima JL, Wihbey KA, Bossert JM, Hope BT, Shaham Y. Neuropharmacology. 2009;56(S1):177-185.

Chemical Biology Research Branch

Mechanisms of Withdrawal-Associated Increases in Heroin Self-Administration: Pharmacologic Modulation of Heroin vs Food Choice in Heroin-Dependent Rhesus Monkeys
Opioid withdrawal can produce a constellation of physiological and behavioral signs, including an increase in opioid self-administration. Different mechanisms mediate different withdrawal signs, and the present study used pharmacologic tools to assess mechanisms underlying withdrawal-associated increases in opioid reinforcement. Five rhesus monkeys were rendered heroin dependent via daily 21-h heroin self-administration sessions. One hour after each heroin self-administration session, monkeys chose between heroin (0-0.1 mg/kg per injection) and food (1 g pellets) during 2-h choice sessions. Under these conditions, heroin maintained a dose-dependent increase in heroin choice, such that monkeys responded primarily for food when low heroin doses were available (0-0.01 mg/kg per injection) and primarily for heroin when higher heroin doses were available (0.032-0.1 mg/kg per injection). Periods of spontaneous withdrawal were intermittently introduced by omitting one 21-h heroin self-administration session, and test drugs were administered during these withdrawal periods. Untreated withdrawal robustly increased heroin choice during choice sessions. Withdrawal-associated increases in heroin choice were completely suppressed by the mu opioid agonist morphine (0.032-0.32 mg/kg/h, i.v.), but not by the alpha-2 noradrenergic agonist clonidine (0.01-0.1 mg/kg/h, i.v.), the dopamine/ norepinephrine releaser amphetamine (0.032-0.1 mg/kg/h, i.v.), or the kappa-opioid antagonist 5'-guanidinonaltrindole (1.0 mg/kg, i.m.). The corticotropin-releasing factor 1 antagonist antalarmin (1.0-10 mg/kg per day, i.m.) produced a morphine-like suppression of withdrawal-associated increases in heroin choice in one of three monkeys. These results suggest that mechanisms of withdrawal-associated increases in the relative reinforcing efficacy of opioid agonists may be different from mechanisms of many other somatic, mood-related, and motivational signs of opioid withdrawal. Negus SS, Rice KC. Mechanisms of withdrawal-associated increases in heroin self-administration: pharmacologic modulation of heroin vs food choice in heroin-dependent rhesus monkeys. Neuropsychopharmacology. 2009;4(4):899-911.

Pharmacological Properties and Discriminative Stimulus Effects of a Novel and Selective 5-HT2 Receptor Agonist AL-38022A [(S)-2-(8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl)-1-methylethylamine]
PAL-38022A is a novel synthetic serotonergic (5-HT) ligand that exhibited high affinity for each of the 5-HT2 receptor subtypes (Ki ˛ 2.2 nM), but a significantly lower (> 100-fold less) affinity for other 5-HT receptors. In addition, AL-38022A displayed a very low affinity for a broad array of other receptors, neurotransmitter transport sites, ion channels, and second messenger elements, making it a relatively selective agent. AL-38022A potently stimulated functional responses via native and cloned rat (EC50 range: 1.9-22.5 nM) and human (EC50 range: 0.5-2.2 nM) 5-HT2 receptor subtypes including [Ca2+]i mobilization and tissue contractions with apparently similar potencies and intrinsic activities and was a full agonist at all 5-HT2 receptor subtypes. The CNS activity of AL-38022A was assessed by evaluating its discriminative stimulus effects in both a rat and a monkey drug discrimination paradigm using DOM as the training drug. AL-38022A fully generalized to the DOM stimulus in each of these studies; in monkeys MDL 100907 antagonized both DOM and AL-38022A. The pharmacological profile of AL-38022A suggests that it could be a useful tool in defining 5-HT2 receptor signaling and receptor characterization where 5-HT may function as a neurotransmitter. May, JA, Sharif, NA, Chen, H-H, Liao JC, Kelly CR, Glennon RA, Young R, Li JX, Rice KC, France C. Pharmacological properties and discriminative stimulus effects of a novel and selective 5-HT2 receptor agonist AL-38022A [(S)-2-(8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl)-1-methylethylamine]. Pharmacol Biochem Behav. 2009;91:307-14.

Corticotropin-Releasing Factor-1 Receptor Antagonists Decrease Heroin Self-Administration in Long- but not Short-Access Rats
Dysregulation of the stress-related corticotropin-releasing factor (CRF) system has been implicated in the development of drug dependence. The present study examined the effects of administering CRF type 1 (CRF1) receptor antagonists on heroin self-administration in animals allowed short (1 hour) or long (8-12 hours) access to intravenous heroin self-administration sessions. The nonpeptide CRF1 antagonists MJL-1-109-2 (1 hour versus 8 hours access) or R121919 (1 hour versus 12 hours access) were systemically injected in both short- and long-access rats. MJL-1-109-2 (10 mg/kg) and R121919 (10 and 20 mg/kg) reduced heroin self-administration in long-access animals without altering heroin intake in short-access animals. Both MJL-1-109-2 and R121919 decreased first-hour intravenous heroin self-administration selectively in long-access rats, with R121919 decreasing cumulative heroin intake across the 12-hour session. The results demonstrate that blockade of the CRF-CRF1 receptor system attenuates the increased heroin intake of rats with extended access to the drug. Greenwell TN, Funk CK, Cottone P, Richardson HN, Chen SA, Rice KC, Zorrilla EP, Koob GF. Corticotropin-releasing factor-1 receptor antagonists decrease heroin self-administration in long- but not short-access rats. Addiction Biology. 2009;14:130-43.

Role of Delta Opioid Efficacy as a Determinant of Mu/Delta Opioid Interactions in Rhesus Monkeys
Delta opioid agonists can selectively enhance the antinociceptive effects of mu opioid agonists without enhancing some other, potentially undesirable mu agonist effects. However, the degree of delta receptor efficacy required to produce this profile of interactions is unknown. To address this issue, the present study examined interactions produced by the mu agonist fentanyl and the intermediate-efficacy delta opioid MSF61 in rhesus monkeys. For comparison, interactions were also examined between fentanyl and the relatively high-efficacy delta agonist SNC243A and the delta antagonist naltrindole, which has negligible efficacy at delta receptors. Two different behavioral procedures were used: (a) a warm-water tail-withdrawal assay of thermal nociception, and (b) an assay of schedule-controlled responding for food reinforcement. Drug interactions within each procedure were evaluated using dose-addition analysis to compare experimental results with expected additivity. Drug interactions across procedures were evaluated using dose-ratio analysis to assess relative potencies to produce antinociception vs. response-rate suppression. As expected, dose-addition analysis found that fentanyl/SNC243A interactions were superadditive in the assay of antinociception but additive in the assay of schedule-controlled responding. Conversely, fentanyl/MSF61 interactions were generally additive in both procedures, and fentanyl/naltrindole interactions were additive or subadditive in both procedures. Dose-ratio analysis found that fentanyl alone produced antinociception and rate suppression with similar potencies. Some fentanyl/SNC243A mixtures produced antinociception with up to 4-fold greater potency than rate-suppression. However, fentanyl/MSF61 and fentanyl/naltrindole mixtures produced antinociception with lower potency than rate suppression. These results suggest that relatively high delta receptor efficacy is required for mu/delta antinociceptive synergy. Negus SS, Bear AE, Folk JE, Rice KC. Role of Delta Opioid Efficacy as a Determinant of Mu-Delta Opioid Interactions in Rhesus Monkeys. Role of Delta Opioid Efficacy as a Determinant of Mu/Delta Opioid Interactions in Rhesus Monkeys. Eur J Pharmacol 2009;602:92-100.

The Selective Non-Peptidic Delta Opioid Agonist SNC80 does not Facilitate Intracranial Self-Stimulation in Rats
Delta opioid receptor agonists are under development for a variety of clinical applications, and some findings in rats raise the possibility that agents with this mechanism have abuse liability. The present study assessed the effects of the non-peptidic delta opioid agonist SNC80 in an assay of intracranial self-stimulation (ICSS) in rats. ICSS was examined at multiple stimulation frequencies to permit generation of frequency-response rate curves and evaluation of curve shifts produced by experimental manipulations. Drug-induced leftward shifts in ICSS frequency-rate curves are often interpreted as evidence of abuse liability. However, SNC80 (1.0-10 mg/kg s.c.; 10-56 mg/kg i.p.) failed to alter ICSS frequency-rate curves at doses up to those that produced convulsions in the present study or other effects (e.g. antidepressant effects) in previous studies. For comparison, the monoamine releaser d-amphetamine (0.1-1.0 mg/kg, i.p.) and the kappa agonist U69,593 (0.1-0.56 mg/kg, i.p.) produced dose-dependent leftward and rightward shifts, respectively, in ICSS frequency-rate curves, confirming the sensitivity of the procedure to drug effects. ICSS frequency-rate curves were also shifted by two non-pharmacological manipulations (reductions in stimulus intensity and increases in response requirement). Thus, SNC80 failed to facilitate or attenuate ICSS-maintained responding under conditions in which other pharmacological and non-pharmacological manipulations were effective. These results suggest that non-peptidic delta opioid receptor agonists have negligible abuse-related effects in rats. Do Carmo GP, Folk JE, Rice KC, Chartoff E, Carlezon WA, Negus SS. The Selective Non-Peptidic Delta Opioid Agonist SNC80 does not Facilitate Intracranial Self-Stimulation in Rats. Eur J Pharmacol. 2009;604:58-65.

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 (pA2) 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-HT2A receptors and not at 5-HT2C 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-HT2A receptors in the discriminative stimulus effects of DOM, 2C-T-7, and DPT in rhesus monkeys. Li J-X, 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;328:976-81.

Presynaptic CRF1 Receptors Mediate the Ethanol Enhancement of GABAergic Transmission in the Mouse Central Amygdala
Corticotropin-releasing factor (CRF) is a 41-amino-acid neuropeptide involved in stress responses initiated from several brain areas, including the amygdala formation. Research shows a strong relationship between stress, brain CRF, and excessive alcohol consumption. Behavioral studies suggest that the central amygdala (CeA) is significantly involved in alcohol reward and dependence. IRP scientists recently reported that the ethanol augmentation of GABAergic synaptic transmission in rat CeA involves CRF1 receptors, because both CRF and ethanol significantly enhanced the amplitude of evoked GABAergic inhibitory postsynaptic currents (IPSCs) in CeA neurons from wild-type (WT) and CRF2 knockout (KO) mice, but not in neurons of CRF1 KO mice. The present study extends these findings using selective CRF receptor ligands, gene KO models, and miniature IPSC (mIPSC) analysis to assess further a presynaptic role for the CRF receptors in mediating ethanol effects in the CeA. In whole-cell patch recordings of pharmacologically isolated GABAAergic IPSCs from slices of mouse CeA, both CRF and ethanol augmented evoked IPSCs in a concentration-dependent manner, with low EC50s. A CRF1 (but not CRF2) KO construct and the CRF1-selective nonpeptide antagonist NIH-3 (LWH-63) blocked the augmenting effect of both CRF and ethanol on evoked IPSCs. Furthermore, the new selective CRF1 agonist stressin1, but not the CRF2 agonist urocortin 3, also increased evoked IPSC amplitudes. Both CRF and ethanol decreased paired-pulse facilitation (PPF) of evoked IPSCs and significantly enhanced the frequency, but not the amplitude, of spontaneous miniature GABAergic mIPSCs in CeA neurons of WT mice, suggesting a presynaptic site of action. The PPF effect of ethanol was abolished in CeA neurons of CRF1 KO mice. The CRF1 antagonist NIH-3 blocked the CRF- and ethanol-induced enhancement of mIPSC frequency in CeA neurons. These data indicate that presynaptic CRF1 receptors play a critical role in permitting or mediating ethanol enhancement of GABAergic synaptic transmission in CeA, via increased vesicular GABA release, and thus may be a rational target for the treatment of alcohol abuse and alcoholism. Nie Z, Zorrilla EP, Madamba SG, Rice KC, Roberto M, Siggins GR. Presynaptic CRF1 receptors mediate the ethanol enhancement of GABAergic transmission in the mouse central amygdala. The ScientificWorld JOURNAL. 2009;9:68-85.

Synthesis of 2-(5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraenamidoethyl-d4 Dihydrogen Phosphate, Tetra-deuterated pAEA
A labile intermediate phospho-anandamide (2-(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenamidoethyl dihydrogen phosphate, pAEA) has been identified in mouse brain and macrophages, but its precise quantitation was difficult because of its low concentration and chemical instability. IRP researchers report the synthesis of tetra-deuterated pAEA from 2-aminoethyl dihydrogen phosphate-1,1,2,2-d4 and (5Z,8Z,11Z,14Z)-2,5-dioxopyrrolidin-1-yl icosa-5,8,11,14-tetraenoate. The compound will be used to quantitate the pAEA necessary for a novel biosynthetic pathway. Cheng K, Saha B, Mahadevan A, Razdan RK, Kunos G, Jacobson AE, Rice KC. Synthesis of 2-(5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraenamidoethyl-d4 dihydrogen phosphate, tetra-deuterated pAEA. J Labelled Comp Radiopharm. 2008;51(12):389-90.

Probes for Narcotic Receptor Mediated Phenomena. 37.(1) Synthesis and Opioid Binding Affinity of the Final Pair of Oxide-Bridged Phenylmorphans, the Ortho- and Para-b-Isomers and their N-Phenethyl Analogues, and the Synthesis of the N-Phenethyl Analogues of the Ortho- and Para-d-Isomers
In the isomeric series of 12 racemic topologically rigid N-methyl analogues of oxide-bridged phenylmorphans, all but two of the racemates, the ortho- and para-b-oxide-bridged phenylmorphans 20 and 12, have remained to be synthesized. The b-isomers were very difficult to synthesize because of the highly strained 5,6-trans-fused ring junction that had to be formed. The authors' successful strategy required functionalization of the position para (or ortho) to a fluorine atom on the aromatic ring using an electron-withdrawing nitro group to activate that fluorine. The racemic N-phenethyl analogues 24 and 16 were moderately potent κ-receptor antagonists in the [35S]GTPϒS assay. IRP researchers synthesized the N-phenethyl-substituted oxide-bridged phenylmorphans in the ortho- and para-d-oxide-bridged phenylmorphan series (51 and 52) which had not been previously evaluated using contemporary receptor binding assays to see whether they also have higher affinity for opioid receptors than their N-methyl relatives 46 and 47. Kurimura M, Liu H, Sulima A, Hashimoto A, Przybyl AK, Ohshima E, Kodato S, Deschamps JR, Dersch C M, Rothman RB, Lee YS, Jacobson AE, Rice KC. Probes for Narcotic Receptor Mediated Phenomena. 37. (1) Synthesis and opioid binding affinity of the final pair of oxide-bridged phenylmorphans, the ortho- and para-b-isomers and their n-phenethyl analogues, and the synthesis of the n-phenethyl analogues of the ortho- and para-d-isomers. J Med Chem. 2008; 51(24):7866-81.

Genetic and Early Environmental Factors Interact to Influence Ethanol's Motivational Effects
To explore these issues, a reciprocal cross-fostering paradigm was applied to Fischer and Lewis rats. The adult female offspring received vehicle or the kappa opioid antagonist nor-BNI (1 mg/kg) followed by assessments of conditioned taste aversion (CTA), blood alcohol concentrations (BACs) and hypothermia induced by 1.25 g/kg intraperitoneal ethanol. CTA acquisition in the in-fostered Fischer and Lewis animals did not differ; however, the Fischer maternal environment produced stronger acquisition in the cross-fostered Lewis rats versus their in-fostered counterparts. CTAs in the Fischer rats were not affected by cross-fostering. In extinction, the in-fostered Lewis animals displayed stronger aversions than the Fischer groups on two trials (of 12) whereas the cross-fostered Lewis differed from the Fischer groups on nine trials. Despite these CTA effects, Lewis rats exhibited higher BACs and stronger hypothermic responses than Fischer with no cross-fostering effects in either strain. No phenotypes were affected by nor-BNI. These data extend previous findings dissociating the aversive and peripheral physiological effects of ethanol in female Fischer and Lewis rats, and highlight the importance of genetic and early environmental factors in shaping subsequent responses to alcohol's motivational effects in adulthood. Roma PG, Rinker JA, Serafine KM, Chen S A, Barr CS, Cheng K, Rice, KC, Riley AL. Genetic and early environmental factors interact to influence ethanol's motivational effects. Pharmacol Biochem Behav. 2008;91(1):134-39.

Discriminative Stimulus Properties of Naloxone in Long-Evans Rats: Assessment with the Conditioned Taste Aversion Baseline of Drug Discrimination Learning
The characterization of the discriminative stimulus properties of naloxone has focused primarily on its actions at the mu opioid receptor, although naloxone also displays an affinity for delta and kappa receptor subtypes. The present study extends this characterization of the naloxone cue by investigating if relatively specific antagonists for the mu (naltrexone: 0.10-0.56 mg/kg), delta (naltrindole: 1-18 mg/kg), and kappa (MR2266: 1.8-10 mg/kg) opioid receptor subtypes will substitute for naloxone in animals trained to discriminate naloxone from its vehicle. The temporal nature of the naloxone cue was examined by varying pretreatment time points (15, 30, 45, 60 min). Finally, various doses of naltrexone methobromide (1-18 mg/kg) were assessed to determine peripheral mediation of the cue. Female Long-Evans rats (N = 30) received an injection of naloxone (1 mg/kg; i.p.) 15 min prior to a pairing of saccharin (20-min access) and the emetic LiCl (1.8 mEq; i.p.; n = 16, group NL) or vehicle (n = 14, group NW); on other days, they were injected with saline prior to saccharin alone. Substitution tests with compounds with various receptor affinities and selective CNS and PNS actions were then assessed. Only naloxone and naltrexone produced dose-dependent decreases in saccharin consumption. Naloxone administered at 15 and 30 min before saccharin produced decreases in consumption similar to that displayed on training days. Naltrexone methobromide substituted only at the highest dose tested (18 mg/kg). Naloxone's stimulus effects appear to be mediated centrally via activity at the mu opioid receptor. Davis CM, Stevenson GW, Ca–adas F, Ullrich T, Rice KC, Riley AL. Discriminative stimulus properties of naloxone in Long-Evans rats: assessment with the conditioned taste aversion baseline of drug discrimination learning. Psychopharmacology. 2009;203(2):421-29.

Synthesis and Pharmacological Effects of the Enantiomers of the N-phenethyl Analogues of the Ortho and Para e- and f-oxide-bridged Phenylmorphans
The N-phenethyl analogues of (1R*,4aR*,9aS*)-2-phenethyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2,3-c]pyridin-6-ol and 8-ol and (1R*,4aR*,9aR*)-2-phenethyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2.3-c]pyridin-6-ol and 8-ol, the ortho- (43) and para-hydroxy e- (20), and f-oxide-bridged 5-phenylmorphans (53 and 26) were prepared in racemic and enantiomerically pure forms from a common precursor, the quaternary salt 12. Optical resolutions were accomplished by salt formation with suitable enantiomerically pure chiral acids or by preparative HPLC on a chiral support. The N-phenethyl (-)- para-e enantiomer (1S,4aS,9aR-(-)-20) was found to be a mu-opioid agonist with morphine-like antinociceptive activity in a mouse assay. In contrast, the N-phenethyl (-)-ortho-f enantiomer (1R,4aR,9aR-(-)-53) had good affinity for the mu-opioid receptor (K(i) = 7 nM) and was found to be a mu-antagonist both in the [(35)S]GTP-gamma-S assay and in vivo. The molecular structures of these rigid enantiomers were energy minimized with density functional theory at the level B3LYP/6-31G* level, and then overlaid on a known potent mu-agonist. This superposition study suggests that the agonist activity of the oxide-bridged 5-phenylmorphans can be attributed to formation of a seven membered ring that is hypothesized to facilitate a proton transfer from the protonated nitrogen to a proton acceptor in the mu-opioid receptor. Zezula J, Singer L, Przyby AK, Hashimoto A, Dersch CM, Rothman, RB, Deschamps, J, Lee YS, Jacobson AE, Rice, KC. Synthesis and pharmacological effects of the enantiomers of the N-phenethyl analogues of the ortho and para e- and f-oxide-bridged phenylmorphans. Org Biomol Chem. 2008;6(16):2868-83.

Integration of In Vivo and In Vitro Approaches to Characterize the Toxicity of Antalarmin, a Corticotropin-Releasing Hormone Receptor Antagonist
Non-clinical studies were conducted to evaluate the toxicity of Antalarmin, a corticotropin-releasing hormone type 1 receptor antagonist being developed for therapy of stress-related pathologies. Antalarmin was not genotoxic in bacterial mutagenesis assays, mammalian cell mutagenesis assays, or in vivo DNA damage assays. In a 14-day range-finding study in rats, Antalarmin doses >or=500 mg/kg/day (3,000 mg/m(2)/day) induced mortality. In a 90-day toxicity study in rats, no gross toxicity was seen at doses of 30, 100, or 300 mg/kg/day (180, 600, or 1,800 mg/m(2)/day, respectively). Antalarmin (300 mg/kg/day) induced mild anemia, increases in serum gamma-glutamyl transferase activity, and microscopic hepatic pathology (bile duct hyperplasia and epithelial necrosis, periportal inflammation). Microscopic renal changes (cortical necrosis, inflammation, hypertrophy, nephropathy) were observed in rats at all Antalarmin doses. In a 14-day range-finding study in dogs, Antalarmin doses >or=50mg/kg/day (1,000 mg/m(2)/day) induced repeated emesis and bone marrow suppression. In a 90-day toxicity study in dogs, Antalarmin (4, 8, or 16 mg/kg/day (80, 160, or 320 mg/m(2)/day, respectively)) induced bone marrow and lymphoid depletion, but no gross toxicity. Comparative in vitro studies using rat, dog, and human neutrophil progenitors demonstrated that canine bone marrow cells are highly sensitive to Antalarmin cytotoxicity, while rat and human bone marrow cells are relatively insensitive. As such, the bone marrow toxicity observed in dogs is considered likely to over-predict Antalarmin toxicity in humans. The hepatic and renal toxicities seen in rats exposed to Antalarmin identify those tissues as the most likely targets for Antalarmin toxicity in humans. Horn TL, Harder JB, Johnson WD, Curry PT, Parchment RE, Morrissey RL, Mellick PW, Calis KA, Gold PW, Rice KC, Contoreggi C, Charney DS, Cizza G, Glaze ER, Tomaszewski JE, McCormick DL. Integration of in vivo and in vitro approaches to characterize the toxicity of Antalarmin, a corticotropin-releasing hormone receptor antagonist. Toxicology. 2008;248(1):8-17.

Non-Stereoselective Reversal of Neuropathic Pain by Naloxone and Naltrexone: Involvement of Toll-Like Receptor 4 (TLR4)
Although activated spinal cord glia contribute importantly to neuropathic pain, how nerve injury activates glia remains controversial. It has recently been proposed, on the basis of genetic approaches, that toll-like receptor 4 (TLR4) may be a key receptor for initiating microglial activation following L5 spinal nerve injury. The present studies extend this idea pharmacologically by showing that TLR4 is key for maintaining neuropathic pain following sciatic nerve chronic constriction injury (CCI). Established neuropathic pain was reversed by intrathecally delivered TLR4 receptor antagonists derived from lipopolysaccharide. Additionally, (+)-naltrexone, (+)-naloxone, and (-)-naloxone, which we show here to be TLR4 antagonists in vitro on both stably transfected HEK293-TLR4 and microglial cell lines, suppressed neuropathic pain with complete reversal upon chronic infusion. Immunohistochemical analyses of spinal cords following chronic infusion revealed suppression of CCI-induced microglial activation by (+)-naloxone and (-)-naloxone, paralleling reversal of neuropathic pain. Together, these CCI data support the conclusion that neuron-to-glia signaling through TLR4 is important not only for initiating neuropathic pain, as suggested previously, but also for maintaining established neuropathic pain. Furthermore, these studies suggest that the novel TLR4 antagonists (+)-naloxone and (-)-naloxone can each fully reverse established neuropathic pain upon multi-day administration. This finding with (+)-naloxone is of potential clinical relevance. This is because (+)-naloxone is an antagonist that is inactive at the (-)-opioid selective receptors on neurons that produce analgesia. Thus, these data suggest that (+)-opioid antagonists such as (+)-naloxone may be useful clinically to suppress glial activation, yet (-)-opioid agonists suppress pain. Hutchinson MR, Zhang Y, Brown K, Coats BD, Shridhar M, Sholar PW, Patel SJ, Crysdale NY, Harrison JA, Maier SF, Rice KC, Watkins LR. Non-stereoselective reversal of neuropathic pain by naloxone and naltrexone: involvement of toll-like receptor 4 (TLR4). Eur J Neurosci. 2008;28(1):20-9.

Evidence for a Mu-Delta Opioid Receptor Complex in CHO Cells Co-Expressing Mu and Delta Opioid Peptide Receptors
Based on non-competitive binding interactions IRP scientists suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. They hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. They used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers. Rutherford JM, Wang J, Xu H, Dersch CM, Partilla JS, Rice KC, Rothman RB. Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors. Peptides. 2008 Aug;29(8):1424-31.

Behavioral Pharmacology of the Mu/Delta Opioid Glycopeptide MMP2200 in Rhesus Monkeys
H(2)N-Tyr-D-Thr-Gly-Phe-Leu-Ser-(O-beta-D-lactose)-CONH(2) (MMP2200) is a novel glycopeptide opioid agonist with similar affinities for mu and delta receptors. Glycosylation promoted brain penetration and production of centrally mediated behavioral effects in mice; however, it is unknown whether the magnitude of enhanced brain penetration is sufficient to permit central mediation of drug effects and production of synergistic mu/delta antinociceptive interactions after systemic administration in primates. To address this issue, the present study compared the effects of MMP2200 and the mu-agonist morphine in four behavioral procedures in rhesus monkeys. In an assay of thermal nociception, morphine (1.0-5.6 mg/kg) produced dose-dependent antinociception, whereas MMP2200 (10-56 mg/kg) was ineffective. In an assay of capsaicin-induced thermal allodynia, both morphine (0.01-1.0 mg/kg) and MMP2200 (0.032-3.2 mg/kg) produced dose-dependent antiallodynic effects. MMP2200-induced antiallodynia was blocked by the moderately mu-selective antagonist naltrexone (0.01 mg/kg), the delta-selective antagonist naltrindole (1.0 mg/kg), and the peripherally selective opioid antagonist quaternary naltrexone (0.32 mg/kg). In an assay of schedule-controlled behavior, both morphine (0.01-1.0 mg/kg) and MMP2200 (10-56 mg/kg) decreased response rates. Morphine effects were antagonized by naltrexone (0.001-0.01 mg/kg); however, the effects of MMP2200 were not antagonized by either naltrexone (0.01 mg/kg) or naltrindole (1.0 mg/kg). In an assay of drug self-administration, morphine (0.0032-0.32 mg/kg/injection) produced reinforcing effects, whereas MMP2200 (0.032-0.32 mg/kg/injection) did not. These results suggest that systemically administered MMP2200 acted as a peripheral, mu/delta-opioid agonist with limited distribution to the central nervous system in rhesus monkeys. These results also suggest the existence of species differences in the pharmacokinetics and brain penetration of glycopeptides. Do Carmo GP, Polt R, Bilsky EJ, Rice KC, Negus SS. Behavioral pharmacology of the mu/delta opioid glycopeptide MMP2200 in rhesus monkeys. J Pharmacol Exp Ther. 2008;326(3):939-48.

Probes for Narcotic Receptor Mediated Phenomena. 38. An Expeditious Synthesis of rac-cis-4a-Ethyl-2-methyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ol and rac-cis-2-Methyl-4a-phenethyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ol
A high-yielding five-step synthesis of cis-benzofuropyridin-6-ols provided an improved route to compounds with low to subnanomolar affinity at opioid receptors and high antinociceptive potency. This synthesis provided the known rac-cis-4a-ethyl-2-methyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ol (1a) in high yield, and the novel rac-cis-2-methyl-4a-phenethyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ol (1b). It was achieved using NBS to prepare the key intermediate 7. Di-demethylation followed by subsequent displacement of the bromine by the phenolic ion in hot Et3N gave the desired 1a. The structure of 1a was confirmed by X-ray crystallography. Iyer MR, Deschamps JR, Jacobson AE, Rice KC. Probes for narcotic receptor mediated phenomena. 38. An expeditious synthesis of rac-cis-4a-Ethyl-2-methyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ol and rac-cis-2-Methyl-4a-phenethyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ol. Heterocycles. 2009;78:1061-72.


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