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Stop Animal Exploitation NOW!
S. A. E. N.
"Exposing the truth to wipe out animal experimentation"

National Institute on Drug Abuse

1 Z01 DA000003-22 BNRB
Drugs On Learned & Spontaneous Behavior Of Experimental Animals

Principal Investigator: Steven R Goldberg, PhD (PP, NIDA)
Lab staff: Sergi Ferre, M.D., Ph.D (PP, NIDA)
Maria Scherma, PhD (PP, NIDA)
Carmen Mazzola, MD (PP, NIDA)
Chanel Barnes (PP, NIDA)
Carrie Wertheim (PP, NIDA)
Julie B Medalie (PP, NIDA)
Joanne Gilman (PP, NIDA)
Charles W. Schindler, PhD (PP, NIDA)
Jessica A Stroik (PP, NIDA)
Eric B Thorndike (PP, NIDA)
NIH Collaborators: Jean Lud Cadet, MD (MNB, NIDA)
Bruce Hope, PhD (BNRB, NIDA)
Jonathan L. Katz, PhD (MDRB, NIDA)
Tsung Ping Su, PhD (CNRB, NIDA)
Extramural Collaborators: Marcello Solinas, PhD (CNRS, University of Poitiers)
Jack Bergman, PhD (ADARC/McLean Hospital, Harvard Medical School)
Rafael Franco, PhD (Department of Biochemistry, University of Barcelona)
Carol Paronis, PhD (ADARC/McLean Hospital, Harvard Medical School)
Daniele Piomelli, PhD (Department of Pharmacology, University of California-Irvine)
Marco Bortolato, PhD (Department of Pharmacology, University of California-Irvine)
Alexandros Makriyannis, PhD (Center for Drug Discovery, Northeastern University)
E Albuquerque (University of Maryland School of Medicine)
P Fadda (University of Cagliari)
I Fattore (University of Cagliari)
W Fratta (University of Cagliari)
J Haller (Institute of Experimental Medicine, Budapest)
E Mikics (Institute of Experimental Medicine, Budapest)
R Schwarcz (Mayland Psychiatric Institute, University of Maryland School of Medicine)
K Vadivel (Northeastern University, Boston)
Sevil Yasar, MD (Johns Hopkins Univ Sch Med)
 from October 01, 2006 to September 30, 2007
Human subject research: Neither Human Subjects nor Human Tissues
Total Staff Years:3.5
Keywords:drug discrimination; schedule-controlled behavior; in-vivo microdialysis; locomotor activity; cannabinoids; anandamide; In-vivo imaging; methamphetamine; nicotine; cocaine; monkeys; adenosine; rats
Goals and Objectives:Our experiments are designed assess the different neuropharmacological and behavioral mechanisms underlying discrimination of abused drugs in order to complement ongoing studies of drug reinforcement or reward. Our overall goal is to understand how abused drugs such as marijuana (cannabinoids), nicotine (tobacco), methamphetamine or cocaine, or drugs that have potential applications as medications for treatment of tobacco smoking or other types of drug abuse have beneficial or detrimental side effects related to disruption of ongoing behavior maintained by food, alterations of emotional responses such as anxiety, or modulation of attention learning and memory processes. Our research is conducted in rats and monkeys and and utilizes behavio;ral, neurochemical (microdialyis), and molecular tehniques. Research is currently focused on nicotine, cannabinoids and endocannabinoids, and methamphetamine. Our main goal is to develop clinical therapeutics without detrimental side effects such as abuse liability and disruption of normal functioning.
Summary:Experiments are being conducted to assess the different neuropharmacological and behavioral mechanisms underlying behavior controlled by drugs as discriminative stimuli in rats and monkeys and the ability of pharmacological or behavioral manipulations to modify discrimination, as well as self-administration, of THC or nicotine, to disrupt ongoing food-maintained behavior to alter emotional responses such as anxiety or to modulate attention learning and memory processes. Currently, studies are focusing on nicotine and a series of cannabinoids, including delta-9-tetrahydrocannabinol (THC), the psychoactive ingredient in marijuana, the cannabinoid CB1 receptor antagonists Rimonabant and AM251, the endogenous cannabinoids anandamide and 2AG, the anandamide uptake inhibitor AM404, and the fatty acid amide hydrolase (FAAH) inhibitor URB597. Studies are also being conducted on methamphetamine, cocaine and heroin.

There is previous data indicating involvement of the cholinergic system in the psychotropic effects of cannabis and systemic administration of the main active ingredient in cannabis, delta-9-tetrahydrocannabinol (THC), has been reported to increase or to decrease extra-cellular levels of acetylcholine in several brain areas. In a recent study Solinas et al., 2007), we investigated whether drugs acting at either nicotinic or muscarinic receptors could modulate the discriminative effects of THC. In rats that had learned to discriminate 3mg/kg of THC from vehicle injections, the nicotinic agonist nicotine and the muscarinic agonist pilocarpine did not produce THC-like effects but they both potentiated the discriminative effects of low doses of THC. Neither the nicotinic antagonist mecamylamine nor the muscarinic antagonist scopolamine altered the discriminative effects of THC but they blocked the potentiation induced by nicotine and pilocarpine, respectively. In order to further investigate the nicotinic receptor subtypes involved in the discriminative effects of THC we then made use of selective antagonists for beta-2 or alpha-7 subunits. The selective alfa-7 nicotinic receptor antagonist MLA but not the selective beta-2 nicotine antagonist DHBE significantly reduced the discriminative effects of THC. However, both MLA and DHBE reversed the potentiation induced by nicotine. Our results suggest that the cholinergic system is involved in the discriminative effects of THC. Furthermore, our results with selective nicotinic antagonists suggest that potentiation or reduction of THCs effects may depend on different mechanisms. Finally, alpha-7 nicotinic antagonists could be useful in the treatment of cannabis intoxication and abuse.

It is known that strong functional interactions exist between endogenous cannabinoid and adenosine systems. The ventral striatum (mostly represented by the nucleus accumbens) forms part of the brain circuitry involved in goal-directed behavior, the conversion of motivation into action. A common denominator in the neurochemical effects of most addictive substances, including opiates, ethanol, amphetamines, cocaine, nicotine in tobacco and G9-tetrahydrocannabinol (THC) in marijuana, is striatal dopamine release, with a preferential effect on the ventral striatum (shell of the nucleus accumbens). Since the ventral striatum contains a high density of adenosine A2A receptors, which strongly modulate excitatory synapses of GABAergic enkephalinergic neurons, adenosinergic modulation of drug-seeking and drug-taking behavior is likely.

We have now demonstrated functional and physical interactions between adenosine A2A and cannabinoid CB1 receptors (Carriba et al., 2007; Ferre et al., 2007). In in-vitro studies we have demonstrated both true A2A-CB1 heteromers using BRET techniques in mammalian co-transfected cells and A2A-CB1 heteromeric receptor complexes in rat striatum using co-immunoprecipitation techniques. In in-vivo studies with squirrel monkeys, the adenosine A2A receptor antagonist MSX-3 markedly potentiated the reinforcing effects of the cannabinoid CB1-receptor agonists anandamide and THC, when drug-taking behavior was studied under a fixed-ratio schedule where every 10th response produced an intravenous injection of drug. This occurred at doses of MDSX-3 with no effect on comparable responding for food. In contrast, adenosine A2A receptor blockade with MSX-3 did not reinstate extinguished anandamide or THC drug-seeking behavior, but immediately and almost completely suppressed drug-seeking behavior maintained by THC under a second-order schedule in which behavioral responses by monkeys intermittently produced brief visual stimuli that were paired with THC injection only at the end of each daily session. Finally, adenosine A2A receptor antagonists can potentiate the reinforcing effects of nicotine and the discriminative effects of nicotine, methamphetamine and cocaine in rats (Munzar et al. 2002; Justinova et al. 2003). Based on these findings, we hypothesize that adenosine A2A receptor antagonists can enhance the direct reinforcing effects of THC and other addictive drugs by potentiating dopaminergic neurotransmission, but can reduce drug-seeking behavior by presynaptic modulation of actions on glutamate release and endocannabinoid signaling.

We have recently reported that nicotine displays high reinforcing efficacy in squirrel monkeys. There is great interest in quantifying brain nicotinic acetylcholine receptors (nAChR) in Squirrel monkeys to study the involvement of these receptors in nicotine dependence. Due to the limitations of existing methods of quantification for PET (need for arterial blood or need of a reference region in the brain), we have now explored the use of an extra-cerebral area for quantification of central receptors (Le Foll et al., 2007). If the ratio between the volume of distribution of the non-displaceable compartment (VDND) in the region of interest and a reference region inside or outside of the brain is constant and known, then theoretically it is possible to use the accumulation of radioactivity over time in a reference region and a region of interest to calculate the binding potential (BPND). To validate this approach, we performed quantitative PET studies of 42* nAChRs with 2 18Ffluoro-A-85380 (2-18FFA) in Squirrel monkeys. VT was measured in several brain areas using a blood input function. BPND values were measured with data from a reference region inside (cerebellum, Cb) or outside (muscle) of the brain using a simplified reference tissue model and PMOD software. When muscle was used as the reference region, BPND values were corrected for the difference between VT in muscle and VDND in brain. VT values were highest in thalamus and lowest in cerebellum and muscle, consistent with the distribution of 42* nAChRs in primates. Limited (94% for Cb) or no displacement (for muscle) of radioligands by nicotine was observed, indicating limited specific binding of 2-18FFA in these regions. A strong correlation (r = 0.9, P<0.001) between BPND values measured using Cb and muscle as reference regions was observed. These results suggest that quantifying central receptors is feasible using an extra-cerebral reference region, providing a novel approach for quantification of brain receptors when no suitable reference region inside the brain is available.
Publications generated by this research:
  1. Borycz J, Pereira MF, Melani A, Rodrigues RJ, Kofalvi A, Panlilio L, Pedata F, Goldberg SR, Cunha RA, Ferre S (2007) Differential glutamate-dependent and glutamate-independent adenosine A1 receptor-mediated modulation of dopamine release in different striatal compartments. J Neurochem 101:355-63. PubMed
  2. Carriba P, Ortiz O, Patkar K, Justinova Z, Stroik J, Themann A, Muller C, Woods AS, Hope BT, Ciruela F, Casado V, Canela EI, Lluis C, Goldberg SR, Moratalla R, Franco R, Ferre S (2007) Striatal Adenosine A(2A) and Cannabinoid CB(1) Receptors Form Functional Heteromeric Complexes that Mediate the Motor Effects of Cannabinoids. Neuropsychopharmacology, in press (e-pub ahead of print). PubMed
  3. Ferre S, Ciruela F, Borycz J, Solinas M, Quarta D, Antoniou K, Quiroz Molina CR, Justinova Z, Lluis C, Franco R, Goldberg SR (2007) Adenosine A1-A2A receptor heteromers new targets for caffeine in the brain. Front Biosci, in press.
  4. Ferre S, Diamond I, Goldberg SR, Yao L, Hourani SM, Huang ZL, Urade Y, Kitchen I (2007) Adenosine A(2A) receptors in ventral striatum, hypothalamus and nociceptive circuitry Implications for drug addiction, sleep and pain. Prog Neurobiol, in press (e-pub ahead of print). PubMed
  5. LeFoll B, Chefer SI, Kimes AS, Shumway D, Goldberg SR, Stein EA, Mukhim AG (2007) Validation of an extra-cerebral reference region approach for quantification of brain nicotinic acetylcholine receptors in squirrel monkeys with PET and 2-[18F]FA. J Nucl Med 48:1492-1500. PubMed
  6. LeFoll B, Goldberg SR (2007) Blocking cannabinoid CB1 receptors for the treatment of nicotine dependence: insights from clinical and preclinical and clinical studies. Addict Biol, in press.
  7. LeFoll B, Justinova Z, Wertheim C, Barnes C, Goldberg SR (2007) Topiramate does not alter nicotine or cocaine discrimination in rats. Eur J Pharmacol, in press.
  8. Quarta D, Ciruela F, Patkar K, Borycz J, Solinas M, Lluis C, Franco R, Wise RA, Goldberg SR, Hope BT, Woods AS, Ferre S (2007) Heteromeric nicotinic acetylcholine-dopamine autoreceptor complexes modulate striatal dopamine release. Neuropsychopharmacology 32:35-42. PubMed
  9. Scherma M, Medalie JB, Fratta W, Makriyannis A, Vadivel SK, Piomelli D, Tanda G, Mikics E, Haller J, Goldberg SR (2007) Motivational anxioloytic and locomotor effects of anandamide alone and after inhibition of fatty acid amide hydrolase (FAAH). Neuropharmacology, in press.
  10. Solinas M, Goldberg SR, Piomelli D (2007) The endogenous cannabinoid system and brain reward processes. Br J Pharmacol, in press.
  11. Solinas M, Scherma M, Fattore L, Stroik J, Wertheim C, Tanda G, Fratta W, Goldberg SR (2007) Nicotinic alpha 7 receptors as a new target for treatment of cannabis abuse. J Neurosci 27:5615-20. PubMed
  12. Solinas M, Scherma M, Tanda G, Wertheim CE, Fratta W, Goldberg SR (2007) Nicotinic facilitation of delta9-tetrahydrocannabinol discrimination involves endogenous anandamide. J Pharmacol Exp Ther 321:1127-34. PubMed
  13. Solinas M, Tanda G, Justinova Z, Wertheim CE, Yasar S, Piomelli D, Vadivel SK, Makriyannis A, Goldberg SR (2007) The endogenous cannabinoid anandamide produces delta-9-tetrahydrocannabinol-like discriminative and neurochemical effects that are enhanced by inhibition of fatty acid amide hydrolase but not by inhibition of anandamide transport. J Pharmacol Exp Ther 321:370-80. PubMed

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