Eliot L. Gardner - US grants

The overall objective of the proposed research is to gain insight into marijuana's actions on brain rewards circuits and on dopaminergic and enkephalinergic mechanisms involved in direct brain reward. This work derives from the hypothesis that drugs of abuse, including marijuana, derive part of their abuse liability from neuropharmacological sensitization of brain reward circuits, and is a direct outgrowth of previous work on drug action on brain reward circuits carried out in the applicants' laboratories. The specific aims are (1) to study the effects of delta-9-tetrahydrocannabinol (Delta9-THC), the major psychoactive ingredient in marijuana, and selected analogs on direct brain reward as measured by the electrical intracranial self-stimulation paradigm in laboratory rats; (2) to study possible neuroanatomic specificity of such effects by directly comparing the effects of Delta9-THC and selected analogs on mesostriatal and mesolimbic brain reward mechanisms; (3) to test for naloxone-reversibility or attenuation of such effects, by analogy to other well-characterized drugs of abuse; (4) to study the effects of Delta9-THC and its analogs on dopamine release, in mesostriatal and mesolimbic forebrain dopaminergic loci associated with direct brain reward, as measured by in vivo electrochemical determination of neurotransmitter release; and (5) to study the effects of Delta9-THC and its analogs on enkephalinergic (delta) receptor density, affinity, and conformation as measured by receptor binding studies and light microscopy autoradiography. By adding to the understanding of marijuana's effects on one of the presumptive neuropharmacologic substrates of drug abuse liability, these studies may well give insight into fundamental brain mechanisms underlying marijuana's abuse potential, and to treatment possibilities for marijuana abuse. The health relatedness of such studies seems clear, given that marijuana is the most widely used illicit drug in the United States, and given the evidence for health consequences of chronic marijuana abuse.

The objective of this research is to gain insight into brain mechanisms of action of the clinically atypical antipsychotic drug clozapine. Unique among anti-schizophrenic drugs, clozapine produces virtually no extrapyramidal side effects or tardive dyskinesia, the often-serious neurological side effects common to other antipsychotic drugs which are thought to result from antipsychotic drug-induced dysfunction in the nigrostriatal dopamine (DA) system. A large body of research, both from this laboratory and elsewhere, strongly suggests that clozapine selectively acts as a functional DA antagonist in the mesolimbic DA system while virtually sparing the nigrostriatal system. This site-specificity explanation of clozapine's unique pharmacological profile is attractive on many grounds, not the least of which is the fact that current pharmacological methods for screening potential new antipsychotic drugs (which do not presently distinguish between mesolimbic and nigrostriatal specificities) could be modified to select only compounds having site-specific mesolimbic profiles. To this end, we have developed an adaptation of the electrical intracranial self-stimulation (ICSS) paradigm which is highly accurate in distinguishing clozapine's mesolimbic site-specificity from the more anatomically diffuse DA antagonism of classical neuroleptics. The paradigm used for these studies, and to be used for the present work, is that of chronic neuroleptic-induced functional DA hypersensitivity in both mesolimbic and nigrostriatal DA loci as assessed by the ICSS model. We now seek funds to extend our previous work by addressing the following question - is clozapine's mesolimbic functional stie-specificity a result of clozapine's powerful intrinsic anticholinergic potency? If "yes", development of new antischizophrenic agents with clozapine-like intrinsic anticholinergic potency would appear warranted. If "no", clozapine's unique atypical profile must logically derive from other neuropharmacological mechanisms, possibly including differential action on mesolimbic versus nigrostriatal presynaptic modulatory mechanisms. Also, if "no", the utility of the ICSS paradigm for screening potential new antipsychotic drugs would be immense, since it would not detect "false-positive" drugs merely on the basis of anticholinergic potency. Thus, either outcome would add significantly to our knowledge of atypical antipsychotic drugs, and aid development of new drugs with significantly lessened side effect liability. The health relatednes of such work is clear and straightforward - the development of better drugs for treating mental illness.

The objective of the proposed research is to gain further insights into marijuana's action on brain reward circuits and on dopamine (DA) and enkephalinergic mechanisms involved in marijuana, derive part of their abuse liability from neuropharmacological facilitation of brain reward circuits. During the period of previous NIDA grant support for this work, we demonstrated that delta-9-tetrahydrocannabinol (delta9-THC), the major psychoactive ingredient in marijuana, does the following: (1) lowers direct brain reward thresholds in the medial forebrain bundle, in a rat strain-specific fashion, (2) enhances presynaptic release of known to support brain reward, and (3) interacts noncompetitively with endogenous brain delta and Mu opioid receptors but not Kappa receptors, and the opioid receptor interaction of a series of cannabinoid analogs correlates crudely but not precisely with psychoactive potency. The general aim of the presently proposed work is to carry forward and extend all these findings. The specific aims are to (1) extend ours studies of delta9-THC and analogs on direct brain reward as measured by electrical intracranial self- stimulation in laboratory rats; (2) extend our studies of the effects of delta9-THC and analogs on DA release, in forebrain DA loci supporting direct brain reward, as measured by both in vivo voltammetric electrochemistry and in vivo intracranial microdialysis; and (3) extend our studies of the interaction of delta9-THC and selected analogs with endogenous brain opioid receptors, as measured by receptor binding and quantitative autoradiography. A major aim running throughout is to learn the specific locus in the brain of the already-demonstrated delta9-TCH effects. This proposal addresses specific research needs stipulated in the NIDA Marijuana Research Announcement and in NIDA Grant Announcement DA-87- 24 ("...studies are needed concerning the neurophysiological effects of marijuana, including potential sites and mechanisms of action.." "...research areas of particular importance include...brain reward mechanisms"). By adding to our understanding of marijuana's effects on one of the presumptive principal neurophysiological substrates of drug abuse liability, these studies should yield additional insight into fundamental brain mechanisms underlying marijuana's abuse potential, and to treatment possibilities for marijuana abuse. The health relatedness is clear, given that marijuana is the most widely used illicit drug in the United States, and given the evidence for health consequences of chronic marijuana abuse.

The objective of this research is to gain further insight into alcohol's action on brain reward circuits and on dopamine (DA) and enkephalinergic mechanisms involved in direct brain reward. This work derives from the hypothesis that abusable substances, including ethanol, derive part of their abuse liability from neuropharmacological facilitation of brain reward circuits. Some workers report that exogenous ethanol robustly lowers brain-stimulation reward thresholds, while others report that experiential factors are additive to ethanol's pharmacological facilitation of brain-reward. We and others have recently found that self-administration of ethanol in laboratory rats more robustly enhances brain reward than exogenous administration. It is also well-established that significant genetic differences exist among rat strains regarding alcohol preference. One alcohol-preferring strain, the Lewis rat, is also unusually sensitive to morphine and cocaine, and shows significantly higher preferences for morphine and cocaine than other rat strains. We have shown that the Lewis rat is also uniquely sensitive to the brain-reward enhancing effects of delta(9)-tetrahydrocannabinol (delta(9)-THC), the psychoactive ingredient in marijuana, and that this unique sensitivity to brain-reward facilitation correlates highly with a unique sensitivity of Lewis rats to facilitation of extracellular DA in reward-relevant loci in the nucleus accumbens, a crucial forebrain limbic convergence of reward-related DA circuitry. The general aim of the presently proposed work is to carry forward and extend all these previous findings, with the hope of identifying forebrain DA mechanisms that may correlate with high ethanol preference, and determine whether it is possible to modulate those mechanisms pharmacologically - thus hopefully opening up new possibilities of pharmacotherapy for alcohol addiction and abuse. The specific aims are to 1) study the effect of exogenously-administered versus self-administered ethanol on extracellular DA efflux (as measured by in vivo brain microdialysis and in vivo voltammetry) in three reward-relevant DA forebrain loci - nucleus accumbens, medial prefrontal cortex, and neostriatum; 2) study the effect of strain differences (alcohol-preferring Lewis rats versus non-preferring F344 rats) on exogenously-administered and self-administered ethanol-induced extracellular DA efflux in these three reward-relevant DA forebrain loci; 3) study effects seen in 1) and 2) in both normal animals and protracted-abstinence animals; and 4) study possible pharmacological modulations (by serotonergic, DAergic, and enkephalinergic manipulations) of effects seen in 1), 2), and 3). This application addresses specific research needs stipulated in NIAAA Request for Applications RFA-AA-92-01 [Research on Pharmacologic Treatments for Alcoholism] and NIAAA Program Announcement PA-91-97 [Exploratory/Developmental Grants (R21) for Research on the Etiology of Alcoholism] ("..Goals include clarifying the basis for alcohol craving so that more effective therapeutic agents, such as dopaminergic agonists and serotonin uptake inhibitors, can be tested.. ..Research is required to specifically delineate the potential of agents that alter dopaminergic function..and..to identify the precise mechanisms by which the opioid system alters drinking behavior..").

During the period 01 Oct 07 to 30 Sept 08, significant progress was made on this research project. We found that blockade of brain dopamine D3 receptors by the novel dopamine D3 receptor antagonist NGB2904 emulates to a very high degree the putative anti-addiction, anti-craving, and anti-relapse efects that we have previously seen with our lead proof-of-concept dopamine D3 receptor antagonist SB277011A. Specifically, we found that NGB2904 attenuates intravenous cocaine self-administration under progressive-ratio (PR) reinforcement (lowers the PR break-point), attenuates relapse to cocaine-seeking behavior (using the reinstatement animal model) triggered by both cocaine itself and by environmental cues (sights, sounds) that were previously associated with cocaine-taking behavior, and significantly attenuates drug-enhanced brain reward (as assessed by electrical brain-stimulation reward electrophysiological techniques) produced by cocaine, nicotine, and heroin. Like the other selective D3 receptor antagonists that we have studied, NGB2904 does not alter cocaine self-administration under low-effort high-payoff fixed-ratio reinforcement. Like SB277011A, NGB2904 has no effect by itself on electrical brain-stimulation reward - a highly promising finding with respect to possible eventual human use of these candidate anti-addiction medications. These findings add further weight to our previous suggestions that highly selective dopamine D3 receptor antagonists may be useful in the treatment of a wide range of drug addictions. We further found that both of our highly-selective D3 receptor antagonists - SB277011A and NGB2904 - significantly attenuate methamphetamine-enhanced brain reward (as assessed by electrical brain-stimulation reward). This finding constitutes the first demonstration that dopamine D3 brain receptors are involved in methamphetamine dependence and addiction, and the first demonstration that highly selective dopamine D3 receptor antagonists may be therapeutically beneficial in the treatment of methamphetamine dependence and addiction. We further compared the effects of SB277011A and NGB2904 with those of BP897 against methamphetamine-enhanced brain reward, and concluded that the anti-methamphetamine effects of SB277011A and NGB2904 are likely attributable to selective D3 receptor antagonism, while the observed effects of BP897 are likely attributable to a combination of D3 and D2 receptor antagonism. We continued our investigation of the anti-addiction effects of SB277011A in an oral ethanol self-administration model, and confirmed our previous findings that SB277011A significantly attenuates ethanol self-administration and reinstatement of ethanol-seeking behavior in laboratory mice. We continued our investigation of the novel anti-addiction compound levo-tetrahydropalmatine (l-THP) on cocaine's rewarding effects, and confirmed that l-THP significantly attenuates cocaine self-administration and cocaine-enhanced brain-stimulation reward. In a series of neurochemical experiments, we found that l-THP likely works via a postsynaptic mechanism, and appears to antagonize dopamine D1, D2, and D3 receptors nonselectively. Additionally, we found that blockade of brain dopamine D3 receptors by SB277011A significantly attenuates incubation of cocaine craving in laboratory rodents. This is the first demonstration of the potential efficacy of selective dopamine D3 receptor antagonists against not only craving itself, but the time-dependent incubation of craving that is such a problem in the clinical management of addictive diseases. Finally, we found that the dopamine D3 receptor antagonist SB277011A significantly inhibits food intake in genetically obese rodents, with significantly lesser effect on genetically non-obese rodents - suggesting a possible utility for selective dopamine D3 receptor antagonists in the treatment of compulsive over-eating and obesity. All of these findings suggest that dopamine D3 receptor antagonists may have anti-addiction, anti-craving, and anti-relapse efficacy in human drug addiction, with additional preliminary suggestive evidence for efficacy in compulsive over-eating and obesity.

During the period 01 Oct 06 to 30 Sept 07, significant progress was made on this research project. We found that the gamma-aminobutyric acid (GABA) transaminase inhibitor gamma-vinyl-GABA (GVG, Vigabatrin) dose-dependently inhibits cocaine-triggered relapse to cocaine-seeking behavior in laboratory rats who have been pharmacologically detoxified and behaviorally extinguished from their prior cocaine-taking habits. We further found that gamma-vinyl-GABA also dose-dependently inhibits sucrose-induced reinstatement of reward-seeking behavior in rats. By using in vivo brain microdialysis, we additionally found that gamma-vinyl-GABA dose-dependently elevates extracellular GABA levels in the nucleus accumbens of the limbic forebrain. However, gamma-vinyl-GABA, when administered either systemically or locally into the nucleus accumbens, fails to inhibit either basal or cocaine-enhanced nucleus accumbens dopamine in either drug-naive rats or in cocaine-extinction rats. We interpret these findings to suggest that: 1) gamma-vinyl-GABA significantly inhibits cocaine- or sucrose-triggered relapse to reward-seeking behavior; and 2) a GABAergic-, but not dopaminergic-, dependent brain mechanism underlies the antagonism by gamma-vinyl-GABA of cocaine-triggered reinstatement of drug-seeking behavior, at least with respect to gamma-vinyl-GABA's action(s) within the nucleus accumbens. In contrast to gamma-vinyl-GABA, systemic administration of gabapentin (another putative GABAmimetic compound claimed in some previous reports from other research groups to have anti-cocaine-addiction properties) was found to have no effect on cocaine-triggered relapse to cocaine-seeking behavior. Gabapentin also failed to alter intarvenous cocaine self-administration under fixed-ratio reinforcement in laboratory rats. In vivo brain microdialysis experiments showed that gabapentin produces a modest (50%) increase in extracellular GABA levels in the nucleus accumbens, but fails to alter either basal or cocaine-enhanced dopamine levels in the nucleus accumbens. When added to our previous extensive findings with gamma-vinyl-GABA in a very wide variety of addiction-related preclinical animal models, the present findings suggest that gamma-vinyl-GABA may have anti-addiction, anti-craving, and anti-relapse efficacy in human drug addiction. However, gabapentin appears to lack significant anti-addiction, anti-craving, or anti-relapse efficacy.