Bartley G. Hoebel - US grants

This is a proposal to investigate neurotensin and cholecystokinin in the function of the mesolimbic dopamine system using behavioral and neurochemical tests. Studies will focus on the ventral tegmental area (VTA) and the nucleus accumbens (NAC). This project grows out of reports that rats will self-inject morphine and neurotensin in the VTA and will self-inject amphetamine, dopamine and cholecystokinin in the NAC. It appears that animals work for chemical control over the mesolimbic system. In addition, it has been reported that the stimulus properties of i.p. amphetamine generalize to NAC amphetamine, and that dopamine and cholecystokinin can act synergistically to induce locomotion. We propose to study neurotensin and cholecystokinin, mprphine and amphetamine, and dopamine by a dose-response analysis of their reinforcing and stimulus generalization properties. Positive results with these agonists will be followed up with (a) receptor antagonists to determine relevance to synaptic neuropharmacology, (b) 6-hydroxy-dopamine to determine dependence on mesolimbic dopamine release, (c) regional assays to measure dopamine turnover, and (d) local dialysis to measure catecholamine and cholecystokinin release. This proposal will clarify the function of neurotensin and cholecystokinin in modulating the mesolimbic system and their relation to morphine and amphetamine.

building /facility design /renovation; animal colony;

Our progress in the three years of this grant shows that microdialysis can be used in behaving animals to monitor dopamine (DA), serotonin (SER), and their metabolites during body weight changes, self-stimulation or injection of drugs. Drugs of abuse were injected systemically and locally, showing that part of the systemic effect of addictive stimulants is an action on presynaptic DA and SER terminals. Experiments were completed for publication showing increased extracellular DA with amphetamine, cocaine, phencyclidine, and nicotine. Systemic haloperidol to block DA receptors caused an acute rise in extracellular DA, but after chronic treatment, DA decreased in the striatum and prefrontal cortex. A nicotinic antagonist blocked nicotine-induced dopamine release in the NAC. Direct infusion of amphetamine by reverse dialysis gave effects like local bolus injection, suggesting that reverse dialysis could be used for self-infusion. In vitro tests showed that a peptide could also pass through the dialysis membrane. This progress suggests the feasibility of local self- infusion of amphetamine or peptides and block with selective receptor antagonists during ongoing measurement of neurotransmitters by microdialysis in the NAC and PFC. In Year 1 we propose to continue development of microdialysis applied to freely moving animals. Microdialysis will be used to measure extracellular DA and SER in the NAC during circadian activities, body weight changes, self-stimulation, and self- infusion of amphetamine. Anatomical specificity will be tested by simultaneous microdialysis in the prefrontal cortex. In Year 2 we will study the effect of locally infused peptides, CCK, neurotensin and met-enkephalin, using microdialysis to simultaneously apply the compound and measure the monoamine response. Repeated samples will be taken to measure the time course and dose-response effects of the peptides. In Year 3 we propose to study the effect of active peptides on amphetamine self-infusion and to test self-infusion of peptides alone. Year 4 will be devoted to blocking the effects of manoamines and peptides. Year 5 is for testing the effect of body weight changes on self-infusion and for assaying the release of peptides by high performance capillary electrophoresis. The uniqueness of this proposal lies in the use of in vivo microdialysis to monitor mesolimbic DA and SER output during ongoing behavior such as local amphetamine self-administration. This research will help clarify the function of selected peptides in modulating the mesolimbic DA system and their role in stimulant addiction. The significance lies (a) in discovering if rats self- titrate their extracellular DA or SER, (b) in finding the role of peptides in controlling the mesolimbic reward system, and (c) in the further development of microdialysis as a means of understanding and screening drugs of abuse.

electrodes; biomedical equipment purchase;

DESCRIPTION (Applicant's Abstract): A new medication for drug abuse and drug craving is proposed. Positive results have been obtained with the combination of properties in two appetite suppressants, the serotonergic drug fenfluramine (Fen) and the dopamineric drug phentermine (Phen). The Fen-Phen combination decreased oral intake of nicotine in mice, decreased i.v. self-administration of cocaine+heroin in rats, and decreased binge eating and food craving in humans. The proposal for animal research is to investigate where, how, and when Fen-Phen works, and for humans, to determine whether it is an effective medication to reduce nicotine and cocaine craving. Fen-Phen probably suppresses appetite via the hypothalamus. Most drugs of abuse act on the mesolimbic dopamine (DA) system that generates conditioned incentives and behavior reinforcement. Therefore, the logic behind Fen-Phen therapy for drug abuse is to activate hypothalamic systems for appetite suppression in a manner that imposes limits on the DA incentive/reinforcement system. This will inhibit conditioned or unconditioned stimuli for drug abuse. [The application is designed to address the following questions:] 1. Where does Fen-Phen work? At the behavioral level, the proposed plan is to compare d- and d,l-fenfluramine and phentermine, separately and together for efficacy in inhibiting drug self-administration in rats and for locomotor side effects, abuse liability, and sites of action in the hypothalamus and a DA terminal region. 2. How does Fen-Phen work? At the neural systems level, our studies suggest that Fen-Phen acts in the hypothalamus to inhibit drug abuse by releasing acetylcholine in nucleus accumbens (NAc), where acetylcholine can limit dopamine's effects. Drugs and neurotransmitters in the brain will be measured by microdialysis. 3. When does Fen-Phen work? Food deprivation and weight loss are known to augment drug abuse. It was discovered that food-restricted rats with low body weight have low DA release in the NAc. Therefore, food restricted animals may be prone to drug abuse as a means to restore DA. Fen-Phen may have special potential in drug abuse prevention or treatment in situations that benefit from appetite suppression. The above three studies will clarify the mechanism and potential of Fen-Phen therapy.

dopamine agonists; smoking cessation; nicotine; fenfluramine; drug addiction; combination chemotherapy; drug abuse chemotherapy; drug addiction antagonist; human therapy evaluation; neurotransmitter transport; body weight; hypothalamus; nucleus accumbens; acetylcholine; self medication; drug interactions; drug adverse effect; psychopharmacology; dopamine receptor; cocaine; clinical research; human subject; laboratory rat; laboratory mouse;

DESCRIPTION (provided by applicant): Alcoholism can be viewed as a motivational disorder that results from alterations in brain systems for ingestive behavior. Evidence supports a strong link between the hypothalamic feeding peptide galanin (GAL) and dietary fat. Our research demonstrated that ethanol increases the expression of GAL and that GAL stimulates ethanol intake. Together, these findings suggest a positive feedback loop between GAL and ethanol, with dietary fat as an initiating factor. The behavioral output of this system releases dopamine (DA) in the nucleus accumbens (NAc). This has led to extensive preliminary studies and the following 5 Aims. Aim 1 is to test the hypothesis that hypothalamic, fat-stimulated peptides, including GAL, orexins (ORX) and opioids, are enhanced by ethanol consumption. These peptides can be distinguished from others, such as, neuropeptide Y and agouti-related protein, which are related to carbohydrate intake and reduced by ethanol consumption. Aim 2 is to inject these fat-stimulated peptides into the hypothalamus to reveal a causal role in ethanol intake and ethanol-seeking behavior. We will also expand on new results suggesting that GAL and ORX facilitate opioid gene expression and function. Aim 3 tests the possibility that dietary fat increases ethanol intake by elevating circulating triglycerides, which in turn enhance expression of fat-stimulated peptides that promote ethanol intake. Aim 4 investigates mice with targeted disruptions of the GAL gene that may alter their ethanol preference and their responsiveness to the stimulatory effect of fat on ethanol intake Aim 5 suggests that hypothalamic peptides act through the NAc to cause an increase in DA and GABA release, which stimulates ethanol intake. These peptides simultaneously reduce NAc acetylcholine and glutamate thereby disinhibiting ethanol-seeking behavior. In summary, this grant tests the novel hypothesis that ethanol, through its impact on circulating triglycerides, stimulates gene expression of hypothaiamic fat- stimulated peptides that potentiate the intake of more ethanol via the release of DA and GABA in the NAc.