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Effects of Alcohol on Brain Reward C.J. Malanga, MD, PhD The University of North Carolina at Chapel Hill January 6, 2009 Background: Prenatal Cocaine Exposure in the Mouse • Timed-pregnant white Swiss-Webster dams • Injected b.i.d. from E8-E18 – – – – COC40: SAL: COC20: SPF: 40 mg/kg/d, s.c. divided b.i.d. Vehicle-injected controls 20 mg/kg/d, s.c. divided b.i.d. Saline pair-fed nutritional controls • Cross-fostered on P0 to non-exposed, matched-parity black Swiss-Webster dams • One pup per litter of each gender used for experiments (“litter effects”) – Holson and Pearce, Neurotoxicol Teratol 14(3), 1992 Mice exposed to cocaine in utero 1. 2. 3. 4. 5. 6. … habituate less to novelty/injection stress than nonexposed controls (Guerriero et al., 2005); … demonstrate less locomotor sensitization to repeated psychostimulant administration than gestational controls (Crozatier et al., 2003; Guerriero et al., 2005); … demonstrate less conditioned place-preference to cocaine than controls (Malanga et al., 2007a); … are more likely to acquire cocaine self-administration than controls (Rocha et al., 2002); … self-administer more cocaine than controls (Rocha et al., 2002); … demonstrate more sensitization of dopamine release in the nucleus accumbens after chronic cocaine administration than gestational controls (Malanga et al., submitted) Mice exposed to cocaine in utero 1. 2. 3. … habituate less to novelty/injection stress than nonexposed controls (Guerriero et al., 2005); … demonstrate less locomotor sensitization to repeated psychostimulant administration than gestational controls (Crozatier et al., 2003; Guerriero et al., 2005); … demonstrate less conditioned place-preference to cocaine than controls (Malanga et al., 2007a); … are more likely to acquire cocaine self-administration than controls (Rocha et al., 2002); … self-administer more cocaine than controls (Rocha et al., 2002); … demonstrate more sensitization of dopamine release in the nucleus accumbens after chronic cocaine administration than gestational controls (Malanga et al., Saline Cocaine Saline Cocaine 5 mg/kg Cocaine 20 mg/kg submitted ) 5 mg/kg Cocaine 20 mg/kg Post Test - Pre Test (sec/min) 20 5. Saline Cocaine 5 mg/kg 10 0 Cocaine 20 mg/kg Conditioning Group 6. -10 -20 -30 COC40 COC20 SPF40 SAL 20 10 0 -10 -20 -30 Saline Cocaine 5 mg/kg Drug Side - Saline Side (sec/min) 4. 30 Drug Side - Saline Side (sec/min) 30 20 COC40 COC20 SPF40 SAL 10 0 Cocaine 20 mg/kg Conditioning Group Conditioning Group 30 -10 -20 -30 Conditioning Group Mice exposed to cocaine in utero 1. 2. 3. 4. 5. 6. … habituate less to novelty/injection stress than nonexposed controls (Guerriero et al., 2005); … demonstrate less locomotor sensitization to repeated psychostimulant administration than gestational controls (Crozatier et al., 2003; Guerriero et al., 2005); … demonstrate less conditioned place-preference to cocaine than controls (Malanga et al., 2007a); … are more likely to acquire cocaine self-administration than controls (Rocha et al., 2002); … self-administer more cocaine than controls (Rocha et al., 2002); … demonstrate more sensitization of dopamine release in the nucleus accumbens after chronic cocaine administration than gestational controls (Malanga et al., In Press) Mice exposed to cocaine in utero 7. 8. 9. 10. 11. … are more sensitive to the reward-potentiating effects of cocaine on ICSS than controls (Malanga et al., 2007b); …are more sensitive to the reward-potentiating effects of selective dopaminergic agonists (Malanga et al., 2007b); … demonstrate robust neuroanatomical, behavioral and neurochemical changes that persist into adult life; … provide a model for some of the clinical observations in cocaine-exposed children, especially state or contextdependent differences; and … demonstrate changes in dopamine release in the nucleus accumbens that correlate directly with changes seen in the effects of cocaine on operant or instrumental behaviors and inversely with associational or Pavlovian behaviors Introduction • Low sensitivity to subjective and motor effects of EtOH is associated with increased EtOH abuse (Schuckit, et al.) • High sensitivity to euphoric or hedonic effects (“liking” or “wanting”) of EtOH is associated with increased EtOH abuse (de Wit, et al.) – Positive hedonic effects are associated with psychomotor stimulation during the rising phase of blood alcohol concentration (BAC) – Higher doses and/or decreasing BAC are associated with sedation and/or negative hedonic effects • Animal studies – “Do you feel it?” (drug-discrimination; self-administration) – “Do you want more?” (self-administration; conditioned place-preference) – “Do you like it?” (intracranial self-stimulation; self-administration) MOTIVATION REWARD Expectancy Consumption ATTENTION WITHDRAWAL TIME Intracranial Self-Stimulation Drug Self-Administration Introduction Genetic contributions to alcohol sensitivity: inbred mouse strains • C57Bl6/J – Less motor stimulation/more sedation with acute EtOH – High levels of oral self-administration of EtOH – Faster acquisition of IV self-administration of EtOH – Less behavioral sensitization to chronic EtOH – Less conditioned place-preference to EtOH – Normal EtOH withdrawal • DBA/2 – High motor stimulation with acute EtOH – No oral self-administration (without sweetening) of EtOH – Normal acquisition of IV self-administration of EtOH – More behavioral sensitization to chronic EtOH – More conditioned place-preference to EtOH – Enhanced EtOH withdrawal How can we utilize these differences to separate the rewarding value of alcohol – how much the animal LIKES and is rewarded by alcohol - from its other behavioral effects in these mouse strains? How can we separate the rewarding properties of alcohol – how much the animal LIKES and is positively motivated to seek alcohol – from its other behavioral effects in mice? How can we measure changes in the rewarding value of alcohol – if the animal LIKES alcohol more or less – after acute or chronic alcohol exposure or consumption at different developmental stages (fetal, adolescent, adult)? What are the pharmacological mechanisms underlying the motivational effects of alcohol in mice, and how do those mechanisms change with exposure or consumption at different developmental time points? Intracranial Self-Stimulation (ICSS) vs. Drug Self-Administration (IVSA) • Psychological: Both methods utilize instrumental conditioning and measure operant behavior; however, in IVSA the drug itself is the reinforcer, while in ICSS rewarding intracranial electrical stimulation (brain stimulation-reward, or BSR) is the primary reinforcer and drugs act by increasing or decreasing its rewarding value. In IVSA, access to drug reinforces behavior during instrumental conditioning, while ICSS measures the potentiating (or depreciating) effects of drugs on reinforcement of a previously learned operant behavior. • Neuroanatomical: IVSA measures effects of drugs on the entire nervous system, while ICSS measures effects of drugs on rewarding electrical stimulation of a discrete set of neural structures, the mesolimbic motor pathways or brain reward system • Pharmacological: In contrast to IVSA, satiety does not develop for BSR and pharmacological antagonism does not increase responding for BSR • ICSS directly measures the positive or negative hedonic effects of drugs on behavior. Because the animal works for BSR and drug delivery is behaviorally non-contingent, ICSS does not measure how much the animal “wants” the drug – ICSS measures how much the animal LIKES the drug. M Meed diiaall P Prreeffrro on nttaall C Co orrtteexx A An ntteerriio orr C Ciin ng gu ullaattee C Co orrtteexx Dorsal Striatum H Hiip pp po occaam mp pu uss A Am myyg gd daallaa N Nu ucclleeu uss A Accccu um mb been nss E IN M A PA AB O G D Substantia Nigra VTA VTA Ventral Tegmental Area E AT M TA U GL Medial Forebrain Bundle 200 Rate of Response (presses/minute) 0 32354045505663717989 112 126 141 158 100 Stimulus Frequency (Hz) 200 Max ½ Max Rate of Response (presses/minute) Slope 0 32 Threshold EF50q0 158 Stimulus Frequency (Hz) 200 Rate of Response (presses/minute) Δ 0 q0 32 158 Stimulus Frequency (Hz) 100 GENETIC MODEL CHRONIC TREATMENT DEVELOPMENTAL EXPOSURE % Change in Reward Threshold 0 0 0.1 1.0 10 Dose of Drug (mg/kg) From: Kornetsky and Bain, NIDA Res Monogr 124, 1992 The release of dopamine in forebrain structures (nAc, PFC) is necessary, but not sufficient, for BSR • Dopamine and EtOH actions – VTA dopaminergic neurons (Brodie and Appel 2000; Brodie 2002) • No difference in in vitro basal firing rate, C57 vs. DBA • EtOH stimulates cell firing in both C57 and DBA, but potency is higher in DBA • Chronic EtOH (7 g/kg/d i.p. x 21d) does not change basal firing but does increase EtOH potency to stimulate firing and decrease GABA potency to inhibit firing in C57 – Dopamine release (Middaugh et al 2003; Zapata et al 2006) • EtOH drinking (12% EtOH x 2 hr, 1.6 g/kg consumed, BAC~60 mg/dl) stimulates DA release in the nAc of C57 mice • No difference in basal extracellular DA levels or acute EtOH (2.0 g/kg i.p.)evoked DA release in nAc, C57 vs. DBA • No difference in basal DA or EtOH-evoked DA release in nAc upon withdrawal after chronic EtOH (vapor chamber 16 hr/d x 4d), C57 vs. DBA The release of dopamine in forebrain structures (nAc, PFC) is necessary, but not sufficient, for BSR • Dopamine and EtOH (continued) – Dopamine content, turnover and receptors (Ng et al 1994; George et al 1995) • Bmax for D1 and D2 in striatum C57>DBA; Bmax for D2 in midbrain DBA>C57 • DA-stimulated adenylyl cyclase activity in striatum C57>DBA • Total tissue DA in striatum and midbrain C57=DBA; DOPAC:DA in striatum and midbrain C57<DBA, and increases in striatum of C57 mice after drinking (10% EtOH 5hr/d x 5d; BAC~100 mg/dl) – Dopamine receptors (McNamara et al 2006) • Bmax for D1 in ventral striatum C57=DBA; Bmax for D3 in ventral striatum DBA>C57 • D1 agonist (SKF 38393) potency to stimulate locomotion C57>DBA • D3 agonist (PD 128907) potency to inhibit locomotion DBA>C57 R01 proposal to NIAAA • Specific Aim 1 – To determine the effect(s) of alcohol on brain stimulationreward (BSR) in mice – To investigate the pharmacological mechanisms mediating the rewarding effects of alcohol in mice • Specific Aim 2 – To determine if acute, intermittent administration changes the rewarding effects of alcohol over time – To determine if chronic alcohol exposure leading to physical dependence changes the rewarding effect of alcohol, particularly after withdrawal • Specific Aim 3 – To determine if adaptations in dopaminergic mechanisms of brain reward are involved in the effects of chronic alcohol exposure and withdrawal on BSR R01 proposal to NIAAA • Specific Aim 1: Preliminary Data – Proof of Concept – Responding for BSR in different inbred/outbred mouse strains -7 Threshold (Coulombs 10 ) Responses/50s 5 100 Swiss C57BL6/J 129S6Ev DBA2/J 80 4 60 3 40 2 20 1 0 0 25 50 100 126 Stimulation Frequency (Hz) Stimulation Frequency (Hz) C57 DBA 129 Swiss Mouse Strain Strain R01 proposal to NIAAA • Specific Aim 1: Preliminary Data – Dose-Dependent Effects – Acute effect of EtOH on BSR in C57 and DBA mice – HYPOTHESIS: Acute administration of EtOH will potentiate the rewarding value of BSR (i.e., will lower reward threshold, q0) in both C57 and DBA mice q0 (% ofq Pre-Injection (% of Baseline)Baseline) 175 Min 16-30 Min 0-15 * 150 125 * Min 31-45 Min 46-60 * * 100 0 75 50 25 * * ** * * DBA (n=7) C57 (n=9) * * * * * = P<0.05 vs. Vehicle 0 V 0.3 0.61.01.7 2.4 V 0.3 1.0 2.4 V 0.3 1.0 2.4 Alcohol Dose(g/kg, (g/kgp.o) p.o.) Alcohol Dose V 0.3 1.0 2.4 R01 proposal to NIAAA • Specific Aim 1: Preliminary Data - Pharmacokinetics – Absorption, distribution and elimination of EtOH in C57 and DBA mice Blood Alcohol (mg/dl) (mg/dl) Concentration Blood Alcohol Blood Alcohol Levels in DBA2/J and C57BL/6J Mice after Gavage with 0.6 g/kg Alcohol 40 30 20 DBA (n=5, 90-Day Old) C57 (n=5, 90-Day Old) 10 0 0 5 15 30 Minutes After Gavage Minutes after Gavage 60 R01 proposal to NIAAA • Specific Aim 1: Preliminary Data - Comparison of Cocaine Effects – Acute effect of cocaine on BSR in C57 and DBA mice – HYPOTHESIS: Acute cocaine administration will potentiate the rewarding value of BSR (i.e., will lower reward threshold, q0) more in DBA than in C57 mice ** 150 175 ** 100 Min 16-30 q0 (% ofq Pre-Injection (% of Baseline)Baseline) * * 50 V 1 3 10 30 0 * * Min 46-60 * * * * ** * ** * * DBA (n=4) (n=7) C57 (n=9) V Min 31-45 * * * ** * *25 * 0 * 100 * 75 * * Min 46-60 ** * 125 50 Min 16-30 150 ** Min 31-45 Min 0-15 0 q0 (% of Pre-Injection Baseline) Min 0-15 C57 (n=9) * * DBA (n=4) ** 1 3 10 30 V 1 3 10 30 * * * * * = P<0.05 vs. Vehicle V 1 3 10 30 V 0.3 1.0 Dose 2.4 (mg/kg) V 0.3 1.0 2.4 V 0.3 1.0 2.4 Cocaine Alcohol Dose(g/kg, (g/kgp.o) p.o.) Alcohol Dose V 0.3 1.0 2.4 R01 proposal to NIAAA • Specific Aim 1: Preliminary Data – Voluntary Consumption – Acute effect of self-administered EtOH on BSR in C57BL6/J mice Consumption of 1.1 ± 0.1(1.1 g/kg EtOH on Affects first Self-Administered Alcohol + 0.1 g/kg) minutes ofMin ICSS session ICSS15 Responding: 0 -test 15 (n=6 C57 Mice) Baseline) Pre-DrinkingBaseline) ofPre-Drinking qq00 (% (%of 120 * 100 80 60 40 20 *= P<0.05 vs. H2O 0 H2O Alc 10% Drinking Solution (1 Hr) Drinking Solution (1 hour) R01 proposal to NIAAA • Specific Aim 2: Preliminary Data – Effects of repeated, intermittent (every other day) administration of a rewardpotentiating dose of EtOH on BSR threshold – HYPOTHESIS: Neither the reward-potentiating nor the reward-depreciating effects of acute EtOH on BSR will change with repeated, intermittent administration * 100 80 60 40 0 Baseline) ofofPre-Injection q0 (% q (% Pre-Injection Baseline) Repeated Administration of Alcohol (0.6 g/kg, p.o): Effects on ICSS in C57 Mice (n= 5-9) 20 *= P<0.05 vs. H2O 0 H2O 1 2 3 Replication Replication 4 5 6 • 40 Specific Aim 2: Preliminary Data – Consumption of modified Lieber-DeCarli liquid diet with EtOH (2.4 and 4.4 % v:v) by C57BL6/J mice 35 30 25 40 20 35 Weight (g) 15 10 17 Control (n=5) 35 Alcohol (n=15) 30 30 25 25 20 20 15 15 0 10 10 5 16 40 5 5 2.4 % v:v 4.8 % v:v 0 0 -1 0 1 2 3 4 5 6 7 8 9 10 Day of Treatment 11 12 13 14 15 16 17 Volume Consumed (ml) Volume Consumed (ml) 5 R01 proposal to NIAAA EtOH consumed (g/kg) Control (body wt) Alcohol (body wt) Control (vol) Alcohol (vol) R01 proposal to NIAAA Research Design • Specific Aim 1 – Experiment 1.1: Acute effect(s) of oral alcohol on BSR in C57 and DBA mice 1.1a. Acute EtOH by oral gavage on BSR 1.1b. Pharmacokinetics of acute EtOH by oral gavage – Experiment 1.2: Dopaminergic pharmacology of alcohol reward in C57 and DBA mice 1.2a. Acute cocaine by i.p. injection on BSR 1.2b. Effect of SKF 82958 / SCH23390 on potentiation / depreciation of BSR by acute EtOH 1.2c. Effects of quinpirole / eticlopride on potentiation / depreciation of BSR by acute EtOH R01 proposal to NIAAA Research Design • Specific Aim 2 – Experiment 2.1: Effect of acute, intermittent alcohol administration on BSR in C57 and DBA mice 2.1a. Effect of repeated 0.6 g/kg EtOH p.o. (gavage) on BSR 2.1b. Effect of repeated 1.7 g/kg EtOH p.o. (gavage) on BSR – Experiment 2.2: Effect of chronic alcohol exposure, dependence and withdrawal on BSR in C57 and DBA mice 2.2a. Effect of acute EtOH withdrawal after chronic exposure on BSR 2.2b. Effect of repeated cycles of re-exposure to and withdrawal from EtOH on BSR 2.2c. Effect of length of withdrawal from chronic EtOH on BSR Experiment 2.2 Experiment 2.2a: Chronic alcohol exposure and withdrawal in C57 and DBA mice BSR without EtOH EtOH 0.6 OR 1.7 g/kg p.o. +BAC BAC 1 wk veh p.o. 4 wk EtOH 13% total calories (2.4% v/v EtOH) 6 12 24 48 Hours after withdrawal Experiment 2.2b: Repeated cycles of chronic alcohol and withdrawal in C57 and DBA mice EtOH EtOH 0.6 OR 1.7 g/kg p.o. 0.6 OR 1.7 g/kg p.o. BAC +BAC 1 wk veh p.o. 4 wk EtOH 13% total calories (2.4% v/v EtOH) ( 1 wk withdrawal 24º EtOH 1 wk withdrawal )x3 Experiment 2.2c: Variable duration of withdrawal from chronic alcohol in C57 and DBA mice EtOH EtOH 0.6 OR 1.7 g/kg p.o. 0.6 OR 1.7 g/kg p.o. BAC +BAC 1 wk veh p.o. 4 wk EtOH 13% total calories (2.4% v/v EtOH) 2 OR 4 wk withdrawal 24º EtOH R01 proposal to NIAAA Research Design • Specific Aim 3 – Experiment 3.1: Dopaminergic mechanisms in the effects of repeated cycles of chronic exposure to and withdrawal from alcohol on BSR 3.1a. Effect of the D1 agonist SKF 82958 on BSR during and after chronic EtOH and repeated cycles of withdrawal and re-exposure 3.1b. Effect of the D2 agonist quinpirole on BSR during and after chronic EtOH and repeated cycles of withdrawal and re-exposure – Experiment 3.2: Dopaminergic mechanisms in the effects of chronic alcohol exposure and variable length of alcohol withdrawal on BSR 3.2a. Effect of the D1 agonist SKF 82958 on BSR during and after chronic EtOH and variable duration of alcohol withdrawal 3.2b. Effect of the D2 agonist quinpirole on BSR during and after chronic EtOH and variable duration of alcohol withdrawal Experiments 3.1 and 3.2 Experiment 3.1: Chronic alcohol exposure and repeated withdrawal in C57 and DBA mice SKF 82958 1.0 mg/kg i.p. OR Quinpirole 3.0 mg/kg i.p. 1 wk veh i.p. SKF 82958 BAC 1.0 mg/kg i.p. OR Quinpirole 3.0 mg/kg i.p. ( 1 wk withdrawal 24º EtOH 1 wk withdrawal 4 wk EtOH 20% of total calories )x3 Experiment 3.2: Variable duration of withdrawal from chronic alcohol in C57 and DBA mice SKF 82958 1.0 mg/kg i.p. OR Quinpirole 1 wk veh i.p. 3.0 mg/kg i.p. 4 wk EtOH 20% of total calories SKF 82958 BAC 1.0 mg/kg i.p. 2 OR 4 wk withdrawal OR Quinpirole 3.0 mg/kg i.p. 24º EtOH Future Directions • Effects of prenatal alcohol exposure on the development and adult functioning of brain reward systems • Effects of adolescent alcohol exposure on the function of brain reward systems in adult mice • Comparison of the effects of voluntary alcohol drinking to those of forced alcohol consumption and non-contingent investigatoradministered alcohol on brain reward function • Investigation of the effects of pharmacological intervention on the development of changes in the brain reward system after alcohol exposure during vulnerable developmental stages – – – – Opioid antagonists (e.g., naltrexone) Calcium acetyl-homotaurinate (acamprosate) Presynaptic glutamate release modulators (e.g., levitiracetam) Atypical neuroleptics / non-selective partial D2 agonists (e.g., aripiprazole) Future Directions GABA DA D2- GABAA CTX D1+ D2- D1- nAChR D2- GLU nAChR 5HT1B- D2- DA GLU GABA AMPA D2- GABAB VTA mGluR1/5 GABAA nAChR GABA NMDA m nAc D1+ D1/D2 MFB mGluR1/5 AMPA D2- k 5HT3+ DA D1/D2 NMDA mAChR1 ACh