Effect of dexfenfluramine and 5-HT3 receptor antagonists on stress-induced reinstatement of alcohol seeking in rats

Anh Dzung Lê, Douglas Funk, Stephen Harding, W Juzytsch, Paul J Fletcher, Yavin Shaham

Introduction Stress is associated with alcohol use and relapse

Stress-induced drug use

(Sinha 2001)

Stress-induced relapse

(Sinha 2001)

An animal model -- reinstatement procedure:

training for drug self-administration extinction of drug-reinforced behavior noncontingent exposure to drugs or nondrug stimuli on reinstatement

Intermittent footshock reinstates alcohol seeking in alcohol- experienced rats.

Serotonin Serotonin system and its interaction

with midbrain and cortical dopamine pathways Pivotal to the rewarding effects of

alcohol 5-HT: tonic inhibitory control of midbrain

DA fibers The median and dorsal raphe and the

centralis posterior the cortico-midbrain regions

(Johnson 2004)

5-HT3 receptor 5-HT has 14 different receptor subtypes 5-HT3 receptor:

ligand-gated ion channel, not linked to G-proteins When activated cation flux depolarizes the

membrane potential 5-HT3 receptors have an important role in the

neural actions of alcohol, alcohol can alter the function of the 5-HT3 receptors: potentiation inhibition

(Lovinger 1999)

5-HT3 receptors are densely distributed in the terminals of mesocorcicolimbic DA-containing neurons and stimulate DA release in these regions.

The interaction between DA and 5-HT3 receptors in the mesocortical and mesolimbic region the rewarding effects of alcohol 5-HT3 receptor antagonist: attenuate

hyperlocomotion induced by DA or ethanol injection into the NAc; suppress neurokinin-induced hyperlocomotion (also diminished by the DA antagonist); reduce alcohol consumption (Johnson 2004)

5-HT3 receptor antagonist: Reduce alcohol craving and increase

abstinence in alcohol-dependent subjects

Decrease anxiogenic-like responses 5-HT3 receptor stress-induced

alcohol seeking

5-HT involved in footshock stress-induced reinstatement of alcohol seeking 8-OH-DPAT (5-HT1A agonist) mimic the

effect of the stressor on reinstatement Fluoxetine (selective serotonin reuptake

inhibitor – SSRI): low doses can attenuate footshock-induced

reinstatement It also reduces general consummatory

behavior, which might at least partially mediate the ethanol consumption.

The effects of fluoxetine on alcohol consumption and feeding behavior are not consistently altered by the manipulation of the 5-HT system

The aim of the study To determine if the inhibitory

effect of fluoxetine on footshock stress-induced reinstatement is mediated by increased 5-HT neurotransmission Dexfenfluramine (5-HT reuptake

inhibitor and releaser): attenuate this reinstatement?

To determine the role of 5-HT3 receptor in footshock-induced reinstatement of alcohol seeking 5-HT3 antagonist: ondansetron,tropisetron

Ondansetron treatment of early-onset alcoholics (Johnson 2004)

SSRIs treatment of late-onset alcoholics (Johnson 2004)

Materials and Methods

Subjects, apparatus and drugs 160 male Wistar rats The self-administration chambers

were equipped with two levers The active lever activate the infusion

pump the delivery of 0.19ml of a 12% alcohol solution into a receptacle

The inactive lever didn’t activate the pump

Drugs Dexfenfluramine: i.p. 1 h before the

start of the test sessions Ondansetron and tropisetron: i.p. 15

min before the start of the test sessions

Procedures Alcohol self-administration training

Two-bottle choice phase: access to alcohol and water in Richter tubes for 30 min/day

3% for 5 days, 6% for 8 days and 12% for 10-12 days

Self-administration Initiated on a fixed ratio-1 (FR-1) 5-s timeout

reinforcement for 10-14 days: 1 h/day a houselight signaled the beginning of the

sessions and was turned off at the end of the sessions.

5-s timeout period: Activation of the pump 5 s infusion During the infusion, a stimulus light above

the active lever was turned on for 6 s Lever presses during this time period were

counted but did not lead to further infusions

Increased to an FR-2 for five sessions Increased to an FR-3 for 8-12 days

until 3 days of stable alcohol seeking check the unconsumed alcohol in

receptacle the volume remaining was taken into account in calculate the number of alcohol reinforcements earned in each session

35 rats were excluded because they didn’t demonstrate reliable self-administration of pharmacologically relevant doses of alcohol

Extinction of the alcohol-reinforced behavior Responding on the active lever didn’t

lead to alcohol delivery 9-10 daily 1 h extinction until fewer

than 12 presses on the active lever Tests for reinstatement (under extinction

conditions) Intermittent footshock was

administered for 10 min immediately before the 1-h test sessions

Drug or vehicle was injected 60 min or 15 min before exposure to shock or no shock conditions

Experiment 1: dexfenfluramine Dexfenfluramine dose: 0, 0.25, 0.50

mg/kg between-subjects factor Stress condition: shock, no shock

within-subjects factor Vehicle or dexfenfluramine was injected

i.p. 60 min before the two test sessions 10 min of intermittent footshock or regular

extinction The two sessions were separated by 24 h

Experiment 2: ondansetron and tropisetron Ondansetron dose (0, 0.001, 0.01 and

0.1 mg/kg) or tropisetron dose (0,0.001,0.01, and 0.1 mg/kg)

Stress condition: shock, no shock Vehicle or drug was injected i.p. 15

min before the two test sessions

Results

Figure 1: alcohol self-administration in experiment 1

A: mean number of alcohol reinforcements

B: mean response on the active levers

The mean estimated alcohol intake during the last 3 days of training: 0.97±0.06 g / kg / h

Figure 2: alcohol self-administration in experiment 2 A: mean number of

alcohol reinforcement

B: mean responses on the active levers

The mean estimated alcohol intake during the last 3 days: 1.05±0.06 g / kg / h

Figure 3: extinction of the alcohol-reinforced behavior

Mean number of lever presses on the previously active lever and on the inactive lever during the extinction phase

Figure 4: the effect of dexfenfluramine injections on footshock-induced reinstatement Dose-dependently

attenuated footshock-induced reinstatement on the previously active lever

No effect on lever responding in the absence of shock

Footshock or dexfenfluramine had no effect on inactive lever responding

Figure 5: the effect of ondansetron and tropisetron injections on footshock-induced reinstatement Both drugs

attenuated footshock-induced reinstatement of responding on the previously active lever

No effect on responding in the absence of shock

Not dose-dependent Footshock or drug

injections had no effect on inactive lever responding

Discussion Dexfenfluramine dose-dependently

attenuated footshock-induced reinstatement of alcohol seeking

The 5-HT3 receptor antagonists, ondansetron and tropisetron, also attenuate footshock-induced reinstatement of alcohol seeking, but the effects of 5-HT3 antagonists were not dose-dependent

Methodological considerations Behavioral depression: unlikely, due to the

absence of effect on locomotor activity and on high rates of lever responding in drug administration procedures

The potential analgesic effects: unlikely, due to either the minimal effect on pain sensitivity or decreasing pain threshold

The magnitude of the effect of footshock reinstatement was greater in Experiment 2 than Experiment 1: individual differences in shock-induced freezing

Effect of dexfenfluramine on footshock stress-induced reinstatement The effect of footshock stress on reinstatement

of alcohol seeking involves decreases in 5-HT transmission and increases in CRF transmission

The drug’s effect on feeding behavior? Unlikely Alcohol not available Food was available during the experiment Footshock is ineffective in reinstating extinguished

responding for a palatable sucrose solution

Effect of 5-HT3 receptor antagonists on footshock stress-induced reinstatement In DRN, stimulation of 5-HT3 receptors

induces the local release of 5-HT Ondansetron and tropisetron inhibit 5-HT release in

the raphe releasing 5-HT neurons from autoreceptor inhibition increasing 5-HT cell firing increasing 5-HT release in terminal regions

Dopamine release The effect of 5-HT3 receptor antogonists on the

release of mesocorticolimbic DA induced by foot-shock stressor

references 1 Lê, A. D., Funk, D., Harding, S., Juzytsch, W., Fletcher, P.

J. and Shaham, Y. (2006) Effects of dexfenfluramine and 5-HT3 receptor antagonists on stress-induced reinstatement of alcohol seeking in rats. Psychopharmacology. 186, 82-92

2 Johnson, B. A. (2004) Role of the serotonergic system in the neurobiology of alcoholism. CNS drugs. 18(15),1105-1118

3 Lovinger, D. M. (1999) 5-HT3 receptors and the neural actions of alcohols: an increasingly exciting topic. Neurochemistry international. 35, 125-130

4 Lê, A. D., Quan, B., Juzytch, W., Fletcher, P. J., Joharchi, N. and Shayam, Y. (1998) Reinstatement of alcohol-seeking by priming injections of alcohol and exposure to stress in rats. Psychopharmacology. 135, 169-174

5 Sinha, R. (2001) How does stress increase risk of drug abuse and relapse? Psychopharmacology. 158, 343-359