1. 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

2. Introduction Stress is associated with alcohol use and relapse
Stress-induced drug use
(Sinha 2001)

3. Stress-induced relapse
(Sinha 2001)

4. 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.

5. 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)

6. 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)

7. 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)

8. 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

9. 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

10. 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?

11. Ondansetron  treatment of early-onset alcoholics (Johnson 2004)
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)

12. Materials and Methods

13. 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

14. 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

15. 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 days

16. Self-administration
Initiated on a fixed ratio-1 (FR-1) 5-s timeout reinforcement for 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

17. 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

18. 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

19. 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

20. 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

21. Results

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. references
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2 Johnson, B. A. (2004) Role of the serotonergic system in the neurobiology of alcoholism. CNS drugs. 18(15),
3 Lovinger, D. M. (1999) 5-HT3 receptors and the neural actions of alcohols: an increasingly exciting topic. Neurochemistry international. 35,
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,
5 Sinha, R. (2001) How does stress increase risk of drug abuse and relapse? Psychopharmacology. 158,