Download Feng Na - USD Biology

Effect of dexfenfluramine and 5HT3 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 earlyonset 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 selfadministration 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 alcoholreinforced 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 footshockinduced 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 footshockinduced 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 footshockinduced 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 shockinduced freezing

Effect of dexfenfluramine on footshock stressinduced 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 5HT 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