article 3 Dopamine D2 receptors in the expression and extinction of contextual and cued conditioned fear in rats Flashcards
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abstract
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- Dopamine mediates fear conditioning through its action on D2 receptors in mesolimbic pathway
- D2R involved in expression of conditioned fear rather than the development of conditioned fear (found using dopaminergic injections)
- To explore ^, D2R-like antagonists Sulpiride and Haloperidol tested on rats
- Rats trained by footshocks (unconditioned stimuli) then exposed to context-CS or light-CS
- 24 hours later, rats re-exposed
- Motor performance tested in open-field & catalepsy test (posture correction task)
- Sulpiride reduced expression of contextual and cued conditioned fear (significantly)
- Haloperidol did not reduce fear expression caused catalepsy and motor impairments
- D2R involved in expression of conditioned freezing rather than its extinction
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introduction
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- Fear and anxiety→Pavlovian aversive conditioning
- Neutral stimulus (light/tone) paired with aversive unconditioned stimulus (US; ex. shock) turning neutral stimulus into conditioned stimulus (CS) eliciting defensive response
- Conditioned responses with repeated CS presentation without US = Extinction
- Cued fear → phobias; Contextual fear → anxiety-related disorders
- “Freezing” response in both fears mentioned above
- Dopamine in VTA→crucial to fear conditioning
- Fear expression→ dopamine increase in Nucleus Accumbens and Basolateral Amygdala
- D2 agonists & antagonists reduce conditioned fear responses
- D2 agonists→VTA; D2 antagonists→Basolateral Amygdala
- Dopamine mediates fear expression through these receptors
- Haloperidol→ D2 antagonist used for schizophrenia but causes parkinsonism and catalepsy
GOAL: replicate Sulpiride effects on fear reduction and test if Haloperidol had similar effects
AIMED: explore dopamine role in extinction process
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method
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Animals:
* 160 rats
* 12/12h dark/light cycle
Drugs:
* Dopaminergice D2 Antagonists Sulpiride and Haloperidol
* Mixed with 2% tween 80 & dissolved saline .9%
* Rats received either Sulpiride (40mg/kg) or Haloperidol (.1 or .25 mg/kg) 15 min before experiment
* Investigator blind to treatment conditions
Contextual Fear Conditioning:
* Three session
a) Training, test and retest
b) 24 hours apart
* Cage with metallic walls and stainless steel bar floor (CS)
* 5 min habituation, then rats get 10 unsignaled foot shocks (1 second) random intervals (30-90 seconds)
* Test and retest in same cage but no footshocks
* Given drug or “vehicle” 15 min before test session
* Freezing behavior→assess conditioned fear (lack of movement except for respiration–6 seconds)
* Data→percentage of freezing in 2 minute blocks & total freezing duration during test/retest
* Fear Extinction Index (FEI) subtract retest freezing from peak freezing during last 2 minutes of training
Cued Fear Conditioning:
* Different group of rats from contextual fear training
a) training , test and retest
b) 24 hours apart
* Training: rats conditioned to light CS paired with footshock in same cage used for contextual fear conditioning
* 5 min habituation phase→ then rats received eight light CS (20 seconds) co-ending with footshocks (1 second)
* Interval between trial→60-120 seconds and each session=20 min
* Test/retest→ new/different cage than training cage
* 24 hours after training, rats got drug or vehicle and tested from freezing after 8 light CS presentations
* Retest with same procedure 24 hours later
* Freezing during light CS presentation→ assess conditioned fear
Results: percentage of freezing during light CS and total session duration
* FEI subtract retest freezing from peak freezing during last training light CS
Motor Performance:
* 2 days after retest, rats did motor tests (catalepsy and open-field tests)
Catalepsy Test:
* 15 & 45 min after retest administration
* In a cage, horizontal acrylic bar 8cm above ground
* Failure to correct posture and duration of deformed posture was measured (delay to step down from horizontal bar)
Open-field Test:
* Locomotor and exploration measured
* 25 min after drug injection; rats placed in middle arena and left 15 to explore
* Measured number of crossings & number of times forelegs located in middle of arena or against walls)
Analysis of results:
* Mean plus or minus SEM
* 2-way ANOVA for repeated measures
a) Percentage of freezing with treatments (contextual: freezing during 2 min blocks or during whole session; cued: freezing during each cue)etc.
b) Treatments→Between subjects factor (drug doses and control)
c) Trials→ Within subjects factor ( 2 min block/cue/test/retest)
* FEI→analyzed with t-tests or one-way ANOVA
* Motor performance→ open-field test with t-tests or one-way anova
* Catalepsy test→ 2-way ANOVA for repeated measure (treatment→BSF & trial→WSF)
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results
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Contextual fear Conditioning
- Rats in same cage froze more than those tested in different cage (p<0.05)
- Sulpiride impaired contextual conditioned freezing
a) Freezing increased during training, no differences between treatment groups
b) Test session sulpiride group froze less than control group
c) No differences in retest sessions - Sulpiride reduced freezing during test; control group reduced freezing between test and retest
d) No differences in FEI - Haloperidol did NOT affect freezing or extinction
a) Freezing increased during training with no treatment effects
b) No differences found in test session for treatment but significan differences observed for time blocks
c) Retest session, freezing decreased over time with no treatment effect
d) Freezing behavior decreased between test and retest for control groupe) No difference in FEI between treatments
Cued Fear Conditioning
* Paired CS-US froze more than unpaired rats
* Sulpiride effects:
a) Freezing increased during training with no treatment differences
b) During test, freezing decreased in later trials, but no significant treatment differences
c) Retest→ differences for trials but not treatments
d) Between sessions, sulpiride reduced freezing during test and reduced in control group between sessions
e) No differences in FEI
Haloperidol effects:
* Freezing increased during training with differences between haloperidol .1 and .25 groups, but no differences compared to control
* Test→ freezing decreased over trials with no treatment differences
* Between sessions, freezing behavior reduced between test and retest with not treatment effects
* No differences in FEI
Motor performance
* Sulpiride did not affect motor performance
* Catalepsy test (2-way ANOVA) not significant effect for treatments, trials, or interaction between treatment and trials
* Open-field test→ no significant differences between number of crossings or earrings
* Haloperidol-induced catalepsy→ decrease in exploring open-field
* Catalepsy test→significant effect for treatments or interaction
* Open-field test, one-way ANOVA→ significant difference for treatments on number of crossings and rearings
* ***
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discussion
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- Sulpiride reduced conditioned freezing w/o affecting motor activity, unlike Haloperidol
- Haloperidol caused motor impairments but did not affect freezing responses (fear expression)
- Both drugs had no effect on extinction recall of conditioned fear
a) D2R involvement in conditioned fear expression, not extinction - Control experiments→ successful fear conditioning and extinction learning in rats
- VTA dopaminergic activity→fear arousal with dopaminergic agent affecting fear response
a) Affects structures like amygdala and nucleus accumbens - D2R in conditioned freezing, Sulpiride targeting mesolimbic and Haloperidol affecting nigrostriatal pathways
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What were the main findings regarding sulpiride and haloperidol in this study?
Sulpiride significantly reduced the expression of contextual and cued conditioned fear without affecting extinction recall.
Haloperidol did not reduce fear expression but caused catalepsy and motor impairments.
How are cued fear and contextual fear related to different disorders?
Cued fear is seen as a model for fear-related disorders like phobias, while contextual fear is considered a better model for anxiety-related disorders.
What were the two main behavioral tests used to assess conditioned fear?
Contextual fear conditioning and cued fear conditioning.
How was motor performance assessed in this study?
Motor performance was evaluated using the open-field test and the catalepsy test.
How did sulpiride affect contextual conditioned freezing?
Sulpiride impaired the expression of contextual conditioned freezing during the test session, but did not affect freezing during the retest session.
What effect did sulpiride have on cued conditioned freezing?
Sulpiride decreased the expression of cued conditioned freezing during the test session, but did not affect freezing during the retest session.
How did haloperidol affect conditioned fear and motor performance?
Haloperidol did not significantly affect the expression or extinction of contextual or cued conditioned freezing, but it induced catalepsy and decreased exploration in the open-field test.
What do the results suggest about the role of D2 receptors in fear conditioning?
The results suggest that D2 receptors are involved in the expression of conditioned freezing rather than in its extinction.
How do the effects of sulpiride and haloperidol differ in terms of dopaminergic pathways?
Sulpiride appears to target the mesolimbic pathway, while haloperidol primarily affects the nigrostriatal pathway.
What is the Fear Extinction Index (FEI) and how was it calculated?
The FEI was calculated by subtracting the percentage of freezing exhibited during the retest session from the percentage of freezing during the final phase of the training session.
What were the drugs and doses used in this study?
Sulpiride (40 mg/kg) and Haloperidol (0.1 or 0.25 mg/kg), administered intraperitoneally 15 minutes before the experiments.
How many rats were used in the study and what was their housing condition?
160 male Wistar rats, housed in groups of four under a 12/12 h dark/light cycle at 23 ± 2 °C.
Describe the apparatus used for contextual fear conditioning.
A cage (26 × 20 × 20 cm) with metallic white walls and ceiling, transparent glass front door, and floor of 13 stainless steel bars.
What was the protocol for the contextual fear conditioning training session?
5 min habituation, followed by ten unsignaled 1 s, 0.6 mA footshocks with intertrial intervals varying randomly between 30 and 90 s.
How was freezing behavior defined and measured?
Total absence of movements, except for respiration, for at least 6 s per episode. Measured as percentage of time spent freezing.
Describe the apparatus used for cued fear conditioning test and retest sessions.
A different cage (26 × 25 × 20 cm) with gray metallic back and side walls, transparent acrylic door and ceiling, and 18 metal bars on the floor.
What was the protocol for the cued fear conditioning training session?
5 min habituation, followed by eight CS-US pairings using a 20-s, 6-W light-CS coterminating with a 1 s, 0.6 mA footshock-US.
How was the catalepsy test performed?
Rats were placed with forepaws on a horizontal acrylic bar 8 cm above the floor. Catalepsy was measured as the latency to step down from the bar.
Describe the open-field test setup and measurements.
Circular enclosure (60 cm diameter, 50 cm height) with floor divided into 12 sections. Measured total number of crossings and rearings over 15 minutes.
