The iTA exercice as a real experiment The context The problem at stake Design of the experiment
Context switch experiment Results?
description
Transcript of Context switch experiment Results?
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• Glutamate receptor antagonist infusions into the basolateral and medial amygdala reveal differential contributions to olfactory vs. context fear conditioning and expression– David L. Walker– Gayla Y. Paschall– Michael Davis
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Context switch experimentResults?
• Page 126 right column on bottom
• During training, background noise was off and a 150 lux houselight was on.
• Olfactory stimulus
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Abbreviations• NBQX……..1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxa-line-
7-sulfonamide disodium salt– 3 μg/side
• AP5……….. DL-2-amino-5-phospho-nopentanoic acid – 2.5 μg/side
• ACSF……..artificial cerebrospinal fluid
• AMPA…….. amino-3-hydroxy-5-methylisoxazole-4-propionic acid• NMDA………N-methyl-D-aspartate
• CS…………conditioned stimulus• 95 db………accustic stimulus
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Classical fear conditioning
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The Amygdala
• key role in auditory and visual stimuli fear conditioning (learning) and fear expression (display)
• exact role in learning is unclear– neural plasticity in aversive learning
paradigms or– a brain area that modulates memory storage
elsewhere
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The Amygdala 2
• Basolateral Amygdala– mediates fear responses to olfactory CSs perhaps via afferents
from the perirhinal cortex– NBQX or AP5
• disrupt the facilitatory effects of sustained bright light on startle
• Medial Amygdala– may participate in olfactory fear conditioning – receives a prominent olfactory input relative to other modalities
• Central Amygdala– NBQX or AP5
• no change
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Visual and auditory stimuli
Olfactory stimuli
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GOALS
• Basolateral amygdala– confirm the involvement in fear expression to
an olfactory CS– determine if if participates in olfactory fear
learning
• Medial amygdala– evaluate the contribution to both
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Materials and methods and methods
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animals
• Male Sprague-Dawley rats – Pre surgery:
• group-housed (4 per cage) in 45 x 20 x 24-cm (depth width height) polycarbonate cages
– Surgery weight:• 275-350g
– Post surgery: • individually housed, in 20 x 19 x 24-cm hanging
wire-mesh cages
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Surgery
• 22-gauge guide cannulae bilaterally into– basolateral amygdala – medial amygdala
• A minimum of 8 d elapsed between surgery and the behavioral procedures
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Histology
• Rats were terminally sampled– 40-μm coronal sections stained with cresyl violet– placement judged by a scorer blind to the animal’s
group assignment and behavioral data– cannula tips were required to be within or no further
than 0.5 mm from the intended target • Experiments 1 & 2
– basolateral amygdala complex
• Experiments 3 & 4– medial amygdala nucleus
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Training cages
• Rats were trained and tested in two identical Plexiglas and wire-mesh cages– The floor: four 6.0-mm
diameter stainless steel bars spaced 18 mm apart.
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Acclimation session(2 consecutive days)
• rats were placed into the test cages– 5 min acclimation
• 30 startle-eliciting noise bursts – (onset-to-onset interstimulus interval = 30
sec).
• rats were sorted into different treatment groups
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Measuring of startle
• Accelerometer at the bottom if the cage
• Startle amplitude– maximal peak-to-peak voltage during the first 300
msec after onset of the startle-eliciting noise burst– evoked by 50-msec white-noise bursts
• Background noise (60-dB wideband white-noise)
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Fear conditioning(Within three days of the final acclimation session)
• 5 min acclimation
• Experiments 1-4A– five odor-shock pairing in a 4 min interval
• Experiment 4B– five light-shock pairings in a 4 min interval
• For each pairing, the 0.5-sec shock was delivered 3.5 sec after onset of the 4.0-sec CS.
• The unconditioned stimulus for all experiments was a 0.5-sec 0.4-mA scrambled floor-bar shock.
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Olfactory stimulus
• continuous flow of air
• stimulus– for 4 sec, scent of 20 ml of 5% amyl acetate in
propylene glycol solution– mixed in a 3:5 ratio with clean air before flowing into
the cage
• The chamber was actively exhausted – Total volume turnover every 25 sec
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Visual stimulus
• Stimulus– A 4-sec light on produced by an 8-W (140 lux)
fluorescent bulb located 10 cm behind each cage
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Drugs and infusion procedure
• Rats were infused– NMDA receptor antagonist AP5– AMPA/kainate receptor antagonist NBQX– ACSF
• Infusions – 0.25 μL/min, 0.5 μL total volume
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Drugs and infusion procedure
• infusion
• Pre-training– 15 min rest?– Animal placed in test cage– 5 min later – first CS-shock pairing
• Pre-test– Animal placed in test cage– Start of test 20 min later
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Fear-potentiated startle test(48 h later returned to the training chamber)
• 5 min acclimation
• Baseline– 30 startle-eliciting noise bursts (every 30 sec)
• Test– 40 test trials consisting of 10 repetitions of a single CS test trial – three noise-alone test trials
• The noise burst was presented 3.5 sec after onset of – the odor (Experiments 1-4A)– light (Experiment 4B)
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Recap Timeline
• Surgery– 8 day recovery
• Baseline sorting (2 days)– 3 day recovery
• Training– 2 day recovery
• Testing
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Recap injections
• Fear conditioning– Injection (AMPA/kainate or NMDA receptor
antagonists) – After 15 min wait moved to training cage– 5 min wait till start
• Fear expression– Injection– Moved to testing cage right afterwards– 20 min wait till start
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Statistical analyses
• mean startle amplitude (%) – noise-alone to CS test
• baseline startle (reflect context conditioning)– pre-conditioning to the post-conditioning
• analyzed using ANOVA and/or two-tailed t-tests
• criterion for significance was P < 0.05.
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Results
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Experiments 1A and 1B
NMDA receptor blockade in the basolateral amygdala
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AP5 prior to training
• Mean footshock reactivity (S.E.M.) during training– ACSF [N = 5] (1188 ±
295) – AP5 [N = 7] (1048 ±
301)
• SIGNIFICANT– t(11) = 2.92.– did not evaluate NBQX
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AP5 prior to testing
• test days separated by 48h
• AP5 day 2 [N = 3]– 25.1 ± 4.9 ACSF– 56.4 ± 27.7 AP5
• AP5 day 1 [N = 5]– 88.7 ± 26.2 AP5 – 91.0 ± 50.2 ACSF
• NOT SIGNIFICANT– t(7) = 0.44,
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What we got till now…
• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus
requires NMDA receptor activity.
– Expression of a conditioned fear stimulus does not require NMDA receptor activity.
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Experiment 2
AMPA/kainate receptor blockade in the
basolateral amygdala
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NBQX prior to testing
• test days separated by 48h
• ACSF on day 1 [N = 7]– 127.6 ± 39.3
• ACSF on day 2 [N = 5]– 89.6 ± 34.4
• The difference was not significant (P = 0.41).
• SIGNIFICANT – t(11) = 4.65.
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What we got till now…
• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus
requires NMDA receptor activity.
– Expression of a conditioned fear stimulus does not require NMDA receptor activity.
– Expression of a conditioned fear stimulus requires AMPA/kainate receptor activity
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Experiment 3
AMPA/kainate and NMDA receptor blockade in the
medial amygdala
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AP5 or NBQX prior to training
• Experiment 3– ACSF [N = 7]– AP5 [N = 8] – NBQX [N = 6]
• NOT SIGNIFCANT– but ACSF is really low – Normal: 92.4 ± 12.
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What we got till now…
• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus requires
NMDA receptor activity.
– Expression of a conditioned fear stimulus does not require NMDA receptor activity.
– Expression of a conditioned fear stimulus requires AMPA/kainate receptor activity.
• MEDIAL AMYGDALA– Acquisition of a conditioned fear stimulus does not
require NMDA or AMPA/kainate receptor activity.
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Experiments 4A and 4B:
AMPA/kainate receptor antagonist into the
medial amygdala
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NBQX prior to odor testing
• Experiment 4a– ACSF [N = 7]– NBQX [N = 10]
• Independet sample t-test
• SIGNIFICANT– t(15) = 2.41.
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What we got till now…
• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus requires NMDA
receptor activity.
– Expression of a conditioned fear stimulus does not require NMDA receptor activity.
– Expression of a conditioned fear stimulus requires AMPA/kainate receptor activity.
• MEDIAL AMYGDALA– Acquisition of a conditioned fear stimulus does not require
NMDA or AMPA/kainate receptor activity.
– Expression of a conditioned fear stimulus does require AMPA/kainate receptor activity.
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NBQX prior to light test
– ACSF [N = 8]– NBQX [N = 13]
• SIGNIFICANT– t(19) = 2.26– Did not do it with AP5
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ACSF vs. DrugSight vs. odor
• ANOVA (Experiment 4A & B):– Significant effect of drug treatment
• F(1,34) = 16.02
– Not significant effect of modality (light vs odor)
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What we got till now…
• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus requires NMDA
receptor activity.
– Expression of a conditioned fear stimulus does not require NMDA receptor activity.
– Expression of a conditioned fear stimulus requires AMPA/kainate receptor activity.
• MEDIAL AMYGDALA– Acquisition of a conditioned fear stimulus does not require
NMDA or AMPA/kainate receptor activity.
– Expression of a conditioned fear stimulus does require AMPA/kainate receptor activity. (olfactory or visual)
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Break?
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Treatment effects on “baseline” startle amplitude
• NBQX – could block the acquisition of the
context in which a stimulus takes place
• Compare 30 noise-alone trials– Pre-training (grouping of baselines) – pre-testing 30 noise alone trials
• ANOVA– significant Treatment effect, F(1,34) =
4.93, not a significant Modality or interaction effect
• The absolute level of startle prior to conditioning was somewhat, although not significantly, lower in the groups that later received NBQX (i.e., vs. the groups that later received ACSF).
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• Comparison– Pre-test NBQX to ACSF– Pre-training AP5 to ACSF
• pre- to post-conditioning changes in baseline startle amplitude
• no obvious effect on the pre- to post-conditioning increases
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Follow up experiments (context switch experiment)
• different chamber– did not show the same pre-training to post-
training increase. • 239.0 ± 37.6 prior to conditioning• 235.4 ± 46.7 after conditioning
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Specific vs. nonselective• NBQX infused into the medial nucleus of the amygdala
– might have specifically disrupted the influence of context on startle– might have depressed startle nonselectively and simply masked the
influence of context
• Evaluated the effect of NBQX infusions in untrained animals. [N = 9]– medial nucleus of the amygdala (One week rest)– 30 95-dB noise bursts– 30 intermixed 110-dB noise bursts.
• were included to ensure that a negative finding could not be attributed to baseline-dependent NBQX effects
• 24 h and 72 h later (the order of treatments was counterbalanced) – ACSF or NBQX
• 24 and 72 h later and were retested
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• NOT SIGNIFICANT
• => NBQX causes a specific disruption of the pre- to post-conditioning increase
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Influence of cannula placement on the magnitude of the NBQX effect
• medial amygdala lies just – medial to the basomedial and cortical amygdala
nuclei– drugs infused into the medial nucleus might diffuse
dorsally along the cannula track where they – might also influence the central nucleus of the
amygdala (where NBQX infusions have previously been shown to disrupt fear-potentiated startle to a visual CS,
• we considered the possibility that the effects of pre-test infusions into the medial nucleus might be mediated by actions elsewhere.
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• Experiment 4A– 10 NBQX-infused rats with
bilateral placements– 4 rats with only a single
cannula in the medial amygdala
• SIGNIFICANT– t(12) = 3.21
• => Effect of NBQX is anatomically specific and require bilateral inactivation of the medial nucleus of the amygdala
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placement-effect correlations rats in Experiment 4A
• the total distance of both cannula tip to either the
– medial, – basomedial, – cortical, or – central amygdala nuclei
• The summed distance was entered into a correlation analysis using percent potentiation as the correlated variable.
• SIGNIFICANT correlation for medial nucleus
– r = 0.82
• NOT SIGNIFICANT for other 3 areas– Central: r = 0.15– Cortical: r = 0.29– Basomedial: r = 0.45
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• No placement correlations in visual experiment– To few animals
• For fear-potentiated startle to context,– No alteration if NBQX was injected
• Bilateral 8 ± 11 [N = 22] % potentiation• Unilateral 1 ± 16 [N = 7] % potentiation
• No correlation between percent potentiation and proximity to any of the four structures examined.
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What we got till now…• BASOLATERAL AMYGDALA
– Acquisition of a conditioned fear stimulus requires NMDA receptor activity, which appears to be involved in connecting contextual events.
– Expression of a conditioned fear stimulus does not require NMDA receptor activity.
– Expression of a conditioned fear stimulus requires AMPA/kainate receptor activity.
• MEDIAL AMYGDALA– Acquisition of a conditioned fear stimulus does not require NMDA or
AMPA/kainate receptor activity.
– Expression of a conditioned fear stimulus does require AMPA/kainate receptor activity. (olfactory or visual)
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BasolateralAmygdala
NMDAAMPA (not NMDA)
MedialAmygdala
(Not NMDA or AMPA)AMPA
BNST
CentralAmygdala
AMPA?AMPA?
primary acoustic startle circuit
•do not require conditioning to evoke fear/anxiety•conditioning to appetitive stimuli
AcquisitionExpression
Conditioned olfactory fear/anxiety stimulus
Visual Stimuli
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Fin(DONE)