Glutamate receptor antagonist infusions into the basolateral and medial amygdala reveal differential contributions to olfactory vs. context fear conditioning

• 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

Context switch experimentResults?

• Page 126 right column on bottom

• During training, background noise was off and a 150 lux houselight was on.

• Olfactory stimulus

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

Classical fear conditioning

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

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

Visual and auditory stimuli

Olfactory stimuli

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

Materials and methods and methods

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

Surgery

• 22-gauge guide cannulae bilaterally into– basolateral amygdala – medial amygdala

• A minimum of 8 d elapsed between surgery and the behavioral procedures

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

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.

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

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)

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.

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

Visual stimulus

• Stimulus– A 4-sec light on produced by an 8-W (140 lux)

fluorescent bulb located 10 cm behind each cage

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

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

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)

Recap Timeline

• Surgery– 8 day recovery

• Baseline sorting (2 days)– 3 day recovery

• Training– 2 day recovery

• Testing

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

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.

Results

Experiments 1A and 1B

NMDA receptor blockade in the basolateral amygdala

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

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,

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.

Experiment 2

AMPA/kainate receptor blockade in the

basolateral amygdala

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.


• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus

requires NMDA receptor activity.


– Expression of a conditioned fear stimulus requires AMPA/kainate receptor activity

Experiment 3

AMPA/kainate and NMDA receptor blockade in the

medial amygdala

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.


• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus requires

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.

Experiments 4A and 4B:

AMPA/kainate receptor antagonist into the

medial amygdala

NBQX prior to odor testing

• Experiment 4a– ACSF [N = 7]– NBQX [N = 10]

• Independet sample t-test

• SIGNIFICANT– t(15) = 2.41.


• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus requires NMDA

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.

NBQX prior to light test

– ACSF [N = 8]– NBQX [N = 13]

• SIGNIFICANT– t(19) = 2.26– Did not do it with AP5

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)


• BASOLATERAL AMYGDALA– Acquisition of a conditioned fear stimulus requires NMDA

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)

Break?

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).

• 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

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

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

• NOT SIGNIFICANT

• => NBQX causes a specific disruption of the pre- to post-conditioning increase

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.

• 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

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

• 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.

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.



• 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)

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

Fin(DONE)

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Glutamate receptor antagonist infusions into the basolateral and medial amygdala reveal differential contributions to olfactory vs. context fear conditioning