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Context switch experiment Results?

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 experiment Results?. Page 126 right column on bottom

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Context switch experiment Results?

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  1. 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

  2. Context switch experimentResults? • Page 126 right column on bottom • During training, background noise was off and a 150 lux houselight was on. • Olfactory stimulus

  3. 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

  4. Classical fear conditioning

  5. 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

  6. 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

  7. Visual and auditory stimuli Olfactory stimuli

  8. 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

  9. Materials and methods and methods

  10. 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

  11. Surgery • 22-gauge guide cannulae bilaterally into • basolateral amygdala • medial amygdala • A minimum of 8 d elapsed between surgery and the behavioral procedures

  12. 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

  13. 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.

  14. 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

  15. 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)

  16. 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.

  17. 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

  18. Visual stimulus • Stimulus • A 4-sec light on produced by an 8-W (140 lux) fluorescent bulb located 10 cm behind each cage

  19. 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

  20. 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

  21. 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)

  22. Recap Timeline • Surgery • 8 day recovery • Baseline sorting (2 days) • 3 day recovery • Training • 2 day recovery • Testing

  23. 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

  24. 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.

  25. Results

  26. Experiments 1A and 1B NMDA receptor blockade in the basolateral amygdala

  27. 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

  28. 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,

  29. 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.

  30. Experiment 2 AMPA/kainate receptor blockade in the basolateral amygdala

  31. 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.

  32. 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

  33. Experiment 3 AMPA/kainate and NMDA receptor blockade in the medial amygdala

  34. 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.

  35. 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.

  36. Experiments 4A and 4B: AMPA/kainate receptor antagonist into the medial amygdala

  37. NBQX prior to odor testing • Experiment 4a • ACSF [N = 7] • NBQX [N = 10] • Independet sample t-test • SIGNIFICANT • t(15) = 2.41.

  38. 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.

  39. NBQX prior to light test • ACSF [N = 8] • NBQX [N = 13] • SIGNIFICANT • t(19) = 2.26 • Did not do it with AP5

  40. 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)

  41. 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)

  42. Break?

  43. 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).

  44. 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

  45. 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

  46. 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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