Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

1. Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala Herry et al European Journal of Neuroscience, Vol. 24 p 261-269, 2006

2. Introduction • Extinction • Form of new learning in which the original memory acquired thru association of a CS with US is inhibited • Occurs after repeated exposures to CS alone • Auditory fear conditioning and extinction of fear conditioning differ • Extinction memories are context dependent and labile giving rise to spontaneous recovery (Quirk, 2002)

3. Intro • Neuronal mechanisms underlying acquisition of extinction still unclear • Emphasis on BLA • Previous studies • Intra-BLA injection of AP5,NMDA antagonist • Extinction prevented (Falls et al 1992)

4. Intro • In contrast • Acquisition of extinction of auditory fear conditioning unaffected by injection of NMDAr antagonist (Santini et al 2001) • Suggested that acquisition of extinction and consolidation of extinction rely on two separate molecular mechanisms

5. Intro • NMDAr can trigger signaling pathways like MAPK/ERK • This pathway implicated in consolidation of auditory and contextual fear conditioning (Trifilieff et al 2006)

6. Goals • Analyze by immunohistochemistry temporal activation of MAPK/ERK in BLA following acquisition of extinction • Directly test if inhibition of MAPK/ERK pathway b/f extinction training prevents acquisition of extinction of auditory fear conditioning

7. Materials and Methods • Adult male mice individually housed in plastic cages w ad libitum food access • 12 h light/dark • Mice handled gently for 2-3 min/day during 5 days to minimize nonspecific stress

8. Materials and Methods • Surgery • Anaesthetized w/ isoflurane in O2 • Secured in sterotaxic frame and bilaterally implanted w/ 24 gauge cannula aimed at BLA • Cannula fixed in place • Mice recovered for 1 week

9. Materials and Methods • Drug infusion • MEK inhibitor, U0126 • Dissolved in DMSO and stored • Animals given bilateral intra-BLA injections of 0.3 µL per site or 0.6µL both in 2% DMSO in artificial CSF • Done in freely moving mice at rate 0.4µL/min

10. Extrecellular ligand binding • RAS phosphorylation • RAF activation • MEK phsphorylation • MAPK activation • MAPK can phosphorylate many proteins • U0126 binds to MEK in a non-competitive manner and prevents the enzyme from phosphorylating MAPK by inhibiting the catalytic activity of the active enzyme MEKInhibitor

11. Materials and Methods • Behavioral Procedures • Conditioning Chamber • Grey plastic cylinder (15.5cm x 14cm) • Shock grid floor • Place in plexiglas box • Speaker w 2.5kHz, 80dB • 1s, 0.9mA foot-shock • Washed w/ 70% ethanol

12. Materials and Methods • Behavioral Procedures • Extinction training • Grey plastic cylinder removed • Grey plastic floor replaced grid • Washed w/ 1% acetic acid before and after session • To maximize discrimination b/w two contexts, light intensity reduced during fear conditioning

13. Materials and Methods • Behavioral procedures • Parallel measurements to score freezing • First, classical time-sampling procedure during which blind experimenter scored freezing/not freezing every 2s • Second, compared values to automatic infrared beam detection system • Two values 95% identical, were averaged for statistical analyses

14. Materials and Methods • Fear conditioning/extinction training • Mice conditioned to acquire fear in response to 30s CS tone paired w/ 1s foot-shock • 5 CS-US pairings, inter-trial interval 20-180 s • For Immunohistochem experiements, CS-US mice divided into 5 groups

15. Materials and Methods • Early Ext • Extinction 5 h after conditioning • Late Ext • Additional extinction 24 h later • Killed at 3 time points • 15min, 1 and 6h after Late extinction

16. Materials and Methods • 2 controls • No Early Ext • Killed 5 h after conditioning, just before early extinction training • No Late Extinction • Submitted to early extinction, killed 24h later just before late extinction training

17. Materials and Methods • CS • To control for possible contextual effect on MAPK/ERK • Submitted to extinction context alone during both sessions • Killed 1 h after late extinction • Naive • Neither submitted to fear conditioning nor extinction • Killed independently

18. Materials and Methods • For pharmacological experiment • Conditioned mice received bilateral intra-BLA injection of either MEK inhibitor (U0126; n=16) or DMSO (n=15) • Ten min before early or late extinction • Long term effect of U0126 on fear expression evaluated by submitting injected mice to final test consisting of 4 CS presentations

20. Materials and Methods • Histology • Mice terminally sampled at completion of experiment • At different times • Brains removed, sectioned and stained to verify cannulae placements

21. Materials and Methods • Immunohistochemistry • At appropriate time (1A) mice submitted to immunohistochemical experiments terminally sampled • Brains removed, section and stained to quantify phospho-MAPK/ERK or total-MAPK/ERK immunoreactivity

22. Materials and Methods • Data analysis • P-MAPK/ERK or total-MAPK/ERK immunoreactivity carried out using 3 coronal serial sections • Number of positive cells quantified using computerized image analysis system • BLA • Two values quantified in each mouse w/in predefined boundaries outlining BLA

23. Materials and Methods • Data analysis • Results expressed as number of positive cells/mm2 • pMAPK/ERK IR restricted to nucleus • tMAPK/ERK IR located w/in cytoplasmic and somatic compartments

24. Results • Preconditioning period CS and CS-US mice showed similar freezing levels • Freezing remained low in CS • CS-US showed progressive increase • From 10-50% freezing

26. Results • Early Extinction (5 hours later) • Pre-CS • Did not display contextual fear generalization in both groups • CS-alone induced high level of freezing in CS-US but not in CS group

28. Results • Late Extinction (24h later) • Induced rapid decrease of freezing response in mice of CS-US group • Shows that protocol promotes complete extinction of conditioned fear responses at end of Late Extinction session

30. Results • Levels of pMAPK/ERK-IR following Early Extinction were not significantly different between Early-Ext and No Early-Ext control groups

31. Results • 60 min after Late Extinction • pMAPK/ERK-IR levels in BLA of Late-Ext group significantly different from control

33. Results • Then compared results of pMAPK/ERK-IR for Early-Ext and Late-Ext groups • Found significant increase in Late-Ext group • Indicates acquisition of extinction during Late-Extinction was associated with an increase of pMAPK/ERK-IR • Also compared results against Ctx, CS, and Naive

35. Results • Immunohistochemical analysis of total MAPK/ERK in subset mice from Late-Ext and No Late-Ext groups • Results show that tMAPK/ERK was not significantly different b/w groups

37. Results • Next, examined pMAPK/ERK-IR in mice killed 15, 60, 360 min in Late-Ext group to No Late-Ext • Results show significant activation of pMAPK/ERK-IR at each time point examined after Late Extinction w/ max at 60 min

39. Photomicrograph showing distribution of pMAPK/ERK positive cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

40. Recap • Extinction learning induces MAPK/ERK phosphorylation in basolateral amygdala

41. Results • Next, tried to determine if MAPK/ERK signaling required for acquisition of extinction • Used MEK inhibitor U0126 • Injected it into one group 10 min before Early or Late Extinction • Control group injected with DMSO

42. Results • Mice underwent fear conditioning • Mice injected before Early Extinction displayed low freezing levels and did not differ from each other during pre-conditioning period

44. Results • After first CS-US paring, both groups of mice (u0126 and DMSO) displayed progressive increase in freezing • Direct comparisons revealed no significant differences b/w u0126 and DMSO groups through whole session

46. Results • U0126 injection prior to Early Extinction did not induce notable changes in freezing level during Early, Late Extinction, or post-extinction in comparison to control • Both groups reach baseline freezing level at end of Late Extinction • Suggests extinction of CF was complete

48. Results • Mice from u0126 and DMSO groups injected before Late Extinction again no difference during pre-conditioning period or during the condition session • Progressive increase in freezing levels • During Early Extinction mice showed elevated levels again

50. Results • When put back in context 24h later mice, during first block of testing both groups showed similar levels of freezing • But significant decrease noted in the control group in later blocks • Mice tested final time 24h after Late Extinction without u0126 • Showed extinction didn’t occur