1 / 53

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

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. Introduction. Extinction

duena
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

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

  20. 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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

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

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

  28. 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

  29. 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

  30. 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

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

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

  33. 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

  34. 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

  35. 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

  36. 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

  37. 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

  38. 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

More Related