Extinction of auditory fear conditioning requires mapk erk activation in the basolateral amygdala
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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

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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
Introduction activation in the basolateral amygdala

  • 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)


Intro
Intro activation in the basolateral amygdala

  • 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)


Intro1
Intro activation in the basolateral amygdala

  • 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


Intro2
Intro activation in the basolateral amygdala

  • NMDAr can trigger signaling pathways like MAPK/ERK

    • This pathway implicated in consolidation of auditory and contextual fear conditioning (Trifilieff et al 2006)


Goals
Goals activation in the basolateral amygdala

  • 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


Materials and methods
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods1
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods2
Materials and Methods activation in the basolateral amygdala

  • 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


  • Extrecellular ligand binding activation in the basolateral amygdala

  • 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


Materials and methods3
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods4
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods5
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods6
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods7
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods8
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods9
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods10
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods11
Materials and Methods activation in the basolateral amygdala

  • Histology

    • Mice terminally sampled at completion of experiment

      • At different times

    • Brains removed, sectioned and stained to verify cannulae placements


Materials and methods12
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods13
Materials and Methods activation in the basolateral amygdala

  • 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


Materials and methods14
Materials and Methods activation in the basolateral amygdala

  • 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


Results
Results activation in the basolateral amygdala

  • 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


Results1
Results activation in the basolateral amygdala

  • 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


Results2
Results activation in the basolateral amygdala

  • 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


Results3
Results activation in the basolateral amygdala

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


Results4
Results activation in the basolateral amygdala

  • 60 min after Late Extinction

    • pMAPK/ERK-IR levels in BLA of Late-Ext group significantly different from control


Results5
Results activation in the basolateral amygdala

  • 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


Results6
Results activation in the basolateral amygdala

  • 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


Results7
Results activation in the basolateral amygdala

  • 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



Recap
Recap cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • Extinction learning induces MAPK/ERK phosphorylation in basolateral amygdala


Results8
Results cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • 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


Results9
Results cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • Mice underwent fear conditioning

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


Results10
Results cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • 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


Results11
Results cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • 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


Results12
Results cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • 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


Results13
Results cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • 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


Summary
Summary cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • All together results indicate that inhibition of MAPK/ERK signaling in BLA before Late but not Early Extinction prevents acquisition of extinction


Summary1
Summary cells in BLA of Late-Ext and No Late-Ext @ 60 min delay

  • Extinction of auditory fear condition induces an increase in MAPK/ERK phosphorylation in BLA

  • Inhibition of MAPK/ERK signaling pathway in BLA prevents acquisition of extinction memory

  • So MAPK/ERK signaling in BLA also necessary for acquisition of fear extinction


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