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

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

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

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

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

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

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

Materials and Methods Surgery Surgery Anaesthetized w/ isoflurane in O 2 Anaesthetized w/ isoflurane in O 2 Secured in sterotaxic frame and bilaterally implanted w/ 24 gauge cannula aimed at BLA Secured in sterotaxic frame and bilaterally implanted w/ 24 gauge cannula aimed at BLA Cannula fixed in place Cannula fixed in place Mice recovered for 1 week Mice recovered for 1 week

Materials and Methods Drug infusion Drug infusion MEK inhibitor, U0126 MEK inhibitor, U0126 Dissolved in DMSO and stored 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 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 Done in freely moving mice at rate 0.4µL/min

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 MEK Inhibitor

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

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

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

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

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

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

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

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

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

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

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

Materials and Methods Data analysis Data analysis Results expressed as number of positive cells/mm 2 Results expressed as number of positive cells/mm 2 pMAPK/ERK IR restricted to nucleus pMAPK/ERK IR restricted to nucleus tMAPK/ERK IR located w/in cytoplasmic and somatic compartments tMAPK/ERK IR located w/in cytoplasmic and somatic compartments

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

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

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

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

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

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

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

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

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

Recap Extinction learning induces MAPK/ERK phosphorylation in basolateral amygdala Extinction learning induces MAPK/ERK phosphorylation in basolateral amygdala

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

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

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

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 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 Both groups reach baseline freezing level at end of Late Extinction Suggests extinction of CF was complete Suggests extinction of CF was complete

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

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

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

Summary Extinction of auditory fear condition induces an increase in MAPK/ERK phosphorylation in BLA 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 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 So MAPK/ERK signaling in BLA also necessary for acquisition of fear extinction