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

53
Extinction of auditory Extinction of auditory fear conditioning fear conditioning requires MAPK/ERK requires MAPK/ERK activation in the activation in the basolateral amygdala basolateral amygdala Herry et al Herry et al European Journal of European Journal of Neuroscience, Vol. 24 p 261- Neuroscience, Vol. 24 p 261- 269, 2006 269, 2006

description

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 - PowerPoint PPT Presentation

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

Page 1: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Extinction of auditory fear Extinction of auditory fear conditioning requires conditioning requires

MAPK/ERK activation in the MAPK/ERK activation in the basolateral amygdalabasolateral amygdala

Herry et alHerry et alEuropean Journal of Neuroscience, European Journal of Neuroscience,

Vol. 24 p 261-269, 2006 Vol. 24 p 261-269, 2006

Page 2: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

IntroductionIntroduction ExtinctionExtinction

Form of new learning in which the original Form of new learning in which the original memory acquired thru association of a CS with memory acquired thru association of a CS with US is inhibitedUS is inhibited

Occurs after repeated exposures to CS aloneOccurs after repeated exposures to CS alone Auditory fear conditioning and extinction of Auditory fear conditioning and extinction of

fear conditioning differfear conditioning differ Extinction memories are context dependent and Extinction memories are context dependent and

labile giving rise to spontaneous recovery labile giving rise to spontaneous recovery (Quirk, 2002)(Quirk, 2002)

Page 3: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

IntroIntro Neuronal mechanisms underlying Neuronal mechanisms underlying

acquisition of extinction still unclearacquisition of extinction still unclear Emphasis on BLAEmphasis on BLA

Previous studies Previous studies Intra-BLA injection of AP5,NMDA Intra-BLA injection of AP5,NMDA

antagonistantagonist Extinction prevented (Falls et al 1992)Extinction prevented (Falls et al 1992)

Page 4: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

IntroIntro In contrastIn contrast

Acquisition of extinction of auditory fear Acquisition of extinction of auditory fear conditioning unaffected by injection of conditioning unaffected by injection of NMDAr antagonist (Santini et al 2001)NMDAr antagonist (Santini et al 2001)

Suggested that acquisition of extinction Suggested that acquisition of extinction and consolidation of extinction rely on and consolidation of extinction rely on two separate molecular mechanismstwo separate molecular mechanisms

Page 5: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

IntroIntro NMDAr can trigger signaling NMDAr can trigger signaling

pathways like MAPK/ERKpathways like MAPK/ERK This pathway implicated in consolidation This pathway implicated in consolidation

of auditory and contextual fear of auditory and contextual fear conditioning (Trifilieff et al 2006)conditioning (Trifilieff et al 2006)

Page 6: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

GoalsGoals Analyze by immunohistochemistry Analyze by immunohistochemistry

temporal activation of MAPK/ERK in temporal activation of MAPK/ERK in BLA following acquisition of BLA following acquisition of extinctionextinction

Directly test if inhibition of MAPK/ERK Directly test if inhibition of MAPK/ERK pathway b/f extinction training pathway b/f extinction training prevents acquisition of extinction of prevents acquisition of extinction of auditory fear conditioningauditory fear conditioning

Page 7: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Adult male mice individually housed Adult male mice individually housed

in plastic cages w in plastic cages w ad libitumad libitum food food accessaccess 12 h light/dark12 h light/dark

Mice handled gently for 2-3 min/day Mice handled gently for 2-3 min/day during 5 days to minimize during 5 days to minimize nonspecific stressnonspecific stress

Page 8: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods SurgerySurgery

Anaesthetized w/ isoflurane in OAnaesthetized w/ isoflurane in O2 2 Secured in sterotaxic frame and Secured in sterotaxic frame and

bilaterally implanted w/ 24 gauge bilaterally implanted w/ 24 gauge cannula aimed at BLAcannula aimed at BLA

Cannula fixed in placeCannula fixed in place Mice recovered for 1 weekMice recovered for 1 week

Page 9: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Drug infusionDrug infusion

MEK inhibitor, U0126MEK inhibitor, U0126 Dissolved in DMSO and storedDissolved in DMSO and stored

Animals given bilateral intra-BLA Animals given bilateral intra-BLA injections of 0.3 injections of 0.3 µL per site or 0.6µL µL per site or 0.6µL both in 2% DMSO in artificial CSFboth in 2% DMSO in artificial CSF Done in freely moving mice at rate Done in freely moving mice at rate

0.4µL/min0.4µL/min

Page 10: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

•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

Page 11: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Behavioral Procedures Behavioral Procedures

Conditioning ChamberConditioning Chamber Grey plastic cylinder (15.5cm x 14cm)Grey plastic cylinder (15.5cm x 14cm) Shock grid floorShock grid floor Place in plexiglas boxPlace in plexiglas box Speaker w 2.5kHz, 80dBSpeaker w 2.5kHz, 80dB 1s, 0.9mA foot-shock1s, 0.9mA foot-shock Washed w/ 70% ethanolWashed w/ 70% ethanol

Page 12: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Behavioral ProceduresBehavioral Procedures

Extinction trainingExtinction training Grey plastic cylinder removedGrey plastic cylinder removed Grey plastic floor replaced gridGrey plastic floor replaced grid Washed w/ 1% acetic acid before and after Washed w/ 1% acetic acid before and after

sessionsession To maximize discrimination b/w two To maximize discrimination b/w two

contexts, light intensity reduced during contexts, light intensity reduced during fear conditioningfear conditioning

Page 13: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Behavioral proceduresBehavioral procedures

Parallel measurements to score freezingParallel measurements to score freezing First, classical time-sampling procedure First, classical time-sampling procedure

during which blind experimenter scored during which blind experimenter scored freezing/not freezing every 2sfreezing/not freezing every 2s

Second, compared values to automatic Second, compared values to automatic infrared beam detection systeminfrared beam detection system

Two values 95% identical, were Two values 95% identical, were averaged for statistical analysesaveraged for statistical analyses

Page 14: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Fear conditioning/extinction trainingFear conditioning/extinction training

Mice conditioned to acquire fear in Mice conditioned to acquire fear in response to 30s CS tone paired w/ 1s response to 30s CS tone paired w/ 1s foot-shockfoot-shock

5 CS-US pairings, inter-trial interval 20-5 CS-US pairings, inter-trial interval 20-180 s180 s

For Immunohistochem experiements, For Immunohistochem experiements, CS-US mice divided into 5 groupsCS-US mice divided into 5 groups

Page 15: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Early ExtEarly Ext

Extinction 5 h after conditioningExtinction 5 h after conditioning Late ExtLate Ext

Additional extinction 24 h laterAdditional extinction 24 h later Killed at 3 time pointsKilled at 3 time points

15min, 1 and 6h after Late extinction15min, 1 and 6h after Late extinction

Page 16: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods 2 controls2 controls

No Early ExtNo Early Ext Killed 5 h after conditioning, just before Killed 5 h after conditioning, just before

early extinction trainingearly extinction training No Late ExtinctionNo Late Extinction

Submitted to early extinction, killed 24h Submitted to early extinction, killed 24h later just before late extinction traininglater just before late extinction training

Page 17: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods CSCS

To control for possible contextual effect on To control for possible contextual effect on MAPK/ERKMAPK/ERK

Submitted to extinction context alone during both Submitted to extinction context alone during both sessionssessions

Killed 1 h after late extinctionKilled 1 h after late extinction NaiveNaive

Neither submitted to fear conditioning nor Neither submitted to fear conditioning nor extinctionextinction

Killed independentlyKilled independently

Page 18: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods For pharmacological experimentFor pharmacological experiment

Conditioned mice received bilateral intra-Conditioned mice received bilateral intra-BLA injection of either MEK inhibitor BLA injection of either MEK inhibitor (U0126; n=16) or DMSO (n=15)(U0126; n=16) or DMSO (n=15)

Ten min before early or late extinctionTen min before early or late extinction Long term effect of U0126 on fear Long term effect of U0126 on fear

expression evaluated by submitting expression evaluated by submitting injected mice to final test consisting of 4 injected mice to final test consisting of 4 CS presentationsCS presentations

Page 19: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 20: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods HistologyHistology

Mice terminally sampled at completion Mice terminally sampled at completion of experimentof experiment

At different timesAt different times Brains removed, sectioned and stained Brains removed, sectioned and stained

to verify cannulae placementsto verify cannulae placements

Page 21: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods ImmunohistochemistryImmunohistochemistry

At appropriate time (1A) mice submitted At appropriate time (1A) mice submitted to immunohistochemical experiments to immunohistochemical experiments terminally sampledterminally sampled

Brains removed, section and stained to Brains removed, section and stained to quantify phospho-MAPK/ERK or total-quantify phospho-MAPK/ERK or total-MAPK/ERK immunoreactivityMAPK/ERK immunoreactivity

Page 22: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Data analysisData analysis

P-MAPK/ERK or total-MAPK/ERK P-MAPK/ERK or total-MAPK/ERK immunoreactivity carried out using 3 immunoreactivity carried out using 3 coronal serial sectionscoronal serial sections

Number of positive cells quantified using Number of positive cells quantified using computerized image analysis systemcomputerized image analysis system

BLABLA Two values quantified in each mouse w/in Two values quantified in each mouse w/in

predefined boundaries outlining BLApredefined boundaries outlining BLA

Page 23: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Materials and MethodsMaterials and Methods Data analysisData analysis

Results expressed as number of positive Results expressed as number of positive cells/mmcells/mm22

pMAPK/ERK IR restricted to nucleuspMAPK/ERK IR restricted to nucleus tMAPK/ERK IR located w/in cytoplasmic tMAPK/ERK IR located w/in cytoplasmic

and somatic compartmentsand somatic compartments

Page 24: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Preconditioning period CS and CS-US Preconditioning period CS and CS-US

mice showed similar freezing levelsmice showed similar freezing levels Freezing remained low in CS Freezing remained low in CS

CS-US showed progressive increaseCS-US showed progressive increase From 10-50% freezingFrom 10-50% freezing

Page 25: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 26: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Early Extinction (5 hours later)Early Extinction (5 hours later)

Pre-CS Pre-CS Did not display contextual fear Did not display contextual fear

generalization in both groupsgeneralization in both groups CS-alone induced high level of freezing CS-alone induced high level of freezing

in CS-US but not in CS groupin CS-US but not in CS group

Page 27: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 28: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Late Extinction (24h later)Late Extinction (24h later)

Induced rapid decrease of freezing Induced rapid decrease of freezing response in mice of CS-US groupresponse in mice of CS-US group

Shows that protocol promotes Shows that protocol promotes complete extinction of conditioned complete extinction of conditioned fear responses at end of Late fear responses at end of Late Extinction sessionExtinction session

Page 29: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 30: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Levels of Levels of

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

Page 31: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults 60 min after Late Extinction60 min after Late Extinction

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

Page 32: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 33: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Then compared results of pMAPK/ERK-IR Then compared results of pMAPK/ERK-IR

for Early-Ext and Late-Ext groupsfor Early-Ext and Late-Ext groups Found significant increase in Late-Ext groupFound significant increase in Late-Ext group Indicates acquisition of extinction during Indicates acquisition of extinction during

Late-Extinction was associated with an Late-Extinction was associated with an increase of pMAPK/ERK-IRincrease of pMAPK/ERK-IR

Also compared results against Ctx, CS, Also compared results against Ctx, CS, and Naiveand Naive

Page 34: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 35: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Immunohistochemical analysis of Immunohistochemical analysis of

total MAPK/ERK in subset mice from total MAPK/ERK in subset mice from Late-Ext and No Late-Ext groupsLate-Ext and No Late-Ext groups

Results show that tMAPK/ERK was Results show that tMAPK/ERK was not significantly different b/w groupsnot significantly different b/w groups

Page 36: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 37: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Next, examined pMAPK/ERK-IR in Next, examined pMAPK/ERK-IR in

mice killed 15, 60, 360 min in Late-mice killed 15, 60, 360 min in Late-Ext group to No Late-Ext Ext group to No Late-Ext

Results show significant activation of Results show significant activation of pMAPK/ERK-IR at each time point pMAPK/ERK-IR at each time point examined after Late Extinction w/ examined after Late Extinction w/ max at 60 minmax at 60 min

Page 38: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 39: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

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

Page 40: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

RecapRecap Extinction learning induces Extinction learning induces

MAPK/ERK phosphorylation in MAPK/ERK phosphorylation in basolateral amygdalabasolateral amygdala

Page 41: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Next, tried to determine if MAPK/ERK Next, tried to determine if MAPK/ERK

signaling required for acquisition of signaling required for acquisition of extinctionextinction

Used MEK inhibitor U0126Used MEK inhibitor U0126 Injected it into one group 10 min before Injected it into one group 10 min before

Early or Late ExtinctionEarly or Late Extinction Control group injected with DMSOControl group injected with DMSO

Page 42: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Mice underwent fear conditioning Mice underwent fear conditioning

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

Page 43: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 44: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults After first CS-US paring, both groups After first CS-US paring, both groups

of mice (u0126 and DMSO) displayed of mice (u0126 and DMSO) displayed progressive increase in freezingprogressive increase in freezing

Direct comparisons revealed no Direct comparisons revealed no significant differences b/w u0126 and significant differences b/w u0126 and DMSO groups through whole sessionDMSO groups through whole session

Page 45: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 46: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults U0126 injection prior to Early U0126 injection prior to Early

Extinction did not induce notable Extinction did not induce notable changes in freezing level during changes in freezing level during Early, Late Extinction, or post-Early, Late Extinction, or post-extinction in comparison to controlextinction in comparison to control

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

Page 47: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 48: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults Mice from u0126 and DMSO groups Mice from u0126 and DMSO groups

injected before Late Extinction again injected before Late Extinction again no difference during pre-conditioning no difference during pre-conditioning period or during the condition session period or during the condition session Progressive increase in freezing levelsProgressive increase in freezing levels

During Early Extinction mice showed During Early Extinction mice showed elevated levels againelevated levels again

Page 49: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 50: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

ResultsResults When put back in context 24h later When put back in context 24h later

mice, during first block of testing both mice, during first block of testing both groups showed similar levels of groups showed similar levels of freezingfreezing

But significant decrease noted in the But significant decrease noted in the control group in later blockscontrol group in later blocks

Mice tested final time 24h after Late Mice tested final time 24h after Late Extinction without u0126Extinction without u0126 Showed extinction didn’t occurShowed extinction didn’t occur

Page 51: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala
Page 52: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

SummarySummary All together results indicate that All together results indicate that

inhibition of MAPK/ERK signaling in inhibition of MAPK/ERK signaling in BLA before Late but not Early BLA before Late but not Early Extinction prevents acquisition of Extinction prevents acquisition of extinctionextinction

Page 53: Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

SummarySummary Extinction of auditory fear condition Extinction of auditory fear condition

induces an increase in MAPK/ERK induces an increase in MAPK/ERK phosphorylation in BLAphosphorylation in BLA

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

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