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 al

European Journal of Neuroscience, European Journal of Neuroscience, Vol. 24 p 261-269, 2006 Vol. 24 p 261-269, 2006

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 Auditory fear conditioning and extinction of fear conditioning differof fear conditioning differ Extinction memories are context dependent Extinction memories are context dependent

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

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)

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

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)

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

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


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


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

•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

http://en.wikipedia.org/wiki/Image:MAPKpathway.png


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


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

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


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


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


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


CSCS To control for possible contextual effect on To control for possible contextual effect on

MAPK/ERKMAPK/ERK Submitted to extinction context alone during Submitted to extinction context alone during

both sessionsboth sessions 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


For pharmacological experimentFor pharmacological experiment Conditioned mice received bilateral Conditioned mice received bilateral

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


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


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


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


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

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

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

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

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

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

ResultsResults

Then compared results of pMAPK/ERK-Then compared results of pMAPK/ERK-IR for Early-Ext and Late-Ext groupsIR for Early-Ext and Late-Ext groups Found significant increase in Late-Ext Found significant increase in Late-Ext

groupgroup 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

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

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

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

RecapRecap

Extinction learning induces Extinction learning induces MAPK/ERK phosphorylation in MAPK/ERK phosphorylation in basolateral amygdalabasolateral 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

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

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

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

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 period or during the condition session 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

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

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

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 pathway in BLA prevents acquisition of extinction memoryof extinction 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

extinction of auditory fear conditioning requires mapk/erk activation in the basolateral amygdala...

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