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 - PowerPoint PPT PresentationTRANSCRIPT
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
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)
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
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
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
•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 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
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
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
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
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
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
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
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
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
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
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
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
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-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
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 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
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 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