Brain-Derived Neurotrophic Factor and Tyrosine Kinase Receptor B Involvement in Amygdala-Dependent Fear Conditioning Lisa M. Rattiner, Michael Davis, Christopher T. French, and Kerry J. Ressler

1
Brain-Derived Neurotrophic Factor and Tyrosine
Kinase Receptor B Involvement in
Amygdala-Dependent Fear ConditioningLisa M.
Rattiner, Michael Davis, Christopher T. French,
and Kerry J. Ressler
2
Introduction

• Brain-derived neurotrophic factor (BDNF) is a
  part of regulating neuronal structure and
  function in the developing and adult CNS.
• BDNF has been shown to be regulated by neuronal
  activity and acutely modify synaptic efficiency
  and induce changes in synaptic morphology.

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Introduction

• The role of BDNF in learning and memory has been
  focused almost exclusively on hippocampal
  long-term potentiation.
• But it has been difficult to demonstrate because
  contributions of some circuits are unclear and
  researchers are limited by their pharmological
  tools.

4
Introduction

• The tyrosine kinase receptors are able to be
  studied by using a dominant-negative truncated
  recombinant protein.
• By removal of cytoplasmic tail of TrkB receptor
  inhibits normal TrkB functions.
• Use virally mediated dominant-negative inhibition
  of TrkB to evaluate role of TrkB in acquisition
  and consolidation of fear memory.

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Introduction

• Circuitry of the amygdala and sensory components
  are tightly coupled to the expression of learned
  fear response makes it an excellent model.
• But, the involvement of trophic factors in
  amygdala-dependent learning and memory has yet to
  be studied.

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Introduction
7
Introduction

• Will be determining role of BDNF in
  amygdala-dependent fear conditioning.
• Findings suggest production of BDNF mRNA is
  amygdala is regulated by neuronal activity during
  fear conditioning.
• Fear conditioning also results in activation of
  Trk receptor in the amygdala.
• Trk receptor blocked with K252a or viral
  expression of dominant-negative TrkB receptor
  impairs learning and memory assessed by
  fear-potentiated startle (FPS).

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Materials and Methods

• 161 male Sprague Dawly rats that weighed between
  300-400 grams
• 12hr light/dark cycle (lights on at 800 a.m.)
• Housed in 45x24x20 cm polycarbonate cage before
  surgery
• 4 rats per cage

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Materials and Methods

• After surgery housed in 20x19x24 cm cages and
  individually in accordance to procedures used at
  Emory University

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Materials and Methods

• Fear conditioning apparatus
• Tested and trained in 4 identical 8x15x15 cm
  Plexiglas and wire mesh cages.
• Cage movement measured by accelerometer
• Startle amplitude defined as maximal peak-to-peak
  accelerometer voltage in first 200 sec.
• Background white noise of 60dB wideband

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Materials and Methods

• Startle response evoked by 50msec 95 dB delivered
  through white noise speakers
• Unconditioned Stimulus (US) was 0.5 sec, 0.4 mA
  foot-shock through cage floor bars.
• Visual conditioned stimulus was 4 sec. Light by a
  8 W bulb behind the cage.
• The odor CS was 5 amyl acetate diluted in
  propylene glycol and delivered for 4 sec through
  a olfactometer.

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Materials and Method

• Behavioral procedures
• Acclimation handled and placed in
  training/testing chamber 5 days before
  conditioning
• Day 3 of pre-exposure measured baseline startle
  with 30 startle stimuli at 30 sec intervals
• Were divided up in groups with equivalent mean
  startle amplitudes

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Methods and Materials

• Experiment 1
• 10 odor-shock pairings were given over 40 min.
• Shock delivered in last 0.5 sec with last 4 sec
  odor stimulus.
• 4 rats were kept for behavioral testing
• 6 rats were killed a varying times afterward
• Context control group placed in chamber for 40
  min with no stimulus and killed 2 hrs after.

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Materials and Methods

• 2nd group was trained with 15 light-shock
  pairings and killed 2 hours after training or
  right from their home cage

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Materials and Methods

• Experiment 2
• Light shock pairings given an avg of 2.5 min
  apart for a 40 min training period.
• Shock (US) delivered during last 0.5 sec and
  terminated with the 4 sec light stimulus
• Light alone group had 15, 4 sec light stimuli
  over 40 min.

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Materials and Methods

• Shock alone group had 15, 0.5sec shocks with 2.5
  min. between shocks for 40 min.
• Animals from all groups were killed 2 hrs later
  and underwent a Western Blot analysis
• 12 were kept for behavioral testing

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Materials and Methods

• K252a Experiment
• Rats were infused with K252a Trk receptor
  antagonist.
• Were placed in chambers and after 5 min presented
  with 15 light-shock pairings with an average of 4
  min between.

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Materials and Methods

• Lentivirus acquisition experiment
• 36 animals infused with lentivirus and 12 days
  later placed in chambers to receive 15
  light-shock pairings for 40 min
• Repeated 24 hours later
• Returned home and waited for behavioral testing

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Materials and Methods

• Performance Experiment with lentivirus
• 23 rats cannulated and give 12 days to recover
• Trained with 15 light-shock pairings for 2 days
• In last 2 min were given a fear-potentiated
  startle test of 5 sound bursts coupled with light
  and 5 in the dark

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Materials and Methods

• Calculated the mean startle amplitude and
  separated into groups
• 4 days later were given injections of
  lenti-TrkB.T1 or lenti-GFP through implanted
  cannulas and recovered for 9 days to allow full
  infection

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Materials and Methods

• Behavioral testing
• 5 min of acclimatization
• 30 startle stimuli of 95 dB sound at 30 sec
  intervals
• Followed by 30 startle-alone trials and 30
  intermixed CS-startle test trials

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Materials and Methods

• Statistical analyses
• Mean startle differences were analyzed by ANOVA

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Methods and Materials

• Surgery and infusions
• Rats anesthetized with sodium pentobarbital and
  had 22 gauge cannulas inserted bilaterally into
  the basolateral amygdala (BLA).
• Dummy cannulas were inserted into each guide
• Allowed 10 days to recover

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Materials and Methods

• Before and after training, rats were infused with
  either K252a diluted in artificial CSF or
  artificial CSF mixed with DMSO
• For lentivirus
• Received 2 micro liters of lenti-TrkB.T1 or
  lenti-GFP bilaterally and recover for 9 days

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Materials and Methods

• Riboprobes
• Used full-length clones from the NIH IMAGE
  database.
• In situ hybridization performed with antisense
  riboprobes for sequence verification of clones.
• Rats killed with chloral hydrate overdose after
  fear conditioning and perfused with 4
  paraformaldehyde in PBS

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Materials and Methods

• Brains were fixed overnight and frozen with dry
  ice to be sectioned.
• Each slide was to contain sections of the
  anterior commissure, anterior amygdala, and
  posterior amygdala.
• Each slide was hybridized with labeled
  riboprobes, placed against film and densities
  were quantitated.

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Materials and Methods

• Immunocytochemistry and immunoblotting
• Brain sections were blocked with normal goat
  serum, bovine serum albumin and Triton X-100
• Incubated in TrkB rabbit polyclonal antibody
• Were then washed with secondary anti-rabbit
  biotinylated antibody
• Visualized with diaminobenzidine peroxidase
  staining

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Materials and Methods

• Rat amygdalas were frozen and homogenized with
  10mM HEPES, 0.5mM EDTA and a protease inhibitor
  mixture
• Samples separated by SDS-PAGE
• Underwent Western Blot analysis

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Materials and Methods

• Recombinant lentiviral vectors
• From HIV backbone
• Lenti-GFP is the pCMO2 vector with a
  cytomegalovirus promoter
• Lenti-TrkB.T1 are from truncated TrkB with BamHI
  inserted on 5 end and a 9 amino acid
  hemagglutinin (HA) eptitope tag on 3 end

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Materials and Methods

• Lentiviral vector constructs

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Materials and Methods

• Viral stock
• Generated by transient cotransfection of
  expression plasmid, pseudotyping construct, and
  packaging construct.

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Results

• Initially examined expression of 6 different
  trophic factors at different points after
  olfactory fear conditioning to determine
  involvement in fear conditioning.
• BDNF, neurotrophin 4/5, NGF, NT3, aFGF and bFGF

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Results

• Received odor-shock pairings or no new stimuli
• Killed at 30 min, 2 hrs, and 4 hrs or tested 48
  hrs later for fear-potentiated startle.
• All context control animals killed at 2 hrs after
  training, because previous experiments suggest 2
  hrs is optimal to observe changes in expression.

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Results
35
Results

• Animals with odor-shock pairing had significant
  fear-potentiated startle
• Expression of the 6 trophic factor genes were
  examined at the 3 points after fear conditioning
  with control group.
• Only BDNF mRNA showed activity-dependent changes
  after fear conditioning.

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Results

• BDNF peaked in the BLA 2 hours after
  conditioning.
• Returned to baseline 4 hours after conditioning.
• Levels of the other factors did not change
  significantly.
• But levels of NGF increased slightly 30 min after
  conditioning in the amygdala.

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Results

• To reinforce the 2 hour optimal time, used larger
  number of rats and performed same experiment.
• Had same results.
• Suggests temporally specific changes in BDNF gene
  expression.

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Results

• Another experiment
• 15 light-shock pairing, light only, or shock only
• Were either killed 2 hours after training or kept
  for 24 hr to test fear-potentiated startle.
• Light-shock had significant startle.
• Light or shock alone had no difference in startle.

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Results
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Results

• Animals that were killed, hybridized with probe
  to determine levels of mRNA.
• Levels suggest that BDNF gene expression shows
  activity-dependent changes after fear
  conditioning, but not after CS alone or US alone.

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Results

• Examined other regions to see if BDNF mRNA is
  selective for BLA.
• Medial nucleus of amygdala
• Ventral posteromedial nucleus of thalamus
• Dorsal hippocampus
• Found no changes in BDNF levels

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Results
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Results

• Examine receptor.
• Large number of TrkB-immunoreactive neurons in
  the BLA.
• Used Western Blot and found significantly
  elevated levels of phospho-Trk receptors after
  light-shock pairing only.

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Results
45
Results

• K252a tyrosine kinase inhibitor before and after
  training.
• K252a infused animals showed significantly lower
  startle amplitudes
• 10 days later with no K252a, same animals showed
  return of normal startle amplitude.
• Missed placed cannulas showed effects were seen
  only when injected directly in BLA

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Results
47
Results

• Lentivirus experiment
• Results showed TrkB.T1 has dominant-negative
  effect on BDNF-mediated signaling.
• Able to visualize HA-epitope tagged TrkB.T1 cells
  with HA antibody

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Results

• Expression of TrkB.T1 resulted in impairment of
  fear learning.
• Tyrosine kinase receptors needed for fear
  learning in the BLA.
• Experiment where rats were injected after
  training showed fear-potentiated startle.
• TrkB.T1 disrupts acquisition and not during
  expression.

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Results
50
Discussion

• BDNF and TrkB signaling in amygdala during fear
  conditioning.
• NGF, NT4/5, NT3, aFGF, and bFGF mRNAs do not
  increase in amygdala.
• Phosphorylated Trk receptors increase levels
  after fear learning
• K252a impairs fear conditioning.

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Discussion

• Dominant-negative TrkB receptor impairs
  acquisition, but not expression.
• TrkB receptor is needed for acquisition of fear
  memory, but not necessary for normal amygdala
  function or expression.
• Also BDNF activation of TrkB has been shown to
  increase NMDA function.

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Discussion

• Recently, BDNF activation of TrkB shown to
  mediate translocation of activated MAPK to the
  nucleus.
• And BDNF may be required for activation of cAMP
  response element-binding protein (CREB) in the
  amygdala that is required for learning.