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

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.

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.

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.

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.

Introduction

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).

Materials and Methods

• 161 male Sprague Dawly rats that weighed between 300-400 grams

• 12hr light/dark cycle (lights on at 8:00 a.m.)

• Housed in 45x24x20 cm polycarbonate cage before surgery

• 4 rats per cage

Materials and Methods

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

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

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.

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

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.

Materials and Methods

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

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.

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

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.

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

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

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

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

Materials and Methods

• Statistical analyses:– Mean startle differences were analyzed by

ANOVA

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

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

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

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.

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

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

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

Materials and Methods

• Lentiviral vector constructs

Materials and Methods

• Viral stock:– Generated by transient cotransfection of

expression plasmid, pseudotyping construct, and packaging construct.

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

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.

Results

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.

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.

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.

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.

Results

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.

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

Results

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.

Results

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

Results

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

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.

Results

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.

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.

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.