Introduction

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.

3. 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.

5. 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.

6. 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).

8. 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

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

10. 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

11. 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.

12. 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

13. 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.

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

15. 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.

16. 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

17. 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.

18. 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

19. 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

20. 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

21. 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

22. Materials and Methods • Statistical analyses: • Mean startle differences were analyzed by ANOVA

23. 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

24. 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

25. 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

26. 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.

27. 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

28. 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

29. 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

30. Materials and Methods • Lentiviral vector constructs

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

32. 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

33. 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.

34. 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.

36. 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.

37. 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.

38. 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.

39. Results

40. 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.

41. 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

42. Results

43. 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.

44. 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

46. 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

48. 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.

49. 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.