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. 2004 May 19;24(20):4796-806.
doi: 10.1523/JNEUROSCI.5654-03.2004.

Brain-derived Neurotrophic Factor and Tyrosine Kinase Receptor B Involvement in Amygdala-Dependent Fear Conditioning

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Free PMC article

Brain-derived Neurotrophic Factor and Tyrosine Kinase Receptor B Involvement in Amygdala-Dependent Fear Conditioning

Lisa M Rattiner et al. J Neurosci. .
Free PMC article

Abstract

Brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine kinase receptor B (TrkB), play a critical role in activity-dependent synaptic plasticity and have been implicated as mediators of hippocampal-dependent learning and memory. The present study is the first to demonstrate a role for BDNF and TrkB in amygdala-dependent learning. Here, the use of Pavlovian fear conditioning as a learning model allows us to examine the concise role of BDNF in the amygdala after a single learning session and within a well understood neural circuit. Using in situ hybridization, mRNA levels of six different trophic factors [BDNF, neurotrophin (NT) 4/5, NGF, NT3, aFGF, and bFGF) were measured at varying time points during the consolidation period after fear conditioning. We found temporally specific changes only in BDNF gene expression in the basolateral amygdala after paired stimuli that supported learning but not after exposure to neutral or aversive stimuli alone. Using Western blotting, we found that the Trk receptor undergoes increased phosphorylation during this consolidation period, suggesting an activation of the receptor subsequent to BDNF release. Furthermore, disruption of neurotrophin signaling with intra-amygdala infusion of the Trk receptor antagonist K252a disrupted acquisition of fear conditioning. To address the specific role of the TrkB receptor, we created a novel lentiviral vector expressing a dominant-negative TrkB isoform (TrkB.T1), which specifically blocked TrkB activation in vitro. In vivo, TrkB.T1 lentivirus blocked fear acquisition without disrupting baseline startle or expression of fear. These data suggest that BDNF signaling through TrkB receptors in the amygdala is required for the acquisition of conditioned fear.

Figures

Figure 1.
Figure 1.
Temporal changes in neurotrophin gene expression after fear conditioning. A, Results of behavioral testing after olfactory fear conditioning. Animals were exposed to odor-shock pairings and tested 48 hr later. Associative odor-shock pairings produced stable fear memories, as assessed by the fear-potentiated startle effect (paired t test; *p < 0.05). Mean startle amplitude (in arbitrary units) on startle-alone and odor-startle test trials and the difference between these two trial types are shown. B, Animals were trained using olfactory fear conditioning and killed 30 min, 2 hr, or 4 hr after training. Relative levels of mRNA expression (in arbitrary units) normalized to control levels are shown for BDNF, NGF, and NT4/5 in the BLA. BDNF mRNA levels rise in the BLA and peak at 2 hr after fear conditioning (t test; *p < 0.05). These levels begin to return to baseline 4 hr after conditioning (ANOVA; post hoc quadratic trend analysis; p < 0.05). Levels of the other trophic factors do not significantly change during this time period. C, Further analysis of the 2 hr time point shows that BDNF mRNA levels increase significantly in the BLA 2 hr after fear conditioning (t test; *p < 0.01), whereas levels of NGF and NT4/5 again do not change. D, In situ hybridization analysis of BDNF mRNA in the amygdala. Da, Region within the temporal lobe analyzed for BDNF mRNA hybridization density. Db, Schematic diagram from Paxinos and Watson (1997) of regions examined. Dc, Dd, Magnified images are shown of amygdala sections that have been hybridized with 35S-labeled antisense riboprobe and exposed to autodradiography film. BDNF mRNA levels rise in the BLA in animals after odor-shock pairings (Dd) but not in animals exposed to context only (Dc).
Figure 2.
Figure 2.
Changes in BDNF gene expression after light-shock fear conditioning. A, Results of behavioral testing after the light-alone, shock-alone, and light-shock training. Light-shock pairings produced stable fear memories in the light-shock associative group, as assessed by the fear-potentiated startle effect (paired t test; *p < 0.05). Light-alone and shock-alone control groups show no appreciable difference between startle in the absence or presence of the light. The difference scores for the light-shock group are significantly different from the difference scores for the light-alone or shock-alone controls (ANOVA; **p < 0.05). B, In situ hybridization analyses of BDNF in the BLA after light-alone, shock-alone, or light-shock presentation. BDNF mRNA expression in the BLA changed significantly across the three groups (ANOVA; *p < 0.05).
Figure 3.
Figure 3.
Anatomical specificity of BDNF signal. In situ hybridization analyses of BDNF in the MeA, VPM, and hippocampus (DG, CA1, CA3) 2 hr after light-shock, light-alone, and shock-alone exposure. There were no changes in BDNF mRNA in any of these regions across the three groups.
Figure 4.
Figure 4.
Changes in levels of phosphorylated-Trk receptor protein in the amygdala after fear conditioning. A, Immunohistochemical analysis of TrkB immunoreactivity (high power) in the amygdala. B, Representative Western blots of amygdala samples probed with phosphorylated-Trk Ab. Samples were taken from animals that had been exposed to light presentations alone, shock-alone, or light-shock pairings.
Figure 5.
Figure 5.
Effect of K252a, a nonselective Trk receptor antagonist, on acquisition of fear conditioning as assessed by fear-potentiated startle. Filled bars represent startle response to startle-alone trials, white bars represent startle response to light-startle trials, and hatched bars represent difference scores. A, Rats received bilateral intra-amygdala infusions of ACSF (vehicle) or K252a immediately before and immediately after light-shock fear conditioning (training) and were tested 48 hr later. Vehicle-infused rats showed fear-potentiated startle during testing (t test; *p < 0.05) that was not seen in the K252a-infused rats. Mean difference scores of K252a-infused animals were significantly lower than difference scores of vehicle-infused animals (t test; **p < 0.05). This difference was not accounted for by a change on the startle-alone trials because there was no significant difference between groups on the startle-alone trials. B, Deficits produced by pretraining infusions of K252a did not impair future acquisition of fear learning. Rats that had previously received vehicle or K252a were retrained without infusions 10 d after the initial training and testing sessions and retested 48 hr after retraining. An ANOVA with repeated measures showed a significant fear-potentiated startle effect by trial type (ANOVA; p ≤ 0.05) but no trial-by-group interaction; thus the previously observed deficits could not be attributed to permanent amygdala damage. C, K252a demonstrates anatomical specificity. Mean difference scores of animals receiving intra-amygdala infusions of K252a or vehicle and animals receiving K252 infusions outside of the amygdala. Rats that received K252a infusions in areas outside of the amygdala showed levels of fear-potentiated startle equivalent to those of animals infused with ACSF.
Figure 6.
Figure 6.
In vitro analysis of lenti-TrkB.T1. A, Schematic representation of lentiviral vector constructs. Lenti-TrkB.T1 and lenti-GFP vectors were constructed downstream of the CMV promoter. The lenti-TrkB.T1 vector construct contained an HA-tagged TrkB.T1 gene. The lenti-GFP construct contained a reporter gene encoding GFP. LTR, Long terminal repeat; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Ba, PC12 cells transfected with TrkB.FL after exposure to BDNF. Bb, PC12 cells cotransfected with TrkB.FL plus TrkB.T1 after exposure to BDNF. Bc, Immunocytochemical analysis of HA immunoreactivity in HEK293T cells infected with lenti-TrkB.T1 virus.
Figure 7.
Figure 7.
Lentiviral expression in vivo. Animals were infected with lentiviral vectors and killed 3 weeks later. a, d, Coronal sections through the amygdala showing lenti-GFP or lenti-TrkB.T1 virus infection as detected by immunohistochemistry using antibodies against GFP or HA. b, e, Cresyl violet staining of parallel sections showing the absence of neuronal damage in infected regions. Arrows indicate regions infected with lentivirus. c, f, High-powered magnification (40×) of other regions with lower-density infection, demonstrating that lentivirus infects individual neurons. CeA, Central amygdala; LA, lateral amygdala; BLA, basolateral amygdala.
Figure 8.
Figure 8.
Effect of lentiviral induced expression of TrkB.T1 in the amygdala on fear conditioning as assessed by fear-potentiated startle. A, Outline of behavioral paradigms for acquisition and expression experiments. B, Filled bars represent startle response to startle-alone trials, white bars represent startle response to light-startle trials, and hatched bars represent difference scores. Mean startle amplitude ± SEM on startle-alone trials, light-startle trials, and the difference between the two are shown for animals receiving lentivirus infusion into the amygdala. Mean difference scores of lenti-TrkB.T1-infused animals were significantly lower than difference scores of lenti-GFP-infused animals (t test; *p < 0.05). C, Anatomical specificity of TrkB.T1. Mean startle amplitude ± SEM on startle-alone trials, light-startle trials, and the difference between the two are shown for animals receiving infusion of TrkB.T1 into the amygdala or areas outside the amygdala (missed placements). Mean difference scores for missed placement animals were significantly higher than difference scores of animals infused with lenti-TrkB.T1 into the amygdala (t test; *p < 0.01), demonstrating that the virus must be present within the amygdala to impair fear learning. D, Effect of amygdala infection with lenti-TrkB.T1 on the expression of fear-potentiated startle. Animals were trained, infected, and tested as in A. When TrkB.T1 virus is present during expression, but not acquisition, of fear learning, there is no difference between fear-potentiated startle with lenti-TrkB.T1 animals compared with lenti-GFP animals. An ANOVA with repeated measures showed a significant fear-potentiated startle effect-by-trial type but no trial-by-group interaction (ANOVA; p ≤ 0.05).

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