TrkB modulates fear learning and amygdalar synaptic plasticity by specific docking sites

Abstract

Understanding the modulation of the neural circuitry of fear is clearly one of the most important aims in neurobiology. Protein phosphorylation in response to external stimuli is considered a major mechanism underlying dynamic changes in neural circuitry. TrkB (Ntrk2) neurotrophin receptor tyrosine kinase potently modulates synaptic plasticity and activates signal transduction pathways mainly through two phosphorylation sites [Y515/Shc site; Y816/PLCgamma (phospholipase Cgamma) site]. To identify the molecular pathways required for fear learning and amygdalar synaptic plasticity downstream of TrkB, we used highly defined genetic mouse models carrying single point mutations at one of these two sites (Y515F or Y816F) to examine the physiological relevance of pathways activated through these sites for pavlovian fear conditioning (FC), as well as for synaptic plasticity as measured by field recordings obtained from neurons of different amygdala nuclei. We show that a Y816F point mutation impairs acquisition of FC, amygdalar synaptic plasticity, and CaMKII signaling at synapses. In contrast, a Y515F point mutation affects consolidation but not acquisition of FC to tone, and also alters AKT signaling. Thus, TrkB receptors modulate specific phases of fear learning and amygdalar synaptic plasticity through two main phosphorylation docking sites.

Figures

Figure 1.

Associative CS–US pairing is impaired in trkBPLC/+ mutants but normal in trkBSHC/+ mutants. A, The percentage of freezing of control (trkBWT/+) and mutant (trkBPLC/+) mice from a typical training trial. Whereas the control mice (trkBWT/+) showed normal acquisition during the training phase, the trkBPLC/+ mutants showed impaired or highly reduced CS–US association (ANOVA; p < 0.001). B, C, The control (trkBWT/+) and the mutant (trkBPLC/+) group of mice were tested 24 h later for contextual fear conditioning (B) followed by cued fear conditioning (C). D, Freezing of trkBSHC/+ mutants during the acquisition phase was similar to that of control mice (trkB+/+). E, F, Twenty-four hours after conditioning, both groups of mice were tested first for contextual fear conditioning (E) and then for cued fear conditioning (F). n = number of mice per group. Bars represent mean ± SEM. **Statistically significant (see Results), p < 0.001; ns, not significant; ITI, intertrial interval; Hb, habituation.

Figure 2.

Synaptic plasticity at the LA–BL synapses is impaired in trkBPLC/+ but not in trkBSHC/+ mutants. A, Schematic representation of stimulating and recording electrodes. B, C, Symbols represent average responses plotted every 2 min. After 20 min of control recordings, an HFS train was presented, marked by the arrow. Data are represented as the mean ± SEM. SLA, Stimulating (lateral amygdala); RBL, recording (basolateral amygdala). B, Normal E-LTP in trkBSHC/+ point mutant mice compared with control trkB+/+ mice (p > 0.1). The number of slices analyzed were five for control mice (trkB+/+) and eight for trkBSHC/+ mice. C, E-LTP is abolished in trkBPLC/+ mice. The difference between trkBPLC/+ mutants and control mice (trkBWT/+) is statistically significant (p < 0.01). Traces show EPSP before (1) and 60 min after (2) LTP protocol was applied. Calibration: 5 ms, 0.5 mV. The number of slices analyzed were 6 for control mice (trkBWT/+) and 10 for trkBPLC/+ mice.

Figure 3.

Normal basal synaptic transmission at BL–LA synapses of trkBPLC/+ mice. A, PPF was measured to determine whether this aspect of synaptic transmission was normal in trkBPLC/+ mice compared with controls. The percentages denote the ratio of the second fEPSP slope to the first fEPSP slope. PPF was tested at 10, 30, 40, 50, 100, and 200 ms ISIs. There was no significant difference between genotypes. Error bars correspond to the SEM. B, AMPA/kainate receptor (CNQX) and NMDA receptor (AP-5) antagonists were used to analyze whether NMDA and AMPA/kainate receptors are functional in trkBPLC/+ mice. Shown are single traces of extracellular recordings for trkBWT/+, trkBPLC/+, and trkBSHC/+ genotypes. Arrows indicate baseline EPSP 15 min after CNQX application or 15 min after AP-5-plus-CNQX application. C, Summary data for all pharmacological experiments. Plotted is the ratio of the EPSP slope 15 min after CNQX application in comparison with baseline recordings. There was no statistically significant difference between genotypes (p > 0.1; Student's t test). D, E, Synaptic fatigue during high-frequency stimulation. Single traces collected during theta-burst stimulation from trkBWT/+ control animal (D) and from trkBPLC/+ mutant mouse (E). The response to the fourth stimulus (indicated by 4) was compared with the response to the first one (indicated by 1). F, Data corresponding to controls and to trkBSHC/+ and trkBPLC/+ mutants during high-frequency stimulation. Plotted is the ratio of the fourth to the first response in a burst of four stimuli during theta-burst stimulation. For LTP induction, three theta bursts were applied, and data for all three are shown. Synaptic fatigue can be observed in all genotypes, and there is no statistically significant difference between control, trkBSHC/+, and trkBPLC/+ mice (p > 0.1; Student's t test).

Figure 4.

Long-lasting LTP is strongly impaired in BL–LA synapses of trkBPLC/+ mutants. A, Schematic representation of stimulating and recording electrodes. SLA, Stimulating (lateral amygdala); RBL, recording (basolateral amygdala). B, C, Slope size of fEPSP recordings before and after the TBS stimulus was plotted. Symbols represent average responses plotted every 4 min. After 20 min of control recordings, HFSs were presented at the time marked by the arrow. Data are represented as the mean ± SEM. B, L-LTP is not significantly different in trkBSHC/+ mutants compared with controls (trkB+/+) (p > 0.1). The number of slices was 5 for control mice (trkB+/+) and 10 for trkBSHC/+ mice. C, L-LTP is abolished in trkBPLC/+ mice. The difference between trkBPLC/+ mutants and control mice (trkBWT/+) was statistically significant (p < 0.01). Traces show EPSP before (1) and 180 min after (2) LTP protocol was applied. Calibration: 5 ms, 0.5 mV. The number of slices was four for control mice (trkBWT/+) and five for trkBPLC/+ mice.

Figure 5.

Synaptic plasticity at the thalamic–lateral amygdala synapses is impaired in both trkBPLC/+ and trkBSHC/+ mutants. A, Schematic representation of stimulating (S) and recording (R) electrodes. B, C, Symbols represent average responses plotted every 2 min. After 20 min of control recordings, an HFS was presented, as marked by the arrow. Data are represented as the mean ± SEM. B, E-LTP was significantly reduced in trkBSHC/+ mutant mice. p < 0.01 compared with control mice (trkB+/+). C, E-LTP was also significantly reduced in trkBPLC/+ mutants. p < 0.01 compared with control mice (trkBWT/+). Traces show EPSP before (1) and 60 min after (2) LTP protocol was applied. Calibration: 5 ms, 0.5 mV. The number of slices was four for trkB+/+ and trkBSHC/+ mice and five for trkBWT/+ and trkBPLC/+ mice. D, PPF was measured to determine whether this aspect of synaptic transmission was normal in trkBSHC/+ and trkBPLC/+ mice compared with controls (trkB+/+ and trkBWT/+, respectively). The percentages denote the ratio of the second fEPSP slope to the first fEPSP slope. PPF was tested at 10, 30, 40, 50, 100, and 200 ms ISI. There was no significant difference between genotypes. Error bars correspond to the SEM. E, Data corresponding to controls and to trkBSHC/+ and trkBPLC/+ mutants during high-frequency stimulation. Plotted is the ratio of the fourth to the first response in a burst of four stimuli during theta-burst stimulation. For LTP induction, three theta bursts were applied, and data for all three are shown. Synaptic fatigue can be observed in all genotypes with no statistically significant difference between controls and trkBSHC/+ and trkBPLC/+ mice (p > 0.1; Student's t test).

Figure 6.

Long-lasting LTP is also reduced in trkBSHC/+ and trkBPLC/+ mutants at thalamic–lateral amygdala synapses. A, Schematic representation of stimulating (S) and recording (R) electrodes. B, C, Slope size of fEPSP recordings before and after the TBS stimulus was plotted. Symbols represent average responses plotted every 4 min. After 20 min of control recordings, HFSs were presented at the time marked by the arrow. Data are represented as the mean ± SEM. B, L-LTP was significantly reduced in trkBSHC/+ mutants compared with control mice (trkB+/+); p < 0.01. C, L-LTP was significantly reduced also in trkBPLC/+ mice compared with control mice (trkBWT/+); p < 0.01. Traces show EPSP before (1) and 180 min after (2) LTP protocol was applied. Calibration: 5 ms, 0.5 mV. Four slices were analyzed for trkB+/+ mice, six for trkBSHC/+ mice, and five for trkBWT/+ and trkBPLC/+ mice.

Figure 7.

Spatial learning is severely impaired in trkBPLC/+ but not in trkBSHC/+ point mutants. A–D, Mice were trained for 10 consecutive days in a fully baited eight-arm radial maze. Learning performance is expressed as the mean number of correct arm choices in the first 8 trials, which is a measure of spatial working memory (A, B), and the percentage of errors until eight correct choices were observed or after the maximal permitted number of trials (15), which reflect long-term spatial memory (C, D). A, C, The performance of the trkBPLC/+ mice was significantly impaired compared with the trkBWT/+ control mice in all measures considered. B, D, In contrast, the performance of the trkBSHC/+ mice was very similar to that of trkB+/+ control mice in both the correct arm choices in the first eight trials and the percentage of errors. *p < 0.05; #p < 0.005.

Figure 8.

Weak pCaMKII at amygdala synapses in trkBPLC point mutants correlates with impaired fear acquisition. A–C, Immunoblots showing pCaMKII (Thr286) levels in synaptosomal preparations from amygdala tissues of trkB+/+ untrained (control, n = 4) versus trained (2 CS–US, n = 5; or 5 CS–US, n = 5), trkBPLC/+ (control, n = 3; 2 CS–US, n = 3; or 5 CS–US, n = 3) and trkBPLC/PLC (control, n = 3; 2 CS–US, n = 3; or 5 CS–US, n = 3) mice. Blots were reprobed with CaMKIIα Abs to visualize CaMKIIα levels. Top, Quantification of pCaMKII levels A, **, ANOVA, five CS–US versus control, p = 0.0059; *, two CS–US versus control, p = 0.047. B, *, ANOVA, five CS–US versus control, p = 0.01; five CS–US versus two CS–US, p = 0.03. C, ***, ANOVA, five CS–US versus control, p = 0.0007; five CS–US versus two CS–US, p = 0.0002. D–F, Immunoblots of PSD95 and synapsin I/II confirmed that the synaptosomal preparation from amygdala tissues contained both post- and presynaptic fractions, respectively.

Figure 9.

Decreased pAKT but not pMAPKs in trkBSHC/+ point mutants. A, Top, Immunoblot showing the time course of MAPK phosphorylation after BDNF stimulation of control and trkBSHC/+ mutant cortical primary neurons using Abs against the phosphorylated forms of ERK1/2, p42/44 (pMAPKs). To control for protein loading, the blots were reprobed with anti-ERK1 Abs. Bottom, Quantification of pMAPK levels. Densitometric analysis was performed on two different blots for each antibody (n = 3 cortices per time point). B–E, Immunofluorescence analysis of amygdala tissues from fear-conditioned (n = 3) versus untrained (n = 2) trkB+/+ and trkBSHC/+ mice by use of pMAPKs Abs. F, Top, Immunoblot showing the time course of AKT phosphorylation after BDNF stimulation of control and trkBSHC/+ mutant cortical primary neurons by using pAKT antibodies. To control for protein loading, blots were reprobed with anti-AKT Abs. Bottom, Quantification of pAKT levels. *, pAKT in trkBSHC/+ versus trkB+/+, p = 0.02, ANOVA. Densitometric analysis was performed on two different blots for each antibody (n = 3 cortices per time point). G, Top, Immunoblot analysis of pAKT in amygdala lysates from trkB+/+ untrained (control) (n = 2) versus trained (fear-conditioned) (n = 3) amygdala lysate and from trkBSHC/+ mice (control) (n = 2) versus trained (fear-conditioned) (n = 3) amygdala lysate. To control for protein loading, blots were reprobed with anti-AKT Abs. Bottom, Quantification of pAKT levels. *p = 0.01. C, Control (untrained); T, trained.