Reversible modification of T-LA synaptic efficacy within a fixed modification range.

<p><b>A</b>. <b>Left</b>, Ceiling of the behaviorally modifiable range assessed with pairing-induced LTP. Robust LTP was observed in the groups for which T-LA synaptic weights were predicted to be at the floor of the range (naïve, extinction, unpaired group 1 & 2), whereas LTP was occluded in the groups for which T-LA synaptic weights were expected to be at the ceiling (conditioned, reconditioned). <b>Right</b>, Floor of the range estimated with depotentiation. Depotentiation was observed in the groups for which T-LA synaptic weights were predicted to be at the ceiling (conditioned, reconditioned), whereas depotentiation was absent in the groups for which T-LA synaptic weights were expected to be at the floor (naïve, extinction, unpaired group 1 and 2). <b>B.</b> Summary of the results shown in Fig. 2A. To avoid possible bias, the experiments in Fig. 2A were performed with the experimenter blind to the behavioral group.</p

Behavioral procedures.

<p><b>A.</b> Schematic diagram for behavioral procedures. Conditioning was carried out for two consecutive days, followed by extinction (three days) and reconditioning (two days) in a distinct context. White and gray tones in the rectangles represent context A and B, respectively. On day eight, brain slices were acquired from rats, while a separate set of animals were tested for fear memory retention. <b>B.</b> Pooled behavioral results for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024260#pone-0024260-g002" target="_blank">Fig. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024260#pone-0024260-g003" target="_blank">3</a>.</p

ABA renewal-inducing stimuli produce a context-dependent enhancement of synaptic efficacy at T-LA synapses.

<p><b>A.</b> The behavioral procedure. On Day 7, brain slices were prepared immediately after a tone test (ABB-tone and ABA-tone group) or context exposure (ABC-context group). In unpaired controls, one set of rats was killed for the preparation of brain slices, while another set was monitored for conditioned freezing to a CS on Day 7. <b>B</b> and <b>C.</b> Pooled behavioral results. **, p<0.01. <b>D.</b> Input-output curves for EPSCs in unpaired controls (n = 7), ABC-context (n = 7), ABB-tone (n = 16) and ABA-tone groups (n = 9). The representative traces are the averages of four responses evoked by input stimulations of 35 µA. Scale bars, 20 ms and 100 pA.</p

The GluR1_S peptide inhibits both low-threshold potentiation and its associated enhancement in the RI.

<p><b>A.</b> Left: Inclusion of GluA1_S in the internal solution inhibited the low-threshold potentiation relative to GluA1_A (GluR1_A, 135.9±7.8% of baseline, n = 12; GluA1_S, 113.3±6.6% of baseline, n = 16). Representative traces are superimposed averages of the EPSCs before and 20 min after pairing. Scale bars, 20 ms and 50 pA. Right: The inclusion of GluA1_S or GluA1_A in the internal solution did not have a significant effect on basal synaptic transmission (GluA1s, 104.1±6.7% of baseline, n = 5, p>0.5; GluA1_A, 109.7±5.8 of baseline, n = 5, p>0.1; paired <i>t</i>-test). <b>B1</b>. The pairing protocol produced an enhancement in the RI and the AMPA EPSC amplitudes when cells were dialyzed with GluA1_A. Left: Sample traces from an individual experiment. Right: Summary data for the RI changes after the induction of low-threshold potentiation (114.9±5.9% of baseline, n = 13). NMDAR-meditated EPSCs did not change significantly after the induction of low-threshold potentiation (113.4±7.4% of baseline, p = 0.0930, paired <i>t</i>-test). <b>B2</b>. The inclusion of GluA1_S in the internal solution attenuated the RI increase associated with the low-threshold potentiation. Left, Sample traces from an individual experiment. Right, Summary data for the RI changes after the induction of low-threshold potentiation (103.9±4.5% of baseline, n = 13). NMDAR-meditated EPSCs did not change (106.9±4.6% of baseline, p = 0.1563, paired <i>t</i>-test). *, p<0.05.</p

The microinjection of a cell permeable form of the GluA1-derived peptides into the LA attenuates ABA renewal.

<p><b>A.</b> The behavioral procedure. The tone stimulus used for renewal was 60<b> </b>sec in duration on Day 6. In this figure, a weaker conditioning protocol was used (<i>see</i> Methods for additional details). <b>B1</b>. Upper, The microinjection of GluA1_S into the LA impaired ABA renewal relative to GluA1_A (GluA1_A, 41.43±2.68%, n = 8; GluA1_S, 21.24±5.63%, n = 9; p<0.01, unpaired <i>t</i>-test). Lower, Schematic representation of the injector cannula tips. Histological plates illustrating the injection site in the LA were adopted from the rat brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100108#pone.0100108-Paxinos1" target="_blank">[50]</a> (○, GluA1_A; •, GluA1_S). <b>B2</b>. Top, GluA1_A peptide injection had no effects on ABA renewal relative to vehicle controls (vehicle, 56.09±6.78%, n = 9; GluA1_A, 53.70±11.05%, n = 10; p>0.5, unpaired <i>t</i>-test). Bottom, Schematic representation of the injector cannula tips. Histological plates illustrating the injection site in the LA were adopted from the rat brain atlas (○, Vehicle; •, GluA1_A). <b>C.</b> Diffusion of the fluorescent dansyl-tat-GluR1_S peptide (1<b> </b>nmol) within 1<b> </b>h after the microinjection, as visualized with a multiphoton microscope (top). The white arrow indicates the end of the injector cannula. Peptide transduction in individual LA neurons at high magnification (bottom). Ce, central amygdala; BA, basal amygdala. <b>D1</b>. Top: Microinjection of GluA1_D into the LA impaired ABA renewal relative to GluA1_A (GluA1_A, 66.82±5.62%, n = 6; GluA1_D, 36.70±6.77%, n = 6; p<0.01, unpaired t-test). Bottom: Schematic representation of the injector cannula tips (○, GluA1_A; •, GluA1_D). <b>D2</b>. Top: GluA1_A injection had no effects on ABA renewal relative to vehicle controls (vehicle, 74.01±5.65%, n = 6; GluA1_A, 77.37±7.24%, n = 4; p>0.05, unpaired t-test). Bottom: Schematic representation of the injector cannula tips (○, Vehicle; •, GluA1_A).</p

T-LA synaptic strength is reversibly modulated by conditioning, extinction, and re-conditioning.

<p>Input-output curves for EPSCs in naïve (n = 6), conditioned (n = 11), extinction (n = 8) and reconditioned (n = 9) groups. The series resistance was not significantly different between the four groups (naïve, 11.94±0.05 MΩ; conditioned, 12.13±0.10 MΩ; extinction, 12.07±0.10 MΩ; reconditioned, 12.05±0.10 MΩ; F_(3,34) = 0.70, p = 0.56; p>0.05 for all pairs, Newman-Keuls posttest). Decay time constants with input stimulation of 25 µA were not significantly different between the four groups (naïve, 5.12±0.48 ms; conditioned, 5.37±0.30 ms; extinction, 5.50±0.66 ms; reconditioned, 5.03±0.43 ms; F_(3,34) = 0.21, p = 0.89; p>0.05 for all pairs, Newman-Keuls posttest). Representative current traces are an average of four consecutive responses with input stimulations of 35 µA. Scale bars, 50 ms and 150 pA.</p

Inhibition of GluR2-lacking AMPAR activity in the LA is required for ABA renewal.

<p><b>A.</b> The behavioral procedure for the experiments. NASPM, a selective blocker of GluA2-lacking AMPARs, was microinjected 15 min before tone presentation in context C on Day 6. In this figure, a weaker conditioning protocol was used (see Methods for additional details). <b>B.</b> Microinjection of NASPM (40 µg) into the LA impaired ABA renewal relative to the microinjection of saline (F_2,23 = 10.44, p = 0.0006, one-way ANOVA; Saline, 66.9±7.0%, n = 12; 10 µg NASPM, 44.1±11.6%, n = 7; 40 µg NASPM, 16.0±3.5%, n = 7; p<0.001, Newman-Keuls post-test). <b>C.</b> Schematic representation of the injector cannula tips. Histological plates illustrating the injection site in the LA were adopted from the rat brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100108#pone.0100108-Paxinos1" target="_blank">[50]</a>.</p

ABA renewal-inducing stimuli enhance Ser-831 phosphorylation of surface GluR1 in the LA synaptosomes.

<p><b>A.</b> The behavioral procedure. On Day 7, the LA synaptosomal membranes were prepared immediately after a tone test (ABA-tone group). In the unpaired and extinction groups, one set of rats was killed for the preparation of LA synaptosomal membranes, while another set was monitored for conditioned freezing to CS on Day 7. <b>B.</b> Representative immunoblot. <b>C.</b> Pooled results showing that Ser-831 phosphorylation was enhanced upon ABA renewal. *, p<0.05, one-way ANOVA (F_2,12 = 5.850, p = 0.0169) followed by Newman-Keuls post-test. <b>D.</b> There was no significant difference across the three groups in terms of the amount of surface GluR1 from the LA synaptosomes. The number of rats used in each group was as follows: unpaired = 20, extinction = 18, renewal = 19.</p

ABA renewal-inducing stimuli enhance the amplitude of AMPAR-mediated mEPSCs at T-LA synapses.

<p><b>A.</b> The behavioral procedure. On Day 7, brain slices were prepared immediately after a tone test (ABA-tone group). In the extinction group, one set of rats was killed for the preparation of brain slices, while another set was monitored for conditioned freezing to CS on Day 7. <b>B.</b> Upper left: Sample traces of evoked EPSCs in the presence of Ca²⁺ or Sr²⁺. Scale bars, 50<b> </b>ms and 10 pA. Note that the representative traces were further processed with a digital Gaussian filter for better display. Lower left: Cumulative amplitude distributions of evoked mEPSCs in the presence of Sr²⁺ (n = 300 events per cell). Upper right: Mean amplitude of mEPSCs evoked in the presence of Sr²⁺ (F_2,16 = 5.896, p = 0.0121; naïve, 14.3±0.9 pA, n = 7; extinction,14.0±0.7 pA, n = 7; ABA-tone, 17.5±0.4 pA, n = 5; *, p<0.05). Lower right: Mean frequency of mEPSCs evoked in the presence of Sr²⁺ (naïve, 8.0±1.1<b> </b>Hz, n = 7; extinction, 7.4±1.2<b> </b>Hz, n = 7; ABA-tone, 9.4±1.7<b> </b>Hz, n = 5).</p

ABA renewal-inducing stimuli enhance GluR2-lacking AMPAR activity at T-LA synapses.

<p><b>A.</b> ABA renewal-inducing stimuli increased the RI of synaptic AMPA currents compared with the extinction group (extinction, n = 13; ABA-tone, n = 12). Superimposed averages of AMPAR-mediated EPSCs recorded at −60 mV and +40 mV are shown in the inset (see Methods for additional details). <b>B.</b> PhTx, a selective blocker for GluA2-lacking AMPARs, inhibited AMPAR-mediated EPSCs in the renewal group, but not in the extinction group. D-AP5 (100 µM) was included in the recording solution. EPSCs were elicited at a frequency of 0.33 Hz. *, p<0.05 (paired t-test).</p