In auditory fear conditioning, experimental subjects learn to associate an auditory conditioned stimulus (CS) with an aversive unconditioned stimulus. With sufficient training, animals fear conditioned to an auditory CS show fear response to the CS, but not to irrelevant auditory stimuli. Although long-term potentiation (LTP) in the lateral amygdala (LA) plays an essential role in auditory fear conditioning, it is unknown whether LTP is induced selectively in the neural pathways conveying specific CS information to the LA in discriminative fear learning. Here, we show that postsynaptically expressed LTP is induced selectively in the CS-specific auditory pathways to the LA in a mouse model of auditory discriminative fear conditioning. Moreover, optogenetically induced depotentiation of the CS-specific auditory pathways to the LA suppressed conditioned fear responses to the CS. Our results suggest that input-specific LTP in the LA contributes to fear memory specificity, enabling adaptive fear responses only to the relevant sensory cue.

To determine the synaptic mechanisms of how discriminative fear memory for a specific CS is encoded in the amygdala, we tested our hypothesis that specific fear memory is encoded by selective LTP in neural pathways defined by presynaptic inputs conveying specific CS information to the amygdala. Using a combined approach of neural activity-dependent behavioral labeling (), optogenetic stimulations (), and electrophysiological recordings (), we found that postsynaptically expressed LTP was induced selectively in the CS-specific ACx/MGN-LA pathways after auditory discriminative fear conditioning in mice, whereas LTP was not detected in randomly selected ACx/MGN-LA pathways. Moreover, optogenetically induced depotentiation of the CS-specific ACx/MGN-LA pathways prevented the recall of fear memory for the auditory CS. Thus, input-specific LTP in the LA could contribute to fear memory specificity, enabling adaptive fear responses only to the relevant sensory cue.

Long-term potentiation (LTP) in the amygdala plays an essential role in the formation of conditioned fear memory (). After fear conditioning, synaptic strength is enhanced in the auditory CS pathways to the lateral nucleus of the amygdala (LA), such that presentation of the CS alone is sufficient to activate the amygdala and its downstream brain areas (), resulting in fear responses to the CS. A specific auditory CS activates only a subset of neurons in the auditory cortex (ACx) and thalamus (medial geniculate nucleus, MGN), which convey CS information to the amygdala for the CS-US association. Thus, LTP may be induced selectively in neural circuits conveying specific CS signals to the amygdala for encoding fear memory for the CS. After fear conditioning with the auditory CS+, auditory-evoked single-unit activity and local field potential in the LA are enhanced more robustly to the CS+ (a relevant stimulus) than to the CS– (an irrelevant stimulus), suggesting a selective increase in the responsiveness of LA neurons to the CS+ (). However, it has not been determined whether LTP is induced selectively in neural pathways conveying specific CS information to the amygdala in discriminative fear learning. If LTP in the CS-specific pathways confers fear memory specificity, fear memory for the CS could be erased selectively by depotentiation, reversing the input-specific LTP. However, it has not been examined whether depotentiation in the CS-specific pathways to the amygdala suppresses fear memory for the CS.

To survive in a dynamic environment, animals develop fear responses to dangerous situations. For these adaptive fear responses to be developed, the brain must discriminate between different sensory cues and associate only relevant stimuli with aversive events. In auditory fear conditioning, an experimental model of fear learning, experimental subjects learn to associate an emotionally neutral auditory conditioned stimulus (CS; e.g., a tone) with an aversive unconditioned stimulus (US; e.g., electric foot shock), displaying conditioned fear responses (e.g., freezing behavior) to the neutral CS (). With sufficient training, animals fear conditioned to an auditory stimulus show fear responses to the same stimulus, but not to irrelevant auditory stimuli. It is poorly understood at the neuronal and synaptic levels how animals discriminate between auditory stimuli to show fear responses selectively to the relevant stimulus.

We also examined how in vivo photostimulations applied to the CS– pathways to the LA affected conditioned fear responses to the CS+. After surgery ( Figures 8 E and 8F), we used our behavioral labeling protocol for the expression of ChR2 or eYFP in ACx/MGN neurons responding to the CS– ( Figure 8 G). Mice were fear conditioned to the CS+ and then received in vivo photostimulations at 1 Hz for 3 days as in previous experiments. Freezing behavior to the CS+ after in vivo photostimulations (day 10) was not significantly different from freezing behavior before photostimulations (day 7) in either the ChR2 or eYFP group ( Figures 8 I, 8J, and S8 F) (main effect of group, p = 0.23; main effect of photostimulation, p = 0.06; groups × photostimulation interaction, p = 0.66; repeated-measures two-way ANOVA). These results indicate that low-frequency photostimulation of the CS– pathways to the LA did not affect conditioned fear response to the CS+. Therefore, observed behavioral effects of photostimulation in the CS+ pathways in our previous experiments are not attributable to the effect of photostimulation in nonspecific input pathways.

We next applied low-frequency photostimulations in vivo to induce depotentiation in the CS-specific ACx/MGN-LA pathways, which had been potentiated after fear conditioning ( Figure 8 D). We injected AAV-pEF1α-DIO-ChR2-eYFP (ChR2-eYFP group) or AAV-pEF1α-DIO-eYFP (eYFP group) into ACx and MGN in Fos-CreERmice ( Figure 8 E). An optical cannula was implanted dorsal to the ipsilateral LA ( Figures 8 E, 8F, S8 A, and S8B), and NMDA was injected into the contralateral amygdala for an excitotoxic lesion ( Figure S8 C). After surgery, mice received behavioral labeling with the auditory CS+ (12 kHz tone) for ChR2-eYFP or eYFP expression in ACx/MGN neurons responding to the CS+. Mice were habituated to optical cable connection on days 1–4 and fear conditioned with CS+/US pairings on days 5 and 6 ( Figures 8 G and S8 D), which resulted in freezing behavior to the CS+ on day 7 in both groups ( Figure 8 H). Mice then received 1 Hz photostimulations for 15 min on days 7–9 ( Figures 8 G and S8 D). After in vivo photostimulations for 3 days, mice in the ChR2-eYFP group displayed significantly reduced freezing behavior to the CS+ (p < 0.01, day 7 versus day 10; Figures 8 H and S8 E), whereas the same photostimulations did not significantly affect fear response to the CS+ in the eYFP control group (p = 0.53, Figures 8 H and S8 E) (groups × photostimulation interaction, p < 0.05; repeated-measures two-way ANOVA with post hoc multiple comparisons). These results indicate that reduced fear responses after photostimulation depended on ChR2 expression in CS+-responding ACx/MGN neurons. Moreover, the AMPA/NMDA ratio of EPSCs in the CS+-specific ACx/MGN-LA pathways was significantly reduced in brain slices from mice that received 1 Hz photostimulation after fear conditioning, compared with that of mice that did not receive 1 Hz photostimulation after fear conditioning (p < 0.05, unpaired t test; Figures 8 K and 8L), indicating that in vivo photostimulation induced depotentiation in the CS-specific ACx/MGN-LA pathways. However, the same 1 Hz photostimulations did not affect the AMPA/NMDA ratio in the CS-specific ACx/MGN-ASt pathways (p = 0.15, unpaired t test; Figures S8 G and S8H). Taken together, our results suggest that depotentiation in the CS+-specific ACx/MGN-LA pathways was sufficient to prevent fear responses to the CS+. Therefore, LTP in the auditory CS+-specific pathways to the LA is necessary for conditioned fear response to the CS+.

Our results demonstrate that postsynaptically expressed LTP is induced selectively in the ACx/MGN pathways conveying CS+ information to the LA in discriminative fear learning. We next determined whether input-specific LTP is necessary for the conditioned fear response by examining how input-specific depotentiation (the reversal of LTP) in the CS-specific ACx/MGN-LA pathways affected fear responses to the CS. To this end, we induced ChR2 expression in ACx/MGN neurons responding to 4 or 12 kHz tone. In brain slices, we then selectively photostimulated tone-specific ACx/MGN-LA pathways and recorded EPSCs in LA neurons ( Figure 8 A). After the baseline recording of EPSC, we applied 1 Hz photostimulations for 5 min with a holding potential of –60 mV, which induced a long-lasting reduction of the EPSC in the tone-specific ACx/MGN-LA pathways (53.1% ± 8.4% of baseline, p < 0.01, paired t test; Figures 8 B and 8C).

(K) Representative traces of EPSCs recorded in brain slices from mice in the ChR2-eYFP group. Left: EPSCs recorded in an LA neuron in mice that were fear conditioned but did not receive 1 Hz photostimulations in vivo (black, FC/photostim–). Right: EPSCs recorded in an LA neuron in mice that received 1 Hz photostimulations in vivo after fear conditioning (blue, FC/photostim+). AMPAR and NMDAR EPSCs recorded as in Figure 3 H.

(J) Quantification of reduction in freezing behavior to the CS+ (% baseline) after 1 Hz photostimulations of the CS+ or CS– pathways to the LA. Two-way ANOVA (groups × pathways interaction, p < 0.05) with post hoc comparisons.

(I) Freezing behavior to the CS+ before and after 3 days of 1 Hz photostimulations of CS– pathways in the ChR2-eYFP group (n = 6 mice, left) and eYFP group (n = 6 mice, right).

(H) Left: freezing behavior to the CS+ before and after 3 days of 1 Hz photostimulations of CS+ pathways, which significantly reduced freezing behavior to the CS+ in the ChR2-eYFP group (n = 6 mice). Right: the same photostimulations did not significantly affect freezing behavior to the CS+ in the eYFP group (n = 6 mice). Open circles indicate freezing responses to the CS+ in each mouse on days 7 and 10. Closed circles are the average of freezing responses.

(G) Experimental setup for (H)–(L). After fear conditioning on days 5 and 6, mice received 1 Hz photostimulations for 15 min through the optical cannula on days 7–9 ( Figure S8 D). On day 10, mice were tested for freezing behavior to the CS+ and electrophysiological recordings were performed in brain slices.

(B) Repeated photostimulation (1 ms pulses) at 1 Hz for 5 min with 10 s pauses every minute induced a lasting reduction of EPSCs in tone-specific ACx/MGN-LA pathways. Top: representative traces of EPSCs recorded at –80 mV in an LA neuron before (1, black) and 30 min after 1 Hz photostimulations (2, blue). Bottom: time course of EPSC changes induced by 1 Hz photostimulations (n = 5 neurons). EPSC amplitude was normalized to the baseline EPSC.

Conditioned fear memory can be extinguished by repeated CS presentations without the US. A previous report suggests the presence of fear extinction-associated synaptic changes in the auditory pathways to the LA (). Because the formation of discriminative fear memory involves LTP selectively induced in the neural pathways conveying CS+ information to the LA, we examined how the extinction of discriminative fear memory affected synaptic efficacy in the CS+-specific pathways to the LA. We induced ChR2 expression in ACx/MGN neurons responding to the CS+ by injecting AAV-pEF1α-DIO-ChR2-eYFP into ACx and MGN in Fos-CreERmice and exposing them to the auditory CS+ (12 kHz tone) ( Figures 7 A and 7B ). Mice in the FC group received discriminative fear conditioning with the CS+ paired with the US and displayed discriminative fear to the CS+ ( Figures 7 A and 7D). In the fear extinction group (EX), we induced extinction of the discriminative fear with repeated CS+ presentations after discriminative fear conditioning ( Figures 7 A and 7C). Fear extinction training for 2 days significantly reduced fear response to the CS+ (p < 0.01, day 5 versus day 8, paired t test; Figure 7 D). Mice in the NS control group received CS+ and CS– without the US and showed no fear to either the CS+ or CS– ( Figures 7 A and 7D). In brain slices, we compared between groups EPSCs that were evoked by photostimulation of axons of CS+-responding ACx/MGN neurons and recorded in LA neurons. In CS+ pathways to the LA, the AMPA/NMDA EPSC ratio was significantly different between behavioral groups (p < 0.01, one-way ANOVA; Figures 7 E and 7F). Post hoc analysis revealed that the AMPA/NMDA ratio was significantly higher in the FC and EX groups than in the NS group (p < 0.01, FC versus NS; p < 0.05, EX versus NS; Figure 7 F), whereas there was no significant difference between the FC and EX groups (p = 1.00), suggesting that synaptic efficacy remains enhanced in the auditory CS+ pathways to the LA after the extinction of discriminative fear memory for the CS+. When ChR2 was globally expressed in ACx and MGN ( Figures 7 G and 7H), the AMPA/NMDA ratio in the EX group was not significantly different from that in the FC group (p = 0.40, unpaired t test; Figures 7 I and 7J), suggesting that synaptic efficacy in nonspecific ACx/MGN-LA pathways was not altered after fear extinction.

(I) Representative EPSC traces in the FC and EX groups. EPSCs were induced by photostimulation of randomly selected ACx/MGN inputs to the LA. Both AMPAR and NMDAR EPSCs were recorded in the same LA neurons as in (E).

(E) Representative traces of EPSCs recorded in LA neurons in the NS, FC, and EX groups. EPSCs were induced by photostimulation of ChR2-expressing ACx/MGN axons in the amygdala. Both AMPAR and NMDAR EPSCs were recorded in the same LA neurons as in Figure 3 H.

(D) Quantification of freezing behavior to the auditory CS+ and CS– in the NS (5 mice, day 5), FC (6 mice, day 5), and EX groups (5 mice, days 5 and 8). ND, not detected.

(C) Time course of freezing behavior to the CS+ during fear extinction learning on days 6 and 7 in the EX group.

(B) Experimental setup for (C)–(F) for recording EPSCs in the CS+ pathways to the LA, which were compared between groups to examine how discriminative fear conditioning and extinction affected synaptic efficacy in these pathways.

We next examined whether discriminative fear learning was associated with enhanced synaptic efficacy in the randomly selected ACx/MGN-LA pathways to LA neurons activated during fear conditioning. To this end, we injected AAV-pCaMKIIα-ChR2-eYFP into ACx and MGN in the Fos-CreER× ROSA-LSL-tdTomato mice for global ChR2 expression in the auditory areas and labeled LA neurons activated during fear conditioning with tdTomato ( Figures 6 I and 6J). After discriminative fear conditioning ( Figure 6 K), we induced EPSCs with photostimulation of randomly selected ACx/MGN inputs and recorded AMPAR and NMDAR EPSCs in LA neurons. There was no significant difference in the AMPA/NMDA ratio between labeled and unlabeled LA neurons (p = 0.80, unpaired t test; Figures 6 L and 6M), suggesting that synaptic efficacy in randomly selected ACx/MGN inputs was not preferentially altered in LA neurons activated during fear conditioning. We also compared sEPSCs and found no significant difference in the amplitude or frequency of sEPSCs between tdTomato-labeled and unlabeled LA neurons (p = 0.73 and p = 0.21 for sEPSC amplitude and frequency, respectively, unpaired t test; Figures S7 G–S7I). Taken together, our results suggest that discriminative fear learning is associated with LTP, which is preferentially induced in presynaptic ACx/MGN inputs conveying CS+ information to a subset of postsynaptic LA neurons activated during fear conditioning.

Our results demonstrate that postsynaptically expressed LTP is selectively induced in the inputs conveying CS+ information to the LA in auditory discriminative fear learning ( Figures 3 4 , and 5 ) and the input-specific LTP was detected in only a small population (approximately 20%) of postsynaptic LA neurons ( Figure 3 I). The activation of presynaptic inputs followed by backpropagating action potentials in postsynaptic neurons with a short time interval induces associative Hebbian plasticity in the ACx/MGN-LA pathways (). Thus, LTP may be induced preferentially in synapses consisting of presynaptic ACx/MGN inputs and postsynaptic LA neurons that are activated during the CS/US pairings. To test this possibility, we injected AAV-pEF1α-DIO-ChR2-eYFP into ACx and MGN in Fos-CreER× ROSA-LSL-tdTomato mice ( Figure 6 A). We employed dual behavioral labeling, in which CS+-responding presynaptic ACx/MGN neurons were labeled with ChR2 and postsynaptic LA neurons activated during fear conditioning were labeled with tdTomato ( Figures 6 A and 6B). After behavioral labeling, mice were trained for discriminative fear responses selectively to the CS+ (12 kHz tone; Figure 6 C). In brain slices, LA neurons activated during fear conditioning were identified with tdTomato expression ( Figure 6 D). We induced EPSCs with photostimulation of CS+-responding ACx/MGN axons and compared EPSCs recorded in tdTomato-labeled and unlabeled LA neurons. The AMPA/NMDA EPSC ratio was significantly higher in tdTomato-labeled neurons than in unlabeled neurons (p < 0.01, unpaired t test; Figures 6 E and 6F), suggesting that CS+ pathway-specific LTP was preferentially induced in a subset of postsynaptic LA neurons activated during fear conditioning. Moreover, we observed more pronounced inward rectification of AMPAR EPSC in tdTomato+ neurons than in unlabeled LA neurons (p < 0.01, unpaired t test; Figures 6 G and 6H), suggesting that postsynaptic increase in GluA2-lacking AMPAR contributes to LTP induced preferentially in the fear engram pathways (). We also found that prior discriminative fear learning occluded additional LTP induced in brain slices in the CS+ pathways to tdTomato+ neurons ( Figures S7 A–S7F). Together, these results suggest that discriminative fear learning preferentially induced LTP in the CS+ pathways to LA neurons that are activated during fear conditioning.

(H) Quantification of the rectification index (RI), which was calculated from the equation RI = (EPSC –80 / 80)/(EPSC +40 / 40), where EPSC –80 and EPSC +40 are peak amplitude of EPSCs recorded at –80 and +40 mV, respectively.

(G) Representative traces of AMPAR-mediated EPSCs recorded in tdTomato– and tdTomato+ neurons. AMPAR EPSCs were induced by photostimulation of ChR2-expressing axons and recorded at –80, 0, and +40 mV in the presence of D-AP5 (50 μM) and SR-95531 (10 μM).

(E) Representative traces of EPSCs recorded in tdTomato– and tdTomato+ neurons. tdTomato+ neurons were identified with red fluorescence within the LA (inset). EPSCs were induced by photostimulation of ChR2-expressing axons. Both AMPAR and NMDAR EPSCs were recorded in the same LA neurons as in Figure 3 H.

(B) Mice were exposed to the auditory CS+ for ChR2-eYFP expression in CS+-responding ACx and MGN as in Figure 1 B. Mice then received the second tamoxifen injection and were presented with six pairings of the CS+ and US for tdTomato (tdT) expression in LA neurons activated during fear conditioning. After LA labeling, mice were trained with the discriminative fear learning protocol as in Figure 3 A.

Consistently, we did not detect a significant difference in the AMPA/NMDA EPSC ratio between groups when we globally photostimulated the ACx/MGN-LA pathways, including the CS+ pathways (p = 0.40, unpaired t test; Figures 5 G–5K). Under this condition, there was no significant difference between groups in the rate of progressive block of NMDAR EPSC by MK-801 (p = 0.83, unpaired t test; Figures 5 L and 5M) or paired-pulse ratio, which is inversely related to the presynaptic release probability () (main effect of groups, p = 0.48; groups × intensity interaction, p = 0.10, repeated-measures two-way ANOVA; Figures 5 N and 5O). These results suggest that LTP by either presynaptic or postsynaptic expression mechanisms was not induced globally in the ACx/MGN-LA pathways. Moreover, we did not detect a significant difference between groups in either the amplitude or frequency of spontaneous EPSCs (sEPSCs), which reflect synaptic responses in nonspecific presynaptic inputs to the LA (p = 0.34 and p = 0.66 for sEPSC amplitude and frequency, respectively, unpaired t test; Figures S6 H–S6L). Taken together, our results suggest that LTP was not induced globally in the ACx/MGN-LA pathways in auditory discriminative fear conditioning.

In discriminative fear learning, postsynaptically expressed LTP was detected in the auditory CS+, but not CS–, pathways to the LA. We next investigated whether discriminative fear conditioning globally induced LTP in the ACx/MGN-LA pathways by examining synaptic efficacy in randomly selected ACx/MGN-LA synapses while excluding the contribution of the LTP in the CS+ pathways for our assay. To this end, we co-injected AAV-pCaMKIIα-ChR2-eYFP and AAV-pEF1α-DIO-eArch3-eYFP into ACx and MGN in Fos-CreERmice. ChR2 was expressed globally in ACx/MGN under the control of the CaMKIIα promoter, whereas eArch3 expression was limited to ACx/MGN neurons responding to the auditory CS+ after behavioral labeling ( Figures 5 A and 5B ). We then trained mice in the FC group for discriminative fear to the CS+ ( Figure 5 C). Mice in the NS group did not show fear responses to either the CS+ or CS–. In brain slices, we globally activated ACx/MGN inputs to the LA with ChR2-activating blue light while silencing the CS+ pathways with eArch3-activating orange light. Illumination with both blue and orange lights significantly reduced EPSC amplitude by 13.1% ± 2.2% (mean ± SEM, n = 36 neurons) compared with that of EPSCs induced by blue light alone (p < 0.001, paired t test; Figure 5 D). The effect was mediated by eArch3 expressed in a subset of the ACx/MGN-LA pathways ( Figures S6 A–S6D). Effective silencing of the CS+ pathways was further confirmed in another experiment, in which both ChR2 and eArch3 were expressed selectively in CS+-responding ACx/MGN neurons ( Figures S6 E–S6G). With this approach, we recorded EPSCs in randomly selected ACx/MGN-LA pathways to the LA, excluding synaptic responses in the CS+-specific synapses. Under this condition, the AMPA/NMDA EPSC ratio was not significantly different between the FC and NS groups (p = 0.91, unpaired t test; Figures 5 E and 5F), suggesting that postsynaptically expressed LTP was not induced globally in the ACx/MGN-LA pathways in discriminative fear conditioning.

(M) Plot showing a gradual decrease of NMDAR EPSCs by MK-801. The peak amplitude of NMDAR EPSCs was normalized to the first NMDAR EPSC induced after MK-801 application for 10–15 min and plotted versus photostimulation number. Inset: quantification of the rate of NMDAR EPSC decrease by MK-801. The decay constant was calculated as in Figure S4 P.

(L) Traces of NMDAR EPSCs evoked by the nth photostimulation after MK-801 application (10 μM), showing progressive block of NMDAR EPSCs by MK-801. NMDAR EPSCs were recorded at +40 mV in the presence of NBQX (10 μM) and SR-95531 (10 μM).

(H) Mice in the FC group were trained with the discriminative fear learning protocol, whereas mice in the NS group received the same CS+ and CS– without the US.

(E) Representative traces of EPSCs recorded in the FC and NS groups. Both blue and orange light illumination was applied to induce EPSCs in the LA as in (D). AMPAR and NMDAR EPSCs were recorded in the same LA neurons, and the AMPA/NMDA EPSC ratio was calculated as in Figure 3 H.

(D) Left: representative traces of EPSCs induced by blue light illumination (blue vertical bars), which globally activated ACx/MGN inputs to the LA. EPSCs were recorded at –80 mV in LA neurons. The EPSC amplitude was reduced when eArch3-activating orange light (590 nm LED, 30 ms duration, orange horizontal bar) was also applied. Right: quantification of the orange light effect on EPSCs induced by blue light.

(B) Mice were exposed to the auditory CS+ for behavioral labeling as in Figure 1 B. Mice in the fear conditioning (FC) group were trained with the discriminative fear learning protocol as in Figure 3 A. Mice in the no shock (NS) group received the same CS+ and CS– without the US.

In discriminative fear learning, mice initially displayed fear to the auditory CS–, which gradually decreased after multiple-trial fear conditioning ( Figures 3 A–3C). We hypothesized that reduced fear to the CS– was due to the lack of LTP in the pathways conveying CS– information to the LA. We tested this hypothesis by examining how synaptic efficacy changed in the CS– pathways in discriminative fear conditioning ( Figure 4 A). For behavioral labeling, we exposed mice to the auditory CS– to induce ChR2 expression in ACx/MGN neurons responding to the CS– ( Figures 4 B and 4C). As in previous experiments, mice in the FC group received the CS+ paired with the US, whereas mice in the NS control group received the CS+ and CS– without the US. After discriminative fear conditioning, mice in the FC group displayed robust freezing behavior to the CS+ but a much lower fear response to the CS– ( Figures 4 D and S3 E). In brain slices, we photostimulated ACx/MGN axons conveying the CS− information and recorded EPSCs in LA neurons ( Figures 4 A and 4E). In the CS– pathways to the LA, there was no difference in the AMPA/NMDA EPSC ratio between the FC and NS groups ( Figures 4 E–4G; main effect of groups, p = 0.95; main effect of tone frequency, p = 1.00; groups × tone frequency interaction, p = 0.86; two-way ANOVA). For presynaptically expressed synaptic changes, we compared the rate of progressive block of NMDAR EPSC by MK-801 and found no significant difference in the decay constant between groups (p = 0.40, unpaired t test; Figures S4 Q–S4S). These results suggest that synaptic efficacy did not change in the CS– pathways to the LA by either presynaptic or postsynaptic mechanisms in discriminative fear learning. We also analyzed the effects of behaviorally labeled auditory pathways (CS+ versus CS– pathways) and behavioral groups (FC versus NS) on the AMPA/NMDA EPSC ratio and found a significant pathways × groups interaction (p < 0.01, two-way ANOVA; Figures 3 J and 4 G; Table S1 ), indicating that postsynaptically expressed LTP was induced in the CS+ pathways, but not in the CS– pathways to the LA in discriminative fear conditioning ( Figure 4 H).

(H) Auditory discriminative fear conditioning induces LTP selectively in the CS+ pathways to the LA, resulting in conditioned fear responses to the CS+, but not to the CS–. Error bars are SEM.

(E) Representative traces of EPSCs recorded in the FC and NS groups. Both AMPAR and NMDAR EPSCs were recorded in each LA neuron, and the AMPA/NMDA EPSC ratio was calculated as in Figure 3 H.

(B) Mice were exposed to the auditory CS– (4 or 12 kHz tone, counterbalanced) for behavioral labeling as in Figure 1 B. Mice in the fear conditioning (FC) group were trained with the discriminative fear conditioning protocol as in Figure 3 A, whereas mice in the no shock (NS) control group received the same CS+ and CS– without the US.

(A) Experimental setup for recording synaptic responses in the auditory CS– pathways to the LA. After behavioral labeling, ACx/MGN neurons responding to the CS– (blue) expressed ChR2-eYFP. Photostimulation selectively activated the CS– pathways and induced postsynaptic responses in LA neurons.

To detect changes in synaptic efficacy by presynaptic expression mechanisms, we examined progressive block of NMDAR EPSC by MK-801, which inhibited NMDAR gradually upon repeated photostimulation ( Figures S4 N and S4O). We compared between groups the decay constant of the NMDAR EPSC, which is inversely related to presynaptic release probability (). There was no significant difference in the NMDAR EPSC decay constant between groups (p = 0.11, unpaired t test; Figure S4 P), indicating that discriminative fear learning did not affect presynaptic release probability in the CS+ pathways to the LA.

Enhanced synaptic efficacy in the CS+ pathways was detected only when the CS+ was presented temporally paired with the US on the training day (p < 0.05, unpaired t test, paired versus unpaired CS+/US; Figures S3 F and S5 A–S5C), suggesting that LTP in these pathways was not due to nonspecific effect of the US. As ACx and MGN inputs can convey distinct information to the LA and play different roles in discriminative fear conditioning (), we examined these pathways separately and found that the AMPA/NMDA ratio was significantly higher in the FC group than in the NS group in the ACx-LA pathway (p < 0.05, unpaired t test; Figures S5 D–S5F), but not in the MGN-LA pathway (p = 0.63, unpaired t test; Figures S5 G and S5H), indicating postsynaptically expressed LTP was selectively induced in the ACx-LA pathway in discriminative fear learning. In the ACx/MGN inputs to the amygdalo-striatal transition area (ASt), we did not detect significant difference in the AMPA/NMDA ratio between groups (p = 0.35, unpaired t test; Figures S5 I–S5K), suggesting that LTP associated with discriminative fear learning is pathway specific.

We next examined how synaptic efficacy changes in the ACx/MGN inputs conveying CS+ information to the LA in discriminative fear conditioning. As we were unable to predict whether LTP would be induced in both the ACx-LA and MGN-LA pathways or would be confined to either of these two pathways in discriminative fear conditioning, we first examined LTP in these pathways altogether. We injected AAV-pEF1α-DIO-ChR2-eYFP into both ACx and MGN in Fos-CreERmice to induce ChR2 expression in ACx/MGN neurons responding to the CS+ ( Figures 3 D–3F). After behavioral labeling, mice in the fear conditioning (FC) group were trained for discriminative fear learning ( Figures 3 A and 3E) and displayed discriminative fear to the CS+ ( Figures 3 G and S3 D). Mice in the no shock (NS) control group received the CS+ and CS– as in the FC group but without the US and did not show fear responses to either the CS+ or CS– ( Figure 3 G). In brain slices from these mice, we recorded EPSCs in principal neurons in the LA, which were differentiated from GABAergic interneurons based on their passive membrane properties ( Figures S4 A–S4D). Photostimulations of ChR2-expressing axons in the LA induced EPSCs, which reflect postsynaptic responses in the CS+ pathways to the LA ( Figures 3 D and 3H). The induction of LTP and long-term depression (LTD) in the ACx/MGN-LA pathways in brain slices was accompanied by changes in the AMPA/NMDA EPSC ratio ( Figures S4 E–S4L), which correlated with the magnitude of LTP and LTD ( Figure S4 M; correlation coefficient r = 0.89; p < 0.001), suggesting that the changes in the AMPA/NMDA ratio reliably reflect long-term synaptic plasticity in the ACx/MGN-LA pathways (). Thus, to detect changes in synaptic strength in the CS+ pathways by postsynaptic expression mechanisms in discriminative fear learning, we compared the AMPA/NMDA ratio between the FC and NS groups. We recorded both AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated EPSCs in the same LA neurons and calculated the AMPA/NMDA ratio ( Figure 3 H). The AMPA/NMDA ratio was significantly higher in the FC group than in the NS group ( Figures 3 H–3J; main effect of groups, p < 0.001; main effect of tone frequency, p = 0.90; groups × tone frequency interaction, p = 0.49; two-way ANOVA), whereas the passive membrane properties of recorded LA neurons were not different between groups ( Table S3 ). In 19.4% of all the LA neurons in the FC group, the AMPA/NMDA ratio was larger than the average AMPA/NMDA ratio in the NS group by more than two SDs ( Figure 3 I), suggesting that these LA neurons underwent LTP in the CS+ pathways ().

To investigate the synaptic mechanisms of fear memory specificity, we developed a behavioral protocol for discriminative auditory fear conditioning, in which mice were trained to show conditioned fear response (e.g., freezing behavior) selectively to an auditory cue, CS+ ( Figures 3 A and S3 A–S3C). After single-trial fear conditioning, in which CS+ (4 or 12 kHz tone, counterbalanced) was presented paired with the US (foot shock, 0.5 mA, 2 s duration), mice displayed non-discriminative fear to both the CS+ and CS–, which was not paired with the US (day 2 in Figures 3 B and 3C). After multiple-trial fear conditioning, however, mice showed fear selectively to the CS+ (day 6 in Figures 3 B and 3C) with better discrimination between the CS+ and CS– (p < 0.001, paired t test; Figure 3 C).

(J) Comparison of the AMPA/NMDA ratio in the CS+ pathways to the LA between groups. Open circles indicate the AMPA/NMDA ratio calculated in each neuron. Numbers within the bars are the number of neurons examined in each group. Error bars are SEM.

(I) Histogram showing the distribution of the AMPA/NMDA ratio in the FC (red) and NS groups (gray). A dotted vertical line indicates the mean + 2 SDs of the AMPA/NMDA ratio in the NS group.

(H) Representative traces of EPSCs recorded in the FC and NS groups. EPSCs were induced by blue light illumination of ChR2-expressing axons and recorded at –80, 0, and +40 mV in voltage-clamp mode in the same LA neurons. AMPAR EPSCs were quantified as the peak amplitude of EPSCs recorded at –80 mV (open circles). NMDAR EPSCs were quantified as the average EPSC amplitude from 47.5 to 52.5 ms after the onset of photostimulation (gray vertical lines and closed circles). SR-95531 (10 μM) was added to block inhibitory postsynaptic currents.

(E) Experimental setup for (F)–(J). Mice were exposed to the auditory CS+ for behavioral labeling as in Figure 1 B. Mice in the fear conditioning (FC) group were trained as in (A). Mice in the no shock (NS) control group received the CS+ and CS– as in the FC group, but the CS+ was not paired with the US.

(D) Diagram showing the experimental approach for recording synaptic responses in the CS+ pathways, which convey auditory CS+ information to the LA. After behavioral labeling, ACx/MGN neurons responding to the CS+ expressed ChR2-eYFP. Photostimulation in the amygdala induces postsynaptic responses in the CS+ pathways to the LA.

(B) Quantification of freezing behavior to the CS+ and CS– in discriminative fear conditioning. Baseline immobility was quantified as the percentage of time when the mice were immobile in the absence of the CS+ or CS–.

(A) Auditory discriminative fear conditioning protocol. Two auditory cues (4 and 12 kHz tone, 20 s duration, 70–75 dB) were used as the CS+ and CS– (counterbalanced). On day 1, mice received six pairings of the CS+ and US in context A ( Figure S3 A). On days 2–5, mice were tested for freezing behavior to the CS+ and CS– in context B ( Figure S3 B). Mice then received a single pairing of the CS+ and US and were also presented the CS– without the US in context A ( Figure S3 C).

The peak amplitude of EPSCs recorded in the tone-specific ACx/MGN-LA pathways was proportional to the light power density ( Figure S2 L). EPSCs recorded in different LA neurons in the same brain slice were heterogeneous ( Figures 2 F–2H), and the distribution of EPSC amplitude was highly skewed ( Figure 2 G). Moreover, robust EPSCs were detected only in a subset of LA neurons, whereas 64.3% of the LA neurons displayed either no synaptic responses or EPSCs with modest amplitude (<100 pA; Figure 2 G). However, when ACx/MGN neurons globally expressed ChR2 and their axons were randomly stimulated as in Figure 2 I, the EPSC amplitude was much larger but normally distributed (p = 0.13, Anderson-Darling normality test), with less variability than EPSCs in tone-specific ACx/MGN-LA pathways ( Figures 2 J–2L, S2 L, and S2M). Together, these results suggest that a subset of LA neurons preferentially receive presynaptic ACx/MGN inputs relaying specific auditory information ( Figures S2 N–S2Q) ().

With our behavioral labeling approach, we examined how each LA neuron received ACx/MGN inputs conveying specific auditory information. We injected AAV-pEF1α-DIO-ChR2-eYFP into ACx and MGN in Fos-CreERmice and exposed them to a 4 or 12 kHz tone after tamoxifen administration ( Figures 2 A and 2B ). Three weeks later, tone-responding ACx/MGN neurons expressed ChR2-eYFP, and eYFP-labeled projections were found in the LA ( Figure 2 C). To induce synaptic responses in the tone-specific ACx/MGN-LA pathways, we applied blue light illumination to activate ChR2-expressing axons in the LA in brain slices and recorded postsynaptic responses in principal neurons of the LA using a whole-cell patch-clamp technique ( Figures 2 A, 2D, and 2E). Short pulses of photostimulation induced monosynaptic excitatory postsynaptic currents (EPSCs) at –80 mV in voltage-clamp mode, which were completely blocked by glutamate receptor antagonists, indicating that these EPSCs were mediated by glutamate (p < 0.01, paired t test; Figures 2 D and S2 A–S2D; Table S2 ). Both ACx and MGN axons labeled with different fluorescent proteins were detected in the LA, suggesting their role in conveying auditory information to the amygdala ( Figures S2 E–S2G). Independent photostimulations of either the tone-labeled ACx or MGN axons induced EPSCs in the same LA neurons, indicating that LA neurons received inputs from both ACx and MGN conveying specific auditory information ( Figures S2 H–S2K).

(L) Comparisons of EPSC amplitude (mean ± SD, left) and its coefficient of variation (CV, right) between tone-specific and nonspecific ACx/MGN-LA pathways. EPSCs in tone-specific ACx/MGN-LA pathways were recorded as in (A) and (F), whereas EPSCs in nonspecific ACx/MGN-LA synapses were recorded as in (I) and (J).

(H) Scatterplot of the peak amplitude of EPSCs recorded in multiple LA neurons in each brain slice. Open circles indicate EPSC amplitude in individual LA neurons. The average amplitude of EPSC recorded in LA neurons in each brain slice (a black curve) was used to sort data along the x axis in increasing order.

(F) EPSCs recorded in four LA neurons in a brain slice. EPSCs were induced by photostimulation of the same intensity and recorded as in (D).

(E) Microscopic image of a principal neuron of the LA loaded with biocytin during whole-cell patch-clamp recording and labeled with streptavidin-Alexa 568 (red, top). A section of dendrites of the labeled neuron is shown below in a higher magnification (bottom).

(D) Left: representative traces of excitatory postsynaptic currents (EPSCs) recorded in a principal neuron of the LA (black). EPSCs were induced by photostimulation (470 nm LED, 20.0 mW/mm 2 , 1 ms duration, blue vertical bar) of ChR2-expressing ACx/MGN axons and recorded at –80 mV in voltage-clamp mode. EPSCs were inhibited completely by NBQX (10 μM) and MK-801 (10 μM) (red). Right: quantification of EPSC inhibition by NBQX and MK-801 (n = 7 cells). Error bars are SEM.

(A) Experimental setup for (B)–(H) and a neural circuit diagram of tone-specific ACx/MGN-LA pathways. After behavioral labeling, tone-responding ACx/MGN neurons expressed ChR2-eYFP (blue). Local blue light illumination in the amygdala activated ChR2-expressing axons and induced postsynaptic responses in LA neurons (Rec). Horizontal lines indicate ACx/MGN axons projecting to the LA, and vertical lines indicate the dendrites of LA neurons.

To examine the specificity of our behavioral labeling method, we labeled ACx/MGN neurons with different fluorescent proteins during the first and second tone exposures. We injected AAV-pEF1α-DIO-mCherry into ACx and MGN in Fos-CreER× Fos-shGFP mice, which express both CreERand short half-life (2 hr) GFP (shGFP) under the control of the c-Fos promoter () ( Figures 1 I and S1 D). After receiving tamoxifen, these mice were exposed to a 4 or 12 kHz tone or left in home cages (HCs) for mCherry expression in ACx and MGN ( Figures 1 J and S1 E). Two weeks later, the mice were exposed to a 4 kHz tone for shGFP expression, and the brain tissues were fixed 90 min later. The proportion of double-labeled ACx/MGN neurons (mCherry+/shGFP+) among all mCherry+ neurons was significantly higher in mice exposed to the same tone (4 kHz-4 kHz tone) than in mice exposed to different tones (12 kHz-4 kHz tone) or in mice of the HC-4 kHz tone group ( Figures 1 J, 1K, S1 E, and S1F; Table S1 ). These results indicate the specificity of our behavioral labeling approach.

Next, we quantified the proportion of behaviorally labeled neurons among ACx/MGN neurons projecting to the LA. We injected AAV-pEF1α-DIO-eYFP to ACx and MGN and retrograde herpes simplex virus (HSV) encoding the mCherry gene into the LA in Fos-CreERmice ( Figure 1 F). After the behavioral labeling, tone-responding ACx/MGN neurons were labeled with eYFP, whereas LA-projecting neurons were labeled with mCherry ( Figures 1 G and S1 C). Our behavioral labeling resulted in eYFP expression in 14.6% ± 2.3% and 12.5% ± 1.9% of ACx and MGN neurons projecting to the LA, respectively (mean ± SEM, 7 mice; Figure 1 H). The proportion was significantly higher in tone-exposed mice compared with mice left in home cages under auditory deprivation ( Figure 1 H; Table S1 ), indicating that behaviorally labeled neurons included tone-responding ACx/MGN neurons.

To examine synaptic changes in the auditory CS-specific pathways to the LA, we employed a behavioral labeling approach with Fos-CreERknockin mice (). We injected adeno-associated virus (AAV) encoding the eYFP gene in a double inverse open reading frame (DIO) (AAV-pEF1α-DIO-eYFP) into ACx and MGN in heterozygous Fos-CreERmice ( Figure 1 A) and exposed them to a 4 or 12 kHz tone (5 s duration, 15 s interval) for 30 min after tamoxifen administration ( Figures 1 B and S1 A). A population of ACx/MGN neurons responding to the tone expressed CreERunder the control of an activity-dependent endogenous c-Fos promoter, which then induced the recombination of the DIO in the presence of tamoxifen, resulting in permanent eYFP expression ( Figure 1 C). Two weeks after the behavioral labeling, eYFP expression was detected in a subset of ACx/MGN neurons ( Figure 1 D). Within the amygdala, eYFP-labeled projections were found predominantly in the LA ( Figure 1 E). Without tamoxifen injection before tone exposure, eYFP expression was detected in only a few neurons in ACx and MGN ( Figure S1 B).

(J) Top: after tamoxifen injection, mice in the 4 kHz-4 kHz tone group were exposed to the 4 kHz tone, and mice in the 12 kHz-4 kHz tone group were exposed to the 12 kHz tone, whereas mice in the HC-4 kHz tone group were left in home cages (HCs). Two weeks later, mice were exposed to the 4 kHz tone. Middle and bottom: representative images showing ACx neurons labeled with mCherry (red) and/or shGFP (green). ACx neurons activated during the first and second tone exposures expressed both mCherry and shGFP (white circles).

(I) Top: experimental setup for (J) and (K). Bottom: ACx neurons responding to the 4 or 12 kHz tone were first labeled with mCherry (red). Mice were then exposed to the 4 kHz tone for shGFP expression (green).

(G) Top: after tamoxifen injection, mice were exposed to the 4 kHz tone as in (B) (tone exposure group), whereas mice in the control group were left in their home cages. Bottom: magnified images of ACx in the tone exposure group. Tone-responding neurons were labeled with eYFP (green), and LA-projecting neurons were labeled with mCherry (red). Neurons expressing both eYFP and mCherry are marked with white circles.

Discussion

Our results demonstrate that LTP in the CS-specific ACx/MGN-LA pathways could contribute to encoding discriminative fear memory for the CS. Our neural activity-dependent behavioral labeling approach enabled the recording of synaptic responses in the CS-specific pathways and revealed a population of LA neurons that preferentially receives presynaptic inputs conveying specific auditory information. With this approach, we found that postsynaptically expressed LTP was induced selectively in the pathways conveying auditory CS+ information to the LA, whereas LTP was not detected in either the CS– pathways or randomly selected ACx/MGN-LA synapses in discriminative fear conditioning. Input-specific LTP was induced preferentially in a subset of LA neurons activated during auditory fear conditioning. CS-specific ACx/MGN-LA synapses remained potentiated after fear extinction. Moreover, depotentiation of the CS-specific ACx/MGN-LA pathways prevented the recall of fear memory for the CS, suggesting that input-specific LTP is necessary for conditioned fear responses to a specific auditory cue.

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Li B. Experience-dependent modification of a central amygdala fear circuit. One of the most commonly used approaches to discover the synaptic correlates of an associative memory is to examine learning-induced changes in synaptic strength in relevant circuits with electrophysiological recordings in brain slices from trained animals. In conventional recordings, synaptic responses are induced by electrical stimulations of presynaptic inputs and recorded in postsynaptic neurons (). Recent advances in optogenetics enable more selective activations of presynaptic inputs and more accurate recordings of synaptic responses at the neural circuit level (). Even with these approaches, however, it was still challenging to examine synaptic function in functionally defined presynaptic inputs and detect input-specific LTP efficiently with sufficient statistical power. This challenge was overcome in our study with a novel combined approach of behavioral labeling, optogenetic stimulation, and electrophysiological recordings. Neural activity-dependent expression of ChR2 by behavioral labeling enabled selective optogenetic stimulation of the CS-specific pathways in brain slices and the analysis of input-specific synaptic changes in discriminative fear learning, which were previously unattainable through conventional approaches. With our novel approach, we identified a population of LA neurons that receives presynaptic inputs from ACx/MGN neurons responding to a specific auditory stimulus. Our study suggests that heterogeneous populations of LA neurons receive ACx/MGN inputs conveying different auditory CS information while the total number of ACx/MGN inputs to each LA neuron is uniform.