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Research Article

Fear extinction reverses dendritic spine formation induced by fear conditioning in the mouse auditory cortex

Cora Sau Wan Lai, Avital Adler, and Wen-Biao Gan
PNAS September 11, 2018 115 (37) 9306-9311; first published August 27, 2018; https://doi.org/10.1073/pnas.1801504115
Cora Sau Wan Lai
aSchool of Biomedical Sciences, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China;
bState Key Laboratory of Brain and Cognitive Sciences, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China;
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  • For correspondence: cora.swlai@hku.hk Wenbiao.Gan@med.nyu.edu
Avital Adler
cSkirball Institute, Department of Neuroscience and Physiology, New York University School of Medicine, New York, NY 10016
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Wen-Biao Gan
cSkirball Institute, Department of Neuroscience and Physiology, New York University School of Medicine, New York, NY 10016
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  • For correspondence: cora.swlai@hku.hk Wenbiao.Gan@med.nyu.edu
  1. Edited by Mu-ming Poo, Chinese Academy of Sciences, Shanghai, China, and approved August 1, 2018 (received for review January 26, 2018)

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Significance

Whether learning-induced changes in neuronal circuits are inhibited or erased during the process of unlearning remains unclear. In this study, we examined the impact of auditory-cued fear conditioning and extinction on the remodeling of synaptic connections in the living mouse auditory cortex. We found that fear conditioning leads to cue-specific formation of new postsynaptic dendritic spines, whereas fear extinction preferentially eliminates these new spines in a cue-specific manner. Our findings suggest that learning-related changes of synaptic connections in the cortex are at least partially reversed after unlearning.

Abstract

Fear conditioning-induced behavioral responses can be extinguished after fear extinction. While fear extinction is generally thought to be a form of new learning, several lines of evidence suggest that neuronal changes associated with fear conditioning could be reversed after fear extinction. To better understand how fear conditioning and extinction modify synaptic circuits, we examined changes of postsynaptic dendritic spines of layer V pyramidal neurons in the mouse auditory cortex over time using transcranial two-photon microscopy. We found that auditory-cued fear conditioning induced the formation of new dendritic spines within 2 days. The survived new spines induced by fear conditioning with one auditory cue were clustered within dendritic branch segments and spatially segregated from new spines induced by fear conditioning with a different auditory cue. Importantly, fear extinction preferentially caused the elimination of newly formed spines induced by fear conditioning in an auditory cue-specific manner. Furthermore, after fear extinction, fear reconditioning induced reformation of new dendritic spines in close proximity to the sites of new spine formation induced by previous fear conditioning. These results show that fear conditioning, extinction, and reconditioning induce cue- and location-specific dendritic spine remodeling in the auditory cortex. They also suggest that changes of synaptic connections induced by fear conditioning are reversed after fear extinction.

  • auditory-cued fear conditioning
  • fear extinction
  • auditory cortex
  • dendritic spine plasticity
  • dendritic spine clustering

Auditory-cued fear conditioning is an associative learning in which a conditioned stimulus (CS; an auditory cue) is paired with the presentation of an unconditioned aversive stimulus (US; a footshock) to elicit a conditioned response (CR; for example a freezing response to CS). Repeated exposures to CS diminish the expression of the CR, resulting in fear extinction (1, 2). Although fear extinction is generally thought to be a new learning process that associates the absence of US with the CS (2⇓⇓⇓⇓⇓–8), several studies suggest that neuronal changes after fear conditioning are partially reversed after fear extinction in the amygdala (9⇓⇓⇓–13). Furthermore, in the mouse frontal association cortex, fear conditioning induces dendritic spine elimination while subsequent fear extinction causes formation of spines near the sites of previously eliminated spines in a cue-specific manner, indicating that fear conditioning and extinction lead to opposing changes at the level of individual synapses (14).

The auditory cortex is the primary cortical region for auditory inputs. Electrophysiological, pharmacological, and optogenetic studies have shown that auditory stimuli paired with footshock or cholinergic stimulation induce robust changes of synaptic strength in the auditory cortex (15⇓⇓–18). Different from the frontal association cortex in which fear conditioning induces dendritic spine elimination (14), recent studies have shown that auditory-cued fear conditioning induces dendritic spine formation in the mouse auditory cortex (19⇓–21). However, the impact of fear extinction on synaptic connections in the auditory cortex has not been examined. Therefore, it remains unclear whether or not fear conditioning-induced synaptic changes might be reversed after fear extinction. Furthermore, several recent studies have suggested that experience-dependent synaptic changes may be clustered along dendritic branches (22⇓⇓–25), but it is not known whether fear conditioning and extinction-induced synaptic changes are distributed along dendritic branches in a clustered fashion.

To better understand experience-dependent synaptic structural plasticity, we examined the formation and elimination of dendritic spines in the auditory cortex in response to auditory-cued fear conditioning and extinction. Consistent with previous studies (19⇓–21), we found that fear conditioning induced the formation of new dendritic spines in the auditory cortex. In addition, new spines induced by fear learning and that persisted over time were clustered along dendritic branches. Importantly, fear extinction preferentially eliminated newly formed spines that were induced by fear conditioning in an auditory cue-specific manner. These findings indicate that clustered changes of synaptic connections induced by fear conditioning are reversed after extinction in the mouse auditory cortex.

Results

Fear Conditioning Induces Dendritic Spine Formation.

Using two-photon microscopy and yellow fluorescent protein-expressing transgenic mice (14, 24, 26), we first examined the effect of fear conditioning on the turnover of dendritic spines located on apical dendrites of layer V pyramidal neurons in the auditory cortex (Fig. 1A and SI Appendix, Fig. S1). In this experiment, mice were first imaged and then subjected to three tones, each paired with a footshock (Fig. 1A). As controls, we used mice subjected to three tones, each temporally unpaired from a footshock (unpaired), three tones only (tone-only), or three shocks only (shock-only). Forty eight hours after training, mice were subjected to a tone-cued recall test to evaluate the conditioned freezing responses, followed by a second imaging session to examine dendritic spine turnover (Fig. 1B). We found that only the paired group, but none of the control groups, showed a significant increase in freezing responses during the recall test, F(3, 24) = 36.82, P < 0.001 (Fig. 1C). Furthermore, only the paired group showed a significant increase in spine formation compared with the unpaired, tone-only, or shock-only groups: 16.44 ± 2.21% versus 9.65 ± 0.56%, 9.15 ± 1.37%, 8.50 ± 1.20%, respectively; F(3, 24) = 35.29, P < 0.001 (Fig. 1D and SI Appendix, Fig. S2A). There was a slight increase of spine elimination in the unpaired group compared with the paired, tone-only, or shock-only groups: 11.99 ± 1.18% versus 9.79 ± 1.69%, 9.65 ± 1.88%, 8.79 ± 1.40%, respectively; χ2(3, 24) = 8.26, P < 0.05. Notably, the percentage of freezing response to the conditioned stimulus was significantly correlated with the percentage of spine formation (r = 0.693, P = 0.003; Fig. 1E), but not spine elimination (r = −0.211, P = 0.432; Fig. 1F). Consistent with previous studies (19⇓–21), these results indicate that fear conditioning induces formation of new spines in the auditory cortex involved in freezing responses.

Fig. 1.
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Fig. 1.

Fear conditioning induces spine formation in the auditory cortex. (A) Experimental design. Postnatal day 30 (P30) mice were imaged and then subjected to fear conditioning [paired, (n = 16), tone-only (n = 4), shock-only (n = 4), and unpaired (n = 4)]. (B) Representative images of dendritic branch. Arrows mark the site of spine formation, while arrowheads mark the site of spine elimination. Asterisks mark filopodia. (Scale bar, 4 µm.) (C and D) Paired group shows significant increase of freezing responses during the recall test and spine formation. (E and F) Spine formation but not spine elimination shows a significant correlation with freezing response. Values represent mean ± SEM (C), mean ± SD (D). One-way ANOVA for C and formation in D, ***P < 0.001. Kruskal–Wallis test for spine elimination in D, *P < 0.05.

Persistent New Spines Induced by Fear Conditioning Are Clustered.

It has been proposed that clustered plasticity allows for more efficient neuronal firing during recall compared with dispersed plasticity (27, 28). Recent study has shown that new spines, formed in response to different motor learning tasks, distribute on different dendritic branches in the primary motor cortex (24). To examine how new spines, formed in response to fear conditioning, are distributed, mice were subjected to fear conditioning with two different auditory cue–US pairings over a period of 4 d (CS1: 4 kHz and CS2: 12 kHz; Fig. 2 A and B). We found that fear conditioning with CS1–US and CS2–US pairings induced dendritic spine formation between days 0 and 2 and between days 2 and 4, respectively (Fig. 2 C and D), F(2, 17) = 6.61, P < 0.01. On the other hand, there was no increase in spine formation between days 2 and 4 if mice were subjected to the same CS1–US pairings as in the previous 2 d (days 0–2) (SI Appendix, Fig. S3). These results suggest that fear conditioning with different auditory cues induce the formation of different new spines in the auditory cortex. Furthermore, when the rates of spine formation on individual dendritic branches induced by CS1–US and CS2–US pairings were examined, the degree of spine formation was not significantly correlated (Fig. 2E; r = −0.015, P = 0.908; 66 branches, ranged 20–68 µm in length, average: 38.83 ± 12.07 µm), indicating that newly formed spines induced by CS1–US and CS2–US pairings are not segregated along different apical dendritic branches of layer V pyramidal neurons.

Fig. 2.
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Fig. 2.

Persisted new spines induced by fear conditioning are clustered within branch segments. (A) Experimental design. Mice were fear conditioned (FC) with CS1 (4 kHz) and CS2 (12 kHz) in two different imaging windows (n = 8). (B) Mice freezing response toward CS1 and CS2 during recall tests (R-1 and R-2). (C) Representative images of dendritic branch after fear conditioning with CS1 and CS2. Arrows mark the site of spine formation, while arrowheads mark the site of spine elimination. Asterisks mark filopodia. (Scale bar, 4 µm.) (D) Fear conditioning with CS1 and CS2 significantly increases the formation rate of dendritic spines. Values represent mean ± SEM (B), mean ± SD (D), one-way ANOVA, *P < 0.05, **P < 0.01. (E) The degree of spine formation on individual branches is not correlated between CS1-FC and CS2-FC. (F) Schematic diagram showing the measurement of distances between newly formed spine pairs (dash lines) induced by CS1-FC and CS2-FC. (G) Cumulative distribution of new spine pair distances between spines formed in response to CS1-FC (observed data, red line) and between simulated spines within each dendritic segment (simulation mean, blue line and SEM, gray envelope). (Inset) Box plot displaying the distribution of simulated new spine pair distances. Red circle marks the median of the observed new spine pair distances in the data. (H–L) Similar to F, cumulative distribution of new spine pair distances between (H) CS2-FC new spines, (I) survived CS1-FC new spines, (J) CS1-FC and CS2-FC new spines, (K) survived CS1-FC new spines and CS2-FC new spines, and (L) eliminated CS1-FC new spines and CS2-FC new spines. The difference between the observed data and the simulation is plotted (black line), showing the deviation peaks at 4 µm in I and at 18 µm in J and K (dashed black line).

Several lines of evidence have shown clustered synaptic plasticity within individual dendrites in vivo (22, 23, 25, 29, 30). To further investigate the distribution of newly formed spines induced by CS1–US or CS2–US pairing in the auditory cortex, we compared the distance measured between new spines formed in response to fear conditioning to the distance measured between simulated new spines independently generated from a random distribution (Fig. 2F and SI Appendix, Materials and Methods). When we examined new spine pair distances in response to CS1 fear conditioning (CS1–CS1), we found that the median values of the observed distances and the simulated distances were not significantly different (observed data, median = 10.1 µm; simulation, median = 10.43 ± 0.03 µm; P = 0.55), as well as their cumulative probabilities (P = 0.29, Fig. 2G and SI Appendix, Fig. S4A). Similar findings were observed for CS2 fear conditioning (CS2–CS2)-induced new spines (observed data, median = 11.1 µm; simulation, median = 11.98 ± 0.03 µm; P = 0.17, Fig. 2H and SI Appendix, Fig. S4B), suggesting new spines are not clustered along dendritic segments in response to auditory-cued fear conditioning.

Interestingly, when we examined CS1–US pairing-induced new spines that survived at day 4, we found they were clustered within 4 μm compared with the simulation (19.1% difference between data and simulation at 4 μm; P = 0.04, Fig. 2I). We also examined the spatial arrangement along individual dendritic segments of new spines formed in response to CS1 fear conditioning with those formed in response to CS2 fear conditioning. The median values of the observed distances and the simulated distances (observed data, median = 12.79 µm; simulation, median = 10.86 ± 0.02 µm; P = 0.002), and their cumulative probabilities (P = 0.02, Fig. 2J and SI Appendix, Fig. S4C) were significantly different, suggesting new spines are formed in a structured manner in response to two cued fear conditionings and are farther apart than expected of a random distribution. Furthermore, when we separately examined the distances between survived or eliminated CS1 new spines to CS2 new spines, we found that CS2 new spines were significantly farther apart from survived CS1 new spines (P = 0.008, Fig. 2K) but not from eliminated CS1 new spine locations (P = 0.78, Fig. 2L) compared with simulated random distribution. Taken together, these data indicate that persistent new spines induced by fear conditioning with one auditory cue are clustered on dendritic branch segments and segregated from new spines induced by fear conditioning with a different auditory cue.

Newly Formed Spines Induced by Fear Conditioning Are Eliminated After Fear Extinction.

To investigate how fear extinction affects dendritic spine plasticity in the auditory cortex, mice were first subjected to fear conditioning as described in the previous experiments. Two days later, these mice were subjected to extinction training through repeated presentation of CS in the absence of footshocks (five trials per day) (Fig. 3A). As expected, the conditioned freezing responses were significantly decreased in the extinction group, compared with the no-extinction group or a control group that was exposed to a different, unconditioned tone (12-kHz tone), F(2, 10) = 21.05, P < 0.001 (Fig. 3B). Notably, we observed that only the extinction group showed a significant increase in spine elimination compared with the no-extinction or unconditioned 12-kHz tone group: 17.48 ± 3.14% versus 8.81 ± 1.81% and 10.60 ± 2.17%, respectively; F(2, 10) = 15.23, P = 0.001 (Fig. 3 C and D and SI Appendix, Fig. S2B). In contrast, there was no significant difference in spine formation among the three groups: 5.87 ± 1.51% versus 8.24 ± 3.53% and 7.86 ± 1.21%, respectively; F(2, 10) = 1.47, P > 0.2 (Fig. 3D). Furthermore, when the elimination of newly formed spines and existing spines were examined separately, we found that extinction training induced no significant difference in the rate of existing spine elimination among the three groups: 6.21 ± 2.21% versus 3.51 ± 1.96% and 5.05 ± 1.10%, respectively; F(2, 10) = 2.34, P > 0.1 (Fig. 3E). However, there was a significant increase in the rate of spine elimination of newly formed spines induced by fear conditioning, compared with the no-extinction and unconditioned 12-kHz tone groups: 11.27 ± 2.29% versus 5.30 ± 0.41% and 5.55 ± 1.17%, respectively; F(2, 10) = 20.61, P < 0.001 (Fig. 3F). Together, these data indicate that fear-extinction training preferentially eliminates newly formed spines induced by fear conditioning.

Fig. 3.
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Fig. 3.

Fear extinction induces elimination of newly formed spine-induced-by-fear conditioning. (A) Experimental design. After fear conditioning and recall, mice were separated into three groups: no-extinction (n = 4), extinction (4 kHz) (n = 5), and unconditioned 12-kHz tone (not paired with footshock, n = 4) groups. (B) The conditioned freezing response significantly decreases in the extinction group, compared with no-extinction group or 12-kHz tone at day 5. (C) Representative images of dendritic branch after fear learning and fear extinction. Arrows mark the site of spine formation, while arrowheads mark the site of spine elimination. Asterisks mark filopodia. (Scale bar, 4 µm.) (D) Extinction significantly increases the rate of spine elimination compared with no-extinction and 12-kHz tone groups. (E) No significant difference in the rate of existing spine elimination among no-extinction, extinction, and 12-kHz tone groups. (F) The rate of newly formed spine elimination is significantly higher in the extinction group compared with no-extinction and 12-kHz tone groups. Values represent mean ± SEM (B), mean ± SD (D–F). One-way ANOVA, **P < 0.01, ***P < 0.001.

Fear Extinction Induces Cue-Specific Spine Elimination.

To further investigate the cue specificity of dendritic spine plasticity induced by fear conditioning and extinction, we fear conditioned mice with two different tone cues (CS1: 4 kHz and CS2: 12 kHz). Subsequently, these mice were subjected to cue-specific extinction training with either CS1 or CS2 (Fig. 4 A and B). We found that mice fear conditioned with two different CS–US pairings and underwent extinction training to either CS1 or CS2 showed reduced freezing response in a cue-specific manner (P < 0.01; Fig. 4 C and D). Notably, when we compared the survival rate of newly formed spines induced by CS1–US or CS2–US pairings after CS1 extinction or CS2 extinction, we found that the survival rate of newly formed spines induced by CS1–US pairing was significantly lower in the CS1 extinction group than in the CS2 extinction group: 30.84 ± 8.07% versus 61.62 ± 6.33% (P < 0.01, Fig. 4E). Similarly, the survival rate of newly formed spines induced by CS2–US pairing was significantly lower in the CS2 extinction group compared with the CS1 extinction group: 35.42 ± 8.84% versus 65.41 ± 16.30% (P < 0.05, Fig. 4E). These data indicate that fear-extinction training preferentially eliminates newly formed spines induced by fear conditioning in a cue-specific manner.

Fig. 4.
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Fig. 4.

Fear extinction induces cue-specific spine elimination. (A) Experimental design. Mice were fear conditioned with CS1 and then conditioned to CS2 over 4 d. Mice were then separated into two groups for either CS1 extinction (n = 4) or CS2 extinction (n = 4). (B) Representative images of dendritic branch after fear conditioning and fear extinction. Arrows mark the site of spine formation, while arrowheads mark the site of spine elimination. Asterisks mark filopodia. (Scale bar, 4 µm.) (C) Freezing response of mice in response to CS1 (R-1), CS2 (R-2), and CS1 or CS2 extinction (Ext-1, Ext-2). (D) Freezing response to CS1 tone or CS2 tone in CS1 extinction and CS2 extinction groups. Fear-extinction training extinguishes freezing response in an auditory cue-specific manner. (E) Survival rate of newly formed spine-induced-by-fear conditioning (CS1-FC and CS2-FC) at day 6 after specific CS extinction. Values represent mean ± SEM (C and D), mean ± SD (E). Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Reconditioning Induces Formation of New Dendritic Spines Near Sites of Spine Formation Induced by the Initial Fear Conditioning.

To investigate cue- and location-specific dendritic spine plasticity induced by fear conditioning and extinction further, we tested the effects of fear reconditioning on dendritic spines in mice subjected to fear extinction (Fig. 5 A and B). In this experiment, mice were first fear conditioned with CS1–US and subsequently underwent CS1 extinction. Next, mice were separated into three groups: (i) reconditioned with CS1–US pairing; (ii) CS1 unpaired with US; and (iii) conditioning with CS2–US. The latter two groups served as the controls. The recall test at day 6 showed that only the reconditioned mice exhibited a significant increase in freezing response to CS1 compared with the unpaired or CS2–US pairing groups (P < 0.01; Fig. 5C and SI Appendix, Fig. S5A). Both reconditioning with CS1–US pairing and conditioning with CS2–US pairing increased the rate of spine formation compared with the unpaired group: 11.82 ± 1.99%, 11.35 ± 1.92% and 7.59 ± 0.76%, respectively; F(2, 9) = 7.75, P < 0.05 (Fig. 5D and SI Appendix, Fig. S5B). There was no significant difference in the rate of spine elimination among the three groups: 7.90 ± 2.07%, 7.39 ± 2.34% and 7.24 ± 1.20%, respectively; F(2, 9) = 0.13, P > 0.8 (Fig. 5D).

Fig. 5.
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Fig. 5.

Reconditioning (Recond.) induces formation of new spine close to the sites of new spine formation induced by initial fear conditioning. (A) Experimental design. Mice were separated into three groups after CS1 fear conditioning and CS1 extinction with imaging sessions in between: unpaired with CS1 (n = 4), reconditioning with CS1 (n = 4), and conditioning with CS2 (n = 4). (B) Representative images of dendritic branch at different imaging time points in three groups: unpaired with CS1, reconditioning with CS1, conditioning with CS2. (Scale bar, 4 µm.) Arrows mark the site of spine formation, while arrowheads mark the site of spine elimination. Asterisks mark filopodia. Red arrowheads mark new spines that were formed after reconditioning and located within 2-µm distance from sites of spine formation induced by the initial fear conditioning. (C) Freezing response to CS1 after CS1 fear conditioning, CS1 extinction, and reconditioning. (D) Reconditioning with CS1 and conditioning with CS2 significantly increase the rate of spine formation but not elimination compared with unpaired group. Values represent mean ± SEM (C) and mean ± SD (D), one-way ANOVA, *P < 0.05, **P < 0.01. (E–G) Cumulative distribution of new spine pair distances between spines formed in response to (E) CS1-FC and unpaired CS1–US, (F) CS1-FC, and new CS2-FC, (G) CS1-FC, and reconditioning of CS1 (observed data, red line) and between simulated spines within each dendritic segment (simulation mean, blue line and SEM, gray envelope), similar to Fig. 2G. The difference between the observed data and the simulation (black line) in G shows the most apparent deviation is for small distances with a peak at 2 µm (dashed black line).

To examine whether reconditioning with CS1–US induces the formation of new spines in close proximity to the location of previously formed new spines, we measured the distances between the newly formed spines from day 4 to day 6 and the newly formed spines induced by the initial CS1–US pairing (from day 0 to day 2) on individual branches (ranging from 15 to 68 µm in length, average length 31.30 ± 9.94 µm) and compared them to randomly distributed simulation data. We found that both the median distances and the cumulative probabilities of the unpaired (Fig. 5E, observed data, median = 8.11 µm; simulation, median = 9.8 ± 0.03 µm; P = 0.57) and the CS2 conditioning (Fig. 5F, observed data, median = 11.6 µm; simulation, median = 10.15 ± 0.03 µm; P = 0.5) groups were not significantly different from randomly distributed simulation data. These data suggest that after fear extinction, unpaired and CS2 conditioning-induced formation of new spines was not clustered along individual dendritic segments relative to previously formed CS1 new spines.

Interestingly, we found that after fear extinction, new spines formed after reconditioning were closer to locations of previously formed CS1 new spines than expected from randomly distributed simulation data (Fig. 5G, observed data, median = 7.32 µm; simulation, median = 9.03 µm; P = 0.031). The difference between the simulation and the observed data for the reconditioning induced new spines and the initial fear conditioning CS1 new spines was evident for shorter distances and peaked at 2 µm, suggesting that reconditioned CS1 new spines were close to the initial fear conditioning-induced CS1 new spines (Fig. 5G, cumulative probabilities P = 0.04). Taken together, these data suggest that fear reconditioning induces reformation of new spines in a cue- and location-specific manner in the auditory cortex.

Discussion

How fear conditioning and extinction cause changes in neuronal circuits and lead to opposing behavioral outcome remains unclear. Consistent with previous studies (19⇓–21), our results show that auditory-cued fear conditioning increases dendritic spine formation of layer V pyramidal neurons in the auditory cortex. We further show that newly formed spines that persisted over time are clustered along individual dendritic branch segments and segregated from new spines induced by fear conditioning with a different auditory cue. The newly formed spines induced by fear conditioning are preferentially eliminated after fear-extinction training in a cue-specific manner. Reconditioning after fear extinction with the same tone cue leads to reformation of spines in close proximity to new spines induced by previous fear conditioning. Together, these findings indicate that fear extinction reverses new spines induced by fear conditioning in a location- and cue-specific manner in the mouse auditory cortex.

It is generally thought that fear extinction represents a form of new learning that inhibits rather than erases memory traces induced by fear conditioning. This view is supported by behavioral evidence of spontaneous recovery and renewal of fear memory after extinction (2⇓⇓⇓⇓⇓–8). In addition, electrophysiological recordings have shown that fear conditioning and extinction modulate activities of nonoverlapping neuronal populations in brain regions, including amygdala (31, 32). On the other hand, it has been shown that fear conditioning and extinction lead to an increase or decrease in neuronal activity in amygdala (9⇓⇓–12). A recent study in the amygdala has provided evidence that a single session of extinction leads to predominantly new learning while multiple sessions of extinction result in the erasure of memory traces (11). Furthermore, in the mouse frontal association cortex, auditory-cued fear conditioning induces dendritic spine elimination, whereas fear extinction causes formation of spines in a location- and cue-specific manner (14). Together, these studies suggest that extinction may cause a partial erasure of fear memory traces (9⇓⇓⇓⇓–14, 33). Our findings that fear extinction preferentially eliminates new spines induced by fear conditioning in the auditory cortex support this view, suggesting that fear conditioning and extinction can lead to opposing remodeling at the level of individual synapses in multiple brain regions.

Clustered structural plasticity of dendritic spines has been observed in multiple brain regions in response to experience (22, 24, 25, 34). For example, an in vivo imaging study has shown that motor training on accelerating rotarod induces branch-specific formation of dendritic spines on layer V pyramidal neurons (24). Furthermore, motor training in a single-pellet reaching task leads to the formation of new spines clustered within dendritic segments (25). Our data show that newly formed spines induced by fear conditioning with different auditory cues are not segregated on different dendritic branches, as opposed to branch-specific new spine formation in response to different motor learning tasks (24). Interestingly, while the initial formation of new spines induced by fear conditioning is not clustered along dendritic segments, those new spines survived over time are clustered on individual dendritic segments. Furthermore, fear reconditioning induces new spine formation near the sites of new spines formed in response to the initial fear conditioning (Fig. 5). Thus, although the initial formation of new spines induced by fear conditioning is not clustered, persistent new spines tend to be clustered within dendritic segments in a cue-specific manner in the auditory cortex.

Recent studies using calcium imaging have shown that functionally related dendritic spines could be clustered or dispersed along dendrites in diverse sensory cortices (29, 30, 35⇓⇓–38). In the primary auditory cortex, dendritic spines tuned to pure-tone stimulation at different frequencies tend to be distributed across dendritic arbors (35). It would be interesting to investigate how new spines induced by fear conditioning are initially distributed and later become clustered in the auditory cortex. A recent study suggests that some of newly formed dendritic spines in the auditory cortex after fear conditioning are specifically connected with axonal projections from the lateral amygdala (21). Thus, factors such as nearby active axonal boutons from amygdala may facilitate the formation of new spines. It is possible that coactivity of new spines and neighboring existing spines may trigger local dendritic signaling to promote the survival of new spines (39⇓–41), leading to the clustering of persistent new spines formed after the CS1–FC training. One important question to be addressed is related to the functionality of new spines induced by fear conditioning (e.g., whether new spines are activated by specific auditory cues used in fear conditioning). The persistent new spines may enhance the responses of layer V pyramidal neurons in the auditory cortex to the inputs from the amygdalocortical projection neurons (21). It has been suggested that the spatial clustering of synaptic inputs can lead to nonlinear input summation on dendrites (27, 42⇓–44) and enhance dendritic computational power and memory recall (27, 34, 45, 46). The clustered new persistent spines induced by fear conditioning in the auditory cortex could therefore represent memory traces and facilitate the process of auditory-cued fear memory recall.

Interestingly, while fear conditioning induces spine formation in the auditory cortex, it leads to spine elimination in the frontal association cortex (14). In contrast to spine formation in the auditory cortex, fear conditioning-induced spine elimination in the frontal association cortex could reduce the activity of layer V pyramidal neurons in response to auditory cues and contribute to the freezing behaviors. Future studies combining calcium and structural imaging of dendritic spines in both cortical regions would be needed to gain insights into the functional significance of spine formation and elimination induced by fear conditioning. Furthermore, spine remodeling induced by fear conditioning is preferentially reversed after fear extinction in a cue- and location-specific manner in both auditory and frontal association cortices (14). Further studies of the mechanisms underlying spine formation and elimination in both cortical regions would provide important understanding of how the reversibility of synaptic connections occurs in specific circuits in response to learning and unlearning.

Materials and Methods

One-month-old male mice expressing YFP (H-line) were used in this study. Mice were purchased from the Jackson Laboratory and group-housed in the Skirball Institute animal facility and Laboratory Animal Unit, University of Hong Kong. All experiments were approved and performed in accordance with New York University School of Medicine’s Institutional Animal Care and Use Committee and University of Hong Kong Committee on the Use of Live Animals in Teaching and Research guidelines. Fear conditioning was conducted with three pairings of a 30-s, 80-dB auditory cue (CS1 = 4,000 Hz or CS2 = 12,000 Hz) coterminating with a 2-s, 0.5-mA scrambled footshock (US). Extinction training was conducted with five CS presentations (each CS lasting 2 min with an intertrial interval of 2 min) per day for 2 d. The procedures of in vivo transcranial two-photon imaging and data quantification were as described previously (14, 24). The Shapiro–Wilk test was used to test for normality of all datasets. For comparison between all datasets with normal distribution, we used either ANOVA with post hoc Tukey’s honestly significant difference test or Student’s t test to compare freezing behavior and spine remodeling among different experimental groups. The Pearson correlation coefficient was used to measure the strength of linear dependence between different variables. For a comparison dataset that does not follow normal distribution, Kruskal–Wallis test followed by post hoc Tukey’s least significant difference test were used. For detailed materials and methods, refer to SI Appendix, Materials and Methods.

Acknowledgments

This work was supported by 5R01NS047325 (to W.-B.G.), Research Grants Council (RGC)/Early Career Scheme (27103715), RGC/General Research Fund (17128816), Health and Medical Research Fund (HMRF) (03143096), and the National Natural Science Foundation of China (NSFC) (31571031) (to C.S.W.L.).

Footnotes

  • ↵1To whom correspondence may be addressed. Email: cora.swlai{at}hku.hk or Wenbiao.Gan{at}med.nyu.edu.
  • Author contributions: C.S.W.L. and W.-B.G. designed research; C.S.W.L. performed research; A.A. contributed new reagents/analytic tools; C.S.W.L., A.A., and W.-B.G. analyzed data; and C.S.W.L., A.A., and W.-B.G. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1801504115/-/DCSupplemental.

Published under the PNAS license.

View Abstract

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Fear extinction reverses dendritic spine formation induced by fear conditioning in the mouse auditory cortex
Cora Sau Wan Lai, Avital Adler, Wen-Biao Gan
Proceedings of the National Academy of Sciences Sep 2018, 115 (37) 9306-9311; DOI: 10.1073/pnas.1801504115

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Fear extinction reverses dendritic spine formation induced by fear conditioning in the mouse auditory cortex
Cora Sau Wan Lai, Avital Adler, Wen-Biao Gan
Proceedings of the National Academy of Sciences Sep 2018, 115 (37) 9306-9311; DOI: 10.1073/pnas.1801504115
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