RE inactivation impairs encoding of extinction

To explore the role of RE in fear extinction, we first examined whether reversible inactivation of the RE with the GABA A agonist muscimol would impair the acquisition and later retrieval of the extinction memory. Because mPFC-HPC circuits have been implicated in contextual processing2,27, we were particularly interested in whether RE inactivation might influence the context-dependence of the extinction memory. To this end, we examined the effects of RE inactivation on freezing during within-subject retrieval tests conducted in the extinction (ABB) and conditioning (ABA) contexts. Rats were first implanted with a single midline cannula targeting the RE (Fig. 1a, Supplementary Figure 1). After recovery from surgery, rats underwent fear conditioning, extinction, and retrieval testing (Fig. 1b). During fear conditioning (Fig. 1c, left), rats exhibited low levels of freezing behavior prior to the onset of the first conditioning trial, and an increase in freezing across the conditioning trials [repeated measures ANOVA, main effect of trial, F (5, 115) = 36.7, p < 0.001]. The levels of freezing did not differ between the drug groups [F < 1.8], indicating that rats in each group acquired similar levels of conditioned fear. The following day, rats received intra-RE infusions of either saline (SAL) or muscimol (MUS) immediately prior to an extinction training session (45 CS-alone trials) that was conducted in a context different from that used for conditioning. During this session (Fig. 1c, middle), both groups of rats exhibited robust conditioned freezing to the CS in the earliest trial block, and saline-treated rats exhibited a within-session decrease in freezing that is typical of extinction learning. However, inactivation of the RE completely eliminated this within-session decrement in freezing [repeated measures ANOVA, main effect of drug, F (1, 23) = 14.86, p = 0.0008; drug × trial interaction F (9, 20) = 6.46, p < 0.0001].

Fig. 1 Muscimol inactivation of RE impairs encoding of fear extinction. a Representative thionin-stained coronal section showing cannula placement in the RE. The darkfield image shows diffusion of TMRx muscimol in the RE. b Schematized behavioral design. Illustrations are original artwork composed by the authors and adapted from ref27. c (Conditioning, left) Percentage of freezing during the 3-min baseline (BL) and 1-min interstimulus interval (ISI) following each CS-US pairing during the fear conditioning session. (Extinction, middle), Percentage of freezing during the 3-min baseline and 30-s ISIs across 9 extinction blocks (each block represents average freezing of 5 extinction trials) for the extinction session. Arrow indicates the timing of saline (SAL; white circles; n = 14) or muscimol (MUS; red circles; n = 11) infusion before the extinction session. (Retrieval tests, right), Average percentage of freezing for 5 CS test trials in the extinction (retrieval) and conditioning (renewal) contexts. All data are means ± s.e.m.s; *p < 0.05; **p < 0.01; one-way factorial and repeated measures ANOVA Full size image

Twenty-four hours after extinction, rats were tested for their fear to the extinguished CS in both the extinction (retrieval) and conditioning (renewal) contexts. As shown in Fig. 1c (right), rats extinguished under muscimol showed greater levels of CS-elicited freezing compared to control rats and this was particularly pronounced in the extinction context; both groups of rats renewed fear to the extinguished CS outside the extinction context. These observations were confirmed in a repeated measures ANOVA, which revealed main effects of drug [F(1,2) = 5.14; p = 0.03] and test context [F (1,23) = 16.85; p = 0.0004]. Although there was not a reliable drug x test interaction [F (1,23) = 0.89; p = 0.36], planned comparisons revealed a significant difference between SAL and MUS groups during the retrieval session (p = 0.011) but not in the renewal session (p = 0.226). These results reveal that RE inactivation causes a deficit in the acquisition of fear extinction.

RE inactivation impairs extinction retrieval, but not fear renewal

Given the critical role of the RE in encoding an extinction memory, we next examined the role of the RE in the retrieval those memories. Rats were implanted with a single midline cannula targeting the RE and, after recovery from surgery, underwent fear conditioning, extinction, and retrieval testing (see Fig. 2a for behavioral design). RE placements were similar to those in Supplementary Figure 1. Freezing behavior during the conditioning session is shown in Fig. 2b. As before, freezing was low before fear conditioning but significantly increased across the conditioning trials [repeated measures ANOVA, main effect of trial, F (5, 145) = 26.3, p < 0.0001]. The following day the rats received extinction training in a different context. Rats showed high levels of CS-elicited freezing early in the session, but it dramatically decreased by the end of the session [repeated measures ANOVA, main effect of trial, F (9, 261) = 53.0, p < 0.0001], indicating successful within-session extinction. There were no group differences observed during the conditioning and extinction sessions [Fs < 1.6].

Fig. 2 Muscimol inactivation of RE impairs the retrieval of fear extinction. a Schematized behavioral design. Illustrations are original artwork composed by the authors and adapted from ref 27. b (Conditioning, left), Percentage of freezing during the 3-min BL and 1-min ISI following each CS-US pairing during fear conditioning. (Extinction, middle), Percentage of freezing during the 3-min BL and 30-s ISIs during extinction. (Test, right), Percentage of freezing averaged across 5 CS test trials in the extinction (retrieval) and conditioning (renewal) contexts (right). Arrows indicate the timing of saline (SAL; white circles; n = 16) or muscimol (MUS; red circles; n = 15) infusions before retrieval testing. All data are means ± s.e.m.s; * p < 0.05; ** p < 0.01; one-way factorial and repeated measures ANOVA Full size image

On subsequent days, the rats received intra-RE infusions of SAL or MUS immediately prior to retrieval tests in the extinction and conditioning contexts; each rat was tested under the same drug condition for both tests. As shown in Fig. 2b, saline-treated rats exhibited significantly lower fear in the retrieval context relative to the renewal context. Importantly, MUS infusions into RE increased CS-elicited freezing in the extinction context, but not the renewal context. These observations were confirmed in a repeated measures ANOVA that revealed significant main effects of drug [F (1,29) = 8.79; p = 0.006], test context [F (1,18) = 20.2; p = 0.0003] and a significant drug × test interaction [F (1,29) = 4.43; p = 0.04]. Post-hoc comparisons revealed a significant difference between SAL and MUS groups during the retrieval session (p = 0.0024) but not in the renewal session (p = 0.28). These results reveal that RE inactivation causes a deficit in the retrieval of extinction memories. Importantly, the increased freezing produced by RE inactivation was not due to nonspecific reductions in locomotor activity insofar as both pre-CS baseline freezing and fear renewal were unaffected by RE inactivation (Supplementary Fig. 2a). These results indicate that the RE is required for the inhibition of conditioned fear to an extinguished CS.

Muscimol-induced extinction impairments are not state-dependent

The previous results reveal that MUS infusions into the RE impair both the encoding and retrieval of fear extinction, but did not affect fear renewal. It is possible that this pattern of results is due to a shift in interoceptive (i.e., drug) context between extinction and retrieval testing that itself causes fear renewal28. To examine this possibility, we conducted an experiment in which RE inactivation occurred before both the extinction and retrieval sessions. If the interoceptive context associated with RE inactivation is critical for the expression of extinction, then animals that are extinguished and tested after RE inactivation should show normal extinction retrieval.

To examine this possibility, rats were implanted with a single midline cannula targeting the RE and after recovery from surgery underwent fear conditioning, extinction, and retrieval testing. Muscimol was infused in RE prior to both extinction and retrieval sessions. During the extinction session (Fig. 3, middle), we replicated our previous observation that RE inactivation impairs within-session extinction compared to saline controls [repeated measures ANOVA, main effect of group, F (2,60) = 12.8; p < 0.001]. During retrieval testing (Fig. 3, right), animals extinguished and tested under RE inactivation continued to exhibit an extinction impairment relative to SAL-treated controls and exhibited levels of fear comparable to that in rats that did not undergo extinction. These observations were confirmed in an ANOVA performed on the average CS-elicited freezing during the test [main effect of group, F (2,60) = 4.8; p < 0.05]. Post-hoc comparison revealed that SAL-treated rats differed from both MUS-treated and No-ext controls, which did not differ from one another. Importantly, these data indicate the extinction retrieval deficits in muscimol-treated rats are not due to a drug-shift induced renewal, because extinction deficits were observed in animals extinguished and tested in the same drug state. These results indicate that encoding and retrieval deficits after MUS infusions into RE are not due to state-dependent generalization deficits.

Fig. 3 Muscimol-induced extinction impairments are not state-dependent. (Conditioning, left), Percentage of freezing during the 3-min BL and 1-min ISI following each CS-US pairing during the fear conditioning session. (Extinction, middle), Percentage of freezing during the 3-min BL and 30-s ISIs across the extinction session. (Retrieval, right), Percentage of freezing for the 5 CS test trials in the extinction (retrieval) context. Arrows indicate the timing of muscimol (MUS; red circles; n = 20) or saline (SAL; white circles; n = 20) infusions into RE before either extinction training or retrieval testing. No-ext rats (gray squares; n = 23) were placed in the extinction context during but did not receive CS presentations. All data are means ± s.e.m.s; *p < 0.05; **p < 0.01; one-way factorial and repeated measures ANOVA Full size image

Another possibility is that extinguishing the animals outside the extinction context more strongly contextualizes the extinction memory than delivering extinction trials in the conditioning context29. This might increase the sensitivity of extinction to RE inactivation. To examine this issue, we compared the effects of RE inactivation on extinction retrieval in rats that underwent extinction in either the conditioning context (COND) or a novel context (NOVEL); all rats were then tested in their respective extinction contexts (AAA or ABB) and then in a novel renewal context (C) (see Supplementary Figure 3a for behavioral paradigm). Animals were first implanted with cannulas targeting RE and, after recovery from surgery, underwent fear conditioning in context A (Supplementary Figure 3b). On Day 2, animals were extinguished in either the conditioning context (COND) or a novel context (NOVEL). During the extinction session, rats showed high levels of CS-elicited freezing early in the session, but it dramatically decreased by the end of the session indicating successful extinction [repeated measures ANOVA, main effect of trial F (1,11) = 13.07; p = 0.0041]. Freezing in rats extinguished in the conditioning context was significantly higher than that in rats extinguished in the novel context [repeated measures ANOVA, main effect of extinction context F (1,11) = 11.24; p = 0.007], which reflects a summation of context and CS fear in the conditioning context.

On subsequent days, animals received infusions of SAL or MUS (counterbalanced, within-S’s design) and a retrieval test in the extinction context followed by a test in a third novel context (renewal test). During the retrieval test (Supplementary Figure 3b, right), MUS infusions into the RE increased freezing to the extinguished CS independent of the extinction procedure; MUS infusions did not affect the renewal of freezing outside the extinction context. These observations were confirmed in a one-way repeated-measures ANOVA that revealed a main effect of drug [F (1,11) = 37.57; p < 0.001], but no effect of extinction context [F (1,11) = 0.79; p = 0.39] or drug × context interaction [F (1,11) = 2.44; p = 0.15]. During the renewal session, a one-way repeated measures ANOVA revealed no main effect of drug [F (1,11) = 2.98; p = 0.12] or extinction context [F (1,11) = 0.21; p = 0.65] and no interaction between the two variables F (1,11) = 2.13; p = 0.13].

Encoding and retrieval of extinction increases Fos expression in RE

The previous results indicate that RE inactivation impairs both the encoding and retrieval of extinction memories. Here we sought to determine whether RE neurons are activated (as indexed by c-Fos immunohistochemistry) during the encoding and retrieval of extinction memories. To this end, we examined Fos expression in the RE after the extinction training session, as well as after extinction retrieval. In the first case (Fig. 4a), rats underwent auditory fear conditioning followed 24 h later by extinction training; the animals were sacrificed 90 min after the end of the extinction session. Conditioning and extinction of fear were similar to previous experiments (Fig. 4c). As shown in Fig. 4b–d, animals that underwent fear extinction exhibited significantly higher number of Fos+ neurons in the RE compared to home control rats [unpaired t test, t (12) = 5.5, p < 0.001]. Retrieval testing also increased Fos expression in the RE. As shown in Fig. 4e, after conditioning and extinction, conditioned freezing to the extinguished CS was suppressed in the retrieval context and elevated in the renewal context. Interestingly, testing in either the retrieval or renewal contexts increased the number of Fos+ neurons in RE relative to home-cage controls (Fig. 4f). A one-way ANOVA revealed a main effect of group [F (2,25) = 3.8, p < 0.05] and post-hoc comparisons revealed that both SAME and DIFF groups were reliably higher than the HOME control and did not differ from one another. These results reveal that RE neurons are recruited during both encoding and retrieval of extinction memories (including extinction memories undergoing renewal).

Fig. 4 Extinction encoding and retrieval increases c-fos expression in the RE. a Schematized behavioral design. Illustrations are original artwork composed by the authors and adapted from ref 27. b Representative coronal sections showing c-fos labeling in each group. c (Conditioning, left), Percentage of freezing during the 3-min baseline (BL) and 1-min interstimulus interval (ISI) after the last CS-US pairing during fear conditioning. (Extinction, middle), percentage of freezing during the first and last extinction blocks (each block represents average freezing of 5 ISIs) for the extinction training session (black circles, no-ext; open circles, extinction). d Number of c-fos-positive neurons (per mm2) in RE of rats that received extinction (EXT; n = 8) or no extinction (No-ext; n = 6). e Percentage of freezing during fear conditioning (left), extinction (middle; each block represents average freezing of 5 ISIs for the first and last session), and the retrieval test (right). The tests show average freezing for 5 CS test trials in the extinction (retrieval; blue circle) and conditioning (renewal, white square) contexts. f Number of c-fos-positive neurons (per mm2) in the RE in rats that received an extinction retrieval test (RET; n = 11), fear renewal test (REN; n = 11) and home control (Home; n = 6). All the data are means ± s.e.m.s; *p < 0.05; ** p < 0.01; one-way factorial and repeated measures ANOVA Full size image

Extinguished CSs increase single-unit firing in the RE

The previous data indicate that extinguished CSs increase Fos expression in the RE in both the extinction and renewal contexts. However, Fos expression has low temporal resolution and integrates neuronal activity elicited by both the context and CS during retrieval testing. It is therefore possible that RE neurons respond differentially to CSs presented in the extinction and renewal contexts. To examine this possibility, we made single-unit recordings from RE neurons in freely behaving rats using a within-subject design. A schematic illustration of the behavioral paradigm is shown in Fig. 5a. Briefly, animals were implanted with a microwire bundle targeting RE (see Fig. 5b for representative electrode placements). After recovery from surgery, animals underwent auditory fear conditioning followed 24 h later by extinction training.

Fig. 5 Extinction retrieval increases CS-elicited spike firing in the RE. a Schematic behavioral design. Illustrations are original artwork composed by the authors and adapted from ref 27. b Representative coronal sections showing electrode placements in RE. Illustrations are original artwork adapted from open access brain atlas templates44. c Average normalized firing rate among RE neurons (n = 7) across five CS presentations in either the retrieval (blue bars) or renewal context (red bars). Firing rate was binned (50 ms) during a 500 ms pre-CS period and a 1-sec post-CS period. d Average firing rate of RE neurons over 5 trials during the first 200 ms of tone onset (black circles) and percentage of freezing for the 5 trials during testing in retrieval and renewal context (gray circles). All data are means ± s.e.m.s; * p < 0.05; ** p < 0.01; repeated measures ANOVA Full size image

Twenty-four hours after extinction, the rats received an unsignaled reminder shock in context A to facilitate the return of freezing during the renewal test. On the subsequent day, rats were subjected to a within-subject testing procedure wherein the extinguished CS was presented in both the extinction context (retrieval) and a novel context (renewal); single-unit recordings were made during both tests and the same neurons were tracked across sessions. During the retrieval tests (Fig. 5d), rats showed lower levels of freezing in the extinction context relative to the renewal context, though this was not statistically reliable [F (1,2) = 17.10; p = 0.053 for trial 1]. During the retrieval tests we recorded from a total of 27 neurons in RE. The basal firing rate of these neurons was significantly higher in the retrieval (2.88 ± 0.17 Hz) than the renewal (2.42 ± 0.22 Hz) [paired t test; t (26) = −2.3, p < 0.03]. Among this population of cells, seven neurons (25%) exhibited significant increases in firing to the tone CS (defined as an increase in firing rate > 1.96 standard deviations above the 500 ms pre-CS baseline). Interestingly, tone-responsive RE neurons exhibited greater CS-evoked firing within 200 ms of CS onset in the extinction context relative to that in the renewal context (Fig. 5c, d). This observation was confirmed in a one-way repeated measures ANOVA that revealed a main effect of test [F (1,6) = 15.67; p = 0.008] indicating that neurons in RE showed greater CS-evoked responses to an extinguished CS in the extinction context relative to the renewal context.

Silencing RE projectors in the mPFC impairs extinction encoding

The mPFC plays a critical role in extinction learning and retrieval. The RE receives heavy input from the mPFC and this may represent a critical functional input regulating fear extinction. Here we sought to determine whether mPFC projections to the RE are involved in the acquisition and retrieval of fear extinction. Rats received injections of AAV5-Cre-GFP in the RE and AAV8-hSyn-DIO-hM4D(G i )-mCherry in the mPFC 4–5 weeks prior to behavioral training (see Fig. 1b for behavioral design and Fig. 6a for viral expression). Twenty-four hours after auditory fear conditioning [repeated measures ANOVA, main effect of trial, F (5,160) = 35.6; p < 0.001] (Fig. 6b, left), rats received systemic injections of either SAL or CNO and underwent fear extinction. As shown in Fig. 6b (middle), CNO administration increased CS-elicited freezing during the extinction session [repeated measures ANOVA, main effect of drug F (1,32) = 4.15; p = 0.05.

Fig. 6 Silencing mPFC→RE projectors impairs encoding and expression of fear extinction. a Representative images of Cre-dependent DREADD expression; AAV8-hSyn-DIO-hM4D(G i )-mCherry in the mPFC and AAV5-Cre-eGFP virus in the RE. Illustrations are original artwork adapted from open access brain atlas templates44. b (Conditioning, left), Percentage of freezing during the 3-min baseline (BL) and 1-min interstimulus interval (ISI) following each CS-US pairing during the fear conditioning session. (Extinction, middle), Percentage of freezing during the 3-min baseline and 30-s ISIs across 9 extinction blocks (each block represents average freezing of 5 extinction trials) for the extinction training session. (Test, right), Percentage of freezing for 5 CS test trials in the extinction (retrieval) and conditioning (renewal) context. The arrow indicates the timing of the CNO (CNO; yellow circles; n = 15) or saline (SAL; white circles; n = 19) injections before the extinction session. c (Conditioning, left), Percentage of freezing during the 3-min baseline (BL) and 1-min interstimulus interval (ISI) following each CS-US pairing during the fear conditioning session. (Extinction, middle), Percentage of freezing during the first block of three extinction sessions (each block represents average freezing of 5 ISIs). (Retrieval, right), Average percentage freezing during 5 CS test trials during extinction retention tests after either SAL or CNO injection. Shaded panel shows average ISI freezing across 5 ISIs and each gray line represents an individual rat (n = 7). Scale bar represent 0.5 mm for mPFC and 0.25 mm for RE histology images. All the data are means ± s.e.m.s; *p < 0.05; **p < 0.01; repeated measures ANOVA Full size image

During retrieval testing (Fig. 6b, right), all animals exhibited low levels of freezing in the extinction context and increased freezing to the CS in the renewal context [repeated measures ANOVA, main effect of test F (1,32) = 17.57; p = 0.0002]. Interestingly, rats that previously received CNO during extinction training showed higher levels of freezing compared to SAL-treated rats during both of the retrieval tests [repeated measures ANOVA, main effect of drug, F (1,31) = 8.23; p = 0.007]. Post-hoc comparisons revealed that CNO-injected animals showed elevated levels of freezing compared to SAL-injected animals during both retrieval (p = 0.031) and renewal (p = 0.011) sessions. These results are consistent with the effects that we previously showed with RE inactivation alone and reveal that projections from the mPFC to the RE are involved in extinction learning. Furthermore, this effect was not simply a performance effect of CNO (e.g., non-specific increases in freezing), insofar as pre-CS baseline freezing during extinction training was not affected by CNO [unpaired t test; t (32) = 0.18; p = 0.85] and the extinction impairments were manifest during the drug-free retrieval tests.

Silencing RE projectors in the mPFC impairs extinction retrieval

Next, we examined whether mPFC projections to RE also mediate the retrieval of extinction memories. Rats received injections of CAV2-Cre in the RE and AAV8-hSyn-DIO-hM4D(G i )-mCherry in the mPFC 4–5 weeks prior to behavioral training (See Fig. 2a for behavioral design). As shown in Fig. 6c, rats underwent auditory fear conditioning [repeated measures ANOVA, main effect of trial, F (5,30) = 9.0; p < 0.001] and three sessions of extinction [repeated measures ANOVA, main effect of session, F (2,12) = 16.0; p < 0.001]. On the following two days after the last extinction session, rats received extinction retrieval tests using a within-subjects design in which each animal served as its own control. That is, rats were tested after receiving either SAL or CNO in two counterbalanced tests in the extinction context, which were conducted over two days. As shown in Fig. 6c (right), CNO administration impaired the retrieval of the extinction memory and increased freezing in the extinction context [repeated measures ANOVA, main effect of drug, F (1,6) = 7.2; p < 0.05]. Importantly, CNO did not increase baseline freezing prior to CS onset [repeated measures ANOVA, no main effect of drug F (1,6) = 0.55; p = 0.49] (Supplementary Fig. 2b). These results indicate that prefrontal projections to the RE are involved in both the encoding and retrieval of extinction memory.

Silencing mPFC terminals in RE impairs extinction retrieval

The previous experiment reveals that mPFC neurons that project to RE are involved in both the encoding and retrieval of extinction memories. However, silencing these projection neurons might influence mPFC output to other brain areas insofar it has been shown that mPFC neurons send collateral projections to medio-dorsal thalamus and reticular thalamus30. To specifically manipulate mPFC projections to RE, we expressed inhibitory DREADDs (or a blank control) in the mPFC and microinfused CNO into the RE to inactivate mPFC terminals31. A schematic illustration of the behavioral paradigm is shown in Fig. 7a. Rats received injections of AAV8-hSyn-hM4D(G i )-mCherry or AAV8-hSyn-GFP in either PL or IL (Fig. 7b) or both and were implanted with cannula targeting the RE five weeks after viral infusions. Viral infusions in the mPFC produced robust terminal expression in the RE (Supplementary Figure 4).

Fig. 7 Silencing mPFC terminals in RE impairs extinction retrieval. a Schematic behavioral design. Illustrations are original artwork composed by the authors and adapted from ref 27. b Representative images of viral expression in the mPFC. Illustrations were adapted from open access brain atlas templates44. c Average percentage of freezing during the retrieval tests. Freezing during each test is averaged across five CS test trials either SAL or CNO infusions in RE. Scale bars represent 0.5 mm. All the data are means ± s.e.m.s; *p < 0.05; **p < 0.01; repeated measures ANOVA Full size image