Experiment 1: the effects of URB597 administered systemically before extinction on fear

We examined the effects of systemically enhancing and blocking the eCB system of rats exposed to shock and reminders on extinction. Drugs were administered immediately following exposure to the second SR (SR2; see Fig. 1a for study design) or 24 h after SR2 (see Fig. 1c for study design). We used a low dose of the CB1r antagonist AM251 (0.3 mg/kg) to block CB1r as previous results have demonstrated that a low dose of this antagonist had no effect on behavior by itself, but it prevented the therapeutic effects of the cannabinoid agonists [6, 13]. Hence, AM251 and URB597 were administered concurrently in order to examine the involvement of CB1rs in the effects of the FAAH inhibitor on fear retrieval and extinction.

Fig. 1 URB597 administered systemically before extinction attenuated fear. a A diagram illustrating the experimental design. Rats were exposed to shock (1.5 mA, 10 s) or not on day 1 and to a situational reminder (SR) on days 3 and 5. One hour after SR on day 5, rats were taken for fear retrieval/first extinction trial (Ret/Ext1). Drugs were i.p. injected 5 min after SR exposure. Additional extinction trials were carried out on days 6 and 7 (Ext2, Ext3). After 72 h another extinction trial was carried out to assess spontaneous recovery (Spon. Recov.). b The Shock-SR Veh, Shock-SR AM and Shock-SR URB + AM groups demonstrated increased latency to enter the dark side on Ret/Ext1 and Ext2 compared to the No Shock-SR Veh and Shock-SR URB groups. Also, the Shock-SR Veh group showed increased latency on Ext3 and Spon. Recov. compared to the No Shock-SR Veh group (*p < 0.05). c A diagram illustrating the experimental design. Rats were exposed to shock (1.5 mA, 10 s) or not on day 1 and to a situational reminder (SR) on days 3 and 5. Twenty-four hours after SR2, rats were i.p. injected with the drugs and taken for fear retrieval/first extinction trial (Ret/Ext1). Additional extinction trials were carried out on days 7 and 8 (Ext2, Ext3). After 72 h another extinction trial was carried out to assess spontaneous recovery (Spon. Recov.). d The Shock-SR Veh group demonstrated increased latency to enter the dark side on Ret/Ext1, Ext2, Ext3, and Spon. Recov. compared to the Shock-SR URB d5 (drugs administered immediately after SR2) and the Shock-SR URB d6 (drugs administered 24 h after SR2) groups (*p < 0.05) Full size image

When drugs were injected immediately after SR2, a two-way ANOVA with repeated measures [(treatment × extinction (5 × 5)] on the latency to enter the dark side revealed significant effects of treatment (F (4,30) = 16.892 , p < 0.001), extinction (F (2,60) = 17.300 , p < 0.001), and interaction (F (8,60) = 2.169 , p < 0.05) (Fig. 1b). Post hoc tests revealed significant differences between the No Shock-SR Veh and Shock-SR URB groups to the other groups on fear retrieval (Ret/Ext1) and Ext2 (p < 0.05). The No Shock-SR Veh group was different from the Shock-SR Veh group on Ext3 and Spon. Recov. (p < 0.05). These findings suggest that URB decreased fear retrieval and this effect persisted throughout extinction training (Ext2 and Ext3), and remained minimal during the spontaneous recovery test. The effects of URB were CB1r-dependent since coadministration of AM251 and URB resulted in a significantly greater latency to enter the dark side than treatment with URB alone on all extinction days. The latency in the Shock-SR AM251 group was not different from the Shock-SR Veh group.

A similar effect was observed when the drugs were administered 24 h after SR2. A two-way ANOVA with repeated measures (3 × 5) on latency revealed significant effects of treatment (F (2,19) = 25.565, p < 0.001), extinction (F (2,38) = 55.987, p < 0.001) and interaction (F (4,38) = 4.856, p < 0.01) (Fig. 1d). Post hoc tests revealed significant differences between the Shock-SR Veh group and the two other groups on Ret/Ext1, Ext2, Ext3, and Spon. Recov. (p < 0.05). Hence, URB decreased fear retrieval and this effect persisted throughout extinction training and did not recuperate during spontaneous recovery. This suggests that administering URB prior to extinction abolished the fear response, even when drug administration was not paired with exposure to the reminder.

As a control experiment we added two groups: (1) injected with vehicle or URB i.p. after SR2 and tested for fear retrieval after 24 h, or (2) exposed to SR2 on day 5, injected i.p. on day 6 and tested for fear retrieval on day 7, 48 h after SR2 (see Fig. S1A for study design).

A two-way ANOVA with repeated measures (3 × 4) on latency revealed significant effects of extinction (F (2,36) = 33.280, p < 0.001), but not of treatment or interaction (Fig. S1B). However, it seems that the Shock-SR URB 24 h group showed decreased latency on Ext2 compared to the two other groups (one way ANOVA: F (2,20) = 3.261, p = 0.05). This suggests that when URB was administered after SR2 and fear retrieval was assessed 24 h later, there was a significant facilitation in extinction with no effect on fear retrieval. When URB was administered 24 h after SR2 and fear retrieval assessed 24 h later (the 48 h group), no effect of URB was observed on fear retrieval or extinction. The findings suggest that URB administered after reminder exposure or prior to fear retrieval facilitate extinction.

Experiment 2: the effects of URB597 microinjected into the hippocampus and amygdala before extinction on fear

We examined the effects of locally microinjecting URB597 and AM251 in the CA1 and BLA of rats exposed to shock and reminders on extinction (see Fig. 2a for study design).

Fig. 2 URB597 microinjected into the hippocampus and amygdala before extinction attenuated fear. a A diagram illustrating the experimental design. Rats were implanted with cannulae into the CA1 or BLA and left for 1-week recovery. Rats were exposed to shock or not on day 1 and to a situational reminder (SR) on days 3 and 5. Immediately after SR exposure on day 5, rats were microinjected with the drugs into the CA1 or BLA and after 1 h taken for fear retrieval/first extinction trial. Additional extinction trials were carried out on days 6 and 7 (Ext2, Ext3). b In the CA1, the No Shock-SR Veh and Shock-SR URB groups demonstrated reduced latency to enter the dark side compared to the Shock-SR Veh, Shock-SR AM251 and Shock-SR URB + AM251 groups on Ret/Ext1 and Ext2. Further, the Shock-SR Veh and Shock-SR URB + AM251 groups demonstrated increased latency compared to the No Shock-SR Veh and Shock-SR URB groups on Ext3 (*p < 0.05). c In the BLA, the No Shock-SR Veh and Shock-SR URB groups demonstrated reduced latency to enter the dark side compared to the Shock-SR Veh, Shock-SR AM251 and Shock-SR URB + AM251 groups on Ret/Ext1 and Ext2. On Ext3, the Shock-SR AM251 group differed from the No Shock-SR Veh and Shock-SR URB groups (*p < 0.05). d Representative schematic drawing of cannula tip positions in the CA1. A coronal view at position −4.30 and −4.16 mm posterior to bregma. e Representative schematic drawing of cannula tip positions in the BLA. A coronal view at position −2.12 and −2.30 mm posterior to bregma Full size image

When drugs were microinjected into the CA1 immediately after SR2, a two-way ANOVA with repeated measures (5 × 3) on latency revealed significant effects of treatment (F (3,36) = 17.351, p < 0.001), extinction (F (2,90) = 62.77, p < 0.001) and interaction (F (6,72) = 5.09, p < 0.001) (Fig. 2b). Post hoc tests revealed significant differences between the Shock-SR Veh and Shock-SR URB groups on all extinction days (p < 0.5). This effect was CB1r-dependent, as co-administration of AM251 and URB resulted in a significantly greater latency than treatment with URB alone on all extinction days (p < 0.05). Latency in the Shock-SR AM251 group was not different from the Shock-SR Veh group.

A very similar and even more profound effect of URB597 was seen when it was injected into the BLA immediately after SR2 (Fig. 2c). Two-way ANOVA with repeated measures (5 × 3) on latency revealed significant effects of treatment (F (4,42) = 22.46, p < 0.001), extinction (F (2,84) = 28.989, p < 0.001), and interaction (F (8,84) = 3.544, p < 0.001). Post hoc tests revealed significant differences in latency during Ret/Ext1 and Ext2 between the Shock-SR URB group and both the Shock-SR Veh and Shock-SR URB + AM251 groups (p < 0.05). The results suggest that intra-BLA URB abolished fear retrieval and that this effect on fear retrieval is CB1 receptor dependent.

Representative schematic drawing of cannula tip positions are shown for the CA1 (Fig. 2d) and BLA (Fig. 2e).

Similar effects were observed when drugs were microinjected 24 h after SR2 (see Fig. S2A for study design). In the CA1, a two-way ANOVA with repeated measures (3 × 4) on latency revealed significant effects of treatment (F (2,18) = 21.932, p < 0.001), extinction (F (2,36) = 15.943, p < 0.001) and interaction (F (4,36) = 5.177, p < 0.01) (Fig. S2B). Post hoc tests revealed a significant increase in latency in the Shock-SR Veh group compared to the other groups on Ret/Ext1, Ext2, and Ext3 (p < 0.05). Hence, URB administered into the CA1 prior to extinction, immediately or 24 h after SR2, attenuated fear.

In the BLA, a two-way ANOVA with repeated measures (3 × 4) on latency revealed significant effects for treatment (F (2,17) = 20.961, p < 0.001), extinction (F (2,36) = 5.749, p < 0.05), and interaction (F (4,36) = 2.994, p < 0.001) (Fig. S2C). Post hoc tests revealed significant differences between the Shock-SR Veh and the other groups on Ret/Ext1 and Ext2 (p < 0.05). As shown in the CA1, URB administered into the BLA prior to extinction, immediately or 24 h after SR2, attenuated fear.

Experiment 3: the effects of URB597 administered systemically before hippocampal LTP induction in rats exposed to shock and reminders

We examined the effects of exposure to shock and reminders on LTP in the CA1 (see Fig. 3a for study design). A three-way ANOVA with repeated measures [(Shock × SR × time (2 × 2 × 12)] on post-HFS EFPs amplitude (Fig. 3b) in the CA1 revealed a significant effect of shock (F (1,32) = 38.608, p < 0.001), SR (F (1,32) = 104.547, p < 0.001) and interaction (F (1,32) = 13.674, p < 0.001). Post hoc analysis revealed a significant difference between the Shock-SR group to all other groups in amplitude (p < 0.05). This indicates that exposure to shock and SRs impaired LTP in the CA1. In addition, the No shock-SR group showed lower amplitude compared with the No shock-No SR and Shock-No SR groups (p < 0.05), suggesting an effect of SR exposure on CA1-LTP.

Fig. 3 The effects of URB597 administered systemically before hippocampal LTP in rats exposed to shock and reminders. a A diagram illustrating the experimental design. Rats were exposed to shock or not on day 1 and to a situational reminder (SR) on days 3 and 5. Immediately after SR exposure on day 5, rats were anesthetized and taken for electrophysiological recording. HFS was induced 1 h after SR. b In the CA1, the Shock-SR group demonstrated significantly reduced amplitude levels compared to all groups post-HFS. The No shock-SR group showed reduced amplitude levels compared with the No shock-No SR and Shock-No SR groups post-HFS (*p < 0.05). c Input–output curve: no significant differences between the groups were found in CA1 EFP amplitude after stimulation of SC input with different stimulus intensities. d Representative signal trace in the CA1 taken before (black line) and 1 h after (gray line) HFS to the SC (calibration: 0.5 mV, 10 ms). e A diagram illustrating the experimental design. Rats were exposed to shock or not on day 1 and to SR on days 3 and 5. Immediately after SR exposure on day 5, rats were anesthetized and taken for electrophysiological recording. Drugs were administered i.p. 30–40 min after SR. HFS was induced 1 h after drug exposure. f In the CA1, the Shock-SR URB group demonstrated significantly increased amplitude levels compared to all groups post-HFS (*p < 0.05). g A diagram illustrating the experimental design. Rats were anesthetized and taken for electrophysiological recording (no shock or SR). Drugs were administered i.p. 30–40 min after SR. HFS was induced 1 h after drug exposure. h In the CA1, the Vehicle group demonstrated significantly increased amplitude levels compared to all groups post-HFS (*p < 0.05). (i) The Shock-SR Veh, Shock-SR AM0.3, and Shock-SR URB + AM0.3 groups demonstrated impaired LTP in the BLA compared to the No Shock-SR Veh group (*p < 0.05) Full size image

A three-way ANOVA with repeated measures on amplitude pre-HFS [shock × SR × time (2 × 2 × 6)] did not reveal any significant effects, suggesting a similar baseline between the groups before HFS was applied.

To examine the effects of stress and SRs on baseline synaptic activity, input–output measurements were taken. A three-way ANOVA with repeated measures [Shock × SR × stimulation intensity (2 × 2 × 5)] revealed a significant effect for stimulation intensity on amplitude (F (4,120) = 49.645, p < 0.001; Fig. 3c). Yet, stimulation of the SC input into the CA1 with different stimulus intensities did not result in any other significant effects on basal EFPs suggesting no effect on baseline synaptic activity. Representative signal traces in the CA1 taken before and 1 h after HFS to the SC are shown (Fig. 3d).

Next, we examined the effects of systemically enhancing and blocking the eCB system in rats exposed to shock and reminders on LTP in the CA1 (see Fig. 3e for study design). Post-HFS analysis (3 × 12) on CA1 EFPs amplitude (Fig. 3f) indicated a significant effect for group (F (3,22) = 20.758, p < 0.001). Post hoc analysis revealed a significant difference between the Shock-SR URB group and the Shock-SR Veh and Shock-SR AM251 groups in amplitude (p < 0.05). These results demonstrate that systemic URB administered immediately after SR2 prevented the shock/SR-induced impairment in LTP.

To evaluate the effects of the drugs on CA1-LTP per se, with no stress exposure, we examined the effects of systemically enhancing (URB 0.3 mg/kg) and blocking (AM251 3 mg/kg) the eCB system on LTP in nonstressed rats (see Fig. 3g for study design). We used a high dose of AM251 (3 mg/kg) to block CB1r activation.

Post-HFS analysis (3 × 12) on EFPs amplitude (Fig. 3h) indicated a significant effect for group (F (2,18) = 11.056, p < 0.001). Post hoc analysis revealed a significant difference between the Vehicle group and the URB and AM251 groups (p < 0.05). This suggests that injecting URB i.p. before HFS attenuated LTP in the CA1, and that the effects of URB on LTP per se is different than the effect observed in stressed rats.

Pre-HFS analysis (3 × 6) on EFPs amplitude did not reveal any significant effects.

Next we examined whether the effects of URB on LTP are CB1r-dependent using a low dose of the CB1r antagonist AM251 (0.3 mg/kg). Post-HFS analysis (3 × 12) on EFPs amplitude (Fig. 3i) indicated a significant effect for group (F (3,24) = 56.888, p < 0.001). Post hoc analysis revealed a significant difference between the No Shock-SR Veh group and all the other groups (p < 0.05), suggesting that AM251 blocked the effects of URB on LTP in the CA1 in stressed rats.

Pre-HFS analysis (3 × 6) on EFPs amplitude did not reveal any significant effects.

As a control experiment, drugs were injected 24 h after SR2 (see Fig. S3A for study design of the Shock-SR URB CA1 d6 group) or immediately after SR2 (see Fig. 3e for study design of the Shock-SR URB CA1 d5 group). Post-HFS analysis (3 × 12) on EFPs amplitude (Fig. S3B) indicated a significant effect of group (F (2,18) = 21.051, p < 0.001). Post hoc analysis revealed a significant difference between the Shock-SR Veh group and all the other groups (p < 0.05), suggesting that URB prevented the effects of shock and reminders on LTP in the CA1 when injected 24 h after the reminder.

Pre-HFS analysis (3 × 6) on EFPs amplitude did not reveal any significant effects.

Experiment 4: the effects of URB597 administered systemically before BLA-LTP induction in rats exposed to shock and reminders

We examined the effects of exposure to shock and SRs on LTP in the BLA (see Fig. 4a for study design). Post-HFS analysis on EFPs amplitude (Fig. 4b) in the BLA revealed significant effects of shock (F (1,22) = 10.039, p < 0.01). Post hoc analysis revealed a significant difference between the Shock-SR group and the other groups (p < 0.05) indicating that the shock-SR group demonstrated higher potentiation levels.

Fig. 4 The effects of URB597 administered systemically before BLA-LTP in rats exposed to shock and reminders. a A diagram illustrating the experimental design. Rats were exposed to shock or not on day 1 and to a situational reminder (SR) on days 3 and 5. Immediately after SR exposure on day 5, rats were anesthetized and taken for electrophysiological recording. HFS was induced 1 h after SR. b In the BLA, the Shock-SR group demonstrated significantly enhanced amplitude levels compared to all groups post-HFS (*p < 0.05). c Input–output curve: no significant differences between the groups were found in BLA EFP amplitude after stimulation of SC input with different stimulus intensities. d Representative signal trace in the BLA taken before (black line) and 1 h after (gray line) HFS to the vSub (calibration: 0.5 mV, 10 ms). e A diagram illustrating the experimental design. Rats were exposed to shock or not on day 1 and to SR on days 3 and 5. Immediately after SR exposure on day 5, rats were anesthetized and taken for electrophysiological recording. Drugs were administered i.p. 30–40 min after SR. HFS was induced 1 h after drug exposure. f In the BLA, the Shock-SR Vehicle group demonstrated significantly increased amplitude levels compared to all groups post-HFS. The Shock-SR AM251 group showed reduced amplitude levels compared to the Shock-SR URB group (*p < 0.05). g A diagram illustrating the experimental design. Rats were anesthetized and taken for electrophysiological recording (no shock or SR). HFS was induced 1 h after drug exposure. h In the BLA, the AM251 group demonstrated significantly reduced amplitude levels compared to all groups post-HFS (*p < 0.05). i The Shock-SR Veh and Shock-SR AM0.3 groups demonstrated enhanced LTP compared to the No Shock-SR Veh group. Further, the Shock-SR Veh group was significantly different from the Shock-SR URB + AM groups (*p < 0.05) Full size image

Pre-HFS analysis on amplitude (2 × 2 × 6) did not reveal any significant effects.

When we examined baseline synaptic activity, a significant effect for stimulation intensity on amplitude was found (F (4,88) = 57.31, p < 0.001; Fig. 4c). Yet, stimulation of the vSub input into the BLA with different stimulus intensities did not result in any other significant effects on basal EFPs suggesting no effect on baseline synaptic activity.

Representative signal traces in the BLA taken before and 1 h after HFS to the vSub are shown (Fig. 4d).

Next, we examined the effects of systemically enhancing and blocking the eCB system in rats exposed to shock and reminders on BLA-LTP (see Fig. 4e for study design).

Post-HFS analysis (3 × 12) on EFPs amplitude (Fig. 4f) indicated a significant effect for group (F (2,22) = 59.55, p < 0.001). Post hoc analysis revealed a significant difference between the Shock-SR Veh group to the Shock-SR URB and shock-SR AM251 groups (p < 0.05). Further, the Shock-SR URB group showed a significant difference in amplitude from the Shock-SR AM251 group (p < 0.05). Thus, URB prevented the effects of the shock/SR on LTP, whereas a high dose of AM251 (3 mg/kg) impaired LTP.

Pre-HFS analysis on amplitude pre-HFS (3 × 6) did not reveal any significant effects.

To evaluate the effects of the drugs on BLA-LTP per se, we examined the effects of systemically enhancing and blocking the eCB system on LTP in nonstressed rats (see Fig. 4g for study design). Post-HFS analysis using two-way ANOVA with repeated measures (3 × 12) on EFPs amplitude (Fig. 3h) indicated a significant effect of group (F (2,17) = 3.982, p < 0.05). Post hoc analysis revealed a significant difference between the Vehicle and the URB groups and the AM251 group (p < 0.05). Hence, injecting a high dose of AM251 (3 mg/kg) before HFS impaired LTP in the BLA.

Pre-HFS analysis on amplitude (3 × 6) did not reveal any significant effects.

Next we examined whether the effects of URB on BLA-LTP are CB1r-dependent using a low dose of AM251 (0.3 mg/kg). Post-HFS analysis (3 × 12) on EFPs amplitude (Fig. 4i) indicated a significant effect of group (F (3,24) = 13.898, p < 0.001). Post hoc analysis revealed a significant difference between the No Shock-SR Veh group and the Shock-SR Veh and Shock-SR AM groups (p < 0.05). Further, the Shock-SR Veh group was significantly different from the Shock-SR URB + AM group (p < 0.05). The results suggest that a low dose of AM251 (0.3 mg/kg) did not block the effects of URB on LTP in the BLA in stressed rats, and that the effects of URB on BLA-LTP in stressed rats is not CB1r-dependent.

Pre-HFS analysis (3 × 6) on EFPs amplitude did not reveal any significant effects.

When drugs were injected 24 h after SR2 (see Fig. S4A for study design), post-HFS analysis (3 × 12) on EFPs amplitude (Fig. S4B) indicated a significant effect for group (F (2,17) = 26.980, p < 0.001). Post hoc analysis revealed a significant difference between the Shock-SR Veh group and all the other groups (p < 0.05), suggesting that URB prevented the effects of shock and reminders on LTP in the BLA when injected 24 h after the reminder.

Pre-HFS analysis (3 × 6) on EFPs amplitude did not reveal any significant effects.

Experiment 5: the effects of exposure to trauma and reminders on endocannabinoid content and hydrolytic enzyme activities in the hippocampus and amygdala

We examined eCB content following exposure to shock and SRs (see Fig. 5a for study design). We used two-way ANOVA [(Shock × SR (2 × 2 × 2)] on eCB content in the CA1, BLA, and serum.

Fig. 5 The effects of exposure to shock and SRs on endocannabinoid content and hydrolytic enzyme activity. a A diagram illustrating the experimental design. Rats were exposed to shock on day 1 and to SR on days 3 and 5. Hundred minutes after SR exposure on day 5, rats were decapitated (i.e., overlapping the time in which HFS was delivered). b In the CA1, the Shock-SR group demonstrated increased 2-AG levels compared to the other groups (*p < 0.05). c In the BLA, no significant effects for 2-AG were found. d In serum, exposure to SRs decreased 2-AG content (*p < 0.05). e In the CA1, exposure to SRs increased AEA content (*p < 0.05). f In the BLA, exposure to shock and/or SRs decreased AEA content (*p < 0.05). g In serum, the Shock-SR group demonstrated decreased AEA levels compared with the No shock-SR group (*p < 0.05). h In the CA1, exposure to Shock increased FAAH activity (*p < 0.05). i In the BLA, exposure to SRs increased FAAH activity (#p = 0.051). j In the CA1, no significant effects for MAGL activity were found. k In the BLA, the Shock-SR group demonstrated decreased MAGL activity compared with the Shock-No SR group (*p < 0.05) Full size image

In the CA1, significant effects were found of shock (F (1,36) = 4.768, p < 0.05), SRs (F (1,36) = 4.193, p < 0.05) and interaction (F (1,36) = 4.309, p < 0.05) on 2-AG content (Fig. 5b). Post hoc revealed a significant difference between the Shock-SR and the other groups (p < 0.05).

In the BLA, no significant effects for 2-AG were found of shock, SR or interaction (Fig. 5c).

In serum, a significant effect of SR (F (1,36) = 14.447, p < 0.001) on 2-AG content was found suggesting that exposure to SRs decreased 2-AG content (Fig. 5d).

In the CA1, AEA content analysis revealed a significant effect of SR (F (1,36) = 6.393, p < 0.05), with no effect of shock or interaction, indicating that exposure to SR increased AEA content (Fig. 5e).

In the BLA, AEA content revealed a significant effect of SR (F (1,36) = 11.064, p < 0.01) and interaction (F (1,36) = 4.501, p < 0.05) with no effect of shock (Fig. 5f). Post hoc revealed a significant difference between the No shock–No SR group and the other groups (p < 0.05).

In serum, AEA content revealed a significant interaction (F (1,36) = 4.199, p < 0.05) with no effect of shock) or SR. Post hoc tests revealed a significant difference between the Shock-SR and No Shock-SR group (p < 0.05; Fig. 5g).

In a different set of rats, eCB enzyme assays were performed following exposure to shock and SRs (see Fig. 5a for study design). We used two-way ANOVA [(Shock × SR (2 × 2)] on FAAH and MAGL activity in the CA1 and BLA.

In the CA1, significant effect of shock (F 1,28) = 4.229, p < 0.05) was found with no effect of SR or interaction on FAAH activity (Fig. 5h).

In the BLA, a significant effect of SR (F (1,37) = 4.043, p = 0.05) was found with no effect of Shock or interaction (Fig. 5i).

In the CA1, no significant effects were found on MAGL activity (Fig. 5j).

In the BLA, a significant interaction was found (F (1,36) = 6.243, p < 0.05) with no effect of Shock or SR. Post hoc revealed a significant difference between the Shock-SR and Shock-No SR group (p < 0.05) (Fig. 5k).

When rats were decapitated 24 h after exposure to SR, no differences were observed in eCB content or activity (data not shown).