Amygdala-dependent fear is regulated by Oprl1 in mice and humans with PTSD

Abstract

The amygdala-dependent molecular mechanisms driving the onset and persistence of posttraumatic stress disorder (PTSD) are poorly understood. Recent observational studies have suggested that opioid analgesia in the aftermath of trauma may decrease the development of PTSD. Using a mouse model of dysregulated fear, we found altered expression within the amygdala of the Oprl1 gene (opioid receptor-like 1), which encodes the amygdala nociceptin (NOP)/orphanin FQ receptor (NOP-R). Systemic and central amygdala infusion of SR-8993, a new highly selective NOP-R agonist, impaired fear memory consolidation. In humans, a single-nucleotide polymorphism (SNP) within OPRL1 is associated with a self-reported history of childhood trauma and PTSD symptoms (n = 1847) after a traumatic event. This SNP is also associated with physiological startle measures of fear discrimination and magnetic resonance imaging analysis of amygdala-insula functional connectivity. Together, these data suggest that Oprl1 is associated with amygdala function, fear processing, and PTSD symptoms. Further, our data suggest that activation of the Oprl1/NOP receptor may interfere with fear memory consolidation, with implications for prevention of PTSD after a traumatic event.

Fig. 1. Immobilization of mice causes long-term impaired declarative memory

(A and B) Mice were exposed for 2 hours using the immobilization to a wooden board (IMO) procedure, 6 days before testing in the water maze test, which is used to evaluate spatial declarative memory. The water maze procedure lasted for 5 days. (A) Training started with six trials in 1 day of the visible platform test. Results showed no difference in the time to reach the platform between groups, which indicated intact motivation and sensorimotor skills. The day after, the invisible platform training started and consisted of four trials a day for three consecutive days, showing no differences in learning evaluated by time to reach the platform. (B) On days 2 (8 days after IMO) and 3 (9 days after IMO) after training, a probe trial was performed to assess short-term memory. Results showed no differences between IMO and control group in the short-term memory tests [***P < 0.001, Target versus Opposite, repeated-measures analysis of variance (ANOVA)]. On day 5 (11 days after IMO), only a probe trial was performed to assess long-term memory. Results showed that IMO animals spent less time than control animals in the target area (*P < 0.05 versus control in target area, repeated-measures ANOVA followed by Bonferroni post-test). IMO mice spent more time than the control mice in the opposite area of the maze (^#P < 0.05 versus control in opposite area, repeated-measures ANOVA followed by Bonferroni post-test). These data replicate previous findings in rats (10).

Fig. 2. Immobilization causes long-term enhanced anxiety-like symptoms in mice

(A and B) Mice were exposed for 2 hours to immobilization to a wooden board (IMO) 6 days before testing in (A) an elevated plus maze or (B) an open-field test. (A) Five-minute exposure to elevated plus maze revealed that IMO mice exhibited long-term enhanced anxiety according to the ratio of the time spent in the open arms (*P < 0.05, two-tailed Student’s t test). (B) Concordantly, 30-min exposure to an open field resulted in mice showing enhanced anxiety when the time that control or IMO mice spent in the center of the apparatus was calculated (**P < 0.01, two-tailed Student’s t test). These data replicate previous findings in rats (49).

Fig. 3. Differential regulation of Oprl1 in the amygdala during cued-fear conditioning and cued-fear expression in a PTSD-like mouse model

(A) Immobilization-fear conditioning (IMO-FC) and control-fear conditioning (Ctrl-FC) groups equally acquired cued-fear conditioning. IMO in the absence of fear conditioning did not elicit freezing (IMO-NoFC). During the cued-fear expression (Exp) test, the IMO-FC group presented enhanced freezing relative to the nonstressed fear conditioned group (***P ≤ 0.001, IMO-FC versus IMO-NoFC, repeated-measures ANOVA followed by Bonferroni post-test; ⁺P < 0.05, ⁺⁺P ≤ 0.01, +++P ≤ 0.001, IMO-FC versus Ctrl-FC, repeated-measures ANOVA followed by Bonferroni post-test) (n = 10 mice per group). IMO alone did not induce freezing in the fear expression test. The term “microarray” and the line (under fear expression) denote that at 2 hours after fear expression, microarray analysis was performed to identify differential gene expression in the IMO-FC and Ctrl-FC groups. (B) During fear conditioning, the control-fear conditioning group (Ctrl-FC) presents discrimination of the conditioned stimulus 5 (CS5) presentation versus the intertrial interval (ITI) (period between stimulus) (**P ≤ 0.01, repeated-measures ANOVA followed by Bonferroni post-test), whereas the IMO-fear conditioning group (IMO-FC) does not. (C) mRNA microarray analysis of amygdala tissue showing 45,281 transcripts of amygdala mRNA, obtained 2 hours after fear expression (n = 4 mice per group). (D) Selection of the 1963 probes (4.34%) that present statistically significant changes in control-fear expression (Ctrl-Exp) group versus the IMO-fear expression (IMO-Exp) group. (E) Oprl1 mRNA is down-regulated in the control-fear expression (Ctrl-Exp) group versus the IMO-fear expression (IMO-Exp) group (*P < 0.05, ANOVA followed by Bonferroni post-test). (F) Oprl1 mRNA is highly expressed in the mouse central amygdala (red arrow) (12). (G) In replication studies, fear conditioning induces down-regulation of Oprl1 mRNA in the amygdala of control-fear conditioned (Ctrl-FC) mice [*P < 0.05 versus home cage group (HC), ANOVA followed by Bonferroni post-test, n = 8 mice per group]. The mice in the home cage group were undisturbed in the vivarium and had compensatory handling the same days that the IMO mice were exposed to stress. (H) The cued-fear expression test also induced down-regulation of Oprl1 mRNA in the amygdala of the control-fear conditioning group (Crl-FC) (*P < 0.05 versus HC, **P ≤ 0.01 versus IMO-FC, ANOVA followed by Bonferroni post-test; n = 8 mice per group).

Fig. 4. SR-8993 is a new specific and potent NOP-R agonist

(A) SR-8993 is characterized by unusually high selectivity for the NOP-R over the closely related opioid receptors. Structure and physical and pharmacokinetic characteristics of SR-8993 are shown. (B to D) SR-8993 dose-response curves in human embryonic kidney (HEK) cells expressing the NOP/NOP-R (B), the μ opioid receptor (C), and the κ opioid receptor (D).

Fig. 5. The NOP-R agonist SR-8993 impairs cued-fear memory consolidation in mice

The NOP-R agonist SR-8993 (3 mg/kg), when systemically injected, impairs cued-fear memory consolidation but has no effects on anxiety, shock reactivity, or fear acquisition. (A) SR-8993 does not elicit locomotor changes evaluated with the open field (n = 8 mice per group). (B) SR-8993 has no effect on anxiety evaluated by the time spent in the center of the apparatus in the open field (n = 8 mice per group). (C) SR-8993 does not induce changes in pain sensitivity to mild electric footshock evaluated in the startle chamber (n = 8 mice per group). (D) SR-8993 does not alter freezing during cued-fear conditioning but impairs fear memory consolidation when evaluated 48 hours later in the fear expression test (**P < 0.01, two-tailed Student’s t test; n = 8 mice per group). (E) Systemic injection of SR-8993 given immediately after cued-fear conditioning. Upon cue fear expression testing 48 hours later, the immediate post-training SR-8993 impaired fear memory consolidation, as shown by reduced freezing in the fear expression test (*P < 0.05, two-tailed Student’s t test; n = 6 to 7 mice per group).

Fig. 6. SR-8993 impairs cued-fear memory when infused in the central amygdala and in a PTSD-like mouse model

(A) SR-8993 bilaterally injected into the central amygdala immediately after fear conditioning causes impaired fear memory consolidation as shown by the degree of freezing in the cued-fear expression test (*P < 0.05, two-tailed Student’s t test; n = 6 mice per group). (B) Histological verification of SR-8993 infusion sites. The dots indicate the lowest point of the injector tip. Bregma is the anatomical point on the skull at which the coronal suture is intersected perpendicularly by the sagittal suture used as a reference point. (C) Systemic SR-8993 given immediately after fear conditioning in mice with a previous IMO exposure impaired fear memory consolidation as determined by reduced freezing in the fear expression test (*P < 0.05, two-tailed Student’s t test; n = 8 mice per group).

Fig. 7. In humans, OPRL1 is associated with PTSD and altered fear processing in PTSD

(A) The significance level of each of the five OPRL1 SNPs examined is shown as “log(P_interaction)” for the interaction of each genotype and level of self-reported child abuse [using the Childhood Trauma Questionnaire (CTQ)]. For a Bonferroni-corrected P value of 0.01 (for five SNPs), the log(P) would be 2. We find that the rs6010719 SNP survives correction for the interaction test at P ≤ 0.005. (B) Location of SNPs within the OPRL1 gene (average 3.5-kb inter-SNP interval) and location of the gene on chromosome 20. (C) Specific interactions of the G allele carriers (GG, GC) versus CC allele carriers of the rs6010719 SNP in OPRL1, demonstrating that G allele carriers who have experienced greater trauma are at higher risk for PTSD symptoms (F_1,1847 = 10.5; P < 0.001, UNIANOVA). Additionally, we found no interactions between this SNP and variables such as sex, age, or substance abuse that predicted PTSD symptoms (all P values >0.1). (D) G allele carriers of the rs6010719 SNP (n = 49) showed no discrimination between CS+ (danger signal) and CS− (safety signal) measured by the FPS response. In contrast, individuals of the CC genotype (n = 70) showed normal discrimination (interaction: *P < 0.05, ***P ≤ 0.001, ANOVA). (E) OPRL1 mRNA is highly expressed in the human central amygdala [picture modified from Allen Brain Atlas (12, 50)]. The arrow indicates the central amygdala. (F) (Left) Within-group random-effects analysis showed enhanced bilateral amygdala activation in response to fearful versus neutral face stimuli in all participants, irrespective of genotype (local maximum for left amygdala: Z = 4.32; x, y, z = −32, −8, −16; P_corr < 0.05; local maximum for right amygdala: Z = 3.12; x, y, z = 28, −4, −28; P_corr < 0.05). (Right) For fearful versus neutral face stimuli, G carriers (n = 10) have increased functional connectivity between amygdala (seed region) and right posterior insula, relative to CC allele carriers (n = 19) (P_corr < 0.05). Results are overlaid on a representative structural anatomical image in standard Montreal Neurological Institute space.