Propranolol Decreases Fear Expression by Modulating Fear Memory Traces

Abstract

Background: Posttraumatic stress disorder can develop after a traumatic event and results in heightened, inappropriate fear and anxiety. Although approximately 8% of the U.S. population is affected by posttraumatic stress disorder, only two drugs have been approved by the Food and Drug Administration to treat it, both with limited efficacy. Propranolol, a nonselective β-adrenergic antagonist, has shown efficacy in decreasing exaggerated fear, and there has been renewed interest in using it to treat fear disorders.

Methods: Here, we sought to determine the mechanisms by which propranolol attenuates fear by utilizing an activity-dependent tagging system, ArcCreER^T2 x eYFP mice. 129S6/SvEv mice were administered a 4-shock contextual fear conditioning paradigm followed by immediate or delayed context reexposures. Saline or propranolol was administered either before or after the first context reexposure. To quantify hippocampal, prefrontal, and amygdalar memory traces, ArcCreER^T2 x eYFP mice were administered a delayed context reexposure with either a saline or propranolol injection before context reexposure.

Results: Propranolol decreased fear expression only when administered before a delayed context reexposure. Fear memory traces were affected in the dorsal dentate gyrus and basolateral amygdala after propranolol administration in the ArcCreER^T2 x eYFP mice. Propranolol acutely altered functional connectivity between the hippocampal, cortical, and amygdalar regions.

Conclusions: These data indicate that propranolol may decrease fear expression by altering network-correlated activity and by weakening the reactivation of the initial traumatic memory trace. This work contributes to the understanding of noradrenergic drugs as therapeutic aids for patients with posttraumatic stress disorder.

Keywords: Arc; Contextual fear conditioning; Engram; Memory; Propranolol; Trace; c-Fos.

Figure 1.. Administration of propranolol prior to delayed, but not immediate, context re-exposure decreases fear expression in 129S6/SvEv mice.

(A) Experimental design. (B) Injection of propranolol following RE1 does not impact fear expression during RE2. (C) Experimental design. (D) Injection of propranolol prior to RE1 decreases fear expression during RE1 but not during RE2. (E) Experimental design. (F) Injection of propranolol following RE1 does not impact fear expression during RE2. (G) Experimental design. (H) Injection of propranolol prior to RE1 does not impact fear expression during RE1 or RE2. (n = 7-10 male mice per group). **p < 0.01, ***p < 0.001. Sal, saline; P, propranolol; CFC, contextual fear conditioning; RE, context re-exposure; min, minutes.

Figure 2.. Administration of propranolol in a delayed re-exposure alters freezing behavior in a cued fear conditioning paradigm in 129S6/SvEv mice.

(A) Experimental design. (B) There are no differences between groups in freezing behavior during cued fear conditioning in context A. (C) Injection of propranolol before tone test in context B reduced freezing behavior during tone presentation in propranolol-administered mice. Freezing presented in bouts of 15s, and 20s for the tones. (D) Mice that were administered propranolol the previous day showed significantly higher freezing behavior than mice that had received saline during exposure to the tone in context B. (E) The mice were tested again in the original FC context and the propranolol group showed higher freezing levels compared to the saline group. (n = 9-10 male mice per group). *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent ± SEM. Sal, saline; P, propranolol; FC, fear conditioning; min, minutes.

Figure 3.. Administration of propranolol does not affect fear generalization, long-term memory retrieval, or social recognition in 129S6/SvEv mice.

(A) Experimental design. (B) Injection of propranolol prior to the re-exposure decreases freezing behavior in the original CFC context (context A) but not in a different context (context B), and no differences are observed in freezing in long term memory retrieval 30 days later. (C) Injection of propranolol prior to the re-exposure decreases average freezing behavior in the original CFC context (context A) but not in a different context (context B), and no differences are observed in average freezing in long term memory retrieval 30 days later. (n = 9-10 male mice per group). (D) Experimental design. (E) The mice spent significantly more time exploring the cup with the novel mouse compared to the cup with the familiar mouse. No differences between mice administered propranolol or saline. (F) Time spent exploring the familiar mouse. (G) Time spent exploring the novel mouse. (n = 5 male mice per group). *p < 0.05. Error bars represent ± SEM. Sal, saline; P, propranolol; CFC, contextual fear conditioning; LTM, long-term memory; min, minutes.

Figure 4.. Administration of propranolol decreases fear expression and alters memory traces in the dorsal dentate gyrus of ArcCreER^T2 x eYFP mice.

(A) Experimental design. (B) Injection of propranolol prior to the RE1 decreases freezing behavior. (C) Representative image of the dorsal hippocampus. The number of (D) eYFP⁺ cells or (E) c-Fos⁺ cells does not differ in dDG, dCA3, or dCA1 following administration of propranolol. The percentage of (F) co-labeled/c-Fos⁺ cells and (G) co-labeled/eYFP⁺ cells significantly decrease in the dDG following administration of propranolol, but not in dCA3 or dCA1. (H) Representative image of the ventral hippocampus. The number of (I) eYFP⁺ cells or (J) c-Fos⁺ cells does not differ in vDG, vCA1, or vCA3 following administration of propranolol. The percentage of (K) co-labeled/c-Fos⁺ cells and (L) co-labeled/eYFP⁺ cells does not differ in dDG, dCA1 or dCA3 following administration of propranolol. (n = 9 male mice per group). *p < 0.05, **p < 0.01. Error bars represent ± SEM. eYFP, enhanced yellow fluorescent protein; 4-OHT, 4-hydroxytamoxifen; CFC, contextual fear conditioning; RE1, context re-exposure; sac, sacrifice; Sal, saline; P, propranolol; dDG, dorsal dentate gyrus; dCA1, dorsal CA1; dCA3, dorsal CA3; vDG, ventral dentate gyrus; vCA1, ventral CA1; vCA3, ventral CA3.

Figure 5.. Administration of propranolol decreases activity in the ILA of the PFC and in the LA and alters memory traces in the BLA of ArcCreER^T2 x eYFP mice.

(A) Representative image of the PFC. (B) The number of eYFP⁺ cells does not differ in PL, ILA, or ACA following administration of propranolol. (C) The number of c-Fos⁺ cells is decreased in the LA of mice that were administered propranolol, but does not differ in PL or ACA. The percentage of (D) co-labeled/c-Fos⁺ cells and (E) co-labeled/eYFP⁺ cells is unchanged in the PLA, ILA, and ACA after propranolol. (F) Representative images of the amygdala. (G) The number of eYFP⁺ cells in amygdalar nuclei does not change following propranolol administration. (H) The number of c-Fos⁺ cells is decreased in the LA for the propranolol group but does not differ in the remaining nuclei of the amygdala. The percentage of (I) co-labeled/c-Fos⁺ cells was altered in the BLA following propranolol administration, but the percentage of (J) co-labeled/eYFP⁺ cells does not differ in any amygdalar nuclei following administration of propranolol. (n = 9 male mice per group). *p < 0.05. Error bars represent ± SEM. eYFP, enhanced yellow fluorescent protein; Sal, saline; P, propranolol; PL, prelimbic area; ILA, infralimbic area; ACA, anterior cingulate area; LA, lateral amygdalar nucleus; BLA, basolateral amygdalar nucleus; BMA, basomedial amygdalar nucleus; PA, posterior amygdalar nucleus; CEA, central amygdalar nucleus; IA, intercalated amygdalar nucleus.

Figure 6.. Propranolol alters correlated activity between hippocampal, prefrontal and amygdalar regions and the correlation of memory trace reactivation with freezing levels.

(A) Correlation analysis of c-Fos⁺ activity across brain regions in the saline group. (B) Correlation analysis of c-Fos⁺ activity across brain regions in the propranolol group. Square color reflects the Pearson correlation coefficient and asterisks represent a significant correlation. Correlations with R>0.5 (in red) or R<−0.5 (in grey) are displayed in (C) for mice that received saline and in (D) for mice that received propranolol, with line thickness being proportional to strength of correlation (R value). (n = 9 male mice per group). Sal, saline; P, propranolol; dDG, dorsal dentate gyrus; dCA1, dorsal CA1; dCA3, dorsal CA3; vDG, ventral dentate gyrus; vCA1, ventral CA1; vCA3, ventral CA3; ILA, infralimbic area; ACA, anterior cingulate area; LA, lateral amygdalar nucleus; BLA, basolateral amygdalar nucleus; BMA, basomedial amygdalar nucleus; PA, posterior amygdalar nucleus; CEA, central amygdalar nucleus; IA, intercalated amygdalar nucleus.

Figure 7.. Correlations between memory reactivation rates across regions and with behavior.

(A) Correlation of percentage of co-labeled/eYFP⁺ cells during context re-exposure after administration of saline. (B) Correlation of percentage of co-labeled/eYFP⁺ cells during context re-exposure after administration of propranolol. (C) Correlation of percentage of co-labeled/c-Fos⁺ cells during context re-exposure after administration of saline. (D) Correlation of percentage of co-labeled/c-Fos⁺ cells during context re-exposure after administration of propranolol. Square color reflects the Pearson correlation coefficient and asterisks represent a significant correlation. (E) Correlation between percentage of co-labeled/c-eYFP⁺ cells in the ACA and freezing levels. (F) Correlation between percentage of co-labeled/eYFP⁺ cells in the LA and freezing levels. (G) Correlation between percentage of co-labeled/c-Fos⁺ cells in the dCA3 and freezing levels. (n = 9 male mice per group). Sal, saline; P, propranolol; dDG, dorsal dentate gyrus; dCA1, dorsal CA1; dCA3, dorsal CA3; vDG, ventral dentate gyrus; vCA1, ventral CA1; vCA3, ventral CA3; ILA, infralimbic area; ACA, anterior cingulate area; LA, lateral amygdalar nucleus; BLA, basolateral amygdalar nucleus; BMA, basomedial amygdalar nucleus; PA, posterior amygdalar nucleus; CEA, central amygdalar nucleus; IA, intercalated amygdalar nucleus.