Witnessed trauma exposure induces fear in mice through a reduction in endogenous neurosteroid synthesis

Abstract

Neurosteroids have been implicated in the pathophysiology of post-traumatic stress disorder (PTSD). Allopregnanolone is reduced in subsets of individuals with PTSD and has been explored as a novel treatment strategy. Both direct trauma exposure and witnessed trauma are risk factors for PTSD; however, the role of neurosteroids in the behavioral outcomes of these unique experiences has not been explored. Here, we investigate whether observational fear is associated with a reduced capacity for endogenous neurosteroidogenesis and the relationship with behavioral outcomes. We demonstrated that mice directly subjected to a threat (foot shocks) and those witnessing the threat have decreased plasma levels of allopregnanolone. The expression of a key enzyme involved in endogenous neurosteroid synthesis, 5α-reductase type 2, is decreased in the basolateral amygdala, which is a major emotional processing hub implicated in PTSD. We demonstrated that genetic knockdown or pharmacological inhibition of 5α-reductase type 2 exaggerates the behavioral expression of fear in response to witnessed trauma, whereas oral treatment with an exogenous, synthetic neuroactive steroid gamma-aminobutyric acid-A receptor positive allosteric modulator with molecular pharmacology similar to allopregnanolone (SGE-516 [tool compound]) decreased the behavioral response to observational fear. These data implicate impaired endogenous neurosteroidogenesis in the pathophysiology of threat exposure, both direct and witnessed. Further, these data suggest that treatment with exogenous 5α-reduced neurosteroids or targeting endogenous neurosteroidogenesis may be beneficial for the treatment of individuals with PTSD, whether resulting from direct or witnessed trauma.

Keywords: GABA; PTSD; allopregnanolone; neurosteroids; observational fear.

FIGURE 1

Observational threat exposure produces a contextual fear response and impairs endogenous neurosteroid signaling. (A) Diagram and schedule of the observational fear paradigm. (B) Normalized freezing times of naïve (N), observer (O) at the 24- and 96-h timepoints, and demonstrator (D) mice at the 24-h time point. n = 6–9 mice per experimental group. (F (3,27) = 37.47, R2 = 0.8063) using a multiway Analysis of Variance (ANOVA) followed by Tukey’s post hoc test. (C) Serum levels of allopregnanolone in observer (O), demonstrator (D) and naïve (N) mice after observational fear exposure. n = 4–13 mice per experimental group. (F (2,23) = 3.555, R2 = 0.4156) using a multiway ANOVA followed by Tukey’s post hoc test. (D) Transcript levels of Srd5a2 in the BLA in observer (O) and demonstrator (D) mice after undergoing observational fear. Fold change difference were calculated using (F (3, 23) = 0.7399, R2 = 0.5147) using a multiway ANOVA followed by Tukey’s post hoc test. (E) Transcript levels of Gabrd in the BLA in observer (O) and demonstrator (D) mice after undergoing observational fear. (F (3, 22) = 4.695, R2 = 0.5351) using a multiway ANOVA followed by Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a one-sample t-test.

FIGURE 2

Impaired endogenous neurosteroidogenesis alters the contextual observational fear response. (A) Illustration of CRISPR Cas9-mediated homologous recombination used to introduce a tdTomato reporter and lox p sites between Exon 3 and Exon 5 in the floxed Srd5a2 (fl5a2) mouse line. (B) Schematic of the generation of the fl5a2 × CaMKII-Cre mouse strain. (C) Representative image of Srd5a2 (RFP) expression in the BLA of the fl5a2 knock-in mouse. (D) Representative image of CaMKII-Cre (GFP) and 5a2 (RFP) expression in the BLA of the fl5a2 × CaMKII-Cre mouse. (E) Protein expression of 5α-reductase type 2 in the BLA of heterozygous floxed (floxed) mice compared to wild type (Cre-) controls (Ctrl). n = 3 animals per group and *p < 0.05 using a one-sample t-test. (F) Floxed Srd5a2 × CamKII mice exhibit an increase in freezing behavior during 24 h recall following observational fear compared to controls. n = 5–13 animals per group and *p < 0.05 using unpaired t-test.

FIGURE 3

Impaired or enhanced endogenous neurosteroid activity alters the contextual observational fear response. (A) Diagram of the experimental paradigm with schedule of finasteride treatment relevant to observational fear exposure. (B) Mice treated with finasteride also exhibit an increase in freezing behavior during 24 h recall compared to controls treated with vehicle injections following observational fear. n = 8–9 mice per experimental group. (C) Schematic of the time course of SGE-516 administration. (D) The average freezing behavior of observer mice dosed with vehicle during the 24- and 96-h recall sessions in vehicle-treated observer mice (O 24 and O 96) and observers treated with SGE-516 (O 24 SGE and O 96 SGE). n = 14 mice per experimental group. (F (2,8) = 0.3580, R2 = 0.02375) using a multiway ANOVA followed by Tukey’s post hoc test. *p < 0.05 and **p < 0.01.