Fig. 2.

Stress-induced and phenotype-dependent freezing behavior is bidirectionally correlated with Orx₁R (Hcrtr1), expressed predominantly in glutamatergic neurons, and Orx₂R (Hcrtr2), more prominent in GABAergic neurons, gene expression in the BLA. A) Phenotype (Escape & Stay) distinctions in SAM-derived socially induced freezing are significant and most pronounced on Day 4 (t₅₆ = 3.8, *p < 0.001). B) In Escape mice, intra-BLA Orx₁R (Hcrtr1) transcription is positively associated with socially induced freezing (social stress-related freezing) in the SAM (F_1,8 = 7.8, R² = 0.4946, p ≤ 0.0233), but this regression in stay mice is not significant (Fig. S2A). C) Conversely, socially induced freezing behavior in the SAM is negatively related to Orx₂R (Hcrtr2) mRNA levels in the BLA of Stay mice (F_1,12 = 9.7, R² = 0.4481, p ≤ 0.0088), and not significant in Escape mice (Fig. S2B). While Orx receptors are associated to phenotype and freezing behavior, D) only a small percentage of the total number of BLA cells contain Orx₁R, Orx₂R, or both receptor subtypes (F_3,44 = 134.0, p < 0.001; significance is by unique symbol, e.g. A is significantly different from B, C, & D). E & F) In the BLA, Orx₁R (green), but not Orx₂R (white), are highly co-expressed with the glutamatergic cell marker CamKIIα (red; some of the observed colocalizations are indicated with solid green arrows = Orx₁R⁺ + CamKIIα⁺, solid white arrow = Orx₂R⁺ +CamKIIα⁺, and unfilled white arrows = Orx₂R⁺ + CamKIIα⁻). (G) The number of BLA Orx₁R⁺ cells expressing CamKIIα is over 60% while Orx₂R⁺ cells co-express the glutamatergic marker ~30% of the time, and about 50% of the small proportion of BLA cells that express both Orx₁R and Orx₂R also express CamKIIα. H & I) Expression of Orx₂R (white) overlaps with GAD67 (red) more than Orx₂R (green) in the BLA (a few observed colocalizations are identified with unfilled green arrows = Orx₁R⁺ + GAD67⁻, solid white arrow = Orx₂R⁺ + GAD67⁺, and unfilled white arrows = Orx₂R⁺ + GAD67⁻). J) Analyses reveal Orx₂R are expressed in GABA neurons in a greater proportion than Orx₁R or cells that co-express Orx₁R & Orx₂R. ⁻p ≤ 0.05 for comparisons to CamKIIα⁻ /GAD67⁻ cells in the same receptor (Orx₁R⁺ or Orx₂R⁺) group; ⁺p ≤ 0.05 for comparisons to Orx₁R⁺ of the same CamKIIα⁺/GAD67⁺ profile; ^#p ≤ 0.05 for comparisons to Orx²R⁺ of the same CamKIIα⁺/GAD67⁺ profile. CeA = central amygdala; ITC = intercalated cells of the amygdala.