IntroductionSocial stress, the most intense and common stressor [1, 2], contributes to widespread expression of anxiety, depression, and post-traumatic stress disorder (PTSD) [3,4,5], highly prevalent, but often ineffectively treated conditions [6,7,8,9,10]. Prospective therapeutic solutions may emerge by targeting specific stress neurocircuitry. The orexin system is intimately involved in stress [11,12,13,14,15,16,17,18,19], motivation [20, 21], sleep/wake cycles [22,23,24,25], feeding [26], and various other attributes associated with psychiatric disorders [27,28,29].Orexin/hypocretin (Orx/Hcrt) neuropeptides, OrxA and OrxB, synthesized in dorsomedial/perifornical (LH-DMH/PeF) hypothalamus [11, 30], innervate broadly [22], contribute to stress neurocircuitry, and influence anxious and depressive responses [31,32,33,34]. In anterior BLA (aBLA), orexin projections promote opposing physiological and behavioral modifications through pro-stress type I receptors (Orx1R) [15] and anti-stress type II receptors (Orx2R) [12, 13, 35,36,37], located respectively on glutamatergic (Camk2α-expressing) and GABAergic (Gad1) neurons [15]. Both OrxRs primarily activate phospholipase C (PLC) signaling cascades, but also potentially modulate cellular systems controlling neural plasticity [18, 38], including extracellular signal-regulated kinase (ERK) [15], cyclic adenosine monophosphate (cAMP), Protein Kinase B (Akt), and mammalian target of rapamycin (mToR) [14,15,16, 22, 39,40,41,42]. However, opposing actions of Orx1R versus Orx2R in aBLA derive mainly from neuronal location [14, 15]. While opposing OrxR functions are not evident in all brain regions [43,44,45], we hypothesize Orx1R versus Orx2R opposition may be predominant in the brain overall, based on previous icv experimental results [13].Distinctive patterns of gene expression for OrxR subtypes (Hcrtr1, Hcrtr2 mRNA) emerge in dorsal hippocampus, implicated in spatial and contextual memory, and crucial components of social stress learning [17, 46]. Furthermore, reciprocal connections between BLA and ventral hippocampus modulate anxiety-related behaviors [47, 48]. The unique neural location of these OrxRs has been hypothesized to balance pro- and anti-stress responses [13,14,15, 18]. Thus, we examine whether Orx1R opposing Orx2R actions are widespread in the brain through actions of systemic or icv treatment, while also probing specific microcircuits within aBLA, hippocampus, and cortex that contain stress-associated genetic markers. These include Wnt-enabler Rspo2 (R-Spondin 2) found in glutamatergic neurons, as well as Akt3 and Mtor, which promote aversive behavioral responses [49,50,51,52], gated by orexinergic activity in BLA microcircuits [53].Inhibition of aBLA Orx1R reduces phenotypic stress-susceptible behaviors, and decreases gene expression of Hcrtr1 and Plcb1, while favoring Hcrtr2, Mapk3, and Bdnf [15,16,17]. Additionally, stimulation of Orx2R balances stress responsiveness through inhibition of pro-stress circuitry, by activating local GABAergic neurons in aBLA [13]. This potential shift of molecular signaling within Hcrtr1 and Hcrtr2 positive neurons, we posit, may lead to the incorporation of Rspo2-Wnt, Akt3, and Mtor activity, and is suggestive of Orx-associated neural plasticity [18]. Thus, both Orx1R inhibition and Orx2R stimulation enhance stress resiliency and limit stress susceptibility [12, 13, 15]. As such, the known circuitry is counterbalanced through OrxR inputs, which modify stress-related physiological and behavioral influences in resilient and vulnerable individuals [14, 15].Pharmacological targets include dual and selective Orx1R and Orx2R antagonists (DORAs and SORAs). Originally designed to treat sleep disorders [54,55,56], but more recently suggested as potential treatments for depression, addiction, and Alzheimer’s disease [27, 29, 55,56,57,58]. However, in some cases, like OrxRs in aBLA and hippocampus [13, 15], antagonism of both receptor subtypes (DORA) may be counterproductive [14] or yield adverse effects [59,60,61,62], limiting their value [58, 60, 62]. Minimal research has addressed the impact of DORAs/SORAs on stress neurocircuitry [15, 16]. Considering functional discrepancies between Orx1R and Orx2R in stress circuitry, and potential complications relative to DORA treatments, we suggest an innovative pharmacological combination of drugs, a selective Orx1R/Orx2R cross-over (SORCO; Orx1R antagonist SB-674042 + Orx2 agonist YNT-185) to target stress-induced disorders [14]. This treatment inhibits pro-stress (via SB-674042) and stimulates anti-stress (via YNT-185) neurocircuitries during social stress, induced through a four-day social defeat/avoidance paradigm, the Stress Alternatives Model (SAM).The potential for stress-vulnerable individuals to be rendered behaviorally resilient is assessed by simultaneously treating pro- and anti-stress microcircuits [15, 63], via cerebrospinal fluid delivery of SORCO drugs [14]. Coincident with behavioral changes following SORCO treatment, we also examined gene expression of Hcrtr1, Hcrtr2, and molecular signaling pathways in aBLA, hippocampus, and cortex. We found Orx1R antagonism and/or Orx2R agonism increases resilient behaviors in stress-vulnerable animals, with potentially synergistic SORCO effects. Behavioral adaptations coincided with an increase in Hcrtr2 gene expression in aBLA, but not hippocampus or cortex, plus increased neuroplasticity-related signaling modulators, such as Wnt signaling activator, Rspo2, along with Akt3 and Mtor in aBLA of stress-vulnerable populations. Thus, a therapeutic potential for SORCO treatment of stress-related behaviors and disorders may devolve from molecular neuroplasticity in limbic neurocircuitry.Materials and methodsAnimalsAdult (8–11 wk) male C57BL6/N mice (Envigo, Indianapolis; N = 100) group housed (5/cage for 7 days) for acclimation, then individually housed on a 12:12 light-dark cycle (lights off 0600) at 22 °C, with ad libitum food and water. Retired Hsd:ICR (CD1) male breeders (Envigo, Indianapolis; N = 73) provided aggression [42] during behavioral experiments, performed in a manner that minimized suffering, the number of animals used, in accordance with NIH Publications No. 80-23 and the IACUC of the University of South Dakota.Intracerebroventricular (icv) stereotaxic surgeriesFollowing acclimation, but prior to handling/exposure to behavioral paradigms, stereotaxic surgery for icv cannula implantation was performed on C57BL/6 N mice. Mice were anesthetized with isoflurane (2% at 1.0 L/min flow rate) and a guide cannula (Plastics One, Roanoke, VA; 26 ga cut to 1.5 mm) was inserted (confirmed, Fig. S1) into the right lateral ventricle (Bregma; AP: -0.50 and ML: -1.0).DrugsGiven via icv guide cannulae with a 1.0 μL syringe (22 ga needle; Hamilton Company, Reno, NV) at a rate of 1 μL/min, 1 h prior to Day 3 SAM social interactions (Fig. 1A), mice received 1.5 nmol/μL Orx1R antagonist (SB-674042, IC50 = 3.76 nM for Orx1R and 531 nM for Orx2R; MedChemExpress, Monmouth Junction, NJ), 50 nmol/μL Orx2 agonist (YNT-185; EC50 = 28 nM; Tocris, Minneapolis, MN), SORCO (SB-674042 [1.5 nmol/μL or 0.0015 M] + YNT-185 [50 nmol/μL or 0.05 M]), or vehicle (artificial cerebrospinal fluid [aCSF] + DMSO), remaining for 90 s after injection. Dose concentrations were adjusted from previous work [13, 15]. Home cage mobility was measured for 3–5 min, 1 h after injection, just prior to SAM exposure. Untreated cage controls allowed comparison of locomotion (Fig. S2), food consumption, or change in body weight (Fig. S3) [13] to vehicle/drug treatments.Fig. 1: Blocking Orx1R and stimulating Orx2R during social defeat increases avoidance behavior in Stay mice.The alternative text for this image may have been generated using AI.Full size imageA Experimental timeline: 4-day SAM interactions followed by SIP/CR Tests (Day 5). The SAM creates two distinctive phenotypes: Escape (stress-resilient) and Stay (stress-vulnerable), which are stabilized by Day 2. Phenotype reversal (Stay to Escape) occurs with anxiolytic drug administration (icv) of Orx1R antagonist (SB-674042; 1.5 nmol/μL), Orx2R agonist (YNT-185; 50 nmol/μL), or combination of Orx1R antagonist and Orx2R agonist (SORCO), given 1 h prior to social interaction on Day 3. B A 50:50 split between phenotype commitment is present on the first day, and more complete on Day 2. C Stay mice reversed phenotype (Stay to Escape) when treated with an Orx1R antagonist on Day 3 (χ 2 = 5.4, *p ≤ 0.020) and Day 4 (χ 2 = 5.4, *p ≤ 0.020), as did Orx2 agonist treatment (Day 3: χ 2 = 6.6, +p ≤ 0.010; Day 4: χ 2 = 6.6, +p ≤ 0.010), and SORCO treatment (Day 3: χ 2 = 13.2, #p