Orexin/hypocretin receptor modulation of anxiolytic and antidepressive responses during social stress and decision-making: Potential for therapy

Abstract

Hypothalmic orexin/hypocretin (Orx) neurons in the lateral and dorsomedial perifornical region (LH-DMH/PeF) innervate broadly throughout the brain, and receive similar inputs. This wide distribution, as well as two Orx peptides (Orx_A and Orx_B) and two Orx receptors (Orx₁ and Orx₂) allow for functionally related but distinctive behavioral outcomes, that include arousal, sleep-wake regulation, food seeking, metabolism, feeding, reward, addiction, and learning. These are all motivational functions, and tie the orexin systems to anxiety and depression as well. We present evidence, that for affective behavior, Orx₁ and Orx₂ receptors appear to have opposing functions. The majority of research on anxiety- and depression-related outcomes has focused on Orx₁ receptors, which appear to have primarily anxiogenic and pro-depressive actions. Although there is significant research suggesting contrary findings, the primary potential for pharmacotherapies linked to the Orx₁ receptor is via antagonists to block anxious and depressive behavior. Dual orexin receptor antagonists have been approved for treatment of sleep disorders, and are likely candidates for adaptation for affect disorder treatments. However, we present evidence here that demonstrates the Orx₂ receptors are anxiolytic and antidepressive. Using a new experimental pre-clinical model of anxious and depressive behavior stimulated by social stress and decision-making that produces two stable behavioral phenotypes, Escape/Resilient and Stay/Susceptible, we tested the effects of intracerebroventricular injections of Orx₂ agonist and antagonist drugs. Over ten behavioral measures, we have demonstrated that Orx₂ agonists promote resilience, as well as anxiolytic and antidepressive behavior. In contrast, Orx₂ antagonists or knockdown kindle anxious and pro-depressive behavior plus increase susceptibility. The results suggest that the Orx₂ receptor may be a useful target for pharmacotherapies to treat anxiety and depression.

Keywords: Anxiety; Depression; Fear conditioning; Orexin 2 receptor; Social defeat; Stress-Alternatives Model.

Fig. 1—

Intracranial injection of shRNA in the BLA produced knockdown (KD) of the Orx₁ or Orx₂ receptors, with scramble shRNA as control [from (Arendt et al., 2014)]. Intra-BLA KD of Orx₁ receptors (dark gray bars) had no effect on measures of anxious behavior such as A) time spent in the center of the open field (OF; Orx₁:t₁₀ =1.0, p ≥ 0.33), B) Susceptibility / Resilience scores (time near container with a social target / time near a novel container lacking a social target × 100; such that a score < 100 indicates susceptibility, and a score > 100 indicates resilience) in the Social Interaction/Preference test (SIP Orx₁:t₁₀ = 0.9, p ≥ 0.39), or EPM (data not shown), but did C) reduce locomotion (Orx₁:t₁₂ = 2.5, p ≤ 0.033) compared to scramble controls (white bars). Conversely, Orx₂ receptor KD in the BLA (light gray bars) was anxiogenic, reducing A) OF center time (Orx₂: t₁₁ = 2.7, p ≤ 0.021),B) Susceptibility / Resilience scores in SIP test (Orx₂: t₁₁ = 2.4, p ≤ 0.037), but C) did not reduce locomotion (Orx₂: t₁₂ =1.5, p ≥ 0.16) compared to scramble shRNA controls.

Fig. 2—

The Stress-Alternatives Model (SAM) social interaction regimen, begins with A) placement of a young adult male C57BL/6 N mouse in the center of the oval arena, but hidden by an opaque cylinder, while a novel and much larger CD1 mouse is also placed in the arena, but outside of the cylinder. After a 30s acclimation period (during which freezing is measured for comparison), a 5s tone is played as a conditioned stimulus (CS), which is followed by a 10s trace period (freezing is also measured during the tone and trace). The opaque cylinder is then removed and social interaction, including vigorous aggression, ensues for 5 min, repeated with a new CD1 aggressor each day for 4 days. During the first 2 days, mice choose a relatively stable behavioral phenotype, either B) Escape, which consists of making use of one of the available tunnels at the ends of the oval interaction arena and shortening the duration of their aggressive interaction, or C) Stay, in which mice remain in the SAM arena for the full 5 minutes and continues to receive attacks from the CD1. Importantly, both phenotypes receive significant aggression, and the phenotypes, while relatively stable can be reversed by anxiolytic or anxiogenic drugs. Drug treatments occur on day 3, after stable phenotypes have been formed.

Fig. 3—

Results from icv Orx₂ agonist and antagonist experiments (Staton et al., 2018) that activation of Orx₂ receptors is anxiolytic and anti-depressive. Behavioral phenotypes derived from social interaction in the Stress-Alternatives Model (SAM) differed, Stay (clear bar) vs Escape (hatched bar; statistical significance between behavioral phenotypes is represented by differing Roman letters atop the bar, such that A is different from B), with more anxious behavior evident in Stay mice by increased A) Mean (± SEM) freezing (t₁₃ = 2.7, P ≤ 0.018) in response to fear conditioning ([Freezing after CS/Freezing before] × 100); B) Freezing during SAM social aggression (% time freezing; t₁₆ = 2.4, P ≤ 0.03); and C) Startle responses (/min; t₁₄ = 2.7, P ≤ 0.019); but enhanced anxiolytic responses in Escape phenotype mice by increased D) Mean (± SEM) % Time attentive to the hole (t₁₆ = 4.67, P ≤ 0.001), and E) Resilience score in the Social Interaction Preference test (SIP; t₁₈ = 3.82, P < 0.001), but F) did not affect locomotion. Activation of the Orx₂ receptor by Ala¹⁵, D-Leu¹¹-Orx_B (light gray bars) reduces A) Freezing during fear conditioning in Stay mice (F_2,31 = 4.86, P ≤ 0.015; statistical significance between Stay groups is represented by differing Greek letters atop the bar, such that α is different from β), B) Conflict induced freezing (F_2,25 = 4.8, P < 0.017), and C) Startle responses (F_2,21 = 3.89, P ≤ 0.036), but increases D) % Time attentive to the hole (F_2,24 =12.37, P < 0.001), which may promote an 11% increase in escape behavior, and E) SIP resilience score “(time near container with a social target / time near a novel container lacking a social target × 100; such that a score < 100 indicates susceptibility, and a score > 100 indicates resilience) in Stay mice (F_2,30 = 4.6, P ≤ 0.018; comparing Escape mice: t₁₁ = 2.6, P ≤ 0.025). Inhibition of the Orx₂ receptor by MK-1064 (dark gray bars) in Stay mice increases B) Conflict freezing on injection day compared to day 2, and plasma corticosterone (data not shown). In Escape phenotype mice the Orx₂ antagonist (dark gray hatched bar) increases E) Susceptibility score in SIP test (statistical significance between Escape groups is represented by differing Italic letters atop the bar, such that Y is different from Z), and reduces the number of escaping mice by half (data not shown), but F) did not affect locomotion (F_2,35 = 0.39, P ≥ 0.68).