Orexin 2 receptor regulation of the hypothalamic–pituitary–adrenal (HPA) response to acute and repeated stress
Introduction
Orexins (also called hypocretins) are peptides generated from the prepro-orexin precursor that is exclusively localized in cells of the lateral and posterior hypothalamic region (de Lecea et al., 1998, Sakurai et al., 1998). Prepro-orexin is cleaved into two highly structurally related and highly conserved peptides, orexin A and orexin B, which bind to two G-protein-coupled orexin receptors, orexin 1 and orexin 2 receptors (OX1R and OX2R, respectively) (de Lecea et al., 1998, Sakurai et al., 1998). Orexin A has nearly equal affinities for both receptors, while Orexin B has higher affinity for OX2R (Gotter et al., 2012b). While many brain regions express both receptors, some regions exhibit differential expression (Marcus et al., 2001). For example, the cingulate cortex and locus coeruleus selectively express OX1R, while the paraventricular nuclei of the hypothalamus (PVN) and shell neurons of nucleus accumbens preferentially express OX2R (Marcus et al., 2001).
Orexins are implicated in a wide variety of neuroendocrine and behavioral responses including arousal, food intake, cognitive function, autonomic responses, emotional memory, and the stress response (Sakurai, 2014). The most salient phenotype of orexin knockouts is reduced arousal resulting in narcolepsy-like behavior, which has led to the identification and development of OXR antagonists for the pharmacological treatment of insomnia (Gotter et al., 2012a). Conversely, i.c.v. administration of exogenous orexin A to rats or orexin receptor agonists to mice promote arousal and wakefulness (Hagan et al., 1999, Nagahara et al., 2015). Orexins are also important in regulating the neurobiological systems that respond to stressful stimuli. For example, orexins promote the hypothalamic–pituitary–adrenal (HPA) axis response (Winsky-Sommerer et al., 2004, Winsky-Sommerer et al., 2005, Berridge et al., 2010, Johnson et al., 2012). Specifically, central orexin administration (either Orexin A or Orexin B) increases HPA hormones (Jászberényi et al., 2000, Kuru et al., 2000, Spinazzi et al., 2006). Conversely, orexin neurons are activated by corticotropin releasing hormone and respond to stressors such as forced swim stress (Winsky-Sommerer et al., 2005, Chang et al., 2007, Furlong et al., 2009, Chen et al., 2013). Both orexin 1 and 2 receptors have been implicated in acute stress; the role of each receptor appears dependent on the paradigm of stress used (Chang et al., 2007, Gozzi et al., 2013, Bonaventure et al., 2015).
While this evidence indicates a role for orexins in activating the HPA activity under conditions of acute stress, the role of orexin signaling in response to repeated stress is not known. With repeated exposure to moderately intense stressors, individuals typically habituate to that stress as indicated by decreasing responsivity in behavioral, HPA and autonomic measures (Grissom et al., 2008, Grissom and Bhatnagar, 2009). Failure to habituate to a stressor is a hallmark of stress-related illnesses such as post-traumatic stress disorder (PTSD) and panic disorder (Johnson et al., 2012). Moreover, patients with panic anxiety symptoms have higher levels of orexin in their CSF (Johnson et al., 2010). If involved in habituation to a stressor, orexins may be a good target for treatment for some of these stress-related psychiatric illnesses.
To determine the role of orexins in acute and repeated restraint, we first characterized orexin neuronal activation and levels in the CSF in both conditions. We found these measures to be increased with acute restraint, but they significantly decreased with repeated restraint. In order to increase orexin activation prior to each of five daily 30-min restraints (to model higher orexin levels as observed in panic disorder), we used Designers Receptors Exclusively Activated by Designer Drugs (DREADDs) driven by the orexin promoter. We found that even with additional orexin stimulation, HPA activation habituated by day 5 of restraint. However, we were able to delineate the roles of orexin receptors in acute and repeated stress using a selective OX2R-specific antagonist (MK-1064) (Roecker et al., 2014). Prior to this, the roles of orexin receptors in the stress response, whether acute or repeated, had not yet been fully characterized. As OX2R is abundant in the PVN and is known to play a role in arousal, it may mediate excitatory effects at this site where the HPA response is initiated (Shirasaka et al., 2001a, Gotter et al., 2012b). In support of this, we found that OX2R receptor promotes the acute stress response, and it partially prevents habituation to repeated stress only in conditions of high orexin release.
Section snippets
Animals
Adult male Sprague–Dawley rats (225–250 g) were obtained from Charles River Laboratories (Wilmington, MA, USA). Rats were singly housed in polycarbonate enclosures with standard bedding and with food and water available ad libitum. Animals were acclimated to a 12-h light–dark cycle with lights on at 06:15 and lights off at 18:15 in a temperature controlled vivarium for at least 5 days prior to administration of any stress protocols. All experiments took place during the inactive phase between
Orexin levels habituate with repeated stress
Habituation of the HPA response to 5 days of restraint stress was assessed by evaluating plasma ACTH levels prior to, during and after 30 min of restraint stress on day 1 and day 5 of restraint (paradigm and data in Fig. 1A). Consistent with previous results (Grissom and Bhatnagar, 2009), male rats in this paradigm habituated to 5 days of repeated restraint with regard to plasma ACTH levels. This effect was significant at 30 min of restraint. However, we did not observe habituation or group
Discussion
While orexins are known to play a role in the acute stress response, it was unclear how they mediated the response to repeated stressors, and the contribution of orexin receptors to both acute and repeated stress was undetermined. With repeated exposure to modest stressors, individuals typically habituate to that stress by decreasing responsivity, particularly in HPA measures (Grissom et al., 2008, Grissom and Bhatnagar, 2009). Failure to habituate is a hallmark of stress-related illnesses such
Disclosures
ALG, CJW and JJR are employees of Merck & Co., Inc., Kenilworth, NJ (known as Merck Sharp & Dohme Corp. (MSD) outside the United States and Canada), have received salary and research support from the company, and may own stock/stock options in the company.
Acknowledgments
This work was supported by Merck & Co., Inc.. LG was supported by award number T32NS007413 from the National Institute of Neurological Disorders and Stroke (NINDS). The content is the sole responsibility of the authors and does not necessarily represent the official views of the NINDS of the National Institutes of Health. We gratefully acknowledge Lauren Wilson and Jane Dobkin for their help in coding struggle behavior, as well as Amanda Cornfeld for validating DREADDs expression by fluorescent
References (49)
- et al.
Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors
Neuron
(2009) - et al.
Influence of psychogenic and neurogenic stressors on endocrine and immune activity: differential effects in fast and slow seizing rat strains
Brain Behav Immun
(1997) - et al.
Hypocretin/orexin in arousal and stress
Brain Res
(2010) - et al.
Inhibitory effects of an orexin-2 receptor antagonist on orexin A- and stress-induced ACTH responses in conscious rats
Neurosci Res
(2007) - et al.
Pharmacosynthetics: reimagining the pharmacogenetic approach
Brain Res
(2013) - et al.
Hypocretin (orexin) neuromodulation of stress and reward pathways
Curr Opin Neurobiol
(2014) - et al.
Orexin receptors as therapeutic drug targets
Prog Brain Res
(2012) - et al.
Habituation to repeated stress: get used to it
Neurobiol Learn Mem
(2009) - et al.
Struggling behavior during restraint is regulated by stress experience
Behav Brain Res
(2008) - et al.
Possible involvement of orexin in the stress reaction in rats
Biochem Biophys Res Commun
(2000)
Orexin, stress, and anxiety/panic states
Prog Brain Res
Influence of psychogenic and neurogenic stressors on neuroendocrine and central monoamine activity in fast and slow kindling rats
Brain Res
Orexin modulation of adipose tissue
Biochim Biophys Acta
Centrally administered orexin-A activates corticotropin-releasing factor-containing neurons in the hypothalamic paraventricular nucleus and central amygdaloid nucleus of rats: possible involvement of central orexins on stress-activated central CRF neurons
Regul Pept
Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior
Cell
Orexin actions in hypothalamic paraventricular nucleus: physiological consequences and cellular correlates
Regul Pept
Hcrtr1 and 2 signaling differentially regulates depression-like behaviors
Behav Brain Res
Distribution and quantification of immunoreactive orexin A in rat tissues
FEBS Lett
Control of sleep and wakefulness in health and disease
Prog Mol Biol Transl Sci
A selective orexin-1 receptor antagonist attenuates stress-induced hyperarousal without hypnotic effects
J Pharmacol Exp Ther
Orexins (hypocretins) contribute to fear and avoidance in rats exposed to a single episode of footshocks
Brain Struct Funct
The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity
Proc Natl Acad Sci U S A
Changes in CSF hypocretin-1 (orexin A) levels in rats across 24 hours and in response to food deprivation
NeuroReport
Hypocretin/orexin contributes to the expression of some but not all forms of stress and arousal
Eur J Neurosci
Cited by (45)
Targeting the orexin/hypocretin system for the treatment of neuropsychiatric and neurodegenerative diseases: From animal to clinical studies
2023, Frontiers in NeuroendocrinologyHow hypocretin agonists may improve the quality of wake in narcolepsy
2023, Trends in Molecular MedicineSleep-wake and arousal dysfunctions in post-traumatic stress disorder: Role of orexin systems
2022, Brain Research BulletinCitation Excerpt :Increased orexin activity has been linked to impaired sleep in several studies (Prober et al., 2006; Tang et al., 2017), potentially by promoting wakefulness and inhibiting REM sleep (Xi et al., 2001; Lee et al., 2005; Herring et al., 2012) and DORAs enhance sleep onset and maintenance. Orexin activity is directly linked to an increased physiological response through the HPA axis, specifically the PVN (Kuru et al., 2000; Kayaba et al., 2003; Chang et al., 2007; Bonnavion et al., 2015; Grafe et al., 2017). The limitations of these findings are that they are found largely in preclinical animal research and not in humans, and there are some conflicting findings (see Table 1).