In the central nervous system (CNS), the cell bodies of orexin neurons are strictly located in the perifornical area and lateral and dorsal hypothalamic areas (Peyron et al., 1998; Date et al., 1999; Nambu et al., 1999). Orexin neurons receive afferents from GABA, serotonin and catecholamine neurons, and interact in the LHA with neurons that produce melanin concentrating hormone (MCH) and with neurons that express the leptin receptor (LepR) (Bayer et al., 2005; Leinninger and Myers, 2008; Schone et al., 2011, 2012; Burdakov et al., 2013; Karnani et al., 2013). Serotonin (5-HT) hyperpolarizes orexin neurons through 5-HT₁A receptor mediated G-protein-coupled inward rectifier potassium channels (GIRK) in hypothalamic slices prepared from orexin neuron/enhanced green fluorescent protein (EGFP) transgenic mice (Muraki et al., 2004). The majority of orexin-immunoreactive (OX-ir) neurons in LHA are surrounded by dense tyrosine hydroxylase-immunoreactive (TH-ir) axons (Yamanaka et al., 2006), but results of studies on the role of catecholamines in orexin neurons are inconsistent. Bayer et al. showed that noradrenaline depolarized and excited orexin neurons (Bayer et al., 2005), while Yamanaka et al. reported that catecholamines directly and indirectly inhibited orexin neurons via α2-adrenoceptor mediated activation of GIRK channels (Yamanaka et al., 2006).

In the LHA, three major types of neurons OX, MCH, and LepRb expressing neurons are tightly intermingled and have a complex interactive relationship. An emerging hypothesis is that OX-MCH-LepR micro-circuits may act to regulate both autonomic function and energy balance (Leinninger and Myers, 2008; Hall et al., 2010; Leinninger, 2011; Burdakov et al., 2013). OX neurons are most active during wakefulness (Hassani et al., 2009), and activation of orexin receptors promotes wakefulness (De Lecea, 2010, 2012; Sakurai et al., 2010), feeding, and energy metabolism (Tsujino and Sakurai, 2009; Teske et al., 2010; Girault et al., 2012; Nixon et al., 2012), excites breathing, and stimulates sympathetic nerve activity (SNA) leading to an increase in blood pressure (Matsumura et al., 2001; Shirasaka et al., 2002; Zhang et al., 2005; Huang et al., 2010; Shahid et al., 2011, 2012; Nattie and Li, 2012). In contrast, MCH neurons are most active during sleep (Hassani et al., 2009), and MCH promotes sleep or physical inactivity (Nahon, 2006; Peyron et al., 2009; Konadhode et al., 2013; Monti et al., 2013), and regulates the autonomic nervous system. Central administration of MCH via chronic intracerebroventricular infusion (Messina and Overton, 2007) or acute injection into the nucleus of the solitary tract (NTS) (Brown et al., 2007) induces bradycardia and decreases blood pressure. Using a fluorescent-tag to identify LepRb positive neurons, Leinninger and Myers (2008); Leinninger (2011) demonstrated that in the LHA LepRb neurons do not co-express OX or MCH, but rather are co-distributed among the OX neurons (Figure 1). Both LepRb and OX neurons are surrounded by the MCH-containing neurons in the LHA (Leinninger and Myers, 2008; Leinninger, 2011). In addition, LHA LepRb neurons have direct synapses with OX neurons suggesting an important modulatory relationship between LepRb and OX neurons in LHA (Louis et al., 2010).

In contract to the very localized property of the OX neurons, OX axons are widely distributed throughout the CNS, with the exception of the cerebellum (Peyron et al., 1998; Date et al., 1999; Nambu et al., 1999). Based on in situ hybridization both OX₁R and OX₂R mRNAs are distributed extensively in the same regions that contain dense OX innervation (Trivedi et al., 1998; Marcus et al., 2001), e.g., the forebrain, the hypothalamus, the brainstem and the spinal cord. Both OX receptors and efferent projections are found in many sites involved in cardiovascular, respiratory and thermo regulation, e.g., the paraventricular nucleus (PVN), NTS, retrotrapezoid nucleus (RTN), locus coeruleus (LC), Kölliker-Fuse nucleus, rostral ventrolateral medulla (RVLM), medullary raphe, lateral paragigantocellular nucleus, midbrain periaqueductal gray, A5 noradrenergic cell group, parabrachial region, area postrema, intermediolateral cell column of the spinal cord and sympathetic pre-ganglionic neurons (Peyron et al., 1998; Trivedi et al., 1998; Date et al., 1999; Nambu et al., 1999; Marcus et al., 2001). In the brainstem, networks of OX-ir fibers and terminals are expressed on the neurons of all major catecholamine cell groups [adrenaline (Adr): C1, C2, and C3 and noradrenaline (NA): locus coeruleus, A1, A2, A4, A5 and A7] (Puskas et al., 2010). Intracerebroventricular (icv) injection of orexin induces c-fos expression in the locus ceruleus, arcuate nucleus, central gray, raphe nuclei, NTS, supraoptic nucleus (SON), and PVN in Wistar rats (Date et al., 1999). The widespread nature and specific connections of the orexin system suggests that orexin may be involved not only in the regulation of the sleep-wake cycle and appetite but also autonomic functions, particularly cardiorespiratory functions. Orexin may act to link the regulation of cardiorespiratory functions to wakefulness and sleep.

Both orexin projections and orexin receptors are enriched in the neuronal sites that are importantly involved in cardio-respiratory regulation, and they are well positioned to participate in the regulation of cardio-respiratory functions.

The sympathetic nervous system (SNS) plays a crucial role in the regulation of circulation and blood pressure (Guyenet, 2006; Fisher and Paton, 2011; Zubcevic et al., 2011), and many neuronal groups in the lateral hypothaluamus and brainstem are critically involved in such regulation. It is known, that in vivo, electrical or chemical stimulation of the perifornical nucleus of the hypothalamus increases blood pressure and heart rate (HR) and activates neurons of the lateral paragigantocellular area (Sun and Guyenet, 1986; Allen and Cechetto, 1992). Soon after orexin and orexin receptors were discovered in 1998, many studies started to examine if orexin in the LHA participation in the regulation of cardiovascular and sympathetic functions (Samson et al., 1999; Shirasaka et al., 1999; Matsumura et al., 2001). In vitro, orexin in a dose-dependent manner, depolarizes neurons that are involved in the regulation of blood pressure and sympathetic nerve activity (SNA), e.g., neurons in the hypothalamic PVN and the RVLM (Shirasaka et al., 2001; Follwell and Ferguson, 2002; Huang et al., 2010), as well as spinal cord sympathetic preganglionic neurons (Antunes et al., 2001). In the RVLM both OX-A and OX-B depolarize neurons in a dose dependent manner, and at 100 nM, orexin excited ~42% of neurons in the area. Application of an OX₂R antagonist (TCS-OX2-29) significantly reduced the number of neurons activated by OX-A, while co-application of OX₁R and OX₂R antagonists completely eliminated orexin A-induced depolarization (Figure 2) (Huang et al., 2010). Furthermore, about 88% of adrenergic, 43% of noradrenergic, and 36–41% of rhythmically firing RVLM neurons can be excited by orexin in the RVLM (Huang et al., 2010).

Functional studies in vivo have demonstrated that orexin participates in blood pressure regulation. Transgenic orexin deficient animals, both prepro-orexin knockout (pp-OX KO) mice (Figure 3) and rats with orexin neurons-genetically ablated (orexin/ataxin-3), have lower resting blood pressure (Kayaba et al., 2003; Schwimmer et al., 2010) relative to their wild type controls. In both anesthetized and unanesthetized normotensive animals, central administration of OX-A or OX-B activates sympathetic activity and increases both arterial blood pressure (ABP) and HR (Samson et al., 1999; Shirasaka et al., 1999, 2002; Chen et al., 2000; Antunes et al., 2001; Matsumura et al., 2001; Machado et al., 2002; Shahid et al., 2011, 2012). In conscious rats and rabbits (Shirasaka et al., 1999; Matsumura et al., 2001; Samson et al., 2007) central administration (icv) of orexin increases ABP (Samson et al., 1999; Shirasaka et al., 1999; Matsumura et al., 2001), and SNA (Shirasaka et al., 1999; Matsumura et al., 2001) in a dose dependent manner, and the increased ABP, HR and SNA induced by orexin is accompanied by an increase in plasma catecholamines (Figure 4) (Shirasaka et al., 1999; Matsumura et al., 2001). Intravenous injection of a ganglionic-blocking agent, pentolinium, can abolish OX-A induced increases in ABP and plasma epinephrine concentrations, which suggests that the pressor response induced by the icv injection of orexin-A can be attributed primarily to enhanced sympathetic outflow (Matsumura et al., 2001). Focal injection of OX-A into the RVLM can also induce a significant increase in ABP and HR in both anesthetized (Chen et al., 2000; Shahid et al., 2012) and conscious (Machado et al., 2002) rats. In anesthetized rats, the increased ABP is accompanied by increased splanchnic SNA(sSNA) and ventilation (phrenic nerve activity) that can be attenuated by blocking OX₁R (Shahid et al., 2012). The peak effects following OX-A injection into the RVLM were observed at a dose of 50 pmol with ~42 mmHg increase in ABP and 45% increase in SNA (Shahid et al., 2012).

Other studies also directly and indirectly support orexin's role in the regulation of blood pressure and SNA, e.g., intrathecal injection of OX-A elicits a dose-dependent increase in ABP, HR (Antunes et al., 2001; Shahid et al., 2011), and sSNA (Shahid et al., 2011), and the effects can be partially attenuated by either beta-adrenergic or alpha-adrenergic receptor antagonists (Antunes et al., 2001) or OX₁R antagonist (Shahid et al., 2011) in anesthetized rats. Microinjection of OX-A into the medullary raphe significantly increases ABP, HR, and body temperature in unanesthetized rats, (Luong and Carrive, 2012). It is well known that orexins are excitatory neuropeptides that also promote locomotion such as chewing and grooming (Shirasaka et al., 1999; Takakusaki et al., 2005; Thorpe and Kotz, 2005). To exclude the possible effects of increased locomotion on the changes in ABP, HR and SNA induced by the exogenous orexin in conscious rats, Shirasaka et al. injected OX-A icv in both anesthetized and conscious rats and tested them under the same experimental conditions. They found that OX-A induced a similar significant increase in ABP, HR and SNA in both anesthetized and conscious conditions in these rats (Shirasaka et al., 1999), which suggests that the sympathoexcitatory effects induced by exogenous orexin in the CSN are not due to the activation of locomotion.

Orexin is involved in the cardio-respiratory responses to acute stress, e.g., panic and fear (Kuwaki et al., 2008; Furlong et al., 2009; Iigaya et al., 2012; Johnson et al., 2012; Xiao et al., 2013). For example, in rats, silencing the hypothalamic pp-OX (Hcrt gene) with RNAi or antagonizing OX₁Rs can block blood pressure and HR responses to acute panic stress (Johnson et al., 2010). And antagonizing both orexin receptors can: (1) significantly reduce the “bicuculline” induced stress responses, e.g., hypertension, tachycardia, and renal sympathoexcitation (Iigaya et al., 2012); (2) decrease the fear induced pressor, tachycardic, and locomotor responses (Furlong et al., 2009); and (3) decrease the hypercapnic-induced respiratory chemoreflex without affecting resting breathing (Li and Nattie, 2010).

In summary, studies in normal animals have shown that the central orexin system participates in regulation of blood pressure, HR, and SNA. Activation of OXRs in the CNS, or only the area of RVLM, by exogenous orexin causes sympathetically mediated hypertension and tachycardia, which can be attenuated by OXR antagonists. Orexin is necessary for blood pressure, HR and SNA responses to certain stresses, e.g., panic and fear.

The observations that: (a) orexin participates in the regulation of cardiovascular homeostasis, (b) exogenous orexins can increase SNA and blood pressure in normal animals, and (c) transgenic orexin deficient animals have lower resting blood pressure, have led two independent groups to test the hypothesis that orexin may participate in the development of neurogenic hypertension in spontaneously hypertensive rats (SHRs) (Lee et al., 2013; Li et al., 2013a). The SHR is one of the most studied animal models of neurogenic hypertension. As in human essential hypertension, the blood pressure of the SHR rises with age, starting at about 6 weeks, accompanied by an overactive sympathetic nervous system (Smith and Hutchins, 1979; Zicha and Kunes, 1999; Simms et al., 2009). We found (Li et al., 2013a,b) in unanesthetized freely moving adult SHRs that: (1) there is a strong trend toward a significant increase in orexin-A mRNA expression in the RVLM (Figure 5A), a projection site for orexinergic LHA neurons; (2) blocking both orexin receptors by oral administration of an antagonist, Almxt: (a) significantly lowers blood pressure in wakefulness and sleep during both the dark and light periods of the diurnal cycle, (b) the largest average decrease of ABP after blocking orexin receptors was in wakefulness during the dark period (−37 mmHg) and the smallest average change in NREM sleep during the light period (−25 mmHg) relative to the pretreatment baseline, (c) one dose of Almxt produced a remarkable and long-lasting (~8 h) reduction of ABP in SHRs (Figure 6); (3) the anti-hypertensive effect was accompanied by: (a) a significantly decreased SNA assessed by power spectral analysis of systolic ABP, and (b) decreased noradrenaline levels in cerebrospinal fluid and plasma (Figures 5B,C); (4) antagonizing orexin receptors had no effect on resting blood pressure in normotensive WKY rats (Figure 6B). Our findings are supported by a recent publication (Lee et al., 2013), which showed that blocking OX₂R centrally by microinjection of an OX₂R antagonist, TCS-OX2-29, into either the cerebral ventricle (icv) or the RVLM in anesthetized SHRs significantly decreased blood pressure (Figure 7) (Lee et al., 2013). The significant decrease in ABP following TCS-OX2-29 was observed at 3, 10, and 30 nmol doses, and the maximum reduction of ABP was ~21 or ~30 mmHg at 30 nmol with icv or RVLM injection respectively. It is important to note that in both studies antagonism of orexin receptor(s) with either Almxt or TCS-OX2-29 had no significant effect on ABP in conscious or anesthetized normotensive Wistar- Kyoto (WKY) control rats.

There are data on cardiovascular effects of the orexin system obtained from transgenic animals and hypertensive animal models that directly and indirectly support an orexin link to hypertension. Two recent genetic analysis studies showed that orexin related genes are altered in both SHRs and mice (Schlager BPH/2J) (Marques et al., 2011a,b; Yamamoto et al., 2013). Like SHRs, the Schlager high blood pressure mouse (BPH/2J) is a genetic model of neurogenic hypertension. Using affymetrix GeneChip mouse gene arrays Marques et al. (2011b) identified many genes that are differentially expressed in the hypothalamus of the hypertensive mouse relative to the normotensive BPN/3J control mice. Among these altered genes, the pp-OX (Hcrt) gene, which encodes orexin/hypocretin, is significantly increased or up-regulated in the hypothalamus in the hypertensive mice. BPH/2J hypertensive mice also exhibit a larger variation in ABP between the active and inactive periods of the day relative to the normotensive BPN/3J mice, and GeneChip mouse gene arrays showed that the pp-OX/Hcrt gene expression is higher during the active period when ABP is highest than in the inactive period when ABP is lowest in BPH/2J hypertensive mice, and is higher than that of in the normotensive control mice during the same period (Marques et al., 2011a). These genetic and functional studies in the neurogenic hypertensive animal models suggest that an up-regulated or overactive central orexin system may play an important role in developing and maintaining high blood pressure in neurogenic hypertension. It is also interesting to note that Yamamoto et al (Yamamoto et al., 2013) reported that the OX₁R (Hcrtr1) gene is down-regulated in the adrenal gland in SHR and stroke-prone SHR (SHRSP) relative to normotensive WKY rats. This raises an interesting question of the role of peripheral orexin on cardiovascular function. As mentioned above, orexin neurons are exclusive located in the lateral hypothalamus and send projections to many brain locations. However orexins are found in many peripheral tissues, and the peripheral orexin distributions and functions have been discussed by recent reviews (Kukkonen et al., 2002; Spinazzi et al., 2006; Heinonen et al., 2008; Leonard and Kukkonen, 2013). Orexin receptors have been detected in various peripheral tissues, e.g., the gastrointestinal tract, pancreas, kidney, lung, adrenal gland, and adipose tissue (Kirchgessner and Liu, 1999; Lopez et al., 1999; Johren et al., 2001; Ouedraogo et al., 2003; Heinonen et al., 2008). In rat, both OX₁R and OX₂R mRNA can be detected in adrenal gland (Lopez et al., 1999; Johren et al., 2001), and the expression of OX₂R mRNA in the adrenal is about eight times higher in male than that of female rats (Johren et al., 2001, 2004). The adrenal gland is importantly involved in stress-related cardiovascular function via the hypothalamic-pituitary-adrenal axis, and the significance of a down-regulated Hcrt1R gene in adrenal gland of SHRs needs to be further investigated.

Psychological stresses, e.g., anxiety, prolonged anger, mental stress, have long been considered as a contributing factor in developing hypertension (Zimmerman and Frohlich, 1990; Boone, 1991; Markovitz et al., 1993), and as discussed above orexin is involved in cardiovascular and respiratory responses to the acute stresses, e.g., panic, fear, drug or foot-shock induced, in animal models. Xiao et al. recently investigated possible role of orexin on developing stress related hypertension in adult Sprague-Dawley (SD) rats (Xiao et al., 2013). A stress induced-hypertensive rat (SIHR) was generated by intermittent electric foot-shocks (75–150 V, 0.5 ms duration) every 2–30 s and a buzzer noises (88–98 dB) for 2 h twice daily for 14 consecutive days. Adult normotensive SD rats develop hypertension after day 6 under these conditions. Using tail-cuff method the authors found the systolic blood pressure at 2 h after stress rose from 110 mmHg at baseline to 142 mmHg at day 14 in these SIHRs, and the number of OX-A immunoreactive (OXA-ir) neurons in the LHA and the protein level of OX₁R in RVLM were significantly greater than that of the control rats. Microinjection of a selective OX₁R antagonist (SB-408124), or a selective OX₂R antagonist (TCS OX2 29) into RVLM can partially block the OX-A induced increased SBP and HR in SIHR (Xiao et al., 2013). It is interesting to note that, similar to the neurogenic hypertensive model SHRs, SIHRs also developed an overactive central orexin system. It would be interesting to see if blocking both OXRs by Almxt or a combination of OX₁R and OX₂R antagonists can decrease blood pressure between days 6 and 14 in these SIHRs and prevent the development of hypertension.

The potential peripheral effects of orexins on blood pressure and SNA have been investigated (Chen et al., 2000; Matsumura et al., 2001). Intravenous injection of OX-A or OX-B at a dose as high as 11 nmol/kg had no significant effect on ABP, HR or SNA in anesthetized rats and conscious rabbits, which suggests that the orexin effects on ABP, HR, and SNA are mediated primarily through the CNS (Chen et al., 2000; Matsumura et al., 2001). At the present time there is little evidence to support a role for peripheral orexins in the regulation of cardiovascular function (Heinonen et al., 2008; Kukkonen, 2013).

In summary: (1) Transgenic orexin deficient animals have a lower resting blood pressure. (2) Blocking OXRs significantly lowers blood pressure and SNA in adult SHRs. (3) PP-OX mRNA and gene expression are upregulated in the CNS in hypertensive rats and mice. Based on these findings we suggest that an overactive orexin system in the CNS may participate in the pathogenesis and maintenance of high blood pressure in certain forms of hypertension. Further, modulation of the orexin system could be a potential target in treating some forms of hypertension.

In normal animals, activation of OXRs by orexin in the CNS or in areas critically involved in SNA regulation, e.g., the RVLM and spinal cord, induces sympathetically mediated hypertension and tachycardia, which can be attenuated by OXR antagonists. Transgenic orexin deficient animals have a lower resting blood pressure. Spontaneously hypertensive rats and mice may have an overactive orexin system as (1) blocking OXRs produces significant anti-hypertensive effects in SHRs, (2) the expression of OX-A mRNA in increased in RVLM in SHRs and (3) the pp-OX/Hcrt gene is upregulated in hypothalamus in hypertensive BPH/2J mice. Orexin is important in the regulation of SNA and blood pressure and an overactive orexin system may be pathologically linked to the development of neurogenic hypertension.

Anatomically the orexin system is well positioned to be involved in the regulation of respiration and central chemoreception. Orexin neurons innervate many brainstem respiratory nuclei including the RTN, medullary raphe, LC, NTS, and pre-Bötzinger complex (Peyron et al., 1998; Date et al., 1999; Kukkonen et al., 2002; Young et al., 2005; Puskas et al., 2010; Lazarenko et al., 2011; Tupone et al., 2011; Nixon et al., 2012). Retrograde tracing studies showed that LHA OX-A positive neurons project to the diaphragm (Young et al., 2005; Badami et al., 2010) and raphe pallidus (Tupone et al., 2011). In Phox2b-eGFP transgenic mice, Lazarenko et al. further showed that the orexin-containing axonal varicosities are closely positioned relative to RTN Phox2b expressing neurons (Lazarenko et al., 2011). The expression of OX₁R and/or OX₂R mRNA is also found abundantly in many respiratory nuclei in the brainstem with a pattern matching that of OX-containing axon terminals (Marcus et al., 2001; Kukkonen et al., 2002).

At the cellular level, using in vitro patch-clamp recordings in visualized orexin neurons in brain slices, Williams et al., showed that the orexin neurons are intrinsically CO₂ (Figure 8A) and pH (Figure 8B) sensitive (Williams et al., 2007). The orexin neurons in LHA increase their firing rate during acidification and decrease their firing rate during alkalization (Figure 8), while non-orexin neurons are not responsive to such changes (Figure 8C) (Williams et al., 2007). These effects are mediated, at least in part, by the TASK-like tandem-pore K⁺ channels (Williams et al., 2007), and possibly by other acid-sensing channels, e.g., ASICs (Song et al., 2012).

In addition to their intrinsic CO₂/pH sensitivity, orexin neurons also modulate the activity of brainstem chemosensitive neurons such as those in medullary raphe nuclei and RTN (Dias et al., 2010; Lazarenko et al., 2011; Tupone et al., 2011; Nattie and Li, 2012). Electrophysiological studies in slices from neonatal (P6-P10) Phox2b-eGFP transgenic mice showed that orexin A excites the acid sensitive eGFP/Phox2b-expressing RTN neurons in a dose dependent manner (ED50 ~250 nM, Figure 9), and these neurons increased activity upon bath acidification, decreased firing with alkalization, and exhibited an ~5 Hz dynamic range of response between pH 7.0 and 7.5 (Figures 9A,B) (Lazarenko et al., 2011). Functional studies have shown that the orexin system participates in the regulation of respiration and the CO₂ central chemoreflex (Dutschmann et al., 2007; Nakamura et al., 2007; Williams et al., 2007; Dias et al., 2009, 2010; Li and Nattie, 2010; Lazarenko et al., 2011). Microinjection of orexin into the RVLM at the level of the pre-Bötzinger complex causes a significant increase in amplitude of integrated phrenic nerve activity (an index of tidal volume) in anesthetized and vagotomized rats (Young et al., 2005; Shahid et al., 2012). Injection of OX-B into the Kölliker–Fuse nucleus significantly increases breathing frequency in P21–42 day rats using the intra-arterially perfused working heart-brainstem preparation (Dutschmann et al., 2007). In the decerebrate cat, OX-A application into the hypoglossal motor nucleus increases genioglossus muscle activity (Peever et al., 2003). The pp-OX knockout mouse with a complete lack of orexin has normal resting breathing but a significantly attenuated respiratory chemoreflex in wakefulness compare to the wide type control mice and supplementation of orexins can partially restore the reflex (Deng et al., 2007; Nakamura et al., 2007). Unilateral administration of an OX₁R antagonist (SB334867) in the RTN significantly reduced the respiratory response to hypercapnia (7%CO₂) with a substantial effect in wakefulness (−30%; Figure 10A) and a much smaller effect in sleep (−9%) (Dias et al., 2009). In the medullary raphe, inhibition of the OX₁R produced a significant reduction of the CO₂ chemoreflex in wakefulness (−16%; Figure 10B) but not in sleep (Dias et al., 2010). Antagonism both OX₁R and OX₂R by orally administrating a dual OXR antagonist, Almxt, significantly decreased the hypercapnic chemoreflex only in wakefulness during the dark period of diurnal cycle (−31%); the CO₂ chemoreflex was not significantly changed in sleep in the dark period and wakefulness and sleep in the light period of the diurnal cycles (Figure 11) (Li and Nattie, 2010). Antagonism of orexin receptors had no effect on resting breathing (Dias et al., 2009, 2010; Li and Nattie, 2010). These in vitro and in vivo experiments demonstrate that the orexin system is significantly involved in the control of breathing, particularly in the central CO₂ chemoreflex.

In summary: (1) Orexin neurons are intrinsically chemosensitive and send projections to other central chemoreceptor sites, e.g., the RTN and medullary raphe. (2) Orexin may regulate breathing and central chemoreception directly and/or indirectly by recruiting other brainstem chemoreceptor sites, e.g., the RTN and medullary raphe. (3) Orexin modulation of the CO₂ chemoreflex may be vigilance state dependent with the strongest effect being during wakefulness in the dark period. (4) Orexin may be a link between breathing and sleep-wake status.

Orexin neurons are intrinsically chemosensitive and their projections and receptors are found densely in all the major respiratory neuronal groups and central chemoreceptor sites. Activation of central chemoreceptors with and without activation of peripheral chemoreceptors leads to significant sympathoexcitation and hypertension in both human and experimental animals. Respiration and the central chemoreflex are involved in regulation of SNA and blood pressure.

A link between neurogenic hypertension and an enhanced carotid body chemoreflex has been more extensively studied in patients and in animal models of sleep apnea (Fletcher et al., 1992; Lesske et al., 1997; Fletcher, 2001; Prabhakar et al., 2001, 2005, 2012; Schultz et al., 2007; Simms et al., 2010; Zoccal and Machado, 2011; Costa-Silva et al., 2012; Moraes et al., 2012a,b; Paton et al., 2013). The peripheral chemoreceptor reflex response has been shown to be significantly enhanced in patients with primary hypertension (Trzebski et al., 1982; Tafil-Klawe et al., 1985a,b; Somers et al., 1988a,b; Sinski et al., 2012) and in animal models of systemic hypertension, e.g., SHRs (Fukuda et al., 1987; Simms et al., 2009; Tan et al., 2010). Rats exposed to chronic intermittent hypoxia (CIH) develop hypertension and persistent sympathetic activation, and elimination of the carotid bodies prevents such CIH-induced hypertension (Fletcher et al., 1992; Lesske et al., 1997). Enhanced carotid body activity has been suggested to result in alterations in respiratory–sympathetic coupling (Simms et al., 2009; Zoccal et al., 2009b) and increased muscle vasoconstrictor activity, which may contribute to the development of hypertension (Trzebski et al., 1982; Somers et al., 1988a). The hyperactive carotid chemoreceptors are accompanied by overexpression of ASIC/TASK (acid-sensing ion channel/2-pore domain acid-sensing K⁺ channel) channels in young pre-hypertensive SHRs (Tan et al., 2010). Abdala et al., further showed that bilateral denervation of the carotid sinus nerves (CSD) in SHRs can significantly lower the resting blood pressure (~25 mmHg), respiratory frequency (transiently) and the low frequency component of the frequency analysis of systolic blood pressure, an index of sympathetic vasomotor tone (Figure 13), and they suggested that the inputs of carotid sinus nerve from the carotid body are partially responsible for increased SNA and blood pressure in SHR (Abdala et al., 2012).

In summary: an enhanced peripheral chemoreceptor reflex is found in both human hypertension and SHRs, and bilateral denervation of the carotid sinus nerves can partially lower blood pressure in SHRs. It is suggested that this enhanced carotid body activity may contribute to the alterations in respiratory–sympathetic coupling in SHRs.

As discussed above it is well established that activation of central chemoreceptors by CO₂ increases breathing, SNA and blood pressure in humans and in anesthetized and conscious animals (Hanna et al., 1981; Lioy and Trzebski, 1984; Somers et al., 1989; Nattie et al., 1992, 1993; Oikawa et al., 2005; Guyenet et al., 2010). In asphyxia, the hypercapnic component is of greater importance than the hypoxic component in causing sympathetically induced increases in ABP and vascular resistance (Morgan et al., 1995; Cooper et al., 2004). However, the links between neurogenic hypertension and the hypercapnic central chemoreflex are not well understood. In human subjects, the hypoxic component of asphyxia reduces the baroreceptor–vascular resistance reflex sensitivity, while the hypercapnic component is responsible for increasing blood pressure (Cooper et al., 2004, 2005). Thus, the effects of both peripheral and central chemoreceptors may contribute to promoting hypertension in patients with obstructive sleep apnea who undergo repeated bouts of asphyxia nightly (Cooper et al., 2005). Combined hypoxia and hypercapnia evoke longer-lasting sympathetic activation in humans than does either hypoxia or hypercapnia alone (Morgan et al., 1995). In patients with heart failure, the central chemoreflex response to hypercapnia is markedly and selectively enhanced (Kara et al., 2003; Yamada et al., 2004), and the enhanced hypercapnic chemosensitivity is correlated significantly with plasma norepinephrine levels suggesting sympathoexcitation (Kara et al., 2003). Administration of 100% oxygen does not lower sympathetic activity in patients with heart failure, providing further evidence against any peripheral chemoreflex potentiation (Kara et al., 2003). In anesthetized SHRs with carotid sinus denervation, systemic hypercapnia increased and hypocapnia decreased the magnitude of both phrenic and sympathetic discharges, and the increased sympathetic discharge during hypercapnia was accompanied by a significant increase in ABP (Czyzyk-Krzeska and Trzebski, 1990). We have recently reported that SHRs have a significantly augmented central hypercapnic chemoreflex relative to the normotensive WKY control rats (Li and Nattie, Neuroscience, 2012). In SHRs, the ventilatory response to normoxic hypercapnia (7% CO₂/21%O₂), or hyperoxic hypercapnia (7%CO₂/93%O₂) when peripheral chemoreceptors are suppressed, is significantly higher in both wakefulness and sleep than that of the normotensive WKY control rats (Li and Nattie, Neuroscience, 2012). It is interesting to note that both peripheral and central chemoreflexes have powerful effects on sympathetic activity (see above), and in SHRs both the peripheral and central chemoreflex are overactive. Asphyxia, a recurring condition in sleep disorders, includes two important components, hypoxia and hypercapnia, which are primarily detected by peripheral and central chemoreceptors, respectively, in normal conditions. Sleep disorders are present in ~ 40% of obese individuals and both sleep disorders and obesity are closely associated with hypertension (Chau et al., 2012). Many studies have focused on the hypoxic effects of sleep disorders and hypertension with little attention to the role of the central CO₂ chemoreflex in sleep disorders and their link to the development of hypertension. It is also interesting to note that while denervation of the peripheral chemoreceptor in SHRs significantly lower ABP (up to ~25 mmHg), ABP remains significantly above the normal even at 21 days post CSD (Abdala et al., 2012) suggesting other possible mechanisms are involved, e.g., an overactive central chemoreflex.

In summary: in SHRs, the respiratory chemoreflex to hypercapnia is exaggerated with and without peripheral carotid chemoreceptor inputs.

That activation of orexin receptors evokes increases in breathing, SNA, and ABP, and that SHRs have augmented central and peripheral chemoreflexes suggest that orexin is a key factor that links the cardiovascular and respiratory systems, and that alterations of the orexin system may contribute to some cardio-respiratory diseases. The following observations support this proposed link: (1) The orexin knockout mouse has a decreased respiratory hypercapnic chemoreflex as well as a lower resting ABP (Kayaba et al., 2003; Nakamura et al., 2007). (2) Orexin injections into the CNS increase ABP, HR, SNA, and breathing in both conscious and anesthetized normotensive animals (Shirasaka et al., 1999; Machado et al., 2002; Huang et al., 2010; Shahid et al., 2011, 2012). (3) The SHRs have: (a) an augmented respiratory–sympathetic coupling in reduced preparations (Simms et al., 2009), (b) a hyperactive peripheral chemoreflex (Zoccal et al., 2008; Simms et al., 2009), and (c) a hyperactive central CO₂ chemoreflex (Li and Nattie, Neuroscience, 2012). (4) Blockade of both OXRs can significantly lower ABP, HR, SNA and the hyperactive central CO₂ chemoreflex in conscious SHRs (Li et al., 2013a). Based on these data, we pose the following questions: (1) What is the role of overactive central chemoreceptors in developing hypertension? (2) What is the role of orexin in this augmented CO₂ chemoreflex and hypertension? (3) Can antagonism of OXRs be a target to treat such an augmented CO₂ response and the associated hypertension in SHR? At present, we have found that antagonism of both OXRs using an orally administered dual orexin receptor antagonist, Almxt: (1) significantly reduced the exaggerated CO₂ chemoreflex in SHR to the same level as measured in the control normotensive WKY rats (Li and Nattie, Neuroscience, 2012), and (2) significantly decreased ABP in both resting and hypercapnic condition (Li et al., 2013a) (Li and Nattie, Neuroscience, 2012). We hypothesize that the source of the enhanced SNA and respiratory-sympathetic coupling may involve neurons in the central chemoreceptor sites, and the overactive central orexin system may play an important role in such an alteration. There is no evidence that orexin projections and orexin receptors are located in the peripheral chemoreceptors, e.g., the carotid body, at the present time and the role of peripheral orexins in regulation of cardio-respiratory functions remain unclear (Johren et al., 2001; Nakabayashi et al., 2003; Heinonen et al., 2008).

In summary: transgenic orexin deficient mice and rats have lower resting blood pressure, and a significantly decreased hypercapnic response (pp-OX KO) while SHRs have severe hypertension, a hyperactive central hypercapnic reflex, and possibly an overactive orexin system. Blocking both orexin receptors in SHR can normalize the exaggerated hypercapnic chemoreflex and significantly lower blood pressure, which suggests the overactive central chemoreceptors may be an important link in neurogenic hypertension with orexin as mediator in SHRs.

Orexin knockout mice have both a severely attenuated hypercapnic chemoreflex and lower resting blood pressure, while SHRs have severely increased blood pressure and enhanced peripheral and central chemoreflexes with a possibly upregulated central orexin system. Antagonism of both orexin receptors can significantly lower SNA and blood pressure and normalize that central hypercapnic chemoreflex in SHRs. We suggest that overactive central chemoreceptors may be an important link to the development and maintenance of high blood pressure in SHRs with orexin as a key mediator.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.