https://orcid.org/0000-0002-2035-4936
Abstract
The hypocretin/orexin (Hcrt/Orx) system in the perifornical lateral hypothalamus has been recognized as a critical node in a complex network of neuronal systems controlling both physiology and behavior in vertebrates. Our understanding of the Hcrt/Orx system and its array of functions and actions has grown exponentially in merely 2 decades. This review will examine the latest progress in discerning the roles played by the Hcrt/Orx system in regulating homeostatic functions and in executing instinctive and learned behaviors. Furthermore, the gaps that currently exist in our knowledge of sex-related differences in this field of study are discussed.
Although hypocretin/orexin (Hcrt/orx) was originally implicated as an appetite-promoting peptide in animals, it is now well established that the Hcrt/orx system regulates a wide array of physiological homeostatic processes (such as energy balance, sleep-wake cycle, and others) and controls complex behaviors (such as reward-seeking and motivation, cognition, stress coping, aggression, and others) through overlapping neuronal circuits in the brain.
Dysregulated Hcrt/orx system resulting from genetic (such as a deficiency in Hcrt/orx peptides or receptors) and environmental factors (such as obesity) leads to neurological and psychiatric diseases and conditions (such as narcolepsy, deficiency in reward-seeking and stress coping, depression and anxiety, panic attack, and others) in animal models and human subjects.
The expression of Hcrt/orx peptides and receptors exhibits sex differences through hormonal and non-hormonal factors. In addition, direct regulation of male and female reproductive functions and maternal behaviors by Hcrt/orx have been emerging. Nevertheless, our understanding of physiological homeostasis and complex behaviors regulated by the Hcrt/orx system has been mostly achieved from investigations in male animals.
The substantial knowledge gaps in the understanding of sex differences of Hcrt/orx-mediated regulation of homeostatic functions and complex behaviors at molecular, cellular and systems levels in animals and humans need to be addressed.
All animals on planet Earth must forage for and consume energy, respond to internal and external stimuli, acclimate to their environment, and produce offspring to secure survival. Within the brain of all vertebrate animals, the hypothalamus (as part of the limbic system), is well known as a link between the central nervous and endocrine systems via the pituitary gland. During the latter half of the 20th century, the hypothalamus was shown to be a critical brain area in vertebrates that regulates essential functions necessary for the survival of individuals and their species (1). This groundbreaking work established the basis for our current understanding of prominent hypothalamic structures. However, the discovery of the hypocretin/orexin (Hcrt/Orx) system slightly more than 2 decades ago and the elucidation of its role in the regulation of brain functions has advanced our understanding of neurobiological processes within the hypothalamus in an unprecedented way. These processes underlie complex behaviors in animals as well as neurological and psychiatric conditions in humans. As summarized in Fig. 1, we now know that the Hcrt/Orx system participates in the homeostatic regulation of many basic physiological functions, such as energy balance, electrolyte-fluid balance, the sleep-wake cycle and circadian rhythm, body temperature and thermoregulation, cardiovascular and respiratory activity, neuro-immune responses, and so on (2-3). In addition to regulating these basic physiological processes, the Hcrt/Orx system integrates information and, thus, controls many complex behaviors in animals, including cognition and learning, motivated and reward-seeking behaviors, stress responses to natural and social stressors, “fight or flight” response, and emotional regulation (3-5). Dysregulations and deficiencies in 1 or several physiological functions and/or complex behaviors mediated by the Hcrt/Orx system can lead to many diverse diseases and conditions in humans, including obesity and eating disorders (Prader-Willi syndrome), addiction to illicit drugs and other natural and social reinforcers, sleep disorders and insomnia, depression and anxiety, and schizophrenia (3, 6).
A diagram depicting major functions of the Hcrt/Orx system in animals. Brain circuits regulating physiological homeostasis and controlling complex behavior converge onto the Hcrt/Orx system, which becomes an essential hub in the brain to mediate the interplay between physiological homeostasis and complex behavior in animals. A dysregulated Hcrt/Orx system resulting from altered homeostasis (such as obesity) may contribute to the development of neurological and psychiatric diseases/conditions (eg, lowered arousal levels, weakened responses to salient stimuli) in animals as reported recently (49).
The neuropeptide hypocretin (orexin) was discovered by 2 groups of researchers independently at the end of the last century (7, 8). Interestingly, a novel clone named “clone 35” was shown to encode a pre-prohormone exclusively expressed in the hypothalamus of rats; it was identified through directional tag PCR subtraction as early as 1996 (9). Eventually, 2 novel peptidergic hormones named hypocretin 1 and 2, specifically synthesized in neurons of the perifornical/lateral hypothalamus, were found to be the products of this pre-prohormone through proteolytic cleavage (7). The naming of hypocretin is the result of this neuropeptide with specific expression in the hypothalamus and aminoacidic similarities to the peptide secretin. At the same time, Sakurai et al (1998) discovered 2 G protein-coupled receptors and their natural ligands that are exclusively expressed in the lateral hypothalamus and promote food intake in animals. Therefore, they named these new peptides as orexins (orexin-a and orexin-b) (8). We now know that hypocretin and orexin are the same peptidergic hormone and that the orexigenic effect merely accounts for a small fraction of the functions governed by this peptide and may be secondary to its arousal- and motivational-promoting effects (Fig. 1).
Genetic Determination and Diversity in Hcrt Cells
The expression of the Hcrt/Orx peptide starts early in development and the neuropeptides dynorphin and nociceptin/orphanin FQ coexist with Hcrt/Orx in LHA neurons (10-12). Genetic analyses have revealed many genes that regulate the expression of Hcrt/Orx under normal and pathological conditions. It was initially shown that genes such as the IGF binding protein 3 (Igfbp3), transcript encoding Tribbles homolog 2 (Trib2), and transcription factor Lhx9 were likely regulators of the Hcrt gene expression, whereas the potassium channel subunit gene Kcnh4a regulated activities in Hcrt/Orx cells in animals. This may account for the loss of function of the Hcrt/Orx system in narcoleptic patients and animal models, although they might not be specific to Hcrt/Orx cells (13-16). Later reports showed that a growing number of genes, including Ahr1, Pcsk1, Pdyn, Peg3, Plagl1, Lhx9, Nek7, Nr2f2, Nptx2, Prrx1, Rfx4, and Six6, were specific markers or fate determinants of Hcrt cells (17, 18). It has been estimated that there are about a few thousand Hcrt/Orx cells in rodents (such as proximately 2000 in male mice) and 50 000 to 90 000 in humans (19-22). The numbers of Hcrt/Orx cells fluctuate throughout the life span of animals. For instance, in C57B6 mice, numbers of Hcrt/Orx cells in both sexes were stable in the first 400 days of life, then started to slowly decline between 400 and 800 days, and with an accelerated rate of loss between 800 and 1000 days of age, at least in males (22). It is also worth noting that male mice exhibits 15% to 20% more Hcrt/Orx-immunopositive cells than females (22). Despite the small number of Hcrt/Orx cells in the brain, nerve fibers containing Hcrt/Orx and distributions of Hcrt/Orx receptors were identified throughout the central nervous system and even in peripheral organs (19, 23-27). Therefore, it is conceivable that Hcrt/Orx cells likely exhibit a heterogeneity in terms of molecular and cellular markers, broad and diverse projections of nerve fibers in the brain, and regulation of a wide array of functions in animals. An emerging body of evidence strongly suggests this possibility. Hcrt/Orx cells are glutamatergic in nature as indicated by early and most recent reports (17, 28-30). However, one-half of these cells also express gamma-aminobutyric acid (GABA) synthetic enzyme gene Gad1 (30). The role of Gad1 in Hcrt/Orx cells is not yet clear because Hcrt/Orx cells do not express an intact vesicular releasing machinery for GABA as typically seen in GABAergic neurons (30); it is intriguing and vitally important to understand the functional implications of the existence of GABA in Hcrt cells. Interestingly, Hcrt/Orx cells can be divided into 2 populations depending on the expression of sex-specific genes Ddx3y and Xist, suggesting possible sex-related dimorphism in these cells (17). The gene Ddx3y, which is located on the Y chromosome and is responsible for male fertility and spermatogenesis, plays a role in neuronal differentiation during development in animals (31). The gene Xist is located on the X chromosome and is responsible for X-inactivation in early development (32). However, it is not clear what effects these genes may have on Hcrt/Orx cells in male and female animals and whether their presence contributes to sexual dimorphism in a hypocretin-dependent way in animals.
Basic Properties of the Hcrt/Orx System
The Hcrt/Orx peptides were demonstrated to be excitatory when they were initially discovered (7). At the cellular level, Hcrt/Orx enhances synaptic transmission and neuronal activity, elevates intracellular calcium levels, and promotes synaptic potentiation in various neuronal types in the brain (33-37). The cellular effects of Hcrt/Orx are mediated by 2 G protein-coupled receptors, which are mediated by the Gs, Gq, and Gi pathways (8, 38). The PLC and PKC pathways downstream to the activation of the Gq protein likely contribute to the excitatory effects of Hcrt/Orx in neurons (38). In addition to G proteins, Hcrt/Orx receptors may also be coupled to β-arrestin, protein tyrosine phosphatase SHP-2, dynein light chain Tctex type 3, and other proteins to transduce broad effects of Hcrt/Orx (38).
Hcrt/Orx-expressing neurons themselves possess many distinctive characteristics compared with other neuronal types in the brain. First, at the single-cell level, Hcrt/Orx cells can be divided into 2 populations (D and H type), depending on their distinct electrophysiological properties (39). H-type cells had a larger 4-AP-sensitive, low-activation threshold A-current than D-type cells (39). H-type cells were sustained hyperpolarized by an elevation of extracellular glucose levels in slices, whereas D-type cells only exhibited a transient hyperpolarization (39). However, it has not been exactly clear whether and how the proposed H-type and D-type cells mediated a diverse array of functions and behaviors in animals. By using conventional and perforated whole-cell patch clamp recordings, Liu and colleagues showed that the intracellular ATP levels were generally higher than in other types of central neurons in the brain and that the intracellular levels of ATP were lower in Hcrt/Orx neurons in sleeping mice than in sleep-deprived animals (40). The cellular responses to changes in internal (intercellular ATP levels) and external cues (glucose, lactate, and amino acid levels) constitute the basis of how the Hcrt/Orx system may serve as a sensor of energy availability in the brain (40-44).
Second, at the synaptic level, Horvath and Gao (2005) showed that the frequency of miniature excitatory postsynaptic currents recorded at the soma of Hcrt/Orx cells was about 10-fold larger than that of miniature inhibitory postsynaptic currents (45). Consistently, there were more asymmetric (putatively excitatory) than symmetric (putatively inhibitory) synapses on cell bodies of Hcrt/Orx neurons (45). These results strongly indicate that their cell bodies are predominately under the innervation of excitatory (likely glutamatergic) but not inhibitory (likely GABAergic) synapses (45, 46). This unique synaptic architecture is distinctively different from other long projection neurons such as pyramidal neurons in the neocortex, in which the numbers of inhibitory (GABAergic) synapses predominate over numbers of excitatory (glutamatergic) synapses at the soma of these neurons; there are more inhibitory synapses than excitatory synapses on soma of pyramidal neurons in the neocortex (47). This unique arrangement of excitatory and inhibitory synapses on Hcrt/Orx neurons is consistent with the reports that the blockade of ionotropic glutamatergic but not GABAA-mediated transmission onto Hcrt/Orx cells with selective glutamatergic antagonists significantly attenuated the spontaneous action potential firing in these neurons (46, 48). Our recent data suggest that predominately excitatory inputs onto Hcrt/Orx cells might be weakened in obese animals compared with their control counterparts (49). Specifically, in male diet-induced obese (DIO) mice, there were relatively more inhibitory inputs onto Hcrt/Orx neuronal soma than in controls fed with a normal diet (49). The release probability of glutamatergic synapses was decreased and expression of spike-timing dependent plasticity at glutamatergic synapses was impaired in Hcrt/Orx cells in DIO mice (49).
Among the glutamatergic synapses on the Hcrt/Orx cells, there are fewer “silent” synapses among them, in that only NMDA-type receptors (NMDARs) but not AMPA-type glutamate receptors (AMPARs) are expressed (50, 51). Our results indicated that the AMPAR-mediated EPSC is significantly larger (> 2-fold) than that mediated by NMDARs (the AMPAR/NMDAR ratio is larger than 1.0) (52-54), strikingly different from what has been reported for the AMPAR/NMDAR ratio (being less than 1.0) in CA1 pyramidal neurons of the hippocampus and dopamine neurons in the ventral tegmental area (VTA) (51, 55). Therefore, it is reasonable to postulate that a small, excitatory input mediated by glutamate may lead to proportionately more activated glutamatergic synapses on Hcrt/Orx neurons than on other central neurons such as CA1 pyramidal neurons; the reason being that silent synapses containing only NMDARs require a sufficient depolarization to remove the Mg2+ blockade of NMDARs to conduct glutamatergic transmission (51) and thus the synaptic transmission would “fail” at these synapses when the postsynaptic component is not adequately excited by other factors, such as a concurrent activation of AMPARs. Therefore, it is expected that glutamatergic transmission onto Hcrt/Orx neurons might be highly efficient without silent synapses, a necessity for the Hcrt/Orx system to mediate its functions.
In addition, GluR2-lacking calcium-permeable AMPARs (Cp-AMPARs) are expressed in Hcrt/Orx cells under basal conditions as indicated by our studies (54). The NMDAR-mediated calcium influx is critical to synaptic plasticity (such as LTP) in central neurons (56). Cp-AMPAR is also recognized as a critical promoter to the development of synaptic plasticity in the brain. In pyramidal cells in the CA1 region of the hippocampus, Cp-AMPARs are not incorporated at postsynaptic sites under basal conditions, but only inserted transiently to postsynaptic sites during the induction phase of expression of NMDAR-dependent LTP (57). In central neurons, such as interneurons of the basolateral amygdala, where Cp-AMPARs are constantly expressed under basal conditions, the expression of LTP is NMDAR-independent and mediated by Cp-AMPARs (58). The existence of Cp-AMPARs at glutamatergic synapses on Hcrt cells implicate a distinctive route for calcium influx during glutamate-induced excitation and expression of synaptic plasticity in these cells.
At the circuit level, emerging evidence has presented a more complicated network of neuronal systems that interact with Hcrt/Orx neurons locally in the LHA than previously reported (Fig. 2). It was originally described that Hcrt/Orx cells had reciprocal synaptic connections with neighboring melanin-concentrating hormone (MCH) containing neurons (59, 60). Exogeneous Hcrt/Orx could directly activate MCH cells, implying a functional role of Hcrt/Orx released from presynaptic boutons onto MCH cells (60). However, exogeneous MCH did not directly modulate activity in Hcrt/Orx cells (53), which is consistent with a report that Hcrt/Orx cells do not express MCH receptor 1 (MCHR1), the only MCH receptor subtype in rodents (61). Because the Hcrt/Orx system could be indirectly suppressed by exogenous MCH in wild-type animals and up-regulated in male MCHR1-deficient mice (53), it is likely that Hcrt/Orx cells are regulated indirectly by MCH through a population of MCHR1-expressing cells. It was originally reported that Hcrt/Orx cells were inhibited by leptin (62). Later, it was shown that Hcrt/Orx cells do not express the long form leptin receptor (LepRb) (63). Instead, a population of GABAergic neurons containing the peptide neurotensin express this specific receptor LepRb in the LHA and directly innervate Hcrt/Orx cells (63, 64). In recent years, another population of GABAergic neurons were identified to express dynorphin but not Hcrt/Orx receptors and to innervate Hcrt cells in the LHA, which provides an additional inhibitory circuit onto these cells locally (65). There was also a report that some populations of GABAergic neurons expressed Hcrt/Orx receptors and innervated MCH cells in the LHA; these cells have been proposed to provide a local inhibitory circuit onto MCH cells (66).
Local neuronal populations that interact with the Hcrt/Orx system. Hcrt/Orx cells and neighboring MCH-containing neurons receive reciprocal synaptic connections between them (59, 60). The Hcrt/Orx system is likely to be regulated indirectly by MCH and leptin through populations of MCHR1-expressing and LepRb-expressing neurons (61, 63, 64). A group of GABAergic neurons expressing dynorphin but not Hcrt/Orx receptors innervate Hcrt cells in the LHA, providing an additional inhibitory circuit onto these cells locally (65). Some other populations of GABAergic neurons expressing Hcrt/Orx receptors innervate MCH cells and may provide an inhibitory pathway onto these cells locally (66).
In summary, the unique glutamatergic and GABAergic synaptic organization onto the somata of Hcrt/Orx neurons, distinctive composition of subtypes of glutamate receptors in these cells, and a complex network of interaction between Hcrt/Orx cells and neighboring neuronal populations provide a functional basis for the role that Hcrt/Orx cells may play in the regulation of critical functions in animals and humans. The predominant innervation by excitatory synapses onto the cell bodies of Hcrt neurons and their putatively high efficiency in glutamatergic transmission are likely to facilitate a rapid activation of this system upon exposure to salient stimuli and environmental cues that, in turn, affects the daily tasks and overall survival of animals. It should be clearly stated, however, that all our knowledge regarding the Hcrt/Orx system described here was predominantly obtained from studies on male animals. There are still significant gaps in our understanding of the basic properties of the Hcrt/Orx system in female animals. Importantly, the influence of estrogen- and non-estrogen-dependent factors on sex differences in the Hcrt/Orx system has not been thoroughly examined. A recent report provides emerging evidence of possible changes in Hcrt/Orx cells in naturally cycling female rodents (67). The authors suggested that both endogenous and exogenous estrogen (E2) might have an inhibitory effect on Hcrt/Orx cells in naturally cycling female rats (67). However, it was not clear which types of estrogen receptors mediated the reported changes in synaptic transmission onto the Hcrt cells by E2. In fact, there was a report that immunolabeling for androgen (AR) and estrogen (ER alpha) receptors revealed no colocalization of Hcrt/Orx with AR and few Hcrt/Orx neurons expressing ER alpha in male rodents, suggesting that hormonal regulation of Hcrt/Orx expression might be via afferents from neurons containing those receptors (68).
Homeostatic Regulation of Physiological Functions by Hcrt/Orx
Physiological functions and behaviors are closely associated because of overlapping neural circuits that mediate both processes. Specifically, the Hcrt/Orx system is among neurocircuits that regulate both homeostatic functions of the brain and complex behaviors in animals. The intake and use of energy and the maintenance of optimal arousal levels are fundamental functions that allow animals to perform daily tasks required for survival. In the following sections, we discuss new developments in the regulation of these essential physiological functions (ie, energy metabolism and the sleep-wake cycle/arousal control) and behaviors (eg, reward seeking, stress coping) by the Hcrt/Orx system with consideration given to sex dimorphism.
Bidirectional Control of Energy Balance
The intake and use of energy are fundamental functions essential to the performance of daily activities in all animals, especially vertebrates. The LHA is a classic structure that participates in the regulation of food intake in animals. The pioneering work of Hetherington and Ranson (69) and Anand and Brobeck (70, 71) led to the characterization of the LHA as a feeding center (72, 73). Although the idea of feeding center has been supported by evidence obtained from electrical stimulations, selective chemical lesions, and in vivo electrophysiological recording in animals (74-78), the exact identities of the neurons responsible for this role of the LHA in positive energy balance was not clear until the discovery of diverse neuronal populations in this region.
Hcrt/Orx promotes both energy intake and expenditure in animals. The intracerebroventricular injection of Hcrt/Orx (either Hcrt 1/Orx A or Hcrt 2/Orx B) led to a short-term increase in food consumption in male rats (8). Local administration of Hcrt 1 to the paraventricular nucleus, dorsomedial nucleus, and lateral hypothalamic/perifornical area triggered food intake in male rats (79, 80). Conversely, the application of a selective Hcrt-1 receptor antagonist, SB-334867, suppressed food intake in male and female rodents (81-83). Consistent with the effects of exogeneous Hcrt/Orx on feeding, the activity in Hcrt neurons is in close alignment with energy and nutrient states in animals. Food deprivation and acute hypoglycemia resulted in up-regulation of Hcrt/Orx mRNA, Hcrt/Orx expression and c-fos expression in Hcrt/Orx neurons in male rodents and female nonhuman primates (8, 84-89), which could be inhibited by leptin treatment (90, 91). In addition to its effects on central mechanisms, Hcrt/Orx also facilitated energy intake in peripheral organs via both central and peripheral pathways. Central administration of Hcrt/Orx increased both gastric acid secretion and gastric motor function in male rodents (92, 93). On the one hand, this is likely mediated by the dorsal motor nucleus of the vagus (DMV), a key region that controls gastric acid secretion and gut motility. Hcrt/Orx neurons innervate this area and directly excite gastrointestinal-projecting DMV neurons (19, 94). Hypoglycemia triggered c-fos expression in the DMV through the activation of Hcrt/Orx neurons and the release of Hcrt/Orx peptide in male rats (95). On the other hand, Hcrt/Orx could directly stimulate contraction of duodenal smooth muscles at the periphery in female mice (96). Hcrt/Orx directly induced depolarization in duodenal longitudinal muscle cells through activation of receptor-operated, store-operated, and voltage-gated Ca2+ channels and inhibition of K+ channels (96). Under voltage clamp, Hcrt/Orx enhanced the amplitude of voltage-gated Na+ currents and lowed the threshold for Na+ channel activation. Similar effects of Hcrt/Orx on voltage-gated L- and T-type Ca2+ channels were observed (96).
Unlike other feeding-promoting neurons, such as neuropeptide Y (NPY) and agouti-related protein (AgRP) in the arcuate nucleus (ARC) and MCH cells in the LHA, which also suppress energy expenditure (97, 98), Hcrt/Orx promotes energy expenditure in animals. Microinjection of Hcrt-1 into the third ventricle, ARC, PVN, and LHA stimulated the metabolic rate in male mice and rats (97, 99-102). The increase in energy expenditure caused by Hcrt/Orx occurred in animals with and without an increase in physical activity. For example, a direct administration of Hcrt-1 into the ARC caused an enhancement in whole-body O2 consumption in male urethane-anesthetized rats (101), suggesting that Hcrt/Orx can boost energy expenditure even without an enhancement in physical activity (equivalent to the rest state) in animals. In conscious rats, Hcrt-1 enhanced energy use by increasing physical activity. Microinjections of Hcrt/Orx into the PVN and LHA significantly increased spontaneous non-exercise-related activities accompanying with an increment of in O2 consumption and thermogenesis in male rats (102-105), suggesting that Hcrt/Orx promotes non-exercise physical activity in animals. Consistent with these results, Hcrt/Orx increased and promoted glucose uptake, insulin-induced glucose uptake, and glycogen synthesis in skeletal muscle, but not in white adipose tissue, through the ventromedial hypothalamus (VMH)-sympathetic nervous system pathway in male mice and rats (106, 107). Furthermore, Hcrt/Orx was required in mediating mobilization of brown fat tissue (108, 109), which was supported by a study showing the direct innervation by Hcrt-containing nerve fibers from the LHA onto the raphe pallidus to promote BAT thermogenesis in male rats (110). Hcrt-mutant mice expressed a higher level of preadipocyte markers, fewer mitochondria, lowered levels of BAT thermogenic proteins (such as PPAR-γ1/γ2, PGC-1α/β, and UCP-1) in brown fat cells, compared with their wild-type littermates (108). These defects led to compromised thermogenesis induced by high-fat diet and cold exposure (108, 111). Based on these results, a recent report showed that Hcrt/Orx signaling was required to enhance energy expenditure and bodyweight loss in patients treated with D2 dopamine receptor agonists (109).
On-demand Control of Arousal by Hcrt/Orx
Higher animals execute many daily tasks, from exploring habitats and foraging for food to escaping from predators, to sustain their survival in the natural environment. During the reproductive season, they compete for mating partners and produce offspring to ensure the survival of the species. The successful performance of these functions likely depends on many well-controlled homeostatic processes based on a complex array of processing and computation of inputs within the brain that encode the internal and external environments of the animal. On the one hand, the promotion of general arousal across the sleep/wake cycle in concert with other wake-promoting centers in the brain is essential for the execution of homeostatic regulation of physiological functions (such as energy metabolism, water and salt balance, cardiovascular and respiratory functions), which is required to support daily tasks necessary for survival. On the other hand, Hcrt/Orx cells may be activated specifically to mediate goal-oriented behaviors beyond the normal regulation of the daily sleep/wake cycle, such as during a food deficit, or stress-inducing situation, and so on.
The involvement of the hypothalamus in sleep/wake regulation was originally proposed by van Economo based on his pioneering observations in human patients that lesions in the posterior hypothalamus and midbrain junction led to sleepiness, whereas anterior hypothalamic inflammation led to insomnia and chorea (112). Later, studies in monkeys, rats, and cats confirmed the sleep-promoting effect of lesions and inhibition of the posterior lateral hypothalamus (113-116). However, it remained unclear how the LHA participates in sleep regulation until the discovery of the neuropeptide Hcrt/Orx in this brain area (117, 118). It has already been shown that the levels of Hcrt/Orx in the cerebrospinal fluid align with the daily sleep/wake cycle: they are low during the resting (sleep) phase and high during the active (wake) phase in male rodents and male/female monkeys (119-122). The concentrations of Hcrt/Orx detected across the sleep/wake cycle are consistent with the activity levels of these cells as revealed by c-fos expression and recordings in vivo in freely moving or head-fixed male rodents (123-125). A direct and specific stimulation of Hcrt neurons by optogenetics increased arousal during the sleep phase, whereas an acute silencing of these cells increased just the slow-wave sleep-like activity during the sleep phase (126-128). The effects of Hcrt/Orx on sleep/wake regulation and arousal maintenance are likely due to its projections to major arousal regions such as the locus coeruleus, basal forebrain, and others (129-133). Although a deficiency in Hcrt peptide and its receptors (Hcrtr2 or OXR2) led to narcolepsy or narcoleptic-like phenotypes in dogs, mice, and human patients (20, 134-137), overexpression of Hcrt/Orx led to insomnia-like phenotypes in zebrafish (138).
In addition to its role in the regulation of the sleep/wake cycle, Hcrt/Orx also acts as a potent arousal promoter beyond the normal maintenance of the sleep/wake cycle in the brain. The activation of Hcrt/Orx cells was detected to be superimposed upon their normal functions of arousal promotion during the active phase. When male rats were required to maintain a prolonged wakefulness because of sleep deprivation, Hcrt/Orx cells were activated as indicated by c-fos expression in these cells (139). When animals were exposed to novel stimuli such as food, the activity in Hcrt/Orx cells increased during the initial period of feeding during the light phase (125, 140). The levels of Hcrt/Orx in the amygdala were maximal during positive emotion, social interaction, and anger in humans (141). Most importantly, activation of Hcrt/Orx neurons was required for the initiation of new locomotion but not ongoing locomotion (142). On top of this, Hcrt/Orx cells underwent synaptic plasticity of glutamatergic (excitatory) synapses induced by sleep deprivation in an activity-dependent way in male mice (52). Interestingly, the number of inhibitory synapses on Hcrt/Orx cells was decreased by sleep deprivation and increased during rebound sleep in zebrafish (143). The results suggest that the Hcrt/Orx system is required to maintain arousal and wakefulness when circumstances demand it, even when animals should be in the resting phase during their normal sleep/wake cycle. Additionally, synaptic plasticity in excitatory and inhibitory synapses upon the Hcrt cells occurs according to the behavioral state of the animal. Overall, these results further supported the idea that the activation of Hcrt/Orx cells is necessary to promote arousal on demand. However, we cannot exclude the possibility that changes in synaptic architectures and functions in Hcrt/Orx cells may result from both circadian and activity-dependent factors. Although there is evidence that the suprachiasmatic nuclei controls the release of Hcrt/Orx in male rodents (144-146), it is still not clear whether and how the circadian clock in the suprachiasmatic nuclei and core clock within Hcrt/Orx cells regulate synaptic plasticity in these cells in a way independent to activity levels in animals. Further investigations along these directions are warranted.
Use-dependent Regulation of Complex Behaviors by Hcrt/Orx
Control of Reward-seeking and Motivational Behaviors by Hcrt/Orx
The perifornical/lateral hypothalamic area has long been known as a brain structure responsible for reward-seeking behaviors. In early studies, electrical stimulation of the LHA induced an act of marked reinforcement and robust self-administration of electric current in rodents (147-150). Drugs of abuse also induce rodents to self-administrate when applied directly to the LHA (151, 152). It is now clear that Hcrt neurons mediate the development of drug addiction in animal models and human patients. These neurons are activated when rodents are exposed to drugs of abuse. The expression of c-fos and pCREB in Hcrt neurons is enhanced by opiates, cocaine, amphetamine, and nicotine in various models of drug-seeking behavior in male animals (54, 153-157). Direct administration of Hcrt into brain reward centers (such as the VTA) promotes drug seeking behavior in animals, including an increase in the breakpoint in a progressive-ratio task for cocaine and reinstatement of extinguished seeking behavior for morphine and nicotine (154, 157-160). The disruption of Hcrt receptor-mediated pathways with the selective Hcrt-1 receptor antagonist, SB334867, or a deficiency in Hcrt peptide and receptors attenuated, blocked, and abolished drug-seeking behavior induced by morphine, cocaine, amphetamine, and nicotine in animals (37,153, 154, 160-163). In human narcoleptic patients with a deficiency in Hcrt peptide or neurons, a significantly lowered tendency to drug abuse was reported (164). The action loci of Hcrt/Orx in mediating drug-seeking behaviors are identified throughout the brain, including the VTA (37, 164), basal forebrain (165), insular cortex (162), nucleus accumbens (166), hypothalamic paraventricular nucleus (PVN) (157), bed nucleus of the stria terminalis (167), paraventricular nucleus of the thalamus (168), and the central amygdala (169). There was evidence that Hcrt/Orx mediated drug-induced plasticity in these brain areas, which may be responsible for the role it plays in these regions. For example, Hcrt/Orx potentiated the NMDAR-mediated transmission by promoting the insertion of these receptors at glutamatergic synapses in VTA dopamine neurons, which was required to facilitate the expression of cocaine-induced plasticity in these cells and in the locomotor sensitization to cocaine in male animals (37). In addition, the Hcrt system per se undergoes synaptic plasticity in male rodents developing cocaine-seeking behaviors (57, 170). The long-lasting synaptic plasticity in Hcrt cells induced by cocaine exposure likely contributes to cue-induced pursuit of cocaine and morphine as well as sleep disorders in drug addicts and animals (54, 154), which has not been well explored. Our most recent data suggest that the impaired expression of activity-dependent synaptic plasticity in Hcrt/Orx cells might underlie the deficiency in the development of reward-seeking behaviors in male obese animals (49).
Another finding regarding the role that Hcrt-mediated motivational circuits play in the regulation of animal behavior is that the Hcrt system is involved in aggressive-like behaviors in animals. Flanigan et al reported that the projections from Hcrt cells to glutamic acid decarboxylase 2-expressing cells in the lateral habenula (LHb) was necessary to mediate intra-sex aggressive behaviors in male animals (171). Because Hcrt/Orx cells receive innervation from the VMH, a brain area well-known to be responsible for the initiation of aggressive behavior in male animals, the authors proposed that the pathway from Hcrt cells to glutamic acid decarboxylase 2 cells in the LHb may provide the link between aggressive and motivational circuits in the hypothalamus (171). The role of Hcrt/Orx in mediating aggressive behaviors in maternal animals has not been identified (172).
Hcrt/Orx-mediated Stress Responses and Stress Coping
The exposure to stressors is critical to both the development of drug addiction and relapse of drug-seeking behavior. The report that the Hcrt/Orx system mediates a stress-induced relapse of cocaine-seeking behavior provided the earliest evidence of the part that Hcrt/Orx cells play in mediating stress-triggered behavioral changes in male animals (158). The Hcrt/Orx system is a potent mediator of stress-induced effects on both physiological functions and behavioral changes in male and female animals (173). Hcrt/Orx cells are activated when animals are exposed to acute and chronic stress (49, 140, 158, 174-176). We now know that Hcrt cells respond to acute stress rather rapidly with a peaked phosphorylation of CREB about 5 minutes after stress exposure (49), which is similar to the activation pattern reported in corticotropin-releasing factor (CRF) neurons in the PVN (177). Considering that the expression of Hcrt/Orx induced by acute stress was still enhanced in the absence of CRF signaling (178), it is likely that the Hcrt/Orx system could serve as a pathway independent of the CRF system in the PVN to mediate certain stress-induced effects, which has not been well explored so far.
The physiological implications of stress-induced activation of Hcrt/Orx cells are emerging. In addition to triggering drug addiction relapses (158), the Hcrt/Orx cells are responsible for coping with acute stressors (49, 179, 180). The activation of Hcrt/Orx cells was required for expressing “claustrophobic” sighing when mice were subjected to confinement in a tiny space (180). The activity levels of Hcrt/Orx cells during stress exposure (such as forced cold water swim) causally contribute to stress-coping strategy (active coping vs passive coping) in male mice (49). Although certain stressors (such as foot shock) induce fear-conditioned learning, the Hcrt/Orx system may not be involved in this fear-associated learning (178, 181) but rather in the elimination of fear-related memories (182). Along this line, the activation of Hcrt/Orx cells is required to mediate anxiety and depressive-like behaviors after stress exposure and panic attack-like behaviors in both male and female animals (67, 176, 183-185). Therefore, the manipulation of the Hcrt/Orx-mediated pathway likely has therapeutic implications. The activation of orexinergic neurons by calorie restriction after social defeat elicited an antidepressant effect, as indicated by prolonged latency to immobility and shortened duration of immobility in the forced swim test (186). This may be mediated by the innervation of the LHb by Hcrt/Orx cells in the LHA (176). Also, an activated Hcrt/Orx system likely promotes adaptive responses to predator-scent stress (187). An early intervention with ORX-A/Hcrt1 reduced the prevalence of the posttraumatic stress disorder phenotype (adaptive responses to predator-scent stress) and increased the prevalence of adaptive phenotypes in animals (187).
Sex Differences in Hcrt/Orx-governed Functions and Behaviors in Animals
Hcrt/Orx specifically regulates reproductive functions and behaviors in males and females. The expression of Hcrt/Orx and its receptors exhibits sex dimorphism (188). In male animals, Hcrt and its receptors are expressed in the male reproductive system (189). In male rats, c-Fos expression was markedly increased in Hcrt/Orx neurons during copulation, whereas a systemic administration of the orexin-1 receptor antagonist, SB 334867, impaired copulatory behavior (68). Castration decreased the Hcrt/Orx neuron count and protein levels in a time course consistent with postcastration impairments in copulatory behavior (68). This effect was mediated by the VTA (68). However, there was also a report that the Hcrt/Orx system might not be critical for male sexual performance or motivation but rather plays a role in arousal and anxiety related to sexual behavior in naive male animals (190).
In female animals, Hcrt expression is increased in the proestrus phase of the estrus cycle (191). Increases in neuronal activity of Hcrt and in HCRT-1 receptor mRNA expression have been identified in association with lactation (192). At intermediate doses, intracerebroventricular injections of Hcrt-1 elevated levels of licking and grooming of pups and the number of nursing bouts. At the highest dose, Hcrt-1 delayed latency to nurse, decreased nursing, increased time off nest, and decreased maternal aggression. IP administration of the Hcrt-1 receptor antagonist, SB-334867, caused a trend toward an increase in low-arched back nursing activity and decrease in licking and grooming of pups during high-arched back nursing. This suggests that endogenously released Hcrt/Orx may be required for the full expression of maternal behaviors independently or dependently with other neuromodulators (192). However, there was also a report that the Hcrt/Orx levels in the medial preoptic area were negatively correlated with the frequency of contact with the litter or the display of erect postures in rats, suggesting possible changes in maternal behavior induced by Hcrt/Orx in specific brain areas in rodents (193).
Except for the direct regulation of reproductive function by Hcrt/Orx in a sex-dependent manner in male and female animals, any sex dimorphism in the regulation of homeostatic functions and complex behaviors by Hcrt/Orx in animals is not entirely clear because most studies to date were performed in males.
The roles of sex-dependent factors playing in the regulation of nonreproductive functions and the development of diseases/conditions resulting from dysregulated physiological functions (such as energy metabolism) are rather complex in humans (194). For example, although obesity is more frequently diagnosed in women than men, type 2 diabetes is more likely diagnosed at lower ages or body mass index in men than women (194). This complexity is consistent with findings in animal studies that brain pathways controlling energy balance exhibit sex dimorphism. The NPY-mediated effects on energy balance have been revealed to be sex-dependent (195). On the one hand, testosterone stimulates NPY expression and release in the hypothalamic nuclei (such as the median eminence, arcuate nucleus, and VMH) in rodents (196, 197). Male rats exhibit more NPY-containing cells than females in the ARC (197). On the other hand, E2 inhibits food intake. The hypothalamic expression of NPY fluctuates across the different phases of the estrus cycle, reaching its lowest levels during proestrus when the plasma estrogen level peaks (198, 199). The cyclic changes in NPY and E2 levels are consistent with the cyclic changes in food intake and body weight in naturally cycling female animals (198, 199). A recent study has identified a sex difference in the response of NPY/AgRP neurons in the development of insulin resistance in mice with DIO. The efficacy of insulin to activate KATP channels in NPY/AgRP neurons was significantly attenuated in male but not female DIO mice, demonstrating that E2 exerted a protective role against insulin resistance based upon the response of the NPY/AgRP neurons in females (200). Data regarding sex-dependent regulation of energy balance by Hcrt/Orx have not been well established thus far. In addition, the effects of gonadal hormones on sleep regulation mediated by Hcrt/Orx and changes in cognitive performance caused by sleep loss because of a dysregulated Hcrt/Orx system are also poorly defined (188).
A growing body of evidence from clinical and preclinical studies have shown that biological processes underlying addictive behaviors are different between male and female animals (201, 202). In humans, women develop substance use disorders more rapidly after initial use and show faster relapse than men at all ages (203, 204). The subjective effects of psychostimulants on women fluctuates across the menstrual cycle (205), with the greatest effects reported when E2 levels are high (206). Consistently, exogenous E2 enhances the subjective effects of psychostimulants (such as cocaine) in women (207), whereas exogenous progesterone and its metabolite, allopregnanolone, attenuate the subjective effects of cocaine and other drugs of abuse in both men and women (208). In animal studies, female rats acquire cocaine self-administration behavior more rapidly and at lower doses than males (209-211). The acquisition of cocaine self-administration behavior was markedly reduced by ovariectomy and then restored by E2 replacement (212, 213). The effects of E2 on brain functions mediated by dopamine production and signaling in the reward pathway has been well documented (214). For instance, the basal neuronal activity (action potential firing) in vivo was higher in VTA dopamine neurons of female mice during estrus than in males or diestrus females, which causally contributed to the facilitated expression of cocaine-seeking behavior in female animals (215). The exact roles played by the Hcrt/Orx system in mediating the sex dimorphism in reward-seeking and addictive behaviors are not yet clear.
Consistent with sex differences in reward-seeking behaviors, females are twice as likely as males to experience stress-related psychiatric disorders. In adult male and female rats exposed to repeated restraint stress, an increase in Hcrt/Orx expression and activation were observed in female rats compared with males (216). Female rats exhibited an impaired habituation to repeated restraints and subsequent deficits in cognitive functions after stress exposure compared to male rats. The inhibition of Hcrt/Orx using designer receptors exclusively activated by designer drugs abolished the heightened hypothalamic-pituitary-adrenal response and reduced stress-induced cognitive impairments in female rats (216). However, it is a different story during early development. Early life stress, such as neonatal maternal deprivation, induced a more remarkable effect on male than female animals (217). Early life stress dampened the responses of Hcrt cells (decreased activation of c-Fos) to restraint stress in both male and female rats in their adulthood. Voluntary exercise during the late adolescence period reversed the deficiency in the Hcrt/Orx system and behavioral deficits in males but not females (218). The data regarding sex differences in Hcrt/Orx-mediated stress-induced actions in animals continue to emerge.
Concluding Remarks
The topic of sex differences in the regulation of physiological functions and animal behaviors has been a highly focused area of research during the past several decades and is critical to the understanding of basic biological mechanisms and the real-world practice of sex equality in human health care. Contributions to our body of knowledge with regard to the essential roles of the Hcrt/Orx system in mediating the sexual dimorphism in physiological functions, complex behaviors, diseases, and pathological conditions is accelerating. However, many gaps remain. For example, it is still not entirely clear how the Hcrt/Orx system mediates sex-related diverse effects on the brain and peripheral organs in animals and humans even while the Hcrt/Orx cells per se do not directly express estrogen and androgen receptors. It is essential to decipher the connections of the Hcrt/Orx cells and their upstream partners that do express estrogen and androgen receptors and are modulated by sex hormones. It is also unclear how the Hcrt/Orx system is regulated genetically and/or epigenetically given the possible influence of various (sex) hormone milieus that occur over the life span of male and female animals. Ultimately, bridging these gaps in our understanding of sex differences in Hcrt/Orx-mediated functions and behaviors will lead to new diagnostic and therapeutic approaches in the advancement of human health care.
Abbreviations
- AgRP
agouti-related protein
- AMPAR
AMPA-type glutamate receptor
- ARC
arcuate nucleus
- Cp-AMPAR
calcium-permeable AMPA-type glutamate receptor
- CRF
corticotropin-releasing factor
- DIO
diet-induced obese
- DMV
dorsal motor nucleus of the vagus
- E2
estrogen
- GABA
gamma-aminobutyric acid
- Hcrt/Orx
hypocretin/orexin
- LepRb
long form leptin receptor
- LHA
lateral hypothalamic area
- LHb
lateral habenula
- MCH
melanin-concentrating hormone
- MCHR1
melanin-concentrating hormone receptor 1
- NMDAR
NMDA receptor
- NPY
neuropeptide Y
- PVN
paraventricular nucleus
- VMH
ventromedial hypothalamus
- VTA
ventral tegmental area
Acknowledgments
The authors would like to pay tribute to their friend and colleague, Dr Anthony N van den Pol, who passed away last year. He was a trailblazer in neuroscience of the hypothalamus who contributed significantly to our understanding of the Hcrt/Orx system. The authors thank Marya Shanabrough for assistance with the manuscript. The graphic abstract was partially created with BioRender.com.
Funding
The authors’ studies have been supported by National Institutes of Health grants DA046160 (T.L.H. and X.B.G.) and DK120891 (X.B.G. and T.L.H.).
Additional Information
Disclosures: The authors have no conflict of interests related with this article to disclose.
Data Availability
Data sharing is not applicable to this article.
