INVITED REVIEW
Functions of the orexinergic/hypocretinergic system

Jyrki P. Kukkonen1, Tomas Holmqvist1, Sylwia Ammoun1, and Karl E. O. Åkerman1,2

1 Laboratory of Cell Physiology, Department of Neuroscience, Division of Physiology, Uppsala University, Biomedical Center, SE-75123 Uppsala, Sweden; and 2 Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, BioTeknia, FIN-70210 Kuopio, Finland


    ABSTRACT
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ABSTRACT
INTRODUCTION
OVERVIEW OF OREXINS AND...
CELLULAR RESPONSES TO OREXINS
DISTRIBUTION OF OREXINERGIC...
SYSTEMIC EFFECTS OF OREXINS
FUTURE PERSPECTIVES
REFERENCES

Orexin A and orexin B are hypothalamic peptides that act on their targets via two G protein-coupled receptors (OX1 and OX2 receptors). In the central nervous system, the cell bodies producing orexins are localized in a narrow region within the lateral hypothalamus and project mainly to regions involved in feeding, sleep, and autonomic functions. Via putative pre- and postsynaptic effects, orexins increase synaptic activity in these regions. In isolated neurons and cells expressing recombinant receptors orexins cause Ca2+ elevation, which is mainly dependent on influx. The activity of orexinergic cells appears to be controlled by feeding- and sleep-related signals via a variety of neurotransmitters/hormones from the brain and other tissues. Orexins and orexin receptors are also found outside the central nervous system, particularly in organs involved in feeding and energy metabolism, e.g., gastrointestinal tract, pancreas, and adrenal gland. In the present review we focus on the physiological properties of the cells that secrete or respond to orexins.

sleep; feeding; calcium; neuron; neuroendocrine regulation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
OVERVIEW OF OREXINS AND...
CELLULAR RESPONSES TO OREXINS
DISTRIBUTION OF OREXINERGIC...
SYSTEMIC EFFECTS OF OREXINS
FUTURE PERSPECTIVES
REFERENCES

OUR KNOWLEDGE of the orexinergic system was initiated in 1998 by two independent groups and approaches (reviewed in Refs. 26, 157). In January 1998, the group of Sutcliffe published a study (37) in which they predicted that a particular rat hypothalamic mRNA species ("clone 35"; Ref. 50) would code for a precursor peptide 130 amino acids (aa) in length, each molecule of which would generate two separate peptides of 39- and 29-aa lengths. The peptides were named hypocretins, on the basis of their hypothalamic localization and their proposed sequence similarity to the secretin family of peptides; however, only orexin B bears any significant resemblance to secretin. The precursor peptide would thus be preprohypocretin and the two final peptides hypocretin (hcrt)-1 and hcrt-2. Simultaneously with the work of the group of Sutcliffe, the group of Yanagisawa (154) isolated a hypothalamic 33-aa peptide that activated the orphan receptor HFGAN72. This receptor was found also to respond to another peptide, 28 aa in length. A second receptor was cloned on the basis of its sequence similarity to HFGAN72, and this receptor also responded to both isolated peptides with Ca2+ elevation. On the basis of the peptide sequences, Sakurai and coworkers (154) cloned the cDNA for the precursor peptide (130 aa in the rat). The peptides were named orexins, because they increased food intake in nonfasted rats when injected into the lateral ventricle. The precursor peptide thus became preproorexin, the 33- and 28-aa final peptides orexin A and orexin B, respectively, and the receptors OX1 and OX2 receptors. Both preprohypocretin and preproorexin were found preferentially in the rat brain and more specifically in the hypothalamus (37, 154). Cellular and systemic responses to the peptides were observed in the rat. Application of hcrt-2 increased postsynaptic current frequency in rat hypothalamic neurons in culture (37), and orexin A and -B increased food intake in nonfasted rats (154).

It soon became clear that the peptides isolated by the two groups were, in principle, identical. However, there was an error in the peptide sequences deduced by de Lecea and coworkers (37), so that most authors refer to the accepted sequences by Sakurai et al. (154) and not to de Lecea et al. (37) even when they are using the hypocretin nomenclature. Therefore, the current praxis makes preproorexin = preprohypocretin, orexin A = hcrt-1, orexin B = hcrt-2, OX1 receptor = hcrtr-1, and OX2 receptor = hcrtr-2. There is an ongoing argument concerning which nomenclature should be used. To avoid confusion with the sequences we use the orexin nomenclature throughout this review.

Today there is a wealth of information concerning the anatomic architecture of the orexinergic system as well as the systemic effects of orexins. The most prominent and well-demonstrated effect of orexins is undoubtedly regulation of sleep/wakefulness (reviewed in Refs. 74, 172, 190). Although many other responses have been shown, their physiological significance is less clear. Effects on feeding behavior have been demonstrated in a multitude of studies, but the results are somewhat contradictory. The current view is that orexins may rather regulate short-term appetite, energy metabolism, and feeding-associated processes (e.g., gastrointestinal functions, mastication). Orexins also affect hormonal secretion, the most well-demonstrated effects being those on the hormones involved in the stress response and energy metabolism such as glucocorticoids and norepinephrine. At the same time, information concerning the regulatory mechanisms of orexinergic systems and their targets on a cellular level is lagging behind. In this review, we try to create a comprehensive picture of orexins and their proven and putative physiological role. The main emphasis is placed on functional properties and interactions between the orexin-producing and orexin-responding cells.


    OVERVIEW OF OREXINS AND OREXIN RECEPTORS
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Genetics and Chemistry of Orexins

Mammalian (human, pig, dog, rat, mouse) preproorexin is composed of 130-131 aa (37, 41, 73, 154, 155). The human preproorexin gene (chromosome 17) has been thoroughly characterized (155). It is composed of two exons, the latter of which includes the whole of the coding region of the final peptides. The 3.2-kb promoter region of the preproorexin gene seems to be enough to direct expression of the downstream gene to the lateral hypothalamus in the mouse (155). This has been utilized to cause selective depletion of orexinergic neurons in transgenic mice expressing preproorexin-ataxin-3 fusion protein (60). In these mice, orexinergic neurons are progressively lost postnatally. For transcription, the most essential part has been suggested to be the 450 bp most proximal to the gene (185).

Cleavage of one molecule of preproorexin and further modification leads to production of one molecule each of orexin A and orexin B; however, HPLC detection of orexins in the human brain suggests two- to fivefold higher levels of orexin B than orexin A in several areas of the central nervous system (CNS) (34, 35, 125, 126). Mammalian orexin A in rat is a 33-aa peptide with two intrachain disulfide bridges, and orexin B is a 28-aa linear peptide (Fig. 1A). There is a substantial sequence identity in the COOH termini of these peptides. In addition, orexin B and secretin contain an identical stretch of seven amino acids. Orexin A and -B are likely to be COOH-terminally amidated, and the NH2-terminal glutamine residue of orexin A may be modified to a pyroglutamoyl residue (154); however, the posttranslational modifications have been experimentally verified only in the rat. Orexin A in human, pig, dog, rat, and mouse are identical (Fig. 1B), whereas orexin B in pig and dog differs by one amino acid and orexin B in rat and mouse by two amino acids from human orexin B (Fig. 1C). The three-dimensional structure of orexin A is not known, but orexin B has been determined to consist of two alpha -helices at a 60-80° angle to each other (100).


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Fig. 1.   Alignment of orexin A and orexin B peptide sequences from different species (A-C). Gray boxes indicate dissimilar amino acids (as compared with human orexin variants). Cystine bridges in orexin A are shown in A with black lines. D: OX1 receptor antagonist SB-334867. hcrt, Hypocretin.

Orexin A seems to have much higher stability than orexin B in the physiological milieu (89), which may explain why orexin A is more readily detected in cerebrospinal fluid (CSF) than orexin B (148). Orexin A also displays much higher lipid solubility than orexin B, probably making orexin A---in contrast to orexin B---blood-brain barrier permeant (89). Orexin immunoreactivity has been found in putative endocrine release sites of hypothalamus, neurohypophysis, and pancreas (see below) offering the interesting possibility that orexins could also act as hormones between the CNS and the periphery. Yet indirect evidence suggests that orexin A in plasma originates from peripheral sources (32), and no brain penetration of intravenous orexin A has been seen in the rat (11).

Orexins from Xenopus laevis have also been described (163). In this species, preproorexin is rather similar to that in humans (overall identity = 56%). Xenopus orexin A is 31 aa long, lacking two NH2-terminal amino acids seen in human orexin A, and it contains 6 aa substitutions compared with human orexin A (Fig. 1B). Xenopus orexin B is 28 aa long, and it contains 7 aa substitutions compared with human orexin B (Fig. 1C). Both Xenopus orexins are supposed to be posttranslationally modified in the same way as the rat orexins (disulfide bridges and COOH-terminal amidation) except that Xenopus orexin A does not contain the NH2-terminal pyroglutamoyl residue.

Orexin Release

Orexin A and preproorexin mRNA concentrations show circadian variation in rats in a 12:12-h light-dark cycle (47, 176, 196). Preproorexin mRNA and orexin A concentration in the hypothalamus reach a maximum around the light onset or somewhat later and a minimum at the light offset or somewhat later. Measurements of orexin A in the pons and in the intracisternal space also show diurnal variation, but the time points are shifted compared with the hypothalamus (47, 176). In contrast, Mondal et al. (126) did not find any circadian variation in hypothalamic orexin A or -B concentration; the time points (5 h after dark and 5 h after light onset) may have been unfortunately chosen (in the light of results in Ref. 176). There appear to be bilateral connections between suprachiasmatic nucleus and orexinergic neurons in the rat posterior hypothalamus (1, 36, 64, 121, 125), which may indicate reciprocal regulation of the circadian rhythms.

Orexin Receptors

Two receptors responding to orexin stimulation have been cloned. These receptors, called OX1 and OX2 receptors, form a subclass of their own under the class A G protein-coupled receptors (class A right-arrow peptide receptor right-arrow orexin and neuropeptide FF receptors right-arrow orexin receptors). Both receptors are rather "average" G protein-coupled receptors with modestly long NH2 and COOH termini and neither very long nor short i3 loops (Fig. 2). OX1 receptor (chromosome 1) is 425 aa long and OX2 receptor (chromosome 6) 444 aa long in humans, and there is an overall 64% sequence identity between them. In each gene, the identity of one amino acid is disputed based on a Entrez search (covering a variety of databases such as SwissProt) and a variety of published references: in OX1 receptor, aa 280 in the i3 loop is either Gly or Ala, and in OX2 receptor, aa 308 in the sixth transmembrane segment is either Val or Ile. Amino acids subject to these polymorphisms/mutations are also marked in Fig. 2 (see also Narcolepsy, a Disorder of the Orexinergic System). The gene transcript for each receptor is composed of a 5'-untranslated region, seven separate exons, each of which codes a part of the final protein, and six introns. High sequence identity [91-98%; comparison made with Blast (http://www.ncbi.nlm.nih.gov/BLAST)] is seen between the cloned mammalian species (human, pig, dog, rat, mouse) variants. Two neuropeptide FF receptors (14, 28) are most closely related to the orexin receptors, but the gross sequence identity is very low (31-35% for the human receptors), no longer stretches of high identity can be found, and identity of a nearly similar degree can be observed between neuropeptide FF receptor and, for instance, some neuropeptide Y (NPY) receptors. Furthermore, orexin receptors are 28-31% identical to many other peptide receptors including NPY Y2, thyrotropin (TSH)-releasing hormone (TRH), and NK2 tachykinin receptors. Therefore, grouping of orexin and neuropeptide FF receptors in the same family is questionable.


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Fig. 2.   Human orexin receptor sequences in putative 7-transmembrane plots. Sequences are from the SwissProt and from Ref. 154. Putative transmembrane domains are represented as in the GPCRDB database (Ref. 68; http://www.gpcr.org/7tm). The putative N-glycosylation sites were calculated with NetNGlyc (R. Gupta, E. Jung and S. Brunak, unpublished; http://www.cbs.dtu.dk/services/NetNGlyc) and the putative phosphorylation sites for protein kinase A (PKA) and C (PKC) and Ca2+/calmodulin kinase II (CaMKII) with PhosphoBase (Ref. 95; http://www.cbs.dtu.dk/databases/PhosphoBase). Possible palmitoylated cysteines are also shown. Amino acids that may represent polymorphisms are marked with *, dagger , or Dagger  (see Orexin Receptors and Narcolepsy, a Disorder of the Orexinergic System).


    CELLULAR RESPONSES TO OREXINS
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OVERVIEW OF OREXINS AND...
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In the original studies on orexin receptors two cellular responses were identified: recombinantly expressed orexin receptor strongly elevated Ca2+ in Chinese hamster ovary (CHO)-K1 cells (154), whereas in the hypothalamic neurons increased action potential frequency was observed (37). Many systemic responses were described thereafter but the molecular mechanisms of these have not in most cases been investigated in detail.

Ca2+ Elevation

Both OX1 and OX2 receptors elevate Ca2+ when recombinantly expressed in CHO cells (67, 107, 138, 154, 168). Because orexin receptors caused Ca2+ elevations of high magnitude in this cell line devoid of voltage-gated Ca2+ channels, they were thought to couple to the phospholipase Cbeta -inositol-1,4,5-trisphosphate (IP3)-Ca2+ release cascade via the Gq family of G proteins. The orexins were subsequently shown to elevate intracellular Ca2+ in neuronal cell cultures from hypothalamus and cortex (183, 184). The Ca2+ elevations in neurons were found to be independent of intracellular release and to require extracellular Ca2+ (Fig. 3). The requirement for extracellular Ca2+ is not a typical property of a Gq-mediated response, which consists of Ca2+ release from intracellular stores followed by a "capacitative" Ca2+ entry to refill the stores. The observed Ca2+ elevation in neurons was blocked by a bisindoylmaleimide protein kinase C inhibitor. It was therefore suggested, that orexin receptors would activate protein kinase C, which would phosphorylate and thereby activate voltage-gated Ca2+ channels (183). Similar findings were described by Uramura et al. (181) in isolated rat ventral tegmental neurons. Orexin A was determined to elevate intracellular Ca2+ via activation of nitrendipine- and omega -conotoxin-sensitive Ca2+ channels. The response was also sensitive to calphostin C (inhibitor of protein kinase C) and D609 (inhibitor of phosphatidylcholine-specific phospholipase C), which suggested the following activation sequence: orexin receptor right-arrow phosphatidylcholine-specific phospholipase C right-arrow protein kinase C right-arrow N-/L-type voltage-gated Ca2+ channels right-arrow influx of Ca2+. Ca2+ elevation has also been measured in cultured rat embryonic spinal cord neurons, although the source of Ca2+ has not been investigated (182). Indirect evidence in rat locus ceruleus and tuberomamillary nucleus also suggests activation of Ca2+ influx (44, 70).


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Fig. 3.   Extracellular Ca2+ dependence of the orexin-stimulated Ca2+ response in rat medial hypothalamic neurons in culture. The data demonstrate the extracellular Ca2+ dependence of the orexin-stimulated Ca2+ response. Cells were stimulated with 1 µM rat hcrt-2 (H) in the presence of 1 mM or ~0 mM (0 Ca2+/EGTA) extracellular Ca2+ concentration ([Ca2+]o), and intracellular Ca2+ levels were monitored with fura 2 methodology. As shown, the response to hypocretin 2 was completely blocked by removal of extracellular Ca2+. The experiment was performed in the continuous presence of 1 µM tetrodotoxin (TTX). Rat hcrt-2 is identical to rat orexin-B2-28. Figure is reproduced from Ref. 183 with permission (Copyright 1998 by the Society for Neuroscience).

What then is the general role of Ca2+ influx in orexin receptor-mediated cellular responses? In neuronal cells, Ca2+ influx has been observed by fura 2 measurements (181, 182-184) and is also suggested by electrophysiological measurements (44, 70). Because orexins may depolarize neurons via block of K+ channels (see Depolarizing actions), some Ca2+ influx may even result from an indirect activation of voltage-gated Ca2+ channels. The situation may be more complex at other sites as discussed below.

In CHO cells, Smart et al. (168) and Lund et al. (107) discovered that removal of extracellular Ca2+ caused a significant (8- to 100-fold) drop in the potency of orexin A for the OX1 receptor (Fig. 4A). In other respects, the results of Smart et al. (168) suggest qualities expected from Gq-dependent signaling, that is, sensitivity to the phospholipase Cbeta inhibitor U-73122, rapid and transient spike, and extracellular Ca2+-dependent secondary phase (capacitative Ca2+ entry). In contrast, a more detailed analysis of Ca2+ signaling in CHO cells indicated that the dependence of the Ca2+ response on extracellular Ca2+ is due to the activation of a Ca2+ influx pathway (107). The Ca2+ response was abolished when the membrane potential was held close to the reversal potential of Ca2+ (Fig. 4B), and a Ca2+ elevation could be observed by returning to a negative holding potential (107). In addition, orexin A activated an influx of Mn2+ ions in conditions where no intracellular release occurred. The primary response to OX1 receptor stimulation in CHO cells is thus receptor-operated Ca2+ influx (107). This Ca2+ influx amplifies phosphatidylinositol-specific phospholipase C, so that in the absence of Ca2+ influx, IP3 production and Ca2+ release occur with a potency 100 times lower than in the presence of Ca2+ influx. Ca2+ elevation alone is not sufficient to activate phospholipase C in these cells, suggesting that Ca2+ influx acts in concert with a G protein-mediated mechanism. The identity of this Ca2+ influx pathway and its activation mechanism by the orexin receptors remains unresolved; however, the potency of pharmacological blockers suggests that it is a different molecular entity than the store-operated Ca2+ channel in CHO cells (96). It thus seems that both in neuronal and nonneuronal cells orexin receptors activate both Ca2+ influx and the phosphatidylinositol-specific phospholipase C pathway, a view that is supported by a study in which orexin receptors are heterologously expressed in a variety of different cell types (Holmqvist, Åkerman, and Kukkonen, unpublished observation). In a recent study, orexin A and -B equipotently increased norepinephrine release from rat cerebrocortical slices. When extracellular Ca2+ was removed, the maximum responses to both peptides were lowered by 50% and the EC50 values shifted to >10-fold higher concentrations (66). Although the response mechanisms have not been clarified with direct Ca2+ measurements, the overall pattern is very similar to what is seen with recombinant CHO cells (107).


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Fig. 4.   Extracellular Ca2+ dependence of the orexin-stimulated Ca2+ response in CHO cells expressing human OX1 receptors (CHO-hOX1). A: reduction of [Ca2+]o to 140 nM shifts the orexin A concentration-response curve 2 orders of magnitude to the right. B: orexin A-mediated Ca2+ response can be blocked by reducing the driving force for Ca2+ entry. A CHO-hOX1 cell was patch-clamped in the whole cell configuration to regulate its membrane potential, and therefore the driving force for Ca2+ entry, and Ca2+ transients were measured with fura 2. Gray boxes and black bars indicate presence of 10 nM orexin A in the bath. Under the light gray box, the cells were exposed to orexin A at the holding potential +60 mV, and under the dark gray box the cells were exposed to orexin A at the holding potential -50 or -40 mV. Inset: voltage application profile. Ca2+ response to orexin A is only seen at the negative holding potential, which indicates that Ca2+ influx is required for the response. Figure adapted from Ref. 107.

Functional experiments indicate that in addition to Ca2+ release and activation of store-operated Ca2+ channels, there are several types of Ca2+ influx pathways activated by G protein-coupled receptors (reviewed in Ref. 7). Although in most cases the mechanisms have not been clarified, these pathways may be activated directly by G protein subunits or second messengers. These pathways may be identical or related to the transient receptor potential (TRP) ion channel family (reviewed in Ref. 29). The TRP genes encode a family of over 20 proteins. Many of the TRP gene products form Ca2+-permeable nonselective cation channels, and some members of the TRP (super)family even have inherent protein kinase or ADP-ribose pyrophosphatase domains. TRP channels are widely expressed in mammalian cells, and they are activated after Gq-coupled receptor stimulation but the activation mechanisms are in many cases unknown. It will be interesting to see whether any of the TRP channels can be held responsible for the orexin receptor-mediated Ca2+ influx.

Electrophysiological Responses

Methodological considerations. Characterization of receptor-mediated electrical responses in neuronal preparations is performed with some common electrophysiological techniques. In many situations in which intracellular recordings are difficult, extracellular electrodes can be more practical to use and they are likely to induce much less cellular damage. On the other hand, these response measurements are usually limited to the registration of action potential frequency. Measurements with intracellular electrodes (normal or microelectrodes) can be performed with feedback amplification allowing voltage or current clamp or in a "free-running" mode, in which case voltage changes are monitored. These latter methods, most importantly, offer better possibilities for investigation of the mechanisms underlying the electrical activity than the extracellular electrodes.

Recordings from neurons are usually performed in slices or similar preparations, in which cells are synaptically coupled. The cell measured can thus be the target for upstream signals rather than responding directly to orexins themselves. The usual way of eliminating the secondary effects far upstream of the measured cell is to use tetrodotoxin (TTX). By blocking voltage-gated Na+ channels this hinders action potential propagation in most neurons. However, TTX will not, for instance, block direct effects of orexins on the presynaptic terminal most immediately upstream of the cell investigated. Therefore, TTX does not exclusively separate pre- and postsynaptic effects. Presynaptic voltage-gated Ca2+ channels---and therefore transmitter release---can be blocked with, for example, Co2+, Cd2+, Ni2+, and more specific drugs or toxins. However, orexin receptors may activate Ca2+ channels not sensitive to these inhibitors (see Ca2+ Elevation). On the other hand, these blockers may also affect, for instance, orexin-activated Ca2+ channels other than the voltage-gated channels. This may lead to a block of both pre- and postsynaptic responses to orexin. As an example of this, Ni2+ blocks voltage-gated Ca2+ channel, some orexin-activated non-voltage-gated Ca2+ channels (96), and even the Na+/Ca2+ exchanger (91). "Synaptic isolation" is performed by using medium with low extracellular Ca2+ (>0.3 mM) and high extracellular Mg2+ (<4 mM). This widely used method appears to block electrically evoked synaptic transmission, but the mechanistic basis for it is unclear. Similarly as above, it is unknown whether this method would affect orexin-mediated presynaptic responses if these were mediated by other than voltage-gated Ca2+ channels or by Ca2+ release. It is also difficult to preclude possible postsynaptic effects.

Some methods give a clearer separation of pre- and postsynaptic responses. Dissociation of the cells removes presynaptic contacts. However, even in this case it is possible, although unlikely, that responses are mediated by some indirect messengers released from neighboring cells. Measurement of synaptic activity is a method often used for studies of receptor-coupled mechanisms. Increased/decreased frequency of excitatory or inhibitory postsynaptic currents (EPSC and IPSC, respectively) by orexins suggests a stimulatory effect on transmitter release, i.e., a presynaptic effect. When the amplitude of a synaptic current is increased, it is difficult to conclude whether the response is pre- or postsynaptic.

Increased synaptic activity. The most common electrophysiological response to orexins appears to be increase in spontaneous or evoked action potential frequency, as observed with extracellular or intracellular electrodes. Increased action potential frequency has been measured in the hypothalamus [rat tuberomamillary nucleus, magnocellular preoptic nucleus, lateral hypothalamic glucose-sensitive, -responsive, and -indifferent neurons (for definition, see Responses of orexinergic cells to feeding stimuli), arcuate nucleus, and paraventricular nucleus; Refs. 8, 42, 44, 103, 147, 159, 164, 165, 195], brain stem (rat locus ceruleus , substantia nigra pars reticulata, and dorsal motor nucleus of vagus and mouse laterodorsal tegmental nucleus; Refs. 15, 19, 56, 70, 75, 93, 170), spinal cord (rat preganglionic sympathetic neurons of intermediolateral cell column of thoracic and lumbar cord; Ref. 3), and peripheral neurons (guinea pig ileal S-type submucosal neurons; Ref. 92). In contrast, orexin A suppresses the firing rate of glucose-responsive neurons in rat ventromedial hypothalamic nucleus, antagonizing the effect of glucose (164). Action potentials and their enhancement with orexins are blocked by TTX (3, 44, 54, 70, 75, 165, 170, 195) but usually not by synaptic isolation with low Ca2+-high Mg2+ (8, 42, 93, 147) or voltage-gated Ca2+ channel block with Cd2+ (170). The latter data therefore suggest that orexins would directly affect the postsynaptic neurons at some sites, with reservations for the methodological shortcomings (see above). It should also be noted that the putative presynaptic block has only been applied in a minority of studies. Interestingly, a TTX-insensitive response is observed in rat locus ceruleus. Hypocretin 2 (approx orexin B) depolarizes and increases spontaneous firing frequency in locus ceruleus slices (70). In the presence of TTX these spontaneous spikes vanish, but orexin B still causes a small depolarization (3.4 mV) and electrical discharges, although these spikes are slower than the spontaneous spikes. These spikes may be caused by Ca2+ channels or TTX-insensitive Na+ channels.

More detailed analysis of individual synaptic currents shows enhancement of synaptic activity by orexins as an increase in spontaneous or evoked EPSC (rat hypothalamus, rat superficial dorsal horn, and mouse laterodorsal tegmental nucleus; Refs. 19, 37, 49, 54, 183, 184) and IPSC (rat hypothalamic and superficial dorsal horn neurons; Refs. 49, 54, 183) frequency. EPSCs and IPSCs are blocked by TTX in rat superficial dorsal horn and mouse laterodorsal tegmental nucleus (19, 54) but not in rat hypothalamus (49, 183). Therefore, the orexin receptor mediating these putative presynaptic responses may lie farther upstream in rat superficial dorsal horn and mouse laterodorsal tegmental nucleus. In both of these sites, depolarization mediated by orexins persists in the presence of TTX, suggesting that orexin receptors are expressed both on the cell investigated and on some upstream neuron. In some cases, the released transmitters causing the postsynaptic currents have been investigated with receptor blockers. In mouse laterodorsal tegmental neurons, the orexin-mediated elevation in the EPSC frequency is attenuated by ionotropic glutamate receptor block, whereas the (postsynaptic) depolarization response is not affected (19). In rat embryonic hypothalamic neurons in culture, hypocretin 2 (approx orexin B) increases IPSC and EPSC frequency in a TTX-insensitive manner (49, 183). The former is blocked by GABAA receptor block and the latter by ionotropic glutamate receptor block. In rat superficial dorsal horn neurons, orexin B-mediated increase in IPSC frequency is inhibited by glycine receptor block (54). Amplitudes of the postsynaptic currents or potentials are also sometimes affected, as reported for guinea pig ileal submucosal ganglia [reduced excitatory postsynaptic potential (EPSP) and inhibitory postsynaptic potential (IPSP) amplitude; Ref. 92], mouse laterodorsal tegmental nucleus (increased EPSC amplitude; Ref. 19), and rat arcuate nucleus (increased IPSP amplitude; Ref. 183). Increased amplitude of postsynaptic potentials or currents does not specify a pre- or postsynaptic mechanism in these cases.

Depolarizing actions. Depolarization or inward current has often been measured in response to orexin application. This depolarization also results, when investigated, without exception in increased action potential frequency, and often the depolarization has been studied in the presence of TTX to block this "disturbance" (Fig. 5, A and B). Depolarizations of a maximum magnitude of 3-10 mV have been observed---mainly with free-running electrodes---in the hypothalamus (rat tuberomamillary nucleus, paraventricular nucleus, and lateral hypothalamic glucose-sensitive neurons; Refs. 8, 44, 103, 159, 165), in the brain stem (rat locus ceruleus and dorsal motor nucleus of vagus; Refs. 56, 70, 75, 80, 170), in the spinal cord (rat intermediolateral cell column; Ref. 3), and in the periphery (guinea pig ileal S-type submucosal neurons; Ref. 92). Inward currents have been observed in rat locus ceruleus (150 pA; Ref. 170), dorsal motor nucleus of vagus (30 pA; Ref. 75), and superficial dorsal horn (35 pA; Ref. 54) and in mouse laterodorsal tegmental nucleus (20 pA; Ref. 19).


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Fig. 5.   Orexin-mediated increase in action potential frequency (A) and depolarization (B) in rat hypothalamic tuberomamillary nucleus neurons in a slice preparation. Whole cell recordings were performed with intracellular microelectrodes in free-running mode. Cells were stimulated with 300 nM orexin A where indicated. Experiment in B was performed in the presence of 300 nM TTX. Figure reproduced from Ref. 44 (Copyright 2001 by the Society for Neuroscience).

The orexin-stimulated increases in current most probably reflect direct effects of orexin. Evidence for this includes the stable currents and lack of effect of TTX, "synaptic block," or other kinds of elimination of EPSCs. What then is the mechanism of this depolarization? In rat lateral hypothalamic glucose-sensitive neurons, locus ceruleus, autonomic preganglionic neurons of intermediolateral cell column, and dorsal motor nucleus of vagus and in guinea pig ileal S-type submucosal neurons, increased input resistance (by 2-85%) has been measured to co-occur with the depolarization (3, 75, 80, 92, 103). This suggests that orexins may inhibit K+ channels, which is also suggested by the reduced afterhyperpolarization amplitude observed in rat locus ceruleus and in guinea pig ileal submucosal ganglia (70, 92). Also, the current-voltage relationship, when measured, suggests K+ channel inhibition (rat locus ceruleus and dorsal motor nucleus of vagus; Refs. 75, 80). On the other hand, orexins may activate Ca2+-dependent K+ channels, as suggested in mouse peritoneal macrophages (76). It is interesting that orexins instead inhibit the slow afterhyperpolarization in locus ceruleus, which is thought to depend on Ca2+-activated K+ channels. It is possible that orexins do not elevate Ca2+ at all sites or that other signals generated by the receptor activation counteract the effect of Ca2+ elevation.

In other cases, reduced input resistance or noise is associated with the orexin-induced depolarization. This has been seen in rat tuberomamillary nucleus (44), rat lumbar spinal cord (54), and mouse laterodorsal tegmental nucleus (19). In hypothalamic histaminergic tuberomamillary nucleus, orexin A and -B both depolarize and increase the firing rate (8, 44, 195) (Fig. 5, A and B). This depolarization is abolished by removal of extracellular Na+ and virtually unaffected by an increase in extracellular K+ (44). Pharmacological block of Na+/Ca2+ exchanger reduces the depolarization, suggesting that it may be mediated by Ca2+ elevation-induced activation of electrogenic Na+/Ca2+ exchange. The residual depolarization is blocked by Ni2+, which may suggest that the putatively underlying Ca2+ current can also slightly depolarize the cells itself. Even in rat dorsal motor nucleus of vagus, a part of the current appears to depend on Na+ and the remaining part may be caused by K+ channel block (75).

Finally, no clear change in the input resistance may be seen [rat hypothalamic paraventricular nucleus (165), locus ceruleus (56, 170), and intermediolateral cell column (3)]. This could occur if the increased conductance caused by opening of some ion channels (Na+, Ca2+?) was compensated by the closure of other channels (K+) or if orexins rather affected transporters. Even in a single cell, orexins may utilize several mechanisms for depolarization (44, 75).

Summary of orexin effects on electrical activity of neurons. Thus orexins most often increase neuronal activity via presynaptic effects (increased transmitter release via unknown mechanisms) and via postsynaptic depolarization (block of K+ channels, activation of cation channels and electrogenic transporters?). Some of the effects such as the putative K+ channel inhibition are typical for Gq-coupled receptors (see, e.g., Refs. 59, 65, 112, 187). Other mechanisms may be secondary to the Ca2+ elevation or may depend on the activation of nonselective cation channels like the TRP channels. On particular neurons, orexins may also cause inhibition via increased inhibitory transmitter release or via decreased postsynaptic effects of excitatory transmitters. It is unclear which direct or indirect roles Ca2+ transients play in electrical responses to orexins, and not much effort has been put into investigation of this to date. Independent of the method of measurement of electrical activity, the effects of orexins usually have a slow onset of action (10 s-4 min) and the recovery is even slower (see, e.g., Refs. 3, 9, 19, 44, 54, 75, 93, 103, 159, 165, 195). Most studies have been performed with slices, a preparation in which perfusion of orexins in and out may be slow. However, because rather superficial neurons are patched, there is no obvious reason why the perfusate would not reach the neurons immediately. Furthermore, similar slow onset (10- to 20-s lag, 1 min to maximum) and delayed offset (>2 min to complete decay after removal) are seen in dissociated neurons (195). Therefore, it is possible that orexins affect ion channels via, e.g., phosphorylation rather than direct G protein interaction. Some results indicate involvement of protein kinase A and -C in orexin responses (93, 181, 183). Orexin B has been reported to reduce N-methyl-D-aspartate (NMDA)-induced current amplitude by 19% and to enhance GABA-induced current amplitude by 48% in dissociated rat nucleus accumbens cells (113); this could also possibly be a phosphorylation-dependent event.

Transmitter Release

As discussed in Increased synaptic activity, orexins may increase transmitter release as indicated by increased/decreased synaptic potential frequency. These are the most direct indications of presynaptic effects of orexins, and in the presence of TTX, rather certainly suggest a presynaptic site for orexin effects. Inhibition of K+-stimulated serotonin release by orexin A and -B from rat hypothalamic synaptosomes also directly shows a presynaptic site of action (140). There are also indirect indications of orexin-stimulated transmitter release from in vitro slice (norepinephrine from rat cerebral cortex; Ref. 66) or in vivo microdialysis (histamine from rat anterior hypothalamus; Ref. 79) studies. In these cases, it is impossible to tell whether orexins directly affect the releasing neurons or some upstream neurons; some complex mechanism is suggested by the delayed (by 6 min) response in rat cerebrocortical slices.

Other Responses

cAMP elevation in response to orexin stimulation was reported in the rat and human adrenal cortices, putatively via OX1 receptors (110, 117). In contrast, in primary cultures of rat hypothalamic neurons, no elevation of cAMP is seen (183). Elevation of cAMP may occur by several mechanisms depending on the isoform of adenylyl cyclase expressed. Adenylyl cyclase isoforms are differentially activated by Ca2+, protein kinase C, and G protein beta gamma -subunits (171), and the expression of adenylyl cyclase isoforms may be different in the adrenal cortex and the hypothalamus. G proteins might also be expressed tissue-specifically, and OX1 and OX2 receptors could couple to different G proteins. OX2 receptors have been reported to couple to Gi and Gs but not Go or Gq proteins in human fetal adrenal glands (88) and to Gi, Gs, and Gq but not Go proteins in adult human adrenal glands (146), as measured with the GTP-azidoanilide labeling method.

Orexin receptors may also couple to protein kinase cascades involved in cell growth, differentiation, and death. These effects are clearly seen when orexin receptors are expressed in CHO cells (S. Ammoun, L. Korhonen, L. Lindholm, K. E. O. Åkerman, and J. P. Kukkonen, unpublished observation). One of the most immediate indications is the rapid activation of mitogen-activated protein kinase (MAPK) pathways seen on OX1 receptor stimulation (Fig. 6).


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Fig. 6.   Mitogen-activated protein kinase (MAPK) activation in response to OX1 receptor stimulation in CHO-hOX1 cells (S. Ammoun, L. Korhonen, L. Lindholm, K. E. O. Åkerman, and J. P. Kukkonen, unpublished observation). Extracellular signal-regulated kinase (ERK)1 and -2 activation was measured by blotting against the respective phosphorylated species with specific antibodies. Both 3 and 100 nM orexin A lead to rapid (3 min) activation of mainly ERK2, a response that is more clearly decreased at the 30 min time point at the lower concentration.

Pharmacology

OX1 and OX2 receptors were already shown in the original study to have different binding affinities for orexin A and -B (154). When expressed recombinantly in CHO cells, OX1 receptor has 10 times higher affinity for orexin A than for orexin B whereas OX2 receptor binds orexin A and -B with equal affinity. The same selectivity is seen with respect to Ca2+ elevation; thus orexin A activates OX1 receptors up to 10 times more potently than orexin B (138, 154, 168). Thus the evidence for selectivity of OX1 receptor and nonselectivity of OX2 receptor between orexin A and -B is based solely on the studies on Ca2+ elevation in recombinant CHO cells. It is difficult to find exclusive expression of one subtype in any tissue, but in some cases support for this selectivity can be found: orexin A is 5.5-fold more potent in increasing electrical activity of rat locus ceruleus, where OX1 expression dominates (170). In isolated human adrenocortical cells, where OX1 mRNA is also predominantly found, orexin A but not orexin B stimulates cortisol secretion (117). In tissues where OX2 expression dominates, such as rat tuberomamillary nucleus, orexin A and -B are essentially equipotent in stimulating the electrical activity. Orexin A and -B are also equally potent in releasing catecholamines from human pheochromocytoma cells, which only express OX2 mRNA (116). On the other hand, the putatively OX2-mediated adrenocorticotropic hormone (ACTH) release is 10-fold less sensitive to orexin B than to orexin A (13, 158). It is generally dangerous to rely on so-called selective agonists, because their efficacies can be very dependent on receptor and G protein expression levels. For these pharmacological reasons and because of the chemical differences between orexin A and -B (see Genetics and Chemistry of Orexins), conclusions on receptor subtype involvement based on potency of orexin variants (42, 113, 138, 158) should probably be avoided. Some studies report higher potency of orexin B than orexin A with respect to some responses (76, 85), which is unexplainable on the basis of recombinant pharmacology. Even if any pharmacological selectivity with respect to the interaction of orexin A and -B with the orexin receptor subtypes was to occur in the tissues, its physiological significance would be unclear.

Molecular determinants required from orexin peptides for binding to and activation of orexin receptors have been investigated in CHO cells. Comparison of structures and activities of orexin A and -B suggests that the NH2-terminal amino acids are less important for receptor binding and activation than the COOH-terminal amino acids (33). Truncation of orexin A to orexin A15-33 reduces its potency for both OX1 and OX2 receptors 20- to 60-fold (33, 138), and further truncation successively abolishes the Ca2+ response, at least for OX1 (33). Mutation of cystine-forming cysteines to alanines in orexin A reduces the potency of the mutant peptides for both OX1 and OX2 receptors ~10-fold (138). However, it is difficult to judge whether this effect is due to the lost disulfide bridges or the exchanged cysteines themselves. One-by-one replacement of each amino acid in orexin-A15-33 with alanine (alanine scan) has revealed regions of particular importance for orexin A activation of the OX1 receptor, in particular leucines 19 and 20 and the most COOH-terminal aa 26-33 seem to be of great importance (33). Recently, we observed (S. Ammoun, T. Holmqvist, R. Shariatmadari, R. Oonk, M. Detheux, M. Parmentier, K. E. O. Åkerman, and J. P. Kukkonen, unpublished observations) that OX1 and OX2 receptors are approximately equally sensitive to NH2-terminal truncation and alanine scan of the orexin peptides. The amino acids of importance are largely the same as those described in Ref. 33. Change/removal of these amino acids affects both OX1 and OX2 receptors, but some interesting differences are seen in the potency of the response. Interestingly, none of the mutated peptides without activity are antagonists.

Xenopus orexin B has been proposed to have 10-fold higher affinity and potency than Xenopus orexin A for human OX2 receptor expressed in CHO cells (163). Both Xenopus orexin A and -B were equipotent on human OX1 receptor. Synthetic peptides (hcrt-1 and hcrt-2) based on the originally described hypocretin sequences (37) have been shown to be ~1,000-fold less potent than the corresponding orexin peptides with respect to Ca2+ elevation in recombinant CHO cells (167).

In more than 50% of the studies on cellular responses, rather high concentrations (>100 nM) of orexins were used to evoke response. Even the EC50 values, when determined, can be rather high [e.g., 200 nM for orexin A in rat locus ceruleus (increased firing); Ref. 170]. On the other hand, even subnanomolar concentrations evoke some responses (e.g., Ca2+ response in rat ventral tegmental neurons; Ref. 130). To resolve the mechanical basis for this, experiments could be performed in recombinant expression systems at different expression levels with measurement of different responses. Another reason for the high concentrations required in some tissues may be the use of orexin B at OX1 receptor or use of the hypocretins based on the original sequences, if the weak potencies reported in recombinant systems hold true in tissues (see above).

At the moment, no reliable in vivo selectivity can be obtained with known orexin receptor agonists. Recently, synthesis of an orexin receptor subtype-selective antagonist was reported (145, 169). This antagonist, SB-334867 (Fig. 1D), displays a 100-fold higher affinity for OX1 than OX2 receptor. This selectivity has been successfully utilized in in vivo experiments to elucidate the role of OX1 receptors in some systemic effects of orexins (see SYSTEMIC EFFECTS OF OREXINS). Unfortunately, no antagonist able to block OX2 receptors is available. Conclusions on the role of OX1 receptors in some physiological processes should not be based solely on SB-334867, because orexin receptor subtypes may have overlapping and interacting roles. However, SB-334867 is a uniquely useful tool in orexin receptor investigations. From this perspective, it is unfortunate that SB-334867 is not widely available for orexin receptor researchers.

Recently, a variety of peptide transmitters, including secretin and NPY variants, were reported to inhibit orexin A binding to membrane preparations from endogenous and recombinant cells expressing OX1 receptors, even with a nanomolar affinity (87). These results were interpreted to indicate that interaction between orexinergic and NPYergic systems (see Connections of orexinergic pathways with other feeding-regulating transmitters/hormones) could occur at the orexin receptor level. These results on binding could not, however, be verified in functional experiments or in binding experiments with intact cells (67, 169). Other peptides [secretin, pituitary adenylate cyclase-activating polypeptide (PACAP), NPY variants] do not display any detectable affinity or activity on OX1 or OX2 receptors (67, 169). As suggested in Ref. 67, it is more likely that the other peptides affect some intracellular process, for instance G protein binding to the receptor, which is required for high-affinity agonist binding, and therefore reduce orexin binding in some particular preparations. Therefore, secretin should not be used to define specific orexin A binding, because this can to lead to possibly erroneous conclusions (86, 188).


    DISTRIBUTION OF OREXINERGIC CELLS AND OREXIN RECEPTORS
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ABSTRACT
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OVERVIEW OF OREXINS AND...
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SYSTEMIC EFFECTS OF OREXINS
FUTURE PERSPECTIVES
REFERENCES

Orexinergic Cells in CNS

The distribution of orexinergic cells has been investigated with molecular biological and immunological methods, which give rather similar results. The main peptide investigated has been orexin A, but comparison of the distribution of orexin A, orexin B, and preproorexin suggests extensive colocalization. Therefore, all the data have been pooled. Most of the data on orexin distribution originate from the rat but, when investigated, the other vertebrate species have given similar results.

Orexinergic Cell Bodies in Rat CNS

In the rat brain, orexinergic cell bodies are found solely in the hypothalamus. Within the hypothalamus these cell bodies reside bilaterally symmetrically in the lateral parts, probably most abundantly in perifornical areas (17, 24, 31, 36, 37, 56, 58, 69, 70, 131). Orexinergic cells are interspersed among melanocyte-concentrating hormone (MCH)-ergic cells (24, 143). However, the overlap of the areas is not complete (17) and MCHergic cells are clearly different from orexinergic cells (17, 43, 58, 143), although they otherwise share some of the properties of orexinergic cells (1, 69, 71). Isolated orexinergic cell bodies have also been reported in median eminence, posterior, dorsal, and dorsomedial hypothalamus, and arcuate and subincertal nuclei (24, 31, 143).

Orexinergic neurons are variable in size (diameter of cell body = 15-40 µm) and shape (spherical, fusiform, multipolar) (24, 31, 36, 131), and they have been assumed to number from 1,100 to 3,400 in the whole rat brain (61, 143). Because orexinergic cells have been shown to project to other orexinergic cells within the hypothalamus (69) there might be different populations of orexinergic cells within the hypothalamus.

Preproorexin mRNA and protein have even been detected in the ependymal cell layer (97, 143), and orexin A has been found in choroid plexi (31). It would be interesting to find out whether this expression occurs in the ependymal cells or, for instance, in neuronal stem cells.

Orexinergic Projections in Rat CNS

Orexinergic fibers have been visualized mainly by using orexin A immunohistochemistry. For orexin A, both smooth and varicose fibers have been observed (31, 143) but varicose fibers appear to be the ones most often seen, although this result might also be caused by the easier detection of this kind of processes. A few studies investigating orexin B distribution report projection sites similar to those for orexin A (31, 36).

Despite the low number of orexinergic cell bodies in the hypothalamus, orexinergic fibers project widely in the CNS. The most important orexinergic projection areas are within the hypothalamus, two thalamic nuclei, brain stem, and the whole length of the spinal cord (Table 1; see also Fig. 7). Interested readers are recommended to consult the original papers, some of which are very detailed (24, 31, 34, 36, 37, 61, 69-71, 97, 125, 126, 154, 182).

                              
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Table 1.   Orexin distribution in rat central nervous system



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Fig. 7.   Orexinergic pathways (according to Refs. 24, 31, 34, 36, 37, 61, 69-71, 125, 126, 154, 182) and orexin receptor mRNA distribution (according to Refs. 111, 180) in the rat. Only a minority of the sites are shown. The density of the hatching (3 different levels) indicates OX1 and OX2 receptor mRNA expression levels. It should be noted that there are subregions within the marked regions that display different expression (e.g., in the hypothalamus), which cannot be shown in the figure. When data in 2 references were diverging or even contradictory, an average was taken. Arcn, arcuate nucleus; CA1-3, areas of hippocampus; cC, cingulate cortex; CMn, centromedial nucleus; dR, dorsal raphe nucleus; LC, locus ceruleus; mE, median eminence; mR, median raphe nucleus; nST, nucleus of solitary tract; olfB, olfactory bulb; olfT, olfactory tubercle; PVn, paraventricular nucleus (in the thalamus and hypothalamus); Rm, nucleus raphe magnus; Ro, nucleus raphe obscurus; SCn, suprachiasmatic nucleus; SOn, supraoptic nucleus; STn, spinal trigeminal nucleus; TMn, tuberomamillary nucleus; VAMn, ventral anteromedial nucleus.

Orexins in CNS of Other Species

Only a few studies have been conducted in species other than the rat. However, when these studies have been performed, the results seem to be in good agreement with studies in the rat. In several vertebrate species (Table 2) orexinergic cells are found in hypothalamic areas similar to those in the rat. The cells have been assumed to number ~20,000 and 50,000-83,000 in dog and human brain, respectively (148, 178). The projection areas also appear similar in the rat and other species (Table 2); however, in most studies only limited areas were investigated, making full conclusions impossible. Also in these species, orexinergic neurites are often determined to be varicose.

                              
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Table 2.   Distribution of orexinergic cells and pathways in other species

Orexin Receptors in CNS

Similarly to orexins, orexin receptor distribution has been investigated with molecular biological and immunological methods. Almost all the data originate from the rat. Altogether, orexin receptors are found at the projection sites of orexinergic neurons (see above), which suggests that orexinergic neurons contact many different transmitter systems (see below). Most of the studies have not specified whether the expression is seen on neurons or other cells, although electrophysiological and other measurements, whenever performed, indicate activation of neurons. Levels of orexin receptors can but do not necessarily fully follow the density of orexinergic innervation. Only in a few cases have synaptic contacts been anatomically shown (48, 55, 70, 195). Localization of some orexinergic cells/processes close to the ventricular surfaces (24, 31, 97, 143) suggests that orexins could act in paracrine or endocrine fashion.

Similar amounts of OX1 and OX2 mRNA are found in the rat brain as a whole, and OX1 and OX2 receptor mRNAs mostly show similar distribution, although some differences are seen (Fig. 7). Most interesting are the areas in which essentially only one subtype is expressed (111, 180). However, mRNA expression may not directly correspond to receptor protein expression, as discussed by van den Pol et al. (184). The correlation of receptor distribution with different functions of the orexinergic system is shown in Table 3. While this article was under review, studies on the expression of OX1 and OX2 receptor protein in the rat CNS were published (30, 64). A particularly interesting finding is the expression of OX2 receptor protein in the molecular and granular layers of the cerebellum (30); earlier studies had failed to detect any orexin receptors or their mRNA in the cerebellum although orexinergic fibers were projecting to the cerebellum. Otherwise, the immunohistochemical studies suggest a distribution generally similar to that from the mRNA measurements, although some differences are seen. For instance, greater overlap of the expression of OX1 and OX2 receptors is suggested on the basis of immunological studies than of in situ hybridization. As discussed by the authors, these differences may in some degree be related to the fact that mRNA measurements visualize the expressing cell bodies whereas immunohistochemistry indicates the actual cellular localization of the protein. Both receptor mRNA and protein measurements are semiquantitative rather than quantitative so they do not allow absolute comparison of subtype expression within any site.

                              
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Table 3.   Orexin receptor mRNA distribution with respect to postulated functions

Coexpression and Connections of Orexins With Other Transmitters

Despite the partial overlap of distribution of orexinergic and MCHergic cell bodies, there is no coexpression of these peptides in the same cells (Refs. 17, 58, 143; Figs. 8 and 9). In contrast, essentially all the orexin and prodynorphin expression colocalizes in the lateral hypothalamus (10, 27). Orexinergic neurons may also coexpress other transmitters such as galanin (58) and glutamate (1).


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Fig. 8.   Some connections between lateral hypothalamic orexinergic neurons and other transmitters/hormones. Orexinergic cells also express dynorphin, and at least some of them also express Ob-Rb (long form of leptin receptor) and the leptin receptor-activated promoter element STAT3. Subpopulations of orexinergic cells may release galanin and glutamate (for references, see text). Thin black arrows indicate input to the orexinergic cells, and thick gray arrows indicate orexinergic output. Anatomic loci: Arcn, LC, laterodorsal tegmental nucleus (LDTn), lateral hypothalamus (LH), hypothalamic periventricular nucleus (PeVn), preoptic area (POA), PVn, raphe nuclei (Raphe n), SCn, substantia nigra (SN), TMn, ventral tegmental area (VTA). ACh, acetylcholine; AGRP, agouti gene-related peptide; AVP, (arginine-) vasopressin; CART, cocaine/amphetamine-regulated transcript; CRF, corticotropin-releasing hormone; DA, dopamine; 5-HT, 5-hydroxytryptamine (serotonin); Glu, glutamate; HA, histamine; MCH, melanocyte-concentrating hormone; NE, norepinephrine; NPY, neuropeptide Y; POMC, proopiomelanocortin; SSt, somatostatin; VIP, vasoactive intestinal peptide.



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Fig. 9.   Some connections between orexins and other appetite-regulating transmitters/hormones in the hypothalamus. Arrows marked + and - indicate stimulation or inhibition, respectively, of neuronal activity or release or synthesis of the hormones in the affected site; ± indicates either stimulatory or inhibitory effects under different conditions or toward similar cells in different anatomic loci. Unmarked arrows indicate connections with unknown effects. As indicated in the text, orexinergic neurons may either themselves respond to glucose or have reciprocal connections with glucose-sensitive (GSN) or glucose-responding (GRN) neurons. Thus it is not clear whether the effect glucose on neurons in LH or Arcn is direct or mediated by other "sensor neurons"; therefore, glucose, GSN, and GRN are grouped in the figure with a dotted line. Solid lines indicate the anatomically defined hypothalamic areas.

Vasoactive intestinal peptide (VIP)-, vasopressin-, and NPY-containing neurons innervate orexinergic neurons in the hypothalamus (Refs. 1, 69, 71; Figs. 8 and 9). On the other hand, orexinergic nerves from lateral hypothalamus innervate neuronal circuits, which utilize many different transmitters, for instance, norepinephrine, dopamine, serotonin, histamine, acetylcholine, vasopressin, VIP, somatostatin, corticotropin-releasing hormone (CRF), NPY/agouti-gene-related peptide (AGRP), proopiomelanocortin (POMC), cocaine/ amphetamine-regulated transcript (CART), GABA, MCH, and glutamate (Figs. 8 and 9; for the anatomy, see, e.g., Refs. 5, 31, 36, 131, 143), and even functional responses to orexins have been shown at some of these sites (e.g., Refs. 9, 18, 19, 42, 44, 49, 56, 70, 80, 93, 113, 130, 159, 165, 170, 183, 195; see CELLULAR RESPONSES TO OREXINS). Orexinergic neurons even make contact with other orexinergic neurons within the hypothalamus (5, 69).

Connections of orexinergic pathways with other feeding-regulating transmitters/hormones. Orexins were first isolated as appetite-increasing peptides, although this view was later disputed. Several hypothalamic sites involved in the regulation of feeding, such as the lateral hypothalamic area, parvocellular paraventricular nucleus, ventromedial nucleus, and arcuate nucleus, are innervated by orexinergic fibers from the lateral hypothalamic area (see e.g., Ref. 36). Both OX1 and OX2 receptors may be expressed at these projection areas (111). There are abundant connections between orexin and other feeding-regulating transmitters/hormones such as leptin, NPY, MCH, POMC, CART, galanin, and AGRP (Figs. 8 and 9). Almost all the orexin-positive cells also express leptin receptor, and even STAT3---the leptin receptor-activated promoter element---and orexin are coexpressed (Refs. 58, 69, Fig. 8; see also Effects of orexins on gastrointestinal tract and other organs of feeding/energy metabolism). On the other hand, not all the leptin receptor-positive cells in the lateral hypothalamic areas where orexinergic cell bodies lie are orexin positive (58). Other cells that express leptin receptors are the MCHergic neurons in the lateral hypothalamus and the NPY-/AGRP-, POMC- and CARTergic cells in the arcuate nucleus (Refs. 69, 57, 58, 119; Fig. 9). Orexinergic fibers project to NPY- and POMCergic cells in the arcuate nucleus (69). On the other hand, NPY/AGRP- and POMCergic fibers, most likely from the arcuate nucleus, project to orexin- and MCHergic cells of the lateral hypothalamus (17, 43, 55, 69, 71). Orexinergic cells of the lateral hypothalamus also contain other feeding-regulating transmitters such as dynorphin and galanin (Refs. 10, 27, 58; Fig. 9), which may enhance the effects of orexins on feeding.

Responses of orexinergic cells to feeding stimuli. Hypoglycemia induced by various means has been shown to increase c-fos (see below), orexin, and preproorexin mRNA expression in a subpopulation of the orexinergic cells of the lateral hypothalamus (16, 20, 22, 53, 99, 105, 127; see also Effects of orexins on gastrointestinal tract and other organs of feeding/energy metabolism). Fasting also abolishes the circadian variation in CSF orexin levels (47). The lateral hypothalamic area, as well as some other hypothalamic regions, contains neurons that are activated by decreased levels of glucose ("glucose-sensitive neurons") and neurons that are activated by elevated levels of glucose ("glucose-responsive neurons") (Ref. 101; Fig. 9). In the study of Muroya et al. (129), almost half of the histochemically defined orexinergic neurons of the rat lateral hypothalamus directly responded to lowering of extracellular glucose with an increase in cytosolic Ca2+; thus some of the glucose-sensitive neurons in lateral hypothalamus may be orexinergic. Liu et al. (103) identified glucose-sensitive, -responsive, or -indifferent populations among rat hypothalamic neurons that responded to orexin stimulation with depolarization and increased firing rate. In contrast, orexin A suppressed the firing rate of glucose-responsive neurons in rat ventromedial hypothalamic nucleus (164). Orexinergic neurons may also make contact and/or be contacted by glucose-sensitive or -responsive neurons in other brain areas such as lateral hypothalamus, ventromedial hypothalamus, arcuate nucleus, and the nucleus of the solitary tract (Ref. 101; see also anatomic studies on orexinergic projections).

Leptin is thought to be one of the major negative regulators of orexin expression in the hypothalamus (see Effects of Orexins on Feeding Behavior), probably both directly via leptin receptors on orexinergic cells and via leptin receptors on other feeding-controlling cells connected to orexinergic cells (see above). Attempts to elucidate leptin-orexin interaction by measurement of orexin and orexin mRNA levels have been made in rodent model systems with defective leptin signaling, such as fa/fa Zucker rats and db/db mice (devoid of functional leptin receptors) and ob/ob mice (devoid of leptin) (reviewed in Ref. 21). The results are, however, contradictory, and if any conclusion can be drawn, it is that leptin is not necessarily required to keep orexin expression low. However, as also often in knockout systems, it is difficult to evaluate the physiological role because of possible multiple disturbances, redundancy in signaling, and compensatory mechanisms.

Orexins and Orexin Receptors Outside CNS

Orexin immunoreactivity or preproorexin mRNA has been found in a variety of peripheral organs, mainly in the rat. The cells expressing orexins have in some cases been identified and include both neurons and endocrine cells. Some projections, e.g., those in the pituitary and pineal gland, may originate from the neurons in hypothalamus, but most peripheral neurons appear to have peripheral origin. Orexin receptors/receptor mRNA have been mainly found in the same organs as orexins. The cell types expressing orexin receptors might include at least endocrine, muscle, and nerve cells (84, 92, 108, 117, 133). Even in the periphery, expression of the subtypes varies in different tissues.

Orexin and orexin receptor immunoreactivity has been found in the gastrointestinal tract and pancreas (rat, guinea pig, human; Refs. 92, 133; Tables 4 and 5). Substantial levels of both orexin A and -B and OX1 and OX2 mRNA and protein are found in the rat pituitary (13, 35, 84), and Jöhren et al. (84) found preproorexin and OX1 mRNA in the rat testis. Both OX1 mRNA and OX2 mRNA or immunoreactivity are found in the rat and human adrenal glands (12, 84, 104, 108, 109), although the subtype expression in different layers is somewhat disputed and there may be differences between species (see, e.g., Ref. 117). Only OX1 mRNA has been found in the kidney and thyroid and only OX2 mRNA in the lung (84). Orexinergic fibers extend from the CNS toward the rat pineal gland, and OX2 mRNA is detected in the pineal gland (121).

                              
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Table 4.   Orexins in periphery


                              
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Table 5.   Orexin receptors in periphery

However, the expression of preproorexin or orexin receptor message in the stomach, gut, pancreas, lung, kidney, pituitary, or adrenal gland has been disputed (31, 84, 154). Some differences may be explained by the less sensitive methods (Northern blotting) failing to detect a signal when more sensitive methods (PCR, in situ hybridization) do. In contrast, it is very difficult to explain opposite receptor subtype expression reported for human adrenal cortex (117, 146).


    SYSTEMIC EFFECTS OF OREXINS
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ABSTRACT
INTRODUCTION
OVERVIEW OF OREXINS AND...
CELLULAR RESPONSES TO OREXINS
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FUTURE PERSPECTIVES
REFERENCES

The location of orexinergic cells, their projections, and their contacts with other transmitter systems suggest a role in eating, sleep/wakefulness, and neuroendocrine and autonomic functions. It is sometimes difficult to judge on the basis of the available experimental evidence whether a particular systemic response to orexins is caused indirectly or directly on the target organs. In some cases, direct effects of orexins on isolated organs or even isolated cells have been demonstrated; we have been trying to highlight this kind of evidence.

Effects of Orexins on Feeding Behavior

In the original publication dealing with orexins, it was shown that intracerebroventricular injection of orexins induces feeding in rats (154). When injected into different hypothalamic sites in the rat, orexin A is effective only in a narrow area (perifornical area, lateral hypothalamus, and dorsomedial and paraventricular nuclei but not in preoptic area or arcuate or ventromedial nucleus) whereas orexin B is inactive (39, 173). Some of the hypothalamic nuclei possibly involved in feeding responses to orexins are listed in Table 3. Intraperitoneally injected OX1 receptor antagonist (SB-334867) inhibits baseline feeding, normal weight gain, and the feeding response to orexin injection in the hypothalamus (62, 150). The feeding-stimulating effect of fasting is blocked by intracisternally administered anti-orexin A antibodies (192) and intraperitoneal SB-334867 (62). This suggests that at least the OX1 receptor is involved in the natural regulation of feeding, but both orexin receptor subtypes are expressed in the nuclei involved in the regulation of feeding (Refs. 111, 180; Table 3). Orexin A increases c-fos expression in areas of energy regulatory sites within the hypothalamus, the limbic region, and the hindbrain (128). As mentioned above (see CELLULAR RESPONSES TO OREXINS), orexins belong to a regulatory network of peptides, including NPY, MCH, AGRP, galanin, POMC products, CART, and dynorphin, which regulate feeding behavior. Orexins appear to act upstream of NPY as antagonists to the NPY Y1 or Y5 receptors block orexin-stimulated feeding (38, 81).

The degree to which orexins regulate long-term food intake is subject to debate; some data indicate rather that orexin-induced food intake is only an acute effect and is evened out by compensatory decrease in food intake within 24 h (see, e.g., Refs. 38, 63, 194). It can be speculated that part of orexin-induced food intake is secondary to increased wakefulness and activity and therefore following increased energy expenditure. A general view emerging from the different results is that orexins may participate in the short-term regulation of energy homeostasis by initiating feeding in response to falls in glucose, but the subsequent increase in glucose, leptin, and visceral signals would decrease orexin, which in its turn would terminate the behavior after food ingestion (Refs. 22, 53; reviewed in Refs. 189, 190). Mapping of orexinergic projections indicates that orexins could also be involved in the overall regulation of feeding. For instance, nucleus of the solitary tract, sensory trigeminal nucleus, and lamina 1 and 10 of the sacral cord receive afferents from the mouth and the gastrointestinal tract and the dorsal motor nucleus of vagus, motor trigeminal nucleus, intermediolateral cell column, and sacral parasympathetic nucleus give rise to nerves that innervate the gastrointestinal tract and muscles of mastication (31, 34, 36, 131, 143, 182, 198). Therefore, it is tempting to suggest that central orexinergic nerves could be involved in the primary eating process via sensory and motor innervation of the mouth and in the passage of food through the gastrointestinal tract via sensory, motor, and secretomotor innervation of the intestines. Peripheral orexinergic neurons and endocrine release of orexins from, e.g., enterochromaffin cells could affect intestinal motility, endo- and exocrine secretion, uptake, and sensory signaling in the gastrointestinal tract (see Effects of orexins on gastrointestinal tract and other organs of feeding/energy metabolism). Some direct data (6, 106, 124, 175, 186, 197) and indirect observations from narcoleptic patients and animal models (23, 60, 162, 193) suggest that orexins may affect metabolic rate or heat loss or related processes. An overall role in energy metabolism is also suggested by the effects of orexins on the release of hormones, especially adrenal steroids (see Orexins in Autonomic/Endocrine Functions).

Involvement of Orexins in Sleep/Wakefulness

Regulation of sleep/wakefulness is the most well-demonstrated systemic effect of the orexinergic pathways. At the anatomic level, orexinergic neurons are present and project to a multitude of nuclei at different levels of CNS involved in sleep/arousal. Orexin receptors/receptor mRNAs are observed in these same areas. OX1 and OX2 receptor mRNA expression within these areas suggests that both receptor subtypes are involved (Table 3).

Injection of orexin A into rat cerebral ventricles or into the more specific CNS sites in the rat or cat increases wakefulness and decreases sleep (15, 45, 56, 72, 120, 144). Most markedly, the effects are seen as decreased rapid eye movement (REM) sleep and slow-wave sleep episode number and duration. A profound role of histaminergic systems in these processes is suggested by results that show that these effects of orexin A in the mouse are blocked by histamine H1 receptor antagonists or gene disruption (72, 195). Narcoleptic dogs with disrupted OX2 receptors have decreased brain histamine content (134), and OX2 receptor seems to be responsible for orexin effects on the tuberomamillary nucleus even in the rat (44, 195). Expression of c-fos has been shown to increase mainly in orexin-positive cells of the hypothalamus at the time of normal waking of rats, and c-fos expression is also increased by sleep deprivation and by the antinarcoleptic drugs metamphetamine and mofanadil (23, 46, 161).

Cellular responses to orexins have been observed in areas involved in sleep. Orexins excite neurons in rat locus ceruleus (56, 70, 80, 170), dorsal raphe nucleus (18), and tuberomamillary nucleus (9, 44) and in mouse laterodorsal tegmental nucleus (19) (see CELLULAR RESPONSES TO OREXINS). Even in these areas OX1 and OX2 receptors seem to have overlapping roles. Orexinergic systems in sleep and wakefulness as well as knockout phenotypes are further discussed in Narcolepsy, a Disorder of the Orexinergic System.

Orexins in Autonomic/Endocrine Functions

Orexinergic efferents project to particular areas in the brain stem and spinal cord, which suggest a direct involvement in autonomic afferents and efferents and pain transmission (11, 31, 34, 122, 182). Autonomic effects are manifested as increased blood pressure, heart rate, and intestinal motility (see below), gastric acid secretion (138, 177), and sympathetic nerve activity (98, 124, 166) in response to intracerebroventricular orexin A. When administered intravenously, orexin A induces analgesia in thermal nociception paradigms (11). This effect of orexin A is blocked by the OX1 receptor antagonist SB-334867---even below the baseline---but not by naloxone, suggesting an opiate-independent mechanism.

Orexins may affect the water balance via effects on drinking, water/salt homeostasis, or mechanisms involved in blood pressure regulation, as suggested by the anatomy of the projections (24, 31, 34, 36, 37, 61, 69-71, 125, 126, 154, 182). In fact, intracerebroventricular orexin A and -B increase water intake in the rat (98, 149). Injection of orexin A or -B into particular brain stem sites increases mean arterial pressure and heart rate in the rat (3, 25, 156, 166). To some extent these effects may be mediated by increased release of hormones, such as (nor)epinephrine (3, 166).

Release of many hormones from hypothalamus has been shown to be affected by orexins. Orexins may regulate hypothalamo-hypophysial hormone secretion indirectly via neuronal circuits, but even a direct effect on the hypophysis is possible: orexins are found in median eminence (35, 125), and mRNA for orexin receptor subtypes is found in anterior and intermediate hypophysial lobes (35). Blanco et al. (13) identify coexpression of growth hormone and OX1 receptors in acidophil cells of adenohypophysis and coexpression of ACTH and OX2 receptors in pars intermedia and basophil cells of adenohypophysis. Orexins and orexin receptors are also found at low levels in the posterior hypophysial lobe. Altogether, both OX1 and OX2 mRNA are expressed in the areas involved in neuroendocrine regulation (111).

Stimulatory effect on the release on multiple levels of the hypothalamo-hypophysio-adrenal axis have been reported (see Orexinergic regulation of hypothalamo-hypophysio-adrenal axis and stress response). Orexin A injected intravenously or directly into the hypothalamic paraventricular nucleus decreases TSH release in the rat (123, 153), but no change has been seen in the plasma levels of thyroid hormones (123, 153). Intracerebroventricular orexin A decreases growth hormone release in the rat (56). Effects on the release of other hormones have also been reported, but the results are equivocal. Some release effects have been shown in hypothalamic explants in vitro: orexin A stimulates release of neurotensin, NPY, VIP, somatostatin, and luteinizing hormone-releasing hormone (LHRH) and decreases the release of TRH but has no effect on the release of dopamine, vasopressin, or MCH (123, 151, 152).

Orexinergic regulation of hypothalamo-hypophysio-adrenal axis and stress response. Intracerebroventricular administration of orexin A increases plasma levels of CRF, ACTH, corticosterone, vasopressin, and epinephrine in the rat (2, 56, 78, 82, 83, 85, 151, 152). CRF release has been suggested to be mediated by elevated NPY acting on Y1 receptors (83, 152), placing NPY downstream of orexins even in this system. Orexin A can even stimulate CRF release in vitro (151). A CRF receptor antagonist has been reported to block orexin-stimulated plasma corticosterone elevation in the rat (82, 159); on the other hand, orexins have been reported to inhibit CRF-stimulated ACTH release from cultured rat adenohypophysial cells (158).

Orexins have also been reported to stimulate release of corticosterone/cortisol (108, 110, 117) and aldosterone (108, 132) from rat, porcine, or human adrenal glands/adrenal cells in vitro. This release is independent of ACTH (110, 117). Corticosterone/cortisol release has been determined to co-occur with cAMP elevation and probably to depend on it [rat (110), human (117)]. This, together with the lack of additivity between orexin A and ACTH, suggests that these peptides utilize the same mechanism for glucocorticoid release (117). In contrast, no direct effect on rat adrenal slices was seen by Jaszberenyi et al. (82). In a study with long-term (7 day) exposure to orexin A or orexin B, plasma levels of both aldosterone and corticosterone but not CRF or ACTH were elevated in the rat (108).

Release of epinephrine and norepinephrine from rat adrenal medulla is increased by intracerebroventricular orexins (166), and a similar although smaller effect is seen in cultured porcine adrenal medullar cells (132). Both OX1 and OX2 receptor mRNA have been suggested to be expressed in the rat adrenal medulla (104), although other studies present opposing views (84).

Orexin-mediated increases in CRF, ACTH, corticosterone, aldosterone, vasopressin, and epinephrine release suggest that orexins could mediate stress responses. Interesting in this context, orexins increase particular stereotypic behavioral patterns, such as grooming, face washing, and burrowing, which may be related to activation of stress response (40, 45, 77, 78). Orexin A-induced grooming is inhibited by OX1 receptor block with SB-334867 (40), and a CRF receptor antagonist inhibits orexin-induced face washing and grooming by ~50% and the shift from rest to locomotor activity by 70-80% (78). Involvement of complex pathways is highlighted by inhibitory effects of dopamine and serotonin receptor antagonists (40, 115, 130). It should be noted that orexins increase wakefulness and/or activity of the rat, and it is therefore important to be able to separate these effects from the stress response.

Effects of orexins on gastrointestinal tract and other organs of feeding/energy metabolism. In the gastrointestinal tract, both neurons and some endocrine cells may express orexins (92, 133). In neurons, orexin immunoreactivity is seen both in the submucosal and myenteric plexi. Orexinergic neurons in these plexi have been suggested to be sensory or secretomotor on the basis of the coexpression of other markers such as VIP and choline acetyltransferase (92, 133). The neurites project to other orexin-containing cells and to mucosa, muscle layers, and submucosal blood vessels. In the guinea pig, all the orexin-positive neurons of submucosal and myenteric plexi express leptin receptors as well (92). Fasting increases the number of orexin A-immunoreactive submucosal neurons in the guinea pig, most of which also become positive for phospho-cAMP response element binding protein (pCREB) (92). This, similar to c-fos expression, indicates activation of the neurons, suggesting that fasting activates both central and peripheral orexinergic systems.

Also, some endocrine cells in the gut and stomach---some of which are enterochromaffin as they express serotonin---display orexin immunoreactivity in mouse, rat, guinea pig, and human (92, 133). Interestingly, some enterochromaffin cells of the rat also 1) express orexin receptors (OX2) and 2) are localized at close proximity to orexinergic nerve fibers (133). At the cellular level, orexin A induces electrical responses in guinea pig submucosal neurons (see CELLULAR RESPONSES TO OREXINS). Thus orexinergic neurons in the gastrointestinal tract could affect intestinal secretion and uptake, endocrine secretion, sensory signaling, and intestinal motility. Different effects on intestinal motility patterns have indeed been measured in mouse and rat small intestine (133, 160) and in guinea pig distal colon (92). Different orexin receptor subtypes have different distribution in the gastrointestinal tract; OX1 receptor immunoreactivity is solely expressed by the submucosal and myenteric neurons, whereas OX2 receptor immunoreactivity is only seen in the enterochromaffin cells in the rat (133).

Energy metabolism is regulated by several hormones, the most important being pancreatic glucagon and insulin, hypophysial growth hormone, glucocorticoids, (nor)epinephrine, and thyroid hormones. Effects of orexins on glucocorticoid and growth hormone release have been discussed above. Insulin release is somewhat increased in the rat by both intracerebroventricular (114) and subcutaneous (137, 174) injection of orexin A, and also in the rat pancreas in vitro (137). Extrinsic neurons in rat and guinea pig pancreatic ganglia display orexin and OX1 and OX2 receptor immunoreactivity (92). Plasma leptin is also increased by subcutaneous injection of orexin A or -B in the rat, although this has been suggested to occur via increased insulin release (174).

Narcolepsy, a Disorder of the Orexinergic System

Narcolepsy is a disorder that in short can be characterized as abnormal inclusion of REM sleep features, such as muscle paralysis and hallucinations in the waking state and in the sleep/waking transition state. In addition, hypersomnia is often observed. Narcolepsy in humans is usually a sporadic disease; its strong association with some major histocompatibility complex antigens (especially HLA-DRB1*15, HLA-DQA1*0102, and HLA-DQB1*0602) has for a long time been suggested to relate it to some autoimmune process. Yet autoantibodies or signs of ongoing or previous inflammation have been hard to find (reviewed in Refs. 74, 94, 102, 141, 179). This could, however, be caused 1) by the fact that postmortem narcoleptic brains are usually investigated years after disease onset and 2) by the dispersed nature of the orexinergic cells. Orexin A is undetectable in the CSF of most human narcoleptic patients (32, 135), the number of orexinergic neurons in postmortem narcoleptic brains is much reduced (142, 178), and even signs of gliosis have been observed (178). At the same time MCHergic neurons, at the same brain areas, are present in normal numbers (142, 178). This, together with the animal model systems (see below), has led to the hypothesis of narcolepsy as a disease of orexinergic neurons. As further support for this, only one clearly debilitating preproorexin or orexin receptor mutation has been found in human subjects (Ref. 142; see below).

In the mouse, depletion of the hypothalamus of orexins results in a narcoleptic phenotype. This has been accomplished by genetic disruption of the preproorexin gene (orexin knockout mouse; Ref. 23), genetic destruction of orexinergic cells in preproorexin-ataxin-3 knockin mouse (60), and toxic destruction of orexinergic cells by hypothalamic injection of orexin B-saporin conjugate (52).

In dogs, narcolepsy is mainly a familial disorder. Recently, familial canine narcolepsy of Doberman pinschers and Labrador retrievers was shown to be caused by mutations in the OX2 receptor gene (102). In both cases, the mutations cause premature amino acid chain termination. When expressed heterologously in HEK293 cells, the mutant receptors mainly remain in the intracellular compartments and neither bind orexin A nor activate Ca2+ mobilization (73). Point mutation Glu54Lys at the NH2 terminus/first transmembrane segment junction of the OX2 receptor also leads to reduced orexin A binding and 400-fold reduced potency for Ca2+ elevation when expressed in HEK293 cells (73). The 10 other reported mutations in preproorexin or OX2 receptor genes are thought to represent benign polymorphisms (73). Sporadic cases of canine narcolepsy have been suggested to be caused by lack of orexins (148).

OX1 or OX2 receptor gene abnormalities are not considered to be responsible for human narcoleptic cases, and the 26 single-base changes (11 aa changes) found in healthy and narcoleptic human subjects (Refs. 139, 142; see also Genetics and Chemistry of Orexins) both in introns and all over the coding regions are supposed to represent benign polymorphisms. One other polymorphism concerns the reported sequence for the OX1 receptors (aa 280; see Genetics and Chemistry of Orexins), although this could be a sequencing error. One mutation, Leu16Arg, found in the 5'-signal peptide region of the human preproorexin gene probably results in early-onset, severe narcolepsy (142). When transiently expressed in Neuro-2A cells, the mutated peptide apparently accumulated in some tubular network, presumably smooth endoplasmic reticulum (ER), in contrast to the wild-type preproorexin, which appeared in vesicles. Another point mutation in the 5'-untranslated region may be involved in disease process in the presence of a particular type of tissue antigen (HLA-DR2; Ref. 51). Insertion of an adenosine at -593 (promoter region) has been found in one narcoleptic patient, although the effect of this on pathogenicity is unclear (51).

Thus there is strong evidence that narcolepsy is caused by reduced orexinergic signaling via either destruction of orexinergic neurons or defective orexin peptides or receptors, although larger population studies are necessary to confirm this. OX2 receptor appears to be especially involved in the dog, which would promote the role of nuclei relying on this receptor subtype, such as tuberomamillary nucleus, although it is possible that OX1 and OX2 receptors have different distributions in rats and humans compared with dogs.

Orexinergic disorders may also provide some interesting information on the role of orexins in feeding. Patients with narcolepsy show somewhat increased body mass index compared with healthy subjects (162). Both of the two previously described kinds of orexin-transgenic mice are hypophagic, but preproorexin knockout mice are lean (23) as opposed to orexinergic neuron-deficient mice, which are obese (60). It is thus possible that preproorexin knockout mice develop some compensatory mechanisms for weight regulation whereas orexinergic neuron-deficient mice, which lose their orexinergic neurons postnatally (see Genetics and Chemistry of Orexins), will not be able to do this. Another interesting possibility was recently proposed by Chou et al. (27). Essentially all the mouse lateral hypothalamic orexinergic neurons express dynorphin (10, 27), removal of which in orexinergic neuron-deficient mice might be responsible for the more pronounced phenotype.


    FUTURE PERSPECTIVES
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ABSTRACT
INTRODUCTION
OVERVIEW OF OREXINS AND...
CELLULAR RESPONSES TO OREXINS
DISTRIBUTION OF OREXINERGIC...
SYSTEMIC EFFECTS OF OREXINS
FUTURE PERSPECTIVES
REFERENCES

Although the anatomic architecture of the orexinergic system in the CNS is well characterized, there are several open questions as to the actual roles of orexins in central and peripheral functions. What are the mechanisms of action of orexins at the cellular level? Have all orexin receptors been identified? Are there presynaptic inhibitory orexin receptors in the brain? The limited access to orexin-responding neurons is an experimental problem. There are several limitations concerning the methods available for the characterization of the functional properties of these cells in slice preparations or acutely isolated cells. Model systems based on cultured neurons or even neuronal stem cells might provide an important improvement for studies of orexin signaling. The CHO cell lines available have provided insight in functions of the orexinergic system and have enabled screening for orexin receptor ligands, but all the data obtained may not be relevant for the nervous system. Recombinant models based on neurons or neuronlike cells might therefore provide further information about the signal pathways used by orexins.

What role do the orexin cells play in the regulation of feeding? Available information suggests that at least one population of the orexinergic cells in the lateral hypothalamus directly monitors and responds to changes in glucose concentration in their environment and may also directly respond to signals inhibiting feeding such as leptin. Are these orexinergic cells the ones that project to areas in brain involved in feeding behavior? Orexin receptors are expressed in high numbers in several regions within the hypothalamus. Do these regulate the release of other feeding-related neurotransmitters? The regulation of feeding seems to be complex, involving many other neuropeptides, and there seems to be a gross redundancy in the system. Blocking of the orexinergic system by various means will reduce feeding, but permanent deletion of the orexinergic system has marginal or controversial effects. Thus even an increase rather than the expected decrease in body mass may occur on deletions of different components of the orexinergic system.

The role of orexins in sleep seems clearer in light of the connection of the narcoleptic phenotype to orexin cell loss and the similar symptomatology occurring in functional orexin or orexin receptor loss. In this context it also appears quite clear that a major part of orexin effects is mediated via monoaminergic systems, which regulate cholinergic pathways involved in the initiation of REM sleep (reviewed in, e.g., Refs. 74, 90). Yet it is unclear how the activity of the orexinergic cells themselves is regulated. Use of orexin antagonists as hypnotics could prove rewarding because many commonly used hypnotics interfere with REM sleep and cause tolerance, addiction, and memory disturbances. An interesting question is whether there is an overlap between cells regulating feeding and sleep in the lateral hypothalamus. Is, for instance, feeding behavior associated with arousal effects of orexins? Possibilities in the future to devise better pharmacological methods for identification of different orexinergic neurons as well as for manipulating their function will hopefully give answers to these questions.

One of the most intriguing questions concerns what roles orexins play in the periphery. Do they act as hormones/paracrine mediators? The presence of orexins and their receptors in many peripheral organs such as the intestine, endocrine pancreas, adrenals, and pituitary suggests that this may be the case. Could orexins even transmit signals from the periphery to the CNS? As discussed above, the few studies available suggest separate "peripheral" and "central" orexinergic systems. In some studies changes in central orexin concentrations are not reflected in the periphery, and in narcoleptics devoid of central orexins, orexins are still found in the plasma. This may indicate that the plasma orexin levels reflect peripherally produced orexins. Are the peripheral orexin receptors similar to the central? At least in the adrenal cortical zona fasciculata/reticularis orexin receptors appear to couple to cAMP elevation and not to Ca2+ elevation. Does this reflect different receptor subtypes---as suggested by some studies---or coupling to different signal pathways? Cell lines and primary cultures from peripheral tissues should provide a lot of the missing information in this context.


    ACKNOWLEDGEMENTS

We acknowledge van den Pol et al. (183) and Eriksson et al. (44), who have allowed reproduction of Figs. 3 and 5.


    FOOTNOTES

This study was supported by European Union Contract QLG3-CT-2002-00826 and grants from the Swedish Medical Research Council, the Cancer Research Fund of Sweden, the Lars Hierta Foundation, the Academy of Finland, and the Sigrid Juselius Foundation.

Address for reprint requests and other correspondence: J. Kukkonen, Dept. of Neuroscience, Div. of Physiology, Uppsala Univ., BMC, PO Box 572, SE-75123 Uppsala, Sweden (E-mail jkukkone{at}fysiologi.uu.se).

10.1152/ajpcell.00055.2002


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
OVERVIEW OF OREXINS AND...
CELLULAR RESPONSES TO OREXINS
DISTRIBUTION OF OREXINERGIC...
SYSTEMIC EFFECTS OF OREXINS
FUTURE PERSPECTIVES
REFERENCES

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