The hypocretin/orexin system

The hypocretins (orexins) are recently described hypothalamic neuropeptides thought to have an important role in the regulation of sleep and arousal states¹. Their discovery was reported independently by two groups using different techniques. de Lecea et al.² identified the pro-hormone pre-prohypocretin, and its peptide products hypocretin-1 (Hcrt-1) and hypocretin-2 (Hcrt-2), by nucleotide sequencing. The discovery of the orexins, orexin-A (Orx-A) and orexin-B (Orx-B), was reported almost simultaneously by Sakurai et al.³ who used the technique of orphan receptor cloning. The terms orexin and hypocretin are synonymous and in this article we will use hypocretin (Hcrt). The finding that cerebrospinal fluid (CSF) levels of these peptides were abnormal in patients with narcolepsy has stimulated research on the potential role of these peptides in human disease. We present here an overview of the pertinent findings from animal studies and a review of the published data from human studies, with a particular emphasis on narcolepsy. Finally, we consider the possible roles of these peptides in neurological and psychiatric disorders.

BACKGROUND

Identification of the peptides

In 1996, a set of neuropeptides related to the hormone secretin were isolated from the rat lateral hypothalamus by the process of directional tag PCR subtraction cloning⁴. The cloning of the gene for these peptides from rat and mouse, the localization of the peptide-producing cell bodies and a description of some of their efferent projections were first presented in 1977^5,6.

The receptors

The receptors for these neuropeptides (Hcrtr1 [Orxr1] and Hcrtr2 [Orxr2]) have been identified as G-protein coupled receptors and shown in the rat brain, by analysis of their mRNA, to display a striking distribution^7,8. The Hcrtr1 receptor has a much higher (100 to 1000-fold) affinity for Hcrt-1 than for Hcrt-2. The Hcrtr2 receptor seems to have equal affinities for both neuropeptides. The distinctive distribution of the receptors has led some authors to hypothesize a sleep-specific role for the Hcrtr1 receptor and a more general role for Hcrtr2 receptor. The receptors have been mapped on human chromosome 1p33 and 6cen, respectively^5,7,8,9.

Projections of the hypocretin system

The hypocretin-producing cell bodies are specific to the hypothalamus and have widespread anatomical projections within the central nervous system of the rat with the densest extra-hypothalamic projection to the noradrenergic locus coeruleus (LC) and lesser projections to the basal ganglia, thalamic regions, the medullary reticular formation, and the nucleus of the solitary tract. There are minor projections to the cortical regions, central and anterior amygdaloid nuclei, and the olfactory bulb^4,10,11. In humans, the localization of hypocretin-producing cell bodies is restricted to the dorso-lateral hypothalamus with extensive dense projections to the locus coeruleus (LC), dorsal raphe nuclei, amygdala, suprachiasmatic nucleus, basal forebrain, cholinergic brainstem^12,13 and spinal cord (Figure 1)¹⁴.

Figure 1.

Projections of the hypocretin (orexin) system (A), to cholinergic neurons, reticular formation and spinal cord; (B), to thalamus and basal ganglia; (C), to basal forebrain; (D), to amygdala and dopaminergic neurons including suprachiasmatic nucleus; (E), to locus coeruleus.

Neurochemical actions of the hypocretins

The hypocretins are thought to act primarily as excitatory neurotransmitters^1,2,7. Systemic and intracerebroventricular administration of hypocretins directly stimulates cells on the LC noradrenergic system in rats and monkeys, suggesting a role for the hypocretins in various central nervous functions related to noradrenergic innervation, including vigilance, attention, learning, and memory¹⁵. Their actions on serotonin, histamine, acetylcholine and dopamine neurotransmission is also thought to be excitatory and a facilitatory role on gamma-aminobutyric acid (GABA) and glutamate-mediated neurotransmission is suggested^16,17. In particular, intravenous administration of Hcrt-1 in rats produces a differential release of GABA and glutamate in the hypocretin-dense amygdala compared with the cerebellum, suggesting that modulation of these neuro-transmitters is dependent on hypocretin innervation¹⁸.

Functions of hypocretin

Apart from their primary role in the control of sleep and arousal^1,7, the hypocretins have been implicated in multiple functions including feeding and energy regulation^{3,16,19,20,21}, neuroendocrine regulation^17,22, gastrointestinal²³ and cardiovascular system²⁴ control, the regulation of water balance, and the modulation of pain¹. A role in behaviour is also postulated²⁵. The cell bodies responsible for hypocretin synthesis are localized to the tuberal part of the hypothalamus, the so-called feeding centre. The observation that Hcrt-1 increases metabolic rate and the demonstration that insulin-induced hypoglycaemia activates up to one-third of hypocretin containing neurons²¹ has led to the suggestion that the hypocretins are mediators of energy metabolism²⁶. The neuroendocrine effects of the hypocretins include a lowering of plasma prolactin and growth hormone and an increase in the levels of corticotropin and cortisol, insulin and luteinizing hormone^1,16,17. Central administration of the hypocretins increases water consumption, stimulates gastric acid secretion and increases gut motility^1,23. The hypocretins increase mean arterial blood pressure and heart rate⁷. The localization of long descending axonal projections containing hypocretin at all levels of the spinal cord¹⁴ suggests a role in the modulation of sensation and pain. Strong innervation of the caudal region of the sacral cord suggests a role in the regulation of both sympathetic and parasympathetic functions.

HYPOCRETIN IN NARCOLEPSY

Narcolepsy is a primary disorder of alertness with an estimated prevalence of 0.03-0.05%. It may develop at any age but peak onset is in adolescence with a secondary peak in the fourth decade. The presenting symptom is usually excessive daytime sleepiness, with irresistible sleep attacks during the day. Other symptoms of this syndrome are cataplexy (brief episodes of muscle weakness or paralysis precipitated by strong emotion, such as laughter or surprise), sleep paralysis, which is a symptom due to the persistence of rapid-eye-movement (REM) sleep atonia on waking, and hypnogogic hallucinations or dream-like images, which characteristically occur at sleep onset. Short periods of automatic behaviour may also occur, a reflection of brief intrusions of sleep (‘micro-sleeps’) into the drowsy state²⁸.

Animal studies

In 1999, Lin et al.²⁹ demonstrated a mutation in the hypocretin receptor 2 gene in canine narcolepsy. The subsequent finding that mice lacking hypocretin receptors show behavioural arrests similar to symptoms of narcolepsy-cataplexy—i.e. direct transitions from wakefulness to REM sleep, gait disturbance preceding and rocking activity during behavioural arrest episodes^30,31—led to a recognition of the potential importance of the hypocretins in sleep, arousal and activation. The animal models of narcolepsy show some variability in the defect causing the narcolepsy-like syndrome. In the mouse model, disruption of both types of hypocretin receptor pathways, Hcrtr1 and Hcrtr2, is necessary to produce the narcoleptic findings^30,31,32,33 whereas in the canine model of narcolepsy the predominant defect is at the Hcrtr-2 receptor²⁹. Intravenous administration of Hcrt-1 to narcoleptic dogs (dobermans) reduces cataplexy and normalizes their sleep and waking durations³⁴.

Hypocretin in cerebrospinal fluid

There have been several studies of hypocretin in human CSF. The published work to date has tested for the presence of Hcrt-1 only and not Hcrt-2. CSF hcrt-1 levels in healthy adults are within a narrow range (250-280 pg/mL)³⁵. A recent study indicated no significant difference in hypocretin levels with respect to gender or age, and concluded that very low or undetectable CSF hypocretin concentrations are an abnormal finding at any age³⁶.

The initial study by Nishino et al.³⁵ found that 7 of 9 patients with narcolepsy-cataplexy had undetectable levels of hypocretin in their CSF. Of the 2 patients with detectable hypocretin, one was within the control range and the other had raised levels. Both these patients were indistinguishable from the other patients with narcolepsy. The authors suggested that these patients might have a hypocretin receptor defect rather than a hypocretin production deficiency. Ripley et al.³⁷ have reported undetectable levels of hypocretin in the CSF from 32 of 36 patients tested. In the remaining 4 the hypocretin levels were below the control range.

There have been two studies examining hypocretin cells post mortem in the brains of patients with narcolepsy^12,13. Both found a striking reduction, to about 10% of the normal number of hypocretin neurons, in narcoleptic brains. In the initial study¹² there was cell loss without gliosis or signs of inflammation. However, in the other study¹³ there was evidence of gliosis in the hypocretin cell region, implying that a degenerative process was the cause of hypocretin cell loss in narcolepsy. Further support for the degenerative hypothesis is their finding of a higher number of astrocytes in the hypothalamus of narcoleptic patients than in controls. The absence of hypocretin neurons can be explained by mechanisms including neurodegeneration, failure of development, reduction in synthesis or release of hypocretins or some mutation in the DNA sequence coding for hypocretin (though only 1 out of the 74 narcoleptic patients screened showed a mutation¹²).

HYPOCRETIN IN NEUROLOGICAL AND PSYCHIATRIC DISORDERS

The role of hypocretin in other neurological illnesses is yet to be established. A recent study³⁸ found that CSF hypocretin levels did not differ significantly between two groups, one with neuroimmunological disease and the other with non-neuroimmunological disease, and normal controls. In a subgroup analysis the investigators found that 4 of 10 patients with Guillain—Barré syndrome had significantly lower Hcrt-1 levels than the controls. Another study³⁷ has demonstrated low CSF hypocretin levels in patients with subarachnoid haemorrhage, acoustic schwannoma and head trauma—perhaps explained by damage to and/or dysfunction of the hypothalamus.

The dense hypocretin projections to the noradrenergic, serotonin, dopaminergic, cholinergic, and GABA/glutamate areas of the brain suggest a possible role in psychiatric and neuropsychiatric disorders^39,40. The hypocretin system may be important in affective disorders such as major depression and bipolar affective disorder. The monoamine hypothesis (biogenic amine hypothesis) of depression suggests that dysfunctional or deficient neurotransmission of noradrenaline and/or serotonin underlies the symptoms of depression^41,42,43. More recently, emphasis has shifted to the possible roles of neuropeptides in the aetiology and treatment of depression^{44,45,46,47,48,49}. Involvement of the hypocretin system in depression is suggested on neuroanatomical and pharmacological grounds. The only substance known to innervate all the relevant areas of the brain implicated in the neurobiology of depression is hypocretin and the excitatory innervation of the LC and dorsal raphe region, the stimulation of dopamine and acetylcholine and the prohistaminergic actions all point to an antidepressant effect. These therapeutic possibilities remain to be clarified by appropriate studies.

CONCLUSION

There is strong evidence that narcolepsy is associated with abnormalities of the hypocretin neurotransmitter system. Low or undetectable levels of hypocretin are found in most patients but some have normal or raised levels. Thus it has been suggested that there are two variants of narcolepsy. In most patients there seems to be a hypocretin deficiency but there may also be a form with ‘hypocretin resistance’ due to abnormal hypocretin receptor/post-receptor dynamics leading to overproduction of hypocretin^9,50. There may be involvement of the hypocretin/orexin system in other disorders of sleep such as primary hypersomnolence, insomnia, and the Kleine-Levin syndrome¹⁰, and a potential role in sleep disorders affecting the ageing population^{7,28,41,51,52}. The role of these peptides in other neurological and psychiatric disorders remains putative.