Hypocretin function in sleep/wake regulation

Hypocretins (also known as orexins) are neuropeptides which are important in the maintenance of wakefulness and arousal.[1] Hypocretin (Hcrt) neurons are localized to the lateral and posterior hypothalamus and are crucial in the regulation of the sleep/wake cycle.[3] They bind to the HcrtR1 and HcrtR2 G-coupled protein receptors (OX1R and OX2R) in midbrain regions associated with sleep regulation.[2][3] Decreased Hcrt neuron function and deficiency of hypocretin has been implicated in narcolepsy, a sleep disorder characterized by the intrusion of sleep phases during waking phase and difficulty maintaining wakefulness.[4][5]

1. Hypocretin overview

1.1 Hypocretin and hypocretin receptor structure and function

Location of hypocretin neurons in rat brain[3]
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A: Hypocretin neurons stained brown in the lateral hypothamalus of the mouse brain
B: magnification of the lateral hypothalamic area[3]

There are two different hypocretin peptides, hypocretin 1 and hypocretin 2 (sometimes known as orexin A and orexin B respectively). [6] There are two hypocretin receptors, HcrtR1 and HcrtR2.[5] The receptors have similar but differentiated functions, and can be found in different areas of the brain (see sections 1.2 and 2).[5] Both receptors are expressed in the dorsal raphe nucleus, while only HcrtR1 receptors are expressed in the locus ceruleus and only HcrtR2 receptors are expressed in the tuberomammillary nucleus (TMN) .[7] Both HcrtR1 and HcrtR2 activity results in rapid eye movement (REM) sleep inhibition, though HcrtR2 has a greater contribution to the maintenance of wakeful states (see 2. Hypocretin function). [7] The HcrtR2 receptor binds to both hypocretin-1 and hypocretin-2 equally, but the HcrtR1 receptor has a much stronger affinity for hypocretin-1 than hypocretin-2.[8]

1.2 Hypocretin neuron circuits and systems

Hypocretin neuron projections in human brain[2]
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A drawing of the projections of hypocretin neurons
based on information gathered from the mouse brain.
PPT: pedunculopontine tegmental nucleus LDT: laterodorsal tegmental nucleus.
Explanations of other acronyms can be found within the text.[2]

Hypocretin neurons are located in the lateral hypothalamus and are sensitive to and respond to an array different transmitters, hormones and peptides, allowing for the perception of a general measure of cortical excitability and for the perception of internal bodily activity.[5][9] The hypocretin system works with the monoaminergic, which is responsible for arousal and wakefulness.[5] The hypocretin system also has targets in the cholinergic system (including the pedunculopontine tegmental nucleus and the laterodorsal tegmental nucleus), which serves to modulate arousal induced by hypocretin function.[5] Other targets include areas of the brain stem and the hypothalamus, including the tuberomammillary nucleus (TMN), the locus ceruleus (LC), the dorsal raphe nuclei (DRN), and the ventral periaqueductal gray (vPAG).[5][9]
Input to the TMN has been found to be responsible for supporting wakefulness.[10] Mochizuki et al. (2011) created transgenic mice with disrupted orexin R2 production (OX2R or HCRTR2), and the mice consequently displayed symptoms of narcolepsy (see section 2.2).[10] When the gene function was recovered selectively in the tuberomammillary nuecleus the orexin function was locally restored and the symptoms of hypersomnolence improved.[10] However, the mice still displayed other symptoms of narcolepsy, suggesting that the TMN is responsible for supporting awake states.[10]
The LC also appears to be especially important in the maintenance of wakefulness.[5] Hypocretin input to the LC increases firing from the LC neurons, and this induced activity activity in the LC in this circuit causes an increase in cortical excitability.[11]

Hypocretin projections to locus ceruleus[11]
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A: a diagram of the hypocretin projections to the locus ceruleus neurons B: Hypocretin neurons stained in the brainstem
C: Locus ceruleus neurons stained in the same area of the brainstem - there is a significant amount of overlap, suggesting the
locus ceruleus is richly innervated with hypocretin neurons[11]

Hcrt neurons appear to receive some form of input from the suprachiasmtic nucleus (SCN), which is involved in the regulation of circadian rhythmicity, which may be why Hcrt activity has different effects during different circadian periods.[5] Hcrt concentration in the brain also appears to be modulated by circadian systems.[5] The functional effects of hypocretin also seem to change over the course of the day. Tsunematsu et al (2011) found that optogenetic inhibition of orexin neurons (achieved by causing their hyperpolarization) during the day while mice were inactive induced slow wave sleep (SWS) and decreased neuronal activity in the dorsal raphe nucleus, while inhibition during the night during the active period had little effect and did not induce SWS. [3]
Input to the hypocretin system also comes from the limbic system, which recruits the hypocretin system to increases arousal when increased alertness is called for.[9] Hcrt neurons are also regulated by metabolic cues and peptides (such as ghrelin), related to the hypocretin functions in homeostasis(see part 3).[9]
Hcrt neurons are hyperpolarized and inhibited by GABAergic projections from the ventrolateral preoptic area (VLPO), which reduces hypocretin-mediated arousal and is associated with the maintenance of sleep states.[9][6] VLPO GABA neurons initiate sleep, and show increased firing during the sleep phase compared to the wake phase.[6]

1.3 Hypocretin activity

Hcrt neuron stimulation is phasic, and this phasic hypocretin activity is especially important during sleep-to-wake transitions.[5] Hcrt neurons have more active firing patterns during periods of wakefulness and display decreased activity during slow-wave sleep (SWS), and are mostly silent during rapid-eye movement sleep (REM).[5] They only fire during REM sleep directly before waking.[5]

2. Function of hypocretin

2.1 Hypocretin function in sleep/wake regulation

Hypocretin neuron activity over the course of the sleep/wake cycle [12]
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A: The spike rate of hypocretin neurons are summed over different sleep stages.
B: hypnogram, and spike rate recordings, EMG, and EEG frequencies recorded over the total 40 recording sessions of 10s.
C: displays the timeline of different stages of sleep cut into 4 graphs [12]

The primary function of hypocretin appears to be to maintain arousal and muscle tone and to inhibit sleep.[11][12] Increases in hypocretin concentration cause decreases in both SWS and REM sleep, and increases in total time spent awake.[1]
Lee et al. (2005) recorded hypocretin neuron activity over the course of the sleep-wake cycle. They found that Hcrt neurons were completely silent during both SWS and REM sleep, and were nearly silent when no movement was occurring.[12] They found that Hcrt neurons fired only during wakefulness and during increases in muscle tone (such as during movement) and arousal.[12] The only period of sleep in which hypocretin activity was found was during the transition from REM sleep to waking.[12]
Hypocretin function is modulated in many different ways, and may compete with separate neural circuits, such as those associated with sleep pressure and sleep induction.[1] Carter et al. (2009) deprived mice of sleepfor 2-4 hours and examined the effects of the resulting sleep pressure (mediated by VLPO GABA neurons) on the effect of Hcrt neuron activity.[1] They found that while stimulation of Hcrt neurons prevented sleep and maintained wakefulness in mice, increases in sleep pressure decreased the effect of Hcrt stimulation and failed to inhibit sleep.[1]

Photostimulation of hypocretin neurons[6]
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Green light photoinhibition illumination caused inhibition of hypocretin neurons. This was induced at 20:00-21:00, at night, or
at 12:00-13:00, during the day. B(c,d,f) shows results of photostimulation during the night, while C(c,d,f) shows results of photostimulation
during the day. SWS was only induced during the night period.[6]

As mentioned previously, circadian systems also have an effect on hypocretin function. Tsunematsu et al. (2011) found that optogenetic inhibition of hypocretin neurons for periods of 1 minute induced SWS during the day but not during the night, highlighting a role for circadian function in the hypocretin system.[3] Tsunematsu et al. (2013) later performed a similar experiment, and inhibited Hcrt neurons for durations of 1 hour rather than 1 minute.[6] This time they found an increase in SWS sleep time during the dark period, when the mice were most active.[6] This evidence suggests that the degree of inhibition (depending on the duration) makes a functional difference in the effects of hypocretin activity. Tsunematsu et al. (2013) suggest that 1 minute of inhibition does not sufficiently block hypocretin neuron activity, allowing for some function to remain and alter the effect of inhibition.[6] They also hypothesize that the reason why 1 hour inhibition of hypocretin failed to have an effect was due to the low levels of hypocretin neuron activity during the day, but fail to explain why inhibition lasting 1 minute produced an effect.[6]

Hcrt neuron photostimulation increases
sleep-to-wake transitions [11]
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mCherry are the control hypocretin neurons, while CHR2-mCherry are hypocretin
neurons containing a protein which allows them to be optogenetically stimulated. Hcrt
optogenetic stimulation of CHR2-mCherry causes an increase in sleep-to-wake transition
compared to control. Inhibition of Hcrt neurons by the Hcrt antagonist SB-334867 erased
this effect.[11]

Part of this discrepancy may be due to the complexity of the hypocretin system and the activity of other areas of the brain. In sleep-to-wake transitions, the hypocretin system appears to recruit the locus ceruleus in order to initiate waking, without which the sleep-to-wake transition would not occur.[11] Carter et al. (2012) optogenetically stimulated Hcrt neurons and found a resulting incresae in cFos expression in neurons in the locus ceruleus.[11] C-Fos is a marker of neuronal activity, and its expression indicates that the locus ceruleus neurons were becoming active in response to input from the Hcrt neurons.[11] Carter et al. 2012 also found that Hcrt caused depolarization and increased firing in the locus ceruleus (Carter et al. 2012). The examined this cicrcuit more closely, and selectively inhibited or enhanced locus ceruleus activity while stimulating the hypocretin system.[11] Optogenetic photoinhibition of locus ceruleus activity with simultaneous optogenetic photostimulation of Hcrt neurons resulted in the cessation of nonREM sleep-to-wake transitions.[11] They found the reverse when they photostimulated locus ceruleus neurons to enhance membrane excitability (while maintaining the same Hcrt neuron photostimulation), and found that sleep-to-wake transitions were facilitated.[11] The concluded that sleep-to-wake transitions depended solely not only on hypocretin activity but also on coordinated activity in the locus ceruleus.[11]

2.2 Hypocretin deficiency and narcolepsy

An individual suffering from narcolepsy describes
the way the sleep disorder has affected her life,
and Dr. Andrew Hall provides and overview of the disorder. [15]

Narcolepsy is a sleep disordercharacterized by hypersomnolence (or oversleeping), a sudden onset of REM (known as paradoxical sleep or PS), and fragmented sleep.[6][12] Another common symptom is cataplexy, when a sudden weakness in the postural muscles causes a disruption in the maintenance of muscle tone, and which is often caused by emotional or stressful stimuli.[6]

The symptoms of narcolepsy are due to a deficiency in hypocretin, and narcoleptics have lower than normal hypocretin concentration in the cerebrospinal fluid (CSF).[5][9] Narcolepsy is a neurodegenerative disease caused by a progressive selective loss of hypocretin neurons.[5][12] With reduced hypocretin input into the sleep regulatory circuit, the system is inbalanced and the cholinergic system input becomes dominant, causing loss of muscle tone and the induction of REM sleep (known as REM sleep intrusions).[9]

Results of Orexin A (Hypocretin-1) intranasal administration [13]
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Orexin A administration caused a decrease in REM sleep and sleep-to-wake transitions and
decreased REM sleep latency regardless of time of day it was administered[13]

Hypocretin-1 deficiency appears to play a stronger role in the development of symptoms of narcolepsy than hypocretin-2 deficiency.[13] Because of this, there is evidence than increasing hypocretin-1 levels in narcoleptics would alleviate many of the symptoms of hypocretin deficiency.[13] Weinhold et al. (2014) administered intranasal orexin A (hypocretin-1) in the morning to individuals with narcolepsy, and found an improvement in many of their symptoms.[13] The participants experienced an increase in daytime wakefulness, a decrease in wake transitions, a decrease in daytime REM sleep and an increase in REM sleep latency.[13]

Mouse displaying narcoleptic attacks
Mouse displaying the abrupt cessation of movement due to cataplexy [16]

4. Other functions of hypocretin

Hypocretin/orexin and obesity
Dr. Devanjan Sikder explains the role of orexin function in obesity. [17]

Hypocretin has many functions outside of the regulation of sleep/wake cycles, and is involved in feeding behaviors, motivated behaviors, and stress response.[11] Hypocretin-mediated increases in arousal occur during non-sleep related events which necessitate increased alertness, such as during food deprivations or acute stress, and Hcrt neurons have targets in the HPA axis and sympathetic nervous system.[1][12]
Hypocretin neurons have targets in the dopaminergicneurons of the ventral tegmental area, and modulate dopamine activity based on the internal environment and metabolic processes occurring.[14] Because of this connection with the dopaminergic system, which is involved in motivation and reward, the hypocretin system has been implicated in addictionand obesity.[14]

1. Carter, M. E., Adamantidis, A., Ohtsu, H., Deisseroth, K, and Lecea, L. Sleep
homeostasis modulates hypocretin-mediated sleep-to-wake transitions. Journal of Neurosceince, 29(35), 10939-10949 (2009).
2. Sakurai, T. The neural circuit of orexin (hypocretin): maintaining sleep and wakefulness. Nature Neuroscience, 8, 171- 181(2007).
3. Tsunematsu, T., Kilduff, T. S., Boyden, E. S., Takahashi, S., Tominaga, M., Yamanaka, A. Acute optogenetic silencing of orexin/hypocretin neurons induces slow-wave sleep in mice. Journal of Neuroscience 31(29), 10529 –10539 (2011).
4. Fuller, P. M., Gooley, J. J., Saper, C. B. Neurobiology of the sleep-wake cycle: sleep architecture, circadian regulation, and regulatory feedback. Sleep, 21(6), 482-493 (2006).
5. Lecea, L. and Huerta, R. Hypocretin (orexin) regulation of sleep-to-wake transitions. Frontiers in Pharmacology, 5, 1-7 (2014).
6. sunematsu, T., Tabuchi, S., Tanaka, K. F., Boyden, E. S., Tominaga, M., Yamanaka, A. Long-lasting silencing of orexin/hypocretin neurons using archaerhodopsin induces slow-wave sleep in mice. Behavioural Brain Research, 255, 64-74 (2013).
7. Dugovi, C., Shelton, J. E., Yun, S., Bonaventure, P., Shireman, B. T., Lovenberg, T. W. (2014). Orexin-1 receptor blockade dysreuglates REM sleep in the presence of orexin-2 receptor antagonism. Frontiers in Neuroscience, 8, 1-7.
8. Arendt, D. H., Hassell, J., Li, H., Achua, J. K., Guarnieri, D. J., DiLeone, R. J., Ronan, P. J., Summers, C. H. (2014). Anxiolytic function of the orexin 2/hypocretin A receptor in the basolateral amygdala. Psychoneuroendocrinology, 40, 17-26.
9. Kilduff, T. S., Lein, E.S., Igleisa, H., Sakurai, T., Fu, Y., Shaw, P. (2008). New developments in sleep research: molecular genetics, gene expression, and systems neurobiology. Journal of Neuroscience, 28(46), 11814-11818.
10. Mochizuki, T., Arrigoni, E., Marcus, J. N., Clark, E. L., Yamamoto, M., Honer, M., Borroni, E., Lowell, B. B., Elmquist, J. K., Scammell, T. E. (2011). Orexin receptor 2 expression in the posterior hypothalamus rescues sleepiness in narcoleptic mice. PNAS, 108(11), 4471-4476.
11. Carter, M. E., Brill, J., Bonnavion, P., Huguenard, J. R., Huerta, R., Lecea, L. (2012). Mechanism for Hypocretin-mediated sleep-to-wake transitions. PNAS, E2635-E2644.
12. Lee, M. G., Hassani, O. K, Jones, B. E. (2005). Discharge of identified orexin/hypocretin nurons across the sleep-waking cycle. Journal of Neuroscience, 25(28), 6716-6720.
13. Weinhold, S. L., Seeck-Hirschner, M., Nowak, A. (2014). The effect of intranasal orexin-A (hypocretin-1) on sleep, wakefulness, and attention in narcolepsy with cataplexy. Behavioral Brain Research, 262, 8-13
14. Thomson, J. L. And Borgland, S. L. (2011). A role for hypocretin/orexin in motivation. Behavioural Brain Research, 217, 446-453.
15. NHS Choices (2010, Jan 29). Narcolepsy Retrieved March 25, 2014, from http://www.youtube.com/watch?v=yg0h6LQDw6o
16. Kalogiannis, Mike (2011, April 29). Mice with narcoleptic attacks. Retrieved March, 25 2014, from http://www.youtube.com/watch?v=L3gf6yzRNHU
17. SanfordBurnam (2011, Au 12). Devanjan Sikder, Ph.D., D.V.M. Retrieved March 26, 2014 from http://www.youtube.com/watch?v=bapVn9ofVSQ

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