VANESSA VILLAMIA SOCHAT

Narcolepsy as a Disorder of the Hypocretin System
How levels of orexins might lead to narcolepsy, & why those levels are off in the first place The Neuroscience of Illusions 3/27/2008

This paper hypothesizes that narcoleptics might have lower levels of hypocretins that leads to an inability to maintain wakefulness and more sporadic activation of monoaminergic neurons that consequently leads to disturbed sleep. To study this hypothesis, this paper suggests using PET scan to measure hypocretin and monamine levels in narcoleptic and normal subjects, and correlate these levels with state of arousal and REM vs NREM sleep as recorded by EEG during sleep onset, during sleep, and waking. These measurements, in context of one another, might reveal patterns between hypocretin levels, monoaminergic activity, and how these trends correlate with the perceptual experience of being awake or asleep. This paper ultimately hypothesizes that narcoleptics will have substantially lower levels of hypocretins than normals, and as a result, monoaminergic activity might fluctuate more often and rapidly to activate cortex and thalamus, leading to observed changes in arousal.

CONTENTS
Part I: Narcolepsy as a Disorder of the Hypocretin System ............................................................................ 3 Background and Significance .................................................................................................................... 3 Physiology of Sleep Regulation .............................................................................................................. 3 The Hypothalamus as Key Player ........................................................................................................... 4 The Hypocretin System .......................................................................................................................... 4 The Hypocretin System and Sleep ......................................................................................................... 4 The Hypocretin System and Narcolepsy ................................................................................................ 5 Hypocretins are Missing in Narcoleptics ............................................................................................... 5 The Reign of the Hypocretin System ..................................................................................................... 6 Hypocretins and Arousal ........................................................................................................................ 7 Description of a Novel Scientific Problem ..................................................................................................... 8 Experimental Synopsis ................................................................................................................................... 8 Specific Aims................................................................................................................................................... 9 Future Research: .................................................................................................................................. 10 Part II: The Underlying Cause of Hypocretin Deficit ..................................................................................... 11 It Probably isn’t Genes ............................................................................................................................ 11 Might it be an Autoimmune Disorder? ................................................................................................... 11 Why a lack of compelling evidence? ....................................................................................................... 12 Bibliography………………………………………………………………………………………………………………………………………..14

PART I: NARCOLEPSY AS A DISORDER OF THE HYPOCRETIN SYSTEM BACKGROUND AND SIGNIFICANCE
Much research has been done to assess genetic and biological explanations of narcolepsy and sleep disorder. Although physiological explanations have been made to discuss these phenomena, the current research lacks an analysis of how this physiology leads to the perceptual phenomena associated with sleep disorder. Low levels of hypocretins are hypothesized to be responsible for sleep disorder, yet the cause of these low levels is still unclear. This paper aims to ultimately address the important points of research related to hypocretins, narcolepsy, and perception, and propose that narcolepsy is a disease of the hypocretin system. There is an enormous gap in answering the question about what physical and perceptual disorders these low levels might cause, and as a subtopic, why certain individuals might possess low levels of hypocretins in the first place. Making connections between the hypocretin system and other parts of the brain, this paper ultimately strives to link the hypocretin system to other sleep and neurological disorders and phenomena to influence future research.

Physiology of Sleep Regulation In order to understand sleep disorder, it is necessary to understand the physiology of normal sleep. During REM sleep, skeletal muscles are inhibited by means of the medulla and spinal cord, and this leads to atonia, which is basically a complete relaxation of all muscles that occurs during sleep. The simplest explanation of narcolepsy and other sleep disorder such as paralysis and cataplexy is the idea of REM sleep atonia intruding into a wakeful state accompanied by other perceptual phenomena. It was first hypothesized that these three sleep disorders are a result of hypoactive dopaminagenic transmission, which would explain why many disorders can be alleviated by modulating dopamine pre-synaptically. Cataplexy, which is often present in individuals with narcolepsy and other sleep disorder, is classified by muscular weakness that ranges from specific groups to the entire body. This is the phenomenon of an individual “falling asleep” with strong emotional stimulation like laughter or surprise. Cataplexy was found to be related to levels of dopamine. A study using localized injection found that inhibitory dopaminergic autoreceptors are linked to cataplexy (Reid et al., 1996).

The Hypothalamus as Key Player Studies (Von Economo) have suggested that sleep disorders like cataplexy and narcolepsy might simply be an activation of this pathway that leads to atonia that is normally inhibited during wakefulness. Lesions of the junction between the hypothalamus and midbrain, also by Von Economo, led patients to feel higher levels of sleepiness, and it was also observed that inflammation of the hypothalamus lead to insomnia. Although this research is more than sixty years old, it lead to the recent discovery that a group of tuberomammillary histamine neurons in the hypothalamus are involved in stimulating wakefulness. This finding explains the significance of using histamine to regulate sleep patterns, and also explored the basal forebrain area as containing wake-on acetylcholine containing neurons that are also important for sleep regulation. This finding brings up the question of how different areas of the brain are networked to the midbrain, specifically the hypothalamus, which seems to be most relevant for the regulation of sleep. Why is the hypothalamus so important for the regulation of sleep? One reason might be that it is the home base of the hypocretin system.

The Hypocretin System Hypocretins are two neuropeptides present in small numbers in the hypothalamus, and they are derived from the same precursor, specifically related to the preprohypocretin gene. It is important to note that hypocretins and orexins are basically the same thing, because they are the same peptide derived from the same gene (Kilduff and Peyron, 2000). It is questionable if these 1,000 to 7,000 or so neurons make up an area in the hypothalamus (Kilduff and Peyron, 2000) that is even large enough so destruction might be observed by the technology that is available today. It seems that the hypothalamus is hugely involved in sleep, and given the location of these neurons, can the hypocretin system be a key player in sleep regulation?

The Hypocretin System and Sleep There are two G protein receptors for hypocretins and they are scattered around the CNS. The distribution of these two types of receptors, called hcrtr-1 and hcrtr-2 are very different in the brain

(Trivedi et al., 1998). The hypocretin fibers themselves project to different areas in the brain, including the olfactory bulb, cerebral cortex, thalamus, hypothalamus, and brainstem, which are involved in sleep (Harrison et al., 1999). The spinal cord is innervated by hypocretin fibers too (Peyron et al., 1998). This is why the hypocretin system is speculated to be involved with maintaining a state of vigilance: it includes homeostatic, limbic, and metabolic regulation. Therefore neurons in the ascending reticular activating system, including the raphe nucleus, pedunculopontine and laterodorsal tegmental nuclei, and tuberomammilary nucleus that are responsible for arousal and much cortical activation, are essential for the delicate balance achieved in normal sleep.

The Hypocretin System and Narcolepsy So if hypocretins are important for arousal and this delicate balance achieved in normal sleep, might a disordered system lead to narcolepsy and the perceptual phenomena associated with it? Neuropathologic studies with a focus on the hypocretin system, performed on humans, point strongly to a yes. Recording specifically from neurons in the perifornical lateral hypothalamus that are highly involved with sleep regulation, it was noted that 38% of these neurons are activated only when hypocretin neurons are in their “awake” phase. If these hypocretin neurons were to be destroyed, this would mean that a portion of the neurons in the hypothalamus important for the maintenance of sleep would not fire, which would arguable lead to sleep disorder. Not surprisingly, these neurons aren’t as prevalent in narcoleptics.

Hypocretins are Missing in Narcoleptics In people without sleep disorder, hypocretin-rich cells are prevalent, and they are MIA in narcoleptics. Hypocretin-rich cells were mapped in the brains of thirteen (dead) control subjects via in situ hybridization in frozen brain tissue (Peyron et al., 2000). In contrast, Hcrt-1 and hcrt-2 peptide levels in the pons and cortex of the same controls and six narcoleptics were measured, and the peptides were completely missing in the narcoleptics. (Peyron et al., 2000). A similar study found equivalent results in

other control and narcoleptic brains (Thannickal et al. 2000). Additionally, Thannickal used immunohistochemistry to observe a reduced number of hypocretin neurons in the hypoalamuses of four narcoleptic patients. This is huge evidence to link hypocretins and narcolepsy, however it might be more telling to observe these patterns in living subjects, as levels of hypocretins change over time between sleep and wakefulness. This correlation between low levels of hypocretins in narcoleptics and an inability to maintain wakefulness leads to question the observation that narcoleptics are able to maintain brief periods of wakefulness. Why might that be so? This paper superficially speculates that other stimuli that lead to activation of cortex such as motor activity and adrenaline, and attention might keep the thalamus sufficiently aroused to allow for wakefulness. Now that we have an understanding of the mechanisms of sleep, hypocretins, and narcolepsy, the next important question to ask is how is the hypocretin system relevant to different regions of the brain, and how this might lead to physical and perceptual phenomena associated with narcolepsy.

The Reign of the Hypocretin System It is clear that the hypocretin system projects to many areas of the brain. Siegel, in 2000, noticed an increased chance of developing cataplexy when hypocretin input to the LC (locus coeruleus) was halted. Kilduff and Peyron (2000) that noticed that a lack of hypocretin input on certain target sites important in the regulation of sleep leads to hyperactive monoaminergic cells, a lower level of excitation to the cortex, more activity of cholinergic cell groups, and consequently more disrupted sleep. Sakurai described these monoaminergic neurons as being activated by hypocretins, leading to increased arousal and wakefulness, and illustrated a system in which hypocretins excite monoaminergic cells, and these cells in turn further excite the thalamus and cortex (Sakurai 2007). These are opposing viewpoints, as Kilduff observed that low hypocretin input causes hyperactive monoaminergic cells and increased cortex activity, and Sakurai implies that low hypocretin input causes less activation of monoaminergic cells, and consequently a smaller increase of activity to cortex. I think to explain this discrepancy it is important to look at Sakurai’s model of a system completely lacking in hypocretins, and additionally question in what

state each researcher recorded monoaminergic neuron activity. In the case of no hypocretin input, Sakurai describes a “mutually inhibitory circuit” between monoaminergic neurons and the VLPO. This implies that activation of these monoaminergic neurons, for example, might completely shut down inhibitory inputs from the VLPO, causing a quick increase in monoaminergic activity, and then disinhibiting its own action. I surmise that a lack of hypocretins might hypersensitize monoaminergic neurons, and in the case of a rapid switch, it might be possible to see hyperactive monoaminergic cells in the absence of hypocretin input. This might lead to the disrupt transition between wakefulness and sleep, Hypocretins may act as a third party in this system to offset this mutually inhibitory circuit.

Sleep disorder has been explained as a loss of “state boundary control,” (Sutcliffe and de Lecea, 2000), so might the hypocretin somehow be involved in this control? Could the hypocretin system, projecting from the hypothalamus, be some sort of controller? It seems that the impact of the hypocretin system can go beyond sleep, and it can be said that these neurons are involved in regulating the body’s internal state based on the external environment. It is clear that a very tiny number of hypocretin neurons are responsible for regulating a ton of physiologic functions, and clearly acting on other neurotransmitters to lead to the perceptual phenomena associated with sleep disorder. These cells that are acted upon by hypocretins include noradrenergic cells of the LC, dopaminergic cells of the VTA, serotonergic cells of the DR, and histaminergic cells of the TMN. Although this network might be responsible for the many phenomena associated with eating, mood and emotion, hormonal regulation, this paper aims to focus on the part of the network associated with sleep, arousal, and wakefulness.

Hypocretins and Arousal Hypocretins are important for the regulation of sleep, as was discussed earlier, but through what medium? They might mediate wakefulness through the histaminergic system, as it was found that injections of hypocretins in rats increases time awake, and this effect was eliminated with a histamine H receptor antagonist (Sakurai 2007). It is also interesting whether or levels of hypocretins might change during the wake/sleep cycle. Studies with mice found that hypocretin neurons need to be activated during awake periods, and switched off during NREM and REM sleep to maintain atonia. Thus, it makes

sense that these studies found higher levels of activity in hypocretin neurons in rats during the dark period (when they are most awake) and lower levels during the light period (when they sleep). There is the interesting correlation between wakefulness and hypocretin neuronal activity. It might be the case that these neurons are responding to the presence of sensory stimulation that is largely absent during sleep, which is an idea that was examined by Lee. Lee found that hypocretin neurons (in the LHA) fire most during wakeful periods with high activity, less during wakeful periods with little activity, and were dormant during REM and NREM sleep. His most interesting observation was the increase in firing of these neurons in the transition phases between sleep and wakefulness. It might be interesting to think about the connection between the firing of these neurons to induce wakefulness, and the limbic system. Perhaps emotional stimuli during sleep might cause hypocretin neurons to fire, and because they receive abundant input from the limbic system, this firing might trigger the transition from sleep to wakefulness and ultimately lead to sleep disorder.

Description of a Novel Scientific Problem The majority of these studies focus on discrete quantities of hypocretins between different subjects as a measurement of narcolepsy as opposed to the fluctuation within one subject, for which there is no discussion. How might this fluctuation differ between narcoleptics and normal subjects? Could the levels of hypocretins be less important than the fluctuation in the levels? Can certain levels of hypocretins be correlated with different levels of arousal and perceptual experience?

Experimental Synopsis This paper proposes an experiment to investigate changes in perceptual experience, mainly the boundary between wakefulness and sleep, correlated with hypocretin fluctuations. It might be helpful to study the relationship between levels of hypocretins throughout the brain and consequent monoaminergic activity via PET scan. Labeled tracers might monitor monoamine levels, and hypocretin peptides might hypothetically be radioactively labeled. However, these recordings have no meaning without being placed in the context of the state of sleep or wakefullness. EEG can be used as a measure

of REM and NREM sleep, and then it will be likely to find patterns between levels of hypocretins and monoamines and the perceptual experience of interest, arousal state. These measurements might all be taken before sleep onset, during sleep, and upon arousal. By matching these levels up with sleep cycles it can be determined what is going on in terms of hypocretin levels and monoaminergic activity during the switch from REM to NREM sleep in normals and narcoleptic subjects. Hypocretin and monoamine systems seem to be intimately related, but I think it would be dangerous to only measure one to make deductions about the other. It would add considerable depth to this experiment to to interview subjects directly after about any perceptual experience, and rate their level of arousal and alertness on a scale of 1-10. Subjects should also be asked about having any hallucinogenic experiences. Humans are essential for this experiment. Studies with rats are troublesome in the sense there is no way to record their perception. If interest is focused on how changing levels of hypocretins contribute to changes in perceptual experience, humans are required. Additionally, it goes without saying that the experiment requires live humans that can react and report on their experience.

Specific Aims • This paper hypothesizes that narcoleptics might have lower levels of hypocretins that leads to an inability to maintain wakefulness and more sporadic activation of monoaminergic neurons that consequently leads to disturbed sleep. • Use PET scan to measure hypocretin and monoamine levels in normal and narcoleptic subjects during sleep onset, during sleep, and arousal. • • Use EEG to correlate these fluctuations in levels with cycles of REM and NREM Look for patterns between hypocretin levels, monoaminergic activity, and how these trends correlate with the perceptual experience of being awake or asleep. • Hypothesized that narcoleptics will have substantially lower levels of hypocretins than normals, and as a result, monoaminergic activity might fluctuate more often and rapidly to activate cortex and thalamus, leading to changes in arousal.

The Main Question It is clear that narcoleptics have lower levels of hypocretins than controls, and these low levels lead to disturbed wakefulness and disorder in the mechanism that switches between wakefulness and sleep. The main question that is the focus of this grant is as follows: Is a narcoleptic’s inability to maintain wakefulness due to low levels of hypocretins in the system, or smaller changes in these levels?

Future Research: Connecting changes in levels of hypocretins might give huge insight into the cause of sleep disorder and its perceptual phenomena. There is much research about the physiological activity of the hypocretin system, but a huge gap in relating hypocretins to hallucinogenic experiences. Is there a connection between frequency and quality of hallucination during sleep paralysis that can be explained by different levels of hypocretins? If hypocretins are involved in the many domains that they are speculated to influence, then sleep paralysis in the absence of narcolepsy might finally be explainable, and more importantly, the illusions that have been coined as out of body, alien abductions, and spiritual experiences, might finally have a physiological explanation.

PART II: THE UNDERLYING CAUSE OF HYPOCRETIN DEFICIT
If narcolepsy is indeed a disorder of the hypocretin system, what mechanism might lead to this imbalance?

It Probably isn’t Genes It’s important to note that as of yet no genetic link to having some sort of mutated hypocretin related gene has been found. The in-situ hybridization studies discussed earlier in this paper noted that the gene responsible for the expression of hypocretin was preserved in both control and narcoleptic subjects, which serves as evidence that the lack of hypocretins is not result of a faulty gene. Lack of a genetic link lead to the speculation that there is nothing wrong genetically, but in fact the hypocretin neurons are somehow getting destroyed. This is where an autoimmune disorder comes in.

Might it be an Autoimmune Disorder? It is highly probable that the underlying cause of the low levels of these hypocretins and the subsequent narcolepsy is due to an autoimmune disorder. Thannickal found a larger number of astrolytes in the hypothalamuses of his narcoleptic patients over the control. As, astrocytes are indicative of a degeneration of neurons, this finding might be evidence for some autoimmune disorder. But why hasn’t anything be found as of now? It seems that the transient nature of most autoimmune disorders coupled with the low number of hypocretin neurons and their tiny presence in the brain might explain this lack of evidence. Perhaps harder examination is needed, and examination in a different way, but first it is important to look at evidence that is out there that might signal the involvement of an autoimmune disorder. One case of a definite autoimmune disorder tied to low levels of Hcrt1 in CSF was found in a disorder called anti-Ma paraneoplastic syndrome. People with this disorder develop encephalitis as a result of having antibodies against Ma proteins in the limbic system, hypothalamus, and brain stem, and these areas are obviously very involved with sleep regulation and linked to hypocretin function.

Additionally, for most people with narcolepsy, the onset of the disease occurs between the ages of ten and thirty, which is a common age of onset for autoimmune disorders. The most compelling evidence is the fact that narcoleptics have these same low levels of Hcrt1 in their CSF. These levels are typically below 100 ng/L, and these levels are most commonly not detectable with radioimmunoassay, as was shown by Thannickal. He also found that in controls and individuals without narcolepsy or sleep or neurological disorder, CSF hypocretin 1 concentrations were usually above 200 ng/L, and given a random subject with a hypocretin concentration of less than 110 ng/L, this individual had a 94% chance of having narcolepsy with cataplexy. The downfall of these experiments is the fact that, as mentioned earlier, hypocretin is very localized and elusive within the brain, and measurements of CSF are lacking in spatial and temporal resolution. This means that where levels were measured can make a difference, and that concentrations of hypocretins for controls and narcoleptics might be different.

Why a lack of compelling evidence? To this date, nobody has found a direct autoimmune cause, but this might be due to the fact that the area being affected in the hypothalamus is so tiny. Other diseases that have some sort of autoimmune damage occurring that also had low levels of hypocretins during the disease are acute disseminated encephalomyelitis and Hashimoto’s thyroiditis (Nishino et al., 2000). Linking low levels of hypocretins with rare autoimmune disorders is telling, but it isn’t enough. An experiment that took sera from narcoleptic patients and put it mice noticed that IgG from these same patients, when given to the mice, enhanced bladder contractile responses to charbachol. It is important to note that IgG is the most common antibody found in bodily fluids that is essential in fighting viral and bacterial invaders. Charbachol activates two types of acetylcholine receptors, one which enhances REM sleep (Hobson et al., 1998). Therefore what this study noticed is that mice that were given an agent to induce REM sleep actually had more muscle contraction as compared to the control when subjected to the immune system of the narcoleptic patients. It is also interesting to note that IgG, that potentially could “pass on” something that might lead to an autoimmune disorder, is the only type of antibody that

can cross the placenta from a mom to her baby, which might explain some genetic links of narcolepsy if it is indeed an autoimmune disorder Clearly, investigation of the causal effect of the destruction of the hypocretin system is in order. It isn’t enough to find subtle evidence linking narcolepsy to low levels of hypocretins and speculating and autoimmune disorder. Perhaps when this connection is solidified, diagnosis of narcolepsy and other sleep disorder can be made based on measurements of hypocretin levels.

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