Welcome back. Now we're going to talk about the peptide, hyporcretin, also called orexin, a relatively newly discovered peptide that's very important for the neurobiology of sleep and wakefulness. The Hypocretin-containing neurons are localized as a lateral hypothalamus shown here. It's a very small group of neurons with extensive projections as indicated by these purple lines and arrows throughout the central nervous system rostrally and caudally. And this system of projections from the lateral hypothalamus in these Hypocretin or orexin-containing neurons was only recognized and published in 1998. So it's very recent addition to our understanding of sleep neurobiology. And the reason, I should say, there's two names is that the peptides, there's actually Hypocretin one and two and orexin A and B, the peptides were discovered pretty much simultaneously by two different groups. One group named these peptides orexin because it was thought that they control appetite, and it's the Greek word for appetite. And another group named these peptides Hypocretin because they are of hypothalamic origin, and with the sequence similarity to the secretin peptides. So that's the origin of the names and the dual names of Hypocretin and orexin. Let's look at where the projections go from these Hypocretin-containing neurons. They said, they project to every major arousal-related nuclei in the brain: the tuberomammillary nucleus in the posterior hypothalamus which we've talked about, that's where the histaminergic neurons are; to the locus coeruleus and dorsal raphe nucleus where the neuronergic and the serotonergic neurons are; to the pontine and medullary reticular formation; and to the LDT, laterodorsal tegmental area, where the cholinergic neurons are. Hypocretin or orexin neurons receive input from arousal-related nuclei. Also they get input from the basal forebrain glutamatergic and GABAergic neurons. They get input from many areas, the ventral tegmental area, locus coeruleus, dorsal raphe, from the suprachiasmatic nucleus which is the circadian pacemaker, and from the limbic system including the amygdala, and the bed nucleus of the stria terminalis. Hypocretin neurons also receive peripheral metabolic cues. They sense glucose levels, and the appetite-regulating peptides ghrelin and leptin. So these neurons are really well poised to integrate a lot of information about metabolism, and behavioral state of arousal and, likewise, they're poised because of their projections to influence arousal. And that is schematized very nicely in this diagram from a review by Sakurai, showing the inputs and outputs to the orexin-containing neurons in the lateral hypothalamus. What the solid lines indicate, pathways that are excitatory. The dotted lines, the bar on the end, indicate pathways that are inhibitory. And so again, you can see mutual projections with the monoaminergic nuclei that we talked about, locus coeruleus, dorsal raphe into medullary nucleus. There are other areas we haven't talked about, the dopaminergic connection with the lateral hypothalamus is very important. Here the cholinergic cells receive input from the hypocretinergic neurons. And this, here, we schematize the peripheral metabolic cues that get integrated and act on hypocretinergic neurons, and the circadian influence coming by way of the dorsal medial hypothalamus directly onto the lateral hypothalamus containing orexin neurons both excitatory and inhibitory input. And the sleep-promoting neurons which we have yet to talk about and the ventrolateral preoptic area, their GABAergic provide inhibitory input to the orexin wake-promoting neurons. So while this interest in Hypocretin? It's extremely clinically relevant. A year after the pathways were mapped, it was shown that canine narcolepsy is caused by mutation in one of the gene for Hypocretin or orexin receptor. There's two kinds of receptors, and the Hypocretin receptor two gene is mutated and dysfunctional in dogs with narcolepsy. And then, similarly, at the same time, another group published a study using knockout mice. They knocked out the peptide, and that these mice show a narcoleptic phenotype that is evidenced by periods of behavioral arrest as narcoleptic dogs have. So these studies provide an excellent example of how preclinical work in animals has led to mechanistic insights about sleep disorder in humans, the disorder of narcolepsy. So we're going to talk, spend a little bit more time talking about this peptide and its role in narcolepsy and its role in normal regulation of sleep and wakefulness. So in the cerebral spinal fluid of people without narcolepsy, there are detectable levels of Hypocretin. But in patients with narcolepsy, you cannot detect Hypocretin indicating an absence. And a really key study was done, thanks to the foresight of Michael Aldridge at the University of Michigan. He banked the brains of narcoleptic patients who had died and post-mortem examination of those brains was discovered that Hypocretin or orexin neurons are gone. They are eliminated in people with narcolepsy. And this has led to a whole series of studies indicating that this may, in fact, be an autoimmune disease because people are born with a normal number of Hypocretin neurons, and that the neurons degenerate and disappear over time. So this may, in fact, be an autoimmune disease. There's a lot of data about that which we won't talk about today. So, we now know that, in specifically there's two groups of narcoleptic patients: those that have cataplexy which is the loss of muscle tone and those that don't. And specifically, there a low Hypocretin levels in the serum of spinal fluid of those patients who have narcolepsy with cataplexy. And this is important because there is a lot of evidence that suggests that Hypocretin plays a role also in motor control. Another, there are progress that was made in the understanding the mechanisms of Hypocretin nergic transmission and its disruption in terms of narcolepsy, was done by creating transgenic mice that lose their Hypocretin containing neurons during development. So again, this is much more similar to what happens in human, it's a better model than a knockout. And so these mice are born with the normal number of Hypocretin neurons and then they are destroyed during development. And similar to humans, these mice develop a phenotype that includes periods of behavioral arrest, they have REM sleep onset periods normally. Remember, that REM sleep arises out of non-REM sleep and that the onset of REM sleep directly from wakefulness is one of the signs of narcolepsy. These mice, as well as people with narcolepsy have fragmented sleep and tend to develop late onset obesity. The symptoms of narcolepsy that we've just reviewed cataplexy, fragmented sleep, and REM sleep onset periods, these symptoms are reversed in these transgenic mice by ectopic production of orexin from a prepro-orexin transgene or if by intracerebroventricular injection when administers orexin-A. So, by replacing Hypocretin, the point of these lies to say that, by replacing Hypocretin, one can rescue the phenotype, which is good evidence that Hypocretin is responsible for these symptoms. In terms of the role of Hypocretin in normal physiology, there have been many studies examining the role of Hypocretin in the promotion of wakefulness. And one of the early key studies show that if you microinject Hypocretin into the locus coeruleus, this key arousal promoting area, see a large increase in wakefulness, and a decrease in sleep. Locus coeruleus has the highest density of Hypocretin receptors in the brain and receives major input from the Hypocretin containing neurons. And we also know Hypocretin or orexin, these are excitatory peptides so they depolarize these wake-promoting neurons in the locus coeruleus, the LDT, posture hypothalamus in the substantia innominata, that's the basil for a brain. So, one of the ways in which hypocretins can promote wakefulness is by depolarizing or stimulating these wake-promoting neurons throughout the networks that we've been discussing. Hypocretin levels have been measured in the cerebral spinal fluid and in experimental animals across the sleep wake cycle and the levels are found to be higher during active waking than during quiet waking. Again, suggesting that this peptide is also involved in the control of movement. And if we look at the discharge pattern of Hypocretin neurons, that data also fits with the released data. And I'm going to show you this pattern in the next four slides. So, we're looking at these state-dependent discharge rate of Hypocretin or orexin neurons. In the unit trace that circled in red, that's the discharge of a single neuron and the trace above the unit that's called rate, that's just the integrated output of that discharge. And so what you see is that, the cell is firing at a high level during active waking. Next, we're going to look at quiet waking or I should say active waking means that the animal is moving around, making motor activity, grooming for example in this case, and you can see in the EMG trace those bursts of activity indicating movement. During quiet waking, where you see there, the EMG tone is much lower and you can see that the unit trace the cell discharge is much slower and much more infrequent when the animal enters slow wave sleep or non-REM sleep, the cell is also firing at a low rate and the same in REM sleep. And if we put these data all together and looking on the right side at the activity of the neurons spikes per second or discharge rate, across states of active waking, quiet waking, transition to sleep, sleep transition into REM, and REM basically, these cells are firing during active waking. And the panel on the left shows the area where these cells were recorded, LH stands for lateral hypothalamus and that's where the red dots indicate where these, one, two, three, four, five, six cells were recorded. So again, the greatest Hypocretin levels in neuronal discharge rates during active waking are consistent with a role for Hypocretin in promoting wakefulness and in activating motor systems. One of the important studies that helped demonstrate that this is a role for Hypocretin in normal wakefulness was another an optogenetic study. These investigators used optogenetic approaches to specifically stimulate Hypocretin containing neurons. And it shows that if they showed, that when you activate these neurons specifically, you decrease the latency to wake onset from both slow-wave sleep and REM sleep. So these experiments are done when the animals are asleep and when the animal is in slow-wave sleep, the Hypocretin neurons are stimulated and the animal wakes up, same from REM sleep, and they wake up much faster than they would without the stimulation or than stimulating in control animals that where the hyper creep neurons are not activated. So the stimulation of Hypocretin neurons also increases the number of transitions from slow-wave sleep and REM sleep, into wakefulness. And importantly, the photostimulation effects were blocked by giving a Hypocretin receptor 1 antagonist. And the pharmacological senses where really nice study combining optogenetic stimulation with classical pharmacology blocking responses by receptor antagonist, a very powerful way to demonstrate that this is a real effect mediated by endogenous receptors. So, what these investigators concluded rightfully so, from this optogenetic study, was that activation of Hypocretin neurons promotes the initiation of wakefulness from sleep, and that this process is mediated by Hypocretin receptors. I told you earlier, in a previous segment that there's another drug for treating insomnia, it's called Suvorexant. It's a dual orexin receptor antagonist, it promotes sleep at doses that do not disrupt cognition. So, that's an important point, both in rats and in rhesus monkeys. And the FDA have news release in August of 2014 saying that this drug suvorexant, which is marketed, as well as Belsomra, was approved for the treatment of insomnia. So this is a new class of drugs, a orexin receptor antagonist, approved for the treatment of insomnia and this drug went on the market in February 2015. So, in addition to the relevance of Hypocretin for insomnia and for narcolepsy, there's also a very interesting relationship with drug abuse. Sleep disruption increases the risk of addiction relapse and so many people who try to withdraw from alcohol or drug abuse have sleep disruption. And, the more their sleep is disrupted, the more they are at risk for relapsing. So when treated with dual orexin receptor antagonists, it's been shown that the genetic and behavioral changes that are caused by these drugs, addictive drugs, such as amphetamine and nicotine can be blocked. And so, this speaks to the potential use of Hypocretin receptor ligands to help treat addiction. And it also points to the important role of Hypocretin in regulating metabolism as well as sleep and wakefulness. So these Hypocretin neurons again, they're in a really, very powerful position to integrate signals, metabolic signals, signals for arousal, and motor activity. That concludes our section on Hypocretin and in the next segment, we're going to review the more classical role of GABAA and that is in the generation of non-REM sleep.
