lecture 33: orexin Flashcards
1
Q
What are hypocretins?
A
- hypothalamus-specific peptides iwth neuroexcitatory activity
2
Q
Where are hypocretins expressed?
A
- in a few thousand cells in the rat brain
- detection of preprohcrt mRNA in large neurons in the dorsal-lateral hypothalamus by in situ hybridization to a coronol section from the rat brain
- not produced anywhere else
3
Q
What was seen after performing reverse pharmacology?
A
- (different group call hypocretins Orexins)
- orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behaviour
- knew there was a receptor and did a lot of purification to find the ligand
4
Q
What was seen in orexin KO mice?
A
- genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity
5
Q
What is seen in treatment with orexin antagonists?
A
- anorexia and weight loss in male rats 24 h following single dose treatment with orexin-1 receptor antagonist SB-334867
- effect of a selective OX1R antagonist on food intake and body weight in two strains of rats that differ in susceptibility to dietary-induced obesty
- but when in clinic no evidence that producing any changes with respect to weight
6
Q
What is seen in orexin KO mice re: narcolepsy?
A
- molecular genetics of sleep regulation
- narcolepsy
- when you stimulate animal (scare or very happy) muscle tone is gone and go straight into REM sleep → this is very unusual
7
Q
Where is the narcoleptic/cataplectic phenotype seen in human narcolepsy recapitulated?
A
- orexin peptide KO and Ox1/Ox2 receptor KO mice
- also seen in dogs
- narcolepsy/cataplexy is commonly seen in orexin KO mice, narcoleptic dogs (defect in Ox2R) and narcoleptic humans with cataplexy (absence of orexin in CSF/LH)
- in mice only Ox1/Ox2 receptor double KO may show narcolepsy/cataplexy
8
Q
What is seen in the human situation?
A
- orexin peptides are absent in narcoleptic human brain
- a mutation in a case of early onset narcolepsy and a generalised absence of hypocretin peptides in human narcoleptic brains
- no traceable orexin in the CSF of patients with narcolepsy with cataplexy
9
Q
What is the flip/flop wake/sleep model?
A
- schematic representation of the switch (flip/flop) model of sleep and the role of the orexin system:
- high during wake
- low during sleep
- engineering applied to biology
- idea that orexin is functioning like a switch: ON and OFF
- when orexin is high, highly activates proteins involved in wake state, inhibits VLPO
- as sleep pressure increases, VLPO becomes very active, inhibition of lateral hypothalamus and TMN and raphe → fall asleep
- if the switch doesn’t work then you keep oscillating between wake and REM
10
Q
What is the role of orexin in wake/sleep balance?
A
- orexin A and B are only produced in the LH
- they act ont wo GPCRs, Ox1 and Ox2 receptors, present in brain regions involved in sleep/wake, feeding and reward
- orexin neurons are active during active wake stage and silent during NREM and REM sleep stages
- orexin is absent in CSF/orexin producing cells in hypothalamus from patients with narcolepsy/cataplexy
- distribution of receptors fairly widespread throughout brain
11
Q
What is the timeline of orexin/hypocretin discovery?
A
- 1998: discovery of orexin system
- 1999:
- discovery of narcolepsy genes in animals
- forward genetics: hypocretin receptor 2 mutation(s) in dogs
- reverse genetics: hypocretin gene ko in muce
- 2000:
- discovery of hypocretin-ligand deficiency in human narcolepsy, 1) CSF, 2) postmortem brain
- 2005:
- CSF hypocretin-1 measures in the international diagnostic criteria
- in the near future
- new treatments: hypocretin replacement gene therapy
- discover aetiology and prevention
12
Q
What is the official nomenclature established in 2008?
A
- agreed upon by IUPHAR and HGO
- OX-A, OX-B / orexin-A and orexin-B
- OX1 Receptor
- OX2 Receptor
- but the genes: HCRT, HCRTR1, HCRTR2
13
Q
How do we get from gene to peptides?
A
- HCRT gene 17q21.1
- HCRT mRNA
- prepro-orexin
- two peptides being produced
- OX-A has double cysteine bond
- OX-A has similar affinity for OX1R and OX2R
- OX-B only binds OX2R
14
Q
What is the structure of orexins in principle?
A
- A has double cysteine bridges
15
Q
What happens when you have orexin deficiency?
A
- oscillating between sleep and wake
- model of orexins’ function in sleep/wake regulation
- left, sleeep/wake activity is homeostatically controlled, with sleep pressure (black solid line) increasing as a function of time awake and subsidising gradually over time asleep until sufficiently reduced to allow waking
- conversely, wakefulness is driven by a circadian pattern, but also phasic bursts during waking as a function of motivational state and adaptive behaviour
- orexins excited wake-active (e.f. LC and dorsal) and inhibit sleep-active brain regions (e.g. medullary REM muscle atonia circuit), so disharge of orexin neurons during wakefulness helps to counteract sleep pressure and decrease the probability of sleep initiation, especially during emotionally arousing situations
- for purposes of clarity, sleep and wake periods have been depicted as uninterrupted, although rodents typically display multiple sleep/wake transitions in both light and dark phases
- right, in orexin-deficient subjects (for example, NC humans, or animal models of NC), even relatively low levels of unopposed sleep pressure can result in inappropriate intrusions of sleep, regardless of circadian phase or motivational state
- conversely, lower peaks of homeostatic sleep pressure (resulting from decreased wake epoch durations) result in short periods of recovery sleep to regain homeostasis, allowing inappropriate intrusion of waking in the rest phase
- this mechanism might account for rapid sleep/wake transition in narcoleptics and orexin-deficient animals
16
Q
What is the relationship between orexin, feeding and arousal?
A
- orexin has effects upon appetitie, cortical arousal, cephalic phase of digestion, reward, motor arousal, emotional arousal
- influenced by metabolic/nutritional signals, circadian influence
- has an effect on certain dopaminergic centres
17
Q
What happens when you do cFos measurements in the brain after a different number of conditions?
A
- orexin neurons located in the lateral hypothalamus (LH) or perifornical and dorsomedial hypothalamic areas (PFA-DMH) show different patters of Fos activation
- percentages of LH orexin neurons that are doubly labelled for orexin and Fos following different stimuli
- LH orexin neurons in conditioned animals expressed significantly more Fos in response to cues associated with morphine, cocaine or food during a test for conditioned place preference than those in non-conditioned animals
- reinstatement of extinguished morphine place preference occured with rat pancreatic polypeptide (rPP), an agonist of Y4 neuropeptide Y receptors and a potent stimulator of orexin neurons, was microinjected into LH, but not when it was microinjected into areas surrounding LH (rPP control)
- effective rPP microinjections significantly elevated Fos levels in LH orexin neurons, but not other orexin neurons
- exposure to footshock did not activate LH orexin neurons
- percentages of PFA-DMH orexin neurons showing double labelling for orexin and Fos
- PFA-DMH orexin neurons showed an elevation in Fos expression above baseline only after footshock and exposure to cocaine-associated cues
- relationship between lateral hypothalamus and drug dependence (Morphine, cocaine, alcohol)
- hopes that orexin receptor antagonists would be good compounds for treating a number of drug addiction disorders
18
Q
What is the relationship between orexin, arousal and reward?
A
- LH
- projections to forebrain and midbrain reward areas:
- VTA
- NAc
- reward-related inputs
- morphine
- cocaine
- food
- PFA-DMH
- projections to brainstem arousal areas:
- LC
- TMN
- PPT-LDT
- arousal related inputs:
- waking
- stress
- projections to brainstem arousal areas:
24
Q
What is the arousal system?
A
- ascending arousal system: a major input to the relay and reticular nuclei of the thalamus (yellow pathway) originates from cholinergic (Ach) cell
- key projections of ventrolateral preoptic nucleus (VLPO) to the main components of the ascending arousal system → gabanergic system gets activated when you have extremely high sleep pressure
- includes monoaminergic cell groups
25
Q
What are the hypothalamic orexin system’s connections?
A
- orexin neurons in the lateral hypothalamic area (LHA) and posterior hypothalamus (PH) are anatomically well placed to provide a link between the limbic system, systems involved in energy homeostasis and monaminergic and cholinergic neurons in the brain stem
- solid arrows show excitatory projections, and broken lines inhibitory ones
- wake-active regions, sleep-active regions and REM-active regions are shown by red, blue and green boxes
- orexin neurons promote wakefulness through the monoaminergic nuclei that are wake-active
- stimulation of dopaminergic centres by orexins can modulate reward systems (purple)
- peripheral metabolic signals such as leptin, ghrelin and glucose influence orexin neuronal activity to coordinate arousal and energy homeostasis
- the nucleus suprachiasmaticus (SCN), the central body clock, sends signals to orexin neurons via the dorsomedial hypothalamus (DMH)
- the DMH acts as a food-entrainable ossilator, and influences orexin neuronal activity
- input from the limbic system (amygdala and bed nucleus of the stria terminalis BST) might regulate the activity of orexin neurons upon emotional stimuli to evoke emotional arousal or fear-related responses
26
Q
What is the mechanism by which the orexin system stabilises sleep and wakefulness?
A
- in awake state orexins are active and have strong influence on monoaminergic neurons → cortex and thalamus gets stimulated
- in sleep state VLPO takes over
- blocks orexin → lowers MA
- in narcolepsy switch is not on off just absent
- mutual inhibition → random movement between wake and sleep states
27
Q
What is the discharge of orexin neurons during sleep/wake cycles?
A
- aW = active wake
- qW = quiet wake
- SWS = slow wave sleep = NREM sleep
- PS = paradoxical sleep = REM sleep
- firing reduced when in deep sleep
28
Q
What is almorexant?
A
- dual Ox1/Ox2 receptor antagonist
- published 9 years after the discovery of the peptides - very fast
- in a dose dependent manner see that active wake is reduced
- locomotion reduced
- REM sleep increasing, whereas Z whatever ‘black night’
- slight increase with non-REM sleep
31
Q
What was written in the prescribing info?
A
- not to be used in the treatment of narcolepsy
- like other sleep medicines, there is a risk from Belsomra of sleep-driving and other complex behaviours while not being fully awake, such as preparing and eating food, making phone calls, or having sex. Chances of such activity increase if a person has consumed alcohol or taken other medicines that make them sleepy. Patients or their families should call the prescribing health care professional if this type of activity occurs
- in dogs, daily oral administration of suvorexant for 4-7 days resulted in behaviour characteristic of cataplexy (e.g. transient limb buckling, prone posture) when presented with food enrichment, a stimulus demonstrated to induce cataplexy in dogs with hereditary narcolepsy
