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Zzzz zzzz zzzz… … …
 Zzzz…..
 Zzzz…
 Zzzzz….
 Zzzz….
 Huh? Wha? Oh… right…
Who Needs Sleep?
• Mammals, birds, reptiles, amphibians, fish, insects,
nematodes, barenaked ladies
• Likely a universal phenomenon for animal kingdom
By James M. Robertson
SLEEP
Repeat.
Sleep features:
 Altered consciousness
 Inhibited sensory activity
 Inhibition of voluntary muscles
 Decreased ability to react to stimuli
 Less so than being awake
 More so than hibernation, comatose
 Heightened anabolic state
 Anabolism = Building Up
Anabolism/Catabolism
How much
should you
sleep?
 From recent
recommendations
of National Sleep
Foundation
Your Biological Clock
 Humans have variable sleep “chronotypes”
 Two hours either side of average
Sleep is a cyclical process
Sleep Stages: NREM
 Non-rapid Eye Movement Sleep
 EEG Neural Oscillations = Engine Noise
 Lower arousal threshold:
 Stage 1 – Between sleep wakefulness
 Muscles active & eyes slowly rolling
 Low voltage / mixed freq.
 Stage 2 – Wave activity
 Strong oscillatory EEG rhythms
EEG
Sleep Stages: NREM
 Higher arousal threshold:
 Stage 3 – Slow Wave Sleep
(SWS)

 20-50% Delta Waves EEG
 Delta wave = high amplitude brain wave
 0-4 hz
 Stage 4 – “Deep Sleep”
 >50% Delta Waves EEG

Sleep Stages: REM
 Rapid Eye Movement Sleep
 Low Muscle Tone
 Difficult to arouse
 Rapid/low voltage EEG
 ↑ Brain Arousal ↑ Brain Oxygen Consumption
 Similar EEG pattern to waking
 20 – 25% Total Sleep
 1.5 – 2 hours per night in humans
 Lack of REM impairs learning ability
Orexin and sleep function
Sakurai 2011
 Sakurai is an O.G.
 His lab discovered Orexin
 Reverse pharmacology methods
 Two orphan GPCR receptors
 Orx A, Orx B
 Molecular cloning techniques reveal prepro-orexin
 De Lecea id’d mRNA for prepro-orexin around same time
 Orx neurons localized to hypothalamus
 3000 neurons - rats
 70,000 neurons - humans
Possibly
wanksta
Orexin and sleep function
Sakurai 2011
 Loss of Orexin Neurons implicated in:
 Narcolepsy
 Excessive daytime sleepiness
 Sleep Attacks
 REM Sleep Disorder
 Cataplexy
 Episodic loss of muscle function
 Ranges from weakness to complete collapse
 Observed in narcoleptic patients
Narcolepsy
Sakurai 2011
 Affects ~ 1 / 2000 in United States
 Notably reduced latency for REM sleep
 Sleep-Onset REM (immediately preceded by waking)
 Sleep Attack = REM sleep intrusion
 Nocturnal sleep highly disturbed
 Vivid dreaming
 Hallucinations
 Sleep Paralysis
Narcolepsy and Cataplexy
Sakurai 2011
 Concomitant disorders
 Cataplexy
 Triggered by emotional stimuli
 i.e. scaring the cute little goats
 Unlike narcolepsy, consciousness is kept
Orexin and Narcolepsy
Sakurai 2011
 Orx involved in narcolepsy
 (-)Orx mice, dogs show similar phenotypes to human condition
 Behavioral arrests similar to cataplexy
 REM sleep attacks
 Human narcoleptic postmortem
 No detectable Orx in cortex or pons (normally present)
 80-100% reduction in neurons containing prepre-orexin mRNA
 Hypothesized to result from selective immune response
 Degeneration of Orx neurons
 Tribbles homolog 2 (Trib2) antigen possible culprit
Orexin and Narcolepsy
Sakurai 2011
 Narcolepsy is two things:
1. Difficulty staying awake
 Abrupt transitions from wakefulness to NREM
 Mouse studies suggest deficiencies in Orx-2
signalling
 Orx-2: Anxiolytic/Antidepressive
2. Intrusion of REM sleep
 Cataplexy, hallucinations, sleep paralysis
 Treated with SSRIs – abnormal monoaminergic
neurotransmission
 Lack of signaling in both receptors associated
with cataplexy
 Orx2 in Limbic System = emotional valence
 Orx1 in PPT = ↓ REM atonia (sleep paralysis)
Orexin and sleep function
Sakurai 2011
 Preoptic area – VLPO
 Region of Hypothalamus
 NREM initiation and maintenance
 Inhibitory GABA to:
 LC
 TMN
 DR
 Orx neurons innervated by GABA cells in
VLPO
 Turns off Orx during sleep
 Disruption of GABAB receptor results in
fragmented sleep-wake cycles
 Orx and inhibitory feedback = feedback loop
Orexin and sleep function
Sakurai 2011
 Feedback loop
important for Orx
regulation
 GABA
 Other hormones,
peptides
 Metabolic cues
Orexin and sleep function
Sakurai 2011
 Orx-2 KO and prepro-Orx KO mice = both have sleep attacks (NREM)
 Orx-2 KO have less disrupted wakefulness
 Only mildly affected by cataplexy, direct REM transitions
Suzuki 2013
 Sleep is regulated by homeostatic mechanisms
 Limited by circadian and environmental arousal
 What is the link between them?
?
 Sleep need is correlated with delta wave power (NREM)
 Correlated with prior wakefulness time
 Conversely, level of arousal ↑ latency to sleep
 Latency varies inversely with delta power
 How does arousal level relate to sleep need?
Suzuki 2013
Methods
 C57 BL/6 Mice
 6 hours sleep deprivation – two methods:
 Gentle handling
 Cage changing
 Evokes emotional, behavioral, physical arousal
 “Frequent Flyer Program” – Gitmo
I would prefer this one
 MSLT – Multiple Sleep Latency Test
 Had their cages jiggled ((every 30 min – 5min) x6)
 Assessed:
 Sleep latency
 Sleep need (via delta power – NREM)
 Biochemical correlates w/arousal, sleep need
Suzuki 2013
Methods
Suzuki 2013
 Most results seen in first 3
MSLT trials
 GH saw decreased latency to
sleep, increased sleep time
comparatively
 CC increased latency to sleep,
decreased time
 Similar to control except in Delta
power (sleep need), gene
expression
 ↑mRNA transcripts known from
brains of SD rodents
Sleep latency ≠ duration of wakefulness
Suzuki 2013
 Biochemical markers
 Dynamin 1
 Formation of new vesicles
 Shows directed cellular
growth
 Reflect biochemical changes
associated with arousal and
sleep need
Suzuki 2013
 Biochemical markers
 NDRG2
 Astrocyte protein
 Role in neurite outgrowth
 Shows directed cellular
growth
 Reflect biochemical changes
associated with arousal and
sleep need
Suzuki 2013
Suzuki 2013
Take-homes
 Sleep homeostatic response not influenced by means of SD
 Not affected by level of arousal
 Rather, means of SD affected subsequent arousal
 CC reduced latency to sleep to control levels
 Delta power similar to GH SD
 DNM1-mediated regulation of presynaptic endocytosis and the level of
arousal
 NDRG2 increase = involvement of glial cells
 Arousal > Sleep need
 Mice with histamine deficiency ≠ prolonged sleep latency
 Histamine subserves Orx-induced arousal
 How does Orx support arousal?
Matsuki 2015
 What is the mechanism that inhibits sleep during arousal?
 How does prolonged waking influence Orx neurons?
 GABA
 Primary (and most abundant) inhibitory neurotransmitter
 Sleep-active GABA Neurons from around the hypothalamus
innervate Orx neurons
 GABA ↑ in LH during NREM sleep
 GABAB receptor
 Knockout led to severe
fragmentation of
vigilance states
 GABAA receptor
 Poorly characterized
 SD upregulates on:
 Basal forebrain cholinergic
neurons
 Perifornical Hypothalamus
 GABAA receptor likely
plays a role in
homeostatic regulation of
sleep
 How does it act on Orx?
Matsuki 2015
Whole cell
patch clamp
Matsuki 2015
Methods
 Orexin-enhanced GFP transgenic mice
 SD with GH for 6 hours
 Tested for immunohistochemistry
 Stained with antibodies for GABAA receptor subunits
 Luminescence observed, intensity measured
 Electrophysiology
 Cells emerged in bath
 Muscimol (GABAA agonist) squirted in
 Response measured
Matsuki
2015
 GABAA R alpha 1
 Enhanced in Orx
neurons following
SD
 HAP1/NLGN2
 Molecules in Orx
neurons
 GABA trafficking and
inhibitory synapse
specialization
Matsuki
2015
 GABAA R agonist
muscimol
 Increased
amplitude in Orx
neurons following
SD
 Orx neurons enhanced
sensitivity to GABA
post-SD
Matsuki
2015

SD increased
freq.,
amplitude of
IPSC in Orx
neurons
Take Home Messages
 Arousal > Sleep need???
 Thanks Orexin…
 Prolonged wakefulness affects Orx through changes in
GABAA receptors
 GABAA receptor subunits upregulated following SD
 Enhanced inhibitory affect
 Post Synaptic Current Changes in Orx neurons
 Altered synapse number, sensitivity to GABA
Take Home Messages
 Change in synaptic plasticity following prolonged
wakefulness
 Previous Studies: Orx neurons maintain activity via
excitatory post-synaptic plasticity changes
 Adapt to homeostatic sleep pressure
 SD alters sensitivity of Orx neurons to inhibitory input
 Modulated by GABA release
 Prepares for sleep onset Under GH SD
Would Cage
Change result
in different
IPSP?