CELL3105 - Sleep 2

Proof VLPO neurons are involved in sleep

- found in von Ecomo's sleep promoting area, the anterior hypothalamus - ablation of these neurons resulted in 40% less sleep. & decreases nREM sleep (Lu, 2000) - Sherin 1966 c-fos

4 Main sleep regulating pathways

1. Ascending Aminergic Arousal System. 2. Ventrolateral Preoptic Nucleus 3. REM-On neurons 4. Posterior/lateral hypothalamic area

Benefits of zebrafish as a sleep model

1. Concerved Hcrt peptide and receptor 2. All drugs that modulate wakefulness in humans modulate wakefulness in zebrafish 3. Concerved orexin expression pattern 4. All the major sleep/wake neurons conserved- only 10 orexin neurons so a much simpler system 5. They project to all the same wake promoting centres. 6. They are transparent so can do calcium imaging

Evidence of Orexin neurons in mice

1. In situ hybridisation showed they were localised in the posterior portion of the lateral hypothalamus. 2. These neurons were active during wake but inactive during sleep- electrophysiology experiments. 3. Optogenetics on orexin expressing neurons found that when activated mice would wake up. (Adamantis, 2007)

What are the 5 sleep criteria

1. Ultimately unavoidable activity 2. Usually 24hr rhythm 3. Elevated arousal threshold 4. Species specific posture and location 5. Compensatory sleep rebound following deprivation

Benzodiazepines + Zdrugs

Activate GABA receptors leading to inhibition of AAS. bare side effects.

What does caffeine do?

Adenosine drives sleep, caffeine inhibits adenosine receptors in the basal forebrain

What is the fly version of galanin and where is it expressed?

Alstatin in the dorsal fan shaped body suggesting a flip/flop circuit in the fly that regulates sleep/wake.

Where do the orexin neurons in the posterior portion of the lateral hypothalamus innervate?

Ascending Arousal System nuclei: - Locus coeruleus - Tuberomamillary Nucleus - Raphe nucleus - Modest input to the ventrolateral preoptic nucleus.

Cortex projecting ascending aminergic arousal pathway

Brainstem: Dorsal raphe nucleus (5-HT) Locus coeruleus (noradrenaline) Posterior hypothalamus: Tuberomamillary Nucleus (Histamine) ventral Periaqueductal Grey (Dopamine) posterior portion of lateral hypothalamus (orexin and melanin concentrating hormone) Basal forebrain (GABA, Acetylcholine) Project to cortex to maintain waking

Thalamus projecting ascending aminergic arousal pathway

Brainstem: Pendunculopontine nucleus (ACh) Laterodorsal tegmental nucleus (ACh) Project to Thalamus to maintain waking (most active during wakefulness and REM sleep

Stanford university narcoleptic dog experiment- Mignot

Found gene in dogs, measured how far a dog would go before falling asleep.

REM-OFF Neurons, where are they found and what do they do?

Found in the ventrolateral periaqueductal grey and in the dorsal pons. Prevent entry into REM through GABAergic inhibitory connections.

Pitolisant

H3 (histamine receptor agonist) inverse agonist. Binds to histamine reuptake channels and prevents histamine being reuptaken. this also prevents histamine production. Promotes wakefulness- suggested as a potential narcolepsy drug.

Gene involved in dog narcolepsy

HcrtR2 (orexin receptor)

Narcolepsy symptoms

Hypnagogic / hypnapompic hallucinations Increased sleep paralysis Rapid sleep onset Cataplexy Quick transition to REM, long REM sleep Lots of sleep awakenings Back to stage 2

describe the flip-flop orexin model in wake

Hypocretin neurons in the anterior portion of the lateral hypothalamus activate the ascending arousal system neurons promoting wakefulness. These inhibit the VLPO (sleep promoting area), which inhibits the lateral inhibition so now there is both demotion of sleep and promotion of wakefulness.

What did the Gal4/UAS experiment uncover and how?

It allowed researchers to identify sleep/wake circuits. This was done by activating and silencing genes in different cells.

What is the VLPO's connectivity?

It inhibits circuits making up the AAS

Neumann 2010

Orexin neurons fire whilst the fish is awake. This was found using Calcium signalling and observing behaviour. As the fish is transparent you can see the illumination.

Benefit of the flip-flop orexin model marital inhibition

Prevents time spent in dangerous intermediate states between sleep and wake.

Dogs with narcolepsy onset

Puppies enter narcoleptic state when excited.

Heat shock experiment

Put hcrt gene under heat shock promoter. heat shock causes the protein to be expressed more. Found that increased hcrt expression the fish is hyperactive for several days and sleep a lot less. hyperactivity could be due to effects of the actual heat shock.

Baron Constantin von Economo, 1930

Studied patients with a rare form of encephalitis in lethargic patients. AKA sleeping sickness If the anterior hypothalamus was damaged patients would have trouble staying asleep. If the posterior hypothalamus was damaged they would have trouble staying awake. Lead to the hypothesis: Anterior hypothalamus promotes sleep and the posterior hypothalamus promotes wake.

REM-ON neurons, located? Role?

Sublaterodorsal nucleus and the preceruleus region. They are either GABAergic of Glutamatergic. Project to the basal forebrain driving eeg desynchronisation. Activate inhibitory interneurons in the medulla and spinal chord, which inhibit motor neurons producing atonia.

What controls the Sleep on/off switch?

The SCN projects to the subparaventricular zone which sends projections to the dorsomedial hypothalamus, containing both gabaergic and glutamatergic neurons that both innervate sleep and wake areas.

What is the sleep promoting area in the drosophila brain?

The dorsal fan shaped body which is inhibited by DA neurons from the PPL1.

What is the region important for REM sleep generation and how was it found?

The dorsolateral pons. Cat brain was bisected and the REM activity in each half measured.

What happens to zebrafish with mutant hcrt gene?

They have unstable sleep/wake states. Do not demonstrate cataplexy Maybe they dont have REM sleep? Maybe there should be a different definition of sleep for zebrafish?

Describe the flip flop orexin model in sleep

VLPO inhibits the hypocretin neurons in the anterior portion of the lateral hypothalamus so there is less excitation in the AAS. VLPO also inhibits the AAS nuclei, thus decreasing their lateral inhibition, and increasing activity in the VLPO. Thus promoting sleep and demoting wakefulness.

What is the role of the VLPO?

Ventrolateral preoptic nucleus: promotes sleep

What are the concequences of orexin innervation to vPAG and dorsal pons?

Without orexin, positive emotions excite the sublaterodorsal nucleus which inhibits the vPAG and dorsal pons, this destabilises the REM-ON/OFF balance, and initiates REM sleep.

How does the Gal4/UAS system work?

- Flies with Gal4 in the areas of interest - Flies with UAS reporters attached to the gene of interest. - Flies mate, in the offspring the UAS will interact with Gal4 in the cells of interest. - This works to either silence or activate the gene in question.

Sherin 1966

- Found a group of neurons in the VLPO that are specifically activated during sleep. - Applied a c-fos antibody stain to detect neuronal activity during sleep

proof VLPO neurons are inhibitory

- In situ hybridisation demonstrated that they express Galanin, an inhibitory neuropeptide.

What are the similarities between the VLPO and dorsal fan shaped body?

- Isoflurane anaesthesia acts on both - engages sleep activity

What are the wake promoting areas in drosophila and what are their connections?

Dopamine neurons in the PPL1 - Extend dopaminergic projections to the sleep centre. Clock neurons I-LNvs - inhibited by GABAergic neurons.

Suvorexant

antagonizes orexin (hypocretin) receptors stimulates sleep but interestingly doesnt induce narcolepsy

Cause of Narcolepsy

decreased hypocretin (orexin) production in lateral hypothalamus