SLEEP L2: Zhang et al. 2016. Nature - Experiment Bills Lab

Question 1

Introduction

Answer 1

• sedatives like DEX target specific receptors & ion channels, but understanding how they induce sedation is challenging
• DEX activates inhibitory metabotropic adrenergic a2 receptors –> induces a state similar to NREM sleep (affecting body temp & brain activitty)
• at circuit level, its unclear how a2 agonists induce sedation
• one theory = inhibit noradrenergic neurons in the LC, which promote wakefulness; however, directly inhibiting LC neurons doesnt reliably induce sleep
• DEX still induces LORR in mice lacking noradrenaline synthesis, suggesting complex effects beyond LC inhibition

Question 1

intro - what did the study hypothesize

Answer 1

• sedation induced by a2 agonists might resemble recovery sleep (deep sleep after deprivation)
• PO hypothalamus as a potential site where these effects converge

Question 2

intro - brief overview of methodology

Answer 2

• using siRNA KD, found that while DEX-induced LORR relied on a2A receptors on LC neurons, sedation did not suggesting involvement of diff neuronal populations
  -TetTaggin w/DREADD pharmacogenetics, identified activated neural ensembles in PO during recovery sleep & DEX-sedation
• reactivating these ensembles induced both NREM & reduced body temp –> both sedation & recovery sleep are induced by activating similar neuronal populations in PO

Question 3

sedation and LORR require distinct neuronal populations

Answer 3

• used siRNA to reduce a2A gene expression in LC of mice
• KD abolished DEX ability to silence LC neurons
• high dose DEX induced LORR in control mice but not those w/ a2A KD, though both groups had increased EEG & delta power
• lower dose of DEX –> equal sedation induced by a2 adrenergic agonists
• suggests that sedation & LORR involve distinct neuronal populations
• DEX induced LORR may involve engaging the spinal cord mechanisms producing muscle atonia, akin to REM sleep or cataplexy
• inhibition of LC neurons by high-dose DEX might release interneuron inhibition of motor neurons, leading to LORR

Question 4

TetTagging neurons in the PO hypothalamus - aim

Answer 4

• aimed to identify brain regions involved in sedative response to a2 receptor activation –> widespread activation of neurons in PO (inc. VLPO) following DEX & during natural/recovery sleep; increased cFos expression in lateral & medial PO areas

Question 5

TetTagging neurons in the PO hypothalamus - TetTagging method

Answer 5

• due to size limitations of AAV genome, generated 2 AAV viruses: one containing Pcfos-tTA transgene & other containing tet-operator promoter (PTRE-tight) linked to hM3Dq-mCherry
• before conducting behavioural experiments, TetTag system was repressed w/doxy in diet –> removing it allows neural activity, driving expression of cFos promoter linked tTA, which then activates hM3Dq-mCherry
• preliminary experiments showed that co-injecting AAV-Pcfos-Tta & AAV-PTRE-tight-hm3Dq-mCherry resulted in co-transduction of neurons;
• consequently both were injected bilaterally in LPO or MnPO;
• mice were maintained on doxy diet for 4 weeks to suppress gene expression;
• after removing doxy, subjected to sedative dose of DEX, saline injection, or 4hrs sleep deprivation then recovery
• examined patterns of activity-inducible mCherry expression in LPO & MnPO; brain samples taken before & after sedation/sleep deprivation
• before DEX or sleep deprivation, low levels of mCherry expression were detected in both LPO & MnPO;
• following DEX, mCherry expression increased significantly in LPO & MnPO , & even stronger during recovery sleep
• CNO induced expression of cFos protein in TetTagged gM3Dq-mCherry-positive neurons, indicating excitatory response;
• patch-clamp recordings of visually identified TetTagged neurons in PO slices after DEX sedation, confirmed that CNO induced excitatory responses in all tested neurons, triggering AP firing;
• sc-qPCR revealed that 84% of these TetTagged neurons were GABAergic, while remaining where glutamatergic

Question 6

Recapitulation of recovery sleep and sedation by CNO

Answer 6

• in LPO- & MnPO-TetTag-hM3Dq mice, DEX sedation resulted in sustained NREM sleep ~90mins, compared to mice given saline
• all DEX mice became immobile but maintained their righting reflex (RR); after 4-days on doxy diet, injected w/CNO
• after CNO injection, LPO mice entered state resembling sustained NREM sleep, characterized by minimal movement & powerful delta activity in neocortical EEG ~90mins; this recaptiulated effects of DEX-sedation
• however, MnPO mice showed substantial delta power in EEG but moved similarly to saline or CNO controls –> discrepancy between EEG & behaviour suggests that while LPO contains neurons sufficient for full adrenergic a2 receptor-induced sedation, MnPO does not
• further 4 weeks on doxy, CNO administration to LPO & MnPO mice had no effect on behaviour or EEG = decay of mCherry protein back to basal levels
• after, mice were subjected to sleep deprivation & recovery; during recovery both mice groups had sustained NREM & minimal movement
• 4 days after sleep deprivation, injected w/CNO or saline –> both groups had sustained delta power & behavioural arrest, indicating activation of an ensemble of neurons sufficient to initiate and sustain recovery sleep
• suggests that DEX-sedation & recovery sleep share similar mechanisms & circuitry in LPO area

Question 7

Recapitulation of hypothermia by CNO

Answer 7

• investigated whether neural circuitries in the LPO or MnPO areas were sufficient to induce sedative or recovery sleep-induced hypothermia;
• saline & CNO injectiosn did not affect body temp;
• 2 days after removing doxy, LPO-TetTag-hM3Dq & MnPO mice were injected w/sedative dose of DEX, leading to significant hypothermia;
• 4 days later, CNO injection in LPO mice replicated temperature drop induced by DEX, but little effect on MnPO mice;
• suggests that following DEX, activated neuronal ensembles in LPO but not MnPO are responsible for hypothermia produced by the drug
• another experiment, both groups of mice were sleep deprived for 4 hrs and allowed recovery sleep;
• sleep deprivation raised their body temp to 38ºC which then fell to 36.5ºC during first few hrs of recovery;
• 4 days later, mice were given CNO; in both groups, substantial grop in body temp –> suggests that both LPO & MnPO contribute equally & in parallel to the induction of hypothermia during recovery sleep

Question 8

Role of GABAergic neurons for the rapid onset of sedation

Answer 8

• to assess whether DEX-induced sedation relied on GABAergic neurons in the LPO, researchers deleted the vesicular GABA transporter (Vgat) gene by injecting AAV-Cre construct into LPO of mice homozygous for a loxP-flanked Vgat gene, generating LPO-ΔVgat mice; control mice were injected w/AAV-GFP to create LPO-GFP mice
• month later, upon receiving sedative doses of DEX, LPO-GFP mice had significant increase in delta pwoer in EEG, sustained NREM sleep ~90mins, and cessation of movement;
• contrast, LPO-ΔVgat mice showed only minor shift in EEG activity towards delta frequencies shortly after DEX injection;
• however, 30mins after, percentage of NREM in those mice increase comapred to saline-injected mice & exhibited sedation similar to DEX-injected LPO-GFP mice; –> indicates that GABAergic neurons in LPO are crucial for rapid-onset DEX sedation

Question 9

Control sof the TetTag-DREADD method

Answer 9

• controls to ensure specificity of the TetTag DREADD system
• administering CNO alone to mice w/o AAV injections = no behavioural effects, EEG changes, or alterations in body temp
• AAV-Pcfos-tTA & AAV-PTRE-tight-hM3Dq-mCherry viruses were co-injected into superior colliculi (area unlikely to be involved in DEX sedation or recovery sleep) –> underwent full experimental procedure yet no behavioural or EEG changes were observed = indicating that effects are specific to PO hypothalamus