L3 - SLEEP: Yu et al. 2022. Science. stress, brain circuits & restorative sleep

Question 1

intro

Answer 1

• activation of hypothalamic-pituitary-adrenal axis (HPA) by acute stress leads to a rapid increase in blood glucocorticoid levels, aiding immediate survival
• however, chronically elevated glucocorticoid levels and memories of stressful experiences can be harmful
• while stress can cause insomnia and raise stress hormone levels, paradoxically, chronic stress can increase REM sleep
• stress (known for its restorative effects) has been proposed as a mechanism for mitigating the negative effects of stress
• although the specific circuit linking stress & sleep is unknown, the VTA in the midbrain is considered a potential link
• specific VTA neurons promote wakefulness, whilst others induce sleep; GABA VTA neurons activated by stressful stimuli may provide a route through which stress induces sleep

Question 2

social defeat stress (SDS) induces sleep

Answer 2

• mice subjected to SDS (consecutive encounters w/ aggressors) show increases levels of corticosterone (a stress hormone)
• this increase was not observed in mice subjected to other stimuli eg. social interaction, physical exercise, exposure to novel object/ environment
• following SDS, mice showed shortened NREMS latency, and increases levels of REMS & NREMS for up to 5 hrs –> not observed in the other conditions or controls, indicating that specifically SDS induces alterations in sleep patterns

Question 3

sleep relieves SDS-induced anxiety and CORT concentrations independently

Answer 3

• sleep relives anxiety induced by SDS, independently of corticosterone (CORT) concentrations
• mice allowed sufficient sleep following SDS showed rapid reduction in anxiety-like behaviours (and raised CORT concentrations returned to baseline within 60 mins) , whilst those subjected to mild sleep deprivation remained anxious (and CORT remained high)
• however, pharmacologically reducing CORT concentrations during sleep deprivation did not alleviate anxiety, suggesting that sleep’s anxiety relieving effects are independent of CORT levels

Question 4

identification of neurons activated by stress

Answer 4

• after subjecting mice to SDS, cfos expression was signficiantly increases in brain regions associated w/ stress responses, incl VTA
• within VTA, cells activated by stress primarily expresses the GABAergic marker Vgat or GABA; fewer cells expressed glutamatergic marker Vglut2 or dopaminergic marker tyrosine hydroxylase (TH)

Question 5

VTAVgat neurons persistently increased activity in response to SDS

Answer 5

• using GCaMP6 fiber photometry, it was osberved that VTAVgat neurons rapidly and strongly responded to SDS, with Ca signal remaining elevated for ~5 hrs
• increased activity correlated w/ prolonged sleep observed after SDS
• in contrast, stimuli such as novel objects, did not elicit a response from VTAVgat neurons

Question 6

subsets of VTAVgat neurons mediate SDS-induced sleep

Answer 6

• subset of VTAVgat neurons (~20%) is excited by SDS
• through cfos-dependent activity tagging coupled w/ expression of DREADD hM3Dq-mCherry, subset was targeted to investigate its role during sleep
• mice experiencing SDS or non-stressful activities (eg. wheel running), had their VTAVgat neurons selectively tagged w/ cre recombinase-dependent vectors
• while only 15% of VTAVgat neurons were captured by activity tagging during SDS, chemogenetic reactivation of these with CNO decreased sleep latencies and increased sleep times
• to assess the necessity of VTAVgat subsets for SDS-induced sleep, chemogenetic inhibition was employed
• SDS-tagged VTAVgat neurons were chemogenetically inhibited using cfos-dep expression of DREADD hM3Di-mCherry
• following SDS, mice became tagged; after given CNO to inhibit tagged neurons, mice were challenged w/ a second bout of SDS –> results showed that SDS-induced sleep diminished after chemogenetically inhibiting tagged VTAVgat neurons

Question 7

circuits linking SDS and VTAVgat-induced sleep

Answer 7

• selectively expresses GCaMP6 in VTAVgat cells and used fiber photometry to measure how VTAVgat terminals in differnet brain regions responded to stress
• only the terminals of VTAVgat cells projecting to the LH showed increase Ca2+ signals after SDS
• to validate previous findings, optogenetics was employed
• mapping of VTAVgat projections showed broad projections, but SDS-tagged VTAVgat cells primarily innervated the LH

Question 8

VTA somatostatin neurons are necessary for SDS-induced sleep

Answer 8

• to identify specific subsets of GABAergic neurons in the VTA responsible for SDS-induced sleep, researchers investigated the molecular identities and activity patterns of SDS-responsive cells
• sc-qPCR revealed that a significant proportion of SDS-activated cells expressed vgat/somatostatin (sst), and some others with vgat/parvalbumin (pv)
• further characterization using reporter mice showed that nearly 40% of VTAsst neurons expressed cfos after SDS, while no induction of cfos was observed in VTApv cells
• giber photometry revealed that both sst and pv populations responded transiently to SDS, but sst neurons showed larger and persistent activation after SDS compared to pv
• VTAsst neurons did not respond to insomnia-inducing stressors such as restraint or cage change, suggesting specifity in their activation by SDS
• calcium photometry revealed that sst neurons were primarily active during spontaneous NREM and REM sleep; while pv neurons were active during wakefulness
• chemogenetic stimulation of VTAsst neurons directly increased sleep
• stress-tagged VTAsst neurons were also primarily active during NREM and REM, and chemogenetic reactivation of these cells promoted both NREM and REM sleep
• lastly, directly confirmed that VTAsst neurons are necessary for SDS-induced sleep: genetic ablation of VTAsst neurons decreased baseline sleep, and when challenged w/SDS, sleep was abolished –> effect was also confirmed through chemogenetic manipulation, showing that inhibition of VTAsst neurons decreased SDS-induced sleep
• in contrast, ablation of VTAPv neurons decreased baseline NREMS, but SDS-induced sleep could still be elicited

Question 9

SDS-induced sleep by VTAsst neurons reduces stress-induced anxiety

Answer 9

• ablation or chemogenetic inhibition of VTAsst neurons or SDS-tagged VTAVgat neruons had no effect on baseline anxiety-like behaviours
• however, mice lacking SDS-induced sleep due to selective lesioning or inhibition of VTAsst or SDS-tagged VTAVgat neurons remained in an anxious state after SDS, similar to the effects of sleep deprivation following SDS
• conversely, when sst neurons were unimpended and mice had sufficient SDS-induced sleep, SDS-induced anxiety like behaviours were reduced to baseline levels

Question 10

activation of VTAsst neurons suppresses corticotrophin-releasing factor (CRF) levels induced by SDS

Answer 10

• VTAsst neurons expressing hM3Dq-mCherry projected numerous axons into the paraventricular nucleus (PVN) (a major site of CRF production)
• after SDS, cells in the PVN were excited, as indicated by strong cfos expression, but stimulation of VTAsst neurons inhibited SDS-activated cells in the PVN
• a genetically encoded CRF sensor was used to monitor CRF release around the PVN
• CRF sensor signals were similar between control and chemogenetic activation of VTAsst, consistent with corticosterone (CORT) concentrations not changing with VTAsst stimulation
• after SDS, there was significant increase in CRF levels
• chemogenetic inhibition of VTAsst furhter increased SDS-induced CRF concentrations, thereby increasing CORT levels after SDS

Question 11

SDS-induced sleep by VTAsst neurons reduces CORT concentrations

Answer 11

• for mice unable to experience SDS-induced sleep due to ablation or inhibition of VTAsst neurons, CORT concentrations remained higher during their home cage sleep after SDS, similar to the effects of sleep deprivation after SDS
• however, when VTAsst neurons were unimpeded, SDS-induced sleep correlated with CORT concentrations returning to baseline
• activation of VTAsst cells during sleep deprivation after SDS partially reduced CORT concentrations, but overall CORT levels still remained elevated, suggesting that sleep after SDS is necessary to effectively reduce CORT concentrations

Question 12

discussion

Answer 12

• study concludes by suggestig that targeting GABA-sst neurons in the VTA could offer a novel approach for treating anxiety disorders, given their role in responding to SDS and inducing restorative sleep