SLEEP L2: Ma et al. 2019. Curr Biol - galanin neurons

Question 1

summary

Answer 1

• this study explores the circuitry underlying sleep homeostasis, the process by which sleep pressure increases with wake, leading to longer/deeper sleep after deprivation
• focus on the PO, known for its role in sleep regulation
• within PO, various neuronal subtypes induce body cooling and NREM sleep
• here they show that mice lesioned with PO galanin neurons show reduced sleep homeostasis, indicating their involvement in regulating recovery sleep after deprivation
• also, DEX fails to induce high-power delta oscillations or sustained hypothermia in these mice
  –> suggests a common mechanism underlying sleep homeostasis and DEX-induced sedation, both reliant on PO galanin neurons

Question 2

ablating galanin neurons in PO hypothalamus

Answer 2

• selectively ablate PO galanin neurons (POGal) implicated in sleep regulation

-achieved by bilaterally injecting a mixture of AAVs expressing cre-activatable caspase-3 (AAV-FLEX-CASP3) and GFP transgenes into the LPO area of Gal-cre mice, resulting in generation of LPO-ΔGal mice
-control mice (LPO-Gal-GFP) were injected only with AAV-FLEX-GFP

• immunohistochemistry with GFP Abs confirmed that after 5 weeks, the AAV-FLEX-CASP3 injections eliminated ~98% of the LPOGal cells, while the number of parvalbumin-expressing cells in LPO remained unaffected, indicating selective deletion of galanin neurons by caspase

Question 3

PO galanin neurons chronically lower body temp

Answer 3

• five weeks after ablation of LPO gal neurons, significant increase in core body temp compared to control mice (elevated to 37ºC compared to 35.5ºC) –> changes in body temp was not attributed to changes in locomotor activity, as they did not exhibit altered activity levels in the open-field test
• higher av body temp in both day and night indicating that LPOGal neurons chronically regulate body temp
• suggests that LPOGal neurons typically lower body temp coinciding with increased sleep pressure –> could explain why LPO-ΔGal mice are more awake compared to control mice at the start of the active phase
• despite the association between NREM sleep and body cooling, its commonly believed that during fever, people tend to sleep more –> circuitry responsible for inducing sleepiness during fever likely involves the PO
• interestingly, LPO-ΔGal mice, which have higher temp, exhibit increased NREM sleep compared to control mice, contrary to the expectation that total sleep time would decrease due to the loss of sleep-promoting gal neurons
• conclude that NREM sleep during hyperthermia does not necessarily require LPO-Gal neurons –> studies in mice and rats have shown mixed results

Question 4

PO galanin neurons are needed for consolidated NREM sleep

Answer 4

• to investigate role of PO Gal neurons in sleep regulation, investigated sleep-wake cycle in LPO-ΔGal mice
• ablation of LPO Gal neurons modestly reduced total wake time and increases total NREM sleep; no changes in REM
• highly-fragmented sleep in LPO-ΔGal mice; increased numbers of wake and NREM episodes w/shortened duration
  -despite sleep fragmentation, LPO gal neurons were found to be dispensable for NREM
  -chemogenetic activation of LPOGal neurons induced NREM sleep
• conversely, in absence of gal neurons, other sleep-promoting neurons likely contribute to sleep initiation, but gal neurons are cucial for maintaining consolidated sleep –> finding is consistent w/human post-mortem studies where number of gal neurons in PO inversely correlate w/sleep-wake fragmentation

Question 5

PO galanin neurons contribute to sleep homeostasis

Answer 5

• sleep homeostasis= longer/deeper sleep characterized by increased delta oscillations, following sleep deprivation
• regulated partly within the PO at the circuit level
• to investigate role of LPOGal neurons in sleep homeostasis, control and deleted mice underwent 5-hr sleep deprivation
• control mice = reduction in wakefulness, and increase in total sleep; recovered ~80% of sleep lost after 19hrs
• LPO-ΔGal mice = no change in wake or total sleep time after deprivation; only recovered ~22%, indicating significantly reduced sleep recovery rate compared to controls
• after 5hr sleep deprivation, delta power of baseline NREM sleep was similar between non-deprived LPO-Gal-GFP and LPO-ΔGal mice
• LPO-Gal-GFP mice had stronger increase in NREM delta power after sleep deprivation, compared to LPO-ΔGal
• suggests that while sleep homeostasis can be regulated globally via the hypothalamus, LPO-ΔGal mice may have a deficiency in entering deep consolidated sleep with high delta power, possibly due to elevated arousal-promoting drive

Question 6

a2- adrenergic agonist-induced sedation and hypothermia require PO gal neurons

Answer 6

• control mice injected w/DEX induced stronger reduction in core body temp from 36-25ºC over 2hrs post-injection, w/hyppthermia persisting beyond 4 hrs
• LPO-ΔGal mice, initial reduction in body temp after DEX lasted only for the first hour and did not reach same low as in controls; body temp returned back to normal levels over the next hour –> suggested that DEX still triggered body cooling, but the extend and duration of hypothermia was reduced compared to control mice, possibly due to differences in the a2A receptor expression on peripheral blood vessels
• after DEX, control mice had significant increase in delta power within 20 mins compared to baseline NREM sleep
  -after DEX, LPO-ΔGal mice had delta power levels similar to baseline NREM sleep –> suggest that ability of DEX to induce an NREM-like sleep state is compromised in absence of LPOGal neurons
• after 3.5hrs post-DEX, there was a rebound in delta power when baseline NREM sleep resumed; rebound was absent in LPO-ΔGal mice, indicating a link between the mechanism of DEX-induced sedation and the sleep homeostasis machinery
• despite common belief that DEX induces sedation by inhibiting noradrenaline release from neurons in LC, evidence suggests otherwise; VLPO lesions in rats blunt the sedative effects of DEX, it induces cfos expression in PO, and can induce sedation even when noradrenlaine release from LC is genetically removed
• DEX is expected to directly excite PO neurons by influencing hyperpolarization-activated cyclic nuclepotide-gated cation channels

Question 7

subtypes of galaning neurons

Answer 7

• single cell profiling and multiplex in situ labelling have revealed multiple subtypes of galanin-expressing neurons dispersed in mouse PO
• most of these are GABAergic, several are glutamatergic, and one subtype expresses tyrosine hydroxylase and the vesicular monoamine transporter
• complexity suggests that single PO galanin neuron type, sensitive to sleep homeostasis factors and having a2A receptors is unliekly to solely induce and maintain NREM sleep and body cooling