SLEEP L2: Harding et al. 2018. Curr Biol - warming stimulus

Question 1

Introduction - background info

Answer 1

• behavioural cues such as nesting or curling up prior to sleep (posture) are recognised precursors to sleep initiation across species
• in mice, warmth has been shown to induce sleep
• human behaviours, hot bath/feet, reduce time to NREM –> suggest that raising skin temp could be a sleep-permissive condition, potentially part of a natural mechanism to induce sleep
• neural circuitry underlying the induction of sleep by external warmth has yet to be fully elucidated
• PO = central role in sleep regulation, temperature control & other homeostatic functions
• while direct heating of PO neurons can induce NREM, its unlikely that this mechanism is responsible for sleep induction following mild external warming
• sustained external warmth also triggers homeostatic response to cool the body –> invovles excitatory glutamatergic pathway from sensory neurons in the skin to cells in spinal cord, relay neurons in lateral parabrachial nucleus, and finally the MPO & MnPO areas of hypothalamus
• PO neurons activate descending pathways to inhibit heat production in brown adipocytes, promote vasodilation, & induce drinking

Question 2

introduction - what they authors did

Answer 2

• used activity tagging to identify MPO-MnPO hypothalamic neurons that respond to warm stimuli
• when reactivated, neurons simultaneously induce sleep & reduction in body temperature, demonstrating the interconnectedness of sleep induction & body cooling

Question 3

mice prefer a warm environment for nesting prior to sleep

Answer 3

• mice were habituated for one week in a cage at 22ºC with bedding material for nesting
• one end of cage was warmed to 32ºC using thermostatically controlled plates; other end remained at 22ºC
• mice were monitored for 2hrs, revealed that they spent 73% +- 4% of their time in warm end of the cage
• preference for warmth was evident in both final position of mice & distribution of bedding material

Question 4

a warm stimulus induces c-fos expression in the PO hyppthalamus

Answer 4

• study aimed to investigate how external warming induced NREM sleep via MPO & MnPO areas in mice
• using c-fos-based activity tagging, researchers selectively drove the expression of hM3Dq-mCherry receptors in these areas during exposure to increased ambient temperature
• transgene system was validated in culture cells, ensuring tight regulation by doxy
• activity-tagging system was then injected to MPO & MnPO areas, to generate Pan-MnPO/MPO-activityTag-hM3Dq mice
• removal of doxy form diet allowed activation of transgene
• during exposure to 32ºC or 22ºC for 2hours, mice showed minimal increase in core body temperature, but significant increase in skin temp, particularly on head & tail
• EEG analysis revealed waking pattern with maxima at theta frequencies under both warm & ambient temperatures –> indicates that external warming leads to c-fos activation in MnPO & MPO, and induces changes in skin temp but not core body temp

Question 5

reactivation of warm-stimulus tagged neurons in MnPO/MPO induces both drop in body temp & sleep

Answer 5

• warm sitmuli robustly induced transgene expression in MnPO & MPO areas compared to ambient temperatures (histological analysis confirmed specificity of transgene induction to MnPO/MPO area)
• after stimuli, mice were placed back on doxy to repress the transgene, allowing for subsequent reactivation using system CNO injection
• reactivated tagged MnPO & MPO neurons with CNO in mic exposed to 32ºC induced sustained hypothermia over several hrs; mice exposed to ambient temp showed no change in core body temp following CNO injection –> suggests that warm stimuli specifically activate MnPO & MPO neurons, leading to hypothermia when reactivated
• mice exposed to warm temperatures exhibited NREM-like sleep shortly after CNO injection; whilst those at ambient temp, showed less sleep induction
• onset of CNO-evoked sleep preceded the maximum temperature decrease
• despite reduction in EEG amplitude & delta F during hypothermia, mice maintained NREM vigilance state
• control experiments confirmed specificity of CNO’s actions, as it did not induce significant NREM sleep in non-AAV injected mice

Question 5

warm stimulus activates nitrergic-glutamatergic & GABAergic neurons in MnPO/MPO area

Answer 5

• neurochemical identity of MnPO/MPO neurons activity tagged with hM3Dq-mCherry protein was investigated –> positive for NOS1, VGLUT2, GAD67
• significant proportion of NOS1 neurons co-stained with VGLUT2
• majority of warm-tagged GABAergic cells were located in the MPO

Question 6

reactivating warm-stmulus tagged nitrergic-glutamatergic neurons in the MnPO/MPO hypothalamys induces both sleep & hypothermia, whereas tagged GABAergic neurons in MPO produce only NREM sleep

Answer 6

• to differentiate the roles of NOS1-expressing and GABAergic neuronal populations in responding to external warmth and inducing hypothermia & NREM sleep, activity tagging was restricted to genetically specified cell types
• NOS1/MnPO/MPO-activityTag-hM3Dq mice = strong & sustained decrease in core body temp & sustained NREM sleep upon reactivation with CNO
• Vgat mice exhibited only small & transient hypothermia but sustained NREM sleep upon CNO

Question 7

discussion - general

Answer 7

• the authors propose that skin warming primarily drives sleep induction & body cooling
• while findings are based on mice, and may not directly apply to humans, they offer insights into the connection between NREM sleep induction & body temperature regulation, consistent with classical data showing lower body & brain temperatures during NREM sleep episodes in humans
• for humans, temperature plays a crucial role in determining sleep duration & timing; higher core body temp during wake and lower during NREM
• prior to sleep onset, distal skin temperature increases, (eg warming of the feet) reduces the latency to NREM sleep

Question 8

discussion - specific pathways

Answer 8

• synaptic pathway for how skin warmth initiates sleep begins with sensory TRPM2 ion channels in the skin stimulated by mild external warming –> initiates an excitatory pathway ascending via the lateral parabrachial nucleus, where glutamatergic neurons convey signals for temperature place preference –> pathway continues to MnPO/MPO hypothalamus where nitrergic/glutamatergic neurons are activated –> excite GABAergic cells in LPO that induce hypothermia & sleep-promoting GABAergic cells in MPO;
• the proposed circuit explains findings that injecting a GABA-A receptor antagonist into the MPO area triggers wakefulness