A Cluster Of Cold-Sensitive DMH^c-Fos+ Neurons Orchestrate The Arousal And Defensive Function

When homothermal animals were exposed to cold environment,cold defense is the vital survival instinct.Cold defense includes rapid perception of cold environment,and then maintenance of core body temperature through autonomic thermoregulation,viligance and defensive behaviors(behavioral thermoregulation).There are serial studies on cold defense,however,little is known about above integarated cold defense process and their neural circuits.Therefore,to expore the integrated cold defense center,which could rapid react to cold exposure and regulate thermoregulation,consciousness and behavior,will not only deepen the understanding of the central mechanism of thermoregulation,but also reveal the interaction of body temperature,consciousness and defensive behavior from the evolutionary perspective.Our previous research verified that the dorsal medial hypothalamus(DMH)participated in the non-rapid eye movement(NREM)sleep and wake transition,as well in general anesthesia recovery.Furthermore,most of cold-sensitive neurons regulated temperature through the DMH,while there are complicated cluster of DMH neurons could be activated by cold exposure,indicating the DMH plays a significant role in cold defense.The DMH neurons were also engaged in defensive behaviors in social and environmental stresses.Therefore,in present study,we applied Fos-TRAP technique,optogenetic and chemogenetic combined with multi-view 3D animal motion-capture system,to explore the cardinal center of integrated cold defense and its cell specificity and functional projection.Part ⅠIdentification and validation of the cold-sensitive neurons in the dorsal medial hypothalamus[Objective]This part of study aims to identify the brain regions activated by cold exposure by 2hour exposure to 4℃ enviornment,followed by Fos-TRAP labelling of the DMH coldactivated neurons for further study.We will further investigate the response of the DMH fos trapped neurons to environmental temperature changes,and clarify their roles in thermoregulation,to screen and preliminary validat the existence of DMH cold-sensitive neurons.The findings of this part will provide the foundation for further study in consciousness and defensive behavior.[Methods]1.Using 8-week-old male C57 mice,the cold exposure model was established by exposing the mice to 4℃ environment for 2 hours.Immediately after cold exposure,brain tissues were collected for immunofluorescence staining,and the c-Fos distribution was observed in the whole brain.2.We microinj ected AAV-DIO-GCaMP6s virus into the DMH of Fos-2A-iCreERT2 mice.Using the cold exposure model,calcium imaging and fos-TRAP strategy,the DMH cold-activated neurons were selectively labeled.Fiber photometry was applied to record the alteration of calcium signals throughout the temperature transition.3.We microinj ected AAV-DIO-hM3D(Gq)-mCherry,AAV-DIO-hM4D(Gi)-mCherry or AAV-DIO-mCherry virus in the DMH of Fos-2A-iCreERT2 mice.After 3 weeks of viral expression,the 2 h cold exposure activated neurons in the DMH were trapped for seven days.Then the wireless sleep recording DSI was implantated.After another 5-7 days recovery,including 3 days habituation,clozapine-N-oxide(CNO,3 mg/kg,)was injected intraperitoneallyn at ZTO(7:00)and ZT12(19:00)respectively,and the mice body temperature was recorded for 24 hours.[Results]1.During cold exposure,the peripheral body temperature of mice decreased rapidly within 15 minutes,followed by a slow and continuous decline.The total decreased temperature in the cold exposure group was statistical greater than those in the control group(-7.6053±0.099℃ vs-0.1507±0.091℃,P<0.001),indicating that 2-hour cold exposure could significantly decrease the peripheral body temperature of mice to establish a cold exposure model.2.The mapping results shown that cold exposure significantly activated the preoptic area(MnPO,LPO,VLPO),the raphes pallidus nucleus,and the DMH.Among them,the cFos+neurons in the DMH increased from 52.8±6.55 at room temperature to 150.4±8.41 after cold exposure(P<0.001),manifesting as the most significant alteration.3.Furthermore,we found that the z-Score of cold-sensitive DMHc-Fos+ neurons calcium signal immediately increased when the mice were transfered from 23℃ to 4℃environment(-0.144±0.4069 vs 3.649±0.697,P=0.0008,n=6),and then gradually returned to baseline level after 180 seconds.However,there was no obvious change in DMHc-Fos+ neurons during the transition from 23℃ to 45℃ environment(-0.729 ±0.691 vs-0.67715±0.679,P=0.8816,n=7).The above results indicate the DMHc-Fos+neurons can be rapidly activated by cold exposure rather than warm environment,meaning that they are cold-sensitive neurons.4.Chemogenetic activation of the DMHc-Fos+ neurons can specifically regulate body temperature time dependently:CNO application at ZT0 significantly elevated core body temperature(CBT)and lasted for about 10 hours(P<0.001),while administration at ZT12 did not show statistically significant effects.[Conclusion]In this study,we preliminarily identify that DMHc-Fos+ neurons are a group of coldsensitive neurons by using cold exposure model and fos-TRAP strategy.Chemogenetic regulation of DMHc-Fos+ neurons have the significant effects on the CBT and movement of mice,laying the foundation for further exploration of the function of cold-sensitive neurons.Part Ⅱ The role of cold-sensitive DMHc-Fos+ neurons in consciousness regulation[Objective]In cold defense,the decline of ambient temperature is often accompanied by the change of consciousness state.However,whether the cold sensitive DMH neurons participate in the regulation of consciousness remains to be resolved.In this part,we also applied Fos-TRAP strategies,combined with optogenetics and chemogenetics,to explore the role of coldsensitive DMHc-Fos+neurons in physiological sleep-wake transition and general anesthesia.Finally,we aimed to reveal the regulatory mechanism of temperature on consciousness state.[Method]1.We microinj ected AAV-DIO-GCaMP6s virus into the DMH of Fos-2A-iCreERT2 mice.After trapping cold-sensitive DMHc-Fos+ neurons as mentioned before,we used fiber photometry to measure the changes in calcium signal during sleep-wake and anesthesiaemergence transitions in mice.2.We microinj ected AAV-DIO-hM3D(Gq)-mCherry,AAV-DIO-hM4D(Gi)-mCherry or AAV-DIO-mCherry virus into the DMH of Fos-2A-iCreERT2 mice.After 3 weeks of viral expression,the cold-sensitive DMHc-Fos+neurons were trapped for further study.Then the wireless sleep recording DSI was implantated.After another 5-7 days recovery,the DMHc-Fos+ neurons were activated or inhibited by CNO intraperitoneal injection,while the sleep recording or anesthetic behaviors were performed.3.We microinjected AAV-DIO-ChR2-mCherry in the DMH of Fos-2A-iCreERT2 mice.After 3 weeks of viral expression and cold-sensitive DMHc-Fos+ neurons trapped,the wireless DSI was implantated.Optical stimulation was administrated randomly in 1315 minutes to investigate the effects of DMHc-Fos+ neurons on sleep-wake transitions.[Result]1.We found that DMHc-Fos+ neurons were wake-and rapid eye movement(REM)-active neurons,as indicated by the increase in z-Score of calcium signals from non-REM sleep(NREM,-0.8217±0.08)to wakefulness(0.8217±0.03,P<0.001),from NREM sleep(－0.3563±0.08)to REM sleep(0.8010±0.03,P<0.001),and the decrease from wakefulness(0.4372±0.192)to NREM sleep(-0.5415±0.03,P<0.001).Meanwhile,there were no significant differences in the transition from REM sleep to wakefulness(P=0.6098).2.Furthermore,chemogenetic activation of DMHc-Fos+ neurons significantly increased wake time(11.0297±0.309 h)companied by great drop in NREM(0.927±0.301 h)or REM(0.0239±0.021 h)sleep.On the other hand,inhibition of DMHc-Fos+ neurons significantly increased NREM sleep time(9.9586±0.552 h),but decreased wake time to 1.820±0.545 h.These results suggest that the cold-sensitive DMHc-Fos+ neurons exhibited strong and stable wake-promoting effect.3.Optical activation of cold-sensitive DMHc-Fos+ neurons could rapidly transfer NREM sleep to wakefulness,which persisted for over 1 minute after the termination of optical stimulation.Though activation of DMHc-Fos+ neurons increased the probability of wakefulness and decreased the probability of NREM sleep,but had no significant effect on REM sleep.4.The z-Score of cold-sensitive DMHc-Fos+ neurons decreased significantly during general anesthesia incuction(Wake:-0.9879±0.5283 v.s.anesthesia:-3.761±1.222,P=0.001),while gradually recovered after isoflurane cessation(anesthesia:0.0854±0.402 v.s.awakening 4.23 0± 1.274,P=0.0023).5.Moreover,chemogenetic activation of the DMHc-Fos+ neurons significantly prolonged the induction time(F(1,7)=26.78,P=0.0024)and shortened the emergence time(F(1,7)=13.83,P=0.0373),while chemogenetic inhibition of the DMHc-Fos+ neurons significantly shortened the induction time(F(1,7)=7.332,P=0.0294),and prolonged emergence time up to 2539±494.1s(F(1,7)=160.5,P<0.0001),indicating that the DMHc-Fos+ neurons significantly promoted arousal even under general anesthesia.[Conclusion]In this part,we confirm that the cold-sensitive DMHc-Fos+ neurons exhibited significant arousal-promoting effect.These neurons not only initiated and sustained arousal during physiological arousal,but also facilitated general anesthesia emergence.Inhibiting the DMHc-Fos+ neurons lead to prolonged sleep and anesthesia emergence time up to nearly seven times.The results suggested that cold-sensitive DMHc-Fos+ neurons may facilitate an alert state during the cold defense,which is beneficial for the instinctive defense behavior from an evolutionary perspective.Part Ⅲ The cold-sensitive DMHc-Fos+ neurons were involved in defensive behavior[Objective]Behavioral thermoregulation,an important component of cold defense,is a survival instinct that helps animal escape from cold exposure.In this part,we applied Fos-TRAP,optogenetics,chemogenetics,and multiple defense behavior tests,including multi-view 3D animal motion-capture system,to explore the role of cold-sensitive DMHc-Fos+ neurons in the instinctive defense behavior.This study aims to provide the further insight for the effects of ambient temperature on animal behaviors,particularly to confirm the involvement of cold-sensitive DMHc-Fos+ neurons in instinctive defense behaviors.[Method]1.We microinjected AAV-DIO-hM3D(Gq)/hM4D(Gi)-mCherry or AAV-DIO-ChR2/NpHR-mCherry viruses into the DMH of Fos-2A-iCreERT2 mice.After 3 weeks of viral expression and cold-sensitive DMHc-Fos+ neurons trapped,we performed serial behavoral tests,including open field test(OFT)and elevated plus maze test combined with chemogenetics,and real-time position preference test(RTPP)with optical stimulation,to reveal the effect of DMHc-Fos+ neurons on behaviors.2.The AAV-DIO-hM3D(Gq)-mCherry,AAV-DIO-hM4D(Gi)-mCherry or AAV-DIOmCherry viruses were microinjected into the DMH of Fos-2A-iCreERT2 mice.After trapping the DMHc-Fos+ neurons,chemogenetics were used to respectively activate or inhibit these cold-sensitive neurons,as well the multi-view 3D animal motion-capture system was involved to preciously analyse the behavioural alterations.3.After microinjection of AAV-DIO-GCaMP6s virus into the DMH in Fos-2A-iCreERT2 mice,the DMHc-Fos+ neurons were labelled by cold exposure as mentioned above.Fiber photometry recording was applied to explore the calcium signals fluctuation of the DMHc-Fos+ neurons during the air puff test,looming test and TMT odor test.[Results]1.The cold-sensitive DMHc-Fos+ neurons statistically enhanced locomotion.In detail,chemogenetic activation of the DMHc-Fos+neurons increased the average speed and total distance traveled in the open field test.As well,the movement parameters improved in the elevated plus maze test,including increase of time spent in the open arms,the number of entries into the open and closed arms,and the speed of movement,but decrease in the time spent in the closed arms.2.Similarly,optogenetic activation of the cold-sensitive DMHc-Fos+ neurons rapidly and significantly increased the speed and distance traveled in the open field test.In the RTPP test,optical activation increased the time spent in the safe room(white chamber),but reduced the time spent in the stimulation room,while the avoidance time of the mice significantly and constantly increased even after the end of optical stimulation.3.The multi-view 3D animal motion-capture system further revealed that activation of the DMHc-Fos+ neurons induced a significant increase in trotting,running,jumping,and rearing,while inhibition led to a crouched posture and significant reduction in speed and distance traveled.4.Last but not least,the cold-sensitive DMHc-Fos+ neurons could be activated by tactile stimulation,visual stimulation and olfactory stimulation in air puff test,looming test and TMT odor test,respectively.It was worth noting that the DMHc-Fos+ neurons could only response immediatedly to the simulated predator approaching.[Conclusion]In this study,we preliminarily confirm that cold-sensitive DMHc-Fos+ neurons not only participated in the regulation of body temperature and consciousness states,but also played an significant role in the instinctive defense behavior.The DMHc-Fos+ neurons could influence the posture and motor abilities of the mice.Therefore,cold-sensitive DMHc-Fos+neurons may be an integrative central hub for the regulation of instinctive defense behaviors.