Awakening Effect Of Alarm Pheromones And Its Neural Circuits

The pheromones are important chemical molecules and perceived by chemoreceptors for communicating signals among organisms.Alarm pheromones(APs),one kind of pheromones,deliver the signals of danger,injury,distress,or the presence of predators to conspecifics.APs play a key role in the animals(e.g.rats and mice)of lower food chains for survival and phylogeny.Sleep and wakefulness are two different brain functional states.Wakefulness is a functional brain state that allows the performance of several “high brain functions” such as diverse behavioral,cognitive and emotional activities.Sleep is a complex physiological and behavioral state that reversibly loses consciousness and response to environmental stimulations,and generally subdivided into non-rapid eye movement(NREM)and rapid eye movement(REM)sleep on the basis of physiological parameters including electroencephalogram(EEG),electromyogram(EMG)and electrooculogram(EOG).The “sensory gating” of olfactory theoretical knowledge indicates that sense of smell to general odors during sleep in human and animals are diminished.It is well known that mice spend half of their lifetime to sleep,are whether they able to perceive the dangerous information released from conspecifics and predators? We still do not know whether APs arouse mice from sleep to escape these dangers.Objective: The present study is designed to reveal the effects and underlying neural circuits of APs on sleep-wake states during NERM and REM sleep in mice.Methods: Firstly,Free Moving Olfaction-Sleep Recording System(FMOSRS)was used to detect the effects of APs and SBT(2-sec-butyl-4,5-dihydrothiazole),a main component of APs,on sleep-wake states,when they were delivered directly to mouse’s nasal cavity during NREM or REM sleep determined by EEG/EMG and digital camera.Emotional behavioral changes induced by APs and SBT were evaluated with behavior test system.Secondly,axotomy of axons of the Grüeneberg ganglion(GGAxo)or excision of the vomeronasal organ(VNOX)were used to identify the sub-sensory organ of SBT-induced arousal.Meanwhile,olfactory behaviors including buried food test and habituation/dishabituation tasks were carried out to determine the influence of main olfactory ability after GGAxo or VNOX.Finally,an anterograde transsynaptic labeling marker of neurotropic virus and dual-immunofluorescences were employed to reveal the neural pathways involved in APs-induced arousal.The c-Fos immunochemistry was used to verify the existence of APs-induced activated neurons in the arousal pathways.Results:APs awaked mice immediately when it was delivered to mouse’s nasal cavity during NREM sleep.SBT also aroused mice from NREM sleep and was much powerful than APs did.The deliveries of APs released from mice in imminent danger,SBT of APs main component synthesized with chemistry and trimethylthiazoline(TMT)released from fox indiscriminately caused anxiety-like behaviors.SBT awaked mice from REM sleep.The mice treated with GGAxo did not induce any alteration in architecture of sleep-wake cycle and main olfactory function,and were awakened from NREM and REM sleep by SBT delivery.The SBT-induced arousal in GGAxo mice was reduction than that in intact mice,that was manifested by declining the amount of wakefulness post SBT-delivery.VNOX in mice showed fragmental sleep and increased wakefulness,and no change in main olfactory function.SBT waked up VNOX mice from NREM and REM sleep,whereas,the SBT-induced arousal effect in VNOX mice was reduction manifested by declining the amount of wakefulness during the period of SBT-delivery compared to intact mice.After microinjection of herpes simplex virus(HSV),an anterograde transsynaptic marker of neurotropic virus,into right olfactory bulb for 72 h,the HSV labeling neurons were found in the ipsilateral basal forebrain(BF)and bilateral locus coeruleus(LC).The percentage of HSV neurons in the ipsilateral of the bilateral LC was 74.14 ± 2.08%.The majority of HSV neurons in the LC was also immunostained with tyrosine hydroxylase(TH),and showed that dual-labeling of HSV+TH neurons in the ipsilateral and contralateral LC respectively was 75.70% ± 3.20% and 72.04 ± 2.82% of HSV neurons.HSV neurons in the BF were not immunostained with choline acetyltransferase(ChAT).SBT significantly enhanced the number of Fos immunoreactive neurons in the LC compared to air delivery.Conclusion: APs wake up mice from NREM and REM sleep and induce anxiety-like behavior.The mice treated with GGAxo or VNOX partly reduce APs-induced arousal without influencing main olfactory function.The neural pathways involved in APs-aroused mice from sleep are probably through GG and VNO sensing to activate the LC-NA neurons,suggesting that rodents retain their olfactory sensory of chemical warnings in NREM and REM sleep,which is beneficial for them to sense danger,awaken and avoid injury hazards.