The Activities And Functions Of Hypothalamus Orexin-sublaterodorsal Tegmental Nucleus Pathway

Sleep and wakefulness are two repeated and alternated physiological states.Both states are necessary for the survival of human body and involve complex neural mechanisms.In1953,Kleitman discovered the repaid eye movement(REM)sleep by using EEG and eye EMG recordings.The REM sleep is characterized with fast activity and low amplitude of EEG and strong inhibition of skeletal muscles.The physiological symptoms and neural mechanisms of the REM sleep are quite different from the slow wave sleep and wakefulness.Therefore,the REM sleep has been regarded as an independent phase with typical characteristics in the sleep state.Remarkably,as an important and complex center in the brain,hypothalamus is composed of diverse neuron populations and integrates internal and external information to coordinate physiological activities.Orexin neurons are exclusively located in the lateral hypothalamus(LH)and the perifornic area.They receive substantial inputs from many different regions of brain and project widely throughout the brain and spinal cord.Thus,the anatomy of orexin neurons strongly suggests their involvement in numerous neural circuits and physiological functions.Similar structures and distributions of orexin neurons also have been found in mammals and model organisms,indicating that orexin neurons are highly conserved throughout the development of animals and play a crucial role in physiological activities coordination.Orexin system directly excites monoaminergic system to promote wakefulness and arousal.Their deficiencies are related to the narcolepsy,which is a sleep disorder characterized with excessive daytime sleepiness and sudden muscle weakness.These results indicate that orexin neurons are involved in maintaining arousal state in mammals.Soon after these findings,subsequent studies also find that orexin neurons can directly project to the sublaterodorsal tegmental nucleus(SLD).The SLD area is considered both necessary and sufficient for the generation and maintenance of the REM sleep.Our previous research also confirmed that orexin fibers distributed in the SLD and the SLD expressed orexin receptors.Furthermore,both optogenetics and chemogenetics proved that the orexin system can directly regulate the SLD and then participate in consolidating brain state activation and muscle tone inhibition in the REM sleep.Although the preceding findings demonstrated that orexin neurons only promote arousal,the activities of the orexin neurons specifically projecting to SLD during sleep/wakefulness cycle remains elusive.Additionally,the exact functions and neural circuit mechanisms of the SLD-projecting orexin neurons relevant to regulation of sleep/wakefulness are also expected to investigate in details.Thus,we firstly used retrograde tracing,c-Fos screening and fiber photometry to explore the main activities of the SLD-projecting orexin neurons precisely during sleep/wakefulness cycle.We next investigated the function and correlative neural circuit of the particular orexin subpopulations by using optogenetics and virus tracing.The main findings in this study are described as follow:1.The activities of the SLD-projecting orexin neurons(1)LH exists a group of orexin neurons specifically innervate SLDWe firstly investigated whether the number and distribution pattern of the SLD-projecting orexin neurons were unique.Chlera toxin subunit B(CTB)-488 and CTB-555were respectively injected into the SLD and its neighboring locus coeruleus(LC),which labeled 7.1±0.8%and 15.2±2.1%of orexin neurons.Intriguingly,the SLD-projecting orexin neurons(orexin^SLD)were only overlapping 1.9±0.3%with the LC-projecting orexin neurons(orexin^LC)(n=6 rats,n=3902 orexin neurons),indicating that there was indeed a group of orexin neurons that could specially project to SLD.These data suggest different orexin neuron subpopulations could be distinguished in the hypothalamus according to their downstream projection or function.We next investigated whether the distribution patterns of orexin neurons projected SLD and LC were specific in anatomical locations.According to the Allen Brain Atlas of rats,the number of the orexin^SLD neurons,orexin^LC neurons and the orexin neuron entirety(orexin^entirety)were counted in the LH from 6 coronal sections with a distance of 200?m(AP:-2.30 to-3.80 mm).Both of them were sporadically distributed across the lateral hypothalamus.(2)The SLD-projecting orexin neurons show REM sleep related activationWe then assessed the REM sleep relevance of the SLD-projecting orexin neurons.Rats with CTB-488 injection in the SLD were subjected to a 72 h REM sleep deprivation.After the following rebound which was abundant with REM sleep,c-Fos screening was performed.Interestingly,we found that the orexin^entirety neurons exhibited no difference in c-Fos expression between the control and RSD groups(control:7.1±1.3%,n=4 rats;RSD:6.0±0.6%,n=7 rats;P=0.435).Nevertheless,c-Fos expression was significantly increased in the orexin^SLD neurons(control:6.8±2.8%,n=4 rats;RSD:28.2±2.9%,n=7 rats;P=9.221×10^-4).These data reveal that the orexin^SLD neurons exhibited strong activation and increased activity in the presence of enriched REM sleep,implying the relevance between REM sleep and orexin SLD neurons.(3)The Ca²⁺signals of the SLD-projecting orexin neurons are enhanced during REM sleepFurthermore,we used fiber photometry to investigate the activities of orexin neurons accurately during the spontaneous sleep/wakefulness cycle.We injected retrograde virus AAV-retro-Enf1?-DIO-GCa MP6s-WPRE-p A into the SLD,and recorded the Ca⁺fluorescence of the SLD-projecting orexin neurons which were fluctuated during sleep/wakefulness cycle.The Ca²⁺signals were significantly higher during the REM sleep(?F/F:0.46±0.05%)than the NREM sleep(?F/F:0.07±0.02%)and gradually increased during the wakefulness(?F/F:1.27±0.17%).Collectively,this part of results indicates that different from the orexin neurons which were activated only during wakefulness in previous studies,the orexin^SLD could be activated during the whole REM sleep state(n=4 mice,one-way repeated-measures ANOVA;F_(2,9)=36.146;P=0.5×10^-4;post hoc LSD comparison test;P REM vs NREM=0.0236;P REM vs wakefulness=3.27×10^-4;P NREM vs wakefulness=0.16×10^-4).2.Preliminary study about the functions and neural circuit mechanisms of the SLD-projecting orexin neurons.(1)The SLD-projecting orexin neurons regulate the stabilization of REM sleepSubsequently,we performed optogenetic inhibition of orexin terminal of the SLD during REM sleep episode.AAV-CAG-FLEX-Arch T-GFP was bilaterally injected into the LH of orexin-Cre mice followed by implantation of optical fibers bilaterally in the SLD.Optogenetic inhibition consistently decreased the theta oscillation power for 7.4%(n=9 mice,P=0.0325),and the REM sleep episode duration for 18.2%(n=9 mice,P=0.0363).Changes in the EMG_REM/NREM ratio was not detected by this inhibition.In addition,we used ta Casp3 virus to damage the SLD-projecting orexin neurons specifically in the long-term.Compared with control group,an increasing trend in the EMG_REM/NREM ratio was observed in ta Casp3 group during REM sleep(n_control=11 mice,n_{ta Casp3}=8 mice,control:0.93±0.01,ta Casp3:1.04±0.01;two-sided unpaired t-test;P=0.09×10^-4).Intriguingly,after 24 h REM sleep deprivation,disrupted REM sleep during rebound was observed in ta Casp3 group.The ability of maintaining REM sleep episode during rebound was significantly reduced in ta Casp3 group(n_control=10 mice,n_{ta Casp3}=10 mice,wildtype:1.77±0.14;ta Casp3:1.34±0.11;two-sided unpaired t-test;P=0.028)and the number of REM sleep episode was obviously increased(wildtype:0.99±0.07;ta Casp3:1.38±0.17;two-sided unpaired t-test;P=0.047).Together,these data demonstrate that the loss of orexin signaling in the SLD or the SLD-projecting orexin neurons had significant influence on the stabilization of REM sleep.(2)The downstream projections of the SLD-projecting orexin neuronsIn order to know which downstream projections were driven by the SLD-projecting orexin neurons to stabilize REM sleep in the sleep/wakefulness regulation.We injected virus into the SLD of orexin-Cre mice,which made the SLD-projecting orexin neurons specially expres m Cherry protein.Therefore,direct downstream projections of this orexin subpopulation could be analyzed by the m Cherry fibers distribution in global.The results initially show that the SLD-projecting orexin neurons only projected to the SLD and its neighbouring LC,but not to other REM sleep related regions such as PNO,LDT and arousal related regions PVT(n=2 mice).(3)The upstream inputs of the SLD-projecting orexin neuronsTo describe the neural circuit of such a unique subpopulation of the SLD-projecting orexin neurons holistically,we used a modified rabies virus to infect and express in the monosynaptic input cells of orexin^SLD of orexin-Cre mice accroding to the method above.The preliminary brain-wide distributions of inputs revealed that the SLD-projecting orexin neurons may receive more inputs from Zona incerta(ZI)and dorsalmedial hypothalamus(DMH),compared with the global input of the orexin neuron entirety(n=2 mice).In conclusion,our present study demonstrates a subpopulation of orexin neurons specifically projects to SLD.They may receive different amount of inputs from the whole brain and carry different information to active the downstream projections so that could regulate REM sleep during sleep/wakefulness cycle.Besides,the specific inhibition or destruction of the SLD-projecting orexin neurons have shown that instabilization of REM sleep may affect the sleep/wakefulness homeostasis.Therefore,this study reveals the activities,functions and neural circuit mechanisms of the SLD-projecting orexin neurons in regulating REM sleep,and provides a new direction for the orexin system to coordinate and regulate homeostasis of sleep/wakefulness cycle.