Roles Of Orexin Signaling In The Caudal Pontine Reticular Nucleus And Sublaterodorsal Tegmental Nucleus On The Regulation Of Muscle Tone

Orexinergic nervous system plays a crucial role in the regulation of sleep/wakefulness.Interestingly,its deficiency also causes clinical symptoms characterizing by various forms of motor impairments.Movement is achieved by the coordination of the motor nervous systems.A plethora of studies show that orexinergic fibers project to all of the motor structures,which suggest that orexin was involved in motor control by direct effects on multiple motor structures.However,the precise pathways,mechanisms and the physiological importance,it involves remain unclear.Moreover,the relationship between dyskinesia and orexinergic deficiency still need further recerch.It has been reported that the rescue of orexin signaling in the pontine region of orexin-knockout mice could improve deficiencies caused by orexin signaling loss,including cataplexy that is characterized by sudden loss of muscle tone.This result suggests a particular important role of the pontine region in the regulation of muscle tone.In the pontine region,the caudal pontine reticular nucleus（PnC）and sublaterodorsal tegmental nucleus（SLD）are two important structures that are involved in muscle tone regulation.It is well-known that the glutamatergic neurons in the PnC help maintain muscle tone during wakefulness,and thus contribute the postural control.In contrast,SLD glutamatergic neurons play a role in the muscle tone inhibition during rapid eye movement（REM）sleep.Interestingly,muscle tone abnormalities were observed during both wakefulness and REM sleep in humans or animal models after loss of central orexin signaling.In particular,the maintenance of muscle tone during wakefulness and muscle atonia during REM sleep are reported to be both severely disrupted after loss of central orexin signaling.It has been reported that dense orexinergic projections reached the pontine area,including both the PnC and the SLD.However,the role of orexin signaling in the PnC and SLD on muscle tone regulation and their contributions to the muscle tone abnormalities after orexin deficiency still remain unknown.The present study has focused on the role of orexin-PnC and orexin-SLD pathways in the regulation of muscle tone.We first carefully mapped the morphological characteristics of these two pathways.Next,through series of circuitry-based manipulations on these pathways,we examined their physiological roles in the regulation of muscle tone.The relationship between these pathways and muscle tone abnormalities after loss of orexin signaling were also investigated.The main results are as follows,1.The morphological characteristics and physiological functions of the orexin-PnC pathway.（1）PnC-projecting orexin neurons are sporadically distributed in the lateral hypothalamus（LH）.We first employed retrograde tracing methods to examine the existence of the orexin-PnC pathway.CTB-555 and CTB-488 were separately injected into the PnC and lateral vestibular nucleus（LVN）,respectively.After successful tagging,orexin-A antibody was applied to enable the visualization of LH orexin neurons.The result confirms a direct projection from the LH orexin neurons to the PnC region.The PnC-projecting orexin neurons were sporadically distributed in the LH region and showed similar distribution pattern to the orexin neuron entirety from causal to rostral axis（number/slice from causal to rostral;PnC-projecting orexin neurons:9±3,11.3±2.0,20.8±2.4,18.8±5.3;number of the orexin neuron entirety:59.5±5.5,70.5±14.1,144.0±32.9,144.3±42.5;n=4）.PnC received bilateral projections from the LH orexin neurons,and no differences were detected between the ipsilateral and contralateral projections（ipsilateral:17.7±7.8%;contralateral:12.3±4.3%;n=4,P>0.05）.We further compared the characteristics between the PnC-projecting and LVN-projecting orexin neurons.The PnC-projecting orexin neurons accounted for 14.4±1.0%（n=4）of the total LH orexin neurons,whereas the LVN-projecting orexin neurons accounted for 6.8±0.9%（n=4）.Interestingly,the PnC-projecting orexin neurons and the LVN-projecting orexin neurons were found to be largely nonoverlapping.（overlap:1.7±0.3%,n=4）.（2）Orexin signaling machineries are widely distributed in the PnC region.We next employed immunostaining methods to examine the distribution of orexin terminals and orexin receptors in the PnC region.The results showed that abundant orexin-A⁺terminals are distributed in the PnC and they exhibited varicose like shape,indicating volume transmission of orexin signaling in the PnC.We also double immunostained the orexin receptors with the PnC glutamatergic neurons,and found that the orexin receptors were abundantly expressed on the PnC glutamatergic neurons.The orexin receptor positive PnC neurons had a cell diameter larger than 35μm,indicating that they were reticulospinal neurons that are involved in muscle tone regulation.These observations are also in consistent with our previous electrophysiological results showing that orexin excites PnC reticulospinal neurons via activation of post-synaptic orexin-1 and orexin-2 receptors.（3）Optogenetic activation of orexin terminals in PnC excites PnC neurons.We then combined optogenetic methods and slice physiology to clarify the electrophysiological influences of the orexin-PnC pathway on PnC neurons.Experiments were conducted on mouse offspring（8-14 days）crossed by orexin-Cre and Ai27D mice.This strategy allows selective expression of the excitatory opsinCh R2 on the LH orexin neurons.Immunostaining results showed that Ch R2 was already expressed on the LH orexin neurons by the age of the used mouse offspring.Under the guidance of the florescence microscope,we observed abundant Ch R2-td Tomato positive terminals in the PnC region.Interestingly,optical activation（473 nm,20 Hz,5 ms pulses,5 s）of the PnC orexinergic terminals excites all tested PnC neurons（n=11）,suggesting the excitation effect of the orexin-PnC pathway.2.The physiological influences of the orexin-PnC pathway on muscle tone regulation.（1）Optogenetic activation of the orexin-PnC pathway facilitate muscle tone.To examine the role of the orexin-PnC pathway in muscle tome regulation,we used optogenetics to selectively activate the PnC orexin terminals in animals performing the rata-rod test（4-40 rpm,180 s）.Light stimulation（473 nm,20 Hz,5 ms pulses,5 s）was randomly applied among trials.Compared to the control trials（no light,106.90±14.41 s）,light activation of the orexin-PnC pathway significantly prolonged the time of mice spent in the rata-rod test（light,125.49±7.49 s,n=6,P<0.05）.It suggests that the orexin-PnC pathway facilitate the muscle tone when animals are engaged in motor tasks,and thereby,enhance their motor performances.（2）Mechanisms underlying the facilitation of orexin-PnC pathway in motor control.We next used fiber photometry recordings to probe the physiological activity of the orexin-PnC pathway during rota-rod test.Interestingly,compared to conditions before the beginning of the rota-rod test（ΔF/F,0.54±0.12%,n=5）,the GCa MP signals were dramatically increased(6.52±1.24%,P_{Ctrl vs Peak}<0.01,n=5)at first peak of test,the lasting signals of the test is also higher than beginning of the test(2.86±0.38%,P_{Ctrl vs Lasting}<0.01,n=5).These observations suggest an elevated activity level of the orexin-PnC pathway in motor tasks.（3）Optogenetic inhibition of the orexin-PnC pathway weakened the motor performance.We further performed optogenetic inhibition of the orexin-PnC pathway in the rota-rod test.Optogentic inhibition of this pathway significantly reduced the time of mice spent in the rota-rod test（control:49.95±5.07 s;light:42.58±9.33 s,n=6,P<0.05）.To probe the underlying mechanism,we also employed the bar grasping test.Interestingly,optogenetic inhibition of the orexin-PnC pathway significantly decreased the bar grasping force（control:125.49±7.49 gf;light:106.90±14.41 gf,n=6,P<0.05）,suggesting the motor enhancement effect of the orexin-PnC pathway is mediated by the facilitated muscle tone.3.The relationship between the motor enhancement of the orexin-PnC pathway and cataplexy.（1）Positive emotions enhanced the physiological activity of the orexin-PnC pathway in motor control.Cataplexy is usually triggered by positive emotions.To probe the emotional integration properties of the orexin-PnC pathway,we performed fiber photometry recordings of the PnC orexin terminals under positive emotions induced by chocolate.We found that positive emotions induced a large increase in the GCa MP signals（chocolate pre 500 ms:3.29±1.13%,chocolate pro 500 ms:9.67±2.47%,P=0.022,n=4）.To further clarify the influence of this positive-emotion induced signals on the motor output of the orexin-PnC pathway,we employed an operant conditioning test which allow us to observe the changes of the orexin-PnC pathway before and after reward conditioning.Interestingly,we found that the activity of the orexin-PnC pathway was significantly increased during the balance beam test after the reward light conditioning（no light:100±32.21%,light:124.56±40.19%,n=4,P=0.023）,suggesting that positive emotions could enhance the motor output of the orexin-PnC pathway.（2）Micro-dialysis of orexin peptide into the PnC rescue cataplexy attacks in orexin-knockout mice.To further confirm the role of the orexin-PnC pathway in cataplexy,we performed micro-dialysis experiments in orexin-knockout mice.Orexin was directly infused into the PnC region during the active phase of orexin-knockout mice to observe the effect on cataplexy.Interestingly,we found that micro-dialysis of orexin into the PnC significantly reduced the number of cataplexy attacks in orexin-knockout mice,compared to the ACSF group（orexin:0.23±0.11/h,ACSF:0.87±0.3/h,n=6,P<0.01）.In addition,the duration of cataplexy attacks was also reduced after infusions of orexin into the PnC（orexin:20.1±10.6 s/h,ACSF:86.2±21.2 s/h,n=6,P<0.01）.These findings further suggest the important role of the orexin-PnC pathway in preventing cataplexy.4.The role of orexin-SLD pathway in muscle tone regulation.（1）The inhibition of muscle tone by the orexin-SLD pathway participate to maintain muscle atonia during REM sleep.We first microinjected orexin into the SLD before animals performed the bar grasping test,to observe its effect on muscle tone regulation.Interestingly,we found that microinjection of orexin into the SLD significantly decrease the maximal bar grasping force（control:159.2±10.0 gf,orexin:131.8±13.7 gf,P=0.019;n=9）.Nevertheless,TCS 1102（a dual orexin receptor antagonist）injections had no effects（control:159.2±10.0 gf,TCS:177.3±14.4 gf,P=0.143;n=9）.In addition,orexin or TCS injections did not affect the time of rats spent in bar grasping（control:1.61±0.14 s,orexin:1.49±0.14 s,P=0.179;control:1.61±0.14 s,TCS:1.65±0.14 s,P=0.685,n=9）.To examine the physiological role of the observed effects of the orexin-SLD pathway on muscle tone inhibition,we microinjected orexin and TCS 1102 into the SLD,and EEG/EMG recordings were performed to monitor the brain states.Interestingly,drug injections only affected the amount of REM sleep,whereas wakefulness or NREM sleep was not affected（One-way repeated measure ANOVA:P<0.05for REM sleep;P=0.121 for wakefulness;P=0.555 for NREM sleep）.Compared to the saline injection（11.7±2.6%）,3 and 30μM orexin-A injections dose-dependently increased REM sleep amount to 14.1±3.0%and 21.2±5.0%,respectively(post-hoc LSD comparison test;Psaline vs 3μM orexin_-A=0.175,Psaline vs 30μM orexin_-A=0.0262)（n=6）.Furthermore,TCS 1102（3μM）alone,a dual orexin receptor antagonist,decreased the REM sleep amount to 9.4±2.5%(post-hoc LSD comparison test;P_{saline vs TCS 3μM}<0.01)（n=6）.These data confirm a routinely-required role of SLD orexin signaling in the maintenance of REM sleep amount.（2）Chemogenetic silencing of the orexin-SLD pathway causes severe disruption of muscle atonia during REM sleep.Since muscle tone inhibition is important for muscle atonia during REM sleep.We next asked whether the muscle tone inhibition effect of the orexin-SLD pathway is involved in REM sleep muscle atonia.We used chemogenetics to selectively silencing the orexin-SLD pathway in orexin-Cre mice and EEG/EMG recordings were performed to monitor the brain states.The chemogenetic methods were validated through immunostaining and slice physiology methods.We found that chemogenetic silencing of the orexin-SLD pathway significantly decreased the EEG theta power（vehicle:409.3±52.3μV²;CNO:372.9±55.6μV²;n=8;P=0.0484）,and the distribution of the power frequency was not affected.Intriguingly,we found that the muscle atonia of REM sleep was severely disrupted after chemogenetic silencing of the orexin-SLD pathway,as the EMG（NREM/REM）ratio was decreased by this manipulation（vehicle:93.4±1.6%;CNO:99.0±1.5%;n=8;P<0.01）.These effects were not observed in control animals that only expressed m Cherry in the PnC-projecting orexin neurons（vehicle:0.93±0.03%,CNO:0.94±0.02%,n=5,P=0.376）.In summary,the present study has first demonstrated the involvement of the orexin-PnC pathway in the facilitation of muscle tone and motor behavior.Importantly,this pathway exhibited emotion-motor integration properties,in which positive emotions could facilitate the motor output of this pathway.This kind of property finally contribute the pathology of cataplexy.On the other side,we also showed that orexin also targeted on another pontine region,the SLD,to inhibit muscle tone.This behavior effect contributes to the muscle atonia during REM sleep.Lack of SLD orexin signaling may lead to disrupted muscle tone inhibition during REM sleep.In total,these results suggest the pontine area,including the PnC and the SLD,are important downstream targets that mediate the orexin’s role in muscle tone regulation.