| Orexins are neurotransmitters produced by a specific group of neurons in the lateral hypothalamus area and perifornical area.Interestingly,orexin fibers are widely distributed throughout the entire central nervous system,such as the cerebral cortex,hypothalamus,limbic system,brainstem,and spinal cord.The same pre-pro-orexin precursor produces two neuropeptides,orexin-A and orexin-B.By activating two kinds of G protein-coupled receptors,orexin-type1 receptor and orexin-type2 receptor,orexins mainly exert excitatory modulation effects on its downstream target region and involve in regulating the region-mediated physiological functions,including the regulation of feeding,thermoregulation,neuroendocrine regulation,sleep-waking function and motivational behaviors.Therefore,different brain circuitries innervated by orexin fibers,the specific modulatory effects of orexin signaling in various downstream target regions as well as the underlying neural mechanisms are essential to study the role of orexin signaling in different functions.Due to dense projections of orexin system on wakefulness-related brain regions,previous studies have paid more attention to their role in wakefulness.Nevertheless,it has also been reported that orexin system participates in the regulation of sleep function.Some studies point that orexin fibers are also detected in the sublaterodorsal tegmental nucleus?SLD?and in the gigantocellular reticular nucleus?Gi?,which are both regarded as necessary and sufficient in REM?rapid eye movement?sleep generation and maintenance,suggesting that orexin system may directly regulate SLD and Gi activities to participate in the regulation of REM sleep.Our previous immunofluorescence experiments proved the distribution of orexin fibers and the expression of orexin-1/2 receptors in the SLD.In addition,in vitro electrophysiological results revealed that there are extensive gap junctions in the SLD.Further researches showed that orexin has a complex modulatory effect on the discharge activity of SLD neuronal network.On one hand,orexin excites individual SLD neuron.With the aid of gap junctions,the orexin-elicited direct and partial excitation is able to spread from orexin receptor positive SLD neurons to affect large-scale of SLD neuronal network.On the other hand,orexin also actively promotes the efficient spreading of the excitation effects in the SLD neuronal network through increasing the conductance of gap junctions.Consequently,orexin affects the discharge activity of the entire SLD neuronal network in an integrated manner,making it more extensive,efficient,and coordinated.However,it is not clear whether the orexin signaling in SLD can exert the above-mentioned modulatory effects in vivo,and how these modulatory effects contribute to brain functional states and REM sleep regulation.Furthermore,we've also observed the distribution of orexin fibers and orexin receptors in Gi and reported that orexin elicited inward currents in Gi neurons through the activation of postsynaptic orexin-1 and orexin-2 receptors.Likewise,the modulations of the above-mentioned excitatory effect on the neuronal firing activities in Gi neuronsare are still unknown.In the present study,the modulatory effects of orexin signaling on the discharge activities in SLD neuronal network and the influences on the brain functional state were firstly studied by using in vivo multi-channel recordings and neuropharmacological techniques.Then,we used a combination of optogenetics and chemogenetics to investigate the effects of SLD orexin signaling on REM sleep and the underlying mechanisms.On the other hand,using whole-cell patch-clamp recordings,the electrophysiological effects and mechanisms of the modulations of orexin on the firings of Gi neurons were observed.The main results are described as follows:1.Orexin signaling exerts synchronized excitation on SLD neuronal network to regulate the brain state?1?Orexin increases SLD neuronal firings and promotes their coincidental spiking to enhance local field potential?LFP?oscillations of hippocampusWe firstly examined the modulatory effects of orexin-A on discharge activities in SLD neuronal network.Orexin-A significantly increased the firing frequency in 67/97?69.1%?recorded SLD neurons in urethane anesthesia?n=67/97 units,P<0.01?.Futhermore,the gap junction activities were reflected in vivo by coincidental spiking within a sharp time window of±1 ms between tested pairs of SLD neurons.We noted that orexin-A?30?M?increased the pairs of units with coincidental spiking?±1 ms?from 8.4%to 13.0%?n=215 pairs?,and produced a 88.0%increase of gap junction activities?n=56 units,P<0.01?.These data suggested that orexin-A not only elicited excitation on individual SLD neurons but also promoted the synchronized spiking to drive synchronized excitation in SLD neuronal network in vivo.Next,to explore the physiological significance of orexin-elicited synchronized excitation in SLD neuronal network,we simultaneously recorded hippocampal local field potential?LFP?activities to reflect the SLD output efficiency and observed changes of brain functional state.In urethane anesthesia,the hippocampal activities were dominated by slow oscillations at?1Hz.We also found that the power of this oscillation?0.3-2.5 Hz?was significantly increased by orexin-A?n=5 rats,P<0.05?.Phase-locking analysis revealed that the SLD spiking activities were still phase-locked to the enhanced hippocampal oscillations with a significantly higher locking-strength after orexin injection?baseline:n=76/97 pairs,orexin:n=84/97pairs,P<0.01?.These results indicated that orexin signaling in SLD could promote its output efficiency by orexin-elicited synchronized excitation,and enhance the activity of the hippocampal network,so as to participate in the regulation of the brain functional state.?2?The increased synchronized spiking induced by orexin in SLD neuronal network is essential for regulating hippocampal LFP oscillationsTo further examine the relationship between the orexin-elicited synchronized excitation and the promoted SLD output efficiency,we tried to use CBX to interfere with the synchronized spiking in the SLD neuronal network.We observed that CBX only partially reduced the coincident spiking probability by 47.7%?n=60 units,P<0.01?in vivo.But it was able to abolish the orexin-elicited elevation of synchronized spiking?n=30 units,P=0.959?,while orexin-elicited increased spiking rate was not affected.In this condition,the orexin-induced increase in phase-locking strength between the SLD spiking activities and hippocampal oscillations?baseline:n=40/72 pairs,orexin-A+CBX:n=55/72 pairs,P=0.488?,and increase in the power of hippocampal oscillations?n=5 rats for each group,P<0.05?were all blunted,demonstrating the necessity of the synchronized spiking induced by orexin in the achievement of SLD output efficiency and in the regulation of brain functional state.2.The behavioral consequences of orexin signaling's effects in SLD neuronal network?1?Activation of SLD orexin signaling induces brain state activation and decreases muscle tone during NREM?non-rapid eye movement?sleep,but does not cause REM sleep transitionIn order to identify the contributions of orexin-enhanced SLD output efficiency in brain state regulation under physiological conditions,we firstly validated that optical stimulation of SLD orexin terminals induced depolarization in all tested SLD neurons using slice physiology?n=6 neurons for each group,P<0.05?.Then,We used optogenetics to activate the SLD orexin terminals and the instant changes in EEG/EMG signals of the brain states were monitored in free-moving animals.During NREM sleep,optical stimulation at different frequencies induced the decrease of total EEG power and EMG amplitude in a frequency-dependent manner(n=6 mice,EEG:P10Hz<0.05,P20Hz<0.01,EMG:P20Hz<0.01).And 20Hz light stimulation caused an immediate?mean latency:2.4±0.3 s?decrease in the total EEG power?n=6 mice,P<0.01?,followed by a late-onset?mean latency:9.4±1.7s?decrease in EMG amplitude?n=6 mice,P<0.01?unless disrupted by wakefulness before the light-off.This phenomenon also happened during physiological NREM to REM sleep transitions.Analysis of the EEG power spectrogram further showed an increased percentage of theta power in the total EEG?n=6 mice,P<0.05?,but consecutive theta oscillations were not induced.These data demonstrated that activation of SLD orexin signaling didn't induce complete REM sleep transitions,but indeed caused the activation of brain state with increased EEG theta component and decreased muscle tone.?2?Manipulating SLD orexin signaling regulates REM sleep episodesWe next examined whether the activated brain states and decreased muscle tone may contribute to REM sleep regulation.Optogenetic activation of SLD orexin terminals was applied during individual REM sleep episodes and the influences on their performances and length were examined.In REM sleep,optical activation of SLD orexin terminals consolidated the EEG theta oscillations,as the theta/delta power ratio increased by 10.5%?n=9 mice,P<0.05?.And the mean duration of REM sleep episodes were significantly prolonged by 12.1%?n=9 mice,P<0.05?.In contrast,optogenetic inhibition of SLD orexin terminals during REM sleep episodes decreased the theta/delta ratio by 8.0%?n=9 mice,P<0.05?,and furthermore,the mean duration of REM sleep episodes were consistently decreased by 18.2%?n=9 mice,P<0.05?.Changes of the EMGREM/NREM ratio were not detected by the optogenetic manipulation.These results showed that the elevated SLD output efficiency induced by orexin signaling is sufficient to prolong mean duration of REM sleep episodes through consolidating brain state activation,and thereby,may contribute to the stabilization of REM sleep.?3?Chemogenetic inhibition of SLD orexin signaling causes instability of REM sleepFinally,we tested the effects of chemogenetic inhibiting SLD orexin signaling on REM sleep.We injected AAV-retro-hSyn-DIO-hM4D?Gi?-mCherry into the bilateral SLD of orexin-Cre mice for the silence of SLD orexin signaling on an hourly time scale.Compared to vehicle injection,CNO caused a 17.0%reduction in total REM sleep amount?n=8 mice,P<0.01?,and the amounts of wakefulness or NREM sleep were not affected.Further analysis showed that the reduction in REM sleep amount after CNO injection was due to a significant decrease in the mean duration of REM sleep episodes?n=8 mice,P<0.05?.Intriguingly,through analyzing the EEG and EMG activities in all REM sleep episodes,an abnormal behavior phenotype that characterized by severe and constant disruption of muscle atonia in the majority of REM sleep episodes was clearly observed after CNO injection.And the physiological muscle tone decrease from NREM to REM sleep(EMGREM/NREM ratio)was abolished by CNO?n=8 mice,P<0.01?.Moreover,the theta/delta power ratio in the EEG activities was also decreased?n=8 mice,P<0.01?.Together,the disruption of these core features in REM sleep behavior after loss of SLD orexin signaling may eventually lead to the instability of individual REM sleep episodes,and thus the reduced REM sleep amount,further suggesting the essential role of SLD orexin signaling in the stabilization of REM sleep.3.The modulatory effects of orexin signaling on neuronal firing activities in the gigantocellular reticular nucleus and the underlying mechnisms?1?Orexin enhances the firing activities in Gi neurons by causing postsynaptic depolarizationFirstly,we studied the regulation of orexin on the firing activities of Gi neurons in the current-clamp mode.Under current-clamp configurations,the recorded Gi neurons held spontaneous firings.We found that bath-application?2-min?of orexin-A?100 nM?significantly increased the firing frequency of these neurons?n=6 neurons,P<0.05?.In addition,TTX?0.1?M?was added into the ACSF to block the spike generations.In this condition,a depolarization was observed after bath-application of orexin-A?n=6 neurons,P<0.01?.These data suggest that orexin enhances the firing activities in Gi neurons,and a postsynaptic depolarization by orexin underlies.?2?Orexin increases the repetitive firing ability and does not affect the input resistance of Gi neuronsNext,we studied whether the excitation effects of orexin-A on Gi neurons is accompanied by the change of the input resistance.The membrane potentials of Gi neurons were held equally?at around-60 mV?by constant current injections before and during the steady state after orexin applications.In this condition,identical hyperpolarized current injections were applied to evoke firing activities in Gi neurons at this equal potential.The input resistance of the neuron were calculated by plotting the changes of the current-voltage relationship obtained in the above tests.We found that orexin-A?100 nM?did not change the input resistance of these neurons.In this circumstance,we further examined whether orexin also affected the firing dynamics evoked by current injections in Gi neurons at equal depolarizing drives.Following the condition set in?2?,we observed that all 9 tested Gi neurons had the ability to fire repetitively in a tonic pattern in response to current injections.Interestingly,an increase in the mean firing frequency of these Gi neurons during the 2-s current injection periods emerged in the presence of orexin-A?100 nM?.Orexin significantly increased the frequency of the evoked repetitive firings when the injected currents are larger than 100 p A?n=9 neurons,P<0.05?.This result suggests that orexin also increases the repetitive firing ability of Gi neurons at equal depolarizing drives.?3?Orexin enhances the excitability of Gi neurons by shortening the inter-spike intervalsFinally,the detailed influences of orexin on the evoked firings of the 9 Gi neurons tested above were further analyzed.We first compared the inter-spike interval distributions of the evoked firings before and after orexin applications.We found that the inter-spike intervals always reached a steady state at the late-half of the current stimulation period.Statistical analysis revealed that orexin-A?100 nM?had no effects on the initial inter-spike interval in Gi neurons.Nevertheless,the inter-spike intervals during the steady state were significantly shortened by orexin?n=9 neurons,P<0.05?,suggesting that orexin increases repetitive firings of the Gi neurons through shortening the inter-spike intervals.We then examined whether this shortened inter-spike interval involved changes of action potentials.The results showed that no differences were found in the action potential waveforms of Gi neurons before and after orexin application.The spike threshold,amplitude and width were all unchanged by orexin.Besides,the amplitude and the time interval of the post-spike AHP also remained unchanged by orexin.These results suggest that the shortened inter-spike intervals by orexin are not caused by changes of action potentials,and reflect facilitated upstroke to the threshold level between individual action potentialsCollectively,the present study found that orexin signaling in SLD can directly excite SLD neurons extensively and promote the synchronized spiking,to drive synchronized excitation in SLD neuronal network.Then,the SLD output efficiency is consequently enhanced to induce brain state activation and decrease muscle tone in vivo.During REM sleep,this mechanism contributes to consolidating EEG theta-band activities and muscle atonia to stabilize REM sleep episodes.The study demonstrates a direct pathway of orexin system in REM sleep regulation and the underlying mechanism.It also provides additional insights to understand the pathogenesis of REM sleep related diseases caused by orexin deficiency.Moreover,the study also found that orexin can directly regulate the firing activities of Gi neurons and promote the repetitive firing ability in Gi neurons by enhancing their excitability.This part of the results revealed the excitatory regulation of orexin on Gi neurons,which provided a foundation for further exploration of the physiological effects and significance of orexin signaling system in Gi and its specific roles in Gi mediated motor behaviors. |
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