| Wakefulness is fundamental to somatosensory,motor,cognition and consciousness.Wakefulness disability is closely associated with poor vigilance state,such as somnolence,stupor and coma state.Wakefulness is controlled by a variety of neural groups,which located in the brainstem,hypothalamus and subcortical regions,including noradrenergic neurons in the locus coeruleus?LC?,hypocretin?Hcrt?/orexin neurons in the lateral hypothalamus?LH?and cholinergic neurons in the basal forebrain?BF?so on.Traditionally,the thalamus was viewed as the major part of the dorsal pathway of the wakefulness-promoting system,which enabled thalamic gating and processing arousal signals to the cortex.Thus,thalamus is necessary for cortical arousal and behavior wakefulness.Patients with localized injuries to the paramedian thalamus show disturbance of consciousness ranging from hypersomnolence to even sleep-like coma when injuries are bilateral,indicating that the paramedian thalamus is a critical node for the controlling of wakefulness.The paramedian thalamus consists of a large number of nuclei with distinct input-output connections and participates in various brain functions.However,the specific nucleus and circuitry controlling wakefulness have not yet been identified.The paraventricular thalamus?PVT?resides in the dorsal part of the midline thalamus and has been considered as part of the non-specific thalamus.The PVT receives diverse inputs from the brainstem and lateral hypothalamus with a particularly strong innervation from Hcrt neurons.In turn,the PVT sends projections to the nucleus accumbens?NAc?,amygdala as well as wide regions of the cerebral cortex.The PVT has been implicated in the regulation of arousal state-dependent behaviors,especially drug addiction and reward,feeding,adaption to chronic stress,mood and emotion.In particular,the activities of PVT neurons are endowed with prominent wakefulness-related diurnal oscillation.Immediate early gene c-fos expression is higher in the subjective active period?dark phase?than in the inactive period?light phase?in rodents.Moreover,action potential firing and intrinsic electrophysiological properties of PVT neurons also display diurnal changes.Thus,PVT could serve as a station of integration of thalamic and hypothalamic wakefulness circuitry and play a critical role in wakefulness regulation.However,direct evidence linking the PVT to wakefulness regulation is still lacking.Here,we firstly evaluated,by fiber photometry,multichannel recordings and electroencephalogram-electromyogram?EEG-EMG?recordings,the in vivo activities of PVT neurons during spontaneous sleep-wake cycles.We next investigated the causal role of the PVT in wakefulness regulation,using a combination of chemogenetic and optogentic methods together with EEG-EMG recordings.We further examined the circuit mechanisms mediating and regulating the wakefulness controlling effects of the PVT.The main results in the present study are listed as follows:1.The activities of PVT neurons are tightly coupled with wakefulnessConsidering the PVT's unique input-output connections and activity patterns,we speculate that the PVT is a key node in the paramedian thalamus controlling wakefulness.We first began by visualizing an unbiased map of c-fos expression in the paramedian thalamus after a period of sleep?zeitgeber time?ZT?6;12:00?,wakefulness?ZT 18;24:00?or extended wakefulness?ZT 0-ZT 6?in mice.We observed an especially higher level of c-fos expression in the PVT at ZT 18 and after extended wakefulness than at ZT 6?one way ANOVA,all P<0.001,n=5?,indicating that PVT neurons were more active during wakefulness state.We next investigated the in vivo dynamics of PVT neurons during the spontaneous sleep-wake cycle by fiber photometry.We injected adeno-associated virus?AAV?expressing the genetically encoded Ca2+sensor GCaMP6f under the control of the CaMKII?promoter and then recorded the population Ca2+signals 4 weeks later.The Ca2+signals of PVT glutamatergic neurons fluctuated during sleep-wake cycles.The population Ca2+activity was significantly higher during wakefulness than during non-rapid eye movement?NREM?sleep and rapid eye movement?REM?sleep(one way RM ANOVA,?F/F?fluorescence intensity?:P Wake vs NREM<0.001,P Wake vs REM=0.042;transient rate:P Wake vs NREM<0.001,P Wake vs REM=0.002;n=7).In addition,the population Ca2+activity gradually decreased when mice fell into sleep whereas began to increase ahead signs of behavioral arousal(paired t test,P Wake?NREM<0.001,P NREM?Wake=0.001,P REM?Wake=0.014,n=7).To directly monitor the spiking activity of individual PVT neurons,we performed in vivo multichannel electrophysiological recordings.Multichannel electrode array was advanced into the PVT and 22 well-isolated neurons were collected.The vast majority?20/22?of recorded PVT neurons exhibited a higher firing rate during wakefulness than during sleep(one-way RM ANOVA,P Wake vs NREM<0.001,P Wake vs REM<0.001,n=20).The dynamics of PVT firing was further analyzed during state transitions.The PVT firing rate gradually decreased before NREM sleep onset(wilcoxon signed rank test,P Wake?NREM<0.001,n=20).However,the firing of PVT dramatically increased during transitions from sleep to wakefulness(paired t test,P NREM?Wake<0.001,P REM?Wake=0.002,n=20).At the onset of behavioral arousal from NREM sleep,the mean firing rate reached 7.1 Hz.In a considerable fraction of neurons?25%?,the firing rate exceeded 10 Hz.The increase of PVT neuronal firing occurred briefly before both cortical activation?1.0±0.3 s?and behavioral arousal?1.4±0.3 s?.2.Chemogenetic inhibition or DTA genetic ablation of the PVT decreased wakefulnessAfter monitoring the activities of PVT neurons at single-cell and population level,we then determined the necessity of PVT activity for wakefulness by inhibiting PVT glutamatergic neurons using chemogenetic method.We injected AAV encoding engineered Gi-coupled hM4D receptor?AAV-CaMKII?-hM4D-mCherry?into the PVT.Functional expression of hM4D was confirmed using whole-cell patch clamp recordings from acute brain slices.The hM4D receptor ligand,clozapine-N-oxide?CNO,5?M?,induced membrane hyperpolarization?unpaired t test,P=0.002,n=5?and a reduction of firing rate?unpaired t test,P=0.001,n=5?of hM4D-expressing PVT neurons.We next tested the effects of chemogenetic inhibition PVT glutamatergic neurons on sleep-wake states.Intraperitoneal injection of CNO at the beginning of the dark phase?ZT 12?induced a significant reduction of wakefulness relative to the mCherry and saline controls(two-way RM ANOVA,Wake:P<0.001;NREM:P<0.001;REM:P hM4D?Saline vs CNO?<0.001,P CNO?mCherry vs hM4D?=0.008;nmCherry=8,nhM4D=13).CNO injection resulted in fragmentation of wakefulness state,as reflected by the shortened wakefulness duration and increased number of wakefulness episodes(two-way RM ANOVA,length of the longest episode:P<0.001;episode duration:P hM4D?Saline vs CNO?<0.001;number of episode:P hM4D?Saline vs CNO?<0.001,P CNO?mCherry vs hM4D?=0.005;nmCherry=8,nhM4D=13),which was primarily due to the increased number of micro-arousals(paired t test,P ZT 13=0.002,P ZT 14<0.001,n=13).In addition,CNO injection decreased latency to sleep(two-way RM ANOVA,NREM:P hM4D?Saline vs CNO?<0.001,P CNO?mCherry vs hM4D?=0.004;REM:P hM4D?Saline vs CNO?=0.009;nmCherry=8,nhM4D=13)and increased the sleep episode(two-way RM ANOVA,P<0.001,nmCherry=8,nhM4D=13),but had no obvious effects on sleep duration.Moreover,spectral analysis of EEG showed that CNO injection increased the high delta power?2-4 Hz?of wakefulness at hour 2 after CNO injection?paired t test,P=0.048,n=10?.However,CNO injection at the beginning of light phase?ZT 0?has no obvious effects on sleep-wake states.Besides reversibly inhibition of the PVT using chemogenetic method,we also explored the role of the PVT in controlling wakefulness by ablating PVT neurons.Genetic ablation of PVT glutamatergic neurons was achieved by injecting a mixture of AAV encoding diphtheria toxin A?DTA??AAV-EF1?-DIO-DTA?and AAV-CaMKIIa-Cre-GFP.After 4 weeks injection,mice with DTA lesion showed a continuous decrease of wakefulness,particularly during the dark phase,but without obvious changes during the light phase(unpaired t test,Wake:P dark=0.014,P 24 h=0.03;NREM:P dark=0.006,P 24 h=0.024;nControl=7,n DTA lesion=9).In addition,DTA lesion lead to fragmentation of wakefulness(mann-whitney rank sum test,number of episode:P=0.02;episode duration:P=0.011;micro-arousal:P=0.015;n Control=7,nDTA lesion=9)and increased NREM sleep episodes(unpaired t test,P=0.002,n Control=7,n DTA lesion=9).DTA lesion also significantly altered the EEG power spectrum of wakefulness during the dark phase.This lesion caused an increase in the high delta power?2-4 Hz?and low theta power?4-7 Hz?whereas decreased alpha power?10-20 Hz?(unpaired t test,P high delta=0.021,P low theta=0.004,P alpha<0.001,n=6).Collectively,these part of results indicated that the PVT was necessary for wakefulness control.3.Optogentic activation of the PVT induced wakefulness from both sleep and general anesthesiaWe then used optogenetics to examine the causal role of the PVT in wakefulness control.AAV expressing channelrhodopsin 2?AAV-CaMKII?-ChR2-mCherry?was injected into the PVT.473 nm laser pulses at 1 to 20 Hz reliably evoked action potential with high fidelity.To test whether optogenetic activation of the PVT induces sleep to wakefulness transitions,we applied optical stimulation lasting 20 s after the onset of stable sleep during the light phase.Optical stimulation of PVT glutamatergic neurons during NREM sleep reliably induced transitions to wakefulness in a frequency-dependent manner in ChR2 but not in mCherry mice(unpaired t test,P 1 Hz=0.005,P 5 Hz<0.001,P 7 Hz<0.001,P 10 Hz<0.001,P 20 Hz<0.001,n=5 for 7 Hz group,n=10 for other groups).Optical stimulation also induced REM sleep to wakefulness transitions(unpaired t test,P 5 Hz<0.002,P 10 Hz<0.001,P 20 Hz<0.001,n=10).Moreover,optical stimulation significantly increased the probability of wakefulness in ChR2 mice,along with a complementary decrease of sleep probability?unpaired t test,NREM:P<0.001;REM:P<0.001;n=10?.To test the capacity of PVT neurons in maintaining wakefulness,we delivered optical stimulation at 10 Hz for 10 s/min lasting 1 hour during the light phase.Such prolonged optical stimulation resulted an overt increase in wakefulness?unpaired t test,P<0.001,n=10?.Prolonged optical stimulation significantly increased the duration of wakefulness episode and shortened NREM sleep episode?unpaired t test,P<0.001,n=10?.Moreover,optical stimulation produced a prominent decrease of EEG delta power?0.5-4 Hz??unpaired t test,P<0.001,n=10?.A typical feature of patients with paramedian thalamic stroke is a loss of consciousness.To investigate whether activation of PVT neurons is sufficient to induce wakefulness from unconsciousness state,we optically activated PVT neurons in mice under general anesthesia induced by isoflurane.Mice were optically stimulated once a stable EEG burst-suppression mode-a marker of anesthetic depth-was recorded in the cortex.10 Hz stimulation lasting 20s induced an immediate increase in total EEG burst activity.The increase in burst activity was accompanied by a prolonged burst duration(ChR2:wilcoxon signed rank test,P=0.016,n=7;10 Hz:unpaired t test,P<0.001,n mCherry=5,n ChR2=7)and a decreased burst-suppression ratio(ChR2:paired t test,P<0.001,n=7;10 Hz:mann-whitney rank sum test,P=0.003,n mCherry=5,n ChR2=7).More importantly,sustained activation of PVT neurons accelerated the emergence from isoflurane-induced unconsciousness,(ChR2:paired t test,P=0.003,n=7;10 Hz:unpaired t test,P=0.0156,n mCherry=5,n ChR2=7).4.The PVT?NAc pathway mediates the wakefulness-controlling effects of the PVTSince the PVT was both necessary and sufficient for wakefulness control,we next searched for the downstream pathways mediating the wakefulness-controlling effects.ChR2-mCherry expression indicated that PVT glutamatergic neurons sent projections to multiple cortical regions,including the medial prefrontal cortex?mPFC?and insular cortex.Bilateral optical stimulation of PVT projections to the mPFC failed to induce rapid transitions from neither NREM nor REM sleep to wakefulness.Optical stimulation of PVT axonal terminals in the insular cortex had no obvious effects on sleep-wake transitions.Together,these results did not sufficiently support a crucial role of the PVT?cortex pathway in controlling of wakefulness.Dense mCherry labeled axon terminals were also observed in the NAc.We then examined the role of PVT?NAc pathway in sleep-wake regulation.10 Hz optical stimulation of the PVT?NAc pathway reliably elicited transitions to wakefulness from both NREM and REM sleep(unpaired t test,P NREM=0.003,P REM<0.001;n=5).In vitro ChR2-assisted circuit mapping was used to verify the functional connection between the PVT and NAc.10 Hz optical stimulation of PVT terminals evoked excitory postsynaptic currents?EPSCs?in the recorded neurons.The short latency of EPSCs indicated a monosynaptic connection.The latency of optogenetic activation of PVT glutamatergic neurons-induced NREM sleep to wakefulness transitions was significantly prolonged after ibotenic acid lesion of NAc neurons.(mann-whitney rank sum test,P 10 Hz=0.002,P 20 Hz=0.002,nControl=5,n NAc lesion=8).To further test the importance of PVT?NAc pathway in wakefulness control,we used pathway specific chemogenetic method to inhibit the NAc-projecting PVT neurons.Retrograde transporting AAVretro-Syn-Cre was injected into the NAc and Cre-dependent AAV-EF1?-DIO-hM4D-mCherry was injected into the PVT.This intersectional strategy effectively targeted NAc-projecting PVT neurons.CNO injection at the beginning of the dark phase significantly reduced wakefulness during 3 h after injection(two-way RM ANOVA,Wake:P hM4D?Saline vs CNO?=0.004,P CNO?mCherry vs hM4D?=0.014;NREM:P hM4D?Saline vs CNO?=0.003;P CNO?mCherry vs hM4D?=0.005;n=7).Taken together,these result suggested that the PVT?NAc pathway mediated the wakefulness-controlling effect of the PVT.5 The wakefulness-controlling function of the PVT is regulated by Hcrt neurons in the LHOne of the most prominent features of the PVT among the midline thalamus is that the PVT receives dense inputs from peptide producing neurons.Members of our group found that PVT glutamateric neurons received monosynaptic inputs from Hcrt neurons in LH(LHHcrt)using rabies virus based-retrograde tracing.We examined the functional role of LHHcrt?PVT pathway in wakefulness control using optogenetic method.We bilaterally injected AAV-EF1?-DIO-ChR2-mCherry into the LH of Hcrt-Cre mice.We implanted optoelectrode into the PVT for optogenetic activation of ChR2 labeled Hcrt neurons'terminals with simultaneously PVT unit and EEG-EMG recordings.20 Hz optical stimulation of Hcrt neurons'terminals significantly increased the firing rate of PVT neurons?wilcoxon signed rank test,P<0.001,n=17?.At behavioral level,optical stimulation induced fast transitions from NREM and REM sleep to wakefulness(unpaired t test,P NREM<0.001,P REM<0.001,n=7).Optical stimulation also increased wakefulness probability(unpaired t test,NREM:P<0.001;REM:P Wake<0.001,P REM=0.001;n=7).To further confirm the importance of the LHHcrt?PVT pathway in wakefulness control,the type 2Hcrt receptor antagonist TCS-OX2-29 was injected into the PVT prior to optical stimulation.TCS-OX2-29 attenuated the optical stimulation-induced sleep to wakefulness transitions(paired t test,P NREM=0.005,P REM=0.044,n=6).Optical stimulation still induced transitions from sleep to wakefulness after blockade of the potential antidromic action potentials by injection of muscimol into the LH.In summary,we found that the population and single-cell activities of PVT neurons were closely correlated with wakefulness state.Chemogentic inhibition and genetic specific ablation of PVT glutamatergic neurons reduced wakefulness,whereas optogenetic activation of PVT glutamatergic neurons not only induced transitions from sleep to wakefulness but also accelerated emergence from general anesthesia.These results suggested that the PVT was both necessary and sufficient for wakefulness.Moreover,the wakefulness controlling function of the PVT was mediated by PVT?NAc pathway and regulated by an input pathway from Hcrt neurons in the LH.Taken together,our findings demonstrated that the PVT was a critical thalamic area for wakefulness. |
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