The Role Of Hypocretins And Melatonin In Regulating Paraventricular Thalamic Nucleus-Associated Wakefulness Control

Sleep/wakefulness is the most prominent biological rhythm of the organism.Wakefulness is very important,and the sensorimotor function,learning and memory,and thinking depend on the maintenance of a good state of wakefulness.Thalamus has long been thought to be highly correlated with the maintenance of wakefulness.The thalamus has complex anatomical connections,however,the specific thalamic nuclei and their neural circuits involved in the regulation of wakefulness have not yet been clarified.Recently,other members of our reaearch group have found that Optogenetic activation of the PVT neurons can rapidly induce sleep-to-wake transition.On the contrary,chemogenetic inhibition of PVT neurons or killing PVT neurons can promote the occurrence of non-rapid eye movement?NREM?sleep.Compared with chemogenetic inhibition and neuron-specific killing methods,electrical leasion can cause more serious damage to nucleus and better simulate the comprehensive damage of nucleus caused by stroke in clinic.To this end,this study used the method of electrical lesion to explore the effect of electrical lesion of the PVT on wakefulness.Additionally,we explore what are regulatory factors underlying the functional activity of PVT neurons.Previous studies found that the hypocretin neurons in the lateral hypothalamus?LH?,representing a prominent arousal-promoting system,innervates the PVT,and the glutamate neurons in the PVT express hypocretin receptors.Moreover,it has been reported that melatonin plays an important role in the occurrence of sleep with high level at night and low level at day.The PVT neurons expressed melatonin receptors in high abundance.On these basis,we speculate that the different functional patterns of PVT may be controlled by the hypocretins neuron inputs and melatonin signal.In this study,using multi-channel recording,patch-clamp recording,optogenetics and morphologic techniques,we investigated the effects of hypocretin and melatonin signals on the excitability of the PVT neuron and sleep/wakefulness behavior.The main results are as follows.1.Further Validation of the Effect of the PVT in wakefulness Control1.1 PVT lesion causes damage of wakefulnessAmong 51 animals that lesion were limited to PVT,51%?n=26?were found to be coma,14%?n=7?to be lethargic,12%?n=6?to be hypersomnia,18%?n=9?to be insensitive to external stimuli,and 18%?n=9?to be normal compared with sham-operated animals.The percentage of coma occurrence was 55%in severe PVT lesion group and 16%in slight PVT lesion group?Chi-square test,P<0.001,n=51?.The results suggested that the severity of PVT damage was positively correlated with the degree of awakening damage.We further developed electroencephalogram?EEG?and electromyography?EMG?recordings to quantitatively observe the effects of PVT lesion on sleep/wakefulness.PVT lesion caused a continuous decrease in the time of wakefulness.The average awakening time of animals with PVT lesion decreased from 45±7%to 21±3%across 12 hours during dark phase?unpaired t test,P<0.01,n=13?.On the contrary,NREM sleep time increased significantly,and the average NREM sleep time increased from 51±6%to 76±4%during the dark phase?unpaired t test,P<0.01,n=13?.PVT lesion did not affect time of REM sleep?4.2±0.6%VS.3.1±1.0%,unpaired t test,P=0.46,n=13?.These results suggest that PVT damage can sustainably cause awakening damage and increase sleep duration during dark phase.1.2 PVT lesion increases the low frequency delta oscillations of EEGIn the animals that continuously monitored EEG for 12 hours during the dark phase,PVT lesion changed the EEG spectrum at awake state.The delta oscillation in low frequency band could be increased by PVT lesion?control:56±3%,n=5;lesion:64±3%,n=8,unpaired t test,P<0.05?.In the sleep state,the PVT lesion also increased the oscillation of delta frequency band?control:54±3%,n=5;lesion:67±3%,n=8,unpaired t test,P<0.01?.In conclusion,the results indicate that PVT can not only reduce the amount of awakening time,but also cause the damage of awakening quality.2.The role of hypocretin neurons in regulating PVT-mediated wakefulness maintenance and its mechanisms2.1 PVT glutamatergic neurons receive mono-synaptic inputs from hypocretin neurons in the lateral hypothalamusIn order to identify the connections between glutamatergic neurons in the PVT and hypocretin neurons in the lateral hypothalamus,a retrograde mono-synaptic tracing technique based on rabies viruses?RV?was used.After injection of AAV helper and RV into PVT,the‘starter cells'were localized within PVT,suggesting that the mono-synaptic inputs of the PVT glutamatergic neurons could be selectively labeled.Hypocretin immunohistochemistry showed that retrogradely labeled LH neurons were co-labeled with hypocretins,indicating that the PVT glutamatergic neurons received mono-synaptic inputs from hypocretin neurons.In addition to RV-labeled hypocretins neurons,we found that LH also contained RV-labeled neurons that were not co-labeled with hypocretins,indicating that PVT glutamatergic neurons also received input from other types of LH neurons.2.2 Chemogenetic inhibition of LH_hypocretins?PVT pathway reduces wakefulnessIn order to study the effects of hypocretin neuron inputs on the wakefulness-promoting function of PVT,we used chemogenetic methods to specifically inhibit the LH_hypocretins?PVT pathway.At the beginning of active phase in mice?ZT 12?,CNO?5?M?was microinjected into PVT through an implanted cannula.Compared with saline and mCherry control groups,CNO injection in the hM4D-mCherry mice significantly decreased the amount of wakefulness(two-way RM ANOVA,P _{hM4D?Saline vs CNO?}<0.001,P _{CNO?mCherry vs hM4D?}<0.001,n=5),increased the NREM sleep time(two-way RM ANOVA,P _{hM4D?Saline vs CNO?}<0.001,P _{CNO?mCherry vs hM4D?}<0.001,n=5)without affecting the time of REM sleep(two-way RM ANOVA,P _{virus vs compoind}=0.103,n=5)?The effects of chemogenetics inhibiting the LH_hypocretins?PVT pathway on the longest duration of wakefulness episodes were further analyzed.Compared with the vehicle group,CNO microinjection significantly shortened longest duration of wakefulness episodes?two-way RM ANOVA,P hM4D?Saline vs CNO?=0.01,P CNO?mCherry vs hM4D?<0.001,n=5?,suggesting that inhibition of LH_hypocretins?PVT pathway decreased the ability to maintain wakefulness.Inhibition of the LH_hypocretins?PVT pathway by CNO injection significantly reduced the NREM sleep latency of hM4D-mCherry mice(two-way RM ANOVA,P _{hM4D?Saline vs CNO?}=0.017,P _{CNO?mCherry vs hM4D?}=0.003,n=5).However,CNO injection had no significant effect on REM sleep latency.CNO injection increased the number of NREM sleep episodes(two-way RM ANOVA,P _{hM4D?Saline vs CNO?}<0.001,P _{CNO?mCherry vs hM4D?}<0.001,n=5).These results suggest that inhibition of LH_hypocretins?PVT pathway reduces wakefulness.2.3 PVT is a critical downstream target for hypocretin arousal system.To further verify the important role of the LH_hypocretins?PVT pathway in controlling wakefulness regulation,we used chemogenetic methods to activate h ypocretin neurons and simultaneously inhibit glutamate neurons in the PVT.In hypocretins-hM3D mice,compared with the vehicle group,the amount of wakefulness was increased significantly after CNO injection?two-way RM ANOVA,P=0.005,n=4?,and the NREM sleep time was decreased?two-way RM ANOVA,P=0.004,n=4?.Compared with solely activating hypocretin neurons,intraperitoneal injection of CNO in hypocretins-hM3D+PVT-hM4D mice to activate hypocretin neurons while inhibiting PVT glutamatergic neurons could still induce an increase in awakening time and decreased sleep time?two-way RM ANOVA,Wake:P hM3D vs hypocretins-hM3D+PVT-hM4D=0.861;NREM:P hM3D vs hypocretins-hM3D+PVT-hM4D=0.568,n=4?.These results suggest the intraperitoneal injection of CNO stimulates hypocretins neuronal terminals in PVT,which induces the release of hypocretins and then excites PVT.Under these conditions,CNO may not effectively inhibit the activity of glutamatergic neurons in PVT through hM4D.In order to further explore the importance of LH_hypocretins?PVT pathway in wakefulness regulation,we used Caspase-3 to specifically kill PVT glutamatergic neurons and simultaneously activated hypocretins neurons by optogenetics.The NREM sleep-to-wakefulness transition was rapidly induced by 1 Hz,10 Hz and 20 Hz light stimulation in control mice.However,in mice with killing PVT glutamatergic neurons by caspase-3,the latency of NREM sleep-to-awakening transition induced by light stimulation of hypocretin neurons was significantly prolonged(unpaired t test,P_{1 Hz}=0.01,P_{10 Hz}=0.02,P_{20 Hz}=0.03,n=5).The results suggested that killing PVT glutamatergic neurons reduced the wakefulness-promoting effect of hypocretin neurons,suggesting that the LH_hypocretins?PVT pathway plays an important role in the regulation of wakefulness.3.The role of melatonin signals in regulating PVT-mediated wakefulness maintenance and its mechanisms3.1 Melatonin inhibits excitability of the PVT glutamate neuronsUsing patch-clamp recording,we found application of melatonin induced hyperpolarization?1?M:2.6±0.8 mV,n=15;100?M:4.7±0.5 mV,n=6;500?M:7.2±1.6 mV,n=7;One Way Analysis of Variance on Ranks,P<0.01?,and significantly inhibited the firing frequency of the PVT neurons?1?M:65±8%,n=15;100?M:83±11%,n=6;500?M:98±2%,n=7;One Way Analysis of Variance on Ranks,P<0.01?.In the presence of luzindole,a MTR1/2 blocker,melatonin could not induce membrane potential hyperpolarization in PVT neurons?melatonin:-42.84±1.98 mV;melatonin+luzindole:-42.75±2.04 mV,n=5,paired t test,P=0.86?.Morphological examination showed that MTR1,but not MTR2,was highly expressed in PVT,suggesting that melatonin inhibits the excitability of the PVT glutamate neurons by acting on MTR1.3.2 Melatonin produces membrane hyperpolarization by activating delayed rectifier potassium channelsThe excitability of neurons largely depends on their intrinsic characteristics.In the presence of glutamate NMDA receptor blockers,AMPA receptor blockers and GABA_A receptor blockers,melatonin could still induce hyperpolarization of PVT neurons?melatonin:4.7±1.5 mV;melatonin+CNQX+AP5+PIC:3.6±0.6 mV,n=6,paired t test,P=0.23?.Voltage-gated K⁺channels are thought to be involved in the regulation of neuronal membrane potential and are closely related to neuronal excitability.Ion exchange experiment was used to replace intracellular K⁺with Cs⁺.Under this condition,perfusion of melatonin could not cause hyperpolarization.?Baseline:-50.3±1.8 mV;Melatonin:-46±2.2 mV,washout:-48.3±2.3 mV,n=6,One Way Repeated Measures ANOVA,P<0.01,?.The results showed that the inhibitory effect of melatonin depended on K⁺channel.Instantaneous activation of K⁺channels and delayed activation of K⁺channels are the main types of voltage-gated potassium channels in neurons.Melatonin significantly increased the amplitude of delayed activation K⁺current,but did not affect the instantaneous activation K⁺current.In conclusion,the results suggest that melatonin can inhibit PVT mainly by enhancing delayed activation of K⁺channels.3.3 Melatonin inhibits excitatory synaptic transmission of PVTThe excitability of neurons is also affected by excitatory and inhibitory synaptic transmission.In the presence of GABA_A receptor blockers,spontaneous excitatory postsynaptic currents?SEPSCs?of neurons were recorded.Melatonin reversibly reduced the frequency of SEPSCs?baseline:1.9±0.2 Hz;melatonin:1.5±0.2 Hz,n=14,RM ANOVA on Ranks,P<0.001?,and decreased the amplitude of SEPSCs?baseline:12.9±0.7 pA;melatonin:11.9±0.7 pA,n=14,RM ANOVA on Ranks,P<0.001?,indicating that melatonin reduces the excitatory synaptic transmission in PVT.In the presence of MTR1/2 receptor blockade,the inhibitory effect of melatonin on SEPSCs frequency?baseline:1.6±0.3 Hz;melatonin:1.5±0.3 Hz,n=9,paired t test,P=0.53?and amplitude?baseline:10.5±0.5 pA;melatonin:10.7±0.6 pA,n=9,paired t test,P=0.29?disappeared.Based on the high expression of MTR1 in PVT,melatonin may inhibit excitatory synaptic transmission by activating MTR1.Spontaneous inhibitory postsynaptic currents?SIPSCs?were isolated in the presence of glutamate NMDA receptor blockers and glutamate AMPA receptor blockers.Melatonin did not affect the frequency of SIPSCs?baseline:1.9±0.3 Hz;melatonin:1.9±0.4 Hz,n=8,One Way Repeated Measures ANOVA,P=0.90?,nor did it affect the magnitude of SIPSCs?baseline:50.0+7.8 pA;melatonin:49±8.5 pA,n=8,One Way Repeated Measures ANOVA,P=0.88?.The changes in excitatory synaptic transmission may be resulted from the alteration of presynaptic neurotransmitter release or postsynaptic glutamate receptors.In the presence of GABA_A receptor blockers and sodium channel blocker TTX,minimal excitatory postsynaptic currents?mEPSCs?were isolated.Melatonin had no significant effect on the frequency of mEPSCs?baseline:1.5±0.4 Hz;melatonin:1.3±0.3 Hz,n=8,One Way Repeated Measures ANOVA,P=0.24?,but significantly inhibited the magnitude of mEPSCs?baseline:12.8±0.6 pA;melatonin:11.5±2.1 pA,n=8,One Way Repeated Measures ANOVA,P<0.01?.These results suggest that melatonin may affect the properties of postsynaptic glutamate receptors,thereby inhibiting excitatory synaptic transmission.3.4 Melatonin inhibits the discharge of PVT neurons and promote the occurrence of sleep in vivo.The effects of melatonin on excitability of the PVT neurons and sleep/wakefulness behavior were investigated by in vivo multi-channel and neuropharmacological techniques.Melatonin reduced the firing frequency of the PVT neurons from 4.9±1.1 Hz to 2.2±0.8Hz?n=41,paired t test,P<0.001?.At the same time,local administration of melatonin in PVT could significantly reduce the awakening time?melatonin:28.0±2.7 min;vehicle:20.2±3.2 min,n=6,paired t test,P<0.01?.On the contrary,administration of melatonin in PVT increased NREM sleep?melatonin:48.5±4.9 min;solvent:55.3±5.5 min,n=6,paired t test,P<0.01?and REM sleep?melatonin:3.4±1.4 min;vehicle:5±1.5 min,n=6,paired t test,P<0.05?.These results suggest that melatonin inhibits the activity of PVT neurons and promotes the transition from wakefulness to sleep.In summary,electrical lesion of PVT caused serious damage to animal awakening,which was mainly manifested in the continuous decrease of wakefulness time and an increase in the EEG delta oscillations.Hypocretins neurons in the lateral hypothalamus can directly innervate PVT neurons.The excitatory inputs from the hypocretins neurons results in a high level of PVT activity during the awakening period and thus promotes the maintenance of wakefulness.Chemogenetic Inhibition of the hypocretins neuron-PVT glutamate neuron pathway decreased awakening time and increased sleep.Additionally,melatonin could reduce the excitability of PVT neurons by enhancing the delayed rectifier potassium current at the neuronal somata and inhibiting glutamate excitatory synaptic input.At the behavioral level,the administration of melatonin in PVT can promote sleep,which was mediated mainly by activation of MTR1.