The Neural Circuit Mechanisms Of The Paraventricular Hypothalamic Nucleus In Regulating Wakefulness

Sleep and arousal is a common biological phenomenon.Sleep can reversibly weaken the functional activities of the brain,and help to maintain the homeostasis of the body,such as the removal of metabolite,the repair of cell and tissue damage,and the processing of memory information in the brain.Arousal is the prerequisite for the realization of brain functions such as sensation,movement,memory and thinking.The brain stem,hypothalamus,thalamus and basal forebrain interact with each other to regulate sleep and wakefulness.The paraventricular hypothalamic nucleus,which is located near the hypothalamus,is mainly composed of a group of neuroendocrine cells and plays an important role in the regulation of autonomic nervous activity.PVN is also an important component of pituitary-hypothalamic-adrenaline axis,which is involved in the stress regulation.However,there are few reports on the role of the PVN in sleep and wakefulness.In this study,we combined oxygen level dependent functional magnetic resonance imaging(f MRI),chemogenetics and optogenetics to elucidate the role of PVN and related neural circuits in the regulation of sleep and wakefulness.Part Ⅰ FMRI study of neural network changes after activation of the glutamatergic neurons in the paraventricular nucleus of hypothalamus in ratsObjective Based on Chemo-f MRI technology to explore the activities of the whole brain and functional connectivity between PVN and other brain regions after chemogenetic activation of the PVN glutamatergic neurons.Methods Seven male rats weighing about 350-500 g were included in the study.MRI scans were performed after 4 weeks of virus injections in the PVN,including the T2-weighted images,EPI sequence and Flash sequence.Significant differences of the fractional low-frequency oscillation amplitude(f ALFF),the degree centrality and the functional connectivity among the groups were tested using paired-T test with a threshold of p < 0.05.Results The result of this part study showed that after activation of PVN glutamatergic neurons,The BOLD signal of the paraventricular nucleus of the hypothalamus,the medial prefrontal cortex(m PFC),the cingulate gyrus(Cg),the paraventricular nucleus of the thalamus(PVT)and the hippocampus increased;while the BOLD signal of the olfactory bulb decreased.The f ALFF and degree centrality of PVN,motor cortex,PAG increased.and the functional connection between PVN and PVT,Cg,motor cortex,hippocampus increased.Conclusions In this part of the experiment,we conclude that after the activation of glutamatergic neurons in the PVN,brain regions that mainly regulate sleep,wake,exercise,emotion and cognition are activated.Part Ⅱ To explore the activity patterns of the PVN glutamatergic neurons during normal sleep-wake state based on fiber photometryObjective To explore the activities of glutamatergic neurons in the PVN in freely mice across spontaneous sleep-wake states using fiber photometry and polysomnography.Methods Five male C57 mice weighing about 20-30 g were included in the study.Adeno-associated virus(AAV)expressing the genetically encoded Ca2+ sensor GCa MP6 s was injected into the PVN,and EEG electrodes were implanted.Behavioral tests were performed for 3 weeks.Each mouse were recorded 12 hours in light period,The number of state transitions between NREM-Wake,NREM-REM,Wake-NREM and REM-Wake of the total 5 mice were counted for analysis.Results During the three sleep state transitions,the activities of PVN glutamatergic neurons began to increase before NREM sleep-to-wake and NREM sleep-to-REM sleep transitions and began to decrease before wake-to-NREM sleep transitions.While during REM sleep-to-wake transition,no significant activity changes were observed.Conclusions The part of this experiment reveals that the population activities of PVN glutamatergic neurons changed on the transitions of sleep-wake states.Part Ⅲ To explore the role of the PVN glutamatergic neurons in regulating arousal based on chemogeneticsObjective To explore the regulation of the PVN glutamatergic neurons on sleep and wakefulness using chemogenetics and polysomnographyMethods The chemogenetic activation group included 12 mice in experiment and control group,respectively;and the chemogenetic inhibition group included 13 mice in experiment and control group,respectively.Male C57 mice weighing about20-30 g were included in the study.3 weeks after virus injection,behavioral tests were performed.The experimental and the control group were given CNO and saline respectively.Sleep analysis software was used to analyze the wake,NREM and REM time.Two-way ANOVA was used for analysis and P<0.05 was considered statistically significant.Results Chemogenetic activation of PVN glutamatergic neurons promoted wakefulness and reduced NREM and REM sleep;chemogenetic inhibition of PVN glutamatergic neurons reduced wake time and increased NREM sleep.Conclusions The part of this experiment reveals that PVN glutamatergic neurons are necessary for the regulation of wakefulness.Part Ⅳ To explore the role of the PVN-PVT neural circuit in regulating arousal based on optogenetics and chemogeneticsObjective Using optogenetics and chemogenetics combined with polysomnography to explore the PVN-PVT neural circuit in regulating wakefulness.Methods The optogenetic group of the PVN-PVT neural circuit included 7 mice in the ChR2-GFP group and the GFP group,respectively.and the chemogenetic group of the PVN-PVT neural circuit included 7 mice in the h M4Di-mcherry group and the mcherry group,respectively.In the optogenetic experiment,mice in the experiment group and the control group were injected with the ChR2-GFP and GFP virus in the PVN respectively,and the ferrule was embedded above the PVT,and the EEG recording electrodes were embedded at the same time.In the chemogenetic experiment,the experiment group and the control group were injected with the h M4Di-mcherry and mcherry virus in the PVN,and electrodes were embedded at the same time,and behavioral tests were conducted 3 weeks later.Two-way ANOVA method was used for analysis and P<0.05 was considered statistically significant.Results Optogenetic stimulation of the PVN-PVT neural circuit promoted wakefulness in the ChR2-GFP group compared with the GFP group under the same light optical stimulation.Chemogenetic inhibition of the PVN-PVT neural circuit decreased wakefulness time and increased the NREM time in the h M4Di-mcherry group compared with the mcherry group in the 6 hour after intraperitoneal injection of CNO.Conclusions The part of this experiment reveals that the PVN-PVT neural circuit is sufficient and necessary for the regulation of wakefulness.