The Mechanism Underlying Melatonin Type 3 Receptor-mediated Homeostatic Restoration Of Synaptic Protein Phosphorylation In The Medial Entorhinal Cortex During Sleep

The superficial layer of the medial entorhinal cortex（s MEC）is an important brain region involved in spatial learning and memory,which has significant changes in rhythmic activity during the sleep-wakefulness cycle.During wakefulness,neurons in s MEC are highly activated and mainly participate in encoding and storing spatial memory through high-frequency theta and gamma oscillations.While during sleep,the excitability of s MEC neurons is decreased,showing a pattern of functional activities mainly with low-frequency delta oscillation.It is worth noting that the homeostatic restoration of synaptic protein phosphorylation mediated by the decrease in neuronal activity during sleep is crucial for learning and memory.During wakefulness,the brain constantly receives environmental stimulation and processes information,leading to increased neuronal activities.Meanwhile,synaptic proteins are required to function in a phosphorylated state and the actin cytoskeleton tends to polymerize for the capability of increasing synaptic volume and synaptic transmission efficiency.Therefore,homeostasis of synaptic protein phosphorylation is gradually destroyed after prolonged awakening.Conversely,during sleep,neuronal activities are decreased,accompanied by dephosphorylation of synaptic proteins,resulting in actin cytoskeleton disaggregation,synaptic volume shrinkage,and homeostatic restoration.But so far,the mechanism underlying the restoration of synaptic protein phosphorylation during sleep is not fully understood.As an important sleep-promoting factor,the secretion of melatonin reaches its peak in the sleep period,which plays an important role in the homeostatic restoration of sleep.It has been revealed that melatonin can regulate sleep’s function of restoring homeostasis by clearing metabolic products,promoting the reduction of oxidative free radicals,and inhibiting neuronal activity.However,it is still unclear whether the melatoni n system regulates the sleep-related synaptic protein phosphorylation homeostasis in s MEC during sleep.Therefore,this study for the first time focuses on the role of the melatonin system in regulating the phosphorylation homeostasis in s MEC during sleep and its role in spatial learning behavior and sleep/wake behavior.We use immunoblotting techniques and chemical genetic manipulation techniques to describe the characteristics of sleep-related s MEC neuron phosphorylation homeostasis.Then,we use CRISPR/Cas9 gene editing technology and neuropharmacological methods to explore the effects of intervening with the melatonin type 3 receptor（MT₃R）signal during sleep on phosphorylation homeostatic restoration.Finally,we observe the effects of intervening MT₃R-mediated sleep-related s MEC phosphorylation homeostatic restoration on post-sleep spatial memory behaviors through animal behavior techniques and sleep/wake behaviors through EEG/EMG recording technique.The main results are summarized as follows:1.Sleep promotes the phosphorylation homeostatic restoration of s MEC synaptic proteins and the depolymerization of the actin cytoskeleton.（1）Using immunoblotting techniques to detect the phosphorylation levels of s MEC synaptic proteins,glutamate receptors and protein kinase in rats during sleep,wakefulness,and sleep deprivation,we found that compared to wakefulness,the phosphorylation levels of Synapsin-1,PSD-95,and Glut R1 significantly decreased during sleep,and after sleep deprivation,the phosphorylation levels of Glut R1,Glut R2,and protein kinases PKA and PKC significantly increased.These results indicate that sleep promotes the phosphorylation homeostatic restoration of s MEC synaptic proteins.（2）Using immunoblotting techniques and ultracentrifugation to separate filamentous actin（F-actin）and globular actin（G-actin）,we detected the expression levels of actin-binding proteins（ABPs）and the functional activity status of the actin cytoskeleton in s MECs during sleep,wakefulness,and sleep deprivation respectively.We found that compared to wakefulness,the expression levels of the s MEC cytoskeleton co-factors Rac1/Cdc42 and AKAP5,as well as ABP Profilin,were significantly decreased during sleep,and the ratio of F/G actin also significantly decreased,indicating a tendency towards depolymerization of the actin cytoskeleton.However,after sleep deprivation,the expression levels of s MEC cytoskeleton co-factors AKAP5 and ABPs Profilin significantly increased,and the ratio of F/G actin significantly increased,indicating a tendency towards polymerization of the actin cytoskeleton.These results suggest that sleep promotes the depolymerization of the s MEC actin cytoskeleton.2.Decreased neuronal excitability s MEC promotes phosphorylation homeostasis restoration of synaptic proteins and decreases expression of cytoskeleton co-factors during sleep.（1）Using chemogenetic methods to inhibit the excitability of s MEC glutamatergic neurons and reduce s MEC neuron excitability,we then detected the phosphorylation levels of synaptic proteins.We found that compared to the control group,the phosphorylation levels of synaptic proteins,glutamate receptors,and protein kinases were significantly reduced after chemogenetic inhibition of s MEC glutamatergic neurons.These results suggest that the downregulation of s MEC neuron excitability during sleep mediates the phosphorylation homeostasis restoration of synaptic proteins.（2）Under the same conditions,we also detected the expression levels of cytoskeleton co-factors and found that compared to the control group,chemogenetic inhibition of s MEC glutamatergic neurons resulted in a significant decrease in the expression levels of cytoskeleton co-factors,indicating that decreased s MEC neuron excitability during sleep mediated the depolymerization of the actin cytoskeleton.3.MT3R promotes the phosphorylation homeostasis restoration of s MEC synaptic proteins and the depolymerization of the actin cytoskeleton during sleep.（1）Using CRISPR/Cas9 gene editing technology to knock out the MT3R gene,we detected the phosphorylation levels of s MEC synaptic proteins during sleep using immunoblotting techniques.We found that compared to the wild-type control group,the phosphorylation levels of s MEC synaptic proteins,glutamate receptors,and protein kinases were significantly increased after knocking out the MT₃R gene,indicating that MT₃R promotes the phosphorylation homeostasis restoration of s MEC synaptic proteins during sleep.（2）Using pharmacological techniques to administer a specific MT₃R blocker S29434to s MECs during sleep and combining it with immunoblotting techniques,we detected the phosphorylation levels of s MEC synaptic proteins.We found that compared to the control group,blocking the MT₃R signal during sleep resulted in a significant increase in the phosphorylation levels of s MEC synaptic proteins,glutamate receptors,and protein kinases,further confirming that MT₃R promotes the phosphorylation homeostasis restoration of s MEC synaptic proteins during sleep.（3）Using CRISPR/Cas9 gene editing technology to knock out the MT₃R gene,we detected the expression levels of cytoskeleton co-factors in s MECs during sleep using immunoblotting techniques.We found that compared to the wild-type control group,the expression levels of cytoskeleton co-factors were significantly increased after knocking out the MT₃R gene during sleep,indicating that MT₃R mediates the depolymerization of the actin cytoskeleton during sleep.（4）Using pharmacological techniques to administer a specific MT₃R blocker S29434to s MECs during sleep and combining it with immunoblotting and ultracentrifugation techniques,we detected the expression levels of cytoskeleton co-factors and ABPs as well as the functional activity status of the actin cytoskeleton.We found that compared to the control group,blocking the MT₃R signal during sleep resulted in a significant increase in the expression levels of cytoskeleton co-factors and ABPs,as well as a significant increase in the F/G actin ratio,indicating a tendency towards polymerization of the actin cytoskeleton.These results suggest that MT₃R mediates the depolymerization of the s MEC actin cytoskeleton during sleep,and blocking MT₃R promotes the polymerization of the actin cytoskeleton.4.MT3R promotes the decrease of s MEC neuron excitability during sleep.（1）Using CRISPR/Cas9 gene editing technology to knock out the MT₃R gene and combining it with in vivo multi-channel recording techniques to observe s MEC neuron firing rate and neural network oscillations during sleep,we found that compared to the wild-type control group,knocking out the MT₃R gene significantly increased the firing rate of s MEC neurons during NREM sleep,and decreased the low-frequencyδoscillation power spectrum while significantly increasing the high-frequencyθ,β,andγoscillation power spectra.These results suggest that knocking out MT₃R increases the excitability of s MEC neurons during NREM sleep,indicating that MT₃R promotes the decrease of s MEC neuron excitability during NREM sleep.（2）Using pharmacological techniques to administer a specific MT₃R blocker S29434 to s MECs during sleep and combining it with in vivo multi-channel recording techniques to observe s MEC network oscillations,we found that compared to the control group,blocking MT₃R signaling during sleep increased neural firing rate,and also a decrease of the low-frequencyδoscillation power spectrum and a significant increase in the high-frequencyθ,β,andγoscillation power spectra in local field potential.These results further confirm that blocking MT₃R increases the excitability of s MEC neurons during NREM sleep,revealing that MT₃R mediates the decrease of s MEC neuron excitability during NREM sleep.5.MT3R-mediated sleep-related synaptic protein homeostasis restoration facilitates spatial memory acquisition after sleep.（1）Using pharmacological techniques to administer a specific MT₃R blocker S29434to s MECs during sleep,combined with spatial learning task training,we found that compared to the control group,blocking MT₃R during sleep significantly impaired the performance of rats in an s MEC-dependent novel location recognition task after sleep,but had no significant effect on a non-s MEC-dependent novel object recognition task.These results suggest that MT₃R-mediated sleep-related synaptic protein homeostasis restoration is crucial for the acquisition of spatial learning and memory after sleep.（2）Using RNAi to knock down MT₃R gene expression specifically in s MECs,combined with spatial learning task training,we found that compared to injectio n of a control virus,knocking down MT₃R specifically in s MECs significantly impaired the performance of rats in an s MEC-dependent novel location recognition spatial learning and memory task after sleep,but had no significant effect on a non-s MEC-dependent novel object recognition task.These results further confirm that MT₃R-mediated sleep-related synaptic protein homeostasis restoration is essential for the acquisition of spatial learning and memory after sleep.（3）Using CRISPR/Cas9 gene editing technology to knock out the MT₃R gene and observing sleep/wake behavior in rats using EEG/EMG recordings,we found that compared to the wild-type control group,there was no significant difference in the 24-hour sleep/wake duration of rats after knocking out the MT₃R gene.Analysis of the brain EEG power spectrum revealed that high-frequencyθwaves during NREM sleep were increased after knocking out the MT₃R gene,further supporting the involvement of MT₃R in regulating sleep-related homeostatic restoration by modulating neuron excitability.Conclusions:Melatonin downregulates the excitability of s MEC neurons during sleep by acting on MT₃R,promoting the restoration of synaptic protein phosphorylation and the depolymerization of the actin cytoskeleton,thereby providing space for the acquisition of novel spatial memory after sleep.Our findings provide new reliable evidence for a deeper understanding of the mechanism by which melatonin participates in regulating synaptic phosphorylation homeostasis restoration.