Mechanism Study On Enriched Environment Against Sleep Deprivation-induced Cognitive Impairments Via PIEZO1/calpain/autophagy Pathway In The Basal Forebrain

ObjectiveAcute sleep deprivation(SD)imposed on individuals in closed isolated environments(CE)seriously impairs the cognitive performance,whereas enriched environments(EE)help mitigate cognitive impairments caused by sleep deprivation.However,there is limited research on the brain regions and signaling mechanisms involved.We have reported that inhibition of basal forebrain PIEZO1 signaling partly counteracted sleep deprivation-induced fear memory impairments.In this study,we proposed and tested this hypothesis that EE ameliorates sleep deprivation-induced fear memory impairments by modulating basal forebrain PIEZO1/calpain/autophagy pathway.Methods1.8-week-old male mice were housed either in either closed isolated environment(CE)or enriched environment(EE)according to grouping.Six-hour total sleep deprivation(SD)was imposed on both CE and EE mice.Short-and long-term fear memory were then examined by step-down inhibitory avoidance test.After fear memory evaluation,mice were sacrificed and changes in basal forebrain PIEZO1 signaling was detected by western blot(WB)and polymerase chain reaction(PCR).2.The PIEZO1 activator Yoda1 was microinjected to bilateral basal forebrain of EE mice.Step-down inhibitory avoidance test detected short-term and longterm fear memory after sleep deprivation,and detected protein changes in the PIEZO1/calpain pathway using WB.3.Gs MTx4,an inhibitor of PIEZO1 signaling,PD151746,an inhibitor of calpain,or vehicle control was microinjected into the basal forebrain of CE mice.Short-term and long-term fear memory were detected by step-down inhibitory avoidance test.WB detected protein changes in the PIEZO1/calpain pathway.4.Autophagy markers LC3 B,p62,TFEB and p-TFEB in the basal forebrain were examined in mice receiving the above-mentioned interventions.PCR detected the m RNA transcription level of p62.Immunofluorescence detected the expression of LC3 B and the nuclear transcription of TFEB in the basal forebrain.5.Rapamycin,an autophagy agonist,or vehicle was microinjected into the basal forebrain of EE mice and 3-MA,an autophagy inhibitor,or vehicle was microinjected into CE mice.WB detected the changes in the expression of autophagy related proteins in the basal forebrain,and step-down inhibitory avoidance test detected fear memory.ResultsMice housed in enriched environment performed better in short-and longterm fear memory test compared with the closed isolated environment mice.Sleep deprivation resulted in increased PIEZO1 protein expression,full-length tropomyocin receptor kinase B(Trk B-FL)degradation and autophagy reflected by increased LC3 Ⅱ / Ⅰ ratio,enhanced p62 degradation,increased TFEB expression and nuclear translocation and decreased TFEB phosphorylation.These molecular changes were partly reversed by enriched environment.Microinjections of the PIEZO1 activator Yoda1 or the autophagy agonist rapamycin into bilateral basal forebrain induced excessive autophagy and eliminated cognitive protective effect of enriched environment.Bilateral basal forebrain microinjection of the PIEZO1 inhibitor Gs MTx4,the calpain inhibitor PD151746 or the autophagy inhibitor 3-MA mimicked the cognitive protective and autophagy inhibitory effects of enriched environment in sleep deprived mice.ConclusionsEnriched environment combated sleep deprivation-induced fear memory impairments in mice by inhibiting the basal forebrain PIEZO1/calpain/autophagy pathway.