Effect Of Constant Light On Hippocampal Neurogenesis And Signal Pathways In Vivo And In Vitro

Appropriate lighting is essential to animal health.In the process of livestock and poultry breeding,animals are often exposed to constant light.For example,the broiler is exposed to light for 23-24 hours a day in the first week after hatching;the heat-retaining lamp in the incubator for newborn piglets is continuously illuminated for 24 hours a day for heating.Although constant light can promote animal growth,excessive light exposure can harm animal health.Studies have shown that constant light significantly reduces the learning and memory abilities of animals,and increases anxiety and depression-like behaviors.In addition,many animal cells have the same photosensitivity as plant cells and bacteria.Cryptochromes（CRY）and cytochrome c oxidase（Cco）can act as photoreceptors to mediate light signal transduction.Hippocampus is a part of the limbic system of the brain.The dentate gyrus（Dentate gyrus,DG）of many animals retains the ability to generate new neurons（neuronogenesis）for life,and hippocampal neurogenesis is closely related to animal behavior.However,the effect of constant light on hippocampal neurogenesis and its mechanism,whether in vivo or in vitro,is still unclear.Therefore,in this study,in vivo and in vitro models were used to reveal the effect of constant light on hippocampal neurogenesis and its signal pathways.The in vivo model used mice exposed to 24 hours of constant light for 3 weeks after weaning;the in vitro model used mouse hippocampal neurons HT-22 cultured in an incubator with 24 hours of light.The in vivo experiment aims to explore the effect of constant light on the stress-related behaviors of mouse hippocampal neurons and its signal pathway;the in vitro experiment aims to reveal the effect and mechanism of constant light on hippocampal neuron proliferation,apoptosis and autophagy.1 Constant light causes behavioral disorders and hippocampal neurogenesis inhibition in miceWeaned C57BL/6 male rats were randomly divided into two groups（20 in each group）.The control group was reared under 12 hours of light and 12 hours of darkness（Control,CON）,and the constant light group was reared under 24 hours of light.（Chronic constant light,CCL）,the light intensity is 300 lux,lasts for 3 weeks,and the weight and feed intake are recorded every week.The results showed that CCL significantly increased（P<0.01）the feed intake of mice,but did not affect body weight（P>0.05）.In morris water maze,the walking distance and the time required to find the platform in CCL group were significantly increased（P<0.05）,suggesting that learning and memory abilities are impaired.In the open field,the number of crossings experiment was significantly increased（P<0.01）,while the number of crossing the middle area,staying time in the middle area,modification behavior and upright behavior significantly decreased（P<0.01）in CCL group.At the same time,in the elevated plus maze experiment,the number and residence time of mice in the open arm were significantly decreased（P<0.01）,while the number and residence time in the closed arm were significantly increased（P<0.01）in CCL group.The freezing time in the forced swimming experiment and the tail suspension experiment in CCL group were significantly increased（P<0.01）.These results suggested that CCL caused anxiety and depression-like behaviors in mice.In addition,the plasma melatonin（MT）level of mice was significantly reduced（P<0.01）,while the corticosterone（CORT）level was significantly increased（P<0.05）,and the hippocampal dentate gyrus（Dentate gyrus,DG）,the expression of DCX,a marker protein of newborn neurons,significantly decreased（P<0.01）in CCL group.In summary,the above results indicate that CCL causes behavioral disorders in mice,which may be related to the decrease of plasma MT level,the increase of CORT level,and the inhibition of hippocampal neurogenesis.2 Constant light blocks the BDNF/TrκB signaling pathway through CRY-mediated FTO/m⁶A pathway and inhibits mouse hippocampal neurogenesisIn order to explore the mechanism of CCL inhibiting mouse hippocampal neurogenesis,this experiment investigated whether the circadian clock gene CRY acts as a photoreceptor to affect the BDNF signaling pathway,a key regulator of hippocampal neurogenesis.The results showed that the expression of CRY 1 and 2（CRY1/2）in the hippocampus of mice in the CCL group were significantly up-regulated（P<0.05）,while BDNF and its downstream TrκB/ERK signaling pathway were significantly inhibited（P<0.05）.At the same time,the level of methylation modification（N⁶-methyladenosine,m⁶A）of hippocampal RNA in CCL mice was significantly increased（P<0.01）,and the expression of RNA demethylase FTO was significantly reduced（P<0.05）.Overexpression of CRY1/2 in hippocampal neurons HT-22 significantly inhibited（P<0.05）FTO expression and significantly increased（P<0.05）m⁶A levels,while knockdown of CRY1/2led to the opposite result.Luciferase reporter gene analysis further confirmed that CRY1/2can inhibit FTO transcription.In addition,knockdown and inhibition of FTO significantly increased（P<0.05）the level of m⁶A modification on TrκB m RNA 3’UTR in HT-22 cells,thereby significantly reducing（P<0.05）the stability of TrκB m RNA and the expression of TrκB protein.In summary,the above results indicate that CCL induces FTO transcriptional inhibition by activating CRY1/2,and inhibits hippocampal TrκB expression through m⁶A-dependent post-transcriptional regulation,thereby inhibiting mouse hippocampal neurogenesis.3 Constant light induces stress-related behaviors in mice through GSK3βmediated GR pathwayChronic stress usually induces stress-related behaviors such as anxiety and depression in mice by inhibiting the activity of hippocampal glucocorticoid receptor（GR）.Therefore,this experiment aims to explore the effect of CCL on hippocampal GR expression and activity and its signaling pathways.The results showed that the content of hippocampus GR phosphorylation was significantly reduced（P<0.01）in CCL group,and it was accompanied by the inactivation（P<0.05）of Glycogen synthesis kinase 3β（GSK3β）.In order to analyze the relationship between GSK3βand GR,HT-22 cells were treated with GSK3βinhibitor CHIR-99021.We found that GR phosphorylation was significantly inhibited（P<0.05）.In turn,the GR inhibitor RU486 could not affect the expression of GSK3β,indicating that GSK3βregulates GR phosphorylation.In addition,hippocampal superoxide anion(O^2-)and malondialdehyde（MDA）levels were significantly increased（P<0.05）,while the activity of superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）were significantly decreased（P<0.05）in CCL group,suggesting that CCL caused oxidative stress in mice hippocampus.In order to further study the mechanism of GSK3βinactivation caused by CCL,HT-22 cells were treated with a combination of melatonin receptor antagonist（luzindole）and H₂O₂ to simulate the decrease of melatonin in the body and the increase of oxidative stress in the hippocampus.The activity of GSK3βwas significantly decreased（P<0.01）,and the phosphorylation level of GR was significantly reduced（P<0.01）.Finally,treatment of mice with melatonin can reverse（P<0.05）GSK3β-mediated hippocampal GR suppression,and significantly alleviate（P<0.05）CCL-induced depression-like behavior.In summary,the above results indicate that CCL can induce depression-like behavior in mice through GSK3β-mediated hippocampal GR inhibition,and it is related to the decline of melatonin and hippocampal oxidative stress.Melatonin can alleviate hippocampal GR suppression and depression-like behaviors induced by CCL.4 Constant light inhibits the proliferation of HT-22 cells and promotes apoptosis through Cco/ROS-mediated IGF-1 and TNF-αsignaling pathwaysLight can directly affect many cells.This experiment aims to explore the effect of constant light on the proliferation and apoptosis of hippocampal neuronal cells cultured in vitro and its underlying mechanism.The results showed that the viability and proliferation activity of HT-22 cells were significantly inhibited（P<0.01）,manifested by significantly cell cycle arrest and apoptosis increased,and at the same time Caspase 3 activity was significantly increased（P<0.01）after light exposure.Meanwhile,mitochondrial cytochrome oxidase（Cco）was significantly activation（P<0.01）,and mitochondrial reactive oxygen species（ROS）levels was significantly increased（P<0.01）,and mitochondrial membrane potential was significantly decreased（P<0.01）after light exposure,suggesting that light causes mitochondrial function damage.In addition,the expression and content of insulin-like growth factor 1（IGF-1）m RNA in cells were significantly reduced（P<0.01）,and tumor necrosis factor-α（Tumor necrosis factor-α,TNF-α）expression and content were significantly increased（P<0.01）after light exposure.Promoter activity analysis found that the activity of the IGF-1 gene promoter was significantly decreased（P<0.01）,the activity of the TNF-αgene promoter was significantlt increased（P<0.01）after light exposure.At the same time,the mitogen activated protein kinase（MAPK）downstream of IGF-1 and the AKT/m TOR pathway were significantly inhibited（P<0.01）.The ROS scavenger N-acetylcysteine（NAC）significantly alleviated（P<0.01）light-induced cell damage,restored cell viability,and the expression of IGF-1 and TNF-α.In summary,the above results indicate that light inhibits the proliferation of hippocampal neurons and induces their apoptosis through mitochondrial Cco/ROS-mediated IGF-1 and TNF-αpathway.5 Constant light promotes HT-22 cell autophagy through GSK3 mediated GR/RORαpathwayAutophagy is the response of cells to stress and is closely related to the survival and death of cells.This experiment aims to explore the effect of constant light on autophagy in HT-22 cells and its mechanism.The results showed that light significantly activated（P<0.01）the expression of autophagy-related genes and proteins in HT-22 cells,and significantly promoted（P<0.01）the formation of autophagosomes.At the same time,constant light damages the rhythm of the expression of circadian clock-related genes,and the rhythm of CLOCK,BMAL1,CRY1 and CRY2 gene expression shows a significant phase shift and the oscillation amplitude was significantly suppressed.The oscillation frequency of PER1,PER2 and PER3 genes were significantly decreased.Retinoid-related orphan receptor alpha（RORα）completely lost its circadian rhythm,and its performance continued to increase（P<0.01）,suggesting that RORαmay play a key regulatory role in light-induced autophagy in HT-22 cells.At the same time,light significantly increased（P<0.01）GSK3βactivity and content of phospho-glucocorticoid receptor（p-GR）.In order to explore the role of GSK3β,GR and RORαin the process of light-induced autophagy and the upstream and downstream relationship between the three,the inhibitors of GSK3β,GR and RORαwere used to treat HT-22 cells respectively,and the RORαinhibitor SR1001 was found.It can significant alleviate（P<0.01）light-induced autophagy activation,but does not affect the expression of GSK3βand GR;GR inhibitor RU486 can significant alleviate（P<0.01）light-induced autophagy activation and the expression of RORα,however,it does not affect the expression of GSK-3β;and the GSK3 inhibitor CHIR-99021 can significant alleviate（P<0.01）light-induced autophagy activation and the expression of GR and RORα,suggesting that GSK3 regulates GR,and GR regulates RORα.The above results indicate that light induces autophagy in hippocampal neurons through GSK3mediated GR/RORαsignaling pathway.In summary,constant light in vivo can significantly inhibit hippocampal neurogenesis in mice;reduce learning and memory ability,and increase anxiety and depression-like behaviors.At the same time,constant light in vitro can directly inhibit the proliferation of HT-22 cells,induce apoptosis,and promote autophagy.The signal pathways in vivo and in vitro are different.In vivo,light on the one hand blocks the BDNF/TrκB signaling pathway through the FTO/m⁶A pathway mediated by the photoreceptor CRY and inhibits hippocampal neurogenesis;on the other hand,it induces depression-like behavior in mice through GSK3β-mediated hippocampal GR inhibition.In vitro,light inhibits HT-22 cell proliferation and induces apoptosis through the IGF-1 and TNF-αpathway mediated by the photoreceptor Cco,on the one hand,and induces its autophagy through GSK3 mediated GR/RORαsignaling pathway.