| In addition to providing illumination,light also affects learning and memory.In the population studies,light exposure increased activity in brain regions associated with cognition to improve memory during cognitive tasks.Based on the improving effect of light on learning and memory,light therapy has been applied to improve memory disorders in clinical practice.Explaining the effect of light on learning and memory and its neural mechanism will help us make better use of light to work lighting environment and clinical phototherapy.Until now,there have been many researches on the mechanism of light regulation on learning and memory,but no consistent conclusion has been reached.The experimental studies of light regulating learning and memory mainly include the acute and chronic effects.Studies on acute effects have used different behavioral paradigms and have not yet reached the same conclusion.It may be that different behavioral paradigms detect different types of memory and involve different brain regions,and the effects of light on each brain region are different,so it is impossible to draw a consistent conclusion.In addition,the memory process can be divided into stages of encoding,storage and retrieval at least.Previous studies have given light at different stages,which is also responsible for the inconsistency of conclusions.In addition,when studying the effects of light on learning and memory in mice,other interference that affect memory behavior in mice should be kept to a minimum.In the fear conditioning or water maze experiment,there are aversive stimuli such as foot shock,sound,drowning.These factors may interact with light,so it is inevitable to have bias to evaluate the effect of light on memory.In order to put the influence of interference factors other than light at the minimum level,our research use Y maze to assess the effect of light on spatial memory in mice,this behavioral paradigm is simple,without any aversive stimulus,and do not produce stress to mice,so it is a very good method used to study the effect of light on spatial learning and memory.We were able to study the effects of light on the encoding and retrieval of spatial memory in mice by using different light regimens in the training stage and the testing stage.With respect to the chronic effects of light,some studies have found that abnormal light cycles,including continuous light or continuous darkness,can cause damage to learning and memory.Other studies have revealed neural pathways in which abnormal light interferes with learning and memory.A recent study explains the neural circuits by which bright light improves learning and memory.Long-term light affects memory through certain neural circuits,and it is bound to have a molecular effect on memory-related brain regions.However,few studies have explored the molecular mechanism of chronic light regulation on memory.It is of great theoretical and practical significance to elucidate the effect of light on memory and reveal its mechanism,which can provide a theoretical basis for the implementation of more effective phototherapy programs to improve cognition in clinical practice,as well as the optimization of lighting parameters in living and working environments to improve work efficiency.In order to clarify the effect of light on spatial memory and its neural mechanism,we conducted the following researches.Part Ⅰ:The effect of different light intensity on spatial memory and anxiety in miceObjective:To investigate the effects of the light with different illuminance on memory encoding and memory retrieval in Y maze spatial memory behavioral test in mice.Methods:Sixty adult male C57BL/6 mice were randomly divided into 6 groups,with 10 animals in each group.Maintaining 50 lux during the test period of Y maze experiment,white light with different brightness(50 lux,100 lux,200 lux,400 lux,800 lux,1200 lux)was applied in the training stage to observe the effect of light with different brightness on the spatial memory of mice in the memory coding stage.In the Y maze experiment,white light with different brightness(50 lux,100 lux,200 lux,400 lux,800 lux,1200 lux)was applied in the test stage,and the light in the training stage was 50 lux.The effects of light with different brightness on the spatial memory of mice in the memory retrieval stage were observed.The effects of white light exposure(50 lux,100 lux,200 lux,400 lux,800 lux,1200 lux)on anxiety in mice were evaluated by open field and elevated plus maze experiments.Results:In the Y maze spatial memory test,the light of different luminance was used in the training period,and 50 lux light was used in the test period,the results showed that the percentage of residence time of mice in the new arm of Y maze under 100 lux,200 lux,400 lux,800 lux and 1200 lux light during the training period did not significantly change compared with the control group(P>0.05).In the Y maze spatial memory test,50 lux light was used in the training period,and light of different brightness was used in the test period,the results showed that the percentage of residence time in the new arm of Y maze of mice under 100 lux and 200 lux light increased gradually compared with the 50 lux control group in the test period,but the difference was not statistically significant(P>0.05).The percentage of residence time of mice in the new arm of Y maze under 400 lux light was significantly higher than that of control group,and the difference was statistically significant(50 lux group:37.99±8.51,400 lux group:40.47±7.46,P<0.01).Under 800 lux and 1200 lux light,the percentage of residence time in the new arm of Y maze was significantly lower than that of control group,and the difference was statistically significant(50 lux vs.800 lux,P<0.05;50 lux vs.1200 lux,P<0.05).In the open field test,compared with the 50 lux control group,the percentage of residence time in the central area of mice under 100 lux and 200 lux light showed a trend of gradual decrease,but the difference was not statistically significant(P>0.05);The percentage of residence time in the central area of mice under 400 lux,800 lux and 1200 lux light was significantly lower than that of control group,and the difference was statistically significant(50 lux vs.400 lux,P<0.05;501ux vs.800 lux,P<0.05;501ux vs.1200 lux,P<0.01).In the elevated plus maze,compared with the 50 lux control group,the percentage of residence time in the open arm of mice under 100 lux and 200 lux light decreased gradually,but the difference was not statistically significant(P>0.05).The percentage of residence time in open arm of mice under 400 lux,800 lux and 1200 lux light were significantly lower than that of control group,and the differences were statistically significant(50 lux vs.400 lux,P<0.05;501ux vs.800 lux,P<0.01;501ux vs.1200 lux,P<0.01).Conclusion:The spatial memory of the mice was not affected by the light with different intensities during the training period while the same light intensities were maintained during the test period.When the same light intensities were maintained during the training period,4001ux during the test period improved the spatial memory of the mice,while 8001ux and 1200lux inhibited the spatial memory of the mice,compared with the 501ux control group.At the same time,the anxiety of mice under 400 lux,800 lux and 1200 lux light increased significantly with the increase of light intensity.Part Ⅱ:Key brain regions involved in the process of spatial memory retrieval promoted by 400 lux lightObjective:To identify the key brain regions involved in the process of spatial memory retrieval promoted by 400 lux light in the Y maze test.Methods:Twenty adult male C57BL/6 mice were randomly divided into control group and intervention group,with 10 mice in each group.Y maze spatial memory behavior test was performed,respectively.The control group was exposed to 50 lux light both in the training period and the test period,the intervention group was exposed to 50 lux light during the training period and 400 lux light during the test period.1.5 h After of Y maze test,animals from each group were used for perfusion,sampling,making frozen sections and whole brain c-Fos staining.The key brain regions determined by c-Fos staining were injected with AAV-hSyn-Gcamp6m virus and embedded with optical fiber,then,in vivo optical fiber recording was used to collect calcium signal changes in the key brain regions induced by 400 lux light during the testing period of Y maze behavioral experiment.Retrograde AAV-hSyn-EGFP was injected into the locus coeruleus nucleus(LC)to observe its expression in LC and CeA;retrograde AAV-hSyn-EGFP was injected in the dentate gyrus(DG)region to observe its expression in DG and LC regions;Using Rabies virus retrograde transsynaptic tracing system,we inject the helper virus of RV into LC region.After 21 days of helper virus expression,RV(RV-ENVA-Δ G-dsRed)was injected into DG region.After 7 days,brain slices were made to observe the expression of the two viruses in brain tissue.Intravenous injection of AAV-CAG-EYFP virus was conducted and retrograde AAV-hSynmCherry virus was injected in CeA to observe the expression of the two viruses in brain tissue and verify the indirect relationship between retina and CeA.Results:The c-Fos expression in CeA(control group:274.80±19.67;intervention group:441.60±23.27;P<0.05),LC(control group:310.20±30.82;intervention group:392.40±25.32;P<0.05)and DG(control group:183.60±15.58;intervention group:411.00±26.63;P<0.01)showed significant differences between the two groups.The results of calcium imaging in CeA,LC and DG showed that the calcium signal activity in the above key brain regions was significantly enhanced under 400 lux compared with the 50 lux during the test period.The results of neural tracing experiment indicated that there was direct projection from CeA to LC and from LC to DG.Meanwhile,the LC neurons receiving CeA could further project into the DG.After intraocular injection of AAV-CAGEYFP virus and intracerebral injection of retrograde AAV-hSyn-mCherry virus in CeA,the simultaneous expression of the two viruses could be observed in the medial amygdala(Me A).This suggests a possible indirect neural pathway through which light affects the function of CeA.Conclusion:The results of c-Fos mapping and in vivo optical fiber recording indicate that the activity of CeA,LC and DG is significantly increased by 400 lux in the Y maze test period,suggesting that these brain regions play a key role in the process of spatial memory retrieval improved by 400 lux.Projection relationship between the above three brain regions was proved by viral tracing method.Meanwhile,we also verified the indirect pathway from retina to CeA.Part Ⅲ:Study of neural circuits involved in the process of spatial memory retrieval promoted by 400 lux lightObjective:To verify the function of CeA-LC-DG neural circuit in the process of spatial memory retrieval promoted by 400 lux light.Methods:Eighty adult male C57BL/6 mice were randomly divided into 4 groups with 20 mice in each group,which were injected with different chemogenetic viruses.AAV-DIOGi virus was injected in CeA or LC,retrograde AAV-cre virus was injected in LC or DG,to express the inhibitory chemogenetic receptor specifically in CeA or LC neurons which project to the LC or DG neurons.Inhibit the function of CeA-LC circuit or LC-DG circuit during the Y maze test under 400 lux,and to observe whether the promoting effect of 400 lux light on the spatial memory retrieval is affected.AAV-DIO-Gq virus was injected in CeA or LC,retrograde AAV-cre virus was injected in LC or DG,so to express the excitatory chemogenetic receptor specifically in CeA or LC neurons which project to the LC or DG neurons.Activate the function of CeA-LC circuit or LC-DG circuit during the Y maze test under 50 lux,and to observe whether the activation of these loop is able to generate the similar effect to that of 400 lux light in the test,which can promote spatial memory retrieval.The excitatory chemogenetic receptor Gq was expressed on the CeA-LC projection neurons and the cannulas were embedded.AAV-CaMKⅡα-Gcamp6m virus was injected into the DG region and the optical fiber was embedded.CNO was administered through the cannula in the CeA to activate the CeA-LC projection,and the activity of DG neurons was recorded by in vivo optical fiber calcium imaging.Results:In the Y maze spatial memory behavioral experiment,inhibition of CeA-LC(Saline group:46.61±10.99;CNO group:34.74±7.96,P<0.05)or LC-DG circuit(Saline group:50.03±10.19;CNO group:35.45±9.574,P<0.05)during the test period(400 lux)decreased the percentage of residence time in the new arm,and hindered the promotion effect of 400 lux light on spatial memory retrieval.In the Y maze spatial memory test,activation of CeA-LC(Saline group:28.64±2.41;CNO group:37.39±6.875,P<0.01)or LC-DG circuits(Saline group:29.55±3.91;CNO group:48.11±14.68,P<0.01)during the test period(50 lux)increased the percentage of residence time of mice in the new arm,which was similar to that of 400 lux light in improving spatial memory retrieval.While conducting chemogenetic activation of CeA-LC projection neurons,enhanced calcium signals of DG neurons were observed by optical fiber recording,suggesting the functional connections among brain regions within the CeA-LC-DG projection.Conclusion:The role of CeA-LC-DG neural circuit in the process of spatial memory retrieval promoted by 400 lux light was demonstrated by chemogenetic method combined with Y maze test under different light conditions.Functional association between brain regions in CeA-LC-DG projection was proved by chemogenetic activation combined with in vivo optical fiber recording.Part Ⅳ:Study on the neuron types and key neurotransmitters in target brain region involved in the process of spatial memory retrieval promoted by 400 lux lightObjective:To identify the key neurotransmitters that play a neuroregulatory role in the process of spatial memory retrieval promoted by 400 lux light.Methods:A total of 20 adult male C57BL/6 mice were randomly divided into 4 groups with 5 animals in each group,which were injected with tracer virus to identify neuron types.The viruses with markers of specific types of neurons as promoters and anterograde/retrograde tracing viruses were simultaneously injected into the upstream or downstream of CeA-LC and LC-DG neural projections to preliminarily determine the types of upstream and downstream neurons.For neurotransmitter dectection,a total of 30 adult male C57BL/6 mice were randomly divided into 3 groups with 10 animals in each group,which were injected with neurotransmitter sensor virus to examine the alternations in neurotransmitter releasing in certain brain regions.AAV-hSyn-CRF virus was injected into LC region,and optical fiber was embedded.In vivo optical fiber recording was used to capture the changing process of CRF signal in LC region under different illumination(50 and 400 lux)during the test period.AAV-hSyn-DA1h or AAV-hSyn-NElh virus were injected into the DG region respectively,and optical fibers were embedded.Signals of CRF neurotransmitters released by the terminals of CRH-positive neurons into LC region and DA or NE neurotransmitter released by the terminals of Th positive neurons into DG region were collected.The cannulas were embedded in LC and DG regions,and CRFR1 receptor blocker was injected into LC region through the cannulas before the Y maze test period(400 lux),to observe whether the promoting effect of 400 lux illumination on spatial memory retrieval was affected after blocking the effect of CRF on the neurons in LC region.Similarly,D1/D5 dopamine receptor blocker or β-type NE receptor blocker was injected into the DG region through the cannulas before the Y maze behavioral test period(400 lux light)to observe whether the promotion of spatial memory retrieval by 400 lux light was affected after blocking the effects of DA or NE on neurons in the DG region.Results:The virus tracing results of neuron type identification indicated that in the projection from CeA to LC,77%of the CeA neurons projecting to LC were CRH positive,and 86%of the LC neurons receiving CeA projection were TH positive.In LC to DG projections,77%of LC neurons that project to DG is TH positive,and 62%of DG neurons that receive LC projections are CaMKⅡα positive.The signal recording of CRF in LC region and DA or NE neurotransmitters in DG showed that the signals of neurotransmitters releasing in the above regions were significantly enhanced under 400 lux light compared with 50 lux light in the test period.After blocking the effect of CRF on LC neurons,the promoting effect of 400 lux light on spatial memory retrieval in mice was inhibited(ACSF(400 lux)group:48.48±6.90;CRFR1 antagonist(400 lux)group:33.96±6.76;P<0.05);After blocking the effect of NE on neurons in DG region,the promotion of spatial memory retrieval by 400 lux light was also inhibited(ACSF(400 lux)group:46.7±3.19;Beta-AR antagonist(400 lux)group:34.52±6.95;P<0.05).After blocking the effect of DA on neurons in DG region,the promotion of spatial memory retrieval by 400 lux light was not affected(ACSF(400 lux)group:45.48±5.11;Beta-AR antagonist(400 lux)group:37.80±4.38;P>0.05).Conclusion:The identification of neuronal types in neural circuit,neurotransmitters detection and neurotransmitter receptor blocking experimental results show that 400 lux light during the Y maze test period activates the CRH positive neurons in CeA,with projection to LC releasing increased CRF,acting on the CRFR1 receptors,so as to activate TH positive neurons in locus coeruleus,the projection of TH positive neurons to DG release more NE in this area,acting on the beta-AR receptors in DG,further activate the CaMKⅡαpositive neurons in the DG area,thus promotes the retrieval process of spatial memory.The neuroregulatory effects of CRF and NE played a key role in the process of spatial memory retrieval promoted by 400 Lux light.Part V:Study on the effect of long-term 400 lux light on spatial memory in mice and its synaptic plasticity mechanismsObjective:To investigate the effects of long-term 400 lux light on spatial memory in mice and explore its synaptic plasticity mechanisms.Methods:Twenty male adult C57BL/6 mice were randomly divided into 2 groups,with ten animals in each group.The dark and the light group are respectively raised under 50 lux and 400 lux,1 day,1 week and 4 weeks later,open field test and elevated plus maze test were used to evaluate the anxiety level of two groups of mice,and Y maze test was used to assess the spatial memory ability of mice.Fifty adults male C57BL/6 mice were randomly divided into two groups according to body weight,with 15 mice in each group.The dark light group and the bright light group were raised under 50 lux and 400 lux respectively.After 4 weeks,the spatial memory ability of the mice was tested by Morris water maze test.Simultaneously,the brains of behaviorally naive animals from the two groups were collected,and Golgi staining was used to observe the effects of different illuminance on the synaptic morphology of neurons in the hippocampal CA1 region.Western blot was used to detect the expression levels of p-αCaMKⅡ/αCaMKⅡ,p-CREB/CREB,BDNF,TrkB,postsynaptic density protein(PSD95)and synaptophysin in the hippocampal tissues of animals in two groups.The expression levels of PSD95,synaptophysin and BDNF in hippocampal tissues of two groups were detected by immunofluorescence assay.Immunohistochemical assay was used to detect the expression levels of p-αCAMKⅡ and p-CREB in hippocampal tissues of the two groups.Results:The results in elevated plus maze and open filed test together indicated that the anxiety level of mice in the bright light group was higher than that in the dark light group after 1 day(percentage of residence time in open arm:dim light group:13.96±0.78,bright light group:9.57±0.64,P<0.001;percentage of residence time in central area:dim light group:24.35±3.36,bright light group:16.33±3.61,P<0.001),but there was no significant difference between the two groups after 1 week and 4 weeks.After 1 week,the Y-maze spatial memory of the bright light group was weaker than that of the dark light group(dim light group:42.29±0.77;bright light group:37.17±0.86,P<0.001).After 1 week,there was no difference in the Y-maze spatial memory between the two groups.After 4 weeks,the Y-maze spatial memory of the bright light group was better than that of the dark light group(dim light group:36.83±1.53;bright light group:45.88±1.24,P<0.001),similar results were found in the water maze experiment.The learning curve during the water maze training period indicated that on days 2,4,and 5 of training,the escape latency of the bright light group was lower than that of the dark light group(F training day(4,72)=77.23,P<0.0001;F light condition(1,18)=48.39,P<0.0001).During the probe test,the bright light group had shorter first escape latency(dim light group:32.63±2.07,bright light group:25.45±1.04,P<0.01),less platform quandrants crossings(dim light group:1.70±0.30,bright light group:2.8±0.25,P<0.05)and shorter time spent in the target quandrants(dim light group:21.57±0.69,bright light group:30.20±1.91,P<0.001).The results of Golgi staining indicated that the density of dendritic spines of pyramidal neurons in hippocampal CA1 region of bright light group was higher than that of dark light group,and the number of mushroom dendritic spines was higher in bright light group than in dark light group.Western blot results showed that the ratio of p-αCaMKⅡ to αCaMKⅡ(1.58±0.07 fold,P<0.001),the ratio of p-CREB to CREB(1.95±0.19 fold,P<0.01),the expression levels of BDNF(1.83±0.18 fold,P<0.05)and the ratio of p-TrkB toTrkB(1.44±0.09 fold,P<0.05),the expression levels of PSD95(1.73±0.08 fold,P<0.001)and Synaptophysin(1.85±0.06 fold,P<0.001)were higher in the bright light group than in the dark light group.The results of immunofluorescence assay indicated that the expression levels of PSD95(1.92 ±0.10 fold,P<0.0001),synaptophysin(1.85±0.06 fold,P<0.001)and BDNF(1.29±0.07 fold,P<0.01)in the bright light group were higher than those in the dark light group.Immunohistochemical results indicated that the expression levels of p-αCAMKⅡ(1.52 ±0.04 fold,P<0.0001)and p-CREB(dim light group:236±5,bright light group:270±6,P<0.01)in the bright light group were higher than those in the dim light group.Conclusion:The anxiety level and spatial memory in mice exposed to bright light change with time.Compared with the control group,4 weeks of bright light exposure can improve spatial memory in mice,and affect the density and morphology of dendritic spines in the hippocampus CA1 region,simultaneously increase the expression of synaptic structural proteins including synaptophysin and PSD95,which may result from the increased expression of the key molecules in the CaMKⅡ-CREB-BDNF/TrkB pathway elicited by bright light.Summary:These above results suggested that 400 lux light exert acute promoting effect on the spatial memory retrieval,with the neural circuit mechanism that light could activate the CRH positive neurons of the CeA,with its projections in LC releasing increased CRF,acting on the LC CRFR1 receptor and inducing the activation of TH positive neurons,which result in the projection of TH positive neurons to DG releasing more NE,further acting on the Beta norepinephrine receptor and leading to the activation of CaMKⅡαpositive neurons,so as to promote the spatial memory retrieval.Meanwhile,long-term daytime light of 400 Lux improved spatial memory and affected the morphology and density of dendritic spines,accompanied by increased expression of synaptic structural proteins including synaptophysin and PSD95,which may be related to the activation of CaMKIICREB-BDNF/TrkB molecular pathway. |
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