Involvement Of BDNF On Memory Extinction And Ischemic Stroke-induced Memory Deficits

Background:Cognitive function is the high-level neural activity of human and animal brain,mainly including mental and intellectual activities,such as feeling,perception,learning,memory,among which the most important is learning and memory.Learning and memory is a complex neural activity process formed by the interaction and interconnections between various neurons and synapses.The study on the molecular mechanisms of learning and memory,especially the memory deficits caused by diseases,will provide new targets for the treatment of disease-related cognitive impairment.Conditional taste aversion(CTA)is a classic conditional reflex.It works like this:Animals learn to associate the novel taste of a new food with the subsequent internal discomfort,such as nausea,vomiting,diarrhea.CTA is formed by single-trial training and is a long-lasting memory.It could be used to study an important memory:memory extinction.Memory extinction is closely related to post traumatic stress disorder,which can be caused by a number of illnesses,including stroke.In addition,the neural circuit of CTA memory is relatively clear.According to neuroanatomy study,thalamus,amygdala,and insular cortex(IC)may be the key brain regions in CTA memory processes.It will be useful for studying the memory mechanism.Stroke is an acute cerebral blood circulation disorder caused by internal artery stenosis,occlusion or hemorrhage.It is one of the three major diseases that threaten human life.It can be divided into hemorrhagic and ischemic according to the pathogenesis.Ischemic stroke accounts for about 80%of all the stroke,and increases at the rate of 8.1%per year.In China,70-80%of the survivors of ischemic stroke suffer from physical and cognitive dysfunction,which brings serious burden to the families of patients and society.However,most current studies focused on promoting motor function recovery and improving language impairment,while the memory impairment after ischemic stroke has not received enough attention.Memory impairments do not only cause daily living disability,but also adversely affects the recovery of somatosensory motor function of ischemic stroke patients.Therefore,it has great medical value and social significance to study the molecular mechanism of memory disorder after ischemic stroke.Brain derived neurotrophic factor(BDNF),the most abundant and widely distributed member of the neurotrophic family in the central nervous system,is highly expressed in cognitive-related brain regions,such as hippocampus,cortex and striatum.BDNF involves in the survival,differentiation,growth and repair of neurons.It plays important roles in learning and memory and other biological functions.Studies showed that neuroplasticity is the basis of functional recovery after stroke.BDNF plays an important role in the regulation of neuronal plasticity.After synthesis,BDNF is usually stored in the dense core vesicle(DCV)of the synapse.The release of BDNF and its binding to tyrosine kinase receptor B(TrkB)can activate a series of intracellular signal transduction pathways,including mitogen activated protein kinase(MAPK),phosphatidylinositol 3-kinase(PI3K)and phospholipase C(PLC).However,the mechanism of BDNF secretion is still unclear.Therefore,finding molecules,regulating secretion of BDNF to effectively promote nerve repair,is expected to be an important treatment for memory disorders caused by ischemic stroke.In conclusion,using rat CTA paradigm,middle cerebral artery occlusion model,real-time PCR,in situ hybridization,IP,western-blot and other molecular biological research methods,we determined the role and mechanism of BDNF in CTA memory extinction and memory impairment after ischemic stroke.Our study will help further the understanding of neurobiological mechanism of BDNF involving in learning and memory,and provide new targets and methods for the diagnosis and treatment of memory disorders in ischemic stroke.Objective:1.To investigate the effects and mechanisms of BDNF secretion in the insular cortex on CTA memory extinction.2.To investigate the effects and mechanisms of BDNF and its regulator in memory deficits induced by ischemic stroke.Methods:1.MicroinjectionMale Wistar rats were anesthetized and restrained in a stereotaxic apparatus,and implanted bilaterally with guide cannula aimed at 1.0 mm above the insular cortex(coordinates in reference to bregma).Animals were allowed one week to recover before being subjected to the following experimental.BDNF antibodies were microinjected immediately after the CTA extinction test for three consecutive days.The effects of BDNF antibodies were evaluated by CTA extinction tests.2.CTA memory extinctionRats were deprived water for 24 h and given water in a fixed time every day.After 3 days adaption,animals get into the habit of drinking water in fixed time.On the 4th day,the water was replaced to equal amount of 0.1%saccharin.40 minutes later,rats received intraperitoneal injection of LiCl,which can induce abdominal discomfort to establish a conditioned reflex.3 days after conditioning,rats were presented with both saccharin and water to test.Rats were tested for 4 consecutive days.The amount of water and saccharin consumed were recorded.Aversion index(AI)were analyzed according to the following method.AI=[water/(water+saccharin)]×100%.The AI of animals gradually decreased,indicating that the animals gradually forgot the aversion memory after extinction training.3.Ischemic Stroke Model After anesthetization,the left common carotid artery and external carotid artery of rats were exposed.A silicon-coated monofilament was injected into the internal carotid artery to block the left middle cerebral artery(MCA).2 h later,the line was drawn out carefully.Sham-operated animals were subjected to the same surgical procedures but the catheter was not inserted into the CCA.4.Real-time PCRRats of experimental group were sacrificed at different time points after the second extinction test and ischemic stroke.Brain tissues were dissected out and then RNA was extracted.The quantitative total RNA was reversely transcribed into cDNA.Finally,different annealing temperatures were selected for real-time quantitative PCR to detect the changes of target genes.5.BDNF ElisaBrain tissues of rats were taken at specific time points after CTA memory extinction and ischemic stroke.Protein concentration was measured after homogenization.The analytical solution,standard protein and samples were added to each hole in turn.Then the samples were incubated for 2 hours at room temperature.After washing,the second antibody was incubated.Then,the substrate working liquid was added.Finally,the reaction was terminated.6.In Situ HybridyzationRats were deeply anesthetized and transcardially perfused with 4%paraformaldehyde 6 hours after the second extinction test.The brains were removed and post-fixed overnight.After equilibration,the brains were sectioned(40 um/section)and in situ hybridization was performed to detect the BDNFmRNA levels.The threshold value was defined as adjacent background area lacking hybridization.For each section,normalized density=(region of interest density-background density).For statistical analysis,the density of hybridization was reported as the average density of all individual animals±standard error of the mean in each group.The mean density of all microphotographs was analyzed with the aid of Image J software.7.ImmunoprecipitationThe rats were sacrificed at different time points after the second extinction test.Brain tissues were dissected and homogenized on ice in radioimmunoprecipitation assay buffer.After centrifugation,the supernatant was collected.The concentration of total protein was determined.Subsequently,5 mg of homogenized lysate was immunoprecipitated overnight at 4? using rabbit anti-TrkB antibodies.Samples coupled with sample buffer were loaded and separated through electrophoresis,and transferred to a polyvinylidene difluoride(PVDF)membrane.After blockage,the blots were separately incubated with the following antibodies:mouse anti-phospho-tyrosine antibodies pY99 or mouse anti-TrkB antibodies.Western blotting signals were detected using the ECL chemiluminescence system.The scanned images were visualized with the Quantity One Manual software.Ratios of immunoprecipitated p-TrkB over total TrkB derived from 0 h were normalized to 1.0.The values of other groups were normalized according to the 0 h group.8.Western blotRats were sacrificed after the second CTA extinction test or MCAO surgery.Brain tissues were dissected and homogenized in radioimmunoprecipitation lysate.Sample buffer was added and boiled to denude the protein.After electrophoresis,membrane transfer and blocking,c-Fos,caspase-3,Erk,phosphorylated Erk,CAPS1,LC3B,TrkB,phosphorylated TrkB,Akt and phosphorylated Akt antibodies were added separately and incubated overnight at 4?.Then,the corresponding secondary antibody was added and incubated at room temperature for 1 hour.Finally,Western blotting signals were detected using the ECL chemiluminescence system.9.ImmunohistochemistryThe ischemic rats were anesthetized at 1 day and 3 days after ischemic stroke.The brains were dissected out after perfusion,and sectioned with a frozen slicer(20?m/section)for immunofluorescence staining.Sections were blocked with 5%goat serum closed for 1 hour.The antibodies of CAPS1 and secretogranin I,the marker of dense core vesicles,were incubated at 4? overnight.After incubation with second fluorescent antibodies,DAPI was added for 5 minutes.The co-location of CAPS1 and DVC was observed under the microscope.10.Morris Water MazeThe water maze experiment is a common test for learning and memory after ischemic stroke.Rats were allowed to recover for 1 week after surgery.The water maze experiment was begun to perform on day 8.A camera connected to the display system was placed above the maze to record the movement track of the rat.Positioning and navigation experiment:rats were put into the water from the four quadrants successively.The time that finding the platform under the water surface was recorded to detect the escape latency.The shorter time reflects stronger learning ability.Space exploration experiment:the platform was removed on the 13th day.The activity time spent in the quadrant in 60 seconds were recorded.Results1.The effects of BDNF in the insular cortex on CTA memory extinction1.1 The involvement of BDNF in the insular cortex in CTA memory extinctionTo examine the biological effects of endogenous BDNF in the IC in CTA extinction,we micro-infused an anti-BDNF antibody into the IC immediately after extinction test.This was repeated for 3 consecutive days.The high aversion index in the first-day test indicated successful CTA conditioning.Rats that underwent treatment with BDNF antibody infusion maintained a high aversion index,whereas the control animals exhibited markedly reduced aversion index,indicating the extinction of CTA by the consumption of saccharin without the association with LiCl injection in the control group.To investigate whether CTA extinction induced neuronal activity after CTA extinction in the IC,expression levels of c-Fos were measured 90 min after the second extinction test using Western blotting.The levels of c-Fos in the IC were significantly increased 90 min after extinction as compared with control group,suggesting induced neuronal activity after the CTA extinction test.These findings suggest that BDNF/TrkB signaling in the IC is functionally involved in CTA extinction.To futher investigate the changes of BDNF in the IC after CTA extinction,rats were sacrificed at different time points after the second extinction test,and the BDNF mRNA and protein levels in the IC were detected.We found that both the mRNA and protein levels of BDNF elevated in the IC during CTA extinction.In addition,the mRNA levels of NGF and NT4 were also detected at different time points after the second extinction test to assess the possibility of these neurotrophin members being altered in the IC during CTA extinction.The mRNA levels of NGF and NT4 did not change after the second extinction test,suggesting that CTA extinction selectively induced the gene expression of BDNF in the IC.Subsequently,in situ hybridization further confirmed the changes in the mRNA levels of BDNF during extinction.Similar to the results obtained from the real-time PCR analysis,the mRNA levels of BDNF increased in the IC after the second extinction test compared with control group.These results indicate that CTA extinction induced high BDNF expression.The changes of BDNF involve in the CTA memory extinction.1.2.CTA memory extinction induces activity-dependent BDNF secretion in the insular cortexThe release and binding of BDNF to its specific receptor(TrkB)triggers its dimerization and autophosphorylation.The levels of phosphorylated TrkB can reflect BDNF secretion.We next investigated whether CTA extinction induced activity-dependent BDNF secretion.The levels of p-TrkB in the IC were examined by immunoprecipitation.We selected the 1 hour and 8 hours after the second extinction test to detect the levels of total TrkB and p-TrkB.The levels of p-TrkB/TrkB were found to be significantly higher at both the 1 hour and 8 hours after CTA extinction test than those reported in the control group in the IC.The levels of BDNF protein and p-TrkB increased significantly at 8 hours.This effect was probably due to the release of newly synthesized BDNF.In addition,the elevated levels of p-TrkB at 1 hour after extinction,when both gene expression and protein levels of BDNF were unaltered,suggest that secretion of BDNF is responsible for this effect.These results indicate that CTA extinction training induces rapid activity-dependent BDNF secretion in advance of the increased synthesis of BDNF in the IC.To further confirm the BDNF release after CTA extinction,expression levels of phosphorylated Erk and total Erk,molecules of the downstream signaling pathway,were detected.Similar to the changes observed in p-TrkB,the expression levels of p-Erk were increased at both the 1 hour and 8 hours after CTA extinction.In contrast,there were no changes found in the levels of total Erk in the IC after CTA extinction.These results further confirmed the release of BDNF after CTA extinction.1.3 CTA extinction attenuates neuronal apoptosisThe apoptosis of neurons is related to memory deficits.To investigate the neuron apoptosis after CTA extinction,the levels of caspase-3 in the IC was detected by western blot.Contrary to the changes observed in the levels of p-TrkB,the expression levels of caspase-3 decreased at both the 1 hour and 8 hours in the IC after CTA extinction.These results further confirmed that CTA extinction inhibits neuronal apoptosis.2.The involvement and mechanisms of BDNF secretion and its regulatory molecules in memory disorders induced by ischemic stroke2.1 Increased BDNF expression and secretion in the hippocampus induced by ischemic strokeRats were sacrificed at 1 day,3 days and 7 days after ischemic stroke.Hippocampus was dissected out and the changes of BDNFmRNA and BDNF protein were detected by real-time PCR and Elisa,respectively.The changes of TrkB phosphorylation and its downstream molecule phosphorylated Akt and total Akt were detected by western blot.The results showed that both BDNFmRNA and BDNF protein in the hippocampus increased after ischemic stroke.p-TrkB and p-Akt also increased at the time points that BDNF increased,indicating BDNF secretion in the hippocampus induced by ischemic stroke.2.2 Increased CAPS1 expression in the hippocampus induced by ischemic strokeIn vitro studies showed that Ca2+-dependent the activator protein for secretion 1(CAPS1)plays an important role in regulating BDNF release,which is a calcium-dependent process.To determine the involvement of CAPS1 in regulating BDNF secretion after ischemic stroke,rats were sacrificed at the time points that BDNF increase induced by ischemic stroke.The expression changes of CAPS1 were detected by western blot.The results showed that the expression of CAPS1 increased at the time point when BDNF and p-TrkB increased,indicating that CAP S1 may be involved in the secretion of BDNF after ischemic stroke.2.3 CAPS1 regulates BDNF secretion by binding with dense core vesicles in the hippocampus after ischemic strokeAfter synthesis,BDNF is stored in dense core vesicles.To investigate the mechanism of CAPS1 regulating BDNF secretion,immunohistochemistry was performed to detect the co-localization of CAPS1 with dense core vesicle.Our results indicate that the colocalization of CAPS1 with dense core vesicles was enhanced at the time point of BDNF increase,suggesting that CAPS1 regulates BDNF secretion by binding with dense core vesicles.2.4 The involvement of CAPS1 in memory deficits after ischemic stroke by regulating BDNF secretion in the hippocampusTo further determine whether CAPS1 is involved in the regulation of memory disorders after ischemic stroke by regulating BDNF secretion,we designed CAPS1 adeno-associated virus.The adeno-associated virus was microinjected into hippocampus.Ischemic stroke procedure was performed 1 month after injection.1 week after stroke,memory disorder was tested by Morris water maze.Compared with the control adeno-associated virus,the injection of CAPS1 adeno-associated virus resulted in decreased expression of CAPS1 and p-TrkB in the hippocampus on 1 day and 3 days after MCAO,and showed no difference with the sham group,indicating that CAPS1 adeno-associated virus could inhibit BDNF secretion.Further application of water maze experiment confirmed that ischemic stroke induced prolonged escape latency,which indicated impaired spatial learning and memory.However,with the extension of training time,the learning and memory ability of stroke rats gradually recovered to a normal level,indicating the gradual recovery of memory deficits.However,the memory deficits could not be recovered in the stroke rats injected with CAPS1 adeno-associated virus in the hippocampus.These results suggest that hippocampal CAPS1 is involved in the recovery of memory damage after ischemic stroke by regulating the secretion of BDNF.Conclusion:1.CTA memory extinction could induce BDNF secretion in the insular cortex,which involves in CTA memory extinction.2.CTA memory extinction inhibits neuron apoptosis in the insular cortex.3.Ischemic stroke increases BDNF expression and secretion in the hippocampus.4.Ischemic stroke increases CAPS1 expression in the hippocampus,which regulates BDNF secretion by binding with DCV.5.CAPS1 participates in ischemic stroke-induced memory deficits by regulating BDNF secretion.Significance:Our study will help further the understanding of the precise role of BDNF and its' regulator in memory-related disease.Moreover,it will provide new ideas and new targets for the treatment of cognitive dysfunction after ischemic stroke.