Endothelin-1-induced mini-stroke in the dorsal hippocampus or lateral amygdala results in deficits in learning and memory

Tao Sheng,Xueting Zhang,Shaoli Wang,Jingyun Zhang,Wei Lu2,,?,Yifan Dai,?

1The Center of Metabolic Disease Research;2Department of Neurobiology,Nanjing Medical University,Nanjing,Jiangsu 210029,China;3The Key Laboratory of Developmental Genes and Human Disease,Institute of Life Sciences,Southeast University,Nanjing,Jiangsu 210096,China.

Shaoli Wang1,Jingyun Zhang4,Tao Sheng3,Wei Lu2,4,?,Dengshun Miao1,?

1The Research Center for Bone and Stem Cells,Department of Human Anatomy;2Department of Neurobiology;3The Center of Metabolic Disease Research,Nanjing Medical University,Nanjing,Jiangsu 210029,China;4The Key Laboratory ofDevelopmental Genes and Human Disease,Institute of Life Sciences,Southeast University,Nanjing,Jiangsu 210096,China.

Endothelin-1-induced mini-stroke in the dorsal hippocampus or lateral amygdala results in deficits in learning and memory

Tao Sheng1,Xueting Zhang3,Shaoli Wang3,Jingyun Zhang3,Wei Lu2,3,?,Yifan Dai1,?

1The Center of Metabolic Disease Research;2Department of Neurobiology,Nanjing Medical University,Nanjing,Jiangsu 210029,China;3The Key Laboratory of Developmental Genes and Human Disease,Institute of Life Sciences,Southeast University,Nanjing,Jiangsu 210096,China.

Functional and structural alterations in brain connectivity associated with brain ischemia have been extensively studied.However,the mechanism whereby local ischemia in deep brain region affect brain functions is still unknown.Here,we first established a mini-stroke model by infusion of endothelin-1(ET-1)into the dorsal hippocampus or the lateral amygdala,and then investigated how these mini-infarcts affected brain functions associated with these regions.We found that rats with ET-1 infusion showed deficit in recall of contextual fear memory,but not in learning process and recall of tone fear memory.In novel object task,ET-1 in the hippocampus also eliminated object identity memory.ET-1 in the lateral amygdale affected acquisition of fear conditioning and disrupted retention of tone-conditioned fear,but did not impair retention of contextual fear.These findings suggest that ET-1-induced mini-infarct in deep brain area leads to functional deficits in learning and memory associated with these regions.

endothelin-1,dorsal hippocampus,lateral amygdala,fear conditioning

Introduction

Occlusions in the blood supply to the brain lead to ischemic stroke[1].Focal stroke is associated with abnormal synaptic activity,morphologic plasticity and neurologic impairments[2-3].Several rodent focal ischemia models are available to study the pathology of stroke[4].The most commonly used one is middle cerebral artery occlusion(MCAO)[5]by insertion of an intraluminal suture[6-7].Mini-ischemia can be produced by coagulation of the MCA,embolism or photothrombosis.In photothrombosis,a photosensitive dye (i.e.,Rose Bengal)is systemically injected into animals where part of the skull has been thinned[8].It can be used to produce small infarcts in any cortical region[9], but it seemed unable to induce deep infarct.In MCAO or MCA embolism animal models,clots can undergo spontaneous thrombolysis,thereby inducing multiple infarcts and high mortality[4,10].To study memory and emotion linked neural circuit remodeling after stroke, it is desirable to establish ischemia models with infarct in deep brain regions(i.e.,the hippocampus and the amygdala).Endothelin-1(ET-1),a potent vasoconstrictor[11],occludes local blood flow to levels that cause ischemia injury[12].ET-1 can be stereotaxically injectedinto brain regions of interest to constrict local vessels. Lesion size can be adjusted by altering the concentration or volume of ET-1 to achieve reproducible or permanent injury.It has been reported that ET-1 injections into the hippocampus caused significant loss of hippocampal tissues[13-14].However,it is still unclear that if ET-1 injections cause deficits in function associated with these brain regions.

In the present study,we infused ET-1 to dorsal hippocampus and lateral amygdala to produce focal deep brain ischemia and then test whether ET-1 can create reliable learning and memory deficits.In earlier studies, lesions caused by pharmacological method(ibotenic acidAPV)or adeno-associated virus injection(expression of tetanus neurotoxin)have confirmed the corresponding dysfunction of the hippocampus[15-20]or lateral amygdala[21-24].Our results demonstrated that mini-stroke in these regions caused deficits in learning and memory.

Materials and methods

Animals

Male Sprague-Dawley(SD)rats weighing approximately 300 g at the time of surgery used in this study were housed in environmentally controlled conditions (23°C±1°C,a 12 hours light/dark cycle with the light cycle from 6:00 to 18:00 and the dark cycle from 18:00 to 6:00).The rats were random ly divided into groups.Before testing,rats were handled for about 4-5 minutes daily for 4 consecutive days.The study protocol was approved by the local institutional review board at the authors’affiliated institutions and animal studies were carried out in accordance with the established institutional guidelines regarding animal care and use.Animal welfare and the experimental procedures were carried out strictly in accordance with the Guide for Care and Use of Laboratory Animals.

Surgical procedures

Rats were initially anesthetized with 3% chloral hydrate,and then 1% chloral hydrate was used to maintain anesthesia.Temperature was maintained from 35.5°C to 37.5°C throughout the surgery.SD rats were p laced in a stereotaxic apparatus(Stoelting,Wood Dale,IL,USA).A gauge syringe was targeted for placement directly into the hippocampus(anteroposterior: 4.50 mm relative to the bregma;mediolateral:±4.0 mm;dorsoventral:3.5 mm from skull)and lateral amygdala(anteroposterior:3.14 mm relative to bregma;mediolateral:±6.2 mm;dorsoventral:5.4 mm from skull).Rats in the experimental group received unilateral(for magnetic resonance imaging, MRI)or bilateral(for behavior test)infusion of ET-1 (15 pmoL,0.8 μL dissolved in sterile saline;Sigma-Aldrich,St Louis,MO,USA).Rats in the sham group received bilateral infusion of saline with the same surgical procedure.The volume and flow rates(10 nl/s, 0.8 μL)were controlled by a microsyringe pump controller(WPI,Florida,USA).The syringe was left in place for 5 minutes after injection.Rats were returned to their home cage after surgery.

Magnetic resonance imaging

Animals underwent focal ischemia using ET-1 and were examined 24 hours(for sample image,but 48 hours for behavior testing of animals’injected locations)after ET-1 administration.MRI was performed using 7.0-T MR imager(Eclipse,Philips Medical Systems,Best,the Netherlands)equipped with a 21-cm bore magnet as previously studied[25].MRI sequences were T2-weighted spin-echo sequences [repetition time(msec)/echo time(msec),500/17.9]. The rats were anesthetized with 1.5% halo thane in a 70:30 mixture of N2O:O2.The imaging slice was placed 3 mm anterior to the bregma using as landmark the anterior commissure obtained at the mid line. Images were obtained with a matrix size of 128×128, two measurements acquired.

Open field task

The apparatus for the open-field consisted of a 100-cm-square open translucent plastic box with 50 cm high.The box was illuminated by ambient fluorescent ceiling lights.We monitored it by an automated video motility system including a camera and tracing software Any-Maze(Stoelting Company,Wood Dale,IL, USA).In the open-field test,rats were placed individually near the center of the box,and their movements were recorded by video for 5 minutes.The imaginary central zone was defined as a 50×50 cm square in the middle of the observation area.We quantified the time spent in center squares and surrounding area,total moving distance,average speed,and number of times that rats reared on their hind paws.

Fear conditioning

Rats were handled for 3 minutes on each of 5 consecutive days before experiments.Training chambers was positioned inside a sound-attenuating isolation box and included a square chamber with 2 plexiglas sides and 2 aluminum sides and a removable controlled electrifiable grid floor(Coulbourn Instruments,Whitehall,PA, USA).We conditioned rats in a previously optimized standard,combined context and tone conditioned fearprotocol in which the rat received 6 shocks as described previously[26].

On day 1,rats were habituated to training background for 12 minutes with no additional operation. On day 2,rats were allowed adapt to the chamber for 4 minutes before the onset of training blocks.Then, rats were presented with 6 consecutive training blocks, each consisting of a 20-second baseline,followed by a 20-second,1KHz and 80 dB tone(conditioned stimulus,CS),followed by a 2-second scrambled 1.2 mA foot shock(unconditioned stimulus,US),followed by a 38-second inter-trial interval(ITI).Rats were returned to their home cage immediately after training.

Two hours later,rats were tested for contextual and tone fear conditioning.Rats were firstly placed in the training chamber for 3 minutes to evaluate contextual fear conditioning,after which they were returned to home cage for 30 minutes.Testing for tone fear conditioning took place in a novel chamber,which differed from the training chamber in texture(solid plastic floor rather than metal grid floor),visual cues(solid walls with corrugated board on each side opposed to 2 clear Plexiglas and 2 plain aluminum sides)and odor.Rats were allowed to acclimate to the novel chamber for 3 minutes prior to tone presentation.To assess the percentage of freezing in the novel chamber,rats that showed indiscriminate freezing(＞50%)in the novel chamber by the end of the adaption period were excluded from further tone fear conditioning analysis[27].A fter 3-minute acclimation period,rats were tested with 4 blocks consisting of a 20-second baseline followed by a 20-second,1 KHz and 80 dB tone,and then followed by a 40-second inter-trial interval.

Percentage of freezing was quantified by automated motion detection software(FreezeFrame,coulbourn instruments,Whitehall,PA,USA).A single set of motion detection parameters was optimized for rats in this study and used for all analyses.

Fig.1 Rats with ET-1-induced DH mini-stroke showed normal spontaneous activity.A:Rats were injected with ET-1 in one side DH and saline in the other side DH.B:Representative T2-weighted MR imaging shows infarct injury of DH as a result of ET-1 injected into this position.C-F: Performance of the open-field task shows similar color-coded time-in-location map(C),the time in the central zone and surrounding zone(D),total moving distance(E)and average speed(F),indicating the same active mode for both treatment groups.In all the animals for behavioral test,ET-1 was injected bilaterally.In(C),same blue-to-red scale is used for each map.

Novel object recognition task

An open-field box(50×50×40 cm)was constructed from high density polyethylene board.Thebox was illuminated as described in the method of open field testing.Before training,rats were individually habituated by driving them to explore the open-field box for 5 minutes per session for 2 sessions. During training stage,2 novel objects(red regular tetrahedron made by corrugated paper)were positioned in the open-field 30 cm away from each other and rats was allowed to explore for 5 minutes,respectively. By tracing the head of rat,exploring behavior was identified when the head of rats facing the object within 3 cm away or any part of the rats excluding the tail touch the object.The time spent for exploring each object was recorded.Rats were returned to their home cages immediately after training.During retention test(2 hours later),the rats were positioned back into the same open-field box again.However,one of the above used objects during training was replaced by a novel object(blue regular hexahedron made by polypropy lene).All objects were balanced and were emotionally neutral.Moreover,the open-field and objects were cleaned by 10% alcohol after each session to avoid possible odorant cues.Discrimination ratio,an index of the time spent to explore the familiar object and the novel object were normalized by the time exploring the familiar and over the total time spent exploring both objects,was used to measure recognition memory.

Data analysis

Data were expressed as mean±SEM.Differences between groups were compared using independent sample t-test(two populations)and ANOVA post-hoc comparisons.Two-way ANOVA(treatment×interval) was performed to detect significant differences between groups,and significant differences at some intervals were established with a Bonferroni post-hoc test.In all other cases,a two-tailed Student’s t-test was used. Differences were considered significant when P was＜0.05.

Results

ET-1 induces mini-stroke in the dorsal hippocampus

To study ET-1 induced lesion in the hippocampus, we in fused ET-1 into the dorsal hippocampus (Fig.1A)[28].We used MRI to examine and confirm the establishment of the mini-stroke model.Twenty-four hours after injection,we used T2-weighted spin-echo MRI sequences to confirm the generation of mini-stroke(Fig.1B)[25,29].The site of local infarct shown by MRI matched ET-1 injection site in the dorsal hippocampus.In contrast to the recession of mobility caused by the damage that results from MCAO),rats receiving ET-1 showed normal spontaneous activity in open field test(Fig.1C),moving distance (Fig.1D)and average speed(Fig.1E)compared to those receiving sham treatment.

Fig.2 ET-1 induced min i-stroke in the dorsal hippocampal(DH)region impairs recall of contextual fear memory.A:Schematic of fear conditioning protocol.B:Rats with ET-1-induced dorsal hippocampal mini-stroke exhibit normal freezing percentage in a 6-trial training protocol. ITI:inter train interval.C:Freezing during 3 minutes of testing in the training context(*P＜0.05,two-tailed Student’s t-test)but not in a novel context (P=0.470,two-tailed Student’s t-test)is significantly impaired in ET-1 infused rats(sham,n=9,DH-stroke,n=9).D:Tone conditioning is unaffected in ET-1 injected rats 2 hours after training(no significant main effect of treatment,F(1,42)=1.991,P=0.878).

ET-1-induced mini-stroke in the dorsal hippocampus impairs recall of contextual fear memory

We investigated the acquisition of the association memory between fear and tone.We trained adult male rats(sham and DH-stroke treatment)using a tone fear conditioning protocol(Fig.2A).Intact hippocampal function is critical for eliciting contextual fear conditioning(freezing in response to training context exposure),but not for tone(freezing in response to tone presentation)fear conditioning in response to this training protocol[24,30-31].In contrast,rats with ET-1 induced mini-stroke in the dorsal hippocampus rats learned fear memory as well as controls did(Fig.2B).When tested 2 hours after training,dorsal hippocampus-stroke resulted in a dramatic reduction in retention of contextual fear memory(Fig.2C)and normal tone fear memory(Fig.2D).These data provide evidence that ET-1 in the dorsal hippocampus induced hippocampal lesion and impaired hippocampus-dependent fear learning in adult rats.

Fig.3 ET-1 in fusion in the dorsal hippocampus impairs recognition memory.A:Schematic of novel object recognition task.B:Color-coded time-in-location map in the training session.The same objects were positioned bottom-left and upper-right.C:The amount of time spent in exploring the two objects was the same for treatments.Dotted line represents performance at chance(50%).(D)Color-coded map and(E)discrimination ratio in testing session show the impairment of object discriminate memory in the ET-1 infused rats(sham,n=9,DH-stroke,n=9,*P＜0.05,two-tailed Student’s t-test).The same blue-to-red scale is used for each map.DH:dorsal hippocampus.

ET-1-induced mini-stroke in the dorsal hippocampus impairs recognition memory

Then,we used the novel-object-recognition task (Fig.3A)to assess recognition memory,which also requires the hippocampus[32-33],in ET-1-infused animals.During training session,2 treatments had no significant difference in time that rats spent in exploring the 2 novel objects(as shown by hot map and exploratory preference;Fig.3Band3C),indicating that both groups of rats had the same motivation and curiosity to explore the objects.During the testing stage,one of the familiar objects used in the training was replaced by a novel object,and rats were allowed to explore for 5minutes.At the 2-hour retention test,rats of the ET-1 infusion dorsal hippocampus-stroke groups showed dramatic reduction in time exploring the novel objects compared to the sham group,indicated by the color-coded map(Fig.3D).In contrast,preference towards the familiar object was similar between the 2 groups(as shown by the discrimination ratio,Fig.3E).This indicated that ET-1 in the dorsal hippocampus impaired capability of rats to identify and recognize novel objects.

ET-1-induced mini-stroke in the lateral amygdala impairs acquisition and retention of tone-conditioned fear memory

We further studied whether and how ET-1 induced mini-stroke in the lateral amygdala would affect brain functions associated with this subcortical region. Twenty-four hours after ET-1 injection,we used T2-weighted MRI to examine the region of ischemia (Fig.4AandB).Rats with bilateral lateral amygdala mini-stroke showed normal exercise capacity (Fig.4C-4F)as the precondition of the following test. The lateral amygdala is imperative for obtaining the association of cued message and emotional memory, and is also critical for tone fear conditioning,but not for contextual fear memory[23-24].We trained rats of both groups using the protocol as shown inFig.2A. Compared to sham rats,lateral amygdala-stroke mice learned the association between shock and tone/context more tardily indicated by a delayed increase in freezing percentage during 6 training trials(Fig.5A).Two hours later,rats were positioned in training context, neither sham nor lateral amygdala-stroke rats showed significant freezing behavior in the training context (Fig.5B).Then,rats were placed in a novel context and presented with only the tone.Lateral amygdala stroke rats showed a dramatic reduction in retention of ton fear memory(Fig.5C).These results indicated that rats with bilateral lateral amygdala mini-stroke show deficits in tone components of aversive conditioning with contextual fear memory kept intact.

Discussion

Fig.4 ET-1 induced mini-stroke in lateral amygdala(LA).A:Rats were injected with ET-1 in one side of LA and saline in the other side of LA.B:Representative T2-weighted MR imaging showing the infarct injury as a result of ET-1 injection into LA.C-F:Performance of the open-field task shows similar color-coded time-in-location map(C),the time in the central zone and surrounding zone(D),total moving distance(E)and average speed (F),indicating the same active mode for both treatment groups.

ET-1 is commonly used to produce focal ischemia in rodents[14,29,34-36].The average lesion size in this study was smaller or larger than the study by Sharkey[37]or Jamshid[14],this may be partly due to the dose of ET-1,stereotaxic coordinates and different rat strain in this study.Previous studies have suggested that MCAOproduces a success rate of only 50%[38].ET-1 induced subcortical nuclei ischemia model showed a lower incidence of sensorimotor deficits and higher percentage of survival com pared to MCAO.Photothrombosis as another model of focal ischemia is similar to the cortical ET-1 model in that the site of infarct can be precisely localized.However,as a permanent ischemia model,photothrombosis can only be used for cortical stroke and its mechanisms of injury are complex[4]. Through T2-weight MRI image,the effect of ET-1 stereotaxical injection in dysfunction of the hippocampus and lateral amygdala was confirmed.Our findings also show that ET-1 in the dorsal hippocampus and lateral amygdala produces a significant and enduring dorsal hippocampus and lateral amygdala-related learning and memory deficits.

Fig.5 ET-1 in the lateral amygdala(LA)impairs acquisition and retention of tone-conditioned fear memory.A:LA-stroke rats exhibit delayed freezing to a six-trial training protocol as described in Fig.2A,but comparable freezing levels to the sham group by the end of the training session.A two-way repeated-measures ANOVA revealed a significant treatments×interval interaction,P＜0.05,F(1,132)=2.828(sham,n=8, LA-stroke,n=8).*P＜0.05,Bonferroni post-hoc test,sham vs.LA-stroke.C:Freezing during the training context(P=0.680)and a novel context (P=0.317,two-tailed Student’s t-test)is intact in LA-stroke rats.D:Tone conditioning during the four tests block is impaired in LA-stroke rats.A two way repeated-measures ANOVA revealed a significant treatments×interval interaction,P＜0.01,F(1,42)=0.125(sham,n=8,LA-stroke,n=8). **P＜0.01,Bonferroni post-hoc test,sham vs.LA-stroke.

Traditionally well-defined synaptic connectivity in the central nervous system is structured during development and is later sculpted by neural activity.However,it has been reported that neurons that participate in intricate brain functions,such as memory engram or trace,are not contributed by a single brain region but are distributed throughout the whole brain[39].Supposing that ischem ia affects some components of circuitry to route sensate signals to the neural system and motor commands out of it, activity dependent,synapse-based and hebbian-valid learning rules can strengthen and refine these circuits. This extensive connectivity,together with multiformity in neuronal processing,might accelerate recovery from stroke damage.

In physiological conditions,some recent studies indicate distinct appetitive and aversive circuits between the hippocampus and the amygdala[40].After special emotional shift task,a change probably occurs in the connectivity between hippocampal and amygdalar memory traces[40-41].

In the model used in our present study,it is still unknown if local infarct changes the connectivity involved dorsal hippocampus and/or the lateral amygdala in the process of recovery.It is tempting in the near future to study on the potential circuit plasticity as the result of ischemia in subcortical nuclei to dissect the correlation between activity-dependent plasticity and functional recovery.

Acknowledgement

This work was supported by Major State Basic Research Program of China(Grant No.2013CB733801).

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Hippocampal ischemia causes deficits in local field potential and synaptic plasticity

Shaoli Wang1,Jingyun Zhang4,Tao Sheng3,Wei Lu2,4,?,Dengshun Miao1,?

1The Research Center for Bone and Stem Cells,Department of Human Anatomy;2Department of Neurobiology;3The Center of Metabolic Disease Research,Nanjing Medical University,Nanjing,Jiangsu 210029,China;4The Key Laboratory of
Developmental Genes and Human Disease,Institute of Life Sciences,Southeast University,Nanjing,Jiangsu 210096,China.

Abstract

The long-term enhancement in glutamate receptor mediated excitatory responses has been observed in stroke model.This pathological form of plasticity,termed post-ischemic long-term potentiation(i-LTP),points to functional reorganization after stroke.Little is known,however,about whether and how this i-LTP would affect subsequent induction of synaptic plasticity.Here,we first directly confirmed that i-LTP was induced in the endothelin-1-induced ischemia model as in other in vitro models.We also demonstrated increased expression of NR2B,CaMKII and p-CaMKII,which are reminiscent of i-LTP.We further induced LTP of field excitatory post-synaptic potentials(fEPSPs)on CA1 hippocampal neurons in peri-infarct regions of the endothelin-1-induced mini-stroke model.We found that LTP of f EPSPs,induced by high-frequency stimulation,displayed a progressive impairment at 12 and 24 hours after ischemia.Moreover,using in vivo multi-channel recording,we found that the local field potential,which represents electrical property of cell ensembles in more restricted regions,was also dampened at these two time points.These results suggest that i-LTP elevates the induction threshold of subsequent synaptic plasticity.Our data helps to deepen the know ledge of meta-synaptic regulation of plasticity after focal ischemia.

Keywords:long-term potentiation,local field potential,ischemia,endothelin-1,multi-channel in vivo recording

Introduction

Ischemic stroke is now one of the major causes of death and disability in the world[1].Evidence shows that focal ischemia in the territory of the middle cerebral artery(MCA)induces widespread neuropathological changes both in the ischemic region and in areas remote from the original infarct[2].The glutamate receptor mediated ischemic long-term potentiation(i-LTP) of ten occurs after ischemic stroke.This neural plasticity plays an important role in ischemic injury and recovery. Therefore,deepening the understanding of the i-LTP mechanism has a great significance for guiding the treatment of ischemic stroke.

LTP,a cellular model of synaptic plasticity that is now widely considered to share similar cellular mechanism with learning and memory,can be reflected by changes in the amplitude or slope of field excitatory postsynaptic potentials(fEPSPs).The low impedance and positioning of the electrode allows the activity of a large number of neurons to contribute to the signal. The unfiltered signal reflects the sum of action potentialsfrom cells within approximately 50-350 μm from the tip of the electrode and represents the electrical property of cell ensembles in more restricted regions.It is generally known that the i-LTP is initiated by excessive calcium influx with the activation of NMDAR after stroke[3].However,whether and how the i-LTP would affect subsequent induction of synaptic plasticity and the local field potential(LFP)remains less known.

Metaplasticity is a concept originally coined by W.C.Abraham and M.F.Bear to refer to synaptic plasticity[4].The idea is that the synapse's previous history of activity determines the plasticity afterwards. Therefore,the metaplastic regulation of i-LTP on subsequent induction of synaptic plasticity may underlie impaired capability of learning and memory after ischemia.The CA 1 area in the hippocampus is one of the most sensitive regions[5-6]to ischemic stroke.In the present study,we first infused endothelin-1(ET-1)to the dorsal hippocampus to establish a mini-stroke model[7-9];we then demonstrated the increase in expression of NR2B,CaMKII and p-CaMKII,which hints the occurrence of i-LTP.Using whole-cell patch-clamp recording,we found that LTP of fEPSPs induced by high-frequency stimulation(HFS)displayed a progressive impairment at 12 and 24 hours after ischemia. Moreover,using in vivo multi-channel recording,we found that the LFP,which represents electrical properties of cell ensembles in more restricted regions,was also dampened at these two time-points.These results point to the notion that i-LTP elevates the induction threshold of subsequent synaptic plasticity.Our data helps to deepen the know ledge on metasynaptic regulation of plasticity after focal ischemia.

Materials and methods

Animals

Four-week-old male Sprague-Dawley(SD)rats were used.All animal studies followed the guidelines of the National Institutes of Health(NIH)for the Care and Use of Laboratory Animals.Before the experiments,animals were randomly divided into two groups:the control group and the ischemia group.To construct the ischemic model,we applied ET-1(15 pmol;Sigma-Aldrich,St. Louis,MO,USA;0.8 μL in saline solution[10];10 nL/s) stereotaxically targeted to the dorsal hippocampus CA1 region(AP:-4.52 mm relative to the bregma;ML: ±3.0 mm;DV:3.0 mm[11-12]).For intracranial injections, rats were deeply anesthetized with 3% chloral hydrate. The rats were then fixed on a stereotaxic frame(RWD Life Science,China)and the injection rate and volume were controlled by a microsyringe pump controller (WPI,USA).The needle was left in place for 5 additional minutes after injection.Rats in the ischemia group received bilateral infusion of ET-1 for all experiments. Rats in the control group received bilateral infusion an equivalent volume of saline through the same surgical procedure.Rats were returned to home cage after surgery.

Magnetic resonance imaging

The infarct size and location were detected by MRI at 6,12 and 24 hours after ischemia.MRI was conducted by using 7.0-T MRI(Eclipse,Philips Medical Systems,The Netherlands)with a 21-cm bore magnet. The MRI sequences were a T2-weighted spin-echo [repetition time(msec)/echo time(msec),500/17.9]. The rats were anesthetized with 3% chloral hydrate.

TTC staining

The infarct size and location were evaluated at 6,12 and 24 hours after ischemia.After anesthetization and euthanasia,the brains were removed directly and frozen at-20°C for 15 min.The whole brains of rats were incised to corresponding slices at 2 mm.The sections were immersed in 1% 2,3,5-triphenyltetrazolium chloride(TTC;Sigma)at 37°C for 20 min including an invert of the slices in light-blocking environment. The pale region representing the focal infarcts was distinctly visible by examining TTC-stained sections.

Electrophysiological recordings

The protocol was adapted from previous studies[13]. Rats were anesthetized with 10% chloral hydrate and decapitated.Ice-cold artificial cerebrospinal fluid (ACSF)containing 126 mmol/L NaCl,2.5 mmol/LKC l,1 mmol/L Mg C l2,1 mmol/L CaCl2,1.25 mmol/L KH 2PO4,26 mmol/L NaHCO3 and 20 mmol/L glucose was bubbled continuously with Carbogen(95%O2/5%CO2).The brain was immediately removed and 350 μm coronal hippocampal brain slices were prepared with a vibrating blade microtome (Leica VT1200S)in ACSF.Fresh slices were incubated in a chamber with carbogenated ACSF and recovered at 30°C for at least 2 h.fEPSP responses were evoked at 0.05 Hz with a 125 μm electrode placed in the middle of the stratum radiatum of CA1. A 2-3 MΩ glass recording electrode filled with 2mol/ L NaCl was positioned orthodromic(200 μm)from the stimulating electrode.High frequency stimulation protocol:Weak HFS(wHFS),1 train,consisting of 10 bursts in 100 Hz;HFS,4 trains,consisting of 100 bursts in 100 Hz with 20 seconds inter-burst interval.Responses were set to-60% max for LTP experiments.

Fig.1 MRI and 2,3,5-triphenyltetrazolium chloride(TTC)staining confirms the ischemic region after local endothelin-1(ET-1) injection.A:Schematic diagram show s the site of injection.B:Methylthionine chloride solution is applied to display the accuracy of the injection site. Left,the area in the red box is the methylthionine chloride solution diffusion region,showing accurate localization of the dorsal hippocampus CA1 region;Right:another sample showing injection sites in magnified image.The line indicates the pyramidal cell layer in CA1 region.C:Sequential brain T2-w MRI in rats with ischemic lesion.The image in the red box and the magnified image shown underneath indicate the infarct regions.D:A series of brain slices stained with TTC after ischemic lesion.Pale staining in the black box and the magnified image shown underneath indicates the infarct region.C and D also show progressive increase of the infarct regions.

Multichannel recordings

Rats were treated in accordance with surgical procedures.Sprague Dawley rats(300-350 g)were anaesthetized with 0.015 mL/g,3% chloral hydrate as needed, and body temperature was maintained with a heating pad.LFP was recorded with an 8-channel electrode array in the hippocampal CA1 pyramidal cell layer.LFP(sampling rate 800 Hz)was filtered online at 0.5-250 Hz[14,15].

Western blotting

Hippocampal slices were prepared from the animals injected with ET-1.We choose the slices with an injection hole and then the slices were homogenized in cold 0.32 mol/L sucrose solution containing 1 mmol/L HEPES,1 mmol/L NaHCO3,1 mmol/L MgCl2,0.2 mmol/L dithiothreitol,20 mmol/Lβ-phosphogrycerol,20 mmol/L sodium pyrophosphate,1 mmol/L EDTA,1 mmol/L EGTA,1 mmol/L Na3VO4,50 mmol/L NaF,1 mmol/ L and p-nitrophenyl phosphate(PNPP)(pH=7.4),in the presence of protease and phosphatase inhibitors. The 40-60 μg samples were subjected to SDS-PAGE and transferred to PVDF membranes.Antibodies against the following proteins were used:CaMKIIα(SC-13141), p-CaMKII(SC-32289),NMDAε2(NR2B,SC-365597), and β-tubulin(CW 0098,Beyotime,China).

Fig.2 The occurrence of i-LTP in ET-1-induced mini-stroke model.A:Weak high-frequency stimulation(HFS)induced i-LTP at 3 hours after ischemia.When a weak HFS(wHFS,1 train,consisting of 10 bursts in 100 Hz)was delivered to Schaffer fibers in control slices,no LTP was induced.In contrast,a persistenpotentiation was induced upon stimulation with wHFS in slices obtained from ET-1-treated animals(A 2,Control: 1.11±0.04,P＜0.001,n=5 at 61-75 minutes;3 hours:1.97±0.08,P＜0.001,n=8).B:HFS faciliates LTP in ET-1-induced stroke model.Compared with normal LTP in control brain slices,LTP in ET-1-treated slices displayed LTP with enhanced potentiation magnitude(B2,Control:1.63±0.06, P＜0.001,n=5 at 61-75 minutes;3 hours:2.75±0.08,P＜0.001,n=5).Data represents mean±SEM.Data represents mean±s.e.m.Statistic differences were compared using independent-sample t-test.

Data analysis

Data were presented as mean±SEM.Differences between groups were compared using independent-sample t-test(two populations)and one-way ANOVA for comparisons.Statistic differences were considered to be significant when P was＜0.05.

Results

MRI and TTC staining reveal progressive infarct regions following ET-1 infusion

We first stereotaxically infused ET-1 with middle concentration and volume(15 pmol,0.8μL dissolved in sterile saline)into the CA1 region of the dorsal hippocampus to establish mini-stroke model(Fig.1A)[12].The location of ET-1 injection sites was then confirmed(Fig.1B)by application of methylthionine chloride solution.We also used the MRI and TTC to examine and confirm the establishment of the mini-stroke model.We found that the region subjecting to ischemic attack was progressively enlarging.At 6 hours post-ischemia,the ischemic region was just around the injection site;12 hours post-ischemia, the infarct region was slightly increased as indicated by MRI and TTC staining.At 24 hours post-ischemia,the infarct region significantly increased in both MRI and TTC staining experiments(Figs.1C,1D).

Post-ischemic long-term potentiation

We then examined,in the ET-1-induced ischemia model,whether i-LTP can be induced as in other in vitro models[16-17].Three hours after ET treatment,acute hippocampal brain slices were obtained.When a weak HFS(wHFS,1 train,consisting of 10 bursts in 100 Hz) was delivered to Schaffer fibers in control slices,no LTP was induced.In contrast,a persistent potentiation was induced upon stimulation with wHFS in slices obtained from ET-1-treated animals(Control:1.11± 0.04,P＜0.001,n=5,at 61-75 minutes;3 hours: 1.97±0.08,P＜0.001,n=8;Fig.2A).Consistently, compared with normal LTP in control brain slices, LTP in ET-1-treated slices displayed LTP with enhanced potentiation magnitude(Control:1.63±0.06,P＜0.001, n=5,at 61-75 minutes;3 hours:2.75±0.08,P＜0.001, n=5;Fig.2B),suggesting that LTP was facilitated in ET-1-treated stroke model.Taken together,these results confirm the occurrence of i-LTP in the ET-1-induced stroke model.

Fig.3 Increased expression level of NR2B,CaMK II and p-CaMKII after ischemia.A:Absence of change in these proteins at 6 hours after ischemia(NR2B,0.97±0.18,P＞0.05,n=4;CaMKII,0.97±0.09,P＞0.05,n=4;p-CaMKII,1.05±0.04,P＞0.05,n=4).B:At 12 hours after ischemia,a slight but not significant increase in the expression of NR2B and CaMKII was detected(NR2B,1.13±0.26,P＞0.05,n=4;CaMKII,1.17±0.14,P＞0.05, n=4).No increases of p-CaMKII was detected(0.94±0.07,P＞0.05,n=4).C:At24 hours after ischemia,a significant increase in the NR2B,CaMKII and p-CaMKII was detected(NR2B,1.65±0.20,P＜0.05,n=4;CaMKII,1.16±0.13,P＜0.05,n=3;p-CaMKII,1.62±0.31,P＜0.05,n=4).Data represents mean±SEM.Statistic differences were compared using one-way ANOVA.CON:control,h:hours.

Post-ischemic increase in expression of NR2B,CaMKII and p-CaMKII

I-LTP is usually accompanied by biochemical changes in LTP-related signaling molecules.Following activation of NMDA receptor[18]and Ca2+influx through the receptor channel,increased intracellular Ca2+/CaMactivates CaMKII[19-21],which in turn activates down-streaming signaling molecules like Ras,PI3K[22-23]and PKCλ[24-25][26-29].As a result,AMPA receptor is phosphory lated and incorporated into postsynaptic membrane[30]which underlies LTP of AMPA receptor-mediated synaptic currents.Therefore,the possible alterations in these LTP-related molecules may be reminiscent of pathological i-LTP.We thus employed Western blotting assays to examine the expression of NR2B,CaMKII and p-CaMKII at 6,12 and 24 hours after ischemia.As shown inFig.3,there was no change in these proteins at 6 hours after ischemia(NR2B, 0.97±0.18,P＞0.05;CaMKII,0.97±0.09,P＞0.05; p-CaMK II,1.05±0.04,P＞0.05,Fig.3A).Twelve hours after ischemia,the expression of NR2B and CaMKII showed a slight but statistically insignificant increase when compared with control(NR 2B, 1.13±0.26,P＞0.05;CaMKII,1.17±0.14,P＞0.05,Fig.3B),while at this time there was no increase of p-CaMKII(0.94±0.07,P＞0.05,Fig.2).However,at 24 hours after ischemia,the expression of NR2B, CaMK II and p-CaMK II increased significantly (NR2B,1.65±0.20,P＜0.05;CaMKII,1.16±0.13, P＜0.05;p-CaMKII,1.62±0.31,P＜0.05,Fig.3C). These results indicate that ischemic attack can increase the expression of these i-LTP-associated molecules and indirectly demonstrate the occurrence of i-LTP.

Fig.4 Ischemia impairs LTP induction in CA1 hippocampal neuorns.A:ET-1 injection sites.B:Schematic diagram showing position of stimulating and recording electrodes.Recording and stimulation sites are at a distance of 150-200 μm from the injection point.C:Sample traces showing LFP recorded in 3 of 8 channels.Scale bar:200 ms,0.5 mV.LTP was slightly but significantly decreased at 12 hours after ischemia(Control: 1.82±0.02 at 61-75 minutes,n=5;12 hours:1.46±0.03,n=5;P＜0.001).D:LTP was completely abolished at 24 hours after ischemia(Control: 1.82±0.02 at 61-75 minutes,n=5;24 hours:0.92±0.01,n=5;P＜0.001).Data represents mean±SEM.C and D share the same data of control group. Statistic differences were compared using independent-sample t-test.

ET-1-induced mini-stroke impairs LTP

To examine whether LTP induction,after i-LTP, upon ischemic attack is altered,we induced LTP of fEPSPs in CA1 neurons of the peri-infarct regions on acute hippocampal slices.The hippocampal slices were prepared at 12 and 24 hours after ischemia(Fig.4A). We only selected the slices containing injection sites and set the recording and stimulation electrode at a distance of 150-200 μm from the injection point (Fig.4B).We found that LTP induced by HFS decreased slightly,but significantly in CA1 hippocampal neurons at 12 hours after ischemia(Control:1.82± 0.02 at 61-75 min;12 hours:1.46±0.03;P＜0.001,Fig.4C).At 24 hours after ischemia,LTP was totally reversed(Control:1.82±0.02 at 61-75 min;24 hours:0.92±0.01;P＜0.001,Fig.4D).In addition,we also found that the maximal amplitude of excitatory postsynaptic potentials that could be achieved gradually decreased(data not shown).Further studies are necessary to examine whether it is a general phenomenon.

Fig.5 Ischemia impairs LFP in dorsal hippocam pus.A:Schematic diagram showing position of rejection and 8-channel recording electrodes. Recording electrodes are surrounded with the injection point.B:Schematic pictures of 8-channel recording electrodes.C:Sample traces showing LFP recorded in 3 of 8 channels.Scale bar:200 ms,0.5 mV.LTP was significantly suppressed at 12 hours and 24 hours time-points after ischemia. D:Power spectral density(PSD) of LFP in A.Note the decrease in the peaks at low frequency rhythm in the PSD of LFP(At the first peak marked with red asterisk:Control:-29.54±0.24,n=8;12 hours:-44.01±0.48,n=8,P＜0.001 compared with control;24 hours:-43.86±1.08,n=8,P＜0.001 compared with control;At the sencond peak marked with green asterisk:Contol:-42.08±0.33,n=8;12 hours:-58.95±0.96,n=8,P＜0.001 compared with control;24 hours:-60.55±1.01,n=8,P＜0.001 compared with control).Statistic differences were compared using one-way ANOVA.

LFP declines after ischemia

Compared to conventional fEPSPs,LFP reflects electrical property of cell ensembles in more restricted regions.To examine possible alterations in LFP,we employed in vivo multi-channel recording to study the effect of ischemia on LFP in hippocampal CA1 pyramidal neurons.We continuously recorded LFP signals with 8-channel electrodes at different time-points after ischemia on anesthesized rats.We found that the amplitude of the LFP decreased dramatically at both 12 and 24 hours after ischemia(Fig.5A).The power spectral density(PSD)analysis of LFP showed that the low frequency rhythms were also impaired significantly at these time points after ischemia(At the first peak: Control:-29.54±0.24;12 hours:-44.01±0.48, P＜0.001 compared with controls;24 hours: -43.86±1.08,P＜0.001 compared with controls;At the second peak:Control:-42.08±0.33;12 hours: -58.95±0.96,P＜0.001 compared with control;24 hours: -60.55±1.01,P＜0.001 compared with controls,Fig.5B).

Discussion

The pathological form of plasticity,i-LTP,is usually accompanied by alterations in plasticity-related proteins.First of all,we directly confirmed that i-LTP can be induced in ET-1-induced ischemia model as described previously in other in vitro models.We monitored the expression of CaMK II,p-CaMK II and NMDA receptor NR2 subtype NR2B.These molecules have been reported to be associated with both LTP and synaptic plasticity.Under ET-1induced mini-stroke condition[31,32],we detected an increase in CaMKII, phospho-CaMKII and NR2B,which is consistent with previous findings showing similar enhancement in NR2B subunit.These results provide both electrophysiological and biochemical evidence that hints the occurrence of i-LTP.

LTP and LTD are widely considered to share similar mechanisms with learning and memory and thus are used to study cellular synaptic plasticity[33-39].The hippocampus is one of the brain regions most associated with learning and memory.However,this region is also very sensitive to ischemic attack.Although i-LTP is indicated as long-term enhancement in AMPA-and NMDA-receptor-mediated excitatory responses,what we actually observed in real life is the impaired capability on hippocampus-dependent learning and memory.This may be caused by the altered induction threshold of subsequent LTP.In the present study,we indeed observed detrimental effect by prior i-LTP.The magnitude of HFS-induced LTP was significantly inhibited at 12 hours after ischemia and was totally abolished at 24 hours after ischemia. Although ET-1 is usually used to mimic mini-stroke, we can still detect deficit on LTP induction. Compared to the MCAO and PT methods that are more of ten used,ET-1 infusion only produces local ischemia in a very restricted region within the dorsal hippocampus.Therefore,LFP,which represents electrical properties of cell ensembles in more restricted regions, seems more suitable to detect slight change in the peri-infarct region.Consistent with the findings on fEPSPs,we also detected suppression of LFP at 12 and 24 hours after ischemia.These results provide solid evidence to the notion that focal ischemia in the hippocampus causes deficits in local field potential and synaptic plasticity.

In summary,we first demonstrated the presence of metaplastic regulation in the mini-stroke model and then demonstrated how the i-LTP affected subsequent synaptic plasticity.These results enrich our understandings of metaplasticity in cerebral ischemia and provide new ideas for potential clinical treatment of cerebral ischemia.

Acknowledgement

This work was supported by Major State Basic Research Program of China(Grant No.2013CB733801).

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?Correspondence to:Yifan Dai,The Center of Metabolic Disease Research,Nanjing Medical University,Nanjing,Jiangsu 210029,China. E-mail:yifandai08@gmail.com;Wei Lu,Department of Neurobiology, Nanjing Medical University,Nanjing,Jiangsu 210096,China.Tel/Fax: +86-25-83789987/86-25-83789987 E-mail:lu@njmu.edu.cn.

Received 13 January 2015,Revised 29 March 2015,Accepted 27 April 2015,Epub 02 June 2015

R338.64 Document code:A

The authors reported no conflict of interests.

?2015 by the Journal of Biomedical Research.All rights reserved.

10.7555/JBR.29.20150008

?Corresponding authors:Pr of .Dengshun Miao,The Research Center for Bone and Stem Cells,Department of Human Anatomy,Nanjing Medical University,Nanjing,Jiangsu 210029,China.Tel/Fax:+86-25-8686-2015, E-mail:dsmiao@njmu.edu.cn.

Prof .Wei Lu,Department of Neurobiology,Nanjing Medical University,Nanjing,Jiangsu 210096,China,E-mail:lu@njmu.edu.cn.

Received 13 January 2015,Revised 16 March 2015,Accepted 10 May 2015,Epub 8 June 2015

CLC number:R743.3,Document code:A

The authors reported no conflict of interests.

?2015 by the Journal of Biomedical Research.All rights reserved. doi:10.7555/JBR.29.20150010