N R2B-containing N-methyl-D-aspartate subtype glutamate receptors regulate the acute stress
e¡ect on hippocampal long-term potentiation/
long-term depression in vivo
Meina Wang a,b,*,Y a Y ang a,b,*,Zhifang Dong a,b,Jun Cao a,b and Lin Xu a,b,c
a Key Laboratory of Animal Models and Human Disease Mechanisms,and Laboratory of Learning and Memory,K unming Institute of Zoology,the Chinese Academy of Sciences,Kunming,
b Graduate School of the Chinese Academy of Sciences,Beijing and
c Mental Health Institute,the2n
d Xiangya Hospital of
Central South University,Changsha,Hunan,PR China
Correspondence and requests for reprints to Professor Lin Xu,PhD,Key Laboratory of Animal Models and Human Disease Mechanisms,and Laboratory of Learning and Memory,Kunming Institute of Zoology,
the Chinese Academy of Sciences,Kunming650223,PR China;Mental Health Institute,
the2nd Xiangya Hospital of Central South University,Changsha410011,PR China
T el:+868715139165;fax:+868715191823;e-mail:lxu@vip.163
Correspondence and requests for reprints also to Jun Cao,Assistant Professor and PhD,Key Laboratory of Animal Models and Human Disease Mechanisms,and Laboratory of Learning and Memory,Kunming Institute of Zoology,the Chinese Academy of Sciences,Kunming650223,Y unnan,PR China
T el:+868715195402;fax:+868715191823;e-mail:juncao@vip.163
*These authors contributed equally to this work.
Sponsorship:This research was supported by grants from the National Natural Science Foundation of China(30530250and30470549to L.X.and
30500150to J.C.),and973Program(2006CB500800to L.X.and J.C.).
Received26April2006;accepted24May2006
Behavioral stress facilitates long-term depression but impairs long-term potentiation in the hippocampus.Recent evidence invitro demonstrates that the NR2B-containing N-methyl-D-aspartate subtype glutamate receptor antagonist Ro25-6981prevents the behavioral stress-facilitated hippocampal long-term depression.It is,however,unknown whether Ro25-6981in£uences hippocampal long-term depression and long-term potentiation induction in vivo under stressful condition.Here,we found that infusion of Ro25-6981(2.3m g in6m l,intracerebroventricular for10min)30min before low-frequency stimulation prevented the facilitation of hippocampal long-term depression by acute stress in anesthe-tized adult rats.Moreover,infusion of Ro25-698130min before high-frequency stimulation reversed stress-impaired hippocampal long-term potentiation.These results suggest that the NR2B-containing N-methyl-D-aspartate subtype glutamate receptors are crucial for the e¡ects of behavioral stress on hippocampal long-term depression and long-term potentiation in vivo.NeuroReport 17:1343^1346 c2006Lippincott Williams&Wilkins.
Keywords:adult,acute stress,NR2B-containing N-methyl-D-aspartate subtype glutamate receptors,anesthetized rats,Ro25-6981,long-term potentiation,long-term depression
Introduction
Activity-dependent or experience-dependent hippocampal synaptic plasticity such as long-term potentiation(LTP)and long-term depression(LTD)is believed to be the mechanism underlying certain types of learning and memory[1–3],in which the N-methyl-D-aspartate subtype glutamate receptor (NMDAR)plays crucial roles.NMDAR comprises heteromers,composed of NR1and at least one NR2subunit [4].Recent evidence in vitro suggests a possibility that the NR2B-containing NMDARs are necessary for LTD induction[5,6].To our knowledge,there is no related study in vivo.
It is known that low-frequency stimulation often fails to induce hippocampal LTD in adult animals[7],but behav-ioral stress facilitates the induction both in vitro and in vivo [8,9].Recent evidence in vitro demonstrates that the hippocampal LTD facilitated by behavioral stress is prevented by the NR2B-containing NMDAR antagonist Ro25-6981[10].It is,however,unclear whether the NR2B-containing NMDARs are involved in stress-facilitated hippocampal LTD in vivo.As behavioral stress is known to impair the induction of hippocampal LTP[11,12],it is unknown whether the NR2B-containing NMDARs also contribute to the stress-impaired hippocampal LTP. Here,we found that the NR2B-containing NMDAR anta-gonist Ro25-6981not only prevented the stress-facilitated hippocampal LTD but also reversed the stress-impaired hippocampal LTP in anesthetized adult rats.
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Methods
Foot shock stress
Experiments were carried out on male Sprague–Dawley rats (inbred strain,Animal House Center,Kunming General Hospital,Kunming,PR China),weighing200–250g(8–10 weeks).The animal care and experimental protocol were approved by the Chinese Academy of Sciences,PR China. Acute behavioral stress was evoked by foot shock as described previously[13].Briefly,the animals were put into the operant chamber(Med Associates Inc.,Georgia(St Albans),Vermont,USA)and received one foot shock (0.8mA,1s duration)per minute for10min.Stressed animals(stressed)were anesthetized immediately after foot shock exposure.This stress protocol did not exclude other factors that could induce stress,such as the novel environ-ment.Nonstressed animals(nonstressed)were taken out of their home cage and anesthetized immediately. Electrophysiological studies
Recordings of the field excitatory postsynaptic potentials (fEPSPs)were made from the CA1stratum radiatum of the hippocampus in response to ipsilateral stimulation of the Schaffer collateral/commissural pathway using techniques similar to those described previously[14,15].Experiments were performed under pentobarbital sodium(50–60mg/kg, intraperitone
al)anesthesia,and the core temperature was maintained at3770.51C.The state of anesthesia was monitored by temperature and toe-pinch response,and supplemental dose(15mg/kg,intraperitoneal)was given if necessary.This procedure did not influence fEPSP recordings[14,15].Animals were ventilated with95%O2 and5%CO2.Recording and stimulating electrodes were made by gluing together a pair of twisted Teflon-coated90% platinum and10%iridium wires(50m m inner diameter, 75m m outer diameter;World Precision Instruments,Saraso-ta,Florida,USA).The recording electrode was inserted 3.8mm posterior to bregma and2.8mm right of the midline and the stimulating electrode was inserted4.8mm posterior to bregma and 3.8mm right of midline,by using the stereotaxic instrument(Stoelting Co.,Wood Dale,Illinois, USA).Test fEPSPs were evoked at a frequency of0.033Hz and at a stimulation intensity adjusted to give an fEPSP amplitude of50%maximum response.For LTD induction, low-frequency stimulation consisting of900pulses at3Hz was performed.The high-frequency stimulation protocol for inducing LTP consists of10trains of20stimuli,interstimu-lus interval5ms(200Hz),intertrain interval2s.
Drug and drug infusion
R-(R,S)-a-(4-hydroxyphenyl)-b-methyl-4-(phenylmethyl)-1-piperidine propranol(Ro25-6981)hydrochloride was pur-chased from Sigma-Aldrich Co.(St Louis,Missouri,USA) and dissolved in s
aline at a concentration of0.376m g/m l. Ro25-6981was applied acutely through intracerebroventri-cular cannula placed into the left lateral ventricle(0.5mm anterior,1mm lateral and4.5mm deep to the dura).After 40min stable baseline recordings,the infusion was performed using an infusion pump at a rate of0.6m l/min for10min(total drug infusion,2.3m g).
Data analysis
All values were reported as the mean percentage7SEM of the baseline fEPSP amplitude recorded over40min baseline periods(the average of40min recordings was set as100%) and n was the number of rats.Synaptic efficacy was the averaged percentage value of fEPSP amplitude55–60min (10sweeps)after low-frequency stimulation or high-frequency stimulation.The effect of Ro25-6981on basal synaptic transmission was analyzed by the averaged percentage value of fEPSP amplitude25–30min after drug infusion.Two-tailed paired t-test was used for comparisons in the same group but unpaired t-test for comparisons between groups.The significance level was set at P o0.05. Results
Effect of the NR2B-containing N-methyl-D-aspartate sub-type glutamate receptor antagonist Ro25-6981on stress-facilitated hippocampal long-term depression
In our experiment,low-frequency stimulation failed to induce LTD in anesthetized adult rats(Fig.1a,nons
truncated bnp是什么tressed, n¼4,101.670.8%,P40.05vs.baseline)but behavioral stress facilitated the induction(Fig.1a,stressed,n¼5,85.573.0%, P o0.05vs.baseline).We further examined the effect of NR2B-containing NMDARs on stress-facilitated LTD in vivo. We found that Ro25-6981(2.3m g in6m l,intracerebroven-tricular for10min)did not affect basal synaptic transmis-sion during the30min postdrug recording(Fig.1b, nonstressed,99.470.1%,stressed,99.670.3%,P40.05vs. baseline)and the LTD induction in nonstressed rats(Fig.1b, nonstressed,n¼3,100.570.9%,P40.05vs.baseline).Ro25-6981,however,completely prevented the stress-facilitated LTD(Fig.1b,stressed,n¼4,104.270.4%,P40.05 vs.baseline).Comparison between stress-facilitated and Ro25-6981-prevented LTD revealed significant difference (unpaired t-test,P o0.05).Our result was consistent with previous in-vitro study[10]and showed that the NR2B-containing NMDARs played an essential role in stress-facilitated LTD in vivo.
Effect of the NR2B-containing N-methyl-D-aspartate subtype glutamate receptor antagonist Ro25-6981on stress-impaired hippocampal long-term potentiation High-frequency stimulation induced LTP in nonstressed rats (Fig.2a,nonstressed,n¼4,126.375.3%,P o0.05vs.base-line),whereas behavioral stress prevented the induction (Fig.2a,stressed,n¼5,100.071.7%,P40.05vs.baseline). Ro25-6981infusion did not affect the induction of hippo-campal LTP in nonstressed animals(Fig.2b,nonstress
ed, n¼4,121.375.4%,P o0.05vs.baseline)but reversed the LTP impairment induced by stress(Fig.2b,stressed,n¼4, 118.674.9%,P o0.05vs.baseline).Comparison between stress-impaired and Ro25-6981-reversed LTP revealed sig-nificant difference(unpaired t-test,P o0.05).This result showed that the NR2B-containing NMDARs were also implicated in stress-impaired LTP in vivo.
Discussion
Our results showed that the NR2B-containing NMDAR antagonist Ro25-6981not only prevented the stress-facilitated hippocampal LTD but also reversed the stress-impaired hippocampal LTP in anesthetized rats. Behavioral stress leads to the activation of a wide variety of neurotransmitter and neuroendocrine systems that can potentially affect the induction of synaptic plasticity[16,17]. The underlying mechanisms are believed mainly due to the
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glucocorticoid receptors activated by glucocorticoids at stress levels [18,19].Recent evidence,however,suggests that behavioral stress or glucocorticoids enhance the expression of NMDAR subtypes in hippocampus,especially the NR2B subunit [20–23].Recent evidence in brain slices demonstrates that the hippocampal LTD facilitated by behavioral stress is prevented by the anta
gonist of NR2B-containing NMDARs [10].Consistent with the in-vitro study,our experiments in anesthetized rats showed that the NR2B-containing NMDAR antagonist Ro25-6981prevented the stress-facilitated hippocampal LTD.
Importantly,we found that Ro25-6981infusion also reversed the LTP impairment induced by behavioral stress,which is somewhat different from previous reports showing that NR2B antagonists prevent the induction of LTD but have no effect on that of LTP [5,6].Kim et al.[9]show that the NMDAR antagonist CGP39551administration (intraperi-toneal)2h before stress can prevent both the stress-facilitated hippocampal LTD and the stress-impaired hippocampal LTP in rat slice.This finding may suggest that behavioral stress via NMDAR can produce long-lasting effect that even obviously influences hippocampal LTP/LTD in vitro .The stress-impaired LTP can also,however,partially be prevented by the glucocorticoid receptor antagonist RU38486[24].A recent report proposes a competitive hypothesis of endogenous LTD and LTP that underlies the storage of emotional memories and stress-induced amnesia [25].As the NR2B-containing NMDAR antagonist Ro25-6981or a glucocorticoid receptor antagonist prevents the stress-facilitated LTD [10],and we found that Ro25-6981
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Fig.2The NR2B-containing N -methyl-D -aspartate subtype glutamate receptor antagonist Ro25-6981reversed the stress-impaired hippo-campal long-term potentiation (L TP).(a)High-frequency stimulation (HFS arrow ,200Hz)induced L TP in nonstressed animals,while behavioral stress impaired the induction.Sample traces were ¢eld excitatory post-synaptic potentials (fEPSPs)recorded before (1)
and during (2)L TP of a nonstressed rat.Calibration:1m V ,10ms.(b)High-frequency stimulation (arrow ,200Hz)induced robust L TP both in stressed and nonstressed groups after Ro25-6981infusion (bar indicating drug infusion period,10min).Sample traces represented baseline (1),after Ro25-6981infusion (2)and after induction (3),respectively .Calibration:1m V ,10ms.
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Fig.1The NR2B-containing N -methyl-D -aspartate subtype glutamate receptor antagonist Ro25-6981prevented the stress-facilitated hippo-campal long-term depression (L TD).(a)Low-frequency stimulation (3Hz;LFS bar)failed to induce L TD in nonstressed animals but induced robust L TD in stressed animals.The traces were ¢eld excitatory post-synaptic potentials (fEPSPs)recorded before (1)and during (2)L TD of a stressed rat.Calibration:1m V ,10ms.(b)Low-frequency stimulation (3Hz;bar)failed to induce L TD either in stressed or in nonstressed groups after Ro25-6981infusion (bar indicating drug infusion period,10min).Sample traces represented baseline (1),after Ro25-6981infusion (2)and after induction (3),respectively .Calibration:1m V ,10ms.
NMDARS REGULA TE STRESS EFFECT ON L TP/L TD
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prevented the effect of stress on both LTP and LTD induction,NMDAR may play important role in regulating stress effect on hippocampal plasticity and functions.On the other hand,Ro25-6981reversed the stress-impaired LTP to control levels,suggesting that it may affect the effect of stress but not the LTP induction.From this point of view,we propose a hypothesis that either the NMDAR or the glucocorticoid receptors may mediate the effect of stress to influence the subsequent induction of synaptic plasticity. Conclusion
We demonstrated for the first time in vivo that the NR2B-containing NMDARs play a crucial role in regulating the effect of behavioral stress on hippocampal synaptic plasticity.
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