脆性X综合征小鼠前额叶可塑性异常的机制研究
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摘要
【目的】
探索FMR1KO小鼠突触可塑性调节异常的机制以及如何恢复ACC区的可塑性，以期对进一步认识脆性X综合症的病理机制，发掘潜在的药物治疗新靶点。
【方法】
1全细胞膜片钳记录
小鼠采用1-2%异氟醚麻醉，取脑。振动切片机横向切取300μmACC脑片，室温下置于混合气饱和的人工脑脊液(ACSF)中，恢复1小时后进行实验。在具有红外线DIC系统的显微镜下观察ACC第II/III层锥体神经元。ACSF灌流液中加入100M picrotoxin，刺激电极放置于ACC第V层，记录电极放置于ACC第II/III层，在锥体神经元中选取状态良好的椎体神经元进行封接。记录兴奋性突触后电流（EPSC）和LTP。LTP诱导方法：80次刺激，频率2Hz，同时神经元钳制于+30mV，然后恢复到基线记录模式，钳制电压恢复在-70mV。记录mEPSC时，灌流液中加入TTX，转换为Gap free记录模式。
2小鼠前额叶皮层神经元的原代培养
取孕18天胎鼠，在无菌条件下断头取脑置于盛有PBS液的平皿中剥去脑膜，取大脑皮层并剪碎，消化后反复吹打制备细胞悬液。接种于涂有多聚赖氨酸的培养皿内，培养箱中进行培养。培养第三天加入阿糖胞苷，以抑制非神经元细胞的继续增长。SKF81297（5μM），DL-AP3（10μM)），和forskolin（5μM）在收集细胞前24小时分组加入培养基，收集细胞待用。
3Western blot检测
采用常规脑组织样本制备方法制备ACC区组织样品。培养神经元细胞膜蛋白的提取，采用细胞膜蛋白生物素试剂盒提取的方法，提取过程同过往文献报道[1]。Western blot检测方法：将蛋白转到NC膜上后，分别用mGluR5，GluR1，NR2A，NR2B，p-NR2B的一抗封闭各自分子量相应的条带，孵育后，漂洗，加入二抗漂洗发光。曝光完成后，经显影和定影，自来水冲洗，最后在室温下晾干。用β-actin或cadherin作为标准定量分子，确定各个蛋白相对量的多少。
4行为学实验
开场实验装置由旷场反应箱和数据自动采集和处理系统两部分组成，实验小鼠置于旷场的中央，记录并分析它的运动情况。高架十字迷宫各有两个对称的开放臂和封闭臂，十字中间为正方形平台。实验小鼠逐个放于中央平台后，记录每个小鼠5min内进入开放臂或封闭臂的次数与时间，并计算进入两臂的总次数。Morris水迷宫定位航行实验历时数天,每天将大鼠面向池壁分别从4个入水点放入水中若干次,记录其寻找到隐藏在水面下平台的时间。动物实验小鼠在实验前分别腹腔注射生理盐水，DL-AP3和/或SKF81297，45分钟后开始实验。
【结果】
1SKF81297和DL-AP3对FMR1基因敲除小鼠LTP的增强
我们用全细胞膜片钳的方法对成年脑薄片的前扣带回区II–III层锥体细胞的电生理活动进行记录，采用突触前电刺激诱导突触后去极化的方法使神经元产生LTP并记录。我们发现在FMR1WT小鼠，该脑区神经元产生了明显的长时程增强现象。但在FMR1KO小鼠，这种增强效应不明显。我们发现，正常和敲除小鼠的LTP效应与各自的空白对照组相比没有显著性差异。接下来，我们检测了D1受体激动剂应用组的LTP诱导情况，发现在D1受体激动剂SKF812975μM孵育十分钟后，正常小鼠脑薄片诱导后的LTP效应明显增强,说明激活D1受体能增强正常小鼠的LTP效应。而在FMR1KO小鼠，应用SKF81297没有增强其诱导后的LTP效应，说明D1受体激动剂的LTP增强作用在FMR1基因敲除小鼠ACC区是受损的。
我们在检测同时使用D1受体激动剂和mGluR1受体拮抗剂对LTP诱导的影响时，发现SKF81297和DL-AP3共同孵育脑片十分钟，能显著增强正常小鼠的LTP效应，这种增强效应与单独应用D1受体激动剂SKF81297的正常小鼠相比没有显著性差异。在FMR1基因敲除小鼠，同时使用D1受体激动剂和mGluR1受体拮抗剂，ACC区LTP效应与单独使用D1受体激动剂有显著性增强。
为了检测合用SKF81297和DL-AP3对FMR1KO小鼠LTP的增强效应是否来源于D1受体激动剂，我们在孵育的液体中同时加入D1受体抑制剂，发现这种LTP增强效应消失。可以认为FMR1KO小鼠LTP的增强效应来源于D1受体激动剂，这种效应是由mGluR1受体拮抗剂恢复的。
2腺苷酸环化酶激动剂恢复D1受体激动剂对FMR1KO小鼠ACC区LTP增强效应
为了确定增加腺苷酸环化酶的量是否能替代D1受体的作用，我们研究了腺苷酸环化酶激动剂forskolin对FMR1KO小鼠ACC区LTP是否有增强作用。发现加入forskolin且有电刺激诱导的情况下，LTP的幅度与未使用forskolin的FMR1WT小鼠对照组相比无显著性差异。与单独使用forskolin的FMR1WT小鼠组相比，DL-AP3与forskolin结合使用并没有引起FMR1WT小鼠LTP幅度增加。单独使用Forskolin并电刺激诱导LTP的FMR1KO未产生明显的LTP增强效应，而联合使用forskolin和DL-AP3的FMR1KO小鼠LTP有增强。
为了确定多巴胺能介导的LTP效应是否需要NMDA受体的激活，我们在灌流液中加入NMDA受体拮抗剂AP5，发现SKF81297介导的LTP增强效应消失。同样，LTP增强效应在灌流液中加入10mM BAPTA后也被阻断，表明多巴胺能介导的LTP效应需要NMDA受体的激活和突触后钙离子浓度增加的。
3mGluR1受体介导的可塑性是经由突触后机制产生的
我们检测了ACC区的双脉冲易化(PPF)情况。在WT和FMR1KO小鼠ACC区，灌流液中同时或分别加入DL-AP3和SKF81297前后，PPF的五个脉冲间隔产生的突触后反应没有显著性差异。结果表明，突触前递质释放没有改变，mGluR1受体介导的可塑性很可能是经由突触后机制产生的。
4mGluR1受体拮抗剂不影响突触的基础谷氨酸能递质释放
我们检测了ACC区AMPA受体介导的微小兴奋性突触后电流(mEPSCs)。在灌流液中同时或分别加入DL-AP3和SKF81297，AMPA受体介导的微小兴奋性突触后电流在WT和FMR1KO小鼠ACC区都没有发生显著性变化。表明mGluR1受体拮抗剂不影响突触的基础性谷氨酸能递质释放。
5AMPA受体的表达
为了确定这种协同作用对AMPA受体的表达和上膜有何影响，我们在培养的PFC细胞中加入D1受体激动剂SKF81297和mGluR1受体拮抗剂DL-AP3。发现单独使用SKF81297或者联合使用DL-AP3都会增加培养的FMR1WT小鼠PFC神经元总的GluR1的表达。而在FMR1KO小鼠，单独使用SKF81297或者DL-AP3都没有改变培养神经元总的GluR1的表达。单独使用SKF81297或者联合使用DL-AP3都会增加培养的FMR1WT小鼠PFC神经元AMPA受体在膜上的表达。SKF81297联合使用DL-AP3增加了培养的FMR1WT小鼠PFC神经元GluR1在膜上的表达。表明mGluR1受体恢复了D1受体激动剂对FMR1KO小鼠PFC细胞AMPA GluR1受体在膜上表达的增强作用。
我们检测了DL-AP3和forskolin对培养神经元GluR1向膜转运过程的影响。发现单独使用Forskolin或联合使用DL-AP3都会增加培养的FMR1WT小鼠PFC神经元总的GluR1的表达。单独使用Forskolin或联合使用DL-AP3都没有增加培养的FMR1KO小鼠PFC神经元总的GluR1的表达。而单独使用Forskolin或联合使用DL-AP3都会增加培养的FMR1WT小鼠PFC神经元GluR1在膜上的表达。forskolin联合使用DL-AP3增加了培养的FMR1WT小鼠PFC神经元GluR1受体在膜上的表达。表明D1受体激动剂SKF81297和mGluR1受体拮抗剂DL-AP3在FMR1KO小鼠上的联合作用是通过抑制mGluR1受体，激活腺苷酸环化酶实现的。
6mGluR5，D1受体和NMDA受体亚型在PFC培养神经元上的表达
通过Western blot检测，发现mGluR5和D1受体以及NMDA受体的两个亚型NR2A和NR2B受体在WT和FMR1KO小鼠ACC区的表达水平都没有显著性差异。因此，mGluR5和D1受体在ACC区突触增强作用受损不是因为mGluR5和D1受体以及NR2A和NR2B受体的基础表达水平差异引起的。且单独使用SKF81297或者SKF81297联合使用DL-AP3都不影响NR2A和NR2B亚型在FMR1WT和KO小鼠PFC神经元上的表达。然而，单独使用SKF81297或者SKF81297联合使用DL-AP3都显著性增加了NR2B亚型磷酸化位点Tyr-1472(p-NR2B-Tyr1472)在FMR1WT小鼠PFC培养神经元上的表达。同时，单独使用SKF81297没有增加NR2B亚型磷酸化位点Tyr-1472(p-NR2B-Tyr1472)在FMR1KO小鼠PFC培养神经元上的表达。而SKF81297联合使用DL-AP3能恢复SKF81297在FMR1KO小鼠PFC培养神经元上p-NR2B-Tyr1472的表达增加作用。
7SKF81297与DL-AP3联合效应对FMR1KO小鼠行为学的影响
通过行为学研究发现，FMR1KO小鼠的自发活动能力增加。腹腔注射SKF81297或者SKF81297联合使用DL-AP345分钟后，FMR1WT小鼠的自发活动的运动距离都没有显著性改变。然而，单独腹腔注射SKF81297或者SKF81297联合使用DL-AP345分钟后，FMR1KO小鼠自发活动的运动距离都有显著性降低。在模拟焦虑行为的高架十字实验中我们发现，FMR1KO与WT组小鼠以及单独应用SKF81297或联合使用DL-AP3组之间焦虑指标没有显著性差异，表明脆性X综合症模型小鼠没有表现出焦虑样行为，且焦虑样行为很可能不是通过D1受体通路起作用的。.
小鼠每天单独腹腔注射SKF81297或者SKF81297联合使用DL-AP35周后，进行Morris水迷宫实验。对此后4天FMR1KO和WT小鼠找到平台所用时间分析发现，在第一天两组之间就有显著性差异，FMR1KO组比WT组小鼠找到平台所用时间长。单独使用SKF81297或DL-AP3均不影响FMR1KO和WT小鼠找到平台所用时间，联合使用SKF81297和DL-AP3在前3天与同基因型组相比没有显著改变，而在第4天，FMR1KO小鼠与联合用药的FMR1KO小鼠组相比有显著性差异，联合使用SKF81297和DL-AP3在第四天显著降低FMR1KO小鼠找到平台所用时间。FMR1KO和WT小鼠在水迷宫实验过程中的平均游泳速度在组间没有显著性差异。
8FMR1KO小鼠ACC区五羟色胺能突触可塑性异常
为了明确FMR1KO小鼠ACC区除多巴胺系统外，其他有关突触可塑性的递质系统是否正常，我们检测了5-HT2A系统在ACC区有关突触可塑性的电生理学和分子生物学表现。我们发现使用5-HT2A受体拮抗剂的WT小鼠能增强LTP效应。然而，5-HT2A受体拮抗剂在FMR1KO没有表现出对LTP的增强效应。电生理实验表明，与多巴胺系统在FMR1KO小鼠ACC区突触可塑性功能受损类似，五羟色胺能系统在FMR1KO小鼠ACC区调节突触可塑性的功能也是受损的。.我们在灌流液中加入NMDA受体拮抗剂AP5(50μM)，发现R-96544(5μM)介导的在FMR1WT小鼠LTP增强效应消失。通过双脉冲易化(PPF)表现，发现在FMR1WT和FMR1KO小鼠ACC区，在灌流液中加入R-96544(5μM)前后，PPF的五个脉冲间隔产生的突触后反应没有显著性差异。结果表明，突触前递质释放没有改变，R-96544介导的可塑性很可能是经由突触后机制产生的。为进一步明确FMR1KO小鼠五羟色胺能受体调节LTP功能受损的机制，我们检测了5-HT2A受体在WT和FMR1KO小鼠ACC区的表达情况，发现5-HT2A受体以及NMDA受体的两个亚型NR2A和NR2B受体在WT和FMR1KO小鼠ACC区的表达水平都没有显著性差异。5-HT2A受体在ACC区突触增强作用受损不是因为5-HT2A受体以及NR2A和NR2B受体的基础表达水平差异引起的。
我们在培养的PFC细胞中加入5-HT2A受体拮抗剂R-96544，发现，在培养液中有1μM五羟色胺的情况下，使用R-96544(5μM)会增加培养的FMR1WT小鼠PFC神经元膜上GluR1的表达。表明5-HT2A受体拮抗剂能增强FMR1WT小鼠PFC细胞GluR1受体在膜上表达，而这种增强作用在FMR1KO小鼠消失，表明FMRP在五羟色胺调控GluR1受体在膜上表达的过程中起着重要作用。
【Objectives】
This research aims to find the mechanism on defect of synaptic plasticity inPFC of a mouse model of Fragile X syndrome and a possible way to rescuesynaptic plasticity in FMR1KO mice. Also, try to find a new way in medicaltreatment for FXS.
【Methods】
1Whole-cell recordings
Coronal prefrontal brain slices (300μm) from FMR1WT and KO mice,containing the anterior cingulate cortex (ACC), were prepared using standardmethods. Slices were transferred to submerged recovery chamber withoxygenated (95%O2and5%CO2) artificial cerebrospinal fluid (ACSF) atroom temperature for at least1h.Experiments were performed in a recordingchamber on the stage of an Axioskop2FS microscope with infrared DIC optics for the visualization of whole-cell patch clamp recording. In the present study,we recorded excitatory postsynaptic currents (EPSCs) from neurons in layer IIto III induced by the stimulation of layer V. AMPA receptor (AMPAR)-mediatedEPSCs were induced by repetitive stimulations at0.02Hz, and neurons werevoltage clamped at-70mV. After obtaining stable EPSCs for at least10min,LTP was induced by80pulses at2Hz, and then paired with postsynapticdepolarization at+30mV. Picrotoxin (100μM) was always present to blockGABAAreceptor-mediated inhibitory synaptic currents. To record the mEPSCs,0.5μM Tetrodotoxin (TTX) was added into the bath solution. The accessresistance was15to30M, which was monitored throughout the experiment.Data were discarded if access resistance changed by over15%during anexperiment.
2Western blot
Primary cultures of prefrontal cortical neurons were prepared fromembryonic18days old (E18) mice as described previously. Cultures were usedfor experiments between10and14days in vitro. SKF81297(5μM), DL-AP3(10μM), and forskolin (5μM) were added in the culture medium for24h beforethe cells harvested. Surface protein samples were detected through abiotinylation assay, followed by Western blot analysis with the antibodies aspreviously described. Western blot analysis was performed as describedpreviously. Equal amounts of protein (50μg) from the cultures were separatedand electrotransferred onto NC membranes, which were probed with antibodiesfor mGluR5, GluR1, NR2A, NR2B, p-NR2B Tyr1472, and with β-actin orcadherin as loading control. The membranes were incubated with horseradishperoxidase conjugated secondary antibodies and bands were visualized using anECL system (Perkin Elmer).
3Behavior test
Horizontal locomotor activity was conducted with an open-field test system (Jiangliang, Shanghai, China). Mice were placed in the center of the box,allowed to freely explore for10min, and videotaped using a camera fixed abovethe floor and then analyzed with a video-tracking system.
The elevated plus maze was tested after Horizontal locomotor activity. Micewere allowed to habituate to the testing room for2days before the test, andpretreated with gentle handling two times per day to eliminate their nervousness.For each test, individual animals were placed in the center square, facing anopen arm, and allowed to move freely for5min. Mice were videotaped using acamera fixed above the maze and analyzed with a video-tracking system. Thenumber of entries and time spent in each arm were recorded.
In the Morris Water Maze test, mice were treated either with SKF81297alone or with SKF81297plus DL-AP3for5weeks. Mice were kept dry betweentrials in a plastic holding cage filled with paper towels. Starting locations werechanged every trial. The time required to escape onto the hidden platform wasrecorded as escape latency.
Forty-five minutes after the subcutaneous injection of saline, DL-AP3,and/or SKF81297, each subject was placed in the center of the open field, andits activity was measured for30min.
【Results】
1Grp1mGluR antagonist rescues LTP facilitation by D1activation inFMR1KO mice
The PFC, including its cingulate region, plays an important role in learningand memory, drug addiction, and pain. First, we performed whole-cellpatch-clamp recordings in visually identified pyramidal neurons in layers II–IIIof cingulate region of PFC slices. LTP was induced by pairing presynapticstimulation with postsynaptic depolarization. The pairing training produced asignificant, long-lasting potentiation of synaptic responses in WT mice, but notin KO mice. Since Grp1mGluR-LTD is exaggerated in the FMR1KO mouse,we next examined the effects of mGluR1antagonist on LTP induction in the PFC. It has been reported that high dose of mGluR1antagonist,DL-2-amino-3-phosphonopropionic acid (DL-AP3) or(+)-alpha-methyl-4-carboxyphenylglycine (MCPG), reduced homosynaptic LTPin the hippocampus. In the present study, the slices were incubated withmGluR1antagonist DL-AP3at low dose of10μM at least for30min before theLTP induction was performed. At the concentration of10μM, DL-AP3did notalter the amplitude of LTP as compared to the pairing training only in the WTand KO mice. We next paired D1agonist application with an LTP inductionprotocol. Bath application of SKF81297for10min paired with the LTPinduction protocol significantly enhanced the amplitude of LTP, suggesting thatD1receptor activation can facilitate LTP induction in the WT mice. However, inFMR1KO mice SKF81297pairing training could not induce LTP,demonstrating that dopaminergic facilitation of LTP is impaired in FMR1KOmice.
Next, D1agonist and mGluR1antagonist were applied simultaneously todetect their synergistic effects on the LTP induction. Bath application ofSKF81297(5μM) and DL-AP3for10min induced a significant LTP. Furthercomparison analysis found that the amplitude of the LTP was similar to the LTPinduced by SKF81297alone in the WT mice. However, simultaneousapplication of DL-AP3markedly rescued the LTP induction by SKF81297inthe FMR1KO mice. Additional D1receptor antagonist SCH23390for10minprior to, and during electrical stimulation totally blocked the LTP which wasrescued by synergistic application of SKF81297and DL-AP3in the KO mice.
SKF81297or DL-AP330min had no effect on basal synaptic responses.Theses data indicate that lower dose of DL-AP3dose not alter the LTPinduction in the FMR1WT and KO mice; however, it rescues the LTPfacilitation of D1receptor activation in the FMR1KO mice.
2Adenylyl cyclase agonist rescues LTP facilitation by D1activation inFMR1KO mice
In our previous study, we found that D1receptor signaling is impaired, i.e.,the increase in cAMP caused by SKF81297is attenuated, accompanied by D1receptor hyperphosphorylation at serine sites in the PFC of FMR1KO mice. Todetect whether or not the increase of cAMP could mimic the function of D1receptor, the adenylyl cyclase activator, forskolin, was used. Many studies haveused forskolin to stimulate AMPAR trafficking and induce chemical LTP. In thepresent study, unlike in the hippocampus, LTP was not induced during theapplication of forskolin, which was not paired with the electrical stimulation inthe ACC, i.e, no chemical LTP was induced in the ACC. The amplitude of LTP issimilar to that of the control when the forskolin was paired with the electricalstimulation in the FMR1WT mice. Combining forskolin with DL-AP3did notresult in an increase in the amplitude of LTP compared with the case, in whichthe forskolin alone was used in the FMR1WT mice. Forskolin paired with theelectrical stimulation was unable to induce LTP in the FMR1WT mice.However, combining forskolin with DL-AP3partially rescued LTP in the FMR1KO mice, even if the amplitude was only118%±2.8%of the base line.
To determine whether or not dopaminergic modulation of LTP requiredNMDAR activation, we applied a selective NMDAR antagonist, AP5, and foundthat LTP facilitation by SKF81297was completely blocked. Similarly, LTP wasabolished by BAPTA in the pipette solution, indicating that dopaminergicmodulation of LTP depended on the activation of NMDARs and elevatedpostsynaptic Ca2+concentrations.
3Grp1mGluRs modulate plasticity by a postsynaptic mechanism
In the different regions of the hippocampus, both presynaptic andpostsynaptic mechanisms have been proposed to contribute to the expression ofLTP. To determine whether presynaptic and/or postsynaptic mechanisms areinvolved in Grp1mGluRs modulation of LTP, we measured paired-pulse facilitation (PPF) in the ACC. PPF is a phenomenon by which a second synapticstimulation of equal magnitude evokes a larger synaptic response than the first,and has been used as a tool to implicate presynaptic probability of transmitterrelease. PPF induced at five different intervals did not differ in WT and FMR1KO mice in the presence of DL-AP3or/and SKF81297in the ACC. The datasuggest that presynaptic mechanisms do not seem to be involved in the Grp1mGluRs modulation of LTP.
4No changing of basal glutamatergic synaptic transmission by Grp1mGluRs inhibition
To determine whether Grp1mGluRs inhibition affects basal glutamatergicsynaptic transmission in the ACC, we measured the miniature AMPAreceptor-mediated EPSCs (mEPSCs). As shown in Fig.4, no significantalteration was detected in the AMPA receptor-mediated mEPSCs frequency andamplitude among the control, DL-AP3or/and SKF81297treated slices fromFMR1WT and KO mice. Results indicate that Grp1mGluRs inhibition andactivation of D1receptor have no effect on the basal excitatory synaptictransmission in the ACC.
5Expression of GluR1
To determine the synergistic effect on the AMPA receptor expression andsurface insertion, we treated cultured PFC neurons with the D1receptor agonistsSKF81297and Grp1mGluRs antagonist DL-AP3. SKF81297alone orcombining with DL-AP3increased the total expression levels of GluR1in thecultures from FMR1WT mice. However, neither SKF81297nor DL-AP3affected the total expression levels of GluR1in cultures from FMR1KO mice.To further investigate the signaling involved in the synaptic plasticity, we testedthe effects of DL-AP3or/and SKF81297on AMPA receptor GluR1subunitssurface trafficking in cultured PFC neurons. Surface expression of GluR1wasincreased after treatment with SKF81297alone or combining with DL-AP3inthe cultures from FMR1WT mice. Contrast to the unchanged total levels of GluR1, surface expression of GluR1was increased after simultaneous treatmentof SKF81297and DL-AP3in the cultures from FMR1KO mice. This findingdemonstrates that inhibition of Grp1mGluRs can rescue the surface expressionof AMPA GluR1receptors by D1receptor in FMR1KO PFC neurons.
Furthermore, the synergistic effects of DL-AP3and forskolin on AMPAreceptor GluR1subunits surface trafficking were detected in cultured PFCneurons. Forskolin alone or combined with DL-AP3increased the totalexpression levels of GluR1in the cultures from FMR1WT mice. However,neither forskolin nor DL-AP3affected the total expression levels of GluR1incultures from FMR1KO mice. The surface expressions of GluR1in the culturesfrom FMR1WT mice increase after treatment with forskolin alone andcombined with DL-AP3. In contrast with the unchanged total levels of GluR1,surface expressions of GluR1in the cultures from FMR1KO mice increasedafter simultaneous treatment of forskolin and DL-AP3. This findingdemonstrated the synergistic effects on the surface expressions of AMPA GluR1receptors in FMR1KO PFC neurons resulting from the inhibition of Grp1mGluRs and activation of adenylyl cyclase.
6mGluR5, D1receptor, and NMDARs expression in the PFC cultures
To further explore the synaptic mechanisms behind the impairment ofdopaminergic modulation of LTP, we examined the expression of mGluR5andD1receptor in the ACC from WT and FMR1KO mice. As shown in Fig.6, therewas no difference in the levels of mGluR5, D1receptor, NMDA receptor NR2Asubunit, and NR2B subunit between the WT and FMR1KO mice. Thus, theimpairment of mGluR5and D1receptor modulation of synaptic potentiation isnot due to defect in levels of mGluR5, D1receptor, and NR2A-or NR2B-containing NMDA receptor.
The function of NMDARs is regulated by its phosphorylation. Given thattyrosine phosphorylation of NMDARs contributes to LTP, we examined thetyrosine phosphorylation levels of NR2B subunits with anti-phosphotyrosine antibody to determine whether or not NMDARs are phosphorylated toSKF81297and DL-AP3. We found that the presence of SKF81297alone orSKF81297plus DL-AP3did not affect total NR2A or NR2B subunit expressionin the cultures from FMR1WT and KO mice. However, SKF81297alone orSKF81297plus DL-AP3significantly increased the level of phosphorylation ofthe NR2B subunit at Tyr-1472(p-NR2B-Tyr1472) in the cultures from FMR1WT mice. Meanwhile, SKF81297alone failed to increase p-NR2B-Tyr1472inthe cultures from FMR1KO mice. Synergistic addition of DL-AP3rescued thep-NR2B-Tyr1472by SKF81297in the cultures from FMR1KO mice. Thesedata implied that the increase of p-NR2B-Tyr1472contributed to the surfaceexpression of GluR1by SKF81297and DL-AP3in the KO cultures.
7Synergistic effect of simultaneous treatments on the behavior of FMR1KO mice
Fragile X patients and animal models exhibit behavioral phenotypes that areconsistent with deficits in PFC function, such as hyperactivity, anxiety, andlearning abnormalities. We analyzed locomotor activity, anxiety, and learningabilities to characterize hyperactivity and learning deficit, the commonphenotypes in fragile X syndrome. We found increased locomotor activity inFMR1KO mice, which was consistent with our previous study. The locomotoractivity was not affected following SKF81297or/and DL-AP3subcutaneousinjection after forty-five minutes in WT mice. However, application of the D1receptor agonist SKF81297or simultaneous treatment of SKF81297andDL-AP3significantly reduced the distance traveled by FMR1KO mice. Thesedata indicate that FMR1KO mice exhibit hyperactivity and that simultaneoustreatment of D1agonist and Grp1mGluRs antagonist can reduce thehyperactivity phenotype.
We then investigated anxiety-like behaviors with the elevated plus-maze test.We found no differences between FMR1KO and WT mice and no effect ofSKF81297alone or plus DL-AP3. These data indicate that anxiety-like behaviors are not detectable in this fragile X mouse model and suggest that thesebehaviors are probably not related to the D1receptor signaling pathway. Thesefindings are consistent with our previous study.
Last, the learning ability was detected in the Morris Water Maze test. Micewere treated with SKF81297alone or plus DL-AP3for5weeks. Swim latencywas compared over the4days of the experiment, and there were significantdifferences in escape latency between genotype at day1. SKF81297or DL-AP3treatment alone did not change the swim latency in the FMR1WT and KO mice.Simultaneous treatment of SKF81297or DL-AP3also did not improve thelearning ability in the FMR1WT mice. However, Simultaneous treatment ofSKF81297and DL-AP3reduced the swim latency on the final day in the FMR1KO mice. Moreover, we compared the swimming speed during the water mazetest and found no differences among the groups. These findings clearly showthat simultaneous treatment of SKF81297and DL-AP3significantly improvesthe spatial leaning of FMR1KO mice.In summary, the present study, together with our previous study, reveals thatmGluR1inhibition is a useful strategy to recover D1receptor signaling in theFMR1KO mice, and that combination of Grp1mGluR antagonist and D1agonist is a potential drug therapy for the FXS.
8Abnormal serotoninergic synaptic plastisty of FMR1KO mice ACC
In order to check if other neurotransmiting related system is normal, wechecked5-HT2A system, another important systern for synaptic plastisty. Wefound that bath application of R-96544or ketanserin significantly enhanced theamplitude of LTP. This suggests that inhibition of5-HT2A receptor canfacilitate LTP induction. However, this facilitation of LTP in WT mice byR-96544or ketanserin was lost in FMR1KO mice, demonstrating thatserotoninergic modulation of LTP is impaired in FMR1KO mice and thatFMRP is required for serotoninergic modulation of LTP in the ACC.
We next applied a selective NMDAR antagonist, AP5(50μM), and found that LTP facilitation by R-96544(5μM) was completely blocked in the WTmice.Similarly, LTP was abolished by10mM BAPTA in the pipette solution,indicating that serotoninergic modulation of LTP is dependent on the activationof NMDARs and elevated postsynaptic Ca2+concentrations.
To determine whether presynaptic and/or postsynaptic mechanisms areinvolved in serotoninergic modulation of LTP, we measured paired-pulsefacilitation (PPF) in the ACC. We found that PPF induced at five differentintervals did not differ in WT and FMR1KO mice in the presence of R-96544inthe ACC. The data suggest that presynaptic mechanisms do not seem to beinvolved in the serotoninergic modulation of LTP.
To further explore the synaptic mechanisms behind the impairment ofserotoninergic modulation of LTP, we examined5-HT2A receptor andglutamate receptor subunits expression in the ACC from WT and FMR1KOmice.There were no differences in5-HT2A receptor expression between the WTand FMR1KO mice. Furthermore, the total GluR1, NMDA receptor NR2A, andNR2B subunits expression were no differences between the WT and FMR1KOmice under basal conditions. Thus, the impairment of serotoninergic modulationof synaptic potentiation is not due to a defect in5-HT2A receptor and glutamatereceptors expression. To investigate the role of5-HT2A receptor signalingpathway in the synaptic plasticity, we tested the effects of R-96544on AMPAreceptor GluR1subunits surface trafficking in cultured PFC neurons in thepresence of1μM serotonin. Surface expression of GluR1was increased aftertreatment with5-HT2AR antagonist R-96544. This finding demonstrates thatinhibition of5-HT2A receptor can facilitate the surface expression of AMPAGluR1receptors. However, the surface expression of GluR1triggered byR-96544was impaired in FMR1KO PFC neurons. These results provide direct evidence that FMRP is involved in serotonin receptor signaling and contributesto GluR1surface expression induced by5-HT2A receptor inactivation.
探索FMR1KO小鼠突触可塑性调节异常的机制以及如何恢复ACC区的可塑性，以期对进一步认识脆性X综合症的病理机制，发掘潜在的药物治疗新靶点。
【方法】
1全细胞膜片钳记录
小鼠采用1-2%异氟醚麻醉，取脑。振动切片机横向切取300μmACC脑片，室温下置于混合气饱和的人工脑脊液(ACSF)中，恢复1小时后进行实验。在具有红外线DIC系统的显微镜下观察ACC第II/III层锥体神经元。ACSF灌流液中加入100M picrotoxin，刺激电极放置于ACC第V层，记录电极放置于ACC第II/III层，在锥体神经元中选取状态良好的椎体神经元进行封接。记录兴奋性突触后电流（EPSC）和LTP。LTP诱导方法：80次刺激，频率2Hz，同时神经元钳制于+30mV，然后恢复到基线记录模式，钳制电压恢复在-70mV。记录mEPSC时，灌流液中加入TTX，转换为Gap free记录模式。
2小鼠前额叶皮层神经元的原代培养
取孕18天胎鼠，在无菌条件下断头取脑置于盛有PBS液的平皿中剥去脑膜，取大脑皮层并剪碎，消化后反复吹打制备细胞悬液。接种于涂有多聚赖氨酸的培养皿内，培养箱中进行培养。培养第三天加入阿糖胞苷，以抑制非神经元细胞的继续增长。SKF81297（5μM），DL-AP3（10μM)），和forskolin（5μM）在收集细胞前24小时分组加入培养基，收集细胞待用。
3Western blot检测
采用常规脑组织样本制备方法制备ACC区组织样品。培养神经元细胞膜蛋白的提取，采用细胞膜蛋白生物素试剂盒提取的方法，提取过程同过往文献报道[1]。Western blot检测方法：将蛋白转到NC膜上后，分别用mGluR5，GluR1，NR2A，NR2B，p-NR2B的一抗封闭各自分子量相应的条带，孵育后，漂洗，加入二抗漂洗发光。曝光完成后，经显影和定影，自来水冲洗，最后在室温下晾干。用β-actin或cadherin作为标准定量分子，确定各个蛋白相对量的多少。
4行为学实验
开场实验装置由旷场反应箱和数据自动采集和处理系统两部分组成，实验小鼠置于旷场的中央，记录并分析它的运动情况。高架十字迷宫各有两个对称的开放臂和封闭臂，十字中间为正方形平台。实验小鼠逐个放于中央平台后，记录每个小鼠5min内进入开放臂或封闭臂的次数与时间，并计算进入两臂的总次数。Morris水迷宫定位航行实验历时数天,每天将大鼠面向池壁分别从4个入水点放入水中若干次,记录其寻找到隐藏在水面下平台的时间。动物实验小鼠在实验前分别腹腔注射生理盐水，DL-AP3和/或SKF81297，45分钟后开始实验。
【结果】
1SKF81297和DL-AP3对FMR1基因敲除小鼠LTP的增强
我们用全细胞膜片钳的方法对成年脑薄片的前扣带回区II–III层锥体细胞的电生理活动进行记录，采用突触前电刺激诱导突触后去极化的方法使神经元产生LTP并记录。我们发现在FMR1WT小鼠，该脑区神经元产生了明显的长时程增强现象。但在FMR1KO小鼠，这种增强效应不明显。我们发现，正常和敲除小鼠的LTP效应与各自的空白对照组相比没有显著性差异。接下来，我们检测了D1受体激动剂应用组的LTP诱导情况，发现在D1受体激动剂SKF812975μM孵育十分钟后，正常小鼠脑薄片诱导后的LTP效应明显增强,说明激活D1受体能增强正常小鼠的LTP效应。而在FMR1KO小鼠，应用SKF81297没有增强其诱导后的LTP效应，说明D1受体激动剂的LTP增强作用在FMR1基因敲除小鼠ACC区是受损的。
我们在检测同时使用D1受体激动剂和mGluR1受体拮抗剂对LTP诱导的影响时，发现SKF81297和DL-AP3共同孵育脑片十分钟，能显著增强正常小鼠的LTP效应，这种增强效应与单独应用D1受体激动剂SKF81297的正常小鼠相比没有显著性差异。在FMR1基因敲除小鼠，同时使用D1受体激动剂和mGluR1受体拮抗剂，ACC区LTP效应与单独使用D1受体激动剂有显著性增强。
为了检测合用SKF81297和DL-AP3对FMR1KO小鼠LTP的增强效应是否来源于D1受体激动剂，我们在孵育的液体中同时加入D1受体抑制剂，发现这种LTP增强效应消失。可以认为FMR1KO小鼠LTP的增强效应来源于D1受体激动剂，这种效应是由mGluR1受体拮抗剂恢复的。
2腺苷酸环化酶激动剂恢复D1受体激动剂对FMR1KO小鼠ACC区LTP增强效应
为了确定增加腺苷酸环化酶的量是否能替代D1受体的作用，我们研究了腺苷酸环化酶激动剂forskolin对FMR1KO小鼠ACC区LTP是否有增强作用。发现加入forskolin且有电刺激诱导的情况下，LTP的幅度与未使用forskolin的FMR1WT小鼠对照组相比无显著性差异。与单独使用forskolin的FMR1WT小鼠组相比，DL-AP3与forskolin结合使用并没有引起FMR1WT小鼠LTP幅度增加。单独使用Forskolin并电刺激诱导LTP的FMR1KO未产生明显的LTP增强效应，而联合使用forskolin和DL-AP3的FMR1KO小鼠LTP有增强。
为了确定多巴胺能介导的LTP效应是否需要NMDA受体的激活，我们在灌流液中加入NMDA受体拮抗剂AP5，发现SKF81297介导的LTP增强效应消失。同样，LTP增强效应在灌流液中加入10mM BAPTA后也被阻断，表明多巴胺能介导的LTP效应需要NMDA受体的激活和突触后钙离子浓度增加的。
3mGluR1受体介导的可塑性是经由突触后机制产生的
我们检测了ACC区的双脉冲易化(PPF)情况。在WT和FMR1KO小鼠ACC区，灌流液中同时或分别加入DL-AP3和SKF81297前后，PPF的五个脉冲间隔产生的突触后反应没有显著性差异。结果表明，突触前递质释放没有改变，mGluR1受体介导的可塑性很可能是经由突触后机制产生的。
4mGluR1受体拮抗剂不影响突触的基础谷氨酸能递质释放
我们检测了ACC区AMPA受体介导的微小兴奋性突触后电流(mEPSCs)。在灌流液中同时或分别加入DL-AP3和SKF81297，AMPA受体介导的微小兴奋性突触后电流在WT和FMR1KO小鼠ACC区都没有发生显著性变化。表明mGluR1受体拮抗剂不影响突触的基础性谷氨酸能递质释放。
5AMPA受体的表达
为了确定这种协同作用对AMPA受体的表达和上膜有何影响，我们在培养的PFC细胞中加入D1受体激动剂SKF81297和mGluR1受体拮抗剂DL-AP3。发现单独使用SKF81297或者联合使用DL-AP3都会增加培养的FMR1WT小鼠PFC神经元总的GluR1的表达。而在FMR1KO小鼠，单独使用SKF81297或者DL-AP3都没有改变培养神经元总的GluR1的表达。单独使用SKF81297或者联合使用DL-AP3都会增加培养的FMR1WT小鼠PFC神经元AMPA受体在膜上的表达。SKF81297联合使用DL-AP3增加了培养的FMR1WT小鼠PFC神经元GluR1在膜上的表达。表明mGluR1受体恢复了D1受体激动剂对FMR1KO小鼠PFC细胞AMPA GluR1受体在膜上表达的增强作用。
我们检测了DL-AP3和forskolin对培养神经元GluR1向膜转运过程的影响。发现单独使用Forskolin或联合使用DL-AP3都会增加培养的FMR1WT小鼠PFC神经元总的GluR1的表达。单独使用Forskolin或联合使用DL-AP3都没有增加培养的FMR1KO小鼠PFC神经元总的GluR1的表达。而单独使用Forskolin或联合使用DL-AP3都会增加培养的FMR1WT小鼠PFC神经元GluR1在膜上的表达。forskolin联合使用DL-AP3增加了培养的FMR1WT小鼠PFC神经元GluR1受体在膜上的表达。表明D1受体激动剂SKF81297和mGluR1受体拮抗剂DL-AP3在FMR1KO小鼠上的联合作用是通过抑制mGluR1受体，激活腺苷酸环化酶实现的。
6mGluR5，D1受体和NMDA受体亚型在PFC培养神经元上的表达
通过Western blot检测，发现mGluR5和D1受体以及NMDA受体的两个亚型NR2A和NR2B受体在WT和FMR1KO小鼠ACC区的表达水平都没有显著性差异。因此，mGluR5和D1受体在ACC区突触增强作用受损不是因为mGluR5和D1受体以及NR2A和NR2B受体的基础表达水平差异引起的。且单独使用SKF81297或者SKF81297联合使用DL-AP3都不影响NR2A和NR2B亚型在FMR1WT和KO小鼠PFC神经元上的表达。然而，单独使用SKF81297或者SKF81297联合使用DL-AP3都显著性增加了NR2B亚型磷酸化位点Tyr-1472(p-NR2B-Tyr1472)在FMR1WT小鼠PFC培养神经元上的表达。同时，单独使用SKF81297没有增加NR2B亚型磷酸化位点Tyr-1472(p-NR2B-Tyr1472)在FMR1KO小鼠PFC培养神经元上的表达。而SKF81297联合使用DL-AP3能恢复SKF81297在FMR1KO小鼠PFC培养神经元上p-NR2B-Tyr1472的表达增加作用。
7SKF81297与DL-AP3联合效应对FMR1KO小鼠行为学的影响
通过行为学研究发现，FMR1KO小鼠的自发活动能力增加。腹腔注射SKF81297或者SKF81297联合使用DL-AP345分钟后，FMR1WT小鼠的自发活动的运动距离都没有显著性改变。然而，单独腹腔注射SKF81297或者SKF81297联合使用DL-AP345分钟后，FMR1KO小鼠自发活动的运动距离都有显著性降低。在模拟焦虑行为的高架十字实验中我们发现，FMR1KO与WT组小鼠以及单独应用SKF81297或联合使用DL-AP3组之间焦虑指标没有显著性差异，表明脆性X综合症模型小鼠没有表现出焦虑样行为，且焦虑样行为很可能不是通过D1受体通路起作用的。.
小鼠每天单独腹腔注射SKF81297或者SKF81297联合使用DL-AP35周后，进行Morris水迷宫实验。对此后4天FMR1KO和WT小鼠找到平台所用时间分析发现，在第一天两组之间就有显著性差异，FMR1KO组比WT组小鼠找到平台所用时间长。单独使用SKF81297或DL-AP3均不影响FMR1KO和WT小鼠找到平台所用时间，联合使用SKF81297和DL-AP3在前3天与同基因型组相比没有显著改变，而在第4天，FMR1KO小鼠与联合用药的FMR1KO小鼠组相比有显著性差异，联合使用SKF81297和DL-AP3在第四天显著降低FMR1KO小鼠找到平台所用时间。FMR1KO和WT小鼠在水迷宫实验过程中的平均游泳速度在组间没有显著性差异。
8FMR1KO小鼠ACC区五羟色胺能突触可塑性异常
为了明确FMR1KO小鼠ACC区除多巴胺系统外，其他有关突触可塑性的递质系统是否正常，我们检测了5-HT2A系统在ACC区有关突触可塑性的电生理学和分子生物学表现。我们发现使用5-HT2A受体拮抗剂的WT小鼠能增强LTP效应。然而，5-HT2A受体拮抗剂在FMR1KO没有表现出对LTP的增强效应。电生理实验表明，与多巴胺系统在FMR1KO小鼠ACC区突触可塑性功能受损类似，五羟色胺能系统在FMR1KO小鼠ACC区调节突触可塑性的功能也是受损的。.我们在灌流液中加入NMDA受体拮抗剂AP5(50μM)，发现R-96544(5μM)介导的在FMR1WT小鼠LTP增强效应消失。通过双脉冲易化(PPF)表现，发现在FMR1WT和FMR1KO小鼠ACC区，在灌流液中加入R-96544(5μM)前后，PPF的五个脉冲间隔产生的突触后反应没有显著性差异。结果表明，突触前递质释放没有改变，R-96544介导的可塑性很可能是经由突触后机制产生的。为进一步明确FMR1KO小鼠五羟色胺能受体调节LTP功能受损的机制，我们检测了5-HT2A受体在WT和FMR1KO小鼠ACC区的表达情况，发现5-HT2A受体以及NMDA受体的两个亚型NR2A和NR2B受体在WT和FMR1KO小鼠ACC区的表达水平都没有显著性差异。5-HT2A受体在ACC区突触增强作用受损不是因为5-HT2A受体以及NR2A和NR2B受体的基础表达水平差异引起的。
我们在培养的PFC细胞中加入5-HT2A受体拮抗剂R-96544，发现，在培养液中有1μM五羟色胺的情况下，使用R-96544(5μM)会增加培养的FMR1WT小鼠PFC神经元膜上GluR1的表达。表明5-HT2A受体拮抗剂能增强FMR1WT小鼠PFC细胞GluR1受体在膜上表达，而这种增强作用在FMR1KO小鼠消失，表明FMRP在五羟色胺调控GluR1受体在膜上表达的过程中起着重要作用。
【Objectives】
This research aims to find the mechanism on defect of synaptic plasticity inPFC of a mouse model of Fragile X syndrome and a possible way to rescuesynaptic plasticity in FMR1KO mice. Also, try to find a new way in medicaltreatment for FXS.
【Methods】
1Whole-cell recordings
Coronal prefrontal brain slices (300μm) from FMR1WT and KO mice,containing the anterior cingulate cortex (ACC), were prepared using standardmethods. Slices were transferred to submerged recovery chamber withoxygenated (95%O2and5%CO2) artificial cerebrospinal fluid (ACSF) atroom temperature for at least1h.Experiments were performed in a recordingchamber on the stage of an Axioskop2FS microscope with infrared DIC optics for the visualization of whole-cell patch clamp recording. In the present study,we recorded excitatory postsynaptic currents (EPSCs) from neurons in layer IIto III induced by the stimulation of layer V. AMPA receptor (AMPAR)-mediatedEPSCs were induced by repetitive stimulations at0.02Hz, and neurons werevoltage clamped at-70mV. After obtaining stable EPSCs for at least10min,LTP was induced by80pulses at2Hz, and then paired with postsynapticdepolarization at+30mV. Picrotoxin (100μM) was always present to blockGABAAreceptor-mediated inhibitory synaptic currents. To record the mEPSCs,0.5μM Tetrodotoxin (TTX) was added into the bath solution. The accessresistance was15to30M, which was monitored throughout the experiment.Data were discarded if access resistance changed by over15%during anexperiment.
2Western blot
Primary cultures of prefrontal cortical neurons were prepared fromembryonic18days old (E18) mice as described previously. Cultures were usedfor experiments between10and14days in vitro. SKF81297(5μM), DL-AP3(10μM), and forskolin (5μM) were added in the culture medium for24h beforethe cells harvested. Surface protein samples were detected through abiotinylation assay, followed by Western blot analysis with the antibodies aspreviously described. Western blot analysis was performed as describedpreviously. Equal amounts of protein (50μg) from the cultures were separatedand electrotransferred onto NC membranes, which were probed with antibodiesfor mGluR5, GluR1, NR2A, NR2B, p-NR2B Tyr1472, and with β-actin orcadherin as loading control. The membranes were incubated with horseradishperoxidase conjugated secondary antibodies and bands were visualized using anECL system (Perkin Elmer).
3Behavior test
Horizontal locomotor activity was conducted with an open-field test system (Jiangliang, Shanghai, China). Mice were placed in the center of the box,allowed to freely explore for10min, and videotaped using a camera fixed abovethe floor and then analyzed with a video-tracking system.
The elevated plus maze was tested after Horizontal locomotor activity. Micewere allowed to habituate to the testing room for2days before the test, andpretreated with gentle handling two times per day to eliminate their nervousness.For each test, individual animals were placed in the center square, facing anopen arm, and allowed to move freely for5min. Mice were videotaped using acamera fixed above the maze and analyzed with a video-tracking system. Thenumber of entries and time spent in each arm were recorded.
In the Morris Water Maze test, mice were treated either with SKF81297alone or with SKF81297plus DL-AP3for5weeks. Mice were kept dry betweentrials in a plastic holding cage filled with paper towels. Starting locations werechanged every trial. The time required to escape onto the hidden platform wasrecorded as escape latency.
Forty-five minutes after the subcutaneous injection of saline, DL-AP3,and/or SKF81297, each subject was placed in the center of the open field, andits activity was measured for30min.
【Results】
1Grp1mGluR antagonist rescues LTP facilitation by D1activation inFMR1KO mice
The PFC, including its cingulate region, plays an important role in learningand memory, drug addiction, and pain. First, we performed whole-cellpatch-clamp recordings in visually identified pyramidal neurons in layers II–IIIof cingulate region of PFC slices. LTP was induced by pairing presynapticstimulation with postsynaptic depolarization. The pairing training produced asignificant, long-lasting potentiation of synaptic responses in WT mice, but notin KO mice. Since Grp1mGluR-LTD is exaggerated in the FMR1KO mouse,we next examined the effects of mGluR1antagonist on LTP induction in the PFC. It has been reported that high dose of mGluR1antagonist,DL-2-amino-3-phosphonopropionic acid (DL-AP3) or(+)-alpha-methyl-4-carboxyphenylglycine (MCPG), reduced homosynaptic LTPin the hippocampus. In the present study, the slices were incubated withmGluR1antagonist DL-AP3at low dose of10μM at least for30min before theLTP induction was performed. At the concentration of10μM, DL-AP3did notalter the amplitude of LTP as compared to the pairing training only in the WTand KO mice. We next paired D1agonist application with an LTP inductionprotocol. Bath application of SKF81297for10min paired with the LTPinduction protocol significantly enhanced the amplitude of LTP, suggesting thatD1receptor activation can facilitate LTP induction in the WT mice. However, inFMR1KO mice SKF81297pairing training could not induce LTP,demonstrating that dopaminergic facilitation of LTP is impaired in FMR1KOmice.
Next, D1agonist and mGluR1antagonist were applied simultaneously todetect their synergistic effects on the LTP induction. Bath application ofSKF81297(5μM) and DL-AP3for10min induced a significant LTP. Furthercomparison analysis found that the amplitude of the LTP was similar to the LTPinduced by SKF81297alone in the WT mice. However, simultaneousapplication of DL-AP3markedly rescued the LTP induction by SKF81297inthe FMR1KO mice. Additional D1receptor antagonist SCH23390for10minprior to, and during electrical stimulation totally blocked the LTP which wasrescued by synergistic application of SKF81297and DL-AP3in the KO mice.
SKF81297or DL-AP330min had no effect on basal synaptic responses.Theses data indicate that lower dose of DL-AP3dose not alter the LTPinduction in the FMR1WT and KO mice; however, it rescues the LTPfacilitation of D1receptor activation in the FMR1KO mice.
2Adenylyl cyclase agonist rescues LTP facilitation by D1activation inFMR1KO mice
In our previous study, we found that D1receptor signaling is impaired, i.e.,the increase in cAMP caused by SKF81297is attenuated, accompanied by D1receptor hyperphosphorylation at serine sites in the PFC of FMR1KO mice. Todetect whether or not the increase of cAMP could mimic the function of D1receptor, the adenylyl cyclase activator, forskolin, was used. Many studies haveused forskolin to stimulate AMPAR trafficking and induce chemical LTP. In thepresent study, unlike in the hippocampus, LTP was not induced during theapplication of forskolin, which was not paired with the electrical stimulation inthe ACC, i.e, no chemical LTP was induced in the ACC. The amplitude of LTP issimilar to that of the control when the forskolin was paired with the electricalstimulation in the FMR1WT mice. Combining forskolin with DL-AP3did notresult in an increase in the amplitude of LTP compared with the case, in whichthe forskolin alone was used in the FMR1WT mice. Forskolin paired with theelectrical stimulation was unable to induce LTP in the FMR1WT mice.However, combining forskolin with DL-AP3partially rescued LTP in the FMR1KO mice, even if the amplitude was only118%±2.8%of the base line.
To determine whether or not dopaminergic modulation of LTP requiredNMDAR activation, we applied a selective NMDAR antagonist, AP5, and foundthat LTP facilitation by SKF81297was completely blocked. Similarly, LTP wasabolished by BAPTA in the pipette solution, indicating that dopaminergicmodulation of LTP depended on the activation of NMDARs and elevatedpostsynaptic Ca2+concentrations.
3Grp1mGluRs modulate plasticity by a postsynaptic mechanism
In the different regions of the hippocampus, both presynaptic andpostsynaptic mechanisms have been proposed to contribute to the expression ofLTP. To determine whether presynaptic and/or postsynaptic mechanisms areinvolved in Grp1mGluRs modulation of LTP, we measured paired-pulse facilitation (PPF) in the ACC. PPF is a phenomenon by which a second synapticstimulation of equal magnitude evokes a larger synaptic response than the first,and has been used as a tool to implicate presynaptic probability of transmitterrelease. PPF induced at five different intervals did not differ in WT and FMR1KO mice in the presence of DL-AP3or/and SKF81297in the ACC. The datasuggest that presynaptic mechanisms do not seem to be involved in the Grp1mGluRs modulation of LTP.
4No changing of basal glutamatergic synaptic transmission by Grp1mGluRs inhibition
To determine whether Grp1mGluRs inhibition affects basal glutamatergicsynaptic transmission in the ACC, we measured the miniature AMPAreceptor-mediated EPSCs (mEPSCs). As shown in Fig.4, no significantalteration was detected in the AMPA receptor-mediated mEPSCs frequency andamplitude among the control, DL-AP3or/and SKF81297treated slices fromFMR1WT and KO mice. Results indicate that Grp1mGluRs inhibition andactivation of D1receptor have no effect on the basal excitatory synaptictransmission in the ACC.
5Expression of GluR1
To determine the synergistic effect on the AMPA receptor expression andsurface insertion, we treated cultured PFC neurons with the D1receptor agonistsSKF81297and Grp1mGluRs antagonist DL-AP3. SKF81297alone orcombining with DL-AP3increased the total expression levels of GluR1in thecultures from FMR1WT mice. However, neither SKF81297nor DL-AP3affected the total expression levels of GluR1in cultures from FMR1KO mice.To further investigate the signaling involved in the synaptic plasticity, we testedthe effects of DL-AP3or/and SKF81297on AMPA receptor GluR1subunitssurface trafficking in cultured PFC neurons. Surface expression of GluR1wasincreased after treatment with SKF81297alone or combining with DL-AP3inthe cultures from FMR1WT mice. Contrast to the unchanged total levels of GluR1, surface expression of GluR1was increased after simultaneous treatmentof SKF81297and DL-AP3in the cultures from FMR1KO mice. This findingdemonstrates that inhibition of Grp1mGluRs can rescue the surface expressionof AMPA GluR1receptors by D1receptor in FMR1KO PFC neurons.
Furthermore, the synergistic effects of DL-AP3and forskolin on AMPAreceptor GluR1subunits surface trafficking were detected in cultured PFCneurons. Forskolin alone or combined with DL-AP3increased the totalexpression levels of GluR1in the cultures from FMR1WT mice. However,neither forskolin nor DL-AP3affected the total expression levels of GluR1incultures from FMR1KO mice. The surface expressions of GluR1in the culturesfrom FMR1WT mice increase after treatment with forskolin alone andcombined with DL-AP3. In contrast with the unchanged total levels of GluR1,surface expressions of GluR1in the cultures from FMR1KO mice increasedafter simultaneous treatment of forskolin and DL-AP3. This findingdemonstrated the synergistic effects on the surface expressions of AMPA GluR1receptors in FMR1KO PFC neurons resulting from the inhibition of Grp1mGluRs and activation of adenylyl cyclase.
6mGluR5, D1receptor, and NMDARs expression in the PFC cultures
To further explore the synaptic mechanisms behind the impairment ofdopaminergic modulation of LTP, we examined the expression of mGluR5andD1receptor in the ACC from WT and FMR1KO mice. As shown in Fig.6, therewas no difference in the levels of mGluR5, D1receptor, NMDA receptor NR2Asubunit, and NR2B subunit between the WT and FMR1KO mice. Thus, theimpairment of mGluR5and D1receptor modulation of synaptic potentiation isnot due to defect in levels of mGluR5, D1receptor, and NR2A-or NR2B-containing NMDA receptor.
The function of NMDARs is regulated by its phosphorylation. Given thattyrosine phosphorylation of NMDARs contributes to LTP, we examined thetyrosine phosphorylation levels of NR2B subunits with anti-phosphotyrosine antibody to determine whether or not NMDARs are phosphorylated toSKF81297and DL-AP3. We found that the presence of SKF81297alone orSKF81297plus DL-AP3did not affect total NR2A or NR2B subunit expressionin the cultures from FMR1WT and KO mice. However, SKF81297alone orSKF81297plus DL-AP3significantly increased the level of phosphorylation ofthe NR2B subunit at Tyr-1472(p-NR2B-Tyr1472) in the cultures from FMR1WT mice. Meanwhile, SKF81297alone failed to increase p-NR2B-Tyr1472inthe cultures from FMR1KO mice. Synergistic addition of DL-AP3rescued thep-NR2B-Tyr1472by SKF81297in the cultures from FMR1KO mice. Thesedata implied that the increase of p-NR2B-Tyr1472contributed to the surfaceexpression of GluR1by SKF81297and DL-AP3in the KO cultures.
7Synergistic effect of simultaneous treatments on the behavior of FMR1KO mice
Fragile X patients and animal models exhibit behavioral phenotypes that areconsistent with deficits in PFC function, such as hyperactivity, anxiety, andlearning abnormalities. We analyzed locomotor activity, anxiety, and learningabilities to characterize hyperactivity and learning deficit, the commonphenotypes in fragile X syndrome. We found increased locomotor activity inFMR1KO mice, which was consistent with our previous study. The locomotoractivity was not affected following SKF81297or/and DL-AP3subcutaneousinjection after forty-five minutes in WT mice. However, application of the D1receptor agonist SKF81297or simultaneous treatment of SKF81297andDL-AP3significantly reduced the distance traveled by FMR1KO mice. Thesedata indicate that FMR1KO mice exhibit hyperactivity and that simultaneoustreatment of D1agonist and Grp1mGluRs antagonist can reduce thehyperactivity phenotype.
We then investigated anxiety-like behaviors with the elevated plus-maze test.We found no differences between FMR1KO and WT mice and no effect ofSKF81297alone or plus DL-AP3. These data indicate that anxiety-like behaviors are not detectable in this fragile X mouse model and suggest that thesebehaviors are probably not related to the D1receptor signaling pathway. Thesefindings are consistent with our previous study.
Last, the learning ability was detected in the Morris Water Maze test. Micewere treated with SKF81297alone or plus DL-AP3for5weeks. Swim latencywas compared over the4days of the experiment, and there were significantdifferences in escape latency between genotype at day1. SKF81297or DL-AP3treatment alone did not change the swim latency in the FMR1WT and KO mice.Simultaneous treatment of SKF81297or DL-AP3also did not improve thelearning ability in the FMR1WT mice. However, Simultaneous treatment ofSKF81297and DL-AP3reduced the swim latency on the final day in the FMR1KO mice. Moreover, we compared the swimming speed during the water mazetest and found no differences among the groups. These findings clearly showthat simultaneous treatment of SKF81297and DL-AP3significantly improvesthe spatial leaning of FMR1KO mice.In summary, the present study, together with our previous study, reveals thatmGluR1inhibition is a useful strategy to recover D1receptor signaling in theFMR1KO mice, and that combination of Grp1mGluR antagonist and D1agonist is a potential drug therapy for the FXS.
8Abnormal serotoninergic synaptic plastisty of FMR1KO mice ACC
In order to check if other neurotransmiting related system is normal, wechecked5-HT2A system, another important systern for synaptic plastisty. Wefound that bath application of R-96544or ketanserin significantly enhanced theamplitude of LTP. This suggests that inhibition of5-HT2A receptor canfacilitate LTP induction. However, this facilitation of LTP in WT mice byR-96544or ketanserin was lost in FMR1KO mice, demonstrating thatserotoninergic modulation of LTP is impaired in FMR1KO mice and thatFMRP is required for serotoninergic modulation of LTP in the ACC.
We next applied a selective NMDAR antagonist, AP5(50μM), and found that LTP facilitation by R-96544(5μM) was completely blocked in the WTmice.Similarly, LTP was abolished by10mM BAPTA in the pipette solution,indicating that serotoninergic modulation of LTP is dependent on the activationof NMDARs and elevated postsynaptic Ca2+concentrations.
To determine whether presynaptic and/or postsynaptic mechanisms areinvolved in serotoninergic modulation of LTP, we measured paired-pulsefacilitation (PPF) in the ACC. We found that PPF induced at five differentintervals did not differ in WT and FMR1KO mice in the presence of R-96544inthe ACC. The data suggest that presynaptic mechanisms do not seem to beinvolved in the serotoninergic modulation of LTP.
To further explore the synaptic mechanisms behind the impairment ofserotoninergic modulation of LTP, we examined5-HT2A receptor andglutamate receptor subunits expression in the ACC from WT and FMR1KOmice.There were no differences in5-HT2A receptor expression between the WTand FMR1KO mice. Furthermore, the total GluR1, NMDA receptor NR2A, andNR2B subunits expression were no differences between the WT and FMR1KOmice under basal conditions. Thus, the impairment of serotoninergic modulationof synaptic potentiation is not due to a defect in5-HT2A receptor and glutamatereceptors expression. To investigate the role of5-HT2A receptor signalingpathway in the synaptic plasticity, we tested the effects of R-96544on AMPAreceptor GluR1subunits surface trafficking in cultured PFC neurons in thepresence of1μM serotonin. Surface expression of GluR1was increased aftertreatment with5-HT2AR antagonist R-96544. This finding demonstrates thatinhibition of5-HT2A receptor can facilitate the surface expression of AMPAGluR1receptors. However, the surface expression of GluR1triggered byR-96544was impaired in FMR1KO PFC neurons. These results provide direct evidence that FMRP is involved in serotonin receptor signaling and contributesto GluR1surface expression induced by5-HT2A receptor inactivation.
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