超极化激活环核苷酸门控（HCN）阳离子通道对大鼠海马突触传递功能及机制研究
摘要
目的
研究超极化激活环核苷酸门控( hyperpolarization-activated cyclic nucleotide-gated,HCN)阳离子通道对穿通纤维通路(perforant pathway,PP)——海马CA3区突触传递的影响及氨基酸释放的调节作用,并探讨二者之间的内在联系。
方法
应用细胞外电生理记录技术记录电刺激PP诱发的CA3区群锋电位(population spike,PS);高效液相色谱(high performance liquid chromatography, HPLC)荧光检测技术测定海马组织及培养海马神经元细胞外液中谷氨酸、天冬氨酸、甘氨酸及γ-氨基丁酸(γ-aminobutyric acid,GABA)含量;透射电子显微镜技术观察大鼠海马组织CA3区突触超微结构的改变;建立新生大鼠海马神经元原代培养技术,应用荧光钙成像系统观察单个海马神经元细胞内钙的变化。
结果
第一部分HCN通道对PP—海马CA3区突触通路正常低频突触传递及海马组织氨基酸释放的影响
HCN通道阻滞剂ZD7288及CsCl对低频刺激PP(0.5Hz)后90 min内海马CA3区PS幅值的的影响及海马组织氨基酸含量的变化。生理盐水对照组(Control组,施与低频刺激,微注射生理盐水1μL), ZD7288 (20,100,200 nmol)组(施与低频刺激,注药容积为1μL)及CsCl(1,5,10μmol)组(施与低频刺激,注药容积为1μL);各组大鼠于电生理记录完毕迅速处死、取材、冻存备测氨基酸含量。结果如下:
①特异性HCN通道阻滞剂ZD7288 20、100及200 nmol可剂量依赖降低CA3区PS幅值。90 min内PS幅值分别为基础值的69.72±1.79 %、45.23±1.5 %、28.07±4.65 %,较给药前平均下降约30.28 %, 54.77%和71.94 %(P < 0.01) ,并显著低于生理盐水对照组(P < 0.01),给药后5 min即起效,作用维持90 min以上。
②非特异性HCN通道阻滞剂CsCl 1和10μmol,亦可显著降低CA3区PS幅值。给药90 min内PS幅值分别为基础值的78.61±2.06 %、44.79±2.11%,较给药前平均下降了约21.38 %和55.2 %(P < 0.01)。微量注射5μmol CsCl,在开始30 min,PS幅值为基础值的58.4±1.37 %,下降显著(P < 0.01),此抑制效应随时间的延长而逐渐减弱,至90 min时,基本上与1μmol CsCl作用接近(P > 0.05)。
③HPLC测定氨基酸含量结果如下:正常组大鼠(未施与任何处理因素)谷氨酸、天冬氨酸、甘氨酸和GABA含量依次为:129.6±31.7, 117.3±22.6, 123.9±34.9和225.3±42.4μmol/g.protein;生理盐水对照组大鼠实验侧海马组织氨基酸含量依次为138.3±34.3 , 101.7±15.1, 113.3±40.5和227.4±41.9μmol/g.protein,与正常组比较无显著差异(P > 0.05)。ZD7288 100 nmol组上述四种氨基酸含量依次为:83. 5±15.6, 63.8±11.2, 73.1±28.2和124.7±23.6μmol/g.protein,均显著低于生理盐水对照组(all P < 0.01, except for glycine P < 0.05) ;CsCl 5μmol组大鼠则依次为75.9±10.3, 66.6±10.7, 80.4±15.2和157.4±26.2μmol/g.protein,亦显著低于对照组(all P < 0.01,except for glycine P < 0.05)。
第二部分HCN通道对PP—CA3区突触通路长时程增强(LTP)的影响
观察ZD7288及CsCl于强直刺激(tetanus stimulation, TS;刺激电压45 V,波宽0.15 ms,频率500 Hz, 4串,每串含50脉冲,串间隔2s)前及后给药对PP—CA3通路LTP PS幅值、起始潜伏期、峰潜伏期的变化,海马CA3区突触超微结构的变化及海马组织氨基酸含量变化。实验动物随机分为:对照组即control组(仅给予测试刺激不施加强直刺激)、LTP诱导组即TS处理组(给予强直刺激诱导LTP后持续观察90 min)、ZD7288—TS组(强直刺激前5 min于海马CA3区局部注入ZD7288 100 nmol)、CsCl—TS组(强直刺激前5 min于海马CA3区局部注入CsCl 5μmol)、TS—ZD7288组(强直刺激后30 min局部注入ZD7288 100 nmol)及TS—CsCl组(强直刺激后30 min局部注入CsCl 5μmol)。结果如下:
①强直刺激PP可诱导CA3区LTP形成,其后90 min内PS幅值持续升高并稳定在基础幅值的281.8±6.6 %水平,显著高于对照组(97.4±1.8 %)(P < 0.01);强直刺激前给予ZD7288、CsCl,LTP的诱导明显被抑制,90 min内PS相对幅值均数分别为89.5±9.4%、62.9±18.0 %,显著低于LTP组(P < 0.01)而与对照组无显著差异(P > 0.05);但CsCl在30 min后抑制LTP PS幅度增加作用呈减弱趋势。
②强直刺激PP诱导LTP形成30 min后,给予ZD7288、CsCl, LTP效应被翻转。5 min时PS幅值即明显下降,给药后1h内相对PS幅值均值分别为95.8±6.4 %、64.2±17.7 %均显著低于LTP组(P < 0.01),前者与对照组接近(P > 0.05)而后者显著低于对照组(P < 0.01);随时间延长,CsCl抑制作用逐渐减弱。
③LTP诱导组大鼠PS起始潜伏期基础值为5.154±0.350 ms,强直刺激后5 min PS起始潜伏期即显著缩短(4.368±0.254 ms,P < 0.01),90 min内起始潜伏期均值为4.386±0.029 ms,较强直刺激前平均缩短约0.768 ms;且各记录时间点PS起始潜伏期值均显著小于对照组(all P < 0.01)。强直刺激前分别给予ZD7288 100 nmol、CsCl 5μmol, PS起始潜伏期缩短幅度较LTP诱导组小,90 min内较强直刺激前基础值平均缩短约0.332 ms、0.267 ms,各时间点PS起始潜伏期值大于LTP诱导组(P < 0.05);而CsCl以20～60 min内作用最为显著。TS—ZD7288组大鼠施与强直刺激后,30 min内PS起始潜伏期较强直刺激前平均缩短约为0.725 ms(P < 0.01);30 min时给予ZD7288 100 nmol,强直刺激导致的PS起始潜伏期的缩短效应迅速被抑制,PS起始潜伏期延长达强直刺激前基础值水平而显著大于给药前值(P < 0.01),给药后5 min即有明显效应(P < 0.01),并可持续稳定60 min;60 min内各时间点PS起始潜伏期值均显著大于LTP诱导组(all P < 0.05)而与对照组接近(P > 0.05)。强直刺激后30 min给予CsCl 5μmol可产生与ZD7288类似的效应。
④LTP诱导组大鼠PS峰潜伏期基础值为7.833±0.585 ms,强直刺激后5 min PS起始潜伏期即显著缩短(6.767±0.578 ms,P < 0.01),90 min内峰潜伏期均值为6.832±0.107 ms,较强直刺激前平均缩短约1.001 ms;各记录时间点PS峰潜伏期值均显著低于对照组( P < 0.05)。强直刺激前给予ZD7288 100 nmol,90 min内PS峰潜伏期平均值较强直刺激前基础值缩短约0.579 ms,缩短幅度小于LTP诱导组(P < 0.05);强直刺激前给予CsCl 5μmol,30 min内PS峰潜伏期仍显著缩短,与LTP诱导组比无差异(P > 0.05),30 min后PS峰潜伏期逐渐延长与强直刺激前基础值水平接近甚至更长,与对照组比较无显著性差异(P > 0.05)。TS—ZD7288组大鼠施加强直刺激后,PS峰潜伏期较强直刺激前(7.291±1.602 ms)显著减小(P < 0.01),并持续稳定在6.415±0.006 ms,30 min内平均缩短约为0.876 ms;30 min时给予ZD7288 100 nmol,强直刺激所致的PS峰潜伏期缩短作用迅速被抑制,PS峰潜伏期延长达强直刺激前基础值水平而显著大于给药前值(P < 0.01),给药后5 min即产生明显效应(P < 0.01),该效应于给药后60 min内可持续稳定维持;强直刺激后30 min给予CsCl 5μmol可产生与ZD7288类似的效应。
⑤HPLC测定氨基酸含量结果如下:对照组(Control)、LTP诱导组、ZD7288—TS组、CsCl—TS组、TS—ZD7288组及TS—CsCl组大鼠海马组织中谷氨酸含量依次为:138.3±34.3、153.7±35.2、112.6±35.6、99.5±36.6、106.6±30.0及132.7±51.9μmol/g.protein,LTP诱导组谷氨酸水平较对照组呈增加趋势,但无统计学差异;ZD7288—TS组、TS—ZD7288组及CsCl—TS组谷氨酸含量显著低于LTP诱导组(P < 0.05);以上各组天冬氨酸含量依次为:96.7±21.0、95.2±31.2、87.3±27.0、70.0±23.0、69.4±15.6及89.2±29.0μmol/g.protein,各组之间天冬氨酸含量无显著差异(P > 0.05);以上各组甘氨酸含量依次为:113.4±40.5、60.1±17.4、105.6±44.5、62.7±19.8、91.8±39.6及88.1±31.7μmol/g.protein,LTP诱导组甘氨酸含量显著低于对照组(P < 0.01),而ZD7288—TS组甘氨酸含量较LTP诱导组增加(P < 0.05)而与对照组接近(P > 0.05);各组GABA含量依次为:206.4±65.4、119.3±30.6、189.6±48.1、176.3±40.0、171.7±10.9及200.6±26.7μmol/g.protein , LTP诱导组GABA含量显著低于对照组(P < 0.01), ZD7288—TS组、CsCl—TS组、TS—ZD7288组及TS—CsCl组GABA含量较LTP诱导组显著增加(P < 0.05 or P < 0.01)。
⑥对照组大鼠海马CA3区突触结构以突触连接面为平直型的突触较为多见,也可见少数凸面突触,突触前膜和突触后膜清晰,突触间隙清楚,突触前终末端聚集较多圆形的突触囊泡,突触前膜胞浆面附有少数锥形雾样致密物质即突触前致密突起(presynaptic dense projection),突触后膜胞浆面有一层浓密的电子致密物质附着即突触后致密物(postsynaptic membrance density, PSD)。LTP诱导组可见平直型突触数目明显增加,同一神经元轴突可与其他同一神经元或多个神经元形成多个突触联系即突触小球形成,突触前胞浆内可见突触囊泡聚集,显示囊泡的轴浆运输活跃;突触后致密物较对照组显著增厚,突触间歇变模糊,似有雾状物存在;凸面突触多见,突触前终末端可见大量圆形囊泡聚集成葡萄串状,突触间歇变模糊,突触后致密物亦明显增厚。与LTP诱导组比较,透射电镜视野下ZD7288—TS组大鼠海马CA3区突触数目减少,可发现较少的平直型突触及凸面突触,未发现突触小球,突触后致密物变薄,突触前囊泡数目减少,轴浆运输受抑制,突触间歇变清晰;CsCl—TS组突触结构呈现与ZD7288—TS组类似变化。
第三部分HCN通道对培养海马神经元氨基酸释放及谷氨酸引起的钙内流的影响
在细胞水平,研究HCN通道对体外培养海马神经元氨基酸释放以及谷氨酸所致钙内流的影响。结果如下:
①正常细胞对照组、cAMP 5μM、50μM组、ZD7288 5μM及50μM组细胞外液谷氨酸含量依次为99.2+17.7、136.1±25.7、188.1±47.7、31.4±7.1及4.4±1.0μmol/L,天冬氨酸含量依次为0.181±0.0517、0.190±0.0884、0.214±0.1243、0.203±0.0905及0.288±0.0747μmol/L,甘氨酸含量依次为13.01±4.32、265.1±37.1、397.8±50.7、3.20±1.56及1.65±0.94μmol/L,GABA含量依次为3.256±0.2925、3.061±0.322,3.227±0.422,0.852±0.297及0.297±0.052μmol/L,显示cAMP可显著增加海马神经元细胞外液中谷氨酸及甘氨酸含量并随剂量增大,作用增强(P < 0.01);对天冬氨酸及GABA含量则无显著影响。ZD7288可使谷氨酸及GABA水平下降并呈现量效关系(P < 0.01);同时降低细胞外液中甘氨酸水平(P < 0.01),对天冬氨酸含量无显著影响。
②海马神经元置于标准外液中,向细胞灌流槽内加入谷氨酸50μM时,数秒内可引起F340/380荧光比值显著增加,并迅速出现尖锐高耸的钙峰,此后较长时间维持在一稳态高钙水平,给药后钙增加率为78.01±10.32%;换为无钙外液时,比值则无明显影响。表明谷氨酸引起培养海马神经元[Ca2+]i上升过程中,细胞外Ca2+的大量内流起着主导作用。
③海马神经元置于标准外液中,荧光值稳定后,分别加入终浓度为25μM、50μM、100μM ZD7288,孵育20 min后荧光值无明显变化; ZD7288分别25μM、50μM、100μM孵育20 min后再加入谷氨酸50μM(终浓度);发现F340/380荧光比值增加较谷氨酸50μM处理组显著降低,钙峰出现缓慢而低平;对应的钙荧光增加率分别为59.22±8.72%,41.35±7.25%,21.01±4.41%,各组之间有显著差异(P < 0.01),表明ZD7288可剂量依赖性抑制谷氨酸诱导的细胞内钙增加。
④海马神经元置于标准外液中,荧光值稳定后,分别加入终浓度为5μM,50μM cAMP孵育5 min后再加入谷氨酸50μM(终浓度),F340/380荧光比值迅速增加且高于谷氨酸50μM处理组(P < 0.01),并在较长时间内维持在较高的水平;对应的钙荧光增加率分别为101.33±9.89,125.36±11.04%(P < 0.01);50μM ZD7288孵育20 min后,加入50μM cAMP, F340/380荧光比值增加幅度、钙峰出现的时间和形状与仅加入谷氨酸50μM时相近,钙荧光增加率为86.23±10.53%,与谷氨酸50μM处理组无显著差异(P > 0.05),表明HCN通道对谷氨酸诱导钙内流具有调节作用。
结论
1 HCN通道参与PP—海马CA3区通路正常低频突触传递过程,其机制可能与其对氨基酸类神经递质释放的调节有关。
2 HCN通道参与PP—海马CA3区突触通路LTP的诱导和形成,其机制可能为:
①促进突触前谷氨酸的释放而抑制GABA及甘氨酸的释放
②促进谷氨酸引起的突触后细胞内钙的增加
③调节突触前、后超微结构的改变。
OBJICTIVE
To study the contribution of HCN channel to synaptic transmission at perforant pathway (PP)---CA3 region pathway and its regulation on the release of amino acids, and the relationship between them.
METHODS
Evoked population spike (PS) was recorded in hippocampal CA3 region in vivo, and high-performance liquid chromatography (HPLC) with fluorescence detection was applied to measure the contents of glutamate (GLU), aspartate (ASP), glycine (GLY) and gamma-aminobutyric acid (GABA) in hippocampal tissue and in the cultured hippocampal neurons. Electron microscopy was applied to explore ultrastructural pathologic features of the CA3 region of hippocampus. Fluorescence calcium imaging technique was used to observe the change of intracellular calcium in cultured hippocampal neurons.
RESULTS
PARTⅠ
Effect of HCN channel on the normal low frequency synaptic transmission at PP --- CA3 region synaptic pathway and release of amino acids in rat hippocampus
To observe the effects of specific HCN cation channel blocker ZD7288 and non-specific blocker CsCl on the synaptic transmission in the patheway from perforant pathway fibers to CA3 region in hippocampus and the contents of amino acid in hippocampal tissue, the experimental rats were divided into different groups as follows: Saline group (receiving low frequency electrical stimulation); ZD7288 group (20,100,200nmol; receiving low frequency electrical stimulation and 1μL different concentration ZD7288); CsCl group(1,5,10μmol; receiving low frequency electrical stimulation and 1μL different concentration CsCl ). After the electrophysiological test, the rats in saline、ZD7288(100 nmol) and CsCl(5μmol) groups were immediately decollated and the experimental isolateral hippocampus were carefully dissociated from the rat’s cerebrain, washed with ice cold saline solution, and frozen in–75 oC, then concentrations of the amino acids were determined by HPLC with fluorescence detection. Results are shown as follows:
①Specific HCN channels blocker ZD7288(20,100,200 nmol) could decrease the amplitudes of population spikes (PS) in CA3 region in hippocampus evoked by stimulating PP fibers in a dose-dependently matter. The average relative PS amplitude was 69.72%, 45.23% and 28.1%, obviously lower than saline group (P < 0.01), decreased by 30.3%, 54.8% and 71.9% respectively (P < 0.01). This inhibition effect began at 5min and could sustain at least 90min.
②Nonspecific HCN channels blocker CsCl 1 and 10μmol could also significantly decrease the amplitudes of PS at hippocampal CA3 region . During the 90 min period after microinjetion of CsCl, the average relative PS amplitude fall to 69.72%、28.1%,decreased by 21.38% and 55.2% respectively than baseline (P < 0.01). Microinjection CsCl 5μmol into CA3 region, at first 30min, the PS amplitude was 58.4 %of baseline and strongly deceased (P < 0.01). The inhibitory effects attenuated with time extension, at the 90min point, were similar to the CsC l 1μmol(P > 0.05).
③After the low frequency stimulating PP and administration drug, the contents of glutamate, aspartate, glycine and GABA in hippocampal tissue were measured by HPLC. The concentrations of glutamate,aspartate,glycine and GABA in normal group(without low frequency electrical stimulation) were 129.6±31.7, 117.3±22.6, 123.9±34.9 and 225.3±42.4μmol/g.protein; which in saline group were 138.3±34.3,101.7±15.1,113.4±40.5 and 227.4±41.9μmol/g.protein respectively and showed no significant difference compared with normal group(P > 0.05). In the rats treated with ZD7288 100nmol, the contents of glutamate, aspartate, glycine and GABA in hippocampus were 83.5±15.6, 63.8±11.2, 73.1±28.2 and 124.7±23.6μmol/g.protein respectively, significantly decreased compared with control group (P < 0.01). In the rats treated with CsCl 5μmol, the glutamate, aspartate,glycine and GABA contents were 75.9±10.3,66.6±10.7, 80.4±15.2 and 157.4±26.2μmol/g.protein, markedly lower than control group (all P < 0.01,except for glycine P < 0.05).
PARTⅡContribution of HCN channel to long-term potentiation at PP ---CA3 region synaptic pathway
To study the contribution of HCN channel on the induction and sustain of LTP in PP-CA3 pathway, the effects of administration of ZD7288 and CsCl before/after the tetanic stimulation (Tetanic stimulation, TS; consisted of 50 trains at 0.5 Hz each composed of 4 pulses at 500Hz) on the PS amplitudes of LTP, the onset and peak latency were observed. The synaptic ultrastructure at hippacampal CA3 region and the concentration of amino acids in hippocampus tissues were also observed. The experimental animals were divided randomly into six groups:①control group, i.e saline group (receiving test stimulation, without TS );②LTP induction group (receiving TS and being observed for 90 minutes);③ZD7288 -TS group (local administration of 100nmol ZD7288 in CA3 region at the 5 min point before TS);④CsCl—TS group(local administration of 5μmol CsCl in CA3 region at the 5 min point before TS );⑤TS—ZD7288 group (local administration of 100nmol ZD7288 in CA3 region at the30 min point after TS);⑥TS—CsCl group(local administration of 5μmol CsCl in CA3 region at the 30 min point after TS). The results are as follows:
①LTP could been induced in CA3 region by stimulating PP fibers, the PS amplitudes sustain at 281.8±6.6% level of the baseline, which last stably at least 90 minutes and was higher than the control group (97.4±1.8%)(P < 0.01). Local administration of 100nmol ZD7288 and CsCl before TS could inhibit the induction of LTP in CA3 region, the PS amplitudes were 89.5±9.4%、62.9±18.0% of the baseline respectively, which were lower than the LTP group(P < 0.01)and has no significant difference compared with the control group(P > 0.05), but the inhibitory effect of CsCl attenuated after 30 min.
②Local administration of ZD7288 and CsCl at the 30min after the induction of LTP could reverse the LTP. The PS amplitudes decreased at the 5 min after the administration of ZD7288 and CsCl. The PS amplitudes sustain at 95.8±6.4%、64.2±17.7% level of the baseline in 1 hour after the administration, the former was similar to the level of the control group(P > 0.05) , the latter was lower than the control group(P < 0.01).With the extension of the time, the inhibition of CsCl was attenuated.
③The baseline of onset latency period was 5.154±0.350ms.5 minutes after the TS, the onset latency , began to decrease(4.368±0.254ms, P < 0.01), the average value was 4.386±0.029 ms in 90 min period, which attenuated averagely 0.768 ms. At the different time point, the onset latency was lower than the control group (all P < 0.01). Local administration of ZD7288 100 nmol and CsCl 5μmol before TS could decrease the onset latency, which attenuated 0.332ms、0.267ms.The onset latency at all time points were higher than LTP induction group(P < 0.05); The effects of CsCl was obvious in the period of 20～60 min. After TS, the onset latency of PS in the TS—ZD7288 group rats reduce 0.725ms(P < 0.01) in 30 minutes; Administration of ZD7288 100 nmol at the 30 min could attenuate the decreasing effects on onset latency, and the onset latency reached the baseline level before TS, which was higher than the value before administration. There were obvious effects at 5 min after administration, and sustain at least 60 minutes. The onset latency of PS at all time points were higher than which in LTP induction group (all P < 0.05) and were similar to the control group (P > 0.05). There were similar effects after administration of CsCl after TS at 30 min, and there was an increasing tendency after 30 minutes.
④The baseline of peak latency were 7.833±0.585ms in LTP induction group. Five minutes after the TS the peak latency begin to decrease (6.767±0.578ms,P < 0.01),and sustain stably at least 90 minutes. In 90 minutes, the average value of the peak latency period were 6.832±0.107 ms,which decreased 1.001 ms compared with the value before TS; The peak latency of different time points was obviously lower than control (all P < 0.05). Administration of 100 nmol ZD7288 before TS could decrease the peak latency, which reduced 0.579 ms compared with the baseline, the decrease extent was less than the LTP induction group (P < 0.05). Administration of 5μmol CsCl before TS could decrease the peak latency in 30 minutes, there were no significant difference compared with LTP induction group. After 30 minutes the peak latency of PS extended and was similar to or longer than the baseline before TS, which had no significant difference compared with control group (P > 0.05). After TS in TS—ZD7288 group, the peak latency of PS were lower than before TS(7.291±1.602 ms)(P < 0.01),which sustained at the level of 6.415±0.006 ms, reduced averagely 0.876 ms; Administration of 100 nmol ZD7288 at the 30 min after TS could reverse the effect of TS-induced peak latency decurtation, which regained to the level before TS and were longer than the level before administration (P < 0.01). Five minutes later, there were obvious effects (P < 0.01), which sustained stably 60 minutes; Administration of CsCl 5μmol after TS at the 30 min could exert similar effects to the ZD7288, and the effects could increase after 15 minutes.
⑤The content of glutamate in control group, LTP induction group,ZD7288-TS group, CsCl—TS group, TS—ZD7288 group and TS—CsCl group were 138.3±34.3,153.7±35.2,112.6±35.6, 99.5±36.6,106.6±30.0 and 132.7±51.9μmol/g.protein, the content of glutamate in LTP induction group was higher than control group, but has no significance difference; The content of aspartate were 96.7±21.0,95.2±31.2,87.3±27.0,70.0±23.0, 69.4±15.6 and 89.2±29.0μmol/g.protein in different group, there were no significance difference between them; The content of glycine were 113.4±40.5,60.1±17.4,105.6±44.5,62.7± 19.8,91.8±39.6 and 88.1±31.7μmol/g.protein in different groups, which in the LTP induction group was lower than the control group (P < 0.01) and in ZD7288—TS group was higher than LTP induction group( P < 0.05),but similar to the control group(P > 0.05); The content of GABA were 206.4±65.4,119.3±30.6,189.6±48.1,176.3±40.0,171.7±10.9 and 200.6±26.7μmol/g.protein, which in LTP induction group was lower than control group(P < 0.01),but in ZD7288—TS group,CsCl—TS group,TS—ZD7288 group and TS—CsCl group which were higher than LTP induction group (P < 0.05 or P < 0.01) .
⑥There were abundant flat synapse conjunction and a few convex synapse conjunctions in the control group. The presynaptic, postsynaptic membrane and synapse gap were clear. In the presynaptic end, there were abundant synaptic vesicles and a few presynaptic dense projection and postsynaptic membrance density (PSD). In the LTP induction group, flat synapse conjunction increased and there were many synaptic glomerulus. There were many synaptic vesicles in presynaptic endochylema, which indicated the active axoplasmic transport. PSD was thicker than that in control groups, and the synapse gap was dim; there were many convex synapses and many synaptic vesicles like thyrsiform in the presynaptic end. The synaptic end was dim and the PSD became thicker. The number of the synaptic vesicles in the ZD7288—TS group decreased compared with the LTP induction group. A few flat synapse conjunction and convex synapse conjunction could be observed, but there were no synaptic glomerulus. The PSD became thinner and the number of synaptic vesicles reduced, which indicated that the axoplasmic transport was inhibited. The synapse gap was clear. The synapse constitution in the CsCl—TS group was similar to that in ZD7288—TS group.
PARTⅢEffects of HCN channel on release of amino acids and calcium influx of hippocampal neuron by glutamate in vivo
To explore the effects of HCN channel on efflux of amino acid and glutamate induced calcium influx of hippocampal neuron in vivo. Cells were treated with HCN channel specific blocker ZD7228 and agonist cAMP; we measured the content of glutamate, aspatate, glycine and GABA in extracellular fluid, and observed the effects of ZD7228 and cAMP on calcium influx induced by glutamate. Results as follows:
①The contents of glutamate in extracellular fluid of control, cAMP 5μM, cAMP 50μM, ZD7288 5μM and ZD7288 50μM were 99.2±17.7, 136.1±25.7, 188.1±47.7, 31.4±7.1 and 4.4±1.0μmol/L individually, the contents of aspartate were 0.181±0.0517, 0.190±0.0884, 0.214±0.1243, 0.203±0.0905 and 0.288±0.0747μmol/L, the contents of glycine were 13.01±4.32, 265.1±37.1, 397.8±50.7, 3.20±1.56 and 1.65±0.94μmol/L, the contents of GABA were 3.256±0.2925, 3.061±0.322, 3.227±0.422, 0.852±0.297 and 0.297±0.052. These data revealed that cAMP could dose-dependently increase the content of glutamate and glycine (P<0.01) and had no significant effect on the content of aspartate and GABA. In addition, ZD7288 could dose-dependently decrease the content of glutamate and GABA (P<0.01), decreased the content of glycine simultaneously, but showed no significant effect on the content of aspartate.
②Adding glutamate (50μM) into hipocampal neurons exposed to standard extracellular fluid, we found that the F340/380 of hippocampal neuron were increased significantly, appeared a steep calcium peak, and maintained a high level of calcium during a long period. The increasing rate of calcium was 78.01±10.32%. While adding glutamate (50μM) into extracellular fluid without calcium, the F340/380 was unvaried. Thus, the influx of extracellar calcium by glutamate-induced played an important role in intracellar calcium increasing.
③There was no change of the F340/380 of hippocampus neuron pretreated with ZD7288 25μM, 50μM, 100μM for 20min. However, adding glutamate 50μM into standard extracellular fluid pretreated with ZD7288 25μM, 50μM, and 100μM for 20min respectively, we found the F340/380 markedly decreased compared with glutamate 50μM group and the calcium peak was flat. The corresponding increasing rates of calcium of different concentrations were 59.22±8.72, 41.35±7.25, 21.01±4.41% (compared with glutamate 50μM group, P < 0.01), which indicated that ZD7288 could dose-dependently inhibit the calcium influx induced by glutamate.
④Adding glutamate 50μM into standard extracellular fluid pretreated with cAMP 5μM and 50μM for 5min respectively, we found the F340/380 significantly increased compared with glutamate 50μM group (P<0.01), the calcium peak was steep, and maintained a high level of calcium during a long period. The corresponding increasing rates of calcium were 101.33±9.89, 125.36±11.04% (compared with glutamate 50μM group, P < 0.01), which indicated that cAMP could dose-dependently reinforce the glutamate-induced calcium influx. However, incubating cells with ZD7288 50μM for 20 min before adding cAMP 50μM, we found that the increasing argument of the F340/380 and the time and the shape of the calcium peak were nearly the same with glutamate 50μM group. The increasing rate of calcium was 86.23±10.53% (compared with glutamate 50μM group, P > 0.05), which indicated that HCN channel may participate in the regulation of calcium influx by glutamate.
CONCLUSIONS
1 HCN channel contributed to the process of lower frequency synaptic transmission in PP-CA3 pathway, the mechanism of which may be associated with facilitating the release of amino acids neurotransmitter.
2 HCN channel participated in the formation of LTP in PP-CA3 pathway, the mechanism of which was associated with the presynaptic composition and postsynaptic composition. The possible pathways are as follows:
①Facilitating the synaptic release of glutamate and inhibiting the release of GABA and glycine;
②Facilitating the release of intracellular calcium caused by glutamate;
③Altering the presynaptic composition and postsynaptic composition through the mechanism above and other possible mechanisms, and furthering to impact the physiological and biochemistry process in the induction and maintenance of LTP.
研究超极化激活环核苷酸门控( hyperpolarization-activated cyclic nucleotide-gated,HCN)阳离子通道对穿通纤维通路(perforant pathway,PP)——海马CA3区突触传递的影响及氨基酸释放的调节作用,并探讨二者之间的内在联系。
方法
应用细胞外电生理记录技术记录电刺激PP诱发的CA3区群锋电位(population spike,PS);高效液相色谱(high performance liquid chromatography, HPLC)荧光检测技术测定海马组织及培养海马神经元细胞外液中谷氨酸、天冬氨酸、甘氨酸及γ-氨基丁酸(γ-aminobutyric acid,GABA)含量;透射电子显微镜技术观察大鼠海马组织CA3区突触超微结构的改变;建立新生大鼠海马神经元原代培养技术,应用荧光钙成像系统观察单个海马神经元细胞内钙的变化。
结果
第一部分HCN通道对PP—海马CA3区突触通路正常低频突触传递及海马组织氨基酸释放的影响
HCN通道阻滞剂ZD7288及CsCl对低频刺激PP(0.5Hz)后90 min内海马CA3区PS幅值的的影响及海马组织氨基酸含量的变化。生理盐水对照组(Control组,施与低频刺激,微注射生理盐水1μL), ZD7288 (20,100,200 nmol)组(施与低频刺激,注药容积为1μL)及CsCl(1,5,10μmol)组(施与低频刺激,注药容积为1μL);各组大鼠于电生理记录完毕迅速处死、取材、冻存备测氨基酸含量。结果如下:
①特异性HCN通道阻滞剂ZD7288 20、100及200 nmol可剂量依赖降低CA3区PS幅值。90 min内PS幅值分别为基础值的69.72±1.79 %、45.23±1.5 %、28.07±4.65 %,较给药前平均下降约30.28 %, 54.77%和71.94 %(P < 0.01) ,并显著低于生理盐水对照组(P < 0.01),给药后5 min即起效,作用维持90 min以上。
②非特异性HCN通道阻滞剂CsCl 1和10μmol,亦可显著降低CA3区PS幅值。给药90 min内PS幅值分别为基础值的78.61±2.06 %、44.79±2.11%,较给药前平均下降了约21.38 %和55.2 %(P < 0.01)。微量注射5μmol CsCl,在开始30 min,PS幅值为基础值的58.4±1.37 %,下降显著(P < 0.01),此抑制效应随时间的延长而逐渐减弱,至90 min时,基本上与1μmol CsCl作用接近(P > 0.05)。
③HPLC测定氨基酸含量结果如下:正常组大鼠(未施与任何处理因素)谷氨酸、天冬氨酸、甘氨酸和GABA含量依次为:129.6±31.7, 117.3±22.6, 123.9±34.9和225.3±42.4μmol/g.protein;生理盐水对照组大鼠实验侧海马组织氨基酸含量依次为138.3±34.3 , 101.7±15.1, 113.3±40.5和227.4±41.9μmol/g.protein,与正常组比较无显著差异(P > 0.05)。ZD7288 100 nmol组上述四种氨基酸含量依次为:83. 5±15.6, 63.8±11.2, 73.1±28.2和124.7±23.6μmol/g.protein,均显著低于生理盐水对照组(all P < 0.01, except for glycine P < 0.05) ;CsCl 5μmol组大鼠则依次为75.9±10.3, 66.6±10.7, 80.4±15.2和157.4±26.2μmol/g.protein,亦显著低于对照组(all P < 0.01,except for glycine P < 0.05)。
第二部分HCN通道对PP—CA3区突触通路长时程增强(LTP)的影响
观察ZD7288及CsCl于强直刺激(tetanus stimulation, TS;刺激电压45 V,波宽0.15 ms,频率500 Hz, 4串,每串含50脉冲,串间隔2s)前及后给药对PP—CA3通路LTP PS幅值、起始潜伏期、峰潜伏期的变化,海马CA3区突触超微结构的变化及海马组织氨基酸含量变化。实验动物随机分为:对照组即control组(仅给予测试刺激不施加强直刺激)、LTP诱导组即TS处理组(给予强直刺激诱导LTP后持续观察90 min)、ZD7288—TS组(强直刺激前5 min于海马CA3区局部注入ZD7288 100 nmol)、CsCl—TS组(强直刺激前5 min于海马CA3区局部注入CsCl 5μmol)、TS—ZD7288组(强直刺激后30 min局部注入ZD7288 100 nmol)及TS—CsCl组(强直刺激后30 min局部注入CsCl 5μmol)。结果如下:
①强直刺激PP可诱导CA3区LTP形成,其后90 min内PS幅值持续升高并稳定在基础幅值的281.8±6.6 %水平,显著高于对照组(97.4±1.8 %)(P < 0.01);强直刺激前给予ZD7288、CsCl,LTP的诱导明显被抑制,90 min内PS相对幅值均数分别为89.5±9.4%、62.9±18.0 %,显著低于LTP组(P < 0.01)而与对照组无显著差异(P > 0.05);但CsCl在30 min后抑制LTP PS幅度增加作用呈减弱趋势。
②强直刺激PP诱导LTP形成30 min后,给予ZD7288、CsCl, LTP效应被翻转。5 min时PS幅值即明显下降,给药后1h内相对PS幅值均值分别为95.8±6.4 %、64.2±17.7 %均显著低于LTP组(P < 0.01),前者与对照组接近(P > 0.05)而后者显著低于对照组(P < 0.01);随时间延长,CsCl抑制作用逐渐减弱。
③LTP诱导组大鼠PS起始潜伏期基础值为5.154±0.350 ms,强直刺激后5 min PS起始潜伏期即显著缩短(4.368±0.254 ms,P < 0.01),90 min内起始潜伏期均值为4.386±0.029 ms,较强直刺激前平均缩短约0.768 ms;且各记录时间点PS起始潜伏期值均显著小于对照组(all P < 0.01)。强直刺激前分别给予ZD7288 100 nmol、CsCl 5μmol, PS起始潜伏期缩短幅度较LTP诱导组小,90 min内较强直刺激前基础值平均缩短约0.332 ms、0.267 ms,各时间点PS起始潜伏期值大于LTP诱导组(P < 0.05);而CsCl以20～60 min内作用最为显著。TS—ZD7288组大鼠施与强直刺激后,30 min内PS起始潜伏期较强直刺激前平均缩短约为0.725 ms(P < 0.01);30 min时给予ZD7288 100 nmol,强直刺激导致的PS起始潜伏期的缩短效应迅速被抑制,PS起始潜伏期延长达强直刺激前基础值水平而显著大于给药前值(P < 0.01),给药后5 min即有明显效应(P < 0.01),并可持续稳定60 min;60 min内各时间点PS起始潜伏期值均显著大于LTP诱导组(all P < 0.05)而与对照组接近(P > 0.05)。强直刺激后30 min给予CsCl 5μmol可产生与ZD7288类似的效应。
④LTP诱导组大鼠PS峰潜伏期基础值为7.833±0.585 ms,强直刺激后5 min PS起始潜伏期即显著缩短(6.767±0.578 ms,P < 0.01),90 min内峰潜伏期均值为6.832±0.107 ms,较强直刺激前平均缩短约1.001 ms;各记录时间点PS峰潜伏期值均显著低于对照组( P < 0.05)。强直刺激前给予ZD7288 100 nmol,90 min内PS峰潜伏期平均值较强直刺激前基础值缩短约0.579 ms,缩短幅度小于LTP诱导组(P < 0.05);强直刺激前给予CsCl 5μmol,30 min内PS峰潜伏期仍显著缩短,与LTP诱导组比无差异(P > 0.05),30 min后PS峰潜伏期逐渐延长与强直刺激前基础值水平接近甚至更长,与对照组比较无显著性差异(P > 0.05)。TS—ZD7288组大鼠施加强直刺激后,PS峰潜伏期较强直刺激前(7.291±1.602 ms)显著减小(P < 0.01),并持续稳定在6.415±0.006 ms,30 min内平均缩短约为0.876 ms;30 min时给予ZD7288 100 nmol,强直刺激所致的PS峰潜伏期缩短作用迅速被抑制,PS峰潜伏期延长达强直刺激前基础值水平而显著大于给药前值(P < 0.01),给药后5 min即产生明显效应(P < 0.01),该效应于给药后60 min内可持续稳定维持;强直刺激后30 min给予CsCl 5μmol可产生与ZD7288类似的效应。
⑤HPLC测定氨基酸含量结果如下:对照组(Control)、LTP诱导组、ZD7288—TS组、CsCl—TS组、TS—ZD7288组及TS—CsCl组大鼠海马组织中谷氨酸含量依次为:138.3±34.3、153.7±35.2、112.6±35.6、99.5±36.6、106.6±30.0及132.7±51.9μmol/g.protein,LTP诱导组谷氨酸水平较对照组呈增加趋势,但无统计学差异;ZD7288—TS组、TS—ZD7288组及CsCl—TS组谷氨酸含量显著低于LTP诱导组(P < 0.05);以上各组天冬氨酸含量依次为:96.7±21.0、95.2±31.2、87.3±27.0、70.0±23.0、69.4±15.6及89.2±29.0μmol/g.protein,各组之间天冬氨酸含量无显著差异(P > 0.05);以上各组甘氨酸含量依次为:113.4±40.5、60.1±17.4、105.6±44.5、62.7±19.8、91.8±39.6及88.1±31.7μmol/g.protein,LTP诱导组甘氨酸含量显著低于对照组(P < 0.01),而ZD7288—TS组甘氨酸含量较LTP诱导组增加(P < 0.05)而与对照组接近(P > 0.05);各组GABA含量依次为:206.4±65.4、119.3±30.6、189.6±48.1、176.3±40.0、171.7±10.9及200.6±26.7μmol/g.protein , LTP诱导组GABA含量显著低于对照组(P < 0.01), ZD7288—TS组、CsCl—TS组、TS—ZD7288组及TS—CsCl组GABA含量较LTP诱导组显著增加(P < 0.05 or P < 0.01)。
⑥对照组大鼠海马CA3区突触结构以突触连接面为平直型的突触较为多见,也可见少数凸面突触,突触前膜和突触后膜清晰,突触间隙清楚,突触前终末端聚集较多圆形的突触囊泡,突触前膜胞浆面附有少数锥形雾样致密物质即突触前致密突起(presynaptic dense projection),突触后膜胞浆面有一层浓密的电子致密物质附着即突触后致密物(postsynaptic membrance density, PSD)。LTP诱导组可见平直型突触数目明显增加,同一神经元轴突可与其他同一神经元或多个神经元形成多个突触联系即突触小球形成,突触前胞浆内可见突触囊泡聚集,显示囊泡的轴浆运输活跃;突触后致密物较对照组显著增厚,突触间歇变模糊,似有雾状物存在;凸面突触多见,突触前终末端可见大量圆形囊泡聚集成葡萄串状,突触间歇变模糊,突触后致密物亦明显增厚。与LTP诱导组比较,透射电镜视野下ZD7288—TS组大鼠海马CA3区突触数目减少,可发现较少的平直型突触及凸面突触,未发现突触小球,突触后致密物变薄,突触前囊泡数目减少,轴浆运输受抑制,突触间歇变清晰;CsCl—TS组突触结构呈现与ZD7288—TS组类似变化。
第三部分HCN通道对培养海马神经元氨基酸释放及谷氨酸引起的钙内流的影响
在细胞水平,研究HCN通道对体外培养海马神经元氨基酸释放以及谷氨酸所致钙内流的影响。结果如下:
①正常细胞对照组、cAMP 5μM、50μM组、ZD7288 5μM及50μM组细胞外液谷氨酸含量依次为99.2+17.7、136.1±25.7、188.1±47.7、31.4±7.1及4.4±1.0μmol/L,天冬氨酸含量依次为0.181±0.0517、0.190±0.0884、0.214±0.1243、0.203±0.0905及0.288±0.0747μmol/L,甘氨酸含量依次为13.01±4.32、265.1±37.1、397.8±50.7、3.20±1.56及1.65±0.94μmol/L,GABA含量依次为3.256±0.2925、3.061±0.322,3.227±0.422,0.852±0.297及0.297±0.052μmol/L,显示cAMP可显著增加海马神经元细胞外液中谷氨酸及甘氨酸含量并随剂量增大,作用增强(P < 0.01);对天冬氨酸及GABA含量则无显著影响。ZD7288可使谷氨酸及GABA水平下降并呈现量效关系(P < 0.01);同时降低细胞外液中甘氨酸水平(P < 0.01),对天冬氨酸含量无显著影响。
②海马神经元置于标准外液中,向细胞灌流槽内加入谷氨酸50μM时,数秒内可引起F340/380荧光比值显著增加,并迅速出现尖锐高耸的钙峰,此后较长时间维持在一稳态高钙水平,给药后钙增加率为78.01±10.32%;换为无钙外液时,比值则无明显影响。表明谷氨酸引起培养海马神经元[Ca2+]i上升过程中,细胞外Ca2+的大量内流起着主导作用。
③海马神经元置于标准外液中,荧光值稳定后,分别加入终浓度为25μM、50μM、100μM ZD7288,孵育20 min后荧光值无明显变化; ZD7288分别25μM、50μM、100μM孵育20 min后再加入谷氨酸50μM(终浓度);发现F340/380荧光比值增加较谷氨酸50μM处理组显著降低,钙峰出现缓慢而低平;对应的钙荧光增加率分别为59.22±8.72%,41.35±7.25%,21.01±4.41%,各组之间有显著差异(P < 0.01),表明ZD7288可剂量依赖性抑制谷氨酸诱导的细胞内钙增加。
④海马神经元置于标准外液中,荧光值稳定后,分别加入终浓度为5μM,50μM cAMP孵育5 min后再加入谷氨酸50μM(终浓度),F340/380荧光比值迅速增加且高于谷氨酸50μM处理组(P < 0.01),并在较长时间内维持在较高的水平;对应的钙荧光增加率分别为101.33±9.89,125.36±11.04%(P < 0.01);50μM ZD7288孵育20 min后,加入50μM cAMP, F340/380荧光比值增加幅度、钙峰出现的时间和形状与仅加入谷氨酸50μM时相近,钙荧光增加率为86.23±10.53%,与谷氨酸50μM处理组无显著差异(P > 0.05),表明HCN通道对谷氨酸诱导钙内流具有调节作用。
结论
1 HCN通道参与PP—海马CA3区通路正常低频突触传递过程,其机制可能与其对氨基酸类神经递质释放的调节有关。
2 HCN通道参与PP—海马CA3区突触通路LTP的诱导和形成,其机制可能为:
①促进突触前谷氨酸的释放而抑制GABA及甘氨酸的释放
②促进谷氨酸引起的突触后细胞内钙的增加
③调节突触前、后超微结构的改变。
OBJICTIVE
To study the contribution of HCN channel to synaptic transmission at perforant pathway (PP)---CA3 region pathway and its regulation on the release of amino acids, and the relationship between them.
METHODS
Evoked population spike (PS) was recorded in hippocampal CA3 region in vivo, and high-performance liquid chromatography (HPLC) with fluorescence detection was applied to measure the contents of glutamate (GLU), aspartate (ASP), glycine (GLY) and gamma-aminobutyric acid (GABA) in hippocampal tissue and in the cultured hippocampal neurons. Electron microscopy was applied to explore ultrastructural pathologic features of the CA3 region of hippocampus. Fluorescence calcium imaging technique was used to observe the change of intracellular calcium in cultured hippocampal neurons.
RESULTS
PARTⅠ
Effect of HCN channel on the normal low frequency synaptic transmission at PP --- CA3 region synaptic pathway and release of amino acids in rat hippocampus
To observe the effects of specific HCN cation channel blocker ZD7288 and non-specific blocker CsCl on the synaptic transmission in the patheway from perforant pathway fibers to CA3 region in hippocampus and the contents of amino acid in hippocampal tissue, the experimental rats were divided into different groups as follows: Saline group (receiving low frequency electrical stimulation); ZD7288 group (20,100,200nmol; receiving low frequency electrical stimulation and 1μL different concentration ZD7288); CsCl group(1,5,10μmol; receiving low frequency electrical stimulation and 1μL different concentration CsCl ). After the electrophysiological test, the rats in saline、ZD7288(100 nmol) and CsCl(5μmol) groups were immediately decollated and the experimental isolateral hippocampus were carefully dissociated from the rat’s cerebrain, washed with ice cold saline solution, and frozen in–75 oC, then concentrations of the amino acids were determined by HPLC with fluorescence detection. Results are shown as follows:
①Specific HCN channels blocker ZD7288(20,100,200 nmol) could decrease the amplitudes of population spikes (PS) in CA3 region in hippocampus evoked by stimulating PP fibers in a dose-dependently matter. The average relative PS amplitude was 69.72%, 45.23% and 28.1%, obviously lower than saline group (P < 0.01), decreased by 30.3%, 54.8% and 71.9% respectively (P < 0.01). This inhibition effect began at 5min and could sustain at least 90min.
②Nonspecific HCN channels blocker CsCl 1 and 10μmol could also significantly decrease the amplitudes of PS at hippocampal CA3 region . During the 90 min period after microinjetion of CsCl, the average relative PS amplitude fall to 69.72%、28.1%,decreased by 21.38% and 55.2% respectively than baseline (P < 0.01). Microinjection CsCl 5μmol into CA3 region, at first 30min, the PS amplitude was 58.4 %of baseline and strongly deceased (P < 0.01). The inhibitory effects attenuated with time extension, at the 90min point, were similar to the CsC l 1μmol(P > 0.05).
③After the low frequency stimulating PP and administration drug, the contents of glutamate, aspartate, glycine and GABA in hippocampal tissue were measured by HPLC. The concentrations of glutamate,aspartate,glycine and GABA in normal group(without low frequency electrical stimulation) were 129.6±31.7, 117.3±22.6, 123.9±34.9 and 225.3±42.4μmol/g.protein; which in saline group were 138.3±34.3,101.7±15.1,113.4±40.5 and 227.4±41.9μmol/g.protein respectively and showed no significant difference compared with normal group(P > 0.05). In the rats treated with ZD7288 100nmol, the contents of glutamate, aspartate, glycine and GABA in hippocampus were 83.5±15.6, 63.8±11.2, 73.1±28.2 and 124.7±23.6μmol/g.protein respectively, significantly decreased compared with control group (P < 0.01). In the rats treated with CsCl 5μmol, the glutamate, aspartate,glycine and GABA contents were 75.9±10.3,66.6±10.7, 80.4±15.2 and 157.4±26.2μmol/g.protein, markedly lower than control group (all P < 0.01,except for glycine P < 0.05).
PARTⅡContribution of HCN channel to long-term potentiation at PP ---CA3 region synaptic pathway
To study the contribution of HCN channel on the induction and sustain of LTP in PP-CA3 pathway, the effects of administration of ZD7288 and CsCl before/after the tetanic stimulation (Tetanic stimulation, TS; consisted of 50 trains at 0.5 Hz each composed of 4 pulses at 500Hz) on the PS amplitudes of LTP, the onset and peak latency were observed. The synaptic ultrastructure at hippacampal CA3 region and the concentration of amino acids in hippocampus tissues were also observed. The experimental animals were divided randomly into six groups:①control group, i.e saline group (receiving test stimulation, without TS );②LTP induction group (receiving TS and being observed for 90 minutes);③ZD7288 -TS group (local administration of 100nmol ZD7288 in CA3 region at the 5 min point before TS);④CsCl—TS group(local administration of 5μmol CsCl in CA3 region at the 5 min point before TS );⑤TS—ZD7288 group (local administration of 100nmol ZD7288 in CA3 region at the30 min point after TS);⑥TS—CsCl group(local administration of 5μmol CsCl in CA3 region at the 30 min point after TS). The results are as follows:
①LTP could been induced in CA3 region by stimulating PP fibers, the PS amplitudes sustain at 281.8±6.6% level of the baseline, which last stably at least 90 minutes and was higher than the control group (97.4±1.8%)(P < 0.01). Local administration of 100nmol ZD7288 and CsCl before TS could inhibit the induction of LTP in CA3 region, the PS amplitudes were 89.5±9.4%、62.9±18.0% of the baseline respectively, which were lower than the LTP group(P < 0.01)and has no significant difference compared with the control group(P > 0.05), but the inhibitory effect of CsCl attenuated after 30 min.
②Local administration of ZD7288 and CsCl at the 30min after the induction of LTP could reverse the LTP. The PS amplitudes decreased at the 5 min after the administration of ZD7288 and CsCl. The PS amplitudes sustain at 95.8±6.4%、64.2±17.7% level of the baseline in 1 hour after the administration, the former was similar to the level of the control group(P > 0.05) , the latter was lower than the control group(P < 0.01).With the extension of the time, the inhibition of CsCl was attenuated.
③The baseline of onset latency period was 5.154±0.350ms.5 minutes after the TS, the onset latency , began to decrease(4.368±0.254ms, P < 0.01), the average value was 4.386±0.029 ms in 90 min period, which attenuated averagely 0.768 ms. At the different time point, the onset latency was lower than the control group (all P < 0.01). Local administration of ZD7288 100 nmol and CsCl 5μmol before TS could decrease the onset latency, which attenuated 0.332ms、0.267ms.The onset latency at all time points were higher than LTP induction group(P < 0.05); The effects of CsCl was obvious in the period of 20～60 min. After TS, the onset latency of PS in the TS—ZD7288 group rats reduce 0.725ms(P < 0.01) in 30 minutes; Administration of ZD7288 100 nmol at the 30 min could attenuate the decreasing effects on onset latency, and the onset latency reached the baseline level before TS, which was higher than the value before administration. There were obvious effects at 5 min after administration, and sustain at least 60 minutes. The onset latency of PS at all time points were higher than which in LTP induction group (all P < 0.05) and were similar to the control group (P > 0.05). There were similar effects after administration of CsCl after TS at 30 min, and there was an increasing tendency after 30 minutes.
④The baseline of peak latency were 7.833±0.585ms in LTP induction group. Five minutes after the TS the peak latency begin to decrease (6.767±0.578ms,P < 0.01),and sustain stably at least 90 minutes. In 90 minutes, the average value of the peak latency period were 6.832±0.107 ms,which decreased 1.001 ms compared with the value before TS; The peak latency of different time points was obviously lower than control (all P < 0.05). Administration of 100 nmol ZD7288 before TS could decrease the peak latency, which reduced 0.579 ms compared with the baseline, the decrease extent was less than the LTP induction group (P < 0.05). Administration of 5μmol CsCl before TS could decrease the peak latency in 30 minutes, there were no significant difference compared with LTP induction group. After 30 minutes the peak latency of PS extended and was similar to or longer than the baseline before TS, which had no significant difference compared with control group (P > 0.05). After TS in TS—ZD7288 group, the peak latency of PS were lower than before TS(7.291±1.602 ms)(P < 0.01),which sustained at the level of 6.415±0.006 ms, reduced averagely 0.876 ms; Administration of 100 nmol ZD7288 at the 30 min after TS could reverse the effect of TS-induced peak latency decurtation, which regained to the level before TS and were longer than the level before administration (P < 0.01). Five minutes later, there were obvious effects (P < 0.01), which sustained stably 60 minutes; Administration of CsCl 5μmol after TS at the 30 min could exert similar effects to the ZD7288, and the effects could increase after 15 minutes.
⑤The content of glutamate in control group, LTP induction group,ZD7288-TS group, CsCl—TS group, TS—ZD7288 group and TS—CsCl group were 138.3±34.3,153.7±35.2,112.6±35.6, 99.5±36.6,106.6±30.0 and 132.7±51.9μmol/g.protein, the content of glutamate in LTP induction group was higher than control group, but has no significance difference; The content of aspartate were 96.7±21.0,95.2±31.2,87.3±27.0,70.0±23.0, 69.4±15.6 and 89.2±29.0μmol/g.protein in different group, there were no significance difference between them; The content of glycine were 113.4±40.5,60.1±17.4,105.6±44.5,62.7± 19.8,91.8±39.6 and 88.1±31.7μmol/g.protein in different groups, which in the LTP induction group was lower than the control group (P < 0.01) and in ZD7288—TS group was higher than LTP induction group( P < 0.05),but similar to the control group(P > 0.05); The content of GABA were 206.4±65.4,119.3±30.6,189.6±48.1,176.3±40.0,171.7±10.9 and 200.6±26.7μmol/g.protein, which in LTP induction group was lower than control group(P < 0.01),but in ZD7288—TS group,CsCl—TS group,TS—ZD7288 group and TS—CsCl group which were higher than LTP induction group (P < 0.05 or P < 0.01) .
⑥There were abundant flat synapse conjunction and a few convex synapse conjunctions in the control group. The presynaptic, postsynaptic membrane and synapse gap were clear. In the presynaptic end, there were abundant synaptic vesicles and a few presynaptic dense projection and postsynaptic membrance density (PSD). In the LTP induction group, flat synapse conjunction increased and there were many synaptic glomerulus. There were many synaptic vesicles in presynaptic endochylema, which indicated the active axoplasmic transport. PSD was thicker than that in control groups, and the synapse gap was dim; there were many convex synapses and many synaptic vesicles like thyrsiform in the presynaptic end. The synaptic end was dim and the PSD became thicker. The number of the synaptic vesicles in the ZD7288—TS group decreased compared with the LTP induction group. A few flat synapse conjunction and convex synapse conjunction could be observed, but there were no synaptic glomerulus. The PSD became thinner and the number of synaptic vesicles reduced, which indicated that the axoplasmic transport was inhibited. The synapse gap was clear. The synapse constitution in the CsCl—TS group was similar to that in ZD7288—TS group.
PARTⅢEffects of HCN channel on release of amino acids and calcium influx of hippocampal neuron by glutamate in vivo
To explore the effects of HCN channel on efflux of amino acid and glutamate induced calcium influx of hippocampal neuron in vivo. Cells were treated with HCN channel specific blocker ZD7228 and agonist cAMP; we measured the content of glutamate, aspatate, glycine and GABA in extracellular fluid, and observed the effects of ZD7228 and cAMP on calcium influx induced by glutamate. Results as follows:
①The contents of glutamate in extracellular fluid of control, cAMP 5μM, cAMP 50μM, ZD7288 5μM and ZD7288 50μM were 99.2±17.7, 136.1±25.7, 188.1±47.7, 31.4±7.1 and 4.4±1.0μmol/L individually, the contents of aspartate were 0.181±0.0517, 0.190±0.0884, 0.214±0.1243, 0.203±0.0905 and 0.288±0.0747μmol/L, the contents of glycine were 13.01±4.32, 265.1±37.1, 397.8±50.7, 3.20±1.56 and 1.65±0.94μmol/L, the contents of GABA were 3.256±0.2925, 3.061±0.322, 3.227±0.422, 0.852±0.297 and 0.297±0.052. These data revealed that cAMP could dose-dependently increase the content of glutamate and glycine (P<0.01) and had no significant effect on the content of aspartate and GABA. In addition, ZD7288 could dose-dependently decrease the content of glutamate and GABA (P<0.01), decreased the content of glycine simultaneously, but showed no significant effect on the content of aspartate.
②Adding glutamate (50μM) into hipocampal neurons exposed to standard extracellular fluid, we found that the F340/380 of hippocampal neuron were increased significantly, appeared a steep calcium peak, and maintained a high level of calcium during a long period. The increasing rate of calcium was 78.01±10.32%. While adding glutamate (50μM) into extracellular fluid without calcium, the F340/380 was unvaried. Thus, the influx of extracellar calcium by glutamate-induced played an important role in intracellar calcium increasing.
③There was no change of the F340/380 of hippocampus neuron pretreated with ZD7288 25μM, 50μM, 100μM for 20min. However, adding glutamate 50μM into standard extracellular fluid pretreated with ZD7288 25μM, 50μM, and 100μM for 20min respectively, we found the F340/380 markedly decreased compared with glutamate 50μM group and the calcium peak was flat. The corresponding increasing rates of calcium of different concentrations were 59.22±8.72, 41.35±7.25, 21.01±4.41% (compared with glutamate 50μM group, P < 0.01), which indicated that ZD7288 could dose-dependently inhibit the calcium influx induced by glutamate.
④Adding glutamate 50μM into standard extracellular fluid pretreated with cAMP 5μM and 50μM for 5min respectively, we found the F340/380 significantly increased compared with glutamate 50μM group (P<0.01), the calcium peak was steep, and maintained a high level of calcium during a long period. The corresponding increasing rates of calcium were 101.33±9.89, 125.36±11.04% (compared with glutamate 50μM group, P < 0.01), which indicated that cAMP could dose-dependently reinforce the glutamate-induced calcium influx. However, incubating cells with ZD7288 50μM for 20 min before adding cAMP 50μM, we found that the increasing argument of the F340/380 and the time and the shape of the calcium peak were nearly the same with glutamate 50μM group. The increasing rate of calcium was 86.23±10.53% (compared with glutamate 50μM group, P > 0.05), which indicated that HCN channel may participate in the regulation of calcium influx by glutamate.
CONCLUSIONS
1 HCN channel contributed to the process of lower frequency synaptic transmission in PP-CA3 pathway, the mechanism of which may be associated with facilitating the release of amino acids neurotransmitter.
2 HCN channel participated in the formation of LTP in PP-CA3 pathway, the mechanism of which was associated with the presynaptic composition and postsynaptic composition. The possible pathways are as follows:
①Facilitating the synaptic release of glutamate and inhibiting the release of GABA and glycine;
②Facilitating the release of intracellular calcium caused by glutamate;
③Altering the presynaptic composition and postsynaptic composition through the mechanism above and other possible mechanisms, and furthering to impact the physiological and biochemistry process in the induction and maintenance of LTP.
引文
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