【作者】 张丽男；

【导师】 王永利；

【作者基本信息】 河北医科大学 ， 药理学， 2010， 博士

【摘要】 5-羟色胺(serotonin, 5-HT)是神经系统内一个非常重要的神经递质,广泛参与各种行为和生理过程,如:情感、睡眠、疼痛、食欲、学习和记忆等多种生理功能的调节。许多研究表明,与年龄有关的认知障碍(如阿尔茨海默病)均存在5-羟色胺能神经紊乱。其紊乱则可导致多种精神类疾病尤其是焦虑、抑郁和创伤后应激综合症等,而这些疾病都伴有学习和记忆的认知障碍。目前,5-羟色胺及其受体在阿尔茨海默病发病机制及治疗应用方面已成为人们关注的重点之一。另外有研究报道,Na+-K+泵除与高血压、糖尿病等多种疾病密切相关外,也参与学习与记忆的形成。有充分的实验证明,兴奋性神经递质谷氨酸激活了N-甲基-D-天门冬氨酸(N-methyl-D-aspartate, NMDA)受体的离子通道,产生长时程增强(long-term potentiation, LTP)对学习与记忆形成促进作用,以及其与NMDA受体之间的关系可能是自1970年以来神经科学的最重要的发现。5-羟色胺能神经系统和NMDA受体都参与正常和病理情况下认知与情感调节,且5-羟色胺和NMDA受体之间相互作用影响海马依赖性的长时程记忆。5-羟色胺能神经系统对NMDA受体的调节很复杂,其在无Mg2+存在的情况下可降低NMDA介导的去极化,而在有Mg2+存在的情况下则可增加NMDA介导的去极化。另外,5-羟色胺能抑制大鼠额前皮质锥体神经元NMDA的反应,但却增强运动神经元NMDA的反应。同时也有研究发现,5-HT和Na+-K+泵之间存在相互作用。其中,5-HT可激活大鼠皮层与海马胶质细胞的Na+-K+泵,也可抑制肾脏和水蛭T感觉神经元的Na+-K+泵活性。而抑制Na+-K+泵则可降低谷氨酸吸收,增加神经递质释放,进而导致谷氨酸累积,也可导致谷氨酸受体的高反应性。除此之外,抑制Na+-K+泵还可直接使NMDA受体变构而降低地佐环平(NMDA非竞争性拮抗药)的结合,增加皮层与海马NMDA受体亚基的表达、聚集和区域化。由此不难看出,Na+-K+泵、5-羟色胺和NMDA在调节学习和记忆功能方面存在着非常复杂的相互作用。本实验拟采用大鼠脑片采用全细胞膜片钳技术,以NMDA电流和Na+-K+泵电流为指标,观察5-HT和Na+-K+泵对海马CA1区神经元NMDA电流的影响和5-HT对大鼠海马CA1区神经元Na+-K+泵电流的影响,以探讨5-HT对学习与记忆功能的调节及其机制。第一部分5-羟色胺与钠钾泵对大鼠海马CA1区神经元NMDA电流的调节目的:本研究旨在观察5-羟色胺和Na+-K+泵对海马CA1区锥体神经元NMDA电流的影响,并进一步探讨相应机制。方法:(1)取出生2周SD大鼠快速断头取脑,置于预先氧饱和(95% O2 +5% CO2)的4℃人工脑脊液(artificial cerebrospinal fluid, ASCF)中,将其按冠状切面用氰丙烯酸盐胶粘在切片机的标本托上,用4℃ACSF浸埋(保持脑组织在低温状态下,以减小由于缺氧而引起的损伤),并持续通气(95% O2+5% CO2)。在4℃氧饱和条件下,用振动切片机切成300μm厚的切片,将切片移至孵育槽内,持续通以95% O2和5% CO2,30-32℃孵育1小时。(2)全细胞膜片钳方法记录NMDA电流:将孵育好的脑片放置于灌流槽中,持续灌流用95% O2和5% CO2饱和的ASCF,流速约为3 ml/min,使脑片浸没于液面下约3～4 mm。利用红外微分干涉相差显微镜,通过图像采集软件观察并选择合适的神经元使其成像于计算机监视器屏幕上。然后通过微电极操纵器引导电极到达记录部位,形成高阻抗(>1G?)封接,再继续施以负压吸破细胞膜,形成whole-cell记录模式,钳制电压设置为-60 mV。当细胞膜电流稳定之后,将灌流液体切换成含有NMDA(此为NMDA受体的特异性激动剂)的ACSF,可记录到膜电流的内向移动变化,此NMDA引起的膜电流内向移动的变化幅度即为NMDA电流(INM)。(3)双氢哇巴因(dihydroouabain, DHO)对INM影响的测定:实验采用自身对照的方法,即在同一细胞上先测定INM幅度作为对照,然后再测定不同浓度DHO干预时的INM幅度,并以对照电流值标化给药后的电流值求得均数。(4)5-HT对INM影响的测定:方法同(3)。结果:(1)若以对照INM作为100%,DHO (10-5, 10-4, 10-3 M)能剂量依赖性地增强INM,并使其分别增强至146.25±10.34%, 195.78±8.94%和154.75±14.18% (P<0.01或0.05),其中DHO 10-4 M增强INM作用最大。(2)在H89(PKA特异性抑制剂)存在的情况下,DHO增强INM的效应分别为202.28±23.28%和175.86±26%,无明显改变(P>0.05)。在十字孢碱(staurosporine, Stau, PKC特异性抑制剂)存在的情况下,DHO增强INM的效应分别为187.46±27.57%和178.32±34.3%,也无明显改变(P>0.05)。H89和Stau本身对INM无明显影响,H89为101.7±11.3%, Stau为108.92±10.77%, (P>0.05)。提示PKA和PKC信号通路不参与介导DHO增强INM的效应。(3)在PP2(Src特异性抑制剂)存在的情况下,DHO增强INM的效应由173±25.66%显著降低到106.8±5.92% (P<0.05)。在PD-98059(MAPK特异性抑制剂)存在的情况下,DHO增强INM的效应也部分被阻断,由210±28.11%降低到135.25±15.60% (P<0.05)。而PP2和PD-98059本身则对INM无明显影响,其中PP2为94.4±12.58%, PD-98059为120.25±12.09%,(P>0.05)。提示Src和MAPK信号转导通路介导DHO增强INM的效应。(4)5-羟色胺0.01,0.1和1 mM可浓度依赖性增强INM,使其分别增强至109.4±4.96% (P>0.05),146.4±13.48%和161.83±13.06% (P<0.05),但5-羟色胺0.01 mM对INM的影响无统计学意义。小结: DHO可通过Src和MAPK信号转导通路浓度依赖性增强大鼠海马CA1区锥体神经元NMDA电流,5-羟色胺也可浓度依赖性增强海马NMDA电流,提示Na+-K+泵和5-羟色胺都可能参与海马NMDA受体对学习记忆的调节。第二部分5-羟色胺对大鼠海马CA1区神经元Na+-K+泵电流的调节目的:研究5-羟色胺对大鼠海马CA1区神经元Na+-K+泵电流的影响,并进一步探讨可能参与这一调节过程的5-羟色胺受体途径。方法:(1)断头取海马脑片:同第一部分。(2)以全细胞膜片钳技术记录Na+-K+泵电流(Ip):将制备好的海马脑片放置于灌流槽中,持续灌流95% O2和5% CO2饱和的ASCF,使脑片浸没于液面下。为了消除钾离子、钙离子、钠离子对记录电流的干扰,整个实验过程中,钳制电压设定为-60 mV,在这一电压下,电压依赖性Na+和Ca2+通道都处于失活状态下。另外,在电极内液中加入了TEACL,以阻断K+电流;在细胞外液中加入了BaCl2、CdCl2、TTX,来分别阻断K+电流、Ca2+电流和Na+电流,从而排除了其它通道电流的影响。在红外微分干涉相差显微镜下,移动电极至细胞上方负压吸引形成封接,再以负压吸破细胞膜,测定该细胞的电容,然后至少稳定5 min使电极内液与细胞内液充分交换,并将细胞钳制在-60 mV,当膜电流稳定时,换为含0.5 mM毒毛旋花子甙(strophanthin, Str)的灌流液,以全细胞膜片钳方式记录海马神经元的Ip (EPC-10,Heka Instruments),并以电流/电容求得该细胞Ip的电流密度。(3)5-羟色胺对Ip的影响:破膜后至少稳定5 min使得电极内液和细胞内液充分交换,首先灌流不同浓度的5-羟色胺(0.01-1 mM)3-4 min,待5-羟色胺引起的膜电流移动不再变化时(表示5-羟色胺作用已经稳定),再用含有相应浓度5-羟色胺和0.5 mM毒毛旋花子甙的ASCF灌流细胞,测定5-羟色胺干预后的Ip,并以电流/电容求得该细胞Ip的电流密度。(4)影响Ip的5-羟色胺受体鉴别:在特异性5-HT1AR与5-HT3R拮抗剂(WAY100635和ondasetron)存在的情况下,分别重复上述(2)和(3)的实验程序,以鉴别可能参与这一调节作用的5-羟色胺受体亚型。结果:(1)对照组Ip的电流密度为0.64±0.04 pA/pF,5-羟色胺0.1和1 mM可显著抑制Ip,使其电流密度分别降低至0.39±0.05和0.35±0.04 pA/pF (P<0.01);(2)在有无WAY100635(5-HT1AR拮抗剂,10μM)存在的情况下,5-羟色胺介导的Ip密度分别为0.38±0.07 pA/pF和0.39±0.05 pA/pF,无明显改变(P>0.05);(3)5-HT3R拮抗剂ondasetron 10μM可使5-羟色胺介导的Ip密度由0.39±0.05 pA/pF显著升高至0.59±0.05 pA/pF (P<0.05)。而WAY100635和ondasetron本身对Ip均无明显影响(P>0.05)。提示5-羟色胺介导的Ip抑制可被ondasetron阻断,而不能被WAY100635阻断;(4)同样,1-(3-Chlorophenyl) biguanide hydrochloride (m-CPBG,5-HT3R激动剂, 0.1 mM)能模拟5-羟色胺(0.1 mM)对Ip的抑制,使其电流密度由0.64±0.04 pA/pF显著降低至0.41±0.05 pA/pF (P<0.01)。这些结果均表明5-羟色胺介导的Ip抑制可能由5-HT3R介导,而与5-HT1AR无关。小结:5-羟色胺可通过激活5-HT3R浓度依赖性抑制大鼠海马CA1区神经元的Na+-K+泵电流,提示Na+-K+泵可能参与5-羟色胺对海马NMDA受体的调节,从而可能参与对学习记忆的调节。结论1 DHO可通过Src和MAPK信号转导通路浓度依赖性增强大鼠海马CA1区锥体神经元NMDA电流,5-羟色胺也可浓度依赖性增强海马NMDA电流,提示Na+-K+泵和5-羟色胺都可能参与海马NMDA受体对学习记忆的调节。2 5-羟色胺可通过激活5-HT3R浓度依赖性抑制大鼠海马CA1区神经元的Na+-K+泵电流,提示Na+-K+泵可能参与5-羟色胺对海马NMDA受体的调节,从而可能参与对学习记忆的调节。更多还原

【Abstract】 5-HT, as a neurotransmitter or neuromodulator in the central nervous system, involves in multiple physiological processes and behaviors. The modulation disorder of the serotonergic system can result in depression, anxiety, posttraumatic stress disorder, et al., which are companied by cognitive disorder of learning and memory. The modulation of the serotonergic system affects LTP and long-term depression (LTD), the likely neurophysiologic derivates of learning and memory formation, which has been involved in the treatment of Alzheimer’s disease. The Na+-K+ pump (also known as sodium pump or Na+, K+-ATPase) is a member of P-ATPase family and present in all animal cells. Na+-K+ pump is a membrane protein responsible for the active transport of sodium and potassium ions in the nervous system necessary to maintain the ionic gradient for neuronal excitability. Changes in Na+-K+ pump activity have been associated with several abnormalities, including epilepsy, hypertension and diabetes. Additionally, Na+-K+ pump might play a relevant role in activity-dependent synaptic plasticity, such as LTP. Evidence also suggests that LTP in the hippocampus is one important phenomenon for memory processing. Na+-K+ pump inhibition impaired learning and memory in Morris water maze and step-through passive avoidance tasks, showing the main role of this enzyme on learning and memory. The sufficient studies have demonstrated that the molecular mechanism of learning and memory in hippocampus is that the activation of NMDA receptor promotes the generation of LTP, synaptogenesis, synaptic plasticity, and neurogenesis, improving the formation of learning and memory. LTP and its relationship with NMDA receptors may be the most important discovery in neuroscience since the early 1970s. Furthermore, 5-HT and NMDA receptor both involve in the regulation of cognition and emotion, and the interaction is complicated. In the absence of Mg2+, 5-HT reduces NMDA-mediated depolarization; whereas in the presence of Mg2+, 5-HT increases NMDA-mediated depolarization. 5-HT inhibits NMDA receptor- mediated response in pyramidal cells of the rat medial prefrontal cortex, while facilitates the effects of excitatory amino acids and enhances NMDA receptor-mediated response on several motoneurone pools. Additionally, 5-HT and NMDA receptors also appeared to interact, which was particularly critical for the expression or retrieval of hippocampal-dependent long-term spatial memory. The serotonin system and NMDA receptors are both critically involved in the regulation of cognition and emotion under normal and pathological conditions. The Na+-K+ pump inhibition leads to hyperstimulation of Glu receptors, Glu accumulation, due to the decrease of the glutamate uptake and the increase of neurotransmitter release, as well as allosterically decreases [3H] dizocilpine, a non-competitive antagonist of the NMDA receptor, binding to NMDA receptor, and increases the expression of NMDA receptor subunits, localization and clustering in cerebral cortex and hippocampus. The Na+-K+ pump inhibition modulates NMDA receptor not only under normal conditions but also in the short-term period that follows the ischemic status. Moreover, 5-HT activates glial Na+/K+ pump activity in rat cerebral cortex and hippocampus, and inhibits it in kidney and T sensory neurons of the leech. Therefore, the interactive modulation of cognitive learning and memory mediated by Na+-K+ pump, 5-HT and NMDA is complicated. Furthermore, more and more attention was paid to the serotonin system involved in the regulation of cognitive function. However, the mechanisms await to be further explored. In the present study, we used the whole-cell patch-clamp technique to address the following questions: (1) whether Na+-K+ pump and 5-HT can modulate NMDA current in hippocampal CA1 pyramidal neurons; (2) whether there lies a possible modulatory effect of 5-HT on Na+-K+ pump current in hippocampal CA1 pyramidal neurons.Part 1 The regulation of Na+/K+ pump and 5-HT on NMDA current in hippocampal CA1 pyramidal neurons Objective: To observe the modulation of NMDA response by Na+/K+ pump and 5-HT in hippocampal CA1 pyramidal neurons and explore the underlying mechanisms.Methods:Transverse hippocampal slices (300μm thick) were obtained by cutting with a vibroslice MA752 in ice-cold ACSF well-saturated with 95% O2 and 5% CO2, and transferred to oxygenated ACSF at 30-32℃for 1-h incubation. Individual slice was transferred to a perfusion chamber and continuously superfused with oxygenated ACSF at a rate of 3 ml min-1 at room temperature (22-25℃). NMDA current was induced by NMDA (30μM), which was applied consecutively 3 times for 1.5 min each, being separated by superfusion periods of 10-15 min with drug-free ACSF. In the present experimental conditions, 30μM NMDA can evoke reproducible inward currents, which were stable, with no evidence of receptor desensitization. The 3-4 min application of DHO (10-7-10-3 M) respectively, finally we applied DHO accompanied by NMDA (30μM) for 1.5 min. The amplitude of the current induced by the superfusion application of NMDA in control was standardized (100%).Results: (1) DHO (10-5, 10-4, 10-3 M) significantly potentiated INM, which were respectively increased to 146.25±10.34%, 195.78±8.94%, and 154.75±14.18% contrast to the control INM as 100% (P<0.01 and 0.05). (2) Pretreatment of hippocampal CA1 pyromidal neurons with H89, a specific inhibitor of PKA, did not antagonize DHO-induced potentiation of INM, which was changed from 202.28±23.28% to 175.86±26% (P>0.05). Pretreatment of hippocampal CA1 pyromidal neurons with Stau, a specific inhibitor of PKC, did not antagonize DHO-induced potentiation of INM, which was changed from 187.46±27.57% to 178.32±34.3% (P>0.05). Extracellular H89 and Stau alone did not significantly affect the control INM (H89 101.7±11.3%, Stau 108.92±10.77%, P>0.05). (3) In the presence of extracellular PP2, a specific inhibitor of Src, DHO-induded potentiation of INM was significantly reduced from 173±25.66% to 106.8±5.92% (P<0.05). Pretreatment of hippocampal CA1 neurons with PD-98059, a specific inhibitor of MAPK, also partly antagonized DHO-induced potentiation of INM from 210±28.11% to 135.25±15.60% (P<0.05). Extracellular PP2 and PD-98059 alone did not significantly affect the control INM (PP2 94.4±12.58%, PD-98059 120.25±12.09%, P>0.05). (4) 5-HT (0.01, 0.1, 1 mM)-mediated INM is 109.4±4.96% (P>0.05), 146.4±13.48% and 161.83±13.06% (P<0.05) contrast to 100% of the first NMDA response of the control. These data showed that 5-HT (0.1, 1 mM) significantly potentiated the INM.Conclusion: DHO potentiates hippocampal NMDA response primarily via tyrosine kinase Src and MAPK, while 5-HT also potentiates hippocampal NMDA response, which discloses novel mechanisms for the function of Na+/K+ pump and 5-HT in learning and memory.Part 2 The regulation of 5-HT on the Na+/K+ pump current in rat hippocampal CA1 neuronsObjective: 5-HT modulates learning and memory, and inhibition of Na+/K+ pump activity in rat hippocampal CA1 cells causes impairment of learning and memory and amnesia. The aim of this study was to investigate whether 5-HT can modulate hippocampal CA1 neurons Na+/K+ pump and then possibly involve in the modulation of learning and memory.Methods: Transverse 300-μm-thick hippocampal slices containing CA1 neurons were obtained by cutting with a vibroslice MA752 in ice-cold ACSF well-saturated with 95% O2 and 5% CO2 (PH 7.3-7.4), then they were pre-incubated in oxygenated ACSF at room temperature (30-32℃) for 1h. The hippocampal slice containing CA1 neurons was transferred to a submerged recording chamber and continuously superfused with oxygenated ACSF at a rate of 2 ml min-1 at room temperature (22-25℃). Whole-cell patch-clamp technique was performed to record the Na+-K+ pump current (Ip): the Na+-K+ pump exchanges 3 intracellular Na+ for 2 external K+ across the cell membrane during each active transport process. This excess positive charge movement generates a net outward current (Ip). With selected external and pipette solutions, membrane currents through K+ channel, Ca2+ channel, and Na+-Ca2+ exchanger were minimized. Under the experimental conditions, Ip was measured as the 0.5×10-3 M strophanthin (Str)-blocked current.Results: (1) The density of the control Ip was 0.64±0.04 pA/pF. 5-HT 0.1 and 1 mM could significantly inhibit Ip, which densities were 0.39±0.05 and 0.35±0.04 pA/pF respectively (P<0.01), demonstrating a dose-dependent inhibition of Ip in hippocampal CA1 neurons. (2) In the presence of WAY100635 (10μM), an antagonist of 5-HT1AR, 5-HT-induced inhibition of Ip did not change, which densities were 0.39±0.05 pA/pF and 0.38±0.07 pA/pF (P>0.05). (3) In the presence of ondasetron (10μM), an antagonist of 5-HT3R, 5-HT-induced inhibition of Ip was significantly recovered from 0.39±0.05 pA/pF to 0.59±0.05 pA/pF (P<0.05). These data showed that 5-HT-induced inhibition of Ip was antagonized by ondasetron not by WAY100635. (4) 1-(3-Chlorophenyl) biguanide hydrochloride (m-CPBG) (0.1 mM), a 5-HT3R specific agonist, mimicked the effect of 5-HT (0.1 mM) on Ip, which was decreased from 0.64±0.04 pA/pF to 0.41±0.05 pA/pF (P<0.01), suggesting that 5-HT-induced inhibition of Ip was mediated by 5-HT3R.Conclusion: 5-HT inhibits neuronal Na+/K+ pump activity via 5-HT3R in hippocampal CA1 neurons, which suggests that Na+/K+ pump may involve in 5-HT-mediated modulation of NMDA current, then modulating learning and memory performance.SUMMARY1 DHO potentiates hippocampal NMDA response primarily via tyrosine kinase Src and MAPK, while 5-HT also potentiates hippocampal NMDA response, which discloses novel mechanisms for the function of Na+/K+ pump and 5-HT in learning and memory.2 5-HT inhibits neuronal Na+/K+ pump activity via 5-HT3R in hippocampal CA1 neurons, which suggests that Na+/K+ pump may involve in 5-HT-mediated modulation of NMDA current, then modulating learning and memory performance.更多还原