【作者】 章小兵；

【导师】 徐天乐；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2008， 博士

【摘要】 γ-氨基丁酸A型受体(GABA_AR)和甘氨酸受体是中枢神经系统两大重要的抑制性受体,根据二者在突触内、外所介导的抑制性电导特性的不同,将其介导的抑制效应分为相位性抑制(phasic inhibition)和紧张性抑制(tonicinhibition)两大类。突触传递过程中,突触前瞬时释放的高浓度神经递质γ-氨基丁酸(GABA)或甘氨酸,作用并激活分布在突触后区域的GABA_AR或甘氨酸受体,导致通道开放而引起氯离子内流,进一步使突触后细胞产生抑制。由于突触释放不是持续存在的,因而突触后受体的激活也是间歇式的。抑制性氨基酸受体的这种间歇式激活所产生的对神经元的抑制称为相位性抑制。抑制性氨基酸受体除了介导由突触内受体产生的相位性抑制以外,近年来的研究越来越关注其介导的另外一种抑制形式——紧张性抑制,紧张性抑制是突触外高亲和力的抑制性氨基酸受体由细胞外本底就持续存在的内源性氨基酸类递质,如GABA、甘氨酸以及牛磺酸等激活而产生。紧张性抑制的特点是,它不像相位性抑制那样严格受突触传递的限制,其介导的抑制效应持续存在,因而,紧张性抑制在控制神经元兴奋性方面被认为比相位性抑制发挥了更为重要的作用。在中枢神经系统海马区域,已经有很多有关GABA_AR介导的紧张性抑制的报道。研究认为紧张性抑制不仅参与了海马学习记忆等功能的调节,还参与了像癫痫、经前期焦虑失调等神经系统疾病的发病过程。在海马的不同区域,其介导紧张性抑制的GABA_AR亚基也不尽相同,如齿状回颗粒细胞上的δ亚基、CA1和CA3锥体神经元上的α5亚基,甚至是仅由α和β亚基(不含任何第三类亚基)组成的功能性GABA_AR等都参与了海马神经元紧张性抑制的调节。现有的研究认为大脑海马组织内没有功能性甘氨酸能突触,然而却有大量的甘氨酸受体亚基表达,并且无论是在急性分离的海马神经元、体外培养的海马神经元,还是在海马脑片上的研究,都能记录到明显的甘氨酸受体激活的电流。此外,海马神经元细胞外有大量的能激活甘氨酸受体的内源性配体如牛磺酸和β-丙氨酸等存在,这也使甘氨酸受体的激活并发挥功能成为可能。近年来的研究也证明了甘氨酸受体在海马中扮演的功能性角色,它能抑制海马的突触传递,并影响海马的短期可塑性。目前认为,甘氨酸受体的这种功能是通过细胞外持续存在的内源性氨基酸配体(甚至一些未知的调节剂)激活突触外甘氨酸受体,产生紧张性抑制而发挥的。虽然紧张性抑制的功能越来越被人们所重视,然而,到目前为止,选择性调节GABA能紧张性抑制的药物并不多见。另外,对高位脑区甘氨酸受体介导的紧张性抑制的功能也在逐渐被揭露。由于紧张性抑制在控制神经元兴奋性方面的重要作用,使得选择性调节紧张性抑制的药物的开发具有很好的前景。因此在本课题中,一方面,我们从天然化合物入手,筛选并研究了以薄荷醇(menthol)为代表的萜类化合物对海马抑制性氨基酸受体介导的紧张性抑制的调节作用。另一方面,通过异源表达甘氨酸受体的方法,发现并研究了环噻嗪(cyclothiazide,CTZ)对含有α2亚基的甘氨酸受体的选择性抑制作用及其分子机制。1.薄荷醇通过增强GABA能紧张性抑制下调中枢神经元兴奋运用电生理方法,我们发现薄荷醇可以降低培养海马神经元的兴奋性。同时行为学实验表明,薄荷醇可以抑制小鼠戊四氮(PTZ)注射以及大鼠电刺激海马点燃所产生的癫痫发作。进一步的机制研究显示,薄荷醇能增强低浓度GABA(与报道的在体动物脑内神经元细胞外空间存在的GABA浓度相接近)在培养的海马神经元上激活GABA_AR所产生的电流。同时相对较高浓度的薄荷醇还可以激活培养海马神经元上的GABA_AR。进一步在海马脑片CA1区锥体神经元上的研究显示,薄荷醇选择性抑制GABA_AR介导的紧张性电流,而对突触部位GABA_AR介导的相位性突触后电流无影响。这些结果因而表明薄荷醇可通过增强GABA_AR介导的紧张性抑制来下调海马神经元的兴奋性。为了进一步排除薄荷醇抑制海马神经元兴奋性是否有GABA_AR以外的机制的参与,我们利用电生理的手段,观察了薄荷醇对海马神经元离子型谷氨酸受体和神经元内在兴奋性的影响。实验结果表明薄荷醇不影响谷氨酸对离子型谷氨酸受体的激活,并对微小兴奋性突触后电流(mEPSCs)无影响;同时对在阻断兴奋性和抑制性氨基酸受体离子通道的情况下,薄荷醇对神经元电流注射所产生的动作电位的发放特性无影响。表明薄荷醇不影响神经元的内在膜兴奋性。此外,胞内游离钙离子螯合实验表明薄荷醇对GABA_AR的增强效应不受细胞内游离钙离子浓度的影响。这些结果进一步显示薄荷醇是通过选择性增强GABA_AR介导的紧张性抑制来发挥对海马神经元的抑制。紧接着对薄荷醇增强GABA_AR功能的结构效应关系的研究表明,薄荷醇的几种旋光异构体均具有明显的增强GABA_AR的作用。而薄荷醇的醇羟基取代物menthyl chloride和JE207对GABA_AR介导的电流无明显增强作用,因而表明醇羟基在薄荷醇增强GABA_AR方面起关键作用。2.薄荷醇对异聚体甘氨酸受体的相对高敏感性抑制通过全细胞电压钳技术,我们发现,薄荷醇浓度依赖性地抑制甘氨酸在海马神经元上激活的电流,并且这种抑制作用具有非竞争性和电压不依赖性的特性。同时,薄荷醇对甘氨酸受体的抑制不依赖于其产生的GABA_AR激活。胞内钙离子螯合实验表明这种抑制作用同时也是细胞内游离钙离子浓度不依赖性的。通过对不同给药模式下,薄荷醇抑制甘氨酸电流的特点进行分析表明,薄荷醇可能同时通过变构调控和开放通道阻断两种方式对甘氨酸受体产生抑制效应。进一步在异源表达甘氨酸受体的HEK293T细胞上的研究提示,薄荷醇对含有α2亚基的甘氨酸受体具有更高的敏感性,而甘氨酸异聚体中β亚基的加入,可以进一步增加薄荷醇的敏感性,导致薄荷醇对α2β异聚体甘氨酸受体具有更强的抑制效应。3.环噻嗪对含有α2亚基的甘氨酸受体的特异性抑制在培养的海马神经元上,致痫剂环噻嗪强有力地抑制甘氨酸激活的电流。其抑制作用具有甘氨酸浓度依赖的特性,对饱和浓度甘氨酸激活的电流,环噻嗪不能产生抑制,因而表明环噻嗪对甘氨酸受体抑制的竞争性特性。在异源表达甘氨酸受体的HEK293T细胞上,我们发现环噻嗪只抑制含有α2亚基的甘氨酸受体所介导的电流,因此表明环噻嗪对甘氨酸受体抑制的α2亚基选择性。由于脊髓神经元甘氨酸受体在发育过程中有着从α2亚基向α1亚基转变的模式,因此,我们在不同培养天数的脊髓神经元上观察了环噻嗪对甘氨酸激活电流的抑制情况,结果表明环噻嗪对体外培养5-7天的脊髓背角神经元上甘氨酸激活的电流具有显著的抑制作用,而随着体外培养时间的延长,到了体外培养两周后,其抑制作用显著减弱。因此进一步证实了环噻嗪对甘氨酸受体抑制作用的α2亚基选择性。通过序列比对和点突变的方法,我们对环噻嗪抑制甘氨酸受体的作用位点进行了研究,结果证实α2亚基细胞外N末端第59位上的苏氨酸在环噻嗪抑制含有α2亚基的甘氨酸受体方面起非常重要的作用。综上所述,本研究有如下创新之处:1)首次研究并发现了日常生活中广泛应用的天然化合物薄荷醇可以通过调节GABA_AR和甘氨酸受体所介导的紧张性抑制而产生中枢效应。并发现一些其他萜类化合物也能调节GABA_AR介导的效应。2)通过药理学的方法进一步凸显了GABA_AR介导的紧张性抑制在中枢神经系统网络调控方面的重要性,并揭示了紧张性抑制可能作为抗癫痫药物治疗具有前景的靶点。3)发现了环噻嗪可选择性抑制含有α2亚基的甘氨酸受体的功能,并找到了其作用位点。环噻嗪对α2亚基的这种特异性抑制将有可能为研究海马等高级脑区甘氨酸受体的功能提供方便。更多还原

【Abstract】 GABA_A receptor（GABA_AR）and glycine receptor（GlyR）are two kinds of important inhibitory amino acid receptor in central nervous system（CNS）.According to the differential characteristic of inhibitory conductance mediated by synaptic and extrasynaptic receptors,the inhibition induced by inhibitory amino acid system is divided into two forms:phasic inhibition and tonic inhibition.During synaptic transmission,postsynaptic GABA_ARs or GlyRs are activated by high concentration of GABA or glycine released from presynaptic neurons and then exert inhibitory conductance on postsynaptic neuron.Because vesicle release of presynaptic neuron is not persistent,the inhibition induced by the activation of postsynaptic GABA_AR or GlyR is intermittent.Therefore,this kind of intermittent inhibition is named of phasic inhibition which is strictly controlled by synaptic transmission.But over the last decade,diffusional inhibitory transmission mediated by GABA_ARs or GlyRs located outside the synapses and activated by endogenous ligand（such as GABA,glycine and taurine）present in the extracellular space has triggered a great deal of interest.This form of inhibition is named of tonic inhibition which is characterized by uninterrupted inhibitory conductance and is not strictly restricted by synaptic transmission.Tonic inhibition thus plays a more important role in controlling neuronal excitability than phasic inhibition.In hippocampus,a lot of studies about GABA_AR mediated tonic inhibition were reported.Tonic inhibition is involved in not only the regulation of learning and memory of hippocampus,but also the nosogenesis of diseases such as epilepsy and premenstrual dysphoric disorder.Different GABA_AR subunit,such asδsubunit in dentate gyrus granule cells andα5 subunit in CA1 and CA3 pyramidal cell, contributes to the tonic conductance in different area of hippocampus.And even, receptors devoid of a third type of subunit,i.e.,only containingαandβsubunits that are highly sensitive to Zn²⁺,have been shown to contribute to the tonic current recorded in hippocampal neurons. Although no functional glycinergic synapse was observed in hippocampus,the wide expression of GlyR subunits was revealed by previous studies.Glycine activated current was recorded not only in acutely isolated and cultured hippocampal neurons but also in neurons of hippocampal slices.In addition,endogenous ligand of GlyRs,such as taurine andβ-alanine were highly spread in extracellular space of hippocampus,supporting the possibility of GlyR activation.Recently,some studies have revealed the functional role of GlyR in synaptic transmission and short-term plasticity of hippocampus.The present idea presumes that tonic activation of extrasynaptic GlyR by some endogenous amino acid ligands and modulators exerts the functional role of GlyR in hippocampus.Although tonic inhibition triggered a great deal of interest in last year,few selective modulator of tonic inhibition by far was found.In addition,the functional role of tonic inhibition mediated by GlyR was gradually revealed.Therefore, selective modulator of tonic inhibition will become promising drugs for treating disease such as epilepsy.In present study,we first investigated the modulatory effects of menthol,a representative terpenoid,on the currents mediated by GABA_AR and GlyR.Besides, through site-directed mutagenesis and exogenously expression of GlyR,we investigatedα2 subunit specificity of cyclothiazide（CTZ）inhibition on GlyR.1.Menthol suppresses central neuronal excitation by enhancing tonic GABA inhibitionUsing electrophysiological recording and behavior study of epileptic animals,we found menthol suppressed the excitability of cultured rat hippocampal neurons and the hyper-excitability of CNS neurons induced by pentylenetetrazole（PTZ）injection and electrical kindling in vivo.Menthol enhanced the response of cultured hippocampal neurons to low concentrations of GABA and also activated GABA_AR with higher concentrations of menthol.Furthermore,in the CA1 region of rat hippocampal slices, menthol enhanced tonic GABAergic inhibition while leaving phasic（synaptic） GABAergic inhibition unaffected.These results thus suggest menthol inhibited neuronal excitability through enhancing GABA_AR mediated tonic inhibition in hippocampus.In order to rule out that menthol inhibited neuronal excitability through other molecular target,we firstly investigated the effect of menthol on the current activated by glutamate in hippocampal neurons.No significant modulation was observed in the experiments.Secondly,we test the effect of menthol on action potential induced by current injection after blocking excitatory and inhibitory amino acid receptor,and found that the firing characteristic and frequency were not affected by menthol.This result suggests that menthol does not alter the intrinsic neuronal excitability.Lastly,we examine the inhibitory effect of menthol on GABA_AR after chelating extracellular free calcium with BAPTA-AM and observed the similar inhibitory effect with that produced in the absence of BAPTA.Taken together,these results further support that menthol inhibited neuronal excitability through selective enhancement of GABA_AR mediated tonic inhibition.Finally,we investigated the effects of menthol isomers and structure-related agents（menthyl chloride,（-）-isopulegol,and JE207）on the current activated by GABA（1μM）in cultured hippocampal neurons.We found that all menthol isomers significantly enhanced GABA-activated current.No significant enhancement was observed by menthyl chloride and JE207 in which the hydroxyl of menthol is substituted by other group.Therefore,the hydroxyl of menthol plays an important role in menthol enhancement of GABA_AR.2.Menthol preferentially inhibitsα2βheteromeric glycine receptorsUsing whole-cell voltage-clamp recording,we firstly investigated the effect of menthol on GlyRs in cultured hippocampal neurons.Menthol noncompetitively inhibited glycine-activated currents(I_Gly)in a concentration-dependent and voltage-independent manner.The inhibitory effect was not influenced by blocking menthol activation of GABA_AR with bicuculline methiodide（BMI）or chelating intracellular free calcium with BAPTA.By analyzing the differential effect of menthol on I_Glywith different sequences of drug application,we found that open channel block and allosteric modulation were all involved in the inhibition, suggesting that menthol may inhibit GlyRs via at least two different sites. Furthermore,we assessed the subunit selectivity of menthol inhibition on recombinant GlyRs expressed in HEK293T cells.Menthol did not significantly affect I_Glymediated by recombinantα1 homomeric GlyRs.Although menthol showed a relatively high inhibitory potency for the recombinantα2 GlyRs,it displayed markedly higher potency atαβheteromeric receptors.In summary,these results indicated that menthol directly inhibited GlyRs via at least one allosteric site and another blocking site on the GlyRs.The inhibitory effect of menthol was not the result of cross-inhibition between GlyRs and GABA_ARs.Moreover,the change of intracellular free calcium concentration did not contribute to menthol inhibition of GlyRs.In addition,menthol preferentially inhibitedα2 subunit-containing GlyRs, and theβsubunit also significantly increased the efficacy of menthol inhibition.3.Alpha 2 subunit specificity of eyclothiazide inhibition on glyeine receptorsIn cultured rat hippocampal neurons,we found that CTZ,an epilepitogenic agent, potently inhibited I_Gly.The inhibition was glycine concentration-dependent, suggesting a competitive mechanism.Notably,when being heterologously expressed in HEK293T cells,GlyRs containing theα2 but not oil orα3 subunit were inhibited by CTZ,indicating subunit specificity of CTZ action.In addition,the degree of CTZ inhibition on I_Glyin rat spinal neurons declined with time in culture,in parallel with a decline ofα2 subunit expression,which is known to occur during spinal cord development.Furthermore,site-directed mutagenesis indicates that a single amino acid threonine at position 59 near the N-terminal of theα2 subunit confers the specificity of CTZ action.Thus CTZ is a potent and selective inhibitor ofα2-GlyRs, and threonine at position 59 plays a critical role in the susceptibility of GlyR to CTZ inhibition.In conclusion,there are several novelties residing in the present studies:1)For the first time,we find that menthol,a widely used natural chemical in daily life, inhibits neuronal excitability of the hippocampus through modulating tonic inhibition mediated by GABA_AR and GlyR.2)Our results underscore the importance of tonic inhibition by GABA_ARs in regulating neuronal activity and reveal that tonic inhibition may be the promising target of treating disease such as epilepsy.3)For the first time,we find that cyclothiazide preferentially inhibitsα2-GlyR which mainly produces tonic inhibition in hippocampus.In addition,the binding site of cyclothiazide on GlyR was also revealed by site-directed mutagenesis.The selective inhibition of cyclothiazide onα2 GlyR may be helpful to explore the functional role ofα2-GlyR in hippocampus.更多还原