【作者】 王海涛；

【导师】 陈林；

【作者基本信息】 中国科学技术大学 ， 生物物理， 2008， 博士

【摘要】 阿斯匹林是临床上广泛运用的解热镇痛药物,由于其具有抗凝血作用,近年来小剂量服用阿斯匹林被用来预防和治疗心脑血管疾病。水杨酸是阿斯匹林的主要代谢产物,也是起药理作用的有效成分。由于大剂量的水杨酸钠可以在动物上可靠地诱导出耳鸣样行为,因此水杨酸钠经常被用于研究耳鸣产生的神经机制。最新的临床结果以及正电子成像实验(PET)表明水杨酸钠诱导的耳鸣往往起源于听觉中枢,即除了外周的作用靶点以外,水杨酸钠诱导的中枢神经系统功能改变亦有可能引发耳鸣。以往的在体电生理实验的确为我们了解水杨酸钠产生耳鸣的中枢神经机制提供了许多有益的资料,但是这些结果并不能说明中枢的这些变化究竟是水杨酸钠直接作用导致的,还是由于外周或低位中枢的改变所继发的。中枢神经系统兴奋性和抑制性的失平衡被认为是耳鸣产生的候选机制。因为水杨酸钠能够通过血脑屏障,并且在具有耳鸣行为学表现的模型动物脑脊液中达到较高的浓度,所以水杨酸钠有可能通过直接作用于中枢听觉系统而改变兴奋性和抑制性的平衡,从而导致耳鸣。听觉中枢GABA能抑制对听觉中枢神经元的反应特性有着重要的影响,而去抑制会导致神经元感受野变宽以及兴奋性升高。中枢GABA能神经元受到许多神经调质系统的重要调控,那么水杨酸钠是否通过影响这些调质系统从而影响到神经元的兴奋性水平?本博士论文运用全细胞膜片钳技术分别在离体的听皮层和下丘上检验了水杨酸钠对抑制性突触传递以及对五羟色胺(5-HT,serotonin)能神经调质系统的影响。主要实验结果如下:1.在急性分离的听觉皮层脑片上,通过刺激周边的抑制性神经元纤维,能够从Ⅱ/Ⅲ层锥体细胞上记录到诱发的抑制性突触后电流(eIPSCs),灌流1.4 mM的水杨酸钠后,神经元的输入阻抗没有改变,但是eIPSCs的幅值表现出明显的压抑,而且这种压抑具有浓度依赖性和可逆性。用1μM TTX阻断神经网络中自发的动作电位,以便记录到微小的抑制性突触后电流(mIPSCs),结果表明水杨酸钠显著地降低了mIPSCs的频率和幅度。这说明突触前和突触后的因素共同参与了水杨酸钠对IPSCs的压抑。该结果提示由于去抑制所导致的听皮层兴奋性升高可能是水杨酸钠诱发耳鸣的神经机制之一。2.因为5-HT能神经调质系统优先性支配中枢神经系统的GABA能神经元,所以水杨酸钠可能影响了5-HT对GABA能突触传递的调控,从而导致了兴奋性和抑制性的失平衡。在离体下丘脑片上,阻断谷氨酸能和甘氨酸能的突触传递后,可以得到药理学分离的GABA能自发的抑制性突触后电流(sIPSCs)。在灌流40μM 5-HT后,GABA能sIPSCs的频率和幅度得到了极为显著的增强。这种5-HT诱发的sIPSCs的动力学特性以及反转电位均无明显改变,说明5-HT诱发的sIPSCs仍然是GABA能的。1.4 mM的水杨酸钠能够显著压抑5-HT所诱发的sIPSCs增加,而0.5μM TTX可以模拟水杨酸钠的作用,这表明水杨酸钠通过压抑GABA能神经元自发的动作电位来压抑5-HT诱发的sIPSCs的增加。该结果表明水杨酸钠可能通过优先性地作用于GABA能神经元,从而在水杨酸钠诱导耳鸣的情况下破坏了由5-HT能系统所维系的正常的GABA能突触传递。以上研究说明水杨酸钠可以直接作用于中枢听觉神经系统,而GABA能神经元的功能改变可能是水杨酸钠引起耳鸣的原因。这将有助于揭示水杨酸钠诱发耳鸣的细胞生物学机制,并为耳鸣治疗提供新的理论依据。更多还原

【Abstract】 Salicytate is a major metabolic component of aspirin.Sodium salicylate(SS)is widely prescribed or sold over the counter as well as aspirin for anti-inflammation and for chronic pain relief with a side effect of tinnitus.Recently,SS is used for prevention and treatment of cardio cerebrovascular diseases for its anticoagulation effect.A large dose of SS could reliably induce the behavior manifestation of tinnitus in laboratory animals,so SS becomes a common drug for investigation into the neural mechanism of tinnitus.Previous studies have revealed that the cochlea is the pharmacological site of SS.The auditory sensitivity and selectivity can be affected by SS thourgh peripheral insults such as damaged electro-motility of outer hair cells. However,studies carried out with positron emission tomography(PET)reveal a possible central origin of tinnitus in patients.These indicate that changed activities in the central auditory system by SS might also contribute to the generation of tinnitus. Many in vivo electrophysiological experiments have indeed provided some insights into our understanding of the neural mechanisms of SS-induced tinnitus,however, these observed changes might inherit alterations occurred in either the cochlea or the central auditory neurons.The auditory system requires a delicate balance between inhibition and excitation for normal auditory perception.One theory holds that tinnitus might be generated if such a balance is altered by hearing loss or ototoxic drugs such as SS.The response properties of central auditory neurons are greatly shaped by GABAergic inhibition,and widen receptive filed and elevated excitability of auditory neurons may be caused by disinhibition.The GABAergic synaptic transmission is powerfully modulated by many neuromodulatory systems such as 5-HT (5-hydroxytryptamine),then whether raised excitability may partially result from likely altered serotonergic system by SS.In the present studies,whole cell recording techniques in the auditory cortex and inferior colliculus were applied to examine whether SS can influence the inhibitory synaptic transmission and whether serotonigeric system is involved in the SS-induced tinnitus.The major findings are as follows:1,Evoked inhibitory postsynaptic currents(eIPSCs)in response to lateral afferent stimulation and miniature postsynaptic inhibitory synaptic currents(mIPSCs)were recorded in layerⅡ/Ⅲpyramidal neurons of auditory cortex slice with whole cell recording technique.The author observed that SS itself didn’t cause a change in the neuronal input resistance,but it could reversibly reduce eIPSCs in a concentration dependent manner.In addition,SS at 1.4 mM significantly reduced the amplitude of mIPSCs and frequency of mIPSCs.These results demonstrate that SS depresses the inhibitory synaptic transmission of the auditory cortex.The depression could be due to both presynaptic and postsynaptic actions of SS given the reduced frequency and amplitude of mIPSCs.This study suggests that the raised excitability of the auditory cortex due to disinhibition is probably one of the mechanisms for tinnitus induced by SS.2,Since serotonin(5-hydroxytryptamine,5-HT)containing fibers preferentially innervate inhibitory GABA neurons,there exists a possibility that SS causes the imbalance between inhibition and excitation through influencing serotonergic modulation of the GABAergic synaptic transmission.The effects of SS on 5-HT-mediated GABAergic spontaneous inhibitory postsynaptic currents(sIPSCs) from neurons of the central nucleus of rat inferior colliculus were examined with whole-cell patch-clamp technique and brain slice preparation.Perfusion of 40μM 5-HT robustly enhanced both frequency and amplitude of GABAergic sIPSCs and this 5-HT-induced enhancement of GABAergic sIPSCs could be suppressed by 1.4 mM SS.Tetrodotoxin at 0.5μM produced a similar effect as SS did,suggesting that SS suppresses the 5-HT-induced enhancement of GABAergic sIPSCs through eliminating spontaneous action potentials of GABA neurons.These findings suggest that SS may preferentially target GABA neurons and consequently interrupt a normal level of GABAergic synaptic transmissions maintained by the serotonergic system in SS-induced tinnitus.In conclusion,these findings indicate that SS has a direct effect on the central auditory system.The impaired functions of GABA neruons are suggested to contribute to SS-induced tinnitus.The present studies may provide insights into understanding the central origin of tinnitus and help us to develop an effective therapeutic strategy for chronic tinnitus.更多还原