电刺激听神经诱发小鼠脑干神经元活动的光信号特征(英文)

Optical mapping of brainstem neuronal activity evoked by auditory electro-stimulation in rats

背景:光学记录技术是以电压敏感染料为介质,以硅光电二极管转换技术为特点的新型电生理检测方法,有助于分析复杂的神经结构中膜电位变化的时间-空间分布。目的:使用光学记录的方法观察小鼠脑干听神经电刺激诱发冲动的时间-空间分布,并分析抑制性神经递质γ-氨基丁酸和γ-氨基丁酸A受体拮抗剂荷包牡丹碱对听觉诱发冲动的影响。设计:随机对照实验。单位:解放军总医院老年医学研究所,日本关西医科大学耳鼻喉科。材料:实验于2002-05/11在日本关西医科大学耳鼻喉科实验室完成。选取出生后0～5d的ddy/ddy小鼠100只,清洁级,雌雄不拘。方法:将全部动物麻醉后断头处死,在冷冻条件下制作有活性的脑干切片。脑片一侧与无损伤的听神经根残端连接。将脑片置于硅胶铺底的有机玻璃平皿中,用直径30μm钨丝固定。用钨丝电极刺激脑片一侧的听神经根残端,在耳蜗核、前庭核记录诱发反应。对照情况下使用正常的人工脑脊液作为灌流液;灌流液中加入50μmol/Lγ-氨基丁酸的人工脑脊液代替正常人工脑脊液孵育脑片20min后,记录γ-氨基丁酸对脑干听觉通路诱发信号的影响;灌流液中加入50μmol/Lγ-氨基丁酸和200μmol/L荷包牡丹碱的人工脑脊液代替含50μmol/Lγ-氨基丁酸的人工脑脊液孵育脑片20min后,记录荷包牡丹碱对脑干听觉通路诱发信号的影响。刺激条件为正相矩形波脉冲,电流强度5mA,持续时间5ms,频率0.1Hz,电刺激起始时间被设置为89.9ms。脑片染色采用电压敏感染料NK3041,使用16×16像素的硅光电二极管阵列设备记录刺激听神经所诱发的光学信号。主要观察指标:①主要结局;电刺激听神经诱发的脑干光学信号及其特点。②次要结局:γ-氨基丁酸和荷包牡丹碱光学反应记录结果。结果:实验纳入ddy/ddy小鼠100只,中途死亡56只,共44只进入结果分析。①电刺激听神经诱发的脑干光学信号及其特征:刺激听神经诱发的光学信号以时间-空间分布的形式被记录。在同侧耳蜗核,光学信号的潜伏期为(4.63±1.01)ms,前庭核的峰潜伏期为(6.00±0.89)ms。每一个光学信号分为两个成分:快的峰电位样反应及慢的长时程反应。快电位的起始相具有突触前性质,晚期相具有突触后性质;慢的长时程反应可能与多突触传递有关。②γ-氨基丁酸和荷包牡丹碱光学反应记录结果:灌流液中加入50μmol/Lγ-氨基丁酸可最大限度地降低听神经诱发的脑干神经元信号的幅度,快反应起始相的潜伏期没有延长,但幅度有所降低,晚期相以及慢反应的幅度被明显抑制;而灌流液中加入50μmol/Lγ-氨基丁酸、200μmol/L荷包牡丹碱后则可部分逆转γ-氨基丁酸对此信号的作用,快的峰电位样反应和慢反应的幅度有部分恢复。结论:多部位的光学记录系统可以收集电刺激听神经的诱发反应,光学信号显示了时间-空间分布的类型。γ-氨基丁酸能够使电刺激听神经诱发的脑干神经元信号的幅度明显降低,而γ-氨基丁酸A受体拮抗剂荷包牡丹碱可以竞争性地对抗部分而非全部的γ-氨基丁酸的抑制作用,提示γ-氨基丁酸能神经元对听神经诱发冲动的抑制作用除部分通过γ-氨基丁酸A受体实现外,还涉及其他亚型的γ-氨基丁酸受体。

BACKGROUND： Optical mapping technique is a novel electrophysiological detection method in which voltage-sensitivity dye is medium and silicon photoelectrical diode transforming technology is characteristic, used for analyzing the spatial-temporal distribution of membrane potential in complex neural system. OBJECTIVE： To observe the spatial-temporal changes of brainstem auditory electro-stimulation evoked potential by using optical mapping technol- ogy, so as to probe into the influence of γ--Aminobutyric acid （GABA） and γ-GABA receptor antagonist-bicuculline （BM1） on auditory evoked potential. DESIGN： Randomized controlled study. SETTING： Aging Medicine Research Institute of Military General Hospital and E.T.N Department of Japanese Kansai Medical University. MATERIALS＇This study was conducted at E.T.N Department of Japanese Kansai Medical University from May to November 2002. Totally 100 ddy/ ddy rats, with age of 0-5 days, clear grade, either gender were selected. METHODS： All rats were put to death after cryo-anaesthetized, andbrainstem was cut into slices under frozen state so as to remain activity. One side of brainstem slice was connected with the residual end of untraumatic auditory nerve, and slices were put on organic glass plate with the bottom covered with siliac gel and fixed by tungsten filament of 30 μm wide. The residual end of untraumatic auditory nerve was stimulated by tungsten electrode, meanwhile the evoke potentials were recorded at cochlea nuclei and vestibule nuclei. In control group slices were incubated in artificial CSF for 20 minutes, which added with 50 μmol/L γ-GABA in experimental group for observing the influence of γ-GABA on brainstem auditory evoked signals; or alternatively incubated with 50 μmol/L g-GA- BA and 200 μmol/L for 20 minutes for observing the influence of BMI on brainstem auditory evoked signals. Stimulation was positive rectangle-shape impulse with electric current of 5 mA and frequency of 0.1 Hz, lasting period of 5 ms, the onset time of electric stimulation was set at 89.9 ms. Brain stem slices were stained with electric-sensitivity dye of NK3041 and 16×16 pixel silicon photoelectrical diode device was used to record the auditory nerve stimulation evoked optical signals. MAIN OUTCOME MEASURES： ① Major outcomes： Character of auditory nerve stimulation evoked optical signals. ② Secondary outcomes： Influence of γ-GABA and BMI on optical signals. RFSULTS： Totally 100 ddy/ddy rats enrolled in this study and 56 died during experience; therefore 44 were remained in the results analysis. ① Character of brainstem auditory electrical-stimulation evoked optical signals： Spatial-temporal changes of auditory evoked optical signals were recorded. The latency of optical signals was （4.63±1.01） ms cochlea nuclei and （6.00±0.89） ms at vestibule fiuclei of the same side. Each optical signal can be divided into two parts： Quick peak-potential and slow long-term potential. The onset phage of quick potential displayed pre-synaptic property, while later phage displayed post-synaptic property; the slow long-term potential might be related to multiple synaptic transmissions. ② Optical reaction due to γ-GABA and BMI administration： 50 μmol/L γ-GABA was added into the perfusion fluid so as to reduce the amplitude of auditory evoked brainstem neuronal signals in maximum degree, the onset latency of quick potential did not prolonged, but amplitude slightly descended; while the latency and amplitude of later phage were obviously reduced. But such changes could be partly resumed due to the 200 μmol/L dicentrine administration. CONCLUSION： Auditory electro-stimulation evoked potentials can be detected by using multiple-site optical mapping system, which displaying spatial-temporal property. γ-GABA can obviously attenuate the amplitude of auditory electro-stimulation evoked brainstem signals, but such effect can be partly but not completely attenuated by γ-GABA A receptor antagonist-BMI, which implying that besides γ-GABA A receptor, such inhibition of γ-GABA energetic neurons on auditory, electro-stimulation evoked potentials can be achieved by other γ-GABA subtype receptors.