【作者】 徐光锦；

【导师】 王伟；

【作者基本信息】 华中科技大学 ， 神经病学， 2009， 博士

【摘要】 第一部分：大鼠海马脑片星形胶质细胞与NG2+胶质细胞电生理特性及形态学的比较研究星形胶质细胞和NG2+胶质细胞是哺乳动物中枢神经系统中主要的两类神经胶质细胞。NG2+胶质细胞过去一直被认为是少突胶质细胞的前体细胞。但随着其特异性识别抗体NG2的发现，该类细胞被划分为第四类神经胶质细胞。NG2+胶质细胞在成年脑内广泛分布，具有不同于星形胶质细胞的独特的电生理学特性。本研究以P14-P25大鼠海马CA1区放射层胶质细胞为研究对象，通过电生理学与免疫组织化学方法，系统地比较了星形胶质细胞与NG2+胶质细胞在形态学和电生理学特性上的差别，并且详细描述了一种由这两类胶质细胞构成的胞体紧密接触的空间排布关系，为以后研究这两种胶质细胞的世系来源及发育分化提供依据。第二部分：大鼠海马脑片星形胶质细胞胞体电压钳误差的直接测量全细胞电压钳技术近年来被广泛应用于脑片星形胶质细胞的电生理研究。并且，这些研究显示海马区成熟星形胶质细胞具有线性电流-电压关系和非常低的膜阻抗。这一过低的膜阻抗必然导致大的钳制误差的存在。量化这一电压钳误差，对于判断星形胶质细胞线性膜电导的“真伪”至关重要。我们首次利用双电极全细胞记录技术在单一星形胶质细胞胞体上直接测量到电压钳误差，平均误差为73．1％；全细胞电容补偿及80％串连电阻补偿后，误差仍高达45．7％。因此，我们通常所记录到的线性膜电导实质上是在未激活电压门控离子通道情况下的静息膜电导。此外，由于全细胞电压钳技术无法对海马脑片星形胶质细胞进行良好钳制，应该注意该技术在星形胶质细胞上应用的局限性。第三部分：大鼠海马脑片生理及缺血条件下星形胶质细胞间电偶联特性的研究哺乳动物脑内原浆型星形胶质细胞通过缝隙连接形成广泛偶联。然而，在生理及缺血情况下，在体星形胶质细胞间缝隙连接电偶联的生物物理特性并不清楚。首先，我们通过细胞内注入Biocytin的方法，分析研究大鼠海马CA1放射层星形胶质细胞合胞体的形态学，结果显示一个星形胶质细胞平均可与11个星形胶质细胞形成直接接触偶联。这11个偶联细胞与记录细胞的胞体间距平均为45μm。其次，我们通过双电极电压钳记录技术对细胞间的电偶联进行测量，结果显示直接接触偶联的两个星形胶质细胞间均可测量到双向的、电压非依赖性的跨缝隙连接电流。而在星形胶质细胞-NG2、星形胶质细胞-中间神经元、NG2-NG2以及中间神经元之间则检测不到这种电流。进一步研究显示，星形胶质细胞间的电偶联率在发育阶段P14组大鼠变异明显(0．5％-12．4％，平均为3．6％)；而在发育成熟的P21组大鼠，则明显趋于一致(0．18％-3．9％，平均为1．6％)。并且，只有在P21组大鼠，电偶联率随着细胞间距的增加而呈现指数下降规律。在脑片缺血研究中发现，短时程OGD不影响电偶联率；细胞外pH6．4的酸性条件对电偶联率起迟发的抑制作用；非常有趣的是，酸化的OGD(OGD+pH6．4)对电偶联率呈现明显加速的抑制作用。综上所述，在大鼠海马CA1放射层区域，相当低的电偶联率提示星形胶质细胞间的缝隙连接对K~+的空间缓冲作用非常微弱；脑缺血并发生酸中毒时，星形胶质细胞间缝隙连接通讯会受到严重抑制。更多还原

【Abstract】 Astrocytes and NG2 glia are two major glial types in the mammalian central nervoussystem (CNS). NG2+ glial cells, previously known as oligodendrocyte precursor cells(OPCs), are distinct by their proteoglycan NG2 expression and have now been classified asthe fourth members in the glial family. They have unique electrophysiological propertiesand extensively distribute in adult brain. Here we systematically studied the difference between astrocytes and NG2+glial cells in basal electrophysiological properties andmorphology. We also described a frequently observed space arrangement with somataphysically attached between astrocytes and NG+ gial cells in rat hippocampal CA1 regionwhich may indicate a lineage relationship between these two types of cells. Over the last twenty years, the somatic whole-cell voltage clamp technique has beenwidely used to investigate the electrophysiology of astrocyte in situ. Hippocampalastrocytes showing a linear current-voltage (I-V) relationship, or electrophysiologicallypassive K~+ membrane conductance and a very low membrane resistance have been reportedby employing this approach. However, the somatic votage clamp will poorly control the membrane potential due to a very low membrane resistance. To quantify this measurementerror is crucial to help us to understand the nature of passive conductance. Here, we directlyquantify the error in the voltage clamp measurement of astrocytes in situ using dual patchwhole-cell recordings from single astrocytes in hippocampal slices. The average voltageescape was 73.1%. However, the compensating for access resistance by 80% failed todecrease the deviation between the recorded membrane potential and the voltage commandsignificantly and the average voltage escape was still as high as 45.7%. Thus, wedemonstrate for the first time that the passive conductance is a resting conductance which isinduced over a narrow range of membrane potentials around the astrocyte restingmembrane potentials. Furthermore, this measurement error indicates that the limitations ofvoltage clamp study on astrocytes in situ should be paid more attention to. Mammalian protoplasmic astrocytes are coupled extensively through gap junctionchannels in vivo. However, the biophysical characterizations of gap junction channelsunder physiological and ischemic conditions are not fully understood. Starting with amorphometrical analysis of astrocytic syncytia in rat hippocampal CA1 stratum radiatumusing intracellular loading of biocytin, we show that on average each astrocyte directlycoupled to another 11 astrocytes 45μm apart. In dual voltage clamp recording,voltage-independent and bidirectional transjunctional currents were always measured fromthe directly coupled astrocytes, but not from astrocyte-NG2 gila or astrocyte-interneuronpairs. The electrical coupling ratio varied significantly among astrocytes in the developingpostnatal day 14 (P14) rats (0.5%-12.4%, mean=3.6%), but became more constant in themature P21 rats (0.18%-3.9%, mean=1.6%). Only in the mature rats, the coupling ratiodeclined exponentially with the increasing pair distance. Electrical coupling was notaffected by a short-term oxygen-glucose deprivation (OGD) treatment, but inhibited in adelayed fashion by the acidic extracellular pH of 6.4. Strikingly. acidic OGD (pH 6.4), acondition that better represents the cerebral ischemia in vivo, accelerated the inhibition ofelectrical coupling markedly. Altogether, a rather low effective electrical coupling ratiosuggests that astrocytic gap junctions conduct little K~+ spatial buffering currents underphysiological condition, and astrocyte gap junctional communication should be severelyaffected as the consequence ofa synergy effect of OGD and acidosis in ischemic brain.更多还原