MEASURING OXYGEN CONSUMPTION OF A SINGLE CELL USING A SELF-REFERENCING ELECTRODE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项研究试图确定与神经递质诱导的去极化相关的可兴奋细胞的代谢成本。用调制电化学技术测量了单电池的耗氧量。为这些研究选择的细胞是视网膜水平细胞，它在视网膜外部的视觉信号处理中发挥着重要作用。酶法分离的细胞具有大约-70 mV的静息电位，并对兴奋性神经递质谷氨酸及其类似物红藻氨酸做出反应而去极化。这些药物诱导的去极化模拟了在暗适应条件下正常情况下在完整视网膜观察到的水平细胞的紧张性去极化。我们的技术允许在静息时测量的代谢状态和紧张性去极化后的代谢状态之间进行直接比较。我们发现，水平电池的平均静息耗氧量为0.024？mo.cm-2.sec-1。应用氧化磷酸化的解偶联剂FCCP(10？M)使氧耗量增加到0.035？mol.cm2.sec-1；这一升高之后在10分钟后急剧下降到低于基础水平。100M海人藻酸盐使耗氧量从基础平均值0.03？mo.cm-2.sec-1增加到0.07？mol.cm-2.sec-1。此外，我们能够跟踪氧耗量在0和-70 mV电压钳电压下的变化。 这个数据是少数几个从单个可识别的细胞来衡量信息处理的代谢成本的例子之一。这一方法的发展对于加深我们对神经元新陈代谢、退行性疾病和衰老的理解有很大的希望。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This study seeks to establish the metabolic cost to an excitable cell associated with neurotransmitter-induced depolarization. Measurements of oxygen consumption from single cells were made using a modulation electrochemical technique. The cell chosen for these studies was the retinal horizontal cell, an important player in the processing of visual signals in the outer retina. Enzymatically isolated cells have a resting potential of approximately -70mV and depolarize in response to the excitatory neurotransmitter glutamate and its analogue, kainate. The depolarization induced by these agents mimics the tonic depolarization of horizontal cells normally observed in the intact retina under dark-adapted conditions. Our technique allows a direct comparison between the metabolic state measured at rest and following tonic depolarization. We found that the average resting O2 consumption for horizontal cells was 0.024 ¿mol.cm-2.sec-1. Application of FCCP (10¿M), an uncoupler of oxidative phosphorylation, elevated oxygen consumption to 0.035 ¿mol.cm2.sec-1; this elevation was followed by a sharp decline to below basal levels after 10min. 100¿M kainate elevated oxygen consumption from a basal average of 0.03 ¿mol.cm-2.sec-1 to 0.07¿mol.cm-2.sec-1. Further, we were able to follow changes in O2 consumption in response to voltage clamp at 0 and -70mV. This data is one of the few examples where the metabolic cost of information processing has been measured from a single identifiable cell. The development of this approach has great promise for furthering our understanding of neuronal metabolism, degenerative diseases and aging.