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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 本研究旨在建立与神经递质诱导的去极化相关的可兴奋细胞的代谢成本。使用调制电化学技术测量单电池的耗氧量。 这些研究选择的细胞是视网膜水平细胞，它是外层视网膜中处理视觉信号的重要参与者。酶促分离的细胞具有约-70mV的静息电位，并且响应于兴奋性神经递质谷氨酸及其类似物红藻氨酸而去极化。由这些试剂诱导的去极化模拟在暗适应条件下正常在完整视网膜中观察到的水平细胞的强直性去极化。我们的技术可以直接比较在休息和紧张性去极化后测量的代谢状态。我们发现，水平细胞的平均静息耗氧量为0.024 <$mol.cm-2.sec-1。 应用FCCP（10 μ M），一种氧化磷酸化解偶联剂，使耗氧量升高至0.035 μ mol.cm2.sec-1; 10分钟后，耗氧量急剧下降至低于基础水平。100 <$M红藻氨酸盐使耗氧量从基础平均值0.03 <$mol. cm-2.sec-1升高至0.07 <$mol.cm-2.sec-1。此外，我们能够跟踪响应于0和-70 mV电压钳位的O2消耗变化。 这个数据是少数几个从单个可识别细胞中测量信息处理代谢成本的例子之一。这种方法的发展对于进一步了解神经元代谢、退行性疾病和衰老具有很大的希望。

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.