Adaptation of microplate-based respirometry for hippocampal slices and analysis of respiratory capacity.
Adaptation of microplate-based respirometry for hippocampal slices and analysis of respiratory capacity.
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DOI：
10.1002/jnr.22650
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发表时间：
2011-12
影响因子：
4.2
通讯作者：
Polster, Brian M.
中科院分区：
文献类型：
--
作者：
Schuh, Rosemary A.;Clerc, Pascaline;Hwang, Hyehyun;Mehrabian, Zara;Bittman, Kevin;Chen, Hegang;Polster, Brian M.
多种神经退行性疾病与线粒体生物能量学改变有关。尽管线粒体O2消耗经常在分离的线粒体、分离的突触神经末梢（突触体）或培养的细胞中测量，但缺乏成熟的脑回路是一个剩余的限制。在这里，我们描述了一种方法的发展，适应海马细胞外通量分析仪（XF 24）的微板为基础的测量海马切片O2消耗。作为该技术的第一个评估，我们比较了整个切片生物能量学与以前的测量与突触体或培养的神经元。我们发现，线粒体呼吸能力和O2消耗耦合到ATP的合成可以估计在培养或急性海马切片保存的神经结构。小鼠器官型海马切片氧化葡萄糖显示线粒体O2的消耗是良好的耦合，确定的ATP合酶抑制剂寡霉素的敏感性。然而，与之前在细胞或突触体中的测量结果相比，解偶联剂对呼吸的刺激是适度的（<基础呼吸的120%），尽管通过急性添加线粒体复合物I连接的底物丙酮酸盐略微增强（至基础呼吸的~150%）。这些发现表明，切片中呼吸能力的基础利用率较高，最大呼吸的葡萄糖衍生底物有限。与传统的基于氧电极的方法相比，基于微板的海马呼吸测定法的通量提高，有利于神经保护药物的筛选。当与细胞类型特异性药理学或遗传操作相结合时，从整个切片中有效测量O2消耗的能力应该会促进我们对线粒体在生理学和神经病理学中作用的理解。
Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O2 consumption is frequently measured in isolated mitochondria, isolated synaptic nerve terminals (synaptosomes), or cultured cells, the absence of mature brain circuitry is a remaining limitation. Here we describe the development of a method that adapts the Seahorse Extracellular Flux Analyzer (XF24) for the microplate-based measurement of hippocampal slice O2 consumption. As a first evaluation of the technique, we compared whole slice bioenergetics to previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O2 consumption coupled to ATP synthesis could be estimated in cultured or acute hippocampal slices with preserved neural architecture. Mouse organotypic hippocampal slices oxidizing glucose displayed mitochondrial O2 consumption that was well-coupled, as determined by the sensitivity to the ATP synthase inhibitor oligomycin. However stimulation of respiration by uncoupler was modest (<120% of basal respiration) compared to previous measurements in cells or synaptosomes, although enhanced slightly (to ~150% of basal respiration) by the acute addition of the mitochondrial complex I-linked substrate pyruvate. These findings suggest a high basal utilization of respiratory capacity in slices and a limitation of glucose-derived substrate for maximal respiration. The improved throughput of microplate-based hippocampal respirometry over traditional O2 electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to efficiently measure O2 consumption from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology.
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影响因子：
7.4
作者：
Gerencser, Akos A.;Neilson, Andy;Choi, Sung W.;Edman, Ursula;Yadava, Nagendra;Oh, Richard J.;Ferrick, David A.;Nicholls, David G.;Brand, Martin D.
通讯作者：
Brand, Martin D.
影响因子：
12.4
作者：
Jekabsons, M. B.;Nicholls, D. G.
通讯作者：
Nicholls, D. G.
DOI：
10.1016/s0034-5687(01)00306-1
发表时间：
2001-11-15
期刊：
RESPIRATION PHYSIOLOGY
影响因子：
--
作者：
Erecinska, M;Silver, IA
通讯作者：
Silver, IA
影响因子：
4.7
作者：
POLI, A;MIGANI, P;BARNABEI, O
通讯作者：
BARNABEI, O
影响因子：
2.5
作者：
Izumi Y;Zorumski CF
通讯作者：
Zorumski CF