Adaptation of microplate-based respirometry for hippocampal slices and analysis of respiratory capacity

Abstract

Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O 2 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 O 2 consumption. As a first evaluation of the technique, we compared whole-slice bioenergetics with previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O 2 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 O 2 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 with previous measurements in cells or synaptosomes, though enhanced slightly (to ~150% of basal respiration) by 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 O 2 electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to measure O 2 consumption efficiently from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology. © 2011 Wiley-Liss, Inc.