title: 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.
authors.affiliation: Research Service, Maryland VA Healthcare System, Baltimore, Maryland, USA.
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Schuh
authors.forename: Pascaline
authors.initials: P
authors.lastname: Clerc
authors.forename: Hyehyun
authors.initials: H
authors.lastname: Hwang
authors.forename: Zara
authors.initials: Z
authors.lastname: Mehrabian
authors.forename: Kevin
authors.initials: K
authors.lastname: Bittman
authors.forename: Hegang
authors.initials: H
authors.lastname: Chen
authors.forename: Brian M
authors.initials: BM
authors.lastname: Polster
mesh_terms: D000818:Animals; D019069:Cell Respiration / Q000502:physiology*; D004734:Energy Metabolism / Q000502:physiology; D006624:Hippocampus / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009924:Organ Culture Techniques / Q000379:methods*; D010100:Oxygen / Q000032:analysis*; D010101:Oxygen Consumption / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D010100:Oxygen