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 to 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 to previous measurements in cells or synaptosomes, although enhanced slightly (tõ 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 O 2 electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to efficiently measure O 2 consumption from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology.

Functional mitochondrial analysis in acute brain sections from adult rats reveals mitochondrial dysfunction in a rat model of migraine

“…Pyruvate was injected first as a supplemental substrate in addition to glucose already within the aCSF for mitochondrial respiration. As seen with previous groups, pyruvate was needed to prevent any substrate-limiting constraints of substrate supply upon injection of other mitochondriatargeting compounds (45). Without it, the FCCP response was not as robust, slower to peak, and would feature a decrease in the plateaued response following FCCP injection that was indicative of limited substrate supply, obscuring accurate quantification of the spare respiratory capacity (data not shown).…”

“…Use of this instrument to study intact tissue is emerging but has proven to be challenging (45,49). Although limited progress has been made with use of this technology in organotypic hippocampal sections from mice (45), until this study, it has not been successful in acutely derived sections in adult animals (45).…”

“…The technology has been validated in isolated mitochondria (43) and has been widely adopted for the study of mitochondrial function in numerous cell types and cellular compartments, including neurons and synaptosomes. Use of this instrument to study intact tissue is emerging but has proven to be challenging (45,49). Although limited progress has been made with use of this technology in organotypic hippocampal sections from mice (45), until this study, it has not been successful in acutely derived sections in adult animals (45).…”

Novel Mitochondrial Targets for Neuroprotection

“…There is evidence for mitochondrial metabolism of both ketone bodies, e.g., b-hydroxybutyrate, and acetyl-L-carnitine after acute brain injury, which is associated with a reduction in oxidative stress and neuroprotection (Rosenthal et al, 1992;Liu et al, 1993;Prins et al, 2005;Scafidi et al, 2010Scafidi et al, , 2011. While there is less evidence that neuroprotection by exogenous pyruvate is a consequence of its oxidative metabolism, support for this mechanism comes from recent measurements of brain slice O 2 consumption indicating that exogenous pyruvate significantly elevates endogenous respiration (Schuh et al, 2011). Other experiments indicate that each of these three neuroprotectants exhibit alternative mechanisms of neuroprotection, including direct scavenging of free radicals (Packer et al, 1991;Varma et al, 1998;Haces et al, 2008).…”

Mechanism of Mitochondrial Dysfunction during Chronic Fatigue

“…Several diseases and conditions are associated with dysfunction of the mitochondrion, such as Cancer, Alzheimer's disease, Parkinson's disease, schizophrenia, diabetes, chronic fatigue syndrome, nonalcoholic steatohepatitis etc. [31][32][33][34][35].…”