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.

Fig. 1

Schematic diagram of hippocampal slices attached to nylon mesh inserts (“Capture Screens”) within XF Islet Capture Microplates. A retractable probe limits O₂ diffusion to the tissue when in the measurement position, enabling estimation of O₂ consumption rates based on the emission of an O₂-sensitive fluorophore. Mixing between measurements allows rapid re-oxygenation of medium surrounding adherent slices.

Fig. 2

Representative brightfield (A) and propidium iodide (PI)-stained fluorescent images (B-D) of organotypic mouse hippocampal slices cultured on nylon mesh inserts for 12-18 DIV. The same slice is depicted in A and B. Scale bars, 50 μm.

Fig. 3

Oligomycin and FCCP titrations for organotypic rat hippocampal slices. Absolute (A) and baseline-normalized (B) O₂ consumption rates (OCR) of 14 DIV organotypic rat hippocampal slices exposed to successive additions of respiratory modulators (arrows) are shown. Slices received three additions of oligomycin (1 μg/ml each, open circles), FCCP (1 μM each, filled gray circles), or control (75 μl of aCSF each, filled black circles) followed by a final addition of antimycin A (10 μM, open and filled gray circles) or control (75 μl of aCSF, filled black circles). Rates in B are normalized to the third baseline measurement point. Data are mean ± SD, n = 4 slices per trace.

Fig. 4

Oligomycin and FCCP titrations for acute rat hippocampal slices. Baseline-normalized O₂ consumption rates (OCR) of acute rat hippocampal slices exposed to successive additions of respiratory modulators (arrows) are shown without (A) and with (B) exclusion criteria (see text). Rates are normalized to the third baseline measurement point. Slices received three additions of oligomycin (1 μg/ml each, open circles), FCCP (1 μM each, open squares or 5 μM each, filled squares), or control (75 μl aCSF each, filled circles) followed by a final addition of antimycin A (10 μM, open circles and open and filled squares) or control (75 μl aCSF, filled circles). Data in A are mean ± SD, n = 3, 3, 2, and 2 slices per trace for filled circles, open circles, filled squares, and open squares, respectively. In B, one slice per trace was excluded with the exception of filled squares.

Fig. 5

Medium O₂ levels (mm Hg) during measurements of O₂ consumption by rat (A) and mouse (B) organotypic slices. In A, representative fast and slow respiring rat slices are depicted (open circles and filled circles, respectively, see text). Arrows represent vehicle additions (75 μl of assay medium equilibrated with air). In B, representative fast and slow respiring mouse slices are depicted (open circles and filled circles, respectively, see text). Arrows represent the successive additions of oligomycin (10 μg/ml), FCCP (5 μM), pyruvate (10 mM) and antimycin A (10 μM).

Fig. 6

The effect of BSA on FCCP-stimulated O₂ consumption rates (OCR). Baseline-normalized initial and peak OCR of organotypic mouse hippocampal slices exposed to FCCP (1 or 5 μM) in the absence (n=4) or presence (n=3) of BSA (4 mg/ml). Initial OCR (black bars) and peak OCR (white bars) were the first or the first or second measurement after FCCP addition, respectively. Data are expressed as mean ± SE and are normalized to the last OCR before FCCP addition. Two slices with baseline OCR below 50 pmol O₂/min were excluded from analysis.

Fig. 7

Estimation of coupling efficiency, maximal respiratory capacity, and the effect of exogenous pyruvate. Absolute (A) and baseline-normalized (B) O₂ consumption rates (OCR) of 17 DIV organotypic mouse hippocampal slices exposed to successive additions of respiratory modulators (arrows) are shown. Slices received successive additions of oligomycin (10 μg/ml), FCCP (5 μM), pyruvate (10 mM), and antimycin A (10 μM, open circles, n=4), or three successive control (con) injections (75 μl of aCSF each) followed by antimycin A (10 μM, filled circles, n=3). Rates in B are normalized to the third baseline measurement point. Data are mean ± SD.

Fig. 8

Assessment of inter-animal variability. Absolute (A) and baseline-normalized (B) O₂ consumption rates (OCR) of 14-15 DIV organotypic mouse hippocampal slices exposed to successive additions of respiratory modulators (arrows) are shown for three different wild type mice from the same litter (mean ± SD, n=4 slices for mouse 1 and for mouse 3 and n=2 slices for mouse 2). Slices received successive additions of control (75 μl of aCSF), oligomycin (10 μg/ml), FCCP (5 μM) + pyruvate (10 mM), and antimycin A (10 μM). Two slices from mouse 2 exhibited antimycin A-insensitive respiration exceeding 30% of the total OCR and were excluded from analysis. Rates in B are normalized to the third baseline measurement point.