Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Summary

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.

Abstract

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca²⁺ concentration control. Mitochondrial dysfunction has been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson's disease (PD), Huntington's disease, and Alzheimer's disease. To study the role of mitochondria in models of these diseases, we can measure mitochondrial respiration via oxygen consumption rate (OCR) as a proxy for mitochondrial function. OCR has already been successfully measured in cell cultures, as well as isolated mitochondria. However, these techniques are less physiologically relevant than measuring OCR in acute brain slices. To overcome this limitation, the authors developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice. The technique is optimized with a focus on the striatum, a brain area involved in PD and Huntington's disease. The analyzer performs a live cell assay using a 24-well plate, which allows the simultaneous kinetic measurement of 24 samples. The method uses circular-punched pieces of striatal brain slices as samples. We demonstrate the effectiveness of this technique by identifying a lower basal OCR in striatal slices of a mouse model of PD. This method will be of broad interest to researchers working in the field of PD and Huntington's disease.

Introduction

Mitochondrial dysfunction has been implicated in several neurological diseases, including Parkinson's disease (PD), Huntington's disease, and Alzheimer's disease^1,2,3. PD models such as PINK1 knockout (KO) mice and rats display impaired mitochondrial function^{4,5,6,7,8,9,10,11}. Mitochondria isolated f....

Protocol

All the procedures including animal work were conducted according to national and international guidelines and were approved by the Animal Care and Use Committee of Thomas Jefferson University. Male FVB/NTac mice at the age of 3 to 14 months were used. The following steps were performed in a non-sterile setting, but caution should be taken to keep everything as clean as possible.

NOTE: The method presented here was established and used in the research reported by Zhi et al.²⁴. The experiments described here used a Seahorse XF24 extracellular flux analyzer (see Table of Materials). These methods can b....

Results

The first step of this study was to optimize the slice thickness and punch size used to excise a section of the striatum from the slice (Figure 3A). A slice at 150 µm thickness and a 1.5 mm punch size gave the best results determined by the coupling efficiency (Figure 3B-C). As shown in Figure 3B, OCR is relatively stable for 5 h with less than 10% run down. In addition, functional measurements were.......

Discussion

The method we developed allowed an XF analyzer to be used for measuring OCR in striatal slices from adult mice over a time span of 4 h. This method provides a new way to measure cellular bioenergetics in punches excised from anatomically defined brain structures. Since the tissue samples being analyzed are rather small, the metabolic parameters of specific brain areas involved in a disease can be investigated. In addition, using acute slices more closely mimics the physiological cellular environment, which cannot be achi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Accumet AB150 pH benchtop meter	Thermo Fisher Scientific	13-636-AB150	To measure pH
Antimycin A from streptomyces sp.	SIGMA	A8674	To inhibit complex III of the mitochondria
Bovine Serum Albumin (BSA)	SIGMA	A6003	To make modified artificial cerebrospinal fluid (BSA-ACSF)
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)	SIGMA	C2920	To uncouple mitochondrial respiration
D-Glucose	SIGMA	G8270	To make artificial cerebrospinal fluid (ACSF)
DMSO	SIGMA	D8418	To dissovle compounds
HEPES	SIGMA	H3375	To make artificial cerebrospinal fluid (ACSF)
Humidified non-CO2 incubator	Fisher Scientific	11-683-230D	To hydrate plates at 37 °C
Oligomycin from Streptomyces diastatochromogenes	SIGMA	O4876	To inhibit mitochondrial ATP synthase
Parafilm	SIGMA-ALDRICH		sealing film
Rotenone	Tocris	3616	To inhibit complex I of the mitochondria
Seahorse XF Calibrant Solution 500 mL	Seahorse Bioscience	103681-100	Solution for seahorse calibration
Seahorse XF Extracellular Flux Analyzer	Seahorse Bioscience		Equipment used to analyze oxygen consumption rate, old generation
Seahorse XFe24 Extracellular Flux Analyzer	Seahorse Bioscience		Equipment used to analyze oxygen consumption rate, new generation
Seahorse XF24 FluxPaks	Seahorse Bioscience	101174-100	Package of flux analyzer sensor cartridges, tissue culture plates, capture screens,  calibrant solution and calibration plates; assay kit.
Sodium pyruvate	SIGMA	P2256	To prevent any substrate-limiting constraints of substrate supply
Stainless steel biopsy punches	Miltex		Device used to punch slices
Sterile cell culture dish, 35 x 10 mm	Eppendrof	0030700102	Used for slice punch
Vibratome	Leica	VT1200	To slice brain tissue
Water bath	Thermo Scientific Precision	282-115	To heat buffer and solutions