In vivo imaging of mitochondrial function in methamphetamine-treated rats
Research highlights
► Administration of methamphetamine induces ATP consumption. ► In response, mitochondria respiratory chain reaction is activated. ► Nitroxyl radical is reduced in the mitochondrial respiratory chain reaction. ► Reduction of nitroxyl radical can be visualized by Overhauser-enhanced MRI.
Introduction
Abuse of the powerfully addictive psychostimulant, methamphetamine, occurs worldwide. Chronic methamphetamine users show signs of psychiatric illness, including a psychotic state and an anxiety-like disorder (Akiyama, 2006, Grelotti et al., 2010, Sato, 1992). Repeated intermittent administration of methamphetamine produces an enduring hypersensitivity to the motor stimulant effect of methamphetamine in humans (Ujike and Sato, 2004) as well as in experimental animals (Shuto et al., 2006), which is termed behavioral sensitization. It is widely accepted that the behavioral sensitization caused by methamphetamine is a characteristic of drug dependence and addiction (Vanderschuren and Kalivas, 2000). Behavioral sensitization by methamphetamine is associated with dopaminergic nerve function (Kalivas and Stewart, 1991).
Energy consumption and supply are tightly linked to neuronal activity in the brain. Depolarizations propagate from the dendritic spines to the dendrites, where they may cause further opening of voltage-gated sodium channels and activation of the Na+/K+ ATPases, leading to an increased demand for energy (ATP). In response, the mitochondrial respiratory chain is rapidly activated, causing a decrease in mitochondrial NADH content (Kasischke et al., 2004). Although this process is physiologically important, the enhancement of mitochondrial respiratory chain function after nerve activation may induce oxidative stress in the brain. Mitochondria are the major cellular source of oxygen free radicals because of the electron leakage from the respiratory chain; the electrons in turn react with molecular oxygen to give superoxide anion radicals (Chen et al., 2005). In fact, lipid peroxidation products accumulate in both the striatum and the prefrontal cortex in chronic methamphetamine users and in animal models (Acikgoz et al., 2000, Fitzmaurice et al., 2006).
During the mitochondrial respiratory chain reaction, electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. To detect redox status in mitochondria, nitroxyl radicals (i.e., nitroxides) are powerful as a redox-sensitive contrast agent coupled with Overhauser-enhanced magnetic resonance imaging (OMRI) (Quintanilha and Packer, 1977, Yamato et al., 2009). OMRI is a double resonance technique that uses the presence of paramagnetic agents to enhance the signal intensity from nuclear spins by a process known as dynamic nuclear polarization, or the Overhauser effect (Krishna et al., 2002, Li et al., 2006, Lurie et al., 1988, Lurie et al., 2005). Recent studies have successfully expanded this technique to obtain functional information, including noninvasive pO2 maps (Krishna et al., 2002, Matsumoto et al., 2009) and simultaneous images of different redox reactions (Utsumi et al., 2006).
Here, we evaluated whether a single administration of methamphetamine induces ATP consumption and overactivation of mitochondria. We measured mitochondrial function using OMRI to monitor the reducing and oxidizing processes noninvasively. For a comprehensive investigation of the mitochondrial function, we also monitored oxygen partial pressure using an oxygen-selective electrode. We also examined the ability of the blood–brain barrier-permeable antioxidant, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) to alter methamphetamine-induced oxidative stress and examined its effect on behavior sensitization.
Section snippets
Chemicals
Methamphetamine was purchased from Dainippon Sumitomo Pharma Co., Ltd., (Osaka, Japan). 3-carboxy-2,2,5,5-tetramethylpyrrolidine-L-oxyl (carboxy-PROXYL), α-methyl-p-tyrosine (α-MT), and TEMPOL were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). ATP, ADP, AMP, lactate dehydrogenase (from pig heart), and NADH were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Methanol and acetonitrile (HPLC grade) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). EDTA was purchased from
Methamphetamine-induced locomotor hyperactivity and dopamine release
A dose-dependent increase in locomotor activity was observed in methamphetamine-treated rats (Fig. 1). Administration of 5 mg/kg methamphetamine significantly affected locomotor activity, and therefore this concentration was chosen for subsequent experiments.
ATP consumption after methamphetamine administration
We examined whether ATP was consumed by methamphetamine-induced dopaminergic nerve activation. ATP levels in the brain were significantly decreased 30 min after methamphetamine administration (Fig. 2A). Simultaneously, ADP and AMP were
Discussion
In the present study, we demonstrated that a single administration of methamphetamine induced dopaminergic nerve activation, ATP consumption and an increase in the mitochondrial respiratory chain reaction. In particular, we detected the reduction of nitroxyl radical in the brain as index of mitochondrial function in vivo. Nitroxyl radical is reduced to the corresponding hydroxylamine form in the mitochondrial respiratory chain reaction, and then the reduction can be visualized by OMRI. Thus,
List of abbreviations
- carboxy-PROXYL
3-carboxy-2,2,5,5-tetramethylpyrrolidine-L-oxyl
- ECD
electrochemical detector
- ESR
electron spin resonance
- METH
methamphetamine
- methoxycarbonyl-PROXYL
3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-L-oxyl
- α-MT
α-methyl-p-tyrosine
- OMRI
Overhauser-enhanced magnetic resonance imaging
- SOD
superoxide dismutase
- TBARS
thiobarbituric acid reactive substance
- TEMPOL
4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl
Acknowledgments
This study was partially supported by a Research Fellowship for Young Scientists and a Grand-in-Aid for Scientific Research (A) from the Japan Society for the Promotion of Science.
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Takeshi Shiba and Mayumi Yamato contributed equally to this work.