In vivo vulnerability of dopamine neurons to inhibition of energy metabolism

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Abstract

In vitro studies indicate that mesencephalic dopamine neurons are more vulnerable than other neurons to impairment of energy metabolism. Such findings may have bearing on the loss of dopamine neurons in Parkinson's disease, in which mitochondrial deficiencies have been identified, but would only be relevant if the selective vulnerability were maintained in vivo. To examine this, rats were stereotaxically administered various concentrations of the succinate dehydrogenase inhibitor, malonate (0.25–4 μmol), either into the left substantia nigra or striatum. One week following injection, dopamine and γ-aminobutyric acid (GABA) levels in the mesencephalon and striatum were measured. Intranigral injection of malonate caused nigral dopamine and GABA to be comparably reduced at all doses tested. The 50% dose level for malonate vs. dopamine and GABA loss was 0.39 and 0.42 μmol, respectively. Tyrosine hydroxylase immunocytochemistry of the midbrains of rats which received an intranigral injection of malonate showed normal staining with 0.25 μmol malonate, but almost complete loss of tyrosine hydroxylase positive nigral pars compacta cells with 1 μmol malonate. Intrastriatal injection of malonate produced a loss of both tyrosine hydroxylase activity and dopamine. In contrast to what was seen in substantia nigra, there was a greater loss of dopamine than GABA in striatal regions nearest the injection site. In striatal regions most distal to the injection site, and which received the lowest concentration of malonate due to diffusion, dopamine levels were significantly reduced with all doses of malonate (0.5–4 μmol), whereas GABA levels were unaffected. Intrastriatal coinfusion of succinate along with malonate completely prevented the loss of dopamine and GABA indicating that succinate dehydrogenase inhibition was the cause of toxicity. These findings indicate that dopamine terminals in the striatum of adult rats are selectively more vulnerable than are the GABA neurons to a mild energy impairment.

Introduction

Dopamine neurons in the substantia nigra pars compacta and their axonal projections in the striatum are the primary neurotransmitter population lost in Parkinson's disease. Recent studies from several laboratories have demonstrated mitochondrial defects in Parkinson's disease. In some studies, the defects were specific to the substantia nigra (Mizuno et al., 1989; Schapira et al., 1989, Schapira et al., 1990; Mann et al., 1992), while in others, a more generalized impairment involving muscle and platelet mitochondria was observed (Parker et al., 1989; Shoffner et al., 1991; Bindoff et al., 1991; Benecke et al., 1993). At present, it is not known whether mitochondrial defects are a cause or an effect of Parkinson's disease. Also not known is the reason for the specific targeting of dopamine neurons should a generalized mitochondrial defect be found to be an underlying cause of the disease. Understanding how mild to moderate metabolic stress affects dopamine neurons, however, may provide insight into the loss of these neurons in Parkinson's disease.

The greater vulnerability of mesencephalic dopamine neurons to impairment of energy metabolism has been suggested by several findings. The loss of high affinity uptake of dopamine in synaptosomal preparations was demonstrated to be more sensitive to acute rotenone (mitochondrial complex I inhibitor), antimycin A (complex III inhibitor) and cyanide (complex IV inhibitor) treatment as compared with norepinephrine, γ-aminobutyric acid (GABA) and serotonin uptake suggesting that a constitutive metabolic deficiency existed in dopamine versus other neurotransmitter populations (Marey-Semper et al., 1993). Dopamine neurons in mouse mesencephalic cultures were three times more sensitive than mesencephalic GABA neurons or striatal GABA or cholinergic neurons to a sequential exposure to rotenone and glutamate (Marey-Semper et al., 1995, Marey-Semper et al., 1995), also suggesting an inherent vulnerability of dopamine neurons to energy impairment. Consistent with the findings of Marey-Semper et al. (1993), Marey-Semper et al. (1995), our laboratory recently reported that dopamine neurons in rat mesencephalic culture were more sensitive to a mild metabolic stress produced by inhibiting the Krebs cycle enzyme succinate dehydrogenase with the reversible, competitive inhibitor, malonate (Zeevalk et al., 1995). The purpose of the present work was to examine the relative vulnerability of dopamine neurons in vivo to energy impairment. Sensitivity to malonate was examined at the level of the cell body in the nigra and at its axonal projection sites in the striatum. In each case, the vulnerability of the dopamine population to malonate was compared with the GABA population.

Section snippets

Animal handling and treatment

Male Sprague-Dawley rats (350–400 g; approximately 3–4 months; Harlan Farms, Indianapolis, IN, USA) were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures were approved by the local Animal Care Committee. Rats were grouped 2 per cage in a room maintained at 20–22°C on a 12-h light-dark cycle with food and water available ad libitum. Animals were anesthetized with Brevital (16.7 mg/kg). Vehicle (saline, pH 7.6) or malonate

Results

Injection of 4-month-old rats with various amounts of malonate into the left substantia nigra produced a similar loss of nigral dopamine and GABA when assayed 1 week after the injection (Fig. 1A). The 50% dose level for malonate-induced loss of dopamine and GABA was 0.39 or 0.42 μmol, respectively. Animals that received greater than 1 μmol malonate had excessive tissue destruction and cavitation in the mesencephalon and were eliminated from the study. Injection of vehicle (saline, pH 7.4) did

Discussion

In the last few years, many laboratories have reported deficiencies in energy metabolism in patients with Parkinson's disease, thus positing inefficient energy metabolism as a possible cause of the loss of nigral dopamine neurons in this disease (Beal, 1992; DiMauro, 1993). In several of these studies (Parker et al., 1989; Shoffner et al., 1991; Bindoff et al., 1991; Benecke et al., 1993), deficits have been reported in the mitochondria isolated from blood platelets and muscle from Parkinson

Acknowledgements

The authors would like to thank Nina Jocnowitz for her technical assistance. This work was supported by a National Parkinson Foundation Grant.

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