An in vitro model of 1‐methyl‐4‐phenyl‐pyridinium (MPP+) toxicity: incubation of rabbit caudate nucleus slices with MPP+ followed by biochemical and functional analysis

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Abstract
1 Slices of rabbit caudate nucleus were preincubated for up to 24 h in vitro in the presence of the neurotoxic compound 1-methyl-4-phenyl-pyridinium (MPP+). Subsequently the levels of endogenous monoamines in the slices were determined by h.p.l.c. with electrochemical detection. MPP+, in concentrations higher than 32 nm significantly diminished the dopamine levels within the slices in a concentration- and time-dependent manner; at 32 μm the depletion was more than 95%. The concentration of the major metabolite of dopamine, dihydroxyphenyl acetic acid (DOPAC) was decreased at concentrations of MPP+ that did not alter dopamine levels. Thus, MPP+ increased the dopamine/DOPAC ratio. 2 In contrast, both 5-hydroxytryptamine (5-HT) levels and 5-HT/5-hydroxyindolacetic acid (5-HIAA) ratios were increased at nanomolar concentrations of MPP +. 5-HT was significantly reduced only at 32 μm. 3 The dopamine uptake inhibitor nomifensine reduced the depletory effect of MPP + on dopamine and DOPAC content. 4 Following 24 h pretreatment with MPP+, the uptake of [3H]-dopamine into rabbit caudate nucleus slices was either enhanced (at 0.32 μm, 1 μm and 3.2 μm MPP+) or reduced (at 32 μm MPP+). 5 Preincubation of slices with 10 μm MPP + for only 1 h increased their 3H-labelling (in contrast to 24 h pretreatment) whereas after 9 h no net increase was detectable. After 1 and 9 h MPP + pretreatment, much less deaminated metabolites of [3H]-dopamine were found in the incubation medium of MPP + treated slices than in the medium of control slices. These findings suggest that MPP + strongly inhibits the enzyme monoamine oxidase (MAO) within dopaminergic (and 5-hydroxytryptaminergic) terminals before destroying them. 6 To validate the proposed in vitro model functionally, the electrically evoked release of [3H]-acetylcholine ([3H]-ACh) was investigated in MPP+ treated slices and controls. MPP+ reduced both the facilitatory effect of the D2-receptor antagonist domperidone and the inhibitory effect of the catecholamine uptake inhibitor nomifensine on [3H]-ACh release; effects compatible with a diminished inhibitory dopaminergic input on cholinergic neurones. 7 These findings also show that the terminal region of dopaminergic neurones, the caudate nucleus, is a site for MPP + toxicity. The present in vitro model may be useful for investigating the effects of MPP + and its interaction with other drugs under defined conditions.