Tyrosine Hydroxylase is the rate-limiting enzyme in the synthesis of dopamine, and as such, it is widely used as a marker of dopaminergic cells. Within the basal ganglia, the dopaminergic cells are located in the substantia nigra pars compacta, and project to the striatum. It is this pathway which degenerates during Parkinson's disease. The data presented here illustrate examples of tyrosine-hydroxylase immunoreactive cells in striatum following intrastriatal injection with the neurotoxin MPP+. We further show by electron microscopy that these cells are, in fact, neurons and that they possess ultrastructural features of interneurons.
Taurine is a sulphur-containing -amino acid found in high (millimolar) concentrations in excitable tissues such as brain and heart. Its suggested roles include osmoregulator, thermoregulator, neuromodulator, and potential neurotransmitter. This amino acid has also been shown to be released in large concentrations during ischaemia and excitotoxin-induced neuronal damage. Here we report a protective effect of taurine against MPP ϩ -induced neurotoxicity in coronal slices from rat brain. Significant protective effects were observed at taurine concentrations of 20 and 1 mM, suggesting a potential role for taurine in cases of neuronal insult. Studies with the synthetic taurine analogues taurine phosphonate, guanidinoethane sulphonate, and trimethyltaurine suggested the observed effect to be mediated via an extracellular mechanism. The use of GABA receptor ligands muscimol and bicuculline indicated the effect to be mediated through activation of GABA A receptors.
The possible protection against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)) afforded by inhibitors of nitric oxide synthase (NOS) and the antagonist of N-methyl-D-aspartate receptor function, MK-801, was studied in a brain-slice superfusion system. Significant decreases in levels of dopamine and its metabolites 3,4-dihyroxyphenylacetic acid (DOPAC) and homovanillic acid were observed following incubation of slices with 25 microM MPP(+). The activity of intracellular lactate dehydrogenase (LDH), a marker of cell viability, was also significantly decreased. These effects were attenuated by preincubation with I mM 7-nitroindazole (7NI), a selective inhibitor of the neuronal isoform of nitric oxide synthase (NOS). In contrast, the nonspecific NOS inhibitor N(omega)-nitro-L-arginine, also at 1 mM, had no effect on levels of dopamine metabolites but did show a small attenuation of the levels of dopamine. 7NI alone caused some increase in levels of dopamine and a decrease in the metabolite DOPAC, which is consistent with it also acting as an inhibitor of monoamine oxidase-B. MK-801 afforded no significant protection of aminergic cells, although changes in LDH activity suggested that there may have been some protection of non-aminergic neurons affected by this, relatively high concentration of MPP(+).
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes selective degeneration of the dopaminergic terminals of the substantia nigra and much research has been carried out on the possible relationship of this compound to idiopathic Parkinson's disease. Originally discovered as a contaminant of a pethidine analogue [ 11, it is now known that the oxidised product 1-methyl4 phenylpyridinium ion (MPP+) is the effective neurotoxin [2]. Briefly, the lipophilic parent compound enters the brain where it is metablised by monoamine oxidase B, ( M A 0 B [EC 1.4.3.41) to the pyridinium ion. MPP+ is a substrate for the dopamine uptake system [3] and is selectively concentrated within these neurons. The toxin is then accumulated in the mitochondrial matrix where it inhibits enzyme complex 1 of the electron transport chain. Subsequent depletion of ATP and associated loss of membrane potential result in neuronal cell death. This process has been described in detail elsewhere [2,4,5l. Potential protection against M P P toxicity is also of constant interest due to the correlation between it and Parlunson's disease.In these experiments, a tissue slicing system was adapted to study MPP+ neurotoxicity by comparing the levels ofdopamine (DA), it's metabolites di-hydroxyphenylacetic acid (DOPAe) and homovanillic acid (HVA), noradrenaline (NA) and serotonin (5HT) from rat striatum. Adult female Wistar rats (-2Mg) were sacrificed by cervical dislocation and brains rapidly removed. Striatal slices of 0.5mm thickness were prepared using a Campden Vibroslice and these were placed singly in a number of open perfusion chambers. The chambers were perfused with oxygenated Krebs bicarbonate solution containing (final concentration) NaCI, 112.5mM; KCI, 4.7mM; KH2P04. 1.2mM; MgSO,, '1.2mM; CaClh 2.5mM; NaHCQ 25mM and glucose 11.5mM. Following a 30min preincubation slices were subjected to 2%M MPP in Krebs solution for a period of l0min and finally returned to normal Krebs solution for a further 30min. Control slices were incubated in Krebs solution for 70min. Following superfusion, slices were homogenised in NE DA W A C YIT HVA Fie. 2: Percent decrease in catecholamines from striatal slices following incubation with 25pM M P P . Values represent mean SEM. 0.4M perchloric acid containing 1mM EDTA, 0.5mM Na&05 and 0. lpglml di-hydroxybenzylamine (DHBA) as internal standard.Homogenates were centrifuged at 13,000 rpm for IOmins at 4OC and supernatants removed. An aliquot (1OOpl) was retained for direct injection while the remainder was treated with l0mg acid-washed alumina (activity grade 1, Sigma) and catecholamines (CA) extracted with 5OOml 0.2M acetic acid. Compounds were separated using reverse-phase ion-pair (C18) HPLC with electrochemical detection (HPLC-ECD). (Fig 1). Recoveries were calculated from the internal standard and results adjusted accordingly, allowing quantitative analysis of the compounds of interest. Depletion of all catecholamines particularly DA was observed (Fig. 2). indicating toxicity to dopaminergic neurons characterist...
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