1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a recently discovered neurotoxin, caused extensive losses of dopamine and its major metabolites after its administration to male Swiss-Webster mice. In contrast, under identical conditions, several MPTP analogues, even those with relatively minor structural changes, were without toxicity. These include compounds with a 1-ethyl and 1-propyl substituent rather than the 1-methyl, the compound lacking the double bond in the tetrahydropyridine ring, as well as the compound with no phenyl substituent. It follows that each part of the MPTP molecule is important in determining its neurotoxic activity.

The rates of decomposition of 3H-dopamine (3H-DA), 3H-apomorphine and 3H-ADTN were determined in Tris buffer at pH 7.4 and in a Tris buffer containing a neostriatal membrane preparation representative of that used in binding experiments. In both the Tris buffer alone and in the neostriatal membrane preparation, 3H-DA was the most stable, 3H-ADTN was intermediate and 3H-apomorphine was the least stable. In the Tris buffer, the extent of decomposition of all three 3H-catechols was greatly retarded by sodium ascorbate. In contrast, in the neostriatal membrane preparation pronounced inhibitory effects of ascorbate were obtained only with 3H-ADTN. Even in the presence of high concentrations of sodium ascorbate (i.e., 0.5 mM), there was an extensive decomposition of 3H-apomorphine in the neostriatal membrane preparation. The data suggest that one exercise great caution in choosing appropriate conditions for binding experiments with these unstable ligands.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) causes a destruction of the nigrostriatal dopamine pathway in humans as well as in monkeys. However, it has been reported that MPTP is inert in several small animal species. We now report that MPTP, given to mice at 30 mg/kg intraperitoneally, causes severe and long-lasting depletions of dopamine and its major metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the neostriatum.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) causes degeneration of the dopaminergic nigrostriatal pathway in several animal species, including humans, monkeys and mice. Changes observed after MPTP administration include marked decrements in the neostriatal content of dopamine and its major metabolites, dihydroxyphenylacetic acid and homovanillic acid, and a greatly diminished capacity of neostriatal synaptosomes to take up 3H-dopamine. In contrast, there is no pronounced loss of serotonin in the neostriatum or of dopamine and its metabolites in other brain areas in MPTP-treated animals. The oxidative metabolism of MPTP to 1-methyl-4-phenyl pyridine, a positively charged species, has been suggested as a critical feature in the neurotoxic process. Moreover, in rat brain preparations, the monoamine oxidase (MAO) inhibitor pargyline and the specific MAO-B inhibitor deprenil can prevent the formation of 1-methyl-4-phenyl-pyridine from MPTP, while the specific MAO-A inhibitor clorgyline has no such effect, suggesting that MAO, and specifically MAO-B, is responsible for the oxidative metabolism of MPTP. We now report that pargyline, nialamide and tranylcypromine, which inhibit both MAO-A and MAO-B, when administered to mice before MPTP, protect against MPTP-induced dopaminergic neurotoxicity. Deprenil is also protective, but clorgyline is not. Our data are consistent with the premise that MAO-B has a crucial role in MPTP-induced degeneration of the nigrostriatal dopaminergic neuronal pathway.

1-Methyl-4-phenyl-1,2,5,6- tetrahydropyri dine ( MPTP ) is known to cause an irreversible destruction of the dopaminergic nigrostriatal pathway and symptoms of parkinsonism in humans and in monkeys. However, MPTP has been reported to act only minimally or not at all in several other animal species. When MPTP (30 milligrams per kilogram of body weight) was administered parenterally to mice, a decrease in concentrations of neostriatal dopamine and its metabolites, a decrease in the capacity of neostriatal synaptosomal preparations to accumulate [3H]dopamine, and a disappearance of nerve cells in the zona compacta of the substantia nigra were observed. In contrast, MPTP administration had no effect on neostriatal concentrations of serotonin and its metabolites. MPTP administration thus results in biochemical and histological changes in mice similar to those reported in humans and monkeys and similar to those seen in Parkinson's disease in humans. The mouse should prove to be a useful small animal with which to study the mode of action of MPTP .

The rate of 3H-dopamine decomposition was determined in buffers and in neostriatal membrane preparations that are commonly used in binding experiments. The rate of decomposition of 3H-dopamine was inhibited considerably by the membrane preparation and by ascorbic acid. Under conditions in which the binding of 3H-dopamine is routinely measured in neostriatal membrane preparations, there was almost no 3H-dopamine decomposition. The data would seem to suggest that the routine addition of very high concentrations of antioxidants is unnecessary when 3H-dopamine biding is being determined.

A considerable controversy exists over whether ascorbic acid should or should not be used in experiments in which the binding of DA agonists to neostriatal membrane preparations is being determined. Some authors claim that its presence is required. In the present study we have determined that sodium ascorbate was a very potent inhibitor of the specific binding of 3H-DA to a rat neostriatal membrane preparation. Under conditions of these binding experiments, there was no decomposition of the 3H-DA as determined by two separate techniques. These data would seem to suggest that when 3H-DA binding is being measured, ascorbate addition is detrimental.

Abstract: Ascorbic acid, sodium ascorbate, and isoascorbic acid (the stereo‐isomer of ascorbic acid) inhibited the stereospecific binding of [3H]spiroperidol to neostriatal membrane preparations. Greater inhibitory effects were obtained at intermediate concentrations of the three ascorbic acid analogs (i.e., 0.06 and 0.6 mM) than at higher (6 mM) or lower (0.006 mM) concentrations. In parallel experiments, the three ascorbic acid analogs induced lipid peroxidation, which was also greater at the two intermediate than at higher or lower concentrations. Several known inhibitors of lipid peroxidation, including propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, α‐naphthol, and cobalt chloride, as well as the iron chelating agents EDTA and DETAPAC (diethylenetriaminepentaacetic acid) were able to counteract the effects of the ascorbic acid analogs on both lipid peroxidation and on [3H]spiroperidol binding. These data strongly suggest that an iron‐catalyzed lipid peroxidation is responsible for the observed inhibitory effects on binding. In other experiments, neostriatal membrane preparations that were preincubated with ascorbic acid (0.6 mM) and subsequently washed still had greatly diminished capacity to bind [3H]spiroperidol, indicating that ascorbic acid need not be physically present during the binding assay in order to affect binding. This experimental procedure also appears to be a way in which [3H]spiroperidol binding sites can be inactivated and washed free of the inactivating agent.

The addition of ferrous sulfate to phosphate buffer at pH 7.4 brought about a large increase in the rate of autoxidation of dopamine or norepinephrine. The iron-chelating agents diethylenetri-aminepentaacetic acid (DETAPAC) and desferroxamine (Desferal) inhibited both the baseline as well as the iron-stimulated autoxidation of these catecholamines. Lesser inhibitory effects were observed with another iron-chelating agent, namely EDTA. In other experiments, DETAPAC or Desferal had little effect on the autoxidation of 6-hydroxydopamine in the absence of added ferrous sulfate, but both inhibited the ferrous sulfate-stimulated autoxidation. In contrast, both the baseline and ferrous sulfate-catalyzed rate of oxidation of 6-hydroxydopamine were greatly stimulated by EDTA addition. DETAPAC and Desferal appear to be useful experimental tools that may be added to solutions containing catecholamines to prevent their degradation.

Mazindol and two homologs of mazindol were tested for their effects as uptake inhibitors in rat tissue slices for [3H]dopamine in the neostriatum, for [3H]norepinephrine in occipital cortex and for [3H]serotonin in whole brain. All three drugs were potent inhibitors of [3H]dopamine uptake (ED50 values between 57 and 280 nM), [3H]norepinephrine uptake (ED50 values less than 19 nM) and were somewhat weaker against [3H]serotonin uptake (ED50 values between 550 and 4100 nM). All three drugs were in contrast very weak as releasing agents for previously accumulated 3H-biogenic amines. Mazindol injection resulted in a large increase in locomotor activity in mice, but its two homologs were without effect. Mazindol was able to counteract amphetamine-induced increases in activity in reserpinized mice, but its homologs were inactive. Mazindol also caused a vigorous ipsilateral rotation in rats with an unilateral 6-hydroxydopamine lesion of the nigrostriatal system, but again the homologs had no such effect. However, all three drugs were potent inhibitors of prolactin secretion in rats (ID50 values 1-2 mg/kg orally). Correlations between the capacities of the drugs to inhibit 3H-biogenic amine uptake and the various in vivo responses are made.

l-Deprenil, a potent inhibitor of type B monoamine oxidase, was a weak inhibitor of 3H-dopamine uptake as well as a weak releasing agent for previously accumulated 3H-dopamine in rat neostriatal tissue slices. In similar experiments d-amphetamine was approximately 100 times as potent as l-deprenil as a releasing agent. When deprenil (20 mg/kg IP) was given to rats with a unilateral 6-hydroxydopamine lesion of the substantia nigra, it brought about a moderate but long-lasting ipsilateral rotational behavior. l-Dopa (20–40 mg/kg, IP) by itself caused a considerably stronger rotation in the opposite direction (contralateral). When l-dopa was given to rats 1 hr after l-deprenil, there was a considerably greater contralateral rotation than after l-dopa alone. Clorgyline, a type A monoamine oxidase inhibitor, which by itself at 20 mg/kg caused no rotation, also potentiated the contralateral rotation seen after l-dopa (5–20 mg/kg). In contrast, d-amphetamine, which by itself caused ipsilateral rotation, failed to potentiate the rotation after l-dopa. Possible mechanisms for these observations will be discussed.

Azathioprine (1mmole/kg), given to male Swiss-Webster mice one hour prior to 75 mg/kg alloxan, was able to protect against the diabetogenic actions of alloxan. Various potential mechanisms for the protective effects of azathioprine, including its scavenging of the hydroxyl radical, which can be generated from alloxan, will be discussed.

Ascorbic acid was a potent inhibitor of the binding of both dopamine agonists (3H-dopamine and 3H-ADTN) and also of dopamine antagonists (3H-spiroperidol and 3H-domperidone) to neostriatal membrane preparations. Against dopamine agonists, ascorbic acid caused a dose-dependent inhibition of binding with about 90% effect at 6 mM ascorbic acid. Against dopamine antagonists there was U-shaped dose response curve for ascorbic acid. That is, 6 mM ascorbic acid caused no significant inhibition, while 0.006 mM caused a slight inhibition, and intermediate concentrations caused extensive inhibition. Almost identical inhibitory effects were obtained with sodium ascorbate. In other experiments, 500 mg/kg of ascorbic acid given to mice 1 hour prior to the dopamine releasing agent d-amphetamine, was able to greatly attenuate the increase in locomotor activity usually seen after amphetamine. These latter data may have important implications for a possible role for ascorbic acid in dopaminergic neurotransmission.