Jan N. Johannessen

Neurochemical and behavioral effects of systematic and intranigral administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the rat☆

At doses of 5-10 mg kg-1, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (NMPTP) produces in rats acute immobility, retropulsion, straub tail, piloerection, exophthalmos, salivation and clonic movements of the forepaws. It does not produce analgesia as measured by the tail test, nor does it produce permanent motor impairment after chronic or intranigral administration. The acute retropulsion and immobilizing effects can be blocked by methysergide. Administered acutely, NMPTP doubles levels of serotonin in the raphe nucleus and substantia nigra. At the same time, levels of dopamine increase in the caudate nucleus and decrease in the substantia nigra. The NMPTP-induced decrease in dopamine content of the substantia nigra persists in chronically treated rats, but there is no significant decrease in striatal dopamine. After chronic administration of NMPTP, striatal levels of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were decreased by about 50%. Intranigral administrations of NMPTP (10 micrograms daily for 5 days) failed to produce a 6-hydroxydopamine-like lesion in the nigrostriatal system. These results indicate that NMPTP in the rat does not cause selective destruction of dopaminergic neurons, but it does produce acute tryptamine-like effects.

IV. Differences in the metabolism of MPTP in the rodent and primate parallel differences in sensitivity to its neurotoxic effects

Primates and rodents show marked differences in sensitivity to the neurotoxic effects of MPTP. We and others have previously shown that the toxic effects of MPTP on nigrostriatal cells are dependent on the oxidative metabolism of MPTP to the quaternary species MPP+. We have therefore compared the distribution and metabolism of MPTP in the monkey and several rodent species. Three major differences have been identified: 1) the primate, but not the rodents, showed a persistently high concentration of MPTP metabolites in the caudate nucleus compared to other brain regions; 2) the rodent brains cleared MPTP and its metabolites much more rapidly than did the monkey, and; 3) the predominant metabolite retained by the monkey brain was MPP+, while MPP+ cannot be detected in rodent brains for more than a few hours after injection. The persistence of MPP+ in the primate brain may explain the heightened toxicity of MPTP in this species.

1-methyl-4-phenylpyridine (MPP+) induces oxidative stress in the rodent

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) produces an irreversible parkinsonism in primates. Recent evidence suggests metabolism of MPTP to 1-methyl-4-phenylpyridine (MPP+) is required for toxicity. We have proposed that MPP+ may play a central role in the toxicity of MPTP, but direct assessment of the effects of MPP+ in brain is difficult. Therefore, we have sought to define the mechanism of peripheral MPP+ toxicity in the rat and mouse. Systemically administered MPP+ produced its major pathology in the lung and was typified by perivascular edema. An increase in plasma glutathione disulfide concentrations also resulted, suggesting that MPP+ in analogy to paraquat produces oxidative stress. In addition, the lethality of MPP+ in the mouse was increased by dietary selenium deficiency. These results define in both pathological and chemical terms the potent systemic toxicity of MPP+ and suggest that MPP+, because of its high concentration in primate brain, has the potential to play an important role in the CNS toxicity of MPTP.

Tryptophan hydroxylase: increase in activity by electrical stimulation of serotonergic neurons

Electrical stimulation of the rat midbrain dorsal raphe nucleus, which contains clusters of 5-HT containing cell bodies, increased the activity of tryptophan hydroxylase prepared in low speed supernatant extracts from cerebral cortex, a region containing 5-HT projections arising from the dorsal raphe nucleus. In contrast, activity of enzyme from hippocampus, which receives its 5-HT innervation primarily from the median raphe was unaffected by dorsal raphe stimulation. The activity of cortical enzyme increased maximally at a stimulation frequency between 10 and 15 Hz. The increase in activity took 20 min to reach maximum at a stimulation frequency of 10 Hz and had returned to control levels 30 min after cessation of stimulation. The increase in enzyme activity is associated with a significant increase in V(max) without any change in K(m) for substrate tryptophan or the artificial reduced pterin cofactor, d-6-methyl-5,6,7,8-tetrahydropterin. These findings may provide a basis for the increase in synthesis of 5-HT which occurs in response to nerve stimulation through the enhanced conversion of tryptophan to 5-hydroxytryptophan.

MPTP treatment combined with ethanol or acetaldehyde selectively destroys dopaminergic neurons in mouse substantia nigra

We have previously reported that ethanol and acetaldehyde potentiate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, enhancing dopamine (DA) depletion in the striatum. The present study was designed to determine whether such enhancement of neurotoxicity was specific for the nigro-striatal DA pathway. In 5-week-old mice acetaldehyde treatment did not enhance DA depletion seen 7 days after MPTP treatment. In 8-week-old animals, however, acetaldehyde or ethanol given with MPTP decreased striatal DA content to about 10% of controls, whereas the depletion was to 43% of controls when MPTP was given alone. In acetaldehyde or ethanol and MPTP-treated mice, changes in DA levels were observed only in the striatum. DA contents in the hypothalamus, olfactory bulb and frontal cortex were similar to that in controls. Contents of norepinephrine and serotonin in striatum, hypothalamus, olfactory bulb and cerebral cortex were not affected by any of the treatments. Three months after MPTP alone, striatal DA recovered to 74% of controls in 8-week-old mice, whereas no recovery occurred in acetaldehyde and MPTP-treated mice. Moreover, both tyrosine hydroxylase (TH) immunocytochemistry and Cresyl violet staining showed an extensive and selective cell loss in the pars compacta of the substantia nigra (SNc) of the mice treated with acetaldehyde or ethanol and MPTP, whereas MPTP alone caused only a limited cell degeneration.

The neurochemical basis of footshock analgesia: the role of spinal cord serotonin and norepinephrine

Previous studies have demonstrated that brief front paw and brief hind paw shock produce potent opiate and non-opiate analgesia, respectively. Additionally, opiate analgesia can be classically conditioned by using either front paw shock or hind paw shock as the unconditioned stimulus. Front paw footshock-induced analgesia (FSIA), hind paw FSIA, and classically conditioned analgesia are similar in that each is mediated by a medullospinal pathway. However, the neurochemistry of these medullospinal connections has never been investigated. One question which arises is whether any of these phenomena are mediated by monoaminergic neurotransmitters at the level of the spinal cord. The present series of experiments examined the effect of depleting spinal serotonin (5-HT) and combined depletion of spinal 5-HT and norepinephrine (NE) on front paw FSIA, hind paw FSIA, and classically conditioned analgesia. Hind paw FSIA and classically conditioned analgesia were not attenuated by either of these neurochemical manipulations. Front paw FSIA was significantly reduced by both 5-HT depletion and combined 5-HT and NE depletion. To assess the relative importance of spinal 5 HT and NE in front paw FSIA, NE and 5-HT antagonists were injected onto the lumbosacral cord prior to shock exposure. Attenuation of front paw FSIA by equimolar doses of the monoamine blockers was much greater following injection of the 5-HT blocker than after the NE blocker. These data indicate that spinal 5-HT and, apparently to a lesser extent, spinal NE mediate front paw (opiate) FSIA whereas neither 5-HT nor NE appears to mediate hind paw FSIA or classically conditioned analgesia.

I. The development of amine substituted analogues of MPTP as unique tools for the study of MPTP toxicity and Parkinson's disease

We are currently developing amino-substituted MPTP analogues as useful probes for understanding the mechanism of MPTP toxicity and Parkinsons disease. One analogue, 4-amino MPTP, induces a loss of striatal dopamine and is thus a suitable substitute for MPTP. This probe will be used as a histologically fixable MPTP which can be used to answer detailed anatomical questions concerning the sites of MPTP, MPP+ uptake and storage. In addition, antibodies have been raised against MPTP and MPP+ in rabbits using diazo-linked bovine serum albumin conjugates. The antibodies have been characterized with regard to their recognition of relevant structural analogues using an enzymelinked immunoassay (ELISA) procedure. Antibodies to MPTP detected MPTP in mouse brain extracts derived from as little as 5 micrograms of tissue. The antibodies will be used for immunohistochemical localization of 4-NH2-MPTP and 4-NH2-MPP+ in brain, as well as probes for the screening of parkinsonian brain tissue for any MPTP- or MPP+-like materials which might exist.

Assessment of the opiate properties of two constituents of a toxic illicit drug mixture

The intravenous use of an illicit synthetic drug preparation has caused permanent parkinsonism in a number of addicts. Chemical analysis has revealed the ingredients to be two related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-4-propionoxypiperidine (MPPP). The opiate properties of these two compounds have been assessed using in vitro receptor binding techniques as well as behavioral tests indicative of opiate action, including analgesia, catatonia, respiratory depression and the loss of righting and corneal reflexes. All opiate activity was found to reside with MPPP, which proved to be a potent mu-type agonist. It is concluded that the opiate properties of MPPP alone explain repeated abuse of MPTP/MPPP mixtures by heroin addicts.

EFFECTS OF MPTP ON THE CEREBROVASCULATURE

The neurotoxin, l‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine, has been shown to cause pooling of blood in the brain microvasculature and decrease the permeability of the blood‐brain barrier. All areas of the brain examined in this study were affected. This study points out the possibility that reduced nutrient uptake, hypoxia and ensuing free radical damage are involved in the mechanism of toxicity of this neurotoxin.

High affinity binding sites for 1-methyl-4-phenyl-pyridinium ion (MPP+) are present in mouse brain.

The possible involvement of 1-methyl-4-phenyl-pyridinium ion (MPP+) in the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prompted us to search for and characterize [3H]MPP+ binding sites in the mouse. Our data show that [3H]MPP+ binds saturably and with high affinity to mouse brain membranes. Scatchard analysis resulted in one straight line. The apparent KD was 15 +/- 1 nM and the Bmax 245 +/- 30 fmol/mg protein. The distribution of [3H]MPP+ binding sites shows a regional variation: the hypothalamus having highest binding and the cerebellum the lowest. Several compounds failed to inhibit [3H]MPP+ binding whereas only analogues of MPP+, MPTP and paraquat were able to antagonize this binding to brain. Specific binding with analogous characteristics also occurs in peripheral tissues. Considering the postulated role of MPP+ in MPTP neurotoxicity, further studies on [3H]MPP+ binding sites might be relevant to elucidate the mechanisms of this toxicity.