Dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine in mice.

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 .

Interference filter microfluorometry of neuromelanin and lipofuscin in human brain.

Following the use of a modified procedure for interference filter microfluorometry, bleached human substantia nigra neuromelanin exhibited a bimodal fluorescence maxima at 435 and 464 nm and human inferior olive lipofuscin, whether oxidized or not, exhibited a peak of fluorescence at 464 nm. Although the fluorescent component of bleached neuromelanin displayed its emission maxima in the spectral region characteristic for lipofuscin, and is regarded as such, its unique bimodality could represent some atypical, but as yet unknown, chemical property. Spectra of three fluorescence standards and a glass blank were also determined.

Further histochemical studies characterizing the lipofuscin component of human neuromelanin.

The residuum of bleached substantia nigra neuromelanin and the nonoxidized and oxidized intraneuronal masses of inferior olive lipofuscin gave sudanophilic staining reactions with oil red O, Sudan black B, and a Sudan III and IV combination stain. The pigments also exhibited oxidation basophilia, each having been stained by Gomori's paraldehyde fuchsin method after bleaching or oxidation with potassium permanganate and by the modified carbol fuchsin method of Barbeito-Lopez after bleaching or oxidation with hydrogen peroxide or potassium permanganate. Confirmation of the presence of lipid substances in bleached neuromelanin was obtained by repeatedly staining, decolorizing, and restaining the residuum of the pigment when utilizing the chemically nonreactive beta naphthol type Sudan combination stain, Sudan III and IV. Not only were tinctorial properties characteristic of lipofuscin clearly demonstrated in the bleached residuum of neuromelanin, but additionally, a partial separation was achieved between the relatively soluble beta naphthol type Sudan-staining lipid component and an insoluble component, possibly lipoprotein, staining with Sudan black B and both basic fuchsin dye-containing mixtures. Consideration was given to the idea that the oxidation basophilia of these neuronal pigments was due to the oxidative formation or exposure of one or more kinds of acidic groups consisting of carboxyls or those containing sulfur or phosphorus.

Neuromelanin and RNA in cells of substantia nigra.

We have found that with accumulation of neuromelanin granules within cell bodies of neurones of the human substantia nigra there is a reduction in cytoplasmic RNA and a decrease in nucleolar volume. These observations imply a gradual decrease in the functional capacity of the cell such that eventually, the cell is unable to produce sufficient protein to maintain its metabolic economy with atrophy and death ensuing. This reduction in protein synthesis may result from the mechanical displacement and disruption of the endoplasmic reticulum by the accumulated pigment granules.

The melanoneurons of the human cerebellum (nucleus pigmentosus cerebellaris) and homologues in the monkey.

Little has been written about the cells here termed cerebellar melanoneurons. This paper describes and illustrates their cytologic features and topographic relationships. In the human brain these large pigmented neurons are scattered in a narrow layer near the lateral wall, dorsal angle and roof of the fourth ventricle. They form an inconspicuous part (group A4) of the system of catecholamine, neuromelanin-containing cells well known in the brain stem. Rostrally, a few of them provide a tenuous continuity with the locus ceruleus but topographically the two nuclei are independent. With ordinary stains the cerebellar cells can be seen as early as the 26th week of gestation (the earliest period examined). Brown neuromelanin granules do not appear until two and a half years of age but argentaffin granules, foreshadowing the production of pigment, are found in increasing numbers in the fetal and postnatal period. Homologues of the human cerebellar cells are reported in two species of monkey, Macaca nemestrina and Lagothrix sp. Neuromelanin, not previously observed in non-human cerebellar cells, occurs in M. mulatta and M. nemestrina. The proximity of the cerebellar melanoneurons to the ventricle raises the possibility that they are related to functions of the ependyma, or that they influence, or are affected by, constituents of the cerebrospinal fluid. The pathologic changes they undergo in Parkinson's disease and other disorders are to be described elsewhere.

Distribution of GABA-immunoreactive neurons in the basal ganglia of the squirrel monkey (Saimiri sciureus).

The distribution of GABA-immunoreactive neurons was visualized in the basal ganglia of the squirrel monkey (Saimiri sciureus), by using a highly specific antiserum raised against GABA-glutaraldehyde-lysyl-protein conjugate and revealed by the indirect peroxidase-antiperoxidase immunohistochemical method. In the dorsal striatum, GABA-immunoreactive nerve cell bodies were small to medium in size (sectional area ranging from 90 to 125 microns2), but some larger ones (500-600 microns2) were also found. These cells displayed no obvious clustering but were significantly more numerous in the caudate nucleus than in the putamen; their number was also markedly greater at caudal than at rostral striatal levels. A moderate number of evenly distributed positive axon terminals were visible in both the caudate nucleus and the putamen. In the ventral striatum, GABA-immunoreactive nerve cell bodies and axon terminals were seen in fair number within the nucleus accumbens and in the deep layers of the olfactory tubercle. Many positive terminals but no somata were found in the islands of Calleja. In the globus pallidus, virtually all nerve cell bodies were GABA-immunoreactive and the neuropil exhibited a multitude of positive terminals. In the substantia innominata, clusters of small, globular GABA-immunoreactive somata were scattered among aggregates of larger, nonimmunoreactive neurons belonging to the nucleus basalis, and the whole region showed a low to moderate number of evenly spread GABA-positive terminals. In the subthalamic nucleus, nerve cell bodies were generally surrounded by several GABA-positive terminals but were not themselves immunoreactive. The substantia nigra showed many GABA-immunoreactive somata, which predominated in the pars lateralis and diminished progressively in number along the lateromedial axis of the pars reticulata. These cells formed a rather pleomorphic group comprising round, fusiform, or polygonal elements of relatively large size (sectional area ranging from 200 to 800 microns2). In the pars compacta and ventral tegmental area, a few GABA-immunoreactive neurons of small size were dispersed among larger, unreactive neurons. In both pars lateralis and pars reticulata of the substantia nigra, the number of GABA-positive terminals was high and their distribution was rather uniform; a smaller number were visible in the pars compacta of the substantia nigra and in the ventral tegmental area. The present results demonstrate that GABA-containing neurons are widely and heterogeneously distributed in the various components of the squirrel monkey's basal ganglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain.

Neurotransmitter-related messenger RNAs were detected by in situ hybridization in sections of rat and mouse brains by using 35S-radiolabelled RNA probes transcribed from cDNAs cloned in SP6 promoter-containing vectors. The distribution of messenger RNAs for glutamic acid decarboxylase, tachykinins (substance P and K), and tyrosine hydroxylase was examined in the striatum, pallidum, and substantia nigra. Dense clusters of silver grains were observed with the RNA probe complementary of the cellular messenger RNA for glutamic acid decarboxylase (antisense RNA) over most large neurons in the substantia nigra pars reticulata and medium-sized to large neurons in all pallidal subdivisions. A few very densely and numerous lightly labelled medium-sized neurons were present in the striatum. Among the areas examined, only the striatum contained neurons labelled with the antisense tachykinin RNA. Most of these neurons were of medium size, and a few were large. With the antisense tyrosine hydroxylase RNA, silver grains were found over neurons of the substantia nigra pars compacta and adjacent A10 and A8 dopaminergic cell groups. No signal was observed with RNAs identical to the cellular messenger RNA for glutamic acid decarboxylase or tachykinin (sense RNA). These results show a good correlation with immunohistochemical studies, suggesting that documented differences in the distribution and the level of glutamic acid decarboxylase, tyrosine hydroxylase, and substance P immunoreactivities in neurons of the basal ganglia are related to differences in the level of expression of the corresponding genes rather than to translation accessibility, stability, or transport of the gene products.

Substance P in human cerebrospinal fluid: reductions in peripheral neuropathy and autonomic dysfunction.

Substance P (SP), a putative peptide neurotransmitter, was measured in human lumbar cerebrospinal fluid (CSF) by radioimmunoassay. Substance P-like immunoreactivity (SPLI) was present in the CSF of 18 neurologically normal adults in concentrations ranging from 2.9 to 11.1 fmol per milliliter, with a mean of 7.0 /+- 0.6 fmol per milliliter (mean /+- SE). Slightly more than half of the CSF-SPLI cochromatographed with synthetic SP on Sephadex G-25. There was no apparent gradient in CSF-SPLI concentration over the first 30 ml of CSF removed by lumbar puncture. Mean concentrations CSF-SPLI in patients with Huntington disease, parkinsonism, miscellaneous dyskinesias, progressive supranuclear palsy, myopathy, and amyotrophic lateral sclerosis did not differ significantly from normal. Patients with neuropathy or multiple-system atrophy (Shy-Drager syndrome) had significantly reduced mean CSF-SPLI concentrations. These observations suggest that lumbar CSF-SPLI arises largely from spinal cord, nerve roots, or dorsal root ganglia, and that pathologic processes affecting these structures may be reflected by reduced levels of CSF-SPLI.

Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains.

We detected large numbers of HLA-DR-positive reactive microglia (macrophages), along with Lewy bodies and free melanin, in the substantia nigra of all cases studied with Parkinson's disease (5) and parkinsonism with dementia (PD) (5). We found similar, but less extensive, pathology in the substantia nigra of six of nine cases of dementia of the Alzheimer type (DAT) but in only one of 11 age-matched nonneurologic cases. All dementia cases with a premortem diagnosis of DAT or PD showed large numbers of HLA-DR-positive reactive microglia and significant plaque and tangle counts in the hippocampus, as well as reduced cortical choline acetyltransferase activity. One of 11 nondemented controls showed mild evidence of similar cortical pathology. These data indicate that HLA-DR-positive reactive microglia are a sensitive index of neuropathologic activity. They suggest a frequent coexistence of DAT- and Parkinson-type pathology in elderly patients.

Brain amino compounds in a Huntington's disease patient on isoniazid therapy.

We describe biochemical abnormalities in autopsied brain of a patient with early Huntington's disease (HD) who died of pentobarbital overdosage while under treatment with isoniazid (INH). The brain contained hydrazine, a terminal metabolite of INH, which inhibits gamma-aminobutyric acid (GABA) aminotransferase. GABA content in the basal ganglia was higher than expected for HD, and GABA content was supranormal in some brain regions. Homocarnosine (GABA-histidine) content was greatly elevated in all brain regions, suggesting chronic GABA elevation in life. Therefore, the increase in brain GABA content observed in experimental animals given INH or hydrazine also occurs in human patients.

Regional distribution of ubiquinones and tocopherols in the mouse brain: lowest content of ubiquinols in the substantia nigra.

C57 black mice of 3 months of age were sacrificed, and their brain regionally dissected according to a protocol that strickly control for the death-freezing interval of each region. HPLC measurements of tocopherols and oxidized and reduced ubiquinones demonstrated significant regional variations. The substantia nigra had the lowest content of Q10 and a skewed ratio in favor of its oxidized form. Forebrain cholinergic nuclei had also more oxydized than reduced Q10 and in addition the lowest content of tocopherols. These findings suggest that nuclei that show neuronal depletion with age are the ones prone to oxidative stress.

Striato-nigral denervation increases type II benzodiazepine receptors in the substantia nigra of the rat.

The degeneration of the striato-nigral projection induced by the injection of kainic acid into the striatum produced a 30% increase in the density of type II benzodiazepine binding sites (measured as the proportion of [3H]flunitrazepam which remained after the addition of 2 X 10(-7) M CL 218872). The lesion did not change the number of type I benzodiazepine binding sites (measured using [3H]ethyl-beta-carboline-3-carboxylate). The increase of type II benzodiazepine binding sites persisted and was markedly enhanced in the substantia nigra, previously lesioned with kainic acid. In fact, the injection of kainic acid into the nigra caused, 3 weeks after the treatment, a 80% decrease in the total number of type I benzodiazepine binding sites, and no change in the number of type II benzodiazepine binding sites. The density of type II sites increased by 70% following a subsequent injection of kainic acid into the striatum, homolateral to the lesioned substantia nigra. The results suggest that type I benzodiazepine binding sites in the nigra are located on kainic acid-sensitive elements (probably intrinsic neurones), while type II benzodiazepine binding sites, the number of which increased after degeneration of the striato-nigral pathway, are localized on kainic acid-resistant structures (probably axons or terminals) that receive an input from striatal afferents and from interneurones in the nigra.

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal and limbic catecholamine neurones in white and black mice. Antagonism by monoamine oxidase inhibitors.

Albino mice and pigmented mice were treated for 6 days with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at the maximum tolerated doses (2 days at 30 mg/kg i.p., 2 days at 40 mg/kg i.p. and 2 days at 50 mg/kg i.p. in white mice, 6 days at 30 mg/kg i.p. in pigmented mice) and the effects of simultaneous treatment with the monoamine oxidase inhibitors, deprenyl (1 mg/kg, i.p.), MDL 72145 (0.5 mg/kg, i.p.) and clorgyline (5 mg/kg, i.p.), determined behaviourally (daily for 6 days and for 4 days after withdrawal) and biochemically (92 hr after withdrawal of drug). In albino mice MPTP caused depletions of dopamine (90%), dihydroxyphenylacetic acid (DOPAC; 82%) and homovanillic acid (HVA; 65%) in the striatum and in dopamine (54%), DOPAC (51%) and HVA (53%) in the nigra. However, MPTP was not selective in its action since the levels of dopamine and its metabolites were also reduced in limbic tissue. Further, MPTP affected the function of noradrenaline, with reduced levels in tissues of the striatum (74%) and nigra (46%). Pigmented mice were as susceptible as albino mice to the actions of MPTP to reduce the levels of dopamine and metabolites in the striatum. However, the limbic areas and substantia nigra of the pigmented mouse were more resistant to the actions of MPTP. Treatment with deprenyl and MDL 72145 (but not clorgyline) could be shown to reduce the biochemical and behavioural consequences of the action of MPTP (although behavioural changes, development of severe motor incapacitation and prostrate appearance, appeared to be non-specific).(ABSTRACT TRUNCATED AT 250 WORDS)

Astroglial response in the excitotoxically lesioned neostriatum and its projection areas in the rat.

The anatomical distribution of the astrocytic glial reactions, following ibotenic acid-induced neuronal degeneration of the neostriatum in the rat, has been studied immunohistochemically using an antibody directed against the astrocytic marker, glial fibrillary acidic protein. The acute astroglial response to the excitotoxic lesion, determined 7 days post lesion, was compared with a sham-operated group and a chronic group that had received the excitotoxic lesion 6 months prior to histological evaluation. Total doses of 16-20 micrograms ibotenic acid injected unilaterally into the head of the neostriatum caused not only a marked neuronal cell loss but was also accompanied by a large increase in the number and size (about 5 times) of glial fibrillary acidic protein-stained astrocytes throughout the neostriatum by 7 days after lesion. Reactive astrocytes were also observed in the major neostriatal projection areas, the globus pallidus and the substantia nigra pars reticulata, at 7 days post lesion, although no neuronal cell loss could be detected in these regions using regular Cresyl Violet staining. Previous studies of lesions identical to the ones used here have shown that globus pallidus and substantia nigra are deafferented as a result of the neostriatal neuronal degeneration. The reactive astrocytes in the striatal projection areas had a 3-5 times larger size than control astrocytes from the same anatomical region. In animals that received a larger dose of ibotenic acid into the neostriatum (25 micrograms), neuronal cell loss was also observed in the neocortex and reactive glial fibrillary acidic-stained astrocytes were found in the entire neocortex of the injected hemisphere. In the chronic group, 6 months after the excitotoxic lesion, the astroglial response was clearly diminished or absent in the major neostriatal projection areas, but was still present within the lesioned neostriatum. The results suggest that focal neuronal destruction can result in widespread astrocytic glial reactions which follow the anatomical connectivity of the lesioned area. This may have implications for the understanding of the multifocal distribution of glial reactions seen in patients with striatal degeneration as a result of Huntington's disease.

The intragranular location of carboxyl groups in neuromelanin and lipofuscin in human brain and in meningeal melanosomes in mouse brain.

The intragranular location of carboxyl groups was tinctorially determined in human substantia nigra neuromelanin granules, human inferior olive lipofuscin granules, and mouse meningeal melanosomes. Soluble and insoluble lipid was stained with beta naphthol Sudans in unoxidized and oxidized frozen and paraffin sections containing neuromelanin or lipofuscin. Nile blue demonstrated carboxyls in unoxidized neuromelanin, lipofuscin, and melanin, and in oxidized neuromelanin and lipofuscin. Carbodiimide demonstrated carboxyls in unoxidized and oxidized lipofuscin and oxidized neuromelanin. In all instances, staining for carboxyls was inhibited by prior mild methylation, and proof of their presence was obtained by a pre-staining, stepwise, alternating, and repetitive mild demethylation, mild methylation sequence. Structurally, carboxyls were demonstrated in the neuromelanin granule's soluble lipid-free lipofuscin component, in the meningeal melanosome's melanin component, and virtually throughout the lipofuscin granule. The following structural and chemical basis was proposed for the different resistance of Nile blue staining of melanosomes and of neuromelanin and lipofuscin to acetone extraction. Nile blue forms an insoluble complex with melanosomal dopa-melanin's quinonoid, diphenolic, and undissociated carboxyl units. Such complex formation does not occur in neuromelanin's carboxyl-free dopamine-melanin component, however. Instead, Nile blue ionogenicly bonds with dissociated carboxyls belonging to the neuromelanin granule's lipofuscin component.

Ultrastructural differentiation within the central nervous system between indolamines and catecholamines by energy dispersive X-ray analysis following paraformaldehyde pretreatment.

Dense bodies containing high amounts of chrome were localized in the perikarya of substantia nigra and dorsal raphe neurons following the cytochemical reaction of endogenous dopamine and serotonin (respectively) with glutaraldehyde-dichromate (GDC). Energy dispersive X-ray analysis of these bodies revealed chrome levels two to four times higher than those recorded from the cytoplasmic background. Pretreatment with paraformaldehyde blocked the GDC reaction within the dense bodies in the substantia nigra (chrome levels similar to background), while the chrome levels in the dense bodies of the raphe neurons remained elevated. This demonstrates that pretreatment with paraformaldehyde allows selective localization of central nervous system serotonin stores by the GDC technique.

Localization of tissue thromboplastin in the human brain.

Monospecific antisera against the purified protein component of tissue thromboplastin (apoprotein-III) from human brain have been raised in goats and rabbits. The antisera neutralized tissue thromboplastin prepared from brain, thyroid gland and pulmonary tissue, indicating that apoproteins in the various preparations cross-reacted immunologically and therefore were similar or identical. Comparison of the activities of tissue thromboplastin preparations from 34 different areas of the brain demonstrated a characteristic distribution pattern and a wide range of activities. White and grey matter from the same areas had similar activities. Bulbus and tractus olfactorius, medulla oblongata, corpus pineale, hippocampus and hypothalamus contained 160-270% of the average activity, whereas cerebellum globus pallidus, nucleus ruber and substantia nigra contained 30-60%. The distinct distribution pattern was unrelated to tissue vascularization, and may suggest that apoprotein-III could serve other functions, apart from the coagulation of blood. The predominance in phylogenetically older brain regions would suggest that it represents a primitive or fundamental feature.

Localization of ARPP-90, a major 90 kiloDalton basal ganglion-enriched substrate for cyclic AMP-dependent protein kinase, in striatonigral neurons in the rat brain.

Cyclic AMP-regulated phosphoproteins with specific cellular localizations in brain represent important targets through which this second messenger system can mediate or modulate distinct neurotransmitter signals. This study reports that two cyclic AMP-regulated phosphoproteins (Mr 90,000 and 93,000) found in brain share several properties, including similar isoelectric points and similar phosphopeptide maps. This protein doublet is particularly enriched in the forebrain basal ganglia, but it can also be found in the substantia nigra, a brainstem region which is a major target for fibers from the forebrain basal ganglia. Quinolinic acid lesions of neurons in the neostriatum decrease the levels of the 90/93 kDa phosphoprotein doublet to about the same extent as they reduce the levels of DARPP-32, a phosphoprotein specifically enriched in striatonigral medium-sized spiny neurons. These reductions are seen in both the neostriatum and the substantia nigra. Therefore, within the basal ganglia, the 90/93 kDa phosphoprotein doublet, termed adenosine 3':5'-monophosphate-regulated phosphoprotein, Mr = 90,000 (ARPP-90), is largely, if not solely, present in striatonigral cells and fibers. The specific localization in these neurons suggests that ARPP-90 could be important in receptor-regulated, cyclic AMP-mediated functions in the striatonigral neurons.

An immunohistochemical demonstration of gastrin-releasing peptide (GRP) in the rat substantia nigra.

The presence of gastrin-releasing peptide-like immunoreactivity in the rat brain was investigated by use of the indirect peroxidase-antiperoxidase technique. A high density of gastrin-releasing peptide-like immunoreactive terminals in the ventral pallidum, the interpenduncular nucleus and in substantia nigra, pars reticulata, was observed. Moreover, gastrin-releasing peptide-like immunoreactive perikarya were observed in the hypothalamic suprachiasmatic nucleus. Antisera raised against gastrin-releasing peptide have been shown to cross-react with substance P, another peptide highly concentrated in the substantia nigra, the ventral pallidum and the interpenduncular nucleus, and gastrin-releasing peptide-immunoreactivity in these areas has therefore been regarded as substance P immunoreactivity. To determine the antigenic epitope recognized by the antiserum raised against gastrin-releasing peptide, specificity studies were performed with known peptides fixed to nitrocellulose filter strips as well as preabsorptions with the same peptides on fixed brain sections containing the substantia nigra. From these experiments, it could be deduced that the antiserum recognizes an epitope within the peptide sequence: Val-Gly-His-Leu-Met-NH2. The antiserum cross-reacts with bombesin and alytesin, but not with substance P, allowing us to conclude that gastrin-releasing peptide or a very closely related peptide is present in areas of the rat central nervous system in which substance P has previously also been shown to be present.

Steady state levels of pro-dynorphin-related end products in the striatum and substantia nigra of the adult rhesus monkey.

Analysis of an acid extract of the striatum of the rhesus monkey revealed that the molar ratio of dynorphin A(1-8)-sized material and dynorphin (A(1-17)-sized material is approximately 1:1. In addition, the molar ratios of the dynorphin A-related end products to both dynorphin B(1-13)-sized material and alpha-neo-endorphin-sized material were approximately 1:1. Fractionation of an acid extract of the substantia nigra by gel filtration and reverse phase HPLC revealed the following molar ratios for pro-dynorphin-related end products. The molar ratio of dynorphin A(1-8) to dynorphin A(1-17) is approximately 6:1. The molar ratios of dynorphin A-related end products to dynorphin B(1-13) and alpha-neo-endorphin were approximately 0.5 and 0.8, respectively. Comparisons between proteolytic processing patterns of pro-dynorphin in the striatum and the substantia nigra of the rhesus monkey are considered. In addition, comparisons between pro-dynorphin processing in the substantia nigra of the rhesus monkey and the substantia nigra of the rat are discussed.