Evaluation of the Biological Activity of Several Analogs of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Several analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were synthesized and screened for their capacity to be oxidized by monoamine oxidase (MAO-A or MAO-B) and their capacity to produce nigrostriatal dopaminergic neurotoxicity in mice. All of the compounds were relatively weak substrates for MAO-A but many of the compounds were found to be good substrates for MAO-B. Only three of the compounds, in addition to MPTP itself, were found to be neurotoxic. These were 1-methyl-4-cyclohexyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine and 1-methyl-4-(3'-methoxyphenyl)-1,2,3,6-tetrahydropyridine. All three of these neurotoxic compounds were found to be substrates for MAO-B; in contrast no compound was found to be neurotoxic that was not oxidized by MAO-B. The capacity of the compounds studied to be oxidized by MAO-B appears to be an important aspect of the neurotoxic process.     

Failure of Glucose and Branched-Chain Amino Acids to Normalize Brain Glucose Use in Portacaval Shunted Rats

Several abnormalities in brain and plasma amino acid concentrations caused by portacaval shunting in rats return toward normal after 4 days of intravenous infusion with either glucose or glucose with branched-chain amino acids. To assess the effect of such treatment on brain energy metabolism, regional brain glucose use was measured using [14C]glucose and autoradiography, 5 weeks after portacaval shunting. In one experiment intravenous glucose or glucose with branched-chain amino acids was given for 4 days. In a separate experiment the treatment was given orally for 2 weeks, and in addition to glucose use, brain monoamines and amino acids were measured. No other food was provided; the rats had free access to water. Normally fed shunted rats and sham-operated rats served as controls. Both types of oral treatment lowered the high concentrations of tyrosine, phenylalanine, and glutamine in plasma and brain. Glucose without amino acids normalized brain tryptophan. Levels of brain norepinephrine, 5-hydroxytryptamine (serotonin), and 5-hydroxyindoleacetic acid were significantly raised after shunting. Treatment had no effect on norepinephrine but the glucose diet brought the indoles into the normal range. In contrast, neither intravenous nor oral treatment affected brain glucose use, which remained depressed by 25-30% in all brain areas examined.     

The distribution of monoamine oxidase and acetylcholinesterase in the brain of Xenopus laevis tadpoles

Summary The distribution of monoamine oxidase (MAO) in the brain of Xenopus laevis tadpoles (stage 52–56) was studied histochemically with a modified Glenner's tryptamine-tetrazolium method. A moderate activity was observed in fibre regions of the striatum and septum (including the medial and lateral forebrain bundles), in the neuropil of the nucleus amygdalae, in the commissura anterior and commissura hippocampi, in the fibre regions of the diencephalon (including the optic chiasma), in the fibre regions of the tectum opticum and the tegmentum of the mesencephalon and in the white substance of the ventral half of the medulla oblongata. A greater MAO activity was found in the neuropil of the entire nucleus praeopticus. In the partes anterior and magnocellularis of this nucleus, MAO positive fibres are present in close contact with the perikarya, indicating a monoaminergic innervation of these neurons. The perikarya themselves did not show MAO activity. In the neurons of the nucleus praeopticus epichiasmaticus, the paraventricular organ (PVO) and nucleus infundibularis dorsalis (NID), only a slight MAO activity has been demonstrated in the perikarya, whereas a strong MAO positivity was found in the intraventricular protrusions and the neuropil. These data indicate the aminergic character of the neurons of these nuclei. From the postoptic fibre region a MAO positive tract was observed towards the developing median eminence and pars intermedia of the hypophysis. The pars nervosa and some cells of the pars distalis also contained MAO. Along the border of the aquaeduct of Silvius and the fourth ventricle, MAO positive liquor-containing neurons are also present.The distribution of acetylcholinesterase (AChE) was investigated in the hypothalamohypophysial region. AChE activity was found in the neuropil of the nucleus praeopticus magnocellularis, in the fibres of the optic chiasma and in the postoptic fibre region. The neurons of the PVO and NID were AChE negative. An AChE positive tract could be traced from the postoptic fibre region to the developing median eminence and pars nervosa. The pars distalis did not show AChE activity. However, in tadpoles reaching the metamorphic climax, ChE activity appeared in certain cells of the pars distalis; this might be related to degenerative phenomena in the acidophilic cells. The absence of AChE activity in the pars intermedia indicates a regulation of MSH release by peptidergic nerves to be unlikely.The stimulating interest and helpful advice of Prof. Dr. P. G. W. J. van Oordt is gratefully acknowledged. Thanks are also due to Mr. H. van Kooten and his co-workers for making the photographs.     

Monoamine oxidase in the hypothalamo-hypophysial region of the teleosts,Anguilla japonica and Oryzias latipes

Summary Distribution of monoamine oxidase (MAO) was histochemically examined in the hypothalamo-hypophysial region of the eel (Anguilla japonica) and the medaka (Oryzias latipes) with a modified Glenner's tryptamine-tetrazolium method. The hypothalamic neurosecretory cells showed very weak MAO activity in their perikarya. MAO-positive fibers were present in close contact with the neurosecretory cells, suggesting that monoaminergic fibers participate in the control of neurosecretory cell activity. The nucleus lateralis tuberis (NLT) contained cells exhibiting strong MAO activity. These cells must be monoaminergic neurons.In the anterior region of the neurohypophysis of both eel and medaka, two bundles of MAO-positive fibers originating from the NLT proceed down along each side of the third ventricle into the pars distalis. This suggests that monoaminergic neurons of the NLT are involved in the release of hormones from the pars distalis. In addition to these tracts, numerous MAO-positive fibers proceed backward from the post-optic area and end around the blood capillaries located between the neurohypophysis and the pars intermedia in both species.I wish to express my gratitude to Prof. H. Kobayashi for his valuable advice during the course of this study. I am indebted to Prof. S. Uchida, Ocean Research Institute, University of Tokyo, for supplying the eels.     

Monoaminhaltige Strukturen im Zentralnervensystem der Trichoptera (Insecta) Teil II

Zusammenfassung Mit Hilfe der fluoreszenzmikroskopischen Methode nach Falck und Hillarp wurden die monoaminhaltigen Strukturen im Zentralnervensystem einiger Trichopteren untersucht (vgl. Klemm, 1968). Im Protocerebrum können vier Gruppen von katecholamin-haltigen Zellkörpern unterschieden werden. Eine weitere unregelmäßig darstellbare Gruppe von globulösen Perikarya liegt im Lobus opticus. Fluoreszierende Varikositäten durchsetzen locker das Cerebralganglion und sind in folgenden Neuropilstrukturen konzentriert: Medulla, Lobula, Corpus centrale, Nodulus, Corpus ventrale, - und -Lobus und Lobus communis. Letzterer verbindet den - und den -Lobus mit dem nicht fluoreszierenden Pedunculus. In der Lamina, in der accessorischen Medulla, in der Pons cerebralis, im Tuberculum opticum, im Tractus olfactorio-globularis, im Pedunculus, im Stielglomerulus und in den Globulizellen ließen sich keine Katecholamine nachweisen. Zwischen dem monoaminhaltigen Lobus communis und dem nicht fluoreszierenden Pedunculus besteht eine scharfe Grenze. Fluoreszierende variköse Fasern und einzelne fluoreszierende Perikarya befinden sich im Deuto- und Tritocerebrum. Zwei fluoreszierende Bahnen können im Cerebralganglion unterschieden werden: 1. Stratum caudale, 2. Tractus ventralis. Letzterer beginnt mit seiner Pars anterior und posterior im dorsalen fluoreszierenden Neuropil des Protocerebrum. Die beiden Teile laufen frontal und caudal um den Zentralkörper herum und vereinigen sich unterhalb des Zentralkörpers in der Pars communis. Von hier aus zieht der paarige Tractus ventralis bis in das Tritocerebrum. Die Frage einer Homologisierung dieser Bahnen wird diskutiert.Ähnlich wie im Ganglion suboesophageale sind in den drei thorakalen und fünf separaten abdominalen Ganglien zwei Paar fluoreszierender Zellkörper vorhanden, in denen Dopamin mikrospektrofluorimetrisch festgestellt werden konnte. Ein Paar dieser Zellen liegt caudoventral, das andere dorso-median bis dorso-caudal. Ihre Zellfortsätze und Abzweigungen werden beschrieben. In einzelnen Fällen konnte noch ein zweites dorsales Paar fluoreszierender Perikarya sichtbar gemacht werden. Das letzte Ganglion der Bauchkette setzt sich aus zwei bis drei Gangliomeren zusammen. Die Anzahl ihrer fluoreszierenden Perikarya ist reduziert. Das Neuropil der Ganglien im Bauchmark ist von monoaminhaltigen Fasern durchsetzt, wobei sich in der dorsalen Hälfte mehr fluoreszierendes Neuropil befindet als in der ventralen. Lateral in den Ganglien sind die monoaminhaltigen varikösen Fasern vorwiegend dorsoventrad angeordnet. Im medianen Teil laufen sie in Längsrichtung, verzweigen sich und setzen sich in die Konnektive fort und verbinden die katecholaminhaltigen Neuropilbereiche der einzelnen Ganglien miteinander.

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Monoamine-containing structures in the central nervous system in Trichoptera (Insecta). Part II

Summary The distribution of monoamine-containing structures in the central nervous system of Trichoptera has been investigated with the histochemical fluorescence method of Falck and Hillarp (cf. also Klemm, 1968). In the protocerebrum, four groups of catecholamine containing perikarya can be distinguished. Another group of irregularly appearing cell bodies is situated in the optical lobe. Fluorescent varicosities occur scattered throughout the cerebral ganglion, being concentrated in the following neuropile areas: In the medulla, the lobula, the corpus centrale, the noduli, the corpora ventralia, and the - and -lobes, as well as in the lobus communis where the - and -lobes join the non-fluorescent pedunculus. In the lamina, the accessory medulla, the pons cerebralis, the tuberculum opticum, the tractus olfactorio-globularis, the pedunculus including its glomerular region, and the globuli-cells, no catecholamines are detectable. There is a sharp borderline between the monoamine-containing lobus communis and the non-fluorescent pedunculus. Fluorescent varicose fibres and single fluorescent perikarya occur in the Deuto- and Tritocerebrum. Two fluorescent tracts can be distinguished in the cerebral ganglion: 1. Stratum caudale. 2. Tractus ventralis. The pars anterior and pars posterior of the tractus ventralis begin in the dorsal fluorescent neuropile of the cerebral ganglion and join underneath the central body into their pars communis. From this, it can be traced as tractus ventralis into the tritocerebrum. The possible homologies of these tracts are discussed. Similar to the ganglion suboesophageale, the three thoracal and the five separate abdominal ganglia contain two paires of fluorescent cell bodies. Microspectrofluorometrically a content of dopamine in these cells could be established. One pair lies caudoventrally. The two other cell bodies are situated dorso-medially to dorso-caudally; their position varies notably, especially in the abdominal ganglia. The cell process and its arborisations are described. Occasionally a second dorsal cellpair could be observed. The last ganglion of the abdominal chain is composed of at least two gangliomeres with a reduced number of fluorescent perikarya. The neuropile of the thoracal- and abdominal ganglia is penetrated by monoamine-containing fibres, with a predominance in the dorsal half. In the ganglia, the fluorescent varicose fibres are mainly oriented dorso-ventrally in the lateral part and longitudinally in the medial part, where they branch and continue into the connectives. In this way, the catecholamine-containing neuropiles of adjacent ganglia are connected to each other.

Die vorliegende Arbeit wurde unterstützt durch The Swedish Medical Research Council (No. B70-14X-56-06 und B70-14X-05).     

Comparison of the Properties of Semipurified Mitochondrial and Cytosolic Monoamine Oxidases from Rat Brain

Mitochondrial and cytosolic monoamine oxidases were purified 220- and 129-fold, respectively, from rat brain. The purification procedure involved extraction (without the use of detergents for mitochondrial monoamine oxidase), ammonium sulfate precipitation, and chromatography on Sephadex G-25 and a DEAE-cellulose column. The properties of both enzymes with kynuramine as substrate, including Km values and pH optima at different kynuramine concentrations; the Rf values on polyacrylamide gel electrophoresis; and the thermal inactivation patterns were different. 2-Mercaptoethanol, together with heat treatment, released the flavin and decreased the enzyme activity differentially for the two enzymes. The absorption spectrum showed a "Red shift" in the absorption maxima when the spectra of the non-Triton-treated purified preparations were compared with those of the Triton-treated ones, thus possibly revealing that the mitochondrial and the cytosolic monoamine oxidases may be two different enzyme entities.     

The Novel Neuropsychotropic Agent Milacemide Is a Specific Enzyme-Activated Inhibitor of Brain Monoamine Oxidase B

The novel neuropsychotropic agent milacemide hydrochloride (2-n-pentylaminoacetamide HCl) is a highly selective substrate of the B form of monoamine oxidase (EC 1.4.3.4; MAO). Under the in vitro conditions used in the present study, milacemide acts as an enzyme-activated, partially reversible inhibitor of MAO-B. A reversible inhibition of MAO-A activity is also observed at high concentrations. The inhibitory activity of milacemide is significantly greater for MAO-B. In vivo, after single or repeated oral administration, a specific inhibition of MAO-B is apparent in brain and liver, with a lack of inhibition of the MAO-A activity. In contrast to the irreversible inhibitory action of L-deprenyl, the recovery of MAO-B activity in vivo after milacemide administration is significantly faster, a result suggesting that it is a partially reversible inhibitor. The selective inhibitory effect of milacemide for MAO-B in vivo is confirmed by its potentiation of phenylethylamine-induced stereotyped behavior, whereas vasopressor responses to tyramine were not affected. These observations suggest that milacemide could enhance dopaminergic activity in the brain and could be used as therapy for Parkinson's disease in association with L-3,4-dihydroxyphenylalanine.     

4-[18F]Fluoro-L-m-Tyrosine: An L-3,4-Dihydroxyphenylalanine Analog for Probing Presynaptic Dopaminergic Function with Positron Emission Tomography

4-[18F]Fluoro-L-m-tyrosine (FMT), a biochemical probe of striatal dopaminergic function, has been synthesized as an L-3,4-dihydroxyphenylalanine analog for positron emission tomography. Biochemical characterization of this compound in the rat 30 min after intrajugular administration indicated that in the brain, selective decarboxylation occurred in the striatum, with the formation of 4-fluoro-3-hydroxyphenylethylamine and its metabolites. Positron emission tomography analysis of brain tissue in monkeys (Macaca nemestrina) after intravenous injection of FMT revealed a true time-dependent, specific accumulation of radioactivity in striatum, with a striatum/cerebellum (nonspecific) ratio of 4 at 180 min. Peripheral metabolism accounted for less than 40% of the total radioactivity in arterial blood samples after 120 min. The amino acid remained as the major component throughout the period of investigation (n = 3; 5 min, 95%; 10 min, 85%; 30 min, 67%; 60 min, 62%; 120 min, 60%), with a plasma clearance t 1/2 of 112 min. 3-O-Methylated metabolites were not observed. The substrate specificity of FMT, coupled with its limited in vivo peripheral metabolism, makes it a useful, new biochemical probe for in vivo, noninvasive evaluation of central dopaminergic mechanisms.     

Bovine Brain Endothelial Cells Express Tight Junctions and Monoamine Oxidase Activity in Long-Term Culture

The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20-fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial-specific characteristics (factor VIII-related antigen, angiotensin-converting enzyme, and nonthrombogenic surface) is shown for brain capillary-derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood-brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity.