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PubMed1977

Transmitter histochemistry of the rat olfactory bulb. I. Immunohistochemical localization of monoamine synthesizing enzymes. Support for intrabulbar, periglomerular dopamine neurons.

The rat olfactory bulb was studied at the light and electron microscopic level with the indirect immunofluorescence technique and the unlabelled antibody enzyme method (PAP-technique), respectively. Antibodies to all 4 enzymes in the catecholamine synthesis were used. In the principal bulb the first two enzymes, tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC), but not dopamine-beta-hydroxylase (DBH), were present in a proportion of periglomerular cell bodies and dendrites indicating that these neurons synthesize dopamine (DA). This amine may therefore be released as a transmitter substance at some of the intraglomerular dendrodendritic synapses which periglomerular cells form with the mitral cells. There is evidence to suggest that some periglomerular cells use GABA as their transmitter. Thus, a morphologically and physiologically homogenous population of neurons can be subdivided on the basis of transmitter histochemical criteria. There was an impression of more DDC-positive than TH-positive fibers in the glomeruli. Such presumably DDC-positive, but TH-negative processes may represent 5-hydroxytryptamine (5-HT) nerve terminals. DBH-positive fibers were seen in the granular, external plexiform, and very rarely, in the glomerular layers, probably representing noradrenaline (NA) nerve terminals ascending from the lower brain stem. Weakly fluorescent DDC-positive fibers may represent nerve terminals of ascending 5-HT neurons. No phenylethanolamine-N-methyltransferase (PNMT)-positive neurons were observed.

Animals↗
PubMed1990

Neuronal markers in rat retinal grafts.

Rat E15 retina was grafted to the retina of adult rat hosts. After varying survival times (1 week-6 months), grafts were stained by immunohistochemistry for neurofilament 160 kDa (NF), HPC-1 (an amacrine cell marker), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and somatostatin-28 (SS-28). The first differentiating graft amacrine cells (cholinergic and dopaminergic) could be seen 1 week after transplantation (corresponding to postnatal day 1 = P1). The inner plexiform layer of the graft started to differentiate at 2 weeks (corresponding to P8) seen by HPC-1 and GAD staining. ChAT, TH and SS-28 immunostaining revealed an abnormal lamination pattern in the graft inner plexiform layer. Also by 2 weeks, the outer plexiform layers of the graft contained NF-immunoreactive horizontal cells. No NF-stained retinal ganglion cells could be observed in the graft. Five and 7 weeks after grafting, the transplants had obtained the same staining intensity with different markers as the host retina.

Animals↗
PubMed1999

Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns.

BACKGROUND: Animal melanin patterns are involved in diverse aspects of their ecology, from thermoregulation to mimicry. Many theoretical models have simulated pigment patterning, but little is known about the developmental mechanisms of color pattern formation. In Drosophila melanogaster, several genes are known to be necessary for cuticular melanization, but the involvement of these genes in melanin pattern evolution is unknown. We have taken a genetic approach to elucidate the developmental mechanisms underlying melanin pattern formation in various drosophilids. RESULTS: We show that, in D. melanogaster, tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are required for melanin synthesis. Ectopic expression of TH, but not DDC, alone was sufficient to cause ectopic melanin patterns in the wing. Thus, changes in the level of expression of a single gene can result in a new level of melanization. The ontogeny of this ectopic melanization resembled that found in Drosophila species bearing wing melanin patterns and in D. melanogaster ebony mutants. Importantly, we discovered that in D. melanogaster and three other Drosophila species these wing melanin patterns are dependent upon and shaped by the circulation patterns of hemolymph in the wing veins. CONCLUSIONS: Complex wing melanin patterns are determined by two distinct developmental mechanisms. Spatial prepatterns of enzymatic activity are established late in wing development. Then, in newly eclosed adults, melanin precursors gradually diffuse out from wing veins and are oxidized into dark brown or black melanin. Both the prepatterning and hemolymph-supplied components of this system can change during evolution to produce color pattern diversity.

Animals↗
PubMed1989

Cell specific enzyme markers as indicators of neurotoxicity: effects of acute exposure to methylmercury.

In order to assess the sensitivity of several cell specific enzyme markers (tyrosine hydroxylase (TH), glutamic acid decarboxylase, choline acetyltransferase, glutamine synthetase (GS), neuron specific and non-neuronal enolase and 2',3'-cyclic nucleotide phosphohydrolase (CNP] as indices of neurotoxicity, changes in their activities were monitored after rats were treated with two doses of the neurotoxic agent, methylmercury chloride (MMC). Comparisons were also made of any histopathological changes occurring in the tissues examined. At the low dose rate (3.36 mg Hg/kg, po, for 14 days), the rats exhibited less body weight gain compared to untreated animals. No change in either the neuronal or noneuronal enzyme markers was observed in brain but a significant increase in the myelin marker, CNP, and total enolase activity was seen in the optic nerve. Morphological evaluation by light microscopy indicated no discernible neuronal lesions in MMC-exposed animals. At the higher MMC dose (7.05 mg Hg/kg, po, for 7 days), there was about a 20% loss in the body weight of treated animals and partial hind limb paralysis was observed. Of all the neuronal marker enzymes examined, only TH was found to be decreased in the striatum. The proliferating astroglial marker, GS, was elevated only in the cerebellum. CNP was found to be decreased in both the optic and sciatic nerve. As in the lower dose group no pathological changes were observed at the light microscopic level in the brain of MMC-treated rats. These data suggest that of the cell specific marker enzymes studied, GS in the cerebellum and TH in the striatum may be useful biochemical markers for the neurotoxic action of MMC.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1997

Role of aromatic L-amino acid decarboxylase for dopamine replacement by genetically modified fibroblasts in a rat model of Parkinson's disease.

Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic L-amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic L-amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic L-amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. L-DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic L-amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic L-amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic L-amino acid decarboxylase produced less L-DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic L-amino acid decarboxylase near striatal grafts producing L-DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1990

Presence of dopamine-immunoreactive cell bodies in the catecholaminergic group A15 of the sheep brain.

Antisera were raised in rabbits against dopamine or noradrenaline conjugated to thyroglobulin with glutaraldehyde. These antisera, tested in enzyme linked immunosorbent assay and immunohistochemistry specifically recognized their homologous antigens. With the aid of anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase, anti-dopamine-beta-hydroxylase, anti-dopamine, and anti-noradrenaline antisera, immunohistochemical reactions were performed on glutaraldehyde fixed sections of sheep diencephalon in order to determine the presence of dopamine in the catecholaminergic group A15. Perikarya of this nucleus were stained with anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase and anti-dopamine, but not with anti-dopamine-beta-hydroxylase or anti-noradrenaline. Both of these latter antisera stained fibers within this area. So as recently found in the rat, we could conclude that dopamine is present in group A15 of the sheep.

Animals↗
PubMed1995

A probe for intracerebral aromatic amino-acid decarboxylase activity: distribution and kinetics of [18F]6-fluoro-L-m-tyrosine in the human brain.

Positron tomography, using [18F]6-fluoro-L-dopa as a tracer, has been used for the study of Parkinson's disease. Unfortunately, the analysis of data obtained with this agent is bedeviled because it readily forms labeled methylated metabolites that enter the brain. We have evaluated [18F]6-fluoro-L-m-tyrosine (FmT) as an alternative tracer to study intracerebral dopamine metabolism with positron tomography. Imaging studies in humans showed specific accumulation of this tracer in the dopamine-rich striatal regions. Reduced striatal uptake of the tracer was demonstrated in a patient suffering from Parkinson's disease. Increased retention of the tracer was demonstrated in a subject pretreated with the peripheral decarboxylase inhibitor carbidopa. Analysis of plasma samples for labeled metabolites of FmT revealed no methylated metabolites. Results of compartmental analysis showed that a two-compartment three rate constant model described adequately the time course of radioactivity in the striatum after an injection of FmT. The FmT decarboxylation rate constant (k21) was found to be 0.0108 min-1. Because the peripheral metabolism of FmT is simpler than that of [18F]6-fluoro-L-dopa, we propose FmT as a superior agent with which to study intracerebral dopamine metabolism in health and disease in humans.

Adolescent↗
PubMed1990

Gene expression of tyrosine hydroxylase in the developing fetal brain.

Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.

Actins↗
PubMedDate not supplied

[Ladasten induces the expression of genes regulating dopamine biosynthesis in various structures of rat brain].

The effect of ladasten (50 mg/kg) on the activity of tyrosine hydrolase (TH) and DOPA-decarboxylase (DDC) gene expression and on the content of dopamine and L-DOPA in the striatum and hypothalamus in rat brain was studied depending on the duration of drug action. In the initial stage (first hours) of the drug action, the dopaminergic effects are related to an increase in the dopamine release. The observed accumulation of L-DOPA and dopamine is correlated with the transcription activity of genes studied. This leads to a conclusion that the pharmacological activity of ladasten is related to activation of de novo synthesis of TH and DDC. There is a certain difference in the ladasten action upon the TH and DDC gene expression of the key enzymes in hypothalamus and striatum of rat brain.

Adamantane↗
PubMed1983

Subcellular fractionation of striatum: sedimentation properties of dopaminergic synaptosomes.

Linear sucrose density gradient centrifugation of a crude synaptosomal-mitochondrial preparation of rat striatum was performed at 82,500g for 7.5, 15 and 30 min and 1, 4 and 20 h. After centrifugation various marker enzyme activities were measured throughout the gradients, viz. tyrosine hydroxylase (TH) and DOPA decarboxylase (DD) as markers of dopaminergic synaptosomes, lactate dehydrogenase (LDH) as a general synaptosomal marker and monoamine oxidase (MAO) as a mitochondrial marker. At all centrifugation times the distribution patterns of TH and DD activity coincided almost perfectly. Notable differences were found between the sedimentation properties of these TH/DD-containing particles and LDH-containing particles: TH and DD were symmetrically distributed in the gradient much sooner than LDH, at all centrifugation times the top of the TH and DD curves was lying deeper in the gradient than the highest LDH activity, and TH and DD became enriched in the gradients to a much greater extent than LDH. It is concluded that rat striatal dopaminergic synaptosomes form a relatively homogeneous population of particles sedimenting faster into the gradients than the bulk of striatal synaptosomes does. This distinct sedimentation behaviour of the dopaminergic synaptosomes can be usefully applied for analytical purposes.

Animals↗
PubMed1999

Neurocircuitries of the basal ganglia studied in organotypic cultures: focus on tyrosine hydroxylase, nitric oxide synthase and neuropeptide immunocytochemistry.

The nigrostriatal and mesolimbic systems of the rat were reconstructed using an organotypic culture model, whereby neonatal brain tissue was grown in vitro for approximately one month. The nigrostriatal system comprised of tissue from the substantia nigra, the dorsal striatum and the frontoparietal cortex, while the mesolimbic system included the ventral tegmental area, ventral striatum (including the fundus striati, accumbens nucleus, olfactory tubercle, lateral septum, ventral pallidum and piriform cortex) and cingulate cortex. These regions were also cultured alone or in pairs. The cultures were monitored in vitro, and after one month fixed in a formalin-picric acid solution, and processed for immunohistochemistry using antibodies raised against tyrosine hydroxylase, nitric oxide synthase, preprocholecystokinin, glutamate decarboxylase, neuropeptide Y, dopamine- and cyclic AMP-regulated phosphoprotein-32 and glial fibrillary acidic protein. The tissue survived in single, double or triple cultures, although differences were found depending upon the source and combination of cultured region. Neurons had localization and shape as in vivo. Local networks were especially prominent in the mesencephalon, where both tyrosine hydroxylase-positive axons spread from the "substantia nigra" to the rest of the tissue, and where nitric oxide synthase-positive networks also surrounded tyrosine hydroxylase-positive neurons. Glutamate decarboxylase-positive nerve terminals formed dense networks around tyrosine hydroxylase-positive neurons. In the striatum, nitric oxide synthase and dopamine- and cyclic AMP-regulated phosphoprotein-32 neurons were surrounded by tyrosine hydroxylase-positive nerve terminals. The nigral and ventral tegmental area dopamine neurons projected to striatal and cortical structures, but the projection from the ventral tegmental area to the cingulate cortex was more prominent. With regard to co-existence, preprochole-cystokinin-like immunoreactivities was found in many tyrosine hydroxylase-positive neurons and neuropeptide Y- and nitric oxide synthase-like immunoreactivity co-existed in striatal and cortical tissues. In general terms, the chemical neuroanatomy in the cultures was similar to that described earlier in vivo. Nitric oxide synthase staining was particularly intense. Taken together, the organotypic model captures many of the morphological and neurochemical features seen in vivo, providing a valuable model for studying neurocircuitries of the brain in detail, where 'normal' and 'pathological' conditions can be simulated.

Animals↗
PubMed1993

Projections of the guinea-pig paracervical ganglion to pelvic viscera.

The uterine cervix, urinary bladder and rectum of guinea pigs were injected with Fast Blue dye for retrograde transport studies. Dye-laden neuronal perikarya were detected for each viscus in the paracervical ganglion. These same perikarya also exhibited immunoreactivities for tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine beta-hydroxylase, neuropeptide Y, or vasoactive intestinal peptide, though the perikarya projecting to the urinary bladder did not exhibit immunoreactivity for aromatic amino acid decarboxylase. The results of this study indicate that the guinea-pig paracervical ganglion projects to viscera in addition to the uterus, and that the ganglion contains a range of immunoreactivities related to adrenergic and non-adrenergic neurotransmitters.

Amidines↗
PubMed1992

Anatomical and functional compartmentalization of the subparafascicular thalamic nucleus in the rat.

The localization and the transmitter phenotype of subparafascicular thalamic nucleus (Spf) neurons projecting to the inferior colliculus (IC) and to the spinal cord (Sp) were studied by using a retrograde fluorescent double labeling technique, and a combined technique of retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD). The cell population of Spf-IC neurons was totally differentiated from that of Spf-Sp neurons which have been reported to be dopaminergic. The former were densely distributed, small to medium sized cells and localized in the central portion of the Spf, while the latter were sparsely distributed, large cells and localized in the marginal portion of the Spf. Spf-IC neurons were completely devoid of TH immunoreactivity and, instead, approximately half of them showed GAD immunoreactivity. From these findings, it is concluded that the Spf is distinctly compartmentalized by the presence at least two separate neuronal subpopulations, which are distinguishable in terms of their cell size, distribution patterns, transmitter phenotypes and trajectories.

Animals↗
PubMed1993

Dopaminergic lateral efferent innervation of the guinea-pig cochlea: immunoelectron microscopy of catecholamine-synthesizing enzymes and effect of 6-hydroxydopamine.

We have used an immunocytochemical approach to gain further data supporting a possible neurotransmitter or neuromodulator function for dopamine at the level of efferent (olivocochlear) innervations of the guinea-pig cochlea. Immunofluorescence screening was first done on cochleas two or seven days after infusion with the neurotoxin 6-hydroxydopamine. Two days after neurotoxin perfusion, the intensity of the tyrosine hydroxylase-like immunoreactivity was decreased in the inner and tunnel spiral bundles of the organ of Corti (and in known noradrenergic sympathetic fibers outside this organ), with respect to the control contralateral cochleas. In cochleas screened seven days after 6-hydroxydopamine infusion, no tyrosine hydroxylase-like immunoreactivity could be found in the organ of Corti. Only occasional faint fluorescence could be detected in sympathetic fibers. In another set of experiments, a pre-embedding immunoperoxidase technique was used to localize tyrosine hydroxylase and aromatic amino acid decarboxylase, another catecholamine-synthesizing enzyme, at the ultrastructural level. With both types of antibody, the same kind of results were observed. Immunoreactivities were only seen in vesiculated fibers within the inner and tunnel spiral bundles, thus are likely in lateral efferent varicosities. These immunostained fibers accounted for approximately half of the efferent profiles in the inner spiral bundle. Within this bundle, the immunoreactive fibers established axodendritic synapses with the radial afferent processes of type I neurons which contacted the inner hair cells. In no case was immunoreactivity to either enzyme observed in the outer hair cell region, at the level of medial efferent terminals. The synaptic localization of tyrosine hydroxylase- and aromatic amino acid decarboxylase-like immunoreactivities in the lateral efferent varicosities of the inner spiral bundle, as well as the effect of 6-hydroxydopamine on the tyrosine hydroxylase-like immunoreactivity in this same bundle, further support the hypothesis that a catecholamine could act as a lateral efferent neurotransmitter or neuromodulator. Based on previous data reporting a lack of dopamine-beta-hydroxylase-like immunoreactivity within the organ of Corti, and the effectiveness of a D2 agonist on the cochlear compound action potential of the auditory nerve, this catecholamine could well be dopamine.

Animals↗
PubMed1979

[The monoamines in molluscs. I. Catecholamines: biosynthesis, disposition and inactivation (author's transl)].

The central nervous system of the mollusc Helix pomatia, like that of other molluscs, contains a very high level of dopamine. However, noradrenaline is weakly represented. These characteristics apply to the peripheral nervous system and more particularly to the heart. The study of the phenomena taking part in the synthesis and inactivation of catecholamines shows that these processes are not different in vertebrates and molluscs. Thus, in the particular case of Helix pomatia the synthesis of catecholamines is carried out by tyrosine hydroxylase, aromatic amino acid decarboxylase and dopamine-beta-hydroxylase. These enzymes are not only active in the ganglia and nerves, but also in the peripheral nervous system. The monoamines are associated with granules. The synthesized enzymes in the pericarya migrate due to the axonal flow and accumulate in the intracardiac nerve endings. In Helix pomatia, the enzymes participate actively in the local synthesis of catecholamines using the precursors tyrosine and DOPA. We have little information on the uptake of dopamine by nervous structures, but it would seem that this phenomenon seems to play an active role in the synaptic inactivation of dopamine. The glial elements also play a part in uptake and inactivation. In most species the nervous system has very little monoamine oxidase, and there is even less in the heart. The enzymic activity depends on substrates and is more active with dopamine than with 5-hydroxytryptamine. The exact localization of monoamine oxidase in the tissues is unknown. However, we believe that it plays a part in the neuronal regulation of dopamine levels and in its synaptic inactivation. The same applies for catechol O-methyltransferase.

Animals↗
PubMed1996

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.

Acetates↗
PubMed2000

Novel fusion proteins in the analysis of diabetes-associated autoantibodies to GAD65 and IA-2.

Assays to detect autoantibodies to glutamic acid decarboxylase (GAD65) and the protein tyrosine phosphatase-like molecule IA-2, which are both present in pancreatic islets, have been used in the diagnosis and prediction of type 1 diabetes. In this study a novel fusion protein combining the entire GAD65 molecule with the 40 kDa intracellular domain of IA-2 (GAD-IA-2) was constructed to detect autoantibodies to both antigens by one single assay. For the same purpose a truncated version of this fusion protein which contained the entire GAD65 linked to the 203 carboxy-terminal amino acids of IA-2 (GAD-dIA-2) was made. A panel of 34 diabetic sera which represented unequivocally positive or negative antibody responses to GAD65 and/or IA-2 as well as 20 serum samples from healthy controls were tested in a radioligand binding assay with the constructed fusion proteins as antigens. Nine of the samples from patients with type 1 diabetes reacted with GAD65 while being negative for IA-2. Six sera were positive for IA-2 only, 11 were double positive, and 8 negative for both antibodies using the standard in vitro transcription translation assay with single antigens. The full-length, as well as the truncated fusion protein detected all samples positive for antibodies either to GAD65 or IA-2 or both, except for one GAD65 antibody positive sample. All samples from healthy controls tested negative in all assays. We conclude that the principle of a combinatorial molecule where a fusion protein expresses both GAD65 and IA-2 epitopes is feasible, and such a fusion protein can be used instead of the single antigens to reduce time and costs of large-scale screening for clinical purposes.

Adolescent↗
PubMed1974

Brain catechol synthesis: control by train tyrosine concentration.

Brain catechol synthesis was estimated by measuring the rate at which brain dopa levels rose following decarboxylase inhibition. Dopa accumulation was accelerated by tyrosine administration, and decreased by treatments that lowered brain tyrosine concentrations (for example, intraperitoneal tryptophan, leucine, or parachlorophenylalanine). A low dose of phenylalanine elevated brain tyrosine without accelerating dopa synthesis. Our findings raise the possibility that nutritional and endocrine factors might influence brain catecholamine synthesis by controlling the availability of tyrosine.

Alanine↗