Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.16.2 (tyrosine hydroxylase)
 14,760 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The injection of the dye Lucifer Yellow (LY) into neurons in slices of fixed brain is used to associate cells displaying a particular dendritic geometry with a specific pattern of neuronal connectivity. In the present report we expand on this technique by combining it at the electron microscopic level with immunocytochemistry and/or degeneration for the study of synaptic relationships. As a model we use the projection neurons of nucleus accumbens. These neurons were retrogradely labeled in vivo with injections or a fluorescent tracer. Fast Blue, into the ventral mesencephalon. Using epifluorescent monitoring, these neurons were located in perfusion-fixed brain slices and intracellularly injected with LY. They were visualized in the light and electron microscope using a peroxidase-antiperoxidase immunocytochemical method. Certain afferent connections of these neurons were identified in the same tissue through the use of either dual-label immunocytochemistry or anterograde degeneration combined with a single-label immunoreaction. In the dual-label procedure, a silver-gold intensification of the diaminobenzidine (DAB) reaction product for the first antigen (LY) was contrasted with a nonintensified reaction product for the second antigen (tyrosine hydroxylase [TH]). Ultrastructurally, metallic gold particles appeared to be dispersed over the immunolabeled perikarya, dendrites, and, occasionally, axonal terminals of LY-injected neurons whereas the flocculent DAB reaction product was present in TH-containing axons and terminals. Following lesions of the ventral subiculum in the hippocampal formation, degenerating axon terminals were detected in nucleus accumbens along with immunoreacted, LY-injected neurons. The techniques outlined in this report should prove invaluable for the study of the synaptic interactions of identified neurons. They can be reliably reproduced with a high yield per experiment.
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PMID:Identification of synaptic interactions of intracellularly injected neurons in fixed brain slices by means of dual-label electron microscopy. 809 37

Dynorphin and other kappa opioid agonists are thought to elicit aversive actions and changes in motor activity through direct or indirect modulation of dopamine neurons in ventral tegmental area (VTA) and substantia nigra (SN), respectively. We comparatively examined the immunoperoxidase localization of anti-dynorphin A antiserum in sections through the VTA and SN of adult rat brain to assess whether there were common or differential distributions of this opioid peptide relative to the dopamine neurons. We also more directly examined the relationship between dynorphin terminals and dopamine neurons in VTA and SN by combining immunoperoxidase labeling of rabbit dynorphin antiserum and immunogold-silver detection of mouse antibodies against tyrosine hydroxylase (TH) in single sections through the VTA and SN. Light microscopy showed dynorphin-like immunoreactivity (DY-LI) in varicose processes. These were relatively sparse in VTA and were unevenly distributed in the SN, with little labeling in the pars compacta (pcSN) and the highest density of DY-LI in the medial and lateral pars reticulata (prSN). Electron microscopy established that the regional differences were attributed to differences in density (number/unit area) of immunoreactive profiles. The profiles containing DY-LI were designated as axon terminals based on having diameters greater than 0.1 micron, few microtubules and many synaptic vesicles. In both the VTA and SN, the dynorphin-labeled terminals contained primarily small (35-40 nm) clear vesicles. These vesicles were rimmed with peroxidase immunoreactivity and were often seen clustered above axodendritic synapses. These synaptic specializations were usually symmetric; however a few asymmetric densities also were formed by immunoreactive terminals in both VTA and SN. Additionally, most of the dynorphin-labeled terminals contained 1-2, but occasionally 7 or more intensely peroxidase positive dense core vesicles (DCVs). Approximately 60% of the DCVs were located near axolemmal surfaces. The axolemmal surfaces contacted by immunoreactive DCVs were more often apposed to dendrites in the VTA; while in the SN other axon terminals were the most commonly apposed neuronal profiles. In both regions, a substantial proportion of the plasmalemmal surface in contact with the labeled DCVs was apposed to astrocytic processes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cellular substrates for interactions between dynorphin terminals and dopamine dendrites in rat ventral tegmental area and substantia nigra. 809 30

Catecholamines in the nucleus tractus solitarii (NTS) have been implicated in autonomic responses to circulating hormones that act on neurons in the area postrema, the most caudal circumventricular organ in brain. We combined immunoperoxidase labeling of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL) with immunogold-silver labeling of tyrosine hydroxylase to determine whether this enzymatic marker for catecholamines was present in efferents from the area postrema or their targets in the rat NTS. At survival periods of 10-12 days after PHAL injections into the area postrema, light microscopy revealed numerous varicose processes containing peroxidase reaction product for PHAL in the dorsomedial, medial, and commissural NTS. Some of these labeled processes were located near neuronal perikarya and processes containing immunogold-silver intensified reaction product for tyrosine hydroxylase. Electron microscopy of the commissural and dorsomedial NTS established that the majority of the labeling for PHAL was in axon terminals, whereas immunogold labeling for tyrosine hydroxylase was mainly in soma and dendrites. Only 3 out of 579 PHAL-labeled terminals also contained detectable tyrosine hydroxylase immunoreactivity. Fifty-eight percent (335/579) of the PHAL-labeled terminals formed synapses with recognized symmetric junctions, whereas the remainder lacked synaptic specializations within the examined series of serial sections. Of those PHAL terminals forming recognized symmetric junctions, 22% were on tyrosine hydroxylase-immunoreactive dendrites, 74% on unlabeled dendrites and 4% on unlabeled axon terminals. From a total of 1,250 observed contacts on tyrosine hydroxylase labeled dendrites, 88 (7%) contained PHAL, 9 (< 1%) contained TH, and 1,180 (93%) lacked detectable immunoreactivity and formed primarily symmetric synapses. We conclude that a few catecholamine, but mainly noncatecholamine efferents from the area postrema provide a monosynaptic, and most likely inhibitory input to target neurons both with and without tyrosine hydroxylase immunoreactivity in the dorsomedial and commissural NTS. Synapses between the efferent terminals from the area postrema and tyrosine hydroxylase labeled and unlabeled dendrites as well as unlabeled axons in these specific subnuclei of the NTS suggest multiple sites for modulation of gastric and cardiovascular reflexes in response to circulating peptides.
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PMID:Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat. 809 27

The relationship between leucine5-enkephalin-containing nerve terminals and midbrain dopaminergic neurons was studied in the adult rat by light and electron microscopy. For light microscopy, alternate midbrain sections were immunostained with rabbit polyclonal antibodies against leucine5-enkephalin and tyrosine hydroxylase, by means of the peroxidase antiperoxidase technique. Leucine5-enkephalin stained fibers and terminals were observed with varying density in the retrorubral field (dopaminergic nucleus A8 region), substantia nigra pars compacta (dopaminergic nucleus A9 region), and ventral tegmental area and related nuclei (dopaminergic nucleus A10 region). For electron microscopy, midbrain sections were immunostained with a mouse monoclonal antibody against leucine5-enkephalin and a rabbit polyclonal antibody against tyrosine hydroxylase, by means of the peroxidase antiperoxidase technique and silver-intensified colloidal gold reactions, respectively. The nucleus A10 area was examined at the electron microscopic level, and there were a) both symmetric (75%) and asymmetric (25%) synapses made between leucine5-enkephalin axon terminals and dopaminergic dendrites, and also synaptic contacts with unlabeled dendrites; b) leucine5-enkephalin synaptic contacts with dopaminergic dendrites that were covered with astrocytic membranes; and c) leucine5-enkephalin appositions with unlabeled nerve terminals that made synaptic contacts with dopaminergic dendrites, suggestive of axo-axonic connections. These findings provide the structural basis for both direct and indirect control of A10 dopaminergic neurons by enkephalin-containing nerve terminals.
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PMID:Leucine5-enkephalin afferents to midbrain dopaminergic neurons: light and electron microscopic examination. 810 Nov 95

We developed a rapid and sensitive radioimmunohistochemical method for the quantification of tyrosine hydroxylase (TH) at both the anatomical and cellular level. Coronal tissue sections from fresh-frozen rat brains were incubated in the presence of a TH monoclonal antibody. The reaction was revealed with a 35S-labeled secondary antibody. TH content was quantified in catecholaminergic brain areas by measuring optical density on autoradiographic films or silver grain density on autoradiographic emulsion-coated sections. Regional TH concentrations determined in the locus ceruleus (LC), substantia nigra pars compacta (SNC), and ventral tegmental area (VTA) were significantly increased by 45% after reserpine treatment in the LC but unchanged in the SNC and VTA. Microscopic examination of TH radioimmunolabeling showed a heavy accumulation of silver grains over catecholaminergic cell bodies. In the LC, grain density per cell was heterogeneous and higher in the ventral than in the dorsal part of the structure. After reserpine treatment, TH levels were significantly increased (57%) in the neurons of the LC but not in those of the SNC or VTA. The data support the validity of this radioimmunohistochemical method as a tool for quantifying TH protein at the cellular level and they confirm that TH protein content is differentially regulated in noradrenergic and dopaminergic neurons in response to reserpine.
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PMID:Cellular quantification of tyrosine hydroxylase in the rat brain by immunoautoradiography. 810 60

The catecholaminergic innervation of neurons that contain oxytocin in the paraventricular nucleus (PVN) of the rat hypothalamus was examined by a combination of methods in the same tissue sections at the electron-microscopic level as follows: (1) Rats were treated with 5-hydroxydopamine (5-OHDA) with peroxidase-antiperoxidase (PAP) staining of sections for oxytocin prior to embedding. (2) Preembedding immunoperoxidase staining with avidin-biotin complexes was used to demonstrate tyrosine hydroxylase (TH) activity, with postembedding staining with immunocolloidal gold for visualization of oxytocin. (3) Prior to embedding, a double-staining technique was used that was based on consecutive staining with silver-gold-intensified PAP complex and 3,3'-diaminobenzidine. We used an antiserum against oxytocin and an antiserum against dopamine-beta-hydroxylase (DBH) for localization of antigens. We found that TH- and DBH-like immunoreactive terminals were distributed throughout the rat hypothalamus and were abundant in all parts of the PVN. Ultrastructural observations revealed 5-OHDA-labeled, TH- or DBH-like immunoreactive axon terminals that contained granular vesicles (70-80 nm in diameter) and small clear synaptic vesicles (30-50 nm in diameter). The terminals appeared at times to be making synapses with cell bodies and with the processes of oxytocin-containing neurosecretory neurons in the PVN. These findings provide morphological evidence for a direct synaptic influence of catecholaminergic elements on the secretory activity of oxytocin-containing neurosecretory neurons in the rat hypothalamic PVN.
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PMID:Catecholaminergic innervation of oxytocin neurons in the paraventricular nucleus of the rat hypothalamus as revealed by double-labeling immunoelectron microscopy. 821 44

Neuroanatomical methods were used to determine if cocaine irreversibly injures neurons. Despite acute and chronic high-dose treatments for months that produced stereotyped behavior and seizures, and the use of a sensitive silver impregnation method, we were unable to find any evidence of neuronal damage anywhere in the brain. Since expression of the inducible 72 kDa heat shock protein (HSP72) is a sensitive indicator of potentially toxic neuronal stress, we next determined if cocaine evoked HSP72 expression. Even high doses of cocaine that evoked seizures did not induce HSP72 immunoreactivity anywhere within the brain, whereas kainic acid produced widespread HSP72 immunoreactivity and irreversible injury. Having failed to find indications of frank neurotoxicity, we examined peptide and protein cell marker immunoreactivities in search of cocaine-induced changes. Although cocaine treatment had no obvious effects on the patterns of hippocampal calbindin-D28K, somatostatin-, tyrosine hydroxylase- and parvalbumin immunoreactivities, cocaine reliably altered neuropeptide Y-like immunoreactivity (NPY-LI). Most notably, NPY-LI was expressed in hippocampal dentate granule cells and pyriform cortical neurons, which do not normally express it. Conversely, we noted decreased NPY-LI in dentate hilar neurons that normally do express it. Since both changes in NPY-LI were seen only in cocaine-treated rats that exhibited seizures, the role of seizure activity per se in producing the NPY changes was addressed in normal rats by electrical stimulation of the perforant path. Like cocaine, perforant path stimulation for as little as 15min evoked NPY-LI in granule cells but did not replicate the cocaine-induced decrease in hilar cell NPY-LI. These results suggest that cocaine does not irreversibly injure neurons in the rat, even at doses that induce seizures. However, cocaine produces long-lasting changes in NPY expression that are of unknown functional significance. Our inability to demonstrate cocaine-induced neuronal damage in rats should in no way be taken as evidence of its safety in humans.
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PMID:Cocaine neurotoxicity and altered neuropeptide Y immunoreactivity in the rat hippocampus; a silver degeneration and immunocytochemical study. 835 18

The history of the nigrostriatal dopamine system may provide a prime example of the two faces of scientific development. First, a given concept is replaced by another simply as a result of methodologies being improved, and second, successive technical improvements make seemingly settled controversies even more complicated and disputable. The nigrostriatal pathway, which had been unrecognizable with Nauta's silver impregnation method, became apparent by use of the more sensitive silver impregnation method of Fink-Heimer. The sensitivity of the latter method, however, was still insufficient to reveal the whole extent of another ascending dopamine system, the mesocortical dopamine system, until its existence was established through the application of glyoxylic acid fluorescent histochemistry. Electron microscopic analysis of nigrostriatal dopamine synapses in properly fixed tissue was initiated by the demonstration of dark type terminal degeneration, which was induced by either electrolytic lesions or chemical destruction with a specific toxin (6-hydroxydopamine) of the substantia nigra and medial forebrain bundle. The degenerating terminal boutons, thus produced, invariably formed postsynaptic membrane specializations of asymmetric type. However, the asymmetric nature of the synaptic morphology, although later confirmed by the combined study of chemical lesions and autoradiographic anterograde tracing, was seriously challenged with the introduction of electron microscopic immunohistochemistry. The latter method has consistently revealed that symmetric en passant synapses or axonal varicosities with no synaptic membrane specializations are the only tissue compartments immunoreactive to antibodies against dopamine and its synthetic enzyme tyrosine hydroxylase. In view of the fact that more than 95% of the nigrostriatal projection neurons are dopaminergic, it is difficult to satisfactorily interpret all the available and seemingly paradoxical fine structural data. In this context, a novel concept has emerged in the process of eliminating all the possible alternative interpretations. The concept is that single nigrostriatal neurons form two chemically distinct types of synapses, one dopaminergic symmetric en passant bouton and another non-dopaminergic (still chemically unclassified) asymmetric terminal bouton. If the concept is a valid one, it contradicts Dale's long standing principle, as defined by Eccles: at all the axonal branches of a neuron there is liberation of the same transmitter substance or substances. Furthermore, a certain population of substantia nigra pars reticulata neurons has recently been recognized to be immunoreactive to both dopamine synthetic tyrosine hydroxylase and GABA synthetic glutamate decarboxylase. These single neurons send projections to both the striatum and superior colliculus by way of axon collaterals.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Conceptual history of the nigrostriatal dopamine system. 839 52

The nucleus accumbens is composed of a core region involved in motor functions and a shell region implicated in emotional and motivational processes. Both of these regions receive serotonin- and dopamine-containing afferents. We examined whether the serotonin innervation or relation to catecholamine (mainly dopamine) axons in the nucleus accumbens shows common features or specializations corresponding to the noted functional differences in core and shell subregions. To address this question, we examined the ultrastructure of serotonin-containing axons and their relation to catecholamine-containing afferents in either the core or shell of the nucleus accumbens. Single coronal sections through the rat forebrain were processed for immunoperoxidase labeling of serotonin and immunogold silver labeling of tyrosine hydroxylase, the catecholamine-synthesizing enzyme. Varicose processes showing peroxidase product for serotonin by light microscopy were confirmed to be axons and terminals by electron microscopy. In a quantitative analysis of serotonin-immunoreactive terminals forming one or more contacts in single sections, some common features were observed. For the core (n = 120) and the shell (n = 82), 41% formed synaptic junctions with unlabeled dendrites, 75% were in apposition with unlabeled terminals, which often formed asymmetric junctions, and 20% were in apposition with axons or terminals containing tyrosine hydroxylase. Thus, in both the core and shell of the nucleus accumbens, serotonin terminals synapse on postsynaptic neurons and are likely to modulate or be modulated by presynaptic interactions with excitatory axons forming asymmetric junctions and by catecholaminergic afferents. Marked differences in the morphology of serotonin axons were also seen in the core versus shell of the nucleus accumbens. By light microscopy, serotonin-immunoreactive axons were thicker and more varicose than those found in the core. Ultrastructural analysis confirmed that, in contrast to the core, serotonin-immunoreactive axons and terminals in the shell were larger in cross-sectional diameter size (0.7 micron vs. 0.3 micron). Additionally, serotonin axon terminals in the shell contained more numerous immunoreactive large dense core vesicles and more frequently formed symmetric as opposed to asymmetric contacts with dendrites. The larger size and more numerous dense core vesicles in serotonin-immunoreactive terminals in the shell support the concept that serotonin or co-existing neurotransmitter may be more tonically released in the shell versus core of the nucleus accumbens.
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PMID:Ultrastructure of serotonin-immunoreactive terminals in the core and shell of the rat nucleus accumbens: cellular substrates for interactions with catecholamine afferents. 840 68

A peroxidase reaction product that can be easily distinguished from standard diaminobenzidine (DAB) reaction products is needed for pre-embedding electron microscopic double-antibody labelling studies. Benzidine dihydrochloride (BDHC) and gold-substituted silver peroxidase reactions are unsatisfactory for double labelling because they lack sensitivity and reliability and/or compromise ultrastructure. We show here that light and electron microscopic immunocytochemistry can be done with a modification of the tungstate-stabilized tetramethylbenzidine (TMB) reaction (Weinberg and Van Eyck 1991) which yields a crystalline reaction product. With this method, we have obtained excellent immunolabelling for a variety of antigens, including tyrosine hydroxylase, enkephalin, serotonin, Fos protein and retrogradely transported cholera toxin B subunit (CTB). The TMB-tungstate reaction is useful for ultrastructural double labelling because the crystals contrast well with the amorphous product of diaminobenzidine reactions. The TMB-tungstate reaction is more sensitive and reliable for immunocytochemistry than the benzidine dihydrochloride reaction and gives better ultrastructure than the gold-substituted silver peroxidase reaction. We also show that neurons filled with biocytin by intracellular injection can be visualized with TMB-tungstate for either light (LM) or electron (EM) microscopy.
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PMID:The tungstate-stabilized tetramethylbenzidine reaction for light and electron microscopic immunocytochemistry and for revealing biocytin-filled neurons. 845 21


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