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L C Schmued

Publications and source records attributed to L C Schmued.

33 records · Page 2Linked to original sources
PubMed1990

A rapid, sensitive histochemical stain for myelin in frozen brain sections.

A staining technique is described whereby frozen brain sections are incubated in a vehicle containing gold chloride. After 2-4 hr in this solution, both large myelinated bundles and fine individually myelinated fibers are darkly stained. Advantages of this technique over conventional myelin stains include speed, sensitivity, metachromatic staining, and compatibility with formalin-fixed and frozen cut sections. Possible histochemical mechanisms are discussed.

Animals↗
PubMed1990

Iontophoretic injection of fluoro-gold and other fluorescent tracers.

Fluoro-gold, a retrograde axonally transported fluorescent tracer, has been successfully iontophoresed into brain regions to yield small injection sites with no detectable dye along the track. Previous difficulties involving iontophoretic application of fluoro-gold were overcome through the use of a cacodylate vehicle. This vehicle was also found to be suitable for iontophoresing other fluorescent tracers, such as propidium iodide or Fast Blue.

Animals↗
PubMed1989

Neurons containing retrogradely transported Fluoro-Gold exhibit a variety of lysosomal profiles: a combined brightfield, fluorescence, and electron microscopic study.

The advantages of axonally transported Fluoro-Gold as a retrograde fluorescent marker are numerous. The objective of the present study was to determine whether transported Fluoro-Gold is visible in either semi-thin sections for light microscopy or thin sections for electron microscopy. Rats received injections of Fluoro-Gold into either the striatum or thoracic spinal cord. After appropriate survival times, labelled neurons were observed with the fluorescence microscope in brain regions that are known to project to the injected areas. Sections that contained labelled cells were embedded in plastic and examined with a fluorescence microscope. Semi-thin sections of unosmicated tissue displayed high-resolution fluorescent labelling of somata and dendrites. In contrast, osmicated tissue did not fluoresce, but numerous dark granules were observed in the dendritic and perikaryal cytoplasm of labelled neurons in toluidine blue stained sections that were examined with brightfield optics. The unosmicated tissue did not display these granules, and this finding suggested that the granules are composed of membranes. Neurons in other brain regions that are known not to project to the injection sites did not contain these dark granules. Adjacent thin sections examined with the electron microscope displayed numerous electron-dense, lysosome-like organelles in the cytoplasm of labelled neurons. The electron density of these organelles was greater than that of lysosomes in unlabelled neurons. Three types of distinctive organelles were observed in these preparations: (1) relatively dense concentric lamellar bodies of various sizes; (2) heterogeneous or lipofuscin-like lysosomes; and (3) coarse grained lysosomes. Control sections and unlabelled neurons did not display these organelles. Therefore, these organelles appear to correlate with Fluoro-Gold localized within the somata and dendrites of retrogradely labelled neurons. It is not known if they are the Fluoro-Gold itself, or represent a physiological effect on membranes. The results of this study indicate that Fluoro-Gold may be useful for tract tracing at the electron microscopic level.

Biological Transport↗
PubMed1987

Neurons of the ventral medulla oblongata that contain both somatostatin and enkephalin immunoreactivities project to nucleus tractus solitarii and spinal cord.

The ventral aspect of the medulla oblongata of colchicine-treated rats was examined immunohistochemically using mouse monoclonal antibodies raised against somatostatin (SOM) and rabbit polyclonal antibodies to methionine enkephalin (ENK). Numerous perikarya showed positive immunostaining for both antisera. For the most part, the double-labelled cells were located (1) along the ventrolateral surface in a region that corresponds to nucleus paragigantocellularis, (2) in the region of nucleus gigantocellularis-nucleus raphe magnus and (3) in a discrete area just above the inferior olivary nucleus. In an attempt to determine the projection sites of the SOM/ENK somata, the retrogradely transported fluorescent dye Fluoro-Gold was injected into either the nucleus tractus solitarii (NTS) or the upper part of the thoracic spinal cord. SOM/ENK cells in all 3 regions were labelled by dye administered into the spinal cord whereas only those SOM/ENK cells located in nucleus paragigantocellularis were stained by dye microinjected into NTS. This is the first evidence of a SOM/ENK projection from the ventral medulla to either the spinal cord or NTS.

Animals↗
PubMed1986

Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties.

A new fluorescent dye, Fluoro-Gold, has been demonstrated to undergo retrograde axonal transport. Its properties include intense fluorescence, extensive filling of dendrites, high resistance to fading, no uptake by intact undamaged fibers of passage, no diffusion from labeled cells, consistent and pure commercial source, wide latitude of survival times and compatibility with all other tested neuro-histochemical techniques.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1986

A GABAergic inhibitory component within the hippocampal commissural pathway.

Previous results from immunocytochemical and retrograde transport studies indicated that some GABAergic neurons in the hilus of the dentate gyrus may have axonal projections through the hippocampal commissure. This study has utilized a combined immunocytochemical and retrograde transport method as well as 2 anterograde electron-microscopic methods to determine the existence of this projection in rats. Combined tracer and immunofluorescence studies showed several double-labeled GABAergic neurons in the hilus contralateral to the injection site. The electron-microscopic studies revealed both degenerating and HRP-labeled commissural axons that formed symmetric synapses, the type shown to be formed by GABAergic terminals in the hippocampus. These data demonstrate a GABAergic component within the hippocampal commissural pathway and add further evidence that cortical GABAergic terminals are not derived exclusively from intrinsic neurons.

Animals↗
PubMed1985

Selective neuronal uptake of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and other related substituted stilbenes in vivo: a fluorescent whole cell staining technique.

The uptake of fluorescent stilbene derivatives including 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) was shown to occur from the blood or cerebrospinal fluid in the adult albino rat. SITS was found to be internalized in select populations of neurons, especially pyramidal neurons in layers V and VI of neocortex, granule, hilar and pyramidal neurons in the hippocampus, Purkinje neurons of the cerebellar cortex and predominantly large multipolar neurons in the oculomotor nuclei, lateral vestibular nucleus, red nucleus and the medial gigantocellular brainstem reticular formation neurons. The extent and quality of labeling of neuronal somata, dendrites and axons can be exploited with this technique to produce selective, 'Golgi' type labeling in the nervous system. The methods utilized are fast, economical, long lasting and compatible with fluorescence tracers and immunofluorescence techniques.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

A retino-mesotelencephalic pathway in the rat.

We have demonstrated a disynaptic pathway in the adult rat that connects retinal ganglion cells with the anteromedial portion of the caudate-putamen and with the prefrontal and anterior cingulate cortex through the ventral midbrain tegmentum. This retino-mesotelencephalic pathway has been revealed by double-labeling methods in which neurons located in the medial portion of the pars compacta of the substantia nigra and the lateral portion of the nucleus paranigralis of the ventral tegemental area have been retrogradely labeled with tracers injected into the anteromedial portion of the caudate-putamen or the prefrontal/anterior cingulate cortex and have been anterogradely, and transynaptically, labeled with [3H]adenosine injected into the eye. The data show a disynaptic connection from retinal ganglion cells to striatal and cortical neurons by which direction and velocity specific visual information may affect visually guided motor behavior.

Animals↗
PubMed1984

Neurons in the ventral tegmentum have separate populations projecting to telencephalon and inferior olive, are histochemically different, and may receive direct visual input.

The connections of the ventral tegmental area (VTA) and medial terminal nucleus (MTN) of the accessory optic system were studied in the albino rat. Using injection of two fluorescent retrograde tracers it was found that individual neurons of the VTA project to frontal cortices or the inferior olive but not both structures. Using combined retrograde fluorescent tracers and glyoxylic acid histochemistry, it was found that although a third of the cells projecting to frontal cortex contained catecholamine, none of the cells projecting to the inferior olive contained catecholamine. Thus, these portions of the ascending and descending VTA systems are independent. In addition, using injections of the anterograde transneuronal tracer [3H]adenosine into one eye, it was found that cells in the VTA, as well as the MTN, contained the tracer. Therefore, there is a basis for direct retino-mesentelencephalic pathways through the VTA.

Animals↗
PubMed1983

Immunohistochemical localization of monoclonal antibodies to the nicotinic acetylcholine receptor in chick midbrain.

We used the indirect immunofluorescence method to determine the crossreactivity of a library of 57 monoclonal antibodies (mAbs) against each of the subunits of the nicotinic acetylcholine receptor (nAcChoR) isolated from Torpedo and Electrophorus electric organs or from fetal calf and human muscle, with specific neural elements in the midbrain of the chick. Out of 17 mAbs that recognized motor end plates on chick muscle, 14 produced a similar pattern of labeling in the midbrain: the neuronal perikarya and dendrites in the lateral spiriform nucleus (SpL) were intensely labeled, and there was moderate labeling of fibers in certain of the deeper layers of the optic tectum, which disappeared after the SpL was destroyed electrolytically. Two lines of evidence suggest that the mAbs may be crossreacting with nAcChoRs in the midbrain. First, all of the mAbs that stained the SpL also stained neuromuscular junctions in skeletal muscle, whereas none of the 40 mAbs that failed to stain end plates crossreacted with the SpL; second, in vitro immunological studies and blocking experiments on tissue sections (in which unlabeled mAbs were used to block the staining of a directly fluorescein-treated mAb) indicated the presence of mAbs specific for unique antigenic determinants on all four of the subunits (alpha, beta, gamma, and delta) from Torpedo nAcChoR in chick midbrain and muscle. On the other hand, the distribution of mAb staining in the optic tectum does not closely parallel that of either acetylcholinesterase staining or of 125I-labeled alpha-bungarotoxin binding; no toxin binding has been observed autoradiographically in the SpL, but the nucleus does contain moderately dense acetylcholinesterase staining. Take together, our observations suggest that there may be a cholinergic input to the SpL and that the projection fibers from the SpL to the optic tectum (which are also stained with an antiserum to [Leu]enkephalin) may contain presynaptic nAcChoRs. It is clear, however, that the distribution of the putative nAcChoRs, alpha-bungarotoxin binding sites, and acetylcholinesterase staining in the avian midbrain are quite different, although they do overlap to some degree in the deeper layers of the optic tectum.

Acetylcholinesterase↗
PubMed1982

Some fluorescent counterstains for neuroanatomical studies.

Methods for counterstaining neural tissue that contains fluorescent markers have been developed. Acridine orange is useful for localizing cells that are retrogradely labelled with the fluorescent tracers true blue, bisbenzimide, and nuclear yellow because at low concentrations it yields a green Nissl stain when excited with blue, but not with ultraviolet, light; since the tracers fluoresce only when exposed to ultraviolet light, they are not masked by the counterstain. In addition, counterstaining at pH 2 increases bisbenzimide fluorescence considerably. Ethidium bromide is useful for immunohistochemistry (IHC) because it yields a bright red Nissl counterstain when excited by green light, and is only faintly visible when the fluorescein marker is excited with blue light, or when ultraviolet excitation is used. Ethidium bromide is therefore a good counterstain for fluorescent retrograde tracer and for combined IHC-retrograde tracer studies as well. Certain dyes are also useful for studies of the normal morphology of neural tissue. For example, bisbenzimide and nuclear yellow at low concentrations produce a brilliant Nissl stain at pH 2, and stain only nuclei at pH 7.2. The latter procedure may be particularly useful for cell counts. Finally, neutral red, astrazone red, and safranin-O differentially stain cells amd myelinated fibers, producing fluorescence analogs of the Klüver-Barrera stain.

Animals↗
PubMedDate not supplied

Evaluation of brain and nerve pathology in rats chronically dosed with ddI or isoniazid.

The antiviral adenosine analog, 2',3'-dideoxyinosine (ddI), and the antitubercular nicotinic acid analogue, isoniazid, have recently received widespread clinical application in the treatment of acquired immunodeficiency syndrome (AIDS). Clinical studies indicate that the primary dose limiting side effect of both drugs is neurological in nature. Most clinical studies are confounded by the fact that the observed neuropathy must be evaluated in the presence of the ongoing disease process associated with human immunodeficiency virus (HIV) infection. The purpose of this study was to develop and validate a rat model of ddI-and isoniazid-induced neuropathy in the absence of any disease-induced pathology. Myelin splitting and intramyelin edema were the most frequent abnormalities observed in the sciatic nerves of ddI-dosed animals, whereas whorls, extracellular debris, macrophages, and reduced myelinated axon number were seen following chronic isoniazid administration. Isoniazid also resulted in myelinopathy of the CNS. Thus, contrary to previous reports, the rodent is a suitable model for ddI- and isoniazid-induced neuropathies.

Animals↗
PubMedDate not supplied

Temporal development of 2',3'-dideoxyinosine (ddI)-induced peripheral myelinopathy.

The anti-HIV therapeutic dideoxyinosine (ddI) has been reported to produce a painful, dose-limiting peripheral myelinopathy in HIV-infected patients after chronic administration. We have previously demonstrated ddI-induced myelinopathy in a non-HIV-infected rat model after 20 weeks of dosing, characterized by myelin splitting and intramyelin edema. The present study examined the time course needed to produce the ddI-induced neuropathy. Adult male Sprague-Dawley rats were gavaged with vehicle or 415 mg/kg ddI twice daily for up to 20 weeks. Groups of treated (n = 6-8) and control (n = 3-5) animals were killed after 5, 10, 15, and 20 weeks of dosing and the distal end of the sciatic nerve was removed. The nerve was postfixed by immersion in neutral phosphate-buffered formalin, dehydrated in graded alcohols, and embedded in plastic embedding media. One-micrometer-thick sections were cut and stained with toluidine blue and basic fuchsin. Plasma levels of ddI on the day the animals were killed were greater than 10 microgram/ml within the first hour after dosing and fell rapidly to less than 1 microgram/ml (clinical range 1-2 microgram/ml) within 3 h after dosing. The abnormalities observed in the sciatic nerve were few, if any, after 5 or 10 weeks, but very prominent after 15 weeks of dosing. Four of the six ddI-treated rats exhibited abnormal morphology as evidenced by myelin splitting and ballooned myelin sheaths. Although abnormal morphology was present at 20 weeks of dosing, the effect was not as robust as at 15 weeks. This suggests that the nerve may partially recover from the effects of ddI with time. Published by Elsevier Science Inc.

Animals↗
PubMedDate not supplied

Fluoro-Jade: novel fluorochromes for detecting toxicant-induced neuronal degeneration.

Two anionic fluorescein derivatives can be used for the simple and definitive localization of neuronal degeneration in brain tissue sections. Initial work on the first generation fluorochrome, Fluoro-Jade, demonstrated the utility of this compound for the detection of neuronal degeneration induced by a variety of well-characterized neurotoxicants, including kainic acid, 3-nitropropionic acid, isoniazid, ibogaine, domoic acid, and dizocilpine maleate (MK-801). After validation, the tracer was used to reveal previously unreported sites of neuronal degeneration associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), methamphetamine, and d-fenfluramine. Preliminary findings with a second generation fluorescein derivative, Fluoro-Jade B, suggest that this tracer results in staining of optimal contrast and resolution in animals dosed with kainic acid. These 2 tracers can be combined with other histologic methods, including immunofluoresence and fluorescent Nissl stains. Recent preliminary findings on a number of specialized applications of Fluoro-Jade include the detection of apoptosis, amyloid plaques, astrocytes, and dead cells in tissue culture.

Animals↗