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At least 505 records · Page 28Linked to original sources
PubMed1991

Loss of AChE- and NGFr-labeling precedes neuronal death of axotomized septal-diagonal band neurons: reversal by intraventricular NGF infusion.

The time course of cellular changes in the medial septum (MS) and vertical limb of the diagonal band area (VDB) after a complete unilateral fimbria-fornix (FF) transection has been studied using prelabeling of the septohippocampal neurons by bilateral hippocampal injections of the fluorescent retrograde tracer Fluoro-Gold (FG), in combination with acetylcholine esterase (AChE) histochemistry and nerve growth factor receptor (NGFr) immunocytochemistry. The results show that the long-term disappearance of AChE-positive and NGFr-positive cells represents a combination of down-regulation of the marker proteins, cell shrinkage, and an actual cell loss. By 4 weeks after lesion the loss of FG-prelabeled cells amounted to 50% in MS and 30% in VDB. A further 25-30% of the MS neurons survived (as indicated by the presence of FG label), but were undetectable by the AChE and NGFr markers. Down-regulation of the marker proteins and cell shrinkage preceded the cell loss by more than a week: while shrinkage and reduced numbers of AChE/NGFr positive cells was evident already by 4-7 days, an actual cell loss (i.e., loss of FG-prelabeled cells) became evident only at 4 weeks after lesion. Continuous intraventricular NGF infusion (0.15 micrograms/day) was capable of counteracting all three types of changes. Infusion over 2 weeks reversed both atrophy and loss of AChE/NGFr staining, whereas infusion over 4 weeks completely prevented the later occurring cell loss. In addition, the NGF infusions induced significant hypertrophy in the undamaged cholinergic neurons in both nucleus basalis and striatum. It is concluded that down-regulation of marker proteins, such as AChE and NGFr, and cellular atrophy precede cell death in the axotomized septohippocampal system and that about 1/3 of the axotomized septal cholinergic neurons may survive for a long time in a down-regulated atrophic state. Exogenous NGF can prevent both the atrophic and the degenerative processes.

Acetylcholinesterase↗
PubMed1990

An in vitro study of the projections of enteric vasoactive intestinal polypeptide-immunoreactive neurons in the human colon.

The anatomical basis of the peptidergic neural control of the human colon is largely unknown. In this study, in vitro retrograde tracing methods have been used on fresh human colon to determine the projection pathways of the enteric nerves and, in particular, those containing vasoactive intestinal polypeptide, one of the most abundant and potent of the gut neuropeptides. Two components of the submucous plexus were identified, the inner one projecting to the lamina propria, and the outer to the circular muscle. The lengths of projections within the submucous plexus were up to 5-14 mm in all directions. Myenteric ganglion cells projected to both longitudinal and circular muscles, for distances of up to only 5 mm. The subpopulation of nerves containing vasoactive intestinal polypeptide arose mainly from the submucous plexus and projected up to 6.5 mm anally, 5 mm orally, and 14 mm within the submucous layer to the mucosa or circular muscle. These findings provide entirely new data on the neuroanatomy of the human colon and may help in the understanding of the neural control of colonic secretion and motility.

Adult↗
PubMed1993

Effects of excitotoxic lesions of the nucleus basalis magnocellularis on conditioned taste aversion and inhibitory avoidance in the rat.

The role of the nucleus basalis magnocellularis (NBM) in a variety of learning tasks is well known. Lesions of this nucleus result in a reduction of cholinergic transmission throughout a vast portion of the cortex. Because cholinergic transmission in the insular cortex seems to be important for the acquisition of conditioned taste aversion, the aim of the present work was to study the effects of bilateral chemically induced lesions of the NBM on this conditioning, as correlated with some cholinergic markers in the insular cortex. The effect on inhibitory avoidance was also studied. Lesions prevented the acquisition of the aversion and disrupted retention of the task in previously trained animals. Learning in the inhibitory avoidance paradigm was also notably affected. Postlesion reductions of choline acetyltransferase and acetylcholinesterase activities and of K(+)-stimulated [3H]acetylcholine release were found in the insular cortex. Further, in intact rats labeling of NBM neurons was observed by retrograde tracing after injection of Fluoro-Gold into the insular cortex. These findings indicate that the NBM is involved in the neural integration of feeding behavior and that its cholinergic projection to the insular cortex is one of the implicated neurotransmitter systems.

Acetylcholinesterase↗
PubMed1988

Combined retrograde and anterograde tracing of neuronal connections: Fluoro-Gold and autoradiography.

We have combined the retrograde Fluoro-Gold (FG) and anterograde autoradiographic (AR) procedures to yield a sensitive high resolution technique by which afferent and efferent connections can be visualized from a single intracerebral injection site. Combined FG/AR sections show excellent results, with no apparent loss of signal compared to performing either procedure alone. Since the FG label is intense, the two labels may be viewed simultaneously by superimposing low level darkfield illumination from below the specimen with fluorescence illumination from above. This combined procedure is useful in the analysis of reciprocal connections involving small spatial domains, such as patchy corticocortical connections. Due to the high signal to noise ratio of both labels, this material is ideally suited for quantitative assessment using automated image analysis.

Afferent Pathways↗
PubMed1989

In vitro and in vivo transplantation of fetal rat brain cells following incubation with various anatomic tracing substances.

Implantation of fetal brain regional anlage into host brains ('brain transplantation') holds promise as a plausible treatment for certain human neurodegenerative disorders. Improvements in experimental brain transplantation techniques include: (1) utilization of brain cells in tissue culture as opposed to freshly prepared cell suspensions as a transplantation source, (2) prelabeling of fetal brain cells with inert, non-toxic tracer substances to allow subsequent (a) unequivocal identification of those cells as being fetally derived, and (b) anatomical and immunohistochemical identification of transplanted neurons, and (3) development of in vitro models for transplantation to allow physiological studies of connections formed between fetal neurons and host brain tissue. We examined the ability of brain cell suspensions derived from rat fetuses 15-17 gestational days old to accumulate and retain anatomic tracing substances, including Phaseolus vulgaris leucoagglutinin (PHA-L), rhodamine-labeled latex microspheres (RLM) and fluorogold (FG). All tracers were rapidly accumulated by fetal brain cells, but only PHA-L and RLM were retained following implantation into adult hosts or in tissue culture in vitro. PHA-L-labeled fetal brain cells transplanted in vivo showed morphological characteristics similar to fetal neurons kept in tissue culture in vitro. RLM- or PHA-L-labeled fetal brain cells can be co-cultured with rat brain slices maintained in long-term roller culture. This in vitro system will allow identification and physiological or immunohistochemical study of interactions between fetally derived and host brain neurons.

Animals↗
PubMed1989

Fluorogold as a retrograde tracer used in combination with immunohistochemistry.

The fluorescent dye Fluorogold has been recently introduced as a retrograde neuronal tracer that can be used in combination with immunohistochemistry. It has, however, lately been reported that immunohistochemical processes induced rapid photobleaching of Fluorogold. In the present study we prove that it is the water in the sections, and not the immunohistochemical processing itself, that is responsible for the photobleaching. This problem is easily corrected by using a 29:1 or 19:1 glycerine-PBS buffer mounting medium.

Animals↗
PubMed1990

Labelling neurons with fluorescent dyes administered via intravenous, subcutaneous or intraperitoneal route.

Fast blue, true blue and fluorogold injected into neonatal and subadult rats via subcutaneous or intraperitoneal route labelled certain forebrain nuclei and the central and peripheral neurons whose axons form the spinal and most cranial nerves. The A1 and A5 noradrenergic neurons, nucleus of the tractus solitarius and some neurons in the nucleus raphe medianus, nucleus reticularis gigantocellularis were also labelled. In addition, occasional fluorescent neurons were seen in the vestibular nucleus, locus coeruleus, fastigial nucleus and Purkinje cell layer of the cerebellum. Some nuclei labelled in neonatal rats hardly showed any labelled neurons in subadult rats. After injection the subarachnoid space, blood vessels in the spinal cord, brain and peripheral ganglia, the circumventricular organs and the choroid plexuses were labelled much faster than the neurons. A similar pattern of labelling was seen in subadult rats receiving intravenous injection of fluorogold. Evans blue and rhodamine injected intravenously failed to label any neurons in the brain or spinal cord.

Amidines↗
PubMed1990

Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold: notes on the application of antibodies to Fluoro-Gold.

Fluoro-Gold has been a very popular fluorescent tracer used recently for retrogradely labeling projection neurons. In this study we described the advantages of using antibodies to Fluoro-Gold in conventional immunohistochemical reaction protocols to further extend the usefulness of this tracer for both light and electron microscopic neuroanatomical studies.

Animals↗
PubMed1990

In vivo staining of the mammalian retina by means of a simple fluorescent method.

Intraocular injections of microliter volumes of a solution containing the fluorescent stain Fluoro-Gold produces an intense and long-lasting in vivo stain of the cells forming the neural retina of rats and mice. The label is incorporated by the neuro-retinal cells, while other intraocular structures such as the lens, iris, ciliary processes and even the optic nerve head exclude it. The fluorophore can be easily demonstrated under epi-illumination by means of commonly used fluorescence filter combinations. The stain has proven to be compatible with cryostat sectioning and with plastic embedding. Furthermore, electron dense lysosomal and lamellar bodies in retinal neurons which have incorporated the stain may provide a means for identification of Fluoro-Gold labeled cells at the electron microscopic level which is consistent with previous results. Intraocular injection of Fluoro-Gold fills the need for a reliable, simple, and robust in vivo and in toto stain of the neural retina.

Animals↗
PubMed1991

Double-labeling of saphenous nerve neuron pools: a model for determining the accuracy of axon regeneration at the single neuron level.

We have developed a labeling procedure which accurately and consistently labels the original sensory pools projecting to their respective nerve branches as a model to quantify the accuracy of nerve regeneration at the single neuron level. Adult and juvenile rats had the saphenous branch of the femoral nerve transected just distal to the bifurcation of the nerve into a sensory branch (saphenous nerve) and a motor branch (nerve to the quadriceps muscle) and exposed to a 3% solution of 1,1'-di-octadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) for 1 h, and then reanastomosed. Two weeks later the sensory branch was redivided proximal to the previous site and labeled with a second tracer (5% fluorogold) using similar procedures. Five days later the animals were killed and cryostat sections were prepared and analyzed with a fluorescence microscope to score single- and double-labeled primary sensory neurons. The results show that the primary sensory neurons which project into the saphenous nerve can be reliably prelabeled by exposure of the saphenous nerve to DiI, and two weeks later approximately 99% of the same population of neurons can be labeled by exposure of the nerve to a second dye, fluorogold. This model system will be very powerful for future studies concerning target reinnervation following nerve regeneration.

Age Factors↗
PubMed1991

Imaging of fluorescent neurons labelled with fluoro-gold and fluorescent axon terminals labelled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope.

This paper describes the implementation of an ultraviolet (UV) laser (Spectra Physics 171-18 with 3 lines: 334, 351 and 364 nm in UV) as light source for fluorescence confocal scanning microscopy. With this instrument it is possible to use fluorophores not previously available for confocal laser microscopical imaging of fluorophores such as fluoro-gold and AMCA. In the study we show confocal laser microscopical imaging of fluorescent motoneurons labelled by retrograde transport of fluoro-gold and AMCA-fluorescent axon terminals labelled with antisera against immunogenes as thyrotropin-releasing hormone (TRH) and calcitonin gene-related peptide (CGRP). These two fluorophores may be recorded simultaneously or separately by using a filter that suppresses the emission of one of the fluorophores. The described instrument should also be useful in applications involving detection of monoamines by the Falck-Hillarp technique, as well as measurements of cytosolic free calcium by indicators such as Fura-2 and Indo-1. Measurements performed in reflected and fluorescence light indicated that the resolution along the optical axis improved by about 25% when UV (351 nm) is used instead of visible light (514 nm). This figure is close to that expected on theoretical basis. There are, however, also serious problems related to the use of UV excitation. Firstly, objectives must be selected based on their UV transmission properties. Secondly, chromatic aberration may cause a substantial focal shift between illuminating and emitted light, calling for a flexible instrumental design in order to allow for compensation. As shown here, this problem can be circumvented by using reflecting objectives but at a price of lower resolution compared with high-aperture refracting objectives.

Animals↗
PubMedDate not supplied

Quadruple labeling of brain-stem neurons: a multiple retrograde fluorescent tracer study of axonal collateralization.

Four different fluorochromes were injected into adjacent cervical spinal cord segments, 1 unique tracer per segment. Each tracer, Fluoro-Gold, Fast Blue, Diamidino Yellow dihydrochloride and Propidium Iodide, was taken up by axonal terminals and transported intra-axonally in a retrograde direction to the cell bodies. Some, though by no means all, of these axons were stem axons with terminals in 2, 3 or 4 of the injected spinal segments. Hence as many as 4 different fluorescent tracers could be discerned simultaneously within individual neuronal somata of origin using fluorescent microscopy. These results extend the possibilities for multiple interconnection determinations within the central nervous system. Specifically, the potential for individual neurons of a nucleus to project collateral branches of a stem axon to as many as 4 different central nervous system nuclei now can be studied simultaneously using these 4 fluorescent tracers.

Afferent Pathways↗
PubMedDate not supplied

Electron microscopic analysis of fluorescent neuronal labeling after photoconversion.

Ultrastructural visualization of non-electron-dense fluorescent retrograde neuronal labeling was attempted by means of photo-oxidation. This procedure was used to convert the fluorescence of neurons labeled by the tracers propidium iodide, rhodamine latex microspheres and fluorogold into a stable diaminobenzidine reaction product. The ultrastructural study revealed an accumulation of electron-dense material in these cells both within lysosomes and scattered in the cytoplasmic matrix. Comparison with several different sets of control samples indicated that this material, on the basis of its amount, electron density and appearance, specifically represents the photoconversion reaction product. The effects of the intensity of the fluorescent labeling and of a prolonged photoconversion on the fine structural features of the reaction product are also described and discussed. The present findings indicate that photoconversion can be effectively applied to ultrastructural study of fluorescent retrogradely labeled neurons. The specificity of the photoconversion reaction product should be tested routinely for each fluorochrome and tissue sample.

Amidines↗
PubMed1992

Fluorogold administration via microdialysis labels neurons terminating within the dialysis region.

The use of microdialysis to monitor the release of neurotransmitters in selected regions of the CNS has increased substantially in the last several years. We describe here a method for retrogradely labeling neurons that terminate within the effective sampling region surrounding the dialysis cannula. This is accomplished by direct infusion of fluorogold through the dialysis cannula. By combining this technique with immunohistochemistry, it is possible to identify neurons that could contribute to the neurotransmitter release measured by microdialysis.

Animals↗