ISSN:1059-910X    

DOI:10.1002/jemt.1070240102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThis article describes methods for photoconverting diaminobenzidine (DAB) into a stable, light and electron microscopically visible dark reaction product in neurons which contain a fluorescent dye. Photoconversion of DAB has been achieved so far with the following fluorescent dyes: rhodamine labeled latex microspheres (RLM), 4,6‐diamidino‐2‐phenylindole (DAPI), 5,7‐di‐hydroxytryptamine (5,7‐DHT), Fast Blue (FB), Nuclear Yellow (NY), Diamidino Yellow (DY), Evans Blue (EB), acridine orange (AO), ethidium bromide (EBR),1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindolcarbocyanine perchlorate, D‐282 (DiI), propidium iodide (PI), and intracellularly injected Lucifer Yellow (LY). The dye is introduced into the neurons by tinctorial staining, retrograde transport, or intracellular injection. Photoconversion is conducted by incubating the tissue with the fluorescent substance‐containing cells in a DAB solution under simultaneous strong illumination with ultraviolet (UV) light. During the formation of the reaction product, the fluorescence disappears from the cell. In all cases, photoconversion provided a stable, nonfading DAB reaction product for light microscopy. In addition, at the electron microscopic level, it appeared that the photoconversion results in a homogeneously distributed, fine granular, dark, intracellularly located reaction product. With most of the retrograde tracers tested, photoconversion led only to staining of the cell bodies and the proximal portions of primary dendrites. Following photoconversion with intracellularly LY‐filled neurons and cells labeled retrogradely with DiI, DiO, and 5,7‐DHT, the reaction product was present throughout the cells, extending from the cell bodies into dendrites and dendritic appendices, and into axons. The high selectivity and methodological simplicity of photoconversion of DAB with fluorescent dyes into a stable, light and electron microscopical dense reaction product provide a promising alternative to classical neuroanatomical techniques and a new useful application of fluorescent neuronal tracers to light and electron mic

ISSN:1059-910X    

DOI:10.1002/jemt.1070240103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractIntracellular Lucifer Yellow filling in fixed tissue has been recently introduced as a novel neuroanatomical approach to reveal the detailed morphology of individual neurons in isolated preparations of the central nervous system. Since dye injections are performed under visual control, the method is characterized by a high degree of inherent staining selectivity, thus circumventing the element of randomness often considered to be the crux of classical Golgi‐impregnation techniques. Moreover, the opportunity to optically monitor the injection procedure renders fixed slice preparations highly advantageous to be used in combination with retrograde fluorescent tracing. Subsequently, dye‐filled neurons may be subjected to a simple photoconversion procedure leading to the intracellular formation of a stable polymer thus obtaining permanent specimens for light microscopy purposes. Due to the osmiophilic nature of the precipitate the photoconverted material is equally suitable for correlated electron microscopy, thus enabling the analysis of neuronal microcircuitry. At the ultrastructural level, sources of afferent input to identified projection neurons may be revealed by lesion‐induced anterograde degeneration of synaptic terminals, therefore enabling the direct demonstration of multisynaptic links. Finally, morphologically identified neurons may be immunocytochemically characterized at the pre‐ and postembedding levels. It is therefore suggested that their methodological versatility and relative technical ease render intracellular fixed‐slice injections a promising complement to the catalogue of anatomical techniques. © 1993 Wiley

ISSN:1059-910X    

DOI:10.1002/jemt.1070240104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe 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. © 19

ISSN:1059-910X    

DOI:10.1002/jemt.1070240105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA variety of intracellular recording and staining techniques has been used to establish structure‐function and, in some cases, structure‐function‐neurochemical correlations in fish, turtle, and cat retinae. Cone photoreceptor‐horizontal cell connectivity has been studied extensively in the cyprinid fish retina by intracellular staining with horseradish peroxidase (HRP) and subsequent electron microscopy. The available data suggest that horizontal cell dendrites around the ridge of the synaptic ribbon are postsynaptic, whilst finger‐like extensions (“spinules”) of lateral dendrites function as inhibitory feedback terminals. An interesting feature of this inter‐action is its plasticity: the feedback pathway is suppressed in the dark and becomes potentiated by light adaptation of the retina.Intracellular recordings and stainings of ganglion cells in both turtle and cat retinae have been possible. Prelabelling of ganglion cells by retrograde transport of rhodamine from the tectum allows ganglion cells to be stained under visual control, and their synaptic inputs determined by electron microscopy. Such studies have been extended to double labelling by using autoradiography or postembedding immunohistochemistry to identify the neurotransmitter content of the labelled cell and/or the neurotransmitter(s) converging upon it. It is envisaged that further applications of intracellular staining followed by double‐ or even triple‐labelling will continue to enhance greatly our understanding of the functional architecture of the vertebrate retina. ©

ISSN:1059-910X    

DOI:10.1002/jemt.1070240106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have assessed the properties of three intracellular markers, horseradish peroxidase, biocytin/Neurobiotin, and Lucifer Yellow, and have compared their usefulness as neuronal markers for light and electron microscopic visualization. Neurons in the acute slice preparation of rat hippocampus were filled with one of these markers, and the marker was converted to an optical and electron‐dense reaction product. Dimethylsulfoxide (DMSO) greatly facilitated penetration of recognition reagents while preserving membrane integrity. The markers were compared with respect to injection parameters, mobility and recognition, stability and visibility, and ultrastructural clarity. Horseradish peroxidase (HRP)‐labeled neurons, recognized histochemically with diaminobenzedine (DAB), were easily visualized by the density of the DAB reaction product; however, the electron density was often so great as to obscure ultrastructural details. Biocytin (BC)‐/Neurobiotin (NB)‐labeled neurons were recognized by avidin‐HRP, followed by histochemical localization of HRP with DAB. The optically dense reaction product gave complete visualization of the soma and processes at the light microscopic level. The electron density was homogeneously distributed throughout the cell, so that ultrastructural features were easily identified. Lucifer Yellow (LY), a fluorescent marker, was converted to an optical and electron‐dense reaction product via immunocytochemical staining with a rabbit anti‐LY antibody, followed by goat anti‐rabbit IgG‐HRP and DAB histochemical localization. Similar to BC/NB, the reaction product was evenly dispersed, providing good light microscopic and ultrastructural clarity. Under our experimental conditions, BC/NB and LY were superior markers that could be used routinely to label neurons, and give excellent visualization not only at the light but also at the electron microscopic level. © 19

ISSN:1059-910X    

DOI:10.1002/jemt.1070240107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThis paper reports on the detailed morphology of inferior olivary neurons in the cat following electrophysiological examination, intracellular injection with horseradish peroxidase, and gamma aminobutyric acid (GABA) immunocytochemistry.The activity of olivary cells was recorded intracellularly in vivo and their response to mesodiencephalic stimulation was tested. In a number of cases their response to stimulation of the contralateral superior cerebellar peduncle was also tested. Mesodiencephalic stimulation resulted in monosynaptic, and superior peduncle stimulation in disynaptic activation of cells in the medial accessory and principal olivary subdivisions. Rebound olivary activity was usually only found after mesodiencephalic stimulation.Light microscopic investigation of osmicated and Araldite embedded Vibratome sections was facilitated considerably when performing the osmication in a glucose solution. Peroxidase labeled olivary cells, like that earlier described for Golgi‐impregnated material, possess a complex globular dendritic geometry. Especially, and unlike Golgi material, the abundance of exceptionally long and complex spiny appendages could be appreciated. The axons usually stemmed from first order dendrites and did not give rise to recurrent axon collaterals.The ultrastructural analysis of this material, mainly from serial sections, was combined with postembedding GABA immunohistochemistry. In this way, GABAergic as well as non‐GABAergic profiles were studied in conjunction with HRP labeled cellular elements.The GABAergic terminals usually contained pleomorphic vesicles and made symmetrical synapses whereas non‐GABAergic terminals nearly always formed asymmetrical synapses and contained round or oval vesicles. Most, if not all, HRP labeled spiny appendages were incorporated in glomeruli. A particular spiny appendage may contribute more than one spine head to a glomerular core, which, on average, consisted of spiny elements of six different neurons. A glomerular core is surrounded by approximately the same amounts of GABAergic and non‐GABAergic boutons. Also, all spiny appendages, and most of their individual spine heads, are contacted by GABAergic as well as non‐GABAergic boutons. Spiny appendages on the axon hillock may be incorporated in dendritic glomeruli, however, most synapses with the hillock were made by GABAergic boutons.The combined physiological and morphological observations imply that (1) the cerebellar nucleican excert an excitatory influence on inferior olivary neurons through a mesodiencephalic relay, (2) the GABAergic nucleo‐olivary input seems to be capable of diminishing the oscillatory tendencies of olivary neurons, and (3) the mesodiencephalic (non‐GABAergic) and cerebellar (GABAergic) input may subserve a timing function since these inputs systematically impinge upon the same olivary spines. © 1993 W

ISSN:1059-910X    

DOI:10.1002/jemt.1070240108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:1059-910X    

DOI:10.1002/jemt.1070240109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:1059-910X    

DOI:10.1002/jemt.1070240101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY