A Light Insensitive Method for Contrast Enhancement of Insect Neurons Filled with a Cobalt-Lysine Complex

Modified protocols for cobalt-filling and silver intensification of neurons in the larval and adult stages of the moth, Manduca sexta, have led to improved neuronal visualization and minimal background staining. In particular, long distance projecting multisegmental in-terneurons. originating in the pterothoracic or terminal abdominal ganglion, were best visualized when a cobalt:lysine complex was used to fill hemi-connectives for several days at 4 C. Ganglia closest to the placement of tracer, which became flooded with cobalt:lysine during the filling period. were removed from the insect. This step eliminated the artifactual filling of neurons that may have taken up the tracer from such pooled regions. This led to a more accurate assessment of whether a multisegmental interneuron projected through the full length of nerve cord to the original site of tracer placement. The protocol for light insensitive silver intensification of cobalt-filled neurons was modified to include an important pH adjustment. NaOH was used to alter the pH of the protective colloid, sodium tungstate, to 10.4 or greater in solution. Especially in larvae. our techniques produced intensely stained cobalt-filled neurons within ganglia that remained transparent and relatively free of nonspecific silver deposition.     

Modified cobalt staining and silver intensification techniques for use with whole-mount gastropod ganglion preparations

While silver intensification of cobalt-filled cells is a common procedure for use with many arthropod preparations, it has not been routinely applied to gastropods. Several modifications in cobalt-staining techniques currently used with gastropods along with adaptations of silver intensification techniques used in insects are described. Cobalt was introduced into cells through axonal filling of cut nerve trunks or by either pressure injection or iontophoresis from intracellular, micropipette electrodes that had previously had their tips etched in dilute hydrofluoric acid. Etching produced consistent, sharp tips with large lumina. Further procedural modifications allowed complete, even intensification of neurons in large gastropod ganglia. These techniques have proved to be reliable and apparently broadly applicable, having been successfully used on three diverse gastropod species.     

Improved Methods for Cobalt Filling and Silver Intensification of Insect Motor Neurons

A Parafilm disk floating on saline and bearing a drop of cobalt solution in which the nerve stump is bathed provides a convenient and versatile method for in vitro or in vivo filling of neurons for cobalt sulfide staining. Silicone grease around the edge of the disk provides an effective seal around the nerve as it passes between the two solutions.

Using a modified developer, silver intensification of cobalt sulfide stained neurons may be done in the light at room temperature, and the time of optimum intensification may be observed under a dissecting microscope.     

A rapid and simple silver enhancement procedure for ultrastructural localization of the retrograde tracer WGAapoHRP-Au and its use in double-label studies with post-embedding immunocytochemistry

WGAapoHRP-Au is a colloidal gold conjugate of wheat germ agglutinin (WGA) coupled to enzymatically inactive (apo) horseradish peroxidase (HRP). This protein-gold complex has proven very useful for retrograde tracing studies in the nervous system (Basbaum and Menétrey: J Comp Neurol 261:306, 1987). To identify retrogradely labeled cells, the colloidal gold is made visible by silver intensification. As the tracer has no HRP enzymatic activity, it can be combined with HRP-based procedures (or with fluorescent methods) in a variety of multiple-label studies. Standard silver intensification procedures, however, are run at low pH and therefore are incompatible with good EM preservation; moreover, osmication of the tissue oxidizes the silver product, which is then lost in subsequent dehydration steps. This report describes a rapid and simple commercially available silver intensification procedure. The procedure is run at neutral pH and can be performed after osmication. The tracer is readily detected at the EM level and tissue preservation is excellent. This report also demonstrates how sections containing retrogradely labeled neurons can be stained with a post-embedding immunocytochemical method so that the transmitter content of synaptic inputs to these neurons can be identified.     

Columnar organization of the optic tectum in the frog

Cobaltous lysine complex was used to label tectal cells. Cobalt soaked into a piece of filter paper and placed onto the surface of the tectum labelled neurons in the whole thickness of the tectum below the filter paper. The labelled area was sharply demarcated from the unlabelled tectal tissue. Focal cobalt injections into different tectal layers labelled small groups of cells and the cobalt-filled structures were perpendicularly oriented to the surface of the tectum. Efferent axons could be followed into layer 7, but other lateral connections were very sparse. These results support the hypothesis that the tectum has columnar organization similar to that of the mammalian neocortex.     

Visualization of the retino-hypothalamic projection in the rat by cobalt precipitation

Summary The technique of cobalt sulfide precipitation combined with Timm's sulfide-silver method for intensification of heavy metals was used to delineate the retino-hypothalamic projection of the rat. Freshly isolated rat brains were dissected and a solution of cobaltous chloride was applied to one of the cut optic nerves. Sixteen hours later, after cobalt ions had passed into the brain along the entire length of the optic fibers, the preparation was treated with ammonium sulfide to precipitate the cobalt as cobalt sulfide. In thick light microscopic sections, cobalt-filled axons were visualized as black fibers against a light gold background. Such fibers were observed to leave the posterior medial portion of the optic chiasm and, after arching dorsally, to project into the posterior fifth of the suprachiasmatic nucleus (SCN), as well as into the rostral part of the arcuate nucleus. Neither bifurcation of these axons nor looping of the axons back to the chiasm was seen. Most fibers projected to the SCN contralateral to the filled nerve, but the projection represented less than 0.1 % of the total number of fibers in one optic nerve. These observations are considered to be graphic evidence of a retino-hypothalamic projection. The interpretation of the cobalt method is discussed, as are the functions of the connections that have been observed.This work was supported by the Nuffield FoundationWe are grateful to Mr. Clifford Jeal of the Department of Pathology for excellent advice on photomicrography     

Retrograde tracing of neural pathways with a protein-gold complex

Summary In this study I have used a tracer complex made of wheat germ agglutinin horseradish peroxidase conjugate (WGA*HRP) coupled to colloidal gold for retrograde tracing of neuronal pathways at the light microscopic level. Visualization of the gold was achieved by silver precipitation (the gold silver intensification method) with gold particles acting as specific cores of nucleation. The presence of horseradish peroxidase in the protein conjugate allowed this method to be compared with classical histochemistry using tetramethylbenzidine as a chromogen. The gold silver intensification method proved to be reliable, specific and sensitive. It has been demonstrated to be useful with fixatives containing a high percentage of paraformaldehyde and compatible with histochemical procedures to show projections of transmitter specific pathways.     

Retrograde tracing of neural pathways with a protein-gold complex. I. Light microscopic detection after silver intensification

In this study I have used a tracer complex made of wheat germ agglutinin horseradish peroxidase conjugate (WGA*HRP) coupled to colloidal gold for retrograde tracing of neuronal pathways at the light microscopic level. Visualization of the gold was achieved by silver precipitation (the gold silver intensification method) with gold particles acting as specific cores of nucleation. The presence of horseradish peroxidase in the protein conjugate allowed this method to be compared with classical histochemistry using tetramethylbenzidine as a chromogen. The gold silver intensification method proved to be reliable, specific and sensitive. It has been demonstrated to be useful with fixatives containing a high percentage of paraformaldehyde and compatible with histochemical procedures to show projections of transmitter specific pathways.     

Metal-catalyzed oxidation renders silver intensification selective. Applications for the histochemistry of diaminobenzidine and neurofibrillary changes

Physical developers can increase the visibility of end products of certain histochemical reactions, such as oxidative polymerization of diaminobenzidine and selective binding of complex silver iodide ions to Alzheimer's neurofibrillary changes. Unfortunately, this intensification by silver coating is generally superimposed on a nonspecific staining originating from the argyrophil III reaction, which also takes place when tissue sections are treated with physical developers. The present study reveals that the argyrophil III reaction can be suppressed when tissue sections are treated with certain metal ions and hydrogen peroxide before they are transferred to the physical developer. The selective intensification of Alzheimer's neurofibrillary changes requires a pre-treatment with lanthanum nitrate (10 mM/liter) and 3% hydrogen peroxide for 1 hr. The diaminobenzidine reaction can be selectively intensified when physical development is preceded by consecutive treatments with copper sulfate (10 mM/liter, pH 5, 10 min) and hydrogen peroxide (3%, pH 7, 10 min). In peroxidase histochemistry, this high-grade intensification may help to increase specificity and reduce the threshold of detectability in tracing neurons with horseradish peroxidase or in immunohistochemistry when the peroxidase-antiperoxidase method is used.     

Protein-gold complexes as neuronal markers for long-term tracing studies

In this study, we have tested the possible use of protein-gold complexes as neuronal markers for long-term tracing studies in rat. The tracer we have used consisted of colloidal gold particles coupled to wheat-germ agglutinin apohorseradish peroxidase conjugate (WGA-apoHRP). The neuronal labeling was studied for survival periods of up to nineteen months following injection in the central nervous system. Maximal visualization of the gold particles was achieved through gold silver intensification. The tracer could be detected throughout the entire range of periods considered. The injection site consisted of a dense black core and retrogradely labeled cells were characterized by round black granules over the cell body. The retrogradely labeled cells were cytochemically characterized by demonstrating their transmitter content. Thus protein-gold complexes may be used as long-term neuronal markers compatible with the persistance of the vital functions of the labeled cells.     

Protein-gold complexes as neuronal markers for long-term tracing studies

Summary In this study, we have tested the possible use of protein-gold complexes as neuronal markers for long-term tracing studies in rat. The tracer we have used consisted of colloidal gold particles coupled to wheat-germ agglutinin apohorseradish peroxidase conjugate (WGA-apoHRP). The neuronal labeling was studied for survival periods of up to nineteen months following injection in the central nervous system. Maximal visualization of the gold particles was achieved through gold silver intensification. The tracer could be detected throughout the entire range of periods considered. The injection site consisted of a dense black core and retrogradely labeled cells were characterized by round black granules over the cell body. The retrogradely labeled cells were cytochemically characterized by demonstrating their transmitter content. Thus protein-gold complexes may be used as long-term neuronal markers compatible with the persistance of the vital functions of the labeled cells.     

Immunogold–silver staining-on-a-chip biosensor based on cross-flow chromatography

Immunogold–silver staining (IGSS) was adopted in cross-flow chromatographic analysis in which immunological reactions and silver intensification were sequentially conducted in the vertical and horizontal directions, respectively. Factors controlling the performance, except the silver substrate solution, were optimized to increase the signal-to-background ratio in measurements of cardiac troponin I as a model analyte. In generating the signal, the size of colloidal gold catalyst was critical; the smallest size (5-nm diameter) in the selected range yielded the highest colorimetric signal. To maintain the low background, two processes, blocking the remaining surfaces of membrane after antibody immobilization and washing the residual tracer after immunological reaction, were necessary. Self-nucleation of silver ions also caused a background signal and was controlled to some degree by decreasing the hydrodynamic force that arose when the substrate solution was supplied in the horizontal direction. Finally, a new chip (IGSS-on-a-chip; IOC) that allowed for convenient, efficient IGSS was produced by injection molding of plastic. This method enhanced the detection capability by 51-fold compared to the conventional rapid test kit using 30 nm-sized colloidal gold as the tracer. The IOC biosensor results also showed that silver intensification yield via cross flow after immunological reaction was 19% higher than that by traditional incubation.     

Improvements for the anatomical characterization of insect neurons in whole mount: the use of cyanine-derived fluorophores and laser scanning confocal microscopy

The optical sectioning capability of the laser scanning confocal microscope was utilized to image dye-filled neurons within whole-mounted insect ganglia. Specific pterothoracic interneurons, in the mothManduca sexta, were retrogradely filled with Neurobiotin and subsequently visualized with a monoclonal anti-biotin conjugated with one of the following fluorophores: fluorescein, and the newly developed cyanines, Cy3.18 (Cy3) and Cy5.18 (Cy5). Overall, the Cy5 fluorophore was best suited for imaging insect neurons within ganglia. This new methodology allowed us to identify and characterize morphologically a collection of descending multisegmental interneurons with large or small diameter somata. A variety of larger molecular weight (10000 daltons) tracers was also used to examine the possibility of nonselective filling of neurons with Neurobiotin, possibly through gap junctions. We also investigated the usefulness of Cy3 and Cy5 as fluorophores for transmitter immunostaining of neurons in whole mount. Neurons immunoreactive for serotonin and the neuropeptides, FMR Famide and SCPb, were imaged in the brain and the pterothoracic ganglion. The central projections of some of these immunoreactive neurons were imaged in their entirety.     

Cytology of large neurons in the guinea pig dorsal cochlear nucleus contacting the inferior colliculus

Large neurons in the dorsal cochlear nucleus of the guinea pig which project to the inferior colliculus were identified after injections of the neural tracer WGA-HRP. Retrograde labelled cells (pyramidal and giant neurons) in the dorsal cochlear nucleus were glycine and GABA immunonegative and showed a similar ultrastructure. Between 30 and 60% of their perimeter was covered by axo-somatic boutons, most of which (>50%) contained pleomorphic synaptic vesicles. Other boutons (about 40% of total) contained flat vesicles and few (5-6%) contained round vesicles, a characteristic of the excitatory cells innervating the inferior colliculus. Immunogold-cytochemistry, coupled to silver intensification, showed that more than 50% of axo-somatic pleomorphic boutons and over 90% of boutons containing flat and pleomorphic vesicles store glycine. Rare WGA-HRP labelled axo-somatic boutons containing flat-pleomorphic vesicles were seen on pyramidal and giant neurons. This suggests that a few inhibitory collicular terminals contact the excitatory large neurons in the dorsal cochlear nucleus.     

昆虫神经元染色的银增感法

NiCl2逆行染色是观察昆虫神经元结构的一个重要方法。这一方法不足之处是使一些神经末梢的分支不清晰,但可以通过神经元染色后的银增感方法来弥补。作者借对双斑蟋蟀Gryllus bimaculatusde Geet产卵瓣内的感觉神经元染色后的银增感介绍了这一方法,包括材料的解剖、染色、固定、增感、脱水、透明等。     

Neuronal maps of the frontal ganglion of the cockroach, Periplaneta americana, prepared by heavy metal iontophoresis.

Neurons in whole mount preparations of the frontal ganglion (FG) of the cockroach, Periplaneta americana, were mapped with the aid of cobalt chloride staining and silver intensification techniques. Eighty-six neurons were counted in the FG after staining with reduced methylene blue. The cell size ranged between 20 to 35 microns in diameter. Of the somata located in the FG, 44 were found to contribute their fibers to the nervus recurrens, 26 to the right frontal commissure, 28 to the left frontal commissure, and 6 to the nervus connectivus. In addition, a few neurons presumably from the tritocerebral region also contribute their fibers in the formation of nervus connectivus. The present study has helped delineate the neuronal connections of the FG with the brain and neuroendocrine system (corpora cardiaca and corpora allata). This information will be useful in facilitating the positioning of microelectrodes in our future electrophysiological experiments.     

Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis

The goal of the present study was to detect as many protein spots as possible in mammalian cells using two-dimensional gel electrophoresis (2-DE). For proteome analysis, it is of importance to reveal as many proteins as possible. A single standard 2-DE gel (pH 3-10, 18 cm x 20 cm, 13.5% gel) could detect 853 spots from proteins of cultured rat hippocampal neurons when visualized by silver staining. To increase the resolution of the separation and the number of detectable proteins by 2-DE, we utilized seven different narrow pH range immobilized pH gradients in the first dimension. In the second dimension, fourteen long SDS polyacrylamide gels were used: seven 7.5% gels for the separation of high molecular mass proteins (> or = 40 kDa) and seven 13.5% gels for the separation of low molecular mass proteins (< or = 40 kDa). Three hundred and sixty microg of proteins from cultured hippocampal neurons were loaded on to individual gels and visualized by silver staining. All 14 gel images were assembled into a 70 cm x 67 cm cybergel that contained 6677 protein spots, thereby indicating that the utilization of the present strategy led to a 783% increase in the number of detected spots in comparison to the standard procedure. Loading double the amount (720 microg) of proteins on to a 13.5% gel led to a 184% increase in the number of detected spots, thereby indicating that the present strategy has a potential to display more protein spots in the cybergels.     

1-naphthol-pyronin B as a novel substrate for silver intensification: application to light and electron microscopic immunocytochemistry of neuroendocrine systems

We describe a modification of silver intensification of immunoperoxidase end-product using 1-naphthol (1N) and 1N enhanced by pyronin B after suppressing nonspecific tissue argyrophilia with a solution of penicillamine and merthiolate buffered near neutral pH. This approach facilitates the preservation of a second antigen sequentially labeled in the same tissue section for light microscopic double immunolabeling experiments and also allows retention of ultrastructural detail. Using this protocol, we obtained rapid and uniform silver intensification of somatostatin (SRIF)-immunoreactive (IR) neuronal perikarya and processes in the rat hypothalamic paraventricular nucleus (PVN). Ultrastructurally, 1N- and 1N-pyronin B-silver intensified reaction product was clearly recognized by the presence of a coarse intracellular precipitate of high electron density. Light microscopic double-immunolabeling studies demonstrated the association between SRIF- and thyrotropin-releasing hormone (TRH)-IR neuronal systems in the PVN. We propose that silver intensification of 1N and 1N-pyronin B is a useful alternative to standard methods of silver intensification of immunoperoxidase reaction product at both light and ultrastructural levels and may be particularly amenable for double-immunolabeling studies.     

Sensory projections of identified coxal hair sensilla of the scorpionHeterometrus fulvipes (Scorpionidae)

The topography of long hair sensilla on the coxae of walking legs and pedipalps of the scorpionHeterometrus fulvipes is described. Identified long hair sensilla are cobalt filled, and central projections of sensory fibres are reported for the first time in the suboesophageal ganglion of this scorpion. The afferent fibres arising from each long hair sensilla segregate into ventral, dorsomedial and dorsal tracts upon their entry into the suboesophageal ganglion. These transverse tracts bifurcate towards the middle of the leg neuromere and form three ipsilateral, plurisegrnental, longitudinal sensory pathways. Filling a pair of bilaterally distributed long hair sensilla shows bilaterally arranged longitudinal afferent tracts interconnected by distinct transverse commissures. Similar patterns of sensory projections are observed when filling homologous hairs on other legs and pedipalps. Numerous fine collaterals arise from the longitudinal sensory trancts that subdivide and end in small blebs presumed to be presynaptic endings. The dorsal and dorsomedial longitudinal tracts and their respective commissures are in close association with the dendritic arborisations of pedipalpal and leg motor neurons, suggesting direct contact between them. The probable functions of these multisegmental hair afferent pathways are discussed.     

Elimination of cobalt from the frog brain introduced into the optic centres through the optic nerve.

One optic nerve in several frogs was filled with cobaltous-lysine complex, and the animals were left to survive from 1 day to 52 days. Degenerated cobalt-filled retinal fibres were phagocytosed by ependymo-glial, and microglial cells. The cobalt appeared in the ependymo-glial cells in the 4th postoperative day, and its amount was greatly reduced by the 52nd day. Within 12 days the labelled axons were replaced by cobalt-loaded microglial cells in the termination sites of optic fibres. By the end of the experimental period, the number of labelled cells increased in the periventricular layers, and decreased in places where retinal fibres had terminated. These processes were accompanied by the appearance of cobalt in the choroid plexus. It is supposed that glial cells dischargd the cobalt into brain ventricles, and the metal left the nervous tissue via the cerebrospinal fluid.