The osmium-ethyl gallate procedure is superior to silver impregnations for mapping neuronal pathways

doi:10.1016/0006-8993(86)91601-X

Brain Research

Volume 367, Issues 1–2, 5 March 1986, Pages 265-272

https://doi.org/10.1016/0006-8993(86)91601-X Get rights and content

Abstract

Ganglia processed through the osmium-ethyl gallate procedure (OEG)¹⁹ retain more structural integrity than those processed through various silver impregnation methods. However, the OEG method continues to be neglected by most neuroanatomists. Both types of procedures have been used to trace large neuronal tracts, but during silver impregnation the neuropils lose many of their identifying characteristics. We demonstrate here the advantages of the OEG procedure by comparing it with two silver techniques, Rowell's¹¹ and Holmes's⁵. The OEG method yields consistent and reliable results and is easier to carry out than silver protocols. Most importantly, the better preservation of the neuropils has led to the discovery and study of regional specializations that were previously undetected from silver preparations.

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Cited by (30)

Outsourcing a visual neuropil – The central visual system of the median eyes of Galeodes granti Pocock, 1903 (Arachnida: Solifugae)
2021, Arthropod Structure and Development
Citation Excerpt :
After dehydration in a graded acetone series (30%, 50% 70%, 80%, 90%, 3 × 100%, 15 min each step), the specimens were embedded in Glycidether 100 and sectioned with a rotary microtome and stainless steel blade in the sagittal planes (referring to the body axis) (14–16 μm). Wigglesworth stains were performed after Leise and Mulloney (1986); Mizunami et al. (1997); Wigglesworth (1957): Three specimens were anaesthetized by using CO2 followed by decapitation. Brains were dissected and fixed overnight in 4% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C.
Only a few studies have examined the central visual system of Solifugae until now. To get new insights suitable for phylogenetic analysis we studied the R-cell (or retinula cell) projections and visual neuropils of Galeodes granti using various methods.
G. granti possesses large median eyes and rudimentary lateral eyes. In this study, only the R-cells and neuropils of the median eyes were successfully stained. The R-cells terminate in two distinct visual neuropils. The first neuropil is located externally to the protocerebrum directly below the retina, the second neuropil lies in the cell body rind of the protocerebrum, and immediately adjacent is the arcuate body.
This layout of the median eye visual system differs from Arachnopulmonata (Scorpiones + Tetrapulmonata). However, there are several similarities with Opiliones. In both, (1) the R-cells are connected to a first and second visual neuropil and not to any other region of the brain, (2) the first neuropil is not embedded in the cell body rind of the protocerebrum, it is rather external to the protocerebrum, (3) the second visual neuropil is embedded in the cell body rind, and (4) the second neuropil abuts the arcuate body. These findings may provide important new characters for the discussion on arachnid phylogeny.
The visual system of Thelyphonida (whip scorpions): Support for Arachnopulmonata
2019, Arthropod Structure and Development
Citation Excerpt :
After dehydration in a graded acetone series, the specimens were embedded in Glycidether 100 and sectioned with a rotary microtome and stainless steel blade in the sagittal planes (14–16 μm). Wigglesworth stains were performed after Wigglesworth (1957); Leise and Mulloney (1986); Mizunami et al. (1997): Specimens (M. giganteus, n = 2) were anaesthetized by using CO2 followed by decapitation. Brains were dissected and fixed overnight in 4% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C.
Only a few studies have examined the central visual system of Thelyphonida (whip scorpions) until now. To obtain new insights suitable for phylogenetic analysis we studied the axonal trajectories and neuropil architecture of the central visual systems of two whip scorpion species (Mastigoproctus giganteus and Typopeltis dalyi) with different neuroanatomical techniques (Cobalt fills, Wigglesworth stains, and μCT).
The central visual system of whip scorpion comprises one pair of median eyes and one pair of lateral eye triplets. The R-cells (or retinula cells) of both eye types each terminate in a first and a second visual neuropil. Furthermore, a few R-cell fibres from the median eyes leave the second median eye visual neuropil and terminate in the second and the first lateral eye neuropil. This means R-cell terminals from the lateral eyes and the median eyes overlap here. Additionally, the arcuate body and mushroom bodies are described.
A detailed comparison of our findings with previously studied chelicerate central visual systems seems to support a monophyly of Arachnopulmonata, i.e. a clade comprising Tetrapulmonata (Thelyphonida, Schizomida, Amblypygi, and Araneae) and Scorpions. Furthermore, the architecture of the central visual systems hints at a close evolutionary relationship of Arachnopulmonata and Xiphosura.
A tiny visual system — retinula axons and visual neuropils of Neobisium carcinoides (Hermann, 1804) (Chelicerata, Arachnida, Pseudoscorpiones)
2018, Zoologischer Anzeiger
Citation Excerpt :
Finally, sections were silver intensified in solution A and B for a second time. Modified after Leise and Mulloney (1986), Mizunami et al. (1997), and Wigglesworth (1957): Specimens were dissected and fixed in 4% glutardialdehyde in 0.1 M cacodylate buffer at 4 °C. After postfixation in 2% OsO4 in 0.1 M cacodylate buffer (3 h at 4 °C) animals were stained for 20 h at 4 °C in a saturated ethyl gallate solution, dehydrated in a graded acetone series, embedded in Glycidether 100, and sectioned with a rotary microtome and stainless steel blade in the sagittal and transversal planes (7–9 μm).
Only a few studies have examined the visual system of Pseudoscorpiones until now. To fill this knowledge gap we analysed the axonal trajectories and neuropil architecture of the visual system of the pseudoscorpion Neobisium carcinoides (Hermann, 1804) with different neuroanatomical techniques. The R-cell axon terminals were identified with Cobalt fills and the morphology of the visual neuropils and the protocerebrum generally is described by means of the osmium-ethyl gallate procedure and TEM. N. carcinoides has two lateral eyes on each side of the prosoma. The R-cells of the eyes per hemisphere are linked to a first and a second visual neuropil, located in the dorsolateral protocerebrum. The first visual neuropil is subdivided into two hemineuropils, one for each lateral eye, while the second neuropil is not. Furthermore, the two-layered arcuate body is found − isolated from the visual neuropils − in the midline of the protocerebrum at a dorsoposterior position. These findings allow a detailed comparison of the pseudoscorpion visual system with that of other previously studied taxa and shed new light on lateral eye evolution in Chelicerata.
Antennal pathways in the central nervous system of a blood-sucking bug, Rhodnius prolixus
2009, Arthropod Structure and Development
Citation Excerpt :
Following the imaginal moult, 2–10 day-old male and female adults were used for dissection. Osmium-ethyl gallate staining was carried out for a global visualisation of the brain anatomy, according to Leise and Mulloney (1986). The head capsules were dissected in PBST buffer and brains were immediately fixed in 2% glutaraldehyde and 1% paraformaldehyde overnight at room temperature.
The haematophagous bug Rhodnius prolixus has been a model system in insect physiology for a long time. Recently, several studies have been devoted to its sensory systems, including olfaction. However, few data are available on the basic organisation of the nervous system in this species. By means of neuronal backfills, histology, confocal microscopy and three-dimensional reconstruction methods, we have characterized the projection patterns of antennal sensory neurons within the central nervous system of this disease-vector insect. We established the first partial three-dimensional map of the antennal lobe (AL) of a hemipteran insect. The ALs of this species are relatively diffuse structures, which nevertheless show a glomerular organisation. Based on computer reconstruction of the AL, 22 glomeruli with a radius of 8–25 μm could be identified. No obvious sexual dimorphism of the glomerular architecture was observed. Antennal afferents project not only into the deutocerebrum, but also some fibres descend through the ventral nerve cord to ganglia belonging to the abdominal segments.
Modular construction of nervous systems: A basic principle of design for invertebrates and vertebrates
1990, Brain Research Reviews
The modular construction of brain tissue is not solely a feature of vertebrate nervous tissue, but is characteristic of many invertebrate nervous systems as well. Modern vertebrate and invertebrate modules vary over several orders of magnitude in volume but vary less in diameter. Although the physiological and anatomical differences between the modules discussed herein are overpowering, their importance to nervous system functions are similar. Modules are the serial and parallel processing units that have allowed large-brained animals to evolve.
Many invertebrate modules are discrete, hemispherical lobes, visible on the surface of the brain or nerve cord, whereas most mammalian modules are columnar or ellipsoidal tissue compartments that can only be visualized with specific anatomical methods. Lobes from the largest invertebrates can be more voluminous than any neocortical compartments, but these large lobes are usually not single modules. Large invertebrate lobes contain internal compartments that are single modules and of similar size to their vertebrate analogs. However, vertebrate cortical modules or columns, are far more numerous than the compartments in invertebrate brains and in several cases are known to be adjoined laterally into slabs of tissue that extend for several millimeters.
Physiological data support the idea that neural modules are not just anatomical entities, but are active local circuits. The specific activities within each type of module will depend upon its neuronal components, both intrinsic and extrinsic, its functional roles and phylogenetic history.
Many cellular and intercellular phenomena common to vertebrates and invertebrates underlie the development of modules. Neuronal and glial interactions and their interplay with the extracellular environment depend upon families of molecules with broad phyletic occurrences. The commonalities of growth mechanisms may to a large degree account for the widespread incidence of neuronal processing units.
The strategy of enlarging a nervous system through the replication of the basic units is thought to be advantageous for several reasons. This plan allows nervous systems to economize on the branch sizes and lengths needed for interconnections, to ensure that appropriate targets are reached during development and to modulate specific circuits within a larger network.
Anatomy of the stemmata in the Photuris firefly larva
2019, Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology

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Abstract

Reference (22)

A low-viscosity epoxy resin embedding medium for electron microscopy

J. Ultrastruct. Res.

Reduced silver impregnations of the Ungewitter type

Reduced silver impregnations derived from the Holmes technique

The Bodian Protargol technique

Fixation for Electron Microscopy

The peripheral nerve biopsy

Recent advances in neuroanatomical methodology

Annu. Rev. Neurosci.

The structure of a hair mechanoreceptor in the antennule of crayfish (Crustacea)

Cell Tissue. Res.

Fine Structure of Synapses of the Sensory Portion of the Circuit for Lateral Giant Fiber Mediated Escape Reactions of Crayfish

Ph.D. Thesis

Local interneurons in the swimmeret system of the crayfish

J. Comp. Physiol.

Cited by (30)

Outsourcing a visual neuropil – The central visual system of the median eyes of Galeodes granti Pocock, 1903 (Arachnida: Solifugae)

The visual system of Thelyphonida (whip scorpions): Support for Arachnopulmonata

A tiny visual system — retinula axons and visual neuropils of Neobisium carcinoides (Hermann, 1804) (Chelicerata, Arachnida, Pseudoscorpiones)

Antennal pathways in the central nervous system of a blood-sucking bug, Rhodnius prolixus

Modular construction of nervous systems: A basic principle of design for invertebrates and vertebrates

Anatomy of the stemmata in the Photuris firefly larva