Staining neurons with Golgi techniques in degenerative diseases of the brain (original) (raw)
Related papers
2018
Golgisilverimpregnation techniques remain ideal methods for the visualization of the neurons as a whole in formalinfixedbrains and paraffinsections, enabling to obtain insight into the morphological and morphometric characters of the dendritic arbor, and the estimation of the morphology of the spines and the spinal density, since they delineate the profile of nerve cells with unique clarity and precision. In addition, the Golgi technique enables the study of the topographic relationships between neurons and neuronal circuits in normal conditions, and the following of the spatiotemporal morphological alterations occurring during degenerativeprocesses. The Golgi technique has undergone many modifications in order to be enhanced and to obtain the optimal and maximal visualization of neurons and neuronal processes, the minimal precipitations, the abbreviation of the time required for the procedure, enabling the accurate study and description of specific structures of the brain. In the visualization of the sequential stages of the neuronal degeneration and death, the Golgi method plays a prominent role in the visualization of degeneratingaxons and dendrites, synaptic " boutons, " and axonal terminals and organelles of the cell body. In addition, new versions of the techniques increases the capacity of precise observation of the neurofibrillary degeneration, the proliferation of astrocytes, the activation of the microglia, and the morphology of capillaries in autopsy material of debilitating diseases of the central nervous system. Abstract Golgi silver impregnation techniques remain ideal methods for the visualization of the neurons as a whole in formalin fixed brains and paraffin sections, enabling to obtain insight into the morphological and mor-phometric characters of the dendritic arbor, and the estimation of the morphology of the spines and the spinal density, since they delineate the profile of nerve cells with unique clarity and precision. In addition, the Golgi technique enables the study of the topographic relationships between neurons and neuronal circuits in normal conditions, and the following of the spatiotemporal morphological alterations occurring during degenerative processes. The Golgi technique has undergone many modifications in order to be enhanced and to obtain the optimal and maximal visualization of neurons and neuronal processes, the minimal precipitations, the abbreviation of the time required for the procedure, enabling the accurate study and description of specific structures of the brain. In the visualization of the sequential stages of the neuronal degeneration and death, the Golgi method plays a prominent role in the visualization of degenerating axons and dendrites, synaptic " boutons, " and axonal terminals and organelles of the cell body. In addition, new versions of the techniques increases the capacity of precise observation of the neurofibrillary degeneration, the proliferation of astrocytes, the activation of the microglia, and the morphology of capil-laries in autopsy material of debilitating diseases of the central nervous system.
Scientific Reports
Analysis of neuronal arborization and connections is a powerful tool in fundamental and clinical neuroscience. Changes in neuronal morphology are central to brain development and plasticity and are associated with numerous diseases. Golgi staining is a classical technique based on a deposition of metal precipitate in a random set of neurons. Despite their versatility, Golgi methods have limitations that largely precluded their use in advanced microscopy. We combined Golgi staining with fluorescent labeling and tissue clearing techniques in an Alzheimer's disease model. We further applied 3D electron microscopy to visualize entire Golgi-stained neurons, while preserving ultrastructural details of stained cells, optimized Golgi staining for use with block-face scanning electron microscopy, and developed an algorithm for semi-automated neuronal tracing of cells displaying complex staining patterns. Our method will find use in fundamental neuroscience and the study of neuronal morphology in disease. Classical histological staining techniques used in neuroscience, such as Nissl stain and many others, indiscriminately visualize all cells or structures of interest. Cell type-specific stains, including antibodies, reveal highly convoluted and entangled networks of axons, dendrites and cell bodies, often making it impossible to fully outline individual neurons or to reliably trace neurites. More recently, several techniques have been developed to allow visualization of single neurons, mostly with the use of advanced fluorescent techniques or genetic labeling methods 1,2. These methods tend to be costly and heavily rely on complex instrumentation and skills. One technique, hailing from the golden age of histology, stands out in that it reveals subsets of cells, rather than all cells of the same type, and works in an all-or-nothing fashion, without much dynamic range, thereby producing images of remarkable contrast and clarity. The "black reaction" method, developed by Camillo Golgi in the late XIX century and progressively refined ever since, is based on the impregnation of neural tissue with heavy metal precipitate 3,4. In contrast to tracing methods based on gene delivery and genetic manipulations 5,6 , Golgi staining does not require special skills or expensive equipment, nor is it costly. In its original form, the Golgi method involves sequential incubation of tissue fragments in solutions of potassium dichromate and silver nitrate, followed by sectioning for light microscopy (LM). Later refinements sought to use chemicals other than salts of silver, e.g. mercury salts, for increased contrast and accelerated staining 7-9. The Golgi method was instrumental for many groundbreaking advances in neurobiology, such as the discovery of dendritic spines 10. Today, Golgi staining techniques are still widely used in research and clinical diagnostics 11 , but they are incompatible with further studies of the subcellular, organellar, architecture of labeled neurons with electron microscopy (EM) due to the formation of large, electron-dense silver deposits, which mask ultrastructural details. The method has been adapted for electron microscopy by replacing silver salts with those of gold, resulting in far smaller particles often deposited at the periphery of neurons 12,13 .
Reliable and durable Golgi staining of brain tissue from human autopsies and experimental animals
Journal of Neuroscience Methods, 2014
Golgi staining techniques make visible the somas and dendritic trees of an apparently random subset of neurons, allowing the identification of the dendritic arbors of individual neurons in thick (>100 microns) tissue sections. These methods have been fundamental to our understanding of the structure of the nervous system. Qualitative studies, usually employing projection into two dimensions and tracing by camera lucida, have been clarifying neuronal organization from Ramon y Cajal's (1968) studies of the hippocampal formation to Marin-Padilla's (2011) studies of motor cortex. More recently, computerassisted tracing in 3 dimensions has allowed quantitative studies of experimental manipulations and human disease, e.g. (Sotrel et al., 1991), although it must be understood that the quantitation is at the cellular level, since the fraction of neurons that are stained usually remains unknown. Over a century after their invention, Golgi stains remain the
Golgi‐Cox Staining of Neuronal Dendrites and Dendritic Spines With FD Rapid GolgiStain™ Kit
Current Protocols in Neuroscience, 2019
The Golgi‐Cox method has been one of the most effective techniques for studying the morphology of neuronal dendrites and dendritic spines. However, the reliability and time‐consuming process of Golgi‐Cox staining have been major obstacles to the widespread application of this technique. To overcome these shortcomings and to promote this invaluable technique, we developed the FD Rapid GolgiStain™ Kit based on the principle of the methods described by Ramón‐Moliner in 1970 and Glaser and Van der Loos in 1981. The kit significantly improves and simplifies the Golgi‐Cox technique. This kit is reliable for visualizing morphological details of neurons, allowing for analysis of various parameters of dendritic morphology—such as dendritic length and branching pattern and dendritic spine number, shape, and size—in both animal and postmortem human brains. A 40‐min instructional video for tissue freezing, cryosectioning, and staining is provided. © 2019 by John Wiley & Sons, Inc.
Frontiers in Neuroanatomy, 2019
The metallic impregnation invented by Camillo Golgi in 1873 has allowed the visualization of individual neurons in their entirety, leading to a breakthrough in the knowledge on the structure of the nervous system. Professor of Histology and of General Pathology, Golgi worked for decades at the University of Pavia, leading a very active laboratory. Unfortunately, most of Golgi’s histological preparations are lost. The present contribution provides an account of the original slides on the nervous system from Golgi’s laboratory available nowadays at the Golgi Museum and Historical Museum of the University of Pavia. Knowledge on the organization of the nervous tissue at the time of Golgi’s observations is recalled. Notes on the equipment of Golgi’s laboratory and the methodology Golgi used for his preparations are presented. Images of neurons from his slides (mostly from hippocampus, neocortex and cerebellum) are here shown for the first time together with some of Golgi’s drawings. The sections are stained with the Golgi impregnation and Cajal stain. Golgi-impregnated sections are very thick (some more than 150 μm) and require continuous focusing during the microscopic observation. Heterogeneity of neuronal size and shape, free endings of distal dendritic arborizations, axonal branching stand out at the microscopic observation of Golgi-impregnated sections and in Golgi’s drawings, and were novel findings at his time. Golgi also pointed out that the axon only originates from cell bodies, representing a constant and distinctive feature of nerve cells which distinguishes them from glia, and subserving transmission at a distance. Dendritic spines can be seen in some cortical neurons, although Golgi, possibly worried about artifacts, did not identify them. The puzzling intricacy of fully impregnated nervous tissue components offered to the first microscopic observations still elicit nowadays the emotion Golgi must have felt looking at his slides.
The Original Histological Slides of Camillo Golgi and His Discoveries on Neuronal Structure
Frontiers in Neuroanatomy
The metallic impregnation invented by Camillo Golgi in 1873 has allowed the visualization of individual neurons in their entirety, leading to a breakthrough in the knowledge on the structure of the nervous system. Professor of Histology and of General Pathology, Golgi worked for decades at the University of Pavia, leading a very active laboratory. Unfortunately, most of Golgi's histological preparations are lost. The present contribution provides an account of the original slides on the nervous system from Golgi's laboratory available nowadays at the Golgi Museum and Historical Museum of the University of Pavia. Knowledge on the organization of the nervous tissue at the time of Golgi's observations is recalled. Notes on the equipment of Golgi's laboratory and the methodology Golgi used for his preparations are presented. Images of neurons from his slides (mostly from hippocampus, neocortex and cerebellum) are here shown for the first time together with some of Golgi's drawings. The sections are stained with the Golgi impregnation and Cajal stain. Golgi-impregnated sections are very thick (some more than 150 µm) and require continuous focusing during the microscopic observation. Heterogeneity of neuronal size and shape, free endings of distal dendritic arborizations, axonal branching stand out at the microscopic observation of Golgi-impregnated sections and in Golgi's drawings, and were novel findings at his time. Golgi also pointed out that the axon only originates from cell bodies, representing a constant and distinctive feature of nerve cells which distinguishes them from glia, and subserving transmission at a distance. Dendritic spines can be seen in some cortical neurons, although Golgi, possibly worried about artifacts, did not identify them. The puzzling intricacy of fully impregnated nervous tissue components offered to the first microscopic observations still elicit nowadays the emotion Golgi must have felt looking at his slides.
A modified Golgi staining protocol for use in the human brain stem and cerebellum
Journal of Neuroscience Methods, 2006
The Golgi silver-impregnation method established itself as an important technique for distinguishing morphology at the individual neuron level. This technique has been especially useful for studying human neuroanatomy because it works on postmortem tissue but it is also unreliable and capricious. In this report, we describe a simple technique that was applied to human autopsy and tissue-bank material yielding useful results for the study of neuronal morphology in the brain stem and cerebellum.
Golgi method is been using for more than 140 years so far for the study of the individual morphological and morphometric characteristics and parameters of the cells of the central nervous system. Although other methods came to light, Golgi method is still unique and one of the most powerful tools in the hands of the neuroscientists. What makes Golgi method unique is the capacity to stain all components of the brain tissue, including neurons, glial cells and the vasculature. The cell soma, the dendritic arborization including the spines and at least a part of the axon are usually visible, providing a panoramic view of the entire neural element. Golgi method can be combined with modern and sophisticated techniques which can reduce the human interference and produce accurate 3D models of the neuronal elements of the central nervous system.
The “single-section” Golgi method adapted for formalin-fixed human brain and light microscopy
Journal of Neuroscience Methods, 2010
The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The "single-section" Golgi method of and the modifications made by are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, "sandwiched" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the "single-section" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results.