Golgi‐Cox Staining of Neuronal Dendrites and Dendritic Spines With FD Rapid GolgiStain™ Kit (original) (raw)
Related papers
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
PLoS ONE, 2012
Characterization of neuronal dendritic structure in combination with the determination of specific neuronal phenotype or temporal generation is a challenging task. Here we present a novel method that combines bromodioxyuridine (BrdU) immunohistochemistry with Golgi-impregnation technique; with this simple non-invasive method, we are able to determine the tridimensional structure of dendritic arborization and spine shape of neurons born at a specific time in the hippocampus of adult animals. This analysis is relevant in physiological and pathological conditions in which altered neurogenesis is implicated, such as aging or emotional disorders.
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.
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.
The dendritic spine story: an intriguing process of discovery
Frontiers in neuroanatomy, 2015
Dendritic spines are key components of a variety of microcircuits and they represent the majority of postsynaptic targets of glutamatergic axon terminals in the brain. The present article will focus on the discovery of dendritic spines, which was possible thanks to the application of the Golgi technique to the study of the nervous system, and will also explore the early interpretation of these elements. This discovery represents an interesting chapter in the history of neuroscience as it shows us that progress in the study of the structure of the nervous system is based not only on the emergence of new techniques but also on our ability to exploit the methods already available and correctly interpret their microscopic images.
Developmental Neuroscience, 2022
Antenatal brain development during the final trimester of human pregnancy is a time when mature neurons become increasingly complex in morphology, through axonal and dendritic outgrowth, dendritic branching, and synaptogenesis, together with myelin production. Characterizing neuronal morphological development over time is of interest to developmental neuroscience and provides the framework to measure gray matter pathology in pregnancy compromise. Neuronal microstructure can be assessed with Golgi staining, which selectively stains a small percentage (1–3%) of neurons and their entire dendritic arbor. Advanced imaging processing and analysis tools can then be employed to quantitate neuronal cytoarchitecture. Traditional Golgi-staining protocols have been optimized, and commercial kits are readily available offering improved speed and sensitivity of Golgi staining to produce consistent results. Golgi-stained tissue is then visualized under light microscopy and image analysis may be co...
Neuroscience, 2013
Because dendritic spines are the sites of excitatory synapses, pathological changes in spine morphology should be considered as part of pathological changes in neuronal circuitry in the forms of synaptic connections and connectivity strength. In the past, spine pathology has usually been measured by changes in their number or shape. A more complete understanding of spine pathology requires visualization at the nanometer level to analyze how the changes in number and size affect their presynaptic partners and associated astrocytic processes, as well as organelles and other intracellular structures. Currently, serial section electron microscopy (ssEM) offers the best approach to address this issue because of its ability to image the volume of brain tissue at the nanometer resolution. Renewed interest in ssEM has led to recent technological advances in imaging techniques and improvements in computational tools indispensable for three-dimensional analyses of brain tissue volumes. Here we consider the small but growing literature that has used ssEM analysis to unravel ultrastructural changes in neuropil including dendritic spines. These findings have implications in altered synaptic connectivity and cell biological processes involved in neuropathology, and serve as anatomical substrates for understanding changes in network activity that may underlie clinical symptoms. This article is part of a Special Issue entitled: Dendritic Spine Plasticity in Brain Disorders.