The Golgi silver impregnation method: commemorating the centennial of the Nobel Prize in medicine (1906) shared by Camillo Golgi and Santiago Ramón y Cajal

The Golgi silver impregnation technique is a simple histological procedure that reveals complete three-dimensional neuron morphology. This method is based in the formation of opaque intracellular deposits of silver chromate obtained by the reaction between potassium dichromate and silver nitrate (black reaction). Camillo Golgi, its discoverer, and Santiago Ramón y Cajal its main exponent, shared the Nobel Prize of Medicine and Physiology in 1906 for their contribution to the knowledge of the nervous system structure, Their successes were largely due to the application of the silver impregnation method. However, Golgi and Cajal had different views on the structure of nervous tissue. According to the Reticular Theory, defended by Golgi, the nervous system was formed by a network of cells connected via axons within a syncytium. In contrast, Cajal defended the Neuron Doctrine which maintained that the neurons were independent cells. In addition, Golgi had used a variant of his "black reaction" to discover the cellular organelle that became known as the Golgi apparatus. Electron microscopy studies confirmed the postulates of the Neuron Doctrine as well as the existence of the Golgi complex and contributed to a resurgence of use of the Golgi stain. Although modern methods of intracellular staining reveal excellent images of neuron morphology, the Golgi technique is an easier and less expensive method for the study of normal and pathological morphology of neurons.     

Gut thoughts on the Golgi complex

The new millennium coincides within 1 year of Camillo Golgi's centennial celebrations. It is quite remarkable that the structure and formation of this organelle is as controversial today as was its mere existence from Golgi's time to the 1950s, when EM approaches were introduced. Since the late 1950s, two opposing models of Golgi structure and function have split the Golgi scientific community, namely vesicular transport versus organelle maturation. Although a few years ago Golgi maturation seemed to be 'out for the count', it has recently seen an almost messianic revival. In this review, I argue that this large-scale desertion from the vesicle transport model to the maturation camp is premature. I propose an alternative, dynamic steady-state model, in which transient tubular connections function in parallel to vesicular transport and that the biosynthetic pathway is made up of three major distinct compartments: the ER, the Golgi and the TGN.     

Molecular basis for Golgi maintenance and biogenesis

The Golgi apparatus contains thousands of different types of integral and peripheral membrane proteins, perhaps more than any other intracellular organelle. To understand these proteins' roles in Golgi function and in broader cellular processes, it is useful to categorize them according to their contribution to Golgi creation and maintenance. This is because all of the Golgi's functions derive from its ability to maintain steady-state pools of particular proteins and lipids, which in turn relies on the Golgi's dynamic character - that is, its ongoing state of transformation and outgrowth from the endoplasmic reticulum. Here, we categorize the expanding list of Golgi-associated proteins on the basis of their role in Golgi reformation after the Golgi has been disassembled. Information gained on how different proteins participate in this process can provide important insights for understanding the Golgi's global functions within cells.     

Brain plasticity and mental processes: Cajal again

The year 2006 marks the 100th anniversary of the first Nobel Prize for Physiology or Medicine for studies in the field of the Neurosciences jointly awarded to Camillo Golgi and Santiago Ramón y Cajal for their key contributions to the study of the nervous system. This award represented the beginning of the modern era of neuroscience. Using the Golgi method, Cajal made fundamental, but often unappreciated, contributions to the study of the relationship between brain plasticity and mental processes. Here, I focus on some of these early experiments and how they continue to influence studies of brain plasticity.     

The sensitive innervation of the proximal sesamoid ligament of the ox

The Authors have studied the sensitive innervation of the proximal sesamoid ligament of the ox and have found capsulated corpuscles only. Among these receptors, besides Pacini and pacini-like corpuscles, Golgi Mazzoni's corpuscles, muscle spindles and Golgi's tendon organs with a typical structure, the Authors have been described, for the first time in the ox, corpuscles constituted by numerous Pacini collected by a single capsule. It must be pointed out that the muscle spindles are always supplied by an annulo-spiral termination which is centrally placed in the equatorial region. A vegetative innervation is also present in this ligament.     

IntraGolgi distribution of the Conserved Oligomeric Golgi (COG) complex

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus.     

Re'COG'nition at the Golgi

The conserved oligomeric Golgi (COG) complex co-ordinates retrograde vesicle transport within the Golgi. These vesicles maintain the distribution of glycosylation enzymes between the Golgi's cisternae, and therefore COG is intimately involved in glycosylation homeostasis. Recent years have greatly enhanced our knowledge of COG's composition, protein interactions, cellular function and most recently also its structure. The emergence of COG-dependent human glycosylation disorders gives particular relevance to these advances. The structural data have firmly placed COG in the family of multi-subunit tethering complexes that it shares with the exocyst, Dsl1 and Golgi-associated retrograde protein (GARP) complexes. Here, we review our knowledge of COG's involvement in vesicle tethering at the Golgi. In particular, we consider what this knowledge may add to our molecular understanding of vesicle tethering and how it impacts on the fine tuning of Golgi function, most notably glycosylation.     

Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi

Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.     

Camillo Golgi and the discovery of the Golgi apparatus

 Camillo Golgi (1843–1926) was born at Corteno, near Brescia, in northern Italy. After graduating in Medicine at the ancient University of Pavia, the former seat of great scientists and naturalists, Golgi continued a long-standing Italian tradition by studying the histology of the nervous system. While working as a modest physician at Abbiategrasso, a small town near Pavia, he developed a silver–osmium technique, the ”reazione nera” (black reaction), for which he was awarded the Nobel Prize in 1906. In the late 1890’s, 25 years after the publication of his black reaction and while Professor of General Pathology in Pavia, Golgi noticed a fine internal network in only partially silver-osmium-blackened Purkinje cells. Following confirmation by his assistant Emilio Veratti, Golgi published the discovery, called the ”apparato reticolare interno”, in the Bollettino della Società medico-chirurgica di Pavia in 1898, which is now considered the birthday of the ”Golgi apparatus”. The discovery of the Golgi apparatus can be added to the long list of accidental discoveries. The man after whom it is named was not a cytologist engaged in studying the inner structure of the cell, but a pathologist searching to prove a neuroanatomical theory. Accepted: 24 October 1997     

A Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 2 1/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50 mum sections are made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

A Golgi-Electron Microscope Method for Insect Nervous Tissue

Golgi's light microscopic method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 21/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50μm sections am made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

How Camillo Golgi became “the Golgi”

On April 1898 Camillo Golgi communicated to the Medical-Surgical Society of Pavia, the discovery of the “internal reticular apparatus”, a novel intracellular organelle which he observed in nerve cells with the silver impregnation he had introduced for the staining of the nervous system. Soon after the discovery it became evident that this cellular component, which was also named the “Golgi apparatus”, was a ubiquitous structure in eukaryotic cells. However the reality of the organelle was questioned for years and many cytologists considered the internal reticular apparatus as an artefact due to the fixation and/or metallic impregnation procedure. The controversy was finally solved in the mid-1950s by electron microscopy when the Golgi apparatus definitely acquired its dignity of being a genuine cell organelle. The designation of “Golgi complex” entered officially in the literature in 1956. Both the terms Golgi apparatus and Golgi complex are currently interchangeable. However a quick “the Golgi” and the introduction of Golgi in adjectival form are now prevalent in the blooming scientific literature on the organelle. Thus Camillo Golgi underwent his final transformation and, becoming the eponym of the organelle he had discovered, he found a way to immortality.     

Cell cycle maintenance and biogenesis of the Golgi complex

How organelle identity is established and maintained, and how organelles divide and partition between daughter cells, are central questions of organelle biology. For the membrane-bound organelles of the secretory and endocytic pathways [including the endoplasmic reticulum (ER), Golgi complex, lysosomes, and endosomes], answering these questions has proved difficult because these organelles undergo continuous exchange of material. As a result, many "resident" proteins are not localized to a single site, organelle boundaries overlap, and when interorganellar membrane flow is interrupted, organelle structure is altered. The existence and identity of these organelles, therefore, appears to be a product of the dynamic processes of membrane trafficking and sorting. This is particularly true for the Golgi complex, which resides and functions at the crossroads of the secretory pathway. The Golgi receives newly synthesized proteins from the ER, covalently modifies them, and then distributes them to various final destinations within the cell. In addition, the Golgi recycles selected components back to the ER. These activities result from the Golgi's distinctive membranes, which are organized as polarized stacks (cis to trans) of flattened cisternae surrounded by tubules and vesicles. Golgi membranes are highly dynamic despite their characteristic organization and morphology, undergoing rapid disassembly and reassembly during mitosis and in response to perturbations in membrane trafficking pathways. How Golgi membranes fragment and disperse under these conditions is only beginning to be clarified, but is central to understanding the mechanism(s) underlying Golgi identity and biogenesis. Recent work, discussed in this review, suggests that membrane recycling pathways operating between the Golgi and ER play an indispensable role in Golgi maintenance and biogenesis, with the Golgi dispersing and reforming through the intermediary of the ER both in mitosis and in interphase when membrane cycling pathways are disrupted.     

Visualizing Biological Membrane Organization and Dynamics

Biological membranes are fascinating. Santiago Ramón y Cajal, who received the Nobel prize in 1906 together with Camillo Golgi for their work on the nervous system, wrote “[…]in the study of this membrane[…] I felt more profoundly than in any other subject of study the shuddering sensation of the unfathomable mystery of life”². The visualization and conceptualization of these biological objects have profoundly shaped many aspects of modern biology, drawing inspiration from experiments, computer simulations, and the imagination of scientists and artists. The aim of this review is to provide a fresh look on current ideas of biological membrane organization and dynamics by discussing selected examples across fields.     

About the presence of interstitial cells of Cajal outside the musculature of the gastrointestinal tract

Santiago Ramon y Cajal observed a special cell type that appeared to function as endstructures of the intrinsic nervous system in several organs. These cells were structurally and functionally further characterized in the gut musculature and named interstitial cells of Cajal (ICC). In recent years, interstitial cells have been identified in the vasculature, urinary tract, glands and other organs. Their morphologies and functions are just beginning to be clarified. It is likely that amongst them, subtypes will be discovered that warrant the classification of interstitial cells of Cajal. This "point of view" continues the discussion on the criteria that should be used to identify ICC outside the musculature of the gut.     

Lucifer yellow - an angel rather than the devil

The fluorescent dye Lucifer yellow (LY) was introduced in 1978, and has been extremely useful in studying cell structure and communications. This dye has been used mostly for labelling cells by intracellular injection from microelectrodes. This review describes the numerous applications of LY, with emphasis on the enteric nervous system and interstitial cells of Cajal. Of particular importance is the dye coupling method, which enables the detection of cell coupling by gap junctions.     

The internal reticular apparatus of Camillo Golgi: a complex, heterogeneous organelle, enriched in acid, neutral, and alkaline phosphatases, and involved in glycosylation, secretion, membrane flow, lysosome formation, and intracellular digestion

Its topography is one of the most characteristic features of the Golgi apparatus and the reticular nature of this organelle is evident in Golgi's first drawings, in light microscopic enzyme cytochemical preparations, and in high voltage electron micrographs of thick sections. Although individual components of the Golgi apparatus may differ in staining characteristics, morphology, contents, and enzymatic activities, they are integrated into a dynamic topographical and functional unit that is closely associated with the endoplasmic reticulum. Modulation of enzymatic activities and morphological and enzymatic heterogeneity are not surprising in an organelle that is the site of both synthetic and digestive events, including glycosylation, sulfation, formation of secretory granules and lysosomes, and the degradation of endocytized material.     

Moving into shape: cell migration during the development and histogenesis of the cerebellum

The enormous expansion the vertebrate nervous system goes through from its first anlage to its adult shape and organization goes along with extensive rearrangements of its constituent cells and typical cellular migrations, often over long distances, and by convoluted pathways. Here, I try to summarize how the cells that form the cerebellum move and migrate during normal cerebellar histogenesis. The cerebellum is made up of a limited set of clearly distinguishable classes of cells, some of which are also readily accessible by genetic tools. Its structure and development have been the focus of studies dating back to at least Ramon y Cajal which have yielded fundamental insights into basic mechanisms of the development of the nervous. During cerebellar histogenesis, several distinct and well-discernable modes of migration may be recognized, some of which have been studied in considerable morphological and molecular detail. Still, often grace to the detail known, a wealth of open questions remains, and the cerebellar anlage remains a highly accessible and promising paradigm for those interested in nervous system development and cell migration in general. I also point out some of the issues that may warrant consideration when results from technically distinct studies are compared and integrated.