The Golgi Stain: invention, diffusion and impact on neurosciences

The black reaction, invented in 1873 by Camillo Golgi (1843-1926, was the first technique to reveal neurons in their entirety, i.e. with all their processes. This important development passed unnoticed at first and only received wide international attention after a long delay. The Golgi stain was widely employed for almost thirty years and was directly responsible for major advances in our knowledge of the microscopic anatomy of the nervous system, as well as in other fields of study. In the hands of other researchers, the black reaction provided vital evidence that helped to establish the neuron theory. The Golgi stain was almost forgotten in the period between the two World Wars, but the introduction of the electron microscope to neurocytological resarch revived its use around the middle of the twentieth century. Today, the black reaction is still used extensively not only in combination with electron microscopy, but also as an autonomous technique in studies on the evolution, ontogeny, and organization of the nervous system.     

The impossible interview with the man of the hidden biological structures. Interview by Paolo Mazzarello

This paper presents an "impossible interview" to Professor Camillo Golgi, placed in time in December 1906. The Italian Professor Golgi from Pavia has been awarded the Nobel Prize for Physiology or Medicine ex aequo with the Spanish anatomist Santiago Ramón y Cajal. Both scientists have obtained the award for their work on the anatomy of the nervous system. However, they have opposite views on the mechanisms underlying nervous functions. Golgi believes that the axons stained by his "black reaction" form a continuous anatomical or functional network along which nervous impulses propagate. Ramón y Cajal is the paladin of the neuron theory, a hypothesis questioned by Golgi in his Nobel lecture of Tuesday, December 11. After the ceremony, an independent journalist has interviewed Professor Golgi in the Grand Hotel in Stockholm. Excerpts about his education, his main scientific discoveries, and his personal life are here given (reconstructing the "impossible interview" on the basis of Golgi's original writings).     

The impossible interview with the man of the neuron doctrine. Interview by Edward G Jones

This paper follows the form of that by Mazzarello that precedes it (Mazzarello, 2006) and presents an imaginary interview with Santiago Ramón y Cajal in December 1906. A few days earlier Cajal had been awarded the Nobel Prize for Physiology or Medicine, an award that he shared equally with Professor Camillo Golgi. Golgi had been recognized for his work as a pioneer into investigations of the nervous system, primarily on account of his discovery of the "black reaction" of silver chromate impregnation of whole nerve cells and their processes. Cajal had been recognized for his implementation of that method and for laying with it the foundations of what was to become modern neuroanatomical science. Paradoxically, the two awardees had been led by their researches to diametrically opposed views of the organization of the nervous system. Golgi believed in a continuous network of axons that formed the basis of all the integrative properties of the nervous system, while Cajal had provided the information that led to the formulation of the neuron doctrine that saw the nervous system as being made up of chains of discontinuous cells joined by polarized functional contacts that we now call synapses. The paper takes the form of an interview with Professor Cajal in the Grand Hotel Stockholm. His responses to questions posed by the imaginary interviewer are all taken from Cajal's own writings.     

Camillo Golgi's Scientific Biography

Born in Corteno, a tiny village in the province of Brescia, Camillo Golgi studied at the University of Pavia where he graduated in medicine in 1865 under the guidance of the psychiatrist Cesare Lombroso who sparked his vocation to study the brain. Golgi then began to learn histological techniques under the direction of the pathologist Giulio Bizzozero. In 1872 he moved to Abbiategrasso as chief of a hospital for chronic diseases. In a rudimentary laboratory he developed the silver-bichromate staining technique, the ‘black reaction’, which was a breakthrough for nervous tissue structure research. While in Abbiategrasso Golgi demonstrated the branching of the axons, and observed striatal and cortical lesions in a case of chorea. He returned to Pavia as Professor of Histology and General Pathology, and made a series of important discoveries that still bear his name: the Golgi tendon organ, the Golgi-Mazzoni corpuscles, another Golgi method to stain nerve cells based on the use of potassium dichromate and mercuric chloride, the canaliculi of the parietal cells of the gastric glands (Müller-Golgi tubules), the Golgi-Rezzonico myelin's annular apparatus (or Golgi-Rezzonico horny funnels), the cycle of malarian parasites (Golgi cycle), the relationship between recurrent malarian fever bouts and the multiplication of the Plasmodium in the blood (Golgi law), the relationship between the vascular pole of the Malpighian glomerulus and the distal tubule, the Golgi's pericellular nets and finally, and most importantly, the cytoplasmic ‘internal reticular apparatus’ (Golgi apparatus). In 1906 Golgi was awarded the Nobel prize for Medicine or Physiology. He died in Pavia on 21 January 1921.     

Some aspects of the history of the law of dynamic polarization of the neuron. From William James to Sherrington, from Cajal and van Gehuchten to Golgi

William James was the first to suggest that propagation of impulses in the nervous system proceeds in one direction, from sensory to motor neurons, but not viceversa. His law of forward direction preceded the formulation of the law of dynamic polarization of van Gehuchten and Cajal, which assumed that nerve impulses are conducted cellulipetally along dendrites and cellulifugally along axons, based on different anatomo-functional properties of these neuronal components. Golgi did not accept the law of dynamic polarization because he believed that dendrites are involved in the nutrition of the neuron rather than in impulse propagation, and that impulses can travel in any direction in the axonal components of the diffuse nerve network. Sherrington in turn experimentally demonstrated that intraneuronic conduction is reversible, whereas, in accord with James's law, propagation of impulses along neuronal chains is irreversible, due to the valve-like action of synapses. The story of the law of dynamic polarization shows that neither Golgi nor Cajal paid much heed to Sherrington's findings and to neurophysiological studies in general, probably because they felt that histology alone could provide the key for understanding the general functioning of the nervous system. It is argued here that this attitude was detrimental to the progress of the neurosciences, because a multidisciplinary approach based on different techniques is inevitably called for in order to develop a plausible theory of the nervous system.     

Camillo Golgi's scientific biography

Born in Corteno, a tiny village in the province of Brescia, Camillo Golgi studied at the University of Pavia where he graduated in medicine in 1865 under the guidance of the psychiatrist Cesare Lombroso who sparked his vocation to study the brain. Golgi then began to learn histological techniques under the direction of the pathologist Giulio Bizzozero. In 1872 he moved to Abbiategrasso as chief of a hospital for chronic diseases. In a rudimentary laboratory he developed the silver-bichromate staining technique, the 'black reaction', which was a breakthrough for nervous tissue structure research. While in Abbiategrasso Golgi demonstrated the branching of the axons, and observed striatal and cortical lesions in a case of chorea. He returned to Pavia as Professor of Histology and General Pathology, and made a series of important discoveries that still bear his name: the Golgi tendon organ, the Golgi-Mazzoni corpuscles, another Golgi method to stain nerve cells based on the use of potassium dichromate and mercuric chloride, the canaliculi of the parietal cells of the gastric glands (Muller-Golgi tubules), the Golgi-Rezzonico myelin's annular apparatus (or Golgi-Rezzonico horny funnels), the cycle of malarian parasites (Golgi cycle), the relationship between recurrent malarian fever bouts and the multiplication of the Plasmodium in the blood (Golgi law), the relationship between the vascular pole of the Malpighian glomerulus and the distal tubule, the Golgi's pericellular nets and finally, and most importantly, the cytoplasmic 'internal reticular apparatus' (Golgi apparatus). In 1906 Golgi was awarded the Nobel prize for Medicine or Physiology. He died in Pavia on 21 January 1921.     

Golgi,Cajal and the Neuron Doctrine

Camillo Golgi and Santiago Ramón y Cajal shared the Nobel Prize in 1906 for their work on the histology of the nerve cell, but both held diametrically opposed views about the Neuron Doctrine which emphasizes the structural, functional and developmental singularity of the nerve cell. Golgi's reticularist views remained entrenched and his work on the nervous system did not venture greatly into new territories after its original flowering, which had greater impact than is now commonly credited. Cajal, by contrast, by the time he was awarded the Nobel Prize, was already breaking new ground with a new staining technique in the field of peripheral nerve regeneration, seeing the reconstruction of a severed nerve by sprouting from the proximal stump as another manifestation of the Neuron Doctrine. Paradoxically, identical studies were going on simultaneously in Golgi's laboratory in the hands of Aldo Perroncito, but the findings did not seem to influence Golgi's thinking on the Neuron Doctrine.     

Colgi, Cajal and the Neuron Doctrine

Camillo Golgi and Santiago Ramon y Cajal shared the Nobel Prize in 1906 for their work on the histology of the nerve cell, but both held diametrically opposed views about the Neuron Doctrine which emphasizes the structural, functional and developmental singularity of the nerve cell. Golgi's reticularist views remained entrenched and his work on the nervous system did not venture greatly into new territories after its original flowering, which had greater impact than is now commonly credited. Cajal, by contrast, by the time he was awarded the Nobel Prize, was already breaking new ground with a new staining technique in the field of peripheral nerve regeneration, seeing the reconstruction of a severed nerve by sprouting from the proximal stump as another manifestation of the Neuron Doctrine. Paradoxically, identical studies were going on simultaneously in Golgi's laboratory in the hands of Aldo Perroncito, but the findings did not seem to influence Golgi's thinking on the Neuron Doctrine.     

Some Aspects of the History of the Law of Dynamic Polarization of the Neuron. From William James to Sherrington,from Cajal and Van Gehuchten to Golgi

William James was the first to suggest that propagation of impulses in the nervous system proceeds in one direction, from sensory to motor neurons, but not viceversa. His law of forward direction preceded the formulation of the law of dynamic polarization of van Gehuchten and Cajal, which assumed that nerve impulses are conducted cellulipetally along dendrites and cellulifugally along axons, based on different anatomo-functional properties of these neuronal components. Golgi did not accept the law of dynamic polarization because he believed that dendrites are involved in the nutrition of the neuron rather than in impulse propagation, and that impulses can travel in any direction in the axonal components of the diffuse nerve network. Sherrington in turn experimentally demonstrated that intraneuronic conduction is reversible, whereas, in accord with James's law, propagation of impulses along neuronal chains is irreversible, due to the valve-like action of synapses. The story of the law of dynamic polarization shows that neither Golgi nor Cajal paid much heed to Sherrington's findings and to neurophysiological studies in general, probably because they felt that histology alone could provide the key for understanding the general functioning of the nervous system. It is argued here that this attitude was detrimental to the progress of the neurosciences, because a multidisciplinary approach based on different techniques is inevitably called for in order to develop a plausible theory of the nervous system.     

The Legacy of Camillo Golgi for Modern Concepts of Brain Organization

The experimental advance made by Camillo Golgi's ‘black reaction’ has been universally recognized as the start of the modern revolution in the study of the nervous system. By contrast, his concepts of nervous organization, particularly his support for the idea of a ‘nervous reticulum’, have been universally rejected. The premise of the present paper is that the ideas of a biologist of this stature deserve re-examination. Golgi's arguments for considering the holistic function of the brain seem to come from his experience as a physician, and presage the views of the gestaltists and, more recently, the conceptual underpinnings of artificial neural networks. His interest in the possible nutritional roles of neuronal dendrites can be seen to anticipate current investigations, at the cellular level, of the metabolic basis of brain imaging. These and other currents in Golgi's thought deserve further study.     

Microscopy in Camillo Golgi's Times

The research performed by Camillo Golgi in histology and pathology dates from 1865, the year in which he obtained his MD degree, to 1923, when his last scientific article was published. Beginning in the mid-1855s, microscope manufacturers in Europe started producing objectives based on the principle of immersion introduced in 1847 by Giovan Battista Amici. The immersion objectives greatly improved the resolution of microscopic observations at high magnifications. From 1860 to 1872, technological improvements in microscope optics and the practicality of their use provided a larger community of investigators effective tools needed to study the structure of the nervous system. This progress in microscopy was associated with the application of new histological techniques, mastered by the chromoargentic reaction introduced by Golgi in 1873. In 1872, further progress in microscopy stemmed from the application of notions of applied physics to the production of microscope optics. These developments in microscopy will be briefly reviewed here.     

The discovery of the Golgi apparatus

The existence of the cell organelle which is now known as Golgi apparatus or Golgi complex, or simply as 'the Golgi", was first reported by Camillo Golgi in 1898, when he described in nerve cells an 'internal reticular apparatus' impregnated by a variant of his chromoargentic staining. It soon became clear that the newly-identified cytoplasmic structure occurred in a variety of cell types. However, the reality of the organelle was questioned for decades, until it was finally ascertained with electron microscopy. The Golgi apparatus was destined to become a protagonist of the research in cytology and cell biology pursued in the second half of the twentieth century.     

Microscopy in Camillo Golgi's times

The research by Camillo Golgi in histology and pathology dates from 1865, the year in which he obtained his MD degree, to 1923, when his last scientific article was published. Beginning in the mid 1855s, microscope manufacturers in Europe started producing objectives based on the principle of immersion introduced in 1847 by Giovan Battista Amici. The immersion objectives greatly improved the resolution of microscopic observations at high magnifications. From 1860 to 1872, technological improvements in microscope optics and the practicality of their use provided a larger community of investigators effective tools needed to study the structure of the nervous system. This progress in microscopy was associated with the application of new histological techniques, mastered by the chromoargentic reaction introduced by Golgi in 1873. In 1872, further progress in microscopy stemmed from the application of notions of applied physics to the production of microscope optics. These developments in microscopy will be briefly reviewed here.     

Painted plaster from Bronze Age Thebes, Boeotia (Greece): a technological study

From the rather difficult excavations that have taken place in Thebes since the beginning of the last century, many fragmentary but often well-preserved paintings on plaster have been unearthed. These belong to several contexts and periods: House of Kadmos (Late Helladic IIIA), Treasury Room (Late Helladic IIIB1) and Oikopedon Spourli (Late Helladic III). Their iconography, style and technology fit well with other paintings from the Late Bronze Age sites found in the vicinity of Thebes (e.g. Gla and Orchomenos). This paper presents a technological study of the Bronze Age painted plaster from Thebes, Greece. The paintings were investigated by means of X-ray diffraction; stereo, reflected light and scanning electron microscopy; some by laser-induced breakdown spectrometry and by micro-Raman spectroscopy, in order to identify the pigments and the composition of the plaster layers, and to determine the painting technique(s) that the artisans may have employed. The plaster layer onto which the paint layer was applied was often the only layer and consisted mainly of calcite, while a clay plaster formed the backing between the lime plaster coat and the actual wall surface. The pigments identified were: yellow ochre, haematite (red), cuprorivaite (Egyptian blue), indigo and possibly riebeckite for blue, charcoal (black), calcite (white), and a combination of black and red for purple. A detailed macroscopic study of the painted surface revealed several features, which indicated that the technique of al fresco painting was employed extensively, a technique already in use since the start of the Late Minoan I phase on Crete. A generally lower level of quality in the appearance of the later paintings (Treasury Room and Oikopedon Spourli) was also noted, but this was not reflected in the materials used or in their overall manipulation.     

The perineuronal net: a weapon for a challenge

Theories and data do not always fit and sometimes are sources of conflicts among scientists. This is the case of a morphological structure, the perineuronal net, which was denied on the basis of an ideological conflict between two giants of neurosciences: Camillo Golgi and Santiago Ramon y Cajal. The perineuronal net is a reticular structure enveloping many neurons. Orginally reported by Golgi in 1893 and 1898 and confirmed by several authors before the turn of the century, the perineuronal net was used by Golgi to support the reticular theory of the organization of the nervous system. Ramon y Cajal, the paladin of the neuronal theory who had also observed this anatomical structure, denied its existence suggesting that it was a fixation artifact. After Cajal's statements, only a few Italian scientists continued to work in this field, and after the 1930s the perineuronal net was forgotten. Only the recent advances in histochemical and immunocytochemical technology confirmed the existence of this structure opening new fields in functional neuroanatomy and neuropathology.     

The Perineuronal Net: A Weapon for a Challenge

Theories and data do not always fit and sometimes are sources of conflicts among scientists. This is the case of a morphological structure, the perineuronal net, which was denied on the basis of an ideological conflict between two giants of neurosciences: Camillo Golgi and Santiago Ramón y Cajal. The perineuronal net is a reticular structure enveloping many neurons. Originally reported by Golgi in 1893 and 1898 and confirmed by several authors before the turn of the century, the perineuronal net was used by Golgi to support the reticular theory of the organization of the nervous system. Ramón y Cajal, the paladin of the neuronal theory who had also observed this anatomical structure, denied its existence suggesting that it was a fixation artifact. After Cajal's statements, only a few Italian scientists continued to work in this field, and after the 1930s the perineuronal net was forgotten. Only the recent advances in histochemical and immunocytochemical technology confirmed the existence of this structure opening new fields in functional neuroanatomy and neuropathology.     

NEW EVIDENCE FOR THE NATURE OF THE ATTIC BLACK GLOSS

New evidence is reported here using transmission electron microscopy, scanning electron microscopy, and laser reflectance for the nature of the black glossy decoration layer present on the Attic black or red figure vases of the sixth to fourth century BC. The black layer, of total thickness about 20 μm, consists mainly of poly crystalline magnetite (Fe₃O₄) particles of dimensions from 0.2 μm down to extremely fine sizes embodied in an amorphous vitreous matrix. On the outer surface of the black paint we discovered for the first time a thin clear glassy film of approximate thickness 0.1 μm which is rich in Al and Fe and poor in Si. This film must be responsible for the characteristic sheen of the surface. This is verified with the reflection of laser light mostly in specular direction, a unique property compared with black paints of other areas and other periods.     

CHARACTERIZATION OF CORAL RED SLIPS ON GREEK ATTIC POTTERY*

Samples of red and black gloss from Greek Attic pottery of the late sixth to fifth centuries bc were examined using scanning electron microscopy (SEM and FIB/STEM). The focus of the study was the chemical and microstructural characterization of the red gloss that was first produced during this period. Two groupings of red gloss were revealed. One red was found to be compositionally similar to the black glosses (labelled ‘LCM coral red’). The other red showed more significant chemical differences, such as higher calcium and magnesium, in comparison to the black (labelled ‘HCM coral red’). The existence of two chemically distinct reds—otherwise identical in colour and texture—suggests that there was more than one source of clay available to the Attic potters for producing red.