Discovering the Structure of Nerve Tissue: Part 2: Gabriel Valentin, Robert Remak, and Jan Evangelista Purkyně.

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Discovering the Structure of Nerve Tissue: Part 2: Gabriel Valentin, Robert Remak, and Jan Evangelista Purkyně Alexandr Chvátal

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Department of Cellular Neurophysiology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic b

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Department of Neuroscience, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic Published online: 03 Feb 2015.

To cite this article: Alexandr Chvátal (2015): Discovering the Structure of Nerve Tissue: Part 2: Gabriel Valentin, Robert Remak, and Jan Evangelista Purkyně, Journal of the History of the Neurosciences: Basic and Clinical Perspectives, DOI: 10.1080/0964704X.2014.977677 To link to this article: http://dx.doi.org/10.1080/0964704X.2014.977677

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Journal of the History of the Neurosciences, 00:1–26, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 0964-704X print / 1744-5213 online DOI: 10.1080/0964704X.2014.977677

Discovering the Structure of Nerve Tissue: Part 2: Gabriel Valentin, Robert Remak, and Jan Evangelista Purkynˇe ALEXANDR CHVÁTAL1,2


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Department of Cellular Neurophysiology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic 2 Department of Neuroscience, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic During the 1830s, the use of improved microscopic techniques together with new histological methods, including tissue fixation, allowed more precise data to be obtained concerning the structure of nerve tissue of animals as well as humans. The present article, based on the translations of original texts never before published, brings together for the first time the discoveries of famous scholars Gustav Valentin, Robert Remak, and Jan Evangelista Purkynˇe, who made their significant discoveries in the field of neuroscience almost simultaneously and shows how their findings affected each other. In addition, this article also contains digitally remastered and reconstructed figures published in the original works of Valentin, Remak, and Purkynˇe and they are displayed for the first time in high quality. Although the fundamental discoveries of these famous scholars did not imply the discovery of nerve cells as we know them today, they were certainly a very important basis for further research of many other eminent scholars during the second half of the nineteenth century. Keywords

nerves, glia, ependymal, brain, structure, history, neuroscience

Introduction Significant discoveries in the study of the nervous system, especially its structure, were made in the 1830s, when the first systematic attempts to clarify basic neurophysiological functions were conducted and the directions for further research into the microscopic structure of nerve tissue were determined. In addition to nerve fibers, it was already possible to observe various cell structures as parts of nerve tissue, including neurons and other elements such as astrocytes, oligodendrocytes, ependymal cells as well as radial glia in the central nervous system, and Schwann and satellite cells in the peripheral nervous system. Interpretation of obtained findings was therefore initially very difficult; however, further and more detailed research, which continues even today, gradually revealed the finest structure of nerve tissue. The present article, based on the translations of original texts never before published, for the first time compiles together the discoveries of famous scholars Gustav Valentin, Robert Remak, and Jan Evangelista Purkynˇe, who made their significant Address correspondence to Assoc. Prof. Alexandr Chvátal, PhD, DSc, MBA, Department of Cellular Neurophysiology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídeˇnská 1083, 142 20 Prague 4 – Krˇc, Czech Republic. E-mail: [email protected]

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discoveries in the field of neuroscience almost simultaneously, and shows how their findings affected each other. While the discoveries of Purkynˇe were described and analyzed in a number of papers (Gibson, 1968; Hykeš, 1936; Kruta, 1969; Posner, 1969; Clarke & O’Malley, 1996), the contributions of Remak and Valentin are less known (Kisch, 1954; Clarke & O’Malley, 1996). The present article using the translations of the original works of these famous scholars uncovers and compares their real contributions to discoveries in the field of neuroanatomy. In addition, this review also contains digitally remastered and reconstructed figures published in the original works of Valentin, Remak, and Purkynˇe and they are displayed in high quality for the first time. Since interest in the history of neuroscientific discoveries is increasing, their review may be particularly useful for historians of neuroscience as well as for neuroscientists, concerning important contributions in the field of neuroanatomy during the first half of the nineteenth century.

Biographical Notes Jan Evangelista Purkynˇe (1787–1869) Biographical data and the scientific legacy of Purkynˇe have been documented in detail in a large number of articles, monographs, and proceedings (Eselt, 1859; Amerling, 1918; Hykeš, 1936; Studniˇcka, 1936, 1953; Borovanský & Weigner, 1937; Gibson, 1968; Kruta, 1969, 1971, 1977; Posner, 1969; Trávníˇcková, 1986; Jay, 2000; Pokorný & Trojan, 2005; Rokyta, 2011; Vožeh, 2011; Žárský, 2012) and will be summarized here. Purkynˇe (should be read as Poorkeynie) was born in 1787 in Libochovice (now a district of Lovosice, Czech Republic), where he also began school. He was then sent for studies to the Piarist monastery in Mikulov in the Moravian region of the Czech Republic, where he completed high school; a vision of a future teaching profession attracted him to join the Piarist order. As a teacher, Purkynˇe worked in the Czech towns of Strážnice and Litomyšl, where he also learned foreign languages, and, after his studies of the philosophical literature, he was motivated to obtain a university education. He moved to Prague where, in 1807 he began his studies in the philosophy faculty at Prague University. To support himself, Purkynˇe also started to work as a home tutor. After some time, he was fortunately hired as a tutor for the family of Baron Ferdinand Hildbrandt, who supported him during his subsequent studies. With the intention of pursuing a scientific career, Purkynˇe moved from the philosophical to the medical faculty. Gross anatomy and physiology were taught at that time by Professor Josef Rottenberger (1760–1834), a native of Mikulov, using the textbooks of Jiˇrí Procháska (1749–1820), who had spent 12 years as a professor of anatomy at the same faculty (Chvátal, 2014b). Passing the relevant examinations, Purkynˇe completed his medical studies during the summer of 1818, and, in the same year, he defended his thesis on the topic of subjective visual phenomena (Purkynˇe, 1819). Subsequently, he became a prosector and assistant in physiology and anatomy at Prague University. His published dissertation attracted interest from the scientific community, even from Johann Wolfgang Goethe (1749–1832), who was also interested in the theoretical hypotheses on color perception in humans. In 1820, Purkynˇe unsuccessfully applied for professorships of pharmacology in Prague and of physiology in Pest (now in the eastern part of Budapest, Hungary) as well as for the position of chair of anatomy and physiology in Graz (Austria) and the chair of anatomy in Ljubljana (Slovenia). The question remains whether these failures were caused by Purkynˇe’s assumed membership in one of the secret societies such as the Illuminati,


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which were opposing the Austrian government in Vienna. Finally in 1821, the professorship position became vacant at the University of Breslau (now Wroclaw in Poland), which at that time was part of Prussia. The Czech native and professor in Berlin, Johan Nepomuk Rust (1775–1840), notified Purkynˇe about this position. Rust urged Purkynˇe to apply for the position of professor in Wroclaw and promised to help him. Such an offer certainly carried weight, since Rust, among others, who held the position of secret medical counselor in Berlin, was a member of the medical department of the ministry of culture, education, and health affairs and served as president of the board of trustees for hospital affairs, which he established (Sajner, 1977). In addition, Rust was “station master” of the Berlin Masonic lodge with extensive contacts with influential officials. Secret societies in Prussia, unlike in Austria, were not persecuted at that time. However, Purkynˇe, as a citizen of Catholic Austria and without even an associate professorship, was in a difficult position. A major benefit for him was his participation in a congress of German naturalists and physicians in Leipzig in 1822, after which he also visited professor Rust in Berlin. Rust introduced young Purkynˇe to the local community and acquainted him with significant scientific personalities, inter alia, with his future father-in-law, the Berlin professor of anatomy and physiology Karl Asmund Rudolphi (1771–1832). Perhaps Rust presumed that the ministry would ask Rudolphi for his opinion of Purkynˇe. In any case, after the completion of all approval procedures, the King of Prussia officially appointed Purkynˇe as professor in the Faculty of Medicine of Wroclaw University in January 1823, and Purkynˇe, with his characteristic zeal, started his research, teaching, and organizing activities in the faculty. The relations between Purkynˇe and Rudolphi remained very good over the years, and, in 1827, Purkynˇe married Rudolphi’s daughter Julie; he even lived in Rudolphi’s house in Berlin for a time during 1828. It is believed that it was Rudolphi, an excellent microscopist and histologist, who convinced Purkynˇe to perform microscopic research (Studniˇcka, 1935). Purkynˇe did not have a good compound microscope at that time, so his initial research in the field of microscopy, including the anatomy of plants, animals, and humans, was carried out using a simple microscope. After great effort, in 1832 Purkynˇe managed to obtain a compound achromatic microscope made by the Austrian optical instrument maker Simon Plössl (1794–1868). At the same time, Purkynˇe suffered a series of deaths of his loved ones. In 1832, his father-in-law Karl Rudolphi died; in the same year, two of Purkynˇe’s daughters died of cholera; in 1834, Purkynˇe’s mother died, and, during the following year, Purkynˇe’s wife, Julie, also died. Despite these tragic events, Purkynˇe remained fully devoted to research, the education of students, and efforts to establish and manage the Institute of Physiology at Wroclaw University. Rudolphi’s position was filled after his death by a relatively young German comparative anatomist and physiologist, Johannes Peter Müller (1801–1858), credited with the further development of the physiological and anatomical institute in Berlin. Contacts between Müller’s institute in Berlin and Purkynˇe’s laboratory presumably continued after Rudolphi’s death. In any case, both Purkynˇe and Müller were well aware of the role of microscopy in the study of human and animal tissues and therefore over the years these two institutions in Berlin and Wroclaw ascended to the top of microscopic research in Europe (Kisch, 1954). In 1849, after a 17-year stay in Wroclaw, Purkynˇe was offered the position of professor of physiology at the medical faculty of Prague University. In the spring of 1850, at the age of 63, he moved to Prague and joined the medical faculty as professor of physiology. In Prague, Purkynˇe very actively integrated into public, cultural, and political life and significantly contributed to the popularization of science through the founding of the journal

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Živa. In addition, due to his efforts, a physiological institute was opened at the Faculty of Medicine in Prague in 1851, the first of its kind in the Austrian Empire, where he continued his research and teaching practice until his death in 1869.


Gabriel Valentin (1810–1883) and Robert Remak (1815–1865) One of the earliest and most gifted of Purkynˇe’s students was Gabriel Gustav Valentin, born in Wroclaw, son of a jeweler and rabbi assistant. He was extremely diligent and systematic, and it is claimed that he was even more systematic in his scientific work and publication activity than Purkynˇe himself (Kruta, 1967). Valentin published the results of his research in extensive and detailed treatises, while Purkynˇe was usually satisfied with just short notices of his lectures. Initially, Valentin worked separately, apparently under the guidance of Purkynˇe, but most likely after 1832, when he finished his studies at the university, he began to have access to the laboratory that Purkynˇe had set up in his apartment after the deaths of his wife and daughters (Studniˇcka, 1935). In 1835, Valentin issued, probably in cooperation with Purkynˇe, a textbook on embryology, the first of its kind. Valentin and Purkynˇe worked in the same room, presumably discussing the results of their observations; in a number of Valentin’s publications, some observations are even explicitly identified as Purkynˇe’s. However, in 1835, the relationship between the two scientists suddenly deteriorated, and they split, either due to issues on the use of books from Purkynˇe’s library or to conflicts on the use of the microscope. Valentin therefore decided to apply for a professorship at several universities in Europe, and shortly thereafter, at the age of 26, he was appointed a position at the University of Bern, where he later moved and worked for the rest of his life. Earlier, in 1836, he visited Müller’s institute in Berlin, where he presented the results of his latest observations. Purkynˇe mentored a number of students, besides Valentin, and included 13 of their dissertations in the bibliography of his own publications, but none of these students achieved Valentin’s fame. A number of young students also worked at the institute of Johannes Müller in Berlin (Finger & Wade, 2002), among them were, for example, Theodor Schwann (1810–1882), Jacob Henle (1808–1883), Rudolph Virchow (1821–1902), and Rudolph Albert von Kölliker (1817–1905). Each became famous in his own field, but one of them, Robert Remak, became world famous in the field of microscopic research of nerve tissue (Kisch, 1954). Robert Remak was born in 1815 in Poznan (Poland) to a Jewish family and studied medicine at the University of Berlin, where he was one of the first students of Johannes Müller. Among his teachers was also Christian Ehrenberg (1795–1876). In his publications, Remak acknowledged these two mentors for their fruitful discussions and for the opportunity to use their microscopes. Based on his famous dissertation, which did not meet with general understanding at that time, he graduated from the University of Berlin in 1838, where he became associate professor in 1859 and remained for the rest of his life; he was not appointed to any higher position.

Contributions of Gabriel Valentin to the Structure of the Nervous System Data on the morphological characteristics of nerve fibers, derived from studies pursued in Purkynˇe’s laboratory, were published by Valentin in 1834. In his work, he referred to the observations of Felice Fontana (1730–1805) and Ehrenberg and tried to morphometrically characterize the mainly segmented (varicose, jointed) nerve fibers with regular globular or spherical extensions, similar to the beaded chains previously described by Ehrenberg (1833). Valentin tried to find a relationship between the thickness of nerve fibers and their



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location in different areas of the central nervous system. He found that the thickest fibers were present in the spinal cord, while thin fibers occurred in virtually all areas of the brain and spinal cord. Valentin was not able to find any obvious physiological significance of the observed differences: Even in minute areas of nerve tissue, hundreds of nerve fibers crossed over without visible organization, which represented a major obstacle in determining their length, course, and interconnection. According to Valentin, the basic structure of fiber tissue in the brain was made up of a fine-grained watery substance, varicose fibers, and globules of different shapes and sizes. Varicose fibers rarely divided and did not anastomose; the fibers were easily recognizable by dark contours that ran in parallel with the much lighter internal parts of the nerve fibers. Valentin could not conclude whether nerve fibers were hollow or not. He observed that the outer part consisted of a double circle from which, when pressed using a compressorium (a device that compressed tissue between two glass plates under a microscope), a milky, translucent, and oily liquid flowed out. In most cases, this space contained no particles or bodies, but occasionally, particularly in the beady, extended portions of the nerve fibers of the brain and spinal cord, small and highly transparent globules could be observed. Their origin was not clear to Valentin, and he wondered whether they were the cause of the beady swellings of the nerve fibers. The globules in the “fibrous areas” of the nerve tissue were spherical or elliptical in shape, often with one process, filled with liquid, probably an oily substance; Valentin hypothesized that they were involved in the formation of nerve fibers. Valentin’s work is also interesting from the methodological point of view. For example, he found that the integrity of varicose nerve fibers in the brain and spinal cord depended on the solution in which the tissue was kept. In alcohol, nerve fibers turned within minutes into an unorganized granular mass, as was the case in a solution of potassium carbonate or ammonium hydroxide. In contrast, after immersion into a saturated solution of sodium chloride or ammonium chloride, the tissue could supposedly be preserved unchanged for several weeks. Finally, Valentin could not forget to mention one historical note: “For those of us who studied at the University of Wroclaw, the fibers described by Ehrenberg were not new, since Purkynˇe had already been showing them in his physiological lectures for many years. I had the opportunity to see them as a student between 1829 and 1830, and each of my former classmates would certainly be happy to confirm this” (Valentin, 1834, p. 409). An additional publication dealing with the microscopic structure of nerve tissue was published by Valentin in 1836, shortly after the insurmountable rift with his mentor and collaborator, Purkynˇe. The publication was relatively long (200 pages) and was quite often cited because it presumably contained the first illustration of brain cells (round or elongated bodies with a nucleus, sometimes with a nucleolus). Based on the large amount of processed material and results, it is most likely that the publication contained data from the relatively long period of joint work between Valentin and Purkynˇe. In many instances, Valentin cited descriptions of Purkynˇe’s methods as well as Purkynˇe’s unpublished results. The publication contained three major sections: The first provided observations of nerve tissue structure in humans and vertebrates accompanied by an extensive set of figures, the second part was an overview of nerve tissue structure based on the results described in the previous section, and the third part, designed as an appendix, contained a description of the structure of nerve tissue of invertebrates. There was no special section summarizing the methods used by Valentin, but it can be assumed that the methods and tools were those commonly used in Purkynˇe’s laboratory. Valentin also mentioned the cutting of thin sections using an adapted knife, and apparently he also often used a compressorium, as well as the same method of freezing the sections as used by Purkynˇe.


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Valentin hypothesized that the nervous system was composed of two types of basic matter, namely isolated spherical bodies and uninterrupted primitive, isolated fibers (Fig. 1B, F). The first type represented the “creative and active”, that is, the “higher” elements, while the second type represented the “conducting and receiving”, that is, the “lower” elements. According to Valentin, each unit was enwrapped by sheaths of cellular tissue, specific for each tissue type, and was responsible for the interactions between the two types of tissue; they were unique and special units of the nervous system. In addition, other general systems occurred, namely the system of blood capillaries and peculiar formations, such as cellular (probably connective) tissue, fat, and deposits of inorganic pigment. Overall, according to Valentin, the central and peripheral nervous systems exhibited an extraordinary similarity. Each nerve bundle, according to Valentin, was composed of larger or smaller quantities of varicose fibers, usually oriented in parallel or intersecting directions, which could be observed with the naked eye or using a weakly magnifying glass. Varicose fibers, as the basic element of the nerve fiber structure, were seen in very thin sections of sufficient translucence. Valentin stated that they were best seen in the velum of the cerebellum, as Purkynˇe had first shown him. In the brain and spinal cord, each nerve fiber formed a continuous, varicose, unbranched fiber that coursed without interruption or division to its end, that is, to the gray matter, being composed of sections of different density, either coated by a sheath of cellular tissue or with a uniformly clear, colorless, and semi-liquid content. This description resembles that of a nerve fiber with its myelin sheath. It is evident from Valentin’s publication that he did not observe nerve fiber endings or fiber connections (i.e., synapses). As he illustrated in his figures, the nerve fibers of muscle tissue, teeth, or brain and spinal cord tissue ended with loops or formed plexuses (Fig. 1A). Valentin also reported that the contents of nerves were quite uniform (light, colorless, transparent, semi-liquid, without particles, globules, bubbles, or fibers), and that the sheaths of the elementary primitive fibers were very simple. According to him, all the other, often visible forms, were the result of the methods of preparation and therefore did not represent the true image of nature but rather documented the observers’ procedures and efforts. Valentin also indicated that there were some disputes as to whether varicose fibers represented hollow channels or not. They contained internal parallel walls and, when pressed, the fiber content did not coagulate but remained oily. Valentin also described the different thickness of nerve fiber sheaths: Peripheral fibers contained thicker and stiffer sheaths than the fibers in the brain and spinal cord. He also described how compression of the fibers may produce beady enlargements on varicose fibers, as he observed on pressed nerve tissue from the spinal cord containing nerve fibers crossing the pia mater. Besides fibers, Valentin observed in the central nervous system a number of globules, in various areas of the brain gray matter, cerebellum, spinal cord, and between nerve fibers. Globules were present in the epithelium-like membrane, the ependyma within the brain ventricles, where they participated in the formation of the choroid plexus. They were also present in the peripheral nervous system, especially in the ganglia, where they created quite specific and characteristic formations. Valentin described in detail the structure of various ganglia and also described their differences in different vertebrate species including humans. He was interested in the passage of nerve fibers through the ganglia and described globules in the ganglia. He observed that in the ganglia these large globules were often coated or woven by nerve fibers coursing in various directions. Each of these globules had an outer smaller or larger cellular sheath and contained its own medullar substance, a nucleus, and a round and transparent core, probably a nucleolus. As Valentin noted, these structures resembled, at a first glance, oocytes


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Figure 1. The structure of nerve tissue by Gustav Valentin (1836). Part A: The nerve endings in the dental sac of the third upper molar of a human: (A) one of the main strands; (a) finer strands; (b) a network of nerve fibers; and (c) looped endings of the primitive fibers. Part B: A small portion from the center of the auditory ganglia (ganglium oticum) of sheep, gently squeezed. It is possible to observe wavy or straight primitive fibers passing through the ganglion and globules of the overlying matters (Belegungsmasse): (a) continuous fibers; (b) twisted primitive fibers; and (c) globules of the overlying matter. Part C: The human choroid plexus epithelium in the cerebellum, where each cell is covered on the outside by spherical pigment particles: (a) one villus; (b) single cells; (c) the nuclei contained in the cells; and (d) the pigment bodies outside the cells. Part D: Single globule of the human yellow matter, terminated by a tail-like process: (a) parenchyma; (b) tail-like process; (c) vesicle-like core similar to the germinal vesicle; and (d) bodies present on the surface. Part E: Globules without ensheathments located at the bottom of the trigeminal ganglia: (a) parenchyma of globules; (b) a body similar to the germinal vesicle; and (c) bodies present on the surface. Part F: Fibers and globules in the sheep spinal cord. A thin section of the spinal cord in the cervical region, medulla oblongata: (a) the undulated longitudinal primitive fibers forming plexuses and (b) globules present in the interstitial space. Note. The original illustrations were digitally restored by the author of the present article.


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in the ovary. The number and location of globules in a ganglion determined whether the ganglion had more of a triangular shape, as seen in humans, elongated, as in most other mammals, or was just a thickened area of nerve, as for most poultry. Valentin observed marked differences among globules and most interesting peculiarities: mostly reddish-gray, fine-grained, slightly rubbery substance, often containing filaments but permeable to light. According to Valentin, these globules formed almost perfect spheres in intact ganglia, but the shape changed quickly after the removal of the ganglia sheaths when they displayed an elongated shape, sometimes with a tail-shaped process. This could lead to the conclusion that such a globular process could initiate the generation of the globule’s own nerve fibers. However, as Valentin highlighted, given that the content of the nerve fibers and globules was significantly different and that cellulous sheaths tightly enclosed the content of the globules, this assumption was incompatible with the existing doctrine of the physics of the nervous system. According to Valentin, globules could be observed in detail at the border between the gray-red and white brain matter. As he indicated, sometimes it was possible to observe individual globules, rounded on one side and with a tail-shaped process on the other (Fig. 1D and 1E). Valentin noted that these formations were first observed by Purkynˇe in sheep and then managed to describe them later in the cerebellum and cerebrum in humans, calves, sheep, pigs, and horses. He explained how it was possible at first sight to observe exactly the same formations that he previously described in the ganglia, namely a bright inner body and a small nucleus close to the surface. Valentin devoted a substantial part of his publication to a description of the ciliary epithelium in the cerebral ventricles (Fig. 1C). He thus compared his findings with those of two papers; the first he had coauthored with Purkynˇe in 1835, in which they had described the ciliated epithelium in the respiratory and genital tracts, and the second was a paper published by Purkynˇe on the presence of ciliated epithelium in the cerebral ventricles (Purkynˇe, 1836). Valentin also described and illustrated in detail the choroid plexus, which consisted of branched capillaries and peculiar epithelium; he emphasized that the choroid plexus was first observed by Purkynˇe in calves and that Valentin himself observed it later in humans, sheep, guinea pigs, geese, and pigeons. According to Valentin, a seemingly simple membrane formed the epithelium, but its structure, as for example in sheep and humans, was very complex. It consisted of villi full of capillary vessels or flakes floating in water and was very similar to chorion villi. The granular membrane was enwrapped by a translucent epithelium consisting of single globules of a hexagonal shape and were colorless and transparent and contained a core. However, in the middle of each core there was a dark, round formation, which reminded Valentin of similar formations that occurred in the cells of the epidermis, in the pistils of flowers, etc. Some globules of the choroid plexus even contained pigmented bodies. In the second part of his publication, Valentin summarized his findings and put forward several hypotheses about the function of the nervous system. The activity of the nervous system was, in earlier times, often compared to the effects of electricity—quite unjustly, as Valentin noted. The performance of physical factors, as well as of the nervous system, was, according to him, based on the same laws. The emerging science of electromagnetism, however, suggested that wires could be something more than mere passive lines. He further noted that the attempts of Faraday and others had shown that wires could be connected without direct contact and that they could affect each other even when separated by an insulating layer. Could these conditions also apply to the nerves, Valentin wondered, and could they have the same subtle effects?



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The publications of Gabriel Valentin are considered significant even today in terms of their contribution to knowledge of the structure of nerve tissue; especially his paper published in 1836 that had already been viewed as significant by the Swiss anatomist and physiologist Albert von Kölliker, who also made significant contributions to the field of neurohistology and described Valentin’s work as “epochal and the first good description of the elements of the nervous system” (Rádl, 1900; Kisch, 1954; Clarke & O’Malley, 1996). Valentin’s globules can be regarded as the cells we know today. The terms “nucleus” and “nucleolus,” as well as illustrations of cell formations containing organelles, were the first of their kind in the scientific literature, despite the fact that the cell theory was formulated and published three years later. Valentin’s hypothesis of the structure of nerve tissue is also very significant. He describes cell bodies and fibers as separated; a concept he stubbornly insisted on for many years. It should also be kept in mind that Valentin’s fundamental observations were made during the time of his close cooperation with Purkynˇe (and most likely under his supervision), and we can assume that the more experienced and respected Purkynˇe, who was 49 years old at that time, was directly involved not only in the observations of his former student, 26-year-old Valentin, but also in the interpretation of the results. Indeed, in Valentin’s publications, the priority of a number of Purkynˇe’s observations was mentioned in many instances, a fact that has since been frequently forgotten. One year later, at the Congress of German Scientists in Prague in 1837, Purkynˇe reported results similar to those of Valentin, though the content and figures were somewhat different. We will probably never know how the two scholars conducted their observations in the same laboratory and using the same microscope, the hypotheses they discussed and conclusions they formulated, or their agreement or disagreement. Incidentally, when Purkynˇe published his memoirs in Živa in 1858, he commented not only on his presentation at the above-mentioned congress but also on the period of his research into ganglionic bodies, about which he wrote the following: “It should be fairly noted here that Prof. Valentin diligently attended this research, especially focusing on the examination of the ganglion nodes. His treatise is published in the Acts of Leopold’s Academy. He communicated there also my main thoughts about the importance of the basic elements of the nervous system, especially there is mentioned (p. 89) my firstly conceived analogy between oocytes and ganglionic bodies” (Purkynˇe, 1858, p. 43).

Contributions of Robert Remak to the Structure of the Nervous System In his first publication, written for a scientific essay contest in 1836, 21-year-old Remak described the various fibers (Fig. 2A) he identified in the rabbit cerebrospinal nerves during development and compared this data with the adult nerve tissue (Remak, 1836). He found that during the third embryonic week, spinal nerves consisted partly of irregular spherical and partially elongated transparent bodies, accompanied by filaments arranged in rows without any fibrous structures. During the fourth and fifth weeks after a rabbit’s birth, Remak distinguished four types of fibers in the cerebrospinal nerves. The first were thick cylindrical fibers (or medullar fibers) ranging in thickness from 5.3 to 12.7 µm. In a longitudinal view, these fibers appeared as irregularly tortuous with jagged edges, inside which he observed subtle contours arranged in parallel with the outer edges. After pressing it, Remak observed that marrow leaked out. In addition, he often observed on transverse sections of the fibers that the outer walls had a kind of content in the form of a double ring, similar to the previous observations of Valentin in varicose fibers. The second fibers

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Figure 2. The structure of nerve tissue by Robert Remak. Panel A: The structure of nerve fibers as published in 1836 (Remak, 1836). Part a: A part of the nerve branching of the subscapularis muscle of a 4-week-old rabbit, magnified 300×: (a) and (b) the place of nerve fiber constriction AA. Part b: Nerve fibers of the same animal and at the same magnification: (a) a site of accidental damage to the fiber; (b) constriction, which most often occurs in all fibers; (c) a regularly varicose fiber; (d) intermediate filaments; (e) an unmyelinated cylindrical fiber; and (f) marrow fibers. Panel B: The structure of nerve fibers as published in 1838 (Remak, 1838). Part a: The figure shows a small part of a cow sympathetic nerve, enlarged about 200×: (a) and (b) irregular primitive fibers; (c), (d), (e), (f), and (h) translucent pale organic fibers. Microscopic organic fibers are visible (g, h, c, d, e), on which oval extensions are present where it seems like fine fibers dissolve. Sometimes crossing fibers can be seen from which emerges a clearly thicker filament (e). Oval extensions sometimes contain nuclei and nucleoli. Part b: Three spherical bodies with nuclei from the calf spinal ganglia underlying the various types of organic fibers, magnified about 200×: (A) two interconnected globules, the smaller contains nuclei. From the larger formation, on whose surface there are filaments and an oval extension, emerges a bundle of organic fibers (a); (B) a globule with a core from which emerges a twisted filament; (C) a globule from which a fiber bundle emerges (b), the fibers passing into oval extensions. Part c: Primitive fiber in the valvula cerebelli, magnified 110×. It is an obvious translucent fiber with thin sheaths. Part d: Several globules with a core in the yellow matter (substantia flava) of a cow brain with long and branched processes, magnified 75×. Part e: Thin and wavy fibers of the lateral area of the lower section of the spinal cord gray matter, magnified 200×. Note. The original illustrations were digitally restored by the author of the present article.



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were thin and cylindrical, 1.7 to 5.3 µm thick, usually without marrow and translucent, never had jagged edges, and always lacked a double ring. The third were fibers containing varicosities along their entire length, 2.4 to 5.3 µm thick, and seldom exceeding 6.6 µm in diameter in places with varicose extensions. Lastly, he distinguished intermediate fibers that changed their cylindrical shape to varicose, were of a thickness greater than 4.5 µm, often observed in the sympathetic nerves and accompanied by small round globules that he had never observed in varicose fibers. In addition, in his paper, Remak compared individual types of fibers in the spinal roots and in sensory and motor nerves and described changes during development. In his subsequent publication, Remak (1837) described the results of his detailed study of myelinated primitive fibers of the cerebrospinal nerves. Just as Valentin, Remak inclined to the view that the natural state of all fibers was cylindrical and that varicosities arose subsequently, for example, during the preparation of nerve tissue and probably also due to the immersion of the tissue in water. Remak summarized his observations in the following conclusions. First, each primitive fiber in the cerebrospinal nerves was enwrapped by a very soft and translucent cellular sheath, with a thickness of the fiber itself. This sheath was very different from that of nerve bundles covered by a fibrous sheath (probably the perineurium). Remak noted that these fibers were specific in the way they expanded along fibers into fine nodules and were partially covered on their edges by variously shaped, usually rounded, bodies. Second, the primitive fibers themselves consisted of thin-walled tubes; they contained a marrow (this is why Remak named them medullar fibers) and were surrounded by numerous lateral lobes forming horizontal lines intermittently interrupted by transverse stripes. He further considered that the observed expansion of the varicose fibers was actually a modification of the described nodule-like extensions. And lastly, primitive fibers did not contain spherical marrow but rather an elongated formation, although Remak admitted that such an observation could be due to nerve compression. The most important of Remak’s publications dealing with the study of the structure of nerve tissue was his dissertation published in 1838. He described in detail the structure of the nerve fibers of the peripheral nervous system, namely the cerebrospinal and sympathetic nerves, and the structure of the central nervous system, where he also included sympathetic and spinal ganglia (Remak, 1838). At that time, Valentin had already published his extensive work on the structure of nerve tissue, and Remak probably also knew the content of Purkynˇe’s communication on the structure of nerve tissue presented at the Congress of German Scientists in Prague in 1837, the abstract of which was published only in 1838. Concerning nerve structure, Remak extended his previous findings, particularly on sympathetic nerves. He was convinced that the color and peculiar shape of sympathetic nerve fibers were not due to interconnected globular formations containing nuclei but to a special structure of primitive fibers arising from the ganglia (Fig. 2Ba, e). According to Remak, these fibers were not surrounded by sheaths, but were naked, transparent, almost gel-like, and much thinner than other primitive myelinated fibers. They expressed on their surfaces longitudinal lines, and it was possible to divide them into small filaments. Remak noted that these fibers were often surrounded by oval formations along their entire length and were covered with small oval or rounded, sometimes irregular, bodies (probably Schwann cells) containing one or more nuclei of the same size as the nuclei of ganglionic bodies. Remak described in detail the structure of sympathetic ganglia. As he noted, Valentin and Purkynˇe were the first who attempted to describe the structure of ganglia. According to Remak, he could only partially confirm Valentin’s observations on the fibers passing through the ganglia. He observed no differences between transiting and wavy fibers; the


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fibers formed bundles, predominantly in the middle of ganglia, so they were very close to each other. In addition, in the middle of such bundles Remak observed only a few ganglionic bodies, while at the outer edges of the ganglia, the fibers were curled and, due to the additional space, were accompanied by a greater number of ganglionic bodies. Valentin, as Remak commented, did not comprehend or did not want to comprehend a very important finding for understanding the nature of the ganglia, namely that the organic, that is, unmyelinated, fibers originated from ganglionic bodies containing a nucleus and that this feature, even if requiring great skill in tissue preparation and examination, was well evident (Fig. 2Bb, d). Thus in describing the sympathetic ganglia, Remak claimed that from the mass of the ganglionic bodies extended either the bundles of very thin nontubular fibers surrounded along their course by oval formations and bodies with nuclei, similar to unmyelinated fibers (Fig. 2Bc), or very thin fibers extending from several areas of the ganglionic bodies, often node-like at their origin and passing directly into the organic fibers. Remak proposed that the sympathetic ganglia should be considered as the true centers of an organic, that is, autonomic, nervous system. According to him, the only difference between the sympathetic and spinal ganglia was the large number of protruding fibers. Consequently, he speculated that even the spinal ganglia were related to the autonomic nervous system. The results obtained by Robert Remak have been of great significance for neurohistology. Although descriptions of myelinated fibers were already known from the works of Procháska, Fontana, Ehrenberg, and Valentin, Remak described myelinated fibers in detail, including the fibers’ transverse constrictions, which in retrospect could be the nodes of Ranvier or Schmidt-Lanterman incisures. In addition, Remak also described unmyelinated fibers (known now as “C-fibers” or “Remak’s fibers”) that are present in postganglionic sympathetic fibers and in some preganglionic sympathetic and parasympathetic fibers (Griffin & Thompson, 2008). These fibers form bundles, sometimes referred to as “Remak bundles,” which are enwrapped by nonmyelinating Schwann cells. Undoubtedly, the most important finding of Robert Remak was the fact that nerve fibers and ganglionic bodies (nerve cells) are linked to each other. However, as noted by Kisch, these findings were doubted not only by Valentin, who believed that Remak’s fibers were incorrect interpretations of artifacts (not to mention the link between ganglionic bodies with fibers), but also by Remak’s teacher Johaness Müller who, although he announced in his yearbook Remak’s description of nerve fibers, considered Valentin to be correct in claiming that the nerve fiber endings were only surrounded by ganglionic bodies without any interconnection (Kisch, 1954). Only in 1842 did Valentin partially admit his mistake, but only regarding the Remak fibers.

Contribution of Jan Evangelista Purkynˇe to the Structure of the Nerve System In contrast to Valentin and Remak, who published their observations in the form of more or less extensive works, Purkynˇe published only a few original scientific publications during the 1830s. This could have been due to personal reasons (distress due to the deaths in his family) and/or to a lack of time due to his management and teaching responsibilities. Therefore, most of the sources on the scientific discoveries of Purkynˇe in that period are represented by the aforementioned references by Valentin, by brief reports on the progress of Purkynˇe’s scientific activities in a newsletter published by the Scientific Section of the Silesian Society in Wroclaw, and by Purkynˇe’s memoirs summarizing his scientific work in the magazine Živa, published during 1857–1858.



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The structure of nerve tissue was in the focus of Purkynˇe’s interests for a long time before the memorable meeting of German naturalists and physicians in Prague in 1837, where, among other topics, he presented his famous hypotheses on the structure of nerve tissue. Archival data reveal that already in 1827 he had lectured on the structure of the brain (Purkynˇe, 1937a), particularly its fiber structure with a special focus on methods for studying brain anatomy. At that time, brain anatomy was described mainly by German scholars, including the neuroanatomist and physiologist Franz Joseph Gall (1758–1828), the physician, physiologist, anatomist, and psychiatrist Johann Christian Reil (1759–1813), and the physiologist Karl Friedrich Burdach (1776–1847). A few years later, Valentin (1834) stated in his publication that Purkynˇe had already shown to students during his lectures in 1829 nerve fibers similar to those described later by Ehrenberg in 1833. Given that Purkynˇe at that time had not yet obtained his Plössl compound microscope, it is very likely that these observations were carried out using a simple one (Kruta, 1977); in any case, one simple microscope was at his estate (Teissler, 1927). Purkynˇe was also interested in the ganglionic bodies. He noted in his memoirs, “I was the first, who was given them to observe. It was in 1833, when together with A. Wendt I worked on investigations of the skin structure and besides that I tried to disassemble the brain into its fiber composition, and looking closer to the cut of the black substance (substantia nigra) hidden in cerebral peduncles, I discovered that it consisted of individual flakes such as husks of bran” (Purkynˇe, 1858, p. 43). Numerous unpublished primary observations of Purkynˇe were also mentioned in Valentin’s publication of 1836. That same year, after a controversy with Gustav Valentin, Purkynˇe described the results of his study of the ciliary ependymal cells along the brain ventricles (Purkynˇe, 1836). He later noted, “I have finally, without any alliance with anybody, reported on the discovery of ciliary movements in the brain cavities, which I observed in the fetus of a fairly mature sheep, and on whose existence many are still skeptical, as it seems that during adulthood they are lost” (Purkynˇe, 1858, p. 37). In sum, he reported that during his study of the stria medullaris near the cerebral ventricles of a sheep fetus about 30 hours after death, he observed ciliary movements, which were visible on all walls of the third and fourth ventricles and also in the aqueductus s. canalis Sylvii that connect these ventricular cavities. The cilia were well visible and relatively long (longer than in the trachea), and it was possible to recognize the layer of bodies where the cilia were anchored, without violating the integrity of the epithelium. Purkynˇe noted that Valentin had observed similar findings in a mature porcine embryo, while in a younger fetus it was not possible to observe anything similar, probably due to the very fine structure of the tissue. Cilia were, according to Purkynˇe, very vulnerable and could be very easily damaged, much more than other parts of the tissue. He failed to observe cilia on the membranes of the choroid plexus in the human brain, even though he assumed that ciliated cells may be present in humans, such as in the fallopian tubes, uterus, or the olfactory epithelium. At the conclusion of his communication, Purkynˇe reported another interesting observation, namely that the whole plexus is covered with granular matter, similar to the ganglionic bodies, with a small particle in the middle of each formation. Therefore, based on his observations, Purkynˇe at first considered the whole matter of the choroid plexus as nerve tissue but later considered it to be epithelial. On December 14, 1836, Purkynˇe communicated in a lecture his latest observations on the structure of the nervous system (Purkynˇe, 1937b). According to him, the results coincided with the observations of Ehrenberg, namely that the entire nervous system consisted of three basic substances. The first, a red-gray substance, intersected by numerous capillaries; the basic building granules of this matter could hardly be measured and, as noted by Purkynˇe, according to Ehrenberg it matched the grains that constituted the central bodies


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of the blood cells. This basic substance was probably the initial substance of the other nervous constituents. The second substance was nerve fibers, partially nude in the brain and at the beginning of certain cranial nerves and partly enwrapped by sheaths of fibrous cellular tissue in real nerves. According to Purkynˇe, once the beginning and the end of a nerve fiber were determined, it could be seen that each nerve fiber had its own course from its beginning to end without branching or creating anastomoses. The third substance was ganglionic bodies. According to Purkynˇe, it was possible to observe them in all brain gray matter and in the nerve ganglia, interwoven with nerve fibers and anchored in “basic” nerve substance. Each ganglionic body contained a small central body inside the center circle, displayed its individual characteristics and was very similar to the embryonic follicles of female eggs. A few months later, on April 5, 1837, Purkynˇe gave a lecture in Wroclaw about his latest research on brain structures, stating that they consisted of ganglionic bodies (Purkynˇe, 1937c). These bodies, supposedly described by Purkynˇe during the previous years, had a certain constant size and shape in different areas of the brain tissue, namely the following: (a) in the brain, most notably in the cerebral peduncles (pedunculus cerebri - A.Ch.), especially in the black matter (substantia nigra - A.Ch.), which was made up mostly of ganglionic bodies with black pigment spots on the surface; (b) in various areas of the thalamus, predominantly in the corpora geniculata, and in quadruplet bodies (corpora quadrigemina - A.Ch.); (c) very clearly in the striped body (corpus striatum - A.Ch.) and in different areas of the gray matter of the brain cortex, especially in the occipital lobes, near the yellow matter (the border between gray and white matter - A.Ch.); (d) in the folds of the hippocampus (stratum pyramidale? - A.Ch.), where the thinnest gray layer was filled with a huge number of rhomboid ganglionic bodies; (e) in the cerebellum on the border between the gray and yellow matters, where there was always a large number of pyriform ganglionic bodies attached inwardly by their wide end and facing outwardly by their thin end; (f) in the fourth ventricle, in its front corner (fossa rhomboidea? - A.Ch.), there was present rust-like matter composed of richly pigmented ganglionic bodies; (g) a gray shell of rhomboid bodies (dentate nucleus? - A.Ch.) in the cerebellum and olives (olivae - A.Ch.) were interspersed with four-sided ganglionic bodies; (h) finally, ganglionic bodies were found in the gray layers of the bridge of Varoli (pons Varolii - A.Ch.), within the medulla oblongata and in the gray matter of the spinal cord from its beginning to its thinnest end. On the 26th of April in the same year, Purkynˇe managed to communicate during his following lecture his observations on the internal structure of nerves (Purkynˇe, 1937d). On very thin slices in the middle of the marrow of individual basic nerve fibers (Nervencylinder), in contrast to the peripheral nerves, he discovered a very bright albuminous (“eiweissartige”) substance; the entire channel seemed to him to be hollow, but this was not confirmed upon closer investigation. To observe the nerves using a microscope on thin sections and to recognize their internal structure, Purkynˇe used wood (oak) vinegar (a red-brown liquid produced by the distillation of wood and containing acetic acid, methanol, acetone, oils, and tars) and potassium carbonate (potash). According to Purkynˇe, anyone could be convinced about the presence of this albuminous substance; the substance itself was similar to a bright tape enwrapping the nerve fibers along their course. Purkynˇe emphasized that Remak in Berlin had been the first who described the tape-like formations in the medullary matter of nerves, and that they were similar to the formations that Purkynˇe himself observed; Purkynˇe also noted that these two observations (his and Remak’s) were complementary. In September 1837, Prague hosted a memorial meeting of German naturalists and physicians, at which Purkynˇe gave a series of lectures on different aspects of his research, including a lecture on the structure of nerve tissue. Summaries of these lectures were



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published simultaneously in the Proceedings of the Meeting (Purkynˇe, 1838a) under the redaction of the Czech nobleman, professor of pharmacy, higher anatomy, and physiology, physician, naturalist, surgeon, botanist, and mycologist Vincent Krombholz (1782–1843), and in the journal Isis (Purkynˇe, 1838b), which had been published since 1816 by the German naturalist, botanist, biologist, and ornithologist Lorenz Oken (1779–1851). Oken’s Isis was initially the only official organ of the meeting of German naturalists and physicians; it was only from the seventh meeting in Berlin in 1828 that the German Society of naturalists and physicians began to publish its own official proceedings of each meeting (Kruta, 1973). Isis, however, continued to publish the abstracts of the meeting in parallel, but in a different version than in Krompholz’s proceedings. In the case of Purkynˇe, his abstracts in the proceedings, in contrast to Oken’s Isis, were truncated. Although Isis was the more popular and more widespread journal, nowadays only the abstract in Krombholz’s proceedings, that is, a shorter version, is cited in the literature. It seems that for a more accurate assessment of this important part of Purkynˇe’s research, the abstract published in Isis is the more decisive source, therefore the following overview is based on a translation of the original unabridged version.

Translation of the Unabridged Purkynˇe’s Abstract Describing Nerve Tissue Structure On the apparent channel-like nature of the elementary nerve fiber (Nervencylinder). As was well known by the oldest medical physiologists who saw an analogy with blood capillaries, nerves are hollow channels; however, this hypothesis was created in order to appease their beliefs about the presence of nerve fluid and spiritus animales. More recently, Bogros has succeeded in mercury injections of nerves up to the finest branching, but this was probably due to the injection of the nerve sheaths and not the nerve fibers themselves. Recently, Ehrenberg determined the latter to be hollow channels in which a thick nervous marrow conveys motion from the center to the periphery. It remains unanswered and still doubted whether the nerve marrow is solid or liquid organic matter, in contrast to the autonomic movements, which mediate metabolism. If it were possible now to discover in the inner matter of nerve marrow or nerve fibers a more liquid substance in the channels, which should have at least a relatively stable form, other hypotheses would not be needed except that this more fluid content circulates. In very fine, translucent, transverse sections of nerve fiber bundles in fresh nerves, it is possible to observe the inner space of the basic nerve fibers. It was found that at the remote outer edge there is a circular double line, in concordance with the idea of a membrane enveloping the nerve fiber and containing capillary fibers; further, inside to the thicker edge follows a layer of nervous marrow and in the middle there is a usually polygonal and completely transparent formation, which could be considered to be the internal channel of the nerve fiber. However, given that such successful cuts could be done only rarely and absolutely randomly, for investigation only, those identically fixed nerves were selected from which it was always possible to cut with great care thin and very translucent slices. Even in this case, a fiber of the same contour occurred in the inner space of each nerve (Fig. 3Aa). When looking at a thin longitudinal cut of a nerve fiber, it was possible to distinguish in the middle of the nerve marrow a very thin and transparent stripe. Something similar was observed in the inner channels after the compression of the cylindrical marrow fibers (Fig. 3Ab). Purkynˇe was once again in doubt about the permanency of these differences in neural marrow; in fresh nerves examined by Burdach’s method in lukewarm water, an internal matter of elementary nerve fibers appeared to be very transparent and there was

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Alexandr Chvátal A

a


c

b

d

e

B

Figure 3. The structure of nerve tissue by J. E. Purkynˇe. Panel A: The channel-like nature of the nerve fibers and the choroid plexus structure in the brain ventricles (Purkynˇe, 1838a, 1838b). Part a: The inner space of the basic nerve fibers in very fine translucent transverse sections of nerve fiber bundles in fresh nerves. Part b: Thin longitudinal section of a nerve fiber. Part c: The choroid plexus papilla of the fourth ventricle of a human. Part d: Isolated granular bodies in the choroid plexus. Part e: Part of the choroid plexus membrane from the lateral cerebral ventricles of a human. Panel B: Granular formations of the sympathetic nerves of a bull’s head (Rosenthal, 1954): (a) a nerve bundle on which appear autonomic nerves with grains; (b) a tubu1e of the cerebrospinal nerves with a double edge; (c) the same, less convoluted tubule; (d) a tubule of the vegetative nerve, separated. Note. The original illustrations were digitally restored by the author of the present article.

no sign of the inner channel. Nevertheless, these observations suggested the presence of a structure of organic nature within the marrow of elementary nerve fibers and it was difficult to believe that such structural relationships could result from exposure to chemicals during the fixation of the tissue. On the granular layer surrounding the choroid plexus of all cerebral ventricles in man and probably in all other vertebrates. The granular bodies were of the same size, semitransparent and oval at their surface and included other granular material. They protruded


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by a pointed end freely from the granular layer to the outer space and were anchored to the inner side by an extensive membrane, which directly surrounded the capillaries of the plexus. Fig. 3Ae shows a part of the plexus membrane of the lateral cerebral ventricles of man; Fig. 3Ad shows isolated granular bodies; Fig. 3Ac is a plexus papilla of the fourth ventricle of a man. These bodies seemed to have an epidermal character; in any case, cilia at the free ends of the bodies cannot be observed. Their translucent nature could lead to the idea that they are of nervous origin, but, because they are not, even seemingly, connected to real nerve fibers, this idea cannot be currently accepted. Probably, this membrane of the cerebral ventricles has a suction function; therefore, excessive secretion of serous fluid from all of the numerous veins in the venous plexuses could have a similar function as the venous plexuses surrounded by granular bodies in the intestinal villi. On the ganglionic nature of some parts of the brain. Since the time during which the concept of ganglia was relatively well defined, nobody has forgotten to consider the gray globular matter of the brain as ganglia. Gall did so and later Reil and others did. This analogy was based only on shape and color and lacked the evidence that can only be provided by the internal structure. Already six years ago, Purkynˇe observed that the so-called black mass in the brain stem is composed of dark-brown flakes visible to the naked eye. Initially, ganglionic bodies in the ganglia were described by Ehrenberg; Purkynˇe soon observed similarities between these bodies with the bodies in the substantia nigra of the brain and started to compile the whole topography of the ganglionic bodies to such an extent in which they are present in the brain. Here are the main preliminary results of his research: 1. The main features of ganglionic bodies, both in the nerve ganglia as well as in the brain, are as follows: They are granular; they have a partly spherical and a partly rounded rectangular shape, sometimes with or without protrusions; they are somewhat rigid and translucent; they consist of loose, probably nerve, matter and they can withstand pressure and chemical agents longer than other nervous matter; in comparison with other microscopic structures, they are large, namely 2.3–79.4 µm; and inside they contain a round and somewhat translucent nucleus enclosed in a spherical ensheathment whose size is in a certain ratio to all ganglionic bodies. In the nerve ganglia these bodies are enclosed in cellulous or fibrous envelopes from which they are released only under great pressure, while in the brain these ensheathments around the ganglionic bodies are not present. In many ganglionic bodies in the brain and in other parts of the nervous system, variously pigmented spots scattered in different shades of brown are present; they are usually located on the edges and leave the central part transparent, through which the central nucleus shines. The pigment itself, as elsewhere, consists of very small particles that exhibit Brownian motion. 2. Concerning the interconnections of the ganglionic bodies with elementary nerve or brain fibers, nothing definite can be said yet. The bodies in the nerve ganglia appear to be substantially isolated in their sheaths; elementary nerve fibers often only form loops around the bodies, without being intergrown into each other. The processes of the ganglionic cells in the brain and in the spinal cord sometimes appear to be related to the large number of surrounding blood vessels, but it has never been proven with certainty, even less could be determined here about the reduction of very fine brain fibers. Mostly it is possible to observe that here the ganglionic bodies are anchored in the fibrous basic matter. 3. The topography of the ganglionic bodies in the brain and in the spinal cord is as follows: They are most evident in the substantia nigra of the brain stem, in the red mass (red nucleus [nucleus ruber]? - A.Ch.) and in the front corners of the fourth ventricle. Here,


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these bodies have a large number of processes that show the most fantastic shapes (Fig. 4b), their pigment is dark-brown and in some bodies is highly accumulated, however, in less developed bodies the amount of pigment is relatively little. In the fourth ventricle the bodies are round and rarely show the presence of processes and their pigment is lighter and reddish-brown. Moreover, the ganglionic bodies themselves can be observed in various locations in the geniculate matter (thalamus? - A.Ch.) and in the corpora geniculata. Here, they are usually very soft, round, and their pigment granules are light brown and rather large (Fig. 4a). Besides this, small, four-sided ganglionic bodies with processes and weak pigment spots are present in the grayish spirally curved layer of the hippocampus. In the occipital lobes of the cerebrum, in the brain near the marrow mass, were also observed elongated ganglionic bodies, similar to the fruits of a fig tree and with processes on the thin end. Similar bodies surrounding a yellow mass could be observed in all layers of the cerebellum, where they are present in large numbers and in rows. Each such body lies with its blunt rounded end submerged inside the yellow mass with a clearly visible central core with a shining edge; its second tail-like end points outwards and is usually in the form of two processes that disappear into the gray matter up to the outer edge, which is covered with a vascular membrane (Fig. 4e). Such a structure is similar everywhere throughout the layered folds of the cerebellum, so that it especially gains the importance as ganglia. This importance is even greater due to the nature of the rhomboid body (dentate nucleus? - A.Ch.) in the medullary matter of the cerebellum, where the core of the corpus rhomboideum surrounding the gray-yellow layer between the sparse brain filaments and more solid basic matter contains foursided pigmented ganglionic bodies throughout. Similar in appearance is the gray-brown matter enveloping the olives in the medulla oblongata (Fig. 4c). Finally, the brain node, or the bridge of Varoli, is of importance as ganglia due to the large number of round ganglionic bodies covered with a gray pigment that alternates in fibrous layers of the gray matter of this node. Similarly, relatively large ganglionic bodies were observed throughout the gray matter of the spinal cord, some circular, others angular. In addition, they can be found in various areas outside the gray matter of the cerebrum and in the gray matter of the striped body (corpus striatum - A.Ch.) and its ganglia, although it is not yet clear whether these are ganglionic bodies, as it was not possible to recognize well in them the central core with a clear outline. 4. In addition to these ganglionic bodies, there are in the brain other structures that do not contain a central core and that belong to a very different kind of group. The gray matter of the brain convolutions presents gray-white granules emerging from the basic matter. In addition, special kinds of round or rounded square bodies are present, which are similar to starch granules of waxy consistency (Fig. 4d) and which are found in large numbers in the lamina cribrosa in front of the chiasma nervorum opticorum (chiasma opticum - A.Ch.) and in the striped corners (stria terminalis? -A.Ch.) on both sides of the thalamus. Another kind of small and very uniform body, together with the elementary brain fibers, creates the yellow inner matter of the cerebellum (nuclei cerebelli? - A.Ch.). 5. As for the significance of the ganglionic bodies concerned, it can be observed that they are similar to the central structures, for they show their whole triple concentric structure and are related to elementary brain and nerve fibers as power connections for the power centers, or as ganglionic nerves to ganglia, or as brain matter to the spinal cord and cranial nerves. They are collectors, producers, and distributors of the nerve organs.

Discovering the Structure of Nerve Tissue: Part 2 a

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b


e

d c

Figure 4. The structure of nerve tissue by J. E. Purkynˇe, continued (Purkynˇe, 1838a, 1838b): (a) ganglionic bodies in the matter of the thalamus and corpora geniculata; (b) ganglionic bodies in the brain (in the substantia nigra, in the nucleus ruber, and in the front corners of the fourth ventricle) and in the spinal cord; (c) angular pigmented ganglion bodies in the gray-brown matter enveloping the olives in the medulla oblongata; (d) round or rounded square-shaped bodies, which are similar to starch granules of waxy consistency; (e) small tetrahedral ganglionic bodies with processes and with weak pigment spots in the gray spirally curved layer (stratum pyramidale) of the hippocampus. Note. The original illustrations were digitally restored by the author of the present article.

Subsequent Purkynˇe’s Studies on the Structure of Nerve Tissue As evident from the above text, Purkynˇe was not quite sure whether the nerve marrow is a solid or a liquid organic matter, although Kisch inaccurately states that Purkynˇe was convinced of its liquid content and that therefore Purkynˇe was supposedly wrong in the interpretation of his results (Kisch, 1954). It is certain that only after a visit to Müller’s institute in Berlin in the autumn of 1837 and after a meeting with Remak, who showed him primitive fibers, Purkynˇe ceased to hold the view of Ehrenberg, who was convinced of the liquid content of the central part of nerve fibers, and accepted the opinion of Remak that the central structure of the nerve, which Purkynˇe named as the “axiscylinder,” was a solid fiber.


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After this meeting in 1839, Purkinˇe’s student Josef Ferdinand Rosenthal (1817–1887) published a dissertation in which he reported the results of further studies of nerve fibers as well as of granular bodies (formatio granulosa) in a variety of tissues, including nerve tissue (Rosenthal, 1954; Vacek, 1986). This was mainly Purkynˇe’s own research he generously passed on for publication to his student (Frankenberger, 1954), which is also evidenced by the fact, that the dissertation figures were drawn personally by Purkynˇe. Under the title “formatio granulosa,” the authors described formations that appeared under microscopic examination to be composed predominantly of nerves, both cerebrospinal and sympathetic, as well as of striated muscles, blood vessels, and connective tissue. They observed that these formations could have diverse shapes, but they always appeared to be composed of finely granular material and contained a distinct nucleus. Rosenthal and Purkynˇe, as mentioned in the dissertation, used acetic acid as the main reagent for staining, after which the granular formations appeared very clearly. Interestingly, for tissue staining (especially the nuclei) they used indigo, about 12 years earlier than the Italian physician and anatomist Alfonso Corti (1822–1876), who is considered to be the first scholar to introduce staining (using carmine) into microscopic anatomy. From the results described, it seems that Rosenthal and Purkynˇe observed cellular formations, mainly the cells of the Schwann sheath (Fig. 3B), as well as connective tissue cells, satellite cells surrounding the ganglionic cells in the ganglia, endothelial cells, and others. However, Rosenthal and Purkynˇe did not fully agree with the views of Remak about the interconnection of nerve cells and fibers, although they confirmed the presence of Remak’s unmyelinated fibers. The authors’ observations of fetal tissues and their comparison with adult tissue were absolutely remarkable. They observed how granular formations could also be found in fetal tissues and even saw that they arise first in the blastema in undifferentiated matter from which all very different tissues develop by “their innate creative effort” (Rosenthan, 1954, p. 647). Rosenthal and Purkynˇe then described how those grains, with their cores partially shrunken at the ends, pass into the fibers to form the cellulous tissue, as well as tendons, how they are partially placed on the surface and between the elementary parts such as the muscles and nerves, and how they change by metamorphoses into these substances. According to the authors, it was quite clear that these formations significantly contributed to the formation of the first elementary parts as well as to their development. In addition, these granular bodies had in the embryonic stage a large plastic productive power that reportedly did not reflect only the development phase as they were present not just in embryonic tissues but also in the tissues of adult animals. Therefore, they wondered whether these granular bodies would have the same functions in the adult organism as in the embryo. From that “plastic matter,” that is, the granular formations, which Rosenthal and Purkynˇe believed had a creative power, developed the elementary part, hence supplementing the losses that the body suffers during life processes. The authors claimed that no one would doubt that organic matter was destroyed and consumed during the life of an organism and that it therefore required renovation. According to the authors, their new idea was that the basic components do not develop directly from the blood or blood fluids but from the granular cell bodies they described. It can therefore be assumed that Rosenthal and Purkynˇe, perhaps as the first, not only formulated the principle of development of animal tissues from granular bodies containing nuclei, which in retrospect are reminiscent of stem cells, but also discovered their presence in mature tissues, where they could play a regenerative role. Undoubtedly, the work of Purkynˇe constituted significant progress in comparison to the previous works of Ehrenberg, Valentin, and Remak (Kruta, 1973). Despite considerable development of the methodology as well as the findings and their evaluation, Purkynˇe’s


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conclusions also documented some of his mistakes, such as his view on the internal structure of nerve fibers. However, Purkynˇe typically tried to stick strictly to his own observations without speculation. He did not insist stubbornly on his opinions, as Valentin, but edited or changed his point of view on the basis of convincing arguments.


The Importance of the Discoveries of Gabriel Valentin, Robert Remak, and Jan Evangelista Purkynˇe The results of studies of the microscopic structure of nerve tissue at the beginning of the nineteenth century show that, despite the improvement of microscopy, progress was slow and laborious. It seems that the main limitation was represented by methods of storing and processing the tissues, especially human tissue, for microscopic observations. At that time, fixation techniques started to be used, by which animal and human tissues could be processed for cutting sections. The works of Valentin, Remak, and Purkynˇe in this regard document significant progress in understanding the structure of nerve tissue. The first evidence of the microscopic structure of nerve fibers was obtained by Procháska, who, convinced of the “globular structure” of nerves, described in 1779 a formation that could probably be neurolemma (see Part 1 of the present article [Chvátal, 2014a]). Further significant progress was achieved by Fontana, who in 1781 described the elementary nerve fibers, when the marrow escaping from the fresh cuts of some fibers led to the concept of channels filled with viscous fluid. A completely transparent, nongrainy, and viscous liquid was described in nerve fibers by Ehrenberg (1837). However, besides classifying the fibers’ distribution according to their thickness and describing varicose extensions, Ehrenberg did not contribute much to the understanding of the detailed structure of the actual nerve tissue. Interestingly, the description of the globular structure of nerve fibers published by Berres was rejected by Valentin and Remak. As mentioned above, a more detailed description of the fibers was provided by Valentin, who also described the first outer nerve lumen, which formed a double circle and from which, when pressed, a milky, translucent, oily liquid was released (Valentin, 1834). However, the most detailed structure of nerve fibers was described by Remak, who identified and described the central channel of myelinated fibers (axons) and illustrated in myelinated fibers also peculiar constrictions (Fig. 2) that could be what are today known as the nodes of Ranvier or Schmidt-Lanterman incisures (Clarke & O’Malley, 1996). Furthermore, Remak first described in detail unmyelinated autonomic fibers, which he named “organic fibers,” now sometimes referred to as “Remak’s fibers.” Purkynˇe also described in detail the structure of nerve fibers. The evaluation of the results obtained by Remak and Purkynˇe, however, differ: While the first published illustrations of Remak and other scholars usually did not reach the scientific level of Purkynˇe’s figures (Kruta, 1973), Kisch (1954) was convinced that Purkynˇe’s figures were less convincing than Remak’s. Although their figures can now be compared in the light of modern knowledge, Purkynˇe was convinced that his and Remak’s observations rather complemented each other. Kruta (1973) also noted that Purkynˇe showed that the nerve fiber marrow maintains its structure even when the fiber’s sheath is broken and stressed that the medullar matter is a permanent component of all nerve fibers. His term “axiscylinder,” often with the addition of “Purkynˇe’s,” was routinely used for a long time in scientific literature. It was only in 1839, when Schwann in his famous book, written together with Schleiden and in which he summarized the principles of his theory of the similar cell formation of plants and animals, also described the cellular structure of the medullary sheaths including nucleated cell elements that they then became known as “Schwann cells.”


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Purkynˇe’s priority was certainly in the description of the ganglionic bodies, which from today’s perspective corresponds to the bodies of various classes of nerve cells in different areas of the brain, spinal cord, and ganglia, and in which he described nuclei and nucleoli (Druga, 1986). Already Stieda stated in 1899 that “undoubtedly Purkynˇe mostly penetrated into the knowledge of the construction elements of the nervous system and he was the first to see the nerve cells of the central organ, as evidenced by Valentin; Purkynˇe claimed that the nerve cells of the central organ lacked ensheathment, in contrast to nerve cells in the ganglia” (Stieda, 1899, p. 113). Although similar bodies had also been observed by Henri Dutrochet (1776–1847) and Ehrenberg, particularly in cephalopods and beetles, Purkynˇe was the first to conduct a systematic classification of these bodies in higher species, including humans, and to describe their regular appearance in different parts of the nervous system. Based on their accumulation in certain areas and typical features, he was convinced that they were an important component of nerve tissue. It is also of interest that the Czech vitalistic biologist and philosopher Emanuel Rádl (1873–1942), after a thorough analysis, positively judged the benefit of Purkynˇe’s research on nerve tissue structure (he was otherwise very critical about Purkynˇe). He stated that “we cannot appoint Purkynˇe as a discoverer of ganglionic cells at all, but at least as a discoverer of certain ganglionic cells (in the brain). However, he deserves credit for the first understanding of their importance, even though their relation to the nerve fibers was still unclear to him” (Rádl, 1900, p. 9). Nowadays, of all the cells of the brain and spinal cord, perhaps one of the more famous kinds are those in the cerebellum called “Purkynˇe cells” (or “Purkynˇe neurons”). Purkynˇe first illustrated not only these cells but also the whole structure of the cerebellar cortex, virtually as it is known today. According to present knowledge, the cerebellar cortex consists of three layers; in the bottom layer (stratum granulosum), there are small granular cells, similar to those described by Purkynˇe. In the middle layer (stratum gangliosum) are Purkynˇe cells. These are the largest cells of the cerebellar cortex (50–80 µm), which have processes (dendrites) appearing as branched antlers at the top, spreading into what is today called the molecular layer (stratum moleculare), in which the so-called basket cells are also located. Purkynˇe’s figure and his description of the cerebellar cortex therefore correspond to current knowledge, and Kruta (1973) already noted that it was epoch making, classic, and the first of its kind. Deserving of priority in Purkynˇe’s findings is also his description of ependymal cells in the brain ventricles, for which he even suggested a function. Interestingly, in human tissue he did not observe the presence of cilia, unlike in animal tissues, this is probably due to the state of the human tissue obtained for examination. This contribution by Purkynˇe was admitted later, in 1855, by the German anatomist Hubert von Luschka (1820–1875; 1855), but nowadays the epithelial cells in the cerebral ventricle walls are not related to Purkynˇe. In terms of our current knowledge, the ependyma consists of cells called ependymocytes (Reichenbach & Wolburg, 2005). They are similar to epithelial cells and coat the ventricles and central canal of the spinal cord. The apical end of the ciliary cells is covered with a layer of cilia, which ensures the circulation of cerebrospinal fluid in the ventricles. These ependymal cells are a glial type of cell of astrocyte origin, populate together with capillaries to form the choroid plexus and are responsible for the production of the cerebrospinal fluid. The structure and proposed function described by Purkynˇe is therefore fully in line with today’s knowledge. Currently forgotten is also Purkynˇe’s priority in describing that the projections of ganglionic cells in the brain and spinal cord are in relationship with nearby blood vessels. Purkynˇe probably could not explain the function of such a relationship, consequently this finding is not included in the abbreviated version of his abstract from the Prague meeting



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in 1837. It is now known that the brain capillaries are surrounded and closely associated mainly with the processes of perivascular astrocytes (Abbott, Rönnbäck, & Hansson, 2006). Since these astrocytes are derived from the neuroepithelium, they share a number of common features, such as transport mechanisms in the perivascular endings that closely associate with the walls of the thinnest capillaries. Purkynˇe was the first to observe the tight contacts of nerve cells (probably of neuroglial origin) with capillaries, 34 years before the Italian pathologist and Nobel Prize winner Camillo Golgi (1843–1926). In 1871, Golgi, based on the use of new techniques of tissue staining (Golgi staining), noted that the astrocyte processes touched the capillaries and suggested the hypothesis that these cellular elements could play a central role in the transport of energy substrates from the blood capillaries into neurons (Rossi & Volterra, 2012). The other formations Purkynˇe first described in the brain, and which he distinguished from the other granular bodies, were those identified as corpora amylacaea. It is now known that corpora amylacea, sometimes called amyloids, are commonly found in the central nervous system of aged mammals and represent glucose polymers, sometimes referred to as “polyglucosan bodies” found in the cytoplasm of astrocytes (Garman, 2011). Corpora amylacea are frequently present in the perivascular space and subpial locations, which also correspond to the cytoplasmic processes of astrocytes. Recent studies have revealed that the major source of cellular corpora amylacea are astrocytes (Nam et al., 2012) and that they could be an indicator of neurodegeneration (Singhrao, Neal, & Newman, 1993). Purkynˇe described in his discovery of these bodies that “this observation remained in ignorance and in obscurity until 1853, when Virchow analyzed it again, and using iodine and sulfuric acid colored those grains in blue and violet color similarly as starch, thus trying to prove that they are composed from real starch identical to cellulose, being of vegetable origin, which is also found elsewhere in lower animals (salpa, ascidians, phallusia etc.), and it would indicate that even in the human brain some sort of lignification can happen” (Purkynˇe, 1856, p. 375). An interesting question is how Valentin, Remak, and Purkynˇe hypothesized the relationships between nerve fibers and cell bodies. Valentin defended the hypothesis according to which nerve fibers and cell bodies were in “juxtaposition,” that is, in the close vicinity without being interconnected. This hypothesis, which he probably invented together with Purkynˇe, is nowadays not mentioned. As presented above, the hypothesis states that isolated cell-like formations represent the creative and active, that is, higher elements, while isolated primitive continuous fibers represent the conductive and receiving, that is, the lower elements. Both of these components did not create united formations but were, according to the hypothesis, interconnected by a kind of electromagnetic induction. Thus, Valentin and Purkynˇe further developed the hypothesis of Henri Dutrochet published in 1824, who suggested that nerve bodies were “producers of nervous energy,” while nerve fibers maintained the transmission of “nervous motion.” On the other hand, Remak was convinced that nerve fibers and ganglionic bodies (nerve cells) were organically joined to each other. Purkynˇe was cautious in his statements, based on the observations the methods of the time enabled. Nevertheless, he described the fine processes of the ganglionic bodies in at least two cases, namely in the description of their close relationship with blood vessels and in the description of the structure of ganglionic cells in the cerebellar cortex (i.e., Purkynˇe cells), when he claimed that their processes divided into very fine fibers that disappeared on the edges of the outer layer. Purkynˇe probably also realized that the bodies of nerve cells most likely represented an important basic structure of the neural tissue and that they were in some relation to nerve fibers, possibly consistent with the hypothesis of electromagnetic induction published

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previously by Valentin. Since Purkynˇe compared this relationship to that of the ganglia to the nerves emanating from them and also to the relationship of the brain to the spinal cord and cranial nerves, it can be assumed that he considered the ganglionic bodies and nerve fibers as essential elements for the function of the nervous system.

Conclusion


Using unabridged translations of the original works of Valentin, Remak and Purkynˇe, the present review uncovered and compared their contributions to neuroanatomy. Although the fundamental discoveries of these famous scholars did not imply the discovery of nerve cells as we know them today, they were certainly a very important basis for further research of eminent scholars such as Albert von Kölliker, Otto Deiters, Camillo Golgi and Santiago Ramón y Cajal, who in the nineteenth century expanded our knowledge of the structure and function of nerve tissue.

Acknowledgements I would like to thank the staff of The Museum of Czech Literature (PNP) in Prague for scans of original illustrations of J. E. Purkynˇe and J. F. Rosenthal. Archival originals are stored in The Museum of Czech Literature (PNP) in Prague—the literary archive, fund of the Purkynˇe Commission. I would also like to thank Prof. Rastislav Druga for valuable comments and James Dutt and Elisa Brann for their critical reading of the manuscript.

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