CHAPTER

19

Cajal and glial cells

Luis M. Garcia-Segura
Institute Cajal (CSIC), Aveaida de1 Doctor Axe 37, 28002 Madrid, Spain

Introduction-Cajal

and glia: an unexplored story

The scientific contributions of Cajal may be placed, without doubt, among the best highlights of the biological sciences of the end of the 19th and the beginning of the 20th centuries. In addition to its own scientific value, Cajals writing is still an enjoyable read. We may find many sources of delight when reading his scientific books and papers. His beautiful drawings, which remain as true masterpieces of art, and his marvelous literary style are also part of the pleasure conveyed to neurobiologists of our time when reading Cajals original work. Cajal, who spent many hours in his laboratory, mastered and improved the scientific technology of his time. He was a fruitful thinker full of curiosity and if this were not enough, he also revealed himself as a gifted artist. Among the many possible approaches to Cajals work, we present here an almost unexplored trail: the contribution of Cajal to our present knowledge of glia. There is no doubt that the name of Cajal is associated with the morphology of neurons and neuronal circuits. However, in very arduous work that included many technical and theoretical di%culties, Cajal and his colleagues were able to start deciphering the glia mystery. Cajal developed new tools that were used by himself and by other members of his laboratory to identify and classify glial cells. This was not an easy task, and reading the papers on glia by Cajal and his colleagues, we can appreciate how difficult it was to identify oligodendroglia and
*C&responding author: Tel.: +34-915854729; Fax: +34-915X54754; E-mail: lmg@cajal.csic.es

micro&a, and the problem of placing the glial cells in the conceptual picture of the brain. The interpretation of the role of glia by the Cajal school was not f&e from intellectual debate among his members and as with all human achievements, it was not free from personal feelings. The fascinating contribution of the Cajal school to the neurobiology of glia remains to be written. The following notes are no more than a brief introduc%ion to the story of this impressive scientific achievement.

Not only neurons

In 1888 Cajal published his hrstpapers on the structure of the cerebellum and the avian brain stud& that represent the starting point of his ftuctiferous scientific career. Before these studies, Cajal was attracted by several different issues. His doctoral dissertation, of 1877, was entitled Patogenia & la inyYamacich (pathogenesis of inflammation),, and his first paper, published in 1880, dealt with experimental studiesof inflammation and the migration of leucocytes. Although his second paper, published in 188 1, was on the branching of nerve endings in striatal muscles, his subsequent studies, in 1884 and 1885, were devoted to microbiology and then, in 1886 and 1887, to the histological characteristics of different tissues. Cajal took more than a decade from finishing his doctoral thesis to discover the wonders of the histological structure of the central nervous system and MS personal path in scientiiic research. However, as soon as he entered the field of neuroscience he started the study of glia. For instance, in a description of the histological structure of the spinal cord published as early as 1889,

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he presented a detailed description of ependymal cells and radial glia, discussing the embryonic origin of neuroglia supporting the concept of the glial cells as individual units in contrast with other reticular theories of his time (Cajal, 1889a; see also Cajal, 1899a). Glia cells appeared in several publications by Cajal between 1888 and 1896. However, it was not until 1896 when Cajal published a paper completely devoted to glia. It was in the first volume of his journal Revista Trimestd Mc~ogd$ca, where he published a short note on the spatial relationship of neurons and glia (Rambn y Cajal, 1896). This is a beautiful study, based on Nissl staining of the rabbit cerebellum and other brain areas, describing several details of the distribution of glial cells around the cell body and the axon of neurons. Although glial cells were mentioned by Cajal in several of his previous studies, this was his first paper in which glial cells were the main player. We should realize that, by this time, Cajal had already put forward many of his ideas about the function of neurons and the organization of neuronal networks. In contrast, no solid functional hypothesis was established for glial cells. Cajal concluded that the specific location of glial cells around neurons was not an arbitrary phenomenon. The peculiar Crown of glial cells around neurons had to be related to their function, but their exact role was still a mystery. Cajal decided to discover that mystery, and it took him just one more year to publish his first paper on the function of glial cells. The second volume of his journal Revista Trimestml Microgkfica contains two important theoretical papers by Cajal. The first one (Ramon y Cajal, 1897a) was a summary of his theories on the laws governing the morphology and function of neurons. In the second paper, entitled: Something on the physiological jktion of neupoglia (Cajal, 1897b), Cajal exposed his first theory on the role of these cells. Using material from this paper, he further developed this subject in his book onthe histology of the nervous system (Cajal, 1899a). .Thebasic ideas presented in his paper of 1897 and in his book of 1899 were the same. of the insulating role of the neuroglia. First we must mention that at that time the distinction among the different types of glial cells, as we know them today, was still emerging. Thus, Cajal made a distinction among myelin and neuroglia in the gray and white matter. Neuroglia for Cajal represented what we call today astroglia. Cells responsible for myelin formation in the central nervous system were still uncharacterized. For Cajal, the processes of neuroglia in the gray matter represented a medium that resisted the passage of nerve impulses, preventing contacts among dendrites and non-myelinated fibers. When Cajal mentioned the processes of neuroglia he was mainly referring to the processes of astrocytes, as the inspection of many of his drawings demonstrate (Fig. 1). Cajal considered that myelin was an improvement in the insulating function of glial cells. For this reason, the presence of neuroglia in the white matter was a puzzle for Cajal. Since axons in the white matter were already insulated by my&n, why was the presence of neuroglia necessary?In addition to the fact that the link between oligodendrocytes and myelin in the central nervous system was still undiscovered, the presence of astrocytes in the white matter was dBicult to explain based on his insulating theory. Although the insulating theory of Cajal on glia was limited and even though we now know many other metabolic (Magistretti and Pellerin, 2000) and signaling (Araque et al., 1999; 2001) functions of glial cells, his basic idea on glia function is still valid. Recent studies in different areas of the brain have shown that the glial covering of neuronal surfaces may regulate the formation of synaptic connections and that the processesof astrocytes and microglia participate in the plasticity of synaptic inputs (Garcia-Segura et al., 1994; Schiefer et al., 1999; Theodosis and Poulain, 1999). Cajal recognized that the idea of the insulating role of neurogha was developed by his brother Pedro. However he supported the theory with his own findings, presented in a medical meeting in Valencia in 1891 (Cajal, 1891), as well as by further studies from his collaborators Claudio Sala y Pons (1894) and Terrazas (1897). It is of interest that the first paper quoted by Cajal in support of his theory was his own communication for the meeting in Valencia, three years before the doctoral dissertation of Sala y Pons on glial cells was published (Sala y Pons, 1894). Thus, although many of the consecutive studies from the laboratory of Cajal on glia were published under

The insulating theory

For Cajal, the main role of glial cells was to insulate neurons to avoid wrong contacts among them and to preserve the function of neuronal circuits. This is his theory

Fig. 1. Neuroglia from the hippocampal formation (stratum radiatum of the Ammon horn) of a human brain. In this drawing Cajal shows astrocytic processes embracing pyramidal neurons as well as astrocytic processes in contact with blood vessels. Reproduced with permission from an originai drawing conserved in the Leg& Cajal.

the name of his collaborators, we have to conclude that Cajal himself showed an early interest in glial cells.

The neurotrophic

hypathesis

Cajals interest with glial cells never declined. It is true that he devoted much more effort to the study of neurons and that the main contributions to understanding glia by Cajals laboratory were published by his collaborators.

If we go through the volumes of the journals originally founded and edited by Cajal, we will encounkr !many relevant papers on glial cells authored by scientists directly related to Cajal or by members of his laboratory. Names such as Sala y Pons, Terrazas,Achticarro, Lafora, Del Rio-Mortega, Ramon y Fafianas, S&nchezy Sanchez, De Castro, Serra, Somoza, Rodriguez-Perez, represent an impressive list, for that time, of scientists contr-ibuting to the glia field. Among these advances we have TV mention the studies by Tell0 on regeneration (Tello, 1911),

particularly because this work led Cajal to postulate a second theory for the function of glial cells: the secretion of trophic factors to support neuronal growth. Tell0 was ahead of his time when he addressed the question of the differences in the regeneration of central and peripheral axons. He considered that Schwann cells may be the cause of these differences and he tested this hypothesis with a revolutionary experiment: he transplanted Schwarm cells in the brain to determine the effect on the regeneration of central axons. Tell0 observed that Schwann cell transplants induced the growth of central axons. This experiment was reviewed and commented by Cajal in his book on the degeneration and regeneration of central nervous system (Cajal, 1914). Cajal postulated that Schwann cells release a trophic factor for neurons, an hypothesis proposed by Tello. Interestingly, Cajal did not consider Schwann cells as glia. However, his hypothesis on Schwann cells represents the most advanced idea that Cajal ever formulated for glia function. For a modern neurobiologist, it is impressive to read the papers by Tell0 and Cajal on central axon regeneration, made many years before the discovery of the first neurotrophin by Rita Levi-Montalcini and also many years before the rediscovery of the ability of Schwann cells to support central axonal growth.

A sustained effort for the study of glia

That Cajal did not wish to co-author the many papers on glia published by members of his laboratory does not reflect a lack of interest on the subject. We should remember that co-authorship of papers was then not so popular as today. Scientific research and discovery was considered to be the result of a personal inquiry rather than the result of the work of an organized scientific team. Apparently at that time the director of a laboratory did not coauthor the papers of his students. Cajal himself profusely contributed to the field of glia and many of his papers mentioned these cells. As a case in point, he was unable to correctly identify microglia which he identified as small neurons in a study on the structure of the human cerebral cortex (Cajal, 1899b), but later rectified his initial observations (Cajal, 1925). Among his more important studies on glia, we should mention an extensive study on the neuroglia in human brain that was published in 1913 in Tmbajos de1 Laboratorio de Investigaciones Bioldgicas de la Universidad de Madrid (Cajal, 1913).

This paper describes many cytological and histological features of astrocytes and their relationships with neurons and blood vessels. The ectodermal origin of astrocytes as well as their capacity for proliferation in the adult brain are recognized. The descriptions of Cajal were based on his own observations as well as in the material previously published by other members of his laboratory. Interestingly, in this study there is a description of oligodendrocytes, under the name of the third element of the nervous centers or small adendritic cells. Cajal, who probably included microglia among adendritic cells, considered that these cells had a mesodermal origin and pointed to its possible homology with Schwann cells. It was his collaborator Del Rio-Hortega who made some years later a complete description of the morphology of microglia (Del Rio-Hortega, 1920) and of oligodendrocytes (Del Rio-Hortega, 1928) and correctly interpreted their function as well as their embryonic origin. Between 1913 and 1916, there were many other essential contributions made by Cajal and colleagues on glial cells. We found several papers by Achircarro, such as his impressive study on the gliotectonics of the cerebral cortex (Achucarro, 1914), Lafora, Del Rio-Hortega, Sanchezy Sanchez and Ramon y Fananas, among others. The year of 19 16 probably marked the main peak in the production of papers on glia from Cajals laboratory. For instance, eight of the 13 papers that composed volume 14 of Trabajos de1Laboratorio de Imstigaciones Bioldgicas de la Universidad de Madrid published in 1916, are on glial cells, including a technical paper by Cajal on the staining of neuroglia (Cajal, 1916) in which Cajai made use of microphotographs to illustrate astrocytes stained with his method. The peak in glia research made by Cajals laboratory coincided with one of the most dramatic situations in European history: the First World War. Fortunately, Spain was a neutral country and Cajal and his collaborators were able to pursue research in Madrid without the restrictions imposed by the devastating war that desolated Europe. However, the war seriously restricted the diffusion of scientific publications. Obviously, this was not the best moment for the publication of the new discoveries made by Cajals laboratory on glial cells. War conditions hampered the distribution of Cajals publications among scientists working in Germany, France, Italy and other European countries. And of course these same scientists were at that moment involved in other more urgent tasks.

The last years: glia are the main player

In the years following the First World War, glia progressively attracted more attention from Cajals laboratory. Cajals collaborators, De Castro, Del Rio-Hortega, Somoza and Rodriguez-Perez were among the main contributors to the understanding of glia at that time. The production of papers on glial cells by Cajals laboratory reached a second peak in 1920. Fernando de Castro published in this year two beautiful and detailed studies on the astroglia in the olfactory bulb (De Castro, 1920a,b). De Castro described many new aspects of the morphology and distribution of astrocytes as well as the process of g&genesis. Based on the relationship of astrocytic processes with blood vessels, De Castro proposed that astrocytes could act as endocrine cells, releasing hormones into the circulation (De Castro, 1920a,b). This hypothesis, not yet confirmed was advanced for its time, considering that the study of endocrinology was at an early stage. It is also interesting that a role of astroglia in the regulation of neuroendocrine events has recently received experimental support (Garcia-Segura et al., 1996; Melcangi et al., 1997; Ojeda et al., 2000). The year of 1920 also saw the discrepancy between Cajal (Cajal, 1920) and Del Rio-Hortega (1920) regarding the interpretation of adendritic cells. It was the opinion of Del Rio-Hortega that Cajal was deceived by adendritic cells due to a technical artifact. Del RioHortega said that these cells had processesthat Cajal was unable to see by a limitation of his method of staining. Cajals reply was in the same journal issue. Even with processes, Cajal argued, these cells would represent a unique population, because these questionable processes were not stained with the same methods that revealed the cell processes of other glial cells. A pure scientific debate or an obscure query among master and disciple? It is hard to give a reliable answer to this question today. We may only guessthat, with all probability, both aspects were mixed in the debate. However, it is clear that Del Rio-Hortega was carrect in his assumption, as we know that oligodendrocytes have cell processes.Another aspect that is now obvious is that the personal relation between Del Rio-Hortega and Cajal will from then suffer a dramatic deterioration. In his last years Cajal was very well aware of the importance of glial cell function in the damaged brain. In 1925, he published an outstanding paper describing reactive astroglia and microglia in the human brain

(Cajal, 1925). As additional proof of the importance that Cajal gave to glial cells at that time is, that he prepared a French version of his Spanish papers of 19 13 and that he published then in the volume corresponding to the years 1931-1932 of his journal ?~a&& del Laboratorio de Imestigaciones Biol&icas de la Universidad de Madr?d. Probably, Cajal considered that these studies, published just before the declaration of the First World War had not reached a wide audience. Furthermore, Cajal continued to support the work of his collaborators on glia. Not only Del Rio-Hortega, who having established his own laboratory comi.rmed the work initiated by Cajal on glia. The other collaborators of Cajal, such as Rodriguez-Perez, Sanchez y Sanchez and Sanz-Ibanez, pursued in the subsequent years work on glia with new original descriptions and modern experimental approaches. In conclusion, it may have been an inevitable consequence of the revolution initiated by Cajal in the study of neurons that his contribution to the understanding of glia remained hidden in second place. However, reading the papers on glia by Cajal and his collaborators, it is impossible to refrain from the admiration of their pioneering exploration into such a difficult field. The exceptional quality and novelty of the studies made by Cajal and his school on glia will remain forever among the most remarkable and outstanding contributions to the history of neuroscience.

Acknowledgments
I want to thank Drs. Julian Taylar and Javier de Felipe for a critical reading of the manuscript.

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