Please cite this article in press as: Serrano-Castro PJ, Garcia-Torrecillas JM. Cajals rst steps in scientic research.

Neuroscience (2012), http:// dx.doi.org/10.1016/j.neuroscience.2012.05.008

Neuroscience xxx (2012) xxxxxx

REVIEW CAJALS FIRST STEPS IN SCIENTIFIC RESEARCH

P. J. SERRANO-CASTRO a* AND J. M. GARCIA-TORRECILLAS b
a

Conclusion References

00 00

Department of Neurology and Neurophysiology, rdenas Hospital Complex, Almera, Spain Torreca Emergency Department, Torrecardenas Hospital Complex, Almera, Spain

AbstractMore than 125 years ago, Santiago Ramon y Cajal was able to draft and prove the neuron doctrine, and later, to develop prophetic theories about neural function and plasticity, many of which have been proven by current neuroscience. It was chance that made Cajal, during his doctorate studies, have his rst contact with histology and force him to study the then current theories about pathogenesis of inammation. Thus, he gained knowledge of the vascular hypothesis, by Julius Cohnheim, a German pathologist who, opposing the opinion of his teacher and father of cellular pathology, Rudolf Virchow, made leukocytes the protagonists of inammation, given their ability to develop ameboid movements directed by chemical signals. Cohnheims chemotactic theory deeply inuenced Cajals conception of biology. So, the basic postulates of chemotaxis can be identied at dierent moments in Cajals research, from the description of the growth cone in embryonic neuroblasts, the origin of the neurotrophic theory, to the proposal of the pathophysiological mechanisms of neuronal plasticity. From Cajals point of view, the neurons move during their development and also adapt to dierent external circumstances. Chemical endogenous substances can stimulate this movement in a similar way to leukocytes during the process of inammation. 2012 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Cajal, doctoral thesis, inammation, Cohnheim, chemotaxis, growth cone. Contents Introduction 00 Cajal after his return from Cuba 00 The debate about inflammation and leukocyte migration in Cajals time 00 Cajals opinion on his doctorate speech 00 Cajal, the experimental researcher 00 Cajal and the reazione nera 00 Cajals neurotrophic theory 00

INTRODUCTION
Santiago Ramon y Cajal (Petilla de Aragon, Spain, 1852. Fig. 1) dedicated nearly 50 years of his life, the period between 1887 and 1934, to undertaking a solitary work that led to proving that the nervous system of humans and vertebrates was comprised of billions of independent interconnected elements that are organized into neural networks, and to drafting several theories about their development, function and plasticity. Many of these theories, elaborated by Cajal in the last part of XIXth century, have been proven by current neuroscience techniques. Currently, there is consensus that this work is the foundation of disciplines such as neuroanatomy, neurophysiology, neuropathology and neuroembriology, and these identify him as the true founder of modern neuroscience (Sotelo, 2002). Our aim in this essay is to analyze the origin of some of his most brilliant ideas, the chemotactic hypothesis or the neurotrophic theory, nowadays regarded as the origin of modern neuroembriology, as well as of concepts on brain plasticity, drafted by Cajal. Although the beginning of Cajals interest for the study of the nervous system can be dated to the year 1887, the moment in which he had his rst contact with silver impregnation techniques, it is logical to assume that a review of his scientic activity over the previous years will allow us to nd some keys that would inuence his subsequent thinking. We set forth on a journey to the origin of Cajals vocation as a researcher, and the implications thereof for his later scientic itinerary.

CAJAL AFTER HIS RETURN FROM CUBA

After spending 2 years in Cuba as a military doctor, Cajal was sent back to Spain in June of 1875, suering from malaria sequels. By then, it was evident for young Cajal that the practice of medicine was not his calling, which, together with his fathers strong will, directed him to fully become a university professor (Lopez Pinero, 2000). However, his progression in the pursuit of a university career was halted by the obstacle of having to hold the title of Doctor. The studies to gain access to the Doctors

*Corresponding author. Address: Department of Neurology and Neurophysiology, Torrecardenas Hospital Complex, Paraje de Tor recardenas S/N, CP: 04009, Almer a, Spain. Tel: +34-671562365. E-mail address: pedro.serrano.c@gmail.com (P. J. Serrano-Castro).

0306-4522/12 $36.00 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.05.008 1

P. J. Serrano-Castro, J. M. Garcia-Torrecillas / Neuroscience xxx (2012) xxxxxx

Fig. 1. Santiago Ramon y Cajal in 1933.

degree in Spain at that time established that it was mandatory to pass an examination in the subject of Normal and Pathological Histology, together with other two examinations on Chemical Analysis and History of Medicine. The Doctors degree would only be granted after nishing this training period, with the reading and defense of a doctorate thesis about a topic chosen by the candidate in any of those subjects. With this perspective, Cajal enrolled as an independent student in all of the three aforementioned subjects (Lazaro Perez, 2007). Cajal was deeply disappointed after studying for the subjects of Chemical Analysis and History of Medicine, which did not meet his expectations. However, his contact with the subject Normal and Pathological Histology was radically dierent. Cajal marveled after visualizing the microscopic preparations to which he had access in the laboratory of Dr. Maestre de San Juan (Cajal, 1981). So, he passed the exam of Normal and Pathological Histology with a notable grade. The next step was the preparation and defence of his doctorate presentation. The chosen topic, as it could not be any other way, was in the subject that had fascinated him. Specically, Cajal decided to study the pathogenesis of inammation.

of Virchow, he defended his teachers concepts on tissues. However, Cohnheims scientic curiosity made him continue analyzing this problem experimentally. This led him to nally publish all of these results in 1867 (Cohnheim, 1867), in a series of studies that entailed a full revision of Virchows ideas and that gave rise to a socalled vascular theory of inammation. For Cohnheim, purulent material comes from white blood cells that, heeding some sort of biological call, thanks to their ability to generate ameboid movements, turn up at the inammation site. Pus, therefore, originates at a place other than the inamed tissue itself. This was the debate that Cajal encountered when he carried out his doctorate presentation. The opinions had become radical. The school of French pathologists, among which were Duval, Picot and Morel, opposed Cohnheims theory, declaring that the alleged extravasation was merely the product of an optical illusion (Picot, 1874). Other prestigious pathologists, mainly German, such as Hayem and Vulpian, found that the migration of leukocytes was an undeniable fact.

CAJALS OPINION ON HIS DOCTORATE SPEECH

Cajals intention when dealing with the topic of inammation for the mandatory drafting of his doctorate presentation was not to intervene in this discussion. This possibility at that time was out of his reach. So, Cajal limited himself to analyze the dierent opinions and to issue a small critical judgment thereof (Rodriguez Quiroga, 2002). The complete manuscript of the doctorate speech is currently available in digital format within the Cajal Digital Collection of the Library of the Complutense University of Madrid (Cajal, 1877). In it, Cajal is decidedly eclectic. Thus, he dares criticize Virchows theory because, from his standpoint, it was not applicable to vascular tissues, where the migration phenomenon seemed irrefutable to him. However, he does not fully agree with Cohnheims hypothesis either. He argued that, even if it was perfectly acceptable in the case of vascular tissues, he considered it too limited and not applicable to other tissues (Cajal, 1877). Cajals contact with Cohnheims theories probably changed his conception of the nature of tissues. From Cohnheim point of view, cellular components of tissues were capable of changing their location purposely directed by internal or external forces. Cajal would not abandon this concept of biology and it would probably impregnate all of his scientic work in the future.

THE DEBATE ABOUT INFLAMMATION AND LEUKOCYTE MIGRATION IN CAJALS TIME

The erce scientic debate that existed during the last quarter of the XIXth century about the genesis of inammation was led by the coexistence of two confronting theories, proposed by two of the most relevant scientic gures of the time: Rudolf Virchow and Julius Cohnheim. The rst theory had dominated medical schools until a few years before. Virchows hypothesis postulated that purulent cells originate as an act of genesis within the affected tissue. Thus, these inammatory cells were not blood cells but cells coming from the inamed tissue itself (Ramon y Cajal Junquera, 2002). This is a theory that is intrinsically linked to the tradition of the great German pathologists, who considered that all pathological processes involved almost exclusively the connective tissue. On the other hand, Julius Cohnheim was a disciple of Rudolf Virchow. In his doctoral thesis, under the tutorship

CAJAL, THE EXPERIMENTAL RESEARCHER

Upon his return to Zaragoza, bearing the title of Doctor and qualied under examination, Cajal was awarded the position of Director of Anatomy Museums of Zaragoza. However, the panorama he encountered at the Faculty of Medicine of Zaragoza was disheartening.
Only in the Physiology Laboratory was there a microscope that was good enough. With this veteran instrument..//..I admired for the rst time the surprising spectacle of the

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Fig. 2. Mesenteric vein impregnated with silver nitrate in a frog. (A) Endothelial cells. (B) Red blood cells perforating the wall. (C) Transmigrated corpuscles. (D) Leukocytes in interstitial position and extremely pale (Literal translation from Cajal, 2006). Heirs of Cajal. With permission.

circulation of blood. Such a suggestive demonstration overwhelmingly contributed to develop in me a drive for micrographic studies (Cajal, 1981).

Using his own resources, he acquired his rst microscope, of the Verick brand, a microtome and other instruments for micrographic technical studies (Rodriguez Quiroga, 2002). With this equipment, he started his rst laboratory at home. Faithful to his known perseverance and scientic curiosity, and unsatised with his eclectic solution about the dilemma of the pathogenesis of inammation set forth in his doctoral thesis, he dedicated the three following years to solving this matter experimentally. These 3 years ended with the publication of his rst study in which, however, Cajal persists in his eclectic opinion. (Cajal, 1880). In Fig. 2 we reproduce a drawing of Cajal from this work representing a mesenteric vein impregnated with silver nitrate in a frog. Many years later, Cajal regrets his lack of independence very eloquently:
Unfortunately, I was immensely inuenced by the ideas of Duval, Hayem and other French histologists (who denied leukocyte extravasation) and was dragged to a syncretic solution or one of transaction, that was erroneous, as usually occurs in science with almost all diagonal opinions (Cajal, 1981).

Cajal did not like to be wrong, and the posterior verication of his perseverant error meant for him a turning point in his scientic mentality. Henceforth, Cajal abandoned the prejudices stemmed from an alleged scientic authority beyond what evidence itself could provide. Cajal and the reazione nera Cajal was appointed to the University of Barcelona at the end of 1887. This move would be the prelude to the most

fascinating and prolic time for Ramon y Cajal in terms of production. There was one name that was especially relevant for this to take place: Dr. Luis Simarro, a great fan of histology who had just returned from a stay in Paris where he had acquired basic notions about a new method for staining the nervous system based on silver nitrate, the Golgi method. This method would allow him for the visualization of its microscopic structures, staining them black (reazione nera or black reaction). Cajal was able to see the stain with the Golgi method for the rst time in Simarros laboratory, and was fascinated with what he could see through the microscope (Cajal, 1981). As had happened before, he went onto setting this technique into practice directly in his own laboratory, now in Barcelona. During the years 1887 and 1888, Cajal forwarded decisive research on the texture of the nervous system by perfecting Golgis stain method and by carrying out a correct election of the study material. Cajals preparations on embryo tissue showed that the terminal ramications of neurons ended in arborisations that juxtaposed the body and the dendrites of other neurons without establishing continuity with them. The study titled Estructura de los centros nerviosos de las aves [Structure of the nerve centres of birds], published on May 1, 1888 in the Quarterly Magazine of Normal and Pathological Histology (Cajal, 1888) may be considered the founding study for the neuron doctrine. Between 1888 and 1892 Cajal published over 30 studies that irrefutably proved the neuron doctrine and placed him at the top of the international scientic research elite. Cajals neurotrophic theory In 1890 Cajal made one of his most fascinating discoveries on the structure and development of neurons: the growth cone. During the months prior to this event he

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Fig. 3. Drawings by Cajal that represent growth cones. From these images, Cajal drafted his theory on neuronal ameboidism. Original publication: Textura del sistema nervioso del hombre y de los vertebrados, Volume I, Fig. 186, pp. 515, 1899. Heirs of Cajal. With permission.

had been impressed by the characteristic arborisations of embryonic nerve cells and their dierences according to the evolutionary stage. These observations were published in Spanish in the Gaceta Sanitaria de Barcelona and in French in the journal Anatomischer Anzeiger (Cajal, 1890a,b). In both papers, Cajal described his observations about the development and rising complexity of neuroblasts in the spinal cord of chicken embryos; he sets forth his own impressions about this dynamic fact that seems to him to be meticulously conducted by an intelligent force. He made drawings of his preparations in detail (see Fig. 3). Cajal carried out the hypotheses on the development of neuroblasts from the static images that his histological methods could provide him. Our hypothesis is that Cajal probably applied at that time all the knowledge he had gathered during his rst years of research on the pathogenesis of inammation. As we have seen, Cajal then acknowledged of the dynamic nature of cells, with elements under constant movement, directed by external forces with a predetermined purpose. Following this line of arguments, in 1892 Cajal formulated his neurotrophic theory, according to which target cells (those that would establish direct connexions with the studied neuroblasts) would be capable of secreting substances with a chemotactic capacity. Growth cones would be provided with sensitivity for these substances and ability for ameboidism, similar to what occurs with leukocytes (Cajal, 1892). In 1994, Serani et al. discovered the rst substances with chemotactic ability, secreted during embryonic development, as Cajal predicted, and called them Netrins. In Cajals mind, the only dierence between the targeted motion of a leukocyte and the expansions of a neuroblast lay on the limitations that the staining techniques imposed in the case of the nervous system, which made the images to be strictly static. It was necessary to imagine a motion that could not be seen directly. A similar idea was pointed out previously by leading gures such as Sherrington (1949). Firmly convinced that this theory is universally applicable, he suggested that chemotactic mechanisms

may also be involved in the processes of nerve cell migration, both in the positive sense (attraction) and in the negative sense (repulsion) (Cajal, 1892). Modern discoveries about the processes regulating neuronal migration have conrmed Cajals theories in this regard. Thus, only until recently has it been possible to demonstrate the existence of a tangential migration that had already been intuitively conceived by Cajal (De Carlos et al., 1996). He took one step further still. Cajal used the term plasticity for the rst time, applying it to the nervous tissue in the International Medical Congress to which he was invited as a lecturer in Rome, in 1894 (De Felipe, 2006). At that time, Cajal used the words dynamism or adaptation to the environment to refer to the structural changes of neurons. At that point in his life, Cajal only saw the brain as being in perpetual motion, and brain structures perpetually changing through their dendritic spines, elements he considered a prototype of this adaptability to their environment (Fig. 4). Cajal then spoke about brain gymnastics as a useful mechanism for multiplying nerve connexions and thus improve the brains functionality (De Felipe, 2007). We are thus faced with a precedent of cognitive stimulation, to which modern neuropsychology attributes evident preventive properties for cognitive impairment. More than 100 years after Cajal spoke of brain gymnastics and proposed the motility of dendritic spines as the anatomical basis of their eects on cognition, the introduction of complex imaging techniques has made it possible to demonstrate that synapses are highly dynamic structures, permitting rapid formation and remodeling of connections in CNS tissues (Marrs et al., 2001). In 1895 Cajal proposed the theory of glial ameboidism (De Felipe, 2006), according to which the changes in the brains activity correlate not only with structural changes in dendritic spines, but also with structural changes in the glia that are capable of disconnecting neuronal synaptic connexions during their expansions. Modern neuroscience corroborates the existence of changes in transient astroglial ramications modulated by brain activity.

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Fig. 4. Drawing of the dendritic spines of the human frontal cortex. Heirs of Cajal. With permission.

CONCLUSION
With the perspective of more than one century, we can arm that there is a common denominator in the group of theories that Cajal proposed for the functioning of the nervous system: the movement. The neurons move during their development and also adapt to dierent external circumstances. Moreover, chemical endogenous substances can stimulate this movement in a similar way to leukocytes during the process of inammation. This is a relevant contribution by Cajal to the History of Neuroscience that is not well known and that we aim to point out in this essay.

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(Accepted 2 May 2012) (Available online xxxx)

Please cite this article in press as: Serrano-Castro PJ, Garcia-Torrecillas JM. Cajals rst steps in scientic research. Neuroscience (2012), http:// dx.doi.org/10.1016/j.neuroscience.2012.05.008