La Fundación de la Neurociencia: _{El trabajo de Santiago Ramón y Cajal y Camillo Golgi}

Budding researchers in a time of technological advances

When Camillo Golgi was born on July 7, 1843, people had been peering through crude microscopes for more than two centuries, but cells of the nervous system were virtually invisible to microscopy. When Santiago Ramón y Cajal was born on May 1, 1852, cell theory was brand new to the world of science. Though proposed 13 years prior, the idea that body tissues were composed of individual cells was not widely accepted among biologists, nor did cell theory even include the basic concept that all cells come from pre-existing cells.

Back in 1665, Robert Hooke had coined the term “cells” after observing tiny compartments in a sample of cork (Figure 1). In the following decades, biologists had observed individual bacteria, and also cells within animal fluids, such as red blood cells and sperm. These observations could not be put into context, however, because the technology of microscopy was extremely primitive. A few simple dyes helped improve cell visibility, but early microscopists lacked stains that could elucidate cell structure. Consequently, cells could not be seen in all biological tissues, and science was not ready for the idea of the cell as the basic unit of life.

By the mid the 19th century, however, due to improvements both stains and microscope lenses, biologists were starting to see cells everywhere. Thus, a series of discoveries by Theodor Schwann and Matthias Jakob Schleiden led to the idea in the 1830s that all organisms were made up of cells. That was the initial cell theory; however, it was flawed with the idea that many of the cells within organisms arose through a crystallization process from material outside the organism. It was not until 1855 that Rudolf Virchow refined the theory with the principle that all cells had to arise from pre-existing cells. That was a major insight, though it would take another decade or so for cell theory to be accepted widely, and still more time before the idea of cells as building blocks would be applied to the most elusive biological structure, the brain. (Read about the development of cell theory in our module Discovery and Structure of Cells.)

Cell theory applied to the brain

The first clear indication that the brain was made of cells came in 1837, six years and fifteen years before the births of Golgi and Ramón y Cajal, respectively. That was when Czech anatomist Johann Purkinje cut into a cerebellum with a new instrument that made very thin slices. Because the brain slices were thin, anatomists could view them under the microscope. Thus, Purkinje was able to see large cells that were later called neurons, and to show their presence throughout the cerebellum. Also, in the mid-19th century, Purkinje and other anatomists were able to define and draw structures visible on very low microscopic magnification, and to the naked eye. The structures were given names such as the thalamus and the substantia nigra, and they appeared consistently in the same locations of brains from different cadavers when the brains were sliced in a consistent, systematic pattern.

This was the beginning of neuroscience, but nobody had any idea what the structures actually did in terms of function. Moreover, despite Purkinje visualizing neurons in the cerebellum, and despite that they would later be called “Purkinje cells,” they did not look like what scientists called “cells” in those days. Unlike blood cells, which are round, or bacterial cells, which can have many shapes but remain small, what Purkinje saw were more like fibers (Figure 2). They were narrow such that they could be seen only with a microscope, but they had long branches that would come to be called dendrites. Thus, while elucidated by microscopy and staining, the idea these long narrow fibers in the brain were cells in the same sense as blood cells or bacterial cells lay decades into the future.

That was the world into which Golgi and Ramón y Cajal were born, the former in the village of Corteno in northern Italy, the latter in Petilla de Aragón in northern Spain. That was the world in which their fathers practiced medicine and surgery and taught anatomy. While the young Golgi always knew he was headed for medicine, Ramón y Cajal spent his boyhood thinking about other things, especially art and gymnastics. Both were connected with his future career, which depended greatly on his ability to draw the biological structures that he observed. For most of his life, those structures would appear in a microscopic field, but as a boy he was inspired by human anatomy on a larger scale, as he saw himself and friends from the gym growing muscle bound. He also sketched bones that he dug up with his father in graveyards.

As a new graduate in medicine at the University of Pavia, Golgi headed for psychiatry. He believed strongly that all mental disorders resulted from physical lesions in neural centers, and soon he grew frustrated with the psychiatric field. In those days, none of the “theories” proposed in connection with mental illness could be subjected to physical tests that might reveal any physical lesions. That was not possible since the nature of the nervous system was completely unknown. Golgi thus left psychiatry and set out instead to figure out the structure of the nervous system and how it worked. This meant focusing largely on histology (microscopic anatomy) of the brain and other structures of the nervous system.

Advances in microscopes and staining

Due to financial difficulties, in 1872 Golgi was forced to accept the position of chief of medicine at Pio Luogo degli Incurabili (Hospital for the Incurably Ill), located near Pavia, Italy. It was a clinical position that he had never desired, but he held it for three years and found a way to pursue research during this period. After converting the hospital’s small kitchen into a makeshift laboratory, Golgi spent the evenings developing new stains and staining techniques that he hoped would elucidate biological tissue, especially nervous tissue, under the microscope. Most of the techniques that he tried didn’t work or helped only a little. But through trial and error, night after night, Golgi soon came up with a procedure that proved remarkably effective. After exposing samples of nervous tissue to potassium dichromate, he immersed the samples in a solution of silver nitrate. This caused precipitation of silver chromate (that is, the formation of crystals resulting from the exchange of ions between the potassium and silver salts), which made features of the nervous tissue – features that today we know as the membranes of nerve cells – stand out as black silhouettes against a golden-yellow background (Figure 3). Under the microscope, this revealed what appeared to be nervous fibers resembling features that Purkinje had seen, but they stood out much better.

In 1873, Golgi wrote up a short paper on the silver staining technique for the journal Gazzetta Medica Italiana. In the paper, he called his discovery the "reazione nera" ("black reaction"). It proved so effective that its use has persisted into our time, although today it is called the "Golgi stain." That same year, Ramón y Cajal graduated from medical school at the University of Zaragoza, where he excelled in anatomic dissection. After a stint in the Spanish army in Cuba, he earned a Ph.D., also at Zaragoza. This launched him into an academic career that moved him from Zaragoza to the universities of Valencia and Barcelona and finally to Central University in Madrid.

After publishing a series of papers with descriptions and drawings of previously unseen structures such as the brain’s hippocampus and olfactory bulb, made visible by his silver staining technique, Golgi was appointed Professor of Histology at the University of Pavia in 1876. Once again, he could conduct full-time research, and now he was also well known in the world of anatomy research.

Meanwhile, Ramón y Cajal was doing his own research over in Spain. Early in his career, the research involved muscle anatomy, inflammation, and microbiology, and it really was completely his own research. Unlike most researchers, he had no mentor, for he was unusually independent-minded. As boy, he’d been rebellious, almost to the point of losing the opportunity to go to medical school, but digging up the bones in the graveyard with his father and sketching them had provided focus. A hint of rebellion would remain throughout life though. Ramón y Cajal disdained dogma, including dogma in the scientific community. Thus, later in life, he would say, "Hypotheses come and go, but data remain." The quote applies perfectly to ideas about the brain that were popular in the 19th century, in particular a belief that the brain was not made of cells.

Though muscle anatomy, microbiology, and inflammation may seem unrelated, as if the young Ramón y Cajal were jumping about science at whim, he published scientific papers on all three topics during the early 1880s. In the course of that decade, he examined and drew muscle tissue on a continuously shrinking scale. He did this until his work focused entirely on histology, that is, anatomy at the microscopic level. Gradually, this is what led him to the central nervous system.

During the 1860s and early 1870s, microscopy had improved greatly, due to rapid advances in optics as well as staining for different types of tissues and parts of cells. By the 1880s, these advances enabled researchers to delve increasingly into the structure of epithelial tissue, the type of tissue that covers body surfaces, cavities, and tubular structures. Epithelial and muscle tissue are present throughout the body, and throughout the animal kingdom, so anyone studying them in those days got an extensive tour of all body systems and many species.

Thus, over the course of their careers, both Golgi and Ramón y Cajal applied their research skills to numerous organ systems, various mammals and insects, and embryos, all in an effort to understand how everything fit together and functioned. Both scientists were prolific writers. Golgi’s publications are collected in the Opera Omnia and are so numerous that they take up volumes. As an author, Ramón y Cajal is listed on several books and manuals dealing with histological techniques. He also published some three hundred scientific papers spanning from 1880 until 1934, the year of his death.

Golgi and Ramón y Cajal were skilled not just in microscopy, but also in drawing (see Figures 2 and 4). That latter ability was vital to microscopists of the era, since cameras were large and could not be integrated with microscopes. Essentially, the researcher had to be a human camera. Thus, for Ramón y Cajal in particular, a lifelong passion was key to his professional success. The unique combination of talents placed both men at the forefront of the research on epithelia and other types of tissue, including tissues of the brain. But the dual talents also set them on course for dueling scientific ideas.

Improving on Golgi’s technique

In biology, when a cell type, a structure within a cell, or a laboratory technique is named for its discoverer, it means that the scientist made major contributions to the field. In the case of neurobiology, there are "Golgi cells" (as well as "Purkinje cells") in the cerebellum of the brain, "Golgi tendon organs" that generate nerve signals in response to muscle tension, and "interstitial cells of Cajal" in the gastrointestinal tract. These cells are named, respectively, for Golgi and Ramón y Cajal (albeit, only for the maternal component of the Spaniard’s full name, which in correct usage must also include his father’s family name, Ramón). The names of both men are also given to other biological structures.

As for the silver staining method, it bears only Golgi’s name, but Ramón y Cajal is also part of its history. After trying the stain in 1887, he started meddling with the procedure, cutting thicker sections of nerve tissue and using the chemicals at concentrations higher than Golgi had prescribed. The younger man found that this made the nervous fibers stand out much better and also noticed that it stained better on tissue from bird brains and mammalian embryos than on the brains of fully developed mammals. He found that unlike fully-grown mammals, embryonic brain tissue and bird brains lacked a fatty substance called myelin, and this accounted for the difference in the ability to hold the modified stain.

Two competing doctrines: Neuron Doctrine and Reticular Theory

In the 1880s, Golgi was appointed chair of General Pathology and Dean of the Faculty of Medicine. He established the University of Pavia’s Institute of Pathology, which greatly increased Pavia’s clout and attracted a host of visiting scientists from within and outside of Italy. All of this catapulted Golgi to the status of mentor for some of biology’s most promising young researchers. But as Golgi was developing into the teacher that every researcher wanted, his Spanish counterpart pressed onward, applying the modified form of the staining technique to a host of different nervous tissues.

Over a six-year period, this led Ramón y Cajal to a major discovery: The brain itself is made of individual cells. It proved an idea that he’d been advocating, called the neuron doctrine. It seems axiomatic today, but it was not so clear in the late 1880s, and indeed, Golgi advocated something entirely different, called the reticular theory. It was an alternative to the neuron doctrine and suggested that the fibers that Golgi, Ramón y Cajal, and others who repeated their experiments were seeing were part of a continuous electrical network, or reticulum. Given his artistic ability, however, Ramón y Cajal was able to draw, almost photographically, what his modified technique showed on the bird brain tissue, and overall the research showed that the fibers were distinct cells. But along the way there were particular discoveries supporting this idea.

Once such discovery came within a year of modifying the Golgi staining technique. In 1888, Ramón y Cajal was able to follow the course of particular structures of dendrites, something that he called "dendritic spines." He could see them extremely well by using a stain called methylene blue, which was part of his modification of the Golgi technique. This led him to see that the axons of what we now know to be nerve cells, or neurons, terminate freely in the retina and cerebellum. That contradicted the reticular theory, which predicted that the same structures should have been woven into a mesh, rather than terminating freely.

Two years later, Ramón y Cajal saw and drew amoeba-like structures at the end of axons of developing neurons of embryos. The positions of the structures changed with respect to the fibers as the embryo developed, leading Ramón y Cajal to propose that the end of the axon was mobile. He called the structure an "axonal growth cone" and decided that it must have functioned to help developing neurons form connections with other neurons. This too did not sway with the reticular theory.

By 1891, Ramón y Cajal decided that the expansion of nerve cells occurred through growth of dendrites, which he referred to as "protoplasmic branches." In other words, he realized that there were separate entities forming what looked like a reticulum, that the dendrites were actually extensions of brain cells. Within three years, he adapted some ideas from other researchers concerning learning, and speculated that intelligence was related to the number and efficiency of connections between different types of cells with the part of the brain known as the cerebral cortex. It was a remarkable idea for the time, and it dovetails with what neuroscientists have learned in recent years.

Up to the mid-1890s, Ramón y Cajal used mostly brains of birds and embryos of mammals for his studies. As methods continued to improve, however, he and other researchers proved that the findings in bird brains carried over to mature mammals, including humans. Just as muscle tissue is made of long, narrow cells, so is nervous tissue made of nerve cells, called neurons (Figure 5). Often these are very long, even a meter or longer, but they are separate cells, not a reticulum, and each brain, spinal cord, and nerve contains billions of them. That conclusion was clear by the close of the 19th century.

One scientist proves the other wrong: Both win the Nobel Prize

The story of Camillo Golgi came to fruition with one of the most ironic twists in the history of science. He and Ramón y Cajal are remembered as the two fathers of neuroscience, but when it came to the nature of the nervous system, only one of them could have been right, and that turned out to be the latter man. Using his modification of Golgi’s technique, Santiago Ramón y Cajal discovered that nerve tissue was composed of individual nerve cells. This overturned reticular theory, the idea that Golgi supported.

By the turn of the 20th century, the cellular nature of the brain was fairly clear, and both scientists were awarded the Nobel Prize in Physiology and Medicine. They received it as a shared prize in 1906 for their work elucidating the structure of the nervous system. The revealed structure included specific features within nervous tissue, but also the cellular nature of that tissue, an idea that Golgi continued to reject even in his Nobel Prize acceptance speech. Whether Golgi agreed with it or not, the revelation that nervous tissue was made of cells was possible largely because of the stain that he had invented and that Ramón y Cajal had modified.