From the Cajal alumni Achúcarro and Río-Hortega to the rediscovery of never-resting microglia

Abstract

Under the guidance of Ramón y Cajal, a plethora of students flourished and began to apply his silver impregnation methods to study brain cells other than neurons: the neuroglia. In the first decades of the twentieth century, Nicolás Achúcarro was one of the first researchers to visualize the brain cells with phagocytic capacity that we know today as microglia. Later, his pupil Pío del Río-Hortega developed modifications of Achúcarro's methods and was able to specifically observe the fine morphological intricacies of microglia. These findings contradicted Cajal's own views on cells that he thought belonged to the same class as oligodendroglia (the so called "third element" of the nervous system), leading to a long-standing discussion. It was only in 1924 that Río-Hortega's observations prevailed worldwide, thus recognizing microglia as a unique cell type. This late landing in the Neuroscience arena still has repercussions in the twenty first century, as microglia remain one of the least understood cell populations of the healthy brain. For decades, microglia in normal, physiological conditions in the adult brain were considered to be merely "resting," and their contribution as "activated" cells to the neuroinflammatory response in pathological conditions mostly detrimental. It was not until microglia were imaged in real time in the intact brain using two-photon in vivo imaging that the extreme motility of their fine processes was revealed. These findings led to a conceptual revolution in the field: "resting" microglia are constantly surveying the brain parenchyma in normal physiological conditions. Today, following Cajal's school of thought, structural and functional investigations of microglial morphology, dynamics, and relationships with neurons and other glial cells are experiencing a renaissance and we stand at the brink of discovering new roles for these unique immune cells in the healthy brain, an essential step to understand their causal relationship to diseases.

Keywords: Achúcarro; Cajal; Río-Hortega; discovery; imaging; microglia; neuroanatomy; phagocytosis.

Figure 1

Unraveling the nature of the “third element.” In the early years of the twentieth century Achúcarro developed a novel method of staining (tannin and amoniacal silver nitrate) that allowed him to visualize what were at the time called “granuloadipose cells” that is, a group of cells that seemed to phagocytose degradation materials in the brain of rabid rabbits (A). Stimulated by the interest of Achúcarro in glia, Cajal developed his famous sublimated gold chloride method that allowed him to beautifully visualize astrocytes but left the remaining neuroglial cells poorly stained (a–h, in B). To describe these apolar, dwarf cells he introduced the term “third element” of the nervous system. Río-Hortega continued the work of Achucarro and in 1919 developed his silver carbonate method, finally enabling him to resolve microglia (C) from what he initially called interfascicular cells and later oligodendrocytes (using his Golgi-Hortega method; D). Achúcarro's images are reprinted with permission from Achúcarro (1909). Cajal's image is reprinted with permission from Garcia-Marin et al. (2007). Río-Hortega's images are reprinted with permission from Río-Hortega (1919c).

Figure 2

Evolution of microglial research. The numbers of published papers per year about microglia was assessed on PubMed Central (www.ncbi.nlm.nih.gov) using the search term “microglia” (All Fields) and setting the Date of Publication from January 1st to December 31st from 1900 until 2014 (black squares). Since the earliest descriptions of Achúcarro and Río-Hortega at the beginning of the twentieth century, there were barely any papers discussing microglia published before 1990 (456 papers in total), from when there was a steady increase until the end of the Century (356 papers per year). It was not until the early 2000s that there was an exponential growth of microglial research (1180 papers per year in 2000–2014). The inflexion point seems to be located around 2005, after the publication of the seminal papers by Davalos and Nimmerjahn on the extraordinary motility of microglia in the adult brain in physiological conditions (Davalos et al., ; Nayak et al., 2014). To detect more specifically papers focused on microglia, we performed another search looking for the term “microglia” in the title (Title Field) (purple dots). Over the period 1990–2014, the percentage of papers with “microglia” in their title over the total number of papers including “microglia” in any field was maintained at a fairly constant rate of 24 ± 0.9% (mean ± SEM). When searching for papers with “microglia” in their title a second inflexion point is also evident around 2010 (155 papers per year in 2000–2004, 231 papers per year in 2005–2009, and 406 papers per year in 2010–2014), coincident with the publication of the influential paper by Ginhoux on the unique origin of microglia from the embryonic yolk sac (Ginhoux et al., 2010). This figure does not intend to be a systematic analysis of the most influential papers on microglia research, but to evidence that the rediscovery of the findings by Achucarro and Río-Hortega shapes our current research in microglial biology.