The History of Spanish Neuroscience: Thematic Papers Issue.

To commemorate the 100th anniversary of the creation of the Instituto Cajal, we bring this thematic issue to honor Spanish neuroscience. The issue includes several papers written by scientists, most of them being direct descendants of those who lived on the front lines of the dawn of Spanish neuroscience. These pages reflect a summary of the main achievements of this unique group of neuroscientists, all of them belonging to the Cajal School. Finally, this school of scientists became the forerunner of the modern worldwide neuroscience. Anat Rec, 303:1179-1180, 2020. © 2020 American Association for Anatomy.

Pedro Ramón y Cajal: The Legacy of a Neurohistologist, a Medical Doctor, and a Pathologist.

The work of Santiago Ramón y Cajal is difficult to understand without knowing the personalities of Justo, his father, and his brother Pedro. His father practically forced the two brothers to study medicine. Thanks to that, Santiago was able to combine his artistic talent with histology and wonderfully describe cerebral architecture. Pedro was a faithful brother and above all a friend of Santiago, and they worked together for years. Pedro was able to demonstrate the theories of his brother in nonmammalian amniotes, concluding that the basic elements of the nervous system are common to these animals and he provided images that served Santiago to formulate the theory of dynamic polarization. Pedro, who decided to remain in the shadow of his brother, was a very complete doctor, pathologist and gynecologist, who made interesting contributions in all these fields and above all was a great humanist who left an important personal and scientific legacy. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists. Anat Rec, 303:1189-1202, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Cajal's Interactions with Sherrington and the Croonian Lecture.

Sherrington was a major proponent of the neuron doctrine and he was inspired by Santiago Ramón y Cajal's theory of dynamic polarization of nerve cells (Ley de la polarización dinámica de la célula nerviosa). Sherrington coined the term "synapse" to name the Cajal description of interneuronal contact and he gave the term, for functional nerve endings, as "Boutons terminaux," still used today. These two giants of neuroscience met only once, but they had a life-long friendship. It was Sherrington who wrote Cajal's Obituary for the Royal Society. We review here some of the scientific exchanges between Cajal and Sherrington, with particular attention to 1894, when the two neuroscientist met in London during Cajal's visit to deliver the Croonian Lecture to the Royal Society. We shall examine not only the scientific exchanges but also their friendship, which was immediate and strong. Anat Rec, 303:1181-1188, 2020. © 2019 American Association for Anatomy.

Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors.

BACKGROUND: Histamine (HA) regulates the sleep-wake cycle, synaptic plasticity and memory in adult mammals. Dopaminergic specification in the embryonic ventral midbrain (VM) coincides with increased HA brain levels. To study the effect of HA receptor stimulation on dopamine neuron generation, we administered HA to dopamine progenitors, both in vitro and in vivo. RESULTS: Cultured embryonic day 12 (E12) VM neural stem/progenitor cells expressed transcripts for HA receptors H1R, H2R and H3R. These undifferentiated progenitors increased intracellular calcium upon HA addition. In HA-treated cultures, dopamine neurons significantly decreased after activation of H1R. We performed intrauterine injections in the developing VM to investigate HA effects in vivo. HA administration to E12 rat embryos notably reduced VM Tyrosine Hydroxylase (TH) staining 2 days later, without affecting GABA neurons in the midbrain, or serotonin neurons in the mid-hindbrain boundary. qRT-PCR and Western blot analyses confirmed that several markers important for the generation and maintenance of dopaminergic lineage such as TH, Lmx1a and Lmx1b were significantly diminished. To identify the cell type susceptible to HA action, we injected embryos of different developmental stages, and found that neural progenitors (E10 and E12) were responsive, whereas differentiated dopaminergic neurons (E14 and E16) were not susceptible to HA actions. Proliferation was significantly diminished, whereas neuronal death was not increased in the VM after HA administration. We injected H1R or H2R antagonists to identify the receptor responsible for the detrimental effect of HA on dopaminergic lineage and found that activation of H1R was required. CONCLUSION: These results reveal a novel action of HA affecting dopaminergic lineage during VM development.

Santiago Ramón y Cajal: The Cajal Institute and the Spanish Histological School.

Santiago Ramón y Cajal was a self-taught researcher. He almost always worked alone, usually in the solitude of his private laboratory installed at his home. He was also a university professor and therefore taught histology and pathology to many students. But because research laboratories were scarce and poorly equipped, he preferred to organize courses and tutor at home as well. For this reason, Cajal left a faint trace of disciples in the three academic chairs that he came to occupy. It may be argued that Cajal formed the histological school when the Spanish government decided to support his investigations and created a scientific laboratory for him, with funding to cover the cost of journals, instruments, materials, personnel, and so forth. This support occurred in the year 1902, after Cajal received the Moscow Prize. Some of his former students accompanied Cajal to the new laboratory. Upon receipt of new awards, including the Gold Medal of von Helmholtz (1905) and the Nobel Prize in Physiology or Medicine (1906), Cajal's popularity increased and a large number of students wanted to learn about the laboratory and work with the great Cajal. This review tells this history. But we realize that this is not an easy task because to be fair to all the people that formed the Spanish Histological School, we would need to write a book. This is not practical. Instead, selection of contributors to the formation of the Spanish Histological School is provided. At the same time, some brushstrokes of the story extend to and include the Cajal Institute, which ran in parallel with the Spanish Histological School.

Epilog: Cajal's unique and legitimated school.

Santiago Ramón y Cajal is recognized as the founder of modern neuroscience, his discoveries representing the fundamental pillars of our current understanding of the nervous system. As Cajal's career spanned a critical period in Spanish history, he witnessed strong social demands for progress in culture, education, and science. Indeed, the life of Santiago Ramón y Cajal can be considered to reflect the gradual development of Spanish science from the last third of the 19th century. Cajal promoted a national movement that had important consequences for Spanish science, mainly triggered by the creation of the "Junta para Ampliación de Estudios e Investigaciones Científicas," an instrument he established to enrich scientific research and that was later to bear such abundant fruit. The school generated by Cajal profited from this development, through which all Cajal's disciples received fellowships to train in laboratories across Europe. Unfortunately, the Spanish Civil War disrupted this revitalization of Spanish science and provoked the diaspora of many Spanish scientists. However, a political impulse, mostly following this spirit, was resumed in Spain during the eighties that successfully led to a renaissance in Spanish science.

Evolution and development of the mammalian cerebral cortex.

Comparative developmental studies of the mammalian brain can identify key changes that can generate the diverse structures and functions of the brain. We have studied how the neocortex of early mammals became organized into functionally distinct areas, and how the current level of cortical cellular and laminar specialization arose from the simpler premammalian cortex. We demonstrate the neocortical organization in early mammals, which helps to elucidate how the large, complex human brain evolved from a long line of ancestors. The radial and tangential enlargement of the cortex was driven by changes in the patterns of cortical neurogenesis, including alterations in the proportions of distinct progenitor types. Some cortical cell populations travel to the cortex through tangential migration whereas others migrate radially. A number of recent studies have begun to characterize the chick, mouse and human and nonhuman primate cortical transcriptome to help us understand how gene expression relates to the development and anatomical and functional organization of the adult neocortex. Although all mammalian forms share the basic layout of cortical areas, the areal proportions and distributions are driven by distinct evolutionary pressures acting on sensory and motor experiences during the individual ontogenies.

Extracortical origin of some murine subplate cell populations.

The subplate layer, the deepest cortical layer in mammals, has important roles in cerebral cortical development. The subplate contains heterogeneous cell populations that are morphologically diverse, with several projection targets. It is currently assumed that these cells are generated in the germinative zone of the earliest cortical neuroepithelium. Here we identify a pallial but extracortical area located in the rostromedial telencephalic wall (RMTW) that gives rise to several cell populations. Postmitotic neurons migrate tangentially from the RMTW toward the cerebral cortex. Most RMTW-derived cells are incorporated into the subplate layer throughout its rostrocaudal extension, with others contributing to the GABAergic interneuron pool of cortical layers V and VI.

Temporal processing in the olfactory system: can we see a smell?

Sensory processing circuits in the visual and olfactory systems receive input from complex, rapidly changing environments. Although patterns of light and plumes of odor create different distributions of activity in the retina and olfactory bulb, both structures use what appears on the surface similar temporal coding strategies to convey information to higher areas in the brain. We compare temporal coding in the early stages of the olfactory and visual systems, highlighting recent progress in understanding the role of time in olfactory coding during active sensing by behaving animals. We also examine studies that address the divergent circuit mechanisms that generate temporal codes in the two systems, and find that they provide physiological information directly related to functional questions raised by neuroanatomical studies of Ramon y Cajal over a century ago. Consideration of differences in neural activity in sensory systems contributes to generating new approaches to understand signal processing.

A further analysis of olfactory cortex development.

The olfactory cortex (OC) is a complex yet evolutionarily well-conserved brain region, made up of heterogeneous cell populations that originate in different areas of the developing telencephalon. Indeed, these cells are among the first cortical neurons to differentiate. To date, the development of the OC has been analyzed using birthdating techniques along with molecular markers and in vivo or in vitro tracking methods. In the present study, we sought to determine the origin and adult fate of these cell populations using ultrasound-guided in utero injections and electroporation of different genomic plasmids into the lateral walls of the ventricles. Our results provide direct evidence that in the mouse OC, cell fate is determined by the moment and place of origin of each specific cell populations. Moreover, by combining these approaches with the analysis of specific cell markers, we show that the presence of pallial and subpallial markers in these areas is independent of cell origin.

The embryonic septum and ventral pallium, new sources of olfactory cortex cells.

The mammalian olfactory cortex is a complex structure located along the rostro-caudal extension of the ventrolateral prosencephalon, which is divided into several anatomically and functionally distinct areas: the anterior olfactory nucleus, piriform cortex, olfactory tubercle, amygdaloid olfactory nuclei, and the more caudal entorhinal cortex. Multiple forebrain progenitor domains contribute to the cellular diversity of the olfactory cortex, which is invaded simultaneously by cells originating in distinct germinal areas in the dorsal and ventral forebrain. Using a combination of dye labeling techniques, we identified two novel areas that contribute cells to the developing olfactory cortices, the septum and the ventral pallium, from which cells migrate along a radial and then a tangential path. We characterized these cell populations by comparing their expression of calretinin, calbindin, reelin and Tbr1 with that of other olfactory cell populations.

Dab1 (Disable homolog-1) reelin adaptor protein is overexpressed in the olfactory bulb at early postnatal stages.

Dab1 mediates reelin signalling and plays critical roles in early brain development such as the stereotypical positioning of neurons in the brain. The olfactory bulb undergoes a prominent layering reorganization, but shows not apparent differences between wild type and reeler in the layer organization. Therefore, an accurate regional and cellular simultaneous analysis of these molecules becomes essential to clarify the role played by Dab1 upon Reelin effect. The present study reveals a strong and consistent Dab1 mRNA and protein expressions, throughout the olfactory bulb layers in both wild type and reeler mice. In addition, noteworthy is the pattern of Dab1 location within cell nuclei in both strains. Furthermore, a temporal increment of Dab1 expression levels is detected from P0 to P15 in both strains, being the protein quantity higher in reeler than in wild type mice. Altogether, our results revealed that Reln acts directly from projection neurons via the production of different Reln fragments. Changes in the pattern of Dab1 expression could reflect an alternative Reln function in postnatal and adult stages, besides a possible regulation of Dab1 by other molecules distinct to Reln.

LIM-homeobox gene Lhx5 is required for normal development of Cajal-Retzius cells.

Cajal-Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here, we show that two closely related LIM-homeobox genes Lhx1 and Lhx5 are expressed in reelin+ cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area, and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem, and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.

A neuronal migratory pathway crossing from diencephalon to telencephalon populates amygdala nuclei.

Neurons usually migrate and differentiate in one particular encephalic vesicle. We identified a murine population of diencephalic neurons that colonized the telencephalic amygdaloid complex, migrating along a tangential route that crosses a boundary between developing brain vesicles. The diencephalic transcription factor OTP was necessary for this migratory behavior.

The influence of the environment on Cajal-Retzius cell migration.

During cerebral cortex development, different cell populations migrate tangentially through the preplate, traveling from their site of origin toward their final positions. One of the earliest populations formed, the Cajal-Retzius (C-R) cells, is mainly generated in different cortical hem (CH) domains, and they migrate along established and parallel routes to cover the whole cortical mantle. In this study, we present evidence that the phenotype of -Retzius cells, as well as some of their migratory characteristics, is specified in the area where the cells are generated. Nevertheless, when implanted ectopically, these cells can follow new migratory routes, indicating that locally provided genetic cues along the migratory path nonautonomously influence the position of these cells emanating from different portions of the CH. This was witnessed by performing CH implants of tissue expressing fluorescent tracers in live whole embryos. In the same way, tracer injections into the hem of Small eye mutant mice were particularly informative since the lack of Pax6 affects some guidance factors in the migratory environment. As a result, in these animals, the C-R cell population is disorganized, and it forms 1 day late, showing certain differences in gene expression that might help explain these disruptions.

Different astroglia permissivity controls the migration of olfactory bulb interneuron precursors.

The rostral migratory stream (RMS) is a well defined migratory pathway for precursors of olfactory bulb (OB) interneurons. Throughout the RMS an intense astroglial matrix surrounds the migratory cells. However, it is not clear to what extent the astroglial matrix participates in migration. Here, we have analyzed the migratory behavior of neuroblasts cultured on monolayers of astrocytes isolated from areas that are permissive (RMS and OB) and nonpermissive (cortex and adjacent cortical areas) to migration. Our results demonstrate robust neuroblast migration when RMS-explants are cultured on OB or RMS-astrocytes, in contrast to their behavior on astroglia derived from nonpermissive areas. These differences, mediated by astrocyte-derived nonsoluble factors, are related to the overexpression of extracellular matrix and cell adhesion molecules, as revealed by real-time qRT-PCR. Our results show that astroglia heterogeneity could play a significant role in migration within the RMS and in cell detachment in the OB.

Synaptogenesis in the mouse olfactory bulb during glomerulus development.

Synaptogenesis is essential for the development of neuronal networks in the brain. In the olfactory bulb (OB) glomeruli, numerous synapses must form between sensory olfactory neurons and the dendrites of mitral/tufted and periglomerular cells. Glomeruli develop from E13 to E16 in the mouse, coincident with an increment of the neuropil in the border between the external plexiform (EPL) and olfactory nerve layers (ONL), coupled to an extensive labelling of phalloidin and GAP-43 from the ONL to EPL. We have tracked synaptogenesis in the OB during this period by electron microscopy (EM) and immunolabelling of the transmembrane synaptic vesicle glycoprotein SV-2. No SV-2 labelling or synapses were detected at E13, although electrodense junctions lacking synaptic vesicles could be observed by EM. At E14, sparse SV-2 labelling appears in the most ventral and medial part of the incipient OB, which displays a ventro-dorsal gradient by E15 but covers the entire OB by E16. These data establish a spatio-temporal pattern of synaptogenesis, which perfectly matches with the glomeruli formation in developing OB.

Early telencephalic migration topographically converging in the olfactory cortex.

Neurons that participate in the olfactory system arise in different areas of the developing mouse telencephalon. The generation of these different cell populations and their tangential migration into the olfactory cortex (OC) was tracked by tracer injection and in toto embryo culture. Cells originating in the dorsal lateral ganglionic eminence (LGE) migrate tangentially along the anteroposterior axis to settle in the piriform cortex (PC). Those originating in the ventral domain of this structure occupy the thickness of the olfactory tubercle (OT), whereas cells from the rostral LGE migrate tangentially into the most anterior telencephalon, at the level of the prospective olfactory bulb (pOB). Neurons from the dorsal telencephalon migrate ventrally, bordering the PC, toward olfactory structures. Two cell populations migrate tangentially from the rostromedial telencephalic wall to the OT and the PC, passing through the ventromedial and dorsolateral face of the telencephalon. Some cells from the germinative area of the rostral telencephalon, at the level of the septoeminential sulcus, migrate rostrally to the pOB or caudally to the OC. Thus, we demonstrate multiple telencephalic origins for the first olfactory neurons and each population following different migratory routes to colonize the OC according to an accurate topographic map.

A historical reflection of the contributions of Cajal and Golgi to the foundations of neuroscience.

In 1906, the Spaniard Santiago Ramón y Cajal and the Italian Camillo Golgi shared the Nobel Prize in Physiology or Medicine, in recognition of their work on the structure of the nervous system. Although both were well-known scientists who had made a large number of important discoveries regarding the anatomy of the nervous system, each defended a different and conflicting position in relation to the intimate organization of the grey matter that makes up the brain. In this communication we will review the importance of Cajal's studies using the method of impregnation discovered by Golgi, as well as the relevant studies carried out by Golgi, the concession of the Nobel Prize and the events that occurred during the Nobel conferences. In summary, we will précis the important contribution of both scientists to the founding of modern Neuroscience.