Cajal’s contributionstogliaresearch

This article was published in an Elsevier journal. The attached copyis furnished to the author for non-commercial research and

education use, including for instruction at the author’s institution,sharing with colleagues and providing to institution administration.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

http://www.elsevier.com/copyright

Author's personal copy

Historical perspective

Cajal’s contributions to glia researchVirginia Garcıa-Marın, Pablo Garcıa-Lopez and Miguel Freire

Museum Ramon y Cajal, Instituto Cajal, CSIC Avda. Doctor Arce 37, Madrid 28002, Spain

In 1906, Santiago Ramon y Cajal was awarded the NobelPrize in Physiology or Medicine in recognition of his workon the structure of neurons and their connections. What isless well known is that he also had a keen interest in gliaand developed specific staining methods for their study.In addition to describing their morphology, he speculatedon a role for glia in sleep and wakefulness and even inexecutive brain functions such as attention. In this article,we focus on Cajal’s histological research into glial cells;this research includes original drawings of astrocytes,oligodendrocytes, microglia and radial glia, as well ashis scientific writings. We aim to show that, concerningglia as well as neurons, Cajal was far ahead of his time.

IntroductionSantiago Ramon y Cajal (1852–1934) is one of thebest-known neuroscientists. He is considered by many tobe one of his time’s most outstanding contributors toknowledge of the nervous system, yet his research wasnot limited to neurons; it also included glial cells. Given therecent exponential increase in research into glial cells, areview discussing Cajal’s important contribution to estab-lishing their importance in the function of the nervoussystem is timely.

The histological data for this review come fromthe Museum Ramon y Cajal (Madrid, Spain), whichholds 4529 original histological preparations from Cajal(Figure 1). About 350 of these preparationsweremadewithspecific glia staining methods developed by Cajal or bycontemporaneous scientists, including Golgi, Weigert andRıo-Hortega. Included in this review are previously unseenimages of glial cells taken from Cajal’s slides. Some ofCajal’s studies of glial cells are summarized in his Histo-logie [1], although other, less well-known papers are alsoincluded in this article.

In present terminology, glial cells are classified into twomain groups: microglia and macroglia [2]. Macroglia aresubdivided into four specialized cell types: ependymalcells, Schwann cells, oligodendroglia and astroglia. Astro-glia include astrocytes, marginal glia, radial glia in thedeveloping brain and spinal cord, Bergmann cells in thecerebellar cortex, Muller cells in the retina, pituicytes inthe neurohypophysis and tanycytes in the hypothalamus[2]. In this review, we will show that Cajal paid specialattention to glial cells, not only from a morphological pointof view but also in relation to their physiological role in thenervous system. Some of the glial functions that Cajal

proposed have been subsequently revised, and for thisreason we hope this article can bring greater clarity tothe history of glial research and contribute to the establish-ment of newworking hypotheses of glial function. AlthoughCajal mentions numerous types of glial cells in his writ-ings, for the purposes of this review we will focus onastrocytes, for which he proposed a functional link betweenmorphology, physiology and function.

Cajal’s early work on astrocytesThe history of glial research began in 1846, when Virchow[3] described a connective substance that forms a sort of‘cement’ in the brain and spinal cord and in which thenervous elements appeared to be embedded. He coined theterm ‘neuroglia’ to describe this interstitial substance.Later, he published the first neuroglia illustrations [4],in which some nuclei appear without protoplasm andothers are shown as small round or lentil-shaped cells[4]. Presumably, these were glial cells whose cytoplasmwas either not stained or incompletely stained (for a recenthistorical review, see [5]). Otto Deiters [6] was the first todescribe the arachnoid-shaped cells that were seen in thewhite matter and were later shown to be glia. Michael vonLenhossek [7] introduced the term ‘astrocyte’ to refer tostar-shaped neuroglial cells. Andriezen [8] and AlbertKoelliker [9] divided glia into two groups, fibrous gliaand protoplasmic glia (for recent historical reviews, see[5,10–12]). Cajal adopted this classification but gave thename ‘astrocyte’ to both cell types [1]. The differencesbetween these two types take into account the configur-ation and number of their processes and their localization.The processes of fibrous astrocytes are fewer and longerand branch less frequently and at a more acute angle thanthose of protoplasmic astrocytes. Protoplasmic astrocytesare found mainly in gray matter, whereas fibrous astro-cytes occur mainly in the white matter of the brain andspinal cord.

Most of the methods available a century ago for stainingastrocytes were complex and most effective at stainingfibrous astrocytes in white matter; they stained protoplas-mic astrocytes poorly [13]. Frustrated by the lack of aneffective stain for protoplasmic astrocytes, Cajal experi-mented with different approaches and succeeded in devel-oping the sublimated gold chloride method [14]. Cajal’smethod allowed the nucleus and other intracellularelements to be better visualized – Golgi’s impregnationtechnique and related approaches did not distinguish theseelements from the dark staining of the background. Onstudying Cajal’s slides, it is clear that if a particular

Review TRENDS in Neurosciences Vol.30 No.9

Corresponding author: Garcıa-Marın, V. ([email protected]).Available online 31 August 2007.

www.sciencedirect.com 0166-2236/$ – see front matter � 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2007.06.008


structure or cell interested him, he became quite driven tofind a way of staining it; he would apply all methodsavailable and modify them if necessary to obtain the bestresult. Table 1 summarizes thesemethods and includes themain components of the chemical reactions used and thekind of cell impregnated. An example of one of Cajal’soriginal slides is shown in Figure 1.

Main functions that Cajal attributed to astrocytesAstrocytes develop many functions in the nervous system(such functions include structural and metabolic support,transmitter reuptake and release, regulation of concen-tration of different ions in the extracellular space, modu-lation of synaptic transmission, blood-flow control, etc.).However, many of these functions, such as the supplying ofnutrients to neurons [23], the removal of toxic wasteproducts of neuronal metabolism [24] and the regulationof synaptic activity [24,25], were proposed more than acentury ago. Cajal proposed other interesting functions for

astrocytes, including their capacity for division and theircontacts with other astrocytes, neurons and blood vesselsthrough their endfeet, but he realized that correctlyanswering the question ‘What is the function of glia?’ wouldrequire improved techniques. In the last few years, suchcellular and physiological techniques have been developedand have confirmed some proposals of these pioneer neu-roscientists.

Division capability of astrocytes

Some histological preparations and drawings show pairs ofastrocytes joined by their soma; Cajal named such pairs as‘astrocitos gemelos’ (twin astrocytes) (Figure 2a). Suchprofiles can be observed in the dentate gyrus of the adulthuman hippocampus. Cajal interpreted these observationsas being of astrocytes that had recently divided, and hespeculated that astrocytes might retain their ability todivide, unlike neurons, which lose this capability duringthe differentiation process [26]. Some decades later, obser-vations in rats sacrificed 1 h after injection ofH3-thymidine(so that actively dividing cells would be stained) revealedsome labeled astrocytes in the corpus callosum and therebyshowed that at least some astrocytes have the ability todivide [27]. However, astrocytes mostly derive from glialcell progenitors of the subventricular zone [28,29]. Astro-cytes generate not only other astrocytes but also neurons.Radial glial-like astrocytes in the subgranular layer (SGL)of the hippocampal dentate gyrus generate dentate gran-ule neurons and possibly some GABAergic neuronsthroughout life [30–32]. This is the same location whereCajal observed his twin astrocytes.

Figure 1. One of Cajal’s original histological preparations. The handwriting on the

label reads: ‘glia plata cerebro y cerebelo gato b’ (glia silver cerebral cortex and

cerebellum cat b) (inventory number (IN) of preparation: 80028).

Table 1. Staining methods that Cajal employed to observe different kinds of glial cells

Method Glial type Figs Refsa

Golgi Protoplasmic and fibrous astrocytes fully impregnated in black. 5b,c [13]

Fixation: osmium acid + potassium dichromate Astrocytic endfeet.

Impregnation: silver nitrate Radial glial cells.

Golgi-Cox Protoplasmic and fibrous astrocytes fully impregnated in black. 2b [14]

Fix.: mercuric chloride + potassium dichromate Astrocytic endfeet.

Impreg: silver nitrate

Golgi-Kenyon Protoplasmic and fibrous astrocytes fully impregnated in dark red. 2a [15]

Fix.: formalin + potassium dichromate Astrocytic endfeet.

Impreg.: silver nitrate Oligodendrocytes impregnated in dark red.

Formol-uranium nitrate Protoplasmic and fibrous astrocytes. 2c, 3a [16]

Fix.: formalin + urano nitrate Endfeet, gliosomes.

Impreg.: silver nitrate (gold toning

recommended)

Gold sublimated method Protoplasmic and fibrous astrocytes. Glial protoplasm, glial filaments and

astrocytic endfeet.

2d [17]

Fix.: formalin ammonium bromide

Impreg.: gold chloride + mercuric bichloride

Silver carbonate Protoplasmic and fibrous astrocytes. Glial protoplasm. 2f, 4i [18]

Fix.: formalin ammonium bromide Oligodendrocytes – only simple types.

Impreg.: silver nitrate + litina carbonate Microglial – all types fully impregnated.

Ammoniacal silver oxide Protoplasmic and fibrous astrocytes. Glial protoplasm. 2e, 4e,g [19]

Fix.: formalin ammonium bromide Endfeet, glial filaments.

Impreg.: ammoniacal silver oxide + pyridine Microglial cells, especially under pathological conditions.

Reduced silver nitrate Microglial cells not fully impregnated. 4h [20]

Fix.: formalin ammonium bromide

Impreg.: silver nitrate + pyridine

Golgi-Rıo-Hortega Oligodendrocytes – all types 4f [21]

Fix.: potassium dichromate + chloral hydrate +

formalin

Impreg.: silver nitrateaReferences indicate the first publication of the method; later modifications are not cited.

480 Review TRENDS in Neurosciences Vol.30 No.9

www.sciencedirect.com


Role of astrocytic endfeet in control of blood-vessel

dilatation

With the development of modern microscopy, it has beenpossible to observe the movement of neurons and glial cells.However, the movement of the nervous system had alreadybeen observed in Cajal’s time by Wiedersheim in 1890 [33],and it was the basis of different theories about neuronalameboidism [34–36] (see recent review in [37]). Cajal alsowas one of thefirst scientists to developadynamic concept ofneurons (1892–1894) [38,39], and later he extended theseconcepts to astrocytes (1895) [25]. Although he obviously

could not observe the movement of glial cells, he couldobserve different lengths of astrocytic processes acrossdifferent astrocytes, and he proposed his theory of gliaameboidism. One of Cajal’s principal values was his prodi-gious imagination. Although he only observed static imagesbyopticalmicroscopy, his descriptionsof thenervous systemare very dynamic and animated and create, in the words ofSherrington, ‘alive scenes’. He applied glial dynamics to twodifferent topics: attention and sleep–wake states.

Astrocytes have numerous endfeet that make contactwith one or more blood vessels. It has been demonstratedthat these endfeet play a role in cerebral metabolic trafficbetween neurons and intracerebral blood vessels [40].Cajal studied astrocytic endfeet with different methods(Figure 3a–e; Figure 4c). Based on the close relationshipbetween astrocytic endfeet and blood vessels and thevariability in the length of astrocytic processes, Cajalproposed that astrocytes could produce either vasodilata-tion or vasoconstriction in arterioles throughmovements oftheir endfeet. Furthermore, he suggested that this mech-anism could explain the process of attention [25](Figure 4a,c). Astrocytes associated with arterioles couldretract their endfeet, resulting in arterial dilatation and anincrease in the supply of nutrients to a specific region of thebrain (such an increase is needed during attentionalswitching) and explaining functional hyperemia, describedyears before by Angello Mosso and Charles Sherrington[41].When neurons are highly activated in a specific area ofthe brain, blood flow and glucose uptake in that regionincrease in a temporally and spatially coordinated manner

Figure 2. (a) Cajal’s drawing of astrocytes (indicated by ‘A’) in the pyramidal layer

of the human hippocampus (indicated by ‘D’), twin astrocytes (indicated by ‘B’)

and a satellite cell called the ‘third element’ by Cajal (indicated by ‘a’). Sublimated

gold chloride method. (b) Different astrocytes (indicated by ‘A’, ‘B’, ‘C’ and ‘D’)

surrounding neuronal somas in the pyramidal layer of the human hippocampus.

Sublimated gold chloride method.

Figure 3. Protoplasmic astrocytes from Cajal’s original slides impregnated by the Golgi-Cox method (a), formol-uranium nitrate method (c), gold chloride sublimated

method (d) and silver carbonated method (f). Fibrous astrocytes impregnated by the Golgi-Kenyon method (b) and ammoniacal silver oxide method (e). Abbreviations:

a = astrocytes, bv = blood vessels, ef = endfeet, gf = gliofilaments, n = neuron, p = processes. The scale bar represents 25 mm (INs: 84511, 84528, 80034, 800155, 80168 and

83231).

Review TRENDS in Neurosciences Vol.30 No.9 481



to meet the enhanced local metabolic demand [42]. Cajalproposed that during periods of increased metabolicdemand, astrocytes dilated vessels by mechanical move-ments. This dilation could be reversible, and astrocytescould cause vasoconstriction when attention is not necess-ary [25]. It has recently been observed that during highsynaptic activity, glutamate provokes a response in astro-cytes, which release vasoactive agents, presumably fromtheir endfeet; this response is mediated by an increase inintracellular Ca2+ concentration [43,44]. This could lead tovasodilation by relaxing the smooth-muscle cells of arter-ioles. By contrast, Mulligan and MacVicar, by using two-photon Ca2+ uncaging, have found that an increase in theavailable Ca2+ concentration within astrocytes produces aconstriction of nearby arterioles and thus decreases localblood flow [45]. These apparently controversial resultscould be caused by differences in the preparation of thebrain slices used for the experiments. The conditions ofbrain slices in vitro (i.e., the presence of blood flow, arterialpressure, synaptic activity and metabolic demands) arevery different from in vivo conditions. Recently, Takanoet al. [46], also using in vivo two-photon Ca2+ uncaging,have described vasodilation of arterioles mediated by pho-tolysis of glutamate in live mice.

Although the contraction or relaxation of blood vessels isprobably not mediated by astrocytic-process contraction orelongation but by release of vasoactive agents from astro-cytes, it is necessary to recognize the merit of Cajal’sproposal of astrocytic control of hyperemia, when he hadonly observed static light-microscopy images.

The astrocyte–neuron relationship

Cajal’s histological slides and drawings clearly illustrateastrocytic processes surrounding the body of neurons

(Figure 2; Figure 3d). Cajal observed that glial processesappeared to be consistently arranged in a fashion thatprevented contact among unmyelinated axons and den-drites at points where functional separation between thoseelements was needed. If contact between dendrites andnon-myelinated fibers were prevented, nerve impulsesbetween those neurons would be blocked [47]. Cajal’sbrother Pedro Ramon had previously suggested that astro-cytes could physically insulate against the passage ofneuronal impulses [48], and this hypothesis receivedstrong support from Cajal [49]. After the discovery ofmyelin as an insulating material, Cajal considered that,in thewhitematter, therewere two ‘insulators’, myelin andastrocytes, whereas in the graymatter, protoplasmic astro-cytes were the only insulating material [49]. Nevertheless,two years later he withdrew this view of astrocytes func-tioning as insulators [1].

Despite changing his opinion on astrocytes asinsulators, Cajal retained the belief that astrocytes werein some way modulating or interfering with neuronalfunction. In a theoretical article published in 1895, Cajalalso proposed a possible role for astrocytes in the sleep–wake cycle; he suggested that astrocytes in the graymatter might represent a switch that connects (wakeful-ness) or disconnects (sleep) the nervous ‘current’(Figure 4b [25]. He proposed that during waking, astro-cytes have their processes retracted, so neurons cancontact each other and the nervous current can flow.However, when astrocytes extend their processes amongneurons, they would act as a sort of circuit breaker,preventing the contact between neurons and thus indu-cing sleep.

Although the function of astrocytes in natural sleephas not been proven, the mechanism that Cajal proposed

Figure 4. (a) Attention mechanism. (i) Astrocytes associated with capillaries could contract their endfeet, resulting in capillary dilatation and thus increasing the amount of

nutrients in a specific region of the brain. (ii) During the relaxation phase, astrocyte processes will elongate, causing vasoconstriction in a brain region when attention is not

necessary. (b) Wakefulness–sleep process. (i) During wakefulness, the astrocytes have their processes contracted so the neurons can contact each other and there will be a flow

of the nervous current. (ii) If astrocytes extend the processes between neurons, they will act as a sort of circuit breaker, preventing contact between neurons and inducing sleep.

(c) Cajal’s drawing of fibrous astrocytes of human cerebral cortex surrounding a blood vessel. The original slide was impregnated by the sublimated gold chloride method.




for these functions (interposition between synapses) hasbeen echoed by modern studies across various brainareas. In the arcuate nucleus of the hypothalamus, estra-diol induces a transient growth of astrocytic processes atthe end of proestrus [50–52]. In the magnocellular nuclei,under conditions of intense neurohypohysial hormonesecretion (e.g. lactation, parturition, chronic dehydrata-tion), the glial coverage of neurons decreases in thevicinity of synapses [53–56]. This results in an alterationof glutamate clearance and also in a decrease of gluta-matergic neurotransmission [57]. Great motility of astro-glial process endings, mediated by lamellipodia andfilopodia, has been observed with two-photon microscopy[58].

In conclusion, the astrocyte–neuron partnership is notstatic but shows dynamic transformations that might beessential for synaptic plasticity and the modulation ofneurotransmission. In recent years, further mechanismsof communication between neurons and astrocytes havebeen observed. It has been shown that astrocytic excit-ability varies with changes in intracellular Ca2+ concen-tration [59]. Astrocytes might sense the activity ofneighboring synapses and respond to neurotransmittersreleased by synaptic terminals. Their response mightinduce an increase in the intracellular Ca2+ concentrationin adjacent glial cells, and this increase might in turnlead to the release of various transmitters, such as glu-tamate [60,61]. These gliotransmitters could mediateintercellular signaling between astrocytes and neurons(for recent review, see [62]). The functional significance ofthese morphological and physiological findings in themodulation of nervous transmission has generated theconcept of ‘tripartite synapse’, whereby the synapse isfunctionally constituted by three elements: the presyn-aptic cell, the postsynaptic neuron and the surroundingastrocytes [63].

Cajal’s ‘third element’The ‘third element’ is a general and ambiguous term thatCajal used to describe a group of adendritic cells thatseemed devoid of processes, probably because of incom-plete staining methods. Rıo-Hortega demonstrated thatthese cells were indeed oligodendrocytes and microglial

cells [67]. Nowadays, it is easy to distinguish betweenmicroglial cells and oligodendrocytes by the applicationof antibodies as specific cell markers [64,65].

In 1896, Cajal observed some small cells that apparentlylacked processes. Because of their close association withneuronal somata, he named them ‘satellite cells’ and con-sidered them to be a new type of glial cell [66] (Figure 2a,b).Cajal applied alternative staining methods to confirm thepresence of the perineuronal adendritic cells and extendedthis observation to the satellite cells of the astrocytes, bloodvessels and other adendritic cells in the white matter [26](see Table 2). Cajal grouped all of them under the generalterm of ‘third element’ of the nervous centers in 1913 [26].Neurons were considered the first element and astrocytesthe second. The drawings and Cajal’s original slides shownin Figure 5a–c summarize some of the main shapes Cajalproposed for his third element [26]. Cajal considered thatall of these elements could have either mesodermic orectodermic origin.

In 1920, Rıo-Hortega obtained clearer images of themorphology of these adendritic cells both in gray matterand white matter by applying a new method [19]. Hedivided Cajal’s third element into two different types ofcells, microglia and interfascicular glia [67]. In microglialcells he included some satellite cells of gray matter andsome of the adendritic cells in white matter, which Cajalhad previously described. Rıo-Hortega also described thetransformation of microglia from an inactive state to thephagocytic ameboid form and their final transformationinto granular cells in response to a threat such as infection.He classified the rows of adendritic cells that Cajal hadpreviously found in white matter (see Table 2) as inter-fascicular glia [67]. With regard to embryonic origin, Rıo-Hortega suggested that microglia had a mesodermal ori-gin, whereas interfascicular glia and astrocytes were ofectodermal descent [67]. Because of their distinct originand function, Rıo-Hortega considered microglia to be thethird element of the nervous system [67].

After Hortega’s work, Cajal applied differentmethods tostudy microglia [19–21], although the best technique forstaining microglia was still Hortega’s method (Figure 5g–i). Cajal confirmed Hortega’s work, but he continued think-ing that the third element comprised different types of

Table 2. The identity of Cajal’s ’third element’

Author Method Identity Observations

Cajal, 1896 - Nissl - Satellite cells - Small cells lacking processes and in close association with neuronal somata

Cajal, 1913 - Formol uranium

nitrate

- Third element - Gray matter (satellite to neurons, astrocytes and blood vessels)

- Sublimated gold

chloride

- White matter (satellite to astrocytes and blood vessels and in rows along nerve

fascicles; star-shaped cells; fusiform cells with an elongated nucleus and

protoplasm; dwarf cells with a pyknotic nucleus)

Rıo-Hortega,

1920

- Silver carbonate - Microglia - Fusiform cells with an elongated nucleus and protoplasm, as described by Cajal

- Interfascicular glia - Rows of cells along nerve fascicles, as described by Cajal

Cajal, 1920 - Modification of

reduced silver nitrate

- Microglia - Adendritic cells (including those close to blood vessels and dwarf cells) and

microglia

- Ammoniacal silver

oxide

- Interfascicular glia

- Silver carbonate

- Other distinct

adendritic cells

Rıo-Hortega,

1921–1928

- Golgi-Hortega - Oligodendrocytes - Oligodendrocytes (including the satellite cells, interfascicular glia, adendritic

cells close to blood vessels and dwarf cells described by Cajal)




cells, including microglia, interfascicular glia and otherdistinct adendritic cells, particularly perivascular adendri-tic cells and the dwarf cells described in white matter (seeTable 2). Because this latter group of adendritic cells couldnot be easily visualized with any known staining tech-niques, Cajal considered them to constitute a special celltype with undiscovered functions and called this cell typethe ‘real third element’ [68,69].

Rıo-Hortega made further contributions to the study ofthis problem in 1921 and 1928. Via a modification of theGolgi-Hortega method, he described the small adendriticcells that Cajal considered the real third element andcalled them ‘oligodendrocytes’ [22,70]. Among oligodendro-cytes (Figure 5f), Rıo-Hortega also included some satellitecells that Cajal had identified adjacent to neurons in graymatter (Figure 5d) and the interfascicular glia found inbetween nerve fibers of the white matter (Figure 5e).

As we have seen, the development of the classification ofdifferent glial cells has been a complex process, involvingnot only methodological (different staining methods),

morphological and functional concerns but also a problemwith scientific terminology.

Radial glial cellsRadial glia (RG) were first fully visualized by Golgi (1885),Magini (1888), Cajal (1889), Kolliker (1896) and otherscientists (for a recent review, see [71,72]). These authorsobserved radial cylindrical epithelial cells covering thewhole section of either the spinal cord or the cerebralcortex and reaching the peripheral border close to thepia mater. Cajal called these cells spongioblasts, whereasother contemporary scientists used different names, suchas epithelial cells, radial cells, and so on. Rakic (1972)introduced the term ‘radial glia’ in his classic descriptionsof neuronal migration in the neocortex of fetal primates[73].

A pivotal role has been suggested for RG in theconstruction of the nervous system: first, providing a scaf-fold for migrating neurons and second, participating inthe generation of diverse brain cells [74]. Some of these

Figure 5. Drawing and microphotographs from Cajal’s original histological preparations. (a) Astrocyte (indicated by ‘A’) and adendritic cells. The sample was taken from a

cat, and the original slide was impregnated by the formol-uranium method. (b) Heterothypical glial cells, big pale cell (indicated by ‘a’) and dwarf cells. The sample was

taken from a man, and the original slide was impregnated by the reduced silver nitrate method. (c) Astrocyte (indicated by ‘A’), perivascular adendritic cell (indicated by ‘a’),

adendritic cell (indicated by ‘b’), fusiform elements (indicated by ‘C’ and ‘D’) and oval nucleus with some rests of glial protoplasm (indicated by ‘d’). The original sample was

taken from a child who died from tuberculosis [26]. Oligodendrocytes impregnated by the Nissl method (d), ammoniacal silver oxide method (e) and Golgi-Hortega method

(f). Microglia impregnated by the ammoniacal silver oxide method (g), reduced silver nitrated method (h) and silver carbonate method (i). Abbreviations: m = microglial

cells; n = neuron; o = oligodendrocytes; p = senile plaque. The scale bar represents 10 mm. (INs: 81206, 80049, 84480, 82127, 80443 and 84467).




functions were first suggested more than a century ago byCajal and his colleagues.

Cajal described the morphology of RG by using theGolgi method in the spinal cord [75] (Figure 6b). Maginiproposed that these cells acted as a scaffold for migratingneurons. He observed that the radial filaments exhibitmany swellings or round varicosities of various sizes ontheir course. He hypothesized that these spherical cells(the varicosities) probably represented the future nervecells of the cerebral cortex (see [72] for a recent review). Inthe early 1970s, Rakic used a combination ofGolgi impreg-nation and reconstructions from electron microscopyserial sections to observe migrating neurons along theRG fibers [73,76].

In Cajal’s Histologie [1], we learn that at that time thespinal cord at its earliest stages was considered to beconstituted of elongated epithelial cells arranged in asingle layer, that is, neuroepithelial cells, by current ter-minology. Among these epithelial cells, there are scatteredspherical elements with mitotic figures, which His hadnamed germinal cells. For His, germinal cells representedspecific elements from which the neuroblasts derive exclu-sively. However, Cajal observed that the number of epi-thelial cells increased notably after the differentiation ofgerminal cells, and he proposed that germinal cells wereundifferentiated forms that could give rise to both epi-thelial cells and neuroblasts. Moreover, Cajal observedtransitional phases between spongioblasts and neuro-blasts in very early stages of development of the chickspinal cord. This led him to consider the possibility thatsome epithelial elements might become neuroblasts. Onlyin recent years has this point been confirmed, when it wasobserved that RG are not only astrocyte progenitors butalso neuronal progenitors in the central nervous system(CNS) [77–80].

Cajal described the differentiation of RG into fibrillaryor protoplasmic astrocytes after they have ceased theirguiding function during neurogenesis (Figure 6a) [1,64].According to Cajal, astrocyte morphology depends on theplace where the RG ends its evolution. Influenced byneuronal activity, astrocytes placed in gray matter devel-oped their processes among the thin processes of neurons;however, an astrocyte situated in white matter acquiredlong and smooth processes [1]. Recent studies have

confirmed that RG transform into astrocytes in most avianand mammalian CNS regions [81–84].

Cajal observed that RG persist through adulthood inlower vertebrates and that they are the dominant glialform in reptiles (Figure 6c) [85]. However, in the adultmammalian stage, most of the RG disappear, with a fewexceptionswhere they adapt to the local functional require-ments and spatial conditions and become specialized astro-cytic cell types (e.g. the Bergmann glial cells of thecerebellum, or the Muller cells of the retina) [74]. Becauseneurogenesis continues in adult cold-blooded vertebrates,a close correlation between the presence of RG and neu-rogenesis has been suggested. Recent studies in avian andmammalian brains also indicate that RG function asneural progenitors and perhaps as stem cells [86].

ConclusionsMore than one hundred years ago, Cajal proposed aphysiological role for glial cells. Astrocytes were initiallyconsidered as ‘brain glue’, providing an inert scaffoldnecessary for neuronal distribution and interactions.However, it is only in the past few years that new functionshave been revealed for astrocytes; such functions includethe control of synapse formation and function, adult neu-rogenesis and brain vascular tone. Some of these ‘new’functions hadalready beenproposed byCajal but couldnotbe tested. Cajal found that the nervous system included anew cell type, which he called the third element. Rıo-Hortega, using specific methods, determined that thisthird element was actually composed of two cell types,oligodendrocytes and microglia. Finally, some of Cajal’smain ideas, that is, the evolution of RG to astrocytes andtheir neurogenic potential, have recently been corrobo-rated. As we have seen in the field of glial research, thescientific work of Cajal is still a rich source of hypotheseswaiting to be tested.

AcknowledgementsWe gratefully acknowledge Adolfo Martinez, and especially Sian Lewisand Helen Barbour, for their invaluable help with editing the manuscript.We also acknowledge Javier DeFelipe and Luis Miguel Garcıa-Segura fortheir critical reading of the manuscript. Original drawings from theMuseum Cajal are reproduced with permission from the Inheritors ofSantiago Ramon y Cajal�. The authors are supported by the SpanishMinistry of Science and the Areces Foundation.

Figure 6. (a) Drawing of radial glial cells from the spinal cord of a 9-day-old chick. (b) RG from the spinal cord of a 3-day-old chick. (‘a’ = dorsal; ‘b’ = ventral; ‘v’ = ventricle;

the scale bar represents 25 mm). (c) RG from the optic lobe of an adult lizard (the scale bar represents 50 mm); impregnation with Golgi method. (INs 84425 and 80624)




References1 Ramon y Cajal, S. (1909) Histologie du systeme nerveux de lhomme et

des vertebres, Maloine2 Garcıa-Segura, L.M. and McCarthy, M.M. (2004) Minireview, Role

of glia in neuroendocrine function. Endocrinology 145, 1082–1086

3 Virchow, R. (1846) Uber das granulierte ansehen der Wandungen derGerhirnventrikel. Allg. Z. Psychiatr. 3, 242–250

4 Virchow, R. (1858) Die Cellularpathologie in ihrer Begrundung augphysiologische und pathologische Gewebelehre, Verlag von AugustHirschwald

5 Somjen, G.G. (1988) Nervenkitt: Notes on the history of the concept ofneuroglia. Glia 1, 2–9

6 Deiters, O. (1865) Untersuchungen uber Gehirn und Ruckenmark desMenschen und der Saugethiere, Vieweg

7 von Lenhossek, M. (1893) Der feinere Bau des Nervensystems im Lichteneuester Forschung, Fischer’s Medicinische Buchhandlung H.Kornfield

8 Andriezen, W.L. (1893) The neuroglia elements of the brain. BMJ 2,227–230

9 Kolliker, A. (1889) Handbuch der Gewebelehre des Menschen, WilhelmEngelmann

10 Kimelberg, H.K. (2004) The problem of astrocyte identity. Neurochem.Int. 45, 191–202

11 Kettenmann, H. and Ranson, B.R. (2005) The concept of neuroglia, ahistorical perspective. In Neuroglia (2nd edn) (Kettenmann, H. andRansom, B., eds), pp. 1–16, Oxford University Press

12 Privat, A. et al. (1995) Morphology of astrocytes. In Neuroglia(Kettenmann, H. and Ransom, B., eds), pp. 3–22, Oxford UnivsersityPress

13 Ramon y Cajal, S. (1913) Sobre un nuevo proceder de impregnacion dela neuroglia y sus resultados en los centros nerviosos del hombre yanimales. Trab. Lab. Invest. Biol. XI, 103–112

14 Ramon y Cajal, S. (1913) Un nuevo proceder para la impregnacion de laneuroglıa. Bol. Soc. Esp. Biol. II, 104–108

15 Golgi, C. (1873) Sulla struttura della sostanza grigia del cervello. Gaz.Med. Lomb. 6, 244–246

16 Cox, W. (1891) Impragnation des centralen Nervensystems mitQuecksilbersalzen. Arch. Mikr. Anat. 37, 16–21

17 Kenyon, F.C. (1897) The optic lobes of the bee’s brain in the light ofrecent neurological methods. Am. Nat. XXXI, 369–376

18 Ramon y Cajal, S. (1912) Formula de fijacion para la demostracion facildel aparato reticular de Golgi. Bol. Soc. Esp. Biol. I, 263–269

19 Rıo-Hortega, P. (1917) Noticia de un nuevo y facil metodo para lacoloracion de la neuroglıa y del tejido conjuntivo. Trab. Lab. Invest.Biol. XV, 367–378

20 Ramon y Cajal, S. (1920) Unamodificacion del metodo de Bielschowshypara la impregnacion de la neuroglia comun y mesoglia y algunosconsejos acerca de la tecnica del oro-sublimado. Trab. Lab. Invest. BiolXVIII, 129–141

21 Ramon y Cajal, S. (1925) Notas tecnicas. Bol. Soc. Esp. Biol. XI, 107–110

22 Rıo-Hortega, P. (1928) Tercera aportacion al conocimiento morfologicoe interpretacion funcional de la neuroglıa. Mem. Soc. Esp. Hist. Nat.XIV, 5–122

23 Golgi, C. (1885-1886) Sulla fina anatomia della sistema nervosa. Riv.Sper. Freniatr. 8, 9 (?) Also appeared in 1903 as Vol. 16 of OperaOmnia,U. Hoepfli, Milano, and in German translation in 1894, Fischer, Jena.Partial English translation by J. Workman in 1883 in Alienist andNeurologist, 4: 236-269

24 Lugaro, E. (1907) Sulle funzioni della nevroglia.Riv. Patol. Nerv. Ment.12, 225–233

25 Ramon y Cajal, S. (1895) Algunas conjeturas sobre el mecanismoanatomico de la ideacion, asociacion y atencion, Imprenta y Librerıade Nicolas Moya

26 Ramon y Cajal, S. (1913) Contribucion al conocimiento de la neurogliadel cerebro humano. Trab. Lab. Invest. Biol. XI, 255–315

27 Mori, S. and Leblond, C.P. (1969) Electron microscopic features andproliferation of astrocytes in the corpus callosum of the rat. J. Comp.Neurol. 137, 197–225

28 Levison, S.W. and Goldman, J.E. (1993) Both oligodendrocytes andastrocytes develop from progenitors in the subventricular zone ofpostnatal rat forebrain. Neuron 10, 201–212

29 Luskin, M.B. and McDermott, K. (1994) Divergent lineages foroligodendrocytes and astrocytes originating in the neonatalforebrain subventricular zone. Glia 11, 211–226

30 Belluzzi, O. et al. (2003) Electrophysiological differentiation of newneurons in the olfactory bulb. J. Neurosci. 23, 10411–10418

31 Seri, B. et al. (2001) Astrocytes give rise to new neurons in the adultmammalian hippocampus. J. Neurosci. 21, 7153–7160

32 Doetsch, F. et al. (1999) Subventricular zone astrocytes are neural stemcells in the adult mammalian brain. Cell 97, 703–716

33 Wiedersheim, R. (1890) Bewegungserscheinungen im gehirn vonleptodora hyalina. Anat. Anz. 5, 673–679

34 Rabl-Ruckhard, H. (1890) Sind die ganglienzellen amoboid? Einehypothese zur mechanik psychischer vorgange. Neurol. Centralbl. 9,199–200

35 Lepine, R. (1894) Sur un cas d’hysterie a form particuliere. Rev. Med.(Paris) 14, 713–728

36 Duval, M. (1895) Hypotheses sur la physiologie des centres nerveux;theorie histologique du sommeil. C. R. Soc. Biol. 47, 74–77

37 DeFelipe, J. (2006) Brain plasticity and mental processes: Cajal again.Nat. Rev. Neurosci. 7, 811–817

38 Ramon y Cajal, S. (1892) El nuevo concepto de la histologıa de loscentros nerviosos. Rev. Cienc. Med. Barc. 18, 361–376, 457–476, 505–520, 529–541

39 Ramon y Cajal, S. (1894) Consideraciones generales sobre lamorfologıade la celula nerviosa. La Veterinaria Espanola 37, 257–260, 273–275,289–291

40 Kacem, K. et al. (1998) Structural organization of the perivascularastrocyte endfeet and their relationship with the endothelial glucosetransporter, a confocal microscopy study. Glia 23, 1–10

41 Roy, C.S. and Sherrington, C. (1890) On the regulatin of the bloodsupply of the brain. J. Physiol. 11, 85–108

42 Bonvento, G. et al. (2002) Does glutamate image your thoughts? TrendsNeurosci. 25, 359–364

43 Zonta, M. et al. (2003) Neuron�to�astrocyte signalling is central to thedynamic control of brain microcirculation. Nat. Neurosci. 6, 43–50

44 Anderson, C. and Nedergaar, M. (2003) Astrocyte-mediated control ofcerebral microcirculation. Trends Neurosci. 26, 340–344

45 Mulligan, S.J. and MacVicar, B.A. (2004) Calcium transients inastrocyte endfeet cause cerebrovascular constrictions. Nature 431,195–199

46 Takano, T. et al. (2006) Astrocyte-mediated control of cerebral bloodflow. Nat. Neurosci. 2, 260–267

47 Ramon y Cajal, S. (1891) Comunicacion acerca de la significacionfisiologica de las expansiones protoplasmicas y nerviosas de lascelulas de la sustancia gris. Rev. Cienc. Med. Barc. 17, 1–15

48 Ramon, P. (1891) El encefalo de los reptiles. Trab. Lab. Histol. Fac.Zarag. 24, 1–31

49 Ramon y Cajal, S. (1897) Algo sobre la significacion fisiologica de laneuroglia. Rev. Trim. Microg. II, 33–47

50 Garcia-Segura, L.M. et al. (1994) Gonadal hormones as promoters ofstructural synaptic plasticity: cellular mechanisms. Prog. Neurobiol.44, 279–307

51 Garcia-Segura, L.M. et al. (1994) Gonadal hormone regulation of glialfibrillary acidic protein immunoreactivity and glial ultrastructure inthe rat neuroendocrine hypothalamus. Glia 10, 59–69

52 Garcia-Segura, L.M. et al. (1994) Gonadal steroids as promoters ofneuro-glial plasticity. Psychoneuroendocrinology 19, 445–453

53 Theodosis, D.T. and Poulain, D.A. (1993) Activity-dependent neuronal-glial and synaptic plasticity in the adult mammalian hypothalamus.Neuroscience 57, 501–535

54 Hatton, G.I. (1997) Function-related plasticity in hypothalamus.Annu.Rev. Neurosci. 20, 375–397

55 Miyata, S. and Hatton, G.I. (2002) Activity-related, dynamic neuron-glial interactions in the hypothalamo-neurohypophysial system.Microsc. Res. Tech. 56, 143–157

56 Theodosis, D.T. (2002) Oxytocin-secreting neurons: a physiologicalmodel of morphological neuronal and glial plasticity in the adulthypothalamus. Front. Neuroendocrinol. 23, 101–135

57 Oliet, S.H.R. et al. (2001) Control of Glutamate Clearance and SynapticEfficacy by Glial Coverage of Neurons. Science 292, 923–926

58 Hirrlinger, J. et al. (2004) Astrogial processes show spontaneousmotility at active synaptic terminals in situ. Eur. J. Neurosci. 20,2235–2239




59 Cornell-Bell, A.H. et al. (1990) Glutamate induces calcium waves incultured astrocytes: long-range glial signaling. Science 247, 470–473

60 Pasti, L. et al. (1997) Intracellular calcium oscillations in astrocytes: ahighly plastic, bidirectional form of communication between neuronsand astrocytes in situ. J. Neurosci. 17, 7817–7830

61 Parpura, V. et al. (1994) Glutamate-mediated astrocyte-neuronsignalling. Nature 369, 744–747

62 Perea, G. and Araque, A. (2006) Synaptic information processing byastrocytes. J. Physiol. (Paris) 99, 92–97

63 Araque, A. et al. (1999) Tripartite synapses: glia, the unacknowledgedpartner. Trends Neurosci. 22, 208–215

64 Szuchet, S. (1995) The morphology and ultrasture ofoligodendrocytes and their functional implications. In Neuroglia(Kettenmann, H. and Ransom, B.R., eds), pp. 24–57, OxfordUniversity Press

65 Streit, W.J. (1995) Microglial cells. In Neuroglia (Kettenmann, H.and Ransom, B., eds), pp. 85–96, Oxford University Press

66 Ramon y Cajal, S. (1896) Sobre las relaciones de las celulas nerviosascon las neuroglicas. Rev. Trimestral Micrografica I, 38–41

67 Rıo-Hortega, P. (1920) Estudios sobre la neuroglia. La microglia y sutransformacion en celulas en bastoncito y cuerpos granulo-adiposos.Trab. Lab. Invest. Biol. XVIII, 37–82

68 Ramon y Cajal, S. (1920) Algunas consideraciones sobre la mesoglia deRoberston y Rıo-Hortera. Trab. Lab. Invest. Biol XVIII, 109–127

69 Ramon y Cajal, S. (1923) Quelques methodes simples pour la colorationde la Neuroglie. Schweiz. Arch. Neurol. Psychiatr. XIII, 187–193

70 Rıo-Hortega, P. (1921) Estudios sobre la neuroglia. La glia de escasasradiacciones (oligodendroglia). Bol. Soc. Esp. Hist. Nat. 10, 63–92

71 Rakic, P. (2003) Elusive radial glial cells: historical and evolutionaryperspectiva. Glia 43, 19–32

72 Bentivoglio, M. and Mazzarello, P. (1999) The history of radial glia.Brain Res. Bull. 49, 305–315

73 Rakic, P. (1972) Mode of cell migration to the sperfical layers of fetalmonkey neorcortex. J. Comp. Neurol. 145, 61–83

74 Rakic, P. (2003) Developmental and evolutionary adaptations ofcortical radial glia. Cereb. Cortex 13, 541–549

75 Ramon y Cajal, S. (1889) Contribucion al estudio de la estructura de lamedula espinal. Rev. Trim. Hist. Norm. Patol. I, 79–106

76 Sidman, R.L. and Rakic, P. (1973) Neuronal migration, with specialreference to adeveloping human brain: a review. Brain Res. 62, 1–35

77 Alvarez-Buylla, A. (1990) Mechanism of neurogenesis in adult avianbrain. Experientia 46, 948–955

78 Miyata, T. et al. (2001) Asymmetric inheritance of radial glial fibers bycortical neurons. Neuron 31, 727–741

79 Hartfuss, E. et al. (2001) Characterization of CNS precursos subtypesand radial glıa. Dev. Biol. 229, 15–30

80 Parnavelas, J.G. and Nadarajah, B. (2001) Radial glial cells. Are theyreally glıa? Neuron 31, 881–884

81 Choi, B.H. and Lapham, W. (1978) Radial glia in the human fetalcerebrum, a combined Golgi, immunofluorescent and electronmicroscopic study. Brain Res. 148, 295–311

82 deAzevedo, L. et al. (2003) Cortical radial gial cells in human fetuses:depth-correlated transformation into astrocytes. J. Neurobiol. 55, 288–298

83 Marin-Padilla, M. (1995) Prenatal development of fibrous (whitematter), protoplasmic (gray matter), and layer I astrocytes in thehuman cerebral cortex: a Golgi study. J. Comp. Neurol. 357, 554–572

84 Pixley, S.K. and de Vellis, J. (1984) Transition between immatureradial glia and mature astrocytes studied with a monoclonal antibodyto vimentin. Brain Res. 317, 201–209

85 Ramon y Cajal, S., (1891) Pequenas contribuciones al conocimiento delsistema nervioso. I. Estructura y conexiones de los ganglios simpaticos.II. Estructura fundamental de la corteza cerebral del los batracios,reptiles y aves. III. Estructura de la retina de los reptiles y batracios.IV. Estructura de la medula espinal de los reptiles. Trab. Lab. Histol.Fac. Med, agosto, 1-56

86 Alvarez-Buylla, A. et al. (2002) Identification of neural stem cells in theadult vertebrate brain. Brain Res. Bull. 57, 751–758

How to re-use Elsevier journal figures in multimedia presentations

It’s easy to incorporate figures published in Trends, Current Opinion or Drug Discovery Today journals

into your multimedia presentations or other image-display programs.

1. Locate the article with the required figure on ScienceDirect and click on the ‘Full text + links’

hyperlink

2. Click on the thumbnail of the required figure to enlarge the image

3. Copy the image and paste it into an image-display program

Permission of the publisher is required to re-use any materials from Trends, Current Opinion or Drug

Discovery Today journals or from any other works published by Elsevier. Elsevier authors can obtain

permission by completing the online form available through the Copyright Information section of

Elsevier’s Author Gateway at http://authors.elsevier.com. Alternatively, readers can access the

request form through Elsevier’s main website at:

www.elsevier.com/locate/permissions



Documents

Cajal’s contributionstogliaresearch