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 Author's personal copy
Review TRENDS in Neurosciences
Historical perspective
Cajal’s contributions to glia research
Virginia Garcı́a-Marı́n, Pablo Garcı́a-López and Miguel Freire
Museum Ramón y Cajal, Instituto Cajal, CSIC Avda. Doctor Arce 37, Madrid 28002, Spain
In 1906, Santiago Ramón y Cajal was awarded the Nobel
Prize in Physiology or Medicine in recognition of his work
on the structure of neurons and their connections. What is
less well known is that he also had a keen interest in glia
and developed specific staining methods for their study.
In addition to describing their morphology, he speculated
on a role for glia in sleep and wakefulness and even in
executive 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 as
his scientific writings. We aim to show that, concerning
glia as well as neurons, Cajal was far ahead of his time.
Introduction
Santiago Ramón y Cajal (1852–1934) is one of the
best-known neuroscientists. He is considered by many to
be one of his time’s most outstanding contributors to
knowledge of the nervous system, yet his research was
not limited to neurons; it also included glial cells. Given the
recent exponential increase in research into glial cells, a
review discussing Cajal’s important contribution to estab-
lishing their importance in the function of the nervous
system is timely.
The histological data for this review come from
the Museum Ramón y Cajal (Madrid, Spain), which
holds 4529 original histological preparations from Cajal
(Figure 1). About 350 of these preparations were made with
specific glia staining methods developed by Cajal or by
contemporaneous scientists, including Golgi, Weigert and
Rı́o-Hortega. Included in this review are previously unseen
images of glial cells taken from Cajal’s slides. Some of
Cajal’s studies of glial cells are summarized in his Histo-
logie [1], although other, less well-known papers are also
included in this article.
In present terminology, glial cells are classified into two
main groups: microglia and macroglia [2]. Macroglia are
subdivided into four specialized cell types: ependymal
cells, Schwann cells, oligodendroglia and astroglia. Astro-
glia include astrocytes, marginal glia, radial glia in the
developing brain and spinal cord, Bergmann cells in the
cerebellar cortex, Müller cells in the retina, pituicytes in
the neurohypophysis and tanycytes in the hypothalamus
[2]. In this review, we will show that Cajal paid special
attention to glial cells, not only from a morphological point
of view but also in relation to their physiological role in the
nervous system. Some of the glial functions that Cajal
Corresponding author: Garcı́a-Marı́n, V. (vgmarin@cajal.csic.es).
Available online 31 August 2007.
www.sciencedirect.com 0166-2236/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2007.06.008
Vol.30 No.9
proposed have been subsequently revised, and for this
reason we hope this article can bring greater clarity to
the history of glial research and contribute to the establish-
ment of new working hypotheses of glial function. Although
Cajal mentions numerous types of glial cells in his writ-
ings, for the purposes of this review we will focus on
astrocytes, for which he proposed a functional link between
morphology, physiology and function.
Cajal’s early work on astrocytes
The 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 the
nervous elements appeared to be embedded. He coined the
term ‘neuroglia’ to describe this interstitial substance.
Later, he published the first neuroglia illustrations [4],
in which some nuclei appear without protoplasm and
others are shown as small round or lentil-shaped cells
[4]. Presumably, these were glial cells whose cytoplasm
was either not stained or incompletely stained (for a recent
historical review, see [5]). Otto Deiters [6] was the first to
describe the arachnoid-shaped cells that were seen in the
white matter and were later shown to be glia. Michael von
Lenhossék [7] introduced the term ‘astrocyte’ to refer to
star-shaped neuroglial cells. Andriezen [8] and Albert
Koelliker [9] divided glia into two groups, fibrous glia
and protoplasmic glia (for recent historical reviews, see
[5,10–12]). Cajal adopted this classification but gave the
name ‘astrocyte’ to both cell types [1]. The differences
between 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 longer
and branch less frequently and at a more acute angle than
those of protoplasmic astrocytes. Protoplasmic astrocytes
are found mainly in gray matter, whereas fibrous astro-
cytes occur mainly in the white matter of the brain and
spinal cord.
Most of the methods available a century ago for staining
astrocytes were complex and most effective at staining
fibrous astrocytes in white matter; they stained protoplas-
mic astrocytes poorly [13]. Frustrated by the lack of an
effective stain for protoplasmic astrocytes, Cajal experi-
mented with different approaches and succeeded in devel-
oping the sublimated gold chloride method [14]. Cajal’s
method allowed the nucleus and other intracellular
elements to be better visualized – Golgi’s impregnation
technique and related approaches did not distinguish these
elements from the dark staining of the background. On
studying Cajal’s slides, it is clear that if a particular
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480 Review TRENDS in Neurosciences Vol.30 No.9

astrocytes, including their capacity for division and their

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).
structure or cell interested him, he became quite driven to
find a way of staining it; he would apply all methods
available and modify them if necessary to obtain the best
result. Table 1 summarizes these methods and includes the
main components of the chemical reactions used and the
kind of cell impregnated. An example of one of Cajal’s
original slides is shown in Figure 1.
Main functions that Cajal attributed to astrocytes
Astrocytes 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 of
nutrients to neurons [23], the removal of toxic waste
products of neuronal metabolism [24] and the regulation
of synaptic activity [24,25], were proposed more than a
century ago. Cajal proposed other interesting functions for
contacts with other astrocytes, neurons and blood vessels
through their endfeet, but he realized that correctly
answering the question ‘What is the function of glia?’ would
require improved techniques. In the last few years, such
cellular and physiological techniques have been developed
and have confirmed some proposals of these pioneer neu-
roscientists.
Division capability of astrocytes
Some histological preparations and drawings show pairs of
astrocytes joined by their soma; Cajal named such pairs as
‘astrocitos gemelos’ (twin astrocytes) (Figure 2a). Such
profiles can be observed in the dentate gyrus of the adult
human hippocampus. Cajal interpreted these observations
as being of astrocytes that had recently divided, and he
speculated that astrocytes might retain their ability to
divide, unlike neurons, which lose this capability during
the differentiation process [26]. Some decades later, obser-
vations in rats sacrificed 1 h after injection of H3-thymidine
(so that actively dividing cells would be stained) revealed
some labeled astrocytes in the corpus callosum and thereby
showed that at least some astrocytes have the ability to
divide [27]. However, astrocytes mostly derive from glial
cell 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 neurons
throughout life [30–32]. This is the same location where
Cajal observed his twin astrocytes.

Table 1. Staining methods that Cajal employed to observe different kinds of glial cells
Method Glial type Figs Refs a
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 2d [17]
astrocytic endfeet.
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-Rı́o-Hortega Oligodendrocytes – all types 4f [21]
Fix.: potassium dichromate + chloral hydrate +
formalin
Impreg.: silver nitrate
a
References indicate the first publication of the method; later modifications are not cited.

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 Author's personal copy
Review TRENDS in Neurosciences
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.
Role of astrocytic endfeet in control of blood-vessel
dilatation
With the development of modern microscopy, it has been
possible to observe the movement of neurons and glial cells.
However, the movement of the nervous system had already
been observed in Cajal’s time by Wiedersheim in 1890 [33],
and it was the basis of different theories about neuronal
ameboidism [34–36] (see recent review in [37]). Cajal also
was one of the first scientists to develop a dynamic concept of
neurons (1892–1894) [38,39], and later he extended these
concepts to astrocytes (1895) [25]. Although he obviously
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).
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Vol.30 No.9 481
could not observe the movement of glial cells, he could
observe different lengths of astrocytic processes across
different astrocytes, and he proposed his theory of glia
ameboidism. One of Cajal’s principal values was his prodi-
gious imagination. Although he only observed static images
by optical microscopy, his descriptions of the nervous system
are very dynamic and animated and create, in the words of
Sherrington, ‘alive scenes’. He applied glial dynamics to two
different topics: attention and sleep–wake states.
Astrocytes have numerous endfeet that make contact
with one or more blood vessels. It has been demonstrated
that these endfeet play a role in cerebral metabolic traffic
between neurons and intracerebral blood vessels [40].
Cajal studied astrocytic endfeet with different methods
(Figure 3a–e; Figure 4c). Based on the close relationship
between astrocytic endfeet and blood vessels and the
variability in the length of astrocytic processes, Cajal
proposed that astrocytes could produce either vasodilata-
tion or vasoconstriction in arterioles through movements of
their endfeet. Furthermore, he suggested that this mech-
anism could explain the process of attention [25]
(Figure 4a,c). Astrocytes associated with arterioles could
retract their endfeet, resulting in arterial dilatation and an
increase in the supply of nutrients to a specific region of the
brain (such an increase is needed during attentional
switching) and explaining functional hyperemia, described
years before by Angello Mosso and Charles Sherrington
[41]. When neurons are highly activated in a specific area of
the brain, blood flow and glucose uptake in that region
increase in a temporally and spatially coordinated manner
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482 Review TRENDS in Neurosciences Vol.30 No.9

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.

to meet the enhanced local metabolic demand [42]. Cajal
proposed that during periods of increased metabolic
demand, astrocytes dilated vessels by mechanical move-
ments. This dilation could be reversible, and astrocytes
could cause vasoconstriction when attention is not necess-
ary [25]. It has recently been observed that during high
synaptic activity, glutamate provokes a response in astro-
cytes, which release vasoactive agents, presumably from
their endfeet; this response is mediated by an increase in
intracellular Ca2+ concentration [43,44]. This could lead to
vasodilation 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 the
available Ca2+ concentration within astrocytes produces a
constriction of nearby arterioles and thus decreases local
blood flow [45]. These apparently controversial results
could be caused by differences in the preparation of the
brain slices used for the experiments. The conditions of
brain slices in vitro (i.e., the presence of blood flow, arterial
pressure, synaptic activity and metabolic demands) are
very different from in vivo conditions. Recently, Takano
et 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 is
probably not mediated by astrocytic-process contraction or
elongation but by release of vasoactive agents from astro-
cytes, it is necessary to recognize the merit of Cajal’s
proposal of astrocytic control of hyperemia, when he had
only observed static light-microscopy images.
The astrocyte–neuron relationship
Cajal’s histological slides and drawings clearly illustrate
astrocytic processes surrounding the body of neurons
www.sciencedirect.com
(Figure 2; Figure 3d). Cajal observed that glial processes
appeared to be consistently arranged in a fashion that
prevented contact among unmyelinated axons and den-
drites at points where functional separation between those
elements was needed. If contact between dendrites and
non-myelinated fibers were prevented, nerve impulses
between those neurons would be blocked [47]. Cajal’s
brother Pedro Ramón had previously suggested that astro-
cytes could physically insulate against the passage of
neuronal impulses [48], and this hypothesis received
strong support from Cajal [49]. After the discovery of
myelin as an insulating material, Cajal considered that,
in the white matter, there were two ‘insulators’, myelin and
astrocytes, whereas in the gray matter, 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 as
insulators, Cajal retained the belief that astrocytes were
in some way modulating or interfering with neuronal
function. In a theoretical article published in 1895, Cajal
also proposed a possible role for astrocytes in the sleep–
wake cycle; he suggested that astrocytes in the gray
matter 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 can
contact each other and the nervous current can flow.
However, when astrocytes extend their processes among
neurons, 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 sleep
has not been proven, the mechanism that Cajal proposed
 Author's personal copy
Review TRENDS in Neurosciences
for these functions (interposition between synapses) has
been echoed by modern studies across various brain
areas. In the arcuate nucleus of the hypothalamus, estra-
diol induces a transient growth of astrocytic processes at
the end of proestrus [50–52]. In the magnocellular nuclei,
under conditions of intense neurohypohysial hormone
secretion (e.g. lactation, parturition, chronic dehydrata-
tion), the glial coverage of neurons decreases in the
vicinity of synapses [53–56]. This results in an alteration
of glutamate clearance and also in a decrease of gluta-
matergic neurotransmission [57]. Great motility of astro-
glial process endings, mediated by lamellipodia and
filopodia, has been observed with two-photon microscopy
[58].
In conclusion, the astrocyte–neuron partnership is not
static but shows dynamic transformations that might be
essential for synaptic plasticity and the modulation of
neurotransmission. In recent years, further mechanisms
of communication between neurons and astrocytes have
been observed. It has been shown that astrocytic excit-
ability varies with changes in intracellular Ca2+ concen-
tration [59]. Astrocytes might sense the activity of
neighboring synapses and respond to neurotransmitters
released by synaptic terminals. Their response might
induce an increase in the intracellular Ca2+ concentration
in adjacent glial cells, and this increase might in turn
lead to the release of various transmitters, such as glu-
tamate [60,61]. These gliotransmitters could mediate
intercellular signaling between astrocytes and neurons
(for recent review, see [62]). The functional significance of
these morphological and physiological findings in the
modulation of nervous transmission has generated the
concept of ‘tripartite synapse’, whereby the synapse is
functionally constituted by three elements: the presyn-
aptic cell, the postsynaptic neuron and the surrounding
astrocytes [63].
Cajal’s ‘third element’
The ‘third element’ is a general and ambiguous term that
Cajal used to describe a group of adendritic cells that
seemed devoid of processes, probably because of incom-
plete staining methods. Rı́o-Hortega demonstrated that
these cells were indeed oligodendrocytes and microglial
Table 2. The identity of Cajal’s ’third element’
Author Method Identity
Cajal, 1896 - Nissl - Satellite cells
Cajal, 1913 - Formol uranium - Third element
nitrate
- Sublimated gold
chloride
Rı́o-Hortega, - Silver carbonate - Microglia
1920 - Interfascicular glia
Cajal, 1920 - Modification of - Microglia
reduced silver nitrate - Interfascicular glia
- Ammoniacal silver - Other distinct
oxide adendritic cells
- Silver carbonate
Rı́o-Hortega, - Golgi-Hortega - Oligodendrocytes
1921–1928
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Vol.30 No.9 483
cells [67]. Nowadays, it is easy to distinguish between
microglial cells and oligodendrocytes by the application
of antibodies as specific cell markers [64,65].
In 1896, Cajal observed some small cells that apparently
lacked processes. Because of their close association with
neuronal 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 the
presence of the perineuronal adendritic cells and extended
this observation to the satellite cells of the astrocytes, blood
vessels and other adendritic cells in the white matter [26]
(see Table 2). Cajal grouped all of them under the general
term of ‘third element’ of the nervous centers in 1913 [26].
Neurons were considered the first element and astrocytes
the second. The drawings and Cajal’s original slides shown
in Figure 5a–c summarize some of the main shapes Cajal
proposed for his third element [26]. Cajal considered that
all of these elements could have either mesodermic or
ectodermic origin.
In 1920, Rı́o-Hortega obtained clearer images of the
morphology of these adendritic cells both in gray matter
and white matter by applying a new method [19]. He
divided Cajal’s third element into two different types of
cells, microglia and interfascicular glia [67]. In microglial
cells he included some satellite cells of gray matter and
some of the adendritic cells in white matter, which Cajal
had previously described. Rı́o-Hortega also described the
transformation of microglia from an inactive state to the
phagocytic ameboid form and their final transformation
into granular cells in response to a threat such as infection.
He classified the rows of adendritic cells that Cajal had
previously found in white matter (see Table 2) as inter-
fascicular glia [67]. With regard to embryonic origin, Rı́o-
Hortega suggested that microglia had a mesodermal ori-
gin, whereas interfascicular glia and astrocytes were of
ectodermal descent [67]. Because of their distinct origin
and function, Rı́o-Hortega considered microglia to be the
third element of the nervous system [67].
After Hortega’s work, Cajal applied different methods to
study microglia [19–21], although the best technique for
staining 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
Observations
- Small cells lacking processes and in close association with neuronal somata
- Gray matter (satellite to neurons, astrocytes and blood vessels)
- 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)
- Fusiform cells with an elongated nucleus and protoplasm, as described by Cajal
- Rows of cells along nerve fascicles, as described by Cajal
- Adendritic cells (including those close to blood vessels and dwarf cells) and
microglia
- Oligodendrocytes (including the satellite cells, interfascicular glia, adendritic
cells close to blood vessels and dwarf cells described by Cajal)
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484 Review TRENDS in Neurosciences Vol.30 No.9

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).

cells, including microglia, interfascicular glia and other
distinct adendritic cells, particularly perivascular adendri-
tic cells and the dwarf cells described in white matter (see
Table 2). Because this latter group of adendritic cells could
not be easily visualized with any known staining tech-
niques, Cajal considered them to constitute a special cell
type with undiscovered functions and called this cell type
the ‘real third element’ [68,69].
Rı́o-Hortega made further contributions to the study of
this problem in 1921 and 1928. Via a modification of the
Golgi-Hortega method, he described the small adendritic
cells that Cajal considered the real third element and
called them ‘oligodendrocytes’ [22,70]. Among oligodendro-
cytes (Figure 5f), Rı́o-Hortega also included some satellite
cells that Cajal had identified adjacent to neurons in gray
matter (Figure 5d) and the interfascicular glia found in
between nerve fibers of the white matter (Figure 5e).
As we have seen, the development of the classification of
different glial cells has been a complex process, involving
not only methodological (different staining methods),
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morphological and functional concerns but also a problem
with scientific terminology.
Radial glial cells
Radial glia (RG) were first fully visualized by Golgi (1885),
Magini (1888), Cajal (1889), Kölliker (1896) and other
scientists (for a recent review, see [71,72]). These authors
observed radial cylindrical epithelial cells covering the
whole section of either the spinal cord or the cerebral
cortex and reaching the peripheral border close to the
pia mater. Cajal called these cells spongioblasts, whereas
other contemporary scientists used different names, such
as epithelial cells, radial cells, and so on. Rakic (1972)
introduced the term ‘radial glia’ in his classic descriptions
of neuronal migration in the neocortex of fetal primates
[73].
A pivotal role has been suggested for RG in the
construction of the nervous system: first, providing a scaf-
fold for migrating neurons and second, participating in
the generation of diverse brain cells [74]. Some of these
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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)
functions were first suggested more than a century ago by
Cajal and his colleagues.
Cajal described the morphology of RG by using the
Golgi method in the spinal cord [75] (Figure 6b). Magini
proposed that these cells acted as a scaffold for migrating
neurons. He observed that the radial filaments exhibit
many swellings or round varicosities of various sizes on
their course. He hypothesized that these spherical cells
(the varicosities) probably represented the future nerve
cells of the cerebral cortex (see [72] for a recent review). In
the early 1970s, Rakic used a combination of Golgi impreg-
nation and reconstructions from electron microscopy
serial sections to observe migrating neurons along the
RG fibers [73,76].
In Cajal’s Histologie [1], we learn that at that time the
spinal cord at its earliest stages was considered to be
constituted of elongated epithelial cells arranged in a
single layer, that is, neuroepithelial cells, by current ter-
minology. Among these epithelial cells, there are scattered
spherical elements with mitotic figures, which His had
named germinal cells. For His, germinal cells represented
specific elements from which the neuroblasts derive exclu-
sively. However, Cajal observed that the number of epi-
thelial cells increased notably after the differentiation of
germinal cells, and he proposed that germinal cells were
undifferentiated forms that could give rise to both epi-
thelial cells and neuroblasts. Moreover, Cajal observed
transitional phases between spongioblasts and neuro-
blasts in very early stages of development of the chick
spinal cord. This led him to consider the possibility that
some epithelial elements might become neuroblasts. Only
in recent years has this point been confirmed, when it was
observed that RG are not only astrocyte progenitors but
also neuronal progenitors in the central nervous system
(CNS) [77–80].
Cajal described the differentiation of RG into fibrillary
or protoplasmic astrocytes after they have ceased their
guiding function during neurogenesis (Figure 6a) [1,64].
According to Cajal, astrocyte morphology depends on the
place where the RG ends its evolution. Influenced by
neuronal activity, astrocytes placed in gray matter devel-
oped their processes among the thin processes of neurons;
however, an astrocyte situated in white matter acquired
long and smooth processes [1]. Recent studies have
www.sciencedirect.com
Vol.30 No.9 485
confirmed that RG transform into astrocytes in most avian
and mammalian CNS regions [81–84].
Cajal observed that RG persist through adulthood in
lower vertebrates and that they are the dominant glial
form in reptiles (Figure 6c) [85]. However, in the adult
mammalian stage, most of the RG disappear, with a few
exceptions where 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 the
cerebellum, or the Müller cells of the retina) [74]. Because
neurogenesis continues in adult cold-blooded vertebrates,
a close correlation between the presence of RG and neu-
rogenesis has been suggested. Recent studies in avian and
mammalian brains also indicate that RG function as
neural progenitors and perhaps as stem cells [86].
Conclusions
More than one hundred years ago, Cajal proposed a
physiological role for glial cells. Astrocytes were initially
considered as ‘brain glue’, providing an inert scaffold
necessary for neuronal distribution and interactions.
However, it is only in the past few years that new functions
have been revealed for astrocytes; such functions include
the control of synapse formation and function, adult neu-
rogenesis and brain vascular tone. Some of these ‘new’
functions had already been proposed by Cajal but could not
be tested. Cajal found that the nervous system included a
new cell type, which he called the third element. Rı́o-
Hortega, using specific methods, determined that this
third element was actually composed of two cell types,
oligodendrocytes and microglia. Finally, some of Cajal’s
main ideas, that is, the evolution of RG to astrocytes and
their neurogenic potential, have recently been corrobo-
rated. As we have seen in the field of glial research, the
scientific work of Cajal is still a rich source of hypotheses
waiting to be tested.
Acknowledgements
We gratefully acknowledge Adolfo Martinez, and especially Sian Lewis
and Helen Barbour, for their invaluable help with editing the manuscript.
We also acknowledge Javier DeFelipe and Luis Miguel Garcı́a-Segura for
their critical reading of the manuscript. Original drawings from the
Museum Cajal are reproduced with permission from the Inheritors of
Santiago Ramón y Cajalß. The authors are supported by the Spanish
Ministry of Science and the Areces Foundation.
 Author's personal copy
486 Review TRENDS in Neurosciences
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