The discovery of dendritic spines by Cajal in 1888 and its relevance

in the present neuroscience

Pablo Garcıa-Lopez, Virginia Garcıa-Marın *, Miguel Freire

Museo Cajal, Instituto Cajal, CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain

Received 27 October 2006; received in revised form 17 February 2007; accepted 3 April 2007

www.elsevier.com/locate/pneurobio

Progress in Neurobiology 83 (2007) 110–130

Abstract

The year 2006 marks the centenary of the Nobel Prize for Physiology or Medicine awarded to Santiago Ramon y Cajal and Camilo Golgi, ‘‘in

recognition of their work on the structure of the nervous system’’. Their discoveries are keys to understanding the present neuroscience, for

instance, the discovery of dendritic spines. Cajal discovered dendritic spines in 1888 with the Golgi method, although other contemporary scientists

thought that they were silver precipitates. Dendritic spines were demonstrated definitively as real structures by Cajal with the Methylene Blue in

1896. Many of the observations of Cajal and other contemporary scientists about dendritic spines are active fields of research of present

neuroscience, for instance, their morphology, distribution, density, development and function. This article will deal with the main contributions of

Cajal and other contemporary scientists about dendritic spines. We will analyse their contributions from the historical and present point of view. In

addition, we will show high quality images of Cajal’s original preparations and drawings related with this discovery.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Cajal; Dendritic spines; Filopodia; Golgi method; Methylene Blue method

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

2. The historical context of the discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

3. Principles of Cajal’s scientific reasoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

4. Principal data contributed by Cajal to the research of the dendritic spines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

4.1. Morphological data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

4.2. Distribution of spines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

4.3. Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

4.4. Physiological role of the dendritic spines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

4.5. Dendritic spines in pathological and poisoning states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

5. The dendritic spines from Cajal to present-day neuroscience. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

5.1. The concept of Sherrington’s synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

5.2. Morphology of dendritic spines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

5.3. Dendritic spines and pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

5.4. Plasticity of dendritic spines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

5.5. Dendritic spines and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Abbreviations: s.s., sensu strictus; CA1, cornu ammonis 1; CA3, cornu ammonis 3; EM, electron microscopy; ER, endoplasmic reticulum; LTP, long-term

potentiation; HIV, human immunodeficiency virus; AMPA, a-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid

* Corresponding author. Tel.: +34 91 585 47 43; fax: +34 91 585 47 53.

E-mail addresses: vgmarin@cajal.csic.es (V. Garcıa-Marın), mfreire@cajal.csic.es (M. Freire).

0301-0082/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pneurobio.2007.06.002

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130 111

1. Introduction

Santiago Ramon y Cajal (1852–1934) was one of the most

outstanding neuroscientists of all time. In 1906 he shared the

Nobel Prize for Physiology or Medicine with Camillo Golgi

(1834–1926) ‘‘in recognition of their work on the structure of

the nervous system’’. We think that the recent centenary of this

Nobel Prize offers a great opportunity to analyze one of Cajal’s

most important discoveries, the dendritic spines.

As proof of his enormous amount of work, 4529 histological

preparations that were personally made by Cajal are preserved

in the Museum Cajal. Of these preparations, 809 are stained

with the Golgi method and 108 with the Ehrlich method; these

methods allow seeing dendritic spines with the light micro-

scope. Apart from his preparations, more than 2500 original

drawings and papers written by Cajal – most of them in Spanish

or French – are conserved almost exclusively in the Cajal

Institute.1 In these funds there is a lot of information for

reinterpreting the discovery of the dendritic spines from a

historical and present-day point of view.

Dendritic spines are one of the most active fields of research

in modern neuroscience, but it took great effort before they

were considered as real structures. This review will deal in

depth with the scientific observations, reasoning, and ideas of

Cajal about dendritic spines, the historical context of the

discovery, and the meaning of Cajal‘s concept of dendritic

spines in current neuroscience.

2. The historical context of the discovery

‘‘. . . the surface . . . appears bristling with points or short

spines . . .’’2 (Cajal, 1888). This quotation, in Spanish, refers to

the dendritic spines of a Purkinje cell (Fig. 1), and marks the

beginning of the research into dendritic spines in the history of

neuroscience. In a footnote Cajal explained:

‘‘At first we believed that these protuberances were the result

of a tumultuous silver precipitation; but the constancy of

their existence . . . inclines us to consider them as normal

structures’’3 (Cajal, 1888).

The Golgi staining (Golgi, 1873) was the method used by

Cajal for visualizing and describing the fine structure of the

nervous system. Cajal was introduced to the Golgi method in

1887 by Luis Simarro La Cabra, the founder of clinical

psychology in Spain. The deep impression caused by this

staining made Cajal focus on the research of the nervous

system. One of the most important reasons for Cajal’s success

in these studies was that he applied the ontogenic method to his

1 Many of the works of Cajal have been tranlated to the English by DeFelipe

and Jones, in their books: Cajal on the Cerebral Cortex (1988) and Cajal’s

Degeneration and Regeneration of the Nervous System (1991).2 ‘‘. . . La superficie . . . aparece erizada de puntas o espinas cortas . . .’’.3 ‘‘Al principio creıamos que estas eminencias eran resultado de una pre-

cipitacion tumultuosa de la plata; pero la constancia de su existencia . . . nos

indica a estimarlas como disposicion normal’’.

research reducing the complexity of the brain by applying the

Golgi staining to embryos and young animals.

But why were the dendritic spines discovered 15 years after

the development of the Golgi method? As we said before, the

Golgi method was developed by Golgi in 1873. Nevertheless,

this method had little repercussion in the scientific community,

probably because of its limited diffusion, the irreproducible and

apparently random impregnation, and something Cajal called

‘‘scholastic discipline’’:

‘‘out of respect for their teachers, students tend to use only

those research methods that have been developed by their

teachers themselves. As far as the great investigators are

concerned, they would feel dishonored working with other

people’s methods’’4 (Cajal, 1899a).

It was Cajal who noticed the value of this technique and

improved it. But the real genius of Cajal was to look with

different eyes than the rest of researchers, and to interpret the

histological observations in the correct manner. In the Golgi

preparations made by Golgi himself it is also possible to

observe dendritic spines (De Felipe, oral communication), but

he made no reference to them until the Nobel lecture in which

he mentioned the dendritic spines without ascribing them any

physiological significance. In addition, we have found only

three drawings of dendritic spines in Golgi’s work (Fig. 1H).

We may conclude from these data that Golgi also saw these

structures but that he did not give any importance to them,

maybe because he thought at first, as other researchers did, that

they were silver artifacts or because he did not think that they

had any physiological meaning.

The discovery of dendritic spines caused some controversy

in the scientific community. Some scientists supported Cajal’s

discovery with new data like Retzius (1891), Schaffer (1892),

Edinger (1893), Berkley (1896), and Monti (1895a,b), while

others denied the reality of these structures like von Kolliker

(1896), Meyer (1896a,b, 1897), and Dogiel (1896). They

argued that what Cajal called ‘‘collateral spines’’ were in reality

a silver precipitate, adhering to the widespread notion that the

Golgi method was not very reliable. It is interesting to note that

this idea was at first also considered by Cajal, but that he

quickly discarded it.

3. Principles of Cajal’s scientific reasoning

The discovery of the dendritic spines by Cajal provides a good

opportunity to analyze the principles of his reasoning in the

discovery of these structures, and his work trying to convince the

other scientists of the reality of the so-called collateral spines.

The following key points summarize the main ideas in

Cajal’s scientific reasoning:

(1) C

4

inve

cree

onstancy of the experimental results: Spines are always

absent from the soma and the origin of thick dendrites, and

‘‘por respeto al maestro, ningun discıpulo suele emplear metodos de

stigacion que no se deban a aquel. En cuanto a los grandes investigadores,

rıanse deshonrados trabajando con metodos ajenos’’.

Fig. 1. Dendritic spines of Purkinje cells: (A) first drawing of Ramon y Cajal showing the dendritic spines of a Purkinje cell of the hen (Cajal, 1888); (B) insert-box

showing the dendritic spines in real size; (C) Purkinje cell of an adult bird (P84186); (D) Purkinje cell of an adult human (P81750); (E) different types of dendritic

spines found on human Purkinje cells of human. From top to bottom: thin, mushroom, sessile, and ramified; (F, G) different segments of dendritic branches of Purkinje

cells of adult bird and human, respectively, showing different densities of dendritic spines; (H) drawing of a Purkinje cell by Golgi (1906), showing dendritic spines.

Golgi proposed that the dendritic tree directs its branches to the blood vessels, according to the nourishing role of the dendrites.

5 ‘

cend

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130112

their distribution is not homogeneous along the dendritic

tree. If it had been a non-specific silver precipitate, it should

have been present on every part of the neuron with the same

intensity.

(2) P

resence of dendritic spines in many species and many cell

types: Spines are observed on pyramidal cells of the

cerebral cortex, Purkinje cells, hippocampal pyramidal

neurons, dentate gyrus granule cells, etc., and in different

species (cat, dog, chicken, pigeon, human, etc.). After his

discovery in chicken Purkinje cells in 1888, Cajal extended

the observation to mammals: 15-day-old cat Purkinje cells

(Cajal, 1889) and rat olfactory granule cells (Cajal, 1890a).

One month later, in November of that same year, he

described dendritic spines in the cerebral cortex of lower

mammals. The first scientific drawing of human dendritic

spines appeared in ‘‘Nuevo concepto de la histologıa de los

centros nerviosos’’ (Cajal, 1892). Cajal concluded that

dendritic spines are a common structure in many species

and many different types of cells, and that they might play

an important role in the functioning of the nervous system.

‘‘it is important to recognize certain morphologic details

on studying the protoplasmic expansions with the Golgi

method, because it is possible that in time they will be

understood to have physiological importance’’5 (Cajal,

1899b).

(3) O

btaining the same results using different staining methods:

Apart from the Golgi method, Cajal used the Golgi–Cox

method, Turnbull Blue method (Cajal, 1890b), and

Methylene Blue method (Cajal, 1896b) to visualize the

collateral spines of Purkinje cells. In the Golgi–Cox method

(Cox, 1891), silver nitrate is replaced by mercury chloride

and the tissue is next exposed to ammonia to darken the

resulting mercury precipitate (Zhang et al., 2003).

‘Conviene conocer . . . porque acaso andando el tiempo alcancen tras-

encia fisiologica’’. 6 ‘

Visualization with Methylene Blue was considered the

definitive demonstration (Cajal, 1896b).

(4) I

mprovement and variations of the staining methods:

Because they were not able to stain them with Methylene

Blue, some researchers like Dogiel and Meyer denied the

reality of dendritic spines, arguing as done originally by von

Kolliker that they were silver artifacts. In fact, Dogiel

stained only just the beginning of the primary Purkinje cell

branches, where no dendritic spines are located, as can be

seen in his lithographic plate (Fig. 2A). Although Meyer

was able to stain the entire dendritic arbor, the color of the

drawing was pale blue (Fig. 2B), which led Cajal to think

that the staining obtained by Meyer was not strong enough.

Cajal had no doubt about the possibility of Methylene Blue

to stain the dendritic spines, because he had observed them

before in ganglion cells of the retina of the frog (Cajal,

1896a). This led Cajal to improve the method of Ehrlich,

and he published a monograph about dendritic spines

stained by Methylene Blue (1896b) (Fig. 2D and E) and

another important and more extended article later that same

year (Cajal, 1896b), in which he applied this staining to

many nerve centers (Fig. 5). The different results achieved

by these three scientists may be due to the different

Methylene Blue staining techniques they employed.

- Semi Meyer employed subcutaneous injection of Methy-

lene Blue into the living animal. Cajal tried to stain the

cortex and the cerebellum with this method, but the color

of the cells was too pale, and only the soma could be

distinguished. In Fig. 2C, a cell is shown from an original

preparation of Cajal impregnated with Methylene Blue,

done with the Meyer method and lubricated afterwards.

On the preparation Cajal wrote: ‘‘Rabbit . . . injection . . .lubrify . . . 1 h . . . ganglia . . . ’’.6 No dendritic spines can

be seen because the staining is too pale. This cell is

comparable to an original drawing of Meyer (Fig. 2B).

‘C

onejo . . . inyeccion . . . lubrifica . . . 1hora . . . ganglios’’.

Fig. 2. Different drawings and photos from preparations stained with Methylene Blue: (A) drawing of Dogiel (1896) of the cerebellum, showing the Purkinje cells

with only the soma and the primary branches stained; (B) drawing of Meyer (1896a) of a pyramidal cell without dendritic spines; (C) Z-projection (five sections) of a

pyramidal cell of a rabbit preparation by Cajal (P81392), stained with the Meyer method followed by lubrication of the tissue; (D) published drawing of Cajal (1896b)

showing pyramidal cells of cortex of adult rabbit stained with Methylene Blue; (E) Z-projection (10 sections) of a fragment of a dendrite with dendritic spines of a

granule cell of the dentate gyrus of rabbit (P81469), made using the Meyer method. On the label of the preparation is written (‘‘1 hora solo de aireacion’’: only 1 h of

aeration); (F) different types of dendritic spines and filopodia seen in drawing (D). From top to bottom: sessile, thin, mushroom, branched, spine with spinule,

dendritic filopodia, and dendritic growth cone filopodia. The image is 1.25 times the real size of the drawing. On the right the same spine shapes found in a Golgi

preparation. (G) Dendritic branch (1.25 times real size) of the drawing compared with a dendritic branch in a Golgi preparation.

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130 113

- Dogiel employed the technique of lubrication, the

Ehrlich–Dogiel method. After leaving pieces of nervous

tissue to the action of air, he applied a diluted solution of

Methylene Blue (Fig. 2A).

- Cajal used the propagation method created by himself.

The pieces were not exposed to the action of air, and the

color, as a pigment or in saturated solution, was applied

directly to the surface of the nerve tissue.

7 ‘‘Ademas en buena logica cientıfica, los hechos negativos de observacion

no invalidan las observaciones positivas. Cuando un metodo no nos permite

confirmar un hecho, facilmente demostrable con otros metodos, lo unico que

legıtimamente podemos afirmar es la insuficiencia del recurso empleado para

hacer la verificacion, a menos que no se pruebe de manera irrecusable (y esto

nadie lo ha probado hasta hoy) que los procedimientos reveladores del detalle

morfologico buscado, provocan en la celula alteraciones artificiales’’.

In the Museum Cajal there are 108 histological preparations

impregnated with Methylene Blue. In these preparations it is

very difficult to see the dendritic spines, probably because the

staining gets paler with time and because of varicose

degeneration. However, there are some preparations where it

is possible to find some dendrites with dendritic spines still

stained (Fig. 2E).

In contrast to von Kolliker, Dogiel, or Meyer there were

other scientists who did not deny the existence of these

structures, but who made a wrong interpretation of them. For

instance, Bethe did recognize their existence, but mistakenly

considered them as insertion points of an enigmatic interstitial

network of grey matter called nervoses Grau by Nissl. Held

also accepted them, but wrongfully assumed that they were the

endings of pericellular nerve fibers, calling them Endfuse

(Endfeet). But, as Cajal said, spines are never stained with

neurofibrillar methods like reduced silver nitrate (Cajal, 1909).

(5) P

ositive science: Negative results cannot invalidate positive

results. Cajal wrote:

‘‘Moreover, in good scientific logic, negative facts of

observation do not invalidate the positive ones. When a

method does not permit us to confirm a fact that can be

easily demonstrated with other methods, the only thing

we can state is that the method employed is not adequate

for the verification, as long as nobody proves in an

irrefutable way (and this has not been proven until now)

that the procedures for revealing the morphological

detail looked for cause artificial alterations in the cell’’7

(Cajal, 1896b).

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130114

(6) U

Fig. 3. Varicose degeneration: (A) original drawing of Cajal (1896a) of the

fascia dentata of a 1-month-old rabbit, based on a preparation stained with

8 ‘

real

revis

que

se of the best technical instruments: Cajal had a

microscope with the best optics of the time. He used very

powerful objectives. Using the Zeiss E 1.30 apochromatic

objective, he realized that the spines did not have the

appearance of crystals or irregular deposits. He also used a

Zeiss camera lucida, as he wrote in the footnotes of some of

his lithographic plates. The Camera Lucida gave Cajal the

opportunity to draw in a very realistic way, although he took

some artistic freedom by grouping cells of different

histological sections. However, contemporaries of Ramon

y Cajal who saw him drawing said that he used to draw by

looking at the histological preparation through the

microscope, while drawing on a piece of paper on his

right-hand side, where the exact reproduction of the nerve

cell appeared, without using the camera lucida that Cajal

considered troublesome (De la Villa, 1952). One possible

explanation of this way of drawing is that Ramon y Cajal

managed to blend the microscope image with the drawing

he was making on the paper seen with his right eye, so that

he did not need the camera lucida that specifically carries

out the function of mixing both images together (Freire,

2003).

Methylene Blue. (B) Z-projection (five sections) of a granule cell of the dentate (7) T gyrus of a rabbit with varicosities on its dendrites (P84169); (C) Z-projection

(11 sections) of dendritic branches with varicose degeneration.

he concept of scientific drawing in the work of Cajal:

‘‘Good drawings, like good microscopic preparations,

are pieces of reality, scientific documents that preserve

their value indefinitely, and their reexamination will

always be profitable no matter the interpretations they

may have elicited’’.8 (Cajal, 1899b).

With this sentence he expressed very well his concept of

scientific drawing. Cajal tried not to act as a filter between what

he observed on his histological preparation and what the

spectator could observe on his scientific drawings. He did not

try to interpret the structures he was watching, but to draw all

structures as realistically as possible, although there are other

schematic drawings where he tried to express a concept or give

an explanation of what he was observing. One proof of this way

of thinking is the drawings of the dendritic spines compared to

the drawings of the dendritic varicosities in the monoliforme

stage (Fig. 3). Cajal drew the varicosities and dendritic spines,

although he thought that the former were a post-mortem

phenomenon. In contrast to the scientific drawings of Cajal,

there are others from contemporary scientists that are more

interpretations by the scientists than a representation of reality.

There is another sentence that could help to understand this

idea.

‘‘. . . to see things for first time, in other words, to

contemplate them, putting aside what we learned from

books, false descriptions and common platitudes, has great

importance in scientific research. We have to free our minds

‘El buen dibujo, como la buena preparacion microscopica, son pedazos de

idad, documentos cientıficos que conservan indefinidamente su valor y cuya

ion sera siempre provechosa, cualesquiera que sean las interpretaciones a

hayan dado origen.’’

of prejudices and other people’s images, and to have the firm

intention to see and judge for ourselves’’9 (Cajal, 1899a).

In the act of drawing it is very important to maintain this

independent and open-minded way of seeing.

4. Principal data contributed by Cajal to the research

of the dendritic spines

The references and data contributed by Cajal about spines

are scattered throughout his different articles and books.

Moreover, the drawings related to dendritic spines help us to

interpret the thinking of Cajal about these structures.

4.1. Morphological data

Cajal noticed the different morphologies of dendritic spines.

This is in contrast to other scientists, who drew the dendritic

spines in a very cloned and symmetrical way like Golgi in his

Nobel lecture (mere sticks without a head). Instead, it is

possible to observe in the drawings of Cajal the three mayor

types of spines described by Peters and Kaiserman-Abramof

(1969): sessile, mushroom, and thin (Fig. 2F), and other

important features like ramified spines or spinules, structures

9 ‘‘. . . ver las cosas por primera vez, es decir, readmirarlas, descartando

reminiscencias librescas, descripciones postizas y frases y topicos comunes,

tienen en la investigacion cientıfica muy senalada aplicacion. Hay que limpiar

la mente de prejuicios y de imagenes ajenas, hacer el firme proposito de ver y

juzgar por nosotros mismos . . .’’.

Fig. 4. Dendritic spines in the hippocampus: (A) original drawing of Cajal (1933) showing the mossy fibers that connect the granule cells of the fascia dentata with the

thorny excrescences of CA3; (B) three pyramidal cells of rabbit CA3 (P83474); (C) thorny excrescences of the apical shaft of a pyramidal cell; (D) different segments

(distal: up, with dendritic spines; and proximal: down, without dendritic spines) of a granule cell of a 6 days old cat hippocampus (P80714).

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130 115

that may be implied in transendocytosis (Spacek and Harris,

2004). The morphology of the spine has great physiological and

functional importance, as we will see in the last section.

Cajal was the first to describe the thorny excrescences

(Cajal, 1893) (Fig. 4). These are clusters of dendritic spines

from CA3 and hilar cells. They are situated along the soma and

the apical dendrites of these pyramidal cells, although they can

be also present on the basal dendrites. They are the postsynaptic

structures of the mossy fibers of the granule cells.

Cajal also was the first to notice the differences in size of

dendritic spines among different areas like cortex and

cerebellum (Cajal, 1896a) (Fig. 5C).

‘‘In A, we show the appendages of a protoplasmic process of

a pyramidal cell of the mouse. It is striking to see the

slenderness and length of the pedicles that support the end

bulbs, and the many directions of the pedicles that sprout out

of the entire cylindric surface of the protoplamic branch. In

B we show the spined branches of the Purkinje cell of the

mouse. Note that the appendages are short and relatively

thick, and that they are close together and regularly spaced,

giving the process a hairy appareance’’.10 (Cajal, 1896a).

‘‘Their wealth, length and width vary among different cell

types: while they are thin and long in pyramidal cells, in

Purkinje cells they are short, thick and numerous’’.11 (Cajal,

1899b).

The differences in size of dendritic spines are not limited to

the cerebellum and the cerebral cortex. There are also

10 ‘‘En A, figuramos los apendices de una rama protoplasmica de una

piramide cerebral del raton. Llama la atencion lo fino y largo de los pedıculos

que sostienen los granos terminales y la diversa direccion de los mismos, los

cuales brotan de toda la superficie cilındrica de la rama protoplasmica. En B,

mostramos las ramas espinosas de las celulas de Purkinje del raton. Observese

la cortedad y espesor relativamente considerable de los apendices y su

proximidad y regularidad, que prestan a la expansion de que nacen un aspecto

festoneado’’.11 ‘‘Su riqueza, longitud y espesor, varıan en los diversos tipos celulares; ası

mientras en las celulas cerebrales, dichas espinas son finas y largas, en los

elementos de Purkinje, se muestran cortas, espesas y numerosas’’.

differences in size between distinct cortical areas suggesting

that the current injected by dendritic spines into the dendrite

changes depending on the cortical region and may have great

relevance in the functional specialization of the different areas

(Benavides-Piccione et al., 2002; Ballesteros-Yanez et al.,

2006)

Cajal also made an observation of great value when he

compared the size of dendritic spines among species (Fig. 1E

and G). Comparing the dendritic spines of human and mouse,

he observed:

‘‘. . . the spines vary somewhat among different species; in

humans they are much longer and have thinner pedicles’’12

(Cajal, 1896a).

Recent studies have confirmed that the the head and the neck

in dendritic spines of human pyramidal cells are bigger and

longer respectively than the dendritic spines in the mouse

(Benavides-Piccione et al., 2002; Ballesteros-Yanez et al.,

2006).

These data have great importance in modern neuroscience,

and form the basis of a great number of studies aimed at

establishing the differences of information processing between

different cortical areas and different species.

4.2. Distribution of spines

Spines are absent from soma and the origin of thick dendrites

(Fig. 4D).

‘‘The small terminal dendritic branches of the pyramids do

not have smooth contours, as the authors appear to represent

them; they bristle with teeth protruding more or less at right

angles (Figs. 5 and 7) and end in a round and slightly

thickened tip. These collateral spines are also found,

although in smaller numbers, on the large ascending shafts

of the medium and large pyramids, from the moment they

12 ‘‘Por lo demas las espinas susodichas varıan algo en las diversas especies;

ası, en el hombre son mucho mas largas y exhiben un pedıculo mas delgado’’.

Fig. 5. Different figures published in the article: ‘‘El azul de Metileno en los centros nerviosos’’ (Methylene Blue in nerve centers; Cajal, 1896a). (A) Fragment of the

Purkinje arborization of the mouse. According to Cajal: (a) opening for a blood vessel; (b) opening for climbing fibers. (B) Details of Purkinje dendritic spines. (C)

According to Cajal: varieties of spines of the cells of the molecular layer of the mouse cerebellum, stained with the Golgi Method: (A) spines of a pyramidal cell; (B)

spines of a Purkinje cell; (C) spines of a nest cell; (D) spines of a Golgi cell; (E) excrescences of a neuroglic cell of the molecular layer of the cerebellum; (a) large

openings for the astrocytes; (b) small openings for the parallel fibers.

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130116

begin emitting small branches in the deep layers of the

cortex’’.13 (Cajal, 1891).

This observation was a proof to Cajal of the reality of these

structures; if they were a non-specific silver precipitate, they

should appear randomly distributed over the entire neuron.

This absence of dendritic spines from the proximal region

of the dendrite has a great physiological importance. On

13 ‘‘Les ramilles protoplasmiques terminales des pyramides nes sont pas lisses

de contour, comme les auteurs semblent les representer; elles sont herissees de

dents naissant a angle droit, ou presque droit, Fig. 7 et 5, et terminees par un

bout rond et un peu eppaissi. On trouve aussi des epines collaterales, quoique

moins nombreuses, dans les grosses tiges ascendantes des moyennes et grandes

pyramides, depuis le moment ou elles commencent a emettre des ramilles dans

les couches profun, depuis le moment ou elles commencent a emettre des

ramilles dans les couches profondes de l ecorce’’.

these proximal regions only inhibitory synapses have been

found (Alonso-Nanclares et al., 2004). The distribution of

dendritic spines has been recently studied in detail by Elston

and DeFelipe (2002) in different cortical areas and species.

They found that: ‘‘. . . normalized cumulative spine distribu-

tion (as a function of relative distance from the soma to the

distal tips of the dendrites) is remarkably constant for all

cells’’ and ‘‘remarkably constant across species of different

orders’’. These authors propose two possible explanations for

the absence of dendritic spines at the origin of thick

dendrites:

- T

he mature membrane is nonpermissive to localization of

excitatory inputs.

- C

haracteristics of the backpropagation potential preclude

excitatory synapse localization on the soma and proximal

dendrites.

in Neurobiology 83 (2007) 110–130 117

Another important observation of Cajal was the increased

density of dendritic spines in areas that are more innervated

(Fig. 5C).

‘‘A priori it is already conceived that, just like the form and

the number of nerve terminal fibers in each center of gray

matter vary, the disposition of the spined appendices

destined to make contacts will also vary in a correlative

way. To be certain that these accomodations are real, we

have made a comparative study of the spined appendices of a

great number of nerve- and neuroglic types’’.14 (Cajal,

1896a).

In this paper, he also made a comparison between different

types of cells and the connections made on these cell types

(Pyramidal cells, Purkinje cells, Nest cells, Golgi cells and

Neuroglic cells). Looking at the density of the spines, Cajal

reaches the following conclusion:

‘‘Such differences seem to indicate that the number of fibers

connected to Purkinje cells is much higher than the number

of fibers connected to nest cells and Golgi cells’’.15 (Cajal,

1896a).

This direct correlation between the number of spines and

the number of fibers is used in present-day neuroscience for the

comparative analysis of pyramidal cells and the analysis of the

complexity of cortical circuits and their evolution. Recent data

show that the cerebral cortex is characterized by regional and

species variations that support the different functions of distinct

cortical areas and the different cognitive capacity across

species. For instance the morphology of the pyramidal cell is

very complex and varies across different areas of the cerebral

cortex. The density of dendritic spines of pyramidal cells of the

prefrontal cortex (PFC) of humans and macaques is higher than

in sensory areas, for instance visual areas (Elston, 2000; Elston

et al., 2001; Elston and DeFelipe, 2002; Elston, 2003). The

increase in the complexity and the number of dendritic spines in

the pyramidal cells of the prefrontal cortex indicates a higher

capacity for the PFC cells to process and integrate information

of different sources that must be essential for the sustained tonic

activity characteristic of these neurons and their role in memory

and cognition. These studies still have a Cajal influence and

recall their statements about the pyramidal cell or psychic cell

(Cajal, 1892, 1894).

Other important data contributed by Cajal are about the

density of dendritic spines across species:

‘‘They vary also with animal species, and we may state

in general terms that a cell with spiny processes in

P. Garcıa-Lopez et al. / Progress

14 ‘‘A priori se concibe ya que, variando como efectivamente varıan en cada

foco de substancia gris la forma y el numero de las fibrillas nerviosas

terminales, variaran tambien, de manera correlativa, la disposicion de los

apendices espinosos destinados a los contactos. Para asegurarnos de la

realidad de estas acomodaciones, hemos estudiado comparativamente los

apendices espinosos de un gran numero de especies nerviosas y neuroglicas’’.15 ‘‘Semejantes diferencias parecen indicar, si, como es muy probable, que el

numero de estas relacionado con las celulas de Purkinje, es muy superior al de

las fibrillas conexionadas con los corpusculos de cesta y los de Golgi’’.

homologous nuclei has more spines, the higher the level of

the subject in the animal serie. Thus, as an example, in

vertebrates the Purkinje cell of birds shows an arborization

less spinous than that of mammals’’.16 (Cajal, 1909)

(Fig. 1F and G).

The variation of the number of spines with the phylogenetic

scale is an issue of research in recent neuroscience. These

studies are important in order to establish how the cortical

microcircuity of the brain across species is conserved, and

which is the anatomical substrate that supports the different

cognitive and mental properties across species. Because the

number of spines represents the number of excitatory inputs

sampled by individual neurons, differences in spine density

represent also differences in patterns of neuronal connectivity:

the more spinous a neuron is the more capacity to process and

integrate information of different inputs. Elston et al. have

confirmed that the density of dendritic spines was consistently

higher in human compared with macaques, and higher in

macaques compared with marmosets for different areas of the

cerebral cortex indicating that the density of dendritic spines is

correlated with the brain size (Elston et al., 2001). Benavides

et al. have confirmed that the spine density of the human

temporal cortical neurons have on average a higher (�30%)

spine density in the basal dendrite with the highest density of

spines than mouse temporal or occipital neurons (Benavides-

Piccione et al., 2002). However the pyramidal cells of the V1-

V2 area of the agouti (a large brain South American rodent

Drasypocta primnolopha) are 3–4-fold more spinous than those

of galagos, monkeys and baboons (Elston et al., 2006). These

differences in density across species and orders might be a clue

of the evolution of the brain in mammals. At least in a same

order the density appears to be higher the larger the brain is.

Across species of different orders the density of dendritic spines

in homologues areas may not be correlated with the size of the

brain.

In the case of the Purkinje cell filogenetic studies about

density in different species have not been done yet. Preliminary

studies based on the observation of the histological preparations

of Cajal of adult bird and human show that the human Purkinje

cells are more spinous than the bird ones as Cajal said (Fig. 1F

and G). However more quantitative data about spine density

within areas across species are essential for studying the

evolution and adaptation of the brain across species in different

orders.

4.3. Development

One of the most important methodological reasons for the

success of Cajal in his study of the nervous system was the

16 ‘‘Elles varient meme avec lespece animale, et nous pouvons, d’e une facon

generale, dire qu’une cellule a prolongements epineux est, pour des foyers gris

homologues, d’autant puls fournie d’epines, qu’elle appartient a un individu

puls eleve dans la serie. Ainsi, pour ne prendre quun exemple chez les vertebras,

la cellule de Purkinje des oiseaus a une ramure moins herissee que celle des

mammiferes’’.

Fig. 6. Dendritic spines and filopodia: (A) original drawing of Cajal (1933) showing filopodia and dendritic spines. According to Cajal: (A) shaft of a pyramid from

the visual region of a nearly mature rabbit; (B) pyramid of the visual region (2-month-old child); (C) shaft of a pyramid of a 1-month-old cat; (D) dendrite of a motor

cell of the spinal cord (1-month-old cat). (B–D) Photomicrographs of dendritic filopodia of the visual cortex of the cat (A, newborn; B, 9 days old; C, 12–15 days old;

E, 1-month-old).

18 ‘‘. . . mostramos en la figura 50 algunos dibujos tomados de tallos de

piramides cerebrales adultas o jovenes. En A presentamos el tallo de una

piramide de la region visual del conejo casi adulto. Notense cuan cortas son las

espinas y como empiezan delgadas y acaban por un bulbo final. Son pocas las

bifurcadas. En C copiamos un tallo de las piramides del gato de un mes.

Confırmase la disposicion mostrada en A; las espinas aparecen un poco mas

largas y con frecuencia incurvadas. En B hemos dibujado otro tallo del nino de

dos meses (piramide de la region visual). Llama la atencion, no solo la mayor

longitud de los apendices, sino su frecuencia con que se dividen y los cambios

de direccion de sus ramillas secundarias. Como termino de comparacion hemos

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130118

application of the ontogenic method. Instead of studying the

complexity of the adult brain, he decided to study the nervous

system of lower animals and embryos, or of young higher

animals. Many of the Golgi impregnated preparations of the

Cajal Museum present filopodia instead of dendritic spines

(Fig. 6B–D). Dendritic filopodia are dendritic appendages that

may be implicated in spinogenesis, synaptogenesis, and

branching. They are usually present during development or

in pathological states.

This influence of age on the size of the dendritic appendages

was also noticed by Cajal:

‘‘Almost all their branches bear thin spines perpendicularly

inserted into their surfaces. These spines are stained in a

light coffee colour, and they are bigger than the terminal

branches of adult cells’’.17 (Cajal, 1889).

In this sentence, the principle of the shortening of the neck of

the spine during development can be recognized, which may

have functional consequences for the compartmentalization of

calcium by the spine.

There is another drawing that appeared in his book

Neuronismo o Reticularismo? (Cajal, 1933) (Neuronism or

Reticularism?) (Fig. 6A), in which it is possible to recognize

spines with long necks that could be filopodia. In the text, Cajal

pointed out the difference in size of the dendritic appendages

depending on age:

‘‘I present in Figure 50 several drawings taken from the

shafts of young or adult cerebral pyramids. A represents the

shaft of a pyramid from the visual region of a nearly mature

rabbit. Note how short the spines are and how they begin

very thin and end in a terminal bulb. Very few of them are

bifurcated. In C the shaft of a pyramid of a one-month-old

17 ‘‘Casi todas sus ramas poseen ligeras espinas perpendicularmente insertas

en su contorno. Estas espinas aparecen tenidas en cafe claro, y son mas grandes

que las de las ramitas terminales de los corpusculos adultos’’.

cat is shown. Here the arrangement described in A is

confirmed; the spines appear a little bit longer and are

frequently curved. In B (two-month-old child) I have drawn

another shaft of a pyramid of the visual region. Here not only

the greater length of the appendages, but also the frequency

with which they divide and change the direction of their

secondary fibrils strike the attention. As a means of

comparison I have included the dendrite of a motor cell

of the spinal cord (D, one-month-old cat). Observe that the

surface is bristling with irregular projections that very

seldomly end in bulbs. It is almost certain that this

arrangement is transitory. Finally, I have drawn several

cruciform or oblique nerve collaterals of the cerebrum. It is

impossible to see any fusion of the latter with the spines’’.18

(Cajal, 1933).

It is possible to observe filopodia in the histological

preparations of Cajal (Fig. 6A). In the case of dendrite D of the

drawing, the appendages are surely filopodia: most of them do

not have a bulbous head and are highly ramified. Cajal thought

that these structures were transient.

In addition to these typical drawings, there are other

drawings (Fig. 7) of the histogenesis of the cerebellum

dibujado en D una dendrita de una celula motriz de la medula (gato de un mes).

Adviertase que la superficie esta erizada de proyecciones irregulares y rara vez

acabadas mediante bulbos. Es casi seguro que esta disposicion es transitoria.

En fin, dibujamos tambien algunas colaterales nerviosas cruciales u oblicuas

(cerebro), sin que sea dable apreciar su fusion con las espinas’’.

Fig. 7. Dendritic filopodia and development in the cerebellum: original

drawing of Cajal (1904) of Purkinje cells at a very embryonic stage from

a newborn puppy. Golgi method. (A) superficial granules; (B) molecular

layer; (C) deep granules; (D) white matter; (a) Purkinje cell; (b) axon

collaterals of this cell; (g) embryonic granule. The Purkinje cells and granule

cells present multiple branches that could evolve from filopodia. In addition

around the soma and near the axon of the Purkinje cells there are other

appendages that we have confirmed like filopodia on the Cajal’s original

slides.

20 ‘‘Que por virtud de las susodichas espinas, la ramificacion protoplasmica

aumenta su superficie colectora y se establecen contactos mas ıntimos entre

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130 119

where it is possible to see some thin appendages that

may be filopodia transforming into branches. About the

development of the granule cells of the cerebellum, Cajal

comments:

‘‘Fine dendrites emerge now from the soma, which becomes

more and more roundish. These dendrites are initially long,

numerous, and poorly branched; finally some of these

appendages reabsorb, others become more regular and

produce the minute and specific arborizations at the tips,

characteristic of a fully developed granule’’.19 (Cajal,

1899a,b).

4.4. Physiological role of the dendritic spines

Ramon y Cajal made some hypothesis about the physio-

logical role of the dendritic spines. The clearest role attributed

by Cajal to dendritic spines was to increase the connective

surface:

‘‘. . . that by virtue of the aforesaid spines, dendritic branches

increase their receptive surface and establish more intimate

19 ‘‘del soma, cada vez mas espeso y redondeado, brotan ahora finas den-

dritas, las cuales son primitivamente largas, numerosas y apenas ramificadas; y

en fin, ulteriormente, algunos de tales apendices se absorben, otros se reg-

ularizan, produciendose en los extremos la diminuta y especıfica arborizacion

del grano perfecto’’.

contacts with the axonal terminal arborizations’’.20 (Cajal,

1899a,b).

However, three-dimensional electron microscopic recon-

structions made by Harris and Stevens (1988) contradict this

view. By graphically removing all spines from their computer

reconstructions of spiny dendrites, they estimated that only 29–

45% of the dendritic membrane area of Purkinje cells would

have been covered by synapses if all spines were deleted and the

associated synapses moved to the dendrites. Applying the same

technique to five reconstructed CA1 pyramidal cell dendrites,

they found that only about 5–9% of the remaining dendritic

surface area would have been covered by the synapses from the

spines. Thus, it does not appear that dendritic spines are required

for lack of dendritic membrane area (Koch and Zador, 1993).

On the other hand, the idea of Cajal that dendritic spines

increase the connective surface allowing connections to axons

that are far way from the dendrite is very interesting:

‘‘In my feelings, the dendritic spines have the principal

function of increasing the surface of connection of the

protoplasmic arborization, going out to contact the nerve

fibres that are distant and cannot contact directly with the

outline of the dendritic process’’.21 (Cajal, 1896).

The dendritic spines may permit an axon to synapse with

dendrites of different neurons without a zig-zag trajectory, and

allow it to course through the neuropil in a relatively straight

path (Peters and Kaiserman-Abramof, 1969). It has been

corroborated that longer spines are occasionally found when

target axons are farther away into adjacent axon bundles in the

reticular nucleus of the thalamus and in the gelatinous

substance of the spinal dorsal horn (Fiala et al., 2002).

In his first papers Cajal thought about dendritic spines as

structures for canalizing the nerve fibers, which would press

down on the open spaces between spines:

‘‘The gulfs between such collateral spines receive impres-

sion from innumerable small fibers of the superficial layer.

Exactly the same disposition is possessed by the terminal

peripheral arborization of the large pyramids’’.22 (Cajal,

1890c).

But there are other statements of Cajal extracted from

‘‘Nuevo concepto de la histologıa de los centros nerviosos’’ that

prove that Cajal saw the contacts between the nerve fibres and

the dendritic spines and thought they might be implicated in

transmission:

aquella y las arborizaciones nerviosas terminales’’.21 ‘‘En mi sentir, las espinas tienen por principal oficio aumentar las super-

ficies de conexion de la arborizacion protoplasmica, saliendo al encuentro de

las fibras nerviosas que, por hallarse a cierta distancia, no pueden establecer

contacto directo con el contorno de las prolongaciones dendrıticas’’.22 ‘‘Los golfos que median entre tales espinas colaterales reciben la impresion

de las innumerables fibrillas de la zona superficial. Exactamente igual dis-

posicion posee la arborizacion periferica final de las grandes piramides.’’

Fig. 8. Similar drawings of (A) Berkley (1896) and (B) Cajal (1933) showing

connections of axons with dendritic spines.

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130120

‘‘. . . The existence of collateral spines on the dendritic

process; and the connection of all [the spines],23 at the level

of the molecular layer, with a tight plexus of little terminal

nerve fibers’’24 (Cajal, 1892, 1894).

Later on in the Textura (Cajal, 1899a,b), Cajal accepted the

function of connection of dendritic spines proposed by Berkley,

because it reconciles well with his hypothesis exposed in other

works:

‘‘Do they represent the lines of charges or absorption of

nerve impulses as stated by Berkley? The latter opinion

appears plausible to us. It reconciles well with our idea

expressed in another publication, namely that by virtue of

the aforesaid spines, dendritic branches increase their

receptive surface and establish more intimate contacts with

the axon terminal arborizations’’.25 (Cajal, 1899a,b).

As Cajal said, Berkley clearly defines dendritic spines as

receptors of currents:

‘‘The function of the gemmule is in all likelihood to receive

nerve impulses from the endings of the numerous terminal

nerve fibers that seem almost to touch them, and carry these

impressions to the dendrite and by its medium on the cell

body. Differences in the function of the gemmule of the

pyramidal and Purkinje cell are probable’’ (Berkley, 1895)

(Fig. 8A).

In his final work Neuronismo o Reticularismo? Cajal did not

clearly point out the possibility of dendritic spines as receptors

of nerve impulses, and he only stated that nerve fibers of the

cerebral cortex rest on the spines.

‘‘Note how these collaterals cross and enter into transversal

or oblique contact with a great number of the dendritic

shafts. It is probable that collaterals rest on the spines wich

cover the protoplasmic surface like down’’.26 (Cajal, 1933)

(Fig. 8B).

We should take into account the contribution of Berkley

clarifying the role of dendritic spines as nervous current

23 ‘‘These words (the spines) do not appear in the spanish edition ‘‘Nuevo

concepto . . .’’, but appear in the french version Les Nouvelles Idees sur la

Structure du Systeme Nerveux chez l’Homme et chez les Vertebres: ‘‘. . .

l’existence d’epines collaterales sur les branches protoplasmiques et la con-

nexion de toutes ces epines au niveau de la zona moleculaire, avec un plexus

serre de fibrilles nerveuses terminales’’ (Cajal, 1894) (see also De Felipe and

Jones, 1988).24 ‘‘La existencia de espinas colaterales en las ramas protoplasmicas, y la

conexion de todas ellas, al nivel de la zona molecular, con un plexo tupido de

fibrillas nerviosas terminales’’.25 ‘‘Representan lıneas de carga o de absorcion de corrientes nerviosas, como

declara Berkley? Plausible nos parece esta ultima opinion, que por otra parte,

se concilia bien con la idea expuesta por nosotros en otro trabajo, a saber: que

por virtud de las susodichas espinas, la ramificacion protoplasmica aumenta su

superficie colectora y se establecen contactos mas intimos entre aquella y las

arborizaciones nerviosas terminales’’.26 ‘‘Notese como dichas colaterales cruzan y entran en contacto transversal y

oblicuo con gran numero de tallos de dendritas, apoyandose quizas en las

espinas que revisten, como un vello, las superficies protoplasmicas.’’

receptors and it was his merit to localize the reception of the

nervous current on the bulbous head of the spines:

‘‘. . . these spherical apparatus (terminal buttons) are closely

adjusted against the bulbous tips of the gemmules, at times

the application being so close as to give the impression of

actual contact . . . the axonal discharges of stimuli overleap

the infinitesimal distance between bulb and gemmule . . .’’(Berkley, 1897)

Cajal did not specify where these contacts were made (head

or neck). Today it is known that the majority of excitatory

synapses are made on the head of the spine, although sometimes

there are synapses on the neck of the head that usually are

inhibitory (Jones and Powell, 1969). In contrast to Berkley,

Cajal’s statements suggest that the contacts could be made also

on dendritic shafts. Today it is known that the majority of

excitatory connections in the cerebral cortex are made on the

dendritic spines (Peters and Kaiserman-Abramof, 1969, 1970),

although excitatory inputs can be made also on dendritic shafts

(Hersch and White, 1981; White and Hersch, 1981), especially

on Layer VI pyramidal cells (Mc Guire et al., 1984). In addition

Berkley thought that nervous conduction was only longitudinal

and occurred between the bulbous terminations of the nerve

fibers that he had described with the silver phosphomolibdate

method and the bulbous heads of the gemmule of the pyramidal

cells (Berkley, 1896). Cajal did not agree with the hypothesis of

dendritic spines as the only place for receiving nerve impulses

because it could not explain the connections, for instance,

between the pericellular axon arborization and the soma or the

origin of axons of the Purkinje cells that lack dendritic spines,

or between the climbing fibers and the primary and secondary

branches of the Purkinje cell. Cajal thought that in these

articulations there might be a horizontal transmission between

the nerve fibers and the soma (Cajal, 1899a,b). Afterwards,

Fig. 9. (A) Original drawing of Cajal (1904) of a section of the olfactory

bulb of a several days old kitten, (I, J, granule cells). (B) Olfactory bulb of 1-

month-old mouse (granule cell and mitral cell). The insert box does not

correspond to this preparation. It corresponds to a granule cell of newborn

dog.

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130 121

apart from these end-bulbous terminals described by Berkley,

en passant varicosities were also described (Colonnier, 1968).

Stefanowska, another contemporary scientist of Cajal,

proposed the idea that what Stefanowska called the pyriform

appendages could modify their form and dimension during life,

loosening their contacts with nerve fibers to a variable degree

and explaining the sleep state by the disappearance of the

pyriform appendages and the interruption of the transmission

(Stefanowska, 1897a,b). This hypothesis was very much

influenced by the theory of neuronal ameboidism of Duval

(Duval, 1895), based on Rabl-Ruckhard’s previous studies

(Rabl-Ruckhard, 1890). This hypothetical retraction of the

spines is the first proposal that the dendritic spines are motile

until the theoretical work of Crick (1982) and the final

definitive observation made by Fischer et al. (1998).

The function and physiological role of dendritic spines

continues being a subject of research. The definitive

demonstration of dendritic spines as postsynaptic units came

from the electron microscopic studies of Gray (1959a,b). Many

other functions have been proposed because excitatory inputs

can be made on dendritic shafts without dendritic spines

(Hersch and White, 1981; White and Hersch, 1981). A great list

of functions have been attributed to these important structures:

synaptic plasticity, biochemical compartmentalization (Ca2+

and other metabolites), neuroprotection, increasing of the

dendritic membrane capacitance, intersynaptic isolation,

attenuation of passive synaptic potential, linear summation

of EPSPs, generation of rapid local EPSPs (for a review see

Shepherd, 1996).

A special case of dendritic spines are those of the olfactory

bulb granule cells where dendritic spines also behave as

presynaptic elements. The dendritic spines of the olfactory

granule cells establish reciprocal synapses with the dendrites of

the mitral cells (Rall et al., 1966; Rall and Shepherd, 1968;

Prince and Powell, 1970; Shepherd and Brayton, 1979). These

spines (gemmules) receive type I excitatory synapses from

mitral cells and have reciprocal type 2 inhibitory synapses onto

those same dendrites. They are considered as the smallest

neuronal compartment capable of performing a complete

input–output operation of a single synapse (Shepherd and

Greer, 1988). It should come as no surprise that this also began

with Cajal. Cajal described the dendritic spines of the olfactory

bulb granule cells in 1890 (Cajal, 1890a). Later on, in the

Textura (1899) Cajal confirms the absence of axon in granule

cells as was first described by Golgi (1875). Describing the

dendrite of the granule cells Cajal wrote:

‘‘. . . the peripheral process has a constant orientation and

connection . . . it terminates in a tuft of very spiny branches

in contact with the secondary dendrites of mitral cells.’’27

(Cajal, 1899a,b) (Fig. 9).

27 ‘‘la expansion periferica de los granos posee una orientacion y conexion

invariables. . ..se termina a favor de un penacho de ramas fuertemente espi-

nosas en contacto con las dendritas secundarias nacidas de las celulas

mitrales’’.

In explaining the current flow in these circuits, he explained:

‘‘The peripheral process would represent, if not morpho-

logically at least dynamically, a functional process since

nerve impulses circulate through it in the cellulifugal sense,

as they do in true axons’’.28

Reciprocal synapses between granule cell spines and mitral

cell dendrites are implicated in mediating feedback and lateral

inhibition of the mitral and tufted cells (Rall et al., 1966; Rall

and Shepherd, 1968). The lateral inhibition through the granule

cell spines enhances the tuning specificity of odor responses

contributing to discrimination of olfactory information (Yokoi

et al., 1995).

4.5. Dendritic spines in pathological and poisoning states

After the verification of dendritic spines as real structures,

many scientists investigated their morphology in different

pathological states. The most typical alteration of dendritic

spines is the monoliforme state or varicose degeneration. It

consists in the formation of swellings along the dendrites and

the absorption of the spines (Fig. 3). Surprisingly, Golgi was the

first to describe varicose degeneration in a case of Chorea

(Golgi, 1877).

This alteration was described in general paralysis (Colella,

1892), melancholia (Azoulay and Klippel, 1894), epileptic

dementia (Colella, 1892), septicemia, haemorrhage (Tirelli,

1895), alcoholism, and serum and ricin poisoning (Berkley,

1895) (Fig. 10C); exposure to chloroform, ether, poison gas,

and electrocution were also found to lead to these alterations

28 ‘‘El apendice periferico representarıa, si no morfologica, dinamicamente,

una expansion funcional, puesto que la corriente nerviosa circula en el en

sentido celulıfugo, como en los axones legıtimos’’.

Fig. 10. (A) Drawing of Monti (1895b) showing varicose degeneration of a pyramidal cell of the dog. The degenerated processes are those directed towards the blood

vessels. Axons never present this varicose degeneration. (B) Drawing of Stefanowska (1897a) showing varicose degeneration because of the action of ether. (C)

Drawing of Berkley (1895) of a Purkinje arborization of a control cell (left) and a cell subject to chronic alcohol poisoning (right). (D) Camera lucida drawings (A, B:

Marın-Padilla, 1972; C, D: Purpura, 1974) of apical dendrites of pyramidal cells from human cerebral cortex. (A) apical dendrite of a newborn girl with D1(13–15)

trysomy (Patau syndrome); (B) apical dendrite of an 18-month-old mentally retarded with 21 trysomy (Down syndrome); (C) a dendrite from normal 6-month-old

infant with no history of neurological disorder with a large number of normal spines; (D) analogous dendrite from a retarded 10-month-old child, showing long,

tortuous spines; (E) schematic of different spine abnormalities in different pathologies (Fiala et al., 2002).

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130122

(Stefanowska, 1897a) (Fig. 10B). Very interesting are the works

of Monti (Fig. 10A), describing varicose degeneration in

embolism and starvation (Monti, 1895a,b). He used these data

to support the hypothesis of Golgi about the nourishing function

of dendrites and the conductive role of the axons, opposing the

theories of Cajal.

For Cajal, varicose degeneration represented a post-

mortem alteration that occurs because the tissue pieces are

fixed too late. This disorganization of the dendrites is

eliminated if the tissue is fixed rapidly, but it is more difficult

to avoid in the Ehrlich staining because it needs the

participation of air in the staining process (Fig. 3). Another

important piece of data is that this degeneration preferen-

tially occurs in thick pieces of nervous tissue in the deep

layers, the areas that are stained later (Cajal, 1899b). Cajal

did not deny the possibility of these alterations under

pathological states, although he was very cautious about it

because he discovered dendritic spines in animals anesthe-

tized by chloroform.

The swelling and loss of spines is nowadays accepted to

occur in epilepsy, hypoxia/ischemia, traumatic injury, edema,

and acute excitotoxicity (for a review, see Fiala et al., 2002)

(Fig. 10E). In addition, loss of spines has also been described in

poisoning, alcohol abuse, and epilepsy. It was difficult to decide

if this varicose atrophy was real or a degenerative post-mortem

process, not related with the disease.

5. The dendritic spines from Cajal to present-day

neuroscience

5.1. The concept of Sherrington’s synapse

It was Sherrington who introduced a concept that would

change neuroscience for years to come, the concept of synapsis.

‘‘So far as our present knowledge goes, we are led to think

that the tip of a twig of the arborescence is not continuous

with but merely in contact with the substance of the dendrite

or cell body on which it impinges. Such a special connection

of one nerve cell with another might be called a synapse’’

(Foster and SherrinGton, 1897; see also Shepherd, 1997).

This proposal united neuroanatomical and physiological

evidence into a single term. Cajal accepted this term and he

used it in his last book Neuronismo o Reticularismo? (Cajal,

1933).

The definitive identification of the synapse was done using

electron microscopy (Palay, 1956). In 1959, Gray identified the

dendritic spines as postsynaptic structures. He also classified

synaptic contacts into type I (made on dendritic trunks and

dendritic spines) and type II (made on dendritic trunks and

soma). The type I synaptic contacts have a thicker postsynaptic

density. These types of synapses correspond to the asymmetric

and symmetric types of Colonnier (1968). The importance of

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130 123

this classification resides in its functional implications:

asymmetric spines are excitatory and glutamatergic, while

symmetric synapses are inhibitory and gabaergic.

5.2. Morphology of dendritic spines

The observation of dendritic spines by electron microscopy

showed that they contain flocculent material and endoplasmic

reticulum (ER). When the ER cisternae are located in groups

longitudinally oriented away from each other, they form the so-

called spine apparatus first described by Gray (1959a,b); the

spine apparatus has many important functions like Ca2+

regulation. In addition some other elements may be present:

sparse Golgi elements (Horton et al., 2005), mitochondria

(Adams and Jones, 1982), endosomes, (Cooney et al., 2002)

proteosome (Ehlers, 2003), and filaments of actin (Fifkova and

Delay, 1982).

After Gray, ultrastructural studies were continued by Jones

and Powell (1969) and by Peters and Kaiserman-Abramof

(1969), who classified the different shapes of the dendritic

spines into thin (more abundant), sessile, and mushroom spines

(Fig. 2F). This classification is still useful for investigating the

structure-function relationships of dendritic spines, although it

has been hypothesized that there may be a continuum of shapes.

In addition, it is difficult to assign some spines to any of these

groups because they have intermediate shapes. Indeed, there

appears to be a clear relationship between the morphology and

the function of the spine, particularly with relation to the size of

the spine head and the length of the neck. For instance, the

volume of the head is directly proportional to the size of the

postsynaptic density (Freire, 1978), to the number of

postsynaptic receptors, to the size of the presynaptic terminal

(Spacek and Hartmann, 1983; Peters, 1987), to the presynaptic

number of docked synaptic vesicles, and to the readily

releasable pool of neurotransmitters (Spacek and Hartmann,

1983; Harris and Stevens, 1988; Nusser et al., 1998; Schikorski

and Stevens, 2001). Recent studies have shown that the number

of AMPA receptors in the postsynaptic density is directly

correlated with the size of the head (Matsuzaki et al., 2001;

Kasai et al., 2003), and that this influences the capacity of being

potentiated by long-term potentiation (LTP).

Important data regarding the structure of the spine and its

function have come from living image studies of the cerebral

cortex of mice (Trachtenberg et al., 2002; Grutzendler et al.,

2002; Matsuzaki et al., 2001, 2004; Kasai et al., 2003). These

studies have shown that big dendritic spines are more stable

than small ones, and that they can persist for months. These

data, combined with the data on AMPA receptor density and

LTP research, have led Kasai to propose the hypothesis of

learning and memory spines. Big spines are stable and

represent the physical trace of long-term memory by forming

stable synaptic connections (memory spines), while thin

spines are motile and instable and form weak connections

implied in learning. It has also been shown that the number of

memory spines or big stable spines is higher on the dendrites

of old animals than on those of young animals (Holtmaat

et al., 2005).

Another important issue is the length and width of the neck.

It has been postulated that the neck of the spine could reduce

the weight of the synapse (Chang, 1952): ‘‘If the end bulbs of

the gemmule (spines) are the receptive apparatus for the

presynaptic impulses, the process of postsynaptic excitation

initiated there must be greatly attenuated during its passage

through the items of the gemmules which probably offers

considerable ohmic resistance because of their extreme

slenderness’’. Especially interesting are the theoretical works

based on computational models made by Rall and Rinzell (Rall,

1964; Rall and Rinzel, 1971a,b; Rall, 1974). They introduced

the idea that the spine might be a site for neuronal plasticity.

‘‘. . . fine adjustments of the stem resistances of many spines . . .could provide an organism with a way to adjust the relative

weights of the many synaptic inputs . . .; this could contribute to

plasticity and learning of a nervous system’’ (Rall and Rinzel,

1971b). Crick also thought that the contraction and extension of

the neck could change the weight of the synapses (Crick, 1982).

However, this role of the neck in the reduction of the weight

of the synapses was questioned by Koch and Zador (1993), who

postulated that the spine neck conductance appears to be too

large relative to the synaptic conductance change to provide

effective modulation of the amplitude of the synaptic current

generated at the spine head. Instead they proposed that spines

create an isolated biochemical microenvironment around the

synapse, so that the spines function as biochemical compart-

ments. Because of its narrowness, the neck can be considered a

barrier for the diffusion of metabolites isolating the dendritic

spines biochemically from the dendrite (Svoboda et al., 1996;

Majewska et al., 2000a; Nimchinsky et al., 2002; Segal, 2005).

One of the more important metabolites in glutamatergic neuronal

transmission is Ca2+ that can act as a second messenger in

different signaling pathways implicated in plasticity functions

such as memory and learning (Yuste and Denk, 1995; Segal,

1995; Korkotian and Segal, 1999; Majewska et al., 2000a,b;

Yuste et al., 1999; Yuste and Holthoff, 2000; reviewed in Yuste

and Bonhoeffer, 2001; Yuste and Bonhoeffer, 2004; Hayashi and

Majewska, 2005; Segal, 2005; Konur and Ghosh, 2005; Oertner

and Matus, 2005; Ethell and Pasquale, 2005). In addition, the

concentration of Ca2+ in the spines can be considered to be under

the control of different buffer systems (Calmodulin; spine

apparatus). It has been observed that spine apparatuses are

present preferentially in the base of the neck of mushroom spines

while they are absent from many of the thin spines, possibly

regulating in this manner the influx of calcium into the spines.

5.3. Dendritic spines and pathology

Data about the possible functions of dendritic spines have

also come from studies of pathological states of the brain. In

these pathological states, as was first proposed by contemporary

scientists of Cajal, there is a significant loss of dendritic spines.

Changes of dendritic spines in pathological states have been

described in many alterations such as malnutrition, brain

edema, traumatic lesions or tumors, alcoholism, drug con-

sumption, poisoning, epilepsy, transmissible diseases (HIV,

Creutzfeldt-Jakob, Kuru, etc.), dementias (Alzheimer’s, Pick’s,

29 ‘‘El notable acrecentamiento intelectual que se observa en los hombres

consagrados a un ejercicio mental profundo y continuado; y la coexistencia de

un talento notable y aun del verdadero genio con cerebros de tamano medio o

inferiores a la dimension y pesos normales. En el primer caso, podrıa suponerse

que la gimnasia cerebral, ya que no puede producir celulas nuevas (las celulas

nerviosas no se multiplican como las musculares) lleva un poco mas alla de lo

corriente el desenvolvimiento de las expansiones protoplasmaticas y colater-

ales nerviosas, forzando el establecimiento de nuevas y mas extensas conex-

iones intercorticales [. . .].’’

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130124

Huntington’s and Parkinson’s diseases), and mental pathologies

(schizophrenia and major depression, etc.) (for a review, see

Fiala et al., 2002). We will focus on only two topics: mental

retardation and hypoxia.

Marın Padilla used the Golgi method to study the motor

cortex of a newborn girl with D1 (13–15) trisomy (Patau

syndrome) and the motor cortex of an 18-month-old girl with

21 trisomy (mongolism or Down syndrome) (Marın-Padilla,

1972) (Fig. 10D). He described loss and distortion of dendritic

spines in the Patau syndrome. Dendritic spines were irregular,

long, and tortuous and appeared disoriented with swellings

along their surfaces that could make immature synaptic

contacts. Thus, they are very similar to immature appendages.

He proposed two explanations for this immaturity: appendages

fail to convert to dendritic spines, or there are too few axons to

mature the appendages (Marın-Padilla, 1974). In Down

syndrome, Marın-Padilla (1976) found three different patterns

of spine pathology in pyramidal neurons of the motor cortex, in

addition to a few essentially normal neurons. Some neurons

were densely covered with long, tortuous spines. Other neurons

were uniformly covered with short, thin spines that were

abnormally small in volume. A third type of neuron exhibited

significant spine loss, with the few remaining spines having

very large heads and thin necks. A pattern of small spines in

neonates and abnormally long spines in older infants has been

observed in more extensive studies of Down’s syndrome

(Takashima et al., 1994). Purpura observed a reduction of the

number of spines and the presence of spines with very long

necks and big heads in children with a normal karyotype but

with deep mental deficiency of unknown etiology (Purpura,

1974) (Fig. 10D). Unusually long, tortuous spines occur in

many other forms of mental retardation, including Fragile X-

Syndrome (Rudelli et al., 1985; Segal et al., 2003), Maple

Syrup Urine Disease (Kamei et al., 1992), and fetal alcohol

syndrome (Ferrer et al., 1987).

The similarity of spine pathologies in different conditions

associated with mental retardation is striking, and led to the

recent suggestion that the different genetic deficits associated

with mental retardation disrupt a common signaling pathway

related to the development of the dendritic cytoskeleton

(Martone et al., 2000).

Hypoxia or ischemia leads to the formation of dendritic

varicosities, with a corresponding loss of spines. This damage is

principally mediated by the high release into the extracellular

space of glutamate, which permanently activates NMDA

receptors causing a massive entrance of Ca2+ into the

intracellular space. However, this phenomenon may be

reversible. Amazingly, spines absorbed in this manner can

recover their original shape after termination of the insult and

elimination of dendritic swelling, suggesting that the synapses

are not lost during this process; this may involve a mechanism

where dendritic spines are not formed from pre-existing

filopodia, but from pre-existing synapses contacted by axons

(Hasbani et al., 2001). However, ischemic lesions due to stroke

in the mature CNS can produce more permanent spine loss (De

Ruiter and Uylings, 1987). As we have seen before, Cajal noted

that it is possible to observe the swelling of dendrites and

resorption of spines during the first half-hour after exitus (Cajal,

1899a,b). Biopsy material, fixed quickly, is surely much better

preserved. In any case, the time interval between withdrawal

and fixation of post-mortem brain tissue is very important to

avoid artifactual swelling.

5.4. Plasticity of dendritic spines

Other interesting lines of research have come from

experiments on plasticity associated with changes in spine

density, initiated by Demoor (1896) and Stefanowska (1897a,b)

and recuperated later by Globus and Scheibel (1967): they

found a loss of dendritic spines in the apical shafts of the

neurons after eye enucleation and lateral geniculate lesions.

Valverde (1967, 1968, 1971) observed using the Golgi method

that light-deprived mice had a reduced number of spines in the

visual cortex that was higher in young animals than in older

animals, although the exponential increase of the dendritic

spines in normal animals was maintained in the light-deprived

animals. Moreover, these changes were completely reversible

in some neurons while other neurons remained damaged

(Valverde, 1971). A possible explanation came from the

realization that there are populations of spines with dimensions

too small to be seen with the light microscope, and that can be

seen only with electron microscopy (Freire, 1978). The

formation and maintenance of dendritic spines depends on

synaptic activity, and these changes may be modulated by

sensory experience (Valverde, 1967, 1968, 1971; Globus and

Scheibel, 1967; reviewed by Lippman and Dunaevsky, 2005;

Matus, 2005; Segal, 2005). Moreover, it has been shown that

early exposure to a rich foreground produces an increase in

dendritic arborization and the number of dendritic spines

(Rosenzweig et al., 1972; Volkmar and Greenough, 1972;

Globus et al., 1973; Greenough et al., 1973).

These conclusions clearly bring to mind Cajal’s concept of

plasticity and mental exercise:

‘‘The remarkable intellectual increase that is observed in

men dedicated to deep and continuous mental exercise; and

the coexistence of a remarkable talent or even of true genius

with medium-sized brains or brains of smaller than normal

dimensions and weights. In the first case, since the brain

cannot produce new cells (nervous cells do not multiply like

muscle cells do), it can be assumed that brain gymnastics

take the unfolding of the protoplasmatic expansions and the

nervous collaterals a little beyond what is common, forcing

the establishment of new and more extensive intercortical

connections [. . .].’’29 (Cajal, 1892).

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130 125

The most important data about the plasticity of these

structures and its possible implication in functions as learning

and memory come from studies on LTP, inspired in the works of

Rall and Rinzel (Rall, 1974; Rall and Rinzel, 1971a,b). During

LTP there is an increase in the volume of the spine head (Van

Harrefeld and Fifkova, 1975; Fifkova and Van Harreveld, 1977)

and a variation of the neck length (Fifkova and Anderson,

1981). Engert and Bonhoeffer (1999) combining a local

superfusion technique with two photon imaging have shown

that new spines can appear on the postsynaptic dendrite after

LTP. In adition during LTP, the number of AMPA type

glutamate receptors at the spine head membrane increases due

to enhanced transport from recycling endosomes (Park et al.,

2004), acting as a mechanism for the potentiation of

transmission. In recent elegant experiments, Park et al.

(2006), using a combination of serial electron microscopy

and live cell fluorescence microscopy, have confirmed that

these recycling endosomes also provide membrane for activity

dependent spine growth and remodeling. Korkotian and Segal

(2007) have shown that chemical induction of LTP increases the

concentration of GluR1 in the head of the spines, preferently

those with short neck, which is F-actin and protein synthesis

dependent. In addition to the intracellular routes, glutamate

receptors can be transported to the spine head by lateral

diffusion along the membrane (Triller and Choquet, 2005;

Ashby et al., 2006). The molecular mechanisms that regulate

the vesicular trafficking to the spine membrane are unknown.

Endosomal transport and vesicular fusion are Ca2+ sensitive

(Khvotchev et al., 2003). CamKII another central intracellular

signal for LTP may regulate other effectors of endosomal traffic

(Karcher et al., 2001). Another signaling molecule for LTP,

cAMP dependent protein kinase (PKA) regulates endosomal

traffic and activity-induced insertion of AMPA receptors in the

spines membrane (Ehlers, 2000). Future research should focus

on the molecular mechanisms on endosomes and endosmal

traffic effectors that sense and respond to NMDA receptor-

mediated Ca2+ influx during LTP.

Another proof of the plasticity of dendritic spines is the fact

that they move, as first demonstrated by Fischer et al. (1998),

using cultures of neurons expressing GFP. Motility of spines

was first proposed by Demoor (1896) and Stefanowska

(1897a,b). In 1982, Crick hypothesized about possible move-

ments of the spines, and its implication for memory in an article

entitled: ‘‘Do spines really twitch?’’ He wondered: ‘‘Suppose

actin was discovered in dendritic spines?’’ This discovery was

in fact made 5 years earlier by Blomberg et al. (1977), who

identified actin in the postsynaptic density fraction isolated

from dog cerebral cortex. The definitive observation was made

by Fifkova and Delay (1982). Recent advances using two

photon microscopy have shown that the movement of spines

also ocurs in brain slices (Dunaevsky et al., 1999) and in intact

brains of anesthetized animals (Lendvai et al., 2000).

The actin cytoeskeleton is essential for the movement of the

spine, its shape and for synaptic plasticity (Matus et al., 2000).

Okamoto et al. (2004) have demonstrated that LTP induces

remodelation of spine actin cytoeskeleton. Tethanic stimulation

causes a rapid, persistent shift of actin equilibrium toward

filamentous actin (F-actin) in the dendritic spines of rat

hippocampus. This enlarges the spines and increases post-

synaptic binding capacity. In contrast, prolonged low frequency

stimulation shifts the equilibrium towards globular actin (G-

actin) resulting in a loss of postsynaptic actin and of structure

(Okamoto et al., 2004). These experiments indicate that F-actin

acts as a support for a larger spine. However, for the increase of

volume observed in LTP apart from the actin cytoeskeleton new

sources of membrane are required. This new membrane could

be provided by the recycling endosomes (Park et al., 2006).

Furthermore, the actin cytoeskeleton could be an ideal

mechanism for the traffic of the postsynaptic proteins and

lipids to their targets. In support of this hypothesis is the

abundant existence of actin-based myosin motors and myosin

regulatory proteins in the dendritic spines, (Osterweil et al.,

2005; Ryu et al., 2006).

The assembly-disassembly of actin filament is regulated by

actin-binding proteins (Arp2/3 complex, Cortactin, ADF/

Cofilin, Drebrin, Profilin II, Gelsolin, Spinophilin, etc.) (for

a review see Ethell and Pasquale, 2005; Tada and Sheng, 2006).

Many signaling pathways that regulate dendritic spine shape

and motility originate at the cell surface and converge on these

actin regulatory proteins like for instance the Rho/Rac

GTPases, Ras GTPases/MAPKinase, Ca2+ signaling (Ethell

and Pasquale, 2005; Tada and Sheng, 2006)

The process of molecular plasticity on dendritic spines is

very complex and dependent of many factors. Furthermore,

local protein syntheis can also contribute to the synaptic

plasticity. Poliribosomes redistribute from dendritic shafts into

spines during LTP (Ostroff et al., 2002; Bourne et al., 2007). In

contrast to the changes in the postsynaptic density, presynaptic

mechanisms (e.g. increase transmitter release) could mediate

the initial phase of potentiation in LTP.

5.5. Dendritic spines and development

As we have seen in the Cajal’s drawing (Fig. 6A), dendritic

appendages often curve and divide. This characteristic is easier

to see during development, when there is more space in the

neuropil for the movement of the appendages. The motility of

the spines decreases in adulthood. In contrast, filopodia are

highly motile structures that appear and disappear in minutes

(Dailey and Smith, 1996), and that are present during

development or in pathological states.

According to Dailey and Smith (1996) the filopodia lack a

differentiated head. In contrast, Fiala et al. (2002) propose that

filopodia can exhibit a bulbous head, where a synapse is

probably localized. Such a filopodium is still distinguishable

from a spine because it is longer than 2 mm and it is filled with a

dense matrix of actin (Fiala et al., 2002). In addition, filopodia

frequently ramify and curve (Morest, 1969; Fiala et al., 1998;

Dailey and Smith, 1996), like the appendages of Cajal’s

drawing (Fig. 6A).

Analyzing other Cajal drawings of developing cerebellum

(Fig. 7A and B), the two principal roles attributed to

filopodia can be seen (for a review, see Yuste and Bonhoeffer,

2004):

Fig. 11. Different models for spinogenesis: (A) spines emerge independently of the afferent axon; (B) first synapses are formed on the dendritic shaft induced by the

axon terminal. At the beginning most of the spines are ‘‘stubbies’’. In the last stage many spines are mushroom-shaped or lollipop-shaped. (C) A dendritic filopodium

captures an axonal terminal and becomes a spine. The diagram has been created with fragments of 3D-reconstructions of neurons from Cajal’s histological

preparations. The design has been made following the diagram of Yuste and Bonhoeffer (2004).

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130126

- s

pinogenesis (Dailey and Smith, 1996) and synaptogenesis

(Harris et al., 1992);

- d

30 ‘‘Por donde se ve que el sinnumero de expansiones y conexiones inter-

celulares ofrecidas por el sistema nervioso adulto, cabe concebirse como

expression morfologica de los innumerables caminos trazados en el espacio

y durante todo el perıodo evolutivo, por las corrientes de las materias reclamo.

La arborizacion entera de una neurona representa, pues, la historia grafica de

los conflictos sufridos durante la vida embrionaria’’.

endritic tree branching (Morest, 1969).

Two different populations of filopodia probably exist

(Portera-Caillau et al., 2003): those of the tip of the dendritic

branch, implicated in branching and modeling of the

dendritic tree; and those distributed along the dendritic

branch, implicated in synaptogenesis (Yuste and Bonhoeffer,

2004).

Although the work of Dailey and Smith (1996) suggests a

role of dendritic filopodia in spinogenesis, it is still not clear

how dendritic spines are formed. Three models have been

proposed: The Sotelo model (1978), the Miller/Peters model

(1981), and the filopodia model (Yuste and Bonhoeffer, 2004)

(Fig. 11).

Konur and Yuste (2004) have confirmed that the presynaptic

terminal is also motile and that the filopodia of the dendrite can

make transient interactions with them indicating that the

filopodia are implicated in sampling the surrounding neuropil

looking for a presynaptic terminal. In contrast, spines and hand-

like spines are less motile and their movement (head morphing)

may be implicated in synaptic competition between two

presynaptic terminals.

As we have seen, the genius of Cajal was not only to describe

the structures he was observing, but also to correctly interpret

them. Cajal also applied his neurotropic hypothesis to

development, especially that of the cerebellum, but moreover

he pointed out the importance of nervous activity in the

maturation of the dendritic tree (Cajal, 1899a,b). In the ideas of

Cajal the basis for the synaptotropic theory of Vaughn (1989)

can be found in the words of Cajal:

‘‘It appears, therefore, that the innumerable processes and

intercellular connections offered by the adult nervous

system can be interpreted as the morphologic expression

of the innumerable routes drawn up in the space by currents

of inducting or positive chemotropic substances during the

entire developmental period. Thus, the total arborization of a

neuron represents the graphic history of conflicts suffered

during its embryonic life’’.30 (Cajal, 1899a,b).

6. Conclusion

Dendritic spines are among the most outstanding topics of

research in present-day neuroscience. The concept of dendritic

spines has evolved in the past century since they were

discovered by Cajal. Technological advances, from electron

microscopy to two photon microscopy, have permitted a change

of the concept of these structures as key elements in

connectivity and synaptic plasticity. However, many of the

recent ideas about dendritic spines were first proposed by Cajal

and other scientists of his time.

Acknowledgement

The authors are supported by The Ramon Areces Founda-

tion.

P. Garcıa-Lopez et al. / Progress in Neurobiology 83 (2007) 110–130 127

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