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Commentary

Editors Note: A number of events have commemorated the centenary of the 1906 Nobel Prize in Medicine to Camillo Golgi and

Santiago Ramon y Cajal in recognition of their work on the structure of the nervous system. Here we present a reanalysis of oneof Cajals original preparations from the collection at the Museo Cajal, fittingly using the Golgi method.

Three-Dimensional Reconstruction and Quantitative Studyof a Pyramidal Cell of a Cajal Histological Preparation

Pablo Garca-Lopez, Virginia Garc a-Marn, and Miguel FreireMuseo Cajal, Instituto Cajal, Consejo Superior de Investigaciones Cientificas, 28002 Madrid, Spain

The year 2006 marks the centenary of the Nobel Prize for Physiology or Medicine awarded to Santiago Ramon y Cajal and Camilo Golgi.We commemorate this centenary with a three-dimensional reconstruction and a quantitative study of a pyramidal cell of a Cajals

histological preparation. This preparation is one of the 4529 histological preparations personally made by Ramon y Cajal and preservedin the Museum Cajal. The three-dimensional reconstruction of the neuron allows visualizing one important discovery of Ramon y Cajal

that constitutes an active field of research in present-day neuroscience: dendritic spines.

Key words: Cajal; pyramidal cell; dendritic spines; filopodia; 3D reconstruction; Golgi method

Dendritic spines were discovered by San-

tiago Ramon y Cajal in 1888, in his firstarticle published with the Golgi Method. . . the surface . . . appears bristling withthorns or short spines. This is the firstreference to dendritic spines in his work,but it is not the only one. He also madeimportant observations that are still top-ics of research. These include the distribu-tion of dendritic spines along the neuro-nal tree (Ramon y Cajal, 1891), thedependence of spine density and size onthe area and the species (Ramon y Cajal,1896a, 1899), and the larger size of den-

dritic appendages during development(Ramon y Cajal, 1889, 1933). Ramon yCajal also proposed a physiological rolefor dendritic spines. In his opinion, den-dritic spines served to increase the recep-tive surface (Ramon y Cajal, 1896a). Healso accepted the hypothesis of Berkley(1895) that dendritic spines were thepoints at which axons connected and dis-

charged nervous impulse: Do they repre-

sent the lines of charge or absorption ofnerve impulses, as stated by Berkley? Thelatter opinion appears plausible to us. Itreconciles well with our idea, expressed inanother publication, namely that by vir-tue of the spines, dendritic branches in-crease theirreceptive surface and establishcloser contacts with the axonal terminalarborization (Ramon y Cajal, 1899). Al-though the work of Gray (1959a,b) con-firmed the hypothesis of Berkley, we stilldo not know the functional role of den-dritic spines because excitatory synapses

can also be made on dendritic shafts. Itseems most likely that dendritic spinesserve as biochemical compartments as hasbeen proposed.

To reconstruct this Cajal neuron, weused an interactive light-microscope com-puter system for the three-dimensional(3D) reconstruction and did quantitativestudy of a neuron from layer III of youngmouse cerebral cortex. Optic sections(1280 1024 pixels) were taken from thepreparation using a digital camera(DXM1200; Nikon, Tokyo, Japan), a mo-

torized stage (ProScan H128; Prior Scien-tific, Rockland, MA), and a light micro-scope (Nikon Eclipse E600) with an oil

immersion objective 100, numericalap-

erture (NA) 1.4. The structures werecoded as 3D coordinates of selected pointsusing the program Neuronal Coding(Freire, 1992). With the program Neuro-nal Quantification, we measured thenumber of dendritic spines, their distancefrom the soma, and the sizes of the headand neck. Dendritic spines were classifiedas sessile, thin, mushroom, or branchedspines. The code was converted with Per-sistence of Vision Raytracer (POV-Ray)for 3D rendering (Arellano and Freire,unpublished results).

The histological preparation (P80001)is a Golgi-impregnated slide from mousecerebral cortex (Fig. 1). Because of thedensity and size of dendritic appendages,we can assume that the mouse was olderthan 20 d. However, the preparation didnot include the superior colliculus, a dis-tinctive morphological feature of adultmouse. Thus, we only can conclude thatthe age of the mouse was between 20 and45 d. The three-dimensional reconstruc-tion of the pyramidal cell is shown in Fig-ure 2. A detail of the dendritic spines of

the original preparation and their codingis shown in Figure 3. A movie of the neu-ron is shown in the supplemental data

Received Aug. 16, 2006; revised Sept. 25, 2006; accepted Sept. 25, 2006.

This work was su pported by the Ramon Areces Foundatio n.

Correspondenceshouldbeaddressedto VirginiaGarca-Marn,Museo Cajal,

InstitutoCajal,ConsejoSuperiorde Investigaciones Cientificas, AvenidaDoctor

Arce37, 28002Madrid, Spain. E-mail:vgmarin@cajal.csic.es.DOI:10.1523/JNEUROSCI.3543-06.2006

Copyright 2006Society forNeuroscience 0270-6474/06/2611249-04$15.00/0

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(availableat www.jneurosci.org as supple-mental material). The total number ofdendritic spines and filopodia codifiedwas 1563.

Distribution and density ofdendritic spines

Spines were absent from soma and the or-igin of thick dendrites, but their numberincreased with distance from soma. Onthe basilar branches on the first 46.5 m,there was an increase of the mean numberof spines from 2.64 spines/15.5 m to12.47 spines/15.5m, after which the dis-tribution reached a plateau. The maxi-mum density 13.66 spines/15.5 m wasfound on the interval 6277.5m.On theapical trunk, there were only three spinesuntil the third oblique branch. Theoblique branches of the apical trunk andthe branches of the apical tuft had a den-sity of dendritic spines similar to the basi-lar branches.

Size of dendritic spines and filopodiaThe dendritic spines differed from filopo-dia especially in their size. The size of thepedunculated appendages was 1.43 0.6(mean SD). Most (81%) had a totallength2 m, 14.36% had a length from2 to 3 m, and only 5.64% of peduncu-lated appendages had a length 3 m,which we considered to be filopodia. Thesessile spines had a mean length of 0.42

0.21 m.

Head and neck sizeThe range of the head size was (0.011.31m2). There was a great heterogeneity ofsizes. The area for the head of the pedun-culated appendages was 0.18 0.20(mean SD). The sessile spines had anarea of 0.17 0.13 (mean SD). Therange of the neck length was from nulllength (sessile spines) to 4.2 m, whichcorresponds to a filopodia. The mean was0.90 0.51. The range of the neck width

was from0.11 to 0.95m (mean of0.230.13).

Dendritic appendages typesWe classified the appendages in dendriticspines (sessile, thin, mushroom, and,branched) and dendritic filopodia (Fig.4). The different percentages were as fol-lows: 64.7% thin, 16.8% sessile, 14.7%mushroom, 2.41% branched, and 1.39%dendritic filopodia. Few dendritic spinesended in an appendage or spinule. Thedistribution of these shapes didnot follow

any known pattern. The mushroomspines were sparsely distributed along thedendrites.

DiscussionDendritic spines receive most of the exci-tatory impulses of a pyramidal cell, con-sistent with the information processingcapacity of the neuron. The first correla-tion between thenumber of afferent fibers

and the density of dendritic spines wasmade by Ramon y Cajal (1896a).

The distribution of dendritic spinesfollows a known pattern. The proximalportions of apicaland basal dendritesgen-erally are devoid of spines and excitatorysynapses, but many symmetric (inhibi-tory) synapses are found (Alonso-Nanclares et al., 2004). Ramon y Cajal(1896b) noted the lack of dendritic spinesin the proximal portion of the dendritesand used it for proving that dendriticspines were real appendages and not silver

nitrate precipitates.The number of dendritic spines in-creases moving away from soma. In our

analysis, the maximum density is lowerthan density found in human temporalcortex 14.19 spines/10 m (Benavides-Piccione et al., 2002), as noted by Ramon yCajal (1909): They vary also with the an-imal species, and we may state in general

terms, that a cell with spiny processes inhomologous nuclei has more spines, thehigher the level of the subject in the ani-mal series. Thus, as an example in verte-brates, the Purkinje cell of birds showsfewer spines than that of mammals. Itimplicates a higher capacity to process in-formation by the pyramidal cells of hu-man cerebral cortex.

We found a great variability in thetotallength of pedunculated appendages. Mostof them (81%) have 2 m. There is apopulation of dendritic appendages

(5.64%) longer than 3m that we consid-ered as filopodia. The rest of the popula-tion (14.36%) had an intermediate length

Figure1. LayerIIIpyramidalcellof cerebralcortexof mousefroma originalpreparationofSantiagoRamon y Cajalimpregnatedwith the Golgi method (P80001). Z-projection (32 sections; z-step, 2.072m). Objective 20, NA 0.75 (NIH ImageJ).

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(23 m); they could be filopodia trans-forming to dendritic spines (Daily andSmith, 1996).

In our data, the percentage of thinspines was 65%. They had small heads(usually0.15 m2). One interesting is-sueis whether this population of thin den-

dritic spines makes stable and functionalsynapses to understand its synaptic plas-ticity. However, the strength of these syn-apses might be less powerful than thosewith big heads because the volume of thespine head is directly proportional to thesize of the postsynaptic density (Freire,1978), the size of the presynaptic terminal(Spaeek and Hartmann, 1983; Peters,1987), the number of AMPA postsynapticreceptors (Nusser et al., 1998; Matsuzakiet al., 2001; Kasai et al., 2003), the presyn-aptic number of docked synaptic vesicles,and the ready available pool of neuro-transmitters (Spac

ek and Hartmann,1983; Harris and Stevens, 1989; Nusser etal., 1998; Schikorski and Stevens, 2001).In addition, small spines are more motileand unstable than bigger ones (Parnass etal., 2000; Grutzendler et al., 2002; Tracht-enberg et al., 2002). Taking these observa-tions, Kasai et al. (2003) proposed thatlarge spines (mushroom and sessilespines) codify synaptic memory, whereassmall spines (filopodia and thin spines)serve as a source to create new synapses(synaptic learning).

Our percentage of spine categories wascomparable with other studies in hip-pocampus, cerebral cortex, and cerebel-lum of rats of different ages (Peters andKaiserman-Abramof, 1970; Harris et al.,1992; Lee et al., 2004). These data suggestthat the distribution of spine shapes ispreserved in different cells and differentareas. Recent experiments (Holtmat et al.,2005) have shown that the proportion ofstable spines (usually mushroom) in-creases with age, whereas the proportionof unstable spines (usually thin spines)

decreases with age. This change in den-dritic spine stability with age might havefunctional consequences for the matura-tion of the neuron such as a reduction insynaptic plasticity.

We are beginning to understand thedendritic spine types in terms of its func-tional activity. Small spines could betransient and contribute to weak synap-tic connections, whereas large spinescould be persistent and contribute tostrong synaptic connections. In the pyra-midal neuron we studied, the percentage

(66.7%) of small spines, thin type was big-ger than the percentage (15.1%) of largespines, mushroom type. This could mean

Figure2. Three-dimensionalreconstructionof a layerIII pyramidal cellof cerebral cortexof mouse. Codificationof theneuronwith neuronal coding and rendered with POV-Ray. The white box outlines the fragment amplified in Figure 3B.

Figure 3. Fragment of an oblique apical branch with thin spines, mushroom spines, filop odia, and branched spines. A,Z-projection (27 sections; z-step, 0.291 m. Objective 100, NA 1.4. B, Three-dimensional reconstruction of the samesegment.

Garca-Lopez et a l. Commentary J. Neurosci., November 1, 2006 26(44):1124911252 11251

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that the studied neuron was involved in afew stable circuits and retained most of itsplastic or learning capabilities. Again, it isinteresting to return to Ramon y Cajal(1894), recalling that he introduced theterm plasticity more than a century ago:. . . Similar plasticity of the cellular ex-

pansions varies, probably at differentages:great in the young, it lowers in the adultand almost disappears in the ancient.

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