Activity-dependent structural plasticity of Purkinje cell spines in cerebellar vermis and hemisphere

ORIGINAL ARTICLE

Activity-dependent structural plasticity of Purkinje cell spinesin cerebellar vermis and hemisphere

P. De Bartolo • F. Florenzano • L. Burello •

F. Gelfo • L. Petrosini

Received: 6 February 2014 / Accepted: 24 June 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract The environmental enrichment (EE) paradigm

is widely used to study experience-dependent brain plas-

ticity. In spite of a long history of research, the EE influ-

ence on neuronal morphology has not yet been described in

relation to the different regions of the cerebellum. Thus,

aim of the present study was to characterize the EE effects

on density and size of dendritic spines of Purkinje cell

proximal and distal compartments in cerebellar vermian

and hemispherical regions. Male Wistar rats were housed

in an enriched or standard environment for 3.5 months

from the 21st post-natal day onwards. The morphological

features of Purkinje cell spines were visualized on calbin-

din immunofluorescence-stained cerebellar vermian and

hemispherical sections. Density, area, length and head

diameter of spines were manually (ImageJ) or automati-

cally (Imaris) quantified. Results demonstrated that the

Purkinje cell spine density was higher in enriched rats than

in controls on both proximal and distal dendrite compart-

ments in the hemisphere, while it increased only on distal

compartment in the vermis. As for spine size, a significant

increase of area, length and head diameter was found in the

distal dendrites in both vermis and hemisphere. Thus, the

exposure to a complex environment enhances synapse

formation and plasticity either in the vermis involved in

balance and locomotion and in the hemisphere involved in

complex motor adaptations and acquisition of new motor

strategies. These data highlight the importance of cere-

bellar activity-dependent structural plasticity underling the

EE-related high-level performances.

Keywords Environmental enrichment � Proximal and

distal dendrites � Dendritic spines � Cerebellum � Rat

Introduction

The brain is strikingly responsive to environmental stim-

ulations to the extent that its morphology is modified by

experience. Interaction between brain and environment

underlies adjustments allowing adaptation to an ever-

changing environment. Brain morphological modifications

triggered by the exposure to increased environmental

complexity may be experimentally investigated through the

environmental enrichment (EE) paradigm (Rosenzweig

et al. 1962; Rosenzweig and Bennett 1996). EE is based on

the enhancement of motor, sensory, cognitive and social

stimulations in housing conditions (Van Praag et al. 2000;

Nithianantharajah and Hannan 2006). Enhanced physical

and cognitive stimulations induce structural modifications

that strengthen neuronal connectivity (Nithianantharajah

and Hannan 2006; Petrosini et al. 2009). The magnitude

and persistence of the changes induced by the enriched

experience depend on the age of the subjects and the length

of the exposure (Tremml et al. 2002; Cutuli et al. 2011; De

Bartolo et al. 2011). Interestingly, phenotypes acquired

P. De Bartolo � L. Burello � F. Gelfo � L. Petrosini

IRCCS Santa Lucia Foundation, Via Del Fosso di Fiorano 64,

00143 Rome, Italy

P. De Bartolo (&) � L. Burello � L. Petrosini

Department of Psychology, University Sapienza of Rome,

Via dei Marsi 78, 00185 Rome, Italy

e-mail: [email protected]

F. Florenzano

Confocal Microscopy Unit, European Brain Research Institute

(EBRI), Via del Fosso di Fiorano 64, 00143 Rome, Italy

F. Gelfo

Department of Systemic Medicine, University Tor Vergata

of Rome, Via Oxford 81, 00133 Rome, Italy

123

Brain Struct Funct

DOI 10.1007/s00429-014-0833-6

from the exposure to enriched environment can even be

transmitted to the next generation (Arai and Feig 2011;

Caporali et al. 2014).

The importance of studying the EE effects on brain

plasticity arises from the evidence that in humans the

exposure to complex-enriched stimulations (as high edu-

cational or occupational attainment, high IQ, and an active

lifestyle) leads to great tolerance of pathological abnor-

malities (Scarmeas and Stern 2003; Whalley et al. 2000,

2004), a concept known as brain (structural) and cognitive

(functional) reserve theory (Stern 2002, 2003, 2006).

Brain stores information by modulating the strength of

the existing synapses and enlarging or shrinking dendritic

spines, principal postsynaptic target of excitatory afferents

(Gray 1959; Harris and Kater 1994; Fiala et al. 2002).

Intrinsic factors, as neurotrophins, and extrinsic factors, as

environmental conditions, modulate number, shape and

function of dendritic spines (Bae et al. 2012; Petrinovic

et al. 2013). In neocortical and hippocampal neurons

increased spine density is demonstrated in relation to EE

exposure (Moser et al. 1994; Kozorovitskiy et al. 2005;

Mandolesi et al. 2008; Liu et al. 2012), neuronal activity

(Bundman and Gall 1994; Hosokawa et al. 1995; Zhao

et al. 2012), learning (Gonzalez-Ramırez et al. 2013;

Muhammad et al. 2013), and physical exercise (Klintsova

and Greenough 1999; Yau et al. 2011).

Even in the cerebellar circuitries, the increased envi-

ronmental stimulation facilitates neuronal remodeling in the

cells directly involved in the experience-dependent activity.

Namely, exposure to EE produces an enlargement of the

Purkinje cell synaptic buttons and an enhancement of the

mossy and climbing fiber terminal density in deep nuclear

neurons (Foscarin et al. 2011). These observations add to

the previously reported EE-dependent structural remodeling

in the cerebellar cortex. Namely, the number of parallel

fiber–Purkinje cell glutamatergic synapses as well as stel-

late cell–Purkinje cell GABAergic synapses is strongly

increased under EE conditions (Lonetti et al. 2010). EE

increases cerebellar production of neurotrophins (Angelucci

et al. 2009; Vazquez-Sanroman et al. 2013) but not of

synaptophysin, as conversely it occurs in forebrain, hippo-

campus, and thalamus (Nithianantharajah et al. 2004).

Additionally, learning complex acrobatic tasks (but not

merely running) increases the number of synapses in Pur-

kinje cells (Federmeier et al. 2002; Kim et al. 2002; Lee

et al. 2007; Gonzalez-Burgos et al. 2011). Purkinje cells

represent an excellent model to investigate EE-induced

synaptic changes given the dynamic changes they exhibit

under diverse conditions, such as physical activity (Pysh

and Weiss 1979; Gonzalez-Burgos et al. 2011 ), block of

electrical activity (Bravin et al. 1999; Morando et al. 2001),

deafferentation (Sotelo et al. 1975), presence of ataxia

(Rhyu et al. 1999a, b) or olivo-ponto-cerebellar atrophy

(Ferrer et al. 1988, 1994). Considering that the responses to

environmental requests may occur through spine changes

regulating in turn synaptic strength and signaling properties,

the present research was aimed at analyzing the effects of a

prolonged exposure to complex environment on density and

size of Purkinje cell spines to provide information on syn-

aptogenesis and circuit complexity (Koch and Poggio 1983;

Turner 1984; Lee et al. 2012). The EE effects on Purkinje

cell spines have been analyzed both in the vermis and

hemisphere, two regions differently involved in the motor

functions of the cerebellum. The vermis plays a primary

role in regulating extensor tone, maintaining dynamic bal-

ance control, modulating the rhythmic flexor and extensor

muscle activity during locomotion (Chambers and Sprague

1955; Thach et al. 1992). The hemispheres have less

importance in controlling balance and walking, but play a

pivotal role in motor control when complex motor adapta-

tions are required, as in novel and attention-demanding

contexts (Manto and Oulad Ben Taib 2010). Given the

functionally different cortico-cerebellar networks in which

vermian and hemispherical regions are engaged (Morton

and Bastian 2004), EE could differently impact on them.

Spine characterization was performed in proximal and

distal dendritic compartments of the same Purkinje cells.

Namely, the proximal compartment consisting of large

dendrites on which the climbing fibers impinge, displays a

rather low number of spines, while conversely, the distal

compartment made up of thin branchlets contacted by the

parallel fibers displays a very high spine density. Accord-

ingly, the two compartments are differently regulated by the

activity of the specific afferents, and thus could differently

respond to environmental drives (Bravin et al. 1999; Mor-

ando et al. 2005; Sugawara et al. 2013). Noteworthy, in

spite of the described differences in cerebellar functioning,

to our knowledge this is the first study addressing the EE-

induced morphological effects in relation to the differential

functional engagement of the cerebellar vermian and

hemispherical regions as well as of the two Purkinje cell

dendritic compartments.

Materials and methods

Animals and experimental groups

Fourteen male Wistar rats (Harlan Laboratories S.r.l.,

Udine, Italy) were used. They were maintained according

to the guidelines for ethical conduct developed by the

European Communities Council Directive of 22 September

2010 (2010/63/EU). All animal protocols used have been

approved by the Ethical Committee on animal experiments

of ‘‘Sapienza’’ University of Rome. All efforts were made

to minimize animal suffering and to reduce the number of

Brain Struct Funct

123

animals used. Animals were randomly assigned to one of

two experimental groups: standard reared animals, used as

controls (group name: C; 4 animals); enriched reared ani-

mals (group name: E; 10 animals).

Rearing conditions

Starting from the 21st post-natal day till the day of killing

(about the 120th post-natal day), the enriched rats were

housed in groups of ten animals in a large cage (100 9

50 9 70 cm). The cage had an additional floor made of a

galvanized wire mesh and connected by ramps of the same

material to create two interconnected levels. The cage

contained wood shavings, a running wheel, shelters and

plastic toys. Throughout the enrichment period, the running

wheel and shelters were always kept in the cage, while the

toys were changed twice a week. Once a week, the feeding

boxes and water bottles were moved to different cage

points to encourage foraging and explorative behavior.

Control rats reared in standard condition were housed in

groups of two animals in standard cages (40 9 26 9

18 cm) containing wood shaving bedding and only a

plastic tube as shelter. Feeding boxes and water bottles

were kept in a fixed position. The animals of both exper-

imental groups received the same type of food. Food and

water were provided ad libitum.

Tissue processing

On the 4th post-natal month, three animals of the E group

and three animals of the C group randomly selected were

deeply anesthetized (33 % chloral hydrate in water) to be

transcardially perfused with saline followed by 4 % para-

formaldehyde in phosphate-buffered saline (PBS; 4 �C, pH

7.5). Brains were removed, cryoprotected in 30 % buffered

sucrose at ?4 �C, frozen to be cut on a freezing microtome.

The posterior part of the brains embracing the cerebellum

was cut into 30-lm sagittal sections for calbindin immu-

nofluorescence to allow visualization of the entire dendritic

tree of Purkinje cells. Namely, sections were collected at

the level of left cerebellar hemisphere (3.90/3.40 mm lat-

eral to the midline) and cerebellar vermis (0.90/0.40 mm

lateral to the midline).

Immunofluorescence

To visualize Purkinje cells, sections were pre-incubated at

room temperature in 0.25 % Triton X-100 (Sigma-Aldrich

Chemie, Steinheim, Germany) and 10 % Normal Donkey

Serum (Vector Laboratories Inc, Burlingame, CA) in PBS

for 1 h. Sections then received a 2-day incubation at ?4 �C

in a solution composed of 0.25 % Triton X-100, 1 %

Normal Donkey Serum and the primary anti-calbindin

antibody (made in mouse, 1:2,000; Sigma-Aldrich Chemie,

Steinheim, Germany). After three rinses in PBS (10 min

each), sections were dark-incubated in a solution of 0.25 %

Triton X-100 and the Cy3-conjugated secondary anti-

mouse antibody (made in donkey, 1:200; Jackson Immu-

noresearch, BA) in PBS. After an incubation of 2 h at room

temperature, sections were mounted on slides and covers-

lipped by the anti-fading mounting medium Fluoromount

(Sigma-Aldrich Chemie, Steinheim, Germany). Specificity

of the staining was checked in sample sections by omitting

the primary antibody.

Confocal imaging

Imaging was performed through a LSM 700 confocal laser

scanning microscope (CLSM; Zeiss, Germany). Two dif-

ferent approaches were used: z-stack series with an

objective 639, allowing the visualization of the entire

dendritic tree of Purkinje cells, and z-stack series with an

objective 1009, allowing the visualization of the dendritic

tree distal portion. For the visualization of the entire den-

dritic tree, the images were acquired starting from the

Purkinje cell body to the pial surface. Such images were

used for spine density analysis on proximal and distal

dendritic compartments. For the visualization of the distal

portion of the dendritic tree, images were acquired starting

immediately below the pial surface. Such images were used

to analyze spine size. The following acquisition settings

were used for the entire dendritic tree: objective 639 oil

immersion NA 1.4; zoom factor 0.5; image format

1024 9 1024; sampled image area was x: 203.03 lm, y:

203.03 lm; pinhole 1 producing an optical section thick-

ness of 1.2 lm; z-spacing 0.5 lm; number of optical sec-

tions was about 10; pixel dwell time 50.4 ls. For the distal

dendritic tree we used: objective 1009 oil immersion NA

1.2; zoom factor 1.5; image format 1024 9 1024; sampled

image area was x: 46 lm, y: 46 lm; pinhole 1 producing an

optical section thickness of 0.8 lm; z-spacing 0.5 lm;

number of optical sections was about 16; pixel dwell time

50.4 ls. To generate projection images, the maximum

intensity algorithm was used, and the first and last images

of the z-series were cut so that only the central images were

used to generate the projection image. This procedure

allowed discarding images of the tissue surfaces eventually

containing broken or disconnected dendritic segments,

allowing a better image definition and a more reliable

analysis. The confocal image acquisitions were performed

so that all samples were imaged using consistent settings

for laser power and detector gain. To increase the resolu-

tion (Vecellio et al. 2000; Wallace et al. 2001; Wallace and

Bear 2004), the images were deconvolved by means of

blind deconvolution by using AutoQuant X3 (Media

Cybernetics Inc., Rockville, MD). For the production of

Brain Struct Funct

123

figures, a .tiff file of projection images was exported,

brightness and contrast of images were adjusted and final

figures were assembled using Adobe Photoshop 6 and

Adobe Illustrator 10. Image analysis was performed by

directly opening the acquisition file (.lsm), which retained

all the information about acquisition setting.

Image analysis

Image analysis was performed on proximal and distal

dendrites of Purkinje cells of both vermis and hemisphere.

Proximal and distal dendrites were identified according to

specific morphological features (Larramendi and Victor

1967; Palay and Chan-Palay 1974), and only those having a

diameter above and below 2 lm, respectively, were

retained for analysis (Bravin et al. 1999). Length and cal-

iber of sampled proximal and distal dendrites were mea-

sured using ImageJ 1.42q software (Abramoff et al. 2004).

Dendritic spines were identified as small protrusions from

the parent dendrite and their density was manually counted

offline in maximum intensity projection of the z-stacks by

using ImageJ 1.42q software. Care was taken to ensure that

each spine was counted only once by following its pro-

jection course through the stack of z-sections. Evaluation

of spine density was performed on segments of about

20 lm in length of dendrites of a single Purkinje cell.

Since significant changes in spine density were found

only in Purkinje cell distal dendrites both in vermis and

hemisphere, the evaluation of spine size was executed only

on distal compartment. Spine size was evaluated using the

Filament Tracer module of the Imaris Suite 7.4� (Bitplane

A.G., Zurich, Switzerland) software to generate spine

reconstructions of the acquired z-stack images. The ana-

lysis procedure was performed under visual control to

determine thresholds subtracting background noise and

taking into account dendritic structures. The spine length

was set at a minimum of 0.2 lm and a maximum of 1.5 lm

(i.e., the shortest quantifiable spines were 0.2 lm and the

longest ones 1.5 lm in length). These thresholds were

settled on the basis of previous not automated manual

measurements performed on the same areas revealing that

no protrusion to be considered as a spine longer than

1.2–1.3 lm was ever observed. In fact, the occasionally

encountered apparently longer protrusions had to be

referred to two or more spines located too closely or even

overlapping on different focus plans, as evidenced by a

detailed visual inspection. During image processing, the

images were compared with the original raw data to make

sure that no structure not seen in the original data was

introduced or vice versa that a structure present in the

original data was erroneously removed. Once determined

thresholds that preserve dendrite and spine features, they

were applied to all images. Projection images were created

by applying maximum intensity projection algorithm to

z-series and volume rendering of the z-stacks were gener-

ated with Filament modules. Analyzed immunofluorescent

images were overlaid with volume rendering images gen-

erated by the filament analysis module to visually verify

the accuracy of the dendrites and spines reconstruction.

Notably, the high spine density in Purkinje cell distal

dendrites creates a challenge for analysis, as single spines

may overlap to form a confounding multi-spine protrusion.

To overcome such a trouble as much as possible, image

analyses were separately performed by two experimenters

blind to the experimental treatment. Measurements of spine

values were considered reliable only when their measure

evaluations were consistent.

As parameters, we evaluated mean spine density (num-

ber of spines per lm of dendritic length), mean spine area

(surface of the spine from the dendritic surface to the spine

ending), mean spine length (extension of the entire spine

from the dendritic surface to the spine ending), and mean

spine head diameter (diameter of the spine bulbous ending).

Statistical analysis

Data are presented as mean ± SEM. Statistical analyses

were performed using the software SPSS 8. Data were first

tested for normality (Will–Shapiro’s test) and homosce-

dasticity (Levene’s test). To verify the appropriateness of

applying nested-design ANOVAs taking into account the

influence of the random factor ‘‘animal’’ on the data, the

significance level of the interaction between the random

factor ‘‘animal’’ and the fixed factor ‘‘group’’ in two-way

ANOVAs was preliminarily evaluated. Since no significant

interaction was found for any parameter, no nested-design

ANOVA was applied. One-way ANOVAs with group (C,

E) as fixed factor were then performed on spine density, for

the cerebellar region (vermis or hemisphere) and neuronal

compartment (proximal or distal dendrites). Size parame-

ters were analyzed by MANOVA with group (C, E) as

fixed factor and spine area and length as dependent vari-

ables for distal dendrites of both cerebellar regions (vermis

or hemisphere). Tukey’s test was used for post hoc com-

parisons. p values B0.05 were considered statistically sig-

nificant. Furthermore, to reveal the magnitude of the

eventual changes, percentage differences of spine values

between E and C groups were calculated.

Results

Methodological approach

Calbindin-immunostained cerebellar sections showed an

intense fluorescence of Purkinje cell bodies, dendrites and

Brain Struct Funct

123

spines. As visible in Fig. 1, the superimposition of the

projection images of z-stack series taken through the

Purkinje cell distal dendritic region with the volume ren-

dering generated by the Filament Analysis module showed

the accuracy of the digital reconstruction relative to the

native image. Broken dendrites or dendritic branches not

perfectly comprised in the acquisition volumes were

excluded from the analysis. Closer analysis of dendrite

small segments (Fig. 1d–f) revealed accurate identification

of spines. In several cases, the immunostained spines

appeared as fluorescent point-like regions not connected to

the dendrite, while the volume rendering showed spines

attached to the dendrite. By increasing the brightness of

the image to fluorescence saturation levels, the spines

were shown to be really connected to the dendrite, indi-

cating that the fluorescence intensity levels of the spines

in the native image were too low to be well appreciated by

the human eye. A clear advantage of the described

approach was the possibility to include in the analysis the

high majority of fluorescent spines present in the imaging

field.

Morphological changes in vermian and hemispheric

regions

Rearing in an enriched environment provoked an

enhancement of spine density on both proximal and distal

compartments of Purkinje cell dendrites in the hemisphere

and only on distal compartment in the vermis (Fig. 2). As

for spine size, a significant increase of area, length and

head diameter was found in the spines on distal dendrites of

Purkinje cells in both vermis and hemisphere (Fig. 3).

Fig. 1 Representative deconvolved images and rendering of distal

dendrites of a calbindin-immunostained Purkinje cell. a Maximum

projection of a confocal z-stack series showing intense spine labeling.

The pial surface is on the left upper part of the image. b Volume

rendering of the maximum projection image created by Filament

Tracer module of Imaris software package. c Overlay of the

maximum projection image and the relative volume rendering. Note

that the areas where the calbindin labeling was not sufficiently intense

or the dendrites that were damaged by cutting procedures were not

included in the analysis. d–f Higher magnification of the boxed area

in a–c to illustrate the accuracy of the automated spine identification

procedure. Scale bars a = 13 lm; d = 0.8 lm

Fig. 2 Effects of the exposure to a standard (C) or an enriched

(E) environment on Purkinje cell spine density. Histograms show the

mean spine density of Purkinje cell proximal and distal dendrites in

the vermis (a1; a2) and the hemisphere (b1; b2). Data are shown as

mean ± SEM. Asterisks indicate significant differences between

groups (*p \ 0.05; ***p \ 0.0005)

Brain Struct Funct

123

Vermis

Manual counts revealed that the mean spine density on the

proximal dendrites of Purkinje cells did not significantly

differ between E and C groups (F(1,58) = 0.96; p n.s.),

being the difference between means of 5 % (Fig. 2a1).

Conversely, on the distal dendrites the mean spine den-

sity significantly increased to 11 % in the E group

(F(1,58) = 14.86; p = 0.0003) (Fig. 2a2). The selectively

increased spine density of distal dendrites appears to be an

index of the specific addition of synapses between parallel

fibers and distal Purkinje cells spines.

To investigate if the increase in the number of synapses

matched a strengthening in synaptic efficacy, three mor-

phological indexes of spine size, such as spine area, length

and head diameter were analyzed. Indeed, an increase in

spine length and largeness may indicate proportionally

enlarged synaptic contacts (Hering and Sheng 2001).

A MANOVA on the mean area, length and head diameter

of distal dendrite spines indicated that all parameters sig-

nificantly increased in the E group (F(3,8) = 49.49;

p \ 0.0001), as showed in Fig. 3a. In particular, the enri-

ched animals exhibited an increase of 28 % in spine area

(Fig. 3a1), of 22 % in spine length (Fig. 3a2), and of 16 %

in spine head diameter (Fig. 3a3).

Hemisphere

On the proximal dendrites, the mean spine density signif-

icantly increased in the E group (F(1,58) = 5.41; p = 0.02)

in comparison to C group. The EE-induced increase was of

36 % (Fig. 2b1). Even on the distal dendrites, the mean

spine density significantly increased of 18 % in the E group

(F(1,58) = 14.91; p = 0.0003) (Fig. 2b2). Notably, the

exposure to environmental complexity induced an increase

in spine density more marked in the hemisphere than in the

vermis in both proximal and distal compartments.

A MANOVA on the mean area, length and head

diameter of distal dendrite spines indicated that once more

all parameters significantly increased in the E group

(F(3,8) = 17.41; p = 0.001), as shown in Fig. 3b. In par-

ticular, the enriched animals exhibited an increase of 43 %

in spine area (Fig. 3b1), of 14 % in spine length (Fig. 3b2),

and of 38 % in spine head diameter (Fig. 3b3).

Discussion

For the first time, to our knowledge, this research analyzes

the differential influence of the exposure to a complex

environment on the two main cerebellar regions, the vermis

and the hemisphere, differently engaged in information

processing. In particular, the EE effects on the morpho-

logical features of Purkinje cells (density, area, length and

head diameter of spines) were evaluated in proximal and

distal dendrite compartments that as known receive dif-

ferent inputs to elaborate. In line with the effects previ-

ously reported on neocortical and hippocampal neurons

(Johansson and Belichenko 2002; Gelfo et al. 2009; Man-

dolesi et al. 2008; Rojas et al. 2013; Gonzalez-Ramırez

et al. 2014), the exposure to an enriched environment

affected density, area, length and head diameter of Purkinje

cell spines. In particular, spine density was higher in

enriched rats than in controls on both proximal and distal

dendrite compartments in the hemisphere, while it

increased only on distal compartment in the vermis, sug-

gesting that the different spinogenesis in the two dendrite

compartments is the expression of the differences in the

inputs they receive. As for spine size, a significant increase

of all parameters analyzed was found in the Purkinje cell

distal dendrites in both vermis and hemisphere. Notewor-

thy, the obtained spine values (density, area, length and

Fig. 3 Effects of the exposure to a standard environment (C) or an

enriched (E) environment on Purkinje cell spine size. Histograms

show the mean area, length and head diameter of the spines of

Purkinje cell distal dendrites in the vermis (a1; a2; a3) and the

hemisphere (b1; b2; b3). Data are shown as mean ± SEM. Asterisks

indicate significant differences between groups (*p \ 0.05;

**p \ 0.005; ***p \ 0.0005)

Brain Struct Funct

123

head diameter) are in agreement with the values reported in

previous studies (Harris and Stevens 1988; Lee et al. 2007;

Sdrulla and Linden 2007; Petrinovic et al. 2013).

As previously demonstrated, the exposure to EE evokes

a different extent of spinogenesis in the apical and basal

arborizations of neocortical pyramidal neurons. Namely,

enriched rats showed an increased spine density in both

apical and basal arborizations of pyramidal neurons of the

layer III in the parietal cortex and mainly in apical arbor-

ization of the pyramidal neurons of layers III and V in

frontal cortex (Gelfo et al. 2009). Thus, the different spine

density in the two dendritic compartments of the vermian

and hemispherical regions seems to be related to the EE-

potentiated locomotor activity, voluntary motility and

motor learning resulting in different morphological effects

at cerebellar level. Rearing in large groups, foraging wide

spaces, experiencing multiple sensorial stimuli require

advanced sensory-motor integration, effective planning of

motor strategies, fine matching of actions with goal, con-

tinuous adaptation of behavior to the multifarious context.

Thus, environmental complexity primarily influences those

neural circuits that have access to sensory information and

at same time modulate motor output. The cerebellar net-

works do meet such a requirement. The strengthening of

the cerebellar synapses has positive repercussions on the

wide cortico-subcortical networks, supporting the EE-

induced high-level performances. Indeed, effective infor-

mation processing, highly coordinated movements and

complex motor adjustments were previously demonstrated

in animals exposed to the very same enrichment protocol.

Namely, the enriched animals quickly acquire tuned navi-

gational strategies, improved procedural competencies

and working memory abilities. Furthermore, when the

requirements of the context change, they promptly are able

to reorganize their strategies by shifting from prevalently

using spatial procedures to applying mnesic competencies

(Leggio et al. 2005; De Bartolo et al. 2008; Mandolesi et al.

2008; Petrosini et al. 2009; Foti et al. 2011). The beneficial

effects of complex experiences appear to be linked to the

strengthening of not only the neocortical and hippocampal

synapses, as already demonstrated, but also of the Purkinje

cell synapses. Specifically, the parallel fiber–Purkinje cell

synapses (distal dendrites) provide contextual information

and the climbing fiber–Purkinje cell synapses (proximal

dendrites) provides signal ‘‘errors’’ adapting the behavior

to requirement of ever-changing environments (Ito 1984;

Hansel and Linden 2000, 2001; Ramnani 2006). It is

intriguing that the exposure to complex environments

enhances synapse formation and plasticity either in the

vermis involved in balance and locomotion and in the

hemisphere involved in complex motor adaptations and

acquisition of new motor strategies. Interestingly, while in

the vermis there was an increased spine density only in

Purkinje cell distal dendrites, in the hemisphere such an

increase was present also in proximal dendrites. This

finding suggests that in the hemisphere, the environmental

complexity specifically strengthened the teaching line

provided by the climbing fibers (Marr 1969; Albus 1971).

The bidirectional connections of cerebellar hemispheres

with neocortical areas, as the prefrontal, premotor, primary

motor and parietal cortices, involved in all facets of motor

function (Voogd 1995; Schmahmann 1996; Morton and

Bastian 2004; Manto and Oulad Ben Taib 2010) are con-

sistent with the striking EE influence on Purkinje cell distal

and proximal dendrites of hemispherical regions. The

present results complement the increased number of syn-

apses per Purkinje cell or the synaptic efficacy changes

following motor learning or classical conditioning previ-

ously described (Sakurai 1987; Black et al. 1990; Schreurs

et al. 1998; Velazquez-Zamora et al. 2011).

As described, the enhanced spine density was accom-

panied by an increased spine size in distal dendrites of both

vermis and hemisphere of enriched rats. An increase in

spine length and largeness may be considered an indication

of proportionally enlarged synaptic contacts (Hering and

Sheng 2001). As evidenced by the findings provided by

biophysics and molecular biology studies, large spines

abundantly express AMPA receptors and are associated

with large postsynaptic density, producing consequently

large AMPA receptor-mediated currents (Matsuzaki et al.

2001, 2004; Kasai et al. 2003; Holtmaat et al. 2005;

Noguchi et al. 2005; Bourne and Harris 2007). On such a

basis, it is intriguing to suggest that the larger spines of

enriched animals are remarkably persistent and function-

ally stronger in their response to glutamate and in local

regulation of intracellular calcium, and that the spine

enlargement may be essential for an increased postsynaptic

response in LTP (Grutzendler et al. 2002; Kasai et al. 2003;

Holtmaat et al. 2005). As advanced by Matsuzaki et al.

(2004), large spines act as ‘‘memory units’’ and are the

structural basis of long-term memory (Kasai et al. 2003;

Matsuzaki et al. 2004; Gonzalez-Burgos et al. 2011). Thus,

enlarged spines can stabilize and retain long-term infor-

mation and become critical for information storage (Kasai

et al. 2003). Spine density and morphology are widely

modulated by environmental inputs in the form of synaptic

activity, which is central to mnesic traces formation and

other adaptive brain changes. The new spines persist at

long last after EE and the performance of enriched animals

is positively associated with the extent of new spine for-

mation, as we previously demonstrated following the

exposure to the same EE paradigm (Mandolesi et al. 2008).

The increase in spine density is obviously related to a

major proximity of the spines. This is particularly inter-

esting, since neighboring spines are retained to function

within the same neural circuit and transmit similar

Brain Struct Funct

123

information to the postsynaptic neuron as it has been pro-

posed to occur in clustered spines (Yang et al. 2009; Fu

et al. 2012).

In conclusion, the interaction with a complex environ-

ment appears to induce tuned neuronal activity that facil-

itates the remodeling of synaptic connectivity mainly in the

structures more directly involved in the experience-

dependent events. In this way, the external stimuli drive a

primarily instructive action that determines the actual

number and size of the adaptive spines. The influence of

external stimuli on the cellular/molecular mechanisms that

regulate spine metabolism has been object of many studies

(Klintsova and Greenough 1999; Lee et al. 2012; Lai and

Ip 2013). In particular, the EE stimulates changes in the

expression of neuronal growth genes, regulatory substances

(as myelin-associated proteins and extracellular matrix

components) and neurotrophins acting on neuronal plas-

ticity and connection patterns. As previously demonstrated,

in enriched rats the cerebellum was the brain region where

BDNF and NGF levels were more increased as compared

to the levels of frontal cortex, hippocampus and striatum

(Angelucci et al. 2009). Thus, the EE-induced effects on

Purkinje cell spines observed in the present research might

be linked also to the modification of neurotrophic factor

levels, dampening of growth-inhibitory mechanisms and

shifting of the balance between synthesis and removal of

matrix components to reorganize the cerebellar output to

the cerebral cortex, as recently described in a different

model of cerebellar plasticity (Foscarin et al. 2011).

Acknowledgments We are grateful to Mr. Maurizio Abbate for the

technical help with images deconvolution. The research was sup-

ported by MIUR fund to LP.

Conflict of interest The authors declare that they have no conflict

of interest.

Ethical standards The animals used in the study described in this

manuscript were maintained according to the guidelines for ethical

conduct developed by the European Communities Council Directive

of 22 September 2010 (2010/63/EU). All animal protocols used have

been approved by the Ethical Committee on animal experiments of

‘‘Sapienza’’ University of Rome. The manuscript does not contain

clinical studies or patient data.

References

Abramoff MD, Magalhaes PJ, Ram SJ (2004) Image processing with

ImageJ. Biophotonics Int 11:36–42

Albus JS (1971) A theory of cerebellar function. Math Biosci

10:25–61

Angelucci F, De Bartolo P, Gelfo F, Foti F, Cutuli D, Bossu P,

Caltagirone C, Petrosini L (2009) Increased concentrations of

nerve growth factor and brain-derived neurotrophic factor in the

rat cerebellum after exposure to environmental enrichment.

Cerebellum 8:499–506

Arai JA, Feig LA (2011) Long-lasting and transgenerational effects of

an environmental enrichment on memory formation. Brain Res

Bull 85:30–35

Bae J, Sung BH, Cho IH, Kim S-M, Song WK (2012) NESH regulates

dendritic spine morphology and synapse formation. PLoS One

7:e34677

Black JE, Isaacs KR, Anderson BJ, Alcantara AA, Greenough WT

(1990) Learning causes synaptogenesis, whereas motor activity

causes angiogenesis, in cerebellar cortex of adult rats. Proc Natl

Acad Sci USA 87:5568–5572

Bourne J, Harris KM (2007) Do thin spines learn to be mushroom

spines that remember? Curr Opin Neurobiol 17:381–386

Bravin M, Morando L, Vercelli A, Rossi F, Strata P (1999) Control of

spine formation by electrical activity in the adult rat cerebellum.

Proc Natl Acad Sci USA 96:1704–2709

Bundman MC, Gall CM (1994) Ultrastructural plasticity of the

dentate gyrus granule cells following recurrent limbic seizures:

II. Alterations in somatic synapses. Hippocampus 4:611–622

Caporali P, Cutuli D, Gelfo F, Laricchiuta D, Foti F, De Bartolo P,

Mancini L, Angelucci F, Petrosini L (2014) Pre-reproductive

maternal enrichment influences offspring developmental trajec-

tories: motor behavior and neurotrophin expression. Front Behav

Neurosci 8:195

Chambers WW, Sprague JM (1955) Functional localization in the

cerebellum. I. Organization in longitudinal cortico-nuclear zones

and their contribution to the control of posture, both extrapy-

ramidal and pyramidal. J Comp Neurol 103:105–129

Cutuli D, Rossi S, Burello L, Laricchiuta D, De Chiara V, Foti F, De

Bartolo P, Musella A, Gelfo F, Centonze D, Petrosini L (2011)

Before or after does it matter? Different protocols of environ-

mental enrichment differently influence motor, synaptic and

structural deficits of cerebellar origin. Neurobiol Dis 42:9–20

De Bartolo P, Leggio MG, Mandolesi L, Foti F, Gelfo F, Ferlazzo F,

Petrosini L (2008) Environmental enrichment mitigates the

effects of basal forebrain lesions on cognitive flexibility.

Neuroscience 154:444–453

De Bartolo P, Gelfo F, Burello L, De Giorgio A, Petrosini L, Granato

A (2011) Plastic changes in striatal fast-spiking interneurons

following hemicerebellectomy and environmental enrichment.

Cerebellum 10:624–632

Federmeier KD, Kleim JA, Greenough WT (2002) Learning-induced

multiple synapse formation in rat cerebellar cortex. Neurosci

Lett 332:180–184

Ferrer I, Kulisevski J, Vazquez J, Gonzalez G, Pineda M (1988)

Purkinje cells in degenerative disease of the cerebellum and its

connections: a Golgi study. Clin Neuropathol 7:22–28

Ferrer I, Genıs D, Davalos A, Bernado L, Sant F, Serrano T (1994)

The Purkinje cell in olivopontocerebellar atrophy. A Golgi and

immunocytochemical study. Neuropathol Appl Neurobiol

20:38–46

Fiala JC, Spacek J, Harris KM (2002) Dendritic spine pathology:

cause or consequence of neurological disorders? Brain Res Rev

39:29–54

Foscarin S, Ponchione D, Pajaj E, Leto K, Gawlak M, Wilczynski

GM, Rossi F, Carulli D (2011) Experience-dependent plasticity

and modulation of growth regulatory molecules at central

synapses. PLoS One 6:e16666

Foti F, Laricchiuta D, Cutuli D, De Bartolo P, Gelfo F, Angelucci F,

Petrosini L (2011) Exposure to an enriched environment

accelerates recovery from cerebellar lesion. Cerebellum

10:104–119

Fu M, Yu X, Lu J, Zuo Y (2012) Repetitive motor learning induces

coordinated formation of clustered dendritic spines in vivo.

Nature 483:92–95

Gelfo F, De Bartolo P, Giovine A, Petrosini L, Leggio MG (2009)

Layer and regional effects of environmental enrichment on the

Brain Struct Funct

123

pyramidal neuron morphology of the rat. Neurobiol Learn Mem

91:353–365

Gonzalez-Burgos I, Gonzalez-Tapia D, Zamora DA, Feria-Velasco A,

Beas-Zarate C (2011) Guided motor training induces dendritic

spine plastic changes in adult rat cerebellar Purkinje cells.

Neurosci Lett 491:216–220

Gonzalez-Ramırez MM, Velazquez-Zamora DA, Olvera-Cortes ME,

Gonzalez-Burgos I (2014) Changes in the plastic properties of

hippocampal dendritic spines underlie the attenuation of place

learning in healthy aged rats. Neurobiol Learn Mem 109:94–103

Gray EG (1959) Electron microscopy of synaptic contacts on dendrite

spines of the cerebral cortex. Nature 183:1592–15933

Grutzendler J, Kasthuri N, Gan WB (2002) Long-term dendritic spine

stability in the adult cortex. Nature 420:812–816

Hansel C, Linden DJ (2000) Long-term depression of the cerebellar

climbing fiber–Purkinje neuron synapse. Neuron 26:473–482

Hansel C, Linden DJ, D’Angelo E (2001) Beyond parallel fiber LTD:

the diversity of synaptic and non-synaptic plasticity in the

cerebellum. Nat Neurosci 4:467–475

Harris KM, Kater SB (1994) Dendritic spines: cellular specializations

imparting both stability and flexibility to synaptic function. Annu

Rev Neurosci 17:341–371

Harris KM, Stevens JK (1988) Dendritic spines of rat cerebellar

Purkinje cells: serial electron microscopy with reference to their

biophysical characteristics. J Neurosci 8:4455–4469

Hering H, Sheng M (2001) Dendritic spines: structure, dynamics and

regulation. Nat Rev Neurosci 2:880–888

Holtmaat AJ, Trachtenberg JT, Wilbrecht L, Shepherd GM, Zhang X,

Knott GW, Svoboda K (2005) Transient and persistent dendritic

spines in the neocortex in vivo. Neuron 45:279–291

Hosokawa T, Rusakov DA, Bliss TVP, Fine A (1995) Repeated

confocal imaging of individual dendritic spines in the living

hippocampal slice: evidence for changes in length and orienta-

tion associated with chemically induced LTP. J Neurosci

15:5560–5573

Ito M (1984) The modifiable neuronal network of the cerebellum. Jpn

J Physiol 34:781–792

Johansson BB, Belichenko PV (2002) Neuronal plasticity and

dendritic spines: effect of environmental enrichment on intact

and post-ischemic rat brain. J Cereb Blood Flow Metab

22:89–96

Kasai H, Matsuzaki M, Noguchi J, Yasumatsu N, Nakahara H (2003)

Structure-stability-function relationships of dendritic spines.

Trends Neurosci 26:360–368

Kim HT, Kim IH, Lee KJ, Lee JR, Park SK, Chun YH, Kim H, Rhyu

IJ (2002) Specific plasticity of parallel fiber/Purkinje cell spine

synapses by motor skill learning. Neuro Rep 13:1607–1610

Klintsova AY, Greenough WT (1999) Synaptic plasticity in cortical

systems. Curr Opin Neurobiol 9:203–208

Koch C, Poggio T (1983) A theoretical analysis of electrical

properties of spines. Proc R Soc Lond B Biol Sci 218:455–477

Kozorovitskiy Y, Gross CG, Kopil C, Battaglia L, McBreen M,

Stranahan AM, Gould E (2005) Experience induces structural

and biochemical changes in the adult primate brain. Proc Natl

Acad Sci USA 102:17478–17482

Lai KO, Ip NY (2013) Structural plasticity of dendritic spines: the

underlying mechanisms and its dysregulation in brain disorders.

Biochim Biophys Acta 1832:2257–2263

Larramendi EM, Victor T (1967) Synapses on the Purkinje cell spines

in the mouse. An electronmicroscopic study. Brain Res 5:15–30

Lee KJ, Jung JG, Arii T, Imoto K, Rhyu IJ (2007) Morphological

changes in dendritic spines of Purkinje cells associated with

motor learning. Neurobiol Learn Mem 88:445–450

Lee KF, Soares C, Beıque JC (2012) Examining form and function of

dendritic spines. Neural Plast 2012:704103

Leggio MG, Mandolesi L, Federico F, Spirito F, Ricci B, Gelfo F,

Petrosini L (2005) Environmental enrichment promotes

improved spatial abilities and enhanced dendritic growth in the

rat. Behav Brain Res 163:78–90

Liu N, He S, Yu X (2012) Early natural stimulation through

environmental enrichment accelerates neuronal development in

the mouse dentate gyrus. PLoS One 7:e30803

Lonetti G, Angelucci A, Morando L, Boggio EM, Giustetto M,

Pizzorusso T (2010) Early environmental enrichment moderates

the behavioral and synaptic phenotype of MeCP2 null mice. Biol

Psychiatry 67:657–665

Mandolesi L, De Bartolo P, Foti F, Gelfo F, Federico F, Leggio MG,

Petrosini L (2008) Environmental enrichment provides a cogni-

tive reserve to be spent in the case of brain lesion. J Alzheimers

Dis 15:11–28

Manto M, Oulad Ben Taib N (2010) Cerebellar nuclei: key roles for

strategically located structures. Cerebellum 9:17–21

Marr D (1969) A theory of cerebellar cortex. J Physiol 202:437–470

Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M,

Kasai H (2001) Dendritic spine geometry is critical for AMPA

receptor expression in hippocampal CA1 pyramidal neurons. Nat

Neurosci 4:1086–1092

Matsuzaki M, Honkura N, Ellis-Davies GC, Kasai H (2004) Structural

basis of long-term potentiation in single dendritic spines. Nature

429:761–766

Morando L, Cesa R, Rasetti R, Harvey R, Strata P (2001) Role of

glutamate delta -2 receptors in activity-dependent competition

between heterologous afferent fibers. Proc Natl Acad Sci USA

98:9954–9959

Morando L, Cesa R, Harvey RJ, Strata P (2005) Spontaneous

electrical activity and structural plasticity in the mature

cerebellar cortex. Ann N Y Acad Sci 1048:131–140

Morton SM, Bastian AJ (2004) Cerebellar control of balance and

locomotion. Neuroscientist 10:247–259

Moser MB, Trommald M, Andersen P (1994) An increase in dendritic

spine density on hippocampal CA1 pyramidal cells following

spatial learning in adult rats suggests the formation of new

synapses. Proc Natl Acad Sci USA 91:12673–12675

Muhammad A, Mychasiuk R, Hosain S, Nakahashi A, Carroll C, Gibb

R, Kolb B (2013) Training on motor and visual spatial learning

tasks in early adulthood produces large changes in dendritic

organization of prefrontal cortex and nucleus accumbens in rats

given nicotine prenatally. Neuroscience 252:178–189

Nithianantharajah J, Hannan AJ (2006) Enriched environments,

experience-dependent plasticity and disorders of the nervous

system. Nat Rev Neurosci 7:697–709

Nithianantharajah J, Levis H, Murphy M (2004) Environmental

enrichment results in cortical and subcortical changes in levels of

synaptophysin and PSD-95 proteins. Neurobiol Learn Mem

81:200–210

Noguchi J, Matsuzaki M, Ellis-Davies GC, Kasai H (2005) Spine-

neck geometry determines NMDA receptor-dependent Ca2?

signaling in dendrites. Neuron 46:609–622

Palay SL, Chan-Palay V (1974) Cerebellar cortex: cytology and

organization. Springer, Berlin

Petrinovic MM, Hourez R, Aloy EM, Dewarrat G, Gall D, Weinmann

O, Gaudias J, Bachmann LC, Schiffmann SN, Vogt KE, Schwab

ME (2013) Neuronal Nogo-A negatively regulates dendritic

morphology and synaptic transmission in the cerebellum. Proc

Natl Acad Sci USA 110:1083–1088

Petrosini L, De Bartolo P, Foti F, Gelfo F, Cutuli D, Leggio MG,

Mandolesi L (2009) On whether the environmental enrichment may

provide cognitive and brain reserves. Brain Res Rev 61:221–239

Pysh JJ, Weiss GM (1979) Exercise during development induces an

increase in Purkinje cell dendritic tree size. Science 206:230–232

Brain Struct Funct

123

Ramnani N (2006) The primate cortico-cerebellar system: anatomy

and function. Nat Rev Neurosci 7:511–522

Rhyu IJ, Oda S, Uhm CS, Kim H, Suh YS, Abbott LC (1999a)

Morphologic investigation of rolling mouse Nagoya (tg(rol)/

tg(rol)) cerebellar Purkinje cells: an ataxic mutant, revisited.

Neurosci Lett 266:49–52

Rhyu IJ, Abbott LC, Walker DB, Sotelo C (1999b) An ultrastructural

study of granule cell/Purkinje cell synapses in tottering (tg/tg),

leaner (tg(la)/tg(la)) and compound heterozygous tottering/leaner

(tg/tg(la)) mice. Neuroscience 90:717–728

Rojas JJ, Deniz BF, Miguel PM, Diaz R, Hermel Edo E, Achaval M,

Netto CA, Pereira LO (2013) Effects of daily environmental

enrichment on behavior and dendritic spine density in hippo-

campus following neonatal hypoxia-ischemia in the rat. Exp

Neurol 241:25–33

Rosenzweig MR, Bennett EL (1996) Psychobiology of plasticity:

effects of training and experience on brain and behavior. Behav

Brain Res 78:57–65

Rosenzweig MR, Krech D, Bennet EL, Diamond MC (1962) Effects

of environmental complexity and train on brain chemistry and

anatomy. J Comp Physiol Psychol 55:429–437

Sakurai M (1987) Synaptic modification of parallel fiber-Purkinje cell

transmission in in vitro guinea-pig cerebellar slices. J Physiol

394:463–480

Scarmeas N, Stern Y (2003) Cognitive reserve and lifestyle. J Clin

Exp Neuropsychol 25:625–633

Schmahmann JD (1996) From movement to thought: anatomic

substrates of the cerebellar contribution to cognitive processing.

Hum Brain Mapp 4:174–198

Schreurs BG, Gusev PA, Tomsic D, Alkon DL, Shi T (1998)

Intracellular correlates of acquisition and long-term memory of

classical conditioning in Purkinje cell dendrites in slices of rabbit

cerebellar lobule HVI. J Neurosci 18:5498–5507

Sdrulla AD, Linden DJ (2007) Double dissociation between long-term

depression and dendritic spine morphology in cerebellar Purkinje

cells. Nat Neurosci 10:546–548

Sotelo C, Hillman DE, Zamora AJ, Llinas R (1975) Climbing fiber

deafferentation: its action on Purkinje cell dendritic spines. Brain

Res 98:574–581

Stern Y (2002) What is cognitive reserve? Theory and research

application of the reserve concept. J Int Neuropsychol Soc

8:448–460

Stern Y (2003) The concept of cognitive reserve: a catalyst for

research. J Clin Exp Neuropsychol 25:589–593

Stern Y (2006) Cognitive reserve and Alzheimer disease. Alzheimer

Dis Assoc Disord 20:112–117

Sugawara T, Hisatsune C, Le TD, Hashikawa T, Hirono M, Hattori

M, Nagao S, Mikoshiba K (2013) Type 1 inositol trisphosphate

receptor regulates cerebellar circuits by maintaining the spine

morphology of Purkinje cells in adult mice. J Neurosci

33:12186–12196

Thach WT, Goodkin HP, Keating JG (1992) The cerebellum and the

adaptive coordination of movement. Annu Rev Neurosci

15:403–442

Tremml P, Lipp HP, Muller U, Wolfer DP (2002) Enriched early

experiences of mice underexpressing the beta-amyloid precursor

protein restore spatial learning capabilities but not normal open

field behavior of adult animals. Genes Brain Behav 1:230–241

Turner DA (1984) Conductance transients onto dendritic spines in a

segmental cable model of hippocampal neurons. Biophys J

46:85–96

Van Praag H, Kempermann G, Gage FH (2000) Neural consequences

of environmental enrichment. Nature Rev Neurosci 1:191–198

Vazquez-Sanroman D, Sanchis-Segura C, Toledo R, Hernandez ME,

Manzo J, Miquel M (2013) The effects of enriched environment

on BDNF expression in the mouse cerebellum depending on the

length of exposure. Behav Brain Res 243:118–128

Vecellio M, Schwaller B, Meyer M, Hunziker W, Celio MR (2000)

Alterations in Purkinje cell spines of calbindin D-28k and

parvalbumin knock-out mice. Eur J Neurosci 12:945–954

Velazquez-Zamora DA, Martınez-Degollado M, Gonzalez-Burgos I

(2011) Morphological development of dendritic spines on rat

cerebellar Purkinje cells. Int J Dev Neurosci 29:515–520

Voogd J (1995) Cerebellum. In: Paxinos G (ed) The rat nervous

system, 2nd edn. Academic press, San Diego, pp 277–308

Wallace W, Bear MF (2004) A morphological correlate of synaptic

scaling in visual cortex. J Neurosci 24:6928–6938

Wallace W, Schaefer LH, Swedlow JR (2001) A workingperson’s

guide to deconvolution in light microscopy. Biotechniques

31:1076–1078 1080, 1082 passim

Whalley L, Starr J, Athawes R, Hunter D, Pattie A, Deary IJ

(2000) Childhood mental ability and dementia. Neurology

55:1455–1459

Whalley LJ, Deary IJ, Appleton CL, Starr JM (2004) Cognitive

reserve and the neurobiology of cognitive aging. Ageing Res

Rev 3:369–382

Yang G, Pan F, Gan WB (2009) Stably maintained dendritic spines

are associated with lifelong memories. Nature 462:920–924

Yau SY, Lau BW, Tong JB, Wong R, Ching YP, Qiu G, Tang SW,

Lee TM, So KF (2011) Hippocampal neurogenesis and dendritic

plasticity support running-improved spatial learning and depres-

sion-like behaviour in stressed rats. PLoS One 6:e24263

Zhao C, Warner-Schmidt J, Duman RS, Gage FH (2012) Electro-

convulsive seizure promotes spine maturation in newborn

dentate granule cells in adult rat. Dev Neurobiol 72:937–942

Brain Struct Funct

123