efectos del enriquecimiento ambiental en el desarrollo del snc

Efectos del enriquecimiento ambiental en el desarrollo del SNC

Una aproximación a estrategias neuroprotectoras y neurorescatadoras

Laboratorio de Neurociencia Clínica y Experimental (LaNCE)Euskal Herriko Unibertsitatea

http://www.ehu.es/ehusfera/lance

viernes 12 de noviembre de 2010

http://www.ehu.es/LaNCE


Efectos del enriquecimiento ambiental en el desarrollo del SNC

Una aproximación a estrategias neuroprotectoras y neurorescatadoras

Laboratorio de Neurociencia Clínica y Experimental (LaNCE)Euskal Herriko Unibertsitatea


Enrike G. Argandoña




Desarrollo cortical

Predeterminado genéticamente

Mediado por experiencia


Efectos del entorno en el desarrollo

Lamarck, Haeckel, Darwin and the giraffe


Cambios mediados por la experiencia

Ramon y Cajal Sherrington



APRENDIZAJE SIMPLEAprendizaje simple

Eric Kandel Aplysia californica

martes 16 de junio de 2009

Aprendizaje simple




jueves 18 de junio de 2009






Aprendizaje simple








Aprendizaje simple










Aprendizaje simple








Aprendizaje simple





Eric Kandel






Aprendizaje simple








Aprendizaje simple







Plasticidad sinápticaTEMA IVMECANISMOS SINÁPTICOS DE PLASTICIDAD

Previo a la experiencia, se forman las vías mediante:

Axones alcanzan la estación relay

Axones alcanzan capa IV cortical

Se forman conexiones aleatorias

Posteriormente se produce el desarrollo influido por la experiencia

Muerte celular programada (neurotrofinas)

Cambios en capacidad sináptica

martes 16 de junio de 2009viernes 12 de noviembre de 2010

Plasticidad sinápticaTEMA IVMECANISMOS SINÁPTICOS DE PLASTICIDAD


Reducción 50%

Reasignación sináptica

Convergencia sináptica

Competencia sináptica

Influencias modulatorias


TEMA IV MECANISMOS SINÁPTICOS DE PLASTICIDAD


Reducción 50%

Reasignación sináptica

Convergencia sináptica

Competencia sináptica

Influencias modulatorias

Locus ceruleus (NA)

Nucleos basales (ACh)



TEMA IVMECANISMOS SINÁPTICOS DE

PLASTICIDAD

La plasticidad sináptica es un fenómeno excitatorio (Glutamato)

NMDA (bloqueado por Mg++ y ligado a canal Ca++)

AMPA Na+



Incremento sinapsis/neurona

Incremento actividad neuronal

Incremento demanda metabólica

Modificaciones arquitectura vascular


Age

Exp

erie

nce

med

iate

d c

hang

es

4. week

1., 2. and 3. weeks 4., 5. and 6. weeks 7. and 8. weeks

Precritical period Critical period Postcritical period

Periodo crítico


PERIODO CRÍTICO3ª - 5ª semanas

Age

Exp

erie

nce

med

iate

d c

hang

es

4. week

1., 2. and 3. weeks 4., 5. and 6. weeks 7. and 8. weeks

Precritical period Critical period Postcritical period

Periodo crítico


Periodo crítico


Sistema visualEstudio efectos de la experiencia


Estudio efectos de la experiencia

Modificaciones sobre las condiciones standard

* Enriquecimiento ambiental

* Empobrecimiento ambiental


Privación visual

Métodos invasivos

Inyección TTX

Sutura párpados

Enucleación mono o bilateral

Extirpación retina


Privación visual

Métodos no invasivos

Cría en oscuridad

Implantación lentillas opacas


Descenso densidad celular

Retraso maduración

Anulación cierre periodo crítico

Privación visual


Cortical parameters


Vascular density


48 E.G. ArgandoZa, J.V, Lafaente/Brain Research 732 (1996) 43-51

3.1. Cortical thickness

3.1.1. Control rats

The thickness of the cortex increased from birth up to

14 dpn when it reached a maximum in control rats; after

14 dpn, this parameter decreased slightly.

Cortical thickness in control rats increased from 300

p,m at birth to 500 pm at 7 dpn, or 70% with very high

statistical significance. The second postnatal week, from 7

to 14 dpn, an increase of 587 pm, or 18’ZO,was registered,

but the difference was not statistically significant. At the

third week there was a slight decrease (3%), from 587 to

568 p,m with no significance. Cortical thickness at 60 dpn

was similar to that of 14 dpn, increasing 3Y0from 21 dpn

to reach 587 pm, but without statistical significance (see

Tables 1 and 2).

3.1.2. Dark-reared rats

This behavior was similar in dark-reared rats, but there

was a quantitative difference at each age, with dark-reared

rats scoring lower at all the considered ages, except at 14

dpn.

From birth to 7 dpn there was a statistically significant

50% increase in cortical thickness in dark-reared rats, from

300 to 443 pm. From 7 to 14 dpn the increase was 56Y0,

from 443 to 691 pm, and was highly significant. From 14

to 21 dpn there was a drop from 691 to 525 (25%), which

was very significant. From 21 dpn to 60 dpn there was an

increase of 3%, similar to the one in controls, which was

not significant and reached 543 pm (see Tables 1 and 2).

At 7 dpn cortical thickness was 12% higher in controls,

at 14 dpn it was 1870higher in dark-reared rats, at 21 dpn

it was 13Y0higher in controls and at 60 dpn it was 870

lower in dark-reared rats. The difference was statistically

significant at all ages except at 14 dpn, being very high at

21 and 60 dpn (see Table 1 and Fig. 4).

3.2. Vascular densi~

3.2.1. Controls

The density of vessels increased massively up to 21 dpn

in controls; at this age it reached the maximum level with

a very weak increase from 21 to 60 dpn.

Vascular density increased 12% at the first week, from

44 to 50 vessels per 40000 kmz. From 7 to 14 dpn there

was almost a 50Y0increase, from 50 to 73 vessels. At 21

dpn vascular density increased 54%, from 73 to 112

vessels. At 60 dpn vascular density was similar to that at

21 dpn (113 vessels) with an imperceptible increase. The

increases from 7 to 14 dpn and from 14 to 21 dpn were

highly significant (see Tables 1 and 2).


The changes in this parameter throughout postnatal

development were similar to the changes in controls, but

there were quantitative differences between the two situa-

tions. Vascular density was higher in controls at all ages

p = o , 0 4 p=o,l p=o,oo2 p=o,ol

0 dpn 7 dpn 14 dpn 21 dpn 60 dpn

Fg.

; ’401 p=o,9 p=o,5 p=o,19 p=o,oo12

1204 T-r T

100

80

160

40

200 0 dpn 7 dpn 14 dpn 21 dpn 60 dpn‘c

~

; 30

z I T T p=o,13 P=0,16 p = o . o o l p = o , o o l

Odpn 7 dpn 14 dpn 21 dpn 60 dpn

! Darkreared

! Controls

Fig. 4. Comparison of average measurements between dark-reared and

control groups at each of the ages considered. Horizontal axes show the

age of the animals, Vertical axes show: CT, cortical thickness in pm;

VD, number of intersections between vessels and the overlying grid per

40000 pmz of visual cortex. Nrad, number of vertically oriented intra-

cortical vascular trunks per mm of cortex.

with the exception of 14 dpn, when it was slightly higher

in dark-reared rats.

At the O to 7 dpn period vascular density increased

11%, from 44 to 49 vessels per measured surface. From 7

to 14 dpn there was a 60Y0increase from 49 to 80 vessels.

At 21 dpn the increase in vascular density slowed to 27%,

from 80 to 100 vessels reaching almost the maximum



3.1.1. Control rats













Tables 1 and 2).





dpn.
















3.2.1. Controls



















Fg.

; ’401 p=o,9 p=o,5 p=o,19 p=o,oo12

1204 T-r T

100

80

160

40


~

; 30



! Darkreared

! Controls
















3.1.1. Control rats













Tables 1 and 2).





dpn.
















3.2.1. Controls



















Fg.

; ’401 p=o,9 p=o,5 p=o,19 p=o,oo12

1204 T-r T

100

80

160

40


~

; 30



! Darkreared

! Controls














BRAIN

RESEARCH

ELSEVIER Brain Research 732 (1996) 43-51

Research report

Effects of dark-rearing on the vascularization of the developmental rat visual

cortex

E.G. Argandoiia a’*,J.V. Lafuente b

aDepartment of Nursing I, School of Nursing, Euskal Herriko Unibertsitatea – University of the Basque Country, E-48940 Leioa, Spain

b Department of Neuroscience, School of Medicine, Euskal Herriko Unibertsitatea – University of the Basque Country, E-48940 Leioa, Spain

Accepted 16 April 1996

Abstract

Cerebral vascular density corresponds to metabolic demand, which increases in highly active areas. External inputs play an important

role in the modeling and development of the visual cortex. Experience-mediated development is very active during the first postnatal

month, when accurate simultaneous blood supply is needed to satisfy increased demand. We studied the development of visuaf cortex

vascularization in relation to experience, comparing rats raised in darkness with rats raised in standard conditions. The parameters

measured were cortical thickness, vascular density and number of perpendicular vessels, constituting the first stage of cortical vascular

development. Vessels were stained using butyryl cholinesterase histochemis~, which labels some neurons and microvascularization

(vesselsfrom5 to 50 km). Animalsfromboth groupswere sampledat O,7, 14, 21 and 60 days postnatal. Vascularization of the brainstarts with vertically oriented intracortical vascular trunks whose density decreases notably after birth in rats reared in standard laboratory

conditions. The most striking finding of our work is the significantly lower decrease in the number of these vessels in dark-reared rats.

Our results also show that cortex thickness and vessel density are significantly lower in dark-reared rats. These results suggest that the

absence of visual stimuli retards the maturation of the visuaf cortex including its vascular bed.

Keywords: Striate cortex; Microvascularization; Development; Butyryl cholinesterase; Histochemistry

1. Introduction

The density of the vascular network corresponds to the

metabolic demand in different brain territories, with the

demand increasing in areas with higher synaptic activity.

Thus vascular density, and especially microvascular den-

sity, becomes higher in these areas [6,7,23,34].

After birth, the neonate is exposed to external stimuli

which modulate cortical development, inducing changes

such as the increase of the dendritic tree, the rise of the

ratio of synapses per neuron and the subsequent increase

of the vascular network. All these changes involve the

thickening of the cortex [14,34,35], Most of the cortical

changes induced by experience occur in what is known as

the critical period [16], which takes place around the 3rd

‘ Corresponding author. Department of Neuroscience, University of the

Basque Country, Apartado 699, E-48080 Bilbao, Spain. Fax: +34 (4)

464-9511; E-mail: [email protected]

postnatal week [1,24,30]. In this period, the higher

metabolic demand due to neuronal plasticity mechanisms

gives art extremely important role to adaptive vascular

changes [34]. That is, the external environment induces

vascular changes by an indirect mechanism: cortical devel-

opment brought about by experience induces vascular plas-

ticity to support the increased metabolism. Under standard

rearing conditions blood vessels are essentially completely

developed by the critical period [30].

Although this kind of change takes place all over the

CNS, most studies of the effects of external inputs have

been performed on the striate cortex. The visual system

has a well-defined hierarchical organization which facili-

tates the study of its structures through the interruption of

pathways at different stages or the deprivation of inputs

using either invasive techniques – tetrodotoxin injection

[29]; surgery, such as eyelid suturing [16,17], bilateral or

unilateral enucleation [3], retina removal [12] – or non-in-

vasive ones, such as dark-rearing [8,10,11], or the use of

opaque contact lenses [24]. While all these methods de-

0006-8993/96/$15.00 Copyright 01996 Elsevier Science B.V. All rights reserved.

PII S0006- 8993(96)00485 -4

Privación visual


Privación visual

� .Brain Research 855 2000 137–142

www.elsevier.comrlocaterbres

Research report

Influence of visual experience deprivation on the postnatal development of

the microvascular bed in layer IV of the rat visual cortex

Enrike G. Argandona a,) , Jose V. Lafuente b˜aDepartment of Nursing I, School of Nursing, Euskal Herriko UnibertsitatearUniÕersity of the Basque Country, Leioa, E-48940, Spain

bDepartment of Neuroscience, School of Medicine, Euskal Herriko UnibertsitatearUniÕersity of the Basque Country, Leioa, E-48940, Spain

Accepted 16 November 1999

Abstract

Cerebral vascular density is correlated with metabolic demands, which increase in highly active brain areas. External inputs are an

essential requirement in the modeling of the visual cortex. Experience-mediated development is very active during the first postnatal

month, when congruous blood supply is needed. We studied the development of visual cortex vascularization in relation to experience,

comparing rats raised in darkness with rats reared in normal conditions. Vascular density, vascular area and their ratio vs. neuronal

density were calculated. Conventionally stained semi-thin sections were used to measure the vascular area by computer assisted� .morphometry. Animals from both groups were sampled at 14, 21, and 60 days postnatal dpn . We found a significantly lower density of

vessels and neurons as well as a smaller vascular area in dark-reared adult rats while no differences were founded at the other ages. Our

results also show no differences between the ratio of vesselsrneuron, and vascular arearneuron, between both groups. The absence ofvisual experience causes decrease of cortical activity which correlates with lower vessels density and vascular area, without their

ratiorneuron being affected. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Dark-rearing; Microvascularization; Blood vessel; Striatal cortex; Computer assisted morphometry; Synaptic activity

1. Introduction

There is a close relationship between metabolic activityw xand vascular network in cerebral cortex 3,9,24 . The

metabolic demand, which increases in areas with higher

synaptic activity is matched by the density of blood vesselsw xin these areas 5,6,16,21 . As we have previously reported,

development of vascularization parallels cortical develop-

ment. Our results showed a decrease in vascular density

and a delay in the maturation of the microvascular patternw xin dark-reared rats 1 .

Some authors have studied the effects of visual experi-

ence on the relationship between neuronal density andw xvascularization 7,21 . When animals are reared under

complex visual experience, there is an increase in the

vascular supply, morphologically detectable as increase in

blood vessels diameter, branching of vessels and increase

in vascular area. There are, however, only a few studies onw xanimals under visual deprivation 1,24 .

)Corresponding author. Fax: q34-94-4649-511; e-mail:

[email protected]

Changes in vascular density are related to the different

stages of development. Most of the changes induced by

experience occur during a defined time-window of postna-

tal life called critical period. In this period, there is reorga-

nization of the cortex correlated with experience, whenw xnon-functioning neurons disappear 12,25 . Studies of neu-

ronal density in animals reared in darkness have found a

relative increase due to a decrease in the neuropil; how-

ever, putative changes in neuronal population have notw xbeen addressed 2,10,23 .

Quantification of neurons and vessels has been

performed exclusively on cortical layer IV, where experi-

ence-induced changes occur as this layer is the terminationw xsite of visual afferents coming from the thalamus 2,18,26 .

In this layer, neurons are smaller and more crowded and

there is a higher density of neuronal somata compared to

the other layers. It can thus be inferred that experience-in-

duced changes in neuronal density should be maximal in

layer IV.

We have studied the relationship between visual experi-

ence and vascularization by measuring vascular density

and vascular area in developing normal, and dark-reared

rats. The purpose was to correlate vascular changes with

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.� .PII: S0006-8993 99 02361-6

( )E.G. Argandona, J.V. LafuenterBrain Research 855 2000 137–142˜ 139

Table 1

Increases and decreases in the measured parameters during the periods

!studied for both dark-reared and control groups percentage of

.increaserdecrease and statistical significance, p value! . ! . ! .Age period dpn Darkness % p Controls % p

Increase of Õascular density14–21 51 -0.001 36.4 0.07

21–60 y21.1 -0.001 20 0.001

Increase of neuronal density

14–21 y9.9 0.02 14.3 0.29

21–60 y21.9 -0.001 y13 -0.001

Increase of Õascular area14–21 y20.6 0.3 28.7 0.2

21–60 25.9 0.01 10.7 0.2

Increase of number of Õessels per neuron14–21 66.7 -0.001 0 0.4

21–60 0 0.9 66.7 -0.001

Decrease of Õascular area per neuron14–21 y27.8 0.08 37.7 0.04

21–60 4.3 0.6 y2.6 0.67

Decrease of aÕerage Õascular area14–21 23.7 0.03 45.8 0.05

21–60 2.1 0.8 26.6 0.03

60 days, there was an increase of 20% which was not

statistically significant.

In dark-reared rats, the number of vessels increased by

51% between 14 and 21 dpn and decreased by 21% from

the 21 to 60 dpn period. Both variations were significant.

Comparing both groups, at 14 dpn, vascular density in

dark-reared rats was 7% higher and at 21 dpn it was 18%

higher. On the other hand, at 60 dpn, vascular density was

22% higher in controls. Differences were statistically sig-! .nificant at 21 and 60 dpn. Fig. 1 .

3.2. Neuronal density

The number of neurons per 2500 mm2 was similar in

all ages in normal rats, being maximal at 21 dpn. Between

14 and 21 dpn, neuronal density increased by 14% but

decreased between 21 and 60 dpn by 13%.

As it happened with the vascular density, the second

increase from 21 to 60 dpn was significant but not the

former from 14 to 21 dpn.


development in the dark-reared group were the opposite of

controls as there was a progressive decrease from 14 to 60

dpn. From 14 to 21 dpn, this parameter decreased by 10%,

from 16 to 15 neurons per 2500 mm2. The decrease was! .more noticeable from 21 to 60 dpn 22% , diminishing

from 15 to 11 neurons. Both differences were statistically

significant.

Comparison between both groups showed a lower neu-

ronal density in dark-reared rats, with the exception of 14



age of the animals. Vertical axes show: Vd, number of vessels per 2500

mm2 of visual cortex; Nd, number of neurons per 2500 mm2 of visual

cortex; Vs, area occupied by vessels per 2500 mm2 of visual cortex.

! ! !


Privación visualDevelopmental Brain Research 141 (2003) 63–69

www.elsevier.com/ locate /devbrainres

Research report

V isual deprivation effects on the s100b positive astrocytic populationin the developing rat visual cortex: a quantitative study

a , b c*˜Enrike G. Argandona , Marco L. Rossi , Jose V. LafuenteaDepartment of Nursing I, School of Nursing, Euskal Herriko Unibertsitatea /University of the Basque Country, Leioako Campusa, Leioa E-48940,

SpainbDepartment of Neuropathology, Walton Centre for Neurology and Neurosurgery, Liverpool L9 7LJ, UK

cDepartment of Neuroscience, School of Medicine, Euskal Herriko Unibertsitatea /University of the Basque Country, Leioa, E-48940 Spain

Accepted 11 December 2002

Abstract

After birth, exposure to visual inputs modulates cortical development, inducing numerous changes of all components of the visual

cortex. Most of the cortical changes thus induced occur during what is called the critical period. Astrocytes play an important role in the

development, maintenance and plasticity of the cortex, as well as in the structure and function of the vascular network. Dark-reared

Sprague–Dawley rats and age-matched controls sampled at 14, 21, 28, 35, 42, 49, 56 and 63 days postnatal (dpn) were studied in order to

elucidate quantitative differences in the number of positive cells in the striate cortex. The astrocytic population was estimated by

immunohistochemistry for S-100b protein. The same quantification was also performed in a nonsensory area, the retrosplenial granular

cortex. S-100b positive cells had adult morphology in the visual cortex at 14 dpn and their numbers were not significantly different in

light-exposed and nonexposed rats up to 35 dpn, and were even higher in dark-reared rats at 21 dpn. However, significant quantitative

changes were recorded after the beginning of the critical period. The main finding of the present study was the significantly lower

astroglial density estimated in the visual cortex of dark-reared rats over 35 dpn as well as the lack of difference at previous ages. Our

results also showed that there were no differences when comparing the measurements from a nonsensory area between both groups. This

led us to postulate that the astrocytic population in the visual cortex is downregulated by the lack of visual experience.

! 2002 Elsevier Science B.V. All rights reserved.

Keywords: Dark-rearing; Immunohistochemistry; Astroglia; Retrosplenial cortex; RSG

1 . Introduction vascular density as well as a delay in the development of

the microvascular pattern [2,3].

Postnatal development of the visual cortex is modulated Astrocytes play an important role in the maintenance of

by experience. Extrinsic cues act as epigenetic factors in the structure and function of the endothelium of the

concert with intrinsic developmental programmes to shape cortex’s microvascular network, including the blood–brain

functional and structural cortical architecture [29]. Ex- barrier [8,20]. Astrocytes are also involved in the develop-

perience-mediated changes induce an increase in neuronal ment, plasticity and maintenance of the cerebral cortical

activity, which leads to increased metabolic demands architecture [10,23,24,27]. Therefore, a crucial role in

[6,40], involving the establishment of adaptive changes to coupling neuronal activity to energy metabolism has been

accomplish new requirements such as changes of the proposed [13]. Inversely, both neuronal activity and vas-

vascular network [5,7]. In previous works, we reported the culature influence glial development in a bidirectional

development of vascularisation of the visual cortex in manner [1,18,22,41].

normal and dark-reared rats showing a decrease in the During development, astrocytes guide cortical organisa-

tion by performing different functions, which are reflected

in morphological, electrophysiological and antigenic dif-

ferences [22]. The behaviour of the glia throughout the*Corresponding author. Tel.: 134-94-601-5595; fax: 134-94-464-

postnatal development of the cerebral cortex can be studied9511.

˜E-mail address: [email protected] (E.G. Argandona). using several antigenic markers. The most frequently used

0165-3806/02/$ – see front matter ! 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0165-3806(02)00643-0

˜E.G. Argandona et al. / Developmental Brain Research 141 (2003) 63–69 65

2formed in a smaller grid (10 000 mm placed exclusively density was higher in dark-reared animals in both visual

on layer IV). For both cases, three horizontally contiguous and retrosplenial cortexes. Furthermore, no differences

measurements were taken from each hemisphere, always were found in the retrosplenial cortex at any of the ages.

on the same layer. Similar results were obtained from areas exclusively

Astrocytes were recognised by their morphological placed on layer IV, with similar differences between both

characteristics, i.e. S100 positive cell bodies with short groups, with the same significance.

positive cytoplasmic processes, and by their nuclear mor- Immunoreactivity for the S-100b antibody had adult

phology. We did not count positive cells that did not fit the morphology at 14 dpn, with strongly stained cell bodies

morphology of astrocytes. and star shaped processes. S-100b positive cells were

Measurements of each slice of the cortex were made in widely present on all cortical layers in opposition to GFAP,

both hemispheres, for each of the ten slices taken per which was almost absent in middle areas while only some

animal (i.e. 60 fields per animal) and the mean value per isolated astrocytes were present in lower and upper areas.

animal was calculated. The average values per group (eight Outside the cortex, both GFAP and S-100b were present,

animals) were compared at each age by statistical analysis especially in regions such as the hippocampus (Figs. 1 and

(ANOVA) on STATVIEW II! Abacus Concepts. 2).

All animal experiments were performed in accordance Comparing both experimental groups at all ages, no

with the European Community Council Directive of 24 morphological differences were found. Thus, we per-

November 1986 (86/609/EEC). formed a quantitative analysis in order to study the

possible differences in the number of astrocytes per area.

The results are shown in Table 1. Quantifying the number

3 . Results of positive cells per cortical surface, the following results

were obtained.

We found a significantly lower number of cells per unit

area in the visual cortex of dark-reared rats at 35 dpn, 3 .1. Visual cortex

whereas no differences were found between both groups in

younger animals, with the exception of 21 dpn, when The density of S-100b positive cells suffered slight

Fig. 1. (a) S100b positivity throughout the visual cortex at 5 weeks postnatal in dark-reared rats. (b) S100b positivity throughout the visual cortex at 5

weeks postnatal in control rats. (c) GFAP positivity throughout the visual cortex at 5 weeks postnatal in dark-reared rats. Positive cells appear mostly in

lower and upper layers being almost absent in middle layers. Scale line is 150 mm.

˜66

E.G

.Argandonaetal.

/DevelopmentalBrainResearch

141 (

2003)

63–69

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Oscuridad Control

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Empobrecimiento ambiental

Corteza somatosensorialCorteza barrel ratas. Afeitado vibrisas produce alteraciones morfologicas y fisiologicas


Empobrecimiento ambiental

* Privación olfativa

* Privación auditivaSimilares efectos al resto de sentidos, pero de mayor intensidad

Un elemento común es la plasticidad compensatoria en los sentidos no empobrecidos

Neuron, Vol. 46, 103–116, April 7, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.neuron.2005.02.016

Activity-Dependent Adjustmentsof the Inhibitory Network in the Olfactory Bulbfollowing Early Postnatal Deprivation

Armen Saghatelyan,1 Pascal Roux,2 environmental conditions. The continuous postnatal sup-ply of newborn inhibitory interneurons to the main olfac-Michele Migliore,3,5 Christelle Rochefort,1,6

tory bulb (MOB) offers an ideal system to study neu-David Desmaisons,1 Pierre Charneau,4ronal adjustment regulated by sensory experiences.Gordon M. Shepherd,3 and Pierre-Marie Lledo1,*Progenitor cells originating from the subventricular zone1Laboratory of Perception and Memory(SVZ) of the lateral ventricle first migrate tangentially toPasteur Institutethe MOB, by way of the rostral migratory stream (RMS),Centre National de la Rechercheand then migrate radially within MOB before they dif-Scientifique (URA 2182)ferentiate into local interneurons (Luskin, 1993; Lois and75015 Paris CedexAlvarez-Buylla, 1994). It has been hypothesized that post-Francenatal neurogenesis is controlled by levels of sensory2Platform of Dynamic Imagingactivity (Frazier-Cierpial and Brunjes, 1989; Corotto etPasteur Instituteal., 1994; Kirschenbaum et al., 1999; Saghatelyan et al.,25 rue du Dr. Roux2003; Lledo et al., 2004). Hence, although proliferation75015 Paris Cedexand tangential migration of neuroblasts are not affectedFranceby bulbar removal (Kirschenbaum et al., 1999) or nostril3Department of Neurobiologyocclusion (Frazier-Cierpial and Brunjes, 1989; but seeYale Medical Schoolalso Corotto et al., 1994), decreased radial migration333 Cedar Street(Saghatelyan et al., 2004) and increased neuronal deathNew Haven, Connecticut 06510(Frazier-Cierpial and Brunjes, 1989; Najbauer and Leon,4Laboratory of Molecular Virology and Vectorology1995; Fiske and Brunjes, 2001) have been both re-Pasteur Instituteported in deprived bulbs. In contrast, odor enrichment28 rue du Dr. Rouxincreases the number of newborn granule cells and im-75724 Parisproves olfactory memory (Rochefort et al., 2002). It isFrancenoteworthy that granule cells provide inhibition ontooutput bulbar neurons (i.e., mitral/tufted cells) that isresponsible for their oscillatory synchronization (Fried-

Summary man and Strowbridge, 2003; Lagier et al., 2004), andthus for information processing (Laurent et al., 2001;

The first-order sensory relay for olfactory processing, Lledo and Gheusi, 2003). Yet, it is unknown whether thethe main olfactory bulb (MOB), retains the ability to newborn interneurons actively participate in the oscilla-acquire new interneurons throughout life. It is there- tory synchronization of principal cells, or what theirfore a particularly appropriate region for studying the functional implications are for overall bulbar activity. Inrole of experience in sculpting neuronal networks. We principle, any variation in the number and/or the mor-found that nostril closure decreased the number of phology of newborn neurons might disrupt the activitynewborn granule cells in the MOB, the complexity of of bulbar output neurons and thus information process-their dendritic arborization, and their spine density, ing. A small number of studies have attempted to ad-without affecting the preexisting population of gran- dress these issues with unexpected results. For in-ule cells. Accordingly, the frequency of miniature syn- stance, measurements of the paired-pulse inhibition ofaptic inhibitory events received by mitral cells was evoked potentials following stimulation of lateral olfac-reduced. However, due to a compensatory increase tory tract in control and odor-deprived bulbs demon-in newborn granule cell excitability, action potential- strated that a reduced number of newborn interneuronsdependent GABA release was dramatically enhanced, surprisingly increased the level of inhibition received bythus counteracting the reduction in spine density and mitral cells (Wilson et al., 1990; Wilson and Wood, 1992;leading to an unaltered synchronization of mitral cell Wilson, 1995).firing activity. Together, this study reveals a unique Here, using unilateral odor deprivation in combina-form of adaptive response brought about exclusively tion with quantitative immunohistological, biphoton,by the cohort of newborn cells and used to maintain and electrophysiological analyses, we explore the func-normal functioning of the MOB. tional consequences of reduced neurogenesis on the

activity of the bulbar network. We show that sensorydeprivation specifically reduces the dendritic length,Introductionspine density, and total number of newborn but not ofpreexisting granule cells. As a consequence, action po-The functional properties of networks in the brain cantential- and glutamate-independent GABA release isbe adjusted to constantly changing developmental andimpaired. Interestingly, however, sensory deprivationalso strengthens action potential-dependent GABA re-

*Correspondence: [email protected] lease, since newborn granule cells were more excit-5 Permanent address: Institute of Biophysics, National Researchable. Such a compensatory effect preserves the overallCouncil, Palermo, Italy.inhibition and thus the spatiotemporal synchronization6 Present address: Max-Planck Institute for Molecular Genetics,

14195 Berlin, Germany. of output neurons. Together, this study demonstrates


Enriquecimiento ambiental

Donald Hebb (1949)

Kresh, Bennett, Rosenzweig, Diamond (60s)

Combinación de complejidad de objetos

inanimados y estimulación social.



* Necesidad de estandarizar

* Super-enriquecimiento

* Rol del ejercicio



Cambios anatómicos

Plasticidad neuronal

Sinaptogénesis

Morfología sináptica

Neurogénesis

Neurotrofinas (BDNF, NGF, NT-3, VEGF)

Gliogénesisviernes 12 de noviembre de 2010


* Corteza auditiva (Greenough, 1973)

* Corteza olfatoria (Roselli-Austin, 1990)

* Corteza somatosensorial (Coq, 1998)

* Hipocampo (Rampon, 2000)

* Amigdala (Nikolaev. 2002)

* Ganglios basales (Comery, 1996)

* Cerebelo (Greenough, 1986)



Mejora aprendizaje y memoria (Dash, 2009)

Reduce deterioro cognitivo fisiologico (Segovia, 2009)

Reduce ansiedad e incrementa actividad exploratoria (Benaroya, 204)

Induce neurogenesis en hipocampo (Kempermann 1997)

Reduce comportamientos adictivos a drogas (Solinas 2010)

Madura y consolida la corteza visual en ratas privadas de luz (Bertoletti 2004)

Acelera el desarrollo de la corteza visual (Cancedda 2004)


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12

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Patología SNC

TCE

Ictus

Tumores

Patologías neurodegenerativas


Patología SNC

TCE

Ictus

Tumores

Patologías neurodegenerativas

Vascularización


Objetivos terapeúticos

Neuroprotección/neurorescate

Incremento vascularización


TCE en Desarrollo

Mayor capacidad de plasticidad

Interferencia en los mecanismos fisiológicos

Apoptosis

Plasticidad sináptica (NMDA)


Reserva Cerebral Cognitiva (Nithianantharajah, 2006)


Enfermedades neurodegenerativas

Alzheimer: reduce deposito ß amiloide (Cracchiolo, 2007), facilita su eliminación (herring, 2008), mejora deterioro cognitivo (Levi, 2003)

Hungtington: disminuye deterioro cognitivo (Hannan, 2008)

Parkinson: aumenta resistencia MPTP, (Thiriet, 2008); reduce deterioro estriado (Bezard, 2003)

S. Rett y Down reduce sintomas motores y cognitivos. (Martinez-Cue, 2005); (Kondo 2008)


Isquemia

Disminuye secuelas (Saucier, 2010)

Facilita migracion celulas SVZ (Hicks, 2007)

Mejora recuperación funcional (Briones, 2009)

Disminuye amiloidogenesis (Briones, 2009)


TCE

Promueve recuperacion funcion cognitiva (Hamm, 1996)

Reduce daño BHE (Ortuzar, 2010)

Disminuye muerte neuronal y mejora angiogenesis (Ortuzar, 2010)

Recuperacion en rehabilitacion postraumática (Penn, 2009)


Tumores

Volume 142, Issue 1, 9 July 2010, Pages 52-64

Article

Environmental and Genetic Activation of a Brain-Adipocyte BDNF/Leptin Axis Causes Cancer Remission and Inhibition


http://www.sciencedirect.com/science/journal/00928674

http://www.sciencedirect.com/science/journal/00928674

http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%237051%232010%23998579998%232204734%23FLA%23&_cdi=7051&_pubType=J&view=c&_auth=y&_acct=C000049823&_version=1&_urlVersion=0&_userid=984461&md5=6bb0c7ac9f4c3f76aeaba7b8d7f43e09

http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%237051%232010%23998579998%232204734%23FLA%23&_cdi=7051&_pubType=J&view=c&_auth=y&_acct=C000049823&_version=1&_urlVersion=0&_userid=984461&md5=6bb0c7ac9f4c3f76aeaba7b8d7f43e09

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anti-VEGF. 25 µg/ml.

PBS.

Untreated rats.


BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE

Combination of intracortically administered VEGFand environmental enrichment enhances brain protectionin developing rats

Naiara Ortuzar • Enrike G. Argandona •

Harkaitz Bengoetxea • Jose V. Lafuente

Received: 8 September 2010 / Accepted: 24 September 2010! Springer-Verlag 2010

Abstract Postnatal development of the visual cortex ismodulated by experience, especially during the critical

period. In rats, a stable neuronal population is only

acquired after this relatively prolonged period. Vascularendothelial growth factor (VEGF) is the most important

angiogenic factor and also has strong neuroprotective,

neurotrophic and neurogenic properties. Similar effectshave been described for rearing in enriched environments.

Our aim is to investigate the vascular and neuronal effects

of combining VEGF infusion and environmental enrich-ment on the visual cortex during the initial days of the

critical period. Results showed that a small percentage of

Cleaved Caspase-3 positive cells colocalized with neuronalmarkers. The lesion produced by the cannula implantation

resulted in decreased vascular, neuronal and Caspase-3

positive cell densities. Rearing under enriched environmentwas unable to reverse these effects in any group, whereas

VEGF infusion alone partially corrected those effects. A

higher effectiveness was reached by combining both theprocedures, the most effective combination being when

enriched-environment rearing was introduced only afterminipump implantation. In addition to the angiogenic

effect of VEGF, applied strategies also had synergic neu-roprotective effects, and the combination of the two strat-

egies had more remarkable effects than those achieved by

each strategy applied individually.

Keywords Critical period ! Enriched environment !Neuroprotection ! Neurovascular unit ! VEGF !Visual system

Introduction

The development of the central nervous system (CNS), and

more specifically of the sensory systems, is modulated by

experience. This leads to an increase in metabolic demand(Black et al. 1990) that is satisfied by the adaptive

remodelling of the vascular network (Argandona and

Lafuente 1996, 2000). Postnatal development of the visualcortex occurs in two stages. The first is genetically pre-

determined and the second modulated by experience. Most

of the cortical changes induced by experience occur duringthe critical period (Hensch 2005). This time window is

specific for each sensory cortex and when experience-mediated reorganization finishes, sensory functions reach

maturity (Bengoetxea et al. 2008). In rats, the critical

period for the visual system is located between the thirdand the fifth postnatal weeks and the maximum peak of

experience-induced changes occurs during the fourth and

the fifth weeks (Fagiolini et al. 1994; Fagiolini and Hensch2000).

During development, more than half of the initially

formed neurons die by programmed cell death (PCD),which is of fundamental importance for the correct devel-

opment of the CNS (Finlay 1992). PCD is highly regulated

during development and is maintained under strict control

N. Ortuzar (&) ! E. G. Argandona ! H. Bengoetxea !J. V. LafuenteDepartment of Neuroscience, Laboratory of Clinical andExperimental Neuroscience (LaNCE), Faculty of Medicine andOdontology, University of the Basque Country UPV/EHU,Barrio Sarriena s/n, E48940 Leioa, Spaine-mail: [email protected]

E. G. ArgandonaDepartment of Nursing I, University of the Basque CountryUPV/EHU, Barrio Sarriena, E48940 Leioa, Spain

123

J Neural Transm

DOI 10.1007/s00702-010-0496-2

Quantitative results

Control group

Vascular density Groups including enriched environment(EE–SC, SC–EE and EE-EE) showed a statistically sig-

nificant difference when compared to the SC–SC group.

Observed differences were 28, 35 and 19% higher,respectively (P = 0.000 for all groups) when an enriched

environment was applied (Fig. 5a).

Neuronal density Only the EE–SC group presented astatistically significant difference when compared to SC–

SC. Neuronal density was 16% (P = 0.011) higher when

the enriched environment was applied. Neither the SC–EEnor the EE–EE group showed significant differences when

compared to SC–SC and the densities obtained were 1%

(P = 1.000) and 10% (P = 0.203) higher, respectively,than in the group reared in standard conditions (Fig. 5b).

Cleaved Caspase-3 density The density of the EE–SC

group was 22% lower than that of the SC–SC group, whichis a statistically significant difference (P = 0.000). The

SC–EE and EE–EE groups also showed lower density but

these differences were not significant (10%, P = 0.184;7%, P = 0.880, respectively) (Fig. 5c).

PBS group

Vascular density Vascular density was 18% (P = 0.091),

9% (P = 1.000) and 8% (P = 1.000) higher in the EE–SC,SC–EE and EE–EE groups, respectively, when compared

to the SC–SC group. None of these differences were sta-

tistically significant (Fig. 5a).

Neuronal density No significant differences were found

between studied groups. Neuronal density was 9%(P = 0.972) lower in the EE–SC group when compared to

SC–SC. A similar tendency was observed in both the SC–

EE and the EE–EE groups where the density was 11%(P = 0.902) and 6% (P = 0.983) lower, respectively

(Fig. 5b).

Cleaved Caspase-3 density Cleaved Caspase-3 densityincreased by about 16% (P = 1.000) in the EE–SC group

and 30% (P = 0.225) in the SC–EE group when compared

to SC–SC, with no statistically significant differences. Onlythe EE–EE group resulted in a statistically demonstrated

change (40%, P= 0.040) (Fig. 5c).

VEGF Group

Vascular density Only the SC–EE group resulted in astatistically significant difference (P = 0.019) when com-

pared to SC–SC. Observed density was 16% higher when

the enriched environment was applied. Not only did the EE–SC and EE–EE groups show any statistically significant

differences (10%, P = 0.724 and 1%, P = 1.000, respec-

tively), but they also showed a lower density when com-pared to rats reared under standard conditions (Fig. 5a). The

SC–EE group had the highest vascular density.

Neuronal density No significant differences wereobserved. Both the EE–SC and the SC–EE groups showed

lower neuronal density than the SC–SC group, with results

that were 15% (P = 0.064) and 7% (P = 0.861) lower,respectively. The EE–EE group showed an 18%

(P = 0.499) higher density when compared to the group

reared in standard conditions (Fig. 5b).

Fig. 4 Primary visual cortex images for quantified vascular, neuronaland Caspase-3 positive cell densities. Sections were stained bybutyryl cholinesterase histochemistry (a, b), NeuN (c, d) and Cleaved

Caspase-3 (e, f) immunohistochemistry. Densities were estimated bythe optical dissector method. Scale bar = 100 lm (a, c, e) and 20 lm(b, d, f)

VEGF and EE enhances brain protection in rats

123

in adulthood. The rat visual system is characterized by

postnatal modulation, and a stable neuronal population isonly acquired after a relatively prolonged postnatal period

(Papadopoulos and Michaloudi 1999). Caspases and their

substrates are essential in the implementation of PCD, asCaspase-3 is the most abundant cysteine protease in the

brain (McLaughlin 2004). Apart from during development,

it has been demonstrated that Caspase-3 is also activatedduring apoptotic cellular death induced by pathological

processes such as neurodegenerative diseases (Mellioset al. 2009), and, to a lesser extent, by traumatic brain

injuries or stroke (Lafuente et al. 2007). One of the most

important mechanisms of neuronal survival consists of themetabolic chains induced by neurotrophins. Recent studies

have shown that both neurotrophins and VEGF play key

roles as antiapoptotic agents (Storkebaum et al. 2004).Vascular endothelial growth factor (VEGF) is the major

angiogenic factor in developmental (Ferrara et al. 2003)

and pathological angiogeneses, and also in pathologicalvascular permeability (Dvorak 2000). The role of VEGF in

nervous tissue is even more extensive. Previous studies

show that VEGF also has strong neuroprotective, neuro-trophic, and neurogenic properties (Jin et al. 2002;

Rosenstein and Krum 2004; Storkebaum et al. 2004). The

neuroprotective function of VEGF includes direct andindirect actions, and appears to be due to a combination of

direct neuroprotective effects and the stimulation of

angiogenesis.Molecules that affect both the neural and the vascular

cell processes have recently been termed angioneurins

(Zacchigna et al. 2008). Although the archetypal angio-neurin is VEGF, neurotrophins like brain-derived neuro-

trophic factor (BDNF) also belong to this family.

Similar effects have been described for enrichedenvironments (EE). Countless studies have reported

changes induced by this paradigm during CNS develop-

ment, not only in standard conditions but also in patho-logical conditions (Nithianantharajah and Hannan 2009).

EE has strong effects on the plasticity of neural con-

nections, especially in the visual cortex, where it hasbeen demonstrated that rearing from birth in an enriched

environment leads to an acceleration of visual develop-

ment (Cancedda et al. 2004). In addition, EE increasesthe expression of some factors referred to above that play

an important role in neuronal trophism, such as VEGF or

BDNF (Bengoetxea et al. 2008; Ickes et al. 2000; Phamet al. 2002).

The aim of this study is to investigate the vascular and

neuronal effects of combined VEGF infusion and envi-ronmental enrichment in the visual cortex during the initial

days of the critical period.

Materials and methods

Animals and housing

5 Long-Evans rats (Harlan, Barcelona, Spain) were used

for each experimental group with different rearing condi-

tions (Fig. 1):

1. Standard condition (SC) rats raised in standard labo-

ratory cages (500 mm 9 280 mm 9 140 mm) with a12-h light/dark cycle.

2. Enriched environment (EE) rats raised in large cages

(720 mm 9 550 mm 9 330 mm) furnished with col-ourful toys and differently shaped objects (shelters,

tunnels) that were changed every 2 days (with a 12-h

light/dark cycle).

All animals had access to food and water ad libitum.

Minipump implantation

Experiments were performed on Long-Evans rats shortly

before the beginning of the critical period (P18). Animalswere anaesthetized with Avertin (1 ml/100 g) and after-

wards underwent a sagittal incision midway on the scalp.

Skin and periosteum were retracted. A subcutaneouspocket was opened in the back for the osmotic minipump

placement (Mod. 1007 D, Alzet, Cupertino, CA, USA).

The brain infusion kit (Mod. Alzet Brain Infusion Kit III,Alzet) was fixed to the skull with cyanoacrylate and the

Fig. 1 Rearing conditions.a Standard condition andb enriched environment

N. Ortuzar et al.

123

Quantitative results

Control group

Vascular density Groups including enriched environment(EE–SC, SC–EE and EE-EE) showed a statistically sig-

nificant difference when compared to the SC–SC group.

Observed differences were 28, 35 and 19% higher,respectively (P = 0.000 for all groups) when an enriched

environment was applied (Fig. 5a).

Neuronal density Only the EE–SC group presented astatistically significant difference when compared to SC–

SC. Neuronal density was 16% (P = 0.011) higher when

the enriched environment was applied. Neither the SC–EEnor the EE–EE group showed significant differences when

compared to SC–SC and the densities obtained were 1%

(P = 1.000) and 10% (P = 0.203) higher, respectively,than in the group reared in standard conditions (Fig. 5b).

Cleaved Caspase-3 density The density of the EE–SC

group was 22% lower than that of the SC–SC group, whichis a statistically significant difference (P = 0.000). The

SC–EE and EE–EE groups also showed lower density but

these differences were not significant (10%, P = 0.184;7%, P = 0.880, respectively) (Fig. 5c).

PBS group

Vascular density Vascular density was 18% (P = 0.091),

9% (P = 1.000) and 8% (P = 1.000) higher in the EE–SC,SC–EE and EE–EE groups, respectively, when compared

to the SC–SC group. None of these differences were sta-

tistically significant (Fig. 5a).

Neuronal density No significant differences were found

between studied groups. Neuronal density was 9%(P = 0.972) lower in the EE–SC group when compared to

SC–SC. A similar tendency was observed in both the SC–

EE and the EE–EE groups where the density was 11%(P = 0.902) and 6% (P = 0.983) lower, respectively

(Fig. 5b).

Cleaved Caspase-3 density Cleaved Caspase-3 densityincreased by about 16% (P = 1.000) in the EE–SC group

and 30% (P = 0.225) in the SC–EE group when compared

to SC–SC, with no statistically significant differences. Onlythe EE–EE group resulted in a statistically demonstrated

change (40%, P= 0.040) (Fig. 5c).

VEGF Group

Vascular density Only the SC–EE group resulted in astatistically significant difference (P = 0.019) when com-

pared to SC–SC. Observed density was 16% higher when

the enriched environment was applied. Not only did the EE–SC and EE–EE groups show any statistically significant

differences (10%, P = 0.724 and 1%, P = 1.000, respec-

tively), but they also showed a lower density when com-pared to rats reared under standard conditions (Fig. 5a). The

SC–EE group had the highest vascular density.

Neuronal density No significant differences wereobserved. Both the EE–SC and the SC–EE groups showed

lower neuronal density than the SC–SC group, with results

that were 15% (P = 0.064) and 7% (P = 0.861) lower,respectively. The EE–EE group showed an 18%

(P = 0.499) higher density when compared to the group

reared in standard conditions (Fig. 5b).

Fig. 4 Primary visual cortex images for quantified vascular, neuronaland Caspase-3 positive cell densities. Sections were stained bybutyryl cholinesterase histochemistry (a, b), NeuN (c, d) and Cleaved

Caspase-3 (e, f) immunohistochemistry. Densities were estimated bythe optical dissector method. Scale bar = 100 lm (a, c, e) and 20 lm(b, d, f)

VEGF and EE enhances brain protection in rats

123


Neuronal density


Neu

rona

l Den

sity

(Op

tica

l dis

sect

or)


Densidad vascular

0

5.500

11.000

16.500

22.000

SC EA Lesion Lesion EA Lesion SC-EA Lesion EA-SC

20061

1844918.344

16.935

21.694

18.149


Densidad neuronal

0

25.000

50.000

75.000

100.000


626426110162.64267.016

90.813

82.161


Densidad Caspasa3

0

5.500

11.000

16.500

22.000


16738

18802

20.254

14.459

19.680

21.110


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