Progress in Brain Research, Vol. 147ISSN 0079-6123Copyright � 2005 Elsevier BV. All rights reserved

CHAPTER 10

Structural plasticity in the developing visual system

Matt Bence and Christiaan N. Levelt*

Netherlands Ophthalmic Research Institute, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands

Abstract: The visual system has been used extensively to study cortical plasticity during development. Seminal

experiments by Hubel and Wiesel (Wiesel, T.N. and Hubel, D.H. (1963) Single cell responses in striate cortex of kittensdeprived of vision in one eye. J. Neurophysiol., 26: 1003–1017.) identified the visual cortex as a very attractive model forstudying structural and functional plasticity regulated by experience. It was discovered that the thalamic projections to

the visual cortex, and neuronal connectivity in the visual cortex itself, were organized in alternating columns dominatedby input from the left or the right eye. This organization was shown to be strongly influenced by manipulating binocularinput during a specific time point of postnatal development known as the critical period. Two chapters in this volume

review the molecular and functional aspects of this form of plasticity. This chapter reviews the structural changes thatoccur during ocular dominance (OD) plasticity and their possible functional relevance, and discusses developments inthe methods that allow the analysis of the molecular and cellular mechanisms that regulate them.

Keywords: plasticity; cortex; ocular dominance; critical period; deprivation; structural; morphology

Structural organization of the visual cortex

The visual system is organized in such a way that

visual information from the left visual field is

processed in the right visual cortex and vice versa

(Fig. 1). To achieve this, projections from the right

sides of both retinas project to the right lateral

geniculate nucleus (LGN) of the thalamus, while

projections from the left sides of both retinas project

to the left LGN (for a review see Casagrande et al.,

2002). Here, the inputs of both eyes are segregated,

giving rise to the layered structure of the LGN

(Fig. 2). Projections to the next relay station, the

stellate neurons in layer 4 of the primary visual

cortex, are also segregated to a large extent and form

columns dominated by the left and the right eye.

Integration of binocular input takes place pre-

dominantly in the pyramidal layers, layers 2/3, 5 and

6, which all receive input from layer 4. Pyramidal

cells in these layers form long-ranging horizontal

connections with each other. Apart from connections

within primary visual cortex (V1), layer 2/3 neurons

also provide output to higher visual areas. Layer 5

pyramidal neurons project back to the superior

colliculus and pulvinar, while layer 6 neurons project

back to the upper part of layer 4 and to the LGN.

Ocular dominance plasticity

The establishment of this circuitry is regulated both

by molecular cues and electrical activity. Although

the initial development of the connectivity does not

seem to depend on visual experience, its maintenance

and adjustment do. For example, when one eye is

closed during the critical period, geniculocortical

projections of the nondeprived eye gain territory

in the primary visual cortex, while the deprived

projections shrink (Wiesel and Hubel, 1963; Hubel

et al., 1977; Shatz and Stryker, 1978). This structural

change also has a functional correlate. Neuronal

responses can be classified according to their*Corresponding author. Tel.: +31-20-5666101;

Fax: +31-20-5666121; E-mail: c.levelt@ioi.knaw.nl

DOI: 10.1016/S0079-6123(04)47010-1 125

responsiveness to stimuli presented to either eye, class

1 being responsive to the contralateral eye only, and

class 7 responsive to the ipsilateral eye only. Most

neurons in layer 4 will be of class 1/2 or 6/7, while in

the extragranular layers, more neurons are detected

in the intermediate classes. Upon monocular depriva-

tion, the majority of neurons in all layers will be

monocular and responsive primarily to the eye that

was open during development (for reviews see Katz

and Shatz, 1996; Bear and Rittenhouse, 1999).

When correlated activity between the inputs from

both eyes is reduced during the critical period by

misalignment of the two eyes (strabismus), responses

of neurons in all layers of the visual cortex become

Fig. 1. Organization of pathways from retina to visual cortex. Right hemiretinae innervate the right LGN, input from different eyes

innervating different layers of the LGN. Inputs from the left hemiretinae innervate the left LGN (not shown) in a similar manner.

Projections from the LGN to layer 4 of the visual cortex terminate maintain the segregation of the inputs from each eye, forming ocular

dominance columns.

Fig. 2. Circuitry of the visual cortex. Thalamic input (green) terminates on spiny stellate cells (S) in layer 4, on pyramidal cells (P) in

layers 2/3, 5, and 6 and on inhibitory basket cells (B). Layer 4 stellate cells project to layer 3 pyramidal cells. Layer 2/3 pyramidal cells

form numerous horizontal connections with other layer 2/3 neurons in addition to projecting to layer 5 and higher visual areas. Layer 5

pyramidal neurons project to the superior colliculus and pulvinar but also form connections with layer 2/3 and layer 6 pyramidal

neurons. Layer 6 pyramidal cells make feedback connections with the LGN and stellate cells in layer 4. Inhibitory basket cells (B)

innervate layer 2/3 and layer 5 pyramidal cells.

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more monocular (Smith et al., 1970). This latter

finding indicates that this connectivity is not just

regulated by levels of activity, but that it is instructed

by correlated activity.

For a very long time it was believed that ocular

dominance shifts were initiated by restructuring of

the geniculocortical projections, followed by reorga-

nization of connections in layers 2/3 and 5 and 6.

Surprisingly, it was recently found that the first

neurons to change their responsiveness are in fact the

neurons in layers 2/3 and 5. Altered responsiveness

in layer 4 and reorganization of geniculocortical

afferents follow several days later (Trachtenberg et al.,

2000).

When animals are reared in the dark, there is a

delay in the development of the visual cortex and the

critical period is postponed (Sherman and Spear,

1982). Monocular deprivation after dark rearing will

induce an ocular dominance shift, also at an age at

which the critical period is already closed in control

animals. Altogether, OD plasticity is a competitive

process that responds to differences in correlated

activity between both eyes. Interestingly, once the

critical period has been induced by visual experience,

it seems to follow an irreversible path that leads to its

own closure (Mower et al., 1983).

Structural changes

As described above, significant structural changes are

induced in the geniculocortical projections by altered

visual experience. In the last few decades, many

studies have been performed in order to obtain a

better understanding of the structural changes that

take place in other components of the visual cortex,

such as the shapes of dendrites and spines of stellate

and pyramidal, horizontal connections in the

pyramidal layers and the synapses formed by

interneurons. Not all of these results are intuitive.

In the next section, we will attempt to integrate them

in a model for structural developmental plasticity.

Developmental phases of the visual cortex

As mentioned above, an ocular dominance shift can

only be induced during the critical period. After that

period, altered visual input has very little effect on the

structural organization of the visual cortex although

some functional changes can still be induced (Sawtell

et al., 2003). Before the critical period, visual input

does induce some structural changes in the visual

cortex, but can not yet incite an ocular dominance

shift (Borges and Berry, 1978; Freire, 1978).

Thus, during postnatal development, the visual

cortex undergoes different phases in which visual

experience has different effects. The phases may be

described as follows:

I. Before the critical period: Spontaneous activity

and a genetic program drive dendritic and

axonal growth and synapse formation of

excitatory neurons in the visual cortex —

visual experience has little influence on the

excitatory network, but is essential for the

development of the inhibitory circuitry.

II. The critical period: Increased inhibition results

in a more critical evaluation of the circuitry and

causes the formation, maintenance or strength-

ening of synapses to depend on visual input.

Less spine formation and more pruning occurs.

Homeostatic mechanisms keep the system in

balance.

III. After the critical period: The system becomes

stabilized, potentially by the maturation of the

extracellular matrix. Visual experience has very

little influence on the plasticity of the circuitry.

Phase I — Before the critical period

Dark rearing animals before the critical period has

little influence on morphological properties of

neurons in the visual cortex, despite the fact that

extensive neuronal morphogenesis occurs during this

time. The main differences that could be detected

between dark-reared and control animals during this

stage are differences in the orientation of the dendritic

fields of stellate neurons in layer 4 (Borges and Berry,

1976, 1978), and a moderate (10–20%) and reversible

decrease in the number of dendritic spines, the main

sites of synaptic contacts on excitatory neurons in the

neocortex (Ruiz-Marcos and Valverde, 1969;

Fifkova, 1970; Valverde, 1971).

What occurs between eye opening and the critical

period and why does dark rearing have so little effect

127

on these events? In the weeks after eye opening and

before the critical period, dendritic growth, synapse

formation and axonal ingrowth occur simultaneously

in the visual cortex (Wise et al., 1979; Zecevic and

Rakic, 1991; Huttenlocher and Dabholkar, 1997).

The concomitant occurrence of these processes in

different parts of the CNS has led to the suggestion

that afferent input drives dendritic growth. More

specifically, Vaughn suggested the synaptotrophic

model of dendritic growth, which hypothesizes that

synapse formation guides dendritic growth and

branching towards areas where synaptic input is

present (Vaughn, 1989). An attractive recent paper

employing in vivo time lapse imaging of growing

dendrites and postsynaptic densities in the tectum of

zebrafish larvae strongly supports this hypothesis

(Niell et al., 2004). It was shown that during

development a reiterative program takes place in

which growing dendrites extend many thin filopodia

that seem to scan the environment for afferents with

which some may form synaptic contacts. This

subsequently results in the growth of the dendrite

towards this newly formed synapse and new branches

are extended there. These branches go through the

same process, resulting in dendritic growth towards

and branching in areas that provide synaptic input. It

is not certain if the same process happens in the

mammalian visual cortex, but some studies support

this notion. For example, very little dendritic growth

of pyramidal neurons occurs in cultures from p6 rat

visual cortices unless afferent ingrowth and the

formation of new synapses is stimulated by cocultur-

ing other pieces of cortex (Baker et al., 1997).

What is the role of activity in this process?

Evidently, the growth and branching of dendritic

arbors is regulated in part by an intrinsic genetic

program and molecular cues from the environment

(for a review, see Dijkhuizen and Ghosh, chapter 2).

But in addition, a wealth of data supports the role

of activity in dendritic and axonal growth and

stabilization in many different experimental settings

(for reviews, please see McAllister, 2000; Cline, 2001;

Wong and Ghosh, 2002; Hua and Smith, 2004). The

mammalian visual cortex seems to be no exception. A

recent report shows that altering levels of inhibition

during early postnatal development changes the

periodicity of ocular dominance columns (Hensch

and Stryker, 2004), indicating that early segregation

of geniculocortical input is regulated by activity. In

slice cultures of p14 ferret visual cortex, dendritic

growth of pyramidal cells is altered significantly when

glutamate dependent activity is blocked (McAllister

et al., 1996). Thus, activity influences morphological

properties of excitatory neurons in the visual system.

However, the exact role of activity remains elusive

and many studies produced counterintuitive and

confusing results. The main difficulty appears to be

that dependent on the nature of the activity and the

developmental stage of the neuron, reduced activity

can produce opposite outcomes. A lack of afferent

activity may be a signal for a developing dendrite to

continue growing or to form more filopodia, in order

to find more active input. At the same time,

correlated activity may be necessary for the

stabilization of newly formed synapses and to guide

further growth and branching. For example, in

isolated slice cultures from p14 ferret visual cortex,

inhibiting glutamate mediated activity initiates

dendritic growth, potentially in search for active

afferents (McAllister et al., 1996). However, in

cocultures of rat visual cortex in which afferent

ingrowth takes place inducing dendritic growth,

inhibiting glutamate mediated activity actually

reduces dendritic and axonal growth, suggesting

that these events are regulated by activity mediated

synapse stabilization (Baker et al., 1997).

Taken together, a picture arises in which a highly

dynamic process takes place in which neurons in

the visual cortex are extending dendrites searching

for afferent input. Successful stabilization of synap-

ses guides the further growth, branching, and

eventual stabilization of these branches. Failure will

lead to their retraction. This process is relatively

promiscuous and spontaneous activity seems suffi-

cient to mediate it. Visual input only influences

this process when spontaneous activity is below

threshold.

Could this scenario explain the data obtained in

dark-reared or enucleated animals? As stated above,

dark rearing up to postnatal day 21 in rats results in a

decrease (10–20%) in total spine number in all layers.

In layer 4 it was observed that this was accompanied

by an increase in the number of fine protrusions,

which could be filopodia or less stable spines

(Valverde, 1971; Borges and Berry, 1976; Freire,

1978). These differences were completely reversed by

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subsequent light rearing. These data seem to suggest

that spontaneous activity is sufficient to set up the

circuitry, but that the maintenance of spines is

somewhat more efficient when activity is stronger or

more correlated.

Another observation made in dark-reared rats, is

that layer 4 stellate neurons show preferred dendritic

growth towards layer 3 (Borges and Berry, 1976,

1978). Within layer 4, stellate neurons form synapses

with geniculocortical afferents, which convey visual

input. Layer 6 pyramidal neurons, which represent

another important source of input to stellate neurons,

connect to them primarily at the border of layers 3

and 4. Dark-rearing may therefore reduce synaptic

activity within layer 4, stimulating stellate neurons to

direct dendritic growth towards layer 3 where they

may find another source of input. No differences in

the morphology of pyramidal neurons could be

detected upon dark-rearing (Tieman et al., 1995).

Possibly, afferents conveying sensory input or

spontaneous activity do not form synaptic contacts

at different locations in these layers.

Few studies have assessed the effect of dark

rearing on geniculocortical connections before the

critical period. The segregation of geniculocortical

projections happens before the critical period

(Crowley and Katz, 1999, 2000; Crair et al., 2001).

Removing one eye before eye opening, thereby

reducing input from spontaneous activity to the

LGN, does not influence segregation (Crowley and

Katz, 1999). Also binocular enucleation seems to

allow the development of geniculocortical segrega-

tion (Crowley and Katz, 2000). Although it is

possible that this is entirely regulated by molecular

cues, it is not excluded that spontaneous activity in

the layered LGN is responsible for the formation of

ocular dominance columns.

The question arises as to how the situation

changes so dramatically during the critical period.

Interestingly, in contrast to the formation of

excitatory synapses, the formation of the inhibitory

circuitry is strongly dependent on visual experience

before the critical period. Dark rearing delays the

maturation of GABA expressing neurons and the

formation of GABAergic synapses, resulting in more

spontaneous activity and prolonged activity in

response to visual stimuli. (Benevento et al., 1992,

1995). The development of the inhibitory circuitry

has been shown to be an important factor in initiating

the critical period (Hensch et al., 1998).

Phase II — The critical period

Studies by the laboratories of Hensch, Stryker,

Tonegawa, Bear and Maffei (Hensch et al., 1998;

Huang et al., 1999; Iwai et al., 2003) have shown

convincingly that the maturation of the inhibitory

circuitry results in the initiation of the critical period.

In GAD65 deficient mice that have reduced

GABAergic input, the critical period is not initiated

until GABAergic transmission is increased by

infusion of Diazepam. In mice in which GABAergic

neurons develop more rapidly due to overexpression

of Brain Derived Neurotrophic Factor (BDNF)

(Huang et al., 1999), the onset of the critical period

is accelerated. As spontaneous activity decreases with

increased inhibition, it is likely that the influence of

visual input becomes a more important factor in the

maintenance, stabilization, and strengthening of

synapses. Furthermore, evaluation of coincident

firing will become more critical, increasing synaptic

competition.

These changes set the stage for adaptation of the

circuitry to the visual environment which is

accompanied by significant structural changes. The

most obvious changes induced by altering binocular

input can be detected in the geniculocortical

projections. By injecting trans-synaptic tracer into

one of the eyes and imaging its geniculocortical

projections to V1, it can be shown that monocular

deprivation causes the deprived afferents to shrink,

while the nondeprived afferents expand. Injection of

lectins such as Phaseolus vulgaris-leucoagglutinin

into the LGN allows more detailed morphological

analyses (Antonini and Stryker, 1996). Using this

method it has been shown that the arbors of the

deprived eye are less branched, have reduced total

length, and show decreased maximal innervation

density while nondeprived arbors show increases in

these parameters. From intraocular tracer injections

it seems that most branch retraction occurs at the

borders between left- and right eye columns. These

are the areas where a lack of correlation between

presynaptic input from the deprived eye and

postsynaptic activity conveyed by the nondeprived

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input will be the strongest and competition is most

severe. In the middle of the column, total activity is

reduced by deprivation, but as this part of the cortex

receives less input from the nondeprived eye,

correlated activity will not be severely affected

(Hubel et al., 1977; Shatz and Stryker, 1978).

Interestingly, in mice, monocular deprivation does

not lead to the retraction of deprived thalamic

afferents, only to reduced growth (Antonini et al.,

1999). One may speculate that this is a consequence

of the lack of a columnar organization in the rodent

visual cortex. In a columnar organization, the

branches of thalamic afferents will form contacts

with stellate neurons of similar OD classes. When an

OD shift is induced, synapses with similar properties

are likely to be lost under similar conditions. Thus, a

deprived geniculocortical branch may lose many

contacts simultaneously, causing its retraction. This

is supported by the finding that the density of

synapses on geniculocortical afferents does not

change much upon monocular deprivation. In the

absence of a columnar organization, a geniculocor-

tical branch may form contacts with neurons of

various OD classes. Induction of an OD shift will

cause this branch to lose some of its contacts, but not

all, keeping the branch in place. If this is true, one

would predict that mice, in contrast to cats, should

show decreased synaptic densities in deprived arbors.

Reduced dependence on retraction and regrowth of

axonal arbors in the mouse may also result in more

residual OD plasticity in the adult visual system,

which is less permissive to structural changes, as was

shown by a recent study (Sawtell et al., 2003).

Apart from the complete loss of synaptic

connectivity or even retraction of axonal arbors,

more subtle changes also occur at geniculocortical

synapses. Upon monocular deprivation, remaining

synapses show a reduction of the size of presynaptic

terminals, and a reduced number of mitochondria in

the terminals (Tieman, 1991). The spines that they

contact are also smaller, and among the deprived

synapses there are reduced numbers of spines with

multiple postsynaptic densities, known as fenestrated

spines. Altogether it seems that a continuum of

reduced connectivity is induced by monocular

deprivation, probably depending on the level of

asynchronicity between the pre- and postsynaptic

partners.

The retraction of geniculocortical afferents hap-

pens faster than the expansion of the nondeprived

afferents. A recent paper by Ruthazer et al. (2003) has

shown that when synaptic competition is induced in

Xenopus tectum by ablation of one tectum, ingrowth

of retinotectal afferents is not specifically regulated by

activity but retraction is. In the visual cortex a similar

type of regulation may occur, in which retraction of

deprived afferents occurs first, followed by ingrowth

of nondeprived afferents that may subsequently be

stabilized.

Due to the excellent correlation between the

functional changes induced by monocular deprivation

and the reorganization of the geniculocortical

afferents, it was believed that the latter was the

structural correlate of an OD shift. The finding that a

functional OD shift occurs first in the pyramidal

layers and not in layer 4, and that the reorganization

of the geniculocortical projections follows several

days later therefore came as a surprise (Trachtenberg

et al., 2000). In search for a better structural correlate,

Trachtenberg and Stryker (2001) analyzed the

reorganization of horizontal connections in the

pyramidal layers after inducing strabismus in cats

during the critical period. They found that connec-

tions between left-eye and right-eye columns were

strongly reduced within 2 days, while connections

between same-eye columns remained. Thus, horizon-

tal connections seem to be a better structural correlate

for the functional changes than geniculocortical

afferents. Several questions remain, however, with

respect to the involvement of the connections between

stellate neurons and pyramidal cells in OD plasticity

and to the mechanism by which the geniculocortical

afferents adjust to the changes in the pyramidal layers.

It is impossible for the extensive reorganization of

the axonal arbors to occur in the absence of struc-

tural changes in postsynaptic neurons but it has been

much more difficult to detect these changes. Mono-

cular deprivation does not seem to affect spine

densities significantly (Lund et al., 1991). Moreover,

dendrites have nearly reached their adult morphol-

ogy, and monocular deprivation barely affects them

except for small changes in dendritic arborization of

stellate neurons (Lund et al., 1991; Kossel et al., 1995).

The answer to the apparent discrepancy between

these observations and the extensive changes in the

presynaptic compartment probably lies in the

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dynamics of the system: the retraction and regrowth

of the afferents is believed to result in enhanced spine

turnover, more so than in changes in absolute spine

numbers. The recent development of multi-photon

imaging of neuronal morphology in live mice

expressing fluorescent proteins in isolated neurons

has made it possible to study these dynamic events. In

line with the idea that most structural changes occur

before and during the critical period, it was found

that in the developing somatosensory cortex spine

motility decreased steadily during development

(Lendvai et al., 2000). This was accompanied by a

decrease in the number of filopodia. Sensory depriva-

tion by whisker trimming resulted in decreased spine

motility but only during the critical period, indicating

that this is a unique window during which sensory

input has the strongest influence on structural

changes, analogous to the visual cortex. The absolute

numbers of spines and their morphology were not

affected by deprivation at any age tested. In the

somatosensory cortex of adult mice, competition

between inputs from different whiskers can be

induced by removing every second one of them.

This procedure resulted in enhanced spine turnover,

but not in the reorganization of axonal or dendritic

structures indicating that their architecture is fixed

after the critical period (Trachtenberg et al., 2002).

The in vivo dynamics of the visual cortex has not

yet been studied in as much detail. However, in vivo

multiphoton imaging in mice expressing enhanced

green fluorescent protein (EGFP) in layer 5

pyramidal neurons has shown that in the visual

cortex also, spine formation and motility and the

numbers of filopodia decrease over age (Grutzendler

et al., 2002). This decrease starts already before the

onset of the critical period. During the critical period,

filopodia are abundant (12% of all protrusions), but

are very unstable and more than 85% are lost within

3 days. Around the critical period and the initial

weeks thereafter, most changes that occur are

associated with the elimination of spines, in line

with the idea that the circuitry is evaluated with more

scrutiny during this time. Only around 80% of spines

present during the critical period are maintained in

the two weeks after, while in adult animals, more

than 95% of all spines are stable during a one month

period. Binocular deprivation of mice from the time

of eye opening results in a small increase of spine

motility during the critical period, and possibly a

small reduction in adulthood (Majewska and Sur,

2003). The increase of spine motility during the

critical period is accompanied by an increased

percentage of filopodia. It remains unclear whether

postponement of the critical period or reorganization

of the circuitry in response to binocular deprivation

caused these changes.

As a whole, the critical period appears to be a time

at which increased inhibition sets the stage for a re-

evaluation of the circuitry in the visual cortex, based

on visual input. This may lead to loss of inefficient or

imprecise synapses and stabilization of more effective

connections. This is accompanied by significant

changes in the structure of geniculocortical and

intracortical afferents and turnover of dendritic

spines. The observation that absolute spine numbers

remain constant during an OD shift and that rapid

ingrowth of axons takes place quickly after retraction

suggests that homeostatic mechanisms keep the

system in balance (Trachtenberg et al., 2002).

Phase III — The end of the critical period

Within the first few days of the critical period, the

cortical circuitry appears to crystallize in a few days

into a balanced and functional system which shows

very little plasticity (Mower et al., 1983). For

example, when the critical period is initiated earlier

by injecting Diazepam or transgenic overexpression

of BDNF (Huang et al., 1999; Iwai et al., 2003), it

also closes earlier. The mechanism by which the

critical period is closed is not entirely clear. It is

unlikely that increased levels of inhibition is the main

cause of this as it has been difficult to reintroduce a

critical period by reducing inhibition experimentally

in adult animals. A more promising explanation

involves the extracellular matrix. The critical period

can be reopened by degradation of so-called

perineuronal nets, formed by extracellular matrix

components known as chondroitin sulphate proteo-

glycans (CSPG), which surround predominantly

Parvalbumin containing interneurons (Pizzorosso

et al., 2002). The mature extracellular matrix thus

inhibits cortical plasticity. Degradation of CSPGs has

been shown to induce axonal sprouting in other

systems, such as the spinal cord, the cerebellum and

131

the superior colliculus (Bradbury et al., 2002; Morel

et al., 2002; Tropea et al., 2003). It is likely that the

development of the extracellular matrix inhibits

structural plasticity by physically constraining the

neuronal architecture and by specific molecular

interactions for example between the extracellular

matrix protein Tenascin-R and NCAM or integrins.

The extracellular matrix may also function as a mesh

which holds secreted proteins such as Wnt factors or

Semaphorins, which stimulate or inhibit synapse

formation. Interestingly Tenascin-R is found prefer-

entially in perineuronal nets around PV interneurons,

which are believed to be the most important players

in regulating the critical period (Fagiolini et al.,

2004). In mice deficient for Tenascin-R, inhibition

seems reduced in the hippocampus (Saghatelyan et al.,

2001). Thus it is possible that the perineuronal nets

suppress cortical plasticity by altering the function-

ality of the inhibitory synapses. How the formation

of the extracellular matrix is influenced by neuronal

activity remains an important question.

Molecular and cellular level

Above we have discussed the principal structural

changes that occur during the development and plas-

ticity of the visual cortex and how visual input influ-

ences them. The activity mediated structural changes

that take place during the critical period are predo-

minantly the formation, stabilization and elimination

of spines and axonal growth and retraction.

In the next section we will discuss some of the

principles by which activity may mediate these

processes. The literature on the molecular

mechanisms that regulate these events is vast and

many excellent reviews have been written on this

subject (for example Sala, 2002; Scheiffele, 2003). We

will therefore only mention some of the molecular

mechanisms involved for illustrative purposes. Many

studies on spine formation and stabilization have

been performed in hippocampal neurons, using long

term potentiation (LTP) by tetanic stimulation as an

experimental paradigm. We would therefore like to

mention, as a caveat, that it remains uncertain

whether some of the mechanisms that regulate spine

formation and stabilization described below will be

applicable to OD plasticity in the visual cortex.

Spine formation

As absolute spine numbers remain stable during

plasticity in the adult somatosensory cortex of mice

and upon induction of an OD shift in the visual

cortex of monkeys (Lund et al., 1991), the formation

of new spines may be regulated by a homeostatic

process (Trachtenberg et al., 2002). Postsynaptic

neurons that lose specific synaptic contacts due to

sensory deprivation may thus start actively searching

for additional input. By extending dendritic filopodia

they may scan the environment for new synaptic

partners, such as newly arriving afferents taking over

the territory of recently retracted axons or afferents

that have remained. In various systems, observations

support such homeostatic mechanisms. For example,

when synaptic activity is blocked in hippocampal

slices, there is an increase in the formation of

dendritic protrusions (Kirov and Harris, 1999). Also,

in organotypic cultures of mouse neocortex more

dendritic shaft protrusions are formed with decreased

synaptic activity (Tashiro et al., 2003).

The mechanisms that could achieve these

responses are unknown, but activity-regulated gene

transcription would be an attractive solution as it

would allow for the monitoring of total neuronal

activity, translating it into a cellular response that

increases spine formation in the entire neuron.

Although various gene products have been shown

to regulate spine formation, few are attractive

candidates for this type of homeostatic regulation.

BDNF has been implicated in homeostatic

mechanisms in the developing visual cortex, but its

method of action may lie more in regulating the

balance between the inhibitory and excitatory

circuitries than in regulating absolute spine numbers

in excitatory neurons (for a review see Turrigiano and

Nelson, 2004). Recently, the kinase SNK was

identified as a gene product whose transcription is

upregulated by neuronal activity, and results in

downregulating overall neuronal spine numbers.

SNK may thus represent an intracellular mechanism

for synaptic scaling, albeit through regulating spine

elimination rather than spine formation (Pak and

Sheng, 2003).

Spine formation may also be induced locally by

synaptic activity. In hippocampal slices production of

dendritic protrusions is induced by local stimulation

132

of NMDA-receptors or the local induction of LTP

(Engert and Bonhoeffer, 1999; Maletic-Savatic et al.,

1999). It has also been shown that upon induction of

LTP in the hippocampus, an increase in the number

of axon terminals contacting two adjacent spines

seems to be caused by the formation of an additional

spine next to a preexisting spine (Fiala et al., 2002).

Some interesting molecular mechanisms have been

identified that could mediate this. For example,

Calcium/Calmodulin dependent Kinase IIb(CamKIIb) has recently been shown to induce the

formation of dendritic protrusions in an activity

dependent fashion (Jourdain et al., 2003). High levels

of Ca2+ that enter through NMDA-receptors may

thus result in the local activation of CamKIIb and

thereby induce the formation of additional spines

nearby. Also, the extracellular domain of the

glutamate receptor GluR2 has been implicated in

the formation of spines (Passafaro et al., 2003). It is

still unknown how this is mediated, but interaction

with a postsynaptic protein interacting with the N-

terminal region of GluR2, similar to the interaction

between the NMDA-receptor and the tyrosine kinase

receptor EphB, may be involved (Henkemeyer et al.,

2003). The structural changes that are induced via

these pathways are mediated by changes in the actin

cytoskeleton. The Rho family of GTPases, such as

RhoA, Rac and Cdc42 are important regulators of

actin dynamics and are the downstream effectors of

many signaling pathways that regulate morphogen-

esis, including EphB (for reviews, see Nakayama and

Luo, 2000; Ramakers, 2002). CamkIIb, however,

interacts with actin itself and may regulate its

dynamics directly (Fink et al., 2003).

Stabilization of spines

It has been shown under various experimental

conditions that spines and synapses can form in the

absence of afferent input (for a review, see Yuste and

Bonhoeffer, 2004). The exact contribution of activity

mediated spine formation to the establishment and

adjustment of the functional circuitry therefore

remains unclear. In contrast, there is abundant

evidence for the need of activity in the maintenance

of spines. Signals through the NMDA-receptor

activate various signaling cascades that result in

spine stabilization. Upon tetanic stimulation of

cultured hippocampal neurons, NMDA-receptor

mediated responses rapidly drive AMPA receptors

in to spines (Shi et al., 1999; Hayashi et al., 2000;

Liao et al., 2001; Lu et al., 2001), especially into small

spines that are previously devoid of AMPA receptors.

The presence of AMPA receptors greatly enhances

the efficacy of synaptic transmission and aids in the

maintenance of dendritic spines (McKinney et al.,

1999). At the same time, NMDA receptor activation

results in rapid enlargement and reduced motility of

spines by modifying actin dynamics (Fischer et al.,

2000). As the actin cytoskeleton is involved in

membrane trafficking and thereby in the insertion or

internalization of AMPA-receptors, these phenom-

ena may be part of the same process. The alterations

in actin dynamics in spines are regulated by the actin

binding protein Profilin which specifically enters

spines upon NMDA receptor stimulation

(Ackermann and Matus, 2003). It has been specu-

lated that Profilin, or other proteins, may ‘‘tag’’

spines for recruitment of newly formed macromole-

cules that help to further potentiate the synapse (Frey

and Morris, 1997; Ackermann and Matus, 2003).

Among those macromolecules may be mRNAs,

which have been shown to enter activated spines of

hippocampal pyramidal neurons. Induction of LTP

in area CA1 of the hippocampus also results in the

recruitment of polyribosomes into spines and may

initiate local protein synthesis. This, in turn would

result in an increase in the postsynaptic density and

more effective synaptic transmission (Ostroff et al.,

2002).

Another mechanism by which spines may become

stabilized is by activity-mediated release of BDNF.

High frequency stimulation of hippocampal neurons

results in the postsynaptic release of BDNF

(Hartmann et al., 2001), which in turn has been

shown to induce mRNA recruitment, local protein

synthesis and synapse stabilization (Messaoudi et al.,

2002).

Spine elimination

The elimination of spines may also be specifically

regulated by electrical activity. Phosphatase activity

plays an important role in long term depression

133

(LTD) induced by low frequency stimulation or

imprecise spike timing (Mulkey et al., 1994; Isaac,

2001; Lisman and Zhabotinsky, 2001; Zeng et al.,

2001). Spinophilin is an anchoring molecule which

interacts with the actin cytoskeleton and brings

protein phosphatase 1 (PP1) in close proximity to its

targets including the AMPA and NMDA receptors.

In spinophilin deficient animals, LTD is impaired

(Feng et al., 2000). This indicates that the depho-

sphorylation of AMPA receptor subunits by PP1,

which down-regulates activity of AMPA receptors, is

regulated by this anchoring protein. Interestingly,

Spinophilin deficient mice also show an increase in

spine density, which suggests a link between spine

elimination and LTD.

Dephosphorylation of AMPA receptors and

down-regulation of their expression at the cell surface

can be observed in the visual cortex of mice upon

induction of LTD but also upon induction of an OD

shift by molecular deprivation (Heynen et al., 2003).

It is therefore likely that induction of LTD and

induction of an OD shift make use of common

mechanisms. It will be interesting to learn if the

elimination of spines is part of the same process.

Axon growth and retraction

As should be clear from the above, the changes in

spine formation and maintenance are regulated by

their interactions with afferent input. Moreover, the

most obvious structural changes that accompany OD

plasticity are rearrangements of axonal arbors.

Recently, presynaptic varicosities have been studied

using real time imaging and the influence of LTP has

been examined. Similar to the situation in the

postsynaptic compartment, induction of LTP results

in an increase in the number of dynamic axonal

varicosities. There is recent evidence for the

contribution of AMPA and Kainate receptors in

presynaptic filopodia formation, and it has been

suggested that feedback mechanisms employing nitric

oxide or BDNF are involved (De Paola et al., 2003;

Muller and Nikonenko, 2003; Nikonenko et al.,

2003).

What are the consequences for the growth and

retraction of axonal branches? The growth of new

axonal branches is believed to be regulated largely by

chemoaffinity and not by activity. However, at least

in Xenopus tectum, axon stabilization or retraction

is regulated by correlated activity and depend on

signals through the NMDA receptor (Ruthazer et al.,

2003). Reorganization of the geniculocortical and

intracortical afferents is therefore likely to be

regulated by specific retraction of branches that

have lost their connectivity, followed by ingrowth of

other axonal branches. Their subsequent stabilization

or retraction will again be dependent on the

formation and maintenance of synaptic contacts.

Technical approaches

The recent developments in time-lapse imaging have

been invaluable for our understanding of structural

changes in neurons during development and plasti-

city. It is clear that we are only beginning to

understand the functional implications of these

structural changes and the cellular and molecular

mechanisms that regulate them. Many fundamental

questions remain unanswered. We hardly understand

how filopodia and the various types of spines are

related and what the functional implications are of

their morphological changes. We know very little of

the specific interactions that take place between pre-

and postsynaptic partners that result in synapse

formation and maintenance and how sensory

information can regulate this.

Therefore it will be essential to analyze these

structural changes dynamically under conditions that

approach physiological conditions as closely as

possible; preferably live animals. An important

approach will be to simultaneously monitor struc-

tural, molecular, and electrophysiological changes

that occur during plasticity and try to uncover their

relationships. Interfering with molecular signaling

pathways that regulate specific structural changes will

allow us to study their functional roles. These

approaches need to be performed in different systems

and during different stages of development.

Several important developments have been taking

place that will allow these types of analyses. Multi-

photon imaging has made it possible to image

significantly deeper into living tissue and to achieve a

high spatial and temporal resolution while minimiz-

ing interference with the biological processes under

134

investigation. Currently, maximal penetration is

around 500mM, but several techniques are being

developed that may allow imaging of brain areas

situated deeper in the brain (Mizrahi et al., 2004;

Theer et al., 2003).

Another important development is the improve-

ment of fluorescent probes and especially the

increasing availability of spectral variants of fluor-

escent proteins (Campbell et al., 2002; Miyawaki,

2002) This allows the genetically encoded tagging of

specific proteins which makes it possible to express

them in the cell types under investigation using

specific promoter sequences and to visualize their

trafficking. A prerequisite is the expression of such

fluorescent proteins in isolated neurons, in a ‘‘Golgi’’-

like pattern. This makes it possible to image neuronal

morphology and to retrieve the same neuron in

successive imaging sessions, enabling the long term

in vivo imaging.

Several methods have been developed that allow

expression of fluorescent proteins in individual

neurons in mammals. The first in vivo multi-photon

imaging studies made use of viral vectors to express

fluorescent proteins in cortical neurons (Lendvai

et al., 2000). Although this technique has the

disadvantage that some tissue is damaged by

injection of the vector, it does allow for effective

expression of fluorescent proteins in isolated neurons,

in the brain area of choice and at a specific

developmental time point. Another tool that has

been very useful for subsequent studies in structural

cortical plasticity has been the mosaic EGFP

expressing mouse lines produced by the Sanes

laboratory (Feng et al., 2000a). Among a large

number of transgenic lines expressing EGFP under a

neuron-specific promoter, several lines expressed the

protein at high levels in a very low number of

pyramidal neurons in the cortex and hippocampus.

These mice can also be used to study the effects of

specific signaling pathways in structural plasticity, by

crossing them to other transgenic or knock-out mice.

In many live imaging studies using neuronal

cultures, investigators make use of transfection

methods that transfect only few neurons, allowing

the examination of the function of the transfected

protein in a cell-autonomous fashion. A significant

advantage of this approach is that one can be certain

that the observed morphological effects are caused by

expression of the protein in the neuron under

investigation, and not because of changes in the

functionality of the surrounding cells. The next

generation of transgenic mice may make use of a

comparable approach. Several transgenic mice have

now been produced that express the Cre recombinase

in a mosaic fashion in the brain (Huang et al., 2002;

Buffelli et al., 2003). These mice can be used to

express specific transgenes that are regulated by Cre-

mediated recombination in individual neurons and

make it possible to study their effects in a cell

autonomous fashion. It is of importance that the

expression of such transgenes can be directed to the

cell types and brain regions under investigation at the

correct developmental time point. For the research

on OD plasticity, expression in cortical pyramidal

and stellate neurons, various subpopulations of

interneurons and thalamic projection neurons will

be essential. The use of inducible Cre variants will

make it possible to regulate the onset and distribution

of expression (Buffelli et al., 2003).

Of course the feasibility of this approach will

depend on the co-expression of fluorescent proteins

together with the functional transgene, which turns

out to be more difficult than expected. For

morphological analyses, the use of EGFP-fusion

proteins is limited to those that will distribute evenly

through the entire neuron or its membrane. And even

for such proteins, it is difficult to express them at

levels sufficient for their detection and in some cases

high expression levels lead to altered distribution of

the fusion protein in the neurons. An alternative

method is the use of internal ribosomal entry sites

allowing the translation of EGFP and a functional

protein from one transcript (Kozak, 2003). However,

disappointing results have been obtained and expres-

sion of the transgenic protein following the IRES

sequence, usually the fluorescent protein, is signifi-

cantly lower than expression of the first protein

resulting in EGFP levels that are too low for

detection.

The expression of multiple fluorescent proteins,

for example PSD95 fused to a red fluorescent protein

for the visualization of postsynaptic densities and

EGFP labeling the entire neuron will allow the

monitoring of molecular and structural changes that

occur simultaneously (Niell et al., 2004). The

development of improved IRES sequences or novel

135

methods that allow the expression of multiple

transgenes upon Cre recombination should provide

better solutions for the future.

Acknowledgments

We would like to thank Sridhara Chakravarthy and

Alexander Heimel for their useful comments on the

manuscript. The authors are funded by the

Netherlands Organization for Scientific Research

(N.W.O.)

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