1 Scuola Normale Superiore, piazza dei Cavalieri 7, 56100 Pisa, Italy
2 Istituto di Neuroscienze del CNR, via G. Moruzzi 1, 56100 Pisa, Italy
Author for correspondence (e-mail:
sibel.naska{at}sickkids.ca)
Accepted 8 April 2004
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SUMMARY |
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Key words: Visual system development, Retinal ganglion cell, Retino-geniculate segregation, ERK pathway
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Introduction |
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Previous findings have indicated that the formation of eye-specific domains
in dLGN occurs through interocular competition
(Lund et al., 1973;
Rakic, 1981
) and it has been
suggested that this process is driven by neural activity
(Shatz and Stryker, 1988
),
particularly by a correlated pattern of spontaneous retinal activity (retinal
waves) present in the developing retina
(Feller et al., 1996
;
Galli and Maffei, 1988
;
Wong et al., 1995
). Indeed,
selective blockade of retinal activity in both eyes prevents formation of the
eye-specific layers in the dLGN, whereas blocking activity in one eye results
in an expansion of the territory occupied by the active untreated eye at the
expense of axons from the treated eye
(Grubb et al., 2003
;
Huberman et al., 2002
;
Penn et al., 1998
;
Rossi et al., 2001
).
Conversely, if one eye is made more active, its layers within the dLGN expand
(Stellwagen and Shatz, 2002
).
However, the role of retinal activity in retino-geniculate development remains
a debated issue (Sengpiel and Kind,
2002
). Some findings favor the hypothesis that correlated retinal
activity plays an instructive rather than a permissive role in the development
of dLGN (Stellwagen and Shatz,
2002
), whereas others suggest that the presence, but not the
spatiotemporal pattern, of retinal activity is required
(Huberman et al., 2003
). In
addition, Cook et al. (Cook et al.,
1999
) have reported that binocular blockade of neural activity
does not prevent segregation of retinal axons but just delays it, implying
that specific cues might be involved in this process
(Huberman et al., 2002
;
Huberman et al., 2003
;
Muir-Robinson et al.,
2002
).
Some molecular systems have been suggested to play a role in the remodeling
of retino-geniculate circuitry (Corriveau
et al., 1998; Menna et al.,
2003
; Pham et al.,
2001
; Ravary et al.,
2003
; Upton et al.,
1999
); however, the intracellular cascades required in shaping
these connections have remained unclear. Extracellular signal-regulated
kinases (ERKs) are signal-transducing enzymes that have been shown to
participate in a diverse array of cellular programs
(Grewal et al., 1999
). They
are activated by phosphorylation on threonine and tyrosine residues and, once
activated, are able to phosphorylate other downstream kinases and
transcription factors (Davis,
1993
; Pizzorusso et al.,
2000
; Seger and Krebs,
1995
). Recently, it has become evident that the ERK pathway can
play multiple roles in the activity-dependent regulation of neuronal
functions. It is involved in cellular models of synaptic plasticity, such as
LTP and LTD (English and Sweatt,
1997
; Kawasaki et al.,
1999
; Martin et al.,
1997
), and in addition, behavioral studies have revealed a
requirement of this pathway in learning and long-term memory
(Atkins et al., 1998
;
Mazzucchelli et al., 2002
).
Recent findings have highlighted the importance of ERKs in visual cortical
plasticity (Di Cristo et al.,
2001
), and in morphological changes in dendritic or axonal
structure (Kim et al., 2004
;
Markus et al., 2002
;
Vaillant et al., 2002
;
Wu et al., 2001
).
In the present study, we examined the role of the ERK pathway in the development of eye-specific domains in dLGN, focusing specifically on the localization of its action along the retino-thalamic circuitry. We blocked ERK phosphorylation in the retina or dLGN during the period in which eye-specific segregation normally occurs and then analyzed the distribution of optic projections in the dLGN. We demonstrate that the active form of ERK is crucial for the refinement of retinogeniculate connections, as its inhibition in dLGN blocks the eye-specific segregation completely. Furthermore, our results provide evidence that, for a correct retino-thalamic pattern formation, ERK signaling is required on both geniculate neurons and retinal ganglion cells. This is demonstrated by the finding that a separate blockade of ERK activation in retinal or geniculo-cortical neurons partially arrests the segregation process, whereas a concurrent blockade at these sites arrests it completely.
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Materials and methods |
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Stock solutions of U0126 or PD98059 (25 mM; Promega and Calbiochem,
respectively) were prepared in 100% DMSO. For lower concentrations we used
dilutions in saline. Fluorescent latex microspheres (Lumafluor Corp.), 50-200
nm in diameter, were incubated overnight at 4°C in a 1:5 mix of
microspheres to U0126, PD98059 or DMSO+saline (vehicle). Beads were then
centrifuged and resuspended in saline
(Riddle et al., 1997;
Riddle et al., 1995
).
The intraventricular injections were performed under ether anesthesia by inserting into the left lateral ventricle a 30 gauge needle connected to a Hamilton syringe. 1 µl of latex beads coated with 500 µM U0126 or 1 mM PD98059 or vehicle was injected. The following coordinates, which vary according to the age of the animal, were used: 3 mm lateral and 1.5-0.2 mm posterior to bregma until age P6, or 1 mm anterior to bregma at P8; 2-3 mm deep from the pial surface. The location of injections was confirmed by the presence of fluorescent latex microspheres throughout the lateral ventricle.
Intraocular injections of fluorescent latex beads coated with 1 mM U0126 were performed using a glass micropipette, with a tip diameter of 30 µm, connected to a microinjector. The micropipette was inserted at the ora serrata, and the drug was slowly released into the vitreous. Intraocular injections of epibatidine (1 mM in DMSO, Sigma) were carried out in the same manner.
For visual cortex injections, the skull was opened on the left side to expose much of the length of area 17. Microspheres (1 µl) coated with 250 µM U0126 were injected at eight different sites using a glass micropipette. Retrograde transport and LGN labeling were obtained 48 hours later. To confirm the presence of beads within dLGN projection neurons, 48 hours after injection of beads, we also labeled the geniculo-cortical neurons by injecting the retrograde tracer FluoroGold (2%; Molecular Probes) into the visual cortex; three days later animals were sacrificed.
Immunohistochemistry
Animals were perfused with ice-cold 4% paraformaldehyde in 0.1 M TBS and 1
mM sodium orthovanadate (pH 7.4). Brains were then removed and cryoprotected
in 30% sucrose overnight. Coronal sections of the brain, 50 µm thick, were
cut on a freezing microtome. After a blocking step, tissue sections were
incubated with pERK (1:1000, Sigma), pAkt (1:50, Promega) or pCaMKII (1:500,
Promega) antibody and then exposed to the appropriate biotinylated secondary
antibody (1:200, Vector). Antibody binding was visualized by the ABC kit
(Vector) and nickel-enhanced diaminobenzidine (DAB) reaction. Images were
acquired with a Zeiss HR Axiocam videocamera connected to a Zeiss Axiophot
microscope and digitalized by Axiovision software.
pERK immunoreactivity in the dLGN was also detected by incubating sections with Oregon-green 488 goat anti-mouse (1:400, Molecular Probes) or biotinylated horse anti-mouse IgG (1:200, Vector), followed by extravidin-Cy3 (1:300 Sigma). Images were then acquired using an Olympus confocal microscope (Fluoview).
For double-labeling experiments, the neuronal-specific nuclear protein, NeuN (1:500, Chemicon), was added after the end of pERK immunohistochemistry, and then pERK and NeuN immunoreactivities were, respectively, detected with Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies (1:400, Molecular Probes). The sections were observed by using an Olympus confocal microscope.
pERK immunostaining in the retina was performed either on whole-mount or coronal retinal sections. For both treatments, animals were perfused with ice-cold 4% paraformaldehyde in 0.1 M TBS and 1 mM sodium orthovanadate (pH 7.4). Whole-mount retinas were dissected, whereas coronal retinal sections (14 µm thick) were cut using a cryostat and then processed for immunostaining. Retinas were permeabilized in 0.3% Triton X-100 and incubated in anti-pERK (1:200 for wholemounts, 1:1000 for retinal sections), then exposed to the Oregon-green 488 goat anti-mouse antibody (1:200 for wholemounts, 1:400 for sectioned retinas). Background staining was observed in control retinas in which the primary antibody was omitted.
Western blotting
Treated or control retinas were pooled in samples containing two equivalent
retinas each and analyzed by immunoblotting. Proteins were extracted with
lysis buffer (1% Triton X-100; 10% Glycerol; 20 mM Tris; 150 mM NaCl; 1 mM
EDTA, 0.5 mM Na3VO4, 10 µg/ml Leupeptin, 10 µg/ml
Aprotinin, 1 mM PMSF, 0.5% Na Deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM
Na2MoO4, 5 mM Na4P2O7)
and the total concentration of the samples was assessed with a protein assay
kit (BioRad) using a bovine serum albumine (BSA)-based standard curve.
Proteins (30 µg) were boiled with sample buffer, loaded on a 12% SDS-PAGE
gel and then electrotransferred to nitrocellulose. Blots were blocked with BSA
(4%, Sigma) and Tween-20 (0.2%) in TBS for 2 hours, and then incubated with
anti-pERK antibody (1:1000, Sigma) overnight with shaking at 4°C. After
washing, blots were incubated with HRP-conjugated secondary antibody (0.3
µg/ml, BioRad) in blocking solution for 2 hours, developed by the ECL
chemiluminescence system (Amersham) and captured on autoradiographic films
(Amersham). Filters were subsequently stripped with stripping buffer
(Chemicon) for 30 minutes at 50°C, reblocked, and then reprobed with
anti-ERK antibody (1:1000, Sigma) using the same immunoblotting procedure as
described above. Films were then digitalized with a camera and densitometric
analysis of the bands was performed with MCID-M4 software. pERK levels in
treated and control retinas were normalized to ERK by measuring the optical
density (OD) of the pERK band and dividing it by the OD of the ERK band, on
the same filter. Finally, data from each sample were normalized with respect
to controls run on the same gel, and results were presented as a percentage of
control values. The mean value for the control eyes was set at 100%
(Cotrufo et al., 2003).
Statistical significance was determined using Student's t-test.
Analysis of pERK-expressing neurons in dLGN
Five animals were used to analyze what type of neurons express pERK in
dLGN. Geniculo-cortical neurons were retrogradely labeled by application of
FluoroGold (2%) in the visual cortex; three days later animals were sacrified.
Brain coronal sections were obtained by using a freezing microtome, collected
through the dLGN and immunostained. pERK immunoreactivity in the dLGN was
detected by incubating sections with appropriate biotinylated secondary
antibody (1:200 Vector) followed by extravidin-Cy3 (1:300 Sigma) in 0.1%
Triton X-100. Images were then acquired at a 60x zoom 3 with an Olympus
confocal microscope. For each animal we selected five sections through the
middle of the dLGN, and for each section six different (60x60 µm)
randomly spaced fields were acquired. Then, pERK, FluoroGold and
pERK-FluoroGold labeled neurons were counted using MetaMorph software.
Analysis of cell density in the retina
Retinas were dissected, fixed with 2.5% glutaraldehyde and then with
formaline-ethanol solution (1:9). The whole-mount retinas were then stained
with 0.1% cresyl violet and visualized with a Zeiss light microscope. For cell
density analysis, two groups of retinas were used: monocular U0126-treated and
monocular vehicle-treated. Injections were performed every 48 hours from P2 to
P8, and the retinas were analyzed at P9. For each retina we evaluated in blind
the density of living cells in the ganglion cell layer (GCL). Specifically,
living cells were counted at 100x magnification by using a Zeiss
computerized microscope (Stereo Investigator software, Microbrightfield) in an
average of 25 fields (80x80 µm). For all measures, fields were
equally spaced throughout the retina.
Retino-geniculate axons labeling
All animals received an intravitreal injection of Cholera Toxin B subunit
(CTB) conjugated with Alexa Fluor 488 (10 µg/µl, Molecular Probes) in
the left eye and CTB-Alexa Fluor 594 in the right eye at least 24 hours before
perfusion. All rats were perfused transcardially with 4% paraformaldehyde in
0.1 M phosphate buffer. Brains were dissected after perfusion and
cryoprotected in 30% sucrose. Coronal sections, 50 µm thick, were cut on a
freezing microtome, collected in a serial order through the entire thalamus
and then visualized using an Olympus confocal microscope.
Analysis of retino-geniculate axon projections
Images were collected with an Olympus Optical confocal microscope using
UPLAPO 10x lens with numerical aperture (NA) 0.4. Settings for laser
intensity, gain, offset and pinhole size were optimized initially and held
constant through the study. For each animal, the entire serial order of
coronal sections of the dLGN was acquired, and for each section, confocal
series of a step size of 2 µm were obtained throughout the whole section
thickness (50 µm). These confocal series were then averaged and visualized
on a single focal plane. Background was measured as the level of
autofluorescence of unstained tissue and was subtracted from the signal
fluorescence. Then, the collected images of the dLGN, corresponding to
contralateral or ipsilateral projections, were imported to the image analysis
system MCID-M4 and used to analyze the areas occupied by the ipsilateral and
the contralateral RGC projections. All image analyses were done blind. For
each animal we analyzed the five largest sections through the middle of the
dLGN, where the two eye-specific domains appear better segregated
(Menna et al., 2003;
Rossi et al., 2001
;
Stellwagen and Shatz, 2002
).
The dLGN boundary and the profile of ipsilateral and contralateral dLGN
projections were drawn on the computer screen excluding the ventral LGN and
extrageniculate optic tract. Great care was exercised to identify the
extension of contralateral projection terminals in the gap area. We defined
the line at which the majority of axon terminals stopped as the border of the
gap. The relative areas occupied by the ipsilateral and contralateral
projections were calculated by dividing the average of the five ipsilateral or
contralateral areas by the average of the five total dLGN areas
(Rossi et al., 2001
). To
determine the extent of overlap between ipsilateral and contralateral
projections to the same dLGN, the ipsilateral and contralateral areas were
measured, then their sum was subtracted from the total dLGN area and expressed
as a percentage of it (Rossi et al.,
2001
; Stellwagen and Shatz,
2002
). The statistical significance of data was evaluated by
Student's t-test.
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Results |
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The inhibition of ERK signaling in dLGN blocks the segregation of retino-geniculate afferents
To assess the role of the ERK pathway in the segregation of retinal
afferents within the dLGN we blocked its signaling by using a potent
MEK-specific inhibitor, U0126 (Favata et
al., 1998). Initially, we tested the capacity of U0126 to block
ERK activation in our in vivo model. P5 rats, which present high levels of
pERK in the dLGN (see Fig. 1M),
were injected in one of the lateral ventricles with U0126 (see Materials and
methods). After one hour, animals were perfused and pERK staining in the dLGN
was examined. Intracerebroventricular (ICV) injection is a well-known
technique for drug delivery into the dLGN and our experiment demonstrated that
1 µl of U0126 at a dose of 500 µM was able to drastically reduce pERK
expression in dLGN (Fig.
2A,B).
|
The drug was injected along with fluorescent latex microspheres to provide
prolonged release. Latex microspheres are able to bind different substances by
passive absorption and to release them gradually
(Penn et al., 1998;
Riddle et al., 1997
). Indeed,
we noted that this method of drug delivery inhibits pERK expression in the
dLGN for 24 hours. pERK expression then recovers gradually until 48 hours
after injection (see Fig. S1 at
http://dev.biologists.org/supplemental/).
Hence, beads-U0126 were injected into the left lateral ventricle of newborn
rats every 48 hours from P2 to P8 and the projections from the two eyes were
examined at P9. Animals injected with fluorescent latex beads coated with the
vehicle (DMSO+saline) were used as controls.
We found that blockade of ERK activation in dLGN completely arrests the process of eye-specific segregation. Indeed, in the dLGN ipsilateral to the U0126-injected side, RGC axons coming from the two eyes were largely overlapped (20.5±2.8%; Fig. 2L,M). The axons from the contralateral eye were present throughout the entire dLGN, occupying 99.2±0.8% of the total area (Fig. 2H,H',I), and the ipsilateral projection expanded (21.3±2.8%) occupying an area twice greater than that ipsilaterally innervated in control animals (Fig. 2J,K). Control animals presented exactly the same distribution of fibers as normal P9 rats (Fig. 2I,K,M and see Fig. 1I,J,K). Quantitative analysis revealed that the relative areas of dLGN occupied by ipsi- or contralateral fibers in U0126-treated animals were comparable to those observed in P4 rats (Fig. 2I,K,M and Fig. 1I,J,K), demonstrating that disruption of ERK signaling results in the total arrest of retinogeniculate segregation.
To test whether this result was specifically due to blockage of ERK activation we repeated the experiment using another MEK inhibitor, PD98059. Under the same experimental conditions as for U0126 treatment, we observed that ICV injections of PD98059 at a dose of 1 mM (the dose able to block ERK phosphorylation in the dLGN, see Fig. 2A,C) produced the same effect: abolishment of eye-specific segregation. The relative contralateral projection area (98.2±0.6%) and ipsilateral projection area (17.9±2.3%) were not statistically different from the corresponding areas in the U0126-treated animals (n=6, Student's t-test, P>0.05 for both ipsi and contra projections).
Geniculo-cortical blockade of pERK causes an expansion of retinal projections in the dLGN
As the ICV delivery of U0126 provides blockade of ERK activation at the
geniculate level, it was difficult to distinguish whether the effect on
eye-specific segregation was due to the blockade of pERK in geniculate
neurons, or in RGC fibers, or in other afferent inputs to the dLGN. To assess
the site of action of pERK we first investigated whether pERK in the dLGN is
expressed by all neurons or only by dLGN projection neurons. We used five P5
rats for this study and for each of them we counted an average of
496.8±44.5 cells stained with pERK in the dLGN (see Materials and
methods for details). Our analysis showed that all of these cells co-localized
with dLGN projection neurons labeled with FluoroGold
(Fig. 3A). Hence, during the
first postnatal week, pERK in the dLGN is expressed only by projection
neurons. The rate between neurons labeled with FluoroGold and those labeled
with FluoroGold-pERK revealed that only half of the projection neurons express
pERK (Fig. 3A).
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Discussion |
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The inhibition of ERK activation in the dLGN through ICV delivery of U0126 or PD98059 results in a complete arrest of the eye-specific segregation process. The RGC axons from the two eyes remain extensively overlapped in their target region, showing a strong similarity with the intermingled pattern of fibers observed in normal P4 rats.
The effect obtained after ICV injections of U0126 potentially originates from the inhibition of pERK on geniculate neurons, or on afferent inputs into the dLGN, or both. To distinguish between these possibilities, ERK activation was separately blocked only on RGCs of both eyes or on dLGN projection neurons. For each treatment, our data demonstrated that fibers from both eyes were only partially segregated within the dLGN. The partial segregation was specific in both experiments and was not due to a partial inhibitory action of U0126. Indeed, the concentration of U0126 in the retina and in the projection neurons of the dLGN was effective, as we have clearly demonstrated a strong reduction of pERK staining at both these sites.
Together, our results demonstrate that for the correct development of
retino-thalamic circuitry, activation of ERK only on RGCs or on geniculate
projection neurons is not sufficient. Importantly, we observed that pERK in
the dLGN is expressed only by the dLGN projection neurons
(Fig. 3A); hence, it is likely
that the complete disruption of the segregation process observed after
ICV-U0126 treatment is caused by a blockade of ERK activation, both on RGC and
dLGN projections cells. One might argue that the effect of ICV treatment could
also involve other afferent inputs to the dLGN. However, the concurrent
inhibition of pERK on geniculo-cortical relay neurons and RGCs faithfully
replicated the results found after ICV-U0126 treatment, making it unlikely
that there was any consistent contribution of pERK present in other afferent
inputs, except RGCs. In summary, these results provide strong evidence that
eye-specific segregation cannot develop without the relative contribution of
pERK on both RGCs and dLGN projection neurons. In view of this, our findings
highlight the role of LGN projection neurons in the formation of eye-specific
domains in the dLGN. It has been previously reported that thalamic NMDA
receptors do not contribute to the eye-specific segregation in the ferret dLGN
(Smetters et al., 1994).
However, although the activity of postsynaptic cells, at least the
NMDA-mediated activity, does not seem to be required, our study indicates that
the postsynaptic cell per se is involved in the segregation of eye-specific
layers in the dLGN.
Retinal activity and ERK signaling: a possible link during eye-specific segregation
A large body of work indicates that spontaneous retinal activity, present
in the developing retina (Feller et al.,
1996; Galli and Maffei,
1988
; Wong et al.,
1995
), shapes the retinogeniculate connectivity during early
development. Our results show that the ERK cascade, both at the retinal and
geniculate level, plays a key role in the segregation of retinal projections
and that ERK activation is affected by retinal activity during this process.
It should be noted that the alterations of retinogeniculate pattern observed
in our experiments after pERK blockade, are very similar to the alterations
due to electrical activity blockade. Indeed, pERK blockade in dLGN by ICV
U0126 treatment (Fig. 2) and
electrical activity blockade in dLGN by intracranial infusion of tetrodotoxin
(TTX) (Shatz and Stryker,
1988
) result in a complete arrest of retinal fiber segregation. In
addition, the monocular blockade of pERK (see
Table 1 and
Fig. 6), or of retinal activity
(Penn et al., 1998
), produces
a retraction of projections of the treated eye and an expansion of the
territory occupied by projections from untreated eye. It is unlikely that
U0126 affected neuronal activity and thus altered segregation through a
non-specific mechanism. Indeed, in vivo studies performed in our laboratory
have demonstrated that, in animals treated with U0126, the
electrophysiological properties of neurons are not affected
(Di Cristo et al., 2001
); this
is in agreement with several in vitro studies
(English and Sweatt, 1997
;
Impey et al., 1998
). Hence, on
the basis of these results, and on the fact that retinal activity drives ERK
phosphorylation in the retina and dLGN, we suggest that retinal activity
signals via ERK to regulate retinal fiber segregation in the RGCs and in the
dLGN projection neurons. The proposed model is consistent with the findings
that binocular blockade of all retinal activity prevents segregation
(Huberman et al., 2002
;
Penn et al., 1998
), but that
only simultaneous blockade of pERK in the retina and the dLGN yields the same
result.
The formation of eye-specific projection patterns in the dLGN occurs, in
different species, during the first postnatal week. Thus far, this process was
considered to reflect eye-specific segregation, whereas recently it has
been proposed that eye-specific segregation can be dissociated from
eye-specific layer formation (Huberman et
al., 2002; Muir-Robinson et
al., 2002
). These studies suggest that spontaneous retinal
activity in the first postnatal week is crucial in establishing eye-specific
layers; hence, the first postnatal week may constitute the crucial period for
eye-specific layer formation. However, it is not yet known what might be the
potential determinants of this crucial period. Because patterns of retinal
activity seem to be insufficient to instruct the development of
retino-geniculate connectivity (Huberman
et al., 2003
), an appealing hypothesis is that the formation of
eye-specific layers might be determined both by the level of endogenous
retinal activity (Stellwagen and Shatz,
2002
) and by the transient expression and recognition of molecular
markers in the retina and/or dLGN (Eglen
et al., 2003
; Huberman et al.,
2002
; Huberman et al.,
2003
; Muir-Robinson et al.,
2002
). In this context, pERK might influence the crucial period
for layer formation as it has two interesting properties that make it a likely
candidate as molecular determinant of the crucial period. First, it is
developmentally regulated, as its pattern of expression correlates with the
timing for layer formation, and second, pERK expression is modified by retinal
activity. We can speculate that during the crucial period electrical activity
may work hand in hand with ERK signaling to activate transcription factors,
and in this way, to regulate function and spatiotemporal distribution of
specific molecular cues.
Neurotrophins as potential activators of ERK
Growth factors such as neurotrophins (NTs) signal via ERKs during many
different neuronal functions, including cell differentiation
(Barnabe-Heider and Miller,
2003), survival (Han and
Holtzman, 2000
; Watson et al.,
2001
), axon growth (Atwal et
al., 2000
) and dendrite formation
(Miller and Kaplan, 2003
;
Vaillant et al., 2002
). These
factors can also mediate the synaptic plasticity of neural connections via the
ERK pathway (Patterson et al.,
2001
). Although there is a wide range of cellular processes in
which NTs are known to be involved, as yet no literature has reported any role
for NTs, except for BDNF, in the formation of eye-specific pattern. Recently,
we showed that endogenous anterogradely transported BDNF is required for the
proper development of retino-geniculate connectivity
(Menna et al., 2003
). In that
study, the binocular blockade of BDNF caused a retraction of retinal
projections rather than an expansion, as we have obtained after binocular pERK
blockade. Moreover, BDNF seems to act independently of retinal activity. These
results may suggest that for the formation of eye-specific domains BDNF
signaling does not converge into ERK; the action of BDNF may proceed in
parallel to that of the ERK pathway. However, a second scenario may exist:
BDNF action might be mediated by ERK. If so, the different alterations in
retino-geniculate connectivity observed in these two studies
(Menna et al., 2003
) (this
paper) might be explained by the different levels of ERK activation, depending
on the BDNF and electrical activity pathways, respectively. Indeed, it is
noted that the level of ERK activity is a crucial component during plastic
processes. For example, it has been reported that either blockade or
overexpression of ERK signaling reduces LTP levels or enhances dendritic
growth (English and Sweatt,
1997
; Kim et al.,
2004
; Komiyama et al.,
2002
). Considering these observations, we may suppose that, during
the formation of a correct retinothalamic pattern ERK signaling may be induced
not only by electrical activity but also by neurotrophins, which together act
in concert to finely regulate the eye-specific segregation.
ERK targets
If the MEK/ERK pathway is crucial in the remodeling of visual system
connectivity, what are the relevant ERK substrates? CREB (cAMP response
element binding protein) has been shown to be one of the targets of ERK
signaling during processes of neuronal plasticity such as learning and memory
(Impey et al., 1999), as well
as during developmental visual cortical plasticity
(Cancedda et al., 2003
).
Moreover, Stryker and colleagues (Pham et
al., 2001
) have shown that the CRE/CREB pathway contributes to the
refinement of retinogeniculate projections. Considering these findings and
ours reported here, we favor the hypothesis that the ERK pathway controls
eye-specific segregation through CREB. However, ERK signaling does not always
converge with CREB. For example, there is a positive coupling between the ERK
cascade and CREB phosphorylation during LTP
(Davis et al., 2000
;
Impey et al., 1998
), but not
during LTD (Thiels et al.,
2002
). Other potential targets of pERK at the cytoplasmic level,
such as cell adhesion molecules, cytoskeletal elements or synaptic proteins
(Bailey et al., 1997
;
Matsubara et al., 1996
;
Suzuki et al., 1995
), may also
be involved in the propagation of ERK signaling during retino-thalamic
development.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: The Hospital for Sick Children, Developmental Biology and
Cancer Research Departments, 555 University Avenue, Toronto, Ontario M5G 1X8,
Canada
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