1 Istituto di Neuroscienze CNR-56100 Pisa, Italy
2 Department of Experimental and Diagnostic Medicine, Section of General
Pathology, and Interdisciplinary Center for the Study of Inflammation (ICSI),
University of Ferrara, 44100 Ferrara, Italy
* Author for correspondence (e-mail: galli{at}in.cnr.it)
Accepted 6 April 2005
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SUMMARY |
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Key words: Retina, Cell death, ATP, P2X7, Amacrine cells, Mosaics, Rat
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Introduction |
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Cell death plays a key role in regulating the number of neurons in the
brain from its earliest stages of development. Death has been described among
proliferating neuroblasts, early postmitotic cells and in many populations of
neurons at the time they form synaptic connections (reviewed by
Davies, 2003;
de la Rosa and de Pablo, 2000
;
Lossi and Merighi, 2003
;
Oppenheim, 1991
;
Pettmann and Henderson, 1998
).
A long established idea is that the survival of developing neurons depends on
trophic factors produced in limited amounts, but recent studies have shown
that neuronal death may also partly be due to the activation of cytotoxic
signaling mechanisms, through cell receptors such as FasL, TNF-R and P75
(reviewed in Davies, 2003
;
Dechant and Barde, 2002
;
Raoul et al., 2000
).
A conserved mechanism of cell death activation has been described in
non-neuronal cells (Falzoni et al.,
1995; Girolomoni et al.,
1993
; Koshlukova et al.,
1999
), whereby extracellular ATP (e-ATP) triggers cell death by
binding the P2X7 receptors. Upon sustained or repeated activation,
the P2X7 receptors induce large non-selective membrane pores, which
eventually lead to cell death (Di Virgilio
et al., 1998
; North,
2002
).
P2X7 receptors have been shown to be expressed in regions of the
nervous system (North, 2002)
and, therefore investigations were undertaken to determine whether endogenous
e-ATP is involved in controlling the number and density of cells in specific
neural populations. The retina is an ideal region to test this, since most of
its neurons are arrayed with precisely controlled density, and there is a
prominent expression of P2X7. Among the developing retinal neurons,
the cholinergic neurons seemed particularly relevant, because they are
regularly spaced from early in development
(Galli-Resta et al., 1997
),
and play a key role in normal development by triggering the waves of
spontaneous neuronal impulse activity controlling the refinement of retinal
projections to the brain (Wong,
1999
).
We found that the retinal cholinergic cells express the P2X7 receptors, and could be sources of endogenous e-ATP. Degrading e-ATP in vivo, or blocking the P2X receptors increases the density of the cholinergic neurons in the developing retina by preventing their naturally occurring death. This was also confirmed by directly monitoring e-ATP-induced death of individual cholinergic neurons in isolated retinas. Death induced by e-ATP in the retina is specific to the cholinergic neurons, normally removing cholinergic cells too close to one another, and thereby contributing to the regular density and spacing of these neurons.
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Materials and methods |
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To control apyrase effectiveness in reducing e-ATP levels in vivo, we
collected 5 µl fluid samples from the posterior eye chamber, reasoning that
changes in ATP levels in this fluid would parallel those in the adjacent
retina. P4 rats were anaesthetized with avertine (10 ml/kg body weight, i.p.;
3.3% tri-bromo-ethanol, 2% tertiary amyl-alcohol in saline) 3 hours after
apyrase or vehicle intraocular injection. The tip (30-50 µm) of a glass
micropipette connected to a 25 µl Hamilton syringe with a micro-driven
piston was inserted into the posterior eye chamber. Samples were collected
under a dissecting microscope to verify the lack of blood or opacity, and
assayed in a luminometer (Perkin Elmer) to measure ATP levels by the
luciferin-luciferase assay. Apyrase-treated animals showed a 50-fold reduction
of e-ATP levels with respect to controls [2±1 µM vehicle injected
(n=5 animals), 40±50 nM apyrase injected (n=5
animals), P<0.05, Student's t-test]. It is important to
stress that these concentrations are lower than those found in the retina,
where endogenous e-ATP reaches local concentrations around 0.1 mM
(Newman, 2001). However, since
it does not diffuse far (approx. 50 µm), and is rapidly degraded
(Newman, 2001
), its final
concentration once diluted in the vitreus humor is much smaller.
Gene-gun labeling of explanted retinas
Imaging of living neurons was performed in retinas explanted from neonatal
rats. Ages were uniformly distributed between P2 and P8. Retinas were quickly
dissected in freshly made artificial cerebrospinal fluid
(Stacy and Wong, 2003),
flattened onto 13 mm Millipore nitrocellulose filters and placed in Petri
dishes containing artificial cerebrospinal fluid (ACSF) within an oxygenated
and humidified incubation chamber at room temperature (25-28°C).
Individual cells were labeled with fluorescent-conjugated dextrans delivered
by shooting dye-coated 1.3 µm tungsten particles into the retina using a
Bio-Rad gene gun (Kettunen et al.,
2002
). Cholinergic cells were identified by their laminar
positioning in the retina, their starburst morphology and their planar
dendritic arrangement, which are already recognizable early in life
(Stacy and Wong, 2003
;
Wong and Collin, 1989
). In
addition, we performed a morphological study combining YFP transfection to
label individual cholinergic cells and immunohistochemistry for choline
acetyltransferase (ChAT) to help identify the cholinergic cells in P2-P13 rat
retinas (unpublished data). Gene-gun labeling with fluorescent dextrans
produced an average of three to four labeled cholinergic neurons every 10
retinas, in accordance with the relatively low frequency of these neurons
(Jeon et al., 1998
).
Cell imaging in isolated retinas
Retinas were analyzed starting 1-2 hours after labeling. Cell images were
obtained with a Zeiss Axioplan fluorescence microscope equipped with a black
and white CCD camera (Chroma1600 DTA, Pisa, Italy). Retinas were placed on the
microscope stage within a small observation chamber containing 4 ml of
constantly oxygenated and frequently replaced ACFS maintained at room
temperature.
Retinas were rapidly screened for labeled cholinergic cells using an Olympus 40x water immersion objective (NA 0.80), keeping excitation light at 10-20% of its maximum intensity. Two images were taken for each cell, focusing on the dendritic tree (3-second exposure), then on the soma (0.5-second exposure). After treatment, images were taken of the dendrites and the soma of previously recorded cells. Reference points within the observation chamber were recorded to identify specific cells in subsequent observations by means of their coordinates. To limit light exposure, treated and control cells were only imaged before and 30 minutes after treatment, unless otherwise specified. In some experiments, cells were transfected with YFP and similar results were obtained as with dextrans (not shown).
Membrane permeability was assessed by adding propidium iodide (PI; 1.5 µM, for 1 minute) to the ACSF. Exposure of phospatidylserine on the outer membrane leaflet was assessed with Cy3-annexin V (3 µg/ml, for 20 minutes). Images were acquired with the FITC (488 nm) filter setting for Oregon-Green-488-dextran, with the TRITC filter setting (568 nm) for PI, Cy-3-annexin V and Alexa-Fluo-568-dextran. Quinacrine (1 µM) was incubated for 10-20 minutes at room temperature and imaged using a Leica TCNS confocal microscope, keeping the laser at minimal power (5-10%). Since quinacrine easily bleaches, a maximum of two cells were scanned per retina, setting the scanning depth for each cell under the TRITC filter (displaying dextran labeling), then scanning with the FITC filter (quinacrine), and finally with the TRITC filter.
Data acquisition and analysis
The cholinergic cell arrays were sampled using a Leica TCNS confocal
microscope. Four 400x400 µm2 samples of either cholinergic
cell arrays were taken in each whole-mount retina at mid-eccentricity along
four perpendicular axes. The density of cholinergic cells does not vary with
eccentricity at these ages (Galli-Resta et
al., 2002). The same proved true in a dedicated analysis of the
treated cases (eight samples per retina taken at two different eccentricities
along four perpendicular axes, two retinas per treatment; not shown). Since in
each case the objective was positioned midway between the papilla and the
retinal margin without prior examination of the cell distribution, each
sampling was unbiased. Sampled fields and retinal images were examined using
an Image analyzer (Imaging Ontario, Canada) to determine cell density, cell
positioning and retinal area. Cell counts and coordinates were obtained by
feeding each sample field to the Imaging system and using an automatic cell
counter based on intensity threshold and size exclusion criteria to eliminate
noise. Total numbers of cells were estimated multiplying average cell density
and retinal area. To investigate death among the cholinergic neurons, the
sampled fields were screened for cellular debris retaining immunoreactivity
for ChAT. ChAT-positive (ChAT+) debris within 15 µm of one another were
counted as a single occurrence. Pycnotic cells in the GCL were counted in four
samples (250x250 µm2) per retina, after staining with
propidium iodide. To sample non-cholinergic cells in the ganglion cell layer
(GCL), whole-mount retinas were immunostained for ChAT (FITC: green), then
labeled with PI (TRITC). The entire thickness of the GCL was scanned with a
confocal microscope under the FITC+TRITC filter setting, taking eight samples
(250x250 µm2) per retina at two regularly spaced
eccentricities along four perpendicular axes. All the PI cells that were not
labeled for ChAT were then counted using Metamorph. Horizontal cells
(tau-immunoreactive), dopaminergic amacrine [inner nuclear layer (INL) cells
immunoreactive for tyrosine hydroxylase (TH)] and AII amacrine cells (Disabled
1 immunoreactive) were also counted: horizontal cells were sampled in four
(400x400 µm2) samples. TH cells were sampled in eight
regularly spaced (400x400 µm2) samples limited to the
dorsotemporal retina where these cells are restricted early in life
(Wu and Cepko, 1993
); AII
cells were sampled in four mid-eccentricity (250x250 µm2)
fields along four perpendicular axes.
Plots, frequency histograms and statistical analysis were made using Origin
7.0 (Microcal). Custom made programs were used to compute the autocorrelation,
the density recovery profile (DRP), and the frequency of cells closer than 15
µm to one of their neighbors. The DRP and the exclusion radius (ER) are
computed as follows. Concentric circles are traced at constant distances (here
2.5 µm) from one another in the autocorrelation, and a histogram (DPR) is
obtained of the density of autocorrelation counts in the annuli delimited by
two consecutive circles. The DRP values are very low close to the center of
the autocorrelation (reflecting the central hole), and then rise to a final
constant density in the bins far from the origin. The ER is obtained as the
radius of the first circle (FC) where the histogram reaches or exceeds its
average final density minus a correction weighing the autocorrelation counts
found in the annuli contained within FC. This correction is computed as the
density of counts within the FC circle divided by the average DRP plateau
value reached away from the origin, and multiplied for the histogram bin size
(Rodieck, 1991). To evaluate
the frequency of cell pairs with an intercellular distance below 15 µm,
data from retinas treated with apyrase and oxidized ATP (oATP) were pooled
together. The frequency of cell pairs was computed as the percentage of the
total number of cell pairs in the field. In a field with n cells
there are n*(n-1)/2 pairs of cells.
Reagents
Fluorescent conjugated dextrans (10 kDa, conjugated with Alexa Fluo 488 or
568 or Oregon Green 488), and fluorescent conjugated secondary antibodies were
from Molecular Probes, Eugene, OR. Suramine was from Calbiochem. Antibodies to
choline acethyltransferase (ChAT) and tyrosine hydroxylase (TH) were from
Chemicon. The BrdU monoclonal antibody was from Roche. The P2X7
polyclonal has been described previously
(Ferrari et al., 1997); the A8
polyclonal to Islet 1/2 was a kind gift from T. Jessell; the antibody to
Disabled 1 was a kind gift from B. Howell. All other chemicals were from
Sigma.
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Results |
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Extracellular ATP participates in the developmental control of the density and number of cholinergic amacrine cells
Neonatal rats subjected on P1 to intraocular injection of oxidized-ATP
(oATP; 300 µM), an irreversible blocker of the P2X purinergic receptors
(Murgia et al., 1993;
North, 2002
), displayed on P2
(i.e. 24 hours after the injection) a significant increase in the density of
retinal cholinergic neurons (Fig.
1A,B). This was about 23% higher than normal for the cholinergic
cells in the GCL, and 22% for the cholinergic cells in the INL (n=8
retinas; Fig. 1C,D). A similar
density increase (+26% in the GCL, +28% in the INL; n=8 retinas) was
observed 24 hours after intraocular injection of suramine (150 µM;
Fig. 1C,D), a generic blocker
of the purinergic P2 receptors (Ralevic
and Burnstock, 1998
), as well as after injections of apyrase (30
U/ml, +30% GCL, +21% INL; n=8 retinas;
Fig. 1C,D), an enzyme that
hydrolyses e-ATP (Komoszynski and
Wojtczak, 1996
). In all cases the increase in the density of
cholinergic neurons was statistically significant (P<0.0001,
t-test).
An increased cell density was not a generalized effect following e-ATP
blockade, since we did not observe any change in retinal area (not shown), in
the thickness of the retinal layers, or in the density of a number of cell
populations that we analyzed as a control. These populations include the
non-cholinergic cells in the GCL [mostly retinal ganglion cells at this age
(Perry et al., 1983;
Rabacchi et al., 1994
)], the
horizontal cells, the long-range dopaminergic amacrine cells and the
short-range AII amacrine cells (Table
1; Fig. S1 in supplementary material).
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e-ATP causes death of the cholinergic neurons in normal retinal development
Since the cholinergic neurons found in treated retinas outnumbered the
maximal number of these cells found in normal development
(Fig. 2A,B), an accelerated
migration of the cholinergic neurons to their layers could not account for the
observed effects. New cell genesis was also excluded, since bromodeoxyuridine
(BrdU), administered to label all the progenitor cells synthesizing DNA in the
interval between treatment and analysis, was not detected in any of the 3000
cholinergic cells analyzed in sections from five treated retinas
(Fig. 2C).
e-ATP can activate cell death in different non-neural cell populations
(Di Virgilio et al., 1998).
Investigations were therefore carried out to determine whether blocking e-ATP
increased the number of cholinergic cells by preventing cell death among these
neurons. In line with this hypothesis we found that cellular debris expressing
cholinergic markers (ChAT+) had an average frequency of 3.7±1.4 every
1000 cholinergic cells in normal retinas (n=8 retinas), and decreased
after e-ATP blockade, being 0.8±0.6 in oATP-treated retinas
(n=8) and 0.7±0.6 in apyrase-treated retinas (n=8).
This decrease was statistically significant (P<0.001,
t-test) in both cases. Conversely, we observed up to a sixfold
increase in the occurrence of ChAT+ debris (20±10 per 1000 cholinergic
cells; n=3 retinas) 90 minutes after injection of 5 mM ATP into the
eye. These changes in the frequency of ChAT+ cellular debris are consistent
with the hypothesis that eATP regulates the basal level of cell death among
the cholinergic neurons. e-ATP blockade did not affect the global occurrence
of pycnotic cells in the GCL which was 6.5±2 every 1000 GCL cells in
normal retinas (n=4) and 7.0±3 in oATP-treated retinas
(n=4).
To test directly whether ATP kills the cholinergic neurons, we monitored
the fate of individually labeled cholinergic cells after the application of
ATP or other agents in isolated neonatal retinas aged between P2 and P8. Since
experiments measuring endogenous e-ATP release in the rat retina have detected
local eATP concentrations around 0.1 mM
(Newman, 2001), we challenged
cholinergic cells with this or higher concentrations of ATP. Developing
cholinergic cells displayed dendritic and soma blebbing within minutes of ATP
application (20/20 cells with 0.1 mM ATP; 5/5 cells with 0.5 mM ATP; 25/25
cells with 1 mM ATP; Fig.
3A-E). P2X7 receptor activation induces membrane
blebbing, membrane permeabilization to large cations such as propidium iodide
(PI), and eventually leads to cell death
(Di Virgilio et al., 1998
;
North, 2002
). ATP-induced
permeability to PI was observed in all individually identified cholinergic
cells we tested (33/33 cells; Fig.
3F). Furthermore, Cy3-annexin V revealed phosphatidylserine
exposure on the external leaflet of the cytoplasmic membrane (10/10 cells;
right inset in Fig. 3C), a
typical early indicator of the activation of a death process
(Reutelingsperger et al.,
2002
).
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The effects of ATP application on the cholinergic cells were the same
throughout the age range we tested (P2-P8; e.g.
Fig. 3A-F, Fig. 4A,B). Any detectable
effect of ATP (1 mM) was prevented by the simultaneous incubation with apyrase
(30 U/ml; 16/16 cells; Fig.
4C,D). Similarly, 2 hours of pre-incubation with the irreversible
P2X antagonist oATP (300 µM) prevented blebbing and PI permeability (ATP 1
mM; 11/11 cells; Fig. 4E,F),
even for ATP incubations as long as 2 hours. Finally, 30 minutes to 1 hour
pre-incubation with Brilliant Blue G [BBG; a selective antagonist of rat
P2X7 when used below the micromolar range
(North, 2002)], prevented
blebbing and membrane permeability induced by ATP (1 mM for 30 minutes) in 95%
of the tested cells (10/10 cells BBG 0.5 µM; 9/10 cells BBG 0.2 µM;
Fig. 4G.H). The protective
effects of BBG were reversible, since 1 mM ATP application after BBG washing
induced membrane blebbing and permeabilization (5/5 cells BBG 0.5 µM; 5/5
cells BBG 0.2 µM; 10-minute washing followed by 1 mM ATP for 30 minutes;
Fig. 4I). Membrane blebbing,
loss of membrane integrity or annexin V labeling were never observed in 30
individually labeled cholinergic control cells, subjected to the same light
exposure as the treated cells.
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To search for e-ATP sources in the neonatal retina we used quinacrine, a
vital green-fluorescent dye labeling high levels of ATP bound to peptides in
large granular vesicles (Bodin and
Burnstock, 2001). In P2-P8 rat retinas where individual cells had
been labeled with Alexa-Fluo-564-dextran (red) to allow cell identification,
we observed quinacrine labeling in the astrocytes of the central retina (not
shown) and in all the cholinergic cells we had labeled (10/10 cells;
Fig. 5B-D), while none of the
individually labeled non-cholinergic amacrine cells or RGCs (0/25 cells)
contained quinacrine (not shown). These data suggest (but do not prove) that
the cholinergic cells could be endogenous sources of e-ATP in developing
retinas, in accordance with previous observations
(Santos et al., 1999
).
Death induced by e-ATP eliminates cholinergic cells getting too close to one another
We reasoned that if the cholinergic cells were able to release e-ATP,
e-ATP-induced death should mostly affect cholinergic cells close to one
another, because e-ATP spreads only short distances (<50 µm) away from
its source (Newman, 2001). In
agreement with this prediction, we found a higher than normal frequency of
cholinergic cells very close to one another after e-ATP blockade: while in
vehicle injected control retinas the frequency of cholinergic cell pairs with
an intercellular distance less than 15 µm is
2.7x10-2±0.8x10-2, this frequency
increased to 4.1x10-2±1x10-2 in oATP-
and apyrase-treated retinas (P<10-5, t-test).
To test this further we used the autocorrelation analysis, which plots the
distances between all pairs of cells in an array. The autocorrelation of the
cholinergic mosaics in normal or vehicle-injected retinas is a uniform
distribution of points with a central empty region (e.g.
Fig. 6A)
(Galli-Resta, 2000
). This
means that each cholinergic cell normally tends to exclude other cholinergic
neurons from a specific region surrounding its soma. Beyond this exclusion
zone cholinergic cells can occupy any position in the field
(Galli-Resta, 2000
). The
autocorrelations of the cholinergic mosaics treated with either oATP or
apyrase differ from control simply because they have smaller empty regions
(e.g. Fig. 6B), revealing a
reduced efficacy of the exclusion mechanisms. This difference can be analyzed
using the density recovery profiles (DRPs), histograms plotting the density of
counts in the autocorrelation as a function of the distance from the center of
the coordinates. In both control (example in
Fig. 6C) and treated cases
(Fig. 6D) the DRPs rise from
zero to a constant density, but do so in much shorter distances in the treated
than in the control cases. The size of the exclusion region is normally
quantified using the exclusion radius (ER; arrows in
Fig. 6C,D), which is calculated
as the distance from the origin where the DRP reaches a constant density minus
a correction factor considering the DRP counts at smaller distances [see
Materials and methods and Rodieck
(Rodieck, 1991
)]. As shown in
Fig. 6E, the average ER is
reduced for the GCL cholinergic cell arrays treated with apyrase or oATP with
respect to control. A similar ER reduction was observed for the INL
cholinergic cell array after e-ATP blockade (not shown).
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Discussion |
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The possibility that e-ATP could also control the cholinergic cell number
by inducing a transdifferentiation of postmitotic cholinergic neurons into
non-cholinergic cells was discounted. To our knowledge transdifferentiation of
postmitotic neurons has never been observed in the retina, where cell fate
appears to be determined around the time of the last cell division (Livesey
and Cepko, 2001). Furthermore, we never observed any indication of
mix-phenotypes (e.g. cells immunopositive for ChAT but not displaying features
typical of the cholinergic amacrine cells such as soma size, dendritic
arrangement, planar organization, Islet immunoreactivity, or displaying
features typical of non cholinergic cells) in vivo in normal retinas or after
eATP blockade. Finally, direct application of ATP in concentrations normally
observed in the rat retina (Newman,
2001) never induced anything but typical cell death indicators in
individually labeled cholinergic cells in isolated retinas.
Future studies will be necessary to investigate the death process activated
by e-ATP in the cholinergic neurons. Strong evidence points to P2X7
as the most likely P2 receptor subtype responsible for the e-ATP cytotoxic
effect, as suggested by the potent killing activity of BzATP, the protective
effect of oATP and Brilliant Blue G, and the high level of expression of this
receptor in the cholinergic neurons. This does not exclude the possibility
that other P2X receptors for which a pore-forming ability has been
demonstrated (e.g. P2X2 or P2X4) (Khakh et
al., 1999; Virginio et al.,
1999
) might also be involved, although a death-inducing activity
for P2X subtypes other than the P2X7 has not been shown
(North, 2002
).
P2X7 receptors have been shown to trigger both apoptosis and
necrosis, depending on the cell type (Di
Virgilio et al., 2001). In the retina we have been unable to label
cholinergic cells using TUNEL or ISEL staining to detect fragmented DNA but
these negative results may simply reflect the loss of cholinergic markers
before these neurons reach advanced stages of death. More interestingly, we
found that broad caspase inhibitors were unable to increase the number of
cholinergic cells (not shown), suggesting that e-ATP induced a
caspase-independent death.
Endogenous e-ATP as a local, possibly cell-type-specific cytotoxic agent for retinal cholinergic cells
Death induced by e-ATP in the developing retina appears specific to the
cholinergic neurons. Blocking e-ATP signaling increased the number of
cholinergic cells, but did not induce any generalized effect on retinal area,
thickness or layering. Furthermore, treatments affecting e-ATP signaling did
not alter cell density in a number of control cell populations. These include
the horizontal cells, the short range amacrine cells AII, the long range
dopaminergic amacrine cells, as well as all the non-cholinergic cells in the
GCL (mostly retinal ganglion cells at these ages), notwithstanding most of
these latter cells express the P2X7 receptors
(Fig. 5A) (Wheeler-Schilling et al.,
2001). These results indicate that P2X7 expression is
not enough to make cells vulnerable to e-ATP-induced death, in line with
previous results showing that susceptibility to ATP may depend on the level of
expression of the P2X7 receptors and the potential coupling of
these receptors to cytoplasmic effectors
(North, 2002
). In addition,
local e-ATP degradation by endogenous ecto-ATPases may selectively protect
specific cells (Zimmermann,
1996
), keeping endogenous e-ATP below the P2X7
activation threshold around these cells. A similar reasoning may explain why
we found that endogenous e-ATP controlled the death of cholinergic neurons
only before P5, while direct ATP application could kill the cholinergic
neurons also at later stages. Our preliminary data on the retinal distribution
of the major ecto-ATPase (CD39)
(Zimmermann, 1996
) are
consistent with this hypothesis, showing a concentration of CD39 around the
ganglion cells between P0 and P5, and a much broader expression pattern
afterwards (not shown).
Muller cells and astrocytes are sources of e-ATP in the adult retina
(Newman, 2001;
Newman, 2003
), but in the
first postnatal days Muller glia has not been generated yet
(Wong and Godinho, 2003
), and
astrocytes are only found in the central retina
(Ling et al., 1989
).
Therefore, we searched for e-ATP sources in the neonatal retina. Using
quinacrine, a vital stain that binds ATP-containing vesicles, we found that
the cholinergic cells store ATP in granules, which suggests that they are
potential sources of e-ATP in the developing retina. Proving that the
cholinergic cells do release ATP however, will require future studies.
Death induced by e-ATP controls the local density and regular spacing of the cholinergic neurons
We have found that the frequency of neighboring cholinergic cells spaced by
less than 15 µm was higher than normal after e-ATP signaling blockade. In
addition, autocorrelation analysis showed that the major effect of e-ATP
blockade on the spacing of the cholinergic cells was a reduced efficacy of the
mechanism by which each cholinergic cell normally excludes other cholinergic
neurons from a limited region surrounding its soma. Thus, death induced by
e-ATP not only controls the density of the cholinergic cells, but also
contributes to the regular spacing that these neurons normally display in the
retina. We do not know how this occurs, but we can propose a working
explanation based on the assumption that the cholinergic cells release ATP:
when two cell sources of e-ATP, which are both vulnerable to e-ATP-induced
death, get too close to one another, if one or both release ATP, this can kill
one, or even both of these cells, thereby reducing local overcrowding.
Most neurons in the retina form arrays, or mosaics, where cells of the same
type are regularly spaced (Cook and
Chalupa, 2000; Galli-Resta,
2002
; Wässle and Riemann,
1978
). This orderly arrangement of neurons of the same type is
thought to ensure an even sampling of the visual field. Previous studies have
shown that lateral cell displacement plays an important role in the formation
of retinal mosaics (Galli-Resta et al.,
1997
; Reese and Galli-Resta,
2002
; Reese et al.,
1995
), but simulation experiments
(Jeyarasasingam et al., 1998
)
and studies of retinal mosaics in death-suppressing transgenic mice
(Raven et al., 2003
) strongly
suggested that cell death also participates in this process. The present study
provides direct evidence of a death control mechanism contributing to regular
cell spacing and density in a neural population.
The selective elimination of cells too close to one another is a potent
mechanism to prevent local overcrowding. However, cell death cannot by itself
create a regular distribution of cells, unless a continuous provision of new
cells is ensured until the final cell density and regular cell spacing are
both achieved. This process would require an enormous amount of cell genesis,
as simulation experiments easily show (e.g. simulating the formation of a
normal cholinergic array by generating cells in random places and eliminating
them whenever they do not obey the minimal spacing rule of 15±2 µm
typical of the neonatal cholinergic arrays, shows that on average as many
cells should be eliminated as finally remain in the array) (see also
Eglen and Willshaw, 2002). This
would not be the case, however, if cells too close to one another could also
move apart: this process would reduce their risk of death, contribute to
regular cell spacing, and reduce the number of new cells necessary to achieve
a final regular density. Tangential cell dispersion could in principle be
enough to space cells appropriately, but we know little of the controlling
mechanisms. Lateral cell migration has been shown to involve dendritic
interactions (Galli-Resta et al.,
2002
), to be limited to a specific developmental time window, and
to displace cells no more than 100-150 µm away from their clone of origin
(reviewed by Reese and Galli-Resta,
2002
). In several situations therefore, it might be more expedient
for misplaced cells to die rather than move around till they are properly
placed.
A dynamic regulation of cell number during cholinergic cell development
We have found that blocking for 24 hours the e-ATP-induced death of retinal
cholinergic neurons in vivo significantly increases the total number of these
cells (Fig. 2A,B). This means
that many cholinergic neurons are normally dying at these ages. However, new
cholinergic cells are also observed to migrate to the cholinergic arrays at
these same times (Galli-Resta,
2000; Galli-Resta et al.,
1997
). The total number of cholinergic neurons reflects the
balance between these two opposite contributions. This is not the first
example of a cell population simultaneously undergoing cell death and cell
replacement in the developing nervous system. This dynamic behavior has
already been shown in the early development of retinal ganglion cells
(Frade et al., 1997
;
Frade et al., 1996
), in the
avian ciliary ganglion (Lee et al.,
2001
) and in populations of retinal and cortical neuroblasts
(reviewed by de la Rosa and de Pablo,
2000
). The simultaneous presence of cell death and new cell
addition makes it very difficult to estimate the real amount of cell death
going on during development, or even to detect cell death as a decrease in the
total number of cells.
Death induced by e-ATP has never been reported in the developing nervous
system. However, a number of non neuronal cell populations, ranging from human
macrophages (Falzoni et al.,
1995) and keratynocytes
(Girolomoni et al., 1993
) to
colonies of fungi (Koshlukova et al.,
1999
) undergo cell death induced by ATP through the
P2X7 receptors, suggesting that the cholinergic neurons are a new
example of an ancient mechanism of cell death control.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/12/2873/DC1
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