 |
 Previous Article | Next Article 
The Journal of Neuroscience, March 15, 2003, 23(6):2363
Glutamate Receptor  2 Subunit in Activity-Dependent
Heterologous Synaptic Competition
Roberta
Cesa,
Laura
Morando, and
Piergiorgio
Strata
Rita Levi Montalcini Center for Brain Repair, Department of
Neuroscience, University of Turin, 10125 Torino, Italy
 |
ABSTRACT |
In the adult cerebellum, the glutamate receptor  2 subunit
(GluR2) is selectively targeted to the spines of the distal Purkinje cell dendrites, the spiny branchlets, that are innervated by the parallel fibers. Although GluR2 has no known channel function, it is
presumed to be involved in the formation and stabilization of these
synapses. After block of electrical activity by tetrodotoxin, GluR2s
appear in the postsynaptic densities of the proximal dendritic spines,
which then lose their contact with climbing fibers and become
ectopically innervated by parallel fibers. This phenomenon suggests
that climbing fiber activity prevents GluR2 targeting to proximal
dendrites and that GluR2s admitted to the postsynaptic density of
the spine cause withdrawal of the silent climbing fiber. To test this
hypothesis, we studied the distribution of GluR2s in the rat
cerebellum by immunoelectron microscopy during the recovery period that
follows removal of the electrical block, and during the sprouting of
climbing fibers that follows subtotal deletion of the parent inferior
olivary neurons by administration of the drug 3-acetylpyridine. We
found that after removal of the electrical block, the climbing fibers
reinnervate proximal spines that bear GluR2s and these subunits are
successively repressed. Similarly, after subtotal lesion of the
inferior olive, reinnervation of denervated Purkinje cells occurs on
spines bearing GluR2s. Thus, GluR2s are not responsible for
displacing silent climbing fibers. We propose instead that GluR2s
are associated with climbing fiber-to-Purkinje cell synapses, during
development or at early stages of climbing fiber regeneration or
sprouting, and are downregulated during the process of synapse maturation.
Key words:
glutamate receptor 2 subunit; afferent fiber
competition; activity-dependent competition; cerebellum; parallel
fiber; climbing fiber
| |
Introduction |
Glutamate receptor (GluR) channels
mediate most of the fast excitatory synaptic transmission in the
vertebrate CNS and play an essential role in various types of
plasticity (Mayer and Westbrook, 1987 ; McDonald and Johnston, 1990).
The 2 subunit (GluR2) belongs to the family of glutamate receptor
channels. It is selectively expressed in Purkinje cells and has no
known channel function (Araki et al., 1993; Lomeli et al., 1993).
During development, GluR2s are expressed in both parallel fiber- and
climbing fiber-innervated spines. In contrast, at the end of the
developmental period, these subunits remain exclusively or nearly
exclusively confined to the parallel fiber synapses (Takayama et al.,
1996; Landsend et al., 1997; Zhao et al., 1998). In the GluR2
knock-out mouse, nearly one-half of the branchlet spines are free of
innervation, although the total spine density is normal (Kashiwabuchi
et al., 1995; Kurihara et al., 1997). In addition, the climbing fiber terminal arbor in this mouse extends its territory of innervation distally to the parallel fiber dendritic domain (Ichikawa et al., 2002). These observations suggest that GluR2s play a role in the
differential distribution and stabilization of the parallel fiber and
climbing fiber synapses on the distal and proximal domains of the
Purkinje dendritic arbor.
Experimental block of electrical activity by tetrodotoxin (TTX) has
also shown that GluR2s are involved in the competition between
parallel fibers and climbing fibers to acquire their postsynaptic domains on the Purkinje cells (Bravin et al., 1999; Morando et al.,
2001). After block of electrical activity, climbing fibers lose their
contacts with the spines of the proximal dendrites. However, before
their withdrawal, GluR2s appear in these spines. This fact suggests
that the active climbing fiber exerts a repressive action on the
expression of GluR2, and that de novo targeting of this
subunit to the spines innervated by the inactive climbing fibers is
responsible for the withdrawal of the terminal afferents. Additionally,
after block of electrical activity the proximal dendritic domain
develops a large number of new spines that also bear a high density of
GluR2s and are ectopically innervated by the parallel fibers.
Therefore, a complementary view is that the active climbing fiber
represses the number of spines of the proximal dendrites and that, in
the absence of electrical activity, the parallel fibers gain a
competitive advantage and displace the synapses of silent climbing
fibers from GluR2-bearing spines.
To test these hypotheses, we decided to study the process of climbing
fiber reinnervation of the proximal Purkinje cell dendrite that takes
place on TTX removal and after 3-acetylpiridine (3-AP)-induced subtotal
deletion of inferior olivary neurons, which are the source of climbing
fibers. Specifically, we set out to ascertain whether during the
recovery period the growing climbing fiber terminals are able to
reinnervate spines bearing GluR2s or whether they form synapses only
with spines devoid of them.
| |
Materials and Methods |
Toxin delivery. We used adult Wistar albino rats
(body weight, 150-250 gm; age, 1.5-3 months; Charles
River, Calco, Italy). TTX (80 µM in 0.12 M phosphate buffer, pH 7.2; Sigma,
St. Louis, MO) was infused for 7 d into lobule VII of the dorsal
vermal cortex by means of an osmotic minipump (Alzet 2002;
Alza, Cupertino, CA) (Bravin et al., 1999). Control
animals were infused with vehicle. Ultrastructural analysis was
performed on vehicle-infused rats (n = 3), TTX-infused
rats killed after 7 d of treatment (n = 3), and
TTX-infused rats that survived 45 d (n = 3) and
135 d (n = 3) after TTX removal. Because TTX
infusion was not successful in every animal, only rats displaying
cerebellar ataxia were selected for our study. All surgical procedures
were performed under general anesthesia by a mixture of ketamine (100 mg/kg, Ketavet; Gellini Farmaceutici, Latina, Italy) and
xylazine (5 mg/kg, Rompum; Bayer, Leverkusen, Germany).
The experimental plan was designed according to the guidelines of
Italian law for care and use of experimental animals (DL 116/92) and
approved by the Italian Ministry of Health.
Inferior olive lesion. We obtained a subtotal lesion of the
inferior olive in another set of adult rats (body weight, 120-170 gm;
age, 35-40 d) by a single intraperitoneal injection of 3-AP (Fluka; Sigma; 65 mg/kg body weight). After
survival periods of 6, 40, 90, 150, and 270 d, 3-AP-treated rats
that were affected by severe ataxia were transcardially perfused with
500 ml of glutaraldehyde (0.1%), formaldehyde (4%), and picric acid
(0.2%) in 0.12 M sodium phosphate buffer, pH
7.4. The extent of the inferior olive lesion was checked in
25-µm-thick serial sections of the medulla oblongata stained
with the Nissl method (Hess et al., 1988). The numbers of surviving
neurons were calculated relative to the control numbers as determined
by Schild (1970). Only the cerebella of rats (n = 3 for
each survival period) with a number of surviving olivary neurons of
<12% were processed for additional study. Three untreated rats were
used as controls.
Electron microscopy. For ultrastructural analysis, sagittal
slices of the cerebellum obtained with a vibrating blade microtome (Leica, Wien, Austria) were embedded in
Epon/Araldite resin (Fluka; Sigma). For
serial reconstructions, 15 series consisting of 10-15 ultrathin
sections were prepared and mounted on single-slot copper grids. After
contrast enhancement with uranyl acetate and lead citrate, photographs
were taken on an electron microscope operated at 80 kV (model 410;
Philips, Eindhoven, The Netherlands) at a magnification of 4000× and
printed at a final magnification of 16,000×. Proximal Purkinje cell
dendrites were identified by their size and typical hypolemmal cisterns
of smooth endoplasmic reticulum (Palay and Chan-Palay, 1974; Bravin et
al., 1999). A proximal dendritic profile was selected in each ultrathin
section series, and all spines in continuity with that profile were
identified. Each spine or spine-like protrusion identified in a given
section was followed until it disappeared downstream and upstream in
the section series.
Freeze substitution and Lowicryl embedding. After perfusion
with 500 ml of glutaraldehyde (0.1%), formaldehyde (4%), and picric acid (0.2%) in 0.12 M sodium phosphate buffer,
pH 7.4, cerebellar blocks were sagittally cut (500 µm) on the
vibrating blade microtome and slammed to a polished copper block cooled
with liquid N2 (MM80 E cryofixation apparatus;
Reichert, Wien, Austria). The morphological analysis of
vehicle- and TTX-treated cerebella was performed in folia on either
side of lobule VII, which was the injection site of the minipump
cannula. Slices were transferred to 0.5% uranyl acetate dissolved in
anhydrous methanol ( 90°C) in a freeze-substitution apparatus (CS
Auto; Leica). The temperature was raised stepwise to
50°C. Samples were infiltrated with Lowicryl HM20 resin
(Chemische Werke Lowi, Waldkraiburg, Germany) and
polymerized by UV light.
Immunoincubation. Ultrathin sections (90-110 nm) of
Lowicryl-embedded blocks were collected on uncoated nickel grids and
processed for immunogold cytochemistry. The sections were etched with a saturated solution of NaOH in absolute ethanol for 2-3 sec, rinsed with double-distilled water, and incubated sequentially in the following solutions (at room temperature): (1) 0.1% sodium borohydride and 50 mM glycine in Tris buffer (5 mM) containing 0.9% NaCl and 0.1% Triton X-100
(TBST; 10 min); (2) 2% human serum albumin (HSA) in TBST (10 min); (3)
primary antibody (rabbit anti-GluR2; kindly provided by M. Watanabe,
Department of Anatomy, Hokkaido University, Sapporo, Japan; 2.5 µg/ml) in TBST containing 2% HSA (overnight); (4) TBST (several
rinses) and 2% HSA in TBST (10 min); and (5) goat anti-rabbit Fab
fragments coupled to 10 nm colloidal gold particles (GFAR10;
British BioCell International, Cardiff, UK), diluted 1:20
in TBST with 2% HSA and 0.05% polyethyleneglycol (1 hr). The nickel
grids were then rinsed several times in double-distilled water,
counterstained with uranyl acetate and lead citrate, and examined in
the electron microscope. The specificity of the GluR2 antiserum was
confirmed by the complete lack of immunostaining in sections incubated
only with TBST omitting the primary antibody.
Quantitative analysis. Quantitative analysis of immunogold
particles representing GluR2 antiserum binding sites was performed on electron micrographs of climbing fiber and parallel fiber synapses identified by standard morphological criteria (Palay and Chan-Palay, 1974). Small clusters of vesicles abutting the synaptic contact characterize parallel fiber terminals. In contrast, climbing fiber boutons contain a high density of uniformly distributed vesicles and
some dense core vesicles. It is possible that the regenerating climbing
fibers have a different morphological profile. This is unlikely,
because it has been demonstrated previously (Rossi et al., 1991a) that
in 3-AP-treated rats the regenerating climbing fibers maintain their
typical morphology. These synapses were sampled throughout the
thickness of the cerebellar molecular layer. The length of the
postsynaptic density was measured, and the number of gold particles was
counted. Then the immunolabeling density was expressed as the number of
gold particles per micrometer of postsynaptic density length.
Statistical evaluation was performed using Student's t
test. Quantitative analysis of GluR2 immunoreaction in the spines
innervated by climbing fiber in both TTX- and vehicle-treated cerebella
was performed on 10 areas selected from 10 grids for each experimental
group. On each grid, composed of 300 squares, we selected, by randomly
shifting the grid, only five squares (for a total area of 34,445 µm2) entirely occupied by molecular
layer. The average number of climbing fiber synapses was expressed per
a unit area of 34,445 µm2.
| |
Results |
To elucidate the role of GluR2s in the competition between
parallel fibers and climbing fibers, we used two different experimental protocols that produce reinnervation of Purkinje cells by the climbing
fibers. In the first model, we topically blocked all electrical
activity in the cerebellar cortex by TTX for 7 d. Under TTX block,
the climbing fiber terminal arbor retracts from its target and becomes
atrophic, the Purkinje cell proximal dendrites undergo a remarkable
increase in spine density, and the parallel fiber input, which is
normally limited to the distal dendritic domain, innervates the newly
formed spines (Bravin et al., 1999; Morando et al., 2001). Four weeks
after removal of the electrical activity block, the climbing fiber
arbor reacquires its normal morphology. It expands its territory to
reinnervate the proximal dendritic domain and, at the same time, it
induces elimination of the competing parallel fiber synapses from the
same territory, thus restoring the original pattern of low spine
density typical of the proximal dendrites.
In the second model, we performed a subtotal lesion of the inferior
olive to eliminate the majority of climbing fibers. A few days after
the lesion, a large number of new spines appear on the Purkinje cell
proximal dendrites, and parallel fibers sprout and contact the newly
formed spines (Sotelo et al., 1975; Rossi et al., 1991a,b). At variance
from the TTX experiments, the new spines develop in the presence of
increased Purkinje cell activity (Benedetti et al., 1984; Savio and
Tempia, 1985; Strata, 1985). In addition, collateral branches of
climbing fibers of surviving inferior olivary neurons reinnervate many
climbing fiber-deprived Purkinje cells. This process is especially
prominent during the first 3 months after the lesion, although it lasts
several months. During the climbing fiber regenerative process, the two
excitatory inputs, climbing fiber and parallel fibers, coexist on the
proximal Purkinje cell dendrite to segregate again when the new
climbing fiber reaches its full maturation and displaces the parallel
fiber synapses (Rossi et al., 1991a,b).
Recovery of climbing fibers after TTX block
Below, we present quantitative data on GluR2 in proximal
dendritic spines in four experimental groups of subjects, each
consisting of three rats, after: (1) vehicle infusion for 7 d
(control group), (2) 7 d of TTX block, and (3) two time points (45 d and 135 d) of recovery from TTX block. In each experimental
group, quantitative analysis was done on 10 randomly selected areas of
the molecular layer measuring 34,445 µm2
each (see Materials and Methods).
In the vehicle group, we counted the number of climbing
fiber-innervated spines per unit area. All of these spines were counted regardless of whether or not they were seen emerging from a dendrite, because we never observed climbing fibers contacting spines of distal
branchlets (see below). We found an
average of 6.9 ± 3.0 SD climbing fiber-innervated spines (Figs.
1a, 2a). Of these
spines, 1.7 ± 1.2 SD (24.6%) expressed GluR2 (Fig.
1a) and the gold particle density was 2.8 per micrometer
(±7.2 SD) of postsynaptic density length (Fig. 1b).
View larger version (19K):
[in this window]
[in a new window]
|
Figure 1.
Quantitative evaluation of proximal and distal
Purkinje cell dendritic spines innervated by climbing fiber
(CF) terminals and by parallel fiber
(PF) varicosities and their GluR2 expression
in the TTX experimental group. Immunogold particle counts were done
after 7 d of infusion with vehicle (v) and
TTX and at 45 and 135 d after TTX removal. a,
Average number ± SD of CF synapses per unit area
of 34,445 µm2. b, c,
Gold particle density per micrometer of postsynaptic density length in
CF- and PF-innervated spines. The total
number of climbing fiber-innervated spines (black
columns) drops under TTX and recovers at 45 and 135 d
(a). The number of GluR2-labeled synapses
(gray columns) is a small fraction in
vehicle-treated animals (a), and they have a very
small gold particle density (b), whereas under
TTX they represent a high fraction (a) and have a
high gold particle density (b). Parallel fiber
synapses on the spiny branchlets maintain similar values of high
GluR2 density in all conditions (c).
|
|
View larger version (109K):
[in this window]
[in a new window]
|
Figure 2.
GluR2 expression in TTX-treated cerebella.
a-c, Climbing fiber-innervated spines. GluR2
expression is virtually absent in vehicle-treated cerebella
(a) and after 135 d of recovery
(c), whereas it is highly expressed at 7 d
of TTX treatment (b). d-f,
Parallel fiber-innervated spines emerging from the branchlets. GluR2
expression is high in all three conditions. Scale bar, 0.23 µm.
|
|
In the 7 d TTX-block group, we made a similar evaluation and found
that the number of climbing fiber-innervated spines per unit area was
significantly (p < 0.002) lower, with an
average value of 2.6 ± 1.6 SD (Fig. 1a). Among these
spines, those bearing GluR2 had a value of 1.8 ± 0.9 SD (Figs.
1a, 2b), not significantly (p > 0.8) different from that of the vehicle
group, but representing 69.2% of the total number of climbing
fiber-innervated spines. In addition, the gold particle density had a
significantly higher value of 28.9 per micrometer (±32.0 SD;
p = 3.49 × 10 4)
relative to the same value in the vehicle group (Fig. 1b).
These results are in line with the description of a decrease in
climbing fiber synapses consequent to the climbing fiber atrophy and an overexpression of GluR2 in the remaining synapses (Morando et al.,
2001).
After removal of TTX block, the climbing fiber terminal arbor grows to
acquire its original extent (Bravin et al., 1999). Therefore, we aimed
to see whether the growing climbing fibers were able to innervate
spines bearing GluR2. In the rats allowed to recover for 45 d, the average number of climbing fiber synapses per unit area
recovered significantly (p < 0.02) with a value of 4.4 ± 1.4 SD (Fig. 1a) and an increase of 69.2%
relative to the value in the 7 d TTX group. Also, the number of
GluR2-labeled synapses increased significantly
(p < 0.025) to a value of 2.7 ± 0.7 SD
(Fig. 1a) with an increase of 50% relative to the same value in the 7 d TTX group. This means that during this recovery period there was a significant number of new GluR2-labeled spines innervated by climbing fibers, which represented 61.4% of the total
number of climbing fiber synapses. In these synapses, the average
density of gold particles was 14.9 per micrometer (±20.0 SD) (Fig.
1b). The significant increase in the climbing fiber GluR2-labeled synapses demonstrates that the climbing fibers are
able to innervate spines bearing GluR2. Nevertheless, it is also
possible that the initial synaptic contact between a newly sprouted
climbing fiber terminal and a dendritic protrusion gives rise to a
synapse that is devoid of GluR2s. Then the GluR2s would appear
while the synapse is still immature and is unable to repress them. In
either case, the expression of GluR2 clearly does not interfere with
the formation of climbing fiber synapses.
In the rats examined after 135 d of recovery, the number per unit
area of climbing fiber synapses was still significantly higher
(p < 0.02) relative to the number found after
7 d of TTX treatment, with a value of 5.3 ± 5.0 SD (Figs.
1a, 2c), which is not significantly different
(p > 0.05) from that of vehicle-treated animals. The number of GluR2-labeled spines per unit area was 1.3 ± 1.6 SD (Fig. 1a), which is not significantly
different from the vehicle-treated and from the 7 d TTX-treated
animals (p > 0.05). In addition, the gold
particle density had a value of 5.7 per micrometer (±12.5 SD)
(Fig. 1b), which is very similar (p > 0.05) to that of the vehicle-treated rats. These data
demonstrate that at 135 d, the effects of the activity block had
been significantly reversed.
In the same material, we aimed to see whether changes in GluR2
expression were limited to the proximal dendrites. Therefore, we
checked the expression of this subunit in the parallel
fiber-innervated spines of the branchlets. To unequivocally
distinguish branchlet spines from those of the proximal dendrites,
we selected only the spines encountered at their point of emergence
from the stems of distal dendrites. All spines had GluR2. Moreover,
the average density of immunogold particles was not significantly
changed compared with findings after activity block and during the
recovery period (all p values >0.13) (Fig.
2d-f). The gold particle density values per
micrometer of postsynaptic density length were 36.2 ± 11.8 SD
(n = 60) in the vehicle-treated cerebella, 38.7 ± 14.4 SD (n = 64) at 7 d after TTX infusion, and
37.7 ± 13.6 SD (n = 56) and 39.7 ± 12.5 SD
(n = 58), respectively, in the rats examined at 45 and
135 d of recovery (Fig. 1c).
Reinnervation of Purkinje cells by climbing fibers
To further confirm that growing climbing fibers in the adult
cerebellum can innervate spines bearing GluR2, we used a second experimental model that is represented by subtotal deletion of the
inferior olivary neurons by intraperitoneal injection of 3-AP. Data
were obtained from six groups (three subjects per group) consisting of
normal rats and rats at 6, 40, 90, 150, and 270 d after 3-AP administration.
The 6 d group was extensively studied by routine and
immunoelectron microscopy to establish a baseline. In these rats, we observed the presence of spines innervated by parallel fibers emanating
from proximal Purkinje cell dendrites, as demonstrated previously
(Sotelo et al., 1975; Rossi et al., 1991a,b). We prepared 15 sets of
serial electron micrographs to count the spines emerging from proximal
Purkinje cell dendrites and to determine: (1) to what extent they were
innervated, and (2) whether their innervation was provided by other
afferents besides the parallel fibers. Of 97 reconstructed spines, 23 (24%) were free of synaptic contacts (Fig.
3). Four of them had a postsynaptic
density (Fig. 3d) that, however, was quite small, because it
was evident only in one section (100 nm thick), whereas the
postsynaptic density of the contacted spines extended in several
sections. The small postsynaptic densities might represent either the
remnants of previous postsynaptic densities innervated by climbing
fibers or new postsynaptic densities formed before ectopic innervation
by parallel fibers. Of the 74 spines provided with well defined
postsynaptic densities, 59 (80%) were innervated by parallel fibers, 3 (4%) by climbing fibers, and 12 (16%) by large terminals synapsing
both with the spine and with the dendritic shaft. Similar large
terminals with double synaptic contacts were identified as being
GABAergic by an anti-GABA antibody in a previous study (Morando et al.,
2001).
View larger version (51K):
[in this window]
[in a new window]
|
Figure 3.
Serial reconstruction of a spine
(asterisk) free of presynaptic terminal that emerges
from the proximal dendrites of a Purkinje cell at 6 d from a
subtotal lesion of the inferior olive. a-e, Consecutive
sections showing the entire spine profile with no presynaptic
terminals. Note that in d (arrowheads)
there is a thickening of the postsynaptic membrane. Scale bar, 0.36 µm.
|
|
Furthermore, we studied the distribution of GluR2s in the spines in
control rats and during the process of Purkinje cell reinnervation. In
the controls, GluR2s were abundant in parallel fiber-innervated
spines and were extremely rare or absent in climbing fiber-innervated
spines (Landsend et al., 1997; Zhao et al., 1998; Morando et al.,
2001). Spines innervated by parallel fibers had an average GluR2
immunogold particle density of 39.0 per micrometer (±14.1 SD;
n = 108) (Figs.
4c,
5e). In contrast, in 25 spines innervated by climbing fibers (Fig. 5a), only one
(4%) was labeled by gold particles, and the average density value was
0.34 per micrometer (±1.7 SD) (Fig. 4a,b). These data were
in line with previous measurements (Landsend et al., 1997; Zhao et al.,
1998).
View larger version (21K):
[in this window]
[in a new window]
|
Figure 4.
Quantitative evaluation of proximal dendritic
spines innervated by climbing fiber (CF) and
distal dendritic spines innervated by parallel fiber
(PF) and their GluR2 expression in rats with
inferior olive lesion at different periods in days. a,
Percentage of climbing fiber-innervated spines bearing GluR2. The
percentage value increases remarkably up to 90 d after the lesion
and then recovers at 150 d and is similar to control
(c) at 270 d. b,
c, Number of gold particles per micrometer of
postsynaptic density length in climbing fiber- and parallel
fiber-innervated spines. The density in the climbing fiber-innervated
spines is increased up to 90 d and then recovers at 150 d and
is similar to control at 270 d, whereas the density in the
parallel fiber-innervated spines is normal in all
conditions.
|
|
View larger version (109K):
[in this window]
[in a new window]
|
Figure 5.
GluR2 expression after a subtotal lesion of the
inferior olive. a-d, Climbing fiber-innervated spine in
control (a) and at 6 (b),
90 (c), and 270 (d) d after
the lesion. A high density of GluR2s is present in b
and c and not in a and d.
e-h, Parallel fiber-innervated spines emerging from the
branchlets at the same periods after lesion are shown. A high density
of GluR2s is visible in all conditions. Scale bar, 0.23 µm.
|
|
Deletion of the inferior olive amounted to between 88 and 99% of its
neurons and was followed within a period of 3 months by a massive
growth of collaterals from the surviving climbing fibers that extended
the territory of a single climbing fiber by 7-10 times, as shown
previously (Benedetti et al., 1983; Rossi et al., 1991a,b). At 6, 40, and 90 d after the lesion, nearly all of the climbing
fiber-innervated spines bore GluR2s (Figs. 4a,
5b,c). Specifically, values were 91.8, 81.3, and 99.3%,
respectively. Given the strong olivary deletion, it is logical to
assume that the number of spines innervated by newly sprouted climbing
fiber collaterals largely surpassed that of spines innervated by
climbing fibers unaffected by the lesion. Although we cannot exclude
the possibility that GluR2s might also be expressed by the latter category of spines (see Discussion), these receptors must belong in
large part to new synapses. It is relevant also that the density of
GluR2 immunogold particles per micrometer of postsynaptic density in
climbing fiber-innervated spines was very high at all three periods
after the lesion, the values of gold particle density being,
respectively, 26.2 ± 14.1 SD (n = 61), 27.6 ± 22.3 SD (n = 77), and 38.4 ± 16.6 SD
(n = 58) (Fig. 4b). All of these values were
significantly higher relative to controls (respectively, p < 3.14 × 1021,
p < 6.88 × 1017,
and p < 4.26 × 1023). The incidence of climbing
fiber-innervated spines bearing GluR2s dropped to 40.3% at 150 d (Fig. 4a) from the lesion, and the gold particle density
also dropped to a value of 9.6 per micrometer (±17.5 SD;
n = 67) (Fig. 4b), which was still
significantly higher (p < 5.46 × 105) than controls. Only at 270 d
from the lesion was the incidence of climbing fiber-innervated spines
bearing GluR2 (13%) (Figs. 4a, 5d) very close
to normal and the gold particle density reduced to a value of 1.3 per
micrometer (±4.7 SD; n = 46) (Fig. 4b), which was not significantly different from controls
(p = 0.22).
Next, we examined whether the inferior olive lesion affects GluR2
expression in branchlet spines, which maintained their parallel fiber
innervation. As for the TTX experimental group, we analyzed only spines
emerging from the branchlets. Figures 4c and
5f-h show that the GluR2 immunogold particle density in all groups of lesioned rats presented values similar to controls (p > 0.6). In control rats, the gold particle
density value per micrometer of postsynaptic density length was
39.0 ± 14.1 SD (n = 108), and in the rats at 6, 40, 90, 150, and 270 d the values were, respectively, 37.6 ± 17.9 SD (n = 103), 40.8 ± 14.9 SD
(n = 91), 37.8 ± 11.8 SD (n = 54), 42.9 ± 14.0 SD (n = 63), and 47.3 ± 17.1 SD (n = 65).
Finally, in one of three rats at 6 d after olivary lesion we found
dark degenerating climbing fiber terminals in contact with spines, all
of which had postsynaptic densities with abundant GluR2s (Fig.
6). The postsynaptic densities of these
spines had an average gold particle density of 38.3 per micrometer
(±14.7 SD; n = 22), which is significantly higher
relative to the climbing fiber-innervated spines of control rats
(p < 4.8 × 1011). The expression of these receptors
in the spines still attached to degenerating terminals is likely the
consequence of the loss of climbing fiber activity, as described
previously (Morando et al., 2001).
View larger version (156K):
[in this window]
[in a new window]
|
Figure 6.
Dark degenerating climbing fiber terminal
innervating three spines at 6 d after subtotal lesion of the
inferior olive. Note the high expression of GluR2. Scale bar, 0.18 µm.
|
|
| |
Discussion |
Postsynaptic GluR2s are considered to represent important cues
for the formation of synapses between the spines of the Purkinje cell
dendrites and their afferents (Guastavino et al., 1990; Kurihara et
al., 1997; Ichikawa et al., 2002). In the adult cerebellum, however,
expression of GluR2s is restricted to the synapses between the
parallel fibers and the spiny branchlets and is virtually lacking in
the synapses between the climbing fibers and the sparse spines of the
proximal dendrites (Landsend et al., 1997; Zhao et al., 1998; Morando
et al., 2001; Strata, 2002). Yet GluR2s appear again in proximal
dendritic spines after loss of the climbing fiber or in the absence of
electrical activity before retraction of the climbing fiber from its
target (Bravin et al., 1999; Morando et al., 2001). We wondered whether
the GluR2s ectopically targeted to the postsynaptic density of
spines innervated by the climbing fibers might be responsible for the
climbing fiber retraction. Two experiments presented here indicate that
this is not the case, and that regrowing climbing fibers are able to
establish new synapses with spines bearing GluR2s, although these
receptors are repressed at later stages. Evidence for this
interpretation is provided by quantitative immunoelectron microscopic
studies on the reinnervation of Purkinje cell dendrites by climbing
fibers during a 135 d period after removal of an electrical
activity block induced by topical infusion of TTX and during a 270 d period after subtotal deletion of inferior olivary neurons by
3-AP.
As shown previously (Bravin et al., 1999), at 7 d of TTX treatment
there was a large drop in the number of climbing fiber-innervated spines, and most of the proximal dendritic spines contained GluR2s localized to the postsynaptic densities. However, at 45 d after removal of the electrical block, there was a significant increase in
spines expressing GluR2s that were innervated by climbing fibers.
This finding indicates that new climbing fiber terminals made contact
with GluR2-bearing spines. At 45 d after removal of the TTX
block, we found that the GluR2 density in climbing fiber-innervated
spines was significantly decreased relative to the condition under
block. A reasonable interpretation of this finding is that, along with
the process of recruiting new spines, the growing climbing fiber exerts
a repressive action on the targeting of these receptors to the spines
with which it establishes synaptic contacts (see also Morando et al.,
2001). After a recovery period of 135 d, the entire picture is
similar to the control. It should be noted that although a significant
recovery of the climbing fiber arbors was demonstrated as early as
28 d (Bravin et al., 1999), a degree of recovery that includes
normalization of the GluR2 pattern requires a much longer time and
it is still incomplete at 45 d.
In the experimental model consisting of subtotal lesion of the inferior
olive by 3-AP, we confirmed in serial sections that at 6 d after
the lesion the proximal dendrites had spines innervated by parallel
fibers (Sotelo et al., 1975; Rossi et al., 1991a). In addition, we
demonstrated that a substantial number of spines were free of
innervation. At this stage, the density of GluR2s in the parallel
fiber-Purkinje cell synapses of the branchlets was similar to that of
control rats. However, GluR2s were present at high density also in
the climbing fiber-innervated spines at this early time point. The
expression of GluR2s in the climbing fiber synapses might be
attributable to an abnormal activity of the surviving inferior olive
neurons. 3-AP is a toxic agent that interferes with the oxidative
metabolism, and it is possible that the surviving neurons undergo a
period of decreased or absent activity. During such a period, a likely
release of the repressive action of climbing fibers on GluR2
expression may explain the significant increase in the receptor
density. Support for this possibility comes from the observation that
many dark degenerating climbing fiber terminals were found in one rat
at 6 d after the lesion, and they had a high GluR2 density.
Because of the difficulty of distinguishing the original climbing
fiber-innervated spines from those that have been formed as a result of
collateral sprouting, we cannot conclude that new synapses have
GluR2, although sprouting is already present 3 d after the
lesion (Rossi et al., 1991a). However, the surviving climbing fibers
undergo an extensive collateral sprouting that leads to the progressive
extension of their target territory, reaching a peak at the end of the
third month after the lesion (Benedetti et al., 1983; Rossi et al.,
1991a,b; Strata and Rossi, 1998). Our experiments show that up to
90 d after the lesion the percentage of climbing fiber-innervated
spines bearing GluR2 is between 81 and 99%. This fact strongly
supports the results of the TTX experiments in which new climbing
fiber-innervated spines were found to express the GluR2s. It was
striking to find such a high incidence of GluR2s several months
after inferior olivary lesion. We expected that as the sprouting
climbing fibers extended their territory during the first 3 months, new
climbing fiber-innervated synapses would lose their GluR2s. During
collateral sprouting, the inferior olivary neurons present phenotypic
alterations aimed at adapting to the burden of innervating a much
larger target territory (Neppi-Modona et al., 1999). It is likely that
full repression of the GluR2s in the reinnervated spines requires that inferior olivary neurons adapt to the new condition. This situation is reminiscent of cerebellar development, during which climbing fiber-innervated spines transiently express GluR2s. Nevertheless, it should be noted that the downregulation of GluR2s achieved at the end of the developmental period (Zhao et al., 1998) may
occupy a time span shorter than that observed in our experimental situation.
Our data show that during the reinnervation process, new climbing fiber
terminals make contact with GluR2-bearing spines. It is possible
that early contact between a climbing fiber and a Purkinje cell spine
takes place in the absence of GluR2s, and that these subunits
emerged after the initial contact. In developing hippocampal neurons,
the process of synapse formation in vivo occurs in <2 hr
(Alsina et al., 2001), and presynaptic differentiation precedes
postsynaptic differentiation (Friedman et al., 2000; Okabe et al.,
2001). However, the addition of GluR2s to the postsynaptic densities
of the spines was unable to displace the active climbing fiber. On the
contrary, during maturation of the synapse, climbing fiber activity
represses the GluR2s. One may argue that the climbing fiber, instead
of repressing the GluR2 in the innervated spines, moves onto a new
spine lacking this subunit, perhaps with the aid of rapid spine
movements (Dunaevsky et al., 1999, 2001). However, the shift of a
climbing fiber terminal onto a new spine lacking GluR2 under TTX
treatment is unlikely. In fact, despite the significant loss of
climbing fiber synapses, the number of those bearing GluR2s does not
decrease relative to vehicle values. In addition, an upregulation of
this subunit is detectable in these remaining synapses. Subsequently,
after a recovery period of 45 d we observed an additional 50%
increase in the number of climbing fiber GluR2-labeled synapses.
Moreover, up to 90 d after 3-AP treatment, the percentage of
climbing fiber-innervated spines bearing GluR2 was between 81 and
99%. Thus, if the climbing fibers moved onto new spines without
GluR2s to stabilize their synapses, almost all of the climbing fiber
branches formed by collateral sprouting would have abandoned the
GluR2-bearing spines in search of spines lacking this subunit. Thus,
we favor the view that climbing fibers can initially contact
GluR2-bearing spines and then repress this subunit.
In conclusion, spinogenesis seems to be an intrinsic Purkinje cell
property (Sotelo et al., 1975; Sotelo, 1978) that is activity independent (Bravin et al., 1999), although a role of a spontaneous release of glutamate from inactive terminals cannot be excluded. All
spines have a constitutive presence of GluR2 (Morando et al., 2001)
that is important for the maturation and stabilization of the parallel
fiber synapses (Kashiwabuchi et al., 1995; Kurihara et al., 1997).
Active climbing fibers are able to innervate GluR2-bearing spines,
limited to their dendritic domain. The present data suggest that after
achieving the mature state, the active climbing fibers downregulate the
GluR2s in the innervated spines. In addition, they displace the
competitor afferents, the parallel fibers, to the distal dendritic
territory (Ichikawa et al., 2002). By this mechanism, the active
climbing fiber established in its dendritic domain the characteristic
synaptic profile that consists of sparse clusters of spine innervated
by a single afferent axon. Such a profile is congruent with the
peculiar function of the climbing fiber to elicit the large all-or-none
synaptic current accompanied by a large increase in
[Ca2+] (Strata et al., 2000).
| |
FOOTNOTES |
Received Sept. 23, 2002; revised Dec. 30, 2002; accepted Jan. 7, 2003.
This work was supported by grants from the Italian Ministero
Istruzione, Università e Ricerca, Consiglio Nazionale
delle Ricerche, and Ministry of Health. We thank Dr. Masahiko Watanabe for kindly providing the GluR2 antibody, Prof. Ferdinando Rossi for
helpful discussion and for critically reading this manuscript, and
Prof. Dario Cantino for the use of the electron microscope laboratory.
Correspondence should be addressed to Dr. Roberta Cesa, Rita Levi
Montalcini Center for Brain Repair, Department of Neuroscience, University of Turin, C.so Raffaello 30, I-10125 Torino, Italy. E-mail:
roberta.cesa{at}unito.it.
| |
References |
-
Alsina B,
Vu T,
Cohen-Cory S
(2001)
Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF.
Nat Neurosci
4:1093-1101[Web of Science][Medline].
-
Araki K,
Meguro H,
Kushiya E,
Takayama C,
Inoue Y,
Mishina M
(1993)
Selective expression of the glutamate receptor channel 2 subunit in cerebellar Purkinje cells.
Biochem Biophys Res Commun
197:1267-1276[Web of Science][Medline].
-
Benedetti F,
Montarolo PG,
Strata P,
Tosi L
(1983)
Collateral reinnervation in the olivocerebellar pathway in the rat.
In: Nervous system regeneration, Vol 19 (Haber B,
Perez-Polo JR,
Hashim G,
Giuffrida Stella AML,
eds), pp 461-464. New York: Liss.
-
Benedetti F,
Montarolo PG,
Rabacchi S
(1984)
Inferior olive lesion induces long-lasting functional modification in the Purkinje cells.
Exp Brain Res
55:368-371[Web of Science][Medline].
-
Bravin M,
Morando L,
Vercelli A,
Rossi F,
Strata P
(1999)
Control of spine formation by electrical activity in the adult cerebellum.
Proc Natl Acad Sci USA
96:1704-1709[Abstract/Free Full Text].
-
Dunaevsky A,
Tashiro A,
Majewska A,
Mason C,
Yuste R
(1999)
Developmental regulation of spine motility in the mammalian central nervous system.
Proc Natl Acad Sci USA
96:13438-13443[Abstract/Free Full Text].
-
Dunaevsky A,
Bazeski R,
Yuste R,
Mason C
(2001)
Spine motility with synaptic contact.
Nat Neurosci
4:685-686[Web of Science][Medline].
-
Friedman HV,
Bresler T,
Garner CC,
Ziv NE
(2000)
Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment.
Neuron
27:57-69[Web of Science][Medline].
-
Guastavino JM,
Sotelo C,
Damez-Kinselle I
(1990)
Hot-foot murine mutation: behavioral effects and neuroanatomical alterations.
Brain Res
523:199-210[Medline].
-
Hess BJ,
Savio T,
Strata P
(1988)
Dynamic characteristics of optokinetically controlled eye movements following inferior olive lesions in the brown rat.
J Physiol (Lond)
397:349-370[Abstract/Free Full Text].
-
Ichikawa R,
Miyazaki T,
Kano M,
Hashikawa T,
Tatsumi H,
Sakimura K,
Mishina M,
Inoue Y,
Watanabe M
(2002)
Distal extension of climbing fiber territory and multiple innervation caused by aberrant wiring to adjacent spiny branchlets in cerebellar Purkinje cells lacking glutamate receptor 2.
J Neurosci
22:8487-8503[Abstract/Free Full Text].
-
Kashiwabuchi N,
Ikeda K,
Araki K,
Hirano T,
Shibuki K,
Takayama C,
Inoue Y,
Kutsuwada T,
Yagi T,
Kang Y,
Aizawa S,
Mishina M
(1995)
Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluR 2 mutant mice.
Cell
81:245-252[Web of Science][Medline].
-
Kurihara H,
Hashimoto K,
Kano M,
Takayama C,
Sakimura K,
Mishina M,
Inoue Y,
Watanabe M
(1997)
Impaired parallel fiber
 Purkinje cell synapse stabilization during cerebellar development of mutant mice lacking the glutamate receptor 2 subunit.
J Neurosci
17:9613-9623[Abstract/Free Full Text]. -
Landsend AS,
Amiry-Moghaddam M,
Matsubara A,
Bergersen L,
Usami S,
Wenthold RJ,
Ottersen OP
(1997)
Differential localization of 2 glutamate receptors in the rat cerebellum: coexpression with AMPA receptors in parallel fiber-spine synapses and absence from climbing fiber-spine synapses.
J Neurosci
17:834-842[Abstract/Free Full Text].
-
Lomeli H,
Sprengel R,
Laurie DJ,
Kohr G,
Herb A,
Seeburg PH,
Wisden W
(1993)
The rat -1 and -2 subunits extend the excitatory amino acid receptor family.
FEBS Lett
315:318-322[Web of Science][Medline].
-
Mayer ML,
Westbrook GL
(1987)
The physiology of excitatory amino acids in the vertebrate central nervous system.
Prog Neurobiol
28:197-276[Web of Science][Medline].
-
McDonald JW,
Johnston MV
(1990)
Physiological and pathophysiological roles of excitatory amino acids during central nervous system development.
Brain Res Brain Res Rev
15:41-70[Medline].
-
Morando L,
Cesa R,
Rasetti R,
Harvey R,
Strata P
(2001)
Role of glutamate -2 receptors in activity-dependent competition between heterologous afferent fibers.
Proc Natl Acad Sci USA
98:9954-9959[Abstract/Free Full Text].
-
Neppi-Modona M,
Rossi F,
Strata P
(1999)
Phenotype changes of inferior olive neurons following collateral reinnervation.
Neuroscience
94:209-215[Medline].
-
Okabe S,
Miwa A,
Okado H
(2001)
Spine formation and correlated assembly of presynaptic and postsynaptic molecules.
J Neurosci
21:6105-6114[Abstract/Free Full Text].
-
Palay SL,
Chan-Palay V
(1974)
In: Cerebellar cortex: cytology and organization. Berlin: Springer.
-
Rossi F,
Wiklund L,
van der Want JJ,
Strata P
(1991a)
Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. I. Development of new collateral branches and terminal plexuses.
J Comp Neurol
308:513-535[Web of Science][Medline].
-
Rossi F,
van der Want JJL,
Wiklund L,
Strata P
(1991b)
Reinnervation of cerebellar Purkinje cells by climbing fibres surviving to a subtotal lesion of the inferior olive in the adult rat. II. Synaptic organization on reinnervated Purkinje cells.
J Comp Neurol
308:536-554[Web of Science][Medline].
-
Savio T,
Tempia T
(1985)
On the Purkinje cell activity increase induced by suppression of inferior olive activity.
Exp Brain Res
57:456-463[Web of Science][Medline].
-
Schild RF
(1970)
On the inferior olive of the albino rat.
J Comp Neurol
140:255-260[Medline].
-
Sotelo C
(1978)
Purkinje cell ontogeny: formation and maintenance of spines.
Prog Brain Res
48:149-170[Medline].
-
Sotelo C,
Hillman DE,
Zamora AJ,
Llinás R
(1975)
Climbing fiber deafferentation: its action on Purkinje cell dendritic spines.
Brain Res
98:574-581[Web of Science][Medline].
-
Strata P
(1985)
Inferior olive: functional aspects.
In: Cerebellar functions (Bloedel JR,
Dichgans J,
Precht W,
eds), pp 230-246. Berlin: Springer.
-
Strata P
(2002)
Dendritic spines in Purkinje cells.
The Cerebellum
1:230-232[Medline].
-
Strata P,
Rossi F
(1998)
Plasticity in the olivocerebellar pathway.
Trends Neurosci
21:407-413[Web of Science][Medline].
-
Strata P,
Morando L,
Bravin M,
Rossi F
(2000)
Dendritic spine density in Purkinje cells.
Trends Neurosci
23:198.
-
Takayama C,
Nakagawa S,
Watanabe M,
Mishina M,
Inoue Y
(1996)
Developmental changes in expression and distribution of the glutamate receptor channel 2 subunit according to the Purkinje cell maturation.
Brain Res Dev Brain Res
92:147-155[Medline].
-
Zhao HM,
Wenthold RJ,
Petralia RS
(1998)
Glutamate receptor targeting to synaptic populations on Purkinje cells is developmentally regulated.
J Neurosci
18:5517-5528[Abstract/Free Full Text].
Copyright © 2003 Society for Neuroscience 0270-6474/03/2362363-08$05.00/0
This article has been cited by other articles:
|
|
|
|
 
R. Cesa, L. Morando, and P. Strata
Transmitter-receptor mismatch in GABAergic synapses in the absence of activity
PNAS,
December 2, 2008;
105(48):
18988 - 18993.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Uemura, S. Kakizawa, M. Yamasaki, K. Sakimura, M. Watanabe, M. Iino, and M. Mishina
Regulation of Long-Term Depression and Climbing Fiber Territory by Glutamate Receptor {delta}2 at Parallel Fiber Synapses through its C-Terminal Domain in Cerebellar Purkinje Cells
J. Neurosci.,
October 31, 2007;
27(44):
12096 - 12108.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Ohtsuki, S.-y. Kawaguchi, M. Mishina, and T. Hirano
Enhanced Inhibitory Synaptic Transmission in the Cerebellar Molecular Layer of the GluR{delta}2 Knock-Out Mouse
J. Neurosci.,
December 1, 2004;
24(48):
10900 - 10907.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
Introduction
