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The Journal of Neuroscience, October 1, 2001, 21(19):7664-7673
Endogenous Activation of Group-I Metabotropic Glutamate Receptors
Is Required for Differentiation and Survival of Cerebellar Purkinje
Cells
M. V.
Catania1,
M.
Bellomo2,
V.
Di
Giorgi-Gerevini3,
G.
Seminara1, 4,
R.
Giuffrida5,
R.
Romeo6,
A.
De
Blasi7, 8, and
F.
Nicoletti3, 8
1 Institute for Bioimaging and Pathophysiology of the
Central Nervous System (IBFSNC), National Research Council
(IBFSNC-CNR), 95123 Catania, Italy, 2 Institute of
Physiology, University of Messina, 98100 Messina, Italy,
3 Department of Human Physiology and Pharmacology,
University of Roma La Sapienza, Rome, Italy, Departments of
4 Chemical Sciences and 5 Physiological
Sciences and 6 Institute of Anatomy, University of Catania,
95100 Catania, Italy, 7 Department of Molecular
Pharmacology and Pathology, "Mario Negri Sud" Institute, 66030 S. Maria Imbaro (Chieti), and 8 I. N. M. Neuromed,
86077 Pozzilli, Italy
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ABSTRACT |
We have applied subtype-selective antagonists of metabotropic
glutamate (mGlu) receptors mGlu1 or mGlu5 [7-(hydroxy-imino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) or
2-methyl-6-(phenylethynyl)pyridine (MPEP)] to mixed rat cerebellar
cultures containing both Purkinje and granule cells. The action of
these two drugs on neuronal survival was cell specific. Although
CPCCOEt (1, 10, 30 µM) reduced the survival of Purkinje
cells, MPEP (3 or 30 µM) selectively reduced the survival
of granule cells. Both effects required an early exposure of cultures
to antagonists [from 3 to 6 d in vitro (DIV) for
CPCCOEt, and from 3 to 6 or 6 to 9 DIV for MPEP]. Addition of MPEP
from 6 to 9, 9 to 13, or 13 to 17 DIV also induced profound morphological changes in the dendritic tree and dendritic spines of
Purkinje cells, suggesting that endogenous activation of mGlu5 receptors is required for the age-dependent refinement of Purkinje cell
phenotype. In in vivo studies, an early blockade of
mGlu1 receptors induced in rats by local injections of LY367385 (20 nmol/2 µl), local injections of mGlu1 antisense oligonucleotides (12 nmol/2 µl), or systemic administration of CPCCOEt (5 mg/kg, s.c.)
from postnatal day (P) 3 to P9 reduced the number and dramatically altered the morphology of cerebellar Purkinje cells. In contrast, mGlu5
receptor blockade induced by local injections of antisense oligonucleotides reduced the number of granule cells but also produced
substantial morphological changes in the dendritic tree of Purkinje
cells. These results provide the first evidence that the development of
cerebellar neurons is under the control of mGlu1 and mGlu5 receptors,
i.e., the two mGlu receptor subtypes coupled to polyphosphoinositide hydrolysis.
Key words:
metabotropic glutamate receptors; Purkinje cells; granule
cells; cerebellum; development; dendrites
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INTRODUCTION |
Activation of glutamate receptors
has been implicated in the regulation of differentiation and survival
of developing CNS neurons. Most of the studies have focused on the role
of NMDA receptors in the induction of neuronal phenotypes, promotion of neuronal survival, and synaptic remodeling during development (Balázs et al., 1988 ; Moran and Patel, 1989; Rabacchi et al., 1992; Burgoyne et al., 1993; Yuzaki et al., 1996; Marini et al., 1998).
However, metabotropic glutamate (mGlu) receptors have also been
implicated in the regulation of developmental plasticity since the
early times of their characterization (Nicoletti et al., 1986; Dudek
and Bear, 1989). mGlu receptors form a family of eight subtypes (named
mGlu1 to mGlu8), of which mGlu1 and mGlu5 receptors, which form the
first subgroup of subtypes, are coupled to polyphosphoinositide (PI)
hydrolysis (Nakanishi, 1982; Pin and Duvoisin, 1995).
Glutamate-stimulated PI hydrolysis in brain slices shows a
developmental peak in the early postnatal life and then progressively
decreases with age (Nicoletti et al., 1986; Schoepp and Johnson, 1989).
This is paralleled by an early peak in the expression of mGlu5
receptors in most of the brain regions, including the cerebellum
(Catania et al., 1994; Minakami et al., 1995; Romano et al., 1996;
Casabona et al., 1997). In the kitten visual cortex, the activity of
mGlu receptors coupled to PI hydrolysis is greater between 3 and 5 weeks of postnatal life, a time that corresponds to the critical period
of synaptic modification in response to monocular deprivation (Dudek
and Bear, 1989). A developmental pattern of expression of group-I mGlu
receptors is also shown in cultured cerebellar granule cells, in which
glutamate-stimulated PI hydrolysis peaks after 4 d in
vitro (DIV) and declines at later stages of maturation (Aronica et
al., 1993). Interestingly, an early activation of mGlu5 receptors
supports the survival of cultured granule cells (Copani et al., 1998),
raising the possibility that the age-dependent reduction in the
expression of mGlu5 receptors contributes to the elimination of
supranumerary granule cells in the intact cerebellum. Pharmacological
activation of mGlu receptors combined with NGF application increases
Purkinje cell survival in culture (Mount et al., 1993), but which
receptor subtype is involved and whether this effect is physiologically
relevant are unknown. Purkinje cells are highly enriched in mGlu1
receptors (Aramori and Nakanishi, 1992; Görcs et al., 1993;
Catania et al., 1994), but mGlu1 knock-out mice show no apparent
changes in the number and only minor changes in the morphology of
Purkinje cells (Aiba et al., 1994). Similarly, the gross anatomy of the cerebellum is unchanged in mGlu5 knock-out mice (Lu et al., 1997). Because compensatory changes might have occurred in knock-out mice, we
decided to use novel subtype-selective mGlu1 or mGlu5 receptor
antagonists or antisense oligonucleotides to examine how endogenous
activation of these receptors affects the development of Purkinje cells.
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MATERIALS AND METHODS |
Materials
7-(Hydroxy-imino) cyclopropa[b]chromen-1a-carboxylate ethyl
ester (CPCCOEt), 2-methyl-6-(phenylethynyl)pyridine (MPEP), and (E)-2-methyl-6-styryl-pyridine (SIB-1893) were purchased from Tocris
Cookson (Bristol, UK). 2-Methyl-4-carboxyphenyglycine (LY367385) and
 -thioxanth-9-ylmethyl-4-carboxyphenylglycine (LY367366) were kind
gifts from Dr. Ann Kingston (Eli Lilly, UK).
Mixed cerebellar cultures
Mixed cerebellar cultures were prepared from rats at postnatal
day (P) 1, as described previously (Furuya et al., 1998). Cells were
plated at a final density of 5 × 106
cells/ml in DMEM/F12 containing putrescine (100 µM),
sodium selenite (30 nM), progesterone (20 nM),
bovine insulin (10 µg/ml), transferrin (100 µg/ml),
tri-iodothyronine (T3) (0.5 ng/ml), bovine serum albumin (0.1 mg/ml),
glutamine (3.9 mM), gentamicin (10 µg/ml), and 1% FCS.
The medium was partially replaced at 9 DIV with fresh serum-free
DMEM/F12 containing the supplements and cytosine arabino- -furanoside (4 µM). Cell cultures were treated daily with MPEP and
CPCCOEt dissolved in serum-free DMEM/F12. Three different types of
treatment were performed: (1) in a first set of experiments, cultures
were treated continuously with drugs from 4 to 13 DIV; (2) in a second set of experiments, cultures were treated only for 3 or 4 d at critical stages of Purkinje cell development. In particular, dishes were divided into four groups and treated from 3 to 5 DIV (and analyzed
at 6 DIV); from 6 to 8 DIV (and analyzed at 9 DIV); from 9 to 12 DIV
(and analyzed at 13 DIV); or from 13 to 16 DIV (and analyzed at 17 DIV). (3) In a third set of experiments, cultures were treated from 6 to 9 DIV with MPEP or CPCCOEt, extensively washed, and then analyzed at
14 DIV. Granule cell counting and morphological analysis of Purkinje
cells were performed on the same dishes.
Animal treatment
Sprague Dawley newborn rats at P3 were injected through the
scalp in the posterior region of the cerebellum (2-3 mm posterior to
the lambda point) under conditions of deep hypothermia. The drugs
LY367385 or LY367366 (both at doses of 20 nmol/2 µl) were injected
once every 2 d until P9. Animals were killed at P9-10. In
different animals we injected methylene blue to validate the site of
injection. The region of injection was invariably located in the vermis
in a region comprising the central lobe (lobules VI, VII, and VIII),
and the dye diffused anteriorly to anterodorsal lobe (lobules IV and V)
and posteriorly to lobule IX as indicated in Altman and Bayer (1996).
"End-capped" antisense oligonucleotides (Oligo Therapeutics Inc.)
directed against mGlu1 or mGlu5 mRNA were injected at doses of 12 nmol/2 µl only at P3 and P5 (animals were killed at P9). The
antisense sequences were as follows: T(s)C(s)AGGCAGGCATCGGTTC(s)A(s)G, corresponding to the position 785-805 of mGlu1 sequence (GenBank accession number M61099) (Houamed et al., 1991), and
G(s)G(s)ATCAACAGAAGGACCA(s)T(s)T, corresponding to position 267-286 of
mGlu5 sequence (GenBank accession number D10891) (Abe et al., 1992). A
scrambled mGlu5 oligonucleotide having the sequence
G(s)G(s)ACTAAACAGGAGACAT(s)C(s)T was used as a control. CPCCOEt (5 mg/kg) was daily injected subcutaneously from P3 to P9.
Western blotting
Western blot analysis of mGlu5 receptors in rat hemicerebella
was performed as described previously (Copani et al., 1998).
Immunocytochemistry
Cultures. Cells were generally fixed with
4% paraformaldehyde/4% sucrose in Tris-buffered saline (TBS; 100 mM Tris, 0.9% NaCl) for 15 min. For mGlu1a staining,
fixation with ice-cold methanol was preferred because it resulted in a
more clearly defined signal than with paraformaldehyde.
Permeabilization was performed in TBS with 0.1% Triton X-100 for 5 min. After blocking with 4% normal goat serum (NGS) for 30 min,
incubation with primary antibodies was performed overnight at 4°C. A
mouse monoclonal antibody against calbindin (1:1000; Swant, Bellinzona,
Switzerland), rabbit polyclonal antibodies anti-glutamate decarboxylase
(GAD)-67 (1:1000; Chemicon International, Temecula, CA), and rabbit
polyclonal antibodies against mGlu1a (1:100; Chemicon International)
and five (1:100; Upstate Biotechnology, Lake Placid, NY) receptors were
used. The following antigenic peptides (one-letter code),
CPNVTYASVILRDYKQSSSTL (corresponding to the C terminus of the mGlu1a
receptors) and KSSPKDTLIIRDYTNSSSSL (corresponding to the C terminus of
mGlu5a/b receptors), were used to test the specificity of
immunolabeling. Both peptides were kindly provided by Dr R. Kuhn
(Novartis Pharma, Basle, Switzerland). After three washes in TBS,
biotin-conjugated goat anti-rabbit or anti-mouse secondary antibodies
(1:200, Vector Laboratories, Burlingame, CA) were added for 1.5 hr.
Immunostaining was revealed by the avidin-biotin-peroxidase method.
In the mGlu5/calbindin double-labeling experiment, a dichlorotriazinyl
amino fluorescein (DTAF)-conjugated goat anti-rabbit IgG (1:200;
Jackson ImmunoResearch, West Grove, PA) and a Cy3-conjugated goat
anti-mouse IgG (1:200; Jackson ImmunoResearch) were used. In the
mGlu5/GFAP double-labeling experiment, staining for mGlu5 was first
developed using the DAB method; cultures were then fixed in
paraformaldehyde for 10 min, blocked with 4% goat serum, and incubated
again overnight with a rabbit anti-cow GFAP antibody (1:1000; Dako)
that was revealed with a secondary DTAF-conjugated goat anti-rabbit IgG
(1:200; Jackson ImmunoResearch). Granule cells were visualized by
nuclear staining with the fluorescent chromatin dye Hoechst 33258 (0.8 µg/ml) for 30 min in 10 mM PBS at 37°C.
Chromatin staining was always performed after DAB/anti-calbindin
immunostaining for the simultaneous visualization of Purkinje and
granule cells.
Intact cerebellum. Brains were removed and
fixed in 4% paraformaldehyde/PBS for 2 d at 4°C and then
cryoprotected in 30% sucrose for 3-7 d. Sagittal sections (20 µm
thick) were cut at the cryostate and serially attached to
gelatin-coated slides. Slices were permeabilized for 30 min in TBS
containing 0.2% Triton X-100. This was followed by a preincubation in
TBS containing 4% NGS for 30 min. Sections were incubated overnight at
4°C in primary antibodies in TBS. Staining was developed as described
above. Sections were rinsed in TBS, dehydrated in increasing
concentrations of ethanol, clarified, and mounted on coverslips in a
xylene-based mounting medium. Sections were also routinely Nissl
stained for histological analysis.
Morphometric analysis
Images were analyzed by using the MCID system (Imaging
Research, St. Catharine's, Ontario) to evaluate the area, the total length of the dendritic tree, the number of branches, the diameter of
primary dendrites, and the length of dendritic spines. Total length of
the dendritic tree was calculated by adding the length of all branches
of a single Purkinje cell. In the computation of spine length, no
distinction was made between headed spines and filopodia-like
processes, although it was clear that MPEP-treated cells bore a
lower density of headed spines compared with control sister cultures.
Granule cells present in a square of fixed area (1700 µm2; three determinations per slice)
were counted in the internal granule layer of comparable sections at
the level of preculminate fissure in the anterior cerebellum (lobules 3 and 4). The width of molecular layer was measured in the same region
from both anti-calbindin and Nissl-stained sections, and comparable
results were obtained.
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RESULTS |
Endogenous activation of group-I mGlu receptors supports
the maturation and survival of cerebellar Purkinje cells in culture
We used primary cultures of cerebellar neurons prepared
from 1-d-old rats and grown in chemically defined medium with 1%
serum. These cultures contained ~65-70% granule cells, 10%
Purkinje cells, and 15-20% glial cells. Purkinje cells were
identified by immunostaining with anti-calbindin antibodies (see
below). Granule cells were identified by their characteristic
morphology (small rounded cell bodies) associated with the absence of
GAD-67 or GFAP immunoreactivity. Purkinje cells were viable for at
least 21 d. The developmental profile of Purkinje cells in our
cultures was identical to that described by Baptista et al. (1994). At
3 DIV, calbindin-stained Purkinje cells showed rounded or fusiform cell
bodies and multiple primary aspiny dendrites elongating from the whole
cell surface (data not shown). At 6 DIV, most of the primary
dendrites were retracted, and Purkinje cells showed a small dendritic
tree (Fig. 1, CONTROL). The
extension of the dendritic tree progressively increased between 9 and
17 DIV, as shown by the formation and elongation of secondary and
tertiary branches (Fig. 1, CONTROL). Spines characterized by
a globoid head and a thin neck were clearly visible on dendrites of
cultured Purkinje cells after 6 DIV (see Fig. 4A).
Under our experimental conditions, the number of Purkinje cells did not
change substantially with age in culture, whereas granule cell number
decreased by ~50% between 6 and 13 DIV (Fig. 3A,B).
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Figure 1.
Morphological differentiation of
cultured Purkinje cells in the absence (CONTROL) or
presence of MPEP or CPCCOEt. Here, the effects of 30 µM
MPEP and 30 µM CPCCOEt are illustrated. Similar effects
are seen with 1 or 10 µM CPCCOEt and 3 µM
MPEP (data not shown). Drugs were administered between 3 and 6 (6 DIV), 6 and 9 (9 DIV), 9 and 13 (13 DIV), or
13 and 17 (17 DIV) DIV, and Purkinje cells were visualized
at the indicated DIV, by immunostaining with an anti-calbindin
antibody. Representative drawings of several Purkinje cells
(left top side) were traced from microphotographs
(right bottom side of each panel). Similar data were
obtained in three experiments from different culture preparations
(n = 3-4). Scale bar, 50 µm.
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We have performed immunocytochemical analysis to study the expression
of mGlu1a and mGlu5 receptors in culture. mGlu1a receptor immunoreactivity was detected in both Purkinje and granule cells but
not in glial cells. At 3 DIV, mGlu1a receptors showed low levels of
expression in both neuronal types (data not shown) but were more
heavily expressed in Purkinje cells than in granule cells at 6 DIV
(Fig. 2A). At 9-13
DIV, Purkinje cells were intensely labeled, and the receptor was
apparently present in both the cell soma and dendrites (data not
shown). In contrast, mGlu5 receptors were expressed in granule cells
and astrocytes but not in Purkinje cells, as demonstrated by double
labeling with anti-GFAP (Fig. 2D,E)
and anti-calbindin (Fig. 2B,C)
antibodies. The expression of mGlu5 receptors did not change between 3 and 9 DIV. No labeling was observed after preadsorbing mGlu1a and mGlu5
antibodies with 100 µM of the respective
antigenic peptide (data not shown).
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Figure 2.
Expression of mGlu1a and mGlu5 receptors in
Purkinje, granule, and glial cells in cerebellar cultures.
A shows the expression of the mGlu1a receptor in granule
(arrowheads) and Purkinje cells (arrow)
in a representative culture at 6 DIV. The receptor is expressed in both
cell types at any developmental stages since at least 3 DIV. Note the
absence of staining in glial cells (asterisks). A double
immunostaining for mGlu5 receptors (green) and
calbindin (red) is shown in B and
C, respectively. Note that mGlu5 receptors are expressed
in granule cells (arrowheads) but not in Purkinje cells
(arrow). A double immunostaining for GFAP
(yellow) and mGlu5 antibodies (DAB staining) is
shown in D and E. Note the presence of
mGlu5 receptors in glial processes. Scale bars: A, 40 µm; B-E, 50 µm.
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To study how endogenous activation of mGlu1 and mGlu5 receptors affects
the development of Purkinje cells, we have treated the cultures with
CPCCOEt or MPEP, which behave as selective noncompetitive antagonists
of mGlu1 and mGlu5 receptors, respectively (Annoura et al., 1996;
Gasparini et al., 1999; Litschig et al., 1999). In a first set of
experiments, cell cultures were treated daily between 4 and 13 DIV and
fixed at 14 DIV. Treatment with either drug dramatically altered the
morphology of Purkinje cells. In the majority of treated Purkinje
cells, the dendritic tree appeared poor and disorganized with short and
thick dendrites, which were always devoid of tertiary branches. No
major morphological feature could distinguish MPEP- and CPCCOEt-treated
cells. However, MPEP-treated Purkinje cells showed less numerous
(7.5 ± 2 branches; n = 15 cells) and slightly
longer secondary branches than CPCCOEt-treated cells (15 ± 2 branches; n = 12 cells).
In other experiments, cultures were exposed to CPCCOEt or MPEP from 3 to 6, 6 to 9, 9 to 13, or 13 to 17 DIV. In each group, morphological
analysis of Purkinje cells and cell counting were performed at
the end of the treatment. We adopted this strategy to examine how
endogenous activation of mGlu1 and mGlu5 receptors regulates the
maturation and survival of cerebellar neurons at particular time
windows. An early blockade of mGlu1 receptor with CPCCOEt (30 µM, from 3 to 6 DIV) substantially reduced the number of
Purkinje cells (counted at 6 DIV) without changing the number of
granule cells. CPCCOEt had no effect on Purkinje and granule cell
survival at any later interval ranging from 6 to 17 DIV. Endogenous
blockade of mGlu5 receptors with MPEP (30 µM) did not affect the survival of Purkinje cells but reduced the number of granule
cells when applied from 3 to 6 or from 6 to 9 DIV (Fig. 3A,B).
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Figure 3.
Effects of MPEP or CPCCOEt treatment on granule
(A) and Purkinje (B) cell
number in cerebellar cultures. Drugs were applied to the cultures as
described in Figure 1. Cells were counted in four random fields per
dish. Values are means ± SEM of four individual culture dishes.
*p < 0.05 (one-way ANOVA + Fisher's PLSD) versus
control values. Similar data were obtained in three experiments on
different culture preparations.
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This protocol of antagonist application was also useful for the study
of how endogenous activation of mGlu1 or mGlu5 receptors affects the
morphology of Purkinje cells (Fig. 1). Purkinje cells exposed to
CPCCOEt at early developmental stages (from 3 to 6 or 6 to 9 DIV)
appeared smaller and bore short dendrites. This effect was
visible with concentrations of 1, 10, and 30 µM of CPCCOEt (the effect of 30 µM is shown in Fig. 1). In
contrast, MPEP had no effect when applied between 3 and 6 DIV but
produced profound changes in Purkinje cell morphology at later
developmental stages. In cultures exposed to MPEP (3 or 30 µM) from 6 to 9 DIV, the majority of Purkinje cells
exhibited longer, thinner, and less branched dendrites (Fig. 1).
This aberrant developmental pattern was even more evident in cultures
exposed to MPEP from 9 to 13 DIV, with Purkinje cell dendrites being
longer and often devoid of secondary and tertiary branches (Fig. 1).
Morphometric analysis indicated that CPCCOEt applied from 6 to 9 DIV
significantly reduced the total length of the dendritic tree and the
number of dendritic branches. MPEP affected all of these parameters
when applied not only from 6 to 9 DIV but also from 9 to 13 DIV. In addition, the thickness of the primary dendrites of Purkinje cells was
significantly reduced in cultures exposed to MPEP at any interval between 6 and 17 DIV (Table 1). The
typical morphological features of Purkinje cells treated from 6 to 9 DIV with MPEP were also observed after a 3 d treatment with
SIB-1893 (30 µM), which also behaves as a selective
noncompetitive mGlu5 receptor antagonist (data not shown). The
morphological changes of Purkinje cells observed at 9 DIV after a
3 d treatment with MPEP were also seen after 24 hr (i.e., between
8 and 9 DIV) but not after 0.5 or 3 hr of drug exposure (data not
shown).
Besides producing a substantial effect on the gross morphology of
Purkinje cells, a 3 d treatment with MPEP also influenced the
morphogenesis of dendritic spines. Dendrites of Purkinje cells treated
with MPEP from 6 to 9 DIV exhibited longer and thinner processes devoid
of heads, which markedly differed from the headed mature spines of
control cultures. (Fig.
4A-C).
CPCCOEt applied from 6 to 9 DIV had no effect on the morphology of
dendritic spines (Fig. 4C).
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Figure 4.
Effects of MPEP or CPCCOEt on the morphology of
dendritic spines in cultured Purkinje cells. Drugs were applied to the
cultures from 6 to 9 DIV. Note that the dendritic branches of control
Purkinje cells at 9 DIV are covered by headed spines
(A), whereas dendrites of Purkinje cells treated
with MPEP show longer protrusions devoid of heads
(B). Kruskal-Wallis one-way ANOVA on ranks
revealed a statistically significant difference between groups
(*p < 0.05 by Dunn's method). C,
Control (CTR; n = 291),
MPEP (n = 220),
CPCCOEt (n = 287); boxes
contain 50% of values; the line dividing the
box represents the median (control = 1.5 µm;
MPEP = 2.3 µm; CPCCOEt = 1.7 µm); bottom
and top lines represent the 25th and 75th
percentile, respectively. The number of spines was obtained from 10-15
cells per group from a single experiment performed in triplicate.
Identical data were observed in three experiments performed on
different culture preparations. Scale bar, 12 µm.
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To examine whether Purkinje cells are able to recover from a transient
pharmacological blockade of mGlu1 or mGlu5 receptors, we have treated
cultures with CPCCOEt or MPEP (both at 30 µM) from 6 to 9 DIV as described previously, but then we have extensively washed the
cultures (Fig. 5, legend) and allowed
them to grow until 14 DIV. Although Purkinje cells in these cultures
showed some morphological abnormalities, their dendrites were more
developed than in cultures examined at the end of the 3 d exposure
to CPCCOEt or MPEP, i.e., at 9 DIV, or at the end of a
continuous exposure to CPCCOEt or MPEP from 6 to 14 DIV (Fig. 5). This
indicates that receptor blockade at a critical time window arrests or
delays, but does not irreversibly impair, Purkinje cell maturation.
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Figure 5.
Purkinje cell morphology in cultures
treated as follows. A, C,
F, Cultures treated with culture medium
(A), 30 µM MPEP (C), or 30 µM CPCCOEt (F) from 6 to 9 DIV and examined at 9 DIV;
B, D, G, Cultures treated
with culture medium (B), MPEP
(D), or CPCCOEt (G) from 6 to 14 DIV and examined at 14 DIV; E, H,
cultures treated with MPEP (E) or CPCCOEt
(H) from 6 to 9 DIV, extensively
washed (wo), and then examined at 14 DIV. Scale bar,
50 µm. Note that Purkinje cells partially recover their morphology
after 5 d of drug washout (E,
H).
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Endogenous activation of mGlu1 and mGlu5 receptors supports the
maturation and survival of cerebellar neurons in
vivo
The compounds LY367385 and LY367366, which behave as competitive
mGlu1 and mGlu1/5 antagonists, respectively (both at the dose of 20 nmol/2 µl), as well as mGlu1 or mGlu5 antisense oligonucleotides (12 nmol/2 µl) were injected into the cerebellar region, whereas the
noncompetitive mGlu1 antagonist CPCOOEt was injected systemically (5 mg/kg, s.c.) in neonate rats (see Materials and Methods).
Pups at P9-10 that had been treated with the mGlu1 receptor antagonist
LY367385 showed a loss of Purkinje cells, which was remarkable in the
vicinity of the injection site. Surviving Purkinje cells showed a
shrunken cell body and a poorly developed dendritic tree, which
appeared shorter and poorly branched (data not shown). A poorly
developed dendritic tree of Purkinje cells was also observed in pups
systemically treated with CPCCOEt (Fig.
6A,B)
or locally injected with antisense oligonucleotides directed against
mGlu1 receptors (Fig.
7C,F).
Antisense treatment was effective in reducing mGlu1a expression in the
Purkinje cell layer, as shown by immunohistochemical analysis (Fig. 7,
compare A, B with D,
E).
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Figure 6.
Calbindin immunostaining of Purkinje cells in
neonate rats systemically injected with saline
(A) or CPCCOEt (5 mg/kg, s.c.)
(B). Note in B a poorly developed
dendritic tree of Purkinje cells. Identical data were obtained in four
treated pups. Scale bar, 60 µm.
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Figure 7.
Effects of mGlu1 and mGlu5 antisense
treatment on the morphology of Purkinje cells. A substantial reduction
of mGlu1a immunostaining is shown in the Purkinje cells of animals
treated with mGlu1 antisense oligonucleotides (D,
E), as compared with animals injected with
saline (A, B) or with animals treated
with mGlu5 antisense oligonucleotides (G,
H). The effect of the treatment on the morphology
of Purkinje cells is shown in C, F, and
I, in which cells are revealed by calbindin
immunostaining. Scale bars: A, D,
G, 1 mm; B-I, 20 µm.
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Pups injected with mGlu5 antisense oligonucleotides were used for the
examination of how mGlu5 receptors affect cerebellar development. To
select the animals in which antisense treatment was effective in
reducing mGlu5 receptor expression, we performed Western blot analysis
of hemicerebella (Fig.
8A). Four of five pups
locally injected with antisense showed a clear-cut reduction in the
expression of mGlu5 receptors, as compared with pups injected with a
scrambled oligonucleotide (Fig. 8A, SCR).
In the other hemicerebella of animals with a proven knockdown of mGlu5
receptors, we observed a reduction in cell number within the granule
cell layer (Fig. 8B,C). Purkinje
cells of these animals showed a stellate configuration resulting from
the presence of short perisomatic dendrites emerging in all directions
(Fig. 7I). There was no reduction in the expression
of mGlu1a receptors in pups treated with mGlu5 antisense
oligonucleotides (Fig. 7G,H).
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Figure 8.
Treatment with mGlu5 antisense oligonucleotides
reduces cell density in the internal granular layer
(IGL) in neonate rats. A, Western blot
analysis of mGlu5 receptors in hemicerebella from neonate rats
treated with mGlu5 antisense oligonucleotides
(ASm5) or with a scrambled oligonucleotide
(SCR). Note the reduction in mGlu5 receptor expression
in four of the five animals treated with antisense
oligonucleotides. An example of the reduction in granule cell
density in animals treated with antisense oligonucleotides as
compared with animals treated with the scrambled oligonucleotides is
shown is C and B, respectively (see
arrows).
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A quantitative assessment of the width of the molecular layer and the
number of granule cells after drug or antisense treatment is shown in
Figure 9, A and B,
respectively. Note that (1) treatment with the mGlu1 receptor
antagonist LY367385 reduced the width of the molecular layer but not
the number of granule cells, (2) treatment with mGlu5 antisense
oligonucleotides significantly reduced both the number of granule cells
and the width of the molecular layer, and (3) a reduction in the cell
number of the granular layer was also observed in pups treated with
LY367366, a mixed mGlu1 and mGlu5 receptor antagonist (Fig.
9A,B). Purkinje cells of animals
treated with LY376366 were smaller in size, and their dendrites were
shorter and not polarized (data not shown).
View larger version (16K):
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|
Figure 9.
Effects of intracerebral injection of
saline, LY367385 (20 nmol), LY367366 (20 nmol), mGlu1, or mGlu5
antisense oligonucleotides (ASm1 or ASm5,
12 nmol) or a scrambled oligonucleotide (SCRm5, 12 nmol)
on the width of molecular layer and granule cell number in the
cerebellar cortex. The control bar refers to values
obtained from noninjected animals. Measurements were taken from the
preculminate fissure in the anterior cerebellum. Values are means ± SEM of three to five animals per group. *p < 0.05 (one-way ANOVA + Fisher's PLSD) versus control or saline.
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|
| |
DISCUSSION |
The major finding of this paper is that endogenous activation of
mGlu1 and mGlu5 receptors contributes to cerebellar development and
that a selective blockade of these receptors within restricted time
windows differentially affects the maturation of Purkinje cells. This
discloses a major neurotrophic activity of group-I mGlu receptors
during early postnatal development that did not emerge from the use of
mGlu1 or mGlu5 knock-out mice (Aiba et al., 1994; Conquet et al., 1994;
Lu et al., 1997). Receptor blockade was achieved by using either
antisense oligonucleotides or a battery of novel subtype-selective
antagonists. CPCCOEt and MPEP (or SIB-1893) behave as potent and
noncompetitive antagonists of mGlu1 and mGlu5 receptors, respectively
(Annoura et al., 1996; Gasparini et al., 1999; Litschig et al., 1999).
LY367385 behaves as a competitive mGlu1 receptor antagonist, whereas
LY367366 inhibits both mGlu1 and mGlu5 receptors with equal potency
(Clark et al., 1997). The latter two drugs are soluble and were
preferred for intracerebral injections.
Mixed cultures of cerebellar Purkinje and granule cells represent a
suitable model to examine how endogenous activation of mGlu1 and mGlu5
receptors affects neuronal development. In these cultures, granule
cells establish synaptic contacts with Purkinje cells as they do in the
intact cerebellum and are known to use glutamate as a neurotransmitter.
Hence, noncompetitive antagonists become optimal tools for the
examination of how endogenous activation of mGlu1 or mGlu5 receptors
affects cell maturation and survival. In culture, pharmacological
inhibition of mGlu1 and mGlu5 receptors selectively impaired the
survival of Purkinje and granule cells, respectively. Expression of
mGlu1a receptors was higher in Purkinje than in granule cells, whereas
mGlu5 receptors were expressed in granule cells but not in Purkinje
cells. Although information on the expression of other mGlu1 splice
variants is lacking, the use of antagonists allows us to conclude that
a molecular cascade supporting cell survival is linked to mGlu1
receptors in Purkinje cells and to mGlu5 receptors in granule cells.
This is consistent with previous findings showing that the
developmental decline in the expression of mGlu5 receptors predisposes
cultured granule cells to
"low-K+"-induced apoptosis (Copani et
al., 1998). Endogenous activation of mGlu1 receptors did not support
granule cell survival, although these receptors are functionally
coupled with ryanodine-sensitive intracellular
Ca2+ stores and L-type voltage-sensitive
Ca2+ channels in granule cells (Chavis et
al., 1996). Both mGlu1 and mGlu5 receptors are coupled to inositol
phospholipid hydrolysis, but they differ in the kinetics of
intracellular Ca2+ response. Activation of
recombinant mGlu1a receptors generates a single peaked increase in
intracellular Ca2+, whereas activation of
mGlu5 receptors produces Ca2+
oscillations. The latter property is attributable to a particular threonine residue that is present only in mGlu5 receptors and is
phosphorylated by protein kinase C (Kawabata et al., 1996). An
attractive hypothesis is that oscillatory increases in intracellular Ca2+ are needed for granule cell viability
but are not required for Purkinje cell survival. The use of receptor
antagonists in cerebellar cultures revealed that mGlu1 or mGlu5
receptors control cell survival within a restricted time window during
early stages of neuronal development. This correlates nicely with
evidence that the expression of mGlu5 receptors in granule cells
decreases with age (Catania et al., 1994; Copani et al., 1998) and that
mGlu1-mediated responses in cultured Purkinje cells peak at 4 DIV and
decline thereafter (Yuzaki and Mikoshiba, 1992).
Interestingly, selective blockade of mGlu5 receptors produced
substantial morphological changes in Purkinje cells, including changes
in the shape of dendritic spines that are consistent with a delay in
their maturation (Zif and Smith, 1996; Jontes and Smith, 2000). Because
Purkinje cells lack mGlu5 receptors, it is conceivable that MPEP acts
primarily on granule cells, which are known to influence Purkinje cell
differentiation and survival by releasing glutamate and/or
neurotrophins (Baptista et al., 1994; Morrison and Mason, 1998; Hirai
and Launey, 2000). Changes in Purkinje cell morphology were
particularly evident after 9 DIV and therefore could not be ascribed to
the reduction in granule cell number induced by MPEP. However, mGlu5
receptor blockade might have reduced the spontaneous
activity of granule cells, thus limiting the major synaptic input to
Purkinje cells. Alternatively, inhibition of mGlu5 receptors present in
astrocytes might have disrupted a trophic mechanism based on a
glial-Purkinje cell interaction. The effects produced by a transient
inhibition of mGlu1 or mGlu5 receptors on dendritic growth of Purkinje
cells were substantial but not irreversible, as indicated by the
partial recovery of the dendritic tree after washing out CPCCOEt or MPEP.
Systemic injections of CPCCOEt and local infusions with LY367385 or
mGlu1 antisense oligonucleotides produced morphological abnormalities
in the neonate rat cerebellum, which are indicative of an arrest or
delay in the maturation of Purkinje cells. These abnormalities (cell
shrinkage and dystrophic neurites) are reminiscent of those seen in
culture treated with CPCCOEt and strongly suggest that activation of
mGlu1 receptors is required for the early development of Purkinje
cells. We did not use MPEP for in vivo studies because high
concentrations of MPEP (>100 µM) may interact
with NMDA receptors (Gasparini et al., 1999), and we could not exclude
the possibility that such concentrations could be reached in the
cerebellum after systemic or local injections. For this reason, we used
antisense oligonucleotides to examine whether mGlu5 receptors have any
role in cerebellar development. Antisense-induced knockdown of mGlu5 receptor was associated with a reduction in granule cell number and
alterations in the polarity and arborization of Purkinje cell dendrites, similar to what we observed in cultures treated with MPEP.
In conclusion, we have shown that endogenous activation of mGlu1 and
mGlu5 receptors is necessary for a proper development of cerebellar
Purkinje cells, with the two receptors operating at different times and
on different cells. mGlu1 receptor blockade impaired Purkinje cell
development only at early stages of maturation, when dendritic
remodeling is presumably independent of innervation by granule cell
axons (Armengol and Sotelo, 1991). Interestingly, the expression and
subcellular distribution of mGlu1a receptors in Purkinje cells is also
independent of synaptic inputs (Takács et al., 1997). Resident
astrocytes are a possible source for the glutamate-activating mGlu1
receptors at these early stages of development. Our scenario is
clearly different from that reported in mGlu1 knock-out mice, where the
only relevant morphological alteration (i.e., the persistence of
multiple climbing fiber synapses on Purkinje cells) depends on
processes that occur late in development (Kano et al., 1997). mGlu5
receptors might be activated primarily on granule cells or astrocytes
and then influence the phenotype of Purkinje cells indirectly, through
a synaptic or a paracrine mechanism. These results raise the
possibility that changes in the activity of mGlu1 or mGlu5 receptors
occurring at critical times of development may contribute to the
pathophysiology of cerebellar disorders.
| |
FOOTNOTES |
Received May 4, 2001; revised July 11, 2001; accepted July 17, 2001.
This work was supported by Telethon Grant 1238. We thank M. Cascone for
excellent technical assistance.
Correspondence should be addressed to Dr. Maria Vincenza Catania,
Bioimaging and Pathophysiology of the Central Nervous System-National Research Council (IBFSNC-CNR), Vl Regina Marherita 6, 95123 Catania, Italy. E-mail: mcatania{at}area.ct.cnr.it.
| |
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ABSTRACT
INTRODUCTION
