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The Journal of Neuroscience, March 1, 1998, 18(5):1735-1742
Effect of Glutamate on Dendritic Growth in Embryonic Rat
Motoneurons
Friedrich
Metzger,
Stefan
Wiese, and
Michael
Sendtner
Klinische Forschergruppe Neuroregeneration, Department of
Neurology, University of Würzburg, 97080 Würzburg,
Germany
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ABSTRACT |
Glutamate is a major excitatory neurotransmitter for spinal
motoneurons. We have investigated its effect on survival and neurite formation in cultures of highly enriched motoneurons from 15-d-old rat
embryos. Whereas the survival of these neurons was not reduced by this
treatment, a distinct and specific effect on dendrite outgrowth could
be observed. Axon outgrowth was not affected by glutamate. Our data
suggest that calcium influx via ionotropic AMPA/kainate (AMPA/KA)
receptors is responsible for the regulation of dendrite outgrowth by
excitatory neurotransmission. This was shown by the use of specific
inhibitors for the different classes of glutamate receptors. The effect
was reduced by continuous depolarization at 35 mM KCl and
by treatment with joro spider toxin (JSTX-3, 3 µM), a
blocker of Ca2+-conducting AMPA receptors. Removal
of glutamate after 5 d of culture led to increased dendrite growth
during the following culture period, and delayed addition resulted in a
reduction in the length of already existing dendrites. Our observation
that the effect is dose-dependent and reversible reflects a potential physiological function of excitatory neurotransmission on dendrite growth and morphology during a developmental period when synaptic contacts from afferent neurons to motoneurons are made in the spinal
cord.
Key words:
glutamate; motoneurons; dendrite growth; development; AMPA/KA receptors; spinal cord
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INTRODUCTION |
In most types of neurons within the
CNS, the excitatory neurotransmitter glutamate activates several
classes of specific receptors. The ionotropic NMDA and AMPA/kainate
(AMPA/KA) glutamate receptors participate in the regulation of synaptic
plasticity (Gu et al., 1996 ) (for review, see Collingridge and Singer,
1990; Gasic and Heinemann, 1991) as well as the formation of neuronal
networks during development (Mayer and Westbrook, 1987), whereas the
G-protein-coupled metabotropic glutamate receptors modulate synaptic
transmission via second messengers like cAMP or inositol triphosphate
(for review, see Schoepp et al., 1990). Besides its function in the transmission of physiological signals, glutamate can be toxic to
neurons of the CNS. It has been hypothesized that developing motoneurons, which express relatively high levels of NMDA receptors, are particularly vulnerable to glutamate (Greensmith et al., 1994; Greensmith and Vrbová, 1996).
Important issues in the development of the nervous system are the
regulation of process outgrowth to establish specific neuronal connections and the stabilization of newly formed synapses. Neurite outgrowth is dependent on several environmental or intrinsic factors, such as extracellular matrix, neurotrophic factors, or electrical activity. The involvement of neurotransmitters, such as glutamate, in
the regulation of process outgrowth has been described for various
neuronal populations. In fetal cat retinal ganglion cells, glutamate
inhibited dendritic stratification selectively via non-NMDA receptors
(Bodnarenko et al., 1995). In cortical brain slices from 14-d-old
ferrets, BDNF, together with electrical activity, was found to induce
dendrite growth in cortical pyramidal neurons (McAllister et al., 1996,
1997). In embryonic rat spinal cord explants the stimulation of
electrical activity, which leads to enhanced excitatory
neurotransmission, modulated the development of motoneuron resting
potential and other specific properties (Xie and Ziskind-Conhaim,
1995). All together, these data suggest that contacting neurons play an
important role in the regulation of dendrite growth. Such influences
also can be observed in cell culture with isolated neurons. For
example, morphological differentiation of Purkinje cells is stimulated
by the presence of granule neurons in coculture (Baptista et al.,
1994). If endogenous electrical activity in cultures of mouse
cerebellar Purkinje cells is blocked by tetrodotoxin (TTX) or high
Mg2+ treatment, then dendrite growth proceeds and
differentiation of these cells apparently is impaired (Schilling et
al., 1991). In cultures of cerebellar granule neurons, activation of
the NMDA receptor inhibited axonal outgrowth (Baird et al., 1996),
whereas in adult hippocampal pyramidal neurons low concentrations of
glutamate (1 µM) decreased dendrite growth (Mattson et
al., 1988).
We have investigated the effect of glutamate on isolated motoneurons
from 15-d-old rat embryos. We observed a specific modulation of
dendritic, but not axonal, growth by glutamate. This plasticity effect
of glutamate is mediated by AMPA/KA receptor activation. To our
knowledge, these data demonstrate for the first time that glutamate
influences the dendritic architecture of motoneurons, thus providing
another example of activity-dependent changes of dendritic structure in
the CNS.
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MATERIALS AND METHODS |
Materials. MC192 hybridoma cells producing a
monoclonal antibody against the low-affinity nerve growth factor
receptor (p75) were obtained from the American Type Culture Collection
(Rockville, MD), and 40.2D6 cells producing the islet-1 antibody were
obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). The growth factors brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) were from Preprotech (Frankfurt, Germany). Neurobasal medium, B27 supplement, glutamine, and
trypsin were obtained from Life Technologies (Eggenstein, Germany).
6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX),
D- -glutamylaminomethane-sulfonic acid (GAMS),
(+)- -methyl-4-carboxyphenylglycine (MCPG), dizocilpine maleate
(MK-801),
1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), NMDA, anti-mouse-Cy3 (goat), and anti-rabbit-Cy2 (goat) were
purchased from RBI/Biotrend (Köln, Germany). JSTX-3 and TTX were
obtained from Molecular Probes (Eugene, OR).
Anti-microtubule-associated protein 2 (MAP2, mouse monoclonal) and
anti-mouse IgG (sheep) were obtained from Boehringer Mannheim
(Mannheim, Germany), goat and horse serum were obtained from Linaris
(Wertheim, Germany), and anti-tau (rabbit) and the other chemicals were
purchased from Sigma (Deisenhofen, Germany).
Rat embryonic motoneuron cultures. Cultures of spinal
motoneurons from embryonic day 15 rats were prepared by an
immunopanning technique as described (Hughes et al., 1993). Briefly,
the ventral parts of the spinal cords were dissected and transferred to
HBSS containing 10 µM  -mercaptoethanol. The
preparation time was kept to a minimum (usually <30 min). After
treatment with trypsin (0.05%, 15 min), tissues were triturated with a
fire-polished Pasteur pipette and passed through a nylon mesh (pore
size, 100 µm) to remove connective tissue and cell clumps. The
resulting suspension containing single cells was transferred to a
polystyrene Petri dish (Falcon, Oxnard, CA) that had been precoated
overnight with sheep anti-mouse IgG (1 µg/ml) and subsequently for at
least 3 hr at room temperature with MC192 hybridoma supernatant (final antibody concentration, 1 µg/ml). After incubation for 1 hr at room
temperature in such pretreated culture dishes, nonadherent cells were
removed by gently washing with HBSS, and the motoneuron-enriched cell
fraction was eluted by adding MC192 hybridoma supernatant. The
motoneurons were plated at a density of 2000 cells/cm2 in four-well culture dishes precoated with
poly-ornithine and laminin as described (Arakawa et al., 1990). Cells
were grown under serum-free conditions in Neurobasal medium
supplemented with B27 and 500 µM glutamine at 37°C in a
humidified atmosphere with 5% CO2. After 1 hr,
neurotrophic factors and the various substances were added to the
cultures and renewed with the medium at day 1 in culture and thereafter
every 2 d. The concentration of BDNF and GDNF used in this study
was 10 ng/ml, reflecting a saturating concentration. EC50
for BDNF was 9 pg/ml, and for GDNF the EC50 was 80 pg/ml.
Maximal survival effects with BDNF and GDNF were observed at 10 ng/ml
(data not shown).
Estimation of survival rates in motoneuron cultures. The
initial cell number per well was determined 3 hr after plating by phase-contrast microscopy (magnification, 125×). Only phase-bright cells larger than 10 µm in diameter were included in the evaluation in predetermined fields corresponding to a total of 21% of the surface
area of each well. We have chosen this technique because it allowed us
to make the determination of cell numbers in the same cultures at
different subsequent time points. Independent experiments in our lab
have shown that the number of surviving neurons determined by counting
phase-bright cells matches >90% with the number determined by a
life/dead kit (Molecular Probes, Leiden, The Netherlands). This kit is
based on the cleavage of calcein AM in life cells and the staining of
DNA by ethidium homodimer-1 in dead cells.
Surviving neurons were counted in the same fields at different time
points for up to 7 d in culture, and the results are presented as
the percentage of the originally plated cell number. To ensure that the
cultures contained mainly motoneurons, we stained the cultures with the
motoneuron-specific islet-1 antibody. Cultures of >90% motoneurons
were obtained consistently.
In some experiments the motoneurons were cultured under depolarizing
conditions in the presence of 35 mM KCl. Therefore, 3 hr
after plating, one-half of the medium was exchanged with isotonic NaCl
solution containing 70 mM KCl. Control cultures were
treated with the same amount of isotonic NaCl solution. Living
motoneurons were counted for up to 5 d in culture, as described
above.
Counting of neurites. The number of neurites on each
motoneuron was determined after 3 and 5 d in culture by
phase-contrast microscopy. At a plating density of 2000 cells/cm2 most motoneurons grew without neurite
contact to other cells. Single motoneurons were photographed
(magnification, 250×), and every neurite longer than 10 µm was
counted. In this part of the study the differences between axons and
dendrites were not taken into account.
Immunostaining of motoneurons for axonal and dendritic proteins.
After 3, 5, or 10 d in culture the axonal and dendritic
processes of the motoneurons were distinguished by staining with
specific markers. Briefly, cells were fixed for 30 min at 37°C with
4% paraformaldehyde. Subsequently, nonspecific binding sites were blocked, and cell membranes were permeabilized for 20 min with 10%
goat serum and 0.01% Triton X-100. Primary antibodies were added for
30 min at 37°C; then the dishes were washed twice, blocked again for
5 min, and incubated for 20 min at 37°C with the secondary antibodies. After being washed, the cells were mounted with
glycerol/PBS (1:1) and observed by fluorescence microscopy. The
motoneurons were double-stained with monoclonal mouse anti-MAP2 (5 µg/ml) and rabbit anti-tau (1:200). MAP2 was visualized by goat
anti-mouse-Cy3 (5 µg/ml), and tau was visualized with goat
anti-rabbit-Cy2 (10 µg/ml).
Estimation of dendrite and axon length. The immunostained
motoneurons were double-exposed under fluorescence light
(magnification, 400×) after 3, 5, and 10 d in culture. Then the
mean length of all MAP2-positive and tau-positive processes per
motoneuron was estimated from photographs by using a scaled overlay. To
estimate the total process length of each motoneuron, we added the
length of all dendrites or axons per cell.
Calculations and statistics. Values from independent
experiments were pooled, and the results were expressed as the
mean ± SEM. Statistical significance of differences was assessed
by ANOVA. Equal variances were tested by Bartlett's test. Differences
between control and individual treatment groups were tested by
Dunnett's multiple comparison test, using the GraphPad Prism software
(San Diego, CA).
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RESULTS |
Glutamate does not induce cell death in embryonic
motoneuron cultures
To investigate whether glutamate can induce cell death in cultured
embryonic motoneurons, we isolated cells from 15-d-old rat embryos,
which were enriched by immunopanning and cultured at a density of 2000 neurons/cm2. Figure 1
illustrates the survival of motoneurons grown either with or without
BDNF or GDNF (10 ng/ml each) in the presence or absence of glutamate
(100 µM). In the absence of neurotrophic factors, only
14% of the originally plated motoneurons survived until day 5 in
culture; survival decreased to <5% by day 7. Glutamate did not
enhance cell death in such cultures in the absence of survival
factors.
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Figure 1.
Survival of cultured embryonic rat motoneurons is
not affected by glutamate. Motoneurons were prepared and cultured
either without survival factors or in the presence of 10 ng/ml BDNF or GDNF. Survival rates were evaluated from cultures in the presence (filled symbols) or absence (open
symbols) of 100 µM glutamate over the total
culture period. At the indicated time points, surviving motoneurons
were counted in predetermined fields corresponding to 21% of the
surface area of each well. These data represent the mean ± SEM of
eight observations from four independent experiments.
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The apparent lack of glutamate toxicity in neurotrophic
factor-supported cultures could be attributable to a specific
alteration of glutamate sensitivity by BDNF or GDNF. Such effects were
observed in the neuronal cell lines PC-12 and HT-22, in which survival factors influenced glutamate-induced neurotoxicity (Schubert et al.,
1992; Davis and Maher, 1994). Moreover, experiments with cultured
retinal ganglion cells as well as other CNS neurons showed that
activation of glutamate receptors was essential for the neurotrophic survival effect (Meyer-Franke et al., 1995; Nichol et al., 1995). Therefore, motoneurons were cultured in the presence of 2% horse serum, which supported their survival in the absence of specific neurotrophic factors. After 5 d in culture, 46.7 ± 2.5%
(n = 3) of the motoneurons were still alive, and the
presence of 100 µM glutamate did not alter their survival
significantly (49.8 ± 2.7%; n = 3).
When the cultures were supplemented with BDNF, 60 ± 3% of the
cells survived after 5 d in the absence and 61 ± 4% in the
presence of glutamate (n = 8). GDNF, another
neurotrophic factor with specific survival-promoting activity in
motoneuron cultures, supported 65 ± 4% of the motoneurons in the
absence and 67 ± 4% in the presence of glutamate after 5 d
in culture (Fig. 1; n = 8). Similar results were
obtained after 1, 3, and 7 d in culture, suggesting that the
addition of 100 µM glutamate is not toxic to motoneuron
cultures derived from 15-d-old rat embryos. Furthermore, the addition
of NMDA (up to 10 µM), JSTX-3 (3 µM), or
TTX (3 µM) did not alter motoneuron survival in the
presence or absence of glutamate (data not shown). Depolarizing culture
conditions (35 mM KCl), which remove the NMDA receptor
block by Mg2+ (Moriyoshi et al., 1991), led to
slightly, but not significantly reduced motoneuron survival (93 ± 13% of control after 5 d in culture; n = 4).
Again, glutamate did not reduce survival under these conditions
(89 ± 12% of control after 5 d in culture;
n = 4).
The addition of glutamate to motoneuron cultures supported with
both BDNF and GDNF also did not affect long-term survival significantly
after a culture period of 10 d (n = 3). At 10 d in culture, survival was 22.6 ± 2.4% without glutamate and
22.2 ± 2.6% with 100 µM glutamate. Removal of
glutamate after a period of 5 d as well as delayed addition of
glutamate from days 5 to 10 led to similar survival rates (24.4 ± 2.7% and 21.0 ± 2.2%, respectively).
Effect of glutamate on neurite number in cultured
rat motoneurons
The number of neurites per cell was determined (Table
1; Fig. 2)
after 3 and 5 d in culture. Glutamate led to a highly significant reduction in neurite numbers both in BDNF- and GDNF-supported cultures.
This effect was already detectable after 3 d. The average number
of neurites in BDNF-supported cultures after 5 d was 2.05 neurites
per motoneuron with 100 µM glutamate and 3.38 neurites in
the absence of glutamate. In GDNF-treated cultures, the number of
dendrites was reduced similarly from 3.45 to 2.11 in the presence of
100 µM glutamate (Table 1). Analysis of the concentration dependence of the glutamate effect (0.1-100 µM) on
neurite growth revealed a maximum effect at 3 µM
glutamate, suggesting an IC50 value in the submicromolar
range (Fig. 2).
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Figure 2.
Glutamate reduces neurite number in embryonic rat
motoneurons. Motoneurons were cultured with 10 ng/ml BDNF
(A) or GDNF (B) and
increasing glutamate concentrations supplemented over the whole culture
period. Neurites were counted after 5 d on phase-contrast micrographs (magnification, 250×). These data represent the mean ± SEM of at least 68 single observations from three to four
independent experiments; asterisks indicate significant
difference (**p < 0.01) from the respective
control (without glutamate) as revealed by ANOVA and Dunnett's
multiple comparison test.
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Characterization of the inhibitory glutamate effect on neurite
growth with specific receptor antagonists
To identify the glutamate receptor subtypes responsible for the
effect on neurite growth, we added NBQX (3 µM), a
specific antagonist of AMPA receptors, CNQX (10 µM), a
blocker of both AMPA and KA receptors, GAMS (100 µM), a
preferential KA receptor blocker (Honoré et al., 1988; Zhou et
al., 1993), and the selective NMDA receptor antagonist MK-801 (10 µM; Moriyoshi et al., 1991) to our cultures. In addition,
involvement of the metabotropic glutamate receptor, which was shown to
sensitize AMPA/KA receptors by prolonged activation in rat dorsal horn
spinal neurons (Cerne and Randic, 1992), was investigated by using the
antagonist MCPG (200 µM; Watkins and Collingridge, 1994).
The effects of these compounds on glutamate-treated motoneurons after
5 d in culture are shown in Figure
3. All antagonists of AMPA and KA
receptors abolished the glutamate effect on neurite growth in cultures
supported by BDNF (Fig. 3A) or GDNF (Fig. 3B). In
contrast, MK-801 and MCPG had no significant effect.
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Figure 3.
Glutamate affects neurite growth via AMPA and KA
receptors. Motoneuron cultures were supplemented over the whole culture
period with 10 ng/ml BDNF (A) or GDNF
(B) and 100 µM glutamate (except control); additionally, the specific glutamate receptor antagonists CNQX (10 µM), NBQX (3 µM), GAMS (100 µM), MK-801 (10 µM), or MCPG (200 µM) were applied. Neurites were counted after 5 d on
phase-contrast micrographs (magnification, 250×). These data represent
the mean ± SEM of at least 101 single observations from four
independent experiments; asterisks indicate significant
difference (*p < 0.05; **p < 0.01) from control (presence of glutamate alone) as revealed by ANOVA
and Dunnett's multiple comparison test.
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Glutamate inhibits dendrite, but not axon growth
To investigate whether glutamate selectively reduced growth of
either axonal or dendritic processes, we double-stained the motoneurons
with antibodies for axonal tau and dendritic MAP2. Figure
4 shows typical stained cells grown with
BDNF for 3 or 5 d either in the absence (Fig.
4A,C) or presence of 100 µM glutamate (Fig. 4B,D). The quantitative evaluation of these
changes is presented in Table 2.
Glutamate treatment selectively inhibited dendrite growth, because the
estimated parameters for tau-positive axons did not differ between
treated and untreated cultures. More than 80% of the motoneurons
contained only one tau-stained axon, and length and number were
unchanged by glutamate (Table 2). On the other hand, glutamate
treatment decreased both the number and length of MAP2-positive
dendrites. The mean number was reduced significantly by glutamate
treatment from 2.46 to 1.20 dendrites per cell, and the mean length of
dendritic processes decreased from 36 to 26 µm (Table 2).
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Figure 4.
Effect of glutamate on dendrite growth in
embryonic rat motoneurons. Motoneurons were cultured for 3 d
(A and B) or 5 d (C and D) with 10 ng/ml BDNF in the presence or absence of
100 µM glutamate. Then the cells were fixed and stained
for axonal tau (green processes) and dendritic
MAP2 protein (orange processes), as described in
Materials and Methods. Shown is a motoneuron cultured for 3 d
without (A) or with (B)
glutamate. C, Control. D,
Glutamate-treated motoneuron after 5 d in culture. Scale bar, 50 µm.
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Table 2.
Characterization of the effect of glutamate on growth of
axons and dendrites in embryonic rat motoneurons
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The modulation of dendrite growth by glutamate is dependent on
Ca2+ influx
As a next step, we investigated whether Ca2+
influx or depolarization via Na+ channels plays a
role in the effect of glutamate on motoneuron dendrite growth.
Therefore, motoneurons were cultured in the presence of BDNF for 5 d under depolarizing conditions (35 mM KCl). Other cultures
were treated with TTX (3 µM), a blocker of
voltage-dependent Na+ channels. To block selectively
the Ca2+-conducting AMPA receptors, we applied
JSTX-3 (3 µM; Blaschke et al., 1993; Iino et al., 1996).
Continuous depolarization by 35 mM KCl was expected to
elevate intracellular Ca2+. This treatment did not
reduce the number of dendrites, but it led to a partial reduction in
their individual length (average length was 30 µm, as compared with
36 µm in control and 26 µm in glutamate-treated cultures).
Interestingly, depolarization also exerted a strong reduction in axon
length (94 µm, as compared with 136 µm in control cultures) that
was not seen in glutamate-treated cultures. This indicates that
depolarization by KCl, but not by glutamate, can reduce axon outgrowth
significantly. Thus the signals that regulate dendrite and axon growth
in isolated motoneurons appear to be different. Treatment with JSTX-3
slightly reduced both dendrite number and length, whereas axon growth
was not changed (Table 2). The reduction of total dendrite length by
glutamate was nearly abolished by depolarization, whereas blockade of
neuronal activity by TTX had no influence on this effect (Table 2; Fig. 5). In contrast, blockade of
Ca2+ influx via AMPA receptors by JSTX-3 provided a
condition under which glutamate could not lead to any further effect on
dendrite number and length (Table 2; Fig. 5).
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Figure 5.
The effect of glutamate on dendrite growth is
abolished by depolarization and JSTX-3. Motoneuron cultures
supplemented with 10 ng/ml BDNF were grown with or without 100 µM glutamate; additionally, the indicated conditions
(depolarization, depol) or substances were
applied. After 5 d, the cells were fixed and stained for tau and
MAP2, and the total dendrite length was estimated from fluorescence
micrographs (magnification, 400×). These data represent the mean ± SEM of at least 91 single observations from three to four
independent experiments; asterisks indicate significant
difference (***p < 0.01) from control without
glutamate as revealed by ANOVA and Dunnett's multiple comparison
test.
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The inhibition of dendrite growth by glutamate is reversible
A further point of investigation was the reversibility and
plasticity of the glutamate effect on process outgrowth. Therefore, motoneurons were cultured in the presence of both BDNF and GDNF to
enhance long-term survival for up to 10 d. Glutamate (100 µM) was added either from days 1 to 5 or from days 5 to
10 in culture. The motoneurons surviving after 10 d were
double-stained for MAP2/tau, and the lengths of dendrites and axons
were determined differentially. At 5 d the motoneurons showed, on
average, 1.03 ± 0.03 axons (length, 136 ± 5 µm) and
2.46 ± 0.13 dendrites (length, 36 ± 1 µm). At 10 d,
on average, 1.12 ± 0.05 axons (length, 160 ± 7 µm) and
2.18 ± 0.15 dendrites (length, 37 ± 1 µm) were observed.
These values did not differ significantly from those obtained after
5 d in culture. The addition of glutamate from days 5 to 10 in
culture strongly reduced total dendrite length (Fig.
6) to levels lower than at 5 d,
suggesting that glutamate addition led to an involution of dendrites
that had already grown out significantly at 5 d. At 10 d, the
dendrite length in these cultures was comparable to those motoneurons
maintained with glutamate over the whole culture period. On the other
hand, removal of glutamate after 5 d led to partial recovery by an
increased growth of dendrites during the following culture period (Fig.
6). Axon growth remained totally unaffected by the removal or addition
of glutamate (data not shown).
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Figure 6.
Plasticity of the glutamate effect on dendrite
growth. Motoneuron cultures were supplemented with 10 ng/ml of both
BDNF and GDNF for the whole culture period. Glutamate
(+glut; 100 µM) was applied over a
10 d culture period, added, or removed after 5 d. After
10 d, the cells were fixed and stained for tau and MAP2, and the
total dendrite length was estimated from fluorescence micrographs
(magnification, 400×). These data represent the mean ± SEM of at
least 77 single observations from three independent experiments.
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DISCUSSION |
We have investigated the effect of glutamate on survival and
outgrowth of dendrites and axons in cultured embryonic rat motoneurons. Our results indicate that glutamate does not induce cell death at a
concentration (100 µM) that saturates all types of
ionotropic receptors. However, glutamate specifically reduces dendrite
outgrowth. This reduction was similar in motoneuron cultures grown in
the presence of BDNF or GDNF and was at least partially reversible when
glutamate was removed. Our data suggest that the modulation of dendrite
outgrowth occurred via activation of AMPA/KA receptors, whereas NMDA
and metabotropic receptors were not involved. Thus AMPA/KA receptors
could regulate highly specific aspects of motoneuron development, in
particular during a period when synaptic connections between
proprioceptive sensory nerve fibers and motoneurons are made in the
embryonic spinal cord.
Glutamate is not toxic to embryonic motoneurons
Glutamate toxicity at high concentrations (IC50 ~300
µM) has been reported in studies that used
motoneuron-enriched cultures from 12- to 14-d-old rat embryos (Estevez
et al., 1995). In organotypic slice cultures derived from 8-d-old
postnatal rats, slow motoneuron degeneration in the presence of
agonists of AMPA/KA receptors or inhibitors of glutamate transporters
has been demonstrated (Rothstein et al., 1993, 1996). Other studies
showed that embryonic motoneurons express NMDA receptors at levels
higher than adult motoneurons (Kalb et al., 1992), and
electrophysiological analysis in rat spinal cord preparations from
embryonic days 15 to 21 indicated that the NMDA receptors on the
motoneurons are functional (Ziskind-Conhaim, 1990). Involvement of NMDA
receptors in lesion-induced motoneuron death has been reported
(Greensmith et al., 1994). Therefore, glutamate toxicity in motoneurons
via NMDA after nerve injury as well as non-NMDA receptors has been
inferred (Greensmith and Vrbová, 1996). Our data suggest that
isolated motoneurons are not susceptible to glutamate toxicity, neither
at embryonic day 15 when they express functional NMDA receptors nor at
periods up to 10 d in culture during which they grow out axons and
dendrites. Therefore, we conclude that highly enriched isolated
motoneurons are much more resistant to glutamate excitotoxicity than
other neuronal cell types, such as hippocampal neurons.
Glutamate inhibits dendrite outgrowth in isolated motoneurons
Motoneurons treated with glutamate show a concentration-dependent
decrease in neurite number (Table 2). This effect was maximal at 3 µM (see Fig. 2), suggesting that specific signaling via
ionotropic glutamate receptors is involved. Using the axon- and
dendrite-specific markers tau and MAP2, we could show that glutamate
treatment does not alter the number and length of axons (Table 2; Fig.
4) but selectively inhibits dendrite growth. Very similar results have been described for subtoxic glutamate concentrations in cultures of
adult hippocampal pyramidal neurons (Mattson et al., 1988) or fetal cat
retinal ganglion cells (Bodnarenko et al., 1995) in which glutamate did
not affect viability but inhibited dendritic stratification in the
retina. These studies suggested an effect of glutamate on the
differentiation of developing neurons via NMDA receptors. Baird et al.
(1996) advanced the hypothesis that NMDA receptor activation in
cerebellar granule neurons can modulate axonal outgrowth after contact
with primary afferents. In contrast, the motoneurons used in our study
showed no change in either number or length of axons by glutamate after
5 or 10 d.
The outgrowth of processes from cultured motoneurons is very fast until
day 5 in culture, but then it slows down dramatically. Little increase
of axon length and virtually no further growth of dendrites could be
observed during the following 5 d in culture. Compensatory
increased growth could be observed from days 5 to 10 in culture (see
Fig. 6) only when glutamate was added transiently from days 1 to 5. The
addition of glutamate to the motoneurons at a time when their dendrites
had reached maximal length (day 5) could induce involution during the
following culture period. This indicates that the effect of glutamate
on dendrite growth is reversible and that glutamate thus modulates the
dendritic architecture of motoneurons during development.
The effect of glutamate on dendrite growth is mediated by
AMPA/KA receptors
The inhibition of dendritic outgrowth by glutamate was abolished
specifically by antagonists of AMPA and KA receptors, but not by
blockers of NMDA or metabotropic receptors (see Fig. 3). Using JSTX-3
(Blaschke et al., 1993), we could show that Ca2+
influx is involved. Moreover, depolarization by elevated
K+ in the culture medium reduces the effect,
probably by desensitization of glutamate receptors or by the elevation
of intracellular Ca2+ in the cytosol of motoneurons
or by both mechanisms. Interestingly, even these conditions have only
very little effect on motoneuron survival. Also, the addition of TTX
does not block the glutamate effect on dendrite growth. This provides
another proof that classical NMDA receptors in which the
Mg2+ block is removed by depolarization probably are
not involved in the observed effects. Theoretically, we cannot exclude
the possibility that NMDA or AMPA receptors or downstream effector systems are desensitized progressively by depolarization or extensive glutamate treatment (Imredy and Yue, 1994; Ballerini et al., 1995). However, this is unlikely, at least for AMPA receptors, because the
inhibition of dendrite growth appears to be strong (Table 2) and is
maintained over periods of at least 10 d in culture.
Ca2+-conducting AMPA/KA receptors have been
reported to trigger long-term effects in dorsal horn spinal neurons (Gu
et al., 1996), but their molecular composition remains to be defined. Motoneurons express the subunits GluR-B and GluR-D at significant levels, mostly the flip isoform. Also, high levels of GluR-C, both in
flip and flop isoform, are detectable (Tölle et al., 1995).
Because these subunits are part of Ca2+-conducting
functional AMPA receptors, it is highly suggestive that such receptors
exist on motoneurons and that they are responsible for mediating the
glutamate effects on dendrite growth observed in our study.
At motoneuron dendrites, glutamate is provided physiologically by
primary sensory afferents from the dorsal root ganglia (Furuyama et
al., 1993). During development of rat embryos the entry of glutamatergic afferents into the gray matter of the spinal cord can be
observed from embryonic day 15 on, and the initial contacts between
afferent projections from the dorsal root ganglia and motoneurons are
formed at embryonic day 16 (Ziskind-Conhaim, 1990). This time course
matches very well with the developmental stage of the embryonic
motoneurons used in our experiments. The presence of glutamate in our
in vitro system thus could mimic a glutamatergic input from
proprioceptive neurons. The postsynaptic ionotropic glutamate receptors
then mediate this regulatory signal for dendrite growth in motoneurons.
This process could contribute to the determination of neuronal
connections in the developing spinal cord.
The establishment and stabilization of synapses plays an important role
for the morphological maturation of the nervous system. Therefore, the
contribution of neurotransmitters in this process is of special
interest. Glutamate has been described to trigger a variety of cellular
responses associated with such changes; some of them are mediated by an
increase in intracellular Ca2+. For example,
postsynaptic phosphorylation of microtubule-associated proteins, such
as MAP2 in hippocampal slices, is influenced by changes in
intracellular Ca2+, mediated via metabotropic and
NMDA-type glutamate receptors (Quinlan and Halpain, 1996). The local
and temporal regulation of MAP protein phosphorylation in response to
glutamate could modulate microtubule stability and bundling during
neuritogenesis (Ávila et al., 1994), thus determining dendrite
arborization (Díez-Guerra and Ávila, 1993). MAP2 is
present only in dendrites and cell somata, which matches the
distribution of Ca2+-conducting GluR-C and GluR-D
subunits in motoneurons (Furuyama et al., 1993). Therefore, we
hypothesize that the activity-dependent regulation of MAP2
phosphorylation via glutamate receptors may trigger the morphological
changes observed in developing motoneurons.
| |
FOOTNOTES |
Received Sept. 25, 1997; revised Dec. 11, 1997; accepted Dec. 15, 1997.
This work was supported by the Deutsche Forschungsgemeinschaft, Grant
To61/8-1. We thank Dr. Jenny Gunnersen for helpful comments on this
manuscript and Dr. Jessell (Columbia University, Hammer Health Sciences
Center, New York) for providing the 40.2D6 hybridoma cells through the
Developmental Studies Hybridoma Bank (Iowa City, IA).
Correspondence should be addressed to Dr. Michael Sendtner, Klinische
Forschergruppe Neuroregeneration, Department of Neurology, University
of Würz burg, Joseph-Schneider-Strasse 11, 97080 Würzburg, Germany.
| |
REFERENCES |
-
Arakawa Y,
Sendtner M,
Thoenen H
(1990)
Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: comparison with other neurotrophic factors and cytokines.
J Neurosci
10:3507-3515[Abstract].
-
Ávila J,
Dominguez J,
Díaz-Nido J
(1994)
Regulation of microtubule dynamics by microtubule-associated protein expression and phosphorylation during neuronal development.
Int J Dev Biol
38:13-25[Web of Science][Medline].
-
Baird DH,
Trenkner E,
Mason CA
(1996)
Arrest of afferent axon extension by target neurons in vitro is regulated by the NMDA receptor.
J Neurosci
16:2642-2648[Abstract/Free Full Text].
-
Ballerini L,
Bracci E,
Nistri A
(1995)
Desensitization of AMPA receptors limits the amplitude of EPSPs and the excitability of motoneurons of the rat isolated spinal cord.
Eur J Neurosci
7:1229-1234[Web of Science][Medline].
-
Baptista CA,
Hatten ME,
Blazeski R,
Mason CA
(1994)
Cell-cell interactions influence survival and differentiation of purified Purkinje cells in vitro.
Neuron
12:243-260[Web of Science][Medline].
-
Blaschke M,
Keller BU,
Rivosecchi R,
Hollmann M,
Heinemann S,
Konnerth A
(1993)
A single amino acid determines the subunit-specific spider toxin block of -amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor channels.
Proc Natl Acad Sci USA
90:6528-6532[Abstract/Free Full Text].
-
Bodnarenko SR,
Jeyarasasingam G,
Chalupa LM
(1995)
Development and regulation of dendritic stratification in retinal ganglion cells by glutamate-mediated afferent activity.
J Neurosci
15:7037-7045[Abstract].
-
Cerne R,
Randic M
(1992)
Modulation of AMPA and NMDA responses in rat spinal dorsal horn neurons by trans-1-aminocyclopentane-1,3-dicarboxylic acid.
Neurosci Lett
144:180-184[Web of Science][Medline].
-
Collingridge GL,
Singer W
(1990)
Excitatory amino acid receptors and synaptic plasticity.
Trends Pharmacol Sci
11:290-296[Medline].
-
Davis JB,
Maher P
(1994)
Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line.
Brain Res
652:169-173[Web of Science][Medline].
-
Díez-Guerra FJ,
Ávila J
(1993)
MAP2 phosphorylation parallels dendrite arborization in hippocampal neurons in culture.
NeuroReport
4:419-422[Web of Science][Medline].
-
Estevez AG,
Stutzmann JM,
Barbeito L
(1995)
Protective effect of riluzole on excitatory amino acid-mediated neurotoxicity in motoneuron-enriched cultures.
Eur J Pharmacol
280:47-53[Web of Science][Medline].
-
Furuyama T,
Kiyama H,
Sato K,
Park HT,
Maeno H,
Takagi H,
Tohyama M
(1993)
Region-specific expression of subunits of ionotropic glutamate receptors (AMPA-type, KA-type, and NMDA receptors) in the rat spinal cord with special reference to nociception.
Mol Brain Res
18:141-151[Medline].
-
Gasic GP,
Heinemann SF
(1991)
Receptors coupled to ionic channels: the glutamate receptor family.
Curr Opin Neurobiol
1:20-26[Medline].
-
Greensmith L,
Vrbová G
(1996)
Motoneuron survival: a functional approach.
Trends Neurosci
19:450-455[Web of Science][Medline].
-
Greensmith L,
Mentis GZ,
Vrbová G
(1994)
Blockade of N-methyl-D-aspartate receptors by MK-801 (dizocilpine maleate) rescues motoneurons in developing rats.
Dev Brain Res
81:162-170.
-
Gu JG,
Albuquerque C,
Lee CJ,
MacDermott AB
(1996)
Synaptic strengthening through activation of Ca2+-permeable AMPA receptors.
Nature
381:793-796[Medline].
-
Honoré T,
Davies SN,
Drejer J,
Fletcher EJ,
Jacobsen P,
Lodge D,
Nielsen FE
(1988)
Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists.
Science
241:701-703[Abstract/Free Full Text].
-
Hughes RA,
Sendtner M,
Thoenen H
(1993)
Members of several gene families influence survival of rat motoneurons in vitro and in vivo.
J Neurosci Res
36:663-671[Web of Science][Medline].
-
Iino M,
Koike M,
Isa T,
Osawa S
(1996)
Voltage-dependent blockage of Ca2+-permeable AMPA receptors by joro spider toxin in cultured hippocampal neurones.
J Physiol (Lond)
496:431-437[Abstract/Free Full Text].
-
Imredy JP,
Yue DT
(1994)
Mechanism of Ca2+-sensitive inactivation of L-type Ca2+ channels.
Neuron
12:1301-1318[Web of Science][Medline].
-
Kalb RG,
Lidow MS,
Halsted MJ,
Hockfield S
(1992)
N-methyl-D-aspartate receptors are transiently expressed in the developing spinal cord ventral horn.
Proc Natl Acad Sci USA
89:8502-8506[Abstract/Free Full Text].
-
Mattson MP,
Dou P,
Kater SB
(1988)
Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons.
J Neurosci
8:2087-2100[Abstract].
-
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].
-
McAllister AK,
Katz LC,
Lo DC
(1996)
Neurotrophin regulation of cortical dendritic growth requires activity.
Neuron
17:1057-1064[Web of Science][Medline].
-
McAllister AK,
Katz LC,
Lo DC
(1997)
Opposing roles for endogenous BDNF and NT-3 in regulating cortical dendritic growth.
Neuron
18:756-778.
-
Meyer-Franke A,
Kaplan MR,
Pfrieger FW,
Barres BA
(1995)
Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture.
Neuron
15:805-819[Web of Science][Medline].
-
Moriyoshi K,
Masu M,
Ishii T,
Shigemoto R,
Mizuno N,
Nakanishi S
(1991)
Molecular cloning and characterization of the rat NMDA receptor.
Nature
354:31-37[Medline].
-
Nichol KA,
Schulz MW,
Bennett MR
(1995)
Nitric oxide-mediated death of cultured neonatal retinal ganglion cells: neuroprotective properties of glutamate and chondroitin sulfate proteoglycan.
Brain Res
697:1-16[Web of Science][Medline].
-
Quinlan EM,
Halpain S
(1996)
Postsynaptic mechanisms for bidirectional control of MAP2 phosphorylation by glutamate receptors.
Neuron
16:257-268.
-
Rothstein JD,
Jin L,
Dykes-Hoberg M,
Kuncl RW
(1993)
Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity.
Proc Natl Acad Sci USA
90:6591-6595[Abstract/Free Full Text].
-
Rothstein JD,
Dykes-Hoberg M,
Pardo CA,
Bristol LA,
Jin L,
Kuncl RW,
Kanai Y,
Hediger MA,
Wang Y,
Schielke JP,
Welty DF
(1996)
Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.
Neuron
16:675-686[Web of Science][Medline].
-
Schilling K,
Dickinson MH,
Connor JA,
Morgan JI
(1991)
Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns.
Neuron
7:891-902[Web of Science][Medline].
-
Schoepp D,
Bockaert J,
Sladezcek F
(1990)
Pharmacological and functional characteristics of metabotropic excitatory amino acid receptors.
Trends Pharmacol Sci
11:508-515[Medline].
-
Schubert D,
Kimura H,
Maher P
(1992)
Growth factors and vitamin E modify neuronal glutamate toxicity.
Proc Natl Acad Sci USA
89:8264-8267[Abstract/Free Full Text].
-
Tölle TR,
Berthele A,
Zieglgänsberger W,
Seeburg PH,
Wisden W
(1995)
Flip and flop variants of AMPA receptors in the rat lumbar spinal cord.
Eur J Neurosci
7:1414-1419[Web of Science][Medline].
-
Watkins JC,
Collingridge GL
(1994)
Phenylglycine derivatives as antagonists of metabotropic glutamate receptors.
Trends Pharmacol Sci
15:333-342[Medline].
-
Xie H,
Ziskind-Conhaim L
(1995)
Blocking Ca2+-dependent synaptic release delays motoneuron differentiation in the rat spinal cord.
J Neurosci
15:5900-5911[Abstract].
-
Zhou N,
Hammerland LG,
Parks TN
(1993)
Gamma-D-glutamylaminomethyl sulfonic acid (GAMS) distinguishes kainic acid- from AMPA-induced responses in Xenopus oocytes expressing chick brain glutamate receptors.
Neuropharmacology
32:767-775[Web of Science][Medline].
-
Ziskind-Conhaim L
(1990)
NMDA receptors mediate poly- and monosynaptic potentials in motoneurons of rat embryos.
J Neurosci
10:125-135[Abstract].
Copyright © 1998 Society for Neuroscience 0270-6474/98/1851735-08$05.00/0
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Top
Abstract
Introduction
Compound via MeSH
