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The Journal of Neuroscience, July 15, 2000, 20(14):5382-5391
Interaction between Metabotropic and Ionotropic Glutamate
Receptors Regulates Neuronal Network Activity
Patrik
Krieger,
Jeanette
Hellgren-Kotaleski,
Petronella
Kettunen, and
Abdel Jabbar
El Manira
Nobel Institute for Neurophysiology, Department of Neuroscience,
Karolinska Institute, S-171 77 Stockholm, Sweden
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ABSTRACT |
Experimental and computational techniques have been used to
investigate the group I metabotropic glutamate receptor
(mGluR)-mediated increase in the frequency of spinal cord network
activity underlying locomotion in the lamprey. Group I mGluR activation
potentiated the amplitude of NMDA-induced currents in identified
motoneurons and crossed caudally projecting network interneurons. Group
I mGluRs also potentiated NMDA-induced calcium responses. This effect was blocked by a group I mGluR-specific antagonist, but not by blockers
of protein kinase A, C, or G. The effect of group I mGluRs activation
was also tested on NMDA-induced oscillations known to occur during
fictive locomotion. Activation of these receptors increased the
duration of the plateau phase and decreased the duration of the
hyperpolarizing phase. These effects were blocked by a group I mGluR
antagonist. To determine its role in the modulation of NMDA-induced
oscillations and the locomotor burst frequency, the potentiation of
NMDA receptors by mGluRs was simulated using computational techniques.
Simulating the interaction between these receptors reproduced the
modulation of the plateau and hyperpolarized phases of NMDA-induced
oscillations, and the increase in the frequency of the locomotor rhythm.
Our results thus show a postsynaptic interaction between group I mGluRs
and NMDA receptors in lamprey spinal cord neurons, which can account
for the regulation of the locomotor network output by mGluRs.
Key words:
locomotion; mGluRs; NMDA; spinal cord; lamprey; calcium; DHPG
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INTRODUCTION |
An understanding of the importance
of synaptic connectivity and membrane properties in producing the final
output of neuronal networks requires knowledge that bridges the gap
between effects at the cellular and network levels. Activation of
metabotropic glutamate receptors (mGluRs) leads to a variety of
cellular responses, including the inhibition of calcium and potassium
currents, presynaptic modulation of synaptic transmission, and
postsynaptic interactions with ionotropic glutamate receptors (iGluRs;
for review see Pin and Bockaert, 1995 ; Pin and Duvoisin, 1995; Anwyl,
1999). In many regions of the CNS, responses to exogenous NMDA
application are potentiated by group I mGluR activation. In some
studies, this potentiation was suggested to be mediated by a
PKC-induced phosphorylation of NMDA receptors because the enhancement
was blocked by PKC inhibitors (Aniksztejn et al., 1991; Kelso, 1992;
but see Harvey and Collingridge, 1993). The functional consequences of
the interaction between mGluRs and iGluRs have been studied in relation
to synaptic plasticity (O'Connor et al., 1994, 1995). The importance
of this interaction has not, however, been analyzed with respect to the
activity of a neural network.
The lamprey spinal locomotor network is a vertebrate system that is
well suited for this type of analysis. In this preparation the synaptic
connectivity and membrane properties of many neurons forming the
locomotor network have been characterized (Buchanan, 1982; Buchanan and
Grillner, 1987; Grillner et al., 1998). Locomotor activity can be
induced in the isolated spinal cord in vitro by activation
of NMDA receptors. NMDA also elicits membrane potential oscillations in
network neurons that persist in the presence of TTX (Sigvardt et al.,
1985; Wallén and Grillner, 1987). These oscillations have been
suggested to be important for maintaining a slow steady rate of
locomotor activity (Brodin and Grillner, 1986; Wallén et al.,
1987). In the lamprey spinal cord, activation of group II and III
mGluRs induces presynaptic inhibition of reticulospinal transmission
(Krieger et al., 1996). Activation of group I mGluRs increases the
frequency of the locomotor rhythm and the amplitude of NMDA-induced
depolarizations (Krieger et al., 1998). These receptors were also shown
to be endogenously activated during fictive locomotion because their
blockade by the specific antagonist CPCCOEt decreases the locomotor
frequency (Krieger et al., 1998). Furthermore, the group I mGluR
agonist DHPG induces presynaptic facilitation in reticulospinal axons,
whereas the antagonist CPCCOEt blocks the facilitation evoked during
trains of action potentials (Cochilla and Alford, 1998). The
presynaptic facilitation was blocked by ryanodine (Cochilla and Alford,
1998).
In the present study we have tested the extent to which
ryanodine-sensitive presynaptic facilitation and the postsynaptic interaction between mGluRs and NMDA receptors contribute to the increase in the locomotor frequency induced by group I mGluRs. Our
results show that both the DHPG-induced increase and CPCCOEt-induced decrease in the locomotor frequency are not blocked by ryanodine. We
also show that group I mGluR activation postsynaptically modulates NMDA-induced currents, calcium responses, and membrane potential oscillations. Finally, computer simulations support the hypothesis that
postsynaptic interaction between mGluRs and NMDA underlies the
mGluR-mediated regulation of the locomotor rhythm.
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MATERIALS AND METHODS |
Electrophysiology. Extracellular measurements of
ventral root activity were performed on spinal cord-notochord
preparations of adult Ichthymyzon unicuspis
(n = 6) and Lampetra fluviatilis (n = 6). Intracellular recordings were made from
isolated spinal cord preparations of adult L. fluviatilis.
The effects of mGluR activation were similar in the different species
(Krieger et al., 1998). The preparation was mounted in a cooled
(8-12°C) Sylgard-lined chamber that was continuously perfused with a
solution of the following composition (in mM):
NaCl 91, KCl 2.1, CaCl2 2.6, MgCl2 1.8, NaHCO3 20, and
glucose 4, bubbled with 95% O2 and 5%
CO2 to pH 7.4 (I. unicuspis) or 138 NaCl, 2.1 KCl, 1.8 CaCl2, 1.2 MgCl2, 4 glucose, 2 HEPES, and 0.5 L-glutamine, bubbled with
O2 and pH adjusted to 7.4 (L. fluviatilis). Fictive locomotion was induced by bath application
of NMDA (100 µM). The cycle duration was
calculated as the time interval between midpoints of two successive bursts and averaged over 60-120 cycles. In these experiments, "n " is the number of animals. Intracellular recordings
were made from gray matter neurons with thin-walled microelectrodes
filled with potassium acetate (4 M). Current
injections were made in discontinuous current-clamp mode (sample rate,
1.5-2.5 kHz; band width, 0.3 kHz) with an Axoclamp 2B amplifier (Axon
Instruments, Foster City, CA). Membrane potential oscillations induced
by NMDA (150 µM) in the presence of
tetrodotoxin (TTX; 0.75-1 µM) are referred to
as NMDA-induced TTX-resistant oscillations (Wallén and Grillner,
1987). The trough potential was defined as the most negative potential
during the hyperpolarized phase. A change in trough potential was
judged to be significant if the change fulfilled two criteria: (1)
p < 0.05, unpaired t test, and (2) change
>1 mV. The detection threshold for measuring the onset and offset of
the plateau duration was set at 50% of the peak amplitude, measured
from trough potential to the peak of the oscillation. Cycle duration
was measured as duration between two successive onset points for the
plateau duration (cycle duration = plateau duration + duration of
hyperpolarized phase). The analysis of the locomotor rhythm and
NMDA-induced TTX-resistant oscillations were performed with DATA-PAC
(Run Technologies, Laguna Hills, CA). The different parameters of
NMDA-induced TTX-resistant oscillations were analyzed over 15-70
cycles in each cell, and the reported "n " is the number
of cells examined.
Cell dissociation. The spinal cord of larval lampreys
(Petromyzon marinus; n = 118) was
dissociated (El Manira and Bussières, 1997) in Leibovitz's L-15
culture medium (Sigma, St. Louis, MO) supplemented with
penicillin-streptomycin (2 µl/ml), and the osmolarity was adjusted to
270 mOsm. Before dissociation, motoneurons were retrogradely labeled by
applying fluorescein-coupled dextran amine (FDA) to the remaining
muscle tissue along the entire length of the preparation after cutting
all dorsal roots, thereby allowing the transport of the dye only
through the ventral roots to label motoneurons. Crossed caudally
projecting (CC) interneurons were labeled by injecting FDA on one side
of the spinal cord and dissociating the contralateral side of the
spinal cord rostral to the injection site. Some of these interneurons
have been shown to be part of the spinal locomotor network. After 24 hr
incubation to allow transport of the dye, the spinal cord was incubated
in collagenase (1 mg/ml, 30 min; Sigma) and then in protease (2 mg/ml,
45 min; Sigma). The tissue was subsequently washed with the culture
medium and triturated through a sterilized pipette. The supernatant
containing the dissociated cells (from two larval spinal cords) was
distributed in 10-12 Petri dishes (35 mm) and incubated at 10-12°C
for 1-7 d.
Whole-cell recordings were performed from neurons in culture using an
Axopatch 200A patch-clamp amplifier (Axon Instruments). Recordings were
done from prelabeled motoneurons (MN; n = 6), contralaterally projecting neurons (CC; n = 7), and
unidentified neurons (n = 19). Neurons were clamped at
a holding potential of  50 mV, and NMDA (200 µM) was applied for 4 sec. Data acquisition and
analysis were performed with pClamp software. The cells were perfused
through a gravity-driven six-barrel microperfusion system with the
nozzle positioned close to the recorded cell. The control solution
contained (in mM): NaCl 124, KCl 2, MgCl2 1.2, CaCl2 5, glucose
10, and HEPES 10, with pH adjusted to 7.6. For whole-cell recordings,
the pipettes were filled with a solution containing (in
mM):
KCH3SO3 102, MgCl2 1.2, CaCl2 1, EGTA
10, glucose 10, HEPES 10, ATP 2, and GTP 0.4, pH 7.6 adjusted with KOH.
Calcium imaging. The dissociated cells were incubated at
room temperature for 1-2 hr with fluo-3/AM (5 µM;
Molecular Probes, Eugene, OR) added to the medium. After removal of the
incubation medium the cells were perfused with a solution containing
(in mM): NaCl 124, KCl 2, MgCl2 1.2, CaCl2 5, glucose 10, and HEPES 10, with pH
adjusted to 7.6. Drugs were added to the perfusing solution. NMDA (200 µM) was added for 10-30 sec. DHPG was applied for 2-3
min before the subsequent application of NMDA + DHPG. The NMDA
antagonist D()-2-amino-5-phosphonopentanoic acid
(D-AP-5) blocked the NMDA-induced calcium response
(n = 5; data not shown). The effect of the following
drugs were tested on cells preincubated with the respective drug: group
I mGluR antagonist
7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester
(CPCCOEt), the protein kinase A and C inhibitor
(-1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), the protein
kinase A and G inhibitor
N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H-8),
and the calcium transport blocker/activator ryanodine. NMDA, DHPG,
CPCCOEt, and D-AP-5 were purchased from Tocris
Cookson (Bristol, UK). H-7, H-8, and ryanodine were purchased from
Research Biochemicals (Natick, MA). TTX and guanosine
5'-O-2-thiodiphosphate (GDP- -S) were purchased from
Sigma. The 488 nm line of an argon laser was used for excitation with
emission filtering passing wavelengths >515 nm. The emitted light was
visualized using a confocal laser scanner (Noran Odyssey) with 10×
(0.25 NA) or 20× (0.40 NA) objectives (Nikon, Tokyo, Japan) attached
to a Nikon Diaphot inverted microscope. Brightness over time plots were
generated by averaging the intensity within manually specified regions
of interest at a sampling rate of 7-15 Hz. Analysis of the brightness over time data were performed with pClamp6 (Axon Instruments). The
imaging experiments were typically done on several cells, in the same
field of view, on several dishes from the same dissociation, which was
done using two larval lampreys. In Table
1 the calcium imaging data are reported
with number of cells and number of dissociations from which the cells
were obtained. Changes in fluorescence ( F), which
is a measure of changed intracellular calcium concentration, were
normalized to resting fluorescence levels
(Frest) subtracted with the
fluorescence from a region not including dye-filled neurons (Fbackground). The fluorescence data
are thus presented as F/F = F/(Frest Fbackground).
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Table 1.
Effect of group I mGluR antagonist and protein kinase
activator and inhibitors on DHPG-induced potentiation of NMDA calcium
response
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Cell model. Compartmentalized Hodgkin-Huxley-type model
neurons were equipped with Na, K, Ca, and KCa
currents (Brodin et al., 1991; Ekeberg et al., 1991; Hellgren et al.,
1992; Tegnér et al., 1998). Two KCa systems
were included, one (called KCaHVA) of which is
activated by calcium influx through high voltage-activated (HVA)
calcium channels and the other (called KCaNMDA)
is activated by calcium entering through the NMDA receptor
(CaNMDA). The latter KCa
system operates with a time constant that is an order of magnitude slower (Brodin et al., 1991; Tegnér et al., 1998). To reproduce NMDA-induced TTX-resistant oscillations,
Na+ conductance was set to zero. The
lamprey spinal CPG is modeled as a population of inhibitory crossing
cells (C) inhibiting all contralateral cells and excitatory cells (E)
exciting ipsilateral C and E cells (Hellgren et al., 1992;
Hellgren-Kotaleski et al., 1999a). The NMDA calcium pool is modeled by
the following equation:
![d[<UP>CaNMDA</UP>]/<UP>dt</UP>=p(&phgr;<SUB><UP>NMDA</UP></SUB>)(E−E<SUB><UP>CaNMDA</UP></SUB>)−&dgr;<SUB><UP>NMDA</UP></SUB>[<UP>CaNMDA</UP>]](/content/vol20/issue14/fulltext/5382/img001.gif)
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(1)
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where  NMDA is the accumulation rate,
and  NMDA is the decay rate (Brodin et al.,
1991). The state variable p is the fraction of NMDA channels
in the open state (Brodin et al., 1991). When the NMDA conductance is
potentiated, there is both an increased depolarizing drive and an
increased calcium inflow.
Statistical analysis. The locomotion experiments were
analyzed with repeated measures ANOVA with post hoc Tukey's
test (rm-ANOVA; GraphPad Prism, Graphpad Software) and two-way ANOVA
(within-subjects design; JMP, SAS Institute). The repeated measurements
in the calcium-imaging experiments with calcium channel blockers did not pass the sphericity test and were thus instead analyzed using paired t tests (GraphPad Prism, Graphpad Software) with
p values adjusted according to the Bonferroni procedure (see
below). A split-plot ANOVA model (JMP, SAS Institute) was used
to determine statistical significance in the experiments comparing the
effect of DHPG on NMDA-induced calcium responses in control cells and in cells preincubated with CPCCOEt, H-7, H-8, or ryanodine. In the
model, the between-subject variable was "group" (control or preincubated), and the within-subject variable was "treatment" (NMDA or NMDA + DHPG). In all cases the main effect of "group" was
not significant whereas the main effect of "treatment" was significant. One-way ANOVA with "treatment" as within factor was performed separately for control and preincubated cells using the mean
square error term from the split-plot ANOVA. The p values were adjusted according to the Bonferroni procedure, i.e., because two
tests are done on each factor, the p values were multiplied with 2 (only applied when unadjusted p < 0.05). In
Figure 5 calcium-imaging results are presented as outlier box plots.
The ends of a box are the 25th and 75th quantiles, also called the
quartiles. The horizontal line across the middle identifies the median
sample value. The ends of the whiskers are the outermost data points from their respective quartiles that fall within the distance computed
as 1.5 *(interquartile range). Any data point that does not fall within
this distance is called an outlier and plotted as a black square.
NMDA-induced oscillations were analyzed using unpaired t
test or two-way ANOVA (within-subjects design). Results are reported as
mean ± SD.
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RESULTS |
The mGluR-induced regulation of the locomotor frequency persists in
the presence of ryanodine
To test if ryanodine-sensitive presynaptic facilitation of
synaptic transmission (Cochilla and Alford, 1998) could account for the
increase and decrease, respectively, in the locomotor frequency by DHPG
and CPCCOEt (Krieger et al., 1998), their effects were examined in the
presence of 20 µM ryanodine, which blocks the
DHPG-induced presynaptic facilitation (Cochilla and Alford, 1998). In
control experiments, DHPG (25-100 µM; applied 30-60 min) increased the frequency of NMDA-induced (100 µM)
locomotor activity from 1.80 ± 0.29 to 2.03 ± 0.33 Hz
(rm-ANOVA; p < 0.01; n = 6; Fig.
1A,B). After addition
of ryanodine (20 µM; 70-120 min), DHPG was
re-applied and again increased the locomotor frequency from 1.85 ± 0.33 to 2.06 ± 0.34 Hz (rm-ANOVA; p < 0.01;
n = 6; Fig. 1A,B). Furthermore, the
relative DHPG-induced frequency increase did not differ in the two
cases (Fig. 1C; p > 0.4; two-way ANOVA). Ryanodine alone had no consistent effect on frequency (rm-ANOVA; p > 0.05; n = 6). Application of the
mGluR antagonist CPCCOEt (100 µM; 30-65 min)
decreased the NMDA-induced (100 µM) locomotor frequency from 1.99 ± 0.41 to 1.77 ± 0.39 Hz (rm-ANOVA;
p < 0.01; n = 6; Fig.
1D). In the presence of ryanodine (20 µM; preapplied for 35-120 min) CPCCOEt still
decreased the locomotor frequency from 1.98 ± 0.47 to 1.72 ± 0.42 Hz (rm-ANOVA; p < 0.01; n = 6; Fig. 1D). The relative decrease was the same in both
cases (Fig. 1E; p > 0.5; two-way ANOVA).
Ryanodine alone had no consistent effect on frequency (rm-ANOVA;
p > 0.05; n = 6). These results thus
show that presynaptic facilitation mediated through ryanodine-sensitive calcium stores cannot explain the changes in locomotor frequency mediated by group I mGluRs. Alternative explanations were thus sought.
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Figure 1.
Ryanodine does not block the effect of group I
mGluR activation on the locomotor rhythm. A, The burst
frequency of 100 µM NMDA-induced locomotion increased
from 1.50 ± 0.22 Hz in control (a) to
1.68 ± 0.29 Hz in the presence of DHPG (25 µM; 34 min) (b). In the presence of ryanodine (20 µM), the burst frequency increased from 1.53 ± 0.20 Hz (c) to 1.78 ± 0.26 Hz with the
application of DHPG (d). B, The
time course of the DHPG-induced frequency increase from one experiment.
Ryanodine was present for 70 min before the application of DHPG Traces
in A are taken from the points indicated.
C, Summary of the effect of DHPG on the locomotor
frequency in control and in the presence of ryanodine from six
different experiments. D, The decrease in burst
frequency induced by CPCCOEt persisted in the presence of ryanodine (20 µM; applied 40 min before CPCCOEt). CPCCOEt decreased the
burst frequency from 1.78 ± 0.24 Hz to 1.47 ± 0.14 Hz in
control and from 1.71 ± 0.20 Hz to 1.46 ± 0.15 Hz in the
presence of ryanodine. C, Summary of the effect of
CPCCOEt on the locomotor frequency in control and in the presence of
ryanodine from six different experiments.
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mGluR activation increases the NMDA current through a
G-protein-mediated mechanism
Because mGluR-mediated presynaptic facilitation could not account
for the regulation of the locomotor frequency, the contribution of
postsynaptic effects was investigated by studying the interaction between mGluRs and NMDA receptors. Whole-cell patch-clamp recordings were made from isolated lamprey spinal cord neurons in culture, including MNs and crossed caudally (CC)-projecting network
interneurons. Application of NMDA (100-200 µM; 4 sec)
induced an inward current that was potentiated by DHPG (100 µM), from 596 ± 122 to 847 ± 218 pA in
magnesium-free solution (n = 17; including MN,
n = 3; CC, n = 4; p < 0.0001, paired t test) and from 241 ± 12 to 360 ± 18 pA (n = 3) in normal solution (Fig.
2A). When the cells
were loaded with GDP--S (1 mM) through the
patch pipette, the potentiation of the NMDA current was blocked (Fig.
2B; Mg-free, 605 ± 127 compared to 621 ± 120 pA; n = 12 including MN, n = 3; CC,
n = 3; p > 0.1; paired t
test; normal solution, 391 ± 202 compared to 391 ± 206 pA;
n = 2). The results were consistent in all cells tested
with no significant difference (p > 0.1;
two-way ANOVA) in the degree of potentiation between the different
types. The potentiation of NMDA current by DHPG does not appear to
depend on intracellular calcium because it was seen in the presence of
the calcium chelator EGTA (10 mM) in the
intracellular solution. These results thus show the existence of a
postsynaptic interaction between group I mGluRs and NMDA receptors that
results in a potentiation of NMDA current through a
G-protein-dependent mechanism.
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Figure 2.
mGluR activation potentiates NMDA currents through
a G-protein-dependent mechanism. A, Whole-cell recording
from an isolated lamprey spinal cord neuron clamped at a holding
potential of 50 mV. In control cells, application of NMDA (200 µM) induced an inward current that increased in the
presence of DHPG (100 µM). B, In cells
loaded with GDP--S (1 mM), the mGluR-mediated increase
of the NMDA current was blocked. The recordings shown in
A and B were made in
Mg2+-free solution. C, Bar diagram
showing the mGluR-mediated potentiation of NMDA current (percentage of
NMDA response) in control cells (n = 17) and in
cells loaded with GDP--S (n = 12).
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mGluR activation increases NMDA-induced calcium responses
To analyze how the interaction between mGluRs and NMDA receptors
affects NMDA-induced calcium responses, calcium imaging was used.
Isolated spinal cord neurons in culture were incubated with fluo-3 AM
(5 µM). Figure 3 shows a
fluo-3-labeled neuron in which application of NMDA (200 µM) induced a calcium response (Fig. 3A).
Application of DHPG (100 µM) alone induced a
transient calcium response (Fig. 3B; 0.68 ± 0.38 F/F; n = 27 of 115 cells), and potentiated the amplitude of NMDA-induced calcium responses (Fig. 3C). DHPG (50-100 µM) increased the
NMDA-induced (200 µM) calcium response from
1.34 ± 0.75 to 1.83 ± 0.88 F/F
(n = 115; p < 0.0001, paired
t test). The DHPG-induced potentiation of NMDA also occurred in cells that did not respond to DHPG alone. As the focus of the present study was on the interaction with NMDA receptors, the DHPG-induced calcium response was not investigated further in the
present study.
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Figure 3.
Activation of group I mGluRs potentiates
NMDA-induced calcium responses. A, Bath application of
NMDA (10 sec; 200 µM) induced a fluorescence increase,
calculated as F/F from the neuron
illustrated below. B, Application of DHPG (100 µM) induced a transient fluorescence increase.
C, When NMDA was reapplied in the presence of DHPG, the
effect of NMDA was potentiated. The images were obtained from the peak
fluorescence created from an average of 30 images with the resting
fluorescence subtracted.
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The NMDA-induced calcium response was measured in the presence of L-
and N-type calcium channel blockers to assess the contribution of these
channels to the potentiation of the NMDA response by DHPG. As above,
application of NMDA in the presence of DHPG increased the calcium
response from 1.45 ± 0.60 to 1.86 ± 0.70 F/F (n = 13; p < 0.0005; paired t test). Blockade of N- and L-type calcium channels by  -conotoxin-GVIA and nimodipine reduced the NMDA-induced calcium response to 0.57 ± 0.25 F/F
(n = 13; p < 0.0005; paired t test). In the presence of the calcium channel blockers,
DHPG increased the calcium response to 0.76 ± 0.37 (F/F;
n = 13; p < 0.05; paired t
test). These results show that N-type and L-type calcium channels
contribute to the calcium response elicited by the application of NMDA,
but that the potentiation of the calcium response by DHPG still
persists after blockade of these channels.
The effect of DHPG was tested on saturating NMDA concentrations to test
if the mGluR-induced potentiation of the NMDA response is different
from simply increasing the NMDA concentration. The dose-response curve
of the NMDA-induced calcium response shows that the maximum response
was reached at an NMDA concentration of 400 µM (one-way
ANOVA; Fig. 4A). DHPG
(100 µM) was still able to potentiate the NMDA
response induced by a saturating concentration (800 µM; Fig. 4B), from 1.72 ± 0.82 F/F to 2.16 ± 0.92 F/F (n = 23 cells;
p < 0.02; paired t test).
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Figure 4.
DHPG potentiates calcium responses to saturating
concentrations of NMDA. A, Dose-response curve of the
calcium responses to increasing concentrations of NMDA. The maximum
response was elicited by 400 µM NMDA (one-way ANOVA).
Each point corresponds to mean ± SD (n = 5-39 cells). B, Application of NMDA (800 µM, 10 sec) induced a transient fluorescence increase,
which was potentiated by 100 µM DHPG.
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The interaction between mGluRs and NMDA receptors is blocked by an
mGluR antagonist but not by protein kinase A, C, and G inhibitors
To determine if the potentiation of the NMDA-induced calcium
response is mediated through activation of group I mGluRs, the specific
group I mGluR antagonist CPCCOEt was used. The experiments were
performed on two parallel series of cultured cells from the same
dissociation; one used as control, and the other preincubated with
CPCCOEt (100 µM; 30 min). In control applications, DHPG
increased the NMDA-induced calcium response from 1.17 ± 0.60 F/F to 1.71 ± 0.89 F/F (n = 34; p < 0.05; Fig. 5A; Table 1). In
cells preincubated with CPCCOEt, application of DHPG did not increase
the NMDA-induced calcium response (NMDA, 1.47 ± 0.71 F/F; NMDA + DHPG, 1.57 ± 0.68 F/F; n = 34; p > 0.05; Fig. 5A; Table 1).
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Figure 5.
Effect of an mGluR antagonist and modulators of
second messenger pathways on the mGluR-induced potentiation of NMDA
receptors. Outlier box plots showing the amplitude of NMDA-induced
calcium responses measured as F/F.
A, The mGluR antagonist CPCCOEt blocked the DHPG-induced
potentiation of NMDA-induced calcium response. B, The
potentiation persisted in the presence of the protein kinase inhibitor
H-7. C, The potentiation persisted in the presence of
the protein kinase inhibitor H-8. D, Ryanodine had no
effect on the potentiation of NMDA response by DHPG.
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In some preparations the potentiation of NMDA receptors by group I
mGluRs has been suggested to be mediated by protein kinase C (Kelso,
1992). To determine if a similar mechanism is responsible for the
potentiation seen in lamprey spinal cord neurons, the effect of DHPG
was tested in the presence of the protein kinase A and C inhibitor H-7.
DHPG significantly increased the NMDA-induced calcium response in both
control cells and cells preincubated with H-7 (Fig. 5B,
Table 1). We also tested H-8, an inhibitor of protein kinase A and G,
and ryanodine, which interacts with intracellular calcium stores. The
potentiation of the NMDA response by DHPG was unaffected by both H-8
and ryanodine (Fig. 5C,D, Table 1). These results
indicate that the interaction between group I mGluRs and NMDA receptors
is mediated through mechanisms independent of protein kinase A, C, and
G, and ryanodine-sensitive calcium stores.
mGluR activation modulates NMDA-induced
TTX-resistant oscillations
We tested the effect of DHPG on NMDA-induced oscillations known to
occur during fictive locomotion (Sigvardt et al., 1985; Wallén
and Grillner, 1987). Modulation of these oscillations can provide
explanations for changes in the locomotor frequency (Tegnér et
al., 1998). Because the shape of the NMDA-induced TTX-resistant
oscillations is voltage-dependent, the effect of DHPG was studied on
individual cells over a range of trough potentials (see Materials and
Methods) adjusted by current injection (Fig. 6A). Cycle duration,
plateau duration, and the duration of the hyperpolarized phase were
analyzed (see Materials and Methods). Comparisons between cells were
made by taking the most hyperpolarized (range, 84 to 60 mV) and
depolarized (range, 74 to 54 mV) trough potential for each
individual cell. In all cases DHPG increased the plateau duration
(p < 0.05; unpaired t test for each
cell; n = 5) at both hyperpolarized (Fig.
6A,B; mean duration increased from 379 ± 155 to
950 ± 290 msec) and depolarized trough potentials (Fig.
6A,B; mean duration increased from 691 ± 369 to
3065 ± 1692 msec). In four of these cells, the relative
increase was larger (p < 0.05; two-way ANOVA)
at depolarized (379 ± 236%) than at hyperpolarized trough
potentials (149 ± 129%). The duration of the hyperpolarized
phase decreased (Fig. 6A,C) in all cells at hyperpolarized trough potentials (mean duration decrease from 2809 ± 1280 to 2015 ± 619 msec; n = 5;
p < 0.05, unpaired t test for each cell),
and in three of five cells at depolarized trough potentials (mean
duration decrease from 1596 ± 649 to 1229 ± 367 msec;
n = 3; p < 0.05; unpaired t
test for each cell). The effect of DHPG on cycle duration was dependent
on trough potential (Fig. 6D). DHPG decreased the
cycle duration at hyperpolarized trough potentials because of the
larger decrease of the hyperpolarized phase compared to the increase of
the plateau duration (Fig. 6B-D). in contrast, DHPG
increased the cycle duration at depolarized trough potentials because
of the larger increase of the plateau duration than the decrease of the
hyperpolarized phase (Fig. 6B-D; see
Discussion).
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Figure 6.
Effect of DHPG on NMDA-induced TTX-resistant
oscillations. A, Application of NMDA (150 µM) in the presence of TTX (1 µM) elicits
membrane potential oscillations (thin traces).
Activation of mGluRs by DHPG (50 µM, thick
traces) increased the plateau duration and decreased the
duration of the hyperpolarized phase. In the presence of DHPG, the
cycle duration was decreased at a trough potential of 80 mV, but was
increased at a trough potential of 74 mV. B, Effect of
DHPG on plateau duration at different trough potentials.
C, DHPG decreased the duration of the hyperpolarized
phase at different trough potentials. D, DHPG had
different effects on the cycle duration depending on trough potential.
The data presented in B-D are from the same neuron as
in A.
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|
Application of DHPG did not affect the trough membrane potential in
nine neurons, induced a hyperpolarization in eight neurons (mean,
2.0 ± 0.6 mV; range, 1.2-2.8 mV), and a depolarization in two
neurons (1.4-2 mV). In the nine neurons in which the trough potential
was unchanged, DHPG significantly (p < 0.05;
unpaired t test) increased the plateau duration (in control:
median, 317 msec; range, 172-5251 msec; in DHPG: median, 842 msec;
range, 379-23,940 msec). In all of these neurons DHPG also decreased the duration of the hyperpolarized phase (in control: median, 2116 msec; range, 777-3688 msec; in DHPG: median, 2036 msec; range, 712-2630 msec). The cycle duration was increased
(p < 0.05 unpaired t test) in six
neurons, decreased in two neurons (p < 0.05), and unchanged in one neuron (p > 0.05;
unpaired t test). The variation in the effects on period is
to be expected from the correlation between change of period and trough
potential (Fig. 6D).
The DHPG-induced increase of the plateau duration was counteracted by
the mGluR antagonist CPCCOEt. DHPG (20-50 µM; applied 4-8 min) increased the plateau duration from 322 ± 142 to
1080 ± 625 msec (n = 6; Fig.
7A,B). The DHPG-induced
increase of the plateau duration was significantly
(p < 0.05; two-way ANOVA) reduced by
application of CPCCOEt (100 µM; 323 ± 190 compared to 517 ± 379 msec; n = 6; Fig.
7A,B). In three neurons, DHPG was re-applied after washout
of CPCCOEt and again increased the plateau duration from 372 ± 101 to 1103 ± 530 msec (n = 3; Fig.
7B). This increase was significantly larger
(p < 0.05; two-way ANOVA) than in CPCCOEt. The
increase in plateau duration by DHPG (20-50
µM; applied 4-7 min) persisted in the presence
of the protein kinase inhibitor H-7 (10 µM;
Parker et al., 1997; applied for 30-40 min before test with H-7 + DHPG; n = 4; p < 0.05; paired
t test). These results show that, like its effect on
NMDA-induced calcium responses (Fig. 5A,B), the effects of
DHPG on NMDA-induced oscillations were blocked by the group I
mGluR antagonist but persisted after inhibition of protein kinase
C.
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|
Figure 7.
The modulation of NMDA-induced oscillations by
DHPG is counteracted by a group I mGluR antagonist. A,
Application of DHPG increased the plateau duration and decreased the
frequency of the NMDA-induced oscillations. These effects were blocked
by the group I mGluR antagonist CPCCOEt (100 µM).
B, Summary of the effect of DHPG on the plateau duration
of the oscillations before (n = 6), during
(n = 6), and after (n = 3)
application of CPCCOEt.
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|
Simulations of mGluR effects on cellular and network behavior
The experimental results show that activation of mGluRs modulates
NMDA-induced currents, calcium responses, and membrane potential oscillations in spinal cord neurons. To determine if these effects can
account for the mGluR-mediated modulation of the locomotor frequency,
the potentiation of NMDA receptors by mGluR activation was included in
a cell model previously developed to analyze NMDA-induced TTX-resistant
oscillations and locomotor network activity (Brodin et al., 1991;
Ekeberg et al., 1991; Hellgren et al., 1992; Wallén et al., 1992;
Tegnér et al., 1998). In Figure 8,
the thin curve represents the control situation, and the thick curve
represents the potentiation of the NMDA conductance, which results in a
proportional increase of calcium influx through NMDA channels
(CaNMDA). We first tested if the model could
reproduce the experimentally obtained effects of DHPG on NMDA-induced
oscillations (Fig. 6). The effect of a potentiation of the NMDA
responses on the simulated membrane potential oscillations was tested
at both a relatively hyperpolarized (Fig. 8A) and a
depolarized trough potential (Fig. 8B), adjusted by a
simulated constant current injection. Potentiation of the NMDA
conductance consists of increased CaNMDA and
depolarizing drive (Fig. 8C,D). Increasing
CaNMDA thus causes a larger activation of
KCa channels, which leads to a more
hyperpolarized trough potential (data not shown). More positive current
was therefore added to achieve the same trough potential as in control
recordings (Fig. 8). At both hyperpolarized (Fig.
8A,E) and depolarized (Fig. 8B,E) trough potentials, the plateau duration increased, whereas the duration
of the hyperpolarized phase decreased (Fig.
8A,B,F). Note that the relative and absolute
decrease of the hyperpolarized phase is larger at more hyperpolarized
trough potentials (Fig. 8A,F), whereas the
relative and absolute increase of the duration of the plateau phase is
larger at more depolarized trough potentials (Fig.
8B,E). The cycle duration decreased at hyperpolarized
trough potentials (Fig. 8A,G), but increased at
depolarized trough potentials (Fig. 8B,G),
corresponding to the experimental findings (Fig. 6).
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Figure 8.
Simulation of the effect of mGluR activation on
NMDA-induced TTX-resistant oscillations. A, Effect of
potentiating NMDA receptors on membrane potential oscillations is shown
in an excitatory neuron. Trough potentials are adjusted by current
injection. In control (thin trace) current is zero, and
with NMDA potentiation, current is 0.025 nA. Potentiation of NMDA
receptors increases the plateau duration, decreases the duration of the
hyperpolarized phase, and decreases cycle duration. B,
At a more depolarized trough potential (0.1 nA; thin
trace), potentiation of NMDA (0.145 nA; thick
trace) again increases the plateau duration and decreases the
duration of the hyperpolarized phase, but instead increases the cycle
duration. C, D, Potentiation of NMDA
receptors [0.1 arbitrary unit (AU)] increases the basal
CaNMDA level. This leads to a slower calcium accumulation,
which increases the plateau duration, and a faster decay, which
decreases the duration of the hyperpolarized phase. E,
Effect of potentiation of NMDA receptors on plateau duration at
different trough potentials. F, The duration of the
hyperpolarized phase decreased at different trough potentials.
G, Potentiation of NMDA receptors had different effects
on the cycle duration depending on the trough potential.
|
|
These results can be understood on the basis of
CaNMDA dynamics (Fig. 8C,D). Calcium
accumulation activates KCa channels, which
terminates the plateau phase and determines the duration of the
hyperpolarized phase (Brodin et al., 1991; Tegnér et al., 1998).
When the NMDA conductance is potentiated (Fig. 8C,D, thick traces), the baseline level of CaNMDA
increases, resulting in a change in calcium dynamics. The accumulation
of calcium becomes slower, whereas the decay becomes faster (Fig.
8C,D). The potentiation of the NMDA conductance delays the
plateau termination because more calcium is necessary to evoke a
sufficient KCa current to overcome the increased
depolarizing drive. The change in CaNMDA kinetics
can thus explain both the increased plateau duration and decreased
duration of the hyperpolarized phase, as well as the effects on cycle
duration (see Discussion; Ekeberg et al., 1991; Tegnér et al.,
1998; Hellgren-Kotaleski et al., 1999b).
Because the results of simulations of the potentiation of NMDA
responses were similar to those obtained experimentally, we next tested
if this potentiation is sufficient to reproduce the group I
mGluR-mediated increase in the locomotor frequency. The mGluR-mediated
increase in burst frequency (Krieger et al., 1998), was also reproduced
in the network model of the lamprey locomotor CPG by potentiating the
NMDA conductance (Hellgren et al., 1992; Hellgren-Kotaleski et al.,
1999b). In a control simulation of the locomotor network (Fig.
9A), activation of NMDA
receptors elicited rhythmic alternating activity. When NMDA receptors
were potentiated on only one side (Fig. 9B), the potentiated
side showed a prolonged burst duration and a shortened interburst
interval, as predicted from the effects on the single cell level.
Potentiation of NMDA receptors on both sides increased the frequency
(Fig. 9C; see also Kepler et al., 1990; Skinner et al.,
1994, Hellgren-Kotaleski et al., 1999a).
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Figure 9.
Simulation of the effect of mGluR potentiation of
NMDA receptors on the locomotor network. A, The network
of a population of excitatory and crossed inhibitory neurons
(left panel) is activated by NMDA (0.45 AU) and
AMPA/kainate (3 AU). Alternating rhythmic locomotor activity is shown
for one left and one right excitatory neuron (right
panel). B, Potentiation of the NMDA
receptors on the left side by 30% shortens the interburst interval
because the potentiation of NMDA receptors leads to an earlier
"escape" from contralateral inhibition. The burst duration is
prolonged because the NMDA potentiation produces a more effective
inhibition of the contralateral side, resulting in later escape from
inhibition. C, Potentiation of both sides (30%) results
in an increased burst frequency of the model network. This is because
the oscillatory mechanism is dominated by an escape mechanism in which
the changes in the duration of the interburst intervals decides the
outcome.
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DISCUSSION |
Interaction between group I mGluRs and NMDA receptors
These results show that activation of postsynaptic group I mGluRs
potentiates NMDA-induced currents through a G-protein-dependent mechanism in a variety of spinal neurons, including motoneurons and
crossed caudally projecting interneurons. The NMDA-induced calcium
response is also potentiated by activation of group I mGluRs. The
interaction between group I mGluRs and NMDA receptors results in a
profound modulation of NMDA-induced TTX-resistant membrane potential
oscillations. The effects of DHPG on NMDA-induced calcium responses,
membrane oscillations, and the locomotor rhythm were all blocked by
CPCCOEt. These results thus indicate that activation of group I mGluRs
potentiates NMDA responses through a postsynaptic interaction. Can this
interaction explain the increase in locomotor frequency induced by
DHPG? In addition to potentiating NMDA receptors, activation of group I
mGluRs mediates presynaptic facilitation of reticulospinal transmission
through a ryanodine-sensitive mechanism (Cochilla and Alford, 1998).
The latter effect could also lead to an increase in the frequency of
the locomotor activity. If this were the case it would be
expected that ryanodine should block, at least partially, the increase
in frequency induced by DHPG. Our results show that ryanodine had no
effect on either the increased frequency induced by DHPG or the
decreased frequency after blockade of endogenously activated group I
mGluRs by CPCCOEt. Ryanodine-sensitive presynaptic facilitation thus
does not seem to be involved in the regulation of locomotor network
activity by group I mGluRs. Although other mechanisms (e.g., calcium
release from internal stores or potentiation of other receptors and ion channels) can be involved in regulating locomotor activity, our results
suggest that the postsynaptic interaction between group I mGluRs and
NMDA receptors can account for this regulation.
mGluR-mediated potentiation of cellular responses to NMDA
The potentiation of NMDA receptors by the group I mGluR agonist
DHPG was blocked by the antagonist CPCCOEt and GDP--S, which causes
an irreversible inactivation of G-proteins, arguing for a specific
effect on mGluRs. Measuring changes in calcium fluorescence showed that
NMDA increased intracellular calcium concentrations. The source of the
NMDA-induced calcium increase is in part caused by the activation of L-
and N-type calcium channels evoked by the depolarization of the neuron.
The remaining part could be attributable to calcium influx through NMDA
channels and/or activation of calcium channels insensitive to the L-
and N-type blockers (El Manira and Bussières, 1997). DHPG could
potentiate calcium responses induced by saturating NMDA concentrations,
suggesting that the interaction between mGluRs and NMDA receptors
changes the properties of the receptor in a way that cannot be achieved by an increased NMDA concentration. NMDA-induced calcium responses were
similar in amplitude in control cells compared to cells preincubated with ryanodine (Table 1), suggesting that ryanodine-sensitive calcium
stores are not involved. The mGluR-mediated potentiation persists in
the presence of protein kinase A, C, and G inhibitors. mGluR-mediated
potentiation of the NMDA current has been shown previously in mammalian
hippocampus (Aniksztejn et al., 1991; Harvey and Collingridge, 1993;
Fitzjohn et al., 1996), spinal dorsal horn neurons (Bleakman et al.,
1992), and other preparations (Kelso, 1992; Collins, 1993; Rahmann and
Neuman, 1996; Wang and Daw, 1996). The signal transduction mechanism
involved in the mGluR-mediated potentiation differs among preparations,
with protein kinase C being involved in the effects of mGluR expressed
in Xenopus oocytes (Kelso, 1992), but with evidence both for
(Aniksztejn et al., 1991) and against (Harvey and Collingridge, 1993)
protein kinase C in hippocampus. Furthermore, results of a recent study have suggested that the group I mGluR agonist DHPG (or a glycine contamination) can act as a coagonist at the glycine site of NMDA receptors and thus potentiate NMDA responses (Contractor et al., 1998).
Our results show that the potentiation of NMDA responses by DHPG is
blocked by the antagonist CPCCOEt and by intracellular injection of
GDP--S. Thus, in the lamprey spinal cord neurons the potentiation of
NMDA receptors by DHPG is mediated through a direct effect of this
agonist on mGluRs rather than on NMDA receptors and that this
interaction is not mediated through activation of protein kinase C.
Mechanism accounting for the mGluR-mediated regulation of
burst frequency
The spinal neuronal network underlying locomotion in the lamprey
can be activated by glutamate or the subtype-specific agonists NMDA,
AMPA, and kainate (Grillner et al., 1998). NMDA also causes membrane
potential oscillations that persist in the presence of TTX (Sigvardt et
al., 1985; Wallén and Grillner, 1987). These oscillations are
thought to contribute to fictive locomotion because their blockade by
perfusion of magnesium-free solution results in weak and less regular
ventral root bursts (Wallén and Grillner, 1987). Furthermore,
NMDA-induced TTX-resistant oscillations have similar amplitude and
frequency to the locomotor oscillatory activity. The oscillations
consist of a rapid depolarization caused by the opening of NMDA
channels, followed by a plateau phase at which voltage-dependent
K+ channels balance the NMDA current. A
gradual increase of KCa current, activated by
calcium influx through NMDA (CaNMDA) and HVA
calcium channels, leads to a rapid repolarization of the membrane potential. The duration of the hyperpolarized phase that follows is
determined by the decay of calcium, which activates
KCa currents (Wallén and Grillner, 1985,
1987; Brodin et al., 1991; Tegnér et al., 1998). The accumulation
and decay rates of CaNMDA are relatively slower
than for the HVA calcium (see Materials and Methods), which more
closely follows the membrane potential. Therefore the level of
CaNMDA tends to decide the duration of the
hyperpolarized phase. Because the properties of the NMDA-induced
TTX-resistant oscillations will change the locomotor burst frequency
(Brodin et al., 1991; Wallén et al., 1992), a modulation of these
oscillations is a possible mechanism for the mGluR-mediated burst
frequency increase. mGluR activation results in an increased plateau
duration, no effect or a decrease in the duration of the hyperpolarized phase, and a membrane potential-dependent change in the frequency (Fig.
6). These results were reproduced in simulations in which calcium
accumulation and decay determines the duration of the plateau and
hyperpolarized phases. In the simulations, the accumulation and decay
of CaNMDA depended on the initial
CaNMDA level. A potentiation of NMDA receptors at
both relatively hyperpolarized (Fig. 8C) and depolarized
trough potentials (Fig. 8D) increases the basal CaNMDA level. The higher the baseline
CaNMDA level, the slower the accumulation and the
faster the decay of calcium. Activation of KCa
channels will terminate the plateau phase through a closure of
voltage-dependent NMDA channels, and thus with a slower calcium accumulation the plateau duration will increase. The duration of the
hyperpolarized phase is also determined by KCa
channels. The faster calcium decay, in combination with the increased
depolarizing drive caused by the NMDA potentiation, leads to a
decreased duration of the hyperpolarized phase. Both experimental and
simulation data show that the oscillation cycle duration is dependent
on the trough potential. At hyperpolarized trough potentials, there is
a more pronounced decrease of the hyperpolarized phase, relative to the
increase of the plateau duration, resulting in an increase in the
frequency. At more depolarized trough potentials the reverse is true.
The network effect of mGluR activation with an increased burst
frequency can thus be accounted for by the cellular effects observed.
| |
FOOTNOTES |
Received Dec. 2, 1999; revised April 14, 2000; accepted April 27, 2000.
This work was supported by the Swedish Medical Research Council
(project 11562), the Swedish Foundation for Strategic Research, Erikssons Stiftelse, Jeanssons Stiftelse, Wiberg Stiftelse, and Karolinska Institute funds. P. Krieger received a fellowship from Knut
and Alice Wallenbergs Stiftelse. We thank Drs. L. Brodin, S. Grillner,
and D. Parker for their comments on this manuscript. We are also
grateful to H. Axelgren and M. Bredmyr for excellent technical assistance.
Correspondence should be addressed to P. Krieger, Nobel Institute for
Neurophysiology, Department of Neuroscience, Karolinska Institute,
S-171 77 Stockholm, Sweden. E-mail: Patrik.Krieger{at}neuro.ki.se
| |
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J. Neurosci.,
November 14, 2007;
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[Abstract]
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H. Huang and A. N. van den Pol
Rapid Direct Excitation and Long-Lasting Enhancement of NMDA Response by Group I Metabotropic Glutamate Receptor Activation of Hypothalamic Melanin-Concentrating Hormone Neurons
J. Neurosci.,
October 24, 2007;
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M. Diaz-Rios, D. A. Dombeck, W. W. Webb, and R. M. Harris-Warrick
Serotonin Modulates Dendritic Calcium Influx in Commissural Interneurons in the Mouse Spinal Locomotor Network
J Neurophysiol,
October 1, 2007;
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R. Levi and A. I. Selverston
Mechanisms Underlying Type I mGluR-Induced Activation of Lobster Gastric Mill Neurons
J Neurophysiol,
December 1, 2006;
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[Abstract]
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G. J. Liu, A. Kalous, E. L. Werry, and M. R. Bennett
Purine Release from Spinal Cord Microglia after Elevation of Calcium by Glutamate
Mol. Pharmacol.,
September 1, 2006;
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[Abstract]
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A. Nistri, K. Ostroumov, E. Sharifullina, and G. Taccola
Tuning and playing a motor rhythm: how metabotropic glutamate receptors orchestrate generation of motor patterns in the mammalian central nervous system
J. Physiol.,
April 15, 2006;
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P. Kettunen, D. Hess, and A. E. Manira
mGluR1, But Not mGluR5, Mediates Depolarization of Spinal Cord Neurons by Blocking a Leak Current
J Neurophysiol,
October 1, 2003;
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S. Alford, E. Schwartz, and G. V. Di Prisco
The Pharmacology of Vertebrate Spinal Central Pattern Generators
Neuroscientist,
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[Abstract]
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L. Mao and J. Q. Wang
Glutamate Cascade to cAMP Response Element-Binding Protein Phosphorylation in Cultured Striatal Neurons through Calcium-Coupled Group I Metabotropic Glutamate Receptors
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September 1, 2002;
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V. Heidinger, P. Manzerra, X. Q. Wang, U. Strasser, S.-P. Yu, D. W. Choi, and M. M. Behrens
Metabotropic Glutamate Receptor 1-Induced Upregulation of NMDA Receptor Current: Mediation through the Pyk2/Src-Family Kinase Pathway in Cortical Neurons
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July 1, 2002;
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G.-Y. Hu, Z. Biro, R. H. Hill, and S. Grillner
Intracellular QX-314 Causes Depression of Membrane Potential Oscillations in Lamprey Spinal Neurons During Fictive Locomotion
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June 1, 2002;
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P. Koulen and J. H. Brandstatter
Pre- and Postsynaptic Sites of Action of mGluR8a in the Mammalian Retina
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[Abstract]
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M. Takahashi and S. Alford
The Requirement of Presynaptic Metabotropic Glutamate Receptors for the Maintenance of Locomotion
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P. Kettunen, P. Krieger, D. Hess, and A. El Manira
Signaling Mechanisms of Metabotropic Glutamate Receptor 5 Subtype and Its Endogenous Role in a Locomotor Network
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March 1, 2002;
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X.-T. Jin, C. J. Beaver, Q. Ji, and N. W. Daw
Effect of the Group I Metabotropic Glutamate Agonist DHPG on the Visual Cortex
J Neurophysiol,
October 1, 2001;
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J.-y. Lan, V. A. Skeberdis, T. Jover, X. Zheng, M. V. L. Bennett, and R. S. Zukin
Activation of Metabotropic Glutamate Receptor 1 Accelerates NMDA Receptor Trafficking
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S. Grillner, P. Wallen, R. Hill, L. Cangiano, and A. E. Manira
Ion channels of importance for the locomotor pattern generation in the lamprey brainstem-spinal cord
J. Physiol.,
May 15, 2001;
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V. Neugebauer, P.-S. Chen, and W. D. Willis
Groups II and III Metabotropic Glutamate Receptors Differentially Modulate Brief and Prolonged Nociception in Primate STT Cells
J Neurophysiol,
December 1, 2000;
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TOP
ABSTRACT
INTRODUCTION
