1Department of Biological Sciences, University of Illinois at Chicago, Chicago 60607; and 2Department of Physiology, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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Schwartz, Neil E. and Simon Alford. Physiological Activation of Presynaptic Metabotropic Glutamate Receptors Increases Intracellular Calcium and Glutamate Release. J. Neurophysiol. 84: 415-427, 2000. Activation of metabotropic glutamate receptors (mGluRs) has diverse effects on the functioning of vertebrate synapses. The cellular mechanisms that underlie these changes, however, are largely unknown. The role of presynaptic mGluRs in modulating Ca2+ dynamics and regulating neurotransmitter release was investigated at the vestibulospinal-reticulospinal (VS-RS) synapse in the lamprey brain stem. Application of the specific Group I mGluRs antagonist 7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) reduced the amplitude of consecutive high-frequency evoked excitatory postsynaptic currents (EPSCs). A series of experiments using techniques of electrophysiology and calcium imaging were carried out to determine the cellular mechanisms by which this phenomenon occurs. Concentration-dependent increases in the pre- and postsynaptic [Ca2+]i were seen with the application of mGluR agonists. Similarly, high-frequency stimulation of axons caused a Group I mGluR-dependent enhancement in presynaptic Ca2+ transients. Application of mGluR agonist caused a depolarization of the presynaptic elements, while thapsigargin decreased the high-frequency stimulus- and agonist-induced rises in [Ca2+]i. These data suggest that both membrane depolarization and the release of Ca2+ from intracellular stores potentially play a role in mGluR-induced Ca2+ signaling. To determine the effect of this modulation of Ca2+ dynamics on spontaneous glutamate release, miniature EPSCs were recorded from postsynaptic reticulospinal neurons. A potent Group I mGluR agonist, (S)-homoquisqualic acid, caused a large increase in the frequency of events. These results demonstrate the presence of presynaptic Group I mGluRs at the VS-RS synapse. Activation of these receptors leads to a rise in [Ca2+]i and enhances the spontaneous and evoked release of glutamate. Taken together, these studies highlight the importance of synaptic activation of these facilitatory autoreceptors in both short-term plasticity and synaptic transmission.
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INTRODUCTION |
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Synaptic strength may be altered by receptor
activation at both pre- and postsynaptic elements. Clearly modulation
of neurotransmitter release is an important mechanism for altering
synaptic efficacy. The small size of the mammalian terminal, however,
has made the direct study of presynaptic cellular mechanisms difficult
in intact preparations. Modulation of release has implications for the
rapid fluctuations in neuronal output that occur with short-term
plasticity (Zucker 1989) as well as for adaptive changes
that take place over longer time scales (Bliss and Collingridge
1993
). Experiments at the vertebrate and invertebrate
neuromuscular junction have shown that Ca2+ entry
from prior action potentials can affect the amount of neurotransmitter released with subsequent spikes (Kamiya and Zucker 1994
;
Katz and Miledi 1968
). Less is known, however, about the
effect that receptor-mediated processes (e.g., autoreceptors) have on
Ca2+ dynamics and transmitter release
particularly at central glutamatergic synapses. Until recently,
autoreceptors were thought to function in only a negative feedback
capacity; it now seems clear, however, that various presynaptic
receptors are able to facilitate transmitter release as well
(Cochilla and Alford 1998
, 1999
; Langer
1997
; Liou et al. 1996
; Miller
1998
). Synaptosomal studies have demonstrated that activation
of metabotropic glutamate receptors (mGluRs) can lead to an enhancement
of transmitter exocytosis (Herrero et al. 1998
).
The amino acid glutamate is the neurotransmitter at
the majority of excitatory synapses in the vertebrate CNS (Mayer
and Westbrook 1987). Glutamate mediates fast synaptic
transmission by binding to ionotropic receptors of both
N-methyl-D-aspartic acid (NMDA) and non-NMDA
subtypes (Collingridge and Lester 1989
). In addition, glutamate acts on metabotropic glutamate receptors that are linked to
various intracellular effector systems. At least eight distinct mGluR
subtypes have been characterized to date (Pin and Duvoisin 1995
; Schoepp and Conn 1993
); these have been
separated into three groups based on sequence homology, transduction
mechanisms, and to some extent, their pharmacological profiles.
Activation of Group I mGluRs (mGluR1 and mGluR5) results in the
production of inositol 1,4,5-trisphosphate
(InsP3), which, by binding to its receptor on the
endoplasmic reticulum, causes the liberation of Ca2+ from internal stores (Anwyl
1995
; Murphy and Miller 1988
). This mechanism
has been demonstrated to play a physiological role at the terminals of
giant axons in the lamprey spinal cord (Cochilla and Alford
1998
)
Lamprey reticulospinal neurons of the posterior and medial
rhombencephalic reticular nuclei (PRRN and MRRN, respectively) provide
descending excitatory inputs to the spinal cord (Buchanan et al.
1987; Ohta and Grillner 1989
). Vestibulospinal
axons from two vestibular nuclei, the nucleus octavomotorii
intermediate (nOMI) and posterior (nOMP), make en passant synapses onto
the dendrites of reticulospinal neurons and contain a chain of
presynaptic elements along their length (Rovainen 1979
;
Shupliakov et al. 1992
). The
vestibulospinal-reticulospinal (VS-RS) synapse is glutamatergic, and
the fast synaptic response comprises AMPA, NMDA, and electrical components (Alford and Dubuc 1993
). The unmyelinated
axons of the vertebrate lamprey are large, and the tissue is optically clear, providing experimental access to presynaptic elements for both
electrophysiological recordings and imaging experiments with Ca2+-sensitive dyes. Here we demonstrate the
presence, properties, and likely mechanisms used by presynaptic Group I
mGluRs in regulating Ca2+ dynamics and glutamate
release. The physiological activation of these receptors has
implications for both short- and long-term plasticity.
Parts of this paper have been published in abstract form
(Schwartz and Alford 1997).
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METHODS |
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In vitro lamprey brain stem preparation
Experiments were performed on the isolated brains of larval
(ammocoete) lampreys, Petromyzon marinus, in accordance with
all local and national guidelines. The animals were anesthetized with 3-aminobenzoic acid ethyl ester methanesulfonate salt (MS-222; 100 mg/l; Sigma Chemical, St. Louis, MO), decapitated and dissected in a
cold saline solution (Ringer) of the following composition (in mM):
100.0 NaCl, 26.0 NaHCO3, 4.0 glucose, 2.6 CaCl2, 2.1 KCl, 1.8 MgCl2,
bubbled with 95%O2-5% CO2
to a pH of 7.4 (modified from Wickelgren 1977). The
skin, musculature, braincase, and choroid plexus were removed; the obex
was opened and the optic tectum/cerebellum cut, fully exposing the
basal and alar plates of the fourth ventricle. For experiments that
required imaging, the tissue was placed dorsal surface upward in a
cooled ~500-µl chamber with a glass coverslip floor that was
inserted onto the stage of an inverted Nikon Diaphot microscope. For
experiments that required only electrophysiology, the tissue was pinned
dorsal surface upward in a cooled ~1-ml recording chamber with a
silicone elastomer (Sylgard) floor. In both cases, the preparations
were continuously superfused with cold oxygenated Ringer solution
(8-10°C) or solutions of pharmacological agents bath-applied at a
rate of approximately 1 ml/min.
Microfluorimetry
Dye filling of the brain stem was achieved by the application of
the dextran-amine-conjugates of Ca2+-sensitive
dyes (McClellan et al. 1994) [3,000 or 10,000 M
Calcium Green-1 or 10,000 M
Oregon Green 488 BAPTA-1; 5 mM; Molecular Probes, Eugene, OR]. Segregation of
vestibulospinal and reticulospinal tracts in the spinal cord enabled
the selective labeling of either presynaptic axons or postsynaptic
somata and dendrites. This technique was used to avoid
contamination of presynaptic Ca2+
transients by postsynaptic Ca2+ signals and
vice versa. The details of this have been previously described (Schwartz and Alford 1998
). Preparations
were superfused with cold oxygenated Ringer solution throughout the 16- to 22-h labeling period.
Imaging was accomplished with a confocal laser scanning microscope
(Bio-Rad MRC-600, Bio-Rad, Hercules, CA) using the 488-nm line of an
argon ion laser as the excitor source. A fluorescein filter set was
used with a ×20 (0.75 NA) objective. For experiments in which agonists
were washed into the bath on retrogradely filled preparations, images
were acquired once per 15 s (at 0.067 Hz) for control periods,
once per 30 s (at 0.033 Hz) for washout periods, and once per
5 s (at 0.2 Hz) during the drug application. For similar
experiments on single patch-clamped cells, images were acquired at
0.067 Hz during control and 0.2 Hz during drug application and washout.
For those studies that explored the modulation of synaptically
activated Ca2+ transients, imaging of axons
and/or dendrites was carried out with a series of 16 frames (at 2 Hz)
acquired before, during, and after a stimulus. Extracellular
stimulations (0.5-20.0 µA) were given with tungsten electrodes
(impedance = 0.5-2.0 M) activated via a stimulus isolation
unit. Three-dimensional reconstruction of image slices from multiple
z planes (at 1-µm intervals) was performed with a Silicon
Graphics Indigo 2 workstation using VoxelView software (Vital Images,
Minneapolis, MN).
Image analysis was performed on a Macintosh computer using NIH Image
software. Identifiable axons or dendrites were selected and an
automated search macro was used to analyze the identically sized region
in all image frames of the trial; the macro optimized the mean
intensity within the defined region of interest within 11 pixels of the
initial location in either (x-y) dimension. For experiments
with tetanic stimuli, increases in fluorescence intensity above
background (F) were normalized to the first three or four frames (prestimulus), giving a baseline value of
F/F = 1.00. For time-series experiments,
responses were normalized to the control period (i.e., pixel intensity
before the addition of drug). All levels of significance are from
Student's t-tests expressed as a probability, P,
that the results are from the same population. Errors are expressed as
standard errors of the mean (SE), which were normalized to the control
(predrug) response for pharmacology experiments.
Electrophysiology
Whole cell patch-clamp recordings were made from reticulospinal
neurons and vestibulospinal axons using a modified "blind" technique (Alford and Dubuc 1993). An intracellular
solution of the following composition was used (in mM): 102.5 potassium
gluconate or methanesulphonate, 5.0 HEPES, 3.0 ATP, 1.0 GTP, 1.0 MgCl2, 1.0 NaCl, and 0.05-5.0 EGTA
(osmolarity = 230 mosmol; pH = 7.20). For some experiments,
cesium fluoride was substituted for potassium gluconate or
methanesulphonate or 1-10 mM
guanosine-5'-O-(2-thiodiphosphate) (GDP-
-S) was
substituted for GTP. For such experiments, the recording was not
started until whole cell access was maintained for at least 20 min,
ensuring adequate time to disable G-protein-mediated signal
transduction systems. Pipettes were pulled from thick-walled borosilicate glass and had an open-tip resistance of 5-10 M
. Most
axons were recorded within the midline cleft just rostral to the PRRN,
where there is a dense plexus of crossing fibers from the nOMP just
under the epithelial surface (see Fig. 5). All cells were held at
70
mV. Extracellular stimulations (0.5-20.0 µA) were achieved with
tungsten electrodes (impedance = 0.5-2.0 M
) activated via a
stimulus isolation unit and controlled with a WPI Pulsemaster A300
stimulator (World Precision Instruments, Sarasota, FL).
Electrophysiological recordings of spontaneous miniature excitatory
postsynaptic currents (mEPSCs) were digitized at 5 kHz and low-pass
filtered at 1 kHz. This level of filtering did not affect the peak
amplitude of the events. The average root mean squared (RMS)
noise (taken from 33 randomly chosen event-free epochs culled from all
the cells used in the mEPSC analysis) was 0.72 pA. Frequency and
amplitude analysis of mEPSCs was done with a Macintosh computer using
Igor Pro software. A macro was written that automated the detection of
mEPSCs, returning the time of occurrence and the amplitude. Raw data
traces were smoothed with a 21-point (±2 ms) box filter,
differentiated, and smoothed again with the same filter. This technique
corrected for any DC shift in baseline. A threshold level was manually
determined for the differentiated control (predrug) data set, and this
threshold was maintained for all files taken from a given cell. All
events that crossed the threshold were detected and local minima
(maximum inward current) were searched for within a window of 2 ms
before and 10 ms after the detected event. This data set was mapped
back onto a smoothed (5-point box filter) set of the raw data for the visual comparison of detected events with mEPSCs. Cumulative and raw
histograms were constructed, and a two-population Kolmogorov-Smirnov goodness-of-fit test reduced to a modified 2
variable (Hays 1988
) was used to determine statistical
significance between control and drug conditions. Evoked EPSCs were
analyzed with pClamp software (Clampfit). Only the fast chemical
(monosynaptic non-NMDA-mediated) component was of interest, and cursor
placement during the analysis was chosen to reflect this bias. The
amplitude of the current at a given time (3-8 ms) after the start of
the rising edge of the response (inward current) was measured for each
average of four responses. The same time point was chosen for all
responses recorded from a given cell.
Combined microfluorimetry and electrophysiology
Calcium signals in reticulospinal dendrites were monitored with
high-affinity Ca2+-sensitive dyes introduced into
the postsynaptic cell via the patch electrode. An internal pipette
perfusion system was used to inject
Ca2+-sensitive dye after obtaining whole cell
access, thus preventing dye leakage into the calcium-rich extracellular
space (Alford et al. 1993). For these experiments 3 µl
of 50 µM Oregon Green 488 BAPTA-1 hexapotassium salt was injected
into the pipette tip with a 10-µl syringe after breaking into the
cell. A complete dye-fill of reticulospinal neurons out to the terminal
dendrites was usually obtained within 20 min of stable whole cell
access. Electrophysiological recordings were made in the whole-cell
patch clamp configuration under voltage clamp as described in the
following text.
Drugs
All drugs were bath-applied in Ringer solution at a superfusion
rate of ~1 ml/min. The compounds 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (AP5), 7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt),
L-quisqualic acid (QA), (S)-homoquisqualic acid
(HQA),
(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid
[(1S,3R)-ACPD], and
(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) were obtained from Tocris Cookson (Ballwin, MO). Tetrodotoxin (TTX), GDP--S, and bicuculline methobromide were obtained from Sigma
Chemical. Hexamethonium dichloride was obtained from Research Biochemicals International (Natick, MA). Thapsigargin was obtained from
Alomone Labs (Jerusalem, Israel).
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RESULTS |
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Effect of Group I mGluR antagonism on the evoked release of neurotransmitter
The synaptic activation of presynaptic Group I mGluRs leads to an
enhancement of glutamate release with repetitive stimulation. This
phenomenon is most easily demonstrated by the use of the specific Group
I mGluR antagonist CPCCOEt (Annoura et al. 1996). The
postsynaptic elements of the VS-RS synapse (reticulospinal neurons of
the PRRN) were voltage clamped under whole cell patch-clamp conditions
while presynaptic vestibulospinal axons were stimulated extracellularly. Monosynaptic glutamate-mediated EPSCs were recorded. The patch solution contained GDP-
-S (1-2 mM) to prevent the
activation of postsynaptic mGluRs; we demonstrate that the inclusion of
this GDP analogue effectively eliminates transduction through
postsynaptic G proteins (Fig. 7). Three stimuli were applied to the
presynaptic axons at an inter-pulse interval of 20 ms (i.e., 50 Hz).
The average amplitudes of the third EPSCs were smaller than those of
the first. [i.e., mean amplitude ratio (1st EPSC/3rd EPSC) = 1.65 ± 0.10; n = 5; Fig.
1]. When CPCCOEt (500 µM) was added to
the superfusate, the amplitude ratio was significantly enhanced (to
2.39 ± 0.16; P < 0.001; n = 5;
Fig. 1), reflecting a reduction in the relative amplitude of the third
EPSC to the first EPSC. There was not a significant effect on the
amplitude of the first response with the addition of CPCCOEt. It has
been demonstrated in the lamprey spinal cord that this compound does
not alter the amplitude of single-fiber-evoked EPSCs (Cochilla
and Alford 1998
).
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Pre- and postsynaptic calcium responses to the application of mGluR agonists
We wished to demonstrate that Group I mGluRs are present on
presynaptic elements at this synapse. We show that activation of mGluRs
by exogenous agonists at the VS-RS synapse raises the presynaptic
Ca2+ concentration. Vestibulospinal axons and
reticulospinal dendrites and somata were retrogradely labeled in
isolation (see METHODS) with high-affinity
Ca2+-sensitive dyes. Vestibulospinal axons are
easily identified as linear processes coursing toward the spinal cord
from the ipsilateral nOMI (Fig.
2A); reticulospinal dendrites
are either punctate or short linear structures found at the level of
the PRRN and aligned perpendicularly to the axons in confocal sections
(Fig. 2C) (Schwartz and Alford 1998).
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To test for the presence of presynaptic mGluRs capable of altering
[Ca2+]i, various mGluR
agonists were applied to preparations with dye-filled vestibulospinal
axons. Figure 2A shows an example of such an experiment in
which the potent nonselective mGluR agonist L-quisqualic
acid (QA; 25 µM) was added to the bathing medium. As QA is also an agonist at AMPA and kainate-sensitive ionotropic glutamate receptors, CNQX (10 µM) was included in the superfusate; this concentration of
CNQX has been shown to effectively block AMPA receptor-mediated transmission in the lamprey brain stem (Alford and Dubuc
1993). Additionally it has been demonstrated that agonist
application of neither AMPA (Schwartz and Alford 1998
)
nor kainate (Cochilla and Alford 1997
, 1999
)
causes a rise in presynaptic
[Ca2+]i. Furthermore TTX
(1 µM) was applied during drug applications to prevent synaptic
transmission and functionally isolate the individual neuronal
processes. The Ca2+ rise began immediately on
superfusion; declines were recorded with washout on a slower time-scale
(Fig. 2B). The variability in the time that the tissue was
exposed to agonist (approximately 8 min), the precise superfusion rate,
the fluid level in the recording chamber, and the depth within the
tissue of the scanned region of interest all preclude the extraction of
meaningful kinetic parameters following agonist application. As such,
only peak amplitudes, which usually occurred at the end of agonist
wash-in or within 1 min of washout, are reported here. QA (1-100 µM)
was applied to 40 preparations, and a total of 88 axons were analyzed.
For most experiments (n = 72 axons), 25 µM QA was
used. This concentration resulted in a substantial increases in
[Ca2+]i (mean peak
F/F = 2.23 ± 0.16). The Group I
mGluR agonist HQA (Porter et al. 1992
) (10-100 µM)
[in the presence of CNQX (10 µM)] also resulted in an increase in
axonal fluorescence (n = 9). Twenty-five micromolar HQA
was used for the majority of experiments (n = 5) and
gave a mean peak
F/F of 2.45 ± 0.66. Likewise application of 1S,3R-ACPD (5-10 µM;
n = 3) caused a presynaptic calcium transient (mean
peak
F/F of 2.48 ± 1.01). The Group II
mGluR agonist L-CCG-I (50-100 µM) did not cause a detectable
increase in presynaptic [Ca2+]i
(n = 2). We conclude that Group I mGluRs are present on
the vestibulospinal axons and that their activation leads to a rise in
presynaptic [Ca2+]i.
Clearly postsynaptic activation of mGluRs could modulate the fast
synaptic response. To test for the presence of such mGluRs that are
capable of altering Ca2+ levels, reticulospinal
dendrites of the PRRN were studied in the same manner as in the
preceding text. These protocols provided similar results. Calcium
transients were recorded in the postsynaptic compartment of
retrogradely labeled neurons in response to mGluR activation. QA
(5-250 µM) was applied to 18 preparations, and analysis was
performed on 32 dendritic processes. A mean peak F/F of 3.17 ± 0.24 (n = 26) was measured in reticulospinal dendrites during wash-in of 25 µM
QA. The application of HQA (25-100 µM; n = 22) also
resulted in large increases in the postsynaptic
Ca2+ signal (mean peak
F/F of 1.88-3.29). Figure 2C shows
an example of dendritic calcium transients in response to 50 µM HQA
in CNQX (10 µM) and TTX (1 µM).
Concentration-response relationships for QA (in 10 µM CNQX and 1 µM TTX) were determined for both the axonal (n = 7) and dendritic (n = 3) calcium responses. Sequential series of drug applications (1-250 µM) were applied to retrogradely labeled preparations, as described in the preceding text. Figure 3 shows examples of a concentration-response relationships for a vestibulospinal axon (Fig. 3A) and reticulospinal dendrites (Fig. 3B). A reversible concentration-dependent increase in [Ca2+]i was seen on both sides of the synapse in response to mGluR activation.
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Effect of metabotropic glutamate receptor antagonists on tetanus-induced calcium transients
It has previously been shown that repetitive stimulation of
vestibulospinal axons leads to a large rise in
[Ca2+]i in both pre- and
postsynaptic elements of the VS-RS synapse, a signal that is due in
part to the activation of ionotropic glutamate receptors
(Schwartz and Alford 1998). To test if the activation of
mGluRs by the physiological release of glutamate might be responsible for a component of the presynaptic-evoked Ca2+
signal, imaging experiments were performed with selectively-labeled vestibulospinal axons filled with high-affinity
Ca2+-sensitive dyes (Schwartz and Alford
1998
). Repetitive stimulation (50 Hz for 1 s; 1.0 ms/stimulus) applied to axons of the ipsilateral nOMI resulted in large
prolonged increases in
[Ca2+]i both pre- and
postsynaptically. The "peak" response during the tetanus (which
occurred at its termination) and the "tail" response at the end of
the trial were used for analysis. The presynaptic tail response
represents a measure of the Ca2+ signal after
termination of the stimulus that will not be dominated by the large
transients that result from presynaptic action potentials (i.e.,
Ca2+ flux through voltage-operated channels). To
eliminate the ionotropic glutamatergic component of the presynaptically
evoked Ca2+ signal, we obtained control responses
in the presence of CNQX (10 µM) and AP5 (100 µM). Application of
CPCCOEt (500 µM) in the presence of the ionotropic glutamate receptor
antagonists caused a significant reversible reduction in the peak and
tail amplitudes of the evoked presynaptic Ca2+
transient [decreased by 25.7% (P < 0.01) and 31.9%
(P < 0.01) from the previous response in CNQX/AP5,
respectively; n = 20; Fig.
4]. These findings indicate that a
component of the Ca2+ signal recorded at the
presynaptic element during repetitive stimulation results from the
activation of presynaptic Group I mGluRs.
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Activation of presynaptic mGluRs leads to a depolarization of the presynaptic element
Metabotropic glutamate receptor-mediated rises in
presynaptic [Ca2+]i could
be caused by either the depolarization of the presynaptic element [and
subsequent Ca2+ influx through voltage-operated
Ca2+ channels (VOCCs)] or via the release of
Ca2+ from intracellular stores.
Electrophysiological recordings were made from vestibulospinal
presynaptic elements using both intracellular and patch electrodes.
These axons form a dense plexus as they cross the midline just under
the epithelium of the fourth ventricle (Fig.
5B). A stimulating electrode
was placed high on the alar plate in the region of the nOMP to identify
putative axons as vestibulospinal; those axons that responded to low
intensity (less than ~3.0 µA) stimulation with an immediate (<1.0
ms) action potential were deemed vestibulospinal. As some preparations
were also retrogradely labeled with
Ca2+-sensitive dyes, it was possible to target VS
axons visually using their confocal image. Experiments were performed
in current clamp, and cells had a resting membrane potential of 63 to
72 mV (n = 3). L-Quisqualic acid (25 µM) in the presence of CNQX (10 µM) was bath-applied to the
preparation and the membrane potential was monitored. In all cases, QA
resulted in a reversible depolarization of the VS axon (10-20 mV); an
example of such an experiment is shown in Fig. 5C. This
depolarization is sufficient to activate high-voltage-activated
Ca2+ channels in lamprey reticulospinal axons
(Cochilla and Alford 1998
). Thus a portion of the
QA-induced increase in
[Ca2+]i may result from
Ca2+ entry through VOCCs secondary to the
activation of mGluRs.
|
Activation of presynaptic mGluRs causes a release of Ca2+ from intracellular stores
Although bath-application of QA leads to the depolarization of the
presynaptic element, the coupling of Group I mGluRs in mammalian
systems to phosphoinositide metabolism (Pin and Duvoisin 1995) suggests that InsP3-sensitive
internal stores may play a role in the Group I mGluR-induced
Ca2+ signal. Bath-application of the endoplasmic
reticulum Ca2+-ATPase inhibitor thapsigargin
(Jackson et al. 1988
) was utilized to test this point
directly. The wash-in of thapsigargin caused a small fast transient
rise in intracellular Ca2+ (in 5 of 7 axons; peak
F/F =1.57 ± 0.19). Experiments were
performed to determine the role of these stores in the agonist
(QA)-induced Ca2+ transient. Dye-labeled VS axons
were challenged with 25 µM QA (in 10 µM CNQX and 1 µM TTX) before
and after thapsigargin (10 µM) was added to the tissue (e.g., Fig.
6Ai).
Incubation with thapsigargin caused a 36.5% reduction in the mean peak
Ca2+ response to QA. This was not, however
significant reflecting the large variability in response amplitude to
these agonist applications (from
F/F = 2.39 ± 0.43 to 1.88 ± 0.28; n = 6;
P < 0.10; Fig. 6Aii.).
|
A more consistent mechanism to activate presynaptic
Ca2+ signals that are significantly reduced by
blockade of group I mGluRs is repetitive stimulation of the axons. It
is also of interest to know if a Ca2+ release
from internal stores can be synaptically activated during repetitive
stimulation of the presynaptic element. Experiments were performed in
an analogous manner to the mGluR antagonist experiments. CNQX (10 µM)
and AP5 (100 µM) were included in the superfusate to eliminate that
portion of the presynaptic Ca2+ transient that is
due to the activation of ionotropic glutamate receptors. Addition of
thapsigargin (10 µM) significantly reduced the tetanically induced
peak presynaptic Ca2+ signal from
F/F = 2.57 ± 0.21 to 2.13 ± 0.25 (a decrease of 36.5%; n = 6; P < 0.05). Likewise, the tail of the Ca2+ transient
was decreased from
F/F = 1.32 ± 0.05 to 1.22 ± 0.03 (a 32.8% reduction; n = 6;
P < 0.05; Fig. 6C). It was not possible to
wash thapsigargin out of the tissue in these experiments.
We have demonstrated the presence of Group I mGluRs on both presynaptic vestibulospinal axons and postsynaptic reticulospinal dendrites. Activation of these receptors by either exogenous agonist or endogenous glutamate release leads to a rise in [Ca2+]i on both sides of the synapse. Presynaptically, this Ca2+ transient may be the result of more than one process, as activation of mGluRs leads to both a depolarization of the presynaptic element as well as a thapsigargin-sensitive Ca2+ signal. In the following sections, we will investigate what effect activation of these presynaptic Group I mGluRs has on synaptic transmission, namely the release of glutamate.
Blockade of postsynaptic G proteins inhibits the mGluR-induced rise in dendritic calcium
In the following electrophysiological experiments, we use
postsynaptic reticulospinal neurons as detectors of glutamate release from vestibulospinal axons. It was demonstrated above that these cells
contain Group I mGluRs, and so it was necessary to eliminate the effect
of their activation to study mGluR-mediated presynaptic mechanisms in
isolation. As mGluRs mediate their signal via heterotrimeric G
proteins, the selective inactivation of these proteins in the reticulospinal cells should isolate the effect of mGluR activation to
the presynaptic element of the synapse. Control experiments were
carried out to demonstrate the feasibility of such a protocol. Simultaneous electrophysiology and microfluorimetry was used to visualize the dendritic Ca2+ signal while
monitoring the postsynaptic current in reticulospinal neurons during
the application of mGluR agonist. Further, this technique allows for
the selective loading of GDP--S into these cells. Reticulospinal
neurons were voltage clamped under whole-cell patch-clamp conditions;
the patch solution (and the injected dye solution) contained GDP-
-S
to inactivate postsynaptic G proteins. Figure
7A shows an example of a live
dye-filled reticulospinal cell. Note that the cell body is just under
the dorsal surface of the brain and that the extensive dendritic tree
passes through its entire extent (200-300 µm). Twenty-five
micromolar HQA (in the presence of 10 µM CNQX, 100 µM AP5, and 1 µM TTX) was bath-applied soon after obtaining whole cell access,
while still allowing adequate time for dye filling. This protocol
evoked a reversible increase in dendritic calcium (mean
F/F = 2.00 ± 0.23;
n = 7; e.g., Fig. 7B), and this transient
was accompanied by an inward current [mean current = 381.8 ± 82.9 pA (relative to baseline); n = 3]. When more
time was allowed for the inactivation of G proteins, the same
application of mGluR agonist resulted in a significantly smaller
Ca2+ signal [mean
F/F = 1.39 ± 0.08 (with G proteins
inactivated) Fig. 7C, left], but the inward
current remained. The application of 100 µM NMDA [in the presence of
CNQX (10 µM) and TTX (1 µM)] was used as a positive control. Under
these conditions, NMDA application evoked a reversible
Ca2+ signal in the dye-filled dendrites that was
insensitive to the intracellular application of GDP-
-S (mean
F/F = 1.81 ± 0.13; n = 6; Fig. 7C, right); the rise
in Ca2+ was accompanied by an inward current
[mean = 272.8 ± 159.5 pA (relative to baseline)]. These
control experiments show that the inclusion of GDP-
-S in the
postsynaptic cell is able to disrupt G-protein-mediated mGluR
signaling, thereby allowing the postsynaptic reticulospinal cell to
serve as a detector for neurotransmitter release in experiments that
use bath applications of mGluR agonists.
|
Whole cell patch-clamp recording of spontaneous mEPSCs from reticulospinal neurons
To examine the effect of the activation of presynaptic Group I
mGluRs on the spontaneous release of glutamate at the VS-RS synapse,
spontaneous mEPSCs were recorded in reticulospinal neurons of the PRRN
held at 70 mV under whole cell patch-clamp conditions (n = 18). The electrode contained either 1-2 mM
GDP-
-S or cesium fluoride (CsF; 102.5 mM) to effectively
inactivate G proteins; thus signal transduction through postsynaptic
mGluRs was prevented. An example of a typical experiment is shown in
Fig. 8. Excitatory events of variable
amplitude were readily seen under control conditions (data not shown).
The application of TTX (1 µM) and strychnine (5 µM) decreased the
frequency of, but did not eliminate, the EPSCs. The bath-application of
a low concentration (5 µM) of the mGluR agonist HQA (in TTX and
strychnine) resulted in a marked increase in the frequency of events
(Fig. 8, A-C). There was no change in the holding current
with the wash-in of HQA. The effect of the mGluR agonist was
reversible. Addition of 10 µM CNQX to the superfusate (in TTX and
strychnine) decreased the frequency of, but did not abolish, the
mEPSCs. The events that remained were insensitive to AP5 (
200 µM),
bicuculline (5 µM), and hexamethonium (
20 µM; data not shown).
|
Histograms of mEPSCs were constructed to quantify the effect of HQA on
the frequency and amplitude of spontaneous events. Application of 5 µM HQA (in TTX and strychnine) resulted in an increased number of
events per time. Figure 8D shows an interval histogram for
the example cell comparing the interval between mEPSCs for a total
8.192 min in both HQA and control conditions, 5,665 and 4,460 events,
respectively. Of all 18 cells tested, the average interval decreased by
21.16% [from 109.72 ms (range 10.00 to 1457.86 ms) to 86.50 ms
(range 10.10
1,187.66 ms)] when the Group I mGluR agonist was added
to the preparation. Similarly each cell recorded showed a significant
increase in mEPSC frequency. A cumulative interval histogram from an
example cell (Fig. 8D, inset) shows a shift toward higher
frequency events in the presence of drug (1,392 and 978 events in a
period of 228 s in HQA and control conditions, respectively:
P < 0.001). There was a small but significant decrease
in the amplitudes of mEPSCs in the presence of 5 µM HQA. The average
mEPSC amplitude decreased from
5.71 to
5.40 pA (a 5.43% reduction)
when Group I mGluRs were activated.
The application of a higher dose of HQA (25 µM; in 1 µM TTX and 5 µM strychnine) resulted in a large inward current
(Iin) recorded in reticulospinal cells
that was insensitive to G-protein inactivation with 1 mM GDP--S
loaded intracellularly. The mean peak
Iin was 383.4 ± 81.2 pA
(relative to baseline; n = 10 cells). This current was
eliminated by the addition of CNQX (10 µM; n = 6/6
cells), and this effect was usually reversible (Fig.
9A; n = 3/4
cells). The inward current was accompanied by a large increase in noise
(Fig. 9Bii). This increase in noise that
occurs with the addition of 25 µM HQA is eliminated by the prior
addition of 10 µM CNQX (Fig. 9Biii);
however, some residual events remain.
|
We have demonstrated that Group I mGluRs are present both pre- and postsynaptically at the VS-RS synapse and that activation of these receptors, both endogenously and exogenously, leads to a rise in [Ca2+]i. Further, we have shown that the increase in presynaptic Ca2+ in response to either agonist or repetitive stimulation of these axons is due in part to the release of Ca2+ from intracellular storage organelles. The activation of these receptors with agonist leads to an enhancement in the frequency of mEPSCs. Taken together, these results suggest that a presynaptic rise in Ca2+ secondary to the activation of Group I mGluRs augments the release of glutamate. Indeed it is likely that the observation that CPCCOEt reduces the amplitude of subsequent EPSCs delivered at high-frequency (Fig. 1) results from the inhibition of this process.
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DISCUSSION |
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Effects of mGluR activation on synaptic transmission are multiple
We have demonstrated that the synaptic activation of presynaptic
Group I mGluRs leads to both an increase in intracellular Ca2+ and an enhancement of spontaneous and evoked
transmitter release. The most parsimonious explanation for the effect
of CPCCOEt on synaptic transmission is that it prevents a rise in
[Ca2+]i, and that this in
turn inhibits the enhancement in glutamate release that occurs when
these presynaptic facilitatory autoreceptors are activated. In many
preparations, mGluRs modulate both synaptic transmission and
spontaneous neurotransmitter release. These effects on release, which
have been variously reported as inhibitory (Baskys and Malenka
1991; Burke and Hablitz 1994
; Dong et al.
1996
; Krieger et al. 1996
; Takahashi et
al. 1996
) and facilitatory (Budd and Nicholls
1995
; Herrero et al. 1992
; Zhang and
Dorman 1993
), could be due to a variety of mechanisms. The
relationship between mGluR activation and Ca2+
dynamics shown here, however, makes the modulation of
[Ca2+]i in the
presynaptic element a likely target for affecting release. Increased
presynaptic Ca2+ entry subsequent to synaptic
mGluR activation can result from either
InsP3-triggered liberation of
Ca2+ from internal stores, direct mGluR-mediated
modulation of VOCCs (Rothe et al. 1994
; Stefani
et al. 1996
), uncoupling of a presynaptic inhibitory receptor
(Budd and Nicholls 1995
; Swartz et al.
1993
), or some combination thereof. At the VS-RS synapse,
activation of presynaptic mGluRs results in depolarization as well as
the liberation of Ca2+ from intracellular storage
organelles. These phenomena allow for a role of mGluRs in the
short-term modulation of glutamate release.
Pre- and postsynaptic mGluR activation leads to an increase in intracellular Ca2+
Bath-application of mGluR agonists evokes
Ca2+ transients in pre- and postsynaptic elements
of the VS-RS synapse. These signals are insensitive to the blockade of
synaptic transmission by TTX. The most robust response was generated by
the nonselective excitatory amino acid agonist QA (in the presence of
CNQX); this agonist has been reported to be the most potent activator
of Group I mGluRs (Anwyl 1995; Tanabe et al.
1992
). The specific Group I mGluR agonist HQA (Porter et
al. 1992
) also produced a large Ca2+
transient both pre- and postsynaptically. Given the essential role of
calcium in neurotransmitter exocytosis, the presence of prejunctional
mGluRs that are capable of affecting
[Ca2+]i is an important
variable to consider for the modulation of synaptic transmission.
Activation of presynaptic mGluRs enhances spontaneous neurotransmitter release
Activation of presynaptic mGluRs affects transmitter release;
however, little is understood of the cellular mechanisms by which this
occurs, and there is marked variability depending on the particular
synapse chosen for study. For example, Group I mGluRs have been shown
to both inhibit Ca2+ channel gating and to open
Ca2+ channels (Chavis et al. 1994;
Lester and Jahr 1990
). In the current study, we have
provided evidence that suggests a causal relationship between the
mGluR-dependent rise in presynaptic Ca2+ and an
increase in glutamate release. Here the postsynaptic reticulospinal neurons were used as detectors of presynaptic activity. The frequency of mEPSCs is dependent on transmitter release probability (Fatt and Katz 1952
; Redman 1990
); this in turn, at
the neuromuscular junction, has been demonstrated to be dependent on
the [Ca2+]i in the
presynaptic element. We have shown that the Group I mGluR agonist HQA
significantly increases the frequency of mEPSCs. Although a change in
the behavior of "silent synapses" could account for such a shift in
mEPSC frequency (Isaac et al. 1995
; Liao et al.
1995
), the inactivation of postsynaptic G proteins in these experiments precludes this as a likely mechanism. The coincidence of
this increase in mEPSC frequency with a rise in presynaptic [Ca2+] makes the presynaptic
Ca2+ signal an attractive candidate for this
mechanism of action of mGluRs. The spontaneous activity was antagonized
by the application of the non-NMDA receptor antagonist CNQX. There was,
however, a component that was insensitive to CNQX, AP5, strychnine,
bicuculline, and hexamethonium; the transmitter(s)/receptor(s)
mediating these events remain to be determined.
Activation of presynaptic mGluRs by the exogenous application of HQA (5 µM) results in an increase in the spontaneous release of
neurotransmitter. With a higher concentration (25 µM) of the same
agonist, a large CNQX-sensitive inward current was recorded in the
postsynaptic cell. We propose that this is due to a sizable release of
glutamate from presynaptic vestibulospinal axons. Several lines of
evidence converge on this conclusion. Firstly, postsynaptic G proteins
were effectively inactivated by GDP--S or CsF; this was confirmed by
the selective elimination of the HQA-mediated postsynaptic
Ca2+ transient in these neurons. In hippocampal
CA1 cells, 500 µM of GDP-
-S included in the patch solution
eliminated the (1S,3R)-ACPD-induced calcium-activated nonspecific cation current (Congar et al.
1997
). Second, it is reasonable to assume that the increase in
spontaneous release seen at low concentrations of HQA would be enhanced
by higher concentrations. Indeed, Ca2+ transients
of increasing magnitude were recorded with application of progressively
higher concentrations of mGluR agonists. As a whole, this result
suggests that activation of Group I mGluRs leads to the action
potential-independent increase in glutamate release from the
presynaptic element.
Sources of the presynaptic Group I mGluR-induced Ca2+ transient are several
Activation of presynaptic Group I mGluRs results in a large
increase in intracellular Ca2+ at the VS-RS
synapse. The Ca2+ signal can potentially arise
from one or more of several different sources. First, phosphoinositide
hydrolysis secondary to phospholipase C (PLC) activation results in an
increase in the formation of InsP3; activation of
InsP3 receptors mobilizes
Ca2+ from distinct intracellular storage pools.
Ca2+ released from intracellular stores may
diffuse throughout the axon within 10 ms (Cochilla and Alford
1998, Schwartz and Alford 1998
) Second, protein
kinase C (PKC) is stimulated by diacylglycerol (DAG) (with or
without Ca2+ as a cofactor) subsequent to PLC
activation. The phosphorylation of ion channels by protein kinases is
thought to play an important role in the regulation of various ionic
conductances. At the neuromuscular junction in Drosophila,
activation of presynaptic mGluRs has been shown to increase glutamate
release via a cAMP-dependent mechanism (Zhang et al.
1999
). In synaptosomes, it is known that activation of Group I
mGluRs facilitates glutamate release by closing
K+ channels secondary to PKC activation; this
facilitation is enhanced by arachidonic acid and desensitizes rapidly
as PKC acts through negative feedback on the mGluR itself
(Coffey et al. 1994
; Herrero et al.
1992
). In this regard, the second potential source of
Ca2+ is extracellular, given its entrance through
VOCCs on membrane depolarization. Such a Group I mGluR-mediated
presynaptic depolarization was seen in our studies; however, the
precise mechanism that underlies it is currently unknown at this synapse.
Release of endogenous glutamate activates presynaptic mGluRs
The activation of presynaptic Group I mGluRs by the application of
an exogenous agonist can evoke a Ca2+ transient
in the presynaptic element, and this in turn alters the frequency of
spontaneous release. We investigated the possibility that the evoked
release of endogenous glutamate might alter presynaptic [Ca2+]i by acting on
these receptors. A high-frequency input to vestibulospinal axons
results in a long-lasting (>6.0 s) Ca2+
transient on both sides of the synapse that far outlasts the duration
of the stimulus. The sources of the calcium are several. First,
Ca2+ flux through VOCCs is expected to contribute
significantly to the presynaptic signal during the stimulus. Second,
NMDA and non-NMDA receptor-activation results in an increase in
[Ca2+]i in both
compartments of the synapse (Schwartz and Alford 1998). Activation of presynaptic NMDA receptors have been shown to alter glutamate release in both mammalian (Glitsch and Marty
1999
) and lamprey (Cochilla and Alford 1998
)
preparations. Finally, axonal and dendritic mGluRs are synaptically
activated and appear to be capable of altering
Ca2+ levels on either side of the synapse.
Application of the Group I mGluR antagonist CPCCOEt reduces the
amplitude of Ca2+ transients evoked by
high-frequency stimulation of vestibulospinal axons. This result
implies that physiological release of glutamate can alter presynaptic
[Ca2+]i by acting at
these mGluRs. Further, this activation of presynaptic receptors and
subsequent Ca2+ entry causes an enhancement of
evoked EPSC amplitudes resulting from repetitive stimulation of the
vestibulospinal terminal. Previously we have shown the recruitment of
ionotropic glutamate receptor-mediated axo-axonic inputs from
vestibulospinal axons onto other vestibulospinal axons during tetanic
stimulus; activation of these receptors can alter the level of
Ca2+ in the presynaptic element (Schwartz
and Alford 1998
). The current study has not distinguished
between mGluR activation by glutamate that is released from axo-axonic
synapses and that released by the axon that is being recorded from
(i.e., autoreceptor activation of mGluRs). Metabotropic glutamate
receptors serve a modulatory role and do not result in a fast
electrophysiological response like their ionotropic counterparts. These
results highlight the importance of these receptor-mediated processes
for synaptic transmission.
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ACKNOWLEDGMENTS |
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We express our gratitude to Drs. E. M. Silinsky, N. T. Slater, A. J. Cochilla, and M. Takahashi and to R. M. Freed for comments on the manuscript and to Dr. J. C. Klapow for advice on statistical analysis. Some batches of (S)-homoquisqualic acid were generously donated by Dr. D. Crawford of Tocris Cookson and Dr. G. L. Collingridge.
This work was supported by National Institute of Neurological Disorders and Stroke Grants RO1 NS-32114 and NS-31713.
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FOOTNOTES |
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Address for reprint requests: S. Alford, Dept. of Biological Sciences (MC 066), College of Liberal Arts and Sciences, University of Illinois at Chicago, 845 W. Taylor St., Chicago, IL 60607-7060 (E-mail: sta{at}uic.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 September 1999; accepted in final form 31 March 2000.
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REFERENCES |
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