Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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ABSTRACT |
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Dubé, G. R. and K. C. Marshall. Activity-Dependent Activation of Presynaptic Metabotropic Glutamate Receptors in Locus Coeruleus. J. Neurophysiol. 83: 1141-1149, 2000. Synaptic activation of metabotropic glutamate receptors (mGluRs) in the locus coeruleus (LC) was investigated in adult rat brain slice preparations. Evoked excitatory postsynaptic potentials (EPSPs) resulting from stimulation of LC afferents were measured with current clamp from intracellularly recorded LC neurons. In this preparation, mGluR agonists (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4) activate distinct presynaptic mGluRs, resulting in an inhibition of EPSPs. When two stimuli were applied to afferents at intervals >200 ms, the amplitude of the second [test (T)] EPSP was identical in amplitude to the first [control(C)]. However, when a stimulation volley was delivered before T, the amplitude of the latter EPSP was consistently smaller than C. The activity-dependent depression (ADD) was dependent on the frequency and duration of the train and the interval between the train and T. ADD was potentiated in the presence of an excitatory amino acid (EAA) uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC, 100 µM), changing the T/C ratio from 0.84 ± 0.05 (mean ± SE) in control to 0.69 ± 0.04 in t-PDC (n = 9). In the presence of t-PDC, the depolarizing response of LC neurons to focally applied glutamate was also increased. Together, these results suggest that accumulation of EAA after synaptic stimulation may be responsible for ADD. To test if ADD is a result of the activation of presynaptic mGluRs, the effect of selective mGluR antagonists on ADD was assessed. In the presence of t-PDC, bath applied (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP4, 500 µM), a mGluR group III antagonist, significantly reversed the decrease in T/C ratio after a train stimulation [from 0.66 ± 0.04 to 0.81 ± 0.02 (mean ± SE), n = 5]. The T/C ratio in the presence of MAP4 was not different from that measured in the absence of a stimulation volley. Conversely, ethyl glutamic acid (EGLU, 500 µM), a mGluR group II antagonist, failed to alter the T/C ratio. Together, these results suggest that, in LC, group III presynaptic mGluR activation provides a feedback mechanism by which excitatory synaptic transmission can be negatively modulated during high-frequency synaptic activity. Furthermore, this study provides functional differentiation between presynaptic groups II and III mGluR in LC and suggests that the group II mGluR may be involved in functions distinct from those of group III mGluRs.
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INTRODUCTION |
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Neurotransmission by excitatory amino acids (EAA)
is mediated via multiple receptors that are divided into two classes,
ionotropic and metabotropic. Ionotropic receptors convey EAA signals
through cation permeable channels, mediating postsynaptic
depolarization within milliseconds after the release of
neurotransmitters (see Clements et al. 1992). Metabotropic glutamate
receptors (mGluRs) mediate EAA signals through a G-protein-dependent
signal transduction pathway. Because of this, their effects are
relatively slower, with an onset >50 ms after EAA binds to the
receptor (Wickman and Clapham 1995
). To date, eight
mGluRs have been cloned and termed mGluR 1-8 (Anwyl
1999
; Conn and Pin 1997
). These can be classified into three groups based on their homology, pharmacology, and
signaling pathways. Functional expression and microscopic localization
demonstrated that mGluRs can be found pre- and postsynaptically and
that they can be localized at the synapse and outside the synaptic zone
(see Li et al. 1997
; Martin et al. 1992
;
Petralia et al. 1996
; Shigemoto et al.
1996
, 1997
). Thus these receptors likely
contribute in different ways to synaptic transmission and modulation.
One of the commonly described effects of specific mGluR agonists is the
reduction of synaptic transmission presynaptically at glutamatergic and
GABAergic synapses (Conn and Pin 1997). All three groups
of mGluRs have been implicated in presynaptic inhibition and, in many
instances, more than one group and subtype can be present at the same
synapse (Pin and Duvoisin 1995
). Recently, activity-dependent endogenous activation of presynaptic group II mGluRs
was demonstrated in hippocampal slices (Min et al. 1998
; Scanziani et al. 1997
). Furthermore, activation of
presynaptic mGluRs resulting from blockage of EAA uptake was reported
in hippocampal cultures, an effect mediated by a group II mGluR
(Fitzsimonds and Dichter 1996
; Maki et al.
1995
).
In the locus coeruleus (LC), excitatory synaptic transmission can be
inhibited after the activation of either a group II or a group III
mGluR (Dubé and Marshall 1997a). Concomitant
activation of both receptors with low doses of agonists results in an
additive response whereas activating them with perimaximal
concentrations of agonists gives a response that is significantly less
than additive, suggesting that both receptors share a common step in
their signal transduction pathway. This is in agreement with
observations in various expression systems that indicate that both
group II and III receptors are coupled to a similar transduction
pathway, i.e., negatively coupled to adenylate cyclase through a
pertussis toxin-sensitive (Gi/o G-proteins)
pathway (Conn and Pin 1997
). Although neither group of
receptors inhibited excitatory postsynaptic potentials (EPSPs) via a
cyclic adenosine monophosphate (cAMP)dependent pathway in
LC, both receptors appeared to be coupled to a Gi/o
G-protein (Dubé and Marshall 1997b
). The LC is the
principal central noradrenergic nucleus, providing innervation
throughout the neuraxis, including exclusive noradrenergic innervation
of the neocortex and the hippocampus. Interestingly, it was recently
demonstrated that administering a mGluR2/3 agonist reduced the
behavioral effects associated with opioid withdrawal
(Vandergriff and Rasmussen 1999
). This was associated with a specific and significant decrease in the glutamate-mediated overactivity of the LC nucleus. Given the apparent involvement of LC
neurons in opioid addiction (Aghajanian 1978
;
Rasmussen et al. 1990
), the modulatory actions of mGluRs
on this nucleus could become therapeutic targets.
In this study, we investigated the role of group II and III mGluRs in
relation to the activity of excitatory synaptic transmission to LC. We
hypothesized that if one or both mGluRs are located presynaptically and
function as feedback receptors, these should inhibit EPSPs in a
synaptic activity-dependent fashion. A portion of this work was
previously presented in abstract form (Dubé and Marshall
1996).
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METHODS |
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All experiments were carried out using a brain stem slice
preparation from young adult male Sprague-Dawley rats (4-8 wk, 50-150 g; Charles River, St. Constant, Quebec) under conditions designed to
minimize animal suffering. A detailed account of these procedures can
be found in Dubé and Marshall (1997a). In brief,
animals were fully anesthetized with oxygen-rich halothane (2%) and
surgically decapitated. The brain was removed, rinsed, and trimmed in
ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl 118.0, KCl 3.0, CaCl 2.5, MgSO4 0.8, NaH2PO4 1.0, D-glucose 10.0, and NaHCO3 20.0, equilibrated to pH 7.4 by continuously bubbling with 95%
O2/5% CO2. Horizontal
slices (350 µM thickness) were cut in ice-cold ACSF using a
Vibroslice (Campden Instruments, London, England). The selected
slice was transferred into the recording chamber, stabilized, and
continuously superfused with warm ACSF (32.0 ± 0.5°C) at a rate
of 1.5 ml/min.
LC neurons were easily identified based on the location and appearance
of the nucleus within the slice, and their electrophysiological properties. Recording electrodes filled with 2 M potassium acetate had
tip resistances ranging from 70 to 100 M. Impaled neurons were
considered to be acceptable when the membrane potential stabilized at a
value less than
50 mV and the amplitude of the action potential was
>60 mV. Potentials measured with current clamp were amplified (Axoclamp 2A, Axon Instruments Inc., Foster City, CA),
displayed by conventional methods, and recorded on a chart recorder
(Gould 2200s).
EPSPs were evoked by electrical stimulation within 1 mm rostrolateral
to the recording site using a bipolar electrode (~10 K). Stimuli
were generated by applying single rectangular pulses (0.4-3.0 V, 0.1 ms) from a Grass S-88 unit through a constant-current stimulus
isolation unit. To prevent cells from reaching threshold during EPSP
recording, the membrane potential of the impaled neurons was
temporarily set to a lower potential (between
70 and
75 mV) by
injecting a hyperpolarizing current pulse. EPSPs were evoked midway
through the pulse. Alternatively, in certain experiments, cells were
hyperpolarized to approximately
75 mV for the duration of the tests.
The resulting EPSP was 95% insensitive to
N-methyl-D-aspartate (NMDA)-receptor
antagonism (50 µM R-CPP) (Dubé and Marshall
1997a
). Bicuculline (10 µM) or picrotoxin (50 µM) (to block
fast GABA-mediated IPSPs) and yohimbine (1 µM) [to block
2-adrenergic inhibitory postsynaptic potentials
(IPSPs)] (Egan et al. 1983
) were included in the ACSF
unless otherwise indicated.
For glutamate-evoked depolarizations, monosodium glutamate (10 mM, pH 7.4; Sigma Chemical Co., St. Louis, MO) was applied by
pressure ejection using single pipettes with tip diameters of 8-12
µm. Pressure electrode tips were placed 50-100 µm away from the
slice. The pressure (3-20 psi, 5-20 ms) applied to the electrode was
adjusted through the pneumatic valve of a Picospritzer II (General
Valve Corp.). All other drugs were dissolved in ACSF and introduced
through the perfusion line by gravity-induced flow. The pH was adjusted
to 7.4 when necessary. Drugs used were
(S)-amino-2-methyl-4-phosphonobutanoic acid (MAP4), ethyl glutamic
acid (EGLU),
L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC; Tocris Cookson),
3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate [(R)-CPP],
(±)--methyl-4-carboxyphenylglycine (MCPG), baclofen, phaclofen, saclophen, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), mianserin, 8-phenyl-theophylline and aminophylline (Research
Biochemical International, Natick, MA), (
)-bicuculline
methiodide, picrotoxin, and yohimbine HCl (Sigma).
Protocol for activity-dependent depression
To assess synaptic depression after high-frequency synaptic transmission, stimulation volleys preceded and followed by single stimuli were delivered to the stimulating electrode. The first single stimulation resulted in a control (C)-evoked EPSP and the last resulted in the test (T) EPSP. Volleys were applied 100 ms after the first stimulation. For each condition, tests were carried out as follows (e.g., Fig. 1): 1) one test evoking the C and T EPSPs without a stimulation volley (Fig. 1A), 2) one test where only the stimulation volley was applied (Fig. 1B), and 3) three consecutive tests where both the single stimuli and the volley were applied (Fig. 1C). The individual tests consisted of four sweeps recorded at 0.2 Hz and were performed at 30 s intervals.
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All acquisition and measurements were made using pClamp 6.0 software (Axon Instruments Inc.). Data from voltage-current protocols were averaged online using four individual sweeps per averaged waveform. Data for EPSPs were averaged offline (with the clampfit module) using four individual sweeps per averaged waveform. For tests where only single EPSPs were evoked, the amplitudes of T and C were measured offline with respect to the baseline a few ms before the stimulus artifact. For the two other tests, the averaged volley waveform was subtracted from the averaged volley + EPSP waveform (Fig. 1D). The resulting waveform, comprising roughly only the C and T EPSPs, was analyzed in the same way. The ratio of T to C amplitudes measured from the resulting waveform (Fig. 1D) was used as an index of the effects of activity on LC excitatory synaptic transmission. Ratios >1 indicated a potentiation and ratios <1 a depression. Averaged data are presented as mean ± SE. For statistical analysis purposes, we tested whether or not the difference between two conditions (obtained by subtracting the respective T/C) was significantly different from zero (one-sample t-test). This eliminated some of the variations observed in the pairing observed when paired Student's t-test analysis was used [i.e., pairing between certain sample groups was not significant (as assessed by the Pearson correlation coefficient)]. For other statistical analysis, where indicated, analysis of variance (ANOVA) was performed followed by a Tukey post hoc test. Differences were considered significant at P < 0.05.
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RESULTS |
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Basic characterization of activity-dependent depression of EPSPs
To determine the conditions leading to activity-dependent depression (ADD), preliminary experiments were designed to delineate the range of durations and frequencies of the stimulation volley and the interval between the end of the volley and T, which resulted in maximal ADD of T with respect to C. Four parameters were examined: 1) the interval between C and T required to avoid paired-pulse facilitation in the absence of a stimulation volley, 2) the interval between the volley and T, 3) the duration of the volley, and 4) the frequency of the volley for optimal ADD. Figure 2 summarizes results pertaining to the first two parameters. Facilitation between a pair of EPSPs was observed when evoked 20-100 ms apart but was not observed at greater intervals (Fig. 2A). When a stimulation volley was applied (300 ms, 70 Hz) and the amplitude of T subsequently compared with C, T was consistently smaller when evoked 200-300 ms after the end of the volley (Fig. 2C). Because noradrenergic-presumptive slow IPSPs inherent to the LC nucleus were often observed during the tests, we assessed the effects of yohimbine on these. As shown in Fig. 2, B and D, 1 µM yohimbine reduced the slow IPSPs observed in both sets of tests but did not significantly affect the amplitudes and ratios of the EPSPs. We included yohimbine in subsequent experiments.
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To test the last two parameters, i.e., the optimal duration and frequency of the stimulation volley, the C and T EPSPs were separated by an 800 ms interval (corresponding to 0 Hz stimulation). Keeping the interval between C and T constant, 100, 200, or 300 ms-long stimulation volleys were delivered to the LC afferents. Frequencies of stimulation between 10 and 90 Hz (increment of 10 Hz) were tested for each time duration, and the amplitude of T was compared with C (Fig. 3A). Results from these experiments showed significant ADD when 300 ms stimulation volleys were delivered to the LC afferents with frequencies ranging from 50 to 70 Hz. Figure 3B shows a representative example displaying how C and T EPSPs varied when 300-ms stimulation volleys of different frequencies (0-90 Hz) were applied. Note that a prolongation of the repolarization phase of the T EPSP, as compared with C, was sometimes observed, although not consistently, even within the same series of tests. This was not NMDA dependent (n = 3, not shown) or mGluR dependent and was not affected by any of the pharmacological treatments performed in this study (listed in Table 1).
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Roles of mGluRs in ADD
To test the hypothesis that presynaptic mGluRs can be activated
after stimulation volleys through the release of high amounts of EAA in
the synaptic cleft, specific mGluR antagonists were tested for their
effects on ADD. First, three mGluR antagonists were tested because of
their actions on mGluR in LC (Dubé and Marshall
1997a). Of these, both (R)-MCPG and EGLU were shown to inhibit the effects of exogenously applied t-ACPD on
excitatory synaptic transmission whereas MAP4 was found to antagonize
selectively the L-AP4 effects. However, results from these
experiments showed that after 15 min perfusion with each antagonist
(500 µM), none caused a significant change in T/C although a tendency
toward higher values was observed with each drug [control T/C vs. drug (mean ± SE) for MCPG: 0.74 ± 0.06 vs. 0.76 ± 0.06, n = 8; EGLU: 0.83 ± 0.04 vs. 0.94 ± 0.04, n = 7; MAP4: 0.67 ± 0.06 vs. 0.70 ± 0.06, n = 9].
We subsequently tested whether or not specifically blocking the uptake of EAA could potentiate ADD. As a control, the ability of t-PDC, an EAA uptake inhibitor, was assessed on focally applied glutamate-evoked depolarizations of LC neurons. As exemplified in Fig. 4, glutamate-evoked depolarizations were markedly potentiated in the presence of t-PDC (100 µM) in all of the five cells tested, suggesting that the clearance of the focally applied glutamate depends, in part, on the activity of the glutamate transporters. Occasionally, the firing frequency and the membrane potential of the impaled neuron were reversibly increased in the presence of t-PDC (Fig. 4). This was presumably caused by the accumulation of glutamate resulting from the clearance block. The changes in membrane potentials were compensated with injection of hyperpolarizing current, but the enhanced glutamate response remained despite the current injection. No overall significant differences in cell input resistance were observed with t-PDC (n = 9).
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Figure 5 displays a representative example of the effect of 100 µM t-PDC on ADD. As depicted in this example, the amplitude of the C was not affected by the uptake inhibitor. Furthermore, when no stimulation volleys were delivered between C and T, the amplitude of T and T/C remained unchanged as compared with control (without t-PDC) (Fig. 5, C and E). Conversely, under the same conditions, when stimulation volleys were applied to the afferents, the amplitude of T was further reduced in the presence of t-PDC (Fig. 5, compare B and D). Thus in the presence of t-PDC, ADD was significantly larger than under control conditions (Fig. 5F) (mean T/C control vs. t-PDC: 0.84 ± 0.05 vs. 0.69 ± 0.04; P < 0.0001; n = 9). The effects of t-PDC were observed in every cell tested and thus were highly reproducible.
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Using t-PDC, we tested the hypothesis that the potentiation of the depression observed with the uptake inhibitor was mediated by the activation of either or both groups of mGluRs characterized in LC. Figure 6A displays representative examples of the effects of MAP4 (500 µM) on the relative amplitude of T in the presence of t-PDC. As expected, t-PDC significantly potentiated ADD. However, in the presence of MAP4, T/C was significantly increased not only as compared with conditions where t-PDC was present but also with respect to the control without stimulation volleys (Fig. 6C). Furthermore, this was not observed when EGLU was tested in the same manner (Fig. 6B and D). Thus application of MAP4, but not EGLU, under these conditions significantly reversed the t-PDC-dependent inhibition of T by stimulation volleys to levels that were not different from conditions where no stimulation volleys were applied. These results support the hypothesis that selective mGluRs can be activated to cause ADD of excitatory synaptic transmission.
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Involvement of other factors in ADD
These tests present some caveats. One of these is that
the focal stimulation applied to our preparations may result in the release of several neurotransmitters in addition to EAA. Two of these,
GABA and noradrenaline (NA), have been addressed by inclusion of antagonists in the perfusion fluid. However, although the
fast IPSPs mediated by GABAA are blocked by inclusion of
specific antagonists in the perfusate, other actions of GABA and other
neurotransmitters could potentially interfere with the measurements.
Therefore the involvement of three likely candidates, GABA (because of
its action on the GABAB receptor), adenosine (a known
neurotransmitter released during high synaptic activity), and serotonin
[shown to depress excitatory synaptic transmission in LC
(Bobker and Williams 1989; Charléty et al.
1993
)] were investigated. Specific antagonists for each of the
three neurotransmitters were tested as for mGluR antagonists. Table 1
summarizes the results obtained for all antagonists tested. Three
specific antagonists of GABAB receptors were tested:
phaclofen, saclofen, and CGP35348. We found that CGP35348 (100 µM)
reversed the GABAB-induced (baclofen, 3 µM) presynaptic
inhibition of excitatory synaptic transmission in LC (unpublished
observations). However, at 100-500 µM concentrations, none of these
antagonists significantly affected ADD. Three distinct antagonists of
adenosine receptors were also tested. Aminophylline and
8-phenyl-theophylline are antagonists of the A1 and
A2 receptor subtypes and DPCPX is a potent and selective
A1 antagonist. Again, none of the antagonists affected ADD.
The broad-range serotonin antagonist mianserin (500 µM) was also
tested and was found to have no effect on ADD. Finally, we tested for
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
desensitization using the desensitization blocker cyclothiazide (100 µM). Pooled results from eight experiments indicated that
desensitization did not contribute significantly to the decrease in
T/C. However, close inspection of the results revealed that when ADD
produced a marked reduction in T/C (<65%), cyclothiazide caused a
significant increase in T/C (P < 0.01). These
results suggest that desensitization of AMPA does occur in our system
under certain conditions. However, the reversal was only partial,
contributing ~10% of the decrease in T/C (4 of 8 cells, Table 1).
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DISCUSSION |
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We previously characterized two pharmacologically
distinct presynaptic mGluRs that modulate excitatory synaptic
transmission In LC (Dubé and Marshall 1997a). The two receptors
belong to groups II and III, respectively, which have been shown in
expression systems to be coupled to the same signal transduction
pathway. We hypothesized that if both receptors carried out identical
functions, then both should be functionally activated in a similar
fashion. However, if these receptors served different functions
resulting in a similar effect, it might be possible to activate one
independently of the other. Our results show that under conditions in
which extracellular EAA levels should be strongly enhanced, the group III mGluR can be endogenously activated. In contrast, under the same
conditions, activation of the group II mGluR was not observed.
Glutamate released into the synaptic cleft may be expected to
activate the various EAA receptors differentially, depending on their
sensitivity to the neurotransmitter and their location in relation to
the synaptic cleft. The average effective concentration for 50% of
maximal response of AMPA receptors for glutamate ranges from
250 to 1500 µM (Clements 1996; Liu et al.
1999
) whereas that of mGluRs is between 3 and 20 µM with the
exceptions of mGluR7, for which it is ~1000 µM, and mGluR8, for
which it is ~0.02 µM (Conn and Pin 1997
). Based on
the time/concentration profile of glutamate released by a single
quantum, as estimated by several laboratories (e.g., Clements et
al. 1992
; Holmes 1995
; Otis et al. 1997
), the concentration of glutamate stays at
AMPA-activating concentrations for only ~1 ms but could be high
enough to act on mGluRs for a relatively long time after release. Thus
it is likely that mGluRs, if located in the synaptic cleft, would be activated. This is supported by the findings of Schrader and
Tasker (1997)
that show tonically active mGluR. However, we
failed to detect any tonically active mGluRs, group II or III, in our
preparation. Different distributions of mGluRs have been shown in
various preparations (e.g., cortex, hippocampus, and cerebellum). Group
I mGluRs appear to be mostly restricted to the postsynaptic side of the
synapse (Luján et al. 1997
; Martin et al.
1992
), although small numbers of terminals staining for mGluR5
have been observed (Romano et al. 1995). Group II mGluRs
have been found to have a relatively broad distribution with rather
diffuse presynaptic staining (Luján et al. 1997
;
Petralia et al. 1996
; Shigemoto et al.
1997
). Conversely, members of group III have been found to be
more selectively associated with presynaptic sites of axon terminals in
the hippocampus (Shigemoto et al. 1997
), providing a
possible morphological correlation for the differences we noted in
these functional studies.
The lack of effect of any of the three mGluR antagonists in the absence
of blockade of glutamate transport indicated that most of the ADD in
this circumstance was caused by other mechanisms. Of the possible
mechanisms, we were able to rule out the participation of other
presynaptic receptors, i.e., GABAB, adenosine
A1/A2, and serotonin. A relatively small but
significant degree of AMPA receptor desensitization was observed when
ADD was pronounced but was negligible otherwise. Thus AMPA
desensitization could not account for most of the ADD. Alternatively,
ADD of EPSPs could be attributed to the intrinsic properties of
synapses. Of these, depletion of vesicles available for evoked release
(Liu and Tsien 1995; Zucker 1989
) or
presynaptic calcium-dependent transient modifications of evoked release
(Hsu et al. 1996
; Mori et al. 1994
) have
been suggested. The lack of effect of mGluR antagonists on ADD suggests
either that the t-ACPD and L-AP4 receptors
in LC are not feedback receptors in normal function or that the
conditions used in our tests were insufficient to produce a significant
level of activation of those presynaptic mGluRs. This is consistent with recent findings in the calyx of Held, which demonstrated that
mGluRs (group II and/or III) contributed only to a small portion
(~6%) of the ADD observed (von Gersdorff et al. 1997
).
We did observe that the glutamate uptake inhibitor t-PDC
caused a significant increase in ADD and that this change was reduced by the group III mGluR antagonists, supporting the idea that increased ambient EAA can produce presynaptic inhibition by mGluR activation. However, t-PDC had no effect on single evoked EPSP
amplitude, suggesting that although the transporters have little effect
on clearance of EAA from the synaptic cleft during relatively slow synaptic transmission, they may play a significant role during robust
presynaptic release of glutamate. Other reports also described activity-dependent endogenous activation of presynaptic mGluRs in the
presence of EAA uptake blockade. In cultured hippocampal neurons, the
frequency-dependent inhibition of EPSPs was not affected by the broad
range antagonist MCPG (Maki et al. 1995)
although depression of EPSPs by 250 µM t-PDC was
blocked by MCPG (Fitzsimonds and Dichter 1996
;
Maki et al. 1994
). In the nucleus of the tractus solitarius (NTS) brain slices, application of low doses (100 µM) of MCPG not only blocked the depression of excitatory synaptic transmission after 20-Hz stimulations, but the size of the EPSPs was
increased over control amplitudes (Glaum and Miller
1993
), suggesting that presynaptic mGluRs were tonically
activated under basal conditions. In hippocampal slices, evidence
indicated that continuous stimulation at 1 Hz resulted in the
activation of presynaptic mGluRs (Min et al. 1998
;
Scanziani et al. 1997
), an effect blocked by MCPG or
(2S,3S,4S)-methyl-2-(carboxycyclopropyl)-glycine (MCCG), suggesting a group II mGluR. Similar to our results,
t-PDC did not affect basal synaptic transmission in the
hippocampal slice (Scanziani et al. 1997
). However,
t-PDC significantly decreased the amplitude of EPSPs
evoked at 1 Hz and, again, MCPG reversed the effects of
t-PDC (Scanziani et al. 1997
).
Release of EAA from the corticostriatal system was enhanced by MCPG in
the presence of t-PDC, but not in its absence
(Lada et al. 1998
). Finally, in the hippocampal slice,
alteration of glutamate clearance by various means, such as changes in
temperature or application of a glutamate-scavenging agent, decreased
presynaptic mGluR activation by 1 Hz stimulation whereas decreasing
clearance (by increasing extracellular viscosity) had the opposite
effect (Min et al. 1998
). Overall, these results
indicate that presynaptic mGluRs can be endogenously activated under
specific conditions but that these conditions may vary between
different systems and protocols, likely because of the heterogeneity
that exists between synapses in different parts of the CNS. These ideas
are dealt with more extensively in a review by Anwyl
(1999)
.
Figure 7 is a schematic providing a
scenario that could explain our results. The
L-AP4-sensitive receptor was placed away from the site of
neurotransmitter release so that it would not be activated after the
relatively low frequency of release. However, it was placed close
enough so that it would be activated during high-frequency synaptic
transmission and spillover of neurotransmitters. Conversely, the
t-ACPD-sensitive receptor was placed far enough away
from the release site so that it would not be activated during high-frequency synaptic transmission. In this schematic, application of
exogenous agonists would result in the activation of either receptor.
Based on the present study, we propose that only the group III mGluR is
a feedback receptor in LC. The function of group II remains
unknown. However, mGluRs have been found away from the synapse (e.g.,
on axon and dendrite shafts) and could be targets for other synapses
(Martin et al. 1992; Petralia et al.
1996
; Shigemoto et al. 1996
). Note that the
glutamate transporters were omitted from the schematic. These have been
found on neurons and glia that envelop the terminals (Rothstein
et al. 1994
). High-density transporters have been shown to be
associated with the postsynaptic terminal (Rothstein et al.
1994
). In a recent study in Purkinje neurons, it was estimated
that there is a 15:1 ratio between EAAT4 alone and AMPA receptors
(Otis et al. 1997
). Therefore, even though the
transporter activity appears to be too slow to cause the termination of
synaptic transmission, the high concentration of transporter on the
postsynaptic membrane could substantially buffer EAA.
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Thus, in LC, MAP4-sensitive mGluRs appear to act as feedback receptors
under conditions where the clearance of EAA is saturated or impaired.
Physiological and pathophysiological implications for these findings
can be better appreciated in light of recent findings implicating EAA
transporters. For example, significant downregulation of the glial
transporter GLAST (assessed by Western blot analysis) was reported in
the piriform cortex/amygdala as early as 24 h after
kindling-induced epilepsy (stage 3 seizure) and persisted through
multiple stage 5 seizure (Miller et al. 1997). In
contrast, the neuronal transporter EAAC-1 was found to be upregulated
in the same areas, but only when the animal had reached stage 5. Therefore the ability for glutamate buffering between stages 3 and 5 was likely to be markedly reduced. Thus, although t-PDC
application may not reflect physiological conditions, it clearly mimics
conditions observed under specific pathologies such as epilepsy and
perhaps other abnormal conditions.
In summary, two groups of presynaptic mGluRs are present on LC afferents, but only those belonging to the group III mGluR were found to be activated under conditions that promoted EAA accumulation and spillover. We conclude that these receptors function as negative feedback receptors in LC. Conversely, the presynaptic group II mGluRs were not activated under the same conditions, suggesting that these receptors may be involved in a negative modulation of presynaptic transmission distinct from group III mGluRs, and ruling out duplication in function at presynaptic terminals innervating LC neurons.
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ACKNOWLEDGMENTS |
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We are grateful to Novartis Pharma Canada for generous provision of a sample of CGP35348.
This research was funded by the Medical Research Council of Canada.
Present address of G. R. Dubé: Center for Learning and Memory and Dept. of Brain and Cognitive Sciences, Massachusetts Institute of Technology, E25-435, Cambridge, MA 02139.
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FOOTNOTES |
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Address for reprint requests: K. C. Marshall, Dept. of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada.
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 16 September 1999; accepted in final form 4 November 1999.
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