1Laboratoire de Neurobiologie de l'Apprentissage et de la Mémoire, Centre National de la Recherche Scientifique, Unité de Recherche Associeé 1491, Université Paris-Sud, 91405 Orsay, France; and 2Postgraduate Studies in Pharmacology, School of Pharmacy, University of Bradford, Bradford BD7 1DP, United Kingdom
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ABSTRACT |
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Jay, T. M., E. Zilkha, and T. P. Obrenovitch. Long-term potentiation in the dentate gyrus is not linked to increased extracellular glutamate concentration. Long-term potentiation (LTP) of excitatory transmission is a likely candidate for the encoding and storage of information in the mammalian brain. There is a general agreement that LTP involves an increase in synaptic strength, but the mechanisms underlying this persistent change are unclear and controversial. Synaptic efficacy may be enhanced because more transmitter glutamate is released or because postsynaptic responsiveness increases or both. The purpose of this study was to examine whether increased extracellular glutamate concentration was associated with the robust and well-characterized LTP that can be induced in the rat dentate gyrus. To favor the detection of any putative change in extracellular glutamate associated with LTP, our experimental strategy included the following features. 1) Two separate series of experiments were carried out with animals under pentobarbital or urethan anesthesia; 2) changes in extracellular concentration of glutamate were monitored continuously by microdialysis coupled to enzyme amperometry; and 3) dialysate glutamate levels and changes in the slope of excitatory postsynaptic potential evoked by activation of the perforant path were recorded precisely at the same site. Tetanic stimulation of the perforant path increased persistently test-evoked responses in the dentate gyrus (by 19 and 14% in barbiturate and urethan group, respectively), but there was no glutamate change either during or after LTP induction and no indication of increased glutamate efflux when low-frequency stimulation was applied. The results do not rule out a possible contribution of enhanced glutamate exocytosis to LTP induction and/or maintenance because such a presynaptic change may not be detectable extracellularly. However, our findings and other data supporting the notion that neurotransmitter glutamate may hardly leak out of the synaptic cleft conflict with the hypothesis that LTP could also involve a broad synaptic spillover of glutamate.
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INTRODUCTION |
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Brief high-frequency stimulation of presynaptic
fibers can induce a long-lasting enhancement of synaptic transmission,
and this phenomenon [i.e., long-term potentiation (LTP)] may be one of the possible cellular mechanisms underlying learning and memory (Bliss and Collingridge 1993). Most of the excitatory
synapses where LTP can be elicited are operated by glutamate, a
transmitter that activates both ionotropic and metabotropic receptors.
Although it is generally acknowledged that induction of LTP involves
depolarization with subsequent postsynaptic Ca2+
influx through
N-methyl-D-aspartate (NMDA)
receptor-operated ion channels, how this initial Ca2+ rise
is converted into sustained enhancement of the synaptic response is
still unresolved. Synaptic strength may be persistently enhanced
because more transmitter glutamate is released or because postsynaptic
responsiveness increases or both (Kullmann and Siegelbaum 1995
). Early in vivo experimental data were interpreted as
indicative of LTP induction resulting in long-lasting enhancement of
transmitter release (Bliss et al. 1986
; Dolphin
et al. 1982
; Lynch et al. 1985
; Skrede
and Malthe-Sorenssen 1981
). For example, samples of
extracellular perfusate collected from a push-pull cannula implanted
into the dentate gyrus contained more glutamate after LTP induction (2- to 3-fold basal levels for 1.5 h after LTP induction) (Bliss et al. 1986
), and the specific NMDA receptor
antagonist D(
)-aminophosphonovalerate (APV) blocked both
LTP induction and glutamate increase (Errington et al.
1987
). However, these data could not be confirmed
(Aniksztejn et al. 1987
, 1989
; Roisin et al.
1990
), and the finding that both glutamate and aspartate
increased in the extracellular fluid after LTP (Bliss et al.
1986
) weakens the notion that these changes indicated enhanced
exocytosis. Indeed, aspartate is not accumulated in synaptic vesicles
(Burger et al. 1989
; Fykse et al. 1992
)
and therefore may not be a neurotransmitter (Nicholls
1993
; Orrego and Villanueva 1993
; however, see
Gundersen et al. 1998
).
Preliminary data obtained by monitoring extracellular glutamate with an
implantable biosensor suggested a sustained increase in basal glutamate
after LTP induction and a twice larger peak of stimulus-dependent (2 Hz) glutamate release measured 60 min after LTP induction
(Galley et al. 1993), but a full report of this study
was not published.
Although the "positive" results outlined previously are often taken
as strong evidence for a presynaptic component of LTP (Richter-Levin et al. 1995), whether increased
extracellular glutamate is associated with LTP induction and
maintenance is still unclear. This is an important issue because
persistently increased extracellular levels of glutamate may suggest
that spillover of transmitter from one synapse to the adjacent ones
contributes to LTP (Asztély et al. 1997
;
Kullmann et al. 1996
) and/or interneuronal spread of LTP
(Harris 1995
). The main purpose of this study was to
examine this question in vivo by application of effective and sensitive methods to the robust and well-characterized dentate gyrus LTP. Glutamate concentration in the dialysate was measured by
enzyme-amperometry (biosensor) as it emerged from a microdialysis probe
implanted into the hippocampus of anesthetized rats. As general
anesthesia may interfere with neurotransmitter release (Miao et
al. 1995
; Schlame and Hemmings 1995
), rats were
anesthetized with either pentobarbital or urethan, two anesthetics
agents with which dentate gyrus LTP was demonstrated in vivo
(Douglas and Goddard 1975
; Winson and Dahl
1986
). Field potentials evoked in the dentate gyrus by
electrical stimulation of the perforant path were recorded precisely at
the microdialysis sampling site. The effects of repetitive low-frequency (2 Hz) stimulation were also examined before and after
LTP induction, as previously performed by Galley and co-workers (1993)
.
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METHODS |
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Animal preparation
Adult male Sprague-Dawley rats (300-400 g; IFFA-Credo, L'Arbresle, France) were anesthetized throughout with either sodium pentobarbitone (65 mg/kg ip, subsequently supplemented as necessary; n = 10) or urethan (1.5 g/kg ip; n = 8) and positioned in a stereotaxic frame. Body temperature was maintained at 36.5-37.5°C by using a heating blanket.
A concentric microdialysis probe with an external 62-µm Ni-chrome
electrode (Fig. 1A) (2-mm
dialysis fiber length; Applied Neuroscience, London, U. K.) was slowly
lowered into the hilus of the dentate gyrus (coordinates: 4.2 mm
posterior to bregma, 2.5 mm lateral to midline) (Paxinos and
Watson 1986). A concentric bipolar stainless steel stimulating
electrode (300 µm OD) was placed ipsilaterally into the angular
bundle of the medial perforant path (coordinates: 8.0 mm caudal to
bregma, 4.3 mm lateral to midline) (Paxinos and Watson
1986
). Recording and stimulating electrodes were lowered under
electrophysiological control, and the depth, adjusted to optimize the
slope of the positive-going population excitatory postsynaptic
potential (EPSP), was generally 3 mm below the cortical surface for
both electrodes. The tip of the recording dialysis electrode was in or
just below the granule cell layer so that the dialysis membrane would
straddle CA1 and the dentate gyrus. The recording electrode was
consistently oriented toward the stimulating electrode relative to the
dialysis fiber to preserve the integrity of the perforant path between
stimulating and recording electrodes (Fig. 1B).
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Recording of extracellular field potentials
Test pulses (100 µs, 0.033 Hz) were delivered via an isolated constant current unit at an intensity that evoked a population spike of 20-40% of its maximum (200-500 µA). Field potentials were amplified, filtered (band-pass 0.1 Hz to 3 kHz), digitized at 20 kHz, and stored to disk as averages of four consecutive responses for off-line analysis. LTP was induced as follows: six series at 2-min intervals of six high-frequency trains (400 Hz for 20 ms) at 0.1 Hz. To increase the number of potentiated synapses the stimulus intensity was increased during the tetanus by 200 µA. Low-frequency stimulation (LFS) protocols (1,200 stimuli at 2 Hz) were conducted at baseline intensity in naive (nonpotentiated) and potentiated synapses.
On-line enzyme-amperometric analysis of dialysate glutamate
Microdialysis probes were perfused with artificial cerebrospinal fluid (composition in mM: 125 NaCl, 2.5 KCl, 1.18 MgCl2, 1.26 CaCl2, and 0.2 NaH2PO4, pH 7.3 adjusted with 1 M NaOH) at 1 µl/min with a syringe pump (CMA/100; CMA/Microdialysis, Stockholm, Sweden).
The outflow tube of the microdialysis probe was directly connected to a
glutamate biosensor (i.e., enzyme-amperometric flow cell). This method
was selected for monitoring changes in dialysate glutamate because it
is so far the most effective in terms of sensitivity, specificity, and
time resolution, and the delay between neurochemical changes and their
detection is reduced to 2-3 min (Zilkha et al. 1995).
Biosensors (Applied Neuroscience) consisted of two blocks of perspex.
One contained the reference (Ag/AgCl) electrode, the auxiliary
electrode (platinum), and the inlet and outlet ports to the cells; the
other incorporated the platinum working electrode. A thin
polytetrafluoroethylene (PTFE) gasket with a 0.5 × 19 mm
slot provided a narrow channel between the working electrode and all
the components of the opposite block listed previously. To eliminate
interference resulting from direct oxidation of ascorbic acid and other
electroactive compounds, a film of 1,2-diaminobenzene (Sigma Chemical,
Poole, U. K.) was deposited on the working electrode by
electropolymerisation. L-Glutamate oxidase (200 U/ml) was
immobilized with 2.5% glutaraldehyde in phosphate buffer. For a
detailed description of the method see Zilkha et al. (1995)
.
The glutamate signal was continuously digitized and stored on disk by using a computer (IBM PC equivalent) equipped with an A/D converter card (Metrabyte DAS-20, Keithley, Reading, U. K.) and running a dedicated program (ASYST laboratory software, Keithley, Reading, U. K.). Glutamate detection was calibrated at the end of each experiment with a 20 µM standard solution of L-glutamate.
Experimental protocol
Test pulses were delivered every 30 s unless otherwise stated. Two-hour postimplantation, animals were subjected to the following procedure: 20-min baseline; 10-min low-frequency (2 Hz) stimulation with test pulse intensity (i.e., frequency of electrical stimulation of the dentate gyrus was increased from 0.033 to 2 Hz for 10 min); 60-min baseline, LTP induction with six series, 2 min apart, of six trains (400 Hz for 20 ms) at 0.1 Hz, delivered at test intensity plus 200 µA; 60-min recording after the end of the tetanic stimulation; 10-min LFS (described previously); and 30-min baseline; death by intraperitoneal administration of anesthetic overdose.
Presentation and analysis of data
The synaptic component of the evoked response was measured by calculating the maximum slope of the early rising phase of the population EPSP. For each individual rat, the data were expressed as a percent change of the mean baseline value obtained before high-frequency stimulation or LFS.
Continuous storage of glutamate data in a digitized form together with
strict adherence to the previous experimental procedure allowed us to
average relevant corresponding sequences from all experiments (Figs.
3-5). Although microdialysis produces repeated measures, as our
statistical analysis was restricted to the comparison of means before
and after a single treatment in the same individuals, we used the
Student's paired t-test (Matthews et al.
1990). Data are means ± SE values throughout
RESULTS. P values not more than 0.05 were deemed
to be statistically significant.
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RESULTS |
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Figure 2 illustrates the potential
of microdialysis coupled to enzyme-amperometric analysis for the
detection of transient changes in extracellular glutamate
concentration. It shows glutamate efflux as the probe was inserted into
the brain (i.e., penetration injury) (Obrenovitch and Urenjak
1997b) and the characteristic biphasic pattern produced by
terminal ischemia (i.e., synchronous exocytosis followed by cytosolic
efflux) (Obrenovitch and Urenjak 1997a
).
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Changes in EPSP slope and extracellular glutamate associated with LTP induction
BARBITURATE ANESTHESIA.
Two experiments were omitted in this series because suitable LTP could
not be induced (technical difficulties with implantation). With
pentobarbital-anesthetized rats, high-frequency stimulation of the
medial perforant pathway consistently resulted in a robust potentiation
of the EPSP recorded in the hilus of the dentate gyrus. As shown in
Fig. 3A (top
graph), the EPSP slope expressed as a percentage of its pretetanus
value increased, reached a plateau a few minutes after the last
tetanus, and stayed at the same level for up to 60 min (19.4 ± 0.5%, n = 8). The population spike component was also
increased after tetanus (226 ± 7% of baseline; data not shown).
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URETHAN ANESTHESIA. Two experiments were omitted in this series, one because LTP could not be induced and the other because of excessive oscillations of the glutamate signal because of microdialysis perfusion irregularities. The magnitude of LTP elicited in the medial perforant pathway of urethan-anesthetized rats was comparable with that measured in pentobarbital-anesthetized rats. After tetanus, EPSP remained significantly potentiated for >60 min (increase by 13.8 ± 0.5%; n = 6; Fig. 3B, top graph), and the population spike component increased to 197 ± 13% of the baseline (data not shown).
In this group the average level of glutamate in the dialysate was 1.14 ± 0.11 µM (n = 6) during the 30 min preceding LTP induction, not significantly different from that measured under barbiturate anesthesia. No consistent change in extracellular glutamate was detected, either during or after LTP induction (Fig. 3B, bottom graph). In one experiment, a small positive shift (0.05-1.0 µM) of dialysate glutamate appeared to develop with LTP induction, but similar changes were observed in another experiment in absence of any electrical stimulation.Effect of LFS (2 Hz) on naive and potentiated synapses and extracellular glutamate
BARBITURATE ANESTHESIA. LFS (2 Hz) of the perforant pathway applied either 1 h before or after tetanus for 10 min produced a similar transient depression in the dentate gyrus synaptic transmission (Fig. 4, A and B). The EPSP slope decreased immediately after the beginning of LFS application, reached a plateau (reduction by 23.2 ± 0.4 and 20.7 ± 0.5% in naive and potentiated synapses, respectively; n = 5), and recovered to near-baseline values after resumption of test stimulation.
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URETHAN ANESTHESIA. The 2-Hz protocol also produced a temporary depression of EPSP (Fig. 5, A and B) in urethan-anesthetized rats. The magnitude of the change in EPSP slope was slightly larger than that observed under barbiturate (reduction by 25.4 ± 0.7 and 29.5 ± 0.4% in naive and potentiated synapses, respectively; n = 7 and n = 5), and the recovery from this change appeared more progressive than with barbiturate. In all cases, LFS produced a transient decrease in the EPSP slope, with complete disappearance of the population spike (data not shown).
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DISCUSSION |
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Methodology
Four circumstances were combined in this study to assist the
detection of any putative change in extracellular glutamate associated with LTP: 1) investigation of dentate gyrus LTP induced by
high-frequency stimulation of the medial perforant pathway because it
is robust and well characterized; 2) careful positioning of
the recording electrode relative to the microdialysis fiber and
stimulating electrode (Fig. 1) to record field potentials at the
sampling site and minimize damage to the afferent fibers; 3)
on-line enzyme-amperometric detection of glutamate, which provides
maximal sensitivity, selectivity, and time resolution (Zilkha et
al. 1995) (Fig. 2); and 4) two series of experiments
with different anesthetics.
Despite our precautions to preserve the integrity of the afferent
fibers between stimulating and recording electrodes, the potentiation
achieved when the recording electrode was adjacent to a dialysis fiber
was approximately one-half to one-third that obtained in previous
experiments with a recording electrode alone (Doyère et
al. 1996), suggesting that microdialysis adjacent to the
recording electrode may be deleterious to LTP induction and/or field
potential recording. Possible detrimental effects of microdialysis
include more pronounced tissue damage and buffering of potential
extracellular changes contributing to interneuronal spread of LTP
(Harris 1995
). However, a consistent and marked LTP was
still recorded precisely at the sampling site of the microdialysis probe (Fig. 3).
The representative changes in dialysate glutamate produced by
penetration injury and death (Fig. 2) and the fact that we were capable
of detecting changes as small as 0.05 µM associated with LFS (Figs. 4
and 5) illustrate the suitability and performance of microdialysis
coupled to enzyme-amperometric detection. In previous studies,
microdialysis coupled to enzyme-amperometric detection allowed us to
detect small increases in extracellular glutamate levels produced by
rising the concentration of K+ in the perfusion medium to
only 15 mM and to resolve very brief changes in glutamate associated
with spreading depression (Obrenovitch and Zilkha 1995a;
Obrenovitch et al. 1996
). As opposed to implantable biosensors (Walker et al. 1995
), flow
enzyme-amperometric analysis allowed us to obtain data even during
electrical stimulation.
The exposed length of the probe extended above and below the tip of the recording electrode (Fig. 1) and therefore a substantial proportion of the collecting area were not in the middle of the molecular layer where the medial perforant path fibers terminates. Despite this inevitable "dilution" of the glutamate signal arising from the molecular layer, the high sensitivity of our detection system should have been capable of recording any relevant changes in extracellular glutamate associated with induction of LTP in this layer.
No detectable change in extracellular glutamate was associated with LTP induction: implications for the presynaptic hypothesis of LTP
Our data (Fig. 3) clearly contradict the notion that LTP induction
in the hippocampal dentate gyrus may be associated with an increase in
extracellular concentration of glutamate. However, these data do not
rule out a possible contribution of enhanced presynaptic glutamate
release to LTP induction and/or maintenance because such a change may
remain undetected extracellularly. This point can be exemplified with
data obtained with 4-aminopyridine (4-AP), a suitable agent for
selective induction of exocytotic glutamate release from synaptosomes
(Tibbs et al. 1996). In contrast to in vitro studies
Segovia et al. (1997)
did not find any increase in dialysate levels of
glutamate when 4-AP (1, 5, and 10 mM) was perfused through the probe
despite a decrease in extracellular glutamine that could reflect
recycling of transmitter glutamate and GABA via the glutamine synthesis
pathway (Hertz 1979
; Westergaard et al.
1995
). In the course of the study reported herein 4-AP (1 and
10 mM) was applied through the probe in three separate experiments.
This agent did not increase the dialysate levels of glutamate despite a
marked enhancement of local EPSPs (Obrenovitch 1998b
).
Similarly, we did not detect any change in extracellular glutamate
levels in the dentate gyrus during tetanic stimulation of the perforant
path (Fig. 3).
In addition, raised extracellular glutamate levels may not necessarily
imply enhancement of its presynaptic release (Miele et al.
1996; Obrenovitch 1998b
; Timmerman and
Westerink 1997
). For example, neuronal activation subsequent to
induced grooming in rats was accompanied by enhanced glutamate efflux,
but this change was not reduced when TTX was present in the
microdialysis perfusion medium (Miele et al. 1996
).
Increased glutamate exocytosis may not lead to glutamate accumulation
in the extracellular space because of one or several of the following
elements: synapse ultrastructure (Edwards 1995), relatively small contribution of synapses to the overall cell membrane
area (Rusakov et al. 1998
), efficient glutamate uptake mechanisms (Eliasof and Werblin 1993
), and changes in
extracellular glutamate of nonneuronal sources masking those much
smaller of synaptic origin.
Whether LTP in the dentate gyrus is associated with increased glutamate
exocytosis might be settled with in vitro studies where glutamate
release is evaluated by whole cell recording of glial glutamate
transport currents, i.e., by taking advantage of the electrogenicity of
glutamate uptake (Barbour et al. 1991). So far
application of this strategy to hippocampal slices suggested that LTP
does not alter the amount of glutamate released on synaptic stimulation
(Diamond et al. 1998
; Luscher et al.
1998
). Other promising experimental strategies may be those
relying on specific pharmacological agents (e.g., selective and
noncompetitive inhibitors of glutamate uptake) and measurements of
changes in exocytotic-endocytotic cycling (Malgaroli et al.
1995
).
No detectable change in extracellular glutamate was associated with LTP induction: implications for the spillover hypothesis of LTP
It was suggested that spillover of transmitter from one synapse to
the adjacent ones, first discovered at lower vertebrate inhibitory
synapse (Faber and Korn 1988), may also contribute to
enhance the size of quantal events in hippocampal cells during LTP,
provided there is an increase of glutamate release that raises its
concentration in the synaptic cleft (Kullmann et al.
1996
). Despite obvious differences in the geometry and
processes governing microdialysis probe/surrounding tissue exchanges on
one hand and putative transmitter spillover from one synapse to the
adjacent ones on the other hand, we consider that our data and other
findings conflict with the spillover hypothesis of LTP.
1) Two different groups presented evidence that LTP spreads
to neighboring cells located within 150 µm of the potentiated cell
(Bonhoeffer et al. 1989
; Schuman and Madison
1994
). As microdialysis should be capable of revealing any
glutamate efflux diffusing over such a distance (Benveniste and
Hüttemeier 1990
), LTP is unlikely to be associated with a
broad spillover of glutamate.
2) One would anticipate that during tetanic stimulation
synchronized exocytosis should result in a generalized spillover from glutamatergic synapses, but this did not occur in these experiments (Fig. 3). Similar, apparently paradoxical observations were made with
drug-induced seizures (Obrenovitch et al. 1996).
3) If synaptic spillover of transmitter glutamate were to
occur and to be functionally relevant, one would expect markedly increased extracellular levels of glutamate, subsequent to local application of exogenous glutamate and/or pharmacological inhibition of
its uptake, to produce obvious abnormalities. This was not the case in
our studies (Obrenovitch et al. 1996, 1997
), even when
high extracellular glutamate was superimposed on ischemia or spreading
depression (Obrenovitch and Zilkha 1995b
;
Obrenovitch et al. 1998
).
LTP induction and increased glutamate efflux in previous studies: critical re-appraisal
We already mentioned that earlier findings, obtained with the
push-pull method and interpreted as indicative of increased glutamate
exocytosis, should not be taken as strong evidence of presynaptic
mechanisms in LTP (Bliss et al. 1986; Errington
et al. 1987
).
Despite our efforts to reproduce as much as possible the experimental
procedure of Galley et al. (1993), we could not confirm their findings
(sustained increase in basal and stimulus-dependent glutamate efflux
during LTP). From our experience with glutamate biosensors, we propose
that the changes in amperometric current obtained with their implanted
biosensors might not have reflected actual changes in glutamate.
Implantable devices are prone to interferences (i.e., changes taking
place in the tissue may alter the device sensitivity and/or
selectivity), and a thorough validation of implantable devices is
necessary, especially when only small changes in extracellular
glutamate are expected (Obrenovitch 1998a
; Obrenovitch and Zilkha 1998
).
In two studies, LTP was induced in vivo, and depolarization-induced
release of endogenous glutamate subsequently tested ex vivo in
potentiated and control hippocampal preparations. Ghijsen et al. (1992)
found that the K+-induced
Ca2+-dependent release of glutamate from
CA1 subslices was increased 150% in rats killed 30 min after LTP
induction. Canevari et al. (1994)
reported that K+ and
veratridine release of glutamate was enhanced in synaptosomes prepared
from potentiated dentate gyri. Both groups concluded that these results
suggest a persistent increase in the presynaptic vesicular pool of
glutamate during LTP, but the following alternative interpretation was
overlooked: enhanced glutamate efflux from tissue previously subjected
to tetanic stimulation may actually reflect its increased sensitivity
to the depolarizing agents used for glutamate release induction. Two
intriguing findings are in line with the latter hypothesis.
1) GABA release from CA1 subslices was also enhanced 30 min
after LTP induction (Ghijsen et al. 1992
); 2)
with 4-AP, a depolarizing agent that triggers exocytosis more specifically than K+ and veratridine (Tibbs et al.
1996
), glutamate release was not significantly enhanced in
synaptosomes prepared from potentiated dentate gyri (Canevari et
al. 1994
).
Effects of low-frequency electrical stimulation
As previously reported, repetitive LFS of the medial perforant
path results in transient but not sustained depression in both the EPSP
and population spike in the dentate gyrus (Errington et al.
1995; Harris et al. 1979
). Such decrements of
the synaptic response under repetitive stimulation could result from
presynaptic or postsynaptic mechanisms. Along with the feedforward
inhibitory influence on granule cells (defined as inhibition driven by
excitatory afferents of inhibitory interneurons) involving
GABA-mediated mechanisms recruited at frequency above 1 Hz
(Sloviter 1991
), a difference in the strength of the
excitatory drive could also contribute to the depression of the
synaptic response. It is also clear that under repetitive stimulation
the releasable transmitter in a limited store is depleted, and the
presynaptic terminals are not instantaneously replenished. Given the
brief time window in which the depression of the signal occurs, one
would have predicted an immediate change in the concentration of glutamate.
We are confident that the slight shifts in amperometric current
associated with 10 min of 2-Hz electrical stimulation reflected changes
in extracellular glutamate (Figs. 4 and 5). A small, transient decrease
was observed in barbiturate-anesthetized rats, whereas a small,
apparently more persistent increase occurred in the urethan group. The
biological significance of these changes and their possible origins are
difficult to assess. One interesting feature, however, is the
difference in pattern of changes obtained with barbiturate and urethan
anesthesia, suggesting that anesthetics may mask or favor different
mechanisms (exocytosis, uptake, or metabolism) influencing glutamate
homeostasis. Although the precise mechanism of the anesthetics, urethan
and pentobarbital are unknown, these results could be explained by a
differential contribution of alteration in the inhibitory response.
Barbiturates like pentobarbital are known to enhance the
GABAA receptor-mediated, chloride-dependent inhibition,
whereas urethan appears to have negligible effects on GABAergic
transmission (Engström et al. 1990).
In conclusion, our data demonstrate that LTP is not associated with increased extracellular concentration of glutamate. This finding does not rule out a possible contribution of enhanced presynaptic glutamate release to LTP induction and/or maintenance but conflicts with the notion that LTP may involve a broad synaptic spillover of glutamate.
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ACKNOWLEDGMENTS |
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The authors thank Dr. H. Kusakabe for a generous gift of L-glutamate oxidase.
This work was supported by grants from Centre National de la Recherche Scientifique, Fondation Cino del Duca, and the Thompson Fund.
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FOOTNOTES |
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Address for reprint requests: T. M. Jay, Laboratoire de Neurobiologie de l'Apprentissage et de la Mémoire, CNRS-URA 1421, Université Paris-Sud, 91405 Orsay, France.
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 8 September 1998; accepted in final form 9 December 1998.
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REFERENCES |
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