Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
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Diamond, Jeffrey S. and Craig E. Jahr. Synaptically Released Glutamate Does Not Overwhelm Transporters on Hippocampal Astrocytes During High-Frequency Stimulation. J. Neurophysiol. 83: 2835-2843, 2000. In addition to maintaining the extracellular glutamate concentration at low ambient levels, high-affinity glutamate transporters play a direct role in synaptic transmission by speeding the clearance of glutamate from the synaptic cleft and limiting the extent to which transmitter spills over between synapses. Transporters are expressed in both neurons and glia, but glial transporters are likely to play the major role in removing synaptically released glutamate from the extracellular space. The role of transporters in synaptic transmission has been studied directly by measuring synaptically activated, transporter-mediated currents (STCs) in neurons and astrocytes. Here we record from astrocytes in the CA1 region of hippocampal slices and elicit STCs with high-frequency (100 Hz) stimulus trains of varying length to determine whether transporters are overwhelmed by stimuli that induce long-term potentiation. We show that, at near-physiological temperatures (34°C), high-frequency stimulation (HFS) does not affect the rate at which transporters clear glutamate from the extrasynaptic space. Thus, although spillover between synapses during "normal" stimulation may compromise the absolute synapse specificity of fast excitatory synaptic transmission, spillover is not exacerbated during HFS. Transporter capacity is diminished somewhat at room temperature (24°C), although transmitter released during brief, "theta burst" stimulation is still cleared as quickly as following a single stimulus, even when transport capacity is partially diminished by pharmacological means.
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INTRODUCTION |
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After its release at excitatory synapses in
the CNS, glutamate is taken up into neurons and glia via
sodium-dependent, plasmalemmal transporters. High-affinity transport is
the primary mechanism by which extracellular glutamate is maintained at
low levels, providing protection against glutamate-induced
excitotoxicity (Rothstein et al. 1996). Transporters
speed the clearance of glutamate from the cleft during the first
millisecond of a synaptic event (Diamond and Jahr 1997
;
Tong and Jahr 1994
) and have been shown at several
synapses to help shape the postsynaptic response (Barbour et al.
1994
; Mennerick and Zorumski 1995
; Otis
et al. 1996
; Takahashi et al. 1995
). Recent
evidence also suggests that glial transporters in the hippocampus may
limit the extent to which glutamate can spill over from one synapse to
another in the CA1 region (Asztely et al. 1997
) and
activate metabotropic glutamate receptors at mossy fiber synapses in
CA3 (Min et al. 1998
; Scanziani et al. 1997
). Although postsynaptic neuronal transporters remove a
significant fraction of transmitter at the cerebellar climbing
fiber-Purkinje cell synapse (Otis et al. 1997
), several
lines of evidence suggest that glutamate is transported primarily into
glia (Bergles and Jahr 1998
; de Barry et al.
1982
; Kojima et al. 1999
; McLennan 1976
; Rothstein et al. 1994
,
1996
; Tanaka et al. 1997
; Wilkin et al. 1982
). The proposed buffering action of transporters
would require a high density of transporters in glial processes, which has been demonstrated with both biochemical (Lehre and Danbolt 1998
) and electrophysiological (Bergles and Jahr
1997
) approaches.
By reducing glutamate spillover, glial transporters may help to
isolate synapses, preserving the signal specificity thought to be
required for efficient information processing. However, recent
physiological evidence suggests that, in the hippocampus, such
isolation may be incomplete (Asztely et al. 1997;
Kullmann et al. 1996
), perhaps due to the fact that many
adjacent synapses in CA1 stratum radiatum have no glial processes
between them (Harris and Ventura 1998
; Lehre and
Danbolt 1998
). Moreover, it is not known to what degree
glutamate transporters are occupied by synaptically released
transmitter, or the extent to which this occupancy is affected by
increased levels of synaptic activity, such as during bursts of
high-frequency stimulation (HFS) that induce long-term potentiation
(LTP) (Bliss and Lomo 1973
). Synapse specificity is
generally assumed to be preserved during HFS, even though the ability
of glutamate transporters to accommodate the increased amounts of
glutamate released during such episodes has not been tested. If
transporters were overwhelmed by glutamate during HFS, transmitter
might diffuse further from its point of release and activate
extrasynaptic metabotropic receptors or even ionotropic glutamate
receptors in neighboring, inactive synapses. The latter effect, which
has become known as "spillover," could lead to non-Hebbian changes
in synaptic efficacy.
The present experiments used synaptically activated, transporter-mediated currents (STCs) recorded in astrocytes located in CA1 stratum radiatum of hippocampal slices to study the time course of glutamate transport under different stimulus conditions. The results indicate that at physiological temperatures transporters are capable of clearing glutamate released during a burst of HFS nearly as quickly as after a single stimulus. At room temperature, transporters appear overwhelmed during longer bursts of HFS (e.g., "tetanic" stimulation), but not during shorter trains (e.g., "theta" stimulation).
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METHODS |
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Slice preparation and extracellular solutions
Hippocampal slices (400 µm) were prepared from 13- to
15-day-old Sprague-Dawley rats, as described (Bergles and Jahr
1997) and in accordance with institutional guidelines. Slices
were prepared in ice-cold artificial cerebrospinal fluid (ACSF)
containing (in mM) 119 NaCl, 2.5 KCl, 1.3 MgCl2,
2.5 CaCl2, 26.2 NaHCO3 and
11 glucose, bubbled with 95% O2-5%
CO2, transferred to identical solution at 34°C
for 30 min, and room temperature thereafter. Immediately before
recording, a cut was made between CA1 and CA3 to reduce the propagation
of epileptiform activity. Experiments were performed with control ACSF
containing (in mM) 119 NaCl, 2.5 KCl, 4 MgCl2, 4 CaCl2, 26.2 NaHCO3, 11 glucose, and 0.1 picrotoxin, equilibrated with 95%
O2-5% CO2 and delivered
via a gravity-fed perfusion system (2-5 ml/min). Except where noted,
the A1 adenosine receptor antagonist
8-cyclopentyl-1,3-dimethylxanthine (8-CPT, 4 µM) was included in all
solutions, to increase release probability (although see
RESULTS) and also to reduce the possibly confounding effects of temperature on adenosine transport (Diao and
Dunwiddie 1998
). For astrocyte experiments, the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
N-methyl-D-aspartate (NMDA) receptor antagonists 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX, 5 µM) and
(RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 5 µM)
were added to the control ACSF to block the neuronal field potential
that obscures the STC. For pyramidal cell experiments, 5 µM CPP was
included to isolate the AMPA receptor-mediated component of the
excitatory postsynaptic current (EPSC).
Electrophysiology
Whole cell (Axopatch 1D) recordings were made from astrocytes in
stratum radiatum and from pyramidal cells in stratum pyramidale of the
CA1 region. Whole cell electrodes (1.5-2.5 M) were filled with (in
mM) 120 K methane sulfonate (for astrocyte recordings) or Cs methane
sulfonate (for pyramidal cell recordings), 10 EGTA, 20 HEPES, 2 MgATP,
and 0.2 NaGTP (pH 7.4). Access resistance, estimated from the peak of
the current transient elicited by a brief 1- to 2-mV test pulse
preceding synaptic stimulation, was typically 5-15 M
and was not compensated.
Astrocytes were identified by their small cell bodies, low (10 M
)
input resistance, and high resting potentials (approximately
95 mV).
Astrocytes were held at their resting potential; pyramidal cells were
held at
70 mV. Stimuli (60-200 µA, 100 µs duration) were
delivered via a bipolar stimulating electrode placed in stratum radiatum ~200 µm from the whole cell electrode.
Salts and glucose were obtained from Mallinckrodt (Paris, KY), and all
other reagents were obtained from Sigma (St. Louis, MO), except NBQX
(RBI, Natick, MA), CPP (RBI), 8-CPT (RBI),
(RS)--cyclopropyl-4-phosphonophenylglycine (CPPG, Tocris Cookson,
St. Louis, MO), and (RS)-
-methyl-4-carboxyphenylglycine (MCPG,
Tocris Cookson).
Analysis
Data acquisition and analysis was performed with custom macros
written in Igor Pro (WaveMetrics). Data were sampled at 10-20 kHz and
filtered at 2 kHz. In whole cell recordings from astrocytes, the
amplitude of the stimulus-activated steady-state potassium current was
subtracted from the peak current to obtain the STC amplitude. For
coefficient of variation (CV) analysis (Fig.
1), the variance of the potassium current
(measured 65 ms after stimulation) was subtracted from the variance of
the peak amplitude before calculating the CV of the STC. In some
experiments, responses elicited in the presence of 300 µM
dihydrokainate (DHK) and 500 µM threo--hydroxyaspartic acid (THA),
which completely blocked the STC, were subtracted from responses in 300 µM DHK +100 µM THA to eliminate the potassium current. This
operation did not affect the time course of the STC decay. Unless noted
otherwise, all data are expressed as means ± SD and P
values were calculated using a paired t-test.
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RESULTS |
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Whole cell recordings were made with patch electrodes in
voltage-clamp mode from astrocytes located in stratum radiatum of the
CA1 region of hippocampal slices, and synaptic responses were elicited
by stimulating the Schaffer collateral/commissural fibers. AMPA and
NMDA receptor antagonists (5 µM NBQX and 5 µM CPP) were applied to
block the postsynaptic field depolarization generated in the
surrounding neurons. Under these conditions, synaptically activated
currents in astrocytes consisted of a small, long-lasting component,
thought to arise from the slow reequilibration of extracellular potassium following stimulation, and a transient component (Fig. 1A). This transient current can be blocked by a cocktail of
the glutamate transporter antagonists DHK and THA (Bergles and
Jahr 1997; Diamond et al. 1998
), identifying it
as a STC (Otis et al. 1997
). Although glutamate
transport is associated with an anion conductance (Fairman et
al. 1995
; Wadiche et al. 1995
), the STCs described here were recorded with an impermeant anion (methane sulfonate) in the patch pipette and therefore primarily reflect the
charge flux arising from the electrogenic glutamate transport cycle.
STCs reflect activation of a large number of synapses
STCs recorded in astrocytes have been shown to follow changes in
release probability (Diamond et al. 1998; Luscher
et al. 1998
), but the number of synapses contributing to an
astrocyte STC (i.e., the quantal content, m) is unknown.
Because postsynaptic responses to single-vesicle events cannot be
resolved in astrocytic recordings, the coefficient of variation
(CV =
/mean) method (Clements 1990
; del
Castillo and Katz 1954
; Faber and Korn 1991
) was
used to obtain a rough estimate of the quantal content of STCs relative
to pyramidal cell EPSCs in the same slice.
Inspection of 50 consecutive STCs recorded in an astrocyte (Fig.
1A) indicated that the responses exhibited very little
trial-to-trial variation. EPSCs elicited by identical stimulation in a
nearby pyramidal cell in the same slice were significantly more
variable (Fig. 1B). In four slices the CV of evoked STCs
(0.006 ± 0.002, mean ± SD) was 15 times less than that of
EPSCs (0.09 ± 0.03) recorded from pyramidal cells located
equidistant from the stimulating electrode (P = 0.012).
If transmitter release is a Poisson process, then the CV of the
response amplitude is inversely proportional to the square root of the
mean quantal content (e.g., m = 1/CV2), suggesting that an astrocyte STC may
reflect release from several hundred times as many synapses as a
pyramidal cell EPSC. Two factors complicate more precise quantitation
of these results. First, release probability at excitatory synapses
onto CA1 pyramidal cells is probably too great to be described
adequately by Poisson statistics (Hjelmstad et al. 1997;
Stevens and Wang 1995
). Second, much of the STC is
likely to be shunted by the low resistance astrocytic membrane,
although this would probably affect the variance and mean to similar
extents (see next section) and therefore exert little effect on the CV.
STCs reliably report changes in response amplitude and time course
The experiments described in this study rely on the ability
to accurately record changes in the size and shape of the STC under
different conditions. To test for this, STCs were elicited by a range
of stimulus intensities, which caused broad changes in response
amplitude (Fig. 2A1).
Normalizing these responses indicated that large differences in
amplitude did not affect the time course of the STC (Fig.
2A2). Similar results were observed in the presence of 300 µM DHK, a competitive inhibitor of a glutamate transporter subtype,
GLT-1, that is expressed in astrocytes (Johnston et al.
1979; Rothstein et al. 1994
) (Fig.
2B). Moreover, varying stimulus intensity to change the size
of the response did not alter the effect of DHK on the STC amplitude
(
, Fig. 2C1), charge transfer (
, Fig. 2C1),
or time course (Fig. 2C2). Hippocampal astrocytes have
extremely low input resistances (
10 M
), which, although severely
compromising space clamp by the recording electrode, would also limit
the membrane depolarization caused by electrogenic transport
(Hausser and Roth 1997
; Spruston et al.
1993
). Therefore, although the current measured at the soma
reflects a large underestimate of synaptic conductance,
proportional changes appear to be recorded faithfully.
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Measuring the occupancy of transporters during synaptic transmission
Our strategy to determine the extent to which transporters are
occupied by synaptically released glutamate was based on the following
rationale: if glutamate released during bursts of high-frequency synaptic stimulation were sufficient to saturate transporters, then the
glutamate released late in the burst would remain in the extrasynaptic
space for a longer time than following a single stimulus because
transporter binding capacity would be overwhelmed. This would result in
a slowing of the STC decay. To test this prediction, STCs were elicited
by single stimuli and 100-Hz trains of 3, 4, 9, or 10 stimuli (Fig.
3A). STCs elicited by single
stimuli decayed exponentially (at 24°C, = 16.3 ± 2.9 ms, n = 25). To extract the response to the fourth
stimulus, the response to a train of three stimuli was subtracted from
a response to a train of four stimuli. The response to the 10th
stimulus was extracted in an analogous manner (Fig. 3B). The
decay time courses of the subtracted responses could then be compared
with the single-stimulus response by fitting the decays with a
single-exponential function and calculating the ratios
4/
1 or
10/
1. In many cases,
responses elicited 15-30 s after HFS were slightly enhanced, due to
residual posttetanic potentiation. This often resulted in small
differences in the amplitudes of responses in trials immediately
following single-stimulus trials and responses in trials immediately
following HFS trials (Fig. 3A). Consequently, subtracted
traces often contained a small offset (subtracted out in Fig.
3B) that did not affect the shape of the STC.
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The rate of glutamate transport is highly temperature dependent
(Q10 ~ 3) (Wadiche and Kavanaugh
1998), suggesting that the occupancy of transporters during the
STC may be sensitive to recording temperature. To account for this
possibility, the experiments described below were conducted both at
room temperature (24°C) and also at a higher, near-physiological
temperature (34°C). At 24°C,
4 was only
slightly greater than
1
(
4/
1 = 1.06 ± 0.03, n = 5, P = 0.008; Fig.
4, A1 and B). When
the bath was warmed to 34°C,
1 of the STC
decreased significantly
(
1,34°C/
1,24°C = 0.57 ± 0.04, n = 9, P < 0.0001),
but
4 and
1 remained
nearly equal (
4/
1 = 1.03 ± 0.04, n = 5, P = 0.2; Fig.
4B). By contrast, a marked difference between
10 and
1 was observed
at 24°C (
10/
1 = 1.82 ± 0.07, n = 5, P = 0.00002;
Fig. 4, A1 and C), but this effect was greatly
reduced at 34°C
(
10/
1 = 1.13 ± 0.06, n = 5, P = 0.007; Fig. 4,
A2 and C).
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The prolonged decay of 10 relative to
1 at 24°C may reflect a slowing in the
clearance of synaptically released glutamate, but it could also be due
to other factors, such as an increase in the asynchrony of release
during a train of HFS. To test for this latter possibility, the above
experiment was repeated in pyramidal cells at 24°C. If the slowing of
10 in the STC were due primarily to an
increase in release asynchrony, a similar effect should also be evident
in AMPA receptor EPSCs elicited by identical stimulation. However,
10 of pyramidal cell AMPA EPSCs (12.1 ± 3.7 ms, n = 6) was much less than
10 in astrocyte STCs (25.1 ± 3.2 ms,
n = 5; Fig. 4D), indicating that the slow STC decay is not due to an increase in release asynchrony.
In the above experiments, A1 adenosine receptors
were blocked by 8-CPT (see METHODS), a manipulation that
increases release probability at these synapses (Brundege and
Dunwiddie 1997). Although 8-CPT increases STC amplitude
(Luscher et al. 1998
), its effect on total release
during a train is complex, due to a concomitant decrease in
facilitation. At 24°C, the total charge transfer during a train
(Q10) relative to that during the response to a
single stimulus (Q1) was significantly reduced in
the presence of 8-CPT (
8-CPT:
Q10/Q1 = 13.7 ± 2.9, n = 6; +8-CPT:
Q10/Q1 = 9.0 ± 1.4, n = 6; P = 0.005, unpaired
t-test). With adenosine-mediated inhibition intact,
10 was still slowed relative to
1
(
10/
1 = 1.48 ± 0.25, n = 6, P = 0.0078), although to a
lesser extent than in the presence of 8-CPT (P = 0.017, unpaired t-test). All subsequent experiments were performed
in the presence of 8-CPT.
We interpret these results to indicate that transmitter released by a 100-Hz barrage of 10 stimuli at 24°C remains at an elevated concentration longer after the 10th stimulus because transporters are unable to clear glutamate as rapidly as after a single stimulus. At 34°C, however, transport appears capacious enough to clear transmitter quickly, even after a burst of 10 stimuli.
Effect of transporter antagonists on STC elicited by stimulus trains
The above results indicate that the glutamate released in response
to the fourth in a 100-Hz train of stimuli is cleared about as quickly
as transmitter released in response to a single stimulus. This suggests
that the capacity of the transporters is not exceeded during a
four-stimulus train. To determine how far transporters are from
saturation under these stimulus conditions, we sought to decrease the
transport capacity of the astrocyte until an increase in
4/
1 was observed.
This was initially attempted by blocking a fraction of transporters
with one of two competitive antagonists. At 300 µM, DHK blocks
transport mediated specifically by the EAAT2/GLT-1 transporter subtype
(Arriza et al. 1994
), which makes up nearly 80% of the
transporters in stratum radiatum (Lehre and Danbolt 1998
). THA does not differentiate between transporter subtypes but, unlike DHK, also acts as a substrate (Arriza et al.
1994
), reducing its efficacy because uptake lowers its
concentration. STC amplitude was reduced by either 300 µM DHK (to
44 ± 4% of control; n = 5; Fig.
5A1) or 300 µM THA (to
41 ± 10% of control; n = 6; Fig.
5B1), in agreement with a previous report (Bergles and Jahr 1997
). In addition, both drugs slowed the decay of the STC (Fig. 5, A1 and B1), possibly indicating that
transporters near active synapses normally take up much of the
synaptically released glutamate but are partially prevented from doing
so in the presence of antagonist, allowing glutamate to diffuse a
greater distance before being transported. It may be that transporters close to the release site actually are saturated by transmitter, whereas those further away remain unoccupied, a three-dimensional analogue of the "saturated disk" phenomenon described at the
neuromuscular junction (Hartzell et al. 1975
). The
slowing of the STC in DHK or THA suggests that the size of the
"saturated sphere" is inversely related to transport capacity.
Expanding the dimensions of this region of saturation with transporter
antagonists might cause neighboring "spheres" to overlap during
high-frequency stimulation, leading to saturation of all the
transporters between release sites and, as a result, slower clearance.
However, neither DHK (Fig. 5A3) nor THA (Fig.
5B3) alone affected
4/
1 (DHK:
4/
1 = 110 ± 24% of control, n = 5, P = 0.4; THA:
4/
1 = 108 ± 10% of control, n = 6, P = 0.1; see
Fig. 7). These results suggest that total transport capacity is not
overwhelmed during a brief HFS train, even when a majority of the
transporters are blocked by a competitive antagonist.
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To reduce the transport capacity even further, 100 µM THA was added
to the bath together with 300 µM DHK (Fig.
6). At 24°C this antagonist cocktail
decreased the STC amplitude to 26 ± 7% (n = 9)
of control and slowed 1 by 277 ± 168%
(n = 9; e.g., Fig. 6A). In addition, the
DHK/THA cocktail increased
4/
1 to 161 ± 49% of control (n = 8; P = 0.008; Fig.
7). At 34°C the DHK/THA cocktail
exerted similar effects on STCs elicited by single stimuli (Fig.
6B), decreasing the STC amplitude to 35 ± 10%
(n = 7) of control and slowing
1 by 350 ± 77% (n = 7),
yet the effect on
4/
1
was completely abolished (100 ± 19% of control,
n = 7, P = 0.97; Fig. 7). These results
indicate that blocking a large majority of transporters can cause a
marked slowing of transmitter clearance at 24°C. At 34°C, however,
it appears that transport capacity is so great that even a small
fraction of transporters is able to take up transmitter released during
a brief train of high-frequency stimulation.
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Effects of metabotropic glutamate receptor antagonists on STCs
Exogenous activation of metabotropic glutamate receptors (mGluRs)
decreases the probability of release at excitatory synapses onto many
cell types in the brain, including CA1 pyramidal cells (Baskys
and Malenka 1991; Forsythe and Clements 1990
).
Synaptic activation of mGluRs reduces transmitter release at CA3 mossy fiber synapses (Min et al. 1998
; Scanziani et al.
1997
) and, to a lesser extent, at a calyceal synapse in the
brain stem (von Gersdorff et al. 1997
), but effects in
CA1 have not been reported. It is also unknown whether activation of
presynaptic mGluRs in CA1 might be restricted by glutamate
transporters, as in CA3 (Min et al. 1998
;
Scanziani et al. 1997
), although DHK has been shown to
increase activation of postsynaptic mGluRs elicited by very strong
stimulation in CA1 pyramidal cells (Cognar et al. 1997
). Blocking the majority of transporters with the DHK/THA cocktail in the
experiments described above (Fig. 6) may have elevated ambient levels
of glutamate or extended the extrasynaptic diffusion of synaptically
released glutamate sufficiently to increase activation of mGluRs and
perhaps affect the shape of the STC during an HFS train. To test this
possibility, the effects of the mGluR antagonists CPPG (300 µM) and
MCPG (1 mM) were measured on STCs elicited by single stimuli and
four-stimulus (100 Hz) trains. At 24°C in the presence of DHK and
THA, the addition of MCPG and CPPG did not affect the response to a
single stimulus (P1 in MCPG/CPPG = 89 ± 11% of control, n = 4, P = 0.2).
This result suggests that blocking transporters with the DHK/THA
cocktail did not elevate ambient glutamate levels sufficiently to
change release probability via activation of mGluRs. CPPG and MCPG
caused only a small increase on
4/
1, either in the
absence (110 ± 5% of control, n = 4, P = 0.026) or in the presence (107 ± 8% of
control, n = 5, P = 0.15) of 300 µM
DHK and 100 µM THA. Similar results were observed at 34°C in the
presence of DHK and THA (114 ± 12% of control, n = 5, P = 0.06). The large increase in
4/
1 observed at
24°C in the presence of DHK and THA (Figs. 6 and 7) therefore appears not to be due to increased activation of mGluRs when glutamate uptake
is reduced.
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DISCUSSION |
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The experiments presented here used transporter-mediated synaptic responses in hippocampal CA1 astrocytes to explore whether glutamate transporters are overwhelmed by HFS. The results indicate that, at 24°C, transporters clear glutamate released during a brief (4-stimulus) burst of HFS quite easily. Significant slowing of transmitter clearance was observed only if a large majority of transporters were blocked with competitive antagonists. At 34°C, even the small remaining fraction of transporters appeared capable of clearing glutamate after a brief burst of HFS. In the absence of antagonists, transporters were overwhelmed by longer (10-stimulus) HFS bursts at 24°C but not at 34°C. Taken together, these results suggest that glutamate transporters do a remarkably good job of clearing synaptically released glutamate over a range of stimulus conditions, particularly at physiological temperatures.
Mechanisms underlying the time course of the STC
Implicit in this interpretation is the assumption that the decay
of the STC reflects the decline in the extrasynaptic glutamate concentration following synaptic stimulation. Theoretical and experimental results suggest that the glutamate concentration inside
the cleft decreases to 10 µM 5-10 ms after the release of a
synaptic vesicle (Clements 1996
; Clements et al.
1992
; Diamond and Jahr 1997
; Wahl et al.
1996
), which is approximately the time required for the STC to
reach a peak (Bergles and Jahr 1997
; see also the
present data). Thus even allowing for concomitant release from
neighboring synapses in response to the same stimulus, the average
extrasynaptic concentration of glutamate during the decay of the STC is
likely to be below the EC50 of glial transporters for glutamate (~20 µM) (Arriza et al. 1994
;
Bergles and Jahr 1997
; Klockner et al.
1994
; Pines et al. 1992
) and, in fact, well
below the concentration of the transporters themselves (140-250 µM) (Lehre and Danbolt 1998
). The decay of the STC is
therefore likely to be proportional to the decrease in the number of
extant glutamate molecules. Accordingly, the STC decay (
~ 16 ms) is much slower than the decay of transporter-mediated currents in
the continuous presence of glutamate (
~ 1 ms) or on removal
of glutamate (
~ 3 ms) in excised patches (Bergles and
Jahr 1997
), as well as the decay of the AMPA receptor EPSC
(
~ 7 ms, data not shown), ruling out major roles for
transporter kinetics and asynchronous release in shaping the STC decay
(see also Bergles and Jahr 1997
).
Spatial extent of glutamate diffusion
The STC is clearly slowed in the presence of DHK or THA (Figs. 2
and 5), yet transport is not slowed further under these conditions during a four-stimulus train (Fig. 5). This suggests that, in the
presence of antagonist and perhaps even in control, transporters in the
region closest to the release site are saturated by transmitter, forcing glutamate to diffuse farther to find available sites. Although
DHK or THA would certainly expand regions of high occupancy ["saturated spheres," analogous to the "saturated disk" at the neuromuscular junction (Hartzell et al. 1975)], neither
DHK nor THA affect
4/
1, suggesting that
there remain unoccupied transporters between active release sites
during a brief train of HFS, even with transport capacity significantly
diminished. It may be that electrical stimulation recruits only a small
fraction of the Schaffer collateral fibers within a given volume and
that release probability remains relatively low, despite our
pharmacological efforts to increase it (see METHODS). As a
result, active release sites may be well separated spatially, even
during brief bursts of HFS, and glutamate may be permitted to diffuse a
significant distance without overlapping significantly with transmitter
released from another synapse. It would be desirable to use the
kinetics of the STC decay to estimate the spatial extent of glutamate
diffusion away from the release site, but such a calculation requires
more information about the diffusion coefficient of glutamate in the neuropil (although see Rusakov and Kullmann 1998
) and
the profile of transporter concentration versus radial distance from
the synaptic cleft.
Astrocytes take up a large majority of synaptically released glutamate
STCs recorded in CA1 stratum radiatum have been used to monitor
changes in synaptically released glutamate under different experimental
conditions (Diamond et al. 1998; Luscher et al.
1998
). STCs reliably reported changes caused by numerous
manipulations of release probability but were unaffected by the
induction of LTP, leading to the conclusion that LTP is not expressed
via any change in the amount of glutamate released in response to
synaptic stimulation (Diamond et al. 1998
;
Luscher et al. 1998
). However, many excitatory synapses
in CA1 stratum radiatum are not immediately adjacent to an astrocytic
process (Harris and Ventura 1998
; Lehre and
Danbolt 1998
), suggesting that astrocytes might not detect glutamate release from all synapses. One possibility is that astrocytes might take up glutamate released from only a particular fraction of
synapses, perhaps a subset that does not undergo LTP. Given the density
and affinity of astrocytic transporters in the CA1 neuropil, however,
it seems likely that transmitter released from all synapses, even those
located some distance from an astrocyte process, could be taken up by
transporters on an astrocyte and contribute to the STC. However, some
other "glutamate sink," including the superfusion saline above the
slice, might prevent transmitter released at some synapses from
reaching astrocyte transporters. We would argue against this
possibility based on the data presented in Fig. 2. At 300 µM, DHK is
a selective and nearly saturating antagonist for the GLT-1 transporter
subtype (Arriza et al. 1994
), which is expressed
exclusively in astrocytes (Rothstein et al. 1994
).
Although DHK decreased the amplitude and slowed the decay of the STC,
it did not significantly reduce the amount of glutamate transported by
the astrocyte, as indicated by the total charge transfer of the STC
(see Fig. 2C1, legend), despite the fact that in hippocampus
~80% of astrocytic transporters are GLT-1 (Lehre and Danbolt
1998
). If another significant source of glutamate uptake
besides the GLAST transporter subtype in astrocytes (Rothstein et al. 1994
) were present, it would have removed some of the
glutamate normally transported by GLT-1, and the charge transfer of the STC in DHK would have been significantly less than in control. Notwithstanding the possibility of a novel, nonastroglial,
DHK-sensitive transporter subtype, we interpret this result to indicate
that astrocytes take up nearly all of the glutamate released in a
region of CA1 stratum radiatum, and that the STC provides a
proportional measure of this uptake. This conclusion is consistent with
recent optical (Kojima et al. 1999
) and physiological
(Bergles and Jahr 1998
) data indicating that neuronal
transporters play, at best, a minor role in glutamate uptake in the hippocampus.
Implications for the input-specificity of LTP
One of the hallmarks of LTP, its input specificity
(Andersen et al. 1977; Lynch et al.
1977
), seems at odds with the evidence for spillover
(Asztely et al. 1997
; Kullmann and Asztely
1998
; Kullmann et al. 1996
) and the
extrasynaptic diffusion of glutamate implicit in STCs. However, if the
spillover said to occur following single-shock stimulation is not
significantly enhanced during HFS required for LTP induction, then
whatever input specificity existed before LTP induction would be
largely preserved afterward. Therefore, although evidence for spillover
may color our interpretation of silent synapses in the hippocampus
(Isaac et al. 1995
; Kullmann 1994
;
Liao et al. 1995
), the input-specific nature of LTP (if not its absolute synapse specificity) might not be compromised unless
spillover were exacerbated during periods of intense stimulation. Previous work has suggested that transporters restrict spillover (Asztely et al. 1997
); the present data suggest that, at
physiological temperatures, transport capacity is not challenged during HFS.
At room temperature, the ability of transporters to limit increased
spillover during induction of LTP may depend on the protocol used. The
four-stimulus, 100-Hz trains used above correspond to theta-burst
stimulation, a potent LTP induction protocol in which such trains are
delivered at 200-ms intervals (e.g., Larson et al.
1986). The results presented here indicate that a four-stimulus train does not result in slowed glutamate clearance, and the 200-ms interval clearly provides enough time for transporters to return the
glutamate concentration back to resting levels. Induction protocols
comprising longer trains may, however, overwhelm transport at 24°C,
possibly leading to a loss of input specificity. Given the remarkable
capacity of transporters at higher temperatures, however, input
specificity seems likely to be preserved in vivo, regardless of
stimulus pattern.
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ACKNOWLEDGMENTS |
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We thank M. Kavanaugh, J. Wadiche, and J. Williams for useful discussions and D. Bergles and J. Dzubay for reading the manuscript.
This work was supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences to J. S. Diamond and National Institute of Neurological Disorders and Stroke Grants NS-10041 to J. S. Diamond and NS-21419 to C. E. Jahr.
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FOOTNOTES |
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Present address and address for reprint requests: J. S. Diamond, National Institutes of Health, National Institute of Neurological Disorders and Stroke, Synaptic Physiology Unit, Bldg. 36, Rm. 2C09, 36 Convent Dr., Bethesda, MD 20892-4066.
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 18 October 1999; accepted in final form 4 February 2000.
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REFERENCES |
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