Download extrasynaptic glutamate does not reach the postsynaptic density

Brain Research 1011 (2004) 195 – 205
www.elsevier.com/locate/brainres
Research report
Protective cap over CA1 synapses: extrasynaptic glutamate does not reach
the postsynaptic density
Natasha Lozovaya, Sergei Melnik, Timur Tsintsadze *, Sergei Grebenyuk,
Yuri Kirichok, Oleg Krishtal
Bogomoletz Institute of Physiology, Kiev, Ukraine
Accepted 23 March 2004
Available online 30 April 2004
Abstract
Numerous data indicate that nonsynaptic release of glutamate occurs both in normal and pathophysiological conditions. When reaching
receptors in the postsynaptic density (PSD), glutamate (Glu) could affect the synaptic transmission. We have tested this possibility in the
hippocampal CA1 synapses of rats, either by applying exogenous Glu to the CA1 neurons or by disruption of Glu transporter activity. LGlu (400 AM) was directly applied to the hippocampal slices acutely isolated from the rats. It produced a strong inhibition of both orthoand antidromically elicited action potentials fired by CA1 neurons while the excitatory postsynaptic current (EPSC) measured in these
neurons remained totally unaffected. The optical isomer D-Glu which is not transported by the systems of Glu uptake inhibited not only
orthodromic and antidromic spikes, but also EPSC. Non-specific glutamate transporter inhibitor DL-threo-h-hydroxyaspartic acid (THA, 400
AM) mimicked the effects of exogenous Glu and produced strong inhibition of both orthodromic and antidromic spikes, without any influence
on the amplitude of EPSCs. Dihydrokainate (DHK, 300 AM), selective inhibitor of GLT-1 subtype of glutamate transporter, exerted a
significant inhibitory action on the orthodromically evoked spikes and also on the EPSC. Our results indicate that extrasynaptic and PSD
membranes of CA1 neurons form separate compartments differing in the mechanisms and efficiency of external Glu processing: the protection
of PSD markedly prevails.
D 2004 Elsevier B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters, and receptors
Topic: Excitatory amino acid: excitotoxicity
Keywords: Extracellular glutamate; CA1 synapse; Glutamate receptor; Hippocampal slice; EPSC; Field potential
1. Introduction
There is growing evidence indicating that Glu is released in the mammalian brain not only from the presynaptic terminals but also from the astroglia [3,26,27,53].
Numerous phenomena that include a neuronal Ca2 + elevation, slow inward currents mediated by extrasynaptic glutamate (Glu) receptors in neighboring neurons and synaptic
* Corresponding author. Department of Cellular Membranology,
Bogomoletz Institute of Physiology, Bogomoletz str. 4, 01024, Kiev,
Ukraine. Tel.: +380-44-2932142; fax: +380-44-2562590.
E-mail address: [email protected] (T. Tsintsadze).
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2004.03.023
modulation have been attributed to bi-directional communications between neurons and glial cells [3]. Extrasynaptic
ionotropic and metabotropic Glu receptors are scattered
over the entire membrane of hippocampal pyramidal neurons surface [44], comprising a target for Glu-mediated
signalling. The extent of activation of extrasynaptic receptors by spillover of Glu in physiological conditions is
negligible [6]. This indicates the effectiveness of specific
machinery, which prevents the escape of Glu from the
synaptic cleft. The question arises, whether the extrasynaptic Glu is capable of eliciting a direct modulatory effect on
the hippocampal glutamatergic synapse. Here we demonstrate that the access of extrasynaptic glutamate to the
receptors in the postsynaptic densities is strictly limited
by the mechanisms of uptake.
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2. Materials and methods
2.1. Preparation of hippocampal slices
This study was carried out on 24 – 28-day-old Wistar
rats (WAG/GSto, Moscow, Russia). After cervical dislocation, rats were rapidly decapitated and brain was
immediately transferred to a Petri dish with chilled
(4 jC) solution of the following composition: 120 mM
NaCl, 5 mM KCl, 26 mM NaHCO3, 2 mM MgCl2 and
20 mM glucose. Calcium salts were omitted to reduce
possible neuronal damage. The solution was constantly
bubbled with 95%O2/5%CO2 gas mixture to maintain
pH = 7.4. Hippocampal slices (300 – 400 Am thick) were
cut manually with a razor blade along the alveolar fibers
to preserve the lamellar structure of excitatory connections. During the preincubation and experiments, the
slices were kept fully submerged in the extracellular
solution: 135 mM NaCl, 5 mM KCl, 26 mM NaHCO3,
1.5 mM CaCl 2 , 1.5 mM MgCl 2 , 20 mM glucose
(pH = 7.4, bubbled with 95% O2/5% CO2) at 30 –31 jC.
Picrotoxin of 25 – 50 AM was also included into the
extracellular solution during experiments to suppress the
inhibitory activity of interneurons. Recording started after
at least 2 h of incubation.
2.2. Electrophysiological recordings
Excitatory postsynaptic currents were recorded by a
standard whole-cell patch clamp technique in the CA1
subfield of the hippocampus in response to stimulation of
the Schaffer collateral/commissural pathway. To prevent
the spread of electrical activity from area CA3, minislices were prepared by making a cut orthogonal to the
stratum pyramidale and extending to the mossy fiber
layer. Intracellular solution for patch pipettes contained
100 mM CsF, 40 mM NaH2PO4, 10 mM HEPES –CsOH,
10 mM Tris –Cl (pH = 7.2). 2– 3 mM N-(2, 6-dimethylphenylcarbamoylmethyl)-triethylammonium bromide (QX314) were routinely added to the intracellular solution to
block voltage-gated sodium conductances. Patch pipettes
were pulled from soft borosilicate glass. When they were
fire-polished and filled with the intracellular solution,
they had a resistance of 2 – 3 MV.
Currents were digitally sampled at 200-As intervals by
a 12-digit ADC board, filtered at 3 kHz, and stored on a
hard disk for further analysis. Access resistance was
monitored throughout the experiments and ranged typically from 6 to 9 MV. The data from cells where access
resistance changed by more than 25% during the experiment were discarded. Extracellular field potentials were
recorded using Ni/Cr electrodes. The population spikes
were digitised and stored on computer disk. The effect of
substance was measured as the mean ratio I/Io where I
was the current under the substances action and Io was
the current in control saline. To stimulate the Schaffer
collateral/commissural pathway input (orthodromic spike),
a bipolar Ni/Cr electrode was positioned on the surface
of the slice. In the case of measurements of antidromic
spikes, stimulating electrode was placed in srtatum oriens/alveus. Recordings were performed in stratum piramidale (Fig. 1A).
Glutamate is effectively taken up by the slice tissue
[23]. To ensure its maximal possible concentration around
the dendrites belonging to the patch clamped neurones,
stratum oriens and alveus were removed by saline jet
from a micropipette as described in Ref. [20] (Fig. 1B).
Current pulses (10 –100 AA) of 0.1 –1-ms duration were
delivered through the isolated stimulator HG 203 (HiMed, London, UK) at 0.066 –0.2 Hz.
2.3. Drugs
Sodium bicarbonate and CsF were obtained from
Merck (Darmstadt, FRG); 4-AP, Lidocaine and picrotoxin
were purchased from RBI (Natick, MA, USA); 6-nitro7-sulphamoylbenzo [ f] quinoxalin-2,3-dione (NBQX)
were obtained from Tocris Cookson (Bristol, UK). All
other chemicals were from Sigma (St. Louis, MO,
USA).
Fig. 1. Schematic representation of the experimental arrangement. Measurements of orthodromically evoked field potentials (OFP) and antidromically evoked
field potentials (AFP) (A); measurements of EPSC (B). Note the removed part of alveus/oriens in the latter case.
N. Lozovaya et al. / Brain Research 1011 (2004) 195–205
2.4. Model
The following calculation is adopted from Ref. [23].
According to this model, the rate of cellular uptake of
Glu is balanced in the steady state by the rate of its
inwardly directed diffusion. The concentration across the
slice thickness can be determined from the following
equation:
1
vx2
2vC0 2
Ce ¼
C0
ð1Þ
2DVx 2DV
where Ce and C0 are the concentration of Glu in the
extracellular medium and superfusing saline, respectively, v is the rate of uptake (1 AM/ml per min), x is
distance from surface and DV is diffusion coefficient
(1.1 10 4 cm2/min). The values of the constants (in
parenthesis) are taken from Garthwaite [22]. The profile
197
of Glu concentration across the slice thickness calculated using Eq. (1) is demonstrated in Fig. 8. Numbers of
rate of uptake and diffusion coefficient are taken from
Garthwaite for slices of cerebellum. The numbers of the
glutamate transporters GLAST (EAAT1) and GLT
(EAAT2) are 3200 and 12,000 per Am3 tissue in the
stratum radiatum of adult rat hippocampus (CA1) and
18,000 and 2800 in the cerebellar molecular layer,
respectively [32]. Since rate of uptake is pro rata the
number of glutamate transporters per Am3 of tissue, we
assumed that rate of uptake in hippocampal slices should
be of about 0.75 AM/ml/min. On the other hand, the total
astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two
regions, respectively, consequently diffusion coefficient in
hippocampal tissue should be significantly higher. This
suppose that numbers for distance of glutamate penetration are the low limits.
Fig. 2. Differential effects of L- and D-glutamate (400 AM) on the CA3 – CA1 synaptic transmission in hippocampus (representative experiments). (A)
Population spikes (a), antidromic spikes (b) and EPSCs (d) in control and under L-glutamate in the moments indicated by arrows in the time-course graph (e).
VHOLD = 50 mV. Note transient shift of baseline due to somatic L-glutamate-activated current. Antidromic spikes (c) were recorded in the continuous
presence of APV, NBQX and mGluR antagonists. (B) The same as A, but for D-glutamate.
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Fig. 3. Modulation of the CA3 – CA1 synaptic transmission by inhibitors of glutamate uptake THA (A) and DHK (B). Population spikes (Aa, Ba), antidromic
spikes (Ab, Bb) and EPSCs (Ad, Bc) were measured in moments indicated by arrows in the time-course graphs (Ae, Bd). Antidromic spikes (Ac) were obtained
in the continuous presence of APV, NBQX and mGluR antagonists. (C) L-Glu-induced responses of the acutely isolated CA1 pyramidal neurons in control
(left) and in the presence of 300 AM of DHK (right).
N. Lozovaya et al. / Brain Research 1011 (2004) 195–205
3. Results
Submillimolar (up to 400 AM) L-Glu was applied to
the hippocampal slices acutely isolated from the rats
(P24 –P28). It produced a strong inhibition of both orthoand antidromically elicited action potentials fired by CA1
neurons, while the amplitude and kinetics of the excitatory postsynaptic currents (EPSC) measured in these
neurons remained totally unaffected (Fig. 2A-a,b,d,e).
The mean numbers for 400 AM L-Glu are as follows:
inhibition of orthodromic field potential to 12 F 6%, n = 14;
inhibition of antidromic spike to 41 F 7%, n = 15, while the
presynaptic fiber volley remained unchanged. Dramatic
inhibition of the orthodromic field potential did not depend
on the position of the electrode: in special trials, we located
the electrode at various depths and obtained the same levels
of inhibition.
In 14 out of 15 in situ patch clamped neurons, the
amplitudes of EPSCs measured at holding potential 70
mV were not affected: (98 F 17%, n = 15). Higher concentrations inhibit EPSC as well: IC50 values for the
inhibition of EPSC and field potentials are 1.6 F 0.14 mM
and 315 F 15 AM, n = 3 correspondingly. In several cases,
even potentiation of EPSCs was observed (115 F9%,
n = 3).
According to Zorumski et al. [62], low micromolar
concentrations of glutamate depress both AMPA and
NMDA components of excitatory autapic currents in
microcultures of rat hippocampal neurons with IC50 of
3.8 and 1.3 AM, respectively, due to the desensitization
of postsynaptic receptors. In our experiments, 2 orders
higher concentrations of Glu failed to produce significant
changes in EPSCs indicating that exogenously applied
glutamate does not access receptors in the postsynaptic
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density (PSD) and activates only extrasynaptic receptors,
modulating the excitability of the neurons.
The inhibition of excitability of the CA1 neurons by
Glu can be related at least partially to the activation of
metabotropic (mGlu) receptors. According to Garaschuk
et al. [20], ACPD, selective agonist of certain subtypes
of these receptors, inhibited population spike in CA1
(but not in CA3) neurons leaving the EPSC unaffected.
The age of the animals is critical for this phenomenology. In the rats younger than P20, ACPD inhibits
EPSCs as well. This action is mediated, at least in
part, by the activation of presynaptic metabotropic
glutamate autoreceptors that are expressed only in
P10 – P20 rats [9]. However, we have found that the
major contribution to the L-Glu-induced inhibition of
focal potentials is produced by the ionotropic glutamate
receptors. Thus, in the presence of NBQX (20 AM) and
APV (150 AM), inhibition of the antidromic spike in
CA1 produced by L-Glu was significantly suppressed
(remaining amplitude was 80 F 8%, n = 4 vs. 41 F 7%,
n = 15). Simultaneous addition of the antagonists of
metabotropic glutamate receptors (antagonist of group I
PHCCC, 100 AM, selective antagonist of group II
APICA, 100 AM and antagonist of group III MSOP,
100 AM) insignificantly decreased the extend of inhibition (85 F 3%, n = 5) (Fig. 2A-c).
D-glutamate is a weak agonist of NMDA receptors (EC50
75 – 80 AM, [42] and a very weak agonist of non-NMDA
receptors: in the presence of APV D-glutamate-activated
current comprised only 3 F 3 % of the current produced
by L-Glu [39]. D-Glu is transported very poorly, if at all, by
the glutamate transporters [5,18]. We have found that, when
applied in the same range of concentrations as its optical
isomer, D-Glu produces not only inhibition of both ortho-
Fig. 4. Differential effect of 100 AM L-glutamate applied in the presence of 200 AM DHK (A) and 200 AM THA (B). The EPSCs were measured at the
moments indicated by corresponding numbers at the time course of experiments (A, B, lower graphs).
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N. Lozovaya et al. / Brain Research 1011 (2004) 195–205
dromic and antidromic spikes (10 F 10%, n = 5 and
53 F 9%, n = 6, respectively), but also significantly inhibits
EPSCs (57 F 17%, n = 5) (Fig. 2B-a,b,d,e).
In the presence of NBQX (20 AM), APV (150 AM)
and antagonists of metabotropic glutamate receptors the
effect of D-Glu on the antidromic spike are insignificant
(90 F 7%, n = 5 vs. 53 F 9%, n = 6) (Fig. 2B-b,c).
It has been demonstrated recently that the basal
activity of glutamate transporters compensates for a
continuous nonvesicular release of Glu from intracellular
(primarily glial) compartments. Correspondingly, this release can be unmasked by the inhibitors of Glu uptake
[26]. Such inhibitors of Glu uptake as THA or tPDC
induce the release of glutamate through the heteroexchange [59]. We have used THA (400 AM) in order to
check whether the endogenous glutamate released by glial
cells is capable of modulating EPSCs. THA mimicked
the effects of exogenous glutamate and produced strong
inhibition of both orthodromic (23 F 6%, n = 4) and
antidromic (46 F 10%, n = 6) spikes without any influence
on the amplitude of EPSCs (105 F 8%, n = 5) (Fig. 2Aa,b,d,e). In the presence of NBQX (20 AM), APV (150
AM) and antagonists of metabotropic glutamate receptors
inhibition of the antidromic spike in CA1 produced by
THA are significantly suppressed (91 F 2%, n = 5 vs.
46 F 10%, n = 6) (Fig. 3A-b,c).
In contrast to THA, dihydrokainate (DHK, 300 AM),
the inhibitor of GLT-1 subtype of glutamate transporter
exerted a significant inhibitory action not only on the
orthodromically evoked spikes (24 F 8%, n = 5), but also
on the EPSC (60 F 12%, n = 5) (Fig. 3B-a,c,d). It should
be specifically noted that antidromic spikes were only
slightly decreased (90 F 6%, n = 5) in the presence of
DHK (Fig. 3B-b).
The effect of DHK on the EPSCs is not mediated by
direct effects of DHK on the AMPA receptors. L-Gluinduced responses in acutely isolated neurons applied on
top of 300 AM of DHK revealed the same amplitudes as
in control (n = 4) (Fig. 3C).
When applied on top of DHK (200 AM) (Fig. 4A), L-Glu
(100 AM) produced significantly stronger inhibition of
the EPSC as compared to the co-application with THA
(200 AM): 58 F 8%, n = 5, vs. 85 F 6%, n = 4 (Fig. 4B).
These observations suggest that the glutamate transporters, specifically GLT-1 and/or GLT-like EAAT2B subtype
[18], form a protective ‘‘cap’’ over the CA1 synapses,
limiting the access of non-synaptically originating glutamate to the synaptic cleft. The summary for the effects
of uptake agonists and inhibitors is presented in Fig. 5.
We have tested the efficiency of the ‘‘cap’’ over the PSD
by using use-dependent blocker of NMDA receptors,
MK801. In the experiments demonstrated in Fig. 6A,
NMDA receptors available for exogenously applied glutamate were blocked by the simultaneous application of
glutamate and MK-801. NBQX was used to block the
AMPA receptor-mediated component of EPSC. The synap-
Fig. 5. The effects of Glu and uptake inhibitors on the synaptic
transmission and electrical excitability. (A) The effects of L-Glu and DGlu (400 AM) on the EPSC, orthodromic field potential (OFP),
antidromic field potential (AFP) and AFP in the presence of
metabotropic receptor antagonists, n = 5, p < 0.05 as compared to control
by two-tailed t-test. (B) The effects of L-Glu (400 AM), THA (400 AM)
and DHK (300 AM) on the EPSC, OFP, AFP and AFP in the presence
of metabotropic receptors antagonists, n = 5, p < 0.05 as compared to
control by two-tailed t-test.
tic input was not stimulated from the start of 10-min bath
application of MK-801 plus Glu. Accordingly, the receptors
belonging to PSDs could be activated and irreversibly
blocked by MK-801 only in cases when the exogenous
glutamate could reach them. However, after the removal of
glutamate, stimulation of Schaffer collaterals (Fig. 6A) still
produced EPSC (32 F 10% of control amplitude, n = 5)
indicating the presence of residual non-blocked NMDA
receptors that were not reached by the exogenously applied
agonist. In contrast, when NMDA which is non-transportable by glutamate uptake was used instead of glutamate in
the same experimental procedure, subsequent stimulation
failed to elicit any trace of the postsynaptic current Fig. 6B
(n = 5). Thus, the receptors in PSD are protected from
activation by the extrasynaptic agonists only when the latter
is transportable by the GLT-type uptake mechanism(s).
N. Lozovaya et al. / Brain Research 1011 (2004) 195–205
201
Fig. 6. EPSCs mediated by NMDA receptors were measured in the control (1) and after 10-min exposure of the slice to Glu (A) and to NMDA (B) in the
presence of MK-801. The agonists and MK-801 were simultaneously applied as indicated by black bars without stimulation of synaptic input. VHOLD = 50
mV. Dashed lines indicate moments of break and restart of electrical stimulation of the synaptic input. Upper lines—traces of EPSC in control (1) and average
of the first five traces under restart of stimulation (2). Paired-pulse facilitation was used to visualize the response under MK-801. When agonist is transportable,
a fraction of postsynaptic receptors is protected from the block (Glu, A), while non-transportable NMDA leads to the complete block (B).
The experiment shown in Fig. 7 demonstrates the
limits of such protection: in conditions where glutamate
release is increased by 4-aminopyridyne (40 AM), the
exogenously applied glutamate inhibits the enhanced
EPSC (from 465 F 16% of control to 320 F 4%, n = 7).
Fig. 7. Modulation of EPSCs by glutamate after preincubation with 4-AP.
EPSCs traces in control and under 4-AP and L-Glu at moments indicated by
corresponding numbers on the time-course. VHOLD = 50 mV.
4. Discussion
Nonsynaptically released Glu has a lot of targets in
the membrane of hippocampal neurons: both ionotropic
and metabotropic Glu receptors are distributed over their
entire surface in large numbers [44]. Numerous data
indicate that nonsynaptic release occurs both in normal
and pathophysiological conditions. The source of nonsynaptic Glu is glia, predominantly astrocytes. Ca2 +dependent glutamate release has been demonstrated both
in cultured astrocytes and in acutely isolated hippocampal
slices [3]. Activation of bradykinin or prostaglandin E2
receptors, co-activation of AMPA and metabotropic
receptors, as well as electrical and mechanical stimuli,
cause the elevation of Ca2 + level in astrocytes which is
followed by the release of glutamate [1,2,10,40,41]. It has
been demonstrated recently that Ca2 +-dependent Glu
release from astrocytes is a SNARE protein-dependent
process, which requires the presence of functional vesicle-associated proteins: astrocytes store Glu in vesicles
and release it by exocytosis. The ability of astrocytes to
release Glu makes them capable of signaling to neurons
[2]. Ca2 +-independent non-synaptic glutamate release is
mediated by glutamate-transporter reversal [53] or by
swelling of the cells [27]. Continuous non-vesicular
release of glutamate has been revealed by the inhibition
of glutamate uptake in the rat organotypic hippocampal
slices [26]. In addition, Warr et al. [60] recently charac-
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N. Lozovaya et al. / Brain Research 1011 (2004) 195–205
terized a mode of glutamate release through cystine/
glutamate exchanger.
In our experiments, L-Glu and THA induced much more
pronounced inhibition of the orthodromic spike than of the
EPSC. We suggested that mechanisms of glutamate uptake
create a protective ‘‘cap’’ specifically over the synaptic
receptors but not over somatic receptors. However, capacious uptake and subsequent processing of Glu lead to a
strong decrease in the concentration of exogenously applied
glutamate in the depth of the slice [22]. One can argue that
somatic receptors are more readily activated by L-Glu than
synaptic receptors buried within the stratum radiatum
neuropil because of bulk uptake, as opposed to specific
uptake, which should be limited to the areas around
synapses. However, our data cannot be accounted for in
the framework of this phenomenology. Using the approach
suggested by Garthwaite et al. [23], we have calculated the
glutamate concentration profile in the depth of slice for 400
AM external Glu. Fig. 8b demonstrates that the Glu
concentration exceeds 10 AM till the distance of 80 Am
from the surface of the slice, while the EC50 for desensitisation of AMPA and NMDA receptors are 3.8 and 1.3
AM, respectively [62]. This means that the 80-Am slice
layers from all sides of the 300-Am slice comprising 64%
of its total volume are in contact with desensitizing concentration of Glu. In the case of a cell located at the equal
distance from the upper and lower boundaries of a 300-Am
slice, the synapses located at the depth below 80 Am will be
‘‘switched off’’ owing to desensitization (Fig. 8a). According to Bannister and Larkman [7], 50% of all spines are
located along a path of 200 AM from the soma. Due to
obvious considerations, these spines represent the closest to
soma least filtered synapses that have the largest contribution to the total somatically measured EPSC [52]. It should
be specifically noted that the kinetics of EPSC was not
altered in the presence of glutamate indicating that NMDA
receptor-mediated component of EPSC which is more
sensitive to glutamate was not affected.
All our observations did not depend either on vertical or
horizontal position of the patch clamped cell. In many
experiments (7 out of 15), the recordings were performed
from the cells just near the surface of the slice (Fig. 8a)
without any difference in the data: 400 AM of Glu did not
affect the EPSCs.
In addition, the following reasoning should be mentioned. In our experiments, THA induced strong inhibition
of orthodromically-induced population spikes. This effect
was significantly suppressed by APV and NBQX, which is
most consistent with the induction of non-synaptic Glu
release by THA [26]. Like in the case of exogenously
administered Glu, EPSCs in the presence of THA were
unaffected. THA is transported just as L-Glu, but is not
processed by specific enzyme, glutamin synthetase. Consequently, when continuously perfused (for up to 20 min),
THA should finally be present in uniform concentration
throughout the slice.
Fig. 8. The estimate for the Glu concentration inside the slice. (a) Schematic
representation of the hippocampal slice preparation. Arrows and dotted lines
indicate the areas of slice where Glu concentration should exceed 10 AM.
For calculations, see Materials and methods. (b) Extracellular glutamate
concentration profiles inside the slice at the steady state calculated according
to Eq. (1). Solid lines represent the profile when the external concentration of
Glu is 400 AM, dotted line is for 300 AM.
Several lines of evidence strongly support the ‘‘cap’’
hypothesis. It was suggested previously that exogenous
glutamate exerts its neurotoxic action by activating exclusively somatic receptors [51]. This hypothesis was further
confirmed by the finding that the selective inhibition of
NR2B receptors that are segregated to cell body effectively
suppresses glutamate-induced, but not NMDA-induced,
neurotoxicity [50]. Moreover, a glutamate transport inhibitor was used to allow glutamate to reach dendritic receptors and become a more potent toxin.
Similar considerations have been recently raised by
Araque et al. In their experiments with mixed neuron –
astrocyte culture, the sustained presence of glutamate was
induced by astrocyte stimulation. It resulted in the appearance of glutamate-dependent slow inward currents in
associated neurons. Clear presynaptic effects, mediated by
glutamate, were not accompanied by any trace of desen-
N. Lozovaya et al. / Brain Research 1011 (2004) 195–205
sitization of miniature synaptic currents. It was concluded
that glutamate released from astrocytes does not act
equally at all sites on the neuron, so that the receptors
in postsynaptic cleft are spared by uptake mechanisms
from desensitization [1].
The qualitative difference in the effects of THA and
DHK indicates that the vulnerability of EPSC to extrasynaptic Glu, either applied extraneously or released by glia,
specifically depends on the activity of GLT-1 or GLT-like
subtypes of Glu transporters. In all probability, these
mechanisms provide the Glu-protective ‘‘cap’’ around the
synaptic cleft. One third of the CA1 synapses totally lack
the glial processes allowing to assume that they are inactive
[57]. In view of our data, the only alternative for such
interpretation is the presence in such synapses of a highly
effective neuronal glutamate transporter [17]. Although
DHK-sensitive subtype of Glu transporters is regarded as
purely glial, there are reports on the existence of GLT
mRNA in hippocampal pyramidal neurons [47] and the
expression of GLT-1 in the neurons at least under appropriate environmental conditions [37]. Presently, it is impossible to distinguish whether glial, neuronal or both
glutamate transporters are responsible for the protective
cap over the CA1 synapses. Anyway, our data are consistent with the indication at the particular role of DHKsensitive transporters in the cross-talk between neighboring
synapses [6].
It is noteworthy that, when applied in the conditions of
enhanced release (Fig. 6), Glu produced significant inhibitory effect on EPSCs. This effect may be due to saturation
of the glutamate transport system. The increased neuronal
activity (repetitive firing induced by 4-AP) should lead to a
significant increase in the extracellular K+ concentration.
Since translocation of glutamate by high affinity transporters is dependent on the concentration gradients for
Na+,K+ and H+ [8,12,61], the changes in these gradients
can reduce or reverse glutamate transport [53].
Alternatively, 4-AP could activate previously silent presynaptic terminals lacking astrocytic processes, as described
by Ventura and Harris [57].
Under conditions of increased neuronal activity, glutamate can escape transporter uptake in sufficient quantities
and activate extrasynaptic, presynaptic [48,58] and postsynaptic[4,6,34] receptors. The phenomenology of ‘‘cap’’
over the synapses is in no way in contradiction with
‘‘spillover’’ hypothesis. As indicated in the Results, the
Glu concentration 400 AM used in our experiments is
limiting for the efficiency of the ‘‘cap’’, and indeed is
substantially lower in the depth of the slice. From the other
hand, in the course of synaptic transmitter release, the
concentration of Glu in the cleft is 1 order of magnitude
higher (1– 5 mM [14]).
Excitatory amino acids are known to play a crucial role in
the development of CNS including synapses elimination, cell
migration, differentiation and death [16,29,30,36,38,43].
The processing of Glu, as well as the expression, localization
203
and subunit composition of Glu receptors, are subjected to
the well-concerted changes in the course of development.
Glu uptake in the rat brain is low at birth and increases to
adult levels during the first few weeks of postnatal development [15,19,46,49]. This increase is correlated with a strong
increase in the synaptogenesis [13,28]. The expression of
GLT, as well as the uptake activity, lags behind the synaptogenesis for 2 days [56].
Recent publications demonstrate that high local glutamate levels result in the down-regulation of postsynaptic
glutamate receptors. For example, in the primary culture
of rat hippocampal neurons, the application of extracellular glutamate causes redistribution of GluR1 subunits
away from the synaptic sites (clusters) within minutes
[33]. Thus, the steady-state Glu level is an important
developmental factor controlling the composition and
clustering of certain glutamate receptor subtypes in postsynaptic density and processes of the formation and/or
elimination of synapses. It is quite probable that isolation
of synaptic cleft compartment from the rest of the CA1
neuron serves for the proper formation and stabilization
of certain synapses, preventing their elimination induced
by external glutamate. Protective cap over the synapses
may be a factor, which determines whether a certain
synapse should be stabilized or eliminated. Indeed, in
support of this assumption, it has been demonstrated
recently that the astrocytes increase the number of mature
functional synapses on the central neurons (in retinal
ganglion cells culture) and are required for synaptic
maintenance in vitro [55]. This implies a new function
for glial cells in the induction and stabilization of CNS
synapses.
The protection of synapses from the extrasynaptic Glu
should also be important in certain pathological conditions,
where the extracellular concentration of glutamate can reach
the submillimolar range due to the reversed glutamate
transport. The brain tissue obtained from the patients with
amyotrophic lateral sclerosis, Huntington’s or Alzheimer’s
diseases has a markedly decreased glutamate uptake [21,24,
25,31,35,54]. The decreased capacity of glutamate clearance
from the extracellular space triggers a cascade of events
leading to neuronal death [11,42,45].
Acknowledgements
This work was supported by the Wellcome Trust and
Howard Hughes Medical Institute. We thank Drs. Dimitri
Kullman and Dmitri Rusakov for their valuable comments.
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