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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. 196 N. Lozovaya et al. / Brain Research 1011 (2004) 195–205 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. 198 N. Lozovaya et al. / Brain Research 1011 (2004) 195–205 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 199 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). 200 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- 202 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. References [1] A. Araque, V. Parpura, R.P. Sanzgiri, P.G. Haydon, Glutamate-dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons, Eur. J. Neurosci. 10 (1998) 2129 – 2142. 204 N. Lozovaya et al. / Brain Research 1011 (2004) 195–205 [2] A. Araque, R.P. Sanzgiri, V. Parpura, P.G. Haydon, Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons, J. Neurosci. 18 (1998) 6822 – 6829. [3] A. Araque, V. Parpura, R.P. Sanzgiri, P.G. Haydon, Tripartite synapses: glia, the unacknowledged partner Trends, Neuroscience 22 (1999) 208 – 215. [4] N. Arnth-Jensen, D. Jabaudon, M. Scanziani, Cooperation between independent hippocampal synapses is controlled by glutamate uptake, Nat. Neurosci. 5 (2002) 325 – 331. [5] J.L. Arriza, W.A. Fairman, J.I. Wadiche, G.H. Murdoch, M.P. Kavanaugh, S.G. Amara, Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex, J. Neurosci. 14 (1994) 5559 – 5569. [6] F. Asztely, G. Erdemli, D.M. Kullmann, Extrasynaptic glutamate spillover in the hippocampus: dependence on temperature and the role of active glutamateuptake, Neuron 18 (1997) 281 – 293. [7] N.J. Bannister, A.U. Larkman, Dendritic morphology of CA1 pyramidal neurones from the rat hippocampus: II. Spine distributions, J. Comp. Neurol. 360 (1995) 161 – 171. [8] B. Barbour, H. Brew, D. Attwell, Electrogenic uptake of glutamate and aspartate into glial cells isolated from the salamander (Ambystoma) retina, J. Physiol. 436 (1991) 169 – 193. [9] A. Baskys, R.C. Malenka, Agonists at metabotropic glutamate receptors presynaptically inhibit EPSCs in neonatal rat hippocampus, J. Physiol. 444 (1991) 687 – 701. [10] P. Bezzi, G. Carmignoto, L. Pasti, S. Vesce, D. Rossi, B.L. Rizzini, T. Pozzan, A. Volterra, Prostaglandins stimulate calcium-dependent glutamate release in astrocytes, Nature 391 (1998) 281 – 285. [11] M. Bouvier, M. Szatkowski, A. Amato, D. Attwell, The glial cell glutamate uptake carrier countertransports pH-changing anions, Nature 360 (1992) 471 – 474. [12] H. Brew, D. Attwell, Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells, Nature 327 (1987) 707 – 709. [13] H. Christensen, F. Fonnum, The ontogeny of the uptake systems for glutamate, GABA, and glycine in synaptic vesicles isolated from rat brain, Neurochem. Res. 17 (1992) 457 – 462. [14] J.D. Clements, Transmitter timecourse in the synaptic cleft: its role in central synapticfunction, Trends Neurosci. 19 (1996) 163 – 171. [15] K.J. Collard, R. Edwards, Y. Liu, Changes in synaptosomal glutamate release during postnatal development in the rat hippocampus and cortex, Brain Res. Dev. Brain Res. 71 (1993) 37 – 43. [16] S. Cull-Candy, S. Brickley, M. Farrant, NMDA receptor subunits: diversity, development and disease, Curr. Opin. Neurobiol. 11 (3) (2001 Jun.) 327 – 335. [17] J.S. Diamond, Neuronal glutamate transporters limit activation of NMDA receptors by neurotransmitter spill over on CA1 pyramidal cells, J. Neurosci. 21 (2001) 8328 – 8338. [18] S. Eliasof, J.L. Arriza, B.H. Leighton, M.P. Kavanaugh, S.G. Amara, Excitatory amino acid transporters of the salamander retina: identification, localization, and function, J. Neurosci. 18 (1998) 698 – 712. [19] S.L. Erdo, J.R. Wolff, Postnatal development of the excitatory amino acid system in visual cortex of the rat. Changes in uptake and levels of aspartate and glutamate, Int. J. Dev. Neurosci. 8 (1990) 205 – 208. [20] O.V. Garaschuk, Y.N. Kovalchuk, O.A. Krishtal, Trans-ACPD selectively inhibits excitability of hippocampal CA1 neurones, Eur. J. Pharmacol. 212 (1992) 305 – 306. [21] M. Garcia-Lopez, Axolemmal transporters for neurotransmitter uptake, Rev. Neurol. 29 (1999) 1056 – 1063. [22] J. Garthwaite, Cellular uptake disguises action of L-glutamate on Nmethyl-D-aspartate receptors. With an appendix: diffusion of transported amino acids into brain slices, Br. J. Pharmacol. 85 (1985) 297 – 307. [23] G. Garthwaite, G.D. Williams, J. Garthwaite, Glutamate toxicity: an experimental and theoretical analysis, Eur. J. Neurosci. 4 (1992) 353 – 360. [24] T. Harkany, T. Hortobagyi, M. Sasvari, C. Konya, B. Penke, P.G. Luiten, C. Nyakas, Neuroprotective approaches in experimental models of beta-amyloid neurotoxicity: relevance to Alzheimer’s disease, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 23 (1999) 963 – 1008. [25] M.E. Harris, Y. Wang, N.W. Pedigo Jr., K. Hensley, D.A. Butterfield, J.M. Carney, Amyloid beta peptide (25 – 35) inhibits Na+-dependent glutamate uptake in rat hippocampal astrocyte cultures, J. Neurochem. 67 (1996) 277 – 286. [26] D. Jabaudon, K. Shimamoto, Y. Yasuda-Kamatani, M. Scanziani, B.H. Gahwiler, U. Gerber, Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 8733 – 8738. [27] H.K. Kimelberg, S.K. Goderie, S. Higman, S. Pang, R.A. Waniewski, Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures, J. Neurosci. 10 (1990) 1583 – 1591. [28] P.E. Kish, S.Y. Kim, T. Ueda, Ontogeny of glutamate accumulating activity in rat brain synaptic vesicles, Neurosci. Lett. 97 (1989) 185 – 190. [29] H. Komuro, P. Rakic, Modulation of neuronal migration by NMDA receptors, Science 260 (1993) 95 – 97. [30] A.S. LaMantia, The usual suspects: GABA and glutamate may regulate proliferation in the neocortex, Neuron 15 (1995) 1223 – 1225. [31] C.M. Lauderback, M.E. Harris-White, Y. Wang, N.W. Pedigo Jr., J.M. Carney, D.A. Butterfield, Amyloid beta-peptide inhibits Na+-dependent glutamate uptake, Life Sci. 65 (1999) 1977 – 1981. [32] K.P. Lehre, N.C. Danbolt, The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain, J. Neurosci. 18 (1998) 8751 – 8757. [33] D.V. Lissin, R.C. Carroll, R.A. Nicoll, R.C. Malenka, M. von Zastrow, Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons, J. Neurosci. 19 (1999) 1263 – 1272. [34] N.A. Lozovaya, M.V. Kopanitsa, Y.A. Boychuk, O.A. Krishtal, Enhancement of glutamate release uncovers spillover-mediated transmission by N-methyl-D-aspartate receptors in the rat hippocampus, Neuroscience 91 (1999) 1321 – 1330. [35] M.P. Mattson, W.A. Pedersen, W. Duan, C. Culmsee, S. Camandola, Cellular and molecular mechanisms underlying perturbed energy metabolism and neuronal degeneration in Alzheimer’s and Parkinson’s diseases, Ann. N. Y. Acad. Sci. 893 (1999) 154 – 175. [36] J.W. McDonald, M.V. Johnston, Physiological and pathophysiological roles of excitatory amino acids during central nervous system development, Brain Res. Brain Res. Rev. 15 (1990) 41 – 70. [37] S. Mennerick, R.P. Dhond, A. Benz, W. Xu, J.D. Rothstein, N.C. Danbolt, K.E. Isenberg, C.F. Zorumski, Neuronal expression of the glutamate transporter GLT-1 in hippocampalmicrocultures, J. Neurosci. 18 (1998) 4490 – 4499. [38] A. Oka, M.J. Belliveau, P.A. Rosenberg, J.J. Volpe, Vulnerability of oligodendroglia to glutamate: pharmacology, mechanisms, and prevention, J. Neurosci. 13 (1993) 1441 – 1453. [39] Z.Z. Pan, G. Tong, C.E. Jahr, A false transmitter at excitatory synapses, Neuron 11 (1993) 85 – 91. [40] V. Parpura, T.A. Basarsky, F. Liu, K. Jeftinija, S. Jeftinija, P.G. Haydon, Glutamatemediated astrocyte-neuron signalling [see comments], Nature 369 (1994) 744 – 747. [41] L. Pasti, A. Volterra, T. Pozzan, G. Carmignoto, Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ, J. Neurosci. 17 (1997) 7817 – 7830. [42] D.K. Patneau, M.L. Mayer, Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors, J. Neurosci. 10 (1990) 2385 – 2399. [43] I.A. Pearce, M.A. Cambray-Deakin, R.D. Burgoyne, Glutamate acting on NMDA receptors stimulates neurite outgrowth from cerebellar granule cells, FEBS Lett. 223 (1987) 143 – 147. N. Lozovaya et al. / Brain Research 1011 (2004) 195–205 [44] D.L. Pettit, G.J. Augustine, Distribution of functional glutamate and GABA receptors on hippocampal pyramidal cells and interneurons, J. Neurophysiol. 84 (2000) 28 – 38. [45] N. Pomara, R. Singh, D. Deptula, J.C. Chou, M.B. Schwartz, P.A. LeWitt, Glutamate and other CSF amino acids in Alzheimer’s disease, Am. J. Psychiatry 149 (1992) 251 – 254. [46] W. Schmidt, G. Wolf, High-affinity uptake of L-[3H]glutamate and D-[3H]aspartate during postnatal development of the hippocampal formation: a quantitative autoradiographic study, Exp. Brain Res. 70 (1988) 50 – 54. [47] A. Schmitt, E. Asan, B. Puschel, T. Jons, P. Kugler, Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry, Neuroscience 71 (1996) 989 – 1004. [48] D. Schmitz, M. Frerking, R.A. Nicoll, Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses, Neuron 27 (2000) 327 – 338. [49] A. Schousboe, V. Lisy, L. Hertz, Postnatal alterations in effects of potassium on uptake and release of glutamate and GABA in rat brain cortex slices, J. Neurochem. 26 (1976) 1023 – 1027. [50] J.D. Sinor, S. Du, S. Venneti, R.C. Blitzblau, D.N. Leszkiewicz, P.A. Rosenberg, E. Aizenman, ENMDA and glutamate evoke excitotoxicity at distinct cellular locations in rat cortical neurons in vitro, J. Neurosci. 20 (2000) 8831 – 8837. [51] E.K. Speliotes, K.A. Hartnett, R.C. Blitzblau, E. Aizenman, P.A. Rosenberg, Comparison of the potency of competitive NMDA antagonists against the neurotoxicity of glutamate and NMDA, J. Neurochem. 63 (1994) 879 – 885. [52] N. Spruston, D.B. Jaffe, S.H. Williams, D. Johnston, Voltage- and space-clamp errors associated with the measurement of electronically remote synaptic events, J. Neurophysiol. 70 (1993) 781 – 802. 205 [53] M. Szatkowski, B. Barbour, D. Attwell, Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake, Nature 348 (1990) 443 – 446. [54] D. Trotti, N.C. Danbolt, A. Volterra, Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol. Sci. 19 (1998) 328 – 334. [55] E.M. Ulian, S.K. Sapperstein, K.S. Christopherson, B.A. Barres, Control of synapse number by glia, Science 291 (2001) 657 – 661. [56] K. Ullensvang, K.P. Lehre, J. Storm-Mathisen, N.C. Danbolt, Differential developmental expression of the two rat brain glutamate transporter proteins GLAST and GLT, Eur. J. Neurosci. 9 (1997) 1646 – 1655. [57] R. Ventura, K.M. Harris, Three-dimensional relationships between hippocampal synapsesand astrocytes, J. Neurosci. 19 (1999) 6897 – 6906. [58] K.E. Vogt, R.A. Nicoll, Glutamate and gamma-aminobutyric acid mediate a heterosynaptic depression at mossy fiber synapses in the hippocampus, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 1118 – 1122. [59] A. Volterra, P. Bezzi, B.L. Rizzini, D. Trotti, K. Ullensvang, N.C. Danbolt, G. Racagni, The competitive transport inhibitor L-trans-pyrrolidine-2, 4-dicarboxylate triggers excitotoxicity in rat cortical neuron-astrocyte co-cultures via glutamate release rather than uptake inhibition, Eur. J. Neurosci. 8 (1996) 2019 – 2028. [60] O. Warr, M. Takahashi, D. Attwell, Modulation of extracellular glutamate concentration in rat brain slices by cystine – glutamate exchange, J. Physiol. 514 (1999) 783 – 793. [61] N. Zerangue, M. Kavanaugh, Flux coupling in a neuronal glutamate transporter, Nature 383 (1996) 634 – 637. [62] C.F. Zorumski, S. Mennerick, J. Que, Modulation of excitatory synaptic transmission by low concentrations of glutamate in cultured rat hippocampal neurons, J. Physiol. (Lond.) 494 (1996) 465 – 477.