Endogenous NMDA-Receptor Activation Regulates Glutamate Release in Cultured Spinal Neurons
Antoine Robert,
Joel A. Black, and
Stephen G. Waxman
Department of Neurology, Yale University School of Medicine, New Haven 06510; and PVA/EPVA Neuroscience Research Center, Veterans Affairs Medical Center, West Haven, Connecticut 06516
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ABSTRACT |
Robert, Antoine, Joel A. Black, and Stephen G. Waxman. Endogenous NMDA-receptor activation regulates glutamate release in cultured spinal neurons. J. Neurophysiol. 80: 196-208, 1998. N-methyl-D-aspartate (NMDA) receptor activation plays a fundamental role in the genesis of electrical activity of immature neurons and may participate in activity-dependent aspects of CNS development. A recent study has suggested that NMDA-receptor-mediated glutamatergic neurotransmission might occur in the developing spinal cord via activation of nonsynaptic receptors, but the details of NMDA-receptor activation in the developing CNS are not yet well understood. We describe here a model of cultured spinal neurons that display ongoing
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activity characterized by spontaneous excitatory postsynaptic currents (EPSCs), with NMDA-receptor activity detectable only as single channel events. DL-2-amino-5-phosphonovaleric acid (100 µM) and tetrodotoxin (TTX) 100 nM each reduced the occurrence of spontaneous AMPA EPSCs; quantal analysis showed a decrease in the number of released quanta but no changes in quantal size, indicating that NMDA-receptor activation and Na+ channel activity affect the generation of spontaneous AMPA EPSCs, at least in part, via mechanisms that impinge on the presynaptic terminal. Once the Mg2+-block was released, activity of NMDA receptors dramatically increased the release of quantal and multiquantal amounts of glutamate, indicating that the NMDA receptors are physiologically coupled to glutamate release. In Mg2+-free solution, TTX application elicited an increase in the number of quantal AMPA EPSCs and a reduction in the number of multiquantal EPSCs, consistent with an effect of NMDA-receptor activation on presynaptic terminals. Our results suggest that endogenous activity at a small number of NMDA receptors can regulate the release of neurotransmitters at developing AMPA synapses.
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INTRODUCTION |
Channels activated by glutamate are responsible for most of the excitatory synaptic transmission in the CNS. The family of ionotropic glutamate receptors (GluRs) fall into three classes. The first, termed the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs), mediate a fast, rapidly desensitizing, current. The second, the N-methyl-D-aspartate (NMDA) receptors (NMDARs), mediate a slowly desensitizing current with a much higher Ca2+ permeability than most AMPARs; for these two reasons, they are involved in second-messenger cascade activation, which follows activation of excitatory pathways. The third class of GluRs, the family of the kainate receptors (KAIRs), carry a fast-desensitizing current, which resembles the AMPA current. It is generally proposed that both AMPARs and NMDARs are localized at postsynaptic membranes in the mature CNS (Kullmann and Siegelbaum 1995
; Kullmann et al. 1996
; Petralia et al. 1994)
.
Such a view of postsynaptic NMDARs and AMPARs focused at excitatory synapses is contradicted in developing neurons by observations showing that the NMDAR subunits are expressed diffusely, rather than focused at synapses, in cultured spinal cord neurons (O'Brien et al. 1997)
or are disseminated in scattered extrasynaptic clusters in cultured hippocampal neurons (Rao and Craig 1997)
. In contrast, AMPAR subunits are clustered at postsynaptic membranes (Baude et al. 1995
; Craig et al. 1993)
. These observations raise the question of whether, at early stages of development of the spinal cord, NMDARs might not participate in excitatory neurotransmission. An active role for NMDARs seems, however, likely for several reasons. First, during development, NMDAR activation plays a crucial role in activity-dependent developmental processes, e.g., in the visual cortex of mammals (Bear et al. 1990
; Carmignoto and Vicini 1992
; Constantine-Paton et al. 1990
; Fox et al. 1989)
and in motor neuron dendritic outgrowth in the spinal cord (Kalb 1994
; Kalb and Hockfield 1994)
, where the NMDA receptors are expressed transiently at a high level during the first weeks after birth (Kalb et al. 1992)
. Second, electrophysiological studies have shown that NMDARs contribute to both spontaneous and induced activity, as evidenced by giant synaptic potentials (Ben-Ari et al. 1989)
, Ca2+ oscillations (Leinekugel et al. 1997)
, and polysynaptic responses in postnatal hippocampus (Crepel et al. 1997
; MacLean et al. 1995). Third, in a number of model systems, before the development of functional AMPA synapses, NMDARs alone support glutamatergic transmission (Durand et al. 1996
; Wu et al. 1996)
.
To study the role of NMDARs in developing spinal neurons, we have used cultures of neonatal spinal cord neurons that display intense spontaneous synaptic activity caused by both
-aminobutyric acid-A (GABAA) and AMPA inputs. Although the cells all expressed functional NMDARs, a direct contribution of NMDARs to the total synaptic input could not be isolated. Very surprisingly, the spontaneous release of glutamate at AMPA synapses was highly dependent on the endogenous activity of NMDARs. Our data indicate that, although some of the effects of NMDAR activation on glutamate release depend on Na+-dependent spiking of the cells, a significant part of the effect of NMDAR activation impinges on presynaptic mechanisms. Our observations suggest a feedback loop, whereby NMDAR activation in pre- or postsynaptic regions leads to further glutamate release.
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METHODS |
Rat spinal cord culture
Postnatal day 0 (P0; within 24 h of birth) Sprague-Dawley rat pups were decapitated. Spinal cords between mid-cervical and lower lumbar levels were dissected free and the meninges removed. The cords were minced and incubated in an enzyme solution containing Earle's salts, 20 units/ml Papain (Worthington), 0.5 (ethylenedinitrilo)tetraacetic acid, disodium salt, dihydrate (EDTA) and 1.65 mM L-cysteine for 20 min at 37°C. The tissue was dissociated by passages through a Pasteur pipette in complete media [Earle's Minimum Essential Medium (MEM) containing 10% fetal bovine serum (Hyclone), penicillin/streptomycin (500 units/each), and 20 mM glucose] containing trypsin inhibitor and bovine serum albumin (BSA) (1.5 mg/ml each). The cells were then centrifuged at 1,000 rpm for 10 min. The supernatant was discarded, the cells resuspended, and plated onto polyornithine/laminin-coated 12-mm circular glass coverslips. The cultures were fed three times a week by changing one-third of the media.
This technique is derived from the mixed glial cell culture technique of Sontheimer et al. (1992)
. The major difference was that 1.5 × 105 cells were seeded per coverslip, against 2.5 × 104 in the original technique. Figure 1 shows typical examples of low-density (Fig. 1A, 1 and 2) and high-density (Fig. 1B, 1 and 2) cultures stained for astrocytic [glial fibrillary acidic protein (GFAP)] and neuronal markers [microtubule-associated protein-2 (MAP-2), NF] after 3 days in vitro (DIV). Figure 1C shows a 7 DIV high-density culture stained with neurofilament (NF) antibodies. In low-density (LD) cultures, GFAP antibodies stained most of the cells. Astrocytes with typical flat or stellate morphology were easily recognized (Fig. 1A1). No NF+ cells were seen, even after 14 DIV (not shown). However, MAP-2 antibodies stained numerous very small cells with short processes at early times in culture (Fig. 1A2). MAP-2 staining was absent at 7 DIV and 14 DIV in LD cultures.

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| FIG. 1.
Mixed cultures of postnatal spinal cord. A: 3 days in vitro (DIV) culture of spinal cells seeded at low-density (2.5 × 104 cells/coverslip). A1: immunostaining with an astrocytic marker [glial fibrillary acidic protein (GFAP)]. A2: immunostaining with a neuronal marker [microtubule-associated protein-2 (MAP-2)]. B: 3 DIV culture of spinal cells seeded at high density (1.5 × 105 cells/coverslip). B1: staining with GFAP. B2: staining with neurofilament antibodies (NF). C: high-density culture after 7 DIV and stained with neurofilament antibodies (NF).
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In high-density (HD) cultures, astrocytes formed a confluent monolayer after ~3 DIV (Fig. 1B1). On top of the astrocyte bed, numerous NF+ cells were identified (Fig. 1B2) and NF+ processes extended throughout the culture. At 7 DIV, the number of NF+ cells was increased and the cultures were covered by a dense network of NF+ processes (Fig. 1C).
Immunocytochemistry
For labeling of GFAP, NF, and MAP-2, the coverslips were rinsed three times with phosphate-buffered saline (PBS) and fixed for 10 min at room temperature with 4% paraformadehyde in 0.14 M Sorensen's phosphate buffer. The coverslips then were incubated sequentially in: PBS three times, 5 min each; blocking solution (PBS/5% normal goat serum/1% BSA) containing 0.1% triton, 30 min; primary antibodies diluted in blocking solution (GFAP 1:100, NF 1:500: MAP-2 1:50), 1 h at room temperature; blocking solution, six times, 5 min each; goat anti-rabbit or goat anti-mouse conjugated to Texas red (1:200, Cappel), 1 h at room temperature; and PBS, six times, 5 min each.
The primary antibodies used were polyclonal rabbit GFAP and NF and monoclonal mouse MAP-2, all purchased from Chemicon. The coverslips were examined on a Leitz Aristoplan microscope with mercury arc and tungsten illumination, equipped with L3 filter cubes.
Electrophysiology
Membrane currents and potentials were recorded with the patch-clamp technique in whole cell configuration (Hammill et al. 1981)
with an EPC9 amplifier (HEKA). Patch electrodes were pulled from thin-walled borosilicate glass with inner filament (Warner) to an open resistance of 2-3.5 M
. Series resistance (RS) and whole cell capacitance were measured by cancellation of the capacitive transients. Cells were rejected if the RS after establishing whole cell configuration did not fall between 2.5 and 5 M
. Series resistance compensation was not used; this permitted us to maintain as high a signal-to-noise ratio as possible. The cells were continuously perfused, permitting application of drugs. Current and voltage traces were filtered at 1 kHz by the low-pass filter of the amplifier. All recordings were performed at room temperature.
The external medium contained (in mM) 154 NaCl, 5 KCl, 2 CaCl2, 0.5 MgCl2, and 5 glucose, buffered with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH adjusted to 7.4 with NaOH). Pipettes were filled either with solution 1, containing (in mM) 90 CsF, 30 tetraethylammonium Cl, 33 CsOH, 2 MgCl2, 1 CaCl2, 1 Na2 ATP, and 11 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or with solution 2, containing 120 KF, 33 KOH, 2 MgCl2, 1 CaCl2, 1 Na2 ATP, and 11 EGTA. pH was adjusted to 7.4 by CsOH (solution 1) or KOH (solution 2). Tetrodotoxin (TTX; Calbiochem), DL-2-amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, ICN), bicucculline, GABA, NMDA, and glycine were added to the normal solution from concentrated solution (1,000×). All chemicals were purchased from Sigma unless otherwise indicated.
Data analysis
The traces were transferred to Origin (Microcal) to perform peak analysis. Multiple peak activation considerably offset some of the events, causing a bias in the amplitude distribution histograms. Thus after a first peak determination giving the values of respectively the beginning, the summit and the end of each peak, a baseline was created. This baseline was subtracted from the traces. A second peak count was then performed and used for numerical analysis. The sensitivity of peak detection was set to 3.5-pA minimal amplitude and 1-ms minimal duration. Peaks also were counted visually on a 250-ms sample from each trace; the results were always within 10% of the computerized count.
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RESULTS |
The cells included in this study were recorded between 4 and 8 DIV. Neurons were recognized easily by their typical triangular shape and thin long processes. Occasionally, however, cells of putative glial origin were patched. To discriminate neurons from glial cells, only those cells with a Na+ current density of >400 pA/pF were considered for further recording. The mean Rinput at
80 mV was 1.32 ± 4.35 G
with solution 1 (n = 12) and 1.19 ± 3.9 G
(n = 35) with solution 2, indicating that at this holding potential the type of internal solution did not influence the measurements. The membrane potential was measured in 17 cells with solution 2 with a mean value of
54 ± 4.9 mV.
GABAAR and AMPAR spontaneous activity
One hundred percent of the neurons displayed some spontaneous activity when recorded in voltage clamp. From cell to cell, this spontaneous activity was variable, ranging from frequent changes of over several hundreds of pA to rare events not exceeding the 100-pA range. However, it remained rather constant for a given cell, even during long-lasting recordings. Figure 2 shows a typical example of current traces recorded at a holding potential of
80 mV from a cell displaying intense spontaneous activity. Each trace lasted 250 ms. Twenty traces were recorded sequentially for each recording, which thus lasted 5 s, and superimposed. This acquisition protocol was used for all the recordings shown in this paper.

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| FIG. 2.
Isolation of -aminobutyric acid-A (GABAA) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) postsynaptic currents. A: typical voltage-clamp traces obtained from a cell with intense spontaneous activity at a holding potential (Vh) of 80 mV. Internal solution contains 36 mM of [Cl ] (ECl = 39 mV). Cell was bathed in bicucculline (50 µM) and then bicucculline + 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 µM) as indicated. This recording displays 20 superimposed traces of 250-ms duration. B: at 80 mV, the 2 currents are inward, at 20 mV, the slow current is outward and the fast current inward, and at +40 mV, both currents are outward. Insets: enlarged fast currents. C: exponential fitting of the slow and fast currents.
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At first sight (Fig. 2A, control), two categories of spontaneous events could be distinguished: first, long-lasting currents of large amplitude and second, short-lasting currents, the amplitude of which was about one order of magnitude smaller. These two classes of events could be differentiated pharmacologically. Bicucculline (50 µM) completely blocked the spontaneous occurrence of the large amplitude currents (n = 6), and CNQX (5 µM) blocked the small amplitude events. Figure 2B shows that the currents also could be separated by their respective reversal potentials. The slow currents always reversed between
30 and
40 mV, and the fast currents near 0 mV. The decaying phase of each of these two currents could be fitted with a single exponential (SD < 1% peak). The
of decay was 32.05 ± 4.04 ms (n = 42, samples taken from 5 cells) for the slow currents and 1.35 ± 0.17 ms (n = 23, samples from 3 cells) for the fast currents. The reversal potential of the slow currents was close to the reversal potential of the current induced by application of GABA (
34.4 ± 3.2 mV, n = 5, not shown), and to the theoretical equilibrium potential for Cl
ions (
39.7 mV for solution 1). The bicucculline blockade, the reversal potential and the inactivation
identified the slow current as caused by spontaneous GABAA receptor activation. The small currents were blocked by CNQX, reversed near 0 mV, and had a fast inactivation
, consistent with the conclusion that they represent EPSCs due to AMPA-receptor activation.
The spontaneous activity thus could be separated into events evoked by GABAA and AMPA input activation, according to respective reversal potentials, sensitivity to antagonists, and inactivation properties. No clearly defined spontaneous events due to NMDAR openings were observed. Nevertheless, application of NMDA (50 µM) and glycine (3 µM), as shown in Fig. 3, induced an inward current in all the cells tested (n = 14) that averaged 48.1 ± 25.4 pA at
40 mV.

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| FIG. 3.
N-methyl-D-aspartate (NMDA) current. NMDA (50 µM) together with glycine (3 µM) were perfused as indicated at different holding potentials. Both the control and the test solution contained tetrodotoxin (TTX; 100 nM) and Cd2+ (200 µM) to prevent synaptic contamination of the NMDA-induced current. Graph shows the I-V relationship of the NMDA-induced current obtained on 5 cells normalized to the current recorded at 40 mV.
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APV and TTX reduce spontaneous AMPAR activity
To examine the roles of NMDARs and Na+ channels in evoking spontaneous EPSCs, we tested the effects of APV and TTX. This approach aimed also at isolating a putative contribution of NMDAR-mediated signals to the spontaneous glutamate activity. The GABAA inhibitory postsynaptic currents (IPSCs) are not visualized on the traces on Fig. 4 because ECl was very close to the holding potential (Vh) (solution 2). Figure 4A shows that APV (100 µM) application dramatically reduced the spontaneous occurrence of the AMPA EPSCs. In these experiments, cells were exposed to the drug for 1 min before the recording was performed. Another recording was taken after 1 min of wash-out. APV was tested on nine cells, which all exhibited spontaneous EPSCs. In one cell, no EPSCs were recorded in the presence of APV; in four cells, a decrease of >80% of the EPSC frequency was observed; in two cells, a decrease of >50% was recorded; and in two cells, APV had no effects. In these two cells, the control number of spontaneous EPSCs was much lower than in the seven where APV had a pronounced effect. In every case, the effects of APV were fully reversible. Figure 4B shows a representative example of the effects of TTX (100 nM) on the spontaneous occurrence of the AMPA EPSCs. This concentration of TTX, which was sufficient to block >95% of the voltage-gated Na+-current on all the cells tested (Fig. 4D), also reduced the number of spontaneous EPSCs. In all cases (n = 7), TTX reduced the number of EPSCs by >60%.

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| FIG. 4.
Blockade of spontaneous AMPA activity with DL-2-amino-5-phosphonovaleric acid (APV) AND TTX. A: voltage-clamp traces of a cell before (control) and during APV (100 µM) application. B: similar recordings of another cell treated with TTX (100 nM). Note the decrease in fast excitatory postsynaptic current (EPSC) occurrence with both treatments. C: amplitude fluctuation histograms of the fast EPSCs recorded in a cell sequentially bathed with TTX and then APV. A 100-ms trace showing small size events is in insets. Histograms extend only to 100 pA maximal amplitude to better focus on the small events. All effects were reversible. D: effect of TTX (100 nM) on the fast inward current elicited by depolarizing pulse to 10 mV from a Vh of 120 mV. In all cases, the inward current was reduced to >95% of the control amplitude.
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We used quantal analysis of EPSCs to determine whether the effects of NMDAR activation or Na+ channel activity were pre- or postsynaptic. Figure 4C shows distribution histograms of the EPSC amplitudes of a cell treated sequentially with TTX and APV. The histograms correspond to a recording period of 5 s. The amplitudes at the peaks were measured and counted using a computerized method (see METHODS). A recording was taken between application of the two drugs, as well as at the end of the experiment, with a result superimposable to the control (not shown). The control histogram showed a peak at ~5 pA with a pronounced tail to the right due to the occurrence of larger amplitude events. In all the cells tested, the peak was between 5 and 7 pA (n = 14). After TTX or APV application, despite the dramatic decrease in the count of events, a peak was clearly still observed at ~5 pA. In studies of CNS glutamate synapses, distribution histograms of spontaneous events amplitude invariably show a peak between 5 and 10 pA (reviewed by Bennett 1995)
, which is thought to correspond to the glutamate quantum size. A change in the quantum size (i.e., a shift of the peak to the left or right) indicates a change in the postsynaptic contribution to EPSCs, whereas a change in the number of released quanta (i.e., a change in the size of the peak and of secondary peaks) indicates a modification of presynaptic mechanisms (reviewed by Kullmann and Siegelbaum 1995)
. Our results thus indicate that spontaneous occurrence of EPSCs is increased by endogenous NMDAR activation by mechanisms that include presynaptic events. Moreover, in addition to NMDAR activation, Na+ channel activity also is required to maintain spontaneous EPSC occurrence.
Effects of 0 Mg2+ solutions on AMPA EPSCs
The mechanisms by which activation of NMDARs contributed to the glutamate release were difficult to further isolate because blocking any steps of the cascade caused a dramatic decrease in the occurrence of the EPSCs. In a further series of experiments, we thus tried to increase the endogenous NMDAR activity by removing Mg2+ block. The effects of 0 Mg2+ solutions are presented in Fig. 5. Omitting Mg2+ in the perfusion solution resulted in a dramatic increase in the number of spontaneous AMPA EPSCs by 519 ± 141% (n = 9). In Fig. 5B, it can be seen that despite the much larger numbers of events, the general characteristics of the amplitude distribution histograms were not affected. The peak at ~5 pA was always preserved together with its skewed aspect. Figure 5 also shows that the effects of the 0 Mg2+ solutions can be accounted for by NMDAR activity because the application of APV always reduced the number of AMPA EPSCs to less than control level (n = 5). The experiments in Fig. 5C show that the effects of the 0 Mg2+ solutions were rapidly and totally reversible. For those experiments (n = 4), cells were exposed to both the 0 Mg2+ and the control solution for 1 min. The EPSCs were counted under each condition and normalized to the initial count. Application of CNQX in the 0 Mg2+ solutions invariably suppressed the spontaneous activity (Figs. 5-7), showing that, even if a large increase in the number of EPSCs was recorded due to removal of Mg2+ block on the NMDARs, these EPSCs were still due to AMPAR activation. These experiments confirm that NMDARs are coupled to the glutamate release. They also show that the coupling mechanism is rapidly and fully reversible and does thus not imply long-term effects.

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| FIG. 5.
Mg2+-free solutions increase the AMPA activity. A: voltage-clamp traces (Vh 80 mV) of a cell sequentially bathed with a Mg2+-free solution, with the same solution together with APV (100 µM), and finally Mg2+-free solution together with CNQX (5 µM). B: amplitude distribution histograms of the fast EPSCs recorded in another cell. Drug sequence was the same. Histogram corresponding to the CNQX solution is not shown because only 9 peaks were counted. C: cells (n = 4) were bathed twice in Mg2+-free solution. Sequence was 1 min each solution. Numbers of EPSCs were counted in each condition and normalized to the original value. Effects of 0 Mg2+ were rapidly and totally reversible.
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Effect of TTX on the NMDAR-induced response
The large increase in the number of AMPA EPSCs in the 0 Mg2+ solution allowed us to better isolate the effects of, respectively, the NMDAR activity and Na+ channel activity. Figure 6 shows how TTX affected the cells' response to 0 Mg2+ solutions. The traces were obtained from a cell that was bathed sequentially in 0 Mg2+, 0 Mg2+ together with TTX, and finally 0 Mg2+ together with both CNQX and TTX. Histograms corresponding to another cell treated with the same protocol are presented in Fig. 6B. The recordings showed that some of the effects of the 0 Mg2+ solution depended on an increase in the Na+ channel activity because the occurrence of large AMPA EPSCs was strongly diminished by the addition of TTX. However, the count of the small events was strikingly increased in TTX as compared with control and 0 Mg2+ solution.

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| FIG. 6.
Effects of TTX on the 0 Mg2+ response. A: voltage-clamp traces of a cell bathed with a Mg2+-free solution, then with the same solution together with TTX (100 nM), and finally with the Mg2+-free solution together with TTX + CNQX (5 µM; Vh 80 mV). B: amplitude distribution histograms of the AMPA EPSCs recorded in another cell. Drug sequence was the same. Histogram corresponding to the CNQX solution is not presented because 5 peaks were detected.
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In four similar experiments, the relative proportion of the small events (<10 pA) to the total count shifted from 47 ± 11% in 0 Mg2+ to 93 ± 3% in 0 Mg2+ together with TTX. One interpretation of these results is that the primary effect of endogenous activation of NMDARs was to increase the number of the small EPSCs (<10 pA), which in turn caused Na+-dependent spiking of the cells and a secondary increase in the number of the large AMPA EPSCs. These experiments thus suggested an effect of the NMDARs on the presynaptic terminals. Such an effect could be mediated directly by presynaptic NMDARs or could be an indirect effect due to retrograde signaling from a postsynaptic element to the presynaptic terminals. It is also possible that intense activity during exposure to 0 Mg2+ triggered a cascade of events producing a sustained increase in the number of small EPSCs, still observed after TTX blockade. A further series of experiments thus were performed in which TTX was added before removal of the Mg2+ block. In those experiments, TTX was introduced as soon as the cells were set for recording. Figure 7 shows that even though no Na+ spiking was allowed to take place, the 0 Mg2+ solution still increased the count of the small EPSCs. The TTX histogram in Fig. 7B was characterized by a peak at 5 pA. The Mg2+-free solutions increased the count of EPSCs by 287 ± 33% (n = 5) but preserved the characteristics of the histogram without a large shift in the peak. Figure 7A also shows that once the AMPA receptors were blocked, NMDAR activity could be observed as single channel events. In Fig. 7C, a representative trace of 5-s duration recorded in 0 Mg2+ together with TTX and CNQX is shown. The single channel NMDAR current at
80 mV was 4.29 ± 0.32 pA (n = 4 cells), which, given a reversal potential of the whole cell current induced by NMDA of + 4.7 mV (see Fig. 3), sets the conductance of the NMDA channels to 50.6 ± 3.8 pS. The APV blockade (Fig. 7A) and the single channel conductance thus clearly identified these currents as arising from NMDA channels.

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| FIG. 7.
Effect of 0 Mg2+ in TTX. A: voltage-clamp traces of a cell treated sequentially with Mg2+-free solution, then with the 0 Mg2+ together with CNQX (5 µM), and finally together with CNQX + APV (100 µM). TTX (100 nM) was present in every solution at all times, beginning when the coverslips were set for recording. Note the single channel openings of NMDA receptors. B: amplitude distribution histograms of the AMPA EPSCs recorded in another cell. Drug sequence was TTX 100 nM, and TTX in Mg2+ free as indicated. Detection threshold of the peak analysis was set to 2 pA because of the low noise level of the recordings. C: 5-s duration recording in Mg2+-free solution together with CNQX and TTX showing the NMDA receptor openings.
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GABA system is inhibitory in cultured spinal neurons
Our results show that sustained NMDAR activity elicits glutamate release at AMPA synapses that in turn triggers spike electrogenesis. According to this schema, cells in these cultures form self-induced positive loops in which basal activity is controlled by the Mg2+ block on NMDAR. We tested the hypothesis that the Mg2+ block is maintained under normal conditions by the hyperpolarizing activity of spontaneous GABAAR activity. It has been shown that GABAAR activation is excitatory in prenatal neurons and during the first days after birth due to the respective values of the cells' resting membrane potential (RMP) and the Nernstian equilibrium potential for Cl
ions (ECl) (reviewed by Ben-Ari et al. 1997)
. To determine whether the GABAA inputs were excitatory or inhibitory in our system, the effects of GABA were tested before breaking into whole cell configuration while the internal concentration of Cl
ions was at its "natural" level. Figure 8A, inset, shows that application of low concentration of GABA (1 or 2 µM) suppressed the spontaneous spiking activity (n = 5). When GABA was washed from the superfusion fluid, a burst of action potentials (APs) was sometimes recorded (3 of 5 cells). These results showed that GABAAR activation resulted in inhibition of activity.

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| FIG. 8.
GABAA-mediated inhibition. A: current-clamp recordings of neurons with an internal solution containing 6 mM of [Cl ]. No current was injected. Application of GABA (2 µM) and bicucculline (50 µM) is indicated. Inset: recording in the cell-attached mode. Upward deflections are spontaneous action potentials. Cell was bathed sequentially with bicucculline and bicucculline + CNQX (B), and bicucculline and bicucculline + APV (C) as indicated; 8 pA of current was injected. D: cell was bathed in Mg2+-free solution, then the same together with APV (100 µM), then the same with CNQX (5 µM).
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To record in the current-clamp mode the consequences on the cells' potential of endogenous activity of the GABAA receptors, we used a pipette solution (solution 2 in METHODS) where the Cl
concentration was 6 mM. The reversal potential of the current induced by exogenous application of GABA was
81.2 ± 2.6 mV (n = 5, not shown). Figure 8, A-C, shows representative examples of the changes in the cells' potential induced by bicucculline and GABA application. Application of GABA (2 µM) caused hyperpolarization as well as a decrease of the spontaneous baseline activity. Conversely, bicucculline application induced a sustained depolarization, firing of action potentials, and marked increase in the baseline activity. These three effects were observed on seven of eight cells tested.
The cell pictured on Fig. 8, B and C, was slightly hyperpolarized by 8 pA of current injection because spontaneous occurrence of bursts of action potentials overshadowed the effects of bicucculline. Figure 8B shows that CNQX completely suppressed the effects of the bicucculline solution (n = 4) because the spiking, the depolarization, and the spontaneous activity were blocked despite the continuous presence of bicucculline in the superfusate. In Fig. 8C, a sustained burst of APs was induced in the same cell by the bicucculline solution. Application of APV (50 µM) resulted in partial blockade of the effects of bicucculline. In the four cells tested, APV application diminished the frequency of the spikes induced by bicucculline by >90%. To further examine the role of NMDARs in this bursting, we removed Mg2+ from the external fluid as shown in Fig. 8D. The cell was hyperpolarized to
70 mV by 15-pA current injection. These current-clamp-mode traces, which are representative of six similar experiments, show that the 0 Mg2+ response was characterized by firing of action potentials and increase in baseline activity, consistent with contribution of NMDAR activity to bursting. This bursting is totally blocked by both APV and CNQX.
These experiments showed that the neurons' membrane potential was set continuously by a balance of opposing stimuli: inhibition by GABAAR activity and, conversely, excitation by AMPAR activity. The spontaneous AMPA synaptic activity was strongly dependent on NMDAR activity, which thus sets the intensity of excitation. Under normal conditions, this is modulated by Mg2+ blockade. Our experiments suggest that, at this stage of synapse formation, GABAA-mediated inhibition achieves this modulation. The observation that the 0 Mg2+-response is totally abolished by CNQX shows that the major result of endogenous NMDAR activation, in this system, is to increase the release of glutamate at AMPA synapses.
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DISCUSSION |
Quantal analysis in the CNS
Quantal analysis, originally used at the neuromuscular junction to describe synaptic transmitter release (Del Castillo and Katz 1954)
, is based on the study of amplitude distribution histograms of spontaneous or evoked postsynaptic events. Such distributions generally are described by a mixture of Gaussians the maxima of which are equidistant. The distance between two maxima, or the position of the first peak if multiple peaks cannot be fitted, defines the quantal size, which is thought to correspond to the smallest packet of transmitter releasable at one time (reviewed by Bennett 1995)
. In the CNS, quantal analysis has been used in various models to study the spontaneous AMPA EPSC amplitude distribution. As compared with the studies at the neuromuscular junction, quantal analysis at CNS AMPA synapses is complicated by multiple innervation, the cable properties of nonisopotential neurons that distort signals, and a much smaller quantum size. The distribution histograms of spontaneous AMPA EPSCs usually are described by a first peak at ~10 pA (Hestrin 1992a
; Jonas 1993
; Silver et al. 1992
; Traynelis et al. 1993)
or 5 pA (Bolshakov and Siegelbaum 1995
; Manabe et al. 1992)
, which is thought to correspond to the size of the glutamate quantum. It reflects simultaneous opening of 10-20 AMPA channels on the postsynaptic side, depending in each case on the single channel conductance. This peak moreover is always skewed to the right by larger events, the occurrences of which are thought to be due to multiquantal release. In the CNS, quantal analysis has been used as a tool to determine the site of changes in synaptic efficacy, such as those induced by long-term potentiation (LTP), as a change in the quantal size indicates a change in the number of receptors stimulated on the postsynaptic membrane by the quantum and a change in the number of quanta released indicates a presynaptic mechanism (Bekkers and Stevens 1990
; Manabe et al. 1992
; reviewed by Kullmann and Siegelbaum 1995)
.
In our study, the distribution histograms of AMPA EPSCs defined a first peak at ~5 pA with a skewed aspect. This skewed aspect was attenuated strongly by TTX application, indicating that Na+-dependent spiking of presynaptic neurons was required for the occurrence of multiquantal events. The observation of a well-defined peak at 5 pA in the TTX solution also showed that a vast majority of the small events were true AMPAR-mediated micro-EPSCs (MEPCS) and thus reflected the glutamate quantum size in our model. The quantal analysis approach used in this study proved very useful for characterization of the mechanisms by which NMDAR activation contributes to spontaneous activity. First, even though glutamate quanta and multiquanta were consistently recorded, a NMDAR-mediated quantum was not recorded despite the fact that we resolved single channel NMDAR currents the magnitude of which (4.29 ± 0.32 pA) predicted a NMDA channel conductance of 50.6 ± 3.8 pS, consistent with values obtained in previous studies (Lo Turco et al. 1991; Silver et al. 1992)
. If the assumptions (receptor density, synaptic cleft size) made to define a glutamate quantum at a AMPA synapse held true for a putative NMDAR-containing synapse, a "NMDA quantum" should be larger than a "AMPA quantum" because the conductance of the NMDA channel is larger than that of the AMPA channel and thus would not likely be overlooked. Such NMDA quanta were recorded in hippocampal neurons, where they average 15 pA of amplitude (Hestrin 1992b)
. In our system, the absence of NMDA quanta indicates that NMDAR activation very likely takes place at nonsynaptic sites or, alternatively, that the density of functional NMDARs in the postsynaptic membranes is very low. Second, endogenous activation of NMDAR increased the frequency of both quantal and multiquantal spontaneous glutamate release without change in the quantal size, indicating that NMDARs are coupled to the release of glutamate at presynaptic sites. Third, AMPAR activity results in Na+ channel-dependent spiking, which in turn induces multiquantal type of events. Our results thus define three steps of a cascade that sustains the spontaneous activity: activation of NMDARs (possibly at nonsynaptic sites), induction of glutamate release at AMPA synapses, and Na+ channel spiking. Blocking any of these three steps, by APV, TTX, or CNQX, resulted in a rapid decrease of the cells' spontaneous activity.
Nonsynaptic NMDARs
Before the establishment of AMPA synapses, glutamatergic transmission is thought to be exclusively dependent on NMDARs. The early glutamate synapses have been called "silent" synapses, because NMDARs are blocked by Mg2+ at RMP (Durand et al. 1996)
. In the developing optic tectal neurons of frog, spontaneous NMDAR activity has been recorded; on the basis of the conductance of a single NMDAR, however, it appears that this activity involved no more than a few channels at a time (Wu et al. 1996)
. Similarly, in the developing rat neocortex, spontaneous NMDAR activity was resolved only as single channel events (Lo Turco et al. 1991). In mature cerebellar granule cells, spontaneous AMPA EPSCs are followed by bursts of openings of NMDARs, also resolved as single channel events (Silver et al. 1992)
. Taken together, these experiments indicate that the release of glutamate at synapses, whether or not endowed with AMPARs, might reflect the activation of a much smaller number of NMDARs than expected from a typical channel density within a synaptic cluster.
Two recent papers argue in favor of a nonsynaptic type of expression of NMDARs in developing neurons, at least under culture conditions. First, studying synaptic development in rat cultured spinal neurons, O'Brien et al. (1997)
showed that between 2 and 4 DIV, the pattern of staining for GluR1 AMPAR subunits changes from diffuse to highly clustered, associated with presynaptic terminals in a majority of cases by 6 DIV. In contrast, the staining for NMDAR subunit NR1 is diffuse. The second study (Rao and Craig 1997)
showed that NMDARs are expressed as clusters that are mostly nonassociated with presynaptic terminals in cultured hippocampal neurons. The number of postsynaptic clusters of NMDARs, however, dramatically increases when the cultures are chronically treated with APV or TTX, indicating that spontaneous activity mediated by NMDAR can prevent synaptic expression of the NMDAR subunits. This mechanism, if generally applicable, could explain the absence of a clearly defined NMDAR-mediated spontaneous EPSC in our system, where spontaneous activity is intense. Finally, activation of nonsynaptic NMDAR has been proposed often because of the much higher sensitivity of NMDAR, compared with AMPAR, to glutamate (Patneau and Mayer 1990)
. In our system, nonsynaptic NMDAR activation could result from glutamate spill-over from adjacent AMPA or silent synapses (Isaac et al. 1997
; Kullmann et al. 1996)
or alternatively from liberation by nonneuronal cells such as astrocytes.
NMDAR activation and glutamate release
At this stage of our investigations, it is not possible to definitively identify the mechanisms that couple activation of the NMDARs to the release of the glutamate at AMPA synapses. Two possibilities can, however, be proposed. First, NMDAR activation might trigger a cascade at postsynaptic loci that, via retrograde signaling, leads to neurotransmitter release. This could occur through a Ca2+-wave that propagates in a retrograde fashion to presynaptic terminals (Leinekugel et al. 1997)
or alternatively to second-messenger activation. The second hypothesis is that NMDARs are expressed at presynaptic membranes and directly provide the Ca2+ necessary for neurotransmitter release; consistent with this suggestion, it has been observed that NR1 subunits can be localized close to presynaptic terminals (Aoki et al. 1994
, 1997
). Neurochemical results already have suggested the involvement of NMDAR in presynaptic mechanisms leading to release of glutamate (Bustos et al. 1992
; Hirsh et al. 1993
; Martin et al. 1991
; Montague et al. 1994)
, norepinephrine (Lehmann et al. 1992
; Montague et al. 1994)
, noradrenaline (Fink et al. 1990
; Wang et al. 1992)
, and dopamine (Krebs et al. 1991)
. It should be noted, however, that many of these studies were carried out either in vivo or in slices, where it is difficult to definitively assess the respective contribution of pre- and postsynaptic mechanisms, and in some of these studies using synaptosomes, varying degrees of postsynaptic elements are included so that a postsynaptic contribution cannot be ruled out. The mechanisms by which NMDAR activation triggers neurotransmitter release may involve corelease of nitric oxide (Hirsh et al. 1993
; Montague et al. 1994)
. Strikingly, these experiments were performed in a wide range of CNS areas, including hippocampus, striatum, cerebral cortex, olfactory bulb and spinal cord, and liberation of many transmitters, including GABA and aspartate as well as the transmitters listed above, was induced. Taken together, these studies suggest a common mechanism shared by many synapses, regardless of their origin.
To our knowledge, no other electrophysiological studies have yet focused on this particular function of NMDAR. Recently, however, an example of receptor-induced glutamate release has been demonstrated at the synapses between sensory neurons and postsynaptic dorsal horn neurons, where activation of presynaptic purinergic P2XRs by ATP induces glutamate release (Gu and MacDermott 1997). Interestingly, ATP not only causes a rapid spike-dependent multiquantal type of release but also a sustained increase in the frequency of quantal event occurrence, very similar to those that we observed. Finally, a presynaptic effect of NMDA receptor activation has been suggested in nociceptive primary afferents, where NMDAR activation induces substance P release onto dorsal horn neurons, causing substance P receptor recycling and pain behavior (Liu et al. 1997)
. Taken together with our findings, these experiments indicate that NMDARs, either expressed at presynaptic membranes or expressed at postsynaptic loci and coupled to the presynaptic element via a retrograde signal, are capable of controlling or modulating neurotransmitter release.
Implications for synaptic development
NMDAR-mediated spontaneous activity is known to play a fundamental role during postnatal development (reviewed by Constantine-Paton et al. 1990)
. In the visual system (Bear et al. 1990
; Cline and Constantine-Paton 1989) and rat spinal cord (reviewed by Hockfield and Kalb 1993)
, NMDAR blockade or TTX application result in a major disruption of the normal synaptic architecture. Long-term synaptic facilitation can be induced in developing neurons (Durand et al. 1996
; Harsanyi and Friedlander 1997
; Mooney et al. 1993)
and may participate in synapse formation, synapse selection, and development of normal brain architecture. However, although the LTP paradigm demonstrates how high-frequency stimulation strengthens certain pathways, it fails to explain how the spontaneous burst of excitation needed for its induction occurs. A striking feature of our experiments is the intensity of spontaneous AMPAR activity in our model system, as spontaneous EPSCs reported in studies of the mature CNS are rarer and smaller (Manabe et al. 1992
; Silver et al. 1992)
. Our results show that this spontaneous excitation is strongly dependent on endogenous NMDAR activity. We thus propose that, in the developing spinal cord, the NMDARs, because of their direct or indirect effects on glutamate release at presynaptic terminals at synapses, act as a potent amplification system of spontaneous synaptic events. As a result, intense spontaneous excitation occurs and could provide, at least in part, the activity needed for maturation of synaptic systems.
The high level of ongoing activity at AMPA synapses reported here has a precedent in GABAA system development. Functional GABAA synapses are present as early as embryonic stages. In prenatal life, as well as in early postnatal life, GABAA inputs depolarize neurons and account for a large part of excitatory neurotransmission (Connor et al. 1987
; Leinekugel et al. 1997
; LoTurco et al. 1995
; Reichling et al. 1994
; reviewed by Ben-Ari et al. 1997)
. These early networks generate spontaneous giant depolarizing potentials (GDPs) (Ben-Ari et al. 1989
; Leinekugel et al. 1997)
, caused by simultaneous activation of GABAA synapses. These GDPs vanish in the presence of TTX or APV and progressively diminish with development to completely disappear at P12. Similar to the AMPA EPSCs that we observed, GDP activation depends on both NMDAR and Na+ channel activity. The efficacy of blockade by APV in both systems suggests that spontaneous synaptic activity caused by NMDARs may be a typical feature of immature synapses and moreover does not depend on the type of receptor involved.
In conclusion, our experiments show that endogenous NMDAR activation is responsible for intense spontaneous activity at AMPA synapses in cultured spinal neurons. Although post- as well as presynaptic mechanisms may be involved in the coupling of NMDARs to the release of glutamate, the present findings, together with the recent results of Gu and McDermott (1997) and Liu et al. (1997)
, strongly suggest a role for NMDARs in controlling or modulating glutamate release in the developing spinal cord.
 |
ACKNOWLEDGEMENTS |
We thank Dr. S. Agulian for technical support and Drs. T. Cummins and A. Bloc for helpful discussions.
This work was supported in part by a grant from the Medical Research Service, Department of Veterans Affairs. A. Robert was supported in part by the Swiss National Fondation and by a Spinal Cord Research Fellowship from the EPVA.
 |
FOOTNOTES |
Address for reprint requests: S. G. Waxman, Neuroscience Research Center (127A), Bldg. 34, VA Medical Center, West Haven, CT 06515.
Received 23 December 1997; accepted in final form 7 April 1998.
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