Development of Glutamatergic Synaptic Activity in Cultured Spinal Neurons

Antoine Robert,1 James R. Howe,2 and Stephen G. Waxman1

 1Department of Neurology and Paralyzed Veterans of America-Eastern Paralyzed Veterans Association Neuroscience Research Center, Yale University School of Medicine, New Haven 06510; Rehabilitation Research Center, Veterans Affairs Medical Center, West Haven 06516; and  2Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Robert, Antoine, James R. Howe, and Stephen G. Waxman. Development of Glutamatergic Synaptic Activity in Cultured Spinal Neurons. J. Neurophysiol. 83: 659-670, 2000. The development of glutamatergic synapses involves a sequence of events that are still not well understood. We have studied the time course of the development of glutamatergic synapses in cultured spinal neurons by characterizing spontaneous synaptic currents recorded from cells maintained in vitro for different times. At short times in culture (2 days in vitro; DIV2), spontaneous synaptic activity consisted almost solely of N-methyl-D-aspartate (NMDA) receptor (NMDAR) openings. In contrast, older neurons (DIV5 to DIV8) displayed clear alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR)-mediated synaptic currents, while the NMDAR-mediated activity remained small. Between 8 and 14 days in vitro there was a large increase in the density of synaptically activated NMDARs, although there was no significant increase in the density of the NMDAR-mediated current activated by exogenous glutamate. The results indicate that there is a switch in NMDAR targeting from somatic to synaptic regions during the course of the second in vitro week. Finally, our results support the conclusion that the spontaneous synaptic activity displayed in culture depends on ongoing NMDAR-mediated activity, even when the expression of synaptic NMDARs is low.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although both alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors are concentrated at postsynaptic membranes in mature systems (Bekkers and Stevens 1989; Craig et al. 1993; Jones and Baughman 1991; Petralia et al. 1994; Siegel et al. 1994), there are indications that clustering of these subtypes of glutamate receptors may occur at different times during development. For example, in situ studies have indicated that, during early development, NMDA receptor (NMDAR)-mediated synaptic currents can be recorded at times when AMPA receptor (AMPAR)-mediated currents are absent (Durand et al. 1996; Wu et al. 1996). Conversely, in cultured spinal neurons, studies have shown that NMDAR expression at postsynaptic sites may be completely lacking, despite extensive synaptic clustering of AMPARs (O'Brien et al. 1997, 1998). These latter studies suggested that the development of glutamatergic synapses in the spinal cord, at least in vitro, may occur in the absence of NMDAR activity. These findings are surprising, however, given the large body of work showing that synaptic organization in the CNS, including the spinal cord, depends critically on NMDAR activity (Hockfield and Kalb 1993; Kalb 1994; reviewed by Constantine-Paton and Cline 1998).

Indeed, recordings from spinal cord slices have shown that NMDAR activity contributes substantially to glutamatergic neurotransmission within the spinal cord as early as postnatal day 0 (P0) (Bardoni et al. 1998; Gao et al. 1998). Thus slice and culture studies of the developing spinal cord have reached opposite conclusions concerning the presence of NMDARs at glutamatergic synapses. Because glutamate-receptor targeting is influenced by the level of spontaneous activity in culture systems (O'Brien et al. 1998; Rao and Craig 1997), differences in the level of ongoing synaptic input might contribute to the noted differences between spinal neurons in vitro and in situ. It is also possible that NMDARs are expressed at synaptic regions on cultured spinal neurons, but only at low density, making detection of NMDAR-mediated synaptic currents difficult and preventing clear evidence of clustering in immunocytochemical studies.

We have recorded spontaneous NMDAR activity in cultured spinal neurons that were maintained for different numbers of days in vitro (DIV). Our results show that spontaneous NMDAR-mediated activity is always present and is largely responsible for maintaining sustained synaptic activity, although during the first week in culture the density of NMDA receptors at postsynaptic regions is probably low. Between DIV8 and DIV14, the density of NMDA receptors in synaptic regions increases markedly, although there is little change in the total number of functional NMDARs per unit cell area. This latter result suggests that the subcellular targeting of NMDARs changes with time in culture and that receptors that are initially distributed largely extrasynaptically are primarily localized at synaptic regions by the end of the second week in vitro.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Embryonic rat spinal cord culture

Spinal cords were removed from embryonic day 17 (E17) Sprague-Dawley rat pups and cultured as described previously (Robert et al. 1998). Briefly, spinal cords were minced and incubated in an enzyme solution containing Earle's salts, 20 units/ml papain (Worthington), 0.5 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 astrocyte-conditioned media [Earle's minimum essential medium (MEM) containing 10% fetal bovine serum (Hyclone), penicillin/streptomycin (500 units/each), and 20 mM glucose] with trypsin inhibitor and bovine serum albumin (BSA; 1.5 mg/ml each). The cells were then centrifuged at 500 r.p.m. for 5 min. The supernatant was discarded, and the cells resuspended and plated onto polyornithine/laminin-coated 12 mm circular glass coverslips at a density of 2 × 104 cells per coverslip. The cultures were fed three times a week with complete astrocyte-conditioned medium.

Astrocyte-conditioned medium

Astrocyte-conditioned medium was prepared as described in Ye and Sontheimer (1998). In brief, the medium used for dissociation and maintenance of the cultures was conditioned for 24 h by confluent spinal cord astrocytes cultured in 75-ml flasks. The astrocytes were derived from P2 rat pups and were maintained in vitro for up to 3 wk.

Electrophysiology

Whole cell patch-clamp recordings (Hamill et al. 1981) were made 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 MOmega . Series resistance (RS) and whole cell capacitance were measured by electronic cancellation of the capacitive currents associated with a small voltage pulse. Cells were rejected if the RS (after establishing a whole cell recording) did not fall between 2.5 and 8 MOmega . Series resistance compensation was not used, which improved the signal-to-noise ratio. The cells were continuously superfused, and drugs were added to the normal external solution. Whole cell currents were analogue low-pass filtered at 1 kHz (4-pole Bessel-type, -3 dB) and were written directly to the hard-drive of the computer at a sampling rate of 30 kHz. All recordings were performed at room temperature.

The external medium was (in mM) 150 NaCl, 3 KCl, 2 CaCl2, 0.5 MgCl2, and 5 glucose, buffered with 10 mM HEPES (pH adjusted to 7.4 with NaOH). Glycine (1 µM) was added to all external solutions. In nominally Mg2+-free medium, the MgCl2 was omitted. Pipettes were filled either with a solution (solution 1) containing (in mM) 120 KF, 33 KOH, 2 MgCl2, 1 CaCl2, and 11 EGTA (pH adjusted to 7.4 with CsOH), or a solution (solution 2) containing 90 KF, 33 KOH, 30 tetraethylammonium Cl, 2 MgCl2, 1 CaCl2, and 11 EGTA. DL-2-Amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 5,7-dinitroquinoxaline-2,3-dione (DNQX), kainic acid, NMDA, tetrodotoxin (TTX), glycine, 1(4-aminophenyl)-3methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3benzodiazepine (GYK1 53655) were added to the normal. GYKI 53655 was kindly provided by David Leander (Eli Lilly). All other chemicals were purchased from Sigma Chemical.

Data analysis

For analysis, the data were imported to Origin (Microcal). Sample point histograms were constructed of single-channel NMDAR activity recorded in the whole cell mode. In some cases, the histograms were fitted with multiple Gaussian components. The multipeak fitting routine of the program was used to obtain the multi-Gaussian repartition of the data points, giving both the mean and the area of each Gausssian. The decay of NMDAR-mediated excitatory postsynaptic currents (EPSCs), and the decay of currents evoked by the exogenous application of agonists, were fitted with biexponential functions to give time constants for the two components (tau 1 and tau 2) present in the decay. Results are given as means ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NMDAR-mediated activity in DIV2 neurons

Embryonic spinal neurons (E17) maintained in culture for 1 or 2 days displayed little spontaneous synaptic activity in Mg2+-containing external solution. The neurons had both high-input resistances (>1 GOmega ) and small capacitances (<10 pF), permitting low noise recordings and allowing the resolution of single-channel activity in the whole cell configuration. To detect the presence of NMDAR -mediated activity, the cells were held at a positive membrane potential, where Mg2+-block of NMDARs is partially relieved. Figure 1A shows continuous channel activity recorded at +40 mV. This activity sharply diminished on the application of APV (5 µM), indicating that it very likely reflected NMDAR openings. Similar results were obtained from seven other cells.



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Fig. 1. N-methyl-D-aspartate receptor (NMDAR) activity in 2 days in vitro (DIV2) neurons. A: baseline noise at +40 mV is decreased by the application of 5 µM DL-2-amino-5-phosphonovaleric acid (APV). Insets show that the noise is likely caused by discrete channel openings. B: typical recordings obtained from a DIV2 spinal neuron in Mg2+-containing and Mg2+-free solutions at -80 mV. C: data obtained in Mg2+-free solution are plotted as a sample-point histogram (the baseline has been set to 0 pA). The multiple Gaussian fit to the results is superposed, showing the main open levels. The mean current value for each peak is indicated.

We also recorded NMDAR activity at -80 mV in Mg2+-free solutions, a potential at which the single-channel currents were larger than at +40 mV and the recordings were less perturbed by voltage-gated channel activity. As shown in Fig. 1B, the application of Mg2+-free solution to an embryonic neuron revealed intense and continuous channel activity. This example is typical of all neurons tested (n = 33). In Fig. 1C, the data obtained in the Mg2+-free conditions are presented as a sample-point histogram (5 s of total recording time), showing that the points were gathered around several peaks and could be fitted with multiple Gaussian components (see METHODS). The mean current values corresponding to each peak are indicated on Fig. 1C. Such histogram plots obtained from seven different neurons showed that the second peak was always separated from the baseline peak by ~4 pA (4.1 ± 0.2 pA, mean ± SD), the third peak was separated from the second by 4.2 ± 0.4 pA, and the fourth from the third by 4.2 ± 0.6 pA. In all recordings analyzed (n = 23), at least two peaks (in addition to the baseline level peak) could be discerned, and in some experiments up to six peaks were discriminated. These experiments indicated that removing Mg2+ from the superfusate revealed spontaneous channel activity whose main open level was ~4 pA at -80 mV. The single-channel conductance and the reversal potential of the currents were determined from sample-point histograms obtained at -100, -80, and -60 mV. The resultant current-voltage plots gave a mean unitary conductance of 42.3 ± 4.3 pS and a reversal potential of 5.2 ± 3.2 mV (n = 4 cells, not shown). These values are close to those reported elsewhere for NMDAR channels by many investigators, including NMDAR openings recorded in the whole cell configuration (Blanton et al. 1990; Cull-Candy et al. 1988; Farrant et al. 1994; Lo Turco et al. 1991).

Sensitivity of the NMDAR activity to APV and TTX in DIV2 neurons

To further confirm that the channel activity recorded in DIV2 neurons was caused by NMDAR openings, we tested the effect of APV, a competitive NMDAR antagonist. Figure 2A shows the effect of the addition of APV (2 µM) on channel activity recorded in Mg2+-free solution. Sample-point histograms from portions (5 s) of the records with and without APV are presented in Fig. 2A (right panels). The area under the peaks arising from NMDAR activity was 16 ± 8% in APV (2 µM) compared with the corresponding area in the absence of the antagonist (n = 4 cells). In higher APV concentrations (50 µM), the NMDAR activity was totally abolished (n = 3 cells). These results support the conclusion that the channel activity consisted principally of NMDAR openings.



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Fig. 2. NMDAR activity is reduced by APV and tetrodotoxin (TTX). A, left: samples of single-channel activity recorded in a DIV2 neuron (Mg2+-free solution) before (top) and after (bottom) the application of APV (2 µM). Right: sample point histograms of the data obtained with and without APV. B, left: single-channel activity recorded in Mg2+-free solution before and after the application of TTX (200 nM). Right: sample-point histograms obtained with and without TTX. Note that in TTX the NMDAR openings are mostly isolated.

As shown in Fig. 2B, the application of TTX (200 nM), a concentration sufficient to completely block voltage-gated Na+ channels in these cells (not shown), resulted in a sharp reduction of the NMDAR activity observed in DIV2 spinal neurons in Mg2+-free solution. Moreover, the vast majority of the events recorded in TTX were restricted to isolated NMDAR openings. In six cells, we found a decrease of >80% in the area of the peaks corresponding to NMDAR activity, and there were no events corresponding to more than two simultaneous NMDAR openings. On average, the area of peaks observed in TTX was 11 ± 8% of the corresponding area without TTX. The results support the conclusion that the majority of the NMDAR activity resulted from the synaptic liberation of transmitter. Although the source of the TTX-insensitive activity is uncertain, it may reflect the spontaneous release of transmitter at synapses where the density of NMDAR channels is low.

NMDAR activity in DIV5 neurons in Mg2+-free solutions

Recordings from DIV5 neurons were characterized by the spontaneous occurrence of large AMPAR-mediated EPSCs (>100 pA). To measure the extent to which NMDAR currents were present, synaptic currents were recorded in Mg2+-free solution. As shown in Fig. 3A, switching to nominally zero external Mg2+ resulted in a severalfold increase in the frequency of spontaneous EPSCs. The frequency of AMPAR-mediated EPSCs in the different conditions are presented in Table 1.



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Fig. 3. NMDAR and alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activity in DIV5 neurons. A: spontaneous synaptic activity recorded in a DIV5 neuron in Mg2+-containing solution (top) and in Mg2+-free solution in the absence (middle) and presence (bottom) of TTX (20 nM). B: typical excitatory postsynaptic currents (EPSCs) obtained from 6 different DIV5 neurons in Mg2+-free solution containing 20 nM TTX. Note that the majority of the NMDAR openings are associated with AMPAR-mediated EPSCs and that all AMPAR-mediated EPSCs are temporally associated with a cluster of NMDAR activity. C: trace obtained by averaging 10 EPSCs recorded in Mg2+-free solution containing 20 nM TTX.


                              
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Table 1. Spontaneous frequency of EPSCs in Hz (>20 pA)

When the results obtained in Mg2+-free solution were plotted as sample-point histograms, peaks corresponding to NMDAR activity could be easily identified (not shown). However, due to the high frequency of synaptic events, it was impossible to determine whether NMDAR openings were temporally correlated with AMPAR-mediated EPSCs.

To assess whether NMDAR activity arose from activation of synaptically located receptors, we tested the effect TTX. When used at a fully effective concentration (200 nM), TTX reduced the size of the AMPAR-mediated EPSCs to an extent that did not allow us to clearly discriminate EPSCs. We found, however, that applications of 20 nM TTX (which reduced voltage-gated sodium currents by ~90%, not shown), greatly reduced the spontaneous synaptic drive while preserving the occurrence of large, isolated, AMPAR-mediated EPSCs (Fig. 3A, bottom trace). Representative records obtained from six additional spinal neurons in Mg2+-free solution and 20 nM TTX are shown in Fig. 3B. These experiments revealed a clear temporal correlation between AMPAR and NMDAR activity, with each AMPAR synaptic current being followed by a cluster of NMDAR openings. It was also evident from examination of the records in 20 nM TTX that some synaptic events, although rare, consisted solely of NMDAR activity. An example of such an event is indicated by the dotted box on the bottom trace in Fig. 3A.

The trace presented in Fig. 3C is the average of 10 spontaneous EPSCs recorded in one spinal neuron and shows that the synaptic activation of NMDA receptors results in a slowly decaying component similar to that found at most mature glutamatergic synapses, although the peak amplitude of the NMDAR component was at most 5-10% of the peak amplitude of the corresponding AMPAR component (n = 3 cells).

Effect of DNQX and CNQX on synaptic activity in DIV5 neurons

Previous studies have failed to detect NMDAR activity after blocking AMPAR-mediated synaptic currents with low concentrations (5-10 µM) of the competitive AMPAR antagonists CNQX or DNQX (O'Brien et al. 1997; Robert et al. 1998). Although this has been taken as evidence that NMDARs are not present at synapses, CNQX and other quinoxaline derivatives are also relatively potent competitive antagonists at the glycine binding site on the NMDAR (Birch et al. 1988; Lester et al. 1989; Randle et al. 1992). We confirmed that CNQX is a potent inhibitor of currents evoked in spinal neurons by the exogenous application of NMDA and glycine (not shown). We therefore reevaluated the effect of DNQX and CNQX on the spontaneous synaptic activity seen in DIV5 neurons. Figure 4A shows traces obtained from a DIV5 neuron in Mg2+-free solution both with and without DNQX (5 µM), as well as with DNQX and glycine (4 µM). This experiment, and eight similar experiments, showed that both AMPAR- and NMDAR-mediated spontaneous activity were markedly suppressed by DNQX or CNQX. However, quinoxaline inhibition of NMDAR-mediated activity could be reversed by elevating the glycine concentration of the external solution (n = 4 cells).



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Fig. 4. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 5,7-dinitroquinoxaline-2,3-dione (DNQX) inhibits both AMPAR- and NMDAR-mediated synaptic events in DIV5 neurons. A: spontaneous activity recorded from a DIV5 spinal neuron in Mg2+-free solution in the absence (top) and presence (middle) of 5 µM DNQX. Bottom trace shows NMDAR-mediated synaptic activity recorded in the presence of DNQX after adding 4 µM glycine to the perfusion solution. B: records from another DIV5 neuron obtained in Mg2+-free solution in the absence of antagonists (top) and in the presence of 5 µM CNQX (middle) or 100 µM GYKI 53655 (bottom). Note that CNQX blocks the vast majority of all synaptic currents, whereas GYKI 53655 selectively blocks fast AMPAR-mediated EPSCs and has little effect on NMDAR-mediated events.

Our findings that quinoxaline inhibition of NMDAR activity, but not inhibition of AMPAR activity, could be surmounted by increasing the glycine concentration, suggest that DNQX and CNQX blockade of NMDAR activity in cultured spinal neurons reflects competitive antagonism at the glycine site on the NMDA receptor. Further support for the idea that the quinoxaline inhibition of NMDAR-mediated events was not secondary to block of AMPAR-mediated synaptic drive was obtained by comparing the effects of CNQX with those of a more selective AMPAR blocker, GYKI 53655. Figure 4B shows traces obtained from a DIV5 neuron (in Mg2+-free solution) with and without CNQX (5 µM), as well as in the presence of GYKI 53655 (100 µM). Although both the NMDAR and AMPAR activity were markedly suppressed by CNQX, the application of GYKI 53655 suppressed AMPAR-mediated activity with little, if any, effect on the NMDAR-mediated activity. Similar results were obtained from three other neurons.

Spontaneous NMDAR and synaptic activity in DIV8 neurons

By eight days in vitro, spontaneous synaptic activity was prominent even in Mg2+-containing external solution. In addition to an increased frequency of EPSCs, inhibitory GABAergic currents, which were rare in DIV5 neurons, were common at DIV8. To record NMDAR-mediated activity in the presence of intact GABAergic activity, yet avoid contamination of the records from GABA-receptor mediated synaptic currents, the holding potential (Vh) was maintained slightly negative to the Nernst potential for Cl-, which was set to -25 mV with the internal solution used for these experiments (solution 2, see METHODS). These conditions partially relieved Mg2+ block of NMDA receptors and ensured that GABA receptor-mediated synaptic currents, which were very prominent at this stage, were small and outward and did not interfere with the analysis. The inset in Fig. 5A shows a portion of a recording obtained in APV, where arrows point to individual outwardly oriented synaptic currents, which are likely postsynaptic events resulting from activation of GABA receptors (Robert et al. 1998).



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Fig. 5. Spontaneous NMDAR and synaptic activity in DIV8 neurons. A: recordings obtained from a DIV8 neuron in Mg2+-containing solution before (top) and after (bottom) the application of APV (50 µM). The neuron was held at -25 mV to partially relieve Mg2+-block of the NMDA receptor. Inset: a portion of the recording during APV at higher gain (calibration bars: 10 pA and 10 ms). Arrows point to outwardly oriented synaptic currents, which correspond to GABAR-mediated synaptic currents. B: recording obtained from another DIV8 neuron showing the reversible shift in the holding current associated with NMDA receptor blockade by APV.

Figure 5A shows a typical example of the spontaneous synaptic activity recorded in a DIV8 neuron (Vh = -27 mV) in the presence of external Mg2+. At this stage, spontaneous EPSCs occurred at much higher frequency than at DIV5 (see Table 1). The contribution of NMDAR-mediated activity to the synaptic activity observed in DIV8 neurons was determined by recording synaptic currents in the absence and presence of APV (50 µM). Figure 5A shows the results of one such experiment. On average, 50 µM APV resulted in a fourfold reduction in the frequency of spontaneous AMPAR-mediated synaptic events (see Table 1). In addition, both the baseline noise and the holding current (Ih) were consistently and substantially reduced by APV. This is illustrated for another neuron in Fig. 5B. The decrease in Ih during APV application ranged from 7 to 55 pA (n = 6 cells). These results suggest that the increased synaptic activity observed at this stage in culture can be sharply reduced by blocking NMDARs.

Glutamatergic EPSCs at DIV8 recorded in TTX (20 nM)

We next sought to determine whether in DIV8 neurons glutamatergic EPSCs were composed of both AMPAR and NMDAR activity. As in DIV5 neurons, addition of a submaximal concentration of TTX (20 nM) preserved large amplitude spontaneous events yet reduced the level of synaptic activity in DIV8 neurons so that isolated EPSCs could be clearly identified. Except for the addition of TTX (20 nM), the conditions were the same as in the other DIV8 experiments (0.5 mM Mg2+, Vh near -25 mV).

Examples of recordings obtained from five different DIV8 cells are presented in Fig. 6A. As can be seen, most EPSCs contained a small NMDAR-mediated component. Ten EPSCs were averaged before and after the application of APV (50 µM) for each of the five cells shown in Fig. 6A. The criteria for the selection of EPSCs were that each event was temporally separated by at least 100 ms from another EPSC, and that the AMPAR-mediated current was larger than 20 pA. The average EPSC obtained from one cell is shown in Fig. 6B, where the NMDAR-mediated component has a slow decay and a small amplitude relative to the AMPAR-mediated component. In five cells, the amplitude of the NMDAR-mediated component was between 1.5 and 2.8 pA, compared with AMPAR components of 20-30 pA in the same cells. Although direct comparison of the amplitude of EPSCs in DIV5 and DIV8 is not possible because of the different recording conditions, at both times in culture the NMDAR synaptic currents are 5-10% the size of the AMPAR-mediated EPSCs.



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Fig. 6. Glutamatergic EPSCs recorded in TTX-containing solution from DIV8 neurons. A: spontaneous EPSCs recorded from 5 different DIV8 neurons in Mg2+-containing solution with TTX (20 nM; Vh = -25 mV). Note that a small NMDAR-mediated component is apparent in the EPSCs. B: averaged traces (10 EPSCs) obtained from a DIV8 neuron before (left) and after (middle) the application of APV (50 µM). Trace in the right column is the difference current obtained by subtracting the record in APV from the control record, showing the NMDAR-mediated EPSC.

In summary, the results obtained with DIV8 neurons indicated that the intense spontaneous synaptic drive observed in the presence of Mg2+ could be substantially reduced by blocking NMDA receptors. A majority of the glutamatergic EPSCs recorded at this time in culture contained both AMPAR- and NMDAR-mediated components, although the small size of the NMDAR-mediated EPSCs suggests that the density of NMDARs in synaptic regions is low. Nevertheless, in the absence of TTX, the high-frequency of glutamate-mediated synaptic events results in temporal overlap of NMDAR currents that is sufficient to result in a substantial and sustained inward current.

Spontaneous NMDAR-mediated EPSCs in DIV14 neurons

In contrast to neurons at early times in culture, the NMDAR component of glutamatergic EPSCs in DIV14 neurons was much more prominent. Figure 7A shows a typical example of spontaneous synaptic activity recorded in a DIV14 neuron in Mg2+-containing solution. At this stage, complex spontaneous polysynaptic events of large amplitude (>400 pA at Vh -25 mV) were always present (n = 12 cells). These events were composed of an initial burst of rapid AMPAR-mediated EPSCs that was invariably followed by a large long-lasting current. Although complete blockade of the slow currents required the addition of 50-200 µM APV, they were substantially reduced by much lower concentrations. The application of 2 µM APV typically reduced the amplitude of the slow component in the EPSCs by ~50% and produced a similar reduction in the frequency of spontaneous synaptic events. Comparison of all the results indicated that, although the frequency and amplitude of NMDAR EPSCs increased markedly from DIV2 to DIV14, there was no substantial difference in the sensitivity of the currents to blockade by APV. Comparison of the ability of APV to inhibit NMDA-evoked currents in neurons at different times in vitro also indicated that the NMDAR affinity of APV did not change with time in culture (not shown).



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Fig. 7. Spontaneous NMDAR and synaptic activity in DIV14 neurons. A: recording obtained from a DIV14 neuron in Mg2+-containing solution before (top) and after (bottom) the application of APV (50 µM). The neuron was held at -25 mV to partially relieve Mg2+-block of the NMDAR. Large complex polysynaptic events are invariably followed by a long-lasting current that is sensitive to APV blockade. B: recordings obtained from another DIV14 neuron showing that the NMDAR-mediated EPSCs could be fitted with a biexponential function.

Figure 7B shows recordings obtained from another DIV14 neuron where the slow decay of a spontaneous complex EPSC was fitted by a biexponential function. The mean time constants, tau 1 and tau 2, of the two components detected in the decay of the slow NMDAR-mediated currents were 97.3 ± 24 ms and 643 ± 356 ms (n = 8 cells, 3 EPSCs fitted per cell). The relative amplitudes of the fast and slow components were 0.71 ± 0.12 and 0.29 ± 0.12, respectively. The tau 1 and tau 2 values, as well as the corresponding amplitudes of each component, are similar to those reported elsewhere for NMDAR-mediated EPSCs (Kirson and Yaari 1996; and references therein).

Glutamatergic mEPSCs in DIV14 neurons

To obtain better estimates of the relative contributions of AMPARs and NMDARs to glutamatergic synaptic currents in DIV14 neurons, we studied miniature synaptic currents (mEPSCs) by recording spontaneous synaptic activity in the presence of a concentration of TTX (200 nM) sufficient to block neuronal firing (Mg2+-containing solutions, Vh approx  -25 mV). A typical example of the results obtained is presented in Fig. 8A, which shows the mEPSCs recorded before and after the application of APV (50 µM). The records obtained by averaging 10 mEPSCs in a neuron in both control and APV conditions are shown in Fig. 8B (criteria for selection were defined as for EPSCs in the previous section). Large NMDAR-mediated components were evident in the majority of mEPSCs in all DIV14 cells tested. The amplitude of the NMDAR-mediated component averaged 8 ± 4 pA, for an AMPAR-mediated component of 23 ± 6 pA (results from 6 cells). This represents a relative increase in the amplitude of the NMDAR-mediated EPSCs, as compared with the AMPAR-mediated EPSCs, of about fivefold between DIV8 and DIV14.



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Fig. 8. Glutamatergic miniature EPSCs (mEPSCs) recorded in DIV14 neurons. A: spontaneous mEPSCs recorded at -25 mV from a DIV14 neuron in Mg2+-containing solution with TTX (200 nM) before (top) and after (bottom) the application of APV (50 µM). B: averaged traces (10 mEPSCs) obtained from a DIV14 neuron before (left) and after (middle) the application of APV (50 µM). Trace in the right column is the difference current obtained by subtracting the record in APV from the control record, showing the NMDAR-mediated EPSC.

Density of the NMDAR-mediated current activated by exogenous glutamate

The above results show that the size of synaptic NMDAR-mediated currents increases substantially during the second week in culture. This difference between DIV8 and DIV14 neurons might simply reflect an overall increase in NMDAR expression. Alternatively, it might reflect a change in the proportion of NMDARs targeted to synaptic regions. To decide between these two possibilities, the amplitude and density of the NMDAR-mediated current activated by exogenous glutamate application were determined for cells maintained in culture for different times. Whole cell currents evoked at -80 mV in DIV5, DIV8, and DIV14 neurons by a brief application of glutamate (10 µM) are shown in Fig. 9A [Mg2+-free solution containing DNQX (5 µM), TTX (200 nM), glycine (10 µM) and strychnine (2 µM)]. The mean results are presented in Fig. 9B. The amplitude of the NMDAR-mediated whole cell current was 815 ± 189 pA at DIV5 (n = 7), 1,594 ± 305 pA at DIV8 (n = 5), and 2,363 ± 353 pA at DIV14 (n = 9). This threefold increase in current amplitude was accompanied, however, by an increase in cell size. When the current amplitudes were divided by the cell's capacitance and expressed as current densities (in pA/pF), similar results were obtained for the three groups (Fig. 9B).



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Fig. 9. NMDAR-mediated current density. A: currents evoked in individual DIV5, DIV8, and DIV14 neurons by the application of glutamate (10 µM) in Mg2+-free solution with DNQX (5 µM), TTX (200 nM), glycine (10 µM), and strychnine (2 µM). B: bar graphs showing the mean values of the whole cell NMDAR current (left), the cell capacitance (middle), and the current density (right, in pA/pF) measured for DIV5, DIV8, DIV14 neurons. Note that the current densities are similar for the 3 groups of neurons. C: biexponential fits of the decay of current evoked by glutamate in DIV5, DIV8, and DIV14 neurons.

Previous mRNA studies have shown that the expression of NMDAR2 subunits changes during spinal cord development (Monyer et al. 1994). In both expression systems and neurons, the set of NMDAR2 subunits that coassemble with NMDAR1 is a key determinant of the decay kinetics of NMDAR-mediated currents (reviewed by McBain and Mayer 1994). To determine whether the apparent redistribution of NMDARs during the second week in vitro was correlated with a change in subunit composition, we performed biexponential fits of the decay of the NMDAR-mediated currents. Figure 9C shows examples of the results obtained for DIV5, DIV8, and DIV14 neurons. We found values for tau 1 and tau 2 of 0.17 ± 0.06 s and 0.74 ± 0.11 s for DIV5 neurons (n = 7), 0.13 ± 0.04 s and 0.82 ± 0.21 s for DIV8 neurons (n = 5), and 0.14 ± 0.03 s and 1.09 ± 0.29 s (n = 7) for DIV14 neurons. The relative amplitude of the fast component was 61 ± 12% for DIV5 neurons, 78 ± 9% for DIV 8 neurons, and 83 ± 8% for DIV 14 neurons. These results suggest that the subunit composition of the NMDARs expressed in spinal neurons does not change markedly during the first 2 wk in culture.

NMDARs are concentrated near the cell body at DIV8

The fivefold increase in the amplitude of the NMDAR-mediated synaptic currents during the second week in vitro, in the absence of a concomitant increase in whole cell NMDAR-mediated current density, suggests that a large proportion of the functional NMDARs present at early times in culture are expressed at extrasynaptic sites. Previous studies in both spinal and hippocampal neurons have shown that most of the synapses occur on processes (Craig et al. 1993; O'Brien et al. 1997; Rao and Craig 1997). We therefore conducted experiments to compare the proportion of AMPARs and NMDARs present at somatic sites in DIV8 neurons.

We recorded from DIV8 neurons while puffing agonist selectively onto the cell body. The bath perfusion was set at a high rate and was run continuously to reduce drug dispersion. Puffs of solutions containing either kainate (500 µM) or NMDA (500 µM) were administered by a picospritzer apparatus through a microperfusion pipette aimed directly at the cell body. The photomicrograph presented in Fig. 10C was taken during the puffing of a trypan blue-containing solution and demonstrates that the effect of the puff was to bath principally the cell body and proximal processes. In each experiment, the size of the current evoked by the puff was compared with the size of the current evoked by agonists perfusion of the entire cell, including its processes.



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Fig. 10. NMDA receptors present at DIV8 are principally confined to the cell body. A: whole cell currents evoked in a DIV8 neuron by 500 µM NMDA (with added glycine, 1 µM) that was either applied with the microperfusion pipette (puff) or via the bath perfusion (perf.). B: the same experiment with kainate (500 µM) C: photomicrograph showing the microperfusion pipette (left-hand side) while a puff of a trypan blue (0.4%) was applied. D: the mean ratio of the current measured by microperfusion application (Ipuff) to the current measured by bath perfusion application (Iperf) of either kainate or NMDA with added glycine. Bars indicate standard deviations.

The results obtained with NMDA and kainate are shown in Fig. 10, A and B. As can be seen, the amplitudes of the currents evoked with NMDA by focal and bath application are similar, whereas kainate application at the soma produced much smaller currents than those evoked by kainate perfusion of the entire cell. Figure 10D shows the mean ratio of the current induced by the puff (Ipuff) to the current induced in the same neuron by bath application (Iperf) for kainate and NMDA. Whereas only about one quarter of the AMPARs expressed in DIV8 neurons are activated by agonist applied to the soma, the somatic application of NMDA is sufficient to activate nearly 80% of the NMDARs present. Thus at times when the majority of AMPARs are expressed at distal processes, most NMDARs are confined to the cell body and proximal processes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have suggested that the activation of NMDARs at glutamatergic synapses in developing spinal neurons differs in situ and in vitro. Whereas experiments on spinal cord slices have shown that NMDAR activity contributes to glutamatergic neurotransmission as early as P0 (Bardoni et al. 1998; Gao et al. 1998), studies on cultured spinal neurons failed to detect the presence of synaptic NMDARs (O'Brien et al. 1997, 1998). In the present paper we have characterized NMDAR activity in spinal neurons maintained in vitro for various times, principally by testing the sensitivity of NMDAR activity to TTX and by determining its relationship to AMPAR-mediated EPSCs. We conclude that NMDAR channel activity is detectable at each time studied and that it primarily results from synaptically activated receptors located on postsynaptic neurons. Our results highlight several aspects of the NMDAR activity in cultured spinal neurons that might explain the failure to detect NMDAR synaptic currents in previous work.

Ongoing NMDAR activity

Our experiments show that the NMDAR activity in cultured spinal neurons appears as ongoing activity rather than identifiable synaptic events. Ongoing NMDAR activity has also been described in slice preparations (Blanton et al. 1990; Lo Turco et al. 1991). The hallmark of the activity observed by others in slices is that it is insensitive to TTX, indicating that the receptors involved are unlikely to be activated by synaptically released glutamate, but rather by ambient glutamate and glycine present in the normal cellular milieu (Sah et al. 1989). In our experiments, however, the neurons were constantly exposed to fresh solution, and steady background accumulation of the transmitters is unlikely to occur. The stable NMDAR channel activity that we observe is therefore probably directly related to spontaneous synaptic activity, a conclusion consistent with our results demonstrating that, as early as DIV2, the activity was markedly reduced by TTX.

The ongoing NMDAR activity present in DIV8 spinal neurons (and DIV5 neurons in Mg2+-free solutions) results in what appears as a substantial standing inward current with no clear evidence of isolated NMDAR synaptic events. However, because the NMDAR shows relatively little desensitization, and because the unbinding of glutamate is slow (Johnson and Ascher 1992), synaptic NMDAR currents may persist for hundreds of milliseconds after the transmitter concentration has fallen to zero (Dzubay and Jahr 1996; Lester et al. 1990). Therefore if synaptic events are frequent, these properties of the NMDAR would be expected to give rise to what appears as a sustained inward current. The results we obtained in the presence of submaximal concentrations of TTX provide evidence that the sustained currents observed in cultured spinal neurons arise from the temporal overlap of NMDAR currents activated by synaptically released glutamate.

Recording spontaneous NMDARs-mediated EPSCs in cultured spinal neurons

At first glance, our results appear at odds with earlier studies on cultured spinal neurons, which failed to detect either NMDAR clustering at postsynaptic regions or electrophysiological evidence of spontaneous NMDAR-mediated activity, despite the presence of large exogenously activated NMDAR-mediated currents (O'Brien et al. 1997, 1998). Several factors might contribute to the differences between our present results and those of previous investigations, but we believe three differences are most important.

First, as we showed previously (Robert et al. 1998), blocking GABA-mediated inhibition with bicuculline results in a large increase in the frequency of excitatory synaptic events. We show here that intense synaptic drive results in the temporal overlap of NMDAR-mediated EPSCs, yielding a noisy sustained inward current that obscures detection of isolated synaptic events. Thus the use of bicuculline in previous studies may have masked the presence of small NMDAR-mediated EPSCs. Second, we provide evidence that, when studying spontaneous synaptic events, neither CNQX nor DNQX selectively blocks AMPARs. This result is somewhat surprising because many studies have shown that CNQX, at the concentrations used here, has little effect on evoked NMDAR-mediated EPSCs, despite the known action of quinoxaline derivatives as competitive antagonists at the glycine binding site of the NMDAR (Lester et al. 1989). It is possible that spinal neurons express NMDARs that are especially sensitive to quinoxaline blockade. Alternatively, spontaneous NMDAR activity may be more sensitive to quinoxaline inhibition than are stimulus-evoked EPSCs, or the addition of glycine to the external solution in other studies may have blunted quinoxaline blockade of the NMDAR. Whichever of these explanations obtains, our results show that the use of CNQX to isolate NMDAR EPSCs in previous studies of cultured spinal neurons (O'Brien et al. 1997; Robert et al. 1998) would in fact have hindered detection of NMDAR-mediated synaptic events.

Although the use of both bicuculline and CNQX in previous studies may have confounded the detection of NMDAR-mediated synaptic currents, it must also be emphasized that, at early times in vitro, the amplitude of the NMDAR-mediated component of EPSCs is very small. Although this may in part reflect the low open probability of synaptic NMDARs (Rosenmund et al. 1993, 1995), our results are consistent with a low density of NMDAR receptors in synaptic regions. Indeed, at early times in culture the size of the NMDAR synaptic events is small enough that they could easily be missed, and it is likely that clear NMDAR clustering would not be detected in immunocytochemical studies. In different systems, such as cultured hippocampal neurons, immunocytochemical data have suggested that pure AMPAR-containing synapses predominate at early times in vitro (Craig et al. 1993; Rao and Craig 1997), although subsequent electrophysiological studies have clearly demonstrated the presence of synaptic NMDARs (Tovar and Westbrook 1999).

Synaptic or extrasynaptic receptors?

Although our results provide evidence that NMDARs contribute to the postsynaptic currents at spinal glutamatergic synapses, our results do not allow a direct estimate of the synaptic density of these receptors, nor do they prove that AMPARs and NMDARs are co-localized. As suggested by O'Brien et al. (1997), it is possible that NMDARs are located on the shafts of dendritic spines and are activated in response to spillover of synaptically released transmitter (Kullmann et al. 1996). Transmitter spillover might be more prevalent at immature synapses where glial investment is incomplete. In addition, our experiments were conducted at room temperature, which slows glutamate uptake, thereby increasing the likelihood of transmitter spillover (Asztely et al. 1997). Given these caveats, it is unclear whether the NMDAR currents we have observed arise from a low density of synaptic receptors or from extrasynaptic receptors. Either possibility is also consistent with the diffuse staining pattern found in previous immunolabeling studies (O'Brien et al. 1997, 1998), especially given our results showing that at DIV8 a majority of the NMDARs are expressed at somatic sites.

In DIV5 and DIV8 neurons, the relative amplitude of the NMDAR-mediated EPSCs, compared with the corresponding AMPAR-mediated EPSCs, was modest. However, by DIV14 this ratio was increased severalfold. At this latter stage, spontaneous NMDAR-mediated EPSCs of 200-400 pA were common, and currents as large as 600 pA were observed (measured at -25 mV in normal external Mg2+). On average, the whole cell currents evoked in DIV14 neurons by saturating agonist concentrations were ~2,400 pA (measured at -80 mV in Mg2+-free solutions). When the difference in holding potential in the two sets of experiments is considered, as well as the residual Mg2+ block present in the synaptic studies, it seems likely that the majority of the NMDARs expressed by DIV14 neurons are targeted to synaptic regions.

The increased expression of synaptic NMDARs that occurs during the second week in culture does not appear to be associated with obvious alterations in subunit composition. The time constants of the two exponential components in the decays of NMDAR-mediated currents suggest that both NR2A and NR2B subunits are present in DIV5, DIV8, and DIV14 neurons (Kirson and Yaari 1996; and references therein), and the relative proportion of the two components does not appear to change substantially. In DIV14 neurons, analysis of the decay of both EPSCs and exogenously evoked currents gave similar results, suggesting that at this stage the subunit ratio at synapses reflects the ratio of the overall receptor population. While the amplitude of the EPSCs in DIV5 and DIV8 neurons was too small to fit their decay kinetics reliably, the brevity of the isolated NMDAR-mediated EPSCs strongly suggest the presence of NR2A subunits. In spinal cord slices from P1 or P10 rats, NMDAR-mediated EPSCs also contain primarily a fast decaying component (Bardoni et al. 1998).

Conclusions

Our results show that synaptic NMDAR events can be detected very early during the in vitro development of glutamatergic spinal synapses, a result consistent with previous in situ studies (Bardoni et al. 1998; Gao et al. 1998). In addition, our findings emphasize the importance of these NMDARs for controlling the spontaneous synaptic activity in spinal cultures. As is the case in many neuronal systems in vivo, NMDAR-mediated synaptic activity is thus likely to play an important part in the organization of the synaptic circuits in the developing spinal cord. Our results also support the conclusion that the targeting of NMDARs to glutamatergic synapses formed in culture lags behind the synaptic clustering of AMPARs. This conclusion agrees with a recent study showing that in DIV7 cultured hippocampal neurons, ~75% of the NMDARs are still expressed at extrasynaptic sites (Tovar and Westbrook 1999). It is presently unclear whether this developmental pattern occurs as a result of culture conditions, or reflects the normal developmental program of spinal neurons.


    ACKNOWLEDGMENTS

We thank Dr. S. Agulian and B. Toftness for technical support and Dr. T. Cummins for helpful discussions.

This work was supported in part by grants from the Medical Research and Rehabilitation Research Service, Department of Veterans Affairs and the Paralyzed Veterans of America/Eastern Paralyzed Veterans Association (PVA/EPVA). A. Robert was supported in part by the Swiss National Foundation (823A-053487) and by a Spinal Cord Research Fellowship from the EPVA.


    FOOTNOTES

Address for reprint requests: S. G. Waxman, Dept. of Neurology, Yale University School of Medicine, 333 Cedar St., LCI 707, New Haven, CT 06510.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 19 August 1999; accepted in final form 25 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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