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. 1998 Apr 15;508(Pt 2):495–502. doi: 10.1111/j.1469-7793.1998.00495.x

Regulation of kinetic and pharmacological properties of synaptic NMDA receptors depends on presynaptic exocytosis in rat hippocampal neurones

Raimund Lindlbauer 1, Ralf Mohrmann 1, Hanns Hatt 1, Kurt Gottmann 1
PMCID: PMC2230880  PMID: 9508812

Abstract

  1. Using whole-cell patch-clamp recordings of NMDA EPSCs from co-cultured rat hippocampal (CA region) neurones, developmental changes in the kinetic and pharmacological properties of synaptic NMDA receptors were investigated. During in vitro differentiation a fast decaying component increasingly contributed to NMDA EPSCs.

  2. Extracellular Mg2+ (1 mm) strongly blocked NMDA EPSCs at all stages in culture. Using the NR2B subunit-specific NMDA receptor antagonist ifenprodil (3 μm), we observed a developmental decrease in ifenprodil sensitivity of NMDA EPSCs. This suggests developmental changes in the expression of NMDA receptor subtypes.

  3. To transiently block presynaptic exocytosis, we incubated presynaptic explants with tetanus toxin (TeTx) prior to cultivation. In TeTx-pretreated cultures the occurrence of fast decaying components of NMDA EPSCs and the developmental decrease in ifenprodil sensitivity was inhibited. Our results indicate a regulatory role of presynaptic exocytosis in the expression of NMDA receptor subtypes.

N-Methyl-d-aspartate (NMDA) receptors play a crucial role in long-term synaptic plasticity (for review see Bliss & Collingridge, 1993). Native NMDA receptors form hetero-oligomeric complexes (Sheng, Cummings, Roldan, Jan & Jan, 1994) consisting of NR1 and NR2 subunits (for review see McBain & Mayer, 1994). In heterologous expression systems the type of NR2 subunit determines the kinetic and pharmacological properties of NMDA receptor subtypes (Williams, Russel, Shen & Molinoff, 1993; Monyer, Burnashev, Laurie, Sakmann & Seeburg, 1994). Developmentally altered expression of NR2 subunits (Watanabe, Inoue, Sakimura & Mishina, 1992; Monyer et al. 1994; Sheng et al. 1994) is thought to underlie developmental changes in the properties of NMDA receptors (Carmignoto & Vicini, 1992; Hestrin, 1992; Williams et al. 1993; Khazipov, Ragozzino & Bregestovski, 1995; Kirson & Yaari, 1996).

These developmental changes in the putative subunit composition of NMDA receptors have been suggested to depend on neuronal activity (Carmignoto & Vicini, 1992; Audinat, Lambolez, Rossier & Crepel, 1994), but the underlying molecular mechanisms are unknown. We show here that presynaptic exocytosis is involved in the regulation of NMDA receptor subtype expression in hippocampal neurones.

METHODS

Hippocampal co-cultures of presynaptic explants and postsynaptic target neurones were obtained and cultured as described previously (Gottmann, Mehrle, Gisselmann & Hatt, 1997). Pregnant Wistar rats were anaesthetized by diethyl ether, decapitated and embryos removed. Explants from the CA region of the immature hippocampus of the rat embryos (embryonic day 19-20) were used as sources of presynaptic fibres. These fibres innervated postsynaptic target neurones that had been obtained by dissociating the isolated CA region. NMDA EPSCs were evoked by electrical stimulation of presynaptic explant fibres and were recorded at room temperature (holding potential, -60 mV) from target neurones using the whole-cell patch-clamp technique, as described (Gottmann et al. 1997). NMDA EPSCs were filtered at 3 kHz and sampled at 4 kHz using pCLAMP 6.0 software (Axon Instruments). Series resistance was not compensated, since maximal currents were < 100 pA (voltage error < 2 mV assuming 20 MΩ access resistance). Potentials given were not corrected for junction potentials. With the stimulus intensities used, the failure rate was < 10%.

To record NMDA EPSCs, patch pipettes with resistances of 3-8 MΩ were filled with a solution containing (mm): 100 potassium gluconate, 10 KCl, 0.25 CaCl2, 10 EGTA and 20 Hepes (pH 7.3). Mg2+-free extracellular solution contained: 130 mm NaCl, 5 mm KCl, 5 mm CaCl2, 10 μm glycine, 10 μm 6,7-dinitro-quinoxaline-2,3-dione (DNQX), 20 μm bicuculline methochloride or 100 μm picrotoxin and 20 mm Hepes (pH 7.3). The decay kinetics of evoked NMDA EPSCs were fitted after averaging ten to twenty individual synaptic responses (failures were excluded) using pCLAMP 6.0 software. The decay kinetics of NMDA EPSCs were considered to be monoexponential if the average deviation between the data and the fit (σ) was decreased by less than 2% after biexponential fitting. Otherwise the decay kinetics of NMDA EPSCs were considered to be biexponential (in these cases σ decreased on average by more than 35% after biexponential fitting). To record AMPA EPSCs (sampled at 33 kHz) potassium gluconate was replaced by KCl and 5 mm N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314; RBI) was added to the intracellular solution. In the extracellular solution we removed DNQX and added 1 mm MgCl2.

To record miniature EPSCs (mEPSCs), patch pipettes were filled with a solution containing (mm): 100 CsCl, 0.25 CaCl2, 20 TEA-Cl, 10 EGTA and 20 Hepes (pH 7.3). Extracellular solution contained: 130 mm NaCl, 20 mm KCl, 3 mm CaCl2, 10 μm glycine, 100 μm picrotoxin, 1 μm TTX and 20 mm Hepes (pH 7.3). mEPSCs were aligned and averaged by means of AUTESP software (H. Zucker, Max-Planck-Institute for Psychiatry, Martinsried, Germany) using the AMPA/kainate receptor-mediated component as a detection signal. The decay kinetics of the NMDA receptor-mediated components were fitted using pCLAMP 6.0 software, after averaging between twenty and fifty individual mEPSCs.

Pharmacological characterization of EPSCs was performed by means of a pressure-driven two-barrel application system combined with a continuously operating suction barrel (Gottmann et al. 1997). Statistical analysis was done by Student's t test. Data represent means ±s.e.m.

RESULTS

To study developmental changes in the properties of synaptic NMDA receptors in hippocampal neurones in vitro, we analysed NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs). At different stages in culture, NMDA EPSCs were elicited by extracellular stimulation of explant fibres and were recorded at a holding potential of -60 mV in nominally Mg2+-free extracellular solution. AMPA/kainate receptors were blocked by DNQX (10 μm) and GABAA receptors were blocked by bicuculline methochloride (20 μm) or picrotoxin (100 μm). The peak amplitudes of NMDA EPSCs were reversibly blocked by 86 ± 2% (n = 3) after addition of 50 μm d-AP-5.

After 5 days in vitro (DIV), NMDA EPSCs were frequently observed in postsynaptic target neurones. No NMDA EPSCs could be elicited during the first 4 days in vitro. At 5-6 DIV the peak amplitudes of NMDA EPSCs were strongly and reversibly blocked by 88 ± 5% (n = 7) after addition of 1 mm Mg2+ at -60 mV holding potential. Similarly, at 8-10 DIV the peak amplitudes of NMDA EPSCs were reversibly blocked by 93 ± 2% (n = 6).

The decay kinetics of NMDA EPSCs showed a strong dependence on time in culture (Fig. 1). At 5 DIV, NMDA EPSCs showed monoexponential decay kinetics with a mean time constant of 288 ± 12 ms in the vast majority of cells (75%). In contrast, at 9-12 DIV, NMDA EPSCs decayed with biexponential kinetics (mean time constants of 63 ± 2 ms and 380 ± 18 ms) in the vast majority of cells (> 90%). The decrease in the percentage of cells showing monoexponentially decaying NMDA EPSCs was paralleled by an increase in the percentage of cells showing biexponentially decaying NMDA EPSCs (Fig. 1C). These results demonstrate developmental changes in the kinetic properties of synaptic NMDA receptors in vitro similar to those described in hippocampal pyramidal neurones in vivo (Khazipov et al. 1995; Kirson & Yaari, 1996).

Figure 1. Developmental changes in the decay kinetics of NMDA EPSCs.

Figure 1

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A, averaged evoked NMDA EPSCs recorded at different stages in culture. Fits of the current decay are shown below current traces. B, averaged mEPSCs recorded at different stages in culture (filtered at 100 Hz for illustration). AMPA receptor-mediated components are truncated. The NMDA receptor-mediated components were blocked by 50 μm d-AP-5 (right traces). Fits of current decay are superimposed on current traces. C, percentage of cells showing monoexponential NMDA EPSC decay kinetics (Inline graphic) and percentage of cells showing biexponential NMDA EPSC decay kinetics (□) at different stages in culture (n is indicated above the bars).

The mean decay time constant of NMDA EPSCs showing monoexponential decay kinetics did not change significantly during the culture period (Table 1A). The mean decay time constants of NMDA EPSCs showing biexponential decay kinetics significantly (P < 0.001) decreased during the culture period (Table 1A), additionally suggesting a developmental change in the kinetic properties of synaptic NMDA receptors.

Table 1.

Decay time constants of evoked NMDA EPSCs

Monoexponential decay kinetics Biexponential decay kinetics
n τ (ms) n τfast (ms) τslow (ms) Ampfast/Ampslow
A.
DIV 5 12 288 ± 12 4 — — —
DIV 6 14 272 ± 16 27 76 ± 3 488 ± 25 1.9 ± 0.2
DIV 7 12 282 ± 24 36 65 ± 3 400 ± 22 1.7 ± 0.2
DIV 8 2 — 32 60 ± 4 389 ± 12 1.7 ± 0.1
DIV 9-12 2 — 55 63 ± 2 380 ± 18 1.7 ± 0.1
B.
TeTx DIV 9-12 12 215 ± 15 14 78 ± 10 468 ± 53 1.0 ± 0.1 *
Control DIV 9-12 1 — 30 69 ± 3 391 ± 24 1.5 ± 0.1
TeTx DIV 14-15 2 — 10 77 ± 9 534 ± 98 1.8 ± 0.3
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A, developmental changes of decay time constants. The decay time constants of biexponentially decaying NMDA EPSCs between day in vitro (DIV) 6 and DIV 9-12 were significantly different (P < 0.001). B, effect of TeTx-preincubation on decay time constants. Values represent means ±s.e.m. No mean values are given for n < 5.

*

Significantly different (P < 0.05). Amp, amplitude.

The mean time to peak of NMDA EPSCs showing monoexponential decay kinetics (16.3 ± 1.1 ms) was slightly larger (P < 0.005) than that of NMDA EPSCs showing biexponential decay kinetics (13.3 ± 0.4 ms). The mean peak amplitude of monoexponentially decaying NMDA EPSCs (14 ± 1 pA) was significantly (P < 0.001) smaller than that of biexponentially decaying NMDA EPSCs (47 ± 3 pA).

NMDA receptor-mediated components of mEPSCs (Fig. 1B) showed a similar developmental change in decay kinetics as found for evoked NMDA EPSCs. At 5 DIV, 57% (n = 23 cells) of averaged mEPSCs lacked a fast decaying (τ < 100 ms) NMDA receptor-mediated component, whereas at 8-11 DIV 86% (n = 35 cells) of averaged mEPSCs showed a fast decaying NMDA receptor-mediated component (τ= 62 ± 4 ms).

The developmental appearance of rapidly decaying NMDA EPSC components might indicate a developmental change in the subunit composition of synaptic NMDA receptors. To further analyse this, we characterized NMDA EPSCs pharmacologically using the NR2B subunit-specific antagonist ifenprodil (Williams et al. 1993). At 5-6 DIV, ifenprodil (3 μm) reversibly blocked the mean peak amplitude of NMDA EPSCs by 77 ± 3% (n = 7; Fig. 2Aa and B). In contrast, at 9-10 DIV, the mean peak amplitude of NMDA EPSCs was reversibly reduced by only 50 ± 5% (n = 10; Fig. 2Ab and B). This age-dependent difference in sensitivity to ifenprodil was statistically significant (P < 0.001).

Figure 2. Effects of ifenprodil on evoked NMDA EPSCs at different stages in culture.

Figure 2

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A, effect of ifenprodil (3 μm) on averaged NMDA EPSCs in early (a) and late (b) stages of cultivation. c, effect of ifenprodil on the decay kinetics of averaged NMDA EPSCs. Amplitude in the presence of ifenprodil was normalized to control amplitude. B, plot of mean NMDA EPSC peak amplitudes in the presence of ifenprodil normalized to peak amplitudes under control conditions at different stages in culture. The ifenprodil block was calculated as the reduction of the mean of control and wash amplitudes, taking into consideration rundown of NMDA EPSCs (n is indicated above the bars). C, effect of ifenprodil on AMPA EPSC amplitudes. Insets show individual AMPA EPSCs.

In addition to blocking NMDA EPSC amplitudes, ifenprodil (3 μm) also affected NMDA EPSC decay kinetics. In eight out of ten cells tested the mean time constant of the fast decaying component of the NMDA EPSCs (9-10 DIV) was significantly (P < 0.05) decreased in the presence of ifenprodil (control: 69 ± 6 ms; ifenprodil: 53 ± 5 ms; wash: 62 ± 6 ms; Fig. 2Ac). This effect of ifenprodil was only partly reversible, presumably due to rundown of NMDA EPSCs. The mean time constant of the slowly decaying component was not significantly affected by ifenprodil (control: 430 ± 16 ms; ifenprodil: 412 ± 50 ms; wash: 385 ± 38 ms).

At 5-6 DIV in four out of five cells tested, ifenprodil (3 μm) did not significantly affect the AMPA receptor-mediated excitatory postsynaptic current (AMPA EPSC) amplitude. In one cell a slight reduction of the mean AMPA EPSC amplitude by 22% was observed. Similarly, at 9-10 DIV in four out of six cells tested ifenprodil did not significantly affect the AMPA EPSC amplitude (Fig. 2C). In two cells a slight reduction (15%, 16%) of the mean AMPA EPSC amplitude occurred. These results demonstrate that the differential effect of ifenprodil on NMDA EPSC amplitudes cannot be attributed to a differential block of presynaptic transmitter release.

To this end, our results demonstrate that during the culture period an additional NMDA receptor subtype with fast offset kinetics and weak sensitivity to ifenprodil (most likely NMDA receptors containing NR2A subunits) increasingly contributed to the NMDA EPSCs. We next addressed the mechanisms involved in the developmental regulation of the putative subunit composition of synaptic NMDA receptors.

To study whether developmental changes in the synaptic expression of NMDA receptor subtypes are dependent on synaptic activity, we transiently blocked presynaptic exocytosis using tetanus toxin. Presynaptic explants were incubated in 20 ng ml−1 tetanus toxin (TeTx) for 10-16 h prior to cultivation. After rinsing, the explants were added to untreated postsynaptic target neurones. TeTx-preincubation did not affect the survival of postsynaptic target cells (10 DIV: sibling control cultures: 43 ± 3 cells mm−2; TeTx-pretreated cultures: 45 ± 3 cells mm−2). To study the effects of TeTx-preincubation on the morphological differentiation of postsynaptic target cells, neurones were filled with Lucifer Yellow (1%) (Fig. 3A). At 9-12 DIV neither the mean length of dendrites (control cultures: 79 ± 12 μm, n = 5 cells; TeTx-pretreated cultures: 75 ± 6 μm, n = 5 cells) nor the mean cell capacitance (control cultures: 16 ± 1 pF, n = 15; TeTx-pretreated cultures: 16 ± 1 pF, n = 19) differed significantly between control cultures and TeTx-pretreated cultures.

Figure 3. Effect of TeTx-preincubation on the expression of NMDA receptor subtypes.

Figure 3

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A, morphology of a target neurone in control culture (a) and in TeTx-pretreated culture (b) after 11 DIV. Cells were filled with Lucifer Yellow. Scale bar represents 15 μm. B, averaged evoked NMDA EPSCs recorded in TeTx-pretreated cultures after 9 DIV (a) and after 14 DIV (b). Fits of the current decay are shown below current traces. C, percentage of cells showing monoexponentially (Inline graphic) and biexponentially (□) decaying NMDA EPSCs in control cultures after 9-12 DIV and in TeTx-pretreated cultures after 9-12 DIV and after 14-15 DIV (n is indicated above the bars). D, plot of mean NMDA EPSC peak amplitudes in the presence of ifenprodil (3 μm) normalized to peak amplitudes under control conditions in control cultures and in TeTx-pretreated cultures after 9-11 DIV.

To study the effect of TeTx-preincubation on presynaptic transmitter release, we evoked AMPA EPSCs at different stages in culture using maximal stimulation of presynaptic explants. At 5 DIV a strong reduction of the mean AMPA EPSC amplitude was observed in TeTx-pretreated cultures (control cultures: 198 ± 35 pA, n = 9; TeTx-pretreated cultures: 43 ± 14 pA, n = 13; P < 0.001). However, at 10 DIV the mean AMPA EPSC amplitude of control cultures (229 ± 52 pA, n = 7) and TeTx-pretreated cultures (129 ± 27 pA, n = 10) was not significantly different. These results indicate that TeTx-preincubation reversibly blocked presynaptic exocytosis without affecting synapse formation.

At 9-12 DIV a second set of NMDA EPSCs was recorded as described and their kinetic properties in TeTx-pretreated cultures were compared with those in untreated sibling cultures. Preincubation of explants in TeTx strongly inhibited the developmental appearance of fast decaying components of the NMDA EPSCs (Fig. 3Ba and C). In TeTx-pretreated cultures 46% (n = 26) of the cells showed monoexponentially decaying NMDA EPSCs with a mean decay time constant of 215 ± 15 ms, whereas only 3% (n = 31) of the cells showed monoexponentially decaying NMDA EPSCs in control cultures. The incidence of cells showing biexponentially decaying NMDA EPSCs in TeTx-pretreated cultures was strongly decreased to 54% (97% in control cultures). Additionally, the amplitude contribution of the fast decaying component of NMDA EPSCs showing biexponential decay kinetics significantly (P < 0.05) decreased in TeTx-pretreated cultures (Table 1B). Chronic action potential blockade by addition of TTX (1 μm) to the culture medium had a similar, slightly weaker effect on the development of NMDA EPSC decay kinetics. In TTX-treated cultures 27% (n = 41) of the cells showed monoexponentially decaying NMDA EPSCs at 9-12 DIV.

To demonstrate differences in the expression of NMDA receptor subtypes in control cultures and TeTx-pretreated cultures, we characterized NMDA EPSCs pharmacologically using the NR2B subunit-specific antagonist ifenprodil. At 9-11 DIV ifenprodil reversibly blocked the mean peak amplitude of NMDA EPSCs in control cultures by 56 ± 3% (n = 12; Fig. 3D). In contrast, at 9-11 DIV the ifenprodil-block of NMDA EPSCs in TeTx-pretreated cultures significantly (P < 0.001) increased to 77 ± 2% (n = 13; Fig. 3D).

Upon prolonged cultivation (14-15 DIV) of TeTx-pretreated cultures the incidence of cells showing biexponentially decaying NMDA EPSCs strongly increased (83%, n = 12; Fig. 3Bb and C), reaching a value similar to control cultures at 9-12 DIV. This result suggests that a transient blockade of presynaptic exocytosis by TeTx led to a strongly delayed appearance of an NMDA receptor subtype showing fast offset kinetics.

In summary, our results demonstrate that the developmental appearance of fast decaying components of NMDA EPSCs and of an NMDA receptor subtype showing weak sensitivity to ifenprodil is dependent on presynaptic exocytosis and suggest that the developmental upregulation of an NMDA receptor subtype with the putative subunit composition NR1/NR2A or NR1/NR2A/NR2B is regulated by synaptic activity.

DISCUSSION

In this study we characterized developmental changes in the properties of synaptic NMDA receptors in co-cultured hippocampal neurones and addressed the underlying molecular mechanisms. The observed effects of blocking presynaptic exocytosis using tetanus toxin indicate an important role of presynaptic exocytosis in the developmental regulation of the putative NMDA receptor subunit composition.

Mg2+ sensitivity and decay kinetics of NMDA EPSCs

The sensitivity of recombinant NMDA receptors to Mg2+ blockade has been shown to depend strongly on their subunit composition (Monyer et al. 1994). In line with previous reports (Khazipov et al. 1995; Kirson & Yaari, 1996) we found a high sensitivity of NMDA EPSCs to Mg2+ blockade at all stages of in vitro development, suggesting that NMDA receptor subtypes containing NR2A or NR2B subunits or both contribute to NMDA EPSCs in our preparation.

We observed a strong developmental change in NMDA EPSC decay kinetics, comparable to developmental changes occurring during hippocampal development in vivo (Khazipov et al. 1995; Kirson & Yaari, 1996). In our co-cultures these developmental changes occurred faster compared to in vivo development, presumably due to the strong spontaneous synaptic activity present in our co-cultures (Gottmann et al. 1997). The decay kinetics of NMDA EPSCs are determined by the offset kinetics of NMDA receptor channels (Hestrin, Sah & Nicoll, 1990; Lester, Clements, Westbrook & Jahr, 1990). Analysis of recombinant NMDA receptor subtypes has revealed that the offset kinetics of NMDA receptor-mediated currents are characteristic of NMDA receptor subunit composition (Monyer et al. 1994; Köhr & Seeburg, 1996). Binary NR1/NR2A receptors show fast offset kinetics (τ≈ 100 ms), while binary NR1/NR2B receptors show much slower offset kinetics (τ≈ 400 ms; Monyer et al. 1994; Köhr & Seeburg, 1996). Ternary NR1/NR2A/NR2B receptors show intermediate offset kinetics (τ≈ 200 ms) (Köhr & Seeburg, 1996). Thus, changes in the decay kinetics of NMDA EPSCs might indicate changes in the NMDA receptor subtypes that contribute to the NMDA EPSCs. Specifically, fast decaying components (τ < 100 ms) of NMDA EPSCs might indicate subsynaptic expression of NR1/NR2A or NR1/NR2A/-NR2B receptors. This interpretation is supported by the close correlation of developmental changes in NMDA EPSC decay kinetics (Carmignoto & Vicini, 1992; Khazipov et al. 1995) and the expression of NR2A subunits at the protein (Sheng et al. 1994) and mRNA levels (Monyer et al. 1994; Flint, Maisch, Weishaupt, Kriegstein & Monyer, 1997) in neocortical and hippocampal neurones. Although subunit composition of NMDA receptors appears to be the major determinant, other factors may also influence the decay kinetics of NMDA EPSCs (Lester & Jahr, 1992).

Ifenprodil sensitivity of NMDA EPSCs

To demonstrate developmental changes in the putative subunit composition of synaptic NMDA receptors, we characterized NMDA EPSCs pharmacologically using the NR2B subunit-specific antagonist ifenprodil (Williams et al. 1993). We found a significant decrease in sensitivity to ifenprodil (3 μm) during the culture period. In line with our results, a decrease in high-affinity ifenprodil binding sites occurred during postnatal development of hippocampal pyramidal neurones in vivo (Kirson & Yaari, 1996). Similar to the results of Kirson & Yaari (1996), 3 μm ifenprodil did not affect AMPA EPSC amplitudes in most cells, thus excluding the possibility that presynaptic effects on high voltage-activated Ca2+ channels (Church, Fletcher, Baxter & MacDonald, 1994) contributed significantly to the observed block of NMDA EPSCs.

At 9-10 DIV, ifenprodil affected the decay kinetics of NMDA EPSCs. The time constant of the fast decaying component was significantly decreased in the majority of cells. This is consistent with the assumption that very fast decaying components of NMDA EPSCs (τ < 100 ms) are mediated by NR1/NR2A receptors and therefore should be more pronounced in the presence of the NR2B subunit-specific antagonist ifenprodil. In an earlier study in hippocampal slices (Kirson & Yaari, 1996) a tenfold lower concentration of ifenprodil did not affect NMDA EPSC decay kinetics. However, it should be taken into consideration that ternary NR1/NR2A/NR2B receptors might significantly contribute to the fast decaying component of biexponentially fitted NMDA EPSCs. Although the sensitivity of these receptors to ifenprodil has not been determined, they might well be blocked by low concentrations of ifenprodil. This could mask effects of ifenprodil on NMDA EPSC decay kinetics.

In conclusion, kinetic and pharmacological analysis of NMDA EPSCs suggests that a developmental increase in the subsynaptic expression of an NMDA receptor subtype with the putative subunit composition NR1/NR2A or NR1/-NR2A/NR2B underlies the observed developmental changes in NMDA EPSC properties.

Effects of tetanus toxin on developmental changes in NMDA EPSC properties

In the developing neocortex the developmental appearance of fast decaying components of NMDA EPSCs depends on neuronal activity (Carmignoto & Vicini, 1992). Furthermore, in cultured cerebellar neurones the expression of NR2A mRNA is regulated by neuronal activity (Audinat et al. 1994). To address the mechanisms involved in activity-dependent regulation of NMDA receptor properties, we studied the effects of blocking presynaptic exocytosis on developmental changes of NMDA EPSC properties.

Tetanus toxin is well known to specifically inhibit presynaptic exocytosis by proteolytically cleaving the synaptic vesicle protein synaptobrevin/VAMP (Niemann, Blasi & Jahn, 1994; Ahnert-Hilger & Bigalke, 1995). In cultured central neurones tetanus toxin does not affect neuronal differentiation with respect to cell survival, axon growth and synapse formation (Osen-Sand et al. 1996). Additionally, we did not observe effects of TeTx-preincubation on the morphological differentiation of target neurones. Here we have shown that preincubating explants with tetanus toxin inhibits the developmental appearance of fast decaying components of NMDA EPSCs and the developmental change in ifenprodil sensitivity of NMDA EPSCs. Upon prolonged cultivation the kinetic properties of NMDA EPSCs in TeTx-pretreated cultures became similar to control cultures, demonstrating that a delay in the developmental maturation of NMDA receptors was induced by the transient blockade of presynaptic exocytosis. Our results therefore suggest that presynaptic exocytosis regulates developmental changes in the properties of synaptic NMDA receptors.

It may be argued that the selective death of a specific type of neurone might underlie the observed developmental changes in the properties of synaptic NMDA receptors. However, similar developmental changes in the properties of NMDA EPSCs have been demonstrated in identified hippocampal pyramidal neurones (Khazipov et al. 1995; Kirson & Yaari, 1996). Furthermore, we did not observe an increased survival of neurones after TeTx-preincubation, although the developmental changes in the properties of NMDA EPSCs were strongly delayed. Thus, selective cell death appears an unlikely explanation for developmental changes in NMDA EPSC properties.

In conclusion, developmental changes in the putative subunit composition of synaptic NMDA receptors, most likely the developmental appearance of NR1/NR2A or NR1/NR2A/NR2B receptors, underlie developmental changes in NMDA EPSC properties. Presynaptic exocytosis seems to play an important regulatory role in determining the putative subunit composition of synaptic NMDA receptors and, as a consequence, might be important in regulating NMDA receptor-dependent synaptic plasticity during development.

Acknowledgments

We would like to thank Dr G. Ahnert-Hilger for the kind gift of tetanus toxin. We further thank H. Jung and H. Bartel for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft.

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