NR2B-Containing NMDA Autoreceptors at Synapses on Entorhinal Cortical Neurons

Gavin Woodhall, D. Ieuan Evans, Mark O. Cunningham, and Roland S. G. Jones

Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Woodhall, Gavin, D. Ieuan Evans, Mark O. Cunningham, and Roland S. G. Jones. NR2B-Containing NMDA Autoreceptors at Synapses on Entorhinal Cortical Neurons. J. Neurophysiol. 86: 1644-1651, 2001. We have previously shown that presynaptic N-methyl-D-aspartate receptors (NMDARs) can facilitate glutamate release onto principal neurons in the entorhinal cortex (EC). In the present study, we have investigated the subunit composition of these presynaptic NMDARs. We recorded miniature alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (mEPSCs), from visually identified neurons in layers II and V of the EC in vitro. In both layers, bath application of the NR2A/B subunit-selective agonist, homoquinolinic acid (HQA), resulted in a marked facilitation of mEPSC frequency. Blockade of presynaptic Ca2+ entry through either NMDARs or voltage-gated Ca2+ channels with Co2+ prevented the effects of HQA, confirming that Ca2+ entry to the terminal was required for facilitation. When the NR2B-selective antagonist, ifenprodil, was applied prior to HQA, the increase in mEPSC frequency was greatly reduced. In addition, we found that an NMDAR antagonist blocked frequency-dependent facilitation of evoked release and reduced mEPSC frequency in layer V. Thus we have demonstrated that NMDA autoreceptors in layer V of the EC bear the NR2B subunit, and that NMDARs are also present at terminals onto superficial neurons.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

N-methyl-D-aspartate (NMDA) receptors have been implicated in synaptic plasticity (Bliss and Collingridge 1993) and in neuropathologies such as epilepsy (Dingledine et al. 1990). The subunits that comprise rat NMDA receptors have been cloned and classified and termed NR1 and NR2 (Ishii et al. 1993; Monyer et al. 1992; Moriyoshi et al. 1991). In addition to these, a third subunit NMDAR-L (Ciabarra et al. 1995; Sucher et al. 1995) has been cloned and shown to function during early development (now termed NR3A) (Das et al. 1998). NMDA receptors (NMDARs) may be either tetra- or pentameric, with the complete NMDAR consisting of two or three different subunit types (hetero- or heterotrimeric complexes) (Dunah et al. 1998; Laube et al. 1998; Luo et al. 1997). Within these complexes, the precise subunit composition of native and recombinant NMDARs is critical in determining receptor pharmacology and function (Köhr and Seeburg 1996; Krupp et al. 1996; Monaghan and Larsen 1997; Pizzi et al. 1999; Vicini et al. 1998). Subunit composition is dynamic, changing during synaptic development (Kew et al. 1998; Tovar and Westbrook 1999) and during other processes of synaptic plasticity (Kiyama et al. 1998; Manabe et al. 2000). Changes in subunit composition may also play a role in maintaining seizures induced by kindling (Al-Ghoul et al. 1997), or in raising seizure threshold (Bengzon et al. 1999).

We have previously demonstrated that NMDA autoreceptors are present on excitatory presynaptic terminals onto principal neurons in layer II of the rat entorhinal cortex (EC), and these act to facilitate glutamate release (Berretta and Jones 1996a). This has been followed by reports of presynaptic NMDA autoreceptors in other in vitro preparations such as cerebellum (Casado et al. 2000; Glitsch and Marty 1999), lamprey spinal cord (Cochilla and Alford 1999), rat spinal cord (Robert et al. 1998), hippocampus (Breukel et al. 1998), suprachiasmatic nucleus (Hamada et al. 1998), and developing Xenopus neuromuscular synapses (Chen et al. 1998; Fu et al. 1995).

Recently, immunocytochemical studies of NMDAR subunit distribution in rat visual cortex have indicated a presynaptic locus for NR1 subunits (Aoki et al. 1994), and in the dorsal horn of rat lumbar spinal cord Boyce et al. (1999) have suggested a presynaptic locus for NR2B containing NMDAR. In addition, a more detailed immunocytochemical study at the electron microscopic level has provided unequivocal evidence that NR1, NR2A, and NR2B subunits of the NMDAR are present in excitatory and inhibitory axon terminals in rat neocortex (DeBiasi et al. 1996). A recent report by Paquet and Smith (2000) has confirmed that NR1 subunit immunoreactivity can be found at GABAergic terminals in many regions of rat brain. NMDAR have also been localized on astrocytic membrane, and their activation has been shown to modulate glutamate/GABA release in hippocampal cultures (Araque et al. 1998).

However, nothing is known yet about the subunit composition of presynaptic NMDARs in mammalian cortex. In the present study, we have utilized subunit-specific NMDAR ligands to probe for the presence of NR2B-containing receptors at excitatory presynaptic terminals on layer V neurons in the EC. In addition, we have examined the possibility that inhibitory synaptic terminals may bear NMDA heteroreceptors. Our data indicate that excitatory synapses on layer V neurons bear NR2B-containing NMDARs, while similar receptors are present at inhibitory synapses on layer II neurons only. The presence of these presynaptic receptors may have implications for the development and maintenance of seizure activity. Some of these studies have been presented in abstract form (Woodhall and Jones 1999).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hippocampal-EC slices were prepared from male Wistar rats (50-110 g) as previously described (Jones and Heinemann 1988). In brief, rats were anesthetized with an intramuscular injection of ketamine (120 mg/kg) plus xylazine (8 mg/kg) and decapitated. The brain was rapidly removed and immersed in oxygenated artificial cerebrospinal fluid (ACSF) chilled to 4°C. Slices (450 µm) were cut using a vibroslice (Campden Instruments) and stored in ACSF continuously bubbled with 95% O2-5% CO2, maintained at 30°C. Following a recovery period of at least 1 h, individual slices were transferred to a recording chamber mounted on the stage of an Olympus upright microscope (BX50WI). The chamber was continuously perfused with oxygenated ACSF at 30-32°C at a flow rate of approximately 2 ml/min. The ACSF contained the following (in mM): 126 NaCl, 4 KCl, 1.25 NaH2PO4, 24 NaHCO3, 2 MgSO4, 2.5 CaCl2, and 25 D-glucose. The solution was continuously bubbled with 95% O2-5% CO2 to maintain a pH of 7.4. Neurons were visualized using differential interference contrast optics and an infrared video camera.

Patch-clamp pipettes were pulled from borosilicate glass (1.2 mm OD, 0.69 ID; Clark Electromedical) and had open tip resistances of 4-5 MOmega . These were used to record either excitatory (EPSC) or inhibitory (IPSC) postsynaptic currents arising spontaneously in neurons of layer V or layer II. When EPSCs were recorded, pipettes were filled with a solution containing the following (in mM): 130 Cs-methanesulphonate, 10 HEPES, 5 QX-314, 0.5 EGTA, 1 NaCl, 0.34 CaCl2, 2-5 MK801, 4 ATP, and 0.4 GTP. The solution was adjusted to 290 mOsmol with sucrose and to pH 7.25-7.3 with CsOH. Whole cell voltage-clamp recordings were made using an Axopatch 200B amplifier (Axon Instruments), and neurons were clamped at -60 mV. MK-801 was included in the patch pipette to block postsynaptic NMDAR in the recorded neuron. To facilitate this blockade, neurons were depolarized to -10 mV for 10 s several times during a 10-min period following breakthrough to whole cell access. Under these experimental conditions, EC neurons in layers II and V exhibited EPSCs mediated by spontaneous release of glutamate acting solely at alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (see Berretta and Jones 1996a).

In some studies we examined evoked EPSCs. A bipolar platinum wire electrode was placed on the surface of the slice and used to deliver electrical stimulation (square-wave pulses, 20 µS duration) to the subiculum at 0.5 or 3 Hz. At each frequency, the mean amplitude of 10 evoked EPSCs was measured, and the number of spontaneous EPSCs occurring in a 350-ms epoch following the stimulus artifact was counted.

Signals were filtered at 2 kHz and digitized at 20 kHz. Access resistance was monitored at regular intervals, and cells were rejected if this parameter changed by more than 15%. Data were recorded directly to computer hard disk using Axoscope software (Axon Instruments). Analysis of spontaneous events was carried out off-line using Minianalysis software (Synaptosoft). Spontaneous events were detected automatically using a threshold-crossing algorithm, and their frequency and amplitude measured. At least 200 events were included in the analysis for each cell under each condition. The nonparametric Kolmogorov-Smirnoff (KS) test was used to assess the significance of shifts in cumulative probability distributions of inter-event interval (IEI) (Van der Kloot 1991). All error values stated in the text refer to standard error of the mean (SE).

Salts used in preparation of ACSF were Analar grade and purchased from Merck/BDH (Lutterworth, UK). Drugs used were NMDA (Tocris, Bristol, UK), ifenprodil tartrate (Sigma-Aldrich, Poole, UK), homoquinolinic acid (gift from Dr. David Jane), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX, Tocris), 2-amino-5-phosphonovalerate (2-AP5, Tocris), MK-801 (Research Biochemicals), and tetrodotoxin (TTX, Alamone Laboratories, Jerusalem, Israel). Unless otherwise stated, drugs were applied by inclusion in the bath perfusion medium.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Presynaptic NMDA receptors mediate frequency-dependent facilitation of glutamatergic transmission in layer V

To determine the effect of presynaptic NMDARs on evoked glutamate release at synapses on EC neurons, we recorded from neurons in layer V and stimulated the subicular input at 0.5 and 3 Hz. All cells were filled with MK-801 (2-5 mM) and repetitively depolarized to prevent postsynaptic NMDAR activation. As shown in Fig. 1, A and D, an increase in stimulus frequency from 0.5 to 3 Hz increased the peak amplitude of the evoked EPSC from 49.24 ± 2.97 to 64.14 ± 3.35 pA (mean ± SE, P < 0.02, Student's t-test; average of 10 currents per cell, 5 cells). In the same cells, we also counted the number of spontaneous events recorded in a 350-ms period following the evoked EPSC. As shown in Fig. 1, B and C, there was an increase in the number of these asynchronous quantal events (AQEs) with increasing frequency, from 8.2 ± 2.59 to 28.20 ± 3.39 events per 10 sweeps (P < 0.02, t-test, n = 5). Both of these effects were reliably attenuated to control levels after bath application of the NMDAR antagonist, 2-AP5 (50 µM). These results demonstrate that even at modest stimulus frequencies, presynaptic NMDARs can be activated and can simultaneously increase both evoked EPSC amplitude and spontaneous EPSC frequency. In subsequent experiments, we used increased activity-independent "miniature" activity as a reporter of presynaptic NMDAR activation or blockade.



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Fig. 1. Frequency-dependent facilitation at synapses on layer V neurons is mediated by presynaptic N-methyl-D-aspartate receptors (NMDARs). A: the traces show groups of 10 evoked excitatory postsynaptic currents (EPSCs) from a layer V neuron. Increasing the frequency from 0.5 to 3 Hz resulted in an increase in the amplitude of the evoked EPSCs, as well as an increase in the number of asynchronous quantal events (AQEs) that follow the evoked event. Both these effects were blocked by bath application of 2-amino-5-phosphonovalerate (2-AP5; 50 µM). B: responses evoked at 0.5 and 3 Hz are shown superimposed on a faster time base. The evoked EPSC at 3 Hz has a larger peak amplitude than that evoked at 0.5 Hz, and is followed by 2 AQEs. C and D: pooled data from 5 neurons illustrating a significant increase in both the evoked EPSC amplitude and the number of AQEs. Both effects are significantly attenuated in the presence of 2-AP5.

NMDAR activation increases mEPSC frequency in deep and superficial layers of EC

To isolate presynaptic effects, the experiments described below were performed under conditions in which action potential-dependent release was suppressed with TTX (1 µM). As Fig. 2 (A and B) shows, under these conditions, bath application of the NMDAR agonist homoquinolinic acid (HQA, 20 µM) resulted in a robust increase in frequency of events recorded in layer V (from mean 1.25 ± 0.08 to 16.00 ± 0.72 Hz, P < 0.0001, KS, n = 5). Figure 2C shows the cumulative probability curve for IEI for pooled data, showing a strong leftward shift indicative of greatly increased frequency. In contrast, there was a much smaller shift in mEPSC amplitude distribution (Fig. 2D), with mean amplitude increased from 8.67 ± 0.23 to 9.75 ± 0.3 pA (P < 0.01, t-test, n = 5). It was possible to block the effects of HQA by prior application of the NMDAR antagonist 2-AP5 (data not shown). Overall, these data suggest that HQA acts at the presynaptic terminal to enhance glutamate release.



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Fig. 2. In layer V, homoquinolinic acid (HQA) increases mEPSC frequency, with little associated change in amplitude. A: mEPSCs in a layer V neuron in the presence of 1 µM TTX. B: in the same cell, HQA application (20 µM) induces a marked increase in mEPSC frequency. C: cumulative probability curves for inter-event interval (IEI) from pooled data (5 cells). D: amplitude plot (2-pA bins, at least 1,000 sEPSCs). There is close overlap between the 2 plots, indicating that mEPSC amplitude changes induced by HQA are minimal in the presence of TTX.

Similar results were obtained in layer II. Thus in the presence of TTX, HQA increased the mean frequency of mEPSCs from 3.35 ± 0.13 to 17.7 ± 0.59 Hz (P < 0.0001, KS, n = 5), as shown in Fig. 3, A and B. Figure 3C shows a pronounced leftward shift in the probability curve for IEI in the presence of HQA. However, Fig. 3D shows that the amplitude plot was skewed toward larger events when HQA was applied. Although an increase in mean amplitude was noted (mean from 10.24 ± 0.28 to 12.11 ± 0.23 pA, P < 0.05, t-test, n = 5), this was not large compared with experiments in the absence of TTX (mean amplitude increased from 8.64 ± 0.21 to 15.71 ± 0.45 pA, P < 0.001, t-test, n = 6, data not shown). It is thought that a change in PSC frequency with no accompanying change in amplitude is an indicator of a presynaptic site of drug action. In the present case, the relative predominance of "bursts" of release onto layer II neurons under these conditions and the slow decay time constant for events in these neurons when compared with layer V cells (Berretta and Jones 1996b) could combine to enhance postsynaptic temporal summation. These factors may be sufficient to enhance mean event amplitude under conditions that cause a marked increase in event frequency. In addition, several other factors point to a predominantly presynaptic locus of action for HQA. 1) The NMDAR antagonist 2-AP5 reduces EPSC frequency in layers II and V, even in the presence of TTX (Berretta and Jones 1996a, and see below). 2) In the presence of TTX, NMDAR-mediated depolarization of the somata of presynaptic neurons during HQA application is unlikely to spread passively throughout the considerable length of the axons in layer II. 3) Depolarization of presynaptic neurons would be similar under conditions when IPSCs are recorded; however, IPSC frequency is unaltered or only slightly enhanced by HQA under these conditions (Woodhall, unpublished observations).



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Fig. 3. mEPSC frequency in layer II is greatly increased by HQA, confirming a presynaptic locus of action. A: a recording from a layer II neuron in the presence of 1 µM TTX, showing baseline alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated mEPSCs. B: in the same cell, HQA application (20 µM) induces a marked increase in frequency. C: again, the cumulative probability plots for IEI from 5 cells show the shift to the left in the presence of HQA, indicative of an increased event frequency. D: amplitude plots (2-pA bins, at least 1,000 mEPSCs). In the presence of HQA (bold line), while the main peak is decreased, the distribution is only slightly skewed to the right. Hence mEPSC amplitude changes induced by HQA are reduced compared with those seen in the absence of TTX.

While the effects of presynaptic NMDAR activation were seen in both deep and superficial layers, the data that follow concern the action of NMDAR in layer V of the EC.

Presynaptic effect of HQA in layer V depends on Ca2+ entry into terminals

Berreta and Jones (1996a) previously suggested that facilitation of glutamate release depended on Ca2+ influx into the terminal via the NMDAR. Recently, Cochilla and Alford (1999) reported that activation of NMDARs on lamprey reticulospinal axons caused an increase in presynaptic Ca2+, thereby increasing transmitter release. If HQA acted at presynaptic NMDA receptors and direct Ca2+ entry (or that subsequent to depolarization) was responsible for the effects described above, we would expect that blockade of the passage of Ca2+ through NMDA receptors or voltage-gated calcium channels would prevent any increase in EPSC frequency. Co2+ has been shown to prevent the passage of Ca2+ through the channel pore of NMDARs (Mercuri et al. 1992; Nagy et al. 1994), although this effect is not specific to NMDARs alone. Figure 4A shows that application of Co2+ (1 mM) markedly reduced the mean sEPSC frequency in layer V neurons (from 10.12 ± 0.33 to 3.27 ± 0.15 Hz, P < 0.001, KS, n = 5), and completely blocked the effect of HQA (3.27 ± 0.15 prior to, and 2.87 ± 0.11 Hz during HQA application, P > 0.05, KS, n = 5). Prolonged washing of the preparation with normal ACSF partially restored both the baseline activity and the response to HQA. Figure 4B shows the cumulative probability curve for IEI before and after Co2+ was applied. A large reduction in event frequency in Co2+ was evident as a rightward shift relative to control. The amplitude histogram in Fig. 4C shows that Co2+ reduced the mean peak amplitude of events. When HQA was applied in the presence of Co2+, there was no change in event frequency (Fig. 4D) or amplitude (Fig. 4E). These data indicate that Ca2+ entry into the terminal is responsible for the enhancement of spontaneous release by HQA.



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Fig. 4. Co2+ blocks the effects of HQA In layer V. A: recordings from a single layer V neuron in the presence of 1 µM TTX. Co2+ (1 mM) greatly reduced the frequency of AMPAR-mediated mEPSCs, and subsequent addition of HQA (20 µM) has very little effect. After prolonged washing of the preparation in normal medium, an increase in mEPSC frequency was elicited on addition of HQA. B: cumulative probability curves for IEI from pooled data (6 cells). In the presence of Co2+, the curve (Co) is shifted to the right compared with control (C), confirming a decrease in mEPSC frequency. C: the amplitude plots show a decrease in event amplitude when Co2+ was applied (thin line). D: cumulative probability curves for IEI (pooled data from 6 cells) showing the effect of Co2+ (Co) on the response to HQA (H). The 2 curves overlap, showing that in the presence of the divalent cation, perfusion with HQA elicited no change in IEI. E: there was also no change in amplitude when HQA (bold line) was applied in the presence of Co2+.

Basal mEPSC frequency in layer V is reduced by 2-AP5 and by the NR2B-specific antagonist, ifenprodil

Since HQA discriminates poorly between NR2B- and NR2A-containing receptors, we utilized the antagonist ifenprodil, which has been reported to have approximately 400-fold selectivity for NR2B over NR2A (Williams 1993). In layer V, ifenprodil at 10 µM induced a small (<25%) decrease in basal mEPSC frequency. However, this effect was variable, being most pronounced in neurons with a high resting mEPSC frequency. Figure 5A shows recordings from a layer V neuron prior to and during ifenprodil application. Although the reduction in basal activity is difficult to see in the raw data, the cumulative probability plot of pooled data (Fig. 5C) shows a small rightward shift, indicating a decrease in event frequency (from a mean of 2.73 ± 0.15 to 2.15 ± 0.09 Hz, P < 0.001, KS, n = 14). There was little discernible change in event amplitude when ifenprodil was applied (control mean amplitude 6.47 ± 0.11 pA in control, vs. 6.13 ± 0.11 pA in ifenprodil, P < 0.05, t-test, n = 14). In experiments using the NMDAR antagonist 2-AP5 (Fig. 6), basal mEPSC frequency in layer V was reduced by approximately 40% (from mean 2.59 ± 0.19 to 1.58 ± 0.09 Hz, P < 0.001, KS, n = 5). Figure 6A shows raw data traces taken before and during perfusion with 50 µM DL-2-AP5. It can be seen that 2-AP5 reduces the frequency of mEPSCs. This is confirmed in the cumulative probability plot (Fig. 6B), in which the curve in the presence of 2-AP5 is shifted to the right, indicating a longer interval between mEPSCs. There was no significant change in mEPSC amplitude (mean 6.93 ± 0.10 to 7.01 ± 0.10 pA; P > 0.05, KS, n = 5, Fig. 6C). These data confirm that tonic presynaptic NMDAR activity in layer V can be reduced by bath application of a competitive receptor antagonist. Similar to the case with ifenprodil, the reduction in mEPSC frequency with 2-AP5 was variable between experiments, being larger in those recordings characterized by a higher initial mEPSC frequency.



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Fig. 5. Ifenprodil (IFN) decreases mEPSC frequency, with no associated change in amplitude in layer V. A: baseline AMPAR-mediated mEPSCs recorded from a layer V neuron in the presence of 1 µM TTX. B: in the same cell, IFN application (10 µM) elicits a small decrease in mEPSC frequency, although this is not readily apparent in these raw traces. However, the cumulative probability curves of pooled data for IEIs in 14 cells shown in C illustrates that in the presence of IFN, the curve (I) is shifted to the right compared with control (C), indicating that there is an increase in mEPSC frequency. D: the overlapping amplitude plots (2-pA bins; IFN, bold line) showing data pooled from not less than 2,800 mEPSCs, indicate that there was no concurrent change in mEPSC amplitude.



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Fig. 6. 2-AP5 decreases mEPSC frequency but not amplitude in layer V. A: mEPSCs recorded in the presence of 1 µM TTX. In the presence of 2-AP5, there is a reduction in the frequency of mEPSCs. B: cumulative probability curve for IEI showing that 2-AP5 increases the interval between events, manifest as a shift to the right in the probability curve. C: plot showing that the amplitude distribution under control conditions (2-pA bins) and during perfusion with 2-AP5 (bold line) are similar. Data pooled from not less than 1,000 mEPSCs.

Ifenprodil blocks the increase in glutamate release induced by HQA

When HQA (20 µM) was applied after 10 min preincubation of slices with ifenprodil (10 µM), the enhancement of glutamate release was greatly reduced (Fig. 7, A and B). In six layer V neurons, the mean frequency of EPSCs increased from 1.98 ± 0.08 to 2.27 ± 0.19 Hz (P < 0.001, KS). These mean data conceal variation in the magnitude of ifenprodil block of HQA, such that the ratio between pre- and post-HQA frequencies ranged from 1 to 1.8 in the presence of ifenprodil. The relatively small increase in mean frequency (<15%) elicited by HQA in the presence of ifenprodil can be seen as shift to the left in the cumulative probability plot for IEI in Fig. 7C. The amplitude plot in Fig. 7D shows that no significant change in amplitude distribution was noted (mean amplitude of 9.77 ± 0.32 pA in ifenprodil vs. 9.88 ± 0.26 pA with subsequent addition of HQA: P > 0.05, t-test, n = 6). The profound blockade of the HQA-induced increase in mEPSC frequency by ifenprodil suggests that NR2B-containing NMDARs are present at presynaptic terminals onto layer V neurons.



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Fig. 7. Ifenprodil substantially reduces the increase in mEPSC frequency, induced by HQA, in layer V. A: AMPAR-mediated mEPSCs recorded in the presence of 1 µM TTX and 10 µM IFN. B: HQA application (20 µM) elicited a small increase in mEPSC frequency (~15%). C: the cumulative probability plots for IEI pooled from 6 cells show only a slight shift to the right when HQA was added in the presence of the antagonist. D: the amplitude plots of pooled data show that there was no change in mEPSC amplitude (2-pA bins).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The use of "pure" postsynaptic AMPA receptor events as reporters of presynaptic activity, uncontaminated by postsynaptic NMDA receptor activity, allowed us to directly assess the effects of activation of presynaptic NMDA receptors at excitatory terminals. Initially, we used the agonist HQA to activate NMDARs. Although HQA discriminates poorly between NR2A and NR2B at micromolar concentrations, it has much lower affinity for NR2C (Brown et al. 1998; Buller and Monaghan 1997; de Carvalho et al. 1996). NR2C comprises the major component of the non-NR2A/B subunits expressed in EC (approximately 7% of NR1 and NR2A expression) (Sun et al. 2000). In addition, it has been reported that heterotrimeric receptors that contain NR2D have a much-reduced affinity for HQA compared with NR1/NR2D dimers (EC50 140 µM vs. 40 µM) (Buller and Monaghan 1997), and that 3H-HQA has a low affinity for NR2D-like binding sites (Brown et al. 1998). Taken together, these data suggest that in our experiments, HQA would have preferentially activated receptors containing NR2A, NR2B, or both subunits. Since the agonist greatly increased the frequency and synchrony of glutamate release, we suggest that excitatory terminals onto both deep and superficial EC layers bear NMDARs that contain NR2A or NR2B subunits.

In layer V neurons, experiments with ifenprodil support this conclusion. There is a possibility that these presynaptic receptors consist of NR1 and NR2B subunits only, with little contribution from NR2A. The concentration of ifenprodil (10 µM) used in these experiments has been reported to have very little activity at recombinant NR1/NR2A-containing receptors, but to exert a powerful blockade of NR2B-contaning dimeric receptors (Williams 1993). However, it is likely that many native NMDARs comprise a heterotrimeric mixture of NR1, NR2B, and NR2A subunits (Dunah et al. 1998; Luo et al. 1997; Sheng et al. 1994). At such heterotrimeric receptors, ifenprodil has been reported to have little (Luo et al. 1997), or intermediate (Tovar and Westbrook 1999) activity when compared with NR2B homomers. While there was some variation in the ability of ifenprodil to block HQA activity in our experiments, overall we observed a profound decrease in the ability of HQA to enhance release. These data suggest that if heterotrimeric receptors are indeed present at excitatory terminals, then they are sensitive to ifenprodil. Alternatively, the relatively strong blockade of HQA activity by ifenprodil may indicate that the presynaptic NMDARs consist predominantly of NR1/NR2B heterodimers.

What are the functional consequences of the presence of presynaptic NMDARs at layer V glutamatergic synapses? In layer II, we have previously demonstrated that these receptors are likely to be tonically active in maintaining glutamate release (Berretta and Jones 1996a), and the effects of ifenprodil and 2-AP5 suggest that this may be the case in layer V. We have also suggested that presynaptic NMDARs contribute to paired-pulse facilitation of evoked released at short (50-100 ms) intervals (Berretta and Jones 1996a). In the present study we have looked further at the role of presynaptic NMDARs at excitatory terminals during repetitive activation of synapses on layer V neurons. When the frequency of stimulation was increased from 0.5 to 3 Hz, we observed an increase in the amplitude of evoked EPSCs, similar to the frequency-dependent enhancement of glutamate-mediated synaptic potentials we have previously reported in sharp electrode studies (e.g., see Cunningham et al. 2000; Jones 1993). In the present experiments, postsynaptic NMDARs were blocked (with intracellular MK-801), but the enhancement was still prevented by the NMDAR antagonist, 2-AP5. The most parsimonious interpretation of these data are that the frequency-dependent, short-term enhancement of evoked synaptic responses is mediated by activation of presynaptic NMDARs leading to increased glutamate release.

We previously showed that when Ca2+ was replaced with Sr2+ in the ACSF, EPSCs evoked at low frequency (0.1 Hz) were succeeded by a series of AQEs, and that the presence of these events was dependent on the activation of presynaptic NMDARs (Berretta and Jones 1996a). In the present study, we found a similar increase in the number of AQEs following the evoked EPSC when stimulation was delivered at a higher frequency in normal Ca2+. Again, this effect was blocked by 2-AP5, indicating a dependence on presynaptic NMDARs. It is conceivable that the increased frequency of spontaneous AQEs engendered by the NMDA autoreceptor could serve to create a relatively large (>300 ms) window in which excitatory tone was increased, and subsequent action-potential dependent events would be more likely to temporally summate with spontaneous events. Overall, these effects could provide a presynaptic basis for an activity-dependent, frequency-facilitating synapse.

What is the mechanism underlying the facilitation of glutamate release by presynaptic NMDARs? We previously speculated that it was dependent on increased Ca2+ entry into the terminal via the receptor-gated ionophore (Berretta and Jones 1996a). This is supported by the study of Cochilla and Alford (1999), which showed that activation of the NMDARs on lamprey reticulospinal axons increased basal Ca2+ levels via the receptor and that this, in turn, increased the evoked release. It seems likely that a similar mechanism could account for the increase in evoked release and AQE frequency seen at entorhinal terminals. We showed that blocking Ca2+ entry at the terminal with Co2+ abolished the increase in frequency of EPSCs elicited by HQA. While Co2+ does not discriminate between NMDAR and voltage-gated calcium channels, the fact that prevention of Ca2+ entry blocks the effect of HQA suggests strongly that a Ca2+-mediated process underlies the increase in frequency of EPSCs elicited by HQA.

Although not reported here, we have also found that HQA increases the frequency of IPSCs in layer II, but not layer V, of the EC. Further work is under way in this laboratory to investigate the action of NMDAR at inhibitory terminals.


    ACKNOWLEDGMENTS

We are very grateful to Dr. David Jane for the gift of homoquinolinic acid.

This work was supported by the Wellcome Trust, the Taberner Trust, and the Biotechnology and Biological Sciences Research Council.


    FOOTNOTES

Address for reprint requests: G. Woodhall (E-mail: g.l.woodhall{at}bristol.ac.uk).

Received 29 September 2000; accepted in final form 13 June 2001.


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ABSTRACT
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society