# The Role of NMDA Receptor Subtypes in Short-Term  Plasticity in the Rat Entorhinal Cortex

Authors: Sophie E. L. Chamberlain; Jian Yang; Roland S. G. Jones
Journal: Neural Plasticity
 (2008)

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## Abstract

We have previously shown that spontaneous release of glutamate in the entorhinal cortex (EC) is tonically facilitated via activation of presynaptic NMDA receptors
(NMDAr) containing the NR2B subunit. Here we show that the same receptors mediate short-term plasticity manifested by frequency-dependent facilitation of evoked glutamate release at these synapses. Whole-cell patch-clamp recordings were made from layer V pyramidal neurones in rat EC slices. Evoked excitatory postsynaptic currents showed strong facilitation at relatively low frequencies (3 Hz) of activation. Facilitation was abolished by an NR2B-selective blocker (Ro 25-6981), but unaffected by NR2A-selective antagonists (Zn2+, NVP-AAM077). In contrast, postsynaptic NMDAr-mediated responses could be reduced by subunit-selective concentrations of all three antagonists. The data suggest that NMDAr involved in presynaptic plasticity in layer V are exclusively NR1/NR2B diheteromers, whilst postsynaptically they are probably a mixture of NR1/NR2A, NR1/NR2B diheteromers and NR1/NR2A/NR2B triheteromeric receptors.


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## Body

## 1. Introduction

A huge amount of research has been devoted to the study of the
physiology, pharmacology, function, and pathology of NMDA receptors (NMDAr). This has been extensively reviewed elsewhere (e.g., [1–6]). Native NMDAr
are heteromeric structures, and consist of NR1 subunits, which are obligatory,
in combination with one or more of four subtypes of NR2 subunit (NR2A-D).
Functional receptors are tetramers, comprising two NR1 subunits and two NR2
subunits, where the functional unit is probably an NR1/NR2 heterodimer. The
functional properties of NMDAr, such as single channel conductance, the degree
of voltage-dependent Mg2+ block, and deactivation kinetics depend on
which of the four NR2 subunits is assembled in the receptor. For example, NR2A
and NR2B-containing channels have a high single channel conductance (40–50 pS) whereas
NR2C and NR2D are lower (15–35 pS). NR2A-containing
receptors display fast decay kinetics (around 100 milliseconds), whereas NR2B
and C are much slower (250 milliseconds), and NR2D slower still (4 seconds) [5, 7]. In addition to functional differences, various subunit combinations display
pharmacological differences in susceptibility to antagonists and regulatory
mechanisms (such as sensitivity to H+, Zn2+, polyamines).Synaptic transmission is a highly dynamic and plastic process,
modified on-demand by a myriad of instantaneous, short, intermediate, and
long-term regulatory mechanisms. Much attention has been devoted to the study
of the role of NMDAr in synaptic plasticity, particularly in long-term
potentiation (LTP) and depression (LTD). These studies have largely focussed on
NMDAr at postsynaptic sites. However, dynamic regulation of synaptic strength
can also involve receptors on presynaptic terminals, which provide a powerful,
synapse-delimited control of transmitter release, and the existence of
presynaptic NMDAr (preNMDAr) is now firmly established. Neurochemical [8–11] and
immunolocalization studies [12–15] provided
early indications for preNMDAr. We
provided the first clear functional demonstration of preNMDAr, showing that the
competitive antagonist, 2-AP5, could reduce the frequency of spontaneous
excitatory postsynaptic currents (sEPSCs) at glutamate synapse in the rat
entorhinal cortex (EC), indicating a tonic facilitatory effect of preNMDAr on
glutamate release [16]. PreNMDAr are now known to modify both glutamate and
GABA release in a wide variety of locations and tissues [17–33].Increasing attention is being paid to the role of preNMDAr as
mediators of both long-term alterations in synaptic strength, and in moment-to-moment
and short-term activity-dependent changes in transmitter release. For example,
a role of preNMDAr in LTD has been demonstrated in cerebellum [34], visual [22, 33], and somatosensory [17] cortex. Conversely, involvement of preNMDAr in LTP
has been demonstrated in amygdala [26, 32]. More intermediate forms of
potentiation of glutamate [30] and
GABA transmission [23], over a time scale of minutes, may
also involve preNMDAr. As noted above, we found that preNMDAr are tonically
activated by ambient glutamate [17, 35], providing instantaneous control over
the level of glutamate release at EC synapses. Similar results have been
reported for other areas [22, 27, 28, 33]. In addition, we found that preNMDAr are activated after action potential-driven synaptic
release of glutamate, increasing the probability of subsequent release and
allowing them to mediate short-term, frequency-dependent facilitation of
glutamate transmission [16, 35].We have also demonstrated that the tonic
facilitatory effect of preNMDAr on spontaneous glutamate release is likely to
be predominantly mediated by NR2B-containing NMDAr, since the increase induced
by 2-AP5 was mimicked [35, 36] by relatively specific blockers of the NR2B
subunit,
ifenprodil [37], and Ro 25-6981 [38]. In addition, an
antagonist with some specificity (albeit weak) for the NR2A subunits,
NVP-AAM077 [39] had little effect. Others have also concluded that preNMDAr are
likely to be predominantly NR2B-containing [27, 33, 40]. Postsynaptically, both
NR2A and NR2B contribute to glutamate transmission, although there is
controversy over whether diheteromeric NR1/NR2A and NR1/NR2B coexist at the
postsynaptic density, or are segregated between synaptic and extrasynaptic
locations, or even in a synapse-specific way [3]. The contribution of
triheteromeric NR1/NR2A/NR2B receptors is also still a matter of debate [3, 41].In the present study, we have extended our
studies in the EC to examine the contribution of NR2A and NR2B receptors to
short-term plasticity of glutamate transmission, by examining the effects of
relatively specific blockers on the preNMDAr mediated, frequency-dependent
facilitation of evoked glutamate release. In addition, we have used the same
agents to determine whether postsynaptic NMDAr may differ from those on
presynaptic terminals.


## 2. Methods

### 2.1. Slice Preparation

Experiments were performed in accordance with the U.K. Animals
(Scientific Procedures) Act 1986, European Communities Council Directive 1986
(86/609/EEC), and the University of Bath ethical review document. Slices
containing EC and hippocampus were prepared from male
Wistar rats
(P28–35), which were
anaesthetized 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 (350–400μm) were cut
using a Vibroslice, and stored in aCSF bubbled with 95% O2/5% CO2,
at room temperature. Following recovery for at least 1 hour, individual slices
were transferred to a recording chamber mounted on the stage of a Zeiss
Axioskop FS or an Olympus BX50WI microscope. The chamber was perfused (2.0 ml/min) with oxygenated aCSF (pH 7.4) at 31–33°C. The aCSF
contained (in mM) NaCl (126), KCl (3), NaH2PO4 (1.4),
NaHCO3 (19), MgSO4 (2), CaCl2 (2), and D-glucose (10). Neurones were visualized
using differential interference contrast optics and an infrared video camera.


### 2.2. Electrophysiological Recording

Patch pipettes were pulled from borosilicate
glass on a Flaming/Brown microelectrode puller. For recording spontaneous
(sEPSCs) or evoked (eEPSCs) excitatory postsynaptic currents, pipettes were
filled with a Cs-gluconate-based solution containing (in mM) D-Gluconate (100),
HEPES (40), QX-314 (1), EGTA (0.6), NaCl (2), MgCl2 (5), TEA-Cl (1),
phosphocreatinine (5); ATP-Na (4), GTP-Na (0.3), MK-801 (2). Solutions were
adjusted to 290 mOsmol, and to pH 7.3 with CsOH. Whole-cell
voltage clamp recordings (holding potential −60 mV unless otherwise stated)
were made from neurones in layer V of the medial division of the EC, using an Axopatch 200B amplifier (Molecular Devices, Calif., USA). Series resistance compensation was not employed, but access resistance (10–30 MΩ) was
monitored at regular intervals throughout each recording and cells were
discarded from analysis if it changed by more than ±10%. Liquid junction
potential (12.3 mV) was estimated using the Junction Potential Calculator
included in pClamp-8 software (Molecular Devices, Calif., USA),
and compensated for in the holding potentials.eEPSCs were elicited by electrical stimulation (bipolar pulses, 10–50 V, 0.02 millisecond
duration) via a bipolar tungsten electrode placed on the surface of the slice
in layer V of the lateral EC. The stimulation intensity was adjusted to give
submaximal (approx. 50–60% maximum
amplitude) responses.


### 2.3. Monitoring Presynaptic NMDAr Activity

In all these experiments, MK-801 (2 mM) was included in the patch
pipette solution to block postsynaptic NMDAr. This allowed us to record
AMPA-receptor mediated responses in isolation, and to monitor activity at
preNMDAr uncontaminated by postsynaptic receptor effects. This approach was
developed by us [16, 35, 42], and has been used successfully
by others to block postsynaptic NMDAr in the recorded neurone [17, 27, 28, 32, 33, 40]. When whole-cell access was gained, neurones were voltage clamped at 0 mV,
and synaptic stimulation was delivered at 2 Hz for 30–40 seconds to
allow blockade of postsynaptic NMDAr by MK-801 dialyzed into the cell via the
patch pipette solution. Membrane potential was then clamped at −60 mV and
single shock stimulation delivered at low frequency (0.05 Hz) to evoke AMPAr
mediated EPSCs. At 2 or 3 minute intervals, the single shock was replaced with
stimulation at 3 Hz for 10 seconds. Such stimulation results in a
frequency-dependent facilitation of the AMPAr-mediated EPSC, which we have
shown previously to be dependent on activation of preNMDAr [35]. We used the
degree of frequency-dependent facilitation of AMPAr-mediated eEPSCs as a
quantitative measure of preNMDAr activation.


### 2.4. Monitoring Postsynaptic NMDAr Activity

In these experiments, MK-801 was omitted from
the patch pipette solution. When whole-cell access was gained, control eEPSCs
were recorded at a holding potential of −60 mV, before addition of the AMPAr antagonist, NBQX,
and the GABAAr-antagonist, bicuculline to the bath perfusion. After
10–12 minutes, the
holding potential was changed to +40 mV to record isolated NMDAr-mediated EPSCs
as positive going currents. These were evoked at low frequency (0.05 Hz) until
stable amplitudes were recorded, before addition of antagonists to the bath.


### 2.5. Data Analysis

Data were recorded to computer hard disk using Axoscope software. Minianalysis (Synaptosoft, Decatur, Ga, USA) was used for
analysis of EPSCs offline. In the studies of preNMDAr, the average peak
amplitude of the 8 responses before each episode of 3 Hz stimulation was
determined. During the period of 3 Hz stimulation, the amplitude of the 8
largest events was determined and normalized to the average amplitude of the
preceding low-frequency events to obtain a quantitative measure of frequency-dependent
facilitation in the presence and absence of antagonists. In these studies, we
also analyzed AMPAr-mediated sEPSCs, by determining interevent interval (IEI),
amplitude, rise (10–90%), and decay
times. sEPSCs were detected automatically using a
threshold-crossing algorithm. Threshold varied from neurone to neurone but was
always maintained at a constant level in any given recording. At least 200
events were sampled during a continuous recording period for each neurone under
each condition. Cumulative probability distributions of IEI were compared using
the Kolmogorov-Smirnoff test. In
experiments on postsynaptic NMDAr, responses were quantified by measuring mean
peak amplitudes of at least 5 NMDAr-mediated eEPSCs evoked at low frequency at
intervals throughout the study. In these studies, the vast majority of sEPSCs
were blocked, as recordings were conducted in the presence of NBQX. Occasional
slow sEPSCs mediated by NMDAr were recorded, their frequency was very low (2-3 per minute) and
precluded meaningful analysis.


### 2.6. Materials

Salts used in preparation of aCSF were “Analar” grade and purchased
from Merck/BDH or Fisher Scientific (Dorset, UK). All drugs were applied by bath perfusion. MK-801,
NMDA, NBQX, D-2-AP5, bicuculline methiodide, and Ro 25-6981 ((αR,βS)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol
hydrochloride) were obtained from Tocris (Bristol, UK). TPEN (N,N,N′, N′-Tetrakis-(2-pyridylmethyl)-Ethylenediamine)
was obtained from Sigma (UK). UBP302
((S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)
pyrimidine-2,4-dione) was a kind
gift from Dr. Dave Jane, University of Bristol, and NVP-AAM077 ((R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-ph- osphonic
acid) was a gift from Dr. Yve
Auberson at Novartis (Basel, Switzerland).


## 2.1. Slice Preparation

Experiments were performed in accordance with the U.K. Animals
(Scientific Procedures) Act 1986, European Communities Council Directive 1986
(86/609/EEC), and the University of Bath ethical review document. Slices
containing EC and hippocampus were prepared from male
Wistar rats
(P28–35), which were
anaesthetized 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 (350–400μm) were cut
using a Vibroslice, and stored in aCSF bubbled with 95% O2/5% CO2,
at room temperature. Following recovery for at least 1 hour, individual slices
were transferred to a recording chamber mounted on the stage of a Zeiss
Axioskop FS or an Olympus BX50WI microscope. The chamber was perfused (2.0 ml/min) with oxygenated aCSF (pH 7.4) at 31–33°C. The aCSF
contained (in mM) NaCl (126), KCl (3), NaH2PO4 (1.4),
NaHCO3 (19), MgSO4 (2), CaCl2 (2), and D-glucose (10). Neurones were visualized
using differential interference contrast optics and an infrared video camera.


## 2.2. Electrophysiological Recording

Patch pipettes were pulled from borosilicate
glass on a Flaming/Brown microelectrode puller. For recording spontaneous
(sEPSCs) or evoked (eEPSCs) excitatory postsynaptic currents, pipettes were
filled with a Cs-gluconate-based solution containing (in mM) D-Gluconate (100),
HEPES (40), QX-314 (1), EGTA (0.6), NaCl (2), MgCl2 (5), TEA-Cl (1),
phosphocreatinine (5); ATP-Na (4), GTP-Na (0.3), MK-801 (2). Solutions were
adjusted to 290 mOsmol, and to pH 7.3 with CsOH. Whole-cell
voltage clamp recordings (holding potential −60 mV unless otherwise stated)
were made from neurones in layer V of the medial division of the EC, using an Axopatch 200B amplifier (Molecular Devices, Calif., USA). Series resistance compensation was not employed, but access resistance (10–30 MΩ) was
monitored at regular intervals throughout each recording and cells were
discarded from analysis if it changed by more than ±10%. Liquid junction
potential (12.3 mV) was estimated using the Junction Potential Calculator
included in pClamp-8 software (Molecular Devices, Calif., USA),
and compensated for in the holding potentials.eEPSCs were elicited by electrical stimulation (bipolar pulses, 10–50 V, 0.02 millisecond
duration) via a bipolar tungsten electrode placed on the surface of the slice
in layer V of the lateral EC. The stimulation intensity was adjusted to give
submaximal (approx. 50–60% maximum
amplitude) responses.


## 2.3. Monitoring Presynaptic NMDAr Activity

In all these experiments, MK-801 (2 mM) was included in the patch
pipette solution to block postsynaptic NMDAr. This allowed us to record
AMPA-receptor mediated responses in isolation, and to monitor activity at
preNMDAr uncontaminated by postsynaptic receptor effects. This approach was
developed by us [16, 35, 42], and has been used successfully
by others to block postsynaptic NMDAr in the recorded neurone [17, 27, 28, 32, 33, 40]. When whole-cell access was gained, neurones were voltage clamped at 0 mV,
and synaptic stimulation was delivered at 2 Hz for 30–40 seconds to
allow blockade of postsynaptic NMDAr by MK-801 dialyzed into the cell via the
patch pipette solution. Membrane potential was then clamped at −60 mV and
single shock stimulation delivered at low frequency (0.05 Hz) to evoke AMPAr
mediated EPSCs. At 2 or 3 minute intervals, the single shock was replaced with
stimulation at 3 Hz for 10 seconds. Such stimulation results in a
frequency-dependent facilitation of the AMPAr-mediated EPSC, which we have
shown previously to be dependent on activation of preNMDAr [35]. We used the
degree of frequency-dependent facilitation of AMPAr-mediated eEPSCs as a
quantitative measure of preNMDAr activation.


## 2.4. Monitoring Postsynaptic NMDAr Activity

In these experiments, MK-801 was omitted from
the patch pipette solution. When whole-cell access was gained, control eEPSCs
were recorded at a holding potential of −60 mV, before addition of the AMPAr antagonist, NBQX,
and the GABAAr-antagonist, bicuculline to the bath perfusion. After
10–12 minutes, the
holding potential was changed to +40 mV to record isolated NMDAr-mediated EPSCs
as positive going currents. These were evoked at low frequency (0.05 Hz) until
stable amplitudes were recorded, before addition of antagonists to the bath.


## 2.5. Data Analysis

Data were recorded to computer hard disk using Axoscope software. Minianalysis (Synaptosoft, Decatur, Ga, USA) was used for
analysis of EPSCs offline. In the studies of preNMDAr, the average peak
amplitude of the 8 responses before each episode of 3 Hz stimulation was
determined. During the period of 3 Hz stimulation, the amplitude of the 8
largest events was determined and normalized to the average amplitude of the
preceding low-frequency events to obtain a quantitative measure of frequency-dependent
facilitation in the presence and absence of antagonists. In these studies, we
also analyzed AMPAr-mediated sEPSCs, by determining interevent interval (IEI),
amplitude, rise (10–90%), and decay
times. sEPSCs were detected automatically using a
threshold-crossing algorithm. Threshold varied from neurone to neurone but was
always maintained at a constant level in any given recording. At least 200
events were sampled during a continuous recording period for each neurone under
each condition. Cumulative probability distributions of IEI were compared using
the Kolmogorov-Smirnoff test. In
experiments on postsynaptic NMDAr, responses were quantified by measuring mean
peak amplitudes of at least 5 NMDAr-mediated eEPSCs evoked at low frequency at
intervals throughout the study. In these studies, the vast majority of sEPSCs
were blocked, as recordings were conducted in the presence of NBQX. Occasional
slow sEPSCs mediated by NMDAr were recorded, their frequency was very low (2-3 per minute) and
precluded meaningful analysis.


## 2.6. Materials

Salts used in preparation of aCSF were “Analar” grade and purchased
from Merck/BDH or Fisher Scientific (Dorset, UK). All drugs were applied by bath perfusion. MK-801,
NMDA, NBQX, D-2-AP5, bicuculline methiodide, and Ro 25-6981 ((αR,βS)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol
hydrochloride) were obtained from Tocris (Bristol, UK). TPEN (N,N,N′, N′-Tetrakis-(2-pyridylmethyl)-Ethylenediamine)
was obtained from Sigma (UK). UBP302
((S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)
pyrimidine-2,4-dione) was a kind
gift from Dr. Dave Jane, University of Bristol, and NVP-AAM077 ((R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-ph- osphonic
acid) was a gift from Dr. Yve
Auberson at Novartis (Basel, Switzerland).


## 3. Results

### 3.1. Presynaptic NMDAar

Figure1(a) shows eEPSCs evoked in a layer V
neurone at 3 Hz, with postsynaptic NMDAr blocked by internally dialyzed MK-801.
The first 6 responses evoked during a train of 30 at 3 Hz are shown and
demonstrate the facilitation seen at this relatively low frequency. As reported
previously [35], the facilitation of the AMPAr-mediated eEPSCs was entirely
dependent on presynaptic NMDAar activation, since it could be abolished by
2-AP5 (n=5, Figure 1(b)). Likewise, the NMDAr channel blocker, MK-801, also
abolished frequency facilitation (n=10, Figure 1(b)). In some neurones,
facilitation was replaced by a weak frequency-dependent depression of eEPSCs in
the presence of the blockers. This can be seen as a reduction in mean amplitude
of eEPSCs in the presence of the blockers (e.g., Figure 1(b)). In a further 5
neurones, we confirmed the specificity of the effect by testing the effects of
GluR5 subunit specific antagonist of kainate receptors (UBP 302, 20μM), since
we have recently shown that these receptors mediate a similar short-term facilitation
of glutamate transmission at 3–5 Hz in layer III
of the EC (Chamberlain S.E.L and Jones R.S.G. unpublished). UBP 302 had no
effect on facilitation in layer V (not shown) confirming its dependence on
NMDAar. Interestingly, 2-AP5 had no effect on frequency facilitation in layer
III of the EC (not shown), so although similar short-term plasticity is seen in
both layers, its underlying mechanism is lamina-specific.Short-term facilitation is
mediated by presynaptic NMDA receptors. (a) First 6 responses evoked by a train
of stimuli (3 Hz, 20 seconds) averaged from 3 neurones. (b) Responses (n=8)
were averaged at low frequency and during 3 Hz stimulation. In the presence of
2-AP5, low-frequency responses were unaltered, but facilitation was abolished.
The bar graphs show the mean results from 5 neurones. (c) Similar results were
seen with MK-801. Stimulation artifacts have been partially blanked for
clarity.
(a)(b)(c)Since neither 2-AP5 nor MK-801 has selectivity
for NR2A v NR2B subunits [5], the data do not indicate the subunit composition
of NMDAr responsible for short-term frequency-facilitation. To determine the
receptor involved, we have examined the effect of more specific antagonists.
First, we tested the effects of Ro 25-6981. This is an allosteric inhibitor of
NMDA receptors, which binds to a site on the N-terminal domain of the NR2
subunit, with a high degree of selectivity (>3000 fold) for NR2B over NR2A
[38]. Figure 2(a) shows that Ro 25-6981 at 500 nM abolished the frequency
facilitation of eEPSCs, again revealing a weak depression. A lower
concentration (200 nM, n=3) of Ro 25-6981 resulted in a mean maximal reduction
in frequency-facilitation of 69  ±  7%. At these concentrations, the drug should
have little or no effect on NR2A subunits [38], strongly suggesting that
NR2B-containing receptors are primarily responsible for this form of short-term
plasticity at layer V synapses. This would agree with previous studies that
have shown the tonic facilitatory effect on spontaneous release is likely to be
NR2B-mediated [16, 35, 43]. Accordingly, Ro 25-6981 resulted in a substantial
increase in IEI of sEPSCs from 277  ±  82 milliseconds (5.5±1.9 Hz) to 764  ±  261 milliseconds
(2.1±0.7 Hz) recorded in the same neurones (cf. [36, 43]). KS analysis of cumulative probability distributions
confirmed a highly significant change. There was no concurrent change in mean
amplitude, rise, or decay time (not shown).Effects of subunit selective
antagonists. (a) Ro 25-6981 abolished frequency-dependent facilitation. In
contrast, neither NVP-AAM077 (b) nor  Zn2+  (c) had any significant effect. (d) Zn2+ also had
little effect on sEPSCs. The records show consecutive sweeps of baseline
recording of sEPSCs and in the presence of Zn2+. The cumulative
probability plots show pooled data from 6 neurones, with 200 events from each
neurone in the presence and absence of the blocker. There was a small shift to
the right in the presence of Zn2+, but this failed to reach
significance (KS test).
(a)(b)(c)(d)Next, we examined the effect of NVP-AAM077 in 5
neurones. This is a competitive antagonist that shows some selectivity for
receptors containing the NR2A subtype. Initial reports indicated a greater than
100 fold selectivity of the compound for NR2A over NR2B [39, 44]. However,
recently, it has been suggested that the selectivity is closer to 10 fold when
the affinity of the two subtypes for glutamate is accounted for ([41], see also
[45, 46]). Thus, at the concentration employed here (400 nM),  we might expect
almost complete blockade of NR2A receptors, but it is possible that substantial
inhibition of NR2B would also occur [41]. Nevertheless,  NVP-AAM077 did not significantly affect the frequency-dependent facilitation of eEPSCs (see Figure 2(b)). If anything, the facilitation was slightly (although not significantly) increased. These data suggest that NVP-AAM077 may have
reasonable selectivity for the NR2A receptor in our preparation, but that these
receptors are not involved in presynaptic short-term plasticity at layer V
synapses. Further support for this was obtained from analysis of sEPSCs. The
mean IEI in control was 443  ±  230 milliseconds (4.0±0.9 Hz), and this decreased
slightly to 377  ±  180 milliseconds (4.5 Hz) with the addition of NVP-AAM077.
Likewise, there was no change in amplitude, rise, or decay times of sEPSCs (not
shown).In view of the controversy over the selectivity
of NVP-AAM077, we also tested (n=5) the effects of Zn2+, which has
been shown to discriminate between NR2A and NR2B receptors. Like Ro 25-6981
at NR2B subunits, Zn2+ binds to the N-terminal domain of the NR2A
subunit to exert a voltage-independent inhibition with >100 fold selectivity
over NR2B [47–49]. However, as
with NVP-AAM077, a relatively high concentration of Zn2+ (300 nM)
failed to alter frequency-dependent facilitation of eEPSCs (see Figure 2(c)).
In addition, it had little effect on the IEI (200  ±  150 ν 298  ±  170 milliseconds, see Figure 2(d)), amplitude (17.7±3.4ν15.4±2.2 pA), rise (1.9±0.3ν2.1±0.4 milliseconds), or decay times
(24.6±1.6ν27.3±1.3 milliseconds) of
sEPSCs (cf. [43]). Thus, the
data from both NVP-AAM077 and Zn2+ studies militate strongly against
a role for NR2A receptors in presynaptic frequency-dependent facilitation in
layer V of the EC. The ability of Ro 25-6981 to block facilitation strongly
indicates that presynaptic plasticity at these synapses is dependent only on
NR2B-containing receptors.A recent paper [50] suggested that activation
of postsynaptic NR2B-containing receptors at a similar frequency (3.3 Hz) to
that employed by us to elicit frequency-dependent facilitation induced a
long-term depression of the NMDAr-mediated currents themselves (primarily by
decreasing fractional Ca2+ currents carried by the receptors). We
were interested to see if the repetitive activation of the presynaptic
NR2B-containing receptors would induce any decrement in frequency facilitation
at layer V synapses. In 5 neurones, we induced facilitation of eEPSCs and
monitored the degree of facilitation but without the addition of any blockers. Overall
there was an initial decrease in the degree of facilitation of AMPAr-mediated
eEPSCs from the first to second episode, but thereafter it was remarkably
consistent (see Figure 3(a)). However, when we looked at absolute amplitude of
eEPSCs, there was a small, but consistent, increase over the course of the
studies. This applied to events evoked at both low and high frequencies (see Figure
3(b)). We also examined the time course of these changes in the neurones tested
with 2-AP5 (see Figure 4). The antagonist appeared to prevent the progressive
increase in amplitude of the low-frequency events at the same time as blocking
the frequency-dependent facilitation. This limited protocol may suggest the
short-term frequency-dependent facilitation could underlie a longer-term
enhancement of glutamate transmission. As the postsynaptic NMDAr were already
blocked (by internal MK-801), this is likely to involve the presynaptic,
NR2B-containing receptors.Progressive changes associated with repeated episodes of stimulation at 3 Hz in the absence of NMDAr blockers. Each point is the degree of facilitation recorded during a 30-second
period of stimulation and is the average from 5 neurones. (a) After an initial
decline in the degree of facilitation, it remained stable throughout the
subsequent 30 minutes of recording. (b) Mean amplitude of responses recorded at
low and high frequency used to assess the facilitation in the neurones shown in
(a). There was a progressive, albeit small increase in amplitude of responses
in both cases. Representative records from one neurone, sampled at the times
indicated, are shown below.
(a)(b)Time course of the effect of 2-AP5 on
eEPSC amplitude and facilitation. (a) The progressive increase in both low- and
high-frequency responses was prevented by the addition of 2-AP5 (n=5 neurones).
The responses at high frequency were progressively reduced to control levels,
in parallel with the degree of facilitation (b). (c) Representative responses
recorded in one neurone at the times indicated.
(a)(b)(c)


### 3.2. Postsynaptic NMDAr

We now wished to determine the contribution of
NR2A/B subunits to NMDAr at postsynaptic sites in layer V of the EC, so we
tested the same antagonists used in the presynaptic experiments for effects on
isolated NMDAr-mediated eEPSCs. As expected, the nonspecific blockers 2-AP5
(n=5) and MK-801 (n=9) both abolished the slow eEPSCs recorded at +40 mV in the
presence of NBQX and bicuculline (not shown). Ro 25-6981 (n=5) also elicited a
concentration dependent reduction in postsynaptic NMDAr responses at
concentrations that would be expected to retain selectivity for NR2B-containing
receptors (see Figure 5(a)). The slow eEPSCs were essentially abolished by
Ro 25-6981 at 500 nM. This suggests that NR1/NR2B receptors dominate at
postsynaptic sites as they do presynaptically. However, when we tested
NVP-AAM077 (n=6), we again found a concentration-related reduction in
postsynaptic responses with around 80% inhibition at 500 nM (see Figure 5(b)).
Comparison with the data of Neyton and Paoletti [41] suggests that the effect
of NVP-AAM077 could be explained by blockade of both NR2B and NR2A receptors
since 500 nM was sufficient to abolish NR2A responses in oocytes, but also to
exert around 60% block of NR2B. However, this is at odds with its failure to
alter preNMDAr-dependent facilitation, which is clearly an NR2B-mediated
response. Studies with Zn2+ (n=6) failed to
substantially clarify the situation. The divalent cation also elicited a
concentration-dependent reduction in slow eEPSCs (see Figure 5(c)). The
concentrations employed exert around an 80% voltage-independent block of NR2A
receptors expressed in oocytes, but retain a considerable degree of selectivity
with regard to block of NR2B receptors [47, 49]. These data do suggest a role
for NR2A receptors at postsynaptic sites, but it is puzzling that Ro 25-6981 essentially also abolished NMDAr EPSC, when
it would be expected to have little effect on NR2A receptors.Effect of subunit selective
antagonists on postsynaptic NMDAr-mediated eEPSCs. Slow eEPSCs were recorded at
+40 mV in the presence of NBQX and bicuculline. Each response is the average of
at least 8 events. (a) The NR2B antagonist, Ro 25-691, induced a
concentration-dependent reduction in slow eEPSCs. They were essentially
abolished at the higher concentration. (b) and (c) show that NR2A selective
blockers induced a very similar blockade of slow EPSCs. (d) A combination of
NR2A and NR2B antagonists also abolished slow EPSCs.
(a)(b)(c)(d)We performed two more sets of experiments to look at this question
further. In 5 neurones, we first perfused a low concentration of Ro 25-6981 (200 nM), to partially block the
NMDAr EPSC.   We then added a low concentration of Zn2+ (100 nM). In
these neurones, Ro 25-6981 resulted in inhibition of around 45%, and with the addition of
Zn2+ there was a further reduction to around 90–100%, which
clearly indicates a role of both NR2A and NR2B in mediating the postsynaptic
response (see Figure 5(d)). Finally, there is evidence that under control
conditions, NR2A-containing receptors may be substantially blocked by Zn2+,
present in the ACSF as a result of contamination of other salts used in its
preparation [47]. Although addition of Zn2+ clearly reduced slow
eEPSCs in our experiments, we also examined whether there was significant
blockade of  the NR2A receptor in control
recordings by testing the effect of the Zn2+-chelator, TPEN (2μM),
in 3 neurones. This had no effect on the mean amplitude of NMDAr eEPSCs (125.3±25.1ν111.9±26.1 pA) suggesting that our
results with antagonists were unlikely to be confounded by Zn2+-contamination.Finally, as noted above, relatively low
frequency, repetitive activation of NR2B receptors has been shown to induce a
depression of postsynaptic NMDA responses per se [50]. In 7 neurones, we determined the effects of a brief
period of repetitive stimulation (3 Hz, 40 seconds) on postsynaptic NMDAr
eEPSCs in 5 neurones. Overall, during the repetitive stimulation there was a
small (15%), progressive decrease in the first 10–15 seconds, and
thereafter the amplitude reached a plateau (see Figure 6(a)). We then recorded
NMDAr eEPSCs at low frequency (0.05 Hz) over the subsequent 30 minutes. There
was an initial period (5 minutes) where responses appeared to be slightly
depressed and thereafter a recovery followed by a slight increase before recovery
to control levels (see Figure 6(b)). However, apart from a brief period around
20 minutes there was no significant difference compared to control.Changes in slow eEPSC amplitudes
during and after repetitive stimulation at 3 Hz for 30 seconds. (a) shows the
average response amplitudes at low frequency (0.05 Hz) recorded during 35
minutes stimulation in 7 neurones. During the period indicated by the arrow,
stimulation was increased to 3 Hz for 30 seconds and the average response
amplitudes (first 37 only for clarity) recorded during this period are shown in
(b). The only significant differences compared to the mean control value are indicated by the
asterisks in (a).
(a)(b)


## 3.1. Presynaptic NMDAar

Figure1(a) shows eEPSCs evoked in a layer V
neurone at 3 Hz, with postsynaptic NMDAr blocked by internally dialyzed MK-801.
The first 6 responses evoked during a train of 30 at 3 Hz are shown and
demonstrate the facilitation seen at this relatively low frequency. As reported
previously [35], the facilitation of the AMPAr-mediated eEPSCs was entirely
dependent on presynaptic NMDAar activation, since it could be abolished by
2-AP5 (n=5, Figure 1(b)). Likewise, the NMDAr channel blocker, MK-801, also
abolished frequency facilitation (n=10, Figure 1(b)). In some neurones,
facilitation was replaced by a weak frequency-dependent depression of eEPSCs in
the presence of the blockers. This can be seen as a reduction in mean amplitude
of eEPSCs in the presence of the blockers (e.g., Figure 1(b)). In a further 5
neurones, we confirmed the specificity of the effect by testing the effects of
GluR5 subunit specific antagonist of kainate receptors (UBP 302, 20μM), since
we have recently shown that these receptors mediate a similar short-term facilitation
of glutamate transmission at 3–5 Hz in layer III
of the EC (Chamberlain S.E.L and Jones R.S.G. unpublished). UBP 302 had no
effect on facilitation in layer V (not shown) confirming its dependence on
NMDAar. Interestingly, 2-AP5 had no effect on frequency facilitation in layer
III of the EC (not shown), so although similar short-term plasticity is seen in
both layers, its underlying mechanism is lamina-specific.Short-term facilitation is
mediated by presynaptic NMDA receptors. (a) First 6 responses evoked by a train
of stimuli (3 Hz, 20 seconds) averaged from 3 neurones. (b) Responses (n=8)
were averaged at low frequency and during 3 Hz stimulation. In the presence of
2-AP5, low-frequency responses were unaltered, but facilitation was abolished.
The bar graphs show the mean results from 5 neurones. (c) Similar results were
seen with MK-801. Stimulation artifacts have been partially blanked for
clarity.
(a)(b)(c)Since neither 2-AP5 nor MK-801 has selectivity
for NR2A v NR2B subunits [5], the data do not indicate the subunit composition
of NMDAr responsible for short-term frequency-facilitation. To determine the
receptor involved, we have examined the effect of more specific antagonists.
First, we tested the effects of Ro 25-6981. This is an allosteric inhibitor of
NMDA receptors, which binds to a site on the N-terminal domain of the NR2
subunit, with a high degree of selectivity (>3000 fold) for NR2B over NR2A
[38]. Figure 2(a) shows that Ro 25-6981 at 500 nM abolished the frequency
facilitation of eEPSCs, again revealing a weak depression. A lower
concentration (200 nM, n=3) of Ro 25-6981 resulted in a mean maximal reduction
in frequency-facilitation of 69  ±  7%. At these concentrations, the drug should
have little or no effect on NR2A subunits [38], strongly suggesting that
NR2B-containing receptors are primarily responsible for this form of short-term
plasticity at layer V synapses. This would agree with previous studies that
have shown the tonic facilitatory effect on spontaneous release is likely to be
NR2B-mediated [16, 35, 43]. Accordingly, Ro 25-6981 resulted in a substantial
increase in IEI of sEPSCs from 277  ±  82 milliseconds (5.5±1.9 Hz) to 764  ±  261 milliseconds
(2.1±0.7 Hz) recorded in the same neurones (cf. [36, 43]). KS analysis of cumulative probability distributions
confirmed a highly significant change. There was no concurrent change in mean
amplitude, rise, or decay time (not shown).Effects of subunit selective
antagonists. (a) Ro 25-6981 abolished frequency-dependent facilitation. In
contrast, neither NVP-AAM077 (b) nor  Zn2+  (c) had any significant effect. (d) Zn2+ also had
little effect on sEPSCs. The records show consecutive sweeps of baseline
recording of sEPSCs and in the presence of Zn2+. The cumulative
probability plots show pooled data from 6 neurones, with 200 events from each
neurone in the presence and absence of the blocker. There was a small shift to
the right in the presence of Zn2+, but this failed to reach
significance (KS test).
(a)(b)(c)(d)Next, we examined the effect of NVP-AAM077 in 5
neurones. This is a competitive antagonist that shows some selectivity for
receptors containing the NR2A subtype. Initial reports indicated a greater than
100 fold selectivity of the compound for NR2A over NR2B [39, 44]. However,
recently, it has been suggested that the selectivity is closer to 10 fold when
the affinity of the two subtypes for glutamate is accounted for ([41], see also
[45, 46]). Thus, at the concentration employed here (400 nM),  we might expect
almost complete blockade of NR2A receptors, but it is possible that substantial
inhibition of NR2B would also occur [41]. Nevertheless,  NVP-AAM077 did not significantly affect the frequency-dependent facilitation of eEPSCs (see Figure 2(b)). If anything, the facilitation was slightly (although not significantly) increased. These data suggest that NVP-AAM077 may have
reasonable selectivity for the NR2A receptor in our preparation, but that these
receptors are not involved in presynaptic short-term plasticity at layer V
synapses. Further support for this was obtained from analysis of sEPSCs. The
mean IEI in control was 443  ±  230 milliseconds (4.0±0.9 Hz), and this decreased
slightly to 377  ±  180 milliseconds (4.5 Hz) with the addition of NVP-AAM077.
Likewise, there was no change in amplitude, rise, or decay times of sEPSCs (not
shown).In view of the controversy over the selectivity
of NVP-AAM077, we also tested (n=5) the effects of Zn2+, which has
been shown to discriminate between NR2A and NR2B receptors. Like Ro 25-6981
at NR2B subunits, Zn2+ binds to the N-terminal domain of the NR2A
subunit to exert a voltage-independent inhibition with >100 fold selectivity
over NR2B [47–49]. However, as
with NVP-AAM077, a relatively high concentration of Zn2+ (300 nM)
failed to alter frequency-dependent facilitation of eEPSCs (see Figure 2(c)).
In addition, it had little effect on the IEI (200  ±  150 ν 298  ±  170 milliseconds, see Figure 2(d)), amplitude (17.7±3.4ν15.4±2.2 pA), rise (1.9±0.3ν2.1±0.4 milliseconds), or decay times
(24.6±1.6ν27.3±1.3 milliseconds) of
sEPSCs (cf. [43]). Thus, the
data from both NVP-AAM077 and Zn2+ studies militate strongly against
a role for NR2A receptors in presynaptic frequency-dependent facilitation in
layer V of the EC. The ability of Ro 25-6981 to block facilitation strongly
indicates that presynaptic plasticity at these synapses is dependent only on
NR2B-containing receptors.A recent paper [50] suggested that activation
of postsynaptic NR2B-containing receptors at a similar frequency (3.3 Hz) to
that employed by us to elicit frequency-dependent facilitation induced a
long-term depression of the NMDAr-mediated currents themselves (primarily by
decreasing fractional Ca2+ currents carried by the receptors). We
were interested to see if the repetitive activation of the presynaptic
NR2B-containing receptors would induce any decrement in frequency facilitation
at layer V synapses. In 5 neurones, we induced facilitation of eEPSCs and
monitored the degree of facilitation but without the addition of any blockers. Overall
there was an initial decrease in the degree of facilitation of AMPAr-mediated
eEPSCs from the first to second episode, but thereafter it was remarkably
consistent (see Figure 3(a)). However, when we looked at absolute amplitude of
eEPSCs, there was a small, but consistent, increase over the course of the
studies. This applied to events evoked at both low and high frequencies (see Figure
3(b)). We also examined the time course of these changes in the neurones tested
with 2-AP5 (see Figure 4). The antagonist appeared to prevent the progressive
increase in amplitude of the low-frequency events at the same time as blocking
the frequency-dependent facilitation. This limited protocol may suggest the
short-term frequency-dependent facilitation could underlie a longer-term
enhancement of glutamate transmission. As the postsynaptic NMDAr were already
blocked (by internal MK-801), this is likely to involve the presynaptic,
NR2B-containing receptors.Progressive changes associated with repeated episodes of stimulation at 3 Hz in the absence of NMDAr blockers. Each point is the degree of facilitation recorded during a 30-second
period of stimulation and is the average from 5 neurones. (a) After an initial
decline in the degree of facilitation, it remained stable throughout the
subsequent 30 minutes of recording. (b) Mean amplitude of responses recorded at
low and high frequency used to assess the facilitation in the neurones shown in
(a). There was a progressive, albeit small increase in amplitude of responses
in both cases. Representative records from one neurone, sampled at the times
indicated, are shown below.
(a)(b)Time course of the effect of 2-AP5 on
eEPSC amplitude and facilitation. (a) The progressive increase in both low- and
high-frequency responses was prevented by the addition of 2-AP5 (n=5 neurones).
The responses at high frequency were progressively reduced to control levels,
in parallel with the degree of facilitation (b). (c) Representative responses
recorded in one neurone at the times indicated.
(a)(b)(c)


## 3.2. Postsynaptic NMDAr

We now wished to determine the contribution of
NR2A/B subunits to NMDAr at postsynaptic sites in layer V of the EC, so we
tested the same antagonists used in the presynaptic experiments for effects on
isolated NMDAr-mediated eEPSCs. As expected, the nonspecific blockers 2-AP5
(n=5) and MK-801 (n=9) both abolished the slow eEPSCs recorded at +40 mV in the
presence of NBQX and bicuculline (not shown). Ro 25-6981 (n=5) also elicited a
concentration dependent reduction in postsynaptic NMDAr responses at
concentrations that would be expected to retain selectivity for NR2B-containing
receptors (see Figure 5(a)). The slow eEPSCs were essentially abolished by
Ro 25-6981 at 500 nM. This suggests that NR1/NR2B receptors dominate at
postsynaptic sites as they do presynaptically. However, when we tested
NVP-AAM077 (n=6), we again found a concentration-related reduction in
postsynaptic responses with around 80% inhibition at 500 nM (see Figure 5(b)).
Comparison with the data of Neyton and Paoletti [41] suggests that the effect
of NVP-AAM077 could be explained by blockade of both NR2B and NR2A receptors
since 500 nM was sufficient to abolish NR2A responses in oocytes, but also to
exert around 60% block of NR2B. However, this is at odds with its failure to
alter preNMDAr-dependent facilitation, which is clearly an NR2B-mediated
response. Studies with Zn2+ (n=6) failed to
substantially clarify the situation. The divalent cation also elicited a
concentration-dependent reduction in slow eEPSCs (see Figure 5(c)). The
concentrations employed exert around an 80% voltage-independent block of NR2A
receptors expressed in oocytes, but retain a considerable degree of selectivity
with regard to block of NR2B receptors [47, 49]. These data do suggest a role
for NR2A receptors at postsynaptic sites, but it is puzzling that Ro 25-6981 essentially also abolished NMDAr EPSC, when
it would be expected to have little effect on NR2A receptors.Effect of subunit selective
antagonists on postsynaptic NMDAr-mediated eEPSCs. Slow eEPSCs were recorded at
+40 mV in the presence of NBQX and bicuculline. Each response is the average of
at least 8 events. (a) The NR2B antagonist, Ro 25-691, induced a
concentration-dependent reduction in slow eEPSCs. They were essentially
abolished at the higher concentration. (b) and (c) show that NR2A selective
blockers induced a very similar blockade of slow EPSCs. (d) A combination of
NR2A and NR2B antagonists also abolished slow EPSCs.
(a)(b)(c)(d)We performed two more sets of experiments to look at this question
further. In 5 neurones, we first perfused a low concentration of Ro 25-6981 (200 nM), to partially block the
NMDAr EPSC.   We then added a low concentration of Zn2+ (100 nM). In
these neurones, Ro 25-6981 resulted in inhibition of around 45%, and with the addition of
Zn2+ there was a further reduction to around 90–100%, which
clearly indicates a role of both NR2A and NR2B in mediating the postsynaptic
response (see Figure 5(d)). Finally, there is evidence that under control
conditions, NR2A-containing receptors may be substantially blocked by Zn2+,
present in the ACSF as a result of contamination of other salts used in its
preparation [47]. Although addition of Zn2+ clearly reduced slow
eEPSCs in our experiments, we also examined whether there was significant
blockade of  the NR2A receptor in control
recordings by testing the effect of the Zn2+-chelator, TPEN (2μM),
in 3 neurones. This had no effect on the mean amplitude of NMDAr eEPSCs (125.3±25.1ν111.9±26.1 pA) suggesting that our
results with antagonists were unlikely to be confounded by Zn2+-contamination.Finally, as noted above, relatively low
frequency, repetitive activation of NR2B receptors has been shown to induce a
depression of postsynaptic NMDA responses per se [50]. In 7 neurones, we determined the effects of a brief
period of repetitive stimulation (3 Hz, 40 seconds) on postsynaptic NMDAr
eEPSCs in 5 neurones. Overall, during the repetitive stimulation there was a
small (15%), progressive decrease in the first 10–15 seconds, and
thereafter the amplitude reached a plateau (see Figure 6(a)). We then recorded
NMDAr eEPSCs at low frequency (0.05 Hz) over the subsequent 30 minutes. There
was an initial period (5 minutes) where responses appeared to be slightly
depressed and thereafter a recovery followed by a slight increase before recovery
to control levels (see Figure 6(b)). However, apart from a brief period around
20 minutes there was no significant difference compared to control.Changes in slow eEPSC amplitudes
during and after repetitive stimulation at 3 Hz for 30 seconds. (a) shows the
average response amplitudes at low frequency (0.05 Hz) recorded during 35
minutes stimulation in 7 neurones. During the period indicated by the arrow,
stimulation was increased to 3 Hz for 30 seconds and the average response
amplitudes (first 37 only for clarity) recorded during this period are shown in
(b). The only significant differences compared to the mean control value are indicated by the
asterisks in (a).
(a)(b)


## 4. Discussion

We originally demonstrated that the presynaptic
NMDAar mediating facilitation of glutamate release in the EC was likely to be
predominantly NR2B-containing, as the frequency of sEPSCs was decreased by the
N2B antagonist, ifenprodil [35]. Other work supports the conclusion that
preNMDAr that facilitate spontaneous glutamate release at cortical synapses are
primarily NR2B-containing. We found that Ro 25-6981 but not NVP-AAM077 or Zn2+ reduced sEPSC frequency ([36], present study), and similar results with Ro
25-6981 and Zn2+ were reported for synapses in layer II/III of the
visual cortex [28]. Jourdain et al. [27] reported that presynaptic NR2B receptors
were responsible for the increase in mEPSC frequency in dentate granule neurones
seen after stimulation of glutamate release from adjacent astrocytes, as it was
blocked by ifenprodil. We now show that the same receptor is likely to mediate
short-term plasticity of evoked glutamate release in layer V of the EC. Thus,
the facilitation of eEPSCs at the relatively low frequency of 3 Hz was blocked by
Ro 25-6981. The lack of effect of NVP-AAM077 and Zn2+ suggests that
NR2A receptors do not contribute to facilitation of either spontaneous or evoked glutamate
release at EC synapses.  We cannot rule out a role of NR2A receptors at higher
frequencies, although Sjöström et al. [33] have reported that frequency facilitation at
30 Hz at layer V synapses in visual cortex is greatly reduced by ifenprodil,
suggesting that NR2B dominate at other presynaptic sites as well.It is somewhat surprising that only presynaptic
NR2B receptors appear to modulate release. Postembedding immunolabeling studies
have shown the presence of NR1 subunits in presynaptic terminals in cortex and
hippocampus [12–14, 51, 52, 53]. Whilst a
host of studies have demonstrated NR2B subunits at presynaptic locations [15, 51, 54–59], similar
studies have also indicated the presence of NR2A subunits [51, 52, 60–62] although, to
date, there are no similar studies specifically related to the EC.The presence of all three subunits suggests
that both NR1/NR2A and NR1/NR2B diheteromeric receptors and possibly also
NR1/NR2A/NR2B triheteromers could be expressed in cortical presynaptic
terminals, and this may well be the case. However, it is clear from the
pharmacological experiments presented here and elsewhere, that NR1/NR2B
receptors are predominantly responsible for short-term NMDAr-mediated facilitation
of glutamate release (but see, [63]). The properties of NR2B subunits differ from
NR2A, in a way that may make them more suited to the task of presynaptic
facilitation (see [6, 7, 64–66]). NR2B
subunits have a higher affinity for both glutamate and glycine, and show less
desensitization. The two subunits confer similar single channel conductance to
diheteromeric receptors (around 50 pS),
but they have very different deactivation kinetics,  with NR1/NR2A receptors
having decay time constants of 50–100 milliseconds,
and NR1/NR2B receptors in the order of 200–400 milliseconds.
Both are Ca2+-permeable, but NR2B receptors exhibit a higher
fractional Ca2+-current than NR2A (see [66, 67]). Both subunits also
display Ca2+-dependent inactivation, but this is more pronounced for
NR2A. The presence of NR2B subunits results in prolonged EPSPs compared to
those seen when NR2A subunits dominate (see [3, 7, 66]). Thus, it seems likely
that activation of presynaptic NR2B-containing receptors would mediate a slowly
deactivating opening of the NMDAr channel and a greater Ca2+-influx
into the presynaptic terminals than any influx mediated by NR2A receptors. Ca2+-influx
via the NMDAr is responsible for instantaneous control of spontaneous glutamate
release [35]. With a deactivation time of around 300 milliseconds, repetitive
activation of NR1/NR2B receptors would readily result in temporal summation of
presynaptic Ca2+-entry leading to the short-term facilitation at
even relatively low-frequency stimulations seen here and previously [35].It  is interesting to speculate on a physiological or pathological
role for short-term plasticity
mediated by preNMDAr. State-dependent rhythms and oscillatory activity at
various frequencies occur in the networks of the EC including ripples and sharp
waves (>100 Hz), gamma (30–80 Hz), theta (4–8 Hz), and slow
waves (0.1–0.5 Hz) [68–71], and these
may be involved in mnemonic processing in temporal lobe structures. There is a
consensus that theta oscillations are intimately involved in declarative memory
and spatial navigation (see [72–74]), and it is
possible that information encoding involved in these processes is reliant on an
increase in entorhinal-hippocampal delta/theta coherence [73]. The facilitation
of glutamate transmission mediated by preNMDAr that we describe is readily
elicited at frequencies in the low theta range. Thus, we could speculate that
these receptors may be involved in the generation of theta activity in the EC,
and the proposed role of this activity in short-term memory and coding of
spatial information (e.g., [72, 74]).At a pathological level, it is noteworthy that, oscillations at
delta (1-2 Hz) and theta
frequency may be associated with epilepsy. In patients with temporal lobe
epilepsy, there is a generalized increase in EEG activity in the delta/theta
range, and the most common pattern of discharges after the initiation of ictal
events is a rhythmic delta/theta activity (e.g., [75, 76]). Also, in rats made
chronically epileptic following kainic acid injection, epileptiform events in
superficial layers of the EC were sometimes followed by spontaneous theta
oscillations in layer V [77]. We recently showed that preNMDAr function
declines in adulthood, but is markedly enhanced in age-matched, chronically
epileptic rats [36] and there is evidence for a similar increased function in
human temporal lobe epilepsy [78]. We could speculate that this increased
preNMDAr function could result in enhanced generation of delta/theta activity in epileptic
conditions. Of further interest in this regard is the observation that
increased delta/theta EEG activity (albeit in patients with generalized
absence/myoclonic seizures) is normalized by the anticonvulsant drugs, valproate, and lamotrigine
[79–81]. We have also
shown that at least one anticonvulsant drug (felbamate) can block the preNMDAr
[42]. This raises the possibility that some anticonvulsants could alter delta/theta
oscillations by targeting preNMDAr.Whatever the function of short-term plasticity,
and the involvement of preNMDAr in it, there is increasing evidence that these
receptors may also contribute to longer term forms of plasticity, apparently
mediating both LTD [17, 22, 33, 34] and LTP [26, 32] at
a variety of synapses. In at least one case, LTD appears to be mediated by NR2B-containing
receptors [33], so both short- and long-term plasticity of glutamate
transmission could involve Ca2+-influx via presynaptic NR2B
receptors. We have also shown recently that preNMDAr are rapidly mobile
and can diffuse between locations near release sites and more distal
locations in the terminal membrane [82]. Trafficking of receptors in the
presynaptic membrane appears to be influenced by ongoing activity levels, and
exerts an intermediate (over 10 seconds of minutes) form of plasticity. Thus,
presynaptic NR2B receptors may be heavily involved in both plasticity and
metaplasticity at glutamate synapses in EC and other cortical synapses.In the present study, we also present evidence for differences in pre-
and postsynaptic NMDAr at layer V synapses. Whilst preNMDAr-mediated effects
are exclusively dependent on NR1/NR2B-containing diheteromers, both NR2B and
NR2A appear to contribute to postsynaptic responses. However, the relative
contributions of the two subunits are not clear. The ability of low
concentrations of both Zn2+ and Ro 25-6981 to reduce postsynaptic
NMDAr responses could suggest that they are dependent on a mix of NR1/NR2A and NR1/NR2B
diheteromeric receptors. However, concentrations of either blocker, that should
largely retain selectivity at the respective subtypes, were able to almost
abolish postsynaptic responses. This could suggest that the postsynaptic
receptors could be largely triheteromeric NR1/NR2A/NR2B receptors. Although
triheteromeric receptors do exhibit high affinity for both NR2A and NR2B
selective blockers, it seems likely that they exhibit a reduced maximal inhibitory
effect to either, and that maximal blockade requires occupation of both sites
[83]. This does not fit well with our finding that combined application of low
concentrations of Zn2+ and Ro 25-6981 could also abolish
postsynaptic responses, which would better support a mediation by a mix of
NR1/NR2A and NR1/NR2B diheteromeric receptors. It should also be noted that the
ability of NMDA antagonists to block the receptors is not just dependent on the
NR2 subunit present, but is also modified by which splice variant of the NR1
subunit with which it combines [47, 49]. We do not know which NR1 subunit(s)
may be present in the EC. Thus, overall it is difficult to define exactly what
the postsynaptic receptor population, but the most likely scenario is a mix of
NR1/NR2A, NR1/NR2B, and NR1/NR2A/NR2B receptors.A number of studies have suggested that NR1/NR2A, NR1/NR2B, and
NR1/NR2A/NR2B receptors may contribute to postsynaptic responses at other
cortical synapses [84–86]. There is
support also for synapse-specific segregation of NR2A and NR2B-containing
receptors (e.g., [87, 88]) and spatial segregation between subsynaptic and
extrasynaptic sites (e.g., [86]). The controversy over whether subunit
composition and spatial location are linked, and the difficulties in defining
the role of triheteromeric receptors has been well reviewed recently [3]. We
cannot make any firm conclusions regarding these aspects in the EC, but our
data do suggest that postsynaptic NR1/NR2A, NR1/NR2B, and NR1/NR2A/NR2B
receptors all contribute to postsynaptic responses at glutamate synapses in
layer V of the EC, in contrast to presynaptic sites where NR1/NR2B receptors
may have exclusive control. Increasing numbers of studies have documented LTP
and LTD at synapses in the EC [89–95]. The EC is
clearly a pivotal site in learning and memory functions resident in the
temporal lobe. We have shown that preNMDAr mediate short-term forms of
plasticity in the EC. In experiments employing a limited protocol of repetitive
activation, we found that this short-term plasticity may lead to longer-term
plasticity (either pre- or postsynaptically), and the aim now is to examine in
detail the relationship between short-term effects and long-term plasticity and
metaplasticity at these synapses.



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*Source: 872456-2008-10-29.xml*