a huge amount of research has been devoted to the study of the 
 physiology , pharmacology , function , and pathology of nmda receptors ( nmdar ) . 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 mg 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 ( 4050 ps ) whereas 
 nr2c and nr2d are lower ( 1535 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 , zn , 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 ) . 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 [ 811 ] and 
 immunolocalization studies [ 1215 ] 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 . prenmdar are now known to modify both glutamate and 
 gaba release in a wide variety of locations and tissues [ 1733 ] . 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 , visual [ 22 , 33 ] , and somatosensory   cortex . conversely , involvement of prenmdar in ltp 
 has been demonstrated in amygdala [ 26 , 32 ] . more intermediate forms of 
 potentiation of glutamate   and 
 gaba transmission , 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 , and ro 25 - 6981 . in addition , an 
 antagonist with some specificity ( albeit weak ) for the nr2a subunits , 
 nvp - aam077   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 . 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 . 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 
 ( p2835 ) , 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 4c . slices ( 350400 m ) 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 3133c . the acsf 
 contained ( in mm ) nacl ( 126 ) , kcl ( 3 ) , nah2po4 ( 1.4 ) , 
 nahco3 ( 19 ) , mgso4 ( 2 ) , cacl2 ( 2 ) , and d - glucose ( 10 ) . 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 ) . 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 . series resistance compensation was not employed , but access resistance ( 1030 m ) 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 . , eepscs were elicited by electrical stimulation ( bipolar pulses , 1050 v , 0.02 millisecond 
 duration ) via a bipolar tungsten electrode placed on the surface of the slice 
 in layer v of the lateral ec . 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 3040 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 , 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 . we used the 
 degree of frequency - dependent facilitation of ampar - mediated eepscs as a 
 quantitative measure of prenmdar activation . in these experiments , 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 
 1012 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 . 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 ( 1090% ) , and decay 
 times . 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 . salts used in preparation of acsf were  analar  grade and purchased 
 from merck / bdh or fisher scientific ( dorset , uk ) . mk-801 , 
 nmda , nbqx , d-2-ap5 , bicuculline methiodide , and ro 25 - 6981 ( ( r,s)--(4-hydroxyphenyl)--methyl-4-(phenylmethyl)-1-piperidinepropanol 
 hydrochloride ) were obtained from tocris ( bristol , 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]-phosphonic 
 acid ) was a gift from dr . figure 1(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 , 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 , 20 m ) , since 
 we have recently shown that these receptors mediate a similar short - term facilitation 
 of glutamate transmission at 35 hz in layer iii 
 of the ec ( chamberlain s.e.l and jones r.s.g . 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 . since neither 2-ap5 nor mk-801 has selectivity 
 for nr2a v nr2b subunits , the data do not indicate the subunit composition 
 of nmdar responsible for short - term frequency - facilitation . to determine the 
 receptor involved 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 
 . 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 , 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 . there was no concurrent change in mean 
 amplitude , rise , or decay time ( not shown ) . 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 ( , 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 . nevertheless ,   nvp - aam077 did not significantly affect the frequency - dependent facilitation of eepscs ( see figure 2(b ) ) . 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 . 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 zn , which has 
 been shown to discriminate between nr2a and nr2b receptors . like ro 25 - 6981 
  at nr2b subunits , zn binds to the n - terminal domain of the nr2a 
 subunit to exert a voltage - independent inhibition with > 100 fold selectivity 
 over nr2b [ 4749 ] . however , as 
 with nvp - aam077 , a relatively high concentration of zn ( 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 v 298      170 milliseconds , see figure 2(d ) ) , amplitude ( 17.7  3.4 v 15.4  2.2 pa ) , rise ( 1.9  0.3 v 2.1  0.4 milliseconds ) , or decay times 
 ( 24.6  1.6 v 27.3  1.3 milliseconds ) of 
 sepscs ( cf . ) . thus , the 
 data from both nvp - aam077 and zn 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   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 ca 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 . 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 . 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   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 zn ( 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 . 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 zn ( 100 nm ) . in 
 these neurones , ro 25 - 6981 resulted in inhibition of around 45% , and with the addition of 
 zn there was a further reduction to around 90100% , 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 zn , 
 present in the acsf as a result of contamination of other salts used in its 
 preparation . although addition of zn 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 zn - chelator , tpen ( 2 m ) , 
 in 3 neurones . this had no effect on the mean amplitude of nmdar eepscs ( 125.3  25.1 v 111.9  26.1 pa ) suggesting that our 
 results with antagonists were unlikely to be confounded by zn - 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 . 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 1015 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 . 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 . 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 zn reduced sepsc frequency ( , present study ) , and similar results with ro 
 25 - 6981 and zn were reported for synapses in layer ii / iii of the 
 visual cortex . 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 zn suggests that 
 nr2a receptors do not contribute to facilitation of either spontaneous or evoked glutamate 
 release at ec synapses . we can not rule out a role of nr2a receptors at higher 
 frequencies , although sjstrm et al . 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 . postembedding immunolabeling studies 
 have shown the presence of nr1 subunits in presynaptic terminals in cortex and 
 hippocampus [ 1214 , 5153 ] . whilst a 
 host of studies have demonstrated nr2b subunits at presynaptic locations [ 15 , 51 , 5459 ] , similar 
 studies have also indicated the presence of nr2a subunits [ 51 , 52 , 6062 ] 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 , ) . 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 , 6466 ] ) . 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 50100 milliseconds , 
 and nr1/nr2b receptors in the order of 200400 milliseconds . 
 both are ca - permeable , but nr2b receptors exhibit a higher 
 fractional ca - current than nr2a ( see [ 66 , 67 ] ) . both subunits also 
 display ca - 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 ca - influx 
 into the presynaptic terminals than any influx mediated by nr2a receptors . ca - influx 
 via the nmdar is responsible for instantaneous control of spontaneous glutamate 
 release . with a deactivation time of around 300 milliseconds , repetitive 
 activation of nr1/nr2b receptors would readily result in temporal summation of 
 presynaptic ca - entry leading to the short - term facilitation at 
 even relatively low - frequency stimulations seen here and previously . 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 ( 3080 hz ) , theta ( 48 hz ) , and slow 
 waves ( 0.10.5 hz ) [ 6871 ] , 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 [ 7274 ] ) , and it is 
 possible that information encoding involved in these processes is reliant on an 
 increase in entorhinal - hippocampal delta / theta coherence . 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 . we recently showed that prenmdar function 
 declines in adulthood , but is markedly enhanced in age - matched , chronically 
 epileptic rats   and there is evidence for a similar increased function in 
 human temporal lobe epilepsy . 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 
 [ 7981 ] . we have also 
 shown that at least one anticonvulsant drug ( felbamate ) can block the prenmdar 
 . 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 , so both short- and long - term plasticity of glutamate 
 transmission could involve ca - 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 . 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 zn 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 . 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 
 . this does not fit well with our finding that combined application of low 
 concentrations of zn 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 [ 8486 ] . 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. , ) . 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 . we 
 can not 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 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 .
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 .