principal neurons of the ca1 region receive input from the entorhinal cortex ( ec ) via two pathways : ( i ) the direct temperoammonic ( ta ) pathway , which consists of afferents from layer iii of ec and forms synapses with ca1 neurons in the stratum lacunosum moleculare ; and ( ii ) the indirect pathway , which originates from layer ii of ec and forms part of the trisynaptic pathway that terminates in schaffer collateral ( sc ) synapses on ca1 neurons in the stratum radiatum .
nmdars are heteromeric assemblies   of two essential glun1 subunits and two glun2 or glun3 subunits . there are four glun2 subunits ( a  d ) of which the glun2a and glun2b predominate in the forebrain .
nmdars are generally thought to be minimally active during basal synaptic transmission , but they have a key role to play during the induction of synaptic plasticity .
although classically regarded as relatively stationary at the synapse , nmda receptors undergo lateral diffusion   and various forms of plasticity [ 5 - 8 ] .
furthermore , the subunit composition of nmda receptors can vary with development [ 9 , 10 ] and following different forms of synaptic activity [ 11 , 12 ] .
recently we have shown that ltd of nmda receptor - mediated synaptic transmission can be induced by theta frequency stimulation at sc but not ta synapses and involves activation of group i mglurs ( termed mglur - ltd ; ) .
the reason for the lack of activity - dependent mglur - ltd at ta - ca1 synapses is not clear but could be due to the afferent stimulation patterns in ta - ca1 not being ideal to activate group i mglurs .
therefore to overcome this issue we have now used pharmacological activation of group i mglurs to investigate whether ltd can be induced at ta - ca1 hippocampal synapses and furthermore to probe the role of glun2b subunits in dhpg - ltdnmda . here
we demonstrate that pharmacological activation of group i mglurs induces synaptic plasticity of nmda receptors at the sc pathway   but fails to induce ltd in the ta - ca1 pathway .
in addition , the nr2b - selective antagonist ro25 - 6981 was used to show that the subunit composition of nmdars remains unchanged following group i mglur - induced ltd .
p14 male wistar rats were killed by cervical dislocation in accordance with united kingdom animal ( scientific procedures ) legislation .
brains were removed and placed in ice - cold acsf consisting of the following ( in mm ) : nacl 124 , kcl 3 , nahco3 26 , nah2po4 1.25 , cacl2 2 , mgso4 1,d - glucose 10 ( bubbled with 95% o2/5% co2 ) .
standard techniques were used to record excitatory postsynaptic potentials ( epsc ) in response to stimulation ( 100 s , 310 v ) of the sc or ta pathways . in those experiments
in which both sc - ca1 and ta - ca1 inputs were studied , alternate stimulation of both pathways was conducted in each slice .
electrodes ( 4 7 m ) were filled with ( in mm ) : csmeso4 , 130 , nacl 8 , mg - atp 4 , na - gtp 0.3 , egta 0.5 , hepes 10 , qx-314 5 , ph adjusted to 7.27.3 using csoh and osmolarity to 275290 mosm with sucrose .
picrotoxin ( 50 m ) and nbqx ( 5 m ) were applied to isolate epscnmda .
( rs)-dhpg ( 100 m ; tocris bioscience ) was used to activate group i mglurs and ro25 - 6981 (; 5 m ; tocris bioscience ) as an antagonist of glunb receptors .
changes in synaptic strength were expressed relative to the normalized baseline ( mean  sem ) and significance tested using student s t - tests 30 min after ltd induction or 50 min following application of ro25 - 6981 . the group ii mglur agonist dcg - iv was applied at the end of experiments to ensure selective stimulation of ta and sc inputs .
after a baseline period of 10 minutes , the group i mglur agonist ( rs)-dhpg ( 100 m ; 10 minutes ) was applied .
an initial depression of nmda receptor - mediated epscs ( epscnmda ) was observed in both pathways ( at the end of dhpg application responses were : sc 53.1  3.3% ; ta 33.5  4.4% below baseline ; p < 0.05 compared to baseline ; n = 18 ; fig .
thirty minutes following washout of dhpg , ltd of epscnmda ( ltdnmda ) was observed at the sc pathway ( 34.9  2.7% depression ; p=0.0012 ; n=18 ) , which was consistent with published findings [ 14 , 18 , 19 ] . however , epscnmda evoked by ta pathway stimulation recovered to baseline levels following dhpg washout ( 15.2  4.2% below baseline ; p = 0.764 ; n = 18 ) .
after a baseline period of 10 minutes , the group i mglur agonist ( rs)-dhpg ( 100 m ; 10 minutes ) was applied .
an initial depression of nmda receptor - mediated epscs ( epscnmda ) was observed in both pathways ( at the end of dhpg application responses were : sc 53.1  3.3% ; ta 33.5  4.4% below baseline ; p < 0.05 compared to baseline ; n = 18 ; fig .
thirty minutes following washout of dhpg , ltd of epscnmda ( ltdnmda ) was observed at the sc pathway ( 34.9  2.7% depression ; p=0.0012 ; n=18 ) , which was consistent with published findings [ 14 , 18 , 19 ] . however , epscnmda evoked by ta pathway stimulation recovered to baseline levels following dhpg washout ( 15.2  4.2% below baseline ; p = 0.764 ; n = 18 ) .
the difference in ltdnmda between sc - ca1 and ta - ca1 might be due to differences in nmdar subunits at the different synapses .
different nmda receptor subunits are associated with different epsc characteristics and the epsc decay time constant (  ) can therefore be used as an indication of receptor subunit population at the synapse .
we have previously shown that the decay constant of epscnmda in ta and sc synapses is similar , indicating a similar population of nmda receptor subunits at the two synapses . in order to clarify this further ,
ro25 - 6981 ( ro ; a potent and selective glun2b receptor antagonist ) was applied ( 1 m ) and a depression of epscnmda was observed in both pathways ( fig .
2 ) . after a period of 1 hour , epscnmda at the sc and the ta pathways
had reduced by similar amounts ( sc : 55.8  7.0% ; ta : 43.3  5.2% ; p = 0.18 , n = 6 ) , confirming our previous work suggesting that a similar population of nmda receptor subunits are expressed at these two synaptic populations .
the above result suggests that it is unlikely that differences in nmdar subunit composition within the ta - ca1 and sc - ca1 synapses explain the difference in ltdnmda between the two inputs .
however , the role of nmda receptor subunits in the induction of ltd was not clear . therefore , to examine directly whether the induction of ltd in the sc pathway was dependent on glun2b activation , dhpg was applied in the presence of ro ( after 1 hour ro application ) .
dhpg again caused a depression of the sc pathway ( 30.5  9.6% ; fig .
3 ) that was not significantly different from dhpg - ltdnmda induced  under control conditions ( p = 0.617 ) .
this suggests that dhpg - ltdnmda in the sc pathway is independent of glun2b subunit activation .
ro25 - 6981 causes a similar depression   of both pathways following dhpg application to examine whether there was a change in nmdar subunit composition following ltd at the sc synapse , we compared the effect of ro before and following dhpg - ltd ; a change in efficacy of glun2b - selective antagonists is often taken as an indication of a change in subunit composition of nmdars ( i.e. an increase or decrease in the complement of glun2b receptors ; e.g. ) .
thus , thirty minutes following dhpg application ( 100 m ; 10 minutes ) when ltd was stably expressed , ro25 - 6981 ( 1 m ; 1 hour ) was bath applied .
application of dhpg caused ltdnmda ( 34.0  4.3% depression ) . following ro25 - 6981 application , a further depression of epscnmda was observed ( 63.5  4.2% of pre - dhpg level ; fig .
4 ) that was not significantly different to the depression caused by ro25 - 6981 under control conditions ( p = 0.589 ) , indicating that a change in glun2b subunit composition does not occur as a result of dhpg - ltdnmda .
recently we have demonstrated a form of ltdnmda that can be induced by synaptic stimulation at sc but not ta synapses in the hippocampus , using a short theta frequency stimulation ( tfs ; ) .
this form of ltd involves activation of group i mglurs and produces metaplasticity such that the induction of subsequent ltp at the sc input is inhibited . in the current study
we have utilised pharmacological activation of group i mglurs using the agonist dhpg to elucidate whether the lack of mglur - ltd of epscnmda at the ta input to ca1 in p14 animals is due to insufficient activation of mglurs by tfs .
we now show that even with agonist activation of group i mglurs , ltd of epscnmda is not seen at the ta input . instead , despite an initial transient depression , responses recover to baseline levels upon washout of the agonist . a previous study , using 4 - 6 week old animals , has shown that ltd can be induced by dhpg at ta - ca1 input ( although of smaller magnitude than at sc synapses ; ) .
this raises the possibility that either developmental changes may impact on the ability to induce mglur - ltd of epscnmda at ta - ca1 synapses or that there is a difference in the ability to induce ltd of epscnmda versus ltd of epscampa at ta - ca1 synapses .
however , it is not possible at this stage to be able to make conclusive statements as to the impact of development on mglur - ltd of epscnmda or differences between ltd of epscnmda versus ltd of epscampa at ta - ca1 synapses .
plasticity of epscnmda may involve a subunit switch in nmdar subunits [ 11 , 21 ] .
as such , the ability to induce such plasticity may be dependent on the subunit composition of nmdars in the basal state .
use of glun2b subunit - selective antagonists , such as ifenprodil and ro25 - 6981 , readily allows comparison of subunit composition [ 11 , 21 ] .
we found that ro25 - 6981 produced the same the magnitude of depression in sc and ta inputs .
similar to our previous work where we found no difference in the decay time constant of epscnmda at the two inputs to ca1 , this suggests that the inability to induce ltdnmda at ta - ca1 synapses is not due to differences in glun2b nmda subunit composition .
the sensitivity of epscnmda to ro25 - 6981 in the sc input was not altered following induction of ltdnmda by dhpg .
thus , a change in relative glun2a / glun2b subunit expression , which is seen in some forms of plasticity , does not underlie ltd .
mglur - ltd of epscampa has been extensively investigated although there is no consensus on the precise mechanism of ltd and multiple signalling cascades have been implicated .
ltdnmda induced by tfs is expected to be postsynaptically expressed , as this form of ltd is specific for epscnmda vs epscampa ; postsynaptic expression of pharmacologically - induced ltdnmda has also been demonstrated [ 14 , 18 ] .
however , it affects subsequent induction of long - term potentiation ( ltp ; ) and is therefore a form of metaplasticity that will regulate the acquisition of hippocampal - dependent spatial information .
future work will aim to elucidate the precise signalling and expression mechanisms underlying this ltd as well as its role in learning and memory .
 nmda receptors are composed of multiple subunits and are crucial in the induction of synaptic plasticity and learning and memory . in this study , 
 application of the group i mglu receptor agonist , dhpg , caused ltd of nmda - epscs ( dhpg - ltdnmda ) of the schaffer collateral , but not of nmda - epscs of the temperoammonic pathway onto ca1 neurons of the hippocampus . 
 dhpg - ltdnmda did not alter the sensitivity of nmda - epsc to the glun2b - antagonist , ro25 - 6981 , indicating that the postsynaptic nmda receptor subunit composition remained unchanged following dhpg - ltdnmda . 
 furthermore , blockade of glun2b receptors did not affect the induction of dhpg - ltdnmda . 
 these results demonstrate a difference in the plasticity of nmda receptors between two synapses onto the same ca1 neuron , but indicate that the subunit composition of nmda receptors does not account for this difference . 
Introduction
Materials and Methods
Results
DHPG Causes LTD of NMDA Receptor-Mediated EPSCs in the SC but not the TA Pathway
The Population of NMDA Receptor Subunits is Similar Between Pathways
DHPG-LTDNMDA of the SC Pathway is Unaffected by RO25-6981
Discussion
CONFLICT OF INTEREST