dedicated research by numerous scientific groups into the causes 
 and treatment of the human demyelinating disease multiple 
 sclerosis ( ms ) , the most common disabling neurological condition 
 of european , north american , and other temperate climates , has been 
 ongoing for many decades .
ms affects relatively young individuals , 
 with a female to male ratio of approximately 2 : 1 .
the disease is 
 considered to involve central nervous system ( cns ) 
 autoantigen - directed t lymphocytes acting in concert with a 
 genetically determined susceptibility and exposure to 
 environmental induction factors . 

progress has been made 
 to advance understanding of the disease process and offer 
 effective methods of control .
however , there remains a lack of 
 fundamental knowledge on the primary aetiology of ms and a paucity 
 of treatments to alleviate symptoms and ultimately improve quality 
 of life for the patient .
the development and refinement of the inducible animal disease 
 experimental autoimmune encephalomyelitis ( eae ) has provided a 
 reliable model for the study of ms offering pathological and 
 neurological features of striking similarity to the human 
 condition .
the principal characteristics in common include 
 immunoregulatory defects , neurological disabilities , blood - brain 
 barrier ( bbb ) damage with associated vasogenic oedema , 
 inflammatory cell invasion of the cns parenchyma and , in the 
 chronic models , demyelination and macroscopic plaque formation 
 . however , 
 the premise that eae is strictly a model for ms 
 must remain , not least because of the obvious species differences 
 and time - scale of disease appearance and progression , but also 
 because of factors such as the divergence in identity of the 
 causative agents and the unpredictable patterns of clinical 
 deficits experienced by patients .
animal counterparts of human disease , whether spontaneous or 
 inducible , have inherent limitations and eae is no exception . 

however , the model does provide an extensively validated and 
 useful in vivo system of immune cell - mediated demyelination 
 complete with quantifiable neurological deficits .
in particular , 
 the model provides the opportunity to evaluate potential new 
 therapies for ms treatment and explore novel approaches to drug 
 design , identify new targets , and add to the growing number of 
 drugs in clinical trials .
the search for compounds with the ability to modify the onset and 
 development of eae have invariably focused on immunomodulatory 
 agents . however , 
 over the last few years
, a group of 
 established compounds have emerged , with the ability to 
 dramatically improve the course of eae but without apparent 
 immunosuppressive activity .
the compounds interact with members of 
 the neuronal ionotropic glutamate receptor family comprising the 
 n - methyl - d - aspartate ( nmda ) , 
 -amino-3-hydroxy-5-isoxazolepropionic acid ( ampa ) , and 
 kainate receptors ( figures 1(a)1(c ) ) .
the 3 
 types of receptors are ligand - gated ion channels , named according 
 to their specific agonists , which control the most rapid synaptic 
 events in the nervous system through receptor - channel 
 complex - mediated events . our original studies of 1994 [ 4 , 
 5 ] were the first to 
 implicate the nmda receptor in the pathogenesis of eae . over the 
 intervening years
, there has been compelling evidence , reviewed 
 below , to confirm an important role for the nmda receptor in the disease .
additional 
 investigations have strongly indicated that ampa receptors play a 
 part in the development of eae and , of particular interest , more 
 recent unpublished studies have shown altered receptor expression 
 in cns tissues from ms patients ( t. smith , personal 
 communication ) .
the amino acid glutamate is the main agonist of 
 the receptors and has been implicated in the pathogenesis of 
 neuroinflammatory disease [ 6 , 
 7 ] .
hence , the discovery of 
 nmda / ampa receptor involvement in both eae and ms offers a 
 plausible association between the receptors , the amino acid , and 
 development of both diseases .
olney , in the late 1960s , 
                                was the first to recognise that 
 the ubiquitous neurotransmitter , glutamate , when present in 
 excess , has the potential to be excitotoxic .
glutamate formation 
 is regulated by the enzyme glutamate dehydrogenase which catalyses 
 the reaction of -oxoglutarate with ammonia .
the 
 agonist concentration can be abnormally increased by accelerating 
 the reversible formative reaction that is controlled by pyridine 
 nucleotide coenzyme activity .
glutamine synthetase controls the 
 incorporation of ammonia into glutamate to form glutamine and the 
 activity of the enzyme can be dramatically increased or decreased 
 in the presence of excess divalent cations , including magnesium 
 ( mg ) .
glutamate is stored in synaptic vesicles and 
 released by calcium ( ca)-dependent exocytosis . 

sodium - dependent , plasma membrane transporter proteins eaac1 
 ( eaat3 ) and eaat4 , present mainly in neurons , and glt-1 ( gaat2 ) and 
 glast ( gaat1 ) , expressed predominantly in glial cells , facilitate 
 cellular uptake of glutamate and accumulation in synaptic vesicles 
 .
several studies have demonstrated glutamate involvement in the 
 pathology of eae , and also ms , offering the clear potential for 
 aberrant ionotropic receptor activation . in particular , the 
 glutamate antagonist amantidine has been shown to reduce the 
 relapse rate in individuals with ms .
also , stover et al 
  have reported elevated glutamate levels in the 
 cerebrospinal fluid from ms patients .
interestingly , the elevation 
 was similar to concentrations recorded in myelopathy and , perhaps 
 more surprisingly , greater than noted during cerebral ischaemia . 
 however , and in contrast to the previous findings , klivenyi et al 
  found no differences between cerebrospinal fluid 
 glutamate concentrations in ms and control samples despite 
 elevated levels in both groups .
enhanced concentrations of the agonist may result from 
 malfunctioning of activated astrocytes normally efficient at 
 controlling excess glutamate through regulation of the 
 metabolising enzymes glutamate dehydrogenase and glutamine 
 synthetase , which become down - regulated during inflammatory 
 conditions such as eae [ 14 , 
 15 ] .
the amount of cns glutamate 
 may also be increased in eae by abnormal changes in neuronal and 
 glial glutamate transporter levels   which , under 
 pathological conditions , are either inoperative or acting reversibly 
 to raise extracellular concentrations of the agonist .
glutamate 
 leakage from the serum across the compromised bbb during eae plus 
 infiltrating inflammatory leukocytes and activated resident 
 microglia with the potential to synthesise and release glutamate 
 would provide a continuous , local supply of the agonist .
also , 
 microglia are known to generate reactive oxygen and nitrogen 
 species that impair glutamate uptake mechanisms .
the constant 
 availability of glutamate would induce upregulation of its 
 receptors and , ultimately , the synthesis of mediators responsible 
 for neuronal dysfunction [ 12 , 
 1619 ] . 

indeed , a recent study in eae found that prophylactic administration of riluzole , 
 an inhibitor of glutamate - dependent neurotransmission , reduced 
 neurological severity , inflammation , demyelination , and axonal 
 damage strongly suggesting a broad role for the enhanced presence 
 of glutamate in the pathology of the disease . in a novel approach to
account for the increase in cns glutamate 
 concentrations during neuroinflammation , rose et al 
 have suggested a mechanism that would operate through the actions 
 of two enzymes , cyclooxygenase-2 ( cox-2 ) and inducible 
 nitric oxide synthase ( inos ) , both of which have been 
 located in ms lesions .
cox-2-derived prostanoids , which 
 exist at high concentrations in eae and ms cns tissues 
 [ 2224 ] , 
 stimulate glutamate release from cns - derived 
 cells [ 25 , 26 ] . 
 additionally , nitric oxide ( no ) , from 
 inos , can increase cox-2 
 , plus reactive 
 oxygen species ( ros ) , to react with 
 no to produce 
 peroxynitrite ( onoo )   that inactivates the 
 glutamate transporters [ 30 , 31 ] . 

in addition , onoo 
 directly damages myelin , oligodendrocytes , and axons , 
 and therefore plays a predominant role in 
 the pathogenesis of eae .
the evidence is unequivocal as to the consequences of excitotoxic 
 glutamate levels in the cns of patients with 
 neuroinflammatory - based disease and that target tissues require 
 protection from the sustained biochemically - mediated attack . 

interestingly , in eae , work by schori et al   supports a 
 t - cell - dependent , self - protective immune mechanism that may , at 
 least in part , reduce the effects of enhanced glutamate levels .
however , the need for greater control of the 
 excitotoxic actions of ionotropic receptor agonists is apparent 
 and has not diminished .
indeed , over the past decade much effort 
 has been diverted to identifying compounds that can negate 
 glutamate - mediated neurotoxicity incurred as a consequence of 
 conditions such as stroke and head injury .
results to date have 
 been largely negative and evidence for a neuroprotective role of 
 glutamate antagonists in neurodegenerative diseases is lacking 
 . similarly , despite efforts to develop compounds that 
 act by altering the metabolism of glutamate , no such drugs have 
 been produced .
the rationale is now strong for assessing compounds 
 designed to limit the possible damaging effects of glutamate in 
 diseases such as ms and , in particular , to employ the animal 
 counterpart eae as the in vivo test system of choice .
the nmda receptor is most abundant in the cortex , basal ganglia , 
 and sensory pathways of the nervous system , and has also been 
 identified in a variety of nonneuronal and peripheral locations 
 . in particular ,
the receptor has been found on the 
 neurovasculature and mast cells derived from the cns 
 [ 3742 ] . 

the receptor consists of several subunits , comprising the ubiquitous nr1 
 subunit and a variety of combinations of nr2a to nr2d and the more recently identified nr3 
 subunit [ 39 , 43 , 
 44 ] .
each subunit has 4 membrane domains , an 
 extracellular amino terminal region and an intracellular carboxy 
 group tail .
the domains 1 , 3 , and 4 transverse the membrane and 
 domain 2 appears to form the reentrant loop which lines the ion 
 channel ( figure 2 ) .
the channel pore is normally 
 blocked by mg to prevent ion flux but , on appropriate 
 ligand stimulation , membrane depolarisation occurs and the 
 mg blockade is removed to cause a functional opening 
 of the receptor channel ( figure 1(a ) ) .
the nmda receptor is of particular interest to pharmacologists as 
 there are a number of ligand binding and modulatory sites that 
 offer potential therapeutic targets for control and points of 
 intervention ( figure 1(a ) ) .
functional nmda receptor 
 complexes are constructs of the nr1 and nr2/nr3 subunits 
 containing the glycine and glutamate recognition sites , 
 respectively [ 4547 ] . 
 agonists , including nmda and glutamate , bind to the glutamate recognition site , whereas 
 competitive antagonists such as selfotel may occupy a single 
 region , distinct from the agonist site , but coupled to provide a 
 competitive interaction .
interestingly , selfotel has been 
 effectively used in vivo to block nmda - induced bbb permeability increases .
glycine and d - serine act as coagonists , through the glycine site , 
 to prevent receptor desensitisation and are prerequisites for the 
 generation of enhanced inward flow of current at the receptor . 

histamine and the polyamines ( pas ) , including spermine and 
 spermidine , act as receptor modulators to both potentiate 
 and inhibit nmda - induced responses through distinct sites 
 [ 4951 ] . 

the receptor can also be modulated by sigma site 
 ligands at a position distinct from the channel - blocking site 

. a clearer understanding of sigma site function in 
 glutamate - mediated responses is required before agents , directed 
 at the target , can be designed to offer therapeutic efficacy .
the 
 current extent of nmda receptor modulatory sites is summarised in 
 figure 1(a ) .
nmda receptors have been extensively studied and show special 
 pharmacological properties that are thought to play a role in 
 pathophysiological mechanisms .
for example , the receptor is highly 
 permeable to ca and other cations , including sodium 
 ( na ) and potassium 
 ( k ) , and is readily 
 blocked by physiological concentrations of mg when the 
 cell is normally polarised . 

the ca 
 permeability of the receptor is controlled by an asparagine 
 residue in the nr1 subunit within the channel pore loop structure 
 of the second membrane domain .
the residue also 
 determines the voltage - dependent mg blockade of the 
 nmda receptor channel .
depolarisation of the receptor 
 leads to loss of mg from the channel pore and an 
 influx of ca with subsequent activation of enzyme 
 systems we , and others , have shown to be pertinent to the 
 inflammatory processes involved in eae , including no and pa 
 production [ 55 , 56 ] . 

indeed , bolton et al first showed 
 elevated no and pa levels in cns tissues from eae - diseased 
 rats prompting the suggestion of an important role for 
 the nmda receptor in the pathogenesis of the disease and , by 
 implication , in ms .
the open channel can be blocked by the uncompetitive nmda receptor 
 antagonist ( + ) mk-801 ( dizocilpine maleate ) 
 ( figures 1(a ) 
 and 3 ) , 
 thereby limiting the flow of ca 
 into the cell and curbing activation of enzyme systems 
 .
our 
 subsequent studies using ( + ) mk-801 confirmed a role for 
 the nmda receptor in eae through the prevention of bbb breakdown 
 and neurological deficits and strongly suggests the involvement of 
 glutamate in the disease . a recent investigation by 
 sharp et al   has described the use of ( + ) mk-801 to 
 confirm nmda receptor involvement in an in vitro model of 
 bbb damage , and our studies with the drug have indicated the existence of the 
 receptor on immortalised bend 3 brain endothelial cells . 

in addition , zhu and liu   used ( + ) mk-801 to attenuate 
 glutamate - induced expression of p - glycoprotein on cns - derived 
 microvessel endothelial cells and further verify the existence of 
 nmda receptors on neuroendothelium .
the precise mechanism of action for ( + ) mk-801 in eae is unclear . 
 in vitro studies by us have shown that the compound does not 
 interfere with mitogen - driven t cell proliferation or affect the 
 inflammatory response made by macrophages ( unpublished data ) . 

however , ongoing studies examining the downstream 
 ca - dependent events triggered as a result of nmda 
 receptor activation may offer some insight into the actions of the 
 drug in models of neuroinflammation .
preliminary 
 work has shown that treatment of bend 3 cells with ( + ) mk-801 
 prevents glutamate - induced release of onoo 
 .
treatment of eae - sensitised animals with 
 ( + ) mk-801 also reduces 
 the disease - associated increase in cns levels of the pa putrescine 
 ( figure 4 ) [ 56 , 
 63 ] .
pas , formed by the 
 rate - limiting ca - dependent enzyme ornithine 
 decarboxylase , act as cell membrane perturbators and 
 vasodisruptors in non - immune - mediated cns diseases [ 64 , 
 65 ] . 

pas and onoo , along with other ros , including 
 superoxide and hydroxyl radicals , closely influence neurovascular 
 changes that are typical during the development and progression of 
 eae and ms .
there is a requirement to clarify pa - mediated events at 
 neurovascular sites with the onset and development of the disease . 

one approach has been to examine the role of the pas in eae by 
 employing enzyme - specific drugs that interrupt the formation of 
 putrescine , spermine , and spermidine , plus compounds that 
 antagonise the pa site on the nmda receptor .
results indicate a 
 complex series of responses to treatment that are dependent upon 
 the compound , dose , and frequency of administration . 

interestingly , the importance of the ornithine decarboxylase - pa 
 pathway in other cns conditions , including stroke , epilepsy , 
 alzheimer 's disease , and schizophrenia , is being realised and will 
 undoubtedly lead to a determined effort to understand the 
 significance of the agents in disease pathogenesis .
studies by paul and bolton   together with 
 earlier experiments by wallstrom et al 
 , using the relatively uncompetitive aminoadamantane nmda 
 receptor antagonist memantine ( 1-amino-3 , 5-dimethyl - adamantane ) 
 ( figure 3 ) , confirmed that pharmacological modulation 
 of receptor function during eae results in disease suppression and 
 restoration of neurovascular function .
importantly , the work by 
 paul and bolton indicates , through the use of specific 
 dosing regimes , that nmda receptor involvement in eae is at , or 
 just prior to , symptom onset and bbb breakdown , rather than 
 earlier , during the induction phase of disease or later at the 
 height of neurological deficits .
furthermore , a significant effect 
 was noted on neuroinflammatory infiltrates which appears distinct 
 from ampa / kainite antagonist activity .
memantine , unlike 
 ( + ) mk-801 , has been reported to differentiate between transient 
 physiological activation and sustained pathological stimulation of 
 the nmda receptor with actions preferentially directed towards the 
 latter state .
the apparent discriminatory profile ascribed to the pharmacology 
 of memantine on abnormal nmda receptor activity makes the drug 
 particularly attractive for use during the onset of clinical 
 episodes in human cns diseases .
indeed , memantine has been 
 reported to provide symptomatic relief to ms patients . 

one current theory suggests that the 
 compound , like mg , occupies the receptor channel and 
 rapidly exits the pore under strong , physiological synaptic 
 depolarisation and in the presence of glutamate fluxes of 
 millimolar concentrations .
in contrast , and under 
 pathological conditions where sustained micromolar concentrations 
 of glutamate preside , memantine , unlike mg , will 
 maintain channel block during prolonged depolarisation .
consequently , total blockade of the receptor , by 
 compounds including ( + ) mk-801 , leads to numerous side effects . 

specific prevention of the pathological activation of nmda 
 receptors with drugs such as memantine reduces unwanted activity 
 and thereby improves clinical tolerance , offering a useful 
 feature in the treatment of neurodegenerative diseases including 
 ms . another approach towards improving specificity and reducing
the 
 unwanted side effects of drug therapy might be to target particular 
 modulatory sites on the nmda receptor .
in addition , an 
 alternative to blocking nmda receptor action completely would be 
 to suppress an exaggerated receptor response .
therefore , 
 targeting inhibitory modulatory sites , such as the pa or 
 neurosteroid binding positions or the poorly defined sigma site 
 ( figure 1(a ) ) , offers the potential to down - regulate 
 rather than completely inhibit nmda receptor - mediated events . 

alternatively , it may be that the subunit - specific modulatory 
 sites mediate features of neuroinflammatory pathology and thereby 
 become primary targets through which to achieve disease control . 

indeed , the nr2b subunit has been recognised as a particular 
 therapeutic target for several neurological conditions 
 and initial studies by wheeler et al have shown an increased 
 expression of the nr2a and nr2b subunits in cns tissues from 
 eae - diseased rats [ 72 , 
 73 ] .
postsynaptic ampa receptors are considered to mediate rapid 
 glutamergic neurotransmission with low ca 
 permeability .
the receptor consists of four subunits , glur1 to 
 glur4 , which are widely distributed throughout the cns 
 [ 74 , 75 ] 
 and each of which can be expressed in two variants 
 originally termed  flip  and  flop  
 ( figure 1(b ) ) . 

ampa receptors are invariably colocalised with nmda receptors 
 indicating a close functional relationship between the 2 
 ligand - gated cation channel - bearing receptors .
indeed , ampa 
 activation causes cellular depolarisation and nmda channel 
 opening with ca influx .
pharmacological studies have 
 provided strong evidence for ampa receptor involvement in several 
 cns conditions including stroke , traumatic brain injury , and 
 parkinson 's disease .
kainate receptors are closely related to ampa receptors and are 
 involved in both pre- and postsynaptic neurotransmission .
the 
 receptor class is comprised of 5 subunits falling into 2 families , 
 glur5 to glur7 and ka1 plus ka2 ( figure 1(c ) ) .
each 
 subunit family shares 70% sequence homology with its members , but 
 only 40% with nonfamily subunits .
weaker identities are shown 
 with ampa ( 3035% ) and nmda ( 1020% ) receptor subunits , 
 although some studies have suggested that glur5 is part of the 
 ampa family [ 7577 ] .
the ampa and kainate positioning of subunits in their respective 
 receptor complex is similar to the arrangement present in the nmda 
 receptor .
the amino terminal portion of each subunit is 
 extracellular ; there are 4 hydrophobic sections , 3 of which are 
 membrane spanning , plus a reentrant membrane loop that contributes 
 to the pore lining .
the cytoplasmic carboxy terminus , in common 
 with the nmda receptor , contains sites for phosphorylation , with a 
 minimum of 12 in the ampa subunits and a suggested involvement in 
 the regulation of channel function [ 78 , 
 79 ] .
fewer modulatory 
 sites have been identified for ampa and kainate receptors compared 
 to the nmda receptor 
 ( figures 1(b ) and 1(c ) ) . 

however , information on the endogenous mechanisms for regulating 
 non - nmda ionotropic receptor function is increasing and a similar 
 capacity for pharmacological modulation of subtype activity can be 
 anticipated .
there is scant information on the subtype selectivity of ampa 
 receptor antagonists ( figure 3 ) and the standard 
 competitive drugs nbqx 
 ( 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f]-quinoxaline-2,3-dione ) 
 and cnqx ( 6-cyano-7-nitroquinoxaline-2,3-dione ) are not selective 
 between non - nmda ionotropic receptors .
nbqx and cnqx 
 have been investigated , with some success , in models of global 
 ischaemia , cns trauma , and parkinson 's disease 
 [ 8183 ] 
 although drug effects mediated through kainate receptor 
 involvement are suspected and therefore can not be excluded 
 [ 8486 ] .
one cardinal feature of ms and the more chronic models of eae is 
 the demyelination of central nerve fibres .
restoration of normal 
 nerve function in ms is dependent , at least in part , upon 
 recruitment of myelin - forming oligodendrocytes to lesioned areas . 

limited remyelination is possible in acute lesions but virtually 
 nonexistent in chronic states due to lack of oligodendrocyte 
 viability and recruitment to damaged areas [ 87 , 
 88 ] .
the 
 oligodendrocyte has been reported to express , exclusively , ampa 
 and kainate receptors , thereby making the cell a target for attack 
 by excitotoxic glutamate in eae 
 [ 8991 ] . however , 
 investigations by wosik et al   indicate a lack of ampa 
 receptors on human oligodendrocytes and a resistance to 
 agonist - mediated toxicity .
furthermore , the work suggests that 
 ampa expression is limited to astrocytes . despite conjecture over 
 the cellular expression of receptor types in brain tissue
, the 
 administration of kainate to the optic nerve causes degenerative 
 toxic lesions , nerve damage in association with inflammation , and 
 demyelination , all of which are strongly suggestive of an 
 ms - related pathology .
interestingly , administration of 
 cnqx prevents kainate - induced lesions whereas the ampa receptor 
 antagonist gyki 53655 
 ( 1-[4-aminophenyl]-3-methylcarbamyl-7 , 
 8-methylenedioxy-3,4-dihydro-5h-2,3-benzodiazepine ) 
 had no significant effect indicating a kainate - specific action and 
 implicating receptor involvement in early ms pathology .
recent investigations in acute and chronic - relapsing eae have 
 demonstrated the effectiveness of nbqx together with mpqx 
 ( [ 1,2,3,4-tetrahydro-7-morpholinyl 
 1,2,3-dioxo-6-(trifluoromethyl)quinoxa lin-1-yl]methylphosphonate ) 
 and the noncompetitive antagonists gyki 52466 
 ( 1-(4-amino - phenyl ) 4-methyl-7,8-methylene 
 dioxy-5h-2,3-benzodiaze - pine ) and gyki 53773 ( ( -)1-(4-aminophenyl ) 
 4-methyl-7,8-methylene - dioxy 4,5-dihydro 3-methylcarbamoyl 
 2,3-benzodiazepine ) ( figure 3 ) in reducing the 
 neurological symptoms of the disease [ 19 , 
 94 , 95 ] . 

interestingly , earlier related work demonstrated that nbqx had 
 anti - oedematous effects at neurovascular sites via a proposed 
 action on glial cells .
eae studies using the competitive 
 antagonists to modify the course of disease can not exclude drug 
 effects on kainate receptor - mediated events .
in contrast , the 
 noncompetitive antagonists do differentiate between the two 
 receptors and therefore indicate a specific ampa involvement in 
 disease development .
more recently , a series of ampa receptor 
 antagonists , with structures based on 2,3-benzodiazepine , have 
 proved effective in reducing the symptoms and morphological 
 changes associated with eae [ 97 , 
 98 ] .
the studies with nbqx have highlighted the ability of competitive 
 receptor antagonists to reduce eae - mediated neuronal death and 
 oligodendrocyte loss despite the uncertainty of ampa or kainate 
 involvement [ 94 , 95 ] . 

however , the extent of oligodendrocyte 
 depletion may be dependent on additional endogenous factors . for 
 example
, testosterone has been shown to amplify both ampa- and 
 kainate - induced toxicity to oligodendrocytes in vitro 
 , suggesting the existence of a steroidal modulatory site 
 on non - nmda receptors .
the studies described by pitt et al   and groom et al 
  also considered the possibility that the competitive and 
 noncompetitive antagonists may operate in eae through an 
 immunosuppressive action .
results showed that competitive 
 antagonists , such as nbqx , did not affect t - cell proliferation 
 rates or reduce perivascular inflammatory cuffs .
however , 
 noncompetitive antagonists did suppress mitogen - induced t cell 
 proliferation , thus offering an alternative explanation for the 
 compounds abilities to modify eae and indicating ampa receptor 
 involvement in immune - mediated inflammation .
glutamate 
 excitotoxicity , together with neuroinflammatory factors in both 
 eae and ms , may be important codeterminants in oligodendrocyte 
 death .
for example , inflammatory mediators , such as interleukin 
 1 and tumour necrosis factor- ( tnf- ) 
 can promote in vitro oligodendrocyte apoptosis and changes 
 in the glutamate buffering system of astrocytes .
moreover , the 
 cytokine - induced effects can be blocked by nbqx and cnqx 
 [ 100 , 
 101 ] .
interestingly , research into the regulation of gene expression 
 during eae has identified a reduction in the important plasma 
 membrane caatpase2 , necessary for cation homeostasis 
 and expressed exclusively in the grey matter , which occurs coincidently with the development of neurological signs 
 .
the studies also found that application of 
 kainate to spinal cord slice cultures significantly lowered the 
 mrna levels of caatpase .
collectively , the results 
 implicate glutamate , particularly via kainate receptors , in the 
 suppression of neuronal plasma membrane 
 caatpase 2 and 
 abnormal ca levels . perhaps of greater significance
is 
 the observation that nbqx can suppress alterations in glutamate 
 transporter expression during eae .
the study found 
 protein and mrna levels of eaac1 to be dramatically increased , 
 while transporters glt-1 and glast were down - regulated together 
 with a concomitant reduction in the incidence of disease .
nbqx , 
 administered semiprophylactically from day 7 postinoculation , 
 suppressed the changes in the expression of transporters 
 suggesting the activation of non - nmda receptors .
undoubtedly , the studies indicate an important role for 
 the ampa receptor in eae and , possibly , in ms .
however , the 
 investigations can not exclude the possible contribution of kainate 
 receptors in the development of disease pathology .
the prospect of 
 ampa / kainate receptor involvement in experimental and human 
 neuroinflammatory conditions offers new targets to focus 
 treatments for the demyelinating diseases with an emphasis on the 
 preservation of oligodendrocyte function .
postsynaptic ampa receptors are considered to mediate rapid 
 glutamergic neurotransmission with low ca 
 permeability .
the receptor consists of four subunits , glur1 to 
 glur4 , which are widely distributed throughout the cns 
 [ 74 , 75 ] 
 and each of which can be expressed in two variants 
 originally termed  flip  and  flop  
 ( figure 1(b ) ) . 

ampa receptors are invariably colocalised with nmda receptors 
 indicating a close functional relationship between the 2 
 ligand - gated cation channel - bearing receptors .
indeed , ampa 
 activation causes cellular depolarisation and nmda channel 
 opening with ca influx .
pharmacological studies have 
 provided strong evidence for ampa receptor involvement in several 
 cns conditions including stroke , traumatic brain injury , and 
 parkinson 's disease .
kainate receptors are closely related to ampa receptors and are 
 involved in both pre- and postsynaptic neurotransmission .
the 
 receptor class is comprised of 5 subunits falling into 2 families , 
 glur5 to glur7 and ka1 plus ka2 ( figure 1(c ) ) .
each 
 subunit family shares 70% sequence homology with its members , but 
 only 40% with nonfamily subunits .
weaker identities are shown 
 with ampa ( 3035% ) and nmda ( 1020% ) receptor subunits , 
 although some studies have suggested that glur5 is part of the 
 ampa family [ 7577 ] .
the ampa and kainate positioning of subunits in their respective 
 receptor complex is similar to the arrangement present in the nmda 
 receptor .
the amino terminal portion of each subunit is 
 extracellular ; there are 4 hydrophobic sections , 3 of which are 
 membrane spanning , plus a reentrant membrane loop that contributes 
 to the pore lining .
the cytoplasmic carboxy terminus , in common 
 with the nmda receptor , contains sites for phosphorylation , with a 
 minimum of 12 in the ampa subunits and a suggested involvement in 
 the regulation of channel function [ 78 , 
 79 ] .
fewer modulatory 
 sites have been identified for ampa and kainate receptors compared 
 to the nmda receptor 
 ( figures 1(b ) and 1(c ) ) . 

however , information on the endogenous mechanisms for regulating 
 non - nmda ionotropic receptor function is increasing and a similar 
 capacity for pharmacological modulation of subtype activity can be 
 anticipated .
there is scant information on the subtype selectivity of ampa 
 receptor antagonists ( figure 3 ) and the standard 
 competitive drugs nbqx 
 ( 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f]-quinoxaline-2,3-dione ) 
 and cnqx ( 6-cyano-7-nitroquinoxaline-2,3-dione ) are not selective 
 between non - nmda ionotropic receptors .
nbqx and cnqx 
 have been investigated , with some success , in models of global 
 ischaemia , cns trauma , and parkinson 's disease 
 [ 8183 ] 
 although drug effects mediated through kainate receptor 
 involvement are suspected and therefore can not be excluded 
 [ 8486 ] .
one cardinal feature of ms and the more chronic models of eae is 
 the demyelination of central nerve fibres .
restoration of normal 
 nerve function in ms is dependent , at least in part , upon 
 recruitment of myelin - forming oligodendrocytes to lesioned areas . 

limited remyelination is possible in acute lesions but virtually 
 nonexistent in chronic states due to lack of oligodendrocyte 
 viability and recruitment to damaged areas [ 87 , 
 88 ] .
the 
 oligodendrocyte has been reported to express , exclusively , ampa 
 and kainate receptors , thereby making the cell a target for attack 
 by excitotoxic glutamate in eae 
 [ 8991 ] .
however , 
 investigations by wosik et al   indicate a lack of ampa 
 receptors on human oligodendrocytes and a resistance to 
 agonist - mediated toxicity .
furthermore , the work suggests that 
 ampa expression is limited to astrocytes . despite conjecture over 
 the cellular expression of receptor types in brain tissue , the 
 administration of kainate to the optic nerve causes degenerative 
 toxic lesions ,
nerve damage in association with inflammation , and 
 demyelination , all of which are strongly suggestive of an 
 ms - related pathology .
interestingly , administration of 
 cnqx prevents kainate - induced lesions whereas the ampa receptor 
 antagonist gyki 53655 
 ( 1-[4-aminophenyl]-3-methylcarbamyl-7 , 
 8-methylenedioxy-3,4-dihydro-5h-2,3-benzodiazepine ) 
 had no significant effect indicating a kainate - specific action and 
 implicating receptor involvement in early ms pathology .
recent investigations in acute and chronic - relapsing eae have 
 demonstrated the effectiveness of nbqx together with mpqx 
 ( [ 1,2,3,4-tetrahydro-7-morpholinyl 
 1,2,3-dioxo-6-(trifluoromethyl)quinoxa lin-1-yl]methylphosphonate ) 
 and the noncompetitive antagonists gyki 52466 
 ( 1-(4-amino - phenyl ) 4-methyl-7,8-methylene 
 dioxy-5h-2,3-benzodiaze - pine ) and gyki 53773 ( ( -)1-(4-aminophenyl ) 
 4-methyl-7,8-methylene - dioxy
4,5-dihydro 3-methylcarbamoyl 
 2,3-benzodiazepine ) ( figure 3 ) in reducing the 
 neurological symptoms of the disease [ 19 , 
 94 , 95 ] . 

interestingly , earlier related work demonstrated that nbqx had 
 anti - oedematous effects at neurovascular sites via a proposed 
 action on glial cells .
eae studies using the competitive 
 antagonists to modify the course of disease can not exclude drug 
 effects on kainate receptor - mediated events .
in contrast , the 
 noncompetitive antagonists do differentiate between the two 
 receptors and therefore indicate a specific ampa involvement in 
 disease development .
more recently , a series of ampa receptor 
 antagonists , with structures based on 2,3-benzodiazepine , have 
 proved effective in reducing the symptoms and morphological 
 changes associated with eae [ 97 , 
 98 ] .
the studies with nbqx have highlighted the ability of competitive 
 receptor antagonists to reduce eae - mediated neuronal death and 
 oligodendrocyte loss despite the uncertainty of ampa or kainate 
 involvement [ 94 , 95 ] . 

however , the extent of oligodendrocyte 
 depletion may be dependent on additional endogenous factors . for 
 example
, testosterone has been shown to amplify both ampa- and 
 kainate - induced toxicity to oligodendrocytes in vitro 
 , suggesting the existence of a steroidal modulatory site 
 on non - nmda receptors .
the studies described by pitt et al   and groom et al 
  also considered the possibility that the competitive and 
 noncompetitive antagonists may operate in eae through an 
 immunosuppressive action .
results showed that competitive 
 antagonists , such as nbqx , did not affect t - cell proliferation 
 rates or reduce perivascular inflammatory cuffs .
however , 
 noncompetitive antagonists did suppress mitogen - induced t cell 
 proliferation , thus offering an alternative explanation for the 
 compounds abilities to modify eae and indicating ampa receptor 
 involvement in immune - mediated inflammation .
glutamate 
 excitotoxicity , together with neuroinflammatory factors in both 
 eae and ms , may be important codeterminants in oligodendrocyte 
 death .
for example , inflammatory mediators , such as interleukin 
 1 and tumour necrosis factor- ( tnf- ) 
 can promote in vitro oligodendrocyte apoptosis and changes 
 in the glutamate buffering system of astrocytes .
moreover , the 
 cytokine - induced effects can be blocked by nbqx and cnqx 
 [ 100 , 
 101 ] .
interestingly , research into the regulation of gene expression 
 during eae has identified a reduction in the important plasma 
 membrane caatpase2 , necessary for cation homeostasis 
 and expressed exclusively in the grey matter , which occurs coincidently with the development of neurological signs 
 .
the studies also found that application of 
 kainate to spinal cord slice cultures significantly lowered the 
 mrna levels of caatpase . collectively , the results 
 implicate glutamate , particularly via kainate receptors , in the 
 suppression of neuronal plasma membrane 
 caatpase 2 and 
 abnormal ca levels . perhaps of greater significance
is 
 the observation that nbqx can suppress alterations in glutamate 
 transporter expression during eae .
the study found 
 protein and mrna levels of eaac1 to be dramatically increased , 
 while transporters glt-1 and glast were down - regulated together 
 with a concomitant reduction in the incidence of disease .
nbqx , 
 administered semiprophylactically from day 7 postinoculation , 
 suppressed the changes in the expression of transporters 
 suggesting the activation of non - nmda receptors .
undoubtedly , the studies indicate an important role for 
 the ampa receptor in eae and , possibly , in ms .
however , the 
 investigations can not exclude the possible contribution of kainate 
 receptors in the development of disease pathology .
the prospect of 
 ampa / kainate receptor involvement in experimental and human 
 neuroinflammatory conditions offers new targets to focus 
 treatments for the demyelinating diseases with an emphasis on the 
 preservation of oligodendrocyte function .
we have reviewed the evidence for ionotropic glutamate receptor 
 involvement in eae and speculated on a role for the 
 receptors in ms .
the finding of both nmda and non - nmda receptor 
 involvement in the pathology of eae is substantiated .
therefore , 
 it is our belief that therapeutic targeting of both receptor 
 groups in models of eae represents a viable proposition for the 
 development of new treatments for ms .
the observation that a variety of nmda , ampa , and kainate receptor 
 antagonists are beneficial in eae corroborates the considerable 
 involvement of glutamate in the pathology of the disease .
aberrant 
 glutamate transporter mechanisms in resident cells of the cns 
 together with altered ionotropic receptor or subunit receptor 
 expression during eae may collectively contribute to excess 
 glutamate levels in target tissues and the gross disturbance to 
 normal homeostasis and nerve function .
figure 5 summarises the involvement of glutamate in 
 eae and , by inference ms , highlights the ways through which 
 excitotoxic levels of the amino acid could be achieved .
direct 
 discharge from resident and infiltrating cells or indirect release 
 resulting from the actions of inflammatory mediators would serve 
 to raise cns glutamate concentrations . the consequences of damage 
 to oligodendrocytes , neurons , and the bbb , plus inflammatory 
 cytokine release from microglia , contribute considerably to the 
 pathology of eae and could account , at least in part , for some of 
 the major central disturbances observed in ms .
the control of glutamate release and metabolism may offer a viable 
 therapeutic approach to limiting the subsequent damage associated 
 with excitotoxicity . suppression of agonist - activated ionotropic 
 receptor function has proved effective in controlling eae , but 
 efficacy in ms remains largely uninvestigated .
regulation of 
 abnormal receptor function rather than total blockade of activity 
 may effectively reduce the results of enhanced cns glutamate 
 levels and allow homeostatic mechanisms to operate thereby 
 reducing unwanted side effects . a clear delineation between the receptor type targeted and the 
 ensuing benefits to limit the disease process appears to exist . 

axonal sparing and oligodendrocyte protection arises 
 from the use of non - nmda receptor competitive antagonists whereas 
 the restriction of bbb dysfunction and reduction of inflammation 
 can be ascribed to drug effects on the nmda receptor .
however , an 
 exclusive action for the compounds at their respective target 
 sites can not be guaranteed .
therefore , coadministration of 
 ionotropic receptor antagonists , with different specificities , 
 offers the real prospect of inhibiting several fundamental 
 parameters of experimental and human demyelinating disease . 

indeed , a recent study by kanwar et al   has indirectly 
 addressed our suggestion by treating eae with nbqx in conjunction 
 with a monoclonal antibody directed against mucosal addressin cell 
 adhesion molecule-1 and the n - terminal tripeptide of insulin - like 
 growth factor .
unremitting disease was ameliorated and 
 oligodendrocyte survival and remyelination were 
 increased . furthermore , cns 
 inflammation , apoptosis , and axonal damage were reduced .
finally , 
 there is a requirement for an increased selectivity of antagonists 
 towards specific receptor subtypes , either through targeting a 
 specific subunit or by targeting a modulatory site , 
 if the true therapeutic potential of ionotropic receptor inhibition is to 
 be realised . a concerted effort to search for drugs with possible efficacy in 
 ms that operate at nonimmunological sites or do not have exclusive , 
 immunosuppressive properties
however , compounds designed to 
 antagonise the agonist actions on nmda and ampa / kainate receptors 
 administered either alone or in combination with other therapies 
 may offer the real prospect of treatment for patients with ms and 
 related disorders of the cns .
modulation of ionotropic glutamate receptor function . ( a ) 
 nmda receptor , ( b ) ampa receptor , and ( c ) kainate receptor .
the 
 main endogenous modulatory sites for the glutamate ionotropic 
 receptors are shown and the sites of key exogenous pharmacological 
 agents are in italics .
( + ) stimulatory / potentiating action , 
 (  ) inhibitory action , ( ss ) subunit - specific action .
additional 
 modifying agents ( not shown ) , where the action has not been 
 specified through a binding site on the receptor , are ( a ) 
 no , ethanol , histamine ( via polyamine site ) ; ( b ) 
 arachidonic acid (  ) , no ; ( c ) ethanol (  ) , arachidonic 
 acid (  ) .
the diagram is intended as a summary overview and 
 provides an indication of modulation at these receptors .
the 
 discovery of new modulatory agents is ongoing , particularly for 
 the ampa and kainate receptors , where significantly less is 
 known compared to the nmda receptor .
abbreviations : h , 
 proton ; no , nitric oxide ; p , phosphorylation site ; 
 pcp , phencyclidine .
( a ) each subunit comprises 4 hydrophobic regions of 
 which 1 , 3 , and 4 are transmembrane domains , while region 2 forms a 
 reentrant loop at the intracellular surface .
( b ) receptor subunit 
 organization : the ion channels are formed from four subunits , 
 which orientate allowing the second membrane domain to form the 
 ion channel pore .
putrescine levels detected in the cns of normal and acute 
 eae rats 13 days postinoculation ( pi ) , with and without mk801 
 treatment .
mk801 was administered intraperitoneally for 6 days 
 from day 7 pi at a concentration of 0.3 mg / kg body weight in sterile 
 phosphate buffered saline .
putrescine levels at day 13 pi are 
 significantly increased in all tissues compared to normal 
 ( # p < .001 ; student t test )
. 
 mk801 significantly reduced elevated putrescine at day 13 pi in all cns areas 
 ( * * p < 0.01 , 
 * * * p < .001 ; 
 student t test )
. schematic diagram summarising the known and proposed role 
 of glutamate and associated relevant mediators in the development 
 of neuroinflammatory and neurodegenerative pathology during eae . 

abbreviations : eae , experimental autoimmune encephalomyelitis ; 
 no , nitric oxide ; onoo , 
 peroxynitrite ; ros , reactive oxygen species ; tnf- , tumour necrosis factor alpha .
 multiple sclerosis ( ms ) is a chronic demyelinating disease of the 
 human central nervous system ( cns ) . 
 the condition predominantly 
 affects young adults and is characterised by immunological and 
 inflammatory changes in the periphery and cns that contribute to 
 neurovascular disruption , haemopoietic cell invasion of target 
 tissues , and demyelination of nerve fibres which culminate in 
 neurological deficits that relapse and remit or are progressive . 
 
 the main features of ms can be reproduced in the inducible animal 
 counterpart , experimental autoimmune encephalomyelitis ( eae ) . 
 the 
 search for new ms treatments invariably employs eae to determine 
 drug activity and provide a rationale for exploring clinical 
 efficacy . 
 the preclinical development of compounds for ms has 
 generally followed a conventional , immunotherapeutic route . 
 
 however , over the past decade , a group of compounds that suppress 
 eae but have no apparent immunomodulatory activity have emerged . 
 
 these drugs interact with the n - methyl - d - aspartate ( nmda ) and 
 -amino-3-hydroxy-5-isoxazolepropionic acid ( ampa)/kainate 
 family of glutamate receptors reported to control neurovascular 
 permeability , inflammatory mediator synthesis , and resident glial 
 cell functions including cns myelination . 
 the review considers the 
 importance of the glutamate receptors in eae and ms pathogenesis . 
 
 the use of receptor antagonists to control eae is also discussed 
 together with the possibility of therapeutic application in 
 demyelinating disease . 
INTRODUCTION
GLUTAMATE IN EXPERIMENTAL AND HUMAN
NEUROINFLAMMATORY DISEASE
THE NMDA RECEPTOR AND ANTAGONISTS
THE CONTROL OF EAE THROUGH LIMITING NMDA RECEPTOR ACTIVATION
AMPA and kainate ionotropic glutamate receptors
AMPA/kainate receptors and antagonists in the
pathology and control of neuroinflammation
SUMMARY
Figures and Tables