cultured cortical neurons were prepared by standard techniques ( 4 , 9 ) and switched to serum - free media at 24 h [ neurobasal with b27 supplement ( gibco )]. the as odn corresponded to nucleotides 435 - 449 of mouse psd - 95 / sap90 mrna ( genebank acc . no . d50621 ). filter - sterilized phosphodiester as se , and ms odns ( 5 μm ) were added in culture medium during feedings at 4 , 6 , 8 and 10 days after plating . cultures were used for all experiments ( fig1 - 4 ) on day 12 . odn sequences exhibited no similarity to any other known mammalian genes ( blast search ( 10 )). immunoblotting was done as described in ref . “ 26 ”. tissue was harvested and pooled from 2 cultures / lane . the blotted proteins were probed using a monoclonal anti - psd - 95 mouse igg1 ( transduction labs , 1 : 250 dilution ), polyclonal anti psd - 93 ( 1 : 1000 dilution ) and anti sap - 102 ( 1 : 2000 dilution ) rabbit serum antibodies ( synaptic systems gmbh ), a monoclonal anti nr1 mouse igg2a ( pharmingen canada , 1 : 1000 dilution ) or a monoclonal anti nnos ( nos type i ) mouse igg2a ( transduction labs , 1 : 2500 dilution ). secondary antibodies were sheep anti - mouse , or donkey anti - rabbit ig conjugated to horseradish peroxidase ( amersham ). immunoblots for psd - 95 were obtained for all experiments ( fig1 - 4 ) from sister cultures , and all gels quantified using an imaging densitometer ( bio - rad gs - 670 ). cgmp determinations were performed 10 min after challenging the cultures with nmda , kainate , or high - k ( fig4 c - e ) with the biotrak cgmp enzymeimmunoassay system according to the kit manufacturer &# 39 ; s instructions ( amersham ). staining for nadph diaphorase ( fig4 b ) was done as described in ref . 12 . electrophysiology . whole cell patch - clamp recordings in the cultured neurons were performed and analyzed as described in ref . 13 . during each experiment a voltage step of − 10 mv was applied from holding potential and the cell capacitance was calculated by integrating the capacitative transient . the extracellular solution contained ( in mm ): 140 nacl , 5 . 4 kcl , 1 . 3 cacl 2 , 25 hepes , 33 glucose , 0 . 01 glycine , and 0 . 001 tetrodotoxin ( ph = 7 . 3 - 7 . 4 , 320 - 335 mosm ). a multi - barrel perfusion system was employed to rapidly exchange nmda containing solutions . the pipette solution contained ( in mm ): 140 csf , 35 csoh , 10 hepes , 11 egta , 2 tetraethylammonium chloride ( tea ), 1 cacl 2 , 4 mgatp , ph 7 . 3 at 300 mosm . lucifer yellow ( ly ; 0 . 5 % w / v ) was included in the pipette for experiments in fig3 d . excitotoxicity and ca 2 + accumulation measurements were performed identically to the methods described and validated in refs . 4 and 14 . we used measurements of propidium iodide fluorescence as an index of cell death , and of radiolabelled 45 ca 2 + accumulation for ca 2 + load determinations in sister cultures on the same day . experimental solutions were as previously described ( 4 ). ca 2 + influx was pharmacologically channeled through distinct pathways as follows : to nmdars by applying nmda (× 60 min ) in the presence of both cnqx ( research biochemicals inc ) and nimodipine ( miles pharmaceuticals ), to non - nmdars by applying kainic acid (× 60 min or 24 h ) in the presence of both mk - 801 ( rbi ) and nimodipine , and to vsccs using 50 mm k + solution (× 60 min ) containing 10 mm ca 2 + and s (−)- bay k 8644 , an l - type channel agonist ( 300 - 500 nm ; rbi ), mk - 801 and cnqx . antagonist concentrations were ( in μm ): mk - 801 10 , cnqx 10 , nimodipine 2 . all three antagonists were added after the 60 mm agonist applications for the remainder of all experiments ( 24 h ). a validation of this approach in isolating ca 2 + influx to the desired pathway in our cortical cultures has been published ( 4 ). whole cell patch - clamp recordings in the cultured neurons were performed and analyzed as described in z . xiong , w . lu , j . f . macdonald , proc nail acad sci usa 94 , 7012 ( 1997 ). during each experiment a voltage step of − 10 mv was applied from holding potential and the cell capacitance was calculated by integrating the capacitative transient . the extracellular solution contained ( in mm ): 140 nacl , 5 . 4 kcl , 1 . 3 cacl 2 , 25 hepes , 33 glucose , 0 . 01 glycine , and 0 . 001 tetrodotoxin ( ph = 7 . 3 - 7 . 4 , 320 - 335 mosm ). a multi - barrel perfusion system was employed to rapidly exchange nmda containing solutions . the pipette solution contained ( in mm ): 140 csf , 35 csoh , 10 hepes , 11 egta , 2 tetraethylammonium chloride ( tea ), 1 cacl 2 , 4 mgatp , ph 7 . 3 at 300 mosm . lucifer yellow ( ly ; 0 . 5 % w / v ) was included in the pipette for experiments in fig3 d . data analysis : data in all figures were analyzed by anova , with a post - hoc student &# 39 ; s t - test using the bonferroni correction for multiple comparisons . all means are presented with their standard errors . fig1 , shows increased resilience of psd - 95 deficient neurons to nmda toxicity in spite of ca 2 + loading . a . immunoblot showing representative effects of sham ( sh ) washes , and psd - 95 as , se and ms odns , on psd - 95 expression . pc : positive control tissue from purified rat brain cell membranes . asterisk : non - specific band produced by the secondary antibody , useful to control for protein loading and blot exposure times . b . densitometric analysis of psd - 95 expression pooled from n experiments . asterisk : different from other groups , one - way anova , f = 102 , p & lt ; 0 . 0001 . odns were used at 5 μm except where indicated ( as 2 μm ). c . representative phase contrast and propidium iodide fluorescence images of psd - 95 deficient ( as ) and control ( se ) cultures 24 h after a 60 min challenge with 30 μm nmda . scale bar : 100 μm . d . decreased nmda toxicity at 24 h in psd - 95 deficient neurons following selective nmdar activation × 60 min ( n = 16 cultures / bar pooled from n = 4 separate experiments ). asterisk : differences from se , ms and sh ( bonferroni t - test , p & lt ; 0 . 005 ). death is expressed as the fraction of dead cells produced by 100 μm nmda in sham - odn - treated controls ( validated in 4 , 14 ). e . no effect of psd - 95 deficiency on nmdar - mediated ca 2 + loading ( n = 12 / bar , n = 3 ; reported as the fraction of 45 ca 2 + accumulation achievable over 60 min in the sham controls by 100 μm nmda , which maximally loads the cells with calcium ( 4 ). fig2 , shows that psd - 95 deficiency does not affect toxicity and ca 2 + loading produced by activating non - nmdars and ca 2 + channels . cultures were treated with sh washes or as or se odns as in fig1 . a . selective activation of ampa / kainate receptors with kainate in mk - 801 ( 10 μm ) and nimodipine ( nim ; 2 μm ) produces toxicity over 24 h ( a1 ) irrespective of psd - 95 deficiency , with minimal 45 ca 2 + loading ( a2 ). b . selective activation of vsccs produces little toxicity ( b1 ), but significant 45 ca 2 + loading ( b2 ) that is also insensitive to psd - 95 deficiency . n = 4 cultures / bar in all experiments . fig3 , shows that there is no effect of perturbing psd - 95 on receptor function . a . immunoblots of psd - 950dn - treated cultures probed for psd - 95 , nr1 , psd - 93 , and sap - 102 using specific antibodies . pc : positive control tissue from purified rat brain cell membranes . b . nmda dose - response curves and representative nmda currents ( inset ) obtained with 3 - 300 μm nmda . c . nmda current density measurements elicited with 300 μm nmda ( as : n = 18 ; se : n = 19 ; sh : n = 17 ; one - way anova f = 1 . 10 , p = 0 . 34 ), and analysis of nmda current desensitization . i ss = steady - state current ; i peak = peak current . as : n = 15 ; se : n = 16 ; sh : n = 16 ( anova f = 0 . 14 , p = 0 . 87 ). time constants for current decay were as : 1310 ± 158 ms ; se , 1530 ± 185 ms ; sh : 1190 ± 124 ms ( anova , f = 1 . 22 , p = 0 . 31 ). d . currents elicited with 300 μm nmda in neurons dialyzed with ly ( insert ) and 1 mm tsxv or control peptide . fig4 , shows the effect of coupling of nmdar activation to nitric oxide signaling by psd - 95 . a . l - name protects against nmda toxicity ( n = 4 , n = 2 ). asterisk : difference from 0 μm l - name ( bonferroni t - test , p & lt ; 0 . 05 ). b . no effect of sh and of psd - 95 as and ms odns on nnos expression in cultures ( immunoblot ) and on nadph diaphorase staining in psd - 95 as and se - treated neurons . pc : positive control tissue from purified rat brain cell membranes . c . effect of isolated nmdar activation on cgmp formation ( n = 12 cultures / bar pooled from n = 3 separate experiments ) d , e . effects of vscc activation ( n = 8 / bar , n = 2 ), and ampa / kainate receptor activation ( n = 4 / bar , n = 1 ) on cgmp formation . data in c - e are expressed as the fraction of cgmp produced in se - treated cultures by 100 μm nmda . asterisk : differences from both sh and se controls ( bonferroni t - test , p & lt ; 0 . 0001 ). f . sodium nitroprusside toxicity is similar in psd - 95 as , se and sh treated cultures . psd - 95 expression was suppressed in cultured cortical neurons to & lt ; 10 % of control levels , using a 15 - mer phosphodiester antisense ( as ) oligodeoxynucleotide ( odn ) ( fig1 a , b ) sham ( sh ) washes , sense ( se ) and missense ( ms ) odns ( 9 ) had no effect . the odns had no effect on neuronal survivability and morphology as gauged by viability assays , herein below , and phase - contrast microscopy ( not shown ). to examine the impact of psd - 95 on nmdar - triggered excitotoxicity , odn - treated cultures were exposed to nmda ( 10 - 100 μm ) for 60 min , washed , and either used for 45 ca 2 + accumulation measurements , or observed for a further 23 h . ca 2 + influx was isolated to nmdars by adding antagonists of non - nmdars and ca 2 + channels ( 4 ). nmda toxicity was significantly reduced in neurons deficient in psd - 95 across a range of insult severities ( fig1 c , d ; ec 50 : as : 43 . 2 ± 4 . 3 ; se : 26 . 3 ± 3 . 4 , bonferroni t - test , p & lt ; 0 . 005 ). concomitantly however , psd - 95 deficiency had no effect on ca 2 + loading into identically treated sister cultures ( fig1 e ). therefore , psd - 95 deficiency induces resilience to nmda toxicity despite maintained ca 2 + loading . i next examined whether the increased resilience to ca 2 + loading in psd - 95 deficient neurons was specific to nmdars . non - nmdar toxicity was produced using kainic acid ( 30 - 300 μm ), a non - desensitizing ampa / kainate receptor agonist ( 15 ), in the presence of nmdar and ca 2 + channel antagonists ( 4 ). kainate toxicity was unaffected in psd - 95 deficient in neurons challenged for either 60 min ( not shown ) or 24 h ( fig2 a 1 ). non - nmdar toxicity occurred without significant 45 ca 2 + loading ( fig2 a 2 ), as & gt ; 92 % of neurons in these cultures express ca 2 + - impermeable ampa receptors ( 4 ). however , ca 2 + loading through vsccs , which is non - toxic ( 4 ) ( fig2 b 1 ), was also unaffected by psd - 95 deficiency ( fig2 b 2 ). thus , suppressing psd - 95 expression affects neither toxicity nor ca 2 + fluxes triggered through pathways other than nmdars . immunoblot analysis ( 11 ) of psd - 95 deficient cultures revealed no alterations in the expression of the essential nmdar subunit nr1 , nor of two other nmdar - associated maguks , psd - 93 and sap - 102 ( fig3 a ). this indicated that altered expression of nmdars and their associated proteins was unlikely to explain reduced nmda toxicity in psd - 95 deficiency ( fig1 c , d ). therefore , i examined the possibility that psd - 95 modulates nmdar function . nmda currents were recorded using the whole - cell patch technique ( 16 ) ( fig3 b ). psd - 95 deficiency had no effect on passive membrane properties , including input resistance and membrane capacitance [ capacitance : as 55 . 0 ± 2 . 6 pf ( n = 18 ); se 52 . 7 ± 3 . 2 pf ( n = 19 ); sh 48 . 1 ± 3 . 4 pf ( n = 17 ; anova , f = 1 . 29 , p = 0 . 28 )]. whole - cell currents elicited with 3 - 300 μm nmda were also unaffected . peak currents were as : 2340 ± 255 pa ( n = 18 ); se : 2630 ± 276 ( n = 19 ); sh : 2370 ± 223 ( n = 17 ) ( fig3 b , inset ; one - way anova , f = 0 . 43 , p = 0 . 65 ). nmda dose - response relationships also remained unchanged ( fig3 b ; ec 50 as : 16 . 1 ± 0 . 8 μm ( n = 7 ); se : 15 . 5 ± 2 . 1 ( n = 6 ); sh : 15 . 9 ± 2 . 9 ; one - way anova , f = 0 . 02 , p = 0 . 98 ), as were nmda current density and desensitization ( fig3 c ). to further examine the effect of psd - 95 binding on nmdar function , a 9 aa peptide , klssiesdv ( seq id no : 1 ) corresponding to the c - terminal domain of the nr2b subunit characterized by the tsxv motif ( 6 ) was injected into the neurons . at 0 . 5 mm , this peptide competitively inhibited the binding of psd - 95 to gst - nr2b fusion proteins ( 6 ), and was therefore predicted to uncouple nmdars from psd - 95 . intracellular dialysis of 1 mm tsxv or control peptide , cskdtmeksesl ( seq id no : 3 ) ( 6 ) was achieved through patch pipettes ( 3 - 5 mω ) also containing the fluorescent tracer lucifer yellow ( ly ). this had no effect on nmda currents over 30 min despite extensive dialysis of ly into the cell soma and dendrites ( fig3 d ). peak current amplitudes were tsxv : 2660 ± 257 pa ( n = 9 ), control : 2540 ± 281 pa ( n = 10 ; t ( 17 ) = 0 . 31 , p = 0 . 76 ). the data is consistent with that obtained from recently generated mutant mice expressing a truncated 40k psd - 95 protein that exhibited enhanced ltp and impaired learning ( 17 ). hippocampal ca1 neurons in psd - 95 mutants exhibited no changes in nmdar subunit expression and stoichiometry , cell density , dendritic cytoarchitecture , synaptic morphology , or nmdar localization using nr1 immunogold labeling of asymmetric synapses . nmda currents , including synaptic currents , were also unchanged ( 16 ). i also found no effects of psd - 95 deficiency on nmdar expression , on other nmdar associated maguks , nor on nmda - evoked currents . in addition , nmdar function gauged by measuring nmda - evoked 45 ca 2 + - accumulation was unaffected . thus , the neuroprotective consequences of psd - 95 deficiency must be due to events downstream from nmdar activation , rather than to altered nmdar function . the second pdz domain of psd - 95 binds to the c - terminus of nr2 subunits and to other intracellular proteins ( 8 ). among these is nnos ( 18 ), an enzyme that catalyzes the production of nitric oxide ( no ), a short - lived signaling molecule that also mediates ca 2 + - dependent nmda toxicity in cortical neurons ( 12 ). although never demonstrated experimentally , the nmdar / psd - 95 / nnos complex was postulated to account for the preferential production of no by nmdars over other pathways ( 8 ). to determine whether no signaling plays a role in nmda toxicity in the present cultures , we treated the cells with n g - nitro - l - arginine methyl ester ( l - name ), a nos inhibitor ( 12 ). l - name protected the neurons against nmda toxicity ( fig4 a ), indicating the possibility that suppressing psd - 95 might perturb this toxic signaling pathway . the effect of suppressing psd - 95 expression on no signaling and toxicity was examined using cgmp formation as a surrogate measure of no production by ca 2 + - activated nnos ( 20 , 21 ). psd - 95 deficiency had no impact on nnos expression ( fig4 b ), nor on the morphology ( fig4 b ) or counts of nadph diaphorase - staining ( 12 ) neurons ( sh : 361 ± 60 , se : 354 ± 54 , as : 332 ± 42 staining neurons / 10 mm coverslip , 3 coverslips / group ). however , in neurons lacking psd - 95 challenged with nmda under conditions that isolated ca 2 + influx to nmdars ( 4 ), cgmp production was markedly attenuated (& gt ; 60 %; fig4 c , one - way anova , p & lt ; 0 . 0001 ). like inhibited toxicity ( fig1 , 2 ), inhibited cgmp formation in neurons lacking psd - 95 was only observed in response to nmda . it was unaffected in neurons loaded with ca 2 + through vsccs ( fig4 d ), even under high neuronal ca 2 + loads matching those attained by activating nmdars ( compare fig1 e and 2 b 2 ) ( 4 ). nnos function therefore , was unaffected by psd - 95 deficiency . ampa / kainate receptor activation failed to load the cells with ca 2 + ( fig2 a 2 ), and thus failed to increase cgmp levels ( fig4 e ). our findings indicate that suppressing psd - 95 selectively reduces no production efficiency by nmdar - mediated ca 2 + influx , but preserves no production by ca 2 + influx through other pathways . bypassing nnos activation with no donors restored toxicity in neurons lacking psd - 95 . the no donors sodium nitroprosside ( 12 ) ( fig4 f ; ec 50 300 μm ) and s - nitrosocysteine ( 17 ) ( not shown ) were highly toxic , irrespective of psd - 95 deficiency . thus , reduced nmda toxicity in psd - 95 deficient cells was unlikely to be caused by altered signaling events downstream from no formation . suppressing psd - 95 expression uncoupled no formation from nmdar activation ( fig4 c ), and protected neurons against nmdar toxicity ( fig1 c , d ) without affecting receptor function ( fig1 e , 3 a - d ), by mechanisms downstream from nmdar activation , and upstream from no - mediated toxic events ( fig4 f ). therefore , psd - 95 imparts nmdars with signaling and neurotoxic specificity through the coupling of receptor activity to critical second messenger pathways . these results have broader consequences , as nmdar activation and no signaling are also critical to neuronal plasticity , learning , memory , and behavior ( 1 , 18 , 19 ). thus , these data provide experimental evidence for a mechanism by which psd - 95 protein may govern important physiological and pathological aspects of neuronal functioning . fig5 shows the utility of tat - peptides in dissociating the nmdar / psd - 95 interaction ( a ) the hypothesis : the nmdar / psd - 95 complex ( left panel ) may be dissociated using tat peptides fused either to the c - terminus of nr2b ( tat - nr2b9c ; middle ) or to the first and second pdz domains of psd - 95 ( ptat - pdz1 - 2 ; right ), thus reducing the efficiency of excitotoxic signaling via ca 2 + - dependent signaling molecules ( b ) intracellular accumulation of tat - nr2b9c - dansyl ( 10 μm ) but not control peptide ( tat - 38 - 48 - dansyl ; 10 μm ) was observed 30 min after application to cortical neuronal cultures using confocal microscopy ( excitation : 360 nm , emission : & gt ; 510 nm ; representative of 5 experiments ). fluorescence of cultures treated with tat - 38 - 48 - dansyl was similar to background ( not shown ). ( c ) time course of tat - nr2b9c - dansyl ( 10 μm ) fluorescence after application to cortical cultures at room temperature ( symbols : mean ± s . e of 4 experiments ). inset : fluorescence images from representative experiment ( d ) tat - nr2b9c , but not control peptides ( see text ), inhibits the co - immunoprecipitation of psd - 95 with nr2b in rat forebrain lysates ( left : representative gel ; right : means ± s . e of 4 experiments , anova , f = 6 . 086 , * p = 0 . 0041 ). in more detail , a conserved tsxv motif at the c - terminus of the nr2b subunit is critical for binding to the pdz2 domain of psd - 95 . i hypothesized that interfering with this interaction might disrupt the coupling between nmdars and psd - 95 . this might be achieved by the intracellular introduction of exogenous peptides that bind to either the nr2b or the pdz2 interaction domains ( fig5 a ). to this end i used a peptide comprised of the nine c - terminal residues of nr2b ( klssiesdv ; nr2b9c ( seq id no : 1 )), which is anticipated to bind the pdz2 domain of psd - 95 . as an alternative means to interfere with the nmdar / psd - 95 interaction i constructed a protein comprised of residues 65 - 248 of psd - 95 encompassing the first and second pdz domains ( pdz1 - 2 ), which contains the principal binding domain in psd - 95 for the c - terminus of nr2b . nr2b9c or pdz1 - 2 on their own did not enter cells ( not shown ) and therefore , i fused each to a peptide corresponding to the cell - membrane transduction domain of the hiv - 1 - tat protein ( ygrkkrrqrrr ( seq id no : 2 ); tat ) to obtain a 20 amino acid peptide ( tat - nr2b9c ) and the fusion protein ptat - pdz1 - 2 . ptat - pdz1 - 2 and ptat - gk fusion proteins were generated by insertion of psd95 residues 65 - 248 encoding the pdz 1 and 2 , and residues 534 - 724 encoding the guanylate kinase - like domains , respectively , into ptat - ha plasmids ( generous gift of s . dowdy , washington university , st . louis , mo .). fusion proteins contain a 6 × his - tag , the protein transduction domain of hiv - 1 tat and a hemagglutinin - tag n - terminal to the insert . plasmids were transformed into bl21 ( de3 ) lyss bacteria ( invitrogen ) and recombinant proteins were isolated under denaturing conditions on a nickle - his column ( amersham - pharmacia ). these are anticipated to transduce cell membranes in a rapid , dose - dependent manner independent of receptors and transporters ( 30 ). to determine whether tat - nr2b9c was able to transduce into neurons , i conjugated the fluorophore dansyl chloride to tat - nr2b9c and to a control peptide comprised of hiv - 1 - tat residues 38 - 48 ( kalgisygrkk ( seq id no : 4 ); tat38 - 48 ) outside the tat transduction domain ( 31 ). electrophysiological recordings were made in 400 μm hippocampal slices from 20 - 36 day old sprague - dawley rats perfused at room temperature with acsf containing ( in mm ) 126 nacl , 3 kcl , 2 mgcl 2 , 2 cacl 2 , 1 . 2 kh 2 po 4 , 26 nahco 3 and 10 glucose and bubbled with 95 % o 2 / 5 % co 2 . whole - cell recordings of ca1 neurons were performed using the “ blind ” method with an axopatch - 1d amplifier ( axon instruments , foster city , calif .) at holding potential − 60 mv . pipettes ( 4 - 5 mω ) were filled with solution containing ( mm ): 135 cscl , 2 mgcl 2 , 0 . 1 cacl 2 , 0 . 5 egta , 10 hepes , 4 mg - atp , 0 . 2 gtp , and 5 qx - 314 , ph 7 . 4 , 310 mosm . field potentials were recorded with glass micropipettes ( 2 - 4 mω ) filled with acsf placed in the stratum radiatum 60 - 80 μm from the cell body layer . synaptic responses were evoked by stimulation ( 0 . 05 ms ) of the schaffer collateral - commissural pathway with a bipolar tungsten electrode in the presence of bicuculline methiodide ( 10 μm ). for i nmda recording , mg 2 + was removed from and 20 μm cnqx was added in acsf . following 10 - 20 min base line recordings of epscs , i nmda and fepsps , tat - peptides were applied in acsf and recordings were continued for 30 min thereafter . i bath applied these to cultured cortical neurons and observed their fluorescence by confocal microscopy . neurons treated with tat - nr2b9c - dansyl ( 10 μm ) exhibited fluorescence in their cytoplasm and processes , indicating intracellular peptide delivery ( fig5 b , left ). sister cultures treated with tat38 - 48 - dansyl ( 10 μm ) exhibited only background fluorescence , indicating no observable peptide uptake in the absence of the tat transduction domain ( fig5 b , right ). tat - nr2b9c - dansyl was detectable in the neurons within 10 min of the start of the application and the peptide accumulated to a maximum level over the next 20 min ( fig5 c ). this level was maintained until the dansyl - tat - nr2b9c was washed from the bath and the peptide remained detectable within the neurons for more than 5 hours thereafter . therefore , the tat transduction domain was able to act as a carrier for nr2b9c and the tat - nr2b9c fusion peptide remained in neurons for many hours after being applied extracellularly . to determine whether tat - nr2b9c may disrupt the interaction between nmdars and psd - 95 i made use of rat brain proteins prepared under weakly denaturing conditions known to permit the nmdar / psd - 95 interaction . adult ( 7 - 8w ) wistar rat forebrains were removed and homogenized in ice - cold buffer ( 0 . 32m sucrose , 0 . 1 mm na3vo4 , 0 . 1 mm pmsf , 0 . 02m pnpp , 0 . 02m glycerol phosphate , and 5 ug / ml each of antipain , aprotinin , and leupeptin ). homogenates were centrifuged at 800 gr for 10 min at 4 ° c . the supernatants were combined and centrifuged at 11 , 000 g at 4 degree for 20 min and the pellet ( p2 ) was resuspended in homogenization buffer . p2 membranes were adjusted 200 ug protein / 90 ul with homogenization buffer with a final concentration of 1 % doc and 0 . 1 % triton x - 100 . the proteins were incubated with tat - nr2b9c or with one of three controls : tat38 - 48 , the tat transduction sequence conjugated to two alanine residues ( tat - aa ), or a tat - nr2b9c peptide in which the c - terminal tsxv motif contained a double point mutation ( tat - klssieada ; tat - nr2baa ) rendering it incapable of binding psd - 95 . i immunoprecipated nmdars , together with associated proteins , with an antibody that selectively recognizes nr2b . the proteins were separated by sds - page and probed with anti - psd - 95 or anti - nr2b antibodies 16 nr2b was precipitated from rat forebrain extracts using a polyclonal rabbit anti - nr2b antibody generated against the c - terminal region encompassing amino acid residues 935 - 1 , 455 of the nr2b protein . proteins were then separated on 8 % sds - page gels and probed with monoclonal anti - nr2b ( clone 13 , transduction laboratories ) or anti psd - 95 antibodies ( clone 7e3 - 1b8 , affinity bioreagents . inc ). detection of proteins was achieved using hrp - conjugated secondary antibodies and enhanced chemiluminescence . i found that tat - nr2b9c reduced the co - immunoprecipitation of psd - 95 with nr2b . on average the optical density signal was reduced by 37 . 6 ± 8 . 2 % as compared with controls ( fig5 d ). in contrast , none of the three control peptides reduced the co - immunoprecipitation of psd - 95 with nr2b . thus , i conclude that tat - nr2b9c disrupts the interaction between nmdars and psd - 95 and that this is dependent upon an intact pdz binding motif in the peptide . fig6 shows neuroprotection and reduction of no signaling by tat - peptides without affecting nmdar function ( a ) effect of tat - nr2b9c ( 50 nm ) on field excitatory post - synaptic currents ( fepsc ) in ca1 neurons in acute hippocampal slices . ( b ) effect of 50 nm tat - nr2b9c or tat - 38 - 48 ( control ) on whole - cell excitatory post synaptic currents ( epsc ). ( c ) effect of tat - nr2b9c on the nmda component of the epsc isolated pharmacologically by applying the ampar antagonist cnqx , and concomitant removal of extracellular mg 2 + . ( d ) effect of 50 nm tat - nr2b9c treatment on nmda - evoked 45 ca 2 + uptake in cortical cultures . tat - peptides were bath - applied 1 h prior to the nmda application . ( e ) effect of 50 nm tat - nr2b9c treatment on nmda - evoked cgmp production in cortical cultures . asterisk : differences from control and tat - nr2b - aa at each nmda concentration ( bonferroni t - test , p & lt ; 0 . 01 ). ( f ) decreased excitotoxicity at 20 h at all nmda concentrations in cultured cortical neurons pre - treated with 50 nm tat - nr2b9c or ptat - pdz1 - 2 for 1 h . asterisk : differences from control , tat - nr2b - aa and ptat - gk at each nmda concentration ( bonferroni t - test , p & lt ; 0 . 005 ). right panels : representative phase contrast and propodium iodide fluorescence images of cultures 20 h after challenge with 100 μm nmda with and without tat - nr2b9c treatment . bars in ( d ), ( e ) and ( f ) indicate the mean ± s . e . for 12 cultures in 3 separate experiments . in more detail , as nmdar - mediated synaptic responses are not altered by the loss of psd - 95 ( 24 ) i predicted that tat - nr2b9c would not affect the function of nmdars . this was tested by examining the effect of tat - nr2b9c on nmdar - mediated currents and on nmda - evoked uptake of 45 ca 2 + . bath - applying tat - nr2b9c ( 50 nm ) to acute rat hippocampal slices had no effect on synaptic responses of ca1 neurons evoked by stimulation of the schaffer collateral - commissural pathway ( fig6 a ) nor on patch recordings of the total excitatory post - synaptic currents ( epsc ) recorded in ca1 neurons , ( fig6 b ) nor on the pharmacologically isolated ampa ( not shown ) or nmda components of the epsc ( fig6 c ). moreover , using cortical cultures i found that pre - treating cultures with tat - nr2b9c or with ptat - pdzi - 2 ( each at 50 nm ) did not alter the uptake of 45 ca 2 + produced by applying nmda ( fig6 d ); cnqx ( 10 μm ) and nimodipine ( 2 μm ) were present in the extracellular solution in these and all subsequent experiments using cultured neurons so as to isolate signaling and thereby preventing secondary activation of ampars or of voltage - gated ca 2 + channels , respectively ( 25 , 32 , 33 ). as the function of nmdars was unaffected by administering tat - nr2b9c , i next determined whether this peptide altered signaling events downstream of nmdar activation . to this end i examined stimulation of nnos , as a key downstream signaling enzyme that mediates the neurotoxic effects of nmdar activation 5 . i measured nmda - evoked changes in the levels of guanosine 3 ′, 5 ′- monophosphate ( cgmp ) as a surrogate measure of no production by nmdar stimulated nnos activity 7 , 20 . cultured cortical neurons were pre - treated for 1 h with tat - nr2b9c ( 50 nm ), the non - interacting tat - nr2b - aa ( 50 nm ) or with sham washes and challenged with nmda ( 0 - 1000 μm ) in the presence of cnqx and nimodipine as above . nmda produced a concentration - dependent increase in cgmp that was significantly suppressed ( average of 39 . 5 ± 6 . 7 %) by pre - treating the cultures with tat - nr2b9c ( fig6 e ). in contrast , nmdar - stimulated elevation of cgmp was unaffected by pre - treatment with tat - nr2b - aa . thus , tat - nr2b9c , but not a mutant peptide incapable of interacting with psd - 95 , depressed nmdar - evoked stimulation of no - cgmp signaling . although tat - nr2b9c and ptat - pdz1 - 2 did not affect nmdar function , tat - nr2b9c was shown to interfere with nmdar / psd - 95 binding and to suppress downstream no signaling . thus , i predicted that tat - peptide treatment should enhance neurons &# 39 ; resilience to nmda toxicity . to test this i pre - treated cortical neuronal cultures with tat - peptides ( 50 nm ) for 1 h , then applied nmda ( 0 - 100 μm ) for 1 h followed by a 20 h observation period ( fig6 f , inset ). control neurons were treated with sham washes , or with the non - interacting control tat - nr2baa . in cultures treated with tat - nr2b9c , cell death was significantly reduced at all concentrations tested ( fig6 f ) whereas pre - treatment with tat - nr2b - aa had no effect on cell death . thus , nmdar - stimulated neurotoxicity is suppressed by pre - treatment with tat - nr2b9c , suppression that is lost by mutating the psd - 95 binding region of the peptide . if tat - nr2b9c suppresses nmda excitotoxicity by interfering with the binding of nr2b to psd - 95 then interfering with this binding by an alternative means should also suppress the toxicity . i tested ptat - pdz1 - 2 , predicted to interfere with psd - 95 binding to nr2b and which permeates into the cells ( not shown ), though without effect on nmda - evoked ca 2 + accumulation ( fig6 d ). pre - treating the cultures with ptat - pdz1 - 2 attenuated the neurotoxicity of nmda to a similar degree as tat - nr2b9c ( fig6 f ). as a control , i made and used ptat - gk , a tat fusion protein containing residues 534 - 724 of psd - 95 comprising the carboxyl - terminal guanylate - kinase homology domain that lacks enzymatic activity 21 . ptat - gk , which is devoid of the necessary domains to bind nr2b , had no effect on the nmda - evoked cell death ( fig6 f ). thus , interfering with the nmdar / psd - 95 interaction using peptides that target either side of the interaction reduces in vitro excitotoxicity produced by nmdar activation . fig7 shows neuroprotection by tat - nr2b9c pretreatment in - vivo . ( a ) detection of tat - nr2b9c - dansyl but not tat38 - 48 - dansyl in the cortex of c57bl / 6 mouse brain 1 h after intraperitoneal injection ( 0 . 5 μmole total dose ). fluorescence of brains from animals treated with tat - 38 - 48 - dansyl was similar to background ( not shown ). ( b ) composite neurological scores ( see text ) during and 24 h after mcao . ( c ) pre - treatment with 3 nmole / g tat - nr2b9c but not mutated tat - nr2b - aa or saline ( control ) significantly reduced ( i ) total infarct area and volume ( inset ), anova ; f = 7 . 3 , p & lt ; 0 . 005 and ( ii ) cortical infarct area and volume ( inset ), anova ; f = 8 . 35 , p & lt ; 0 . 005 measured 24 h after transient mcao . ( n = 6 animals per group ; symbols and bars indicate mean ± s . e ). infarct volume was calculated by analyzing the infarct area in 8 stereotactic coordinates of the brain as shown at right inset . agents that block nmdar activity were initially deemed as promising neuroprotectants for stroke and other neurological disorders involving excitotoxic mechanisms , but were later shown to be deleterious or ineffective in animal and human studies ( 27 , 28 , 29 ). however , tat - peptides that target the nmdar / psd - 95 interaction protect against nmda toxicity without blocking nmdars . therefore i reasoned that treatment with tat - nr2b9c in vivo could serve as an improvement on nmda blockers in the treatment of ischemic brain damage . before testing this i determined whether tat - nr2b9c could be delivered into the brain in the intact animal . i injected 25 g c57bl / 6 mice intraperitoneally with a 500 μmole dose of either tat - nr2b9c - dansyl , or with tat38 - 48 - dansyl as a non - transducing control . 40 μm cryostat coronal brain sections taken 1 h after injection 22 were examined for peptide uptake using dansyl fluorescence detection by confocal microscopy . the mice were perfused with fixative solution ( 3 % paraformaldehyde , 0 . 25 % glutaraldehyde , 10 % sucrose , 10 u / ml heparin in saline ) 1 hour after peptide injection . brains were removed , frozen in 2 - methylbutane at − 42 ° c . and 40 m sections were cut using a leitz cryostat . brain sections from animals injected with tat - nr2b9c exhibited strong fluorescence in the cortex ( fig7 a , right ), and in all other areas examined ( hippocampus , striatum ; not shown ), whereas signal from controls remained at background levels ( fig7 a , left ). similar results were obtained using intravenous injection in rats ( not shown ). thus , tat - nr2b9c enters the brain upon peripheral administration . next , i examined whether pretreatment with tat - peptides would reduce stroke damage . experiments were carried out in adult male sprague - dawley rats subjected to transient middle cerebral artery occlusion ( mcao ) for 90 minutes by the intraluminal suture method ( 36 , 37 ). animals were fasted overnight and injected with atropine sulfate ( 0 . 5 mg / kg ip ). after 10 minutes anesthesia was induced with 3 . 5 % halothane in a mixture of nitrous oxide and oxygen ( vol . 2 : 1 ) and maintained with 0 . 8 % halothane . rats were orally intubated , mechanically ventilated , and paralyzed with pancuronium bromide ( 0 . 6 mg / kg iv ). body temperature was maintained at 36 . 5 - 37 . 5 ° c . with a heating lamp . polyethylene catheters in the femoral artery and vein were used to continuously record blood pressure and to sample blood for gas and ph measurements . transient mcao was achieved for 90 min by introducing a poly - l - lysine - coated 3 - 0 monofilament nylon suture ( harvard apparatus ) into the circle of willis via the internal carotid artery , effectively occluding the middle cerebral artery . this produces an extensive infarction encompassing the cerebral cortex and basal ganglia . animals were pretreated with either saline , the tat - nr2b - aa control , or with tat - nr2b9c by a single intravenous bolus injection 45 min prior to mcao ( 3 nmoles / g ). physiological parameters ( body temperature , blood pressure , blood gases ) were monitored and maintained throughout the experiment ( table 1 ). all experimental manipulations and analyses of data were performed by individuals blinded to the treatment groups . the extent of cerebral infarction was measured 24 h after mcao onset ( fig7 c inset ). the postural reflex test ( 38 ), and the forelimb placing test ( 39 ) were used to grade neurological function on a scale of 0 to 12 ( normal = 0 ; worst = 12 ) during mcao ( at 50 minutes ) and 24 h thereafter . pretreatment with tat - nr2b9c produced a trend toward improvement in 24 h neurological scores in animals treated with tat - nr2b9c ( fig7 b ). moreover , the treatment reduced the volume of total cerebral infarction by 54 . 6 ± 11 . 27 % as compared with stroke volume in controls ( fig7 c ,; anova , f = 7 . 289 , p = 0 . 0048 ). this effect was largely accounted - for by a 70 . 7 ± 11 . 23 % reduction in cortical infarction ( fig7 c , anova , f = 8 . 354 , p = 0 . 0027 ), which is thought to be largely caused by nmdar - dependent mechanisms . a treatment for stroke with a single - bolus drug injection would be most therapeutically valuable if effective when given after the onset of ischemia . i thus first evaluated whether treatment with tat - peptides could be neuroprotective when applied post - insult in vitro . fig8 shows neuroprotection by post - treatment with tat - nr2b9c in - vitro and in - vivo ( a ) decreased excitotoxicity at 20 h in cultured cortical neurons post - treated with 50 nm tat - nr2b9c or ptat - pdz1 - 2 at 1 h after nmda application . bars indicate the mean ± s . e . for 12 cultures in 3 separate experiments . asterisk : differences from control , tat - nr2b - aa and ptat - gk at each nmda concentration ( bonferroni t - test , p & lt ; 0 . 005 ). right panels : representative phase contrast and propodium iodide fluorescence images of cultures 24 h after challenge with 100 μm nmda with and without tat - nr2b9c treatment . ( b ) composite neurological scores ( see text ) during and 24 h after mcao . asterisk : difference from control and tat - nr2b - aa ( anova ; f = 17 . 25 , p & lt ; 0 . 0001 ). ( c ) post - treatment with 3 nmole / g tat - nr2b9c ( 9 animals ) but not mutated tat - nr2b - aa ( 8 animals ) or saline controls ( 10 rats ) significantly reduced ( i ) total infarct area and volume ( inset ), anova ; f = 12 . 0 , p & lt ; 0 . 0005 and ( ii ) cortical infarct area and volume ( inset ), anova ; f = 12 . 64 , p = 0 . 0001 as measured 24 h after transient mcao . symbols and bars indicate mean ± s . e ( d ). representative appearance of h & amp ; e stained rat brain sections from which the infarct areas were analyzed . cultured cortical neurons were exposed to an nmda challenge ( 0 - 100 μm ) for 1 h and were then treated with the tat - peptides ( all at 50 nm ) described in the pre - treatment study ( fig6 f ). cell death was gauged 20 h thereafter ( fig8 a — inset ). post - treatment with tat - nr2b9c or with ptat - pdz 1 - 2 significantly reduced the vulnerability of neurons to nmda toxicity as compared with control cultures post - treated with sham washes , with tat - nr2baa , or with ptat - gk ( fig8 a ). thus , when administered 1 hr after the start of the nmda insult each of the tat fusion constructs that target the nmdar / psd - 95 interaction significantly reduced neuronal cell death in vitro . finally , i examined whether treatment with tat - nr2b9c could attenuate ischemic neuronal damage in - vivo when given after stroke onset . a post - treatment study was conducted in which the rats were subjected to transient mcao for 90 minutes as before , but the intravenous saline or tat - peptide bolus ( tat - nr2b9c or tat - nr2b - aa ; 3 nmole / g ) was injected 1 h after mcao onset ( fig8 c — inset ). infarction volume and neurological outcome measurements were performed at times identical to the pre - treatment study . body temperature , blood pressure and blood gases were monitored throughout the 24 h experiment and maintained equivalent between groups ( table 2 ). post - treatment with tat - nr2b9c , but not with tat - nr2b - aa or saline , resulted in animals exhibiting a significant improvement in 24 h neurological scores as compared with controls ( fig8 b ; anova , f = 17 . 25 , p & lt ; 0 . 0001 ). most strikingly , post - treatment with tat - nr2b9c reduced the volume of total cerebral infarction by 67 . 0 ± 3 . 75 % as compared with stroke volume in controls ( fig8 c ; anova , f = 11 . 99 , p = 0 . 0002 ). similar to the previous study , this reduction was accounted - for by a 86 . 97 ± 4 . 38 % reduction in cortical infarction volume ( fig8 c , 4 d ; anova , f = 12 . 64 , p & lt ; 0 . 0001 ). the aforesaid description demonstrates that introducing into cells an exogenous peptide containing the c - terminal nine amino acids of the nr2b nmdar subunit has profound effects on signaling pathways downstream of nmdar activation , on in vitro excitotoxicity , and on in vivo ischemic brain damage . the effects of this peptide are lost by mutating amino acids that are essential for mediating pdz binding to psd - 95 . in addition , a protein comprising pdz1 - 2 of psd - 95 shares the effects of the nr2b c - terminal peptide . together these findings imply that the downstream signaling from nmdars that leads to negative consequences for neuronal viability may be interrupted by interfering with the interaction between nr2b and psd - 95 . i have discovered that the strategy of treating neurons with tat - fusion peptides is effective in reducing vulnerability to excitotoxicity in vitro and stroke damage in vivo . as this occurs without affecting nmdar activity then adverse consequences of blocking nmdars are not expected . efficacy after the insult onset suggests that targeting the nmdar / psd - 95 interaction is a practical future strategy for treating stroke . it is also likely that targeting other intracellular proteins using the same approach could be used to modulate additional signaling mechanisms mediated by protein - protein interactions that lead to other human diseases . although this disclosure has described and illustrated certain preferred embodiments of the invention , it is to be understood that the invention is not restricted to those particular embodiments . rather , the invention includes all embodiments which are functional or mechanical equivalence of the specific embodiments and features that have been described and illustrated .
a method of inhibiting the binding between n - methyl - d - aspartate receptors and neuronal proteins in a neuron the method comprising administering to the neuron an effective inhibiting amount of a peptide replacement agent for the nmda receptor or neuronal protein interaction domain that effect said inhibition of the nmda receptor neuronal protein . the method is of value in reducing the damaging effect of injury to mammalian cells . postsynaptic density - 95 protein couples neuronal n - methyl - d - aspartate receptors to pathways mediating excitotoxicity and ischemic brain damage . this coupling was disrupted by transducing neurons with peptides that bind to modular domains on either side of the psd - 95 / nmdar interaction complex . this treatment attenuated downstream nmdar signaling without blocking nmdar activity , protected cultured cortical neurons from excitotoxic insults and dramatically reduced cerebral infarction volume in rats subjected to transient focal cerebral ischemia . the treatment was effective when applied either before , or one hour after , the onset of excitotoxicity in vitro and cerebral ischemia in vivo . this approach prevents negative consequences associated with blocking nmdar activity and constitutes practical therapy for stroke .