Di erential modulation of AMPA receptor mediated currents by Evans Blue in postnatal rat hippocampal neurones

Transcription

1 British Journl of Phrmcology (1997) 121, 237 ± Stockton Press All rights reserved 0007 ± 1188/97 $12.00 Di erentil modultion of AMPA receptor medited currents y Evns Blue in postntl rt hippocmpl neurones Britt SchuÈ rmnn, Xueqing Wu, Irmgrd D. Dietzel & 1 Volkmr Leûmnn Lehrstuhl fuè r Molekulre Neuroiochemie, Ruhr-UniversitÈ t Bochum, NC7/ Bochum, Germny 1 The modultion of non-n-methyl-d-sprtte (NMDA) receptor-medited whole cell currents nd of glutmtergic synptic trnsmission y puri ed Evns Blue () ws investigted in rt cultured postntl hippocmpl neurones y use of ptch clmp recordings nd fst drug ppliction system. 2 Three di erent groups of neurones could e distinguished with respect to the type of modultion otined with 10 mm : ws either predominnt inhiitor of desensitiztion (13% of the neurones), predominnt inhiitor of current mplitudes (42%) or mixed inhiitor of oth properties (45%). Both e ects were not use-dependent nd reched mximl levels fter 30 s of pre-equilirtion with the dizo dye. 3 Dose-response curves otined from glutmte ctivted whole cell currents yielded n IC 50 vlue for of 13.3 mm (Hill coe cient: 1.3) for the inhiition of desensitiztion, nd n IC 50 vlue of 10.7 mm (Hill coe cient: 1.2) for the inhiition of current mplitudes. 4 Chicgo cid SS (100 mm) which is one of the synthesis precursors of hd no e ect on current mplitudes of glutmte ctivted whole cell currents ut ws wek inhiitor of desensitiztion in ll hippocmpl neurones investigted, irrespective of the type of modultion otined with in the sme neurone. 5 Oxidtively modi ed (the so-clled VIMP (10 mm)) hd no e ect on the kinetics ut ws prtil inhiitor of glutmte-ctivted whole cell currents in ll hippocmpl neurones investigted. 6 (10 mm) inhiited the mplitudes of non-nmda receptor medited utptic currents to the sme extent (to 39+19% of control) s oserved for glutmte ctivted whole cell currents (to 41+17% nd 56+20%). However, the decy of the utptic responses remined unin uenced upon ppliction, indicting tht either receptor desensitiztion does not dominte the time course of the synptic response or tht the non-nmda receptors sensitive to modultion of desensitiztion y re not present in the postsynptic memrne. 7 In conclusion, di erentilly modultes -mino-3-hydroxy-5-methyl-4-isoxzole propionic cid (AMPA) receptor gting in di erent susets of neurones. Upon identi ction of the cellulr determinnts for the di erentil modultion (e.g. AMPA receptor suunit composition) could ecome useful tool to investigte receptor sutypes during electrophysiologicl recordings. Keywords: Desensitiztion; glutmte receptors; AMPA receptors; non-nmda receptors; Evns Blue; utpses; Chicgo cid SS; hippocmpl cell culture; whole cell currents Introduction 1 Author for correspondence. Glutmte is the mjor excittory neurotrnsmitter in the mmmlin centrl nervous system nd detiled knowledge of the properties of glutmte receptors is essentil to understnd excittory synptic trnsmission in the rin. Glutmtergic trnsmission t excittory synpses is medited y three different clsses of receptor gted ion chnnels: (i) AMPA (mino-3-hydroxy-5-methyl-4-isoxzole propionic cid) prefering receptors consisting of (hetero)oligomeric complexes of the glutmte receptor suunits GluR1-4 (lso nmed GluR A-D), (ii) kinte prefering receptors consisting of the suunits GluR 5-7 nd KA 1-2 nd (iii) N-methyl-D-sprtte (NMDA) receptors ssemled from heterooligomeric complexes of the suunits NR1 nd NR2A-D, respectively (for reviews see Seeurg, 1993; Hollmnn & Heinemnn, 1994). An dditionl degree of complexity of possile suunit compositions of AMPA receptors results from lterntive processing of ech of the 4 AMPA receptor suunit mrnas (GluR1-4), yielding either ip or op splice vrints (Sommer et l., 1990). A numer of electrophysiologicl studies indicte tht neurones from di erent rin res possess distinct kinetic properties of non-nmda receptor medited currents (see e.g. Colquhoun et l., 1992; Leûmnn & Gottmnn, 1994), suggesting the presence of distinct types of non-nmda receptors in these rin regions. These receptor sutypes could di er either in their receptor suunit composition, in their posttrnsltionl modi ctions (e.g. phosphoryltion, glycosyltion), in intrcellulr protein-protein interctions or in their lipid microenvironment. Among these possiilities, thus fr, only receptor suunit composition hs een proven to modulte the kinetics of AMPA receptor-medited currents: comined electrophysiologicl nd moleculr iologicl studies hve reveled tht speci c AMPA receptor suunits nd suunit splice vrints di erentilly in uence the kinetic properties of AMPA receptor medited currents (Sommer et l., 1990; Moscher et l., 1994; Lomeli et l., 1994). However, receptor sutype speci c ntgonists nd modultors of AMPA receptors (whether they sense di erentil suunit compositions or other di erences in AMPA receptor properties) would e ene cil to identify directly AMPA receptor sutypes during electrophysiologicl recordings. Severl drugs (Evns Blue, cyclothizide, concnvlin A) hve een shown previously to hve the potentil to discriminte etween different non-nmda glutmte receptor complexes: Evns Blue () ws found to reduce desensitiztion nd prtilly lock the pek mplitudes of non-nmda-receptor medited currents in rt thlmic neurones (Leûmnn et l., 1992), nd to lock kinte evoked currents of heterologously expressed recominnt AMPA receptors contining either suunit

2 238 B. SchuÈrmnn et l GluR1 or GluR2 (Keller et l., 1993). On the other hnd, cyclothizide nd concnvlin A selectively inhiit desensitiztion of either AMPA or kinte receptors, respectively (Myer & Vyklicky, 1989; Ymd & Tng, 1993; Prtin et l., 1993; Wong & Myer, 1993). Cyclothizide ws shown to hve higher potency t the ip splice vrints s compred to the respective op suunits of AMPA receptors (Prtin et l., 1994). In ddition, Joro spider toxin hs een shown to lock speci clly current mplitudes of heterologously expressed AMPA receptors lcking the GluR2 suunit (Blschke et l., 1993). However, drug tht shows distinct types of e ects on di erent clsses of AMPA receptors hs so fr not een descried. Previous experiments on thlmic neurones hd shown tht the ntgonism y t non-ndma receptors is purely noncompetitive wheres the reduction of desensitiztion shres competitive nd non-competitive spects (V.L., unpulished). These dt suggested tht either (i) two di erent compounds of the commercilly ville impure dye (tht hs een used in former studies; purity580%) independently modulted desensitiztion nd current mplitudes, (ii) di erent residues of the molecule medite the two distinct e ects t non- NMDA receptors or (iii) cn medite oth e ects vi inding to di erent clsses of non-nmda receptors. Our study of the modultion of non-nmda receptormedited currents y puri ed now revels tht the dye medites oth, the reduction of pek current mplitudes nd the inhiition of desensitiztion t AMPA receptors. Secondly, di erent susets of cells cn e distinguished with respect to the type of modultion oserved with, leding either to predominnt modultion of desensitiztion, predominnt inhiition of current mplitudes or to mixture of oth e ects. Finlly, since does not chnge the kinetics of glutmtergic synptic currents, we provide evidence tht in cultured hippocmpl neurones either desensitiztion does not dominte the decy of excittory postsynptic currents or tht the non- NMDA receptors sensitive for modultion of desentiztion y re not locted in the postsynptic memrne. Methods Cell culture Hippocmpl cultures were prepred from postntl Wistr rts (P0 ± P5) essentilly s descried previously (Leûmnn et l., 1994). In rief, hippocmpi were dissected in ice-cold phosphte u ered sline with C 2+ nd Mg 2+ (PBS+/+) nd incuted for 10 ± 15 min t 378C in 0.25% trypsin. After eing rinsed with PBS+/+ the tissue ws dissocited with plstic pipettes of decresing tip dimeters nd the cell suspension ws injected into three fold excess of DMEM/10% FCS. Cells were centrifuged t 3006g (10 min, 48C) nd resuspended in 3 ± 5 ml DMEM/10% FCS. Vile cells were counted nd 50,000 cells per dish were plted in 3.5 cm dishes (Nunc) coted with polyornithine (0.5 mg ml 71, overnight). After 3 h the medium ws chnged to serum-free DMEM/B18 medium (Brewer & Cotmn, 1989). Cultures were mintined in humidi ed incutor with 10% CO 2 t 378C. Microislnd cultures: we modi ed (V.L., unpulished) the microislnd technique initilly introduced y Segl & Furshpn (1990). In rief, corticl hemispheres (Wistr rts P1 ± P5) were trypsinized nd dissocited s descried ove. Depending on the ge of the rt pups 80,000 ± 200,000 corticl cells were plted in uncoted plstic dishes. Glil cells preferentilly ttched to the plstic dishes nd the superntnt ws removed fter 1 ± 2 h. These glil cells were cultured for 7 dys in DMEM/10% FCS, nd supplemented with 10 mm cytosine-- D-rinofurnoside (ARAC) strting t 4 dys in vitro (DIV). Under these conditions netly spced strocytic islnds of 100 ± 300 mm dimeter were formed. At 7 DIV 30,000 ± 70,000 dissocited hippocmpl neurones (see ove) were plted into these dishes. On the next dy the medium ws exchnged to AMPA receptor modultion y Evns Blue DMEM/B18, or to Neurosl medium supplemented with B27 (Gico). Cultures were mintined in n incutor with 5% CO 2 t 378C. All experiments were performed t room temperture (i.e. 228C). Electrophysiologicl recording Whole cell ptch clmp recordings of gonist-induced currents were otined from hippocmpl cultures t 7 ± 12 DIV y employing n EPC-7 ptch clmp mpli er (List Medicl Ins.). Autptic currents were recorded in the whole cell con gurtion of the ptch clmp technique t 10 ± 14 DIV. Somtic voltge ctivted N + currents were elicited with short (1 ms) depolrizing current pulses injected through the ptch pipette. This prdigm evoked ction potentils in more distl prts of the cell leding to trnsmitter relese (Bekkers & Stevens, 1991). Ptch electrodes were fricted from orosilicte glss (GB150-8P, Science Products) nd hd typicl resistnces of 4±7 MO when lled with the stndrd pipette solution (in mm): K-gluconte 140, NCl 5, MgCl 2 1, CCl 2 0.2, EGTA 2, HEPES 15, ph djusted to 7.3 with KOH. Stndrd extrcellulr solution ws (in mm): NCl 145, KCl 4, CCl 2 3, MgCl 2 2, HEPES 10 nd glucose 10, ph djusted to 7.3 with NOH. The holding potentil ws corrected for junction potentil of 710 mv resulting from this comintion of intrnd extrcellulr solutions. The ccess resistnce in the whole cell con gurtion ws in the rnge of 9 ± 13 MO nd ws compensted to 40 ± 60% y feedck circuit of the ptch clmp mpli er. For recordings of current voltge reltionships K-gluconte in the intrcellulr solution ws replced y CsCl (120 mm). During recordings of gonist-induced currents 1 mm TTX ws dded to ll extrcellulr solutions to lock synptic ctivity. Agonists were dissolved in extrcellulr solution nd pplied s descried elow. Currents were ltered t 3 khz, digitized (Digidt 1200, Axon Ins.) nd stored on the hrd disk of personl computer. Dt nlysis ws performed y AUTESP softwre (Version 0.93, Grching Ins.). If not stted otherwise, ll current trces shown re the verge of 3 ± 6 consecutive gonist pplictions or evoked utptic currents, respectively. If not stted otherwise, ll dt re given s men+s.d. Signi cnce of di erences of men vlues were nlysed y unpired Student's t- test t P levels s indicted in the text nd gure legends. Drug ppliction Fst trnsmitter ppliction ws chieved y superfusion system consisting of 3 glss cpillries s descried in detil previously (Leûmnn et l., 1992; Leûmnn & Dietzel, 1995). In rief, opposing in ow nd out ow pipettes creted lminr strem of gonist solution lterl to the cell. The strem ws rpidly extended to superfuse the cell som y trnsiently incresing the in ow pressure. Solution exchnge mesured s di usion current t the tip of ptch pipette ws 41 ms. Solution exchnge t the cell during whole cell recordings ws 520 ms s judged from the relxtion current otined when cell ws depolrized to elicit voltge ctivted K + currents nd the extrcellulr K + concentrtion ws switched to 20 mm y the superfusion system. Cells were equilirted with the modultors (e.g., VIMP) y mens of second in ow pipette. This second strem of solution ws replced y the fst extending jet of gonist solution during trnsmitter ppliction. Removl of solution ws chieved y sudden ppliction of negtive pressure in the in ow pipette. The concentrtion of L-glutmte ws 1 mm in ll experiments shown in this study. This fcilittes the direct comprison of the results otined from gonist-induced whole cell currents with the utptic recordings, since the pek glutmte concentrtion in the synptic cleft of hippocmpl neurones hs een estimted to e 4300 mm (Jons & Skmnn, 1992) nd 51 mm(clements et l., 1992). In ddition 1 mm glutmte is close to the EC 50 vlues (0.4 ± 1.1 mm) for glumteinduced whole cell nd outside-ptch currents in rt centrl

3 neurones when using fst trnsmitter pplictions systems (see e.g. Kiskin et l., 1986; Hestrin, 1992; Jons & Skmnn, 1992; Leûmnn & Gottmnn, 1994). Dye puri ction nd nlysis Since commercilly ville Evns Blue () is indicted y the mnufcturers to contin impurities of *10% we developed puri ction method, to ssure tht the modultory e ects of were medited y the min compound of this rw mteril. Brie y, 1 g of ws dissolved in 4 ml H 2 O, mixed with n equl mount of silicgel nd dried. This solid ws plced on top of silicgel column (4620 cm, silicgel, 70 ± 230 mesh) tht hd een equilirted with ultrpure cetone (regent grde). The seprtion ws performed y elution with successive cetone:wter dilutions of 50 : 1, 20 : 1 nd 6 : 1, respectively ( detiled description of the puri ction protocol is ccessile upon request). This procedure yielded one mjor compound (490%) spectroscopiclly identi ed s Evns Blue. The purity of this frction ws determined y nucler mgnetic resonnce (n.m.r.) spectroscopy to e 599%. This puri ed ws used in ll experiments for the preprtion of recording solutions. The only impurity present in the commercilly ville in sizele mounts ws violet nd mounted to out 5% of the rw. This violet impurity (VIMP) ws shown to e generted from y oxidtion with dissolved moleculr oxygen nd occurred during extensive stirring of solutions or in the presence of surfce ctlysts (see Figure 5). VIMP ws not generted during preprtion or use of our recording solutions s judged y silicgel chromtogrphy tht ws regulrly performed t the end of recording session. As judged from n.m.r. spectroscopic dt, VIMP is n symmetric molecule creted most likely y oxidtion of the C-5 nd/or C- 8 toms (see sterisks in Figure 5) of the Chicgo cid residues in (B.S., unpulished). The moleculr weight of this oxidized ws estimted to 1000 g mol 71 (compred to 960 g mol 71 for ) nd the concentrtions for VIMP given in this study re sed on this estimted vlue. A more detiled chemicl nlysis of VIMP ws hindered y the inccessiility of the molecule to the di erent protocols of mss spectroscopy tht we tried so fr. In ddition, VIMP still contined some minor impurities tht, nevertheless, hindered the chemicl nlysis y 2-D-n.m.r. spectroscopy or X- ry-crystllogrphy. L-Glutmte, kinte nd Evns Blue were purchsed from Sigm, AMPA from Tocris Cookson (U.K.), surmin from RBI nd Chicgo cid SS ws gift kindly provided y Dr Oerkirch from Byer AG (Leverkusen, Germny). Results B. SchuÈrmnn et l Non-NMDA receptor medited whole cell currents The ppliction of 1 mm L-glutmte t holding potentil of 770 mv evoked whole cell currents with men rise time (0 ± 100% of pek current) of ms nd men pek mplitude of pa (n=58). At this negtive holding potentil nd in the sence of glycine nd the presence of 2mM Mg 2+ in the extrcellulr solution, glutmte evoked whole cell currents re lmost exclusively medited y non- NMDA receptors. This ws con rmed in suset of experiments where 10 mm of the non-nmda receptor speci c ntgonist 6,7-dinitroquinoxline-2,3-dione (DNQX) reversily locked the glutmte evoked currents to % (n=7) of control mplitudes. The glutmte evoked responses showed pronounced desensitiztion to stedy stte vlue (mesured 180 ms fter the pek current) of pa (i.e. 17% of pek mplitudes). Pek currents showed liner dependence on the holding potentil in the rnge of 750 to +30 mv, with men reversl potentil of mv (n=12, not shown). The desensitizing phse of the whole cell currents could e tted monoexponentilly with men time AMPA receptor modultion y Evns Blue 239 constnt of ms (n=58). The time constnt of desensitiztion vried considerly from cell to cell (Figure 1; rnge 12 ± 45 ms). This vriility could in principle result from di erent time courses in the rise of the glutmte concentrtion t the cell memrne, leding to more or less synchronous ctivtion of glutmte receptors depending on the speci c cell under investigtion. In this cse cells showing lrger rise time should lso show lrger time constnt of desensitiztion. This is not the cse under our experimentl conditions s shown y plot of the rise time versus the corresponding time constnt of desensitiztion for ech of 50 τ of desensitiztion (ms) τ = 17 ms τ = 40 ms Glutmte 1 mm Glutmte 1 mm 200 pa Rise time (0 100%) (ms) Figure 1 Sctter in time constnts of desensitiztion in cultured hippocmpl neurones. L-Glutmte-induced (1 mm) whole cell currents otined from 2 di erent cultured postntl hippocmpl neurones (). Although the rise time (0 ± 100% of pek current) ws 510 ms for oth cells the time constnts of mcroscopic desensitiztion (t) show mrked di erences. The stippled lines show the results of the monoexponentil t to the desensitizing phse of the currents. A plot of the time constnt of desensitiztion vs the respective rise time () for 58 di erent hippocmpl cells shows the wek correltion (solid line, correltion coe cient=0.042) etween oth prmeters, suggesting cell speci c di erences in the time course of desensitiztion.

4 240 B. SchuÈrmnn et l 58 cells (Figure 1). Since no correltion etween these two prmeters is evident (r=0.042), we ssume tht the vriility of the time constnt of desensitiztion re ects t lest in prt cell speci c di erences in the time course of desensitiztion. E ects of puri ed on ntive non-nmda receptors in hippocmpl cultures L-Glutmte (1 mm for 360 ms) ws pplied to the cells in 15 ± 30 s intervls strting 1 min fter otining the whole cell con gurtion. Severl glutmte-ctivted currents were elicited to ssure stle mplitudes nd kinetics under control conditions. Preincution of the neurones with 10 mm puri ed (purity 599%) di erentilly modulted non- NMDA receptor medited currents in di erent susets of cells: (A) 22 out of 52 cells investigted showed prtil lock of pek current mplitudes to 41+17% of control vlues with recovery to 73+17% of the initil mplitude upon wshout of (Figure 2). Wheres the time constnt of desensitiztion (t) remined un ected, the stedy stte current vlue (residul current fter 180 ms) slightly decresed (Tle 1) nd the rise times signi cntly incresed to ms (control ms, P50.05). (B) Seven cells showed nerly complete inhiition of desensitiztion (t= ms compred to 26+5 ms under control) ccompnied y incresed mximl current mplitudes (126+18% of control; Figure 2, Tle 1). In ddition the AMPA receptor modultion y Evns Blue rise time of the whole cell currents ws reversily prolonged to ms (P50.001). (C) The remining 23 cells displyed comintion of the e ects descried under (A) nd (B): pek current mplitudes were reduced to 56+20% (rnge: 28 ± 91%) of control wheres stedy stte current mplitudes were incresed three fold. In ddition, the time constnt of desensitiztion ws prolonged to ms (see Tle 1) nd the rise time ws incresed signi cntly to ms (P50.01). Our criterion to ssign neurone to group (C) insted of (A) ws chnge of 510% in the time constnt of desensitiztion. As n lterntive to the t of desensitiztion time constnts, desensitiztion of glutmte receptor currents cn e qunti ed y clculting the per cent desensitiztion (i.e. 1006(pek current7stedy stte current)/(pek current 7seline current). A plot of the normlized pek current inhiition vs the normlized inhiition of the percent desensitiztion y for the cells clssi ed in the three groups (A), (B) nd (C) is shown in Figure 2e. The dominnt modultion of either desensitiztion (group B) or pek current mplitudes (group A) y is clerly visile. As expected, the vlues for the group C neurones re scttered long line tht would connect the two extreme groups, re ecting the cell speci c vrition in the proportions of the e ects of on desensitiztion nd current mplitudes. Both e ects of could lso e oserved when using lower gonist concentrtions (e.g. 100 mm L-glutmte) nd the reltive inhiition of desensitiztion nd current mplitudes y c Glutmte 1 mm Glutmte 1 mm Glutmte 1 mm W C 100 pa C W W C d Glu 1mM5 s 5 s 5 s 5 s 30 s e 20 s 500 pa 200 ms 100 pa 5 s 30 s Pek current mplitude under (% of control) (B) (C) (A) Per cent desensitizton under (% of control) Figure 2 Distinct types of modultion of glutmte ctivted whole cell currents y Evns Blue (). L-Glutmte (1 mm)-induced currents from three di erent hippocmpl neurones ( ± c, correspond to groups A, B nd C in text). Upon pre-equilirtion of the neurones with 10 mm Evns Blue (, 20 s) either predominnt lock of current mplitudes (; 42% of the cells), predominnt inhiition of desensitiztion (; 13%) or mixed lock of mplitude nd desensitiztion (c; 45%) were oserved. C, control;, 10 mm ; W, wsh. Pre-equilirtion (20 s) of neurone with 10 mm in the sence of chnnel openings (d, lower trce) ws su cient to otin the full e ect. Shorter incution interrupted y glutmte pplictions resulted in the successive development of the full e ect (d, upper trce). In either cse the recovery ws complete within 30 s of wshout. In (d) nonverged originl currents re shown. (e) Plot of the pek current mplitude seen in the presence of vs the lock y of the per cent desensitiztion for individul hippocmpl neurones given s per cent of control (i.e. in the sence of ). The neurones with either the predominnt lock of desensitiztion (&, group B), the predominnt lock of current mplitudes (~, group A) or the mixed e ect (*, group C) re indicted y the di erent symols.

5 B. SchuÈrmnn et l AMPA receptor modultion y Evns Blue 241 Tle 1 Modultion of AMPA receptor medited whole cell currents y Evns Blue nd relted sustnces Test Amplitude t (desensitiztion) Stedy stte sustnce (% of control) (ms) (% of control) (mm) n Test Wsh Test Wsh Test Wsh (A) (10) (B) (10) (C) (10) VIMP (10) CASS (100) Surmin (10) AMPA+ (10) KA+ (10) * * 56+20* 57+16* * 50+20* * * * * * * * 60+34* * * Neurones were equilirted (530 s) with either Evns Blue (), oxidized (VIMP), Chicgo cid SS (CASS) or surmin. L-Glutmte (1 mm), S-AMPA (200 mm) or kinte (KA, 500 mm) evoked whole cell current trces were verged (n=3 ± 6). Amplitudes of pek currents, stedy stte currents (180 ms fter the pek) nd the time constnt of desensitiztion (t) were clculted from this verge currents (`test'). Current mplitudes re given s percentge of `control' efore the ppliction of modultor. The extent of recovery (`wsh') is indicted. Dt re given s mens+s.d. *Signi cntly di erent from control with P<0.01 (unpired Student's t test). ws comprle to the e ects oserved when using 1 mm glutmte (not shown). Except for the only prtil recovery of current mplitudes, ll modultory e ects of were reversile upon wshout. Either of the 3 e ects hd reched mximl levels fter 20 s of preincution of the neurones with the dye nd ws independent of chnnel opening (see Figure 2d). All e ects were lso independent of the presence of during the glutmte ppliction. No correltion etween the cell speci c desensitiztion time constnts (Figure 1) nd the type of modultion otined with ws oserved. The recovery fter wshout of ws lso non use-dependent nd ws complete within 20 ± 40 s. We lso investigted the e ect of on non-nmda receptor medited currents recorded from outside-out ptches. Glutmte (1 mm) ctivted currents showed men mplitude of pa nd men time constnt of desensitiztion of ms (n=6, not shown). Upon ppliction of (10 mm) we oserved either pure inhiition of current mplitudes (n=3) or mixed inhiition of current mplitudes nd desensitiztion (n=3), indicting tht the two distinct effects of cn e oserved independent of the presence of n intct intrcellulr environment of the receptors. We next investigted the potency of for the inhiition of desensitiztion nd current mplitudes y determining dose-response reltionships. The dt otined were tted to the Hill eqution y lest squres tting procedure. The IC 50 vlue for the inhiitory ction on pek current mplitudes ws mm (men+s.e.). The corresponding Hill coe cient ws (Figure 3). The dose-response curve for for the per cent desensitiztion yielded n IC 50 vlue of mm nd Hill coe cient of (Figure 3). Thus, the two seprte e ects of on glutmte ctivted currents re elicited t very similr concentrtions. We wnted to nd out whether the two distinct e ects of cn lso e oserved when only AMPA receptors re ctivted. Therefore, we performed n dditionl series of experiments with AMPA, which speci clly ctivtes chnnels ssemled of GluR1-4 suunits. (S)-AMPA (200 mm) ws pplied to neurones preincuted with 10 mm. Out of the 6 cells investigted 2 neurones displyed predominnt lock of current mplitudes (Figure 4) wheres the remining 4 cells showed the comined desensitiztion/mplitude e ect (Figure 4; Tle 1). This strongly suggests tht the speci c interction of with di erent clsses of AMPA receptors ccounts for the 2 distinct modultory e ects of the dye oserved with glutmte s the gonist. Kinte-evoked (500 mm) currents in the sence of never displyed ny detectle sign of desensitiztion which would hve een expected if lrge mounts of kinte receptors (composed of the suunits GluR5-7 nd KA1-2) were present in our cultures. The kinte (500 mm)- induced whole cell currents were reversily inhiited to 50+20% of control in the presence of 10 mm (see Tle 1). This vlue lmost exctly mirrors the extent of pek current reduction otined in more thn 80% of the neurones when glutmte ws the gonist. Thus, the inhiition of current mplitudes y ws independent of the gonist used to ctivte the non-nmda receptors. Evns Blue nd surmin hve een shown to e potent ntgonists t P 2X -purinoceptors (BuÈ ltmnn & Strke, 1993, BuÈ ltmnn et l., 1995). Hence, if the e ects of on non- NMDA receptors were medited vi inding to these purinoceptors, surmin should hve comprle e ects. In contrst, preincution of neurones with 10 mm surmin left L- glutmte induced whole cell currents unltered (Figure 4d, Tle 1). Likewise, ATP (500 mm) which is the nturl gonist of the P 2X -purinoceptors hd no signi cnt e ect on non- NMDA receptor medited whole cell currents (dt not shown). E ects of derivtives of on whole cell currents VIMP The puri ction of (see Methods) yielded 2 min compounds: which mde up *95% of the unpuri ed dye nd violet frction (VIMP) which mounted to *5% of the unpuri ed dye. By use of n.m.r.-spectroscopy nd silicgel chromtogrphy we found tht VIMP is n oxidtively modi ed derivtive of most proly resulting from oxidtion of the lterl Chicgo cid SS moieties of y moleculr oxygen (see Figure 5). Previous studies of -medited e ects on glutmte receptors (Leûmnn et l., 1992; Keller et l., 1993) were performed with the unpuri ed dye nd, thus, in the presence of VIMP. Therefore, we sought to determine, whether VIMP hs n e ect on non-nmda-receptor medited currents on its own. Preincution (30 s) of neurones with 10 mm VIMP (the concentrtion of VIMP ws clculted sed on the estimted moleculr weight of 1000 g mol 71, see Methods) resulted in reversile reduction of pek (57+16% of control) nd stedy stte (60+34% of control) current mplitudes (Tle 1) in ll neurones investigted (n=9). Activtion nd desensitiztion time courses of the glutmte-ctivted currents were un ected. As expected, the inhiitory e ect of VIMP on AMPA (200 mm)-evoked currents ws indistinguishle from the e ect on glutmte-evoked currents (not shown). The IC 50 vlue for the pek reduction y VIMP ws estimted to e elow 3 mm since 10 nd 3 mm VIMP showed comprle ntgonistic potency (not shown). In contrst to the di erentil modultion y, identicl e ects of VIMP were oserved in cells showing either predominnt mplitude lock or predominnt inhiition of desensitiztion upon ppliction (compre Figure 6 nd ). This suggests tht the oxidtion of the Chicgo cid residues in VIMP prevents the inhiitory e ect on desensitiztion.

6 242 B. SchuÈrmnn et l Chicgo cid SS (CASS) The results otined with VIMP indicted tht the CASS moieties of re criticl for the modultion of desensitiztion y. Consequently, we determined the e ects of free CASS on non-nmda receptormedited currents. Normlized pek current AMPA receptor modultion y Evns Blue In ll cells investigted (n=16) preincution (60 s) of the neurones with 100 mm CASS signi cntly prolonged the time constnts of desensitiztion of glutmte-evoked whole cell currents (from ms to ms, P50.01) nd incresed stedy-stte current mplitudes to % of control vlues (Tle 1). The rise time nd pek current mplitudes were not ected y CASS (Figure 6). Preincution of neurones for 1 min ws oligtory to otin the stedy stte e ect of CASS. For incution times 55 min the modultory e ect of CASS ws fully reversile. The e ect of CASS ws the sme irrespective of the type of modultion otined with in the sme cell. Thus, CASS modulted desensitiztion even in cells tht showed the predominnt mplitude lock with (see Figure 6). Preincution of the neurones with only 10 mm CASS hd no detectle e ect on ny of the inspected properties of glutmte-induced currents. Hence, lthough the modultion of desensitiztion y seems to depend on the presence of unltered Chicgo cid moieties in the dye molecule, free CASS only wekly modulted desensitiztion. Thus, the chemicl con gurtion of CASS in the molecule most likely improves the potency nd confers the di erentil modultion of non-nmda receptors on. Normlized per cent desensitiztion (µm) Modultion of glutmtergic synpses y nd relted compounds We wnted to determine whether the modultion of synptic non-nmda receptors y re ected the phrmcologicl pro le otined for glutmte-evoked whole cell currents. Evoked glutmtergic utptic currents were investigted in microislnd cultures (see Methods) of postntl rt hippocmpl neurones fter 10 ± 14 DIV. At holding potentil of c AMPA 200 µm AMPA 200 µm Wsh 50 pa Kinte 500 µm d Wsh Glutmte 1 mm (µm) Figure 3 Dose-response reltionship for the two modultory e ects of Evns Blue (). () Plot of the reduction of current mplitudes of L-glutmte (1 mm) -induced whole cell currents vs the concentrtion. Ech point represents the men (n54 cells; ll cells from either group (A) or (C), compre Tle 1) pek current mplitude otined in the presence of the indicted concentrtion divided y the respective control current mplitude of the sme cell. () Plot of the men (n54 cells, ll cells from group (C)) per cent desensitiztion (i.e. 1006(pek current7stedy stte current)/(pek current7seline current) in the presence of vrious concentrtions of divided y the respective control vlue (in the sence of ) of the sme cell. Dt points were tted y the Hill eqution (n H =Hill coe cient, IC 50 =concentrtion producing hlf mximl inhiition). In (): IC 50 =10.7 mm, n H =1.2; in (): IC 50 =13.3 mm, n H =1.3. Insets in oth () nd (): fmily of currents otined from two typicl cells t indicted concentrtions (in mm). Brs represent 40 ms nd 20 pa, respectively. Wsh Wsh Surmin 75 pa Figure 4 Binding to AMPA receptors medites the two distinct e ects of Evns Blue (). AMPA (200 mm) evoked whole cell currents in postntl rt hippocmpl neurones in the presence of 10 mm (,). Similr to glutmte, AMPA evoked currents showed the predominnt lock of pek currents () nd the mixed lock of mplitudes nd desensitiztion () in di erent susets of cells. Kinte (500 mm) induced whole cell currents (c) showed no indiction of mcroscopic desensitiztion. Following pre-equilirtion of the cells with 10 mm, kinte-induced currents were reversily reduced in mplitude. The ntgonism of t P 2X - purinoceptors does not ccount for the modultion of AMPA receptors since nother such ntgonist (surmin 10 mm) left glutmte induced whole cell currents unltered (d).

7 B. SchuÈrmnn et l AMPA receptor modultion y Evns Blue 243 Evns Blue () Chicgo cid SS (CASS) Thus, wheres the potencies of nd VIMP to lock the mplitude of utptic currents were nerly identicl to the respective results oserved for the glutmte-evoked currents, neither nor CASS chnged the decy of the utptic responses. If one ssumes tht the non-nmda receptors sensitive to modultion of desensitiztion y re locted postsynpticlly, these results suggest tht desensitiztion does not contriute signi cntly to the decy of glutmtergic synptic currents in cultured hippocmpl neurones. Discussion VIMP 1 2 Figure 5 Chemicl structure of the AMPA receptor modultors nd silic gel chromtogrphy. () Wheres medites oth e ects on glutmte ctivted currents (i.e. lock of desensitiztion nd mplitude lock), Chicgo cid SS (CASS, sme compound s lterl residues in ) is pure inhiitor of desensitiztion (see text). The sterisks in the formul indicte the cndidte C-toms of tht re oxidized in VIMP. () Silic gel chromtogrm of puri ed mnipulted in the presence (1) or sence (2) of moleculr oxygen. In the presence of surfce ctlysts, such s silic gel, moleculr oxygen oxidizes the CASS residues in, yielding VIMP (see Methods). 770 mv somtic voltge-ctivted N + currents were elicited to trigger trnsmitter relese. Under control conditions evoked glutmtergic utptic currents hd men current mplitude of pa (n=14) nd monoexponentilly decyed with men time constnt of ms (rnge: 3.4 ± 6.5 ms). Autptic currents were elicited in regulr intervls (0.1 Hz) until stle seline vlues were otined. Superfusion of the microislnd cultures with (10 mm) resulted in reversile reduction of the utptic currents to 39+19% of control vlues in ll cells investigted (Figure 7; n=12), which is close to the mplitude lock otined for glutmte-evoked whole cell currents (inhiition to 41+17% nd 56+20% of control; see Tle 1). Interestingly, the time constnt of decy of the utptic responses remined unchnged in ll cells investigted (see Tle 2), lthough in the sme preprtion more thn hlf of the cells showed pronounced modultion of desensitiztion of glutmte-evoked whole cell currents y. Superfusion of the utpses with VIMP (10 mm, Figure 7) cused reversile reduction of the utptic responses to the sme extent s oserved for glutmte-evoked currents (see Tle 2). Likewise, the kinetic properties of utptic responses were lso not ffected. Finlly, unlike the smll reduction of desensitiztion in whole cell recordings, superfusion of neurones with 100 mm CASS left the decy of utptic currents unchnged. The lck of e ect of CASS on mplitudes of utptic currents is in greement with the respective results otined for its e ect on glutmte-evoked currents (Figure 7c; Tle 2). Neither of the derivtives ected the mplitude or time course of voltge-ctivted somtic N + or C 2+ currents (n=15, not shown). AMPA receptor sutype speci c ntgonists or modultors would e vlule tool to chrcterize the receptors involved in cellulr responses to glutmte ppliction nd in glutmtergic synptic trnsmission in di erent neuronl popultions. The dizo dye Evns Blue () hs een shown previously to e promising cndidte to modulte speci clly non-nmda receptors (Leûmnn et l., 1992; Keller et l., 1993). Con icting results existed with respect to the exct mode of ction of. Our results now show tht hs two distinguishle e ects on AMPA receptor medited currents, nmely lock of current mplitudes nd inhiition of desensitiztion. Secondly, we provide evidence tht the mjor impurity (VIMP) of the commercilly ville unpuri ed potently inhiits glutmte evoked whole cell currents in ll cells investigted. Finlly we show tht the decy of glutmtergic synptic currents remins un ected upon or CASS ppliction, suggesting tht either receptor desensitiztion does not contriute signi cntly to the termintion of the synptic response or the non-nmda receptors sensitive to modultion of desensitiztion y re not locted in the post-synptic memrne. Non-NMDA receptor-medited whole cell currents The properties of non-nmda receptor-medited whole cell currents in this study re comprle to previously pulished results from postntl rt hippocmpl neurones. More speci clly, our men time constnt of desensitiztion ( ms) reltes to the 40 ms (Kiskin et l., 1986), 70 ms (Thio et l., 1991) nd 10 ± 30 ms (Ptneu & Myer, 1991) found y others. However, direct comprison of such dt is complicted y the fct tht di erent (concentrtions of) gonists were employed in ll these studies. In ddition, the time constnt of desensitiztion in the whole cell recording mode is lwys limited y the speed of solution exchnge t the cell memrne ecuse delyed ctivtion of neighouring receptors rti clly prolongs mcroscopic desensitiztion. This interprettion is corroorted y the nding tht the sme experimentl setup llowed us to oserve two fold fster desensitiztion time constnts in outside-out ptch recordings (i.e ms) from the sme neurones, which is lmost identicl to the respective desensitiztion time constnts descried y Colquhoun et l. (1992). However, the reltively lrge sctter in the whole cell desensitiztion time constnts etween di erent hippocmpl neurones in our cultures (12 ± 45 ms) still seems to re ect, t lest in prt, cell speci c differences in non-nmda receptor properties, since we could not detect correltion of the time constnts of desensitiztion with the speed of the glutmte ppliction (see Figure 1). In fvour of this interprettion Fleck et l. (1996) hve recently otined similr sctter in the time constnts of desensitiztion for hippocmpl neurones. Cell speci c di erences in glutmte uptke kinetics re not likely to ccount for the heterogeneity of desensitiztion, since the fstest time constnts of desensitiztion in our whole cell recordings (12 ms) re close to the vlues oserved in outside-out ptch recordings from non-nmda receptor chnnels (see e.g. Colquhoun et l., 1992), nd glutmte uptke should decrese rther thn increse the time constnts of desensitiztion in the whole cell con gurtion s is oserved here. Whether the heterogeneity in

8 244 B. SchuÈrmnn et l AMPA receptor modultion y Evns Blue A (10 µm) CASS (100 µm) VIMP (10 µm) Glutmte 1mM c,c,c 100 pa 50 pa 100 pa B (10 µm) CASS (100 µm) VIMP (10 µm),c 50 pa 50 pa c 50 pa c Figure 6 Modultion of glutmte induced currents y VIMP nd CASS. Hippocmpl neurones showing lock of desensitiztion with 10 mm (A, sme cell for ll currents) showed reduced desensitiztion with CASS (Chicgo cid SS, 100 mm) nd lock of pek current mplitudes in the presence of 10 mm VIMP (violet impurity, oxidtively modi ed in the CASS residues); : control, : in the presence of the modultor; c: wsh. Hippocmpl neurones showing lock of current mplitudes with 10 mm (B, sme cell for ll currents) lso showed reduced desensitiztion with 100 mm CASS nd lock of pek currents with 10 mm VIMP. Thus, CASS ws pure modultor of desensitiztion nd VIMP pure modultor of current mplitudes irrespective of whether ws n inhiitor of desensitiztion (A) or n inhiitor of current mplitudes (B) of glutmte ctivted currents in the sme neurone. the time constnts of desensitiztion re ects di erentil expression of AMPA receptor suunits in di erent cells, s hs een shown directly with single-cell PCR nlysis in hippocmpl nd corticl neurones in vitro (Bochet et l., 1994; Jons et l., 1994; Geiger et l., 1995; Lmolez et l., 1996), cnnot e deduced from our experiments. E ects of nd derivtives on whole cell currents The modultion of glutmte ctivted whole cell currents y s shown here is in greement with the e ects found for thlmic neurones (Leûmnn et l., 1992). However, in this previous study the mixed desensitiztion/mplitude e ect of (5 mm) ws found in ll cells investigted. This di erence corroortes the nding tht thlmic nd hippocmpl non- NMDA receptor medited currents di er with respect to their kinetic properties (Colquhoun et l., 1992; Leûmnn & Gottmnn, 1994), nd could result from distinct AMPA receptor suunit compositions in neurones from these rin regions. Since even puri ed (purity 499%) still exerts oth e ects on non-nmda receptors, the di erentil e ects of on desensitiztion nd current mplitudes cnnot e explined y the presence of di erent components in the unpuri ed dye. In the present study, the neurones were ssigned to di erent groups (A through C) depending on the type of e ect oserved with (see Figure 2 nd Tle 1). This necessrily tends to e ritrry t the edges of the groups. The criterion to ssign neurone to group C (mixed e ect) insted of B (predominnt desensitiztion e ect) ws reduction of the pek current mplitude. The criterion to ssign neurone to group C insted of A (predominnt pek inhiition) ws chnge in the time constnt of desentitiztion. As is evident from Figure 2e the qunti ction of desensitiztion y di erent procedure (per cent desensitiztion) would justify the ssignment of some neurones to the neighouring group s well. However, the presence of 2 clerly distinguishle types of e ects produced y (groups A nd B) nd n intermedite group C etween these extremes is evident from our experiments whtever method of nlysis of the currents is selected. It could e rgued tht even in the group B neurones there still ws pek current reduction y tht ws msked y the lrge inhiition of desensitiztion y. Single chnnel recordings will hve to e performed to solve this issue de nitely. However, the fct tht the proportions of the e ects of on desensitiztion versus current mplitudes vried etween di erent neurones is not questioned y this hypothesis. Irrespective of the ssignment of the neurones to the groups A through C, prolonged the rise time of the glutmte induced currents in ll neurones investigted. A prolongtion of the rise time is n expected nding for drug tht ects the gting of the non- NMDA receptors. Whether the modultion of rise times is third independent e ect of or whether it is correlted with its e ects on desensitiztion nd current mplitudes ws not investigted systemticlly here. This kind of correltion hs to e investigted in outside-out ptch recordings, where the solution exchnge is fst enough to resolve the ctivtion kinetics of the non-nmda receptor chnnels relily nd will hve to e ddressed in future studies. The di erentil modultion of non-nmda receptors y showed no correltion with distinct properties of glutmtectivted control currents (e.g. slow or fst desensitiztion time constnts; see ove). In contrst, Fleck et l. (1996) hve shown recently tht the sensitivity of AMPA receptors for cyclothizide directly correltes with the time constnt of desensitiztion. Tken together, oth results suggest tht the receptor properties sensed y re di erent from those distinguished y the di erentil sensitivity to cyclothizide, suggesting tht more thn two distinguishle groups of non- NMDA receptor sutypes re present in hippocmpl neurones. Under our experimentl conditions glutmte ctivted predominntly AMPA receptors nd AMPA-induced whole

9 B. SchuÈrmnn et l AMPA receptor modultion y Evns Blue 245 Tle 2 Modultion of AMPA receptor medited utptic currents y Evns Blue nd relted sustnces Test Amplitude t (decy) sustnce (% of control) (ms) (mm) n Test Wsh Test Wsh (10) VIMP (10) CASS (100) * 64+24* * Neurones were equilirted (530 s) with either Evns Blue (), oxidized (VIMP) or Chicgo cid SS (CASS). Evoked glutmtergic utptic currents (n=3 ± 6) were verged. Amplitudes of pek currents nd the time constnt of the decy of utptic currents (t) were clculted from this verge currents (`test'). Current mplitudes re given s percentge of `control' efore the ppliction of modultor. The extent of recovery (`wsh') is indicted. Dt re given s mens+s.d. *Signi cntly di erent from control with P<0.01 (unpired Student's t test). c τ=4.7 ms τ=4.8 ms 10 µm τ=4.9 ms VIMP 10 µm τ=3.9 ms CASS 100 µm Wsh τ=4.9 ms Wsh τ=3.9 ms Wsh τ=5.1 ms τ=4.6 ms τ=4.6 ms 100 pa 100 pa 15 ms 150 pa Figure 7 Modultion of glutmtergic utptic currents y Evns Blue () nd relted sustnces. Autptic currents were elicited y short depolrizing current pulses leding to somtic voltge ctivted N + currents (truncted downwrd de ections). The hippocmpl microcultures were preincuted for t lest 1 min with either of the indicted modultors ( ± c). Ech current is the verge of 3 individul trces otined efore the ppliction of modultor (), fter equilirtion with modultor (middle trce) nd fter wshout (Wsh) of the respective sustnce. The decy time constnts (t) represent the result of monoexponentil t to ech verge current. The resulting t curves re shown s stippled lines in (), nd were omitted in () nd (c). Wheres the lock of pek current mplitudes y nd VIMP ws comprle to the corresponding e ects on whole cell currents, neither nor CASS chnged the decy of the glutmtergic utptic currents. cell currents were lso di erentilly modulted y. Thus the simplest explntion for the cell speci c e ects of is the di erentil expression or ssemly of the AMPA receptor suunits (GluR1-4) in di erent susets of cells. This interprettion could in principle e supported y single cell RT-PCR nlysis, relting di erent ptterns of AMPA receptor suunit compositions to the distinct modultory e ects of. However, the di erentil modultion of desensitiztion nd current mplitudes y oserved in the present study ppers to e grded phenomenon (compre Figure 2e). Thus, it seems likely tht the di erences in glutmte receptors sensed y rther result from quntittive thn from qulittive vritions in AMPA receptor suunit expression. To otin convincing results from quntittive single cell PCR nlysis is very dif- cult tsk nd ws eyond the scope of the present study. Our future experiments will focus on heterologously expressed homomeric nd heteromeric recominnt AMPA receptors to revel possile involvement of speci c AMPA receptor suunits in the di erentil modultion y. Amplitude reduction y of kinte-induced currents through heterologously expressed AMPA receptors contining either GluR1 or GluR2 suunits hs een shown to occur with n IC 50 of *0.5 mm (Keller et l., 1993). The higher potency of in the oocyte expression system s compred to the ntive hippocmpl receptors nlysed here (IC 50 *10 mm) might result from inherent di erences in these distntly relted expression systems. In spite of these di erences in IC 50 vlues the mplitude locking e ect of in our study could in principle e ttriuted to the presence of GluR1 nd/or GluR2 suunits in the ntive hippocmpl AMPA receptors. As expected y the presence of either GluR1 or GluR2 suunits in virtully ll hippocmpl neurones in vitro (Crig et l., 1993; Eshhr et l., 1993) nd in vivo (Sto et l., 1993), we found the mplitude lock of in the mjority of cells (45 of 52 cells=87%). Interestingly, the reduced desensitiztion in response to ws found in only 30 out of 52 cells (58%), suggesting tht less prominent AMPA receptor suunits or suunit splice vrints could confer the sensitivity of desensitiztion to on AMPA receptors. A receptor suunit speci c modultion of desensitiztion of non-nmda receptors hs een shown for cyclothizide nd concnvlin A (Prtin et l., 1993; Wong & Myer, 1993). These two gents discriminte etween AMPA receptor- (GluR1-4, sensitive to cyclothizide) nd kinte receptor-suunits (GluR5-7 nd KA1-2, sensitive to concnvlin A). In ddition, the desensitiztion properties of the ip splice vrints of the heterologously expressed AMPA receptor suunits GluR1-4 in HEK293 cells re more sensitive to cyclothizide thn the respective op splice vrints (Prtin et l., 1994). Provided tht speci c AMPA receptor suunit(s) sensitive to the desensitiztion nd/or mplitude e ect of will e identi ed, the dye could fcilitte the identi ction of AMPA receptor suunits during electrophysiologicl recordings. The di erentil modultion of AMPA receptors y must not necessrily result from di erences in receptor suunit expression. Di erences in the phosphoryltion or glycosyltion sttus of AMPA receptors, in the lipid microenvironment or in the interction with ttched proteins could eqully well explin the di erentil e ects of. However, to our knowledge such di erences hve not yet een descried to modulte the kinetics nd mplitudes of AMPA receptor medited currents. In n ttempt to deduce the residues of which re criticl for the 2 modultory e ects, we performed experiments with

10 246 B. SchuÈrmnn et l VIMP nd CASS. VIMP, which is closely relted derivtive of, oxidized in the lterl CASS residues, retined only the locking e ect of on current mplitudes. This suggests tht unltered CASS residues in re criticl for the modultion of desensitiztion. However, isolted CASS modultes desensitiztion less e ectively nd with lower potency thn. In ddition, CASS does not show di erentil modultion of desensitiztion. Thus, the chemicl environment in confers higher potency nd the cell speci c e ects on the CASS residues. E ects of nd derivtives on glutmtergic synptic trnsmission The time constnt of the decy of postsynptic currents in our preprtion ( ms; rnge: 3.4 ± 6.5 ms) closely mtches the respective vlues tht cn e otined from hippocmpl neurones in situ (3 ± 9 ms (Keller et l., 1991); 3 ± 7 ms (Jons et l., 1993); 4 ± 8 ms (Hestrin et l., 1990)). Although it is likely tht the decy time constnts in our study (s in others) re slowed y dendritic ltering, we elieve tht our results otined with justify the conclusion tht non-nmda receptor desensitiztion does not dominte the synptic decy in our prepriton: we show tht signi cntly increses the time constnt of desensitiztion y fctor of 3 ± 20 nd increses the stedy stte current component of glutmte-induced whole cell currents in 58% of the hippocmpl neurones (see Tle 1). If desensitiztion does contriute signi cntly to the termintion of the glutmtergic synptic current in our preprtion, the synptic current decy should e slowed to comprle extent. However, does not show ny sign of prolongtion of the decy of glutmtergic utptic currents in the sme preprtion lthough the ccessiility of the susynptic non-nmda receptors to is indicted y the pek inhiition of the synptic responses, tht occurs to the sme extent s the inhiition of the glutmte-evoked whole cell responses (compre Tles 1 nd 2). Tken together, these results indicte tht non-nmda receptor desensitiztion does not contriute signi cntly to the decy of glutmtergic synptic currents in hippocmpl neurones. This conclusion is in line with previous results otined from the comprison of the time constnts of desensitiztion of glutmte-induced currents in outside-out ptch recordings with the decy of synptic responses in the hippocmpus (Colquhoun et l., 1992). Alterntively, we cnnot exclude the possiility tht the non- NMDA receptor sutypes, sensitive for modultion of desensitiztion y re not locted in the postsynptic memrne. In ddition, we cnnot de nitely rule out tht lso hs some presynptic e ects, lthough voltge ctivted N + nd C 2+ currents were not ected nd the synptic mplitude lock cn e entirely explined y the e ect of on postsynptic non-nmda receptors. hs een shown to inhiit vesiculr glutmte uptke in rt centrl neurones (Roseth et l., 1995). Since synptosoml uptke ws not ected in this study nd does not penetrte the plsm memrne, presynptic e ects on glutmte uptke re lso not likely to hve in uenced our results. At rst glnce, the results presented here seem to e contrdictory to the slowing of the decy of glutmtergic synptic currents y 5 mm in thlmic neurones (Leûmnn et l., 1992). AMPA receptor modultion y Evns Blue However, it hs een shown previously tht the contriution of desensitiztion to the decy of glutmtergic synptic responses vries gretly, depending on the prticulr neuronl circuit investigted. For exmple, the desensitiztion kinetics in outside-out ptches from neurones of di erent rin regions in response to step ppliction of glutmte were compred with the respective decy time constnts of synptic currents from the sme rin re: wheres in hippocmpl nd neocorticl neurones desensitiztion proceeds too slowly to contriute to the decy of the synptic response (Colquhoun et l., 1992; Hestrin, 1992), desensitiztion in thlmic neurones, spinl cord neurones nd neurones from the nucleus mgnocellulris is fst enough to contriute signi cntly to the decy of synptic trnsmission (Trussell & Fischch, 1989; Trussell et l., 1993; Leûmnn & Gottmnn, 1994). By use of di erent chnnel modultors (nircetm, cyclothizide, ) desensitiztion of non-nmda glutmte receptors hs een shown to contriute to the termintion of synptic trnsmission in vrious preprtions (cultured hippocmpl neurones: Tng et l., 1991; Ymd & Tng, 1993; hippocmpl slice: Iscson & Nicoll, 1991; nmag: Trussell et l., 1993; cultured thlmic neurones: Leûmnn et l., 1992). The modultory ction of nircetm nd cyclothizide t hippocmpl synpses might t lest in prt e explined y the dditionl slowing of chnnel closure y oth drugs (Tng et l., 1991; Hestrin, 1992; Ymd & Tng, 1993). In fvour of this interprettion recent phrmcologicl study (Rmmes et l., 1996) hs shown tht the slowing of glutmtergic synptic responses y cyclothizide t lest in prt re ects delyed chnnel closure upon removl of glutmte from the synptic cleft. If desensitiztion does not ect the decy of glutmtergic synptic currents in our hippocmpl preprtion (s suggested y the results otined with ), dectivtion of non-nmda receptors must e responsile for the synptic decy. Provided tht the non-nmda receptor complexes sensitive for the modultion of desensitiztion y re expressed susynpticlly, this could e interpreted such tht, t the concentrtions used here, does not modulte the dectivtion of non- NMDA receptors. However, to nd out whether hs n e ect on non-nmda receptor dectivtion, future studies with very short (1 ms) glutmte pulses (tht cnnot e chieved with our superfusion system) pplied to outside-out ptches must e performed. In conclusion, the dizo dye Evns Blue () modultes desensitiztion nd current mplitudes of AMPA receptormedited currents to vrious extents in di erent hippocmpl neurones. The chemicl con gurtion of the lterl Chicgo cid residues of is crucil for this di erentil modultion to e oserved. Glutmtergic utptic currents show only sensitivity to the mplitude lock exerted y, thus suggesting tht desensitiztion does not signi cntly ect the decy of the synptic currents. This work ws supported y the Deutsche Forschungsgemeinschft (DFG) nd the Hochschulsonderprogrmm II. We would like to thnk Prof. R. Heumnn for helpful discussions nd generous support. We wnt to thnk Sine Hoppe for skillful technicl ssistnce, P. Wol nd C. Ludwig for help with the n.m.r. spectr, nd Dr K. Gottmnn for criticl comments on the mnuscript. References BEKKERS, J.M. & STEVENS, C.F. (1991). Excittory nd inhiitory utptic currents in isolted hippocmpl neurones mintined in cell culture. Proc. Nt. Acd. Sci. U.S.A., 88, 7834 ± BLASCHKE, M., KELLER, B.U., RIVOSECCHI, R., HOLLMANN, M., HEINEMANN, S.F. & KONNERTH, A. (1993). A single mino cid determines the suunit-speci c spider toxin lock of -mino-3- hydroxy-5-methylisoxzole-4-propionte/kinte receptor chnnels. Proc. Nt. Acd. Sci. 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