from slices of rat olfactory cortex after anoxia

Transcription

1 Br. J. Phrmcol. (1994), 111, '." Mcmilln Press Ltd, 1994 NMDA ntgonists increse recovery of evoked potentils from slices of rt olfctory cortex fter noxi Mged Yssin & IC.N. Scholfield School of Biomedicl Science, Queen's University, 97 Lisurn Rod, Belfst, BT9 7BL 1 The role of glutmte in producing tissue dmge during cererl noxi ws investigted in rin slices using ntgonists to the NMDA nd AMPA receptor types. 2 Tissue function ws ssessed y field recordings of the synpticlly evoked potentils elicited y stimulting the min fferent input to the olfctory cortex, the lterl olfctory trct. ws produced y thing the slice in glucose-free solution equilirted with 95% N2/5% CO2. 3 The mount of recovery of the evoked potentil ws inversely dependent on the period of noxi nd temperture: t 24 C, 15 min of noxi followed y reoxygention produced 14.6 ± 4.1 % recovery wheres there ws no recovery t 35 C. 4 Dizocilpine nd ketmine hd no effect on synptic trnsmission in oxygented medi ut following noxi they produced n incresed recovery of the responses: from 14.6 ± 4.1% to 48.3 ± 7.8% for dizocilpine (10 AM) nd 21.6 ± 7.7% to 87.2 ± 7.1% for ketmine (200,M); the tissue endurnce to noxi ws incresed y round 5 min. 5 Blockde of the AMPA receptors did not influence recovery in spite of the depressed synptic trnsmission. A similr synptic ttenution produced y lignocine provided some increse in postnoxic recovery. 6 The NMDA receptor ntgonist, AP5, ntgonized NMDA t 50 AM y 3.7 fold nd t 200 AM y 15 fold ut only 200 AM incresed post-noxic recovery. This suggests tht sustntil degree of NMDA ntgonist is required efore noxic tissue dmge due to NMDA receptor ctivtion cn e nullified. The ntgonist to the glycine inding site, 7-chlorokynurenic cid lso incresed recovery. 7 These in vitro experiments confirm the ide tht NMDA receptor ctivtion mkes sustntil contriution to cererl tissue dmge nd tht this cn e reduced y sustntil lockde of these receptors. Keywords: Glutmte; NMDA; noxi; rin; neuroprotection; rt; olfctory cortex; dizocilpine Introduction A vriety of chnges tke plce in tissues during oxygen deprivtion. The centrl nervous system is prticulrly vulnerle to ischemi prtly ecuse of glutmte relese (Benveniste et l., 1984; Rothmn, 1984; Drejer et l., 1985; Snchez-Prieto & Gonzlez, 1988; Nicholls & Attwell, 1990) which cuses cell deth (Choi, 1988; Frndsen et l., 1989; Meldrum & Grthwite, 1990). Under pthologicl conditions, glutmte cts on severl receptor systems. For cell pthology, the most importnt of these is the N-methyl-Dsprtte receptor type (NMDA) since it is coupled to voltge-dependent chnnels tht re C2+perment (Grthwite & Grthwite, 1986; McDermott et l., 1986; Choi, 1987; Michels et l., 1990) producing C2" loding of the cell. This loding in turn ctivtes severl vigorous Cctivted enzymes including phospholipse, proteses nd nucleses leding to cell deth. There hs een n ssessment of severl strtegies tht might provide some protection of neurl tissue during ischemi nd one of these is sed on NMDA ntgonists. Severl studies hve found NMDA ntgonists to offer some protection ginst ischemi/noxi (Prk et l., 1988; Kochlr et l., 1988; Bielenerg, 1989) whilst in others, the protection hs een smll or sent (Perkins et l., 1988; Aitkin et l., 1988; Fleischer et l., 1989; Sterz et l., 1989; Lnter et l., 1990; McDonld et l., 1990). Most studies on the phrmcology of neuroprotection hve een in vivo. Such studies re est suited to the ssessment of sustnces tht might hve immedite clinicl ppliction. However, there re lrge numer of vriles thn cn contriute to the tissue dmge so the mechnism of ction of protective gents is ' Author for correspondence. more difficult to nlyse. For exmple, in cererl ischemi, tissue dmge might result from oxygen lck directly or from ccumulted metolites. Furthermore, since most in vivo experiments hve een conducted over severl dys, the postischemic chnges might e secondry to gliosis or ltertions in the lood rin rrier (Giulin, 1993). Some of the prolems encountered in vivo re circumvented in experiments with isolted tissues. Although most studies hve concentrted on the microscopic chnges fter glutmte gonist insult some hve looked to the effects of noxi on evoked potentils in the hippocmpus (Clrk & Rothmn, 1987; Rder & Lnthorn, 1989; Pps et l., 1993). In the present study, we hve ssessed the effects of noxi y following the chnges in electricl ctivity in slices of rt olfctory cortex mintined in vitro. Two gols were in mind () to elucidte the mechnisms underlying the noxiinduced cell deth, nd () to find sustnces tht might provide tissues with some protection during noxi. We show tht some NMDA ntgonists produce sustntil increse in the recovery of tissues following short periods of noxi. A preliminry ccount of this work hs een pulished (Scholfield & Yssin, 1992). Methods Femle Sprgue-Dwley rts of g weight were decpitted nd their rins removed. The rin ws isected long the mid-line nd pil surfce slices of olfctory cortex were cut to thickness of either 350 pm for studies t 35 C or 450 ym for 24 C. The time etween decpittion nd plcement of the slices in oxygented thing solution t 24 C ws 3 min, period too short to produce permnent

2 1222 M. YASSIN & C.N. SCHOLFILD chnges in tissue responsiveness to electricl stimultion (see results). The two olfctory cortex slices were ech sudivided long the xonl rditions into three, such tht ech hd length of lterl olfctory trct (LOT). The slices were plced in 50 ml th of Kres solution contined within sintered glss Buchner funnel. 95% 02/5% CO2 ws supplied to the ottom of the funnel llowing the slices to e continully gitted in the thing medium. Individul slices were plced on nylon mesh within recording chmer (effective volume of 0.5 ml) nd superfused with Kres solution equilirted with 95% 02/5% CO2 t 5 ml min-' y recircultion from 50 ml reservoir. The connections were through silicon ruer tuing the length of which ws minimized to void gseous de-equilirtion. The gs mixture ws lso lown over the surfce of the recording chmer to void gseous exchnge with ir. The reservoir ws wter-jcketed nd the flow line immeditely djcent to the recording chmer contined het exchnger to mintin the recording chmer t either 240 or 350C. A pir of stimulting electrodes ws plced on the LOT nd recording pipette plced on the pil surfce 2 mm wy from the stimulting electrodes. The electrode ws connected to premplifier nd the wveforms were digitized, stored nd nlysed y computer. The LOT ws continully stimulted with 10 V, 0.2 ms pulses t 0.05 Hz nd ech response stored. The tissue ws llowed to equilirte for 30 min in the th efore the collection of records. Antgonist ws dded to the reservoir nd recirculted for 30 min. The recirculting solution ws then chnged for glucose-free solution equilirted with 95% N2/5% CO2. This nitrogen gs mixture ws lso lown over the surfce of the recording chmer. After the period of noxi, the thing solution ws chnged ck to norml solution equilirted with 95% 02/5% CO2. Most experiments were performed t 24 ± 10C (mximum rnge) ut we lso looked t some effects t temperture nerer to tht in vivo (380C in the rt). However, t this temperture, the responses ecome unstle (Scholfield, 1980) presumly ecuse of greter oxygen demnds. Therefore to void the possiility of metolic insufficiency nd 'sensitiztion' to noxi, experiments were conducted t 35C rther thn 38TC. Test sustnces were present for 30min efore nd during the noxi period. They were not present during the wshout period. The concentrtion of NMDA ntgonist ws ssessed in nother series of experiments. The mplitude of the evoked potentil ws monitored during the ppliction of NMDA t M in norml solution nd fter 30 min equilirtion in comintion with vrious ntgonists. Between nimls, sustntil mount of vriility ws oserved. To reduce the effects of vriility, control experiment ws performed in norml solution on one slice nd then the test ntgonist ws tested on nother slice from the sme niml. The next pir of slices, the test experiment preceded the control. Some slices were used within 2 h of cutting wheres others were used up to 6 h fter preprtion. There ws no time-dependent difference in control effects of noxi. ch slice ws sujected to one period of noxi only. In some experiments, rnge of noxic periods ws used. For most experiments, one time period ws used to produce 5-30% control recovery, level chosen to llow for ny enhncement in the recovery of test slices. The pek mplitudes of the synptic responses were mesured nd the mount of recovery expressed s proportion of the response immeditely efore the noxic period. Test nd control experiments were compred y Student's pired t test (two tiled). All vlues re mens ± s.e. of men nd significnce ws tken s P<0.05. The Kres solution hd the following composition (mm): N+ 144, K+ 5.0, C2` 2.5, Mg2+ 1.3, Cl- 128, HCO3-25, D-glucose 2.0 nd it ws equilirted with 95% 02/5% CO2- The rther low glucose concentrtion ws necessry ecuse previous work (Donghy & Scholfield, 1990) hd shown delyed response to noxi when slices were stored with 11 mm. During noxi, glucose ws removed since it hd een shown lso tht responses could e sustined in glucose. The drugs used were from the following sources: dizocilpine (MK801) Merck, Shrp nd Dohme, Hrlow, UK; 5,7- dinitroquinoxline-2,3-dione (DNQX), DL-2-mino-5-phosphopentnoic cid (AP5) nd 7-chlorokynurenic cid, Tocris Neurmin, Bristol, UK; 2-[2-chlorophenyl]-2-[methylmino]- cyclohexnone (ketmine), Sigm, Poole, UK; lignocine, Astr Lortories, Wtford, UK. Results Stimultion of the LOT elicited surfce negtive wveform which is the result of the relese of cidic mino-cid (Collins, 1979) cting on x-mino-3-hydroxy-5-methyl-4- isoxzoleproprionic cid (AMPA) type receptors (Collins & Buckley, 1989). NMDA receptors re present in olfctory cortex (Tcconi et l., 1993) nd they my lso e involved in synptic trnsmission (Collins, 1991) ut in our experiments, this component did not contriute to the evoked potentil (see elow). When recordings were mde from n re of the slice close to the LOT, the synptic potentil ws preceded y shorter trnsient response due to the ctivity of the xons underlying the recording electrode. This fire response is not pprent in Figure 1 ut cn e seen with oth preprtions in Figure 2. The fire potentil is close to the stimulus rtefct ut it cn e distinguished y compring the response lelled 'noxi' in Figure 2 where ll the iologiclly generted responses hd een lost. Without ny experimentl intervention, ll the responses were mintined constnt over period of severl hours. (Scholfield, 1980). ffect of noxi When the thing solution ws chnged to one equilirted with 95%N2/5%CO2 there ws rpid nd complete loss of oth the pre- nd postsynptic responses (Figures 1 nd 2). The response strted to decline when the new solution rrived t the slice nd this ws normlly complete within 4-6 min (Figure 3). When oxygention ws restored, the response recovered over period of 30min. In 5 slices, we followed the responses for further 5 h fter noxi ut there ws no further recovery. Therefore, routinely, we studied only the initil 30 min recovery period. In some experiments, noxi ws tested t 240 nd 350C. After 15 min of noxi the recovery ws 14.6 ± 4. 1% nd 3.5 ± 1.4% respectively. With some responses, postsynptic popultion spike could e resolved nd the reltion etween this nd the excittory postsynptic potentil (e.p.s.p.) cn e used to gin insight into the chnges in memrne potentil (Scholfield, 1980; Richrds, 1972). During the onset of noxi, there ws lrge depression of the e.p.s.p. with little chnge in the popultion spike. If the e.p.s.p. ws reduced y decresing the stimulus voltge, the e.p.s.p. nd popultion spike were reduced in sympthy. These oservtions indicte tht noxi cused postsynptic depolriztion. At 35 C, slices were exposed to vrying periods of noxi etween 5 nd 15 min nd the mount of recovery is shown in Figure 4. For short periods (5 min), recovery ws complete ut diminished with longer periods such tht little recovery ws seen fter 12 min. ffect of NMDA Appliction of NMDA (5-50gM) produced depression in the evoked potentil. The following NMDA ntgonists (Kemp et l, 1987) were tested for their ility to reduce the ction of NMDA: AP5 (50 nd 200 gm), 7-chlorokynurenic cid (50 gm), dizocilpine (10 gm) nd ketmine (200 gm). NMDA ws not locked immeditely y dizocilpine or ketmine ut ntgonism developed over period of 30 min

3 NMDA AND ANOXIA 1223 wheres AP5 nd 7-chlorokynurente showed full effect within 10min of ddition. The concentrtions of NMDA which produced 50% depression of the evoked response in the presence of the ove ntgonists re shown in Tle 1. All the sustnces tested produced sustntil ntgonism of NMDA nd AP5 ws dose-dependent. 3 :' I 20 ms S 02_ 3 I k-\t-w- j I- I I Dizocilpine 10 p.m Figure 1 Single synpticlly evoked potentils recorded from slices of rt olfctory cortex. In the upper trces (), the slices ws thed in norml solution ('norml') followed 15 min in solution equilirted with 95% N2/5% CO2 ('') followed y normlly oxygented solution ('wshout'). A time-course for similr experiment is seen in Figure 3. In the lower trces (), nother slice from the sme niml ws thed in dizocilpine (10 pm) for 30 min efore the period of noxi nd during the noxi. 'S' indictes the point of stimulus ppliction. On these trces, negtive is upwrds. 4- ) 3- - r 20 ms 1O 0 - S 4- > 3 ) = Ketmine 200 F- FLM W Figure 2 An experiment on evoked responses from nother pir of slices compring the effect of norml solution nd incution with ketmine (200 gm). In this experiment, the presysnptic ction is seen s trnsient etween the stimulus rtifct (rrow lelled 'S') nd the synptic response. This trnsient hs disppered from the records lelled ''.

4 1224 M. YASSIN & C.N. SCHOLFILD 5 ) t._ o 4. > 3. ~ 0 2-._ < 1-20 min 0- No l -1 Dizocilpine 10 jlm Figure 3 Time-courses for chnges in the mplitude of the evoked response. The preprtion ws stimulted t 0.05 Hz nd for ech response, the pek mplitude of the evoked potentil ws mesured nd plotted on the time-course s single point. ch point ws joined to produce the continuous trce shown. In () the slice ws thed successively in norml (oxygented) solution, in solution equilirted with N2/CO2 (12 min) nd then re-oxygented ('wshout'). () Shows time-course of slice from the sme niml ut with preincution period of 30 min in 1OIM dizocilpine. ffect of dizocilpine Appliction of 101M dizocilpine in norml oxygented solution hd no effect on the synptic response (Figures 1 nd 3) confirming tht ny involvement of NMDA receptors in this pthwy is smll (Collins, 1991). The presence of dizocilpine mde no difference to onset nd the decline response during noxi. However, the speed of recovery nd level of recovery ttined were sustntilly ugmented y this gent (Figure I nd 3). Thus t 24C, 15 min period of noxi produced recovery in norml solution which ws 14.6% compred to 48.3% in 10 LM dizocilpine (Tle 2). The sme effect ws seen t 350C for 12 min of noxi (6.3% recovery in norml solution nd 86.9% in dizocilpine) * - 80 >o 60{ Q T 40 20t \t (>O Period of noxi (min) Figure 4 ffect of vrying the period of noxi (sciss scle) on the recovery of the evoked potentil (ordinte scle) in norml solution (0) nd in the presence of 10 gtm dizocilpine (@). The ordinte scle is the rtio of the mplitude of the evoked potentil 30 min fter the period of noxi to tht efore the noxi. The lines were drwn y 'eye'. Tle 1 Concentrtions of N-methyl-D-sprtte (NMDA) which produced 50% depression of the evoked potentil Antgonist AP5 50gpM AP5 200 gm 7-Chlorokynurente 50g1M Dizocilpine 10 (m Ketmine 200 LM D50 for NMDA (~lm) Test 21.3 ± ± ± ± ± ± ± ± 55 > ± 13 Rtio 3.7 ± ± ± 3.7 > ± 1.3 n P-vlue 4 < The rtios re the vlues in ntgonist ('Test') divided y tht in norml solution ('') for ech slice. The n-vlues re the numers of pirs of slices nd the P-vlue clculted using the two-tiled t test. Tle 2 ffect of glutmte ntgonists on recovery fter period of noxi 0/ recovery % recovery xperiment control test Chnge Lignocine DNQX AP5, 50jM AP5, 200 gm 7-Chlorokynurente Dizocilpine Dizocilpine t 35 C Ketmine 8.1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 9.2 n P-vlue < < The chnge ws clculted y sutrcting the % recovery in norml solution (control) from tht in the experimentl solution (test) for pired slices from the sme nimls nd verging these. The P vlues were clculted from the pired two tiled t test for the control nd test groups. All experiments were done t 24 C with period of noxi of 15 min except those t 35 C where the period of noxi ws 12 min.

5 NMDA AND ANOXIA 1225 In one group of experiments, slices were exposed to vrying periods of noxi. For ll periods of noxi tested, there ws n enhnced recovery in the presence of dizocilpine (Figure 4). Thus in effect, the endurnce of the tissue ws incresed y 5-6 min. As in norml solution, there ws no further chnge in the mplitude of the response fter 30 min in the recovery solution. ffect of ketmine Ketmine t 200 jm hd no effect on synptic trnsmission (Figure 2): higher concentrtions were not tested since they depress xonl responses (McGivern & Scholfield, 1990). Nor did ketmine influence the depression produced y noxi ut like dizocilpine, the rte nd the mount of recovery ttined were sustntilly incresed (Tle 2). ffect of APS Both dizocilpine nd ketmine lock the chnnels ssocited with the NMDA receptor wheres AP5 ws tested s sustnce tht competes with the NMDA inding site. It ws tested t two concentrtions, 50 nd 200 jm. The lower concentrtion hd no effect on responses in normoxic nd noxic conditions nor ws the recovery significntly ffected y its presence (Tle 2). AP5 200 gm hd two cler effects: () lone it cused depression of the response from 1.45 ± 0.23 to 1.24 ± 0.22 mv ( 16 ± 1% depression, n = 14); () following noxi, it permitted greter recovery in the response Tle 2). ffect of 7-chlorokynurenic cid 7-Chlorokynurenic cid is n ntgonist t the glycine inding site (Kemp et l., 1988). Under normoxic conditions, it produced depressnt effect on its own (Figure 5) which ws concentrtion-dependent etween gm. To test its effect on recovery, 50 jm ws used nd this depressed the evoked potentils from 2.35 ± 0.20 to 1.34 ± 0.12 mv ( depression of 42.3%: n = 14). 7-Chlorokynurenic cid (50 pm) lso permitted sustntil increse in the recovery fter noxi (Tle 2). ffect of DNQX Neurotoxicity my lso rise from non-nmda receptors (Koh et l., 1990). Therefore n ntgonist to the AMPA receptor (DNQX) ws lso tested. At concentrtion of 10lM it depressed the synpticlly medited potentil confirming the role of AMPA receptors in norml trnsmission (Figure 6). After equilirtion for 30 min with this ntgonist, noxi completely olished the residul response. The DNQX ws wshed out t the sme time s reoxygention nd the response recovered to level similr to tht in slices without DNQX pretretment (Figure 6; Tle 2). ffect of other gents In some experiments ove, there ws depression of the response y the test gent (e.g., DNQX nd 7-chlorokynurenic cid) efore the period of noxi. We therefore tested the effect of reltively non-selective depressnt gent, lignocine. As shown in Tle 2, there ws smll ut significnt enhncement in the recovery provided y this gent ) I--T --.' l ~~~~ I ~0 ' > 4. 0) 3. I2I 20 s > l o 4 X~~~~V < 1' 01 7-Chlorokynurente 50 1AM Figure 5 A similr time course to Figure 3 during the ppliction of 50 gm 7-chlorokynurenic cid present during the period indicted y the solid r. Note tht there is some depression of response on dding the 7-chlorokynurenic cid. ' '' DNQX Figure 6 Another time-course using 5,7-dinitroquinoxline-2,3- dione (DNQX, 10 JAM) s the test sustnce. Note tht this gent on its own lso depressed the synptic response. The DNQX ws wshed out t the end of the period of noxi. Without the intervening period of noxi, the response would normlly recover completely from the DNQX.

6 1226 M. YASSIN & C.N. SCHOLFILD Discussion Tissue noxi produces complex series of events nd in the present study, we hve looked t one of these: short-term chnges in synptic function. This is prticulrly vulnerle to noxi ecuse the presynptic nerve terminls re very fine ( < 0.2 tm in olfctory cortex, Grcey & Scholfield, 1990). Cesstion of ATP production will cuse run down of ionic grdients t rte, dependent on the surfce re to volume rtio nd on ctivity. Thus ionic run-down would e rpid in fine terminls nd rther slower in postsynptic neurones. Thus the cell odies my survive noxi, ut in the long term (in vivo studies) the effective defferenttion my induce their uto-degenertion. In olfctory cortex, trnsmission is glutmte-medited (see results) nd the xonl depolriztion cused glutmte to e relesed from the nerve terminls (see Introduction). The sustined rise in extrcellulr glutmte stimultes NMDA receptors nd since the ssocited chnnels re depolriztion-dependent, ny depolriztion due to metolic insufficiency will ccentute NMDA chnnel opening Ṡeverl glutmte lockers were tested ut none of them influenced the time course for the loss of excitility during the onset of noxi. This would suggest tht synptic function is not lost s consequence of ny NMDA- or AMPAmedited process. Recovery ws sustntilly enhnced y dizocilpine, ketmine, the higher AP5 concentrtion nd 7- chlorokynurenic cid. This would suggest tht NMDA receptors re ctivted during noxi nd tht these contriute to tissue dmge. The lower AP5 concentrtion provided little protection lthough it did produce some ntgonism of NMDA. The stronger protective ction of dizocilpine, ketmine nd 200 gm AP5 reflect the greter effectiveness of these s NMDA chnnel lockers t the concentrtions used. This implies tht sustntil lockde of NMDA receptor/ chnnel is required efore ny protective effect is otined. This requirement for high degree of lock my explin why some studies hve filed to show ny effect of NMDA ntgonists with noxi nd ischemi. On the other hnd, AMPA receptor lockde y DNQX did not id recovery. This indictes tht AMPA receptors re not importnt in priming the depolriztion for NMDA chnnel opening nd it would suggest tht depolriztion results from other cuses. Although the NMDA chnnel ntgonists were the most effective protectnts, the degree of protection only extended the endurnce of the tissue y round 5 min. Clerly, NMDA receptor ctivtion is only one of proly multiplicity of degrdtive functions in neurl tissue. However, the present results do confirm the ide tht NMDA receptors ply n importnt prt in the pthogenesis of cererl noxi. References AITKIN, P.G., BALSTRINO, M. & SOMJN, G.C. (1988). 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