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Neuroprotective effect of 5-HT3 receptor antagonist on ischemia-induced decrease in CA1 field potential in rat hippocampal slices
A~tb#~~~~a variety of subtypes of 5HT receptors arc prcscnt in the brain (Radja ct aI*, IWl; Frazcr ct al., IWU), little is known about their role in ischemiainduced ncuronal damage, lschemic neuronal damage in viva was found to be prevented, not only by treatment with 5-HT,, receptor ~~on~~t~~~jelenber~ and Bnrkhardf, iY4& ~~-~re~~~ et &, i99tOf,but by treatment with S-HT, receptor ~~t~~o~i~t~~~~j~knr~et ai., 398%!&feud& I 5W%~~~~~~~~we d~~~~~s~r~~~~d a ~c~r~~r~~~~~~~~effect of the: 5-HT2 reucptor antagu-
nists, cyprohcptadinc, mianscrin and ketanserin, on the i~c~l~rni~-~~d~ceddecrease in 2~d~~xy~lucoscuptake and CA1 field potential in hippoc~rnp~~lslices (Shibata et ;rl., 1992hI. However the ncurcrprutcctive effect of 5-HT, receptor antagonists has not been elucidated. A~t~r~djo~r~phic studies have dem~nstr~~tcdthat a mode~te level of S-HI+>~~ndj~~sites exists in the CA1
Malo W&r rats weighing 250-300 g were used. ‘The mimals wc’rt’decapitated and the brain was quickly removed. Using a tissue chopper, parasagital hippocampaI slices f&it&pm thickness) were prepared from abe hippocampus of each animal. The composition of the control Krchs-Ringer solution which was equilibrated with a 95% 0,-S% CO, gas mixture. was (in mM): NaCl 129, MgSO, 1.3. NaHCO, 22.4. KH, PO, I.‘. KC1 4.2, glucose 10.0. CaCI, 1.5. For ~~~(~~ly~~rni~the glueosc in the incubation medium was replaced by lfl mM sucrose. The hypoxia solution was equilibrated with a ‘),sR, N,-5% CO, gas mixture for at least I h. The buffer had a pH of 7.3-7.4 and the temperature was 3PC. Pr~~r~tions wcrc preincubated with normal Krebs-Rinser solution for I h in a rccircuIation chamber. Our chamber design, slice transfer methods. and incubation procedures have been described previously (Newman et al.. 1989; Shibata et al., IZr’)&Lh).
gium), 2-mcthyI-5-hydroxytry~tami~le mafeate G!methyl-S-HT, Research Biochemicals Incorporated, USA), dcsipramine hydrochloride (Sigma) and 5.7-dihydroxytryptamine (5,7-DHT, Sigma). Y-25 130 and 2methyl-S-HT were dissolved in distilled water. 5,7-DHT was dissolved in saline containing 0.1% ascorbic acid.
C.iWSiki
The field potential was recorded through glass micwpipcttcs filled with normal Krebs-Ringer solution (DC resistance, ft.S-1 MR) 3 h after l5-min ischemia. Tho hipp~mpal CA1 field potential evoked by Schaffer collateral stimulation was recorded in a recording chamber at 37°C. The sficcs were kept submerged in the solution in the recording chamber and were perfused continuously with 4 ml/min normal oxygenated Krebs-Ringer solution. Stimulation pulses, O.tI5 ms and 11.2Hz. had a mean intensity which produced a supramaximal response of 0.9 + 0.1 mA (n = 5) in the normal non-&hernia-treated group. Extracellular recordings of ~~uIation responses in the stratum pyramidale of the CA1 region were made and the latency of the negative portion of the population spike was fixed at 2 ms. The amplitude of the population spike elicited by the stimulation (0.05 ms in duration and 0.3 mA in intensity) of the Schaffer collateral was measured from the crest of the population excitatory postsynaptic potentials to the most negative portion of the population spike.
The drugs used in this study were Y-25130 f(-t-INfl-azabicyclof2.2.2joct-3-yI~-6-~hloro-4-mcthyl-3-oxo3~4-dihydro-2H-l,4-benzoxazine-~-carboxamidc hydrochloride, Yoshitomi, Japan), kctanscrin (Jansscn, Bet-
The rats were anesthetized with pentobarbital sodium (40 mg/kg i.p.1 and desipramine (10 mg/kg i.p.1 was given. 5,7-DHT 10 ~1 (10 hg/pI) was infused into the lateral ventricles via a stainless steel cannula (anterior: - 1.0 mm ventral from bregma; 4.0 mm from the surface of the skull, lateral: 2.0 mm from middle line) 30 min after desip~mi~e. This injection was carried out over a 5min period, The rats were used for in vitro experiments I4 days after 5,7-DHT injection.
The data were expressed as the means f S.E. The significance of differences between groups was determined using an analysis of variance followed by Student’s t-test for individuai comparisons.
We had determined the time course of the reeove~ rate of synaptic function in hippo~mpal slices fohowing ischcmic conditions for IO-20 min in earlier experiments, The amplitude of CAI field potentials decreased markedly depending upon the duration of ischcmia (Shihata et al., 1992bl. The CA1 field potential of slices exposed to lo-min i’schemia was decreased by about 70% after a 3-h washout (Shibata et al., 1492b). Therefore, the slices were exposed to normal MrcbsRinger solution containing the drug for IO min before the induction of hypoxia/ h~~Iy~ern~a ~ischemia~ and were then removed. They were then placed for I5 min in ischemic solution containing the drug. FoIlowing this procedure the slices were removed from the ischemic solution and placed in normal Krebs-Ringer sohttion for 3 h. Some slices were exposed to normal KrebsRinger solution containing the drug for 25 min then placed in normal buffer for 3 h. The percent recovery caused by the drugs was calculated as: percent recovery(%) = 100 X (C - Bl/fA - B), using the CA1 field potential in non-ischemic slices (A), CA1 field potcntiaI in ischcmic slices 03) and CA1 fis!d potrutiaI in the dru~trettted ischemic slices (Cl. The values of IX& fur the drugs were assessed from the con~cnt~tion of drug which produced 50% recovery of the CAf field potential.
hypoxia + hypoglycemia 15 min
3. Results
The amplitude of CA1 field potentials elicited by the stimulation of Schaffer collate~ls (0.9 mA) in normal slices (3.2 rt 0.11 mV, n = 8) was IQ0 + 3.5%, n = 8 (fig. 2). Thus there were small differences within experiments. The amplitude of CA1 field ~~otentials in slices exposed to 15-min iscltemia ranged from 23% (fig. 4) to 33% (fig. 3) of the level seen in control slices, thus there were also small differences between expcriments. In contrast, hypoxia for 15 min did not decrease the amplitude of CA1 field pot~ntiaIs (90 it 3.8%, n = 3 of control slices). The effect of Y-25130 and ketanserin on the ischemia-induced reduction in CA1 field potential elicited by the stimulation of Schaffer collaterals was cxamincd. Rcpresentativc examples of CA1 rreld potentials elicited by the stimulation of Schaffer collaterals are shown in fig. 1. The reduction of CAI field potential induced by ischemia (fig. 1BI was attcnuatcd by treatment with Y-25130 (1 i.~hilI Ifig. IC), but was potentiated by treatment with I&methyl-5-HT (1 @haf (fig. ID). Co-treatment with 2-methyl-5-HT and Y25 130 attenuated the Y-25 13O~induced protection against the ischemia-induced reduction in CA1 field potential (fig. 1E). Treatment with Y-25130, ketanserin or 2-methyl-5HT for 25 min in normal non-ischemic solution did not significantly change the CA1 field ~tentia~ after a 3-h washout, The amplitude of CA1 field potentials in hippocampai slices treated with Y-25130 (100 @MI,
A
e-J
B
c
.
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(4, I.
50
100
100 z
89
Z
+I p 20
0
control
0.01 0.1
V-25130
•j
1
10
100 (PM)
katenserln
Fig. 2. Effect uf Y -25 1311 and ketzmserin on the decrease in CA1 field potential iuduced by 15min ischcmia. The amplitude of CA1 field potentials in normal slices is regarded us 100~~ (0. All observ;ttians were recctrded 3 h after washout. Numbers in pur~nth~s~s indicate the number of animals. “P < Wl vs. normal contn)l group icctntntl). * * P < 0.01 vs. ischemic vehicle-treated group (Student’s t-test).
ketanscrin (100 @MI or 2-methyl-5-HT (10 FM) for 25 min was 80 rtr 7.2% (n = 61, 87 & 6.2% (n = 5) or 518c 3.5% (n = 31, respectively, of the level seen in normal vehicle-treated slices (figs. 2 and 3). The concentration-response relationship of the effect of Y-25130 or ketanscrin on the ~schemia-induced reduction in CA1 field potential is summarized in fig. 2. The magnitude of recovery of the CA1 field potentials in the Y-25130 (0.1 PM)-treated group was 27%. When the concentration of Y-25130 was increased to 1 ir_M, the pcrccnt recovery was 48% and protection was ,,c\ .3:cc, _..... :..-L....-1.. sigiiiftcantlji rn 41 x, I\\P.IIJJ ualL;IL;:IL fKXii itiai iii mcac;I*I~titreated slices, Furthermore, at 10 and 100 FM, Y25130.induced rccovcry was maximal and the values became 86%: (P < Cl.01) and ‘91% (P < 0.011, rcspectivcly, The vahtcs of KS,, were I .8 !kB- 12.9) PM for Y-2.5130 and 33 (7.3-148) PM for ketanserin. Thus the ncuroprot~ctive effect was about 20 times more potent in Y-ZSl30-trcat~d slices than in kctanscrin-treated
f?)
Fig. 1. Effect of Y-25130 and Z-methyl-fi-HT on ~hc decrease in ~~h~~ffer-CAl field pl)t~ntiul induced by Smin ischemia. All &WTvaticms wore rctxrdcd 3 h nftr’r 1%min ischcmie. (A) CA 1 field potential evakud by the stimulution of S~Iiilffer collaterals (0.05 ms, 0.9 mA) under normal conditions. (13) Chl field potential in hipnrW!lmntll ,.‘._ _...r-_. ~!i~~~ 3 h after tfi,mjfi ighgni:l. [Cl Effect of Y-25 !?(I !! pM). (D) Effect nf 2-methyl-S-HT (IO &MI. (E) Effect of co.trcalmcnt with 2-rnetl~yl-S-~~T(10 FM) utld Y-25:31) (1 &MI. l-i~~t)(~;~rnpiti slia3i were cxpc3scdto Krehs-Ringer dirtiim ~~ln~~iil~ii~~ llw drw for 10 min lwforc lhc imfuctian of isclvda nnct during the 1%min
(5) 0’
hypoxia + hypo#ycemla
15 mln
(3) -
2-meiyt-!&HT Fig. 3. Effect of 2-methyl-S-HT on the decrease in CA1 field poten!i~! indu& by t$min is&pmi~!.The ;rmp!i!udr r?fC.A! fie!i! potet!tiuls in contr<,l slice# was regarded 3s ltlO%~.All obscrvntions WCK rccclr&d .7 11;dkr 1fi-min i~~iI~nl~t. Numhcrs in pi~tei~tl~~s~s indicute the ntmtlrer ctf ;tnim;Ils. #*I’ < O&l vs. nmtritl gnmp tcuntr0lb. * P i group 6tudent’s 0.1)5. * * P < (1.01 vs. ischemic vclricbtreated I-tcstt.
0
control
Fig.
;I&I~
4.
on Y-?513tt-produced protection E&xl of ~-nwt1~$5-lU il 1%mire i~chrmia-induced decreac in CA1 fidci potrntial.
‘The amplitude of CA1 field potentials in conlnd slices ~95 regarded a, IOO’;.
Nl
ohwrwtions wcrc rscordcJ 3 h :~ftcr 15.min ischcmia.
I-h&d~-5IIT
and Y-5l.ItI
irduction rd irhumia
H’LXCco-dminiatcrcd
10 min I>elorc !hc
and during the 15.min ischcmie. Numhw
in ptircnthcsr$ indicate the number of ;mim& ‘#P < O.tI! vs. control group (control). * P c 0.05. “’ P i 0.01 vs. ischcmic vchick-trcatcd group (Student‘s t-lest).
On the other hand, the 5HT agonist, 2-methyl-S-HT ~1MI. potcntiatcd an ischomia-induced deficit of CAI field potentials in a dose-dependent manner Uig. 3). To d&zrmine whether 5-HT,$ rcccptors arc inwlvcd in the ncuroprotuctiun by Y-2,5;1.70,WCexamined the effect of co-treatment with 2-methyl-5-HT (1 g&l) and Y-25130 on the ischcmia-induced dccrcasc in CA1 field potentials (fig. 4). The protective effect of Y-251330was attenuated by co-treatment with 2-methyl1 Ll\ -‘i-l-n. . x lfi ,..&* . ,. kcausc the S-HT, rcccptor antagonist, kctanscrin, and the S-HT, receptor antagonist, Y-25130, wcrc
(11.l- 10
Fig. h. Effect of pretreatment with S,7-dihydro~t~pt~minc on the decrease in Schaffer-CA1 field potential induced by 15min ischemia. The amplitude of CA1 field potentials in normal slices from control rats was regarded as IOO?~. All observations were recorded 3 h aftcr 1.5min ischemk. Numbers in parentheses indicate the number of animals. Open bars. nonkchemic group: c!osed bars. ischemic group. ##P < O.Oi vs. non-ischrmic group (control). * * P < O.OI (Student’s l-test).
found to exert a neuroprotective effect against the ischemia-induced decrease in CA1 field potential, the cffcct of destruction of S-HT neurons on the ischemiainduced decrease in CA1 field potential was also examined (figs. 5 and 6). Treatment with SJ-DHT did not significantly affect the CA1 field potential under normal non-ischemic conditions (figs. 5B and 6). Pretreatment with 5,7-DHT gave significant protection (fig. SD) fP < 0.01 vs. control rats, fig. 61 against the ischcmia-induced reduction in CA1 field potential (fig. 5C).
4. Discussion The present results demonstrated that antagonists at 5-HT, or 5-HT, receptors attenuate the ischemia-induced dccreasc in CA1 field potential elicited by stimulation of Schaffer collaterals. Activation of 5-HT, receptors stimulates the increase of phosphoinositide hydrolysis turnover and the release of arachidonic acid (Felder
et
al.,
1990;
Stmsherg;
1991);
Recently
Ed-
Fig. 5. Effect of pretreatment with S.7.dihydnvxytrypt;tminc f5,7DHTI on the decrease in Schaffer-C’A1 field potential induced hy 1%min &hernia. All ubsetations were recorded 3 h after IS-min
wards et al. t 1991) demonstrated that phosphoinositide hydrolysis was linked to both S-HT, and 5-HT, receptors in rat brain. Thus both 5-HT, and 5-HT, receptor agonists increase phosphoinositide hydrolysis. This may stimulate the release of intracdlular Ca** and protein kinase C (Wolf et al,, 1986; Conn and Sanders-Bush, 1986; Nishizuka, 1988). An increase in the release of cellular Cal+ is thought to be an important trigger in ischemic ceil death (Deshpande and Wieloch, 1986; Choi, 1988; Lipton et al., 1988). S-NT, receptor antagonists naftidrofuryi tFujikura it a!,, 1989? and cmopamil
kiici~lia.
iD&uiiis,
iAi
CAi
ikid
pr&xdai
cVitkuu’ iBy iiir
sihuiaihln
rif
Schaffer collateral (0.M ms. 0.0 mA) from iI normal rat. fBf CA1 field potential from a 5.7-DHT-treated rat. (Cl <‘Al field potential from a normal rut 3 h after IS-min ischemia. (D) CA1 field pdentkl from the S-f-DIiT-trc;rtcxl rat 3 h after 1%min ischcmia. Ench tr;tcc is an ilVtXiigt! of 16 sweeps.
isag),
&ib&
gji;lii&kii\re
&f&s
rjrj
is_
~hemia-induced neuronal damage in vivo. These findings, together with the present results, suggest that the neuroprotective action of 5-HT, or 5-HT, rcccptor antagonists against the ischcmi&nduccd decrease in
CA1 field potential may be mediated through a blocking effect of these compounds on 5-HT, or 5-HT, receptors which are coupled to increase phosphoninositide hydrolysis turnover. 5-HT and 2-methyl-%HT induce a transient depolarization in neuroblastoma NIE-I 15 cells (Ncijt et al., 1988). Excitatory responses to S-HT or 2-methyl-5HT are blocked by selective 5-HT, receptor antagonists. The 5-HT, receptor agonists, 2-methyl-5-HT and phenylbiguanide, mimic the action of 5-HT and dose dependently produce a significant increase in phosphoinositide hydrolysis (Edwards et al., 1991). The stimulatory action of 2-methyl-5-HT was completely blocked by 5-HT, receptor antagonists. A facilitatory effect of 2-methyl-5-HT on the ischemia-induced decrease in CA1 field potential may be involved in its depolarizing effect on membrane potential and/or its effect to increase phosphoinositide hydrolysis. Stimulation of CAMP formation by 5-HT in mouse embryonic colliculi neurons is blocked by 5-HT, receptor antagonists but not by S-HT, receptor Emtagonists (Dumuis et al., 1988), Thus inhibition of 5-HT-induced CAMP production by 5-PIT, receptor antagonists may result in the neuroprotective action of 5-HT, receptor antagonists, l[n tict we found that treatment with CAMP analogues exacerbated the ischemia-induced decrease in CA1 field potential (Shibata et al., 1992~1. Treatmcm with 5,7-DHT gave significant protection against the isch~!mi~-induced reduction in CA1 field potential. Since prctreatmcnt with dcsipramine and 5,FDHT prcferentialfy decreases the 5-HT content more than the noradrenaline or dopaminc contents in the brain (Baumgarten et al., 1975), S-HT neurons in the hippocampus may play a dctrimcntal role in the development of ischcmir damage. :n summary, the present results demonstrated that stimulation of S-FIT, receptors plays a detrimental role in the development of ischemia damage, whcrcas the blockade of 5-HT, rcccptors plays a neuroprotcctivc role in ischemia-induced damage. Thcsc results suggest a facilitatory role of 5-HT neurons in the ischemia-induccd ncuronal deficit.
Rgfe~nees &umgar!cn, H.G., .A. Bj~~rktund, L. Lar!!enmzyer and .A. N&in. 1975, Evulu~;til~n of the effect of ~.7-dihydr~)~t~pt;lmin~ on semtonin and c~it~chnlurni~y~ neurons in thz rat CNS. A&I Physinl. Sand. (Suppi,) 391, 1, i3ictcnbcrp. G.W, and M. Burkhardt. 1?100,S-I ly~r~)~yt~plurnill~~~ sgonists :I new theritpoutic principle for stroke trcatmunt. Stroke 21, 101. Btrde-Grcuci. K.M., J. Klisch, E. llorvath, T. Glascr ;md J. Trahcr. l99tk Effects of 5-llydro)x~~trypt:lnlillc,~-rccuptor agonists cm hip
PocamPai dW?EIgr after lransirnt fort&tin gc,ban gerbil. Stroke 21. 164.
ischemia i;, the mcm_
Choi. D.W., 19xX. Glut~lm~t~ n~ur{~toxi~ity and disease (,f lhc nL1r_ vous system. Neuron 1. 62.3. Corm. P.J. and E. ~Ind~rs.Bu~t;. I%%. Regulation ‘,f %cqtonin_ stimuktted ph(~spti[~in~~~itid~ hydrttlyxis: ReIatitm 10 the serolclnin 5-HT, binding site. J. Neurosci. 6. 3hh9. Defeudis. F.V.. I’IXY. The C;I-‘” channel and ~-HT, recept,,r antago. nistfsbemnpamit in cerebral ischemia. Trends Pharm:icot. Sci. lt). 215. neshpande. J.K. and T. Wit&h, 10X6. Flunarizine. a c;llcium cn;rq’ blocker. umcliorates ischcmic brain damage in the rat. Ancsthesiology 64, 215. Dumuis. A.. R. Bouhelal. M. Sehhcn and J. Bockart. IOXX. A $HT receptor in the central IIL~IVOUS system. positively coupled with :ldenylate cyclasr. is tintagonized by ICS2t)SY30. Eur. J. Pharmacol. 146, 1x7. Edward>, C.. K. Harkins. C.R. Ashhy and Y. Wang. 1901. Effect of S-hyclroxytryptaminc, receptor agonists on phosphoinositides hydrotysis in the rat fronlo-eingulate and entorhinal cortices. J, Phormacol. Exp. Thor, 2%. 103. Felder. CC., R.Y. Kuntremon. A.L. Ma and J. Axelrod, 19Y11. Serotonin stimulales phnsphotipasc A2 and the release of urachidonic acid in hippoccampalneurons hy a type 2 scmtonin receptor that is independent of inositol ph~splll~lipid hydrolysis. Proc. NatI. Acad. Sci. U.S.A. X7, 21X7, Frazer, 4.. S. Maaytmi and B.B. Wolfe, 19%). Subtypes of receptors for s~r~~tunin,Ann. Rev. Pharmaatl. Toxicol. 30. 307. Fujikura. H., H. Kate. S. Nakano and K. Kagure. lYX9, A ~~r~)t~)nin S2 ~lnt~~~nni~t.nuftidr~)fuiyl, exhibited a protective effect on ischemic Il~ur~~n~ldamage in the gerbil. Brain Res. 4Y4. 3X7. Fukuda, T., M Setoguchi, K.-l. In&a, M. Shoji and T. Tahara, IWI. The anticmetic profile of Y-251 130, it new solectivc S-HT, rcccp tor antagonist. Eur. J, Phurmacol, 1%. 2% Kilpatrick. G.J., B.J. Jones ond M.B. Tyers. IOXX. The distrihutinn of specific binding of the S-HT, receptor ligund [3H]GRhSh30 in rat brain using quentitativr: autoradio~rnphy, Ncurosci. Lett. 94. 156. Lipton, P.. K.M. Raley and II. Lohner. 1OXX.Long-term inhibition of synaptic transmission ;md macromolecul;tr synthesis following anoxin in the r;lt hippoc;nnpal slice: intcr:lction hctwecn C? + and NMDA rcccptors. in: Mechanisms of Ccrchral I lypoxia and Stroke. cd. G. Somjen (Plenum, New York) p. 2?J. Ncijt. I1.C.. I.J. D&s and H.P.M. Vijvorhcrp. IOXX. Ph;lrm;tcolagical ch;lractrriz;ltiol1 of suroionin S-HT, receptor-mediated electrical response in cultured mouse t~e~~rohl;~stonla cells. Ncumphnrmncology 27, 301. Newman, G.C.. F.E. Hospr)d iIIld P, WU, 19X9. GIUCO~Cutiliz:~lirm of ischrmic l~ippocampal slices. J. Ncurosci. Muth. 2X. 73. NishizukiI. Y., IOXX. The molecuhir hcterogaicity of prntcin kinnse C and its implications for cellular rugulutian, Nature 334. 661. Pulsinclli. W.A., J.B. Bricrlcy and F. Plum. 10X2. Temporal Profile of neurtmal damage in ii model of transient forebrain ischemia. Ann. Neural. I I. 4YI. R;tdja, F.. A.-M. Luportc, Ci. DaViil. II. Verge. ft. Go&n and M. Hamon. 1991, Aut~~r~~di(~~r~lphy of serotonin receptor sui~typc~in the central nervous system, Ncurochcm. lnt. IX. I. Shihnta. S., Y. Ki~~~~~ni-isl~i. S. Ueki and b, Wut~in~ll~~.i’tY&. Nwroprute&ve effect of WEB IXHI FU cm an ~scll~n~i;i-induceddeficit of glua)s~ uplake and CAI field potential in rat hit~p~~c~trnp~tl slices, Jap. J. Pl~~lrmil~)l. 5% 243. Sgh&;i, S., Y. K~l~~lnli-istli.K. Tolnin~t~:t, K. Kadnnta, S. Ucki and S. Wstunullc. 10921). Iscl~~lnkI-induc~d impairmcnl of 2-dec)xvgtucase uptake itnd CAI field . .,tenti;il in rat hippocampal slices: ncuroprotcctive itction of S-1IT,,, receptor agonists and S-lfT, rcceptar ;mt;tgonists, Eur. J. Pl~;trmncol. 3). 21. Shilr:ltu, S., K. K<)d;lm;l, K. Tominugu, S. Ueki and S. W:ltoIl;thc.
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helonging proteins. Wolf.
B.A.,
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InIracellular AD.,
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Chem. Xl.
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Eur. J. Biochem. C‘a’ 3501.
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1986, J. Biol.
nists, cyprohcptadinc, mianscrin and ketanserin, on the i~c~l~rni~-~~d~ceddecrease in 2~d~~xy~lucoscuptake and CA1 field potential in hippoc~rnp~~lslices (Shibata et ;rl., 1992hI. However the ncurcrprutcctive effect of 5-HT, receptor antagonists has not been elucidated. A~t~r~djo~r~phic studies have dem~nstr~~tcdthat a mode~te level of S-HI+>~~ndj~~sites exists in the CA1
Malo W&r rats weighing 250-300 g were used. ‘The mimals wc’rt’decapitated and the brain was quickly removed. Using a tissue chopper, parasagital hippocampaI slices f&it&pm thickness) were prepared from abe hippocampus of each animal. The composition of the control Krchs-Ringer solution which was equilibrated with a 95% 0,-S% CO, gas mixture. was (in mM): NaCl 129, MgSO, 1.3. NaHCO, 22.4. KH, PO, I.‘. KC1 4.2, glucose 10.0. CaCI, 1.5. For ~~~(~~ly~~rni~the glueosc in the incubation medium was replaced by lfl mM sucrose. The hypoxia solution was equilibrated with a ‘),sR, N,-5% CO, gas mixture for at least I h. The buffer had a pH of 7.3-7.4 and the temperature was 3PC. Pr~~r~tions wcrc preincubated with normal Krebs-Rinser solution for I h in a rccircuIation chamber. Our chamber design, slice transfer methods. and incubation procedures have been described previously (Newman et al.. 1989; Shibata et al., IZr’)&Lh).
gium), 2-mcthyI-5-hydroxytry~tami~le mafeate G!methyl-S-HT, Research Biochemicals Incorporated, USA), dcsipramine hydrochloride (Sigma) and 5.7-dihydroxytryptamine (5,7-DHT, Sigma). Y-25 130 and 2methyl-S-HT were dissolved in distilled water. 5,7-DHT was dissolved in saline containing 0.1% ascorbic acid.
C.iWSiki
The field potential was recorded through glass micwpipcttcs filled with normal Krebs-Ringer solution (DC resistance, ft.S-1 MR) 3 h after l5-min ischemia. Tho hipp~mpal CA1 field potential evoked by Schaffer collateral stimulation was recorded in a recording chamber at 37°C. The sficcs were kept submerged in the solution in the recording chamber and were perfused continuously with 4 ml/min normal oxygenated Krebs-Ringer solution. Stimulation pulses, O.tI5 ms and 11.2Hz. had a mean intensity which produced a supramaximal response of 0.9 + 0.1 mA (n = 5) in the normal non-&hernia-treated group. Extracellular recordings of ~~uIation responses in the stratum pyramidale of the CA1 region were made and the latency of the negative portion of the population spike was fixed at 2 ms. The amplitude of the population spike elicited by the stimulation (0.05 ms in duration and 0.3 mA in intensity) of the Schaffer collateral was measured from the crest of the population excitatory postsynaptic potentials to the most negative portion of the population spike.
The drugs used in this study were Y-25130 f(-t-INfl-azabicyclof2.2.2joct-3-yI~-6-~hloro-4-mcthyl-3-oxo3~4-dihydro-2H-l,4-benzoxazine-~-carboxamidc hydrochloride, Yoshitomi, Japan), kctanscrin (Jansscn, Bet-
The rats were anesthetized with pentobarbital sodium (40 mg/kg i.p.1 and desipramine (10 mg/kg i.p.1 was given. 5,7-DHT 10 ~1 (10 hg/pI) was infused into the lateral ventricles via a stainless steel cannula (anterior: - 1.0 mm ventral from bregma; 4.0 mm from the surface of the skull, lateral: 2.0 mm from middle line) 30 min after desip~mi~e. This injection was carried out over a 5min period, The rats were used for in vitro experiments I4 days after 5,7-DHT injection.
The data were expressed as the means f S.E. The significance of differences between groups was determined using an analysis of variance followed by Student’s t-test for individuai comparisons.
We had determined the time course of the reeove~ rate of synaptic function in hippo~mpal slices fohowing ischcmic conditions for IO-20 min in earlier experiments, The amplitude of CAI field potentials decreased markedly depending upon the duration of ischcmia (Shihata et al., 1992bl. The CA1 field potential of slices exposed to lo-min i’schemia was decreased by about 70% after a 3-h washout (Shibata et al., 1492b). Therefore, the slices were exposed to normal MrcbsRinger solution containing the drug for IO min before the induction of hypoxia/ h~~Iy~ern~a ~ischemia~ and were then removed. They were then placed for I5 min in ischemic solution containing the drug. FoIlowing this procedure the slices were removed from the ischemic solution and placed in normal Krebs-Ringer sohttion for 3 h. Some slices were exposed to normal KrebsRinger solution containing the drug for 25 min then placed in normal buffer for 3 h. The percent recovery caused by the drugs was calculated as: percent recovery(%) = 100 X (C - Bl/fA - B), using the CA1 field potential in non-ischemic slices (A), CA1 field potcntiaI in ischcmic slices 03) and CA1 fis!d potrutiaI in the dru~trettted ischemic slices (Cl. The values of IX& fur the drugs were assessed from the con~cnt~tion of drug which produced 50% recovery of the CAf field potential.
hypoxia + hypoglycemia 15 min
3. Results
The amplitude of CA1 field potentials elicited by the stimulation of Schaffer collate~ls (0.9 mA) in normal slices (3.2 rt 0.11 mV, n = 8) was IQ0 + 3.5%, n = 8 (fig. 2). Thus there were small differences within experiments. The amplitude of CA1 field ~~otentials in slices exposed to 15-min iscltemia ranged from 23% (fig. 4) to 33% (fig. 3) of the level seen in control slices, thus there were also small differences between expcriments. In contrast, hypoxia for 15 min did not decrease the amplitude of CA1 field pot~ntiaIs (90 it 3.8%, n = 3 of control slices). The effect of Y-25130 and ketanserin on the ischemia-induced reduction in CA1 field potential elicited by the stimulation of Schaffer collaterals was cxamincd. Rcpresentativc examples of CA1 rreld potentials elicited by the stimulation of Schaffer collaterals are shown in fig. 1. The reduction of CAI field potential induced by ischemia (fig. 1BI was attcnuatcd by treatment with Y-25130 (1 i.~hilI Ifig. IC), but was potentiated by treatment with I&methyl-5-HT (1 @haf (fig. ID). Co-treatment with 2-methyl-5-HT and Y25 130 attenuated the Y-25 13O~induced protection against the ischemia-induced reduction in CA1 field potential (fig. 1E). Treatment with Y-25130, ketanserin or 2-methyl-5HT for 25 min in normal non-ischemic solution did not significantly change the CA1 field ~tentia~ after a 3-h washout, The amplitude of CA1 field potentials in hippocampai slices treated with Y-25130 (100 @MI,
A
e-J
B
c
.
&__I/
(4, I.
50
100
100 z
89
Z
+I p 20
0
control
0.01 0.1
V-25130
•j
1
10
100 (PM)
katenserln
Fig. 2. Effect uf Y -25 1311 and ketzmserin on the decrease in CA1 field potential iuduced by 15min ischcmia. The amplitude of CA1 field potentials in normal slices is regarded us 100~~ (0. All observ;ttians were recctrded 3 h after washout. Numbers in pur~nth~s~s indicate the number of animals. “P < Wl vs. normal contn)l group icctntntl). * * P < 0.01 vs. ischemic vehicle-treated group (Student’s t-test).
ketanscrin (100 @MI or 2-methyl-5-HT (10 FM) for 25 min was 80 rtr 7.2% (n = 61, 87 & 6.2% (n = 5) or 518c 3.5% (n = 31, respectively, of the level seen in normal vehicle-treated slices (figs. 2 and 3). The concentration-response relationship of the effect of Y-25130 or ketanscrin on the ~schemia-induced reduction in CA1 field potential is summarized in fig. 2. The magnitude of recovery of the CA1 field potentials in the Y-25130 (0.1 PM)-treated group was 27%. When the concentration of Y-25130 was increased to 1 ir_M, the pcrccnt recovery was 48% and protection was ,,c\ .3:cc, _..... :..-L....-1.. sigiiiftcantlji rn 41 x, I\\P.IIJJ ualL;IL;:IL fKXii itiai iii mcac;I*I~titreated slices, Furthermore, at 10 and 100 FM, Y25130.induced rccovcry was maximal and the values became 86%: (P < Cl.01) and ‘91% (P < 0.011, rcspectivcly, The vahtcs of KS,, were I .8 !kB- 12.9) PM for Y-2.5130 and 33 (7.3-148) PM for ketanserin. Thus the ncuroprot~ctive effect was about 20 times more potent in Y-ZSl30-trcat~d slices than in kctanscrin-treated
f?)
Fig. 1. Effect of Y-25130 and Z-methyl-fi-HT on ~hc decrease in ~~h~~ffer-CAl field pl)t~ntiul induced by Smin ischemia. All &WTvaticms wore rctxrdcd 3 h nftr’r 1%min ischcmie. (A) CA 1 field potential evakud by the stimulution of S~Iiilffer collaterals (0.05 ms, 0.9 mA) under normal conditions. (13) Chl field potential in hipnrW!lmntll ,.‘._ _...r-_. ~!i~~~ 3 h after tfi,mjfi ighgni:l. [Cl Effect of Y-25 !?(I !! pM). (D) Effect nf 2-methyl-S-HT (IO &MI. (E) Effect of co.trcalmcnt with 2-rnetl~yl-S-~~T(10 FM) utld Y-25:31) (1 &MI. l-i~~t)(~;~rnpiti slia3i were cxpc3scdto Krehs-Ringer dirtiim ~~ln~~iil~ii~~ llw drw for 10 min lwforc lhc imfuctian of isclvda nnct during the 1%min
(5) 0’
hypoxia + hypo#ycemla
15 mln
(3) -
2-meiyt-!&HT Fig. 3. Effect of 2-methyl-S-HT on the decrease in CA1 field poten!i~! indu& by t$min is&pmi~!.The ;rmp!i!udr r?fC.A! fie!i! potet!tiuls in contr<,l slice# was regarded 3s ltlO%~.All obscrvntions WCK rccclr&d .7 11;dkr 1fi-min i~~iI~nl~t. Numhcrs in pi~tei~tl~~s~s indicute the ntmtlrer ctf ;tnim;Ils. #*I’ < O&l vs. nmtritl gnmp tcuntr0lb. * P i group 6tudent’s 0.1)5. * * P < (1.01 vs. ischemic vclricbtreated I-tcstt.
0
control
Fig.
;I&I~
4.
on Y-?513tt-produced protection E&xl of ~-nwt1~$5-lU il 1%mire i~chrmia-induced decreac in CA1 fidci potrntial.
‘The amplitude of CA1 field potentials in conlnd slices ~95 regarded a, IOO’;.
Nl
ohwrwtions wcrc rscordcJ 3 h :~ftcr 15.min ischcmia.
I-h&d~-5IIT
and Y-5l.ItI
irduction rd irhumia
H’LXCco-dminiatcrcd
10 min I>elorc !hc
and during the 15.min ischcmie. Numhw
in ptircnthcsr$ indicate the number of ;mim& ‘#P < O.tI! vs. control group (control). * P c 0.05. “’ P i 0.01 vs. ischcmic vchick-trcatcd group (Student‘s t-lest).
On the other hand, the 5HT agonist, 2-methyl-S-HT ~1MI. potcntiatcd an ischomia-induced deficit of CAI field potentials in a dose-dependent manner Uig. 3). To d&zrmine whether 5-HT,$ rcccptors arc inwlvcd in the ncuroprotuctiun by Y-2,5;1.70,WCexamined the effect of co-treatment with 2-methyl-5-HT (1 g&l) and Y-25130 on the ischcmia-induced dccrcasc in CA1 field potentials (fig. 4). The protective effect of Y-251330was attenuated by co-treatment with 2-methyl1 Ll\ -‘i-l-n. . x lfi ,..&* . ,. kcausc the S-HT, rcccptor antagonist, kctanscrin, and the S-HT, receptor antagonist, Y-25130, wcrc
(11.l- 10
Fig. h. Effect of pretreatment with S,7-dihydro~t~pt~minc on the decrease in Schaffer-CA1 field potential induced by 15min ischemia. The amplitude of CA1 field potentials in normal slices from control rats was regarded as IOO?~. All observations were recorded 3 h aftcr 1.5min ischemk. Numbers in parentheses indicate the number of animals. Open bars. nonkchemic group: c!osed bars. ischemic group. ##P < O.Oi vs. non-ischrmic group (control). * * P < O.OI (Student’s l-test).
found to exert a neuroprotective effect against the ischemia-induced decrease in CA1 field potential, the cffcct of destruction of S-HT neurons on the ischemiainduced decrease in CA1 field potential was also examined (figs. 5 and 6). Treatment with SJ-DHT did not significantly affect the CA1 field potential under normal non-ischemic conditions (figs. 5B and 6). Pretreatment with 5,7-DHT gave significant protection (fig. SD) fP < 0.01 vs. control rats, fig. 61 against the ischcmia-induced reduction in CA1 field potential (fig. 5C).
4. Discussion The present results demonstrated that antagonists at 5-HT, or 5-HT, receptors attenuate the ischemia-induced dccreasc in CA1 field potential elicited by stimulation of Schaffer collaterals. Activation of 5-HT, receptors stimulates the increase of phosphoinositide hydrolysis turnover and the release of arachidonic acid (Felder
et
al.,
1990;
Stmsherg;
1991);
Recently
Ed-
Fig. 5. Effect of pretreatment with S.7.dihydnvxytrypt;tminc f5,7DHTI on the decrease in Schaffer-C’A1 field potential induced hy 1%min &hernia. All ubsetations were recorded 3 h after IS-min
wards et al. t 1991) demonstrated that phosphoinositide hydrolysis was linked to both S-HT, and 5-HT, receptors in rat brain. Thus both 5-HT, and 5-HT, receptor agonists increase phosphoinositide hydrolysis. This may stimulate the release of intracdlular Ca** and protein kinase C (Wolf et al,, 1986; Conn and Sanders-Bush, 1986; Nishizuka, 1988). An increase in the release of cellular Cal+ is thought to be an important trigger in ischemic ceil death (Deshpande and Wieloch, 1986; Choi, 1988; Lipton et al., 1988). S-NT, receptor antagonists naftidrofuryi tFujikura it a!,, 1989? and cmopamil
kiici~lia.
iD&uiiis,
iAi
CAi
ikid
pr&xdai
cVitkuu’ iBy iiir
sihuiaihln
rif
Schaffer collateral (0.M ms. 0.0 mA) from iI normal rat. fBf CA1 field potential from a 5.7-DHT-treated rat. (Cl <‘Al field potential from a normal rut 3 h after IS-min ischemia. (D) CA1 field pdentkl from the S-f-DIiT-trc;rtcxl rat 3 h after 1%min ischcmia. Ench tr;tcc is an ilVtXiigt! of 16 sweeps.
isag),
&ib&
gji;lii&kii\re
&f&s
rjrj
is_
~hemia-induced neuronal damage in vivo. These findings, together with the present results, suggest that the neuroprotective action of 5-HT, or 5-HT, rcccptor antagonists against the ischcmi&nduccd decrease in
CA1 field potential may be mediated through a blocking effect of these compounds on 5-HT, or 5-HT, receptors which are coupled to increase phosphoninositide hydrolysis turnover. 5-HT and 2-methyl-%HT induce a transient depolarization in neuroblastoma NIE-I 15 cells (Ncijt et al., 1988). Excitatory responses to S-HT or 2-methyl-5HT are blocked by selective 5-HT, receptor antagonists. The 5-HT, receptor agonists, 2-methyl-5-HT and phenylbiguanide, mimic the action of 5-HT and dose dependently produce a significant increase in phosphoinositide hydrolysis (Edwards et al., 1991). The stimulatory action of 2-methyl-5-HT was completely blocked by 5-HT, receptor antagonists. A facilitatory effect of 2-methyl-5-HT on the ischemia-induced decrease in CA1 field potential may be involved in its depolarizing effect on membrane potential and/or its effect to increase phosphoinositide hydrolysis. Stimulation of CAMP formation by 5-HT in mouse embryonic colliculi neurons is blocked by 5-HT, receptor antagonists but not by S-HT, receptor Emtagonists (Dumuis et al., 1988), Thus inhibition of 5-HT-induced CAMP production by 5-PIT, receptor antagonists may result in the neuroprotective action of 5-HT, receptor antagonists, l[n tict we found that treatment with CAMP analogues exacerbated the ischemia-induced decrease in CA1 field potential (Shibata et al., 1992~1. Treatmcm with 5,7-DHT gave significant protection against the isch~!mi~-induced reduction in CA1 field potential. Since prctreatmcnt with dcsipramine and 5,FDHT prcferentialfy decreases the 5-HT content more than the noradrenaline or dopaminc contents in the brain (Baumgarten et al., 1975), S-HT neurons in the hippocampus may play a dctrimcntal role in the development of ischcmir damage. :n summary, the present results demonstrated that stimulation of S-FIT, receptors plays a detrimental role in the development of ischemia damage, whcrcas the blockade of 5-HT, rcccptors plays a neuroprotcctivc role in ischemia-induced damage. Thcsc results suggest a facilitatory role of 5-HT neurons in the ischemia-induccd ncuronal deficit.
Rgfe~nees &umgar!cn, H.G., .A. Bj~~rktund, L. Lar!!enmzyer and .A. N&in. 1975, Evulu~;til~n of the effect of ~.7-dihydr~)~t~pt;lmin~ on semtonin and c~it~chnlurni~y~ neurons in thz rat CNS. A&I Physinl. Sand. (Suppi,) 391, 1, i3ictcnbcrp. G.W, and M. Burkhardt. 1?100,S-I ly~r~)~yt~plurnill~~~ sgonists :I new theritpoutic principle for stroke trcatmunt. Stroke 21, 101. Btrde-Grcuci. K.M., J. Klisch, E. llorvath, T. Glascr ;md J. Trahcr. l99tk Effects of 5-llydro)x~~trypt:lnlillc,~-rccuptor agonists cm hip
PocamPai dW?EIgr after lransirnt fort&tin gc,ban gerbil. Stroke 21. 164.
ischemia i;, the mcm_
Choi. D.W., 19xX. Glut~lm~t~ n~ur{~toxi~ity and disease (,f lhc nL1r_ vous system. Neuron 1. 62.3. Corm. P.J. and E. ~Ind~rs.Bu~t;. I%%. Regulation ‘,f %cqtonin_ stimuktted ph(~spti[~in~~~itid~ hydrttlyxis: ReIatitm 10 the serolclnin 5-HT, binding site. J. Neurosci. 6. 3hh9. Defeudis. F.V.. I’IXY. The C;I-‘” channel and ~-HT, recept,,r antago. nistfsbemnpamit in cerebral ischemia. Trends Pharm:icot. Sci. lt). 215. neshpande. J.K. and T. Wit&h, 10X6. Flunarizine. a c;llcium cn;rq’ blocker. umcliorates ischcmic brain damage in the rat. Ancsthesiology 64, 215. Dumuis. A.. R. Bouhelal. M. Sehhcn and J. Bockart. IOXX. A $HT receptor in the central IIL~IVOUS system. positively coupled with :ldenylate cyclasr. is tintagonized by ICS2t)SY30. Eur. J. Pharmacol. 146, 1x7. Edward>, C.. K. Harkins. C.R. Ashhy and Y. Wang. 1901. Effect of S-hyclroxytryptaminc, receptor agonists on phosphoinositides hydrotysis in the rat fronlo-eingulate and entorhinal cortices. J, Phormacol. Exp. Thor, 2%. 103. Felder. CC., R.Y. Kuntremon. A.L. Ma and J. Axelrod, 19Y11. Serotonin stimulales phnsphotipasc A2 and the release of urachidonic acid in hippoccampalneurons hy a type 2 scmtonin receptor that is independent of inositol ph~splll~lipid hydrolysis. Proc. NatI. Acad. Sci. U.S.A. X7, 21X7, Frazer, 4.. S. Maaytmi and B.B. Wolfe, 19%). Subtypes of receptors for s~r~~tunin,Ann. Rev. Pharmaatl. Toxicol. 30. 307. Fujikura. H., H. Kate. S. Nakano and K. Kagure. lYX9, A ~~r~)t~)nin S2 ~lnt~~~nni~t.nuftidr~)fuiyl, exhibited a protective effect on ischemic Il~ur~~n~ldamage in the gerbil. Brain Res. 4Y4. 3X7. Fukuda, T., M Setoguchi, K.-l. In&a, M. Shoji and T. Tahara, IWI. The anticmetic profile of Y-251 130, it new solectivc S-HT, rcccp tor antagonist. Eur. J, Phurmacol, 1%. 2% Kilpatrick. G.J., B.J. Jones ond M.B. Tyers. IOXX. The distrihutinn of specific binding of the S-HT, receptor ligund [3H]GRhSh30 in rat brain using quentitativr: autoradio~rnphy, Ncurosci. Lett. 94. 156. Lipton, P.. K.M. Raley and II. Lohner. 1OXX.Long-term inhibition of synaptic transmission ;md macromolecul;tr synthesis following anoxin in the r;lt hippoc;nnpal slice: intcr:lction hctwecn C? + and NMDA rcccptors. in: Mechanisms of Ccrchral I lypoxia and Stroke. cd. G. Somjen (Plenum, New York) p. 2?J. Ncijt. I1.C.. I.J. D&s and H.P.M. Vijvorhcrp. IOXX. Ph;lrm;tcolagical ch;lractrriz;ltiol1 of suroionin S-HT, receptor-mediated electrical response in cultured mouse t~e~~rohl;~stonla cells. Ncumphnrmncology 27, 301. Newman, G.C.. F.E. Hospr)d iIIld P, WU, 19X9. GIUCO~Cutiliz:~lirm of ischrmic l~ippocampal slices. J. Ncurosci. Muth. 2X. 73. NishizukiI. Y., IOXX. The molecuhir hcterogaicity of prntcin kinnse C and its implications for cellular rugulutian, Nature 334. 661. Pulsinclli. W.A., J.B. Bricrlcy and F. Plum. 10X2. Temporal Profile of neurtmal damage in ii model of transient forebrain ischemia. Ann. Neural. I I. 4YI. R;tdja, F.. A.-M. Luportc, Ci. DaViil. II. Verge. ft. Go&n and M. Hamon. 1991, Aut~~r~~di(~~r~lphy of serotonin receptor sui~typc~in the central nervous system, Ncurochcm. lnt. IX. I. Shihnta. S., Y. Ki~~~~~ni-isl~i. S. Ueki and b, Wut~in~ll~~.i’tY&. Nwroprute&ve effect of WEB IXHI FU cm an ~scll~n~i;i-induceddeficit of glua)s~ uplake and CAI field potential in rat hit~p~~c~trnp~tl slices, Jap. J. Pl~~lrmil~)l. 5% 243. Sgh&;i, S., Y. K~l~~lnli-istli.K. Tolnin~t~:t, K. Kadnnta, S. Ucki and S. Wstunullc. 10921). Iscl~~lnkI-induc~d impairmcnl of 2-dec)xvgtucase uptake itnd CAI field . .,tenti;il in rat hippocampal slices: ncuroprotcctive itction of S-1IT,,, receptor agonists and S-lfT, rcceptar ;mt;tgonists, Eur. J. Pl~;trmncol. 3). 21. Shilr:ltu, S., K. K<)d;lm;l, K. Tominugu, S. Ueki and S. W:ltoIl;thc.
“h PW2c.
h\\e\\nlcnl
~~hcmwinduccd tirld
powntisl
of
.I
role
impairments
of
adrenoceplor
t>f 2-deoxyglucose
in rat hippocampal
function
in
uptake and CAI
slices. Eur. J. Pharmacol.
72!.
35. >tnrkrg.
helonging proteins. Wolf.
B.A.,
IO the J. Turk.
InIracellular AD.,
1YYi. Struclure/tunctwn
relationship
of prolems
Chem. Xl.
farnib: of receptors
Eur. J. Biochem. C‘a’ 3501.
W.R.
’
coupled
to GTP-binding
190. 1. Sherman
mohilizaticm
and
M.L.
hy qxhidonic
McDaniel. acid,
1986, J. Biol.