188

BIOL PSYCHIATRY 1992;32: ! 88-190

BRIEF REPORTS

Unaltered Hippocampal Dihydropyridine and -Conoto×in GVIA Binding Sites After Repeated Electroconvulsive Shock in Rats David J. Dooley

Although the efficacy of electroconvulsive treatment (ECT) in human depression is indisputaI;le, the relevant physiologic changes effected by this therapeutic approach remain controversial and elusive (Green lC.J88;Gleiter and Nutt 1989). Since ECT undoulrtedly affects some of the processes underlying calcium-dependent neu~transmitter release, I hypothesized ~at ECTinduced alterations in hippocampal voitage..sensitive calcium channels (VSCCs) may comribute to the antidepressant effect. I addressed ~his possibilit~ by assessing the binding of i31-il-isradipiae ([:~HI-ISR) to L-type VSCCs a:ld the binding of l~2:ql-~-coilotoxin GVIA (I~:511-o~CT) to N-type VSCCs of hippocampal membranes prepared from rats treated with repeated electroconvulsive shock (ECS). The efficacy of ECS was determined by quantitating hipptx'ampal {3-adrenoceptors with [~Hl-dihy,:lroalpreno!ol ([3H]-DHA).

Methods Repeated ECS (ear-clip electrodes; 50 m~, 0.5 sec, 50 Hz sinusoidal; I x 10 days) or control treatm.znt (placing the ear-clip electrodes but not applying current) was administered to male rats [Sprague-Dawley, 120-130 g], killin~ t|~ese an-

imals (10 control, 10 ECS) by decapitation 24 hr after the last placement of electrodes, pooling the hippocampi from each of two rats, and preparing crude hippocampai membrane suspensions in 50 mM HEPES-NaOH buffer (pH 7.4) (Dooley e~ al 1988) tot use in the following filtration binding assays (Dooley and Bittiger 1q87; Dooley et aI 1988): [3HI-ISR (10-1000 pM or 100 pM; 3.15 TBq/mmol, Amersham) with unlabeled isradipine (100 nM; Sandoz) detieing nom'pecitic binding, [~2sI]-m-CT(0.1-10 pM or 4 pM; 76 TBq/mmol, Amersham) with unlabeled o~-CT (10 nM; Peninsula) defining nonspecific binding, and [-~H]-DHA (0.25 nM; 3.33 TBqtmmol, Dulxmt) in combination with alprenolol hydrochloride (I p,M; Ciba-Geigy) defining nonspecific binding and the specific 13~adrenocel',r~r antagonist CGP 20712 A (100 nM; Ciba-Geigy) al!owing a differentiation of binding to 151-and {~2-adrenoceptors. Results of point concentration, saturation, and interaction experiments, generated using the specific binding of each radiolabeled ligand, were analyzed by nonlinear curve-fitting algorithms if applicable, and expressed as mean values with standard errors. Tiae final ~su!ts were compared using the t-statistic for group means; the minimal level of significance was p ~< 0.05,

Results Dcparmlent of Neurosctence, P~ke-Davi~ Pharmaceutical Research Division, Warner-,l.ambert Co,, Ann Arbor, Michigan. Address reprint requests to David J. Dooley. Department of Neuroscience. P',ake-Davis Pharmaceutical Research, Warner-Lambert Co, Ann Arbor, MI 48106-1047, Received Ma:'ch 10, 1992; revised April 17, 19q2. © 1992 Society of Biological Psychiatry

Using |~HioDHA at a I~int concentration (0.25 nM) to label 13-adrenoceptors (X ___ SE, n = 4), [3~-adrenoceptor binding was decreased in the ECS group by 11 fmol/mg protein [33 __. 1 0006-3223192/$05.00

BriefReports

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BlOt. PSYCHIATRY 1992;32:188-190

Table 1. Binding Parameters of [3H]-lsradipine and [~251]-to-ConotoxinGVIA for Hippocampal Membranes of Control- and ECS-Treated Rats Bma~ [..igand

Ko (pM)

Treatment

[3H]-ISR

Control ECS Control ECS

['2Sll-t0-CT

58 62 0.40 0.46

-+ ± +_ .4-

(fmol/mg protein)

5 5 0.04 0.01

1041 1009 630 t~i9

± ± ± ±

24 10 23 48

Values given are X _-A-SE, n = 3. There was no significant difference in binding parameters between m:atments.

(control) vs 22 + I fmol/mg protein (ECS) (p < 0.001)], whereas ~2-adrenoceptor binding was unchanged [22 -+ 1 (control) vs 23 --. 1 fmol/mg protein (ECS)]. The saturation experiments with [3H]-ISR and [ t 2 5 | ] - t 0 - C T indicated that the Kz~(apparent dissociation constant) and Bmax (maximal binding capacity) values of these two VSCC radioligands were unchanged between the control and ECS groups (Table 1). Interaction experiments, which attempted to detect alterations in the inhibition of [3H]-ISR binding (I00 pM) and [12sI]-to-CT binding (4 pM) by various substances after ECS treatment, gave essentially negative results as based on a comparison of ICso (concentration of a substance required to displace or inhibit radiolabeled ligand binding by 50%) and n# (Hill coefficient) values between the control and ECS groups (Table 2). In addition, fostedil (10 IxM) and MDL 12330 A (10 ttM) enhanced [3H]-ISR binding by 12% and 20%, respectively, in both

control and ECS groups, lsradipine (10 IxM) did not inhibit [12Sl]-to-CT binding in either group.

Discussion Neuronal L- and N-type VSCCs, like various neurotransmitterreceptors,have been shown to exhibit plasticity under certain experimental conditions (Fen'ante and Triggle 1990; Czyrak et al 1992). In response to ECS, however, the affinityof [3H]-ISR and [t25I]-e0-CTfor these VSCCs and their density in the hippocampus appears unchanged, a finding confirming and extending previous observations (Gleiteret al 1989; Silverstone and Grahame-Smith 1991). This is in contrastto the positivecontrol result of decreased [3~-adrenoceptorbinding by 33%, an effectdemonstrating the efficacyof repeated ECS treatment and also documented elsewhere (Dooley et al 1987). Moreover, characterization of [3H]-ISR bindin~ using the L-type VSCC

Table 2. Pharmacologic Characterization of [3H]-Isradipine and ['2Sl]-to-ConotoxinGVIA Binding for Hippocampal Membranes of Control- and ECS-Treated Rats Ligand [3H]-I,~R

Sllbst~,nce f + 1-202-791 Runarizine

[1251].to..CT

Calciam Cadmium Neomycin

Treatment f'nntml ECS Control ECS Control ECS Control ECS Control ECS

- plC5o (M) 6.~0 6.82 5.68 5.64 3.17 3.38 3.82 3.80 6.96 7.02

"*" ± "" _+ ± ± ± ± ± ±

~.03 0,03 0.03 0.03 0.09 0.10 0.03 0.09 0.08 0.03

Values given are X ± SE, n -- 4. There was no significe~ltdifference in IC~o and nss between treatments.

nH !.00 0,99 1.19 1.24 1.24 1.24 1.84 1.61 1.38 1.42

± ± ± ± ± ± ± -+ ± __

0.03 0.03 0.04 0.04 0.23 0.22 0.39 0.15 0.20 0.08

190

BIOLPSYCHIATrcY 1992;32:188-190

Brief Reports

Dooley DJ, Licker M, Lupp A, Osswald [] (1988): Distribution of [nSl-to-conotoxinGVIA and [3H]isradipine binding sites in the central nervous system of raLsof differentages. NeurosciLeu 93:318323. Ferrante J, Triggle DJ (1990): Drug- and diseaseinduced regulation of voltage-dependent calcium channels. Pharmacol Rev 42:29-44. Gleiter CH, Cain CI, Weiss SRB, Post RM, Marangos PJ (1989): Differential effects of acute and repeated electrically and chemically induced seizures on [3H]-nimodipineand [nSl]-to-conotoxin GVIA binding in rat brain. Epilepsia 30:487-492. Gleiter CH, Nt:tt DJ (1989): Chronic electroconvulsire shock and neurotransmitter receptors--an update. Life $ci 44:985-1006. Green AR (1988): The mechanism of action of antidepressant treatments: Basic aspects. Pharmaco. psychiatry 21:3-5. Hof RP, Ruegg UT, Hof A, Vogel A (1985): Stereoselectivity at the calcium Channel:Opposite action of the e~-antiomers of a 1,4-dihydropyridine. J Cardiovasc Pharmacol 7:689--693. Mintz IM, Venema VJ, Swiderek KM, Lee TD, Bean BP, Adams ME (1992): P-type calcium channels blocked by the spider toxin ~o-Aga-lVA. Nature References 355:82"/-829. Czyrak A, Dooley DJ, Jones GH, RobbinsTW (1992): Rampe D, Triggle DJ 0990): New ligands for LSocial isolation increases the density of [~2Sl]-totype Ca:+ channels.Trends PharmacolSci 1I:112conotoxin GVIA binding sites in the rat frontal i!5. cortex and caudate nucleus. Brain Res 583:189- Silverstone PH, Grahame-Smith DG (1991): Effects 193. of chronic lithium, amitriptyline, and electroconDooley DJ, Bittiger H (1987): Quantitative assessvulsive shock on calcium channel binding in a rat mentof central 13t-and {32-adrenoceptorregulation brain homogenate.Psychopharmacology 105:132using CGP 20712 A. J Pharmacol Meth 18: ! 3 !133. 136. Staudinger R, Knaus H-G, Glossmann [] (1991): PosDoolcy DJ, Heal DJ, Goodwin GM (1987): Repeated itive heterotropic allosteric regulators of dihydroelectroconvulsive shock prevcn,.s increased ricopyridine binding increase the Ca2~ ¢tffii~,[~",~f the cortical flt-adrenoceptor binding after DSP-4 L-type Ca2. channel. J Biol Chem 266:10787treatment in rats. Eur d Pharmacol 134:333-337. 10795.

activator (+)-2~-791 (Hof et al 1985), the dual sodium and calcium channel modulator flunarizine, and the allosteric enhancers foste,.,d and MDL 12330 A (Rampe and Triggle 1990; Staudinger et al 1991) failed to detect alterations of the ¢fihydropyridine binding site associated with hippot.ampal L-type VSCCs. This was also the case for [~251]-e0-CTbinding to Ntype VSCCs as characterized with the divalent cations Ca 2+ and Cd 2+ and the aminoglycoside antibiotic neomycin. These results suggest that hippocampal L- and N-~ype VSCCs, as assessed in [3H]-ISR and [t2Sl]-co-CT binding assays, are unaltered by a standard ECS procedure, and that changes in these VSCCs are not a prerequisite for the antidepressant effect of ECT. Thb: conclusion should, however, be tempered by considering the possible regional effects of ECS, subtle changes in VSCC function, and the existence of other VSCCs such as the P-type (Mintz et al 1992).

Unaltered hippocampal dihydropyridine and omega-conotoxin GVIA binding sites after repeated electroconvulsive shock in rats.

188 BIOL PSYCHIATRY 1992;32: ! 88-190 BRIEF REPORTS Unaltered Hippocampal Dihydropyridine and -Conoto×in GVIA Binding Sites After Repeated Electroc...
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