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Diazepam restores the increased [3H]glutamate binding to hippocampal synaptic membranes in the amygdaloid-kindled rat
Neuroscience Research, 4 (1987) 185-194 Elsevier Scientific Publishers Ireland Ltd.
NSR 00165
Diazepam restores the increased [3H]glutamate binding to hippocampal synaptic membranes in the amygdalcid-kindled rat Jun-Ichiro Oka, Mari Yamada, Masayoshi Goto and Hideomi Fukuda Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Btmkyo-ku. Tokyo (Japan)
(Received 24 March 1986; Revised version received 16 July 1986; Accepted 28 July 1986) Key words: Diazepam; [3H]Glutamate binding; CaCI2; Hippocampus; Amygdala; Kindling; Rat
[3HlGlutamic aeid binding to hippocampi was increased after amygdaloid kindling in rats, and diazepam inhibited this increased binding, without any effect on the enhanced binding by CaCI2 or the binding in control rats. By inducing kindlingin the same way as that used in the binding experiment, the inhibitingeffects of diazepam on kindled seizures, the afterdischargeand the developmentof kindlingwere observed.
Kindling, described originally by Goddard et al. in 19695, is one of the animal models of epilepsy, and may provide a model of learning or information storage in the brain, because seizure susceptibility in kindled animals can last for a long time. This phenomenon has features in common with the other model of synaptic memory, the so-called long-term potentiation (LTP) in hippocampi 3'4.~ t. Savage et al. 19 reported the selective increase in [ 3H ]glutamate binding to a quisqualate-sensitive site in hippocampi after angular bundle kindling, suggesting a similar phenomenon to that in the case of LTP tl. There has been some evidence which may suggest the existence of a neuronal connection between the amygdala and the hippocampus 2. The amygdala projects to the entorhinal cortex 9 from which originates the perforant path; the perforant path impinges on hippocampal neurons 7. This excitatory input may become stimulated in the process Correspondence: H. Fukuda, Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,Tokyo 113, Japan.
186 of amygdaloid kindling, and may, in turn, induce an increase in the glutamate receptor. There are many reports dealing with the anticonvulsant effects of benzodiazepines 6, which prevent or abolish the seizures, and the electroencephalogram afterdischarge induced by chemical convulsants or electroshock, and also those induced in kindled animals 6.17-19. In order to explore a new profile of the anti-kindling effects of benzodiazepines, we examined the effect of diazepam on [3H]glutamate binding to hippocampal synaptic membranes in the amygdaloid-kindled rat, after determining the conditions necessary for the induction of kindling and examining [ 3H]glutamate binding in the absence or presence of Ca 2+ and/or CI-. Here we show that diazepam selectively restored the increased [3H]glutamate binding in kindled rats, but did not influence the effect of CaCI 2.
Materials Diazepam (Sumitomo Chem.), ~,-aminobutyric acid (GABA), L-glutamic acid, ethyleneglycol-bis-(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, Wako Pure Chem.), leupeptin (Sigma Chem., Lot. 42F-0525), bovine serum albumin (Nakarai Chem.), L-[2,3-aH]glutamic acid (New England Nuclear, 21.6 Ci/mmol), [N-methyl-3H ]diazepam (The Radiochemical Centre, Amersham, 85 Ci/mmol) and Ro 15-1788 (Roche) were used.
Procedure for #lduction of kindling Under anaesthesia with sodium pentobarbital (30 mg/kg, i.p.), male Wistar-strain rats weighing 190-250g were stereotaxically implanted with 5 pairs of bipolar electrodes made of two twisted stainless steel wires coated with Teflon, each 279 ILm in diameter (A-M systems, No. WSS 8T); olae pair was positioned in the left basolateral part of the amygdala (AP = + 3.75, ML = + 3.4, DV = - 3.0 mm according to KOnig and KlippeF) for stimulation, and the other pairs were positioned in the bilateral central parts of the amygdala (AP--- + 4.62, ML = + 3.45, DV = -2.4) and the bilateral hippocampi (AP = + 2.58, ML-- + 3.4, DV = + 1.5), respectively, for recording. Seven days after surgery, the animals were stimulated once or twice per day with a rectangular pulse train (400/~A, 1 ms duration, 60 Hz) for 1 s (Nihon Kohden, SEN-3201 and SS-320J). The afterdischarge was recorded by each pair of bipolar electrodes and amplified by the conventional method (Nihon Kohden, RB-2 and RM-150; time constant = 0.3 s). Behavioural changes were evaluated according to the classification of Racine ~6. When a rat showed the Stage 5 behavioural signs of Racine over 3 consecutive days, the animal was considered to have been kindled. In order to determine the position of the electrodes histologically, brains were removed after the end of the whole experiment, fixed in 10~ formalin for at least 24 h, and then stained with
both Luxol fast blue and Cresyl violet as 4-1(m paraffin sections. Diazepam was first dissolved in a minimum amount of 0.1 N HC! followed by dilution with saline to obtain an intraperitoneal injection volume of I ml/kg with an adequate concentration.
Preparation of hippocampal crude synaptic membranes and their binding assay Three days after the last of three Stage 5 seizures, bilateral hippocampi from kindled rats or electrode-implanted, unstimulated rats (sham-stimulated rats) were rapidly removed. The crude synaptic membrane was prepared, as described by Zukin et al. 21 with some modification. These differences were: that all sucrose, distilled water and buffer used included 1 mM EGTA; that 50 mM Tris-acetate buffer (pH 7.4) was used as the buffer; and that before the binding assay the crude synaptic membrane was washed 3 times with centrifugation at 48,000 x g for 10 rain at 4 ~ The final pellet was suspended in the buffer and diluted to keep the protein concentration in the range from 1.0 to 1.2 mg/ml following protein measurement according to the method of Lowry et al. io. In the case of [ 3H ]glutamic acid binding, the tissue suspension (final concentration: about 0.I mg) was preincubated for 15 min at 30 ~ [3H]glutamic acid solution (final concentration: 100 or 500 nM) was added, and the incubation was continued for an additional 30 min at 30 ~ Four ml of ice-cold buffer were added to each sample immediately before filtering through Whatman GF/B glass fiber filters. The filters were washed immediately with an additional 4 ml of ice-cold buffer, dried thoroughly, shaken with 6 ml of scintillation cocktail in a counting vial, and assayed for SH using a Packard Tri-Carb model 3255. Specific binding was determined in duplicate as the difference between total binding and that in the presence of L-glutamic acid (final concentration: 100 itM). The binding constants (Ko and B ...... ) were estimated by computer analysis (Statistical Analysis with Least-Squares Fitting; Program Library Laboratory, Computer Centre, The University of Tokyo) of the specific binding at different concentrations of [3H]glutamic acid (final concentrations: 2, 5, 10, 20, 50, 100, 300, 600 and I000 nM). In the case of [ 3H]diazepam binding, the procedure was the same as that described above, except that [3H]diazepam (final concentration: 0.5 nM) and diazepam (3 #M, in non-specific binding) were used, and the incubation temperature was 4 ~ The whole process described above was carried out on the same day.
Evahlation of statistical significance Statistical analysis was carried out using the two-tailed Student's t-test, or Welch's modification in cases where the variances of two compared groups were different.
Effects of diazepam on behaviour and afterdischarge The mean numbers o f stimulations needed to induce Stage 1 and Stage 5 seizures were 6.9 + 0.4 (the m e a n of 61 rats, with S.E.M. indicated) and 15.5 + 0.7 (63 rats), respectively. During this period, afterdischarge developed and increased in duration in the ipsilateral amygdala, the ipsilateral hippocampus, the contralateral amygdala and the contralateral hippocampus, in turn. It was observed that, once a rat had been kindled, the Stage 5 seizure could be triggered electrically, even after 6 months without any additional stimulation. As shown in Table I, diazepam inhibited the occurrence of kindled seizures, and decreased the duration as well as the amplitude of afterdischarge in all of the recorded areas o f fully kindled rats (neither data recorded in the right h i p p o c a m p u s nor those related to the afterdischarge amplitude are shown in Table I). Table II indicates the inhibitory effect of diazepam on the development of kindling. All of the 6 rats given diazepam (1 mg/kg, i.p.) 15 min before stimulation every day showed only Stage 1 seizures even on the 21st day of stimulation. When Stage 5 seizures
TABLE I THE EFFECTS OF DIAZEPAM ON KINDLED SEIZURE AND AFTERDISCHARGE DURATION Diazepam was administered 15 rain before electrical stimulation of the left amygdala. Saline with added HCI (the same pH as the corresponding diazepam solution) was injected in the control on the previous day to diazepam administration. Each value represents the mean of 3-5 rats, with S.E.M. indicated. * P < 0.01. compared with control. Note: although the value of seizure stage is essentially an integer (0 to 5), we show it here as a continuous value as a result of the arithmetical mean in order to show clearly the potency of the effect of diazepam. mg/kg (i.p.)
Control Diazepam
Afterdischarge duration (s) Seizure stage
Left amygdala
Left hippocampus
Right amygdala
78.3 + 8.6 64.3 + 23.8
79.5 + 13.5 66.0 11.8
72.0 + 11.5 68.7 + 13.9
86.3 + 5.4 60.3 8.6
80.0 + 6.6 71.0 10.7
59.5 + 9.1 50.5 + 10.3
0.1
5 3.2 + 1.1
Control Diazepam
0.3
5 3.0 + 1.0
Control Diazepam
1.0
5 0.2 + 0.2
77.0 + 6.7 12.3 7.1'
92.0 + 3.4 29.3 10.3"
79.5 + 12.3 11.0 ! 1.0"
3.0
5 0.8 + 0.2
79.8 +_ 10.7 13.5 7.0*
96.5 + 27.9 12.3 _~ 6.9
69.0 + 8.2 0.0(*)
Control Diazepam
TABLE 11 THE NUMBERS OF STIMULATIONS REQUIRED TO INDUCE STAGE 5 SEIZURE Diazepam, or saline with added HCI in the control, was administered 15 rain before stimulation every day for 21 days, or until the completion of kindling. Also indicated is the number of stimulations required to induce the Stage 5 seizure, after 21-day diazepam treatment had been discontinued (non-diazepam retest). Each value represents the mean, with S.E.M. indicated. Numbers in parentheses are the numbers of rats used. Number of stimulations Control Diazepam (I mg/kg) Non-diazcpam retest
15.6 ~ 0.77 (5) >21 (6) 7.0 ~ 1.3 (4)
TABLE 11I [3H]DIAZEPAM BINDING TO HIPPOCAMPAL CRUDE SYNAPTIC MEMBRANES IN THE ABSENCE OR PRESENCE OF 100/~M GABA Each value represents the mean binding in fmol/mg protein, with S.E.M, indicated. Numbers in parentheses are the numbers of rats used.
Intact (6) Sham-stimulated (3) Kindled (6)
Basal
+ 100 ltM GABA
133.3 ~+20.8 101.3 24.8 129.7 ~ 14.8
240.6 + 31.3 192.9 40.7 222.5 + 21.1
were observed in a non-diazepam retest, the afterdischarge duration in the left hippoc a m p u s was shorter than that in the control.
[3H]Diazepam binding to hippocampi of khldled rats As shown in Table III, there was no difference in either the basal [ 3 H ] d i a z e p a m (0.5 n M ) binding or in the enhancing effect o f G A B A on the binding to hippocampi of intact, sham-stimulated, and kindled rats.
Effects of diazepam on [~H]glutamate bhlding to hippocampi (a) Intact rats. As described in Materials and Methods, we used an incubation temperature of 30 ~ because it was preliminarily shown that: the specific binding at 30 ~ was 8 times as large as that at 0 ~ and was also larger than that at 24 ~ or 37 ~ and, the enhancing effect o f CaC12 on the binding was m o s t obvious at 30 ' C . Also, we used a protein concentration of 0.1 mg/assay, because around this concentration the binding showed a linear correlation with the protein concentration.
190 TABLE IV THE BINDING CONSTANTS OF [3H]GLUTAMIC ACID BINDING TO HIPPOCAMPAL CRUDE SYNAPTIC MEMBRANES OF INTACT RATS IN THE ABSENCE OR PRESENCE OF 10 mM CaCI: Values represent Ka in nM, and B..... in pmol/mg protein.
Kd. B..... ~ /Ca: B.... 2
22.6 0.506 1340 33.0
6.4 0.407 898 102.7
[~H ]Glutamic acid binding to hippocampal crude synaptic m e m b r a n e s from intact rats was specific and saturable, and was increased by CaC! 2 in a concentrationdependent manner (0.01-30 raM). The kinetic studies indicated the existence of two affinity states. Table IV shows the typical binding constants in the absence and presence of 10 mM CaCI 2, where C a C I : showed a maximal effect (i.e. 9 times), indicating that K d values were decreased and the B ...... , value was increased by CaCI z. The enhanced binding by CaCI_, was retained, when CaCI 2 was removed after a 30-rain preincubation (data not shown). In intact ral.s (n = 3-10), C I - (as Tris-HCl, 0.01-20 raM) enhanced [3H]glutamic acid binding; Ca "-+ (as calcium acetate, 0.01-30 m M ) did not enhance the binding, except in the presence of 20 m M C I - ; a protease inhibitor, leupeptin (100 #g/ml), inhibited this effect o f Ca-" ~ in the presence o f C I - but not the effect of C I - itself (data not shown). Diazepam ( 0 . 1 - 5ILM) showed no effect either on the basal [3H]glutamic acid binding or on the enhancing effect of CaCI, (0.01-30 m M ) .
TABLE v [-IHIGI.UTAMIC ACID BINDING (100 nM) TO tilPPOCAMPAL CRUDE SYNAPTIC MEMBRANES OF KINDLED OR SHAM-STIMUI.ATED RATS AND THE EFFECI" OF DIAZEPAM Each value represents the mean binding in pmol/mg protein, with S.E.M. indicated. Numbers in parentheses arc the numbers of rats used. * P < 0.05, compared with the binding in the absence of diazepam.
Sham-stimulated
+5/rim diazepam
1.27 + 0.44 (4) I.II _~0.52 (4)
3.53 + 0.61 (5) 3.52+0.51 (5}
7.20 + 0.61 (5) 6.60_~0.31 (5)
Kindled
+5/IM diazepam
i.22 + 0.31 (5) 1.01 0.34* (5)
3.72 + 0.46 (6) 3.97 + 0.51 (6)
7.22 __.0.86 (6) 7.51 0.76 (6)
A
B
1o.o
3.o
,,,,"'
o.ol
o.1
1.o
10
loo
o.ol
o.1
1.o
lo
Fig. I. Effects ofCaCI2 (A) or CI - (B) on [3lglutamie acid binding (100 nM) to hippocampal crude synaptic membranes of kindled or sham-stimulated rats. 0 , sham-stimulated rats; O, kindled rats. A: each point represents the mean of 4-6 rats, with S.E.M. indicated. Solid and broken horizontal lines represent the binding in sham-stimulated and kindled rats, respectively, in the absence of CaCI:. B: each point represents the mean of 3-4 rats, with S.E.M. indicated. Solid and broken horizontal lines represent the binding in sham-stimulated and kindled rats, respectively, in the absence of CI-.
(b) Kindled and sham-sthmdated rats Fig. 1A and B s h o w the effects o f CaCI2 and C i - , respectively, indicating that [ ~ H ] g l u t a m i c acid binding in kindled rats was increased by even lower c o n c e n t r a t i o n s o f CaCI z or C I - c o m p a r e d with that in sham-stimulated rats, even t h o u g h this was not statistically significant b e c a u s e o f large variances. As s h o w n in T a b l e V, there were no differences in the basal binding and maximally increased binding by C I - or C a C I , between sham-stimulated a n d kindled rats. TABLE VI [aHIGLUTAMIC ACID BINDING (500 riM) TO HIPPOCAMPAL CRUDE SYNAPTIC MEMBRANES OF KINDLED OR SHAM-STIMULATED RATS AND THE EFFECT OF DIAZEPAM Each value represents the mearr binding in pmol/mg protein, with S.E.M. indicated. Numbers in parentheses are the numbers ofrats used. ** P < 0,05, compared with the binding in sham-stimulated rats. * P < 0.01, compared with the binding in the absence of diazepam. Basal Sham-stimulated
5 #M diazepam
Kindled
+ 5/~M diazepam
3.49 + 0.46 3.75 + 0.77
+ 10 mM CaCI: (10) (9)
29.7 + 0.69 (8) 27.6 0.99 (7)
4.54.4- 0.51"* (10) 3.65 0.50" (9)
30.2 4- 0.84 (8) 34.3 2.9 (4)
However, diazepam slightly decreased only the basal binding in kindled rats. This effect of diazepam is made more obvious in Table VI, which shows the data for a higher [ 3H ]glutamic acid concentration of 500 nM. The basal binding increased in kindled rats in comparison with that in sham-stimulated rats, and only this increased binding was significantly inhibited by diazepam. Preliminary studies by the present authors have shown that this inhibiting effect of diazepam was enhanced by 100 #M GABA in 2 out of 3 kindled rats, and antagonized by 50 I~M Ro 15-1788, a benzodiazepine antagonist, in one kindled rat.
Our principal findings are that [3H]glutamic acid binding to hippocampi was increased after amygdaloid kindling, and that diazepam restored this increased binding, without any other effect on the binding. It should be noted that this increased binding was not induced by the seizure itself, because the binding assay was carried out 3 days after the last stimulation. By inducing kindling in the same way as that used in the binding experiment, we confirmed that the afterdischarge duration in hippocampi was increased after kindling, and that diazepam inhibited the development of kindled seizures as well as the seizure and afterdischarge in fully kindled rats, similar to results reported previously6"~7-t'~. Although we examined [ 3H]glutamic acid binding only to hippocampi, it is possible that increased binding may have been observed wherever the increased afterdischargc was recorded after kindling. As to the mechanism of the inhibiting effect of benzodiazepines on kindling, it has been suggested that benzodiazepines suppress the impulse propagation and generalization of epileptic activity in the central nervous system, both in developing and fully kindled states, by enhancing the GABAergic inhibitory mechanism6. According to our findings, diazepam probably inhibits the increase of glutamate receptors capable of binding the excitatory transmitter, glutamic acid, in the process of the development of kindling as well as in the fully kindled state, thus this mechanism contributes considerably to the anti-kindling effect of diazepam. Certainly, in a future study, we should examine whether glutamate receptors increase during the process of kindling development and whether they are inhibited by diazepam. In the present study, we determined the conditions best suited for examination of glutamate binding, particularly in order to assess clearly the increasing effect of Ca 2 § and/or CI-, because such conditions have not yet been confirmed, although some related studies have been reported using somewhat different conditions 1"4't3'~4. A wide range of K d values has been reported4. Our values described here lie in this range. We used [ 3H ]glutamic acid at 100 nM and 500 nM for the present work, since the binding in these concentrations is considered to be representative of that on "medium affinity sites''a and to be dependent on CI - and Ca 2§ ions 4. It should be noted that no inhibiting effect ofdiazepam on the glutamate binding in kindled rats could be detected whenever
CI- and/or Ca 2 § were included in the experimental process. Contamination with a small amount of CI- and/or Ca 2 § partly accounted for the large variances observed in the data. The present study indicates that glutamate binding may be increased by even lower concentrations of CI- in kindled rats, suggesting an increase in the receptor subtype capable of binding ligands CI- dependently, as reported by Savage et al. w. Diazepam did not have any influence on this CI- effect. According to our results related to the effects of diazepam, the increase in glutamate binding observed after kindling is probably a different phenomenon from that induced by CaCI2, even though both changes may be considered to be induced through some biochemical process, this assumption being contrary to that of Savage et ai.~, who did not examine any effect of diazepam. As to the mechanism of the effect of diazepam, we are not yet able to explain this clearly at the present stage of our studies. It is, however, suggested that this effect may occur after the binding of diazepam to its specific receptor coupled with the GABA receptor, and that there may be no change in the characteristics of diazepam receptors under the conditions used in the glutamate binding experiments, although some previous reports have shown changes of diazepam receptors12'ls, using different conditions. In conclusion, we demonstrated the restoring effect of diazepam on the increased [3H]glutamic acid binding to hippocampi of kindled rats. These results lead us to studies on the mechanism of diazepam effects following its binding to the receptor, and on the biochemical mechanism responsible for the increase in the glutamate receptor in relation to information storage in the brain.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (57480382). We are greatly indebted to Sumitomo Chem. and Nippon Roche for providing diazepam and Ro 15-1788, respectively.
REFERENCES 1 Baudry,M., Siman,R., Smith, E.K. and Lynch,G., Regulationby calciumions of glutamate receptor binding in hippocampal slices, Eur. d. PharmacoL, 90 (1983) 161-168. 2 Ben.Ari, Y., Tremblay, E., Ottersen, O.P. and Meldrum, B.S., The role of epileptic activity in hippocampal and 'remote' cerebral lesions induced by kainic acid, Brahl Res., 191 (1980) 79-97. 3 Bliss,T.V.P. and Lomo,T., Long-lastingpotentiationof synaptietransmissionin the dentate area ofthe unanaesthetized rabbit followingstimulation of the perforant path, J. Physiol. (London), 232 (1973) 357-374. 4 Foster, A.C. and Fagg, G., Acidicamino acid binding sites in mammalianneuronal membranes:their characteristics and relationship to synaptic receptors,Brain Res. Rev., 7 (1984) 103-164.
194 5 Goddard, G.K., Mclntyre, D.C. and Leech, C.K., A permanent ehal~ge in brain function resulting from daily electrical stimulation, Exp. NeuroL, 25 (1969) 295-330. 6 H aefely, W., Pole, P., Pieri, L., Sehaffner, R. and Laurent, J.-P., Neuropharmacology ofbenzodiazepines: synaptie nleehanisms and neural basis of action. In E. Costa (Ed.), The Benzodiazepines:from Molecular Biology to Cl01icalPractice, Raven Press, New York, 1983, pp. 21-66. 7 Hjorth-Simonsen, A., Projection of the lateral part of the entorhinal area to the hippoeampus and facia dentata, J. Comp. Neurol., 146 (1972) 219-232. 8 K6nig, J.F.R. and Klippel, R.A., The Rat Bra#z: a Stereotoxic Atlas of the Forebrain and Lower Parts of tile Brain Stem, Krieger, Huntington, New York, 1967. 9 Krettek, J.E. and Price, J.L., Projections from the anlygdaloid complex and adjacent olfactory structures to the entorhinal cortex and to tile subiculum in the rat and eat, J. Comp. Neurol., 172 (1977) 723-752. 10 Lowry, O.1-1., Rosebrough, R.N., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. I 1 Lynch, (3., Halpain, S. and Baudry, M., Effects of high-frequency synaptic stimulation on glutamate receptor binding studied with modified in vitro hippoc.'nllpal slice preparation, Brain Res., 244 (1982) 1(11-111. 12 McNamara, J.O., Peper, A.N. and Patrone, V., Repeated seizures induce long-term increase in hippocampal benzodiazepine receptors, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 3029-3032. 13 Mena, E.E., Whittemore, S.R., Monaghan, D.T. and Cotman, C.W., Ionic regulation of glutamate binding sites, Life Sci., 35 (1984) 2427-2433. 14 Nadler, J.V., Wang, A. and Werling, L.L., Binding sites lbr I.-[31-1]glutamateon hippocampal synaptic membranes: three populations differentially affected by chloride and calcium ions, d. Nearochem., 44 (1985) 1791-1798. 15 Niznik, H.B., Burnham, W.M. and Kish, S.J., Benzodiazcpine receptor binding following amygdalakindled convulsions: differing results ill washed and unwashed brain membranes, d. Neuroehem., 43 (1984) 1732-1736. 16 Racine, R., Modification of seizure activity by electrical stimulation: Ii. Motor seizure, Electroenc,'l,halogr. Clin. Neurophysiol., 32 (1972) 281-294. 17 Racine, R., Livingston, K. and Joaquin, A., Effects of procaine hydroehloride, diazepam, and diphenyl hydantoin on seizure development in cortical and subcortical structures in rats, Electroencephalogr. Clin. Neuroplo,sioL, 38 (1975) 355-365. 18 Robertson, H.A. and Riives, M., A benzodiazepine antagonist is an anticonvulsant in an animal model for limbic epilepsy, Bra#l Res., 270 (1983) 380-382. 19 Savage, D.D., Werling, L.L., Nadler, J.V. and MeNamara, J.O., Selective increase in L-[3H]glutamate binding to a quisqualate-sensitive site on hippocampal synaptie membranes after angular bundle kindling, Eur. J. Phannacol., 85 (1982) 255-256. 20 Wise, R.A. and Chinerman, J.. Effects ofdiazepam and phenobarbital on electricallyinduced amygdaloid seizures and seizure development, Exp. Neurot., 45 (1974) 355-363. 21 Zukin, S.R., Young, A.B. and Snyder, S.H., Gamma-aminobutyric acid binding to receptor sites in the rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 4802-4807.
NSR 00165
Diazepam restores the increased [3H]glutamate binding to hippocampal synaptic membranes in the amygdalcid-kindled rat Jun-Ichiro Oka, Mari Yamada, Masayoshi Goto and Hideomi Fukuda Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Btmkyo-ku. Tokyo (Japan)
(Received 24 March 1986; Revised version received 16 July 1986; Accepted 28 July 1986) Key words: Diazepam; [3H]Glutamate binding; CaCI2; Hippocampus; Amygdala; Kindling; Rat
[3HlGlutamic aeid binding to hippocampi was increased after amygdaloid kindling in rats, and diazepam inhibited this increased binding, without any effect on the enhanced binding by CaCI2 or the binding in control rats. By inducing kindlingin the same way as that used in the binding experiment, the inhibitingeffects of diazepam on kindled seizures, the afterdischargeand the developmentof kindlingwere observed.
Kindling, described originally by Goddard et al. in 19695, is one of the animal models of epilepsy, and may provide a model of learning or information storage in the brain, because seizure susceptibility in kindled animals can last for a long time. This phenomenon has features in common with the other model of synaptic memory, the so-called long-term potentiation (LTP) in hippocampi 3'4.~ t. Savage et al. 19 reported the selective increase in [ 3H ]glutamate binding to a quisqualate-sensitive site in hippocampi after angular bundle kindling, suggesting a similar phenomenon to that in the case of LTP tl. There has been some evidence which may suggest the existence of a neuronal connection between the amygdala and the hippocampus 2. The amygdala projects to the entorhinal cortex 9 from which originates the perforant path; the perforant path impinges on hippocampal neurons 7. This excitatory input may become stimulated in the process Correspondence: H. Fukuda, Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,Tokyo 113, Japan.
186 of amygdaloid kindling, and may, in turn, induce an increase in the glutamate receptor. There are many reports dealing with the anticonvulsant effects of benzodiazepines 6, which prevent or abolish the seizures, and the electroencephalogram afterdischarge induced by chemical convulsants or electroshock, and also those induced in kindled animals 6.17-19. In order to explore a new profile of the anti-kindling effects of benzodiazepines, we examined the effect of diazepam on [3H]glutamate binding to hippocampal synaptic membranes in the amygdaloid-kindled rat, after determining the conditions necessary for the induction of kindling and examining [ 3H]glutamate binding in the absence or presence of Ca 2+ and/or CI-. Here we show that diazepam selectively restored the increased [3H]glutamate binding in kindled rats, but did not influence the effect of CaCI 2.
Materials Diazepam (Sumitomo Chem.), ~,-aminobutyric acid (GABA), L-glutamic acid, ethyleneglycol-bis-(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, Wako Pure Chem.), leupeptin (Sigma Chem., Lot. 42F-0525), bovine serum albumin (Nakarai Chem.), L-[2,3-aH]glutamic acid (New England Nuclear, 21.6 Ci/mmol), [N-methyl-3H ]diazepam (The Radiochemical Centre, Amersham, 85 Ci/mmol) and Ro 15-1788 (Roche) were used.
Procedure for #lduction of kindling Under anaesthesia with sodium pentobarbital (30 mg/kg, i.p.), male Wistar-strain rats weighing 190-250g were stereotaxically implanted with 5 pairs of bipolar electrodes made of two twisted stainless steel wires coated with Teflon, each 279 ILm in diameter (A-M systems, No. WSS 8T); olae pair was positioned in the left basolateral part of the amygdala (AP = + 3.75, ML = + 3.4, DV = - 3.0 mm according to KOnig and KlippeF) for stimulation, and the other pairs were positioned in the bilateral central parts of the amygdala (AP--- + 4.62, ML = + 3.45, DV = -2.4) and the bilateral hippocampi (AP = + 2.58, ML-- + 3.4, DV = + 1.5), respectively, for recording. Seven days after surgery, the animals were stimulated once or twice per day with a rectangular pulse train (400/~A, 1 ms duration, 60 Hz) for 1 s (Nihon Kohden, SEN-3201 and SS-320J). The afterdischarge was recorded by each pair of bipolar electrodes and amplified by the conventional method (Nihon Kohden, RB-2 and RM-150; time constant = 0.3 s). Behavioural changes were evaluated according to the classification of Racine ~6. When a rat showed the Stage 5 behavioural signs of Racine over 3 consecutive days, the animal was considered to have been kindled. In order to determine the position of the electrodes histologically, brains were removed after the end of the whole experiment, fixed in 10~ formalin for at least 24 h, and then stained with
both Luxol fast blue and Cresyl violet as 4-1(m paraffin sections. Diazepam was first dissolved in a minimum amount of 0.1 N HC! followed by dilution with saline to obtain an intraperitoneal injection volume of I ml/kg with an adequate concentration.
Preparation of hippocampal crude synaptic membranes and their binding assay Three days after the last of three Stage 5 seizures, bilateral hippocampi from kindled rats or electrode-implanted, unstimulated rats (sham-stimulated rats) were rapidly removed. The crude synaptic membrane was prepared, as described by Zukin et al. 21 with some modification. These differences were: that all sucrose, distilled water and buffer used included 1 mM EGTA; that 50 mM Tris-acetate buffer (pH 7.4) was used as the buffer; and that before the binding assay the crude synaptic membrane was washed 3 times with centrifugation at 48,000 x g for 10 rain at 4 ~ The final pellet was suspended in the buffer and diluted to keep the protein concentration in the range from 1.0 to 1.2 mg/ml following protein measurement according to the method of Lowry et al. io. In the case of [ 3H ]glutamic acid binding, the tissue suspension (final concentration: about 0.I mg) was preincubated for 15 min at 30 ~ [3H]glutamic acid solution (final concentration: 100 or 500 nM) was added, and the incubation was continued for an additional 30 min at 30 ~ Four ml of ice-cold buffer were added to each sample immediately before filtering through Whatman GF/B glass fiber filters. The filters were washed immediately with an additional 4 ml of ice-cold buffer, dried thoroughly, shaken with 6 ml of scintillation cocktail in a counting vial, and assayed for SH using a Packard Tri-Carb model 3255. Specific binding was determined in duplicate as the difference between total binding and that in the presence of L-glutamic acid (final concentration: 100 itM). The binding constants (Ko and B ...... ) were estimated by computer analysis (Statistical Analysis with Least-Squares Fitting; Program Library Laboratory, Computer Centre, The University of Tokyo) of the specific binding at different concentrations of [3H]glutamic acid (final concentrations: 2, 5, 10, 20, 50, 100, 300, 600 and I000 nM). In the case of [ 3H]diazepam binding, the procedure was the same as that described above, except that [3H]diazepam (final concentration: 0.5 nM) and diazepam (3 #M, in non-specific binding) were used, and the incubation temperature was 4 ~ The whole process described above was carried out on the same day.
Evahlation of statistical significance Statistical analysis was carried out using the two-tailed Student's t-test, or Welch's modification in cases where the variances of two compared groups were different.
Effects of diazepam on behaviour and afterdischarge The mean numbers o f stimulations needed to induce Stage 1 and Stage 5 seizures were 6.9 + 0.4 (the m e a n of 61 rats, with S.E.M. indicated) and 15.5 + 0.7 (63 rats), respectively. During this period, afterdischarge developed and increased in duration in the ipsilateral amygdala, the ipsilateral hippocampus, the contralateral amygdala and the contralateral hippocampus, in turn. It was observed that, once a rat had been kindled, the Stage 5 seizure could be triggered electrically, even after 6 months without any additional stimulation. As shown in Table I, diazepam inhibited the occurrence of kindled seizures, and decreased the duration as well as the amplitude of afterdischarge in all of the recorded areas o f fully kindled rats (neither data recorded in the right h i p p o c a m p u s nor those related to the afterdischarge amplitude are shown in Table I). Table II indicates the inhibitory effect of diazepam on the development of kindling. All of the 6 rats given diazepam (1 mg/kg, i.p.) 15 min before stimulation every day showed only Stage 1 seizures even on the 21st day of stimulation. When Stage 5 seizures
TABLE I THE EFFECTS OF DIAZEPAM ON KINDLED SEIZURE AND AFTERDISCHARGE DURATION Diazepam was administered 15 rain before electrical stimulation of the left amygdala. Saline with added HCI (the same pH as the corresponding diazepam solution) was injected in the control on the previous day to diazepam administration. Each value represents the mean of 3-5 rats, with S.E.M. indicated. * P < 0.01. compared with control. Note: although the value of seizure stage is essentially an integer (0 to 5), we show it here as a continuous value as a result of the arithmetical mean in order to show clearly the potency of the effect of diazepam. mg/kg (i.p.)
Control Diazepam
Afterdischarge duration (s) Seizure stage
Left amygdala
Left hippocampus
Right amygdala
78.3 + 8.6 64.3 + 23.8
79.5 + 13.5 66.0 11.8
72.0 + 11.5 68.7 + 13.9
86.3 + 5.4 60.3 8.6
80.0 + 6.6 71.0 10.7
59.5 + 9.1 50.5 + 10.3
0.1
5 3.2 + 1.1
Control Diazepam
0.3
5 3.0 + 1.0
Control Diazepam
1.0
5 0.2 + 0.2
77.0 + 6.7 12.3 7.1'
92.0 + 3.4 29.3 10.3"
79.5 + 12.3 11.0 ! 1.0"
3.0
5 0.8 + 0.2
79.8 +_ 10.7 13.5 7.0*
96.5 + 27.9 12.3 _~ 6.9
69.0 + 8.2 0.0(*)
Control Diazepam
TABLE 11 THE NUMBERS OF STIMULATIONS REQUIRED TO INDUCE STAGE 5 SEIZURE Diazepam, or saline with added HCI in the control, was administered 15 rain before stimulation every day for 21 days, or until the completion of kindling. Also indicated is the number of stimulations required to induce the Stage 5 seizure, after 21-day diazepam treatment had been discontinued (non-diazepam retest). Each value represents the mean, with S.E.M. indicated. Numbers in parentheses are the numbers of rats used. Number of stimulations Control Diazepam (I mg/kg) Non-diazcpam retest
15.6 ~ 0.77 (5) >21 (6) 7.0 ~ 1.3 (4)
TABLE 11I [3H]DIAZEPAM BINDING TO HIPPOCAMPAL CRUDE SYNAPTIC MEMBRANES IN THE ABSENCE OR PRESENCE OF 100/~M GABA Each value represents the mean binding in fmol/mg protein, with S.E.M, indicated. Numbers in parentheses are the numbers of rats used.
Intact (6) Sham-stimulated (3) Kindled (6)
Basal
+ 100 ltM GABA
133.3 ~+20.8 101.3 24.8 129.7 ~ 14.8
240.6 + 31.3 192.9 40.7 222.5 + 21.1
were observed in a non-diazepam retest, the afterdischarge duration in the left hippoc a m p u s was shorter than that in the control.
[3H]Diazepam binding to hippocampi of khldled rats As shown in Table III, there was no difference in either the basal [ 3 H ] d i a z e p a m (0.5 n M ) binding or in the enhancing effect o f G A B A on the binding to hippocampi of intact, sham-stimulated, and kindled rats.
Effects of diazepam on [~H]glutamate bhlding to hippocampi (a) Intact rats. As described in Materials and Methods, we used an incubation temperature of 30 ~ because it was preliminarily shown that: the specific binding at 30 ~ was 8 times as large as that at 0 ~ and was also larger than that at 24 ~ or 37 ~ and, the enhancing effect o f CaC12 on the binding was m o s t obvious at 30 ' C . Also, we used a protein concentration of 0.1 mg/assay, because around this concentration the binding showed a linear correlation with the protein concentration.
190 TABLE IV THE BINDING CONSTANTS OF [3H]GLUTAMIC ACID BINDING TO HIPPOCAMPAL CRUDE SYNAPTIC MEMBRANES OF INTACT RATS IN THE ABSENCE OR PRESENCE OF 10 mM CaCI: Values represent Ka in nM, and B..... in pmol/mg protein.
Kd. B..... ~ /Ca: B.... 2
22.6 0.506 1340 33.0
6.4 0.407 898 102.7
[~H ]Glutamic acid binding to hippocampal crude synaptic m e m b r a n e s from intact rats was specific and saturable, and was increased by CaC! 2 in a concentrationdependent manner (0.01-30 raM). The kinetic studies indicated the existence of two affinity states. Table IV shows the typical binding constants in the absence and presence of 10 mM CaCI 2, where C a C I : showed a maximal effect (i.e. 9 times), indicating that K d values were decreased and the B ...... , value was increased by CaCI z. The enhanced binding by CaCI_, was retained, when CaCI 2 was removed after a 30-rain preincubation (data not shown). In intact ral.s (n = 3-10), C I - (as Tris-HCl, 0.01-20 raM) enhanced [3H]glutamic acid binding; Ca "-+ (as calcium acetate, 0.01-30 m M ) did not enhance the binding, except in the presence of 20 m M C I - ; a protease inhibitor, leupeptin (100 #g/ml), inhibited this effect o f Ca-" ~ in the presence o f C I - but not the effect of C I - itself (data not shown). Diazepam ( 0 . 1 - 5ILM) showed no effect either on the basal [3H]glutamic acid binding or on the enhancing effect of CaCI, (0.01-30 m M ) .
TABLE v [-IHIGI.UTAMIC ACID BINDING (100 nM) TO tilPPOCAMPAL CRUDE SYNAPTIC MEMBRANES OF KINDLED OR SHAM-STIMUI.ATED RATS AND THE EFFECI" OF DIAZEPAM Each value represents the mean binding in pmol/mg protein, with S.E.M. indicated. Numbers in parentheses arc the numbers of rats used. * P < 0.05, compared with the binding in the absence of diazepam.
Sham-stimulated
+5/rim diazepam
1.27 + 0.44 (4) I.II _~0.52 (4)
3.53 + 0.61 (5) 3.52+0.51 (5}
7.20 + 0.61 (5) 6.60_~0.31 (5)
Kindled
+5/IM diazepam
i.22 + 0.31 (5) 1.01 0.34* (5)
3.72 + 0.46 (6) 3.97 + 0.51 (6)
7.22 __.0.86 (6) 7.51 0.76 (6)
A
B
1o.o
3.o
,,,,"'
o.ol
o.1
1.o
10
loo
o.ol
o.1
1.o
lo
Fig. I. Effects ofCaCI2 (A) or CI - (B) on [3lglutamie acid binding (100 nM) to hippocampal crude synaptic membranes of kindled or sham-stimulated rats. 0 , sham-stimulated rats; O, kindled rats. A: each point represents the mean of 4-6 rats, with S.E.M. indicated. Solid and broken horizontal lines represent the binding in sham-stimulated and kindled rats, respectively, in the absence of CaCI:. B: each point represents the mean of 3-4 rats, with S.E.M. indicated. Solid and broken horizontal lines represent the binding in sham-stimulated and kindled rats, respectively, in the absence of CI-.
(b) Kindled and sham-sthmdated rats Fig. 1A and B s h o w the effects o f CaCI2 and C i - , respectively, indicating that [ ~ H ] g l u t a m i c acid binding in kindled rats was increased by even lower c o n c e n t r a t i o n s o f CaCI z or C I - c o m p a r e d with that in sham-stimulated rats, even t h o u g h this was not statistically significant b e c a u s e o f large variances. As s h o w n in T a b l e V, there were no differences in the basal binding and maximally increased binding by C I - or C a C I , between sham-stimulated a n d kindled rats. TABLE VI [aHIGLUTAMIC ACID BINDING (500 riM) TO HIPPOCAMPAL CRUDE SYNAPTIC MEMBRANES OF KINDLED OR SHAM-STIMULATED RATS AND THE EFFECT OF DIAZEPAM Each value represents the mearr binding in pmol/mg protein, with S.E.M. indicated. Numbers in parentheses are the numbers ofrats used. ** P < 0,05, compared with the binding in sham-stimulated rats. * P < 0.01, compared with the binding in the absence of diazepam. Basal Sham-stimulated
5 #M diazepam
Kindled
+ 5/~M diazepam
3.49 + 0.46 3.75 + 0.77
+ 10 mM CaCI: (10) (9)
29.7 + 0.69 (8) 27.6 0.99 (7)
4.54.4- 0.51"* (10) 3.65 0.50" (9)
30.2 4- 0.84 (8) 34.3 2.9 (4)
However, diazepam slightly decreased only the basal binding in kindled rats. This effect of diazepam is made more obvious in Table VI, which shows the data for a higher [ 3H ]glutamic acid concentration of 500 nM. The basal binding increased in kindled rats in comparison with that in sham-stimulated rats, and only this increased binding was significantly inhibited by diazepam. Preliminary studies by the present authors have shown that this inhibiting effect of diazepam was enhanced by 100 #M GABA in 2 out of 3 kindled rats, and antagonized by 50 I~M Ro 15-1788, a benzodiazepine antagonist, in one kindled rat.
Our principal findings are that [3H]glutamic acid binding to hippocampi was increased after amygdaloid kindling, and that diazepam restored this increased binding, without any other effect on the binding. It should be noted that this increased binding was not induced by the seizure itself, because the binding assay was carried out 3 days after the last stimulation. By inducing kindling in the same way as that used in the binding experiment, we confirmed that the afterdischarge duration in hippocampi was increased after kindling, and that diazepam inhibited the development of kindled seizures as well as the seizure and afterdischarge in fully kindled rats, similar to results reported previously6"~7-t'~. Although we examined [ 3H]glutamic acid binding only to hippocampi, it is possible that increased binding may have been observed wherever the increased afterdischargc was recorded after kindling. As to the mechanism of the inhibiting effect of benzodiazepines on kindling, it has been suggested that benzodiazepines suppress the impulse propagation and generalization of epileptic activity in the central nervous system, both in developing and fully kindled states, by enhancing the GABAergic inhibitory mechanism6. According to our findings, diazepam probably inhibits the increase of glutamate receptors capable of binding the excitatory transmitter, glutamic acid, in the process of the development of kindling as well as in the fully kindled state, thus this mechanism contributes considerably to the anti-kindling effect of diazepam. Certainly, in a future study, we should examine whether glutamate receptors increase during the process of kindling development and whether they are inhibited by diazepam. In the present study, we determined the conditions best suited for examination of glutamate binding, particularly in order to assess clearly the increasing effect of Ca 2 § and/or CI-, because such conditions have not yet been confirmed, although some related studies have been reported using somewhat different conditions 1"4't3'~4. A wide range of K d values has been reported4. Our values described here lie in this range. We used [ 3H ]glutamic acid at 100 nM and 500 nM for the present work, since the binding in these concentrations is considered to be representative of that on "medium affinity sites''a and to be dependent on CI - and Ca 2§ ions 4. It should be noted that no inhibiting effect ofdiazepam on the glutamate binding in kindled rats could be detected whenever
CI- and/or Ca 2 § were included in the experimental process. Contamination with a small amount of CI- and/or Ca 2 § partly accounted for the large variances observed in the data. The present study indicates that glutamate binding may be increased by even lower concentrations of CI- in kindled rats, suggesting an increase in the receptor subtype capable of binding ligands CI- dependently, as reported by Savage et al. w. Diazepam did not have any influence on this CI- effect. According to our results related to the effects of diazepam, the increase in glutamate binding observed after kindling is probably a different phenomenon from that induced by CaCI2, even though both changes may be considered to be induced through some biochemical process, this assumption being contrary to that of Savage et ai.~, who did not examine any effect of diazepam. As to the mechanism of the effect of diazepam, we are not yet able to explain this clearly at the present stage of our studies. It is, however, suggested that this effect may occur after the binding of diazepam to its specific receptor coupled with the GABA receptor, and that there may be no change in the characteristics of diazepam receptors under the conditions used in the glutamate binding experiments, although some previous reports have shown changes of diazepam receptors12'ls, using different conditions. In conclusion, we demonstrated the restoring effect of diazepam on the increased [3H]glutamic acid binding to hippocampi of kindled rats. These results lead us to studies on the mechanism of diazepam effects following its binding to the receptor, and on the biochemical mechanism responsible for the increase in the glutamate receptor in relation to information storage in the brain.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (57480382). We are greatly indebted to Sumitomo Chem. and Nippon Roche for providing diazepam and Ro 15-1788, respectively.
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