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Kindling and electrode effects on the benzodiazepine receptors density of olfactory bulb and hippocampus after olfactory bulb kindling
~;euro.wiencc L~,r*~'r.~, 143 11992) 74 7,~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940'92/$ 05.0~
74
NSL 08862
Kindling and electrode effects on the benzodiazepine receptors density of olfactory bulb and hippocampus after olfactory bulb kindling M. Ben Attia a, P. N ' G o u e m o b, M. Belaidi b, G. R o n d o u i n b and R. Chicheportiche a alNSERM U.336, Laboratoire de Biochimie GOn~rale, Ecole Nationale Sup~rieure de Chimie de Montpellier, Montpellier (France) and bCNRS UPR.8402-1NSERM U.249, Laboratoire de MOdecine Expkrimentale, Institut de Biologie, Montpellier (France) (Received 9 December 1991; Revised version received l0 April 1992; Accepted 18 May 1992)
Key words." Kindling; Olfactory bulb; Hippocampus; Epilepsy; Benzodiazepine receptor; Electrode effect; Rat The olfactory bulb (OB) kindling is a model of limbic secondary generalized epilepsy. Ten days after the completion of OB kindling, we have studied the long term effects of both electrode insertion and kindling on the binding of [3H]diazepam to crude mitochondrial fractions. On the one hand, we have shown that electrode implantation in sham-operated controls induced an obvious increase in benzodiazepine (BZD) receptor density (Bm~) only at the site of the electrode in comparison to sham-unoperated rats. These results might indicate an additional mechanism extending earlier observations reported by others, who have shown that prolonged electrode implantation induced changes in sham-operated and kindled rats. On the other hand, the long lasting effect of OB kindling on the binding parameters of [3H]diazepam was examined in the focus and in the hippocampus. The results indicate a bilateral increase of BZD receptors in the OB and an ipsilateral increase in the hippocampus. These changes might be a regulation phenomenon in response to a hyperexcitability state and to focal stimulations.
The kindling phenomenon is induced by repetitive low level electrical stimuli to susceptible regions of the brain until the apparition of electrical seizure activity [17]. Full convulsion is associated with behavioural changes which are divided into 5 behavioural stages as described by Racine [30]. The seizures that occur in this model are intermittent and paroxymal and share many other similarities with human epilepsy [16, 25]. Among the brain areas, the limbic system is the most susceptible to kindling [17]. While Goddard et al. [17] showed that amygdala was a brain structure of great susceptibility, other groups have found that the olfactory bulb (OB) was more sensitive to electrical stimulations [12], with a slightly higher afterdischarge threshold [5, 12], but without occurrence of oscillatory phenomena [5]. Moreover, the OB as the site of kindling stimulations offers several advantages in comparison with the other cerebral structures. Indeed, its rostral anatomical position permits a direct implantation of the electrode with respect to the integrity of forebrain areas or pathways. The electrical kindling has been increasingly used as Correspondence: R. Chicheportiche, INSERM U.336, Laboratoire de Biochimie G6n6rale, Ecole Nationale Sup~rieure de Chimie de Montpellier, 8 rue de l'Ecole Normale, 34053 Montpellier Cedex 1, France.
an excellent tool for the neurochemical studies. It is the most extensively investigated among animal models of epilepsy. But the precise physiological and neurochemical mechanisms by which this occurs remains unknown. In the present study, we carried out a series of multifactorial experiments in order to determine whether kindling and/or electrode implantation effects caused any dysfunction of benzodiazepine (BZD) receptors at the site of the electrode and at the hippocampus 10 days after the last electrical stimulation to avoid a short term effect of the seizure itself on neurochemical studies. It should be emphasized, however, that factors other than seizures, kindling, or both may be also involved. Male Sprague-Dawley rats (250-300 g) were used throughout the study. Rats were randomly divided into 3 sets of 15 animals, each set being subdivided into 3 groups of 5 rats which are designated as follows. Control I group: sham-non-implanted rats were daily handled but they were neither implanted nor stimulated. Control II group: sham-implanted rats with one electrode implanted in the right OB as described below, were daily handled and not submitted to the stimulation process. Kindled group: the rats of this group were implanted in the right OB and both handled and stimulated daily. During surgery, all rats of control II and kindled groups were anaesthetized by Equithesin (0.4 ml/100 g,
75 i.p.) and placed in a stereotaxic apparatus. A bipolar electrode made from two strands of nickel-chrome wires (100/,tin) twisted together, and insulated except at the tip, was stereotaxically implanted into the right OB (A, + 15.0 mm; L, 1.5; H, 2.5). At least 2 weeks were allowed for recovery from surgery before experimentation was initiated. The rats of kindled groups were submitted to stimulation sessions. Electrical stimulations were given according to the parameters described by Goddard et al. [17]: a pulse duration 1 ms, biphasic square waves, train duration 2 s, frequency 60 Hz. The initial intensity, delivered through the electrode, was set to 50 ~A peak to peak and increased by 50/,tA steps until an afterdischarge (AD) was triggered. The kindling proceeded twice daily until appearance of the sixth generalized seizure (stage 5). The electrical activity was recorded on an electroencephalograph and the behavioural stages scored as described by Racine [30]. Ten days after the last stimulation of the kindled group, the rats of the same set were killed by decapitation. After remaining and washing procedures of brains, only the OBs and hippocampus with attached area dentata were dissected from both sides and stored at - 8 0 ° C until further analysis. On the day of the analysis the previously frozen tissues were thawed at room temperature and homogenized using a teflon homogenizer in 10 vols. of ice-cold 0.32 M sucrose and centrifuged at 1,000 x g for 10 min. The supernatants were then centrifuged 20 min at 17,000 x g to yield the crude synaptosomal membranes (P2 fraction). [3H]Diazepam (70 Ci/mmol) was obtained from the Service des Molecules Marquees (C.E.A., Saclay, France). The P~ fractions were resuspended in ice-cold 50 mM Tris-HCl, pH 7.4, for binding assay. The membrane preparations were incubated with ice-cold buffer (50 mM Tris-HC1, pH 7.4) containing [3H]diazepam at the desired concentration. After 1 h of incubation at 4°C, 0.5ml aliquots of the suspension were carefully filtered through GF/B filters which were rapidly washed twice with 5 ml ice-cold incubation buffer. The radioactivity on the filters was assayed by liquid scintillation counting in 4 ml of liquid scintillant (ACS I, Amersham). The nonspecific binding was performed in the presence of unlabelled flunitrazepam (10 -s M). Protein was measured by the technique of Hartree [11]. Statistical analysis was computed by a one-way ANOVA followed by Newman Keuls multiple range test. Significance was considered at P < 0.05. There is evidence that the site of the electrode [12, 17] and the prolonged electrode insertion [7, 8] influenced the kindling rate. The question one must ask is, does this electrode have effects on the neurochemical studies? We
decided to determine if the influence of the implanted electrode and/or OB kindling, in each side of OB, affected the binding characteristics of [~H]diazepam. The binding parameters of [3H]diazepam in the right OB (site of the electrode implantation) were subjected to one-way analysis of variance. The difference between B..... values was significant (~,6 = 47.28: P < 0.001 ) while no difference was found between the KD values (Table I). Student Newman Keuls test indicated significant increase between control I and kindled groups ( P < 0.01), between control II and kindled groups (P < 0.01), and between control II and control 1 groups (P < 0.01 ). Focusing our attention on the left OB, we investigated both the effect of OB kindling and a possible artifact due to the electrode implantation in the controlateral side. A oneway analysis of variance of the number of [3H]diazepam binding sites (B ..... ) was significant (F,.~, -- 23.65: P < 0.01). This difference was subjected to Student Newman Keuls test as mentioned earlier. We showed a significant increase between control I and kindled groups ( P < 0 . 0 1 ) , between control II and kindled groups (P < 0.01 ), but there was no difference between control I and control II. These results demonstrate that electrode implantation produced a significant increase of B...... (+25 _+ 6%) only in the ipsilateral side and not in the contralateral side, while the kindling increased significantly the B...... values in each side of the OB (+27 + 6% at right and +30 _+ 7% at left) 10 days after the last kindled seizure. In contrast, in both sides of the OB the KD for [3H]diazepam did not change after kindling effect or electrode implantation, according to the same statistical analysis used for the B....... To be sure that the results, concerning the effect of the electrode, observed in the focal area (OB) were not seen in other areas, not crossed by the electrode, we have chosen the hippocampus for the same multiple comparisons used for OB (Table I). In the right side, the B ...... values of the 3 groups of rats compared by analysis of variance, indicated a significant difference (~,~, = 6.50: P < 0.05). Student Newman-Keuls test showed a significant difference between control I and kindled groups (P < 0.05), and between control lI and kindled groups (P < 0.05). No significant difference in B...... values was seen between control I and control II. In the left side, analysis of variance revealed that the B..... values of these 3 groups of rats were not significantly different from each other. These results demonstrate: first, the lack of such an artifact or effect of electrode implanted in OB in each side of the hippocampus: second, the OB kindling produced a significant increase in the B ...... value of the right side (~16%) 10 days after the last kindled convulsion: third, in the left side no significant change was seen, although the data suggest an increasing trend (10% of control). It was
76 TABLE I REGIONAL DISTRIBUTION OF [3H]DIAZEPAM BINDING IN BRAIN 10 DAYS AFTER THE COMPLETION OF OB KINDLING IN RATS Rats were sacrificed 10 days after the completion of kindling. Crude synaptosomal membranes were assayed in 50 mM Tris-HCl buffer pH 7.4 and incubated with 10 different concentrations of [3H]diazepam (range 0.5-10 nM) at 4°C for 60 min. The [3H]diazepambinding sites to various areas of brain was determined in triplicate assays, non-specific binding was measured in the presence of 10 ktM flunitrazepam. The [3H]diazepam binding densities (Bin,x)are expressed as fentomoles per milligramof protein; dissociation constants (KD)are expressedin nanomolar. Kd and Bma~were calculated by least squares linear regression analysis of the Scatchard data. Values are reported as the mean +_SD of 3 separate experiments. Brain region
[3H]Diazepambinding
KD (nM) Right
Left
Bmax (fmol/mg protein) Right
Left
Olfactory bulb ControlI 4.29 + 0.66 4.19_+0.62 1305 _+69 1299 +_ 85 ControllI 4.48 + 0.47 4.32_+0.54 1631 _+93°*" 1403+_ 96 Kindled 5.16_+0.55 4.72_+0.50 2070+_ 121**'b 1830_+117**.b Hippocampus Control I 3.67 + 0.16 3.73 _+0.29 895 +_51 Control II 3.71 _+0.35 3.74 _+0.32 898 _+56 Kindled 3.92 _+0.52 4.01 + 0.47 1040_+63*h
905 _+ 71 897 _+ 59 996 + 53
*P < 0.05; "P < 0.01. aControl II vs. control I; bKindled vs. both control I and control II.
also shown that no significant changes of the KD values occurred between groups, earlier mentioned, in each side of the hippocampus, according to the statistical analysis used for the Bmax. This result shows that OB kindling and the implantation of an electrode in OB do not change the KD values for either the right or the left hippocampus. A few studies have concerned the role of the electrode, in both the kindling rate [7, 8] and the neurochemical studies [3, 4, 32], but to our knowledge none have considered the part played by the electrode at the focal area. Our results indicate that the electrode implantation increases the Bmax values of [3H]diazepam binding only in the ipsilateral side of OB. This present work supports and extends our earlier results [4, 32] performed on pooled samples o f both ipsi- and controlateral sides. In contrast, in each hippocampus, there were no differences in [3H]diazepam binding between sham-operated (control II) and age-matched (control I) rats. This result revealed the lack of effect of the electrode in the areas not injured by the electrode (e.g. the hippocampus). Until recently there was little information on the possi-
ble involvement of the electrode in the kindling or epilepsy phenomenon; however, 3 important findings have been made. First, Blackwood [7] and Blackwood et al. [8] have reported that prolonged electrode insertion in the amygdala of a set of rats increased the rate of kindling development. Second, however, the apparent absence of focal lesions in the kindling model may not rule out the molecular changes at the level of electrode site or in the perifocal areas traversed by the electrode, since Ashton et al. [3] have observed a significant decrease (30%) of the [3H]spiperone binding in the frontal cortex of sham-operated amygdala rats in comparsion with sham-unoperated animals. Third, implantation of electrodes in one of the rhinencephalic structures (e.g. either in the amygdaloid complex, A m m o n ' s horn, or gyrus hippocampi), both in animal [22, 29] and in human [2, 21, 28] set up an 'injury discharge' with the electrical features of an epileptiform activity. The up-regulation of BZD receptors may explain part but not all neurochemical events which occurred alter the prolonged implantation of an electrode in the focus of sham-operated rats (control II). It is possible that a presynaptic denervation, perhaps of inhibitory synapses, set up an increase of BZD receptors in the focus. It seemed at the time that such changes occurred exclusively at both the electrode site (present result) and perifocal areas traversed by the electrode [3]. The enhancement of the rate of kindling development after prolonged electrode implantation [7, 8] may be the consequence of these neurochemical changes. On the other hand, the present results show an increase in BZD receptor density after OB kindling at the site of the electrode and in the hippocampus. In amygdala-kindled rats an increase in BZD binding sites has been reported by several other groups in the focus [26, 37] and in the hippocampus [24, 26, 35-38]. In contrast to these observations, in cortical-kindled rats B Z D receptors are unchanged in all structures investigated [33]. It is not unlikely that the divergences are related to differences in kindling site and in biochemical and/or autoradiographical techniques used. But, in general, it seemed that B Z D binding sites are unchanged or up-regulated in the majority of the brain areas of kindled rats [10]. Since diazepam binds to central-type (CBR) and peripheral-type (PTBR) B Z D receptors [23], the increase of [3H]diazepam binding previously described could be due to one or both of the B Z D receptors. In the site injured by electrode implantation (right OB), glial proliferation could account for an increase of the PTBR as it was observed in m a n y models [6, 14, 27, 34]. However, in the OB, a rich P T B R region, a decrease of the number of P T B R was described after lesion of the olfactory nerve
77
[1, 9]. The increase of BZD receptors observed after OB kindling (this article) does not appear to be related to an increase of the PTBR for many reasons. First, for the range of concentrations used, diazepam labelled the CBR (high affinity, 4~5 nM) rather than the PTBR (low affinity sites in the micromolar range) of the rodent brain [15, 18, 31]. Second, in the hippocampus, there was no direct effect of the electrode implantation. Third, no modification of the binding of [3H]Ro 5-4864, a specific ligand of the PTBR, was observed in the hippocampus after amygdala kindling [11, 26], but these experiments were performed only with one concentration of ligand (2.5 nM). However, recently it was shown that neuronal loss [13] or increase in the concentration of glial fibrillary acidic protein (GFAP), a marker protein of astrocytes [19] was observed in the hippocampus after hippocampal kindling. Consequently, binding experiments with [-~H]PK 14105, the high specific PTBR ligand, by autoradiography are necessary to conclude definitively about possible subtle cellular modifications due to OB kindling. In summary, some previous reports have taken neurochemical or kindling development changes as evidence of prolonged electrode implantation in sham-operated and kindled rats, respectively [3, 7, 8]. This situation arises in the present study and the conspicuous increase of BZD receptors density in the electrode location of sham-operated controls provides additional evidence. All these observations might be related to a biological response of adaptation, associated with conditions of novelty. The authors thank Mr. Jean Bayl6 for the excellent technical assistance, Dr. B. Lokhart for careful reading of the manuscript, and acknowledge the assitance of Mr. Maurice Fauquier in the photographic preparations, and of Mr. Fernand Redal in carefully maintaining the animals. This work was supported in part by a grant from INSERM (contrat de recherche externe Neurotransmission GABAergique et 6pilepsie limbique CNAMTS816034), and a research grant from CNRS. I Anholt, R.R.H., Murphy, K., Mack. G. and Snyder, S.H., Peripheral type benzodiazepine receptors in the central nervous system: localization to ol[itctory nerves, J. Neurosci., 4 (1984) 593 603. 2 Arfel, G., Albe-Fessard, D., Guiot, G., Derome, P., De La Herran, J., Hertzog. E. and Vourc'h, G., Activities characteristic of certain deep structures in man, Electroencephalogr. Clin. Neurophysiol., 17 (19641473 478. 3 Ashton, D.. Leysen, J.E. and Wauquier, A., Neurotransmitters and receptor binding in amygdaloid kindled rats: serotonergic and noradrenargic modulatory effects, Life Sci., 27 (19801 1547 1556. 4 Ben Atria, M., Rondouin, G., Lerner-Natoli, M., Heaulme, M. and Baldy-Moulinier, M., Benzodiazepine receptor changes in kindled states, J. Cerebr. Blood Flow Metab., 5 Suppl. I (1985) $373 $374. 5 Ben Atria, M., N'Gouemo, E, Belaidi. M., Lerner-Natoli, M., Ron-
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78 23 Marangos, EJ., Patel, J., Boulenger, J.E and Clark-Rosenberg, R., Characterization of the peripheral-type benzodiazepine sites using [3H]Ro 5-4864, Mol. Pharmacol., 22 (1982) 26--32. 24 McNamara, J.O., Peper, A.M. and Patrone, V., Repeated seizures induced long-term increase in hippocampal benzodiazepine receptors, Proc. Natl. Acad. Sci. USA, 77 (1980) 3029-3032. 25 McNamara, J.O., Bonhaus, D.W., Crain, B.J., Gellman, R.L. and Shin, C., Biochemical and pharmacological studies of neurotransmitters in the kindling model. In P.C. Jobe and H.E. Laird II (Eds.), Neurotransmitters and Epilepsy, Humana, Clifton, NJ. 1987, pp. 115 160. 26 Niznik, H.B., Burnham, W.M. and Kish, S.J., Benzodiazepine receptor binding following amygdala-kindled convulsions: differing results in washed and unwashed brain membranes, J. Neurochem., 43 (1984) 1732--1736. 27 Owen, F., Poulter, M., Waddington, J.L., Mashal, R.D. and Crow, T.J., [3H]RO-4864 and [3H]flunitrazepam binding in kainate lesioned rat striatum and in temporal cortex of brains from patients with senile dementia of the Alzheimer type, Brain Res., 278 (1983) 373 375. 28 Pagni, C.A. and Massero, M.D., Some observations on the human rhinencephalon: a stereo-electroencephalographic study, Electroencephalogr. Clin. Neurophysiol., 18 (1965) 260-270. 29 Passouant, P. and Cadilhac, J., Physiologie normale et pathologique de l'hippocampe. In T. Alajouanine (Ed.), Les Grandes Activit6s du Rhinenc6phale, Masson Editeur, Paris, 1961, pp. 145-189. 30 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294.
31 Rampe, D. and Triggle, D.J., Benzodiazepine and calcium channel function, Trends Pharmacol. Sci., 7 (1986) 461 464. 32 Rondouin, G., Chicheportiche, R., Lerner-Natoli, M., Ben Attia, M., Privat, A. and Baldy-Moulinier, M., Inhibitory processes ira limbic kindling. In J.A. Wada (Ed.), Kindling 3. Raven, New York, 1986, pp. 361-374. 33 Santori, E.M and Collins, R.C., Effects of chronic cortical seizures on GABA and benzodiazepine receptors within seizure pathways, Brain Res., 442 (1988) 261 --269. 34 Schoemaker, H., Morelli, M., Deshmukh, P. and Yamamura, H.I., [3H]Ro 5-4864 benzodiazepine binding in the kainate lesioned striaturn and Huntington's diseased basal ganglia, Brain Res., 248 (1982) 396-~,01. 35 Shin, C., Pedersen, H.B. and McNamara, J.O., ?'-Aminobutyric acid and benzodiazepine receptors in the kindling model of epilepsy: a quantitative radiohistochemical study, J. Neurosci., 5 (1985) 2696-2701. 36 Tietz, E.I., Gomez, F. and Berman, R.F., Amygdala kindled seizures stage is related to altered benzodiazepine binding density, Life Sci., 36 (1985) 183-190. 37 Tuff, L.E, Racine, R.J. and Mishra, R.K., The effects of kinding on GABA-mediated inhibition in the dentate gyrus of the rat. II. Receptor binding, Brain Res., 277 (1983) 91 98. 38 Valdes, F., Dasheiff, R.M., Birminham, F., Crutcher, K.A. and McNamara, J.O., Benzodiazepine receptor increases after repeated seizures: evidence for localization to dentate granule cells, Proc. Natl. Acad. Sci. USA, 79 (1982) 193--197.
74
NSL 08862
Kindling and electrode effects on the benzodiazepine receptors density of olfactory bulb and hippocampus after olfactory bulb kindling M. Ben Attia a, P. N ' G o u e m o b, M. Belaidi b, G. R o n d o u i n b and R. Chicheportiche a alNSERM U.336, Laboratoire de Biochimie GOn~rale, Ecole Nationale Sup~rieure de Chimie de Montpellier, Montpellier (France) and bCNRS UPR.8402-1NSERM U.249, Laboratoire de MOdecine Expkrimentale, Institut de Biologie, Montpellier (France) (Received 9 December 1991; Revised version received l0 April 1992; Accepted 18 May 1992)
Key words." Kindling; Olfactory bulb; Hippocampus; Epilepsy; Benzodiazepine receptor; Electrode effect; Rat The olfactory bulb (OB) kindling is a model of limbic secondary generalized epilepsy. Ten days after the completion of OB kindling, we have studied the long term effects of both electrode insertion and kindling on the binding of [3H]diazepam to crude mitochondrial fractions. On the one hand, we have shown that electrode implantation in sham-operated controls induced an obvious increase in benzodiazepine (BZD) receptor density (Bm~) only at the site of the electrode in comparison to sham-unoperated rats. These results might indicate an additional mechanism extending earlier observations reported by others, who have shown that prolonged electrode implantation induced changes in sham-operated and kindled rats. On the other hand, the long lasting effect of OB kindling on the binding parameters of [3H]diazepam was examined in the focus and in the hippocampus. The results indicate a bilateral increase of BZD receptors in the OB and an ipsilateral increase in the hippocampus. These changes might be a regulation phenomenon in response to a hyperexcitability state and to focal stimulations.
The kindling phenomenon is induced by repetitive low level electrical stimuli to susceptible regions of the brain until the apparition of electrical seizure activity [17]. Full convulsion is associated with behavioural changes which are divided into 5 behavioural stages as described by Racine [30]. The seizures that occur in this model are intermittent and paroxymal and share many other similarities with human epilepsy [16, 25]. Among the brain areas, the limbic system is the most susceptible to kindling [17]. While Goddard et al. [17] showed that amygdala was a brain structure of great susceptibility, other groups have found that the olfactory bulb (OB) was more sensitive to electrical stimulations [12], with a slightly higher afterdischarge threshold [5, 12], but without occurrence of oscillatory phenomena [5]. Moreover, the OB as the site of kindling stimulations offers several advantages in comparison with the other cerebral structures. Indeed, its rostral anatomical position permits a direct implantation of the electrode with respect to the integrity of forebrain areas or pathways. The electrical kindling has been increasingly used as Correspondence: R. Chicheportiche, INSERM U.336, Laboratoire de Biochimie G6n6rale, Ecole Nationale Sup~rieure de Chimie de Montpellier, 8 rue de l'Ecole Normale, 34053 Montpellier Cedex 1, France.
an excellent tool for the neurochemical studies. It is the most extensively investigated among animal models of epilepsy. But the precise physiological and neurochemical mechanisms by which this occurs remains unknown. In the present study, we carried out a series of multifactorial experiments in order to determine whether kindling and/or electrode implantation effects caused any dysfunction of benzodiazepine (BZD) receptors at the site of the electrode and at the hippocampus 10 days after the last electrical stimulation to avoid a short term effect of the seizure itself on neurochemical studies. It should be emphasized, however, that factors other than seizures, kindling, or both may be also involved. Male Sprague-Dawley rats (250-300 g) were used throughout the study. Rats were randomly divided into 3 sets of 15 animals, each set being subdivided into 3 groups of 5 rats which are designated as follows. Control I group: sham-non-implanted rats were daily handled but they were neither implanted nor stimulated. Control II group: sham-implanted rats with one electrode implanted in the right OB as described below, were daily handled and not submitted to the stimulation process. Kindled group: the rats of this group were implanted in the right OB and both handled and stimulated daily. During surgery, all rats of control II and kindled groups were anaesthetized by Equithesin (0.4 ml/100 g,
75 i.p.) and placed in a stereotaxic apparatus. A bipolar electrode made from two strands of nickel-chrome wires (100/,tin) twisted together, and insulated except at the tip, was stereotaxically implanted into the right OB (A, + 15.0 mm; L, 1.5; H, 2.5). At least 2 weeks were allowed for recovery from surgery before experimentation was initiated. The rats of kindled groups were submitted to stimulation sessions. Electrical stimulations were given according to the parameters described by Goddard et al. [17]: a pulse duration 1 ms, biphasic square waves, train duration 2 s, frequency 60 Hz. The initial intensity, delivered through the electrode, was set to 50 ~A peak to peak and increased by 50/,tA steps until an afterdischarge (AD) was triggered. The kindling proceeded twice daily until appearance of the sixth generalized seizure (stage 5). The electrical activity was recorded on an electroencephalograph and the behavioural stages scored as described by Racine [30]. Ten days after the last stimulation of the kindled group, the rats of the same set were killed by decapitation. After remaining and washing procedures of brains, only the OBs and hippocampus with attached area dentata were dissected from both sides and stored at - 8 0 ° C until further analysis. On the day of the analysis the previously frozen tissues were thawed at room temperature and homogenized using a teflon homogenizer in 10 vols. of ice-cold 0.32 M sucrose and centrifuged at 1,000 x g for 10 min. The supernatants were then centrifuged 20 min at 17,000 x g to yield the crude synaptosomal membranes (P2 fraction). [3H]Diazepam (70 Ci/mmol) was obtained from the Service des Molecules Marquees (C.E.A., Saclay, France). The P~ fractions were resuspended in ice-cold 50 mM Tris-HCl, pH 7.4, for binding assay. The membrane preparations were incubated with ice-cold buffer (50 mM Tris-HC1, pH 7.4) containing [3H]diazepam at the desired concentration. After 1 h of incubation at 4°C, 0.5ml aliquots of the suspension were carefully filtered through GF/B filters which were rapidly washed twice with 5 ml ice-cold incubation buffer. The radioactivity on the filters was assayed by liquid scintillation counting in 4 ml of liquid scintillant (ACS I, Amersham). The nonspecific binding was performed in the presence of unlabelled flunitrazepam (10 -s M). Protein was measured by the technique of Hartree [11]. Statistical analysis was computed by a one-way ANOVA followed by Newman Keuls multiple range test. Significance was considered at P < 0.05. There is evidence that the site of the electrode [12, 17] and the prolonged electrode insertion [7, 8] influenced the kindling rate. The question one must ask is, does this electrode have effects on the neurochemical studies? We
decided to determine if the influence of the implanted electrode and/or OB kindling, in each side of OB, affected the binding characteristics of [~H]diazepam. The binding parameters of [3H]diazepam in the right OB (site of the electrode implantation) were subjected to one-way analysis of variance. The difference between B..... values was significant (~,6 = 47.28: P < 0.001 ) while no difference was found between the KD values (Table I). Student Newman Keuls test indicated significant increase between control I and kindled groups ( P < 0.01), between control II and kindled groups (P < 0.01), and between control II and control 1 groups (P < 0.01 ). Focusing our attention on the left OB, we investigated both the effect of OB kindling and a possible artifact due to the electrode implantation in the controlateral side. A oneway analysis of variance of the number of [3H]diazepam binding sites (B ..... ) was significant (F,.~, -- 23.65: P < 0.01). This difference was subjected to Student Newman Keuls test as mentioned earlier. We showed a significant increase between control I and kindled groups ( P < 0 . 0 1 ) , between control II and kindled groups (P < 0.01 ), but there was no difference between control I and control II. These results demonstrate that electrode implantation produced a significant increase of B...... (+25 _+ 6%) only in the ipsilateral side and not in the contralateral side, while the kindling increased significantly the B...... values in each side of the OB (+27 + 6% at right and +30 _+ 7% at left) 10 days after the last kindled seizure. In contrast, in both sides of the OB the KD for [3H]diazepam did not change after kindling effect or electrode implantation, according to the same statistical analysis used for the B....... To be sure that the results, concerning the effect of the electrode, observed in the focal area (OB) were not seen in other areas, not crossed by the electrode, we have chosen the hippocampus for the same multiple comparisons used for OB (Table I). In the right side, the B ...... values of the 3 groups of rats compared by analysis of variance, indicated a significant difference (~,~, = 6.50: P < 0.05). Student Newman-Keuls test showed a significant difference between control I and kindled groups (P < 0.05), and between control lI and kindled groups (P < 0.05). No significant difference in B...... values was seen between control I and control II. In the left side, analysis of variance revealed that the B..... values of these 3 groups of rats were not significantly different from each other. These results demonstrate: first, the lack of such an artifact or effect of electrode implanted in OB in each side of the hippocampus: second, the OB kindling produced a significant increase in the B ...... value of the right side (~16%) 10 days after the last kindled convulsion: third, in the left side no significant change was seen, although the data suggest an increasing trend (10% of control). It was
76 TABLE I REGIONAL DISTRIBUTION OF [3H]DIAZEPAM BINDING IN BRAIN 10 DAYS AFTER THE COMPLETION OF OB KINDLING IN RATS Rats were sacrificed 10 days after the completion of kindling. Crude synaptosomal membranes were assayed in 50 mM Tris-HCl buffer pH 7.4 and incubated with 10 different concentrations of [3H]diazepam (range 0.5-10 nM) at 4°C for 60 min. The [3H]diazepambinding sites to various areas of brain was determined in triplicate assays, non-specific binding was measured in the presence of 10 ktM flunitrazepam. The [3H]diazepam binding densities (Bin,x)are expressed as fentomoles per milligramof protein; dissociation constants (KD)are expressedin nanomolar. Kd and Bma~were calculated by least squares linear regression analysis of the Scatchard data. Values are reported as the mean +_SD of 3 separate experiments. Brain region
[3H]Diazepambinding
KD (nM) Right
Left
Bmax (fmol/mg protein) Right
Left
Olfactory bulb ControlI 4.29 + 0.66 4.19_+0.62 1305 _+69 1299 +_ 85 ControllI 4.48 + 0.47 4.32_+0.54 1631 _+93°*" 1403+_ 96 Kindled 5.16_+0.55 4.72_+0.50 2070+_ 121**'b 1830_+117**.b Hippocampus Control I 3.67 + 0.16 3.73 _+0.29 895 +_51 Control II 3.71 _+0.35 3.74 _+0.32 898 _+56 Kindled 3.92 _+0.52 4.01 + 0.47 1040_+63*h
905 _+ 71 897 _+ 59 996 + 53
*P < 0.05; "P < 0.01. aControl II vs. control I; bKindled vs. both control I and control II.
also shown that no significant changes of the KD values occurred between groups, earlier mentioned, in each side of the hippocampus, according to the statistical analysis used for the Bmax. This result shows that OB kindling and the implantation of an electrode in OB do not change the KD values for either the right or the left hippocampus. A few studies have concerned the role of the electrode, in both the kindling rate [7, 8] and the neurochemical studies [3, 4, 32], but to our knowledge none have considered the part played by the electrode at the focal area. Our results indicate that the electrode implantation increases the Bmax values of [3H]diazepam binding only in the ipsilateral side of OB. This present work supports and extends our earlier results [4, 32] performed on pooled samples o f both ipsi- and controlateral sides. In contrast, in each hippocampus, there were no differences in [3H]diazepam binding between sham-operated (control II) and age-matched (control I) rats. This result revealed the lack of effect of the electrode in the areas not injured by the electrode (e.g. the hippocampus). Until recently there was little information on the possi-
ble involvement of the electrode in the kindling or epilepsy phenomenon; however, 3 important findings have been made. First, Blackwood [7] and Blackwood et al. [8] have reported that prolonged electrode insertion in the amygdala of a set of rats increased the rate of kindling development. Second, however, the apparent absence of focal lesions in the kindling model may not rule out the molecular changes at the level of electrode site or in the perifocal areas traversed by the electrode, since Ashton et al. [3] have observed a significant decrease (30%) of the [3H]spiperone binding in the frontal cortex of sham-operated amygdala rats in comparsion with sham-unoperated animals. Third, implantation of electrodes in one of the rhinencephalic structures (e.g. either in the amygdaloid complex, A m m o n ' s horn, or gyrus hippocampi), both in animal [22, 29] and in human [2, 21, 28] set up an 'injury discharge' with the electrical features of an epileptiform activity. The up-regulation of BZD receptors may explain part but not all neurochemical events which occurred alter the prolonged implantation of an electrode in the focus of sham-operated rats (control II). It is possible that a presynaptic denervation, perhaps of inhibitory synapses, set up an increase of BZD receptors in the focus. It seemed at the time that such changes occurred exclusively at both the electrode site (present result) and perifocal areas traversed by the electrode [3]. The enhancement of the rate of kindling development after prolonged electrode implantation [7, 8] may be the consequence of these neurochemical changes. On the other hand, the present results show an increase in BZD receptor density after OB kindling at the site of the electrode and in the hippocampus. In amygdala-kindled rats an increase in BZD binding sites has been reported by several other groups in the focus [26, 37] and in the hippocampus [24, 26, 35-38]. In contrast to these observations, in cortical-kindled rats B Z D receptors are unchanged in all structures investigated [33]. It is not unlikely that the divergences are related to differences in kindling site and in biochemical and/or autoradiographical techniques used. But, in general, it seemed that B Z D binding sites are unchanged or up-regulated in the majority of the brain areas of kindled rats [10]. Since diazepam binds to central-type (CBR) and peripheral-type (PTBR) B Z D receptors [23], the increase of [3H]diazepam binding previously described could be due to one or both of the B Z D receptors. In the site injured by electrode implantation (right OB), glial proliferation could account for an increase of the PTBR as it was observed in m a n y models [6, 14, 27, 34]. However, in the OB, a rich P T B R region, a decrease of the number of P T B R was described after lesion of the olfactory nerve
77
[1, 9]. The increase of BZD receptors observed after OB kindling (this article) does not appear to be related to an increase of the PTBR for many reasons. First, for the range of concentrations used, diazepam labelled the CBR (high affinity, 4~5 nM) rather than the PTBR (low affinity sites in the micromolar range) of the rodent brain [15, 18, 31]. Second, in the hippocampus, there was no direct effect of the electrode implantation. Third, no modification of the binding of [3H]Ro 5-4864, a specific ligand of the PTBR, was observed in the hippocampus after amygdala kindling [11, 26], but these experiments were performed only with one concentration of ligand (2.5 nM). However, recently it was shown that neuronal loss [13] or increase in the concentration of glial fibrillary acidic protein (GFAP), a marker protein of astrocytes [19] was observed in the hippocampus after hippocampal kindling. Consequently, binding experiments with [-~H]PK 14105, the high specific PTBR ligand, by autoradiography are necessary to conclude definitively about possible subtle cellular modifications due to OB kindling. In summary, some previous reports have taken neurochemical or kindling development changes as evidence of prolonged electrode implantation in sham-operated and kindled rats, respectively [3, 7, 8]. This situation arises in the present study and the conspicuous increase of BZD receptors density in the electrode location of sham-operated controls provides additional evidence. All these observations might be related to a biological response of adaptation, associated with conditions of novelty. The authors thank Mr. Jean Bayl6 for the excellent technical assistance, Dr. B. Lokhart for careful reading of the manuscript, and acknowledge the assitance of Mr. Maurice Fauquier in the photographic preparations, and of Mr. Fernand Redal in carefully maintaining the animals. This work was supported in part by a grant from INSERM (contrat de recherche externe Neurotransmission GABAergique et 6pilepsie limbique CNAMTS816034), and a research grant from CNRS. I Anholt, R.R.H., Murphy, K., Mack. G. and Snyder, S.H., Peripheral type benzodiazepine receptors in the central nervous system: localization to ol[itctory nerves, J. Neurosci., 4 (1984) 593 603. 2 Arfel, G., Albe-Fessard, D., Guiot, G., Derome, P., De La Herran, J., Hertzog. E. and Vourc'h, G., Activities characteristic of certain deep structures in man, Electroencephalogr. Clin. Neurophysiol., 17 (19641473 478. 3 Ashton, D.. Leysen, J.E. and Wauquier, A., Neurotransmitters and receptor binding in amygdaloid kindled rats: serotonergic and noradrenargic modulatory effects, Life Sci., 27 (19801 1547 1556. 4 Ben Atria, M., Rondouin, G., Lerner-Natoli, M., Heaulme, M. and Baldy-Moulinier, M., Benzodiazepine receptor changes in kindled states, J. Cerebr. Blood Flow Metab., 5 Suppl. I (1985) $373 $374. 5 Ben Atria, M., N'Gouemo, E, Belaidi. M., Lerner-Natoli, M., Ron-
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