Various hippocampal lesions induced by multi-fractional ibotenic acid injections and amygdala kindling in rats

Brain Research, 559 (1991) 154-158 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124842Y

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Various hippocampal lesions induced by muki-fractionai ibotenic acid injections and amygdala kindling in rats Tatsuya Tanaka 1, Shinji Kondo 2, Tomokatsu Hori 2, Shigeya Tanaka I and Yukichi Yonemasu 1 1Department of Neurosurgery, Asahikawa Medical College, Nishikagura, Asahikawa (Japan) and 2Departmentof Neurosurgery, Tottori University School of Medicine, Nishikimachi, Yonago (Japan) (Accepted 18 June 1991)

Key words: Kindling effect; Ibotenic acid; Amygdala; Hippocampus; Degenerative lesion

Hippoeampal degenerative lesions were made bilaterally by means of multiple ibotenic acid injections and development of amygdala kindling was studied. In groups with lesions in either bilateral dorsal or ventral hippocampus, stimulations required for kindling were almost the same as those of controls. In the group with lesion of the entire hippoeampus, kindling development was remarkably slow especially in the early stage of the kindling process. However, kindling effect was finally established in all groups. Among the limbic structures, the amygdala (AM) and hippocampus (HIPP) have a close relationship. Especially, each structure has a significant influence upon the development of AM or H I P P kindling 2'4'9'13'17'25'27. Our previous report 1° suggested that intra-amygdaloid application of quisqualic acid (QA) remarkably accelerated the development of hippocampal kindling in cats. Feldblum and Ackermann 6 reported an increased susceptibility to AM or HIPP kindling following intrahippocampal application of kainic acid (KA). However, injection of a strong excitant such as K A or Q A into the limbic structure itself may induce primary epileptogenicity before the establishment of kindling 1,3,7,8,21,22 On the other hand, ibotenic acid (IBO) is regarded as a good tool to make a degenerative lesion without an excessive epileptic excitation 12'14'24'26'28. In the present study, IBO was injected in the segmental manner into bilateral HIPP in order to make various degrees of hippocampal lesions and the development of amygdala kindling was examined. Thirty-two male Wistar descendant rats (250-350 g) were allowed free access to food and water and were housed on a 12-h light/dark cycle. Rats were anesthetized with pentobarbital (45 mg/kg, i.p.) and placed in a stereotaxic instrument. Then, rats were separated into 4 groups. Three groups received IBO solution into bilateral HIPP. IBO (Sigma: 1/~g) was dissolved in 1/tl of phosphate buffer solution (0.2 M at pH 7.4). The solu-

tion was sterilized through Millex-HA microfilters (0.45 #m filter unit) and each injection was done under aseptic conditions. Injection speed was kept at the rate of 0.5 /A/min. Group A (8 rats). An external stainless steel guide cannula (0.6 mm in diameter) with an internal stainless steel delivery cannula (0.3 mm in diameter, 0.5 mm longer than the external cannula) was stereotaxically placed in bilateral dorsal hippocampus (DH, A: 4.0, L: 2.0, D: 2.5) using a stereotaxic atlas 16. The inner cannula was replaced with the delivery cannula connected with a microinjector and 2/~1 of IBO solution was injected into each DH. The cannula was left in place at least 15 min for the diffusion of the solution. Group B (8 rats). The injection cannula was placed in bilateral ventral hippocampus (VH, A: 2.8, L: 5.0, D: -2.5) and 2/~1 of IBO solution was injected into each VH. Group C (8 rats). HIPP was divided into 4 parts, and 8 injection cannulae were inserted into VH, D H , posterior part of D H (A: 2.8, L: 3.3, D: 2.2) and caudal HIPP (A: 1.6, L: 5.2, D: -1.0), bilaterally. IBO solutions were injected via all cannulae (2/zl each, total 16 ktg).

Group D (8 rats) received phopshate buffer solution (PBS: 4-16 kd) for the control study. Two rats received injections into the bilateral D H , 3 rats received injections into the bilateral V H and 3 rats received 4 injections bilaterally (2 #1 each, total 16 #1 of phosphate

Correspondence: T. Tanaka, Department of Neurosurgery, Asahikawa Medical College, Nishikagura, Asahikawa 078, Japan. Fax: (81) 16665-8560.

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Fig. 1. Chronologicaldevelopment of amygdaloid kindling in each group. Note the remarkable delay of kindling development in group C with total lesion of HIPP. Group A: bilateral dorsal hipIx)campus destroyed. Group B: bilateral ventral hippocampus destroyed. Group D: controls. buffer solution). Following these IBO injections, bipolar twisted electrodes (0.2 mm in diameter), insulated except for a 1-mm tip, were implanted in the left amygdala (A: 5.0, L: 5.0, D: -3.0) and left dorsal hippocampus (A: 3.0, L: 3.2, D: 1.5). A stainless steel screw was placed on the dura over the left sensorimotor cortex (LCx). An additional screw in the frontal sinus was placed for the indifferent electrode. The electrodes were connected to the plug, which was fixed to the skull using dental cement. After recovery from the anesthesia, none of them demonstrated epileptiform behavior. Some of them were subjected to E E G recording. However, epileptic paroxysm was not observed even in Group C. All rats were left free from 14 to 25 days for the recovery from the operation. The mean interval between IBO treatment and the first kindling stimulation was 20 days. Kindling. AM stimulation (biphasic square pulse; 1 ms pulse duration, 250 #A, 62.5 c/s and 2 s duration) was delivered every 12 h. Generalized seizure threshold (GST) was not employed in this study in order to deliver enough current intensity to the AM. This strong current intensity for amygdala kindling is known to elicit AM discharges without exception as previously reported as a high intensity kindling 23. During the course of kindling in rats, Racine's scale 17 was employed in order to describe the progress of the seizure development. Class 5 motor seizure pattern involves in sequential facial clonus, head nodding, forelimb clonus, rearing and falling. This class 5 motor seizure is commonly referred to as full or maximal seizure. When rats demonstrated 6 successive class 5 motor seizures, they were perfused with 10% formalin solution under deep pentobarbital anesthesia (60 mg/kg, i.p.). Their brains were removed and processed for microscopic study. After fixation and embedding in paraffin, coronal sections (10 #m in thickness)

were serially made. The sections were stained with Cresyl violet or hematoxylin and eosin. The extent of HIPP lesions was carefully examined in all cats. Fig. 1 shows the average number of stimulations required for kindling development in each group. Controls (group D) showed usual kindling development and required 11.2 --4.3 stimulations for a fully kindled seizure (class 5 motor seizure). Group A (DH-lesioned) and Group B (VHlesioned) required 15.1 +- 4.6 and 16.2 - 5.7 stimulations, respectively. However, statistical analysis revealed no significant changes between group A or group B and controls. Kindling development of group C (totally lesioned) was very slow and required 24.1 --- 6.8 stimulations for a stage 5 seizure. Afterdischarge (AD) durations of class 5 motor seizures were studied in each group. The AD duration of groups A, B, C, and D were 33.0 --- 10.1, 44.0 ___ 8.2, 55.5 --- 18.5 and 33.0 --- 7.3, respectively. The characteristics of AD of a class 5 motor seizure were analogous in group A, B and D. However, electrographic seizure activities of the HIPP in group C were not active and the amplitude was less than one-third of those of the groups A or D (Fig. 2). In group B, hippocampal seizure activities were not constant and 3 rats demonstrated low amplitude activities as shown in Fig. 2. Histological study revealed extensive degenerations of bilateral D H in group A and bilateral VH in group B (Fig. 3). A single IBO injection (2 #g) into HIPP resulted in a focal degenerative lesion about 25 mm in diameter and degenerations of the pyramidal cells were observed in CA3 and CA4 but not in CA1 or CA2 or dentate granule cells. HIPP degenerations were maximal in group C. Neuronal cell loss and remarkable pyknosis were observed in CA1, CA3 and CA4 of both entire HIPP. The granule cell was partly affected and cell loss was partly observed. The size of the HIPP was small suggesting some atrophic process. However, extra-hippocampal regions such as amygdala, thalamus, septal nuclei or pyriform cortex were intact and degenerative lesions were limited within the hippocampus. Zaczek and Coyle23 first described the hippocampal degenerative lesions after intra-hippocampal IBO injection. Our results were almost the same as their observations. No degenerative change was observed in the HIPP of controls (Group D). In the present study, multi-fractional IBO administrations were made into bilateral HIPP. AM kindling was significantly suppressed only when extensive degenerations were observed in bilateral HIPP (Group C). However, partial destruction of bilateral D H or VH resulted in no modification of the kindling process. Various attempts were made in order to make a circumscribed lesion within the HIPP. However, anatomical and structural presentations of the HIPP were quite complicated

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Fig. 2. Electroencephalogram demonstrates class 5 motor seizures in each group, The propagation of the electrographic seizure activities to the hippocampus were not prominent in the group with VH lesion and total lesion,

because of its elongated C-like form (Fig. 3). Several reports d e m o n s t r a t e d the influences of H I P P lesions upon kindling 4'6'19'27. The m e t h o d of destruction was electrolytic, kainate-induced or colchicine-induced lesions of

HIPP. Their results are controversial because their lesions were limited to unilateral D H , V H or bilateral D H . Regional differences in the H I P P should be taken into account TM. M o r e o v e r , electrolytic lesion was often

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Fig. 3. A schematic illustration of the rat brain demonstrates injected sites of IBO in each group and extents of hippocampal lesions of groups A, B and C. (C.C.: corpus callosum, HIPP: hippocampus.)

157 excessive seizure input from the perforant path or another circuit from the A M , (2) the booster effect of H I P P on the A M seizure was abolished by the destruction of the pyramidal cell layer or a part of the dentate gyrus, (3) in the development of focal A M seizures, an interaction of H I P P seizure was required during initial development of kindling process. However, amygdala kindling was finally established even in group C. This may be explained as follows: (1) IBO-induced degenerative lesion was not complete in the destruction of the H I P P functions, (2) H I P P functions may not be inevitable in the final process of A M kindling establishment. In surgical treatment of intractable complex partial seizure, resection of cortical epileptogenic focus of the temporal lobe may not be sufficient and radical H I P P resection may be required in order to obtain favorable results ~5'2°. Our experimental study may support this facilitatory action of the H I P P upon A M seizure development. Further study should be made to understand the function of H I P P in the development of A M seizures.

expanded to the surrounding structures of the HIPP. Racine et al. TM reported that bilateral aspiration lesions of the dorsal hippocampus or large bilateral direct current lesions of the ventral hippoeampus had no effect on A M kindling. These results go the same way as our findings. However, they did not attempt total H I P P lesions upon A M kindling. Kainate L3 or quisqualate 1° injection possibly induced an epileptogenic focus at the injected site. These factors might have modified the true effect of H I P P lesions upon kindling. I B O has been proven to be a good tool to make focal lesions in the deep brain without prominent epileptic excitation of the H I P P 28, A M 26 or substantia nigra 24. In the present study, H I P P was extensively destroyed by I B O injections in 4 segments. However, these lesions did not completely abolish the development of amygdala kindling but strongly suppressed the development. The extent of the H I P P lesion is well correlated with this finding. These I B O injections made it possible to destroy total H I P P by the multi-fractional injections of I B O (Group C). This method of H I P P destruction may be useful not only in studies of epilepsy but also in those of neurophysiological and neuropharmacological experiments. Explanation of the inhibitory effect of the total H I P P lesions upon A M kindling is very difficult. We assumed that: (1) multifractional I B O injection resulted in an interruption of

This research was supported in part by a grant from the Epilepsy Research Foundation, 1991. The authors wish to thank Mr. Hironari Isobe and Miss Hitomi Saitou for their excellent technical assistance.

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