- Email: [email protected]
Kainic acid-induced substantia nigra seizure in rats: behavior, EEG and metabolism
Brain Research 911 (2001) 89–95 www.elsevier.com / locate / bres
Research report
Kainic acid-induced substantia nigra seizure in rats: behavior, EEG and metabolism Atsushi Sawamura*, Kiyotaka Hashizume, Katsunari Yoshida, Tatsuya Tanaka Department of Neurosurgery, Asahikawa Medical College, 2 -1, Midorigaoka-Higashi, Asahikawa 078 -8510, Japan Accepted 22 June 2001
Abstract Rationale: In order to clarify the role of substantia nigra pars reticulata (SNr) upon the development of epileptic seizure, kainic acid (KA) was injected into a unilateral SNr. Materials and methods: Wistar rats weighing 250–350 g were used. A stainless-steel cannula and depth electrode were inserted stereotaxically into the left substantia nigra pars reticulata (SNr). At 7 days after surgery, 1.0 mg of KA was injected into the left SNr. Experiment 1: In eight rats, behavior and electroencephalograms (EEG) were continuously recorded for about 30 h, and intermittently monitored following 1 month. Experiment 2: Two hours after KA injection into SNr, rats demonstrated status epilepticus. Then, 100 mCi / kg of [ 14 C]2-deoxyglucose (2-DG) was intravenously injected in seven rats, and the rats were processed for autoradiographic study. Results: Changes in behavior and EEG: On EEG, a secondary generalized seizure status was observed at about 70 min after KA injection. In video, limbic seizure manifestations such as salivation were observed as a initial symptom and followed by rolling and generalized tonic seizures. [ 14 C]deoxyglucose autoradiographic study demonstrated increased local cerebral glucose metabolism in the medial and lateral septal nucleus, substantia nigra, hippocampus, parietal cortex, piriform cortex, medial and lateral geniculate nucleus, anterodorsal, lateral and ventral nucleus of the thalamus, amygdala and midbrain reticular formation. Summary: The result suggested that the substantia nigra played an important role in the secondary generalization in the substantia nigra seizure model due to the decreased function of the GABAergic projection system induced by an excessive epileptic excitation of SNr. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: basic mechanisms Keywords: Substantia nigra pars reticulata; Electroencephalogram; Cerebral glucose metabolism; Kainic acid; GABA; Experimental epilepsy
1. Introduction
2. Materials and methods
Substantia nigra pars reticulata (SNr) is an important structure in the mechanism of the GABA system, and the SNr plays an important role as the center of the inhibitory system of epileptic phenomena [4,12]. In order to clarify the role of the SNr in the development of epileptic seizure, kainic acid was injected into a unilateral SNr. After induction of SNr seizure, behavior, electroencephalograms (EEG) and local cerebral glucose utilization (LCGU) were studied.
2.1. Experiment 1: Kainic acid ( KA)-induced substantia nigra seizure
*Corresponding author. Tel.: 181-166-68-2594; fax: 181-166-682599. E-mail address: [email protected] (A. Sawamura).
Eight Wistar rats weighing 250–350 g were used. Under intraperitoneal pentobarbital anesthesia (45 mg / kg), a stainless-steel cannula (0.6 mm, O.D.) with an inner stainless-steel needle (0.3 mm in diameter) was inserted stereotaxically into the left SNr (A512.8 mm, L512.5 mm, D523.5 mm) for KA injection according to the atlas of Pellegrino et al. [9]. For recording EEG, a depth electrode was inserted into the left SNr, just posterior to the tip of the injection guide cannula. Electrodes were also inserted into the left sensorimotor cortex, globus pallidus, and hippocampus. The electrodes were connected to a socket with six channels, and the guide cannula was fixed
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02732-9
90
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
Fig. 1. (A) About 10 min after KA injection, continuous low-voltage spikes were observed in the left substantia nigra and globus pallidus on EEG at stage 1. (B) On EEG, the intermittent multiple spikes and wave complexes began to propagate to the left hippocampus and globus pallidus at about 30 min after KA injection at stage 2. (C) On EEG, continuous spikes and wave complexes began to propagate to the left sensorimotor cortex at about 50 min after KA injection at stage 3. (D) On EEG, a secondary generalized seizure was observed at about 70 min after KA injection at stage 4.
to the skull with resin dental cement. The rats were kept for 7 days until the brain had recovered from the surgical damage. At 7 days after surgery, 1 mg of KA (Sigma) was dissolved in a 1 ml phosphate buffer solution (0.2 M, pH 7.4), and 1.0 ml of the KA solution (1.0 mg / ml) was injected into the left SNr. The inner needle was removed and a stainless-steel injection cannula (0.3 mm in diameter) was inserted into the guide cannula. Then 1.0 ml of the KA solution was injected at 0.4 ml / min. These procedures prevent leakage of KA solution into the surrounding structures. The effectiveness of this method was
preliminarily confirmed using KA solution including 0.2% Methylene Blue. It was also histologically confirmed that diffusion of the dye with KA solution was 1.0 mm in diameter and limited within the left SNr. Behavior of the rats and EEG were continuously monitored and recorded by video for about 30 h and then intermittently monitored for 1 month by the use of a video EEG monitoring system. EEG setting: filter setting is the following. The high cut filter was 50 Hz and the low cut filter was 1.5 Hz. The time-constant was 0.1. Alternating current (a.c.) filters (50 Hz) were on. Video–EEG monitorings were employed for
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
91
the present study. At 1 month after KA injection, the rats were sacrificed and subjected to histopathological examination.
3. Results
2.2. Experiment 2: Cerebral glucose metabolism during substantia nigra seizure
On EEG, continuous low-voltage spikes were observed in the left substantia nigra and globus pallidus at about 10 min after KA injection (Fig. 1A). On video, limbic seizure manifestations such as salivation were observed as initial symptoms. On EEG, intermittent multiple spikes and wave complexes were observed in the left substantia nigra, globus pallidus and hippocampus at about 30 min after KA injection (Fig. 1B). On video, standing and jumping manifestations such as wet dog shake just like a limbic seizure were observed. On EEG, continuous spikes and wave complexes began to propagate to the left sensorimotor cortex at about 50 min after KA injection (Fig. 1C). On video, contralateral head turning and contralateral forelimb clonus were observed. On EEG, a secondary generalized seizure was observed at about 70 min after KA injection (Fig. 1D). On video, rolling and generalized tonic seizures were observed. Substantia nigra seizures recurred every 5–7 min and lasted for 3–4 h. However, the seizures gradually subsided and became normal within 12 h. No spontaneous seizure was detected for the next 30 days. The type of epilepsy is secondary generalized seizure. However, standing, jumping and rotational movement of the body axis were the characteristic movements during generalized seizure. This model is similar to brainstem seizure and secondarily generalized seizure.
Two hours after KA injection, seven rats demonstrated status epilepticus. Then, 100 mCi / kg of [ 14 C]2-deoxyglucose (2-DG) was intravenously injected, and the rats were processed for an autoradiographic study. Arterial blood samples were collected at 2, 3, 5, 10, 15, 20, 25, 30, 35 and 45 min after the radioactive tracer injection. The rats were decapitated as soon as possible after the last blood collection and the brain was removed quickly. Brain specimens were immediately frozen and sliced in a cryostat into 20-mm-thick serial coronal sections. These were dried at 608C and [ 14 C]methyl methacrylate standards (Amersham) were consecutively placed on X-ray film (Kodak, SB-5) in an X-ray cassette to be exposed for 7 days. Blood samples were centrifuged and plasma glucose concentration were measured. The optical density of the autoradiogram was measured by a densitometer (Sakura, PDA15) to calculate the tissue 14 C concentration. LCGU was calculated by employing the equation given by Sokoloff et al. [11]. LCGU in various brain structures was determined in seven rats with seizures and seven shamoperated rats used as the control. Comparative analysis was made by using the Mann–Whitney U-test.
3.1. Changes in behavior and EEG
Fig. 2. A small gliotic lesion with neuronal cell loss, caused by KA infiltration, was noted around the cannula tip in the SNr. Pyramidal cell loss was observed in the CA3 region of the left hippocampus.
92
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
Fig. 3. [ 14 C]deoxyglucose autoradiograms. Autoradiograms prepared from coronal sections of the rat brain. (A–D) are from a control rat and (E–H) were obtained from a representative rat of the KA group. Increased LCGU in the medial and lateral septal nucleus, substantia nigra, hippocampus, parietal cortex, piriform cortex, medial and lateral geniculate nucleus, anterodorsal, lateral and ventral nucleus of the thalamus, amygdala and midbrain reticular formation was observed. L, left.
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
93
3.2. Histopathological findings
3.3. [ 14 C] deoxyglucose autoradiograms
Coronal sections were stained by Hematoxylin–Eosin and Cresyl Violet. A small gliotic lesion with neuronal cell loss, caused by KA infiltration, was noted around the cannula tip in the SNr. Pyramidal cell loss was observed in the CA3 of the left hippocampus (Fig. 2).
In a [ 14 C]deoxyglucose autoradiogram, increased LCGU in the medial and lateral septal nucleus, substantia nigra, hippocampus, parietal cortex, piriform cortex, medial and lateral geniculate nucleus, anterodorsal, lateral and ventral nucleus of the thalamus, amygdala and midbrain reticular
Table 1 Local cerebral glucose utilization in substantia nigra seizure versus control rats Structures
Substantia nigra seizure (n57) Ipsilateral
Cerebral cortex Frontal cortex Parietal cortex Visual cortex Auditory cortex Sensorimotor cortex Entorhinal cortex Piriform cortex Cingulate cortex Corpus callosum Extrapyramidal Caudate nucleus Globus pallidus Substantia nigra Thalamus Anterodorsal n. Ventral n. Lateral n. Centromedian n. Medial geniculate n. Lateral geniculate n. Paraventricular n. Hypothalamus Dorsal n. Ventral n. Paraventricular n.
Control (n57) Contralateral
Ipsilateral
Contralateral
88.664.9 94.565.7 97.863.8 85.364.3 88.265.1 79.663.7 99.364.6 83.565.5
77.963.3 80.062.4 90.064.1 83.763.5 87.763.1 65.562.6 70.260.6 76.366.3
77.262.4 79.263.0 96.562.8 93.966.0 84.563.1 66.563.7 70.260.7 75.963.0
32.163.2
23.661.7
23.561.7
95.868.7 82.466.4 215.7610.8**
88.464.3 70.563.8 66.465.4
74.664.6 60.963.4 56.063.0
76.164.9 62.763.6 56.063.0
185.4616.3* 189.3615.7* 198.4614.8* 88.669.5 158.4614.3* 146.2613.6* 78.567.5
120.4613.8 102.1611.4 116.9610.6 87.368.1 72.567.4 80.668.5 69.566.3
84.564.6 77.163.3 79.064.6 79.564.8 65.362.1 63.462.4 60.264.0
83.664.2 77.863.2 79.164.4 79.665.1 65.662.3 64.361.7 61.664.3
72.365.6 76.466.9 80.564.3
65.262.5 63.163.6 52.562.9
52.362.8 46.263.2 49.765.4
52.762.6 46.563.0 53.664.2
114.866.8 211.5612.6** 103.564.7 96.465.4 104.665.8 129.864.6 179.2611.6** 93.169.5 33.662.4
Limbic system Medial septal n. Lateral septal n. Accumbens Hippocampus CA1 Hippocampus CA3 Dentate gyrus Amygdala Mamillary body
152.3611.4* 173.4612.3* 177.9616.2* 131.7615.7* 161.4614.6* 152.6615.1* 216.8616.2** 125.4610.3
70.666.8 75.467.1 64.267.2 73.566.9 80.368.6 85.267.3 64.266.5 103.8611.5
54.763.5 51.562.8 52.162.1 54.063.2 54.862.9 56.162.9 48.762.4 99.065.0
54.663.6 52.363.2 52.762.2 53.663.0 55.363.4 55.263.4 49.462.6 99.964.9
Brainstem MRF Periaqueductal gray Tegmentum Superior colliculus Inferior colliculus
169.5614.3** 66.566.8 62.866.9 134.6615.4 110.2613.6
72.667.3 64.266.1 61.165.8 84.969.6 122.3613.4
55.164.3 48.163.8 61.362.4 72.361.0 83.461.0
55.063.9 49.264.4 61.662.6 72.360.8 83.561.5
53.265.2
48.962.1
48.562.0
Cerebellar cortex
54.665.6
Values: mean6S.E.M. (Fmol / 100 g / min). *P,0.05; **P,0.01 (Mann–Whitney U -test).
94
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
formation (MRF) was observed (Fig. 3; Table 1). In the preliminary study, we injected KA into bilateral SNr. The elicited seizures were so severe that animals did not tolerate the procedure. All animals died in severe seizure status within 30–60 min after the bilateral injection. In the present study, the elicited seizures were mainly localized in the ipsilateral hemisphere. However, hypermetabolic areas were already observed in the contralateral hemisphere, especially in the inferior colliculus, anterodorsal nucleus of the thalamus, frontal and parietal cortices, and entorhinal and piriform cortices as indicated in Table 1.
4. Discussion The substantia nigra (SN) is thought to play a major role in the control of motor function. A major efferent of the SN is the dopaminergic nigrostriatal system. In our present study, a microinjection of KA into SNr induced characteristic generalized seizure status. It is very difficult to explain why excessive excitation of the SNr can cause epileptic manifestation. But, LCGU during seizure provided important information. A remarkable increase in LCGU was observed not only in the SNr but also in the MRF, thalamus and cerebral cortex. In such an abnormal condition, the anticonvulsant effect of GABAergic output of SNr became inactive upon the MRF, thalamus and cerebral cortex, which demonstrated excessive epileptic excitation. Another explanation may be the enhancement of the excitatory dopaminergic circuit of nigrostriatal projection from substantia nigra pars compacta due to hyperexcitation of the SNr. Most of the GABA content in the SNr is associated with GABAergic nerve terminals arising from cell bodies located in the striatum. GABA in the SNr exerts a control over nigrostriatal and mesolimbic dopaminergic neurons [3]. The central amygdala receives direct connections from the substantia nigra [1]. Le Gal La Salle et al. [7] found that bilateral administration of a GABA-elevating substance (gamma vinyl-GABA) in the vicinity of the substantia nigra greatly shortens the duration of amygdaloid kindled seizures without significantly altering the motor convulsions. Tanaka et al. [12] reported that bilateral destruction of the SNr by ibotenic acid injections induced remarkable aggravation of limbic seizure induced by KA injection into the amygdala. Iadarola et al. [6] found that protection from seizures induced by maximal electroshock or by convulsant drugs was obtained as a result of GABA elevation or direct stimulation of GABA receptors within a restricted region of the ventral midbrain tegmentum, the substantia nigra. The concentration of substance P (SP) in the SN is the highest of all brain areas, and it is localized in nerve terminals deriving from cell bodies in the
striatum. The fact that the blockade of nigral SP transmission had a similar action to that of GABA-mediated inhibition in the SNr is consistent with the proposed excitatory role of nigral SP [5]. Garant et al. [4] found that GABA terminals and SPcontaining terminals exert reciprocal actions on nigral output, and they provided the first evidence of an effect of SP antagonists on the seizure process. According to Vitek et al. [13] there are many excitatory and inhibitory motor circuits between the cerebral cortex, striatum, globus pallidus, thalamus, substantia nigra and subthalamic nucleus. In addition to decreased inhibition from the globus pallidus pars externa (GPe), increased activity in the subthalamic nucleus (STN) could also occur through increased activity in excitatory projections to the STN from the cortex, thalamus, or midbrain extrapyramidal area. The reduced rate of discharge of the GPe would also be expected to increase rates in the GPi via its direct projection to the GPi. In a sense, a window for the development of altered patterns and phasic responses of thalamic neuronal activity that is dependent on the mean discharge rate of neurons in the GPi may exist. Previous investigations have demonstrated that neocortical epileptiform activity is modulated by the basal ganglia and is particularly inhibited by stimulation of the globus pallidus pars interna (GPi) [2,4,8,10] which together with the SNr is an important output station. The basal ganglia are considered to play a role in the genesis and / or spread of epileptic activity. The SN may present an anticonvulsant region influencing epileptiform activity through its GABAergic projections, showing an action synergic to that of the internal pallidum [6]. In the present study, the inhibitory role of the SNr and GPi became inactive by the excessive epileptic excitation and brainstem seizures and secondarily generalized seizures were elicited. The results suggested that an epileptogenic focus of the substantia nigra demonstrated a rapid evolution from the focal seizure status to the secondarily generalized seizure status. Thus the substantia nigra may play a key role as a relay nucleus for the secondary generalization of a focal seizure.
5. Conclusions A microinjection of kainic acid into a unilateral SNr resulted in limbic seizure manifestations as initial symptoms. These seizures finally evolved into a generalized seizure status. The results demonstrated that the substantia nigra plays an important role in secondary generalization in a substantia nigra seizure model due to excessive excitation of the nigrostriatal pathways and decreased function of the GABAergic projection system.
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
References [1] R.M. Beckstead, V.B. Domesick, W.J.H. Nauta, Efferent connections of the substantia nigra and ventral tegmental area in rat, Brain Res. 175 (1979) 191–217. [2] A.R. Cools, G. Hendriks, J. Korten, The acetylcholine–dopamine balance in the basal ganglia of rhesus monkeys and its role in dynamic, dystonic, dyskinetic, and epileptoid motor activities, J. Neural Transm. 36 (1975) 91–105. [3] K. Gale, M.J. Iadarola, GABAergic denervation of rat substantia nigra: functional and pharmacological properties, Brain Res. 183 (1980) 217–223. [4] D.S. Garant, K. Gale, Lesions of substantia nigra protect against experimentally induced seizures, Brain Res. 273 (1983) 156–161. [5] D.S. Garant, M.J. Iadarola, K. Gale, Substance P antagonists in substantia nigra are anticonvulsant, Brain Res. 382 (1986) 372–378. [6] M.J. Iadarola, K. Gale, Substantia nigra; site of anticonvulsant activity mediated by gamma-aminobutyric acid, Science 218 (1982) 1237. [7] G. Le Gal La Salle, M. Kaijima, S. Feldblum, Abortive amygdaloid kindled seizures following microinjection of gamma-vinyl-GABA in the vicinity of substantia nigra in rats, Neurosci. Lett. 36 (1983) 69–74.
95
[8] E.J. Neafsey, C.M. Chuman, A.A. Ward Jr., Propagation of focal cortical epileptiform discharge to the basal ganglia, Exp. Neurol. 66 (1979) 97–108. [9] L.J. Pellegrino, A.S. Pellegrino, A.J. Cushman, A Stereotaxic Atlas of the Rat Brain, 2nd Edition, Plenum Press, New York, 1979. [10] M. Sabatino, G. Gravante, G. Ferraro, V. Savatteri, V. La Grutta, Inhibitory control by substantia nigra of generalized epilepsy in the cat, Epilepsy Res. 2 (1988) 380–386. [11] L. Sokoloff, M. Reivich, C. Kennedy, M.H. Des Rosiers, C.S. Patlak, K.D. Pettigrew, O. Sakurada, M. Shinohara, The [ 14 C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat, J. Neurochem. 28 (1977) 897–916. [12] T. Tanaka, S. Tanaka, M. Kaijima, Y. Yonemasu, Ibotenic acidinduced nigral lesion and limbic seizure, Brain Res. 498 (1989) 215–220. [13] J.L. Vitek, J. Zhang, M. Evatt, K. Mewes, M.R. Delong, T. Hashimoto, S. Triche, R.A.E. Bakay, GPi pallidotomy for dystonia: clinical outcome and neuronal activity, Adv. Neurol. 78 (1998) 211–219.
Research report
Kainic acid-induced substantia nigra seizure in rats: behavior, EEG and metabolism Atsushi Sawamura*, Kiyotaka Hashizume, Katsunari Yoshida, Tatsuya Tanaka Department of Neurosurgery, Asahikawa Medical College, 2 -1, Midorigaoka-Higashi, Asahikawa 078 -8510, Japan Accepted 22 June 2001
Abstract Rationale: In order to clarify the role of substantia nigra pars reticulata (SNr) upon the development of epileptic seizure, kainic acid (KA) was injected into a unilateral SNr. Materials and methods: Wistar rats weighing 250–350 g were used. A stainless-steel cannula and depth electrode were inserted stereotaxically into the left substantia nigra pars reticulata (SNr). At 7 days after surgery, 1.0 mg of KA was injected into the left SNr. Experiment 1: In eight rats, behavior and electroencephalograms (EEG) were continuously recorded for about 30 h, and intermittently monitored following 1 month. Experiment 2: Two hours after KA injection into SNr, rats demonstrated status epilepticus. Then, 100 mCi / kg of [ 14 C]2-deoxyglucose (2-DG) was intravenously injected in seven rats, and the rats were processed for autoradiographic study. Results: Changes in behavior and EEG: On EEG, a secondary generalized seizure status was observed at about 70 min after KA injection. In video, limbic seizure manifestations such as salivation were observed as a initial symptom and followed by rolling and generalized tonic seizures. [ 14 C]deoxyglucose autoradiographic study demonstrated increased local cerebral glucose metabolism in the medial and lateral septal nucleus, substantia nigra, hippocampus, parietal cortex, piriform cortex, medial and lateral geniculate nucleus, anterodorsal, lateral and ventral nucleus of the thalamus, amygdala and midbrain reticular formation. Summary: The result suggested that the substantia nigra played an important role in the secondary generalization in the substantia nigra seizure model due to the decreased function of the GABAergic projection system induced by an excessive epileptic excitation of SNr. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: basic mechanisms Keywords: Substantia nigra pars reticulata; Electroencephalogram; Cerebral glucose metabolism; Kainic acid; GABA; Experimental epilepsy
1. Introduction
2. Materials and methods
Substantia nigra pars reticulata (SNr) is an important structure in the mechanism of the GABA system, and the SNr plays an important role as the center of the inhibitory system of epileptic phenomena [4,12]. In order to clarify the role of the SNr in the development of epileptic seizure, kainic acid was injected into a unilateral SNr. After induction of SNr seizure, behavior, electroencephalograms (EEG) and local cerebral glucose utilization (LCGU) were studied.
2.1. Experiment 1: Kainic acid ( KA)-induced substantia nigra seizure
*Corresponding author. Tel.: 181-166-68-2594; fax: 181-166-682599. E-mail address: [email protected] (A. Sawamura).
Eight Wistar rats weighing 250–350 g were used. Under intraperitoneal pentobarbital anesthesia (45 mg / kg), a stainless-steel cannula (0.6 mm, O.D.) with an inner stainless-steel needle (0.3 mm in diameter) was inserted stereotaxically into the left SNr (A512.8 mm, L512.5 mm, D523.5 mm) for KA injection according to the atlas of Pellegrino et al. [9]. For recording EEG, a depth electrode was inserted into the left SNr, just posterior to the tip of the injection guide cannula. Electrodes were also inserted into the left sensorimotor cortex, globus pallidus, and hippocampus. The electrodes were connected to a socket with six channels, and the guide cannula was fixed
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02732-9
90
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
Fig. 1. (A) About 10 min after KA injection, continuous low-voltage spikes were observed in the left substantia nigra and globus pallidus on EEG at stage 1. (B) On EEG, the intermittent multiple spikes and wave complexes began to propagate to the left hippocampus and globus pallidus at about 30 min after KA injection at stage 2. (C) On EEG, continuous spikes and wave complexes began to propagate to the left sensorimotor cortex at about 50 min after KA injection at stage 3. (D) On EEG, a secondary generalized seizure was observed at about 70 min after KA injection at stage 4.
to the skull with resin dental cement. The rats were kept for 7 days until the brain had recovered from the surgical damage. At 7 days after surgery, 1 mg of KA (Sigma) was dissolved in a 1 ml phosphate buffer solution (0.2 M, pH 7.4), and 1.0 ml of the KA solution (1.0 mg / ml) was injected into the left SNr. The inner needle was removed and a stainless-steel injection cannula (0.3 mm in diameter) was inserted into the guide cannula. Then 1.0 ml of the KA solution was injected at 0.4 ml / min. These procedures prevent leakage of KA solution into the surrounding structures. The effectiveness of this method was
preliminarily confirmed using KA solution including 0.2% Methylene Blue. It was also histologically confirmed that diffusion of the dye with KA solution was 1.0 mm in diameter and limited within the left SNr. Behavior of the rats and EEG were continuously monitored and recorded by video for about 30 h and then intermittently monitored for 1 month by the use of a video EEG monitoring system. EEG setting: filter setting is the following. The high cut filter was 50 Hz and the low cut filter was 1.5 Hz. The time-constant was 0.1. Alternating current (a.c.) filters (50 Hz) were on. Video–EEG monitorings were employed for
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
91
the present study. At 1 month after KA injection, the rats were sacrificed and subjected to histopathological examination.
3. Results
2.2. Experiment 2: Cerebral glucose metabolism during substantia nigra seizure
On EEG, continuous low-voltage spikes were observed in the left substantia nigra and globus pallidus at about 10 min after KA injection (Fig. 1A). On video, limbic seizure manifestations such as salivation were observed as initial symptoms. On EEG, intermittent multiple spikes and wave complexes were observed in the left substantia nigra, globus pallidus and hippocampus at about 30 min after KA injection (Fig. 1B). On video, standing and jumping manifestations such as wet dog shake just like a limbic seizure were observed. On EEG, continuous spikes and wave complexes began to propagate to the left sensorimotor cortex at about 50 min after KA injection (Fig. 1C). On video, contralateral head turning and contralateral forelimb clonus were observed. On EEG, a secondary generalized seizure was observed at about 70 min after KA injection (Fig. 1D). On video, rolling and generalized tonic seizures were observed. Substantia nigra seizures recurred every 5–7 min and lasted for 3–4 h. However, the seizures gradually subsided and became normal within 12 h. No spontaneous seizure was detected for the next 30 days. The type of epilepsy is secondary generalized seizure. However, standing, jumping and rotational movement of the body axis were the characteristic movements during generalized seizure. This model is similar to brainstem seizure and secondarily generalized seizure.
Two hours after KA injection, seven rats demonstrated status epilepticus. Then, 100 mCi / kg of [ 14 C]2-deoxyglucose (2-DG) was intravenously injected, and the rats were processed for an autoradiographic study. Arterial blood samples were collected at 2, 3, 5, 10, 15, 20, 25, 30, 35 and 45 min after the radioactive tracer injection. The rats were decapitated as soon as possible after the last blood collection and the brain was removed quickly. Brain specimens were immediately frozen and sliced in a cryostat into 20-mm-thick serial coronal sections. These were dried at 608C and [ 14 C]methyl methacrylate standards (Amersham) were consecutively placed on X-ray film (Kodak, SB-5) in an X-ray cassette to be exposed for 7 days. Blood samples were centrifuged and plasma glucose concentration were measured. The optical density of the autoradiogram was measured by a densitometer (Sakura, PDA15) to calculate the tissue 14 C concentration. LCGU was calculated by employing the equation given by Sokoloff et al. [11]. LCGU in various brain structures was determined in seven rats with seizures and seven shamoperated rats used as the control. Comparative analysis was made by using the Mann–Whitney U-test.
3.1. Changes in behavior and EEG
Fig. 2. A small gliotic lesion with neuronal cell loss, caused by KA infiltration, was noted around the cannula tip in the SNr. Pyramidal cell loss was observed in the CA3 region of the left hippocampus.
92
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
Fig. 3. [ 14 C]deoxyglucose autoradiograms. Autoradiograms prepared from coronal sections of the rat brain. (A–D) are from a control rat and (E–H) were obtained from a representative rat of the KA group. Increased LCGU in the medial and lateral septal nucleus, substantia nigra, hippocampus, parietal cortex, piriform cortex, medial and lateral geniculate nucleus, anterodorsal, lateral and ventral nucleus of the thalamus, amygdala and midbrain reticular formation was observed. L, left.
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
93
3.2. Histopathological findings
3.3. [ 14 C] deoxyglucose autoradiograms
Coronal sections were stained by Hematoxylin–Eosin and Cresyl Violet. A small gliotic lesion with neuronal cell loss, caused by KA infiltration, was noted around the cannula tip in the SNr. Pyramidal cell loss was observed in the CA3 of the left hippocampus (Fig. 2).
In a [ 14 C]deoxyglucose autoradiogram, increased LCGU in the medial and lateral septal nucleus, substantia nigra, hippocampus, parietal cortex, piriform cortex, medial and lateral geniculate nucleus, anterodorsal, lateral and ventral nucleus of the thalamus, amygdala and midbrain reticular
Table 1 Local cerebral glucose utilization in substantia nigra seizure versus control rats Structures
Substantia nigra seizure (n57) Ipsilateral
Cerebral cortex Frontal cortex Parietal cortex Visual cortex Auditory cortex Sensorimotor cortex Entorhinal cortex Piriform cortex Cingulate cortex Corpus callosum Extrapyramidal Caudate nucleus Globus pallidus Substantia nigra Thalamus Anterodorsal n. Ventral n. Lateral n. Centromedian n. Medial geniculate n. Lateral geniculate n. Paraventricular n. Hypothalamus Dorsal n. Ventral n. Paraventricular n.
Control (n57) Contralateral
Ipsilateral
Contralateral
88.664.9 94.565.7 97.863.8 85.364.3 88.265.1 79.663.7 99.364.6 83.565.5
77.963.3 80.062.4 90.064.1 83.763.5 87.763.1 65.562.6 70.260.6 76.366.3
77.262.4 79.263.0 96.562.8 93.966.0 84.563.1 66.563.7 70.260.7 75.963.0
32.163.2
23.661.7
23.561.7
95.868.7 82.466.4 215.7610.8**
88.464.3 70.563.8 66.465.4
74.664.6 60.963.4 56.063.0
76.164.9 62.763.6 56.063.0
185.4616.3* 189.3615.7* 198.4614.8* 88.669.5 158.4614.3* 146.2613.6* 78.567.5
120.4613.8 102.1611.4 116.9610.6 87.368.1 72.567.4 80.668.5 69.566.3
84.564.6 77.163.3 79.064.6 79.564.8 65.362.1 63.462.4 60.264.0
83.664.2 77.863.2 79.164.4 79.665.1 65.662.3 64.361.7 61.664.3
72.365.6 76.466.9 80.564.3
65.262.5 63.163.6 52.562.9
52.362.8 46.263.2 49.765.4
52.762.6 46.563.0 53.664.2
114.866.8 211.5612.6** 103.564.7 96.465.4 104.665.8 129.864.6 179.2611.6** 93.169.5 33.662.4
Limbic system Medial septal n. Lateral septal n. Accumbens Hippocampus CA1 Hippocampus CA3 Dentate gyrus Amygdala Mamillary body
152.3611.4* 173.4612.3* 177.9616.2* 131.7615.7* 161.4614.6* 152.6615.1* 216.8616.2** 125.4610.3
70.666.8 75.467.1 64.267.2 73.566.9 80.368.6 85.267.3 64.266.5 103.8611.5
54.763.5 51.562.8 52.162.1 54.063.2 54.862.9 56.162.9 48.762.4 99.065.0
54.663.6 52.363.2 52.762.2 53.663.0 55.363.4 55.263.4 49.462.6 99.964.9
Brainstem MRF Periaqueductal gray Tegmentum Superior colliculus Inferior colliculus
169.5614.3** 66.566.8 62.866.9 134.6615.4 110.2613.6
72.667.3 64.266.1 61.165.8 84.969.6 122.3613.4
55.164.3 48.163.8 61.362.4 72.361.0 83.461.0
55.063.9 49.264.4 61.662.6 72.360.8 83.561.5
53.265.2
48.962.1
48.562.0
Cerebellar cortex
54.665.6
Values: mean6S.E.M. (Fmol / 100 g / min). *P,0.05; **P,0.01 (Mann–Whitney U -test).
94
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
formation (MRF) was observed (Fig. 3; Table 1). In the preliminary study, we injected KA into bilateral SNr. The elicited seizures were so severe that animals did not tolerate the procedure. All animals died in severe seizure status within 30–60 min after the bilateral injection. In the present study, the elicited seizures were mainly localized in the ipsilateral hemisphere. However, hypermetabolic areas were already observed in the contralateral hemisphere, especially in the inferior colliculus, anterodorsal nucleus of the thalamus, frontal and parietal cortices, and entorhinal and piriform cortices as indicated in Table 1.
4. Discussion The substantia nigra (SN) is thought to play a major role in the control of motor function. A major efferent of the SN is the dopaminergic nigrostriatal system. In our present study, a microinjection of KA into SNr induced characteristic generalized seizure status. It is very difficult to explain why excessive excitation of the SNr can cause epileptic manifestation. But, LCGU during seizure provided important information. A remarkable increase in LCGU was observed not only in the SNr but also in the MRF, thalamus and cerebral cortex. In such an abnormal condition, the anticonvulsant effect of GABAergic output of SNr became inactive upon the MRF, thalamus and cerebral cortex, which demonstrated excessive epileptic excitation. Another explanation may be the enhancement of the excitatory dopaminergic circuit of nigrostriatal projection from substantia nigra pars compacta due to hyperexcitation of the SNr. Most of the GABA content in the SNr is associated with GABAergic nerve terminals arising from cell bodies located in the striatum. GABA in the SNr exerts a control over nigrostriatal and mesolimbic dopaminergic neurons [3]. The central amygdala receives direct connections from the substantia nigra [1]. Le Gal La Salle et al. [7] found that bilateral administration of a GABA-elevating substance (gamma vinyl-GABA) in the vicinity of the substantia nigra greatly shortens the duration of amygdaloid kindled seizures without significantly altering the motor convulsions. Tanaka et al. [12] reported that bilateral destruction of the SNr by ibotenic acid injections induced remarkable aggravation of limbic seizure induced by KA injection into the amygdala. Iadarola et al. [6] found that protection from seizures induced by maximal electroshock or by convulsant drugs was obtained as a result of GABA elevation or direct stimulation of GABA receptors within a restricted region of the ventral midbrain tegmentum, the substantia nigra. The concentration of substance P (SP) in the SN is the highest of all brain areas, and it is localized in nerve terminals deriving from cell bodies in the
striatum. The fact that the blockade of nigral SP transmission had a similar action to that of GABA-mediated inhibition in the SNr is consistent with the proposed excitatory role of nigral SP [5]. Garant et al. [4] found that GABA terminals and SPcontaining terminals exert reciprocal actions on nigral output, and they provided the first evidence of an effect of SP antagonists on the seizure process. According to Vitek et al. [13] there are many excitatory and inhibitory motor circuits between the cerebral cortex, striatum, globus pallidus, thalamus, substantia nigra and subthalamic nucleus. In addition to decreased inhibition from the globus pallidus pars externa (GPe), increased activity in the subthalamic nucleus (STN) could also occur through increased activity in excitatory projections to the STN from the cortex, thalamus, or midbrain extrapyramidal area. The reduced rate of discharge of the GPe would also be expected to increase rates in the GPi via its direct projection to the GPi. In a sense, a window for the development of altered patterns and phasic responses of thalamic neuronal activity that is dependent on the mean discharge rate of neurons in the GPi may exist. Previous investigations have demonstrated that neocortical epileptiform activity is modulated by the basal ganglia and is particularly inhibited by stimulation of the globus pallidus pars interna (GPi) [2,4,8,10] which together with the SNr is an important output station. The basal ganglia are considered to play a role in the genesis and / or spread of epileptic activity. The SN may present an anticonvulsant region influencing epileptiform activity through its GABAergic projections, showing an action synergic to that of the internal pallidum [6]. In the present study, the inhibitory role of the SNr and GPi became inactive by the excessive epileptic excitation and brainstem seizures and secondarily generalized seizures were elicited. The results suggested that an epileptogenic focus of the substantia nigra demonstrated a rapid evolution from the focal seizure status to the secondarily generalized seizure status. Thus the substantia nigra may play a key role as a relay nucleus for the secondary generalization of a focal seizure.
5. Conclusions A microinjection of kainic acid into a unilateral SNr resulted in limbic seizure manifestations as initial symptoms. These seizures finally evolved into a generalized seizure status. The results demonstrated that the substantia nigra plays an important role in secondary generalization in a substantia nigra seizure model due to excessive excitation of the nigrostriatal pathways and decreased function of the GABAergic projection system.
A. Sawamura et al. / Brain Research 911 (2001) 89 – 95
References [1] R.M. Beckstead, V.B. Domesick, W.J.H. Nauta, Efferent connections of the substantia nigra and ventral tegmental area in rat, Brain Res. 175 (1979) 191–217. [2] A.R. Cools, G. Hendriks, J. Korten, The acetylcholine–dopamine balance in the basal ganglia of rhesus monkeys and its role in dynamic, dystonic, dyskinetic, and epileptoid motor activities, J. Neural Transm. 36 (1975) 91–105. [3] K. Gale, M.J. Iadarola, GABAergic denervation of rat substantia nigra: functional and pharmacological properties, Brain Res. 183 (1980) 217–223. [4] D.S. Garant, K. Gale, Lesions of substantia nigra protect against experimentally induced seizures, Brain Res. 273 (1983) 156–161. [5] D.S. Garant, M.J. Iadarola, K. Gale, Substance P antagonists in substantia nigra are anticonvulsant, Brain Res. 382 (1986) 372–378. [6] M.J. Iadarola, K. Gale, Substantia nigra; site of anticonvulsant activity mediated by gamma-aminobutyric acid, Science 218 (1982) 1237. [7] G. Le Gal La Salle, M. Kaijima, S. Feldblum, Abortive amygdaloid kindled seizures following microinjection of gamma-vinyl-GABA in the vicinity of substantia nigra in rats, Neurosci. Lett. 36 (1983) 69–74.
95
[8] E.J. Neafsey, C.M. Chuman, A.A. Ward Jr., Propagation of focal cortical epileptiform discharge to the basal ganglia, Exp. Neurol. 66 (1979) 97–108. [9] L.J. Pellegrino, A.S. Pellegrino, A.J. Cushman, A Stereotaxic Atlas of the Rat Brain, 2nd Edition, Plenum Press, New York, 1979. [10] M. Sabatino, G. Gravante, G. Ferraro, V. Savatteri, V. La Grutta, Inhibitory control by substantia nigra of generalized epilepsy in the cat, Epilepsy Res. 2 (1988) 380–386. [11] L. Sokoloff, M. Reivich, C. Kennedy, M.H. Des Rosiers, C.S. Patlak, K.D. Pettigrew, O. Sakurada, M. Shinohara, The [ 14 C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat, J. Neurochem. 28 (1977) 897–916. [12] T. Tanaka, S. Tanaka, M. Kaijima, Y. Yonemasu, Ibotenic acidinduced nigral lesion and limbic seizure, Brain Res. 498 (1989) 215–220. [13] J.L. Vitek, J. Zhang, M. Evatt, K. Mewes, M.R. Delong, T. Hashimoto, S. Triche, R.A.E. Bakay, GPi pallidotomy for dystonia: clinical outcome and neuronal activity, Adv. Neurol. 78 (1998) 211–219.