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Selective bilateral destruction of substantia nigra has no effect on kindled seizures induced from stimulation of amygdala or piriform cortex in rats
Neuroscience Letters, 113 (1990) 205-210
205
Elsevier Scientific Publishers Ireland Ltd.
NSL 06867
Selective bilateral destruction of substantia nigra has no effect on kindled seizures induced from stimulation of amygdala or piriform cortex in rats Ulrich Wahnschaffe and Wolfgang LSscher Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Hannover (F.R.G.) (Received 23 November 1989; Revised version received 19 January 1990; Accepted 27 January 1990)
Key words: Substantia nigra; Kindling; Epilepsy; y-Aminobutyric acid Enhancement of GABAergic transmission in the substantia nigra has been shown to attenuate motor manifestations of diverse seizure models, including kindling. Similar anticonvulsant effects were reported after bilateral lesions of the substantia nigra, supporting the view that the nigra efferents constitute a critical gating mechanism in the propagation of seizure activity. However, in the lesion studies reported so far the nigra was not destroyed selectively so that regions destroyed in addition to the nigra could have been involved in the anticonvulsant effects observed. We destroyed the nigra selectively in fully kindled rats by bilateral microinjection of small amounts of the neurotoxin ibotenic acid. Two groups of rats were studied; one was kindled from stimulation of the basolateral amygdala, the other from stimulation of the piriform cortex. In both groups, there was no indication of a reduction in seizure susceptibility, seizure severity or seizure duration after bilateral destruction of the nigra. The data thus indicate that, at least in kindled rats, the substantia nigra might be less important for seizure generation and/or propagation than previously thought.
The search for anatomical and neurochemical substrates of seizure generation and propagation in the brain has been the major goal of epilepsy research over the last decades. A substantial body of evidence suggests that the substantia nigra (SN), particularly the pars reticulata (SNR) region, is a critical site involved in seizure propagation [4,5]. Bilateral intranigral microinjections of either muscimol, a GABA agonist, or gamma-vinyl GABA (GVG), a GABA-elevating drug, were shown to attenuate or block motor seizures in different animal models of epilesy, including amygdaloid kindling [8,10]. Because GABA has been shown to inhibit nigral efferents, it was proposed that the anticonvulsant effect of intranigral application of muscimol or GVG relates to an inhibition of nigral projections that are permissive or facilitative to seizure propagation [4]. In support of this, bilateral destruction of the SN by Correspondence." W. L6scher, Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Biinteweg 17, D-3000 Hannover 71, F.R.G. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
206
electrocoagulation or microinjection of the neurotoxin kainic acid attenuated bicuculline-induced clonic and tonic seizures as well as tonic electroshock seizures [6]. In amygdala-kindled rats, bilateral destruction of SN by N-methyl-o,L-aspartate (NMDA) not only suppressed motor seizures but also reduced after-discharges recorded at the amygdala, suggesting that the nigra not only transmits seizure activity from rostral to caudal sites but is actively involved in the generation of limbic seizures at the site of origin [10]. However, in the lesion studies reported by McNamara and colleagues [10] the SN was not lesioned selectively, but the lesions involved several other regions. We were therefore interested to investigate the effects of selective lesions of the SN on kindled seizure parameters. For this purpose, rats were kindled either from stimulation of the amygdala or the piriform cortex, the latter region thought to be an important generator in the subcortically kindled seizure mechanisms [9]. All experiments were carried out in female Wistar rats, weighing 210-230 g. Fifty rats were anesthetized with chloral hydrate (360 mg/kg i.p.) and received stereotaxic implantation of one bipolar electrode in either the right basolateral amygdala (n = 15) or the right piriform cortex (n = 35). Coordinates for electrode implantation derived from the atlas of Paxinos and Watson [11] were AP - 0 . 8 , L -4.8, V - 8 . 5 for the piriform cortex and AP - 2 . 2 , L - 4 . 8 , V - 8 . 5 for the basolateral amygdala. In all rats, guide cannulae were implanted over the area of the SN bilaterally (AP - 4 . 9 , L +2.2, V - 1.5; for microinjection V was - 7 . 5 ) . Skull screws served as the indifferent reference electrode. After a post-operative period of 2 weeks, constant current stimulations (500/~A, I ms, monophasic square-wave pulses, 50/s for 1 s) were delivered to the piriform cortex or amygdala at intervals of 1 day until at least 10 reproducible stage-5 seizures were elicited. The electrical activity of the stimulated region was recorded before and after each stimulation. In fully-kindled rats, after-discharge threshold (ADT) was determined by using an ascending method of limits with 20% increments of the current (initial current 10/IA) at interstimulation intervals of 1 min [3]. Since all animals exhibited generalized seizures (stages 4-5) at the ADT current, it was not necessary to determine the threshold for generalized seizures (GST) separately. The ADT determined with interstimulation intervals of 1 min was not different from ADT values determined with interstimulation intervals of 1 day, thus demonstrating that the short interstimulation interval did not bias ADT determinations. In each fully kindled animal, the ADT was determined at least twice prior to the SN lesion. ADTs thus determined were stable and reproducible in all rats used for this study. Severity and duration of the seizures and after-discharge duration (ADD) were recorded both during threshold determinations and at supra-threshold stimulation with 500/iA as described elsewhere [7]. For lesioning of SN, the injection cannulae were inserted through the guide cannulae and 5 min later the neurotoxin ibotenic acid [2] was injected at a dose of 5/~g in 0.5/tl of buffered saline at a rate of 0.1 /tl/min. This dose of ibotenic acid was chosen on the basis of dose-effect and time course experiments in rats showing neuron degeneration in the SN with a minimum destruction of surrounding regions
207
within 2-3 days after injection of this dosage. The injection cannula was withdrawn 5 min after completion of the injection. All injections were made unilaterally; the SN of the other hemisphere was destroyed 3 days after the first lesion. The major behavioural symptom after unilateral injection of ibotenic acid was contralateral circling for several hours, but animals rapidly recovered thereafter and it was not necessary to tube-feed them. In order to examine the effect of SN lesions on kindled seizure parameters, the ADT was determined ! and 5 weeks after bilateral destruction of the SN and was compared to the pre-lesion values. Furthermore, stimulations with 500 pA were carried out before and after the lesions for recording of seizure parameters. After termination of the last threshold determination, placement of stimulating electrodes and injection cannula tips and the extent of the SN lesions were examined histologically in all animals and only animals with correct placement of the kindling electrodes and selective near-complete (at least 80%) bilateral lesions of the SN were used for evaluation of the kindling experiments described below. Fourteen rats fulfilled the selection criteria. Histological examination of the SN in these animals demonstrated an almost complete loss of neurons, which were replaced by glia cells. Axonal bundles in the lesioned SN were preserved. The neuronal damage was largely restricted to the SN and zona incerta (Fig. 1). Kindled seizure parameters before and after the SN lesions are shown in Table I. With respect to prelesion values, the ADT was considerably lower in piriform cortex than in amygdala-kindled rats. The bilateral SN lesions had no influence on susceptibility of the rats to electrical stimulation of amygdala or piriform cortex. With respect to seizure severity determined at ADT stimulation, all animals exhibited generalized seizures before and one week after the lesion without any indication of differences in the behavioural characteristics of the seizures. The only significant difference was an increased seizure duration in the amygdala-kindled group one week after the lesion (Table I). When animals were stimulated at supra-threshold current (500 pA), no differences were found between pre- and post-lesion seizure parameters (not illustrated). McNamara et al. [10] have reported previously that bilateral destruction of the SN markedly reduced the duration of motor seizures and after-discharges in amygdalakindled rats stimulated with currents 10% above the generalized seizure threshold. In some lesioned animals, motor seizures were abolished and brief or no after-discharges were obtained. In these animals, administration of additional stimuli in which both current intensity and train duration were doubled resulted in long afterdischarges accompanied by generalized seizures, thus suggesting that the lesions had increased seizure threshold rather than eliminated the ability to generate seizures. This suggestion is not substantiated by the present experiments, which demonstrate that selective lesions of the SN in kindled rats do not reduce the duration of focal or generalized seizures and have no influence on seizure threshold. The findings of McNamara et al. [10] thus might relate to the fact that, in contrast to our experiments, the lesions produced in their study by 20/zg of the neurotoxin NMDA were not selective but included several structures other than the SN, e.g. parts of the thalamus, which were not damaged in our animals. Interestingly, a subsequent study of
208
Bregma -
4.80 mm
'
"~
~"_b--,mt
,'
'
z',
.'
j.
--5.20
mm
Fig. I. Schematic reconstruction of ibotenic acid-induced lesions of the SN as plotted on the drawings of coronal brain sections according to the stereotaxic atlas of Paxinos and Watson [11]. Shaded areas represent the lesioned area as determined by histological examination. The animal with the largest lesions of the group of 14 rats used for evaluation of the kindling experiments is represented by the drawings on the left side, whereas the drawings on the right represent the animal with the smallest lesion, cp, cerebral peduncle; ml, medial lemniscus; SNR, substantia nigra pars reticulata; SNC, substantia nigra pars compacta; SNL, substantia nigra pars lateralis; VTA, ventral tegmental area: ZI, zona incerta.
M c N a m a r a ' s g r o u p [13] showed t h a t m o r e selective lesions o f the SN by N M D A or t h e r m o c o a g u l a t i o n facilitated rather t h a n inhibited kindling d e v e l o p m e n t in rats. Thus, the i n h i b i t o r y effects o f lesions in fully k i n d l e d rats r e p o r t e d by M c N a m a r a et al. [10] might have been due to d e s t r u c t i o n o f structures o t h e r t h a n the S N o r to a c o m b i n e d effect o f the SN lesions a n d lesions o f s u r r o u n d i n g regions. In this respect, it should be n o t e d that in the studies r e p o r t e d by G a r a n t a n d G a l e [6], k a i n a t e or t h e r m o c o a g u l a t i v e lesions o f S N were also not selective b u t included d a m a g e to the cerebral peduncles a n d / o r medial lemniscus, which were not lesioned in o u r experiments. W i t h respect to lesions induced by excitotoxins, it has been d e m o n s t r a t e d recently for o t h e r b r a i n regions that ibotenate, which, at least in part, is t h o u g h t to act by s t i m u l a t i o n o f N M D A receptors, generally p r o d u c e s m u c h m o r e direct a n d u n i f o r m lesions t h a n k a i n a t e o r N M D A itself [2], thus sugesting that the choice o f excitotoxin might influence d a t a o b t a i n e d from lesion studies. The fact that, in c o n t r a s t to o t h e r n e u r o t o x i n s such as N M D A o r kainate, i b o t e n a t e is d e c a r b o x y lated to f o r m the G A B A r e c e p t o r agonist m u s c i m o l within the brain [2] might be one factor that limits the extent o f d a m a g e (especially d i s t a n t d a m a g e ) p r o d u c e d . W i t h respect to the effect o f the S N lesions, it is n o t likely that i b o t e n a t e - d e r i v e d mus-
209
TABLE I ADT A N D SEIZURE P A R A M E T E R S AT ADT BEFORE A N D A F T E R BILATERAL DESTRUCTION OF THE SN Values are means + SE of 8 piriform cortex-kindled and 6 amygdala-kindled rats. The data were determined 1 day before and 1 and 5 weeks after bilateral destruction of the SN. Significance of differences was calculated by the Wilcoxon signed rank test for paired replicates. Values significantly different (P < 0.05) from pre-lesion data are marked by asterisks. Group
ADT
Seizure parameters at ADT
~uA) Seizure severity (score)
Seizure duration (sec)
After-discharge duration (sec)
32.0+ 8.5 33.6+ 6.8 33.8+11.1
4.9+0.12 5.0+0 4.9+0.12
48.5+5.5 52.9+4.8 53.2+6.1
83.8+ 5.0 73.8+ 8.5 76.1+ 9.3
115 +27.4 107 __+29.1 86.3 _ 24.5
4.5+0.22 4.7+0.52 3.3 + 1.6
27.7+9.3 55.3+9.1" 33.3 + 8.9
37.3+14.2 63.8+12.4 40.0_ 13.6
Piriform cortex-kindled Prelesion l weekpost-lesion 5 weeks post-lesion
Amygdala-kindled Pre-lesion 1 weekpost-lesion 5 weeks post-lesion
cimol alters seizure sensitivity in a direction opposing the lesion effect, because muscitool is eliminated very rapidly (within 3 h) from rat brain regions [1], whereas the effects of the nigra lesions on kindled seizure parameters were measured 1-5 weeks after the ibotenate injection. With respect to the SN lesions, it should be noted that it was not possible to destroy only the SNR, but, as in other studies with excitotoxic SN lesions [6,10,13,14], the pars compacta (SNC) and, at least in part, the pars lateralis were also destroyed by microinjection of ibotenate (Fig. 1). More recent studies indicate that the role of the SNC in modulation of seizures may be opposite to that of the SNR [16], which might be important for results of SN lesions studies. The present findings do not substantiate previous suggestions [10] that the SN regulates propagation of motor seizures and might be involved in the generation of limbic seizures in the kindling model. With respect to other seizure models, a recent study with selective SN lesions in cats showed that the ibotenate-induced lesions facilitated rather than inhibited seizures induced by kainate [14]. These data seem to be in contrast to the role of the SN indicated by effects of intranigral microinjections of GABAergic drugs in seizure models (see above). However, it should be noted that intranigral microinjections of GABAergic, such as muscimol or GVG, drugs markedly reduce muscle tone [12,15], which could explain, at least in part the attenuation of motor seizures observed in several models, including kindling, after bilateral injection of such drugs into the SN (see above). Muscle relaxation would not explain the finding that intranigral injection of GVG in kindled rats reduces after-discharge duration [10]; however, this finding could not be confirmed by another study [8].
210 T a k e n t o g e t h e r , t h e s e d a t a m i g h t s u g g e s t t h a t , a t l e a s t in t h e k i n d l i n g m o d e l , t h e S N is less i m p o r t a n t t b r s e i z u r e i n i t i a t i o n a n d / o r p r o p a g a t i o n t h a n p r e v i o u s l y t h o u g h t . The study was supported
by grants from the Deutsche Forschungsgemeinschaft
(Lo 274/3-1). We thank Dr. Turski (Schering, Berlin, F.R.G.) for helpful discussions a n d a d v i c e . W e t h a n k M r s . C. B a t t l i n g f o r t e c h n i c a l a s s i s t a n c e . l Baraldi, M., Grandison, L. and Guidotti, A., Distribution and metabolism of muscimol in the brain and other tissues of the rat, Neuropharmacology, 18 (1979) 57 62. 2 Coyle, J.T., Excitotoxins. In H.Y. Meltzer (Ed.), Psychopharmacology: The Third Generation of Progress, Raven Press, New York, 1987, pp. 333 340. 3 Freeman, F.G. and Jarvis, M.F., The effect of interstimulation interval on the assessment and stability of kindled seizure thresholds, Brain Res. Bull., 7 (1981) 629-633. 4 Gale, K., Role of substantia nigra in GABA-mediated anticonvulsant actions. In A.V. DelgadoEscueta, A.A. Ward, D.M. Woodbury and R.J. Porter (Eds.), Basic Mechanisms of the Epilepsies Molecular and Cellular Approaches, Raven Press, New York, 1986, pp. 343 364. 5 Gale, K., Progression and generalization of seizure discharge: anatomical and neurochemical substrates, Epilepsia, 19 (Suppl. 2) (1988) S15 $34. 6 Garant, D.S. and Gale, K., Lesions of substantia nigra protect against experimentally-induced seizures, Brain Res., 273 (1983) 156 161. 7 L6scher, W. and H6nack, D., High doses of memantine (1-amino-3,5-dimethyladamantane) induce seizures in kindled but not in non-kindled rats, Naunyn-Schmiedeberg's Arch. Pharmacol., in press. 8 L6scher, W., Czuczwar, S.J., Jfickel. R. and Schwarz, M., Effect of microinjections of ~,-vinyl-GABA or isoniazid into substantia nigra on the development of amygdala kindling in rats, Exp. Neurol., 95 (1987) 622 638. 9 Mclntyre, D.C., Kindling and the piriform cortex, In J.A. Wada (Ed.), Kindling 3, Raven Press, New York, pp. 249 260, 1986. 10 McNamara, J.O., Galloway, M.T., Rigsbee, L.C. and Shin, C. Evidence implicating substantia nigra in regulation of kindled seizure threshold, J. Neurosci., 4 (1984) 2410-2417. 11 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, Sydney, 1986. 12 Schwarz, M., L6scher, W., Turski, L. and Sontag, K.-H., Disturbed GABAergic transmission in mutant Han-Wistar rats: further evidence for basal ganglia dysfunction, Brain Res., 347 (1985) 258 267. 13 Shin, C., Silver, J.M., Bonhaus, D.W. and McNamara, J.O., The role of substantia nigra in the development of kindling: pharmacologic and lesion studies, Brain Res., 412 (1987) 311 317. 14 Tanaka, T., Tanaka, S., Kaijima, M. and Yonemasu, Y., Ibotenic acid-induced nigral lesion and limbic seizure in cats, Brain Res., 498 (1989) 215-220. 15 Turski, L., Schwarz, M., Klockgether, T. and Sontag, K.H., Substantia nigra: a site of action of muscle relaxant drugs, Neurosci. Abstr., 11 (1985) 1162. 16 Turski, L., Cavalheiro, E.A., Bortolotto, Z.A., Ikonomidou-Turski, C., Kleinrok, Z. and Turski, W.A. Dopamine-sensitive anticonvulsant site in the rat striatum, J. Neurosci., 8 (1988) 4027 4037.
205
Elsevier Scientific Publishers Ireland Ltd.
NSL 06867
Selective bilateral destruction of substantia nigra has no effect on kindled seizures induced from stimulation of amygdala or piriform cortex in rats Ulrich Wahnschaffe and Wolfgang LSscher Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Hannover (F.R.G.) (Received 23 November 1989; Revised version received 19 January 1990; Accepted 27 January 1990)
Key words: Substantia nigra; Kindling; Epilepsy; y-Aminobutyric acid Enhancement of GABAergic transmission in the substantia nigra has been shown to attenuate motor manifestations of diverse seizure models, including kindling. Similar anticonvulsant effects were reported after bilateral lesions of the substantia nigra, supporting the view that the nigra efferents constitute a critical gating mechanism in the propagation of seizure activity. However, in the lesion studies reported so far the nigra was not destroyed selectively so that regions destroyed in addition to the nigra could have been involved in the anticonvulsant effects observed. We destroyed the nigra selectively in fully kindled rats by bilateral microinjection of small amounts of the neurotoxin ibotenic acid. Two groups of rats were studied; one was kindled from stimulation of the basolateral amygdala, the other from stimulation of the piriform cortex. In both groups, there was no indication of a reduction in seizure susceptibility, seizure severity or seizure duration after bilateral destruction of the nigra. The data thus indicate that, at least in kindled rats, the substantia nigra might be less important for seizure generation and/or propagation than previously thought.
The search for anatomical and neurochemical substrates of seizure generation and propagation in the brain has been the major goal of epilepsy research over the last decades. A substantial body of evidence suggests that the substantia nigra (SN), particularly the pars reticulata (SNR) region, is a critical site involved in seizure propagation [4,5]. Bilateral intranigral microinjections of either muscimol, a GABA agonist, or gamma-vinyl GABA (GVG), a GABA-elevating drug, were shown to attenuate or block motor seizures in different animal models of epilesy, including amygdaloid kindling [8,10]. Because GABA has been shown to inhibit nigral efferents, it was proposed that the anticonvulsant effect of intranigral application of muscimol or GVG relates to an inhibition of nigral projections that are permissive or facilitative to seizure propagation [4]. In support of this, bilateral destruction of the SN by Correspondence." W. L6scher, Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Biinteweg 17, D-3000 Hannover 71, F.R.G. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
206
electrocoagulation or microinjection of the neurotoxin kainic acid attenuated bicuculline-induced clonic and tonic seizures as well as tonic electroshock seizures [6]. In amygdala-kindled rats, bilateral destruction of SN by N-methyl-o,L-aspartate (NMDA) not only suppressed motor seizures but also reduced after-discharges recorded at the amygdala, suggesting that the nigra not only transmits seizure activity from rostral to caudal sites but is actively involved in the generation of limbic seizures at the site of origin [10]. However, in the lesion studies reported by McNamara and colleagues [10] the SN was not lesioned selectively, but the lesions involved several other regions. We were therefore interested to investigate the effects of selective lesions of the SN on kindled seizure parameters. For this purpose, rats were kindled either from stimulation of the amygdala or the piriform cortex, the latter region thought to be an important generator in the subcortically kindled seizure mechanisms [9]. All experiments were carried out in female Wistar rats, weighing 210-230 g. Fifty rats were anesthetized with chloral hydrate (360 mg/kg i.p.) and received stereotaxic implantation of one bipolar electrode in either the right basolateral amygdala (n = 15) or the right piriform cortex (n = 35). Coordinates for electrode implantation derived from the atlas of Paxinos and Watson [11] were AP - 0 . 8 , L -4.8, V - 8 . 5 for the piriform cortex and AP - 2 . 2 , L - 4 . 8 , V - 8 . 5 for the basolateral amygdala. In all rats, guide cannulae were implanted over the area of the SN bilaterally (AP - 4 . 9 , L +2.2, V - 1.5; for microinjection V was - 7 . 5 ) . Skull screws served as the indifferent reference electrode. After a post-operative period of 2 weeks, constant current stimulations (500/~A, I ms, monophasic square-wave pulses, 50/s for 1 s) were delivered to the piriform cortex or amygdala at intervals of 1 day until at least 10 reproducible stage-5 seizures were elicited. The electrical activity of the stimulated region was recorded before and after each stimulation. In fully-kindled rats, after-discharge threshold (ADT) was determined by using an ascending method of limits with 20% increments of the current (initial current 10/IA) at interstimulation intervals of 1 min [3]. Since all animals exhibited generalized seizures (stages 4-5) at the ADT current, it was not necessary to determine the threshold for generalized seizures (GST) separately. The ADT determined with interstimulation intervals of 1 min was not different from ADT values determined with interstimulation intervals of 1 day, thus demonstrating that the short interstimulation interval did not bias ADT determinations. In each fully kindled animal, the ADT was determined at least twice prior to the SN lesion. ADTs thus determined were stable and reproducible in all rats used for this study. Severity and duration of the seizures and after-discharge duration (ADD) were recorded both during threshold determinations and at supra-threshold stimulation with 500/iA as described elsewhere [7]. For lesioning of SN, the injection cannulae were inserted through the guide cannulae and 5 min later the neurotoxin ibotenic acid [2] was injected at a dose of 5/~g in 0.5/tl of buffered saline at a rate of 0.1 /tl/min. This dose of ibotenic acid was chosen on the basis of dose-effect and time course experiments in rats showing neuron degeneration in the SN with a minimum destruction of surrounding regions
207
within 2-3 days after injection of this dosage. The injection cannula was withdrawn 5 min after completion of the injection. All injections were made unilaterally; the SN of the other hemisphere was destroyed 3 days after the first lesion. The major behavioural symptom after unilateral injection of ibotenic acid was contralateral circling for several hours, but animals rapidly recovered thereafter and it was not necessary to tube-feed them. In order to examine the effect of SN lesions on kindled seizure parameters, the ADT was determined ! and 5 weeks after bilateral destruction of the SN and was compared to the pre-lesion values. Furthermore, stimulations with 500 pA were carried out before and after the lesions for recording of seizure parameters. After termination of the last threshold determination, placement of stimulating electrodes and injection cannula tips and the extent of the SN lesions were examined histologically in all animals and only animals with correct placement of the kindling electrodes and selective near-complete (at least 80%) bilateral lesions of the SN were used for evaluation of the kindling experiments described below. Fourteen rats fulfilled the selection criteria. Histological examination of the SN in these animals demonstrated an almost complete loss of neurons, which were replaced by glia cells. Axonal bundles in the lesioned SN were preserved. The neuronal damage was largely restricted to the SN and zona incerta (Fig. 1). Kindled seizure parameters before and after the SN lesions are shown in Table I. With respect to prelesion values, the ADT was considerably lower in piriform cortex than in amygdala-kindled rats. The bilateral SN lesions had no influence on susceptibility of the rats to electrical stimulation of amygdala or piriform cortex. With respect to seizure severity determined at ADT stimulation, all animals exhibited generalized seizures before and one week after the lesion without any indication of differences in the behavioural characteristics of the seizures. The only significant difference was an increased seizure duration in the amygdala-kindled group one week after the lesion (Table I). When animals were stimulated at supra-threshold current (500 pA), no differences were found between pre- and post-lesion seizure parameters (not illustrated). McNamara et al. [10] have reported previously that bilateral destruction of the SN markedly reduced the duration of motor seizures and after-discharges in amygdalakindled rats stimulated with currents 10% above the generalized seizure threshold. In some lesioned animals, motor seizures were abolished and brief or no after-discharges were obtained. In these animals, administration of additional stimuli in which both current intensity and train duration were doubled resulted in long afterdischarges accompanied by generalized seizures, thus suggesting that the lesions had increased seizure threshold rather than eliminated the ability to generate seizures. This suggestion is not substantiated by the present experiments, which demonstrate that selective lesions of the SN in kindled rats do not reduce the duration of focal or generalized seizures and have no influence on seizure threshold. The findings of McNamara et al. [10] thus might relate to the fact that, in contrast to our experiments, the lesions produced in their study by 20/zg of the neurotoxin NMDA were not selective but included several structures other than the SN, e.g. parts of the thalamus, which were not damaged in our animals. Interestingly, a subsequent study of
208
Bregma -
4.80 mm
'
"~
~"_b--,mt
,'
'
z',
.'
j.
--5.20
mm
Fig. I. Schematic reconstruction of ibotenic acid-induced lesions of the SN as plotted on the drawings of coronal brain sections according to the stereotaxic atlas of Paxinos and Watson [11]. Shaded areas represent the lesioned area as determined by histological examination. The animal with the largest lesions of the group of 14 rats used for evaluation of the kindling experiments is represented by the drawings on the left side, whereas the drawings on the right represent the animal with the smallest lesion, cp, cerebral peduncle; ml, medial lemniscus; SNR, substantia nigra pars reticulata; SNC, substantia nigra pars compacta; SNL, substantia nigra pars lateralis; VTA, ventral tegmental area: ZI, zona incerta.
M c N a m a r a ' s g r o u p [13] showed t h a t m o r e selective lesions o f the SN by N M D A or t h e r m o c o a g u l a t i o n facilitated rather t h a n inhibited kindling d e v e l o p m e n t in rats. Thus, the i n h i b i t o r y effects o f lesions in fully k i n d l e d rats r e p o r t e d by M c N a m a r a et al. [10] might have been due to d e s t r u c t i o n o f structures o t h e r t h a n the S N o r to a c o m b i n e d effect o f the SN lesions a n d lesions o f s u r r o u n d i n g regions. In this respect, it should be n o t e d that in the studies r e p o r t e d by G a r a n t a n d G a l e [6], k a i n a t e or t h e r m o c o a g u l a t i v e lesions o f S N were also not selective b u t included d a m a g e to the cerebral peduncles a n d / o r medial lemniscus, which were not lesioned in o u r experiments. W i t h respect to lesions induced by excitotoxins, it has been d e m o n s t r a t e d recently for o t h e r b r a i n regions that ibotenate, which, at least in part, is t h o u g h t to act by s t i m u l a t i o n o f N M D A receptors, generally p r o d u c e s m u c h m o r e direct a n d u n i f o r m lesions t h a n k a i n a t e o r N M D A itself [2], thus sugesting that the choice o f excitotoxin might influence d a t a o b t a i n e d from lesion studies. The fact that, in c o n t r a s t to o t h e r n e u r o t o x i n s such as N M D A o r kainate, i b o t e n a t e is d e c a r b o x y lated to f o r m the G A B A r e c e p t o r agonist m u s c i m o l within the brain [2] might be one factor that limits the extent o f d a m a g e (especially d i s t a n t d a m a g e ) p r o d u c e d . W i t h respect to the effect o f the S N lesions, it is n o t likely that i b o t e n a t e - d e r i v e d mus-
209
TABLE I ADT A N D SEIZURE P A R A M E T E R S AT ADT BEFORE A N D A F T E R BILATERAL DESTRUCTION OF THE SN Values are means + SE of 8 piriform cortex-kindled and 6 amygdala-kindled rats. The data were determined 1 day before and 1 and 5 weeks after bilateral destruction of the SN. Significance of differences was calculated by the Wilcoxon signed rank test for paired replicates. Values significantly different (P < 0.05) from pre-lesion data are marked by asterisks. Group
ADT
Seizure parameters at ADT
~uA) Seizure severity (score)
Seizure duration (sec)
After-discharge duration (sec)
32.0+ 8.5 33.6+ 6.8 33.8+11.1
4.9+0.12 5.0+0 4.9+0.12
48.5+5.5 52.9+4.8 53.2+6.1
83.8+ 5.0 73.8+ 8.5 76.1+ 9.3
115 +27.4 107 __+29.1 86.3 _ 24.5
4.5+0.22 4.7+0.52 3.3 + 1.6
27.7+9.3 55.3+9.1" 33.3 + 8.9
37.3+14.2 63.8+12.4 40.0_ 13.6
Piriform cortex-kindled Prelesion l weekpost-lesion 5 weeks post-lesion
Amygdala-kindled Pre-lesion 1 weekpost-lesion 5 weeks post-lesion
cimol alters seizure sensitivity in a direction opposing the lesion effect, because muscitool is eliminated very rapidly (within 3 h) from rat brain regions [1], whereas the effects of the nigra lesions on kindled seizure parameters were measured 1-5 weeks after the ibotenate injection. With respect to the SN lesions, it should be noted that it was not possible to destroy only the SNR, but, as in other studies with excitotoxic SN lesions [6,10,13,14], the pars compacta (SNC) and, at least in part, the pars lateralis were also destroyed by microinjection of ibotenate (Fig. 1). More recent studies indicate that the role of the SNC in modulation of seizures may be opposite to that of the SNR [16], which might be important for results of SN lesions studies. The present findings do not substantiate previous suggestions [10] that the SN regulates propagation of motor seizures and might be involved in the generation of limbic seizures in the kindling model. With respect to other seizure models, a recent study with selective SN lesions in cats showed that the ibotenate-induced lesions facilitated rather than inhibited seizures induced by kainate [14]. These data seem to be in contrast to the role of the SN indicated by effects of intranigral microinjections of GABAergic drugs in seizure models (see above). However, it should be noted that intranigral microinjections of GABAergic, such as muscimol or GVG, drugs markedly reduce muscle tone [12,15], which could explain, at least in part the attenuation of motor seizures observed in several models, including kindling, after bilateral injection of such drugs into the SN (see above). Muscle relaxation would not explain the finding that intranigral injection of GVG in kindled rats reduces after-discharge duration [10]; however, this finding could not be confirmed by another study [8].
210 T a k e n t o g e t h e r , t h e s e d a t a m i g h t s u g g e s t t h a t , a t l e a s t in t h e k i n d l i n g m o d e l , t h e S N is less i m p o r t a n t t b r s e i z u r e i n i t i a t i o n a n d / o r p r o p a g a t i o n t h a n p r e v i o u s l y t h o u g h t . The study was supported
by grants from the Deutsche Forschungsgemeinschaft
(Lo 274/3-1). We thank Dr. Turski (Schering, Berlin, F.R.G.) for helpful discussions a n d a d v i c e . W e t h a n k M r s . C. B a t t l i n g f o r t e c h n i c a l a s s i s t a n c e . l Baraldi, M., Grandison, L. and Guidotti, A., Distribution and metabolism of muscimol in the brain and other tissues of the rat, Neuropharmacology, 18 (1979) 57 62. 2 Coyle, J.T., Excitotoxins. In H.Y. Meltzer (Ed.), Psychopharmacology: The Third Generation of Progress, Raven Press, New York, 1987, pp. 333 340. 3 Freeman, F.G. and Jarvis, M.F., The effect of interstimulation interval on the assessment and stability of kindled seizure thresholds, Brain Res. Bull., 7 (1981) 629-633. 4 Gale, K., Role of substantia nigra in GABA-mediated anticonvulsant actions. In A.V. DelgadoEscueta, A.A. Ward, D.M. Woodbury and R.J. Porter (Eds.), Basic Mechanisms of the Epilepsies Molecular and Cellular Approaches, Raven Press, New York, 1986, pp. 343 364. 5 Gale, K., Progression and generalization of seizure discharge: anatomical and neurochemical substrates, Epilepsia, 19 (Suppl. 2) (1988) S15 $34. 6 Garant, D.S. and Gale, K., Lesions of substantia nigra protect against experimentally-induced seizures, Brain Res., 273 (1983) 156 161. 7 L6scher, W. and H6nack, D., High doses of memantine (1-amino-3,5-dimethyladamantane) induce seizures in kindled but not in non-kindled rats, Naunyn-Schmiedeberg's Arch. Pharmacol., in press. 8 L6scher, W., Czuczwar, S.J., Jfickel. R. and Schwarz, M., Effect of microinjections of ~,-vinyl-GABA or isoniazid into substantia nigra on the development of amygdala kindling in rats, Exp. Neurol., 95 (1987) 622 638. 9 Mclntyre, D.C., Kindling and the piriform cortex, In J.A. Wada (Ed.), Kindling 3, Raven Press, New York, pp. 249 260, 1986. 10 McNamara, J.O., Galloway, M.T., Rigsbee, L.C. and Shin, C. Evidence implicating substantia nigra in regulation of kindled seizure threshold, J. Neurosci., 4 (1984) 2410-2417. 11 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, Sydney, 1986. 12 Schwarz, M., L6scher, W., Turski, L. and Sontag, K.-H., Disturbed GABAergic transmission in mutant Han-Wistar rats: further evidence for basal ganglia dysfunction, Brain Res., 347 (1985) 258 267. 13 Shin, C., Silver, J.M., Bonhaus, D.W. and McNamara, J.O., The role of substantia nigra in the development of kindling: pharmacologic and lesion studies, Brain Res., 412 (1987) 311 317. 14 Tanaka, T., Tanaka, S., Kaijima, M. and Yonemasu, Y., Ibotenic acid-induced nigral lesion and limbic seizure in cats, Brain Res., 498 (1989) 215-220. 15 Turski, L., Schwarz, M., Klockgether, T. and Sontag, K.H., Substantia nigra: a site of action of muscle relaxant drugs, Neurosci. Abstr., 11 (1985) 1162. 16 Turski, L., Cavalheiro, E.A., Bortolotto, Z.A., Ikonomidou-Turski, C., Kleinrok, Z. and Turski, W.A. Dopamine-sensitive anticonvulsant site in the rat striatum, J. Neurosci., 8 (1988) 4027 4037.