Intrastriatal N-methyl-d-aspartate prevents amygdala kindled seizures in rats

Brain Research, 377 (1986) 173-176 Elsevier

173

BRE 21627

Intrastriatal N-methyI-D-aspartate prevents amygdala kindled seizures in rats ESPER A. CAVALHEIRO1and LECHOSLAW TURSKI2 1Department of Neurology and Neurosurgery, Laboratory of Experimental Neurology, Escola Paulista de Medicina, BR-04023 Sao Paulo (Brazil) and 2Max-Planck-lnstitute for Experimental Medicine, D-3400 G6ttingen (F. R. G.)

(Accepted March 4th, 1986) Key words: epilepsy - - N-methyl-D-aspartate- - striatum - - kindling

Microinfusion of an excitatory amino acid, N-methyl-D-aspartate, into the ventral mid-striatum in the rat protects from amygdala kindled seizures. This result demonstrates that excitatory activity in the striatum modulates the threshold for seizures in the limbic forebrain.

A basic tenet of modern epileptology is that specific morphologic substrate underlies the mechanism of motor convulsions 2,6,1°,12. The basal ganglia are part of a neuronal network whose participation is necessary for the expression of motor seizures 3. Recent studies in rats have revealed that the substantia nigra is essential in the spread of seizures 6'9'14'16. The substantia nigra has been proposed as a critical site at which anticonvulsant activity related to 7-aminobutyrate (GABA)-mediated inhibition may be mediated 6. G A B A is believed to subserve the neurotransmitter function in the striatal efferent pathways directed to the substantia nigra and entopeduncular nucleus 4. We have established that microinfusions of an excitatory amino acid, N-methyl-D-aspartate (NMDA), into the ventral mid-striatum prevents rats from seizures produced by pilocarpine 15. To test the validity of this finding we decided to investigate whether NMDA, when given into the ventral mid-striatum, blocks amygdala kindled seizures in rats. Male Wistar rats, 250-300 g in weight, were stereotaxically implanted with a bipolar electrode aimed at the right amygdala (AP 4.6: L +4.2; V -3.6) 7 under pentobarbitone anesthesia (Nembutal; Ceva, Neuilly-sur-Seine, France; 50 mg/kg i.p.). The bipolar electrodes were constructed of twisted ni-

chrome wire, 100/~m in diameter, and insulated with Teflon except at the cut tips. For microinjections into the striatum bilateral guide cannulae (21 gauge stainless-steel) were chronically implanted. After surgery animals were housed individually and were allowed to recover for a period of 5 - 7 days. Coordinates for microinjections were derived from the atlas of K6nig and Klippel7: AP 7.9; L _+2.4; V -1.0. The drugs were delivered in a volume of 0.5 ~tl at a rate of 0.1/A/min. Electrical stimulation consisted of a 2 s train of 60 Hz biphasic square waves each 1 ms in duration and was delivered daily to the amygdala from a DC stimulator through a constant current output. The electroencephalogram (EEG) was recorded before and after each stimulation between the tips of the bipolar electrode. The measure of the seizure activity was (a) an assessment of behavioral severity of convulsions according to the ranking scale defined by Racine13: (0) no behavioral response to amygdala stimulation; (1) facial clonus; (2) head nodding; (3) forelimb clonus; (4) rearing; and (5) rearing and falling and (b) a measurement of the after-discharge duration. The rats were considered kindled when 3 consecutive stage 5 seizures were observed after stimulation of the amygdala. Upon completion of the experiment, the rats were

Correspondence: L. Turski, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Str. 3, D-3400 G6ttingen, F.R.G.

0006-8993/86/$03.50 ~) 1986 Elsevier Science Publishers B .V. (Biomedical Division)

174 sacrificed and their brains were prepared for histological verification of electrode and cannulae tip placement. N-methyI-D-aspartic acid (Tocris Chemicals, Buckburst Hill, Essex, U.K.) was dissolved in saline and administered bilaterally into the striatum of fully kindled rats in the dose of 0.002 Hmol 15 min prior to the amygdala stimulation. This dose of NMDA blocked seizures produced in rats by pilocarpine, 380 mg/kg, when given into the ventral mid-striatum 15. Five rats received NMDA into the ventral mid-striatum and 15 min later were electrically stimulated at an individually predetermined threshold current. On the following day, the animals received intrastriatal injection of saline and subsequently electrical stimulation of the amygdala. Five reference rats received intrastriatal microinjection of saline 15 min prior to the amygdala stimulation. Twenty-four h later the same rats underwent bilateral intrastriatal microinjection of NMDA followed by amygdala stimulation. The mean after-discharge threshold was determined as 90.3 + 12.8#A (n = 10), while the number of stimulations necessary to evoke a stage 5 motor seizure was 16.5 + 3.5 (n = 10). The mean after-discharge duration in fully kindled rats was 96.6 + 7.5 s

VENTRALSTRIATUM(A7890) 51

-

]-

-

[SCONTROL

~4

~SALINE mNMOA

C

1

2

L 1J C

2

Fig. 1. Effect of intrastriatal microinjection of N-methyl-o-aspartate (NMDA) or saline on seizure severity in amygdala kindled rats. NMDA, 0.002 Mmol, was microinjected into the ventral mid-striatum (AP 7890) 15 min prior to the amygdala stimulation. Two groups of 5 rats were used for the experiments shown in the graph. Open bars represent the control response (C); darkened bars represent the response after intrastriatal application of NMDA, while hatched bars indicate the response after microinjection of saline into the striatum. The graph data represent means _+S.E.M. ***P < 0.001 vs control (Student's t-test).

(n = 10). Fig. 1 shows that bilateral microinjection of NMDA, 0.002 Hmol, into the ventral mid-striatum blocks motor seizures in amygdala kindled rats. E E G recordings revealed that the intrastriatal injection of NMDA and not that of saline blocked after-discharges evoked by amygdala stimulation (Fig. 2). The mean after-discharge duration in rats pretreated with NMDA in the ventral mid-striatum was 7.5 +_ 3.7 s (n -- 10) (P < 0.001 vs saline treated rats, Student's t-test). Microinjection of saline into the striatum of animals treated with N M D A (24 h) did not affect the seizure severity and after-discharge duration (Fig. 2). Reversal of the experimental protocol in reference rats (n -- 5) (Fig. 1) revealed no effect of bilateral in-. trastriatal microinjections of saline on amygdala kindling. Subsequent (after 24 h) treatment with NMDA, 0.002 Hmol, prevented the development of motor seizures and blocked after-discharges evoked by amygdala stimulation (Fig. 1). Histological examination revealed the distribution of electrode tips in the basolateral regions of the amygdala. The examination of injection sites of NMDA in the striatum showed the cannula tip to be located in the ventral part of mid-striatum (AP 74707890) (ref. 7). The studies presented here represent an initial attempt to characterize the function of excitatory amino acid transmission in the striatum in modulating the seizure threshold of the limbic forebrain. There is general agreement that abnormal activity in the striaturn can play a critical role in the pathogenesis of movement disorders. Although there is considerable evidence on the relation of basal ganglia to epilepsy3'6'9, very little is known (a) of the function of basal ganglia in the spread of seizures, (b) of morphological requirements necessary for propagation of seizures, (c) of changes in neurotransmitters and (d) of electrophysiological properties of neuronal networks engaged in spreading and control of the threshold for seizures. The present study represents the first evidence of an anticonvulsant effect related to enhanced excitation in the striatum in the kindling model of epilepsy. NMDA is a neurotoxin and a potent convulsant when injected into the rat hippocampus, amygdala, cortex or cerebral ventricles TM. In contrast, microinjections of NMDA into the striatum prevent amygdala

175 AMG

.,,,,,1~ r,,,I a , i t I. I~tt l J, ......

• p m . . . . . . . . ~"tIl'T[Irt""

~1

~

q

. . . . .

,

tl

I

I

I

Jl

'

t

cx-

t

......

,,50ov

5s

Fig. 2. Effect of intrastriatal microinjection of N-methyl-D-aspartate (NMDA) or saline on after-discharge duration in amygdala kindled rats. Upper trace: the EEG record registered after amygdala stimulation 15 min following microinjection of NMDA, 0.002 gmol, into the ventral mid-striatum (AP 7890) in a fully kindled rat. Lower trace: typical after-discharge registered after amygdala stimulation 15 min following microinjection of saline into the ventral mid-striatum and 24 h after microinjection of NMDA. Arrows indicate stimulus artifacts. AMG, amygdala; CX, neocortex.

kindled seizures in rats. The anticonvulsant effect of NMDA in the striatum is topographically specific and confined to ventral mid-striatum 15. This is in agreement with the striatal topography of anticonvulsant action of NMDA against seizures produced by pilocarpine ~5. These results are consistent with electrical stimulation studies which suggest a role for the caudate nucleus in the control of the seizure threshold in the amygdala and hippocampus 1"8. The topographic specificity of the action of NMDA in the striatum forms an interesting parallel to anatomical findings on distinct topographical organization of the striatal output-input compartments 5. Focal injection

studies show that the balance of inhibitory and excitatory activity within the two output relay nuclei of the striatum, the substantia nigra and the entopeduncular nucleus, modulates the limbic seizure threshold 11AaA6. The results reported here imply that either the excitatory corticostriatal input or intrinsic excitatory activity in the striatum participate in a neuronal network regulating the seizure threshold of the limbic forebrain.

1 Amato, G., Crescimanno, G., Sorbera, F. and La Grutta, V., Relationship between the striatal system and amygdaloid paroxysmal activity, Exp. Neurol., 77 (1982) 492-504. 2 Collins, R.C. and Olney, J.W., Focal cortical seizures cause distant thalamic lesions, Science, 218 (1982) 177-179. 3 Engel, J., Kuhl, D.E. and Phelps, M.E., Patterns of human local cerebral glucose metabolism during epileptic seizures, Science, 218 (1982) 64-66. 4 Fonnum, F., Gottesfeld, Z. and Grofova, I., Distribution of glutamate decarboxylase, choline acetyltransferase and aromatic amino acid decarboxylase in the basal ganglia of normal and operated rats. Evidence for striatopallidal, striatoentopeduncular and striatonigral GABAergic fibers, Brain Research, 143 (1978) 125-138. 5 Gerfen, C.R., The neostriatal mosaic: compartmentaliza-

tion of corticostriatal input and striatal output systems, Nature (London), 311 (1984) 461-464. Iadarola, M.J. and Gale, K., Substantia nigra: site of anticonvulsant activity mediated by 7-aminobutyric acid, Science, 218 (1982) 1237-1240. K6nig, J.F.R. and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. La Grutta, V., Sabatino, M., Gravante, G. and La Grutta, G., Effects of caudate nucleus on paroxysmal activity in hippocampus of cat, Electroencephalogr. Clin. Neurophysiol., 61 (1985) 416-421. 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.

This work was supported by CNPq and FAPESP of Brazil (E.A.C.).

6

7 8

9

176 10 Mirski, M.A. and Ferrendelli, J.A., Interruption of the mammillothalamic tract prevents seizures in guinea pigs, Science, 226 (1984) 72-74. 11 Patel, S., Millan, M.H., Mello, L.M. and Meldrum, B.S., 2-Amino-7-phosphonoheptanoic acid (2-APH) infusion into entopeduncular nucleus protects against limbic seizures in rats, Neurosci. Lett., 64 (1986) 226-230. 12 Piredda, S. and Gale, K., A crucial epileptogenic site in the deep prepiriform cortex, Nature (London), 317 (1985) 623-625. 13 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 14 Turski, L., Cavalheiro, E.A., Schwarz, M., Turski, W.A., de Moraes Mello, L.E.A., Bortolotto, Z.A., Klockgether, T. and Sontag, K.-H., Susceptibility to seizures produced by pilocarpine in rats after microinjection of isoniazid or 7-

vinyl-GABA into the substantia nigra, Brain Research, 372 (1986) 294-309. 15 Turski, L., Cavalheiro, E.A., Turski, W.A. and Meldrum, B.S., Anticonvulsant action of excitatory amino acid in the rat striatum, Epilepsia, 26 (1985) 507. 16 Turski, L., Cavalheiro, E.A., Turski, W.A. and Meldrum, B.S., Excitatory neurotransmission within substantia nigra pars reticulata regulates threshold for seizures produced by pilocarpine in rats: effects of intranigral 2-amino-7-phosphonoheptanoate and N-methyl-o-aspartate, Neuroscience, 18 (1986) 61-77. 17 Turski, L., Meldrum, B.S. and Collins, J.F., Anticonvulsant action of fl-kainic acid in mice. Is fl-kainic acid an Nmethyl-o-aspartate antagonist?, Brain Research, 336 (1985) 162-166. 18 Zaczek, R. and Coyle, J.T., Excitatory amino acid analogues: neurotoxicity and seizures, Neuropharmacology, 21 (1982) 15-26.