Brain Research, 528 (1990) 223-230 Elsevier

223

BRES 15872

Selective stimulation of kainate but not quisqualate or N M D A receptors in substantia nigra evokes limbic motor seizures Roberto Maggio, Ulla Liminga* and Karen Gale Department of Pharmacology, Georgetown University Medical Center, Washington, DC20007 (U.S.A.) (Accepted 20 March 1990)

Key words: Kainic acid; Substantia nigra; Seizure; Quisqualic acid; N-Methyl-D-aspartate receptor; L-Homocysteic acid

Bilateral microinjection of kainic acid (30-117 pmol) into the substantia nigra induced convulsive seizures resembling those elicited from limbic system structures. The convulsive seizures, which consisted of facial and forelimb clonus with rearing and falling, developed after a latency of more than 30 min and were preceded by wet dog shakes and non-convulsive seizure activity registered electroencephalographically. The convulsant effect of intranigral kainic acid was strictly dose-dependent (EDso = 60 pmol) and anatomically site-specific. Stimulation of nigral neurons by focal application of agonists for NMDA or quisqualate receptors, or by focal application of the GABA antagonist, bicuculline, was without convulsant effects. The convulsant action of intranigral kainic acid was prevented by the focal application of kynurenic acid (100 nmol) but not by 2-amino-7-phosphonoheptanoic acid (AP-7) (25 nmol) or 7-chlorokynurenic acid (20 nmol), suggesting that the convulsant effect of kainic acid in the substantia nigra does not depend upon activation of NMDA receptors in this region. INTRODUCTION Systemic or intracerebroventricular administration of kainic acid (KA) has been found to selectively induce limbic seizures 2°'22 as well as neuronal cell damage in the limbic system 3. The behavioral pattern evoked by systemic administration of K A generally consists of 4 phases 21, including staring and abnormal breathing, wet dog shakes (WDS), automatisms and mild limbic convulsions, and finally, severe limbic convulsions. Seizures induced by intracerebroventricular KA can be prevented by ~-D-glutamylaminomethylsulphonate (y-D-GAMS), an antagonist of K A receptors, but not by antagonists selective for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors 37. When high concentrations of KA are applied directly into brain regions, neurotoxicity results, often accompanied by convulsive seizure activity. However such doses of KA (1-3 nmol) induce convulsive activity with little or no anatomic site specificity. In contrast, unilateral application of pmol amount of K A directly into an epileptogenic region within the deep prepiriform cortex, 'area tempestas', can trigger bilateral convulsive seizures; this effect shows a high degree of anatomic site specificity 31. Unlike convulsions induced by intracerebroventricular KA, however, seizures induced by K A in the area tempestas are sensitive to blockade by

N M D A receptor antagonists placed at that site 32. This suggests that some other site or sites at which K A acts independently of N M D A mediated transmission must be involved in initiating convulsions in response to KA. Recently, in the course of investigating the role of excitatory amino acid receptors in the substantia nigra (SN) for influencing seizure propagation, we observed convulsive responses to the local application of pmol amounts of KA. This was an unexpected finding because, in our experience, numerous excitatory manipulations in the SN were without convulsant action. While blockade of excitatory amino acid receptors in the SN or stimulation of G A B A transmission has been shown to be anticonvulsant in many different experimental seizure models 5"8'9'11'13"15'19'23-26"34'35, neither bilateral application of G A B A antagonists 2,14,28, electrical stimulation 12' 18, nor cobalt injection into the nigra 7 provoke convulsant activity. In the present study, the convulsive effect of intranigral KA was characterized and compared with the effects of other excitatory amino acid receptor agonists acting on the N M D A and quisqualate receptor subtypes. Moreover, interactions with antagonists for excitatory amino acid receptors were examined in an effort to determine the role of N M D A and n o n - N M D A receptors for the convulsant action of K A in the SN.

* Visiting scientist from Psychiatric Research Center, University of Uppsala, S-750 17 Uppsala, Sweden. Correspondence: R. Maggio, Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, N.W., Washington, DC 20007, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

224 MATERIALS AND METHODS

Animals Experiments were performed on male Harlan Sprague-Dawley albino rats (300-350 g). The animals were housed in groups of 5 per cage under environmentally controlled conditions (12 h light/dark cycle; light on between 08.00-20.00 h), with food and water 'ad libitum'. All experiments were conducted during the light cycle, in awake freely moving animals. Each animal was tested only once.

Surgery The rats were anesthetized with Equithesin (i.p.) and placed in a Kopf stereotaxic apparatus. Two 22-gauge stainless-steel guide cannulae directed to the SN were bilaterally implanted and fixed to the skull with dental acrylic and jewelers screws. The coordinates used for the final injection site were: 5.6 mm caudal to bregma, 2.3 mm lateral to the midline and 7.8 mm ventral to the skull, with the incisor bar 3.3 mm below the interaural line according to atlas of Paxinos and Watson 3°. In 4 additional animals in which EEG recording was performed, 4 epidural electrodes were implanted at the level of the frontal and parietal cortex of both hemispheres and secured in place with dental acrylic. This implantation was performed at the same time that the cannula implantation took place.

Experimental procedures Two 28-gauge internal cannulae were inserted through the guide cannulae (the tips protruding 2 mm below the guide cannula tips) while the rat was gently hand held. The internal cannulae were connected via polyethylene tubing to two 10-~1 Hamilton syringes driven simultaneously by a Sage infusion pump. After each infusion the internal cannulae remained in place for 1 min before removal. In all experiments, the rats were observed for convulsive activity over a 2-h period following intranigral infusions of drugs. Seizure severity was scored using the following scale modified from Racine33: 0.5, jaw clonus; 1, facial myoclonus; 2, facial and mild forelimb clonus lasting at least 5 s; 3, severe forelimb clonus lasting at least 15 s; 4, rearing in addition to severe forelimb clonus; 5, rearing and falling in addition to severe forelimb clonus. For EEG recording, the epidural electrodes protruding from the skull were connected through a shielded coaxial cable to a Grass 8-channel polygraph. The EEG was continuously recorded in freely moving rats, beginning 30 min before the drug injection, and continuing after drug treatment for at least 2 h.

7-Chlorokynurenic acid (7-CLKYN) (Tocris Neuramin) was dissolved in a small volume of 1 N NaOH and diluted with water to a concentration of 23.3 mg/ml. The pH was adjusted to 7.4 with H3PO 4. A dose of 20 nmol in 0.20/~1 was infused over a period of 3 min. Kynurenic acid (KYN) (Sigma) was dissolved in a small volume of 1 N NaOH and diluted with water to a concentration of 23.6 or 94.6 mg/ml. The pH was adjusted to 7.4 with HaPO 4. Doses of 25 or 100 nmol in 0.2/~l were infused over a period of 3 min. Quisqualic acid (QA) (Cambridge Research Biochemicals) was dissolved in 0.1 N NaOH and diluted with saline to 0.5 or 1.9 mg/ml. Doses of 0.5 nmol in 0.2/A or 2.5 nmol in 0.25/~l were infused over a period of 3 min or 3 min 20 s, respectively.

Histology Animals were sacrificed by decapitation and their brains were removed and placed in 10% formalin. Brains were sectioned (50 /~m) on a freezing-stage microtome, and injection sites were verified in Nissl-stained sections. The location of the injection sites are shown in Fig. lc.

Evaluation of KA diffusion A solution of KA (117 pmol) and [3H]KA 0.64 pCi (final spec. act.: 5.47 t~Ci/mmol) in 0.25/~1 was injected into the right substantia nigra. KA (25 ng) without radioactive tracer was simultaneously injected into the left SN so that the convulsant effect could be monitored in the same rats in which diffusion was evaluated. Rats were decapitated by guillotine at 1 h after injection. This time was selected to allow for the convulsant activity to be manifest. Following the removal of the brain from the skull, a 3-mm-thick coronal section was cut, with the rostral and caudal ends of SN defining the rostral and caudal limits of the slice. The slice was then cut into several pieces (as shown in Fig, 2a,b). The pieces were then homogenized in 0.5 ml of water, placed in vials containing 7 ml of Safety-Solve cocktail (Research Products International Corp.), and counted in an automatic liquid scintillation counter. Other regions of the brain adjacent to but not included in the coronal slice were also assayed for radioactivity, but as those areas were not yielding counts above background they were omitted from analysis.

Statistics The EDso was estimated by fitting the data by linear regression analysis of the probit transformed percentage vs the log dose of KA. For statistical analysis, the Mann-Whitney U-test for non-parametric data was used.

Drugs a-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) (Tocris Neuramin) was dissolved in a small volume of 0.5 N NaOH and diluted with saline to 0.5 mg/ml or 1.0 mg/ml. The pH was adjusted to 7.4 with 0.1 N HCI. Doses of 0.55 or 1.1 nmol in 0.2/~1 were infused over a period of 3 min. 2-Amino-7-phosphonoheptanoic acid (AP-7) (Cambridge Research Biochemicals) was dissolved in a small volume of 1 N NaOH, diluted with water to a concentration of 11.3 mg/ml and the pH was adjusted to 7.4 with H3PO 4. A dose of 25 nmol of AP-7 in 0.5/A was infused over a period of 7 min. Bicuculline methiodide (BIC) (Sigma) was dissolved in saline at a concentration of 0.2 mg/ml or 0.8 mg/ml and 98 pmol/0.25/11 or 393 pmol/0.25/~1 were infused over a period of 3 min 20 s. L-Homocysteic acid (HCA) (Sigma) was dissolved in water at a concentration of 76.3 or 146.5 mg/ml. Doses of 50 nmol/0.12 /d, 100 nmol/0.24/~1, 200 nmol/0.48/~1 or 400 nmol/0.5 /~1 were infused during 3 min, 3 rain 20 s, 6 min 45 s or 7 min, respectively. KA (Sigma) was dissolved in saline at the following concentrations: 12, 20, 30, 40, 60, 80 and 100/~g/ml. Doses of 14, 23, 35, 47, 70, 94 or 117 pmol in a volume of 0.25/~1 were infused into each nigra over a period of 3 min 20 s. NMDA (Sigma) was dissolved in water (with sufficient NaOH to adjust the pH to 7.4) at a concentration of 6.1 mg/ml. Doses of 10 nmol in 0.24/~1 were infused over a period of 3 min 20 s.

RESULTS T h e b i l a t e r a l i n t r a n i g r a l a d m i n i s t r a t i o n o f K A (117 pmol)

resulted

in s t r o n g

response

to i n t r a n i g r a l

inactivity

with

motor

KA

increased

seizures.

consisted

respiration.

l a t e n c y o f 24 + 2 m i n (n =

The

initial

o f staring After

a

and mean

34), an i s o l a t e d W D S

a p p e a r e d , f o l l o w e d by a d d i t i o n a l W D S a f t e r a n o t h e r 10 min. D u r i n g t h e p e r i o d b e t w e e n 35 and 50 m i n f o l l o w i n g i n j e c t i o n , W D S i n c r e a s e d in f r e q u e n c y . Signs o f c o n v u l sive activity s t a r t e d at a b o u t 40 m i n ( r a n g e 3 0 - 6 0 m i n ) with j a w and facial c l o n u s (score 0 . 5 - 1 ) , t o g e t h e r with i n c r e a s e d l o c o m o t o r activity. S e v e r e clonic c o n v u l s i o n s c h a r a c t e r i z e d by r e a r i n g , f o r e l i m b a n d facial c l o n u s a n d falling, o c c u r r e d a f t e r a m e a n l a t e n c y o f 55 + 3 rain (n = 32) and w e r e r e c u r r e n t o v e r at least a 4 5 - m i n p e r i o d . T h e r e was a p o s i t i v e c o r r e l a t i o n b e t w e e n t h e d o s e o f K A and i n c i d e n c e o f c o n v u l s i o n s as s h o w n in Fig. 1. T h e

225 convulsant ED5o was found to be 60 pmol (Fig. 1). No significant differences between doses were found for latency to onset of convulsive effect or the convulsive profile. The location of the injections is indicated in Fig. 2c. Bilateral infusions of K A (117 pmol) 1 m m dorsal to the nigral injection site (which resulted in placement outside of the SN) did not induce convulsive activity in 9 out of 10 rats tested (see Fig. 2c). Bilateral infusions of K A (117 pmol) into the ventral region of the SN (1.0 mm below the site used to generate the data in Fig. 1) induced convulsions that occurred after a latency (mean = 124 _+ 20 min; range 97-165 min; n = 5) that was considerably longer than the latency for those elicited from the dorsal SN (see Fig. 2c). The diffusion pattern of K A was evaluated using [3H]KA tracer unilaterally in 3 rats receiving bilateral nigral K A injections and this was compared to the diffusion pattern after injections placed 1 mm dorsal to SN in another group of 3 rats. All 3 rats receiving nigral injections exhibited convulsive activity, while none of the rats with dorsal injections exhibited convulsive signs. Fig. 2a,b shows the percent of total injected radioactivity in each of 4 pieces of tissue adjacent to the injection site and cannula tract, as well as in the hemisphere contralateral to the radiolabeled injection, in a 3-mm-thick coronal section at 1 h after injection. The highest amount of radioactivity was measured in the tissue piece containing the injection site. The next highest region in terms of radioactivity was located immediately dorsal to (in the case of nigral injections) or ventral and dorsal to (in the case of injections above SN) the injected region; these areas always contained less than half as many counts as the region of injection. Negligible levels of radioactivity were detected at distances greater than 1 m m lateral, or

a

I ',

Inleraural 3.7 mm

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b

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Inleraural 3.7 ram

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Fig. 2. Coronal sections of rat brain showing diffusion pattern of [3H]KA following mieroinjection into the SN (a), or i mm dorsal to

80

§

7o

p~ ~ ~

6O 5O aO

~

?D

g

213 10 0

14

23

35 4 7

94

117

pmol

DOSE (log scale)

Fig. 1. Dose-response function for the convulsant effect of bilateral intranigral KA. n = 5, 16, 10, 9, 12, 37 rats tested, respectively, for each dose shown. Percent of rats exhibiting convulsions equivalent to score 3, 4 or 5 is indicated on the Y axis.

the SN (b). All injections of [3H]KA were placed on right side and unlabeled KA was injected on left side. The data in a represents the mean + S.E.M. of 3 rats, all showing convulsions; the data in b represents the mean + S.E.M. of 3 rats, none of which exhibited convulsions. Numbers shown correspond to the amount of total radioactive counts recovered from the tissue piece, expressed as a percent of total radioactive counts injected. Protein content of the tissue pieces within the subcortical regions was between 1.5 and 1.7 mg; protein content of the ventral piece of cortex (containing hippocampus and entorhinal cortex) was between 4.0 and 5.0 mg, and the dorsal piece of cortex was between 3.0 and 4.0 mg. The underlined value represents the amount recovered from the tissue piece containing the injection site. * Significantly greater than the value obtained in the corresponding piece of tissue in b. In c the dotted area delineates the region containing injection sites from which KA elicited bilaterally generalized convulsions, striped areas delineate the regions containing injection sites ineffective for eliciting convulsions.

226 a

TABLE I

LF-RF , , . ~ , ~ . ~ ~ k - x ~ j

Incidence of convulsions induced by excitatory amino acid agonists and a GA BA antagonist microinjected into SN

LP-RP ~,,,,r~.~¢%,.,',~'$, , , , ~ , - ~ , , . ~ e ' ~ ? ' ~ , ~ w ~ , ~ ' ~ ~ RF-RP

~,~'%',~,"~%"~,,v"~,'¢'~%",\~',~f,:C,~.~,~.",',~m,~'~'~,,~',,~ b i

I

t

'i,

'

Drug (intranigral)

Dose (nmol)

Incidence of clonic seizures*

Kainic acid

0.035 0.117 50 100 200 400 10 0.5 2.5 0.55 1.1 0.098 0.393

2/10 32/37 0/4 0/2 0/2 0/5 0/5 0/3 0/6 0/2 0/6 0/6 0/6

L-Homocysteic acid

LF-RF LP-RP RF-RP

N-Methyl-D-aspartate Quisqualic acid

LF-LP

AMPA

C

Bicuculline methiodide

* Scores 3, 4 or 5; denominator equals number of rats tested in each group.

o. zsec

Fig. 3. EEG recording from a representative rat (a) before, (b) 20 min after, and (c) 40 min after KA (117 pmoi) bilaterally in the SN. Note the similarity between the pattern in b during which no behavioral convulsions occurred and c during which score 5 clonic convulsions were present. Leads: L, left; R, right; F, frontal cortex; P, parietal cortex.

greater than 2 mm dorsal, medial, rostral or caudal to the injection site in the SN. When the pattern of radioactivity following intranigral injections was compared with that following the injection dorsal to the SN, the only region showing a significantly greater content of isotope was the region of the SN as indicated by the asterisk in Fig. 2a. The injection dorsal to SN (which did not cause convul-

sions) resulted in a significantly greater amount of isotope in the injected region as well as in the regions medial and lateral to this region when compared with nigral injections (Fig. 2b). To e v a l u a t e the c o n v u l s a n t effect of u n i l a t e r a l K A , we selected a dose of K A a b o v e t h e EDso for p r o d u c i n g c o n v u l s i o n s following b i l a t e r a l i n t r a n i g r a l i n j e c t i o n . U n i lateral i n f u s i o n s of 70 p m o l K A i n t o the left o r right SN i n d u c e d W D S in two of the 4 a n i m a l s tested b u t n o a n i m a l s e x h i b i t e d a n y signs of c o n v u l s i v e activity. A l l a n i m a l s s h o w e d t u r n i n g ipsiversive to the t r e a t e d side. A f t e r a d m i n i s t r a t i o n of the highest dose of K A (117 p m o l ) u n i l a t e r a l l y , o n l y two o u t of eight rats e x h i b i t e d signs of c o n v u l s i v e activity. E E G studies r e v e a l e d that the first signs of modification of the p a t t e r n o c c u r r e d 2 0 - 2 5 m i n after a d m i n i s t r a -

TABLE II

Interactions between excitatory amino acid receptor antagonists and KA (117pmol) in SN In all experiments the drugs were infused 20 min after kainic acid infusions, except for 2a where the quisqualic acid was infused 5 min before kainic acid. No significant differences were found between groups on measures of latency or frequency.

lntranigral drug treatments (nmol)

Incidence of clonic convulsion*

Mean convulsion score

Latency of clonic convulsion (rain)*

Frequency of convulsion**

(1) (2a) (2b) (2c) (3a) (3b) (4) (5)

17/19 5/6 5/5 5/5 4/5 0/6 4/5 5/5

4.2 4.2 4.6 4.6 3.6 0. 3.4 4.4

50 + 3 65 + 15 48 + 5 55 + 5 52 _+ 12 . . 50 + 6 41 _+7

5.8 + 0.4 5.0 + 0.6 4.4 + 0.7 5.6 _+0.5 5.0 + 0.7

KA QA (0.5) KA KA KA KA KA KA

Saline (0.2/~!) KA QA (0.5) QA (2.5) KYN (25) KYN (100) AP-7 (25) 7-CLKYN (20)

.

.

* Score 3, 4, or 5; denominator equals the number of rats tested in each group. ** The frequency represents the number of seizure episodes occurring during the 45 min following the first clonic seizure. *** Significantly different from group 1 (control with saline): P < 0.01.

5.0 + 0.4 5.0 + 0.3

227 tion of KA (117 pmol) into the SN. At this time, continuous high-voltage spiking was present in all leads (see Fig. 3b). This activity was not associated with behavioral convulsions. Behavioral clonic convulsions started at 40 rain after KA, and were accompanied by E E G seizure activity as shown in Fig. 3c. The latter E E G pattern was similar to that observed in the absence of convulsive behavior (compare Fig. 3b and c). Once convulsive activity started, all convulsive episodes were associated with E E G seizure activity. KYN, an antagonist of NMDA and non-NMDA (KA and QA) receptors, was examined for its effect on the convulsions induced by intranigral KA. Infusion of 100 nmol of KYN into SN 20 min after KA (117 pmol) resulted in a complete blockade of the convulsive effects of KA in all animals (Table II). A lower dose of KYN (25 nmol) did not significantly change the incidence, frequency or latency of KA-induced convulsions. Sniffing and intermittent gnawing behavior similar to that previously observed after intranigral muscimo115 were observed following both doses of KYN in the SN. Bilateral intranigral infusions of NMDA (10 nmol) or the NMDA receptor agonist, HCA (50, 100, 200 or 400 nmol), the QA receptor agonists AMPA (0.55 or 1.1 nmol) and QA (0.5 or 2.5 nmol), and the G A B A A receptor antagonist, BIC (98 or 393 pmol), were all ineffective for inducing convulsive activity (Table I). Moreover, no signs of WDS or myoclonic activity were evident following these treatments. Reduction of locomotor activity was observed following all of these intranigral treatments, and vacuous chewing movements, as previously described after intranigral bicuculline TM occurred during the initial 30 min following injection. These chewing movements could only be observed in a completely quiet environment, because even the slightest sounds or movements suppressed their occurrence. In order to determine whether NMDA receptor activation is necessary for the convulsive effect of KA in the SN, the selective NMDA receptor antagonist, AP-7 (25 nmol), was applied into SN 20 min after KA, prior to the onset of intranigral KA-induced convulsions. This treatment did not alter either the incidence, severity, latency to onset, or frequency of convulsive activity induced by KA (Table II). In addition, a relatively selective and high potency antagonist of the NMDAassociated glycine site, 7-CLKYN 17, did not attenuate the response to KA (Table II). Stereotyped sniffing and intermittent gnawing behavior occurred within a few minutes after injection of AP-7 or 7-CLKYN and lasted for more than 60 min. Finally, the effect of QA on the response to KA was examined in view of several lines of evidence indicating that Q A can exert an inhibitory action on KA receptor

function 1°'16'27'29'38. QA was given in a dose (0.5 nmol), previously found effective for preventing convulsions induced by KA (117 pmol) in area tempestas 4°. As shown in Table II, when QA was given either 5 min prior to KA or 20 min following KA (and prior to seizure onset), no reduction in either incidence, severity, latency to onset, or frequency of convulsions was obtained. In addition, because QA alone did not exert convulsant actions, we were able to examine the effect of a high dose of QA in combination with KA. No change in the convulsant effect of intranigral KA occurred in the presence of the higher dose (2.5 nmol) of QA (Table II). DISCUSSION Our study has demonstrated that bilateral application of low doses of KA directly into the SN induces convulsive seizures that resemble those evoked from limbic structures. Furthermore, the profile of convulsive responses were limited to this type of seizure (facial and forelimb clonus with rearing and falling) and in no cases did other types of convulsions, such as explosive runningbouncing clonus or tonic extension of the limbs, occur. The convulsive effect of intranigral KA showed a strict dose-dependency and restricted anatomical site specificity. Moreover, it appears that bilateral KA stimulation in the SN was required to obtain consistent responses in the dose range studied. The occurrence of WDS was a highly consistent feature of the response to bilateral intranigral KA. This response appeared to be evoked by the lowest doses of KA tested, even in the absence of any signs of convulsant effects. WDS always occurred with a shorter latency of onset than that required for convulsive behavior. This response profile is remarkably similar to that observed following systemic KA administration which also induces WDS at shorter latencies and lower doses than required for convulsive activity21. It was clear from our E E G studies that the convulsive activity produced by intranigral KA was associated with pronounced electrographic seizure activity. Interestingly, seizure activity in the E E G consistently appeared 15-20 min prior to the first convulsive seizures. The electrographic seizures were not apparently different in the presence or absence of behavioral convulsions. This raises the possibility that intranigral KA may exert a biphasic action on behavioral convulsions: an early antagonism which may wear off to reveal the convulsant action. It is not clear why there is at least a 20-min latency following intranigral KA before the onset of electroencephalographic seizure or convulsive activity. It is unlikely that it is due to drug diffusion to a site distant to

228 SN because placement of KA as little as 1 mm away from the SN had no convulsant action. Moreover, our studies of [3H]KA diffusion demonstrate that the region in which significantly more [3H]KA is found following effective (convulsant) injections in the SN as compared with ineffective injections (dorsal to the SN), corresponds only to the ventral tegmental area containing the SN. In addition, injections placed medial to the SN were less effective than those within the SN with respect to inducing convulsions (see Fig. 2c), ruling out diffusion to this region as responsible for the effect we have obtained from the SN. Thus, it appears that the SN is in fact the site of action for KA in terms of its convulsant effect following microinjections into this region. The latency to onset of convulsions following intranigral KA also does not appear to be related to the dose of KA, as there was no significant relationship between dose and latency to convulsive effect. The long latency in this situation stands in sharp contrast to the rapid (within 5 min) onset of convulsive action of KA (117 pmol) applied to area tempestas 31, and the immediate induction of ipsiversive turning behavior upon unilateral KA application in the SN. It is probable that the seizure activity evoked by intranigral KA does not originate from the SN itself; instead KA in the SN may indirectly produce alterations in excitability of limbic circuitry which in turn allows seizures to be initiated from the forebrain. The latency to seizure onset could therefore reflect the time required for limbic excitability to reach a level permissive for evoking seizures. Clearly, additional studies will be needed in order to determine the mechanism(s) responsible for the observed time course. It is likewise difficult to explain our finding that KA alone and no other agonist for excitatory amino acid receptors was capable of evoking WDS and convulsive seizures after intranigral administration. Moreover, in agreement with previous observations 14"28, blockade of G A B A inhibition in the SN was not effective for inducing WDS or any signs of convulsive response. The lack of convulsant effect of GABA blockade or NMDA receptor stimulation in the SN is not a result of a lack of activation of nigral outputs, since these treatments are quite effective for inducing behavioral signs of stimulation of nigral outputs, such as ipsiversive turning behavior with unilateral application 1,28. It would appear that under our experimental conditions, KA is uniquely capable of affecting nigral transmission in a fashion that evokes convulsions resembling those following activation of limbic circuits. Our results with the NMDA receptor antagonist, AP-7, indicate that stimulation of NMDA receptors in the SN does not contribute in any significant way to the convulsant action of intranigral KA. This is further

supported by the finding that 7-CLKYN, an agent that is a relatively potent antagonist of the NMDA-associated glycine site 17, was ineffective for protecting against convulsions evoked by intranigral KA. In contrast, intranigral KYN, a broad spectrum excitatory amino acid receptor antagonist known to block both NMDA and non-NMDA receptors 4, albeit with relatively low potency, completely prevented the WDS and the convulsions induced by intranigral KA. These observations support the conclusion that in the SN, stimulation of KA receptors, but not QA or NMDA receptors, is sufficient for inducing limbic-type convulsions, and that only non-NMDA receptors are necessary for this action. The pharmacologic profile described above for the intranigral treatments stands in marked contrast to that previously reported for another convulsant site of action of KA, the area tempestas. In the area tempestas, unilateral application of HCA (150 nmol), AMPA (500 pmol), QA (2.5 nmol) and BIC (50 pmol) are effective for eliciting full convulsive responses, whereas the same and much higher doses of these drugs bilaterally in the SN were without convulsant action. The dose of KA (117 pmol) associated with 90% incidence of convulsions is the same in area tempestas 31 and in the SN. The convulsant action of this dose in area tempestas is completely antagonized by AP-7 (100 pmol) 32 while in the SN, it is resistant to AP-7 even when this antagonist is given in a dose 250 times higher (i.e. 25 nmol). Similarly, in the area tempestas, 7-CLKYN at a dose of 2.5 nmol blocks convulsions induced by KA 38 whereas in the SN, 20 nmol of this drug was without effect on convulsions induced by KA. The ability of the NMDA antagonists to block KA actions in area tempestas is consistent with an action of KA to stimulate the release of endogenous glutamate (or aspartate) via a presynaptic action 3z which then activates postsynaptic NMDA receptors. Thus, while KA in the area tempestas induces convulsions, probably via presynaptic KA receptors and in a strictly NMDA-receptor dependent fashion, KA in the SN appears to work directly via postsynaptic KA receptors, independent of any NMDA receptor-mediated activity within the SN. The different mechanisms of action for KA in the SN vs the area tempestas may underlie the contrasting effects of QA in the two systems. In the area tempestas, a subconvulsant dose of QA (0.5 nmol) prevented the convulsant action of KA (117 pmol) 4°. This ability of QA to antagonize the actions of KA has been characterized in in vitro preparations and may be due to an allosteric negative modulation of the KA receptor by QA 27'39. The fact that QA did not alter the convulsant action of KA in the SN, raises the possibility that the negative modulation by QA may be selectively associated with presynaptic KA receptors (as are operative in area tempestas), but not

229 with the postsynaptic K A receptors that mediate the convulsive response elicited from the SN. The sensitivity to the convulsant action of K A in the SN appears to vary dramatically across different strains of rats. Turski et al. 36 administered K A to Wistar rats in the same doses that we have used and higher (23-234 pmol) and found no signs of convulsive activity. Instead, they report increased muscle tone and marked catalepsy. In pilot studies with Wistar rats, we have been able to confirm their findings and, in addition, we found that still higher doses of intranigral K A (at and above 300 pmol) were required to elicit convulsive activity in this strain. On the other hand, we have observed little or no cataleptic responses in the S p r a g u e - D a w l e y rats that we have used in our studies (including those with injections of K A in the ventral SN), even at doses below those required for inducing convulsions. Curiously, the Wistar rat appears to respond to intranigral N M D A by exhibiting limbic m o t o r seizures 35. It will be interesting in future studies to determine the neural mechanisms in the SN that account for this strain-related differential neurological response to focal K A and N M D A . Finally, we can only speculate on the relationship between the convulsions evoked by application of K A into the SN and those evoked in response to systemic

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Acknowledgements. This work was supported by HHS Grants NS20576 and NS28130, and a Research Scientist Development Award, MH00497 (K.G.). The authors wish to thank Eugenia Sohn for technical assistance in the preparation of histological samples, and Dr. Marino Massotti for his help in the interpretation of the EEG recordings.

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