Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures

Developmental Brain Research, 65 (1992) 227-236

227

1992 Elsevier Science Publishers B.V. BRESD 51408

Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures C a r l E . S t a f s t r o m , J a m e s L. T h o m p s o n a n d G r e g o r y L. H o l m e s Department of Neurology, The Children's Hospital, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 22 October 1991)

Key words: Developing brain; Flurothyl; Kainic acid; Seizure; Spontaneous recurrent seizure; Status epilepticus

Acute and chronic effects of seizures induced by intraperitoneal (i.p.) injection of kainic acid (KA) were studied in developing rats (postnatal days (P) 5, 10, 20, 30, and adult 60). For 3 months following KA-induced status epilepticus, spontaneous recurrent seizure (SRS) occurrence was quantified using intermittent video monitoring. Latency to generalized seizures was then tested using flurothyl, and brains were histologically analyzed for CA3 lesions. In P5-10 rats, KA caused generalized tonic-clonic ('swimming') seizures. SRS did not develop, and there was no significant difference between control and KA-treated rats in latency to flurothyl-induced seizures. In contrast, rats P20 and older exhibited limbie automatisms followed by fimbic motor seizures which secondarily generalized. Incidence and frequency of SRS increased with age. P20--30 rats with SRS had shorter latencies to flurothyl seizures than did KA-treated P20-30 rats without SRS or controls. KA-treated P60 rats (with or without SRS) had shorter latencies than controls to flurothyl seizure onset. SRS in P60 rats occurred sooner after KA than in P20-30 rats. CA3 lesions were seen in P20-60 rats with and without SRS, but not in P5-10 rats. These data suggest that there are developmental differences in both acute and chronic responses to KA, with immature animals relatively protected from the long-

term deleteriouseffectsof this convulsant.

INTRODUCTION

Kainic acid (KA), an analog of the excitatory amino acid (EAA) transmitter glutamate, is a potent convulsant and neurotoxin. When administered systemically or intracerebrally to adult rats, KA produces a characteristic seizure-brain damage syI~drome consisting of progressive limbic seizures (culminating in status epilepticus (SE)) and neuronal necrosis in limbic structures (especially hippocampal CA3 and dentate hilus)23'24. KA seizures are of considerable interest since the ictal manifestations and subsequent pattern of neuronal damage are similar to those of human temporal lobe epilepsy3'23. The tendency of KA-treated animals to develop spontaneous recurrent seizures (SRS) weeks to months following SE supports its relevance as a model of chronic epilepsy7'8'29'4°. Studies of SRS in developing animals have not yet appeared. Behavioral and electrographic characteristics of KAinduced seizures have been described in both adult2°'z3 and immature rats l'4,ga°'~6,28'3s'43. Although KA causes SE at all ages, clinical features of KA seizures vary with age. Before the end of the third week of life, KA-induced behavioral changes consist of immobility and

ataxia, then repetitive scratching and finally generalized clonic or tonic/clonic seizures; at this age, KA does not produce significant neuronal damage1't6'26'3~. However, beginning at about 18 days of age, KA causes 'limbic motor seizures' with staring and wet-dog shakes (WDS) followed by automatisms (blinking, chewing, head bobbing), clonus of one or more extremities, and eventual progression to continuous clonus of all limbs accompanied by rearing and falling. After the third week of life, systemic KA leads to widespread neuronal damage with predilection for limbic structures (especially CA3)23'24. These age-dependent variations in response to KA have important implications for seizure ontogenesis. In order to more fully describe the age-dependent features of KA-induced SE and its sequelae, we administered KA intraperitoneally to rats of different ages who had implanted hippocampal electrodes. We then monitored behavioral changes, seizure manifestations, and electroencephalograms (EEG) as a function of age. Later, SRS frequency was quantified using intermittent video monitoring, and long-term seizure susceptibility was assessed using the convulsant flurothyP~'37. Finally, brains were examined histologically for CA3 cellular damage.

Correspondence: C.E. Stafstrom, Clinical Neurophysiology Unit, HunneweU 2, The Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, U.S.A.

228 MATERIALS AND METHODS

"fABLE I

Animals Male Sprague-Dawley rats (Charles River Laboratories, Cam. bridge, MA) of postnatal (P) ages P5, P10, P20, P30 and P60 (with P0 defined as the day of birth) were used in this study. Animals were weaned at age 21 days and were housed in plastic cages with a 12/12 h light/dark cycle. Animals had free access to food and water.

KA-induced status epilepticus* in rats of different ages

Electrode placement Two days prior to KA administration (i.e. at Pg, PI8, P28 and P58), rats were anesthetized with a mixture of i.p. ketamine hy. drochloride and xylazine (50-70 mg/kg and 2-5 mg/kg, respectively, depending on age). Bipolar electrodes were then stereotaxically implanted into the left ventral hippocampus, using coordinates according to Sherwood and Timiras34. Electrodes consisted of insulated 0.25 mm diameter stainless steel wires twisted together except at the tips. Electrodes were anchored to the skull using dental acrylic and two stainless steel screws. Following surgery, 1:'8 and Pl8 rats were returned to cages with their littermates, while P28 and P58 animals were placed in individual cages. Since P5 animals were too young for stable long-term electrographic recording, these animals did not have electrodes implanted; instead, some were anesthetized by immobilization on ice followed by stereotaxic insertion then immediate withdrawal of a 25-gauge needle into the left ventral hippocampus, in order to disrupt the blood-brain bartier in an analogous way to the older rats. Other P5-10 rats were immobilizedon ice only, without blood-brain barrier disruption. KA injection and recording Two days after surgery, animals were divided into age- and weight-matched control and KA-treated groups. Kainic acid (Sigma) was prepared in phosphate-buffered saline (pH 7.4). In pilot studies3s we determined doses of KA that produced SE (~20 win) yet resulted in a mortality rate of 25% or less: P5, 1-2 mg/kg; PI0, 2-3 mg/kg; 1:'20, 8 mg/kg; P30, 10-11 mg/kg and P60, 10-12 mg/kg, Control animals received an equal i,p. volume of phosphate-buffered saline. EEG activity was recorded from the hippocltmpal electrode for 5-10 win prior to KA administration and then intermittentl~ tot 4-8 h afterwards, Clinical behavior and seizure activity were con. tinuously observed during this period. SRS occurrence was evaluated using intermittent closed circuit video monitoring from several days after KA-induced SE umil sa '. rifice at 100-130 days of age. A wide angle lens permitted video. taping of up to 9 animals simultaneously. Each animal was videotaped for 60-120 h (in 6-hour recorc:ingsessions) evenly distributed over 3 months following KA administration, An equal number of day time and evening recording sessions were included to control for diurnal variation in SRS frequency, Observers were blind to the treatment status of each rat. Several SRS also occurred during routine laboratory handling (not recorded on videotape). These were included in some parts of the quantitative analysis, as stated in Results. Seizure susceptibility to flurothyl At 100-130 days of age, seizure susceptibility was tested using flurothyl ether (Indoklon), a volatile convulsant that causes a stereotyped progression of generalized seizures when inhaled, Seizure susceptibilitywas defined as the latency to the first component of fiurothyl seizures: myoclonic jerks. Rats were placed in an airtight clear plastic chamber (12 x 12 × 5.25 in.) containing 4 compartments of equal size separated by a wire mesh screen. Liquid flurothyl (bis-2,2,2-trifluoroethyl) was delivered through a plastic tube onto a 2-irt. piece of filter paper in the center of the chamber, at a constant rate of 38 pl/min via a constant infusion pump (Harvard Apparatus). Four animals (includingboth controls and experimentals) were tested simultaneously in each trial. Observers were

Age (days)

n

KA dose (mglkg)

% SE

Latency to % mortality to SE (s)** (mean + S.D.)

P 5 PI0 P2~ P3"0 P00

24 49 26 23 54

1-2 2-3 8 10-11 10-12

~00 ]00 100 87 93

45.9 + 22.2 44.0 + 14.0 66.3 _+ 25.1 80.4 __ 21.7 80.3 + 24.1

25 24 0 13 7

*Status epil,~pticas (SE) is defined as continuous seizure activity for >~20win. **Latencies t~ SE in P20, P30, and P60 animals were significantly different .from P5 and P10 rats (Fisher PLSD, P < 0.05).

blind to the treatm~cntstatus of each rat.

Pathology After flurothyi testing, animals were sacrificed and their brains prepared for histology. Animals were injected with a lethal dose of pentobarbital, then their hearts were perfused with Ringer's lactate followed by 10% formalin phosphate. Brains were removed and placec., in formalin for at least 2 months. Twenty-micron sections were cut and stained with hematoxylin/eosin (H/E). Electrode placement was verified in all animals. Sections were analyzed for major ¢~:ll loss with particular attention to hippocampal subfield CA3. Data analysis Differences between means of two g~oups were compared using t-tests. Differences between means of more than two groups were compared using either one-way ANOVA with post-hoe Fisher PLSD or with two.way ANOVA, Signifi~ance level was defined as 0,05 for ~!1 comparisons,

RESULTS

K A - i n d u c e d behavioral changes and S E For each age group, Table I shows the n u m b e r of rats which developed SE when K A was administered at a dose chosen to produce SE yet result in a mortality rate of ~<25%. SE was operationally defined as continuous seizure activity for at least 20 win. Clinical responses to K A differed with age. P 5 - 1 0 animals became immobile within 5 win of K A administration, with loss of tone and splaying of the hindlimbs, ataxia and falling. Vigorous side-to-side head wagging lasting several seconds appeared about 5 w i n after KA. P10 pups sometimes exhibited shudders of the entire body, similar to WDS but slower and more prolonged. Repetitive scratching began within 10 win of K A injection at P5 and w i t h i n 15 w i n at P10. Pups then rolled onto their sides, with rapid elonie movements of all extremities ('cycling' or 'swimming'), beginning with the hindlimbs. Cycling was interrupted by tonic seizures with extension of one or more limbs and increased axial tone. A high-frequency generalized tremor often superimposed

229 TABLE II Abnormal behaviors after KA in 1>5 rats

All values in rain -4- S.D. BBB, blood-brain barrier; GTC, generalized tonic-clonic.

Latency to: Immobility Scratching First GTC seizure SE duration

Intact BBB

Disrupted BBB

(n =8)

(n =16)

Significance*

4.2 + 3.3 4.1 + 2.7 14.3 _+ 10.2 14.9 + 8.6

n.s. n.s.

22.0 __+_17.0 25.1 + 19.6 150 + 66 174 +- 78

n.s. n.s.

*n.s., not significantat P = 0.05 (t-test)

on this tonus. Continuous ictai behavior (SE) began within 1 h of KA (Table I) and persisted up to 6 h, interspersed with behaviors seen during the pre-ictal phase (ataxia, scratching, head wags). To investigate the role of the blood-brain barrier in KA seizures in pups, KA was administered to P5 and P10 animals that had not undergone stereotaxic surgery or electrode implantation (i.e., intact blood-brain barrier). There was no significant difference in latency to onset of abnormal behaviors, ictal features or seizure duration between animals with intact vs disrupted bloodbrain barriers (Table II; t-test, P > 0.05). In older rats, the features of KA seizures differed and behavioral changes had later onset. At P20, immobility

accompanied by staring occurred first, followed by myoclonic jerks, repetitive scratching or wet dog shakes. Unilateral forelimb clonus began 20-40 rain after KA, followed by clonus of the other forelimb and/or hindlimbs~ Facial clonus (repetitive chewing, salivation, twitching of vibrissae and ears, blinking) usually appeared shortly after forelimb cienus, but sometimes preceded it. Eventually, animals developed SE with rearing, loss of posture, and clonus of all limbs (but forelimb clonus was predominant). Occasionally, tonic limb extension occurred, but much less frequently than in younger animals. KA-induced behavioral changes in pubescent and adult animals (P30 and P60) were similar to those observed at P20 but had later onset. These rats first exhibited immobility and staring !5-30 min after KA injection. While scratching was rare, WDS invariably occurred and were more vigorous and frequent than in younger animals. Facial and unilateral forelimb clonus occurred next, quickly progressing to involve the opposite for~limb and bJndlimbs. Rearing and falling occurred as the seizure generalized. Tonus was infrequent and often heralded death. Control animals had no seizures at any age. The latency to SE differed significantly by age at the time of KA administration (Table I; one-way ANOVA, F4,74 - 10.5, P < 0.0001). Post hoc means testing revealed significant differences in SE latencies between P5 and P10 groups compared to all older age groups (Fisher PLSD, P < 0,05).

After KA

Before KA PlO

A2 cycling , A. mo~.v le.~v ., i c . ~ m ~ v

A3 90 min

,,Nil tonus :;;. . . . . . . - ' ; ' ~ ¢ ~

I see

90rain P20 B2

C2 bilateral forelimbdonus

circling

Fig. 1. Representativeintrahippocampal recordingsbefore and after i.p. KA. A: tracings from a P10 rat before, 60 and 90 rain after KA. B: P20 rat before and 90 rain after KA. C: P60 rat before and 180 rain after KA. Clinical signs corresponding to various EEG changes are noted.

230

A 1

fomllmband fadal donus

I

Ise¢

[ A.aoe~v s. 2eogv

~mnob~ty, staring

B 1

rear--fall 2

immobility,ehewin&blinking

immobility Fig. 2. lntrahippocampalrecordingsduringelectrographicseizuresin rats, showingpoor correlationof clinical and electrographicchanges. A: similartracingsfrom two P60 rats with dissimilarclinical features. B: similartracingsfrom P60 (BI), P30 (B2) and P20 (B3) rats during electrographicSE, each disl~layingdifferentclinical signs.

KA.induced electrographic changes Baseline hippocampal EEOs of freely moving rats of different ages prior to KA injection are shown in Fig. 1. At PlO, the baseline EE(3 consisted of low voltage (generally <50 /~V) mixed frequencies (mostly ~ 8 Hz). Baseline EEOs of older animals showed higher amplitudes and a wider frequency range, as well as some sharp waveforms. Ictal discharges with clear onset and termination followed KA in rats of all ages. EEG changes preceded all clinical changes except immobility. Initial EEG changes at P10 consisted of an increase in low voltage, sharply contoured fast activity with intermixed higher amplitude sharp waves and spikes (Fig. IA2). Irregular semi-rhythmic spikes were seen during repetitive ictal scratching and cycling. Voltage attenuation was often seen during generalized tonus. Well-organized, regular, high amplitude ictal EEG activity was uncommon in Pl0 animals. Several ictal EEG patterns were seen at P20-60 (Figs, 1 and 2). During the period of immobility/staring, there was progressive build-up of high amplitude rhythmic spikes and spike-wave complexes, As iimbic seizure signs (WDS, automatisms) generalized to bilateral clonic motor activity, the periodic ictal discharges coalesced into faster frequency, high amplitude spikes and spike/wave complexes, often persisting for several minutes (e.g. Fig. 2Bt). It was not always possible to correlate specific din-

ical features with EEG changes. For example, Fig, 2A shows ictal traces from two P60 animals about 1 h after KA injection, Despite nearly identical rhythmic spike and slow wave patterns, one animal had forelimb and facial clonus, whi[e the other was immobile, Similar EEGs were recorded during circling and falling (Fig. 2BI) and during immobility with chewing and eye blinking (Fig. 2Ba). The same clinical state (e.g. immobility) could be associated with quite different EEG manifestations (compare Fig. 2Az, B z and B3). Intermittent clinical and electrographic seizures often persisted for more than 8 h. As clinical seizures waned, progressively fewer abnormal EEG changes were noted. EEG recordings over several days following KA-induced SE showed rare isolated spikes, without prominent epileptiform activity.

Spontaneous Recurrent Seizures To quantify SRS frequency, duration, and age-dependence we utilized intermittent dosed-circuit video monitoring for 3 months following KA administration. SRS were quite stereotyped, resembling stage 5 limbic motor seizures induced by kindling 32, with initial rearing, followed by bilateral forelimb clonus, repetitive chewing movements, loss of posture and falling. This sequence could repeat several times during a given SRS. SRS duration averaged about 40 s, and did not significantly differ with the age at which KA was admini.~,',.~red(Fig, 3A;

231 TIMING OF SRS OCCURRENCE

A

NUMBER OF $R8

SRS Duration

14r

G0.SRS Duration

12 ~ ~

s0-

]

KA at PSO

10

403020100

PS

Pl0 P20 P30 Age at time of KA administration

P60

/

8

Percentage of rats with SRS

B

o

!~

KA at P30

6

SO ~o of rats with SRS In each ewe ~roup 4

1

4O

2 0

30

I

'f

20

6

10

4

1

I

I

I

I

i

4

S

8

KA st p i e

0 P8

Pl0

P20

P30

pe0

Age L~:time of KA administration C

0

'|RSlday

2.0 °

°

1.0

°

O.S

"

0.0

PS

2

3

7

8

9

10

11

12

Fig. 4. Timing of SRS occurrenceafter KA administration.Data includeboth video-recordedand non-recordedSRS.

2,S'

I.S

1

WEEK AFTER KA ADMINISTRATION

SRS Frequency

Pl0

P20

P30

P60

Age at time of KA administration

Fig, 3. Age-dependent characteristics of SRS, which occur only in animals who received KA at ~>P20. A: mean (+ S.D.) SRS duration does not significantly vary with age (one-way ANOVA, F2.48 = 0.535, P = 0.593). B: percentage of rats with SRS increases with age at the time of KA administration. C: mean SRS frequency (seizures/day) increases with age at the time of KA administration. Data in A and B include boti: video-recorfied and non-recorded SRS.

one-way ANOVA, F2,4s = 0.535, P = 0.593). Postically, animals often appeared less alert, hypoactive or hostile for several minutes. SRS often occurred during awakening or handling. The occurrence and frequency of SRS varied according to the age at which an animal underwent KA-induced SE (Fig. 3B,C). SRS were never observed in rats which received KA at P5 or P10, despite the fact that these animals received 120 h of video monitoring. Fom~eeJ~ percent of I)20 rats, 30% of P30 rats and 44% of P60 rats had SRS (Fig. 3B). SRS frequency also increased sharply with the age at which animals received KA. P20 ra~s with SRS averaged 1.0 SRS/day, while P30 and P60 rats with SRS had 1.2 and 2.4 SRS/day, respectively (Fig. 3C). There was no diurnal variation in SRS frequency. Fig. 4 indicates when SRS occurred after KA. The earliest SRS occurred in a I)60 rat 4 days after KA, with

232 a peak incidence in this group 5 weeks after KA (mean _+ S.D., 4.84 -+ 2.35 weeks), when animals were 90-100 days old. SRS in I'20-30 rats began later, with most occurring after the sixth post-KA week. In P30 animals SRS frequency peaked 9-10 weeks after KA (mean _+ S.D., 9.37 _+ 1.26 weeks), which also corresponded to 90-100 days of age. Means for P60 and P30 rats were significantly different (t-test, P < 0.0001). Since so few SRS were observed in P20 rats, it is difficult to make conclusions about their timing (mean _+ S.D., 9.17 _+ 2.99 weeks). Among P'20-60 animals who had SRS while not being videotaped, the latest observation of an SRS was 121 days after KA.

A 200 T

ff'l

z

S O ~

100

>z

5O

0

P5

Pathological changes Neuronal damage (CA3 cell loss and glial scar) was seen in the majority (14/19) of KA-treated rats P20 and older. Fig. 6 shows H/E-stained sections through CA3 of KA-treated P60 rats which did (Fig. 6A) and did not

P10

P20

P30

P60

AGE AT KA ADMINISTRATION

Long-term seizure susceptibility To assess whether the age at the time of KA treatment alters seizure susceptibility later in life, flurothyl ether was administered at P100-130. The first stage of a flurothyl-induced seizure is myoclonic jerks, followed rapidly by bilateral forelimb clonus, loss of posture and wild running behavior with rapid clonus of all limbs3~. In Fig. 5A, latency to myoclonus is plotted against age at KA administration for control and treated rats. Twoway ANOVA shows no effect of age (F4.mo4ffi 1.36, P = 0.256), b~tt significant effects of KA treatment (Fi.26 -8.77, P :: 0.007 and age × treatment (F4,104 = 4.66, P ffi 0.002). Post hoe t-tests using the Bonferroni correction reveal a significant difference only between the P60 controls and KA-treated rats (P < 0.005). To determine whether rats with SRS had different flurothyl seizure latencies than KA-treated rats without SRS, these groups are considered separately in Fig. 5B. For each age group with SRS (P20, P30, P60), one-way ANOVA shows a significant effect of KA treatment (P20, F2,21 ffi 6.07, P ffi 0.008; P30, F2.24 = 4.18, P = 0.03; P60, F2.22 = 6.02, P -- 0.008). Post hoc comparisons show that in the P20 and P30 groups, animals with SRS had shorter flurothyl seizure latencies than controls or KA-treated animals without SRS (Fisher PLSD, P < 0.05). In the P60 group, the KA-treated rats without SRS also significantly differed from controls (Fisher PLSD, P < 0.05). Therefore, in adult rats (P60), KA treatment confers enhanced susceptibility to generalized non-limbic seizures. Only prepubescent rats (P20-30) with SRS have shorter flurothyl seizure latencies. In pups (P5-10), KA treatment does not affect future generalized seizure susceptibility.

control KA-treoted

150

B 200 u~ z

m ~ 150

. ~

(J

0 t).. (.J z

control KA without SRS KA with SRS

100 ,

0

~

P20

1=30

P60

AGE AT KA ADMINISTRATION

Fig. 5. Latency to flurothyl-induced myoclonus in seconds (s) versus age at the time of KA administration. Flurothyl testin 8 was performed in all rats as adults (P100-130). A: control vs KAtreated rats. There are significant effects of KA treatment and age by treatment (two-way A N O V A ; see text). Ry post hoc t-test with Bonferroni correction, only the P60 group shows a significant difference between controls and experimentals ( * P < 0.005). B: control vs KA-treated rats subdivided into animals which did or did

not have SRS. One-way ANOVA is significant at each age. At P20 and P30, KAotreated rats with SRS differed from controls and KAtreated rats without SRS (Fisher PLSD, P < 0.05; see text). At P60, both groups of KA-treated rats (with and without SRS) differed from controls. Error bars = S.D. *Significant difference from controls. P < 0.05.

(Fig. 6B) have recorded SRS, necrosis and gliosis are seen in both cases. Although a quantitative analysis of cell number was not performed, lesion severity and extent appeared similar in rats with and without SRS. No control rat had hippocampal damage. In P5-10 rats, no significant CA3 damage was noted in either control or KA-treated animals.

233

Hg. 6. Photomicrographs of H/E-stained hippocampal CA3 subfields of rats treated with KA at P60. A: rat with SRS. B: rat without SRS. Cell loss and gliosis (arrowheads) are similar whether or not a rat had SRS.

DISCUSSION As previously reported ta°'te'2s'43 we noted distinct differences between KA seizure:; in P5-10 rats versus those which received KA at P20 and older. Younger rats lacked WDS and other features characteristic of limbic system involvement, yet they developed severe, prolonged generalized tonic-clonic convulsions. Immature rats developed seizures at lower doses and had higl'er mortality rates, probably because of greater KA penetration through an underdeveloped blood-brain barrier; the lack of significant differences in latencies to abnormal behaviors after KA in P5-10 re ts with and without an intact blood-brain barrier supports this notion. P30 and P60 rats had full limbic motor seizures which resembled stage 5 kindled seizures 2°'32. P20 appears to represent an intermediate stage in the evolution ot KA-induced seizure manifestations, with seizures at this age sharing some features of P5-10 rats (scratching) and some more typical of older animals (WDS, facial and limb clonus). At all ages, ictal discharges from the hippocampus preceded clinical seizure activity. In rats ~P20, our data generally agrees with Treiman et al. 42, in that the pattern of KA electrographic seizures evolves from discrete seizures with interictal slowing to continuous ictal discharges to periodic epileptiform discharges on a flat background. Below P20, ictal d~'~charges were briefer, less rhythmic and less well organized, and the electrographic sequence less closely follows Treimen's scheme. In human neonates, several seizure types may be seen, including 'subtle' seizures (which include 'swimming' movements of the upper limbs or 'pede!ing' movements

of the lower limbs) and multifocal clonic or tonic seizures with migrating extremity involvement ~5. Electrographically, multiple independently discharging areas can be seen, perhaps reflecting incompletely myelinated pathways and immature synaptic organization and function. In our experiments, it was intriguing that at P5-10 (corresponding to the human neonatal period) rats displayed intermittent, independent tonic extension of individual limbs as well as more generalized cycling movements. Reasons for distinct age.specific seizure patterns after KA have not been fully explained. Maturation of limbic pathways (especially glutaminergic), which occurs at about 3 weeks of age in the rat 2, is a prerequisite for limbic signs and subsequent limbic neuronal damage 4'43. Similarly, striatal t~eurotoxicity is minimal at P7 in the rat, but by 3 weeks of age (when glutarninergic innerva. tion is established), KA lesions are indistinguishable from adults e. It is possible that ontogenetic differences in the number, distribution or affinity of KA receptors in different brain regions or within different limbic structures could partly explain the age discrepancy 5. In the immature rat, CA3 KA receptors increase sharply during the third week of life, whereas KA receptors in the inner layers of neocortex are transiently over-expressed at this age and thereafter gradually decline to adult levels n'22. Some authors have postulated that KA seizures in immature rats may originate primarily in neocortex 1°'43 although in the first 3 weeks of life seizure-induced metabolic activation, as measured by 2-deoxyglucose (2-DG) uptake 1'43 is restricted to hippocampus. There is also some evidence 'that KA seizures may be partly mediated by re-

234 ceptors for other EAA (such as NMDA) which have developmental profiles distinct from KA receptors 2t. While SRS following KA have been reported by several authors~'s'll'ts't6.29'4° this is the first report to quantify SRS using video monitoring and describe their developmental features. Animals which received KA at 5 or 10 days of age had no recorded SRS, and the frequency of SRS increased with age from P20 to adulthood. It is possible that our video recording protocol missed SRS in younger animals or underestimated the true frequency in older animals. However, the P5 and Pl0 groups were videotaped for more than 1400 rathours, without detecting a single SRS. Also, no SRS was ever witnessed in a P5-10 rat while not being videotaped, whereas non-recorded SRS was commonly observed in older animals. These data support the hypothesis that limbic system maturation is necessary for the development of SRS. SRS were more frequent and occurred earlier when KA was administered at P60 than at earlier ages. Our data differs somewhat from previous reports in that we first observed an SRS only 4 clays after KA administration, i.e. there was little or no 'silent period '7'29'4°. SRS occurrence peaked 5 weeks after KA administered at P60 and 10 weeks after KA when the drug was given at P30. It is intriguing that in both groups, peak SRS occurred when animals were 90-100 days old; perhaps there is some developmental process governing the maximal expression of SRS. Since so few SRS were observed at P20, it is difficult to define their natural history, except to speculate that their frequency may have been increasing when video monitoring was discontinued 3 months after KA. Longer monitoring is needed to resolve this issue. In our data and that of others 7, it appears that SRS wane with time; our latest observed SRS was 121 days after KA, similar to previous reports ~5. Flurothyl ether has often been used as a test of susceptibility to non-limbic generalized seizures 17'al,37. Our data show that KA-treated adult animals (P60) have increased flurothyl seizure susceptibility (defined as shorter latency to flurothyl-induced myoclonus) when tested later in life. P20-30 animals with SRS also had reduced flurothyl seizure thresholds compared to controls, suggesting that whatever anatomic or functional alteration produces SRS also increases the susceptibility to generalized seizures. However, KA-treated P5-10 rats and P20 rats which did not exhibit SRS showed no difference from controls in flurothyl seizure latency when tested as adults, suggesting that the immature brain is somehow protected from the long-term deleterious effects of KA. Our preliminary his~.ologicanalysis confirmed predominant loss of neurons in the CA3 region of hippocampus

in KA-treated aninals ~>P20. KA-treated P5-10 rats did not have detectable CA3 damage, confirming previous reports LS'14a6.26.36. Older KA-treated animals with and without recorded SRS had similar CA3 cell loss. Since some rats with SRS had minimal or no CA3 damage, a lesion in this area is not a prerequisite for the development of SRS. In addition to CA3 pathology, lesions elsewhere (dentate hilus, posterior thalamus) could be important in SRS pathogenesis25. KA treatment causes sprouting of dentate mossy fibers onto granule cell dendrites, which is associated with altered granule cell excitability35"36'4~. One mechanism of spontaneous seizures after KA might involve synaptic reorganization with recurrent excitatory feedback of dentate granule cells onto themselves, as has been described in kindled seizures ~q and suggested for human epilepsy ~s' 33. However, KA seizures in the immature brain do not appear to cause synaptic reorganization36. Cronin and Dudek n have shown that KA-treated adult rats with SRS had significantly more sprouting than those without SRS. However, 31% of their rats with spontaneous seizures had no demonstrable sprouting, so at least this form of synaptic reorganization may not be necessary for SRS. One of our P60 rats had an SRS only 4 days after KA, which is probably too short a time for sprouting to become established, but corresponds to a period of abnormal hyperexcitability in the dentate gyrus27'3s. Therefore, other mechanisms, such as KA-induced loss of dentate hilar cells that normally activate inhibitory interneurons a5 may be primarily responsible for generation of spontaneous seizures. It is also possible that KA-induced alterations of membrane and synaptic properties 13'44'46 or immediate early gene expression 19'~ could promote the development of SRS and enhanced susceptibility to generalized seizures. In conclusion, KA produces age-specific patterns of seizures and brain damage. It appears that maturation of limbic pathways, occurring about 3 weeks of age, is essential for the full spectrum of deficits following KA (SRS, enhanced susceptibility to generalized seizures, characteristic limbic pathology). The pathophysiology of acute KA seizures in younger animals is still unclear, making this an important model for study of the mechanisms of epilepsy during development.

Acknowledgements. We thank Antonia Chronopoulos and Samuel Thurber for technical assistance and Dr. David Amato for statistical consultation. This project was sponsored by the American Epilepsy Society with support from the Milken Family Medical Foundation (C.E.S., G.L.H.), an EpilepsyFoundationof America Medical Student Research Stipend (J.L.T.), and the Steven Linn Research Fund (G.L.H.).

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