Chronic epileptogenicity following focal status epilepticus

BRAIN RESEARCH ELSEVIER

Brain Research 655 (1994) 33-44

Research report

Chronic epileptogenicity following focal status epilepticus Yukiyoshi Shirasaka, Claude G. Waster]ain

*

Epilepsy Research, Neurology Serl'ice (127), Veterans Affairs Medical (_~'nter, 16111 Plurnrner Street, Sepuh'eda, CA 91343, USA Department of Neurology, RNRC, UCLA School of Medicine, 710 Westwood Plaza, Los Angeles, CA 90024-17~59. USA Accepted [0 May 1994

Abstract

We examined chronic epileptogenicity in the perforant path stimulation model of focal status epilcpticus. After 24 h of perforant path stimulation, every stimulation elicited multiple population spike discharges, and this phenomenon persisted more than 2-3 months after stimulation. Short (10-100 ms) interstimulus interval-dependent paired-pulse inhibition was almost completely lost right after stimulation, but recovered progressively over the following month. Long (200-1(100 ms) interstimulus interval-dependent paired-pulse inhibition decreased, and in spite of a partial recovery, remained significantly reduced 4 wceks after stimulation. Frequency-dependent paired-pulse inhibition was lost immediately after stimulation. One month later, inhibition at 2 Hz remained significantly reduced, although in individual rats recovery ranged from poor to complete. Input/output response curves showed increased population spike amplitude but no change of the slope of excitatory postsynaptic potentials. 3-4 weeks after stimulation, spontaneous generalized motor convulsions were observed in half of the stimulated rats. In all of the stimulated rats, the kindling phenomenon was significantly accelerated compared with non-stimulated controls, and class 5 convulsions were elicited in 3.3 _+ 1.0 trials in stimulated rats, against 11.0 _+ 2.5 trials in controls. Key words: Status epilepticus; Kindling; Neurophysiology; Chronic epileptogenicity; Perforant path; Dentate gyrus: Spontaneous generalized convulsion

I. Introduction

Recent studies suggest that seizure-induced brain damage causes secondary chronic epileptogenicity [6,7,10,13,22,23,35,53]. Spontaneous amygdaloid seizures appeared from 20 to 40 days, and generalized convulsions from 30 to 60 days after kainic acid (KA) seizures [10,53]. Kindling was greatly accelerated and elicited generalized motor convulsions after a few stimulations [13]. In pilocarpine-injected rats, secondarily generalized convulsions began to occur 2 weeks after injection [8]. One to 2 months after electrical stimulation-induced status epilepticus (SE) [21], the rats showed interictal spikes and electrographic seizures [6,22]. Sloviter et al. found that sustained stimulation to

* Corresponding author. Fax: (1) (818) 895-9554. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 6 1 4 - I

the perforant path induced neuronal injury morphologically similar to that induced by KA [30,40,45]. Light microscopic analysis showed a highly reproducible pattern of hippocampal damage to a selected population of dentate hilar interneurons [41,42], and CA1 and CA3 pyramidal neurons [40]. This model is focal, induces a more restricted lesion than other types of experimental SE, and is free from the adverse effects of chemical convulsants. It was reported that spontaneous limbic seizure-like symptoms occasionally occurred following perforant path stimulation, and that decreased frequency-dependent paired-pulse (pp) inhibition was observed in that model and might be the cause of its epileptogenicity [42]. However, a systematic study is needed to provide definitive evidence of a chronic epileptic state. In this study, we report the results of kindling studies and sequential electrophysiological examinations of the dentate gyrus in the perforant path stimulation model of SE [42].

34

K Shtrasaka, (.~;. H,~/sterh~in /' Brain Research 655 (1994) 33-.44

2. Materials and methods

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2.1. Animals and procedure of anesthesia 18-week-old male Wistar rats (400-500 g; Simonsen Labs) were housed on a 12 h light/dark cycle (06.0018.00 h) and given free access to food and water. Rats were placed in a metofane (methoxyflurane; 3-4 ml) jar until they lost consciousness, injected with urethane intravenously through the tail vein (loading dose, 1000 m g / k g in saline), and placed in a stereotaxic apparatus. Intravenous injection of urethane was continued until the end of experiments to maintain anesthesia, and the rate of infusion was changed according to the response to sensory stimulation. Rectal temperature was monitored continuously and maintained at 37.0 + 0.5°C with a warm water coil placed under the animal during anesthesia.

Fig. 1. Schematic presentation of relative positions of electrodes. Black dots in A and B show relative positions of stimulating and recording electrodes, respectively. Background figure was from [31].

2.2. Surgery discharges. Electrode positions are displayed in Fig. 1. Chromium non-coated wires (Consolidated Wire and Associated Co.) were connected to screws on the skull and used as ground electrodes. The skin was cleaned with Povidine solution (Rugby Laboratories Inc.) and 95% alcohol, and 100 mg of chloramphenicol (Warner Lambert Co.) was injected subcutaneously once per day for three days after surgery.

Two holes were drilled on the left side of the skull to accommodate the stimulating and recording electrodes. The center of the hole for the stimulating electrodes was 8.1 mm posterior and 4.4 mm lateral to the bregma, and that for the recording electrodes was 3.5 mm posterior and 2.2 mm lateral to the bregma. Four jewelery screws (two anterior, one right, and one posterior) were drilled into skull holes. Bipolar electrodes made of twisted Teflon-coated 0.005 inch diameter stainless steel wires (California Fine Wire Co.; tip separation < 1.0 mm for recording electrodes and < 0.5 mm for stimulating electrodes) were cemented by dental acrylate. Final electrodes locations were determined by maximizing the amplitude of the characteristic potential recorded in the dentate gyrus in response to ipsilateral perforant path stimulation. The final electrode depth was 2.4 + 0.1 mm from brain surface for stimulating electrodes, and 3.1 + 0.1 mm for recording electrodes (mean + S.E.M.). In these locations, evoked potentials in dentate gyrus induced by perforant path stimulation showed positive excitatory postsynaptic potentials (EPSP) with negative population spike (PS)

2wkx 1

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We used a Grass $88 stimulator and stimulus isolation units (Grass SIU5, Grass PSIU6) to stimulate the perforant path, a type 502 Dual-Beam Oscilloscope to measure stimulation voltage, and a R2 Digital Oscilloscope Software (Rapid Systems Inc.) to record and analyze evoked potentials. Fig. 2 shows the time course of the entire experiment. Four weeks after surgery, we investigated interstimulus interval (ISI)-dependent pp responses at intervals of 15, 25, 40, 60, 100, 200, 300, 400, 1000, 3000, and 5000 ms (0.1 Hz, 0.1 ms duration, 30 V monophasic stimulation); input/output ( I / O ) responses at 250,

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Fig. 2, Timeline for entire experiment. Timeline for entire experiment for rats given 24 h perforant path stimulation (PfPS) is shown. For control rats, timetable is same except for PfPS. Abbreviations: surgery, surgery for placement of electrodes; wk, week; pp exam, paired-pulse and input/output examinations; awake, in the awake state; anesthetized, under urethane anesthesia.

Y. Shirasaka, C.G. Wasterlain/Brain Research 655 (1994) 33 44 500, 750, 1000, 1250, 1500, and 5000 /zA (0.1 Hz, 0.2 ms duration, biphasic stimulation); frequency-dependent pp responses [43] at 0.1, 1, and 2 Hz (40 ms apart, 0.1 ms duration, 30 V monophasic stimulation), in the awake state and 30 rain after the start of urethane anesthesia (1000 m g / k g i.v. as loading dose followed by continuous intravenous injection). A 30-min interval between examinations and the beginning of anesthesia was used in all examinations in the anesthetized state. To minimize the effect of 1 and 2 Hz stimulation on other examinations, frequency-dependent studies were performed at the end of the experiments. The awake state recordings were performed while the rats were resting quietly with their eyes open. Ten consecutive waveforms were recorded after waveforms had become stable. In the frequency-dependent studies we started recording after 10 stimulations at 2 Hz in the awake state, because in preliminary studies, prolonged 2 Hz stimulation in this situation sometimes caused seizurelike symptoms similar to those seen in kindling trials. In preliminary studies, we investigated only frequency-dependent effects, 30 min after cementing the electrodes under urethane anesthesia. However, in the sequential examination of evoked potentials, it became necessary to examine the awake state to rule out potential effects of anesthesia. Therefore, we examined evoked potentials 4 weeks after cementing the electrodes. A long time interval between examination and surgery minimizes the effects of electrode insertion.

2.4. 24 hours perforant path stimulation (PfPS)

35

state i week before kindling (4-5 weeks after PfPS). Since frequency-dependent examination at 2 Hz in the awake state induced seizure-like symptoms, we examined evoked potentials at the same times in control rats to exclude the effect of examinations on kindling studies. Whenever spontaneous convulsions were observed, examinations were postponed by 24 h.

2.6. Kindling One week after electrophysiological examinations under urethane anesthesia, rats received 60 Hz biphasic square-wave pulses (1 ms pulse duration and 2 s total duration) at intensities which sequentially increased 50 /zA at 3 min intervals until an afterdischarge (AD) was induced. A D was defined as high amplitude spike or polyspike epileptiform activity visible for 3 s or more after completion of the stimulus in either recording site. Stimulation was given via the perforant path electrode, and E E G was recorded via both electrodes. A D threshold (ADT) was determined as the lowest intensity of current necessary to elicit AD, and A D duration ( A D D ) as the time elapsed from the end of the stimulus until the organized A D terminated. Starting the following day, kindling stimulation was given 5 d a y s / w e e k . Kindling stimulation consisted of 2 s trains of 60 Hz, 1 ms, biphasic square-wave pulses and was administered three times per day (at least 3 h apart) at an intensity 100 p~A above ADT. If an animal demonstrated A D at a given intensity on three consecutive trials, the intensity was reduced in steps of 50 p~A. And if an animal failed to show an AD, the intensity was increased in steps of 100 # A . In the latter case, interval of each trial was 3 rain or longer. Seizures were classified according to Sutula and Osteward [50] and kindling trials were continued until rats experienced three class 5 seizures. Rats which needed over 1500 /xA to induce A D were excluded from this study.

Immediately after electrophysiological studies under urethane anesthesia, PfPS was started using a modified Sloviter method [40]. Perforant path was stimulated at 2 Hz (paired pulses, ISI 40 ms apart, 20 V, 0.1 ms duration) continuously with intermittent 10 s trains of single stimuli at 20 Hz (20 V, 0.1 ms duration) delivered once per minute throughout the 24 h period under continuous intravenous injection of urethane. During PfPS the evoked potentials were checked frequently, and if PS discharges disappeared, the rats were excluded from the stimulated group. Because rats given PfPS often died with increased salivary secretions and dyspnea on effort in preliminary studies, 0.1 mg atropine was injected subcutaneously at the end of PfPS. Thirty minutes after PfPS, evoked potentials were examined as described above. Control rats were anesthetized in the same manner as the stimulated group, but not stimulated except for electrophysiological examinations.

Electrophysiological examinations were performed until 1-4 weeks after kindling trials, and thereafter rats were deeply anesthetized with m e t o p h a n e and were perfused transcardially with heparinized 0.9% saline followed by 4% paraformaldehyde in p H 6.5 phosphate-buffered saline (PBS). The brains were embedded in paraffin, sectioned horizontally at 8 /zm, and stained with Cresyl violet for determination of electrode placement.

2.5. Electrophysiological examination after PfPS

2.8. Data analysis

Evoked potentials were examined in the awake state 2 weeks after PfPS, and in the awake and anesthetized

PS amplitude and EPSP slope were measured from the waveforms made from the average of 10 consecu-

2. 7. Anatomical studies

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tive evoked potentials. PS amplitude was calculated as: [(field potential at the beginning of PS) + (field potential at the end of PS)]/2 - (field potential at the peak of PS), and EPSP slope was measured between two fixed points after the onset of the EPSP and before the onset of PS. In multiple PS discharges, we used the first PS to measure PS amplitude. In pp examination, inhibition score, which was calculated as: [ 1 0 0 - (PS amplitude of the 2rid pulse)/(PS amplitude of the 1st p u l s e ) × 100], was used as a measure of the pp responses of each experiment. In I / O response examinations, the proportion of the PS amplitude and EPSP slope at each stimulus intensity to the maximal response in the same experiment was used as an indicator of the number of granule cells firing and of the 120

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3. l. 24 hours perforant path stimulation and its £[fect on behat'ior Four of 15 stimulated rats died within 48 h after PfPS. Among surviving rats, only one rat lost PS dis-

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strength of synaptic response [12]. The data after PfPS or after kindling were compared with the data before PfPS or kindling in the same state (awake or anesthetized), and the data of the stimulated group were also compared with those of the control group at the same time point from surgery, by analysis of variance (ANOVA), and P < 0.05 was considered significant.

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Fig. 3. Sequential changes of paired-pulse inhibition and granule cell excitability. A: sequential changes of inhibition score [100 (PS amplitude of the 2nd pulse)/PS amplitude of the 1st pulse) x 100] in interstimulus interval (ISl)-dependent paired-pulse (pp) examinations, at 25 ms ISI in stimulated rats and control rats is shown. B: those at 300 ms ISI. C: sequential changes of inhibition scores in frequency-dependent pp examinations, at 2 Hz in stimulated and control rats. D: sequential changes of PS ratio ((PS amplitude at 250 tzA stimulus intensity)/(maximal PS amplitude in the same experiment) x 100) in stimulated rats and control rats. All the data shown in this figure were examined in the awake state except for 30 minutes after 24 h perforant path stimulation (PfPS) or 24 h anesthesia, which was examined under urethane anesthesia. Abbreviations: PfPS or Kindling with arrows, the time point at which PfPS or Kindling stimulation was given; stimulated, rats given PfPS; control, non-stimulated control rats; before, before PfPS in stimulated rats and before 24 h anesthesia in control rats; 30 min after, 2 weeks after, 4 weeks after, 30 rain, 2 weeks, and 4 weeks after PfPS or 24 h anesthesia, respectively. *'*Significantly different from before PfPS (stimulated group) or before kindling (control group) in the same state (awake or anesthetized), at P < 0.05 and P < 0.01, respectively; . . . . 'significantly different from control at P < 0.05 and P < 0.01, respectively.

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charge during PfPS, and the results of kindling studies of this rat are shown as 'stimulated control' in Table 4. 2182 _+ 63 m g / k g (mean _+ S.E.M.) of urethane was used during an average 24 h experiment. No systematic analysis of behavior was carried out in this experiment, however, spontaneous limbic seizurelike symptoms were sometimes observed [42], and in addition, spontaneous generalized convulsions (GCs) were observed in 6 of 12 rats 3 to 4 weeks after PfPS. GCs consisted of bilateral clonic activities of the head and forepaws and rearing with/without falling, and in one case of a tonic convulsion. They occurred under stressful circumstances, such as before attachment of electrode leads or during handling. In the awake state 4 weeks after PfPS, 10 s 2 Hz pp stimulations induced GCs in 6 of 10 stimulated rats, although none of them showed GCs in response to the same stimulus 2 weeks after PfPS.

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3.2. ISI-dependent pp examination

Naive rats showed marked pp inhibition at short ISis both in the awake and anesthetized states (Table 1). Thirty minutes after PfPS, the inhibition scores significantly decreased at all ISis other than at 100 ms ISI. At short ISis (10-60 ms), pp inhibition was completely lost (Fig. 3a), and so was the much milder pp inhibition at ISis longer than 200 ms (Fig. 3b). Two weeks after PfPS, some inhibition returned, however, short ISI-dependent pp inhibition was still reduced significantly. Four weeks after PfPS, the inhibition scores increased further. With short ISis, significant differences disappeared in the awake state but remained in the anesthetized state, although the results varied among animals. With long ISis (200-1000 ms), the return of inhibition was only partial and significant differences with the naive state persisted over 4 weeks. After kindling, long ISI-dependent inhibition showed a non-significant increase compared to its value before kindling (4 weeks after PfPS), however, the inhibition score was still significantly lower than before PfPS. Fig. 4 shows examples of ISI-dependent pp examination of stimulated rats. Pp disinhibition at short ISis persisted even 4 weeks after PfPS in the rat in Fig. 4a. In contrast, in the rat shown in Fig. 4b, disinhibition at short ISis had disappeared completely 4 weeks after PfPS. In this rat, spontaneous GCs were observed before kindling in spite of the complete return of short ISI pp inhibition. A prominent finding in these tracings was the multiple PS discharges in all waveforms after PfPS. Multiple PS discharges were present even in rats which showed recovery of short ISI-dependent pp inhibition, as in Fig. 4b, and these discharges remained until perfusion in 9 of 10 stimulated rats (up to 3 months after PfPS). One rat in which multiple PS discharges almost disap-

Fig. 4. Interstimulus interval-dependent paired-pulse examination in two stimulated rats. The waveforms show the average of L0 consecutive waves. The first positive deflection in each trace is the stimulation artefact, and the EPSP with/without PS discharge follows. Pp stimulations (0.1 ms duration, 30 V monophasic) were given at 0.1 Hz. Abbreviations: A, before PfPS in awake state; B, before PfPS under urethane anesthesia; C, 30 rain after PfPS; D, 2 week after PfPS in awake state; E, 4 weeks after PfPS in awake state; F, 4 weeks after PfPS under urethane anesthesia; 1st, 1st PS waveform; 25, 40, 100, 300, and 1000 indicates the ISI (ms) for that waveform. Vertical scale bar = 1 mV; horizontal scale bar = 10 ms.

peared at 4 weeks after stimulation did not show spontaneous seizures. We could not examine kindling in this rat because of electrode cap detachment. Multiple PS discharges were never observed in control rats even after kindling. In control rats, prolonged anesthesia did not affect ISI-dependent responses nor other evoked potential examinations (Figs. 3a-d). The possibility that some of the changes in pp inhibition represented a kindling-like effect of PfPS was examined by studying pp inhibition before and after perforant path kindling in control rats not previously subjected to PfPS. In that population, there was no decrease in pp inhibition after kindling. On the contrary, pp inhibition significantly increased at ISis of 40-100 ms, demonstrating that loss of pp inhibition in PfPS rats is not the result of perforant path kindling during the 24 h stimulation.

Y Shirasaka, C.G. Wasterlain/Brain Research 655 (19941 33-44 0.1Hz

3.3. Frequency-dependent pp examination Table 2 shows the results of frequency-dependent pp examination in PfPS rats, and in non-stimulated controls before and after kindling. Thirty minutes after PfPS, the inhibition scores significantly decreased at all frequencies. Two weeks later, they increased at all frequencies, however, were still significantly lower than before PfPS. Four weeks after PfPS, the inhibition scores increased again, but were still significantly reduced at 2 Hz while awake and anesthetized (Fig. 3c). The inhibition score showed a non-significant increase after kindling, and significant difference with values before PfPS disappeared. Fig. 5 shows frequency-dependent pp examination of the same rat used in Fig. 4b. The 2nd PS amplitudes at all frequencies increased immediately after PfPS indicating complete loss of frequency-dependent inhibition. Two weeks after PfPS, the amplitude of the 2rid PS at 0.1 Hz markedly decreased; however, at higher frequencies, the high amplitude of the 2nd PS persisted and the ratio of 2nd PS to 1st PS remained high. Such increases in 2nd PS amplitude at higher stimulation frequencies were never observed before PfPS or in control rats. Four weeks after PfPS, significant disinhibition remained. Multiple PS discharges appeared enhanced at higher frequencies in most stimulated rats. In control rats, frequency-dependent pp inhibition increased with kindling, and there was no enhancement of 2rid PS discharges at higher frequencies. 3. 4. I / 0 response Table 3 shows the results of I / O responses at 250 and 500 /~A in stimulated rats before and after PfPS, and in control rats before and after kindling. In stimulated rats, the proportion of PS amplitude at each stimulus intensity to maximal PS amplitude did not increase immediately after PfPS, but showed delayed increase at 2 weeks after PfPS (Fig. 3d). This

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ratio also rose significantly under urethane anesthesia (Table 3). After PfPS multiple PS discharges appeared whenever PS were elicited, in all rats. The proportions of EPSP slope to maximal EPSP

Table 2 Frequency-dependent paired-pulse examination Frequency

0.1 Hz

1 Hz

2 Hz

(7.0) (10.21 (4.5) ** (21.9) * (12.0) (13.6) (12.9)

78.2 (7.9) 72.9 (7.1) - 4 . 9 (3.4)** 21.0(14.2) ** 56.1 (13.4) 54.4 (10.3) 72.4(12.7)

93.0 (5.4) 84.5 (5.11 - 1 1 . 6 (3.7)** 33.4 (4.8) ** 55.9(12.51 * 41.6 (12.21 ** 80.0 (6.t;)

75.0 (4.2) 95.2 (2.8) **

59.5 (9.5) 97.7 (2.3)**

91.5 (8.5) 99.2 (0.8)

Stimulated Before, Awake (101 Before, Anesth (10) 30 min after, Anesth (10) 2 weeks after, Awake (5) 4 weeks after, Awake (10) 4 weeks after, Anesth (8) After kind, Awake (6)

78.0 57.1 3.3 29.8 74.0 50.0 76.0

Control Before kind, Awake (7) After kind, Awake (4)

Frequency-dependent pp examinations in experimental rats before and after PfPS, and in control rats before and after kindling. Pp stimulations (0.1 ms duration, 30 V monophasic) were given at 40 ms ISI. Abbreviations and symbols as in Table 1.

411

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Table 3 I n p u t / o u t p u t response to perforant path stimulation Stimulation: ( ~ A )

Spike a m p l i t u d e / m a x

EPSP s l o p e / m a x

250

500

250

500

28.11 (7.5) 11.4 (11.31 5.7 (3.9) 74.4 (9.6) ** 55.0 112.1t) 17.3 (7.91" 67.4 (I 1.81 *

74.8 39.8 42.3 93.8 88.4 59.4 95.8

(7.2) (8.2) (9.4) (4.5) (4.11 (8.3) (2.5)

24.8 (5.3) 10.6 (2.9) 8.7 (3.9) 23.11 (10.1) 34.4 16.8/ 14.8 (6.0) 37.2 (8.9)

54.~ 42.8 42.7 55.0 61.3 50.9 64.2

10.0 13.31 58.0 (14.91 *

36.7 ( 19.1 ) 77.8 (11.51

1.3 ( 1.3) 44.3 (18.71

42.7 ( 17. I ) 61/./I (10.01

Stimulated

Before, Awake(10) Before, A n e s t h ( 1 0 ) 30 rain after, Anesth (10) 2 weeks after, Awake (5) 4 weeks after, Awake (10) 4 weeks after, A n e s t h ( 8 ) After kind, Awake (6)

(~.11 (6.4) (5.6) 19.3! (6.4) (8.8) (4.8)

Control

Before kind, Awake (7) After kind, Awake (4)

I n p u t / o u t p u t ( I / O ) responses in 24 h perforant path stimulated rats before and after PfPS, and in control rats before and after kindling. Single stimulations (0.2 ms duration, biphasic) were given at 0.1 Hz. N u m b e r s represent the m e a n s and (S.E.M.) of ((PS amplitude or EPSP slope at each stimulus intensity)/(maximal PS amplitude or EPSP slope in the same experiment) x 100). The responses are compared before and after PfPS in the same state (awake or anesthetized), using A N O V A . Abbreviations as in Table 1.

slope showed no significant changes during the course of the experiments. In stimulated rats, there was no significant difference in PS amplitude or EPSP slope as a result of kindling. In control rats, PS ratio increased significantly (Fig. 3d), and EPSP ratio also showed a non-significant increase after kindling (Table 3).

3.5. Kindling Table 4 shows the results of kindling stimulation. As there was no difference between the group which were given PfPS immediately after cementing the electrodes and the group stimulated 4 weeks later, we combined the data. In the PfPS group, first A D T and number of trials to induce the first class 5 convulsion and three class 5 convulsions were significantly smaller compared with the non-stimulated control group. 6 of 12 stimulated rats showed class 5 convulsions on the first trial, and 3

of them showed three successive class 5 convulsions starting with the first trial. Class 5 convulsions occurred at the first trial in both rats shown in Figs. 4 and 5. Unstimulated controls took 3 times as many trials to reach criterion and never showed generalized convulsions on the first trial. The stimulated control kindled slowly, and showed relatively low first A D T and relatively short first A D D , but overall was similar to unstimulated controls.

3.6. Correlation between the results o/" evoked potentials and kindling We calculated the Pearson correlation coefficiency between the kindling results (first ADT, first A D D , number of trials necessary to induce first or three class 5 convulsions) and six indexes derived from each result of pp and I / O response examination in stimulated rats. These indexes include the awake state data at 4 weeks after PfPS (A), the anesthetized state data at 4

Table 4 Effect of perforant path stimulation on kindling rate

Stimulated (12) Control (7) Stimulated control (1)

First A D T

First A D D

First C5

Third C5

283.3 (35.0) * (100-500) 458.3 171.21 (250-700) 301/.0 ( - )

88.3 (13.2) (13-15(11 52.3 ( 13.61 (24-112) 20.0 ( - )

3.3 (1.0) ** ( I - 10) 11.0 (2.5) (4-15) 18.0 ( - )

6.3 (1. l ) ** (3-16) 18.0 ( 1.11 (14-211 26.0 ( - )

Stimulations were given via electrodes in the perforant path, and responses were recorded via electrodes in the dentate gyrus and perforant path. An afterdischarge (AD) was defined as high amplitude spike or polyspike epileptiform activity visible for 3 s or more after completion of the stimulus. A D threshold (ADT) was the lowest intensity of current necessary to elicit AD, and A D duration ( A D D ) as the time lapsed from the end of the stimulus until the organized A D terminated at either recording site. Data depicts mean (S.E.M.), and (minimum-maximum). Stimulated rats were compared with non-stimulated controls by A N O V A . Abbreviations: First ADT, A D T at first trial; First A D D , A D D at first trial; First C5, n u m b e r of trials necessary to induce the first class 5 convulsion; Third C5, n u m b e r of trials necessary to induce three class 5 convulsions; Stimulated control, the rat in which perforant path was stimulated for 24 h but spike discharges were lost; other abbreviations as in Table I.

E Shirasaka, C.G. Wasterlain/ Brain Research 055 (1994) 33-44

weeks after PfPS (B), and their calculated ratio to the awake state data (C) and anesthetized state data (D) before PfPS, that is, ( A / C ) , ( B / D ) , ( A - C ) / ( A + C), and (B - D ) / ( B + D). T h e r e was no consistent correlation between the results of kindling and these indexes in stimulated rats (data not shown). We also examined these relationships in individual animals. Of the 8 rats which showed complete recovery of ISI-dependent early phase pp inhibition (Fig. 4b), all but one kindled rapidly, and 3 of them showed spontaneous GCs. Two rats showed complete recovery of frequency-dependent pp inhibition, and one of them kindled relatively slowly compared with other stimulated rats (10 ADs were necessary to induce the first class 5 convulsion in this rat); however, the other one kindled rapidly and showed a spontaneous GC.

4. Discussion 4.1. Chronic epileptogenicity

Previous studies have shown that the PfPS paradigm used in our studies causes extensive hippocampal damage [30,4(I,41]. In our laboratory, this damage includes widespread selective necrosis of neurons in the ipsilateral dentate gyrus, and occasional individual neuronal loss in the contralateral hilus, or in C A 3 - 4 or CAI pyramidal cells [32,33]. The present study shows that this damage in turn can lead to chronic hippocampal hyperexcitability and to chronic epilepsy. In spite of the fact that no effort was made to search for chronic epilepsy, half of our chronic animals had spontaneous generalized seizures during handling or during pairedpulse testing, and we have never observed such seizures in unstimulated animals. Given the very focal, unilateral nature of the damage in the current model, it is very likely that the generalized convulsive seizures that we observed were focal with secondary generalization. Thus in this model, seizure-like activity in the perforant path causes ipsilateral hilar damage which results in chronic epilepsy, providing a final proof to Gowers' adage that 'seizures beget seizures' [16]. Furthermore, the massive facilitation of kindling observed in our stimulated animals confirms the epileptogenicity of their hippocampal lesions, and suggests a possible mechanism for their spontaneous seizures. We have previously noted how paradoxical it is that spontaneous kindling never occurs, while some CA3 neurons physiologically burst at frequencies similar to those used for inducing kindling. We speculate that a 'filter' function which normally prevents kindling in response to physiological activity [57] is lost as a result of PfPS. This could lead over a period of a few weeks to spontaneous seizures and to exaggerated responses to perforant path stimulation (GCs induced by 2 Hz pp examina-

41

tion) which appear 3 - 4 weeks after the insult in our model and in animals with brain damage for KA [13,53], pilocarpine seizures [8] or continuous hippocampal stimulation [6,22]. 4.2. Relation between pfPS and kindling

Spontaneous GCs and facilitated kindling indicate chronic epileptogenicity in PfP stimulated rats. As electrical stimulations in the anesthetized state have been reported to have a kindling effect [46,47], we suspected that the PfPS paradigm in our experiments might have a kindling-like effect. However, PfPS consists of continuous 2 Hz stimulation and of 20 Hz stimulation 50 s apart. This PfPS paradigm is dissimilar to the intermittent stimulation paradigm of kindling [15]. The effects of PfPS on evoked potentials were completely different from those of kindling. Multiple PS discharges in dentate gyrus elicited by single stimulation of perforant path, which were observed in all of the 24 h stimulated rats, were never observed in kindled rats. The increase of 2nd PS in ISl-dependent and in frequency-dependent pp examinations observed in 24 h-stimulated rats is the opposite of the effect of kindling in our control rats and in other reports [12,29,56,59]. Although Kamphuis ct al. reported that the 2nd PS amplitude in early phase of 1Sl-dependent pp examination increased after kindling when examined at medium stimulus intensity [18], our stimulus intensity (30 V) is high, and w)[tage-dependcnt pp examinations in our rats (data not shown) showed that ISl-dependent early phase pp inhibition saturated at 20-30 V stimulation (examined at 40 ms ISI). In 1 / O responses, after PfPS the EPSP slope showed no change, while it usually increases after kindling [36,38]. Spontaneous seizures which were observed in our stimulated rats, rarely occur in the brief kindling paradigm used here [34,50]. In addition, the rat which was given PfPS but lost spike discharges before completion of PIPS kindled slowly, showing that PfPS itself had no kindling effect. These facts indicate that the pathophysiological changes induced by PfPS are different from those induced by kindling, and that PfPS-induccd chronic epilcptogenicity does not result from a kindling-like change. 4.3. Relationship between PJPS and sproutin,~

After KA [10,11,14,44,48,52], pilocarpine SE [2~)] or kindling [17,51] as well as in the human epileptic temporal lobe [4,24,49], sprouting of Timm positive mossy fibers has been observed in the molecular layer of the dentate gyrus, and has suggested the development through misdirected regeneration of a recurrent granule cell-to-granule cell excitatory circuit that might be responsible for chronic epilepsy [4,9,10,11,14,17,24,44,

42

}'..~,'hirasaka, ( '. (;. Waster&in/" Hrain Researcll 055 ( / 9q4) 33-44

48,49,51,52]. Such a scenario could not account tbr the hippocampal hyperexcitability seen in our stimulated rats. Loss of inhibition and hyperexcitability were always maximal at the end of PfPS, at a time where no sprouting has had time to occur. Indeed, over a period of time some types of inhibition returned, possibly in a time frame compatible with sprouting and regenerative efforts (Tables 1 and 2, Figs. 3-5). On the other hand, spontaneous GCs were first observed 3 - 4 weeks after PfPS and a relation with mossy fiber sprouting cannot be ruled out.

4.4. Role of short ISI-dependent pp inhibition Among individual animals, there was no correlation between the degree of disinhibition and the kindling rate, or the occurrence of spontaneous convulsions. For example, the rat in Fig. 4b showed complete recovery of short ISI-dependent inhibition 4 weeks after PfPS, but this rat showed spontaneous GCs and consecutive class 5 convulsions from the first kindling trial. These facts indicate that ISI-dependent early phase inhibition in dentate gyrus, which is thought to be GABAA-mediated [3,25,56], has no relation with chronic epileptogenicity in PfPS rats. In fact, the time course of recovery of early phase ISI-dependent inhibition in these rats coincides with mossy fiber sprouting, as it does in KA models [10,11,48,52].

4.5. Role of long ISI-dependent pp inhibition In dentate gyrus, ISI-dependent late phase inhibition is thought to be mediated by G A B A ~ receptor activation [37] a n d / o r CaZ+-mediated increase in potassium conductance [54]. G A B A B receptor agonists might have some anticonvulsant effects [28], but the proconvulsant action of antagonists is doubtful [19]. In our experiments, there was no correlation between this type of inhibition and susceptibility to kindling. The meaning of the significant but relatively small loss of late phase ISI-dependent inhibition in PfPS animals is unclear at present.

4.6. Role of frequency-dependent pp inhibition in chronic epileptogenicity Sloviter [43] suggested that decreased GABA Amediated inhibition might explain the changes in frequency-dependent pp inhibition in the 24 hour-stimulated rats [41,42]. In our experiments, pp inhibition at 2 Hz was completely lost right after stimulation and recovered only partially by 4 weeks, confirming Sloviter's findings [42]. However, at the same time, short ISI-dependent inhibition, which is also thought to be G A B A A mediated [3,25,56], recovered completely,

demonstrating that two processes which may both bc GABA A mediated can evolve in different directions. After PfPS, the inhibition scorc dccreascd with stimulation frequency (Table 2, Figs. 3c and 5), possibly because higher frequency increased pp facilitation a n d / o r because of malfunction of inhibitory, systems in the damaged dentate hilus. When given the 2 Hz pp stimulation while awake 4 weeks after PIPS, many rats showed class 5 convulsions. However, correlations between kindling and loss of frequency-dependent inhibition were poor, because one rat with complete recovery of frequency-dependent pp inhibition showed spontaneous GCs and facilitated kindling, and because spontaneous GCs began to appear while this inhibition was recovering.

4.7. Delayed increase in granule cell excitability after

Pp's In I / O responses, the PS ratio did not increase immediately after PfPS; however, it had increased significantly 2 weeks after PfPS, similar to the response to kindling stimulation in control rats (Fig. 3d). This delayed increase of dentate granule cell excitability might be explained by a spontaneous kindling effect due to loss of a 'filter' function [57] as a result of PfPS, or might have some relation with mossy fiber sprouting, which usually appears several weeks after neuronal damage [48]. However, this hyperexcitability of granule cells which peaked 2 weeks after PfPS cannot alone be responsible for the spontaneous seizures and hypersensitivity to 2 Hz stimulation which did not appear until 3 - 4 weeks after PfPS.

4.8. Significance of multiple PS discharges Multiple PS discharges were observed at all stimulation frequencies after PfPS. Similar discharges are elicited in dentate gyrus [44] and in CA1 [3,39] of KA treated rats, in dentate gyrus in the presence of bicuculline [20], and in CA3 of rats treated with N M D A [1] and other convulsants [39,55,58], and could result from facilitated excitatory a n d / o r decreased inhibitory synaptic mechanisms [1,2,39,55]. Although GABA-tike immunoreactive neurons were reported to be spared after PfPS [41,42], involvement of specific subpopulations [27] or their functional disconnection [5] have not been ruled out.

Acknowledgements We wish to express our thanks to Mr. Roger A. Baldwin for his skillful assistance. This work was supported in part by the VA research service, by research grant NS13515, and by EFA fellowship(YS).

Y. Shirasaka, C.G. Wasterlain /Brain Research 655 (19941 33 44 References

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