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Arousal, performance and absence seizures in rats
430
Electroencephalography and clinical Neurophysiology , 79 (1991) 430-434 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0013-4649/91/$03.50 ADONIS 001346499100162D
EEG 90538
Arousal, performance and absence seizures in rats E.L.J.M. Van Luijtelaar, S.J. Van der Weft, J.M.H. Vossen and A.M.L. Coenen Department of Psychology, Unit,ersity of Nijmegen, Nijmegen (The Netherlands)
(Accepted for publication: 10 May 1991)
Summary Rats of the WAG/Rij strain show bilateral symmetrical spontaneous spike-wave discharges in the EEG, with clinical concomitants. The present experiment investigated whether, during a learning task, the number of discharges would be diminished compared to a period of rest. Additionally, it was investigated whether behavioural differences would be noticed within the task in trials with and without spike-wave discharges. The length of the post reinforcement pause in a fixed interval task was used as a performance index. Eleven rats were extensively trained to press a lever for food in a fixed (60 sec) interval task until a stable response pattern emerged: a post-reinforcement pause of about half the interval. Next, EEG electrodes were implanted and baseline EEGs were made, before and after the first and fifth test sessions. In addition, the behavior of the animals in the task was monitored when an EEG was recorded. During the task, a significantly smaller number of spike-wave discharges was found, compared to the preceding and succeeding baseline hours. This reduction is probably related to a higher level of vigilance during the task compared to the rest hour. Furthermore, the post-reinforcement pause was significantly enhanced in trials with spike-wave discharges compared to trials without discharges, indicating a clear change in performance. Both results are in agreement with what could be expected in patients with absence epilepsy and provide further evidence for the validation of the spike-wave discharges as genuine epileptic phenomena. Key words: Spike-wave discharge; Performance; Arousal; Absence epilepsy; Fixed interval; WAG/Rij strain; (Rats)
B r i e f l a p s e s in p a t i e n t s ' ability to m a i n t a i n c o n t a c t with the e n v i r o n m e n t form the p r i m a r y s y m p t o m o f a b s e n c e epilepsy. G e n e r a l a g r e e m e n t exists with res p e c t to the d i u r n a l i n c i d e n c e o f absences. T h e y app e a r w h e n the level o f vigilance is not high (see for review V i e t h 1986). A n a b s e n c e is less likely to occur w h e n a p a t i e n t is e n g a g e d in high m e n t a l activity. O n the contrary, d u r i n g s h o r t p e r i o d s of relaxation, t h e c h a n c e for spike-wave d i s c h a r g e s to b r e a k t h r o u g h increases. This is also the case just b e f o r e failing a s l e e p a n d d u r i n g s h o r t a r o u s a l s from s l e e p ( T o m k a 1985). V a r i o u s n a m e s have b e e n assigned to this key symptom of a b s e n c e epilepsy, such as a loss o f c o n s c i o u s n e s s o r a r e d u c t i o n in the ability to p r o c e s s i n f o r m a t i o n . I n c r e a s e s in s i m p l e a n d choice r e a c t i o n times (e.g., S c h w a b 1941; T i z a r d a n d M a r g e r i s o n 1963; S e l l d e n 1971; P o r t e r et al. 1973), as well as o m i s s i o n s in continuous p e r f o r m a n c e tasks a n d in stimulus d e t e c t i o n , a r e f r e q u e n t l y r e p o r t e d to a c c o m p a n y the c h a r a c t e r i s t i c spike-wave d i s c h a r g e s (for a r e c e n t review see Mirsky 1989). C h a n g e s in m e n t a l f u n c t i o n i n g m a y even be p r e s e n t w i t h o u t e v i d e n t c h a n g e s in clinical b e h a v i o r ( A a r t s et al. 1984). W i t h o u t any d o u b t the p r e s e n c e of Correspondence to: Dr. E.L.J.M. Van Luijtelaar, Department of Psychology, University of Nijmegen, P.O. Box 9104, Nijmegen (The Netherlands). Tel.: 31-80-612544.
the above m e n t i o n e d s y m p t o m s , which can be s u m m a rized as cognitive deficits, a r e i m p o r t a n t f e a t u r e s in epilepsy, including a b s e n c e e p i l e p s y (e.g., L e n n o x 1960; M a j k o w s k i 1981). S o m e y e a r s ago it was f o u n d that rats o f the W A G / R i j strain show n o n - i n d u c e d but s p o n t a n e o u s l y o c c u r r i n g clinical signs of a b s e n c e e p i l e p s y t o g e t h e r with spike-wave d i s c h a r g e s in the E E G ( V a n L u i j t e l a a r a n d C o e n e n 1986, 1988, 1989; C o e n e n a n d V a n Luijtelaar 1987). B o t h the clinical a n d E E G signs m i m i c k e d t h e W i s t a r m o d e l , d e s c r i b e d by V e r g n e s et al. (1982, 1987) a n d M a r e s c a u x et al. (1984) a n d the m o d e l p r o p o s e d by Buszaki et al. (1989). S u b s e q u e n t p h a r m a cological a n d e l e c t r o p h y s i o l o g i c a l s t u d i e s in the W A G / R i j strain, o r in V e r g n e s a n d M a r e s c a u x ' s Wistar selection line, s h o w e d that spike-wave d i s c h a r g e s w e r e s u p p r e s s e d by the typical drugs a n d a g g r a v a t e d by typical a n t i - c o n v u l s a n t drugs ( M i c h e l e t t i et al. 1985; P e e t e r s et al. 1988). T h e results from all t h e s e studies strongly suggest that, next to o t h e r models, rats from the W A G / R i j strain a r e a suitable g e n e t i c m o d e l for g e n e r a l i z e d a b s e n c e e p i l e p s y in man. In c o n t r a s t to m o d e l s for focal e p i l e p s y in which cognitive d e t e r i o r a t i o n has b e e n the subject of various p a p e r s (e.g., M a j k o w s k i 1981, 1986), the r e l a t i o n bet w e e n spike-wave d i s c h a r g e s in rats a n d cognitive malf u n c t i o n i n g has not b e e n studied. T h e r e is one p a p e r d e v o t e d to this subject, in which cats w e r e u s e d
SPIKE-WAVE I)IS('I IAR(;ES AND P E R F O R M A N C E
(Taylor-Courwd and Gloor 1984). These authors induced spike-wave discharges by injecting penicillin and noticed a selectively impaired responsiveness to sensory stimuli during spike-wave discharges. They suggested that this response failure was either attributable to a cognitive deficit or to a motor impairment, associated with temporary amnesia. These results were used to support the penicillin model as an acceptable model of human primary generalized epilepsy. Some authors have doubts about the epileptic nature of rodent spike-wave discharges (Kaplan 1085). If the performance is deteriorated during spike-wave discharges in rats, howevcr, this can bc considered as additional evidence for the view that spike-wave discharges in rats represent genuine epileptic phenomena. An index for performance is derived from a temporal regulation task: fixed interval (FI) responding (Richelle and l,ejeunc 1980, 1984). In this task, rats press a levcr and only the first response after the passage of 60 scc will be reinforced. Rats develop during training a characteristic response pattern, a long period of inactivity callcd the post-reinforcement pause, which is followed by accelerated responding. The response latency is used as the performance index. The purposc of the present study is twofold. First it investigates whether thc execution of a task influences the number of spikc-wavc discharges; viewed against the increased level of arousal or activity, a decrease in the number of spike-wave discharges is anticipated. ]'he second purpose is to investigate whether spikewave discharges affect the pcrformance in a learning task: it is expected that thesc discharges will influence the characteristic FI behavior and the length of the post-rein forcement pause.
Methods and materials
131
the chamber was left open to allow free movements of the E E G cable. Both the lever (3 × 2 cm) and a non-recessed food tray were placed only 4 mm above the floor, in order to avoid entanglement of the E E G cable. The experimcntal subjects were provided with standard subdural E E G electrodes (Plastics One, MS 333,/2-A). Surgery was performed under Nembutal anesthesia (60 m g / k g ) . One cortical electrode was placed in the frontal area (A 2.0, l~3.5), the other in the parietal region (A 6.0, L4.0). The rcference electrode was placed above the cerebellum. The Elema-Sch6nandcr polygraph and the steering equipment for the adapted opcrant chamber were placed in an adjacent room. On the polygraph the following data were registercd (paper spced 1 cm/scc): the E E G (frequencies between 1 and 70 Hz), the levcr presses, the presentation of the food pellets, and the start and finish of each new interval. Procedu re
Rats were first deprived of food until they reached 90% of their pre-deprivation body weight. After getting acquainted with the pellet-feeder system, magazine training, continuous reinforcement training whereby each lever press is food rewarded, was started. Next day this was followed by the actual FI 60 sec training. This implies that food reward is made contingent upon the first lever press only when 60 scc has elapsed. Earlier lever presses are without consequence (see e.g., Reynolds 1968). The experimental protocol is schematically presented in Fig. 1. During training, a response pattern will develop which is characteristic for FI schedule of reinforcement: a period without any re-
I
a)
fixed interval
J
I
timing of the interval
1
Subjects
Subjects were 11 experimentally naive male W A G / Rij rats. The age varied between 4 and 12 months, the body weight ranged between 262 and 350 g. Rats were individually housed and maintained on a 12 light/12 dark regime, with lights on at 21.00 h at all times. Material
Training was carried out in eight identical standard operant conditioning chambers (inner sizes 24 cm wide, 23 cm deep, 22 cm high). The left side wall contained an intelligence panel with three 2.8 W bulbs, a lever, and a recessed food tray. Standard 45 mg food pellets (Campden Instruments) were used as reinforcers. A PDP 11/03 computer was used to control the experimental equipment and to collect data. The simultaneous operant task and E E G recording necessitated an adapted operant chamber. The top of
responses accelerated leverpressing c) l presentation of food pellet
reinforcers
presentation of food pellet
Fig. 1. Schematic representation of an FI (fixed interval) task. a: timing of the 60 sec interval: a next interval is initiated by the presentation of a food pellet, b: lever press responding, typical l'or an FI schedule of reinforcement. After having received a pellet, the animal is not likely to re.spond. This period ot non-responding is called the post-reinforcement pause (PRP). Only after about 50-80% of the interval has elapsed, lever pressing slowly begins. The chance that the interval has been exceeded increases with time. This explains the accelerated lever pressing towards the end of the interval. c: the first lever press after 60 sec will be folMwed (reinforced) by the presentation of a food pellet.
432
sponding (PRP, post-reinforcement pause) is followed by accelerated lever pressing. The duration of the FI session was 1 h. The F1 training was continued until one of the following two criteria was met: a mean PRP of 30 sec over three subsequent sessions or no significant increase in the P R P during ten subsequent sessions. When a subject fulfilled one of these criteria, it was moved to the E E G registration room where three FI sessions were presented in the adapted chamber. The animals were allowed to eat at liberty and surgery took place after the third session. Next, the animals were allowed to recover and to eat at liberty for another 5 days. The new body weight was adjusted and at postoperative days 6 and 7 the animals were slightly deprived of food, to 95% of their body weight. On day 7, the animals were placed in an E E G recording cage and on day 8 a first baseline (B1) E E G was recorded for 1 h. After this, the animals were moved to the operant chamber for the first of a series of five (one daily, T I ) FI sessions. After the first session, a second baseline E E G (B2) was recorded in the recording cage for 1 h on the next days followed by 4 daily sessions (T2-T5). Also before and after the last session (T5), baseline E E G s were made (B3 and B4, respectively) for 1 h. All FI sessions took place at the same time of day, between 13.00 and 14.00 h. A spike-wave discharge was identified as such if it included a train of sharp spikes and slow waves, with an amplitude of at least twice the background E E G amplitude and a duration of at least 1 sec. The number of trains of spike-wave discharges and their mean duration were noted. Examples of spike-wave discharges can be found elsewhere (Van Luijtelaar and Coenen 1986).
Results
F1 responding The duration of the PRP increased during training and, after 40-80 FI sessions, the characteristic response pattern for FI had developed and remained unchanged over successive days. The grand mean and S.D. of the P R P of the last training session in the standard boxes was 27.5 _+ 3.2 sec (n = 1l). The grand mean of the P R P of T 1 - T 5 in the adapted chamber was 35.9 _+ 1.4 sec (n = 11).
E.L,J.M. VAN LUIJTELAAR ET AL. 20
u
O
O
'-
0 B1 T1 B2 T2
T3
T4
B3 T5 B4
Fig. 2. Mean and S.D. of the number of spike-wave discharges during baseline (B1-B4) and Fixed Interval 60 task (TI-T5).
indicated that the 5 days were not statistically different. However, within the first ( F = 14, 72; df 2, 9; P < 0.01) and fifth ( F = 16, 39; df 2, 9; P < 0.01) day, significant (t test for correlated data) differences between B1 and T1 (t = 5, 15; dr= 8; P < 0.01), B2 and T1 (t = 4, 29; dr= 8; P < 0.01), B3 and T5 (t = 8, 49; df= 8; P < 0.0l), B4 and T5 (t = 4, 41; d r = 8; P < 0.01) were found. During the task (T1 and T5) the number of spike-wave discharges was always lower than during rest (B1 to B4).
F1 responding and spike-wace discharges For this analysis the results of the trials of all 5 sessions in which spike-wave discharges were noticed were used. This was allowed since no significant day effect was noticed. Eight of the 11 rats showed one or more spike-wave discharges during one of the sessions. One animal was disregarded because the spike-wave discharges occurred after the end of the PRP. The mean and S.D. of the duration of the spike-wave discharges was then 6.2 + 2.7 sec. While inspecting the E E G s made during the FI task, it was noticed that microsleeps ( > 1 and < 5 sec) emerged incidentally. Since microsleeps might influence the P R P as well, it was decided to discriminate also between trials with and without microsleeps and between trials with and
TABLE I Grand mean of the post-reinforcement pauses (PRPs) during the EEG controlled FI sessions in which spike-wave discharges occurred. A distinction is made between trials with and without spike-wave discharges and between trials with and without microsleeps. Between brackets the number of trials and the number of rats is given (n t and ns, respectively). First the mean PRP was obtained for each subject, then the grand mean was obtained.
Spike-wace discharges The numbers of spike-wave discharges in the 4 baseline and 5 FI sessions are presented in Fig. 2. An analysis of variance with repeated (days) measure for within subjects showed no day effect for T 1 - T 5 . This
10
Spike-wave activity
No sleep Sleep
No
Yes
37.2 (nt = 600, ns = 7) 85.8 (n t = 17, ns = 3)
58.4 (nt = 30, ns = 7) 92.6 (n t = 18, n s = 4)
SPIKE-WAVE 1)IS('ttARGKS ANt) PERFORMANCE without spike-wave discharges. In Table 1 the results are presentcd. Thirty trials of 7 animals contained an unambiguous waking E E G with spikc-wave discharges. The PRP for each subjcct in trials with spike-wave discharges was h)nger compared to all trials without discharges. In these sessions, the grand mean of the PRP was significantly prolonged comparcd to the grand mean of all trials without spike-wave discharges (58.4 +_ 6.8 vs. 37.2 +_ 7.1 sec: P < 0.05). Sleep also prolonged the PRP in trials without spike-wave dischargcs (85.8 ± 31 vs. 37.2 ± 7.1 scc: I' <0.05). The increase in the PRP in sessions with spike-wave discharges (21.2 + 12.1 sec) was longer than thc moan duration of the spike-wave dischargcs (~).2 _+ 2.7 scc).
Discussi4m
Two major results were obtained. The first is that during the execution of thc task, a significantly smaller number of spikc-wave discharges was found compared to the prcceding lind succeeding baseline hours. Thc dccreasc m the number of spikc-wave dischargcs during the task hours was present on both days during which control mcasurcments were taken. It can be safely concluded that the number of spike-wave discharges dccreased during the task. This result seems in line with the well documented relationship between vigilance, arousal and absence epilepsy in man in which it is also rcporlcd that high vigilance is accompanied by a low number of spike-wave discharges and low vigilance by a high number of spike-wave discharges (see for rcviews Vieth lclS(~ and Coenen et al. 1991). A simihir phenomenon might have been present in the results dcscribed by Aarts ct a[. (1984). They pointed out that a task in itself can dcpress epileptic activity in children with gencralized absence epilepsy. It seems that in the present experiment a low level of vigilance during the baseline sessions and a high arousal or vigilance lcvcl during: the task, influences the number of spike-wavc discharges. It can therefore be concluded that a reversc relationship exists between the levcl of vigilance and the number of spike-wave discharges. The second major result was that the occurrence of spike-wave discharges changed the performance c)f epileptic rats in the t:'l task. The PRP was prolonged in trials with spike-wavc discharges compared to trials without discharges and the prolongation was longer than the duration of thc spike-wave discharge itself. Additionally, it was found that microsleeps also prolong thc duration of the PRPs. This finding helps to interprct thc prolonged PRP. It is reasonablc to assume that during sleep information processing is s o m e -
133 what diminished. In the present experimcnt it was found that microsleeps and spike-wave discharges had in common that both of them increase the PRP. Thcrcfore it is thought thai information processing, as measured by the change in PRP, is also diminished during the prescncc of spike-wave discharges. In fact, spikcwavc discharges might have reduced amncsia, comparable to amnesia induced by clectroconvulsive shocks, but to a much smaller degree. Amnesia has been reported to occur after gencralizcd 3 Hz spikc-wavc discharges in man (Jus and Jus 1%2: Ounstead et al. 1963). The possibility cannot bc excluded that part of the fixed interval has been x~ipcd out by thc excessive electrical activity and consequcntly, an overestimation of the interval occurs, In line with thc vicw that information processing is disruptcd during trials with spikewavc discharges, lnoue ct al. (1991)havc found that both primary lind secondary componcnts of thc visual evoked potcntial arc differentially disruptcd during spikc-wave discharges compared to wakcfulncss, slow wave sleep lind REM sleep. Inouc's preliminary restllts togcther with the present data strongly suggest that information processing might be disrupicd during the presence of spike-wave discharges. Further studics will bc directed towards possible dcficits in other typcs of cognitive activity such as pcrccption, central processing and short-term mcmol3' during spike-wave discharges. The prolongation of the PRP was longer than the duration of the spike-wave dischargc, suggesting that more time is missed by the animal than the actual duration of the aberrant activit$. It is often thought that only during the presence of the spike-wave arc behavioral changcs prescnt in absence cpilcpsy, ttowever, Mirsky anti Grady (lq88) found a significant incrcasc in the power of 3 - 4 ttz KEG frcqucncy bands as early as 20 sec preceding the epileptic bursts, monotonically increasing until cumulating in the fully developed spike-wave bursts, h can be spcculatcd that the long lasting pcrformance changes are rclalcd to clcctrophysiological antecedcnts prcccding the fully developcd spikc-wavc discharge. It can be concluded that the tinting of the first responsc in a fixed interval schedulc ot rcinforcemcnt is changed by a spiko.wavc discharge. Furthermorc, thc decrcasc of the numbcr of spikc-wavc discharges during the execution of the task is in line with what could be cxpected from the extensive literature on the relationship between vigilance, activity, arousal and itbsencc epilcpsy m man. P,oth typcs of rcsults form additional evidence for the view that spike-wave dischargcs arc genuinc cpilcptic phenomena. It sccms that thc prescnt results positivcly contribute in the debate about the cpileptic naturc of the spike-wavc discharges in gcneral and in particular to the validity of the W A G / R i j strain as a model for gcncralized ilbsencc epilepsy in man.
434 Dr. E.L.J.M. Van Luijtelaar is supported by the Royal Dutch Academy of Sciences (K.N.A.W.).
References Aarts, J.H.P., Binnie, C.D., Stair, A.M. and Wilkins, A.J. Selective cognitive impairment during focal and generalized epileptiform EEG activity. Brain, 1984, 107: 293-308. Buszaki, G., Smith, A., Roskies, A.L., Fisher, L.J. and Gage, F.H. Parkinsonian tremor and petit mal epilepsy: hypothesis of a common pacemaker. Neuroscience, 1989, 36: 1-14. Coenen, A.M.L. and Van Luijtelaar, E.LJ.M. The WAG/Rij model for absence epilepsy: age and sex factors. Epilepsy Res., 1987, 1: 297-301. Coenen, A.M.L., Drinkenburg, W.H.I.M., Peeters, B.W.M.M., Vossen, J.M.H. and Van Luijtelaar, E.L.J.M, Absence epilepsy and the level of vigilance in rats of the WAG/Rij strain. Neurosci. Behav. Rev., in press. Inoue, M., Van Luijtelaar, E.L.J.M., Vossen, J.M.H. and Coenen, A.M.L. Visual evoked potentials during absence seizures in the rat. Electroenceph. clin. Neurophysiol., submitted. Jus, A, and Jus, K. Retrograde amnesia in petit mal. Arch. Gen. Psychiat., 1962, 6:71 75. Kaplan, B.J. The epileptic nature of rodent polyspiking is still unproven. Exp. Neurol., 1985, 88: 425-426. Lennox, W.G. Epilepsy and Related Disorders. Little and Brown, Boston, MA, 1960. Majkowski, J. Brain electrical stimulation: kindling and memory aspects. Acta Neurol. Scand., 1981, 64: 101-108. Majkowski, J. Kindling: a model for epilepsy and memory. Acta Neurol. Scand., 1986, 74: 97-108. Marescaux, C., Vergnes, M., Micheletti, G., Depaulis, A., Reis, J., Rumbach, L., Warter, J.M. and Kurtz, D. Une forme g~n6tique d'absences petit mal chez le rat Wistar. Rev. Neurol. (Paris), 1984, 140: 63-66. Micheletti, G., Vergnes, M., Marescaux, C., Reis, J., Depaulis, A., Rumbach, L. and Warter, J.M. Antiepileptic drug evaluation in a new animal model: spontaneous petit real epilepsy in the rat. Arzneim.-Forsch.,/Drug Res., 1985, 35: 483-485. Mirsky, A.F. Information processing in petit mal epilepsy, ln: B.P. Hermann and M. Seidenberg (Eds.), Childhood Epilepsies. Neuropsychological, Psychosocial and Intervention Aspects. Wiley, Chichester, 1989: 51-70. Mirsky, A.F. and Grady, C.L. Towards the development of alternative treatments in absence epilepsy. In: M.S. Myslobodsky and A.F. Mirsky (Eds.), Elements of Petit Mal Epilepsy. Lang, New York, 1988: 285-310.
E.L.J.M. VAN LUIJTELAAR ET AL. Ounstead, C., Hutt, S.J. and Lee, D. The retrograde amnesia of I~etit mal, Lancet, 1963, i: 671. Peeters, B.W.M.M., Spooren, W.P.J.M., Van Luijtelaar, E.L.J.M. and Coenen, A.M.L. The WAG/Rij model for absence epilepsy: anticonvulsant drug evaluation. Neurosci. Res. Comm., 1988, 2: 93-97. Porter, R.J., Penry, J.K. and Dreifuss, F.E. Responsiveness at the onset of spike-wave bursts. Electroenceph. clin. Neurophysiol., 1973, 34: 239-245. Reynolds, G.S. A Primer of Operant Conditioning, Scott, Foresman and Co., Glenview. 1968. Richelle, M. and Lejeune, H. Time in Animal Behavior, Pergamon Press, Oxford, 1980. Richelle, M. and Lejeune, H. Timing competence and timing performance: a cross-species approach. In: J. Gibbon and L. Allan (Eds.), Timing and Time Perception. Vol. 423. Ann. NY Acad. Sci., 1984: 254-268. Schwab, R.S. Method of measuring consciousness in attacks of petit real epilepsy. Arch. Neurol. Psychiat., 1941, 41: 215-217, Sellden, U. Psychotechnical performance related to paroxysmal discharges in the EEG. Clin. Electroenceph., 1971, 2: 18-27. Taylor-Courval, D. and Gloor, P. Behavioral alterations associated with generalized spike-and-wave discharges in the EEG of the cat, Exp, Neurol., 1984, 83:167 186. Tizard, B. and Margerison, J.H. Psychological functions during wave-spike discharge. Br. J. Soc. Clin. Psychol., 1963, 3: 6-15. Tomka, I. Zirkadiane Ph~inomene bei der Petit Mal-Epilepsie. Z. EEG-EMG, 1985, 16: 10-16. Van Luijtelaar, E.L.J.M. and Coenen, A.M.L. Two types of electrocortical paroxysms in an inbred strain of rats. Neurosci. Lett., 1986, 170: 393-397. Van Luijtelaar, E.L.J.M. and Coenen, A.M.L. Circadian rhythmicity in absence epilepsy in rats. Epilepsy Res., 1988, 2: 331-336. Van Luijtelaar, E.L.J.M. and Coenen, A.M.L. The WAG/Rij model for generalized absence seizures. In: J. Manelis, E, Bental, J.N. Loeber and F.E. Dreifuss (Eds.), Advances in Epileptology Vol. 17. Raven Press, New York, 1989: 78-83. Vergnes, M., Marescaux, C., Micheletti, G., Reis, J., Depaulis, A., Rumbach L. and Warter, J. Spontaneous paroxysmal electroclinical patterns in rat: a model for generalized non-convulsive epilepsy. Neurosci. Lett., 1982, 33: 97-101. Vergnes, M., Marescaux, C., Depaulis, A., Micheletti, G. and Wafter, J.M. Spontaneous spike and wave discharges in thalamus and cortex in a rat model of genetic petit real-like seizures. Exp. Neurol., 1987, 96: 127-136. Vieth, J. Vigilance, sleep and epilepsy. Eur. Neurol,, 1986, 2: 128-133.
Electroencephalography and clinical Neurophysiology , 79 (1991) 430-434 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0013-4649/91/$03.50 ADONIS 001346499100162D
EEG 90538
Arousal, performance and absence seizures in rats E.L.J.M. Van Luijtelaar, S.J. Van der Weft, J.M.H. Vossen and A.M.L. Coenen Department of Psychology, Unit,ersity of Nijmegen, Nijmegen (The Netherlands)
(Accepted for publication: 10 May 1991)
Summary Rats of the WAG/Rij strain show bilateral symmetrical spontaneous spike-wave discharges in the EEG, with clinical concomitants. The present experiment investigated whether, during a learning task, the number of discharges would be diminished compared to a period of rest. Additionally, it was investigated whether behavioural differences would be noticed within the task in trials with and without spike-wave discharges. The length of the post reinforcement pause in a fixed interval task was used as a performance index. Eleven rats were extensively trained to press a lever for food in a fixed (60 sec) interval task until a stable response pattern emerged: a post-reinforcement pause of about half the interval. Next, EEG electrodes were implanted and baseline EEGs were made, before and after the first and fifth test sessions. In addition, the behavior of the animals in the task was monitored when an EEG was recorded. During the task, a significantly smaller number of spike-wave discharges was found, compared to the preceding and succeeding baseline hours. This reduction is probably related to a higher level of vigilance during the task compared to the rest hour. Furthermore, the post-reinforcement pause was significantly enhanced in trials with spike-wave discharges compared to trials without discharges, indicating a clear change in performance. Both results are in agreement with what could be expected in patients with absence epilepsy and provide further evidence for the validation of the spike-wave discharges as genuine epileptic phenomena. Key words: Spike-wave discharge; Performance; Arousal; Absence epilepsy; Fixed interval; WAG/Rij strain; (Rats)
B r i e f l a p s e s in p a t i e n t s ' ability to m a i n t a i n c o n t a c t with the e n v i r o n m e n t form the p r i m a r y s y m p t o m o f a b s e n c e epilepsy. G e n e r a l a g r e e m e n t exists with res p e c t to the d i u r n a l i n c i d e n c e o f absences. T h e y app e a r w h e n the level o f vigilance is not high (see for review V i e t h 1986). A n a b s e n c e is less likely to occur w h e n a p a t i e n t is e n g a g e d in high m e n t a l activity. O n the contrary, d u r i n g s h o r t p e r i o d s of relaxation, t h e c h a n c e for spike-wave d i s c h a r g e s to b r e a k t h r o u g h increases. This is also the case just b e f o r e failing a s l e e p a n d d u r i n g s h o r t a r o u s a l s from s l e e p ( T o m k a 1985). V a r i o u s n a m e s have b e e n assigned to this key symptom of a b s e n c e epilepsy, such as a loss o f c o n s c i o u s n e s s o r a r e d u c t i o n in the ability to p r o c e s s i n f o r m a t i o n . I n c r e a s e s in s i m p l e a n d choice r e a c t i o n times (e.g., S c h w a b 1941; T i z a r d a n d M a r g e r i s o n 1963; S e l l d e n 1971; P o r t e r et al. 1973), as well as o m i s s i o n s in continuous p e r f o r m a n c e tasks a n d in stimulus d e t e c t i o n , a r e f r e q u e n t l y r e p o r t e d to a c c o m p a n y the c h a r a c t e r i s t i c spike-wave d i s c h a r g e s (for a r e c e n t review see Mirsky 1989). C h a n g e s in m e n t a l f u n c t i o n i n g m a y even be p r e s e n t w i t h o u t e v i d e n t c h a n g e s in clinical b e h a v i o r ( A a r t s et al. 1984). W i t h o u t any d o u b t the p r e s e n c e of Correspondence to: Dr. E.L.J.M. Van Luijtelaar, Department of Psychology, University of Nijmegen, P.O. Box 9104, Nijmegen (The Netherlands). Tel.: 31-80-612544.
the above m e n t i o n e d s y m p t o m s , which can be s u m m a rized as cognitive deficits, a r e i m p o r t a n t f e a t u r e s in epilepsy, including a b s e n c e e p i l e p s y (e.g., L e n n o x 1960; M a j k o w s k i 1981). S o m e y e a r s ago it was f o u n d that rats o f the W A G / R i j strain show n o n - i n d u c e d but s p o n t a n e o u s l y o c c u r r i n g clinical signs of a b s e n c e e p i l e p s y t o g e t h e r with spike-wave d i s c h a r g e s in the E E G ( V a n L u i j t e l a a r a n d C o e n e n 1986, 1988, 1989; C o e n e n a n d V a n Luijtelaar 1987). B o t h the clinical a n d E E G signs m i m i c k e d t h e W i s t a r m o d e l , d e s c r i b e d by V e r g n e s et al. (1982, 1987) a n d M a r e s c a u x et al. (1984) a n d the m o d e l p r o p o s e d by Buszaki et al. (1989). S u b s e q u e n t p h a r m a cological a n d e l e c t r o p h y s i o l o g i c a l s t u d i e s in the W A G / R i j strain, o r in V e r g n e s a n d M a r e s c a u x ' s Wistar selection line, s h o w e d that spike-wave d i s c h a r g e s w e r e s u p p r e s s e d by the typical drugs a n d a g g r a v a t e d by typical a n t i - c o n v u l s a n t drugs ( M i c h e l e t t i et al. 1985; P e e t e r s et al. 1988). T h e results from all t h e s e studies strongly suggest that, next to o t h e r models, rats from the W A G / R i j strain a r e a suitable g e n e t i c m o d e l for g e n e r a l i z e d a b s e n c e e p i l e p s y in man. In c o n t r a s t to m o d e l s for focal e p i l e p s y in which cognitive d e t e r i o r a t i o n has b e e n the subject of various p a p e r s (e.g., M a j k o w s k i 1981, 1986), the r e l a t i o n bet w e e n spike-wave d i s c h a r g e s in rats a n d cognitive malf u n c t i o n i n g has not b e e n studied. T h e r e is one p a p e r d e v o t e d to this subject, in which cats w e r e u s e d
SPIKE-WAVE I)IS('I IAR(;ES AND P E R F O R M A N C E
(Taylor-Courwd and Gloor 1984). These authors induced spike-wave discharges by injecting penicillin and noticed a selectively impaired responsiveness to sensory stimuli during spike-wave discharges. They suggested that this response failure was either attributable to a cognitive deficit or to a motor impairment, associated with temporary amnesia. These results were used to support the penicillin model as an acceptable model of human primary generalized epilepsy. Some authors have doubts about the epileptic nature of rodent spike-wave discharges (Kaplan 1085). If the performance is deteriorated during spike-wave discharges in rats, howevcr, this can bc considered as additional evidence for the view that spike-wave discharges in rats represent genuine epileptic phenomena. An index for performance is derived from a temporal regulation task: fixed interval (FI) responding (Richelle and l,ejeunc 1980, 1984). In this task, rats press a levcr and only the first response after the passage of 60 scc will be reinforced. Rats develop during training a characteristic response pattern, a long period of inactivity callcd the post-reinforcement pause, which is followed by accelerated responding. The response latency is used as the performance index. The purposc of the present study is twofold. First it investigates whether thc execution of a task influences the number of spikc-wavc discharges; viewed against the increased level of arousal or activity, a decrease in the number of spike-wave discharges is anticipated. ]'he second purpose is to investigate whether spikewave discharges affect the pcrformance in a learning task: it is expected that thesc discharges will influence the characteristic FI behavior and the length of the post-rein forcement pause.
Methods and materials
131
the chamber was left open to allow free movements of the E E G cable. Both the lever (3 × 2 cm) and a non-recessed food tray were placed only 4 mm above the floor, in order to avoid entanglement of the E E G cable. The experimcntal subjects were provided with standard subdural E E G electrodes (Plastics One, MS 333,/2-A). Surgery was performed under Nembutal anesthesia (60 m g / k g ) . One cortical electrode was placed in the frontal area (A 2.0, l~3.5), the other in the parietal region (A 6.0, L4.0). The rcference electrode was placed above the cerebellum. The Elema-Sch6nandcr polygraph and the steering equipment for the adapted opcrant chamber were placed in an adjacent room. On the polygraph the following data were registercd (paper spced 1 cm/scc): the E E G (frequencies between 1 and 70 Hz), the levcr presses, the presentation of the food pellets, and the start and finish of each new interval. Procedu re
Rats were first deprived of food until they reached 90% of their pre-deprivation body weight. After getting acquainted with the pellet-feeder system, magazine training, continuous reinforcement training whereby each lever press is food rewarded, was started. Next day this was followed by the actual FI 60 sec training. This implies that food reward is made contingent upon the first lever press only when 60 scc has elapsed. Earlier lever presses are without consequence (see e.g., Reynolds 1968). The experimental protocol is schematically presented in Fig. 1. During training, a response pattern will develop which is characteristic for FI schedule of reinforcement: a period without any re-
I
a)
fixed interval
J
I
timing of the interval
1
Subjects
Subjects were 11 experimentally naive male W A G / Rij rats. The age varied between 4 and 12 months, the body weight ranged between 262 and 350 g. Rats were individually housed and maintained on a 12 light/12 dark regime, with lights on at 21.00 h at all times. Material
Training was carried out in eight identical standard operant conditioning chambers (inner sizes 24 cm wide, 23 cm deep, 22 cm high). The left side wall contained an intelligence panel with three 2.8 W bulbs, a lever, and a recessed food tray. Standard 45 mg food pellets (Campden Instruments) were used as reinforcers. A PDP 11/03 computer was used to control the experimental equipment and to collect data. The simultaneous operant task and E E G recording necessitated an adapted operant chamber. The top of
responses accelerated leverpressing c) l presentation of food pellet
reinforcers
presentation of food pellet
Fig. 1. Schematic representation of an FI (fixed interval) task. a: timing of the 60 sec interval: a next interval is initiated by the presentation of a food pellet, b: lever press responding, typical l'or an FI schedule of reinforcement. After having received a pellet, the animal is not likely to re.spond. This period ot non-responding is called the post-reinforcement pause (PRP). Only after about 50-80% of the interval has elapsed, lever pressing slowly begins. The chance that the interval has been exceeded increases with time. This explains the accelerated lever pressing towards the end of the interval. c: the first lever press after 60 sec will be folMwed (reinforced) by the presentation of a food pellet.
432
sponding (PRP, post-reinforcement pause) is followed by accelerated lever pressing. The duration of the FI session was 1 h. The F1 training was continued until one of the following two criteria was met: a mean PRP of 30 sec over three subsequent sessions or no significant increase in the P R P during ten subsequent sessions. When a subject fulfilled one of these criteria, it was moved to the E E G registration room where three FI sessions were presented in the adapted chamber. The animals were allowed to eat at liberty and surgery took place after the third session. Next, the animals were allowed to recover and to eat at liberty for another 5 days. The new body weight was adjusted and at postoperative days 6 and 7 the animals were slightly deprived of food, to 95% of their body weight. On day 7, the animals were placed in an E E G recording cage and on day 8 a first baseline (B1) E E G was recorded for 1 h. After this, the animals were moved to the operant chamber for the first of a series of five (one daily, T I ) FI sessions. After the first session, a second baseline E E G (B2) was recorded in the recording cage for 1 h on the next days followed by 4 daily sessions (T2-T5). Also before and after the last session (T5), baseline E E G s were made (B3 and B4, respectively) for 1 h. All FI sessions took place at the same time of day, between 13.00 and 14.00 h. A spike-wave discharge was identified as such if it included a train of sharp spikes and slow waves, with an amplitude of at least twice the background E E G amplitude and a duration of at least 1 sec. The number of trains of spike-wave discharges and their mean duration were noted. Examples of spike-wave discharges can be found elsewhere (Van Luijtelaar and Coenen 1986).
Results
F1 responding The duration of the PRP increased during training and, after 40-80 FI sessions, the characteristic response pattern for FI had developed and remained unchanged over successive days. The grand mean and S.D. of the P R P of the last training session in the standard boxes was 27.5 _+ 3.2 sec (n = 1l). The grand mean of the P R P of T 1 - T 5 in the adapted chamber was 35.9 _+ 1.4 sec (n = 11).
E.L,J.M. VAN LUIJTELAAR ET AL. 20
u
O
O
'-
0 B1 T1 B2 T2
T3
T4
B3 T5 B4
Fig. 2. Mean and S.D. of the number of spike-wave discharges during baseline (B1-B4) and Fixed Interval 60 task (TI-T5).
indicated that the 5 days were not statistically different. However, within the first ( F = 14, 72; df 2, 9; P < 0.01) and fifth ( F = 16, 39; df 2, 9; P < 0.01) day, significant (t test for correlated data) differences between B1 and T1 (t = 5, 15; dr= 8; P < 0.01), B2 and T1 (t = 4, 29; dr= 8; P < 0.01), B3 and T5 (t = 8, 49; df= 8; P < 0.0l), B4 and T5 (t = 4, 41; d r = 8; P < 0.01) were found. During the task (T1 and T5) the number of spike-wave discharges was always lower than during rest (B1 to B4).
F1 responding and spike-wace discharges For this analysis the results of the trials of all 5 sessions in which spike-wave discharges were noticed were used. This was allowed since no significant day effect was noticed. Eight of the 11 rats showed one or more spike-wave discharges during one of the sessions. One animal was disregarded because the spike-wave discharges occurred after the end of the PRP. The mean and S.D. of the duration of the spike-wave discharges was then 6.2 + 2.7 sec. While inspecting the E E G s made during the FI task, it was noticed that microsleeps ( > 1 and < 5 sec) emerged incidentally. Since microsleeps might influence the P R P as well, it was decided to discriminate also between trials with and without microsleeps and between trials with and
TABLE I Grand mean of the post-reinforcement pauses (PRPs) during the EEG controlled FI sessions in which spike-wave discharges occurred. A distinction is made between trials with and without spike-wave discharges and between trials with and without microsleeps. Between brackets the number of trials and the number of rats is given (n t and ns, respectively). First the mean PRP was obtained for each subject, then the grand mean was obtained.
Spike-wace discharges The numbers of spike-wave discharges in the 4 baseline and 5 FI sessions are presented in Fig. 2. An analysis of variance with repeated (days) measure for within subjects showed no day effect for T 1 - T 5 . This
10
Spike-wave activity
No sleep Sleep
No
Yes
37.2 (nt = 600, ns = 7) 85.8 (n t = 17, ns = 3)
58.4 (nt = 30, ns = 7) 92.6 (n t = 18, n s = 4)
SPIKE-WAVE 1)IS('ttARGKS ANt) PERFORMANCE without spike-wave discharges. In Table 1 the results are presentcd. Thirty trials of 7 animals contained an unambiguous waking E E G with spikc-wave discharges. The PRP for each subjcct in trials with spike-wave discharges was h)nger compared to all trials without discharges. In these sessions, the grand mean of the PRP was significantly prolonged comparcd to the grand mean of all trials without spike-wave discharges (58.4 +_ 6.8 vs. 37.2 +_ 7.1 sec: P < 0.05). Sleep also prolonged the PRP in trials without spike-wave dischargcs (85.8 ± 31 vs. 37.2 ± 7.1 scc: I' <0.05). The increase in the PRP in sessions with spike-wave discharges (21.2 + 12.1 sec) was longer than thc moan duration of the spike-wave dischargcs (~).2 _+ 2.7 scc).
Discussi4m
Two major results were obtained. The first is that during the execution of thc task, a significantly smaller number of spikc-wave discharges was found compared to the prcceding lind succeeding baseline hours. Thc dccreasc m the number of spikc-wave dischargcs during the task hours was present on both days during which control mcasurcments were taken. It can be safely concluded that the number of spike-wave discharges dccreased during the task. This result seems in line with the well documented relationship between vigilance, arousal and absence epilepsy in man in which it is also rcporlcd that high vigilance is accompanied by a low number of spike-wave discharges and low vigilance by a high number of spike-wave discharges (see for rcviews Vieth lclS(~ and Coenen et al. 1991). A simihir phenomenon might have been present in the results dcscribed by Aarts ct a[. (1984). They pointed out that a task in itself can dcpress epileptic activity in children with gencralized absence epilepsy. It seems that in the present experiment a low level of vigilance during the baseline sessions and a high arousal or vigilance lcvcl during: the task, influences the number of spike-wavc discharges. It can therefore be concluded that a reversc relationship exists between the levcl of vigilance and the number of spike-wave discharges. The second major result was that the occurrence of spike-wave discharges changed the performance c)f epileptic rats in the t:'l task. The PRP was prolonged in trials with spike-wavc discharges compared to trials without discharges and the prolongation was longer than the duration of thc spike-wave discharge itself. Additionally, it was found that microsleeps also prolong thc duration of the PRPs. This finding helps to interprct thc prolonged PRP. It is reasonablc to assume that during sleep information processing is s o m e -
133 what diminished. In the present experimcnt it was found that microsleeps and spike-wave discharges had in common that both of them increase the PRP. Thcrcfore it is thought thai information processing, as measured by the change in PRP, is also diminished during the prescncc of spike-wave discharges. In fact, spikcwavc discharges might have reduced amncsia, comparable to amnesia induced by clectroconvulsive shocks, but to a much smaller degree. Amnesia has been reported to occur after gencralizcd 3 Hz spikc-wavc discharges in man (Jus and Jus 1%2: Ounstead et al. 1963). The possibility cannot bc excluded that part of the fixed interval has been x~ipcd out by thc excessive electrical activity and consequcntly, an overestimation of the interval occurs, In line with thc vicw that information processing is disruptcd during trials with spikewavc discharges, lnoue ct al. (1991)havc found that both primary lind secondary componcnts of thc visual evoked potcntial arc differentially disruptcd during spikc-wave discharges compared to wakcfulncss, slow wave sleep lind REM sleep. Inouc's preliminary restllts togcther with the present data strongly suggest that information processing might be disrupicd during the presence of spike-wave discharges. Further studics will bc directed towards possible dcficits in other typcs of cognitive activity such as pcrccption, central processing and short-term mcmol3' during spike-wave discharges. The prolongation of the PRP was longer than the duration of the spike-wave dischargc, suggesting that more time is missed by the animal than the actual duration of the aberrant activit$. It is often thought that only during the presence of the spike-wave arc behavioral changcs prescnt in absence cpilcpsy, ttowever, Mirsky anti Grady (lq88) found a significant incrcasc in the power of 3 - 4 ttz KEG frcqucncy bands as early as 20 sec preceding the epileptic bursts, monotonically increasing until cumulating in the fully developed spike-wave bursts, h can be spcculatcd that the long lasting pcrformance changes are rclalcd to clcctrophysiological antecedcnts prcccding the fully developcd spikc-wavc discharge. It can be concluded that the tinting of the first responsc in a fixed interval schedulc ot rcinforcemcnt is changed by a spiko.wavc discharge. Furthermorc, thc decrcasc of the numbcr of spikc-wavc discharges during the execution of the task is in line with what could be cxpected from the extensive literature on the relationship between vigilance, activity, arousal and itbsencc epilcpsy m man. P,oth typcs of rcsults form additional evidence for the view that spike-wave dischargcs arc genuinc cpilcptic phenomena. It sccms that thc prescnt results positivcly contribute in the debate about the cpileptic naturc of the spike-wavc discharges in gcneral and in particular to the validity of the W A G / R i j strain as a model for gcncralized ilbsencc epilepsy in man.
434 Dr. E.L.J.M. Van Luijtelaar is supported by the Royal Dutch Academy of Sciences (K.N.A.W.).
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