159
Epilepsy Res., 11(19(n) 159-166
Elsevier EPIRES 00466
Positive transfer of audiogenic kindling to electrical hippocampal kindling in rats
Edouard HirschaTb, Bruno Matonayb , Mar~e~tc Vergnesb, Antoine Depaulisb and Christian Marescauxa*b “Clirdque ~eurologiq~,
C. H. U., 67091 Strasbourg Cedex and bCentre de Neurochimie,
UPR 419,6708$
Strasbourg Cedex (Frartce)
(Received 1 November 1991; revision received 17 January 1992; accepted 20 January 1992) Key words: Audiogenic seizures; Kindling; Hippocampus;
Animal model; Epiiepsy
Audiogenic seizures in genetically susceptible rodents are provoked by intense acoustic stimulations which result in a tonic seizure associated with a short flattening of the EEG. These seizures have been shown to involve primarily brainstem structures. Daily exposure to sound for 30-40 days produced a permanent change in the evoked seizure with development of facial myoclonias, rearing and falling, or of tonic-clonic seizures accompanied by high amplitude cortical spike-and-wave discharges. Kindled audiogenic seizures appear similar to seizures kindled from amygdala or hippocampus, suggesting that repeated auditory stimulations cause a progressive propagation of the epileptic discharge toward limbic structures. To verify this hypothesis, the behavioral and EEG development of electrical hippocampal kindling has been studied in 7 non epileptic controls (NE), 8 acoustic susceptible (AS), and 8 audiogenic kindled rats (KAS). The behavioral and EEG development of the electrical hippocampal kindling was similar in the AS and the NE rats. However, 2 animals in the AS group but no controls exhibited behavioral running and bouncing during the course of hippocampal kindling. In the KAS group, the ~p~c~p~ kinking was clearly facilitated as compared to NE and AS: behavioral stage 3 5 was reached in a mean of 4 stimulations in KAS versus 30 and 22 stimulations respectively in NE and AS groups. This positive transfer phenomenon suggests that during kindling of audiogenic seizures, epileptic discharge spreads from the brainstem to the forebrain and progressively involves the hippocampus.
INTRODUCTION A strain of Wistar rats was inbred in our laboratory for its susceptibility to sound. In this strain, a prolonged high intensity acoustic stimulus induces audiogenic seizures (AS) characterized by 1 or 2 wild running episodes, followed by a tonic phase with dorsal hyperextension. The tonic phase is associated with a short flattening of the EEG preCorrespondence
C.H.U.,
to: C. Marescaux, Clinique Neurologique, 1 place de I’H6pital,67091 StrasbourgCedex, France.
0920-i211/92/$05.00~
ceding a regular low voltage theta-like activity11V22. Similar results are reported in other rodent strains29*‘2~‘5.The absence of cortical spikes or spike-and-wave is explained by the fact that AS originate in brainstem structures (inferior colliculus and reticular formation) and do not involve the CorteX’A8.24.
When sound susceptible Wistar rats are subjected to 40 daily acoustic stimulations, facial and forelimb clonus, rearing and falling, or tonic-clonic seizures appear progressively. The myoclonic or tonic-clonic seizures are accompanied by high am-
1992 Elsevier Science Publishers B.V. Al1 rights reserved
plitude spike or spike-and-wave discharges, for 40-120 s. These behavioral and EEG modifications, which can be elicited by a renewed acoustic stimulation several months after discontinuation of acoustic stimulations, evoke a phenomenon similar to kindling4~‘0~1’~22. The EEG and motor symptoms of kindled audiogenie seizures (KAS) appear very similar to those of seizures kindled from amygdala or hippocampus, suggesting that repeated auditory stimulations cause a progressive propagation of the epileptic discharge toward limbic structures. To verify this hypothesis, this study was designed to examine a possible positive transfer of audiogenic kindling to electrical hippocampal kindling. MATERIAL
AND METHODS
Three groups of male Wistar rats (300-400 g) were used. They were chosen from a non epileptic control strain, insensitive to acoustic stimuli, and from a strain susceptible to audiogenic seizures. These 2 strains were inbred in our laboratory”.“. All rats were housed in individual cages on a standard 12/12 h light-dark cycle with free access to food and water. (1) Non epileptic control group (NE): 7 rats selected from the control strain failed to show any behavioral response during 2 acoustic stimuli (white noise, 120 dB, lO,~-20,~ Hz, 90 s). (2) Audiogenic seizure group (AS): 8 rats selected from our strain susceptible to acoustic stimuli exhibited a typical audiogenic seizure during each of 2 acoustic stimuli. (3) Kindled audiogenic seizure group (KAS): 8 rats were selected from our sound susceptible strain. Forty daily acoustic stimuli were delivered. All animals exhibited at least 5 fully kindled myoclonic or tonic-clonic audiogenic seizures. Surgery Under pentobarbital anesthesia (40 mg/kg i.p.), the 3 groups were implanted with 3 frontal and parietal single contact cortical electrodes made of stainless steel screws secured in the skull. Bipolar electrodes made of twisted, enameled stainless steel wires (tip diameter 100 pm, vertical inter-
electrode distance 1 mm) were placed in the right dorsal hippocampus (anteroposterior 4 mm, dorsoventral 4 mm, m~diolateral 2 mm; reference lambda). The single contact and the depth electrodes were attached to a microconnector secured to the cranium with dental acrylic cement. EEG was recorded from cortical and bipolar electrodes with an Aivar Minidix EEG apparatus. Bipolar electrodes were connected to a stimulator (AlvarPhysiovar) through a switch box for the duration of the stimulation. Procedure
Three and 4 days after surgery, EEG was recorded while the animals underwent acoustic stimuli”. One week after the second acoustic stimulus, the threshold for elicitation of after-discharges was defined. The electrical stimulus consisted of a 2-s burst of monophasic 1 ms duration square pulses at 50 Hz. The initial current intensity, 20 PA, was increased by 20% steps every 3 min until a hippocampal after-discharge was recorded. One week after the threshold determination, a stimulus twice higher than threshold was delivered daily between 8 a.m. and 10 a.m., for a total of 40 stimulations. For each electrical stimulus, motor seizures were scored using Racine’s scale modified by PineIi6,i7 as described in Table I. In each group, the number of animals demonstrating at least one seizure of stage 3-4, 5-6 and/or 7-8, and the mean number of stimuli necessary to reach each of these stages for the first time were noted (Table 11). The total duration of the after-discharge was measured. When 2 distinct aver-disch~ges were recorded, the total duration represented the sum of both. For each group of rats, the mean duration of the first after-discharge, the mean duration of the longest after-discharge, the number of animals exhibiting after-discharges 5100, 2150 and 2200 s, and the number of stimuli necessary to induce these after-discharges for the first time were measured (Table III). Statistical analyses were performed using Mann-Whitney and chi-square tests. Two days after the last electrical stimulation, 5 acoustic stimuli were delivered at 48-h intervals with simultaneous EEG recording.
161
TABLE I Kindled motor seizure score designation Hip? Motor
Behavioral response
seizure score 0
Animals exhibit a stationary stance with head in a fixed forward posture
1
Facial clonus, noted by bristling whiskers, nose twitching and jaw movements Repetitive vertical head and neck movement Unilateral forelimb clonus Bilateral forelimb clonus with rearing onto hindlimbs
5
Bilateral forelimb clonus with rearing followed by loss of balance
6
Bilateral forelimb clonus and multiple sequence of rearing and falling
7
Running and bouncing
8
Running, bouncing, followed by a tonic phase
At the end of the experiment, the rats were killed with an overdose of pentobarbital. The brain was removed and processed for histological control of electrode placement.
Fig. 1. Electrographic recording of the first electrical hippocampal stimulus in a non epileptic rat. Initial and recurrent electrical after-discharges are clearly separated. CX, cortex; Hip,_hippocampus. Calibrations: 1 s, 4OOpV.
TABLE II Behavioral development of hippocampal kindling in control and audiogenic ratr
(A) Number of rats reaching at least one stage 3-4, 5-6 and 7-8 and number of stimuli necessary to reach these stages for the first time. NE
AS
KAS
number of rats number of stimuli median range
6i-7
6/%
8k.F
22.5 19-39
22c 10-37
40
number of rats number of stimuli median range
9-7
518’
30 22-36
22 lo-32
4a,b
number of rats number of stimuli median range
Oi7
2/g=
6Wb
17.5* 10-25
8.5~ 3-13
Stage
3-4
RESULTS Histological analysis confirmed that the stimulating electrode was always located in the right dorsal hippocampus. In the NE control group (n = 7), the mean (f SEM) after-discharge threshold was 116 f 17 PA. Initially, 2 EEG after-discharges occurred, separated by lo-30 s desynchronized low-voltage activity (Fig. 1). After 18-40 days, the 2 after-discharges which had increased in duration (Table III) finally joined up. When the 2 after-discharges are clearly separated, the behavioral motor symptoms occurred during the initial one, while the recurrent one was asymptomatic. The first stimuli failed to induce any behavioral response. With repetition of stimuli, a classical kindling phenomenon developed slowly in 6/7 rats, which reached the first stage 23 after a mean of 19-39 stimulations (Table II). The duration of after-discharges
i
F/p
L
5-6
7-8
1-16
1-16
(B) Total number of seizures 2 5 during the 40 daily stimulations. NE
AS
KAS
(n=7)
(n=B)
(n=B)
Median
3
6.5’
19.PJ
Range
o-13
O-14
9-33
“P < 0.01 vs. NE; bP < 0.01 vs. AS; WS vs. NE.
*Number of rats in the control groups too small to allow comparison.
162
and their behavioral concomitant varied from one stimulation to the other in a same rat and therefore kindling developed irregularly (Fig. 2). Acoustic stimuli failed to induce audiogenic seizures before and after hippocampal kindling in these animals. In the AS group (n = 8), and audiogenic seizure was recorded prior to electrical hippocampal stimuli in all animals. During the wild running episodes, no EEG paroxysmal abnormalities were observed. During tonic seizures, cortical EEG recordings were characterized by a flattening with rapid (20-30 cps) low amplitude rhythms for a few seconds, followed by a low voltage regular lo-12 cps activity, whereas hippocampal recordings showed high amplitude, lo-12 cps activity (Fig.
--,
cxt
Fig. 3. Electrographic recording of a non kindled audiogenic seizure. Arrow, beginning of acoustic stimulus. CX, cortex; Hip, hippocampus; W.R., wild running; T.S., tonic seizure. Calibrations: 1 s, 4OOpV.
3).
In the AS group, hippocampal kindling developed in a way similar to the NE control group. The
4
.
NE
-.
AS
--.- KAS
Fig. 2. Behavioral response during hippocampal kindling in NE, AS and KAS. Stages defined according to Racine and Pine1 (see text) are represented as mean f SEM.
mean (k SEM) after-discharge threshold was 135 _+30 ,uA. A classical kindling appeared in 618 rats which reached stage 23 after lo-37 stimulations (Fig. 2, Tables II and III). As in the NE group, when 2 after-discharges were clearly separated, the behavioral symptoms occurred during the first one. However, 2 animals from the AS group differed from the NE group: after a few hippocampal stimulations, one AS rat exhibited stage 7 (wild running, bouncing) and one exhibited stage 8 (tonic phase), whereas no NE rats reached stage 7 or 8 (Table II). As in NE animals, high daily variability in seizures occurred for each rat (Fig. 2). Following hippocampal kindling, the sensitivity of AS rats to acoustic stimuli decreased. The first post-kindling acoustic stimulus induced no response in 3 rats, wild running only in 4 rats, and a complete typical tonic seizure in 1 rat. During the 5th post-kindling acoustic stimulus, 6/8 rats again exhibited a typical AS accompanied with cortical and hippocampal lo-12 cps activity, without spikes or spike-and-waves. In the KAS group (n = 8), prior to any hippocampal stimulation, acoustic stimuli induced tonicclonic or clonic seizures accompanied by cortical and hippocampal spike, or spike-and-wave discharges for 50-120 s (Fig. 4). In KAS rats, the mean (& SEM) after-discharge threshold was 131 + 29 PA. Electrical stimuli induced a rapid kindling phenomenon compared to the NE and AS groups. Following the first stimulus, 4 rats exhibited a stage 2,1 rat a stage 4, and 2
163
TABLE III
cx
EEG development of hippocampal kindling in control and audi-
Hip
ogenic rats
t
W.R.
T.S.
(A) Number of rats displaying at least one after-discharge of 2 100; 150 and 200 s and number of stimuli applied to reach these durations for the first time. NE
AS
KAS
number of rats number of stimuli median range
7fl
6kY
8/v
19 4-25
20s 9-40
l.Y,b 1-17
number of rats number of stimuli median range
Ii7
2J8c
8wb
22 -
28.5’ 21-36
6’ 1-17
Ii7
0/g
7wb
22 -
-
8* 1-17
After-dbcharge duration (s)
2100
B 150
2200
number of rats number of stimuli median range
(B) Duration of the first after-discharge, of the longest afterdischarge and number of stimuli needed to induce the longest after-discharge. AS
KAS
54 31-74
34= 27-51
87.5a*b 41-215
125 100-215
KZY 77-156
212.5’~~ 185-365
22 19-32
28.5c 12-40
7.5”*b 1-17
NE
First after-discharge (s) median range Longest after-discharge (s) median range Number of stimuli median range
Fig. 4. Electrographic recording of kindled audiogenic seizure (40 stimulations). Arrow, beginning of acoustic stimulus. CX, cortex; Hip, hippocampus; W.R., wild running; T.S., tonic seizure; C.S., clonic seizure. 30 s separate the 1st and the 2nd, and the 2nd and the 3rd pair of lines. Calibrations: 1 s, 4oopV.
ability of seizure stage and duration was extremely high (Fig. 2). When 2 after-discharges were clearly differentiated, the behavioral seizures induced by hippocampal stimulation occurred during the recurrent after-discharge (Fig. 5) and not during the initial one, in contrast with the NE and AS groups. After completion of hippocampal kindling, during the first acoustic stimulus, 1 rat did not respond
‘P < 0.01 vs. NE; bP < 0.01 vs AS; =NS vs. NE.
* Number of rats in the control groups too small to ahow comparison.
rats a stage 6 seizure. The first stage 35 was reached by 8/8 rats after 1-16 stimulations. The mean number of stimuli necessary to reach the first stage 5 was more than 4-fold smaller in KAS compared to AS and NE groups. Five animals repeatedly exhibited stage 7 seizures with wild running and bouncing (Fig. 2, Table II). The mean duration of the first EEG after-discharges and the mean maximum duration of after-discharges were longer than in the NE and AS groups (Table III). As in the previous groups, the individual vari-
Fig. 5. Electrographic recording of the first electrical hippocampal stimulus in a audiogenic kindled rat. C.S., clonic seizure. The recording is interrupted 70 s before the end of the seizure. Calibrations: 1 s, 4OOrV.
164
at all, 2 exhibited only wild running, and 5 had a typical non kindled AS, accompanied by a lo-12 cps cortical and hippocampal activity, without spikes or spike-and-waves. During the 5th postkindling acoustic stimulus, all rats exhibited classical audiogenic seizures accompanied in 5/S rats by myoclonias and spike-and-wave discharges. DISCUSSION Audiogenic seizure is a ‘brainstem’ seizure, related to a genetically determined dysfunction of the auditory pathway’*24. Strong acoustic stimuli induce a paroxysmal response in the inferior colliculus accompanied by a wild running phase. Spread of the paroxysmal discharges to the nucleus reticularis pontis oralis (RPO) of the mesencephalic reticular formation results in a tonic seizure’.“,‘.‘“. Experimental data have demonstrated that the forebrain is not involved in this mechanism. Cortical spreading depression, or transcollicular section (‘endphale isole’), fail to modify the behavioral symptomatology of audiogenic seizures”‘-‘“. Moreover, during the tonic phase of audiogenic seizures, cortical EEG recordings do not reveal any epileptic discharges, but only an electrodecremental activity followed by a rhythmic lo-12 cps activity likely due to simultaneous recording through the cortex of a high amplitude hippocampal theta rhythm. Indeed, simultaneous hippocampal depth recordings during the tonic phase shows a rhythmic high amplitude lo-12 cps activity (Fig. 3). This hippocampal theta activity does not seem to be related to an epileptic discharge. Similar lo-12 Hz hippocampal theta waves are recorded under physiological conditions during REM sleep, in ‘cerveau isole’ preparation? and following a non convulsant electrical stimulation of the RP02’. In the NE group, the hippocampal stimulationinduced kindling was slow and progressed irregularly. In 1 animal, stage 3 was not reached, and only in 2 rats were stage 5 seizures consistently elicited during the course of the experiment. Similar results were obtained by others: among limbic structures, the dorsal hippocampus was shown to have the lowest kindling rate, with a mean of 40-80 stimulations, according to the intensity ap-
plied4,14. The high daily variations of the responses observed in our experiments are in accordance with previously described data, when daily stimulations were applied. This variability, which may be related to temporary inhibitions or refractoriness, can be reduced by increasing the inter-> stimulus interval to 2 or 3 days, as soon as stage 3 seizures are elicited14.“. The development of the behavioral and EEG concomitants of hippocampal kindling were not found to be accelerated in the AS group when compared to NE group (Fig. 2, Tables II and III), although different results have been reported in other strains of rodents susceptible to sound: electrical angular bundle kindling in the genetically epilepsy-prone rats20 and olfactory bulb kindling in audiogenic mice were reported to be accelerated6. However, 2 animals in the AS group exhibited behavioral stages 7 and 8 during the course of hippocampal kindling. Running, bouncing, and tonic fits did not occur in NE rats and are reported after more than 120 electrical stimulations in amygdala kindling l6. These data sugg est that the spread of hippocampal seizures may follow different pathways in AS and in NE animals. This unusual spread of seizure might be related to a genetic predisposition, or to the prior modification of the excitability of some pathways due to the 4 control audiogenic seizures induced prior to the electrical hippocampal kindling. After repeated auditory stimuli, a kindling of audiogenic seizures occurred characterized by clonic or tonic-clonic seizures associated with high amplitude cortical and hippocampal spike-and-wave discharges (Fig. 4). In these rats (KAS group), subsequent hippocampal kindling was clearly facilitated (Fig. 2, Tables II and III). This facilitation seems to be related to an increase of the recurrent after-discharge (Fig. 5). There is no clear explanation for this finding. Similarly, McIntyre (personal communication) observed behavioral seizures during recurrent after-discharge in a strain genetically hypersensitive to hippocampal kindling. Goddard et al.” demonstrated that following kindling of a primary brain site, the number of electrical stimuli necessary to induce a kindling phenomenon at a secondary brain site was re-
165
duced. This effect, defined as positive transfer, was usually reported from one site to another in the forebrainr4Tu. Sato et a1.19 reported that following unilateral electrical hippocampal stimulation in cats, a positive transfer phenomenon was observed in the contralateral hippocampus, in both amygdalae, and in globus pallidus in both sides. They also reported transfer to the ipsilateral midbrain reticular formation in 1 animal. Racine et al.” suggested that spread of after-discharges from the primary site to the secondary site is a crucial factor for a positive transfer. In the present experiment, a positive transfer was observed between audiogenic kindling and hippocampal kindling, suggesting that during repeated auditory stimuli, the audiogenic seizure spread from the brainstem to the hippocampus and forebrain. This hypothesis was confirmed in the current study by the recordings of hippocampal and cortical epileptic discharges during kindling of audiogenic seizures (Fig. 4). Following electrical hippocampal kindling, AS animals did not exhibit an accelerated audiogenic
REFERENCES 1 Browning, R.A., Nelson, D.K., Mogharreban, N., Jobe, P.C. and Laird, H.E., Effect of midbrain and pontine tegmental lesions on audiogenic seizures in genetically epilepsy-prone rats, Epilepsia, 26 (1985) 175-183. 2 Collins, R.L., Audiogenic seizures. In: D.P. Purpura, J.K. Penry, D.B. Tower, D.M. Woodbury and R. Walter (Eds.), Experimental Models of Epilepsy, Raven Press, New York, NY, 1972, pp. 347-372. 3 Furlow, T.W., A comparison of shortened-latency auditory-evoked potentials in two strains of mice: possible neurophysiological correlates of susceptibility to audiogenic seizures?, Brain Res., 220 (1981) 378-385. 4 Goddard, G.V., McIntyre, C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 5 Gottesmann, C., What the cerveau isole preparation tells us nowadays about sleep-wake mechanisms?, Neurosci. Behay. Rev., 12 (1988) 39-48. 6 Green, R. and Seyfried, T., Kindling susceptibility and ge-
netic predisposition in inbred mice, Epilepsia, 32 (1991) 23-26. 7 Jobe, P.C., Pharmacology of audiogenic seizures. In: R.D. Brown and E.A. Daigneault (Eds.), The Pharmacology of Hearing. Experimental and Clinical Bases, Wiley Intersci-
kindling, but showed’less susceptibility to acoustic stimulations. Moreover, following electrical hippocampal kindling, an acoustic stimulus induced a typical audiogenic seizure without electrocortical discharge in KAS animals previously kindled to sound. Goddard et a1.4 showed that following a positive transfer from a primary to a secondary site, the first restimulation at the primary site loses its ability to trigger convulsion, and several trials are necessary to reestablish the response. In the current study, this negative post-transfer interference might also be related to the negative control that the hippocampus exerted on audiogenic seizure susceptibility. Hippocampal electrical stimulations were shown to inhibit audiogenic seizure, while ablation of the hippocampus potentiated audiogenic seizures18. In conclusion, electrical hippocampal kindling was accelerated after audiogenic kindling in acoustic susceptible animals. This positive transfer phenomenon confirms that during audiogenic kindling, epileptic discharges spread from the brainstem to limbic structures.
ence, New York, NY, 1981, pp. 271-304. 8 Kesner, R.P., Subcortical mechanisms of audiogenic seizures, Exp. Neurol., 15 (1966) 192-205. 9 Krushinsky, L.V., Etude physiologique des differents types de crises convulsives de l’tpilepsie audiogbne du rat. In: Psychophysiologie, Crke Audiogene,
Neuropharmacologie
et Biochimie de la
Colloques Intemationaux du C.N.R.S., No. 112, Paris, 1963, pp. 71-92. 10 Krushinsky, L.V., Molodkina, L.N., Fless, D.A., Dobrokhotova, L.P., Steshenko, A.P., Semiokhina, A.F., Zorina, Z.A. and Romanova, L.G., The functional state of the brain during sonic stimulation. In: B.L. Welch and AS. Welch (Eds.), Physiological Effects of Noise, Plenum Press, New York, NY, 1970, pp. 159-183. 11 Marescaux, C., Vergnes, M., Kiesmann, M., Depaulis, A., Micheletti, G. and Warter, J.M., Kindling of audiogenic seizures in Wistar rats: an EEG study, Exp. Neural., 97 (1987) 160-168. 12 Maxson, S.C. and Cowen, J.S., Electroencephalographic correlates of the audiogenic seizure response of inbred mice, Physiol. Behav., 16 (1976) 623-629. 13 McCown, T.J., Greenwood, R.S., Frye, G.D. and Breese, G.R., Electrically elicited seizures from the inferior colliculus: a potential site for the genesis of epilepsy, Exp. Neurot., 86 (1984) 527-542. 14 McIntyre Burnham, W., Primary and ‘transfer’ seizure de-
166 velopment in the kindled rat. In: J.A. Wada and R.T. Ross (Eds.), Kindling, Raven Press, New York, NY, 1976, pp. 61-83. 15 Niaussat, M.M. and Laget, P., Etude Clectroenc&phalographique de la crise audiogene de la souris. In: Psychophysiologic, Neurophamtacologie et Biochimie de la Crise Audiogtne, Colloques Internationaux du C.N.R.S., No. 112, Paris, 1963, pp. 181-197. 16 Pine], J.P. and Rovner, L.I., Experimental epileptogenesis: kindling-induced epilepsy in rats, Exp. Neurol., 58 (1978) 190-202. 17 Racine, R., Okujava, V. and Chipasvili, S., Modification of seizure activity by electrical stimulation, Electroenceph. Clin. Neurophysiol.,
32 (1972) 269-299.
18 Reid, H.M., Mamott, B.D. and Bowler, K.J., Hippocam-
pal lesions render SJL/J mice susceptible to audiogenic seizures, Exp. Neural., 82 (1983) 237-240. 19 Sato, M. and Nakashima, T., Kindling: secondary epilepto-
genesis, sleep and catecholamines, Can. J. Neural. Sci., 2 (1975) 439-446. 20 Savage, D.D., Reigel, C.E. and Jobe, P.C., Angular bundle kindling is accelerated in rats with a genetic predisposition to acoustic stimulus-induced seizures, Brain Res., 376 (1986) 412-415. 21 Vertes, R.P., An analysis of ascending brain stem systems
involved in hippocampal synchronization and desynchronization, J. Neurophysiol., 46 (1981) 1140-1159. 22 Vergnes, M., Kiesmann, M., Marescaux, C., Depaulis, A.. Micheletti, G. and Warter, J.M., Kindling of audiogenic seizures in the rat. Int. J. Neurosci., 36 (1987) 167- 176. 23 Wada. J.A., Secondary cerebral function alterations examined in the kindling model of epilepsy. In: A. Mayersdorf and R.P. Schmidt (Eds.), Secondary Epileptogenesis, Raven Press, New York, NY, 1982, pp. 45-87. 24 Willot, J.F. and Ming Lu, S., Midbrain pathways of audiogenie seizures in DBAiA mice, Exp. Neural.. 70 (1980) 288-299.
Epilepsy Res., 11(19(n) 159-166
Elsevier EPIRES 00466
Positive transfer of audiogenic kindling to electrical hippocampal kindling in rats
Edouard HirschaTb, Bruno Matonayb , Mar~e~tc Vergnesb, Antoine Depaulisb and Christian Marescauxa*b “Clirdque ~eurologiq~,
C. H. U., 67091 Strasbourg Cedex and bCentre de Neurochimie,
UPR 419,6708$
Strasbourg Cedex (Frartce)
(Received 1 November 1991; revision received 17 January 1992; accepted 20 January 1992) Key words: Audiogenic seizures; Kindling; Hippocampus;
Animal model; Epiiepsy
Audiogenic seizures in genetically susceptible rodents are provoked by intense acoustic stimulations which result in a tonic seizure associated with a short flattening of the EEG. These seizures have been shown to involve primarily brainstem structures. Daily exposure to sound for 30-40 days produced a permanent change in the evoked seizure with development of facial myoclonias, rearing and falling, or of tonic-clonic seizures accompanied by high amplitude cortical spike-and-wave discharges. Kindled audiogenic seizures appear similar to seizures kindled from amygdala or hippocampus, suggesting that repeated auditory stimulations cause a progressive propagation of the epileptic discharge toward limbic structures. To verify this hypothesis, the behavioral and EEG development of electrical hippocampal kindling has been studied in 7 non epileptic controls (NE), 8 acoustic susceptible (AS), and 8 audiogenic kindled rats (KAS). The behavioral and EEG development of the electrical hippocampal kindling was similar in the AS and the NE rats. However, 2 animals in the AS group but no controls exhibited behavioral running and bouncing during the course of hippocampal kindling. In the KAS group, the ~p~c~p~ kinking was clearly facilitated as compared to NE and AS: behavioral stage 3 5 was reached in a mean of 4 stimulations in KAS versus 30 and 22 stimulations respectively in NE and AS groups. This positive transfer phenomenon suggests that during kindling of audiogenic seizures, epileptic discharge spreads from the brainstem to the forebrain and progressively involves the hippocampus.
INTRODUCTION A strain of Wistar rats was inbred in our laboratory for its susceptibility to sound. In this strain, a prolonged high intensity acoustic stimulus induces audiogenic seizures (AS) characterized by 1 or 2 wild running episodes, followed by a tonic phase with dorsal hyperextension. The tonic phase is associated with a short flattening of the EEG preCorrespondence
C.H.U.,
to: C. Marescaux, Clinique Neurologique, 1 place de I’H6pital,67091 StrasbourgCedex, France.
0920-i211/92/$05.00~
ceding a regular low voltage theta-like activity11V22. Similar results are reported in other rodent strains29*‘2~‘5.The absence of cortical spikes or spike-and-wave is explained by the fact that AS originate in brainstem structures (inferior colliculus and reticular formation) and do not involve the CorteX’A8.24.
When sound susceptible Wistar rats are subjected to 40 daily acoustic stimulations, facial and forelimb clonus, rearing and falling, or tonic-clonic seizures appear progressively. The myoclonic or tonic-clonic seizures are accompanied by high am-
1992 Elsevier Science Publishers B.V. Al1 rights reserved
plitude spike or spike-and-wave discharges, for 40-120 s. These behavioral and EEG modifications, which can be elicited by a renewed acoustic stimulation several months after discontinuation of acoustic stimulations, evoke a phenomenon similar to kindling4~‘0~1’~22. The EEG and motor symptoms of kindled audiogenie seizures (KAS) appear very similar to those of seizures kindled from amygdala or hippocampus, suggesting that repeated auditory stimulations cause a progressive propagation of the epileptic discharge toward limbic structures. To verify this hypothesis, this study was designed to examine a possible positive transfer of audiogenic kindling to electrical hippocampal kindling. MATERIAL
AND METHODS
Three groups of male Wistar rats (300-400 g) were used. They were chosen from a non epileptic control strain, insensitive to acoustic stimuli, and from a strain susceptible to audiogenic seizures. These 2 strains were inbred in our laboratory”.“. All rats were housed in individual cages on a standard 12/12 h light-dark cycle with free access to food and water. (1) Non epileptic control group (NE): 7 rats selected from the control strain failed to show any behavioral response during 2 acoustic stimuli (white noise, 120 dB, lO,~-20,~ Hz, 90 s). (2) Audiogenic seizure group (AS): 8 rats selected from our strain susceptible to acoustic stimuli exhibited a typical audiogenic seizure during each of 2 acoustic stimuli. (3) Kindled audiogenic seizure group (KAS): 8 rats were selected from our sound susceptible strain. Forty daily acoustic stimuli were delivered. All animals exhibited at least 5 fully kindled myoclonic or tonic-clonic audiogenic seizures. Surgery Under pentobarbital anesthesia (40 mg/kg i.p.), the 3 groups were implanted with 3 frontal and parietal single contact cortical electrodes made of stainless steel screws secured in the skull. Bipolar electrodes made of twisted, enameled stainless steel wires (tip diameter 100 pm, vertical inter-
electrode distance 1 mm) were placed in the right dorsal hippocampus (anteroposterior 4 mm, dorsoventral 4 mm, m~diolateral 2 mm; reference lambda). The single contact and the depth electrodes were attached to a microconnector secured to the cranium with dental acrylic cement. EEG was recorded from cortical and bipolar electrodes with an Aivar Minidix EEG apparatus. Bipolar electrodes were connected to a stimulator (AlvarPhysiovar) through a switch box for the duration of the stimulation. Procedure
Three and 4 days after surgery, EEG was recorded while the animals underwent acoustic stimuli”. One week after the second acoustic stimulus, the threshold for elicitation of after-discharges was defined. The electrical stimulus consisted of a 2-s burst of monophasic 1 ms duration square pulses at 50 Hz. The initial current intensity, 20 PA, was increased by 20% steps every 3 min until a hippocampal after-discharge was recorded. One week after the threshold determination, a stimulus twice higher than threshold was delivered daily between 8 a.m. and 10 a.m., for a total of 40 stimulations. For each electrical stimulus, motor seizures were scored using Racine’s scale modified by PineIi6,i7 as described in Table I. In each group, the number of animals demonstrating at least one seizure of stage 3-4, 5-6 and/or 7-8, and the mean number of stimuli necessary to reach each of these stages for the first time were noted (Table 11). The total duration of the after-discharge was measured. When 2 distinct aver-disch~ges were recorded, the total duration represented the sum of both. For each group of rats, the mean duration of the first after-discharge, the mean duration of the longest after-discharge, the number of animals exhibiting after-discharges 5100, 2150 and 2200 s, and the number of stimuli necessary to induce these after-discharges for the first time were measured (Table III). Statistical analyses were performed using Mann-Whitney and chi-square tests. Two days after the last electrical stimulation, 5 acoustic stimuli were delivered at 48-h intervals with simultaneous EEG recording.
161
TABLE I Kindled motor seizure score designation Hip? Motor
Behavioral response
seizure score 0
Animals exhibit a stationary stance with head in a fixed forward posture
1
Facial clonus, noted by bristling whiskers, nose twitching and jaw movements Repetitive vertical head and neck movement Unilateral forelimb clonus Bilateral forelimb clonus with rearing onto hindlimbs
5
Bilateral forelimb clonus with rearing followed by loss of balance
6
Bilateral forelimb clonus and multiple sequence of rearing and falling
7
Running and bouncing
8
Running, bouncing, followed by a tonic phase
At the end of the experiment, the rats were killed with an overdose of pentobarbital. The brain was removed and processed for histological control of electrode placement.
Fig. 1. Electrographic recording of the first electrical hippocampal stimulus in a non epileptic rat. Initial and recurrent electrical after-discharges are clearly separated. CX, cortex; Hip,_hippocampus. Calibrations: 1 s, 4OOpV.
TABLE II Behavioral development of hippocampal kindling in control and audiogenic ratr
(A) Number of rats reaching at least one stage 3-4, 5-6 and 7-8 and number of stimuli necessary to reach these stages for the first time. NE
AS
KAS
number of rats number of stimuli median range
6i-7
6/%
8k.F
22.5 19-39
22c 10-37
40
number of rats number of stimuli median range
9-7
518’
30 22-36
22 lo-32
4a,b
number of rats number of stimuli median range
Oi7
2/g=
6Wb
17.5* 10-25
8.5~ 3-13
Stage
3-4
RESULTS Histological analysis confirmed that the stimulating electrode was always located in the right dorsal hippocampus. In the NE control group (n = 7), the mean (f SEM) after-discharge threshold was 116 f 17 PA. Initially, 2 EEG after-discharges occurred, separated by lo-30 s desynchronized low-voltage activity (Fig. 1). After 18-40 days, the 2 after-discharges which had increased in duration (Table III) finally joined up. When the 2 after-discharges are clearly separated, the behavioral motor symptoms occurred during the initial one, while the recurrent one was asymptomatic. The first stimuli failed to induce any behavioral response. With repetition of stimuli, a classical kindling phenomenon developed slowly in 6/7 rats, which reached the first stage 23 after a mean of 19-39 stimulations (Table II). The duration of after-discharges
i
F/p
L
5-6
7-8
1-16
1-16
(B) Total number of seizures 2 5 during the 40 daily stimulations. NE
AS
KAS
(n=7)
(n=B)
(n=B)
Median
3
6.5’
19.PJ
Range
o-13
O-14
9-33
“P < 0.01 vs. NE; bP < 0.01 vs. AS; WS vs. NE.
*Number of rats in the control groups too small to allow comparison.
162
and their behavioral concomitant varied from one stimulation to the other in a same rat and therefore kindling developed irregularly (Fig. 2). Acoustic stimuli failed to induce audiogenic seizures before and after hippocampal kindling in these animals. In the AS group (n = 8), and audiogenic seizure was recorded prior to electrical hippocampal stimuli in all animals. During the wild running episodes, no EEG paroxysmal abnormalities were observed. During tonic seizures, cortical EEG recordings were characterized by a flattening with rapid (20-30 cps) low amplitude rhythms for a few seconds, followed by a low voltage regular lo-12 cps activity, whereas hippocampal recordings showed high amplitude, lo-12 cps activity (Fig.
--,
cxt
Fig. 3. Electrographic recording of a non kindled audiogenic seizure. Arrow, beginning of acoustic stimulus. CX, cortex; Hip, hippocampus; W.R., wild running; T.S., tonic seizure. Calibrations: 1 s, 4OOpV.
3).
In the AS group, hippocampal kindling developed in a way similar to the NE control group. The
4
.
NE
-.
AS
--.- KAS
Fig. 2. Behavioral response during hippocampal kindling in NE, AS and KAS. Stages defined according to Racine and Pine1 (see text) are represented as mean f SEM.
mean (k SEM) after-discharge threshold was 135 _+30 ,uA. A classical kindling appeared in 618 rats which reached stage 23 after lo-37 stimulations (Fig. 2, Tables II and III). As in the NE group, when 2 after-discharges were clearly separated, the behavioral symptoms occurred during the first one. However, 2 animals from the AS group differed from the NE group: after a few hippocampal stimulations, one AS rat exhibited stage 7 (wild running, bouncing) and one exhibited stage 8 (tonic phase), whereas no NE rats reached stage 7 or 8 (Table II). As in NE animals, high daily variability in seizures occurred for each rat (Fig. 2). Following hippocampal kindling, the sensitivity of AS rats to acoustic stimuli decreased. The first post-kindling acoustic stimulus induced no response in 3 rats, wild running only in 4 rats, and a complete typical tonic seizure in 1 rat. During the 5th post-kindling acoustic stimulus, 6/8 rats again exhibited a typical AS accompanied with cortical and hippocampal lo-12 cps activity, without spikes or spike-and-waves. In the KAS group (n = 8), prior to any hippocampal stimulation, acoustic stimuli induced tonicclonic or clonic seizures accompanied by cortical and hippocampal spike, or spike-and-wave discharges for 50-120 s (Fig. 4). In KAS rats, the mean (& SEM) after-discharge threshold was 131 + 29 PA. Electrical stimuli induced a rapid kindling phenomenon compared to the NE and AS groups. Following the first stimulus, 4 rats exhibited a stage 2,1 rat a stage 4, and 2
163
TABLE III
cx
EEG development of hippocampal kindling in control and audi-
Hip
ogenic rats
t
W.R.
T.S.
(A) Number of rats displaying at least one after-discharge of 2 100; 150 and 200 s and number of stimuli applied to reach these durations for the first time. NE
AS
KAS
number of rats number of stimuli median range
7fl
6kY
8/v
19 4-25
20s 9-40
l.Y,b 1-17
number of rats number of stimuli median range
Ii7
2J8c
8wb
22 -
28.5’ 21-36
6’ 1-17
Ii7
0/g
7wb
22 -
-
8* 1-17
After-dbcharge duration (s)
2100
B 150
2200
number of rats number of stimuli median range
(B) Duration of the first after-discharge, of the longest afterdischarge and number of stimuli needed to induce the longest after-discharge. AS
KAS
54 31-74
34= 27-51
87.5a*b 41-215
125 100-215
KZY 77-156
212.5’~~ 185-365
22 19-32
28.5c 12-40
7.5”*b 1-17
NE
First after-discharge (s) median range Longest after-discharge (s) median range Number of stimuli median range
Fig. 4. Electrographic recording of kindled audiogenic seizure (40 stimulations). Arrow, beginning of acoustic stimulus. CX, cortex; Hip, hippocampus; W.R., wild running; T.S., tonic seizure; C.S., clonic seizure. 30 s separate the 1st and the 2nd, and the 2nd and the 3rd pair of lines. Calibrations: 1 s, 4oopV.
ability of seizure stage and duration was extremely high (Fig. 2). When 2 after-discharges were clearly differentiated, the behavioral seizures induced by hippocampal stimulation occurred during the recurrent after-discharge (Fig. 5) and not during the initial one, in contrast with the NE and AS groups. After completion of hippocampal kindling, during the first acoustic stimulus, 1 rat did not respond
‘P < 0.01 vs. NE; bP < 0.01 vs AS; =NS vs. NE.
* Number of rats in the control groups too small to ahow comparison.
rats a stage 6 seizure. The first stage 35 was reached by 8/8 rats after 1-16 stimulations. The mean number of stimuli necessary to reach the first stage 5 was more than 4-fold smaller in KAS compared to AS and NE groups. Five animals repeatedly exhibited stage 7 seizures with wild running and bouncing (Fig. 2, Table II). The mean duration of the first EEG after-discharges and the mean maximum duration of after-discharges were longer than in the NE and AS groups (Table III). As in the previous groups, the individual vari-
Fig. 5. Electrographic recording of the first electrical hippocampal stimulus in a audiogenic kindled rat. C.S., clonic seizure. The recording is interrupted 70 s before the end of the seizure. Calibrations: 1 s, 4OOrV.
164
at all, 2 exhibited only wild running, and 5 had a typical non kindled AS, accompanied by a lo-12 cps cortical and hippocampal activity, without spikes or spike-and-waves. During the 5th postkindling acoustic stimulus, all rats exhibited classical audiogenic seizures accompanied in 5/S rats by myoclonias and spike-and-wave discharges. DISCUSSION Audiogenic seizure is a ‘brainstem’ seizure, related to a genetically determined dysfunction of the auditory pathway’*24. Strong acoustic stimuli induce a paroxysmal response in the inferior colliculus accompanied by a wild running phase. Spread of the paroxysmal discharges to the nucleus reticularis pontis oralis (RPO) of the mesencephalic reticular formation results in a tonic seizure’.“,‘.‘“. Experimental data have demonstrated that the forebrain is not involved in this mechanism. Cortical spreading depression, or transcollicular section (‘endphale isole’), fail to modify the behavioral symptomatology of audiogenic seizures”‘-‘“. Moreover, during the tonic phase of audiogenic seizures, cortical EEG recordings do not reveal any epileptic discharges, but only an electrodecremental activity followed by a rhythmic lo-12 cps activity likely due to simultaneous recording through the cortex of a high amplitude hippocampal theta rhythm. Indeed, simultaneous hippocampal depth recordings during the tonic phase shows a rhythmic high amplitude lo-12 cps activity (Fig. 3). This hippocampal theta activity does not seem to be related to an epileptic discharge. Similar lo-12 Hz hippocampal theta waves are recorded under physiological conditions during REM sleep, in ‘cerveau isole’ preparation? and following a non convulsant electrical stimulation of the RP02’. In the NE group, the hippocampal stimulationinduced kindling was slow and progressed irregularly. In 1 animal, stage 3 was not reached, and only in 2 rats were stage 5 seizures consistently elicited during the course of the experiment. Similar results were obtained by others: among limbic structures, the dorsal hippocampus was shown to have the lowest kindling rate, with a mean of 40-80 stimulations, according to the intensity ap-
plied4,14. The high daily variations of the responses observed in our experiments are in accordance with previously described data, when daily stimulations were applied. This variability, which may be related to temporary inhibitions or refractoriness, can be reduced by increasing the inter-> stimulus interval to 2 or 3 days, as soon as stage 3 seizures are elicited14.“. The development of the behavioral and EEG concomitants of hippocampal kindling were not found to be accelerated in the AS group when compared to NE group (Fig. 2, Tables II and III), although different results have been reported in other strains of rodents susceptible to sound: electrical angular bundle kindling in the genetically epilepsy-prone rats20 and olfactory bulb kindling in audiogenic mice were reported to be accelerated6. However, 2 animals in the AS group exhibited behavioral stages 7 and 8 during the course of hippocampal kindling. Running, bouncing, and tonic fits did not occur in NE rats and are reported after more than 120 electrical stimulations in amygdala kindling l6. These data sugg est that the spread of hippocampal seizures may follow different pathways in AS and in NE animals. This unusual spread of seizure might be related to a genetic predisposition, or to the prior modification of the excitability of some pathways due to the 4 control audiogenic seizures induced prior to the electrical hippocampal kindling. After repeated auditory stimuli, a kindling of audiogenic seizures occurred characterized by clonic or tonic-clonic seizures associated with high amplitude cortical and hippocampal spike-and-wave discharges (Fig. 4). In these rats (KAS group), subsequent hippocampal kindling was clearly facilitated (Fig. 2, Tables II and III). This facilitation seems to be related to an increase of the recurrent after-discharge (Fig. 5). There is no clear explanation for this finding. Similarly, McIntyre (personal communication) observed behavioral seizures during recurrent after-discharge in a strain genetically hypersensitive to hippocampal kindling. Goddard et al.” demonstrated that following kindling of a primary brain site, the number of electrical stimuli necessary to induce a kindling phenomenon at a secondary brain site was re-
165
duced. This effect, defined as positive transfer, was usually reported from one site to another in the forebrainr4Tu. Sato et a1.19 reported that following unilateral electrical hippocampal stimulation in cats, a positive transfer phenomenon was observed in the contralateral hippocampus, in both amygdalae, and in globus pallidus in both sides. They also reported transfer to the ipsilateral midbrain reticular formation in 1 animal. Racine et al.” suggested that spread of after-discharges from the primary site to the secondary site is a crucial factor for a positive transfer. In the present experiment, a positive transfer was observed between audiogenic kindling and hippocampal kindling, suggesting that during repeated auditory stimuli, the audiogenic seizure spread from the brainstem to the hippocampus and forebrain. This hypothesis was confirmed in the current study by the recordings of hippocampal and cortical epileptic discharges during kindling of audiogenic seizures (Fig. 4). Following electrical hippocampal kindling, AS animals did not exhibit an accelerated audiogenic
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