Kindling and Its Consequences on Learning in Rats - Science Direct

BEHAVIORAL AND NEURAL BIOLOGY 57, 37--43 (1992)

Kindling and Its Consequences on Learning in Rats AXEL BECKER, 1 GISELA GRECKSCH, HEIDE-LINDE RUTHRICH, WULF POHLE, BERNHARD MARX, AND HANSJURGEN MATTHIES

Medical Academy Magdeburg, Institute of Pharmacology and Toxicology, Leipziger Strasse 44, 3090 Magdeburg, Germany

the stimulus culminating in a generalized convulsion. Especially the increasing susceptibility to convulsive stimulation m a y reflect some aspects of the progressive character of epileptogenesis. Recently, in kindled animals an impairment of cognitive processes was described, e.g., passive avoidance (Stone & Gold, 1988) and spatial memory deficits (Lopes da Silva et al., 1986). With the intention to investigate the consequences of repeated convulsions and the particular method of kindling on learning and memory performance we tested kindled rats in three different learning models: short-term memory was investigated in the response-to-change model, acquisition and retention performance for brightness discrimination was tested in a Y-chamber, and for a conditioned reaction we tested in a two-way shuttle-box. The learning and memory performances of amygdala- and pentylenetetrazol-kindled rats were compared.

To study the learning performance of pentylenetetrazoland amygdala-kindled Wistar rats we used the following ].earning tests: short-term memory was tested in the response-to-change model, brightness discrimination was tested in a Y-chamber, and two-way active avoidance learning was tested in a shuttle-box. Short-term memory was not impaired by both kindling procedures. Considering two-way active avoidance learning the performance of pentylenetetrazol (PTZ)-kindled rats was significantly diminished. This effect persists over a period of 4 weeks. However, amygdala (AMY)-kindled rats acquired this task like the controls. In brightness discrimination reaction (BDR) the learning performance of PTZ-kindled animals was not influenced. Although the acquisition of BDR was nearly identical, the 24-h retention was remarkably diminished in AMY-kindled rats. It was hypothesized that the different kindling procedures interfere in different ways and extent with neuronal circuits resulting in different functional impairments. ©1992Academic Press, Inc.

Clinical observations demonstrate that epileptic patients often show impairments in a number of cognitive functions (Dodrill, 1986; Levin et al., 1986). Despite a quantity of papers on this field the relation between repeated seizures or the corresponding drug therapy and such impairments is far from understanding. To investigate this probl~em an animal model involving occurrence of repeated seizures is necessary. Such a model could be kindling, a widely employed technique for studying seizure mechanisms and, moreover, a commonly accepted model of epilepsy. Kindling is induced by repeated exposure to electrical stimuli (Goddard, 1967) or repeated administration of convulsant drugs (Mason & Cooper, 1.972) resulting in an increased responsiveness to

MATERIALS AND M E T H O D S

Animals Experiments were performed with adult male Wistar rats from our own breeding stock. The animals were kept under controlled laboratory conditions under a lighting regime of LD 12:12 (light on at 6:00 A.M.), temperature 20 ° +_ 2°C, and relative air humidity 55-60%. They had free access to commercial rat pellets (R !3) and tap water. The rats were housed in groups of six to eight.

Chemical Kindling For PTZ kindling an initially subeffective dose of 45 m g / k g body weight PTZ was injected intraperitoneally once every 48 h. After each injection the convulsive behavior was observed for 20 min. The resultant seizures were classified as follows:

Address correspondence and reprint requests to Dr. Axel Becker, Medical Academy Magdeburg, Institute of Pharmacology and Toxicology, Leipziger Str. 44, 3090 Magdeburg, Germany. 37

0163-1047/92 $3.00 Copyright © 1992 by AcademicPress, Inc. All rights of reproductionin any form reserved.

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BECKER ET AL.

Stage 0: no response Stage 1: ear and facial twitching Stage 2: convulsive waves through the body Stage 3: myoclonic jerks, rearing Stage 4: turn over into side position Stage 5: turn over into back position, generalized clonic-tonic seizures. To acutely evoke generalized seizures, 60 mg/kg PTZ was injected intraperitoneally. For control the solvent (0.9% saline) was administered.

Experimental Design In principle, the rats were tested in the three different learning models 24 h after the last PTZ injection. The animals were considered to be "kindled" after having received 10 PTZ injections and after having reached at least Stage 4 seizures. In the first experiment the response-to-change model was used to compare the learning performance of kindled and saline-injected controls, whereas the second experiment was based on brightness discrimination. As described below, both of these learning tasks did not reveal any differences between kindled animals and controls. Therefore, in our further experiments special consideration was given to active avoidance learning. The third experiment was concerned with learning impairments in the active avoidance in the shuttle-box as a result of kindling. One group received 10 PTZ injections, whereas controls received the same number of saline applications and after kindling development the animals were tested in the shuttle-box. In the fourth test the persistance of kindling induced deficits on shuttle-box learning was investigated. For this purpose rats received 10 PTZ injections. The animals were left untreated and sheltered for 4 weeks. After this period, the rats were tested in the shuttle-box. The influence of one acute seizure induced by an injection of 60 mg/kg PTZ 24 h before the learning experiment was investigated in the last experiment. For each learning model separate experimental groups of animals were used.

Electrical Kindling Surgery. The rats were anesthetized with a mixture of hexobarbital (100 mg/kg) and ethyl urethan (600 mg/kg) injected intraperitoneally and a bipolar electrode (Teflon-coated stainless wire, 0.005 in., Medwire Corp.) was implanted in the right amyg-

dala. The following coordinates were used: 1.7 mm posterior and 4.2 mm lateral from bregma and 8.0 mm ventral from skull.

Kindling procedure. After a recovery period of 1 week the rats were separated into two groups. The first group was subjected to the kindling procedure. To this end, each rat was stimulated once daily by a current impulse. The first stimulation was necessary to determine the individual susceptibility of each rat. The initial current evoking eye blinking or vibrissae erection was used for continuing the kindling procedure. The impulse consisted of a 1-s train of 60-Hz monophasic constant current square wave at an intensity of 200-500/uA. Seizure severity was graded into five classes according to Racine (1972) (see above). Duration of the behavioral seizures after stimulation was also measured. Rats were considered to be fully kindled when developing stable Stage 4 or Stage 5 seizures with a duration of minimal 60 s after 15 stimulations. The other group which was also implanted with electrodes received identical handling without electrical stimulations. This group was used as controls in the learning experiments. LEARNING EXPERIMENTS The learning experiments using the models response-to-change, two-way active avoidance learning, and brightness discrimination reaction were performed as described (see above) 24 h after the last seizure induction.

Learning Procedures Short-term memory--Response-to-change. If a rat in a T-maze is exposed to two arms differing in brightness of the walls (black or white) it will enter the arm in which brightness has been changed in a subsequent test trial. It responds to a stimulus change reflecting exploratory motivation (Lukaszewska, 1978; Lukaszewska & Dlawichoska, 1982). The experiment was conducted in an enclosed Tmaze and consisted of two trials. During Trial No. 1 the rat was allowed to explore the white-black T-maze arms for 3 min. Then the animal was removed from the maze for 60 min while the brightness of one arm was changed, so that both arms were either white or black. In the following test trial it was registered whether the animal entered the changed or the unchanged arm.

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KINDLING AND LEARNING

Brightness discrimination--Y-chamber.

In this experiment rats were trained in a foot shock-motivated brightness discrimination using a semiautomatic Y-chamber (Ott et al., 1972). The training session involved 31 runs. The animals had to learn to run into the illuminated alley of the chamber immediately after application of an electrical starting stimulus. Entering the dark alley of the chamber (error) was punished by the foot shock (1 mA). Only escape into the illuminated alley was counted as a correct run. The retention performance was estimated by a relearning test performed in the same manner 24 hr later. The savings percentage was calculated according to the formula

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In order to preclude directional training, the program was designed such that the illuminated goal alley was to the left of the starting alley and then to the right for three successive runs each.

Histology Upon the completion of testing the rats received an overdose of anesthetic and were decapitated after death. Following formaline fixation the brains were removed and postfixed in formaline. After soaking in 10% saccharose solution freezing sections of 25/~m thickness were cut and stained with toluidine blue. Placement of the electrode tips was verified microscopically by another person blind to the kindling data. Animals with correct placement of the electrodes were considered for evaluation of learning results only.

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Two-way active avoidance--Shuttle-box.

The automatic shuttle-box was divided into two compartments 0.25 x 0.25 x 0.6 m separated by a 5-cm hurdle. The conditioned stimuli were 40-W bulbs located on the central ceiling of each compartment and a sound produced by a buzzer. The unconditioned stimulus was an electric foot shock of 1 mA delivered through stainless rods forming the floor. The conditioned stimuli-unconditioned stimulus interval lasted 4 s. One trial was limited to 20 s. Each session consisted of 20 trials and was repeated on 4 consecutive days. Sessions were performed during the light part of the 12:12-h cycles at about the same time _+lh. Prior to the first session, the rats were allowed to explore the box for 5 min, and on the following days 1-min explorations were provided.

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FIG. 1. Brightness discrimination in kindled rats. Pentylenetetrazol: control group (n = 14), open columns; kindled group (n = 14), hatched columns. Amygdala-kindling: sham-stimulated group (n = 12), open columns; kindled group (n = 13), hatched columns. TE = training errors; RE = relearning errors. Means -+ SEM. *P < 0.05 (U test).

Statistics Data were analyzed for significance by the M a n n Whitney U test. RESULTS

1. Chemical Kindling The parameters determined for the saline-treated and the PTZ-kindled animals by means of brightness discrimination did not vary (Fig. 1). There were also no differences between kindled and saline-injected animals in the response-to-change test (Table 1). However, studies dealing with the PTZ kindlinginduced impairments on shuttle-box learning revealed significant differences after 10 PTZ injections (Fig. 2). This impairment is still ascertainable 4 weeks after the last treatment (Fig. 3). To investigate possible interactions between the progressive intensification of seizures during kindling development and the impairment of shuttlebox learning we compared the number of condi-

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BECKER ET AL.

TABLE

1

Short.Term Memory Tested in the "Response-toChange" Model in Kindled Rats and Controls

Pentylenetetrazol-kindled Controls Kindled Amygdala-kindled Controls Kindled

Changed

Unchanged

16 (76%) 10 (72%)

5 (24%) 4 (28%)

16 (67%) 11 (58%)

8 (33%) 8 (42%)

Note. Number and percentage of rats choosing the visually changed and the unchanged maze arm.

tioned reactions in dependence of seizure severity. For this purpose rats were injected 10 times with PTZ. The mean seizure stage of the last 3 test days was calculated and the animals were divided into two groups according to this value. The first group consisted of animals with a seizure score between 1 and 3.5, the other had a score of 3.5 and more. Figure 4 shows a clear impairment of shuttle-box performance in dependence of seizure severity. Rats with low seizure scores reached a significantly higher shuttle-box score on the third and fourth test day. Their learning performance resembles that of the control animals. On the other hand seizures induced by a single dose of 60 mg/kg PTZ 24 h before the learning experiment did not influence the performance in the shuttle-box (Fig. 5).

Electrical Kindling When comparing short-term memory in AMYkindled rats with controls no significant differences

could be found (Table 1). Similarly, two-way active avoidance learning in both groups is nearly identical (Fig. 2). In BDR the number of training errors made in both groups was similar, presenting a reliable basis for comparison of retention. However, in the relearning test AMY-kindled rats produced more errors thus yielding a significantly diminished savings percentage (Fig. 1). DISCUSSION The term kindling refers to repeated administration of initially subeffective chemical or electrical stimulation resulting in progressively intensified seizure activity. This effect is long lasting and persists throughout the animal's lifetime (McNamara, 1988). Although the mechanisms underlying kindling are poorly understood, it is an accepted model of epileptogenesis (McNamara et al., 1986), neuronal plasticity (Morrell & de Toledo-Morrell, 1986), and related processes (Schmutz, 1988). The evolution of kindling depends on numerous intrinsic and extrinsic factors. It was found that mice showing a low retention in an avoidance task needed more stimulation for developing the kindling syndrome. On the other hand several impairments of cognitive processes in animals were described as a result of kindling (Stone & Gold, 1988; Lopes da Silva et al., 1986; Voigt & Morgenstern, 1990). The comparison of these intrinsic factors sharing kindling and learning, e.g., lasting changes as a result of periodic stimulation, implication of the limbic system, and trans-synaptic alteration in function

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FIG. 2. Shuttle-box learning in kindled animals. Pentylenetetrazol: rats having been treated with 10 injections ofpentylenetetrazol. Open circles, saline-treated controls (n = 14); solid circles, pentylenetrazol-treated animals (n = 12). Amygdala-kindling: open circles, sham-stimulated controls (n = 12); solid circles, kindled animals (n = 12). Means _+ SEM. *, P < 0.05 (U test).

41

KINDLING AND LEARNING

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FIG. 3. Persistence of pentylenetetrazol kindling induced impairment of shuttle-box learning over a period of 4 weeks. Open circles, saline-treated controls (n = 12); solid circles, pentylenetetrazol-treated animals (n = 12). Means -+ SEM. *, P < 0.05 (U test). and transfer effects (Schmutz, 1988) reveals connections between both processes. In epileptic h u m a n s various intellectual, emotional, and psychosocial impairments have been described (Dodrill, 1986; Lesser et al., 1986; Levin et

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F I G . 4. Shuttle-box l e a r n i n g in pentylenetetrazol-kindled rats in comparison to controls. Connection between learning performance and seizure severity. O, Saline-injected controls (n = 12); V, pentylenet~trazol-kindled rats, mean seizure score 1-3.3 (n = 11); " , pentylenetetrazol-kindled rats, mean seizure score 3.5-5 (n = 11). ( + , *) P < 0.05 in comparison to animals with a mean seizure stage of 3.5-5 (U test).

FIG. 5. Shuttle-box performance of rats after acute generalized seizures induced by a single injection of 60 m g / k g pentylenetetrazol. Open circles, saline-treated controls (n = 12); solid circles, pentylenetetrazol-injected animals (n = 8). Means -+ SEM.

al., 1986). Only minor attention in pharmacotherapy is put on this aspect of epilepsy. Considering the relation between kindling as a model of epileptogenesis (McNamara et al., 1986) and cognitive processes on the other hand we tried to investigate patterns of cognitive impairments after termination of kindling. Furthermore, we compared the impairments following chemical (using pentylenetetrazol) and electrical (amygdata stimulation) kindling. Considering short-term memory we could not detect any differences between kindled (PTZ or AMY) animals and controls. This result implicates that this form of memory is not affected by the kindling procedures used (Table 1). This is supported by the normal acquisition of active avoidance and BDR on the first test day (Figs. 1 and 2). In PTZ-kindled animals the retention of active avoidance learning was considerably impaired (Fig. 2) but no effect on BDR could be detected (Fig. 1). The effects of PTZ on avoidance learning were published by Krivanek (1971) who discovered that different doses of PTZ are able to produce facilitation, disruption, or no effect at all. These experiments, however, were carried out to investigate the acute effect of an acutely induced convulsion. The induction of an acute seizure 24 h before shuttle-box learning did not influence acquisition or retention in this learning task (Fig. 5). These results suggest that the impairment of shuttle-box learning in the kindling experiment is not the result of the last production of seizures. The learning impairment became obvious after about 10 PTZ injections, thus suggesting the relation to long-lasting func-

42

BECKER ET AL.

tional changes as described by Schmutz (1988). This is underlined by the fact, that the shuttle-box impairment is still detectable 4 weeks after the last kindling stimulation (Fig. 3). Regarding the performance of the controls in this experiment the relatively low retention is noticeable. Perhaps this m a y be due to the age and/or the reduced handling during the period of persistence. In BDR no differences between PTZ-kindled animals and controls were found. In contrast, the retention performance in BDR was significantly deteriorated in AMY-kindled rats (Fig. 1). Due to the fact that the basic processes underlying kindling are poorly understood it is not easy to explain the differences found in the learning experiments between PTZ- and AMY-kindled rats. First, it cannot be ruled out that the learning deficits in kindled animals observed in the shuttle-box test and BDR are due to microseizures elicited by the 1-mA foot shock used in the course of the experimental procedure. But Boast and McIntyre (1977) had never seen any indication of foot shock-dependent epileptiform activity in EEG recordings of kindled animals. Furthermore, if microseizures would be the very reason, one could expect nearly identical results in two-way active avoidance learning and BDR since both learning models are foot shock-motivated. But as shown, shuttle-box learning was impaired in PTZ-kindled animals, and BDR in AMY-kindled animals only. Although the convulsive component of PTZ and AMY kindling share common features, both methods interfere differently with learning abilities. It seems not very likely that modulations of the basic mechanisms underlying both kindling models are the reason for the differences in learning performance. With regard to the well-known phenomenon of "kindling transfer" (Cain, 1982, 1986, 1987) we m a y speculate t h a t chemical and electrical kindling interfere in comparable fashion with these basic mechanisms. Therefore, we would like to focus on the functional structures differently involved in both kindling procedures, although no clear-cut morphological features underlying kindling have been identified as yet. Modifications of neuronal circuits remain a promising hypothesis (Morrell & de Toledo-Morrell, 1986; Schmutz, 1986) to explain the influence of kindling on mechanisms responsible for learning and memory storage. Consequently, different kindling procedures lead to different modifications of neuronal circuits resulting in specific impairments of learning and memory. Several studies have shown a variety of changes in synaptic neurotransmission in kindled animals

(for review see McNamara et al., 1986). It is wellknown t h a t learning and memory can be modified by pharmacological interventions affecting the neurotransmitter systems (McGaugh, 1973; Grecksch et al., 1978a,b; Bischoff et al., 1979, Grecksch & Matthies, 1981, Gold & Zornetzer, 1983). Thus, a different modulation of neurotransmitter systems could be another possible explanation for the effects described above. Sleep, especially paradoxical sleep, is an important aspect of learning and memory storage (Fishbein & Gutwein, 1977; Hennevin & Leconte, 1977; Pearlman, 1979; Smith, 1985). Electrical kindling leads to several changes in sleep of cats and rats (Shouse & Sterman, 1983; Stone & Gold, 1988). On the other hand, deficits in paradoxical sleep may be connected with deficits in cognitive processing. First indications concerning alterations in sleep of PTZkindled rats were observed in our laboratory which have to be investigated thoroughly in the future. In addition, pharmacological intervention in these impairments are possibly helpful for explaining their fundamental mechanisms in more detail. In summary, kindling can be regarded as a suitable model for studying basic mechanisms of epileptogenesis and cognitive deficits associated with epileptogenesis. This model allows us not only to test the effects of anticonvulsive drugs but also to investigate different possibilities to prevent and to reduce such impairments. REFERENCES Bischoff, S., Scatton, B., & Korf, J. (1979).~Biochemicalevidence for a transmitter role of dopamine in the rat hippocampus. Brain Research, 165, 161-165. Boast, C. A., & McIntyre, D. L. (1977). Bilateral kindled foci and inhibitory behavior in rats. A functional lesion effect.Physiology and Behavior, 18, 25-28. Cain, D. P. (1982). Bidirectional transfer of intracerebrally administered pentylenetetrazol and electrical kindling. Pharmacology, Biochemistry, and Behavior, 17, 1111-1113. Cain, D. P. (1986). The transfer phenomenonin kindling. In J. A. Wada (Ed.), Kindling 3 (pp. 231-245). New York: Raven Press. Cain, D. P. (1987). Kindling by repeated intraperitoneal or intracerebral injectionsof picrotoxintransfers to electricalkindling. Experimental Neurology, 97, 243-254. Dodrill, C. B. (1986). Correlates of generalized tonic-clonicseizures with intellectual, neuropsychological,emotional, and social functionsin patients with epilepsy.Epilepsia, 27, 399411. Fishbein, W., & Gutwein, B. M. (1977). Paradoxical sleep and memorystorage processes.Behavioral Biology, 19, 425-464. Goddard, G. V. (1967). Developmentof epileptic seizuresthrough brain stimulation at low intensity. Nature, 214, 1020-1021.

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remember the spatial arrangement of visual stimuli? Acta

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