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Potentiation effects in the dentate gyrus of pentylenetetrazol-kindled rats
Physiology & Behavior, Vol. 60, No. 2, pp. 455-462, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/96 $15.00 + .00 ELSEVIER
PII S0031-9384(96) 00074-1
Potentiation Effects in the Dentate Gyrus of Pentylenetetrazol-Kindled Rats HEINZ
RUETHRICH, l GISELA
GRECKSCH,
AXEL
BECKER
AND MANFRED
KRUG
Otto-von-Guericke-University Magdeburg, Medical Faculty, Institute of Pharmacology and Toxicology, Leipziger Str. 44, D-39120 Magdeburg, Germany R e c e i v e d 21 M a r c h 1995 RUETHRICH, H., G. GRECKSCH, A. BECKER AND M. KRUG. Potentiationeffects in the dentate gyrus ofpentylenetetrazolkindled rats. PHYSIOL BEHAV 60(2) 455-462, 1996.--The study examines changes in the function of perforant pathway dentate granule cell synapses after pentylenetetrazol (PTZ) kindling. Field potentials evoked in the dentate area by test stimuli to the perforant pathway were recorded in freely moving rats at different times after injection of PTZ. In fully kindled animals, but not in sham-kindled controls, subconvulsive test doses of PTZ induced long-lasting potentiation of the population spike. Also, potentiation was not induced in naive controls injected with equieffective doses of the convulsant. The slope function of the field EPSP was depressed 90-120 rain after PTZ administration, in both kindled and control animals, indicating that this was an effect of acute-injected PTZ. Later on, only in kindled animals that showed seizure stages 4 or 5 did it increase in parallel with the population spike potentiation. Finally, when compared to controls the kindled animals showed a greater pop spike potentiation induced by moderate tetanization of the perforant pathway. The model offers the possibility of differentiating between acute effects of the convulsant drug and kindling-related changes in neuronal plasticity. Chemical kindling Pentylenetetrazol PTZ-related potentiation
Rat
Hippocampus
K I N D L I N G , originally described by Goddard et al. (18) is considered an animal model of epileptogenesis (10,18) and, more generally, of neuronal plasticity (45), in which the periodic administration of initially subconvulsive electrical stimuli to relevant brain structures results in generalized electroencephalographic and behavioral signs of seizures (40,51,61). Kindling can also be induced by repeated administration of convulsant drugs such as pentylenetetrazol ( P T Z ) and other substances (22,37,65,66). Even now the neuronal processes leading to the establishment of the kindled state and the neuronal networks involved are not fully understood. Decreased functioning of inhibitory G A B A e r g i c systems (6,43) and changes in glutamatergic transmission systems, mainly at the level of the N-methyl-D-aspartate ( N M D A ) receptor complex, have been discussed (39,43). The similarities between the procedures that initiate long-term potentiation ( L T P ) and kindling behavior by electrical stimulation have also led to the assumption that potentiation phenomena play a crucial role in induction and maintenance of the kindling state ( 7 ) . Indeed, kindling-induced potentiation ( K I P ) has frequently been described during development and after successful electrical kindling procedures, but the results are still conflicting (52,62). Kindling by perforant pathway ( P P ) stimulation resulted in different changes of the field excitatory postsynaptic potential ( E P S P ) and population spike of the monosynaptic evoked field potential ( M E F P ) in the dentate gyrus evoked by test
Field potentials
LTP
stimulation of the same pathway ( 10,19,34-36,63 ). Although acute effects of convulsants on hippocampal electrical activity are relatively well documented, there is little information on the occurrence of potentiation phenomena in the course of chemical kindling. For instance, various alterations of single neuron electrical membrane parameters in CA1 and dentate gyrus after electrical kindling have been reported. However, in PTZ kindling, similar changes were not observed (21). Other authors found enhanced "burst firing" in the CA3 region without concurrent occurrence of potentiation in the dentate gyrus (50). On the other hand, data have been reported that indicate long-lasting alterations in G A B A e r g i c and excitatory amino acid-mediated neurotransmission, gene expression, and pro-nerve growth factor-like immunoreactivity even after PTZ kindling (2,23,26,30,58), which parallels those seen after electrical kindling (12,16,25,27,28,42,46). These data, which signal an altered and enduring state either of glutamatergic receptors and associated ionic channels or intracellular regulation mechanisms, gave rise to the question of whether the essential change occurring during the kindling procedure might be a sustained but latent increase in the ability to produce potentiation phenomena upon repetitive strong stimulation. Such an altered state might explain the spontaneous occurrence of seizures every time an unusual excitation of the subject occurs. Also, the " l e a r n i n g deficit" that can be observed in fully kindled rats (3) could be due to overexpres-
To whom requests for reprints should be addressed. 455
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RUETHRICH ET AL.
A
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FIG. 1. Schematic diagram of electrophysiologicalarrangement. (A) Electrode placements for electrical stimulationof the perforant pathway (pp) and recording of monosynapticevoked field potentials in the hilus of dentate area. (B) A typical evoked potential consisting of the initial positive going field EPSP onto which a negative going population spike is superimposed. The measurements of the slope function of field EPSP, and the amplitude of the population spike as well as its latencies are indicated by SF, PS, T1, and T2. sion of repeated potentiation in the hippocampus interacting with LTP-like potentiation, which also occurs during a learning procedure (4,38,64). To test this assumption and to elucidate further the mechanisms of PTZ kindling, fully kindled rats as well as sham-kindled and naive animals were chronically implanted with recording electrodes in the dentate gyrus and stimulation electrodes in the angular bundle and subjected to the following experiments: i) long-term registration of MEFPs upon perforant path (pp) stimulation with single pulses after a subconvulsive dose of PTZ, and ii) induction of LTP in the dentate gyrus with a moderate tetanization procedure. METHOD
Animals The subjects were 70 male Wistar rats [Mol:Wist (Shoe), Moellegard Breeding Centre, Deutschland GmbH ] aged 8 weeks at the beginning of the experiments. During the kindling procedure the animals were kept under controlled laboratory conditions, of constant temperature (20 +__2°C ), relative humidity 5 5 60%, and maintained on a 12 h/12 h light/dark cycle (light period between 0600 and 1800 h). During the time of kindling the animals were housed in groups of five, but after surgery they were housed individually in transparent plastic cages. Food (AItromin 1326) and water were freely available.
Kindling Procedure To produce the kindling state, an initially subconvulsive dose of 45 mg/kg PTZ was injected IP once every 48 h. The animals received 10 PTZ injections during the time of kindling. Kindling controls were treated with the same number of injections of 0.9% saline. After each injection the convulsive behavior was observed for 20 min and classified according to Racine (51 ) in a modified five-stage scale (3). Rats were considered to be fully kindled when presenting stable seizure stage 4 or 5, reflecting complete generalization of convulsive activity.
Electrode Implantation Fully kindled animals and saline-injected kindling controls as well as naive animals were chronically implanted with stainless steel electrodes (125 #m diameter, polyurethane coated) under
deep nembutal/urethane anaesthesia (40 mg/kg, 500 mg/kg). The animals were mounted in a David Kopf Inc. stereotaxic instrument with bregma 1 mm above lambda. Bipolar stimulation electrodes were inserted in the right angular bundle and monopolar recording electrodes in the hilus of the dentate gyrus slightly below the granular cell layer of the dorsal blade (Fig. 1A). The stereotaxic coordinates for the stimulating electrodes were AP 6.8, lat. 4.1 and for the recording electrode AP 2.8, lat. 1.8 [according to Skinner (59)]. The exact electrode placement, especially in depth, was controlled by monitoring the MEFP during implantation procedure as described previously (38). Two stainless steel watchscrews driven into the bone above the ipsilateral olfactorius and the contralateral parietal cortex served as indifferent and ground electrodes. All electrodes were connected to a miniature plastic socket and fixed together with them to the skull by acrylic dental cement. The wounds were treated with a long-acting local anaesthetic (Lidocain) and a sulphonamidecontaining powder (Tonil). After finishing the experiments, the animals were anaesthetized and sacrificed by an overdose of hexobarbital and perfused intracardially with physiological saline followed by 5% formalin solution. The electrode positions were subsequently verified histologically.
Recording of Monosynaptically Evoked Field Potentials The animals were allowed to recover from surgery for 8 - 1 0 days. At least 4 weeks after the last kindling injection they were subjected to the electrophysiological experiments. The rats were placed in a recording chamber and connected to a DISA amplifier (frequency range 0.2 Hz to 10 kHz) and a constant current stimulation unit through a shielded flexible cable and a commutator system, thus allowing full freedom of movement. All evoked potential data were obtained from awake animals. A Robotron A7100 personal computer was used to generate rectangular pulses for driving the constant current stimulation unit, to trigger calibration signals at the input of the amplifier, and to record, average, and store the MEFPs. For recording of test potentials, series of 10 biphasic square wave pulses with a duration of 0.1 ms per half cycle were generated with a frequency of 0.2 Hz. The MEFPs were amplified and digitized with a resolution time of 100 #s. Eight to 10 single potentials were averaged and stored on disk. From the averaged potentials the slope function (Sf) of the field excitatory postsynaptic potential (EPSP in mV/ms) was calculated at the steepest 400 #s segment between two markers
POTENTIATION A N D PTZ KINDLING
from the onset of the EPSP and Tl. The amplitude of the population spike (PS in mV) was measured by taking the voltage difference between the onset and peak of the PS ( T I , T2). Figure I B shows a typical MEFP together with the indication of the parameters that were calculated. LTP was induced by moderate tetanization of the perforant pathway with three trains of biphasic impulses having the same intensity and duration as the test pulses. Each of the trains consisted of 15 impulses (together 45 impulses). The frequency within the trains was 200 Hz, and the intervals between the trains were 5 s.
Experimental Schedule
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Statistics The data were statistically analyzed using Witcoxon's sign rank test for determination of differences during the experimental period in each group and the M a n n - W h i t n e y U-test for differences between the groups. For all cases the threshold for significance was fixed at p < 0.05. RESULTS
Changes in Behavior and Electroencephalogram After Acute Administration of PTZ The administration of PTZ induced dose-dependent behavioral effects, which differed in their intensity in nonimplanted and chronically implanted rats. Generally, chronically implanted rats responded more sensitively to PTZ than animals without electrodes. As Fig. 2 shows, nonimplanted and nonkindled ani-
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The electrophysiological experiment lasted for 4 days. On the first day the animals were habituated to the experimental conditions for 20 min and the main parameters for stimulation (e.g., stimulus polarity, threshold for occurrence of the EPSP, and the population spike) were estimated. On the second day an input/ output ( I / O ) curve was determined. By means of this curve the test stimulus intensity was calculated which evoked a population spike of 40% of its maximal amplitude. Neither EEG suppression nor epileptiform discharges or long-lasting potentiation effects were induced under these experimental conditions. On the following day, the baseline values were initially determined. To evaluate the effect of PTZ on the evoked potentials, 20, 30, and 45 m g / k g PTZ or physiological saline were administered IP. To register changes of the MEFPs, 10, 20, 90, 120, 180, 240, 360 rain, and 24 h after the PTZ injection, series of test potentials were recorded again. Our aim was to compare the correlates of the same seizure stage 5 in controls and kindled animals, induced by different PTZ doses. Therefore, we injected 20 m g / k g PTZ to induce seizure stages 1 to 2, and 30 m g / k g in kindled animals or 45 m g / k g in controls to induce stages 4 to 5. After PTZ or saline administration and during the recording of the test potentials the EEG was continuously monitored. The results were expressed as means + SEM of the differences to the basic values. In a separate series of experiments an I / O curve was initially recorded using five stimulus intensities starting with a stimulus intensity (50 # A ) that induced a just detectable population spike in the MEFP and a doubling of the stimulus current from step to step. From this curve the stimulus intensities for the test pulses and the tetanic train were calculated. On the following day, after registration of three control potential series, the animals were tetanized by a moderate tetanizing train of three groups of stimuli. The extent of potentiation of the population spike was measured at appropriate times until 24 h after tetanization and expressed as percent deviation from control values.
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FIG. 2. Mean seizure stage of untreated and nonimplanted animals (n = 16) of saline-injected kindling controls and PTZ-kindled rats (both implanted after the injection schedule) induced by different challenge doses of PTZ; lower line: mg/kg PTZ IP. +p < 0.05 between kindling controls (n - 14) and kindled animals (n = 16), challenge dose: 20 mg/kg PTZ. *p < 0.05 between kindling controls (challenge dose: 30 mg/kg PTZ; n = 12 and challenge dose: 45 mg/kg PTZ; n = 19) and kindled animals (challenge dose: 30 mg/kg PTZ; n = I l). mals reacted to a dose of 45 m g / k g PTZ with seizure stage 1. Implanted, saline-injected control animals from the kindling experiment developed after administration of only 20 or 30 m g / k g PTZ seizure stages 1 or 2, and after 45 m g / k g PTZ seizures of the stages 4 or 5. There was no difference in the reaction to PTZ between naive rats and saline controls from the kindling experiment after implantation of chronic electrodes (data are not shown). Fully PTZ-kindled and implanted animals developed seizure stages 1 or 2 after injection of 20 m g / k g PTZ whereas 30 m g / k g PTZ induced seizures stage 4 or 5. This was followed in several animals by repeated seizure stages 2 or 3, automatisms and hyperactivity with climbing behavior. Two to three minutes after PTZ administration the continuously monitored EEG showed the first seizure activity progressing in the following time in the frequency of occurrence and intensity. During the PTZinduced electroencephalographic events different epileptic behavior phenomena were observed in the animals, such as grooming, chewing, sniffing, and frozen posture. During seizure stage 4 or 5 in the kindled animals the EEG showed rhythmic synchronous discharges followed by EEG suppression similar to a spreading depression.
Evoked Responses in the Dentate Gyrus to Test Stimuli in the Perforant Pathway As described in the Method section, I / O curves were recorded starting with an intensity that evoked a just detectable population spike in the field potential record. As seen in Fig. 3, with a stimulus intensity of 50 pA the population spike amplitude was 0.84 + 0.12 mV in the group of naive, noninjected animals, 0.88 _+ 0.20 mV in that of the kindling controls, and 0.74 + 0.15 mV in that of the PTZ-kindled animals. The differences between the groups were not statistically significant. Also, the I / O curves constructed for each group did not differ from each other in a significant manner. Furthermore, the E / S curves that relate the field EPSP values to the amplitude of the population spike also showed no significant differences between the three groups (data not shown).
Dose-Dependent Changes in MEFPs of Kindled Animals and Kindling Controls After a Challenge Dose of PTZ Figure 4 shows analogue examples of evoked potentials registered from fully kindled rats before and after injection of two
458
RUETHRICH ET AL.
4 or 5 (as produced in kindled animals using 30 m g / k g ) no potentiation was found in the controls.
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different challenge doses of PTZ, indicating the induction of long-lasting changes in the slope function and population spike amplitude by the substance. However, the application of PTZ induced different time courses for the changes in evoked potentials in saline-injected kindling controls and in kindled animals, as represented in Figs. 5 and 6. Figure 5 demonstrates the changes in the amplitude of the population spike after injection of a challenge dose of PTZ compared to the basal values before injection. After application of 20 m g / k g PTZ to the kindled animals the amplitude increased over 24 h and was enhanced by approximately 2 - 3 mV (Fig. 5, top). These changes in the population spike were influenced by the severity and duration of the seizures, induced by the challenge dose. Its amplitude was gradually diminished if convulsion stages 4 or 5 occurred. In general, this reduction was detectable 5 - 7 min after injection of 30 m g / k g PTZ. However, if the long-lasting potentiation was also evident, it occurred with a delay (Fig. 5, bottom). In this case the amplitude of the population spike was significantly increased 180 min after injection and this increase lasted longer than 24 h. In contrast to that, kindling controls showed only a very short-lasting increase in the amplitude, which was dependent on the dosage of PTZ. A dose of 20 m g / k g caused an amplitude increase lasting only on average appoximately 6 0 - 9 0 min. The increase induced by 30 m g / k g PTZ lasted between 120 and 180 min. A longerlasting increase was never observed in control animals, even when using higher PTZ doses inducing seizure stages 4 or 5. The field EPSPs were influenced in a more complex way (Fig. 6). In kindled animals as well as in kindling controls 20 m g / k g PTZ caused an initial decrease of the slope function of the field EPSP, which lasted only 9 0 - 1 2 0 minutes. Later on, in both groups, no further changes were observed (Fig. 6, top). After application of 30 m g / k g PTZ and if convulsion stages 4 or 5 occurred in kindled animals, the slope function showed a biphasic alteration. The initial depression changed to a considerable long-lasting potentiation, which was significant from 120 min to 24 b after application (Fig. 6, bottom). In kindling controls the initial depression was not followed by any potentiation effects. Also, when using 45 m g / k g PTZ to induce seizure stages
When inducing LTP by a relatively moderate tetanization procedure using only three groups of tetanizing impulses, marked differences in the extent of potentiation of the population spike occurred between naive animals, kindling controls, and kindled animals. As Fig. 7 demonstrates, in all groups of animals a potentiation of the population spike was induced. In both control groups an increase of the population spike amplitude by 39 _+ 10% and 55 _+ 15%, respectively, was observed, which lasted at least 5 h, but declined to near control values within 24 h. On the contrary, in kindled animals the increase was 114 _+ 13%. The difference in amplitude increase between the two control groups and the kindled rats was statistically significant at all points up to 24 h after tetanization. DISCUSSION
The present in vivo study describes specific long-lasting excitability changes after a chemical kindling with PTZ. PTZ kindling is generally assumed as a model for primarily generalized epilepsy. In contrast to electrical kindling, the changes induced by repeated administration of a convulsive drug resulting in a kindling state similar to that seen after electrical kindling have not been sufficiently characterised until now. Here, we report on changes in the hippocampus, a brain formation that is directly involved in the development of epilepsy and other types of adaptive behavior such as learning and memory formation (4,38,64). In particular, the ability to produce long-term potentiation is well documented ( 1,5,13,53 ). Because the procedures for electrical kindling resemble those for inducing LTP and because some authors have described excitability changes similar to those after LTP induction, a connection between LTP and kindling has frequently been proposed such that potentiation mechanisms may also play a role in the development of kindling (7,13,17). However, the relationships between LTP and kindling-related potentiation phenomena have not been clarified (52,62). Notably, the altered ability not only to respond to stimulation but also to produce potentiation upon short strong stimulation when the kindling or epileptic state is established has not been extensively examined before either in electrical or chemical kindling models. Various articles have described changes in neuronal respon~ siveness during the course of electrical kindling that were visible as changes in field potential size or enhanced "burst firing " of single neurons and that tended to outlast the kindling procedure (I 1), although alternative results have also been reported (35,36,50). However, a growing body of evidence indicates that mainly after electrical kindling but also after chemical kindling the glutamatergic N M D A receptor ionic channel complex and intracellular second messenger systems, which are also involved in induction and maintenance of LTP, undergo lasting changes (i.e., they are upregulated) (2,12,16,25,27,46,58). This might imply that in kindled animals potentiation phenomena can be induced more easily and can last for a long time, and this might be one of the reasons for increased seizure susceptibility in the epileptic state. Therefore, we investigated whether a chemical stimulus (PTZ injection) of a challenge dose that at the beginning of the kindling procedure and in naive and sham-kindled animals in-
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FIG. 4. Examples of field potentials evoked in the dentate gyrus by test stimulation of the perforant pathway in fully kindled rats before (control potentials) and at different times after administration of a challenge dose of PTZ. On the left side potentials are shown of an animal that reached seizure stage 2 after the challenge dose of 20 mg/kg PTZ; on the right side potentials are shown of an animal which reached seizure stage 5 after 30 mg/kg PTZ. Each trace represents an averaged response to ten test pulses at the same stimulus intensity. The long-lasting response enhancement of the field EPSP and the population spike is obvious.
duced only a short increase of MEFPs in the dentate gyms was able to produce potentiation (i.e., enhancement of the field potential that exceeds the stimulus action) (56). Additionally, it was tested whether LTP induced by tetanic stimulation was also changed in time course and extent in PTZ-kindled animals. From the results obtained some conclusions can be drawn. Firstly, the chronic implantation by itself enhanced the responsiveness of the animals to PTZ. Naive controls, sham-kindled, and kindled animals implanted with electrodes responded with higher seizure stages than nonimplanted animals. This confirms results (33) that additionally demonstrated marked changes in amino acid contents, especially glycine, in relevant brain structures. This effect was interpreted as a "prokindling effect." Thus, implantation can induce enduring changes that must be considered in interpreting results obtained in long-term experiments. The reason for such changes, at this time, seems unexplained, but perhaps reorganization processes may play a role (8,49). Secondly, the responsiveness of the dentate gyms to single test stimuli seems to be unaltered in fully kindled animals. Four weeks after the last kindling session neither the amplitude of the population spike elicited by a near threshold intensity nor the I/ O curve or E / S curve obtained from evoked potentials in kindled animals differed from that obtained in sham-kindled or naive animals. This result is in good agreement with other studies (30,50) and was also confirmed in the CA1 region in an in vitro experiment (Krug, in preparation). However, our results provide clear evidence for the development of enduring changes in the potency of granule cells to react with plastic reactions (response potentiation to PTZ) as a consequence of a chemical kindling. A test dose of PTZ that was subconvulsive in naive and sham-kindled animals and induced only a short-lasting increase in the population spike amplitude upon test stimulation of the perforant pathway provoked in kindled animals not only seizures but also
drastic changes in reaction to test stimuli, which were seen up to 24 h after injection. These changes in reaction cannot be explained by an enhanced PTZ concentration in the brains of kindled animals because after a single test dose of 25 m g / k g PTZ no differences in PTZ content were found between kindled and sham-kindled animals until 6 h after administration (Rauca, in preparation). Thus, PTZ kindling induces a novel type of response enhancement that can be described as a long-lasting potentiation. This potentiation effect is related to the kindling status and not to the severity of seizures, because naive animals showing the same seizure stage as kindled animals but with a higher PTZ dose show only shortlasting amplitude increases. However, in evaluating the different effects of PTZ on the population spike amplitude and the slope function of the field EPSP in the animals, the interaction of acute PTZ effects and the occurrence of seizures with the development of the longlasting response potentiation must be considered. For instance, the test doses of PTZ depressed the field EPSP for 2 - 3 h in all animals and, therefore, this depression must be considered as an acute PTZ effect that perhaps interacts with the development of potentiation. It is also of interest that after this initial depression of the field EPSP only in fully kindled animals that react with a seizure stage 4 or 5 a long-lasting EPSP potentiation was observed. This might indicate that a critical level of activation must be reached to induce potentiation and that seizure-induced depression influences the potentiation development. Furthermore, our results show that the induction and maintenance of LTP induced by tetanic stimulation is also facilitated. The procedure used produces a true LTP of both the population spike and the field EPSP, but as was discussed in chronic implanted animals (14,29), EPSP potentiation cannot be measured with the same accuracy as population spike potentiation. This is especially true under conditions of a very
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T i m e a f t e r PTZ i n j e c t i o n (rain / h) FIG. 5. Time course of changes of the amplitude of the population spike in saline-injected kindling controls (filled triangles) and kindled animals (filled circles). Top: changes after application of the same dose of PTZ (20 mg/kg) in controls (n = 10) and kindled animals (n = 19), respectively. Bottom: changes following induction of seizure stage 4 to 5 after application of 30 mg/kg PTZ in kindled animals (n = 12) and 45 mg/kg PTZ in controls (n = 9). (The difference of the actual values to baseline values before PTZ is given with p < 0.05 vs. *baseline, and +between the groups).
m o d e r a t e tetanization protocol. In our opinion the results indicate further that the r e s p o n s e potentiation seen after PTZ application in kindled animals is not due to an altered metabolism o f PTZ but rather is the result o f an enduring change in plastic properties o f the neurons after kindling. It may further indicate that in both cases related cellular m e c h a n i s m s are involved. C o n c e r n i n g the m e c h a n i s m s that underlie the p h e n o m enon described here, data on changes in passive m e m b r a n e properties are not conclusive as yet ( 4 1 ) . H o w e v e r , several results described in the literature as well as the results o f our p h a r m a c o l o g i c a l observations point to lasting changes in the glutamatergic transmitter system. For instance, both, c o m p e t itive and n o n c o m p e t i t i v e N M D A antagonists prevent or reduce kindling in different m o d e l s (14,15,20,44,54,57). An e n h a n c e d binding o f [3H]glutamate to h i p p o c a m p a l synaptic m e m b r a n e s ( 5 8 ) and altered properties o f the N M D A receptor ionic channel c o m p l e x as well as lasting e n h a n c e m e n t o f N M D A receptor sensitivity (27,28,46,48) have also been reported. Changes in the inhibitory G A B A system may be also partially r e s p o n s i b l e for the increased ability to produce potentiation because the m o s t p r o m i n e n t m e c h a n i s m of PTZ is a
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FIG. 6. Time course of changes of the field EPSP (slope function) in saline-injected kindling controls (&) and kindled animals (O). Top: changes after application of the same dose of PTZ (20 mg/kg) in controls (n = 10) and kindled animals (n = 19), respectively. Bottom: changes following induction of seizure stage 4 to 5 after application of 30 mg/kg PTZ in kindled animals (n = 12) and 45 mg/kg PTZ in controls (n = 9). (The difference to the actual values to baseline values before PTZ is given with p < 0.05 vs. *baseline, and +between the groups).
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FIG. 7. Time course of potentiation of the population spike induced by tetanic stimulation of the perforant pathway in naive animals, kindling controls, and kindled animals. Abscissa: time after tetanization; ordinate: potentiation as percent of control potentials before tetanization _+ SEM. Naive animals (filled triangles; n = 8); kindling controls (open circles, n = 7); kindled animals (filled circles, n = 9); **p < 0.01 between kindled rats and kindling controls or naive animals.
P O T E N T I A T I O N A N D PTZ K I N D L I N G
461
reduction of G A B A e r g i c i n h i b i t i o n ( 9 , 3 1 , 6 0 ) and k i n d l i n g induces d y s f u n c t i o n s in the G A B A e r g i c s y s t e m ( 2 4 , 3 1 , 4 7 ) . H o w e v e r , b e n z o d i a z e p i n e s in a n t i c o n v u l s i v e dosages prev e n t e d i) P T Z k i n d l i n g d e v e l o p m e n t , ii) k i n d l i n g - i n d u c e d seizures, and iii) acute seizures ( m o t o r i c and e l e c t r o e n c e p h a l o g r a p h i c ) but they did not suppress the d e v e l o p m e n t o f the p o t e n t i a t i o n reaction to P T Z in the case of k i n d l i n g and did not d i m i n i s h P T Z - i n d u c e d p o t e n t i a t i o n in kindled a n i m a l s in acute e x p e r i m e n t s ( 5 5 ) . On the other hand, p r e l i m i n a r y exp e r i m e n t s have s h o w n in our m o d e l that N M D A a n t a g o n i s t s can p r e v e n t the d e v e l o p m e n t of P T Z k i n d l i n g and kindlingrelated p o t e n t i a t i o n ( 4 ) .
Our results show that a novel form of response potentiation in the hippocampus of chemical-kindled rats and a facilitation of long-term potentiation has been observed. From these results it might be concluded that an essential feature of kindled or epileptic nervous tissue is an enhanced ability to produce potentiation effects, perhaps via an upregulation of postsynaptic glutamatergic mechanisms. This alteration may not be reflected in normal glutamate-mediated excitation because N M D A receptorcoupled mechanisms are normally not involved in excitatory transmission, but it would be of high importance for the development of lasting plastic changes in excitability of neurons after strong and repetitive activation.
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19. Grace, G. M.; Corcoran, M. E.; Skelton, R. W. Kindling with stimulation of the dentate gyms, II. Effects on evoked field potentials. Brain Res. 509:257-265; 1990. 20. Grecksch, G.; Becker, A.; R~ithrich, H. NMDA-receptor antagonists interfere specifically with kindling development and related cognitive deficits. Neuropsychopharmacology 10:241S; 1994. 21. Heinemann, U.; Stabel, J.; Mody, J.; Stanton, P. K. Long-term changes in cellular and synaptic properties in the hippocampus following kindling-induced long-term plasticity. In: Rahmann, H., ed. Stuttgart: Gustav-Fischer Verlag; 1989:220-228. 22. Ho, T.; Moil, M.; Yoshida, K.; Shimizu, M. Effects of anticonvulsants on seizures developing in the course of daily administration of pentetrazol to rats. Eur. J. Pharmacol. 45:165-172; 1977. 23. Humpel, C.; Ebendal, T.; Cat, Y.; Olson, L. Pentylenetetrazol seizures increase pro-nerve growth factor-like immunoreactivity in the reticular thalamic nucleus and nerve growth factor mRNA in the dentate gyrus. J. Neurosci. Res. 35:419-427; 1993. 24. Iadarola, M.; Gale, K. Substantia nigra: A site of anticonvulsant activity mediated by gamma-amino butyric acid. Science 218:12371240; 1980. 25. Kamphuis, W.; Monyer, H.; DeRijk, T. C.; Lopes da Silva, F. H. Hippocampal kindling increases expression of glutamate receptor A flip and B flop mRNA in dentate granular cells. Neurosci. Lett. 148:51-54; 1992. 26. Kiessling, M.; Gass, P. Immediate early gene expression in experimental epilepsy. Brain. Pathol. 3:381-393; 1993. 27. K6hr, G.; Mody, I. Kindling increases N-methyl-D-aspartate potency at single N-methyl-D-aspartate channels in dentate gyrus granule cells. Neuroscience 62:975-981; 1994. 28. K6hr, G.; DeKoninck, J.; Mody, J. Properties of NMDA receptor channels in neurons acutely isolated from epileptic (kindled) rats. J. Neurosci. 13:3612-3627; 1993. 29. Krug, M.; Manhies, R.; Wagner, M.; Br6demann, R. Nonopioid antitussives and methadone differentially influence hippocampal longterm potentiation in freely moving rats. Eur. J. Pharmacol. 231:355361; 1993. 30. Leung, L. St.; Zhao, D.; Shen, B. Long-lasting effects of partial hippocampal kindling on hippocampal physiology and function. Hippocampus 4:696-704; 1994. 31. Lewecke, F. M.; Louvel, J.; Rausche, G.; Heinemann, U. Effects of pentetrazol on neuronal activity and on extracellular calcium concentration in rat hippocampal slices. Epilepsy Res. 6:187- 198; 1990. 32. Lewin, E.; Bleck, V.; Dildy-Mayfield, J. E.; Harris, R. A. Gaba(A) and glutamate receptor subunit mRNAs in cortex of mice chemically kindled with FG 7142. Mol. Brain Res. 22:320-322; 1994. 33. LOscher, W.; HOrstermann, D.; H6nack, D.; Rundfeldt, Ch.; Wahnschaffe, U. Transmitter amino acid level in rat brain regions after amygdala-kindling or chronic electrode implantation without kindling: Evidence for a pro-kindling effect of prolonged electrode implantation. Neurochem. Res. 18:775-781; 1993. 34. Maru, E.; Goddard, G. V. Alteration in dentate neural activities associated with perforant path kindling. I. Long-term potentiation of excitatory synaptic transmission. Exp. Neurol. 96:19-32; 1987.
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35. Maru, E.; Goddard, G. V. Alteration in dentate neural activities associated with perforant path kindling. II. Decrease in granule cell excitability. Exp. Neurol. 96:33-45; 1987. 36. Maru, E.; Tatsuno, J.; Okamoto, J.; Ashida, H. Development and reduction of synaptic potentiation induced by perforant path kindling. Exp. Neurol. 78:409-424; 1982. 37. Mason, C. R.; Cooper, R. M. A permanent change in convulsive threshold in normal and brain-demaged rats with repeated small doses of pentylenetetrazol. Epilepsia 13:663-674; 1972. 38. Matthies, H.; Rtithrich, H.; Ott, T.; Matthies, H. K.; Matthies, R. Low frequency perforant path stimulation as a conditioned stimulus demonstrates correlations between long-term synaptic potentiation and learning. Physiol. Behav. 36:811-821 ; 1986. 39. McNamara, J. O. Pursuit of the mechanisms of kindling. Trends Neurosci. 11:33-36; 1988. 40. McNamara, J. O.; Bonhaus, D. W.; Shin, Ch.; Crain, B. J.; Gellmann, R. L.; Giacchino, J. L. The kindling model of epilepsy: A critical review. Crit. Rev. Clin. Neurobiol. 1:341-371; 1985. 41. Mody, I.; Stanley, K. J. Cell properties in the epileptic hippocampus. Hippocampus 4:275-280; 1994. 42. Morgan, J. I.; Curran, T. Proto-oncogene transcription factors and epilepsy. Trends Neurosci. 12:343-349; 1991. 43. Morimoto, K. Seizure-triggering mechanisms in the kindling model of epilepsy: Collapse of GABA-mediated inhibition and activation of NMDA receptors. Neurosci. Biobehav. Rev. 13:253-260; 1989. 44. Morimoto, K.; Katayama, K.; Inoue, K.; Sato, K. Effects of competitive and noncompetitive NMDA receptor antagonists on kindling and LTP. Pharmacol. Biochem. Behav. 40:893-899; 1991. 45. Morrell, F.; deToledo-Morrell, I. Kindling as a model of neuronal plasticity. In: Wada, J. A., ed. Kindling 3. New York: Raven Press; 1986:17-35. 46. Morriset, R. A.; Chow, C.; Nadler, J. V.; McNamara, J. O. Biochemical 'vidence for enhanced sensitivity to N-methyl-D aspartate in the hippocampal formation of kindled rats. Brain Res. 496:2528; 1989. 47. Myslobodsky, M. R.; Ackermann, R. F.; Engel, J. Effects of gammaacetylenic GABA and gamma-vinyl GABA on metrazol-activated and kindled seizures. Pharmacol. Biochem. Behav. 11:265-271; 1979. 48. Nadler, J. V.; Thompson, M. A.; McNamara, J. O. Kindling reduces sensitivity of CA3 hippocampal pyramidal cells to competitive NMDA receptor antagonists. Neuropharmacology 33:147-153; 1994. 49. Nitsch, R.; Bader, S.; Frotscher, M. Reorganisation of input synapses of parvalbumin containing neurons in the rat fascia dentata following entorhinal lesions. Neurosci. Lett. 135:33-36; 1992. 50. Piredda, S.; Yonekawa, W.; Whittingham, T. S.; Kupferberg, H. J. Enhanced bursting activity in the CA3 region of the mouse hippocampal slice without long-term potentiation in the dentate gyrus after
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PII S0031-9384(96) 00074-1
Potentiation Effects in the Dentate Gyrus of Pentylenetetrazol-Kindled Rats HEINZ
RUETHRICH, l GISELA
GRECKSCH,
AXEL
BECKER
AND MANFRED
KRUG
Otto-von-Guericke-University Magdeburg, Medical Faculty, Institute of Pharmacology and Toxicology, Leipziger Str. 44, D-39120 Magdeburg, Germany R e c e i v e d 21 M a r c h 1995 RUETHRICH, H., G. GRECKSCH, A. BECKER AND M. KRUG. Potentiationeffects in the dentate gyrus ofpentylenetetrazolkindled rats. PHYSIOL BEHAV 60(2) 455-462, 1996.--The study examines changes in the function of perforant pathway dentate granule cell synapses after pentylenetetrazol (PTZ) kindling. Field potentials evoked in the dentate area by test stimuli to the perforant pathway were recorded in freely moving rats at different times after injection of PTZ. In fully kindled animals, but not in sham-kindled controls, subconvulsive test doses of PTZ induced long-lasting potentiation of the population spike. Also, potentiation was not induced in naive controls injected with equieffective doses of the convulsant. The slope function of the field EPSP was depressed 90-120 rain after PTZ administration, in both kindled and control animals, indicating that this was an effect of acute-injected PTZ. Later on, only in kindled animals that showed seizure stages 4 or 5 did it increase in parallel with the population spike potentiation. Finally, when compared to controls the kindled animals showed a greater pop spike potentiation induced by moderate tetanization of the perforant pathway. The model offers the possibility of differentiating between acute effects of the convulsant drug and kindling-related changes in neuronal plasticity. Chemical kindling Pentylenetetrazol PTZ-related potentiation
Rat
Hippocampus
K I N D L I N G , originally described by Goddard et al. (18) is considered an animal model of epileptogenesis (10,18) and, more generally, of neuronal plasticity (45), in which the periodic administration of initially subconvulsive electrical stimuli to relevant brain structures results in generalized electroencephalographic and behavioral signs of seizures (40,51,61). Kindling can also be induced by repeated administration of convulsant drugs such as pentylenetetrazol ( P T Z ) and other substances (22,37,65,66). Even now the neuronal processes leading to the establishment of the kindled state and the neuronal networks involved are not fully understood. Decreased functioning of inhibitory G A B A e r g i c systems (6,43) and changes in glutamatergic transmission systems, mainly at the level of the N-methyl-D-aspartate ( N M D A ) receptor complex, have been discussed (39,43). The similarities between the procedures that initiate long-term potentiation ( L T P ) and kindling behavior by electrical stimulation have also led to the assumption that potentiation phenomena play a crucial role in induction and maintenance of the kindling state ( 7 ) . Indeed, kindling-induced potentiation ( K I P ) has frequently been described during development and after successful electrical kindling procedures, but the results are still conflicting (52,62). Kindling by perforant pathway ( P P ) stimulation resulted in different changes of the field excitatory postsynaptic potential ( E P S P ) and population spike of the monosynaptic evoked field potential ( M E F P ) in the dentate gyrus evoked by test
Field potentials
LTP
stimulation of the same pathway ( 10,19,34-36,63 ). Although acute effects of convulsants on hippocampal electrical activity are relatively well documented, there is little information on the occurrence of potentiation phenomena in the course of chemical kindling. For instance, various alterations of single neuron electrical membrane parameters in CA1 and dentate gyrus after electrical kindling have been reported. However, in PTZ kindling, similar changes were not observed (21). Other authors found enhanced "burst firing" in the CA3 region without concurrent occurrence of potentiation in the dentate gyrus (50). On the other hand, data have been reported that indicate long-lasting alterations in G A B A e r g i c and excitatory amino acid-mediated neurotransmission, gene expression, and pro-nerve growth factor-like immunoreactivity even after PTZ kindling (2,23,26,30,58), which parallels those seen after electrical kindling (12,16,25,27,28,42,46). These data, which signal an altered and enduring state either of glutamatergic receptors and associated ionic channels or intracellular regulation mechanisms, gave rise to the question of whether the essential change occurring during the kindling procedure might be a sustained but latent increase in the ability to produce potentiation phenomena upon repetitive strong stimulation. Such an altered state might explain the spontaneous occurrence of seizures every time an unusual excitation of the subject occurs. Also, the " l e a r n i n g deficit" that can be observed in fully kindled rats (3) could be due to overexpres-
To whom requests for reprints should be addressed. 455
456
RUETHRICH ET AL.
A
B
FIG. 1. Schematic diagram of electrophysiologicalarrangement. (A) Electrode placements for electrical stimulationof the perforant pathway (pp) and recording of monosynapticevoked field potentials in the hilus of dentate area. (B) A typical evoked potential consisting of the initial positive going field EPSP onto which a negative going population spike is superimposed. The measurements of the slope function of field EPSP, and the amplitude of the population spike as well as its latencies are indicated by SF, PS, T1, and T2. sion of repeated potentiation in the hippocampus interacting with LTP-like potentiation, which also occurs during a learning procedure (4,38,64). To test this assumption and to elucidate further the mechanisms of PTZ kindling, fully kindled rats as well as sham-kindled and naive animals were chronically implanted with recording electrodes in the dentate gyrus and stimulation electrodes in the angular bundle and subjected to the following experiments: i) long-term registration of MEFPs upon perforant path (pp) stimulation with single pulses after a subconvulsive dose of PTZ, and ii) induction of LTP in the dentate gyrus with a moderate tetanization procedure. METHOD
Animals The subjects were 70 male Wistar rats [Mol:Wist (Shoe), Moellegard Breeding Centre, Deutschland GmbH ] aged 8 weeks at the beginning of the experiments. During the kindling procedure the animals were kept under controlled laboratory conditions, of constant temperature (20 +__2°C ), relative humidity 5 5 60%, and maintained on a 12 h/12 h light/dark cycle (light period between 0600 and 1800 h). During the time of kindling the animals were housed in groups of five, but after surgery they were housed individually in transparent plastic cages. Food (AItromin 1326) and water were freely available.
Kindling Procedure To produce the kindling state, an initially subconvulsive dose of 45 mg/kg PTZ was injected IP once every 48 h. The animals received 10 PTZ injections during the time of kindling. Kindling controls were treated with the same number of injections of 0.9% saline. After each injection the convulsive behavior was observed for 20 min and classified according to Racine (51 ) in a modified five-stage scale (3). Rats were considered to be fully kindled when presenting stable seizure stage 4 or 5, reflecting complete generalization of convulsive activity.
Electrode Implantation Fully kindled animals and saline-injected kindling controls as well as naive animals were chronically implanted with stainless steel electrodes (125 #m diameter, polyurethane coated) under
deep nembutal/urethane anaesthesia (40 mg/kg, 500 mg/kg). The animals were mounted in a David Kopf Inc. stereotaxic instrument with bregma 1 mm above lambda. Bipolar stimulation electrodes were inserted in the right angular bundle and monopolar recording electrodes in the hilus of the dentate gyrus slightly below the granular cell layer of the dorsal blade (Fig. 1A). The stereotaxic coordinates for the stimulating electrodes were AP 6.8, lat. 4.1 and for the recording electrode AP 2.8, lat. 1.8 [according to Skinner (59)]. The exact electrode placement, especially in depth, was controlled by monitoring the MEFP during implantation procedure as described previously (38). Two stainless steel watchscrews driven into the bone above the ipsilateral olfactorius and the contralateral parietal cortex served as indifferent and ground electrodes. All electrodes were connected to a miniature plastic socket and fixed together with them to the skull by acrylic dental cement. The wounds were treated with a long-acting local anaesthetic (Lidocain) and a sulphonamidecontaining powder (Tonil). After finishing the experiments, the animals were anaesthetized and sacrificed by an overdose of hexobarbital and perfused intracardially with physiological saline followed by 5% formalin solution. The electrode positions were subsequently verified histologically.
Recording of Monosynaptically Evoked Field Potentials The animals were allowed to recover from surgery for 8 - 1 0 days. At least 4 weeks after the last kindling injection they were subjected to the electrophysiological experiments. The rats were placed in a recording chamber and connected to a DISA amplifier (frequency range 0.2 Hz to 10 kHz) and a constant current stimulation unit through a shielded flexible cable and a commutator system, thus allowing full freedom of movement. All evoked potential data were obtained from awake animals. A Robotron A7100 personal computer was used to generate rectangular pulses for driving the constant current stimulation unit, to trigger calibration signals at the input of the amplifier, and to record, average, and store the MEFPs. For recording of test potentials, series of 10 biphasic square wave pulses with a duration of 0.1 ms per half cycle were generated with a frequency of 0.2 Hz. The MEFPs were amplified and digitized with a resolution time of 100 #s. Eight to 10 single potentials were averaged and stored on disk. From the averaged potentials the slope function (Sf) of the field excitatory postsynaptic potential (EPSP in mV/ms) was calculated at the steepest 400 #s segment between two markers
POTENTIATION A N D PTZ KINDLING
from the onset of the EPSP and Tl. The amplitude of the population spike (PS in mV) was measured by taking the voltage difference between the onset and peak of the PS ( T I , T2). Figure I B shows a typical MEFP together with the indication of the parameters that were calculated. LTP was induced by moderate tetanization of the perforant pathway with three trains of biphasic impulses having the same intensity and duration as the test pulses. Each of the trains consisted of 15 impulses (together 45 impulses). The frequency within the trains was 200 Hz, and the intervals between the trains were 5 s.
Experimental Schedule
457
Controls ® 4 Gq l-
Statistics The data were statistically analyzed using Witcoxon's sign rank test for determination of differences during the experimental period in each group and the M a n n - W h i t n e y U-test for differences between the groups. For all cases the threshold for significance was fixed at p < 0.05. RESULTS
Changes in Behavior and Electroencephalogram After Acute Administration of PTZ The administration of PTZ induced dose-dependent behavioral effects, which differed in their intensity in nonimplanted and chronically implanted rats. Generally, chronically implanted rats responded more sensitively to PTZ than animals without electrodes. As Fig. 2 shows, nonimplanted and nonkindled ani-
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The electrophysiological experiment lasted for 4 days. On the first day the animals were habituated to the experimental conditions for 20 min and the main parameters for stimulation (e.g., stimulus polarity, threshold for occurrence of the EPSP, and the population spike) were estimated. On the second day an input/ output ( I / O ) curve was determined. By means of this curve the test stimulus intensity was calculated which evoked a population spike of 40% of its maximal amplitude. Neither EEG suppression nor epileptiform discharges or long-lasting potentiation effects were induced under these experimental conditions. On the following day, the baseline values were initially determined. To evaluate the effect of PTZ on the evoked potentials, 20, 30, and 45 m g / k g PTZ or physiological saline were administered IP. To register changes of the MEFPs, 10, 20, 90, 120, 180, 240, 360 rain, and 24 h after the PTZ injection, series of test potentials were recorded again. Our aim was to compare the correlates of the same seizure stage 5 in controls and kindled animals, induced by different PTZ doses. Therefore, we injected 20 m g / k g PTZ to induce seizure stages 1 to 2, and 30 m g / k g in kindled animals or 45 m g / k g in controls to induce stages 4 to 5. After PTZ or saline administration and during the recording of the test potentials the EEG was continuously monitored. The results were expressed as means + SEM of the differences to the basic values. In a separate series of experiments an I / O curve was initially recorded using five stimulus intensities starting with a stimulus intensity (50 # A ) that induced a just detectable population spike in the MEFP and a doubling of the stimulus current from step to step. From this curve the stimulus intensities for the test pulses and the tetanic train were calculated. On the following day, after registration of three control potential series, the animals were tetanized by a moderate tetanizing train of three groups of stimuli. The extent of potentiation of the population spike was measured at appropriate times until 24 h after tetanization and expressed as percent deviation from control values.
Kindled
5
non
45
implanted
20
30
implanted
45
20
30
FIG. 2. Mean seizure stage of untreated and nonimplanted animals (n = 16) of saline-injected kindling controls and PTZ-kindled rats (both implanted after the injection schedule) induced by different challenge doses of PTZ; lower line: mg/kg PTZ IP. +p < 0.05 between kindling controls (n - 14) and kindled animals (n = 16), challenge dose: 20 mg/kg PTZ. *p < 0.05 between kindling controls (challenge dose: 30 mg/kg PTZ; n = 12 and challenge dose: 45 mg/kg PTZ; n = 19) and kindled animals (challenge dose: 30 mg/kg PTZ; n = I l). mals reacted to a dose of 45 m g / k g PTZ with seizure stage 1. Implanted, saline-injected control animals from the kindling experiment developed after administration of only 20 or 30 m g / k g PTZ seizure stages 1 or 2, and after 45 m g / k g PTZ seizures of the stages 4 or 5. There was no difference in the reaction to PTZ between naive rats and saline controls from the kindling experiment after implantation of chronic electrodes (data are not shown). Fully PTZ-kindled and implanted animals developed seizure stages 1 or 2 after injection of 20 m g / k g PTZ whereas 30 m g / k g PTZ induced seizures stage 4 or 5. This was followed in several animals by repeated seizure stages 2 or 3, automatisms and hyperactivity with climbing behavior. Two to three minutes after PTZ administration the continuously monitored EEG showed the first seizure activity progressing in the following time in the frequency of occurrence and intensity. During the PTZinduced electroencephalographic events different epileptic behavior phenomena were observed in the animals, such as grooming, chewing, sniffing, and frozen posture. During seizure stage 4 or 5 in the kindled animals the EEG showed rhythmic synchronous discharges followed by EEG suppression similar to a spreading depression.
Evoked Responses in the Dentate Gyrus to Test Stimuli in the Perforant Pathway As described in the Method section, I / O curves were recorded starting with an intensity that evoked a just detectable population spike in the field potential record. As seen in Fig. 3, with a stimulus intensity of 50 pA the population spike amplitude was 0.84 + 0.12 mV in the group of naive, noninjected animals, 0.88 _+ 0.20 mV in that of the kindling controls, and 0.74 + 0.15 mV in that of the PTZ-kindled animals. The differences between the groups were not statistically significant. Also, the I / O curves constructed for each group did not differ from each other in a significant manner. Furthermore, the E / S curves that relate the field EPSP values to the amplitude of the population spike also showed no significant differences between the three groups (data not shown).
Dose-Dependent Changes in MEFPs of Kindled Animals and Kindling Controls After a Challenge Dose of PTZ Figure 4 shows analogue examples of evoked potentials registered from fully kindled rats before and after injection of two
458
RUETHRICH ET AL.
4 or 5 (as produced in kindled animals using 30 m g / k g ) no potentiation was found in the controls.
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different challenge doses of PTZ, indicating the induction of long-lasting changes in the slope function and population spike amplitude by the substance. However, the application of PTZ induced different time courses for the changes in evoked potentials in saline-injected kindling controls and in kindled animals, as represented in Figs. 5 and 6. Figure 5 demonstrates the changes in the amplitude of the population spike after injection of a challenge dose of PTZ compared to the basal values before injection. After application of 20 m g / k g PTZ to the kindled animals the amplitude increased over 24 h and was enhanced by approximately 2 - 3 mV (Fig. 5, top). These changes in the population spike were influenced by the severity and duration of the seizures, induced by the challenge dose. Its amplitude was gradually diminished if convulsion stages 4 or 5 occurred. In general, this reduction was detectable 5 - 7 min after injection of 30 m g / k g PTZ. However, if the long-lasting potentiation was also evident, it occurred with a delay (Fig. 5, bottom). In this case the amplitude of the population spike was significantly increased 180 min after injection and this increase lasted longer than 24 h. In contrast to that, kindling controls showed only a very short-lasting increase in the amplitude, which was dependent on the dosage of PTZ. A dose of 20 m g / k g caused an amplitude increase lasting only on average appoximately 6 0 - 9 0 min. The increase induced by 30 m g / k g PTZ lasted between 120 and 180 min. A longerlasting increase was never observed in control animals, even when using higher PTZ doses inducing seizure stages 4 or 5. The field EPSPs were influenced in a more complex way (Fig. 6). In kindled animals as well as in kindling controls 20 m g / k g PTZ caused an initial decrease of the slope function of the field EPSP, which lasted only 9 0 - 1 2 0 minutes. Later on, in both groups, no further changes were observed (Fig. 6, top). After application of 30 m g / k g PTZ and if convulsion stages 4 or 5 occurred in kindled animals, the slope function showed a biphasic alteration. The initial depression changed to a considerable long-lasting potentiation, which was significant from 120 min to 24 b after application (Fig. 6, bottom). In kindling controls the initial depression was not followed by any potentiation effects. Also, when using 45 m g / k g PTZ to induce seizure stages
When inducing LTP by a relatively moderate tetanization procedure using only three groups of tetanizing impulses, marked differences in the extent of potentiation of the population spike occurred between naive animals, kindling controls, and kindled animals. As Fig. 7 demonstrates, in all groups of animals a potentiation of the population spike was induced. In both control groups an increase of the population spike amplitude by 39 _+ 10% and 55 _+ 15%, respectively, was observed, which lasted at least 5 h, but declined to near control values within 24 h. On the contrary, in kindled animals the increase was 114 _+ 13%. The difference in amplitude increase between the two control groups and the kindled rats was statistically significant at all points up to 24 h after tetanization. DISCUSSION
The present in vivo study describes specific long-lasting excitability changes after a chemical kindling with PTZ. PTZ kindling is generally assumed as a model for primarily generalized epilepsy. In contrast to electrical kindling, the changes induced by repeated administration of a convulsive drug resulting in a kindling state similar to that seen after electrical kindling have not been sufficiently characterised until now. Here, we report on changes in the hippocampus, a brain formation that is directly involved in the development of epilepsy and other types of adaptive behavior such as learning and memory formation (4,38,64). In particular, the ability to produce long-term potentiation is well documented ( 1,5,13,53 ). Because the procedures for electrical kindling resemble those for inducing LTP and because some authors have described excitability changes similar to those after LTP induction, a connection between LTP and kindling has frequently been proposed such that potentiation mechanisms may also play a role in the development of kindling (7,13,17). However, the relationships between LTP and kindling-related potentiation phenomena have not been clarified (52,62). Notably, the altered ability not only to respond to stimulation but also to produce potentiation upon short strong stimulation when the kindling or epileptic state is established has not been extensively examined before either in electrical or chemical kindling models. Various articles have described changes in neuronal respon~ siveness during the course of electrical kindling that were visible as changes in field potential size or enhanced "burst firing " of single neurons and that tended to outlast the kindling procedure (I 1), although alternative results have also been reported (35,36,50). However, a growing body of evidence indicates that mainly after electrical kindling but also after chemical kindling the glutamatergic N M D A receptor ionic channel complex and intracellular second messenger systems, which are also involved in induction and maintenance of LTP, undergo lasting changes (i.e., they are upregulated) (2,12,16,25,27,46,58). This might imply that in kindled animals potentiation phenomena can be induced more easily and can last for a long time, and this might be one of the reasons for increased seizure susceptibility in the epileptic state. Therefore, we investigated whether a chemical stimulus (PTZ injection) of a challenge dose that at the beginning of the kindling procedure and in naive and sham-kindled animals in-
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duced only a short increase of MEFPs in the dentate gyms was able to produce potentiation (i.e., enhancement of the field potential that exceeds the stimulus action) (56). Additionally, it was tested whether LTP induced by tetanic stimulation was also changed in time course and extent in PTZ-kindled animals. From the results obtained some conclusions can be drawn. Firstly, the chronic implantation by itself enhanced the responsiveness of the animals to PTZ. Naive controls, sham-kindled, and kindled animals implanted with electrodes responded with higher seizure stages than nonimplanted animals. This confirms results (33) that additionally demonstrated marked changes in amino acid contents, especially glycine, in relevant brain structures. This effect was interpreted as a "prokindling effect." Thus, implantation can induce enduring changes that must be considered in interpreting results obtained in long-term experiments. The reason for such changes, at this time, seems unexplained, but perhaps reorganization processes may play a role (8,49). Secondly, the responsiveness of the dentate gyms to single test stimuli seems to be unaltered in fully kindled animals. Four weeks after the last kindling session neither the amplitude of the population spike elicited by a near threshold intensity nor the I/ O curve or E / S curve obtained from evoked potentials in kindled animals differed from that obtained in sham-kindled or naive animals. This result is in good agreement with other studies (30,50) and was also confirmed in the CA1 region in an in vitro experiment (Krug, in preparation). However, our results provide clear evidence for the development of enduring changes in the potency of granule cells to react with plastic reactions (response potentiation to PTZ) as a consequence of a chemical kindling. A test dose of PTZ that was subconvulsive in naive and sham-kindled animals and induced only a short-lasting increase in the population spike amplitude upon test stimulation of the perforant pathway provoked in kindled animals not only seizures but also
drastic changes in reaction to test stimuli, which were seen up to 24 h after injection. These changes in reaction cannot be explained by an enhanced PTZ concentration in the brains of kindled animals because after a single test dose of 25 m g / k g PTZ no differences in PTZ content were found between kindled and sham-kindled animals until 6 h after administration (Rauca, in preparation). Thus, PTZ kindling induces a novel type of response enhancement that can be described as a long-lasting potentiation. This potentiation effect is related to the kindling status and not to the severity of seizures, because naive animals showing the same seizure stage as kindled animals but with a higher PTZ dose show only shortlasting amplitude increases. However, in evaluating the different effects of PTZ on the population spike amplitude and the slope function of the field EPSP in the animals, the interaction of acute PTZ effects and the occurrence of seizures with the development of the longlasting response potentiation must be considered. For instance, the test doses of PTZ depressed the field EPSP for 2 - 3 h in all animals and, therefore, this depression must be considered as an acute PTZ effect that perhaps interacts with the development of potentiation. It is also of interest that after this initial depression of the field EPSP only in fully kindled animals that react with a seizure stage 4 or 5 a long-lasting EPSP potentiation was observed. This might indicate that a critical level of activation must be reached to induce potentiation and that seizure-induced depression influences the potentiation development. Furthermore, our results show that the induction and maintenance of LTP induced by tetanic stimulation is also facilitated. The procedure used produces a true LTP of both the population spike and the field EPSP, but as was discussed in chronic implanted animals (14,29), EPSP potentiation cannot be measured with the same accuracy as population spike potentiation. This is especially true under conditions of a very
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T i m e a f t e r PTZ i n j e c t i o n (rain / h) FIG. 5. Time course of changes of the amplitude of the population spike in saline-injected kindling controls (filled triangles) and kindled animals (filled circles). Top: changes after application of the same dose of PTZ (20 mg/kg) in controls (n = 10) and kindled animals (n = 19), respectively. Bottom: changes following induction of seizure stage 4 to 5 after application of 30 mg/kg PTZ in kindled animals (n = 12) and 45 mg/kg PTZ in controls (n = 9). (The difference of the actual values to baseline values before PTZ is given with p < 0.05 vs. *baseline, and +between the groups).
m o d e r a t e tetanization protocol. In our opinion the results indicate further that the r e s p o n s e potentiation seen after PTZ application in kindled animals is not due to an altered metabolism o f PTZ but rather is the result o f an enduring change in plastic properties o f the neurons after kindling. It may further indicate that in both cases related cellular m e c h a n i s m s are involved. C o n c e r n i n g the m e c h a n i s m s that underlie the p h e n o m enon described here, data on changes in passive m e m b r a n e properties are not conclusive as yet ( 4 1 ) . H o w e v e r , several results described in the literature as well as the results o f our p h a r m a c o l o g i c a l observations point to lasting changes in the glutamatergic transmitter system. For instance, both, c o m p e t itive and n o n c o m p e t i t i v e N M D A antagonists prevent or reduce kindling in different m o d e l s (14,15,20,44,54,57). An e n h a n c e d binding o f [3H]glutamate to h i p p o c a m p a l synaptic m e m b r a n e s ( 5 8 ) and altered properties o f the N M D A receptor ionic channel c o m p l e x as well as lasting e n h a n c e m e n t o f N M D A receptor sensitivity (27,28,46,48) have also been reported. Changes in the inhibitory G A B A system may be also partially r e s p o n s i b l e for the increased ability to produce potentiation because the m o s t p r o m i n e n t m e c h a n i s m of PTZ is a
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P O T E N T I A T I O N A N D PTZ K I N D L I N G
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reduction of G A B A e r g i c i n h i b i t i o n ( 9 , 3 1 , 6 0 ) and k i n d l i n g induces d y s f u n c t i o n s in the G A B A e r g i c s y s t e m ( 2 4 , 3 1 , 4 7 ) . H o w e v e r , b e n z o d i a z e p i n e s in a n t i c o n v u l s i v e dosages prev e n t e d i) P T Z k i n d l i n g d e v e l o p m e n t , ii) k i n d l i n g - i n d u c e d seizures, and iii) acute seizures ( m o t o r i c and e l e c t r o e n c e p h a l o g r a p h i c ) but they did not suppress the d e v e l o p m e n t o f the p o t e n t i a t i o n reaction to P T Z in the case of k i n d l i n g and did not d i m i n i s h P T Z - i n d u c e d p o t e n t i a t i o n in kindled a n i m a l s in acute e x p e r i m e n t s ( 5 5 ) . On the other hand, p r e l i m i n a r y exp e r i m e n t s have s h o w n in our m o d e l that N M D A a n t a g o n i s t s can p r e v e n t the d e v e l o p m e n t of P T Z k i n d l i n g and kindlingrelated p o t e n t i a t i o n ( 4 ) .
Our results show that a novel form of response potentiation in the hippocampus of chemical-kindled rats and a facilitation of long-term potentiation has been observed. From these results it might be concluded that an essential feature of kindled or epileptic nervous tissue is an enhanced ability to produce potentiation effects, perhaps via an upregulation of postsynaptic glutamatergic mechanisms. This alteration may not be reflected in normal glutamate-mediated excitation because N M D A receptorcoupled mechanisms are normally not involved in excitatory transmission, but it would be of high importance for the development of lasting plastic changes in excitability of neurons after strong and repetitive activation.
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