Neuroscience Letters, 141 (1992) 101-105 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
101
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Hippocampal kindling leads to different changes in paired-pulse depression of local evoked field potentials in CA! area and in fascia dentata Willem Kamphuis a, Jan A. Gorter b, Wytse J. W a d m a n a and Fernando H. Lopes da Silva a aDepartment of Experimental Zoology, University of Amsterdam, Amsterdam (Netherlands) and bNetherlands Institute for Brain Research, Amsterdam (Netherlands) (Received 16 March 1992; Revised version received 7 April 1992; Accepted 9 April 1992)
Key words: Kindling; Epilepsy; Field potential; Inhibition; Hippocampus; CA1; Fascia dentata Monosynaptic evoked field potentials (EPs) in response to paired-pulse stimulation (20 ms interval) were recorded in area CA1 and fascia dentata of the same animal in the course of development of a kindled focus in the CA 1 region. A significant reduction of paired pulse depression in response to medium and high stimulation intensity was found in CAl. A similar change was found in the fascia dentata in response to medium intensity stimulation of the angular bundle. In contrast, at high intensity, paired pulse depression was enhanced in the fascia dentata in the course of kindling. These results indicate that kindling epileptogenesis is accompanied by regionally different changes in recurrent inhibition: a reduction in CA1 and intensity dependent changes in fascia dentata.
Repeated application of short, high-frequency, electrical stimuli results in a progressive increase of the duration of epileptiform afterdischarges and of the severity of behavioural seizures. This gradual process of epileptogenesis, called 'kindling', is considered to be a model of certain aspects of human epilepsy of focal onset [4, 8]. Changes in excitability of neuronal networks, induced by kindling, have been investigated by recording local evoked field potentials (EPs) in the course of kindling acquisition [21]. Field potentials are the extracellular reflection of the population postsynaptic potential (pPSP), which is a sequence of an excitatory component (EPSP) followed by a ~'-aminobutyric acid (GABA)-mediated inhibitory component (IPSP) due to activation of recurrent inhibitory pathways [1, 11-13, 24]. When a second stimulation pulse is given during the IPSP, the amplitude of the pPSP evoked in response to this second pulse is depressed; this is referred to as paired-pulse depression (PPD). In previous experiments we have shown that kindling of the Schaffer collateral/commissural pathway leads to a reduced PPD of the pPSP amplitude in CAI area of the Correspondence: W. Kamphuis, Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, Netherlands.
rat dorsal hippocampus [7]. These alterations indicate an impairment of recurrent inhibition in this brain region [10, 11, 16, 26, 27]. In contrast, it has been reported that in the fascia dentata and in the piriform cortex of the rat, kindling stimulation resulted in an enhanced PPD which is interpreted as an increased inhibition [2, 15, 19, 22-25]. These results suggest that kindling stimulations may lead to opposite changes in different brain areas. However, a definite conclusion is hampered by the fact that the experimental data reported above were obtained using different experimental protocols, and the kindling stimulations were applied in different brain sites. The aim of the experiments described here was to investigate whether regional differences exist in kindling-induced field potential changes. For this purpose, we have measured simultaneously the alterations in PPD of monosynaptic evoked field potentials both in area CA1 and in fascia dentata during kindling of the Schaffer collateral/commissural pathway. Male Wistar rats (350-400 g), under anesthesia, were stereotaxically implanted with 4 electrodes: 2 were used for stimulation and 2 for recording. Reference electrodes were screws placed in the skull. The first stimulation electrode was placed in stratum radiatum/stratum lacunosum-moleculare in CA1 area of the dorsal hippocampus to stimulate the Schaffer collaterals/commissural fibers
102 (coordinates: 3.2 mm posterior to the bregma and 2.5 mm left lateral to the midline). The first recording electrode was placed in the stratum radiatum of CAI area (3.0 mm posterior to the bregma and 2.4 mm left lateral). The second stimulation electrode was placed in the angular bundle (7.8 mm posterior to bregma, 4.4 mm left lateral to the midline) and the second recording electrode was positioned in the hilar region of the fascia dentata (3.0 mm posterior to the bregma and 2.4 mm left lateral). The precise position of the electrodes was guided by the depth profiles of the local evoked field potentials (EPs) in response to test stimuli. The electrodes were fixed in position and connected to a plug which was secured to the skull with acrylic cement. After a postoperative period of 2 weeks, monosynaptic EPs in stratum radiatum (SR) of area CA1 were recorded in response to stimulation of the Schaffer collaterals/ commissural pathway (SCH). In the hilus (HL) of the fascia dentata, monosynaptic EPs were elicited by stimulation of the angular bundle (AB). In the following, these stimulation-recording positions are indicated as SCHSR and AB-HL. EPs were elicited using a double pulse stimulation paradigm with two 0.1 ms biphasic stimulus pulses at 20 ms interval delivered at a sweep interval of 8 s. Responses were sampled (3.3 kHz), averaged over 8 sweeps and stored on disk. Before the animals were used in the kindling experiment described here, they were used in another experiment designed to study the effects of sleep deprivation on local EPs [5]. Animals (n = 8) were only included in the experiment described here when the form and stability of the elicited field potentials showed no drift during 6 weeks, namely 4 during the sleep deprivation experiment and 2 weeks of rest that elapsed before kindling was initiated. The peak amplitudes of the averaged pPSP were measured against the baseline value determined 1 ms preceding the stimulation. In order to quantify the paired-pulse interaction, the peak amplitude in response to the second pulse of the pair was divided by the amplitude of the evoked response to the first pulse. This paired pulse index (PPI) depends on the stimulation intensity used. At low intensities of SCH or AB stimulation PPI is larger than 1.0 (paired-pulse facilitation), while at high intensities a PPI smaller than 1.0 was apparent (paired-pulse depression or PPD) [7]. On the basis of these control measurements, three stimulation intensities (L, M, H) were selected for further study during a pre-kindling period of 3 days and a subsequent period in which kindling stimuli were given: a low intensity (L), just above the threshold for evoking field potentials, gave an PPI value of approximately 1.3 for SCH-SR and 1.1 for AB-HL. The stimulus intensity for SCH-SR was within the range
of 30 150 HA and for AB-HL 20-180/IA. A mid-range intensity (M) gave a PPI value of 1.0 for SCH-SR and 0.7 for AB-HL. The stimulus intensity for SCH-SR was within the range of 100-200/IA and for AB-HL 40-250 HA. A high intensity (H) gave the maximum field potential amplitude. At this high intensity the value of PPI showed maximal PPD. A notable feature was that the maximal PPD reached in these two areas was rather different: for SCH-SR this was 0.87 while in AB-HL it was 0.44. Stimulus intensity range for SCH-SR was 200-400 /IA and for AB-HL 100 500/,tA. In a typical recording session the animal was connected to the stimulation/recording unit and placed in a plexiglass observation box. The animal remained sitting quietly in an alert state during the period of EP measurements. The recording session started with the AB-HL stimulations at the pre-selected intensities followed by the SCH-SR stimulations. Two recording sessions were carried out on each day, at least 5 h apart. During the pre-kindling period (3 days), baseline measures were taken. At the end of the 6th recording session, the first kindling stimulation was given through the SCH stimulation electrode. Thereafter, the kindling stimulation (1-2 s, 50 Hz, biphasic square pulses 0.1 ms, of an intensity ranging from 300 to 500 HA. supra-threshold for eliciting an afterdischarge), was given at the end of each session where EPs were recorded. Alter the 12th kindling session, EPs were recorded once a day while kindling stimulations were continued at a rate of twice a day. All animals (n=8) responded to the first tetanic stimulation with an afterdischarge. After 22 sessions, when all animals showed class 5 seizures [20], kindling stimulations were ended. Subsequently, the animals were anesthetized and perfusion fixed for histological verification of the electrode positions. Histology confirmed a correct position of the electrodes. The EPs of 8 animals with stable responses during the pre-kindling period were studied in detail. Kindling induced changes were analyzed by comparing the value of PPI at a particular kindling session with the mean value of the 6 measurements made in the pre-kindling period. The difference between these values was tested with the paired Student's t-test. In addition, the trend of PPI change as function of session number after the start of the kindling protocol was tested with the non-parametric Spearman's correlation test. The mean PPI for SCH-SR and AB-HL for the H and M stimulation intensity during the pre-kindling period and in the course of kindling is presented in Fig. 1. No significant changes in the mean value of PPI were found in response to the L intensity double pulse stimulation in either region (results not shown). The mean PPI of the SCH-SR response to the H stimulus intensity was significantly enhanced immedi-
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Fig. 1. The development of the paired-pulse index (PPI) in response to (a) the high (H) and (b) the medium (M) stimulus intensity for the CA1 SCH-SR response, and to (c) the high (H) and (d) the medium (M) stimulus intensity for the fascia dentata AB-HL response in the course of kindling. The first 6 closed symbols represent the measurements in the pre-kindling period and the open symbols indicate the recordings during the kindling period. The horizontal dotted line indicates the mean pre-kindling value of PPI. Values represent means + S.E.M. (n=8).
ately from session 1 onwards to session 22 compared to the pre-kindling period (Student's t-test, P < 0.05). PPI increased with session number from session 0 to session 6 (P < 0.006, Fig. la). A significant increase of PPI was also found for the SCH-SR response for the M intensity from session 2 to session 19. PPI increased with session number from session 0 to session 6 (P < 0.002, Fig. lb). Increasing the stimulus intensity above the H intensity did not restore the PPD. In contrast to the increase found for SCH-SR, the mean PPI of the AB-HL response to H stimulus intensity was significantly decreased at kindling sessions 5 till 17 compared to the pre-kindling period (P < 0.05). PPI decreased with session number from session 0 to session 6 (P < 0.026, Fig. lc). The Spearman's correlation test
showed that PPI in response to M stimulus intensity increased significantly with session number from session 0 to session 8 (P < 0.05; and to session 11, P < 0.001). Since the changes at this intensity were not so robust, the Student's t-test did not reveal a significant difference in PPI value between pre-kindling period and the later kindling sessions (Fig. ld). Since the value of the PPI is also dependent on the amplitude of the conditioning response, we determined whether changes took place in the amplitude o f the first, or conditioning EP (El). The mean El of the 6 pre-kindling sessions was used as a reference set at 100%. For SCH-SR, at H stimulus intensity, the mean E1 decreased only after session 7 to a stable value o f approximately 90% from session 9 to 22. For the A B - H L response, the
104 mean value o f E1 at high intensity stimulation increased after the first and second session and remained a r o u n d a value o f 110% during the rest o f the kindling period. F r o m this follows that the opposite changes in PPI could partly be caused by opposite changes in the response to the conditioning stimulus. This is unlikely, however, since not all animals showed the changes in E l , while clear changes in PPI were observed in all animals. Moreover, the development o f the changes in E 1 in the course of kindling was clearly different f r o m the development o f PPI alterations. In view o f the hypothesis that an imbalance o f excitatory over inhibitory transmission underlies epileptogenesis, it was anticipated that kindling would lead to specific modifications o f the m o n o s y n a p t i c EPs that would indicate either a gradual reduction o f synaptic inhibition and/or a strengthening o f excitation. The observations in C A 1 described here confirm those o f our previous report [7] and are in agreement with the results o f other studies o f changes in paired pulse interaction after kindling in CA1 [10, 11, 16, 26, 27]. In support o f the hypothesis that a reduction o f GABAA-mediated recurrent inhibition contributes to the development o f kindling epilepsy in this brain area, we have recently shown that in fully kindled animals the pyramidal neurons in CA1 were less sensitive to the suppressive action o f iontophoretically applied G A B A [9], suggesting a decrease in GABAA-receptor function. In contrast to the observations in CA1 at the high stimulation intensity, P P D in the fascia dentata at high stimulus intensities became more pronounced in the course o f kindling, which confirms the increase in paired-pulse depression in this region observed in vivo [2, 15, 21, 23, 24, 25] and in slices from kindled animals [11, 19, 25]. However, a decrease in P P D was found at the mid-range stimulus intensity. Therefore, at a comparable pre-kindling level o f P P D (0.7 1.0) a decrease in P P D was found in both C A I and in fascia dentata. The enhancement of P P D in the fascia dentata occurs at at level o f P P D that could not be obtained in C A 1. A n elevated n u m b e r o f benzodiazepine and G A B A binding sites after kindling, uniquely f o u n d in the fascia dentata [17, 18] and perhaps only activated by strong stimuli, m a y underly the enhanced G A B A e r g i c inhibition. On the basis o f the evidence presented here it can be excluded that the observed differences between the changes in EPs recorded in C A 1 and in fascia dentata are caused by methodological differences in the protocol o f EPs recording. We conclude that regionally different alterations, possibly related to different modifications o f G A B A e r g i c inhibition, underlie the different changes in paired pulse interaction in C A 1 area and fascia dentata. Electrophysiological and biochemical evidence for a kindling related decrement o f G A B A e r g i c functions has
been f o u n d for several other brain areas in addition to CA1 [3, 6, 14]. This suggests that a reduced G A B A e r g i c inhibition m a y be the prevailing modification underlying the kindling induced enhanced seizure susceptibility. We thank Ton Juta, Liesbeth Clason and Simon van Mechelen for their contribution. This work was supported by the D u t c h Committee for Epilepsy Research ( C L E O - T N O , G r a n t s A-67 and A-77). 1 Andersen, P.,Organization ofhippocampalneurons andtheir interconnections. In R.L. Isaacson and H. Pribram (Eds.), The Hippocampus, Vol. 1, Plenum, New York, 1975, pp. 155-175. 2 De Jonge, M. and Racine, R.J., The development and decay ot" kindling-induced increases in paired-pulse depression in the dentate gyrus, Brain Res.~ 412 (1987) 318 328. 3 Gean, P.-W., Shinnick-Gallager, E and Anderson, A.C., Spontaneous epileptiform activity and alteration of GABA- and of NMDAmediated neurotransmission in amygdala neurons kindled in vivo, Brain Res., 494 (1989) 177 181. 4 Goddard, G.V., Mclntyre, EC. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295 330. 5 Gorter, J.A., Kamphuis, W. and Coenen, A.M.L., Paradoxical sleep deprivation does not affect neuronal excitability in the rat hippocampus, Brain Res., 476 (1989) 16-20. 6 Hernandez, T.D., Rosen, J.B. and Gallagher, D.W., Long-term changes in sensitivity to GABA in dorsal raphe neurons following amygdala kindling, Brain Res.. 517 (1990) 294 300. 7 Kamphuis, W., Wadman, W.J. and Lopes da Silva, F.H., Changes in local evoked potentials in the rat hippocampus (CAI) during kindling epileptogenesis, Brain Res., 440 (1988) 205 215. 8 Kamphuis, W. and Lopes da Silva, F.H., The kindling model of epilepsy: the role of GABAergic inhibition, Neurosci. Res. Commun., 6 (1990) 1 10. 9 Kamphuis, W., Gorter, J.A. and Lopes da Silva, F.H., Long-term decreased sensitivity of hippocampal pyramidal cells to GABA induced by kindling epileptogenesis, Neuroscience, 41 (1991) 425431. 10 Kapur, J., Michelson. H.B.. Buterbaugh, G.F. and Lothman, E.W., Evidence for a chronic loss of inhibition in the hippocampus after kindling: electrophysiological studies, Epilepsy Res., 4 (1989) 90 99. 11 King, G.L., Dingledine, R., Giacchino, J.L. and McNamara, J.O., Abnormal neuronal excitability in hippocampal slices from kindled rats, J. Neurophysiol., 54 (1985) 1295- 1304. 12 Leung, L.S., Hippocampal CA1 region-demonstration of antidromic dendritic spike and denditic inhibition, Brain Res., 159 (1978) 219-222. 13 Leung, L.S., Orthodromic activation of the hippocampal CAI region in the rat, Brain Res., 176 (1979) 49 63. 14 L6scher, W. and Schwark, W.S., Further evidence for abnormal GABAergic circuits in amygdala-kindled rats, Brain Res., 420 (1987) 385 390. 15 Maru~ E. and Goddard, G.V.. Alteration in dentate neuronal activities associated with perforant path kindling, lII Enhancement of synaptic inhibition, Exp. Neurol., 96 (1987) 46 -60. 16 Michelson, H.B., Kapur, J. and Lothman, E.W., Reduction of paired-pulse inhibition in the CA1 region of the hippocampus by pilocarpine in naive and amygdala-kindled rats, Exp. Neurol., 104 (1989) 264 271.
105 17 Nobrega, J.N., Kish, S.J. and Mclntyre Burnham, W., Autoradiographic analysis of benzodiazepine binding in entorhinal kindled rat brains, Brain Res., 498 (1989) 315 322. 18 Nobrega, J.N., Kish, S.J. and Mclntyre Burnham, W., Regional brain [3H]muscimol binding in the kindled rat brain: a quantitative autoradiographic examination, Epilepsy Res., 6 (1990) 102-109. 19 Oliver, M.W. and Miller, J.J., Alterations of inhibitory processes in the dentate gyrus following kindling-induced epilepsy, Exp. Brain Res., 57 (1985) 443445. 20 Racine, R.J., Modification of seizure activity by electrical stimulation. 11 Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 21 Racine, R.J., Burnham, W.M., Gilbert, M. and Kairiss, E.W., Kindling mechanisms: I Electrophysiological studies. In J.A. Wada (Ed.), Kindling, Raven, New York, 1986, pp. 263-282. 22 Racine, R.J., Moore, K.-A. and Evans, C., Kindling induced potentiation in the piriform cortex, Brain Res., 556 (1991) 218-225.
23 Robinson, G.B., Sclabassi, R.J. and Berger, T.W., Kindling-induced potentiation of excitatory and inhibitory inputs to hippocampal dentate granule cells. I. Effects on linear and non-linear response characteristics, Brain Res., 562 (1991) 17-25. 24 Tuff, L.P., Racine, R.J. and Adamec, R., The effects on GABAmediated inhibition in the dentate gyrus of the rat. I. Paired pulse depression, Brain Res., 277 (1983) 79-90. 25 Voskuyl, R.A. and Albus, H., Enhancement of recurrent inhibition by angular bundle kindling is retained in hippocampal slices, Int. J. Neurosci., 36 (1987) 153-166. 26 Yamada, N. and Bilkey, D.K., Kindling-induced persistent alterations in the membrane and synaptic properties of CA1 pyramidal neurons, Brain Res., 561 (1991) 324-331. 27 Zhao, D. and Leung, L.S., Effects of hippocampal kindling on paired-pulse response in CA1 in vitro, Brain Res., 564 (1991) 220229.
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Hippocampal kindling leads to different changes in paired-pulse depression of local evoked field potentials in CA! area and in fascia dentata Willem Kamphuis a, Jan A. Gorter b, Wytse J. W a d m a n a and Fernando H. Lopes da Silva a aDepartment of Experimental Zoology, University of Amsterdam, Amsterdam (Netherlands) and bNetherlands Institute for Brain Research, Amsterdam (Netherlands) (Received 16 March 1992; Revised version received 7 April 1992; Accepted 9 April 1992)
Key words: Kindling; Epilepsy; Field potential; Inhibition; Hippocampus; CA1; Fascia dentata Monosynaptic evoked field potentials (EPs) in response to paired-pulse stimulation (20 ms interval) were recorded in area CA1 and fascia dentata of the same animal in the course of development of a kindled focus in the CA 1 region. A significant reduction of paired pulse depression in response to medium and high stimulation intensity was found in CAl. A similar change was found in the fascia dentata in response to medium intensity stimulation of the angular bundle. In contrast, at high intensity, paired pulse depression was enhanced in the fascia dentata in the course of kindling. These results indicate that kindling epileptogenesis is accompanied by regionally different changes in recurrent inhibition: a reduction in CA1 and intensity dependent changes in fascia dentata.
Repeated application of short, high-frequency, electrical stimuli results in a progressive increase of the duration of epileptiform afterdischarges and of the severity of behavioural seizures. This gradual process of epileptogenesis, called 'kindling', is considered to be a model of certain aspects of human epilepsy of focal onset [4, 8]. Changes in excitability of neuronal networks, induced by kindling, have been investigated by recording local evoked field potentials (EPs) in the course of kindling acquisition [21]. Field potentials are the extracellular reflection of the population postsynaptic potential (pPSP), which is a sequence of an excitatory component (EPSP) followed by a ~'-aminobutyric acid (GABA)-mediated inhibitory component (IPSP) due to activation of recurrent inhibitory pathways [1, 11-13, 24]. When a second stimulation pulse is given during the IPSP, the amplitude of the pPSP evoked in response to this second pulse is depressed; this is referred to as paired-pulse depression (PPD). In previous experiments we have shown that kindling of the Schaffer collateral/commissural pathway leads to a reduced PPD of the pPSP amplitude in CAI area of the Correspondence: W. Kamphuis, Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, Netherlands.
rat dorsal hippocampus [7]. These alterations indicate an impairment of recurrent inhibition in this brain region [10, 11, 16, 26, 27]. In contrast, it has been reported that in the fascia dentata and in the piriform cortex of the rat, kindling stimulation resulted in an enhanced PPD which is interpreted as an increased inhibition [2, 15, 19, 22-25]. These results suggest that kindling stimulations may lead to opposite changes in different brain areas. However, a definite conclusion is hampered by the fact that the experimental data reported above were obtained using different experimental protocols, and the kindling stimulations were applied in different brain sites. The aim of the experiments described here was to investigate whether regional differences exist in kindling-induced field potential changes. For this purpose, we have measured simultaneously the alterations in PPD of monosynaptic evoked field potentials both in area CA1 and in fascia dentata during kindling of the Schaffer collateral/commissural pathway. Male Wistar rats (350-400 g), under anesthesia, were stereotaxically implanted with 4 electrodes: 2 were used for stimulation and 2 for recording. Reference electrodes were screws placed in the skull. The first stimulation electrode was placed in stratum radiatum/stratum lacunosum-moleculare in CA1 area of the dorsal hippocampus to stimulate the Schaffer collaterals/commissural fibers
102 (coordinates: 3.2 mm posterior to the bregma and 2.5 mm left lateral to the midline). The first recording electrode was placed in the stratum radiatum of CAI area (3.0 mm posterior to the bregma and 2.4 mm left lateral). The second stimulation electrode was placed in the angular bundle (7.8 mm posterior to bregma, 4.4 mm left lateral to the midline) and the second recording electrode was positioned in the hilar region of the fascia dentata (3.0 mm posterior to the bregma and 2.4 mm left lateral). The precise position of the electrodes was guided by the depth profiles of the local evoked field potentials (EPs) in response to test stimuli. The electrodes were fixed in position and connected to a plug which was secured to the skull with acrylic cement. After a postoperative period of 2 weeks, monosynaptic EPs in stratum radiatum (SR) of area CA1 were recorded in response to stimulation of the Schaffer collaterals/ commissural pathway (SCH). In the hilus (HL) of the fascia dentata, monosynaptic EPs were elicited by stimulation of the angular bundle (AB). In the following, these stimulation-recording positions are indicated as SCHSR and AB-HL. EPs were elicited using a double pulse stimulation paradigm with two 0.1 ms biphasic stimulus pulses at 20 ms interval delivered at a sweep interval of 8 s. Responses were sampled (3.3 kHz), averaged over 8 sweeps and stored on disk. Before the animals were used in the kindling experiment described here, they were used in another experiment designed to study the effects of sleep deprivation on local EPs [5]. Animals (n = 8) were only included in the experiment described here when the form and stability of the elicited field potentials showed no drift during 6 weeks, namely 4 during the sleep deprivation experiment and 2 weeks of rest that elapsed before kindling was initiated. The peak amplitudes of the averaged pPSP were measured against the baseline value determined 1 ms preceding the stimulation. In order to quantify the paired-pulse interaction, the peak amplitude in response to the second pulse of the pair was divided by the amplitude of the evoked response to the first pulse. This paired pulse index (PPI) depends on the stimulation intensity used. At low intensities of SCH or AB stimulation PPI is larger than 1.0 (paired-pulse facilitation), while at high intensities a PPI smaller than 1.0 was apparent (paired-pulse depression or PPD) [7]. On the basis of these control measurements, three stimulation intensities (L, M, H) were selected for further study during a pre-kindling period of 3 days and a subsequent period in which kindling stimuli were given: a low intensity (L), just above the threshold for evoking field potentials, gave an PPI value of approximately 1.3 for SCH-SR and 1.1 for AB-HL. The stimulus intensity for SCH-SR was within the range
of 30 150 HA and for AB-HL 20-180/IA. A mid-range intensity (M) gave a PPI value of 1.0 for SCH-SR and 0.7 for AB-HL. The stimulus intensity for SCH-SR was within the range of 100-200/IA and for AB-HL 40-250 HA. A high intensity (H) gave the maximum field potential amplitude. At this high intensity the value of PPI showed maximal PPD. A notable feature was that the maximal PPD reached in these two areas was rather different: for SCH-SR this was 0.87 while in AB-HL it was 0.44. Stimulus intensity range for SCH-SR was 200-400 /IA and for AB-HL 100 500/,tA. In a typical recording session the animal was connected to the stimulation/recording unit and placed in a plexiglass observation box. The animal remained sitting quietly in an alert state during the period of EP measurements. The recording session started with the AB-HL stimulations at the pre-selected intensities followed by the SCH-SR stimulations. Two recording sessions were carried out on each day, at least 5 h apart. During the pre-kindling period (3 days), baseline measures were taken. At the end of the 6th recording session, the first kindling stimulation was given through the SCH stimulation electrode. Thereafter, the kindling stimulation (1-2 s, 50 Hz, biphasic square pulses 0.1 ms, of an intensity ranging from 300 to 500 HA. supra-threshold for eliciting an afterdischarge), was given at the end of each session where EPs were recorded. Alter the 12th kindling session, EPs were recorded once a day while kindling stimulations were continued at a rate of twice a day. All animals (n=8) responded to the first tetanic stimulation with an afterdischarge. After 22 sessions, when all animals showed class 5 seizures [20], kindling stimulations were ended. Subsequently, the animals were anesthetized and perfusion fixed for histological verification of the electrode positions. Histology confirmed a correct position of the electrodes. The EPs of 8 animals with stable responses during the pre-kindling period were studied in detail. Kindling induced changes were analyzed by comparing the value of PPI at a particular kindling session with the mean value of the 6 measurements made in the pre-kindling period. The difference between these values was tested with the paired Student's t-test. In addition, the trend of PPI change as function of session number after the start of the kindling protocol was tested with the non-parametric Spearman's correlation test. The mean PPI for SCH-SR and AB-HL for the H and M stimulation intensity during the pre-kindling period and in the course of kindling is presented in Fig. 1. No significant changes in the mean value of PPI were found in response to the L intensity double pulse stimulation in either region (results not shown). The mean PPI of the SCH-SR response to the H stimulus intensity was significantly enhanced immedi-
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Fig. 1. The development of the paired-pulse index (PPI) in response to (a) the high (H) and (b) the medium (M) stimulus intensity for the CA1 SCH-SR response, and to (c) the high (H) and (d) the medium (M) stimulus intensity for the fascia dentata AB-HL response in the course of kindling. The first 6 closed symbols represent the measurements in the pre-kindling period and the open symbols indicate the recordings during the kindling period. The horizontal dotted line indicates the mean pre-kindling value of PPI. Values represent means + S.E.M. (n=8).
ately from session 1 onwards to session 22 compared to the pre-kindling period (Student's t-test, P < 0.05). PPI increased with session number from session 0 to session 6 (P < 0.006, Fig. la). A significant increase of PPI was also found for the SCH-SR response for the M intensity from session 2 to session 19. PPI increased with session number from session 0 to session 6 (P < 0.002, Fig. lb). Increasing the stimulus intensity above the H intensity did not restore the PPD. In contrast to the increase found for SCH-SR, the mean PPI of the AB-HL response to H stimulus intensity was significantly decreased at kindling sessions 5 till 17 compared to the pre-kindling period (P < 0.05). PPI decreased with session number from session 0 to session 6 (P < 0.026, Fig. lc). The Spearman's correlation test
showed that PPI in response to M stimulus intensity increased significantly with session number from session 0 to session 8 (P < 0.05; and to session 11, P < 0.001). Since the changes at this intensity were not so robust, the Student's t-test did not reveal a significant difference in PPI value between pre-kindling period and the later kindling sessions (Fig. ld). Since the value of the PPI is also dependent on the amplitude of the conditioning response, we determined whether changes took place in the amplitude o f the first, or conditioning EP (El). The mean El of the 6 pre-kindling sessions was used as a reference set at 100%. For SCH-SR, at H stimulus intensity, the mean E1 decreased only after session 7 to a stable value o f approximately 90% from session 9 to 22. For the A B - H L response, the
104 mean value o f E1 at high intensity stimulation increased after the first and second session and remained a r o u n d a value o f 110% during the rest o f the kindling period. F r o m this follows that the opposite changes in PPI could partly be caused by opposite changes in the response to the conditioning stimulus. This is unlikely, however, since not all animals showed the changes in E l , while clear changes in PPI were observed in all animals. Moreover, the development o f the changes in E 1 in the course of kindling was clearly different f r o m the development o f PPI alterations. In view o f the hypothesis that an imbalance o f excitatory over inhibitory transmission underlies epileptogenesis, it was anticipated that kindling would lead to specific modifications o f the m o n o s y n a p t i c EPs that would indicate either a gradual reduction o f synaptic inhibition and/or a strengthening o f excitation. The observations in C A 1 described here confirm those o f our previous report [7] and are in agreement with the results o f other studies o f changes in paired pulse interaction after kindling in CA1 [10, 11, 16, 26, 27]. In support o f the hypothesis that a reduction o f GABAA-mediated recurrent inhibition contributes to the development o f kindling epilepsy in this brain area, we have recently shown that in fully kindled animals the pyramidal neurons in CA1 were less sensitive to the suppressive action o f iontophoretically applied G A B A [9], suggesting a decrease in GABAA-receptor function. In contrast to the observations in CA1 at the high stimulation intensity, P P D in the fascia dentata at high stimulus intensities became more pronounced in the course o f kindling, which confirms the increase in paired-pulse depression in this region observed in vivo [2, 15, 21, 23, 24, 25] and in slices from kindled animals [11, 19, 25]. However, a decrease in P P D was found at the mid-range stimulus intensity. Therefore, at a comparable pre-kindling level o f P P D (0.7 1.0) a decrease in P P D was found in both C A I and in fascia dentata. The enhancement of P P D in the fascia dentata occurs at at level o f P P D that could not be obtained in C A 1. A n elevated n u m b e r o f benzodiazepine and G A B A binding sites after kindling, uniquely f o u n d in the fascia dentata [17, 18] and perhaps only activated by strong stimuli, m a y underly the enhanced G A B A e r g i c inhibition. On the basis o f the evidence presented here it can be excluded that the observed differences between the changes in EPs recorded in C A 1 and in fascia dentata are caused by methodological differences in the protocol o f EPs recording. We conclude that regionally different alterations, possibly related to different modifications o f G A B A e r g i c inhibition, underlie the different changes in paired pulse interaction in C A 1 area and fascia dentata. Electrophysiological and biochemical evidence for a kindling related decrement o f G A B A e r g i c functions has
been f o u n d for several other brain areas in addition to CA1 [3, 6, 14]. This suggests that a reduced G A B A e r g i c inhibition m a y be the prevailing modification underlying the kindling induced enhanced seizure susceptibility. We thank Ton Juta, Liesbeth Clason and Simon van Mechelen for their contribution. This work was supported by the D u t c h Committee for Epilepsy Research ( C L E O - T N O , G r a n t s A-67 and A-77). 1 Andersen, P.,Organization ofhippocampalneurons andtheir interconnections. In R.L. Isaacson and H. Pribram (Eds.), The Hippocampus, Vol. 1, Plenum, New York, 1975, pp. 155-175. 2 De Jonge, M. and Racine, R.J., The development and decay ot" kindling-induced increases in paired-pulse depression in the dentate gyrus, Brain Res.~ 412 (1987) 318 328. 3 Gean, P.-W., Shinnick-Gallager, E and Anderson, A.C., Spontaneous epileptiform activity and alteration of GABA- and of NMDAmediated neurotransmission in amygdala neurons kindled in vivo, Brain Res., 494 (1989) 177 181. 4 Goddard, G.V., Mclntyre, EC. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295 330. 5 Gorter, J.A., Kamphuis, W. and Coenen, A.M.L., Paradoxical sleep deprivation does not affect neuronal excitability in the rat hippocampus, Brain Res., 476 (1989) 16-20. 6 Hernandez, T.D., Rosen, J.B. and Gallagher, D.W., Long-term changes in sensitivity to GABA in dorsal raphe neurons following amygdala kindling, Brain Res.. 517 (1990) 294 300. 7 Kamphuis, W., Wadman, W.J. and Lopes da Silva, F.H., Changes in local evoked potentials in the rat hippocampus (CAI) during kindling epileptogenesis, Brain Res., 440 (1988) 205 215. 8 Kamphuis, W. and Lopes da Silva, F.H., The kindling model of epilepsy: the role of GABAergic inhibition, Neurosci. Res. Commun., 6 (1990) 1 10. 9 Kamphuis, W., Gorter, J.A. and Lopes da Silva, F.H., Long-term decreased sensitivity of hippocampal pyramidal cells to GABA induced by kindling epileptogenesis, Neuroscience, 41 (1991) 425431. 10 Kapur, J., Michelson. H.B.. Buterbaugh, G.F. and Lothman, E.W., Evidence for a chronic loss of inhibition in the hippocampus after kindling: electrophysiological studies, Epilepsy Res., 4 (1989) 90 99. 11 King, G.L., Dingledine, R., Giacchino, J.L. and McNamara, J.O., Abnormal neuronal excitability in hippocampal slices from kindled rats, J. Neurophysiol., 54 (1985) 1295- 1304. 12 Leung, L.S., Hippocampal CA1 region-demonstration of antidromic dendritic spike and denditic inhibition, Brain Res., 159 (1978) 219-222. 13 Leung, L.S., Orthodromic activation of the hippocampal CAI region in the rat, Brain Res., 176 (1979) 49 63. 14 L6scher, W. and Schwark, W.S., Further evidence for abnormal GABAergic circuits in amygdala-kindled rats, Brain Res., 420 (1987) 385 390. 15 Maru~ E. and Goddard, G.V.. Alteration in dentate neuronal activities associated with perforant path kindling, lII Enhancement of synaptic inhibition, Exp. Neurol., 96 (1987) 46 -60. 16 Michelson, H.B., Kapur, J. and Lothman, E.W., Reduction of paired-pulse inhibition in the CA1 region of the hippocampus by pilocarpine in naive and amygdala-kindled rats, Exp. Neurol., 104 (1989) 264 271.
105 17 Nobrega, J.N., Kish, S.J. and Mclntyre Burnham, W., Autoradiographic analysis of benzodiazepine binding in entorhinal kindled rat brains, Brain Res., 498 (1989) 315 322. 18 Nobrega, J.N., Kish, S.J. and Mclntyre Burnham, W., Regional brain [3H]muscimol binding in the kindled rat brain: a quantitative autoradiographic examination, Epilepsy Res., 6 (1990) 102-109. 19 Oliver, M.W. and Miller, J.J., Alterations of inhibitory processes in the dentate gyrus following kindling-induced epilepsy, Exp. Brain Res., 57 (1985) 443445. 20 Racine, R.J., Modification of seizure activity by electrical stimulation. 11 Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 21 Racine, R.J., Burnham, W.M., Gilbert, M. and Kairiss, E.W., Kindling mechanisms: I Electrophysiological studies. In J.A. Wada (Ed.), Kindling, Raven, New York, 1986, pp. 263-282. 22 Racine, R.J., Moore, K.-A. and Evans, C., Kindling induced potentiation in the piriform cortex, Brain Res., 556 (1991) 218-225.
23 Robinson, G.B., Sclabassi, R.J. and Berger, T.W., Kindling-induced potentiation of excitatory and inhibitory inputs to hippocampal dentate granule cells. I. Effects on linear and non-linear response characteristics, Brain Res., 562 (1991) 17-25. 24 Tuff, L.P., Racine, R.J. and Adamec, R., The effects on GABAmediated inhibition in the dentate gyrus of the rat. I. Paired pulse depression, Brain Res., 277 (1983) 79-90. 25 Voskuyl, R.A. and Albus, H., Enhancement of recurrent inhibition by angular bundle kindling is retained in hippocampal slices, Int. J. Neurosci., 36 (1987) 153-166. 26 Yamada, N. and Bilkey, D.K., Kindling-induced persistent alterations in the membrane and synaptic properties of CA1 pyramidal neurons, Brain Res., 561 (1991) 324-331. 27 Zhao, D. and Leung, L.S., Effects of hippocampal kindling on paired-pulse response in CA1 in vitro, Brain Res., 564 (1991) 220229.
