Spontaneous epileptiform discharges in hippocampal slices induced by 4-aminopyridine

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Spontaneous Epileptiform Discharges in Hippocampal Slices Induced by 4-Aminopyridine R. A. VOSKUYL I--'and H. ALBUS 1,2 1Department of Physiology, University of Leiden, P.O. Box 9604, 2300 RC Leiden; and 21nstituut voor Epilepsiebestrijding. Meer en Bosch-De Cruquiushoeve, P.O. Box 21, 2100 AA Heemstede (The Netherlands) (Accepted December 3rd, 1984) Key words: 4-aminopyridine - - hippocampal slices - - epileptogenesis

4-Aminopyridine (4-AP) induced 2 types of spontaneous field potentials (SFPs) in the hippocampal slice, Type I resembled spontaneous activity induced by other convulsants. They occurred at a rate of approximately 1 Hz. started in the CA2/CA3 region and spread at a velocity of 0.3 m/s to area CA1. Transsection experiments and laminar profiles indicated that they spread synaptically along the Schaffer collateral pathway. Synaptic blockade by low Ca2+/high Mg2+ or kynurenic acid reversibly abolished type I SFPs. Increasing [Ca2+]olowered the rate and slightly increased the amplitude. Possibly, increased spontaneous transmitter release, and not disinhibition, is responsible for the generation of type I SFPs. Type If occurred at a rate of about 0.15 Hz and travelled in the same direction, but a factor 10 slower. They could not be blocked by separation of the CA1 and CA3 region: coupling remained until stratum moleculare was severed. Type II could not be suppressed by blockade of synaptic transmission. The laminar profile is similar in shape to that of type I but not identical. Increasing [Ca2+]ohad the same but stronger effect as on type I. Type II SFPs depressed evoked population spikes up to a second and delayed the next type I SFP. The mechanisms involved remain largely speculative: further analysis is needed to help understand the epileptogenic action of 4-AP. INTRODUCTION 4-Aminopyridine (4-AP) has attracted much attention as a pharmacological tool to study such diverse subjects as potassium channels, synaptic transmission and mechanisms of epileptogenesis. It has been shown that in invertebrate22,37 and vertebrate 33 axons, 4-AP blocks the delayed rectifying K + channel. In molluscan neurones32 and in hippocampal pyramidal cells12.29 it blocks even more efficiently a fast transient potassium current (IA), a current which possibly plays a role in regulating n e u r o n a l excitability. At the neuromuscular junction and a large n u m b e r of other types of synapses it strongly enhances the release of transmitters 31. A possible explanation for the facilitatory action on synaptic transmission is that blockade of delayed rectifier channels prolongs the action potential in the nerve terminals, allowing a greater calcium influx and thus a larger transmitter release 24. However, prolonged action potentials have been

demonstrated only in u n m y e l i n a t e d fibres 333,18, In myelinated fibres~9 this effect was absent, except in lateral olfactory tract fibres 8. Alternatively, there could be a direct effect of 4-AP on voltage-dependent calcium channels21,26. 4-AP has a powerful convulsam action if it is applied to the vertebrate central nervous system. The question how the aforementioned actions of 4-AP are related to its convulsant effects is as yet not completely resolved. Recently, the actions of 4-AP have been investigated in hippocampal and olfactory cortex slices 4-9-34. In both preparations evoked excitatory and inhibitory synaptic transmission was e n h a n c e d , either in duration (hippocampal IPSP and olfactory cortex EPSP) or in amplitude (hippocampal EPSP). Spontaneous synaptic potentials increased in amplitude and frequency in both preparations. In the olfactory cortex seizure-like discharges of long duration were observed at intervals of 30 s or moreg. In hippocampal slices relatively simple spontaneous field potentials

Correspondence: R. A. Voskuyl, Department of Physiology, University of Leiden. P.O. Box 9604. 2300 RC Leiden. The Netherlands. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

at 0.2-2 rain intervals and after prolonged application also more complex, seizure-like potentials have been briefly described 4. We have analyzed spontaneous activity in hippocampal slices in further detail, to understand the convulsant action of 4-AP more clearly. 4-AP provides an interesting case, because it is unlikely that it acts in the same way as convulsants such as penicillin, which have been claimed to act primarily by suppressing synaptic inhibition (see ref. 1). Furthermore, it might provide a useful tool for future studies on the mechanism of action of anti-epileptic drugs. We have found that 4-AP induces 2 types of spontaneous activity, one which resembles that induced by other convulsants in terms of site of origin and spread to other regions and a second that has a less restricted site of origin, spreads more slowly and seems independent of synaptic transmission. MATERIALS AND METHODS The results in this study were derived from 78 slices of 57 rats. Wistar rats of both sexes (200-300 g) were anesthetized with ether, decapitated and the brain was quickly removed and placed in cold saline solution. The hippocampus was dissected and placed on moistened filterpaper with the alvear side upwards. Transverse slices of 400 gm were made either with an array of 4 equally spaced razor blades or with a modified MclIwain chopper. Six to 8 slices were transferred to an incubation chamber which was continuously perfused at a rate of 1.4 ml/min. The standard solution contained (mM): NaC1, 122.5: KC1, 3; CaC12, 1.5; MgSO4, 1.3: NaH2PO 4, 1.25; NaHCO 3, 25; glucose 10, gassed with 5% CO2 + 95% 0 2, and kept at 35 °C. Moistened O, + CO 2 (95% + 5%) was blown over the partially submerged slices. Drugs were applied to the slices via bath application. 4-Aminopyridine solutions contained (/.05 or 0.1 mM 4-AP (Sigma), kynurenic acid (Sigma) was added in 1 mM concentration. Low Ca2+/high Mg :+ solutions were made in concentrations of 0.1 mM Ca :+ and 5 or 10 mM Mg2+; to compensate for changes in osmolarity NaCI was reduced. In some slices cuts were made with fine scissors or a microknife to disrupt synaptic connections between certain areas. Extracellular recordings were made with glass mi-

croelectrodes filled with 4 M NaCI (5 Mf~). When slices were stimulated, rectangular pulses (0.03-0.07 ms, 6-10 V, 0.1-0.2 Hz) were delivered to stratum radiatum in the CA2/CA3 area via bipolar Pt-electrodes (50 um diameter). Data were stored on magnetic tape (D.C.-2.5 kHz) and could be digitized via a transient recorder (Biomation 802) for further analysis on a PDP 11/70 computer. With a computer program peaks could be detected in the digitized records and the corresponding amplitude to baseline and latency to the stimulus were stored for plotting. Also, the slope of the straight part of the curve between 2 peaks was stored. The first 'peak', i.e. a point at the curve at a fixed latency (e.g. start of the EPSP) is determined by the experimenter (Fig. 1A). When evoked potentials were analyzed, the initial slope was used as a measure for the excitatory postsynaptic potential (EPSP): for a population spike (PS) the difference between the peaks indicating the start and the maximum of the PS were taken (see Fig. 1A). Spontaneous field potentials were also recorded on a 2-channel chart recorder to facilitate detection of synchronization in different areas of the slice and to monitor long term changes in their occurrence. RESULTS Field potentials in stratum pyramidale of area CA1 evoked by stimulation of stratum radiatum consisted of a positive excitatory postsynaptic potential (EPSP) and a single superimposed population spike (PS), indicating synchronous discharge of pyramidal cells. Within 5 min after switching to 4-AP the evoked field potentials started to change -~4(Fig. 1A). The population EPSP and population spike increased in amplitude (Fig. IB) and 2 or 3 additional PSs appeared. Also, in a number of slices a second slow positive wave emerged. 5-10 Min later spontaneous potentials started to appear (Fig. 2A). This was found in 40 out of 50 slices (80%). When these effects had fully developed they remained essentially stable as long as 4-AP was applied. When perfusion was returned to control solution, the effects were reversible but recovery was very slow (60-90 rain). These resuits agree with those reported earlier by Buckle and Haas 4. The spontaneous field potentials have been inves-

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displayed on a chart recorder (Figs. 2A, 3A). These deviating SFPs and intervals occurred approximately every 7 s. Thus, it seemed possible that there were 2 types of SFPs, one occurring at a fast rate and one at a slow rate. In order to be able to investigate both types we preferred to select slices displaying fast activity. For practical reasons we have t e r m e d SFPs occurring at fast rates type I and SFPs at lower rates type 1I.

50

Fig. 1. A: field potentials recorded in stratum pyramidale in CA1 after stimulation of stratum radiatum fibres in control solution and during application of 4-AP. In the control response the peaks are indicated that are detected with a computer program. 'Peak 1' is determined by the experimenter, The slope of the straight part of the response between peak 1 and 2 is used as a measure for the EPSP; for the amplitude of the first population spike the potential difference between peak 2 and 3 is taken. Positivity is upwards. B: time course of the effect of 4-AP on the EPSP and the first population spike. Raising 4-AP from 0.1 to 0.5 mM did not further increase these components. tigated in further detail. S p o n t a n e o u s activity was seen in slices that were not stimulated or only at a low rate (e.g. 0.1 Hz). Increasing the stimulus frequency tended to suppress spontaneous activity. Spontaneous field potentials (SFPs) were observed in all parts of the slice, but were largest in area CA1 and CA3. SFPs occurred either at intervals of 7 - 2 0 s or at about 1 s. In most slices displaying SFPs at 1/s, longer intervals of 1.5-2.5 s were periodically observed, p r e c e d e d by an SFP of a slightly different shape (Fig. 2C). This difference in shape could also be detected, sometimes even better, when the SFPs were

The shape of type I SFPs was the most consistent and there were no essential differences between the CA1 and C A 3 region (Fig. 2B). In stratum pyramidale it consisted of 3 c o m p o n e n t s , a small initial negativity, a slow positive potential and a late negativity (Fig. 2B, C). The first 2 components had a mean duration of 70 ms (S.D. 11 ms, n = 11). The maximal amplitude of the positive wave varied per slice and recording site between 0.5 and 8 inV. tn individual slices, however, amplitude, duration and shape of the SFP were rather constant for each recording site. Large amplitude SFPs often had negative peaks sup e r i m p o s e d on the rising slope (Fig. 2B). These negative peaks were identified as population spikes. The evidence was based on extracellular recordings of single cell activity and on simultaneous intra- and extracellular recordings (unpublished results h Histograms of interval distributions showed that SFPs occurred in a regular fashion, with a characteristic rate for each slice (Fig. 3B. C). The mean rate for type I SFPs. d e t e r m i n e d in 9 slices, was ~h9 H z (S. D. 0.2). The periodical occurrence of longer intervals was often indicated by a second p e a k in the histogram at about twice the interval of the main p e a k (Fig. 3C). Rates differed between slices of one hipp o c a m p u s and differences can, therefore, not exclusively be ascribed to variability between animals.

Type H SFPs The second SFP type was more variable in shape, In 19 out of 25 slices the main differences c o m p a r e d to type I SFPs were a more p r o n o u n c e d late negativity (Fig. 2C) and the lack of the initial negativity in some experiments (5 out of 19). The amplitude of the positive wave was usually smaller than type I SFPs and showed more variation in the course of an experiment. In other experiments the total duration of this

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Fig. 2. Examples of spontaneous field potentials (SFPs) recorded in stratum pyramidale in area CAI and CA3. A: chart recording in CA3 displaying both types of SFPs. Due to the low frequency response of the recorder, the high frequency components are filtered. B: type I SFPs in CA1 and CA3 are not essentially different. Examples of SFPs displaying population spikes on the rising phase of the positive wave. C: type I and II recorded in CA1. Note the more pronounced late negative wave of the type II SFP. D: example of a long duration type It SFP with a long series of population spikes.

SFP was considerably longer than of type I SFPs, up to one second, and the 3 components could no longer be distinguished (Fig. 2D). In 4 slices type II SFPs exhibited cyclic activity (cf. ref. 35). They decreased periodically in amplitude and even disappeared; during those periods there were no long intervals between type l SFPs. In the CA3 region the amplitude of the positive wave of type II SFPs could be considerably smaller than that of type I SFPs. In such cases detection of the presence of type II SFPs was facilitated by making chart recordings (cf. Figs. 2A, 3A) or recording in the region of the apical dendrites, where the amplitude of the SFPs is larger (see below). Also, the presence of type II SFPs could be indicated by the periodic occurrence of long intervals between type I SFPs. Histograms of the interval distributions provided the best criterion to distinguish between the 2 SFP types. The mean rate of occurrence of type II SFP was 0.15 Hz (S.D. 0.03, n = 12) and showed often very little variation (Fig, 3B, C). In slices showing

only slow activity we observed the same rates as for the type II SFPs in slices showing both types, and the same variability in shape. A histogram made of all main rates that have been observed in all slices, showed that the main rates of slow and fast spontaneous activity did not overlap.

Spread of activity In simultaneous recordings in different regions of a slice, both types of SFPs appeared to occur with slight phase shifts in all parts of the slice. This suggested that SFPs might originate in a special region of the slice and spread to other areas along synaptic pathways, as is the case with for example penicillininduced spontaneous activity that starts in the CA2/CA3 region and triggers similar activity in CA1 via the Schaffer collaterals 2s. To determine where SFPs start, delays between SFPs recorded simultaneously in 2 different areas were measured. Furthermore, laminar profiles were made of both SFP types. If excitatory pathways are involved in the spread of spontaneous activity, this would give rise to an active

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Fig. 3. A: SFPs recorded simultaneously with electrodes in CA1 and CA3, displayed on a chart recorder: Type II SFPs in CA3 are small, but their presence is indicated by the small negative waves and the periodically occurring long intervals: B: intervals of type I (black dots) and type II (open circles) SFPs plotted as a function of time, showing that the interval distribution remains constant over time. C: interval histogram of type I (black bars) and type II (open bars) SFPs. The bimodal distribution of intervals of type I SFPs is an extreme example of the delays that can be induced by type II SFPs.

current sink at the level of the excitatory synapses and this would be reflected in the laminar profile. F o r the delay m e a s u r e m e n t s , we a v e r a g e d at least 20 SFPs, simultaneously r e c o r d e d at 2 different locations, as it was difficult to d e t e r m i n e the start of individual SFPs. W e used the same trigger for both averaging series to m a k e sure that no artificial delays could be introduced. The start of the SFP was considered the time at which the variance (i.e. square of S.D.) increased to 3 times the baseline variance. Determination of the delay by comparison of the time of peak of the SFPs gave the same values in all but one experiment. Type I SFPs in area C A 3 p r e c e d e d those in area CA1 by 4 - 1 5 ms, d e p e n d i n g on the electrode

distance (8 slices). This c o r r e s p o n d e d to a propagation speed of 0 . 1 - 0 . 5 m/s. Type II SFPs travelled in the same direction. Delays between C A 3 and CA1 were 2 0 - 4 0 ms. which meant a p r o p a g a t i o n s p e e d of approximately 0.01 m/s (5 slices). This suggests that type I. but not type lI. activity m a y be transferred from C A 3 to CA1 along the Schaffer collaterals. which have a conduction velocity of 0.3 rots (see ref. 2). L a m i n a r profiles were made in the CA1 region by moving the recording electrode along the dendritic axis with 50 a m steps, starting from the b o r d e r between the alveus and stratum oriens, The recordings were m a d e at a fixed d e p t h under the surface (20 urn) and at least 5 SFPs were averaged at each recording

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site. A second recording electrode was placed somewhat closer to area CA3. Triggering on the SFPs recorded with this electrode provided a fixed reference point in time. The profiles of type I SFPs were the most consistent, When the amplitude of the second component was measured at a fixed latency, approximately coinciding with the peak (Fig. 4A), it was found to be maximally positive in stratum pyramidale and to reverse polarity at 135 _+ 9 #m (+ S.D., n = 6) from the soma layer (Fig. 4B). Maximal negativity was reached in stratum radiatum at 315 _+ 60 #m from the soma layer. The amplitude was larger than in stratum pyramidale. It reversed again in stratum moleculare, but the reversal points scattered widely (430-730/zm from the soma layer). Profiles of type II SFPs showed more variation. In 4 out of 6 experiments the profiles were similar to those of type I SFPs (Fig. 4A, B), but the reversal point scattered more. Maximal positivity was found slightly more towards the apical dendrites (10Hm from the soma layer), whereas the reversal point and the maximal negativitity were found closer to the soma layer than for type I (85 + 48Hm and 250 + 50 Hm, respectively). In the other 2 experiments the profiles were irregular.

The extracellular EPSP, evoked by stimulation of the Schaffer collaterals in stratum radiatum, is positive in stratum pyramidale and negative in stratum radiatum. Thus, in analogy, the second component of type I SFPs may reflect synchronous activity of excitatory synapses in stratum radiatum. Considering these data the same might be true for type II SFPs, though it seems unlikely that the same synapses would be involved.

Blockade of synaptic transmission To determine whether SFPs originated solely in area CA3 and triggered SFPs in other parts of the slice via synaptic pathways, or could originate independently in several areas, we investigated the effect of mechanical interruption of synaptic pathways and pharmacological blockade of synapses. In 8 slices in which both types of SFPs occurred, a cut was made between area CA1 and CA3 from alveus to stratum moleculare (Fig. 5A). A stimulus at the CA3 side of the cut was used to verify that the transsection of the stratum radiatum fibres was complete. Stimulation at the CA1 side was used to show that the cut fibres were still functionally intact. Type I and type II SFPs continued at the same rate in area CA3, but in area

6(i CA1 type I SFPs had disappeared, while type II SFPs still occurred synchronously with type Ii SFPs in CA3 (Fig. 5C). The rate of occurrence of type I SFPs sometimes changed in CA3, but both increases and decreases have been observed. If the cut was extended to include stratum moleculare or further (Fig. 5A), the type II SFPs were no longer coupled in CA1 and CA3. However, the rate of occurrence did not change. Type II SFPs at the CA1 side still travelled in the direction of the subiculum. Synaptic transmission was blocked by applying a 4AP solution containing low Ca :+ (0.1 mM) and high Mg2+ (5 or 10 mM) after SFPs had been induced with 4-AP (5 experiments). The rate of type I SFPs was reduced and after about 20 min they disappeared corn-

pletely, in area CA1 as well as m area CA3, an effect which was completely reversible upon washout (Fig. 6A). Type I SFPs remained coupled in CA i and CA3 up to the moment that the SFPs failed altogether. The amplitude was hardly affected. The slices were stimulated once every minute to monitor the time course of suppression of synaptic transmission (Fig. 6B). Reduction of the amplitude of the evoked EPSP and of the rate of the SFPs started simultaneously but the SFPs disappeared before the EPSP was completely suppressed. Type I1 SFPs were not blocked by application of low CaZ+/high Mg:~, though the rate became more irregular and the amplitude was reduced. Reduction of calcium may not only suppress syn-

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line up to the arrow. This point can be recognized as the border of a slightly whiter band. if the cut was extended along tile interrupted line up to the hippocampal fissure or further, type II SFPs in CA1 and CA3 became uncoupled.

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Fig. 6. A: effect of 0.1 mM Ca2-/10 mM Mg2+ on the intervals between type I SFPs. After about 15 min, the type I SFPs are completely suppressed. The effect of 1 mM kynurenic acid on type I SFPs was identical (not shown). B and C: time course of the effect of low Ca2+/high Mg2+ and kynurenic acid at the EPSP recorded in CAl. Note that type I SFPs are abolished before synaptic transmission is completely suppressed. aptic transmission, but also affects excitability and various v o l t a g e - d e p e n d e n t currents. T h e r e f o r e , it is possible that o t h e r mechanisms may be involved as well in the effects of low Ca2+/high Mg2+, in particular because the effects are observed before synaptic transmission is completely blocked. T h e r e f o r e we l o o k e d for a m e t h o d to block excitatory synaptic transmission in a different way. F o r this we have used kynurenic acid. Kynurenic acid is a relatively unspecific antagonist of excitatory amino acid receptorsl~L Since the transmitter of the m a j o r pathways in the hippocampus is p r o b a b l y glutamate or aspartate, kynurenic acid might be expected to block effectively the spread of neuronal activity along synaptic pathways in the hippocampus. It blocks amino acid-induced excitation of cortical cells 25, reduces sponta-

neous synaptic potentials 5 and suppresses synaptic field potentials at a n u m b e r of central synapses, including the hippocampus 5.m. The Schaffer collateralCA1 pathway was relatively unsensitive to kynurenic acid: 2.5 m M was n e e d e d for complete blockade of the EPSP. The time course of blockade was the same as for low Ca:+/high Mg 2+ (Fig. 6C). 1 mM was sufficient to suppress type I SFPs in CA1 and C A 3 , i.e. before synaptic transmission was blocked completely. Type I I SFPs were not suppressed and the rate and amplitude did not change. W h e n kynurenic acid was washed out, type I SFPs r e - a p p e a r e d . The fact that the 2 different m e t h o d s give the same results, makes it very likely that reduction of synaptic transmission is responsible for this effect. Taken together, these results indicate that type I

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We have carried out 2 further series of experiments to investigate the properties of the 2 SFP types. [Ca2+]o was varied between 0.5 and 3 mM in 6 slices. Increasing calcium lowered the rate of both type I and type II SFPs. Histograms showed that the variability of interval length increased in high calcium. Increasing calcium slightly enhgnced the amplitude of the second component of type 1 SFPs (Fig. 7A). The effect on the amplitude of type 1I SFPs was much stronger, in particular on the late part. The effects were not completely reversible (Fig. 7B), but upon a second application of high or low calcium the rate and amplitude changed in the same direction as with the first application. The long intervals to the next SFP after the occurrence of a type 1I SFP (Fig. 3) suggested that, at least type II, SFPs are followed by a period in which neuronal activity is suppressed. To test this, single stimuli were given to stratum radiatum at various intervals

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are the average of 6 experiments for type I SFPs and 4 experiments for type II SFPs. The bars represent 1 S.D.

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SFPs originate in the C A 2 / C A 3 area and spread via synaptic pathways to area CA1. The fact that low CaZ+/high Mg 2÷ and kynurenic acid blocks it both in CA3 and CA1 suggests that synaptic transmission may also be involved in the initiation, O n the other hand, these experiments suggest that synaptic pathways are not required for the initiation and spread of type II SFPs.

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Fig. 8. Influence of SFPs on field potentials in CA1 evoked by stimulation of stratum radiatum fibres. The stimulator was activated at increasing intervals after an SFP. Each point represents the mean of 5 responses. A: influence of a type I SFP on the EPSP and the first population spike. B: influence of a type II SFP.

63 after an SFP in 10 slices. The slope of the EPSP and the amplitude of the first population spike in CA1 were plotted as a function of the interval. There was little effect of either type of SFP on the EPSP (Fig. 8). There was a slight reduction during the first 50 ms, but the measurement of the slope was not reliable because the SFP and the field potential were superimposed. However, population spikes were depressed. They were depressed for 50-100 ms after a type 1 SFP, but after a type II SFP depression lasted for more than a second (Fig. 8). The depression seemed to vary with the amplitude of the type II SFPs. This was difficult to test because these variations could not be controlled experimentally. Indirect support for such an effect was given in the few experiments in which the type II SFP periodically disappeared and re-appeared. Such an effect was accompanied by disappearance of the periodically occurring long intervals. This will be further investigated by altering [Ca2+]o. DISCUSSION The main finding of this study is that 4-AP induces 2 types of spontaneous activity in the hippocampal slice. Both types can occur with a remarkable degree of regularity. The two types differ with respect to rate of occurrence, propagation speed and sensitivity to synaptic blockade. Type 1 SFPs, which occur at a high rate, share a number of properties with spontaneous activity induced by other convulsants; type II has so far not been described in the literature. Cells in the CA2/CA3 region serve as pacemakers for the generation of type I SFPs, which spread along synaptic pathways to cells in the CA1 region. This is suggested by the abolishment of spontaneous activity in CA1 after transsection of fibres running in stratum radiatum and after pharmacological reduction of synaptic transmission, even though the latter experiments could not discriminate whether this was due to partial reduction of synaptic excitation or to lack of input from CA3, because in both regions spontaneous activity failed simultaneously. Furthermore, the propagation speed of type 1 SFPs was equal to the conduction velocity of the fibres running in stratum radiatum2 and the second component of the SFP and evoked population EPSPs in the CA1 region had similar profiles along the dendritic axis. Thus, these re-

suits are consistent with the notion that this component is an excitatory synaptic potential generated by nerve terminals in stratum radiatum, which gives rise to repetitive discharges of pyramidal cells as indicated by the superimposed population spikes. Spontaneous activity induced by penicillin, bicuculline, picrotoxin and kainic acid starts preferentially in the same subregion and spreads in the same way. It has been proposedl that depression of G A B A - m e diated inhibitory synaptic transmission is the crucial factor for the onset of epileptiform activity and indeed this action has been demonstrated for all these convulsants 6.7,14,27. Nevertheless, there are differences between the effects of these drugs, indicating that the exact nature of the induced epileptiform activity is not only determined by neuronal properties and interconnections. For example, picrotoxin is the only one capable of producing spontaneous bursts in CA1 after severance of Schaffer collaterals, though at a lower rate than in CA3 (ref. 14). Kainic acid in doses over 1/~M produces spontaneous bursts only transiently, due to a secondary developing reduction of synaptic excitation7. 4-AP is possibly the first example of a convulsant that does not act primarily by reduction of synaptic inhibition in the hippocampal slice. Both in the hippocampal and olfactory cortex slice 4-AP enhanced synaptic inhibition 4,~. The fact that repetitive firing of the pyramidal cells was much less pronounced with 4-AP than with the other convulsants (cf. refs. 6, 11, 14 and 35) could well be the result of enhanced inhibition. The reduction of population spikes in field potentials evoked by stimulation shortly after an SFP also indicates that synaptic inhibition is still intact. Buckle and Haas reported that the spontaneous potentials contained a component that could be blocked by bicuculline 4. They did, however, not distinguish different types of spontaneous activity. On the other hand, it was shown in the olfactory cortex slice that synaptic inhibition could temporarily fail 9. Such failure was always followed by long epileptiform discharges. If a similar mechanism could occur in the hippocampal slice is still unknown. The blockade of type I SFPs in CA3 by low Ca2+/high Mg 2+ and kynurenic acid strongly suggests that a synaptic component is involved not only in the spread but also in the triggering of type I SFPs. This could well be explained by the strong increase in fre-

64 quency of spontaneously occurring synaptic potentials induced by 4-AP (refs, 4, 9 and unpublished resuits). This may also be the reason that SFPs occur at a much higher rate with 4-AP than with other convulsants. The rate of spontaneous activity with other convulsants is at least a factor 3 lower 6,11A4,23.35. (The use of lower temperatures or different concentrations of extracellular calcium or potassium may, however, have contributed as we1116,20.) Considering the literature, increased spontaneous transmitter release is not a prominent feature of other convulsants. It is easily conceivable that this increase in excitatory influence can accelerate the triggering of a burst. For example, during penicillin-induced bursting, a local K + pulse, which depolarized neighbouring cells, could decrease the delay of the next burst 36. The lowering of the rate with increasing [Ca2+]o is probably not mediated via an effect on spontaneous transmitter release, because in that case an increased rate would have been expected 15. Suppression of convulsant induced bursting by high Ca 2+ could be due to an increased threshold36 or to a role in processes that are involved in termination of bursting, e.g. recurrent inhibition and/or calcium-dependent potassium conductance (see ref. 14). The present results do not allow a definitive conclusion on the origin of type II SFPs. It is unlikely that synaptic pathways are involved in this phenomenon, because type II SFPs could not be abolished by blockade of synaptic transmission. Also, the results of the transsection experiments and the laminar profiles would be contradictory, if synaptic pathways would be involved in the spread of activity. Synchrony of type II SFPs in the CA1 and CA3 region depended on the integrity of stratum moleculare. However, the laminar profile shows a maximal negativity rather close to the soma layer, which is inconsistent with a current sink generated by synapses terminating distally on the apical dendrites. In the intact slice, type I1 SFPs propagate from CA3 to CA1, suggesting initiation in CA3. However, they are also observed in slices lacking the CA3 region and the type II SFPs are often quite small in the CA3 region in intact slices. Therefore, the CA1 region may be the most sensitive region for the initiation of type II SFPs. Type II SFPs are clearly different from the epileptiform discharges that can be induced in the absence

of synaptic transmission by lowering [Ca 2~],, (refs. 17, 30). Such discharges are negative in the soma layer, propagate at a lower speed and in the opposite direction. Nevertheless, the mechanisms that have been suggested for the synchronization of such discharges, e.g. ephaptic interaction, electrotonic coupling, may explain the synchronization of type II SFPs as well. One possible explanation for the generation of type II SFPs could be that it is the result of blockade of K + channels (see Introduction). If there would be a resting Na + or Ca 2+ conductance, this could eventually lead to a regenerative inward current and set the sequence of events in motion. The evidence that 4-AP may have a direct effect on calcium influx~6 and the fact that [Ca2+]o strongly influences the amplitude of type II SFPs would support a role for calcium. Ephaptic interactions and electrotonic coupling could account for synchronization and spread. If so, this would point to a special role for stratum moleculare, possibly due to a different extracellular resistance or to local contacts between pyramidal cells. Alternatively, there could be a disturbance of the regulation of extracellular ions. For example, a (local) increase in extracellular K ~ could depolarize a group of cells, thereby activate Na* or Ca 2+ conductances and so on. One should also take into account, however, that there is apparently little interaction between the 2 SFP types. It is striking for example that the rate of type II SFPs is not affected in the transsection experiments or by kynurenic acid. Neither did we observe consistent changes in the rate of type I SFPs in the transsection experiments. The only obvious interaction is the period of inhibition following a type lI SFP, which delays the next type I SFP. Whether this is due to recurrent inhibition or some other mechanism, for example a calcium-activated potassium conductance, remains to be determined. Clearly, further study is needed to clarify the mechanisms involved in the generation of type II SFPs, in particular at the cellular level. The present results show that it is possible by transsection, blockade of synaptic transmission or manipulation of extracellular ions to optimize conditions to study the 2 SFP types in isolation. The influence of type II SFPs on type i SFPs and on evoked potentials, the persistence of the phenomenon, the ubiquitous occur-

65 rence in the h i p p o c a m p a l slice and the fact that in

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the C h r i s t e l i j k e V e r e n i g i n g v o o r de V e r p l e g i n g van Lijders aan E p i l e p s i e .

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