Epilepsia, 46(4):509–516, 2005 Blackwell Publishing, Inc. C 2005 International League Against Epilepsy
Electrical and Chemical Long-term Depression Do Not Attenuate Low-Mg2+–induced Epileptiform Activity in the Entorhinal Cortex ∗ J¨org Solger, †Uwe Heinemann, and ∗ Joachim Behr ∗ Neuroscience Research Center of the Charit´e, and †Johannes-Mueller-Institue of Physiology, Humboldt University Berlin, Berlin, Germany
Summary: Purpose: Low-frequency electrical and magnetic stimulation of cortical brain regions has been shown to reduce cortical excitability and to decrease the susceptibility to seizures in humans and in vivo models of epilepsy. The induction of longterm depression (LTD) or depotentiation of a seizure-related long-term potentiation has been proposed to be part of the underlying mechanism. With the low-Mg2+ -model of epilepsy, this study investigated the effect of electrical LTD, chemical LTD, and depotentiation on the susceptibility of the entorhinal cortex to epileptiform activity. Methods: The experiments were performed on isolated entorhinal cortex slices obtained from adult Wistar rats and mice. With extracellular recording techniques, we studied whether LTD induced by (a) three episodes of low-frequency paired-pulse stimulation (3 × 900 paired pulses at 1 Hz), and by (b) bathapplied N-methyl-D-aspartate (NMDA, 20 µM) changes timeto-onset, duration, and frequency of seizure-like events (SLEs) induced by omitting MgSO4 from the artificial cerebrospinal fluid. Next we investigated the consequences of depotentiation
on SLEs themselves by applying low-frequency stimulation after onset of low-Mg2+ –induced epileptiform activity. Results: LTD, induced either by low-frequency stimulation or by bath-applied NMDA, had no effect on time-to-onset, duration, and frequency of SLEs compared with unconditioned slices. Low-frequency stimulation after onset of SLEs did not suppress but induced SLEs that lasted for the time of stimulation and were associated with a simultaneous increase of the extracellular K+ concentration. Conclusions: Our study demonstrates that neither conditioning LTD nor brief low-frequency stimulation decreases the susceptibility of the entorhinal cortex to low-Mg2+ – induced epileptiform activity. The present study does not support the hypothesis that low-frequency brain stimulation exerts its anticonvulsant effect via the induction of LTD or depotentiation. Key Words: Long-term depression—Lowfrequency stimulation—Entorhinal cortex—NMDA receptor— Low Mg2+ —Epileptiform activity—Seizure-like events.
Deep brain stimulation and transcranial magnetic stimulation are considered promising new technologies for the treatment of pharmacoresistant epilepsy (1). Both treatments have been shown to reduce cortical excitability (2,3), to decrease epileptic spike frequency in complexpartial epilepsy with mesiobasal limbic onset (4), and to improve focal temporal and extratemporal epilepsy (5– 8). In pentylenetetrazol-treated rats, an in vivo model of epilepsy, low-frequency repetitive transcranial magnetic stimulation increased the latency to seizure onset (9). In the kindling model of epilepsy, low-frequency stimulation (LFS) of the kindling focus was shown to block or delay the development and progression of afterdischarges and
seizures in adult and immature rats (10,11) (but see ref. 12). In hippocampal slices, LFS reduced the frequency of spontaneous interictal-like activity induced by the γ -aminobutyric acid (GABA)-A receptor antagonist bicuculline (13). In all studies, long-term depression (LTD) or depotentiation of a seizure-related long-term potentiation (LTP) has been proposed to be part of the underlying mechanisms. LTD comprises a persistent activitydependent decrease in synaptic strength, which can be produced reliably by the appropriate experimental manipulations of presynaptic and postsynaptic activity. LTD is usually induced by prolonged LFS (1–5 Hz) of afferent fibers. The most common forms of LTD require the activation of postsynaptic metabotropic glutamate receptors and N-methyl-D-aspartate (NMDA) receptors for induction (14). In the superficial layers of the entorhinal cortex (EC), LFS was recently shown by us and others to induce a stable and lasting NMDAR-dependent LTD (15–17).
Accepted December 8, 2004. Address correspondence and reprint requests to Dr. J. Behr at Neuroscience Research Center of the Charit´e, Humboldt University Berlin, Schumannstr. 20/21, 10117 Berlin, Germany. E-mail
[email protected]
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Interestingly, in CA1, not only LFS but also pharmacologic activation of NMDA receptors was shown to induce LTD (18,19). Little is known about the effect of LTD on the generation of epileptic activity. In this study, we investigated the conditioning effect of LFS-induced electrical LTD and NMDA-induced chemical LTD on low-Mg2+ – induced seizure-like events (SLEs) in the EC. The shortterm effect of LFS on the generation of SLEs was evaluated by applying LFS after onset of low-Mg2+ –induced repetitive SLEs. The induction of SLEs by lowering the extracellular Mg2+ concentration is a widely used in vitro model of epilepsy that is largely due to a relief of the physiologic Mg2+ block of the NMDA receptor (20). Isolated EC slices were used for several reasons: First, an increasing number of studies have presented evidence for the participation of the EC in the genesis of temporal lobe epilepsy (21–25). Second, in vitro, the EC generates SLEs independent of input from the hippocampus (26), most likely because of its complex intrinsic connectivity. Third, in the EC, this form of activity is characterized by single repetitive events of synchronized field excitatory postsynaptic potentials (EPSPs), underscoring the clustering of action-potential firing that shifts in the bursting pattern from fast and regular discharges (tonic phase) to slower and clustered discharge (clonic phase) (see inset in Fig. 2A). In contrast to low-Mg2+ –induced epileptiform activity generated in the hippocampus, which is characterized by interictal-like recurrent discharges, the activity in the EC keenly mimics tonic and clonic electrographic seizures (27). Recordings were performed in the superficial layers (II and III) of the EC that relay most of the input from cortical associational areas to the hippocampus (28) and were shown to be the main region of seizure onset in this model of temporal lobe epilepsy (29,30). METHODS All experiments on the conditioning effect of electrical and chemical LTD on SLEs were performed on isolated horizontal EC slices obtained from adult Wistar rats (older than 8 weeks). The short-term effect of LFS on the generation of SLEs was investigated in combined hippocampalEC slices from adult Wistar rats and adult CD1 mice (older than 4 weeks). In a subset of experiments, the effect of LFS on the SLE was examined in slices in which the Schaffer collaterals were sectioned. Animals were decapitated under deep ether anesthesia, the brains were quickly removed, and 400-µm-thick slices were prepared with a Campden vibroslicer (Loughborough, U.K.). The slices were transferred to an interface recording chamber continuously perfused with an aerated (95% O2 , 5% CO2 ), ◦ prewarmed (34 C) artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 129, Na2 PO4 1.25, NaHCO3 26, KCl 3, CaCl2 1.6, MgSO4 1.8, glucose 10, at a pH Epilepsia, Vol. 46, No. 4, 2005
of 7.4. To induce repetitive SLEs, MgSO4 was omitted from the aCSF. To provide the same time courses of Mg2+ washout, conditioned (slices that expressed LTD) and unconditioned slice were recorded simultaneously during perfusion with low-Mg2+ aCSF. SLEs and evoked field potentials (fEPSPs) were recorded in the superficial layers of the medial EC with aCSF-filled microelectrodes. Field potentials were evoked by using 500-µs pulses delivered through bipolar electrodes placed on the lateral portion of superficial layers of the medial EC adjacent to the recording electrode (for details, see Fig. 1A). Stimulus intensity (2–4 V) was adjusted to 60% of maximum response to induce reliable synaptic plasticity. To induce electrical LTD, paired-pulse LFS was administered at 1 Hz, consisting of 900 paired pulses (50-ms interstimulus interval). Chemical LTD was induced by perfusing the slices with NMDA (20 µM) for 3 min. Under control conditions, both types of LTD were stable and lasted for >80 min (see Results). All low-Mg2+ experiments were performed within this time window. To determine changes of the extracellular K+ concentration during SLEs, we used doublebarreled K+ -selective reference microelectrodes (Fluka 60031 ionophore, reference electrode 150 mm NaCl) prepared and tested as described by Lux and Neher (31). Electrodes were accepted if they responded to a 10-fold change in K+ concentration with a potential shift of 58 ± 2 mV. Duration and frequency of repetitive SLEs were calculated from the average of the first four succeeding events after washout of Mg2+ in each slice. Signals were filtered at 3 kHz, sampled at 8–10 kHz by an ITC16 interface (Instrutech Corp., Great Neck, NY, U.S.A.), and subsequently stored on an IBM-compatible computer. Peak amplitudes of fEPSPs were measured from an average of 20 responses recorded over a 10-min period (5 min after conditioning), and analyzed off-line by using TIDA software (HEKA, Lambrecht/Pfalz, Germany). All data were normalized to the averaged baseline response of each experiment. Statistical evaluation was performed by applying Student’s t test (Excel, Microsoft); data are expressed as mean ± SEM. Significance level was set to p < 0.05. The following drugs were applied: d,l-2-amino-5-phosphonovaleric acid (D,L-APV), 60 µM; and NMDA, 20 µM (both from Tocris, Bristol, U.K.). RESULTS Effect of electrical LTD on epileptiform activity In rat EC slices, LFS of cortical afferents caused a stable LTD of synaptic efficacy in EC superficial layers that lasted ≥80 min (75.8 ± 5.8% of control; n = 6; p < 0.01; Fig. 1B). Three episodes of 1-Hz LFS (3 × 900 paired pulses, 20-min interepisode intervals) were given to saturate LTD. After the second episode, the amplitude of the fEPSP declined from 78.0 ± 2.9% to 64.7 ± 3.8% (n = 16; p < 0.05; Fig 1C) of control; after the third episode,
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FIG. 1. Electrical long-term depression (LTD) in superficial layers of the entorhinal cortex (EC). A: Experimental configuration: placement of the recording electrode in layers II–III of the isolated medial EC. Stimulation electrode was placed on the lateral portion of the superficial layers (∗). mEC, medial entorhinal cortex; Sub, subiculum; DG, dentate gyrus; LD, lamina dissecans. B: Low-frequency stimulation (LFS)induced LTD caused a stable LTD that lasted ≥80 min (n = 6). The inset shows representative excitatory field postsynaptic potentials (fEPSPs) before and after induction of LTD, taken at the indicated time points. C: Three episodes of 1-Hz LFS (3 × 900 paired pulses, 20-min interepisode intervals) were given to saturate LTD (n = 16). Paired-pulse facilitation (PPF) was not altered after each episode of LFS.
the response was not significantly different (60.3 ± 4.7%; p = 0.085). In unconditioned EC slices, lowering the extracellular Mg2+ concentration for 23.6 ± 2.5 min (time to onset) induced repetitive SLEs lasting 35.4 ± 5.2 s at a frequency of 0.27 ± 0.03/min (n = 11 slices). In slices that experienced three episodes of LFS (n = 11 slices), we observed a similar time to onset (24.9 ± 3.3 min; p = 0.76), duration (38.4 ± 6.1 s; p = 0.71), and frequency (0.29 ± 0.04/min; p = 0.69) of repetitive SLEs as compared with unconditioned slices (Fig. 2A–C). Effect of chemical LTD on epileptiform activity To induce LTD at synapses throughout the entorhinal slice instead of being confined to only those synapses that are within reach of the stimulating electrode, we tried to induce LTD by pharmacologically activating NMDA receptors. Application of 20 µM NMDA for 3 min consistently induced LTD of evoked responses in the superficial layer of the isolated EC (Fig. 3A): Bath-applied NMDA initially caused a marked decrease of synaptic responses owing to depolarization of the neurons. Field EPSPs recovered slowly until a stable but depressed response was reached (70.1 ± 5.7%; n = 7; p < 0.01). The magnitude of NMDA-induced synaptic depression was not significantly different from the one recorded after LFS. To exclude NMDA-induced unspecific effects, which may cause a
long-lasting depression of evoked responses, NMDA was applied in the presence of the NMDA-receptor antagonist APV (60 µM). As shown in (Fig. 3B), NMDA-induced establishment of LTD was prevented by APV (102.5 ± 1.6%; n = 5; p = 0.19), but still could be induced after washout of the antagonist (78.9 ± 5.3%; n = 5; p < 0.05). As the NMDA-induced depression of evoked responses may simply be a reflection of the excitotoxic actions of NMDA, we tested whether saturation of electrical LTD occludes the induction of chemical LTD at the same synapses. Three episodes of 1-Hz LFS (3 × 900 paired pulses, 20-min interepisode intervals) were given to saturate electrical LTD. After the third episode, the amplitude of the fEPSP declined to 62.4 ± 7.7% (n = 6) of control. After application of NMDA and relaxation to a stable response, we observed no significant further depression of synaptic transmission (59.3 ± 6.9%; p = 0.5; Fig. 3C). These data indicate that LFS-induced and NMDAinduced LTD share a common mechanism and that the NMDA-induced synaptic depression is not caused by excitotoxicity. After characterizing the NMDA-induced form of LTD, we studied its effect on low-Mg2+ –induced SLEs. As in slices that experienced three episodes of LFS, we observed no significant difference in time to onset (27.4 ± 3.4 min; n = 11 slices; p = 0.38), duration (29.0 ± 4.7 s; p = 0.38), and frequency (0.29 ± 0.04/min; p = 0.68) Epilepsia, Vol. 46, No. 4, 2005
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FIG. 2. Effect of electrical and chemical long-term depression (LTD) on epileptiform activity (A–C). Comparison of the effect of electrical LTD (3 × low-frequency stimulation) and chemical LTD (N-methyl-D-aspartate, 20 µM for 3 min) on time to onset, duration, and frequency of low-Mg2+ –induced repetitive seizure-like events (SLEs). Both forms of LTD had no significant effect on the investigated parameters compared with unconditioned slices (n = 11 slices in each experimental group). Representative recording of single SLE and the associated local [K+ ] increase in an unconditioned slice is shown in (A).
of repetitive SLEs as compared with unconditioned slices (Fig. 2A–C). Short-term effect of LFS on epileptiform synaptic activity Synaptic plasticity that is necessary for the rapid acquisition of memory can potentially underlie epilepsy. Highfrequency stimulation, as occurs during SLEs, may induce LTP, causing an enhancement of synaptic strength (32). Prior induction of LTP is known to facilitate subsequent LTD induction. This facilitated LTD has been called depotentiation (33). We investigated the short-term effect of Epilepsia, Vol. 46, No. 4, 2005
FIG. 3. Chemical long-term depression (LTD) in superficial layers of the entorhinal cortex (EC). A: Application of N-methylD-aspartate (NMDA; 20 µM) for 3 min caused a transient and marked decrease of synaptic responses owing to depolarization of the neurons followed by a stable but depressed excitatory postsynaptic field potential (fEPSP; n = 7). The inset shows representative fEPSPs before and after induction of LTD, taken at the indicated times. B: NMDA-induced LTD was prevented in the presence of 2-amino-5-phosphonopentanoic acid (APV; 60 µM), but could be induced after washout of the antagonist (n = 5). C: Saturation of electrical LTD by three episodes of low-frequency stimulation (3 × 900 paired pulses, 20-min interepisode intervals) occluded NMDA-induced chemical LTD (n = 6).
LFS on SLEs in combined hippocampal-EC slices. Stimulating electrodes were placed on the lateral portion of superficial layers of the medial EC adjacent to the recording electrode to activate superficial layer neurons or in the alveus (or stratum radiatum of CA1) to stimulate CA1
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FIG. 4. Short-term effect of low-frequency stimulation (LFS) on epileptiform activity. A: Experimental configuration: Placement of the recording electrode in layers II–III of the medial entorhinal cortex (EC) in a combined hippocampal-EC slice. Stimulation electrode was placed on the lateral portion of the superficial layers (∗), in the alveus (+), or in stratum radiatum of CA1 (#). mEC, medial entorhinal cortex; Sub, subiculum; DG, dentate gyrus; LD, lamina dissecans; CA1, hippocampal area CA1. B: After onset of repetitive seizure-like events (SLEs), each input was stimulated at frequencies between 0.25 and 10 Hz for 60–300 s in slices obtained from rats (n = 26) and mice (n = 10). Independent of the input stimulated, all frequencies failed to block SLEs in superficial layers of the EC. As shown in the representative recording, LFS induced epileptiform discharges in mouse slices lasting for the time of stimulation. The expanded trace illustrates that this activity had typical features of SLEs consisting of tonic–clonic discharges. The activity was associated with a simultaneous increase of the extracellular K+ concentration, proving ictogenesis in the EC and excluding far field effects.
efferents that terminate in the deep layers of the EC (Fig. 4A). As shown in previous studies (27,34), 15– 30 min after onset, SLEs progressively occurred at higher frequencies and with shorter duration (Fig. 4B). At this stage, LFS was applied at 0.25–10 Hz for 60–300 s (n = 26 slices). All frequencies tested failed to suppress SLEs. Rather we observed the induction of epileptiform events that lasted for the time of stimulation. Like SLEs, these events were characterized by synchronized field EPSPs that shifted in their bursting pattern from fast and regular discharges (tonic phase) to slower and clustered discharges (clonic phase) (see expanded trace in Fig. 4B). A previous study showed that either 1-Hz stimulation of CA1 efferents or interictal CA1 discharges block low-Mg2+ – induced SLEs generated in the EC of adult mice (35). As this effect might be due to the enhanced CA1–EC connectivity in brain slices obtained from mice, we turned to mice preparations. However, as in rat slices, LFS at 1–10 Hz of deep and superficial EC layers never blocked SLEs (n = 10 slices) (Fig. 4B). Rather, we observed the induction of SLEs, which was always associated with a simultaneous increase of the extracellular K+ concentration, excluding far-field effects of epileptiform activity generated in other areas of the combined hippocampal-EC slice. In addition, preventing the propagation of hippocampal interictal ac-
tivity to the EC by cut of the Schaffer collaterals in both rat (n = 2) and mouse slices (n = 4) did not increase the frequency of SLEs in the EC (data not shown). DISCUSSION In the present study, we investigated (a) whether LTD changes ictogenesis threshold and (b) the consequences of LFS on SLEs. By using an in vitro approach, we demonstrated that neither electrical LTD nor chemical LTD has an effect on time to onset, duration, and frequency of lowMg2+ –induced epileptiform activity in isolated EC slices. LFS after onset of epileptiform discharges did not suppress but induced tonic–clonic SLEs that lasted for the time of stimulation. The present study does not support the hypothesis that LFS exerts its anticonvulsant effect via the induction of LTD or depotentiation. We used the low-Mg2+ model of epilepsy, as this model reliably induced long-lasting SLEs not only in intact hippocampal-EC slices but also in isolated EC minislices. We also used the GABA receptor–antagonist bicuculline as an alternative in vitro model of epilepsy. However, in all investigated EC minislices, we observed predominantly interictal-like discharges (an example is given in Fig. 5) and only rarely ictal-like events that lasted ≤10 seconds. Epilepsia, Vol. 46, No. 4, 2005
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FIG. 5. γ -Aminobutyric acid (GABA)-receptor antagonist in entorhinal cortex (EC) minislices. In EC minislices (n = 5), application of the GABA-receptor antagonist bicuculline (5 µM) induced predominantly interictal-like discharges lasting between 0.5 and 3 s.
Hence to investigate the conditioning effect of LFS on time to onset, duration, and frequency of ictal events, in the present study, we preferred the low-Mg2+ model. However, as a variety of convulsant drugs or altered aCSF can be used to produce brief epileptic activity in brain slices (20), we cannot rule out that certain forms of epileptic activity might be attenuated by LFS. Electrical LTD induced by LFS was shown by us and others to induce NMDA receptor–dependent LTD in the superficial layers of the medial EC (15–17). A major concern with electrical LTD is that only a small fraction of synapses are modulated by LFS. Epileptiform activity, however, is a network phenomenon characterized by synchronized synaptic activity throughout the slice preparation. To circumvent this limitation, we induced LTD in the isolated EC slice preparation by pharmacologically activating NMDA receptors. Here we present evidence that brief bath application of NMDA produces LTD in superficial layers of the EC. Like electrical LTD, this form of LTD is sensitive to the NMDA-receptor antagonist APV, suggesting an NMDA receptor–mediated expression mechanism. In addition, saturated LFS-induced LTD completely occluded further induction of chemical LTD, indicating that the same signaling processes are involved. Most important, the latter finding demonstrates that, as in previous studies (18,19), the NMDA concentration and duration of application used (20 µM, 3 min) do not cause irreversible neuronal damage. In spite of using this pharmacologic approach to induce a robust LTD at synapses throughout the superficial layer of the EC, we failed to modulate time to onset, duration, and frequency of low-Mg2+ –induced repetitive SLEs. These results are reminiscent of a study on the effect of LFS on epileptiform activity in CA1 hippocampal brain slices obtained from kainic acid–lesioned rats (36). The authors showed that LFS-induced LTD decreases the number of epileptiform discharges. However, the probability for the cells to discharge in synchrony is increased, and a smaller depolarization is sufficient to reach the firing threshold. This effect might be due to a decreased firing threshold of the pyramidal cells or to a decrease in the inhibitory drive from the interneurons. In our study, an alternative mechanism must be considered: LFS has been shown to facilitate LTP induction (33). In keeping with the sliding-threshold model of synaptic plasticity (37), a previous history of low levels of synaptic activation, as occurs during LFS, produces a leftward shift in threshold Epilepsia, Vol. 46, No. 4, 2005
that makes it more likely for input to elicit LTP. Hence, prior LTD induction primes the EC network for synaptic potentiation by low-Mg2+ –induced epileptiform activity. Considering these findings, LTD not necessarily reduces the propensity of a neuronal network to increase its cellular excitability and synaptic strength in the presence of a convulsant agent. High-frequency synaptic activation, as occurs during seizures, is known to increase synaptic strength (32). Hence it is feasible that LFS-induced depotentiation after development of SLEs may decrease the excitability of layer II–III neurons. A previous study showed that interictal hippocampal discharges as well as interictal-like LFS (1 Hz) of hippocampal output blocks the occurrence of low-Mg2+ –induced SLEs in the EC of adult mice (35). The authors suggested that CA3-driven rhythmic inputs may perturb the ability of the EC network to reverberate and to express ictal activity. In our hands, however, stimulation of CA1 efferents at frequencies between 0.25 and 10 Hz did not prevent SLEs in the EC in slices from adult rats and mice. Rather, we recorded epileptiform events with a tonic–clonic discharge pattern. However, this activity does not necessarily indicate active generation of SLEs in the EC. When using slow field potentials as indicators for the generation of SLEs, one must ascertain that these potentials are not generated elsewhere. It is well established that during SLEs, K+ concentration increases to levels of ∼12 mM in the EC (38). Likewise, in the present study, SLEs in the EC were always associated with a similar increase of the extracellular K+ concentration, proving the generation of SLEs in the EC and excluding far field effects. We do not have a conclusive explanation for the discrepancy between our study and the one by Barbarosie and Avoli (1997). As the topology of the connections between CA1, the subiculum, and the EC is characterized by selective and restricted origin and termination along the transverse or proximodistal axis of CA1 and the subiculum (39), differences in the stimulation site might account for the discrepant results. In conclusion, by using low-Mg2+ –induced epileptiform activity in the EC as an in vitro model for limbic seizures, we demonstrated that neither LFS-induced LTD nor NMDA-induced LTD decreases the susceptibility of the medial EC to SLEs. Acute LFS after onset of repetitive SLEs does not block but induces SLEs that last for the time of stimulation. The present study does not support the hypothesis that low-frequency brain stimulation
LTD DOES NOT ATTENUATE EPILEPTIFORM ACTIVITY exerts its anticonvulsant effect via the induction of LTD or depotentiation. Of course, we must consider that, in contrast to in vivo studies that demonstrated an anticonvulsant effect of LFS, we did not use pathologic brain tissue but physiologic tissue in epileptogenic conditions. Nonetheless, our study posed the question whether the successful treatment of focal epilepsy by low-frequency electrical or transcranial magnetic stimulation might require alternative mechanisms than the induction of LTD. In particular, the activation of certain brain regions and transmitter systems that subsequently exert a lasting inhibitory effect on the epileptic focus must be taken into account. This concept is supported by the fact that, in addition to stimulation of the epileptic focus itself, activation of various brain regions including the cerebellum, the caudate nucleus, the thalamus, and the vagus nerve was shown to reduce seizure frequency (1). Even more intriguing, highfrequency stimulation of the hippocampus in patients with intractable temporal lobe epilepsy—a procedure that induces LTP of synaptic transmission—rather than LFS, was shown to suppress epileptogenesis (40). These results indicate that the induction of LTD not necessarily represents the major underlying mechanism of LFS in the successful treatment of focal epilepsy. Acknowledgment: This study was supported by a grant from the German Research Foundation DFG to J. Behr (TR3). We appreciate S. Walden’s technical assistance.
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