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Department of Physiology and Pharmacology, State University of New York, Brooklyn, New York
Submitted 24 February 2004; accepted in final form 23 July 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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120 min after the induction of LTP). Renewed tetanic stimulation re-established LTP. SDP was remarkably specific: baseline transmission and other forms of hippocampal plasticity, i.e., Ca2+-induced LTP and two forms of LTD [(RS)-3,5-dihydroxyphenyglycine (DHPG) mediated and low-frequency stimulation mediated] were not affected by the same type of seizure activity. SDP was blocked in the presence of the group I mGluR antagonist (S)-4-carboxyphenylglycine. The mGluR1 antagonist (S)-(+)-
-amino-methylbenzeneacetic acid blocked 
80%, the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)-pyridine 30% of SDP. Most efficient implementation of SDP was observed during seizures in the combined presence of the group I mGluR agonist DHPG and the GABAA antagonist bicuculline. However, similar ictal activity generated in the presence of DHPG alone did not lead to SDP in the vast majority of recordings. Complete disinhibition and at least partial activation of group I mGluR were necessary conditions for the induction of SDP. The depotentiating pharmacological conditions were accompanied by tonic membrane depolarization of CA1 pyramidal cells. Since hyperpolarization (by negative current injection) prevented intracellular SDP under depotentiating pharmacological conditions and depolarization (by positive current injection) led to selective intracellular SDP in the non-depotentiating seizure protocol of DHPG, it is concluded that cell depolarization was a sufficient condition for seizure-like activity to reverse hippocampal LTP. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Lynch et al. 2000; Thompson 1991) or therapeutic electroconvulsive stimulation (Squire 1986). Although seizure-induced memory loss can persist for several months, it can occur without other neurological deficits or structural brain damage (Weeks et al. 1980). Seizure-induced amnesia is especially frequent in patients with temporal lobe epilepsy, and a particularly strong link between seizure activity and memory loss can be found in the hippocampal formation due to the hippocampus’ prominent role in memory consolidation processes (Halgren et al. 1991; Milner 1966; Zola-Morgan and Squire 1990).
Long-term potentiation (LTP) and its counterpart, long-term depression (LTD), are widely regarded as cellular models of learning mechanisms (Bear 1996; Bliss and Collingridge 1993; Huang and Kandel 1994; Kandel et al. 1986; Morris 1989; Tsien et al. 1996). Given the prominent role attributed to LTP/LTD as cellular models of memory on the one hand and clinical observations of memory-impairing effects of seizures on the other, surprisingly few studies have examined seizure effects on synaptic plasticity. A reversible loss of LTP was seen in CA1 in vivo after stimulation-induced seizure activity (Hesse and Teyler 1976). It was not clear, however, whether the loss of LTP was caused by seizures per se or spreading depression that followed seizures (Hesse and Teyler 1976). The hippocampal slice (Alger 1984) was the preparation of choice in more recent investigations of seizure effects on plasticity. LTP-like effects were observed in the disinhibited slice during interictal-type of epileptiform activity (Ben Ari and Represa 1990; Schneiderman et al. 1994). Both LTP and LTD effects were reported using the potassium model of in vitro epilepsy (Contzen and Witte 1994). The LTP induction process was impaired during postictal depression (Barr et al. 1997; Moore et al. 1993).
In this study, we used the recently described disinhibition model of electroencephalographic seizure-like activity in the isolated CA1 slice preparation (Karnup and Stelzer 2001) to examine effects of seizures on hippocampal plasticity. The first objective was to examine whether seizures exerted specific effects: did seizures affect excitatory postsynaptic potentials (EPSPs) in general (including control EPSPs) or more specifically (i.e., only potentiated or depressed EPSPs)? Specific effects on synaptic plasticity are an important criterion for the validity of a given cellular model of seizure-induced amnesia. It was shown that the specific loss of LTP—by brief perfusion of high [K+] plus glutamate—was transient. In contrast, lasting (>1 h) depression by longer high-[K+]/glutamate perfusion was not specific as it was accompanied by a general failure of axonal responsiveness (Harrison and Alger 1993). A specific depression of potentiated EPSPs, however, was reported in an earlier in vivo study in CA1: the seizure-induced complete depression of potentiated EPSPs recovered to- or above-pretetanization baseline, suggesting a specific reversal of potentiated EPSPs (Hesse and Teyler 1976). Another question concerning specificity is which type of plasticity would be affected by seizures. Besides stimulation-induced LTP (Bliss and Collingridge 1993)—which is most frequently linked to learning and memory mechanisms—several other forms of hippocampal plasticity have been described in recent years, e.g., Ca2+-induced LTP (Turner et al. 1982) and two forms of LTD, one evoked by low-frequency stimulation (Dudek and Bear 1992; Mulkey and Malenka 1992), another by (RS)-3,5-dihydroxyphenyglycine (DHPG) application (Anwyl 1999; Kemp and Bashir 2001). The question whether ictal events affected hippocampal synaptic plasticity in general or more selectively was addressed by subjecting different forms of hippocampal plasticity to the same seizure protocol. We show that the reversal of stimulation-induced LTP, termed spontaneous depotentiation (SDP), was the only effect of seizures in this model: baseline EPSPs and other forms of hippocampal plasticity, i.e., Ca2+-induced LTP and two forms of LTD [DHPG and low-frequency stimulation (LFS) mediated] were not affected by ictal activity. This remarkable specificity indicates that SDP may serve as a useful in vitro model of seizure-induced amnesia.
The second objective was to examine cellular mechanisms of SDP. Although the pharmacological blockade of GABAA receptor function was the only, and thus sufficient, experimental means for the induction of SDP, a strong activation of glutamate receptors (by tetanic stimulation and spontaneous ictal activity) was an integral part of the SDP protocol. We examined a possible role of group I mGluR in the induction of SDP. Group I mGluRs were shown to undergo long-term activation by seizure-like activity (Galoyan and Merlin 2000; Lee et al. 2002; Wong et al. 1999; Zhao et al. 2004) and LTP-inducing tetanization (Bortolotto et al. 1994; Fitzjohn et al. 1996; Rammes et al. 2003). In particular, a long-term synaptic activation of group I mGluR was shown under similar experimental conditions, i.e., during prolonged epileptiform discharges induced by bicuculline and 4-aminopyridine (4-AP) (Lee et al. 2002). The diversity of mGluR subtypes and their different effects on neuronal excitability present a complex and often controversial picture, notably in the study of synaptic plasticity (Anwyl 1999). The eight cloned subtypes of mammalian mGluRs are divided into three groups based on their respective primary structures, transduction pathways, and pharmacological properties (Nakanishi et al. 1998; Pin and Duvoisin 1995). Hippocampal function is regulated by all three groups of mGluRs (Anwyl 1999). We focused on group I mGluR. The two main subtypes of group I mGluR, mGluR1 and mGluR5, share similar transduction pathways—leading to the activation of phospholipase C and phosphoinositide hydrolysis—but their cellular effects are different (Mannaioni et al. 2001). The rationale for focusing on group I mGluR was twofold. First, the pharmacological blockade of group I mGluR does not affect SDP-triggering ictal activity itself (frequency of events, ictal duration or any other parameter) in the applied seizure model (Karnup and Stelzer 2001). In contrast, the general blockade of mGluR by the broad-spectrum antagonist MCPG compromised ictal activity by reducing the frequency of epileptiform events and impairing the development of ictal components, notably the second and third burst component (Karnup and Stelzer 2001). The second reason to study effects of group I mGluR lies in its profound impact on CA1 neuronal excitability via cell depolarization and increased firing of CA1 neurons in both principal cells and interneurons (Charpak et al. 1990; Davies et al. 1995; Desai et al. 1994; Mannaioni et al. 2001). We show that group I mGluR activation and its depolarizing effect on CA1 neurons played a critical role in the implementation of SDP: depolarization of CA1 neurons promoted SDP in a non-depotentiating seizure model. In contrast, cell hyperpolarization prevented SDP in a depotentiating seizure model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Transverse hippocampal slices were obtained from adult guinea-pigs (Hartley, from Harlan Sprague Dawley, Indianapolis, IN; 150–200 g). Guinea pigs were anesthetized by inhalation of halothane (2-bromo-2-chloro-1,1,1,-trifluoroethane) before decapitation with an animal guillotine (in conformation with the guidelines of the Institutional Animal Care and Use Committee, Protocol 9808069). After removal of the brain and isolation of the hippocampus, slices of 450 µm thickness were cut on a vibrotome (Series 1000, TPI, St. Louis, MO) in ice-cold ACSF. CA1 "mini" slices were created by dissecting CA2/3 and the subiculum under microscopic control. Slices were superfused in an interface recording chamber (Fine Science Tools, Belmont, CA) with a solution saturated with 95% O2-5% CO2 (temperature: 30–32°C) of the following composition (in mM): 118 NaCl, 3 KCl, 25 NaHCO3, 1.2 NaH2PO4, 1.7 MgCl2, 2.0 CaCl2, and 11 D-glucose.
Recordings
Recording electrodes (World Precision Instruments, Sarasota, FL.) were pulled by a Brown-Flaming electrode puller (Model P-87, Sutter Instrument, Novato, CA). Extracellular recordings were carried out in stratum radiatum of CA1. Sharp-electrode intracellular recordings were performed in CA1 pyramidal cell somata (n = 41) and apical dendrites (n = 18). Dendritic pyramidal cell recordings were identified by the recording site in s. radiatum (100–250 µm perpendicular to s. pyramidale) and the burst response to suprathreshold current injection (Wong et al. 1979). Tracking was performed using manually controlled hydraulic stepping micromanipulators (Narashige). Electrodes were filled with potassium acetate (2–3 M) yielding electrode resistances of 42–97 M
. EPSPs were elicited by single stimuli delivered to the Schaffer collateral-commissural pathway at 30-s intervals through a pair of insulated tungsten bipolar electrodes (stimulation range: 15–50 µA). In most experiments, EPSPs were measured in response to stimulation of two independent afferent pathways (both in s. radiatum, but opposite with respect to the recording electrode). Signals were recorded and amplified with an Axoprobe-1A (Axon Instruments), fed into an A/D converter (Digidata 1200, Axon Instruments) digitized, stored, and analyzed off-line using "pCLAMP8" software from Axon Instruments in a Pentium PC computer.
Data analysis
The strength of synaptic excitatory responses was assessed by measuring the slope (20–80%) of the EPSP rising phase. Data were pooled through averaging and normalization. Control values were recorded for 20–40 min prior to tetanization. Comparisons of synaptic strength at stated points of time after tetanic stimulation (e.g., 120 min after tetanization) are based on 10 measurements over 5-min periods (5 measurements were obtained before and 5 measurements were obtained after the stated point of time). Values are depicted as means ± SE. Statistical comparisons of EPSPs were performed by Student’s t-test (2 groups) or ANOVA (
3 groups). Statistical significance was accepted for all P < 0.05.
Seizures
Seizure-like activity in the CA1 minislice preparation was triggered by the competitive GABAA receptor antagonist bicuculline-methiodide (Bic, 50–100 µM), the chloride channel blocker picrotoxin (PTX, 100–200 µM), or the group I mGluR stimulator DHPG (30–60 µM) as recently described in detail (Karnup and Stelzer 2001). Extracellular calcium ([Ca2+]e) was used to control epileptiform activity. Ictal events were reliably observed in the presence of control [Ca2+]e (2 mM) but completely blocked during elevated [Ca2+]e (6 mM).
LTP
LTP was normally triggered by theta burst stimulation (TBS, 3–4 trains of 4 pulses at 100 Hz separated by 200 ms repeated 2–4 times in 30-s intervals (Barrionuevo et al. 1980; Larson et al. 1986). In some recordings (Figs. 3Bb and 6), high-frequency stimulation (HFS, 1–2 trains, 1 s, 100 Hz, at test pulse strength) was applied to induce LTP. The term "tetanic stimulation" was used for both TBS and HFS. Experiments were designed to ensure proper induction of LTP (or LTD) uninfluenced by impairing effects of postictal depression (Barr et al. 1997; Moore et al. 1993). For example, when ictal activity was present during pretetanization controls, tetanic stimulation was only applied after full recovery from postictal depression. Ca2+-induced LTP was implemented by increasing [Ca2+]e from 2 mM controls to 6 mM (Fig. 4D).
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LTD was induced by low-frequency stimulation (LFS, 1 Hz for 10 min at test pulse strength; Fig. 7B). mGluR-LTD was induced through transient application of DHPG (30–60 µM; Fig. 7A).
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Bic, PTX (from Sigma), DHPG, (S)-4-carboxyphenylglycine (4-CPG), (S)-(+)--amino-methylbenzeneacetic acid (LY367385), and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (from Tocris Cookson, Ballwin, MO) were applied by bath perfusion.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Individual recordings in Figs. 1 and 2 illustrate the basic finding of this study: seizure-like events that occurred spontaneously during the blockade of GABAA-receptor function in the isolated CA1 subfield (Karnup and Stelzer 2001) caused a stepwise reversal of LTP. LTP was elicited at the Schaffer collateral/CA1 synapse by theta-burst stimulation (if not otherwise stated). The most frequently observed pattern of depotentiation (in >90% of recordings) is illustrated in Fig. 1: each ictal event was followed by a large but transient (1–5 min) postictal depression (in some cases below pretetanization controls). The recovery from postictal depression was not complete, however, leaving a small but long-lasting depression. Only these lasting decreases of field EPSPs (fEPSPs) after recovery from transient postictal depression are referred to as SDP or ictal-induced LTP reversal in the following. EPSP peaks (Fig. 1Aa) and slopes (Fig. 1Ab) exhibited the same time courses of depotentiation.
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A different depotentiation pattern is shown in Fig. 2. Fewer seizures caused larger depotentiation steps. Although less frequently observed (in only 5 of 67 recordings), this depotentiation pattern highlights three important properties of SDP. First, it illustrates more clearly the temporal link between ictal events and individual depotentiation steps. Only two ictal events (marked by arrows at t = 40 min and 47 min, respectively) led to a combined 64% reversal of potentiated fEPSPs. After each event, fEPSP remained at the partially depotentiated levels (in the absence of further ictal events). The coincidence of seizures and depotentiation steps indicates that seizures were instrumental in the reversal of LTP. Second, transient postictal depression was absent in the recording in Fig. 2. Thus lasting depotentiation was not contingent on effects of postictal depression or even spreading depression as suggested in an earlier in vivo study (Hesse and Teyler 1976). A third property featured in Fig. 2 is that seizure-induced depotentiation could be reversed by renewed tetanic stimulation in agreement with previous reports (Contzen and Witte 1994; Harrison and Alger 1993; Hesse and Teyler 1976; Moore et al. 1993). Reversal of SDP was seen after partial depotentiation (Figs. 2 and 4C) but also after complete depotentiation (not shown). Renewed LTP exhibited the same behavior as LTP established by the first TBS, i.e., it was maintained in the absence of ictal activity but exhibited depotentiation during ictal activity (not shown).
Specificity of SDP
Two-pathway experiments demonstrate that only potentiated EPSPs were affected by seizures (see summary graphs in Fig. 3A): EPSPs were evoked at two independent stimulation sites (both in s. radiatum) but opposite with respect to the recording electrode (termed paths 1 and 2, respectively). Only path 1 was tetanized. Similar to the individual recordings of Figs. 1 and 2, a lasting decrease of potentiated EPSPs was observed after each ictal event. On average, depotentiation after each ictal event was 5.9 ± 1.2% (based on n = 281 ictal events in 16 different recordings). Depotentiation steps accumulated until pre-TBS baseline values were reached and remained at pretetanization baseline regardless of whether seizure activity was present (e.g., Fig. 3Aa) or discontinued (Fig. 4Ab after t = 160 min). Ictal activity had no long-term effect on all controls, i.e., EPSPs in both pathways before tetanization and also EPSPs of the nontetanized paths 2 throughout (Fig. 3A). Depotentiated EPSPs of paths 1 became statistically equal with those of the nonpotentiated paths 2 at t = 82 ± 9 min. At t = 120 min, averaged and normalized EPSP slopes were 1.05 ± 0.08 in paths 1 and 1.04 ± 0.06 in path 2 (P = 0.34, n = 16). The strength of afferent input was unchanged as shown by the amplitude of afferent volleys in the tetanized paths (Fig. 3Ad). In summary, we demonstrate one component of specificity—arguably the most critical one—in that seizures affected potentiated EPSPs but not baseline EPSPs or EPSPs after complete depotentiation.
Tetanization had increased the overall duration of ictal activity by 46% on average (Fig. 3Ac). This increase was due to an increase of the frequency of ictal events as the average duration of a given ictal event was the same before and after TBS (6.3 ± 1.8 s, n = 379 episodes in 16 recordings, pre- and post-TBS ictal events lumped together; Fig. 3Ab). As a function of the nth ictal event, SDP was completed after 17 ictal events on average (Fig. 5B).
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Data in Fig. 3B show that potentiation was maintained (>2 h) when ictal activity was blocked (in the presence of 6 mM [Ca2+]e; see METHODS): fEPSPs slopes were 1.87 ± 0.14 of pre-TBS controls at t = 120 min (n = 8; P < 0.0001 compared with depotentiated values obtained in the presence of 2 mM [Ca2+]e; Fig. 3Ba). Figure 3Bb illustrates the same behavior for LTP induced by HFS (1–3 trains at 100 Hz, each 1-s duration, 20 s apart, test-pulse strength). Similar to TBS-induced potentiation (Fig. 3Ba), HFS-induced potentiation was reversed during ictal activity (2 mM [Ca2+]e, 50–100 µM Bic present throughout) but maintained (>2 h) in the absence of ictal activity (6 mM [Ca2+]e, 50–100 µM Bic, present throughout; Fig. 3Bb). EPSPs were 1.96 ± 0.06 at t = 120 min (n = 5) in the absence of ictal activity and 0.83 ± 0.12 in the presence of ictal activity (n = 7; P < 0.00001). No further attempts were made to examine possible mechanisms of the small depression after complete SDP in the HFS protocol.
Initiation of SDP at different stages of LTP consolidation
The question was asked whether seizures would impact different phases of LTP consolidation. Clinical observations show that memories occurring in close proximity to seizures are disproportionately impaired. Analogously, it can be expected that earlier phases in the LTP consolidation process would be more vulnerable to seizures. Previous studies had demonstrated that seizures interfered with the LTP induction process (Barr et al. 1997; Moore et al. 1993). But reversal of LTP after seizures—shown in vivo—was also effective, at least, partially, at various intervals (measured 60 min) after the initial induction of LTP (Hesse and Teyler 1976).
To examine whether different stages of LTP consolidation were vulnerable to seizure activity, ictal activity was initiated at various points of time after LTP induction (Figs. 4, 5A, and 8A). Control and potentiated EPSPs were initially recorded in the absence of ictal activity (in 6 mM [Ca2+]e) during which LTP was maintained (as shown in Fig. 3B). Ictal activity was then initiated at different points of time after TBS, i.e., 30 min (Figs. 5A and 8A), 40 min (Fig. 4C), 60 min (Fig. 4A), and 120 min (Fig. 4B) by lowering [Ca2+]e to 2 mM. Such delayed initiation of ictal activity led to SDP in 35 of 39 recordings. The properties of delayed SDP initiated after maintained potentiation (Figs. 4, 5A, and 8A) were similar to those of immediate SDP (Figs. 1–3): small depotentiation steps after each ictal event accumulated leading to complete depotentiation. Figure 4C illustrates that—similar to immediate SDP—tetanic stimulation after delayed SDP led to maintained LTP in the absence of ictal activity. The times from seizure onset to SDP completion in Bic were 63 ± 10 min when seizure onset was delayed by 30 min (n = 16, Fig. 8A), 64 ± 11 min when seizures were delayed by 60 min (n = 7, Fig. 4A), and 62 ± 13 min when seizures were initiated 120 min after LTP induction (n = 5, Fig. 4B). These data demonstrate that seizures were capable of disrupting different phases of LTP consolidation with equal efficacy. This observation corresponds to stimulation studies using strong stimulation paradigms: theta bursts at high stimulation intensities were shown to reverse LTP at later stages of consolidation (Barr et al. 1995), whereas depotentiation by weaker low-frequency stimulation protocols were only effective within a very narrow time window after LTP induction (see Huang et al. 2001).
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Reversal of LTP during PTX-induced ictal activity
Bic-methiodide-containing solution was routinely used because it prompted ictal activity reliably and more frequently than other antagonists of GABAA receptor function (Karnup and Stelzer 2001). But Bic-methiodide was linked to several GABAAR-unrelated effects, notably block of Ca2+-activated K+ conductances (Seutin et al. 1997). To examine whether seizures caused by the blockade of GABAA receptor function were responsible for SDP, stimulation-evoked LTP was subjected to ictal activity generated by the chloride-channel blocker PTX (instead of Bic; Fig. 5) (Karnup and Stelzer 2001). The delayed SDP protocol (as in Fig. 4, A–C) was applied. Figure 5Ab illustrates that PTX-induced ictal activity (initiated at t = 30 min) led to progressive SDP similar to previously featured experiments during Bic-induced seizures. The time course toward the completion of SDP, however, was considerably longer during PTX compared with recordings during Bic: fEPSPs of the potentiated and nontetanized paths became statistically indistinguishable for t = 140 min, i.e., 110 min after ictal onset as opposed to t = 71 min after ictal onset in Bic (n = 28; this number is based on lumped recordings after immediate and delayed SDP. But when depicted as function of the nth ictal event, EPSPs were statistically the same after each ictal event, (except the 17th) in the presence of Bic and PTX, respectively (Fig. 5B). On average, complete reversal of LTP in PTX was seen after the 18th event. The most probable explanation for the longer time course of SDP during PTX-induced ictal activity can be found in the lower frequency of ictal activity (compared with Bic-containing solution; Fig. 5Aa). The depotentiating efficacy of a given ictal event, however, was the same in the presence of the competitive GABAAR blocker Bic and the chloride-channel blocker PTX. These data support the notion that SDP was triggered by disinhibition-induced ictal activity. In addition, these data demonstrate that SDP was implemented by seizures occurring at considerably lower frequency (compared with the Bic protocol).
Reversal of potentiation induced in ACSF
Experiments were carried out to test whether LTP induced under physiological conditions (i.e., with inhibition intact) would be reversed by ictal activity (Fig. 6). Controls were established in two pathways in the absence of pharmacological treatment. HFS (2 trains, 100 Hz, 1 s each, 20 s apart, test pulse strength) were applied to path 1. The stronger HFS paradigm was used to compensate for the absence of disinhibition-mediated facilitation of LTP induction (Wigström and Gustafsson 1983). Tetanization resulted in fEPSP increases to 173 ± 2 over controls (n = 6; measured at t = 50 min; Fig. 6Ab, path 1). At t = 50 min, Bic (50–100 µM) was applied which prompted ictal activity shortly thereafter (Fig. 6Aa). EPSP slopes in both paths were enhanced by Bic (Karnup and Stelzer 1999). The Bic-induced potentiation of path 1 was transient followed by a progressive decline leading to the complete erasure of LTP. This conclusion is based on the comparison with EPSPs of the nontetanized path 2, which remained elevated after Bic: EPSPs of path 1 stabilized at 123 ± 8 (n = 6; P = 0.09 compared with 134 ± 7 of path 2, measured at t = 170 min).
The same protocol as in Fig. 6A was used in the individual recording shown in Fig. 6B. In addition, this recording shows the return to pre-tetanus/pre-disinhibition values following Bic washout (at t = 140 min). EPSP values measured after Bic washout were 91 ± 8 in path 1 and 102 ± 8 in path 2 (values from t = 180 to 200 min). In both pathways, values were statistically the same compared with respective pre-HFS controls; P = 0.22 and P = 0.19, respectively). In summary, based on the comparison between the tetanized path 1 and nontetanized (control) path 2, it is shown that ictal events were effective in reversing LTP induced by tetanic stimulation under physiological conditions.
Ca2+-induced LTP is not affected by ictal activity
The question was asked whether SDP-generating ictal activity affected other forms of plasticity. We examined Ca2+-induced LTP (Turner et al. 1982) (see Fig. 4D) and two types of LTD (see following text, Fig. 7). Data depicted in Fig. 4D illustrate that Ca2+-induced LTP was not affected by ictal activity. Switching [Ca2+]e from 2 to 6 mM (at t = 0 min) led to potentiation of fEPSPs to 1.55 ± 0.03 (measured from t = 40 to t = 80 min; P < 0.001 compared with controls in 2 mM [Ca2+]e). EPSPs remained potentiated during ictal activity after the re-introduction of 2 mM control [Ca2+]e at t = 80 min (1.52 ± 0.06, measured from t = 100 to 220 min, P = 0.12 compared with values during 6 mM [Ca2+]e).
The observation that Ca2+-induced LTP was not affected by ictal activity is critical for our interpretation of delayed SDP given that the pretetanization controls in Fig. 4, A–C (obtained in the presence of 6 mM [Ca2+]e) represented Ca2+-potentiated EPSPs. In these recordings, TBS-induced potentiation was implemented on top of Ca2+-mediated potentiation. The ictal-induced depression of EPSPs potentiated by TBS (Fig. 4, A–C) ended at the pre-TBS baseline. If Ca2+-induced potentiation had been affected by ictal activity, the ictal-induced depression in Fig. 4, A–C, would have continued below the pretetanization baseline to the lower values recorded in the presence of 2 mM [Ca2+]e. These data allow the conclusion that Ca2+-potentiated fEPSPs were not affected by ictal activity. The comparison demonstrates that ictal activity had—specifically—affected the stimulation-induced form of LTP.
Two types of LTD were not reversed by ictal activity
The question was asked whether ictal activity would affect the opposite type of plasticity, i.e., LTD. Two LTD forms can be distinguished at the CA3–CA1 synaptic junction, homosynaptic LTD induced by LFS (Dudek and Bear 1992; Mulkey and Malenka 1992) and LTD induced by the group I mGluR agonist DHPG (Anwyl 1999; Kemp and Bashir 2001). Ictal activity did not reverse either form of LTD. Data shown in Fig. 7A illustrate that ictal activity did not change the DHPG-induced form of LTD. LTD was induced by a 40-min application of DHPG (30–60 µM, applied from t = 40 to 80 min) in the absence of ictal activity (6 mM [Ca2+]e; Bic, 50–100 µM, was present throughout). The onset of ictal activity (after switching to ictal promoting 2 mM [Ca2+]e) did not change DHPG-depressed EPSPs: fEPSPs during DHPG-LTD were 53 ± 2 before ictal activity (measured between 90 and 120 min, n = 8 recordings) and 54 ± 4 during ictal activity (measured between 160 and 240 min; P = 0.53).
LFS-induced LTD was also not reversed by Bic-induced ictal activity (Fig. 7B). Controls were established in the presence of Bic (50–100 µM) and 6 mM [Ca2+]e (to block ictal activity) in two-pathway experiments. LTD was induced by LFS in pathway 1 (1 Hz for 10 min; marked in Fig. 7Bb, 
). It is inferred that the LTD-inducing LFS stimulation pattern (1 Hz for 10 min) induced the homosynaptic, N-methyl-D-aspartate (NMDA)-dependent, possibly postsynaptic form of LTD (see Rammes et al. 2003), but no efforts were made to characterize this LTD type further. The second pathway was not conditioned. LFS-induced depression (68 ± 11, n = 6, measured at t = 60 min) was not affected by ictal activity (introduced by 2 mM containing [Ca2+]e at t = 60 min). At t = 160 min (i.e., 100 min after ictal onset), EPSP slopes were 71 ± 6 in path 1 and 101 ± 5 in path 2 (P > 0.05, n = 6, compared with respective values before ictal onset). It would have been desirable to use a potentiated control (path 2) in combination with LFS-induced LTD to demonstrate the specificity of SDP. However, such potentiation was shown to induce (heterosynaptic) reversal of the LFS-treated fiber pathway (Muller et al. 1995), thus precluding the experimental objective.
SDP is blocked by group I mGluR antagonists
Although the blockade of GABAA-receptor function was sufficient to generate ictal-like events (Karnup and Stelzer 2001) and subsequently SDP in the isolated CA1 slice (Figs. 1–6), a strong activation of glutamate receptors by tetanic stimulation or ictal activity can be inferred (Bortolotto et al. 1994; Fitzjohn et al. 1996; Galoyan and Merlin 2000; Lee et al. 2002; Raymond et al. 2000; Wong et al. 1999). We examined whether the activation of group I mGluR had contributed to SDP. A possible role of group I mGluR was examined by adding group I mGluR antagonists to the standard, ictal- and SDP-generating solution containing high concentrations of Bic (50–100 µM; Fig. 8). We had shown previously (Karnup and Stelzer 2001) that the pharmacological blockade of group I mGluR had no impact on Bic-induced ictal activity itself (frequency of events, ictal duration, shape, or burst components).
Pooled data in Fig. 8 demonstrate that SDP was blocked or considerably impaired during the pharmacological blockade of group I mGluR. Group I mGluR antagonists were introduced together with seizure-generating 2 mM [Ca2+]e 30 min after LTP induction to allow for proper induction and consolidation of LTP ("delayed SDP" as shown in Fig. 4). In the presence of the specific group I mGluR antagonist 4-CPG (100 µM; Fig. 8Bb), path 1 EPSPs remained potentiated at 1.56 ± 0.07 (n = 9) at t = 120 min, i.e., 90 min after onset of ictal activity. In contrast, the control recordings in which ictal activity was generated at t = 30 min in the absence of mGluR antagonists (Fig. 8Ab) exhibited a complete depotentiation of path 1 EPSPs at t = 87 min, i.e., 53 min after ictal onset. At t = 120 min, these path 1 control EPSPs were 0.99 ± 0.06 (n = 16; P < 0.0005 comparing EPSPs during Bic alone and during Bic +4-CPG). In the presence of the mGluR1-specific antagonist LY367385 (100 µM), path 1 EPSPs remained at 1.42 ± 0.04 at t = 120 min (i.e., 90 min after ictal onset; n = 8; P < 0.0001 compared with Bic alone; Fig. 8Db). In the presence of the mGluR5-specific antagonist MPEP (50 µM), path 1 EPSPs remained at 1.25 ± 0.09 at t = 120 min (n = 5, P < 0.05 compared with Bic alone; Fig. 8Cb). The superimposed summary graphs in Fig. 8E illustrate the SDP-preventing efficacies of various group I mGluR antagonists in direct comparison, as function of time (Fig. 8Ea) and as function of the nth ictal event (Fig. 8Eb).
Properties of ictal activity, i.e., overall duration (Fig. 8, Aa–Da) and frequency of ictal activity, duration of individual ictal events, burst duration, etc., were similar in the depotentiating controls of Bic alone and in the presence of different group I mGluR antagonists (Karnup and Stelzer 2001). Data in Fig. 8Eb (potentiated EPSPs depicted as a function of the nth ictal event) and Table 1 (percentage of depotentiation mediated by a single ictal event) show that the depotentiating strength of a given ictal event was reduced in the presence of group I mGluR antagonists. After 17 ictal events—at which SDP was completed in the presence of Bic alone—EPSPs remained at 1.57 ± 0.07 potentiation in the presence of 4-CPG (P < 0.0005 compared with Bic alone), 1.54 ± 0.04 in the presence of the mGluR1 antagonist LY367385 (P < 0.001 compared with Bic alone), and 1.27 ± 0.13 in the presence of the mGluR5 antagonist MPEP (P < 0.05 compared with Bic alone).
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The results shown in Fig. 8 and Table 1 demonstrate that the synaptic activation of group I mGluR was a critical step in the implementation of SDP. Based on these observations, we then hypothesized that ictal activity triggered by the pharmacological stimulation of group I mGluR (Galoyan and Merlin 2000; Karnup and Stelzer 2001; Lee et al. 2002; Wong et al 1999) would be most efficient in the induction of SDP. This hypothesis was tested by the same basic experimental approach as shown in Fig. 8A, except that DHPG (30–60 µM) was used as the source of ictal activity (Karnup and Stelzer 2001). Contrary to the working hypothesis, however, DHPG-induced ictal activity did not result in significant depotentiation (Fig. 9A). During DHPG-induced ictal activity (initiated at t = 30 min after TBS), potentiated EPSPs remained at 1.58 ± 0.07 at t = 120 min, i.e., 90 min after ictal onset (n = 14; P < 0.001 compared with Bic alone; see superimposed graphs in Fig. 9B). After 17 DHPG-induced ictal events after tetanic stimulation, fEPSPs remained at 1.60 ± 0.06 of pretetanized controls (P < 0.001 compared with experiments during Bic; Fig. 10D).
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). A breakdown of individual recordings indicates that SDP was possible but far less probable during DHPG-induced ictal activity: potentiation was completely maintained in 8 of 14 recordings (fEPSPs were 1.87 ± 0.09 at t = 120 min; P < 0.001 compared with Bic). Gradual SDP (with variable time courses) was observed in 6 of 14 recordings: EPSPs in these six recordings were 1.27 ± 0.09 at t = 120 min (P < 0.05 compared with Bic alone). SDP was most effective when disinhibition was combined with group I mGluR stimulation
Two explanations are conceivable as to why DHPG-induced ictal events were less effective in mediating SDP than those generated during Bic. First, it could be argued that DHPG-mediated LTD (Anwyl 1999; Kemp and Bashir 2001; Mannaioni et al. 2001) had occluded SDP: the pretetanization controls obtained in the presence of DHPG (Fig. 9A) represented DHPG-depressed EPSPs as illustrated in the individual recording of Fig. 10B. An alternative explanation would be that both disinhibition and group I mGluR activation were required for the successful implementation of SDP. Data shown in Fig. 10 clearly demonstrate that the latter hypothesis is correct. SDP was readily implemented in the combined presence of DHPG+Bic (Fig. 10). Because the DHPG+Bic protocol also relied on DHPG-depressed EPSPs as controls, it can be ruled out that DHPG-mediated LTD had occluded SDP. In the combined presence of Bic and DHPG, ictal events not only reversed LTP but did so far more effectively compared with other seizure protocols, Bic alone and—even more so—DHPG alone. This is best illustrated by the depiction of SDP as a function of the nth ictal event (Fig. 10D). The potentiation of the tetanized path 1 was completely reversed after the ninth ictal event in the presence of DHPG+Bic (compared with an average of 17 events required for complete depotentiation during Bic alone; Fig. 10D). The time to completion of SDP (Fig. 10, A–C and E) is another indicator of the higher depotentiating efficacy of the DHPG+Bic protocol: on average, SDP was complete 27 ± 6 min after ictal onset in the presence of DHPG+Bic (n = 11; Fig. 10A) compared with 63 ± 10 min in the presence of Bic alone.
The experimental protocol in Fig. 10, C (pooled data) and E (individual recording), illustrates the critical role of disinhibition most directly: maintained potentiation of intracellular EPSPs during DHPG-induced ictal activity was followed by fast depotentiation (in path 1) after Bic was added to the DHPG-containing solution (at t = 40 min). EPSPs of the potentiated path 1 and nontetanized path 2 became statistically equal (at Bic-induced elevated levels) for all t > 64 min, i.e., 24 min after Bic application (Fig. 10Cb). A similarly fast time course of SDP completion can be seen in the individual recording (Fig. 10Eb). In sum, most expedient SDP was observed when disinhibition was combined with pharmacological group I mGluR stimulation (Table 1).
Cell depolarization during depotentiating pharmacological conditions (DHPG+Bic)
Examination of ictal activity did not reveal properties that could have accounted for the observed differences in SDP efficacy during various pharmacological models (DHPG+Bic >> Bic >> DHPG; Table 1). Neither the overall duration (Fig. 10Ca) nor the duration of individual ictal events (Fig. 10Ea) was changed after Bic was added to the DHPG-containing solution (Karnup and Stelzer 2001). On average, the duration of a given ictal event was 5.1 ± 0.7 s (n = 22) during DHPG and 5.3 ± 0.9 after Bic was added (n = 35; P = 0.1). Other properties of the individual ictal event (shape and amplitude, duration of the entire episode, duration of burst components) (Traub et al. 1996) were similar in the three seizure models used in this study.
Intracellular recordings revealed tonic cell depolarization as a main difference between the non-depotentiating seizure model of DHPG alone and the depotentiating seizure model of DHPG+Bic (Figs. 10Ec and 11). The rapid implementation of SDP in the combined presence of DHPG+Bic was accompanied by an average depolarization shift of +7.8 ± 1.1 mV [from –64.8 ± 1.1 to –57.0 ± 1.9 mV; P < 0.001, t-test; compared with maintained potentiation during DHPG alone; n = 9 pyramidal cell recordings: somatic (n = 5) and dendritic (n = 4) lumped together]. In the individual recording shown in Fig. 10E, the cell’s membrane potential was tonically depolarized by +8 to –58 mV during rapid SDP in the combined presence of DHPG+Bic (from –66 mV before onset of SDP, i.e., during maintained potentiation in the presence of DHPG alone; Fig. 10Ec).
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The application of DHPG alone did not lead to significant depolarization (+0.7 ± 0.5 mV; from –64.3 ± 1.3 mV in the untreated slice to –63.6 ± 1.5 mV; P = 0.12, n = 8). Our data suggest that—although DHPG was responsible for tonic cell depolarization as previously shown (Charpak et al. 1990; Davies et al. 1995; Desai et al. 1994; Mannaioni et al. 2001)—significant depolarization in pyramidal cells embedded in the CA1 network was only observed when fast synaptic inhibition was completely blocked. The concomitant intra- and extracellular recordings shown in Fig. 11A depict the depolarization shift—caused by adding Bic to a DHPG-containing solution—in a complete cycle. Ictal activity—initially in the presence of DHPG alone—occurred in a remarkably regular rhythm in this particular recording (every 1.2 min). Intracellularly recorded ictal events exhibited long AHPs and pre-ictal plateau phases. Vrest (determined during pre-ictal plateaus) was about –65 mV. The addition of Bic induced a tonic depolarization shift of roughly +9 mV. The depolarization persisted as long as Bic was present but was reversed after washout. The addition of Bic did not affect ictal properties, e.g., expression of burst components, frequency of occurrence, duration, or periodicity.
Cell depolarization promoted ictal-induced SDP
Was cell depolarization a critical mechanism in the implementation of SDP? The correlation of SDP efficacy and Vm (Fig. 10F)—albeit highly significant—does not establish a causal effect. Two series of concomitant extra/intracellular recordings were performed to examine whether the tonic cell depolarization during depotentiating pharmacological conditions (Figs. 10Ec and 11) was an essential mechanism in the induction of SDP or merely a byproduct. First, it was asked whether experimental depolarization of the recorded cell (via positive current injection) would result in selective intracellular SDP during the non-depotentiating seizure protocol of DHPG alone (Fig. 12). Similar to fEPSPs, intracellular EPSPs remained potentiated during the DHPG-induced ictal activity as long as the cells were held at respective Vrest (–64.5 ± 1.4 mV) from t = 0 to 60 min. Starting at t = 60 min, positive current (between +0.2 and +0.5 nA) was injected between test pulses for 28 s (of 30 s as illustrated in Fig. 12A). Test EPSPs were recorded at Vrest as before. The experimental objective was to keep the recorded cells at depolarized Vm as long as possible to ensure that random ictal events were initiated at depolarized levels. The rationale of this experimental protocol was to mimic the condition of tonic cell depolarization observed during the depotentiating pharmacological protocol of DHPG+Bic (Figs. 10E and 11). Positive current injection between test pulses led to an average depolarization shift of +6.7 ± 1.4 mV (to 58.2 ± 1.1 mV). Figure 12Bb illustrates that the injection of positive DC between test pulses led to fast SDP: potentiated intracellular EPSPs (1.64 ± 0.02, n = 6; recorded before cell depolarization at t = 60 min) became completely depotentiated within 8 and 19 min after experimental cell depolarization: pooled EPSPs were 1.02 ± 0.03 measured at t = 80 min, i.e., 20 min after tonic cell depolarization in these six intracellular recordings. In contrast, concomitantly recorded fEPSPs remained potentiated (1.70 ± 0.05; P < 0.0001 comparing intra- and extracellular EPSPs at t = 80 min). The cells’ input resistances (measured at respective Vrest, Fig. 12Bc) were unchanged during SDP indicating that the depression of intracellular EPSPs was not due to cell deterioration. Depotentiation was brought to a halt when current injection between test pulses was discontinued (not shown).
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Cell hyperpolarization prevented ictal-induced SDP
Experiments in which the reverse protocol was applied (Fig. 13) confirmed the notion that cell depolarization was a sufficient condition for seizure-induced reversal of LTP. It was asked whether cell hyperpolarization would prevent SDP under depotentiating pharmacological conditions. Concomitant intra/extracellular recordings (n = 8) were carried out. Depotentiating conditions were provided at t = 30 min by adding Bic (n = 6) or PTX (n = 2) to a DHPG-containing solution (as shown before, see Fig. 10C). SDP of intracellular EPSPs was prevented by negative current injection (as schematically shown in Fig. 13A) from t = 30 min to t = 90 min. Intracellular EPSPs were 1.96 ± 0.03 at 60 min and 1.94 ± 0.05 at t = 90 min (P = 0.09). In contrast, fEPSPs exhibited rapid SDP after a brief Bic or PTX-induced potentiation. SDP was complete within 30 min of Bic or PTX application: fEPSPs were 1.29 ± 0.02 at t = 60 and 1.30 ± 0.02 at t = 90 min (P = 0.14). The discontinuation of negative current injection (for t > 90 min) led to rapid SDP of intracellular EPSPs. SDP was complete within 26 min: intracellular EPSPs between t = 116 and t = 120 min were 1.33 ± 0.03 and fEPSPs were 1.29 ± 0.03 (P = 0.35; Fig. 13Bb). These data demonstrate that hyperpolarizing the recorded cell resulted in selective (intracellular) protection from depotentiation during the depotentiating pharmacological protocol of DHPG+Bic.
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Tonic cell depolarization was measured in both somatic and apical dendritic CA1 pyramidal cell recordings during the depotentiating seizure protocol of DHPG+Bic. In apical dendritic recordings (n = 4, recorded at 100–200 µm distances from soma), Vm in the combined presence of DHPG and Bic was –58.3 ± 2.7 mV (up from –65.0 ± 1.9 mV in the same recordings in the native slice, P < 0.001). In somatic recordings (n = 5), Vm during DHPG + Bic was –56.3 ± 2.3 mV (compared with –64.1 ± 1.8 mV, P < 0.001). In contrast to the rather homogeneous membrane potential distributions in the pharmacological protocols of depotentiation, considerable passive membrane potential gradients—away from the respective current-injection sites—may have existed in the current-injection protocols (Figs. 12 and 13). Passive responses to local current injections illustrate the gradients in concomitant somatic and dendritic recordings in the same CA1 pyramidal cell. Only 37% of the voltage response was preserved in the passively coupled recording site over a ca. 150 µm distance: current pulses (–0.4 nA) injected through the somatic recording electrode led to a membrane potential change of –8 mV at the somatic injection site (Fig. 14Aa), whereas the membrane deflection in the passively coupled dendritic recording was only –3 mV (Fig. 14Aa). The same numbers of membrane deflections, i.e., 
V(m) = –8 mV at the dendritic recording site and V(m) = –3 mV at the passively coupled somatic recording site were obtained through the reverse current pulse application (i.e., –0.4 nA through the dendritic recording electrode; Fig. 14Ab). Similar gradients were in effect in response to subthreshold positive current injections. The larger resistances to axial current flow in smaller-diameter branches yields considerably smaller passive V(m) in more distant dendritic branches. Thus passive responses per se were unlikely a factor in the control of SDP during the current-injection protocols (Figs. 12 and 13). Instead, local changes of Vm could have been involved in the control of the propagation and amplification of active, suprathreshold responses. Figure 14B illustrates this notion: the somatic responses to the same dendritic input were recorded at two different holding potentials. A +0.3-nA current pulse through the dendritic electrode led to a modest burst response (Wong and Stewart 1992) containing a spikelet followed by a full action potential (Fig. 14B, top). The passive somatic response at Vrest (–60 mV, "soma 1", Fig. 14B) was a single AP in close temporal vicinity to the dendritic AP. In contrast, a relatively modest tonic depolarization (+4 mV; "soma 2", Fig. 14B) of the cell soma led to AP doublets riding on a much larger depolarizing envelope. These data illustrate the control exerted by local membrane potential changes over propagating responses. But it remains to be seen if the local control of active responses was indeed instrumental in the promotion (Fig. 12) or prevention of SDP (Fig. 13).
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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We show that spontaneous ictal events—caused by the removal of GABAA receptor-mediated inhibition—led to a stepwise depression of potentiated fEPSPs in the isolated CA1 slice. The ictal-induced depression of EPSPs ended at the pretetanization control level (Figs. 1–5) or in few cases slightly below control levels (Figs. 3Bb and 6B). Ictal activity had no long-term effects on baseline EPSPs (Figs. 1–6). Based on criteria established in stimulation studies (Barr et al. 1995; Bashir and Collingridge 1994; Fujii et al. 1991; Huerta and Lisman 1995; Larson et al. 1993; O’Dell and Kandel 1994; Staubli and Lynch 1990), it can be concluded that the depression of EPSPs by ictal activity shown here represents a clear form of depotentiation as opposed to forms of long-term depression of EPSPs at naïve synapses (Bear 1996; Oliet et al. 1997). The efficacy of spontaneous depotentiation did not decrease over time (shown 120 min after the induction of LTP; see Fig. 4, A and B). In analogy to stimulation-induced depotentiation, SDP resembles depotentiation achieved by high-intensity stimulation (Barr et al. 1995; Bashir and Collingridge 1994). In contrast, weaker stimulation protocols were only effective within a short window of LTP induction (see (Huang et al. 2001). Spontaneous seizure-like events were causal in triggering depotentiation steps as evidenced by the close temporal correlation of the two phenomena (Figs. 1 and 2). SDP was also observed in cases where transient postictal depression was minimal or completely absent (see Fig. 2). Spreading depression—which was suggested as the cause of seizure-induced loss of LTP in vivo (Hesse and Teyler 1976)—was not involved in SDP. Taken together, our data suggest that seizures per se led to the reversal of LTP.
A common feature of seizure-induced forms of depotentiation is their heterosynaptic nature (Harrison and Alger 1993; Hesse and Teyler 1976; Moore et al. 1993). The strongest evidence that ictal events in our model activated a different—although partially overlapping—set of excitatory synapses than the ones potentiated by tetanic stimulation derives from the finding that the stimulation of the Schaffer collaterals (even at highest intensities) not only failed to trigger ictal activity but in fact blocked it (Karnup and Stelzer 2001). Similar to CA3 (Miles and Wong 1986), recurrent excitatory connections between CA1 principal cells (Deuchars and Thomson 1996) may have become functional after their inhibitory control was removed. This notion is also supported by the properties of developing synchronization in the CA1 disinhibition model of epilepsy (Karnup and Stelzer 2001) that closely resembled the synchronization mechanisms in the recurrently connected CA3 network (Traub and Wong 1982). Dependent on the subcellular sites of recurrent excitatory synapses, ictal events could have affected sites of potentiation through direct activation of glutamatergic synapses at pyramidal cell dendrites (Deuchars and Thomson 1996) or through back-propagating action potentials after suprathreshold activation of somatic or proximal dendritic glutamatergic receptors (Stuart et al. 1997). In either case, SDP was mediated by a heterosynaptic mechanism.
Specificity of SDP
Seizure effects on excitatory transmission in this model were remarkably specific. We demonstrate the reversal of stimulation-induced potentiation, whereas baseline EPSPs and other forms of plasticity, Ca2+-induced LTP, and both forms of LTD (Figs. 4D and 7), were not affected by ictal activity. Types of plasticity spared included those with postulated postsynaptic loci of expression [e.g., Ca2+-induced LTP (Turner et al 1982) or LFS-induced LTD (Rammes et al. 2003)] (Fig. 7) but also with a likely presynaptic locus of expression (e.g., DHPG-mediated LTD) (Anwyl 1999). In addition, we did not observe LTP-like effects by seizures seen in the disinhibition model (Ben Ari and Represa 1990; Schneiderman et al. 1994) nor a dual expression of both LTP and LTD effects observed for example in the potassium model of in vitro epilepsy (Contzen and Witte 1994) or in several protocols of electrical stimulation (Barr et al. 1995; Bashir and Collingridge 1994; Harrison and Alger 1993; Huerta and Lisman 1995; O’Dell and Kandel 1994). The lack of some forms of plasticity—such as LTP-like effects seen in the disinhibited slice during interictal-type of epileptiform activity from the CA3 subfield (Ben Ari and Represa 1990; Schneiderman et al. 1994)—can be explained by the specific experimental conditions applied in this study: the isolation of the CA1 region and application of high concentrations of GABAA antagonists (Bic: 50–100 µM, PTX 100 µM) precluded CA3-derived interictal events and led to a state of exclusive ictal activity as shown previously (Karnup and Stelzer 2001). Similarly, the (pharmacological) conditions for bi-directional plasticity contingent on the phase angle of an underlying theta wave (Huerta and Lisman 1995) were absent in this study. In analogy to TBS, which induced LTP at low-intensity stimulation but reversed LTP when applied at 10 times higher intensity (Barr et al. 1995), it might be inferred that ictal activity was consistently strong enough to reverse but not generate LTP. Taken together, ictal events in this study—despite their complex composition of three distinct burst components and wide range of firing frequencies (from 100 to 3 Hz) (Traub et al. 1996)—were extremely consistent in their exclusive action of reversing stimulation-induced LTP.
Our data show that LTP-inducing tetanic stimulation had increased the frequency of ictal events and thus the overall duration of ictal activity by 46% on average (Fig. 3Ac). Was the higher volume of seizure activity responsible for the specific effects on EPSPs after tetanization? SDP in the presence of PTX (Fig. 5) indicates that this was not the case. During PTX, the frequency of events and thus the overall duration of ictal activity was even lower than the seizure duration during controls and other forms of plasticity that were not affected by seizures (compare Figs. 5, Aa and D, and 7, Aa and Ba). No differences were seen between Bic- and PTX-induced SDP as a function of the nth ictal event (Fig. 5). Thus the lower frequency of ictal events per se was unlikely responsible for the lack of seizure effects on baseline excitation, Ca2+-induced LTP (Fig. 4D) and both forms of LTD during ictal activity (Fig. 7). But a higher ictal volume is only one consequence of the different network dynamics introduced by tetanic stimulation. In addition to its augmenting effect on seizure frequency, the LTP-inducing tetanic stimulation—absent during baseline transmission and during Ca2+-induced LTP and both forms of LTD—could have generated other cellular conditions that were responsible for the specific erasure of stimulation-induced LTP by seizures. An example is the critical role of synaptic activation of group I mGluR, possibly through tetanic stimulation, in the induction of SDP (Fig. 8). This notion is also not valid: when tetanic stimulation was applied prior to the induction of Ca2+-LTP or both forms of LTD, ictal events were equally ineffective in reversing these forms of plasticity (not shown). In addition, the fact that EPSPs returned to—and remained at—pretetanization baseline levels after completion of SDP is further evidence that the specific reversal of potentiated EPSPs after tetanic stimulation was not contingent on network dynamics introduced by LTP-inducing stimulation.
Depolarization promoted seizure-induced depotentiation
Results from two experimental protocols show that cell depolarization was a pivotal mechanism for the induction of SDP: current injection promoted or prevented SDP on a single-cell level (Figs. 12 and 13) and depotentiating pharmacological protocols were accompanied by tonic depolarization shifts (Fig. 10Ec; Table 1). The current-injection protocols demonstrate a postsynaptic induction mechanism. Correlation analysis (Fig. 10F) suggests that ictal-induced depotentiation was not an all-or-none but a graded event: the size of depotentiating steps was functionally linked to the amount of cell depolarization (Fig. 10F). These data can explain observations that seemingly identical ictal events triggered individual depotentiation steps of vastly variable sizes ranging from no depotentiation (e.g., in the presence of DHPG alone, Figs. 9 and 10F) to 50% depotentiation mediated by a single ictal event (see Fig. 2).
It is inferred that group I mGluR activation was responsible for the tonic depolarization (Mannaioni et al. 2001) seen in depotentiating pharmacological protocols. Group I mGluR-mediated depolarization can be traced to several conductance mechanisms, e.g., the impairment of (diverse) Ik and resulting membrane resistance increases or activation of cationic conductance increases (for review, see INTRODUCTION in Chuang et al. 2002). However, our data demonstrate that group I mGluR-mediated depolarization—and subsequently SDP—required the complete block of GABAA-mediated inhibition (Figs. 10, E and F, and 11). Previous reports have shown changes of the inhibitory efficacy by group I mGluR activation (Desai et al. 1994; Mannaioni et al. 2001; McBain et al. 1994; Miles and Poncer 1993). Our data point to a reverse interaction in that fast synaptic inhibition controlled DHPG-mediated depolarization in CA1 pyramidal cells. We demonstrate one particular functional consequence of the inhibitory control of group I mGluR in that seizure-induced reversal of LTP was prevented unless inhibition was completely blocked. Partial disinhibition did not suffice. The DHPG protocol of seizure activity exemplifies this notion. During DHPG, only minimal cell depolarization (<2 mV on average) was expressed and SDP was absent in the vast majority of recordings (Fig. 9). In these recordings, fast synaptic inhibition was at least partially blocked by the action of DHPG itself (Desai et al. 1994; Mannaioni et al. 2001). After LTP-inducing tetanic stimulation, an additional component of disinhibition was added (Desai et al. 1994; Stelzer et al. 1987, 1994). Even the addition of lower, submaximal concentrations of Bic (1–5 µM) to a DHPG-containing solution did not reveal significant cell depolarization or SDP (data not shown). In contrast, only saturating concentrations of Bic (50–100 µM)—added to the DHPG-containing solution—led to significant depolarization and most expedient SDP (Fig. 10, Table 1). Depotentiating efficacies during various pharmacological protocols (DHPG+Bic >> Bic alone >> DHPG alone; Table 1) were a reflection of the expression of the cellular conditions leading to cell depolarization, i.e., complete disinhibition and—at least partial—group I mGluR stimulation. In the presence of Bic alone, complete disinhibition was paired with (synaptic) activation of mGluR1 (Fig. 8, Table 1); this was arguably weaker than the pharmacological stimulation by DHPG. The largest depolarization and the highest depotentiating efficacy (Fig. 10, Table 1) was obtained under saturating pharmacological conditions in the presence of DHPG plus Bic.
The question is whether such stringent conditions, notably the complete block of inhibition, were to occur in vivo. Two main scenarios are feasible. First, depotentiating cellular conditions need to be implemented in very small parts of brain tissue: we have previously shown that spontaneous ictal events could be generated in ca. 1 mm3 CA1 tissue (80% of the CA1 slice) (Karnup and Stelzer 2001). SDP can occur in far smaller neuronal units, in fact in a single neuron (Figs. 12 and 13). Second, far more permissible circumstances for cell depolarization are conceivable. Tonic depolarization protocols in this study were applied out of experimental necessity. Because seizure activity in most recordings occurred infrequently and randomly in time (except in very few recordings; see Fig. 10), tonic depolarization protocols best ensured the presence of depolarization at the time of ictal onset. But it is possible that transient depolarization shifts coinciding with ictal onset may suffice to reverse LTP by seizures.
We cannot conclusively comment on how tonic (or possibly phasic) cell depolarization preceding ictal events promoted SDP. Ictal firing—for several seconds at various frequencies from 3 to 100 Hz on top of a large depolarization envelope (Traub and Jefferys 1994)—may have led to the same phasic activation of voltage-gated conductances (e.g., NMDAR) regardless of the presence or absence of underlying tonic depolarization. NMDARs were also activated by LTP-inducing/reversing tetanic stimulation (Barr et al. 1995; Bliss and Collingridge 1993) and possibly potentiated by the stimulation of mGluR5 (Fitzjohn et al. 1996; Mannaioni et al. 2001). But a role of NMDAR in SDP remains to be established. The simple approach of using antagonists is precluded by the fact that NMDAR antagonists (even at low concentrations) blocked SDP-triggering ictal activity (Karnup and Stelzer 2001). A more likely contribution of depolarization prior to ictal activity may be found in activity before the ictal event especially if the preictal depolarization shift was above firing threshold (see Fig. 11Bb). Priming effects by voltage-gated conductances are conceivable. But exact mechanisms remain to be elucidated.
SDP as a model of seizure-induced amnesia
In contrast to stimulation-induced forms of depotentiation that are generally discussed within the context of a physiological mechanism, akin to "forgetting," seizure-induced depotentiation represents undoubtedly a cellular model of a pathological form of "memory loss." A number of properties of SDP conforming with clinical observations—its clear distinction from the transient, general postictal depression of excitation, its specificity, renewed LTP after partial or complete depotentiation—point to a useful model of seizure-induced amnesia. LTP, however, is not equivalent to learning as LTP-inducing stimulation does not create but rather eliminates the particular spatial pattern of synaptic weights of memory engrams (Brun et al. 2001). The LTP/memory analogy presumes that memory processes and LTP share the same cellular mechanisms. In this context, both the reversal and the creation of plasticity by seizures (Ben Ari and Represa 1990; Contzen and Witte 1994; Schneiderman et al. 1994) represent forms of interference with memory processes.
Several questions emerge from our findings that SDP could be implemented in a single neuron (Figs. 12 and 13)—possibly even at subsets of synapses in which potential gradients were maintained (Fig. 14)—but also in neuronal populations (during the pharmacological protocols; Figs. 1–6 and 10). Considering that information is stored in distributed patterns of synaptic weights in the hippocampal formation (Brun et al. 2001), how did depotentiation in a single cell influence or even erase the particular spatial pattern of synaptic weights created by tetanic stimulation? In contrast to single-cell SDP, SDP under pharmacological conditions was more likely to engage populations of neurons. How small were independently depotentiated subpopulations and what determined their size? Assuming that the stability of the newly consolidated (potentiated) state was preserved by cross-talk between neurons, how did SDP interfere with the binding of neuronal assemblies? It is conceivable that the elucidation of the mechanisms underlying selective depotentiation independently in individual cells or small subpopulations may also bear relevance for the physiological erasure of information stored in the hippocampal network.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Stelzer, Dept. of Physiology and Pharmacology, State University of New York, 450 Clarkson Ave., Box 29, Brooklyn, New York, 11203 (E-mail: armin.stelzer{at}downstate.edu)
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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