Modest Increase in Extracellular Potassium Unmasks Effect of Recurrent Mossy Fiber Growth

Jeremy L. Hardison, Maxine M. Okazaki, and J. Victor Nadler

Department of Pharmacology and Cancer Biology and Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hardison, Jeremy L., Maxine M. Okazaki, and J. Victor Nadler. Modest Increase in Extracellular Potassium Unmasks Effect of Recurrent Mossy Fiber Growth. J. Neurophysiol. 84: 2380-2389, 2000. The recurrent mossy fiber pathway of the dentate gyrus expands dramatically in many persons with temporal lobe epilepsy. The new connections among granule cells provide a novel mechanism of synchronization that could enhance the participation of these cells in seizures. Despite the presence of robust recurrent mossy fiber growth, orthodromic or antidromic activation of granule cells usually does not evoke repetitive discharge. This study tested the ability of modestly elevated [K+]o, reduced GABAA receptor-mediated inhibition and frequency facilitation to unmask the effect of recurrent excitation. Transverse slices of the caudal hippocampal formation were prepared from pilocarpine-treated rats that either had or had not developed status epilepticus with subsequent recurrent mossy fiber growth. During superfusion with standard medium (3.5 mM K+), antidromic stimulation of the mossy fibers evoked epileptiform activity in 14% of slices with recurrent mossy fiber growth. This value increased to ~50% when [K+]o was raised to either 4.75 or 6 mM. Addition of bicuculline (3 or 30 µM) to the superfusion medium did not enhance the probability of evoking epileptiform activity but did increase the magnitude of epileptiform discharge if such activity was already present. (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (1 µM), which selectively activates type II metabotropic glutamate receptors present on mossy fiber terminals, strongly depressed epileptiform responses. This result implies a critical role for the recurrent mossy fiber pathway. No enhancement of the epileptiform discharge occurred during repetitive antidromic stimulation at frequencies of 0.2, 1, or 10 Hz. In fact, antidromically evoked epileptiform activity became progressively attenuated during a 10-Hz train. Antidromic stimulation of the mossy fibers never evoked epileptiform activity in slices from control rats under any condition tested. These results indicate that even modest changes in [K+]o dramatically affect granule cell epileptiform activity supported by the recurrent mossy fiber pathway. A small increase in [K+]o reduces the amount of recurrent mossy fiber growth required to synchronize granule cell discharge. Block of GABAA receptor-mediated inhibition is less efficacious and frequency facilitation may not be a significant factor.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The dentate granule cells of the hippocampus are believed to function as a buffer against seizure propagation through the limbic circuit (Collins et al. 1983; Lothman et al. 1992; Stringer et al. 1989). Studies of animal models of epilepsy suggest that seizures that originate from or invade the entorhinal cortex normally reach the hippocampus only if they are strong enough to activate the dentate granule cells synchronously (Stringer and Lothman 1992). These cells resist seizure propagation for several reasons, including a highly negative resting membrane potential, strong tonic inhibition from GABA interneurons (Freund and Buzsáki 1996; Otis et al. 1991), low Ca2+ conductance (Fricke and Prince 1984), and lack of a synaptic mechanism for synchronization of cell discharge. With respect to the latter point, spontaneous cellular bursts can be induced by increasing the K+ concentration or reducing the Ca2+ concentration in the medium bathing a hippocampal slice, but synchronized granule cell activity appears only under extreme conditions (Pan and Stringer 1996, 1997; Patrylo et al. 1994; Schweitzer and Williamson 1995; Schweitzer et al. 1992). Granule cell synchrony is difficult to achieve, in part, because the few synaptic connections among these cells present in normal brain (Molnár and Nadler 1999; Okazaki et al. 1999) are insufficient to support recurrent synchronous discharge. However, in many persons with temporal lobe epilepsy the recurrent mossy fiber pathway of the dentate gyrus expands dramatically (Babb et al. 1991; Franck et al. 1995; Represa et al. 1989; Sutula et al. 1989). These new connections among granule cells could reduce their threshold for synchronization, thus enhancing the participation of these cells in seizures.

Despite formation of new recurrent excitatory connections, granule cells still remain difficult to recruit into epileptiform activity. Stimulation of the perforant path in vivo does not usually evoke reverberating excitation (Buckmaster and Dudek 1997). Similarly, responses of the granule cell population usually remain unaltered during low-frequency stimulation in hippocampal slices superfused with standard artificial cerebrospinal fluid (ACSF). For example, antidromic stimulation of the mossy fibers seldom evokes extracellularly recorded granule cell activity beyond the antidromic population spike (Cronin et al. 1992; Patrylo and Dudek 1998; Tauck and Nadler 1985). Under what circumstances then will these novel connections mediate synchronous activity? Previous research suggested that several factors present during the period just before seizure initiation may combine to strengthen recurrent excitatory circuitry and enhance its ability to support epileptiform activity. Among the most obvious candidate factors are increased [K+]o (Patrylo and Dudek 1998; Traub and Dingledine 1990) and reduced GABA-mediated inhibition (Cronin et al. 1992; Miles et al. 1984; Patrylo and Dudek 1998). With respect to the recurrent mossy fiber pathway, use-dependent forms of synaptic plasticity may also play an important role. Mossy fiber synapses on CA3 pyramidal cells exhibit marked frequency facilitation. Increasing the stimulus frequency from 0.0125 to 0.33 Hz increases the size of the excitatory postsynaptic current (EPSC) by more than fivefold (Salin et al. 1996). The range of presynaptic activity that provokes the greatest facilitation (0.1-1 Hz) corresponds to the normal firing frequencies of granule cells in vivo (Jung and McNaughton 1993). If mossy fiber-granule cell synapses also exhibit this form of plasticity, then the recurrent pathway might support synchronous granule cell discharge preferentially when driven at particular frequencies or in particular patterns. Thus we tested, alone and in combination, the ability of modestly elevated [K+]o, reduced synaptic inhibition, and frequency facilitation to unmask the effect of recurrent mossy fiber circuitry.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

Pilocarpine-induced status epilepticus

Adult male Sprague-Dawley rats (175-200 g; Zivic-Miller Laboratories, Allison Park, PA) were treated with pilocarpine hydrochloride (335-365 mg/kg ip) 30 min after receiving 2 mg/kg ip each of scopolamine methyl bromide and terbutaline hemisulfate. Status epilepticus, defined as a continuous limbic motor seizure of stage 2 or higher (Racine 1972), usually began within 90 min and was terminated 3.5 h after onset with a single injection of phenobarbital sodium (50 mg/kg ip). No systematic behavioral monitoring was performed after recovery from status epilepticus. However, all rats that develop status epilepticus after administration of pilocarpine exhibit chronic spontaneous limbic motor seizures after a latent period of 1-3 wk (Lemos and Cavalheiro 1996; Mello et al. 1993).

Some pilocarpine-treated rats exhibited only a few brief seizures that never progressed to status epilepticus. Previous work demonstrated the absence of both neuronal degeneration and supragranular mossy fiber growth in these animals (Okazaki et al. 1999). They were therefore used as controls.

Preparation and incubation of hippocampal slices

Animals were studied >= 10 wk after status epilepticus because previous studies suggested that the recurrent mossy fiber pathway required this period of time to become fully mature (Okazaki et al. 1995). The rat was decapitated under ether anesthesia, the brain was removed, and 400-µm-thick transverse slices were cut from the caudal third of the hippocampal formation with a vibratome. Slices used for electrophysiological recording corresponded to horizontal plates 98-100 of Paxinos and Watson (1986). These slices were trimmed and transferred to a beaker of standard ACSF, which contained (in mM) 122 NaCl, 25 NaHCO3, 3.1 KCl, 1.8 CaCl2, 1.2 MgSO4, 0.4 KH2PO4, and 10 D-glucose, pH 7.4. They were continuously gassed at room temperature with 95% O2-5% CO2.

Beginning 1.5 h after preparation, a slice was transferred to a small experimental chamber, placed on a nylon net and submerged in ACSF equilibrated with 95% O2-5% CO2 at 35°C. The superfusion rate was 1 ml/min. A monopolar stimulating electrode fashioned from 25-µm-diam nichrome wire and insulated to the tip with polymerized polyvinyl resin (Formvar) was placed in stratum lucidum of area CA3b >100 µm from the opening of the dentate hilus. Then an extracellular recording electrode fashioned from borosilicate glass and filled with 0.15 M NaCl (2-8 MOmega ) was placed in the granule cell body layer of the dentate gyrus. The recording electrode was positioned at numerous locations in this layer until the location was found at which the antidromic population spike was of maximal amplitude. The stimulating electrode was then moved perpendicular to the pyramidal cell body layer until stimulation evoked an antidromic population spike of the greatest possible amplitude. The final optimization of antidromic spike amplitude was achieved by adjusting both electrodes along the z axis.

Constant-current stimuli (rectangular pulses, 100-µs duration) were varied from 100 to 800 µA at 100-µA intervals to determine the smallest current that evoked a maximal antidromic response. This stimulus current was used for the rest of the experiment. After allowing 1 h for the response to stabilize, a train of 10 pulses was presented at a frequency of 0.2 Hz and then again at a frequency of 1 Hz. Then 10 trains of 10 pulses each were presented at a frequency of 10 Hz with a 20-s intertrain interval. Responses were filtered below 2 kHz, digitized at 10 kHz, and stored to disk with use of a Digidata board and Axoscope (Axon Instruments, Foster City, CA) running on a Micron P-100 microcomputer.

The superfusion medium was then modified, and 45 min later the stimulation protocol was carried out as before. In some experiments, a second change of medium and third round of stimulation were performed.

Data analysis

Antidromically evoked responses were inspected visually for the presence of epileptiform activity. This judgment was based on the presence or absence of any activity on the trace after the antidromic population spike. The magnitude of antidromically evoked epileptiform activity was quantitated by measurement of the coastline index (Dingledine et al. 1986). For this purpose, the Axoscope files were transferred to Microsoft Excel and the Pythagorean theorem was used to determine the change in response between successive data points. These changes were then summed for the 30-ms period that began at the end of the antidromic population spike. Unless stated otherwise, quantitative analyses were performed on recordings made at a stimulus frequency of 0.2 Hz, because all 10 responses recorded during such trains were practically identical. The coastline index was determined for each response, and indices for the 10 responses in the train were averaged. It should be noted that the presence or absence of epileptiform activity was determined only from visual inspection of the traces and not from the value of the coastline index. The coastline index is not useful for this purpose because differences in baseline noise could alter the coastline index by as much as brief, but clearly recognizable, epileptiform activity.

The percentage effect of (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) on antidromically evoked epileptiform activity was calculated by subtracting the coastline index after addition of DCG-IV to the superfusion medium from the coastline index before addition of DCG-IV and dividing this value by the difference of the coastline index before the addition of DCG-IV and the expected baseline. The expected baseline was the value of the coastline index in the absence of epileptiform activity. It was calculated by averaging the coastline indices obtained from all slices in the status epilepticus group that had no visible epileptiform response during recording in standard ACSF. This calculation yielded a value of 2.54.

Timm histochemistry

After the last set of recordings, the slice was immersed in a solution of 0.1% (wt/vol) Na2S, 0.1 M sodium phosphate buffer, pH 7.3, for 90 min. Then it was fixed in phosphate-buffered 10% formalin/0.9% (wt/vol) NaCl at 4°C for 1-2 days. After being embedded in albumin-gelatin, the slice was cut into 30-µm-thick sections with a Vibratome. Slide mounted sections were stained for the presence of heavy metals as described by Danscher (1981) and lightly counterstained with cresyl violet. The density of Timm staining in the supragranular zone of the dentate gyrus was estimated on an ordinal scale of 0-3 as previously described (Okazaki et al. 1995; Tauck and Nadler 1985). Sections were scored without knowledge of how the animal had been treated or of the electrophysiological results. In slices from control rats, scattered clusters of mossy fiber-like Timm stain were usually present in the supragranular zone (Molnár and Nadler 1999; Okazaki et al. 1999), qualifying them for a Timm score of 1. Some slices from the status epilepticus group were also assigned a score of 1. In these instances, there were more clusters of supragranular stain than were present in controls, but not enough to qualify for a score of 2.

Materials

DCG-IV was purchased from Tocris Cookson (Bristol, UK) and bicuculline methiodide was from Research Biochemicals (Natick, MA). Phenobarbital sodium, pilocarpine hydrochloride, (-)scopolamine methyl bromide, and terbutaline hemisulfate were obtained from Sigma Chemical (St. Louis, MO).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Control group

Antidromic stimulation of the mossy fibers did not evoke epileptiform activity in slices from control rats under any condition tested (15 slices from 12 animals; Table 1). Conditions tested included 4.75 mM [K+]o followed by 4.75 mM [K+]o + 3 µM bicuculline (n = 2), 6 mM [K+]o (n = 1), 3 µM bicuculline (n = 4), and 6 mM [K+]o + 30 µM bicuculline (n = 8). The coastline index measured under the most extreme condition tested, 6 mM [K+]o + 30 µM bicuculline, was not significantly different from the coastline index measured when the same slice was superfused with standard ACSF (98 ± 4%, mean ± SE). No epileptiform activity emerged when the stimulus frequency was increased from 0.2 to 1 or 10 Hz.


                              
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Table 1. Antidromic stimulation of the mossy fibers evoked epileptiform activity only in slices from the status epilepticus group

Status epilepticus group

EFFECTS OF INCREASING [K+]O. In slices from rats that had developed status epilepticus, antidromic stimulation of the mossy fibers during superfusion with standard ACSF (3.5 mM K+) usually evoked only a single population spike (Fig. 1, Table 1). We observed epileptiform activity in only 14% of these slices.



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Fig. 1. Effects of elevated [K+]o and bicuculline (Bic) on field potentials evoked by antidromic stimulation of the mossy fibers. *, antidromic population spike. A: increasing [K+]o to 4.75 mM did not cause the appearance of antidromically evoked epileptiform activity in this slice. The further addition of bicuculline also had no effect. B: in a slice from a different rat, antidromic stimulation evoked poorly-synchronized epileptiform activity when [K+]o was increased to 4.75 mM. The further addition of bicuculline increased the magnitude of the response without altering the waveform. C: in this slice, antidromic stimulation evoked multiple population spikes when [K+]o was increased to 6 mM. Again, the further addition of bicuculline increased the magnitude (peak amplitude and duration) of the response.

In contrast, when [K+]o was raised to 4.75 or 6 mM antidromic mossy fiber stimulation evoked epileptiform discharge in 48% of the slices. After the change to 4.75 mM [K+]o, the additional granule cell activity that followed the antidromic population spike was typically poorly synchronized (Fig. 1B). Occasionally, more synchronized discharges in the form of repetitive population spikes were evident. Well-formed repetitive population spikes were more frequently observed when [K+]o was increased to 6 mM (Fig. 1C). Antidromic stimulation evoked epileptiform activity in 45% of the slices exposed to 4.75 mM K+ and 54% of the slices exposed to 6 mM K+ (Table 2). In 10 of 22 experiments where epileptiform activity was not observed during superfusion with standard ACSF, epileptiform activity appeared when [K+]o was increased. These results were consistent across all stimulus frequencies tested. Coastline indices for slices first superfused with standard ACSF and then with elevated K+ showed significant effects of both K+ concentrations (Fig. 2; P < 0.02 for both 4.75 and 6 mM K+, Wilcoxon signed ranks test).


                              
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Table 2. Elevated K+, but not bicuculline, increased the probability of evoking epileptiform activity



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Fig. 2. The magnitude of antidromically evoked epileptiform activity was greatest when [K+]o was increased and GABAA receptor-mediated inhibition was reduced by addition of bicuculline (Bic) to the superfusion medium. There was no consistent difference between additions of 3 or 30 µM bicuculline. Exposure of hippocampal slices from control rats (Con) to 6 mM K+ and 30 µM bicuculline failed to change the coastline index, and antidromic stimulation never evoked epileptiform activity.

EFFECTS OF BICUCULLINE. To test the hypothesis that block of GABA inhibition alone could unmask the effect of recurrent excitation in the dentate gyrus, slices were superfused with the GABAA receptor antagonist bicuculline. Two concentrations were used, 3 and 30 µM. At a concentration of 3 µM, bicuculline reduces monosynaptic inhibition onto dentate granule cells by ~40%, whereas 30 µM bicuculline abolishes the response (P. Molnár and J. V. Nadler, unpublished observations). However, there was no consistent difference between additions of 3 and 30 µM bicuculline in the present study.

Addition of bicuculline to standard ACSF never caused antidromic stimulation to evoke epileptiform activity (n = 8; Table 2). Similar results were obtained when the slice was first exposed to 4.75 or 6 mM K+ provided that epileptiform activity had not been observed in the presence of elevated K+ alone (n = 3; Fig. 1A, Table 2).

If epileptiform activity was already present, addition of bicuculline to the superfusion medium increased the amplitude and duration of the response without changing the waveform (Fig. 1, B and C). Similar effects were observed with [K+]o concentrations of 3.5 (n = 2), 4.75 (n = 3), and 6 (n = 3) mM. These effects were reflected in a substantial enhancement of the coastline index (Fig. 2; P < 0.02, Wilcoxon signed ranks test).

In some experiments, bicuculline was added to the superfusion medium at the same time [K+]o was increased from 3.5 to either 4.75 or 6 mM. The proportion of experiments in which such treatment caused antidromic stimulation to evoke epileptiform activity was no greater than that obtained by increasing [K+]o alone (Table 2). However, the elevation of [K+]o with addition of bicuculline produced the greatest average increase in coastline index of any treatment studied (Fig. 2).

OCCURRENCE OF EPILEPTIFORM ACTIVITY DID NOT CORRELATE WITH ANTIDROMIC SPIKE AMPLITUDE. Antidromic stimulation of the mossy fibers evoked epileptiform activity in at most about half the slices tested, even under conditions expected to maximize excitability (6 mM [K+]o + 30 µM bicuculline). In several instances, epileptiform activity was evoked in one slice from an animal and not in another. One potential explanation for these findings is that different numbers of mossy fibers were activated by the stimulus. The more mossy fibers are activated, as indicated by increased amplitude of the antidromic population spike (Andersen et al. 1971), the more recurrent excitatory circuitry should be recruited. There was, however, no significant difference between the size of the antidromic spike in slices with (3.9 ± 0.3 mV; n = 30) and without (4.1 ± 0.3 mV; n = 33) evoked epileptiform activity. In fact, spike amplitude tended to decline when [K+]o was increased. Yet the smaller antidromic spike was frequently followed by epileptiform activity that was not present before.

CORRELATION WITH TIMM SCORE. The Timm score serves as a valid measure of recurrent mossy fiber growth (Okazaki et al. 1995). Antidromic stimulation evoked epileptiform activity in nine slices during superfusion with standard ACSF. All but one of these slices was assigned a Timm score of 3 (Fig. 3). The likelihood of evoking epileptiform discharge correlated significantly with the value of the Timm score (P < 0.05, chi 2 test). For each value of the Timm score, raising [K+]o increased the percentage of slices that responded with epileptiform activity. This effect was particularly dramatic in slices with the lowest density of supragranular mossy fibers, but there was still a trend toward higher probability of epileptiform activity with more robust mossy fiber growth. The magnitude of epileptiform activity, however, showed no relationship to the Timm score.



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Fig. 3. In standard ACSF (3.5 mM K+), antidromic stimulation evoked epileptiform activity only in the presence of robust recurrent mossy fiber growth. For each value of the Timm score, raising [K+]o increased the percentage of slices that responded with epileptiform activity. In slices with a Timm score of 1, epileptiform activity appeared only when [K+]o was increased.

DCG-IV REDUCES ANTIDROMICALLY EVOKED EPILEPTIFORM ACTIVITY. Mossy fiber terminals express a type II metabotropic glutamate receptor (Shigemoto et al. 1997). Activation of this receptor inhibits synaptic transmission (Kamiya et al. 1996). We tested the ability of DCG-IV, an agonist of type II metabotropic glutamate receptors (Conn and Pin 1997), to attenuate antidromically evoked epileptiform activity. The results of one such experiment are shown in Fig. 4, A-C. In this example, raising [K+]o to 4.75 mM with addition of 30 µM bicuculline converted the antidromically evoked response from a single population spike to multiple population spikes. Addition of a maximal concentration of DCG-IV (1 µM) to the superfusion medium strongly depressed this response. A similar result was obtained in each of six experiments (Fig. 4D). DCG-IV reduced the coastline index by 79 ± 8%.



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Fig. 4. (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) markedly depressed antidromically evoked epileptiform activity. It was assumed that inhibiting recurrent mossy fiber transmission would be effective or ineffective regardless of the condition under which epileptiform activity was observed. Therefore slices selected for this test had been exposed to several different modifications of the superfusion medium. Results from a representative slice are shown in A-C. A: during superfusion with standard ACSF, there was little evidence of antidromically evoked epileptiform activity. B: antidromic stimulation evoked multiple population spikes in the presence of 4.75 mM [K+]o and 30 µM bicuculline (Bic). C: addition of DCG-IV to the superfusion medium strongly attenuated this response. D: DCG-IV reduced the magnitude of the epileptiform response in 6 experiments. In the experiment with 30 µM bicuculline alone, some epilepiform activity was visible during superfusion with standard ACSF. The magnitude was quite small, and it did not appreciably increase the coastline index above the expected baseline value.

EFFECTS OF REPETITIVE STIMULATION. When 10 antidromic stimulus pulses were presented at a frequency of 0.2 or 1 Hz, all responses were essentially superimposable on one another. The coastline index did not change significantly during the train (Fig. 5D). This result was obtained at all values of [K+]o tested and in either the presence or absence of bicuculline. There was no consistent evidence of frequency facilitation, even when we excluded results from experiments in which antidromic stimulation failed to evoke epileptiform activity. For example, in the 11 positive experiments with 6 mM [K+]o and 30 µM bicuculline, the ratio of coastline indices for response 10 compared with response 1 was 1.07 ± 0.04 for stimulation at 0.2 Hz and 1.08 ± 0.07 for stimulation at 1 Hz.



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Fig. 5. Repetitive antidromic stimulation at frequencies of 0.2, 1, or 10 Hz did not increase the magnitude of antidromically evoked epileptiform activity. Repetitive stimulation at a frequency of 10 Hz reduced, and sometimes abolished, the epileptiform response. Results shown were obtained in the presence of 6 mM K+ and 30 µM bicuculline. Representative responses to the first, third, and eighth stimuli in a train of 10 pulses delivered at 10 Hz are shown in A-C. *, antidromic population spike. D: change in the coastline index during the train. Results from all experiments performed under these conditions are included, whether or not epileptiform activity was visible. To obtain coastline indices for stimulation at 10 Hz, corresponding responses from each of the 10 stimulus trains presented at 10 Hz were averaged. Values are means ± SE for 22 experiments. Effect of a 10-Hz stimulus train differed from that of a 0.2- or 1-Hz train at P < 0.001 (Newman-Keuls test after 2-way ANOVA [stimulus frequency × stimulus number] yielded P < 0.001).

The response to 10-Hz stimulus trains was quite different. Repetitive stimulation at this frequency reduced, and sometimes even abolished, the epileptiform response (Fig. 5, A-C). In the presence of 6 mM K+ and 30 µM bicuculline, the coastline index dropped by an average of 15% between responses to the first and fourth stimulus in the train and then remained constant throughout the rest of the train (Fig. 6D).



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Fig. 6. Repetitive antidromic stimulation at 10 Hz reduced the coastline index only when the 1st stimulus evoked epileptiform activity, and the reduction was greatest under conditions that produced epileptiform activity of the greatest magnitude. Values are means ± SE for the number of slices as follows: 4.75 mM K+ with epileptiform activity, n = 5, and without epileptiform activity, n = 6; 6 mM K+ with epileptiform activity, n = 7, and without epileptiform activity, n = 6; 6 mM K+ + 30 µM bicuculline (Bic) with epileptiform activity, n = 11, and without epileptiform activity, n = 11. Under all 3 conditions, the effect of repeated stimulation depended on the presence of epileptiform activity (P < 0.001, Newman-Keuls test after 2-way ANOVA [stimulus frequency × stimulus number] yielded P < 0.001).

The degree of inhibition during a 10-Hz stimulus train depended on [K+]o and whether GABAA receptors were blocked. Comparisons of experiments in which antidromic stimulation either did or did not evoke epileptiform activity suggested that response depression during the train reflected the initial magnitude of epileptiform activity. The coastline index changed little, if at all, during the train when epileptiform activity was absent, regardless of the extracellular K+ concentration or the presence of bicuculline (Fig. 6). In contrast, the coastline index declined significantly during the train whenever the first stimulus evoked epileptiform activity. The effect was greatest in experiments with 6 mM K+ and 30 µM bicuculline (~25%) and least in experiments with 4.75 mM K+ (~10%).

We recorded no evoked or spontaneous seizure-like events, such as those described by Patrylo and Dudek (1998), under any condition tested in this study.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results provide insights into the factors that regulate expression of recurrent excitation in the dentate gyrus of epileptic brain. They demonstrated that antidromic stimulation of the mossy fibers in standard ACSF evoked epileptiform granule cell discharge only in hippocampal slices from pilocarpine-treated rats with recurrent mossy fiber growth, increasing [K+]o from 3.5 to 4.75 or 6 mM increased the proportion of slices in which epileptiform discharge was evoked, increased [K+]o also reduced the extent of recurrent mossy fiber growth required to support epileptiform activity, depression or block of GABAA receptor-mediated inhibition only increased the magnitude of antidromically evoked epileptiform activity not the probability of evoking such activity, facilitation of epileptiform activity did not occur during antidromic stimulation at frequencies reported to enhance the EPSC at mossy fiber-CA3 pyramidal cell synapses, and repetitive stimulation at 10 Hz reduced, and sometimes abolished, the epileptiform response.

Recurrent mossy fiber growth as a prerequisite for antidromically evoked epileptiform activity

Pilocarpine-induced status epilepticus triggers in the dentate gyrus several pathologic sequelae that could facilitate epileptiform activity. Besides expansion of the recurrent mossy fiber pathway, some hilar GABA neurons degenerate (Obenaus et al. 1993) and possible disturbances of inhibitory synaptic transmission from other causes have also been described (Gibbs et al. 1997; Isokawa 1996). Any of these changes could, in principle, have contributed to antidromically evoked epileptiform activity. However, two lines of evidence suggest that expansion of the recurrent mossy fiber pathway played a key role. First, the occurrence of antidromically evoked epileptiform activity was clearly related to the presence and extent of recurrent mossy fiber growth. Antidromic stimulation never evoked epileptiform activity in slices from control rats. Furthermore in slices from the status epilepticus group, robust recurrent mossy fiber growth was essentially required to evoke epileptiform activity during superfusion with standard ACSF. These observations closely resemble results obtained with hippocampal slices from kainic acid-treated rats (Cronin et al. 1992; Patrylo and Dudek 1998; Tauck and Nadler 1985). Second, DCG-IV, an agonist of type II metabotropic glutamate receptors, markedly reduced the magnitude of antidromically evoked epileptiform activity. Within the rat hippocampal formation, type II receptors have a restricted distribution. Immunoreactivity is found only on the preterminal axons of the mossy fiber pathway and the medial perforant path (Shigemoto et al. 1997). The concentration of DCG-IV used in the present study (1 µM) is expected to depress mossy fiber transmission maximally (IC50 = 44 nM) (Kamiya et al. 1996) and has no detectable effect on type I or type III metabotropic glutamate receptors. We have confirmed with whole cell patch-clamp recordings that 1 µM DCG-IV inhibits transmission at mossy fiber synapses on dentate granule cells (P. Molnár and J. V. Nadler, unpublished observations), as it does at all other mossy fiber synapses studied to date (Doherty and Dingledine 1998; Kamiya et al. 1996; Maccaferri et al. 1998). DCG-IV can also activate N-methyl-D-aspartate receptors, but this action is significant only when it is applied at higher concentrations (EC50 = 144 µM) (Wilsch et al. 1994). Thus our data support the involvement of recurrent mossy fiber synapses in antidromically evoked epileptiform activity.

The only other excitatory input to the dentate granule cells reported to be activated by stimulation of the CA3 area is the disynaptic dentate associational pathway (CA3 pyramidal cells-hilar mossy cells-granule cells) (Scharfman 1994). Although the associational pathway may have contributed in some way to antidromically evoked epileptiform activity, several considerations suggest that its contribution, if any, was much less than that of the recurrent mossy fiber pathway. 1) Limbic status epilepticus kills the majority of hilar mossy cells (Buckmaster and Jongen-Rêlo 1999; Sloviter 1987). Nevertheless, epileptiform activity was observed only in slices from rats that had developed status epilepticus. 2) We could not evoke an EPSC in granule cells by uncaging glutamate within the dentate hilus in the presence of bicuculline (Molnár and Nadler 1999). This result suggests that most mossy cells either do not survive slice preparation under our conditions or are not connected to granule cells within the plane of our slices. 3) Associational terminals are not immunoreactive for type II metabotropic glutamate receptors (Shigemoto et al. 1997). 4) Associational terminals do express mGluR4, a type III metabotropic glutamate receptor (Shigemoto et al. 1997). However, L-AP4, an agonist of this receptor, does not significantly reduce the magnitude of antidromically evoked epileptiform activity (Okazaki and Nadler 1999).

Effect of modestly increasing [K+]o

Of the three variables examined in the present study, [K+]o was clearly of greatest significance with respect to unmasking the effect of recurrent excitation. Even a modest elevation of [K+]o (by 1.25 mM) more than tripled the probability of observing antidromically evoked epileptiform activity of dentate granule cells in slices with recurrent mossy fiber growth. Increases in [K+]o of this magnitude have been observed in response to low-frequency electrical stimulation, intense natural stimuli, and rhythmic neuronal activity in vivo (Krnjevic' et al. 1982; Walz and Hertz 1983). Thus the K+ concentrations we tested are within the physiological range. They were also below the concentrations achieved in the dentate gyrus during a seizure (Lux et al. 1986; Stringer and Lothman 1989). It is not clear, however, which stimuli raise [K+]o to 4.75 or 6 mM in the dentate gyrus of epileptic brain. Studies of this issue are needed.

Elevated [K+]o can itself precipitate epileptiform discharge (Korn et al. 1987; Rutecki et al. 1985; Traynelis and Dingledine 1988). In addition, elevated [K+]o facilitates epileptiform discharge in hippocampal slices prepared from animal models of epilepsy (Patrylo and Dudek 1998; Stringer and Lothman 1988). Patrylo and Dudek (1998) used hippocampal slices from kainic acid-treated rats to study the effect of >= 6 mM [K+]o in the presence of recurrent mossy fiber growth. They reported evidence of hyperexcitability to "hilar" stimulation, ranging from multiple granule cell population spikes to negative field potential shifts with superimposed spikes, in 82% of slices from kainic acid-treated rats that were superfused with 6 mM K+. These abnormal discharges were shown to depend on the activation of glutamate synapses. The incidence of hyperexcitability in their study was greater than that observed in the present study with hippocampal slices from pilocarpine-treated rats, despite comparable recurrent mossy fiber growth. Although we cannot rule out the possibility that kainic acid-induced status epilepticus and pilocarpine-induced status epilepticus differentially alter granule cell excitability, a more likely explanation lies in the different sites of stimulation. Stimulation of mossy fibers within the dentate hilus brings more granule cells to threshold than does stimulation in area CA3b, as evidenced by much larger antidromic population spikes. The more granule cells are activated, the more recurrent mossy fiber circuitry can potentially be recruited and the easier it is to detect unmasking by elevated [K+]o. However, hilar stimulation is more likely to involve other elements of dentate gyrus circuitry. Both excitatory mossy cells and inhibitory GABA neurons may be activated, either synaptically or by DC application. Hilar stimulation should also activate more effectively the sparse mossy fiber-granule cell synapses that are normally present in the rat dentate gyrus.

It should be noted that Timm histochemistry visualizes only mossy fiber boutons and provides no information about connectivity within the recurrent mossy fiber circuit. It is possible that antidromic stimulation failed to evoke much recurrent excitation in some of our experiments because key segments of the circuit were not present in that particular slice. This may account for a few instances in which epileptiform activity was evoked in one slice but not in another slice from the same rat tested under the same experimental conditions.

Kindling reduces the value of [K+]o at which stimulation of the Schaffer collateral-commissural projection evokes epileptiform bursting of CA1 pyramidal cells (Stringer and Lothman 1988). The concentration of extracellular K+ required depends critically on [Ca2+]o; the greatest incidence of epileptiform activity is observed with a combination of high K+ and low Ca2+ concentrations. Increased [K+]o and reduced [Ca2+]o also promote spontaneous cellular bursts in the normal dentate gyrus (Pan and Stringer 1997). The concentrations of these ions required to promote cellular bursting are more extreme than those used in the present study, and there is still no evidence of epileptiform activity in the field recording. Our results demonstrate that, similar to the effect of kindling on excitability in area CA1, pilocarpine-induced status epilepticus reduces the threshold ionic conditions for epileptiform activity in the dentate gyrus. Antidromic stimulation evoked epileptiform activity in the presence of 1.8 mM Ca2+ and 4.75 mM K+. The recurrent mossy fiber pathway provided the substrate for synchronization of the cellular bursts.

[K+]o regulates granule cell excitability in several ways. The membrane potential of these neurons varies linearly with [K+]o up to ~6 mM (Pan and Stringer 1997). Raising [K+]o from 3.5 to 4.75 or 6 mM is expected to depolarize dentate granule cells by 8 and 16 mV, respectively. In addition, increasing [K+]o can shift ECl to a more positive value, thus reducing the magnitude of GABAA receptor-mediated inhibition (Chamberlin and Dingledine 1988), depress K+-dependent hyperpolarizations produced by activation of postsynaptic receptors (e.g., GABAB and adenosine A1 receptors), reduce K+-mediated afterhyperpolarizations (Jensen et al. 1994), increase the frequency of spontaneous EPSPs (Traub and Dingledine 1990) and induce glial swelling, thus increasing extracellular resistance (Lux et al. 1986; Traynelis and Dingledine 1989; Walz 1987). Further studies are needed to assess the relative contributions of each factor in the presence and absence of recurrent excitatory circuitry.

Effect of blocking GABAA receptor-mediated synaptic inhibition

Blocking GABAA receptor-mediated inhibition never caused the emergence of antidromically-evoked epileptiform activity, although it did enhance the magnitude of such activity. Thus the effect of the recurrent mossy fiber pathway was not masked by such inhibition. This result appears at odds with the study of Patrylo and Dudek (1998), in which addition of 10 µM bicuculline to the superfusion medium increased the proportion of slices in which hilar stimulation evoked epileptiform activity from 27 to 84%. Again this discrepancy may be explained by the difference in stimulation sites. Stimulation within the dentate hilus would be expected to activate greater synaptic inhibition than stimulation in area CA3b, due both to greater driving of inhibitory neurons through mossy fiber connections and to direct activation of interneurons by the stimulus current. The recurrent mossy fiber pathway may be more effectively regulated by GABA inhibition under these conditions; thus blockade of GABA inhibition would have a more dramatic effect. Hilar stimulation may also activate more effectively the excitatory associational-commissural pathway, especially when GABA inhibition is reduced. Even in slices from previously untreated rats, hilar stimulation can sometimes evoke multiple population spikes in the presence of bicuculline (Fournier and Crepel 1984; Jackson and Scharfman 1996; Patrylo and Dudek 1998). No such response was observed in slices from the control group when we stimulated the mossy fibers outside the dentate hilus.

Our results suggest that GABA inhibition regulates the recurrent mossy fiber pathway less strongly than [K+]o. They do not, however, exclude the possibility that status epilepticus produced some deficit in GABA inhibition that allowed epileptiform activity to emerge when [K+]o was increased. The existence of such a deficit is largely hypothetical at this time. Studies of synaptic inhibition in the dentate gyrus of epileptic brain have yielded mixed results as to the direction of any change (Okazaki et al. 1999).

Effect of stimulus trains

If mossy fiber synapses on dentate granule cells operate similarly to mossy fiber synapses on CA3 pyramidal cells (Salin et al. 1996), one would expect to observe marked frequency facilitation during stimulation at rates of 0.1-1 Hz. This increase in synaptic strength should then manifest itself as facilitation of epileptiform activity evoked by similar stimulus trains. Our results did not confirm this expectation. No significant change in response magnitude occurred during stimulus trains delivered at a frequency of 0.2 or 1 Hz, and stimulation at a frequency of 10 Hz caused a progressive and profound loss of the orthodromic component of the response. Perhaps mossy fiber synapses on dentate granule cells differ from those on CA3 pyramidal cells in having little frequency facilitation. There is precedent in this pathway for regulation of presynaptically mediated plasticity by the postsynaptic cell; long-term potentiation, which is induced through a presynaptic change, occurs at mossy fiber synapses on CA3 pyramidal cells but not at mossy fiber synapses on CA3 interneurons (Maccaferri et al. 1998). With respect to both synaptic size (Okazaki et al. 1995) and unitary EPSC amplitude (Molnár and Nadler 1999), mossy fiber synapses on dentate granule cells are intermediate between mossy fiber synapses on CA3 pyramidal cells and interneurons. If these synapses do exhibit frequency facilitation, this process must have been masked by some inhibitory effect of repeated stimulation.

Further research is needed to explain the profound suppression of epileptiform discharge observed during stimulation at 10 Hz. The magnitude of this effect appeared to depend on the initial size of the discharge. One possibility is that glutamate released from the recurrent mossy fiber boutons inhibited subsequent glutamate release by activating type II metabotropic receptors. This mechanism is significant only at high rates of presynaptic activity; transport processes clear extracellular glutamate adequately during low frequency activity (Scanziani et al. 1997). Other possible contributory factors include buildup of extracellular adenosine, activation of Ca2+- dependent K+ conductance, and synaptic fatigue. Whatever the cause, our results suggest that transmission in the recurrent mossy fiber circuit fails at rates of activity similar to that occurring during the tonic phase of seizures.

Implications for seizure propagation through the dentate gyrus

Recurrent mossy fiber growth appears not to be required for pilocarpine-treated rats to become epileptic, because suppression of mossy fiber growth with cycloheximide does not prevent the development of spontaneous seizures (Longo and Mello 1997). This finding does not exclude a role for recurrent mossy fiber growth in epileptogenesis, however. Pilocarpine-induced status epilepticus severely damages many forebrain regions (Clifford et al. 1987; Okazaki et al. 1999; Turski et al. 1983), which may result in sprouting of residual excitatory pathways, loss of synaptic inhibition, and altered expression of many genes. Thus development of epilepsy in this model probably involves multiple changes in several regions of the brain. Removal of any single pro-epileptic consequence of the insult would not by itself be expected to prevent spontaneous seizures.

Our results reinforce the suggestion of Patrylo and Dudek (1998) that elevation of [K+]o increases seizure susceptibility in the dentate gyrus when a significant recurrent mossy fiber pathway is present. The dramatic expansion of this pathway could contribute to episodic spontaneous seizures in both pilocarpine-treated rats and humans with temporal lobe epilepsy by the following mechanism. During interictal periods, when [K+]o is at resting levels, the recurrent mossy fiber pathway is seldom activated and dentate granule cells behave normally. That is, they discharge asynchronously at low frequency. Seizures are presumably triggered by excessive discharge in the entorhinal cortex that is propagated to the dentate gyrus through the perforant path. Based on studies in other regions of the brain, [K+]o might rise to a level that would unmask recurrent excitation if the granule cells were driven by perforant path activity at a rate near the upper limit of normal (~1 Hz) (Jung and McNaughton 1993). However, the entorhinal cortex may discharge abnormally in the epileptic brain. In both human temporal lobe epilepsy and animal models of this condition, the entorhinal cortex suffers damage (Du et al. 1993, 1995), which is presumably followed by synaptic reorganization and perhaps other changes. Whatever the mechanism, entorhinal cortical discharge assumes a prolonged burst-like morphology (Scharfman et al. 1998). This abnormally strong excitatory input may not only serve as a more effective stimulus for granule cell activation but might easily raise [K+]o in the dentate gyrus by enough to facilitate reverberating granule cell excitation. Importantly, our results support a synergistic interaction between [K+]o and recurrent mossy fiber growth; the combination of a relatively small increase in the number of mossy fiber-granule cell synapses with a relatively small increase in [K+]o appears sufficient to facilitate the recruitment of granule cells into synchronous epileptiform activity. Once the granule cell population has been engaged, prolonged field bursts appear. The recurrent mossy fiber pathway becomes nonoperational during the high-frequency activity associated with these bursts and synchronization then depends entirely on non-synaptic mechanisms (Pan and Stringer 1996; Schweitzer and Williamson 1995; Schweitzer et al. 1992). The field bursts then propagate into and through the hippocampus. This hypothesis does not exclude the involvement of additional abnormalities that may be present in the dentate gyrus of epileptic brain, including weakened synaptic inhibition, reduced calcium buffering, or enhanced glutamate receptor function. To the extent that recurrent mossy fiber growth reduces seizure threshold, however, it seems rational to pursue an approach to pharmacotherapy of temporal lobe epilepsy based on depression of activity in that pathway.


    ACKNOWLEDGMENTS

We thank Dr. J. O. McNamara for timely assistance and K. Gorham for secretarial help.

This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-17771 (J. V. Nadler) and a Four Schools Research Scholarship (J. L. Hardison).


    FOOTNOTES

Address for reprint requests: J. V. Nadler, Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710 (E-mail: nadle002{at}acpub.duke.edu).

Received 27 March 2000; accepted in final form 14 July 2000.


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