The Journal of Neurophysiology Vol. 78 No. 4 October 1997, pp. 1860-1868
Copyright ©1997 by the American Physiological Society

Spontaneous Excitatory Currents and -Opioid Receptor Inhibition in Dentate Gyrus Are Increased in the Rat Pilocarpine Model of Temporal Lobe Epilepsy

Michele L. Simmons¹, Gregory W. Terman², and Charles Chavkin¹

¹ Department of Pharmacology and ² Department of Anesthesiology, University of Washington, Seattle, Washington 98195-7280

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Simmons, Michele L., Gregory W. Terman, and Charles Chavkin. Spontaneous excitatory currents and -opioid receptor inhibition in dentate gyrus are increased in the rat pilocarpine model of temporal lobe epilepsy. J. Neurophysiol. 78: 1860-1868, 1997. Temporal lobe epilepsy is associated with a characteristic pattern of synaptic reorganization in the hippocampal formation, consisting of neuronal loss and aberrant growth of mossy fiber collaterals into the dentate gyrus inner molecular layer. We have used the rat pilocarpine model of temporal lobe epilepsy to study the functional consequences of mossy fiber sprouting on excitatory activity and -opioid receptor-mediated inhibition. Using the whole cell voltage-clamp technique, we found that abnormal excitatory activity was evident in granule cells of the dentate gyrus from pilocarpine-treated rats. The frequency of spontaneous excitatory postsynaptic currents (EPSCs) was increased greatly in cells from tissue in which significant mossy fiber sprouting had developed. In the presence of bicuculline, giant spontaneous EPSCs, with large amplitudes and long durations, were seen only in association with mossy fiber sprouting. Giant EPSCs also could be evoked by low-intensity stimulation of the perforant path. Mossy fibers release not only excitatory amino acids, but also opioid peptides. -Opioid receptor-mediated inhibition in normal Sprague-Dawley rats was seen only in hippocampal sections from the ventral pole. In pilocarpine-treated rats, however, kappa receptor-mediated effects were seen in both ventral and more dorsal sections. Thus in this model of temporal lobe epilepsy, several types of abnormal excitatory activity were observed, thereby supporting the idea that mossy fiber sprouting leads to recurrent excitatory connections. At the same time, inhibition of excitatory activity by -opioid receptors was increased, perhaps representing an endogenous anticonvulsant mechanism.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Temporal lobe epilepsy (TLE) is one of the most common seizure disorders, and the histopathologic changes in the hippocampus associated with TLE have been well characterized (see Armstrong 1993). The cell death underlying TLE is limited largely to the pyramidal cells in CA3 and CA1, hilar mossy cells, and certain inhibitory interneurons (Sloviter 1987). In addition, sprouting of the mossy fiber axons is a hallmark neuropathological change evident in TLE (Armstrong 1993; Houser et al. 1990; Nadler et al. 1980). In the normal hippocampus, the granule cell axons (i.e., mossy fibers) form a major projection to the CA3 pyramidal cells, and they also have collaterals that synapse with neurons in the hilar region of the dentate gyrus. In TLE, newly sprouted mossy fibers cross the granule cell layer and terminate in the inner molecular layer and stratum granulosum of the dentate gyrus (Franck et al. 1995; Houser et al. 1990).

Although the histology of TLE within the hippocampal formation is well described, the physiological consequences of these changes are less clear. Sloviter (1992) proposed that the sprouted mossy fibers reinnervate inhibitory interneurons that become disconnected when the hilar mossy cells, which normally drive them, die. On the other hand, there is evidence that the mossy fiber terminals in the inner molecular layer form excitatory synapses with granule cell dendrites (Franck et al. 1995; Okazaki et al. 1995; Represa et al. 1993; Wuarin and Dudek 1996), which would promote, rather than inhibit, abnormal excitatory activity. Indeed, both possibilities may occur (Cronin et al. 1992).

Synaptic reorganization may affect not only excitatory amino acid circuitry, but also neuropeptide transmission. Mossy fibers are known to contain opioid peptides, enkephalins and dynorphins (Chavkin et al. 1985b), as well as other peptides including cholecystokinin (Gall 1988). The dynorphin peptides, which are the preferred endogenous ligands for the -opioid receptor, are of particular interest for their potential anticonvulsant actions (Tortella et al. 1986). Dynorphins, like other neuropeptides, are released in response to high-frequency stimulation within the hippocampus (Wagner et al. 1991), such as may occur during seizures. Dynorphins have been shown to inhibit excitatory transmission in the guinea pig hippocampus (Simmons et al. 1995; Wagner et al. 1992; Weisskopf et al. 1993). Further, we have found recently that -opioid agonists inhibit ventral but not dorsal hippocampal granule cell excitation and pilocarpine-induced seizures (Bausch et al. 1996). Thus these opioid peptides may serve to limit seizure frequency and duration in TLE.

The pilocarpine model of epilepsy is useful for studying the physiological consequences of mossy fiber sprouting (Mello et al. 1993). Rats treated with pilocarpine endure a period of status epilepticus and thereafter continue to have brief, spontaneous seizures indefinitely. Furthermore, the neuropathologic changes seen in human TLE patients, including cell death and mossy fiber sprouting, are reproduced by pilocarpine treatment. In the present experiments, pilocarpine-treated rats were studied 6-7 wk after treatment, when mossy fiber sprouting was well developed. The excitatory input to dentate granule cells was studied by recording both spontaneous and perforant path-evoked excitatory postsynaptic currents (EPSCs) with whole cell voltage clamp. To compare the electrophysiologic findings with the histological findings, mossy fiber sprouting was assessed by neo-Timm staining of the same slices from which voltage-clamp recordings were taken. In addition, using extracellular recording techniques, we tested whether -opioid effects were altered in the epileptic hippocampus as would be predicted by an expansion of a -opioid-sensitive pathway.

	METHODS

Abstract Introduction Methods Results Discussion References

Pilocarpine treatment

Male Sprague-Dawley rats (100-140 g; Bantin and Kingman, Bellevue, WA) were treated with either pilocarpine or saline according to previously published protocols (Mello et al. 1993). Rats were pretreated with 1 mg/kg methylscopolamine (0.1% wt/vol in saline, ip) to block peripheral muscarinic receptors. After 30 min, rats were injected with either 375 mg/kg pilocarpine (37.5% wt/vol in saline, ip) or an equivalent volume of saline. Seizure activity usually began within 20 min after pilocarpine injection. About 80% of the pilocarpine-treated rats reached status epilepticus, usually within 45 min after injection. Rats endured status epilepticus, i.e., continual generalized seizures, for 1 h, and then they were given 4 mg/kg diazepam (5 mg/ml ip). Additional doses of diazepam were administered 2 and 4 h after the first dose, if needed, to control seizures. Rats were offered rat chow soaked in a sucrose-Gatorade solution (~10% wt/vol) for 48 h after treatment to avoid dehydration. Animals were killed for electrophysiological experiments 6-7 wk after treatment. In the pilocarpine group, only animals that endured 1 h of status epilepticus were used for further experiments. The care and handling of the rats followed institutional guidelines.

Electrophysiology

Animals were anesthetized deeply with halothane and/or pentobarbital before decapitation, and hippocampal slices were prepared according to standard techniques. Briefly, transverse hippocampal slices (500 µm) were made using a Campden Vibroslicer. Slices then were submerged in the recording chamber, warmed to 34°C, and continuously perfused with oxygenated Krebs-bicarbonate buffer [which contained (in mM) 120 NaCl, 3.5 KCl, 1.3 MgCl₂, 2.5 CaCl₂, 1.25 NaH₂PO₄, 25.6 NaHCO₃, and 10 glucose]. Slices were allowed to equilibrate for ~1 h in the recording chamber before beginning experiments.

Spontaneous and evoked EPSCs were recorded in the whole cell mode with glass microelectrodes (2-4 M) filled with an intracellular solution that blocked postsynaptic K⁺ currents and maximized recording stability (modified from Langdon et al. 1993). This solution contained (in mM) 120 CsF, 10 CsCl, 10 ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, and 5 Mg-ATP; pH adjusted to 7.2-7.3 with CsOH; osmolarity 260-270 mOsm. Cells were held at 70 mV, except in a few experiments, as noted. EPSCs were evoked with a bipolar stimulating electrode (SNE-100; Kopf) placed in the outer two-thirds of the dentate molecular layer to stimulate perforant path fibers. Stimuli were delivered as 0.3-ms square waves 10-50 µA, at ~0.2 Hz. At various times during each experiment, current-voltage (I-V) relationships were measured to monitor the stability of the recording.

Cells were allowed to stabilize for ~10 min after establishing the whole cell configuration. EPSCs were digitized and stored with pClamp 6.0. Spontaneous activity was recorded in 60 consecutive 500-ms sweeps. Events were detected and analyzed using AxoGraph 3.0. The baseline was subtracted in each sweep. The threshold for the event detector was set at the amplitude of the noise, as judged by the amplitude of the positive deflections. Criteria for synaptic events were further defined in the presence of 10 µM bicuculline, 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 50 µM ±2-amino-5-phosphonovaleric acid (±APV), when fast synaptic currents were blocked. Specifically, "events" marked by the event detector under the above conditions had a characteristic low amplitude and shallow slope, and thus such "events" detected in the experimental sweeps were deleted from analysis. EPSC duration was measured as the width of the response at 50% of the peak amplitude. In the case of evoked EPSCs, three consecutive responses were averaged for display and analysis.

In the extracellular recording experiments, a distinction was made between ventral and more dorsal hippocampal slices. Brain slices (500 µm) were made starting from the ventral surface of the brain; the first three hippocampal slices from the temporal pole were collected as "ventral" slices; slices from the next 1 mm were discarded, and then the next three slices were collected from the middle third of the hippocampus and labeled "dorsal" for the comparisons made in this study. Extracellular population spikes were recorded with a glass microelectrode (3-5 M) filled with 3 M NaCl, placed in the granule cell body layer. As in the voltage-clamp studies, a bipolar electrode was placed in the dentate molecular layer and perforant path fibers were stimulated. Stimuli of 0.3 ms duration were applied at 1-min intervals at current intensities sufficient to evoke responses at half the maximal population spike amplitude. Amplitudes were measured as the difference between the first peak and the nadir of the population spike waveform. Baseline amplitude values were recorded for 5 min, then the selective kappa₁ agonist U69593 (1 µM; Research Biochemicals International, Natick, MA) was bath-applied. Amplitude values were again recorded 15-20 min after beginning drug application. The drug effect was reversed by washing out the drug for 40-60 min or by 15 min bath application of the selective kappa₁ antagonist norbinaltorphimine (nBNI; 100 nM; Research Biochemicals International).

Neo-Timm histochemistry

The neo-Timm staining protocol was modified from that of Holm and Geneser (1991). Hippocampal slices used for electrophysiological experiments were removed from the recording chamber and placed in a sodium sulfide solution [0.1% wt/vol in 150 mM sodium phosphate buffer (PB)] for 30-40 min at room temperature. Slices then were placed in 4% paraformaldehyde-30% sucrose in 100 mM PB at 4°C for 24 h and then stored in 30% sucrose in 100 mM PB at 4°C for 1 wk. Subsequently, slices were cut into 40-µm sections on a freezing sliding stage microtome and mounted onto gelatin-coated slides. At least 24 h after mounting, slides were postfixed in ethanol, rehydrated, and dipped in 0.5% gelatin.

The Timm staining solution was prepared freshly as follows, per 100 ml stain: 2.55 g citric acid, 2.35 g Na citrate, 0.85 g hydroquinone, 0.11 g silver lactate, and 60 ml 50% gum arabic. Hydroquinone and silver lactate were dissolved in water and added to the Timm staining solution while protected from light. The slides were placed in the Timm stain and allowed to react for 60-80 min, always protected from light. The reaction was terminated by washing the slides in water continuously for 30 min. Slides were counterstained with Neutral red and coverslipped with Permount.

Statistics

Most data were statistically analyzed using analysis of variance with Newman-Keuls tests for appropriate post hoc comparisons. Comparisons of proportions was accomplished using Fisher Exact Test for nonparametric data. An  of 0.05 was used to determine statistical significance.

	RESULTS

Abstract Introduction Methods Results Discussion References

Spontaneous EPSCs

Spontaneous postsynaptic currents were recorded in granule cells from pilocarpine- and saline-treated rats 6-7 wk after treatment. Granule cells from both groups exhibited spontaneous inward currents when the cell was held at 70 mV (Fig. 1). To determine whether these currents were glutamate receptor-mediated excitatory currents carried by Na⁺ and Ca²⁺ or -aminobutyric acid (GABA)-mediated inhibitory currents carried by Cl, spontaneous activity also was recorded at a holding potential of 50 mV. The reversal potential for Cl was calculated to be near 70 mV, whereas Na⁺/Ca²⁺ currents should reverse near 0 mV. Spontaneous events from saline-treated rats were observed as outward currents at 50 mV (Fig. 1B), and therefore they were primarily GABAergic inhibitory postsynaptic currents (IPSCs). The very low frequency of spontaneous EPSCs in normal dentate granule cells is in agreement with previous studies (Staley and Mody 1991). Granule cells from pilocarpine-treated rats often exhibited both outward and inward currents at 50 mV (Fig. 1D). After pilocarpine treatment, spontaneous EPSCs, in addition to IPSCs, were evident in 10 out of 25 cells (40%) studied.

View larger version (29K): [in this window] [in a new window]   FIG. 1. Spontaneous excitatory postsynaptic currents (EPSCs) occur in granule cells from pilocarpine-treated rats. A: spontaneous activity recorded from a granule cell from a saline-treated rat held at 70 mV. All synaptic currents were inward. B: spontaneous activity recorded from a granule cell from a saline-treated rat held at 50 mV. All synaptic currents were reversed, indicating that they were GABAergic chloride currents. C: spontaneous activity recorded from a granule cell from a pilocarpine-treated rat, held at 70 mV. All synaptic currents were inward. D: spontaneous activity recorded from a granule cell from a pilocarpine-treated rat held at 50 mV. Some synaptic currents were reversed and some remained inward; *, traces with both inward and outward currents. Therefore, both EPSCs and inhibitory postsynaptic currents occurred in this recording. Scale bars indicate 100 pA, 100 ms; scales apply to all traces.

To confirm that some of the inward currents recorded at a holding potential of 70 mV were GABAergic IPSCs, spontaneous activity was recorded in the presence of bicuculline, a GABA_A receptor antagonist. In granule cells from saline-treated animals, nearly all spontaneous currents were abolished by 10 µM bicuculline (data not shown). In contrast, spontaneous inward currents occurred even in the presence of bicuculline in granule cells from pilocarpine-treated animals (Fig. 2). These results confirm that spontaneous currents in normal granule cells are usually GABAergic IPSCs and that after pilocarpine treatment, spontaneous EPSCs are also evident.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Small and large EPSCs occurred in granule cells from pilocarpine-treated rats in the presence of bicuculline. A: in this representative pilocarpine-treated granule cell, small EPSCs occurred in the presence of bicuculline (10 µM). B and C: in other granule cells from pilocarpine-treated rats, both small EPSCs and giant EPSCs occurred. Scale bars indicate 100 pA, 100 ms in all panels.

In cells from pilocarpine-treated rats, giant spontaneous EPSCs often were seen during bicuculline treatment (Fig. 2, B and C). The size and shape of the giant EPSCs were somewhat variable, but they were distinguished easily from the typical events by their longer duration and higher complexity (e.g., Fig. 2B). The giant EPSCs were usually >100 pA in amplitude (range 76-1711 pA), whereas the typical EPSCs were rarely >100 pA. Likewise, the giant EPSCs were usually >20 ms in duration (measured at 50% peak; range 12-176 ms), whereas the duration of the typical EPSCs was usually <10 ms. The giant EPSCs were of very low frequency, as less than five were usually seen in any 30-s recording period. Giant EPSCs were seen in 16 of 30 (53%) pilocarpine-treated rats. In slices from these 16 rats, at least one cell, but not necessarily every cell tested, exhibited a giant EPSC. Giant EPSCs were never observed in cells from saline-treated rats (n = 16 animals, 4-6 cells/animal).

View larger version (82K): [in this window] [in a new window]   FIG. 3. Photomicrographs of hippocampal slices after neo-Timm staining of mossy fibers. Two to three 40-µm sections per 500-µm slice were evaluated on a scale of 0-3, similar to the scale described by Tauck and Nadler (1985), and the scores for each 500-µm slice were averaged. Timm scores were based on staining specifically in the inner molecular layer and were given as follows: 0 if no Timm granules were seen or if only a few scattered granules were seen; 1 if more granules were present but did not form a distinct band; 2 if granules were sufficiently dense as to form a continuous band and the band was less dense than the staining in the hilus; 3 if granules formed a band that was equally as dense as the staining in the hilus. Timm scores were assigned without knowledge of the corresponding treatment group or electrophysiologic results. All images were taken from the genu of the dentate gyrus. m, molecular layer; g. granule cell layer; h, hilus. A: Timm group 0, with dense Timm staining in the hilus but no Timm granules in the inner molecular layer. B: Timm group 1, with scattered Timm staining in the inner molecular layer (). Note the Timm-stained fiber coursing through the granule cell layer. C: Timm group 2, with a continuous band of light Timm staining in the inner molecular layer (). D: Timm group 3, with a continuous band of dense Timm staining in the inner molecular layer (). Scale bar indicates 0.1 mm for all panels.

Unlike typical spontaneous EPSCs, the giant EPSCs were not seen in the absence of bicuculline. We tested whether partial disinhibition caused by the mu opioid receptor agonist[ D - A l a ² , N - M e - P h e ⁴ , G l y - o l ] - e n k e p h a l i n   ( D A M G O )   a l s ocould unmask giant EPSCs. Mu opioid receptors inhibit GABA release from a subpopulation of interneurons, thereby reducing, but not eliminating, GABA_A tone (Bausch and Chavkin 1997; Cohen et al. 1992). DAMGO (1 µM) was applied to granule cells from pilocarpine-treated rats, in the absence of bicuculline. DAMGO reduced the frequency of small spontaneous currents, presumably those currents that were IPSCs (Bausch and Chavkin 1997; Cohen et al. 1992). However, DAMGO did not unmask giant EPSCs (n = 8; data not shown). These results suggest that either the interneurons that express mu opioid receptors do not control these giant EPSCs or that partial disinhibition was not sufficient to unmask them.

We further tested whether the giant EPSCs were synaptic and glutamatergic or whether they were intrinsically generated. Because granule cells were clamped at a membrane potential of 70 mV, large intrinsic currents would have to be generated from a region of the cell that escaped the voltage clamp (e.g., distal dendrites). To test the source of the currents pharmacologically, the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor antagonist CNQX (10 µM) was applied to cells that exhibited giant spontaneous currents. In all cases (n = 5; data not shown), no EPSCs of any size were observed after CNQX application, confirming that both the giant currents and the typical EPSCs were indeed synaptic and glutamatergic. Moreover, all giant spontaneous EPSCs were blocked by application of 1 µM tetrodotoxin (n = 4; data not shown), indicating a requirement for action potentials in the afferent fibers releasing the excitatory amino acid.

We next asked whether the granule cell giant EPSCs could have been produced by increased excitability of normal excitatory afferents, i.e., perforant path and mossy cell terminals, or whether they depended on axonal sprouting. Slices from saline-treated rats were made acutely hyperexcitable with the K⁺ channel antagonist 4-aminopyridine (4-AP). In CA3, 5-10 µM 4-AP causes pyramidal cells to burst in an epileptiform manner (Rutecki et al. 1987). In our experiments in the dentate gyrus, however, 100 µM 4-AP (in addition to bicuculline) increased the baseline noise and the frequency of small spontaneous EPSCs, but it did not produce giant EPSCs (n = 6; data not shown). This result supports the idea that the giant EPSCs were not produced solely by increased excitability of the normal circuitry, but rather, the giant EPSCs required synaptic reorganization.

The degree of mossy fiber sprouting was assessed in each slice used for these electrophysiological experiments using the neo-Timm stain as a marker of mossy fibers (Fig. 3). Timm scores were given to each slice on a scale of 0 (no Timm granules in the inner molecular layer) to 3 (dense band of Timm staining in the inner molecular layer), as described by Tauck and Nadler (1985). Slices were assigned Timm scores without knowledge of treatment group or electrophysiologic results. All slices from saline-treated rats had Timm scores of 0 (n = 16). Slices from pilocarpine-treated rats had Timm scores of 1 (n = 4), 2 (n = 19), or 3 (n = 7). There was no overlap in the distribution of Timm scores between the two treatment groups, indicating that pilocarpine treatment always produced some degree of mossy fiber sprouting.

View larger version (15K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   FIG. 4. A and B: amplitude histograms for all typical spontaneous EPSCs from Timm group 0 (n = 298 events from 16 cells; left) and Timm group 2 (n = 1499 events from 19 cells; right). All cells in Timm group 0 were from saline-treated rats and all cells in Timm group 2 were from pilocarpine-treated rats. C: cumulative probability distributions for the data presented in the histograms. This graph demonstrates that more relatively large spontaneous EPSCs were recorded from cells in Timm group 2 compared with Timm group 0.

Data were grouped according to the Timm score for the slice from which the electrophysiological measures were derived. Amplitude histograms were constructed for typical spontaneous EPSCs recorded in the presence of 10 µM bicuculline from cells in Timm groups 0 and 2. Figure 4A shows that cells in Timm group 0 (saline controls) exhibited relatively few spontaneous EPSCs; the mean event frequency was 0.22 Hz. The mean event frequency was increased fourfold in Timm group 2, to 0.88 Hz (Fig. 4B). The cumulative probability graph for the typical EPSCs (Fig. 4C) demonstrates that there were more relatively large currents in Timm group 2 than in Timm group 0 (see also Table 1). The larger mean amplitude in Timm groups 2 and 3 compared with Timm group 0 (P < 0.05) could be the result of increased activity at synaptic sites more proximal to the soma (and therefore less attenuated), increased transmitter release from afferent terminals, and/or increased postsynaptic sensitivity to excitatory amino acids. The mean rise time for the events was not different between Timm groups (Table 1), which argues against the possibility that events in Timm groups 2 and 3 were more proximal in origin than events in Timm group 0. Nevertheless, the frequency and amplitude of small spontaneous EPSCs was associated with the degree of mossy fiber sprouting.

In Fig. 5A, the numbers of giant EPSCs recorded in each cell during three 30-s periods are plotted as a function of Timm score. This graph indicates that large-amplitude EPSCs were very rare at Timm scores of 1, and EPSCs reached maximal frequency at a Timm score of 2. The histogram in Fig. 5B illustrates the amplitude distribution of giant EPSCs, as defined previously, recorded from cells in Timm group 2. Table 1 summarizes the frequencies, amplitudes, and rise times for both typical and giant EPSCs grouped according to Timm score.

View larger version (15K): [in this window] [in a new window]   FIG. 5. A: number of giant spontaneous EPSCs per cell is plotted as a function of Timm score. Frequency of giant spontaneous EPSCs was increased greatly in cells from Timm groups 2 and 3 compared with those from Timm groups 0 and 1. Each point represents the number of giant spontaneous EPSCs observed in three 30-s recordings per cell vs. Timm score for the slice containing the recorded cell. B: amplitude histograms for all giant spontaneous EPSCs from Timm group 2.

Giant EPSCs were observed in a significantly greater proportion of cells in Timm group 2 (11 of 19 rats; 58%) and Timm group 3 (4 of 7 rats; 57%), compared with cells in Timm group 0 (0 of 16 rats). Among cells that exhibited giant EPSCs, cells in Timm group 3 did not exhibit significantly more frequent or larger giant EPSCs than cells in Timm group 2. Thus the degree of mossy fiber sprouting appeared to be correlated with the observed spontaneous excitatory activity and particularly the giant EPSC frequency, which was 0 in tissue with no sprouting. These results support the hypothesis that the spontaneous EPSCs are due to excitatory synaptic activity from sprouted mossy fibers.

Perforant path-evoked EPSCs

Granule cell synaptic responses evoked by perforant path stimulation also showed signs of increased excitatory activity in cells from pilocarpine-treated rats compared with saline-treated controls. In the absence of bicuculline, 13 of 23 (57%) synaptic currents evoked in cells from pilocarpine-treated rats (Fig. 6A) were similar in size and shape to currents evoked in cells from saline-treated rats (n = 7). Notably, a substantial proportion (10 of 23; 43%) of evoked responses in cells from pilocarpine-treated rats had multiple components suggestive of polysynaptic inputs (Fig. 6B), although only a few could be considered giant currents. In bicuculline, however, 22 of 25 (88%) EPSCs evoked in cells from pilocarpine-treated rats were giant currents resembling the spontaneous giant EPSCs. This proportion is significantly different (P < 0.05) from control, because no giant currents were evoked in cells from saline-treated rats (n = 18). Evoked giant EPSCs had variable morphologies, including currents with very large amplitudes and long durations (Fig. 6C) and large currents with multiple components (Fig. 6D). Thus the evoked giant EPSCs may result from polysynaptic transmission, initiated orthodromically by perforant path stimulation and perpetuated by recurrent mossy fiber collaterals. In both treatment groups, evoked responses were abolished by 10 µM CNQX and 50 µM ± APV (pilocarpine,n = 3; saline, n = 5; data not shown).

View larger version (9K): [in this window] [in a new window]   FIG. 6. Representative examples of perforant path-evoked EPSCs from pilocarpine-treated rats. A: this evoked EPSC recorded in the absence of bicuculline had a normal waveform. Stimulus was 40 µA. Scale bars indicate 50 pA, 20 ms. B: in a different granule cell, the evoked EPSC recorded in the absence of bicuculline was much greater in amplitude and duration than a typical EPSC from a normal granule cell, and the EPSC had multiple components suggestive of polysynaptic inputs. Stimulus was 25 µA. Scale bars indicate 100 pA, 20 ms. C: in a different granule cell, the evoked EPSC recorded in bicuculline had a very large amplitude and long duration with smooth rising and falling phases. Stimulus was 30 µA. Scale bars indicate 100 pA, 100 ms. D: in a different granule cell, the evoked EPSC recorded in bicuculline had a complex waveforms with multiple components. Stimulus was 30 µA. Scale bars indicate 100 pA, 50 ms. Stimulus artifacts are truncated it all traces.

-Opioid effects

The correlation between mossy fiber sprouting and the increased granule cell excitability evident in pilocarpine-treated animals suggests that mossy fiber collaterals mediate the enhanced excitability. A characteristic of mossy fiber collaterals that we previously reported using guinea pig hippocampal slices is a sensitivity to -opioid receptor-mediated presynaptic inhibition (Terman et al. 1994). Additionally, there is anatomic evidence for -opioid receptors on perforant path terminals in the ventral molecular layer of rats (McGinty et al. 1994). Granule cell responses to perforant path stimulation are likely to include a component attributable to mossy fiber collaterals, as suggested by the evoked EPSCs in the current study (Fig. 6) and our previous work (Terman et al. 1994). Therefore, perforant path-evoked responses could be inhibited by -opioids at multiple sites. We examined whether the enhanced granule cell excitability after pilocarpine treatment was sensitive to modulation by -opioids.

As previously seen in untreated rats (Bausch et al. 1996), in slices from control animals (6-7 wk after saline treatment), the selective kappa₁ agonist U69593 (1 µM) inhibited granule cell population spike amplitudes in ventral, but not in more dorsal hippocampal slices (Fig. 7). This inhibition was reversed by washout or by the kappa₁ opioid receptor antagonist nBNI (100 nM). In ventral slices from pilocarpine-treated rats, U69593 also reversibly inhibited population spike amplitudes to a similar degree as seen in control slices. However, in more dorsal hippocampal slices from pilocarpine-treated animals, population spike amplitudes also were inhibited reversibly by U69593 (Fig. 7). These results support the idea that the sprouted mossy fiber collaterals, which grow into the inner molecular layer at all dorso-ventral levels (Mello et al. 1993) express -opioid receptors. Because the mossy fiber collaterals also contain the endogenous -opioid peptide dynorphin, these results suggest that endogenous -opioids may provide an autoinhibitory regulation of excitability in temporal lobe epilepsy.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Reversible population spike inhibition by U69,593 in ventral hippocampal slices from pilocarpine- and saline-treated rats and more dorsal slices from pilocarpine-treated rats. In slices from saline-treated animals, U69,593 (1 µM) inhibited granule cell population spike amplitudes in ventral but not more dorsal slices. This inhibition was reversed by washout (n = 2) or norbinaltorphimine (nBNI; n = 2). U69,593 inhibited spike amplitudes in both dorsal and ventral slices from pilocarpine-treated rats. This inhibition also was reversed by washout (dorsal, n = 8; ventral, n = 3) or nBNI (dorsal, n = 4; ventral, n = 2). *, significant difference from the amplitude of the response after reversal. Percent inhibition was calculated as the (mean predrug amplitude  mean postdrug amplitude)/mean predrug amplitude). Inset: a representative pair of oscilloscope traces taken before (control) and after treatment with 1 µM U6,9593. Downward deflection at the left of the trace marks the stimulation artifact produced by orthodromic activation of the perforant path input at an intensity sufficient to evoke a half-maximal response (60 µA).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The present study includes the first whole cell voltage-clamp analysis of the excitatory inputs to granule cells after seizure-induced synaptic reorganization. Cells from tissue with mossy fiber sprouting showed several types of increased excitatory activity. First, cells from pilocarpine-treated rats exhibited spontaneous EPSCs in the absence of bicuculline, whereas cells from saline-treated rats rarely, if ever, exhibited such currents. Second, the frequency of spontaneous EPSCs in bicuculline was dramatically increased (more than fourfold) in cells from pilocarpine-treated rats compared with cells from saline-treated rats. Third, in the presence of bicuculline, many cells from tissue with substantial mossy fiber sprouting had spontaneous giant EPSCs, which were often several hundred picoamps in amplitude. Fourth, perforant path-evoked EPSCs in pilocarpine-treated cells were increased greatly in amplitude and duration especially in bicuculline.

Previous studies of granule cells in epileptic tissue have been done using extracellular and intracellular sharp electrode techniques (reviewed by Dudek et al. 1994). Recordings done within a few days after convulsant treatment, before mossy fiber sprouting has occurred, show increased excitability (Sloviter 1992). This finding is attributed partly to the death of inhibitory interneurons and the mossy cells, which normally drive them, which creates, in effect, a disinhibited hippocampus (Sloviter 1991). Recordings done weeks to months after convulsant treatment, when mossy fiber sprouting has developed, can show relatively normal synaptic responses in normal medium (Cronin et al. 1992; Sloviter 1992). These results suggest that sprouted mossy fibers may innervate inhibitory interneurons, thereby reestablishing inhibitory tone in the dentate gyrus.

Morphological evidence has suggested that sprouted mossy fibers in the inner molecular layer synapse with granule cells (Franck et al. 1995; Okazaki et al. 1995; Represa et al. 1993). These excitatory granule cell-granule cell connections have been demonstrated physiologically, both with GABA_A antagonists (Cronin et al. 1992) and even without them (Masukawa et al. 1992; Tauck and Nadler 1985). In these studies, antidromic activation of granule cells by mossy fiber stimulation produced orthodromic population spikes, which were sometimes epileptiform. In the present experiments, similar epileptiform responses were evoked by perforant path stimulation. These responses likely were caused by orthodromic activation of granule cells and consequent activation of recurrent excitatory loops formed by sprouted mossy fibers.

Spontaneous EPSCs occurred frequently in cells from pilocarpine-treated rats, both with and without bicuculline, but were seen rarely in cells from saline-treated rats. Although the physiological changes evident correlated with the anatomic changes in Timm staining, the axonal source of the small spontaneous events is not known, and the source could be either sprouted mossy fiber collaterals, recurrent inputs from CA3 pyramidal cells, expanded perforant path afferents, or some other unidentified change. The results presented are consistent with a mossy fiber collateral source but alternatives are not excluded. For example, an increased probability of excitatory amino acid release from perforant path terminals could account for the increased frequency of EPSCs. In support of this alternative, increasing the excitability of normal slices by treatment with 4-AP was found to increase the frequency of the small spontaneous currents. Moreover, hyperexcitability of CA3 axon terminals in kindled rats has been reported (Stasheff et al. 1993). On the other hand, if sprouted mossy fiber collaterals had a higher rate of spontaneous excitatory amino acid release than the mossy cell terminals that occupy the inner molecular layer in normal tissue, an increased EPSC frequency also would be observed. Another explanation is that spontaneous excitatory amino acid release was not changed, but the granule cells became more sensitive to the transmitter. The increase in the mean amplitude of the typical EPSCs in cells from pilocarpine-treated animals supports this possibility. Thus further studies are required to define the axonal source of the increased EPSCs evident in this model of TLE.

Disinhibition with bicuculline unmasked another form of abnormal excitatory activity, the giant EPSCs. These large epileptiform currents are likely to be the voltage-clamp correlate of the paroxysmal depolarization shifts (PDSs) that have been described with current-clamp recording. Previous studies have described evoked and spontaneous PDSs in granule cells from kainate-treated rats with inhibition blocked (Cronin et al. 1992; Wuarin and Dudek 1996). PDSs also are observed in CA3 pyramidal cells under disinhibitory conditions even in nonepileptic tissue (Chamberlin et al. 1990; Hablitz 1984; Johnston and Brown 1981; Lee and Hablitz 1989; Miles et al. 1984; Rutecki et al. 1987).

PDSs in CA3 and these giant EPSCs in the dentate gyrus share several common characteristics. 1) Both responses occur spontaneously and can be evoked by afferent stimulation. 2) Both responses are synaptic and glutamatergic, because they are sensitive to glutamate receptor antagonists (Lee and Hablitz 1989; Miles et al. 1984). 3) Both responses are associated with recurrent circuitry. CA3 pyramidal cells have numerous collateral fibers that synapse with neighboring pyramidal cells and thereby facilitate synchronous discharges of an entire population of neurons (Miles and Wong 1983; Traub and Wong 1982). Similarly, granule cell giant EPSCs were observed only in slices with substantial recurrent mossy fiber sprouting. Even when control slices were treated with the convulsant 4-AP, giant EPSCs were not observed, presumably because control tissue lacked the requisite collateral connections. 4) Both responses require disinhibition to be unmasked. The requirement for GABA blockade suggests that the recurrent circuitry in both regions is under strong inhibitory control most likely to prevent the large epileptiform events that are seen when inhibition is blocked.

The axonal source of the spontaneous giant EPSCs in the dentate gyrus is also unclear. It is known that granule cells are normally quiescent; they do not fire spontaneously nor do they burst under most disinhibitory conditions (Fricke and Prince 1984). There is no evidence to suggest that granule cells from epileptic tissue are more prone to intrinsic bursting than normal granule cells (Mody and Staley 1994). CA3 pyramidal cells and hilar mossy cells intrinsically burst (Scharfman 1994), but it is unlikely that these cells are responsible for the giant EPSCs (Cronin et al. 1992; Wuarin and Dudek 1996) Alternatively, it is possible that a giant spontaneous event may be initiated by summation of small spontaneous EPSCs (Chamberlin et al. 1990). If a granule cell were stimulated sufficiently by a barrage of small spontaneous EPSCs, it may fire and induce the synchronous recurrent activation that leads to the giant current.

Coincident with the expansion of excitatory circuitry in the dentate gyrus, pilocarpine treatment also caused an expansion of the -opioid receptor regulation of dentate granule cell excitability. As in our previous studies (Bausch et al. 1996) with younger animals, in these studies in age-matched controls, the kappa receptor agonist U69593 reduced perforant path-evoked dentate granule cell excitation only in slices from the ventral pole of the rat hippocampus. This finding is consistent with the anatomic data showing kappa receptor-like immunoreactivity in the ventral hippocampus in the middle molecular layer (McGinty et al. 1994) and with previous findings of field excitatory postsynaptic potentials (fEPSP) inhibition by U69593 in ventral, but not dorsal slices from younger rats (Bausch et al. 1996). In slices from pilocarpine-treated rats, U69593 reduced the population response amplitude in slices from both ventral and more dorsal regions of the hippocampus. The expansion of -opioid sensitivity could be due to expansion of mossy fiber collateral innervation in the molecular layer, because these fibers are thought to contain both opioid peptides and receptors. Additionally, increased expression of kappa receptors on perforant path terminals, particularly at dorsal levels, could produce the observed results. Because kappa receptor activation inhibits excitatory amino acid release from both perforant path and mossy fiber collateral afferents to granule cells (Simmons et al. 1994; Terman et al. 1994), this expansion could serve to limit excitatory transmission.

The results presented support the conclusion that mossy fiber sprouting after pilocarpine-treatment results in an increase in the excitatory drive to the granule cells of the dentate gyrus. In addition, the demonstration that the activation of -opioid receptors in the dentate gyrus effectively reduced the hyperexcitatory input, supports the hypothesis that the endogenous dynorphin peptides may have an anticonvulsant role and may function to reduce seizure frequency and duration in temporal lobe epilepsy.

	ACKNOWLEDGEMENTS

  We thank Dr. Suzanne Bausch and T. Esteb for assistance.

  This work was supported by the National Institute of Neurological Disorders and Stroke Grant NS-33898 to C. Chavkin and a Merck Distinguished Scholars Fellowship (predoctoral) to M. L. Simmons.

	FOOTNOTES

  Address reprint requests to C. Chavkin.

  Received 20 March 1997; accepted in final form 25 June 1997.

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