Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patrylo, Peter R. and F. Edward Dudek. Physiological unmasking of new glutamatergic pathways in the dentate gyrus of hippocampal slices from kainate-induced epileptic rats. J. Neurophysiol. 79: 418-429, 1998. In humans with temporal lobe epilepsy and kainate-treated rats, the mossy fibers of the dentate granule cells send collateral axons into the inner molecular layer. Prior investigations on kainate-treated rats demonstrated that abnormal hilar-evoked events can occasionally be observed in slices with mossy fiber sprouting when -aminobutyric acid-A (GABAA)-mediated inhibition is blocked with bicuculline. However, these abnormalities were observed infrequently, and it was unknown whether these rats were epileptic. Wuarin and Dudek reported that in slices from kainate-induced epileptic rats (3-13 mo after treatment), hilar stimulation evoked abnormal events in most slices with mossy fiber sprouting exposed simultaneously to bicuculline and elevated extracellular potassium concentration [K+]o. Using the same rats, extracellular recordings were obtained from granule cells in hippocampal slices to determine whether 1) hilar stimulation could evoke abnormal events in slices with sprouting in normal artificial cerebrospinal fluid (ACSF), 2) adding only bicuculline could unmask hilar-evoked abnormalities and glutamate-receptor antagonists could block these events, and 3) increasing only [K+]o could unmask these abnormalities. In normal ACSF, hilar stimulation evoked abnormal field potentials in 27% of slices with sprouting versus controls without sprouting (i.e., saline-treated or only 2-4 days after kainate treatment). In bicuculline (10 µM) alone, hilar stimulation triggered prolonged field potentials in 84% of slices with sprouting, but not in slices from the two control groups. Addition of the N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5), either blocked the bursts or reduced their probability of occurrence. The
-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), always eliminated the epileptiform bursts. In kainate-treated rats with sprouting, but not in saline-treated controls, abnormal hilar-evoked responses were also revealed in 6-9 mM [K+]o. Additionally, 63% of slices with sprouting generated spontaneous bursts lasting 1-40 s in ACSF containing 9 mm [K+]o; similar bursts were not observed in controls. These results indicate that 1) mossy fiber sprouting is associated with new glutamatergic pathways, and although NMDA receptors are important for propagation through these circuits, AMPA receptor activation is crucial, 2) modest elevations of [K+]o, in a range that would have relatively little effect on granule cells, can unmask these new excitatory circuits and generate epileptiform bursts, and 3) this new circuitry underlies an increased electrographic seizure susceptibility when inhibition is depressed or membrane excitability is increased.
The kainate-treated rat is an animal model that appears to reproduce many of the clinical and neuropathological attributes of temporal lobe epilepsy. In both human temporal lobe epilepsy (Babb et al. 1991 Kainate treatment and behavioral monitoring
Male Sprague-Dawley rats (170-300 g; Harlan) were initially injected with kainate (5 mg/kg ip; Sigma, St. Louis, MO) or saline every hour for ~2-4 h. By the third to fourth injection, most kainate-treated rats started having behavioral class III, IV, and V seizures. Abnormal behavior and seizure activity were rated according to a modified Racine's scale (Ben-Ari 1985 Slice preparation
Rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and then decapitated. The brains were rapidly removed and stored for 1 min in ice-cold artificial cerebrospinal fluid (ACSF; composition, in mM: 124 NaCl, 3 KCl; 1.3 CaCl2, 26 NaHCO3, 1.3 MgSO4, 1.25 NaH2PO4, and 11 glucose equilibrated with 95% O2-5% CO2, pH 7.2-7.4). The brains were then bisected along the midline and trimmed into a block containing the hippocampus, dentate gyrus, subicular complex, and entorhinal cortex. Using a vibraslicer (Campden Instruments), 400- to 500-µm slices were cut parallel to the base of the brain in ice-cold ACSF and then placed in a storage chamber; the precise order along the septal-temporal axis was maintained. Slices were trimmed to isolate the dentate gyrus and hippocampus, and placed on the ramp of a gas-liquid interface chamber humidified with 95% O2-5% CO2 and maintained with a constant flow (2 ml/min) of ACSF at 32-34°C. Slices were equilibrated for at least 90 min before recording. All slices were from the temporal half of the hippocampus.
Electrophysiological recording and stimulation
Because field potentials can arise from a variety of sources, including shifts in ion concentration and synchronous synaptic currents, multiple-unit recordings were also used to assess action-potential activity. The tips of the field-potential and multiple-unit electrodes were positioned in the granule cell layer as close together as possible with the field-potential electrode usually 50-100 µm below the multiple-unit electrode. Therefore the activity recorded with both electrodes was presumably from the same population of neurons. Multiple-unit activity was recorded using metal electrodes made of 90% platinum/10% iridium wire. The signals were amplified with a differential amplifier, and a band-pass filter between 0.3 and 3-10 kHz was used. Field-potential activity was recorded using glass microelectrodes (resistance 1-10 M Protocols and solutions
Hilar-evoked responses were examined in 1) normal ACSF (i.e., containing 3 mM [K+]o), 2) ACSF containing the GABAA-receptor antagonist bicuculline methiodide (10 µM; Sigma, St. Louis, MO), or 3) ACSF with elevated [K+]o (6-9 mM). Approximately 30 min of bath application was allotted for equilibration of bicuculline or elevated [K+]o (i.e., from 3 to 6 mM, and/or 6 to 9 mM). Only slices in which a population spike Anatomy
A modified neo-Timm's protocol (Babb et al. 1991
Seizures
All kainate-injected rats 3-13 mo after treatment (n = 27 rats) had spontaneous class III, IV, and V seizures during the 6 wk before euthanasia, and no seizures were observed in the saline-treated controls (n = 20 rats). The seizure frequency of these kainate-treated rats ranged from 0.08 to 1.00 seizure/h of observation (0.49 ± 0.23, mean ± SD) during a 28- to 55-h monitoring period (39 ± 6 h). Occasionally, seizures were also induced in these kainate-treated rats while changing their cages and/or injecting them with lactated Ringer, but these seizures were not used in the analysis of spontaneous seizure frequency. No seizures were noted among the kainate-injected rats 2-4 days after treatment, even on handling. Most kainate-treated rats were relatively lethargic and motionless for the first 1-3 days after treatment. In 12 kainate-injected rats that were studied 3-13 mo after treatment and were also monitored behaviorally from the day of treatment, spontaneous recurrent seizures were first observed after a delay of 34-118 days. Therefore it is unlikely that the kainate-injected rats studied 2-4 days after treatment had seizures, other than those that derived directly from the initial kainate treatment. Finally, to ensure that the kainate injections performed on the rats studied 2-4 days after treatment were sufficient to induce chronic epileptogenesis, four of their cage mates that underwent the same treatment were allowed to survive. These rats were observed to have spontaneous recurrent class III, IV, and V seizures >1 mo after treatment.
Anatomy
Timm's staining was used to assess the presence of mossy fibers within the inner molecular layer in 85 slices from 27 kainate-injected rats 3-13 mo after treatment, 55 slices from 20 saline-treated controls, and 24 slices from 7 kainate-injected rats 2-4 days after treatment. Compared with saline-treated controls and kainate-injected rats 2-4 days after treatment, most slices from the kainate-induced epileptic rats 3-13 mo after treatment had robust Timm's staining in the inner molecular layer (i.e., grade 2.5-3.0, Figs. 1 and 2). Grade 2.5-3.0 sprouting was seen in 76% of all slices from kainate-injected rats 3-13 mo after treatment and in 85% of the rats. This grade of Timm's staining in the inner molecular layer was never seen in saline-treated controls or kainate-injected rats 2-4 days after treatment. Most slices from the saline-treated controls and kainate-injected rats 2-4 days after treatment had no Timm's staining in the inner molecular layer. Grade 0.5-1 staining was observed in the inner molecular layer in only 13% of the slices from saline-treated controls and 4% of the slices from kainate-injected rats 2-4 days after treatment. Of the remaining slices from kainate-injected rats 3-13 mo after treatment, 12% showed grade 2 sprouting, 8% showed grade 1.5 sprouting, and 4% showed grade 0.5 Timm's staining. In total, 96% of slices from kainate-injected rats 3-13 mo after treatment had mossy fiber sprouting into the inner molecular layer. No obvious correlation was observed, however, between Timm's staining score within an animal and the frequency of generalized behavioral seizures (P = 0.226 Sperman's rank order correlation) [also see Buckmaster and Dudek (1997)
Hilar stimulation in normal ACSF
Hilar stimulation, even at maximal intensity, only evoked 1 population spike in 97% of 33 slices from saline-treated controls (Fig. 3A; n = 15 rats); 1 control slice had 2 population spikes. Similarly 80% of the 15 slices from kainate-injected rats 2-4 days after treatment (Fig. 3B; n = 7 rats) had 1 population spike and 20% had 2 population spikes. Evidence of abnormal or hyperexcitable responses in normal ACSF was observed in 27% of the 33 slices from kainate-treated rats with mossy fiber sprouting (i.e., 3-13 mo after treatment; Fig. 3C; n = 16 rats). These abnormal responses ranged from three to six population spikes in 9% of the slices to prolonged negative field-potential shifts (1.2-4.0 mV; 100 ms to 1.6 s) in 18% of the slices with sprouting (Table 1). In three slices, two short-latency population spikes were followed after a variable delay with the prolonged negative field potential and variable population spike activity (Fig. 3C). Increasing the stimulus intensity decreased the time between the initial population spikes and the subsequent negative shift; at maximal intensity, the population spikes led directly into the negative shift. In the other three slices, the responses were similar but lacked the delay. In the remaining 73% of slices from kainate-treated rats with mossy fiber sprouting, however, the responses to hilar stimulation in normal medium were similar (i.e., 1-2 population spikes) to those observed in saline-treated controls and kainate-injected rats 2-4 days after treatment (Fig. 3C1). Therefore, in ~30% of the kainate-induced epileptic rats with sprouting (i.e., 3-13 mo after kainate treatment), hilar stimulation in normal medium could evoke prolonged negative field-potential shifts with concurrent bursts of multiple-unit activity.
Hilar stimulation in bicuculline
In bicuculline, hilar stimulation evoked prolonged negative field-potential shifts with variable superimposed population spike activity (Fig. 4; Table 1) in 84% of 25 slices in 93% of the kainate-treated rats with sprouting (n = 15 rats). A concurrent burst of multiple-unit activity usually lasted at least until the peak of the field-potential shift and was almost always characterized by synchronized spikes (Fig. 4C3). In 76% of the slices, a threshold stimulus intensity was found that yielded a prolonged negative field potential after a variable delay. As was seen in normal ACSF, the magnitude of this delay decreased and eventually disappeared (i.e., multiple population spikes led directly into the negative shift) when the stimulus intensity was increased. At the threshold stimulus intensity to elicit the prolonged field potentials (i.e., with delay), their amplitude was 1.2-8.0 mV and their duration was 140 ms to 2 s. Bursts lasting longer than 1 s occurred in 32% of 25 slices (47% of rats). As seen in Fig. 4C3, additional negative field-potential shifts with superimposed population spikes and concurrent bursts of multiple-unit activity were frequently observed. In the two slices without a delay, the prolonged field potentials (1.5-8 mV; 150 ms to 1 s) also had variable spike activity. In the remaining 16% of slices from the kainate-injected rats with sprouting, hilar stimulation evoked one or two (Fig. 4C1; 8%) or multiple (12-24) population spikes (Fig. 4C2; 8%). In one of the slices in which a single antidromic population spike was evoked, a positive field-potential shift with small superimposed spikes (i.e., spikes <1 mV) and a concurrent increase in multiple-unit activity followed the antidromic population spike. Furthermore, in 1 slice with 24 population spikes, a negative shift (2.8 mV; ~800 ms) followed the initial spikes, but only a small and brief increase in multiple-unit activity was observed (i.e., 2-3 times increase in baseline activity).
Effects of glutamate-receptor antagonists
If new recurrent excitatory circuits underlie the prolonged negative field-potential shifts revealed by bicuculline treatment in slices from kainate-treated rats with mossy fiber sprouting, then glutamate-receptor antagonists should block these events. Furthermore, NMDA-receptor antagonists would be expected to block propagation through the multisynaptic circuits characteristic of recurrent excitation in cortical networks. When the NMDA receptor antagonist AP5 was added to the ACSF containing bicuculline, the hilar-evoked bursts were completely blocked in 40% of 10 slices (Fig. 5A). The effect occurred in 5-15 min, and was at least partially reversible (Fig. 5A3; n = 4/4 slices) within 45-90 min. In six other experiments with AP5, the probability of hilar-evoked bursts was decreased (Fig. 5B), and/or their duration was shortened. In five of these six experiments, the kainate/AMPA receptor antagonist DNQX was subsequently added to the bathing medium, and the combination of AP5 and DNQX completely blocked the hilar-evoked field-potential shifts in 6-14 min (Fig. 5B3). This effect was reversible in 3-5 h (Fig. 5B4; n = 3 slices). In DNQX alone (n = 5 slices), hilar-evoked bursts were completely blocked in 4-18 min (Fig. 6), and this effect was partially to completely reversible in 2-3 h (Fig. 6C, n = 3 slices). That DNQX consistently blocked these hilar-evoked bursts suggests that synaptic mechanisms are altered in slices with mossy fiber sprouting, and that AMPA receptor activation is necessary for the epileptiform bursts. Therefore these data are consistent with the hypothesis that glutamatergic circuits mediate the hilar-evoked abnormalities seen in slices from kainate-treated rats with mossy fiber sprouting. Additionally, the data with AP5 indicate that NMDA receptors can play an important role in propagation through the excitatory synaptic circuits that are responsible for generating these network bursts, although their activation is not always necessary.
Effect of elevated [K+]o
We also tested the hypothesis that elevated [K+]o, which depolarizes neurons and reduces inhibition, also unmasks abnormal hilar-evoked and spontaneous field potentials in slices from kainate-treated rats with sprouting. In slices from saline-treated controls (n = 12 rats), hilar stimulation evoked 1-2 population spikes in 96% of 25 slices and 3 population spikes in 1 slice when [K+]o was raised to 6 mM (Fig. 7A). In 9 mM [K+]o, hilar stimulation evoked 1-2 population spikes in 93% of 14 slices (Fig. 7B). In one of these slices, a 1.1-mV field-potential shift with 90 ms of small-amplitude multiple-unit activity was observed after the antidromic population spike. In the remaining slice, six population spikes were elicited.
Spontaneous activity in elevated [K+]o
Robust spontaneous epileptiform activity was also observed in slices with mossy fiber sprouting from kainate-injected rats 3-13 mo after treatment, and similar activity was never seen in slices from saline-treated controls. On bath application of 9 mM [K+]o, only short-duration (range: 40-370 ms), small-amplitude field-potential shifts (range: 0.1-0.5 mV) with concurrent multiple-unit activity were observed in saline-treated controls (Fig. 8A; 91% of 23 slices; n = 12 rats). These events may result from relatively sparse excitatory feedback from CA3 and/or the hilus to the granule cell layer (Jackson and Scharfman 1996
Relationship between Timm's score and abnormal field potentials
To determine whether Timm's score was directly correlated with the abnormal field potentials, a full range of Timm's scores would be needed. Because most slices had a Timm's score of either 0-1 (controls) or 2-3 (kainate-injected rats 3-13 mo after treatment), a correlation analysis was not possible. However, as shown in Table 1, the prolonged hilar-evoked field potentials were only observed in slices with mossy fiber sprouting prepared from kainate-injected rats 3-13 mo after treatment (P These experiments revealed that 1) hilar stimulation could evoke prolonged negative field potentials in normal ACSF in approximately one-quarter of the preparations from kainate-induced epileptic rats with robust mossy fiber sprouting, but not in slices prepared from controls; 2) depression of GABAA-receptor-mediated inhibition greatly increased the proportion of preparations that showed hilar-evoked bursts (i.e., increased from 27 to 84%); 3) glutamate-receptor antagonists blocked the hilar-evoked bursts revealed by depressing GABAA-receptor-mediated inhibition; 4) abnormal, prolonged hilar-evoked events could also be unmasked in most slices from kainate-treated rats with sprouting by elevating [K+]o from 3 mM to 6-9 mM; and 5) ictallike events (>1 s in duration) could occur spontaneously in some slices from kainate-treated rats with sprouting when [K+]o was raised to 9 mM. These hilar-evoked and spontaneous bursts in bicuculline or high [K+]o were not seen in controls. The data strongly suggest that mossy fiber sprouting forms a network of new glutamatergic circuits that underlie an increased seizure susceptibility, which can be unmasked by reductions in inhibition or physiological elevations in [K+]o.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; de Lanerolle et al. 1989
; Houser et al. 1990
; Sutula et al. 1989
) and the kainate model (Cronin and Dudek 1988
; Mathern et al. 1992
, 1993
; Nadler et al. 1980
), the mossy fibers of the granule cells sprout new axon collaterals and appear to form abnormal circuits in the inner molecular layer of the dentate gyrus. However, the role that mossy fiber sprouting plays in epileptogenesis is unclear.
provided evidence that mossy fiber sprouting in kainate-treated rats could lead to hilar-evoked paired-pulse potentiation and occasionally was associated with multiple population spikes in the dentate gyrus. This led to the hypothesis that the mossy fibers form a new recurrent excitatory circuit, which could underlie epileptogenesis. Sloviter (1992)
subsequently reported that the granule cells of anesthetized rats appeared hyperexcitable to perforant path stimulation 2-7 days after kainate treatment, but were relatively normal after mossy fiber sprouting had occurred; these observations suggested the hypothesis that mossy fiber sprouting was restorative (i.e., new excitatory synapses were formed with inhibitory interneurons, which restored recurrent inhibition).
; Dichter and Spencer 1969a
,b
; Miles and Wong 1986
, 1987
). Cronin et al. (1992)
investigated whether recurrent inhibition in kainate-treated rats with mossy fiber sprouting masked newly formed recurrent excitatory circuits; they found that in normal solution responses to hilar stimulation were relatively normal, butwhen bicuculline was used to block
-aminobutyric acid-A(GABAA) receptor-mediated inhibition, some preparations with mossy fiber sprouting had delayed and variable-latency bursts. The presence of a long and variable latency at lower stimulus intensities is believed to represent the time required for the activation of polysynaptic pathways of recurrent excitatory circuits (Cronin et al. 1992
; Traub and Wong 1982
). It should be noted that in the study by Cronin et al. (1992)
, rats were used 1-4 mo after kainate treatment (single systemic injection of kainate; 18 mg/kg), and it was unknown whether these kainate-treated rats had spontaneous generalized seizures (i.e., were epileptic). Anatomic and electrophysiological tests revealed that robust mossy fiber sprouting was present in 58% of slices from the kainate-treated rats, and hilar stimulation could evoke bursts in only 28% of these slices with mossy fiber sprouting. A similar unmasking of hyperexcitable responses has been reported in dentate granule cells of humans with medial temporal sclerosis following exposure to bicuculline (Franck et al. 1995
).
recently reported that hilar stimulation could evoke prolonged seizurelike events in nearly all slices from kainate-treated rats with mossy fiber sprouting in solutions containing both bicuculline and 6 mM [K+]o, but not in slices from control animals treated identically. Furthermore, glutamate-microdrop application in the granule cell layer evoked abrupt increases in the frequency of excitatory postsynaptic potentials (EPSPs) in granule cells in slices with mossy fiber sprouting. In comparison to the rats used by Cronin et al. (1992)
, the rats used in this study had longer survival times (3-13 mo vs. 1-4 mo) following kainate treatment with a modified protocol (multiple low-dose injections) and were known to be epileptic. Although these studies support the hypothesis of new recurrent excitatory circuits, pharmacological experiments are needed to determine whether the abnormal field potentials from hilar stimulation involve glutamatergic neurotransmission. If glutamatergic synaptic mechanisms produce these hilar-evoked events, then adding a kainate/
-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist should block them. Furthermore, experiments with N-methyl-D-aspartate (NMDA)-receptor antagonists would be expected to block polysynaptic excitatory circuits that generate network bursts in dentate granule cells after mossy fiber sprouting. Several laboratories have reported an increase in the NMDA component of the EPSP in the dentate gyrus of kindled animals (Mody and Heinemann 1987
; Mody et al. 1988
) and in CA1 of kainate-treated rats (Turner and Wheal 1991
); although the experiments in this investigation do not specifically address whether such changes occur in the dentate gyrus of kainate-induced epileptic rats, such alterations could increase the probability of neuron-to-neuron communication within newly formed polysynaptic circuits.
; Rutecki et al. 1985
; Traynelis and Dingledine 1988
). In contrast, the dentate gyrus normally appears relatively resistant to the generation of seizurelike activity when GABAA-receptor antagonists are added or [K+]o is elevated up to 12 mM (Fricke and Prince 1984
; Patrylo et al. 1994
; Schwartzkroin and Prince 1978
). In the studies reported here, we hypothesized that the formation of recurrent excitatory circuits would make the dentate gyrus prone to seizure generation during physiological increases in [K+]o (i.e., 6-9 mM). Elevations in [K+]o cause neuronal depolarization and depression of GABA-mediated inhibition, which would facilitate neuron-to-neuron communication through local excitatory circuits; therefore the effects of increased [K+]o provide the basis for a hypothetical mechanism by which the dentate gyrus, after mossy fiber sprouting had occurred, would behave in a relatively normal manner when activity levels are low and [K+]o is normal, but would display an increased seizure susceptibility when activity and [K+]o were elevated within otherwise physiological levels.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Racine 1972
). Individual injections were occasionally delayed once an animal showed 1) exaggerated running or jumping, 2)
10 class III, IV, or V seizures/hour, or 3) excessive lethargy, because we have observed that these behavioral manifestations often precede death. Subsequent injections (i.e., after the initial 2-4) of kainate or saline were reduced to 2.5 mg/kg per 30 min, and depending on the behavior, were again occasionally delayed. Kainate or saline treatment was continued until class IV/V seizures were elicited in the kainate-treated rats for at least 3.5-6 h. A total dose of 20-50 mg/kg was given to each kainate-treated rat. The rats were then injected subcutaneously with lactated Ringer (~0.6-4.0 ml for a 200-g rat) and were given moistened rat chow for the next week.
.
) and a DC amplifier. Multiple-unit and field-potential activity was digitally stored on videotape for later analysis. Evoked responses and spontaneous activity were analyzed using pClamp 6 software (Axon Instruments, Foster City, CA); spontaneous activity was also studied on a chart recorder.
5 mV could be evoked were used in this investigation. Several different recording and stimulating positions were examined per slice, and the most robust or "hyperexcitable" response was used for analysis. When counting the number of evoked population spikes, only spikes
1 mV were included. The amplitude of field-potential shifts was measured from the baseline preceding the shift to the peak of the negativity. Field-potential shifts were analyzed and reported only if they were
1 mV, and there was a concurrent increase in multiple-unit activity. Similarly, the multiple-unit recording was analyzed only if there was a corresponding change in the field-potential recording that was
1 mV.
) was used to visualize mossy fiber sprouting, and was performed with blind procedures. The slices that were studied electrophysiologically (as well as some adjacent slices) were placed in fixative containing 3-4% glutaraldehyde, 0.1% sodium sulfide, and 0.002% calcium chloride in 0.12 M Millonig's buffer (pH 7.2) for 24-48 h. Sections were rinsed twice in phosphate-buffered saline (pH 7.4) and then placed in 30% sucrose in the saline until they sank. Sections were then cut on either a sliding microtome or cryostat at 25-30 µm, mounted on slides, and allowed to air dry overnight. Mounted sections were reacted with the developer for ~1.5 h in the dark, and the degree of staining was monitored during this time. The Timm's reaction was continued until equivalent levels of staining were observed in the hilus (Fig. 1). The developer was made by adding a 30-ml aqueous solution of 7.65 g citric acid and 7.05 g sodium citrate to 180 ml of gum arabic. Just before use, 5 g of hydroquinone in 90 ml of water and 1.5 ml of 15% silver nitrate solution were added in the dark. After sections were reacted, they were rinsed with distilled water, counterstained with cresyl violet, dehydrated in a series of alcohols, and coverslipped. Sections were coded, analyzed, and scored with blind procedures based on the subjective 0-3 rating scale of Tauck and Nadler (1985)
. If there was variability in the degree of Timm's staining between the blades of the dentate granule cell layer (e.g., the lower blade had grade 3 sprouting while the upper blade was grade 2), an average score (e.g., 2.5) was used.
View larger version (80K):
[in a new window]
FIG. 1.
Photomicrographs of Timm's staining in the dentate gyrus of control and kainate-treated rats. In saline-treated controls (A) and kainate-injected rats 2-4 days after treatment (B), Timm's staining was present in the hilus of the dentate gyrus and the stratum lucidum of CA3. In contrast, in kainate-treated rats 3-13 mo after treatment (C), robust Timm's staining was found additionally in the inner molecular layer of the dentate gyrus.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
, in which this issue has been examined more quantitatively].
View larger version (26K):
[in a new window]
FIG. 2.
Percent of slices (top graph) and rats (bottom graph) showing abnormal Timm's staining. These graphs illustrate that the presence of robust Timm's staining in the inner molecular layer was only observed in slices from kainate-treated rats 3-13 mo after treatment. Note the distinct separation of the 3 groups of rats into 2 categories: little or no Timm's staining in the inner molecular layer (saline-treated controls and 2-4 days after kainate-treatment) and robust sprouting (epileptic rats 3-13 mo after kainate-treatment; P < 0.0001; 2). The 1 kainate-treated rat with a 0.5 sprouting score in the 3-13 mo group (3 slices, only 1 hippocampus studied) was observed to have 3 spontaneous seizures during 40 h of monitoring, and an episode of status epilepticus. The slices had normal responses in bicuculline and in 6 mM [K+]o, and the electrophysiological data from this rat were not included in RESULTS.
View larger version (23K):
[in a new window]
FIG. 3.
Comparison of hilar-evoked responses in the dentate granule cell layer of saline-treated controls (A) and kainate-injected rats 2-4 days (B) and 3-13 mo (C) after treatment in normal ACSF. Hilar stimulation evoked a single antidromic population spike in slices from saline-treated controls (A) and kainate-injected rats 2-4 days after treatment (B). Although hilar stimulation evoked only a single population spike in 73% of the slices from kainate-injected rats 3-13 mo after treatment (C1), multiple population spikes (C2) or delayed negative field-potential shifts (C3) were elicited in 9 and 18% of slices, respectively. Note in C3 that there was a delay between the initial antidromic response and the onset of the negative field-potential shift. The dashed horizontal line represents baseline. Concurrent multiple-unit recordings (C3a) during the negative field-potential shift (C3b) revealed an increase in spike activity. Trace C3c is an expansion of the area marked by the arrow in C3b. Traces in A, B, C1, and C2 are of similar scale (calibration: amplitude = 10 mV and duration = 10 ms).
View this table:
TABLE 1.
Percent slices showing prolonged negative field potentials to hilar stimulation
demonstrated that reduced population spike amplitude correlated with the extent of neuronal cell loss (i.e., smaller population spikes were observed in slices with greater cell loss) in slices from patients with temporal lobe epilepsy.
View larger version (31K):
[in a new window]
FIG. 4.
In 10 µM bicuculline, hilar stimulation evoked bursts in 84% of the slices from kainate-injected rats 3-13 mo after treatment. A and B: antidromic stimulation in slices from saline-treated controls (A) and kainate-injected rats 2-4 days after treatment (B) evoked either a single population spike or multiple (2-4) population spikes. C: hilar-evoked responses in slices from kainate-injected rats 3-13 mo after treatment. Hilar stimulation evoked a prolonged negative field-potential shift with variable superimposed population spikes in most slices (C3, 84%). The 10-mV and 10-ms calibrations apply to A, B, and C1. The multiple-unit recording in C3 is clipped, and the stimulus artifact in some traces is truncated.
; Jackson and Scharfman 1996
). Therefore hilar stimulation in bicuculline could evoke abnormal field potentials only in kainate-treated rats with mossy fiber sprouting, but not in kainate-injected rats 2-4 days after treatment without sprouting.
View larger version (24K):
[in a new window]
FIG. 5.
DL-2-Amino-5-phosphonopentanoic acid (AP5) blocked (A) or reduced the probability (B) of hilar-evoked prolonged negative field-potential shifts in the presence of bicuculline in slices from kainate-injected rats 3-13 mo after treatment. In this and the subsequent figure, the multiple-unit recordings are the top traces, and the field-potential recordings are the bottom traces. A: AP5 blocked hilar-evoked burst. A1: 10 of 10 hilar stimuli (1 stimulus/min) evoked a burst (100%) before bath application of AP5. A2: after bath application of 30 µM AP5, hilar-evoked bursts were blocked (15-25 min). Ten consecutive stimuli failed to evoke a burst. A3: partial recovery of hilar-evoked bursts occurred 45 min after removal of AP5. Five of 10 stimuli evoked a burst (50%). B: AP5 partially blocked hilar-evoked bursts. B1: in bicuculline, hilar stimulation evoked prolonged negative field-potential shifts. Ten of 10 stimuli (1 stimulus/min) elicited such a burst. B2: after bath application of 30 µM AP5, 3 of 10 stimuli (30%) still evoked a prolonged field-potential shift (15-25 min). B3: adding 30 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX) completely blocked the hilar-evoked bursts. Approximately 10 min was required for this effect. B4: recovery of hilar-evoked bursts 2.5 h after removing AP5 and DNQX from the bathing medium.
View larger version (22K):
[in a new window]
FIG. 6.
DNQX blocked hilar-evoked prolonged negative field-potential shifts in the presence of bicuculline in slices from kainate-injected rats 3-13 mo after treatment. A: hilar-evoked burst in a slice from a kainate-treated rat, in the presence of bicuculline. Ten of 10 stimuli evoked such a burst (100%). B: hilar-evoked bursts were blocked 12 min after bath application of 10 µM DNQX. Ten consecutive stimuli (1 stimulus/min) failed to evoke a burst. C: recovery of hilar-evoked bursts ~11/2 h after removal of DNQX.
View larger version (25K):
[in a new window]
FIG. 7.
Elevating [K+]o to 6-9 mM increased the probability of hilar stimulation evoking an abnormal response in slices from kainate-injected rats 3-13 mo after treatment vs. controls. A: in 6 mM [K+]o, hilar stimulation evoked a single population spike in slices from saline-treated controls. When [K+]o was further elevated to 9 mM, hilar stimulation still evoked 1 population spike in 93% of the control slices B. In contrast, in most slices from kainate-induced epileptic rats, hilar stimulation evoked either multiple population spikes (C2; 14%) or a prolonged negative shift (C3; 68%). A response similar to that observed in controls was evoked in only 18% of 23 slices from kainate-treated rats 3-13 mo after treatment (C1).
). In contrast, prolonged field-potential shifts (1.1-4.2 mV) lasting 1-40 s were generated in 63% of 19 slices from kainate-injected rats 3-13 mo after treatment (Fig. 8B; n = 11 rats). Variable population spike activity was usually superimposed on these field-potential shifts. In six of the remaining seven slices (32% of slices examined) from the kainate-induced epileptic rats, the spontaneous field-potential shifts were less robust (0.2-4.6 mV; 87-869 ms). No discernable activity was observed in 9% of slices from controls and 5% of slices from kainate-induced epileptic rats. Although we did not investigate whether spontaneous events were generated in elevated [K+]o in slices from kainate-injected rats 2-4 days after treatment, Wuarin and Dudek (1996)
did not observe any spontaneous epileptiform events in slices from these rats in 6 mM [K+]o and 10 µM bicuculline.
View larger version (36K):
[in a new window]
FIG. 8.
Multiple-unit and field-potential recordings of spontaneous activity recorded in the dentate granule cells of saline-treated control (A) and kainate-treated rats 3-13 mo after treatment (B) in 9 mM [K+]o. Top traces: multiple-unit recordings. Bottom traces: field-potential recordings. Note the robust "ictallike" activity generated in slices from kainate-treated rats (B) compared with that elicited in the controls (A). Traces in B1 and B2 are from different rats to illustrate the variability of the long-duration spontaneous events.
), hilar stimulation in bicuculline and 6 mM [K+]o evoked prolonged negative field-potential shifts in the single kainate-treated rat with sprouting in which no robust abnormalities were observed in 6-9 mM [K+]o. Thus elevated [K+]o can lead to spontaneous epileptiform events in the dentate gyrus of kainate-treated rats with sprouting.
0.01;
2). Additionally, although prolonged field-potential shifts could be observed in slices with less than grade 3 sprouting (i.e., grade 2), in each of the conditions examined, the most extreme electrophysiological abnormalities (i.e., longest-duration field potentials in normal ACSF, ACSF with bicuculline, or 9 mM [K+]o) were always observed in slices with grade 3 sprouting.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
reported similar findings in slices from kainate-treated rats with sprouting. Cronin et al. (1992)
did not observe hilar-evoked abnormalities in such slices in normal solution, however, and the reason for this difference is unknown. One possible explanation is that in the present investigation, versus the study of Cronin et al. (1992)
, kainate-treated rats were examined at longer survival times following systemic kainate administration (3-13 mo vs. 1-4 mo), and Timm's staining in the inner molecular layer (i.e., mossy fiber sprouting) is apparently greater with longer survival times after kainate treatment (Mathern et al. 1992
). On the other hand, survival time was relatively short for many of the kainate-treated rats in the Tauck and Nadler (1985)
study. Nevertheless, although hilar stimulation evoked abnormal responses in some slices with sprouting in normal solution, adding the GABAA-receptor antagonist bicuculline unmasked abnormalities in nearly all of the slices.
; Wuarin and Dudek 1996
), the new data show that glutamate-receptor antagonists blocked these hilar-evoked bursts. The rationale for this experiment was that dentate granule cells release glutamate from their mossy fiber terminals (Jonas et al. 1993
; Kneisler and Dingledine 1995
; Neumann et al. 1988
), and hilar stimulation should antidromically activate the mossy fibers, thus leading to glutamate secretion from the sprouted axon collaterals. The NMDA-receptor antagonist AP5 blocked the bursts in some preparations and decreased the probability of bursts in others, which is expected for electrophysiological events that involve polysynaptic recurrent excitatory networks (e.g., Kim et al. 1993
; Tasker et al. 1992
). The AMPA/kainate-receptor antagonist DNQX not only blocked bursts when AP5 was not completely effective, but also blocked them when added alone, suggesting that synaptic mechanisms are altered in slices with mossy fiber sprouting. Therefore one can conclude that new glutamatergic synapses working primarily through AMPA/kainate receptors mediate these abnormal network bursts. The observation that AP5 could decrease the probability of or block hilar-evoked bursts suggests that NMDA receptors can play an important role in the spread of electrical activity through these glutamatergic circuits, although their activation is not always essential. Additional experiments are needed to determine whether there are alterations in the NMDA receptors of dentate granule cells of kainate-treated rats, similar to those reported in CA1 pyramidal cells (Turner and Wheal 1991
). Although synaptic reorganization of other glutamatergic circuits (e.g., those involving the hilar mossy cells or CA3 pyramidal cells) may also occur after kainate treatment, these changes are unlikely to play a major role in producing the hilar-evoked field potentials, because the number of neurons within these regions is drastically reduced after kainate treatment (Buckmaster and Dudek 1997
; Mathern et al. 1992
; Nadler 1981
; Tauck and Nadler 1985
). Additionally, Wuarin and Dudek (1996)
found that glutamate microapplication in the hilar region did not evoke bursts of EPSPs in the dentate granule cells of kainate-treated rats. Thus these data suggest that the explanation for these abnormal glutamate-mediated responses is that mossy fiber sprouting forms recurrent excitatory circuits between dentate granule cells. Further support for this hypothesis is the inability to generate hilar-evoked bursts in either bicuculline or 6 mM [K+]o in three slices without sprouting (i.e., Timm's score = 0.5) from a kainate-induced epileptic rat. It should be noted, however, that our data do not rule out the possibility that sprouted mossy fiber collaterals may also synapse onto inhibitory interneurons (Sloviter 1992
). Although experiments with dual intracellular recording are needed to demonstrate directly new recurrent excitatory circuits, electron microscopic evidence (Babb et al. 1991
; Repressa et al. 1993; Wenzel et al. 1995
; Zhang and Houser 1995
) and data from other field-potential studies (Golarai and Sutula 1996
) and glutamate-microstimulation experiments (Wuarin and Dudek 1996
) independently support this hypothesis.
; Dichter and Spencer 1969a
,b
; Miles and Wong 1986
, 1987
; Traub and Wong 1982
). It is therefore not surprising that bicuculline unmasks recurrent excitatory circuits in kainate-treated rats with mossy fiber sprouting. This effect of inhibition on recurrent excitation probably accounts for why hilar-evoked responses appeared normal in most preparations with mossy fiber sprouting, and may explain why mossy fiber sprouting in epileptic animals does not continuously generate hippocampal seizures. However, if mossy fiber sprouting leads to new excitatory circuits that underlie epileptic seizure activity, these circuits may also be evident under more physiological conditions. In this investigation, abnormal responses to hilar stimulation were observed in some slices in normal medium, and raising [K+]o to levels known to occur during synaptic activation (Benninger et al. 1980
; Heinemann and Lux 1977
; Krnjevic et al. 1982
) significantly increased the probability of hilar-evoked bursts. This unmasking by increased [K+]o may arise because elevated [K+]o depresses inhibitory postsynaptic potentials by shifting the chloride reversal potential to more positive levels (Chamberlin and Dingledine 1988
; Korn et al. 1987
; Somjen 1979
). Although elevating [K+]o to 6 mM may not compromise inhibition to the same extent as adding bicuculline, the depolarizing effect of elevated [K+]o would also increase the probability of neuronal firing and thus neuron-to-neuron communication within polysynaptic excitatory circuits. Nonsynaptic mechanisms of neuronal communication may, however, also play a role in the generation of the seizurelike events in conditions where the [K+]o is elevated (Patrylo et al. 1994
; Traynelis and Dingledine 1988
). Although the ability to block hilar-evoked bursts in elevated [K+]o with DNQX suggests the importance of a synaptically mediated mechanism, we cannot rule out the possibility that there also are alterations in nonsynaptic mechanisms of neuronal communication in kainate-induced epileptic rats. In conclusion, these results suggest that physiological mechanisms that depress synaptic inhibition and raise excitability might unmask the effects of recurrent excitation and initiate electrographic seizures in the dentate gyrus of kainate-treated rats with mossy fiber sprouting.
). Third, Mathern et al. (1993)
have reported observing seizures in some kainate-treated rats at a time period when they did not observe mossy fiber sprouting. Fourth, in preliminary experiments Longo and Mello (1996)
reported that they could retard mossy fiber sprouting but not the development of spontaneous recurrent seizures after pilocarpine treatment if they administered cycloheximide before the pilocarpine. Finally, the possible pathways between mesial temporal structures, such as the temporal regions of the dentate gyrus, and the neural circuits likely responsible for the motor convulsions observed here are indirect. Therefore alterations in other structures may be responsible for the increased propensity for generalized seizures. The formation of new synaptic circuits is probably not limited to the mossy fibers of the dentate granule cells. Synaptic reorganization has been proposed to occur in the CA1 area of the hippocampus (Meier and Dudek 1996
) and also in other cortical regions (Prince and Tseng 1993
; Salin et al. 1995
), as well as within other neurotransmitter/neuromodulator systems (de Lanerolle et al. 1989
; Mathern et al. 1995
). Thus, after an initial insult, synaptic reorganization could hypothetically occur in many regions in addition to or instead of the dentate gyrus, with one or several of these sites contributing to the generation of spontaneous behavioral seizures (Dudek and Spitz 1997
).
; Cronin and Dudek 1988
; Nadler et al. 1980
; Sperk 1994
). Therefore the kainate-treated rat is thus a useful animal model for testing hypotheses concerning the physiological consequences of anatomic abnormalities in the dentate gyrus, because appropriate controls are available. Although it has not been extensively studied, available data suggest that direct excitatory connections between granule cells do not exist in the normal rat or human dentate gyrus (Cronin et al. 1992
; Fricke and Prince 1984
; Tauck and Nadler 1985
; Wuarin and Dudek 1996
). Several studies on hippocampal tissue resected from humans with intractable epilepsy have reported abnormal (i.e., "hyperexcitable") electrophysiological responses in the dentate gyrus with sprouting in normal ACSF or in ACSF with bicuculline (Franck et al. 1995
; Isokawa and Fried 1996
; Masukawa et al. 1989
, 1991
, 1992
; Williamson et al. 1995
). Although the formation of new recurrent excitatory circuits could account for the hyperexcitability, alterations in NMDA receptors and/or a loss of inhibition may also be an underlying mechanism. In the study by Franck et al. (1995)
, however, evidence was presented that bicuculline unmasked hyperexcitable responses in human granule cells. Although the authors hesitated to attribute the electrophysiological abnormalities to recurrent excitation, the results do support this hypothesis. Additional experiments on hippocampal slices resected from patients with intractable epilepsy are needed to test this hypothesis more directly.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Welton for technical assistance, A. Bienvenu for word processing, and Dr. A. Williamson for comments on the manuscript.
This research was supported by National Institute of Neurological Disorders and Stoke Grant NS-16683.
![]() |
FOOTNOTES |
---|
Present address of P. R. Patrylo: Section of Neurosurgery, Yale University School of Medicine, 333 Cedar St., TMP 4, New Haven, CT 06520.
Address reprint requests to F. E. Dudek.
Received 30 January 1997; accepted in final form 9 September 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|