NMDA Receptor-Dependent Plasticity of Granule Cell Spiking in the Dentate Gyrus of Normal and Epileptic Rats

M. Lynch,1,3 Ü. Sayin,1 G. Golarai,1,3 and T. Sutula1,2,3

 1Department of Neurology,  2Department of Anatomy, and  3The Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53792


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INTRODUCTION
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REFERENCES

Lynch, M., Ü. Sayin, G. Golarai, and T. Sutula. NMDA Receptor-Dependent Plasticity of Granule Cell Spiking in the Dentate Gyrus of Normal and Epileptic Rats. J. Neurophysiol. 84: 2868-2879, 2000. Because granule cells in the dentate gyrus provide a major synaptic input to pyramidal neurons in the CA3 region of the hippocampus, spike generation by granule cells is likely to have a significant role in hippocampal information processing. Granule cells normally fire in a single-spike mode even when inhibition is blocked and provide single-spike output to CA3 when afferent activity converging into the entorhinal cortex from neocortex, brainstem, and other limbic regions increases. The effects of enhancement of N-methyl-D-aspartate (NMDA) receptor-dependent excitatory synaptic transmission and reduction in gamma -aminobutyric acid-A (GABAA) receptor-dependent inhibition on spike generation were examined in granule cells of the dentate gyrus. In contrast to the single-spike mode observed in normal bathing conditions, perforant path stimulation in Mg2+-free bathing conditions evoked graded burst discharges in granule cells which increased in duration, amplitude, and number of spikes as a function of stimulus intensity. After burst discharges were evoked during transient exposure to bathing conditions that relieve the Mg2+ block of the NMDA receptor, there was a marked increase in the NMDA receptor-dependent component of the EPSP, but no significant increase in the non-NMDA receptor-dependent component of the EPSP in normal bathing medium. Supramaximal perforant path stimulation still evoked only a single spike, but granule cell spike generation was immediately converted from a single-spike firing mode to a graded burst discharge mode when inhibition was then reduced. The induction of graded burst discharges in Mg2+-free conditions and the expression of burst discharges evoked in normal bathing medium with subsequent disinhibition were both blocked by DL-2-amino-4-phosphonovaleric acid (APV) and were therefore NMDA receptor dependent, in contrast to long-term potentiation (LTP) in the perforant path, which is induced by NMDA receptors and is also expressed by alpha -amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. The graded burst discharge mode was also observed in granule cells when inhibition was reduced after a single epileptic afterdischarge, which enhances the NMDA receptor-dependent component of evoked synaptic response, and in the dentate gyrus reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. NMDA receptor-dependent plasticity of granule cell spike generation, which can be distinguished from LTP and induces long-term susceptibility to epileptic burst discharge under conditions of reduced inhibition, could modify information processing in the hippocampus and promote epileptic synchronization by increasing excitatory input into CA3.


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ABSTRACT
INTRODUCTION
METHODS
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The N-methyl-D-aspartate (NMDA) subclass of glutamate receptors has been implicated in activity-dependent plasticity of neural circuits in both the developing and adult nervous system. In the developing brain, activation of the NMDA receptor by neural activity regulates the formation of synaptic connections and circuit organization (Ben-Ari et al. 1997; Constantine-Paton and Cline 1998; Hanse et al. 1997). In the adult brain, the NMDA receptor plays an important role in the induction of use-dependent synaptic plasticity such as long-term potentiation and depression, and in several forms of seizure-induced plasticity (Bear and Malenka 1994; Meldrum et al. 1999; Mody 1998). For example, repeated seizures evoked by kindling increase the NMDA receptor-dependent current in granule cells of the dentate gyrus (Köhr et al. 1993; Mody and Heinemann 1987; Mody et al. 1988; Sayin et al. 1999) and induce progressive, permanent susceptibility to additional seizures (Goddard et al. 1969). The induction of permanent seizure susceptibility by repeated kindled seizures is NMDA-receptor dependent (Bengzon et al. 1999; Sprengel et al. 1998).

The initial seizure-induced increase of the NMDA receptor-dependent current, while not permanent (Sayin et al. 1999), nevertheless appears to play an important long-term role by initiating structural and functional modifications which contribute to long-lasting seizure susceptibility in hippocampal circuitry (Sutula et al. 1996). For example, repeated seizures induce sprouting by the mossy fiber axons of granule cells, which reorganize synaptic connectivity in the dentate gyrus and form recurrent excitatory circuits that could contribute to increased excitability and susceptibility to seizures (Buckmaster and Dudek 1997; Lynch and Sutula 2000; Molnár and Nadler 1999; Simmons et al. 1997; Wuarin and Dudek 1996). Both mossy fiber sprouting (Sutula et al. 1996) and the associated progressive increase in seizure susceptibility induced by kindling can be prevented by the NMDA receptor antagonist MK-801 administered at doses that do not suppress evoked kindled seizures (Gilbert and Mack 1990; McNamara et al. 1988; Sutula et al. 1996). Because activation of NMDA receptors only transiently enhances synaptic transmission in granule cells but is required for the progression of long-term seizure-induced alterations in the structural organization and functional properties of hippocampal circuitry, it was of interest to further examine how the initial seizure-induced increase in the NMDA receptor-dependent component of synaptic current and more slowly evolving NMDA receptor-dependent cellular alterations may sequentially or independently contribute to the generation of epileptic discharges in the dentate gyrus.

The effects of activation of NMDA receptors by transient exposure to Mg2+-free conditions and seizures on susceptibility to burst discharge and repetitive firing of granule cells were examined in the normal dentate gyrus, and in circuitry reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. The results demonstrated that activation of NMDA receptors by transient relief of Mg2+ block or seizures can induce alterations in granule cell firing patterns when inhibition is reduced, an action that potentially promotes epileptic synchronization and could modify information processing in hippocampal circuitry.


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INTRODUCTION
METHODS
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Surgical procedures

Adult male Sprague-Dawley rats (250-350 g) were anesthetized with a combination of ketamine (80 mg/kg im) and xylazine (10 mg/kg im) and were stereotaxically implanted with an insulated stainless steel bipolar electrode for stimulation and recording. The electrode was implanted in either the perforant path (8.1 mm posterior, 4.4 mm lateral, 3.5 ventral with respect to bregma) or the olfactory bulb (9.0 anterior, 1.2 lateral, 1.8 ventral with respect to bregma) and was fixed to the skull with acrylic.

Kindling procedures

After a 2-wk recovery period following electrode placement, the unrestrained awake animals in the kindling group received twice-daily kindling stimulation (5 days per wk) with a 1-s train of 62-Hz biphasic constant current 1.0-ms-square wave pulses. The stimulation was delivered at the lowest intensity that evoked an afterdischarge according to standard procedures (Cavazos et al. 1991). The electroencephalogram was recorded from the bipolar electrode, which was switched to the stimulator for the delivery of kindling stimulation. Evoked behavioral seizures were classified according to standard criteria (Racine et al. 1975; Sutula and Steward 1986) and ranged from Class I (behavioral arrest) to Class V (bilateral tonic-clonic motor activity with loss of postural tone, which are comparable to partial complex seizures with secondary generalization).

Administration of kainic acid

Adult male Sprague-Dawley rats (250-350 g) were injected with kainic acid (9-12 mg/kg ip or sc) and were observed for signs of behavioral seizure activity, which typically consisted of altered responsiveness to environmental stimuli, irregular tonic-clonic movements of the extremities, and alterations in postural tone. The injected rats were observed for 2-3 h, when the most severe behavioral alterations usually diminished. In previous studies, kainic acid produced initially intense electrographic seizures, which gradually diminished after 4-5 days (Sutula et al. 1992) and are eventually followed by spontaneous recurrent seizures (Hellier et al. 1998). Rats that did not experience status epilepticus in response to the kainic acid injection were not used in this study. Rats were killed 30-60 days after kainic acid treatment.

Preparation of hippocampal slices

Normal, kindled, and kainic acid-treated rats were decapitated after induction of anesthesia by ether. The brains were rapidly removed and placed into ice-cold artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 124 NaCl, 4.4 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and 10 glucose, which was saturated with 95% O2-5% CO2 at pH 7.4. Transverse hippocampal slices were cut from the septal half of the hippocampus with a vibratome or McIlwain tissue chopper at a thickness of 400 µm. The slices were maintained for at least 1 h in ACSF at 20-22°C before transfer to a submersion or interface recording chamber containing oxygenated ACSF at 31-32°C. Granule cells were impaled with tapered borosilicate glass micropipettes (100-150 MOmega ; 1.0/0.58 mm OD/ID) filled with 2 M potassium acetate, adjusted to pH 7.4. Some micropipettes also contained biocytin (2% wt/vol) for intracellular staining and morphological analysis that has been previously reported (Sutula et al. 1998). Granule cells were identified by morphological and physiological criteria such as highly negative resting membrane potential, strong spike frequency adaptation, and absence of a voltage "sag" in response to hyperpolarizing current injection (Lübke et al. 1998; Scharfman 1992; Staley et al. 1992). Granule cells were included in the study only when stable impalements were obtained for at least 1 h, the resting membrane potential was at least -60 mV, the input resistance was at least 40 MOmega , and the action potential amplitude exceeded 50 mV. A conventional bridge circuit was used for intracellular recording and current injection; bridge balance was routinely monitored. Extracellular recording electrodes (2-10 MOmega ) containing 2 M NaCl were positioned within 200 µm of the intracellular electrode. Responses were amplified, digitized, and stored on optical disks for off-line analysis.

Orthodromic synaptic responses were evoked in granule cells of the dentate gyrus by monopolar constant-voltage stimuli (0.05 ms) delivered by electrodes placed in the stratum moleculare of the dentate gyrus in the region of the perforant path. Input-output curves were generated using a sequence of increasing stimulation intensities. Based on input-output curves, subsequent stimuli were delivered at the minimal intensity that evoked the maximal response, unless otherwise indicated. In an attempt to assess the contribution of other regions of hippocampal circuitry to the burst responses recorded in the dentate gyrus, transections were made by razor cuts in some slices to isolate the dentate gyrus from the entorhinal cortex, CA3a,b, and CA1 (illustrated in Fig. 3D). In some experiments, the NMDA component of the synaptic response was studied by replacing MgSO4 in the ACSF with Na2SO4. We have referred to ACSF with no added MgSO4 as "Mg2+-free," but such solutions may still contain trace concentrations of Mg2+. Kainic acid, bicuculline methiodide(-), picrotoxin, and DL-2-amino-5-phosphonovaleric acid (APV) were obtained from Sigma.

Evoked responses were recorded, stored, and analyzed using software developed in the laboratory, pCLAMP 6.02, and Clampfit 6.02 (Axon Instruments).

Statistical methods

Group differences were evaluated for statistical significance by analysis of variance (ANOVA) or Student's t-test. When data were not normally distributed, analyses were performed using a nonparametric ANOVA by ranks, or the Mann-Whitney rank sum test. Averages are reported as mean ± SE.


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Passive membrane properties of granule cells from normal and epileptic rats

Data were collected from 32 control rats, 19 kainic acid-treated rats killed 30-60 days after kainic acid-induced status epilepticus, and 41 kindled rats killed 1-90 days after kindling stimulation that evoked a range of one afterdischarge to 120 Class V generalized tonic-clonic seizures. The average resting membrane potential of granule cells from normal rats was -73.6 ± 1.4 mV (n = 61 cells) and did not differ in kainic acid-treated rats (-73.1 ± 1.2 mV, n = 48 cells) or kindled rats (-73.3 ± 0.8 mV, n = 96 cells). Average input resistance calculated from the voltage deflection produced by 50-ms hyperpolarizing and depolarizing current pulses was 59.1 ± 2.9, 61.0 ± 2.7, 62.7 ± 1.9 MOmega in granule cells from control, kainic acid-treated, and kindled rats, respectively. There were no significant differences in membrane properties as a function of kindling site or number of evoked seizures, or in the threshold for action potential generation in normal controls, kindled, or kainic acid treated groups (Table 1).


                              
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Table 1. Intrinsic properties of granule cells

Synaptic responses and firing patterns of granule cells in normal ACSF

In hippocampal slices from normal rats, orthodromic stimulation of the perforant path evoked an excitatory postsynaptic potential (EPSP) and inhibitory postsynaptic potential (IPSP) in granule cells of the dentate gyrus. The EPSP increased in amplitude with increasing stimulus strength and was accompanied by an action potential when the EPSP exceeded spike threshold, which was -53.3 ± 1.8 mV (Fig. 1A, Table 1). The EPSP was followed by an IPSP, which was usually depolarizing at a typical resting membrane potential in the range of -68 to -75 mV in the recording conditions of these experiments (see Fig. 1A) and became hyperpolarizing at less negative membrane potentials. In agreement with previous reports (Fricke and Prince 1984; Lambert and Jones 1990; Scharfman 1992), stimulation of the perforant path typically evoked a single-action potential (Figs. 1A and 2A). Only 4 of 58 granule cells from normal rats demonstrated two action potentials in normal ACSF. Burst discharges, defined as three or more action potentials evoked by an EPSP, were never evoked by perforant path stimulation of granule cells from normal rats in standard ACSF (n = 58 cells). During bath application of the gamma -aminobutyric acid-A (GABAA) receptor antagonist bicuculline (10 µM) or picrotoxin (100 µM), supramaximal stimulation of the perforant path in hippocampal slices from normal rats still evoked an EPSP and only one or two action potentials (Fig. 1B; n = 28 of 28 granule cells).



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Fig. 1. Single-spike discharge mode in granule cells from normal rats. A: a 30-V perforant path stimulus pulse in a transverse hippocampal slice obtained from a normal rat and bathed in standard artificial cerebrospinal fluid (ACSF) evoked an excitatory postsynaptic potential (EPSP) in a granule cell. Higher stimulation intensities of 60 and 90 V evoked an EPSP and a single-spike discharge. Supramaximal stimulation pulses in hippocampal slices from normal rats typically evoked only 1-2 action potentials. Only 4 of 58 control cells demonstrated 2 action potentials in standard ACSF. Repetitive spikes and burst discharges, defined as EPSPs with 3 or more spikes, were never observed in hippocampal slices from normal rats. B: in a hippocampal slice from a normal rat, a supramaximal perforant path stimulus pulse in standard ACSF evoked an EPSP and single-action potential in a granule cell, and bath application of 10 µM bicuculline did not significantly alter the response to stimulation of the same intensity. Calibration bars: 15 mV, 100 ms.

Synaptic responses and firing patterns of granule cells during and after transient exposure to Mg2+-free ACSF

The NMDA receptor channel in granule cells at resting membrane potential is normally blocked by Mg2+ and plays a relatively minor role in synaptic transmission (Lambert and Jones 1990; Mody and Heinemann 1987). Effects of enhancing the NMDA receptor-dependent component of the synaptic current in granule cells from normal rats were assessed by comparing the perforant path-evoked synaptic response before, during, and after bath application of Mg2+-free ACSF. In contrast to granule cells in normal ACSF, perforant path stimulation of hippocampal slices in Mg2+-free ACSF evoked a burst discharge that increased in duration and amplitude as a function of stimulus strength and was accompanied by multiple granule cell action potentials (Fig. 2B; n = 34 of 38 cells). The evoked burst discharges in Mg2+-free ACSF were field events (Fig. 3A) and were blocked by application of the NMDA receptor antagonist APV (30 µM) (Fig. 3B; n = 5 of 6 cells).



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Fig. 2. Induction of the N-methyl-D-aspartate (NMDA) receptor-dependent graded burst discharge mode in granule cells in hippocampal slices from normal rats by transient exposure to Mg2+-free ACSF. A: low-intensity perforant path stimulation evoked an EPSP, and higher stimulation intensities evoked only a single-action potential in a granule cell from a normal rat. B: when Mg2+ was removed from the ACSF, perforant path stimuli at the same intensities evoked a graded burst discharge in the same granule cell as in A. The bursts increased in duration and evoked an increasing number of spikes as a function of increasing stimulation intensity. C: when the hippocampal slice was returned to ACSF containing 1.3 mM Mg2+, the same stimulus intensities evoked only a single spike. D: when the hippocampal slice was then exposed to 10 µM bicuculline, perforant path stimulation at the same series of stimulation intensities now evoked graded burst discharges which increased in duration and were accompanied by increasing numbers of action potentials as a function of increasing stimulus intensity. E: the graded burst discharges were suppressed by the NMDA receptor antagonist APV (30 µM). Calibration bars: 15 mV, 100 ms.



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Fig. 3. A: simultaneous intracellular and extracellular recordings demonstrated that burst discharges evoked in granule cells from normal rats by a perforant path pulse in Mg2+-free ACSF were population events. B: the granule cell burst discharges evoked in Mg2+-free ACSF were suppressed by the NMDA receptor antagonist APV (30 µM). C: burst discharges evoked in ACSF containing 10 µM bicuculline and 1.3 mM Mg2+ after transient exposure to Mg2+-free ACSF were population events, as shown by simultaneous intra- and extracellular recordings. Calibration bars: 15 mV (intracellular), 20 mV (extracellular), 100 ms. D: burst discharges were also evoked in Mg2+-free ACSF in transected hippocampal slices containing only a small sector of the granule cell layer, molecular layer, the subgranular hilus of the dentate gyrus, and the stratum lacunosum-moleculare of CA1, which suggested that the burst discharges were generated in local circuitry of the dentate gyrus. A representative example of the regions included in these transected slices is schematically indicated by broken lines. Scale bar, 250 µm.

After transient exposure to Mg2+-free ACSF for 5-10 min followed by return to normal ACSF containing 1.3 mM Mg2+, supramaximal perforant path stimulation again evoked only a maximum of one or two action potentials (Fig. 2C). In striking contrast to normal granule cells that were not exposed to Mg2+-free ACSF, subsequent stimulation of the perforant path in ACSF containing 1.3 mM Mg2+ and bicuculline (10 µM) or picrotoxin (100 µM) evoked a prolonged burst with repetitive granule cell discharges (Fig. 2D; n = 27 of 30 cells, compare to Fig. 1B). The burst discharges were also field events (Fig. 3C) and increased in duration and amplitude as a function of stimulus intensity (Fig. 2D, n = 27 of 27 cells). The bursts evoked during reduction of GABAA receptor-mediated inhibition after transient exposure to Mg2+-free ACSF were APV sensitive (Fig. 2E; n = 19 of 20 cells).

Bursts evoked during transient exposure to Mg2+-free ACSF, and then during exposure to ACSF containing 1.3 mM Mg2+ and antagonists of GABAA receptor-mediated inhibition, were both suppressed by APV, which indicated that both the induction and the expression of granule cell bursts were NMDA receptor dependent. Burst discharges were also evoked in these conditions in transected hippocampal slices containing only a small sector of the granule cell layer, molecular layer, the subgranular hilus of the dentate gyrus, and the stratum lacunosum-moleculare of CA1, which suggested that the burst discharges were generated in local circuitry of the dentate gyrus (Fig. 3D; n = 6 of 6 slices). There was no difference in the action potential threshold during or after exposure to Mg2+-free ACSF (see Table 1), which indicates that the repetitive granule cell spiking was not caused by an alteration in the threshold for spike generation. These findings demonstrate that enhancement of the NMDA receptor-dependent component of synaptic transmission by transient exposure to Mg2+-free ACSF is sufficient to increase susceptibility to epileptic burst discharge when GABAA receptor-mediated inhibition is blocked.

The acquired susceptibility to burst discharge under conditions of reduced inhibition was not simply a consequence of exposure to Mg2+-free ACSF, as granule cells which did not demonstrate burst discharges in response to perforant path stimulation in Mg2+-free ACSF did not generate bursts when GABAA receptor-mediated inhibition was subsequently blocked (n = 4/4). This observation suggested that the induction of susceptibility to burst discharge under conditions of reduced inhibition was also a consequence of synaptic activity and was therefore activity-dependent.

The effects of evoked bursting during transient exposure to Mg2+-free ACSF on the NMDA receptor-dependent and non-NMDA receptor-dependent component of the evoked synaptic response were evaluated in standard ACSF by comparing intracellular and extracellular evoked responses before and after bath application of 30 µM APV. As reported previously (Lambert and Jones 1990; Mody and Heinemann 1987), there was a small NMDA receptor-dependent component of the perforant path-evoked EPSP in granule cells examined in hippocampal slices from normal rats (n = 4/5, Fig. 4A). After evoked bursting during transient exposure to Mg2+-free ACSF, there was a significant increase in the NMDA receptor-dependent component of the evoked EPSP (n = 3/4, Fig. 4A), which was also observed in the evoked extracellular population EPSP (Fig. 4B, n = 4/4, P < 0.01). Comparison of the non-NMDA receptor-dependent component of the evoked intracellular EPSP before and after exposure to Mg2+-free ACSF, obtained by subtraction of the EPSPs evoked in 30 µM APV, revealed only a minor increase in the non-NMDA receptor-dependent component (Fig. 4A). The extracellular population EPSP also demonstrated only a trend toward an increase in the non-NMDA receptor-dependent component, which did not reach statistical significance (Fig. 4B).



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Fig. 4. NMDA receptor-dependent component of evoked synaptic transmission in granule cells from normal rats before and after transient exposure to Mg2+-free ACSF. A1: addition of 30 µM APV to normal ACSF produced a slight decrease of the intracellular EPSP evoked in a granule cell by 10-V perforant path stimulation in a hippocampal slice from a normal rat. A2: after transient exposure to Mg2+-free ACSF, 10-V stimulation of the perforant path in normal ACSF evoked a larger EPSP that was significantly attenuated by addition of 30 µM APV, demonstrating enhancement of the NMDA receptor-mediated component of the EPSP. All traces are averages of EPSPs evoked by of 5 consecutive stimulations of the same intensity. A3 and A4: subtraction of the average EPSP evoked in APV prior to transient exposure to Mg2+-free ACSF from the average EPSP evoked in APV after transient exposure to Mg2+-free ACSF demonstrated the increase in the NMDA component, but only a minimal enhancement of the non-NMDA receptor-dependent component. Calibration bars: 10 mV, 20 ms. B: prior to exposure to Mg2+-free ACSF, the extracellular population EPSP evoked by perforant path stimulation in hippocampal slices from normal rats demonstrated only a small component that was sensitive to 50 µM APV (left inset). After transient exposure to Mg2+-free ACSF, there was a significant increase in the APV-sensitive component of the extracellular EPSP in normal ACSF (right inset). *P < 0.01, normal ACSF after exposure to Mg2+-free ACSF vs. normal ACSF before exposure to Mg2+-free bathing medium. **P < 0.01, ACSF plus APV vs. normal ACSF after exposure to Mg2+-free bathing medium. The population EPSPs were compared by measuring the area under the EPSP curve. Calibration bars: 1 mV, 10 ms.

Synaptic responses and firing patterns of granule cells in epileptic kainic acid-treated and kindled rats in normal ACSF

In hippocampal slices from kindled or kainic acid-treated rats in normal bathing medium, orthodromic stimulation of the perforant path also evoked an EPSP and a depolarizing IPSP at resting membrane potential, but did not evoke a burst response (Figs. 5A and 6, A1 and B1). Previous studies in granule cells have demonstrated an increase in the NMDA receptor-dependent synaptic current following status epilepticus or repeated brief seizures (Köhr and Mody 1994; Köhr et al. 1993; Okazaki et al. 1999; Patrylo and Dudek 1998; Sayin et al. 1999), but stimulation of the perforant path with a single pulse of supramaximal intensity still typically evoked only one action potential in granule cells from epileptic kainic acid-treated or kindled rats in normal bathing medium (Figs. 5A and 6, A1 and B1; n = 122 of 132 cells).



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Fig. 5. Granule cell burst discharge mode demonstrated in disinhibited granule cells from kainic-acid treated rats. A: in a hippocampal slice from a kainic acid-treated rat, supramaximal perforant path stimulation in normal ACSF evoked only a single action potential. Supramaximal perforant path stimulation in normal ACSF typically evoked 1-2 action potentials in granule cells from kainic-acid treated rats. Only 4 of 44 granule cells from kainic acid-treated demonstrated 2 action potentials in response to a supramaximal perforant path stimulus pulse; bursts and repetitive spiking were never observed. B: in another granule cell from a kainic-acid treated rat, a supramaximal perforant path stimulus evoked an EPSP with a single action potential (left trace), and in 10 µM bicuculline the same intensity evoked a burst discharge that was a population event, as demonstrated by simultaneous intra- and extracellular recordings. C: evoked burst discharges in disinhibited granule cells from kainic acid-treated rats were graded events, as increasing the stimulation intensity increased the duration of the burst discharge and number of action potentials. D: in a granule cell from another kainic acid-treated rat, the burst evoked by a supramaximal perforant path stimulation pulse in 10 µM bicuculline was suppressed by bath application of 30 µM APV. Calibration bars: 15 mV (intracellular), 10 mV (extracellular), 100 ms.



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Fig. 6. Perforant path stimulation in hippocampal slices from kindled rats killed 24 h after the last evoked seizure. A1: supramaximal perforant path stimulation in normal ACSF evoked an EPSP and a single discharge in a granule cell from a kindled rat that experienced 50 Class V seizures (left trace). Supramaximal intensity perforant path stimulation in normal ACSF typically evoked only a single-spike discharge. Two action potentials were evoked in 6 of 88 granule cells from kindled rats in normal ACSF; bursts and repetitive spiking were never observed (n = 88). In 10 µM bicuculline, a perforant path pulse of the same stimulus intensity evoked a population burst discharge, as demonstrated by simultaneous intra- and extracellular recordings (right traces). A2: a perforant path-evoked granule cell burst discharge in 10 µM bicuculline from a kindled rat that experienced 65 Class V seizures was suppressed by bath application of 30 µM APV. B1: in a hippocampal slice from a rat killed 24 h after a single kindled afterdischarge, perforant path stimulation evoked an EPSP and a single action potential in a granule cell in normal ASCF (left trace), and a population burst discharge after bath application of 10 µM bicuculline (right traces). B2: in a hippocampal slice from another rat that experienced a single kindled afterdischarge, the granule cell burst evoked in 10 µM bicuculline was blocked by bath application of 30 µM APV. Calibration bars: 15 mV (intracellular), 15 mV (extracellular), 100 ms.

Effects of GABAA receptor blockade on synaptic responses and firing patterns of granule cells from epileptic kainic acid-treated rats

In contrast to normal rats, when GABAA receptor-mediated inhibition was blocked with bicuculline or picrotoxin in hippocampal slices from rats examined 30-60 days after kainic acid-induced status epilepticus, stimulation of the perforant path evoked granule cell burst discharges characterized by prolonged depolarization and repetitive spikes (n = 38 of 39 cells; compare Fig. 5B to Fig. 1B). There was no difference in the action potential threshold in normal and kainic acid-treated rats (see Table 1). Simultaneous extracellular recording demonstrated that the discharges were synchronous field events (Fig. 5B). Perforant path-evoked bursts were reliably elicited in transected hippocampal slices (as in Fig. 3D, n = 5 of 5 slices), which suggested that the burst discharges were generated in local circuitry of the dentate gyrus. Increasing the intensity of the perforant path pulse increased the amplitude and duration of the EPSP, and the number of evoked granule cell discharges (Fig. 5C). In contrast to the "all-or-none" character of epileptiform events in other regions of hippocampus or neocortex (Courtney and Prince 1977; Schwartzkroin and Prince 1978), the evoked epileptiform discharges in the dentate gyrus were graded and increased in duration with increasing stimulus strength. Spontaneous granule cell burst discharges were not observed. The evoked repetitive granule cell discharges were blocked by addition of APV (30 µM) and were therefore dependent on the NMDA receptor (Fig. 5D; n = 31 of 33 cells).

Effects of GABAA receptor blockade on synaptic responses and firing patterns of granule cells from kindled rats

In hippocampal slices from kindled rats that had experienced 3-95 generalized Class V seizures, perforant path stimulation in normal bathing medium evoked an EPSP with a single-action potential (Fig. 6A1). Supramaximal intensity perforant path stimulation in hippocampal slices obtained from kindled rats and bathed in normal ACSF typically evoked only a single-spike discharge. Two action potentials were evoked in only 6 of 88 granule cells from kindled rats in normal ACSF; bursts and repetitive spiking were never observed. In ACSF-containing bicuculline or picrotoxin, perforant path stimulation evoked an APV-sensitive granule cell burst discharge (n = 46 of 49 cells, Fig. 6A). As in hippocampal slices from kainic-acid treated rats, the evoked bursts increased in amplitude and duration, and the number of spikes per burst also increased as a function of increasing stimulus intensity (data not shown). There were no differences in the resting membrane potential, input resistance, action potential threshold, or firing patterns as a function of kindling site or number of evoked seizures. In ACSF-containing bicuculline or picrotoxin, burst discharges were evoked in hippocampal slices obtained as long as 12 wk after the last kindled seizure.

The chronic epileptic state induced by kindling or treatment with kainic acid is associated with the development of mossy fiber sprouting in the dentate gyrus, which may contribute to generation of epileptic burst discharges by formation of recurrent circuits (Sutula et al. 1988; Tauck and Nadler 1985). Histological evidence of mossy fiber sprouting induced by kindling first becomes apparent by 7 days after the initial kindling stimulation and increases with continued stimulation, but is not seen within the first 3 days after initial stimulation (Cavazos et al. 1991). It was therefore of interest to assess effects of the initial evoked kindled seizures on susceptibility to granule cell burst discharge prior to the development of sprouting. In hippocampal slices prepared from rats 1 day after a single-evoked afterdischarge, perforant path stimulation evoked NMDA receptor-dependent granule cell burst discharges in ACSF containing 10 µM bicuculline (n = 9 cells from 4 rats), which were field events (Fig. 6B1) and were blocked by APV (Fig. 6B2). This observation demonstrates that mossy fiber sprouting is not necessary for generation of epileptic burst discharges in the dentate gyrus because bursts can be evoked in granule cells within 24 h after an afterdischarge before mossy fiber sprouting has developed. Perforant path stimulation also evoked NMDA receptor-dependent granule cell burst discharges in ACSF containing 10 µM bicuculline in hippocampal slices obtained after 1 wk of kindling stimulation (n = 12 cells from 5 rats that experienced 5-8 afterdischarges; data not shown).

NMDA receptor-dependence of burst discharges in the chronic epileptic state

The increase in the NMDA receptor-dependent component of the evoked synaptic current in kindled rats diminishes by 2.5-3 mo after the last evoked seizure and becomes comparable to the NMDA receptor-dependent current in granule cells from age-matched normal rats (Sayin et al. 1999). The NMDA receptor-dependence of evoked burst discharges was compared in hippocampal slices obtained within 1 wk after 50-120 evoked Class V kindled seizures, and 3 mo after the last of >100 Class V seizures, when the NMDA component of the synaptic current has diminished to normal. When GABAA receptor-dependent inhibition was blocked by 10 µM bicuculline in these groups, supramaximal perforant path stimulation evoked epileptic burst discharges (Fig. 7, A and B; n = 18 of 20 cells).



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Fig. 7. NMDA receptor dependence of burst discharges evoked in kindled rats as a function of time since the last evoked seizure. A: a representative example of a burst evoked by a perforant path pulse at the lowest stimulus intensity that evoked the maximal response in a granule cell from a kindled rat examined at 4 days after the last of 103 Class V seizures, in ACSF containing 10 µM bicuculline. The burst duration and the number of granule cell spikes associated with the burst were reduced by bath application of 50 µM APV. B: a representative example of a perforant path-evoked burst in a granule cell from a kindled rat examined at 3 mo after the last of 108 Class V seizures, in ACSF containing 10 µM bicuculline. The amplitude and number of spikes were reduced by the addition of 50 µM APV, but the effect appears to be less than in the granule cell from a kindled rat examined within 1 wk after the last seizure (compare to A). Calibration bars for A and B: 20 mV, 20 ms. C: action potentials per burst in granule cells from kindled rats examined within 1 wk after the last of 50-120 evoked Class V seizures, and 3 mo after the last of more than 100 Class V seizures. The number of action potentials per burst was decreased in hippocampal slices from rats examined at 3 mo after the last seizures compared with kindled rats killed within 1 wk after the last evoked seizure (compare open bars, **P < 0.001). There was prominent NMDA receptor dependence of the number of spikes per burst in granule cells examined within 1 wk of the last evoked seizure as assessed by effects of 50 µM APV (compare open and filled bars, *P < 0.001). In granule cells from kindled rats examined at 3 mo after the last evoked seizure, there was no significant reduction in the number of action potentials per burst or burst duration after APV. D: there was a significant decrease in burst duration in hippocampal slices from kindled rats studied at 3 mo after the last evoked seizure compared with kindled rats killed within 1 wk after the last evoked seizure (compare open bars, **P < 0.001). Burst duration was significantly decreased by application of 50 µM APV in rats examined within 1 wk of the last seizure (compare open and filled bars, *P < 0.01) but not in granule cells from rats studied at 3 mo after the last seizure.

It was of interest to compare the duration, number of action potentials, and NMDA dependence of the burst discharges in these groups. For this analysis, the perforant path stimulus intensity was standardized by using the lowest stimulus intensity required to evoke a maximal response as defined by input-output curves. At this standardized stimulus intensity, the burst duration, measured as the interval from initial depolarizing deflection of the EPSP to the return of the evoked depolarization to baseline resting membrane potential, was shorter in granule cells from rats examined at 3 mo after the last evoked seizure compared with rats examined within 1 wk after similar numbers of kindled seizures; there were also fewer action potentials per evoked burst compared with granule cells from acutely kindled rats (Fig. 7, C and D). The duration of the evoked bursts was strongly correlated with number of action potentials in all groups (r = 0.77, P < 0.0001, Spearman rank-order correlation). The number of action potentials per burst and burst discharge duration in rats examined at 3 mo after the last evoked seizure were less sensitive to APV (Fig. 7), which is in agreement with voltage-clamp recordings in granule cells studied at 2.5-3 mo after the last evoked seizure (Sayin et al. 1999).


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Granule cells in the dentate gyrus, which normally generate a single spike even when inhibition is reduced and thereby restrict the flow of afferent activity into CA3, converted to a graded burst discharge spike mode in conditions of reduced inhibition after burst discharge was evoked during transient relief of the Mg2+ block of the NMDA in vitro, or after preceding epileptic afterdischarges in vivo. We have referred to this rapidly induced transformation in the pattern of granule cell spike output to CA3 as "granule cell spike plasticity." Both the induction and expression of this plasticity in granule cell spike generation were prominently NMDA receptor dependent and could therefore be distinguished from other forms of activity-dependent synaptic plasticity in granule cells such as LTP, which depends on the NMDA receptor for induction, but is also expressed through enhancement of the AMPA receptor-dependent component of the synaptic response (Maren et al. 1992; O'Connor et al. 1995; Wang et al. 1996; Xie et al. 1992). This NMDA receptor-dependent transformation of granule cell firing patterns was observed in normal granule cells and in the epileptic dentate gyrus reorganized by mossy fiber sprouting. The transformation of granule cell spike generation from a single-spike firing mode to a graded burst discharge mode could modify information processing and epileptogenesis in the hippocampus by increasing susceptibility to epileptic synchronization and altering the "filtering" properties of the dentate gyrus.

Features of granule cell spike plasticity

Granule cells acquired susceptibility to burst discharge when inhibition was reduced in hippocampal slices examined: 1) immediately after burst discharge evoked in Mg2+-free ACSF, 2) at 24 h after a single epileptic afterdischarge evoked in vivo, which induces synaptic potentiation (Sutula and Steward 1986), 3) in kindled rats which have enhanced NMDA receptor-dependent synaptic currents after repeated seizures (Mody and Heinemann 1987; Sayin et al. 1999), and 4) in kainic acid-treated rats.

Because reduction of inhibition, which was required for expression of the acquired capacity for burst discharge, occurs during use-dependent fading of IPSPs (Deisz and Prince 1989; McCarren and Alger 1985), in the initial period after status epilepticus induced by kainic acid or pilocarpine (Franck et al. 1988; Hellier et al. 1999; Michelson et al. 1989; Sloviter 1992), and in kindled rats experiencing spontaneous seizures (Rutecki et al. 1996), the sequence of use-dependent increases in NMDA receptor-dependent currents followed by reduced strength of inhibition is likely to be encountered in realistic settings of ongoing neural activity and pathologies (Franck et al. 1995; Isokawa 1996; Isokawa et al. 1997; Masukawa et al. 1996; Mathern et al. 1999; Williamson et al. 1999).

The NMDA receptor dependence and underlying mechanisms of granule cell spike plasticity in the dentate gyrus: contrasts to LTP

There were no differences in the threshold for spike generation between granule cells in normal controls and any of the experimental groups, which suggests that the observed effects were more likely to be caused by alterations in the functional properties of neural circuitry in the dentate gyrus rather than alterations in the intrinsic neuronal mechanisms of spike generation.

The induction of graded burst discharges in Mg2+-free conditions, and the expression of burst discharges evoked with subsequent disinhibition, were both blocked by APV. While these observations implicate the NMDA receptor in the induction and expression of alterations in granule cell spike generation, the specific cellular mechanisms which underlie the induction process, and the subsequent burst generation when inhibition is reduced, remain uncertain. Enhancement of NMDA receptor-dependent synaptic transmission has been observed to contribute to burst discharges also in pyramidal cells of the hippocampus and cortex (Anderson et al. 1987; Ben-Ari and Gho 1988; Bush et al. 1999; DeLorenzo et al. 1998; Hoffman and Haberly 1989; Telfeian and Connors 1999).

The requirement for synaptically evoked burst activity during exposure to Mg2+-free conditions implies that cellular events resulting from neural activity, not merely removal of Mg2+ or relief of the Mg2+ block of the NMDA receptor, play a role in the induction of subsequent susceptibility to burst discharges when inhibition is reduced. The cellular events induced by neural activity during Mg2+-free conditions could include Ca2+ influx, activation of other types of receptors and second messenger systems, conformational changes related to rapid activity-dependent processes such as phosphorylation or dephosphorylation, or other unrecognized cellular alterations (Meldrum et al. 1999; Mody 1998).

Additional experiments will be required to understand how burst discharges during Mg2+-free conditions induce long-lasting susceptibility to burst discharge in granule cells. Whatever the specific mechanisms underlying the induction, however, the overall induction process is influenced by the NMDA receptor, as APV blocks the process. Furthermore, it is unlikely that gene expression is required for the induction of susceptibility to burst discharge, as the conversion of granule cell spike generation from a single-spike mode to the graded burst discharge mode is immediately apparent in normal bathing conditions when inhibition is reduced. If the process of induction required transcription and translation, a longer time interval would be expected for the conversion.

The mechanisms underlying the expression of the burst discharges in disinhibited granule cells after burst discharges have been induced during transient Mg2+-free conditions are also NMDA receptor dependent, as the bursts are blocked by APV. Analysis of the perforant path-evoked EPSP by both intracellular and extracellular recordings in normal bathing conditions after the induction process demonstrated a marked enhancement of the NMDA receptor-dependent component of the synaptic response, but no significant increase in the non-NMDA dependent component, which is likely to be mediated by AMPA receptors. AMPA and kainate receptors, which contribute to synaptic plasticity in other regions of the hippocampus and neocortex (Bortolotto et al. 1999; Malenka and Nicoll 1999), appear to play a minor role in the expression of activity-dependent plasticity of spike generation in the dentate gyrus.

The prominent effects of the NMDA receptor on the induction and expression of granule cell spike plasticity described in these experiments can be distinguished from other forms of activity-dependent synaptic plasticity such as long-term potentiation. In contrast to long-term potentiation in granule cells, which depends on NMDA receptors for induction but also induces a prominent increase in non-NMDA, AMPA receptor-dependent component of the evoked excitatory synaptic response (Maren et al. 1992; O'Connor et al. 1995; Wang et al. 1996; Xie et al. 1992), both the induction and the expression of the granule cell spike plasticity observed in these experiments were NMDA receptor dependent, but produced little alteration in the non-NMDA receptor-dependent component of the evoked EPSP.

Functional effects of granule cell spike plasticity: implications for filtering properties of the dentate gyrus

The concept that the dentate gyrus acts as a "filter" for input to the hippocampus is supported by 2-deoxyglucose autoradiography and electrophysiological observations demonstrating that seizures in the normal rat do not propagate from the dentate gyrus to CA3, but do so after seizures (Heinemann et al. 1992; Lothman et al. 1992). The cellular and circuit properties that are responsible for the filtering function of the dentate gyrus are uncertain, but this study provides some insights into how the filtering property of the dentate gyrus could be dynamically impaired by synchronous neural activity both in normal and epileptic rats. The single-spike firing mode of granule cells in the normal dentate gyrus serves to filter or restrict flow of afferent activity passing from the entorhinal cortex to CA3 by way of the dentate gyrus, as output to CA3 is "fixed" in the normal dentate gyrus even when GABAA receptor-dependent inhibition is blocked. In the burst discharge mode, granule cells fire in a pattern of repetitive spikes that increase in number as a function of stimulus strength, and the filtering of the spike output from the dentate gyrus is thereby reduced or lost, with potential effects on the flow of activity from the entorhinal cortex to CA3.

The activity-dependent transformation from the single-spike mode to the graded burst discharge mode could potentially modify the balance of excitation and inhibition in hippocampal pathways with consequences for epileptogenesis. By enhancing propagation of neural activity through the dentate gyrus into CA3, where pyramidal neurons demonstrate both spontaneous burst discharges and high propensity for evoked epileptic bursts (Johnston and Brown 1981; Schwartzkroin and Prince 1978), conversion to the graded burst discharge mode could increase excitatory drive in CA3 and promote synchronization in this highly epileptogenic region of hippocampal circuitry.

Mossy fiber sprouting is not necessary for burst discharge in the dentate gyrus

It has been suggested that the resistance of granule cells in the dentate gyrus to burst discharge may be caused by absence of recurrent excitatory circuits (Fricke and Prince 1984; Golarai and Sutula 1996; Lynch and Sutula 2000; Patrylo and Dudek 1998; Wuarin and Dudek 1996) which are normally found in regions such as CA3 and CA1, where spontaneous and evoked bursts are observed when GABAA receptor-dependent inhibition is blocked (Dingledine et al. 1986; Hablitz 1984; MacVicar and Dudek 1980; Miles and Wong 1987; Tancredi et al. 1990; Traub et al. 1994). In the present study, bursts were evoked in disinhibited granule cells from normal rats immediately following enhancement of the NMDA receptor-dependent component of the EPSP during transient exposure to Mg2+-free ACSF, and at 24 h after a single subconvulsive afterdischarge, when mossy fiber sprouting has not yet developed. These findings demonstrate that recurrent circuitry formed by mossy fiber sprouting is not necessary for epileptic burst discharge in the dentate gyrus.

In epileptic kindled rats studied at 3 mo after the last evoked seizure, when the dentate gyrus is reorganized by sprouting and other seizure-induced alterations but the NMDA receptor-dependent synaptic current is comparable to normal (Sayin et al. 1999), disinhibited granule cells continue to fire in the graded burst mode, which is consistent with direct observation of enhanced propagation from the entorhinal cortex to CA3 and reduced filtering by the dentate gyrus in kindled rats (Behr et al. 1996, 1998). Because the NMDA receptor-dependent component of the synaptic current in these rats is comparable to controls at this time interval following a seizure, seizure-induced cellular alterations other than enhancement of the NMDA receptor-dependent synaptic response must be contributing to the burst-firing mode. The long-term seizure-induced structural and functional alterations in the reorganized dentate gyrus, which include mossy fiber sprouting and other seizure-induced cellular alterations, are sufficient to generate epileptic bursts when inhibition is reduced. It should be noted, however, that burst duration is shorter and spiking is reduced in epileptic rats examined at 3 mo after the last seizure compared with granule cells from kindled rats that recently experienced a seizure. This observation suggests that enhancement of seizure-induced NMDA receptor-dependent synaptic transmission may potentially have an additional proconvulsant effect in the reorganized dentate gyrus, which has clinical implications in regard to the importance of seizure control in people with epilepsy.

NMDA receptor-dependent plasticity: implications for relationships between synapse formation in development, information storage and memory, and epileptogenesis

NMDA receptors not only influence plasticity of granule cell spike generation, but also play a role in several forms of activity-dependent plasticity that influence epileptogenesis. Repeated induction of LTP in the dentate gyrus, which also is an NMDA receptor-dependent process, increases the rate of kindling development in response to perforant path stimulation (Sutula and Steward 1987). In addition, about 30 s of synchronous afterdischarge activates NMDA receptors, signal transduction, and gene expression, which eventually results in permanent structural and functional alterations including mossy fiber sprouting and increased susceptibility to evoked and spontaneous seizures (McNamara 1999; Shin et al. 1990). The importance of NMDA receptors in the induction of mossy fiber sprouting and the associated progressive increase in seizure susceptibility induced by kindling is indicated by the observation that administration of the NMDA receptor antagonist MK-801 at doses that do not suppress evoked kindled seizures blocks both the sprouting and the associated progression of kindling (Sutula et al. 1996).

NMDA receptor-dependent plasticity also regulates circuit formation in normal development, and information storage in the adult nervous system that plays a role in learning and memory (Bliss and Collingridge 1993; Scheetz and Constantine-Paton 1994), but also can be epileptogenic in the dentate gyrus. NMDA receptor-dependent mechanisms of plasticity confer an important capacity for experience-dependent modification of synapses and circuits that undoubtedly has significant adaptive advantages in the developing and adult nervous systems, but with the potentially undesirable consequence that this plasticity in the dentate gyrus also enhances susceptibility to synchronous neural activity and paroxysmal behavioral dysfunction during epileptic seizures.


    ACKNOWLEDGMENTS

The authors appreciate the helpful comments of L. Haberly and P. Rutecki.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-25020.

Present address of G. Golarai: Rm. 145, BMSB, Dept. of Neuroscience, University of New Mexico, Albuquerque, NM 87131.


    FOOTNOTES

Address for reprint requests: T. Sutula, Dept. of Neurology, H6/570, University of Wisconsin, Madison, WI 53792 (E-mail: sutula{at}neurology.wisc.edu).

Received 27 January 2000; accepted in final form 28 August 2000.


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