Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth

A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.     

Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid-treated rats

Repeated seizures induce mossy fiber axon sprouting, which reorganizes synaptic connectivity in the dentate gyrus. To examine the possibility that sprouted mossy fiber axons may form recurrent excitatory circuits, connectivity between granule cells in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by kindling and kainic acid. The experiments were designed to functionally assess seizure-induced development of recurrent circuitry by exploiting information available about the time course of seizure-induced synaptic reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When recurrent inhibitory circuits were blocked by the GABA(A) receptor antagonist bicuculline, focal application of glutamate microdrops at locations in the granule cell layer remote from the recorded granule cell evoked trains of excitatory postsynaptic potentials (EPSPs) and population burst discharges in epileptic rats, which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 microM tetrodotoxin and were therefore dependent on network-driven synaptic events. Excitatory connections were detected between blades of the dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after > or = 1 wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting. Excitatory connectivity between blades of the dentate gyrus was also assessed in slices deafferented by transection of the perforant path, and bathed in artificial cerebrospinal fluid (ACSF) containing bicuculline to block GABA(A) receptor-dependent recurrent inhibitory circuits and 10 mM [Ca(2+)](o) to suppress polysynaptic activity. Low-intensity electrical stimulation of the infrapyramidal blade under these conditions failed to evoke a response in suprapyramidal granule cells from normal rats (n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 +/- 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation of monosynaptic excitatory connections between blades of the dentate gyrus. Recurrent excitatory circuits developed in the dentate gyrus of epileptic rats in a time course that corresponded to the development of mossy fiber sprouting and demonstrated patterns of functional connectivity corresponding to anatomic features of the sprouted mossy fiber pathway.     

Reassessment of the effects of cycloheximide on mossy fiber sprouting and epileptogenesis in the pilocarpine model of temporal lobe epilepsy

A feature of animal models of temporal lobe epilepsy and the human disorder is hippocampal sclerosis and Timm stain in the inner molecular layer (IML) of the dentate gyrus, which represents synaptic reorganization and may be important in epileptogenesis. We reassessed the hypothesis that pre-treatment with cycloheximide (CHX) prevents Timm staining in the IML following pilocarpine (PILO)-induced status epilepticus (a multifocal model of temporal lobe epilepsy), but allows epileptogenesis (i.e., chronic spontaneous seizures) after a latent period. Hippocampal slices from PILO-treated rats without Timm stain in the IML after CHX treatment were hypothesized to lack the electrophysiological abnormalities suggestive of recurrent excitation. The primary experimental groups were as follows: 1) CHX (1 mg/kg) 30-45 min prior to administration of PILO (320 mg/kg ip, 2) only PILO, and 3) only saline (0.5 ml, IP). The CHX pre-treatment significantly decreased the number of rats that responded to PILO with status epilepticus compared to rats that received only PILO. Pre-treatment with CHX did not significantly alter the spontaneous motor seizure rate post-treatment compared to treatment with PILO alone in those animals from each group that developed status epilepticus during PILO treatment. Timm stain in the IML was not significantly different between the PILO- and PILO+CHX-treated rats. Using quantitative methods, CHX did not prevent hilar, CA1, or CA3 neuronal loss compared to the PILO-treated rats. Extracellular responses to hilar stimulation in 30 microM bicuculline and 6 mM [K(+)](o) demonstrated all-or-none bursting in both the CHX+PILO- and PILO-treated rats but not in control rats. Whole cell recordings from granule cells, using glutamate flash photolysis to activate other granule cells, showed that both the CHX+PILO- and PILO-treated rats had excitatory synaptic interactions in the granule cell layer, which were not found after saline treatment. Some rats responded to PILO (with or without CHX pre-treatment) with only one or a few seizures at treatment, and some of these animals (n = 4) demonstrated spontaneous motor seizures within 2 mo after treatment. Timm staining and neuron loss in this group were not clearly different from saline-treated rats. These results suggest that in the PILO model, pre-treatment with CHX does not affect mossy fiber sprouting in the IML of epileptic rats and does not prevent the formation of recurrent excitatory circuits. However, the develoment of spontaneous motor seizures, in a small number of rats, could occur without detectable hippocampal neuron loss or mossy fiber sprouting, as assessed by the Timm stain method.     

Electrophysiological evidence of monosynaptic excitatory transmission between granule cells after seizure-induced mossy fiber sprouting

Mossy fiber sprouting is a form of synaptic reorganization in the dentate gyrus that occurs in human temporal lobe epilepsy and animal models of epilepsy. The axons of dentate gyrus granule cells, called mossy fibers, develop collaterals that grow into an abnormal location, the inner third of the dentate gyrus molecular layer. Electron microscopy has shown that sprouted fibers from synapses on both spines and dendritic shafts in the inner molecular layer, which are likely to represent the dendrites of granule cells and inhibitory neurons. One of the controversies about this phenomenon is whether mossy fiber sprouting contributes to seizures by forming novel recurrent excitatory circuits among granule cells. To date, there is a great deal of indirect evidence that suggests this is the case, but there are also counterarguments. The purpose of this study was to determine whether functional monosynaptic connections exist between granule cells after mossy fiber sprouting. Using simultaneous recordings from granule cells, we obtained direct evidence that granule cells in epileptic rats have monosynaptic excitatory connections with other granule cells. Such connections were not obtained when age-matched, saline control rats were examined. The results suggest that indeed mossy fiber sprouting provides a substrate for monosynaptic recurrent excitation among granule cells in the dentate gyrus. Interestingly, the characteristics of the excitatory connections that were found indicate that the pathway is only weakly excitatory. These characteristics may contribute to the empirical observation that the sprouted dentate gyrus does not normally generate epileptiform discharges.     

Kindling induces transient NMDA receptor-mediated facilitation of high-frequency input in the rat dentate gyrus

To elucidate the gating mechanism of the epileptic dentate gyrus on seizure-like input, we investigated dentate gyrus field potentials and granule cell excitatory postsynaptic potentials (EPSPs) following high-frequency stimulation (10-100 Hz) of the lateral perforant path in an experimental model of temporal lobe epilepsy (i.e., kindled rats). Although control slices showed steady EPSP depression at frequencies greater than 20 Hz, slices taken from animals 48 h after the last seizure presented pronounced EPSP facilitation at 50 and 100 Hz, followed by steady depression. However, 28 days after kindling, the EPSP facilitation was no longer detectable. Using the specific N-methyl-D-aspartate (NMDA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptor antagonists 2-amino-5-phosphonovaleric acid and SYM 2206, we examined the time course of alterations in glutamate receptor-dependent synaptic currents that parallel transient EPSP facilitation. Forty-eight hours after kindling, the fractional AMPA and NMDA receptor-mediated excitatory postsynaptic current (EPSC) components shifted dramatically in favor of the NMDA receptor-mediated response. Four weeks after kindling, however, AMPA and NMDA receptor-mediated EPSCs reverted to control-like values. Although the granule cells of the dentate gyrus contain mRNA-encoding kainate receptors, neither single nor repetitive perforant path stimuli evoked kainate receptor-mediated EPSCs in control or in kindled rats. The enhanced excitability of the kindled dentate gyrus 48 h after the last seizure, as well as the breakdown of its gating function, appear to result from transiently enhanced NMDA receptor activation that provides significantly slower EPSC kinetics than those observed in control slices and in slices from kindled animals with a 28-day seizure-free interval. Therefore, NMDA receptors seem to play a critical role in the acute throughput of seizure activity and in the induction of the kindled state but not in the persistence of enhanced seizure susceptibility.     

Contributions of mossy fiber and CA1 pyramidal cell sprouting to dentate granule cell hyperexcitability in kainic acid-treated hippocampal slice cultures

Axonal sprouting like that of the mossy fibers is commonly associated with temporal lobe epilepsy, but its significance remains uncertain. To investigate the functional consequences of sprouting of mossy fibers and alternative pathways, kainic acid (KA) was used to induce robust mossy fiber sprouting in hippocampal slice cultures. Physiological comparisons documented many similarities in granule cell responses between KA- and vehicle-treated cultures, including: seizures, epileptiform bursts, and spontaneous excitatory postsynaptic currents (sEPSCs) >600 pA. GABAergic control and contribution of glutamatergic synaptic transmission were similar. Analyses of neurobiotin-filled CA1 pyramidal cells revealed robust axonal sprouting in both vehicle- and KA-treated cultures, which was significantly greater in KA-treated cultures. Hilar stimulation evoked an antidromic population spike followed by variable numbers of postsynaptic potentials (PSPs) and population spikes in both vehicle- and KA-treated cultures. Despite robust mossy fiber sprouting, knife cuts separating CA1 from dentate gyrus virtually abolished EPSPs evoked by hilar stimulation in KA-treated but not vehicle-treated cultures, suggesting a pivotal role of functional afferents from CA1 to dentate gyrus in KA-treated cultures. Together, these findings demonstrate striking hyperexcitability of dentate granule cells in long-term hippocampal slice cultures after treatment with either vehicle or KA. The contribution to hilar-evoked hyperexcitability of granule cells by the unexpected axonal projection from CA1 to dentate in KA-treated cultures reinforces the idea that axonal sprouting may contribute to pathologic hyperexcitability of granule cells.     

Mossy fiber-granule cell synapses in the normal and epileptic rat dentate gyrus studied with minimal laser photostimulation.

Dentate granule cells become synaptically interconnected in the hippocampus of persons with temporal lobe epilepsy, forming a recurrent mossy fiber pathway. This pathway may contribute to the development and propagation of seizures. The physiology of mossy fiber-granule cell synapses is difficult to characterize unambiguously, because electrical stimulation may activate other pathways and because there is a low probability of granule cell interconnection. These problems were addressed by the use of scanning laser photostimulation in slices of the caudal hippocampal formation. Glutamate was released from a caged precursor with highly focused ultraviolet light to evoke action potentials in a small population of granule cells. Excitatory synaptic currents were recorded in the presence of bicuculline. Minimal laser photostimulation evoked an apparently unitary excitatory postsynaptic current (EPSC) in 61% of granule cells from rats that had experienced pilocarpine-induced status epilepticus followed by recurrent mossy fiber growth. An EPSC was also evoked in 13-16% of granule cells from the control groups. EPSCs from status epilepticus and control groups had similar peak amplitudes ( approximately 30 pA), 20-80% rise times (approximately 1.2 ms), decay time constants ( approximately 10 ms), and half-widths (approximately 8 ms). The mean failure rate was high (approximately 70%) in both groups, and in both groups activation of N-methyl-D-aspartate receptors contributed a small component to the EPSC. The strong similarity between responses from the status epilepticus and control groups suggests that they resulted from activation of a similar synaptic population. No EPSC was recorded when the laser beam was focused in the dentate hilus, suggesting that indirect activation of hilar mossy cells contributed little, if at all, to these results. Recurrent mossy fiber growth increases the density of mossy fiber-granule cell synapses in the caudal dentate gyrus by perhaps sixfold, but the new synapses appear to operate very similarly to preexisting mossy fiber-granule cell synapses.     

Opposing modifications in intrinsic currents and synaptic inputs in post-traumatic mossy cells: evidence for single-cell homeostasis in a hyperexcitable network

Recent experimental and modeling results demonstrated that surviving mossy cells in the dentate gyrus play key roles in the generation of network hyperexcitability. Here we examined if mossy cells exhibit long-term plasticity in the posttraumatic, hyperexcitable dentate gyrus. Mossy cells 1 wk after fluid percussion head injury did not show alterations in their current-firing frequency (I-F) and current-membrane voltage (I-V) relationships. In spite of the unchanged I-F and I-V curves, mossy cells showed extensive modifications in Na(+), K(+) and h-currents, indicating the coordinated nature of these opposing modifications. Computational experiments in a realistic large-scale model of the dentate gyrus demonstrated that individually, these perturbations could significantly affect network activity. Synaptic inputs also displayed systematic, opposing modifications. Miniature excitatory postsynaptic current (EPSC) amplitudes were decreased, whereas miniature inhibitory postsynaptic current (IPSC) amplitudes were increased as expected from a homeostatic response to network hyperexcitability. In addition, opposing alterations in miniature and spontaneous synaptic event frequencies and amplitudes were observed for both EPSCs and IPSCs. Despite extensive changes in synaptic inputs, cannabinoid-mediated depolarization-induced suppression of inhibition was not altered in posttraumatic mossy cells. These data demonstrate that many intrinsic and synaptic properties of mossy cells undergo highly specific, long-term alterations after traumatic brain injury. The systematic nature of such extensive and opposing alterations suggests that single-cell properties are significantly influenced by homeostatic mechanisms in hyperexcitable circuits.     

Enhanced but fragile inhibition in the dentate gyrus in vivo in the kainic acid model of temporal lobe epilepsy: a study using current source density analysis

Temporal lobe epilepsy is related to many structural and physiological changes in the brain. We used kainic acid in rats as an animal model of temporal lobe epilepsy, and studied the neural interactions of the dentate gyrus in urethane-anesthetized rats in vivo. Our initial hypothesis was that sprouting of mossy fibers, the axons of the granule cells, increases proximal dendritic excitatory currents in the inner molecular layer of the dentate gyrus. Extracellular currents were detected in vivo using current source density analysis. Backfiring the mossy fibers in CA3 or orthodromic excitation of the granule cells through the medial perforant path induced a current sink at the inner molecular layer. However, the sink or inferred excitation at the inner molecular layer was not increased in kainic acid-treated rats and the sink actually correlated negatively with the degree of mossy fiber sprouting. It is inferred that the latter sink was mediated mainly by association fibers and not by recurrent mossy fibers. After kainic acid treatment, paired-pulse inhibition of the population spikes in the dentate gyrus was increased. In contrast, reverberant activity that involved looping around an entorhinal-hippocampal circuit was increased in kainic acid-treated rats, compared to control rats. The increase of inhibition in kainic acid-treated rats was readily blocked by a small dose of GABA(A) receptor antagonist bicuculline. The latter dose of bicuculline induced paroxsymal spike bursts in kainic acid-treated but not control rats, demonstrating that the increased inhibition in dentate gyrus was fragile.In conclusion, after kainic acid induced seizures, the dentate gyrus in vivo showed an increase in inhibition that appeared to be fragile. The hypothesized increase in proximal dendritic excitation due to mossy fiber sprouting was not detected. However, the fragile inhibition could explain the seizure susceptibility in patients with temporal lobe epilepsy.     

Synaptic connections from multiple subfields contribute to granule cell hyperexcitability in hippocampal slice cultures

Limbic status epilepticus and preparation of hippocampal slice cultures both produce cell loss and denervation. This commonality led us to hypothesize that morphological and physiological alterations in hippocampal slice cultures may be similar to those observed in human limbic epilepsy and animal models. To test this hypothesis, we performed electrophysiological and morphological analyses in long-term (postnatal day 11; 40-60 days in vitro) organotypic hippocampal slice cultures. Electrophysiological analyses of dentate granule cell excitability revealed that granule cells in slice cultures were hyperexcitable compared with acute slices from normal rats. In physiological buffer, spontaneous electrographic granule cell seizures were seen in 22% of cultures; in the presence of a GABA(A) receptor antagonist, seizures were documented in 75% of cultures. Hilar stimulation evoked postsynaptic potentials (PSPs) and multiple population spikes in the granule cell layer, which were eliminated by glutamate receptor antagonists, demonstrating the requirement for excitatory synaptic transmission. By contrast, under identical recording conditions, acute hippocampal slices isolated from normal rats exhibited a lack of seizures, and hilar stimulation evoked an isolated population spike without PSPs. To examine the possibility that newly formed excitatory synaptic connections to the dentate gyrus contribute to granule cell hyperexcitability in slice cultures, anatomical labeling and electrophysiological recordings following knife cuts were performed. Anatomical labeling of individual dentate granule, CA3 and CA1 pyramidal cells with neurobiotin illustrated the presence of axonal projections that may provide reciprocal excitatory synaptic connections among these regions and contribute to granule cell hyperexcitability. Knife cuts severing connections between CA1 and the dentate gyrus/CA3c region reduced but did not abolish hilar-evoked excitatory PSPs, suggesting the presence of newly formed, functional synaptic connections to the granule cells from CA1 and CA3 as well as from neurons intrinsic to the dentate gyrus. Many of the electrophysiological and morphological abnormalities reported here for long-term hippocampal slice cultures bear striking similarities to both human and in vivo models, making this in vitro model a simple, powerful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis.     

NMDA-dependent currents in granule cells of the dentate gyrus contribute to induction but not permanence of kindling

Single-electrode voltage-clamp techniques and bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV) were used to study the time course of seizure-induced alterations in NMDA-dependent synaptic currents in granule cells of the dentate gyrus in hippocampal slices from kindled and normal rats. In agreement with previous studies, granule cells from kindled rats examined within 1 wk after the last of 3 or 30-35 generalized tonic-clonic (class V) seizures demonstrated an increase in the NMDA receptor-dependent component of the perforant path-evoked synaptic current. Within 1 wk of the last kindled seizure, NMDA-dependent charge transfer underlying the perforant path-evoked current was increased by 63-111% at a holding potential of -30 mV. In contrast, the NMDA-dependent component of the perforant-evoked current in granule cells examined at 2.5-3 mo after the last of 3 or 90-120 class V seizures did not differ from age-matched controls. Because the seizure-induced increases in NMDA-dependent synaptic currents declined toward control values during a time course of 2.5-3 mo, increases in NMDA-dependent synaptic transmission cannot account for the permanent susceptibility to evoked and spontaneous seizures induced by kindling. The increase in NMDA receptor-dependent transmission was associated with the induction of kindling but was not responsible for the maintenance of the kindled state. The time course of alterations in NMDA-dependent synaptic current and the dependence of the progression of kindling and kindling-induced mossy fiber sprouting on repeated NMDA receptor activation are consistent with the possibility that the NMDA receptor is part of a transmembrane signaling pathway that induces long-term cellular alterations and circuit remodeling in response to repeated seizures, but is not required for permanent seizure susceptibility in circuitry altered by kindling.     

Optical recording study of granule cell activities in the hippocampal dentate gyrus of kainate-treated rats

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.     

Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy

Human mesial temporal lobe epilepsy is characterized by hippocampal seizures associated with pyramidal cell loss in the hippocampus and dispersion of dentate gyrus granule cells. A similar histological pattern was recently described in a model of extensive neuroplasticity in adult mice after injection of kainate into the dorsal hippocampus [Suzuki et al. (1995) Neuroscience 64, 665-674]. The aim of the present study was to determine whether (i) recurrent seizures develop in mice after intrahippocampal injection of kainate, and (ii) the electroencephalographic, histopathological and behavioural changes in such mice are similar to those in human mesial temporal lobe epilepsy. Adult mice receiving a unilateral injection of kainate (0.2 microg; 50 nl) or saline into the dorsal hippocampus displayed recurrent paroxysmal discharges on the electroencephalographic recordings associated with immobility, staring and, occasionally, clonic components. These seizures started immediately after kainate injection and recurrid for up to eight months. Epileptiform activities occurred most often during sleep but occasionally while awake. The pattern of seizures did not change over time nor did they secondarily generalize. Glucose metabolic changes assessed by [14C]2-deoxyglucose autoradiography were restricted to the ipsilateral hippocampus for 30 days, but had spread to the thalamus by 120 days after kainate. Ipsilateral cell loss was prominent in hippocampal pyramidal cells and hilar neurons. An unusual pattern of progressive enlargement of the dentate gyrus was observed with a marked radial dispersion of the granule cells associated with reactive astrocytes. Mossy fibre sprouting occurred both in the supragranular molecular layer and infrapyramidal stratum oriens layer of CA3. The expression of the embryonic form of the neural cell adhesion molecule coincided over time with granule cell dispersion. Our data describe the first histological, electrophysiological and behavioural evidence suggesting that discrete excitotoxic lesions of the hippocampus in mice can be used as an isomorphic model of mesial temporal lobe epilepsy.     

Potentiation of spontaneous synaptic activity in rat mossy cells.

Recent studies have demonstrated the vulnerability of dentate mossy cells to seizure-induced damage. One source of potentially damaging synaptic input are spontaneously active granule cell terminals ('mossy terminals'.) We sought to test whether there were activity-dependent changes in the spontaneous excitatory input to mossy cells. Using the in vitro slice preparation, we examined the frequency and amplitude of spontaneous excitatory postsynaptic potentials (EPSPs) after intracellular current injection designed to mimic the extreme depolarization these neurons receive during repetitive afferent stimulation. In 4 of 7 neurons, depolarization with trains of current pulses resulted in a significant and persistent increase in frequency of spontaneous synaptic depolarizations (to an average of 178% of the initial baseline rate). In 3 of these affected neurons, an increased frequency of large amplitude, fast-rising EPSPs accounted for the majority of this change. Injection of hyperpolarizing current pulses failed to alter spontaneous activity in 3 other mossy cells. These results suggest spontaneous synaptic input to mossy cells in plastic and can be potentiated by depolarization of a single postsynaptic mossy cell. The ability of mossy cells to potentiate their excitatory input may be relevant to their vulnerability to excitotoxic injury during repetitive afferent stimulation.     

Survival of dentate hilar mossy cells after pilocarpine-induced seizures and their synchronized burst discharges with area CA3 pyramidal cells.

The clinical and basic literature suggest that hilar cells of the dentate gyrus are damaged after seizures, particularly prolonged and repetitive seizures. Of the cell types within the hilus, it appears that the mossy cell is one of the most vulnerable. Nevertheless, hilar neurons which resemble mossy cells appear in some published reports of animal models of epilepsy, and in some cases of human temporal lobe epilepsy. Therefore, mossy cells may not always be killed after severe, repeated seizures. However, mossy cell survival in these studies was not completely clear because the methods did allow discrimination between mossy cells and other hilar cell types. Furthermore, whether surviving mossy cells might have altered physiology after seizures was not examined. Therefore, intracellular recording and intracellular dye injection were used to characterize hilar cells in hippocampal slices from pilocarpine-treated rats that had status epilepticus and recurrent seizures ('epileptic' rats). For comparison, mossy cells were also recorded from age-matched, saline-injected controls, and pilocarpine-treated rats that failed to develop status epilepticus.Numerous hilar cells with the morphology, axon projection, and membrane properties of mossy cells were recorded in all three experimental groups. Thus, mossy cells can survive severe seizures, and those that survive retain many of their normal characteristics. However, mossy cells from epileptic tissue were distinct from mossy cells of control rats in that they generated spontaneous and evoked epileptiform burst discharges. Area CA3 pyramidal cells also exhibited spontaneous and evoked bursts. Simultaneous intracellular recordings from mossy cells and pyramidal cells demonstrated that their burst discharges were synchronized, with pyramidal cell discharges typically beginning first.From these data we suggest that hilar mossy cells can survive status epilepticus and chronic seizures. The fact that mossy cells have epileptiform bursts, and that they are synchronized with area CA3, suggest a previously unappreciated substrate for hyperexcitability in this animal model.     

High ratio of synaptic excitation to synaptic inhibition in hilar ectopic granule cells of pilocarpine-treated rats

After experimental status epilepticus, many dentate granule cells born into the postseizure environment migrate aberrantly into the dentate hilus. Hilar ectopic granule cells (HEGCs) have also been found in persons with epilepsy. These cells exhibit a high rate of spontaneous activity, which may enhance seizure propagation. Electron microscopic studies indicated that HEGCs receive more recurrent mossy fiber innervation than normotopic granule cells in the same animals but receive much less inhibitory innervation. This study used hippocampal slices prepared from rats that had experienced pilocarpine-induced status epilepticus to test the hypothesis that an imbalance of synaptic excitation and inhibition contributes to the hyperexcitability of HEGCs. Mossy fiber stimulation evoked a much smaller GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in HEGCs than in normotopic granule cells from either control rats or rats that had experienced status epilepticus. However, recurrent mossy fiber-evoked excitatory postsynaptic currents (EPSCs) of similar size were recorded from HEGCs and normotopic granule cells in status epilepticus-experienced rats. HEGCs exhibited the highest frequency of miniature excitatory postsynaptic currents (mEPSCs) and the lowest frequency of miniature inhibitory postsynaptic currents (mIPSCs) of any granule cell group. On average, both mEPSCs and mIPSCs were of higher amplitude, transferred more charge per event, and exhibited slower kinetics in HEGCs than in granule cells from control rats. Charge transfer per unit time in HEGCs was greater for mEPSCs and much less for mIPSCs than in the normotopic granule cell groups. A high ratio of excitatory to inhibitory synaptic function probably accounts, in part, for the hyperexcitability of HEGCs.     

Activation of NMDA receptors in rat dentate gyrus granule cells by spontaneous and evoked transmitter release

Activation of N-methyl-D-aspartate (NMDA) receptors by synaptically released glutamate in the nervous system is usually studied using evoked events mediated by a complex mixture of AMPA, kainate, and NMDA receptors. Here we have characterized pharmacologically isolated spontaneous NMDA receptor-mediated synaptic events and compared them to stimulus evoked excitatory postsynaptic currents (EPSCs) in the same cell to distinguish between various modes of activation of NMDA receptors. Spontaneous NMDA receptor-mediated EPSCs recorded at 34 degrees C in dentate gyrus granule cells (DGGC) have a frequency of 2.5 +/- 0.3 Hz and an average peak amplitude of 13.2 +/- 0.8 pA, a 10-90% rise time of 5.4 +/- 0.3 ms, and a decay time constant of 42.1 +/- 2.1 ms. The single-channel conductance estimated by nonstationary fluctuation analysis was 60 +/- 5 pS. The amplitudes (46.5 +/- 6.4 pA) and 10-90% rise times (18 +/- 2.3 ms) of EPSCs evoked from the entorhinal cortex/subiculum border are significantly larger than the same parameters for spontaneous events (paired t-test, P < 0.05, n = 17). Perfusion of 50 microM D(-)-2-amino-5-phosphonopentanoic acid blocked all spontaneous activity and caused a significant baseline current shift of 18.8 +/- 3.0 pA, thus identifying a tonic conductance mediated by NMDA receptors. The NR2B antagonist ifenprodil (10 microM) significantly reduced the frequency of spontaneous events but had no effect on their kinetics or on the baseline current or variance. At the same time, the peak current and charge of stimulus-evoked events were significantly diminished by ifenprodil. Thus spontaneous NMDA receptor-mediated events in DGGC are predominantly mediated by NR2A or possibly NR2A/NR2B receptors while the activation of NR2B receptors reduces the excitability of entorhinal afferents either directly or through an effect on the entorhinal cells.     

Epileptogenesis is associated with enhanced glutamatergic transmission in the perforant path

The perforant path provides the main excitatory input into the hippocampus and has been proposed to play a critical role in the generation of temporal lobe seizures. It has been hypothesized that changes in glutamatergic transmission in this pathway promote the epileptogenic process and seizure generation. We therefore asked whether epileptogenesis is associated with enhanced glutamatergic transmission from the perforant path to dentate granule cells. We used a rat model of temporal lobe epilepsy in which spontaneous seizures occur after an episode of pilocarpine-induced status epilepticus. Whole cell patch-clamp recordings were obtained from dentate granule cells in hippocampal slices from control and epileptic animals 3 wk after pilocarpine-induced status epilepticus. The paired pulse ratio of perforant path-evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) was reduced in tissue obtained from epileptic rats. This is consistent with an increase in release probability. N-methyl-D-aspartate (NMDA) receptor-mediated EPSCs were also prolonged. This prolongation could not be accounted for by decreased activity of glutamate transporters or by a change in NMDA receptor subunit composition in dentate granule cells, implying a change in NMDA receptor kinetics. This change in NMDA receptor kinetics was associated with the emergence of significant synaptic cross-talk, detected as a use-dependent block of receptors activated by medial perforant path synapses after lateral perforant path stimulation in MK-801. Enhanced glutamatergic transmission and the emergence of cross-talk among perforant path-dentate granule cell synapses may contribute to lowering seizure threshold.     

Neuropeptide-Y immunoreactivity in the pilocarpine model of temporal lobe epilepsy

 Neuropeptide-Y (NPY) is expressed by granule cells and mossy fibres of the hippocampal dentate gyrus during experimental temporal lobe epilepsy (TLE). This expression may represent an endogenous damping mechanism since NPY has been shown to block seizure-like events following high-frequency stimulation in hippocampal slices. The pilocarpine (PILO) model of epilepsy is characterized by an acute period of status epilepticus followed by spontaneous recurrent seizures and related brain damage. We report peroxidase-antiperoxidase immunostaining for NPY in several brain regions in this model. PILO-injected animals exhibited NPY immunoreactivity in the region of the mossy fibre terminals, in the dentate gyrus inner molecular layer and, in a few cases, within presumed granule cells. NPY immunoreactivity was also dramatically changed in the entorhinal cortex, amygdala and sensorimotor areas. In addition, PILO injected animals exhibited a reduction in the number of NPY-immunoreactive interneurons compared with controls. The results demonstrate that changes in NPY expression, including expression in the granule cells and mossy fibres and the loss of vulnerable NPY neurons, are present in the PILO model of TLE. However, the significance of this changed synthesis of NPY remains to be determined. Received: 19 August 1996 / Accepted: 21 March 1997