Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser-scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked EPSCs in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared with controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy.     

Detection of Increased Local Excitatory Circuits in the Hippocampus during Epileptogenesis Using Focal Flash Photolysis of Caged Glutamate

Summary:  Purpose: Local synaptic circuits, particularly recurrent excitation, are hypothesized to contribute to the generation and synchronization of epileptiform activity. The present study tested whether local excitatory circuits in the hippocampus are increased in an animal model of temporal lobe epilepsy, and thus may contribute to epileptic seizures.
Methods: Rats were given hourly injections of kainic acid to induce status epilepticus, which led to chronic epilepsy with spontaneous recurrent seizures. Whole-cell recording was performed in hippocampal slices, and focal flash photolysis of caged glutamate was used to detect local excitatory circuits.
Results: In the dentate gyrus of rats with kainate-induced epilepsy and mossy fiber sprouting, focal stimulations with caged glutamate at many different sites in the granule cell layer consistently evoked repetitive excitatory postsynaptic currents (EPSCs) in normal medium and prolonged bursts of action potentials in bicuculline; these responses were not observed in similarly treated slices from control rats. In CA1, focal flash photolysis of caged glutamate in stratum pyramidale revealed significantly more excitatory connections between CA1 pyramidal cells in rats with kainate-induced epilepsy than saline-treated control animals.
Conclusion: Focal flash photolysis of caged glutamate revealed that new local excitatory circuits are formed in both the dentate gyrus and CA1 area of rats with kainate-induced epilepsy, which supports the hypothesis that the progressive formation of new local excitatory circuits occurs in many locations during epileptogenesis.     

Neuroprotective effects of a postischemic treatment with a bradykinin B2 receptor antagonist in a rat model of temporary focal cerebral ischemia

Recurrent mossy fiber synapses in the dentate gyrus of epileptic brain facilitate the synchronous firing of granule cells and may promote seizure propagation. Mossy fiber terminals contain and release zinc. Released zinc inhibits the activation of NMDA receptors and may therefore oppose the development of granule cell epileptiform activity. Hippocampal slices from rats that had experienced pilocarpine-induced status epilepticus and developed a recurrent mossy fiber pathway were used to investigate this possibility. Actions of released zinc were inferred from the effects of chelation with 1 mM calcium disodium EDTA (CaEDTA). When granule cell population bursts were evoked by mossy fiber stimulation in the presence of 6 mM K(+) and 30 microM bicuculline, CaEDTA slowed the rate at which evoked bursting developed, but did not change the magnitude of the bursts once they had developed fully. The effects of CaEDTA were then studied on the pharmacologically isolated NMDA receptor- and AMPA/kainate receptor-mediated components of the fully developed bursts. CaEDTA increased the magnitude of NMDA receptor-mediated bursts and reduced the magnitude of AMPA/kainate receptor-mediated bursts. CaEDTA did not affect the granule cell bursts evoked in slices from untreated rats by stimulating the perforant path in the presence of bicuculline and 6 mM K(+). These results suggest that zinc released from the recurrent mossy fibers serves mainly to facilitate the recruitment of dentate granule cells into population bursts.     

Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats: Visualization after retrograde transport of biocytin

In complex partial epilepsy and in animal models of epilepsy, hippocampal mossy fibers appear to develop recurrent collaterals, that invade the dentate molecular layer. Mossy fiber collaterals have been proposed to subserve recurrent excitation by forming granule cell-granule cell synapses. This hypothesis was tested by visualizing dentate granule cells and their mossy fibers after terminal uptake and retrograde transport of biocytin. Labeling studies were performed with transverse slices of the caudal rat hippocampal formation prepared 2.6–l70.0 weeks after pilocarpine-induced or kainic acid-induced status epilepticus. Light microscopy demonstrated the progressive growth of recurrent mossy fibers into the molecular layer; the densest innervation was observed in slices from pilocarpine-treated rats that had survived 10 weeks or longer after status epilepticus. Thin mossy fiber collaterals originated predominantly from deep within the hilar region, crossed the granule cell body layer, and formed an axonal plexus oriented parallel to the cell body layer within the inner one-third of the molecular layer. When sprouting was most robust, some recurrent mossy fibers at the apex of the dentate gyrus reached the outer two-thirds of the molecular layer. The distribution and density of mossy fiber-like Timm staining correlated with the biocytin labeling. When viewed with the electron microscope, the inner one-third of the dentate molecular layer contained numerous mossy fiber boutons. In some instances, biocytin-labeled mossy fiber boutons were engaged in synaptic contact with biocytin-labeled granule cell dendrites. Granule cell dendrites did not develop large complex spines (“thorny excrescences”) at the site of synapse formation, and they did not appear to have been permanently damaged by seizure activity. These results establish the validity of Timm staining as a marker for mossy fiber sprouting and support the view that status epilepticus provokes the formation of a novel recurrent excitatory circuit in the dentate gyrus. Retrograde labeling with biocytin showed that the recurrent mossy fiber projection often occupies a considerably greater fraction of the dendritic region than previous studies had suggested. © 1995 Wiley-Liss, Inc.     

Physiologic and Morphologic Characteristics of Granule Cell Circuitry in Human Epileptic Hippocampus

Summary: Morphological and electrophysiological techiques were used to examine granule cells and their mossy fiber axons in nine surgically resected hippocampal specimens from temporal lobe epilepsy (TLE) patients. Timm histochemistry showed mossy fiber sprouting into the inner molecular layer (IML) of the dentate in a subset of tissue samples. In slices from five tissue samples, stimulus-induced bursting activity could be induced with a low concentration (2.5 μM) of bicuculline; bursts were sensitive to the N -methyl- d -aspartate (NMDA) blocker, APV. There was a general correlation between such sprouting and experimentally induced yperexcit ability. Fourteen granule cells from five tissue samples were intracellularly stained [with lucifer yellow (LY) or neurobiotin]. Axons from a subset of these neurons showed axon collaterals reaching into the IML, but this axon projection pattern for single cells was not directly correlated with degree of mossy fiber sprouting shown grossly by Timm staining. Electron microscopic examination of intracellularly stained elements showed mossy fiber axon terminals making asymmetric synaptic contacts (including autapses on the granule cell dendrite) with dendritic shafts and spines in both apical and basal domains. These data are consistent with the hypothesis that mossy fiber sprouting provides a structural basis for recurrent excitation of granule cells, but does not provide direct support of the hypothesis that mossy fiber sprouting causes hyperexcitability. The data suggest that granule cell bursting activity is at least in part a function of compromised synaptic inhibition, since levels of γ-aminobutyric acid (GABA) blockade that are generally subthreshold for burst induction were epileptogenic in some tissue samples from human epileptic hippocampus.     

Kindling induces transient fast inhibition in the dentate gyrus--CA3 projection

The granule cells of the dentate gyrus (DG) send a strong glutamatergic projection, the mossy fibre tract, toward the hippocampal CA3 field, where it excites pyramidal cells and neighbouring inhibitory interneurons. Despite their excitatory nature, granule cells contain small amounts of GAD (glutamate decarboxylase), the main synthetic enzyme for the inhibitory transmitter GABA. Chronic temporal lobe epilepsy results in transient upregulation of GAD and GABA in granule cells, giving rise to the speculation that following overexcitation, mossy fibres exert an inhibitory effect by release of GABA. We therefore stimulated the DG and recorded synaptic potentials from CA3 pyramidal cells in brain slices from kindled and control rats. In both preparations, DG stimulation caused excitatory postsynaptic potential (EPSP)/inhibitory postsynaptic potential (IPSP) sequences. These potentials could be completely blocked by glutamate receptor antagonists in control rats, while in the kindled rats, a bicuculline-sensitive fast IPSP remained, with an onset latency similar to that of the control EPSP. Interestingly, this IPSP disappeared 1 month after the last seizure. When synaptic responses were evoked by high-frequency stimulation, EPSPs in normal rats readily summate to evoke action potentials. In slices from kindled rats, a summation of IPSPs overrides that of the EPSPs and reduces the probability of evoking action potentials. Our data show for the first time that kindling induces functionally relevant activity-dependent expression of fast inhibition onto pyramidal cells, coming from the DG, that can limit CA3 excitation in a frequency-dependent manner.     

Synaptic reorganization in explanted cultures of rat hippocampus

Due to loss of afferent innervation, synaptic reorganization occurs in organotypic hippocampal slice cultures. With extra- and intracellular recordings, we confirm that the excitatory loop from the dentate gyrus (DG) to CA3 and further to CA1 is preserved. However, hilar stimulation evoked antidromic population spikes in the DG which were followed by a population postsynaptic potential (PPSP); intracellularly, an antidromic spike with a broad shoulder or EPSP/IPSP sequences were induced. Synaptic responses were blocked by glutamate receptor antagonists. Stimulation of CA1 induced a PPSP in DG. Dextranamine stained pyramidal cells of CA1 were shown to project to DG. After removal of area CA3, DG's and mossy fibers' (MF) stimulation still elicited PPSPs and EPSP/IPSP sequences in area CA1 which disappeared when a cut was made through the hippocampal fissure. During bicuculline perfusion, hilar stimulation caused EPSPs in granule cells and spontaneous and evoked repetitive firing appeared even after its isolation from areas CA3 and CA1. Collateral excitatory synaptic coupling between granule cells was confirmed by paired recordings. Besides the preservation of the trisynaptic pathway in this preparation, new functional synaptic contacts appear, presumably due to MF collateral sprouting and formation of pathways between areas CA1 and DG.     

Abnormal responses to perforant path stimulation in the dentate gyrus of slices from rats with kainate-induced epilepsy and mossy fiber reorganization.

Previous electrophysiological studies have demonstrated that in a subset of hippocampal slices from tissue resected from patients with mesial temporal lobe epilepsy, perforant path stimulation can elicit prolonged negative field-potential shifts in the dentate granule cell layer (Masukawa et al., 1989. Brain Res. 493, 168-174; Isokawa and Fried, 1996. Neuroscience 72, 31-37). In this investigation, hippocampal slices were prepared from rats: (1) 2-4 days following kainate treatment, when little or no reorganization of the mossy fibers would be present and (2) 3-13 months after kainate treatment, when mossy fiber reorganization would have occurred. In saline-treated controls, perforant path stimulation typically evoked a single population spike. In contrast, perforant path stimulation could evoke 3-12 population spikes in nearly all slices from kainate-injected rats 2-4 days and 3-13 months after treatment. The majority of slices from kainate-injected rats 3-13 months after treatment had qualitatively similar responses to perforant path stimulation as that observed in slices from kainate-injected rats 2-4 days after treatment. However, in 17% of the slices from kainate-treated rats 3-13 months after treatment (29% of rats), the multiple population spikes were followed by a prolonged negative field-potential shift (duration: 140 ms-1.5 s) with variable superimposed population spike activity. This type of epileptiform activity was only observed in slices with robust Timm's staining in the inner molecular layer and similar responses could also be evoked in these slices with hilar stimulation. Furthermore, pharmacological depression of inhibition by adding the GABA(A) receptor antagonist bicuculline unmasked hilar-evoked prolonged negative field-potential shifts in most slices from kainate-treated rats 3-13 months following treatment, and these slices had robust Timm's staining in the inner molecular layer. Such events were not observed in slices from saline-treated controls or kainate-injected rats 2-4 days after treatment. In conclusion, the prolonged negative field-potential shifts evoked to perforant path stimulation in normal ACSF were associated with mossy fiber reorganization, but the relative contribution of altered inhibition, increased synaptic excitation, or even non-synaptic mechanisms is unknown.     

Opioid modulation of recurrent excitation in the hippocampal dentate gyrus.

kappa opioid receptor activation inhibits granule cell-mediated excitatory neurotransmission in the hippocampal formation via a decrease in glutamate release from both perforant path and mossy fiber terminals. We now report a third, anatomically and pharmacologically distinct site of such kappa opioid inhibition within the hippocampus. Granule cell population responses to selective stimulation of an excitatory hilar pathway were decreased by the kappa(1) opioid receptor agonist U69,593, an effect blocked by the kappa(1) antagonist norbinaltorphimine. U69,593 also inhibited hilar path induced long-term potentiation (LTP) of granule cell responses. LTP in this pathway was also blocked by the NMDA receptor antagonist d-2-amino-5-phosphonovalerate, unlike granule cell mossy fiber LTP in CA3. The kappa opioid peptide dynorphin is present in hilar mossy fiber collaterals. Ultrastructural analysis of these collaterals demonstrated dynorphin-containing vesicles in asymmetric synapses formed between axon terminals and granule cell dendrites, suggesting direct granule cell-granule cell connections. Evoked release of endogenous dynorphin within the hilus was effective in reducing hilar excitation of granule cells, although this release, in contrast to the release of dynorphin in the dentate molecular layer, was not dependent on L-type calcium channels. No hilar path excitation was observed in the absence of bicuculline, suggesting a strong GABA(A)-mediated inhibition of this pathway. However, hilar path activity could be seen after LTP, with or without bicuculline. Thus, kappa opioids can inhibit granule cell recurrent excitation, likely via effects on excitatory mossy fiber collaterals. Such collaterals are thought to be important in mediating temporal lobe epilepsy.     

Recurrent excitation in the dentate gyrus of a murine model of temporal lobe epilepsy

Similar to rats, systemic pilocarpine injection causes status epilepticus (SE) and the eventual development of spontaneous seizures and mossy fiber sprouting in C57BL/6 and CD1 mice, but the physiological correlates of these events have not been identified in mice. Population responses in granule cells of the dentate gyrus were examined in transverse slices of the ventral hippocampus from pilocarpine-treated and untreated mice. In Mg(2+)-free bathing medium containing bicuculline, conditions designed to increase excitability in the slices, electrical stimulation of the hilus resulted in a single population spike in granule cells from control mice and pilocarpine-treated mice that did not experience SE. In SE survivors, similar stimulation resulted in a population spike followed, at a variable latency, by negative DC shifts and repetitive afterdischarges of 3-60 s duration, which were blocked by ionotropic glutamate receptor antagonists. Focal glutamate photostimulation of the granule cell layer at sites distant from the recording pipette resulted in population responses of 1-30 s duration in slices from SE survivors but not other groups. These data support the hypothesis that SE-induced mossy fiber sprouting and synaptic reorganization are relevant characteristics of seizure development in these murine strains, resembling rat models of human temporal lobe epilepsy.     

Kainic acid-induced recurrent mossy fiber innervation of dentate gyrus inhibitory interneurons: possible anatomical substrate of granule cell hyper-inhibition in chronically epileptic rats

Kainic acid-induced neuron loss in the hippocampal dentate gyrus may cause epileptogenic hyperexcitability by triggering the formation of recurrent excitatory connections among normally unconnected granule cells. We tested this hypothesis by assessing granule cell excitability repeatedly within the same awake rats at different stages of the synaptic reorganization process initiated by kainate-induced status epilepticus (SE). Granule cells were maximally hyperexcitable to afferent stimulation immediately after SE and became gradually less excitable during the first month post-SE. The chronic epileptic state was characterized by granule cell hyper-inhibition, i.e., abnormally increased paired-pulse suppression and an abnormally high resistance to generating epileptiform discharges in response to afferent stimulation. Focal application of the gamma-aminobutyric acid type A (GABA(A)) receptor antagonist bicuculline methiodide within the dentate gyrus abolished the abnormally increased paired-pulse suppression recorded in chronically hyper-inhibited rats. Combined Timm staining and parvalbumin immunocytochemistry revealed dense innervation of dentate inhibitory interneurons by newly formed, Timm-positive, mossy fiber terminals. Ultrastructural analysis by conventional and postembedding GABA immunocytochemical electron microscopy confirmed that abnormal mossy fiber terminals of the dentate inner molecular layer formed frequent asymmetrical synapses with inhibitory interneurons and with GABA-immunopositive dendrites as well as with GABA-immunonegative dendrites of presumed granule cells. These results in chronically epileptic rats demonstrate that dentate granule cells are maximally hyperexcitable immediately after SE, prior to mossy fiber sprouting, and that synaptic reorganization following kainate-induced injury is temporally associated with GABA(A) receptor-dependent granule cell hyper-inhibition rather than a hypothesized progressive hyperexcitability. The anatomical data provide evidence of a possible anatomical substrate for the chronically hyper-inhibited state.     

Residual granule cells can maintain susceptibility of CA3 pyramidal cells to kainate-induced epileptiform discharges

Slices of adult rat hippocampus made from animals exposed neonatally to X-ray irradiation were studied with electrophysiological techniques. A single dose of 6 Gy irradiation of the pup's head significantly but unevenly reduced the number of granule cells in the dentate gyrus. A larger reduction was detected in the septal than in the temporal hippocampus. The number of hilar cells decreased also. Effects of irradiation were confirmed with histological techniques. Field potential responses to mossy fiber stimulation in the pyramidal layer of the CA3 subfield was smaller in irradiated than in normal rats. Superfusion of the slices with kainic acid (KA, 300–500 nM ) induced spontaneously recurrent paroxysmal activity (SRPA) in about 40% of irradiated slices in contrast with nearly 90% of slices cut from nonirradiated rats. Intracellular recordings from CA3 pyramidal cells in irradiated rats revealed recurrent bursts of action potentials on top of large depolarizing waves after KA application. Cells impaled in slices from the septal half of hippocampus of irradiated rats failed more often to respond with bursts to KA than cells in slices cut from the temporal half. Removal of mossy fiber input can therefore reduce KA induced hyperexcitability of CA3 pyramidal cells, but quantitative factors such as proportional loss of granule and hilar cells may explain the considerable differences found among cells and slices. Removal of 80% of granule cells reduces hyperexcitability consistently, while SRPA can be found in slices where as much as 50% of granule cells are missing. Intracellular findings suggest that failures of detection of SRPA following KA application to hippocampal slices of irradiated rats does not necessarily mean that CA3 pyramidal cells are no longer responding to KA with epileptiform bursting. Hippocampus 1998;8:548–561. © 1998 Wiley-Liss, Inc.     

Hippocampal granule cell activity and c-Fos expression during spontaneous seizures in awake, chronically epileptic, pilocarpine-treated rats: implications for hippocampal epileptogenesis

The process of postinjury hippocampal epileptogenesis may involve gradually developing dentate granule cell hyperexcitability caused by neuron loss and synaptic reorganization. We tested this hypothesis by repeatedly assessing granule cell excitability after pilocarpine-induced status epilepticus (SE) and monitoring granule cell behavior during 235 spontaneous seizures in awake, chronically implanted rats. During the first week post-SE, granule cells exhibited diminished paired-pulse suppression and decreased seizure discharge thresholds in response to afferent stimulation. Spontaneous seizures often began during the first week after SE, recruited granule cell discharges that followed behavioral seizure onsets, and evoked c-Fos expression in all hippocampal neurons. Paired-pulse suppression and epileptiform discharge thresholds increased gradually after SE, eventually becoming abnormally elevated. In the chronic epileptic state, interictal granule cell hyperinhibition extended to the ictal state; granule cells did not discharge synchronously before any of 191 chronic seizures. Instead, granule cells generated only low-frequency voltage fluctuations (presumed "field excitatory postsynaptic potentials") during 89% of chronic seizures. Granule cell epileptiform discharges were recruited during 11% of spontaneous seizures, but these occurred only at the end of each behavioral seizure. Hippocampal c-Fos after chronic seizures was expressed primarily by inhibitory interneurons. Thus, granule cells became progressively less excitable, rather than hyperexcitable, as mossy fiber sprouting progressed and did not initiate the spontaneous behavioral seizures. These findings raise doubts about dentate granule cells as a source of spontaneous seizures in rats subjected to prolonged SE and suggest that dentate gyrus neuron loss and mossy fiber sprouting are not primary epileptogenic mechanisms in this animal model.     

Mossy fiber sprouting as a potential therapeutic target for epilepsy

Hippocampal mossy fibers, axons of dentate granule cells, converge in the dentate hilus and run through a narrow area called the stratum lucidum to synapse with hilar and CA3 neurons. In the hippocampal formation of temporal lobe epilepsy patients, however, this stereotyped pattern of projection is often collapsed; the mossy fibers branch out of the dentate hilus and abnormally innervate the dentate inner molecular layer, a phenomenon that is termed mossy fiber sprouting. Experimental studies have replicated this sprouting in animal models of temporal lobe epilepsy, including kindling and pharmacological treatment with convulsants. Because these axon collaterals form recurrent excitatory inputs into dendrites of granule cells, the circuit reorganization is assumed to cause epileptiform activity in the hippocampus, whereas some recent studies indicate that the sprouting is not necessarily associated with early-life seizures. Here we review the mechanisms of mossy fiber sprouting and consider its potential contribution to epileptogenesis. Based on recent findings, we propose that the sprouting can be regarded as a result of disruption of the molecular mechanisms underlying the axon guidance. We finally focus on the possibility that prevention of the abnormal sprouting might be a new strategy for medical treatment with temporal lobe epilepsy.     

“Epileptic” brain damage in rats induced by sustained electrical stimulation of the perforant path. I. Acute electrophysiological and light microscopic studies

Sustained electrical stimulation of the perforant path in urethane-anesthetized rats evoked hippocampal granule cell population spikes and epileptiform discharges. After stimulation, recurrent inhibition in the granule cell layer was abolished. Light microscopic analysis revealed a highly reproducible pattern of hippocampal damage to dentate pyramidal basket cells, hilar cells in general and CA3 and CA1 pyramidal cells. CA2 pyramidal cells and dentate granule cells were relatively unaffected. When perforant path stimulation on one side of the brain evoked bilateral granule cell discharges, damage was bilateral. Unilateral hippocampal seizures were associated with unilateral hippocampal damage. Rapid Golgi-stained hippocampi exhibited spherical dendritic swellings at the sites of termination of excitatory entorhinal afferents to the hippocampus and in the mossy fiber region. Electrical stimulation of a single excitatory afferent to the hippocampus appears to reproduce the “epileptic” pattern of hippocampal damage without using convulsant drugs and without causing motor convulsions. It is suggested that seizure-associated brain damage in caused by excessive pre-synaptic release of excitatory transmitter that induces intracellular post-synaptic changes that lead to dendritic swelling and cell death.     

Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry

Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus-induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine-treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells.     

Long‐lasting modulation of synaptic plasticity in rat hippocampus after early‐life complex febrile seizures

A small fraction of children with febrile seizures appears to develop cognitive impairments. Recent studies in a rat model of hyperthermia‐induced febrile seizures indicate that prolonged febrile seizures early in life have long‐lasting effects on the hippocampus and induce cognitive deficits. However, data on network plasticity and the nature of cognitive deficits are conflicting. We examined three specific measures of hippocampal plasticity in adult rats with a prior history of experimental febrile seizures: (i) activity‐dependent synaptic plasticity (long‐term potentiation and depression) by electrophysiological recordings of Schaffer collateral/commissural‐evoked field excitatory synaptic potentials in CA1 of acute hippocampal slices; (ii) Morris water maze spatial learning and memory; and (iii) hippocampal mossy fiber plasticity by Timm histochemistry and quantification of terminal sprouting in CA3 and the dentate gyrus. We found enhanced hippocampal CA1 long‐term potentiation and reduced long‐term depression but normal spatial learning and memory in adult rats that were subjected to experimental febrile seizures on postnatal day 10. Furthermore, rats with experimental febrile seizures showed modest but significant sprouting of mossy fiber collaterals into the inner molecular layer of the dentate gyrus in adulthood. We conclude that enhanced CA1 long‐term potentiation and mild mossy fiber sprouting occur after experimental febrile seizures, without affecting spatial learning and memory in the Morris water maze. These long‐term functional and structural alterations in hippocampal plasticity are likely to play a role in the enhanced seizure susceptibility in this model of prolonged human febrile seizures but do not correlate with overt cognitive deficits.     

Glutamate receptor involvement in dentate granule cell epileptiform activity evoked by mossy fiber stimulation

In many persons with temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network. This recurrent mossy fiber circuit mediates reverberating excitation that may facilitate seizure propagation by synchronizing granule cell discharge. The involvement of specific glutamate receptors in granule cell epileptiform activity evoked by stimulating the mossy fibers was investigated with use of rat hippocampal slices superfused with bicuculline, with or without increasing [K+](o) to 6 mM. The occurrence of short-latency mossy fiber-evoked granule cell epileptiform activity in slices from pilocarpine-treated rats correlated with the presence and extent of recurrent mossy fiber growth. Blockade of AMPA receptors nearly abolished the orthodromic component of the response; subsequent antagonism of kainate receptors as well appeared to have no further action. Antagonism of NMDA receptors reduced the duration of epileptiform discharge, but increased the amplitude of population spikes within the evoked burst. Thus AMPA and NMDA, but perhaps not kainate, receptors play an important role in this type of epileptiform activity. Activation of type II metabotropic glutamate receptors, which inhibits the release of glutamate from mossy fiber boutons, reduced the magnitude of epileptiform discharge. This action was reversed by a partial agonist of these receptors. However, neither an agonist nor an antagonist of type III metabotropic glutamate receptors significantly altered the response. Considering the importance of synchronous granule cell discharge for seizure propagation from the entorhinal cortex to the hippocampus, agonists of type II metabotropic glutamate receptors may be useful in suppressing such discharge both experimentally and clinically.     

Mossy fiber synaptic reorganization induced by kindling: time course of development, progression, and permanence

Recent studies have revealed that mossy fiber axons of granule cells in the dentate gyrus undergo reorganization of their terminal projections in both animal models of epilepsy and human epilepsy. This synaptic reorganization has been demonstrated by the Timm method, a histochemical technique that selectively labels synaptic terminals of mossy fibers because of their high zinc content. It has been generally presumed that the reorganization of the terminal projections of the mossy fiber pathway is a consequence of axonal sprouting and synaptogenesis by mossy fibers. To evaluate this possibility further, the time course for development of Timm granules, which correspond ultrastructurally to mossy fiber synaptic terminals, was examined in the supragranular layer of the dentate gyrus at the initiation of kindling stimulation with an improved scoring method for assessment of alterations in Timm histochemistry. The progression and permanence of this histological alteration were similarly evaluated during the behavioral and electrographic evolution of kindling evoked by perforant path, amygdala, or olfactory bulb stimulation. Mossy fiber synaptic terminals developed in the supragranular region of the dentate gyrus by 4 d after initiation of kindling stimulation in a time course compatible with axon sprouting. The induced alterations in the terminal projections of the mossy fiber pathway progressed with the evolution of behavioral kindled seizures, became permanent in parallel with the development of longlasting susceptibility to evoked seizures, and were observed as long as 8 months after the last evoked kindled seizure. The results demonstrated a strong correlation between mossy fiber synaptic reorganization and the development, progression, and permanence of the kindling phenomenon.     

Actions of brain-derived neurotrophic factor in slices from rats with spontaneous seizures and mossy fiber sprouting in the dentate gyrus.

This study examined the acute actions of brain-derived neurotrophic factor (BDNF) in the rat dentate gyrus after seizures, because previous studies have shown that BDNF has acute effects on dentate granule cell synaptic transmission, and other studies have demonstrated that BDNF expression increases in granule cells after seizures. Pilocarpine-treated rats were studied because they not only have seizures and increased BDNF expression in granule cells, but they also have reorganization of granule cell "mossy fiber" axons. This reorganization, referred to as "sprouting," involves collaterals that grow into novel areas, i.e., the inner molecular layer, where granule cell and interneuron dendrites are located. Thus, this animal model allowed us to address the effects of BDNF in the dentate gyrus after seizures, as well as the actions of BDNF on mossy fiber transmission after reorganization. In slices with sprouting, BDNF bath application enhanced responses recorded in the inner molecular layer to mossy fiber stimulation. Spontaneous bursts of granule cells occurred, and these were apparently generated at the site of the sprouted axon plexus. These effects were not accompanied by major changes in perforant path-evoked responses or paired-pulse inhibition, occurred only after prolonged (30-60 min) exposure to BDNF, and were blocked by K252a. The results suggest a preferential action of BDNF at mossy fiber synapses, even after substantial changes in the dentate gyrus network. Moreover, the results suggest that activation of trkB receptors could contribute to the hyperexcitability observed in animals with sprouting. Because human granule cells also express increased BDNF mRNA after seizures, and sprouting can occur in temporal lobe epileptics, the results may have implications for understanding temporal lobe epilepsy.