Increased densities of AMPA GluR1 subunit proteins and presynaptic mossy fiber sprouting in the fascia dentata of human hippocampal epilepsy

In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of ‘excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P<0.000001) and GluR1 protein densities were significantly higher (P<0.0005) than those of the non-HS group. Unlike supragranular mossy fiber sprouting, which was limited to the IML, the increased GluR1 stainings were distributed throughout the whole molecular layer. For all cases, MF synaptic reorganization in the supragranular ML was correlated with GluR1 subunit protein densities in the IML (R=0.784, P<0.0093). These data demonstrate that in the human epileptic fascia dentata, there are significantly increased AMPA GluR1 subunit proteins associated with aberrant MF synaptic reorganizations. This suggests that the hyperexcitability of sclerotic hippocampus occurs, at least in part, from the associated changes of both presynaptic mossy fiber glutamatergic neoinnervation and increased GluR1 subunit proteins in the dendritic domains of the FD.     

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.     

Stereological analysis of GluR2-immunoreactive hilar neurons in the pilocarpine model of temporal lobe epilepsy: correlation of cell loss with mossy fiber sprouting

Mossy fiber sprouting and the genesis of ectopic granule cells contribute to reverberating excitation in the dentate gyrus of epileptic brain. This study determined whether the extent of sprouting after status epilepticus in rats correlates with the seizure-induced degeneration of GluR2-immunoreactive (GluR2+) hilar neurons (presumptive mossy cells) and also quantitated granule cell-like GluR2-immunoreactive hilar neurons. Stereological cell counting indicated that GluR2+ neurons account for 57% of the total hilar neuron population. Prolonged pilocarpine-induced status epilepticus killed 95% of these cells. A smaller percentage of GluR2+ neurons (74%) was killed when status epilepticus was interrupted after 1-3.5 h with a single injection of phenobarbital, and the number of residual GluR2+ neurons varied among animals by a factor of 6.2. GluR2+ neurons were not necessarily more vulnerable than other hilar neurons. In rats administered phenobarbital, the extent of recurrent mossy fiber growth varied inversely and linearly with the number of GluR2+ hilar neurons that remained intact (P=0.0001). Thus the loss of each GluR2+ neuron was associated with roughly the same amount of sprouting. These findings support the hypothesis that mossy fiber sprouting is driven largely by the degeneration of and/or loss of innervation from mossy cells. Granule cell-like GluR2-immunoreactive neurons were rarely encountered in the hilus of control rats, but increased 6- to 140-fold after status epilepticus. Their number did not correlate with the extent of hilar cell death or mossy fiber sprouting in the same animal. The morphology, number, and distribution of these neurons suggested that they were hilar ectopic granule cells.     

Granule Cell Neurogenesis After Status Epilepticus in the Immature Rat Brain

Summary: Purpose : Several experimental paradigms of seizure induction that produce epilepsy as a consequence have been shown to be associated with the proliferation of dentate granule cells. In developing animals, the acute sequela of hilar damage and the chronic sequelae of spontaneous seizures and mossy fiber synaptic reorganization, in response to status epilepticus, occur in an age-dependent manner. We investigated seizure-induced granule cell neurogenesis in developing rat pups to study the association between hilar injury, granule cell neurogenesis, and epilepsy.
Methods : Rat pups of 2 and 3 weeks postnatal age were subjected to lithium-pilocarpine status epilepticus (LiPC SE). Rats were given bromodeoxyuridine (BrdU; 50 mg/kg intra-peritoneal) twice daily for 4 days beginning 3 days after SE to label dividing cells. Routine immunocytochemistry and quantification of BrdU labeling by image analysis were performed. Results were compared with previously reported data on cellular injury, mossy fiber sprouting, and spontaneous seizures in rat pups of these ages after LiPC SE.
Results : In 3-week-old pups, which demonstrate SE-induced hilar damage and develop spontaneous seizures accompanied by mossy fiber sprouting, the BrdU-immunoreactive area (percent) in the subgranular proliferative zone increased to 10·6 ± 2·5 compared with 1·4 ± 0·5 in the control animals (p < 0·05). The 2-week-old animals, which show neither hilar damage nor sprouting and rarely develop spontaneous seizures, also showed a comparable extent of SE-induced neurogenesis [8·0 ± 1·4 (LiPC SE) versus 0·4 ± 0·2 (control), p < 0·05].
Conclusions : Seizure-induced granule cell neurogenesis does not appear to be a function of seizure-induced hilar cellular damage. Granule cell neurogenesis induced by SE does not determine epileptogenesis in the developing rat.     

Stereologic estimation of hippocampal GluR2/3- and calretinin-immunoreactive hilar neurons (presumptive mossy cells) in two mouse models of temporal lobe epilepsy

Purpose: Hippocampal mossy cells receive dense innervation from dentate granule cells and, in turn, mossy cells innervate both granule cells and interneurons. Mossy cell loss is thought to trigger granule cell mossy fiber sprouting, which may affect granule cell excitability. The aim of this study was to quantify mossy cell loss in two animal models of temporal lobe epilepsy, and determine whether there exists a relationship between mossy cell loss, mossy fiber sprouting, and granule cell dispersion. Methods: Representative hippocampal sections from p35 knockout mice and mice with unilateral intrahippocampal kainate injection were immunolabeled for GluR2/3, two subunits of the amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor and calretinin to identify mossy cells. Mossy fibers were immunostained against synaptoporin. Key Findings: p35 Knockout mice showed no hilar cell death, but moderate mossy fiber sprouting and granule cell dispersion. In the kainate‐injected hippocampus, there was an 80% and 85% reduction of GluR2/3‐ and GluR2/3/calretinin‐positive hilar neurons, respectively, and dense mossy fiber sprouting and significant granule cell dispersion. In the contralateral hippocampus there was a 52% loss of GluR2/3‐, but only a 20% loss of GluR2/3‐calretinin‐immunoreactive presumptive mossy cells, and granule cell dispersion; no mossy fiber sprouting was observed. Significance: These results indicate a probable lack of causality between mossy cell death and mossy fiber sprouting.     

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.     

Growth-associated Protein 43 Expression in Hippocampal Molecular Layer of Chronic Epileptic Rats Treated with Cycloheximide

Summary:  Purpose: GAP43 has been thought to be linked with mossy fiber sprouting (MFS) in various experimental models of epilepsy. To investigate how GAP43 expression (GAP43-ir) correlates with MFS, we assessed the intensity (densitometry) and extension (width) of GAP43-ir in the inner molecular layer of the dentate gyrus (IML) of rats subject to status epilepticus induced by pilocarpine (Pilo), previously injected or not with cycloheximide (CHX), which has been shown to inhibit MFS.
Methods: CHX was injected before the Pilo injection in adult Wistar rats. The Pilo group was injected with the same drugs, except for CHX. Animals were killed between 30 and 60 days later, and brain sections were processed for GAP43 immunohistochemistry.
Results: Densitometry showed no significant difference regarding GAP43-ir in the IML between Pilo, CHX+Pilo, and control groups. However, the results of the width of the GAP43-ir band in the IML showed that CHX+Pilo and control animals had a significantly larger band (p = 0.03) as compared with that in the Pilo group.
Conclusions: Our current finding that animals in the CHX+Pilo group have a GAP43-ir band in the IML, similar to that of controls, reinforces prior data on the blockade of MFS in these animals. The change in GAP43-ir present in Pilo-treated animals was a thinning of the band to a very narrow layer just above the granule cell layer that is likely to be associated with the loss of hilar cell projections that express GAP-43.     

皮质发育障碍大鼠小脑回形态学和海马苔藓纤维发芽的研究

目的 探讨液氮损伤诱导局灶性皮质发育障碍大鼠海马形态学及苔藓纤维发芽的情况。方法 实验随机分为正常对照组、假手术组和液氮损伤组，建立局灶性皮质发育障碍动物模型，察看其行为改变；采用常规HE染色、Nissl染色和Timm’s硫化银组织化学方法染色，肉眼和光镜下观察大鼠脑皮质形态变化，光镜下评估海马苔藓纤维发芽情况，各组数据取苔藓纤维发芽评分，采用非参数秩和Kruskal—Wallis H检验，组间两两比较用Nemenyi法。结果 液氮损伤组大鼠行为轻微改变，鼠脑嘴尾方向形成了一小的脑回，同侧海马CA3区有苔藓纤维发芽．而正常对照组和假手术组却没有。结论 幼鼠早期液氮损伤可导致小脑回形成及海马CA3区苔藓纤维发芽。小脑回周围异常兴奋性突触环路和海马CA3区苔藓纤维发芽形成推测是局灶性皮质发育障碍导致癫痫发生的重要机制。     

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.     

GAP-43 mRNA and protein expression in the hippocampal and parahippocampal region during the course of epileptogenesis in rats

In order to reveal axonal rewiring in the hippocampal and parahippocampal regions after status epilepticus, we investigated the temporal evolution of growth-associated protein-43 (GAP-43) mRNA and protein expression in two rat models of mesial temporal lobe epilepsy (MTLE). Status epilepticus (SE) was induced by electrical stimulation of the angular bundle or by intraperitoneal kainic acid (KA) injections. Despite increased GAP-43 mRNA expression in dentate granule cells at 24 h after SE, GAP-43 protein expression in the inner molecular layer (IML) of the dentate gyrus decreased progressively after 24 h after SE in both models. Nevertheless robust mossy fiber sprouting (MFS) was evident in the IML of chronic epileptic rats. Remaining GAP-43 protein expression in the IML in chronic epileptic rats did not correlate with the extent of MFS, but with the number of surviving hilar neurons. In the parahippocampal region, GAP-43 mRNA expression was decreased in layer III of the medial entorhinal area (MEAIII) in parallel with extensive neuronal loss in this layer. There was a tendency of GAP-43 mRNA up-regulation in the presubiculum, a region that projects to MEAIII. With regard to this parahippocampal region, however, changes in GAP-43 mRNA expression were not followed by protein changes. The presence of the presynaptic protein GAP-43 in a neurodegenerated MEAIII indicates that fibers still project to this layer. Whether reorganization of fibers has occurred in this region after SE needs to be investigated with tools other than GAP-43.     

Sprouting of GABAergic and mossy fiber axons in dentate gyrus following intrahippocampal kainate in the rat.

The present study examined the bilateral synaptic rearrangements of presumed gamma-aminobutyric acid (GABAergic) inhibitory axons and mossy fiber (presumed excitatory) recurrent collaterals following intrahippocampal kainic acid (KA) injection. Glutamate decarboxylase immunoreactivity (GAD-IR) was used to study inhibitory axon terminal sprouting, following 0.5 microgram KA/0.2 microliter injected unilaterally into the posterior hippocampus of rats (n = 16), with survival periods of 14, 28, and 120 days. The age-matched control animals (n = 9) received intrahippocampal 0.2 microliter saline (sham, n = 4) or no injection (normal, n = 5). To study mossy fiber synaptic rearrangements, 0.5 microgram KA/0.2 microliter volumes were injected unilaterally into the posterior hippocampus of rats (n = 10), with survival periods from 14, 28, and 120 days, and Timm sulfide-stained tissue sections were compared to age-matched sham (n = 4) or normal controls (n = 4). At 14 through 120 days after posterior KA injection, GAD-IR puncta were significantly increased in the ipsi- and contralateral inner molecular layers (IML) of the fascia dentata (FD) when compared to sham or normal controls. KA lesion also induced mossy fiber recurrent collateral sprouting into the ipsi- and contralateral FD IMLs. The loss of both the commissural and ipsilateral associational afferents to the FD apparently induced sprouting into their ipsi- and contralateral termination zones by granule cell mossy fibers and GAD-IR axons, thus establishing an abnormal circuitry near the observed pathology in the kainate model of epilepsy. Although reactive synaptogenesis of mossy fibers producing monosynaptic excitation may be one mechanism for KA epileptogenicity, the concurrent sprouting of GABAergic terminals in the same IML zone of the FD suggests that anomalous inhibitory synapses may contribute to chronic KA hippocampal hyperexcitability.     

Electrophysiology of dentate granule cells after kainate-induced synaptic reorganization of the mossy fibers.

Morphological data from humans with temporal lobe epilepsy and from animal models of epilepsy suggest that seizure-induced damage to dentate hilar neurons causes granule cells to sprout new axon collaterals that innervate other granule cells. This aberrant projection has been suggested to be an anatomical substrate for epileptogenesis. This hypothesis was tested in the present study with intra- and extracellular recordings from granule cells in hippocampal slices removed from rats 1-4 months after kainate treatment. In this animal model, hippocampal cell loss leads to sprouting of mossy fiber axons from the granule cells into the inner molecular layer of the dentate gyrus. Unexpectedly, when slices with mossy fiber sprouting were examined in normal medium, extracellular stimulation of the hilus or perforant path evoked relatively normal responses. However, in the presence of the GABAA-receptor antagonist, bicuculline, low-intensity hilar stimulation evoked delayed bursts of action potentials in about one-quarter of the slices. In one-third of the bicuculline-treated slices with mossy fiber sprouting, spontaneous bursts of synchronous spikes were superimposed on slow negative field potentials. Slices from normal rats or kainate-treated rats without mossy fiber sprouting never showed delayed bursts to weak hilar stimulation or spontaneous bursts in bicuculline. These data suggest that new local excitatory circuits may be suppressed normally, and then emerge functionally when synaptic inhibition is blocked. Therefore, after repeated seizures and excitotoxic damage in the hippocampus, synaptic reorganization of the mossy fibers is consistently associated with normal responses; however, in some preparations, the mossy fibers may form functional recurrent excitatory connections, but synaptic inhibition appears to mask these potentially epileptogenic alterations.     

Kainic acid induction of mossy fiber sprouting: dependence on mouse strain

After seizures caused by kindling or kainic acid (KA), hippocampal granule-cell axons, the mossy fibers, sprout into the supragranular layer of the rat. The mechanisms underlying this phenomenon remain elusive, but excitotoxic loss of hilar cells, which project to this supragranular layer, is suspected to be a critical determinant. Consistent with this hypothesis, we previously reported that while rats show mossy fiber sprouting after kainate, ICR mice do not. This may be associated with the observation that ICR mice, unlike rats, do not appear to show hilar cell death after KA (McNamara et al., Mol Brain Res 1996;40:177-187). Other strains of mice, however, such as 129/SvEMS, do show hilar cell death after KA (Schauwecker and Steward, Proc Natl Acad Sci USA 1997;94:4103-4108). We examined the possibility that the 129/SvEMS mouse strain would show granule-cell sprouting, in contrast to ICR mice. After administration of KA, mossy fiber sprouting was indeed observed in strain 129/SvEMS, but only in animals displaying evident hilar cell death. In contrast, neither hilar cell death nor mossy fiber sprouting was observed in ICR mice, confirming previous results. Both mouse strains demonstrated comparable behavioral seizures. These results strengthen the view that hilar cell death, together with epileptogenesis, triggers reactive synaptogenesis and mossy fiber sprouting.     

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.     

Alterations of Neuronal Connectivity in Area CA1 of Hippocampal Slices from Temporal Lobe Epilepsy Patients and from Pilocarpine-Treated Epileptic Rats

Summary: Purpose : Neuronal network reorganization might be involved in epileptogenesis in human and rat limbic epilepsy. Apart from aberrant mossy fiber sprouting, a more wide-spread fiber rearrangement in the hippocampal formation might occur. Therefore, we studied sprouting in area CA1 because this region is most affected in human temporal lobe epilepsy.
Methods : In slices from hippocampi of patients operated on for temporal lobe epilepsy (n = 134), from pilocarpine-treated rats (n = 74), and from control rats (n = 15), viable neurons were labeled with fluorescent dextran amines.
Results : In human hippocampi as well as in pilocarpine-treated rats, the degree of nerve cell loss varied. In 67 of 134 slices from human specimens with distinct Ammon's horn sclerosis and in 23 of 74 slices from pilocarpine-treated rats, a severe shrunken area CA1 presented with a similar picture: few damaged neurons were labeled, and aberrant fiber connections were not visible. This was in contrast to human resected hippocampi and hippocampi from pilocarpine-treated rats with no or moderate loss of neurons. In these cases, pyramidal cells remote from the injection site were labeled (human tissue, n = 59 of 134; pilocarpine-treated rats, n = 39 of 74). In human resected hippocampi without obvious pathology and in control animals, no pyramidal neurons were labeled apart from the injection site.
Conclusions : Axon collaterals of CA1 pyramidal cells are increased in human temporal lobe epilepsy and in pilocarpine-treated rats. Adjacent CA1 pyramidal cells project via aberrant collaterals to the stratum pyramidale and the stratum radiatum of area CA1. This network reorganization can contribute to hyperexcitability via increased backward excitation.     

Effects of early-life LiCl-pilocarpine-induced status epilepticus on memory and anxiety in adult rats are associated with mossy fiber sprouting and elevated CSF S100B protein

Purpose: This study investigated putative correlations among behavioral changes and: (1) neuronal loss, (2) hippocampal mossy fiber sprouting, and (3) reactive astrogliosis in adult rats submitted to early‐life LiCl‐pilocarpine‐induced status epilepticus (SE). Methods: Rats (P15) received LiCl (3 mEq/kg, i.p.) 12–18 h prior pilocarpine (60 mg/kg; s.c.). At adulthood, animals were submitted to behavioral tasks and after the completion of tasks biochemical and histological analysis were performed. Results: In SE group, it was observed an increased number of degenerating neurons in the CA1 subfield and in the hilus of animals 24 h after SE. At adulthood, SE group presented an aversive memory deficit in an inhibitory avoidance task and the animals that presented lower latency to the step down showed a higher score for mossy fiber sprouting. In the light‐dark exploration task, SE rats returned less and spent less time in the light compartment and present an increased number of risk assessment behavior (RA). There was a negative correlation between the time spent in the light compartment and the score for mossy fiber sprouting and a positive correlation between score for mossy fiber sprouting and number of RA. LiCl‐pilocarpine‐treated animals showed higher levels of S100B immunocontent in the CSF as well as a positive correlation between the score for sprouting and the GFAP immunocontent in the CA1 subfield, suggesting an astrocytic response to neuronal injury. Conclusions: We showed that LiCl‐pilocarpine‐induced SE during development produced long‐lasting behavioral abnormalities, which might be associated with mossy fiber sprouting and elevated CSF S100B levels at adulthood.     

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.     

Mossy fiber sprouting in the dentate gyrus in a newly developed epileptic mutant, Ihara epileptic rat

We examined the correlation between seizure activity and development of mossy fiber sprouting in the hippocampal formation using Timm staining in a newly developed Ihara epileptic rat (IER). The sprouting of mossy fibers were clearly shown in the inner molecular portion of the dentate gyrus and in the stratum oriens of CA3 pyramidal cell layer with repeated seizures. A positive correlation between the frequency of generalized tonic and clonic convulsions and the Timm staining score in molecular layer of dentate gyrus was revealed. Sprouting of mossy fiber in IER seems to be linked with seizure activities resulting from epileptic bursts, not to the genetic mutation.     

Locus Coeruleus and Neuronal Plasticity in a Model of Focal Limbic Epilepsy

Summary:  Purpose: A lesion of the noradrenergic nucleus Locus Coeruleus (LC) converts sporadic seizures evoked by microinfusion of bicuculline into the anterior piriform cortex (APC) of rats into limbic status epilepticus (SE). The purpose of this study was to evaluate the chronic effects of this new model of SE on the onset of secondary epileptogenesis. We further related the loss of noradrenaline (NE) with hippocampal mossy fiber sprouting.
Methods: Male Sprague Dawley rats were treated with systemic saline or DSP-4 (a neurotoxin selective for noradrenergic terminals originating from the LC), microinfused with bicuculline into the APC three days later, and sacrificed after 45 days. Naïve and DSP-4 pretreated sham-operated rats served as respective controls. The following evaluations were performed: (a) monitoring of acute seizures and delayed occurrence of spontaneous recurrent seizures (SRS); (b) NE levels in the hippocampus, frontal and olfactory cortex; (c) occurrence of mossy fiber sprouting into the inner molecular layer of the dentate gyrus of the dorsal hippocampus.
Results: In 30% of rats lacking noradrenergic terminals, SE evoked from the APC was followed by SRS. Conversely, seizures evoked in intact rats did not result in chronic epileptogenesis. Seizures/SE did not modify NE levels as compared with baseline levels both in naïve and DSP-4-pretreated rats. Rats undergoing SE following DSP-4 + bicuculline developed SRS which were accompanied by hippocampal mossy fiber sprouting.
Conclusions: Noradrenergic loss converts focally induced sporadic seizures into an epileptogenic SE, which is accompanied by mossy fiber sprouting within the dentate gyrus.     

Characterization of Target Cells for Aberrant Mossy Fiber Collaterals in the Dentate Gyrus of Epileptic Rat

Previous studies have demonstrated formation of recurrent excitatory circuits between sprouted mossy fibers and granule cell dendrites in the inner molecular layer of the dentate gyrus (9, 28, 30). In addition, there is evidence that inhibitory nonprincipal cells also receive an input from sprouted mossy fibers (39). This study was undertaken to further characterize possible target cells for sprouted mossy fibers, using immunofluorescent staining for different calcium-binding proteins in combination with Timm histochemical staining for mossy fibers. Rats were injected intraperitoneally with kainic acid in order to induce epileptic convulsions and mossy fiber sprouting. After 2 months survival, hippocampal sections were immunostained for parvalbumin, calbindin D28k, or calretinin followed by Timm-staining. Under a fluorescent microscope, zinc-positive mossy fibers in epileptic rats were found to surround parvalbumin-containing neurons in the granule cell layer and to follow their dendrites, which extended toward the molecular layer. In addition, dendrites of calbindin D28k-containing cells were covered by multiple mossy fiber terminals in the inner molecular layer. However, the calretinin-containing cell bodies in the granule cell layer did not receive any contacts from the sprouted fibers. Electron microscopic analysis revealed that typical Timm-positive mossy fiber terminals established several asymmetrical synapses with the soma and dendrites of nonpyramidal cells within the granule cell layer. These results provide direct evidence that, in addition to recurrent excitatory connections, inhibitory circuitries, especially those responsible for the perisomatic feedback inhibition, are formed as a result of mossy fiber sprouting in experimental epilepsy.