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.     

Localized overexpression of FGF-2 and BDNF in hippocampus reduces mossy fiber sprouting and spontaneous seizures up to 4 weeks after pilocarpine-induced status epilepticus

Purpose: We have recently reported that viral vector–mediated supplementation of fibroblast growth factor‐2 (FGF‐2) and brain‐derived neurotrophic factor (BDNF) in a lesioned, epileptogenic rat hippocampus limits neuronal damage, favors neurogenesis, and reduces spontaneous recurrent seizures. To test if this treatment can also prevent hippocampal circuit reorganization, we examined here its effect on mossy fiber sprouting, the best studied form of axonal plasticity in epilepsy. Methods: A herpes‐based vector expressing FGF‐2 and BDNF was injected into the rat hippocampus 3 days after an epileptogenic insult (pilocarpine‐induced status epilepticus). Continuous video–electroencephalography (EEG) monitoring was initiated 7 days after status epilepticus, and animals were sacrificed at 28 days for analysis of cell loss (measured using NeuN immunofluorescence) and mossy fiber sprouting (measured using dynorphin A immunohistochemistry). Key Findings: The vector expressing FGF‐2 and BDNF decreased both mossy fiber sprouting and the frequency and severity of spontaneous seizures. The effect on sprouting correlated strictly with the cell loss in the terminal fields of physiologic mossy fiber innervation (mossy cells in the dentate gyrus hilus and CA3 pyramidal neurons). Significance: These data suggest that the supplementation of FGF‐2 and BDNF in an epileptogenic hippocampus may prevent epileptogenesis by decreasing neuronal loss and mossy fiber sprouting, that is, reducing some forms of circuit reorganization.     

Resistance of neurofilaments to degradation, and lack of neuronal death and mossy fiber sprouting after kainic acid-induced status epilepticus in the developing rat hippocampus

Neurofilament (NF) proteins, the major constituent of intermediate filaments in neurons, have an important role in cellular stability and plasticity. We have now studied the short-term (hours) and long-term (up to 1 week) effects of kainic acid (KA)-induced status epilepticus (SE) on the reactivity of NF proteins, and mossy fiber (MF) sprouting and neuronal death up to 4 weeks in 9-day-old rats. In Western blotting, the expression of the phosphorylation-independent epitopes of NF-L, NF-M, and NF-H rapidly but transiently increased after the treatment, whereas the phosphorylated NF-M remained elevated for 7 days. However, the treatment did not change the immunoreactivity of NF proteins, and no neuronal death or mossy fiber sprouting was detected at any time point. Our findings indicate seizure-induced reactivity of NF proteins but their resistance to degradation, which could be of importance in neuronal survival and may also prevent MF sprouting in the developing hippocampus.     

Prolonged infusion of tetrodotoxin does not block mossy fiber sprouting in pilocarpine-treated rats

PURPOSE: Mossy fiber sprouting is a common abnormality found in patients and models of temporal lobe epilepsy. The role of mossy fiber sprouting in epileptogenesis is unclear, and its blockade would be useful experimentally and perhaps therapeutically. Results from previous attempts to block mossy fiber sprouting have been disappointing or controversial. In some brain regions, prolonged application of the sodium channel blocker tetrodotoxin prevents axon sprouting and posttrauma epileptogenesis. The present study tested the hypothesis that prolonged, focal infusion of tetrodotoxin would block mossy fiber sprouting after an epileptogenic treatment. METHODS: Adult rats were treated with pilocarpine to induce status epilepticus. Several hours to 3 days after pilocarpine treatment, a pump with a cannula directed toward the dentate gyrus was implanted to deliver 10 microM tetrodotoxin or vehicle alone at 0.25 microl/h. This method blocks local EEG activity in the hippocampus (Galvan et al. J Neurosci 2000; 20:2904-16). After 28 days of continuous infusion, rats were perfused with fixative, and their hippocampi analyzed anatomically with stereologic techniques. RESULTS: Tetrodotoxin infusion was verified immunocytochemically in tetrodotoxin-treated but not vehicle-treated hippocampi. Tetrodotoxin-infused and vehicle-infused hippocampi displayed similar levels of hilar neuron loss. The Timm stain revealed mossy fiber sprouting regardless of whether hippocampi were treated with tetrodotoxin infusion, vehicle infusion, or neither. CONCLUSIONS: Prolonged infusion of tetrodotoxin did not block mossy fiber sprouting. This finding suggests that sodium channel-mediated neuronal activity is not necessary for mossy fiber sprouting after an epileptogenic treatment.     

Is mossy fiber sprouting present at the time of the first spontaneous seizures in rat experimental temporal lobe epilepsy?

The contribution of mossy fiber sprouting to the generation of spontaneous seizures in the epileptic brain is under dispute. The present study addressed this question by examining whether sprouting of mossy fibers is present at the time of appearance of the first spontaneous seizures in rats, and whether all animals with increased sprouting have spontaneous seizures. Epileptogenesis was induced in 16 rats by electrically stimulating the lateral nucleus of the amygdala for 20-30 min until the rats developed self-sustained status epilepticus (SSSE). During and after SSSE, rats were monitored in long-term by continuous video-electroencephalography until they developed a second spontaneous seizure (8-54 days). Thereafter, monitoring was continued for 11 days to follow seizure frequency. The density of mossy fiber sprouting was analyzed from Timm-stained preparations. The density of hilar neurons was assessed from thionin-stained sections. Of 16 rats, 14 developed epilepsy. In epileptic rats, the density of mossy fiber sprouting did not correlate with the severity or duration (115-620 min) of SSSE, delay from SSSE to occurrence of first (8-51 days) or second (8-54 days) spontaneous seizure, or time from SSSE to perfusion (20-63 days). In the temporal end of the hippocampus, the sprouting correlated with the severity of neuronal damage (ipsilateral: r = -0.852, P < 0.01 contralateral: r = -0.748, P < 0.01). The two animals without spontaneous seizures also had sprouting. Increased density of sprouting in animals without seizures, and its association with the severity of neuronal loss was confirmed in another series of 30 stimulated rats that were followed-up with video-EEG monitoring for 60 d. Our data indicate that although mossy fiber sprouting is present in all animals with spontaneous seizures, its presence is not necessarily associated with the occurrence of spontaneous seizures.     

Sprouting of mossy fibers and the vacating of postsynaptic targets in the inner molecular layer of the dentate gyrus

Aberrant mossy fiber sprouting, which presumably results from hilar mossy cell death after status epilepticus (SE), is a frequently studied feature of temporal lobe epilepsy. Although mossy fiber sprouting can be suppressed by the protein synthesis inhibitor cycloheximide, spontaneous seizures remain unaltered. We have investigated the mechanisms underlying the ability of cycloheximide to block SE-induced mossy fiber sprouting in the inner molecular layer of dentate gyrus (IML). Pilocarpine-induced SE in the presence of cycloheximide resulted in a reduced number of injured hilar cells compared to rats not pretreated with cycloheximide. Presumed mossy cells, identified by calcitonin gene related peptide (CGRP) immunohistochemistry, were not significantly reduced in either group 60 days after SE. Whereas controls had a strong band of CGRP-positive fibers (putative mossy cell axons) and no neo-Timm stained fibers in the IML, pilocarpine-treated rats had no CGRP fibers and strong neo-Timm staining. Cycloheximide-pilocarpine-treated animals, in contrast, had CGRP and neo-Timm staining similar to controls. Cycloheximide might protect hilar CGRP-positive cells during SE and, by allowing those cells to retain their normal axonal projection, prevent mossy fiber sprouting. The recently suggested "irritable" mossy cell hypothesis relies on the survival of mossy cells for network hyperexcitability. We hypothesized that CGRP may be a marker for a subpopulation of relatively resistant mossy cells in rats, which, if they survive injury, may become irritable and contribute to hyperexcitability. We suggest that cycloheximide prevents SE-induced mossy fiber sprouting by preventing the loss of hilar CGRP-positive cells (putative mossy cells).     

Epileptogenic roles of astroglial death and regeneration in the dentate gyrus of experimental temporal lobe epilepsy

Recent studies have demonstrated that blockade of neuronal death in the hippocampus cannot prevent epileptogenesis in various epileptic models. These reports indicate that neurodegeneration alone is insufficient to cause epilepsy, and that the role of astrocytes in epileptogenesis should be reconsidered. Therefore, the present study was designed to elucidate whether altered morphological organization or the functionalities of astrocytes induced by status epilepticus (SE) is responsible for epileptogenesis. Glial responses (reactive microgliosis followed by astroglial death) in the dentate gyrus induced by pilocarpine-induced SE were found to precede neuronal damage and these alterations were closely related to abnormal neurotransmission related to altered vesicular glutamate and GABA transporter expressions, and mossy fiber sprouting in the dentate gyrus. In addition, newly generated astrocytes showed down-regulated expressions of glutamine synthase, glutamate dehydrogenase, and glial GABA transporter. Taken together, our findings suggest that glial responses after SE may contribute to epileptogenesis and the acquisition of the properties of the epileptic hippocampus. Thus, we believe that it is worth considering new therapeutic approaches to epileptogenesis involving targeting the inactivation of microglia and protecting against astroglial loss.     

Amygdala kindling develops without mossy fiber sprouting and hippocampal neuronal degeneration in rats

Repeated electrical stimulation of limbic structures has been reported to produce the kindling effect together with morphological changes in the hippocampus such as mossy fiber sprouting and/or neuronal loss. However, to argue against a causal role of these neuropathological changes in the development of kindling-associated seizures, we examined mossy fiber sprouting in amygdala (AM)-kindled rats using Timm histochemical staining, and evaluated the hippocampal neuronal degeneration in AM-kindled rats by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labelling (TUNEL). Amygdala kindling was established by 10.3 +/- 0.7 electrical stimulations, and no increase in Timm granules (neuronal sprouting) was observed up to the time of acquisition of a fully kindled state. However, the density and distribution of Timm granules increased significantly in the dentate gyrus compared with unkindled rats after 29 after-discharges or more than 10 kindled convulsions. In addition, no significant increase in TUNEL-positive cells was found in the hilar polymorphic neurons or in CA3 pyramidal neurons of the kindled rats that had fewer than 29 after-discharges. However, a significant increase of TUNEL-positive cells was found in the granule cell layer in the dentate gyrus of the stimulated side after 18 after-discharges or 10 kindled convulsions. Our result show that AM kindling develops without evidence of mossy fiber sprouting, and that mossy fiber sprouting may appear after repeated kindled convulsions, following death of the granule cells in the dentate gyrus.     

Epilepsy after early-life seizures can be independent of hippocampal injury

Prolonged early-life seizures are considered potential risk factors for later epilepsy development, but mediators of this process remain largely unknown. Seizure-induced structural damage in hippocampus, including cell loss and mossy fiber sprouting, is thought to contribute to the hyperexcitability characterizing epilepsy, but a causative role has not been established. To determine whether early-life insults that lead to epilepsy result in similar structural changes, we subjected rat pups to lithium-pilocarpine-induced status epilepticus during postnatal development (day 20) and examined them as adults for the occurrence of spontaneous seizures and alterations in hippocampal morphology. Sixty-seven percent of rats developed spontaneous seizures after status epilepticus, yet only one third of these epileptic animals exhibited visible hippocampal cell loss or mossy fiber sprouting in dentate gyrus. Most epileptic rats had no apparent structural alterations in the hippocampus detectable using standard light microscopy methods (profile counts and Timm's staining). These results suggest that hippocampal cell loss and mossy fiber sprouting can occur after early-life status epilepticus but may not be necessary prerequisites for epileptogenesis in the developing brain.     

Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model

The rodent pilocarpine model of epilepsy exhibits hippocampal sclerosis and spontaneous seizures and thus resembles human temporal lobe epilepsy. Use of the many available mouse mutants to study this epilepsy model would benefit from a detailed neuropathology study. To identify new features of epileptogenesis, we characterized glial and neuronal pathologies after pilocarpine-induced status epilepticus (SE) in CF1 and C57BL/6 mice focusing on the hippocampus. All CF1 mice showed spontaneous seizures by 17-27 days after SE. By 6 h there was virtually complete loss of hilar neurons, but the extent of pyramidal cell death varied considerably among mice. In the mossy fiber pathway, neuropeptide Y (NPY) was persistently upregulated beginning 1 day after SE; NPY immunoreactivity in the supragranular layer after 31 days indicated mossy fiber sprouting. beta2 microglobulin-positive activated microglia, normally absent in brains without SE, became abundant over 3-31 days in regions of neuronal loss, including the hippocampus and the amygdala. Astrogliosis developed after 10 days in damaged areas. Amyloid precursor protein immunoreactivity in the thalamus at 10 days suggested delayed axonal degeneration. The mortality after pilocarpine injection was very high in C57BL/6 mice from Jackson Laboratories but not those from Charles River, suggesting that mutant mice in the C57BL/6(JAX) strain will be difficult to study in the pilocarpine model, although their neuropathology was similar to CF1 mice. Major neuropathological changes not previously studied in the rodent pilocarpine model include widespread microglial activation, delayed thalamic axonal death, and persistent NPY upregulation in mossy fibers, together revealing extensive and persistent glial as well as neuronal pathology.     

Effects of vigabatrin on epileptiform abnormal discharges in hippocampal CA3 neurons of spontaneously epileptic rats (SER)

Vigabatrin, a γ-amino butyric acid (GABA) transaminase inhibitor, is known to inhibit partial epilepsy in humans. The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsion and absence-like seizures from the age of 8 weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single stimulus is delivered to the mossy fibers in slice preparations. The effects of vigabatrin on the abnormal excitability of hippocampal CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action of the drug. Intracellular recordings were performed in 24 hippocampal slice preparations of 20 SER aged 8–17 weeks old. Bath application of vigabatrin (1 mM) inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation in 15 min without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. A higher dose of vigabatrin (10 mM) sometimes inhibited the first spike. However, vigabatrin at doses up to 10 mM did not significantly affect the single action potential elicited by stimulation of the mossy fibers in the hippocampal CA3 neurons of age-matched Wistar rats. In addition, application of vigabatrin (10 mM) did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER, nor the miniature excitatory postsynaptic potential (mEPSP) recorded in the CA3 neurons of SER. The inhibitory effect of vigabatrin (1 mM) on the mossy fiber stimulation-induced depolarization shift with repetitive firing was blocked by concomitant application of bicuculline (10 μM), a GABA_A receptor antagonist. These findings strongly suggested that GABA increased by inhibition of GABA transaminase with vigabatrin inhibits abnormal excitation of hippocampal CA3 neurons of SER via GABA_A receptors, although the possibility that the drug acted directly on the GABA_A receptors of CA3 neurons could not be completely excluded.     

Loss of input from the mossy cells blocks maturation of newly generated granule cells

The objective of this work is to check whether the input from the mossy cells to the inner molecular layer is necessary for the integration and maturation of the newly generated granule cells of the dentate gyrus (DG) in mice, and if after status epilepticus the sprouting of the mossy fibers can substitute for this projection. Newly generated cells were labeled by administration of 5-bromo-deoxyuridine either before or after pilocarpine administration. The neuronal loss in the hippocampus after administration of pilocarpine combined with scopolamine and diazepam seemed restricted to the hilar mossy cells. The maturation of the granule cells was studied using immunohistochemistry for calretinin and NeuN in combination with detection of 5-bromo-deoxyuridine. The sprouting of the mossy fibers was detected using Timm staining for zinc-rich boutons. In normal conditions, granule cells took about 2 weeks to lose the immature marker calretinin. After the loss of the mossy cells, newly generated granule cells remained expressing calretinin for more than a month, until the sprouting of the mossy fibers substituted for the projection of the mossy cells in the inner molecular layer of the DG. Therefore, a proper pattern of connectivity is necessary for the normal development and integration of newly generated granule cells in the adult brain. In a changed environment they cannot adapt transforming in other cell types; simply they are unable to mature. The sprouting of the mossy fibers, although aberrant and a probable source of epileptic activity, may be important for the correct physiology of the granule cells, restoring a likeness of normality in their connective environment. The survival of granule cells incorporated as mature neurons was increased after pilocarpine when compared with normal conditions. Thus, it is likely that the reorganization of the circuitry after the loss of the mossy cells facilitates the survival and incorporation of the newly generated granule cells.     

Vigabatrin extracellular pharmacokinetics and concurrent γ-aminobutyric acid neurotransmitter effects in rat frontal cortex and hippocampus using microdialysis

Purpose:   To investigate the pharmacokinetic interrelationship of vigabatrin in blood and the brain (frontal cortex vs. hippocampus) and to ascertain the relationship between brain extracellular vigabatrin concentrations and concurrent γ-aminobutyric acid (GABA) concentrations.
Methods:   Sprague-Dawley rats were implanted with a jugular vein catheter for blood sampling, and microdialysis probes in the frontal cortex and hippocampus for extracellular fluid (ECF) sampling. Vigabatrin was administered intraperitoneally at two different doses (500 and 1,000 mg/kg), and blood and ECF were collected at timed intervals up to 8 h. Rats were freely moving and behaving. Vigabatrin (sera and ECF) and GABA (ECF) concentrations were measured with use of high performance liquid chromatography (HPLC).
Results:   Vigabatrin concentrations in blood rose linearly and dose-dependently, and vigabatrin rapidly appeared in the brain as evidenced by the detection of vigabatrin in the ECF of both the frontal cortex and hippocampus at time of first sampling (15 min). However, frontal cortex concentrations were twofold greater than those of the hippocampus. Furthermore, GABA concentrations increased five-fold in the frontal cortex but were unaffected in the hippocampus. In addition, GABA concentrations began to increase approximately 3 h after vigabatrin administration at a time when vigabatrin concentrations were in exponential decline.
Conclusions:   Vigabatrin distribution in the brain is region specific, with frontal cortex concentrations substantially greater than those seen in the hippocampus. Elevation of GABA concentrations did not reflect the concentration profile of vigabatrin but reflected its regional distribution.     

Strain differences affect the induction of status epilepticus and seizure-induced morphological changes

Genetic deficits have been discovered in human epilepsy, which lead to alteration of the balance between excitation and inhibition, and ultimately result in seizures. Rodents show similar genetic determinants of seizure induction. To test whether seizure‐prone phenotypes exhibit increased seizure‐related morphological changes, we compared two standard rat strains (Long–Evans hooded and Wistar) and two specially bred strains following status epilepticus. The special strains, namely the kindling‐prone (FAST) and kindling‐resistant (SLOW) strains, were selectively bred based on their amygdala kindling rate. Although the Wistar and Long–Evans hooded strains experienced similar amounts of seizure activity, Wistar rats showed greater mossy fiber sprouting and hilar neuronal loss than Long–Evans hooded rats. The mossy fiber system was affected differently in FAST and SLOW rats. FAST animals showed more mossy fiber granules in the naïve state, but were more resistant to seizure‐induced mossy fiber sprouting than SLOW rats. These properties of the FAST strain are consistent with those observed in juvenile animals, further supporting the hypothesis that the FAST strain shares circuit properties similar to those seen in immature animals. Furthermore, the extent of mossy fiber sprouting was not well correlated with sensitivity to status epilepticus, but was positively correlated with the frequency of spontaneous recurrent seizures in the FAST rats only, suggesting a possible role for axonal sprouting in the development of spontaneous seizures in these animals. We conclude that genetic factors clearly affect seizure development and related morphological changes in both standard laboratory strains and the selectively bred seizure‐prone and seizure‐resistant strains.     

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.     

Coenzyme Q10 Ameliorates Neurodegeneration, Mossy Fiber Sprouting, and Oxidative Stress in Intrahippocampal Kainate Model of Temporal Lobe Epilepsy in Rat

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults and the most resistant type to treatment. Novel treatment approaches are strongly required to prevent or even reverse the cellular and molecular mechanisms of epileptogenesis. In this study, we investigated the possible neuroprotective effect of coenzyme Q10 (CoQ10) in an intrahippocampal kainate model of TLE in rat. Kainate injection caused a higher seizure severity during status epilepticus and spontaneous seizure phases, and CoQ10 pretreatment significantly attenuated its severity and incidence rate. Intrahippocampal kainate also led to elevation of malondialdehyde (MDA) and nitrite, and CoQ10 significantly attenuated the increased MDA and nitrite content. In addition, intrahippocampal kainate induced a significant degeneration of neurons in CA1, CA3, and hilar regions of the hippocampus, and CoQ10 significantly attenuated these changes in CA1 and CA3 regions. Timm’s staining data showed a robust mossy fiber sprouting (MFS) in dentate gyrus of kainate-lesioned rats and CoQ10 significantly lowered MFS intensity. These data suggest that CoQ10 pretreatment could attenuate spontaneous recurrent seizures and inhibit hippocampal neuronal loss and aberrant MFS in kainate-induced model of TLE in rat, and part of its beneficial effect is due to its potential to mitigate oxidative stress.     

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.     

Synaptic connections of hilar basal dendrites of dentate granule cells in a neonatal hypoxia model of epilepsy

Numerous animal models of epileptogenesis demonstrate neuroplastic changes in the hippocampus. These changes occur not only for the mature neurons and glia, but also for the newly generated granule cells in the dentate gyrus. One of these changes, the sprouting of mossy fiber axons, is derived predominantly from newborn granule cells in adult rats with pilocarpine-induced temporal lobe epilepsy. Newborn granule cells also mainly contribute to another neuroplastic change, hilar basal dendrites (HBDs), which are synaptically targeted by mossy fibers in the hilus. Both sprouted mossy fibers and HBDs contribute to recurrent excitatory circuitry that is hypothesized to be involved in increased seizure susceptibility and the development of spontaneous recurrent seizures (SRS) that occur following the initial pilocarpine-induced status epilepticus. Considering the putative role of these neuroplastic changes in epileptogenesis, a critical question is whether similar anatomic phenomena occur after epileptogenic insults to the immature brain, where the proportion of recently born granule cells is higher due to ongoing maturation. The current study aimed to determine if such neuroplastic changes could be observed in a standardized model of neonatal seizure-inducing hypoxia that results in development of SRS. We used immunoelectron microscopy for the immature neuronal marker doublecortin to label newborn neurons and their HBDs following neonatal hypoxia. Our goal was to determine whether synapses form on HBDs from neurons born after neonatal hypoxia. Our results show a robust synapse formation on HBDs from animals that experienced neonatal hypoxia, regardless of whether the animals experienced tonic-clonic seizures during the hypoxic event. In both cases, the axon terminals that synapse onto HBDs were identified as mossy fiber terminals, based on the appearance of dense core vesicles. No such synapses were observed on HBDs from newborn granule cells obtained from sham animals analyzed at the same time points. This aberrant circuit formation may provide an anatomic substrate for increased seizure susceptibility and the development of epilepsy.     

Resistance of the immature hippocampus to seizure-induced synaptic reorganization.

Temporal lobe epilepsy is a common form of epilepsy in human adults and is associated with a unique pattern of damage in the hippocampus. The damage includes cell loss of the CA3 and CA4 areas and synaptic growth (sprouting) of mossy fibers in the supragranular layer of the dentate gyrus. Experimental evidence indicates that in adult rats the excitatory amino acid, kainic acid, induces a similar pattern of changes in hippocampal circuitry associated with alterations in perforant path excitation and inhibition. It has been suggested that, in humans, this type of damage may be a result of seizures early in life. In this study we examined the effects of kainic acid-induced status epilepticus on synaptic reorganization and paired-pulse electrophysiology in developing rats and adults. Kainic acid induced more severe seizures in 15-day-old rat pups than in adults. In contrast to adult rats, these seizures did not produce CA3/CA4 neuronal loss, mossy fiber sprouting or changes in paired-pulse excitation or inhibition in the hippocampus of rat pups tested 2-4 weeks after status epilepticus. Our results provide evidence that the immature hippocampus may be more resistant to seizure-induced changes than the mature hippocampus.     

N-cadherin在颞叶癫痫海马苔藓纤维出芽和突触重组中的作用

目的 探讨神经性钙粘附分子(N-cadherin)在癫痫状态后海马苔藓纤维出芽和突触重组中的作用。方法取锂一匹罗卡品诱导大鼠癫痫持续状态及慢性自发性颞叶癫痫发作期的大鼠脑片，用Timm染色和免疫组化的方法分别检测苔藓纤维出芽和N-cadherin在大鼠海马组织中的表达。结果癫痫状态后第2周和第4周的实验组大鼠可见到苔藓纤维出芽，穿越齿状回颗粒细胞层到达内分子层，并在此形成一条致密的层状带(Timm染色)。免疫组化染色发现实验组大鼠在第2周和第4周，海马齿状回内分子层可以看到强染色，并形成一条致密带，与Timm染色时观察到的条带一致。结论癫痫状态后在海马齿状回内分子层N-cadherin的表达上调．N-cadherin可能参与了癫痫后苔藓纤维出芽和突触重组过程。