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Granule cell dispersion in two mouse models of temporal lobe epilepsy and reeler mice is associated with changes in dendritic orientation and spine distribution

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Abstract

Temporal lobe epilepsy is characterized by hippocampal neuronal death in CA1 and hilus. Dentate gyrus granule cells survive but show dispersion of the compact granule cell layer. This is associated with decrease of the glycoprotein Reelin, which regulates neuron migration and dendrite outgrow. Reelin‐deficient (reeler) mice show no layering, their granule cells are dispersed throughout the dentate gyrus. We studied granule cell dendritic orientation and distribution of postsynaptic spines in reeler mice and two mouse models of temporal lobe epilepsy, namely the p35 knockout mice, which show Reelin‐independent neuronal migration defects, and mice with unilateral intrahippocampal kainate injection. Granule cells were Golgi‐stained and analyzed, using a computerized camera lucida system. Granule cells in naive controls exhibited a vertically oriented dendritic arbor with a small bifurcation angle if positioned proximal to the hilus and a wider dendritic bifurcation angle, if positioned distally. P35 knockout‐ and kainate‐injected mice showed a dispersed granule cell layer, granule cells showed basal dendrites with wider bifurcation angles, which lost position‐specific differences. Reeler mice lacked dendritic orientation. P35 knockout‐ and kainate‐injected mice showed increased dendritic spine density in the granule cell layer. Molecular layer dendrites showed a reduced spine density in kainate‐injected mice only, whereas in p35 knockouts no reduced spine density was seen. Reeler mice showed a homogenous high spine density. We hypothesize that granule cells migrate in temporal lobe epilepsy, develop new dendrites which show a spread of the dendritic tree, create new spines in areas proximal to mossy fiber sprouting, which is present in p35 knockout‐ and kainate‐injected mice and loose spines on distal dendrites if mossy cell death is present, as it was in kainate‐injected mice only. These results are in accordance with findings in epilepsy patients.

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Dysregulated adult hippocampal neurogenesis occurs in many temporal lobe epilepsy (TLE) models. Most dentate granule cells (DGCs) generated in response to an epileptic insult develop features that promote increased excitability, including ectopic location, persistent hilar basal dendrites (HBDs), and mossy fiber sprouting. However, some appear to integrate normally and even exhibit reduced excitability compared to other DGCs. To examine the relationship between DGC birthdate, morphology, and network integration in a model of TLE, we retrovirally birthdated either early-born [EB; postnatal day (P)7] or adult-born (AB; P60) DGCs. Male rats underwent pilocarpine-induced status epilepticus (SE) or sham treatment at P56. Three to six months after SE or sham treatment, we used whole-cell patch-clamp and fluorescence microscopy to record spontaneous excitatory and inhibitory currents from birthdated DGCs. We found that both AB and EB populations of DGCs recorded from epileptic rats received increased excitatory input compared with age-matched controls. Interestingly, when AB populations were separated into normally integrated (normotopic) and aberrant (ectopic or HBD-containing) subpopulations, only the aberrant populations exhibited a relative increase in excitatory input (amplitude, frequency, and charge transfer). The ratio of excitatory-to-inhibitory input was most dramatically upregulated for ectopically localized DGCs. These data provide definitive physiological evidence that aberrant integration of post-SE, AB DGCs contributes to increased synaptic drive and support the idea that ectopic DGCs serve as putative hub cells to promote seizures.
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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. 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. 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. These results indicate a probable lack of causality between mossy cell death and mossy fiber sprouting.
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Temporal lobe epilepsy (TLE) is often accompanied by granule cell dispersion (GCD), a migration defect of granule cells in the dentate gyrus. We have previously shown that a decrease in the expression of reelin, an extracellular matrix protein important for neuronal positioning, is associated with the development of GCD in TLE patients. Here, we used unilateral intrahippocampal injection of kainate (KA) in adult mice which is also associated with GCD formation and a decrease of reelin expression. In this mouse epilepsy model we aimed to prevent GCD development by the application of exogenous reelin. As a prerequisite we analyzed whether the reelin signaling transduction cascade was preserved in the KA-injected hippocampus. Using in situ hybridization and Western blot analysis we found that the expression of the reelin signaling components, apolipoprotein E receptor 2, the very-low-density lipoprotein receptor and the intracellular adaptor protein disabled 1, was maintained in dentate granule cells after KA injection. Next, recombinant reelin was infused into the KA-injected hippocampus by osmotic minipumps over a period of 2 weeks. Quantitative analysis of granule cell layer width revealed a significant reduction of GCD in reelin-treated, but not in saline-infused animals when compared to KA injection alone. Our findings highlight the crucial role of reelin for the maintenance of granule cell lamination in the dentate gyrus of adult mice and show that a reelin deficiency is causally involved in GCD development.
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The morphology of the hippocampus and dentate gyrus in normal and reeler mice has been studied in Nissl, myelin, Golgi, Timm's sulfide silver and gold chloride-sublimate preparations. It is evident form both cell-and fiber-stained sections that despite the obvious defect in the positioning of the hippocampal pyramidal and dentate granule cells in the reeler mouse with in the radial dimension, the hippocampal formation as a whole shows a normal and consistent progression of cytoarchitectonic fields along its transverse axis, and a normal and consistent progression of changes in the structure of the hippocampus and dentate gyrus along their longitudinal axes. Thus, at least in these structures, the reeler gene seems to exert its effect only in the radial dimension.
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In this study the Golgi/electron microscopy (EM) technique has been used for an analysis of the fine structure, specific synaptic connections, and differentiation of neurons in the hippocampus and fascia dentata of rodents. In a first series of experiments the specific synaptic contacts formed between cholinergic terminals and identified hippocampal neurons were studied. By means of a variant of the section Golgi impregnation procedure, Vibratome sections immunostained for choline acetyltransferase, the acetylcholine-synthesizing enzyme, were Golgi-impregnated in order to identify the target neurons of cholinergic terminals in the hippocampus. It could be shown with this combined approach that cholinergic septohippocampal fibers form a variety of synapses with different target structures of the Golgi-impregnated and gold-toned hippocampal neurons. In this report cholinergic synapses on the heads of small spines, the necks of large complex spines, dendritic shafts, and cell bodies of identified dentate granule cells are described. The variety of cholinergic synapses suggests that cholinergic transmission in the fascia dentata is a complex event.
Article
The distribution of granule cells in the dentate gyrus of the hippocampal formation was studied in control autopsy and temporal lobe epilepsy (TLE) specimens. In control tissue, the granule cell somata were closely approximated and formed a narrow lamina with a distinct, regular border with the molecular layer. In 11 of 15 TLE specimens, the granule cell somata were dispersed and formed a wider than normal granule cell layer. The granule cell somata extended into the molecular layer to varying extents, creating an irregular boundary between the lamina. The dispersed granule cells were frequently aligned in columns, and many of these neurons displayed elongated bipolar forms. The extent of granule cell dispersion appeared to be related to the amount of cell loss in the polymorph layer of the dentate gyrus. Granule cell dispersion was not consistently associated with granule cell loss although 5 of the 11 specimens with granule cell dispersion also showed moderate to marked granule cell loss. The most common features in the histories of the TLE cases with granule cell dispersion were severe febrile seizures or seizures associated with meningitis or encephalitis during the first 4 years of life. The dispersion of the granule cells suggests that there has been some alteration in the patterns of cell migration in a subpopulation of cases with severe TLE. The resultant ectopic positions of the granule cells could lead to changes in both the afferent and efferent connections of these neurons and, thus, contribute to the altered circuitry of the hippocampal formation in TLE.
Article
The distribution of the mossy fiber synaptic terminals was examined using the Timm histochemical method in surgically excised hippocampus and dentate gyrus from patients who underwent lobectomy of the anterior part of the temporal lobe for refractory partial complex epilepsy. The dentate gyrus of epileptic patients demonstrated intense Timm granules and abundant mossy fiber synaptic terminals in the supragranular region and the inner molecular layer. In contrast, the dentate gyrus of presenescent nonepileptic primates demonstrated no Timm granules in the supragranular region. In nonepileptic senescent primates, occasional very sparse supragranular Timm granules were results are morphological evidence of mossy fiber synaptic reorganization in the temporal lobe of epileptic humans, and suggest the intriguing possibility that mossy fiber sprouting and synaptic reorganization induced by repeated partial complex seizures may play a role in human epilepsy.
Article
The adult mammalian cortex is characterized by a distinct laminar structure generated through a well-defined pattern of neuronal migration. Successively generated neurons are layered in an "inside-out" manner to produce six cortical laminae. We demonstrate here that p35, the neuronal-specific activator of cyclin-dependent kinase 5, plays a key role in proper neuronal migration. Mice lacking p35, and thus p35/cdk5 kinase activity, display severe cortical lamination defects and suffer from sporadic adult lethality and seizures. Histological examination reveals that the mutant mice lack the characteristic laminated structure of the cortex. Neuronal birth-dating experiments indicate a reversed packing order of cortical neurons such that earlier born neurons reside in superficial layers and later generated neurons occupy deep layers. The phenotype of p35 mutant mice thus demonstrates that the formation of cortical laminar structure depends on the action of the p35/cdk5 kinase.
Article
Dendritic morphology was studied in human hippocampal dentate granule cells (DGCs) by intracellularly-injecting biocytin in slice preparations that were obtained from temporal lobe epilepsy patients who underwent a surgical treatment for medically-intractable seizures. These DGCs had a fan-shaped dendritic domain of 54.1 degrees +/- 4.1 S.E.M. with 13.8 +/- 1.1 branch points and an estimated total dendritic length of 11535.6 microns +/- 3045.4. Dendritic spines were counted, and spine density was calculated to be 0.25 spines/microns +/- 0.16 S.E.M.. However, when the cells were categorized into two groups based on the presence or absence of the aberrant mossy fiber collaterals, the number of dendritic branches was significantly lower and spine density was significantly higher in DGCs that had aberrant collaterals. In particular, in the proximal dendrite, the spine density was 5 times higher in DGCs whose own mossy fibers were reorganized sending aberrant collaterals to this dendritic region (0.750 spines/microns +/- 0.203 S.E.M.: P < 0.01) than the DGCs without such collaterals (0.082 spines/microns +/- p.021 S.E.M.). These results suggest that the axonal reorganization may have an effect on the morphology of DGC dendrites directly or indirectly in such a way that dendritic structure and spines could be protected from seizure-induced excitotoxic cell damage.
Article
This study determined fascia dentata anatomy and hippocampal neuron densities in patients with different epileptic syndromes. Based on presurgical data, patients were classified into: (a) pediatric patients (n=19); (b) temporal mass lesion cases (n=14); and (c) hippocampal sclerosis patients (n=31). Surgically removed hippocampi and autopsies (n=34) were studied for: (a) hippocampal neuron densities; (b) stratum granulosum (SG) widths and lengths; and (c) hilar areas. The number of granule cells and hilar neurons per tissue section were estimated from the neuron densities and fascia dentata area measurements. Results showed that compared with autopsies (p<0.05): (a) pediatric patients had similar SG and hilar areas; granule cell density was lower (but not hilar neuron density); and the estimated number of granule cells was lower (but not the number of hilar neurons); (b) the widths of SG and hilar areas were greater in mass lesion cases; the density of granule cells and hilar neurons was lower; and the total estimated numbers of granule cells and hilar neurons were similar to those of the autopsies; and (c) hippocampal sclerosis patients had wider, yet shorter SG; hilar areas were smaller; granule cell and hilar densities were lower; and the total estimated numbers of granule cells and hilar neurons were lower than those of the autopsy cases. The duration of the seizures did not correlate with lower fascia dentata neuron densities or estimates of total granule cell and hilar neurons. Furthermore, greater SG widths correlated with lower hilar and CA4 neuron densities, but not with age at first seizure or duration of epilepsy. These results indicate that the size of the fascia dentata SG and hilus along with hippocampal neuron densities differ between surgical patients with different epileptic syndromes, and a wider SG was associated with a lower density of end folium neurons. These findings support the hypothesis that hippocampal sclerosis and granule cell dispersion are not the consequence of repetitive seizures beginning at an early developmental age, but seem to differ depending on the type of epileptic syndrome.
Article
Several examples of structural plasticity in the adult brain have been provided in the hippocampus, among which the most striking concerns axonal remodeling of the dentate gyrus granule cells. We have recently demonstrated that a single injection of kainic acid into the dorsal hippocampus of adult mice triggers a conspicuous morphogenetic response of granule cells. Cellular labeling with biocytin 1, 2, and 4 weeks after injection of kainate revealed a progressive increase in dendritic thickness and length (up to 2.5-times), combined with an increase in the number of dendritic spines. This correlation resulted in the conservation of total spine density. No modifications of the dendritic arborization pattern were noted. In addition to dendritic changes, the number of axonal profiles observed within the hypertrophied granular layer and the inner part of the molecular layer appeared dramatically increased. Timm staining and anterograde labeling of two of the main extra-hippocampal afferent systems (i.e., septal, entorhinal) evidenced sprouting of mossy fibers and of septal afferents. Entorhinal fibers were not obviously modified. As revealed by calretinin-immunohistochemistry, commissural afferents also responded by an extensive sprouting. In addition, increases of dendritic spine number and dendritic length were noticeably greater in portions of dendrites that receive mossy fiber collaterals and septal and hypothalamic afferents, than in the external portion which receives entorhinal afferents. Although qualitative, this correlation suggests a relationship between kainate-induced structural plasticity of mature granule cells and the specific capacities of afferent systems to elaborate axon collaterals.
Article
Hilar mossy cells of the mouse were shown recently to display calretinin immunoreactivity (Liu et al. [1996] Exp Brain Res 108:389-403). The morphological and connectional characteristics of these cells are poorly understood. In the present study, we used immunohistochemical, electron microscopic, and neuronal tracing techniques to describe their distribution, morphology, and connectivity. The distribution of calretinin-immunoreactive mossy cells varied significantly along the dorsoventral axis of the hilus. At dorsal levels, calretinin immunoreactivity was limited largely to a subpopulation of interneurons. At mid-dorsoventral and ventral levels, however, most if not all mossy cells displayed calretinin immunoreactivity. We found that most hilar mossy cells are calretinin immunoreactive but lack gamma-aminobutyric acid, as demonstrated by postembedding immunostaining of alternate semithin sections. Calretinin-immunoreactive mossy cells typically had two to three thick dendrites covered with complex spines (thorny excrescences). Electron microscopy revealed that these spines received multiple asymmetric contacts from mossy fibres. Axons arising from these cells formed a strong belt of calretinin immunoreactivity restricted to the inner third of the dentate molecular layer. This immunoreactivity was equally dense throughout the dorsoventral length of the dentate gyrus, suggesting that axons of calretinin-immunoreactive mossy cells located in the ventral levels diverge greatly and are capable of innervating distant regions of the dentate gyrus. Ultrastructural examination showed that calretinin-immunoreactive boutons made asymmetric synaptic contacts primarily on spines and, occasionally, on dendritic shafts of granule cells and accounted for the majority of asymmetrical synapses in the inner molecular layer. Injections of the retrograde tracer wheatgerm agglutinin-gold into the dentate gyrus demonstrated that calretinin-immunoreactive mossy cells concentrated in the ventral hilus project massively to both the dorsal and ventral aspect of the contralateral dentate gyrus. A small proportion of retrogradely labelled cells showed immunoreactivity for neuropeptide Y or somatostatin. If mossy cells of the ventral hilus receive the majority of their input from ventral granule cells, one may expect ventral granule cells to be more efficient in recruiting large numbers of granule cells during synchronous activity patterns than dorsal granule cells. Spontaneous activity originating from granule cells in the ventral dentate gyrus can be propagated throughout the dorsoventral length of the dentate gyrus bilaterally via the dorsoventrally divergent and contralaterally projecting axons of the mossy cells. This organization may explain why the ventral dentate gyrus is frequently involved in pathological phenomena.
Article
Human mesial temporal lobe epilepsy is characterized by hippocampal seizures associated with pyramidal cell loss in the hippocampus and dispersion of dentate gyrus granule cells. A similar histological pattern was recently described in a model of extensive neuroplasticity in adult mice after injection of kainate into the dorsal hippocampus [Suzuki et al. (1995) Neuroscience 64, 665–674]. The aim of the present study was to determine whether (i) recurrent seizures develop in mice after intrahippocampal injection of kainate, and (ii) the electroencephalographic, histopathological and behavioural changes in such mice are similar to those in human mesial temporal lobe epilepsy. Adult mice receiving a unilateral injection of kainate (0.2 μg; 50 nl) or saline into the dorsal hippocampus displayed recurrent paroxysmal discharges on the electroencephalographic recordings associated with immobility, staring and, occasionally, clonic components. These seizures started immediately after kainate injection and recurred for up to eight months. Epileptiform activities occurred most often during sleep but occasionally while awake. The pattern of seizures did not change over time nor did they secondarily generalize. Glucose metabolic changes assessed by [¹⁴C]2-deoxyglucose autoradiography were restricted to the ipsilateral hippocampus for 30 days, but had spread to the thalamus by 120 days after kainate. Ipsilateral cell loss was prominent in hippocampal pyramidal cells and hilar neurons. An unusual pattern of progressive enlargement of the dentate gyrus was observed with a marked radial dispersion of the granule cells associated with reactive astrocytes. Mossy fibre sprouting occurred both in the supragranular molecular layer and infrapyramidal stratum oriens layer of CA3. The expression of the embryonic form of the neural cell adhesion molecule coincided over time with granule cell dispersion.
Article
Hilar mossy cells represent an important excitatory subpopulation of the hippocampal formation. Several studies have identified this cell type as particularly vulnerable to seizure activity in rat models of limbic epilepsy. Here we have subjected hilar mossy cell loss in the hippocampus of patients with chronic temporal lobe epilepsy (TLE) to a systematic morphological and immunohistochemical analysis. Hippocampal specimens from 30 TLE patients were included; 21 patients presented with segmental neuronal cell loss [Ammon's horns clerosis (AHS)] and 8 with focal lesions (tumors, scars, malformations) not involving the hippocampus proper. In one additional TLE patient, no histopathological alteration could be observed. Surgical specimens from tumor patients without epilepsy (n = 2) and nonepileptic autopsy brains (n = 8) were used as controls. Hilar mossy cells in the human hippocampus were visualized using a novel polycloncal antiserum directed against the metabotropic glutamate receptor subtype mGluR7b or by intracellular Lucifer Yellow injection, confocal laser scanning microscopy, and three-dimensional morphological reconstruction. Compared with controls, a significant loss of mGluR7 immunoreactive mossy cells was observed in patients with AHS (p < 0.05). In contrast, TLE patients with focal lesions but structurally intact hippocampus demonstrated only a discrete, nonsignificant reduction of this neuronal subpopulation. This observation was confirmed by analysis of 62 randomly injected hilar neurons from AHS patients, in which we were unable to detect neurons with a morphology like that of hilar mossy cells. Our present data indicate significant hilar mossy cell loss in TLE patients with AHS. In contrast, hilar mossy cells appear to be less vulnerable in patients with lesion-associated TLE. Although the significance of mGluR7 immunoreactivity in mossy cells remains to be studied, loss of this cell population is compatible with alterations in hippocampal networks and regional hyperexcitability as pathogenic mechanism of AHS and TLE.
Article
Cortical dysplasia is a major cause of intractable epilepsy in children. However, the precise mechanisms linking cortical malformations to epileptogenesis remain elusive. The neuronal-specific activator of cyclin-dependent kinase 5, p35, has been recognized as a key factor in proper neuronal migration in the neocortex. Deletion of p35 leads to severe neocortical lamination defects associated with sporadic lethality and seizures. Here we demonstrate that p35-deficient mice also exhibit dysplasia/ heterotopia of principal neurons in the hippocampal formation, as well as spontaneous behavioral and electrographic seizures. Morphological analyses using immunocytochemistry, electron microscopy, and intracellular labeling reveal a high degree of abnormality in dentate granule cells, including heterotopic localization of granule cells in the molecular layer and hilus, aberrant dendritic orientation, occurrence of basal dendrites, and abnormal axon origination sites. Dentate granule cells of p35-deficient mice also demonstrate aberrant mossy fiber sprouting. Field potential laminar analysis through the dentate molecular layer reflects the dispersion of granule cells and the structural reorganization of this region. Similar patterns of cortical disorganization have been linked to epileptogenesis in animal models of chronic seizures and in human temporal lobe epilepsy. The p35-deficient mouse may therefore offer an experimental system in which we can dissect out the key morphological features that are causally related to epileptogenesis.
Article
The fascia dentata of the hippocampal formation is characterized by the nonoverlapping and lamina-specific termination of afferent fibers: entorhinal fibers terminate in the outer molecular layer and commissural/associational fibers terminate in the inner molecular layer. It has been proposed that this fiber lamination depends on the presence of the correct postsynaptic partner at the time of fiber ingrowth during development. Pioneer neurons that guide afferent fibers to their correct layers as well as signals located on granule cells have both been implicated. To study the role of granule cells for the lamina-specific ingrowth of afferents, the cyto- and fiberarchitecture of three mouse mutants (very low density lipoprotein receptor knockout mouse, apolipoprotein E receptor 2 knockout mouse, and reeler mouse) that show different degrees of granule cell migration defects were analyzed. Anterograde tracing with Phaseolus vulgaris-leucoagglutinin was used to visualize the afferent fiber systems, and immunohistochemistry was used to determine the position of their putative target cells. In controls, granule cells are packed in a single layer. This laminar organization is mildly altered in very low density lipoprotein receptor knockout mice, moderately disturbed in apolipoprotein E receptor 2 knockout mice, and severely disrupted in reeler mice. These changes in granule cell distribution are mirrored by the distribution of commissural fibers. In contrast, changes in granule cell distribution do not severely affect the laminar termination of entorhinal fibers. These data provide further evidence for a role of granule cells in the laminar termination of commissural/associational afferents to the fascia dentata.
Article
We have studied the organization and cellular differentiation of dentate granule cells and their axons, the mossy fibers, in reeler mutant mice lacking reelin and in mutants lacking the reelin receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). We show that granule cells in reeler mice do not form a densely packed granular layer, but are loosely distributed throughout the hilar region. Immunolabeling for calbindin and calretinin revealed that the sharp border between dentate granule cells and hilar mossy cells is completely lost in reeler mice. ApoER2/VLDLR double-knockout mice copy the reeler phenotype. Mice deficient only in VLDLR showed minor alterations of dentate organization; migration defects were more prominent in ApoER2 knockout mice. Tracing of the mossy fibers with Phaseolus vulgaris leukoagglutinin and calbindin immunolabeling revealed an irregular broad projection in reeler mice and ApoER2/VLDLR double knockouts, likely caused by the irregular wide distribution of granule cell somata. Mutants lacking only one of the lipoprotein receptors showed only minor changes in the mossy fiber projection. In all mutants, mossy fibers respected the CA3-CA1 border. Retrograde labeling with DiI showed that malpositioned granule cells also projected as normal to the CA3 region. These results indicate that ( 1 ) reelin signaling via ApoER2 and VLDLR is required for the normal positioning of dentate granule cells and (2) the reelin signaling pathway is not involved in pathfinding and target recognition of granule cell axons.
Article
The most common type of epilepsy in adults is temporal lobe epilepsy. After epileptogenic injuries, dentate granule cell axons (mossy fibers) sprout and form new synaptic connections. Whether this synaptic reorganization strengthens recurrent inhibitory circuits or forms a novel recurrent excitatory circuit is unresolved. We labeled individual granule cells in vivo, reconstructed sprouted mossy fibers at the EM level, and identified postsynaptic targets with GABA immunocytochemistry in the pilocarpine model of temporal lobe epilepsy. Granule cells projected an average of 1.0 and 1.1 mm of axon into the granule cell and molecular layers, respectively. Axons formed an average of one synapse every 7 microm in the granule cell layer and every 3 microm in the molecular layer. Most synapses were with spines (76 and 98% in the granule cell and molecular layers, respectively). Almost all of the synapses were with GABA-negative structures (93 and 96% in the granule cell and molecular layers, respectively). By integrating light microscopic and EM data, we estimate that sprouted mossy fibers form an average of over 500 new synapses per granule cell, but <25 of the new synapses are with GABAergic interneurons. These findings suggest that almost all of the synapses formed by mossy fibers in the granule cell and molecular layers are with other granule cells. Therefore, after epileptogenic treatments that kill hilar mossy cells, mossy fiber sprouting does not simply replace one recurrent excitatory circuit with another. Rather, it replaces a distally distributed and disynaptic excitatory feedback circuit with one that is local and monosynaptic.
Article
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Article
There is a high correlation between pediatric epilepsies and neuronal migration disorders. What remains unclear is whether there are intrinsic features of the individual dysplastic cells that give rise to heightened seizure susceptibility, or whether these dysplastic cells contribute to seizure activity by establishing abnormal circuits that alter the balance of inhibition and excitation. Mice lacking a functional p35 gene provide an ideal model in which to address these questions, because these knock-out animals not only exhibit aberrant neuronal migration but also demonstrate spontaneous seizures. Extracellular field recordings from hippocampal slices, characterizing the input-output relationship in the dentate, revealed little difference between wild-type and knock-out mice under both normal and elevated extracellular potassium conditions. However, in the presence of the GABA(A) antagonist bicuculline, p35 knock-out slices, but not wild-type slices, exhibited prolonged depolarizations in response to stimulation of the perforant path. There were no significant differences in the intrinsic properties of dentate granule cells (i.e., input resistance, time constant, action potential generation) from wild-type versus knock-out mice. However, antidromic activation (mossy fiber stimulation) evoked an excitatory synaptic response in over 65% of granule cells from p35 knock-out slices that was never observed in wild-type slices. Ultrastructural analyses identified morphological substrates for this aberrant excitation: recurrent axon collaterals, abnormal basal dendrites, and mossy fiber terminals forming synapses onto the spines of neighboring granule cells. These studies suggest that granule cells in p35 knock-out mice contribute to seizure activity by forming an abnormal excitatory feedback circuit.
Article
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.
Article
Reeler mice are a model of cortical malformation with enhanced seizure susceptibility. Data suggest that the propensity to anesthesia-induced seizures may be enhanced in animal models with developmental anomalies. Consequently, reeler mice were monitored behaviorally before, during and after isoflurane anesthesia. During recovery, 12% of reeler homozygotes had class I/II seizures while the remaining 88% exhibited convulsive seizures entailing opisthotonus and forepaw drumming. Similar behavior was not observed in controls. These data reveal that reeler mice display isoflurane-induced seizures and provide support for the hypothesis that developmental anomalies may predispose the central nervous system to anesthesia-induced seizures.
Article
Granule cell dispersion (GCD) in the dentate gyrus is a frequent feature of Ammon's horn sclerosis (AHS) which is often associated with temporal lobe epilepsy (TLE). It has been hypothesized that GCD may be caused by an abnormal migration of newly born granule cells. To test this hypothesis, we used markers of proliferation and neurogenesis and immunocytochemical methods as well as quantitative Western blot and real-time RT-PCR analyses in surgically resected hippocampi from TLE patients and controls. Below the age of 1 year, Ki-67-immunopositive nuclei were detected in the subgranular zone of the dentate gyrus, but not in the dentate of TLE patients independent of age. The expression of the proliferation marker minichromosome maintenance protein 2 (mcm2) and of doublecortin (DCX) decreased significantly with age in controls and in TLE patients, but the expression of both proteins was independent of the degree of AHS and GCD. Quantitative real-time RT-PCR confirmed these findings at the level of gene expression. In contrast, immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin as well as Golgi staining revealed a radially aligned glial network in the region of GCD. GFAP-positive fiber length significantly increased with the severity of GCD. These results indicate that epileptic activity does not stimulate neurogenesis in the human dentate gyrus and that GCD probably does not result from a malpositioning of newly generated granule cells, but rather from an abnormal migration of mature granule cells along a radial glial scaffold.
Article
Although cortical malformations (CMs) are often associated with epilepsy, the underlying mechanisms are unknown. The reeler mouse is a model of CM with enhanced susceptibility to epileptiform activity, including the in vitro dentate gyrus, a region normally resistant to seizures. In this study, field potential recordings in hippocampal slices and the Timm stain were used to examine mossy fiber distribution in the dentate gyrus. In artificial cerebrospinal fluid containing bicuculline, 100% of reeler slices and 0% of control slices had spontaneous and antidromic evoked prolonged negative field potential shifts that were blocked by glutamate receptor antagonists. Sections from reeler mice, but not controls, exhibited a dark band of Timm's stain at the molecular layer/granule cell layer border. These data reveal that mossy fiber distribution is altered in reeler mice and coincides with the presence of an abnormal proconvulsive glutamatergic circuit.