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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.
The maturation of adult-born granule cells and their functional integration into the network is thought to play a key role in the proper functioning of the dentate gyrus. In temporal lobe epilepsy, adult-born granule cells in the dentate gyrus develop abnormally and possess a hilar basal dendrite (HBD). Although morphological studies have shown that these HBDs have synapses, little is known about the functional properties of these HBDs or the intrinsic and network properties of the granule cells that possess these aberrant dendrites.
We performed patch-clamp recordings of granule cells within the granule cell layer "normotopic" from sham-control and status epilepticus (SE) animals. Normotopic granule cells from SE animals possessed an HBD (SE+ HBD+ cells) or not (SE+ HBD- cells). Apical and basal dendrites were stimulated using multiphoton uncaging of glutamate. Two-photon Ca2+ imaging was used to measure Ca2+ transients associated with back-propagating action potentials (bAPs).
Near-synchronous synaptic input integrated linearly in apical dendrites from sham-control animals and was not significantly different in apical dendrites of SE+ HBD- cells. The majority of HBDs integrated input linearly, similar to apical dendrites. However, 2 of 11 HBDs were capable of supralinear integration mediated by a dendritic spike. Furthermore, the bAP-evoked Ca2+ transients were relatively well maintained along HBDs, compared with apical dendrites. This further suggests an enhanced electrogenesis in HBDs. In addition, the output of granule cells from epileptic tissue was enhanced, with both SE+ HBD- and SE+ HBD+ cells displaying increased high-frequency (>100 Hz) burst-firing. Finally, both SE+ HBD- and SE+ HBD+ cells received recurrent excitatory input that was capable of generating APs, especially in the absence of feedback inhibition.
Taken together, these data suggest that the enhanced excitability of HBDs combined with the altered intrinsic and network properties of granule cells collude to promote excitability and synchrony in the epileptic dentate gyrus.
The hippocampus is reciprocally connected with the entorhinal cortex. Although several studies emphasized a role for the entorhinal cortex in mesial temporal lobe epilepsy (MTLE), it remains uncertain whether its synaptic connections with the hippocampus are altered. To address this question, we traced hippocampo-entorhinal and entorhino-hippocampal projections, assessed their connectivity with the respective target cells and examined functional alterations in a mouse model for MTLE. We show that hippocampal afferents to the dorsal entorhinal cortex are lost in the epileptic hippocampus. Conversely, entorhino-dentate projections via the medial perforant path (MPP) are preserved, but appear substantially altered on the synaptic level. Confocal imaging and 3D-reconstruction revealed that new putative contacts are established between MPP fibers and dentate granule cells (DGCs). Immunohistochemical identification of pre- and postsynaptic elements indicated that these contacts are functionally mature synapses. On the ultrastructural level, pre- and postsynaptic compartments of MPP synapses were strongly enlarged. The length and complexity of postsynaptic densities were also increased pointing to long-term potentiation-related morphogenesis. Finally, whole-cell recordings of DGCs revealed an enhancement of evoked excitatory postsynaptic currents. In conclusion, the synaptic rearrangement of excitatory inputs to DGCs from the medial entorhinal cortex may contribute to the epileptogenic circuitry in MTLE.
The dentate gyrus (DG) is important to many aspects of hippocampal function, but there are many aspects of the DG that are incompletely understood. One example is the role of mossy cells (MCs), a major DG cell type that is glutamatergic and innervates the primary output cells of the DG, the granule cells (GCs). MCs innervate the GCs as well as local circuit neurons that make GABAergic synapses on GCs, so the net effect of MCs on GCs - and therefore the output of the DG - is unclear. Here we first review fundamental information about MCs and the current hypotheses for their role in the normal DG and in diseases that involve the DG. Then we review previously published data which suggest that MCs are a source of input to a subset of GCs that are born in adulthood (adult-born GCs). In addition, we discuss the evidence that adult-born GCs may support the normal inhibitory 'gate' functions of the DG, where the GCs are a filter or gate for information from the entorhinal cortical input to area CA3. The implications are then discussed in the context of seizures and temporal lobe epilepsy (TLE). In TLE, it has been suggested that the DG inhibitory gate is weak or broken and MC loss leads to insufficient activation of inhibitory neurons, causing hyperexcitability. That idea was called the "dormant basket cell hypothesis." Recent data suggest that loss of normal adult-born GCs may also cause disinhibition, and seizure susceptibility. Therefore, we propose a reconsideration of the dormant basket cell hypothesis with an intervening adult-born GC between the MC and basket cell and call this hypothesis the "dormant immature granule cell hypothesis."
Purpose: Aberrant plastic changes among adult‐generated hippocampal dentate granule cells are hypothesized to contribute to the development of temporal lobe epilepsy. Changes include formation of basal dendrites projecting into the dentate hilus. Innervation of these processes by granule cell mossy fiber axons leads to the creation of recurrent excitatory circuits within the dentate. The destabilizing effect of these recurrent circuits may contribute to hyperexcitability and seizures. Although basal dendrites have been identified in status epilepticus models of epilepsy associated with increased neurogenesis, we do not know whether similar changes are present in the intrahippocampal kainic acid model of epilepsy, which is associated with reduced neurogenesis.Methods: In the present study, we used Thy1‐YFP–expressing transgenic mice to determine whether hippocampal dentate granule cells develop hilar‐projecting basal dendrites in the intrahippocampal kainic acid model. Brain sections were examined 2 weeks after treatment. Tissue was also examined using ZnT‐3 immunostaining for granule cell mossy fiber terminals to assess recurrent connectivity. Adult neurogenesis was assessed using the proliferative marker Ki‐67 and the immature granule cell marker calretinin.Key Findings: Significant numbers of cells with basal dendrites were found in this model, but their structure was distinct from basal dendrites seen in other epilepsy models, often ending in complex tufts of short branches and spines. Even more unusual, a subset of cells with basal dendrites had an inverted appearance; they completely lacked apical dendrites. Spines on basal dendrites were found to be apposed to ZnT‐3 immunoreactive puncta, suggestive of recurrent mossy fiber input. Finally, YFP‐expressing abnormal granule cells did not colocalize Ki‐67 or calretinin, indicating that these cells were more than a few weeks old, but were found almost exclusively in proximity to the neurogenic subgranular zone, where the youngest granule cells are located.Significance: Recent studies have demonstrated in other models of epilepsy that dentate pathology develops following the aberrant integration of immature, adult‐generated granule cells. Given these findings, one might predict that the intrahippocampal kainic acid model of epilepsy, which is associated with a dramatic reduction in adult neurogenesis, would not exhibit these changes. Herein we demonstrate that hilar basal dendrites are a common feature of this model, with the abnormal cells likely resulting from the disruption of juvenile granule cell born in the weeks before the insult. These studies demonstrate that postinjury neurogenesis is not required for the accumulation of large numbers of abnormal granule cells.
Accumulation of abnormally integrated, adult-born, hippocampal dentate granule cells (DGCs) is hypothesized to contribute to the development of temporal lobe epilepsy (TLE). DGCs have long been implicated in TLE, because they regulate excitatory signaling through the hippocampus and exhibit neuroplastic changes during epileptogenesis. Furthermore, DGCs are unusual in that they are continually generated throughout life, with aberrant integration of new cells underlying the majority of restructuring in the dentate during epileptogenesis. Although it is known that these abnormal networks promote abnormal neuronal firing and hyperexcitability, it has yet to be established whether they directly contribute to seizure generation. If abnormal DGCs do contribute, a reasonable prediction would be that the severity of epilepsy will be correlated with the number or load of abnormal DGCs. To test this prediction, we used a conditional, inducible transgenic mouse model to fate map adult-generated DGCs. Mossy cell loss, also implicated in epileptogenesis, was assessed as well. Transgenic mice rendered epileptic using the pilocarpine-status epilepticus model of epilepsy were monitored continuously by video/EEG for 4 weeks to determine seizure frequency and severity. Positive correlations were found between seizure frequency and (1) the percentage of hilar ectopic DGCs, (2) the amount of mossy fiber sprouting, and (3) the extent of mossy cell death. In addition, mossy fiber sprouting and mossy cell death were correlated with seizure severity. These studies provide correlative evidence in support of the hypothesis that abnormal DGCs contribute to the development of TLE and also support a role for mossy cell loss.
The functional impact of adult-generated granule cells in the epileptic brain is unclear, with data supporting both protective and maladaptive roles. These conflicting findings could be explained if new granule cells integrate heterogeneously, with some cells taking neutral or adaptive roles and others contributing to recurrent circuitry supporting seizures. Here, we tested this hypothesis by completing detailed morphological characterizations of age- and experience-defined cohorts of adult-generated granule cells from transgenic mice. The majority of newborn cells exposed to an epileptogenic insult exhibited reductions in dendritic spine number, suggesting reduced excitatory input to these cells. A significant subset, however, exhibited higher spine numbers. These latter cells tended to have enlarged cell bodies, long basal dendrites, or both. Moreover, cells with basal dendrites received significantly more recurrent mossy fiber input through their apical dendrites, indicating that these cells are robustly integrated into the pathological circuitry of the epileptic brain. These data imply that newborn cells play complex--and potentially conflicting--roles in epilepsy.
The extracellular matrix protein Reelin, secreted by Cajal-Retzius (CR) cells in the marginal zone (MZ) of the cerebral cortex, is important for neuronal migration during development. Two lipoprotein receptors for Reelin have been identified, apolipoprotein E receptor 2 (ApoER2) and the very low-density lipoprotein receptor (VLDLR). The binding of Reelin to these receptors induces tyrosine phosphorylation of an adapter protein, disabled 1 (Dab1) by src family kinases (SFKs). In the Reelin-deficient mutant reeler, cortical lamination is inverted with many neurons invading the marginal zone and others that are unable to migrate to their destinations and accumulate underneath their predecessors, suggesting a role for Reelin signaling in dynamic cytoskeletal reorganization. At present these effects of Reelin are poorly understood. In our recent study, we showed that Reelin induces serine3 phosphorylation of n-cofilin, an actin-depolymerizing protein promoting the disassembly of F-actin. Phosphorylation of cofilin renders it unable to depolymerize F-actin, thus stabilizing the cytoskeleton. We provided evidence for ApoER2, Dab1, SFKs and phosphatidylinositol-3-kinase (PI3K) to be involved in Reelin-induced cofilin phosphorylation. We found that phosphorylation of cofilin occurs in the leading processes of radially migrating neurons as they grow towards the Reelin-containing marginal zone. By cofilin phosphorylation, Reelin may act as a stop signal for radially migrating neurons.
Dynorphin A(1-17), an opioid peptide that is normally present in the hippocampal mossy fiber system, was localized immunocytochemically in the hippocampal formation of control autopsy and temporal lobe epilepsy (TLE) specimens. In control tissue, dynorphin-like immunoreactive (Dyn-IR) structures were confined to the mossy fiber path and were most highly concentrated in the polymorph (hilar) region of the dentate gyrus. Very few Dyn-IR structures were present in the molecular and granule cell layers of the dentate gyrus. In contrast, in all TLE specimens, Dyn-IR elements were present in these layers. The extent of aberrant staining varied among the TLE specimens, and 2 major patterns were observed. The first was a relatively wide band of reaction product in the inner one-third to one-fourth of the molecular layer (8 cases), and the second was a more limited distribution of immunoreactive fibers and presumptive terminals in the granule cell and immediately adjacent supragranular regions (2 cases). The extent of aberrant Dyn-IR structures appeared to be related to the amount of cell loss in the polymorph and CA3 fields and to dispersion of the granule cell somata. Specimens processed with the Timm's sulfide silver method for heavy metals provided independent evidence for the distribution of mossy fibers. In both control and TLE specimens, the patterns of labeling were virtually identical to those of dynorphin localization. These findings suggest that sprouting of mossy fibers or their axon collaterals has occurred in hippocampal epilepsy and that the reorganized fibers contain at least one of the neuropeptides that are normally present in this system. Such fibers could form recurrent excitatory circuits and contribute to synchronous firing and epileptiform activity, as suggested in studies of experimental models of epilepsy.
Previous histological and immunocytochemical studies suggest that reorganization of the dentate granule cell axons, the mossy fibers, can occur in epileptic human hippocampus (Sutula et al., 1989; Houser et al., 1990; Babb et al., 1991) and in animal models of epilepsy (Tauck and Nadler, 1985; Sutula et al., 1988; Cronin et al., 1992). However, neuroanatomical analyses of the trajectory and morphology of reorganized axons are not yet available. The present study was conducted to investigate single dentate granule cell axonal systems in human epileptic hippocampus. Individual mossy fibers were directly visualized by injecting a tracer (biocytin or Lucifer yellow) intracellularly in hippocampal slices prepared from temporal lobes that were surgically removed from patients for treatment of intractable epilepsy. Two major arborization patterns were identified: (1) the parent axons extended to and coursed through the hilus toward CA3, leaving collaterals along their paths in the hilus (N = 19 neurons); (2) in addition to the aforementioned axonal system, collateral(s) branched from the parent axon near the soma and projected to the granule cell layer and molecular layer, forming an aberrant axonal pathway (N = 9 neurons). These aberrant collaterals bore large boutons similar to those of the hilar axons and formed extensive plexuses in the granule cell layer and/or in the molecular layer. The summed length of collaterals in the granular/molecular layers was 1110.8 microns on average, which was one-fourth of the total summed length of the mossy fibers (3698.5 microns on average). The size of the somata in neurons that had aberrant collaterals was significantly larger than that of neurons without such collaterals (p < 0.025). In four cases, filopodium-like fine processes were present near the axon hillock and proximal parts of the parent axon, suggesting that the aberrant collateral formation might be an ongoing process in these tissues. The lack of control slices from normal living human hippocampus makes it difficult to assess to what extent the present findings are epilepsy associated. However, the presence of aberrant mossy fiber collaterals in the hippocampi used in the present study has been confirmed by Timm's staining and/or dynorphin immunohistochemistry in comparison with nonepileptic autopsy material, indicating its relation to epilepsy (Babb et al., 1991, 1992). At present, there seems to be a consensus that the projection of mossy fiber collaterals to the supragranular layer is a rare occurrence in normal rats (Lorento de Nó, 1934; Claiborne et al., 1986; Seress et al., 1991; present study), normal monkeys (Seress et al., 1991), and normal humans (Houser et al., 1990).(ABSTRACT TRUNCATED AT 400 WORDS)
The reelin signaling pathway plays a crucial role during the development of laminated structures in the mammalian brain. Reelin, which is synthesized and secreted by Cajal-Retzius cells in the marginal zone of the neocortex and hippocampus, is proposed to act as a stop signal for migrating neurons. Here we show that a decreased expression of reelin mRNA by hippocampal Cajal-Retzius cells correlates with the extent of migration defects in the dentate gyrus of patients with temporal lobe epilepsy. These results suggest that reelin is required for normal neuronal lamination in humans, and that deficient reelin expression may be involved in migration defects associated with temporal lobe epilepsy.
Mesio-temporal lobe epilepsy (MTLE) is often accompanied by granule cell dispersion (GCD), a widening of the granule cell layer. The molecular determinants of GCD are poorly understood. Here, we used an animal model to study whether GCD results from an increased dentate neurogenesis associated with an abnormal migration of the newly generated granule cells. Adult mice were given intrahippocampal injections of kainate (KA) known to induce focal epileptic seizures and GCD, comparable to the changes observed in human MTLE. Ipsilateral GCD progressively developed after KA injection and was paralleled by a gradual decrease in the expression of doublecortin, a marker of newly generated granule cells, in the dentate subgranular layer. Staining with Fluoro-Jade B revealed little cell degeneration in the subgranular layer on the KA-injected side. Labeling with bromodeoxyuridine showed an early, transient increase in mitotic activity in the dentate gyrus of the KA-injected hippocampus that gave rise to microglial cells and astrocytes but not to new neurons. Moreover, at later time points, there was a virtually complete cessation of mitotic activity in the injected hippocampus (where GCD continued to develop), but not on the contralateral side (where no GCD was observed). Finally, a significant decrease in reelin mRNA synthesis in the injected hippocampus paralleled the development of GCD, and neutralization of reelin by application of the CR-50 antibody induced GCD. These results show that GCD does not result from increased neurogenesis but reflects a displacement of mature granule cells, most likely caused by a local reelin deficiency.
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.
Hippocampal sclerosis (HS) is the most prevalent pathology in temporal lobe epilepsy (TLE) characterized by segmental neuronal cell loss in the cornu ammonis (CA) 1-4. In addition, migration of granule cells and reorganization of their axons is observed, known as granule cell dispersion (GCD) and mossy fiber sprouting (MFS). The loss of mossy fibers` (MF) target cells in CA4 and CA3 was considered to be causative for MFS. The ILAE HS (International League Against Epilepsy) classification identifies three subtypes with different cell loss patterns in CA1-4. We studied the relation of ILAE HS subtypes to GCD and MFS to corroborate clinico-pathological subgroups in a large retrospective single-center series.
Material and methods:
Hippocampal specimen of 319 patients were screened, 214 could be used for analysis. Immunohistochemical stainings for semi-quantitative analysis of neuronal cell loss (NeuN) and MFS (synaptoporin) were performed. Presurgical data were available from patient files and seizure outcome was classified according to Engel score after surgery.
In 39 patients (18%) no neuronal cell loss (ILAE no-HS), no GCD and no MFS was observed. In 154 patients (72%) severe neuronal cell loss was seen in CA1, CA4 and CA3 (ILAE HS 1, typical HS); in addition extensive GCD and MFS was observed. In 17 patients (8%) cell loss was seen predominantly in CA1 (ILAE HS 2); despite different cell loss pattern these hippocampi also showed GCD and MFS. In 4 patients (2%) cell loss was predominately detected in CA3 and CA4 (ILAE HS type 3), consecutively GCD and MFS were observed. Longer epilepsy duration and younger age at surgery was more often associated with ILAE HS 2 and febrile convulsions were completely absent in ILAE no-HS. Yet, seizure onset, age at initial precipitating injury and postsurgical seizure outcome did not show any significant association with ILAE HS subtypes.
GCD and MFS might develop independently from the neuronal cell loss of MF target cells.
The most frequent finding in temporal lobe epilepsy is hippocampal sclerosis, characterized by selective cell loss of hippocampal subregions CA1 and CA4 as well as mossy fiber sprouting (MFS) towards the supragranular region and granule cell dispersion. Although selective cell loss is well described, its impact on mossy fiber sprouting and granule cell dispersion remains unclear.
Materials and methods:
In a single center series, we examined 319 human hippocampal specimens, collected in a 15-years period. Hippocampal specimens were stained for neuronal loss, granule cell dispersion (Wyler scale I-IV, Neu-N, HE) and mossy fiber sprouting (synaptoporin-immunohistochemistry). For seizure outcome Engel score I-IV was applied.
In Wyler I and II specimens, mossy fibers were found along their natural projection exclusively in CA4 and CA3. In Wyler III and IV, sprouting of mossy fibers into the molecular layer and a decrease of mossy fibers in CA4 and CA3 was detected. Mean granule cell dispersion was extended from 121μm to 185μm and correlated with Wyler III-IV as well as mossy fiber sprouting into the molecular layer. Wyler grade, mossy fiber sprouting and granule cell dispersion correlated with longer epilepsy duration, late surgery and higher preoperative seizure frequency. Parameters analyzed above did not correlate with postoperative seizure outcome.
Mossy fiber sprouting might be a compensatory phenomenon of cell death of the target neurons in CA4 and CA3 in Wyler III-IV. Axonal reorganization of granule cells is accompanied by their migration and is correlated with the severity of cell loss and epilepsy duration.
Febrile seizures (FS) are fever-associated convulsions, being the most common seizure disorder in early childhood. A subgroup of these children later develops epilepsy characterized by a hyperexcitable neuronal network in the hippocampus. Hippocampal excitability is regulated by the hippocampal dentate gyrus (DG) where postnatal neurogenesis occurs. Experimental FS increase the survival of newborn hippocampal dentate granule cells (DGCs), yet the significance of this neuronal subpopulation to the hippocampal network remains unclear. In the current study, we characterized the temporal maturation and structural integration of these post-FS born DGCs in the DG.
Experimental FS were induced in 10-day-old rat pups. The next day, retroviral particles coding for enhanced green fluorescent protein (eGFP) were stereotactically injected in the DG to label newborn cells. Histochemical analyses of eGFP expressing DGCs were performed one, 4, and 8 weeks later and consisted of the following: (1) colocalization with neurodevelopmental markers doublecortin, calretinin, and the mature neuronal marker NeuN; (2) quantification of dendritic complexity; and (3) quantification of spine density and morphology.
At neither time point were neurodevelopmental markers differently expressed between FS animals and normothermia (NT) controls. One week after treatment, DGCs from FS animals showed dendrites that were 66% longer than those from NT controls. At 4 and 8 weeks, Sholl analysis of the outer 83% of the molecular layer showed 20-25% more intersections in FS animals than in NT controls (p < 0.01). Although overall spine density was not affected, an increase in mushroom-type spines was observed after 8 weeks.
Experimental FS increase dendritic complexity and the number of mushroom-type spines in post-FS born DGCs, demonstrating a more mature phenotype and suggesting increased incoming excitatory information. The consequences of this hyperconnectivity to signal processing in the DG and the output of the hippocampus remain to be studied.
Aberrant plastic changes among adult-generated hippocampal dentate granule cells are hypothesized to contribute to the development of temporal lobe epilepsy. Changes include formation of basal dendrites projecting into the dentate hilus. Innervation of these processes by granule cell mossy fiber axons leads to the creation of recurrent excitatory circuits within the dentate. The destabilizing effect of these recurrent circuits may contribute to hyperexcitability and seizures. Although basal dendrites have been identified in status epilepticus models of epilepsy associated with increased neurogenesis, we do not know whether similar changes are present in the intrahippocampal kainic acid model of epilepsy, which is associated with reduced neurogenesis.
In the present study, we used Thy1-YFP-expressing transgenic mice to determine whether hippocampal dentate granule cells develop hilar-projecting basal dendrites in the intrahippocampal kainic acid model. Brain sections were examined 2 weeks after treatment. Tissue was also examined using ZnT-3 immunostaining for granule cell mossy fiber terminals to assess recurrent connectivity. Adult neurogenesis was assessed using the proliferative marker Ki-67 and the immature granule cell marker calretinin.
Significant numbers of cells with basal dendrites were found in this model, but their structure was distinct from basal dendrites seen in other epilepsy models, often ending in complex tufts of short branches and spines. Even more unusual, a subset of cells with basal dendrites had an inverted appearance; they completely lacked apical dendrites. Spines on basal dendrites were found to be apposed to ZnT-3 immunoreactive puncta, suggestive of recurrent mossy fiber input. Finally, YFP-expressing abnormal granule cells did not colocalize Ki-67 or calretinin, indicating that these cells were more than a few weeks old, but were found almost exclusively in proximity to the neurogenic subgranular zone, where the youngest granule cells are located.
Recent studies have demonstrated in other models of epilepsy that dentate pathology develops following the aberrant integration of immature, adult-generated granule cells. Given these findings, one might predict that the intrahippocampal kainic acid model of epilepsy, which is associated with a dramatic reduction in adult neurogenesis, would not exhibit these changes. Herein we demonstrate that hilar basal dendrites are a common feature of this model, with the abnormal cells likely resulting from the disruption of juvenile granule cell born in the weeks before the insult. These studies demonstrate that postinjury neurogenesis is not required for the accumulation of large numbers of abnormal granule cells.
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.
Reelin controls the migration of neurons and layer formation during brain development. However, recent studies have shown that disrupting Reelin function in the adult hippocampus induces repositioning of fully differentiated neurons, suggesting a stabilizing effect of Reelin on mature neuronal circuitry. Indeed, Reelin was recently found to stabilize the actin cytoskeleton by inducing cofilin phosphorylation. When unphosphorylated, cofilin acts as an actin-depolymerizing protein that promotes the disassembly of F-actin. Here, a novel hypothesis is proposed whereby decreased Reelin expression in the mature brain causes destabilization of neurons and their processes, leading to aberrant plasticity and aberrant wiring of brain circuitry. This has implications for brain disorders, such as epilepsy and schizophrenia, in which deficiencies in Reelin expression occur.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Hilar mossy cells of the mouse were shown recently to display calretinin immunoreactivity (Liu et al.  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.
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.
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.
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.
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.
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.
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.
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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.
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.
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.
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.
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.