Restrained Dendritic Growth of Adult-Born Granule Cells Innervated by Transplanted Fetal GABAergic Interneurons in Mice with Temporal Lobe Epilepsy.

The dentate gyrus (DG) is a region of the adult rodent brain that undergoes continuous neurogenesis. Seizures and loss or dysfunction of GABAergic synapses onto adult-born dentate granule cells (GCs) alter their dendritic growth and migration, resulting in dysmorphic and hyperexcitable GCs. Additionally, transplants of fetal GABAergic interneurons in the DG of mice with temporal lobe epilepsy (TLE) result in seizure suppression, but it is unknown whether increasing interneurons with these transplants restores GABAergic innervation to adult-born GCs. Here, we address this question by birth-dating GCs with retrovirus at different times up to 12 weeks after pilocarpine-induced TLE in adult mice. Channelrhodopsin 2 (ChR2)-enhanced yellow fluorescent protein (EYFP)-expressing medial-ganglionic eminence (MGE)-derived GABAergic interneurons from embryonic day (E)13.5 mouse embryos were transplanted into the DG of the TLE mice and GCs with transplant-derived inhibitory post-synaptic currents (IPSCs) were identified by patch-clamp electrophysiology and optogenetic interrogation. Putative synaptic sites between GCs and GABAergic transplants were also confirmed by intracellular biocytin staining, immunohistochemistry, and confocal imaging. 3D reconstructions of dendritic arbors and quantitative morphometric analyses were carried out in >150 adult-born GCs. GABAergic inputs from transplanted interneurons correlated with markedly shorter GC dendrites, compared to GCs that were not innervated by the transplants. Moreover, these effects were confined to distal dendritic branches and a short time window of six to eight weeks. The effects were independent of seizures as they were also observed in naïve mice with MGE transplants. These findings are consistent with the hypothesis that increased inhibitory currents over a smaller dendritic arbor in adult-born GCs may reduce their excitability and lead to seizure suppression.

Proliferative hippocampal activity in a group of patients with Rasmussen's encephalitis: Neuronal, glial, and BDNF tissue expression correlations.

Rasmussen's encephalitis (RE) is a rare and devastating unilateral inflammatory brain disease that causes severe and intractable partial epilepsy. It has been shown that epilepsy and subsequent inflammation have deleterious influence on hippocampal cell survival and neurogenesis, but this still has not been systematically explored in human tissue. In this study, we investigated the correlation between inflammation and epilepsy as well as the rates of hippocampal gliogenesis and neurogenesis in a pediatric group of six patients with RE and six control cases. The dentate gyrus (DG) samples were obtained from patients who underwent surgery for intractable RE. Sections were processed for immunohistochemistry using antibodies against sex determining region Y-box 2 (Sox2), nestin, human protein encoded by MKI67 gen (Ki67), and brain-derived neurotrophic factor (BDNF). There was an increase in the number of Ki67-positive granule cells in the DG of patients with RE in comparison with the autopsy control group, but no statistical difference for Sox2-positive cells was observed between these groups. Nestin immunolabeling was less intense in the RE group while BDNF expression was increased. Neurons that were BDNF-positive were found in DG from patients with RE but not in the control group. In patients with RE, few nestin-positive cells in DG were also positive for BDNF, unlike in controls which showed no colocalization for these two markers. These results suggest a proliferation activity in the DG subfield of patients with RE, and also future studies are necessary to address the role of new cells in the hippocampus of patients with RE.

MGE-derived nNOS+ interneurons promote fear acquisition in nNOS-/- mice.

Neuronal nitric oxide synthase (nNOS) 1, mainly responsible for NO release in central nervous system (CNS) 2, plays a significant role in multiple physiological functions. However, the function of nNOS+ interneurons in fear learning has not been much explored. Here we focused on the medial ganglionic eminences (MGE) 3-derived nNOS+ interneurons in fear learning. To determine the origin of nNOS+ interneurons, we cultured neurons in vitro from MGE, cortex, lateral ganglionic eminence (LGE) 4, caudal ganglionic eminences (CGE) 5 and preoptic area (POA) 6. The results showed that MGE contained the most abundant precursors of nNOS+ interneurons. Moreover, donor cells from E12.5 embryos demonstrated the highest positive rate of nNOS+ interneurons compared with other embryonic periods (E11.5, E12, E13, E13.5 and E14). Additionally, these cells from E12.5 embryos showed long axonal and abundant dendritic arbors after 10 days culture, indicating the capability to disperse and integrate in host neural circuits after transplantation. To investigate the role of MGE-derived nNOS+ interneurons in fear learning, donor MGE cells were transplanted into dentate gyrus (DG) 7 of nNOS knock-out (nNOS-/-) or wild-type mice. Results showed that the transplantation of MGE cells promoted the acquisition of nNOS-/- but not the wild-type mice, suggesting the importance of nNOS+ neurons in fear acquisition. Moreover, we transplanted MGE cells from nNOS-/- mice or wild-type mice into DG of the nNOS-/- mice and found that only MGE cells from wild-type mice but not the nNOS-/- mice rescued the deficit in acquisition of the nNOS-/- mice, further confirming the positive role of nNOS+ neurons in fear learning.

Differential gene expression in dentate granule cells in mesial temporal lobe epilepsy with and without hippocampal sclerosis.

OBJECTIVE: Hippocampal sclerosis is the most common neuropathologic finding in cases of medically intractable mesial temporal lobe epilepsy. In this study, we analyzed the gene expression profiles of dentate granule cells of patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis to show that next-generation sequencing methods can produce interpretable genomic data from RNA collected from small homogenous cell populations, and to shed light on the transcriptional changes associated with hippocampal sclerosis. METHODS: RNA was extracted, and complementary DNA (cDNA) was prepared and amplified from dentate granule cells that had been harvested by laser capture microdissection from surgically resected hippocampi from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis. Sequencing libraries were sequenced, and the resulting sequencing reads were aligned to the reference genome. Differential expression analysis was used to ascertain expression differences between patients with and without hippocampal sclerosis. RESULTS: Greater than 90% of the RNA-Seq reads aligned to the reference. There was high concordance between transcriptional profiles obtained for duplicate samples. Principal component analysis revealed that the presence or absence of hippocampal sclerosis was the main determinant of the variance within the data. Among the genes up-regulated in the hippocampal sclerosis samples, there was significant enrichment for genes involved in oxidative phosphorylation. SIGNIFICANCE: By analyzing the gene expression profiles of dentate granule cells from surgically resected hippocampal specimens from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis, we have demonstrated the utility of next-generation sequencing methods for producing biologically relevant results from small populations of homogeneous cells, and have provided insight on the transcriptional changes associated with this pathology.

Prognostic value of CA4/DG volumetry with 3T magnetic resonance imaging on postoperative outcome of epilepsy patients with dentate gyrus pathology.

PURPOSE: Hippocampal sclerosis (HS), the most common feature of mesial temporal lobe epilepsy (MTLE), is widely accepted as surgical indication for refractory epilepsy. Pathological hallmarks in hippocampal dentate gyrus (DG), including granule cell loss (GCL) and granule cell dispersion (GCD), are known to be closely related to the status epilepticus and spontaneous seizure. Our aim was to assess the association between volumetric changes in the hippocampal CA4/DG determined with 3-Tesla (3T) magnetic resonance imaging (MRI) and the postoperative seizure outcomes in MTLE patients with or without dentate gyrus pathology (DGP). METHODS: High-resolution T2- and T1-weighted three-dimensional (3D) MRI scans were performed on 39 MTLE patients before surgery with a 3T Philips scanner. ITK-SNAP software was used for segmentation and volumetry of the CA4/DG segment, and NASP software was used for 3D reconstructions of the CA4/DG region. Immunostaining for Neuronal Nuclei (NeuN) was performed on resected hippocampal specimens after surgery to verify the accuracy of CA4/DG segmentation and histopathological changes in DG. RESULTS: The CA4/DG subfield could be precisely segmented with high-resolution 3T MRI and confirmed by comparison of NeuN-immunoreactive slices with MRI results. MTLE patients with DGP showed smaller CA4/DG volume and favorable postoperative seizure outcomes. CONCLUSION: The volumetry of CA4/DG was associated with the pathological changes in DG in MTLE patients. The volumetry of CA4/DG with preoperative 3T MRI could predict the postoperative seizure outcomes in those patients.

Human temporal lobe epilepsy analyses by tissue proteomics.

Although there are many types of epilepsy, temporal lobe epilepsy (TLE) is probably in humans the most common and most often studied. TLE represents 40% of the total epilepsy form of the disease and is difficult to treat. Despite a wealth of descriptive data obtained from the disease history of patients, the EEG recording, imaging techniques, and histological studies, the epileptogenic process remains poorly understood. However, it is unlikely that a single factor or a single mechanism can cause many changes associated with this neuropathological phenomenon. MALDI mass spectrometry imaging (MSI) coupled to protein identification, because of its ability to study a wide range of molecules, appears to be suitable for the preparation of molecular profiles in TLE. Seven neuropeptides have been have been identified in Dental gyrus regions of the hippocampus in relation with TLE pathology. Shot-gun studies taking into account gender influence have been performed. Tissue microextraction from control (10) toward 10 TLE patients have been analyzed after trypsin digestion followed by separation on nanoLC coupled to LTQ orbitrap. From the shot-gun analyses, results confirmed the presence of specific neuropeptides precursors and receptors in TLE patients as well as proteins involved in axons regeneration including neurotrophins, ECM proteins, cell surface proteins, membrane proteins, G-proteins, cytoskeleton proteins and tumor suppressors. Among the tumor suppressors identified, the Leucine-rich glioma inactivated 1 (LGI1) protein was found. LGI1 gene recently been demonstrated being implicated in heritability of TLE. We have also demonstrate the presence a complete profile of tumor suppressors in TLE patients, 7 have been identified. Refining this analysis taken into account the gender influence in both control and in TLE reflected the presence of specific proteins between male and female and thus mechanisms in pathology development could be completely different.

Upregulation of APP, ADAM10 and ADAM17 in the denervated mouse dentate gyrus.

The disintegrin and metalloproteinases ADAM10 and ADAM17 are regarded as the most important α-secretases involved in the physiological processing of amyloid precursor protein (APP) in brain. Since it has been suggested that processing of APP by α-secretases could be involved in the reorganization of the brain following injury, we studied mRNA expression of the two α-secretases Adam10 and Adam17, the ß-secretase Bace1, and the App-gene family (App, Aplp1, Aplp2) in the dentate gyrus of the mouse following entorhinal denervation. Using laser microdissection, tissue was harvested from the outer molecular layer and the granule cell layer of the denervated dentate gyrus. Expression levels of candidate genes were assessed using Affymetrix GeneChip Mouse Gene 1.0 ST arrays and reverse transcription-quantitative PCR, revealing an upregulation of Adam10 mRNA and Adam17 mRNA in the denervated outer molecular layer and an upregulation of Adam10 mRNA and App mRNA in the dentate granule cell layer. Immunolabeling for ADAM10 or ADAM17 in combination with markers for astro- and microglia revealed an increased labeling of ADAM10 and ADAM17 in the denervated outer molecular layer that was associated with reactive astrocytes but not with microglia. Collectively, these data show that denervation affects the expression level of APP and its two most important α-secretases. This suggests that APP-processing could be shifted towards the non-amyloidogenic pathway in denervated areas of the brain and, thus, towards the formation of neuroprotective APP cleavage products, such as APPsα.

Differentiation and functional incorporation of embryonic stem cell-derived GABAergic interneurons in the dentate gyrus of mice with temporal lobe epilepsy.

Cell therapies for neurological disorders require an extensive knowledge of disease-associated neuropathology and procedures for generating neurons for transplantation. In many patients with severe acquired temporal lobe epilepsy (TLE), the dentate gyrus exhibits sclerosis and GABAergic interneuron degeneration. Mounting evidence suggests that therapeutic benefits can be obtained by transplanting fetal GABAergic progenitors into the dentate gyrus in rodents with TLE, but the scarcity of human fetal cells limits applicability in patient populations. In contrast, virtually limitless quantities of neural progenitors can be obtained from embryonic stem (ES) cells. ES cell-based therapies for neurological repair in TLE require evidence that the transplanted neurons integrate functionally and replace cell types that degenerate. To address these issues, we transplanted mouse ES cell-derived neural progenitors (ESNPs) with ventral forebrain identities into the hilus of the dentate gyrus of mice with TLE and evaluated graft differentiation, mossy fiber sprouting, cellular morphology, and electrophysiological properties of the transplanted neurons. In addition, we compared electrophysiological properties of the transplanted neurons with endogenous hilar interneurons in mice without TLE. The majority of transplanted ESNPs differentiated into GABAergic interneuron subtypes expressing calcium-binding proteins parvalbumin, calbindin, or calretinin. Global suppression of mossy fiber sprouting was not observed; however, ESNP-derived neurons formed dense axonal arborizations in the inner molecular layer and throughout the hilus. Whole-cell hippocampal slice electrophysiological recordings and morphological analyses of the transplanted neurons identified five basic types; most with strong after-hyperpolarizations and smooth or sparsely spiny dendritic morphologies resembling endogenous hippocampal interneurons. Moreover, intracellular recordings of spontaneous EPSCs indicated that the new cells functionally integrate into epileptic hippocampal circuitry.

Dentate gyrus and hilus transection blocks seizure propagation and granule cell dispersion in a mouse model for mesial temporal lobe epilepsy.

Epilepsy-associated changes of the anatomical organization of the dentate gyrus and hilus may play a critical role in the initiation and propagation of seizures in mesial temporal lobe epilepsy (MTLE). This study evaluated the role of longitudinal projections in the propagation of hippocampal paroxysmal discharges (HPD) in dorsal hippocampus by performing a selective transection in a mouse model for MTLE obtained by a single unilateral intrahippocampal injection of kainic acid (KA). Full transections of the dentate gyrus and hilus were performed in the transverse axis at 22 days after KA injection when spontaneous HPD were fully developed. They: (i) significantly reduced the occurrence of HPD; (ii) increased their duration at the KA injection site; (iii) abolished their spread along the longitudinal axis of the hippocampal formation and; (iv) limited granule cell dispersion (GCD) of the dentate gyrus posterior to the transection. These data suggest that: (i) longitudinal projections through the dentate gyrus and hilus are involved in HPD spread; (ii) distant hippocampal circuits participate in the generation and cessation of HPD and; (iii) GCD requires continuous HPD to develop, even when seizures are established. Our data reveal a critical role for longitudinal projections in the generation and spread of hippocampal seizures.

Survival of mossy cells of the hippocampal dentate gyrus in humans with mesial temporal lobe epilepsy.

OBJECT: Hippocampal sclerosis can be identified in most patients with mesial temporal lobe epilepsy (TLE). Surgical removal of the sclerotic hippocampus is widely performed to treat patients with drug-resistant mesial TLE. In general, both epilepsy-prone and epilepsy-resistant neurons are believed to be in the hippocampal formation. The hilar mossy cells of the hippocampal dentate gyrus are usually considered one of the most vulnerable types of neurons. The aim of this study was to clarify the fate of mossy cells in the hippocampus in epileptic humans. METHODS: Of the 19 patients included in this study, 15 underwent temporal lobe resection because of drug-resistant TLE. Four patients were used as controls because they harbored tumors that had not invaded the hippocampus and they had experienced no seizures. Histological evaluation of resected hippocampal tissues was performed using immunohistochemistry. RESULTS: Mossy cells were identified in the control as well as the epileptic hippocampi by using cocaine- and amphetamine-regulated transcript peptide immunohistochemistry. In most cases the number of mossy cells was reduced and thorny excrescences were smaller in the epileptic hippocampi than in controls; however, there was a significant loss of pyramidal cells and a partial loss of granule cells in the same epileptic hippocampi in which mossy cell loss was apparent. The loss of mossy cells could be correlated with the extent of hippocampal sclerosis, patient age at seizure onset, duration of epilepsy, and frequency of seizures. CONCLUSIONS: In many cases large numbers of mossy cells were present in the hilus of the dentate gyrus when most pyramidal neurons of the CA1 and CA3 areas of the Ammon's horn were lost, suggesting that mossy cells may not be more vulnerable to epileptic seizures than the hippocampal pyramidal neurons.

Towards a clinico-pathological classification of granule cell dispersion in human mesial temporal lobe epilepsies.

The dentate gyrus (DG) plays a pivotal role in the functional and anatomical organization of the hippocampus and is involved in learning and memory formation. However, the impact of structural DG abnormalities, i.e., granule cell dispersion (GCD), for hippocampal seizure susceptibility and its association with distinct lesion patterns in epileptic disorders, such as mesial temporal sclerosis (MTS) remains enigmatic and a large spectrum of pathological changes has been recognized. Here, we propose a clinico-pathological classification of DG pathology based on the examination of 96 surgically resected hippocampal specimens obtained from patients with chronic temporal lobe epilepsy (TLE). We observed three different histological patterns. (1) A normal granule cell layer was identified in 11 patients (no-GCP; 18.7%). (2) Substantial granule cell loss was evident in 36 patients (referred to as granule cell pathology (GCP) Type 1; 37.5%). (3) Architectural abnormalities were observed in 49 specimens, including one or more of the following features: granule cell dispersion, ectopic neurons or clusters of neurons in the molecular layer, or bi-lamination (GCP Type 2; 51%). Cell loss was always encountered in this latter cohort. Seventy-eight patients of our present series suffered from MTS (81.3%). Intriguingly, all MTS patients displayed a compromised DG, 31 (40%) with significant cell loss (Type 1) and 47 (60%) with GCD (Type 2). In 18 patients without MTS (18.7%), seven displayed focally restricted DG abnormalities, either cell loss (n = 5) or GCD (n = 2). Clinical histories revealed a significant association between DG pathology patterns and higher age at epilepsy surgery (p = 0.008), longer epilepsy duration (p = 0.004), but also with learning dysfunction (p < 0.05). There was no correlation with the extent of pyramidal cell loss in adjacent hippocampal segments nor with postsurgical seizure relief. The association with long-term seizure histories and cognitive dysfunction is remarkable and may point to a compromised regenerative capacity of the DG in this cohort of TLE patients.

Exposure of the vertebrobasilar artery junction with traction of the dentate ligament for the treatment of large vertebral artery aneurysms: technical note.

A technique for exposing the vertebrobasilar junction with traction of the dentate ligament is described for treatment of large vertebral artery (VA) aneurysms via the far lateral suboccipital approach with partial condylar resection. The most rostral attachment of the dentate ligament is divided above the site where the VA pierces the dura mater. A traction suture is placed into the dentate ligament and gently retracted using mosquito forceps. As a result, the medulla oblongata is lifted dorsally and slightly rotated by the divided and retracted dentate ligament, allowing an approach from a more superior or inferior direction. The present technique is useful for the treatment of large thrombosed VA aneurysms.

Modeling behavioral recovery following lesion induction in the rat dentate gyrus.

Unilateral entorhinal lesions have enjoyed immense popularity as a model of recovery from damage. In part, the popularity has been supported the laminar organization of the hippocampal formation, which allows for the dissection of the contribution of individual afferent pathways to the recovery process. The commissural/associational pathway is of particular interest, since electrophysiological and gross anatomical data, although limited, have correlated sprouting in this pathway with behavioral recovery. Unfortunately, information relating recovery to synaptic structure is lacking. Addressing this issue, two analyses were conducted. Initially, a quantitative review of the literature reporting behavioral recovery following this type of lesion was conducted using meta-analytic techniques. Using this detailed information across decades of research, multiple linear regression analysis was conducted to address whether the morphological correlates of recovery could predict behavioral recovery. This resulted in an equation relating morphology and recovery that stood up well to several diagnostic tests. Moreover, this model suggests that synapse structure (in particular, synapse size and curvature, as well as terminal compartmentalization and the density of multi-synaptic terminals) holds a greater potential to predict behavioral recovery than increases in synapse number, which is typically seen as the optimal anatomical measure of recovery. This initial attempt to identify, quantify, and validate a model of lesion recovery is an important initial step in understanding how synaptic morphology may help mediate recovery of function.

Transplantation of neurospheres after granule cell lesions in rats: cognitive improvements despite no long-term immunodetection of grafted cells.

EGF-responsive C17 murine-derived neural stem cells (neurospheres) were grafted into the dentate gyrus of adult male rats after dentate granule cells lesions produced by colchicine injections. Behavioural performance was evaluated over two post-grafting periods, using tests sensitive to hippocampal dysfunctions. The first period began 1 month after grafting and testing conducted in the water maze and the radial maze distinguished working- and reference-memory performance. The second period began 9 months after grafting and learning performance was also evaluated in a Hebb-Williams maze, in addition to both other tests. The lesions induced lasting deficits in all tests. During the first period, the grafts had no effect in either test. Conversely, during the second period, grafted rats showed a weak improvement in the water maze and a significant increase of reference memory performance in the radial maze. In the Hebb-Williams maze, performance of grafted rats was close to normal. Strengthening the idea that dentate gyrus granule cells play an important role in the acquisition of new (perhaps more configural than only spatial) information, our results, moreover, suggest that neurosphere grafts may foster recovery after damage to point-to-point connection systems in the adult brain.

A retrospective analysis of hippocampal pathology in human temporal lobe epilepsy: evidence for distinctive patient subcategories.

PURPOSE: This study is a retrospective analysis of the pathology of the hippocampus from patients with medically intractable temporal lobe epilepsy. We attempted to relate neuronal density, immunohistochemistry, electrophysiologic data, and surgical outcome. METHODS: Immunostaining patterns for neuropeptide Y, somatostatin, substance P, and dynorphin defined the immunohistochemical characteristics of the hippocampi. Neuronal densities were determined by microscopic cell counts. Sharp electrode recordings from dentate granule cells determined measures of inhibition and excitation. RESULTS: Patient hippocampi without evidence of sclerosis generally resembled autopsy controls on the basis of neuronal densities of hippocampal subfields and patterns of immunostaining. The nonsclerotic hippocampi were divisible into two subgroups on the basis of neuronal density correlations between hippocampal subfields, the excitability of dentate granule cells, etiology, and surgical outcome. Hippocampi with sclerosis were divisible into those with significant neuronal loss confined to area CA1 and those with neuronal loss throughout the hippocampus and dentate gyrus. In the former, the dentate gyrus resembled in morphology the nonsclerotic hippocampi but with slightly increased excitability of the dentate granule cells. The hippocampi with more extensive neuronal loss had changes in immunostaining patterns associated with the dentate gyrus, correlated with significant hyperexcitability of dentate granule cells. The surgical outcome, with the exception of one group, was good in approximately 70-90%. CONCLUSIONS: Hippocampi from patients with intractable temporal lobe epilepsy can be assigned to several groups on the basis of pathophysiology. Different pathologies may represent differing causative mechanisms of intractable temporal lobe epilepsy and be predictive of surgical outcome.

Visualizing changes in circuit activity resulting from denervation and reinnervation using immediate early gene expression.

We describe a novel strategy to evaluate circuit function after brain injury that takes advantage of experience-dependent immediate early gene (IEG) expression. When normal rats undergo training or are exposed to a novel environment, there is a strong induction of IEG expression in forebrain regions, including the hippocampus. This gene induction identifies the neurons that are engaged during the experience. Here, we demonstrate that experience-dependent IEG induction is diminished after brain injury in young adult rats (120-200 gm), specifically after unilateral lesions of the entorhinal cortex (EC), and then recovers with a time course consistent with reinnervation. In situ hybridization techniques were used to assess the expression of the activity-regulated cytoskeleton-associated protein Arc at various times after the lesion (4, 8, 12, 16, or 30 d). One group of rats was allowed to explore a complex novel environment for 1 hr; control operated animals remained in their home cage. In unoperated animals, exposure to the novel environment induced Arc mRNA levels in most pyramidal neurons in CA1, in many pyramidal neurons in CA3, and in a small number of dentate granule cells. This characteristic pattern of induction was absent at early time points after unilateral EC lesions (4 and 8 d) but recovered progressively at later time points. The recovery of Arc expression occurred with approximately the same time course as the reinnervation of the dentate gyrus as a result of postlesion sprouting. These results document a novel approach for quantitatively assessing activity-regulated gene expression in polysynaptic circuits after trauma.

Neuropathological findings in a patient with epilepsy and the Parry-Romberg syndrome.

PURPOSE: The Parry-Romberg syndrome is an unusual disorder frequently associated with epilepsy. The origin of this disease, and the cause of epilepsy, are unknown. This study is the first reported case of the Parry-Romberg syndrome, with intractable temporal lobe epilepsy, in which detailed microanatomic analyses have been performed on resected brain tissue obtained after surgical intervention. METHODS: Standard histopathologic methods and correlative light and electron microscopy, combined with immunocytochemical techniques, were used to study in detail the synaptic microorganization of the resected hippocampal formation. RESULTS: After surgery, the patient was seizure free (follow-up period of 4 years and 7 months). The resected temporal lobe showed a variety of dramatic microanatomic alterations (small groups of ectopic cells, neuronal loss, gliosis, and activated microglial cells) in mesial structures, including the entorhinal cortex, subiculum, and dentate gyrus. At the electron-microscopic level, we found that in the dentate gyrus, the number of synapses in the cell-sparse region adjacent to the ectopic mass of neurons was almost twice that found in the molecular and polymorph cell layers, indicating the intrusion of neuritic processes and synapse formation. In addition, the symmetrical axosomatic synapses characteristically found on granule cells, which are likely derived from gamma-aminobutyric acid (GABA)ergic inhibitory basket cells, were not observed. CONCLUSION: The complete seizure relief after surgery suggests that the pacemaker region(s) of seizure activity were within the resected tissue. However, we do not know which of the multiple neuropathologic findings reported here were the primary cause of seizure activity. Nevertheless, the changes found in the dentate gyrus circuitry appear to be among the most important alterations that would lead to epilepsy.

Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus.

The adult rat spinal cord contains cells that can proliferate and differentiate into astrocytes and oligodendroglia in situ. Using clonal and subclonal analyses we demonstrate that, in contrast to progenitors isolated from the adult mouse spinal cord with a combination of growth factors, progenitors isolated from the adult rat spinal cord using basic fibroblast growth factor alone display stem cell properties as defined by their multipotentiality and self-renewal. Clonal cultures derived from single founder cells generate neurons, astrocytes, and oligodendrocytes, confirming the multipotent nature of the parent cell. Subcloning analysis showed that after serial passaging, recloning, and expansion, these cells retained multipotentiality, indicating that they are self-renewing. Transplantation of an in vitro-expanded clonal population of cells into the adult rat spinal cord resulted in their differentiation into glial cells only. However, after heterotopic transplantation into the hippocampus, transplanted cells that integrated in the granular cell layer differentiated into cells characteristic of this region, whereas engraftment into other hippocampal regions resulted in the differentiation of cells with astroglial and oligodendroglial phenotypes. The data indicate that clonally expanded, multipotent adult progenitor cells from a non-neurogenic region are not lineage-restricted to their developmental origin but can generate region-specific neurons in vivo when exposed to the appropriate environmental cues.

Carbamazepine effects on Na+ currents in human dentate granule cells from epileptogenic tissue.

PURPOSE: Carbamazepine (CBZ) is a well-established drug in the therapy of temporal lobe epilepsy (TLE). The anticonvulsant action of CBZ has been explained mainly by use-dependent effects on voltage-dependent Na+ channels in various nonhuman cell type. However, it is unclear whether Na+ currents in neurons within the focal epileptogenic area of patients with medically intractable TLE show similar characteristics. METHODS: Therefore we used the whole-cell patch-clamp technique to investigate the effects of CBZ on voltage-dependent Na+ currents in 23 acutely isolated dentate granule cells (DGCs) from the resected hippocampus of eight patients with medically intractable TLE. RESULTS: As in findings in animal preparations, CBZ significantly reduced the amplitude of the Na+ current and significantly shifted the current-voltage dependence of the steady-state inactivation in the hyperpolarizing direction. In contrast, the rapid component of the recovery from inactivation of the Na+ currents was not affected by CBZ. In addition, the reduction of the Na+ current amplitude observed during repetitive stimulation with depolarizing pulses was not significantly altered by CBZ. CONCLUSIONS: In summary, CBZ strongly affects the voltage-dependent steady-state inactivation, with no effects on the removal of inactivation in Na+ currents of human DGCs. In spite of the lack of suitable control material, the CBZ insensitivity of the removal of inactivation may be an interesting concept to explain the medically intractable TLE in these patients.

Voltage-dependent Ca2+ currents in epilepsy.

Voltage-dependent Ca2+ channels (VCCs) represent one of the main routes of Ca2+ entry into neuronal cells. Changes in intracellular Ca2+ dynamics and homeostasis can cause long-lasting cellular changes via activation of different Ca2+ dependent signalling pathways. We have investigated the properties of VCCs in human hippocampal dentate granule cells (DGCs) using the whole-cell configuration of the patch-clamp method. Classical high-threshold Ca2+ currents were composed mainly of omega-CgTx-sensitive N-type and nifedipine-sensitive L-type currents that were present in similar proportions. In addition, a Ca2+ current component that was sensitive to low concentrations of Ni2+, but not to nifedipine or omega-conotoxin GVIA (omega-CgTx GVIA) was present. This latter component showed a half-maximal inactivation at more hyperpolarized potentials than high-threshold currents and a more rapid time-dependent inactivation. This current was termed T-type Ca2+ current. Current components with similar pharmacological and kinetic characteristics could be elicited in acutely isolated control rat DGCs. The current density of high threshold and T-type Ca2+ components was significantly larger in human DGCs and in the kainate model compared to DGCs isolated from adult control rats. These differences in current density were not accompanied by parallel differences in the voltage-dependence of VCCs. Taken together, these data suggest that an up-regulation of Ca2+ current density may occur in hippocampal epileptogenesis without consistent changes in Ca2+ current properties.