Ex vivo study of dentate gyrus neurogenesis in human pharmacoresistant temporal lobe epilepsy

M. Paradisi, M. Fernández, G. Del Vecchio, G. Lizzo, G. Marucci, M. Giulioni, E. Pozzati, T. Antonelli, G. Lanzoni, G. P. Bagnara, L. Giardino and L. Calzà (2010) Neuropathology and Applied Neurobiology 36, 535–550
Ex vivo study of dentate gyrus neurogenesis in human pharmacoresistant temporal lobe epilepsy Aims: Neurogenesis in adult humans occurs in at least two areas of the brain, the subventricular zone of the telencephalon and the subgranular layer of the dentate gyrus in the hippocampal formation. We studied dentate gyrus subgranular layer neurogenesis in patients subjected to tailored antero‐mesial temporal resection including amygdalohippocampectomy due to pharmacoresistant temporal lobe epilepsy (TLE) using the in vitro neurosphere assay. Methods: Sixteen patients were enrolled in the study; mesial temporal sclerosis (MTS) was present in eight patients. Neurogenesis was investigated by ex vivo neurosphere expansion in the presence of mitogens (epidermal growth factor + basic fibroblast growth factor) and spontaneous differentiation after mitogen withdrawal. Growth factor synthesis was investigated by qRT‐PCR in neurospheres. Results: We demonstrate that in vitro proliferation of cells derived from dentate gyrus of TLE patients is dependent on disease duration. Moreover, the presence of MTS impairs proliferation. As long as in vitro proliferation occurs, neurogenesis is maintained, and cells expressing a mature neurone phenotype (TuJ1, MAP2, GAD) are spontaneously formed after mitogen withdrawal. Finally, formed neurospheres express mRNAs encoding for growth (vascular endothelial growth factor) as well as neurotrophic factors (brain‐derived neurotrophic factor, ciliary neurotrophic factor, glial‐derived neurotrophic factor, nerve growth factor). Conclusion: We demonstrated that residual neurogenesis in the subgranular layer of the dentate gyrus in TLE is dependent on diseases duration and absent in MTS.     

Preservation of dendrites with the presence of reorganized mossy fiber collaterals in hippocampal dentate granule cells in patients with temporal lobe epilepsy

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°±4.1 S.E.M. with 13.8±1.1 branch points and an estimated total dendritic length of 11535.6 μm±3045.4. Dendritic spines were counted, and spine density was calculated to be 0.25 spines/μm±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/μm±0.203 S.E.M.: P<0.01) than the DGCs without such collaterals (0.082 spines/μm±0.021 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.     

Long- and short-term plasticity at mossy fiber synapses on mossy cells in the rat dentate gyrus

Mossy cells give rise to the commissural and associational pathway of the dentate gyrus, and receive their major excitatory inputs from the mossy fibers of granule cells. Through these feed-back excitatory connections, mossy cells have been suggested to play important roles in both normal signal processing in learning and memory, as well as in seizure propagation. However, the nature of the activity-dependent modifications of the mossy fiber inputs to mossy hilar cells is not well understood. We studied the long- and short-term plasticity properties of the mossy fiber-mossy cell synapse, using the minimal stimulation technique in slices in whole cell recorded mossy cells retrogradely prelabeled with the fluorescent dye DiO from the contralateral dentate gyrus. Following tetanic stimulation, mossy fiber synapses showed significant NMDA receptor-independent long-term potentiation (LTP), associated with increased excitatory postsynaptic currents (EPSC) amplitude and decreased failure rates. Coefficient of variance and failure rate analyses suggested a presynaptic locus of LTP induction. Mossy fiber synapses on mossy cells also showed activity-dependent short-term modification properties, including both frequency-dependent facilitation (stimuli at higher frequencies evoked larger EPSCs with lower failure rates) and burst facilitation (each EPSC in a burst had a larger amplitude and higher probability of occurrence than the preceding EPSCs within the burst). The data show that mossy fiber-mossy cell synapses exhibit both long- and short-term plasticity phenomena that are generally similar to the mossy fiber synapses on CA3 pyramidal cells.     

Circadian dentate gyrus excitability in a rat model of temporal lobe epilepsy

In human mesial temporal lobe epilepsy (mTLE), seizure occurrence peaks in the late afternoon and early evening. This temporal binding of seizures has been replicated in animal models of mTLE following electrically-induced status epilepticus (SE). We hypothesized that in chronic epilepsy, alterations of circadian excitatory and inhibitory functions of the dentate gyrus (DG), which is believed to regulate the generation of limbic seizures, pathophysiologically contribute to the temporal binding of ictogenesis. We performed electrophysiological single and paired pulse measurements hourly over 24h in the DG of epileptic rats (n=8) 8 weeks after electrically induced SE. Results were compared to individual data obtained before induction of SE and to those of control animals (n=3). Pre and post SE data were analyzed in two distinct phases of the day, i.e. a high-seizure phase between 2p.m. and 10p.m. and a low-seizure phase between 10p.m. and 2p.m. In chronic epileptic animals, latency of evoked potentials was significantly reduced in the high-seizure phase (p=0.027) but not in the low-seizure phase. Compared to baseline values, paired pulse inhibition was significantly increased during the low-seizure phase (interpulse interval (IPI) 25ms, p=0.003; IPI 30ms; p<0.001) but not in the high-seizure phase. Similarly, when compared to controls, inhibition at IPI 20ms was diminished only in the high-seizure phase (p=0.027). Thus, in chronic epileptic animals, DG excitability is increased in the afternoon and early evening possibly contributing to the time of day-dependency of spontaneous seizures in this model system of mTLE. Alterations of circadian DG excitability in epileptic animals may be influenced by changes in hypothalamus-regulated superordinate functions such as excretion of endocrine hormones but further studies are needed.     

Recurrent excitation in the dentate gyrus of a murine model of temporal lobe epilepsy

Similar to rats, systemic pilocarpine injection causes status epilepticus (SE) and the eventual development of spontaneous seizures and mossy fiber sprouting in C57BL/6 and CD1 mice, but the physiological correlates of these events have not been identified in mice. Population responses in granule cells of the dentate gyrus were examined in transverse slices of the ventral hippocampus from pilocarpine-treated and untreated mice. In Mg(2+)-free bathing medium containing bicuculline, conditions designed to increase excitability in the slices, electrical stimulation of the hilus resulted in a single population spike in granule cells from control mice and pilocarpine-treated mice that did not experience SE. In SE survivors, similar stimulation resulted in a population spike followed, at a variable latency, by negative DC shifts and repetitive afterdischarges of 3-60 s duration, which were blocked by ionotropic glutamate receptor antagonists. Focal glutamate photostimulation of the granule cell layer at sites distant from the recording pipette resulted in population responses of 1-30 s duration in slices from SE survivors but not other groups. These data support the hypothesis that SE-induced mossy fiber sprouting and synaptic reorganization are relevant characteristics of seizure development in these murine strains, resembling rat models of human temporal lobe epilepsy.     

癫痫大鼠海马出芽苔藓纤维突触的超微结构特征

目的:探讨匹罗卡品颞叶癫痫大鼠海马出芽苔藓纤维突触的超微结构特征及其在颞叶癫痫发病机制中的作用。方法:采用Timm组化染色标记出芽苔藓纤维突触末端,在电镜下观察新生突触的类型、比例、定位、以及突触后靶成分。结果:颞叶癫痫大鼠齿状回内分子层可见到银标记的突触末端,出芽苔藓纤维突触主要是轴棘型非对称性突触,其次是轴树型非对称性突触,偶可看到出芽轴突和颗粒细胞体形成突触联系。结论:轴棘型非对称性突触是颞叶癫痫大鼠海马出芽苔藓纤维突触的主要类型,出芽苔藓纤维突触的超微结构特性支持重组突触形成重复的兴奋性环路,而且形成的新的兴奋性环路可能在颞叶癫痫的发生与发展中起重要作用。     

颞叶癫痫患者海马苔藓纤维发芽的实验研究

目的观察颞叶癫痫病人海马齿状回和CA3区苔藓纤维出芽情况。方法癫痫组样本来自12例颞叶癫痫病例的手术切除标本包含海马齿状回和CA3区的脑组织，对照组脑组织样本来自4例非癫痫病的尸检脑组织。应用Timm组织化学染色方法在光镜和电镜水平进行海马结构苔藓纤维发芽的研究。结果光镜下癫痫组可见苔藓纤维穿越海马齿状回颗粒细胞层到达内分子层．CA3区也可见明显的苔藓纤维发芽。癫痫组CA3区和齿状回内分子层苔藓纤维发芽评分高于对照组．统计学上差异有显著性意义。电子显微镜下观察显示癫痫组患者齿状回内分子层可见到银标记的突触末端，主要和树突形成突触连接，所形成的突触为非对称性突触。结论颞叶癫痫可致海马齿状回和CA3区苔藓纤维发芽增加，这可能是难治性癫痫形成的重要机制。     

The functional relationship between antidromically evoked field responses of the dentate gyrus and mossy fiber reorganization in temporal lobe epileptic patients

Field recordings from the dentate granule cell layer of in vitro brain slices of temporal lobe epileptic patients were evoked by antidromic stimulation. Tissue from the same specimen was stained by the Timm-sulfide method to assess the pattern and degree of mossy fiber reorganization into the supragranular layer. A wide range of physiological responses and Timm staining patterns was present across patients. A significant correlation was observed between the abnormality of antidromic responses, reflected by multiple secondary population spikes, and the degree of Timm staining of the supragranular layer. This relationship lends support to the hypothesis that mossy fiber synapses located in the supragranular layer may have functional implications for granule cell excitability in human epileptic tissue.     

海马苔藓纤维发芽与颞叶癫痫的研究进展

颞叶癫痫是部分性发作癫痫的最主要类型，约有40％的人类癫痫起源于颞叶，这与海马结构独特的神经元环路及纤维联系有关。海马结构中异位突触的形成被认为与颞叶癫痫发作有着密切的联系。而在海马结构的突触重排中，研究最多而且被充分证明的就是苔藓纤维发芽（mossy fiber sprouting，MFS）。现就海马MFS的形态学特性、诱发因素、出芽机理及其与颞叶癫痫发作之间的关系作一综述。     

Granule cell dispersion in the dentate gyrus of humans with temporal lobe epilepsy.

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.     

Ultrastructural alterations in hippocampal mossy fiber synapses in schizophrenia: a postmortem morphometric study

Synapses formed between mossy fibers, the axons of hippocampal dentate granular cells, and the dendrites of CA3 pyramidal neurons are important links within the trisynaptic circuitry. Abnormalities in this circuitry are associated with the failure of schizophrenics to integrate affective experience with higher cognitive function, and with disturbances in memory and spatial learning processes. The abnormalities include reduced size and altered dendritic arborization of CA3 pyramidal neurons. In addition, decreased expression and binding activity of glutamate receptors have been reported, predominantly in the CA3 region of the hippocampus. These findings suggest that there are disturbed neuronal processes and connections in the hippocampus of schizophrenics. An electron microscope morphometric study of synaptic contacts between mossy fiber axon terminals (MFT) and branched dendritic spines of pyramidal neurons in stratum lucidum of the CA3 region of the hippocampus was performed in 10 normal controls and 9 age-matched chronic schizophrenics (postmortem delay 3-9 h). Schizophrenic cases with predominantly positive symptoms had a significantly reduced volume fraction of spines (-35%, P < 0.05), total number of invaginated spines (-47%, P < 0.01), and number of spines forming synapses (-32%, P < 0.05) per MFT compared with the control group. No effects of postmortem delay, age, duration of disease, or neuroleptic exposure were found. These data may reflect decreased efficacy of mossy fiber synapses in the CA3 hippocampal region in schizophrenics with predominantly positive symptoms. These data are in line with the neurodevelopmental hypothesis of schizophrenia.     

Impairment of dentate gyrus neuronal progenitor cell differentiation in a mouse model of temporal lobe epilepsy

Unilateral intrahippocampal injection of kainic acid (KA) in adult mice induces an epileptic focus replicating major histopathological features of temporal lobe epilepsy (TLE). In this model, neurogenesis is impaired in the lesioned dentate gyrus, although cell proliferation transiently is increased bilaterally in the subgranular zone (SGZ). To investigate further the relationship between epileptogenesis and neurogenesis, we compared the differentiation of cells born shortly before and after KA injection. Immunohistochemical staining for doublecortin and PSA-NCAM, two markers of young neurons, revealed a rapid downregulation of both markers ipsilaterally, whereas they were increased transiently on the contralateral side. To determine whether KA treatment directly affects neural progenitors in the SGZ, dividing cells were prelabeled with 5'-bromo-2'deoxyuridine (BrdU) treatment before unilateral injection of KA. Double staining with the proliferation marker PCNA showed that prelabeled BrdU cells survived KA exposure and proliferated bilaterally. Unexpectedly, the neuronal differentiation of these cells, as assessed after 2 weeks with doublecortin and NeuN triple-staining, occurred to the same extent as on the contralateral side. Only 5% of pre-labeled BrdU cells were GFAP-positive within the lesion. Therefore, SGZ progenitor cells committed to a neuronal phenotype before KA treatment complete their differentiation despite the rapid down-regulation of doublecortin and PSA-NCAM. These findings suggest impaired fate commitment and/or early differentiation of proliferating cells in the lesioned dentate gyrus. Loss of neurogenesis in this TLE model likely reflects an irreversible alteration of the SGZ germinal niche during development of the epileptic focus and may therefore be relevant for human TLE.     

Altered theta coupling between medial entorhinal cortex and dentate gyrus in temporal lobe epilepsy

Purpose: Temporal lobe epilepsy is often accompanied by neuron loss and rewiring in the hippocampus. We hypothesized that the interaction of subnetworks of the entorhinal–hippocampal loop between epileptic events should show significant signatures of these pathologic changes. Methods: We combined simultaneous recording of local field potentials in entorhinal cortex (EC) and dentate gyrus (DG) in freely behaving kainate‐injected mice with histologic analyses and computational modeling. Key Findings: In healthy mice, theta band activity was synchronized between EC and DG. In contrast, in epileptic mice, theta activity in the EC was delayed with respect to the DG. A computational neural mass model suggests that hippocampal cell loss imbalances the coupling of subnetworks, introducing the shift. Significance: We show that pathologic dynamics in epilepsy encompass ongoing activity in the entorhinal‐hippocampal loop beyond acute epileptiform activity. This predominantly affects theta band activity, which links this shift in entorhinal‐hippocampal interaction to behavioral aspects in epilepsy.     

匹罗卡品癫痫模型中突触素Ⅰ的表达及苔藓纤维出芽的动态变化

目的探讨癫痫发生过程中突触素Ⅰ(SYNⅠ)在海马和齿状回的表达及齿状回苔藓纤维出芽的动态变化。方法建立匹罗卡品癫痫持续状态模型,用图像分析系统测定海马和齿状回不同时点SYNⅠ免疫反应吸光度值．Neo-timms’染色观察齿状回苔藓出芽的演变。结果SYNⅠ在海马和齿状回的表达于癫痫状态后2d、7d出现降低,14d开始升高,30d、60d表达明显增高；齿状回内分子层于14d开始出现苔藓纤维出芽,大鼠在同期开始出现自发发作。结论在癫痫状态后2d即出现了突触可塑性的变化,14d后由于神经轴突的再生,齿状回内分子层出现苔藓纤维出芽,形成了兴奋性的环路,可能是癫痫反复自发发作的病理基础,SYNⅠ及苔藓纤维出芽较好的反应了神经可塑性的变化。     

Ultrastructural localization of neurotransmitter immunoreactivity in mossy cell axons and their synaptic targets in the rat dentate gyrus

Electrophysiologically identified and intracellularly biocytin-labeled mossy cells in the dentate hilus of the rat were studied using electron microscopy and postembedding immunogold techniques. Ultrathin sections containing a labeled mossy cell or its axon collaterals were reacted with antisera against the excitatory neurotransmitter glutamate and against the inhibitory neurotransmitter γ-aminobutyric acid (GABA). From single- and double-immunolabeled preparations, we found that 1) mossy cell axon terminals made asymmetric contacts onto postsynaptic targets in the hilus and stratum moleculare of the dentate gyrus and showed immunoreactivity primarily for glutamate, but never for GABA; 2) in the hilus, glutamate-positive mossy cell axon terminals targeted GABA-positive dendritic shafts of hilar interneurons and GABA-negative dendritic spines; and 3) in the inner molecular layer, the mossy cell axon formed asymmetric synapses with dendritic spines associated with GABA-negative (presumably granule cell) dendrites. The results of this study support the view that excitatory (glutamatergic) mossy cell terminals contact GABAergic interneurons and non-GABAergic neurons in the hilar region and GABA-negative granule cells in the stratum moleculare. This pattern of connectivity is consistent with the hypothesis that mossy cells provide excitatory feedback to granule cells in a dentate gyrus associational network and also activate local hilar inhibitory elements. Hippocampus 1997;7:559–570. © 1997 Wiley-Liss, Inc.     

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

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

Ultrastructural localization of somatostatin-like immunoreactivity in the rat dentate gyrus

Neurons containing somatostatin (SOM) are enriched in the dentate gyrus. We sought to establish the ultrastructural localization of this peptide in the dentate gyrus of the rat brain with a double-bridged peroxidase-antiperoxidase (PAP) method localizing antisera directed against somatostatin (SOM)-28 and SOM-28. Initial light microscopic observations confirmed that the majority of perikarya and thick varicose processes with intense SOM-like immunoreactivity (SOM-LI) were observed in the hilus. Fine varicose processes with SOM-LI were found throughout all layers of the dentate gyrus but were most intense in the outer third of the molecular layer (ML), where an occasional perikaryon with SOM-LI was seen. By electron microscopy, SOM-LI was found in neuronal perikarya, dendrites, axons, and axon terminals. Two types of SOM-containing perikarya were observed. The first type was small (6-10 microns), round or avoid, and had a labeled cytoplasma with abundant Golgi complexes and a dense accumulation of PAP-reaction product. The second type of perikarya was larger (11-16 microns) and had a more abundant cytoplasm than the first type, but the Golgi complexes did not appear labeled. Most (96% of 374) of the synapses on the SOM-labeled perikarya and dendrites were from terminals without SOM-LI which formed nearly equal proportions of asymmetric and symmetric junctions. The remainder of the presynaptic terminals contained SOM-LI and made primarily symmetric synapses. Synaptic junctions from both unlabeled and labeled terminals were primarily on the shafts of the small (0.5-1.5 microns) SOM-immunoreactive dendrites. The terminals with SOM-LI (0.25-1.3 microns) contained many small, clear vesicles and from zero to four large dense-core vesicles. Terminals with SOM-LI were associated 1) with one unlabeled perikaryon or dendrite (49% of 215 in the hilus; 76% of 326 in the ML); 2) with two unlabeled perikarya or dendrites simultaneously (5% hilus; 4% ML); and 3) with one SOM-containing perikaryon or dendrite (6% hilus; 3% ML). In all three types of associations, synaptic contacts on perikarya were few while the majority were with small (distal) dendrites. Moreover, most of the terminals with SOM-LI formed symmetric junctions or lacked membrane specializations but were without any apparent glial intervention in the plane of section analyzed. The remaining SOM-labeled terminals (40% hilus; 17% ML) were without any apparent synaptic relations. However, a few of these terminals were in direct apposition to other terminals, some of which were also SOM-immunoreactive.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tracing the dentate gyrus mossy fiber system with horseradish peroxidase histochemistry

We have traced the mossy fiber projections of the dentate gyrus of the rat and mouse using a new anatomical procedure involving horseradish peroxidase histochemistry. Granule cells take up injected horseradish peroxidase and transport it to their dendrites and axon terminals. As shown by this method, the mossy fibers project in a very narrow septo-temporal plane at right angles to the longitudinal axis of the hippocampus. In addition, discrete loci of the dentate gyrus project to nearly the entire length of the regio inferior. We also consider the detailed organization of the mossy fiber system and the value of horseradish peroxidase histochemistry as a neuroanatomical tool.     

Neurogenesis in temporal lobe epilepsy: Relationship between histological findings and changes in dentate gyrus proliferative properties

Objective

The relationship between hippocampal histopathological abnormalities, epileptogenesis and neurogenesis remains rather unclear.

Methods

Tissue samples including the subgranular zone of dentate gyrus (DG) were freshly collected for tissue culture for neurospheres generation in 16 patients who underwent surgery for drug-resistant temporal lobe epilepsy. Remaining tissues were histologically examined to assess the presence of mesial temporal sclerosis (MTS) and focal cortical dysplasia.

Results

MTS was detected in 8 cases. Neurospheres were formed in 10/16 cases. Only three out of these 10 cases exhibited MTS; on the contrary 5/6 cases lacking neurosphere proliferation presented MTS. There was a significant correlation between presence of MTS and absence of proliferation (p = 0.0389). We also observed a correlation between history of febrile seizures (FS) and presence of MTS (p = 0.0004) and among the 6 cases lacking neurosphere proliferation, 4 cases (66.6%) had experienced prolonged FS. Among “proliferating” cases the percentage of granular cells pathology (GCP) was lower (20% vs 50%) compared to “non proliferating” cases.

Conclusion

A decreased potential to generate neurosphere from the SGZ is related to MTS and to alterations of dentate gyrus granule cells, especially in MTS type 1b and GCP type 1. These histological findings may have different prognostic implications, regarding seizure and neuropsychological outcome, compared to patients with other epileptogenic lesions (such as FCD, glioneuronal tumours, vascular lesions).