Heat-Shock Induces Granule Cell Dispersion and Microgliosis in Hippocampal Slice Cultures

Granule cell dispersion (GCD) has been found in the dentate gyrus (dg) of patients with temporal lobe epilepsy (TLE) and a history of febrile seizures but was also recently observed in pediatric patients that did not suffer from epilepsy. This indicates that GCD might not always be disease related, but instead could reflect normal morphological variation. Thus, distribution of newborn granule cells within the hilar region is part of normal dg development at early stages but could be misinterpreted as pathological GCD. In turn, pathological GCD may be caused, for example, by genetic mutations, such as the reeler mutation. GCD in the reeler mutant goes along with an increased susceptibility to epileptiform activity. Pathological GCD in combination with epilepsy is caused by experimental administration of the glutamate receptor agonist kainic acid in rodents. In consequence, the interpretation of GCD and the role of febrile seizures remain controversial. Here, we asked whether febrile temperatures alone might be sufficient to trigger GCD and used hippocampal slice cultures as in vitro model to analyze the effect of a transient temperature increase on the dg morphology. We found that a heat-shock of 41°C for 6 h was sufficient to induce GCD and degeneration of a fraction of granule cells. Both of these factors, broadening of the granule cell layer (gcl) and increased neuronal cell death within the gcl, contributed to the development of a significantly reduced packaging density of granule cells. In contrast, Reelin expressing Cajal–Retzius (CR) cells in the molecular layer were heat-shock resistant. Thus, their number was not reduced, and we did not detect degenerating CR cells after heat-shock, implying that GCD was not caused by the loss of CR cells. Importantly, the heat-shock-induced deterioration of dg morphology was accompanied by a massive microgliosis, reflecting a robust heat-shock-induced immune response. In contrast, in the study that reported on GCD as a non-specific finding in pediatric patients, no microglia reaction was observed. Thus, our findings underpin the importance of microglia as a marker to distinguish pathological GCD from normal morphological variation.

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Synaptic Connections From Multiple Subfields Contribute to Granule Cell Hyperexcitability in Hippocampal Slice Cultures

Journal of Neurophysiology ◽

10.1152/jn.2000.84.6.2918 ◽

2000 ◽

Vol 84 (6) ◽

pp. 2918-2932 ◽

Cited By ~ 51

Author(s):

Suzanne B. Bausch ◽

James O. McNamara

Keyword(s):

Dentate Gyrus ◽

Granule Cell ◽

Hippocampal Slice ◽

Granule Cells ◽

Granule Cell Layer ◽

Synaptic Connections ◽

In Vivo Models ◽

Hippocampal Slice Cultures

Limbic status epilepticus and preparation of hippocampal slice cultures both produce cell loss and denervation. This commonality led us to hypothesize that morphological and physiological alterations in hippocampal slice cultures may be similar to those observed in human limbic epilepsy and animal models. To test this hypothesis, we performed electrophysiological and morphological analyses in long-term ( postnatal day 11; 40–60 days in vitro) organotypic hippocampal slice cultures. Electrophysiological analyses of dentate granule cell excitability revealed that granule cells in slice cultures were hyperexcitable compared with acute slices from normal rats. In physiological buffer, spontaneous electrographic granule cell seizures were seen in 22% of cultures; in the presence of a GABAA receptor antagonist, seizures were documented in 75% of cultures. Hilar stimulation evoked postsynaptic potentials (PSPs) and multiple population spikes in the granule cell layer, which were eliminated by glutamate receptor antagonists, demonstrating the requirement for excitatory synaptic transmission. By contrast, under identical recording conditions, acute hippocampal slices isolated from normal rats exhibited a lack of seizures, and hilar stimulation evoked an isolated population spike without PSPs. To examine the possibility that newly formed excitatory synaptic connections to the dentate gyrus contribute to granule cell hyperexcitability in slice cultures, anatomical labeling and electrophysiological recordings following knife cuts were performed. Anatomical labeling of individual dentate granule, CA3 and CA1 pyramidal cells with neurobiotin illustrated the presence of axonal projections that may provide reciprocal excitatory synaptic connections among these regions and contribute to granule cell hyperexcitability. Knife cuts severing connections between CA1 and the dentate gyrus/CA3c region reduced but did not abolish hilar-evoked excitatory PSPs, suggesting the presence of newly formed, functional synaptic connections to the granule cells from CA1 and CA3 as well as from neurons intrinsic to the dentate gyrus. Many of the electrophysiological and morphological abnormalities reported here for long-term hippocampal slice cultures bear striking similarities to both human and in vivo models, making this in vitro model a simple, powerful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis.

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Contributions of Mossy Fiber and CA1 Pyramidal Cell Sprouting to Dentate Granule Cell Hyperexcitability in Kainic Acid–Treated Hippocampal Slice Cultures

Journal of Neurophysiology ◽

10.1152/jn.01028.2003 ◽

2004 ◽

Vol 92 (6) ◽

pp. 3582-3595 ◽

Cited By ~ 35

Author(s):

Suzanne B. Bausch ◽

James O. McNamara

Keyword(s):

Dentate Gyrus ◽

Kainic Acid ◽

Granule Cell ◽

Hippocampal Slice ◽

Granule Cells ◽

Mossy Fiber ◽

Mossy Fibers ◽

Axonal Sprouting ◽

Mossy Fiber Sprouting ◽

Hippocampal Slice Cultures

Axonal sprouting like that of the mossy fibers is commonly associated with temporal lobe epilepsy, but its significance remains uncertain. To investigate the functional consequences of sprouting of mossy fibers and alternative pathways, kainic acid (KA) was used to induce robust mossy fiber sprouting in hippocampal slice cultures. Physiological comparisons documented many similarities in granule cell responses between KA- and vehicle-treated cultures, including: seizures, epileptiform bursts, and spontaneous excitatoty postsynaptic currents (sEPSCs) >600pA. GABAergic control and contribution of glutamatergic synaptic transmission were similar. Analyses of neurobiotin-filled CA1 pyramidal cells revealed robust axonal sprouting in both vehicle- and KA-treated cultures, which was significantly greater in KA-treated cultures. Hilar stimulation evoked an antidromic population spike followed by variable numbers of postsynaptic potentials (PSPs) and population spikes in both vehicle- and KA-treated cultures. Despite robust mossy fiber sprouting, knife cuts separating CA1 from dentate gyrus virtually abolished EPSPs evoked by hilar stimulation in KA-treated but not vehicle-treated cultures, suggesting a pivotal role of functional afferents from CA1 to dentate gyrus in KA-treated cultures. Together, these findings demonstrate striking hyperexcitability of dentate granule cells in long-term hippocampal slice cultures after treatment with either vehicle or KA. The contribution to hilar-evoked hyperexcitability of granule cells by the unexpected axonal projection from CA1 to dentate in KA-treated cultures reinforces the idea that axonal sprouting may contribute to pathologic hyperexcitability of granule cells.

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Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures

Brain Research ◽

10.1016/j.brainres.2004.11.059 ◽

2005 ◽

Vol 1035 (2) ◽

pp. 111-119 ◽

Cited By ~ 13

Author(s):

Matthew Paul Sadgrove ◽

John E. Chad ◽

William Peter Gray

Keyword(s):

Cell Proliferation ◽

Cell Death ◽

Kainic Acid ◽

Granule Cell ◽

Hippocampal Slice ◽

Cell Layer ◽

Granule Cell Layer ◽

Hippocampal Slice Cultures ◽

Organotypic Hippocampal Slice Cultures

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Cerebellar proteoglycans regulate sonic hedgehog responses during development

Development ◽

10.1242/dev.129.9.2223 ◽

2002 ◽

Vol 129 (9) ◽

pp. 2223-2232 ◽

Cited By ~ 4

Author(s):

Joshua B. Rubin ◽

Yoojin Choi ◽

Rosalind A. Segal

Keyword(s):

Heparan Sulfate ◽

Sonic Hedgehog ◽

Granule Cell ◽

Granule Cells ◽

Cerebellar Granule Cells ◽

Cell Layer ◽

Heparan Sulfate Proteoglycans ◽

Granule Cell Layer ◽

Conserved Sequence ◽

External Granule Cell Layer

Sonic hedgehog promotes proliferation of developing cerebellar granule cells. As sonic hedgehog is expressed in the cerebellum throughout life it is not clear why proliferation occurs only in the early postnatal period and only in the external granule cell layer. We asked whether heparan sulfate proteoglycans might regulate sonic hedgehog-induced proliferation and thereby contribute to the specialized proliferative environment of the external granule cell layer. We identified a conserved sequence within sonic hedgehog that is essential for binding to heparan sulfate proteoglycans, but not for binding to the receptor patched. Sonic hedgehog interactions with heparan sulfate proteoglycans promote maximal proliferation of postnatal day 6 granule cells. By contrast, proliferation of less mature granule cells is not affected by sonic hedgehog-proteoglycan interactions. The importance of proteoglycans for proliferation increases during development in parallel with increasing expression of the glycosyltransferase genes, exostosin 1 and exostosin 2. These data suggest that heparan sulfate proteoglycans, synthesized by exostosins, may be critical determinants of granule cell proliferation.

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BDNF stimulates migration of cerebellar granule cells

Development ◽

10.1242/dev.129.6.1435 ◽

2002 ◽

Vol 129 (6) ◽

pp. 1435-1442 ◽

Cited By ~ 3

Author(s):

Paul R. Borghesani ◽

Jean Michel Peyrin ◽

Robyn Klein ◽

Joshua Rubin ◽

Alexandre R. Carter ◽

...

Keyword(s):

Granule Cell ◽

Granule Cells ◽

Cerebellar Granule Cells ◽

Cell Layer ◽

Time Lapse ◽

Granule Cell Layer ◽

Boyden Chamber ◽

Cerebellar Granule ◽

Proliferative Zone ◽

On Line

During development of the nervous system, neural progenitors arise in proliferative zones, then exit the cell cycle and migrate away from these zones. Here we show that migration of cerebellar granule cells out of their proliferative zone, the external granule cell layer (EGL), is impaired in Bdnf–/– mice. The reason for impaired migration is that BDNF directly and acutely stimulates granule cell migration. Purified Bdnf–/– granule cells show defects in initiation of migration along glial fibers and in Boyden chamber assays. This phenotype can be rescued by exogenous BDNF. Using time-lapse video microscopy we find that BDNF is acutely motogenic as it stimulates migration of individual granule cells immediately after addition. The stimulation of migration reflects both a chemokinetic and chemotactic effect of BDNF. Collectively, these data demonstrate that BDNF is directly motogenic for granule cells and provides a directional cue promoting migration from the EGL to the internal granule cell layer (IGL). Movies available on-line

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Hippocampal morphometry in sudden and unexpected death in epilepsy

Neurology ◽

10.1212/wnl.0000000000007969 ◽

2019 ◽

Vol 93 (8) ◽

pp. e804-e814 ◽

Cited By ~ 3

Author(s):

Alyma Somani ◽

Anita-Beatrix Zborovschi ◽

Yan Liu ◽

Smriti Patodia ◽

Zuzanna Michalak ◽

...

Keyword(s):

Granule Cell ◽

Hippocampal Sclerosis ◽

Qualitative Evaluation ◽

Granule Cell Layer ◽

Parahippocampal Gyrus ◽

Unexpected Death ◽

Cell Dispersion ◽

Autonomic Networks ◽

Left And Right ◽

The Right

ObjectiveTo determine hippocampal morphometric measures, including granule cell dispersion and features of malrotation, as potential biomarkers for sudden unexpected death in epilepsy (SUDEP) from an archival postmortem series.MethodsIn a retrospective study of 187 archival postmortems from 3 groups, SUDEP (68; 14 with hippocampal sclerosis [HS]), non-SUDEP epilepsy controls (EP-C = 66; 25 with HS), and nonepilepsy controls (NEC = 53), Nissl/hematoxylin & eosin–stained sections from left and right hippocampus from 5 coronal levels were digitized. Image analysis was carried out for granule cell layer (GCL) thickness and measurements of hippocampal dimensions (HD) for shape (width [HD1], height [HD2]) and medial hippocampal positioning in relation to the parahippocampal gyrus (PHG) length (HD3). A qualitative evaluation of hippocampal malrotational (HMAL) features, dentate gyrus invaginations (DGI), and subicular/CA1 folds (SCF) was also made.ResultsGCL thickness was increased in HS more than those without (p < 0.001). In non-HS cases, increased GCL thickness was noted in EP-C compared to NEC (p < 0.05) but not between SUDEP and NEC. There was no difference in the frequency of DGI, SCF, measurements of hippocampal shape (HD1, HD2, or ratio), or medial positioning among SUDEP, EP-C, and NEC groups, when factoring in HS, coronal level, and age at death. Comparison between left and right sides within cases showed greater PHG lengths (HD3) on the right side in the SUDEP group only (p = 0.018).ConclusionsNo hippocampal morphometric features were identified in support of either excessive granule cell dispersion or features of HMAL as definitive biomarkers for SUDEP. Asymmetries in PHG measurements in SUDEP warrant further investigation as they may indicate abnormal central autonomic networks.

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Optical Recording Study of Granule Cell Activities in the Hippocampal Dentate Gyrus of Kainate-Treated Rats

Journal of Neurophysiology ◽

10.1152/jn.2000.83.4.2421 ◽

2000 ◽

Vol 83 (4) ◽

pp. 2421-2430 ◽

Cited By ~ 12

Author(s):

Yo Otsu ◽

Eiichi Maru ◽

Hisayuki Ohata ◽

Ichiro Takashima ◽

Riichi Kajiwara ◽

...

Keyword(s):

Dentate Gyrus ◽

Granule Cell ◽

Granule Cells ◽

Mossy Fiber ◽

Molecular Layer ◽

Mossy Fibers ◽

Optical Recording ◽

Granule Cell Layer ◽

Gabaergic Inhibition ◽

Mossy Fiber Sprouting

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca2+. The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizurelike activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.

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Deficits in Motor Coordination with Aberrant Cerebellar Development in Mice Lacking Testicular Orphan Nuclear Receptor 4

Molecular and Cellular Biology ◽

10.1128/mcb.25.7.2722-2732.2005 ◽

2005 ◽

Vol 25 (7) ◽

pp. 2722-2732 ◽

Cited By ~ 54

Author(s):

Yei-Tsung Chen ◽

Loretta L. Collins ◽

Hideo Uno ◽

Chawnshang Chang

Keyword(s):

Nuclear Receptor ◽

Granule Cell ◽

Motor Coordination ◽

Granule Cells ◽

Molecular Layer ◽

Granule Cell Layer ◽

Abnormal Development ◽

Cerebellar Development ◽

Morphological Development ◽

Orphan Nuclear Receptor

ABSTRACT Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4−/−) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4−/− cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4−/− cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4−/− cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4−/− cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.

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