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

Abstract

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.

Tetrodotoxin prevents heat-shock induced granule cell dispersion in hippocampal slice cultures

“…In some experimental animal models and in humans with TLE, GCD has been linked with the loss of reelin-expressing cells. We recently introduced an in vitro heat-shock model ( Weninger et al, 2021 ) wherein subjecting OHSCs to elevated temperatures induced GCD, but no significant reductions in Cajal-Retzius cells were observed after heat-shock. Here, we attempted to trace the causes that might underlie heat-shock induced GCD.…”

“…To examine the potential role of fever on the formation of GCD, we established a model wherein OHSCs were subjected to increased temperature ( Weninger et al, 2021 ). In this model, we found that transiently elevated temperature alone caused GCD, and that this phenomenon was not accompanied by changes in the number of Cajal-Retzius cells.…”

Considering the Role of Extracellular Matrix Molecules, in Particular Reelin, in Granule Cell Dispersion Related to Temporal Lobe Epilepsy

“…One difference from many other studies is that the study ( Roy et al, 2020 ) examined mainly pediatric specimens. During development, early generated GCs migrate from the hilus of the DG towards the marginal zone (MZ) to their final positions ( Förster et al, 2006 ; Hayashi et al, 2015 ), so a transient distribution of newborn GCs in the hilus could reflect a natural stage of early postnatal development of the DG, which, conversely, could be rapidly mistaken for pathological GCD ( Weninger et al, 2021 ). Figure 3 illustrates the histological organization of the DG.…”

“…In this regard, epileptic states induce the upregulation of endogenous TIMP-1, thereby inhibiting matrix metalloproteinase activity, ultimately resulting in the extracellular accumulation of un-cleaved reelin ( Tinnes et al, 2013 ). In a more recent study, the association between the occurrence of GCD and FS was investigated, and a transient increase in temperature led to the induction of GCD in vitro , accompanied by partial degeneration of GCs, whereas reelin-expressing CR cells were preserved in the ML, although it could not be ruled out that reelin processing was abnormal ( Weninger et al, 2021 ). In the aforementioned publication, the appearance of GCD after temperature elevation was accompanied by severe microgliosis, suggesting an immune response of the tissue, and it was proposed that microglia could serve as markers to distinguish pathological GCD from normal variation ( Roy et al, 2020 ; Weninger et al, 2021 ).…”

“…In a more recent study, the association between the occurrence of GCD and FS was investigated, and a transient increase in temperature led to the induction of GCD in vitro , accompanied by partial degeneration of GCs, whereas reelin-expressing CR cells were preserved in the ML, although it could not be ruled out that reelin processing was abnormal ( Weninger et al, 2021 ). In the aforementioned publication, the appearance of GCD after temperature elevation was accompanied by severe microgliosis, suggesting an immune response of the tissue, and it was proposed that microglia could serve as markers to distinguish pathological GCD from normal variation ( Roy et al, 2020 ; Weninger et al, 2021 ). Concordant with this, the results of a transcriptome signature study indicate that macrophages and microglia in particular play a critical role in epileptogenesis ( Chen et al, 2020 ).…”