The extracellular matrix protein Reelin is an essential regulator of neuronal migration and lamination in the developing and mature brain. Lack of Reelin causes severe disturbances in cerebral layering, such as the reeler phenotype and granule cell dispersion in temporal lobe epilepsy. Reelin is synthesized and secreted by Cajal-Retzius cells and GABAergic interneurons, and its function depends on proteolytic cleavage after secretion. The mechanisms regulating these processes are largely unknown. Here, we used rat hippocampal slice cultures to investigate the effect of neuronal activation and hyperexcitation on Reelin synthesis, secretion, and proteolytic processing. We show that enhanced neuronal activity does not modulate Reelin synthesis or secretion. Moreover, we found that intracellular Reelin resides predominantly in the endoplasmic reticulum before it is constitutively secreted via the early secretory pathway. Epileptiform activity, however, impairs the proteolytic processing of Reelin and leads to accumulation of Reelin in the extracellular matrix. We found that both conditions, epileptiform activity and impaired proteolytic cleavage of Reelin, cause granule cell dispersion via inhibition of metalloproteinases. Taken together, our results strongly suggest that secretion of Reelin is activity-independent and that proteolytic processing of Reelin is required for the maintenance of granule cell lamination in the dentate gyrus.

Action potential (AP) responses of dentate gyrus granule (DG) cells have to be tightly regulated to maintain hippocampal function. However, which ion channels control the response delay of DG cells is not known. In some neuron types, spike latency is influenced by a dendrotoxin (DTX)-sensitive delay current (ID) mediated by unidentified combinations of voltage-gated K+ (Kv) channels of the Kv1 family Kv1.1–6. In DG cells, the ID has not been characterized and its molecular basis is unknown. The response phenotype of mature DG cells is usually considered homogenous but intrinsic plasticity likely occurs in particular in conditions of hyperexcitability, for example during temporal lobe epilepsy (TLE). In this study, we examined response delays of DG cells and underlying ion channel molecules by employing a combination of gramicidin-perforated patch-clamp recordings in acute brain slices and single-cell reverse transcriptase quantitative polymerase chain reaction (SC RT-qPCR) experiments. An in vivo mouse model of TLE consisting of intrahippocampal kainate (KA) injection was used to examine epilepsy-related plasticity. Response delays of DG cells were DTX-sensitive and strongly increased in KA-injected hippocampi; Kv1.1 mRNA was elevated 10-fold, and the response delays correlated with Kv1.1 mRNA abundance on the single cell level. Other Kv1 subunits did not show overt changes in mRNA levels. Kv1.1 immunolabeling was enhanced in KA DG cells. The biophysical properties of ID and a delay heterogeneity within the DG cell population was characterized. Using organotypic hippocampal slice cultures (OHCs), where KA incubation also induced ID upregulation, the homeostatic reversibility and neuroprotective potential for DG cells were tested. In summary, the AP timing of DG cells is effectively controlled via scaling of Kv1.1 subunit transcription. With this antiepileptic mechanism, DG cells delay their responses during hyperexcitation.

Neuronal ectopia, such as granule cell dispersion (GCD) in temporal lobe epilepsy (TLE), has been assumed to result from a migration defect during development. Indeed, recent studies reported that aberrant migration of neonatal-generated dentate granule cells (GCs) increased the risk to develop epilepsy later in life. On the contrary, in the present study, we show that fully differentiated GCs become motile following the induction of epileptiform activity, resulting in GCD. Hippocampal slice cultures from transgenic mice expressing green fluorescent protein in differentiated, but not in newly generated GCs, were incubated with the glutamate receptor agonist kainate (KA), which induced GC burst activity and GCD. Using real-time microscopy, we observed that KA-exposed, differentiated GCs translocated their cell bodies and changed their dendritic organization. As found in human TLE, KA application was associated with decreased expression of the extracellular matrix protein Reelin, particularly in hilar interneurons. Together these findings suggest that KA-induced motility of differentiated GCs contributes to the development of GCD and establish slice cultures as a model to study neuronal changes induced by epileptiform activity.

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Temporal lobe epilepsy is frequently associated with granule cell dispersion (GCD), an abnormal widening of the granule cell layer in the dentate gyrus. There is increasing evidence that a loss and the functional inactivation of the positional signal Reelin is involved in GCD formation. Reelin is synthesized and released by Cajal-Retzius cells and interneurons, and its function depends on proteolytic cleavage after secretion. Epileptic conditions impair Reelin processing by inhibition of matrix metalloprotease (MMP) activity and cause the extracellular accumulation of unprocessed Reelin. Here we investigated how epileptic conditions inhibit MMP activity. We used kainate (KA) treatment of organotypic hippocampal slice cultures as an epilepsy model and found a significant increase of tissue inhibitor of metalloproteases 1 (TIMP-1) levels and strongly enhanced TIMP-1 immunolabeling in hippocampal neurons. Functional inhibition of TIMP-1 prevented the KA-induced impairment of Reelin cleavage indicating that TIMP-1 inhibits MMP activity. Moreover, application of recombinant TIMP-1 alone was sufficient to impair Reelin processing and to induce GCD, similar to that observed after KA treatment. In summary, we present evidence that epileptic conditions inhibit MMP activity by up-regulation of endogenous TIMP-1, which in turn leads to extracellular accumulation of uncleaved and inactive Reelin and thereby to GCD.