Shining Light on the Neuropathology of Temporal Lobe Epilepsy

Abstract

Chronic Optogenetic Stimulation of Dentate Gyrus Granule Cells in Mouse Organotypic Slice Cultures Synaptically Drives Mossy Cell Degeneration

Koretz CC, Schneider R, Jungenitz T, Drakew A, Roeper J, Deller T. Epilepsia. 2025;66:1734–1746.

Objective: Degeneration of hilar mossy cells in the dentate gyrus is an important hallmark of hippocampal sclerosis and is often observed in patients with temporal lobe epilepsy. To understand the pathogenesis of hippocampal sclerosis and develop novel neuroprotective treatments, it is critical to determine the mechanistic processes of mossy cell degeneration and factors that influence cell vulnerability or resilience. However, suitable in vitro approaches are currently lacking. We have developed and validated an organotypic slice culture-based in vitro model that facilitates mechanistic studies of activity-dependent mossy cell vulnerability and resilience. Methods: A model was developed using entorhino-hippocampal slice cultures. Dentate gyrus granule cells were transduced with adeno-associated viruses to express channelrhodopsin2. Transduced cultures were chronically stimulated by light, and resulting cell damage was assessed by propidium iodide staining. Spontaneous synaptic activity before and after optical stimulation was recorded using whole-cell patch-clamp. Results: Selective and dose-dependent hilar neuron degeneration was observed following chronic optogenetic stimulation of organotypic slice cultures expressing channelrhodopsin-2 in granule cells. Treatment with the anticonvulsant retigabine reduced stimulation-induced hilar neuron loss in a dose-dependent manner. This demonstrates the suitability of our optogenetic in vitro model for drug screening. Patch-clamp recordings verified strong synaptic activation of mossy cells during optical stimulation and a reduction in spontaneous excitatory synaptic activity after stimulation. Significance: The role of mossy cells in the context of epileptic seizures has been a controversial topic of discussion. The presented in vitro model allows the study of mossy cell vulnerability on a single-cell level and provides the first evidence for changes in synaptic activity after stimulation. This model will facilitate our mechanistic understanding of temporal lobe epilepsy, providing a foundation for novel therapeutic interventions aimed at preserving mossy cell function in epilepsy patients.

Commentary

One of the hallmarks of acquired temporal lobe epilepsy (TLE) is its neuropathology. A common pattern is mesial temporal sclerosis (MTS), and another is endfolium sclerosis. ¹ In each case, the hippocampus shows a pattern of neuronal loss which includes death of neurons in the hilus of the dentate gyrus (DG; Figure 1). Within the hilus, one of the cell types that is vulnerable is the mossy cell, named for its numerous large spines, which give the cell an appearance of being “mossy.” ² In contrast, the primary cell type of the DG, granule cells, and area CA2 pyramidal cells, are relatively resistant. One of the puzzles of MTS is why mossy cells are vulnerable. The selective vulnerability is consistent across various types of insults or injuries that lead to TLE, such as status epilepticus, traumatic brain injury, and ischemia. ³

Figure 1.

The classic neuropathological pattern in temporal lobe epilepsy is called MTS. (A) Neuronal loss occurs in the dentate gyrus hilus, areas CA3 and CA1. The granule cells of the dentate gyrus and area CA2 pyramidal cells are relatively spared. In endfolium sclerosis, only hilar neurons are lost. (B) In the dentate gyrus, granule cells are the main cell type and make excitatory synapses on hilar neurons, including GABAergic neurons that co-express somatostatin and mossy cells (MCs). (C) In MTS, neuronal loss of SOM cells and MCs is accompanied by additional changes, including dispersion of the granule cells, abnormal granule cells, and sprouting of the granule cell axons into the sublayer where the granule cell proximal dendrites are located (mossy fiber sprouting). The hilus is typically invaded by astrocytes, and there are additional effects on gene expression and circuitry of surviving cells (not shown). Of note for this commentary is the loss of hilar MCs. MCs are densely innervated by the unusually large glutamatergic terminals of granule cells, and these terminals have very large numbers of glutamatergic vesicles. It has been suggested that MCs die after an insult or injury because the granule cells become extremely active during the insult and release excitotoxic concentrations of glutamate on MCs.

Rodents also show an MTS-like pathology when status epilepticus, traumatic brain injury, or ischemia are simulated experimentally. ³ Research using rodent models has led to several hypotheses for the reasons underlying mossy cell vulnerability. One of the hypotheses is that the mossy cells die shortly after the insult because granule cells become very active, and they have unusually large terminals which synapse on mossy cells and release high concentrations of glutamate, ⁴ potentially inducing excitotoxic injury.

In the work of Koretz et al, ⁵ the authors set out to develop a reduced preparation to study this hypothesis for mossy cell vulnerability. They selected organotypic slice cultures because that preparation allows them excellent visualization of the circuitry and survives for weeks, providing the time for adeno-associated virus to infect granule cells and express the light-activated opsin channelrhodopsin-2 (ChR2). When ChR2 is activated by the appropriate wavelength of light, depolarization of granule cells occurs because the opsin opens an ion channel that allows cations to enter the cell. Using a light-emitting diode to emit the correct wavelength of light to the cultures, they could depolarize granule cells at intensities and durations under their control. This specificity provided a more selective activation of granule cells than in the past^6,7 to ask if selective excitation of granule cells could lead to mossy cell death.

They selected a pattern of light stimulation that simulated prior studies using afferent stimulation of granule cells. It is important to note that it is not known how active granule cells are during an insult or injury, but experimental models that lead to hilar cell loss suggest that many minutes or hours of intermittent stimulation are required, so a pattern lasting 6 h was selected by the authors. To ensure phototoxicity was absent, cultures without ChR2 were exposed to light, and wavelengths of light were selected that were outside the peak wavelengths typically used for ChR2. After light stimulation, the investigators used propidium iodine staining of the cultures to define the extent of damage, and the majority appeared to be mossy cells based on calretinin co-expression. However, those nonmossy cells that died were interesting because hilar neurons other than mossy cells are also vulnerable in MTS. For example, hilar GABAergic neurons that co-express the neuropeptide somatostatin are vulnerable (Figure 1). The loss of these neurons and the loss of mossy cells have both been implicated in TLE, although their roles are debated.^8,9

Another advantage of the cultures is the ability to record with patch clamp methods, and the authors did such recordings to confirm that granule cells and mossy cells were activated by light. However, there are limitations to organotypic cultures. Like any preparation, the entire circuitry is not present. Another potential limitation is that the inferior blade of the DG did not develop in cultures in the past, but in the work of Koretz et al, ⁵ the DG structure appears intact, which is a strength of the study.

Another aspect of the study that is notable is that it provides a new model system to screen drugs to ask what drugs might be neuroprotective. Interestingly, they found that 1 antiseizure medication, retigabine, protects hilar cells. Thus, exposure of cultures to retigabine decreased the propidium iodide staining of the hilus after prolonged ChR2 stimulation of granule cells. This result is intriguing because retigabine is a drug that opens Kv7 potassium channels, the subtype of potassium channels responsible for the M current, a depolarization-activated potassium current that reduces firing evoked by sustained depolarization. One reason retigabine might be neuroprotective is by inhibiting granule cell action potentials during the sustained activity accompanying the ChR2 stimulation, but recordings of granule cells were not studied. An alternative hypothesis is based on the understanding that retigabine not only influences Kv7, but also inhibits Kv2.1, a delayed rectifier potassium channel which normally triggers an apoptotic cascade. ¹⁰ Thus, retigabine may reduce excitotoxic as well as apoptotic cell death, leading to more neuroprotection than just activating Kv7 channels alone. Indeed, apoptosis is important to consider as a potential contribution to the neuronal loss in MTS. Some investigators only use apoptotic markers to characterize hilar cell death after experimental status epilepticus, ¹¹ raising the question of just how much hilar cell death is excitotoxic and how much is apoptotic. Other types of cell death may also be important, but the mechanisms of cell death can be hard to dissociate. ¹²

In summary, Koretz et al ⁵ provide an update on organotypic cultures as a tool to study MTS and also add support to the hypothesis that granule cell activity leads to hilar cell vulnerability in TLE. The results strongly support the hypothesis that mossy cell death is directly mediated by the activity of granule cells, helping explain the pattern of cell loss in TLE (Figure 1). One would expect the work to help change our culture, paving the way to more advances in understanding TLE and its potential treatment.