Chronic optogenetic stimulation of dentate gyrus granule cells in mouse organotypic slice cultures synaptically drives mossy cell degeneration

Abstract

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.