Ryanodine receptors drive neuronal loss and regulate synaptic proteins during epileptogenesis

Status epilepticus (SE) is a clinical emergency that can lead to the development of temporal lobe epilepsy (TLE). The development and maintenance of spontaneous seizures in TLE are linked to calcium (Ca⁺²)-dependent processes such as neuronal cell loss and pathological synaptic plasticity. It has been shown that SE produces an increase in ryanodine receptor-dependent intracellular Ca⁺² levels in hippocampal neurons, which remain elevated during the progression of the disease. However, the participation of ryanodine receptors (RyRs) in the neuronal loss and circuitry rewiring that take place in the hippocampus after SE remains unknown. In this context, we first investigated the functional role of RyRs on the expression of synaptic and plasticity-related proteins during epileptogenesis induced by pilocarpine in Wistar rats. Intrahippocampal injection of dantrolene, a selective pharmacological blocker of RyRs, caused the increase of the presynaptic protein synapsin I (SYN) and synaptophysin (SYP) 48 h after SE induction. Specifically, we observed that SYN and SYP were regulated in hippocampal regions known to receive synaptic inputs, revealing that RyRs could be involved in network changes and/or neuronal protection after SE induction. In order to investigate whether the changes in SYN and SYP were related to neuroplastic changes that could contribute to pathological processes that occur after SE, we evaluated the levels of activity-regulated cytoskeleton-associated protein (ARC) and mossy fiber sprouting in the dentate gyrus (DG). Interestingly, we observed that although SE induced the appearance of intense ARC-positive cells, dantrolene treatment did not change the levels of ARC in both western blot and immunofluorescence analyses. Accordingly, in the same experimental conditions, we were not able to detect changes in the levels of both pre- and post-synaptic plasticity-related proteins, growth associated protein-43 (GAP-43) and postsynaptic density protein-95 (PSD-95), respectively. Additionally, the density of mossy fiber sprouting in the DG was not increased by dantrolene treatment. We next examined the effects of intrahippocampal injection of dantrolene on neurodegeneration. Notably, dantrolene promoted neuroprotective effects by decreasing neuronal cell loss in CA1 and CA3, which explains the increased levels of synaptic proteins, and the apparent lack of positive effect on pathological plasticity. Taken together, our results revealed that RyRs may have a major role in the hippocampal neurodegeneration associated to the development of acquired epilepsy.