Abstract
Hypersynchronous neuronal excitation manifests clinically as seizure (ictogenesis), and may recur spontaneously and repetitively after a variable latency period (epileptogenesis). Despite tremendous research efforts to describe molecular pathways and signatures of epileptogenesis, molecular pathomechanisms leading to chronic epilepsy remain to be clarified. We hypothesized that epigenetic modifications may form the basis for a cellular memory of epileptogenesis, and used a primary neuronal cell culture model of the rat hippocampus to study the translation of massive neuronal excitation into persisting changes of epigenetic signatures and pro-epileptogenic target gene expression. Increased spontaneous activation of cultured neurons was detected 3 and 7 days after stimulation with 10 μM glutamate when compared to sham-treated time-matched controls using calcium-imaging in vitro. Chromatin-immunoprecipitation experiments revealed short-term (3 h, 7 h, and 24 h) and long-term (3 d and 2 weeks) changes in histone modifications, which were directly linked to decreased expression of two selected epilepsy target genes, e.g. excitatory glutamate receptor genes Gria2 and Grin2a. Increased promoter methylation observed 4 weeks after glutamate stimulation at respective genes suggested long-term repression of Gria2 and Grin2a genes. Inhibition of glutamatergic activation or blocking the propagation of action potentials in cultured neurons rescued altered gene expression and regulatory epigenetic modifications. Our data support the concept of a cellular memory of epileptogenesis and persisting epigenetic modifications of epilepsy target genes, which are able to turn normal into pro-epileptic neurons and circuits.