Altered excitability and distribution of NMDA receptor subunit proteins in cortical layers of rat pups following multiple perinatal seizures

Abstract

During a critical period of postnatal development the epileptogenic focus is thought to be of cortical origin. We used immunohistochemistry and Western blotting to elucidate potential mechanisms underlying an increased state of susceptibility to seizures in immature animals. Distribution patterns of N-methyl- d-aspartic acid (NMDA) (NR1 and NR2A/B) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) (GluR1 and GluR2) subunits were analyzed in retrosplenial, parietal and temporal cortices during the first two postnatal weeks following three episodes of status-epilepticus. Rat pups were injected three times with kainic acid (3× KA) on P6, P9, and P13 and subsequently sacrificed 48 h after the third seizure. Cortical electroencephalography (EEG) showed increased number of spikes and bursts of longer duration after 3× KA. Immunodensity measurements after 3× KA revealed a robust increase in NR2A/B labeling specific to cortical layer V throughout the retrosplenial, parietal, and temporal cortices, with no changes noted in piriform cortex. NR1 layer V immunoreactivity was also simultaneously increased in serial sections but to a lesser degree; heightened immunodensities were specific to retrosplenial and temporal cortices. The NR1:NR2 ratio was decreased in cortical layer V of the temporal and retrosplenial cortices but not in parietal cortex despite elevated immunoreactivity. Steady levels of GluR1 and GluR2 subunits were noted in all cortical areas studied in the same animals. Thus, recurrent perinatal seizures led to selective and layer-specific increases in NMDA receptor proteins. These changes may be responsible for lowering the seizure threshold in deeper cortical areas and eventually contribute to the cortical epileptogenic focus.