Abstracts

Rationale: Early-life seizures are a prevalent pediatric neurological insult, and can be associated with long-term deficits in cognition and the development of epilepsy. In a rat model of hypoxia-induced seizures (HS) at postnatal day(P)10, we have shown similar development of behavioral deficits and spontaneous recurrent seizures (Lippman-Bell et al., Epilepsia, 2013), plus deficits in long-term plasticity (LTP) of hippocampal synapses 48hrs after HS (Zhou et al., J. Neurosci., 2011). We have shown that one week post-HS, but not earlier, rats that have had seizures maintain a higher number of dendritic spines on the proximal portion of the primary apical dendrites in hippocampal CA1 compared to controls. Here we examine whether the increase in spines represents an increase in synapses, and begin to examine potential mechanisms, specifically the role of cofilin and FMRP, that can account for the post-HS spine density increase. Methods: To identify excitatory synapses, 40µm thick floating sections containing the middle third of the hippocampus from rats 1 week post-HS and littermate controls were immunolabeled with glutamatergic presynaptic markers vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2), postsynaptic marker PSD-95, and dendritic marker MAP2. A different set of sections were also stained for GABAergic neuronal marker GAD67, and a third group with FMRP. Z-stacks were obtained by imaging on a Leica SP5 confocal microscope. Images were analyzed using the JACoP plugin for ImageJ to determine the Mander's colocalization coefficient (Bolte & Cordelieres, J Microscopy, 2006) of PSD-95 staining with VGluT1/2, within MAP2 staining in the same area of CA1 s. radiatum as we have shown increased dendritic spine density. To determine the expression of cofilin and FMRP, rat brains were removed at 3, 6, 12, 24, and 48hrs and 1 week following hypoxia-induced seizures at P10 (hypoxia=7%O2 for 8 mins, 5% for 6 mins, and 4% for 1 min), and western blots were performed on whole cell hippocampal lysates. Results: In the same area of dendrite we first observed increased spine density (as determined by MAP2 labeling) in rats one week post-HS, we see an increase in synapses (colocalization coefficient median=0.27±0.02; n=7 fields from 3 rats) compared to age-matched littermates (0.19±0.02; n=6 fields from 2 rats; p=0.035 by Mann-Whitney). Preliminary data shows no increase in overall GABAergic neurons, as assessed with GAD67 immunostaining in this area. Preliminary data show an early increase in the cofilin protein expression in post-HS compared to control rats the hippocampus beginning at 48hrs post-HS (n=2/group). FMRP expression increases beginning 24hrs post-HS (p=19 control, 16 post-HS; p=0.04) accompanied by subcellular localization changes. Conclusions: We show here that the excess spines we have previously reported one week post-HS appear to be forming functional excitatory synapses. This could therefore contribute to the increased excitability during this silent period of epileptogenesis. We are currently investigating the roles of cofilin and FMRP pathways.