Abstracts

Rationale: Periventricular nodular heterotopia (PNH) is highly correlated with early and difficult to control seizure activity; however, we have little understanding of if, why, or how these PNH abnormalities affect seizure activity. We have used a rat model of PNH, in which injection of methylazoxymethanol (MAM) into pregnant rat dams produces offspring with nodular heterotopia-like brain abnormalities, to assess nodule interactions with surrounding neocortex and hippocampus. However, our acute slice and intact animal studies are limited by interruption of connectivity (slices) and inadequate spatial coverage (intact brain). We have therefore employed organotypic hippocampal slice cultures (OHSCs) to determine baseline levels of neuronal activity and activity levels in response to treatment with a seizure-inducing agent (bicuculline methiodide (BMI)), as reflected in c-fos activation. Methods: OHSCs from postnatal day 7-8 rat pups (from dams treated with MAM) were grown to 8-10 days in vitro and then exposed for 60 minutes to either artificial cerebrospinal fluid (ACSF) or 40µM BMI (in ACSF). OHSCs were then fixed and stained for c-fos immunoreactivity; photomicrographs of representative regions of the cortex (CTX), hippocampus (CA1) and nodule (PNH) were taken at 40x. For each region, the number of activated neurons and the number of pixels within each neuron expressing c-fos were quantified using ImageMeterPro (software kindly supplied by Joachim Schnier - see http://flashscript.biz/AIR/imagemeter/ImageMeterPro.html), and neuronal cell density calculated based on NeuN staining. Results: Multivariate statistical analysis showed a significant difference in number of neurons activated and average activated pixels per activated neuron between BMI- and ACSF-treated cultures, for all three regions. Within BMI-treated cultures, the CTX showed a significantly higher number of activated neurons than the PNH and CA1. Neuron counts based on NeuN labeling showed no significant difference in cell density (within the counting frame) across regions, confirming that a higher percentage of neurons in CTX showed c-fos activation. While the CA1 was not significantly different from the PNH in the number of neurons activated, it did show a statistically significant higher number of activated pixels per activated neuron (compared to both the PNH and CTX), suggesting a greater intensity of activation in the hippocampus. Conclusions: These results show that neuronal activation, as measured with c-fos, can be quantified in OHSCs, and confirm that BMI does cause broad neuronal activation. At least at the time point sampled, PNH activation was not higher than activation in CTX or CA1, in either number of activated neurons or in average activated pixels per activated neuron. These data are consistent with our slice and intact animal results (Tschuluun et al., 2011, Epilepsia 52:2304-14), suggesting that the PNH is not more easily or more intensely activated than CTX or CA1. Supported by grant NS057209 from NIH