Abstracts

Cortical malformations are a common feature in early-onset epilepsy disorders and an area of considerable research interest. Our laboratory has developed and characterized a unique rodent model of cortical malformations (prenatal MAM exposure) that features loss of lamination and hippocampal heterotopia. In recent years we demonstrated that MAM-exposed offspring exhibit hyperexcitability [italic]in vivo [/italic]and [italic]in vitro[/italic]. Hyperexcitability is observed in adolescent MAM offspring and is likely related to loss of a fast, transient I[sub]A[/sub]current on heterotopic neurons (Castro et al. 2001). However, it is currently unknown when in development heterotopic cells first exhibit this distinct sign of hyperexcitability, and hence when a malformed brain is first vulnerable to epilepsy. Here we examine this issue using rats exposed to MAM [italic]in utero[/italic].
Pregnant S-D rats were injected with MAM (25 mg/kg) on E15. Rat pups (P0-P15) were anesthetized and brains rapidly removed, chilled in ice-cold, oxygenated sucrose artificial cerebrospinal fluid and sectioned (420 [mu]m for extracellular recordings, 300 [mu]m for patch-clamping). Field recordings were made in heterotopia and adjacent normotopic CA1 regions (MAM) or CA1 (controls) . In slices at P0 and P1, where there is no heterotopia, recordings were made along normotopic CA1. To induce bursting, [K⁺][sub]o[/sub] was raised sequentially from 3 to 6 to 8.5 mM. To study I[sub]A[/sub], whole-cell voltage-clamp techniques were used in combination with IR-DIC. 4-AP (10 mM) and TEA (20 mM) were used to confirm the identification of early and late K current components. Non-radioactive [italic]in situ[/italic] hybridization was performed on fixed tissue sections.
Slices prepared from early postnatal MAM-treated rats ([italic]n [/italic]= 3-5 per age) were hyperexcitable in comparison to untreated controls ([italic]n [/italic]= 3-5 per age). Specifically, we observed small epileptiform burst discharges (0.1-0.5 mV) at P5 and both 6 mM [K⁺][sub]o[/sub] in slices from MAM-treated animals; infrequent discharges could be observed occasionally at younger ages (P3-P5). In contrast, burst discharges were not observed until P5 and 8.5 mM [K⁺][sub]o [/sub]in controls. In voltage-clamp studies, hippocampal neurons with prominent I[sub]A[/sub] and I[sub]DR[/sub] components were observed as early as P1 in CA1 pyramidal cells (Control; [italic]n [/italic]= 5). Heteroptic neurons lacked the I[sub]A[/sub]at early postnatal ages (P3-P5). Potassium current results were confirmed using [italic]in situ[/italic] hybridization to examine the expression of Kv4.2 (I[sub]A [/sub]channel subunit) at various embryonic and early postnatal ages.
Here we describe the development of hyperexcitability in MAM-treated rats. Previous work established that heterotopiae are evident by P3 (Castro et al. 2002) and the present work demonstrates that seizure-like activity can be observed at this early postnatal timepoint. Therefore, our findings address when molecular changes (loss of Kv 4.2 in heterotopic cells) first lead to a functional outcome and may provide insight into how epilepsy develops in children with brain malformations.
[Supported by: National Institutes of Health.]