Abstracts

RATIONALE: Malformations of cortical development (MCD) can be associated with mental retardation, dyslexia, and intractable forms of epilepsy. With better detection capabilities, there has been increased interest in patients with MCD and in developing animal models that mimic the human pathology. The origin of these malformations could be a defect in cell differentiation, specification, migration, or a combination of all three. As such, determining the process by which a malformed brain develops would aid in creating better treatment options for patients with MCD. In rats, prenatal exposure to methyazoxymethanol (MAM) yields offspring with loss of cortical lamination, microencephaly, and nodular heterotopias in the hippocampus. MAM-exposed rats are hyperexcitable both in vitro and in vivo, possibly due to an altered potassium current on heterotopic cells, and heterotopic neurons exhibit abnormal inhibitory synaptic function. Recent work from our laboratory [Castro et al., Neuroscience 2002] demonstrated that hippocampal heterotopic cells share molecular and functional characteristics of supragranular cortical neurons from layers II/III. The question remains of how abnormal cell clusters arise in the hippocampi of MAM-exposed animals. Here we describe our early efforts to understand the mechanism(s) by which heterotopiae develop by characterizing, anatomically, the timespan in which they appear.
METHODS: Pregnant Sprague-Dawley rats were injected with 25 mg/ml methylazoxymethanol acetate (MAM, i.p.) on day 15 of gestation. For preparation of hippocampal rat tissue sections, the mother was sacrificed and rat pup brains were removed at days 16 and 19 of gestation (E16 & E19), the day of birth (P0) and postnatal day 3 (P3). Brains were fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose solution, frozen rapidly on dry ice, and then cut into 40 [mu]m coronal sections on a vibrating tissue slicer. Hippocampal sections were subsequently stained with cresyl violet dye.
RESULTS: Gross analysis of rat brain sections at E16, E19, and P0 (day of birth) all show a global effect of microcephaly with MAM prenatal exposure. At the light microscopic level, hippocampal morphology in tissue sections from MAM-exposed animals was comparable to that of hippocampi from un-treated control animals. In particular, heterotopic cells were not observed in these sections. However, analysis of tissue sections at P3 revealed the presence of distinct clusters of displaced neurons (heterotopia) and loss of hippocampal lamination. At P3, we also observed prominent expression of Id-2 mRNA in the upper layers of cortex and in a [dsquote]stream[dsquote] of displaced cells leading into the hippocampus. Further immunohistochemical and in situ hybridization analysis of these tissue sections will yield additional information on how these heterotopiae develop.
CONCLUSIONS: By following, in time, the emergence of heterotopia in hippocampi of MAM-exposed rats, we ascertained that abnormal cell clusters appear postnatally. Our studies establish the time of their appearance to between birth and the third postnatal day. These studies will assist in determining the process by which experimental heterotopiae appear and may, ultimately, yield insight into the human condition of MCD.
[Supported by: National Institutes of Health and Parents Against Childhood Epilepsy.]