A genetic animal model of human neocortical heterotopia associated with seizures

Abstract

Malformations of the human neocortex are commonly associated with developmental delays, mental retardation, and epilepsy. This study describes a novel neurologically mutant rat exhibiting a forebrain anomaly resembling the human neuronal migration disorder of double cortex. This mutant displays a telencephalic internal structural heterotopia (tish) that is inherited in an autosomal recessive manner. The bilateral heterotopia is prominent below the frontal and parietal neocortices but is rarely observed in temporal neocortex. Neurons in the heterotopia exhibit neocortical-like morphologies and send typical projections to subcortical sites; however, characteristic lamination and radial orientation are disturbed in the heterotopia. The period of neurogenesis during which cells in the heterotopia are generated is the same as in the normotopic neocortex; however, the cells in the heterotopia exhibit a "rim-to-core" neurogenetic pattern rather than the characteristic "inside-out" pattern observed in normotopic neocortex. Similar to the human syndrome of double cortex, some of the animals with the tish phenotype exhibit spontaneous recurrent electrographic and behavioral seizures. The tish rat is a unique neurological mutant that shares several features with a human cortical malformation associated with epilepsy. On the basis of its regional connectivity, histological composition, and period of neurogenesis, the heterotopic region in the tish rat is neocortical in nature. This neurological mutant represents a novel model system for investigating mechanisms of aberrant neocortical development and is likely to provide insights into the cellular and molecular events contributing to seizure development in dysplastic neocortex.

Fig. 1.

Top. Coronal sections of atish brain stained for AChE. Low-magnification photomicrographs (A, B) show the heterotopia to be a large bilateral structure intercalated between the neocortex and underlying white matter. A thin layer of white matter separates the heterotopia from the overlying neocortex, and a thicker layer of white matter separates it from lower structures. InA and C, the heterotopia is located above the darkly stained striatum and below the neocortex. In the more caudal sections (B, D), the heterotopia is located between the hippocampus (or lateral ventricle) and the neocortex. The heterotopia has a multinodular appearance attributable to the passage of one or more large fiber tracts through the structure.

Fig. 3.

MR image of the tish brain. In this proton density image, the heterotopia can be observed bilaterally as an area isodense to the overlying normotopic cortex (arrowhead indicates structure on the right side of the brain). MRI was performed at the Small Aperture Magnetic Imaging and Spectroscopy Center at the University of Virginia using a 4.7 Tesla 40 cm bore 200/400 MR imager.

Fig. 4.

Histological appearance of the normotopic neocortex and heterotopic region of a tish animal. Photomicrographs of Nissl-stained sections are shown of the normotopic neocortex (A) and heterotopia (B). Cellular somata in the normotopic neocortex exhibit a laminar organization, whereas cells in the heterotopia do not exhibit strict lamination. Golgi-stained sections of the normotopic neocortex (C) and heterotopic region (D) demonstrate the presence of similar cell types in the two structures. In the normotopic neocortex, the apical dendrites of pyramidal neurons are oriented radially, and their somata are located in appropriate laminae. In contrast, the neurons in the heterotopia do not exhibit a strict laminar pattern and their apical dendrites are not oriented uniformly. Numbers indicate layers of the neocortex. W, White matter;T, heterotopia. Scale bars: A, B, 200 μm; C, D, 170 μm.

Fig. 5.

Neurogenetic patterns in the forebrain of the tish rat. Photomicrographs are shown of coronal sections of tish brains in which immunostaining was performed on animals labeled with BrDU on E15 (A) or E18 (B); these animals survived to P33 and P30, respectively. Darkly stained (BrDU-positive) cells are found primarily in the deep aspect of the normotopic neocortex in the E15-injected animal (A) and in the superficial aspect of the normotopic neocortex in the E18-injected animal (B). Labeled cells are located primarily in the rim of the heterotopia in the E15-injected animal (A) and in the core of the heterotopia in the E18-injected animal (B). Arrowsindicate the white matter surrounding the heterotopic region. Scale bar, 200 μm.

Fig. 6.

Subcortical connectivity of thetish forebrain. Fluorescence micrographs show retrogradely labeled projection neurons in both the normotopic neocortex and neighboring heterotopia after injection of Fluorogold into either the thalamus (A) or spinal cord (B). After an injection into the VPL of the thalamus (A), the somata of labeled neurons in the normotopic neocortex are located primarily in layer VI, and their apical dendrites are radially oriented. Labeled cells in the heterotopic region are similar in size and appearance to those in the normotopic neocortex but are more widely dispersed and tend to be concentrated along the rim area of the heterotopia. Injection of Fluorogold into the cervical spinal cord labels large pyramidal cells in layer V of the normotopic neocortex; the apical dendrites of these cells exhibit a typical radial orientation. The heterotopia also contains retrogradely labeled pyramidal neurons; however, these cells are not laminated and their apical dendrites are not consistently oriented toward the surface of the brain. Arrowsindicate the white matter surrounding the heterotopia. Scale bar, 200 μm.

Fig. 7.

Electroencephalographic recordings of a seizure in a tish rat. The four lines represent a continuous EEG recording from a single electrode positioned in the normotopic neocortex. Seizure activity can be observed as changes in the frequency and amplitude of the EEG; this event lasted ∼63 sec. The animal exhibited convulsive behavioral seizures during the electrographic seizure event (arrow indicates seizure onset).