Development of cortical malformations in BCNU-treated rat, model of cortical dysplasia.

Cortical dysplasia (CD) comprises a wide range of cerebral cortex alterations ranging from severe brain malformations to local disruption of the cortical structure. Most hypotheses focused on the role of embryonic/perinatal development insults as the main cause for the majority of CD. Rats with prenatal exposure to BCNU (1-3-bis-chloroethyl-nitrosurea) represent an injury-based model and reproduce many anatomical features seen in human patients with CD, such as altered cortical layering and the presence of heterotopia and dysmorphic/heterotopic neurons. With the aim to investigate the formation and evolution of CD during development, we analysed the expression of a panel of layer-specific genes (Nurr1, Er81, Ror-ß and Cux2, markers of layers VI, V, IV and superficial layers, respectively) in BCNU-treated cortices from E17 to postnatal day 14. By means of appropriate immunohistochemical markers, we also analysed the structural organization of embryonic ventricular zone and of glial and axonal fibres, substrates supporting radial and tangential migration, respectively. The main results of the present study are: (i) the ventricular zone appeared disorganized and the neuroependyma was partially disrupted; (ii) radial glia scaffold and tangential fibres were deeply disarranged, thus explaining the neuronal migration defects; (iii) cortical heterotopia were detectable by E19, whereas periventricular heterotopia were detectable after birth; (iv) both cortical and periventricular heterotopia showed a pseudo-laminar structure, with cells of the upper cortical layers in the core of the nodules and cells of layer IV and V at their border; (v) the distribution of GABAergic cells was altered since the embryonic stages, as a consequence of the derangement of tangential fibres. Our analysis sheds light on how a malformed cortex develops after a temporally discrete environmental insult and adds additional knowledge on specific aspects of the etiopathogenesis of CD.

Similar binding to glutamate receptors by Rasmussen and partial epilepsy patients' sera.

Immunoreactivity of sera from patients with Rasmussen encephalitis (RE) and patients with partial epilepsy (PE) was analyzed by immunohistoblot on rat brain sections and the staining pattern compared with that obtained with antibodies to a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and NMDA receptors. Staining for anti-glutamate receptor 3 (GluR3) was found in 82% of patients with RE and 64% of patients with PE. Histoblot analysis showed a positive staining in GluR3- and NMDA-specific regions of rat brain, providing a comprehensive CNS immunolocalization.

Morphological organization of somatosensory cortex in Otx1(-/-) mice.

Knock-out Otx1 mice show brain hypoplasia, spontaneous epileptic seizures and abnormalities of the dorsal region of the neocortex. We investigated structural alterations in excitatory and inhibitory circuits in somatosensory cortex of Otx1(-/-) mice by immunocytochemistry using light, confocal and electron microscopy. Immunostaining for non-phosphorylated neurofilament SMI311 and subunit 1 of the NMDA receptor - used as markers of pyramidal neurons - showed reduced layer V pyramidal cells and ectopic pyramidal cells in layers II and III of the mutant cortex. Immunostaining for calcium-binding proteins calbindin, calretinin and parvalbumin - markers of non-overlapping types of GABAergic interneurons - showed no differences between wild-type and knock-out cortex for calbindin and calretinin neurons, while parvalbumin neurons were only patchily distributed in Otx1(-/-) cortex. The pattern of positivity of the GABAergic marker glutamic acid decarboxylase in Otx1(-/-) cortex was also altered and similar to that of parvalbumin. GABA transporter 1 immunoreactivity was greater in Otx1(-/-) than wild-type; quantitation of structures immunoreactive for this transporter in layer V showed that they were increased overall in Otx1(-/-) but the density of inhibitory terminals on pyramidal neurons in the same layer labeled with this transporter was similar to that in wild-type mice. No differences in the distribution or intensity of the glial markers GABA transporter 3 or glial fibrillary acidic protein were found. The defects found in the cortical GABAergic system of the Otx1(-/-) mouse can plausibly explain the cortical hyperexcitability that produces seizures in these animals.

Potentially epileptogenic dysfunction of cortical NMDA- and GABA-mediated neurotransmission in Otx1-/- mice.

Knockout Otx1 mice present a microcephalic phenotype mainly due to reduced deep neocortical layers and spontaneous recurrent seizures. We investigated the excitable properties of layer V pyramidal neurons in neocortical slices prepared from Otx1-/- mice and age-matched controls. The qualitative firing properties of the neurons of Otx1-/- mice were identical to those found in wild-type controls, but the proportion of intrinsically bursting (IB) neurons was significantly smaller. This is in line with the lack of the Otx1 gene contribution to the generation and differentiation of neurons destined for the deep neocortical layers, in which IB neurons are located selectively in wild-type rodents. The pyramidal neurons recorded in Otx1-/- mice responded to near-threshold electrical stimulation of the underlying white matter, with aberrant polysynaptic excitatory potentials often leading to late action potential generation. When the strength of the stimulus was increased, the great majority of the Otx1-/- neurons (78%) responded with a prominent biphasic inhibitory postsynaptic potential that was significantly larger than that observed in the wild-type mice, and was often followed by complex postinhibitory depolarizing events. Both late excitatory postsynaptic potentials and postinhibitory excitation were selectively suppressed by NMDA receptor antagonists, but not by AMPA antagonists. We conclude that the cortical abnormalities of Otx1-/- neocortex due to a selective loss of large projecting neurons lead to a complex rearrangement of local circuitry, which is characterized by an excess of N-methyl-d-aspartate-mediated polysynaptic excitation that is counteracted by GABA-mediated inhibition in only a limited range of stimulus intensity. Prominent postsynaptic inhibitory potentials may also act as a further pro-epileptogenic event by synchronizing abnormal excitatory potentials.

Synaptic properties of neocortical neurons in epileptic mice lacking the Otx1 gene.

PURPOSE: The murine homeobox-containing Otx gene is required for correct nervous system and sense organ development. Otx1-/1 mice obtained by replacing Otx with the lac Z gene show developmental abnormalities of the cerebellum, mesencephalon, and cerebral cortex associated with spontaneous epileptic seizures (1). The epileptogenic mechanisms accounting for these seizures were investigated by means of electrophysiological recordings made from neocortical slices. METHODS: The 400-microm slices were prepared from the somatosensory cortex of Otx1-/- and Otx1+/+ mice, and the current clamp intracellular recordings were obtained from layer V pyramidal neurons by means of pipettes containing K+ acetate 1.5 mol/L and biocytin 2% (pH 7.3). RESULTS: Synaptic responses could be evoked in the neocortical pyramidal neurons by electrically stimulating the underlying white matter. gamma-Aminobutyric acid A/B-mediated inhibitory postsynaptic potentials were more pronounced in the Otx1-/- than in the control pyramidal neurons from the earliest postnatal period; multisynaptic excitatory postsynaptic potentials were significantly more expressed in the Otx1-/- mice also at the end of the first postnatal month, when they were only rarely encountered in controls. CONCLUSION: Excessive excitatory amino acid-mediated synaptic driving may lead to a hyperexcitable condition that is responsible for the epileptic manifestations occurring in Otx1-/- mice. This excess of excitation is not counteracted by well-developed gamma-aminobutyric acid activity, which seems to be involved in the synchronization of cell discharges. Our ongoing and more extensive comparative analysis of the mutants and controls should help to clarify the way in which the putative rearrangement taking place in Otx1-/- neocortex may lead to the excitatory hyperinnervation of layer V pyramidal neurons.