Alfonso Represa

The clinicopathologic spectrum of focal cortical dysplasias: A consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission†

Purpose:  Focal cortical dysplasias (FCD) are localized regions of malformed cerebral cortex and are very frequently associated with epilepsy in both children and adults. A broad spectrum of histopathology has been included in the diagnosis of FCD. An ILAE task force proposes an international consensus classification system to better characterize specific clinicopathological FCD entities.

Kainate-induced apoptotic cell death in hippocampal neurons

We have examined the role apoptosis plays in epileptic brain damage using intra-amygdaloid injection of kainate. With the silver staining technique of Gallyas, argyrophylic (dying) neurons were observed, a few hours after the injection, in the amygdala and in the vulnerable pyramidal neurons of the hippocampal CA3 region. In both areas, cell death has apoptotic features, including: (i) nuclear chromatin condensation and marginalization with light and electron microscopy; (ii) DNA fragmentation with a typical ladder pattern on agarose gel electrophoresis; (iii) positive nuclear labelling with a selective in situ DNA fragmentation staining method. Combined in situ DNA labelling and silver staining showed that the DNA fragmentation occurred in dying neurons. CA1 or granule cells which do not degenerate following intra-amygdaloid injection of kainate were not stained with the in situ DNA labelling or the argyrophylic technique. Administration of diazepam blocked the kainate-induced seizures and prevented DNA fragmentation in CA3 but not in the amygdala. Therefore, apoptosis contributes to the local and distant damage induced by kainate.

GABAergic Hub Neurons Orchestrate Synchrony in Developing Hippocampal Networks

Coordinating Neuronal Assemblies Theoretical models predict the existence of so-called hub neurons—highly connected cells that strongly influence the synchronization of spiking activity in a large group of neurons. However, experimental evidence for the existence of these neuronal hubs is lacking. Bonifazi et al. (p. 1419) used high-resolution, two-photon calcium imaging to measure spontaneous calcium fluctuations in hundreds of neurons simultaneously and determined the relative timing of these fluctuations. Examination of functional connectivity maps, based on temporal correlation measurements, revealed a subpopulation of GABAergic hub neurons displaying a remarkably widespread axonal arborization that orchestrated network synchrony in developing hippocampal networks. Spontaneous network synchronizations in developing hippocampus caused giant depolarizing potentials in individual neurons, and manipulating the spike activity in potential hub cells influenced network activity. A model for the topology of brain networks incorporates a morpho-functional description of neuronal hubs. Brain function operates through the coordinated activation of neuronal assemblies. Graph theory predicts that scale-free topologies, which include “hubs” (superconnected nodes), are an effective design to orchestrate synchronization. Whether hubs are present in neuronal assemblies and coordinate network activity remains unknown. Using network dynamics imaging, online reconstruction of functional connectivity, and targeted whole-cell recordings in rats and mice, we found that developing hippocampal networks follow a scale-free topology, and we demonstrated the existence of functional hubs. Perturbation of a single hub influenced the entire network dynamics. Morphophysiological analysis revealed that hub cells are a subpopulation of γ-aminobutyric acid–releasing (GABAergic) interneurons possessing widespread axonal arborizations. These findings establish a central role for GABAergic interneurons in shaping developing networks and help provide a conceptual framework for studying neuronal synchrony.

Trophic actions of GABA on neuronal development

During brain development, transmitter-gated receptors are operative before synapse formation, suggesting that their action is not restricted to synaptic transmission. GABA, which is the principal excitatory transmitter in the developing brain, acts as an epigenetic factor to control processes including cell proliferation, neuroblast migration and dendritic maturation. These effects appear to be mediated through a paracrine, diffuse, non-synaptic mode of action that precedes the more focused, rapid mode of operation characteristic of synaptic connections. This sequential operation implies that GABA is used as an informative agent but in a unique context at an early developmental stage. This sequence also implies that by altering these effects, drugs acting on the GABA system could be pathogenic during pregnancy.

Apoptosis and Necrosis After Reversible Focal Ischemia: An In Situ DNA Fragmentation Analysis

Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain (Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385–388). We now show that a significant number of striatal and cortical neurons exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery.

We report a signaling mechanism in rats between mother and fetus aimed at preparing fetal neurons for delivery. In immature neurons, γ-aminobutyric acid (GABA) is the primary excitatory neurotransmitter. We found that, shortly before delivery, there is a transient reduction in the intracellular chloride concentration and an excitatory-to-inhibitory switch of GABA actions. These events were triggered by oxytocin, an essential maternal hormone for labor. In vivo administration of an oxytocin receptor antagonist before delivery prevented the switch of GABA actions in fetal neurons and aggravated the severity of anoxic episodes. Thus, maternal oxytocin inhibits fetal neurons and increases their resistance to insults during delivery.

Transient increased density of NMDA binding sites in the developing rat hippocampus

Using quantitative autoradiography and membrane preparations, the density of specific glutamate and N-methyl-D-aspartic acid (NMDA) binding sites have been determined in the developing rat hippocampus. We found an abrupt reduction in the density of NMDA binding sites after P8 (postnatal day) without change in affinity. The transient expression of NMDA receptors during maturation suggests that they may play a particularly important role in synaptogenesis.

NCAM is essential for axonal growth and fasciculation in the hippocampus.

The neural cell adhesion molecule (NCAM), probably the best characterized and most abundant cell adhesion molecule on neurons, is thought to be a major regulator of axonal growth and pathfinding. Here we present a detailed analysis of these processes in mice deficient for all NCAM isoforms, generated by gene targeting. The hippocampal mossy fiber tract shows prominent expression of polysialylated NCAM and the generation of new axonal projections throughout life. Focusing on this important intrahippocampal connection, we demonstrate that in the absence of NCAM, fasciculation and pathfinding of these axons are strongly affected. In addition we show alterations in the distribution of mossy fiber terminals. The phenotype is more severe in adult than in young animals, suggesting an essential role for NCAM in the maintenance of plasticity in the mature nervous system.

Kainate binding sites in the hippocampal mossy fibers: Localization and plasticity

The regional distribution of high affinity binding sites for kainic acid has been determined in rat hippocampi by quantitative autoradiography. Selective lesions were made in order to determine the exact localization of these sites in the mossy fiber system, and to evaluate whether the sprouting and synaptic reorganization of the mossy fibers are associated with alterations in the distribution of these binding sites. The results show that kainate binding sites in the stratum lucidum are more vulnerable to destruction of the granules and their mossy fibers by intrahippocampal colchicine injections than to destruction of the CA3/CA4 pyramidal cells by injection of kainate into the amygdala. This suggests that a substantial proportion of the kainate binding sites is associated with the mossy fiber terminals (i.e. the presynaptic elements). Furthermore, in keeping with an earlier study, destruction of the pyramidal neurons of CA3 by intracerebral kainate produced a dark Timm positive band in the supragranular zone which is due to the sprouting of mossy fibers. This was associated with an increase in the density of kainate binding sites, which further stresses the parallelism between the distribution of these sites and mossy fiber terminals.

Mutations in the [beta]-tubulin gene TUBB2B result in asymmetrical polymicrogyria

Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. Here we report de novo mutations in a β-tubulin gene, TUBB2B, in four individuals and a 27-gestational-week fetus with bilateral asymmetrical polymicrogyria. Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that TUBB2B is required for neuronal migration. We also show that two disease-associated mutations lead to impaired formation of tubulin heterodimers. These observations, together with previous data, show that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly and pachygyria, but also polymicrogyria malformations.