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Over the last years, the critical role of cytoskeletal proteins in cortical development including neuronal migration as well as in neuronal morphology has been well established. Inputs from genetic studies were provided through the identification of several mutated genes encoding either proteins associated with microtubules (DCX, LIS1, KIF2A, KIF5C, DYNC1H1) or tubulin subunits (TUBA1A, TUBB2B, TUBB5 and TUBG1), in malformations of cortical development (MCD). We also reported the identification of missense mutations in TUBB3, the postmitotic neuronal specific tubulin, in six different families presenting either polymicrogyria or gyral disorganization in combination with cerebellar and basal ganglial abnormalities. Here, we investigate further the association between TUBB3 mutations and MCDs by analyzing the consequences of Tubb3 knockdown on cortical development in mice. Using the in utero-electroporation approach, we demonstrate that Tubb3 knockdown leads to delayed bipolar morphology and radial migration with evidence, suggesting that the neuronal arrest is a transient phenomenon overcome after birth. Silenced blocked cells display a round-shape and decreased number of processes and a delay in the acquisition of the bipolar morphology. Also, more Tbr2 positive cells are observed, although less cells express the proliferation marker Ki67, suggesting that Tubb3 inactivation might have an indirect effect on intermediate progenitor proliferation. Furthermore, we show by rescue experiments the non-interchangeability of other beta-tubulins which are unable to rescue the phenotype. Our study highlights the critical and specific role of Tubb3 on the stereotyped morphological changes and polarization processes that are required for initiating radial migration to the cortical plate.

Mutations in the TUBB3 gene, encoding β-tubulin isotype III, were recently shown to be associated with various neurological syndromes which all have in common the ocular motility disorder, congenital fibrosis of the extraocular muscle type 3 (CFEOM3). Surprisingly and in contrast to previously described TUBA1A and TUBB2B phenotypes, no evidence of dysfunctional neuronal migration and cortical organization was reported. In our study, we report the discovery of six novel missense mutations in the TUBB3 gene, including one fetal case and one homozygous variation, in nine patients that all share cortical disorganization, axonal abnormalities associated with pontocerebellar hypoplasia, but with no ocular motility defects, CFEOM3. These new findings demonstrate that the spectrum of TUBB3-related phenotype is broader than previously described and includes malformations of cortical development (MCD) associated with neuronal migration and differentiation defects, axonal guidance and tract organization impairment. Complementary functional studies revealed that the mutated βIII-tubulin causing the MCD phenotype results in a reduction of heterodimer formation, yet produce correctly formed microtubules (MTs) in mammalian cells. Further to this, we investigated the properties of the MT network in patients' fibroblasts and revealed that MCD mutations can alter the resistance of MTs to depolymerization. Interestingly, this finding contrasts with the increased MT stability observed in the case of CFEOM3-related mutations. These results led us to hypothesize that either MT dynamics or their interactions with various MT-interacting proteins could be differently affected by TUBB3 variations, thus resulting in distinct alteration of downstream processes and therefore explaining the phenotypic diversity of the TUBB3-related spectrum.

A new method of instantaneous EEG analysis based on amplitude modulation (AM-EEG) was applied to analyze the AM-EEG changes in the alpha frequency band (8.20-12.89 Hz) for successive 5 ms epochs. Repeated auditory tone-burst stimuli (of 220 ms duration) were presented at fixed 2.56 second intervals to 12 attending right-handed young female volunteers, who were EEG-recorded over 19 EEG channels at 200 Hz sampling frequency. The time-course of functional activation was characterized in terms of percent decrease in instantaneous amplitude modulation, as compared to baseline, in analogy with the classical event-related desynchronization paradigm. A dynamic sequence of the successive 122 AM-EEG maps obtained for the 610 ms from beginning of tone-burst was stored and later animated on PC microcomputer. Topological changes among successive maps were extracted into 40 specific AM-EEG maps. Early left temporal and centro-temporal activation was observed, followed first by strong bilateral frontal, and then by left temporal activation. These changes induced by a warning tone-burst are discussed in terms of functional neurophysiology. The present method therefore allows an improved time-resolution for functional brain activation paradigms.

The aim of this study was to analyze the timing and topography of brain activity in relation to the cognitive processing of different types of auditory information. We specifically investigated the effects of familiarity on environmental sound identification, an issue which has been little studied with respect to cognitive processes, neural substrates, and time course of brain activity. To address this issue, we implemented and applied an electroencephalographic mapping method named event-related desynchronization, which allows one to assess the dynamics of neuronal activity with high temporal resolution (here, 125 ms); we used 19 recording electrodes with standard positioning. We designed an activation paradigm in which healthy subjects were asked to discriminate binaurally heard sounds belonging to one of two distinct categories, "familiar" (i.e., natural environmental sounds) or "unfamiliar" (i.e., altered environmental sounds). The sounds were selected according to strict preexperimental tests so that the former should engage greater semantic, and the latter greater structural, analysis, which we predicted to preferentially implicate left posterior and right brain regions, respectively. During the stimulations, significant desynchronizations (thought to reflect neuronal activations) were recorded over left hemisphere regions for familiar sounds and right temporofrontal regions for unfamiliar sounds, but with only few significant differences between the two sound categories and a common bilateral activation in the frontal regions. However, strongly significant differences between familiar and unfamiliar sounds occurred near the end of and following the stimulations, due to synchronizations (though to reflect deactivations) which appeared over the left posterior regions, as well as the vertex and bilateral frontal cortex, only after unfamiliar sounds. These unexpected synchronizations after the unfamiliar stimuli may reflect an awareness of the unfamiliarity of such sounds, which may have induced an inhibition of semantic and episodic representations because the latter could not be associated with meaningless sounds.