Visual stimulus-dependent changes in interhemispheric EEG coherence in humans.

We analyzed the coherence of electroencephalographic (EEG) signals recorded symmetrically from the two hemispheres, while subjects (n = 9) were viewing visual stimuli. Considering the many common features of the callosal connectivity in mammals, we expected that, as in our animal studies, interhemispheric coherence (ICoh) would increase only with bilateral iso-oriented gratings located close to the vertical meridian of the visual field, or extending across it. Indeed, a single grating that extended across the vertical meridian significantly increased the EEG ICoh in normal adult subjects. These ICoh responses were obtained from occipital and parietal derivations and were restricted to the gamma frequency band. They were detectable with different EEG references and were robust across and within subjects. Other unilateral and bilateral stimuli, including identical gratings that were effective in anesthetized animals, did not affect ICoh in humans. This fact suggests the existence of regulatory influences, possibly of a top-down kind, on the pattern of callosal activation in conscious human subjects. In addition to establishing the validity of EEG coherence analysis for assaying cortico-cortical connectivity, this study extends to the human brain the finding that visual stimuli cause interhemispheric synchronization, particularly in frequencies of the gamma band. It also indicates that the synchronization is carried out by cortico-cortical connection and suggests similarities in the organization of visual callosal connections in animals and in man.

Fraser syndrome: epidemiological study in a European population.

Fraser syndrome is a rare autosomal recessive disorder characterized by cryptophthalmos, cutaneous syndactyly, laryngeal, and urogenital malformations. We present a population-based epidemiological study using data provided by the European Surveillance of Congenital Anomalies (EUROCAT) network of birth defect registries. Between January 1990 and December 2008, we identified 26 cases of Fraser syndrome in the monitored population of 12,886,464 births (minimal estimated prevalence of 0.20 per 100,000 or 1:495,633 births). Most cases (18/26; 69%) were registered in the western part of Europe, where the mean prevalence is 1 in 230,695 births, compared to the prevalence 1 in 1,091,175 for the rest of Europe (P = 0.0003). Consanguinity was present in 7/26 (27%) families. Ten (38%) cases were liveborn, 14 (54%) pregnancies were terminated following prenatal detection of a serious anomaly, and 2 (8%) were stillborn. Eye anomalies were found in 20/24 (83%), syndactyly in 14/24 (58%), and laryngeal anomalies in 5/24 (21%) patients. Ambiguous genitalia were observed in 3/24 (13%) cases. Bilateral renal agenesis was present in 12/24 (50%) and unilateral in 4/24 (17%) cases. The frequency of anorectal anomalies was particularly high (42%). Most cases of Fraser syndrome (85%) are suspected prenatally, often due to the presence of the association of renal agenesis and cryptophthalmos. In the European population, a high proportion (82%) of pregnancies is terminated, thus reducing the live birth prevalence to a third of the total prevalence rate.

PAX6 aniridia and interhemispheric brain anomalies.

PURPOSE: To report the clinical and genetic study of patients with autosomal dominant aniridia. METHODS: We studied ten patients with aniridia from three families of Egyptian origin. All patients underwent full ophthalmologic, general and neurological examination, and blood drawing. Cerebral magnetic resonance imaging was performed in the index case of each family. Genomic DNA was prepared from venous leukocytes, and direct sequencing of all the exons and intron-exon junctions of the Paired Box gene 6 (PAX6) was performed after PCR amplification. Phenotype description, including ophthalmic and cerebral anomalies, mutation detection in PAX6 and phenotype-genotype correlation was acquired. RESULTS: Common features observed in the three families included absence of iris tissue, corneal pannus with different degrees of severity, and foveal hypoplasia with severely reduced visual acuity. In Families 2 and 3, additional findings, such as lens dislocation, lens opacities or polar cataract, and glaucoma, were observed. We identified two novel (c.170-174delTGGGC [p.L57fs17] and c.475delC [p.R159fs47]) and one known (c.718C>T [p.R240X]) PAX6 mutations in the affected members of the three families. Systemic and neurological examination was normal in all ten affected patients. Cerebral magnetic resonance imaging showed absence of the pineal gland in all three index patients. Severe hypoplasia of the brain anterior commissure was associated with the p.L57fs17 mutation, absence of the posterior commissure with p.R159fs47, and optic chiasma atrophy and almost complete agenesis of the corpus callosum with p.R240X. CONCLUSIONS: We identified two novel PAX6 mutations in families with severe aniridia. In addition to common phenotype of aniridia and despite normal neurological examination, absence of the pineal gland and interhemispheric brain anomalies were observed in all three index patients. The heterogeneity of PAX6 mutations and brain anomalies are highlighted. This report emphasizes the association between aniridia and brain anomalies with or without functional impact, such as neurodevelopment delay or auditory dysfunction.

13q deletion syndrome and retinoblastoma in identical dichorionic diamniotic monozygotic twins.

PURPOSE: To report the case of identical dichorionic diamniotic female twins with unilateral retinoblastoma in 13q deletion syndrome. METHODS: Clinical and ophthalmoscopic evaluation, combination of multiple ligation-dependent probe amplification, array-comparative genomic hybridization analyses, and magnetic resonance imaging were performed. RESULTS: Peculiar facial features, marked hypotonia, gastroesophageal reflux, interatrial septal defect with left to right shunt and light dilatation of right chambers, 5th finger hypoplasia, 3rd-5th toes clinodactyly, 2nd toe overlapped to 3rd toe, and cutis marmorata were found. Ophthalmoscopic evaluation revealed unilateral retinoblastoma in both girls. Magnetic resonance imaging detected corpus callosum hypoplasia in both twins. A 34.4-Mb deletion involving bands 13q13.2-q21.33 and including the RB1 gene was identified in both twins. The deletion was not present in the DNA of their parents and older brother. CONCLUSIONS: Dysmorphic features in children must be always suspicious of 13q deletion syndrome and a short ophthalmoscopic follow-up is necessary to detect the presence of a retinoblastoma.

SOURCE, FATE AND FUNCTION OF EARLY GLIAL CELLS EXPRESSING NKX2.1 IN MOUSE EMBRYONIC BRAINS

The brain tissue is made of neuronal and glial cells generated in the germinal layer bordering the ventricles. These cells divide, differentiate and migrate following specific pathways. The specification of GABAergic interneurons and glutamatergic neurons has been broadly studied but little is known about the origin, the fate and the function of early glial cells in the embryonic telencephalon. It has been commonly accepted since long that the glial cells and more particularly the astrocytes were generated after neurogenesis from the dorsal telencephalon. However, our work shows that, unlike what was previously thought, numerous glial cells (astroglia and polydendrocytes) are generated during neurogenesis in the early embryonic stages from E14.5 to E16.5, and originate from the ventral Nkx2.1-expressing precursors instead. NK2 homeobox 1 (Nkx2.1) is a member of the NK2 family of homeodomaincontaining transcription factors. The specification of the MGE precursors requires the expression of the Nkx2.1 homeobox gene. Moreover, Nkx2.1 is previously known to regulate the specification of GABAergic interneurons and early oligodendrocytes in the ventral telencephalon. Here, in my thesis work, I have discovered that, in addition, Nkx2.1 also regulates astroglia and polydendrocytes differentiation. The use of Nkx2.1 antibody and Nkx2.1 riboprobe have revealed the presence of numerous Nkx2.1-positive cells that express astroglial markers (like GLAST and GFAP) in the entire embryonic brain. Thus, to selectively fate map MGE-derived GABAergic interneurons and glia, we crossed Nkx2.1-Cre mice, Glast-Cre ERT+/- inducible mice and NG2-Cre mice with the Cre reporter Rosa26-lox-STOP-lox-YFP (Rosa26-YFP) mice. The precise origin of Nkx2.1-positive astroglia has been directly ascertained by combining glial immunostaining and focal electroporation of the pCAG-GS-EGFP plasmids into the subpallial domains of organotypic slices, as well as, by using in vitro neurosphere experiments and in utero electroporation of the pCAG-GS-tomato plasmid into the ventral pallium of E14.5 Nkx2.1-Cre+/Rosa-YFP+/- embryos. We have, thus, confirmed that the three germinal regions of the ventral telencephalon i.e. the MGE, the AEP/POA and the triangular septal nucleus are able to generate early astroglial cells. Moreover, immunohistochemistry for several astroglial cells and polydendrocyte markers, both in the Nkx2.1-/- and control embryos and in the neurospheres, has revealed a severe loss of both glial cell types in the Nkx2.1 mutants. We found that the loss of glia corresponded to a decrease of Nkx2.1-derived precursor division capacity and glial differentiation. There was a drastic decrease of BrdU+ dividing cells labeled for Nkx2.1 in the MGE*, the POA* and the septal nucleus* of Nkx2.1 mutants. In addition, we noticed that while some remaining Nkx2.1+ precursors still succeeded to give rise to post-mitotic neurons in vitro and in vivo in the Nkx2.1-/-, they completely lost the capacity to differentiate in astrocytes. Altogether, these observations indicate for the first time that the transcription factor Nkx2.1 regulates the proliferation and differentiation of precursors in three subpallial domains that generate early embryonic astroglia and polydendrocytes. Furthermore, in order to investigate the potential function of these early Nkx2.1- derived glia, we have performed multiple immunohistochemical stainings on Nkx2.1-/- and wild-type animals, and Nkx2.1-Cre mice that were crossed to Rosa-DTA+/- mice in which the highly toxic diphtheria toxin aided to selectively deplete a majority of the Nkx2.1-derived cells. Interestingly, in these two mutants, we observed a drastic and significant loss of GFAP+, GLAST+, NG2+ and S100ß+ astroglial cells at the telencephalic midline and in the medial cortical areas. This cells loss could be directly correlated with severe axonal guidance defects observed in the corpus callosum (CC), the hippocampal commissure (HIC), the fornix (F) and the anterior commissure (AC). Axonal guidance is a key step allowing neurons to form specific connections and to become organized in a functional network. The contribution of guidepost cells inside the CC and the AC in mediating the growth of commissural axons have until now been attributed to specialized midline guidepost astroglia. Previous published results in our group have unravelled that, during embryonic development, the CC is populated in addition to astroglia by numerous glutamatergic and GABAergic guidepost neurons that are essential for the correct midline crossing of callosal axons. Therefore, the relative contribution of individual neuronal or glial populations towards the guidance of commissural axons remains largely to be investigated to understand guidance mechanisms further. Thus, we crossed Nkx2.1-Cre mice with NSE-DTA+/- mice that express the diphtheria toxin only in neurons and allowed us to selectively deplete Nkx2.1-derived GABAergic neurons. Interestingly, in the Nkx2.1-/- mice, the CC midline was totally disorganized and the callosal axons partly lost their orientation, whereas in the Nkx2.1Cre+/Rosa-DTA+/- and the Nkx2.1Cre+/NSE-DTA+/- mice, the axonal organization of the CC was not affected. In the three types of mice, hippocampal axons of the fornix were not properly fasciculated and formed disoriented bundles through the septum. Additionally, the AC formation was completely absent in Nkx2.1-/- mice and the AC was divided into two/three separate paths in the Nkx2.1Cre+/Rosa-DTA+/- mice that project in wrong territories. On the other hand, the AC didn't form or was reduced to a relatively narrower tract in the Nkx2.1Cre+/NSE-DTA+/- mice as compared to wild-type AC. These results clearly indicate that midline Nkx2.1-derived cells play a major role in commissural axons pathfinding and that both Nkx2.1-derived guidepost neurons and glia are necessary elements for the correct development of these commissures. Furthermore, during our investigations on Nkx2.1-/- and Nkx2.1Cre+/Rosa-DTA+/- mice, we noticed similar and severe defects in the erythrocytes distribution and the blood vessels network morphology in the embryonic brain of both mutants. As the Cre-mediated recombination was never observed to occur in the blood vessels of Nkx2.1-Cre mice, we inferred that the vessels defects observed were due to the loss of Nkx2.1-derived cells and not to the cells autonomous effects of Nkx2.1 in regulating endothelial cell precursors. Thereafter, the respective contribution of individual Nkx2.1-regulated neuronal or glial populations in the blood vessels network building were studied with the use of transgenic mice strains. Indeed, the use of Nkx2.1Cre+/NSE-DTA+/- mice indicated that the Nkx2.1-derived neurons were not implicated in this process. Finally, to discriminate between the two Nkx2.1-derived glial cell populations, the GLAST+ astroglia and the NG2+ polydendrocytes, an NG2-Cre mouse strain crossed to the Rosa-DTA+/- mice was used. In that mutant, the blood vessel network and the erythrocytes distribution were similarly affected as observed in Nkx2.1Cre+/Rosa-DTA+/- animals. Therefore, this result indicates that most probably, the NG2+ polydendrocytes are involved in helping to build the vessels network in the brain. Taken altogether, these observations show that during brain development, Nkx2.1- derived embryonic glial cells act as guidepost cells on the guidance of axons as well as forming vessels. Both Nkx2.1-regulated guidepost GABAergic neurons and glia collaborate to guide growing commissural axons, while polydendrocytes are implicated in regulating brain angiogenesis. - Le tissu cérébral est composé de cellules neuronales et gliales générées dans les couches germinales qui bordent les ventricules. Ces cellules se divisent, se différencient et migrent selon des voies particulières. La spécification des interneurones GABAergiques et des neurones glutamatergiques a été largement étudiée, par contre, l'origine, le destin et la fonction des cellules gliales précoces du télencéphale embryonnaire restent peu élucidées. Depuis longtemps, il était communément accepté que les cellules gliales, et plus particulièrement les astrocytes, sont générés après la neurogénèse à partir du télencéphale dorsal. Toutefois, notre travail montre que de nombreuses cellules gliales sont générées à partir de précurseurs ventraux qui expriment le gène Nkx2.1, entre E14.5 et E16.5, c'est-à dire,à des stades embryonnaires très précoces. Le gène NK2 homéobox 1 (Nkx2.1) appartient à une famille de facteurs de transcription appelée NK2. Il s'agit de protéines qui contiennent un homéo-domaine. La spécification des précurseurs de la MGE requiert l'expression du gène homéobox Nkx2.1. De plus, la fonction du gène Nkx2.1 dans la régulation de la spécification des interneurones GABAergiques et des oligodendrocytes dans le télencéphale ventral était déjà connue. Au cours de mon travail de thèse, j'ai également mis en évidence que, Nkx2.1 régule aussi les étapes de prolifération et de différenciation de divers sous-types de cellules gliales soit de type astrocytes ou bien polydendrocytes. L'utilisation d'un anticorps contre la protéine Nkx2.1 ainsi qu'une sonde à ribonucléotides contre l'ARN messager du gène Nkx2.1 ont révélé la présence de nombreuses cellules positives pour Nkx2.1 qui exprimaient des marqueurs astrocytaires (comme GLAST et GFAP) dans le télencéphale embryonnaire. Afin de déterminer de manière sélective le sort des interneurones GABAergiques, des polydendrocytes et des astrocytes dérivés de la MGE, nous avons croisé soit des souris Nkx2.1-Cre, des souris Glast-Cre ERT+/- inductibles ou bien des souris NG2-Cre avec des souris Rosa26-lox-STOP-lox-YFP (Rosa26-YFP) Cre rapportrices. L'origine précise des astroglies positives pour Nkx2.1 a été directement établie en combinant une coloration immunologique pour les glies et une électroporation focale d'un plasmide pCAG-GS-EGFP dans les domaines subpalliaux de tranches organotypiques, puis également, par des cultures de neurosphères in vitro et des expériences d'électroporation in utero d'un plasmide pCAG-GS-tomato dans le pallium ventral d'embryons Nkx2.1-Cre+/Rosa- YFP+/- au stade E14.5. Nous avons donc confirmé que les trois régions germinales du télencéphale ventral, c'est-à-dire, la MGE, l'AEP/POA et le noyau triangulaire septal sont capables de générer des cellules astrogliales. D'autre part, l'immunohistochimie pour plusieurs marqueurs d'astrocytes ou de polydendrocytes, dans les embryons Nkx2.1-/- et contrôles ainsi que dans les neurosphères, a révélé une sévère perte de ces deux types gliaux chez les mutants. Nous avons trouvé que la perte de glies correspondait à une diminution de la capacité de division des précurseurs dérivés de Nkx2.1, ainsi que l'incapacité de ces précurseurs de se différencier en cellules gliales. Nous avons en effet observé une diminution importante des cellules BrdU+ en division exprimant Nkx2.1dans la MGE*, la POA* et le noyau septal* des mutants pour Nkx2.1. D'autre part, nous avons pu mettre en évidence aussi bien in vitro, qu'in vivo, que certains précurseurs Nkx2.1+ chez le mutant gardent la capacité à se différencier en neurones tandis qu'ils perdent celle de se différencier en cellules gliales. Prises dans leur ensemble, ces observations indiquent pour la première fois que le facteur de transcription Nkx2.1 régule les étapes de prolifération et de différentiation des précurseurs des trois domaines subpalliaux qui génèrent les astroglies et polydendrocytes embryonnaires précoces. Par la suite, dans le but de comprendre la fonction potentielle de ces glies précoces, nous avons procédé à de multiples colorations immunohistochimiques sur des animaux Nkx2.1-/- et sauvages, ainsi que sur des souris Nkx2.1-Cre croisées à des souris Rosa-DTA+/- dans lesquelles la toxine diphthérique hautement toxique a permis de supprimer sélectivement la majorité des cellules dérivées de Nkx2.1. De manière intéressante, nous avons observé dans ces deux mutants, une perte drastique et significative de cellules astrogliales GFAP+, GLAST+ et polydendrocytaires NG2+ et S100ß+ dans le télencéphale, à la midline et dans les aires corticales médianes. Ces pertes ont pu être directement corrélées avec des défauts de guidage axonal observés dans le corps calleux (CC), la commissure hippocampique (HIC), le fornix (F) et la commissure antérieure (AC). Le guidage axonal est une étape clé permettant aux neurones de former des connections spécifiques et de s'organiser dans un réseau fonctionnel. La contribution des cellules « guidepost » dans le CC et dans la AC comme médiateurs de la croissance des axones commissuraux à jusqu'à aujourd'hui été attribuée spécifiquement à des astroglies « guidepost » de la midline. Des résultats publiés précédemment dans notre groupe, ont permis de montrer que, pendant le développement embryonnaire, le CC est peuplé en plus de la glie par de nombreux neurones « guidepost » glutamatergiques et GABAergiques qui sont essentiels pour le croisement correct des axones callosaux à la midline. Ainsi, la contribution relative des populations individuelles neuronales ou gliales pour le guidage des axones commissuraux demande à être approfondie afin de mieux comprendre les mécanismes de guidage. A ces fins, nous avons croisé des souris Nkx2.1-Cre avec des souris NSE-DTA+/- qui expriment la toxine diphthérique uniquement dans les neurones et ainsi, nous avons pu sélectivement supprimer les neurones dérivés de domaines Nkx2.1+. Dans les souris Nkx2.1-/-,nous avons découvert que le CC était désorganisé avec des axones callosaux perdant partiellement leur orientation, alors que dans les souris Nkx2.1Cre+/Rosa-DTA+/- et Nkx2.1Cre+/NSE-DTA+/-, l'organisation axonale n'était pas affectée. De plus, les faisceaux hippocampiques du fornix étaient défasciculés dans les trois types de mutants. Par ailleurs, la formation de la commissure antérieure (AC) était complètement absente dans les souris Nkx2.1-/- d'une part, et d'autre part, celle-ci était divisée en deux à trois voies séparées dans les souris Nkx2.1Cre+/Rosa-DTA+/-. Finalement, la AC était soit absente, soit réduite de manière ne former plus qu'un faisceau relativement plus étroit dans les souris Nkx2.1Cre+/NSE-DTA+/- en comparaison avec la AC sauvage. Ces derniers résultats indiquent clairement que les cellules dérivées de Nkx2.1 à la midline, jouent un rôle majeur dans le guidage des axones commissuraux et que, autant les neurones, que les astrocytes « guidepost » dérivés de Nkx2.1, sont des éléments nécessaires au développement correct de ces commissures. En outre, lors de nos investigations sur les souris Nkx2.1-/- et Nkx2.1Cre+/Rosa-DTA+/-, nous avons remarqués des défauts sévères et similaires dans la distribution des erythrocytes et dans la morphologie du réseau de vaisseaux sanguins dans le cerveau embryonnaire des deux mutants précités. Puisque nous n'avons jamais observé de recombinaison de la Cre recombinase dans les vaisseaux sanguins des souris Nkx2.1Cre, nous en avons déduit que les défauts de vaisseaux observés étaient dus à la perte de cellules dérivées de Nkx2.1. Il existerait donc en plus de la fonction cellulaire autonome de Nkx2.1 reconnue pour régulée directement la spécification des cellules endothéliales, une fonction indirecte de Nkx2.1. Afin de déterminer la contribution respective des populations individuelles neuronales ou gliales régulées par Nkx2.1 dans la construction du réseau de vaisseaux sanguins, nous avons utilisé diverses lignées de souris transgéniques. L'utilisation de souris Nkx2.1Cre+/NSE-DTA+/- a indiqué que les neurones dérivés de Nkx2.1 n'étaient pas impliqués dans ce processus. Finalement, afin de discriminer entre les deux populations de cellules gliales dérivées de Nkx2.1, les astroglies et les polydendrocytes, nous avons croisé une lignée de souris NG2-Cre avec des souris Rosa-DTA+/-. Dans ce dernier mutant, le réseau de vaisseaux sanguins du cortex ainsi que la distribution des erythrocytes étaient affectés de la même manière que dans le cortex des souris Nkx2.1Cre+/Rosa-DTA+/-. Par conséquent, ce résultat indique que très probablement, les polydendrocytes NG2+ sont impliqués dans la mise en place du réseau de vaisseaux dans le cerveau. Prises dans leur ensemble, ces observations montrent que durant le développement embryonnaire du cerveau, des sous-populations de glies régulées par Nkx2.1 jouent un rôle de cellules « guidepost » dans le guidage des axones, ainsi que des vaisseaux. Les polydendrocytes sont impliquées dans la régulation de l'angiogenèse tandis que, autant les neurones GABAergiques que les astrocytes collaborent dans le guidage des axones commissuraux en croissance.

Medium-sized neurofilament protein related to maturation of a subset of cortical neurons.

Neurofilaments are typical structures of the neuronal cytoskeleton and participate in the formation and stabilization of the axonal and dendritic architecture. In this study, we have characterized a murine monoclonal antibody, FNP7, that is directed against the medium-sized neurofilament subunit NF-M. This antibody identifies a subset of neurons in the cerebral cortex of various species including human and in organotypic cultures of rat cortex. In the neocortex of all species examined, the antibody labels pyramidal cells in layers III, V, and VI, with a distinctive laminar distribution between architectonic boundaries. In comparison with other antibodies directed against NF-M, the FNP7 antibody identifies on blots two forms of NF-M that appear relatively late during development, at the time when dynamic growth of processes changes to the stabilization of the formed processes. Dephosphorylation with alkaline phosphatase unmasks the site, making it detectable for the FNP7 antibody. The late appearance suggests that the site is present during early development in phosphorylated form and with increasing maturation becomes dephosphorylated, mainly in dendrites. This event may relate to changes in cytoskeleton stability in a late phase of dendritic maturation. Furthermore, mainly corticofugal projections and only few callosal axons are stained, suggesting a differential phosphorylation in a subset of axons. The antibody provides a useful marker to study subsets of pyramidal cells in vivo, in vitro, and under experimental conditions.

Topographic organization of V1 projections through the corpus callosum in humans.

The visual cortex in each hemisphere is linked to the opposite hemisphere by axonal projections that pass through the splenium of the corpus callosum. Visual-callosal connections in humans and macaques are found along the V1/V2 border where the vertical meridian is represented. Here we identify the topography of V1 vertical midline projections through the splenium within six human subjects with normal vision using diffusion-weighted MR imaging and probabilistic diffusion tractography. Tractography seed points within the splenium were classified according to their estimated connectivity profiles to topographic subregions of V1, as defined by functional retinotopic mapping. First, we report a ventral-dorsal mapping within the splenium with fibers from ventral V1 (representing the upper visual field) projecting to the inferior-anterior corner of the splenium and fibers from dorsal V1 (representing the lower visual field) projecting to the superior-posterior end. Second, we also report an eccentricity gradient of projections from foveal-to-peripheral V1 subregions running in the anterior-superior to posterior-inferior direction, orthogonal to the dorsal-ventral mapping. These results confirm and add to a previous diffusion MRI study (Dougherty et al., 2005) which identified a dorsal/ventral mapping of human splenial fibers. These findings yield a more detailed view of the structural organization of the splenium than previously reported and offer new opportunities to study structural plasticity in the visual system.

RAX and anophthalmia in humans: Evidence of brain anomalies.

PURPOSE: To report the clinical and genetic study of two families of Egyptian origin with clinical anophthalmia. To further determine the role of the retina and anterior neural fold homeobox gene (RAX) in anophthalmia and associated cerebral malformations. METHODS: Three patients with clinical anophthalmia and first-degree relatives from two consanguineous families of Egyptian origin underwent full ophthalmologic, general and neurologic examination, and blood tests. Cerebral magnetic resonance imaging (MRI) was performed in the index cases of both families. Genomic DNA was prepared from venous leukocytes, and direct sequencing of all the exons and intron-exon junctions of RAX was performed after PCR amplification. RESULTS: Clinical bilateral anophthalmia was observed in all three patients. General and neurologic examinations were normal; obesity and delay in psychomotor development were observed in the isolated case. Orbital MRI showed a hypoplastic orbit with present but rudimentary extraocular muscles and normal lacrimal glands. Cerebral MRI showed agenesis of the optic nerves, optic tracts, and optic chiasma. In the index case of family A, the absence of the frontal and sphenoidal sinuses was also noted. In the index case of family B, only the sphenoidal sinus was absent, and there was significant cortical atrophy. The three patients carried a novel homozygous c.543+3A>G mutation (IVS2+3A>G) in RAX. Parents were healthy heterozygous carriers. No mutations were detected in orthodenticle homeobox 2 (OTX2), ventral anterior homeobox 1 (VAX1), or sex determining region Y-box 2 (SOX2). CONCLUSIONS: This is the first report of a homozygous splicing RAX mutation associated with autosomal recessive bilateral anophthalmia. To our knowledge, only two isolated cases of anophthalmia, three null and one missense case affecting nuclear localization or the DNA-binding homeodomain, have been found to be caused by compound heterozygote RAX mutations. A novel missense RAX mutation was identified in three patients with bilateral anophthalmia and a distinct systemic and neurologic phenotype. The mutation potentially affects splicing of the last exon and is thought to result in a protein that has an aberrant homeodomain and no paired-tail domain. Functional consequences of this change still need to be characterized.

Difference in distribution of microtubule-associated proteins 5a and 5b during the development of cerebral cortex and corpus callosum in cats: dependence on phosphorylation.

MAP5, a microtubule-associated protein characteristic of differentiating neurons, was studied in the developing visual cortex and corpus callosum of the cat. In juvenile cortical tissue, during the first month after birth, MAP5 is present as a protein doublet of molecular weights of 320 and 300 kDa, defined as MAP5a and MAP5b, respectively. MAP5a is the phosphorylated form. MAP5a decreases two weeks after birth and is no longer detectable at the beginning of the second postnatal month; MAP5b also decreases after the second postnatal week but more slowly and it is still present in the adult. In the corpus callosum only MAP5a is present between birth and the end of the first postnatal month. Afterwards only MAP5b is present but decreases in concentration more than 3-fold towards adulthood. Our immunocytochemical studies show MAP5 in somata, dendrites and axonal processes of cortical neurons. In adult tissue it is very prominent in pyramidal cells of layer V. In the corpus callosum MAP5 is present in axons at all ages. There is strong evidence that MAP5a is located in axons while MAP5b seems restricted to somata and dendrites until P28, but is found in callosal axons from P39 onwards. Biochemical experiments indicate that the state of phosphorylation of MAP5 influences its association with structural components. After high speed centrifugation of early postnatal brain tissue, MAP5a remains with pellet fractions while most MAP5b is soluble. In conclusion, phosphorylation of MAP5 may regulate (1) its intracellular distribution within axons and dendrites, and (2) its ability to interact with other subcellular components.

PAX6 mutations in Egyptian families with aniridia

Methods Ten patients with aniridia from 3 families of Egyptian origin underwent full ophthalmologic, general and neurological examination, and blood drawing. Cerebral MRI was performed in the index case of each family. Genomic DNA was prepared from venous leukocytes and direct sequencing of all the exons and intron-exon junctions of the PAX6 gene was performed after PCR amplification. Results Common features observed in the three families included absence of iris tissue, corneal pannus with different degrees of severity and foveal hypoplasia with severely reduced visual acuity. In families 2 and 3, additional findings such as lens dislocation, lens opacities or polar cataract and glaucoma were observed. We identified two novel (c.170-174delTGGGC [p.L57fs17] and c.475delC [p.R159fs47]) and one known (c.718C>T) PAX6 mutations in the affected members of the 3 families. Systemic and neurological examination was normal in all ten affected patients. Cerebral MRI showed absence of the pineal gland in all three index patients. Severe hypoplasia of the brain anterior commissure was associated to the p.L57fs17mutation, absence of the posterior commissure to both p.R159fs47 and p.R240X, and optic chiasma atrophy and almost complete agenesis of the corpus callosum to p.R240X. Conclusions We identified two novel PAX6 mutations in families with severe aniridia from Northern Egypt, an ethnic group which is not well studied. In addition to common phenotype of aniridia and despite normal neurological examination, absence of the pineal gland was observed in all 3 index patients. The heterogeneity of brain anomalies related to PAX6 mutations is underexplored and is highlighted in this study.

Auditory Neurons with Transitory Axons to Visual Areas Form Short Permanent Projections.

The kitten's auditory cortex (including the first and second auditory fields AI and AII) is known to send transient axons to either ipsi- or contralateral visual areas 17 and 18. By the end of the first postnatal month the transitory axons, but not their neurons of origin, are eliminated. Here we investigated where these neurons project after the elimination of the transitory axon. Eighteen kittens received early (postnatal day (pd) 2 - 5) injections of long lasting retrograde fluorescent traces in visual areas 17 and 18 and late (pd 35 - 64) injections of other retrograde fluorescent tracers in either hemisphere, mostly in areas known to receive projections from AI and AII in the adult cat. The middle ectosylvian gyrus was analysed for double-labelled neurons in the region corresponding approximately to AI and AII. Late injections in the contralateral (to the analysed AI, AII) hemisphere including all of the known auditory areas, as well as some visual and 'association' areas, did not relabel neurons which had had transient projections to either ipsi- or contralateral visual areas 17 - 18. Thus, AI and AII neurons after eliminating their transient juvenile projections to visual areas 17 and 18 do not project to the other hemisphere. In contrast, relabelling was obtained with late injections in several locations in the ipsilateral hemisphere; it was expressed as per cent of the population labelled by the early injections. Few neurons (0 - 2.5%) were relabelled by large injections in the caudal part of the posterior ectosylvian gyrus and the adjacent posterior suprasylvian sulcus (areas DP, P, VP). Multiple injections in the middle ectosylvian gyrus relabelled a considerably larger percentage of neurons (13%). Single small injections in the middle ectosylvian gyrus (areas AI, AII), the caudal part of the anterior ectosylvian gyrus and the rostral part of the posterior ectosylvian gyrus relabelled 3.1 - 7.0% of neurons. These neurons were generally near (<2.0 mm) the outer border of the late injection sites. Neurons with transient projections to ipsi- or contralateral visual areas 17 and 18 were relabelled in similar proportions by late injections at any given location. Thus, AI or AII neurons which send a transitory axon to ipsi- or contralateral visual areas 17 and 18 are most likely to form short permanent cortical connections. In that respect, they are similar to medial area 17 neurons that form transitory callosal axons and short permanent axons to ipsilateral visual areas 17 and 18.

Spectrum of congenital anomalies in pregnancies with pregestational diabetes.

BACKGROUND: Maternal pregestational diabetes is a well-known risk factor for congenital anomalies. This study analyses the spectrum of congenital anomalies associated with maternal diabetes using data from a large European database for the population-based surveillance of congenital anomalies. METHODS: Data from 18 population-based EUROCAT registries of congenital anomalies in 1990-2005. All malformed cases occurring to mothers with pregestational diabetes (diabetes cases) were compared to all malformed cases in the same registry areas to mothers without diabetes (non-diabetes cases). RESULTS: There were 669 diabetes cases and 92,976 non diabetes cases. Odds ratios in diabetes pregnancies relative to non-diabetes pregnancies comparing each EUROCAT subgroup to all other non-chromosomal anomalies combined showed significantly increased odds ratios for neural tube defects (anencephaly and encephalocele, but not spina bifida) and several subgroups of congenital heart defects. Other subgroups with significantly increased odds ratios were anotia, omphalocele and bilateral renal agenesis. Frequency of hip dislocation was significantly lower among diabetes (odds ratio 0.15, 95% CI 0.05-0.39) than non-diabetes cases. Multiple congenital anomalies were present in 13.6 % of diabetes cases and 6.1 % of non-diabetes cases. The odds ratio for caudal regression sequence was very high (26.40,95% CI 8.98-77.64), but only 17% of all caudal regression cases resulted from a pregnancy with pregestational diabetes. CONCLUSIONS: The increased risk of congenital anomalies in pregnancies with pregestational diabetes is related to specific non-chromosomal congenital anomalies and multiple congenital anomalies and not a general increased risk.

Prioritizing genetic testing in patients with kallmann syndrome using clinical phenotypes.

Context: The complexity of genetic testing in Kallmann syndrome (KS) is growing and costly. Thus, it is important to leverage the clinical evaluations of KS patients to prioritize genetic screening. Objective: The objective of the study was to determine which reproductive and nonreproductive phenotypes of KS subjects have implications for specific gene mutations. Subjects: Two hundred nineteen KS patients were studied: 151 with identified rare sequence variants (RSVs) in 8 genes known to cause KS (KAL1, NELF, CHD7, HS6ST1, FGF8/FGFR1, or PROK2/PROKR2) and 68 KS subjects who remain RSV negative for all 8 genes. Main Outcome Measures: Reproductive and nonreproductive phenotypes within each genetic group were measured. Results: Male KS subjects with KAL1 RSVs displayed the most severe reproductive phenotype with testicular volumes (TVs) at presentation of 1.5 ± 0.1 mL vs 3.7 ± 0.3 mL, P < .05 vs all non-KAL1 probands. In both sexes, synkinesia was enriched but not unique to patients with KAL1 RSVs compared with KAL1-negative probands (43% vs 12%; P < .05). Similarly, dental agenesis and digital bone abnormalities were enriched in patients with RSVs in the FGF8/FGFR1 signaling pathway compared with all other gene groups combined (39% vs 4% and 23% vs 0%; P < .05, respectively). Hearing loss marked the probands with CHD7 RSVs (40% vs 13% in non-CHD7 probands; P < .05). Renal agenesis and cleft lip/palate did not emerge as statistically significant phenotypic predictors. Conclusions: Certain clinical features in men and women are highly associated with genetic causes of KS. Synkinesia (KAL1), dental agenesis (FGF8/FGFR1), digital bony abnormalities (FGF8/FGFR1), and hearing loss (CHD7) can be useful for prioritizing genetic screening.

Holt Oram syndrome: a registry-based study in Europe.

BACKGROUND: Holt-Oram syndrome (HOS) is an autosomal dominant disorder characterised by upper limb anomalies and congenital heart defects. We present epidemiological and clinical aspects of HOS patients using data from EUROCAT (European Surveillance of Congenital Anomalies) registries. METHODS: The study was based on data collected during 1990-2011 by 34 registries. The registries are population-based and use multiple sources of information to collect data on all types of birth using standardized definitions, methodology and coding. Diagnostic criteria for inclusion in the study were the presence of radial ray abnormalities and congenital heart disease (CHD), or the presence of either radial ray anomaly or CHD, with family history of HOS. RESULTS: A total of 73 cases of HOS were identified, including 11 (15.1%) TOPFA and 62 (84.9%) LB. Out of 73 HOS cases, 30.8% (20/65) were suspected prenatally, 55.4% (36/65) at birth, 10.7% (7/65) in the first week of life, and 3.1% (2/65) in the first year of life. The prenatal detection rate was 39.2% (20/51), with no significant change over the study period. In 55% (11/20) of prenatally detected cases, parents decided to terminate pregnancy. Thumb anomalies were reported in all cases. Agenesis/hypoplasia of radius was present in 49.2% (30/61), ulnar aplasia/hypoplasia in 24.6% (15/61) and humerus hypoplasia/phocomelia in 42.6% (26/61) of patients. Congenital heart defects (CHD) were recorded in 78.7% (48/61) of patients. Isolated septal defects were present in 54.2 (26/48), while 25% (12/48) of patients had complex/severe CHD. The mean prevalence of HOS diagnosed prenatally or in the early years of life in European registries was 0.7 per 100,000 births or 1:135,615 births. CONCLUSIONS: HOS is a rare genetic condition showing regional variation in its prevalence. It is often missed prenatally, in spite of the existence of major structural anomalies. When discovered, parents in 45% (9/20) of cases opt for the continuation of pregnancy. Although a quarter of patients have severe CHD, the overall first week survival is very good, which is important information for counselling purposes.

Interhemispheric visual integration in humans explored by multimodal brain imaging

Résumé: Les récents progrès techniques de l'imagerie cérébrale non invasives ont permis d'améliorer la compréhension des différents systèmes fonctionnels cérébraux. Les approches multimodales sont devenues indispensables en recherche, afin d'étudier dans sa globalité les différentes caractéristiques de l'activité neuronale qui sont à la base du fonctionnement cérébral. Dans cette étude combinée d'imagerie par résonance magnétique fonctionnelle (IRMf) et d'électroencéphalographie (EEG), nous avons exploité le potentiel de chacune d'elles, soit respectivement la résolution spatiale et temporelle élevée. Les processus cognitifs, de perception et de mouvement nécessitent le recrutement d'ensembles neuronaux. Dans la première partie de cette thèse nous étudions, grâce à la combinaison des techniques IRMf et EEG, la réponse des aires visuelles lors d'une stimulation qui demande le regroupement d'éléments cohérents appartenant aux deux hémi-champs visuels pour en faire une seule image. Nous utilisons une mesure de synchronisation (EEG de cohérence) comme quantification de l'intégration spatiale inter-hémisphérique et la réponse BOLD (Blood Oxygenation Level Dependent) pour évaluer l'activité cérébrale qui en résulte. L'augmentation de la cohérence de l'EEG dans la bande beta-gamma mesurée au niveau des électrodes occipitales et sa corrélation linéaire avec la réponse BOLD dans les aires de VP/V4, reflète et visualise un ensemble neuronal synchronisé qui est vraisemblablement impliqué dans le regroupement spatial visuel. Ces résultats nous ont permis d'étendre la recherche à l'étude de l'impact que le contenu en fréquence des stimuli a sur la synchronisation. Avec la même approche, nous avons donc identifié les réseaux qui montrent une sensibilité différente à l'intégration des caractéristiques globales ou détaillées des images. En particulier, les données montrent que l'implication des réseaux visuels ventral et dorsal est modulée par le contenu en fréquence des stimuli. Dans la deuxième partie nous avons a testé l'hypothèse que l'augmentation de l'activité cérébrale pendant le processus de regroupement inter-hémisphérique dépend de l'activité des axones calleux qui relient les aires visuelles. Comme le Corps Calleux présente une maturation progressive pendant les deux premières décennies, nous avons analysé le développement de la fonction d'intégration spatiale chez des enfants âgés de 7 à 13 ans et le rôle de la myelinisation des fibres calleuses dans la maturation de l'activité visuelle. Nous avons combiné l'IRMf et la technique de MTI (Magnetization Transfer Imaging) afin de suivre les signes de maturation cérébrale respectivement sous l'aspect fonctionnel et morphologique (myelinisation). Chez lés enfants, les activations associées au processus d'intégration entre les hémi-champs visuels sont, comme chez l'adulte, localisées dans le réseau ventral mais se limitent à une zone plus restreinte. La forte corrélation que le signal BOLD montre avec la myelinisation des fibres du splenium est le signe de la dépendance entre la maturation des fonctions visuelles de haut niveau et celle des connections cortico-corticales. Abstract: Recent advances in non-invasive brain imaging allow the visualization of the different aspects of complex brain dynamics. The approaches based on a combination of imaging techniques facilitate the investigation and the link of multiple aspects of information processing. They are getting a leading tool for understanding the neural basis of various brain functions. Perception, motion, and cognition involve the formation of cooperative neuronal assemblies distributed over the cerebral cortex. In this research, we explore the characteristics of interhemispheric assemblies in the visual brain by taking advantage of the complementary characteristics provided by EEG (electroencephalography) and fMRI (Functional Magnetic Resonance Imaging) techniques. These are the high temporal resolution for EEG and high spatial resolution for fMRI. In the first part of this thesis we investigate the response of the visual areas to the interhemispheric perceptual grouping task. We use EEG coherence as a measure of synchronization and BOLD (Blood Oxygenar tion Level Dependent) response as a measure of the related brain activation. The increase of the interhemispheric EEG coherence restricted to the occipital electrodes and to the EEG beta band and its linear relation to the BOLD responses in VP/V4 area points to a trans-hemispheric synchronous neuronal assembly involved in early perceptual grouping. This result encouraged us to explore the formation of synchronous trans-hemispheric networks induced by the stimuli of various spatial frequencies with this multimodal approach. We have found the involvement of ventral and medio-dorsal visual networks modulated by the spatial frequency content of the stimulus. Thus, based on the combination of EEG coherence and fMRI BOLD data, we have identified visual networks with different sensitivity to integrating low vs. high spatial frequencies. In the second part of this work we test the hypothesis that the increase of brain activity during perceptual grouping depends on the activity of callosal axons interconnecting the visual areas that are involved. To this end, in children of 7-13 years, we investigated functional (functional activation with fMRI) and morphological (myelination of the corpus callosum with Magnetization Transfer Imaging (MTI)) aspects of spatial integration. In children, the activation associated with the spatial integration across visual fields was localized in visual ventral stream and limited to a part of the area activated in adults. The strong correlation between individual BOLD responses in .this area and the myelination of the splenial system of fibers points to myelination as a significant factor in the development of the spatial integration ability.