Proteomic analysis of cerebellar radial glia/astrocyte differentiation by comparative differential fluorescence in-gel electrophoresis

Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603

541

[P106]

[P107]

Development of the serotonergic system in the central nervous system of the sea lamprey

Anatomical narrowing without categorical specialization: Developmental changes in brain activity during face and object processing in 5–11 year olds and adults

X. Abalo ∗ , B. Villar-Cheda, A. Barreiro-Iglesias, M. Mel´endez-Ferro, E. P´erez-Costas, R. Anad´on, C. Rodicio

A.D. Gathers ∗ , R.S. Bhatt, C.R. Corbly, A.B. Farley, J.E. Joseph

University of Santiago de Compostela, Spain

University of Kentucky, USA

Lampreys belong to the Agnathans, the sister group of jawed vertebrates. Accordingly, characterization of neuronal groups and their development may provide useful information for understanding early evolution of the vertebrate nervous system. Here, the development of the CNS serotonergic system of the sea lamprey from embryos to adults was investigated immunocytochemically with an antibody against serotonin. The appearance of the different serotonin-immunoreactive neuronal populations occurs between the embryonic and metamorphic stages. The earliest serotonergic neurons appear in the basal plate of the isthmus of late embryos. In prolarvae, there appear progressively new serotonergic cell groups: first in the spinal cord, followed by those of the pineal organ, tuberal region, zona limitans intrathalamica, and then in the caudal rhombencephalon. In early larvae, a new serotonergic population appears in the mammillary region, whereas serotonergic cells of the pretectal region and parapineal organ first appear in middle and late larval stages, respectively. The first serotonergic fibers grow in early prolarvae, ascending and descending from the isthmic cell group, and the number of immunoreactive fibers progressively increases until the adult period. During metamorphosis, serotonin-immunoreactive cells appear in the retina. Comparison of the spatiotemporal sequence of appearance of serotonergic cells in agnathans and gnathostomes stresses the evolutionary trend for disappearance of rostral serotonergic cell groups in tetrapods, and for increase of differentiated groups in the brainstem. It also reveals that the developmental pattern of early ascending and descending serotonergic pathways is remarkably similar among vertebrates. Some striking differences between development of serotonergic structures in lampreys and other vertebrates are in close relation to the peculiar development of the lamprey visual system.

Because of its importance and complexity, visual processing has been the subject of many adult functional imaging studies; however, relatively little is known about the development of the neural organization of visual processing. The current project used functional magnetic resonance imaging (fMRI) to identify maturational changes in neural substrates of face and object recognition in 5–8 years old, 9–11 years old and adults. From fMRI data collected while subjects passively viewed grayscale photos of faces, manufactured objects and natural objects, we conducted comprehensive quantitative and qualitative analyses of extent of activation, signal magnitude and hemispheric lateralization. Results showed that the extent of activation decreased with age as children recruited a greater number of spatially scattered regions than adults. Signal strength did not change systematically across age groups. However, signal strength showed regional age-related changes with decreases in frontal regions and increases in parietal and ventral regions with age. Analysis of lateralization also revealed developmental changes. Children produced primarily bilateral activity whereas adult brain activity was lateralized to the right hemisphere. None of these developmental changes were category-specific. This functional honing of activation with development corresponds with underlying neuroanatomical changes and may reflect experience with the process or task.

Acknowledgements Supported by MCYT (BMC2000-0283) and Xunta de Galicia (PGIDT01PX120006PN). Keywords: Sea lamprey; Serotonin; Development; Immunohistochemistry doi:10.1016/j.ijdevneu.2006.09.168

Keywords: fMRI; Visual processing; Extent of acdtivation; Signal magnitude doi:10.1016/j.ijdevneu.2006.09.169 [P108] Proteomic analysis of cerebellar radial glia/astrocyte differentiation by comparative differential fluorescence in-gel electrophoresis P. Leprince ∗ , J. Joris, A. Courtois, F. Guillonneau, E. Depauw, B. Rogister University of Li`ege, Belgium Radial glial cells function during central nervous system development as both a scaffolding along which postmitotic neurons migrate and as neuronal and glial progenitors. However, even when they generate neurons, radial glial cells show some typical features of astrocytes. To investigate how radial glial cells and mature astrocytes really differ, we have compared some of their properties in cultures of each cell type. Radial

542

Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603

glial cells are obtained from E16 cerebellum and, when grown in the presence of FGF and EGF, assume a bipolar morphology and maintain a very immature phenotype, expressing typical progenitors markers, such as Nestin, RC2, Glast and BLBP. Astrocytes are obtained from P3 cerebellum at a time when all radial glial cells have translocated to the Bergmann glia formation. Those cells grow slowly, express GFAP and assume a flat epithelioid morphology. A comparison of the proteomes of the two cell types has been realized using the 2D-DIGE technology, which allows a very precise and sensitive determination of differential protein expression. Statistical analysis of the protein spots distribution and abundance revealed 222 and 128 protein spots that were increased >1.5-fold in radial glia and astrocytes, respectively. After tryptic digestion and Maldi-TOF sequencing, 88 and 60 unique proteins were identified as being expressed between 1.5- and 29-fold more in radial glia and astrocytes respectively. Radial glial cells express in particular a large array of chaperones, including 7 forms of the T-complex protein 1, 17 different cytoskeletal proteins including 4 dihydropyrimidinaserelated proteins and 4 tubulin chains, several enzymes of lipid metabolism, signaling molecules, such as FABP and Immunophilins, several AA-tRNA synthase and 8 different splicing-involved hnRNPs. Astrocytes generally express more mature forms of chaperones and cytoskeletal proteins, such as Actin, GFAP, Moesin, Vimentin and Vinculin, enzymes of intermediary metabolism, Peroxiredoxins and 5 forms of Annexins. Information from this study will be useful to uncover the molecular mechanisms of astroglial differentiation and maturation. Keywords: Proteomic; Differentiation; Astrocyte; DIGE doi:10.1016/j.ijdevneu.2006.09.170 [P109] Neural progenitor cells are impaired in extremely preterm infants with ischemic brain injuries K. Deguchi 1,∗ , S. Takashima 3 , D. Armstrong 4 , K. Inoue 2 1 Deguchi Pediatric Clinic, Japan; 2 National Center of Neurol-

ogy & Psychiatry, Japan; 3 International University of Health & Welfare, Japan; 4 Baylor College of Medicine, USA Extremely preterm infants (EPIs), birth less than 28 weeks of gestation is associated with cognitive and neurological impairment, however, its exact pathophysiology remains unknown. Brain pathology of MRI in EPIs shows loss of brain tissue with periventricular white matter injury (PVWI) and ventricular zone irregularity. At the time of birth for EPIs, neurogenesis and gliogenesis are still in process, and proliferating neural progenitor and precursor cells are present in ventricular and subventricular zones (VZ/SVZ). We thus hypothesized that brain injury in EPIs may disrupt VZ/SVZ and concomitantly affect neural progenitor and precursor cells, thereby interrupting subsequent brain development and contributing to cortical dysfunction. To test this hypothesis, we neuropathologically examined a

series of 45 autopsy brains from EPI with PVWI in comparison with a developmental series of 40 control brains. We observed prominent disorganization in the VZ/SVZ and reduction of neural progenitor and precursor cells expressing neural stem cell markers, Musashi1 and Nestin, in all EPI brains with PVWI. Residual neural progenitors in the VZ/SVZ of short-term survivors revealed TUNEL and caspase3-reactivity, suggesting an involvement of apoptosis in the neural progenitor injury. Accordingly, an examination in long-term survivors showed a complete loss of postnatal neural progenitor cell lineage, i.e., ependymal and adult neural stem cells. Together, our observations suggest that the brain injuries to the neural progenitors and precursors in VZ/SVZ are common in EPIs with PVWI and may be associated with delayed cortical development, which possibly contribute to cognitive dysfunction in EPIs. Keywords: Neural progenitor cells; Ischemia; Immature human brain; Periventricular white matter injury doi:10.1016/j.ijdevneu.2006.09.171 [P110] Identification of genes necessary for the development of cranial motor neurons G.S. Walsh 1,∗ , A. Carmany-Rampey 1 , P.K. Grant 1 , A. Wark 1 , L. Margaretha 1 , C. Moens 2 1 Fred Hutchinson Cancer Research Center, USA; 2 HHMI and

Fred Hutchinson Cancer Research Center, USA Despite the complexity of the vertebrate central nervous system (CNS), considerable progress is being made in elucidating the mechanisms that transform the neural plate into the complex network of cells that constitutes the functional circuitry of the brain. We have undertaken a mutagenesis-based forward genetic screen using the isl1-green fluorescent protein (GFP) transgenic line of zebrafish, which express GFP in a subset of cranial neurons, including hindbrain branchiomotor neurons. This line affords the possibility of screening for new mutations that affect hindbrain segmentation, neuronal differentiation, neuronal migration and axonal pathfinding. To date, we have found 28 mutant lines that exhibit defects in some aspect of branchiomotor neuron development. By far, the largest group consists of mutants that display a defect in the tangential caudal migration of facial branchiomotor neurons (FBM). FBM neurons in wild-type isl1-GFP transgenic embryos migrate posteriorly from rhombomere (r)4 to establish clusters in r6 and r7, leaving behind them axons that exit from lateral r4 to extend into the second branchial arch and innervate jaw support muscles. Mutants with defects in FBM neuron migration fall into several classes, including those with full block (i.e., FBM neurons are completely retained in r4), a partial block (i.e., FBM neurons are partially retained in r4 with scattered neurons migrating to r5 and r6), and those that display a defect in the final lateral migration of FBM neurons in r6. The majority of mutant lines with an impairment in FBM migration have no other gross mor-