Oral Abstracts

Int. J. Devl Neuroscience 22 (2004) 601–632 www.elsevier.com/locate/ijdevneu

Oral Abstracts Plenary Lecture [O1] Forebrain patterning—a new perspective A. Lumsden King’s College London, UK The formation of cell-tight compartments and their maintenance by lineage restriction boundaries has long been recognised as a key feature of insect development. The more important functions of compartment boundaries include maintaining the position of local signalling centres and the prevention of cell mingling, which might disrupt emergent spatial organization in an otherwise homogeneous tissue. More recently, the existence of compartments has been recognized also in the vertebrates, specifically in the hindbrain region of the CNS, although neither of the above functions for compartment boundaries has yet been satisfactorily demonstrated for rhombomere boundaries, nor have the cell segregation mechanisms involved in their maintenance been fully elucidated. It has been suggested that the forebrain also is a segmented region of the neuraxis, with a compartmental organisation of prosomeres analogous to the rhombomeres. However, our data on molecular marker expression and the dispersal of labelled cell clones in chick are not in agreement with the ‘prosomere model’. Nevertheless, one of the putative interprosomeric boundaries, the zona limitans intrathalamica (zli), itself constitutes a bone fide compartment that may also have an important local signalling function. The zli forms at the border between epichordal and prechordal regions. Lineage labelling reveals that the zli arises from a wedge-shaped region, narrow ventrally, broad dorsally and amounting to around one-third of the entire prosencephalon. This extensive region narrows to a thin stripe of cells later in development. From the first appearance of the wedge region at HH stage 14 though later stages of the definitive zli, both anterior and posterior borders of the structure are lineage restriction boundaries. The zli is thus not a simple boundary but an individual 0736-5748/$30.00 # 2004 Published by Elsevier Ltd on behalf of ISDN. doi:10.1016/j.ijdevneu.2004.12.004

compartment in its own right. It is also the only compartment so far detected in the avian embryo forebrain. Gene expression analysis at sequential stages of chick development reveals that L-fng is expressed throughout the forebrain except in the wedge of cells that is the zli precursor. Wnt8b is expressed in the wedge, with Wnt8b and L-fng domains being mutually exclusive. Both anterior and posterior borders of the wedge correspond with interfaces between fringe-positive and fringe-negative cells. Ectopic expression of L-fng throughout the wedge prevents the formation of a zli. The role of L-fng and Notch signalling in zli formation will be discussed. Once the definitive zli has narrowed to a stripe of cells, the signal molecule Sonic hedgehog (SHH) is expressed strongly within it. The effects of interfering with SHH signalling from the zli and of over-expressing Shh in neighbouring thalamic progenitor regions will be described. Symposium 1: Cell Fate Determination in the CNS [O2] Cell fate specification in the mouse telencephalon F. Guillemot1*, O. Britz1, C. Parras1, C. Schuurmans2 1

National Institute for Medical Research, UK; 2University of Calgary, Canada E-mail address: [email protected] (F. Guillemot). The mammalian telencephalon is the part of the brain most concerned with thoughts, emotions and voluntary movements. It is also by far the most complex region of the brain, with the two major components of the telencephalon, the cerebral cortex and basal ganglia, having very distinct architectures, connectivity, and cellular composition. Despite this complexity, the telencephalon is a good model system to study how a neural structure is generated through cellular diversification and morphogenesis, owing to the relatively simple organization of the telencephalic primordium and the extensive analysis of the properties of its stem cells and progenitors. To begin addressing the mechanisms underlying cell fate specification in the telencephalon, we have focused on the

602

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

role of transcription factors of the proneural bHLH family. These factors, which include Mash1 and Neurogenins, play essential roles in neurogenesis, by committing multipotent progenitors to the neuronal fate, promoting cell cycle exit and initiating cell differentiation. Interestingly, Mash1 and Neurogenins are mostly expressed in distinct populations of telencephalic progenitors giving rise to different types of neurons, suggesting that these proteins may be involved in the diversification of cell fates. I will present evidence that proneural proteins are indeed required for the specification of multiple features of telencephalic neurons. In particular, the analysis of Neurogenin mutant mice demonstrates that these genes coordinately specify the regional (cortical) character, neurotransmitter (glutamatergic) phenotype and temporal (laminar) identity of a subset of cortical neurons. In parallel, Neurogenins repress an alternative, Mash1-dependent, subcortical GABAergic neuronal phenotype. Thus, proneural genes control a binary switch between the two major neuronal identities of the telencephalon, indicating that they play an essential role in generating neuronal diversity in this part of the brain. Keywords: Neuronal subtypes; Neocortex; Neurotransmitter phenotype; Proneural proteins [O3] Glial cells generate neurons: cellular and molecular mechanisms of neurogenesis M. Go¨ tz GSF—National Research Center for Environment and Health, Germany During development and in adulthood, neurons arise from multipotent precursors by hierarchical fate restriction. Thus, multipotent precursors do not directly give rise to neurons but first generate intermediate sets of precursors restricted to the generation of neurons only. The molecular cues regulating this transition from a multipotent to a neuronal precursor are not well understood. To elucidate these fate determinants we used fluorescent-activated cell sorting to separate neuronal from multipotent precursors for differential expression analysis. I will discuss the functional analysis of several transcription factors involved in multipotent or neuronal fate specificiation. For example, the transcription factor Pax6 is strongly enriched in neuronal precursors, both in the developing forebrain as well as in neuronal precursors of the neurogenic zones in the adult forebrain. Interestingly, prior to neurogenesis multipotent precursors up-regulate a splice variant of Pax6 exhibiting a different DNA-binding activity than the form of Pax6 that predominates in neuronal precursors. Gain-of-function experiments confirm that Pax6 without the splice insert acts as major neurogenic determinant that is sufficient to drive not only almost all adult neural stem cells towards neurogenesis, but also is

sufficient to instruct astrocytes from non-neurogenic brain regions towards neurogenesis in vitro and in vivo. We could further demonstrate that the splice variant of Pax6 affects proliferation, most likely instructing asymmetric cell division, but fails to instruct neurogenic fate. Thus, the targets of the unspliced paired domain of Pax6 are crucial for the potent neurogenic activity of Pax6. Taken together, these results raise considerable hope for reconstitution of neurons from endogenous astrocytes in the affected brain regions. [O4] Generation of temporal identity in the Drosophila CNS B. Pearson, R. Grosskortenhaus, A. Marusich, C.Q. Doe* University of Oregon, USA E-mail address: [email protected] (C.Q. Doe). The CNS of higher organisms exhibits extraordinary cellular diversity, due to complex spatial and temporal patterning mechanisms. The role of spatial patterning in generating neuronal diversity is well known; here I will discuss how neural progenitors change over time to contribute to cell diversity. The embryonic Drosophila CNS develops from progenitors called neuroblasts. Each neuroblast forms at a specific time and position, expresses characteristic molecular markers, and generates a stereotyped cell lineage. All neuroblasts divide asymmetrically to ‘bud off’ a series of smaller daughter cells into the embryo, called ganglion mother cells (GMC-1, GMC-2, etc. based on their birth-order), which differentiate into a characteristic pair of neurons or glia. What determines the fate of each successive GMC, i.e. its temporal identity— within each neuroblast lineage? We previously showed that nearly all Drosophila neuroblasts in each segment sequentially express the transcription factors Hunchback ! Kru¨ ppel ! Pdm ! Castor. Here I will focus on the zinc finger protein Hunchback, which is necessary and sufficient to specify early-born temporal identity. Hunchback function is sufficient in mitotic progenitors to confer heritable early-born fates, but post-mitotic neurons are not competent to respond to Hunchback. Progressively older neuroblasts show a gradual restriction in their ability to generate early-born cell fates in response to Hunchback. I will also discuss the mechanism of the ‘‘clock’’ that regulates the successive expression of different temporal identity genes within single neuroblasts. We have shown that the gene expression sequence occurs normally in single neuroblasts cultured in vitro, that these transitions in gene expression are regulated at the transcriptional level, and that at least the first step in this pathway requires neuroblast cytokinesis to advance (i.e., neuroblasts that undergo DNA replication but not cytokinesis remain Hunchback-positive and never express later genes). Keywords: Drosophila; Hunchback; Neuroblast; Progressive restriction; Cell fate; Cytokinesis

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

603

Symposium 2: Cognitive Dysfunction of Aging

[O7]

[O6]

Characterization of nuclear factor kb binding site in the rat ChAT and BACE1 promoters

Transcriptional events involved in neurogenesis are recapitulated after stroke and implicated in neuroplasticity T.A. Kent1, M.E. DeBakey2 1

Baylor College of Medicine, USA; 2VAMC, USA

Cortical remodeling appears to be a basis for recovery following a variety of brain insults and is a process influenced by aging. We have documented widespread changes in expression of synaptic and neurite-related proteins after experimental stroke. The pre-synaptic protein, synaptophysin, increases dramatically in the peri-infarct region, homotopic contralateral cortex, commissural fibers and relay nuclei, suggesting that activitydependent signals are involved in these changes. Interestingly, the superoxide dismutases (SODs) also demonstrate widespread increases in similar regions. Given the involvement of CCAAT-enhancer binding proteins (C/ EBPs) in the transcriptional response of SOD to various signals, we examined the expression pattern of C/EBP family members following photothrombotic stroke in adult rats. Multi-labeled immunostaining of brain slices and Western blot analysis of nuclear extracts revealed that the 40 Kd and 35 Kd isoforms of C/EBP b increased within 24 h in cortical pyramidal neurons, both peri-infarct and in the homotopic cortex. Isoform-specific C/EBP b constructs under control of a CMV promoter were generated and overexpressed in differentiating PC12 cells. Both isoforms significantly increased neurite extension compared to empty vector or untransfected cells. Interestingly, C/EBP b is essential for growth factor-driven cortical neurogenesis, suggesting an important role for this protein in both development as well as response to injury. The interaction of aging, C/EBP functional activity, and potential for recovery will be discussed from a mechanistic perspective.

K. Bourne1, S. Roßner2, T. Toliver-Kinsky1, J. Regino PerezPolo1 1

University of Texas Medical Branch, USA; 2Paul Flechsig Institute for Brain Research, Germany The aged nervous system displays both impaired cognitive functions and recovery from challenges to its ability to maintain homeostasis. Our hypothesis is that agingassociated chronic oxidative stress serves to alter the dynamic ability for the nervous system to respond to acute insults. The transcription factor NF-kB has been shown to regulate transcription of many genes that play a role in inflammation and recovery from acute or chronic trauma. The aged-associated changes in NF-kB action may account for some changes in cognitive function. Choline acetyltransferase (ChAT), the rate-limiting enzyme in the synthesis of acetylcholine, is expressed in the basal forebrain cholinergic neurons that are important for memory and cognition. Significant reductions in ChAT activity are frequently associated with aging and Alzheimer’s disease. Significant increases in transcription factor nuclear factor kappa B (NF-kB) activity have been detected in brains of aged animals and Alzheimer’s disease patients. The ChAT promoter contains a potential binding site for NF-kB within a region that responds to nerve growth factor as an enhancer element. We tested the hypothesis that NF-kB is a negative regulator of the mouse choline acetyltransferase promoter. Our data indicate that NF-kB is a negative regulator of nerve growth factor-induced expression of the mouse ChAT gene that may contribute to age-associated decreases in cholinergic function. The pathogenic processing of the amyloid precursor protein (APP) into the bamyloid protein gives rise to b-amyloid plaques in the brains of Alzheimer’s disease patients and requires the enzymatic activity of the beta-site APP-cleaving enzyme 1 (BACE1). Our data supports the hypothesis that NF-kB is involved in BACE1 down-regulation. Supported in part by SHC grant 8710 and an NIEHS Training Grant Award to KB.

References [O8] Bolanos, S.H., Saito, H., Papaconstantinou, J., High, K.W., Perez-Polo, J.R., Kent, T.A., 2003. Differential C/EBP expression after stroke. Mol. Biol. Cell 13 (Suppl.), 394a. Menard, C., Hein, P., Paquin, A., et al., 2002. An essential role for a MEK-C/EBP pathway during growth factor regulated cortical neurogenesis. Neuron 36, 597–610. Stroemer, R.P., Kent, T.A., Hulsebosch, C.E., 1995. Neocortical neural sprouting and synaptogenesis and behavioral recovery after neocortical infarction in rats. Stroke 26, 2135–2144.

Multiple effects of fruits and vegetables in neuronal and behavioral aging J.A. Joseph, B. Shukitt-Hale Tufts University, USA Research from our laboratory has suggested that dietary supplementation with fruit or vegetable extracts high in antioxidants (e.g., blueberry, BB and spinach) can decrease the enhanced vulnerability to oxidative stress (OS) that

604

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

occurs in aging and these reductions are expressed as improvements in behavior. It also appears that there are additional multiple mechanisms involved in the beneficial effects observed from these supplementations including: enhancement of neuronal communication (i.e., signaling and neurogenesis). Recent work shows that BB supplementation from 4 to 12 months of age (APP/PS1) mice prevents behavioral deficits by increasing extracellular signal regulated kinase (ERK) and protein kinase C (PKC), two important signaling factors in learning and memory. Findings also indicate that one of the most striking effects of BB supplementation may involve increases in neurogenesis. The results showed that aged BB-supplemented rats, tested in the radial arm water maze (RAWM) showed a greater number of dentate gyrus—BrdU-labeled cells than control animals which were inversely correlated with the number of errors in the RAWM performance (i.e., the greater the number of BrdU-labeled cells, the fewer the errors) (e.g., reference memory errors: r = 0.654; p < .05). A subsequent study showed that errors on a Morris water maze were inversely correlated with the number of the anthocyanins localized in the cortex in the BB-supplemented animals (r = 0.74). Additional studies, suggest that hippocampal ERK and PKC levels are increased in both young and old BB-supplemented rats and that these increases are correlated significantly with both RAWM and motor behavioral performance in both old and young animals. Taken together, these findings, suggest that antioxidant-rich fruits such as BBs may improve cognitive and motor function by enhancing neuronal signaling and ultimately, neuronal communication. They further suggest that the antioxidant/anti-inflammatory effects of the berryfruit polyphenols may only represent a small aspect of their beneficial effects.

kinase C, a DAG/PE receptor, regulates APP processing. However, in addition to PKC there are DAG/PE receptors present in neurons, which may participate in the modulation of APP processing. Munc13-1, a presynaptic protein with an essential role in synaptic vesicle priming, represents such a potential target of the DAG second messenger pathway. Using Munc13-1 knock-out mice and knock-in mice with a Munc13-1(H567K) variant deficient in DAG/PE binding, we determined the relative contributions of PKC and Munc13-1 to PE-stimulated secretory APP processing. We report a reduction in basal b-secretase enzymatic activity and in the concentration of secretory APP fragments in brains of Munc13-1 knock-out mice. PE-stimulated b-secretase activity is compromised in brain homogenate of both Munc13-1 knock out mice and knock-in mice, while bsecretase enzymatic activity was unaltered as compared to wild-type mice. In organotypic brain slice cultures from Munc13-1 knock-out and Munc13-1 H567K knock-in mice, the PE-evoked APP-secretion appeared to be Munc13-1dependent. Additionally, the PE-stimulated APP secretion from human BE-2(C) cells was twice as high after transfection of Munc13-1 but not of dysfunctional Munc13-1 (H567K) constructs. Our data identify Munc13-1 as a novel regulator of PE-stimulated secretory APP processing and argue against an exclusive role of PKC in this pathway.

Keywords: Aging; Nutrition; Signaling; Neurogenesis behavior

Generation of regional and structural identity in the developing mammalian forebrain

[O9]

J. Corbin*

Secretory APP processing is dependent on Munc13-1mediated vesicle priming S. Rossner1*, K. Fuchsbrunner1, C. Lange-Dohna1, M. Hartlage-Ru¨ bsamen1, V. Bigl1, A. Betz2, K. Reim2, N. Brose2 Universita¨ t Leipzig, Germany; 2Max Planck Institut fu¨ r Experimentelle Medizin, Germany

1

E-mail address: [email protected] (S. Rossner). The amyloid precursor protein (APP) may give rise to bamyloid peptides, which are the main constituents of senile plaques in brains of Alzheimer’s disease patients. Nonamyloidogenic processing of the APP can be stimulated by phorbol esters (PEs) and by intracellular diacylglycerol (DAG) generation. This led to the hypothesis that protein

Keywords: Amyloid precursor protein; Synaptic vesicle priming; Phorbol ester; Alzheimer’s disease Symposium 3: Progenitor Cell Migration into the Embryonic Telencephalon [O10]

Georgetown University Medical Center, USA In the embryonic mammalian telencephalon different cell populations are specified within distinct progenitor zones. As a means to generate regional cellular heterogeneity within mature brain structures, many of these cells undergo diverse routes of long-range tangential migration. A major source of these migrating cells is the ventral ganglionic eminences (MGE, LGE and CGE). Recent studies have revealed that each of these progenitor zones is distinct and contributes cells to unique mature brain structures. Preliminary data also indicates that these, and other unique progenitor zones, may also give rise to distinct subsets of interneurons that are widely dispersed throughout the mature brain. Keywords: Cell migration; Interneurons; Telencephalon; Progenitor

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

[O11] Patterns of neurogenesis and migration in the developing neocortex S.C. Noctor, V. Martı´nez-Cerden˜ o, L. Ivic, A.R. Kriegstein* Columbia University Medical Center, USA E-mail address: [email protected] (A.R. Kriegstein). The cerebral cortex is composed of carefully regulated numbers of excitatory and inhibitory neurons. These two principle cell types arise in different proliferative regions of the embryonic dorsal and ventral telencephalon, and subsequently migrate considerable distances to converge in specific cortical layers where they form the characteristic intracortical circuits of the neocortex. Establishment of cortical function thus depends on precise patterns of cell division and neuronal migration within distinct cortical and subcortical proliferative regions. We have examined the patterns of neurogenesis and migration of excitatory neurons arising within the proliferative epithelium of the dorsal cortex. We find that neurons arise from neurogenic radial glial cells within the cortical ventricular zone through asymmetric cell division, and are generated both directly and by way of an intermediate progenitor cell. Intermediate progenitor cells derived from radial glia undergo symmetric division within the subventricular zone to produce pairs of neurons or, at times, additional progenitor cells. This pattern of division resembles that for transit amplifying cells observed in the CNS of invertebrates and in the mammalian brain regions that exhibit adult neurogenesis. The distinct niches for symmetric and asymmetric neurogenic division in the cortex suggest local environmental factors may influence cell division mode. We also find that pyramidal neurons exhibit four distinct phases of migration. These include a phase of migratory arrest in the subventricular zone and a phase of retrograde migration toward the ventricle before neurons reverse polarity to recommence radial migration to the cortical plate. We propose that a number of neuronal migration disorders in humans may represent a failure of migrating neurons to transition between specific phases of migration. Keywords: Radial glia; Asymmetric division; Ventricular zone; Stem cell [O12] Lhx6 does not specify the GABA phenotype of cortical interneurons but regulates their migration from the ventral telencephalon A. Liapi, P. Alifragis, J.G. Parnavelas University College London, UK E-mail address: [email protected] (J.G. Parnavelas). The LIM–homeodomain family of transcription factors in involved in many processes in the developing CNS

605

ranging from cell phenotype determination to connectivity. Lhx6 is member of this family that is specifically expressed in the first branchial arch and in the developing mammalian telencephalon. In the latter, Lhx6 is confined to the medial ganglionic eminence (MGE), the primordium of the globus pallidus of the basal ganglia, where the majority of the cortical interneurons are generated. The expression of this transcription factor in the GABA-containing MGE cells that migrate to the cortex has prompted speculation that it plays a role in the neurochemical identity and migration of these neurons. We performed loss of function studies for Lhx6 in mouse E13.5 brain slices and dissociated MGE neuronal cultures using Lhx6-targeted siRNA produced by a U6 promoter driven vector. We found that silencing Lhx6 impeded the tangential migration of interneurons into the cortex, although it did not obstruct their dispersion within the ganglionic eminence. Blocking Lhx6 expression in dissociated cultured neurons taken from E13.5 mouse MGE did not interfere with the production of GABA or the expression of GAD65/67 in these cells. Similarly, overexpression of Lhx6 in ganglionic eminence neurons did not affect the GABA phenotype, but promoted their differentiation, as identified with MAP2 immunostaining. These results indicate that Lhx6 does not specify the neurochemical identity of cortical interneurons, but regulates their differentiation and migration to the cortex. Keywords: Cortical interneurons; Migration; GABA; Lhx6 [O13] Tangential migration of a subset of neuronal and glial progenitor cells in the developing cortex W.B. Macklin*, Y. Fujita, Q. Hao, B.S. Mallon Cleveland Clinic Foundation, USA E-mail address: [email protected] (W.B. Macklin). Oligodendrocytes are terminally differentiated cells that produce the myelin that is essential for normal neuronal function. In order to investigate how oligodendrocyte progenitor cells populate the cortex, we analyzed mice expressing enhanced green fluorescent protein (EGFP), driven by the myelin proteolipid protein (PLP) promoter (Mallon et al., 2002). The highest level of PLP gene expression occurs postnatally, and the PLP protein has generally been considered a mature myelin marker. Nevertheless, PLP gene expression has also been detected during early embryonic development, well before myelination. In the embryonic telencephalon, PLP–EGFP cells emerged in restricted regions of ventral telencephalon, the lateral ganglionic eminence, the medial ganglionic eminence and the anterior entopeduncular area at E11.5–13.5 and then migrated tangentially to the cortex by E16.5. The anterior entopeduncular area is known to be the primary source of oligodendrocyte progenitors in the cortex, and this tangential migration pathway from ventral to dorsal is

606

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

consistent with that of GABAergic interneuron progenitors and later oligodendrocytes. Two pathways of migration of PLP–EGFP cells were seen. Some cells migrated into the cortex in the intermediate zone, while others migrated into the cortex through the subventricular zone. PLP–EGFP cells in the embryonic subventricular zone were non-proliferative, and expressed few classic progenitor markers. PLP– EGFP cells in the E14.5 intermediate zone of the telencephalon had a migratory morphology and were GABA-positive cells, which are likely the progenitor cells of cortical GABAergic interneurons. By E16.5, the PLP– EGFP cells in the intermediate zone no longer expressed GABA. PLP–EGFP was expressed in cells with a ramified morphology, which expressed oligodendrocyte progenitor markers. These oligodendrocyte lineage cells proliferated in situ, and continued to express PLP–EGFP in the postnatal cortex. These findings indicate that in early telencephalon, the PLP gene is expressed in progenitor cells that can become neurons or oligodendrocytes. It is possible that the earliest PLP-expressing cells may be the bipotential progenitor cells identified by others, which can give rise to GABAergic neurons and/or oligodendrocytes in the developing telencephalon. Supported by NIH grant NS25304. Keywords: Oligodendrocyte; Proteolipid protein; Gaba; Progenitor Symposium 4: Molecular Regulation of Neural Crest Development [O14] Early induction of the neural crest R. Mayor, C. Araya, J. deCalisto University College London, UK The neural crest (NC) segregates from the dorsal portion of the neural tube and migrates through the embryo to generate a highly pluripotent cell population, able to generate a variety of cells. The differentiation of each particular crest cell will depend on its own internal program and on signals received from the surrounding environment. The identification of genes in Xenopus, chick and zebrafish expressed early in prospective NC cells has challenged the previous view that the NC is induced during the closure of the neural tube. Initial NC induction is dependent on a gradient of BMP activity established in the ectoderm. This gradient is established by the secretion of BMPs from the ectoderm and of anti-BMP molecules, such as noggin, chordin, cerberus, nodal, follistatin and FrzB, from the organizer. The neural plate border, induced at a precise location within the medio-lateral axis of the ectoderm, has an anterior character. At a later stage additional signals originating from the dorsolaterla mesoderm transform a region of the previously

induced anterior neural plate border into prospective NC cells. These signals correspond to Wnts, FGFs and RA. The anterior neural plate border either does not receive such signals or these are inhibited by other agents produced by the anterior regions of the embryo, such as cerberus, Dkk1 or Dan, three known Wnts inhibitors, and as a consequence, this region does not develop into NC. [O15] Cell intrinsic control of neural crest induction J. Briscoe National Institute for Medical Research, UK A defining feature of vertebrates is the neural crest, a population of migratory multipotent progenitor cells generated at the border between the neural plate and nonneural ectoderm. Premigratory cells of the neural crest initiate a distinct programme of gene expression, undergo an epithelial–mesenchymal transition, delaminate from the neuroepithelium and migrate into the periphery. Despite the identification of a number of genes expressed by prospective neural crest cells, how these act to specify and integrate the diverse properties of neural crest cells is unclear. Our data indicate that neural crest induction involves the co-ordinated activity of the HMG transcription factor Sox9, the zinc finger protein, Slug/Snail, and the winged helix protein, FoxD3. Alone, none of these proteins are sufficient to induce the full complement of neural crest features; however, combined expression of the three genes induces the characteristics of early migratory neural crest cells. Moreover, in the absence of Sox9, spinal neural cells commence neural crest differentiation but undergo apoptosis, indicating that Sox9 acts at a critical step to co-ordinate neural crest specification with an epithelial mesenchymal transition. Together these data suggest a model in which neural crest development is initiated by the independent but integrated induction of a combination of transcription factors that control and co-ordinate the acquisition of the diverse properties of neural crest cells. [O16] Lineage decisions in early neural crest cells L. Sommer Swiss Federal Institute of Technology, Switzerland E-mail address: [email protected] (L. Sommer). Because of its broad developmental potential, the neural crest represents an ideal model system to study stem cell biology. Neural crest cells generate various neural cell types of the vertebrate peripheral nervous system as well as several

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

non-neural tissues, including pigment cells, smooth muscle, and craniofacial bones and cartilage. As revealed by clonal analysis in culture, early emigrating neural crest cells are a fairly homogeneous population of stem cells with respect to their developmental potential. They are multipotent and able to adopt specific fates in response to instructive growth factors such as Wnt, BMPs and TGFbeta. Conditional manipulation of these signaling pathways demonstrates a role of these signals in neural crest stem cell development in vivo. In particular, Wnt/beta-catenin signaling induces sensory neurogenesis in virtually all neural crest stem cells, while it regulates cell cycle progression in other types of stem cells. Thus, intrinsic differences determine how different types of stem cells respond to Wnt signaling. In addition, the biological activity of Wnt/beta-catenin is modulated by the cross-talk with other signal transduction pathways, promoting either neurogenesis or stem cell maintenance in neural crest stem cells. Keywords: Neural crest; Stem cell; Wnt; BMP [O17] Signals the control fate decisions in Schwann cell development K. Jessen University College of London, UK The Schwann cell lineage shows three major developmental transitions. First, the generation of Schwann cell precursors from neural crest cells, second, the generation of immature Schwann cells from the precursors and, third, the reversible formation of the two distinct Schwann cell types, the myelinating and non-myelinating cells. Since crest cells can generate several cell types and since immature Schwann cells can form two different Schwann cell variants, the first and last of the transitions involves a fate choice. The most dramatic of these transitions in terms of phenotypic change is the transition from immature Schwann cells to myelinating cells. This transition involves withdrawal from the cell cycle, acquisition of death resistance, activation of myelin genes and suppression of genes expressed by immature Schwann cells. In vivo, these changes require the function of the transcription factor Krox-20 as shown by genetic inactivation in mice. We find that enforced expression of Krox-20 in Schwann cells cell-autonomously inactivates the proliferative response of Schwann cells to the major axonal mitogen Neuregulin and blocks the death response to TGFbeta, a developmental Schwann cell death signal. Krox-20 also induces protein expression appropriate for myelination. Even in 3T3 fibroblasts, Krox-20 not only blocks proliferation and death but also activates the myelin genes periaxin and protein zero, showing properties in common with master regulatory genes in other cell types. Significantly, a major function of Krox-20 is to suppress the c-Jun NH2 terminal

607

protein kinase (NK) pathway. We find that activation of this pathway is required for proliferation and death, but that it blocks myelin gene. By suppressing the JNK pathway Krox20 co-ordinately suppreses proliferation and death and lifts a brake on myelin gene expression. Krox-20 also up-regulates the scaffold protein JIP-1. We propose this as a possible component of the mechanism by which Krox-20 regulates JNK activity. Symposium 5: Mitochondrial Alterations in the Immature and Aging Brain [O18] Mitochondrial bioenergetics in aging D.G. Nicholls Buck Institute for Age Research, USA E-mail address: Nicholls).

[email protected]

(D.G.

The varied hypotheses relating the aging process to attenuated mitochondrial function can be evaluated based on our relatively detailed knowledge of the bioenergetic behavior of isolated mitochondria. Some concepts, such as the ‘rate of living’ hypothesis, that rapidly respiring mitochondria generate more reactive oxygen species than ‘resting’ mitochondria, are contrary to the experimental observation that superoxide generation is low under the conditions of lowered mitochondrial membrane potential that accompany rapid respiration. Other concepts have a firmer theoretical basis. Thus a number of reports suggest that expression of novel uncoupling proteins, that allow proton re-entry while bypassing ATP synthesis, may decrease oxidative stress by lowering membrane potential and may even be associated with increased longevity. One of the current uncertainties concerns the mechanisms by which such uncoupling proteins might be regulated, since a constitutive proton leak would adversely affect the capacity of tissues to generate ATP. In addition to the existence of identified uncoupling proteins, all mitochondria possess an endogenous proton leak that is regulated by membrane potential. This ‘non-ohmic leak’ may serve the purpose of limiting reactive oxygen species generation in resting mitochondria without compromising maximal ATP generation. Finally, a decrease in the total glutathione pool is predicted on thermodynamic and kinetic grounds to result in a substantial oxidative shift in thiol redox potential in the cell. Such a reduction may occur in aging models and the consequences will be discussed. Acknowledgements Supported by NIH grants NIH: R01 AG21440 and R01 NS41908.

608

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

[O19] Mitochondria, oxidative stress and the neurodegenerative process J.B. Clark University College London, UK E-mail address: [email protected] (J.B. Clark). It is now clear that mitochondrial dysfunction may play a key role in the aetiology of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, motor neuron disease, multiple sclerosis and aging (1) (Stewart, V., et al., 2002). Central to these proposals is the concept that oxidative stress may be a critical component of the pathogenic mechanisms of these conditions either through enhanced free radical production or impaired antioxidant defences. In the brain, glial cells exceed neurons by a ratio of 9:1 and the astrocytes are crucial to the maintenance of neuronal function (2). Using nitric oxide (NO) as a naturally occurring free radical we have studied its effects on mitochondrial function in cultured neurons and the role that cocultured astrocytes have on protecting the neuron from oxidative stress. We conclude that astrocytes are critical to the maintenance of neuronal glutathione (GSH) and that a complex pattern of 2-way signalling between astrocyte and neuron controls this process involving the upregulation of key enzymes of the GSH synthetic pathway (3). Impairment of this neuronal/astrocytic cooperation may be part of the aging/neurodegenerative process. Acknowledgements Supported by MRC, Wellcome, Brain Research Trust, Hospital Savings Association & Worshipful Company of Pewterers. References Fields, R.D., 2004. Sci. Am. (April), 54–61. Gegg, M., et al., 2003. J. Neurochem. 86, 228–237. Stewart, V., et al., 2002. J. Neurochem. 83, 984–991. Keywords: Mitochondria; Neurodegeneration; Oxidative stress; Glutathione [O20] Age-related mitochondrial responses to calcium and proapoptotic proteins G. Fiskum*, C. Robertson

chondria to stressful stimuli, e.g., high levels of Ca2+, oxidative stress, and pro-apoptotic proteins. Our studies focused on the differences between the responses of free plus synaptosomal brain mitochondria from 7-, 17-, and 90day old rats to Ca2+ and to BH3 death domain only proteins, e.g., tBid. Isolated mitochondria were incubated at 378 in a KCl-based medium containing physiological concentrations of Mg2+ and phosphate, and respiratory substrates, e.g., pyruvate and malate. Experiments were performed at pH 7.0 and at 6.5, and at high and low [ATP] to compare responses under a range of conditions that exist following hypoxia and trauma. Measurements were made of O2 consumption, H2O2 production, Ca2+ uptake, and cytochrome c (CytC) release. Brain mitochondria isolated from immature rats had lower maximal, respiration-dependent Ca2+ uptake capacity than brain mitochondria isolated from adult rats in the presence of ATP at both a pH of 7.0 and 6.5. However, in the absence of ATP, immature brain mitochondria exhibited greater Ca2+ uptake capacity at pH 7.0 and 6.5, indicating a greater resistance of immature brain mitochondria to Ca2+induced dysfunction under conditions relevant to those that exist during acute ischemic and traumatic brain injury. Acidosis reduced the maximal Ca2+ uptake capacity in both immature and adult brain mitochondria. Cytochrome c was released from both immature and adult brain mitochondria in response to Ca2+ exposure. This release was not affected by cyclosporin A but was reduced by 2-APB, inhibitors of the mitochondrial membrane permeability transition (MPT). CytC release and associated H2O2 production induced by tBid was greater with mitochondria from immature compared to mature rats and is due to a higher endogenous level of mitochondrial Bax. While MPT inhibitors had no effect on the response to tBid, dibucaine and propranolol were effective. Developmental changes in mitochondrial response to Ca2+ loads and pro-apoptotic proteins may promote apoptotic vs. necrotic neural cell death in the neonate, and have important implications in the treatment of hypoxic, ischemic, and traumatic brain injury in young children. Keywords: Cytochrome c; Apoptosis; Permeability transition; Ischemia Acknowledgements Supported by NIH grants R01 NS34152 and P01 HD 16596. [O21]

University of Maryland School of Medicine, USA

Mitochondrial control of Ca2+ homeostasis in normal neuronal ageing

E-mail address: [email protected] (G. Fiskum).

E.C. Toescu

While changes in the activities of several metabolic enzyme activities in brain mitochondria during early postnatal development are well documented, little is known of the developmental influence on the response of brain mito-

University of Birmingham, UK A detailed characterization of the cellular mechanisms that define the functional changes associated with normal

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

609

neuronal brain ageing is an ongoing task, complicated by the multi-factorial nature of the ageing process. Central to neuronal homeostasis is the relationship between mitochondrial status and Ca2+ regulation. Calcium transients generated by neuronal activity selectively trigger different signaling pathways with exquisite precision depending on the temporal, spatial, and amplitude characteristics of the Ca2+ transient and the source of Ca2+ entry. Because of the ubiquitous role of Ca2+ at the initiation point of many signalling cascades, dysregulation of Ca2+ responses could readily have far-reaching and multiplying effects, which led to the proposal of the ‘‘Ca2+ hypothesis of ageing’’, that sees alterations in Ca2+ homeostasis as an early and crucial event in the ageing process. However, although some of the Ca2+ homeostatic systems, such as the voltage-operated Ca2+ channels, are affected by ageing (in hippocampus, in particular), in resting conditions, the intracellular free Ca2+ concentration ([Ca2+]i) is the same irrespective of the age, as shown for cerebellar, hippocampal and neocortical neurones in brain slices. The differences between the young and old neurones become apparent only during stimulation and the most consistent age-dependent change in Ca2+ homeostasis in neurones is a delayed recovery of [Ca2+]i following stimulation, a process that most likely underlies the increased vulnerability of aged neurones. The features of this age-dependent reduction in the Ca2+ homeostatic capacity are: (i) is dependent on the level of the metabolic challenge, ie, is not apparent at lower levels of stimulation, and (ii) is associated with changes in the mitochondrial status. In the aged neurones, the mitochondria are chronically depolarised, and simultaneous recording of [Ca2+]i and mitochondrial membrane potential shows that the mitochondrial repolarization process that follows neurotransmitter- or depolarization-induced stimulation is also significantly delayed. The possibility that the mitochondrial permeability transition pore (PTP) is involved in the age-dependent changes in mitochondrial status is suggested by the fact that its inhibition enhances both the rate of mitochondrial repolarization and the rate of [Ca2+]i recovery following stimulation.

rise to copies of themselves and to EGF dependent neural stem cells. The separate FGF2 and EGF dependent neural stem cells increase greatly in numbers later in neurogenesis, but by E14 appear identical to adult neural stem cells. The adult mammalian neural stem cells are mostly quiescent, dividing asymmetrically only once every few weeks. The numbers of adult neural stem cells are maintained into old age mammals. Mouse ES cells cultured in low cell density, completely defined media adopt a neural identity. Using a clonal colony-forming assay, we identify a novel primitive neural stem cell stage as a component of neural lineage specification, which is negatively regulated by TGFbrelated signaling. These results are consistent with a default mechanism for neural fate specification. Primitive neural stem cells are formed directly from single ES cells in an exogenous LIF- and endogenous FGF-dependent manner, express multiple neural precursor markers and give rise to neurons and glia. The primitive neural stem cells undergo a survival challenge in the minimal media conditions, but survival factors allow up to 35% of single ES cells to from primitive neural stem cells that will proliferate into self-renewable neural colonies. Moreover, in vivo mouse chimera experiments reveal that these primitive ES-derived neural stem cells have a broad range of neural and non-neural lineage potential. These results support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.

Keywords: Ageing; Ca2+ homeostasis; Mitochondria; PTP

1

[O22] Building a brain D. van der Kooy University of Toronto, Canada The earliest mammalian neural stem cells differentiate from pluripotent embryonic stem (ES) cells. These primitive neural stem cells emerge in response to LIF. These primitive neural stem cells also may be isolated from the mouse embryonic day (E) 5.5 to E8.0 epiblast. By E8.5, true FGF2 dependent neural stem cells emerge from the primitive neural stem cells and by embryonic day 14 they have given

Keywords: ES cells; Neural stem cells; Default Symposium 6: Emerging Concepts on Neural Stem Cells [O23] Conversion of pluripotent embryonic stem cells into tissue-specific CNS stem cells S. Pollard1, L. Conti1,2, E. Reitano, Q. Ying1, T. Gorba1, E. Cattaneo2, A. Smith1 University of Edinburgh, UK; 2University of Milano, Italy

Our laboratory seeks to elucidate the molecular machinery of stem cell self-renewal, the process whereby a stem cell suppresses differentiation during a given round of cell division but retains that potential for future divisions. We are studying this process in pluripotent embryonic stem (ES) cells capable of producing all three germ layers, and in lineage-restricted neural stem (NS) cells that can form neurons and glia but not other tissue cell types. We have developed conditions for conversion of ES cells into NS cells. Mammalian neural stem and progenitor cells are conventionally propagated ex vivo in aggregates known as neurospheres. The heterogeneously differentiated composition of neurospheres is considered to provide a niche that sustains self-renewal. However, the

610

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

identity of the stem cells remains unclear and initial neuronal differentiation capacity typically declines during serial propagation. In contrast NS cells can be expanded in adherent monoculture with retention of neuronal and astrocyte differentiation potential. On transplantation into mouse brains, NS cells differentiate into neurons and astrocytes, without tumour formation. NS cultures thus appear to constitute an enriched, expandable, and nontumourigenic tissue stem cell resource. [O24] The determination of stem cells into neurons versus astrocytes Y.E. Sun1, H. Wu1, F. He1, K. Martinowich1, W. Ge1, G. Fan2 UCLA Medical School, CA, USA E-mail address: [email protected] (Y.E. Sun). During development of the mammalian central nervous system (CNS) multipotent neural progenitor cells first go through a neurogenic phase, then switch into a gliogenic state. Neural stem cells isolated from the neurogenic zones of the developing and adult CNS or derived from embryonic stem cells are also initially neurogenic and gradually decrease their neurogenic activities and become gliogenic following in vitro expansion. The sequential differentiation of neurons and glia appears to be intrinsically programmed in neural progenitor/stem cells. By studying the regulation and function of the pro-neural bHLH genes and the astrogliogenic JAK-STAT pathway, we attempted to delineate the molecular mechanisms underlying the sequential activation of the neurogenic and gliogenic differentiation programs. Our studies indicate that during the initial neural induction period the proneural bHLH genes are not heavily methylated and are situated in more open chromatin structures. By simply inhibiting histone de-acetylases (HDACs), the pro-neural genes can be readily turned on, which marks the initiation of the neuronal differentiation program. On the contrary, the glial differentiation genes are more heavily methylated during this period. They are associated with rather inactive chromatin structures, which disables HDAC inhibitors from directly activating the genes. In addition, the proneural bHLH genes, once expressed, further suppress the gliogenic JAK-STAT pathway through pre- and posttranscriptional regulations. As a result, no astrocytes are generated during the neurogenic period. As the CNS development proceeds, a strong suppression of the proneural gene expression occurs. At this time, the glial genes become less methylated. This event together with the decreased expression of pro-neural genes and the gradually increased activation of the JAK-STAT pathway, all participate in the robust activation of the astroglial differentiation program, which marks the entry of a gliogenic state of neural progenitor/stem cells.

[O25] Selective specification of neural stem cells into oligodendrocytes or neurons J. de Vellis, A. Espinosa, S. Becker, C.A. Ghiani, S. Kumar UCLA, USA E-mail address: [email protected] (J. de Vellis). The determination of stem cells into different cell lineages generally requires the expression of transcription factors and specific epigenetic factors. We first developed serum-free culture media to address the requirement for epigenetic factors in vitro. Neural stem cells (NSCs) were isolated from embryonic day 16 Sprague–Dawley rats and cultured in a novel serumfree stem cell medium that selected for the growth of NSCs and against the growth of GFAP+ astrocytes. NSCs maintained in culture for extended periods of time retained immunoreactivity for both nestin and PSA-NCAM, two markers characteristic of the stem cell phenotype. Moreover, using an oligodendrocyte (OL) specification medium, NSCs differentiated into OL as evidenced by their morphology and expression of multiple oligodendrocyte/myelin-specific markers. As expected, NSCs were capable of giving rise to astrocytes in a medium favoring this phenotype. In addition, NSCs were capable of acquiring a neuronal phenotype as evidenced by expressing neuronal markers, such as neurofilament (NF) and NeuN when cultured in a defined medium for neurons indicating that these cells are also a good source of neuroblasts, which could be used to replace neuronal populations in the brain. We also showed successful propagation and differentiation of NSCs into OL after cryostorage, allowing for the later use of stored NSCs. The long-term goal of culturing NSCs and committed oligodendrocyte progenitors (OLP) is to obtain homogeneous populations for transplantation with the goal of remyelinating the myelin-deficient CNS. The role of epigenetic factors on oligodendrogenesis in vivo in dysmyelinating mutants was also investigated by injection of growth factors postnatally. Keywords: Neural stem cells; Epigenetic factors; Neurons; Oligodendrocytes [O26] Manipulating the differentiation potential of neural stem cells by ectopic expression of basic helix–loop–helix factors E.R. Bongarzone San Raffaele Hospital, Italy Damage to the adult central nervous tissue is generally accompanied by poor and incomplete—if any at all— recovery of the lesion. Endogenous repair of demyelinated lesions, for example, has been seen in some cases although

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

with limited efficacy. The identification of reservoirs of cells in the postnatal central nervous system with stemness characteristics and the improvement to isolate and culture them in vitro have been fundamental to fuel the idea of their use to treat certain neurodegenerative disorders. Neural stem cells have the capacity to generate the main three neural cell types in vitro and in vivo, after transplantation. Facilitating their endogenous ability to adopt a particular fate might improve the potential application of these cells in particular neurological conditions. In this respect, numerous signals and transcription factors have been described as fundamental for the timely generation of different cell types from neuroepithelial cells during the development of the neural tube. Some of them appear as attractive candidates to modulate the differentiation potential of stem cells in vitro and in vivo. I will present recent data describing our studies to promote the formation of oligodendrocytes after genetic ex vivo manipulation of neural stem cells produced from the adult mouse subventricular zone. I will show that ectopic expression of certain basic helix–loop–helix transcription factors such as those from the Olig family led to an increase in the number of cells with oligodendrocyte characteristics after induction of in vitro differentiation by switching neural stem cells from mitogen-rich medium to serum-containing medium. Further, genetically-modified stem cells do not appear to have altered their cell proliferation and selfrenewal capacity, allowing their in vitro expansion for long periods of time. Finally, I will explore the therapeutic outcome of their use in experimental models affected by myelin breakdown. [O27] Functional properties of neural stem cells and their therapeutic potential A.L. Vescovi San Raffaele Hospital, Italy The finding that the central nervous system (CNS) embodies neurogenetic regions enriched of neural stem cells (NSCs) has spurred a flurry of studies which investigate on both the basics of NSCs’ biology as well as on their perspective application for the therapy of neurological disorders. NSCs are multipotential precursors that grow and self-renew in culture in response to growth factors for extensive time. It has recently been argued that NSCs undergo rapid transformation in vitro and that, in turn, this would represent an obstacle to the use of cultured NSCs in therapeutic applications for neurological disorders. Furthermore, it has been concluded that the capacity of neural stem cells for transdifferentiation may not be an inherent property of these cells but may rather arise from their random transformation. I shall be discussing recent data in our hand, which describe the systematic investigation of the functional

611

properties of NSCs upon long-term culturing and show their lack of transformation and their actual ability to give rise to non-neural cells. NSCs do not display any sign of transformations neither at early or at late culture stages. The self-renewal capacity of NSCs, i.e. their ability to generate new stem cells—does not change over time and no chromosomal abnormalities are observed up to passage 30 in human NSCs. Following removal of mitogens, human NSCs display steady growth potential for many months and stop dividing and differentiate into neurons and glia with highly reproducible frequency. I will then show how we can confirm previous findings showing how NSCs do possess the ability to give rise to non-neural-derivatives, i.e. mesodermal cells—a phenomenon that, therefore, does not depend on the in vitro transformation of NSCs. Finally, I will illustrate the lack of any tumorigenic potential of NSCs and their striking capacity for engraftment in the CNS and their utmost clinical efficacy in the context of multiple sclerosis (MS). Critical issues exist regarding the therapeutic use of focal cell transplantation in MS, in which multifocal demyelination represents the main pathological feature. We have recently established an effective experimental therapeutic protocol based on NSCs and consisting of transplantation of syngenic adult NSCs in mice affected by experimental autoimmune encephalomyelitis (EAE), the elective animal model of MS. The peculiarity of this protocol arises from the fact that the NSCs are not transplanted directly into the CNS damage parenchyma, but are rather injected into the cerebrospinal fluid (i.c.) or intravenously (i.v.). By this technique, NSCs enter and distribute in the CNS parenchyma in a multifocal fashion, trigger and carry out remyelination and significantly ameliorate the clinical course and neuropathological signs of EAE. Symposium 7: Reciprocal Glial-Neuronal Interaction in Migration, Axon Guidance and Myelination [O28] Prospero maintains the mitotic potential of glial precursors enabling them to respond to neurons R. Griffiths, A. Hidalgo* University of Birmingham, UK E-mail address: [email protected] (A. Hidalgo). During central nervous system development, glial cells need to be in the correct number and location, at the correct time, to enable axon guidance and neuropile formation. Repair of the injured or diseased central nervous system will require the manipulation of glial precursors, so that the number of glial cells is adjusted to that of neurons, enabling axonal tracts to be rebuilt, remyelinated and functional. Unfortunately, the molecular mechanisms controlling glial precursor proliferative

612

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

potential are unknown. We show here that glial proliferation is regulated by interactions with axons and that the Drosophila gene prospero is required to maintain the mitotic potential of glia. During growth cone guidance, Prospero positively regulates cycE promoting cell proliferation. Neuronal Vein activates the MAPKinase signalling pathway in the glia with highest Prospero levels, coupling axon extension with glial proliferation. Later on, Prospero maintains glial precursors in an undifferentiated state by activating Notch and antagonising the p27/p21 homologue Dacapo. This enables prospero expressing cells alone to divide further upon elimination of neurons and to adjust glial number to axons during development. Keywords: Prospero; Glial proliferation; Precursor; Oligodendrocyte; Axon guidance [O29] Dynamic cell–cell interactions control migration behaviour during zebrafish sensory nervous development D. Gilmour*, F. van Bebber, P. Haas, C. Nu¨ sslein-Volhard Max Planck Institut fu¨ r Entwicklungsbiologie, Germany Essentially all cells in the developing nervous system take up their final position through an active migration process. While recent work by a number of labs has lead to the identification of many classes of cell and axon guidance molecules, we still have no idea how the expression of these guidance cues is coordinated to allow functionally related cells to coalesce at the right time and place. We have developed the zebrafish lateral line as model system for the study of such complex cell movements in vivo. The lateral line is a series of mechanosensory organs, termed neuromasts, dispersed throughout the skin of fish and amphibia that allow them to feel pressure changes in the surrounding water. During the formation of this sensory system there is a highly synchronous migration of three distinct cell types, namely a migrating primordium that deposits cells fated to become the neuromast organs, the nerve that innervates these organs and peripheral glia ensheathing the nerve. We have used the combination of genetics and in vivo imaging to show that the migration of these three cell types is coordinated by a relay of distinct guidance cues from one cell type to the next. In an attempt to identify the molecules that regulate these dynamic cell–cell interactions, we have isolated a number of mutants that affect lateral line migration and the genes underlying several of these have been cloned. Such genetic analysis has revealed an unexpected role for peripheral glia in regulating sensory organ development. Keywords: Cell migration; Lateral line; Glia; Chemokine signaling

[O30] Border controls at the spinal cord: a novel role for the neural crest J. Cohen1*, M. Vermeren1, R. Bron1, G. Maro2, P. Topilko2, P. Charnay2 King’s College London, UK; 2E´ cole Normale Supe´ rieure, France

1

E-mail address: [email protected] (J. Cohen). In nervous system development the phases of neuron migration and axon extension are processes that are rarely temporally distinct but frequently overlap. How then are they separately regulated? Clues to the underlying mechanisms have recently been provided in the case of somatic motor neurons by our studies of boundary cap (BC) cells, a population of neural crest derivatives transiently located at spinal cord motor exit points (Vermeren et al., 2003). Surgical or targeted genetic ablation of BC cells leads, remarkably, to the emigration of many spinal motor neuron soma into the periphery, by a process involving translocation along their axons within ventral nerve roots. Likewise at the dorsal root entry zone (drez), where primary sensory afferents penetrate the spinal cord, mouse mutants lacking drez BC cells display ectopic DRG neuron soma within nerve roots and the dorsal funiculus. These findings imply that the settling positions of projection neuron soma in the CNS and PNS are determined in part by signals received from strategically positioned BC cells at CNS: PNS interfaces. To identify the signaling molecules responsible we have adopted a loss of function approach using small inhibitory RNAs (siRNA) electroporated in chick embryos. Targeted knock down of Neuropilin-2 (NP-2) but not NP-1, the major receptors for class 3 semaphorins, on both spinal motor and DRG neurons generates ectopic neuronal soma comparable to those seen in BC cell-ablated embryos (Bron et al., 2004). Moreover the NP-2 ligand sema 3B is expressed at high levels in BC cells. Together these data support the idea that in developing spinal cord and sensory ganglia, signaling from BC cells is required to confine the soma of long distance projection neurons to their appropriate compartments. Keywords: Motor neuron; Neural crest; Neuron migration; Neuropilin-2 Reference Bron, et al., in press. Dev. Dyn. Vermeren, et al., 2003. Neuron 37, 403–415. [O31] Axo-glial junctions at the node P. Brophy University of Edinburgh, UK

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

Paranodal axoglial junctions play an important role in ensuring normal nerve impulse transmission and there has been much recent progress in identifying their molecular composition. Two proteins, Caspr and Contactin, form a cis adhesion complex on the axon and the ligand for this complex is a glial member of the L1 family of adhesion molecules, Neurofascin. The assembly of this complex is providing important new insights into how myelinated nerves acquire their specilised functions during development. Symposium 8: Roles of TGFbeta Family Members in the Nervous System (Sponsored, in part, by an award from the March of Dimes) [O32]

613

unidentified fate are labelled. GDF-15 knockout mice reveal an approximately 30% loss of facial motoneurons and an approximately 12% loss of nigrostriatal dopaminergic neurons suggesting that GDF-15 plays a role in the development and/or maintenance of these neuron populations. Acknowledgement Supported by a grant from the Deutsche Forschungsgemeinschaft to J.S. and K.U. [O33] BMP signaling in the autonomic nervous system N. Tsarovina1, F. Mu¨ ller1, M. Stanke1, J. Stubbusch1, A. Pattyn2, J.-F. Brunet2, J. v.d. Wees3, H. Rohrer1* 1

Neural functions of GDF-15, a novel TGF-ß K. Unsicker*, O. v. Bohlen, A. Strzelczyk, S. Subramaniam, A. Schober, J. Strelau University of Heidelberg, Germany E-mail address: (K. Unsicker).

[email protected]

Growth/differentiation factor-15 (GDF-15) is a novel member of the TGF-ß superfamily discovered in our and other laboratories. GDF-15 mRNA and protein are widely distributed in embryonic and adult tissues of mouse and rat, most notably in placenta, kidney, and exocrine glands. In the nervous system, highest levels of mRNA expression and protein are found in the choroid plexus; the protein is secreted into the cerebrospinal fluid. Low to medium levels of GDF-15 mRNA expression are found in all regions of the CNS. Following cortical lesioning GDF-15 mRNA is clearly detectable in neurons in the vicinity of the lesion site. Microglial cells are another source of GDF-15. GDF-15 is abundantly expressed in cultured astroglia, but not in the oligodendrocyte lineage. Concerning putative functions of GDF-15 in the nervous system, we have found that GDF-15 is a potent survival promoting factor for cultured embryonic and 6-OHDA lesioned nigrostriatal dopaminergic neurons in vivo. When applied in vivo GDF-15 also prevents abnormal motor behavior. Using cultured cerebellar granule cells we have found that GDF-15 can promote survival by activation of the Akt pathway and preventing c-jun phosphorylation and formation of reactive oxygene species. GDNF-15 does not signal through any of the known TGF-ß receptors or the Smad pathway, respectively; its receptor(s) remain to be identified. We have generated a GDF-15 knockout/lacZ knockin, which is viable and fertile. In the CNS, a novel site of GDF15 expression is a region in the lateral wall of the embryonic spinal cord central canal, where progenitor cells of yet

MPI for Brain Research, Germany; 2ENS Paris, France;3Erasmus MC, The Netherlands E-mail address: Rohrer).

[email protected]

(H.

The generation of noradrenergic sympathetic neurons from neural crest progenitors is induced by extrinsic signals (BMPs) which elicit the expression of a network of transcription factors that in turn control sympathetic neuron differentiation. This network includes Mash1, Phox2b and Phox2a, dHand, and the Zn-finger transcription factors Gata2/3. Phox2 proteins, acting in concert with dHand, bind to the promotor of the subtype-specific noradrenergic marker genes tyrosine hydroxylase (TH) and dopamine-bhydroxylase (DBH) and activate their transcription. The Gata3 transcription factor has been implicated, in addition to dHand, in the selective control of noradrenergic characteristics. Our recent functional analysis of Gata factors establish Gata2 and Gata3 in the chick and mouse, respectively, as essential signals in sympathetic neuron development, affecting type-specific as well as generic neuron differentiation. For the generation of cholinergic parasympathetic neurons this scheme is modified in several ways. Using the chick ciliary ganglion as model, we demonstrate that ciliary neurons are generated in a BMP-dependent manner. However, developing ciliary neurons are devoid of of dHand and Gata2 and express the noradrenergic markers TH and DBH only transiently. As TH and DBH are maintained by ectopic expression of dHand in the ciliary ganglion, a similar function for dHand in sympathetic neurons is implicated. The different composition of the transcription factor network in sympathetic and ciliary precursors suggests that BMPs act on neural crest precursor cells that display specific, location-dependent differences which determine the response to BMP signaling and subsequently the autonomic neuron subtype. Keywords: Sympathetic; Parasympathetic; Gata2; dHand

614

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

[O34] Activin in sensory neuron differentiation and response to injury A.K. Hall*, B.A. Cruise, P. Xu Case Western Reserve University, USA E-mail address: [email protected] (A.K. Hall). Regulated expression of neuropeptide transmitters in the sensory nervous system results in the ability to feel pain and modulate inflammation. These functions are carried out by sensory neurons that express calcitonin gene-related peptide (CGRP) and project to skin, muscle or gut target tissues. Activin and members of the bone morphogenetic protein (BMP) family contribute to the induction of CGRP in embryonic dorsal root ganglion neurons. Bioassays using embryonic tissues as well as cell lines derived from these tissues clearly implicate a target derived factor in the induction of CGRP, and these tissues express BMPs and activin during the period of neuronal contact in vivo. These data support the hypothesis that BMPs and activin regulate sensory neuron phenotypic differentiation. Furthermore, activin increases in skin injury paradigms associated with increased pain sensation and swelling, including skin wound, inflammation, or in the skin disorder psoriasis. Following a skin wound the number of CGRP expressing neurons in sensory ganglia increases, suggesting some adult sensory neurons retain plasticity. The overall size of CGRP immunoreactive neurons is increased after inflammation, suggesting that some larger diameter neurons newly acquire CGRP expression. Isolectin B4 that identifies predominantly small light touch mechanoreceptors is present on some newly CGRP-expressing neurons suggesting this population is one source of mutable neurons. These data suggest that activin is an important regulator of CGRP in sensory development and skin disease and injury.

commitment. Bacterial two-hybrid and co-immunoprecipitation studies demonstrate that ID4 and ID2 complex with OLIG1 and OLIG2. Further, exposure of cultured progenitor cells to BMP4 changes the intracellular localization of OLIG1 and OLIG2 from a predominantly nuclear to a predominantly cytoplasmic localization. These observations suggest that the induction of ID4 and ID2 and their sequestration of OLIG proteins mediate inhibitory effects of BMP signaling on OL lineage commitment and contribute to the generation of astrocytes. BMP signaling also induces expression of Hesr3. Hesr3 stimulates transcription of Hes5 and Hes1 but inhibits transcription from its own promoter. Conversely Hes5 and Hes1 repress transcription from their own promoters but activate Hesr3 expression. In addition to cross regulation at the transcriptional level, Hesr3 binds to other members of the Hes family, and coexpression of Hesr3 inhibits the effects of Hes5, Hes1, and Hesr1. BMP4 treatment of cultured neural progenitor cells results in oscillating expression of Hes5, Hes1, Hrt1, and Hrt3 with Hrt3 exactly out of phase with the other transcription factors. These findings suggest a model in which crossregulatory interactions between Hes and Hesr3 proteins in response to BMP signaling create a molecular oscillator that regulates neural stem cell responses to other epigenetic signals. Keywords: BMP; Oligodendroglia; ID; Hes Symposium 9: Development of the Olfactory System (Sponsored, in part, by an award from the Conferences Committee of the International Society for Neurochemistry) [O36] Cellular and molecular properties of olfactory neural cells

[O35]

S.C. Barnett, L. Chang

BMP regulation of glial development

Glasgow University, UK

J. Samanta, A. Bassuk, N. Israsena, J.A. Kessler*

E-mail address: [email protected] (S.C. Barnett).

Northwestern University Medical School, USA

The peripheral olfactory system exhibits a remarkable regenerative capacity; a capacity which is demonstrated both by the continuous neurogenesis of olfactory receptor neurons in the olfactory epithelium and by the fact that new olfactory axons are able to grow into the adult CNS environment of the olfactory bulb. It is thought that these properties are in part due to basal stem cells in the olfactory epithelium that generate new olfactory receptor neurons, and also specialised glial cells that reside in the olfactory mucosa and bulb, termed olfactory ensheathing cells (OECs) which direct the newly generated axons to their target in the olfactory bulb. We have examined the differentiation capacity of olfactory neural cells in culture from embryonic olfactory mucosa and olfactory bulb tissue. Using a panel of

E-mail address: [email protected] (J.A. Kessler). Bone morphogenetic protein (BMP) signaling inhibits the generation of oligodendroglia (OLs) and enhances generation of astrocytes by neural progenitor cells both in vitro and in vivo. These effects are mediated, in part, by the ID proteins and Hesr3. BMP signaling induces expression of all four ID proteins. Overexpression of Id4 or Id2 in cultured progenitor cells reproduces both the inhibitory effects of BMP4 treatment on OL lineage commitment and the stimulatory effects on astrogliogenesis. Conversely decreasing levels of ID4 mRNA by RNA interference enhances OL differentiation and inhibits effects of BMP4 on glial lineage

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

markers we have identified two types of olfactory ensheathing cells, one Schwann cell-like the other flatter and more astrocyte cell-like, as has previously been described from P7 animals. Comparison between cells obtained from the olfactory mucosa and olfactory bulb will be described. Specifically neurospheres generated from the olfactory mucosa are distinct from those formed from the olfactory bulb, supporting the view that both tissues contain cells capable of forming neurospheres which possess different differentiation potentials. Experiments will also be described that demonstrate that removal of the Schwann cell-like OEC by complement kill results in an increase in ENCAM expressing cells illustrating cellular interactions between these cells in culture influence their differentiation capacity. Keywords: Olfactory; Neural cells; Glial cells; Differentiation [O37] Stem cells of the adult olfactory epithelium J.E. Schwob Tufts University School of Medicine, USA E-mail address: [email protected] (J.E. Schwob). The peripheral olfactory system exhibits a remarkable capacity to recover after injury: the olfactory epithelium (OE) reconstitutes itself, nascent axons reinnervate the olfactory bulb, and near-normal olfactory function is restored. Are there distinct stem cells responsible for replenishing each of the different cell types that were destroyed? Or is there a type of broadly multipotent stem cell that is reminiscent, in a functional sense, of the cells of the olfactory placode that produce the multiple cell types of the nasal epithelium and the olfactory nerve during embryonic development? Our data demonstrate clearly that among the population of globose basal cells (GBCs) are very broadly multipotent ones, thus satisfying a criterion for ‘‘stemness’’. The lines of evidence include marker expression studies after epithelial injury, retroviral lineage tracing experiments, and, most powerfully, a colony forming unittransplantation assay. In the latter, FACS-sorted GBCs from normal OE engraft into the regenerating OE of a host after nasal infusion and give rise to GBCs and neurons, as well as sustentacular, gland/duct, and even respiratory epithelial cells. The other FACS-sorted cell types engraft in host OE but are limited to self-replication. Moreover, infectability with retroviral vectors ex vivo prior to transplantation indicates that the multipotent GBCs are mitotically active in the unlesioned epithelium and choosing continuously between neuro- (their fate in the normal OE) and multipotency. From our analysis of gene expression during the recovery after epithelial lesion, members of the bHLH family of transcription factors likely act during the execution of that choice, while signaling in the Notch pathway may be driving it. The broad multipotency of GBCs and their easy

615

accessibility make the OE a fertile ground for harvesting stem cells that may generate other therapeutically useful populations, including the ensheathing glia of the olfactory nerve. Keywords: Regeneration; Stem cells; Transplantation; bHLH transcription factors [O38] Modelling ORN axonal targetting of olfactory glomeruli T.C. Pearce University of Leicester, UK E-mail address: [email protected] (T.C. Pearce). Generating organised neuronal connectivity between the olfactory epithelium and bulb represents a significant wiring problem. In mammals, around 108 olfactory receptor neurons (ORNs), each expressing one of up to 900 distinct GPCRs, send axons towards the bulb and correctly target one out of a few thousand glomeruli. Although the ORNs expressing a given receptor protein are restricted to one of four epithelial zones, so far no other topological order has been discovered within the ORN population. Yet the disorganised axons from this mixed receptor population have been shown to target specific glomeruli over large distances with extraordinary accuracy. Contrary to pre-existing hypotheses relating to olfactory development, recent experimental results suggest that stimulus-dependent activity may play an important role in this precise axonal targeting process, which continues throughout the life of the animal. I will discuss a simple model that uses Hebbian plasticity and is capable of generating an organised chemotopic map of receptor input using stimulus-dependent activity alone. The wiring behaviour of this model is compared to that seen in two important development studies. Finally, the model generates a prediction for the role of periglomerular cells in orchestrating olfactory development that is yet to confirmed by experimentation. Keywords: Olfactory bulb; Olfactory receptor; Axonal targeting [O39] Formation and refinement of olfactory sensory neuron projections during development J. Strotmann*, S. Conzelmann, K. Schwarzenbacher, J. Fleischer, H. Breer University of Hohenheim, Germany E-mail address: strotmann@uni-hohenheim (J. Strotmann). Olfactory information is conveyed from the nasal neuroepithelium to the main olfactory bulb, the first relay station in the brain, via axons of the olfactory sensory neurons. Each neuron extends a single axon to the bulb

616

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

where it synapses onto second order neurons in a spherical region of neuropil, called glomerulus. A glomerulus represents the site of convergence for neurons expressing the same olfactory receptor. The glomeruli from neurons expressing the highly homologous receptors of the mOR37 subfamily are located in immediate vicinity, nevertheless each glomerulus receives input with very high precision from only one, receptor-subtype specific population of neurons. To monitor the unfolding of this precise olfactory map during development, the onset of receptor expression, outgrowth of axons as well as glomerulus formation for two neuron populations expressing different mOR37 subtypes was investigated on transgenic mouse lines. The data indicate a synchronous onset of receptor expression at about embryonic day 10 (E10). From E15, axons of both populations terminate in a common, small area of the presumptive olfactory bulb. During a short postnatal phase, the two axon populations segregate into distinct, protoglomerular structures. Between E11 and E16, populations of cells expressing distinct olfactory receptor types are located in the cribriform mesenchyme, between the prospective olfactory epithelium and the developing telencephalon. Molecular phenotyping demonstrated that these ‘extraepithelial’ cells co-express key elements characteristic for neurons in the nasal epithelium. Studies on transgenic mice showed that they are positioned along the axon tracts, and each population expressing a given receptor gene is specifically associated with the axons of those olfactory sensory neurons with the same receptor type. The data suggest that they either might be guide posts for the outgrowing axons or migrate along the axons into the brain. Keywords: Olfactory receptor; Projection; Development; Olfactory bulb The IBRO Symposium: Emerging Topics in Neural Function [O40] Cloning and characterization of a novel glial cell linederived neurotrophic factor receptor a-like gene Z. Li, B. Wang, X. Wu, M. Yin, L. Xu, J. Zhou* Shanghai Institutes for Biological Sciences, China Members of the glial cell line-derived neurotrophic factor (GDNF) family are crucial for the development and maintenance of distinct sets of central and peripheral neurons. All ligands signal through the GPI-linked GFRa and transmembrane cRet receptor complexes. Here we described the cloning of a novel gene that may be related to the GFRa receptor family. Using bioinformatics tools and RACE (Rapid Amplification of cDNA Ends), a full-length cDNA (2123 bp) was cloned from mouse brain. DNA sequence analysis showed moderate homology of this gene

to GFRa3 receptor. The gene contained a putative signal peptide at its N-terminus and shares conserved spacing of cystine residues which are also present in the GFRa receptors family. At least two splice variants of the gene existed in mouse brain, differing at their respective COOH termini. One of the splice variants encoded a transmembrane form of the gene, while the other encoded a secreted one. The putative transmembrane variant was localized on plasma membrane, determined by fluorescence immunohistochemistry. RT-PCR analysis indicated that its mRNA was expressed primarily in the central nervous system of adult mouse, albeit at low levels. It was relatively abundant in the substantia nigra, hippocamus and the spinal cord. Their presence, however, was not detectable in peripheral organs by RT-PCR. In situ hybridization analysis confirmed the results obtained from RT-PCR experiments. Further investigations on its biological function using stabletransfection PC12 cells indicated that the gene may participate in neuronal differentiation and anti-apoptotic processes. Taken all these results together, we have named the novel gene GRAL (GDNF receptor alpha-like gene). Keywords: Neurotrophic factor; Novel gene; Neuronal differentiation; Transmembrane receptor complex [O41] Role of oxidative stress in the programmed death of cultured cerebellar neurons J. Mora´ n* National University of Mexico, Mexico Cerebellar granule neurons (CGN) require being depolarized to survive in culture. When CGN maintained in a medium containing 25 mM K+ (K25) are transferred to a medium with 5 mM KCl (K5), caspase-3 activity is induced and cells die apoptotically. Under these conditions CGN death is triggered by a [Ca2+]i reduction that is followed by an increase in reactive oxygen species (ROS) production. The generation of both cytosolic and mitochondrial ROS occurs at different times, but only those ROS produced after 3–4 h seems to be involved in the process of cell death. When CGN are treated with antioxidants like superoxide dismutase or catalase, the activity of caspase, chromatin condensation and cell death is markedly diminished. Caspase activation and chromatin condensation are downstream of ROS production. When CGN are exposed to superoxide anion in the culture, cells die apoptotically; while treatment with hydrogen peroxide induces cell death with necrotic features. Some preliminary results suggest that a possible source involved in the generation of superoxide anion in response to K5 could be the enzyme NADPH oxidase. All the subunits of this enzyme are expressed in CGC and its inhibition significantly reduces cell death induced by K5. We conclude that ROS and NADPH oxidase

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

could play a role in the initiation of the apoptotic process in these cells. Keywords: Oxidative stress; Cerebellar granule neurons; Reactive oxygen species; NADPH oxidase Acknowledgements This work was partially supported by CONACYT 36235 project and DGAPA IN222303-3 project. [O42] Glutamate uptake by hippocampal slices decreases in neonatal rats submitted to hypoxic-ischemic insult: reversal by guanosine D.O. Souza1*, M.B. Moretto2, N.S. Arteni1, D. Lavinsky1, C.A. Netto1, S. Wofchuk1, J.B.T. Rocha2 1

Universidade Federal do Rio Grande do Sul, Brazil; 2Universidade Federal de Santa Maria, Brazil E-mail address: [email protected] (D.O. Souza). Brain injury secondary to hypoxic-ischemic disease is relevant form in the perinatal period. The impact of neonatal hypoxia-ischemia (HI) in 7-day-old pups on the high-affinity [3H] glutamate uptake by hippocampal slices at different times after insult were examined. Immediately and 1 day after the insult there was no effect. However, at 3–5 days after the HI insult, glutamate uptake by hippocampus slices was markedly reduced. After 30 or 60 days returning to control levels. Also, this study demonstrated that guanosine (Guo) reversed [3H] glutamate uptake maintaining the [3H] glutamate uptake on control levels when injected before and after insult HI. We conclude that neonatal HI influenced the glutamate uptake and that guanosine reverted this action.

617

the cell death in this model appears to result from excitotoxic injury and apoptosis, and that nigrostriatal dopaminergic neurons are affected. The aims of the present work were to determine whether cycad exposure induces oxidative damage to biomolecules in relevant brain areas, and to explore if other neuronal populations are also vulnerable to cycad neurotoxicity. For this purpose, the brains of control and cycad-fed mice were processed for the immunohistochemical analysis of neurotransmitter-synthesizing enzymes and oxidation products of lipids (malondialdehyde), protein (3-nitrotyrosine) and DNA (8-hydroxydeoxyguanosine). Cycad-fed animals, compared with age-matched controls, show a higher level of 8-hydroxydeoxyguanosine in the CA1 hippocampal region and also a higher immunoreactivity of 3-nitrotyrosine in the striatum. In this area, cholinergic and gabaergic neurons remained unaffected by the neurotoxin exposure, while the neuronal nitric oxide synthase isoform (nNOS) showed an increased expression. In addition, cycad induced a loss of both CA1 neurons and cortical gabaergic cells, but spared septal cholinergic nucleus. The finding that not all the neuronal populations and brain areas are equally affected further confirms the validity of cycad-fed mice as a model of neurodegeneration. The description of the temporal profile of changes in oxidant status and the elucidation of the molecular basis of the neuronal vulnerability will suggest potential stages for therapeutic intervention on other neurodegenerative disorders, in which environmental agents could also play a triggering role. Keywords: Neurodegeneration; Environmental toxins; Oxidative stress; Neurotransmitter [O44]

Keywords: Hypoxic-ischemia; Glutamate uptake; Guanosine

Evidence for the involvement of neurosteroids with GABA-modulatory properties in the pathogenesis of hepatic encephalopathy

[O43]

S. Ahboucha1*, N. Chatauret1, G. Pomier-Layrargues2, O. Mamer3, G.B. Baker4, R.F. Butterworth1

Oxidative damage and differential neuronal vulnerability in a mouse model of amyotrophic lateral sclerosisparkinsonism-dementia complex R. Cruz-Aguado*, J. Schulz, E. Hawkes, C.A. Shaw University of British Columbia, Canada E-mail address: [email protected] (R. Cruz-Aguado). Amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC) is a neurological variant found on Guam, wherein symptoms of the three major neurodegenerative disorders can co-occur. The neurotoxins contained in cycad seeds, a food staple in Guam, have been proposed as a causal environmental agent. Mice fed with cycad flour show behavioral and morphobiochemical outcomes that are consistent with the symptoms and neuropathological pattern of ALS-PDC. The previous results from our group show that

1

Neuroscience, Canada; 2CHUM (Hoˆ pital Saint-Luc), Canada; 3McGill University, Canada; 4University of Alberta, Canada Electrophysiological and biochemical studies suggests that GABAergic tone is increased in brain in liver failure and that this is a major cause of hepatic encephalopathy (HE). However, the precise mechanisms responsible have eluded researchers in this area for the last two decades. Studies in brain tissue from both human and experimental animals with liver failure have consistently shown that GABA synthesis, GABA receptors and their associated benzodiazepine modulatory sites are unchanged. On the other hand, there is emerging evidence to suggest that neurosteroids (NS), a novel class of compounds with potent GABA agonist properties are increased in brain in liver failure. Evaluation of the total levels of NS with positive allosteric modulatory

618

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

properties on the GABA-A receptor in brain material from patients who died in hepatic coma revealed up to 12-fold increases of NS as compared to appropriately matched controls. Subsequent analysis by gas chromatography-mass spectrometry showed that the major component NS was allopregnanolone which increases up to 7-fold in HE patients. In animals with liver failure, onset of encephalopathy was accompanied by a 15-fold increase of allopregnanolone, and a 7-fold increase in brain concentrations of a second NS, tetrahydrodeoxycorticosterone (THDOC). Moreover, brain extracts from rats with liver failure significantly increased the binding of the GABA-A receptor agonist 3H-muscimol to brain membrane preparations suggesting that the concentrations observed in brains of these animals were of pathophysiologic significance. Plasma allopregnanolone levels were also increased suggesting that its synthesis in the periphery may contribute to increased brain levels of this NS in animals with liver failure. Neither patient nor animal material contained significantly increased concentrations of either GABA or benzodiazepines. Moreover, administration of indomethacin, an inhibitor of the NS synthetic enzyme 3-hydroxysteroid dehydrogenase resulted in improvement in locomotor activity scores in liver failure animals. These findings provide evidence that NS with positive allosteric modulatory properties are generated in brain in both human and experimental liver failure and that these substances contribute to the ‘‘increased GABAergic tone’’ characteristic of HE.

mining. First, there was a robust and reproducible decrease in the group of transcripts encoding proteins regulating presynaptic function (PSYN), although the pattern of altered gene expression within the PSYN group varied across subjects. Second, we discovered that the most changed gene, never associated previously with the disease, was regulator of G-protein signaling 4, a protein that modulates the extent of postsynaptic responsiveness of neurons following activation of G-protein coupled neurotransmitter receptors. Third, a small subset of genes encoding proteins involved in cellular metabolism was altered. These findings suggest fundamental problems in the efficacy of synaptic transmission, and may reflect both primary genetic defects and molecular adaptations that define the pathophysiology of the disease. Neuroanatomical data demonstrate decreased cortical neuropil and modified pre- and postsynaptic structures. We have hypothesized that the neurodevelopmental origin of schizophrenia, suggested by Weinberger almost 20 years ago, reflects a fundamental disruption of early synaptic function that may alter the normal course of exuberant synapse formation and pruning during childhood and adolescence. Disease threshold may be reached when compensatory mechanisms are no longer available, such as subsequent to pruning post-puberty. The heterogeneity of the gene defects and molecular adaptations in schizophrenia may reflect distinct patterns of clinical onset, the course of the disease over the lifetime and the pharmacotherapeutic responsiveness of different individuals with the disease.

Keywords: Nurosteroids; Hepatic encephalopathy; GABAergic tone; GABA agonist; Allopregnanolone

Symposium 10: Animal Models for Autism

Acknowledgement

[O46]

Funded by CIHR Canada.

Modeling genetic susceptibility to autism in mouse models of Fragile-X and Smith–Lemli–Opitz syndrome

[O45]

J.M. Lauder*, H. Waage-Baudet, S.S. Moy

Schizophrenia as a neurodevelopmental disorder of synapse formation and plasticity

University of North Carolina, USA

P. Levitt Vanderbilt University, USA The level of cellular and molecular complexity of the nervous system, compounded by the polygenic and epigenetic etiology of most neuropsychiatric disorders, creates unique challenges for scientists utilizing new approaches in functional genomics to investigate brain diseases. In particular, how does one integrate neuroanatomical and molecular findings from postmortem and clinical studies to understand the pathogenesis of a disorder such as schizophrenia? Using gene microarrays, we demonstrated altered patterns of gene expression in dorsolateral prefrontal cortex (DLPFC) between subjects with schizophrenia and controls. DLPFC dysfunction is associated with the cognitive symptoms of schizophrenia. Three seminal findings were obtained using functional data

This symposium discusses different animal models currently being used to model behavioral, neuroanatomic or physiologic aspects of this developmental disorder, as well as possible gene-environment interactions in its etiology. As part of ongoing studies of gene-behavior interactions relevant to autism, behavioral phenotypes and gene expression profiles have been determined in mice from different background strains with targeted disruption of Fmr1, the gene mutated in Fragile-X Syndrome (FXS) or Dhcr7, the last enzyme in cholesterol biosynthesis, the gene mutated in Smith–Lemli–Opitz Syndrome (SLOS). These studies provide evidence for significant differences in behavioral phenotypes and gene expression profiles resulting from the Fmr1 mutation on different genetic backgrounds, which could bear relevance to gene-gene interactions in susceptibility to autism. The SLOS mouse has been recently been characterized neuroanatomically using antibodies to serotonin (5-HT), and found to exhibit

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

619

hypermorphic development of the raphe nuclei (WaageBaudet et al., 2004). Interestingly, preliminary comparisons of behavioral phenotypes and gene expression profiles between these mouse models have found evidence of similarities in behavioral deficits and altered expression of common genes relating to regulation of cholesterol homeostasis, neurodevelopment, and neurodegenerative disease. These findings suggest that important insights into neural and genetic substrates of autism may be gained by behavioral phenotyping together with gene expression profiling in appropriate animal models.

Keywords: Autism; Organophosphates; Paraoxonase; Reelin

Keywords: Autism; Behavior; Microarray; Gene

A.M. Thompson1*, J.C. Shih2, I. Seif3, J.M. Lauder4

Acknowledgements

University of Oklahoma Health Sciences Center, USA; University of Southern California, USA; 3Universite Paris-Sud, France; 4University of North Carolina at Chapel Hill, USA

Supported by grants from NIH, NIMH and NIAAA. [O47] Autism and the Reelin pathway: evidence for potential gene-environment interactions M. D’Amelio, I. Ricci, A.M. Persico* University ‘‘Campus Bio-Medico’’, Italy E-mail address: [email protected] (A.M. Persico). Autism is a severe neuropsychiatric disorder, whose incidence has apparently risen during the last decade from 2–5 to 15–20/10,000 children. Segregation analyses and genome scan studies support complex genetic contributions to autism, with several major loci, complex epistasis and possibly gene-environment interactions. We have described an association, replicated in at least some independent studies, between autism and ‘‘long’’ alleles of a polymorphic GGC repeat located in the 50 untranslated region (UTR) of the gene encoding Reelin, a pivotal protein for neuronal migration during neurodevelopment. We have also found that long GGC alleles yield reduced gene expression, both in vitro and in vivo. Reelin exerts a proteolytic activity potently inhibited by organophosphates (OPs), compounds routinely used as pesticides in agriculture and as insecticides within the household particularly in North America. A subgroup of genetically-vulnerable individuals producing lower amounts of Reelin, if prenatally exposed to OPs during critical periods in neurodevelopment, could thus undergo altered neuronal migration resulting in an autistic syndrome. Additional evidence supporting this pathogenetic mechanism comes from our recent analyses of the PON1 gene encoding paraoxonase, the enzyme responsible for OP detoxification. Assessments carried out on 278 complete trios with primary autistic probands using functional SNPs located in the promoter and coding sequence of PON1 yield a significant association in Caucasian-American (allelic frequencies in cases versus controls: P < 0.01; transmission/ disequilibrium test x2 = 4.85, 1d.f., P < 0.05) and not in Italian families, as predicted. Animal models based on this gene-environment interactive mechanism will be discussed.

Acknowledgements Supported by grants from Telethon-Italy, the Cure Autism Now Foundation (Los Angeles, CA), and the Fondation Jerome Lejeune (Paris, France). [O48] Serotonin and auditory brainstem development: implications for autism

1

2

E-mail address: Thompson).

[email protected]

(A.M.

Pervasive developmental disorders such as autism have been associated with a broad range of auditory disorders during development including deficits in discriminating the temporal order of auditory signals and impaired binaural processing. The sites of impairment leading to these deficits are poorly understood, but it has been shown that the responses and morphology of the auditory brainstem may be abnormal. Here we review the evidence from a hyperserotonergic model of autism, the monoamine oxidase A knock-out (MAO A KO) mouse, suggesting that auditory brainstem development relies on proper levels of serotonin (5-HT). In the MAO A KO mouse, the gene encoding MAO A is disrupted leading to excess levels of 5-HT in the early postnatal period. The increase in 5-HT is believed responsible for disrupted neural projection patterns as in the abnormal clustering of thalamocortical afferents to the somatosensory cortex (barrel fields). Also, the growth cones of developing thalamocortical axons have been shown to accumulate 5-HT and blocking this uptake disrupts barrel field pattern formation. These observations indicate that 5HT helps to establish precise projection patterns of sensory pathways. It has been shown in the MAO A KO that auditory brainstem neurons accumulate 5-HT indicating that their development too may be regulated by 5-HT. Like those of other sensory systems, the projections of the auditory brainstem are highly organized in order to faithfully analyze and process the spectral and temporal features of the acoustic signal that originates in the cochlea. The processed information is used to help localize and attend to sound sources, and also provides the temporal and spectral cues utilized by higher centers. Therefore, evaluating auditory brainstem development in serotonergic models may be useful in understanding abnormalities in central auditory processing that may contribute to the communicative disorders associated with autism.

620

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

Keywords: 5-Hydroxytryptamine; MAO A knockout; Topography; Ascending auditory system

Stromland, et al., 1994. Dev. Med. Child Neurol. 36, 351– 356.

[O49]

[O50]

Eyeblink conditioning in autism and a rodent model

Modeling the autism behavioral phenotype in mice

M.E. Stanton1, P.M. Rodier2

S.S. Moy1*, J.N. Nadler1, G.E. Duncan1, J.N. Crawley1,2

1

University of Delaware, USA; 2University of Rochester Medical Center, USA

University of North Carolina, USA; 2National Institute of Mental Health, USA

E-mail address: [email protected] (M.E. Stanton).

E-mail address: [email protected] (S.S. Moy).

Brain injury caused by exposure to thalidomide around the time of neural tube closure is associated with autism (Stromland et al., 1994). Rats exposed to valproic acid (VPA) on Gestational day (GD) 12 show brainstem-cerebellar anomalies that resemble those found in autism (Rodier et al., 1996; Ingram et al., 2000). Early gestational exposure to VPA and thalidomide in the rat is also associated with abnormal levels of serotonin and/or dopamine in prefrontal cortex, hippocampus, and cerebellum (Narita et al., 2002). These findings, together with recent reports that valproate use during human pregnancy increases the incidence of autism (e.g., Moore et al., 2000), suggest that the GD12-VPA-exposed rat may serve as a useful rodent model of autism. Eyeblink conditioning is altered in autism (Sears et al, 1994). A preparation for studying eyeblink conditioning in developing rats (Stanton, 2000) has been used with the G12-VPA-exposed rat. Autistic individuals and VPA-treated rats both show normal responses to an auditory conditional stimulus (CS) and eyeblink-evoking unconditional stimulus (US); show more rapid acquisition of eyeblink conditioned responses (CRs); and show CRs that are abnormally large and inappropriately timed in relation to US onset. When tested on discriminative eyeblink conditioning and reversal, VPA-exposed rats show deficits in reversal suggestive of altered forebrain function. Findings with spatial reversal tasks also suggest that perseverative responding during reversal is another behavioral feature of autism that is also observed in the VPA-exposed rat. Further studies of eyeblink conditioning in developing humans and in rodent models may help elucidate the developmental neurobiology of autism.

Clinical profiles in children include deficits in social interaction and communication, as well as repetitive and inflexible behavioral patterns, as core symptoms for autism. Familial studies on the incidence of autism have indicated a strong genetic component for this developmental disorder. Animal models of autism would allow the investigation of specific genes that might mediate the social, motoric, and cognitive components of the aberrant behavioral phenotype. The following studies compared mice from several different inbred strains, including C57BL/6J, DBA/2J, FVB/NJ, and A/J, on measures for sociability and preference for social novelty, as well as measures for activity, motor ability, and sensory function. The results showed strain differences in the social choice task, with the A/J mice least likely to choose proximity to an unfamiliar conspecific. Further work with selected transgenic mouse lines, including animals with deficient NMDA receptor function, has provided additional evidence for an association between genetic background and aberrant social behavior. Overall, these studies demonstrate a methodology for modeling elements of the autism behavioral phenotype in mice.

1

Keywords: Autism; Mouse; Inbred strain; Social interaction Acknowledgements Supported by grants from the National Institutes of Health (MRDDRC P30 HD03110 and STAART U54 MH66418.

Keywords: Autism; Cerebellum; Prefrontal cortex; Learning and memory

Symposium 11: Advances in Retrograde Transport: Functions and Mechanisms of Retrograde Neurotrophin Signaling

Acknowledgement

[O51]

Supported by NIH PO1HD35466. References Ingram, et al., 2000. Neurotoxicol. Teratol. 22, 319–324. Moore, et al., 2000. J. Med. Genet. 37, 489–497. Narita, et al., 2002. Pediatr. Res. 52, 576–579. Rodier, et al., 1996. J. Comp. Neurol. 370, 247–261. Sears, et al, 1994. J. Aut. Dev. Disord. 24, 737–751. Stanton, 2000. Beh Brain Res. 110, 25–37.

Neurotrophins coordinate sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling R. Kuruvilla, L.S. Zweifel, N. Glebova, B.E. Lonze, H. Ye, D.D. Ginty* The Johns Hopkins University School of Medicine, USA A fundamental question in developmental biology is how a limited number of growth factors and their cognate

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

621

receptors coordinate the formation of tissues and organs endowed with enormous morphological complexity. The neurotrophins NGF and NT-3 and their receptors TrkA and p75 are crucial for formation of the sympathetic nervous system, but how they collaborate to control the processes of axon growth, target innervation, and retrograde survival of sympathetic neurons is not known. We found that both NGF and NT-3, acting through TrkA, are required for growth of sympathetic axons and innervation of target fields, and yet NGF, but not NT-3, supports retrograde TrkA signaling and retrograde survival. Interestingly, retrograde NGF/TrkA signaling but not NT-3/TrkA signaling promotes upregulation of p75, which, in turn, attenuates the sensitivity of axons to intermediate target-derived NT-3. NGF and NT-3 differ in their ability to support retrograde TrkA signaling, survival and gene expression because while NGF promotes internalization and retrograde trafficking of TrkA, NT-3 cannot. Our findings indicate that a hierarchical neurotrophin signaling cascade coordinates sympathetic axon growth, innervation of targets, and survival in a manner dependent on the differential control of TrkA internalization, trafficking and retrograde axonal signaling.

accumulated with considerable (12 h) delay in lysosomes when compared with BDNF or NT-3. Since ION neurons do not express trkA, this NGF binds exclusively to the p75 neurotrophin receptor. The tyrosine/serine/threonine kinase inhibitor K252a failed to prevent the rapid degradation of BDNF and NT-3, but consistently accelerated the degradation of NGF. Does phosphorylation of p75 constitute a trafficking signal that delays degradation? P75 contains multiple serines, and their phosphorylation by protein kinase C is diminished by K252a. Thus we postulate that serine/ threonine phosphorylation of p75 via protein kinase C acts as an intracellular trafficking signal for its ligand, NGF. Recycling of internalized and trafficked proteins may be widespread in neurons, and this mechanism may contribute to a wider range of functions than is currently appreciated.

Keywords: Sympathetic neuron; NGF; TrkA; Retrograde signaling

Going the distance with neurotrophin signals: mechanisms and implication

Acknowledgements Supported by NIH grants TW 05700, EY 12841. [O53]

W.C. Mobley

[O52] Fates of retrogradely transported proteins: degradation or recycling? *

C.S. von Bartheld , H.B. Rind, E.S. Guglielmo, R. Butowt University of Nevada School of Medicine, USA E-mail address: Bartheld).

Keywords: Trafficking; Recycling; Neurotrophin; Lysosome

[email protected]

(C.S.

von

Internalized proteins may be degraded, or they may be recycled. Little is known about recycling of proteins in neurons. We analyzed the fate of internalized and retrogradely transported neurotrophic factors in two model systems. In the postnatal rat hypoglossal motor system, we tracked the fate of radiolabeled GDNF, BDNF, and cardiotrophin-1 (CT-1), and compared their distribution at the ultrastructural level in motoneuron cell bodies and dendrites. Interestingly, GDNF and BDNF accumulated at dendritic synapses, while CT-1 did not associate with synapses. Significant fractions of internalized GDNF and BDNF escaped degradation, and instead were targeted to postsynaptic sites in dendrites, possibly for functions related to synaptic plasticity. CT-1, however, followed a strict degradation pathway after retrograde transport to the soma. Recycling with synaptic trafficking thus is restricted to particular neurotrophic factors such as GDNF and BDNF. We compared fates of retrogradely transported neurotrophins in a second model system, the isthmo-optic nucleus (ION) of chick embryos. Using quantitative autoradiography at the electron microscopic level, we found that NGF

Stanford University, USA Elucidating the cellular mechanisms that build, maintain and modify synaptic contacts is required for understanding normal and abnormal nervous system function. Neurotrophic factors (NTFs) are small proteins that act through specific receptors to guide the development and maintenance of neuronal connections. The neurotrophins (NTs) are a family of NTFs whose actions influence neurons of both the peripheral nervous system (PNS) and central nervous system (CNS). Understanding the physiological roles played by NTs requires studies to define how NT-mediated activation of signaling pathways results in specific, well-coordinated biological responses. It has long been known that nerve growth factor (NGF) and other members of the NT family are produced and released in target tissues and that this source of trophic support is required for development and maintenance of synapses. Among the most important unanswered questions is how NT signals generated in the target of innervation are moved to the cell bodies of innervating neurons. We and others hypothesized that the NGF signal is transmitted via endocytosis of complexes containing NGF bound to its activated TrkA receptors followed by retrograde transport of the ‘‘signaling endosome’’ thus formed. We will review the studies that have tested the signaling endosomes hypothesis. In particular, we discuss recent studies in which we isolated the organelle predicted by the hypothesis, the signaling endosome, and have characterized the movement of signaling endosomes in vitro and in vivo. Important insights from our studies on

622

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

NGF and TrkA signaling are that: (1) signaling endosomes arise from membranes in which many of the component signaling molecules are preassembled; (2) these endosomes contain NGF bound to TrkA and feature on their cytosolic surface a membrane-based array of signaling molecules; (3) they arise in clathrin-coated membranes and are moved via dynein and microtubules from clathrin-coated vesicles to early endosomes; (4) subclasses of signaling endosomes harbor different signaling pathways; (5) early endosomes carry NGF signals from axon terminals to the cell bodies of neurons by fast retrograde transport. These new data offer compelling support for the signaling endosome hypothesis and provide a model for how to efficiently move NTF signals that are long-lived, robust and of high-fidelity. Testing the ‘‘signaling endosome hypothesis’’ must now feature studies that detail the signaling events generated by signaling endosomes. Suggesting that these events are critical, in studies on basal forebrain cholinergic neurons (BFCNs) in the Ts65Dn mouse model of Down syndrome, we discovered that failed retrograde transport of NGF was linked to neurodegeneration. The view that the defect in NGF transport, and presumably of the NGF signal, was causative was the ability to reverse the neurodegenerative phenotypes by delivering NGF directly to the cell bodies of BFCNs. We will review these data and other recent observations that point to the importance of axonal transport for the survival and function of neurons. We will discuss recent findings from other laboratories that point to a role for failed retrograde transport in neurodegeneration and will discuss strategies to reverse or prevent degeneration due to abnormalities in trafficking NTF signals. Keywords: Neurotrophin; Neurodegeneration; Endosome; Down syndrome

label varied across the organelle suggesting that it contained subpopulations of vesicles consistent with a multivesicular body. Small GTP-ase Rab proteins characterise the stages of the endocytic pathway and may play a role in the internalisation, targeting and retrograde transport of the NGF containing signalling organelle. Using antibodies against Rab 4, 5a, 5b, 7 and 11, we demonstrated the retrogradely transported signalling organelle containing fluorescent NGF also contains retrogradely transported Rab 4, 5a and 5b. Since these Rab proteins are restricted to early endosomes, the multivesicular body containing NGF originating from the nerve terminal has characteristics of an early endosome. We examined the retrograde axonal transport of two populations of vesicles in the sympathetic nervous system. Those containing NGF, NT-3 or NT-4 were significantly lighter than those containing dopamine beta-hydroxylase (DBH). Latrunculin A, Jasplakinolide, LY294002 and wortmannin inhibited the transport of both DBH and neurotrophins. The retrograde axonal transport of NGF is inhibited by AG879 and phenylarsine oxide but not by bistyrphostin, FK506, dibutyryl-cAMP, thapsigargin, cadmium and nickel whereas the transport of DBH is the reverse. Thus, for these proteins, there are significant differences in the mechanism of vesicular endocytosis and/or nerve terminal regulatory processes that regulate the targeting of signalling vesicles for retrograde axonal transport. Keywords: Retrograde axonal transport; Nerve growth factor; Endosome Symposium 12: Basic Studies on the Essentiality of DHA for Brain Development [O55]

[O54] Proteins associated with the neurotrophin retrograde transport organelle L. Harvey, M.W. Weible II, S.K. Kaasinen, A.J. Reynolds, I.A. Hendry JCSMR, ANU, Australia E-mail address: [email protected] (I.A. Hendry). Nerve growth factor (NGF) is released from target tissues, internalised by sympathetic nerve terminals via receptor mediated endocytosis and targeted for retrograde axonal transport within a multivesicular body representing signalling organelle. We used biotinylated NGF (bNGF) and fluorescently labelled receptors p75 and TrkA to positively identify the signalling organelle. bNGF accumulated on the distal side of a sciatic nerve ligature, indicating that it was retrogradely transported. When injected into the anterior chamber of the eye all 3 labelled molecules were retrogradely transported to the superior cervical ganglion in vesicle like organelles. However, the proportion of each

Essentiality of docosahexaenoic acid for the nervous system N. Salem Jr.*, S.-Y. Lim, A. Ahmad, B. Lefkowitz, T. Moriguchi NIAAA, NIH, USA It has long been known that the nervous system concentrates and retains the n-3 fatty acid, docosahexaenoate (DHA). Our studies have attempted to ascertain the meaning of this biological adaptation by inducing a dietary deficiency in sources of n-3 fatty acids and thereby decreasing the concentrations of brain and retinal DHA. Behavioral and anatomical measures of structure and function can then be associated with measured decreases in CNS DHA content. Purified diets are constructed based on the AIN-93 formulation but modified to remove sources of essential fatty acids. Fat sources are then formulated to supply linoleic acid alone (n = 3 deficient) or linoleic acid plus alpha-linolenic acid (n-3 adequate), producing a one variable study.

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

Diets are fed thru two generations so that the pups born to dams on these controlled diets are the subjects of the study. In this manner, pups suckled to n-3 deficient dams and weaned to the same diets had an approximately 80% or greater decline in brain and retinal DHA content relative to those fed the n-3 adequate diet. These pups exhibited spatial task deficits on the Morris water maze but had no decrease in locomotor activity or changes in behavior on the elevated plus maze. Quantification of hippocampal neurons indicated no significant change in cell number or layer thicknesses but a decline in the cell soma size in the n-3 deficient rats. Repletion studies indicate that spatial task performance is directly correlated with the brain content of DHA and recovers along with brain DHA. Metabolic studies in humans and rats indicate that preformed DHA is the preferred source of brain DHA rather than via linolenate metabolism. These studies suggest that DHA is an essential nutrient and component of the nervous system. [O56] Essentiality of polyunsaturated fatty acids during cell differentiation and under intrauterine oxidative stress E. Yavin Weizmann Institute of Science, Israel E-mail address: [email protected] (E. Yavin). During the perinatal period a rapid acquisition of polyunsaturated fatty acids (FA) such as docosahexaenoic acid (DHA, 22:6 n3) and arachidonic acid (AA, 20:4 n6) takes place. DHA is found mainly in phosphatidylethanolamine (PE), plasmalogen PE (pPE) and phosphatidylserine phospholipid (PL) species in the inner membrane bilayer, while AA is most abundant in phosphatidylcholine (PC) in the outer bilayer although some is also present in PE and PS. Both polyunsaturated FA are potential candidates for lipid peroxidation, yet, some circumstantial evidence suggests that DHA unlike AA, appears to be neuroprotective. To examine this working hypothesis we have manipulated the FA composition of primary hippocampal neurons (PHN) devoid of glia cells, and of a highly proliferating oligodendroglia cell line, OLN 93, by feeding with various combinations of DHA, AA and dimethyl ethanolamine (dE), a choline base analog. PHN in the absence of glia cells, accumulate predominantly Mead acid (20:3 n9) but rapidly substitute it following AA and DHA supplements. Unlike AA, DHA supplements protect cells from genotoxic stress. A possible PL candidate for non-harmful DHA oxidation was identified in OLN 93 cells after DHA and dE supplements. Based on FA composition by liquid chromatography/mass spectrometry analysis, a number of distinct PL molecular species disappeared while others appeared in the course of FA manipulation. In particular, diacyl PE and pPE were highly enriched in DHA. Following dE supplements PE, pPE and oxidized pPE exhibited a highly unusual

623

FA composition. It is suggested that at least part of the beneficial effect of DHA supplements on fetal brain subjected to oxidative episodes during intrauterine life may result from enhanced free radical scavenging ability of the vinyl ether bond and enriched DHA of brain pPE species. This work was supported by a grant from the Gulton Foundation, NY. [O57] EFA and cerebral development: a study of neurotransmission systems in a model of n-3 PUFA deficiency S. Chalon Lab. Biophysique Me´ dicale et Pharmaceutique, France E-mail address: [email protected] (S. Chalon). During the prenatal and early postnatal periods, longchain polyunsaturated fatty acids are actively accumulated in the brain where they are involved in neurogenesis and synaptogenesis. In rodent models, a chronic deficiency in n3 PUFA greatly modifies the FA composition of cerebral membrane phospholipids, impairs performance in a variety of learning tasks, alters several sensory processes, and affects the dopaminergic and serotonergic functions. We studied the effects of reversion of the diet-deficiency at different developmental stages on these neurochemical functions. Studies were performed on 2-month-old male Wistar rats. The reference groups (deficient and control) were the progeny from females which received either a deficient diet (<6 mg a-linolenic acid/100 g diet) or an equilibrated diet (200 mg a-linolenic acid/100 g). Animals from D0, D7 and D14 groups went from deficient females that received the control diet at 0, 7 or 14 days after parturition. Rats from the D21 group went from deficient females and received the control diet at weaning. At weaning, all rats received the same diet as their dams. The study of FA composition of the main phospholipid classes (PC, PE and PS) was performed in different brain regions. The tyramine-stimulated release of dopamine in the prefrontal cortex and accumbens nucleus and the fenfluramine-stimulated release of serotonin in the hippocampus were measured by microdialysis. The main results showed that when given during the lactating period (D0, D7 and D14), the reversal diet was able to restore both the FA composition of brain membranes and the pharmacological-stimulated release of dopamine and serotonin. By contrast, when given from weaning, this diet allowed partial recovery of biochemical parameters but no recovery of neurochemical factors. The occurrence of profound n-3 PUFA deficiency during the prenatal and post-natal lactating period is an environmental insult that could lead to irreversible damage to specific brain functions. Keywords: a-Linolenid acid diet deficiency; Dopamine; Microdialysis; Serotonin

624

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

[O58] Zellweger’s syndrome: a docosahexaenoic acid deficiency M. Martinez Stauros Children’s Hospital Barcelona, Spain

Symposium 13: Development of Seratonin-Modulated Behaviors [O59] Mid-hindbrain organizer controls dopaminergic and serotonergic neurons W. Wurst

E-mail address: [email protected] (M. Martinez). Zellweger’s syndrome and related peroxisomal disorders are metabolic diseases characterized by the absence of peroxisomes and multiple enzyme deficiencies involving lipid metabolism. Peroxisomal disorders are most severe diseases, leading to death very early in life. Patients have liver involvement, and profound neurological damage, progressing quickly to demyelination, blindness and deafness. Typically, a-oxidation of phytanic, acid as well as boxidation of pristanic acid and the very long chain fatty acids (VLCFA) 24:0, 26:0 and 26:1, are defective, and these compounds increase in plasma. Bile acids and plasmalogen levels are decreased because of defective synthesis. Most significantly, the DHA levels are drastically decreased in the blood and tissues of peroxisomal patients. Generally, damage is attributed to accumulation of VLCFA in the brain. However, given the crucial role of DHA in the CNS and retina, the DHA deficiency is a more probable damaging factor. Thus, since 1991, we have being trying to correct this deficiency by treating patients with highly purified (>90% pure) DHA ethyl ester (DHA-EE), usually in daily oral doses of 200 mg. To date, we have treated 34 patients with different Zellweger variants and ages for varying periods of time. For ethical reasons, treatment has been provided to all patients, disregarding severity and degree of involvement. In contrast to the generally recommended low fat diet, complete nutrition has been provided to all patients. In spite of this, no increase in the VLCFA has ever been observed. On the contrary, a significant decrease in plasma VLCFA has been the rule, together with a rapid normalization of blood DHA and a significant increase in erythrocyte plasmalogen levels. From the clinical point of view, liver function improved in all cases, very often showing a spectacular decrease in liver enzymes. Patients generally became more alert and active, with better social contact, and their vision and muscle tone clearly improved in most cases. In virtually all patients controlled by MRI brain myelination progressed. In one patient, whose brain was actively demyelinating before treatment started, the process was halted and no further myelin loss has been observed to date (she is now 15-year-old). Two peroxisomal twins subjected to different diets and treatment showed how DHA and good nutrition is fundamental in the therapy of peroxisomal disorders. Keywords: DHA; Zellweger’s syndrome; Peroxisomes; Nutrition

Max-Planck Institute of Psychiatry, Germany Midbrain dopaminergic and hindbrain serotonergic neurons play an important role in the modulation of behavior and are involved in a series of neuropsychiatric disorders. Despite the importance of these cells, little is known about the molecular mechanisms governing their development. During embryogenesis, midbrain dopaminergic neurons are specified rostral to the midbrain-hindbrain organizer (MHO), and hindbrain serotonergic neurons are specified caudal to it. We report that in transgenic mice in which Otx2 and accordingly the MHO are shifted caudally, the midbrain dopaminergic neuronal population expands to the ectopically positioned MHO and is enlarged. Complementary, the extension of the hindbrain serotonergic cell group is decreased. These changes are preserved in adulthood, and the additional, ectopic dopaminergic neurons project to the striatum, which is a proper dopaminergic target area. In addition, in mutants in which Otx2 and the MHO are shifted rostrally, dopaminergic and serotonergic neurons are relocated at the newly positioned MHO. However, in these mice, the size ratio between these two cell populations is changed in favor of the serotonergic cell population. To investigate whether the position of the MHO during embryogenesis is also of functional relevance for adult behaviour, we tested mice with a caudally shifted MHO and report that these mutants show a higher locomotor activity. Furthermore, we provide evidence that Wnt1 is involved in mediating the activity of Otx2 in midbrain cell type specification. [O60] Specification of cell fate in the ventral brainstem J. Ericson Karolinska Institute, Sweden Neuronal cell diversity is established by mechanisms that operate in space and over time during central nervous system (CNS) development. Insight has been provided into the initial steps of spatial patterning of neurons along the dorsalventral (DV) and anterior-posterior (AP) axes of the neural tube, whereas less is known about the molecular determinants that control how neural progenitor cells can produce distinct types of neurons in a specific order. We have examined the sequential generation of visceral motor neurons and serotonergic neurons from a common pool of

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

neural progenitors located in the ventral hindbrain. We find that the temporal specification of these neurons varies along the AP axis of the hindbrain, and that the timing of their generation critically depends on the integrated activities of Nkx and Hox class homeodomain proteins (previously implicated in DV and AP patterning, respectively). A primary function of these proteins is to coordinate the spatial and temporal activation of the homeodomain protein Phox2b, which in turn acts as a binary switch in the selection of motor neuron or serotonergic neuronal fate. These findings assign roles for Nkx, Hox and Phox2 proteins in the control of temporal neuronal fate determination, and link spatial and temporal patterning of CNS neuronal fates. [O61] The role of Lmx1b in the development of serotonergic (5-HT) neurons Z.-F. Chen1*, Y.-Q., Ding 1, Z.-Q. Zhao1, M. Scott2, R.L. Johnson3, E. Deneris2 Washington University, USA; 2Case Western Reserve University, USA; 3University of Texas, USA

1

E-mail address: [email protected] (Z.-F. Chen). The specification and differentiation of 5-HT neurons require both extrinsic and intrinsic transcription factors to work in concert or in cascade. A major determinant in a genetic cascade that governs the development of 5-HT neurons is a LIM homeodomain-containing transcription factor, Lmx1b. Lmx1b is expressed in all 5-HT neurons of the central nervous system (CNS). The onset of its expression coincides with the birth of 5-HT neurons, and precedes Pet-1, a gene required for terminal differentiation of 5-HT neurons. In Lmx1b-null embryos, Pet-1 is completely lost despite its transient expression at E11.5. As a result, no central 5-HT neurons are found in Lmx1bnull embryos. Forced expression of Lmx1b in the hindbrain fails to induce the ectopic 5-HT neurons, suggesting that Lmx1b itself is necessary but not sufficient for specifying the fate of 5-HT neurons. To further define the role of Lmx1b in the differentiation and maturation of 5-HT neurons, we have generated Lmx1b mutants using conditional knockout approach. This method should allow us to delete Lmx1b only in the hindbrain and at later stages of development. We are currently analyzing the molecular, cellular and behavior changes of these mutant mice, and the results will be presented and discussed. Keywords: 5-HT neuron; Lmx1b; Development; Mice [O62] Transcriptional control of serotonin modulated behaviors E.S. Deneris*, J.T. Erickson

625

Case Western Reserve University, USA Despite the prominence of serotonin in central neuromodulation and psychiatric disorders the genetic mechanisms governing the generation of serotonin neurons are poorly understood and it is not known how these mechanisms are linked to eventual serotonergic modulation of behavior. We have combined mouse molecular genetic, physiological and behavioral approaches to investigate the function of the Pet-1 ETS gene in embryonic serotonin neuron development and to determine the impact of its function on postnatal behavior. In Pet-1 null mouse embryos serotonin neuron precursors appear to be generated in normal numbers but most of them fail to synthesize serotonin. Consequently, serotonin levels are severely reduced throughout the developing and adult null brain. We obtain Mendelian numbers of Pet-1 null mice at weaning if they are born and nurtured in a pathogen free environment. However, if they are born and nurtured in a non-pathogen free environment approximately one third of the nulls die during the first few days of life. This suggests that Pet-1 nulls suffer from a defect in homeostatic responsiveness to environmental stressors during an early critical period due to a genetic vulnerability that impacts serotonin signaling. Significantly, during this early critical period Pet-1 null neonates have highly abnormal breathing patterns, which are often interrupted by apnea. Our behavioral analyses in null adults indicated that the defective development of serotonin neurons is followed by heightened aggressive and anxiety-like behavior. Together our findings suggest the existence of a Pet-1-dependent genetic program that selectively couples steps in serotonin neuron differentiation to serotonergic modulation of behavior later in life. It will be important to determine whether the Pet-1-dependent genetic program operates in humans and if so whether genetically transmitted variation within it exists and contributes to interindividual differences in behavioral and physiological traits related to aggression, anxiety and homeostatic function. Keywords: Serotonin; Pet-1; Aggression; Breathing Symposium 14: Control of Oligodentrocyte Development [O63] Control of oligodendrocyte development by transcription factors in the vertebrate CNS H. Arnett1, M. Kitada1, S. Fancy2, K. Ligon1, J. Alberta1, R. Franklin2, C. Stiles1, D. Rowitch1* Harvard Medical School, Boston, USA; 2Cambridge University, UK

1

Vertebrate oligodendrocytes have diverse roles in the maintenance of neurological function. Until recently, many aspects of the oligodendrocyte developmental program were poorly understood. It is now clear that mechanisms of

626

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

oligodendroglial development in vivo show striking similarity with those for neuronal subtypes including emergence from localized regions of the neural tube, involvement of common signalling pathways and downstream transcription factors. For instance, function of the bHLH transcription factors, Olig1 and Olig2, is required for development of motor neurons and oligodendrocytes in the developing spinal cord. This talk will focus on regulation of Olig protein activity and coordinated actions of other transcription factors with roles during oligodendrocyte development. Data will be presented in support of the view that mechanisms of embryonic oligodendrogenesis are conserved in stem cell populations of the normal adult brain as well as in neurological disease states. [O64] Fate-mapping the rodent forebrain and spinal cord: complex origins of oligodendrocytes N. Kessaris*, M. Fogarty, M. Grist, W.D. Richardson University College London, UK E-mail address: [email protected] (N. Kessaris). Oligodendrocytes in the central nervous system are derived from proliferating migratory progenitor cells (OLPs). In the rodent telencephalon, the first OLPs appear in the medial ganglionic eminence (MGE) and anterior entopeduncular area about a week before birth (E12.5 in the mouse). OLPs first arrive in the cerebral cortex some days later, apparently migrating from ventral forebrain territories. A dramatic expansion of the cortical OLP population then occurs shortly before birth. We wished to determine whether this population explosion results from rapid proliferation of inwardly-migrating OLPs or else by activation of an additional source of OLPs within the developing cortex or elsewhere. To address this question we used the Cre-lox approach to mark different precursor populations within the forebrain of transgenic mice. Our results show that the first OLPs to appear in the embryonic cortex do indeed migrate from the MGE. However, this immigrant population is rapidly replaced during postnatal life by a second wave of OLPs that originates outside the MGE, so that the MGEderived population is essentially displaced from the cortex within the first postnatal week. We are now investigating the origin of the second wave of OLPs in the forebrain. We are taking a similar approach to fate map the spinal cord neuroepithelium. There is a major source of OLPs in the ventral cord, in a region of the neuroepithelium that initially gives rise to motor neurons. We found that a minor population of OLPs is generated more dorsally, within the Dbx1/Dbx2 expressing region of the neuroepithelium (spanning the dorso-ventral midline). These latter OLPs (approximately 2% of the total) seem to undergo more restricted migration than the majority and mainly populate the dorsolateral white matter.

Keywords: Oligodendrocytes; Forebrain; Spinal cord; Cre [O65] Integrins regulate the time and space of growth factor signalling in oligodendrocyte development C. ffrench-Constant*, H. Colognato, W. Baron, J. Relvas, M. Olsen, L. Decker University of Cambridge, UK The control of myelination in the developing CNS requires precise regulation of proliferation and apoptosis in both oligodendrocyte precursors and newly-formed oligodendrocytes. There is considerable evidence that soluble growth factors regulate these facets of behaviour, but much less is known about the necessary cell–cell interactions that ensure growth factor responsiveness only occurs in appropriate locations ie adjacent to axons to be myelinated. I will describe experiments showing that integrins control both the space and time of signalling by two growth factors, platelet-derived growth factor (PDGF) and neuregulin, both known to act as mitogens and survival factors in the oligodendroglial lineage. These experiments show that, at physiological concentrations of growth factors, signalling requires the association of ligand-bound integrins with the growth factor receptors. The identity of the associated integrin then determines the response to the growth factor— alphaVbeta3 stimulates proliferation while alpha6 beta1 promotes cell survival. Equally, the availability of integrin ligand determines in which cells these responses occur, with the laminin-alpha2 chain-containing laminins expressed on the surface of axons prior to myelination ensuring the survival of only those oligodendrocytes that establish axonal contact and so providing a mechanism for target-dependent survival. An important mechanism of integrin/growth factor interaction is integrin activation by signalling from the growth factor receptor, with the activated integrin then triggering the downstream signalling pathways that promote proliferation or survival. The two integrins are found in distinct lipid microdomains, with the PDGF receptor and a6b1 integrin present in lipid rafts that are required for normal survival signalling, so providing a mechanism by which the integrin/growth factor interactions can be regulated independently during development. Keywords: Apoptosis; Myelin; PI3K; MAPK [O66] Differential expression of FGF receptors 1, 2 and 3 in the embryonic and postnatal mouse brain: comparison to Olig2, Pdgfra and Plp/dm20, PLP–EGFP. Implications for OL development Y. Kaga1, V. Lakhina2, R. Remedio2, S. Tole2, R. Bansal1* University of Connecticut Medical School, USA; 2Tata Institute for Fundamental Research, India 1

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

FGF receptors FgfR1, FgfR2 and FgfR3 are differentially regulated during oligodendrocyte (OL) maturation in vitro: FgfR3 is expressed by OL progenitors whereas FgfR2 is expressed by differentiated OLs, Here we have used in situ hybridization to investigate the expression patterns of FgfR1-3 and compare them to the putative OL progenitor markers Olig2, Pdgfra and Plp/dm20 as a function of development in vivo, in particular at sites of OL specification, migration or differentiation in the mouse forebrain and cerebellum. In addition, we compared them with enhanced green fluorescent protein (EGFP) expression driven by PLP gene promoter in a transgenic mouse. We show that at early stages, FgfR1-3 expressions overlaps with that of Olig2 in the embryonic ventricular zone of the lateral and medial ganglionic eminences. Further, a scattered population of cells expressing FgfR3 (but not FgfR1 or FgfR2) in the ventral telencephalon appear to arise from the ventricular zone, and at later stages are found more dorsally in the cortex, in an overall pattern similar to Olig2 and/or Pdgfra. Plp/dm20 expressing cells are also EGFP+ at all time points. Postnatal expression of FgfR2 increases with age, more prominently in specific regions, including the cortical and cerebellar white matter and optic nerve. Thus, the differential expression pattern of FgfR2 and FgfR3 observed in vivo suggests that their expression is developmentally regulated in a manner consistent with the pattern of their expression in culture. These data provide further insights into role of FgfRs in OL development, and they emphasize that these receptors are positioned both spatially and temporally to impact OL generation in vivo. We are currently investigating the role of each FGF receptor through the application of FGF receptor null mice. Supported by NIH grant NS 38878 (RB). [O67] Oligodendrocyte precursors development requires vascular endothelial growth factor VEGF-C B. LeBras1, M. Karkkainen 2, L. Yuan3, J. Homman-Ludiye 1, P. Keiko2, M.-J. Barallobre1, B. Zalc1, A. Eichmann3, K. Alitalo2, J.-L. Thomas1* 1

2

University PM. Curie, France; Biomedicum, Helsinki, Finland; 3Colle`ge de France, France

627

[O68] Using embryonic stem cells to study neural development M. Bibel1, N. Plachta2, Y.-A. Barde2* 1

Novartis Pharma Basel, Switzerland; 2Biocenter Basel, Switzerland The isolation of mouse embryonic stem (ES) cells has opened the possibility to generate unlimited numbers of any cell type. This is of special interest to neurobiologists, as it is essentially impossible to isolate from wild-type and mutant animals homogeneous cell populations in sufficient quantities to biochemically characterize brain neurons. However, still very little is known about the in vitro conditions required for the generation of defined neuronal precursors that may give rise to specified neuronal phenotypes. We recently found that by selecting for rapidly dividing ES cells, homogenous populations of neuronal precursors can be generated with the characteristics of Pax-6-positive radial glial cells. In vitro, they rapidly become neurons with the biochemical and functional characteristics expected for a lineage recently described in the rodent cortex by Go¨ tz and co-workers. This finding also offered the possibility to test the differentiation potential of homogenous, defined neuronal precursors participating in normal brain development. We implanted them in place of a portion of the chick neural tube and found after 6 days very large numbers of donor-derived neurons in the spinal cord. The analysis of the cell types generated by these precursors revealed that they can interpret local cues to generate the cell types expected for the Pax-6-positive radial glial cell lineage. In the DRG, the donor-derived neurons failed to extend axons and to express the expected markers. We conclude that Pax-6 positive radial glial cells generated from ES cells have a restricted developmental potential. Like their neuronal progeny in vitro, wild-type and mutant cells can now be studied with regard to their patterns of gene and protein expression, and their response to specific growth factors. Keywords: Stem cells; Radial glial cells; Pax-6; Transplantation Symposium 15: Axonal Development to Regeneration

Vascular endothelial growth factor C (VEGF-C) was initially identified in the vascular system, where it is selectively required for development of lymphatic endothelial cells. We here investigated possible actions of VEGF-C on nervous system cells. VEGF-C showed a potent effect on the proliferation, survival and migration of OPCs in vitro. Mice lacking one allele of VEGF-C showed a dramatic loss of OPCs in the embryonic and neonatal optic nerve, while retinal ganglion cells and astroglial cells were unaffected. VEGF-C thus represents a novel growth factor selectively required for OPC development. Interestingly, VEGF-C and VEGF-A target complementary cell populations in the nervous as well as in the vascular system.

(Sponsored, in part, by an award from the Conferences Committee of the International Society for Neurochemistry) [O69] Survival and outgrowth capabilities of injured brainstem-spinal neurons W. Tetzlaff, B.K. Kwon, J. Liu, K. Khodarahmi, R. Lane, D. Sutherland, et al. University of British Columbia, Canada

628

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

E-mail address: [email protected] (W. Tetzlaff) The survival and neuronal cell body response to axonal injury plays an important role in the failure of CNS neurons to regenerate. In higher vertebrates, an injury to the spinal cord often induces massive atrophy of the axotomized brainstem-spinal neurons, which in case of rubrospinal neurons lead to reports of 40–50% death. We found that rubrospinal neurons fail to (re)-express a variety of genes and signaling factors after axotomy that are seen at increased levels in regenerating peripheral neurons, e.g. Brain Derived Neurotrophic Factor (BDNF) or cAMP. The exogenous application of BDNF to the rubrospinal neuron cell bodies prevented their atrophy, promoted the expression of regeneration-associated genes (e.g. GAP-43) and stimulated their regeneration into free ending peripheral nerve transplants. Importantly, this treatment was still successful when initiated 1 year after spinal cord injury fully reversing the chronic atrophy and revealing that there was no significant rubrospinal neuron loss. Application of BDNF to the spinal cord was no longer successful at 2 months. After acute treatment (chronic not yet tested) this cell body treatment with BDNF promoted the regeneration of rubrospinal neurons across peripheral nerve bridges, with some sprouting into the distal spinal cord. This combination regimen correlated with significant improvements in a foodpellet reaching test and cylinder righting test. The application of cAMP to acutely axotomized rubrospinal neurons also prevented their atrophy and stimulated the expression of GAP-43. Interestingly, several cAMP treated rats displayed pronounced sprouting of rubrospinal axons into the site of injury and beyond; this was never observed in controls. Some axons regenerated for over a 1 mm within the caudal dorsolateral funiculus. Thus, targeting the neuronal cell bodies is a promising approach to resuscitate chronically injured neurons and to increase their outgrowth capacities after both acute and chronic injury.

tory reaction in the eye, many RGCs survive axotomy and regenerate axons into the inhibitory optic nerve environment. In dissociated cultures, we found that this regeneration is stimulated by a 14 kDa protein that is secreted by macrophages (MDP14), acting in combination with the carbohydrate mannose, which is constitutively present in the eye, and requiring elevated intracellular cAMP. When delivered together with cAMP analogs into the vitreous, MDP14 stimulates optic nerve regeneration in vivo. The profile of gene expression induced by these factors was investigated in RGCs isolated via fluorescent-activated cell sorting. Whereas axotomy alters the expression of numerous genes, macrophage-derived factors stimulate the expression of a relatively small number of additional genes, including GAP-43, SPRR1A, and several transcription factors, that transform RGCs into a regenerative state. The intracellular transduction pathway leading to these changes involves a purine-sensitive protein kinase that we have identified. In vivo, activation of this kinase with inosine promotes the growth of new brain connections and improves functional outcome after a cortical stroke. Finally, we show that axon regeneration can be enhanced further with gene therapy. We have used AAV to transfect RGCs with a dominant-negative form of NgR (NgRdn), a receptor that mediates the inhibitory effects of 3 myelin proteins (MAG, NogoA, OMgp). NgRdn expression increased regeneration dramatically when RGCs were in a growth-activated state, but not otherwise. In contrast, overexpression of wild-type NgR prevented RGCs from regenerating axons. These studies point to novel ways of inducing axon regeneration in the CNS, and demonstrate that successful regeneration will probably require both activation of neurons’ intrinsic growth state and overcoming inhibitory signals. [O71] Extrinsic and intrinsic control of axon regeneration J. Fawcett

Keywords: Neuronal survival; Axonal regeneration; Spinal cord injury; Rubrospinal

Cambridge University Centre for Brain Repair, UK

Acknowledgements

E-mail address: [email protected] (J. Fawcett).

Supported by CIHR, BC-Neurotrauma, and Christopher Reeve Paralysis Foundation.

Whenever the CNS is injured a reactive process is initiated known as glial scar formation which acts as a barrier to the regeneration of damaged axons. Various lines of evidence suggest that the main inhibitory molecules in the glial scar are chondrointin sulphate proteoglycans (CSPGs), most of which have axon growth inhibitory properties, and are upregulated after CNS injury. Not only are the protein cores upregulated, but also there is more glycosaminoglycan (GAG) attached to them, and these GAGs are responsible for much of the inhibition. The final stage of GAG synthesis is sulfation, which can occur in three positions, mediated by seven sulfotransferases. 6-Sulfated GAG, which is particularly inhibitory to axon growth is upregulated after injury. All CSPGs possess GAG chains of similar structure

[O70] Axon regeneration in the mature CNS: extrinsic signals and intracellular signaling pathways L.I. Benowitz1,2, Y. Yin1,2, D. Fischer1,2, N. Irwin1,2 1

Children’s Hospital, Boston, USA; School, USA

2

Harvard Medical

As in other parts of the mature CNS, retinal ganglion cells (RGCs) do not normally regenerate injured axons, and soon undergo apoptosis. However, by stimulating an inflamma-

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

629

produced by the same enzymes, and these can be removed by chondroitinase. We therefore tested to see whether GAG digestion by chondroitinase would promote axon regeneration in vivo. We first treated mechanical lesions of the nigrostriatal tract, and saw regeneration of about 4% of axons back to their target. Next dorsal column lesions of the spinal cord at C4 were treated. Both sensory and corticospinal axons regenerated in treated cords, and there was rapid return of function in beam and grid walking tests. CSPGs are also involved in the control of plasticity by coating many neurones and their dendrites in perineuronal nets. Treatment of the visual cortex of adult animals can reactivate ocular dominance plasticity, which normally terminates at the end of the critical period. Axons vary greatly in their regenerative response after damage. Their regenerative ability in vivo correlates well with their ability to regenerate their growth cones in vitro. This ability is greater in axons that contain abundant protein synthesis machinery, and can be inhibited by blockers of translation.

phy. At PD 25, D4-selective antagonists, CP-293,019, L745,870 and U-101,958 dose-dependently reversed lesioninduced hyperactivity. Other DA or serotonin receptor antagonists did not attenuate hyperactivity in juvenile lesioned rats. Autoradiographic analysis indicated that D4 receptors in lesioned rats were substantially increased in caudate-putamen when the animals at PD 25. Neonatal lesions also led to minor changes in D1 and D2 receptor binding in forebrain regions. However, behavioral hyperactivity most closely paralleled the temporal pattern of D4, and not D1 or D2 receptor expression in basal forebrain after lesioning. These neurochemical and pharmacological findings further support the genetic linkage studies and indicate that D4 receptors play a pivotal role in mediating behavioral hyperactivity in juvenile rats. In addition, these results suggest that D4-selective antagonists may represent much-needed innovative therapy for ADHD.

Symposium 16: Neurobiological Bases of Developmental Disorders

[O74]

(Sponsored, in part, by an award from the March of Dimes)

Keywords: ADHD; Dopamine D4; 6-Hydroxydopamine; Motor hyperactivity

Developmental role of neuroligins in synapse formation: implications for autism A. El-Husseini

[O73]

University of British Columbia, Canada

ADHD: new genes, novel mechanisms and innovative therapy

Autism is one of the most genetically determined neuropsychiatric disorders. The risk for first degree relatives is approximately 100-fold higher than the risk in the general population and the concordance in monozygotic twin (60–90%) is much higher than the concordance in dizygotic twins (3%). Although specific genes implicated in autism have not been firmly identified, mutations in two members of the neuroligin (NLG) family of cell adhesion molecules implicated in synapse formation, namely NLG-3 and NLG-4, have been identified in two independent French families. These findings suggest that mutations in NLGs may result in the manifestation of autism, possibly through malfunction of the synapse. Recently, we have obtained new exciting results which demonstrate that appropriate interactions between NLG and other associated proteins, such as the postsynaptic density protein PSD-95, are critical for maintaining an appropriate excitatory/inhibitory synaptic ratio, an effect that is critical for regulating neuronal excitability. These results indicate that defects in NLG functions and/or association with synaptic proteins important for building synaptic contacts may result in abnormal neuronal excitability. Our new findings are consistent with a recent model which proposes that an imbalance in neuronal excitation/inhibition is a major cause for autism.

F.I. Tarazi1,2*, K. Zhang1,2, R.J. Baldessarini1,2 Harvard Medical School, USA; 2Mailman Research Center, USA 1

E-mail address: [email protected] (F.I. Tarazi). Attention deficit-hyperactivity disorder (ADHD) is a neuropsychiatric syndrome characterized by hyperactive, inattentive, and impulsive behavior. Recent genetic association studies indicate that the 7-repeat dopamine (DA) D4 receptor allele (D4.7) is more prevalent in patients with ADHD. Features of ADHD are modeled in juvenile rats following lesioning of pups with the neurotoxin 6hydroxydopamine (6-OHDA) to destroy DA projections to forebrain. Juvenile lesioned rats exhibit age-specific locomotor hyperactivity that is dose-dependently antagonized by stimulants commonly used to treat clinical ADHD. We investigated the role of DA D4 receptors in behavioral hyperactivity in juvenile rats with neonatal 6OHDA lesions produced by intracisternal injections of 6OHDA or vehicle on postnatal day (PD) 5; their locomotor activity was measured on PD 25. D4-selective agents (10– 30 mg/kg) were given immediately prior to behavioral testing, rats were sacrificed 24 h after the testing, and brains were processed for in vitro receptor autoradiogra-

Keywords: Synapse development; Cell adhesion molecules; Autism; Postsynaptic density

630

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

[O75] Prenatal stress and developmental insults as concurrent factors underlying neurobiology of schizophrenia V.G. Barros, M.C. Antonelli* Universidad de Buenos Aires, Argentina E-mail address: [email protected] (M.C. Antonelli). Increased vulnerability to psychiatric and neurological disorders, such as schizophrenia, depression, Parkinson’s disease and Tourette’s syndrome, has been associated with high levels of stress. Several lines of evidence have consistently observed that the ability of an individual to cope with a stressful event in adult life is greatly influenced by previous early experience with stress, especially during the prenatal period. Conversely, postnatal stimulation might counteract the biological effects observed after prenatal stress. In our laboratory, repeated restraint during the last week of pregnancy of the rat was used as a model of prenatal stress, and adoption at birth was used to change the postnatal environment. The densities of dopamine D2-like, glutamate ionotropic NMDA and Group III metabotropic glutamate, and benzodiazepine receptors were mesasured by quantitative autoradiography in several forebrain regions of adult male offsprings. Moreover, the ontogeny of dopamine receptors were determined and various behavioral paradigms were tested in adult prenatally stressed rats. Taken together our results will try to demonstrate that restraint stress exerted on the gestant mother produces long-lasting effects on receptor expression in several forebrain areas of the adult offsprings as well as behavioral changes, that can however be reversed by adoption procedures. Moreover, changes in dopamine receptors are only observed after puberty indicating that the effect of prenatal stress is not directly acting on the developing dopamine receptors. Alternatively, our observations may suggest that the insult received prenatally is modifying the sensitivity of the receptors to the modulation of sex steroids during puberty. The gestational experience of the mother was important in the outcome of the pup receptor levels denoting the importance of maternal behaviour in the normal maturation of synaptic structures and function. The complex pattern of DA and Glu receptor changes reflects the high vulnerability of DA and Glu systems to variations in the pre and postnatal environment. Keywords: Dopamine receptors; Glutamate receptors; Prenatal stress; Cross-fostering [O76] Developmental neurobiology of Tourette’s syndrome W. Zheng1, P. Kalanithi1, M. DiFiglia2, H. Grantz1, C.B. Saper3, J.F. Leckman1, F.M. Vaccarino1* Yale University, USA; 2Massachusetts General Hospital, USA; 3Harvard Medical School, USA 1

Tourette’s syndrome (TS) is a common childhood neurological disorder characterized by motor and vocal tics. Both genetic and environmental factors figure in TS etiology. Anatomical imaging studies comparing TS patients to age-matched controls have found that lenticular nucleus volumes are reduced in TS adults, while caudate nucleus volumes are reduced in both adults and children with TS. To understand cellular and circuitry alterations in the basal ganglia, we have examined postmortem material from TS patients and age-matched controls using unbiased stereological techniques. The examination of 3 cases of TS revealed an altered distribution of parvalbumin (PV)containing neurons, which are inhibitory GABAergic neurons, in the basal ganglia. There was a 2-fold increase in density of PV cells the internal segment of the globus pallidus (GPi) and a decrease in the PV neuron density in the putamen, without any change in volume in these regions. No changes in PV neurons were present in the external segment of the globus pallidus (GPe), and no changes in either number or density of glial cells were found in any of these regions. Total neuron density in the GPi, GPe, or the putamen was not different between control and TS subjects, suggesting that significant basal ganglia change seems restricted to interneurons. Consistently, enkephalin and substance P staining indicated no change in density of striatal projection neurons to the GPe and GPi. The imbalance in PV neuron distribution suggests a developmental change in cortico-striato-cortical circuitry. Studies in mouse have shown that the medial ganglionic eminence (MGE) generates all the striatal interneurons in addition to the PV projection neurons of the GPi. Thus, a possible explanation for our data is an altered development or migration of neurons arising from the MGE in TS patients. The small sample size precludes firm conclusions; future postmortem studies should clarify these findings. Keywords: Basal ganglia; Interneuron; Tourette’s Syndrome; Human Symposium 17: Genetic and Epigenetic Metabolic Dysfunctions with Consequences for Neurodegeneration and Neurogenesis [O77] Characterization of ASPA knockout mice brain: a novel observation of parallel neural cell death and genesis of progenitor cells in canavan diseased mice brain S. Kumar1, R. Somyalakshmi1, C. Ghiani1, S.L. Daniels1, R. Chang1, J.C. Biancotti1, S. Surendran2, R. Matalon2, J. de Vellis1 1

University of California, USA; 2University of Texas, USA

Canavan disease (CD) is widely distributed, despite its known prevalence among Ashkenazi Jews. CD is caused by an autosomal recessive mutation in enzyme aspartoacylase

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

(ASPA) gene, resulting in build up of its substrate, Nacetyl-aspartate (NAA), and lack of acetate, an important component of myelin sheath. The pathophysiology includes leukodystrophy, a spongy degeneration of the white matter and a short life expectancy. Recent study places ASPA gene expression in oligodendrocyte lineage. In human, a much needed study of early developmental events due to mutation in ASPA was not possible, until a recent generation of ASPA KO mouse, which mimics CD symptoms. The level of ASPA enzyme activity was reported undetectable in the knockout mice with a sharp elevation of NAA in the sub-cortical region of KO mice. Our observation shows the lack of ASPA in oligodendrocytes leads to a massive cell death in white matter regions of cerebellum, corpus callosum and lateral ventricle. In addition, cell death could be seen in other regions of the brain, including large projection neurons in the striatum. Interestingly, a simultaneous robust mitotic activity is detected in the cortical tissue of diseased mice. An array of progenitor neural cell markers, such as NG2, GD3, PSA-NCAM, Nestin, and Vimentin, have been identified in adjacent sections of the brain where cell death was also detected, suggestive of a concurrent trans-lineage expression of neural progenitors in KO mice Brain. Preparation and characterization of neural cell cultures from embryonic brain, E15, and 6-month-old diseased mice have provided further insights into early developmental profile of these mice. Keywords: Canavan disease mice; Oligodendrocytes; Apoptosis; DNA synthesis [O78] Heme oxygenase-1 induction and thiol homeostasis in aging rat brain V. Calabrese*, A. Ravagna, C. Colombrita, F. Spadaro, G. Tomaselli, M. Sapienza, E. Guagliano G. Scapagnini, A.M. Giuffrida Stella University of Catania, Italy Oxidative damage plays a crucial role in the brain aging process and induction of heat shock proteins (HSPs) is utilized by brain cells in the repair process following various pathogenic insults (Calabrese, 2004). In the present study we investigated changes in heat shock protein expression and antioxidant status in rats 6-, 12- and 28-month-old. In all brain regions examined mRNA and protein synthesis of heme oxygenase-1 (HO-1) and Hsp90 increased significantly and progressively with age up to 28 months; at this age the maximum induction was observed in the hippocampus and S. Nigra followed by cerebellum, cortex, and striatum. Interestingly, a significant positive correlation between decrease in GSH/GSSG ratio and increase in HO-1, Hsp90, hydroxynonenal (HNE) and protein carbonyl levels was observed in all brain regions examined during aging.

631

Our results sustain a role for GSH redox state in Hsps synthesis. Increased expression of Hsps promotes the functional recovery of oxidatively damaged proteins and protects cells from progressive age-related cell damage (Morimoto, 2004). Conceivably, heme oxygenase signal pathway, by increasing bilirubin levels, may represent a crucial mechanism of defence against free radical-induced damage occurring in aging brain and in neurodegenerative disorders. References Calabrese, V., et al., 2004. Mech. Age Dev. 125, 325–335. Morimoto R.I., et al., 2004. Mol. Biol. Cell. 15, 657–664. [O79] pH and mitochondrial dysfunction in myelin proteolipid protein gene overexpression Z. Zhang, M. Franklin, R.P. Skoff*, D. Bessert, C. Mullins Wayne State University School of Medicine, USA E-mail address: [email protected] (R.P. Skoff). The majority of proteolipid protein (PLP) gene mutations in humans are caused by overexpression of the native PLP gene. PLP duplications in humans and in rodents are usually lethal. In humans and rodents, the hallmarks of PLP duplications are dysmyelination, oligodendrocyte death, and axonal abnormalities. The pathways by which overexpression of native PLP induce apoptosis and cause axonal abnormalities have not been investigated. In an in vitro study (J. Neurosci., 02), we found that death of dorsal root ganglion cells was greater when co-cultured with non-neural cells expressing the PLP than with cells expressing the DM20 isoform. Co-culture of media from PLP specific expressing cells caused neuronal loss. The media from the PLP expressing cells was acidic, suggesting acidity contributes to neuronal loss. We investigated in vivo whether the pH of extracellular space (ECS) of PLP overexpressing mice was acidic using a time lapse proton flux assay. The increase in protons between PLP overexpressors and wildtype mice is 0.4  1014. We estimate the pH of the ECS of the PLP overexpressors to be 5.63 compared to 7.2 for wildtype mice. The increase in pH of the ECS contributes to the phenotype in the PLP overexpressors. We investigated the integrity of mitochondria (mt) from PLP overexpressors as cytochrome c release leads to H+ influx first into the cytosol and then into the ECS. Mitochondrial/cytosolic preps show that COX1, an inner mtl membrane protein, remains in the mt but cytochrome c and other outer mtl membrane proteins, are abundant in the cytosol of overexpressors. These findings indicate the outer but not the inner mtl membrane is compromised in these mutants. We hypothesize that native PLP is mistargeted to the mtl outer membrane because (1) PLP is abundant in the mtl fraction of overexpressors and (2) electron microscopic gold immunocytochemistry reveals

632

Oral Abstracts / Int. J. Devl Neuroscience 22 (2004) 601–632

PLP in mt of oligodendrocytes but not in mt of other neural cell types. Our findings show a molecular pathway by which overexpression of PLP causes autocrine and paracrine abnormalities in mammals. [O80] Regulation of neurogenesis in the adult brain by cytokine and growth factors P.F. Bartlett* The University of Queensland, Australia E-mail address: [email protected] (P.F. Bartlett). The production of neurons in the adult brain is now well documented to occur in a number of regions and to be influenced by external and disease processes. Although a precise functional role for these neurons has not been established, mounting evidence suggests it may underpin some higher-brain functions and has the potential to restore function following disease and mental illness. Thus, finding the key molecular regulators of neuronal

production in the adult brain will provide both new insights onto this process and identify drug-candidates for the treatment of degenerative, traumatic and psychiatric diseases. To identify these candidates we have purified the stem cell in the adult brain of rodents to near homogeneity (Rietze et al., 2001. Nature) and analysed their expression profile by Affymetrix gene-microarray. We have identified two receptors expressed on the surface of the stem cell, LIFreceptor and the p75NTreceptor, and one on the progenitor cell, Growth Hormone receptor (Turnley, et al., 2002. Nat. Neurosci.) which appear to be involved in the regulation of neuronal production. The role of these receptors in regulating stem cell differentiation and neuron production will be discussed in detail. Keywords: Neural stem cell; Adult neurogenesis; Gene expression; Flow cytometry References Rietze et al., 2001. Nature. Turnley, et al., 2002. Nat. Neurosci.