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It was hypothesized that superparamagnetic nanoparticles encapsulated in a silica shell with a fluorescent dye could be functionalized with axonal tracers and could be used for serial, non-invasive imaging with magnetic resonance imaging (MRI) for axon tract tracing. Nanoparticles functionalized with amine, octadecyl, silica, and biotinylated dextran amine were manufactured and characterized with MRI, scanning electron microscopy, and UV-visible, infrared, and fluorescence spectroscopy. Nanoparticle concentrations of 10 mM were not toxic to adult rat neural progenitor cells (NPCs) and labeled approximately 90% of cells. Nanoparticles were assessed for anterograde and retrograde tract tracing in adult rat models. With MRI and microscopy, the nanoparticles did not appear to trace axons but did provide an MRI signal for up to 3 weeks post implantation. While functionalized nanoparticles did not appear to trace axons, they are not toxic to NPCs and may be used as a MRI contrast agent in the neural axis.

An important mechanism during nervous system development is the guidance of axons by chemical gradients. The structure responsible for responding to chemical cues in the embryonic environment is the axonal growth cone -- a structure combining sensory and motor functions to direct axon growth. In this thesis, we develop a series of mathematical models for the gradient-based guidance of axonal growth cones, based on the idea that growth cones might be optimised for such a task. In particular, we study axon guidance from the framework of Bayesian decision theory, an approach that has recently proved to be very successful in understanding higher level sensory processing problems. We build our models in complexity, beginning with a one-dimensional array of chemoreceptors simply trying to decide whether an external gradient points to the right or the left. Even with this highly simplified model, we can obtain a good fit of theory to experiment. Furthermore, we find that the information a growth cone can obtain about the locations of its receptors has a strong influence on the functional dependence of gradient sensing performance on average concentration. We find that the shape of the sensitivity curve is robust to changes in the precise inference strategy used to determine gradient detection, and depends only on the information the growth cone can obtain about the locations of its receptors. We then consider the optimal distribution of guidance cues for guidance over long range, and find that the same upper limit on guidance distance is reached regardless of whether only bound, or only unbound receptors signal. We also discuss how information from multiple cues ought to be combined for optimal guidance. In chapters 5 and 6, we extend our model to two-dimensions, and to explicitly include temporal dynamics. The two-dimensional case yields results which are essentially equivalent to the one dimensional model. In contrast, explicitly including temporal dynamics in our leads to some significant departures from the one-dimensional and two-dimensional models, depending on the timescales over which various processes operate. Overall, we suggest that decision theory, in addition to providing a useful normative approach to studying growth cone chemotaxis, might provide a framework for understanding some of the biochemical pathways involved in growth cone chemotaxis, and in the chemotaxis of other eukaryotic cells.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The ophthalmic, maxillary and mandibular axon branches of the trigeminal ganglion (TG) provide cutaneous sensory innervation to the vertebrate face, and multiple families of guidance cues amalgamate to direct the navigation of these branches. However, target tissue specific guidance cues that discriminately guide the three TG axon branches are unknown. Prior work demonstrated that EphAs and ephrin-As could discriminately direct dorsal versus ventral motor axon projections into the hindlimb. Similarly, do EphA tyrosine kinases and ephrin-A ligands discriminately guide trigeminal ganglion ophthalmic (TGop) lobe versus maxillomandibular (TGmm) axon projections into the chick embryo face? The aims of this work were two-fold: (1) to identify candidate EphA and ephrin-A molecules during TG axon guidance, and (2) to detennine the functional significance of TG axon EphA and ephrin-A signalling in vitro. This study identified EphA3, EphA4, ephrin-A2 and ephrin-A5 at stages 13, IS and 20, as putative guidance cues to TG axons. TG-EphA3 and -ephrin-A5 were identified as putative receptors to guidance cues expressed in the target fields. EphA3 receptor was differentially expressed, with the TGop lobe expressing higher levels compared to the TGmm lobe. However, ephrin-A5 transcript was not differentially expressed between the two ganglion lobes. In a substratum choice in vitro assay, ephrin-AS-Fc was found to repel approximately 50% of axons growing from stage 20 whole TG explants. This population of axons was identified to be from the TGop lobe. The in vitro data supports the contention that during facial development there may be trigeminal ganglion lobe specific guidance of TGop in comparison to TGmm peripheral sensory axonal projections to target fields coordinated through EphA3 and ephrin-A2/A5 repulsive interactions. In vitro, EphA4-Fc caused morphological changes to TG growth cones, which is likely mediated through TG ephrin-A5 reverse signaling. Furthermore, this study provided in vitro evidence that trigeminal ganglion axons were not responsive to EphA4-Fc, possibly implying that EphAs expressed in the target fields were not repulsive to ganglionic axons during pathfinding. The data suggests that EphN ephrin-A interactions may specifically guide TGop projections into the ophthalmic process similar to lateral motor axon guidance into the hindlimb. For the first time, a model of how EphN ephrin-A interactions and other families of guidance cues may act in concert to guide trigeminal ganglion axons is suggested. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1179603 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005

Hannover, Univ., Diss., 2007

xv, 83 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / This dissertation describes research to identify genes involved in specification, patterning and development of zebrafish primary motoneurons. We first examined the spatiotemporal expression patterns of retinoic acid and retinoid X receptor mRNAs to determine whether particular ones might be involved in motoneuron specification or patterning. Retinoic acid and retinoid X receptor mRNAs are expressed at the right time to pattern motoneurons, but the expression patterns did not suggest roles for particular receptors. In contrast, netrin mRNAs are expressed in specific motoneuron intermediate targets and knockdown experiments revealed an important role in development of VaP motoneurons. Two identified motoneurons, CaP and VaP, initially form an equivalence pair. CaPs extend long axons that innervate ventral muscle. VaPs extend short axons that stop at muscle fibers called muscle pioneers; VaPs later typically die. Previous work showed that during extension, CaP axons pause at several intermediate targets, including muscle pioneers, and that both CaP and muscle pioneers are required for VaP formation. We found that mRNAs for different Netrins are expressed in intermediate targets before CaP axon contact: netrin 1a in muscle pioneers, netrin 1b in hypochord, and netrin 2 in ventral somite. We show that Netrins are unnecessary to guide CaP axons but are necessary to prevent VaP axons from extending into ventral muscle. Netrin 1a is necessary to stop VaP axons at muscle pioneers, Netrin 1a and Netrin 2 together are necessary to stop VaP axons near the hypochord, and Netrin 1b appears dispensable for CaP and VaP development. We also identify Deleted in colorectal carcinoma as a Netrin receptor that mediates the ability of Netrin 1a to cause VaP axons to stop at muscle pioneers. Our results suggest Netrins refine axon morphology to ensure final cell-appropriate axon arborization. To learn whether Netrin proteins diffuse away from their sources of synthesis to function at a distance, we are developing Netrin antibodies. If successful, the antibodies will provide the research community at large with a new tool for understanding in vivo Netrin function. This dissertation includes both my previously published and unpublished coauthored material. / Committee in charge: Monte Westerfield, Chairperson, Biology Judith Eisen, Advisor, Biology; Victoria Herman, Member, Biology; John Postlethwait, Member, Biology; Clifford Kentros, Outside Member, Psychology

xi, 56 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / The development of a functional nervous system is paramount for the ability of animals to interact with their environments. Minor defects in nervous system function compromise the effectiveness of sensing and responding to stimuli. Severe defects in nervous system function often lead to extreme sensory, cognitive and motor skill impairment. The nervous system is a complex network of connections, with each neuron making functional contacts with several other neurons. Any single animal species generally exhibits a stereotyped pattern of neuronal connectivity, but the specific intrinsic and extrinsic signals that impart to a neuron its unique connective properties have only recently begun to be identified. In this study, we use the Drosophila visual system to examine neuronal connectivity. Our screen for genes involved in R7 photoreceptor connectivity led to the identification of the RhoGAP domain-containing protein dsyd-1 and the transcriptional repressor tramtrack . Flies harboring homozygous mutant dsyd-1 R7s fail to phototax towards UV light, an innate behavior mediated by the R7s. Subsequent analysis of axons of dsyd-1 R7s showed abnormal morphology in the region of presynaptic sites, suggesting that similar to its role in C. elegans , dsyd-1 is involved in presynaptic assembly. Further analysis demonstrated a requirement for dsyd-1 function in docking presynaptic components to terminal sites of contact. R7 axons are restricted to non-overlapping columns in the optic neuropil, thereby preserving spatial visual information in the retintopic map. The axon terminals of tramtrack mutant R7s exhibit overgrowth, similar to that observed in R7s that have loss of function mutations in genes involved in the activin signaling pathway. Previous studies have shown that activin signaling is involved in restricting R7 axons to their appropriate columns, and our results demonstrate that tramtrack may be functioning in the same pathway. One of two tramtrack isoforms, ttk69 , is expressed in photoreceptors after they have differentiated, and expression of ttk69 is specifically required for R7 axon targeting. / Committee in charge: Eric Johnson, Chairperson, Biology; Victoria Herman, Advisor, Biology; Bruce Bowerman, Member, Biology; Christopher Doe, Member, Biology; Tom Stevens, Outside Member, Chemistry

O maior nicho neurogênico no encéfalo adulto está ao redor dos ventrículos laterais, mas a identificação das células que iniciam tal formação é controversa. Há uma inervação do peptídeo CART que pode abrir perspectivas para o entendimento de seu papel na modulação da neurogênese. Propormos estudar a citoarquitetura ultra-estrutural das células proliferativas na região periventricular e descrever a organização dessa região e sua inervação por axonios imunorreativos ao CART. Utilizamos ratos e camundongos adultos, preparados para análise ultraestrutural e neuroquímica em microscópios eletrônicos de transmissão e varredura de alta-resolução, de luz e laser confocal. O estudo da proliferação e inervação ocorreu com a administração do marcador de fase S BrdU e anticorpos anti-BrdU, anti-CART, anti-DCX, anti-GFAP e anti-GFP. Cada tipo celular do nicho neurogênico apresentou uma densidade própria de ir-BrdU. Identificamos células de revestimento ventricular inervadas por axônios. A maior densidade de inervação ir-CART ocorre ao longo do trajeto dos neurônios em formação. / The major neurogenic niche in adult brains surrounds the lateral ventricles, but the identity of the cell that initiates this process in controversial. There is an innervation made by the CART peptide that may lead to perspectives for understanding its role in modulation of neurogenesis. We propose to study the ultrastructural cytoarchitecture of proliferative cells in this region and its innervation by CART immunoreactive axons. We used adult rats and mice, prepared for ultrastructural and neurochemical analysis by transmission and high-resolution scanning electron, light and laser confocal microscopes. The proliferation and innervation studies occured with the S-phase marker BrdU and anti-Brdu, anti-CART, anti-DCX, anti-GFAP, anti-GFP antibodies. Each sort of cells in neurogenic niche presented a proper density of BrdU staining. We identified the cells lining the ventricle being innervated by axons. The major density of CART innervation occurs along the pathway of neurons in maturation process.

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth. / Medicine, Faculty of / Graduate

It was hypothesized that superparamagnetic nanoparticles encapsulated in a silica shell with a fluorescent dye could be functionalized with axonal tracers and could be used for serial, non-invasive imaging with magnetic resonance imaging (MRI) for axon tract tracing. Nanoparticles functionalized with amine, octadecyl, silica, and biotinylated dextran amine were manufactured and characterized with MRI, scanning electron microscopy, and UV-visible, infrared, and fluorescence spectroscopy. Nanoparticle concentrations of 10 mM were not toxic to adult rat neural progenitor cells (NPCs) and labeled approximately 90% of cells. Nanoparticles were assessed for anterograde and retrograde tract tracing in adult rat models. With MRI and microscopy, the nanoparticles did not appear to trace axons but did provide an MRI signal for up to 3 weeks post implantation. While functionalized nanoparticles did not appear to trace axons, they are not toxic to NPCs and may be used as a MRI contrast agent in the neural axis.