In contrast to mammals, adult zebrafish are able to regenerate motor neurons and regain swimming ability within 6 weeks after a spinal cord injury. During this regenerative process, a range of developmental signals such as dopamine and serotonin are found to be re-deployed. This makes the research of embryonic signals become essential for the promotion of regeneration in the future. In my research, I am interested in identifying genes that are important for motor neuron development and motor axon differentiation. I also aimed to study the ability of zebrafish larvae to regenerate spinal motor neurons, and whether they can be used to study the essential developmental cues and the mechanisms underlying successful functional recovery. Motor axons grow out of the spinal cord in a motor neuron subtype specific manner and innervate different muscle groups to facilitate locomotor movements. To find genes and important pathways involved in motor neuron generation and axon development in zebrafish, we conducted an ENU-induced mutagenesis screen in islet-1:GFP transgenic zebrafish, in which a subset of dorsally projecting motor neurons are labelled. We have discovered 6 mutants displaying delayed or inhibited appearance of secondary motor neurons and/or motor axon deficits among 111 F2 families screened. Through subsequent mutant phenotypical analysis, I focused my study in two mutant lines manifesting a lack of islet-1:GFP motor neurons, and an absence of islet-1:GFP motor axons. I used various molecular markers to characterise the mutant phenotypes and observed several additional anatomical defects. I also initiated the study of causative mutation analysis based on the candidate gene list generated from Next Generation Sequencing (NGS). To gain an insight of the genes’ role in motor neuron development and axonal differentiation, I started functional analyses in order to confirm genes that are responsible for the observed motor neuron/axon phenotypes, and I have achieved some promising preliminary results. Motor neurons are generated from the motor neuron progenitor domain (pMN). This neurogenesis process sharply declines at 48 hours post-fertilisation (hpf), while pMN progenitor cells continue to proliferate to produce oligodendrocytes. By inflicting a mechanical lesion in the spinal cord of zebrafish larvae, we demonstrated that they are capable of regenerate new motor neurons and achieve full functional recovery within 48 hours following the injury, sharing similar mechanisms to that of the adult zebrafish. I further studied oligodendrocyte generation and found that pMN domain is able to switch from oligodendrogenesis to motor neuron generation after a spinal lesion. This demonstrates the high plasticity of the pMN domain. Interestingly, the generation of dorsal Pax2-positive interneurons was not altered after the lesion, suggesting that the regenerative potential differs in different progenitor domains. This study showed that the motor neuron regenerative process in zebrafish larvae is robust and they can be used for studying motor neuron regeneration. Taken together, the discovery of the genes from our screen will provide insights to the developmental cues that are involved in motor neuron generation and axon growth. Furthermore, spinal cord lesion in larval zebrafish larvae is established as a regenerative model that can be utilized to dissect the roles and mechanisms of these signals and pathways in the promotion of motor neuron regeneration.

El posicionament de les neurones en el sistema nerviós central i el creixement dels seus axons són processos essencials que s'han de desenvolupar degudament per assolir un funcionament correcte del cervell. La gran varietat de processos migratoris que es produeixen durant tot el desenvolupament desemboquen en la formació de les estructures cerebrals. La Reelina, així com d'altres proteïnes secretables, és una senyal extracel·lular que controla els processos migratoris que es produeixen durant el desenvolupament en estructures laminades com l'escorça, l'hipocamp, el cerebel i el bulb olfactori. També en el cervell adult la Reelina pot intervenir en processos de sinaptogènesi i plasticitat neuronal, a més de mantenir el control de la migració de neurones al bulb olfactori. L'estudi detallat de la transducció de senyal que es desencadena en les neurones estimulades amb Reelina permet identificar les proteïnes que hi participen. Així, els receptors VLDLR i ApoER2, la proteïna adaptadora mDab1, les proteïnes quinasa de la família Src, la PI3K, la Akt1, la GSK3â i la MAP1B formen part d'aquest sistema de senyalització intracel·lular que acabarà donant resposta a l'estímul originat per la Reelina. En aquesta tesi doctoral es fa un estudi descriptiu de la distribució d'mDab1, que participa en els primers passos de la transducció de la senyalització induïda per la Reelina, i s'analitza la seva implicació en axogènesi i en l'establiment del patró de projeccions que innerven l'hipocamp. Els resultats obtinguts indiquen que l'mDab1 s'expressa en les regions d'origen de les fibres de la via perforant i de les connexions comissurals durant l'etapa de formació d'aquestes connexions. Els axons procedents de l'escorça entorínica i de CA3 contenen la proteïna mDab1 en els seus tractes axonals i també a nivell dels cons de creixement. Els ratolins mdab1 -/- presenten el mateix fenotip que els ratolins reeler en aquestes connexions. L'mDab1 és important tant en les regions d'origen de les fibres de la via perforant com en les regions diana d'aquesta connexió. D'altra banda, la Reelina indueix col·lapse axonal sobre els axons de CA3 i incrementa la ramificació axonal en neurones hipocàmpiques. El cultiu d'explants de les regions CA1/CA3, EC i DRG sobre substrats que contenen la Reelina en promou una disminució del creixement axonal. A continuació s'estudien nous processos de senyalització que s'activen en resposta a la Reelina, centrant l'estudi en la via de senyalització d'ERKs. La Reelina indueix la fosforilació de les MAPKs Erk1 i Erk2, i no en canvi de les Jnk1/2/3 ni de la p38. La fosforilació de les Erk1/2 es correspon amb l'activació d'aquestes proteïnes. L'increment d'activitat de les Erk1/2 és depenent de la prèvia activació de les SFKs, de l'mDab1 i de la via de PI3K; i no requereix la participació de la proteïna Ras. A conseqüència de l'activació de la via d'ERK, s'indueix un increment de la transcripció gènica de l'egr-1 per acció de l'estímul de la Reelina. També s'estudia la implicació de l'activació d'ERK en models de migració in vitro de la regió SVZ. L'activació de la via d'ERK és necessària per a la desadhesió neuronal induïda per la Reelina sobre les neurones de la SVZ. Finalment es genera un model animal per a l'estudi de la funció de la proteïna Reelina en processos de desenvolupament i plasticitat de l'edat adulta. La sobreexpressió regulada de Reelina en cervell anterior d'animals postnatals es limita a les regions on s'expressa el promotor CamKIIá. Per aconseguir-ho es generen animals dobles transgènics d'expressió de la Reelina mitjançant un sistema Tet-Off. Els ratolins dobles transgènics expressen la Reelina en neurones principals de l'estriat, de CA1 de l'hipocamp i del gir dentat. / To study the participation of Reelin and its signaling pathway in the development of the central nervous system, different experimental approaches were performed. First, we analyzed the participation of mDab1 and Reelin in the development of hippocampal connections. Second, we reported the activation of ERK pathway by Reelin stimulus to induce egr-1 upregulation and SVZ detachment. And third, we generate a new mouse model to study Reelin functions.The results indicate that mDab1, which participates in the first step of intracellular transduction of Reelin signaling, is expressed in the regions of origin of the perforant and comissural pathways. Entorhinal and CA3 axons contain mDab1 protein in their axonal tracts and also in the growth cones. mdab1 -/- mice show the same phenotype as reelers in the development of those connections. mDab1 is required in the regions where perforant axons originate and in the terminal areas. Moreover, Reelin induces CA3 growth cone collapse, increased axonal branching in hippocampal primary cultures and reduced outgrowth of CA1/CA3, EC and DRG explants.Reelin induces the phosphorylation and activation of MAPKs Erk1 and Erk2, but not of Jnk1/2/3 or p38. The activation of Erk1/2 occurs downstream of the sequential activation of SFKs/mDab1 and PI3K pathway without Ras participation. The activation of the ERK pathway induces the increased transcription of egr-1 after Reelin stimulation, and the detachment of neurons from the SVZ.Finally, to study adult processes of development and plasticity, transgenic mice was designed and generated to express Reelin in postnatal forebrain under the control of CamKIIá promoter. Using the Tet-Off binary system, the double transgenic animals express Reelin in principal neurons of the striatum, CA1 hippocampal region and dentate gyrus.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Neurosciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Multiple sclerosis is a progressive autoimmune disease where myelin is gradually stripped from axons. Axon degeneration inevitably follows protracted myelin loss ultimately leading to irreversible neurological decline. To better understand the cellular mechanisms associated with the axon loss phase of the disease, spinal cord axons from the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis were examined using correlated in vivo time-lapse microscopy and serial section transmission electron microscopic (ssTEM) reconstruction. A novel technique, termed near infrared burning (NIRB), was developed that took advantage of a femtosecond-pulsed mode locked laser’s ability to create photoconvertable fiducial markers for routine identification of previously imaged axons for ssTEM reconstruction. This combination of imaging techniques revealed the subcellular milieu that underlies axon degeneration at both the light and electron microscopic level. In particular, paranodal regions of axons in EAE animals contained a significantly higher population of mitochondria with large rounded, electron lucid, vesiculated mitochondria with unorganized cristae compared to controls. This effect was largely restricted to the paranodal region and was not always associated with direct immune cell interaction or myelin loss. Together, these results suggest a novel mechanism for axon degeneration that is not only focal in nature, but decoupled with myelin loss in the EAE animal model of multiple sclerosis. / Department of Biology

The nervous system, including the brain, is a complex network with billions of complex neurons. Ion channels mediate the electrical signals that neurons use to integrate input and produce appropriate output, and could thus be thought of as key instruments in the neuronal orchestra. In the field of neuroscience we are not only curious about how our brains work, but also strive to characterize and develop treatments for neural disorders, in which the neuronal harmony is distorted. By modulating ion channel activity (pharmacologically or otherwise) it might be possible to effectively restore neuronal harmony in patients with various types of neural (including channelopathic) disorders. However, this exciting strategy is impeded by the gaps in our understanding of ion channels and neurons, so more research is required. Thus, the aim of this thesis is to improve the understanding of how specific ion channel types contribute to shaping neuronal dynamics, and in particular, neuronal integration, excitability and memory. For this purpose I have used computational modeling, an approach which has recently emerged as an excellent tool for understanding dynamically complex neurophysiological phenomena. In the first of two projects leading to this thesis, I studied how neurons in the brain, and in particular their dendritic structures, are able to integrate synaptic inputs arriving at low frequencies, in a behaviorally relevant range of ~8 Hz. Based on recent experimental data on synaptic transient receptor potential channels (TRPC), metabotropic glutamate receptor (mGluR) dynamics and glutamate decay times, I developed a novel model of the ion channel current ITRPC, the importance of which is clear but largely neglected due to an insufficient understanding of its activation mechanisms. We found that ITRPC, which is activated both synaptically (via mGluR) and intrinsically (via Ca2+) and has a long decay time constant (τdecay), is better suited than the classical rapidly decaying currents (IAMPA and INMDA) in supporting low-frequency temporal summation. It was further concluded that τdecay varies with stimulus duration and frequency, is linearly dependent on the maximal glutamate concentration, and might require a pair-pulse protocol to be properly assessed. In a follow-up study I investigated small-amplitude (a few mV) long-lasting (a few seconds) depolarizations in pyramidal neurons of the hippocampal cortex, a brain region important for memory and spatial navigation. In addition to confirming a previous hypothesis that these depolarizations involve an interplay of ITRPC and voltage-gated calcium channels, I showed that they are generated in distal dendrites, are intrinsically stable to weak excitatory and inhibitory synaptic input, and require spatial and temporal summation to occur. I further concluded that the existence of multiple stable states cannot be ruled out, and that, in spite of their small somatic amplitudes, these depolarizations may strongly modulate the probability of action potential generation. In the second project I studied the axonal mechanisms of unmyelinated peripheral (cutaneous) pain-sensing neurons (referred to as C-fiber nociceptors), which are involved in chronic pain. To my knowledge, the C-fiber model we developed for this purpose is unique in at least three ways, since it is multicompartmental, tuned from human microneurography (in vivo) data, and since it includes several biologically realistic ion channels, Na+/K+ concentration dynamics, a Na-K-pump, morphology and temperature dependence. Based on simulations aimed at elucidating the mechanisms underlying two clinically relevant phenomena, activity-dependent slowing (ADS) and recovery cycles (RC), we found an unexpected support for the involvement of intracellular Na+ in ADS and extracellular K+ in RC. We also found that the two major Na+ channels (NaV1.7 and NaV1.8) have opposite effects on RC. Furthermore, I showed that the differences between mechano-sensitive and mechano-insensitive C-fiber types might reside in differing ion channel densities. To conclude, the work of this thesis provides key insights into neuronal mechanisms with relevance for memory, pain and neural disorders, and at the same time demonstrates the advantage of using computational modeling as a tool for understanding and discovering fundamental properties of central and peripheral neurons. / <p>QC 20120914</p>

Teleost retinal ganglion cells can regenerate severed axons following injury, something their mammalian counterparts cannot do. In the teleost, successful regeneration has been attributed in part to microglial cell activities including the phagocytosis of myelin. Although the regulation of microglial phagocytosis has been studied in mammals, in the teleost it is largely unexamined. The present study was designed to identify mediators of microglial phagocytosis released by injured goldfish optic nerve during the course of regeneration. We found that microglial phagocytosis was significantly enhanced in the presence of a 7 day regenerating nerve or medium conditioned by the nerve (CM). When either nerve or CM was incubated with microglia along with an antibody against tumour necrosis factor alpha (TNFalpha), this effect was neutralized. The L929 cell cytotoxicity assay further demonstrated TNFalpha activity in the CM. However, Western blot analysis did not confirm this result. Therefore, further work is necessary to clearly establish the presence of TNFalpha.

Access to and subsequent control of spinal cord function are critical considerations for design of optimal therapeutic strategies for SCI patients. Electrical stimulation of the spinal cord is capable of activating behaviorally-relevant populations of neurons for recovery of function, and is therefore an attractive target for potential devices. A promising method for accessing these spinal circuits is through their axons, which are organized as longitudinal columns of white matter funiculi along the cord exterior. For this thesis, I hypothesized that these funiculi can be selectively recruited via electrodes appropriately placed on the surface of the spinal cord, for functional activation of relevant motor circuitry in a chronically-transected spinal cord. My tandem design goal was to fabricate and implement a conformable multi-electrode array (MEA) that would enable this selective stimulation. To accomplish this design goal, I participated in the design, fabrication, and electromechanical testing of a conformable MEA for surface stimulation of spinal tracts. I then assessed the fundamental capability of this MEA technology to stimulate white matter tracts in a precise, controlled, and functionally-relevant manner. This was accomplished via in vitro experiments that explored the ability of this MEA to locally activate axons via single- and dual-site surface stimulation. The results from these evaluation studies suggest that spinal-cord surface stimulation with this novel MEA technology can provide discrete, minimally-damaging activation of spinal systems via their white matter tracts. To test my hypothesis that surface stimulation can be used to recruit distinct populations in the spinal cord, I performed studies that stimulated lateral funiculi in both chronically-transected and intact in vitro spinal cords. Results from these studies reveal that selective surface stimulation of white matter tracts in the ventrolateral funiculus (VLF) elicit motor outputs not elicited in intact cords. In addition, I was able to demonstrate that the spinal systems activated by this surface stimulation involve synaptic components and are responsive to spatial, temporal, and pharmacologic facilitation. Corresponding labeling of the axonal tracts projecting through the T12 VLF indicate that, after chronic transection, the remaining spinal neurons whose axons travel through the VLF include those with cell bodies in both the intermediate region and dorsal horn. These electrophysiological results show that surface-stimulating technologies used to control motor function after injury should include focal activation of interneuronal systems with axons in the ventrolateral funiculus. As a whole, these studies provide essential starting points for further use of conformable MEAs to effectively activate and control spinal cord function from the surface of the spinal cord.

Graduation date: 1993

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.