Injury and degenerative disease of the central nervous system (CNS) are among the major causes for disabilities in humans. They result in permanent damage that is not repaired by regenerative processes. In contrast, anamniotes like fish and amphibia display a striking potential for successful regeneration in the CNS. The zebrafish (Danio rerio) has been established as a model for successful regeneration after spinal cord injury. However, it is yet unknown which factors are involved in regeneration after spinal lesions and other insults to the CNS. Focusing on motor neurons, I asked whether regeneration can also be observed in larval zebrafish. This would allow to take advantage of their accessibility to live imaging, pharmacological and genetic manipulation. It is unknown, whether the loss of a specific cell type in the absence of injury, which is reminiscent of the pathological change observed in neurodegenerative diseases, would be sufficient to induce regeneration. Comparing the regenerative response after spinal lesion to that after selective neuronal cell loss would allow to identify factors that act as a trigger for regeneration, e.g. mechanical injury signals, the extent of cell death or microglia activation. To address these questions, an experimental paradigm in which motor neurons can be selectively ablated without the need to inflict tissue damage would prove useful. Key findings of this work are: · Motor neuron generation ceases during early larval developmental stages. · The Nitroreductase system can be used for successful ablation of motor neurons in the larval spinal cord. · New motor neurons are generated in a regenerative response to both targeted ablation of motor neurons and spinal lesion in larval zebrafish after cessation of developmental generation of MNs. To test whether larval zebrafish can be used to analyse motor neuron regeneration, I carried out a birthdating study to establish a developmental time line for motor neuron generation in the spinal cord. The end of developmental motor neuron generation at an early time point, at around 54 hours post fertilisation, allows for the use of larval zebrafish to assess the regenerative response after insults to the spinal cord. In addition, I could show a time dependent role for Hedgehog signalling during the generation of a motor neuron subpopulation. The influence of Hedgehog is diminished before the end of motor neurogenesis. Utilizing the Gal4/UAS system to combine the Nitroreductase‐mCherry fusion protein expressing Tg(UAS:nfsB‐mCherry) with the motor neuron specific driver Tg(hb9:Gal4), I generated a new transgenic zebrafish line for the genetically targeted ablation of motor neurons. In the resulting transgenic fish, the administration of the prodrug Metronidazole induces apoptotic cell death in ~25% of spinal motor neurons leading to impaired motor performance and increased numbers of microglia in the spinal cord. My work shows that larval animals subjected to motor neuron ablation or spinal lesion display a regenerative response detected by increased numbers of newborn motor neurons. Importantly, this happens after developmental production of motor neurons has ceased, suggesting that progenitor cells are reverting to the generation of motor neurons. The data presented shows that in larval zebrafish, the selective loss of motor neurons is sufficient to induce a regenerative response in the spinal cord. The increased numbers of microglial profiles in the spinal cord after both spinal lesion and targeted cell ablation indicates a role for the immune system in mediating a regenerative response. This new targeted cell ablation paradigm in larval zebrafish will allow to identify and characterize the progenitor cell population forming new motor neurons. One can then further investigate how specific loss of motor neurons is sensed and which factors contribute to the activation of the endogenous stem cell populations. Using larval zebrafish has many benefits, as they are accessible to pharmacological testing with small molecules and live imaging. Moreover, the combination of additional transgenic reporter lines will allow for the investigation of single cell behaviour during regeneration.

Interacting cellular systems with nano-interfaces has shown great promise in promoting differentiation, regeneration, and stimulation. Functionalized nanostructures can serve as topological cues to mimic the extracellular matrix network to support cellular growth. Nanostructures can also generate signals, such as thermal, electrical, and mechanical stimulus, to trigger cellular stimulation. At this stage, the main challenges of applying nanostructures with biological systems are: (1) how to mimic the hierarchical structure of the ECM network in a 3D format and (2) how to improve the efficiency of the nanostructures while decreasing its invasiveness. To enable functional neuron regeneration after injuries, we have developed a 2D nanoladder scaffold, composed of micron size fibers and nanoscale protrusions, to mimic the ECM in the spinal cord. We have demonstrated that directional guidance during neuronal regeneration is critical for functional reconnection. We further transferred the nanoladder pattern onto biocompatible silk films. We established a self-folding strategy to fabricate 3D silk rolls, which is an even closer system to mimic the ECM of the spinal cord. As demonstrated by in vitro and in vivo experiments, such a scaffold can serve as a grafting bridge to guide axonal regeneration to desired targets for functional reconnection after spinal cord injuries. Benefited from the robust self-folding techniques, silk rolls can also be used for heterogeneous cell culture, providing a potential therapeutic approach for multiple tissue regeneration directions, such as bones, muscles, and tendons. For achieving neurostimulation, we have developed photoacoustic nanotransducers (PANs), which generate ultrasound upon excitation of NIR II nanosecond laser light. By surface functionalize PAN to bind to neurons, we have achieved an optoacoustic neuron stimulation process with a high spatial and temporal resolution, proved by in-vitro and in-vivo experiments. Such an application can enable non-invasive, optogenetics free and MRI compatible neurostimulation, which provides a new direction of gene-transfection free neuromodulation. Collectively, in this thesis, we have developed two systems to promote functional regeneration after injuries and stimulate neurons in a minimally invasive manner. By integrating those two functions, a potential new generation of the bioengineered scaffold can be investigated to enable functional and programmable control during the regeneration process.

Olfactory sensory neurons in the olfactory epithelium are responsible for detecting the odors we smell and are constantly dying. However, in order for the sense of smell to be maintained, the olfactory system has the unique ability to generate new neurons. After an olfactory sensory neuron is born in the olfactory epithelium, it must extend an axon towards the olfactory bulb in the central nervous system. Within the olfactory bulb, these axons make specific synaptic contacts with the dendritic processes of mitral cells, which are the main projection neurons from the olfactory bulbs into higher cortical areas in the brain. In addition to regeneration due to normal turnover, the olfactory system is also capable of recovery after an injury. The olfactory system’s ability to recover is remarkable because it is capable of regeneration after a mild injury (a portion of olfactory epithelium is removed) or a severe injury (in which the entire olfactory epithelium is removed.) A well-established model for producing a severe type of injury in the olfactory epithelium is by inducing a chemical ablation by a single injection of the drug methimazole. A specific interest in the regenerative process after injury is reestablishment of synaptic connections. We hypothesized that expression of synaptic markers will allow for establishing a timeline of functional recovery of the olfactory system after injury. Our lab has studied three synaptic vesicle associated proteins, vesicular glutamate transporter -1 (VGlut-1), vesicular glutamate transporter-2 (VGlut-2), and synaptophysin, as well as one activity-regulated protein, tyrosine hydroxylase. These studies found specific temporal expression profiles at 2, 7 and 14 days post injury. Our initial data show that VGlut-1 and VGlut-2 are decreased after injury, indicative of a reduction in synaptic connectivity in both olfactory sensory neuron axons and in dendrites of mitral cell neurons. These changes in synaptic connectivity help in understanding functional connectivity after an injury and can further be used to correlate histological axonal tracing with behavioral studies.