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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21453号 / 医博第4420号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 浅野 雅秀, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Thesis advisor: Laura Anne Lowery / Thesis advisor: Eric Folker / In order to form the exact synaptic connection required for proper neurological function, the growing tip of the neuron hosts an orchestra of hundreds of different proteins interacting with extracellular cues to steer neuron growth in the right direction. The goal of our current research is to study several of the components of this pathway, known as the TACC family. Here, we present a detailed structure/function analysis of the TACC family in regards to binding and activity with other proteins in the growth cone. We investigate the function of TACC3 in mediating neuron outgrowth and guidance in vivo. We have found structural elements of the TACC family that enable their activity. Studying these conserved structures and functions of the TACC family will enable greater understanding of the entire process of cytoskeletal regulation and neurodevelopment. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.

Ca2+-dependent transcription is a fundamental process by which neurons translate activation experience into cellular level adaptations. The nuclear factor of activated T-cells (NFAT) family of proteins comprise four Ca2+/CaN-dependent transcription factors that are widely expressed throughout virtually all tissues. Within neurons, NFAT dependent signaling is critical for axonal development, regulation of synapse number and efficacy, and survival. Furthermore, NFAT is implicated in activity dependent regulation of genes involved in synaptic transmission, learning and memory, mood, and pain sensation. NFAT is activated upon elevations in intracellular Ca2+, which results in CaN -dependent dephosphorylation of multiple serine residues within an N-terminal regulatory region. NFAT dephosphorylation permits NFAT translocation to the nucleus, where it can regulate gene expression, frequently co-operatively with other transcription factors, including AP-1 and MEF2. NFAT activation is opposed or terminated by several kinases, including CK1 and GSK3. Despite the importance of NFAT proteins as regulators of Ca2+-dependent transcription, little is known about the regulation and function of specific NFAT isoforms within neurons. In Aim 1 of this thesis I characterized the differential activation of NFATc3 and NFATc4 in DRG neurons. While NFATc3 rapidly translocates the nucleus upon Ca2+-influx through voltage-gated calcium channels, NFATc4 remained remarkably intransient. Modular substitution of NFATc3 regulatory elements increased the rate or retention of NFATc4, whereas converse substitutions of NFATc4 regulatory elements into NFATc3 decreased NFATc3 nuclear translocation. The activation of NFATc4 appears to be inhibited by preferential phosphorylation by kinases, such as GSK3, which counteract CaN-dependent dephosphorylation. In Aim 2 I investigated the role of NFATc3 in hippocampal neurons. While the majority of NFAT reports in neurons have focused on NFATc4, my data suggest that NFATc3 is the predominantly expressed isoform in hippocampal neurons and is critical for depolarization-induced NFAT target gene expression. I further characterized NFATc3 KO mice in a battery of behavioral assays to test whether loss of NFATc3 expression would affect the baseline anxiety/depression state of the animal, or if NFATc3 was critical for learning and memory. Taken together, my data suggest that NFATc3 is important for NFAT-dependent gene expression in central and peripheral neurons and that distinct regulation of NFAT isoforms within neurons may underlie isoform-specific effects on gene expression.

Visual environments may contain a complex combination of object motion. Animals respond to features of complexity by generating adaptive behavioural responses. One important feature of a complex visual environment is a rapidly expanding object in the visual field (looming) which may represent an approaching predator or an object on a collision path. Many animals respond to looming objects by generating avoidance behaviours (Maier et al. 2004; Santer et al. 2005; Oliva et al. 2007) and neurons involved in the detection and relay of looming stimuli are present in birds (Sun and Frost 1998) and many insects (Simmons and Rind 1992; Hatsopoulos et al. 1995; Wicklein and Strausfeld 2000). One of the most widely studied visual pathways is found in the locust. This visual pathway, which includes the lobula giant motion detector (LGMD) and its post-synaptic target, the descending contralateral motion detector (DCMD), signals the approach a looming visual stimulus (Schlotterer, 1977; Simmons and Rind, 1992; Hatsopoulos et al., 1995). The DCMD descends through the ventral nerve cord and synapses with motorneurons involved in predator evasion and collision avoidance (Simmons, 1980; Simmons and Rind, 1992; Santer et al., 2006).<p> Previous studies have suggested that this pathway is also affected by more complicated movements in the locusts visual environment. For example, Guest and Gray (2006) demonstrated that the approach of paired objects in the azimuthal position and approaches at different time intervals affect DCMD firing rate properties. In my first objective of this thesis (Chapter 2), I tested locusts with computer-generated discs that traveled along a combination of non-colliding (translating) and colliding (looming) trajectories and demonstrate how distinctly different DCMD responses result from different trajectory types. In addition to estimating the time of collision and direction of object travel, the presence of a discernable peak associated with the time of object deviation suggests that DCMD responses may contain information related to changes in motion.<p> Previous studies suggest that LGMD/DCMD encodes approaching objects using rate coding; edge expansion of approaching objects causes an increased rate of neuronal firing (Schlotterer, 1977; Hatsopoulos et al., 1995; Judge and Rind, 1997; Gabbiani et al., 1999). Based on observations of DCMD responses to simple looming objects that showed oscillations in DCMD responses (for example, Fig. 1D Santer et al., 2006) and the fact that bursting occurs in many other sensory systems (Yu and Margoliash, 1996; Sherman, 2001; Krahe and Gabbiani, 2004; Marsat and Pollack, 2006), it was hypothesized that the DCMD may show bursting activity. In my second objective of this thesis (Chapter 3), I tested locusts with simple looming stimuli known to generate behavioural responses in order to identify and quantify bursting activity. Results show that the highest frequency of bursts occurred at intervals of 40-50 ms (20-25 Hz). The behavioural significance of this frequency is related to the average wingbeat frequency of the locusts forewing during flight (~25 Hz; Robertson and Johnson, 1993). Based on previous evidence of DCMD flight-gating (see, for example, Santer et al., 2006), bursting may gate information into the flight circuitry, thereby providing visual feedback that may be modified to generate an avoidance response during flight. Single spiking and bursting occurred throughout object approach up until the late stage of approach, where burst frequency rapidly increased. Results predict that the DMCD may use a bimodal coding strategy to detect looming visual stimuli, where single spiking at the beginning of approach may result in subtle course changes during flight and bursting near the time of collision may initiate an evasive glide.<p> Taken together, these results illustrate that the encoding of visual stimuli in single neurons is dynamic and likely much more complicated than previously thought.

Visual environments may contain a complex combination of object motion. Animals respond to features of complexity by generating adaptive behavioural responses. One important feature of a complex visual environment is a rapidly expanding object in the visual field (looming) which may represent an approaching predator or an object on a collision path. Many animals respond to looming objects by generating avoidance behaviours (Maier et al. 2004; Santer et al. 2005; Oliva et al. 2007) and neurons involved in the detection and relay of looming stimuli are present in birds (Sun and Frost 1998) and many insects (Simmons and Rind 1992; Hatsopoulos et al. 1995; Wicklein and Strausfeld 2000). One of the most widely studied visual pathways is found in the locust. This visual pathway, which includes the lobula giant motion detector (LGMD) and its post-synaptic target, the descending contralateral motion detector (DCMD), signals the approach a looming visual stimulus (Schlotterer, 1977; Simmons and Rind, 1992; Hatsopoulos et al., 1995). The DCMD descends through the ventral nerve cord and synapses with motorneurons involved in predator evasion and collision avoidance (Simmons, 1980; Simmons and Rind, 1992; Santer et al., 2006).<p> Previous studies have suggested that this pathway is also affected by more complicated movements in the locusts visual environment. For example, Guest and Gray (2006) demonstrated that the approach of paired objects in the azimuthal position and approaches at different time intervals affect DCMD firing rate properties. In my first objective of this thesis (Chapter 2), I tested locusts with computer-generated discs that traveled along a combination of non-colliding (translating) and colliding (looming) trajectories and demonstrate how distinctly different DCMD responses result from different trajectory types. In addition to estimating the time of collision and direction of object travel, the presence of a discernable peak associated with the time of object deviation suggests that DCMD responses may contain information related to changes in motion.<p> Previous studies suggest that LGMD/DCMD encodes approaching objects using rate coding; edge expansion of approaching objects causes an increased rate of neuronal firing (Schlotterer, 1977; Hatsopoulos et al., 1995; Judge and Rind, 1997; Gabbiani et al., 1999). Based on observations of DCMD responses to simple looming objects that showed oscillations in DCMD responses (for example, Fig. 1D Santer et al., 2006) and the fact that bursting occurs in many other sensory systems (Yu and Margoliash, 1996; Sherman, 2001; Krahe and Gabbiani, 2004; Marsat and Pollack, 2006), it was hypothesized that the DCMD may show bursting activity. In my second objective of this thesis (Chapter 3), I tested locusts with simple looming stimuli known to generate behavioural responses in order to identify and quantify bursting activity. Results show that the highest frequency of bursts occurred at intervals of 40-50 ms (20-25 Hz). The behavioural significance of this frequency is related to the average wingbeat frequency of the locusts forewing during flight (~25 Hz; Robertson and Johnson, 1993). Based on previous evidence of DCMD flight-gating (see, for example, Santer et al., 2006), bursting may gate information into the flight circuitry, thereby providing visual feedback that may be modified to generate an avoidance response during flight. Single spiking and bursting occurred throughout object approach up until the late stage of approach, where burst frequency rapidly increased. Results predict that the DMCD may use a bimodal coding strategy to detect looming visual stimuli, where single spiking at the beginning of approach may result in subtle course changes during flight and bursting near the time of collision may initiate an evasive glide.<p> Taken together, these results illustrate that the encoding of visual stimuli in single neurons is dynamic and likely much more complicated than previously thought.

Locomotion is controlled in large part by neural circuits (CPGs) that generate rhythmic stereotyped outputs in the absence of descending or sensory inputs. The output of a neural circuit is determined by the configuration of the circuit, synapse properties, and the intrinsic properties of component neurons. In order to understand how a neural circuit functions component neurons, their connections, and their intrinsic properties must be characterized. Motoneurons are a useful cell in which to begin investigation of CPG function because they are accessible and provide a measure of the cumulative activity of the circuit. Drosophila is a potentially useful model system for the study of motoneuron intrinsic properties, their contribution to locomotion, and of locomotor CPGs because the genetic and molecular techniques available in Drosophila are surpassed in no other organism and because the Drosophila nervous system is small in comparison to vertebrate nervous systems. Further, whole-cell in situ patch clamp recordings from adult and larval motoneurons in relatively intact preparations are possible. Therefore, the first goal of this work was to investigate whether the firing behavior and recruitment of identified Drosophila 1b and 1s motoneurons is analogous to the recruitment of high-threshold, phasic and low-threshold, tonic motoneurons in other organisms. The second goal was to determine whether active conductances influence motoneuron recruitment in response to synaptic input. The final aim was to investigate how these factors influence CPG output to muscles. Findings from current clamp studies indicate that1b motoneurons are more easily recruited than 1s motoneurons, in agreement with the hypothesis that 1b motoneurons are analogous to low-threshold motoneurons described in other organisms. Further, orderly recruitment of Drosophila 1b motoneurons before 1s motoneurons is not a result of passive properties. Instead, the Shal channel that encodes a large portion of IA in motoneuron somatodendritic regions is a critical determinant of delay-to-spike in larval Drosophila motoneurons. These findings are behaviorally-relevant because the same recruitment order is seen in simultaneous recordings from motoneuron pairs recruited by synaptic input.

Spiral ganglion neurons (SGNs) within the cochlea degenerate following the loss of the auditory sensory epithelium, the auditory hair cells. Since these neurons are the target cells of the cochlear implant, which bypasses damaged or lost hair cells to stimulate the SGNs directly, enhanced SGN number and integrity may provide enhanced outcomes for cochlear implant patients. Improved contact between the cochlear implant electrode array and the auditory nerve fibres is also likely to enhance the benefits received by cochlear implant patients. Therefore, the identification of molecules with the capacity to support SGN survival and stimulate axonal growth has significant clinical implications. Based on their roles in the development and maintenance of the auditory system, some neurotrophic factors are expected to play an important role in enhancing the survival of auditory elements following deafening. This thesis investigates various molecules for their capacity to stimulate and guide the growth of SGN axons, and also investigates the survival-promoting effects of specific neurotrophic factors on SGN survival in clinically relevant animal models of deafness. Two neurotrophic factors were identified specifically to stimulate axonal growth from SGNs in an in vitro model of deafness.

Cell membranes are made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as neuronal differentiation, cell membranes undergo dramatic structural changes induced by proteins such as ARC and Cofilin among others in the case of synaptic modification. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. As expected, NIH3T3 cells have more rigid membrane at earlier stages of their development. On the other hand neurons tend to have the highest membrane fluidity early in their development emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.