With the recent discoveries of phenomena such as plateau potentials, bistability, and synaptic amplification the focus of motoneuron research has been directed to the dendritic processes giving rise to these latent behaviors. The common consensus is that the mechanism behind bistability (an L-type calcium channel generating a persistent inward current, PIC; Schwindt and Crill 1980, Hounsgaard and Kiehn 1985, 1989) is also responsible for the amplification of synaptic input in motoneurons. However, modeling studies utilizing only calcium-based PICs (Powers 1993, Booth et al. 1997, Elbasinouy et al. 2005) have been unable to reproduce the high degree of synaptic amplification observed in experimental preparations (Prather et al. 2001, Lee et al. 2003, Hultborn et al. 2003). The present work examines a theoretical amplification mechanism (electrotonic compression), based on a sodium PIC of dendritic origin, which acts to supplement the synaptic amplification due to the calcium PIC. The current goal is to test the "goodness-of-fit" of electrotonic compression with established mechanisms and behaviors. The findings of this modeling study support the concept of a dendritic sodium PIC which acts to reduce the attenuation of synaptic currents enroute to the motoneuron soma. Furthermore, it is suggested that the ratiometric expression of ion channels giving rise to this mechanism takes the form of a distribution "rule" applied ubiquitously across the dendritic tree, while the plateau-producing L-type calcium channels undergo a more discretized or regional distribution. This study demonstrates the power inherent to the controlled expansion of morphological complexity in an already complex model. While modeling studies are suitable testbeds for the evaluation of theoretical and/or experimentally intractable facets of physiology, great care and consideration should be given to the specification of models with high dimensionality. With the continual progression of our knowledge-base and computational capabilities, we can expect that more and more empirical observations will find their way into models of increasing complexity wherein the layers of embedded hypotheses are frequently implicit. It is therefore imperative that the neural modeling discipline adopt more rigorous methodologies to both accommodate and rein-in this growing complexity.

Chitosan is widely investigated for biomedical applications due to its excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial, etc. In the field of neural engineering, it has been extensively studied in forms of film and hydrogel, and has been used as scaffolds for nerve regeneration in the peripheral nervous system and spinal cord. One of the main issues in neural engineering is the incapability of neuron to attach on biomaterials. The present study, from a new aspect, aims to take advantage of the bio-adhesive property of chitosan to develop chitosan-based materials for neural engineering, specifically in the fields of brain repair and neuroprosthetics. Neuronal responses to the developed biomaterials will also be investigated and discussed.In the first part of this study (Chapter II), chitosan was blended with a well-studied hydrogel material (agarose) to form a simply prepared hydrogel system. The stiffness of the agarose gel was maintained despite the inclusion of chitosan. The structure of the blended hydrogels was characterized by light microscopy and scanning electron microscopy. In vitro cell studies revealed the capability of chitosan to promote neuron adhesion. The concentration of chitosan in the hydrogel had great influence on neurite extension. An optimum range of chitosan concentration in agarose hydrogel, to enhance neuron attachment and neurite extension, was identified based on the results. A “steric hindrance” effect of chitosan was proposed, which explains the origin of the morphological differences of neurons in the blended gels as well as the influence of the physical environment on neuron adhesion and neurite outgrowth. This chitosan-agarose (C-A) hydrogel system and its multi-functionality allow for applications of simply prepared agarose-based hydrogels for brain tissue repair.In the second part of this study (Chapter III), chitosan was blended with graphene to form a series of graphene-chitosan (G-C) nanocomposites for potential neural interface applications. Both substrate-supported coatings and free standing films could be prepared by air evaporation of precursor solutions. The electrical conductivity of graphene was maintained after the addition of chitosan, which is non-conductive. The surface characteristic of the films was sensitively dependent on film composition, and in turn, influenced neuron adhesion and neurite extension. Biological studies showed good cytocompatibility of graphene for both fibroblast and neuron. Good cell-substrate interactions between neurons and G-C nanocomposites were found on samples with appropriate compositions. The results suggest this unique nanocomposite system may be a promising substrate material used for the fabrication of implantable neural electrodes. Overall, these studies confirmed the bio-adhesive property of chitosan. More importantly, the developed chitosan-based materials also have great potential in the fields of neural tissue engineering and neuroprosthetics.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 200-224). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 200-224). Also available online.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2004. / Vita. Includes bibliographical references (leaves 109-114).

published_or_final_version / Anatomy / Master / Master of Philosophy

MicroRNAs are short RNA molecules composed of 20-22 nucleotides. They highly accurately indicate cell identity and hence they are useful in labeling cells and tacking lineage commitment. However, this requires accurate microRNA profiling of cells in individual developmental stages. Since microRNAs are important negative regulators of eukaryotic gene expression, microRNA profiling allows better understanding of molecular regulatory networks of important cellular events, such as adult neurogenesis. Adult neurogenesis is the process in which neurons, as well as glia, are generated from neural stem cells. It was found to be responsible for brain regeneration, olfactory discrimination, memory formation and learning. Depression was suggested to be related to dysregulation of neurogenesis. Thus, knowledge in cellular and molecular mechanisms of adult neurogenesis will lay solid foundation to develop therapies to regenerate neural cells after injuries or onsets of neurodegenerative diseases and to understand the cognitive ability, memory formation and learning of the brain. In spite of its importance, investigation into the miRNA profiles and functions in neurogenesis is still infant. This project aimed to establish a preliminary microRNA profile on neurogenesis. Although this was not completed, the project could be extended to a large-scale microRNA profiling in neurogenesis. This would enable future workers to track the lineage commitment, the migration, and the distribution of NSCs and their derived cells accurately by in situ hybridization. Also, the future workers may construct a 2D representation of the changes in miRNA profiles and this may lead to discovery of previously unknown molecular and cellular differences among cells of same cell identity. / published_or_final_version / Anatomy / Master / Master of Medical Sciences

μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to detect in rat liver mitochondria. In a mitochondrial fraction from SH-SY5Y cells, incubation with 5 mM Ca2+ resulted in the activation of mitochondrial μ-calpain but not in AIF truncation. Finally, in hippocampal neurons calpain activation did not induce AIF processing or nuclear translocation and AIF translocation to nucleus was calpain independent. The localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive. We observed that mitochondrial μ-calpain required high Ca2+ for activation. We examined the hypothesis that the endogenous calpain inhibitor, calpastatin, may be present in the neuronal mitochondria. Calpastatin was detected in the mitochondriaenriched fraction obtained from rat cerebral cortex and SH-SY5Y cells. The mitochondrial calpastatin was resistant to proteinase K digestion, indicating localization internal to the outer mitochondrial membrane. Submitochondrial fractionation revealed that the calpastatin was localized to the mitochondrial intermembrane space and mitoplasts (inner mitochondrial membrane and matrix) but not to the mitochondrial outer membrane fraction. Mitochondrial calpastatin was not detected when mitoplasts were incubated with proteinase K, suggesting that calpastatin is not present in the matrix. The N-terminus of XL domain of calpastatin, when fused to GFP and transfected to SH-SY5Y cells showed mitochondrial localization and thus confirmed the presence of a mitochondrial targeting sequence in calpastatin. Together, these results demonstrate the presence of calpastatin in the neuronal mitochondrial intermembrane space, the same submitochondrial compartment as mitochondrial μ-calpain. This finding explains the high Ca2+ requirements for mitochondrial μ-calpain activation.

Epilepsy is a neurological disorder characterized by recurrent seizures. Sleep problems can cooccur with epilepsy, and adversely affect seizure diagnosis and treatment. In fact, the relationship between sleep and seizures in individuals with epilepsy is a complex one. Seizures disturb sleep and sleep deprivation aggravates seizures. Antiepileptic drugs may also impair sleep quality at the cost of controlling seizures. In general, particular vigilance states may inhibit or facilitate seizure generation, and changes in vigilance state can affect the predictability of seizures. A clear understanding of sleep-seizure interactions will therefore benefit epilepsy care providers and improve quality of life in patients. Notable progress in neuroscience research—and particularly sleep and epilepsy—has been achieved through experimentation on animals. Experimental models of epilepsy provide us with the opportunity to explore or even manipulate the sleep-seizure relationship in order to decipher different aspects of their interactions. Important in this process is the development of techniques for modeling and tracking sleep dynamics using electrophysiological measurements. In this dissertation experimental and computational approaches are proposed for modeling vigilance dynamics and their utility demonstrated in nonepileptic control mice. The general framework of hidden Markov models is used to automatically model and track sleep state and dynamics from electrophysiological as well as novel motion measurements. In addition, a closed-loop sensory stimulation technique is proposed that, in conjunction with this model, provides the means to concurrently track and modulate 3 vigilance dynamics in animals. The feasibility of the proposed techniques for modeling and altering sleep are demonstrated for experimental applications related to epilepsy. Finally, preliminary data from a mouse model of temporal lobe epilepsy are employed to suggest applications of these techniques and directions for future research. The methodologies developed here have clear implications the design of intelligent neuromodulation strategies for clinical epilepsy therapy.

Lipid peroxidation is a key contributor to the pathophysiology of traumatic brain injury (TBI). Traditional antioxidant therapies are intended to scavenge the free radicals responsible for either the initiation or propagation of lipid peroxidation (LP). However, targeting free radicals after TBI is difficult as they rapidly react with other cellular macromolecules, and thus has a limited post-injury time window in which they may be intercepted by a radical scavenging agent. In contrast, our laboratory has begun testing an antioxidant approach that scavenges the final stages of LP i.e. formation of carbonyl-containing breakdown products. By scavenging breakdown products such as the highly reactive and neurotoxic aldehydes (often referred to as “carbonyls”) 4-hydroxynonenal (4-HNE) and acrolein (ACR), we are able to prevent the covalent modification of cellular proteins that are largely responsible for posttraumatic neurodegeneration. Without intervention, carbonyl additions render cellular proteins non-functional which initiates the loss of ionic homeostasis, mitochondrial failure, and subsequent neuronal death. Phenelzine (PZ) is an FDA-approved monoamine oxidase (MAO) inhibitor traditionally used for the treatment of depression. Phenelzine also possesses a hydrazine functional group capable of covalently binding neurotoxic carbonyls. The hypothesis of this dissertation is that carbonyl scavenging with PZ will exert an antioxidant neuroprotective effect in the traumatically injured rat brain mechanistically related to PZ’s hydrazine moiety reacting with the lipid peroxidation (LP)-derived reactive aldehydes 4-hydroxynonenal (4-HNE) and acrolein (ACR). Data from our ex vivo experiments demonstrate that the exogenous application of 4-HNE or ACR significantly reduced respiratory function and increased markers of oxidative damage in isolated non-injured rat cortical mitochondria, whereas PZ pre-treatment significantly prevented mitochondrial dysfunction and oxidative modification of mitochondrial proteins in a concentration-related manner. Additionally, PZ’s neuroprotective scavenging mechanism was confirmed to require the presence of a hydrazine moiety based on experiments with a structurally similar MAO inhibitor, pargyline, which lacks the hydrazine group and did not protect the isolated mitochondria from 4-HNE and ACR. Our in vivo work demonstrates that subcutaneous injections of PZ following TBI in the rat are able to significantly protect brain mitochondrial respiratory function, decrease markers of oxidative damage, protect mitochondrial calcium buffering capacity, and increase cortical tissue sparing without decreasing neuronal cytoskeletal spectrin degradation. These results confirm that PZ is capable of protecting mitochondrial function and providing neuroprotection after experimental TBI related to scavenging of neurotoxic LP degradation products.