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Effective movement is central to survival and it is essential for all animals to react in response to changes around them. In many animals the rhythmic signals that drive locomotion are generated intrinsically by small networks of neurons in the nervous system which can be switched on and off. In this thesis I use a very simple animal, in which the behaviours and neuronal networks have been well characterised experimentally, to explore the salient features of such networks. Two days after hatching, tadpoles of the frog Xenopus laevis respond to a brief touch to the head by starting to swim. The swimming rhythm is driven by a small population of electrically coupled brainstem neurons (called dINs) on each side of the tadpole. These neurons also receive synaptic input following head skin stimulation. I build biophysical computational models of these neurons based on experimental data in order to address questions about the effects of electrical coupling, synaptic feedback excitation and initiation pathways. My aim is better understanding of how swimming activity is initiated and sustained in the tadpole. I find that the electrical coupling between the dINs causes their firing properties to be modulated. This allows two experimental observations to be reconciled: that a dIN only fires a single action potential in response to step current injections but the population fires like pacemakers during swimming. I build on this hypothesis and show that long-lasting, excitatory feedback within the population of dINs allows rhythmic pacemaker activity to be sustained in one side of the nervous system. This activity can be switched on and off at short latency in response to biologically realistic synaptic input. I further investigate models of synaptic input from a defined swim initiation pathway and show that electrical coupling causes a population of dINs to be recruited to fire either as a group or not at all. This allows the animal to convert continuously varying sensory stimuli into a discrete decision. Finally I find that it is difficult to reliably start swimming-like activity in the tadpole model using simple, short-latency, symmetrical initiation pathways but that by using more complex, asymmetrical, neuronal-pathways to each side of the body, consistent with experimental observations, the initiation of swimming is more robust. Throughout this work, I make testable predictions about the population of brainstem neurons and also describe where more experimental data is needed. In order to manage the parameters and simulations, I present prototype libraries to build and manage these biophysical model networks.

Three-dimensional (3D) in vitro culture systems that provide controlled, biomimetic microenvironments would be a significant technological advance for both basic cell biology research and the development of clinical therapeutics (e.g., as in vivo cell delivery constructs). A variety of signals determine cell phenotype, including those from soluble factors, immobilized biomolecules, mechanical substrates, and culture geometry. My research seeks to create hydrogel culture systems that incorporate these signals in a defined, controllable manner. Specifically, I have focused on developing hydrogels based on the extracellular matrix (ECM) component hyaluronic acid (HA) with precisely specified mechanical, chemical and geometrical microenvironments. For example, the mechanical environment presented by HA hydrogels was tuned to span the threefold range measured for neonatal brain and adult spinal cord by modifying HA with varying numbers of photocrosslinkable methacrylate groups. These hydrogels were used to evaluate the effects of mechanical properties of a 3D culture paradigm on the differentiation of ventral midbrain-derived neural progenitor cells (NPCs) and results demonstrated that the mechanical properties of these scaffolds can assert a defining influence on differentiation. In addition, whole fibronectin was incorporated into HA hydrogels as an adhesive factor to encourage angiogenesis in 3D cultures, as interplay between endothelial cells and neurons is an important determining factor during NPC development and axonal regeneration after injury. To create spatially defined neuronal cultures in three-dimensions, multiphoton excitation (MPE) was used to photocrosslink protein microstructures within HA hydrogels. This method can be used to create complex, 3D architectures that provide both chemical and topographical cues to direct cell adhesion and guidance on size scales relevant to in vivo environments. Using this approach, both dorsal root ganglion cells (DRGs) and hippocampal NPCs could be guided along user-defined, 3D paths. In future studies, these strategies can be combined into a single hydrogel to create a culture microenvironment with multiple types of highly specified cues (i.e., chemical, topographical, and mechanical). / text

The study of the mechanism of traumatic brain injury (TBI) processes at the cellular level is vital to obtain characterization of nerve cell damage after mechanical deformation. This understanding is needed to find feasible therapeutic targets for mechanically damaged neurons. To study the cellular level of TBI damage, development of a new in vitro cellular model of TBI might be done to simulate in vivo cellular TBI. In this research, two studies were performed: (1) the design and construction of an in vitro cell stretching device to mechanically injure cells and (2) the characterization of the molecular and cellular level of the TBI mechanism. The cell stretching device design allows for the precise control of cell strain and duration of stretching cells such that TBI can be mimicked. Analysis of the cellular and molecular level mechanisms of TBI in the proposed in vitro model might help in the design of therapeutic strategies for the treatment of TBI. Our proposed mechanism of injury due to TBI is as follows: after the cell is stretched, a cellular signaling molecule is released to activate the cellular signaling pathway. The activated cell signal may activate kinases which phosphorylate proteins and initiate new protein synthesis. Newly phosphorylated and synthesized proteins may activate the apoptotic process. Using a variety of pharmacological agents, one could block steps in the hypothesized mechanism and examine the effect of those agents on downstream cellular processes and cell apoptosis. For example, the inhibitions of calcium transport, protein synthesis, and caspases were performed to examine the initial activation of the signaling pathway and the role of both in the apoptosis process. Proteomics of TBI may help the understanding of the mechanism of TBI related protein expression. This work will contribute to the discovery of new therapeutic targets and better treatments for TBI.

Ion channel regulation is key to the control of excitability and behaviour. In the bag cell neurons of Aplysia californica, a voltage- and Ca2+-dependent nonselective cation channel drives a ~30-minute afterdischarge, culminating in the release of egg-laying hormone. Using excised, inside-out single channel patch-clamp, this study tested the hypothesis that inositol 1,4,5-trisphosphate (IP3), which is produced during the afterdischarge, and channel-associated protein kinase C (PKC), which is activated throughout the afterdischarge, cause a left-shift (enhancement) in both the voltage- and Ca2+-dependence of the cation channel. Kinetic analysis of bag cell neuron cation channel voltage-dependence revealed that, with depolarization, the channel remained open longer and reopened more often. A cation channel subconductance was also observed, and found to be 13 pS vs. the typical 23 pS full-conductance. The cytoplasmic face of cation channel-containing patches was exposed to 1 mM ATP, as a phosphate source for channel-associated PKC, and/or 5 uM IP3. Apparent PKC-dependent phosphorylation left-shifted voltage-dependence by -3 mV, although this effect was more prominent at negative voltages (between -90 and -30 mV). Conversely, IP3 right-shifted voltage-dependence (change in V1/2 of 6 mV). Cation channel Ca2+-dependence was similar to that previously reported, with a control EC50 of 3-5 uM. This was right-shifted by PKC (EC50 = 30 uM) and even more so by IP3 (apparent EC50 = 20 M). PKC largely rescued the Ca2+ responsiveness in the presence of IP3 (EC50 = 20 uM). Unexpectedly, IP3 plus ATP resulted in an increase in channel unitary conductance at more positive voltages. The multi-faceted regulation of the bag cell neuron cation channel suggests sophisticated modulatory control. Upregulation, such as depolarization and the left-shift in voltage-dependence with PKC, would drive the afterdischarge, while counteracting effects, such as IP3 right-shifting voltage-dependence, as well as PKC and IP3 suppressing Ca2+-dependence, would simultaneously or subsequently attenuate the channel, thus preventing an interminable afterdischarge. / Thesis (Master, Physiology) -- Queen's University, 2008-08-26 13:20:16.528