Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 60 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: James E. Hoffman, Dept. of Psychology. Includes bibliographical references.

Thesis (M.Sc.) - Swinburne University of Technology, Brain Sciences Institute, 2008. / A thesis submitted for M.Sc by Research, Brain Sciences Institute, Swinburne University of Technology - 2008. Typescript. Includes bibliographical references (p. 117-153)

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The glucagon-secreting α-cell is vital for the maintenance of glucose homeostasis and prevention of hypoglycaemia. Despite its importance many aspects of α-cell physiology are disputed. Thus, in this thesis, I aimed to elucidate several features of α-cell function - exploring how autonomic signals are integrated by the cell and how diabetes leads to its dysfunction. The autonomic response to hypoglycaemia results in increased acetylcholine and adrenaline in the islet vicinity, which stimulate glucagon secretion. The mechanisms underlying these effects were investigated using live [Ca²⁺]_i imaging and patch-clamp electrophysiology. Adrenaline was found to target the α-cell via a Î2-adrenergic mechanism, inducing TPC2-mediated Ca²⁺ release from the (endo)lysosomal stores, which triggered calcium-induced calcium release from the endoplasmic reticulum (ER). Acetylcholine also induced ER Ca²⁺-release via muscarinic G_q signalling. However, a component of the effect resulted from activation of a nicotinic pathway that evoked P/Q-type Ca²⁺ channel influx. The glucagon response to hypoglycaemia is lost in diabetes. To investigate the effect of hyperglycaemia on α-cell function at low glucose, the Fh1Î2KO type 2 diabetic mouse model was used. In these mice, prolonged hyperglycaemia led to blunted glucagon secretion at low glucose. Using live pH imaging, it was shown that this was caused by hyperglycaemia increasing flux through Na⁺ coupled glucose transporters (SGLTs), disrupting Na⁺-dependent acid extrusion and inducing cytoplasmic acidification. The resulting build-up of protons was speculated to compromise mitochondrial ATP production leading to the observed glucagon secretory defects. The effects of insulin-induced hypoglycaemia on δ-cell [Ca²⁺]i activity were also investigated. Increased SGLT2 transport and low [K⁺]_o, features of insulin-induced hypoglycaemia, were both shown to increase [Ca²⁺]i activity in δ-cells, stimulating somatostatin secretion and consequently suppressing glucagon secretion. Together these data suggest that glucagon secretion at low glucose is lost due to the combined effects of hyperglycaemia-driven intrinsic dysfunction and insulin-induced somatostatin secretion.

According to Piaget and Bartlett, learning involves both assimilation of new memories into networks of preexisting knowledge and alteration of existing networks to accommodate new information into existing schemas. Recent evidence suggests that the hippocampus integrates related memories into schemas that link representations of separately acquired experiences. In this thesis, I first review models for how memories of individual experiences become consolidated into the structure of world knowledge. Disruption of consolidated memories can occur during related learning, which suggests that consolidation of new information is the reconsolidation of related memories. The accepted role of the hippocampus during memory consolidation and reconsolidation suggests that it is also involved in modifying appropriate schemas during learning. To study schema development, I trained rats to retrieve rewards at different loci on a maze while recording hippocampal calls. About a quarter of cells were active at multiple goal sites, though the ensemble as a whole distinguished goal loci from one another. When new goals were introduced, cells that had been active at old goal locations began firing at the new locations. This initial generalization decreased in the days after learning. Learning also caused changes in firing patterns at well-learned goal locations. These results suggest that learning was supported by modification of an active schema of spatially related reward loci. In another experiment, I extended these findings to explore a schema of object and place associations. Ensemble activity was influenced by a hierarchy of task dimensions which included: experimental context, rat's spatial location, the reward potential and the identity of sampled objects. As rats learned about new objects, the cells that had previously fired for particular object-place conjunctions generalized their firing patterns to new conjunctions that similarly predicted reward. In both experiments, I observed highly structured representations for a set of related experiences. This organization of hippocampal activity counters key assumptions in standard models of hippocampal function that predict relative independence between memory traces. Instead, these findings reveal neural mechanisms for how the hippocampus develops a relational organization of memories that could support novel, inferential judgments between indirectly related events.

Voltage-gated calcium channels (CaV) are key regulators of cellular excitability; they translate electrical information into biochemical responses in excitable cells such as nerve and muscle cells. CaV are separated in three families: CaV1, CaV2 and CaV3. CaV1 and CaV2 typically comprise a pore-forming alpha1 with auxiliary β and alpha2delta subunits. The alpha2delta enhances surface expression and modulates the biophysical properties of CaV. It has been implicated in pain and epilepsy, and the target for anti-epileptic and anti-nociceptive gabapentinoid drugs. Despite its clinical significance, the relationship between the structure and function of this subunit remains poorly understood. Fitzgerald and co-workers recently showed that the N-terminal region of alpha2delta-1, termed the R domain (Rd), is both necessary and sufficient to replicate the effects of full-length alpha2delta on CaV2.2 channels. In order to understand the functional role(s) of Rd and the regions downstream of it, the biochemical and cell biological properties of alpha2delta were explored producing a set of alpha2delta-truncated proteins, in which the delta protein was inserted into an inert type-1 transmembrane reporter protein (PIN-G). The construct was then extended towards the N-terminal of the alpha2delta-1 (C- to N- PIN-constructs). Other sets of constructs, lacking the delta protein, were prepared after successive additions of stop codons (TGA) in the alpha2delta (N- to C- PIN-constructs). The MIDAS motif within the VWA domain of alpha2delta-1/-2 has been suggested to be critical for trafficking of alpha2delta to the cell surface. Whilst the present study supports a role for MIDAS in surface expression of alpha2delta, it is the Rd that appears essential. Mutation of MIDAS reduced expression, whereas the removal of Rd completely abolished the presence of alpha2delta at the cell surface. Examination of the electrophysiological effects of N- to C- terminal truncated constructs (PIN-Rd, PIN-Rd-VWA and PIN-alpha2) on CaV2.2/β1b channels revealed that, in contrast to the full functionality of Rd alone, extension to the end of the VWA domain, or the alpha2 region, abolished typical alpha2delta-mediated current enhancement. Nevertheless, both constructs increased rate of voltage-dependent inactivation, indicating that they interact with the channel via Rd. Thus, Rd appears to contain all the machinery required to support the electrophysiological and trafficking effects of alpha2delta. Preliminary work has generated tools that could be used to conduct competition-based assays to identify the extracellular loops of the CaV2.2 alpha1 subunit that interact with the Rd. Such an approach could be applied to other alpha1 subtypes to determine discrete alpha2-Rd interactions, information that is critical for further therapeutic exploitation of alpha2delta. Finally, the data from this thesis and the existing literature have been used to propose a revised model of how alpha2delta interacts with CaV.

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