The ability to respond rapidly with spatial precision is required in a number of facets of everyday life, whether catching a falling object, reacting to other drivers on a busy freeway or recovering one’s balance following an unexpected perturbation. The sophisticated central nervous system (CNS) control of these reactions is often overlooked until the speed of such reactions becomes delayed, either due to ageing or brain injury, wherein the individual becomes at risk of injury. Surprisingly, little is known regarding the control of these ‘temporally urgent’ movements. Therefore, the primary objectives of this dissertation were to develop an understanding of the control of these movements by exploring the factors that may be involved in the generation of temporally urgent movements in the healthy CNS, locating the areas within the CNS that such modulation occurs and identifying the relative weighted importance of those modulators based on the initial conditions of stimulus delivery. Specific characteristics of stimulus properties, such as intensity and modality were particularly influential in the latency of motor reactions and physiological electrodermal skin responses fluctuated in accordance with input stimulus parameters. Importantly, outcomes from this dissertation identified that rapid reactions likely utilize a CNS network that includes higher cortical regions such as somatosensory cortex and primary motor cortex, which may be modulated by physiological arousal, rather than the solitary involvement of subcortical structures. The findings from this dissertation have important implications for individuals with disordered speed of processing and indicate the potential modifiability of factors that influence reaction time.

eneurin C-terminal associated peptides (TCAPs) are a novel family of peptides encoded on the last exon of the teneurin genes. The predicted peptide sequences are highly conserved across metazoans and possess the structural hallmarks of a cleavable bioactive peptide that are 40 or 41 amino acid residues. One of the peptides in the family, TCAP-1, is a potent regulator of neurite outgrowth and dendritic spine density in the hippocampus and inhibits corticotropin-releasing factor (CRF)-associated stress-induced and cocaine-seeking behaviours. The effects of TCAP-1 are long lasting, suggesting that TCAP-1 plays a significant role in the regulation of cell-to-cell communication and cellular plasticity. Moreover, TCAP-1 regulates cellular energy, metabolism and cell survival and may, therefore, possess functional attributes outside of the CNS. However, the molecular mechanisms associated with TCAP-1-mediated trophic effects are not known. My research was aimed to 1) determine whether TCAP-1 exerts its effects as part of a direct teneurin-1 function, whereby TCAP-1 represents a functional region of the large teneurin-1 protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin-1; 2) map the distribution of TCAP-1-immunoreactivity and TCAP-1 binding sites in mouse; 3) elucidate the molecular mechanism by which TCAP-1 regulates cytoskeletal dynamics; and 4) investigate a role for TCAP-1 in male reproduction. My research establishes that the C-terminal region of teneurin-1, corresponding to TCAP-1, can be both structurally and functionally independent from teneurin-1 in both the brain and testis of the adult mouse. Furthermore, I provide novel evidence that functionally links the teneurin-TCAP-1 system with the dystroglycan complex and provide new insight into the molecular and signaling mechanisms by which TCAP-1 regulates cytoskeletal dynamics. These studies implicate the teneurins in a broader range of neuroendocrine and trophic functions than previously thought and furthers our understanding of the mechanisms associated with TCAP-1-mediated function in the body.

eneurin C-terminal associated peptides (TCAPs) are a novel family of peptides encoded on the last exon of the teneurin genes. The predicted peptide sequences are highly conserved across metazoans and possess the structural hallmarks of a cleavable bioactive peptide that are 40 or 41 amino acid residues. One of the peptides in the family, TCAP-1, is a potent regulator of neurite outgrowth and dendritic spine density in the hippocampus and inhibits corticotropin-releasing factor (CRF)-associated stress-induced and cocaine-seeking behaviours. The effects of TCAP-1 are long lasting, suggesting that TCAP-1 plays a significant role in the regulation of cell-to-cell communication and cellular plasticity. Moreover, TCAP-1 regulates cellular energy, metabolism and cell survival and may, therefore, possess functional attributes outside of the CNS. However, the molecular mechanisms associated with TCAP-1-mediated trophic effects are not known. My research was aimed to 1) determine whether TCAP-1 exerts its effects as part of a direct teneurin-1 function, whereby TCAP-1 represents a functional region of the large teneurin-1 protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin-1; 2) map the distribution of TCAP-1-immunoreactivity and TCAP-1 binding sites in mouse; 3) elucidate the molecular mechanism by which TCAP-1 regulates cytoskeletal dynamics; and 4) investigate a role for TCAP-1 in male reproduction. My research establishes that the C-terminal region of teneurin-1, corresponding to TCAP-1, can be both structurally and functionally independent from teneurin-1 in both the brain and testis of the adult mouse. Furthermore, I provide novel evidence that functionally links the teneurin-TCAP-1 system with the dystroglycan complex and provide new insight into the molecular and signaling mechanisms by which TCAP-1 regulates cytoskeletal dynamics. These studies implicate the teneurins in a broader range of neuroendocrine and trophic functions than previously thought and furthers our understanding of the mechanisms associated with TCAP-1-mediated function in the body.

Microglia, the resident immune cells of the central nervous system, can become activated following injury, disease, or infection. In vitro, they can be activated by stimuli, which determine the inflammatory phenotype they will develop. In this thesis, stimulating microglia with tumor necrosis factor- and interferon- resulted in classical activation, characterized by proliferation, increased transcription of complement receptor 3 and major histocompatibility class II molecules, and elevated production and transcription of interleukin-1 and nitric oxide. Stimulation with TNF and IFN also changed the intensity of phosphorylated (activated) cyclic adenosine monophosphate response element binding protein immunoreactivity in microglia. Specifically, cells differentiated into populations with high or low pCREB intensity. This was the first example of such a response in microglia and was representative of what occurred in vivo, after ICH. Thus, the characterization of this model will be useful for future studies of this and other intracellular pathways of classically activated microglia.

Hedgehog (HH) signaling controls renal development. Mutations in PTC1, the HH receptor, cause cancer in non-renal tissues. We hypothesized that constitutively active HH signaling is deleterious to renal development in mice with PTC1 deficiency targeted to the metanephric mesenchyme (MM)(Rarb2-Cre;Ptc1 loxP/-, termed Ptc1 mutants). Increased HH signaling in MM of mutant mice was confirmed by qRT-PCR for Ptc1. A decrease in NCAM-positive nephrogenic precursors at E13.5 and WT1-positive glomeruli at E18.5 was found. Increased cortical expression of Foxd1 was observed. At E13.5, a cluster of ectopic cells expressing Raldh2, Ptc2 and Bmp4 accumulated at the presumptive uretero-pelvic junction (UPJ). Magnetic resonance imaging demonstrated an increase in pelvic volume. Constitutive expression of GLI3 repressor via the Gli3Δ699 allele in Ptc1 mutants increased nephron number comparable to wild type mice and decreased pelvic volume compared to Ptc1 mutants. Thus repression of HH activity is required for proper nephrogenesis and patterning of the UPJ.

Calcium ions (Ca2+) are admitted into presynaptic nerve terminals through voltage gated calcium channels and diffuse to bind and activate the secretory vesicle discharge mechanism. Current research favors a highly ‘modal’ organization where the release sites are activated by one or a few closely apposed channels (Stanley, 1997). To fully understand the nanophysiology of transmitter release site activation, it is necessary to determine the rate of Ca2+ flux through individual channels at normal physiological external concentrations. OBJECTIVE: To explore the relationship between CaV2.2 channel conductance and external Ca2+ across the physiological range. CONCLUSION: The conductance of the CaV2.2 channel was determined across the range of 1-100 mM [Ca2+]EXT . With 2 mM [Ca2+]EXT, the conductance was determined to be 2.76 ± 0.24 pS.

A dominant ENU screen produced mouse line 7238 with large platelets. Sequence capture and Next Generation sequencing identified a mutation in Myh9 at Q1443L [1]. Mice were tested for aspects of MYH9-Related Disease (MYH9RD), a rare human condition caused by mutations within MYH9; macrothrombocytopenia and neutrophil inclusions are found in almost all cases, while deafness, cataracts and renal disease have variable penetrance and severity. Myh9Q1443L/+ and Myh9Q1443L/Q1443L animals have neutrophil inclusions [1] and increased cataracts at 2, 6 and 12 months; Myh9Q1443L/Q1443L animals at 12 months have changes in kidney output [2]. Immunofluoresence showed changes in protein expression in glomeruli at two months. This is the first ENU mouse model identified by a sequence capture mechanism, and the first mouse line to produce a point mutation within the Myh9 gene [1,2]. This mouse models MYH9RD, and is an invaluable tool to understand the role of this protein, and to determine mechanisms underlying this disease.

Rationale: Activation of the platelet-derived growth factor receptors-α and -β (PDGF-Rα and -Rβ) is critical in the formation of secondary crests/septa during alveologenesis, and its regulation has been found to be disrupted in rat lung injury models. Objective: To determine whether the process of secondary septation can occur after transient pharmacologic inhibition of PDGF-R action during postnatal days (P)1 – 7 in rats. Hypothesis: The initial process of secondary crest formation is time-limited and, if missed, will result in a permanent loss of alveoli. Methods: Imatinib mesylate, a PDGF-R inhibitor, was injected intraperitoneally from P1 – 7. Pups were sacrificed on P2, 4, 8, 14, 28 and 65 for studies of alveolar development. Main results: The injection of imatinib inhibited PDGF-R action, resulting in a permanent decrease in alveolar number in treated rats. Conclusions: Inhibition of secondary septation during the first 7 days of life resulted in a decrease in alveolar number lasting into early adult life. This is consistent with a critical time window for secondary septation, which, if disrupted, results in long-term adverse effects on lung development.

Calcium ions (Ca2+) are admitted into presynaptic nerve terminals through voltage gated calcium channels and diffuse to bind and activate the secretory vesicle discharge mechanism. Current research favors a highly ‘modal’ organization where the release sites are activated by one or a few closely apposed channels (Stanley, 1997). To fully understand the nanophysiology of transmitter release site activation, it is necessary to determine the rate of Ca2+ flux through individual channels at normal physiological external concentrations. OBJECTIVE: To explore the relationship between CaV2.2 channel conductance and external Ca2+ across the physiological range. CONCLUSION: The conductance of the CaV2.2 channel was determined across the range of 1-100 mM [Ca2+]EXT . With 2 mM [Ca2+]EXT, the conductance was determined to be 2.76 ± 0.24 pS.

Rationale: Activation of the platelet-derived growth factor receptors-α and -β (PDGF-Rα and -Rβ) is critical in the formation of secondary crests/septa during alveologenesis, and its regulation has been found to be disrupted in rat lung injury models. Objective: To determine whether the process of secondary septation can occur after transient pharmacologic inhibition of PDGF-R action during postnatal days (P)1 – 7 in rats. Hypothesis: The initial process of secondary crest formation is time-limited and, if missed, will result in a permanent loss of alveoli. Methods: Imatinib mesylate, a PDGF-R inhibitor, was injected intraperitoneally from P1 – 7. Pups were sacrificed on P2, 4, 8, 14, 28 and 65 for studies of alveolar development. Main results: The injection of imatinib inhibited PDGF-R action, resulting in a permanent decrease in alveolar number in treated rats. Conclusions: Inhibition of secondary septation during the first 7 days of life resulted in a decrease in alveolar number lasting into early adult life. This is consistent with a critical time window for secondary septation, which, if disrupted, results in long-term adverse effects on lung development.