Cerebral ischemia and intracerebral hemorrhage (ICH) are both characterized by a prolonged inflammatory response and secondary injury phase, yet the spatial/temporal relationships between inflammation and white matter (WM) damage were largely unknown. Thus, I quantified the development of WM damage and inflammation over 7 days after ischemia, and 14 days after ICH. Following ischemia, myelin and axons were progressively damaged, and myelin damage coincided with neutrophil infiltration. Activated microglia/macrophages increased dramatically in the lesion core and edge, and selectively infiltrated damaged WM tracts while surrounding undamaged ones. To investigate the involvement of neutrophils in WM damage and inflammation after ICH, rats were rendered neutropenic before performing ICH. Neutrophil depletion reduced peri-hematomal axonal damage, BBB breakdown, and MMP-9 production at early times, and lessened microglia/macrophage and astrocyte responses at later times. Activated microglia/macrophages infiltrated peri-hematomal WM tracts, correlating with myelin fragmentation and axonal loss, and this was reduced with neutrophil depletion.

The anterior cingulate cortex (ACC) expresses high density of Gastrin-releasing peptide (GRP) and GRP receptor mRNA. To address possible function, this investigation used patch clamp recordings in mouse brain slice preparations to evaluate intrinsic properties of ACC neurons and neuronal responses to bath-applied GRP peptide. The ACC neurons were divided according to their morphology, the properties of action potentials and their firing pattern in response to depolarizing current pulses. Two physiological groups of interneurons and three groups of pyramidal neurons were defined. Application of the GRP induced depolarization and increased firing of the interneurons while hyperpolarization and reduced firing in pyramidal neurons. Moreover, activation of GRP receptor facilitated GABAergic neurotransmission via a postsynaptic mechanism. The results suggest that GRP receptor is an important regulator of neuronal circuits in the ACC and may consequently play an important role for ACC neurons in the central processing of high brain function.

The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na+ and glucose. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the first project, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Charged and polar residues and glucose/galactose malabsorption (GGM) missense mutations in TM IV were replaced with cysteine. Mutants exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. The results from mutants T156C and K157C suggest that TM IV participates in sugar interaction with SGLT1. This work has been published in Am J Physiol Cell Physiol 295 (1), C64-72, 2008. The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) was recently published (1) and showed discrepancy with the predicted topology of mammalian SGLT1 in the region surrounding transmembrane segments IV-V. Therefore, in the second project, we investigated the topology in this region, thirty-eight residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were individually replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. Based on the results from biotinylation of mutants in intact COS-7 cells, MTSES accessibility of cysteine mutants expressed in COS-7 cells, effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells, and characterization of cysteine mutants in TM V expressed in Xenopus laevis oocytes, we suggest that the region including residues 143-180 forms part of the Na+- and sugar substrate-binding cavity. Our results also suggest that TM IV of mammalian SGLT1 extends from residue 143-171 and support the crystal structure of vSGLT. This work has been published in Biochem Biophys Res Commun 378 (1), 133-138, 2009 Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C following reaction with methanethiosulfonate derivatives, but Na+ and sugar binding remain intact. Therefore, in the third project, we explored the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 expressed in Xenopus laevis oocytes were studied using chemical modification, the two-electrode voltage-clamp technique and computer model simulations. Our results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. This work has been published in Biophysical Journal 96 (2), 748-760, 2009. Taken together our study along with previous biochemical characterization of SGLT1 and crystal structure of vSGLT, we propose a limited structural model that attempts to bring together the functions of substrate binding (Na+ and sugar), coupling, and translocation. We propose that both Na+ and sugar enter a hydrophilic cavity formed by multiple transmembrane helices from both N-terminal half of SGLT1 and C-terminal half of SGLT1, analogous to all of the known crystal structures of ion-coupled transporters (the Na+/leucine transporter, Na+/aspartate transporter and lactose permease). The functionally important residues in SGLT1 (T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites.

The role of placental VEGFA in placental development and maternal pregnancy adaptations is unknown. In this thesis, one or both copies of Vegfa was knocked out from the placental spongiotrophoblast region using a mouse Cre-loxP system. Surprisingly, in pregnancies carrying 100% conceptuses with a single deletion of Vegfa from the spongiotrophoblast layer, maternal circulating VEGFA increased by 20% accompanied by a 15% decrease in arterial pressure while no impairment in embryo growth was found. In pregnancies carrying 50% conceptuses with both copies of Vegfa deleted from spongiotrophoblast, 17% of conceptuses had been reabsorbed by late gestation suggesting a function for spongiotrophoblast VEGFA in sustaining early pregnancy. In conclusion, spongiotrophoblast VEGFA affects maternal function during pregnancy but the exact mechanism remains to be defined.

Long term memory (LTM) formation is a complex process involving signalling cascades, new protein synthesis and gene regulation. Increasing evidence demonstrates a role of intrinsic plasticity in memory formation, but the underlying molecular mechanisms remain relatively unknown. LTM was established using an aversive operant conditioning model in Lymnaea stagnalis. Using intracellular electrophysiology in an isolated preparation, increased gain of firing frequency was observed in the RPeD1 neuron after LTM. This provides the first demonstration of intrinsic plasticity after operant conditioning in RPeD1, a neuron required for the conditioned behaviour and LTM. I also determined the contribution of the transcription factor men1 to plasticity. Using in vivo RNAi silencing, I found that men1 is required for LTM and increasing RPeD1 excitability during consolidation, demonstrating men1-mediated intrinsic plasticity is critical for LTM. I propose a new model of memory formation in which men1-dependent increase of excitability during consolidation is required for LTM.

Long term memory (LTM) formation is a complex process involving signalling cascades, new protein synthesis and gene regulation. Increasing evidence demonstrates a role of intrinsic plasticity in memory formation, but the underlying molecular mechanisms remain relatively unknown. LTM was established using an aversive operant conditioning model in Lymnaea stagnalis. Using intracellular electrophysiology in an isolated preparation, increased gain of firing frequency was observed in the RPeD1 neuron after LTM. This provides the first demonstration of intrinsic plasticity after operant conditioning in RPeD1, a neuron required for the conditioned behaviour and LTM. I also determined the contribution of the transcription factor men1 to plasticity. Using in vivo RNAi silencing, I found that men1 is required for LTM and increasing RPeD1 excitability during consolidation, demonstrating men1-mediated intrinsic plasticity is critical for LTM. I propose a new model of memory formation in which men1-dependent increase of excitability during consolidation is required for LTM.

The anterior cingulate cortex (ACC) expresses high density of Gastrin-releasing peptide (GRP) and GRP receptor mRNA. To address possible function, this investigation used patch clamp recordings in mouse brain slice preparations to evaluate intrinsic properties of ACC neurons and neuronal responses to bath-applied GRP peptide. The ACC neurons were divided according to their morphology, the properties of action potentials and their firing pattern in response to depolarizing current pulses. Two physiological groups of interneurons and three groups of pyramidal neurons were defined. Application of the GRP induced depolarization and increased firing of the interneurons while hyperpolarization and reduced firing in pyramidal neurons. Moreover, activation of GRP receptor facilitated GABAergic neurotransmission via a postsynaptic mechanism. The results suggest that GRP receptor is an important regulator of neuronal circuits in the ACC and may consequently play an important role for ACC neurons in the central processing of high brain function.

The role of placental VEGFA in placental development and maternal pregnancy adaptations is unknown. In this thesis, one or both copies of Vegfa was knocked out from the placental spongiotrophoblast region using a mouse Cre-loxP system. Surprisingly, in pregnancies carrying 100% conceptuses with a single deletion of Vegfa from the spongiotrophoblast layer, maternal circulating VEGFA increased by 20% accompanied by a 15% decrease in arterial pressure while no impairment in embryo growth was found. In pregnancies carrying 50% conceptuses with both copies of Vegfa deleted from spongiotrophoblast, 17% of conceptuses had been reabsorbed by late gestation suggesting a function for spongiotrophoblast VEGFA in sustaining early pregnancy. In conclusion, spongiotrophoblast VEGFA affects maternal function during pregnancy but the exact mechanism remains to be defined.

The present study examined the hemodynamic response to recreational hockey (n= 22) in middle-aged men (53±6 yrs). Study participants were equipped with ambulatory blood pressure and heart rate monitoring equipment prior to a weekly hockey games. Participants were monitored throughout the duration of their hockey game for “On-Ice” responses and during seated bench time (“Bench”), and for a brief period afterwards. On-Ice HR’s and blood pressures were significantly higher than values obtained during maximal cycle exercise (HR 174±8.9 vs. 163±11.0 bpm) (SBP 17%; DBP 15%) (p<0.05), Blood pressures decreased throughout the duration of the game while HR increased significantly. The On-Ice endocardial viability ratio (EVR), an index of myocardial oxygen supply and demand, did not change from early (1.56±0.05) to late (1.44± 0.06) in the game. In conclusion, recreational hockey is an extremely vigorous form of interval exercise that produces cardiovascular responses exceeding intensities commonly recommended for continuous training.

Hypoglycemia is a severe side effect of insulin overdose in the diabetic population and can result in various neurological sequalae including seizures, coma, and brain death. There is still a limited understanding of the generation and propagation of hypoglycemic seizures, which may exacerbate hypoglycemia-induced neuronal damage. Moreover, glucose reperfusion after a period of transient hypoglycemia has been shown to cause neuronal hyperexcitability which can have further damaging effects. Gap junctional communication can be involved in the spread of hypoglycemic injury in two ways: 1) by providing a cytoplasmic continuity in which seizures can easily propagate and 2) by engaging the astrocytic network in metabolic compensation as well as enhancing astrocytic buffering of K+. This study aims to investigate the role that gap junctions play during brain energy deprivation. Results from these experiments show that gap junction blockade can have a neuroprotective role during hypoglycemia and glucose reperfusion.