Hypoglycemia is one of the most serious acute complications in intensively treated diabetes. Recurrent hypoglycemia predisposes individuals to subsequent hypoglycemia, and diminished counterregulatory hormone responses increase this threat. Elevated pancreatic and/or circulating somatostatin has been reported in diabetic humans and animals, and we postulated that excessive somatostatin contributes to the attenuation of counterregulatory hormone release during hypoglycemia in diabetes. It is known that somatostatin suppresses stimulated secretion of glucagon, epinephrine, and corticosterone. We hypothesized that selective somatostatin receptor type 2 (SSTR2) antagonism would: (Study 1) improve hormone counterregulation to hypoglycemia, and (Study 2) ameliorate hypoglycemia in recurrently hypoglycemic rats. Using both high (10 U/kg) and low (5 U/kg) dose insulin to induce hypoglycemia, we demonstrate that inhibiting the action of somatostatin on SSTR2 normalizes the severely attenuated glucagon and corticosterone responses to acute hypoglycemia in diabetic rats. These improvements were specific to diabetes since SSTR2 antagonism did not increase these hormones in non-diabetic rats in response to hypoglycemia. In the absence of hypoglycemia, SSTR2 antagonist neither markedly alters glycemia nor causes sustained elevations in counterregulatory hormones in diabetic animals. Diabetic rats exhibit up to 65% and 75% more pancreatic and plasma somatostatin than non-diabetic rats following hypoglycemia, respectively. Despite improvements of glucagon and corticosterone, expression of gluconeogenic enzymes PEPCK1 and G6Pase was unaltered. SSTR2 antagonism reduced the glucose requirement during a hypoglycemic clamp induced with a lower dose of insulin. In recurrently hypoglycemic diabetic rats, we demonstrate that SSTR2 antagonist treatment reduces the depth and duration of hypoglycemia and promotes the recovery to euglycemia, without affecting the glycemia-lowering effect of insulin. This amelioration of hypoglycemia by SSTR2 antagonism may be attributable in part to the observed modest improvements of glucagon, epinephrine, and corticosterone counterregulation following recurrent hypoglycemia. These results implicate an important role for increased pancreatic, and possibly circulating, somatostatin in defective hypoglycemic counterregulation in diabetes.

This thesis consists of four chapters. In this thesis we explored associative plasticity of human motor cortex with the use of noninvasive transcranial magnetic stimulation (TMS). Paired Associative Stimulation (PAS) has grown in popularity because of its potential clinical applications. We used TMS techniques in combination with electromyographic (EMG) measurements to study cortical excitability and kinematic features of arm movement. This work has focused in a cohesive approach to answer certain fundamental questions about a) the rules of cortical plasticity and mechanism of PAS, b) the interaction between the state of neuronal excitability at the targeted cortical network and the effects of PAS, and c) translation of these effects into obvious measurable kinematic changes starting from network level changes and ending up with the behavioral modulation of arm movement. First we explored the role of GABAergic intracortical networks and intracortical facilitation on modulation of cortical excitability by showing for the first time that PAS can be conditioned by these inhibitory and facilitatory intracortical networks. Next, using standard indirect approaches utilizing peripheral EMG measures, we showed a graded excitability response for the PAS technique and showed that interactions of PAS with motor learning depends on the degree as well as the state of cortical excitability. Rules governing the interactions of brain stimulation techniques and motor learning are important because brain stimulation techniques can be used to modify, improve or disrupt motor adaptation and skill learning with great potential for clinical applications such as facilitation of recovery after stroke. TMS provide us with a unique opportunity to study the rules of plasticity at a systems level, which is a combination of synaptic and nonsynaptic (metaplastic) changes. These changes can occur either in the direction to limit the physiological range of neuronal functioning (homeostatic) or against the direction established state of neurons.

There is a large sex difference in the prevalence of attention deficit disorder; yet, little is known about sex differences in prefrontal attention circuitry. We investigated sex differences in the developmental nicotinic excitation of corticothalamic layer VI neurons, which play an important role in attention. Using whole cell recording in prefrontal brain slices, we examined the inward currents elicited by nicotinic stimulation in rodents. We found a prominent sex difference in the currents during the first postnatal month when males had significantly greater α4β2* nicotinic currents. Immunohistochemical analysis of α4YFP mice revealed no sex difference in the pattern or proportion of YFP-positive neurons in layer VI. Further electrophysiological experiments revealed that progesterone is able to rapidly and significantly suppress nicotinic currents in layer VI neurons. This is the first illustration at a cellular level that prefrontal attention circuitry is differently excited by nicotinic stimulation in males and females during development.

Some animals have adapted to hypoxia by increasing their haemoglobin affinity for oxygen, but in vitro studies have not shown any change of haemoglobin affinity for oxygen in human high altitude natives or lowlanders acutely acclimatized to high altitude. We conducted the first in vivo study of the oxyhaemoglobin dissociation curve by progressively reducing arterial PO2 while maintaining normocapnia in lowlanders at sea level, lowlanders sojourning at 3600m for two weeks and native Andeans at the same altitude. We found that the in vivo PO2 at which haemoglobin is half-saturated (P50) is higher in lowlanders at sea level (32 mmHg) than that measured in vitro (27 mmHg) and that lowlanders and highlanders do significantly increase the in vivo affinity of their haemoglobin for oxygen with exposure to high altitude. These results indicate the value of an in vivo approach for studying the oxyhaemoglobin dissociation curve.

The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.

Parkinson’s disease (PD) is characterized by the loss of dopamine in the basal ganglia and leads to paucity of movements, rigidity of the limbs, and rest tremor. Synaptic plasticity was characterized in the substantia nigra pars reticulata (SNr), a basal ganglia output structure, in 18 PD patients undergoing implantation of deep brain stimulating electrodes. Field evoked potentials (fEPs) in SNr were measured with one microelectrode using single pulses from a second microelectrode ~ 1 mm away. High frequency stimulation (HFS – 4 trains of 2s at 100Hz) in the SNr failed to induce a lasting change in test fEPs amplitudes in patients OFF medication. Following L-Dopa, HFS induced a potentiation of the fEPs that lasted more than 150s. Our findings suggest that extrastriatal dopamine modulates activity dependent synaptic plasticity at basal ganglia output neurons. Dopamine medication state clearly impacts fEP amplitude, and the lasting nature of the increase is reminiscent of LTP-like changes, indicating that aberrant synaptic plasticity may play a role in the pathophysiology of PD.

Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Regulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.

The exocyst is thought to tether secretory vesicles to specific sites on the plasma membrane. As a member of the exocyst, Sec5 is implicated in cell survival and membrane growth in Drosophila. Little is known of the exocyst function in mammals, with previous work suggesting involvement of exocyst in GTP-dependent exocytosis. Using RNA interference, we stably down-regulated Sec5 in PC12 cells. We found that these knockdown cells exhibit decreased GTP- and Ca2+-dependent exocytosis of dense-core vesicles (DCVs), and contain less proportion of docked vesicles. Expression of Sec6/8 is also slightly reduced in Sec5 knockdown cells. Our results suggest that Sec5 is involved in both GTP- and Ca2+-dependent exocytosis, possibly through the regulation of DCV docking. We also established doxycycline-inducible knockdown system for Sec5 in PC12 cells which may be more appropriate to study development-related proteins. Efforts were also made to re-introduce Sec5 into the Sec5 knockdown cells for rescue purposes.

The molecular mechanisms that coordinate postnatal brain enlargement, synaptic properties and cognition remain an enigma. This study demonstrates that neuronal complexity controlled by p21-activated kinases (PAKs) is a key determinant for postnatal brain enlargement and synaptic properties. Double knockout (DK) mice lacking both PAK1 and PAK3 were severely impaired in postnatal brain growth, resulting in a dramatic reduction in brain volume at maturity. Remarkably, the reduced brain was accompanied by minimal changes in total cell count, due to a significant increase in cell density. However, the DK neurons have smaller soma, markedly simplified dendritic arbors/axons and reduced synapse density. Surprisingly, the DK mice were elevated in basal synaptic responses due to enhanced individual synaptic potency, but severely impaired in bi-directional synaptic plasticity. The PAK1/3 action is likely mediated by cofilin-dependent actin regulation because the activity of cofilin and the properties of actin filaments were specifically altered in the DK mice.