Locomotion is controlled in large part by neural circuits (CPGs) that generate rhythmic stereotyped outputs in the absence of descending or sensory inputs. The output of a neural circuit is determined by the configuration of the circuit, synapse properties, and the intrinsic properties of component neurons. In order to understand how a neural circuit functions component neurons, their connections, and their intrinsic properties must be characterized. Motoneurons are a useful cell in which to begin investigation of CPG function because they are accessible and provide a measure of the cumulative activity of the circuit. Drosophila is a potentially useful model system for the study of motoneuron intrinsic properties, their contribution to locomotion, and of locomotor CPGs because the genetic and molecular techniques available in Drosophila are surpassed in no other organism and because the Drosophila nervous system is small in comparison to vertebrate nervous systems. Further, whole-cell in situ patch clamp recordings from adult and larval motoneurons in relatively intact preparations are possible. Therefore, the first goal of this work was to investigate whether the firing behavior and recruitment of identified Drosophila 1b and 1s motoneurons is analogous to the recruitment of high-threshold, phasic and low-threshold, tonic motoneurons in other organisms. The second goal was to determine whether active conductances influence motoneuron recruitment in response to synaptic input. The final aim was to investigate how these factors influence CPG output to muscles. Findings from current clamp studies indicate that1b motoneurons are more easily recruited than 1s motoneurons, in agreement with the hypothesis that 1b motoneurons are analogous to low-threshold motoneurons described in other organisms. Further, orderly recruitment of Drosophila 1b motoneurons before 1s motoneurons is not a result of passive properties. Instead, the Shal channel that encodes a large portion of IA in motoneuron somatodendritic regions is a critical determinant of delay-to-spike in larval Drosophila motoneurons. These findings are behaviorally-relevant because the same recruitment order is seen in simultaneous recordings from motoneuron pairs recruited by synaptic input.

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

Levetiracetam (LEV) is an antiepileptic drug (AED) that has been shown to mainly enhance synaptic depression and modulate certain voltage and ligand-gated channels, after it gains entry into neurons through endocytosis. Since synaptic terminals and distal axons are the first compartments exposed to LEV, we utilized a crayfish motor axon preparation to investigate whether LEV modulates axonal excitability. Two electrode current clamp from the inhibitor axon of the crayfish opener showed that LEV reduced action potential amplitude (APamp) and enhanced synaptic depression, although these events did not occur at the same time, the latter occurred later than the reduction in APamp. Further examinations of these effects and comparison of antidromic and orthodromic conducting action potentials in LEV suggests that this drug preferentially reduces excitability of the proximal axon despite the expectation that it enters the axon at terminals and reaches distal branches first. Results presented here demonstrate that LEV modulates axonal excitability, which may also contribute to its antiepileptic effects.

Introduction: Excitability in cardiomyocytes is dependent on the subthreshold current required to raise transmembrane potential to the activation threshold and subsequent recruitment of voltage gated sodium channels to trigger an action potential. Conduction in cardiomyocytes is dependent on the robustness and speed of action potential propagating through tissue. Both are equally important for normal heart function and claim to be linear correlated (i.e if conduction decreases, excitability decreases) Cardiac sodium channels are densely expressed in the intercalated disc within the perinexus, which is two orders of magnitude narrower than bulk extracellular interstitium. The biphasic relationship between conduction and perinexus is well-researched and consistent between computations models. We hypothesized a biphasic relationship between Excitability and perinexal width (Wp). In addition, we hypothesize that the relationship between excitability and conduction is not linear but dependent on the original width of the perinexus. Methods/and Results: Ex vivo guinea pig hearts were epicardially paced and optically mapped to assess ventricular conduction and excitability. Strength-duration curves were constructed for pacing stimuli to measure rheobase (inversely correlated to excitability). Computation models incorporating ephaptic coupling and sodium channel localization to cleft widths between cardiomyocytes demonstrate these findings. Conclusion: Models and experiments reveal that the excitability and perinexus relationship is biphasic where narrowing and widening perinexus decreases conduction and excitability thus showing a linear relationship between excitability and conduction. However, the excitability and conduction become overly complex in the transition phase from release of self-attenuation to reduced self-activation. Therefore, targeting ephaptic coupling and monitoring plasma ions may be a novel strategy for increasing the efficacy and efficiency of cardiac pacemakers. / Doctor of Philosophy / The heart is a muscular organ that uses electrical impulses to function. The heart is made of cells called cardiomyocytes that allow for electricity to flow through the cells. They are connected via different junctions such as gap junctions, adherens, etc. Any loss of electrical coordination leads to irregular heartbeats which can lead to heart death. There are two ways to study electrical coordination, excitability, how easy is for the current to start in the tissue, and conduction, how easy can that current travel through the tissue. Since the 1900s researchers have stated that if excitability decreases conduction decreases. In other words, if you need more current to start the heart (excitability decreases) then that current will travel slower through the tissue (conduction decreases) thus increasing one chances of irregular heartbeats. However, the understanding of how conduction works has changed but not of excitability. For example, originally current was thought to travel through channels called gap junctions. If you have limited availability of gap junctions, current increases (aka excitability decreases) and conduction decreases. However, other species such as frogs, fishes have limited number of gap junctions and can survive. Therefore, a new mechanism was proposed called ephaptic coupling. There is space next to the gap junctions called perinexus which is rich in a channel called Na channels, which is the main driving force for excitability and conduction. The lab has shown that if you change that space between cells, you can change the conduction response. In other words, if you decrease the space between the cells, conduction will not change therefore reducing the chances of irregular heartbeats. Therefore, my project is to understand if by changing this space between cells, is excitability and conductions are still correlates of each other. Using mathematical and animal models, this dissertation shows excitability and conduction have a very complicated relationship.

Diversity in the expression of ion channel proteins among neurons allows a wide range of excitability, growth and functional regulation. Ether-a-go-go (EAG), a member of the voltage-gated K+ channels, was characterized by spontaneous firing in nerve terminals and enhanced neurotransmitter release. In situ whole-cell patch-clamp recordings performed from the somata of Drosophila larval thoracic aCC motoneurons revealed spontaneous spike-like events in eag mutants. Spontaneous events were absent in wild type motoneurons. Spikes evoked by somatic current injection in to the cell body were not altered and comparable to wild type. Spontaneous spike-like events could be due to increased synaptic drive or altered intrinsic excitability of the motoneuron. Reduction of EAG function with selective expression of eag double stranded RNAi transgene in motoneurons only did not cause spontaneous spike-like events or alter evoked firing. This suggests increased synaptic drive contributes to spontaneous events.Both transient and sustained voltage-activated K+ currents, each with Ca++-sensitive (IA(Ca) and IK(Ca)) and Ca++ -insensitive components (IA and IK), were isolated in thoracic aCC motoneurons. In wild type motoneurons, IA was larger than IA(Ca). Conversely, IK(Ca) was larger than IK. Both eag mutants and eag RNAi expression resulted in a decrease in IA , IK and a slow sustained K+ current. Further, EAG and Shal demonstrate a potential functional interaction and contribute to IA. The voltage sensitivity for inactivation was reduced in Shal only and EAG-Shal double knock down compared to controls and EAG only knock down. In addition, a Ca++ sensitive EAG dependent K+ current was blocked by cAMP. Thus, both voltage-dependent and modulatory functions of EAG influence excitability in motoneurons.Firing properties and K+ currents distinguish aCC motoneurons in thoracic segments, T1 and T3. T3aCC had a shorter delay to spike, higher input resistance and were more easily recruited than T1aCC. T1aCC had a larger IA than T3aCC, but comparable IA(Ca). IK(Ca) was larger in T3aCC compared to T1aCC. These differences reflect cell-specific ion channel distribution that could contribute to patterned segmental motor output.

AIM The aim of this study was to identify the immediate effects on cortical excitability and grip control of a short intervention of EMG-triggered neuromuscular electrical stimulation, compared to voluntary activation of the finger flexor muscles, in people with chronic stroke. STUDY DESIGN This experimental study used a within-subject design with experimental and control interventions. PARTICIPANTS Fifteen people with chronic stroke participated in the study. INTERVENTION Participants performed a simple force tracking task with or without EMG-triggered neuromuscular electrical stimulation of the finger flexor muscles. MAIN OUTCOME MEASURES Cortical excitability was measured by single and paired-pulse transcranial magnetic stimulation. Multi-digit grip control accuracy was measured during ramp and sine wave force tracking tasks. Maximal grip strength was measured before and after each intervention to monitor muscle fatigue. RESULTS No significant increases in cortico-motor excitability were found. Intracortical inhibition significantly increased following the EMG-triggered neuromuscular electrical stimulation intervention immediately post-intervention (t = 2.466, p = .036), and at 10 minutes post-intervention (t = 2.45, p = .04). Accuracy during one component of the force tracking tasks significantly improved (F(1, 14) = 4.701, p = .048), following both EMG-triggered neuromuscular electrical stimulation and voluntary activation interventions. Maximal grip strength reduced significantly following both interventions, after the assessment of cortical excitability (F(1, 8) = 9.197, p = .16), and grip control (F(1, 14) = 9.026, p = .009). CONCLUSIONS EMG-triggered neuromuscular electrical stimulation during short duration force tracking training does not increase cortical excitability in participants with chronic stroke. Short duration force tracking training both with and without EMG-triggered neuromuscular electrical stimulation leads to improvements in training-specific aspects of grip control in people with chronic stroke.

AIM The aim of this study was to identify the immediate effects on cortical excitability and grip control of a short intervention of EMG-triggered neuromuscular electrical stimulation, compared to voluntary activation of the finger flexor muscles, in people with chronic stroke. STUDY DESIGN This experimental study used a within-subject design with experimental and control interventions. PARTICIPANTS Fifteen people with chronic stroke participated in the study. INTERVENTION Participants performed a simple force tracking task with or without EMG-triggered neuromuscular electrical stimulation of the finger flexor muscles. MAIN OUTCOME MEASURES Cortical excitability was measured by single and paired-pulse transcranial magnetic stimulation. Multi-digit grip control accuracy was measured during ramp and sine wave force tracking tasks. Maximal grip strength was measured before and after each intervention to monitor muscle fatigue. RESULTS No significant increases in cortico-motor excitability were found. Intracortical inhibition significantly increased following the EMG-triggered neuromuscular electrical stimulation intervention immediately post-intervention (t = 2.466, p = .036), and at 10 minutes post-intervention (t = 2.45, p = .04). Accuracy during one component of the force tracking tasks significantly improved (F(1, 14) = 4.701, p = .048), following both EMG-triggered neuromuscular electrical stimulation and voluntary activation interventions. Maximal grip strength reduced significantly following both interventions, after the assessment of cortical excitability (F(1, 8) = 9.197, p = .16), and grip control (F(1, 14) = 9.026, p = .009). CONCLUSIONS EMG-triggered neuromuscular electrical stimulation during short duration force tracking training does not increase cortical excitability in participants with chronic stroke. Short duration force tracking training both with and without EMG-triggered neuromuscular electrical stimulation leads to improvements in training-specific aspects of grip control in people with chronic stroke.

No description available.

Transcranial magnetic stimulation (TMS) is commonly used to measure corticospinal and intracortical excitability in basic and clinical neuroscience. However, the effect of glucose on TMS-based measures is not well defined, despite a potentially impactful influence on precision and reliability. Here, a double-blinded placebo-controlled study was used to test the effects of glucose on two commonly used TMS measures: short-interval intracortical inhibition (SICI), and the area under the motor evoked potential recruitment curves (AURC). SICI and AURC are thought to reflect inhibitory (GABAergic) and excitatory (glutamatergic) neurotransmission respectively. Healthy males (N=18) each participated in four sessions. Session 1 involved TMS familiarization and acquisition of an individualized blood glucose response curve. During sessions 2, 3 and 4, dependent measures were taken before (T0) and twice after (T1 & T2) drinking 300 mL of solution containing glucose (75 g), sucralose-sweetened placebo (control for sweetness) or plain water (control for time). The T1 and T2 measurements were started 5 minutes prior to the blood glucose peak observed during Session 1. Blood glucose and mean arterial pressure (MAP) were also monitored. Sucralose, but not water or glucose increased AURC and none of the treatments altered SICI. There was no association between blood glucose level and TMS measures, but in all three conditions MAP rose after consumption of the drink. There was a positive correlation between the rise in blood pressure and the relative increase in AURC at the higher stimulus intensities. Eleven participants returned for a fifth session to quantify the smallest detectible change in the AURC measurements and it was confirmed that significant changes were real while non-significant differences in measurement means fell within the range of expected measurement error. This study also suggests a relationship between corticospinal excitability and autonomic tone. Additional investigation is required to understand the mediating factors of this association. / Thesis / Master of Science (MSc)

As a central component of the hypothalamic-pituitary-adrenal (HPA) axis, the anterior pituitary corticotrophs play an important role in the regulation of HPA axis function and the neuroendocrine response to stress. Pituitary corticotrophs integrate stress-induced stimulatory signals (CRH and AVP) from the brain together with the negative feedback control from circulating glucocorticoid hormones to coordinate adrenocorticotrophin hormone (ACTH) secretion. Previous studies have classified pituitary corticotrophs as both endocrine and electrically excitable cells with a number of ion channels and signaling pathways implicated in the control of their electrical properties and ACTH secretion. However, the mechanisms involved in native corticotrophs are poorly understood partly due to the current limitations of identifying physiological intact corticotrophs. To address the electrophysiological properties of native murine corticotrophs, a lentiviral transduction system was developed, using a minimal pro-opiomelanocortin (POMC) promoter to drive the expression of enhanced yellow fluorescent protein (eYFP), to allow highly efficient and specific labeling and identification of corticotrophs in vitro. This approach, with patch clamp electrophysiological investigations, revealed metabolically intact native murine corticotrophs displayed spontaneous action potentials with highly heterogeneous firing patterns including single spikes and variable “pseudo plateau bursting” action potentials. The resting membrane potential of native murine corticotrophs was maintained by a TTXresistant background sodium conductance. Physiological concentrations of CRH/AVP rapidly depolarized native murine corticotrophs resulting in a sustained increase in the frequency of action potentials. Native murine corticotrophs express multiple outward potassium conductances with two major components mediated by intermediate-conductance calcium-activated (SK4) potassium channels and A-type potassium channels. Inhibition of SK4 channels with TRAM-34 lead to an increase in corticotroph excitability with firing pattern transition from single spikes to “pseudo plateau bursting”. When A-type potassium channels were blocked, the afterhyperpolarization amplitude of single spikes was decreased in some corticotrophs. In native murine corticotrophs, outward potassium current carried by large conductance calcium- and voltage- activated potassium (BK) channels was very low, which is in contrast with that in the mouse pituitary adenoma cell line (AtT20 cell line). Corticotroph cells from wild type (WT) mice and mice with a genetic deletion of the BK channel (BK-/-) were compared. The only potassium current that showed significant difference between WT and BK-/- corticotrophs was carried via the barium-sensitive inwardly rectifying (Kir) potassium channel. However, the blockage of Kir channels displayed no clear effect on corticotroph cell electrical excitability. Similar heterogeneous spontaneous firing patterns were found in WT and BK-/- corticotrophs. Taken together, the lentiviral-mediated expression of eYFP, driven by a minimal POMC promoter, provides an efficient method to identify physiological intact native murine anterior pituitary corticotrophs. These findings demonstrate that native murine anterior pituitary corticotrophs are spontaneous excitable cells that display significant heterogeneity of firing patterns. Results also reveal an important role of a background TTX-insensitive sodium conductance in controlling spontaneous and CRH/AVP evoked action potentials. Furthermore, an unexpected role for SK4 calcium-activated potassium channels in corticotroph excitability was revealed. In all, these studies give new insight into the physiology of corticotroph excitability and ACTH secretion, and provide the basis for understanding the roles of these ion channels in HPA axis function.