abstract: Neural interfacing applications have advanced in complexity, with needs for increasingly high degrees of freedom in prosthetic device control, sharper discrimination in sensory percepts in bidirectional interfaces, and more precise localization of functional connectivity in the brain. As such, there is a growing need for reliable neurophysiological recordings at a fine spatial scale matching that of cortical columnar processing. Penetrating microelectrodes provide localization sufficient to isolate action potential (AP) waveforms, but often suffer from recorded signal deterioration linked to foreign body response. Micro-Electrocorticography (μECoG) surface electrodes elicit lower foreign body response and show greater chronic stability of recorded signals, though they typically lack the signal localization necessary to isolate individual APs. This dissertation validates the recording capacity of a novel, flexible, large area μECoG array with bilayer routing in a feline implant, and explores the ability of conventional μECoG arrays to detect features of neuronal activity in a very high frequency band associated with AP waveforms. Recordings from both layers of the flexible μECoG array showed frequency features typical of cortical local field potentials (LFP) and were shown to be stable in amplitude over time. Recordings from both layers also showed consistent, frequency-dependent modulation after induction of general anesthesia, with large increases in beta and gamma band and decreases in theta band observed over three experiments. Recordings from conventional μECoG arrays over human cortex showed robust modulation in a high frequency (250-2000 Hz) band upon production of spoken words. Modulation in this band was used to predict spoken words with over 90% accuracy. Basal Ganglia neuronal AP firing was also shown to significantly correlate with various cortical μECoG recordings in this frequency band. Results indicate that μECoG surface electrodes may detect high frequency neuronal activity potentially associated with AP firing, a source of information previously unutilized by these devices. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2018

The study of cardiac action potentials has many medical applications. Dr. Dennis Noble first used mathematical models to study cardiac action potentials in the 1960s. We begin our study of cardiac action potentials with one form of the Fitzhugh-Nagumo partial differential equation. We use the non-classical method to produce a closed form solution for the decoupled Fitzhugh Nagumo equation. Using voltage recording data of action potentials in a cardiac myocyte and in purkinje fibers, we estimate parameter values for the closed form solution with standard linear and non-linear regression methods. Results are limited, thus leading us to perturb the solution to obtain a better fit. We turn to singular perturbation theory to justify our pole-based approach. Finally, we test our model on independent action potential data sets to evaluate our model and to draw conclusions on how our model can be applied.

Auditory physiology is nearly unique in the human body because of its small-diameter neurons. When considering a single node on one neuron, the number of channels is very small, so ion fluxes exhibit randomness. Hodgkin and Huxley, in 1952, set forth a system of Ordinary Differential Equations (ODEs) to track the flow of ions in a squid motor neuron, based on a circuit analogy for electric current. This formalism for modeling is still in use today and is useful because coefficients can be directly measured. To measure auditory properties of Firing Efficiency (FE) and Post Stimulus Time (PST), we can simply measure the depolarization, or "upstroke," of a node. Hence, we reduce the four-dimensional squid neuron model to a two-dimensional system of ODEs. The stochastic variable m for sodium activation is allowed a random walk in addition to its normal evolution, and the results are drastic. The diffusion coefficient, for spreading, is inversely proportional to the number of channels; for 130 ion channels, D is closer to 1/3 than 0 and cannot be called negligible. A system of Partial Differential Equations (PDEs) is derived in these pages to model the distribution of states of the node with respect to the (nondimensionalized) voltage v and the sodium activation gate m. Initial conditions describe a distribution of (v,m) states; in most experiments, this would be a curve with mode at the resting state. Boundary conditions are Robin (Natural) boundary conditions, which gives conservation of the population. Evolution of the PDE has a drift term for the mean change of state and a diffusion term, the random change of state. The phase plane is broken into fired and resting regions, which form basins of attraction for fired and resting-state fixed points. If a stimulus causes ions to flow from the resting region into the fired region, this rate of flux is approximately the firing rate, analogous to clinically measuring when the voltage crosses a threshold. This gives a PST histogram. The FE is an integral of the population over the fired region at a measured stop time after the stimulus (since, in the reduced model, when neurons fire they do not repolarize). This dissertation also includes useful generalizations and methodology for turning other ODEs into PDEs. Within the HH modeling, parameters can be switched for other systems of the body, and may present a similar firing and non-firing separatrix (as in Chapter 3). For any system of ODEs, an advection model can show a distribution of initial conditions or the evolution of a given initial probability density over a state space (Chapter 4); a system of Stochastic Differential Equations can be modeled with an advection-diffusion equation (Chapter 5). As computers increase in speed and as the ability of software to create adaptive meshes and step sizes improves, modeling with a PDE becomes more and more efficient over its ODE counterpart.

Action potentials of striated muscle are created through movement of ions through membrane ion channels. ATP-sensitive potassium (KATP) channels are the only known channels that are gated by the intracellular energetic level ([ATP]/[ADP] ratio). KATP channels are both effectors and indicators of cellular metabolism as part of a negative feedback system. Decreased intracellular energetic level alters the gating of KATP channels, which is reflected in alterations of the action potential morphology. These changes protect the cell from exhaustion or injury by altering energy-consuming processes that are driven by membrane potential. Assessing the effects of KATP channel activation on resting membrane potential and action potential morphology, and the relationship to cellular stress is important to the understanding of normal cellular function. To better understand how muscle cells adapt to energetic stress, the monophasic action potential (MAP) electrode and floating microelectrode were used to record action potentials in intact hearts and skeletal muscles, respectively. Intact organs provide a more physiological environment for the study of energetics and membrane electrical phenomena. Utilizing these techniques, a stress on the intracellular energetic state resulted in greater and faster shortening of the duration of cardiac action potentials, and hyperpolarization of the membrane of skeletal muscle in a KATP channel dependent manner. Motion artifacts are a limitation to studying transmembrane action potentials, but the MAP and floating microelectrode techniques uniquely allow for reading of action potential morphology uncoupled from motion artifacts. The use of the floating microelectrode in skeletal muscles is a novel approach that provides previously unavailable data on skeletal muscle membrane potentials in situ.

Proper heart function results from the periodic execution of a series of coordinated interdependent mechanical, chemical, and electrical processes within the cardiac tissue. Central to these processes is the action potential - the electrochemical event that initiates contraction of the individual cardiac myocytes. Many models of the cardiac action potential exist with varying levels of complexity, but none account for the electrophysiological role played by caveolae - small invaginations of the cardiac cell plasma membrane. Recent electrophysiological studies regarding these microdomains reveal that cardiac caveolae function as reservoirs of 'recruitable' sodium ion channels. As such, caveolar channels constitute a substantial and previously unrecognized source of sodium current that can significantly influence action potential morphology. In this thesis, I formulate and analyze new models of cardiac action potential which account for these caveolar sodium currents and provide a computational venue in which to develop and test new hypotheses. My results provide insight into the role played by caveolar ionic currents in regulating the electrodynamics of cardiac myocytes and suggest that in certain pathological cases, caveolae may play an arrhythmogenic role.

<p>Excess cardiac myofibroblasts in fibrotic heart diseases as well as cell-based therapies involving implantation of stem cells or genetically engineered somatic cells in the heart may all lead to a situation where a cardiomyocyte becomes electrically coupled to an unexcitable cell. In these settings, electrotonic loading of cardiomyocytes by unexcitable cells can affect cardiac action potential generation, propagation, and repolarization depending on the properties of both cardiomyocytes and unexcitable cells. The objective of this dissertation was to advance our understanding of the electrical interactions between cardiomyocytes and unexcitable cells using a variety of electrophysiological, molecular, and cell culture techniques.</p><p>First, we utilized aligned cardiomyocyte monolayers covered with unexcitable cardiac fibroblasts or human embryonic kidney-293 (HEK) cells that expressed similar levels of the gap junction protein connexin-45. These cells weakly coupled to cardiomyocytes and marginally slowed cardiac conduction only at high coverage density, while producing no other measurable electrophysiological changes in cardiomyocytes. In contrast, unexcitable HEK cells genetically engineered to stably express the more conductive connexin-43 channels (Cx43 HEK) strongly coupled to cardiomyocytes, depolarized cardiac resting membrane potential, significantly slowed impulse propagation, decreased maximum capture rate, and increased action potential duration (APD) at high coverage density. None of the studied unexcitable cells significantly altered conduction velocity anisotropy ratio or the relatively low incidence of pacemaking activity of cardiac monolayers at any coverage density.</p><p>Next, we utilized individual micropatterned cell pairs consisting of a cardiomyocyte and an unexcitable Cx43 HEK cell with or without stably overexpressed inward rectifier potassium channels (Kir2.1+Cx43 HEK). By systematically varying the relative sizes of micropatterned cells, we showed that Cx43 HEK cells significantly depolarized cardiomyocytes, reduced maximum upstroke velocity and action potential amplitude, prolonged APD, and modulated beating rate as a function of HEK:CM area ratio. In contrast, in cell pairs formed between cardiomyocytes and Kir2.1+Cx43 HEK cells we observed significant reduction in cardiomyocyte action potential amplitude, duration, and maximum upstroke velocity, but no change in other measured parameters.</p><p>Finally, we utilized a hybrid dynamic clamp setting consisting of a live micropatterned cardiomyocyte coupled in real time to a virtual model of capacitive and/or ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells. We found that coupling of cardiomyocytes to the ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells was sufficient to reproduce the dependence of cardiomyocyte maximal diastolic potential and pacemaking behavior on HEK:CM area ratio observed in micropatterned cell pairs, but did not replicate the observed changes in action potential upstroke or duration. The pure capacitance model with no ionic current, on the other hand, significantly decreased cardiomyocyte maximum upstroke velocity and prolonged cardiomyocyte APD as function of HEK:CM area ratio without affecting maximal diastolic potential or pacemaking behavior. When the unexcitable cell model containing both capacitive and ionic currents was connected to cardiomyocytes, all changes in action potential shape observed in micropatterned cell pairs were accurately reproduced. </p><p>These studies describe how coupling of unexcitable cells to cardiomyocytes can alter cardiomyocyte electrophysiological properties dependent on the unexcitable cell connexin isoform expression, ion channel expression, and cell size. This knowledge is expected to aid in the design of safe and efficient cell and gene therapies for myocardial infarction, fibrotic heart disease, and cardiac arrhythmias.</p> / Dissertation

This thesis presents a neuron emulator and headstage circuit for patch clamp setups and provides simulation, measurement and verification results. The circuit implemented on a printed circuit board (PCB) is battery powered and portable. The emulator provides both passive (resting potential) and active (action potential) electrical properties of a live neuron as seen from a single electrode by using the headstage circuit. It can be used to test electrophysiological equipment such as current-clamp, voltage-clamp or patch-clamp amplifiers. The action potentials (APs) are generated with a voltage-dependent frequency controlled by a microcontroller implementing a firing range from -60 mV to -30 mV and firing frequency from 1 Hz to10 Hz. The charge released by firing the neuron is initially stored on a 110 pC capacitor. Compared to directly using a current or voltage source, this design results in a more realistic simulation of the APs generated by ionic currents in a live neuron. The measured results from a prototype demonstrate that the neuron emulator meets the design specifications and it is capable of performing voltage clamp and rate responsive current clamp functionality. Measured results using a commercial clamp amplifier are provided to confirm the emulator operation in a practical recording environment.

In the heart, electrical signaling is responsible for its rhythmicity and is necessary to initiate muscle contraction. The net electrical activity in a cardiac myocyte during a contraction cycle is observed as the action potential (AP), which describes a change in membrane potential as a function of time. In ventricular cardiac myocytes, voltage-gated sodium channels (Nav) and voltage-gated potassium channels (Kv) play antagonistic roles in shaping the AP with the former initiating membrane depolarization and the latter repolarizing it. Functional changes in the primary cardiac Nav isoform, Nav 1.5, or any one of the many Kv isoforms expressed in the ventricle, as evidenced by those characterized in various congenital and/or acquired etiologies, can lead to severe cardiac pathologies. Nav and Kv are large transmembrane proteins that can be extensively post-translationally modified through processes that include glycosylation. The sequential glycosylation process typically ends with negatively charged sialic acid residues added through trans-Golgi sialyltransferase activity. Sialyltransferases belong to a much larger group of glycogene products that number in the hundreds and are responsible for creating a complex and variable glycan profile (glycome) unique to different cell types and tissues. Sialic acids impact Nav and Kv function likely by contributing to the extracellular surface potential and thereby causing channels to gate following smaller depolarizations. Additionally, developmentally regulated sialylation contributes to cardiac myocyte excitability in the neonatal mouse atria. However, little is understood concerning how the glycosylation machinery (glycogene products) influences cell and tissue electrical signaling. The sialytransferase Β-galactoside α-2,3-sialyltransferase 4 (ST3Gal4) adds sialic acids to galactose residues of N- and O-linked glycans through α-2,3-linkgages. ST3Gal4 is uniformly expressed throughout the chambers and developmental stages of the heart and therefore is likely a useful target to question whether and how glycosylation impacts these events. Additionally, diseases of glycosylation often cause symptoms that are consistent with changes in excitability that include arrhythmias and seizures. Congenital disorders of glycosylation lead to variably reduced glycoprotein and glycolipid glycosylation. However, because sialic acids are typically the terminal residues added to glycan structures, disease-related reduced glycosylation often leads to fewer sialic acids being attached. In addition, Chagas disease, which results in pathological changes in cardiac electrical function, may reduce sialic acids directly. Because of this, the ST3Gal4-/- strain was also used to investigate the role of glycosylation in the pathological cardiac electrical remodeling often associated with these diseases. The methodologies included cellular, tissue and whole-animal electrophysiology as well as biochemical assays. The data indicate that deletion of ST3Gal4 significantly affects Nav sialylation and gating with no change in maximum current density or protein expression. ST3Gal4 deletion also depolarizes the activation gating of both voltage-dependent kinetic components of repolarization found in the mouse ventricle: Ito and IKslow; however unlike the effect on INa, ST3Gal4 gene deletion causes a reduction in the peak IK density. Protein expression of the putative Kv isoforms responsible for Ito and IKslow was variably affected by ST3Gal4 gene deletion with Kv1.5 and Kv4.2 demonstrating no differences in protein densities. Contrastingly, a small but significant reduction in Kv2.1 protein from ST3Gal4-/- ventricular tissue was observed. In addition to effects on Nav and Kv activity, ST3Gal4 expression is necessary for normal cellular electrical signaling as demonstrated by a reduction in cellular refractory period and alterations in AP waveforms that include a slowing of cellular conduction and an extension of AP duration in ventricular myocytes from ST3Gal4-/- mice. Concurrent with aberrant excitability at the cellular level, the ST3Gal4-/- left ventricular epicardium demonstrated a reduced refractory period and was more susceptible to arrhythmias as observed through optical mapping studies. Additionally, ECGs of ambulatory ST3Gal4-/- mice demonstrated that deletion of the gene causes modest aberrant conduction under basal conditions and, in preliminary studies, appears to increase susceptibility to arrhythmias following a cardiac challenge, in the form of a low dosage of the Β-adrenergic agonist isoproterenol, suggesting a reduction in repolarization reserve in ST3Gal4 hearts. Based on the data reported here, it is apparent that relatively minor perturbations in the cardiac glycome cause significant changes in cardiac electrical signaling. These data highlight the role of glycosylation in normal physiology and underscore it as an important mediator in diseases where it may be altered.

Annotation Herta Gurskaitė The title of dissertation: Effects of new sulfonylcarbamide modulators of potassium channels (KACh and KATF) on the electromechanical activity of the heart. The aim of this study was to investigate the influence of new sulfonylcarbamide modulators of potassium channels (KACh and KATP) on the electromechanical activity parameters of guinea pig myocardium and the heart rhythm. The objectives of the scientific work were as follows: 1. To estimate the influence of carbachol, an activator of the muscarinic receptor-operated KACh channels, and pinacidil, an activator of KATP channels, and their blockers (atropine and glybenclamide) on the action potential (AP) duration and contraction force in guinea pig myocardium. 2. To investigate the effect of procainamide and its new sulfonylcarbamide derivatives on the AP duration and contraction force in guinea pig myocardium under the KACh and KATP channel activation. 3. To investigate the influence of 2-, and 4-aminopyridines and their new sulfonylcarbamide derivatives as well as 2-aminopyrimidine derivatives on the AP duration and contraction force in guinea pig myocardium under the KACh and KATP channel activation. 4. To determine the anticholinergic effect of the most potent new sulfonylcarbamide derivatives on the sinus rate in isolated guinea pig heart. 5. To determine, does the anticholinergic effect of the most effective sulfonylcarbamide compound was induced via blockade of M2-receptors and/or it... [to full text]

Little is known regarding the neurophysiological mechanisms by which the neuropeptide prokineticin 2 (PK2) regulates circadian rhythms. Using whole-cell electrophysiology, we have investigated a potential role for regulation of neuronal excitability by PK2 on neurons of the area postrema (AP), a medullary structure known to influence autonomic processes in the central nervous system. In current-clamp recordings, focal application of 1µM PK2 reversibly influenced the excitability of the majority of dissociated AP cells tested, producing both depolarizations (38%) and hyperpolarizations (28%) in a concentration-dependent manner. Slow voltage ramps and ion substitution experiments revealed a PK2-induced Cl- current was responsible for membrane depolarization, while hyperpolarizations were the result of inhibition of an inwardly rectifying non-selective cation current. In contrast to these differential effects on membrane potential, nearly all neurons that displayed spontaneous activity responded to PK2 with a decrease in spike frequency. These observations are in accordance with voltage-clamp experiments showing that PK2 caused a leftward shift in Na+ channel activation and inactivation gating. Lastly, using post hoc single cell RT-PCR technology, we have shown that 7 out of 10 AP neurons depolarized by PK2 were enkephalin-expressing cells. The observed actions on enkephalin neurons indicate PK2 may have specific inhibitory actions on this population of neurons in the AP acting to reduce their sensitivity to incoming signals. These data suggest that PK2 regulates the level of AP neuronal excitability and may impart a circadian influence on AP autonomic control. / Thesis (Master, Physiology) -- Queen's University, 2009-08-18 11:18:05.977