Browsing by Subject "Electrophysiology"
Now showing 1 - 12 of 12
Results Per Page
Sort Options
Item Analysis of sodium pump gene expression in microdissected nephrons using competitive RT-PCR and a novel HPLC technique(Texas Tech University, 1996-12) Hayward-Lester, Amanda LHypertension is determined by the interaction of multiple genetic and environmental factors, making it difficult to elucidate any single genetic determinant. Biochemical markers such as intraerythrocytic sodium concentration, erythrocytic ouabain binding-site density and passive sodium leak indicate that abnormal membrane cation flux segregates with some hypertension subtypes. The sodium pump, Na+, K+ ATP-ase (NKA) may therefore participate in the development of hypertension. It is a multi-subunit cell membrane protein which translocates sodium and potassium ions with hydrolysis of ATP. It is inhibited by ouabain and regulated by phosphorylation. The alpha subunit (of which there are four isoforms encoded by different genes) is currently ascribed all catalytic function, while the beta and gamma subunits may have regulatory roles. We examined NBCA alpha and gamma subunit gene expression in spontaneously hypertensive rats (SHR) and Wistar Kyoto controls (WKY). Both prehypertensive and adult SHR exhibit abnormal renal sodium retention. Solution hybridization studies in adult SHR revealed a decrease in alpha 1 expression in kidney. Kidney is a heterogeneous tissue whose functional unit, the nephron, may be divided into 12 distinct segments. To examine NKA expression in individual segments requires an assay allowing quantitation of NKA alpha and gamma isoform RNA in microdissected tissue samples. We combined competitive RT-PCR with a novel ion-paired reversed phase HPLC to produce rapid, accurate and precise measurement of gene expression in a single-tube assay. The ability of HPLC to resolve heteroduplex molecules formed between native and competitor products proved essential. Assay validation confirmed absolute quantification is possible if competitors have identical reverse-transcription efficiency to the native RNA. We used the assay to examine qualitative and quantitative expression of NKA subunits in normotensive Sprague-Dawley, prehypertensive and adult SHR and WKY. Qualitative analysis revealed alpha 1 and gamma expression in all segments examined. Expression of the other alpha isoforms was not detected. Quantitative analysis in the prehypertensive SHR revealed that a selective alteration in alpha 1 expression in proximal convoluted tubule may explain the results obtained in whole kidney and suggest an attempted feedback response by SHR to reduce sodium reabsorption.Item Application of parametric sensitivity analysis to calcium handling in cardiac myocytes(Texas Tech University, 2003-08) Smith, Charles NHeart failure disease (HF) kills 220,000 people in America every year. HF is characterized by the ineffective handling of calcium ions by the mechanisms present in cardiac myocytes resulting in decreased contractile force in the heart. This inefficiency leads to necrosis of the myocardium, cardiac hypertrophy, and eventual death of the patient. The mechanisms affected include various ion channels and active transport mechanisms. Numerous technologies and techniques have been developed recently to apply genetic and pharmacological therapies to these cells. Unfortunately, there are a large number of target parameters that can be manipulated in the myocytes. Mathematical models have been developed that accurately reproduce the calcium mechanisms. These models are reproducible and rapidly evaluated on desktop computers using current mathematical software. Sensitivity analysis is useful in determining the best targets for manipulation by external sources in the hopes of restoring the proper calcium ion handling by ion channels and calcium transporters. Sensitivity analysis uses matrix algebra and calculus to determine the normalized response of a control variable to a change in a parameter. More specifically, this technique calculates the responses of all control variables to changes in all of the parameters individually. This generates a matrix of values that can easily be analyzed. This method will save valuable time and resources that would have been spent on performing multiple experiments or simulations to determine the best targets for manipulation. The cytosolic calcium concentration is targeted as it is directly related to the contractile force of the heart. Reduction of complex models is also possible using the results of sensitivity analysis. Rota et al. describe a method of normalizing fluxes and sensitivities against their greatest magnitude as a characteristic for suggesting unimportant fluxes and parameters. Two models are examined: the Tang-Othmer (T-0) model and the Winslow, Rice, and Jafri (WRJ) model. Both of these models seek to describe the handling of calcium ions within cardiac myocytes; however the level of their complexities differs greatly. The T-O model contains much fewer state variables, parameters, and fluxes than the WRJ model. The greater incorporation of states and the related parameters and fluxes leads the WRJ model to yield a much greater complexity than that presented with the T-O model. This complexity is magnified upon formation of the sensitivity matrices for these models, where the T-O model yields a fifty-five element matrix and the WRJ model yields 1,881 elements in its sensitivity matrix. The T-O model is used to develop the application method whereas the WRJ model is used to determine the best targets for manipulation and to illustrate the efficacy of model reduction. Sensitivity analysis upon the T-O model yields the sodium-calcium exchanger parameters and the sarcolemmal leak coefficient as the optimal targets for manipulation to restore proper cytosolic calcium concentration. The best targets for genetic or pharmacological manipulation according to the WRJ model are the Na+ and Ca2+ background currents, the maximum current for the sodium-potassium pump, and the half saturation constants for the sodium-potassium pump. These parameters are not present in the T-O model and have greater sensitivity magnitudes than those that carry over into the WRJ model. Model reduction by the method of Rota et al. reveals that the Tang-Othmer model is irreducible in its present state. The WRJ model was found to have no reducibility with regards to the number of fluxes in the model. However the integration of some sensitivities was unattainable, and some of these parameters may be found to be removable upon further analysis of the model once these sensitivities are obtained. Further integration methods will be attempted, such as the use of a hard-coded implicit integrator. Sensitivity analysis also revealed a crossover phenomenon in both models. This phenomenon describes the change in sign of a sensitivity of a state variable to a parameter during the course of a heartbeat. When this occurs the desired effect of manipulating the parameter yields the opposite effect upon the state variable. This phenomenon may generate interesting side effects that require further study. The results of sensitivity analysis provide future direction for physical experiments. These experiments will both confirm the calculated sensitivities, and investigate their application to failing myocytes. The crossover phenomenon will provide interesting avenues of research into the side-effects of parameter manipulation based on the magnitude and location in the cycle of the crossover. Model reduction may play a key role in simplifying models for easier computation and analysis.Item Cys-loop receptors: Investigating the nicotinic acetylcholine receptor transmembrane domain and overexpression of a prokaryotic homolog (Gloeobacter violaceus Ion Channel)(2012-05) Pauwels, Jonathan; Jansen, Michaela; Cuello, Luis G.; Knaff, David B.We investigated the thermal motion of the transmembrane domain in murine muscle type nicotinic acetylcholine receptors (nAChR) and developed a bacterial expression system for the overexpression and purification of a prokaryotic ligand-gated ion channel from Gloeobacter violaceus (GLIC). nAChR, along with receptors for γ-aminobutyric acid, serotonin, glycine and glutamate, is a member of the superfamily of pentameric ligand-gated ion channels known as cys-loop receptors and GLIC is a closely related bacterial homolog. In the first set of experiments, we investigated differences measured by disulfide trapping at the 20’ position in the M2 transmembrane helices of nAChR and GABAA receptors. Due to their homology, it is commonly assumed these receptors have a great deal of structural and functional similarity. At the 20’ position in GABAA receptors disulfide trapping has shown inter-subunit crosslinking between both adjacent and non-adjacent (across the channel) residues. Whereas, similar experiments at the 20’ position in nAChR only showed inter-subunit crosslinking between adjacent subunits. In this study, we attempted to elucidate the fundamental difference between nAChR and GABAA receptors that led to these divergent findings. Specifically, we engineered neutralizing residues at the 20’ position in nAChR in an attempt to promote a biochemical environment more favorable to the formation of disulfides. Next, we developed a procedure for the overexpression and purification of GLIC. As a bacterial homolog, GLIC is an invaluable asset in the study of the basic structure and function of cys-loop receptors. After comparison of BL21(DE3), C41(DE3) and C43(DE3) chemically competent cells in the transformation and expression of GLIC, we determined C41(DE3) provided the best expression system for our construct. We used centrifugation and chromatographic techniques to purify GLIC. Our procedure provides large amounts of pure protein and can easily be adapted to chimeric GLIC proteins for downstream functional and structural studies.Item Decision-making signals in the primate parietal cortex(2012-05) Meister, Miriam Lucia Roth; Huk, Alexander; Geisler, Wilson; Priebe, Nicholas; Mauk, Michael; Seidemann, EyalLateral intraparietal area (LIP) neurons are thought to compute the decision of where to look. Specifically, their firing rate is thought to represent accumulated evidence for a decision by ramping up to a high level, the putative decision bound, before an eye movement to a given location. However, LIP neurons are also highly responsive to visual stimuli. Because all previous research put a visual stimulus (a saccadic target) in the response field (RF) location of a neuron during decision formation, it is unknown if LIP neurons can still show decision computation without this visual drive. We therefore recorded the spiking activity of LIP neurons in a conventional decision task where a monkey decides the direction of a noisy motion stimulus and indicates his decision with a saccade. On half the trials, the Choice Targets remained on for the whole trial, as is conventionally done. On the remaining trials, targets were flashed at the beginning of the trial and absent during motion-viewing. Furthermore, we recorded the activity of any neuron with an RF, instead of only neurons exhibiting persistent memory activity before an instructed saccade. This enabled us to also test the long-held assumption that only cells with persistent memory activity show decision signals. Our results show that 1) cells without persistent activity indeed show decision signals, 2) population response drops without RF stimulation (although individual neurons were affected in diverse ways), 3) distinct, repeating response “motifs” exist among cells, 4) a signal exists where neural response is lower for stronger motion strength stimuli, regardless of direction. These results prove that contrary to dogma, a neuron’s ability to show a long “memory” response is not related to an ability to accumulate evidence over time for a decision. Also, LIP firing rate cannot be interpreted as a pure decision variable because it simultaneously represents decision-irrelevant, visual stimuli. Finally, diverse, but repeating responses among cells suggest the existence of cell types in LIP. These results demonstrate that LIP acts as a bank of potentially useful signals, and raises the question of how they might be used for a decision.Item Electrophysiological analysis of cerebellar serotonergic mechanisms in thiamine deficiency(Texas Tech University, 1983-08) Lee, Rong-shengNot availableItem Intracellular calcium and pacemaker activity in the sinoatrial node(Texas Tech University, 1997-05) Li, JinThe importance of the sinoatrial (S-A) node as the pacemaker of the heart is well known; however, little is known about calcium homeostasis and excitation-contraction coupling in the pacemaker cells. Thus, the major objectives of this dissertation research were: (1) to quantitatively measure intracellular calcium (Cai^2+) in isolated pacemaker cells; (2) to examine the relationship between Cai^2+ and electrical activity; and (3) to measure Ca^2+ release from the sarcoplasmic reticulum (SR) and determine its effect on pacemaker activity. We used the fluorescent Ca^2+ indicator, Indo-1, to assess the changes in Cai^2+ and perforated-patch whole-cell recordings to monitor the electrical activity of single, cultured pacemaker cells that had been isolated from the rabbit S-A node. Indo-1 fluorescence ratios were calibrated both in vitro and in vivo. The contributions to action potential-induced Cai^2+ transients from Ca^2+ entry through voltage-gated channels and Ca^2+ release from the SR were demonstrated. The results indicate the existence of an internal Ca^2+ store, the SR, and that action potential-induced Cai^2+ transients are produced mainly by Ca^2+ entry through voltage-gated Ca^2+ channels, rather than by Ca^2+ release from the SR. Simultaneous recordings of membrane potentials and the Indo-1 fluorescence ratio showed that automaticity can be modified by isoproterenol and caffeine, agents that modulate Cai^2+. Changes in beat rate were correlated with changes in Cai^2+; however, Cai^2+ transients were not essential for cells to generate spontaneous firing. Using the perforated-patch voltage-clamp technique, we studied the effects of ryanodine and thapsigargin, inhibitors of SR Ca^2+ release, on pacemaker activity, hyperpolarization-activated inward current, time-independent inward current, L- and T-type Ca ^2+ currents, and inward Na^+-Ca^2+ exchange current. Both ryanodine and thapsigargin slowed pacemaker activity significantly, in part by reducing inward Na^+-Ca^2 exchange current; ryanodine also had a direct inhibitory effect on T-type Ca^2+ current. In summary, rabbit S-A node pacemaker cells display a complex set of positive and negative feedback mechanisms that control intracellular Ca^2+ and pacemaker activity.Item Iron-overload toxicity in central nervous system-associated cells and its attenuation by calcium channel blockers(2006-05) Gaasch, Julie Ann; Van der Schyf, Cornelis J.; Abbruscato, Thomas J.; Allen, David D.; Bickel, Ulrich; Lockman, Paul R.; Weidanz, Jon A.Iron is essential to many vital biological pathways within the brain through its participation in oxidation-reduction reactions. Conversely, conditions of iron overload such as hemorrhagic stroke, or iron accumulation as in neurodegenerative disorders, can lead to saturation of iron transport and storage proteins to increase free iron levels within the brain. Free iron is highly reactive and in cases of overload can induce oxidative stress by producing hydroxyl free radicals that cause damage to cellular DNA, membranes, and proteins. Traditionally, iron entry into cells occurs by receptor-mediated endocytosis of transferrin-bound iron. However, studies in heart cells have demonstrated an alternate route of iron entry under conditions of iron overload through the L-type voltage-gated calcium channel. This dissertation investigates two important concepts related to iron-overload toxicity in brain, i.e. 1) the existence of differential responses of select central nervous system cells to iron-induced toxicity, and 2) the existence of an alternative route of entry for iron into brain cells in iron overload conditions via voltage-gated calcium channels. Our studies indicate that iron competes with calcium for entry via voltage-gated calcium channels and that iron uptake is inhibited in a dose-dependent manner by nimodipine, a calcium channel blocker, in neuronal cells. Studies measuring iron toxicity in astrocytes, neurons, and endothelial cells demonstrate that these three cell types respond differentially to iron-induced toxicity when cultured in isolation, and that calcium channel blockers are effective in attenuating the toxicity observed in neuronal and endothelial cells, but not in astrocytic cells. NGP1-01, a polycyclic cage compound, has been shown to be a dual blocker of neuronal voltage gated calcium channels as well as N-methyl-D-aspartate (NMDA) receptors. Our studies investigated the neuroprotective effect of NGP1-01 in iron-loaded brain endothelial cells. Similar to nimodipine, NGP1-01 demonstrated a significant attenuation of iron-induced cellular toxicity. Taken as a whole, these studies suggest that calcium channel blockers may be utilized as neuroprotective agents in conditions of brain iron overload, particularly at the blood brain barrier where brain endothelial cells are intimately affected.Item Mobility of the M2 segments of the transmembrane domain in muscle type nicotinic acetylcholine receptors(2011-08) Rajan, Robin; Jansen, Michaela; Artigas, Pablo; Knaff, David B.Ionotropic receptors such as the ligand gated ion channels mediate fast synaptic transmissions of impulses in the central nervous system. The Cys loop superfamily includes ligand gated ion channels that act as receptors for neurotransmitters acetylcholine (nACh), gamma aminobutyric acid (GABAA), glycine (Gly) and serotonin (5-HT3A). The muscle type nicotinic acetylcholine receptor assembles from five homologous subunits to form a heteropentamer. All subunits share a three domain topology and structure (1) An extracellular domain with two antiparallel ?-sheets housing the ligand binding site (2) A transmembrane domain that crosses the membrane with four distinct ?-helical segments (M1-M4) (3) and a long loop between M3 and M4 forms the intracellular domain. The channel is lined by five M2 segments contributed by individual subunits. This receptor has a known subunit arrangement of ????? in the clockwise direction when viewed from the extracellular side. Disulfide trapping between engineered pairs of cysteines is a method to assess the separation of the involved cysteines as well as give us a measure of extent of proximity and flexibility of the region containing the residues. We monitored inter-subunit disulfide bond formation between Cys pairs in different M2 segments in muscle nAChR expressed in Xenopus laevis oocytes by two electrode voltage clamp experiments. Chemically, cystine bond formation can be reversed by the reducing agent dithiothreitol (DTT). We investigated ?M2 Cys mutants in the ?C192S-C193S background with and without the corresponding ?M2 Cys mutants to determine which positions in M2 can form disulfide bonds and compared it to previously published studies in the homologous GABAA receptor. This study will help to understand the thermal mobility of the M2 segment and how different subunits move relative to one another during gating transitions.Item The relationship between glycine receptor agonist efficacy and allosteric modulation(2014-05) Kirson, Dean; Mihic, S. JohnThe glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine.Item The relationship between glycine receptor agonist efficacy and allosteric modulation(2014-05) Kirson, Dean; Mihic, S. JohnThe glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine.Item Simultaneous electricity, bioethanol, and algal biodiesel production using microbial fuel cell(2012-05) Kerls, Marci; Karim, M. Nazmul; Vaughn, Mark W.With an ever increasing world population, the energy needs for this population will only grow. A renewable sustainable energy is needed to maintain the world’s energy consumption. A study of a device that would be capable of simultaneously producing bioelectricity and bioethanol and algae oil was conducted. The microbial fuel cell was catalyzed with Chlorella vulgaris and Saccharomyces bayanus in the cathode and anode, respectively. A series of four experimental runs were conducted, along with three different control experiments. The algae growing in the microbial fuel cell was also monitored, and the algae was harvested for lipid content. The yeast in the anode was run in batch and continuous conditions over the course of the experiments and samples were taken to measure ethanol concentrations. Polarization curves were obtained to calculate the power density and current density, and intermittent energy harvesting was performed. The MFC with yeast and algae in the anode and cathode, respectively, achieved a power density of 70.61 ± 12.41 mW/m2, and a current density of 155.02 ± 31.23 mA/m2. The volumetric productivity of the ethanol was found to be 0.49±0.37 g l-1 hr-1, and an algae oil content of 13 ± 1.7 grams of lipids per grams of algae biomass