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Thesis (Ph. D.)--University of Chicago, Dept. of Pharmacological and Physiological Sciences, August 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 105-120).

Voltage-gated ion channels are fundamental for electrical signaling in living cells. They are composed of four subunits, each holding six transmembrane helices, S1-S6. Each subunit contains a voltage-sensor domain, S1-S4, and a pore domain, S5-S6. S4 contains several positively charged amino-acid residues and moves in response to changes in membrane voltage. This movement controls the opening and closing of the channel. The structure of the pore domain is solved and demonstrates principles of channel selectivity. The molecular mechanism of how the voltage sensor regulates the opening of the channel is still under discussion. Several models have been discussed. One of the models is the paddle model where S3b and S4 move together. The second one is the helical-twist where S4 makes a small rotation in order for the channel to open. The third one is the helical-screw model where S4 twists around its axis and moves diagonally towards the extracellular side of the channel. The aim of this PhD project was to study the molecular movement of the voltage sensor in the depolarization-activated Shaker K channel. Cloned channels were expressed in Xenopus laevis oocytes, and investigated with several electrophysiological techniques. 1. We show that S4 moves in relation to both S3b and S5. The formation of some disulfide bonds between S4 and neighboring positions, in only the open state, shows that the paddle model cannot be correct. Furthermore, electrostatic and steric effects of residues in S3b suggest that S3b is tilted, with the intracellular part close to S4. 2. We show that the relatively Mg-sensitive Shaker K channel is changed into the less Mg-sensitive Kv2.1 K channel with respect to its sensitivity to extracellularly applied Mg2+ by changing the charge of three extracellularly positioned amino acid residues. One of the residues, F425C, mediates its effect through the neighboring residue K427. 3. We show that oxaliplatin, an anti-cancer drug, has no effect on the Shaker K channel. It has been suggested that a negatively charged monochloro complex of oxaliplatin is the active substance, and also causes the neurotoxic side effects. Neither this complex shows any effect on the channel. Our experiments point towards the helical-screw model. The other models for voltage-sensor movements are incompatible with the results in this study.

Whole-cell patch clamp electrophysiology of neurons in vivo enables the recording of electrical events in cells with great precision, and supports a wide diversity of morphological and molecular analysis experiments important for the understanding of single-cell and network functions in the intact brain. However, high levels of skill are required in order to perform in vivo patching, and the process is time-consuming and painstaking. Robotic systems for in vivo patching would not only empower a great number of neuroscientists to perform such experiments, but would also open up fundamentally new kinds of experiment enabled by the resultant high throughput and scalability. We discovered that in vivo blind whole cell patch clamp electrophysiology could be implemented as a straightforward algorithm and developed an automated robotic system that was capable of performing this algorithm. We validated the performance of the robot in both the cortex and hippocampus of anesthetized mice. The robot achieves yields, cell recording qualities, and operational speeds that are comparable to, or exceed, those of experienced human investigators. Building upon this framework, we developed a multichannel version of “autopatcher” robot capable establishing whole cell patch clamp recordings from pairs and triplets of neurons in the cortex simultaneously. These algorithms can be generalized to control arbitrarily large number of electrodes and the high yield, throughput and automation of complex set of tasks results in a practical solution for conducting patch clamp recordings in potentially dozens of interconnected neurons in vivo.

Diss. Stockholm : Karol. inst., 2004.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

by Cheng Wai Ming. / Thesis submitted in: October, 1998. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 54-68 (2nd gp.)). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iv / ABSTRACT --- p.v / ABSTRACT (Chinese) --- p.vii / Chapter CHAPTER 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Ionotropic glutamate receptors --- p.1 / Chapter 1.1.1 --- AMP A receptor --- p.3 / Chapter 1.1.1.1 --- Structure of AMP A receptor --- p.3 / Chapter 1.1.1.2 --- Electrophysiological properties of AMPA receptor --- p.4 / Chapter 1.1.1.3 --- Pharmacology of AMPA receptors --- p.6 / Chapter 1.1.1.4 --- Kinetics of AMPA receptors --- p.8 / Chapter 1.1.2 --- NMDA receptor --- p.9 / Chapter 1.1.2.1 --- Structure of NMDA receptor --- p.9 / Chapter 1.1.2.2 --- Electrophysiological properties of NMDA receptor --- p.10 / Chapter 1.1.2.3 --- Pharmacology of NMDA receptor --- p.11 / Chapter 1.1.2.4 --- Kinetics of NMDA receptor --- p.12 / Chapter 1.2. --- The basal ganglia and the SNR --- p.12 / Chapter 1.3 --- Excitatory glutamatergic inputs on SNR --- p.16 / Chapter 1.4 --- Aim of study --- p.17 / Chapter CHAPTER 2 --- Electrophysiological properties of SNR neurones --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.2 --- Methods --- p.19 / Chapter 2.2.1 --- In vitro slice preparation and maintenance --- p.19 / Chapter 2.2.2 --- Whole-cell patch-clamp recording --- p.20 / Chapter 2.2.3 --- Solutions and drugs --- p.21 / Chapter 2.2.4 --- Histological methods --- p.21 / Chapter 2.2.5 --- Data analysis --- p.22 / Chapter 2.3 --- Results --- p.22 / Chapter 2.3.1 --- Passive membrane properties of SNR neurones --- p.22 / Chapter 2.3.2 --- Firing rate and action potential characteristics --- p.23 / Chapter 2.3.3 --- Firing patterns --- p.23 / Chapter 2.3.4 --- Weak hyperpolarization activated inward rectification --- p.24 / Chapter 2.3.5 --- Slow aflerhyperpolarization --- p.25 / Chapter 2.3.6 --- Current-frequency relationship --- p.25 / Chapter 2.3.7 --- Morphology of labelled SNR neurones --- p.25 / Chapter 2.4 --- Discussion and conclusion --- p.26 / Chapter CHAPTER 3 --- AMPA and NMDA induced membrane responses --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Methods --- p.31 / Chapter 3.2.1 --- In vitro slice preparation and maintenance --- p.31 / Chapter 3.2.2 --- Whole-cell patch-clamp recording --- p.31 / Chapter 3.2.3 --- Solutions and drugs --- p.31 / Chapter 3.2.4 --- Drug application --- p.32 / Chapter 3.2.5 --- Immunocytochemistry --- p.32 / Chapter 3.2.6 --- Data analysis --- p.33 / Chapter 3.3 --- Results --- p.33 / Chapter 3.3.1 --- AMPA induced responses in SNR GABA neurones --- p.33 / Chapter 3.3.1.1 --- AMPA induced membrane depolarization --- p.33 / Chapter 3.3.1.2 --- AMPA induced membrane current --- p.34 / Chapter 3.3.1.3 --- Current-voltage relationship --- p.34 / Chapter 3.3.1.4 --- Effect of NBQX --- p.35 / Chapter 3.3.1.5 --- Effects of JSTX and spermine --- p.35 / Chapter 3.3.2 --- NMDA-induced response in SNR GABA neurones --- p.36 / Chapter 3.3.2.1 --- NMDA induced membrane depolarization --- p.36 / Chapter 3.3.2.2 --- NMDA induced membrane current --- p.36 / Chapter 3.3.2.3 --- APV blocked NMDA-induced current --- p.36 / Chapter 3.3.2.4 --- Effect of glycine on NMDA induced response --- p.37 / Chapter 3.3.2.5 --- Mg2+-sensitivity --- p.37 / Chapter 3.3.2.6 --- Current-voltage relationship --- p.38 / Chapter 3.3.3 --- GluR2 subunit immunostaining --- p.38 / Chapter 3.4 --- Discussion and conclusion --- p.39 / Chapter 3.4.1 --- AMPA receptors in SNR neurones --- p.39 / Chapter 3.4.2 --- NMDA receptors in SNR neurones --- p.41 / Chapter 3.4.3 --- Functional significance --- p.41 / Chapter CHAPTER 4 --- Glutamate-mediated synaptic currents in SNR --- p.43 / Chapter 4.1 --- Introduction --- p.43 / Chapter 4.2 --- Methods --- p.44 / Chapter 4.2.1 --- In vitro slice preparation and maintenance --- p.44 / Chapter 4.2.2 --- Electrophysiological recordings --- p.44 / Chapter 4.2.3 --- Electrical stimulation --- p.45 / Chapter 4.2.4 --- Solutions and drugs --- p.45 / Chapter 4.2.5 --- Data analysis --- p.46 / Chapter 4.3 --- Results --- p.46 / Chapter 4.3.1 --- Characteristics of spontaneous EPSCs --- p.46 / Chapter 4.3.1.1 --- General characteristics --- p.46 / Chapter 4.3.1.2 --- Kinetics --- p.47 / Chapter 4.3.1.3 --- Pharmacology --- p.47 / Chapter 4.3.2 --- Characteristics of evoked EPSCs --- p.48 / Chapter 4.3.2.1 --- General characteristics --- p.48 / Chapter 4.3.2.2 --- Pharmacological characterization --- p.49 / Chapter 4.3.2.3 --- Effects of bicuculline --- p.50 / Chapter 4.4 --- Discussion and conclusion --- p.50 / Chapter 4.4.1 --- Excitatory transmission onto SNR neurones --- p.50 / Chapter 4.4.2 --- Source of excitatory drive --- p.51 / Chapter 4.4.3 --- Interaction with GABA inputs --- p.52 / Chapter 4.4.4 --- Functional significance --- p.52 / REFERENCES --- p.54

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.