Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 77-82).

Thesis (Ph.D. in Pharmacology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 145-170). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Superficial layers of the dorsal horn receive a dense plexus of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide (PACAP). In vivo experiments were conducted in the mice to evaluate the effects of PACAP-38, herein referred to as PACAP, PACAP receptor antagonist PACAP(6-38) and PACAP-antiserum on nociceptive behaviors induced by radiant heat, intrathecally administered N-methyl-D-aspartate (NMDA) or intraplantarly administered formalin. PACAP (0.05-0.5μg) dose-dependently decreased the paw-withdrawal latencies induced by thermal stimulation and enhanced the aversive licking and biting behaviors induced by intrathecally injected NMDA. Pretreatment with the PACAP receptor antagonist PACAP(6-38) (0.5-2μg) or PACAP-antiserum (1:500-2000 dilution) dose-dependently attenuated the second phase, but not the first phase, of nociceptive responses to formalin. Next, the effects of PACAP on NMDA- and kainate-induced currents evoked in single dorsal horn neurons were studied. Whole-cell patch recordings were made from superficial dorsal horn neurons of spinal cord slices from 14- to 20-day-old mice. PACAP at the concentrations of 100 and 200nM, which caused no significant change of holding currents, increased NMDA-but not kainate-induced currents in superficial dorsal horn neurons. Our results suggest that exogenously applied PACAP sensitizes the dorsal horn neurons to formalin stimulation, and facilitates NMDA receptor-mediated nociceptive response. As a corollary, PACAP, which may be released from primary afferent fibers potentiates nociceptive transmission to the dorsal horn by interacting primarily with NMDA receptors.

Clinically relevant concentrations of ethanol modulate the function of a number of ion channel proteins. A fundamental question regarding the effects of alcohol is whether the drug modifies ion channels by directly binding to the protein, indirectly by perturbing the surrounding membrane lipid, or some combination of both. This thesis further characterized ethanol's site of action by examining the effects of ethanol on N-methyl-D-aspartate (NMDA) receptor/channels and large conductance Ca2+-activated K+ (BK) channels at a number of levels using direct electrophysiological methods. In Chapter One, the magnitude of ethanol's inhibition of a number of cloned heteromeric NMDA receptor/channels in the absence or presence of a number of modulators was compared. The rank order of ethanol sensitivity for the subunit combinations studied was NR1b/NR2A > NR1b/NR2B > NR1b/NR2C > NR1b/NR2D. Modulation of the receptor with Mg2+, Zn2+, the glycine antagonist 7-Chlorokynurenic Acid, or after reduction or oxidation of the redox regulatory site did not alter the ethanol sensitivity of heteromeric NMDA receptors. Therefore, the ethanol sensitivity of NMDA receptor/channels is dependent upon which NR2 subunit is present, and ethanol's site of action is unrelated to these modulatory sites on the receptor/channel protein. In Chapter Two, ethanol's site of action at cloned BK channels was characterized using of a number of 1-alkanols. Ethanol, butanol, hexanol, and heptanol reversibly and dose-dependently increased the current carried through BK channels. Longer chain 1-alkanols, such as octanol had no effect on channels. In Chapter Three, the action of ethanol on BK channels reconstituted in a number of model planar bilayers was studied. Ethanol increased the activity of BK channels incorporated in bilayers composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) or PE alone by decreasing the average amount of time channels dwelled in the closed state. There was no significant effect of alcohol on either channel conductance or unitary current. Taken together, these data suggest that ethanol action on BK channels does not require the complex membrane architecture found in native membranes, and does not require freely diffusible cytoplasmic factors or proteins.

No description available.

No / Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex. Cortical spreading depression promotes lesion progression in experimental stroke, and may contribute to the initiation of migraine attacks. The purpose of this study was to investigate the roles of the marked increase of nitric oxide (NO) formation that occurs with CSD. Microdialysis electrodes were implanted in the cortex of anesthetized rats to perform the following operations within the same region: (1) elicitation of CSD by perfusion of high K+ medium; (2) recording of CSD elicitation; (3) application of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME); and (4) recording of dialysate pH changes. The primary effect of L-NAME (0.3 to 3.0 mmol/L in the perfusion medium) was a marked widening of individual CSD wave, resulting essentially from a delayed initiation of the repolarization phase. This change was due to NO synthase inhibition because it was not observed with the inactive isomer D-NAME, and was reversed by L-arginine. This effect did not appear to be linked to the suppression of a sustained, NO-mediated vascular change associated with the superposition of NO synthase inhibition on high levels of extracellular K+. The delayed initiation of repolarization with local NO synthase inhibition may reflect the suppression of NO-mediated negative feedback mechanisms acting on neuronal or glial processes involved in CSD genesis. However, the possible abrogation of a very brief, NO-mediated vascular change associated with the early phase of CSD cannot be ruled out.

Little is known about higher order chromatin structures in the human brain and their function in transcription regulation. We employed chromosome conformation capture (3C) to analyze chromatin architecture within 700 Kb surrounding the transcription start site (TSS) of the NMDA receptor and schizophrenia susceptibility gene, GRIN2B, in human and mouse cerebral cortex. Remarkably, both species showed a higher interaction between the TSS and an intronic sequence, enriched for (KRAB) Krueppel associated Box domain binding sites and selectively targeted by the (H3K9) histone 3 lysine 9 specific methyltransferase ESET/SETDB1. Transgenic mice brain cortical nuclei over-expressing Setdb1 showed increased heterochromatin-protein 1 signal at the interacting regions coupled with decreased Grin2b expression. 3C further revealed three long distant chromatin loop interactions enriched with functional enhancer specific (H3K27Ac) histone 3 lysine 27 acetylation signal in GRIN2B expressing tissue (human cortical nuclei and Human Embryonic Kidney - HEK cells). Doxycycline-induced SETDB1 over-expression decreased 2 out of 3 loop interaction frequencies suggesting a possible SETDB1-mediated transcription repression. We also report a specific looping interaction between a region 50Kb upstream of the (GAD1) Glutamic Acid Decarboxylase – 1 gene TSS and the GAD1 TSS in human brain nuclei. GAD1 catalyzes the rate limiting step in (GABA) gamma amino-butyric acid synthesis and is quintessential for inhibitory signaling in the human brain. Clinical studies in schizophrenia brain samples reveal a decreased looping interaction frequency in correspondence with a decrease in gene expression. Our findings provide evidence for the existence of transcription relevant higher order chromatin structures in human brain.

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) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.