Browsing by Subject "Other Neuroscience and Neurobiology"
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Item [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors(2000-05-01) Yagle, Monica A.; Dillon, Glenn; Martin, Michael; de Fiebre, ChristopherYagle, Monica A., [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors. Master of Science (Pharmacology), May 2000, 59 pp., 3 tables, 7 illustrations, bibliography, 75 titles. Modulation of the GABAA receptor has been studied with noncompetitive convulsant ligands such as tert-butylbicyclophosphorothionate (TBPS) and picrotoxin (PTX). EBOB is a more recently developed ligand that appears to bind in the same region of the channel at TBPS, but with a higher affinity. While only a few studies have examined the binding of EBOB to vertebrate brain tissue and insect preparations, none have examined potential subunit-dependent binding of EBOB. We have thus examined [3H] EBOB binding in rat cerebellum and HEK293 cells stably expressing human α1β2γ2, human α2β2γ2, and rat α6β2γ2 GABAA receptors. For comparison, [35S] TBPS binding was also examined in α1β2γ2 receptors. Saturation and Scatchard analyses revealed saturable [3H] EBOB binding at one site in all tissue preparations with Kd values ranging from 3 to 9nM. [3H] EBOB binding, like [35S] TBPS binding was inhibited by the CNS convulsants dieldrin, lindane, tert-butylbicyclophosphorothionate (TBOB), PTX, TBPS, and pentylenetetrazole (PTZ) at one site in a concentration dependent fashion. Affinities were in the high nM to low μM range for all compounds except PTZ (low mM range). GABA modulated [3H] EBOB binding in a biphasic manner in α1β2γ2 receptors with a 100-fold difference between stimulatory and inhibitory affinities. Inhibition of GABA-mediated current by TBOB in α1β2γ2 receptors resulted in a functional IC50 of 0.2 μM, in agreement with binding study results. Differences seen in binding between the different receptor subtypes examined suggest that some characteristics of EBOB binding are subunit dependent. In addition, we have shown that [3H] EBOB is a useful ligand in the study of recombinant GABAA receptors and that results obtained with [3H] EBOB are comparable to those obtained with [35S] TBPS.Item Cellular and Molecular Mechanisms that Distinguish the Effects of Progestorone and Medroxyprogesterone Acetate on Neuroprotection(2006-07-28) Kaur, Paramjit; Goldfarb, Ronald; Singh, Meharvan; Agarwal, NeerajKaur, Paramjit. Cellular and Molecular Mechanisms That Distinguish the Effects of Progesterone and Medroxyprogesterone Acetate on Neuroprotection., Doctor of Philosophy, (Pharmacology and Neuroscience), July, 2006, 203 pp., 5 illustrations, 20 figures and bibliography. Women have a higher prevalence for Alzheimer’s disease (AD) than men, suggesting that the precipitous decline in gonadal hormone levels following the menopause may contribute to the risk of developing AD. However, principal results from the Women’s Health Initiative concluded that women taking conjugated equine estrogens combined with medroxyprogesterone acetate (MPA, tradename: Prempro) incurred more harmful than beneficial outcomes versus the placebo group (Rossouw et al., 2002). This dissertation was aimed at determining if the discrepancy between basic science reports and these clinical studies could have been due to the synthetic progestin, MPA. I hypothesized that P4 and MPA differed in their ability to protect against the excitotoxic/oxidative insult, glutamate. Further, I proposed that this difference in neuroprotective potential would be reflected in the difference in the ability of these hormones to elicit key effectors of two neuroprotection-associated signaling pathways, the ERK/MAPK and P13-Kinase pathways. Finally, studies were initiated to evaluate the potential importance of BDNF (brain-derived neurotrophic factor) in mediating the protective effects of P4. I used organotypic explants of the cerebral cortex, and found that both P4 and MPA elicit the phosphorylation of ERK and Akt, two signaling pathways implicated in neuroprotection, with maximal phosphorylation occurring at a concentration of 100 nM. Interestingly, P4 protected against glutamate- induced toxicity however, while an equimolar concentration of MPA (100nM) did not. Further, P4 resulted in an increase in BDNF, while MPA did not. Our data bring into question the relevance of using MPA as a component of hormone therapies in postmenopausal women, and instead, argue that the relevant progestin for use in treating brain-related disorders is progesterone. Collectively, the data presented here suggest that P4 is protective via multiple, and potentially related mechanism, and importantly, its neurobiology is different from the clinically used progestin, MPA.Item Corticotropin-Releasing Factor and Corticosterone Modulate the Anxiogenic-Like Effects of mCPP(1998-06-01) Jenkins, Jennifer A.; Michael Forster; Robert Luedtke; Patricia GwirtzJenkins, Jennifer A., Corticotropin-Releasing Factor and Corticosterone Modulate the Anxiogenic-Like Effects of mCPP. Doctor of Philosophy (Pharmacology), June 1998, 119 pp., 2 tables, 29 figures, bibliography, 100 titles. The administration of PTZ or mCPP produces anxiety-like behavior as measured by an increase in the percentage of entries into the open arms and the time spent on the open arms of the elevated plus maze (Prunell et al., 1994). Reportedly, PTZ and mCPP substitute for each other in the drug discrimination paradigm (Wallis and Laz, 1998). It is therefore suggested that commonality exists among anxiogenic drugs as perceived by trained animals. Andrews and Stephen (1990) suggested that this overall parallelism is an indication that anxiogenic agents may possess similar properties. Therefore, the question posed is as follows: Is there a common denominator anxiety? The global hypothesis is that the core component of anxiety produced by anxiogenic agents or processes involves stimulation of the HPA axis to release CRF, ACTH and/or CORT. Long Evans rats were trained to discriminate either mCPP (1.4 mg/kg) or PTZ (16mg/kg) from saline in a two-lever choice procedure (FR10) which is food reinforced. Animals were pretreated with CRF, α-helical CRF (a CRF antagonist), two steroid synthesis inhibitors (ketoconazole, KETZ and aminoglutethimide, AMG), CORT or underwent an adrenalectomy prior to behavioral testing in order to test the hypothesis that the release of CRF and/or CORT are components of the discriminate stimulus of the mCPP and/or PTZ. Pretreatment with CRF, KETZ, AMG and an adrenalectomy facilitated mCPP level selection. However in the absence of mCPP neither drug nor adrenalectomy produced drug lever selection. In addition CORT did not alter the mCPP dose response curve. However, CORT replacement therapy returned the does response curve to baseline in adrenalectomized animals. Alpha-helical CRF did not block mCPP discrimination. Unlike mCPP-trained animals, KETZ and AMG decreased PTZ-lever selection in PTZ-trained animals. In addition, CORT enhanced and partially substituted for the discriminative stimulus of PTZ. However, adrenalectomy completely abolished drug lever selection in PTZ animals. To compare the discriminative stimulus effects of mCPP and PTZ, PTZ-trained animals were injected with cumulative doses of mCPP. mCPP-trained animals were injected with cumulative doses of PTZ. mCPP and PTZ minimally substituted for each other. The results suggested that neither CRF nor CORT are components of the discriminative stimulus of mCCP and that the role of the HPA axis in mCPP discrimination maybe be a modulator of the stress response. However, CORT is a component of the discriminative stimulus of PTZ such that CORT is necessary for drug lever selection in PTZ trained animals.Item Effect of Progesterone on Calcium Signaling of Hippocampal Neurons(2006-05-01) Hwang, Ji-Yeon; Koulen; Singh, Meharvan; Yang, ShaohuaJi-yeon Hwang, Effects of Progesterone on Calcium Signaling of Hippocampal Neurons. Master of Science (Pharmacology and Neuroscience), May 2006, 74 pp., 18 Figures. Progesterone (P4) is one of the steroid hormones responsible for female sexual behavior. It has been recently show that P4 plays also multiple roles in the central nervous system (CNS) including neuroprotection. Calcium (Ca2+) is involved in numerous cellular processes in nerve cells such as neurotransmitter release and cell death. In the present studies, we present evidence that P4 increases the activity of IP3R-mediated Ca2+ release within nerve cells leading to cell survival and neuroprotection. The purpose of the present study is to identify the subcellular distribution of all IP3Rs and other signaling proteins including Akt and phosphor-Akt, in the primary hippocampal neuron and to test the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca2+ signaling in neurons. We observed that primary hippocampal neurons express predominantly IP3R type 1, 2, and 3. The cellular distribution of all IP3R isoforms as well as Akt and phospho-Akt was increased in primary hippocampal neurons by P4 treatment. In addition, phospho-Akt was translocated to nucleus in response to P4. P4-pretreated neurons showed potentiated IP3R-mediated intracellular Ca2+ responses. Acute application of P4 resulted in transient elevations of intracellular Ca2+ concentrations. Our results will contribute to establishing potential pharmacological approaches for the treatment of pathological conditions characterized by a dysregulation of cellular Ca2+ concentrations such as Alzheimer’s disease.Item Endothelin-1-Induced Signaling Involved in Extracellular Matrix Remodeling(2006-12-01) He, Shaoqing; Thomas Yorio; Neeraj Agarwal; Peter KoulenET-1-Induced Signaling in ECM Remodeling in Astrocytes. Shaoqing He, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107. ET-1 levels are elevated under pathophysiological conditions, including glaucoma, however, ET-1’s ocular functions are not fully documented. Therefore, ET-1-induced signaling and ECM remodeling in astrocytes and at the optic nerve head were determined in this study. Three signaling pathways, including ERK1/2, PKC, and P13 kinase, were involved in ET-1-medicated cell proliferation of U373MG astrocytoma cells. Blocking one of these pathways completely abolished cell proliferation. It appeared that ERK1/2 activation was involved, but was independent of PKC and P13 kinase activation by ET-1. It was also determined that the ETB receptor was the dominant receptor involved in ERK1/2 phosphorylation and cell proliferation. In addition, ERK1/2 phosphorylation was not transactivated by the EGF receptor by ET-1. The studies also indicated that there was no activation of c/nPKC, although PKC was involved in cell proliferation. In U373MG astrocytoma cells, MAPK-ERK, PKC and P13K pathways appear to exert their roles in parallel without a direct, apparent “cross-talk”. Based on the signaling pathways obtained from U373MG astrocytoma cells, the regulation of MMPs/TIMPs and fibronectin in ET-1-activated human optic nerve head astroctyes (hONAs) was also determined. ET-1 not only induced rapid phosphorylation of ERK1/2 and PKC βI/ βII/δ but also increased the activity of MMP-2 and the expression of TIMP=1 and 2. The activity of MMP-2 was enhanced in the presence of inhibitors of MAPK or PKC in hONAs, whereas the expression of TIMP-1 and 2 was abolished. ET-1 increased the soluble fibronectin (FN) expression as well as FN matrix formation, however, the expression and deposition of FN were MAPK- and PKC-independent, whereas expression and activity of MMps and TIMPs were MAPK- and PKC-dependent. Therefore, ET-1 shifted the balance of MMPs/TIMPs and substrates that altered the ECM composition and subsequently let to ECM remodeling in activated hONA cells. ET-1’s effects on ECM remodeling at the optic nerve head were also examined following intravitreal administration of ET-1 in rats. The increased expression of MMP-9 and collagen VI was detected in both ETB deficient rats and wildtype Wistar rats post ET-1 intravitreal injection for 2 and 14 days, whereas the deposition of FN and collagen IV was unchanged. There was no significant difference in staining of MMP-9 and collagen VI between ETB deficient rats and wildtype Wistar rats. In this study, ECM remodeling was demonstrated in rats injected with ET-1 into the vitreous. Such changes in the ECM seen in the current study provide additional insight into the mechanisms that might explain the glaucomatous changes observed in ET-1-injection or perfusion models. In summary, ET-1 not only activated several signaling pathways in cell proliferation of astrocytes, but also modulated the expression of ECM molecules in vitro and in vivo, indicating that ET-1 plays a regulatory role in ECM remodeling. These effects coupled with observations that ET-1 levels are elevated in glaucoma patients, suggests that ET-1 may be involved in glaucomatous optic neuropathy.Item Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes(2008-05-01) Fuller, John Anthony; Wordinger, Robert J.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Fuller, John Anthony Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes. Doctor of Philosophy (Biomedical Sciences), May, 2008, 140 pp., 2 tables, 25 illustrations, bibliography, 218 titles. Primary Open Angle Glaucoma (POAG) is a family of heterogeneous optic neuropathies characterized by progressive retinal ganglion cell (RGC) death that leads to peripheral vision loss and eventually blindness. Various risk factors are associated with glaucoma, however the molecular mechanisms leading to RGC cell death remain unknown. The optic nerve serves as the conduit for the transmission of retinal ganglion action potentials to the brain. The cells that compromise the optic nerve form a scaffold that forms a physical support for the RGC axons. One cell type found throughout the optic nerve and associated with the RGC axon is the optic nerve astrocyte (ONA). Astrocytes are a predominant cell throughout the CNS and are believed to play crucial roles in metabolic, growth factor, and structural support, and respond to protect neurons during injury. The neuronal-glial interface in the optic nerve is poorly understood and believed to plan an important role in POAG pathophysiology, as unmyelenated RGC axons have direct contact with astrocyte processes. IN this study, the subtilisin-like Proprotein Convertases, (SPC) a family of proteases responsible for cleaving a wide variety of protein substrates, were examined in the retina and optic nerve head. PACE4, an SPC found to be secreted and active in the extracellular matrix was found to be highly expressed in the optic nerve, and colocalized to Mϋller cells in the retina and astrocytes in the optic nerve. Exposure of primary optic nerve astrocytes to oxygen-glucose deprivation (OGD) induces an increase in PACE4 mRNA. Furthermore, protein levels of extracellular, processed PACE4 increase following transient ODG, whereas the pro form of the molecule is degraded, and is believed to be chaperoned by the cleaved cysteine rich domain, a product found at high levels in the optic nerve in situ and the ONA in vitro. Due to the extracellular activity of PACE4, we hypothesized that it may regulate the bioactivity of TGF-β2, a growth factor believed to be involved in glaucoma-associated ONH remodeling by inducing the production of extracellular matrix (ECM). When PACE4 is inhibited via siRNA-mediated knockdown, as well as extracellular inactivation, TGF-β2 levels decrease. In addition, fibronectin, a major component of the ECM, is decreased. Furthermore, there is an increase in latent TGF-β2 secreted from the cell. It is therefore possible that PACE4 plays an active role in extracellular growth factor maturation, and may be a central mediator for growth factor bioactivity in the glaucomatous ONA.Item Genetic Modulation of β-Amyloid Neurotoxicity and Protection by Nicotinic Agents(2007-05-01) Martin, Shelley E.; Basu, Alakananda; Forster, Michael; Singh, MeharvanMartin, Shelley E., Genetic Modulation of β-Amyloid Neurotoxicity and Protection by Nicotinic Agents. Master of Science (Pharmacology and Neuroscience), May, 2007, 53 pp., 7 figures, 2 tables, bibliography, 95 titles. Β-amyloid1-42 (Aβ42) has been implicated in the pathogenesis of Alzheimer’s disease (AD); however, the amount of this peptide in the brain does not correlate well with the presence or severity of AD. This project tested the hypothesis that individual differences exist in susceptibility to Aβ42 neurotoxicity arising from the differences in the expression of α7 nicotinic acetylcholine receptors α7 nACHRs). This hypothesis was tested in primary neuronal cultures derived from inbred mouse strains which differ in expression of α7 nAChRs. Also, the ability of nicotinic agents to modulate Aβ42 toxicity was examined. Significant strain differences in susceptibility to Aβ42 toxicity were found; however, these were not related to levels of α7 nAChRs. Additionally, strain differences were found in the ability of α7-selective partial agonist, an α7-selective antagonist and a α4β2 nAChR-selective antagonist to protect against this toxicity. Inbred strains of mice may be useful in uncovering the pathophysiology of AD.Item Identification of Actin Binding Proteins Associated with Cross-Linked Actin Networks(2006-12-01) Mills, Christy E.; Clark, Abbot F.; Yorio, Thomas; Wordinger, Robert J.Mills, Christy E., Identification of Actin Binding Proteins Associated with Cross-Lined Actin Networks. Master of Science (Pharmacology and Neuroscience), December 2006, 95 pp., 9 tables, 16 figures, references, 122 titles. Glucocorticoid therapy can leady to ocular hypertension and glaucoma. The purpose of this study is to examine mechanisms contributing to increased intraocular pressure using tissue culture models of steroid-induced ocular hypertension through identification of specific actin-binding proteins associated with cross-linked actin network (CLANs). Human trabecular meshwork ™ cells were cultured to confluence and treated with dexamethasone or vehicle for 14 days. Total RNA was extracted for gene expression analysis to confirm steroid-induced expression of actin binding proteins in human TM cells. Western blots confirmed expression of actin binding proteins and demonstrated the specificity of selected antibodies. Fluorescence microscopy of treated TM cells showed cytoskeleton rearrangements from linear actin stress fibers to cross-linked actin networks and the position of candidate proteins in relation to CLANs. Dexamethasone treatment of TM cells altered the expression of actin-associated proteins that may be important in the formation of CLANs and increased outflow resistance.Item Inhibitory Rib-Raising and Microneurographic Measurement of Sympathetic Nervous System Activity(2007-05-01) Kinzler, Damien W.; Michael Smith; Russell Gamber; Hollis KingThe clinical effectiveness of osteopathic manipulative therapy (OMT) techniques that are designed to address the autonomic nervous system (ANS) are untested to current research standards. As the concept of “autonomic imbalance” is frequently ascribed as the etiology of various pathologic conditions, it is paramount to undertake basic research into not only efficacy but also possible mechanistic actions and origins. Osteopathic physicians often utilize treatment regimens and techniques for which the given mechanism of action is simply attributed to “balancing the autonomics”. This intuitive concept may finally be at the threshold where enough basic science exists to justify clinical investigations. Osteopathic manual manipulative techniques have shown effectiveness in the treatment of various musculoskeletal conditions and have been shown to lower perceived pain; supporting the use of manual therapy as an effective treatment modality. A brief review yields the following within just the last four years: Eisenhart showed positive range-of-motion outcomes after ankle sprain in the emergency department. Biondi reviews the usefulness of cervical manipulation for tension headache and McReynolds demonstrated an equivalent decrease in acute neck with OMT versus intramuscular ketolac in an emergency department setting, although the dosing was not maximal. German researchers have shown effectiveness in chronic epicondylopathia humeri radialis and research has led to the demonstration of lowered post-operative pain in hip or knee arthroplasty. There has also been decreased post-operative pain medication reported in hysterectomy when compared with a control group. OMT has demonstrated a decrease in fibromyalgia symptoms when used with standard care over standard care alone. Low back pain, perhaps the most extensively studied diagnosis in which OMT has been evaluated, has reported numerous positive outcomes including lower levels of narcotic use and decreased pain in both double-blinded and meta-analysis studies, although there is still considerable debate within this area. There has also been favorable outcomes associated with the management of gain in Parkinson’s disease and preliminary work has shown the efficacy in treatment of carpal tunnel syndrome. Most of the aforementioned musculoskeletal conditions are not amenable to traditional therapies and have a high-cost burden on the economy. Traditional treatments generally have a “wait and see” approach combined with analgesics which may not cause harm, but hampers quality of life and income in the interim. The cost effectiveness of OMT is still in the preliminary stages, but there is evidence supporting a superior cost benefit ratio when compared to standard care and since many of these conditions have no other proven treatment modality available patients will often try anything over nothing. The evaluation of OMT addressing clear autonomic dysfunction is limited. This study closes a small part of that gap by examining the proposed physiologic mechanism of OMT and its’ interaction with the ANS. Small studies have documented changes, namely heart rate variability, in autonomic processes in healthy individuals while other, older studies have found benefit in clinical variables. With few exceptions however, most of these studies lacked a particular technique protocol. Operators were free to use whatever intervention that they chose and most of these studies were not performed under rigorous testing methods with a randomized design. The technique that was evaluated (inhibitory rib-raising) has a documented history from the origins of osteopathic medicine in the United States, and is currently taught to students in osteopathic medical schools as part of their medical education curriculum. Rib-raising is most often taught to enhance the mechanical motion of the ribs, but other paradigms utilize this technique to either enhance or inhibit sympathetic nervous system (SNS) activity. The evaluation of inhibitory rib-raising or its’ proposed mechanism of action has never been rigorously scrutinized to modern scientific standards. The current study was designed to address that gap with both direct and indirect measurement of SNS variable in healthy individuals with the hypothesis that there would be a time-dependent, graded reduction in measured sympathetic nervous system activity (MSNA) in healthy individuals undergoing cold-pressor stimulus.Item Leucine-Enkephalin and Sympathetic Control of Heart Rate(2001-12-01) Stanfill, Amber; Caffrey, James L.; Downey, H. Fred; Shi, XiangrongStanfill, Amber A., Leucine-enkephalin and Sympathetic Control of Heart Rate. Master of Science (Biomedical Sciences), December, 2001, 51 pp., 1 table, 4 figures, references, 48 titles. The following study examined the role of leucine-enkephalin in the sympathetic regulation of the cardiac pacemaker. Leucine-enkephalin (0.3 mM) was administered, by microdialysis into the interstitium of the sinoatrial node in 10 mongrel dogs in conjunction with either sympathetic nerve stimulation or infused norepinephrine. In study one, the right cardiac sympathetic nerves were isolated as they exit the stellate ganglion and stimulated to produce graded (low, 20-30; high 40-50 bpm) increases in heart rate. Once stimulation frequencies were determined, leucine-enkephalin (0.3mM) was added to the dialysis inflow and perfused at 5: 1/min thereafter. The sympathetic stimulations were repeated after 5 and 20 min exposure to leucine-enkephalin. The resulting increases in heart rate during sympathetic stimulation were attenuated at both low (18.2 ±1.3 to 11.4 ±1.4 bpm) and high (45 ±1.5 to 22.8 ±1.5 bpm) frequency stimulation. The degree of inhibition was nearly identical after 20 minutes exposure providing no evidence for a progressively evolving response and for desensitization. Vagal function was also evaluated at 5 and 20 min by stimulating the right cervical vagus at 1 and 3 Hz. Leucine-enkephalin reduced the vagal bradycardia approximately 50% at both time intervals. The administration of the delta-selective opioid antagonist, naltrindole, restored only one third of the sympathetically medicated tachycardia. The same dose of naltrindole completely reversed the coincident vagolytic of leucine-enkephalin. These observations suggested that the sympatholytic effect was either non-opioid or mediated by a different opioid receptor subtype. Study two was conducted to determine if the sympatholytic effect was prejunctional and post-junctional in character. Norepinephrine was added to the dialysis inflow into the SA node in a concentration (6-9 μM) sufficient to produce an intermediate increase in heart rate. The average increase in heart rate was 35.2 ±1.8 bpm. Leucine-enkephalin was then combined with norepinephrine and sympathetic and parasympathetic responses were recorded at 5-min intervals for 20 minutes. The tachycardia mediated by added norepinephrine was unaltered by leucine-enkephalin or the subsequent addition of naltrindole. At the same time intervals, vagal control of heart rate was reduced by more than 50% and then completely restored by naltrindole. When combined with observations in study one, the data support the conclusion that the local nodal sympatholytic effect of leucine-enkephalin was the result of a reduction in the effective interstitial concentration of norepinephrine and not the result of a post-junctional interaction between leucine-enkephalin and norepinephrine.Item Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed(2000-08-01) Pearlman, Eric Brian; Barbara Barron; Patricia A. Gwirtz; Michael L. SmithPearlman, Eric B., Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed. Master of Science (Biomedical Science), August, 2000, 37 pp., 3 tables, 11 figures, references, 20 titles. Methionine enkephalin arginine phenylalanine (MERF) has been shown to be co-stored with catecholamines in vesicles. The catecholamines appear to decrease the degradation rate of 3H-MERF in vitro. The aim of this study is to investigate the spillover and metabolism of MERF across the canine heart vascular bed. I hypothesize that 3H-MERF is either degraded in the plasma or taken up and degraded by the heart. I further hypothesize that the exogenous catecholamine, isoproterenol, inhibits or reduces the rate of MERF degradation. Mongrel dogs were anesthetized and instrumented to record cardiovascular parameters, infuse 3H-MERF, and obtain blood samples across the heart. Blood samples were taken before and after stopping 3H-MERF infusion to evaluate kinetics, show steady state, and test the effect of treatments. Steady state concentration of 3H-MERF was observed after 30 min of infusion. Chromatography separated intact from degraded 3H-MERF. Three experimental groups were used: control, propranolol plus isoproterenol, and propranolol only. Blockade of β-receptors was necessary to prevent changes in coronary blood flow. Propranolol bolus (0.2 mg/kg) was administered IV at 50 min. 3 μg/min isoproterenol or 0.5 ml/min normal saline was infused starting at 70 min until the end of sample collection. The 3H-MERF venous-arterial (V-A) difference prior to treatment was negative, indicating degradation in the plasma or uptake and degradation by the heart. The 75 min V-A difference was used to calculate the effect of the infusions on the degradation or uptake of the 3H-MERF; this value was unchanged by any treatment. Spillover of 3H-MERF was significantly lower in the propranolol + isoproterenol dogs (p [less than] 0.05) compared to propranolol only treatment at 75 min. Heart rate was significantly lower for the propranolol only group compared to control. Blood pressure and change in coronary flow were unchanged. In conclusion, isoproterenol does not affect the metabolism of 3H-MERF across the canine heart vascular bed. Propranolol, however, does increase the intact 3H-MERF in the plasma, but additional β adrenergic blockade agents need to be investigated to determine the mechanism by which this takes place.Item Modulation of GABAA Receptor Function by Tyrosine Phosphorylation(1998-05-01) Fang, Mingjun; Glenn Dillon; Thomas Yorio; Eugene E. QuistMingjun, Fang. Modulation of GABAA Receptor Function by Tyrosine Phosphorylation. Master of Science (Biomedical Sciences), May, 1998, 32 pp., 6 illustrations, bibliography, 42 titles. The goal of this study was to determine the modulation of GABAA receptor function by tyrosine kinase phosphorylation, and to detect which subunit is phosphorylated to alter the GABA-induced chloride currents. From previous studies, we suggested that protein tyrosine phosphorylation may maintain GABAA receptor function. Here we tested the hypothesis that tyrosine phosphorylation modulates other GABAA receptor subtypes e.g., α1β2γ2 and α6β2γ2, and subsequently attempted to determine which subunit(s) may be phosphorylated. Our results support the hypothesis that PTK phosphorylation may maintain GABAA receptor function. In addition, we suggest this tyrosine phosphorylation occurs at the γ2 subunit of the receptor.Item N-Acylethanolamine Signaling in Neurons(2008-12-01) Duncan, Raymond Scott; Koulen, Peter; Simpkins, James; Forster, MichaelDuncan, Raymond S., N-acylethanolamine signaling in neurons. Doctor of Philosophy (Biomedical Sciences), December 2008, 356 pp., 1 table, 70 illustrations, bibliography, 576 titles. Neurodegenerative diseases including Alzheimer’s disease are and will continue to be significant health problems as the aging population increases. The maintenance of neuronal calcium homeostasis has been a focus in degenerative disease research for many years. Within the last several years, lipids that activate cannabinoid receptors, and thus called cannabinoids, have gained recognition as neuroprotectants in models of neurodegenerative diseases. A subset of these cannabinoids, the N-acylethanolamines (NAEs), includes the well characterized neuroprotective lipid, arachidonylethanolamine. Other NAEs, such as palmitoylethanolamine (PEA), are more abundant in neurons and do not activate cannabinoid receptors, suggesting other targets for these lipids exist. Since non-cannabinoid NAEs rapidly accumulate after neuronal injury, it is likely they play a role in cellular responses to injury. Interestingly, some NAEs can alter intracellular Ca2+ signaling, but the underlying mechanism of action remains unclear. I hypothesized that the non-cannabinoid NAEs, such as PEA, protect the hippocampal cell line, HT22, from oxidative stress in part by reducing intracellular calcium release. I determined that HT22 cells and cultured mouse cortical neurons express proteins involved in NAE signaling, thus warranting the use of pharmacological inhibitors of these proteins in subsequent neuroprotection studies. Using HT22 cells, I determined that PEA exhibitis antiproliferative effects and neuroprotects against oxidative stress. In addition, I determined that PEA facilitates the nuclear translocation of putative protective proteins that can be regulated by Ca2+ through a mechanism not involving cannabinoid receptor activation. These findings led me to hypothesize that PEA alters release of Ca2+ from intracellular stores. To test this hypothesis, I determined that our cell models express inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) both of which are intracellular Ca2+ channels elevated in response to oxidative stress. I determined that treatment of HT22 cells with PEA reduced intracellular Ca2+ release elicited by chemical depolarization with KCI. My results suggest that non-cannabinoid NAEs, such as PEA, protect the hippocampal cell line, HT22, from oxidative stress in part by activating putative neuroprotective signaling proteins and by reducing intracellular calcium release.Item Neuroprotective effects of peroxisome proliferator-activated receptor-gamma (PPAR-y) ligands against oxidative stress(2003-12-01) Aoun, Paul; Simpkins, James W.; Dillon, Glenn; Gracy, RobertAoun, Paul Neuroprotective effects of peroxisome proliferator-activated receptor-gamma (PPAR-y) ligands against oxidative stress. Doctor of Philosophy (Pharmacology and Neurosciences), December, 2003, 254 pp., 37 figures. Diabetes mellitus is a significant public health problem in the United States and the world resulting in substantial morbidity and mortality. Diabetes complications, i.e., neuropathy, are common and almost triple the annual cost of managing diabetes. In our studies, we investigated the role that insulin sensitizers currently used for the treatment of diabetes, the PPAR-y ligands, might play in protecting neurons against oxidative stresses. We showed that two PPAR-y ligands, 15 deoxy-PGJ2 and troglitazone, protected, in a dose-dependent manner, HT-22 mouse hippocampal and RGC-5 retinal ganglion cell lines against various oxidative insults. Further, we demonstrated that neuroprotection by 15deoxy-PGJ2 and troglitazone was independent of the PPAR-y receptor. Our findings brought to light an important role of PPAR-y ligands in preventing neuronal complications from diabetes. Moreover, the studies reported herein provide valuable insights into the development of novel therapeutic compounds that improve insulin sensitivity while preventing neurological, and possibly other complications of diabetes.Item Opioid Receptors in Aging and Oxidative Stress(2007-01-01) Raut, Atul M.; Ratka, Anna; Simpkins, James W.; Dillon, GlennRaut, Atul M., Opioid Receptors in Aging and Oxidative Stress. Doctor of Philosophy (Pharmacology and Neuroscience), January 2007, 181 pp, 4 illustrations, 21 figures, 159 titles. Oxidative stress has been implicated in aging and neurodegenerative disorders. Pain sensitivity and responses to opioids change with aging. The effect of aging and oxidative stress on opioid receptor system is not yet well understood. To study the effects of aging on pain sensitivity and opioid-induced antinociception, and to determine the possible association of oxidative stress with these pain parameters, in vivo studies were conducted. To further elucidate the effects of oxidative stress on opioid receptor proteins and their function, in vitro studies were carried out. The effects of aging on pain sensitivity and opioid-induced antinociception were studied in male Fischer 344 rats. Oxidative stress markers in cerebral cortex, hippocampus, striatum and midbrain of these rats were estimated. It was concluded that sensitivity to nociceptive stimulus increases and responses to opioids decrease with aging and age-related oxidative damage is negatively correlated with opioid-induced antinociception. To characterize the effects of oxidative stress on function of opioid receptors, changes in intracellular cyclic adenosine monophosphate (cAMP) was measured in human SK-N-SH neuronal cells under oxidative stress conditions. It was found that oxidative stress decreased the function of mu opioid receptor (MOR) but not that of delta or kappa opioid receptors (DOR and KOR respectively). Antioxidant intervention preserved the function of MOR. Western immunoassays revealed that MOR but not DOR and KOR proteins were decreased under oxidative stress conditions. Thus, these findings show a selective impairment of the MOR function and reduction in MOR protein under conditions of oxidative stress. The results from the in vivo and in vitro projects demonstrate the involvement of aging and oxidative stress in modulation of pass sensitivity, opioid-induced antinociception and opioid receptor function and expression.Item Sexually Dimorphic Anxiety-Like Interoceptive Discriminative Stimuli(1997-12-01) Jung, Marianna E.; Walls, Cleatus; Downey, H. Fred; Forster, MichaelJung, Marianna E., Sexually Dimorphic Anxiety-Like Interoceptive Discriminative Stimuli. Doctor of Philosophy (Biomedical Sciences), December 1997, 150 pp, introduction, 2 chapters, discussion, bibliography, 109 titles. This study compared gender differences in the anxiogenic stimuli induced by either a GABA-A antagonist, pentylenetetrazol (PTZ) or by a 5-HT1b/2 agonist, m-chlorophenylpiperazine (m-CPP) before and during ethanol withdrawal (EW). Rats were trained to discriminate either PTZ (16mg/kg, IP) or m-CPP (1.2 mg/kg, IP) from saline in a two lever choice task for food reward. Male and female rats were gonadectomized or sham-operated, and ovariectomized (OVX) female rats were tested during replacement treatment with 17β estradiol (2.5 mg, 21 day release, sc). The dose-response for the discrimination of the interoceptive stimulus (IDS) produced by PTZ (0-16 mg/kg) or m-CPP (0 to 1.2 mg/kg) was measured under all hormonal conditions. For m-CPP trained rats, latency to first lever-press response was also tested. Results: sham and estradiol-replaced female rats had higher ED50s for discrimination of the PTZ or m-CPP IDS than intact males or OVX rats. There is a dose-related impairment of operant responding after mCPP injection. Sham and estradiol replaced OVX rats showed an increased delay to the initiation of response after m-CPP injection as compared to sham or castrated male rats or OVX rats that showed no effect at the doses tested. Rats then received a chronic ethanol diet (6.5%) for 10 days. At twelve hours of ethanol withdrawl, they were tested for lever selection after saline injection. Fewer sham female and estradiol-replaced female rats responded on the drug lever during acute EW as compared to sham male, castrated or OVX rats. In general, the anxiogenic drug lever selection of OVX rats resembled that of male rats but was restored toward that of sham female rats by estradiol replacement. Castration did not alter the response of male rats to either PTZ or mCPP. Serum β –estradiol concentrations were determined by radioimmunoassay for sham, OVX, and estradiol-replaced female rats. The concentration was significantly higher in hormone-replaced female rats than in OVX. The estradiol concentration in sham female rats showed a cyclic pattern over 4 consecutive days, but this pattern did not correlate with any difference in IDS. Blood ethanol concentration (BEC) was determined using head space gas chromatography. BEC was higher in intact female rats than in intact male rats after ethanol injection (2 g/kg, ip), but did not differ during EW. Conclusions: females produce less anxiogenic IDS in response to either GABA inhibition or 5-HT1b/2 activation, but are more impaired by m-CPP in their ability to initiate operant responses than male rats. In addition, fewer intact females developed a spontaneous IDS during EW than males which is not the result of lower BEC. Estrogen appears to play a trophic role in altering responsiveness to anxiogenic stimuli.Item The Effect of Late-Life Antioxidant Supplementaion on Brain Function(2007-10-01) Shetty, Ritu A.; Forster, Michael J.; Sumien, Nathalie; Singh, MeharvanShetty, Ritu A., The effect of late-life antioxidant supplementation on brain function. Doctor of Philolosophy (Biomedical Sciences), October, 2007, 229 pp., 5 tables, 18 figures, bibliography, 284 titles. Purpose: Aging is associated with mild to moderate loss in brain function over time. These functional losses are thought to involve reversible changes disrupting important cellular signaling processes. One of the theories that proposes to explain the reversible losses of function is the ‘oxidative stress’ hypothesis of aging. According to the oxidative stress hypothesis, there is an inherent cellular imbalance between production of oxidants and antioxidative defenses that increases with age and that leads to an increase in oxidative damage to macromolecules that are involved in crucial cell functions. Previous studies have established a link between these cellular changes associated with aging and the impairments in cognitive and psychomotor function. Further it has also been suggested that dietary interventions can modulate the level of oxidative stress, reducing oxidative damage and perhaps even ameliorate age-related dysfunction. Most interventions have been implemented relatively early in life and maintained until old age. However, the current studies were based on the rationale that interventions initiated in late-life could potentially lower oxidative damage and thereby alter cellular components responsible for functional impairments. Methods: In study I, separate groups of young (4 months) and old mice male C57BL/6 (18 months) were fed a control diet or a diet supplemented with low (105 mg/kg/day) or high (368 mg/kg/day) concentrations of CoQ10 for a period of 15 weeks. After 6 weeks on the diets, the mice were subjected to a battery of age-sensitive behavioral tests. In study II, separate groups of male C57BL/6 young mice aged 3-4 months and old mice 17-18 months (total of n=124) were fed ad libitum either a control diet (cyclodextrin in base diet), or the same diet supplemented with D- α-tocopheryl acetate (Toc) (200 mg/kg body wt/day), or with CoQ10 (148 mg/kg body wt/day) or a diet containing a combination of CoQ and Toc (200 mg/kg body wt/day + 148 mg/kg body wt/day) for a period of 13-14 weeks. In both studies mice were subjected to a battery of behavioral tests that required utilization of various component of memory and learning and sensorimotor reflexes. Results: In study I, low CoQ10 failed to improve cognitive and psychomotor function in old mice. However, the high CoQ10 marginally helped the old mice to navigate in the swim maze task with greater efficiency than control mice but did not affect their performance in probe trials. Conversely, the high CoQ10 diet selectively impaired the spatial performance in young mice in probe trials. The results from study I indicated that intake of CoQ10 initiated in late-life had minimal beneficial effects on behavior function. In study II, an age-associated decline of behavioral functioning was observed; however CoQ10 treatment failed to improve the performance of mice in any of the age-sensitive tests. Moreover, young mice supplemented with a high CoQ diet performed poorly in the probe trial in a swim maze task, suggesting a possible deleterious effect. The results from study II indicated that there was a significant improvement in performance of old mice in the coordinated running and the learning ability in discriminated avoidance task when supplemented with Toc or with a combination of CoQ10 and Toc. Conclusions: In conclusion, these studies suggest that benefits of single antioxidant supplementation when initiated late in life are limited; however dietary supplementation with a combination of antioxidants has a greater impact in reversing age-related decline in behavioral function.Item The Effects of Short-Term Intermittent Hypoxic Apneas on Sympathetic Nerve Activity and the Chemoreflex Control of Sympathetic Nerve Activity in Humans(2004-05-01) Cutler, Michael J.; Smith, Michael L.; Raven, Peter B.; Downey, H. FredCutler, Michael J., The Effects of Short-Term Intermittent Hypoxic Apneas on Sympathetic Nerve Activity and the Chemorelex Control of Sympathetic Nerve Activity in Humans. Doctor of Philosophy (Integrative Physiology), May 2004. Obstructive sleep apnea is associated with sustained elevation of muscle sympathetic nerve activity (MSNA) and altered chemoreflex control of MSNA both of which likely play an important role in the development of hypertension in these patients. Hypoxia is postulated to be primary stimulus for elevated daytime MSNA and altered chemoreflex control of MSNA both of which likely play an important role in the development of hypertension in these patients. Hypoxia is postulated to be the primary stimulus for elevated daytime MSNA and altered chemoreflex control of MSNA in OSA patients. Recently, short-term exposure to hypoxia was shown to produce sustained elevation of MSNA. Therefore, we studied the effects of 20 min of intermittent voluntary hypoxic apneas (to mimic OSA) on MSNA and the chemoreflex control of MSNA during 180 min post exposure. Also, we compared MSNA and chemoreflex control of MSNA for 180 min following either 20 min of intermittent voluntary hypoxic apneas, hypercapnic hypoxia, or isocapnic hypoxia. Consistent with our hypotheses, both total MSNA and MSNA burst frequency were elevated following 20 min of intermittent hypoxic apnea compared to baseline (p [less than] 0.05). Both total MSNA and MSNA burst frequency remained elevated throughout the 180 min recovery period and were statistically different from time control subjects throughout this period (p [less than] 0.05). Additionally, a significant main effect for chemoreflex control of SNA was observed following 20 min of intermittent hypoxic apneas (p [less than] 0.001). Specifically, the MSNA response to a single hypoxic apnea was attenuated 1 min post exposure compared to baseline (p [less than] 0.001), became augmented within 30 min of recovery, and remained augmented through 165 min of recovery (p [less than] 0.05). Finally, comparison of treatment groups (hypoxic apnea, hypercapnic hypoxia, and isocapnic hypoxia) revealed no differences in resting MSNA (p=0.50) and the chemoreflex control of MSNA (p=0.69) during recovery. Therefore, these data support the hypothesis that short-term exposure to intermittent hypoxic apneas resulted in sustained elevation of MSNA and altered chemoreflex control of MSNA. Furthermore, these responses appear to be mediated by hypoxia.Item The Role of Advanced Glycation End Products in Brain Aging(2007-10-01) Thangthaeng, Nopporn; Michael J. Forster; Tina MachuThangthaeng, Nopporn, The Role of Advanced Glycation End Products in Brain Aging. Doctor of Philosophy (Biomedical Sciences), October, 2007, 178 pp., 9 tables, 6 figures, bibliography, 213 titles. Glycoxidation is a process of post-translational modification of proteins, involving both glycation and oxidation that ultimately generated advanced glycation end products (AGEs). Glycoxidation, which pay promote oxidative stress and disrupt protein structure and function, is hypothesized to be responsible for pathological conditions related to aging, diabetes, neurodegenerative diseases, and degenerative ophthalmic diseases. Previous studies have demonstrated that AGEs accumulate in the brains of aged animals and humans, yet few studies have directly addressed the possibility that AGEs are a cause of age-related brain dysfunction. Therefore, the overall purpose of the present studies was to examine the role AGEs in normal brain again and the associated decline in cognitive and psychomotor function. In order to achieve the goals, two different approaches were taken. The first approach involved (i) determining whether or not AGEs accumulated in different regions of the brain as a function of age and (ii) determining whether these changes were correlated with individual differences in the ability of old mice to perform in tests of cognitive and psychomotor function. Age-associated accumulation of CML, a predominant form of AGEs in vivo, and expression of receptor for AGEs (RAGE) protein, inferred from densitometry quantification of immunoblots in different regions of the brain, were assessed by comparing groups of 8-or 25-month old mice. The 25-month-old mice were administered a series of behavioral tests to assess cognitive and psychomotor function prior to assessment of glycation status. In the second approach, groups of mature (6 mos) and older mice (18 mos) were fed with a control diet or a diet enriched with galactose (49% of caloric content), an intervention that was expected to promote formation of AGEs. The mice were subsequently tested for impairment of their cognitive and psychomotor functions after 8 weeks on the assigned diet. Upon completion of the behavioral tests (after 14 weeks on diet), amounts of CML and RAGE protein were assessed through densitometric analyses of the immunoblots. The main findings from the first approach were that (i) there was a robust increase in CML content and expression of RAGE protein in the aged mouse brain that occurred in a region-specific manner; (ii) the relative amounts of CML and RAGE were not closely associated with the degree of age-related impairment of mice tested for brain function. The main findings from the second approach were that high dietary galactose: (i) failed to induce aged-like behavioral impairments in young/mature mice; (ii) exacerbated age-related impairment of some psychomotor functions and (iii) had no significant effects on glycation status or oxidative damage. Comparison of the experimental outcomes from the first and second approaches was complicated by a difference in the fat content of the diets fed to the mice in the two studies, which had an apparent effect on the amounts of AGEs and protein oxidation present in young mice. However, considering the results of the two studies independently warrants the following conclusions: (i) Amounts of AGEs do not predict individualized brain aging as assessed by neurobehavioral impairment and may instead by largely reflective of chronological age. (ii) Diets enriched with galactose may produce deleterious effects in older mice that do not involve a change in oxidative damage or glycation status. Overall, these studies provide little support for a specific role of glycoxidation in normal brain aging. It is impossible that the extent of accrual of AGEs in the normally aging brain is insufficient to affect cellular function, whereas larger accumulations of AGEs may be associated with various pathological conditions discussed in the literature.Item The Role of Dopamine, Nicotine Acetylcholine, Opioid and Sigma Receptors in Ketamine Self-Administration and Reward(2000-05-01) Stoffel, Stephen A.; Michael Forster; Glenn Dillon; Robert LuedtkeStoffel, Stephen A., The Role of Dopamine, Nicotinic Acetylcholine, Opioid and Sigma Receptors in Ketamine Self-Administration and Reward. Doctor of Philosophy in Pharmacology, May 2000, 114 pp 15 figures, bibliography. The rewarding effects of ketamine were postulated to involve dopaminergic neural tracts modulated by nicotinic, sigma, or opioid receptor mechanisms. In support of the hypothesized involvement of dopamine, an increase in extracellular dopamine was detected in the nucleus accumbens using electrochemical chronoamperometry following intravenous ketamine self-administration. When rats were permitted unlimited access to ketamine via self-administration, a greater concentration of dopamine was detected in the nucleus accumbens than was detected in the nucleus accumbens than was detected when self-administration was limited. In a subsequent set of experiments, the effects of agonists or antagonists of dopaminergic, nicotinic, sigma, or opioid receptors were examined for their effect on ketamine self-administration. Decreases in the rate of self-administration following treatment were interpreted to represent an increase in rewarding effect, whereas increases in self-administered were interpreted as a decrease in rewarding effect. The rate of self-administered intraperitoneally prior to ketamine self-administration sessions, but intravenous BMS181-100 would not substitute for ketamine in the self-administration occurred following intraperitoneal (i.p.) administration of: ketamine, SCH23390 (a D1 receptor antagonist), naloxonazine (a mu opioid receptor antagonist), and mecamylamine, a central nicotinic acetylcholine receptor antagonist. An increase in the rate of ketamine self-administration followed nicotine and dihydrexidine (a D1 receptor agonist) intraperitoneal injection. In previous studies, published in the literature, SCH23390 increased the rate of self-administration of amphetamines and cocaine, indicating a competitive effect on drug reward. However, the current studies indicate that the rewarding effects of ketamine were facilitated by SCH23390. The results are consistent with the hypothesis that the rewarding effects of ketamine are mediated through dopaminergic neural pathways. The rewarding effects of ketamine were facilitated by SCH23390. The results are consistent with the hypothesis that the rewarding effects of ketamine are mediated through dopaminergic neural pathways. The rewarding effects of ketamine may be modulated, in an inhibitory fashion, via sigma receptors, presynaptic D1 receptors, nicotinic acetylcholine receptors, and/or μ opioid receptors. Ligands at nicotinic acetylcholine and dopamine receptors yielded effects opposite to that hypothesized based on their ability to modulate the rewarding effects of other abused chemicals.