by Tung Sin Yi, Cindy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 117-132). / Acknowledgements --- p.I / List of Abbreviations --- p.II / Abstract --- p.IV / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Astrocytes in the Central Nervous System (CNS) l / Chapter 1.1.1 --- Characteristics of astrocytes / Chapter 1.1.2 --- Functions of astrocytes / Chapter 1.1.2.1 --- General functions of astrocytes / Chapter 1.1.2.2 --- Effects of neuroactive peptides on astrocytes / Chapter 1.1.3 --- Gliomas and the rat C6 glioma cells / Chapter 1.2 --- C-Type natriuretic peptide (CNP) in the CNS --- p.9 / Chapter 1.2.1 --- Structure and distribution of natriuretic peptides in the CNS / Chapter 1.2.2 --- Actions of CNP / Chapter 1.2.3 --- Natriuretic peptide receptors and signal transduction in astrocytes / Chapter 1.3 --- Endothelin-3 (ET-3) in the CNS --- p.18 / Chapter 1.3.1 --- Structure and distribution of endothelins (ETs) in the CNS / Chapter 1.3.2 --- Actions of ET-3 / Chapter 1.3.3 --- Endothelin receptors and signal transductionin astrocytes / Chapter 1.4 --- cGMP second messenger system in astrocytes --- p.28 / Chapter 1.4.1 --- Second messenger systems in astrocytes / Chapter 1.4.2 --- cGMP as second messenger in astrocytes / Chapter 1.4.3 --- Post cGMP cascade effects / Chapter 1.5 --- The aims of this project --- p.33 / Chapter Chapter 2 --- Methods / Chapter 2.1 --- In vitro culture of rat C6 glioma cells --- p.36 / Chapter 2.1.1 --- Preparation of reagents / Chapter 2.1.2 --- Culture of C6 glioma cells / Chapter 2.1.3 --- "Cell plating in 6-well, 24-well and 96-well plastic trays" / Chapter 2.2 --- Determination of cGMP --- p.40 / Chapter 2.2.1 --- Measurement of cGMP / Chapter 2.2.2 --- Data analysis / Chapter 2.3 --- Determination of the effect of CNP on cGMP productionin C6 cells --- p.41 / Chapter 2.4 --- Determination of the effect of ET-3 on the action of CNPin C6cells --- p.44 / Chapter 2.4.1 --- Measurement of intracellular cGMP levels affected by ET-3 / Chapter 2.4.2 --- Measurement of intracellular cGMP levels affected by CNP with ET-3 pretreatment / Chapter 2.5 --- Determination of the effects of PKC activator and inhibitor on CNP-treated C6 cells --- p.46 / Chapter 2.5.1 --- Measurement of intracellular cGMP levels affected by PKC activator or inhibitor / Chapter 2.5.2 --- Measurement of intracellular cGMP levels affected by CNP with PKC activator or inhibitor pretreatment / Chapter 2.5.3 --- Measurement of intracellular cGMP levels affected by CNP with PKC inhibitor antagonized PMA or ET-3 pretreatment / Chapter 2.6 --- Determination of the effect of arachidonic acid on the action of CNP in C6 cells --- p.49 / Chapter 2.7 --- Determination of the effects of ET-3 and CNP on calcium uptake in C6 cells --- p.50 / Chapter 2.8 --- Determination of the effects of CNP and ET-3 on cell volume change in C6 cells --- p.51 / Chapter 2.9 --- Determination of the effects of CNP and ET-3 on glucose and amino acids uptake in C6 cells --- p.53 / Chapter 2.9.1 --- Measurement of glucose uptake in CNP - and/or ET- 3-treated C6 cells / Chapter 2.9.2 --- Measurement of amino acids uptake in CNP - and/or ET-3-treated C6 cells / Chapter 2.10 --- "Determination of thymidine, uridine and leucine incorporation in CNP - and/or ET-3- treated C6 cells" --- p.55 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of CNP and ET-3 on cGMP production in cultured rat C6 glioma cells --- p.56 / Chapter 3.1.1 --- Effect of CNP on cGMP production in cultured C6 glioma cells --- p.57 / Chapter 3.1.1.1 --- The time course of CNP on cGMP production / Chapter 3.1.1.2 --- Dosage-response of CNP on cGMP production / Chapter 3.1.2 --- Effect of ET-3 on cGMP production in C6 glioma cells --- p.61 / Chapter 3.1.2.1 --- Effect of ET-3 on basal cGMP production / Chapter 3.1.2.2 --- Effect of pre-exposure duration to ET-3 on CNP-induced cGMP formation / Chapter 3.1.2.3 --- Dosage-response of ET-3 on CNP-induced cGMP production / Chapter 3.1.3 --- Effect of PMA on cGMP production in C6 glioma cells --- p.65 / Chapter 3.1.3.1 --- Effect of PMA on basal cGMP production / Chapter 3.1.3.2 --- Effect of pre-exposure duration to PMA on CNP-induced cGMP formation / Chapter 3.1.3.3 --- Dosage-response of PMA on CNP-induced cGMP production / Chapter 3.1.4 --- Effects of PKC inhibitors on cGMP production in C6 glioma cells --- p.73 / Chapter 3.1.4.1 --- Effects of PKC inhibitors on basal cGMP production / Chapter 3.1.4.2 --- Effects of PKC inhibitors on CNP-induced cGMP formation / Chapter 3.1.4.3 --- Antagonism of PKC inhibitors on the action of PMA on CNP-induced cGMP formation / Chapter 3.1.4.4 --- Antagonism of PKC inhibitors on the action of ET-3 on CNP-induced cGMP formation / Chapter 3.1.5 --- Effect of arachidonic acid on CNP-induced cGMP production in C6 glioma cells --- p.82 / Chapter 3.2 --- Effects of CNP and ET-3 on cellular metabolism in cultured rat C6 glioma cells --- p.83 / Chapter 3.2.1 --- Effects of CNP and ET-3 on calcium uptake in C6 glioma cells --- p.86 / Chapter 3.2.2 --- Effects of CNP and ET-3 on cell volume changes in C6 glioma cells --- p.89 / Chapter 3.2.3 --- Effects of CNP and ET-3 on glucose and amino acids uptake in C6 glioma cells --- p.91 / Chapter 3.2.4 --- Effects of CNP and ET-3 on C6 cell proliferation --- p.98 / Chapter 3.2.5 --- Effects of CNP and ET-3 on RNA synthesis --- p.101 / Chapter 3.2.6 --- Effects of CNP and ET-3 on protein synthesis --- p.103 / Chapter Chapter 4 --- Discussion and Conclusion --- p.105 / References --- p.117

by Josephine Wing Sze Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 84-112). / Abstracts in English and Chinese. / Acknowledgment --- p.iii / Abstract --- p.iv / List of Abbreviations --- p.viii / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Methamphetamine (METH) --- p.1 / Chapter 1.1.1 --- Historical Background and Epidemiology --- p.1 / Chapter 1.1.2 --- Physical Effects of METH --- p.4 / Chapter 1.1.3 --- Neurochemical Alternation of METH --- p.6 / Chapter 1.2 --- Mechanisms of METH Toxicity / Chapter 1.2.1 --- Oxidative Stress --- p.8 / Chapter 1.2.1.1 --- Superoxide (O2-) and Superoxide Dismutase (SOD) --- p.10 / Chapter 1.2.1.2 --- "Hydrogen Peroxide (H202), Catalase and Glutathione (GSH)" --- p.11 / Chapter 1.2.1.3 --- Hydroxyl Radical (OH.) --- p.12 / Chapter 1.2.1.4 --- Nitric Oxide (NO) --- p.13 / Chapter 1.2.2 --- Apoptosis --- p.16 / Chapter 1.2.3 --- Excitotoxicity --- p.17 / Chapter 1.2.4 --- Mitochondrial Dysfunction --- p.18 / Chapter 1.2.5 --- Hyperthermia --- p.21 / Chapter 1.2.5.1 --- Cyclooxygenase-2 (COX-2) --- p.23 / Chapter 1.2.5.2 --- Heme-oxygenase-1 (HO-1) --- p.25 / Chapter 1.2.5.3 --- The Effects of Nitric Oxide (NO) on COX-2 and HO-1 Expressions --- p.27 / Chapter 1.3 --- Astrocytes / Chapter 1.3.1 --- Characteristics of Astrocytes --- p.29 / Chapter 1.3.2 --- Astrocyte Functions --- p.30 / Chapter 1.3.3 --- The Role of Astrocytes in METH-induced Neurotoxicity --- p.34 / Chapter 1.4 --- Aim of Project --- p.37 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Cell Cultures / Chapter 2.1.1 --- Astrocyte Cultures --- p.42 / Chapter 2.1.2 --- CATH.a Cell line and Astrocytes Co-cultures --- p.43 / Chapter 2.2 --- Treatment / Chapter 2.2.1 --- METH Treatment --- p.44 / Chapter 2.2.2 --- Inhibition of Cyclooxygenase-2 (COX-2) and Inducible Nitric Oxide Synthase (iNOS) --- p.44 / Chapter 2.3 --- Lactate Dehydrogenase (LDH) Assay --- p.45 / Chapter 2.4 --- Assay for Reactive Oxygen Species (ROS) Formation --- p.47 / Chapter 2.5 --- Assay for Adenosine Triphosphate (ATP) Content --- p.48 / Chapter 2.6 --- Determination of Mitochondrial Membrane Potential (Δ Ψm) --- p.50 / Chapter 2.7 --- Determination of Nitrite Levels in Cultured Astrocytes --- p.51 / Chapter 2.8 --- Western Blot Analysis --- p.52 / Chapter 2.8.1 --- COX-2 --- p.53 / Chapter 2.8.2 --- HO-1 --- p.53 / Chapter 2.9 --- Viability Assay of CATH.a-Astrocyte Cocultures --- p.54 / Chapter 2.10 --- Statistics --- p.55 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- The Effects of METH Treatment on Cultured Astrocytes / Chapter 3.1.1 --- Lactate Dehydrogenase (LDH) Activities --- p.56 / Chapter 3.1.2 --- Morphological Changes --- p.56 / Chapter 3.1.3 --- The Production of Reactive Oxygen Species / Chapter 3.1.3.1 --- Rate of change (0-120 min) --- p.57 / Chapter 3.1.3.2 --- Time course (0 - 48 h) --- p.57 / Chapter 3.1.4 --- Change in ATP Content --- p.58 / Chapter 3.1.5 --- Change in Mitochondrial Membrane Potential (Δ Ψm) --- p.59 / Chapter 3.1.6 --- Nitrite levels after METH treatment / Chapter a) --- Striatal astrocytes --- p.59 / Chapter b) --- Mesencephalic astrocytes --- p.60 / Chapter c) --- Cortical astrocytes --- p.60 / Chapter 3.1.7 --- The Effects of Aminoguanidine (AG) on Nitrite Levels / Chapter a) --- Striatal astrocytes --- p.61 / Chapter b) --- Mesencephalic astrocytes --- p.62 / Chapter c) --- Cortical astrocytes --- p.62 / Chapter 3.1.8 --- The Effects of Indomethacin (INDO) on Nitrite Levels / Chapter a) --- Striatal astrocytes --- p.63 / Chapter b) --- Mesencephalic astrocytes --- p.64 / Chapter c) --- Cortical astrocytes --- p.64 / Chapter 3.1.9 --- Change in Cyclooxygenase-2 (COX-2) Protein Levels / Chapter a) --- Striatal astrocytes --- p.65 / Chapter b) --- Mesencephalic astrocytes --- p.65 / Chapter c) --- Cortical astrocytes --- p.66 / Chapter 3.1.10 --- Change in Heme-oxygenase-1 (HO-1) Protein Levels / Chapter a) --- Striatal astrocytes --- p.66 / Chapter b) --- Mesencephalic astrocytes --- p.66 / Chapter c) --- Cortical astrocytes --- p.67 / Chapter 3.2 --- Cell Viability on CATH.a-Astrocyte Cocultures After METH Treatment --- p.67 / Chapter CHAPTER FOUR: --- DISCUSSION AND CONCLUSION --- p.69 / REFERENCES --- p.84

Mutação na proteína <font face = \"symbol\">b-glucuronidase Klotho tem sido associada com envelhecimento prematuro e disfunção cognitiva. Embora altamente expressa em regiões específicas do encéfalo, as funções da proteína Klotho no sistema nervoso central ainda são desconhecidas. Aqui, mostramos que animais com gene mutado e hipomórfico para a proteína Klotho possuem regulação glicêmica alterada, sugerindo maior sensibilidade à insulina. No sistema nervoso central, vias relacionadas à sinalização intracelular de insulina apresentam-se mais ativadas no hipocampo, com maior ativação de AKT e mTOR e inativação dos fatores de transcrição FOXO. Neurônios hipocampais em cultura parecem responder à estimulação por insulina e glutamato aumentando os níveis de Klotho. De forma coerente, antagonismo de receptor AMPA ou NMDA suprime a expressão neuronal de Klotho. Também mostramos aqui que a forma solúvel da Klotho é capaz de induzir a glicólise aeróbica de astrócitos impedindo a metabolização do piruvato pela mitocôndria, e estimulando seu processamento pela lactato desidrogenase. Inibição farmacológica de FGFR1, fosforilação da ERK e de transportadores de ácidos monocarboxílicos previne a liberação de lactato induzida por Klotho em astrócitos. Inibição da AKT pelo tratamento com Klotho induz a atividade transcricional dos fatores de transcrição FOXO e promove proteção antioxidante em astrócitos pelo aumento de expressão de catalase. De forma similar, tratamento com a Klotho apresentou propriedades antiinflamatórias em astrócitos, impedindo ativação do fator de transcrição NF-<font face = \"symbol\">kB após estímulo com LPS. Em neurônios, tratamento com a proteína Klotho induz ubiquitiniação e degradação proteassomal de PFKFB3. Por fim, provamos que a quantidade de Klotho diminui em hipocampo de animais envelhecidos. Genes e proteínas relacionados ao metabolismo energético e ao acoplamento metabólico de neurônios e astrócitos tem padrão de expressão alterados no hipocampo com o envelhecimento, mostrando que essa pode ser uma característica importante para explicar o declínio cognitivo comum ao processo de envelhecimento. Em conjunto, esses dados sugerem que a Klotho pode ser um novo participante do acoplamento metabólico entre neurônios e astrócitos. / Mutations in the <font face = \"symbol\">b-glucuronidase protein Klotho have been associated with premature aging and cognitive dysfunction. Although highly expressed in specific regions of the brain, Klotho actions in the central nervous system are still largely unknown. Here we show that animals with a mutated hypomorphic Klotho gene have altered glycemia regulation, suggestive of a higher insulin sensitivity. In the central nervous system, pathways related to insulin intracellular signaling were shown to be up-regulated in the hippocampus, with higher AKT and mTOR activation, and inactivation of transcription factor FOXO. Here, we show that cultured hippocampal neurons respond to insulin and glutamate stimulation by elevating Klotho protein levels. Conversely, APA and NMDA antagonism suppress neuronal Klotho expression. We also provide evidence that soluble Klotho enhances astrocytic aerobic glycolysis by hindering pyruvate metabolism through the mitochondria, and stimulating its processing by lactate dehydrogenase. Pharmacological inhibition of FGFR1, ERK phosphorylation, and monocarboxylic acid transporters prevents Klotho-induced lactate release from astrocytes. AKT inhibition by Klotho treatment induces transcriptional activity of FOXO transcription factors and promote antioxidant defense in astrocytes by inducing catalase expression. Similarly, Klotho treatment has anti-inflammatory properties, as shown by its ability to hinder NF-<font face = \"symbol\">kB activation in astrocytes after LPS stimulation. In neurons, Klotho treatment induces PFKFB3 ubiquitination and degradation through the proteasome. Lastly, in the hippocampus, we show that Klotho is less present in hippocampi of aged mice. Genes and proteins related to energy metabolism and metabolic coupling between neurons and astrocytes show an altered expression pattern with aging, suggesting that this could be a crucial characteristic that explains cognitive decline commonly seen during the aging process. Taken together these data suggest Klotho as a potential new player in the metabolic coupling between neurons and astrocytes.

Approximately 16 million people in America are diagnosed with Alcohol Use Disorder (AUD) but no efficacious medical treatments exist. Alcohol-related behaviors can be studied in model organisms, and changes in these behaviors can be correlated with either (i) a risk for alcohol dependence or (ii) a symptom/feature of AUD itself. Although AUD is a disease of the central nervous system, a majority of research has focused on the neuronal underpinnings, leaving glial contributions largely undescribed. We used Drosophila melanogaster (fruit fly) to identify genes whose expression in glia regulates alcohol sedation. Mammals and Drosophila have conserved behavioral responses to alcohol and functionally similar adult glial cells, especially astrocytes. Since previous research in mammals and flies has demonstrated that glia respond to alcohol administration, we hypothesized that glia are important regulators of alcohol-related behaviors. To pursue this, we characterized a pan-glial steroid-inducible GeneSwitch transgenic fly, which allows gene manipulation within glia during adulthood. We performed a targeted screen and manipulated genes that were known to be expressed within Drosophila glia and measured their alcohol sedation sensitivity using the ethanol sedation assay. We identified the genes Cysteine proteinase 1 (Cp1) and Tyramine decarboxylase 2 (Tdc2). Knocking down Cp1 in cortex glia, as well as all glia during adulthood, increased alcohol sedation sensitivity and may also enhance rapid tolerance development. We could not identify what pathway Cp1 was functioning within to mediate this response, suggesting that Cp1 may have a unique function within glia. Knockdown or overexpression of Tdc2 in glia increased or decreased alcohol sedation sensitivity, respectively. Tdc2 functions upstream of the vesicular monoamine transporter (VMAT) and the SNARE complex to regulate alcohol sedation. These results were specific to astrocytes, as well as all glia during adulthood. These results suggest that tyramine synthesis via Tdc2 and its release via vesicular exocytosis regulates alcohol sedation. Taken together, these results suggest that glia are important regulators of alcohol-related behaviors in flies. Interestingly, fly cortex glia and astrocytes are functionally similar to mammalian astrocytes, indicating that these results may be translatable to mammals.

Astrocytes display a complex, stellate morphology in the brain, commensurate with their role in regulating nearly every functional aspect of the central nervous system. Innate activation of astrocytes is a classic hallmark of neurologic injury and disorder, characterized by proliferation, process hypertrophy, and transition toward polygonal morphology. However, the extent and consistency of these morphological changes, especially in the context of distinct pathologies, remain largely unknown. Here, changes in the fine neuroanatomy of astrocytes in rhesus macaque monkeys were characterized under a variety of conditions with relevance to translational research, including autistic-like behavior, bacterial and viral infections. Brain slices from paraffin embedded tissue archives were stained for glial fibrillary acidic protein, a reactive astrocyte marker, and imaged for both grey and white matter astrocytes. These cells were 3D-reconstructed in Neurolucida software and morphometric data including soma size, process length, volume and branching were analyzed and aggregated by animal and condition. It was found that process complexity in white matter astrocytes atrophied during lentiviral infection and depressive behavior, with decreased arbor length and tip quantity observed in SIV-infected and self-injuring animals. However, the opposite was seen during a bacterial infection, with increased arbor complexity and process length seen in Brucella-infected animals. Additionally, astrocytes from animals receiving oral naltrexone treatment for self-injurious behavior displayed a reversion from atrophied cell phenotypes back to original, control morphology. Thus, astrocyte activation and subsequent remodeling is not consistent, but rather context-dependent on the specific pathology and cellular subtype, and may be a driving factor in neurocognitive disorder. / acase@tulane.edu

Abstract not available

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, degenerative disorder of the CNS. The hallmark of this disease is the premature and selective death of upper and lower motor neurons (MNs) in the brain and spinal cord, leading to fatal paralysis. Although the archetypal vision of neurotoxicity in neurodegenerative diseases is based on the idea that a specific neuronal population is particularly vulnerable to a cumulative toxic event (protein aggregation, mitochondria dysfunction, compromised axonal transport etc…), experimental evidence illustrate that ALS possibly does not arise strictly from damage within MNs. There is now convincing data supporting a non-cell autonomous mechanism in which neurodegeneration is influenced by the toxicity of non-neuronal cells in the vicinity of neurons such as astrocytes and microglia. Considering the accumulation of data implicating astrocytes in the pathogenesis of ALS (loss of GLT-1, secretion of toxic factor, enhanced inflammation, etc…), approaches aiming at replacing astrocytes at site of lesions constitute promising therapeutic strategies. Rapid progresses in the characterization of adult stem cell biology have generated considerable enthusiasm for the development of therapeutic strategies for CNS insults. Several observations support the hypothesis that stem cells may display a valuable influence on diseased host tissues by exerting a protective “chaperone” effect to neurons after differentiation in glial cells. Hence, we decided to study the neuroprotective potential of adult mesenchymal stem cells (MSCs) in ALS. In contrast to neural stem cells (NSCs) which localization in the central nervous system complicates their isolation, MSCs are easily isolated from the bone marrow. The relevance of using on MSCs in stem cell therapies of neurodegenerative disorders is also justified by their capacity to (trans)differentiate into neural cells. For this purpose, we exposed MSCs to growth factors involved in the astroglial differentiation of NSCs. The differentiation of MSCs was characterized by the acquisition of astrocyte morphology in addition to an increased expression of gene related to NSCs (nestin) and astrocytes (glutamine synthetase). The astroglial differentiation of MSCs is associated with the acquisition of a glial-like specific regulation of the production of GDNF, a potent neurotrophic factor for neurons. Then, we characterized the glutamate uptake in differentiated MSCs, a critical function of astrocytes. Our data demonstrate that the differentiation of MSCs is associated with an increased expression of the high affinity glutamate transporter, GLT-1. Thus, our in vitro results confirm the astrocytic differentiation potential of MSCs and we decided to use then in stem cell therapy of ALS. Indeed, we demonstrated that mechanism of stem cell recruitment is present in the spinal cord during the development of the disease by the secretion of stem cell factor (SCF). We injected MSCs derived from healthy animals into the cerebrospinal fluid of a transgenic rat model of familial ALS (expressing a mutated form of the human superoxide dismutase-1, hSOD1G93A) at disease onset. MSCs were found to infiltrate the nervous parenchyma and migrate substantially into the ventral grey matter by interacting with the SCF. At the site of lesion, MSCs differentiated massively into astrocytes around MNs. The intrathecal delivery of MSCs preserved motor functions and extended the survival of hSOD1G93A rats. Investigation of the lumbar spinal cord 35 days after graft demonstrated that the generation of healthy astrocytes from MSCs decreased motor neuron loss. However, this beneficial effect is not related to a decreased excitotoxicity by the rescue of GLT-1 expression but rather a decreased inflammation around MNs. Together, the data presented in this thesis highlight the protective capacity of adult MSC-derived astrocytes in the treatment of ALS.

Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. I used the technique of serum-free cell culture to develop a serum-free mouse embryo (SFME) cell line in which serum was replaced by a set of defined supplements. SFME cells, cultured in a nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin, have maintained a diploid karyotype with no detectable chromosomal abnormalities for more than 200 generations. The cells did not undergo growth crisis and remain in culture today. SFME cells were dependent on EGF for survival and were reversibly growth inhibited by serum or platelet-free plasma. Treatment of SFME cells with serum or transforming growth factor beta led to the appearance of glial fibrillary acid protein (GFAP), a specific marker for astrocytes, identifying SFME cells as proastroblasts. Following the derivation of SFME cells my research focussed on (1) defining more precisely the growth response of SFME cells to medium supplements, (2) investigating the relationship between the nonsenescent nature of SFME cells and their responses to serum and EG1., and (3) applying the serum-free cell culture methods to the multipassage culture of human embryonic astrocytes. SFME cells in serum-containing medium arrested in the G1 phase of the cell cycle with greatly reduced DNA replication activity. A portion of the inhibitory activity of serum was extracted by charcoal, a procedure that removed steroid and thyroid hormones. However, the effect of serum on untransformed SFME cells could not be prevented by addition of antiglucocorticoid, and ras-transformed clones of SFME cells, which are not growth inhibited by serum, retained inhibitory responses to glucocorticoid and thyroid hormone T3. These results suggest that glucocorticoid or thyroid hormones may contribute to the inhibitory activity of serum on SFME cells, but additional factors are involved. SFME cell death resulting from EGF deprivation exhibited characteristics associated with apoptosis or programmed cell death. Ultrastructural analysis showed cells became small and vacuolated, with pyknotic nuclei. The cultures contained almost exclusively G1- phase cells. Chromatin exhibited a pattern of degradation into oligonucleosome-length fragments generating a regularly spaced ladder. I applied the serum-free approach used to derive SFME cells to the multipassage culture of human embryonic astrocytes. Cells were cultured in nutrient medium supplemented with insulin, transferrin, EGF, HDL, fibronectin, basic fibroblast growth factor and heparin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized GFAP. / Graduation date: 1992

Thèse de doctorat : Aspects moléculaires et cellulaires de la biologie. Neurosciences : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. f. 97-116.

The research of functional stimulation of neural tissue is of great interest within the field of clinical neuroscience to further develop new neural prosthetics. A technique which has gained increased interest during the last couple of years is the stimulation of nervous tissue using infrared laser light. Successful results have been reported, such as stimulation of cells in both the central nervous system, and in the peripheral nervous system, and even cardiomyocytes. So far, the details about the stimulation mechanism have been a question of debate as the mechanism is somewhat hard to explain. The mechanism is believed to have a photo-thermal origin, where the light from the laser is absorbed by water, thus increasing the temperature inside and around the target cell. Despite the mechanism questions, the technique holds several promising features compared to traditional electrical stimulation. Examples of advantages are that it is contact free, no penetration is needed, it has high spatial resolution and no toxic electrochemical byproducts are produced during stimulation. However, since the laser pulses locally increase the temperature of the tissue, there is a risk of heat induced damage. Therefore, the effect of increased temperatures must be investigated thoroughly. One method of examining the changes in temperature during stimulation is to model the heating. This thesis is based on the work from four papers with the main aim to investigate and describe the response of heating, caused by laser pulses, on central nervous system cells. In paper one, a model of the heating during pulsed laser stimulation is established and used to describe the dynamic temperature changes occurring during functional stimulation of cerebral cortex cells. The model was used in all four papers. Furthermore, single cell responses, as action potentials, as well as network responses, as activity inhibition, were observed. In paper two, the response of rat astrocytes exposed to laser induced hyperthermia was investigated. Cellular migration was observed and the migration limit was used to calculate the kinetic parameters for the cells, i.e., the reaction activation energy, Ea (321.4 kJ⋅mol-1), and the frequency factor, Ac (9.47 x 1048 s-1). Furthermore, a damage signal ratio (DSR) for calculating a threshold for cellular damage was defined, and calculated to six percent. In paper three, the response of hyperthermia to cerebral cortex cells was investigated, in the same way as in the second paper. Fluorescence staining of the metabolic activity was used to reveal the heat response, and by using the limit of the observed increased fluorescence the kinetic parameters, Ea (333.6 kJ⋅mol-1), and Ac (9.76 x 1050 s-1), were calculated. The DSR for the cells was calculated to five percent. In paper four, the behavior of action potentials triggered by laser stimulation was investigated. More specifically, the time delay from the start of a laser pulse to the detection of an action potential, delta-t, were investigated. Two different behaviors for the initial action potentials were observed: fast decreasing delta-t and slow decreasing delta-t. The results show the dynamic behavior of action potential responses to infrared light. The work of this thesis show the dynamic changes of the temperature during optical stimulation, using an infrared laser working at 1,550 nanometers. It also shows how the changes cause astrocytes to migrate for pulses several seconds long, and neurons to fire action potentials for pulses in the millisecond range. Furthermore, a damage signal ratio was defined and calculated for the cell systems.