Representing a major public health concern, suicide is a leading cause of death worldwide. Generally regarded as a behavior with a multitude of state and trait dependent risk factors (e.g. psychiatric disorders, substance abuse, genetics), explanations as to why certain individuals commit suicide while others do not are complex. Of interest is in studying potential trait dependent variables involved in the neurobiology of suicide, particularly at the cellular level. Knowledge of the cellular integrity may aid in explaining the observed macroscopic alterations and ultimately the behavioral correlates associated with suicidality. Therefore we set out to summarize extant knowledge of the cellular alterations occurring in the brains of major depressive and suicide individuals. Following this, we conducted our own cellular investigation in a region known to be altered in major depression and suicide, a supracallosal area of BA24a. Neuronal and glial cell densities as well as neuronal cell sizes were assessed in upper and lower cortical layers between sudden-death controls and MDD suicide subjects. Secondary analyses were also conducted to examine the effect of alcohol on depressed suicides. Analyses of cell densities and neuronal soma sizes between controls and MDD suicide subjects did not reveal any significant differences. Further analyses showed increased glial cell densities in alcoholic depressed suicides. Future studies are necessary to examine explicit changes in the cellular compositions occurring in alcoholic dependent individuals. Staining techniques aimed at targeting specific subtypes of neurons and glial cells will help determine if these cell populations do in fact have an influential role in suicide and MDD.

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Tam Wing-yip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 91-113). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese Abstract --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Hirschsprung's disease --- p.1 / Chapter 1.2 --- Neural crest cells and enteric nervous system --- p.3 / Chapter 1.3 --- Genetics of Hirschsprun´gةs disease --- p.10 / Chapter 1.3.1 --- RET/GDNF/NTN signaling pathway --- p.10 / Chapter 1.3.2 --- EDNRB/EDN3/ECE-1 signaling pathway --- p.13 / Chapter 1.3.3 --- Dominant megacolon and Sox10 --- p.15 / Chapter 1.3.4 --- Other genes involved in intestinal aganglionosis --- p.16 / Chapter 1.4 --- Objectives of the present study --- p.19 / Chapter Chapter 2 --- Enteric Neural Crest Cells Migration in Dominant Megacolon Mouse Embryos --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Animal --- p.26 / Chapter 2.2.2 --- Preparation of rat serum --- p.26 / Chapter 2.2.3 --- Isolation of embryos from pregnant mice --- p.27 / Chapter 2.2.4 --- Preparation of wheat germ agglutinin-gold (WGA-Au) --- p.28 / Chapter 2.2.5 --- Microinjection of WGA-Au conjugate --- p.28 / Chapter 2.2.6 --- Whole embryo culture --- p.29 / Chapter 2.2.7 --- Examination of cultured embryos --- p.30 / Chapter 2.2.8 --- Histological preparation of WGA-Au injected embryos --- p.30 / Chapter 2.2.9 --- Silver enhancement staining and histological examination of the sections --- p.31 / Chapter 2.2.10 --- Genotyping by polymerase chain reaction --- p.32 / Chapter 2.2.11 --- TUNEL assays --- p.33 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- In vivo development of Dominant megacolon mouse embryos of different genotypes --- p.35 / Chapter 2.3.2 --- In vitro development of embryos in control and experimental groups --- p.35 / Chapter 2.3.3 --- Migration of vagal neural crest cells in Dom embryos --- p.36 / Chapter 2.3.4 --- Apoptotic cells detection at the vagal region by TUNEL assay --- p.37 / Chapter 2.3.5 --- Migration of sacral neural crest cells in Dom embryos --- p.37 / Chapter 2.3.6 --- Apoptotic cells detection at the sacral region by TUNEL assay --- p.38 / Figures and Tables / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- In vitro culture system supporting the normal development of mouse embryos --- p.40 / Chapter 2.4.2 --- WGA-Au as a cell marker for tracing the NCCs migration --- p.41 / Chapter 2.4.3 --- Vagal neural crest cells migration in Dom mouse embryos --- p.42 / Chapter 2.4.4 --- Apoptotic cell death does not contribute to the total aganglionosis in Dom homozygous embryos --- p.43 / Chapter 2.4.5 --- Sacral neural crest cells migration in Dom mouse embryos --- p.45 / Chapter 2.4.6 --- NCCs migration in zebrafish colourless mutant --- p.47 / Chapter 2.4.7 --- Limitation of the method used in this study --- p.49 / Chapter 2.4.8 --- Conclusions --- p.49 / Appendices / Chapter Chapter 3 --- Migration of Enteric Neural Crest-derived Cells in the Developing Gut of Dominant Megacolon Mouse Embryos --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.55 / Chapter 3.2.1 --- Isolation of the gut from Dom mouse embryos --- p.55 / Chapter 3.2.2 --- Whole mount immunohistochemistry --- p.55 / Chapter 3.3 --- Results --- p.57 / Chapter 3.3.1 --- PGP9.5 immunoreactivity in the 12.5 d.p.c. Dom embryos --- p.57 / Chapter 3.3.2 --- TH immunoreactivity in the 12.5 d.p.c. Dom embryos --- p.58 / Chapter 3.3.3 --- PGP9.5 immunoreactivity in the 14.5 d.p.c. Dom embryos --- p.59 / Figures and Tables / Chapter 3.4 --- Discussion --- p.61 / Chapter 3.4.1 --- The use of PGP9.5 and TH antibodies as markers for studying the migration of enteric neural crest-derived cells --- p.61 / Chapter 3.4.2 --- Incomplete migration of neural crest-derived cells within the gut of Dom heterozygous embryos --- p.62 / Chapter 3.4.3 --- Failure of sacral NCCs to invade the hindgut of Dom heterozygous embryos --- p.63 / Chapter 3.4.4 --- PGP9.5 and TH positive signals in the gut of Dom homozygous embryos --- p.64 / Chapter 3.4.5 --- Early differentiation of neural crest-derived cells into neurons due to haploinsufficiency of Sox10 --- p.65 / Chapter 3.4.6 --- Conclusions --- p.66 / Chapter Chapter 4 --- Localization of Interstitial Cells of Cajal in the Gut of Dominant Megacolon Mice --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2. --- Materials and Methods --- p.72 / Chapter 4.2.1 --- Isolation of the gut from mouse embryos and adult mice --- p.72 / Chapter 4.2.2 --- Cryosection and immunohistochemistry --- p.73 / Chapter 4.2.3 --- Whole-mount immunohistochemistry --- p.73 / Chapter 4.2.4 --- Total RNA extraction --- p.74 / Chapter 4.2.5 --- Reverse transcription for the first strand cDNA synthesis --- p.75 / Chapter 4.2.4 --- Reverse transcription-Polymerase chain reaction (RT-PCR) --- p.76 / Chapter 4.3 --- Results --- p.77 / Chapter 4.3.1 --- PGP9.5 and c-kit immunoreactivity in the Dom wild type colon --- p.77 / Chapter 4.3.2 --- c-kit immunoreactivity in the Dom heterozygous adult colon --- p.78 / Chapter 4.3.3 --- c-kit and SCF expression during gut development --- p.78 / Figures and Tables / Chapter 4.4 --- Discussion --- p.80 / Chapter 4.4.1 --- The importance in studying the development of ICCs in aganglionic gut --- p.80 / Chapter 4.4.2 --- ICCs development in Dominant megacolon mice --- p.81 / Chapter 4.4.3 --- The relationship between enteric neurons and ICCs development --- p.83 / Chapter 4.4.4 --- Advantages of using confocal microscopy and whole- mount preparations to study the ICCs development --- p.85 / Chapter 4.4.5 --- Conclusions --- p.86 / Chapter Chapter 5 --- General Discussion and Conclusions --- p.87 / References --- p.91

Yung, Him Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Publications based on work in this thesis --- p.vii / Abbreviations --- p.viii / Contents --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Objectives and overview of this study --- p.1 / Chapter 1.2 --- Rat pheochromocytoma (PC12) cells --- p.3 / Chapter 1.3 --- Prostanoids and their receptors --- p.4 / Chapter 1.4 --- Roles of prostanoids --- p.7 / Chapter 1.5 --- Nerve growth factor (NGF) and its receptors --- p.9 / Chapter 1.6 --- Change of gene expressions by NGF in PC12 cells --- p.10 / Chapter 1.7 --- Signaling pathways involved in NGF-induced differentiation of PC12 cells --- p.12 / Chapter 1.8 --- Classification of adenylyl cyclases --- p.14 / Chapter 1.9 --- Methods to study differentiation of PCI 2 cells --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.19 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.2 --- Cell culture medium and buffers --- p.25 / Chapter 2.3 --- Buffers and solutions for assay of [3H]inositoI phosphates ([3H]IP) production --- p.25 / Chapter 2.4 --- Buffers and solutions for assay of [3H]cAMP production --- p.27 / Chapter 2.5 --- Buffers and solutions for Western blotting --- p.28 / Chapter 2.6 --- Methods --- p.30 / Chapter 2.6.1 --- Maintenance of PC12 cells --- p.30 / Chapter 2.6.2 --- General culture condition of PCI2 cells for NGF treatment --- p.31 / Chapter 2.6.3 --- Determination of phospholipase C activity in PC12 cells --- p.31 / Chapter 2.6.3.1 --- Principle of assay --- p.31 / Chapter 2.6.3.2 --- Column preparation --- p.32 / Chapter 2.6.3.3 --- Measurement of [3H]IP production --- p.33 / Chapter 2.6.3.4 --- Data analysis --- p.34 / Chapter 2.6.4 --- Determination of adenylyl cyclase activity in PC12 cells --- p.35 / Chapter 2.6.4.1 --- Principle of assay --- p.35 / Chapter 2.6.4.2 --- Column preparation --- p.35 / Chapter 2.6.4.3 --- Measurement of [3H]cAMP production --- p.36 / Chapter 2.6.4.4 --- Data analysis --- p.37 / Chapter 2.6.5 --- Determination of neurofilament protein expression in PC12 cells by Western blotting --- p.38 / Chapter 2.6.6 --- Determination of adenylyl cyclase isoform expression in PC12 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.39 / Chapter 2.6.6.1 --- Isolation of total cellular RNA --- p.39 / Chapter 2.6.6.2 --- Synthesis of first strand cDNA by reverse transcription (RT) --- p.40 / Chapter 2.6.6.3 --- Polymerase Chain Reaction (PCR) --- p.41 / Chapter 2.6.6.4 --- Agarose gel electrophoresis --- p.41 / Chapter 2.6.7 --- Neurite quantification --- p.42 / Chapter 2.6.8 --- Trypan blue exclusion test --- p.42 / Chapter Chapter 3 --- Results --- p.45 / Chapter 3.1 --- Characterization of prostanoid receptor expression in PC12 cells . --- p.45 / Chapter 3.1.1 --- Study of the presence of Gq-coupled prostanoid receptors --- p.45 / Chapter 3.1.2 --- Study of the presence of Gs-co»pled prostanoid receptors --- p.47 / Chapter 3.1.3 --- Study of the presence of Gi-coupled prostanoid receptors --- p.48 / Chapter 3.1.4 --- Further proof of EP3 expression in PC12 cells --- p.50 / Chapter 3.1.5 --- Discussion --- p.51 / Chapter 3.2 --- Time course effect of NGF on PC12 cells --- p.65 / Chapter 3.2.1 --- Effect of NGF on PGE2-mediated inhibition of forskolin-stimulated [3H]cAMP production --- p.65 / Chapter 3.2.2 --- Effect of NGF on basal and forskolin-stimulated [3H]cAMP production --- p.67 / Chapter 3.2.3 --- Acute effect of NGF on [3H]cAMP production --- p.70 / Chapter 3.2.4 --- Effect of NGF withdrawal on basal and forskolin-stimulated [3H]cAMP production --- p.71 / Chapter 3.2.5 --- Effect of NGF on adenylyl cyclase gene expression --- p.72 / Chapter 3.2.6 --- Discussion --- p.74 / Chapter 3.3 --- Quantification of the degree of differentiation of PC12 cells --- p.89 / Chapter 3.3.1 --- Expression of neurofilament protein as a marker of differentiation --- p.89 / Chapter 3.3.2 --- Neurite assays --- p.90 / Chapter 3.3.2.1 --- Manual assessment of PC12 cells --- p.90 / Chapter 3.3.2.2 --- Quantification of images of PC1 2 cells --- p.91 / Chapter 3.3.3 --- Discussion --- p.93 / Chapter 3.4 --- Adenosine A2a receptor activity in PC12 cells --- p.106 / Chapter 3.4.1 --- Effect of NGF on A2Areceptor-mediated [3H]cAMP production --- p.106 / Chapter 3.4.2 --- Synergistic activation of adenylyl cyclase by A2A receptor and forskolin --- p.108 / Chapter 3.4.3 --- Chronic and acute effect of ADA and ZM241385 on [3H]cAMP production --- p.109 / Chapter 3.4.3.1 --- Chronic effect of ADA and ZM241385 --- p.110 / Chapter 3.4.3.2 --- Acute effect of ADA and ZM241385 --- p.111 / Chapter 3.4.4 --- Discussion --- p.112 / Chapter Chapter 4 --- Discussion and future perspectives --- p.121 / Chapter 4.1 --- Discussion --- p.121 / Chapter 4.2 --- Future perspectives --- p.131 / References --- p.133

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-alpha4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

Der wichtigste exzitatorische Neurotransmitter Glutamat spielt eine grosse Rolle in der Gehirnentwicklung, wie neuronale Migration und Synaptogenese. Ob glutamaterge Stimulation für das Überleben entwickelnder Neuronen notwendig ist, war bislang jedoch unbekannt. Um zu untersuchen, ob eine Hemmung von Glutamatrezeptoren im unreifen Gehirn zu Neurodegeneration führt, wurden Ratten im Alter von 1 bis 31 Tagen für 24 Stunden mit dem N-Methyl-D-Aspartat-(NMDA) Glutamatrezeptorantagonisten Dizocilpin (MK801) behandelt. Die Dichte neuronaler Degeneration wurde mikroskopisch in Kupfer-Silber- und TUNEL- gefärbten Hirnschnittpräparaten ermittelt und Unterschiede mittels ANOVA analysiert (Signifikanzniveau p / The predominant excitatory neurotransmitter glutamate plays a major role in certain aspects of neural development. However, whether developing neurons depend on glutamate for survival remains unknown. To investigate if deprivation of glutamate stimulation in the immature mammalian brain causes neuronal cell death (apoptosis), rat pups aged 0 to 30 days were treated for 24 hours with dizocilpine maleate (MK801), an N-methyl-D-aspartate-(NMDA) glutamate receptor antagonist. Density of neural degeneration was evaluated by a stereological dissector method in cupric-silver and TUNEL-stained brain slices. Groups were compared by ANOVA and significance considered at p