In 1985, Mendelson and Gorzalka proposed the dual role hypothesis of serotonergic modulation of lordosis behaviour. In this hypothesis it was proposed that serotonergic activity can either inhibit or facilitate lordosis behaviour. Specifically it was suggested that the lordosis-inhibiting effects of serotonin are mediated by activity at 5-HT₁ receptors, whereas lordosis-facilitating effects of serotonin are mediated by activity at 5-HT₂ receptors. The purpose of the following series of studies was both to confirm and to extend the dual role hypothesis. The intraperitoneal administration of the 5-HT2 antagonists pizotefin (1 mg/kg), cyproheptadine (1 mg/kg), metitepine (1 mg/kg), and ketanserin (1 mg/kg) were found to inhibit lordosis behavior in ovariectomized rats that had been primed with estradiol benzoate (EB) and progesterone (P). Pipamperone was ineffective. The 5-HT₂ agonist guipazine (3 mg/kg) was ineffective alone, but it reversed the inhibitory effects of pizotefin, cyproheptadine, and ketanserin. It did not reverse the effects of metitepine. The highly selective 5-HT₂ antagonist LY53857 (0.3 mg/kg) was also found to inhibit lordosis behaviour in female rats that had been primed with EB and P. The lordosis-inhibiting effect of LY53857 (1 mg/kg) in females primed with EB and P was reversed by quipazine (3 mg/kg). The nonselective 5-HT antagonist methysergide (7 mg/kg) was found to inhibit lordosis behavior 30 min after intraperitoneal administration to females treated chronically with EB, or with EB and P. However, methysergide was found to facilitate lordosis behavior 200 and 300 min after administration to female rats treated acutely with EB. In an analysis of dose response it was found that methysergide (0.02 - 7 mg/kg) administered 30 min prior to behavioural testing produced no facilitation of lordosis in females primed with EB. However, when administered 200 min prior to testing, methysergide (1 mg/kg) produced a significant facilitation of lordosis. The administration of the 5-HT₁ A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) inhibited lordosis behavior in ovariectomized rats primed with EB. 8-OH DPAT was ineffective at 0.01 mg/kg, whereas inhibition occurred at the 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg doses. In an evaluation of the effects of 8-OH DPAT on the expression of male sexual behaviour by females treated chronically with testosterone, 8-OH DPAT ( 1 mg/kg) increased the number of females mounting and significantly increased mount frequency. The 5-HT₁ A agonists ipsapirone (0.1 mg/kg) and gepirone (0.3 mg/kg) facilitated lordosis in females treated with EB. When administered at higher doses, ipsapirone (3.0 mg/kg) and buspirone (3.0 mg/kg) inhibited lordosis in rats treated with EB. In females treated with EB and P, ipsapirone (> 1.0 mg/kg), gepirone (> 0.3), and buspirone (> 0.3) inhibited lordosis behaviour. The newly developed 5-HT₁ A antagonist BMY 7378 (0.2 mg/kg) facilitated lordosis behaviour in females treated with EB. However, this facilitation was no longer apparent at the 5 mg/kg dose. BMY 7378 (0.04 - 5 mg/kg) was ineffective in females primed with EB and P. The 5-HTTB agonist 1 -(3-trifluoromethylphenyl)piperazine (TFMPP, 0.2 -5 mg/kg) was found to facilitate lordosis in females treated with EB. In females primed with EB and P, TFMPP (5 mg/kg) produced a significant inhibition of lordosis. The 5-HT₁ B agonist m-chlorophenylpiperazine (MCPP, 0.04 - 5 mg/kg) was ineffective in females primed either with EB or with EB and P. The 5-HT₃ Antagonist ICS 205-930 (5 mg/kg) was found to facilitate lordosis behaviour, whereas the 5-HT₃ Antagonist MDL 72222 (0.05 - 5 mg/kg ) was found to be ineffective in females primed with EB. The results of these studies tend to confirm that serotonergic activity can either inhibit or facilitate lordosis behaviour. It is suggested that the lordosis-inhibiting effects of serotonin are mediated by activity at postsynaptic 5-HTTA and possibly 5-HT₃ Receptors. The lordosis-facilitating effects of serotonin are mediated by activity at 5-HT₂ and possibly presynaptic 5-HT₁ B receptors. Finally, it is suggested that activity at somato-dendritic 5-HT₁ A autoreceptors may mediate facilitatory effects of low doses of 5-HT₁ A agonists. In closing, there is a discussion of the implications these results might hold for the understanding of the effects of serotonergic drugs on human behaviour. / Arts, Faculty of / Psychology, Department of / Graduate

A wealth of data supports the hypothesis that the neurotransmitter serotonin regulates the intake of ethanol, and is involved in the development of alcoholism in humans. Reduced functioning of the serotonergic system hypothetically increases alcohol intake in both animals and humans. In this thesis, it was proposed that the effect of lowered serotonergic function on alcohol intake is mediated by an increase in disinhibition. The hypothesis that lowered serotonin increases disinhibition was tested in separate groups of individuals at high risk for the development of psychopathology: nonalcoholic young men with a strong family history of paternal alcoholism, and adolescent men with previous histories of physically aggressive behavior. Lowered serotonergic synthesis (and thus presumably function) was experimentally induced through a transient dietary reduction in the availability of the amino add precursor of serotonin, tryptophan. Disinhibition was quantified using a go/no-go task previously shown to characterize psychopaths and children with attention deficit hyperactivity disorder as disinhibited. In the first study, acute tryptophan depletion had no effect on aggressive responding on a modified competitive reaction time aggression task, but increased disinhibition in young men at risk for alcoholism. This effect was independent of the tryptophan depletion-induced mood alterations. The effect tryptophan depletion on disinhibition was not replicated in the second study with previously aggressive adolescent men. A number of explanations for this were posited, including the presence of a ceiling effect. An association between disinhibition and executive functioning (cognitive abilities associated with proper functioning of the prefrontal cortex, such as working memory, planning abilities) was demonstrated in the second study. In a third preliminary study, no association between disinhibition on the go/no-go task and allelic polymorphisms of the dopamine D4 receptor

No description available.

Rats are nocturnal animals, their ingestive behaviours show circadian rhythms and the suprachiasmatic nuclei (SCN) of the hypothalamus are the primary pacemaker of these rhythms. Serotonin (5-HT) is one of the most abundant neurotransmitter in the SCN and it is involved in the regulation of ingestive behaviour. In this study, we compared food and water intakes of male adult Wistar rats infused during 7 days with serotonin (2.5 nmol/24h) or artificial cerebrospinal fluid (CSF) in the SCN in a three-way selection of macronutrients. Within 5-HT infusion, nocturnal and diurnal water intakes, and the nocturnal caloric intake from the protein diet were significantly lower than those of the CSF infused group. Decrease in water consumption was most significant in the early and middle dark phases. Besides, increased diurnal energy intake and decreased nocturnal energy intake were observed during 5-HT and CSF infusion. In conclusion, this first chronic and continuous infusion work on 5-HT in the SCN specifically disrupted the circadian rhythmicities in water and protein consumptions.

No description available.

Previous studies have focused on gastric dysmotility and delayed emptying in diabetes mellitus. There is little information about the effects of hyperglycemia on colonic motility and secretion. 5-HT was reported to mediate contractile activity by activating receptors on both enteric neurons and smooth muscle cells. The aim of this study was to investigate and compare the effects of 5-HT on circular contractile activity coordinated with secretion in streptozocin-induced diabetic and non-diabetic rats. Sonomicrometry and voltage clamping techniques were used to measure motility and secretion simultaneously in isolated whole thickness colonic sheets. Male Sprague Dawley rats were injected with streptozocin (65 mg/kg body weight) in 0.1 M sodium citrate buffer, into the tail veins. Glucose levels of 300-400 mg/dl and above were achieved. The control rats were injected with the same volumes of vehicle (0.1 M sodium citrate buffer). Animals were sacrificed 10-12 days following the induction of hyperglycemia. Flat sheets of colon were mounted serosal side up in Ussing chambers. 1 mm piezocrystals were glued to the serosal surface 4-5 mm apart to measure circular contractions as decrease in inter-crystal distances (ICD). Voltage-clamping the tissues at 0 mV was used ix to measure short circuit current (Isc), indicative of chloride secretion. In diabetic rats 50 gM 5-HT caused mean amplitude of contractions of 174 ± 26 gm (n=4), which was significantly reduced as compared to the response in non-diabetic rats of 970 + 243 gm (n=4; p<0.05). The secretory response in diabetic rats paralleled the reduction in ICD (diabetic: 23 +1 gA/cm2, controls: 57 + 18 gA/cm2). Neural blockade with 0.1 gM TTX revealed a decreased myogenic contractile activity in diabetic rats. The mean amplitude of contractions after TTX in diabetic rats was 162 ± 45 gm verses controls of 612 ± 86 gm. These results suggest that the reduction of the 5-HT contractile response in diabetic rats may be a composite of direct effects on the smooth muscle as well as indirect effects on the neurons. / Department of Physiology and Health Science

Serotonin syndrome (SS) is a drug-induced toxicity caused by an excess of serotonin (5-HT) in the central nervous system (CNS). The symptoms of the disorder range from mild to severe, with the severe state evoking life-threatening hyperthermia. Autonomic dysfunction is controlled in part by serotonin receptors, with the 5-HT2A receptor responsible for increasing core body temperature (Tcor). Our results show that the 5-HT2A receptors on the preoptic/anterior hypothalamus (PO/AH) and prefrontal cortex (PFC), in particular, are sensitive to changes in ambient temperature (Tamb). The toxic increase of 5-HT is postulated to occur due to the temperature-dependent activation of these receptors that promotes a positive feedback mechanism. Our results suggest that changes in Tamb can either exacerbate or alleviate the symptom and that this is mediated by the 5-HT2A receptors. Understanding the mechanism involved in elevating Tcor is imperative in treating and preventing the disorder. / by Swapna Krishnamoorthy. / Vita. / Thesis (M.S.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

In this study, I investigate how serotonergic modulation of the medial prefrontal cortex (mPFC) affects neuronal activity and impacts cognitive flexibility, anxiety, and fear extinction (Figure 1). I begin in Chapter 1 with general information on the PFC with a focus on the mPFC, then discuss the role and complexity of serotonin and how manipulation of serotonin affects behavior. I, finally, introduce what is understood about how serotonin modulates the mPFC, the significance of which has implications for cognitive and emotional behaviors. In Chapter 2, I studied the role of serotonin in cognitive flexibility. Specifically, I used retrograde tracing to determine the origin of mPFC and assessed how terminal release of 5-HT affects mPFC pyramidal neuron activity using whole-cell electrophysiology in acute brain slices. Furthermore, through in vivo fiber photometry, I evaluated the activity of 5-HTergic neurons projecting to the mPFC during cognitive flexibility behavior. Lastly, by selectively increasing or decreasing mPFC 5-HTergic terminal release through in vivo optogenetics, I assessed the modulatory role of 5-HTergic input into the mPFC on intradimensional rule reversal and extradimensional rule shift performance in the cognitive flexibility task. Furthermore, I evaluated the activity of 5-HTergic neurons projecting to the mPFC during an open field task using in vivo fiber photometry and, in Chapter 3, used in vivo optogenetics to determine the role 5-HT in the mPFC plays in modulating fear-related behavior. In Chapter 4, I examined a pharmacological screen of a psychedelic drug in the cognitive flexibility task outlined in Chapter 2. I conclude in Chapter 5 with a discussion of the study implications and future directions.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke. / Li, Jin. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts in English. / ACKNOWLEDGEMENTS --- p.i / LIST OF PUBLICATIONS --- p.ii / ABSTRACT --- p.iii / ABSTRACT [in Chinese] --- p.v / TABLE OF CONTENT --- p.vi / LISTS OF FLOWCHARTS --- p.xii / LISTS OF FIGURES --- p.xiii / LIST OF TABLES --- p.xvi / LIST OF EQUIPMENTS --- p.xvii / LIST OF ABBREVATIONS --- p.xvii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- Central nervous system disorder --- p.1 / Chapter 1.1.1 --- Stroke --- p.1 / Chapter 1.1.2 --- Spinal cord injuries --- p.4 / Chapter 1.1.3 --- Parkinson's disease --- p.6 / Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8 / Chapter 1.2 --- Stem cell therapy --- p.10 / Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11 / Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11 / Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15 / Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16 / Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18 / Chapter 1.3 --- Stem cells --- p.20 / Chapter 1.3.1 --- Embryonic stem cells --- p.21 / Chapter 1.3.1.1 --- Derivation and characterization --- p.21 / Chapter 1.3.1.2 --- Biology of ES cells --- p.21 / Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21 / Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24 / Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25 / Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27 / Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28 / Chapter 1.3.2 --- Neural stem cells --- p.30 / Chapter 1.3.2.1 --- Derivation and characterization --- p.30 / Chapter 1.3.2.2 --- Biology of NS cells --- p.32 / Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34 / Chapter 1.3.4 --- Mesenchymal stem cells --- p.35 / Chapter 1.4 --- Serotonin (5-HT) --- p.36 / Chapter 1.4.1 --- Distribution --- p.37 / Chapter 1.4.2 --- Metabolism --- p.37 / Chapter 1.4.3 --- Biological effects of 5-HT --- p.38 / Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40 / Chapter Chapter2 --- Aim --- p.43 / Chapter 2.1 --- Hypothesis and study objectives --- p.43 / Chapter Chapter3 --- Materials and Methods --- p.49 / Chapter 3.1 --- Chemicals and Reagents --- p.49 / Chapter 3.1.1 --- Cell culture --- p.49 / Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51 / Chapter 3.1.3 --- Cell proliferation assay --- p.52 / Chapter 3.1.4 --- Cell apoptosis assay --- p.52 / Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52 / Chapter 3.1.6 --- Western blotting --- p.55 / Chapter 3.1.7 --- Molecular biology --- p.56 / Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58 / Chapter 3.1.9 --- MAO activity assay --- p.58 / Chapter 3.1.10 --- Endogenous ROS production assay --- p.58 / Chapter 3.2 --- Consumable --- p.58 / Chapter 3.3 --- Cells --- p.60 / Chapter 3.3.1 --- Feeder cell --- p.60 / Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60 / Chapter 3.3.2 --- ES cells --- p.61 / Chapter 3.3.2.1 --- ES cell D3 --- p.61 / Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61 / Chapter 3.3.3 --- NS cells --- p.61 / Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61 / Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61 / Chapter 3.4 --- In-house prepared solutions --- p.62 / Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63 / Chapter 3.4.2 --- Gelatin solution 01% --- p.62 / Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62 / Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63 / Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63 / Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63 / Chapter 3.4.7 --- Laminin solution --- p.64 / Chapter 3.4.7 --- MEF Maintenance medium --- p.64 / Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64 / Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65 / Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65 / Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65 / Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66 / Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66 / Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67 / Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67 / Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67 / Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68 / Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68 / Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68 / Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69 / Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69 / Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69 / Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70 / Chapter 3.4.22 --- RIPA --- p.70 / Chapter 3.4.23 --- Resolving buffer (8X) --- p.70 / Chapter 3.4.24 --- Stacking buffer (4X) --- p.71 / Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71 / Chapter 3.4.26 --- Transfer buffer (10X) --- p.72 / Chapter 3.4.27 --- Transfer buffer (IX) --- p.72 / Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72 / Chapter 3.4.29 --- TBS (10X) --- p.73 / Chapter 3.4.30 --- TBS-T (IX) --- p.73 / Chapter 3.4.31 --- Stacking gel --- p.73 / Chapter 3.4.32 --- Resolving gel --- p.74 / Chapter 3.5 --- Methods --- p.75 / Chapter 3.5.1 --- Cell culture --- p.75 / Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75 / Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75 / Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76 / Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76 / Chapter 3.5.1.5 --- Passage of culture --- p.77 / Chapter 3.5.1.5 --- 6 Cell count --- p.78 / Chapter 3.5.1.7 --- Cytospin --- p.78 / Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78 / Chapter 3.5.1.9 --- Cryopreservation --- p.79 / Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79 / Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81 / Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83 / Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84 / Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85 / Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85 / Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86 / Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87 / Chapter 3.5.2 --- Cell proliferation assay --- p.87 / Chapter 3.5.2.1 --- Cell morphology --- p.87 / Chapter 3.5.2.2 --- WST-1 assay --- p.88 / Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88 / Chapter 3.5.2.4 --- NCFC assay --- p.89 / Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89 / Chapter 3.5.3.1 --- RNA extraction --- p.89 / Chapter 3.5.3.2 --- RNA quantitation --- p.90 / Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90 / Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91 / Chapter 3.5.3.5 --- RNA Integrity Check --- p.91 / Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91 / Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92 / Chapter 3.5.4 --- Microarray --- p.94 / Chapter 3.5.5 --- Immunofluoresent staining --- p.94 / Chapter 3.5.6 --- Western blot --- p.95 / Chapter 3.5.6.1 --- Harvesting samples --- p.95 / Chapter 3.5.6.2 --- Protein extraction --- p.96 / Chapter 3.5.6.3 --- Protein quantification --- p.96 / Chapter 3.5.6.4 --- SDS-PAGE --- p.97 / Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97 / Chapter 3.5.6.6 --- Blocking the membrane --- p.97 / Chapter 3.5.6.7 --- Immunoblotting --- p.97 / Chapter 3.5.6.8 --- Signal detection --- p.98 / Chapter 3.5.7 --- Cell apoptosis assay --- p.98 / Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98 / Chapter 3.5.7.2 --- TUNEL --- p.99 / Chapter 3.5.8 --- Endogenous ROS assay --- p.100 / Chapter 3.5.9 --- In vivo studies --- p.101 / Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101 / Chapter 3.5.9.2 --- Transplantation --- p.101 / Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102 / Chapter 3.5.10 --- Histological analysis --- p.103 / Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103 / Chapter 3.5.10.2 --- Cryosectioning --- p.103 / Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104 / Chapter 3.6 --- Data analysis --- p.104 / Chapter Chapter4 --- Results --- p.113 / Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113 / Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113 / Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115 / Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115 / Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115 / Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117 / Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119 / Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119 / Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119 / Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120 / Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121 / Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122 / Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122 / Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123 / Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123 / Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127 / Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130 / Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130 / Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130 / Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131 / Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132 / Chapter 4.7.2 --- Cell labelling --- p.132 / Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133 / Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135 / Chapter Chapter5 --- Discussion --- p.180 / Chapter Chapter6 --- Conclusions --- p.192 / References --- p.195

M.A. / Irritable bowel syndrome [IBS] has been described as a chronic relapsing condition, characterised by a change in bowel habit and abdominal pain, that cannot be explained by an organic disease. Some research indicates that IBS may be psychogenic in origin, however, the aetiology of this complex syndrome is still unclear. Some researchers have postulated that IBS is primarily a motility disorder of the gut, while others have indicated that the symptoms of IBS are mediated by the central nervous system. Thus it is not surprising that the care of patients with IBS poses a particular challenge to physicians, especially because of its biologic and symptomatic heterogeneity and, particularly for patients with refractory symptoms, its association with psychological disturbances. The literature study indicates that there that there may be a possible connection between the ingestive disorders, the menstrual cycle fluctuations associated with premenstrual dysphoric disorder and IBS. All three of these disorders also appear to be mediated, to some extent, by the neurotransmitter serotonin. In terms of these suggested correlations one of the aims of this study was to determine whether blood-serotonin levels significantly influence the symptomatology of IBS. Extensive literature exists documenting the potential role that serotonin plays in gastrointestinal functioning. However, none of the existing studies refer specifically to blood-serotonin levels. Thus the present study attempted to address this problem. A second aim of the present study was to determine the possible serotonergic connection in the ingestive disorders and premenstrual dysphoric disorder in women with IBS. All the subjects were required to go for a blood test in order to determine whether their serotonin levels were low, normal, or high. In addition, three self-report questionnaires were used in this investigation. The Irritable Bowel Syndrome Client Questionnaire; The Eating Disorder Inventory -2, of which four subscales out of 11 subscales were included, namely the Drive for Thinness, Bulimia, Body Dissatisfaction and Introceptive Awareness subscales; and the Premenstrual Assessment Form, of which six subscales out of 18 were included, namely Endogenous Depressive Features, Atypical Depressive Features, Signs of water Retention, General Physical Discomfort, Autonomic Physical Changes and Miscellaneous Physical Changes. In order to address the above mentioned aims, research was conducted at the Research and Counselling Centre for Psychogastroenterology at the Rand Afrikaans University. The Research and Counselling Centre for Psychogastroenterology is a facility developed to investigate the psychological constituents of IBS. Researchers at the centre are aiming to explore the multidimensional components of IBS with the purpose of gaining some understanding into the development and maintenance of this syndrome. A variety of topics are being investigated at the Research and Counselling Centre for Psychogastroenterology, including the role that stress, depression and coping styles play in IBS. Initially a sample group of (N = 60) women with IBS were selected for this research from a population of South Africans who were referred from gastroenterologists and general practitioners to the Centre for Gastroenterology at the Rand Afrikaans University. A number of women (N = 40) without IBS, from the north eastern suburbs of Johannesburg, were also asked to participate in this study in order to compile the comparison group.