The midline glia (MG) perform an active role in the pioneering and morphogenesis ofthe commissural axons of the central nervous system (CNS) during Drosophila embryogenesis. Following the establishment of these commissural axon pathways, a subset of the MG undergoes apoptosis and the surviving MG remain to ensheath the commissures. Previous studies demonstrated that the pattern ofthis developmental apoptosis is stochastic. The surviving MG exhibit intersegment variability in their final number and position relative to the commissures. Interestingly, argos, a gene which encodes a diffusible extracellular factor with an epidermal growth factor (EGF) motif, is only expressed in MG which survive. argos expression in this subset of MG is initiated at the onset of apoptosis, reflecting the temporal pattern of cell death of the other MG. In argos loss-of-function mutants, there are extra surviving MG in each segment while ectopic over-expression of argos results in increased apoptosis among the MG. Therefore, Argos has a negative regulatory effect on MG survival. Its effects are opposite to the spitz group/Drosophila EGF receptor (DER) pathway, a cassette of genes required for MG survival. However, argos is hypostatic to the spitz group/DER pathway function and its expression requires a certain threshold of spitz group and DER pathway activity. Argos is postulated to act as an EGF receptor antagonist. It attenuates signaling of the DER pathway. Therefore, the regulation of MG survival is mediated by a balance of extracellular inductive and inhibitory signals. How can this signaling pathway be reconciled with the stochastic pattern of MG survival and the observation that only argos expressing MG survive? We propose a model in which the adherens junction along with possible accessory proteins like Rhomboid and Star mediate the close apposition of MG and promote intense Spitz mediated DER pathway signaling. The MG with the most concentrated coupling of this adhesion and signaling pathway network achieve sufficient DER signaling to express argos. These MG are in tum fated to assume a final differentiated identity and will remain to ensheathe the commissures. Argos is secreted and it mediates the apoptosis of MG with reduced levels of adhesion contacts and lower DER signaling (MG not expressing argos). The other mesectodermal cell (MEC) lineages appear to develop independently of argos function. The repercussions of HSargos elicited MG loss to the commissural axons and CNS cytoarchitecture was also examined. Removal of the MG through ectopic expression ofargos results in a loss of ensheathment and the commissural axon tracts are exposed to the haemolymph. Furthermore, the commissural axons are wider and they misexpress Fasciclin II, a phenotype reminiscent of mutations in roundabout. / Thesis / Master of Science (MSc)

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A protocol for culturing mammalian type 1 astrocytic cells, using female post-natal rat cerebral cortical tissue, was established and refined for use in steroidogenic metabolic studies incorporating progestin radioisotopes. Cultures were characterised for homogeneity using standard morphological and immunostaining techniques. Qualitative and quantitative studies were conducted to characterise the progesterone (P) metabolic pathways present in astrocytes in vitro. Of particular interest was the formation of the P metabolite, 5á-pregnan-3á-ol-20-one (THP). THP is a GABA(A) receptor agonist, believed to play a vital role in neural functioning and CNS homeostasis. One aim of this study was to observe any modulatory effects selected neuroactive ligands have on the conversion of P into THP, in an attempt to link astrocytic steroidogenesis with neuronal control. In qualitative studies, chromatographic procedures were used to establish the progestin profile of cerebral cortical astrocytes. Tritiated P, DHP (5á-pregnan-3,20-dione) and THP incurbates were preliminary fractionated by either normal phase (NP) or reverse phase (RP) high performance liquid chromatography (HPLC). The radiometabolites associated with each fraction were further chromatographed, before and/or after chemical derivatistation, by the aforemention HPLC procedures and thin layer chromatography (TLC). Steroid radiometabolites were tentatively identified by comparing their chromatographic mobility with authentic steroids. The identity of the main putative 5á-reduced P metabolities, DHP, THP and 5á-pregnan-3á,20á-diol (20áOH-THP) were further confirmed by isotopic dilution analysis. Their conclusive identification, along with the tentative identification of 20á-hydroxypreg-4-en-3-one (20áOH-P) and 20á-hydroxy-5á-pregnan-3-one (20áOH-DHP), verify the localisation of 5á-reductase, 3á-hydroxy steroif oxidoreductase (HSOR), and 20á-HSOR activity in the cultured astrocytes utilised in this study programme. Other minor metabolites detected were tentatively identified, including 5á-pregnan-3á,21-diol-20-one (THDoc), indicating the presence of 21-hydroxylase enzymatic activity. THDoc, like THP, is a GABA(A) receptor agonist. The chemical and physical characterisation of several yet unidentified progestin metabolites, associated with a highly polar RP HPLC fraction (designated RP peak 1*), indicate the presence of one or more extra hydroxylase enzymes. Quantitative analysis included a preliminary study. In this study, the percentage yields of radiometabolites formed in cultures incubated with increasing substrate concentrations of (3)H-P for 24 hours were determined. At the lower concentrations examined (ie 0.5 to 50nM), the metabolites associated with the polar RP HPLC fraction (RP peak 1*) collectively have the highest percentage yield. They are subsequently considered metabolic end products of degradative catabolic P pathways. The percentage yield of THP peaks in the medium concentration ranges (ie 5 to 500nM), whereas DHP remains fairly static at a low level with increasing concentration. Both DHP and THP are considered metabolic pathway intermediates. The percentage yield of 20áOH-THP continues to increase with increasing concentration over 5nM, superseding THP approaching the highest concentration examined (5000nM). This indicated the formation of 20áOH-THP does not occur entirely via THP. 20áOH-THP also possibly serves as the direct intermediate in the formation of the main radiometabolites associated with RP peak 1*. A time/yield study incorporating incubation times from one to 24 hours was also conducted. The full array of radiometabolites (individually or in groups) formed in astrocyte cultures incubated with 50nM tritiated P, DHP of THP, were assayed. Cultures were observed to rapidly convert any DHP into THP, showing astrocytic 3á-HSOR activity is very high. The study also showed 5á-reduction (ie the conversation of P into DHP) is the rate limiting reaction in the two step conversion of P into THP. 5á-Reduction also appears to be a rate limiting step in the formation of 20á-hydroxylated metabolites in astrocytes. Cultures incubated with the tritiated 5á-reduced pregnanes from one to four hours form greater quantities to 20á-hydroxylated radiometabolites compared to cultures incubated with (3)H-P. The time yield/studies also provided further evidence the unidentified polar radiometabolites associated with RP peak 1* are metabolic end products. For the P and DHP incubates, the collective formation of the aforementioned polar radiometabolites initially lags behind the formation of THP. As the formation of the latter begins to plateau with increasing time between four to 24 hours, the net yield of radiometabolites associated with RP peak 1* continues to rise. The time/yield studies also indicate 5á-reduction and perhaps 3á-hydroxylation are pre-requisite steps in the formation of the polar metabolites. Cultures incubated with the 5á-reduced progestins from one to four hours form higher yields of the radiometabolites associated with RP peak 1* compared to cultures incubated with P as substrate. The net yields of the radiometabolites associated with RP peak 1* for cultures incubated with THP were substantially higher compared to cultures incubated with DHP after equivalent times. The effect selected neuroligands have on the yield of radiometabolites formed by cultured astrocytes incubated with 50nM (3)H-P was also examined. Dibutyryl cyclic adenosine monophosphate (DBcAMP), not actually a neuroligand per se, but an analog of the intracellular secondary messenger cAMP, was also utilised in these studies. The inhibitory neurotransmitter ã-amino-nbutyric acid (GABA), DBcAMP and isoproterenol (a â-adrenergic receptor agonist) all quickly induce a transient but substantial increase in 20á-HSOR activity in cultured astrocytes. Cultures pretreated with these three compounds (10, 20 and 1µM respectively) form substantially higher yields of 20á-hydroxylated metabolites, including 20áOH-THP (between 200 to 580% greater), when incubated with 50nM (3)H-P for one to four hours. These increases also coincide with increases in the net yield of metabolites formed (by 16 to 48%). The same pre-treated cultures form significantly lower yields of THP, by 25 to 41%, after one hour. This is most likely due to the increased metabolism of any formed THP into 20áOH-THP. Octopamine (an á-adrenergic agonist) only induces a slight increase in 20á-HSOR activity, having relatively little effect on the yield of 20áOH-THP formed. Pretreatment with octopamine induces a significant increase in the yield of THP for cultures incubated with (3)H-P for four hours (by 24%). The increase in THP formation appears to be due to an increase in 3á-HSOR activity, as judged by the concomitant drop in the yield of the 5á-reduced, 3-keto substrates. An increase in 5á-reductase activity cannot be excluded however. Isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol one and four hour incubates have higher yields of DHP. This is in contrast to the other three incubates. After 12 hours, all incubates have higher yields of THP (15-30%).

Over the recent years, olfactory ensheathing cells (OECs) have gained world-wide attention due to their reputed potential in promoting spinal cord regeneration and repair. In order to isolate, identify, and characterize OECs in vitro and following implantation, researchers have used three OEC markers: p75NTR, GFAP, and S100. The downfall with using these specific proteins is that Schwann cells, which are located within the olfactory system, as well as migrate into the damaged spinal cord, also express these proteins. It is therefore impossible to distinguish OECs from phenotypically similar Schwann cells using these molecular markers. Recently proteomic analyses have revealed that OECs (derived from embryonic rat olfactory bulbs), but not Schwann cells (derived from adult rat sciatic nerves) express a variety of proteins. The main aim of this project is to determine if heat shock protein-27 (Hsp27), carbonic anhydrase-III (CA-III), and annexin-A3 (Anx3) markers label OECs but not Schwann cells, both in vivo and in vitro. Additional analyses were also done to determine if smooth muscle α-actin (SMA) and calponin (two smooth muscle-related markers previously shown to label mucosal OECs of adult rats) label bulbar OECs of adult rats and OECs of adult cats. Using immunohistochemistry we found that SMA labeled olfactory mucosal and bulbar OECs of adult rats and adult cats, Hsp27 labeled olfactory mucosal and bulbar OECs of adult rats and olfactory mucosal OECs of adult cats, while calponin labeled only olfactory mucosal OECs of adult rats. In addition, calponin and SMA did not label Schwann cells (in vivo and in vitro), while Hsp27 labeled this peripheral glial cell. Finally, CA-III did not label OECs of adult rats or adult cats, in vivo or in vitro, and Anx3 did not label OECs in vivo, but showed immunopositive labeling of OECs and Schwann cells in vitro. In conclusion, Hsp27, CA-III, and Anx3 cannot be used as OECs markers either because of their expression in both OECs and Schwann cells or their lack of expression in OECs. Discovering new molecular markers expressed only by OECs is essential in order to determine the properties, fate, and overall potential of OECs in promoting spinal cord regeneration. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-29 09:50:09.869

In the peripheral nervous system the myelin sheath is produced by the spiral wrappings of the Schwann cell (SC) membrane around the axon. This provides insulation and increases the velocity of impulse propagation. The structure of myelin is maintained by a group of myelin proteins. Peripheral myelin protein-22 (PMP-22) is a 22 KDa glycoprotein, originally identified following nerve crush injury, that is found within SCs and is identical to the growth arrest specific protein GAS-3. The PMP-22 gene is regulated by two alternative promoters immediately upstream of two alternative non-coding exons. In order to study temporal and spatial expression of the PMP-22 gene and regulation of SC ensheathment and myelination, a transgenic mouse expressing the green fluorescent protein (GFP) driven by the myelin specific PMP-22 promoter was produced. To achieve this the PI promoter isolated from genomic DNA was initially incorporated into a plasmid containing the EGFP gene. In vitro transfection studies demonstrated appropriate expression of EGFP fluorescence. Microinjection of the transgene into pre- implantation fertilised embryos gave rise to three transgenic lines as confirmed by Southern blot and PGR. One founder expressed the transgene in a tissue specific manner. Mosaicism of expression both within an individual and between individuals was noted. In vitro manipulations showed that the expression patterns observed were independent of axonal contact and myelination but could be influenced by the extracellular matrix. These GFP expressing transgenic mice potentially provide a means to determine the dynamics of SC-axon interactions during myelination and the behaviour of transplanted SCs into myelin deficient regions and the SCs response to injury. Preliminary reports of this work are found in abstract form: British Neurosci. Assoc. Abstr., Vol 15, pi04, 1999.

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