published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

Not available / text

(Uncorrected OCR) ABSTRACT Abstract of thesis entitled 'Chondroitin sulphate proteoglycans produced by astrocytes modulate neuron adhesion and neurite outgrowth' submitted by HO Yeung for the degree of Master of Philosophy at the University of Hong Kong in 2000 Several types of proteoglycans were known to be associated with the glial boundary during CNS development and glial scar following CNS injury. The aim of this study is to investigate the role of astroglial proteoglycans, particularly chondroitin sulfate proteoglycans (CSPGs), in modulating neuron adhesion and neurite growth. Neonatal rat cerebral cortical astrocytes as well as an astrocyte cell line DI TNCI were studied for the expression of CSPGs and their effects on neuron adhesion and neurite outgrowth. Immunocytochemical staining with CS-56, a monoclonal antibody against epitopes in the CS moiety of CSPGs, demonstrated the localization of CSPGs on both primary astrocytes and DI TNCI cell line. Immunostaining with anti-neurocan (a neuronal CSPG originally thought) showed pericellular and intracellular distribution in confluent cultures of primary astrocytes but only intracellular distribution in the DI TNCI cells, both being positive for glial fibrillary acidic protein (GFAP). Neurocan was also found deposited by primary astrocytes on the culture substratum. Western blots of the conditioned medium of primary astrocytes indicated a distinct 220 kDa neurocan core protein band. Five immunoreactive bands were also found on Western blots detected with stub-sensitve antibody against C-6-S-PG. Furthermore, RT-PCR analysis indicated the expression of neurocan mRNA in the primary astrocytes. Cortical neural cells (from E18 rats) selectively attached and extended neurites on the primary astrocytes but not on the non-cellular, neurocan immuoreactive gaps left by the astrocytes nor on the DI TNCI cells. Neuronal distribution on astrocytes remained similar even after digestion with chondroitinase or neutralization with antibody, CS-56 or antineurocan. Neurite length on the chondroitinase ABC (20mU/ml) treated astrocytes was however increased. Taken together, these in vitro observations suggest that apart from neurons, astrocytes are another cellular source of brain-specific CSPG, neurocan. They also suggest that CS components expressed on the surface of astrocytes are supportive of neuronal attachment and survival but limiting towards neurite extension. Evidence is also provided for the selective development of embryonic neurons directly on astrocytes and not on neurocan immunoreactive non-cellular routes traversed by astrocytes. 11 / abstract / toc / Biochemistry / Master / Master of Philosophy

No description available.

The formation of patterns of functionally appropriate chemical synapses is one of the key aspects of nervous system development. I have investigated the cellular interactions that culminate in the formation of an inhibitory synapse between the R and P neurons of the leech. These neurons may be isolated and maintained in culture, where they reform synaptic connections under easily manipulable conditions. An early event in the formation of this synapse is the loss from sites of contact in the postsynaptic P cell of an excitatory response to the transmitter serotonin. The loss of this response was triggered specifically by contact with the presynaptic R neuron, and not by contact with other leech neurons, including other serotonergic neurons. Furthermore, contact with the R neurons of the reproductive ganglia, which do not innervate P cells, was also ineffective in causing the loss of response. This highly specific cellular interaction was prevented by treatment of the R cell surface with the proteolytic enzyme trypsin or with the lectin wheat germ agglutinin (WGA), suggesting involvement of an R cell surface glycoprotein. WGA blocked not only the loss of the excitatory response, but also prevented the formation of the R-P synapse. An antibody library against the R cell generated using a novel phage-display system produced antibodies which bound to subsets of leech neurons, including the R neurons, but none of these was specific for the R cells. I conclude that an early event in the formation of the R-P synapse is the recognition by the P cell of its correct synaptic partner through an R cell-specific surface molecule.

I explored the relationship between neurite outgrowth and the onset of synaptic activity in the central neuropil of the leech embryo. In order to follow changes in early morphology and the onset of synaptic activity in the same identified neuron, whole cell patch clamp recordings and fluorescent dye fills were obtained from dorsal pressure-sensitive (P) cells, the first neurons that could be reliably identified in the early embryo. I followed the development of the P cell from the first extension of neurites to the elaboration of an adult-like arbour. Following the growth of primary neurites, we observed a profuse outgrowth of transient neurites within the neuropil that retracted to form spurs. Following a dormant period, stable secondary branches grew from the spurs and became tipped with terminals. At this time, neurites of the Retzius (R) cell, a known presynaptic partner in the adult, were observed to contact the terminals. Although voltage-dependent currents were seen in the P cell at the earliest stage, spontaneous synaptic activity was only observed when terminals had formed. Spontaneous release was observed before evoked release could be detected from the R cell. The results suggest that transient neurites are formed during an exploratory phase of development, whereas the more precisely timed outgrowth of stable neurites from the spurs signals functional differentiation during synaptogenesis. As spurs have also been observed in neurons of the mammalian brain, they may constitute a primordial synaptic organizer.

No description available.

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.