Neurotransmitter release at central synapses is sustained by the synaptic vesicle cycle. It has been assumed that vesicle replenishment operates autonomously at individual presynaptic terminals. In this study the classical model of a compartmentalized synaptic vesicle cycle was tested by using a novel combination of FRAP (fluorescence recovery after photobleaching) and CLEM (correlative light and electron microscopy) in cultured hippocampal neurons. The stability of vesicle clusters labelled with fluorescent styryl dye at individual synapses were assessed by photobleaching using a confocal laser microscope and monitoring fluorescence recovery over time. The observed fluorescence recovery which was abolished by inhibitors of vesicular transport, suggested that synaptic vesicles recycled at sites outside the bleach region were transported along axons into bleached synapses. These newly-imported vesicles could undergo exocytosis upon stimulation, demonstrating that they formed part of the functional recycling pool. The spatial organization of imported vesicles in presynaptic boutons was examined using CLEM and FM dye photoconversion techniques. Imported vesicles were distributed throughout the native vesicle cluster, indicating that they become morphologically integrated into the synapse. The ability of imported vesicles to mix well with native vesicles highlights the dynamic nature of vesicle clusters at resting synapses. The departure of fluorescent packets from boutons into axons was observed by time- lapse microscopy. Ultrastructural analysis confirmed the mature state of donor synapses and showed these mobile packets to be loose aggregates of synaptic vesicles. Mobile vesicle clusters were comprised of vesicles from both the recycling and resting pools of the synapse, thus demonstrating no preference for mobility of any one vesicle population.

An HPLC method has been established for the determination of ubiquinone (C0Q9 and C0Q10) levels in biological samples. This necessitated the synthesis of a novel internal standard for C0Q9 and C0Q10 measurement in rodent tissue. Rat glial cell lines contained C0Q9 as the predominant ubiquinone isoform this was C0Q10 in human tissue. Comparison with primary cultures of rat astrocytes showed that these human and rat glial cancer cell lines had relatively less CoQ9+i0 than primary glial cultures, possibly reflecting a lower dependence of transformed cells upon OXPHOS for ATP generation. Lovastatin decreased C0Q9 (but not C0Q10) in primary astrocytes and glial cell lines. Moreover, glial cell lines displayed an approximately 10-fold higher sensitivity to lovastatin or its P-hydroxy acid isoform than primary rat astrocyte cultures. Cellular C0Q9 levels did not appear to be limiting for mitochondrial complex II+III activity, thus it is possible that C0Q10 is more intimately involved in OXPHOS than C0Q9. Primary cultures of rat astrocytes and neurons contained approximately equal levels of C0Q9. However C0Q10 levels were significantly higher in neuronal cultures. In contrast to transformed cell lines, the neuron's reliance on mitochondrial OXPHOS to synthesise ATP may manifest as higher cellular availability of C0Q10 Activation of iNOS in rat primary astrocytes to generate nitric oxide (NO) for 24h did not alter C0Q9 or C0Q10 levels. Following 36h exposure, activation of iNOS significantly decreased C0Q9 and C0Q10. An NO-donor decreased astrocyte C0Q9 and C0Q10 after 24h exposure, while in neurons, both CoQ isoforms were maintained. Additionally 36h exposure of astrocytes to DETA-NO appeared to cause a recovery in the amount of C0Q9 and C0Q10, possibly representing a protective effect in response to RNS exposure. Additionally, preliminary data demonstrated that small interfering RNA (siRNA) may decrease C0Q9 but not C0Q10 in rat primary astrocytes although not HEK293T cells.

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