Synaptophysin sustains presynaptic performance by preserving vesicular synaptobrevin-II levels

The two most abundant molecules on synaptic vesicles (SVs) are synaptophysin and synaptobrevin-II (sybII). SybII is essential for SV fusion, whereas synaptophysin is proposed to control the trafficking of sybII after SV fusion and its retrieval during endocytosis. Despite controlling key aspects of sybII packaging into SVs, the absence of synaptophysin results in negligible effects on neurotransmission. We hypothesised that this apparent absence of effect may be because of the abundance of sybII on SVs, with the impact of inefficient sybII retrieval only revealed during periods of repeated SV turnover. To test this hypothesis, we subjected primary cultures of synaptophysin knockout neurons to repeated trains of neuronal activity, while monitoring SV fusion events and levels of vesicular sybII. We identified a significant decrease in both the number of SV fusion events (monitored using the genetically encoded reporter vesicular glutamate transporter-pHluorin) and vesicular sybII levels (via both immunofluorescence and Western blotting) using this protocol. This revealed that synaptophysin is essential to sustain both parameters during periods of repetitive SV turnover. This was confirmed by the rescue of presynaptic performance by the expression of exogenous synaptophysin. Importantly, the expression of exogenous sybII also fully restored SV fusion events in synaptophysin knockout neurons. The ability of additional copies of sybII to fully rescue presynaptic performance in these knockout neurons suggests that the principal role of synaptophysin is to mediate the efficient retrieval of sybII to sustain neurotransmitter release

Optimization of Nafion Polymer Electrolyte Membrane Design and Microfabrication

Nafion is a solid electrolyte polymer that can be used as a sensor membrane in microfabricated electrochemical oxygen sensors. It allows ions to be transported between the sensor electrodes and removes the need for a liquid electrolyte. Here we used a series of small square Nafion test structures, fabricated on a variety of materials using standard thin-film patterning techniques, to optimize the design and processing of Nafion membranes. Measurements showed that the choice of photoresist developer is critical. Use of diluted MF-26A developer provided the most effective and manufacturable process. The underlying material also had an influence on robustness, with silicon dioxide and platinum giving the longest membrane lifetime under simulated conditions of use. Membrane size had no clear effect on lifetime, and under optimal processing conditions there were minimal failures even under continuous mechanical agitation for up to six weeks. We also developed test electrodes covered by Nafion, and showed that they were effective at supporting electrochemical oxygen detection

Test structures for optimizing polymer electrolyte performance in a microfabricated electrochemical oxygen sensor

Test structures were produced for optimizing the design and fabrication of a patterned solid polymer electrolyte in an electrochemical oxygen sensor. Measurements showed that choice of photoresist developer and the underlying insulator material affected durability of the polymer structures. Test electrodes covered by the polymer were effective at supporting electrochemical oxygen detection