| dc.description.abstracteng | This thesis described the development of a novel, versatile microfluidic platform for studying the ionic mechanisms of vesicular neurotransmitter transporters in rodent synaptic vesicles (SVs). The level of filling of an SV with neurotransmitters is relevant as it is an influential factor in neuronal communication. The quantal load of a single SV does not saturate postsynaptic receptors (Barberis et al., 2004; Ishikawa et al., 2002). Changes in the expression of the proteins responsible for neurotransmitter uptake (vesicular neurotransmitter transporters, e.g. VGLUT, VGAT, VNUT) changed the amount of neurotransmitter that was stored and released and thereby changed the behavior of the animal (Fon et al., 1997; Takahashi et al., 1997).
Neurotransmitter filling in SVs has two main components; the vacuolar ATP-driven proton pump (V-ATPase) and the uptake of neurotransmitters. The role of the V-ATPase is the generation of the electrochemical gradient (ΔμH+) across the vesicle membrane, which the vesicular neurotransmitter transporters use to load neurotransmitters into the vesicular lumen (Forgac, 2007). These two processes are intricately connected and co-occur in SVs in physiological conditions. Each of the currently available approaches to study these mechanisms is limited in their way: neuronal cell culture or brain slices based methods have limited control of the environment to decipher the details of the ionic mechanism, whereas isolated SVs or proteoliposomes in spectrofluorometers and the single vesicle assay (Farsi et al., 2016; Upmanyu et al., 2021) only allow for the addition of compounds, with no option of removal of a compound once added. Many of these approaches have a static nature in common; once a compound is added to the experiments, it cannot be removed. The microfluidic platform developed during my PhD addresses this limitation.
An assay capable of dynamically and repetitively changing the environment of SVs has to fulfill specific requirements. First of all, antibody immobilization is required in a manner that can sustain the shear rate in the microfluidic device. Secondly, these antibodies have to be able to stably immunocapture SVs. For accurate quantification, antibodies and SVs have to be immobilized at a sufficiently high density. Lastly, the microfluidic device has to be capable of solution changes faster than 1 second. This new assay can then be used to study the unanswered questions on the loading and maintenance of neurotransmitter filling in synaptic vesicles.
In this thesis, three different surfaces (2D epoxy, siPEG/siPEG-biotin, copoly(DMA-NAS-MAPS)) were used to functionalize the glass surface. First antibody immobilization in static conditions was established on each surface, followed by immunocapture of SVs in static conditions. All three surfaces performed well. A microfluidic device was developed in collaboration with Dr. Eleonora Perego (Sarah Köster group, X-ray laboratory), which fulfilled the requirements. Together with a home-built pressure clamping holder (compatible with the biological material on the surface), these parts finalized the microfluidic platform.
Immunocaptured SVs on each surface were subjected to the flow in the microfluidic channel, resulting in the wash off of the previously immobilized SVs on the 2D epoxy and siPEG/siPEG-biotin coverslips. Only SVs immobilized on the copoly(DMA-NAS-MAPS) coverslips showed stable immunocapture under 30 minutes of 3mm/s flow conditions. The copoly(DMA-NAS-MAPS) surface was therefore chosen as the final surface for the assay. Preliminary experiments were executed to test the ability of the assay to measure acidification in SVs and the de-acidification upon removal of ATP from the buffer, which was successful.
This assay combines the following advantages compared to other existing methods; no need for highly pure SV preparations, possible capture of a wide range of targets, simple analysis of micrometer-sized spots containing concentrated vesicles, possible SV capture from small samples, and fast and repetitive exchange of solutions. Overall, the system allows for a high degree of experimental flexibility. The ability to change solutions with a high temporal resolution allows us to address the long-standing questions on, for example, glutamate efflux and clarify how neurotransmitter concentration in the SV is maintained. | de |