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    • The replenishment time course of already tethered vesicle to

    2022-02-09

    The replenishment time course of already-tethered vesicle to became release ready, was examined by TIRFM imaging combined with calcium uncaging. By UV-flash photolysis of caged calcium, intracellular calcium concentration raises rapidly and homogenously throughout the cell (Ellis-Davies, 2008). Upon calcium uncaging, a fast component of vesicle fusions occurred instantly, and the slow component of vesicle fusions occurred continuously thereafter. The time constant of the slow component (∼300 ms) is consistent with the rate of replenishment to the RRVs. None of newly tethered vesicles participated in the slow component, and they were mediated by already-tethered vesicles before the flash. Therefore, fast replenishment of releasable vesicles is mediated not by tethering of new vesicles to the plasma membrane, but rather by the molecular transition of the already-tethered vesicles from no-primed state to primed state. Such information cannot be achieved without live visualization of the synaptic vesicle dynamics before exocytosis, which is feasible only by TIRFM imaging so far. These features of synaptic vesicle activities at calyx of Held terminal, a model terminal for conventional synapse, were visualized since we succeeded in isolating nerve terminals enzymatically and to preserve them in a functional state. Measuring dynamics of synaptic vesicles at the calyx of Held terminal is exciting not only because it is mammalian conventional central nerve system (CNS) neuron, but also because the kinetics of exocytosis have been extensively studied with high-resolution electrophysiological techniques (Borst and Soria van Hoeve, 2012), and direct comparison between kinetics of transmitter release and vesicle fusion is possible there. TIRFM imaging of single synaptic vesicle is recently applied to another mammalian CNS presynaptic terminal, hippocampal mossy fiber boutons (hMFBs) (Midorikawa and Sakaba, 2017). hMFBs are large presynaptic terminal of the granule Dovitinib Lactate synthesis at the dentate gyrus of the hippocampus, which form synapses mainly against CA3 pyramidal cells (Nicoll and Schmitz, 2005). Hippocampus is well known to play an important role in learning and memory through long-term plasticity mechanisms. Among several synapses that show long-term plasticity in hippocampus, hMFBs-CA3 pyramidal cell synapse has a unique characteristic, which is that the origin of long-term plasticity relies largely on presynaptic side (Nicoll and Malenka, 1995). At this presynaptic preparation, exocytosis and pre-exocytotic movements of FM-labeled single synaptic vesicles was also visualized using TIRFM. By comparing data before and after the chemically induced potentiation by cAMP, and also by combining TIRFM imaging and electrophysiology, it was shown that potentiated exocytosis from hMFBs are mediated by increased release probability of releasable pool of synaptic vesicles, not by increase of the number of readily releasable vesicles. Furthermore, it was strongly suggested that the increase of the release probability was due to tighter coupling distance between Ca2+ channels and synaptic vesicles.
    Further perspectives TIRFM imaging can be applied to monitor synaptic activities other than exocytosis of synaptic vesicles. Presynaptic active zones of the large terminals (such as retinal photoreceptor, bipolar cell, calyx of Held, and hMFBs) can be adhered tight enough to the coverslip to apply TIRFM simply by acute dissociation, but the postsynaptic membrane-like structures can also be formed after culturing on the coverslip coated with a cell adhesion molecule neurexin (Tanaka et al., 2013). Because of the culture period, application of genetic manipulations is much easier, and the types of synapses are not limited to the large terminal as long as they grow on the culture dish. By visualizing postsynaptic sites by TIRFM, one can examine the mobility of postsynaptic proteins during the synaptic activity, or during the plastic change. TIRFM imaging could provide not only detailed spatio-temporal information within the single synaptic site, but also an opportunity to compare different sites under different conditions (i.e. synapses with or without stimulation or drug application, e.t.c.). It is also interesting to investigate the special organization of endocytosis, but so far visualizing endocytotic sites at the synapse by TIRFM is technically demanding and there are only limited number of studies (Pelassa et al., 2014). Another interesting direction is to visualize the kinetics of functional synaptic membrane proteins. Recent developments of the super-resolution microscopy (e.g. STED, STORM, PALM) have provided detailed distributions of numbers of synaptic proteins (Willig et al., 2006; Kittel et al., 2006; Sieber et al., 2007; Dani et al., 2010; Tang et al., 2016). Although live TIRFM imaging cannot provide sub-diffraction limit image in X-Y directions, its Z direction resolution (tens of nms) and high temporal resolution are still useful enough to examine the dynamics of events occurring just beneath the plasma membrane.