Developmental and activity-dependent modulation of coupling distance between release site and Ca2+ channel.

Synapses are junctions between a presynaptic neuron and a postsynaptic cell specialized for fast and precise information transfer. The presynaptic terminal secretes neurotransmitters via exocytosis of synaptic vesicles. Exocytosis is a tightly regulated reaction that occurs within a millisecond of the arrival of an action potential. One crucial parameter in determining the characteristics of the transmitter release kinetics is the coupling distance between the release site and the Ca2+ channel. Still, the technical limitations have hindered detailed analysis from addressing how the coupling distance is regulated depending on the development or activity of the synapse. However, recent technical advances in electrophysiology and imaging are unveiling their different configurations in different conditions. Here, I will summarize developmental- and activity-dependent changes in the coupling distances revealed by recent studies.

Pathway-specific maturation of presynaptic functions of the somatosensory thalamus.

Neural circuits are the bases of brain function, and signal transmission between neurons is mediated by synapses. However, neural circuits and synapses are not fully functional at the time of birth. In the nervous system of newborn animals, neurons form an extensive number of redundant synapses that are targeted to construct neural circuits, of which 40-50% are subsequently eliminated during adolescence before circuit maturation. It is widely understood that the maturation of synaptic function differs between surviving and eliminated synapses before their eventual selection; however, direct evidence is currently lacking because of technical limitations. We recently acquired direct electrical recordings from single synapses destined for survival and elimination in the rodent somatosensory thalamus. Results demonstrated detailed presynaptic functional development both in surviving and eliminated pathways. Our work not only revealed the functional properties of surviving and eliminated synapses but also provided a new model system to elucidate the mechanisms that underlie mature neural circuit formation.

Dysfunction of parvalbumin-expressing cells in the thalamic reticular nucleus induces cortical spike-and-wave discharges and an unconscious state.

Spike-and-wave discharges and an accompanying loss of consciousness are hallmarks of absence seizure, which is a childhood generalized epilepsy disorder. In absence seizure, dysfunction of the cortico-thalamo-cortico circuitry is thought to engage in abnormal cortical rhythms. Previous studies demonstrated that the thalamic reticular nucleus has a critical role in the formation of normal cortical rhythms; however, whether thalamic reticular nucleus dysfunction leads directly to abnormal rhythms, such as epilepsy, is largely unknown. We found that expressing the inhibitory opsin, archaerhodopsin, including in the thalamic reticular nucleus, caused abnormal cortical rhythms in Pvalb-tetracycline transactivator::tetO-ArchT (PV-ArchT) double transgenic mice. We validated the PV-ArchT line as a new mouse model of absence seizure through physiological and pharmacological analyses, as well as through examining their behavioural features. We then discovered that archaerhodopsin expression exclusively in thalamic reticular nucleus parvalbumin-positive neurons was sufficient to induce cortical spike-and-wave discharges using adeno-associated virus-mediated thalamic reticular nucleus targeting. Furthermore, we found that archaerhodopsin expression impaired rebound burst firing and T-current in thalamic reticular nucleus parvalbumin-positive cells by slice physiology. Although T-current in the thalamic reticular nucleus was impaired, the T-current blocker ethosuximide still had a therapeutic effect in PV-ArchT mice, suggesting a gain of function of T-type calcium channels in this absence seizure model. However, we did not find any over- or misexpression of T-type calcium channel genes in the thalamus or the cortex. Thus, we demonstrated that thalamic reticular nucleus dysfunction led to an absence seizure-like phenotype in mice. In a final set of experiments, we showed that the archaerhodopsin-mediated absence seizure-like phenotype disappeared after the removal of archaerhodopsin by using a time-controllable transgenic system. These data may provide a hint as to why many absence seizures naturally regress.

Distinct functional developments of surviving and eliminated presynaptic terminals.

For neuronal circuits in the brain to mature, necessary synapses must be maintained and redundant synapses eliminated through experience-dependent mechanisms. However, the functional differentiation of these synapse types during the refinement process remains elusive. Here, we addressed this issue by distinct labeling and direct recordings of presynaptic terminals fated for survival and for elimination in the somatosensory thalamus. At surviving terminals, the number of total releasable vesicles was first enlarged, and then calcium channels and fast-releasing synaptic vesicles were tightly coupled in an experience-dependent manner. By contrast, transmitter release mechanisms did not mature at terminals fated for elimination, irrespective of sensory experience. Nonetheless, terminals fated for survival and for elimination both exhibited developmental shortening of action potential waveforms that was experience independent. Thus, we dissected experience-dependent and -independent developmental maturation processes of surviving and eliminated presynaptic terminals during neuronal circuit refinement.

Direct imaging of rapid tethering of synaptic vesicles accompanying exocytosis at a fast central synapse.

A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.

Real-time imaging of synaptic vesicle exocytosis by total internal reflection fluorescence (TIRF) microscopy.

Synaptic vesicles are one of the smallest organelle in the cell with their sizes far below the diffraction limit of the light microscopy. Exocytosis at the synapse is tightly regulated reaction which typically occurs within a millisecond after the arrival of an action potential. It has been assumed that synaptic vesicles have to be ready for immediate exocytosis upon the arrival of final trigger before exocytosis. But direct observation of the pre-exocytotic synaptic vesicle dynamics have been lacking. Total internal reflection microscopy (TIRFM) is a fluorescence microscopy which has best z-axis resolution (∼100 nm) as a light microscopy, and is close to that of the ultrathin section used for electron microscopy. Although its application is limited to the objects just beneath the plasma membrane, TIRFM has revealed dynamics of various organelles and proteins. We recently managed to dissociate mammalian neuronal presynaptic terminals and let the exocytotic sites adhere tightly to the coverslip. There, TIRFM revealed the detailed dynamics of pre-exocytotic vesicles. Our work opened up the way to visualize dynamics of sub-diffraction limited sized organelle in a real time, and will be useful for direct visualization of various synaptic components in the future.

Kinetics of Releasable Synaptic Vesicles and Their Plastic Changes at Hippocampal Mossy Fiber Synapses.

Hippocampal mossy fiber boutons (hMFBs) are presynaptic terminals displaying various forms of synaptic plasticity. The presynaptic mechanisms underlying synaptic plasticity still remain poorly understood. Here, we have combined high temporal resolution measurements of presynaptic capacitance and excitatory postsynaptic currents (EPSCs) to measure the kinetics of exocytosis. In addition, total internal reflection fluorescence (TIRF) microscopy was employed to directly visualize dynamics of single synaptic vesicles adjacent to the plasma membrane at high spatial resolution. Readily releasable vesicles mostly consisted of already-tethered vesicles in the TIRF field. Vesicle replenishment had fast and slow phases, and TIRF imaging suggests that the fast phase depends on vesicle priming from already-tethered vesicles. Application of cyclic AMP (cAMP), a molecule crucial for LTP, mainly increases the vesicular release probability rather than the number of readily releasable vesicles or their replenishment rate, likely by changing the coupling between Ca2+ channels and synaptic vesicles. Thus, we revealed dynamic properties of synaptic vesicles at hMFBs.

Distinct modes of endocytotic presynaptic membrane and protein uptake at the calyx of Held terminal of rats and mice.

Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, compensatory retrieval of membranes and vesicular proteins is essential. We combined capacitance measurements and pH-imaging via pH-sensitive vesicular protein marker (anti-synaptotagmin2-cypHer5E), and compared the retrieval kinetics of membranes and vesicular proteins at the calyx of Held synapse. Membrane and Syt2 were retrieved with a similar time course when slow endocytosis was elicited. When fast endocytosis was elicited, Syt2 was still retrieved together with the membrane, but endocytosed organelle re-acidification was slowed down, which provides strong evidence for two distinct endocytotic pathways. Strikingly, CaM inhibitors or the inhibition of the Ca(2+)-calmodulin-Munc13-1 signaling pathway only impaired the uptake of Syt2 while leaving membrane retrieval intact, indicating different recycling mechanisms for membranes and vesicle proteins. Our data identify a novel mechanism of stimulus- and Ca(2+)-dependent regulation of coordinated endocytosis of synaptic membranes and vesicle proteins.

Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses.

Developmental changes in Ca2+ channel subtypes regulating endocytosis at the calyx of Held.

At the mammalian central synapse, Ca(2+) influx through Ca(2+) channels triggers neurotransmitter release by exocytosis of synaptic vesicles, which fuse with the presynaptic membrane and are subsequently retrieved by endocytosis. At the calyx of Held terminal, P/Q-type Ca(2+) channels mainly mediate exocytosis, while N- and R-type channels have a minor role in young terminals (postnatal days 8-11). The role of each Ca(2+) channel subtype in endocytosis remains to be elucidated; therefore, we examined the role of each type of Ca(2+) channel in endocytosis, by using whole-cell patch-clamp recordings in conjunction with capacitance measurement techniques. We found that at the young calyx terminal, when R-type Ca(2+) channels were blocked, the slow mode of endocytosis was further slowed, while blocking of either P/Q- or N-type Ca(2+) channels had no major effect. In more mature terminals (postnatal days 14-17), the slow mode of endocytosis was mainly triggered by P/Q-type Ca(2+) channels, suggesting developmental changes in the regulation of the slow mode of endocytosis by different Ca(2+) channel subtypes. In contrast, a fast mode of endocytosis was observed after strong stimulation in young terminals that was mediated mainly by P/Q-type, but not R- or N-type Ca(2+) channels. These results suggest that different types of Ca(2+) channels regulate the two different modes of endocytosis. The results may also suggest that exo- and endocytosis are regulated independently at different sites in young animals but are more tightly coupled in older animals, allowing more efficient synaptic vesicle cycling adapted for fast signalling.

Different roles of ribbon-associated and ribbon-free active zones in retinal bipolar cells.

Synaptic ribbons with a halo of synaptic vesicles are seen at the active zones of sensory neurons that release transmitter tonically. Thus, ribbons are assumed to be a prerequisite for sustained exocytosis. By applying total internal reflection fluorescence microscopy to goldfish retinal bipolar cell terminals, we visualized Ca2+ entry sites, ribbons, and vesicle fusion events. Here we show that the main Ca2+ entry sites were located at ribbons, and that activation of the Ca2+ current induced immediate and delayed vesicle fusion events at ribbon-associated and ribbon-free 'hot spots', respectively. The activation of protein kinase C (PKC) specifically potentiated vesicle fusion at ribbon-free sites. Electron microscopy showed that PKC activation selectively increased the number of docked vesicles at ribbon-free sites, which faced neuronal processes with the postsynaptic density. Retinal bipolar cells have both ribbon-associated and ribbon-free active zones in their terminals and might send functionally distinct signals through ribbon-associated and ribbon-free synapses to postsynaptic neurons.

Increase in the pool size of releasable synaptic vesicles by the activation of protein kinase C in goldfish retinal bipolar cells.

Secretion from neurons and neuroendocrine cells is enhanced by the activation of protein kinase C (PKC) in various preparations. We have already reported that transmitter (glutamate) release from Mb1 bipolar cells in the goldfish retina is potentiated by the activation of PKC. However, it is not yet settled whether the potentiation is ascribed to the increase in the pool size of releasable synaptic vesicles or in release probability. In the present study, Ca2+ influx and exocytosis were simultaneously monitored by measuring the presynaptic Ca2+ current and membrane capacitance changes, respectively, in a terminal detached from the bipolar cell. The double pulse protocol was used to estimate separately the changes in the pool size and release probability. The activation of PKC by phorbol 12-myristate 13-acetate (PMA) specifically increased the pool size but not the release probability. PKC was activated by PMA even after the Ca2+ influx was blocked by Co2+. In bipolar cells the releasable pool can be divided into two components: one is small and rapidly exhausted, and the other is large and slowly exocytosed. To identify which component is responsible for the increase in the pool size, the effects of PMA and a PKC-specific inhibitor, bisindolylmaleimide I (BIS), on each component were examined. The slow component was selectively increased by PMA and reduced by BIS. Thus, we conclude that the activation of PKC in Mb1 bipolar cells potentiates glutamate release by increasing the pool size of the slow component.