Ryota Fukaya

Bio: Ryota Fukaya is an academic researcher from Doshisha University. The author has contributed to research in topics: Synapse & Hippocampal formation. The author has an hindex of 1, co-authored 1 publications receiving 5 citations.

Rapid Ca2+ channel accumulation contributes to cAMP-mediated increase in transmission at hippocampal mossy fiber synapses

Abstract: The cyclic adenosine monophosphate (cAMP)-dependent potentiation of neurotransmitter release is important for higher brain functions such as learning and memory. To reveal the underlying mechanisms, we applied paired pre- and postsynaptic recordings from hippocampal mossy fiber-CA3 synapses. Ca2+ uncaging experiments did not reveal changes in the intracellular Ca2+ sensitivity for transmitter release by cAMP, but suggested an increase in the local Ca2+ concentration at the release site, which was much lower than that of other synapses before potentiation. Total internal reflection fluorescence (TIRF) microscopy indicated a clear increase in the local Ca2+ concentration at the release site within 5 to 10 min, suggesting that the increase in local Ca2+ is explained by the simple mechanism of rapid Ca2+ channel accumulation. Consistently, two-dimensional time-gated stimulated emission depletion microscopy (gSTED) microscopy showed an increase in the P/Q-type Ca2+ channel cluster size near the release sites. Taken together, this study suggests a potential mechanism for the cAMP-dependent increase in transmission at hippocampal mossy fiber-CA3 synapses, namely an accumulation of active zone Ca2+ channels.

Calcium dependence of neurotransmitter release at a high fidelity synapse.

Abstract: The Ca2+-dependence of the priming, fusion, and replenishment of synaptic vesicles are fundamental parameters controlling neurotransmitter release and synaptic plasticity. Despite intense efforts, these important steps in the synaptic vesicles' cycle remain poorly understood due to the technical challenge in disentangling vesicle priming, fusion, and replenishment. Here, we investigated the Ca2+-sensitivity of these steps at mossy fiber synapses in the rodent cerebellum, which are characterized by fast vesicle replenishment mediating high-frequency signaling. We found that the basal free Ca2+ concentration (<200 nM) critically controls action potential-evoked release, indicating a high-affinity Ca2+ sensor for vesicle priming. Ca2+ uncaging experiments revealed a surprisingly shallow and non-saturating relationship between release rate and intracellular Ca2+ concentration up to 50 μM. The rate of vesicle replenishment during sustained elevated intracellular Ca2+ concentration exhibited little Ca2+-dependence. Finally, quantitative mechanistic release schemes with five Ca2+ binding steps incorporating rapid vesicle replenishment via parallel or sequential vesicle pools could explain our data. We thus show that co-existing high- and low-affinity Ca2+ sensors mediate priming, fusion, and replenishment of synaptic vesicles at a high-fidelity synapse.

Prolonged development of long-term potentiation at lateral entorhinal cortex synapses onto adult-born neurons.

Abstract: Critical period plasticity at adult-born neuron synapses is widely believed to contribute to the learning and memory functions of the hippocampus. Experience regulates circuit integration and for a transient interval, until cells are ~6 weeks old, new neurons display enhanced long-term potentiation (LTP) at afferent and efferent synapses. Since neurogenesis declines substantially with age, this raises questions about the extent of lasting plasticity offered by adult-born neurons. Notably, however, the hippocampus receives sensory information from two major cortical pathways. Broadly speaking, the medial entorhinal cortex conveys spatial information to the hippocampus via the medial perforant path (MPP), and the lateral entorhinal cortex, via the lateral perforant path (LPP), codes for the cues and items that make experiences unique. While enhanced critical period plasticity at MPP synapses is relatively well characterized, no studies have examined long-term plasticity at LPP synapses onto adult-born neurons, even though the lateral entorhinal cortex is uniquely vulnerable to aging and Alzheimer's pathology. We therefore investigated LTP at LPP inputs both within (4-6 weeks) and beyond (8+ weeks) the traditional critical period. At immature stages, adult-born neurons did not undergo significant LTP at LPP synapses, and often displayed long-term depression after theta burst stimulation. However, over the course of 3-4 months, adult-born neurons displayed increasingly greater amounts of LTP. Analyses of short-term plasticity point towards a presynaptic mechanism, where transmitter release probability declines as cells mature, providing a greater dynamic range for strengthening synapses. Collectively, our findings identify a novel form of new neuron plasticity that develops over an extended interval, and may therefore be relevant for maintaining cognitive function in aging.

Mechanisms of Synaptic Vesicle Exo- and Endocytosis

Abstract: Within 1 millisecond of action potential arrival at presynaptic terminals voltage–gated Ca2+ channels open. The Ca2+ channels are linked to synaptic vesicles which are tethered by active zone proteins. Ca2+ entrance into the active zone triggers: (1) the fusion of the vesicle and exocytosis, (2) the replenishment of the active zone with vesicles for incoming exocytosis, and (3) various types of endocytosis for vesicle reuse, dependent on the pattern of firing. These time-dependent vesicle dynamics are controlled by presynaptic Ca2+ sensor proteins, regulating active zone scaffold proteins, fusion machinery proteins, motor proteins, endocytic proteins, several enzymes, and even Ca2+ channels, following the decay of Ca2+ concentration after the action potential. Here, I summarize the Ca2+-dependent protein controls of synchronous and asynchronous vesicle release, rapid replenishment of the active zone, endocytosis, and short-term plasticity within 100 msec after the action potential. Furthermore, I discuss the contribution of active zone proteins to presynaptic plasticity and to homeostatic readjustment during and after intense activity, in addition to activity-dependent endocytosis.

Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca2+ channels drives sustained active zone potentiation

Abstract: At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca2+ channels (VGCCs). While AZs can potentiate SV release in the minutes range, we lack an understanding of how AZ scaffold components and VGCCs engage into potentiation. We here establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel’s intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. Our single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation.

Increased vesicle fusion competence underlies long-term potentiation at hippocampal mossy fiber synapses

Abstract: Presynaptic long-term potentiation (LTP) is thought to play an important role in learning and memory. However, the underlying mechanism remains elusive because of the difficulty of direct recording during LTP. Hippocampal mossy fiber synapses exhibit pronounced LTP of transmitter release after tetanic stimulation and have been used as a model of presynaptic LTP. Here, we induced LTP by optogenetic tools and applied direct presynaptic patch-clamp recordings. The action potential waveform and evoked presynaptic Ca2+ currents remained unchanged after LTP induction. Membrane capacitance measurements suggested higher release probability of synaptic vesicles without changing the number of release-ready vesicles after LTP induction. Synaptic vesicle replenishment was also enhanced. Furthermore, stimulated emission depletion microscopy suggested an increase in the numbers of Munc13-1 and RIM1 molecules within active zones. We propose that dynamic changes in the active zone components may be relevant for the increased fusion competence and synaptic vesicle replenishment during LTP.