No description available.

No description available.

No description available.

NMDARs are indispensable for developmental plasticity in the mammalian brain, but their roles in vivo are difficult to ascertain as NMDAR-knockouts are lethal. To circumvent this problem, we utilized NR1-knockdown mice to examine plasticity at the calyx of Held-MNTB synapse in the auditory brainstem. Previous work shows NMDARs at this synapse are rapidly down-regulated following the onset of hearing (P12), leading to the hypothesis that transient NMDAR expression is indispensable for modulating functional and morphological remodelling during development. We tested this by performing electrophysiological recordings, fluorescence tracing in vitro, and auditory brainstem responses in vivo, and surprisingly found that reducing postsynaptic NMDARs appears to promote functional maturation via presynaptic mechanisms in the absence of morphological and acoustic transmission differences in the mature calyx. This suggests a novel role for NMDARs to function as an activity-dependent control for setting the pace of consolidation and maturation in the calyx of Held synapse.

NMDARs are indispensable for developmental plasticity in the mammalian brain, but their roles in vivo are difficult to ascertain as NMDAR-knockouts are lethal. To circumvent this problem, we utilized NR1-knockdown mice to examine plasticity at the calyx of Held-MNTB synapse in the auditory brainstem. Previous work shows NMDARs at this synapse are rapidly down-regulated following the onset of hearing (P12), leading to the hypothesis that transient NMDAR expression is indispensable for modulating functional and morphological remodelling during development. We tested this by performing electrophysiological recordings, fluorescence tracing in vitro, and auditory brainstem responses in vivo, and surprisingly found that reducing postsynaptic NMDARs appears to promote functional maturation via presynaptic mechanisms in the absence of morphological and acoustic transmission differences in the mature calyx. This suggests a novel role for NMDARs to function as an activity-dependent control for setting the pace of consolidation and maturation in the calyx of Held synapse.

The effects of alpha1A subunit mutations on presynaptic Ca2+ channel activity and functional development of synaptic properties remain elusive. The calyx of Held/medial nucleus of the trapezoid body synapse is an ideal model for studying the developmental effects of presynaptic voltage-gated Ca2+ channel (VGCC) impairment on synaptic function since simultaneous voltage-clamp recordings can be made directly from the pre- and postsynapse. The alpha1A subunit leaner (tgla/la) mutation induced a profound reduction in synaptic transmission after hearing onset (> postnatal day 12; P12), with relatively preserved relationship between presynaptic Ca2+ current (Pre-ICa) and release and G-protein-mediated inhibition. Some synaptic properties were more reflective of an immature state, while other properties displayed a delay in maturation after P12. Direct presynaptic recordings from P15/16 tgla/la nerve terminals revealed a decrease in the density of Pre-ICa, elevated activation threshold and slowing in the kinetics of VGCCs, all of which contribute to the deficit in transmitter release. Fractional contribution of P/Q-type channels to total Pre-ICa and their role in vesicle release was markedly reduced. N-type Ca2+ channels and close association of VGCCs to release sites was not sufficient to fully compensate for impaired P/Q-type channel function. The extent to which compensatory mechanisms preserve synaptic transmission at tgla/la synapses was further constrained by the developmental narrowing of the action potential waveform. Activation of the cAMP pathway by forskolin or direct modulation of VGCCs by cdk inhibitors rescued deficits in transmitter release at P15/16 tgla/la synapses. The major effect of roscovitine was a slowing of presynaptic VGCC deactivation kinetics accompanied by a leftward shift in the activation curve. Activation of the cAMP pathway or direct modulation of presynaptic VGCCs may serve as two potential pathways to facilitate release and improve neuronal communication at synapses normally compromised by impaired P/Q-type channel function. While significant for the tgla/la mutant, these studies provide an important advancement in our understanding of the crucial developmental and functional roles of P/Q-type Ca2+ channels in driving the maturation of synaptic properties at central synapses. These findings may improve our understanding of the pathophysiology of presynaptic VGCCs and elucidate essential mechanisms underlying the tgla/la phenotype.

The effects of alpha1A subunit mutations on presynaptic Ca2+ channel activity and functional development of synaptic properties remain elusive. The calyx of Held/medial nucleus of the trapezoid body synapse is an ideal model for studying the developmental effects of presynaptic voltage-gated Ca2+ channel (VGCC) impairment on synaptic function since simultaneous voltage-clamp recordings can be made directly from the pre- and postsynapse. The alpha1A subunit leaner (tgla/la) mutation induced a profound reduction in synaptic transmission after hearing onset (> postnatal day 12; P12), with relatively preserved relationship between presynaptic Ca2+ current (Pre-ICa) and release and G-protein-mediated inhibition. Some synaptic properties were more reflective of an immature state, while other properties displayed a delay in maturation after P12. Direct presynaptic recordings from P15/16 tgla/la nerve terminals revealed a decrease in the density of Pre-ICa, elevated activation threshold and slowing in the kinetics of VGCCs, all of which contribute to the deficit in transmitter release. Fractional contribution of P/Q-type channels to total Pre-ICa and their role in vesicle release was markedly reduced. N-type Ca2+ channels and close association of VGCCs to release sites was not sufficient to fully compensate for impaired P/Q-type channel function. The extent to which compensatory mechanisms preserve synaptic transmission at tgla/la synapses was further constrained by the developmental narrowing of the action potential waveform. Activation of the cAMP pathway by forskolin or direct modulation of VGCCs by cdk inhibitors rescued deficits in transmitter release at P15/16 tgla/la synapses. The major effect of roscovitine was a slowing of presynaptic VGCC deactivation kinetics accompanied by a leftward shift in the activation curve. Activation of the cAMP pathway or direct modulation of presynaptic VGCCs may serve as two potential pathways to facilitate release and improve neuronal communication at synapses normally compromised by impaired P/Q-type channel function. While significant for the tgla/la mutant, these studies provide an important advancement in our understanding of the crucial developmental and functional roles of P/Q-type Ca2+ channels in driving the maturation of synaptic properties at central synapses. These findings may improve our understanding of the pathophysiology of presynaptic VGCCs and elucidate essential mechanisms underlying the tgla/la phenotype.

化学シナプスにおいて、シナプス小胞内に蓄えられた神経伝達物質の開口放出によってシナプス伝達が起こる。開口放出後、シナプス小胞はエンドサイトーシスによって細胞内へ回収されて再利用される。膜容量測定法とpHイメージングを併用して、開口放出とエンドサイトーシスに伴う小胞膜および小胞タンパク質の動態を同時測定した。Ca2+-カルモジュリン-Munc13シグナリングが小胞タンパク質の回収に関与していることが示唆された。 / Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, membranes and vesicular proteins has to be retrieved for reuse. I have combined capacitance measurements and pH-imaging via a pH-sensitive vesicular protein marker, and compared the retrieval kinetics of membranes and synaptotagmin2 (Syt2) at the calyx of Held presynaptic terminal. Data in this thesis identifies a novel mechanism of stimulus- and Ca2+-dependent regulation of coordinated endocytosis of synaptic membranes and Syt2. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

The auditory brainstem is comprised of a number of synapses specialized for the transmission of high-fidelity synaptic signals. Within the first three postnatal weeks, these pathways acquire the ability to process high-frequency signals without compromising timing information. However, little is known regarding developmental adaptations which confer this ability. Situated in the sound localization pathway, the calyx of Held-medial nucleus of the trapezoid body synapse provides an ideal model for investigating such adaptations as both the pre- and postsynaptic neurons are accessible to electrophysiological experimentation. Using this synapse, we have shown herein that the spatial coupling between voltage-gated calcium channels (VGCCs) and synaptic vesicles (SVs) tightens during development. Immature synapses use a loosely-coupled arrangement of many N- and P/Q-type VGCCs (“microdomain” modality) while mature synapses use a tightly-coupled arrangement of fewer P/Q-type VGCCs, to release SVs (“nanodomain” modality). As a consequence of this tightening, synaptic delay (SD) shortens. By fluorescence- and electron microscopy of SVs near active zones, we further identified the filamentous protein septin 5 as a molecular substrate, differentiating the two release modalities, which may act as a spatial barrier separating VGCCs and SVs in immature synapses. Finally, we have demonstrated that changes in release modality affect the nature of short-term plasticity observed at this synapse. Using trains of action potentials as presynaptic voltage-commands, we showed that, downstream of calcium influx, the microdomain modality promotes short-term facilitation in excitatory postsynaptic currents (IEPSC), and calcium-dependent decreases in SD, with these being absent in synapses employing the nanodomain modality. In contrast, we found that as a result of depletion of SVs, short-term depression of IEPSC dominates in synapses using the nanodomain modality, and correlates with calcium-dependent increases in SD. These findings imply that the type of release modality has a significant impact on the strength and timing of synaptic responses. The microdomain modality imparts greater dynamic range in timing and strength, but does so at the cost of efficiency and fidelity, while the nanodomain modality is a key accomplishment consolidating the high-fidelity abilities of this synapse.

The auditory brainstem is comprised of a number of synapses specialized for the transmission of high-fidelity synaptic signals. Within the first three postnatal weeks, these pathways acquire the ability to process high-frequency signals without compromising timing information. However, little is known regarding developmental adaptations which confer this ability. Situated in the sound localization pathway, the calyx of Held-medial nucleus of the trapezoid body synapse provides an ideal model for investigating such adaptations as both the pre- and postsynaptic neurons are accessible to electrophysiological experimentation. Using this synapse, we have shown herein that the spatial coupling between voltage-gated calcium channels (VGCCs) and synaptic vesicles (SVs) tightens during development. Immature synapses use a loosely-coupled arrangement of many N- and P/Q-type VGCCs (“microdomain” modality) while mature synapses use a tightly-coupled arrangement of fewer P/Q-type VGCCs, to release SVs (“nanodomain” modality). As a consequence of this tightening, synaptic delay (SD) shortens. By fluorescence- and electron microscopy of SVs near active zones, we further identified the filamentous protein septin 5 as a molecular substrate, differentiating the two release modalities, which may act as a spatial barrier separating VGCCs and SVs in immature synapses. Finally, we have demonstrated that changes in release modality affect the nature of short-term plasticity observed at this synapse. Using trains of action potentials as presynaptic voltage-commands, we showed that, downstream of calcium influx, the microdomain modality promotes short-term facilitation in excitatory postsynaptic currents (IEPSC), and calcium-dependent decreases in SD, with these being absent in synapses employing the nanodomain modality. In contrast, we found that as a result of depletion of SVs, short-term depression of IEPSC dominates in synapses using the nanodomain modality, and correlates with calcium-dependent increases in SD. These findings imply that the type of release modality has a significant impact on the strength and timing of synaptic responses. The microdomain modality imparts greater dynamic range in timing and strength, but does so at the cost of efficiency and fidelity, while the nanodomain modality is a key accomplishment consolidating the high-fidelity abilities of this synapse.