Kohgaku Eguchi

Developmental shift to a mechanism of synaptic vesicle endocytosis requiring nanodomain Ca2

Ca2+ is thought to be essential for the exocytosis and endocytosis of synaptic vesicles. However, the manner in which Ca2+ coordinates these processes remains unclear, particularly at mature synapses. Using membrane capacitance measurements from calyx of Held nerve terminals in rats, we found that vesicle endocytosis is initiated primarily in Ca2+ nanodomains around Ca2+ channels, where exocytosis is triggered. Bulk Ca2+ outside of the domain could also be involved in endocytosis at immature synapses, although only after extensive exocytosis at more mature synapses. This bulk Ca2+-dependent endocytosis required calmodulin and calcineurin activation at immature synapses, but not at more mature synapses. Similarly, GTP-independent endocytosis, which occurred after extensive exocytosis at immature synapses, became negligible after maturation. We propose that nanodomain Ca2+ simultaneously triggers exocytosis and endocytosis of synaptic vesicles and that the molecular mechanisms underlying Ca2+-dependent endocytosis undergo major developmental changes at this fast central synapse.

Vesicular glutamate filling and AMPA receptor occupancy at the calyx of Held synapse of immature rats

At central glutamatergic synapses, neurotransmitter often saturates postsynaptic AMPA receptors (AMPARs), thereby restricting the dynamic range of synaptic efficacy. Here, using simultaneous pre‐ and postsynaptic whole‐cell recordings, at the calyx of Held synapse of immature rats, we have investigated the mechanism by which transmitter glutamate saturates postsynaptic AMPARs. When we loaded l‐glutamate (1–100 mm) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration‐dependent manner. At physiological temperature (36–37°C), the qEPSC amplitude increased when intraterminal l‐glutamate concentration was elevated from 1 mm to 10 mm, but it reached a plateau at 10 mm. This plateau persisted after bath‐application of the low affinity AMPAR antagonist kynurenate, suggesting that it was caused by saturation of vesicular filling with glutamate rather than by saturation of postsynaptic AMPARs. In contrast to qEPSCs, action potential‐evoked EPSCs remained unchanged by increasing intraterminal l‐glutamate from 1 mm to 100 mm, even at room temperature, indicating that multi‐quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 μm). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 nm; this was far below the concentration required for AMPAR desensitization. We conclude that rapid AMPAR desensitization, caused by glutamate released from multiple vesicles during synaptic transmission, underlies postsynaptic AMPAR saturation at this immature calyceal synapse before the onset of hearing.

Rho-Kinase Accelerates Synaptic Vesicle Endocytosis by Linking Cyclic GMP-Dependent Protein Kinase Activity to Phosphatidylinositol-4,5-Bisphosphate Synthesis

Rho-kinase plays diverse roles in cell motility. During neuronal development, Rho-kinase is involved in neuronal migration, and in neurite outgrowth and retraction. Rho-kinase remains highly expressed in mature neurons, but its physiological roles are poorly understood. Here we report that Rho-kinase plays a key role in the synaptic vesicle recycling system in presynaptic terminals. Vesicles consumed by excessive exocytosis are replenished by accelerating vesicle endocytosis via a retrograde feedback mechanism involving nitric oxide released from postsynaptic cells. This homeostatic control system involves presynaptic cyclic GMP-dependent protein kinase (PKG) and a plasma membrane phospholipid, phosphatidylinositol-4,5-bisphophate (PIP2). We found that application of a Rho-kinase inhibitor, a PKG inhibitor or both, reduced the PIP2 content in Wistar rat brainstem synaptosomes to a similar extent. Likewise, application of the Rho-kinase inhibitor into the calyx of Held presynaptic terminal slowed vesicle endocytosis to the same degree as did application of the PKG inhibitor. This endocytic slowing effect of the Rho-kinase inhibitor was canceled by coapplication of PIP2 into the terminal. By contrast, a RhoA activator increased the PIP2 content and reversed the effect of the PKG inhibitor in brainstem synaptosomes. The RhoA activator, when loaded into calyceal terminals, also rescued the endocytic slowing effect of the PKG inhibitor. Furthermore, intraterminal loading of anti-PIP2 antibody slowed vesicle endocytosis and blocked the rescuing effect of the RhoA activator. We conclude that Rho-kinase links presynaptic PKG activity to PIP2 synthesis, thereby controlling the homeostatic balance of vesicle exocytosis and endocytosis in nerve terminals.

Reconstitution of Giant Mammalian Synapses in Culture for Molecular Functional and Imaging Studies

Giant presynaptic terminal brain slice preparations have allowed intracellular recording of electrical signals and molecular loading, elucidating cellular and molecular mechanisms underlying neurotransmission and modulation. However, molecular genetic manipulation or optical imaging in these preparations is hampered by factors, such as tissue longevity and background fluorescence. To overcome these difficulties, we developed a giant presynaptic terminal culture preparation, which allows genetic manipulation and enables optical measurements of synaptic vesicle dynamics, simultaneously with presynaptic electrical signal recordings. This giant synapse reconstructed from dissociated mouse brainstem neurons resembles the development of native calyceal giant synapses in several respects. Thus, this novel preparation constitutes a powerful tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development. SIGNIFICANCE STATEMENT We have developed a novel culture preparation of giant mammalian synapses. These presynaptic terminals make it possible to perform optical imaging simultaneously with presynaptic electrophysiological recording. We demonstrate that this enables one to dissect endocytic and acidification times of synaptic vesicles. In addition, developmental elimination and functional maturation in this cultured preparation provide a useful model for studying presynaptic development. Because this giant synapse preparation allows molecular genetic manipulations, it constitutes a powerful new tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development.

Impact of vesicular glutamate leakage on synaptic transmission at the calyx of Held

It is controversial whether glutamate can leak out of vesicles in the nerve terminal. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate or by blocking vacuolar ATPase activity using bafilomycin A1. In the absence of vesicular glutamate uptake, both spontaneous and nerve‐evoked EPSCs underwent a rundown, suggesting that vesicular glutamate can leak out of vesicles. However, the rundown of evoked EPSCs was caused mainly by accumulation of unfilled vesicles after exocytic release of glutamate, suggesting a minor influence of glutamate leakage on synaptic transmission.

Wild-Type Monomeric α-Synuclein Can Impair Vesicle Endocytosis and Synaptic Fidelity via Tubulin Polymerization at the Calyx of Held

α-Synuclein is a presynaptic protein the function of which has yet to be identified, but its neuronal content increases in patients of synucleinopathies including Parkinsons disease. Chronic overexpression of α-synuclein reportedly expresses various phenotypes of synaptic dysfunction, but the primary target of its toxicity has not been determined. To investigate this, we acutely loaded human recombinant α-synuclein or its pathological mutants in their monomeric forms into the calyces of Held presynaptic terminals in slices from auditorily mature and immature rats of either sex. Membrane capacitance measurements revealed significant and specific inhibitory effects of WT monomeric α-synuclein on vesicle endocytosis throughout development. However, the α-synuclein A53T mutant affected vesicle endocytosis only at immature calyces, whereas the A30P mutant had no effect throughout. The endocytic impairment by WT α-synuclein was rescued by intraterminal coloading of the microtubule (MT) polymerization blocker nocodazole. Furthermore, it was reversibly rescued by presynaptically loaded photostatin-1, a photoswitcheable inhibitor of MT polymerization, in a light-wavelength-dependent manner. In contrast, endocytic inhibition by the A53T mutant at immature calyces was not rescued by nocodazole. Functionally, presynaptically loaded WT α-synuclein had no effect on basal synaptic transmission evoked at a low frequency, but significantly attenuated exocytosis and impaired the fidelity of neurotransmission during prolonged high-frequency stimulation. We conclude that monomeric WT α-synuclein primarily inhibits vesicle endocytosis via MT overassembly, thereby impairing high-frequency neurotransmission. SIGNIFICANCE STATEMENT Abnormal α-synuclein abundance is associated with synucleinopathies including Parkinsons disease, but neither the primary target of α-synuclein toxicity nor its mechanism is identified. Here, we loaded monomeric α-synuclein directly into mammalian glutamatergic nerve terminals and found that it primarily inhibits vesicle endocytosis and subsequently impairs exocytosis and neurotransmission fidelity during prolonged high-frequency stimulation. Such α-synuclein toxicity could be rescued by blocking microtubule polymerization, suggesting that microtubule overassembly underlies the toxicity of acutely elevated α-synuclein in the nerve terminal.

Culture of Mouse Giant Central Nervous System Synapses and Application for Imaging and Electrophysiological Analyses

Primary neuronal cell culture preparations are widely used to investigate synaptic functions. This chapter describes a detailed protocol for the preparation of a neuronal cell culture in which giant calyx-type synaptic terminals are formed. This chapter also presents detailed protocols for utilizing the main technical advantages provided by such a preparation, namely, labeling and imaging of synaptic organelles and electrophysiological recordings directly from presynaptic terminals.