Yoshiaki Kidokoro

Exocytosis and Endocytosis of Synaptic Vesicles and Functional Roles of Vesicle Pools: Lessons from the Drosophila Neuromuscular Junction

To maintain synaptic transmission during intense neuronal activities, the synaptic vesicle (SV) pool at release sites is effectively replenished by recruitment of SVs from the reserve pool and/or by endocytosis. The authors have studied dynamics of SVs using a fluorescence dye, FM1-43, which is incorporated into SVs during endocytosis and released by exocytosis. Drosophila is one of the most suitable preparations for genetic and pharmacological analyses, and this provides a useful model system. The authors found at the neuromuscular junctions of Drosophila that exocytosis and endocytosis of SVs are triggered by Ca2+ influx through distinct routes and that selective inhibition of exocytosis or endocytosis resulted in depression of synaptic transmission with a distinct time course. They identified two SV pools in a single presynaptic bouton. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low-frequency stimulation and locates close to release sites in the periphery of boutons, whereas the reserve pool (RP) is loaded and unloaded only during high-frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the quantal content of evoked release, suggesting that SVs in the ECP are primarily involved in synaptic transmission. SVs in the RP are recruited to synaptic transmission by a process involving the cAMP/PKA cascade during high-frequency stimulation. Cytochalasin D blocked this recruitment process, suggesting involvement of filamentous actin. Endocytosed SVs replenish the ECP during stimulation and the RP after tetanic stimulation. Replenishment of the ECP depends on Ca2+ influx from external solutions, and that of the RP is initiated by Ca2+ release from internal stores. Thus, SV dynamics is closely involved in modulation of synaptic efficacy and influences synaptic plasticity.

Experience-Dependent Formation and Recruitment of Large Vesicles from Reserve Pool

The sizes and contents of transmitter-filled vesicles have been shown to vary depending on experimental manipulations resulting in altered quantal sizes. However, whether such a presynaptic regulation of quantal size can be induced under physiological conditions as a potential alternative mechanism to alter the strength of synaptic transmission is unknown. Here we show that presynaptic vesicles of glutamatergic synapses of Drosophila neuromuscular junctions increase in size as a result of high natural crawling activities of larvae, leading to larger quantal sizes and enhanced evoked synaptic transmission. We further show that these larger vesicles are formed during a period of enhanced replenishment of the reserve pool of vesicles, from which they are recruited via a PKA- and actin-dependent mechanism. Our results demonstrate that natural behavior can induce the formation, recruitment, and release of larger vesicles in an experience-dependent manner and hence provide evidence for an additional mechanism of synaptic potentiation.

Inhibition of nerve- and agrin-induced acetylcholine receptor clustering on Xenopus muscle cells in culture

During an early stage of neuromuscular junction formation the nerve induces acetylcholine (ACh) receptors to accumulate at the contact area. To elucidate the induction process we tested various glycosaminoglycans for their ability to inhibit nerve-induced receptor accumulation. The potency sequence was found as follows: fucyoidin > dextran sulfate > heparin = heparan sulfate > chondroitin sulfate type A and B. This sequence is similar to that for agrin-induced receptor clustering in chick myotubes [J. Neurosci., 10 (1990) 3576-3582], suggesting that agrin-like molecules are involved in nerve-induced receptor accumulation in the Xenopus system. We further tested whether agrin in the culture medium competes with the endogenous inducing substance and found that agrin partially inhibited nerve-induced receptor accumulation. We compared nerve- and agrin-induced receptor accumulation under various experimental conditions. Generally, they behaved similarly except in the presence of heparin. Heparin in the culture medium partially blocked nerve-induced receptor accumulation, whereas it totally inhibited agrin-induced receptor clustering. Our observations are consistent with the hypothesis that an agrin-like molecule released by the nerve is the induction signal for receptor accumulation in the Xenopus system.

Adenylyl cyclase encoded by AC78C participates in sugar perception in Drosophila melanogaster

In gustatory receptor neurons (GRNs) in Drosophila melanogaster, Gr5a and one of the Gr64s encode sugar receptors with seven transmembrane domains. Previously, we have shown that the responses to various sugars are depressed in DGsα mutant flies ( Ueno et al., 2006 ). Because DGsα is a homolog of Gs, we hypothesized that the sugar receptors are coupled to adenylyl cyclase (AC) in Drosophila. The aim of this study was to identify the AC that participates in sugar perception. Here, we found that an AC inhibitor, MDL‐12330A, depressed the response in GRNs to trehalose as well as sucrose; that an AC gene, AC78C, was expressed in the sugar‐sensitive GRNs; that RNAi against AC78C depressed the electrical response in GRNs to sucrose; and that the sugar response in GRNs, as well as sugar intake in a behavioral assay in an AC78C mutant, was depressed at low sugar concentrations. We conclude that AC78C, via cAMP, participates in the sugar‐taste signaling pathway at the low concentration range.

Two types of Ca2+ channel linked to two endocytic pathways coordinately maintain synaptic transmission at the Drosophila synapse

Endocytosis at the presynaptic terminal is initiated by Ca2+ influx through voltage‐gated Ca2+ channels. At the Drosophila neuromuscular junction, we demonstrated two components of endocytosis linked to distinct Ca2+ channels. A voltage‐gated Ca2+ channel blocker, (R)‐(+)‐Bay K8644 (R‐BayK), selectively blocked one component (R‐BayK‐sensitive component) without affecting exocytosis, while low concentrations of La3+ preferentially depressed the other component (La3+ ‐sensitive component). In a temperature‐sensitive mutant, shibirets, at non‐permissive temperatures, dynamin clusters were found immunohistochemically at the active zone (AZ) during the R‐BayK‐sensitive endocytosis, while they were detected at the non‐AZ during the La3+‐sensitive endocytosis. Immunostaining of the Ca2+ channel α2δ subunit encoded by straightjacket (stj) was found within the AZ, and a mutation in stj depressed the R‐BayK‐sensitive component but enhanced the La3+ ‐sensitive one, indicating that the α2δ subunit is associated with the R‐BayK‐sensitive Ca2+ channel. Filipin bound to the non‐AZ membrane and inhibited the La3+ ‐sensitive component, but not the R‐BayK‐sensitive one. We concluded that the R‐BayK‐sensitive component of endocytosis occurred at the AZ and termed this AZ endocytosis. We also concluded that the La3+ ‐sensitive component occurred at the non‐AZ and termed this non‐AZ endocytosis. These two types of endocytosis were modulated by various drugs towards opposite directions, indicating that they were differentially regulated. During high‐frequency stimulation, AZ endocytosis operated mainly in the early phase, whereas non‐AZ endocytosis operated in the late phase. Thus, intense synaptic transmission is coordinately maintained by synaptic vesicle recycling initiated by Ca2+ influx through the two types of Ca2+ channel.

Action potentials and sodium inward currents of developing neurons in Xenopus nerve-muscle cultures

Action potentials and voltage-gated Na+ inward currents from cultured embryonic neurons of Xenopus laevis were recorded using the patch-clamp technique in the whole cell configuration. Neurons together with muscle cells were dissociated from embryos shortly after completion of gastrulation. Under the voltage-clamp condition the voltage-gated Na+ inward current was isolated from other currents by pharmacological means and by ion substitution. A small Na+ current was observed in round cells without neurites (presumptive neurons). The mean amplitude of the peak Na+ current was 2.5 times larger in neurons with short processes than in presumptive neurons. As they developed further by extending longer processes, the maximum amplitude of the Na+ inward current recorded at the soma decreased. In varicosities, the Na+ inward current density was greater than that at the soma of neurons with extended neurites but kinetic properties and voltage-dependency were similar.

Vesicle Trafficking and Recycling at The Neuromuscular Junction: Two Pathways for Endocytosis

Publisher Summary Two types of endocytoses have been demonstrated by electron microscopy (EM) at the Drosophila neuromuscular junction (MNJ): (1) active zone endocytosis that occurs at the presynaptic active zone facing the specialized postsynaptic membrane and (2) nonactive zone endocytosis that operates at the area away from the active zone. It has been found that two separate types of Ca2+ channels, with different pharmacological properties, support these two types of endocytoses. Nonactive zone endocytosis is blocked by low concentrations of La3+, while active zone endocytosis is inhibited by a spider toxin—plectreurys toxin II (PLTXII). Once synaptic vesicles (SVs) are formed by endocytosis, they are delivered to two SV pools: (1) the exo/endo cycling pool (ECP) that consists of the functionally defined readily releasable pool (RRP) and the immediately releasable pool (IRP) and (2) the reserve pool (RP). With low-frequency nerve stimulation, SVs in the ECP are released and endocytosed vesicles are delivered to the same pool. During high-frequency tetanic stimulation, SVs from the RP are recruited and maintain synaptic transmission.

Developmental Changes in Acetylcholine Receptor Channel Properties of Vertebrate Skeletal Muscle

In 1972 Katz and Miledi reported by analyzing the acetylcholine (ACh)-induced voltage noise in frog skeletal muscle that the elementary voltage changes were greater in denervated muscle than in innervated muscle. This observation was interpreted as an increased duration of the underlying elementary conductance change following denervation. This was later substantiated by noise analysis of ACh-induced membrane current (Neher and Sak-mann, 1976a). The duration of the elementary current calculated from ACh-induced current noise at the extrajunctional membrane was about four times longer and somewhat smaller in the unitary size than the junctional counterpart. Thus, this was the first indication that two types of channels exist in adult frog muscle following denervation. Subsequently, embryonic rat muscle, which has receptors in the extrajunctional region as well as at the junction, was found to have two types of ACh receptor channels in noise analysis studies (Sakmann and Brenner, 1978; Schuetze, 1980). It was found that embryonic-type channels (which were similar to the extrajunctional channels on denervated muscle) were either replaced by or modified into adult-type channels with a shorter apparent mean open time.

Initial uncoordinated expression of three types of voltage-gated currents in cultured Xenopus myocytes

The expression of three types of voltage-gated ionic currents, namely the Na+ inward current, K+ outward rectifier current and K+ inward rectifier current, was examined in cultured developing Xenopus myocytes. In the population of myocytes, the Na+ inward current and K+ outward rectifier current appeared at around 32 h after fertilization (stage 27) and gradually increased during the period of observation, up to more than 44 h after fertilization (stage 33/34). The developmental time course of these two types of currents was similar. However, during the transition period individual myocytes did not necessarily express these two types of currents in a coordinated fashion. Some myocytes had a large Na+ inward current and small K+ outward rectifier current or vice versa. The K+ inward rectifier current was observed in some cells earlier than stage 27 and also gradually increased during the observation period. The initial expression of this current was not correlated with the K+ outward rectifier current or with the Na+ inward current.