[My research life: from synaptic transmission to behavior].

I have studied signal transmission at synapses and the effects of drugs on it at the molecular and cellular levels. Specific areas of research interest are outlined here. 1) Electrophysiological experiments in cats and rabbits suggested that a new type of analgesic, the phenothiazine derivative levomepromazine, exerts analgesic effects by depressing emotional responses accompanying the sensation of pain.

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 Ca(2+) influx through voltage-gated Ca(2+) channels. At the Drosophila neuromuscular junction, we demonstrated two components of endocytosis linked to distinct Ca(2+) channels. A voltage-gated Ca(2+) channel blocker, (R)-(+)-Bay K8644 (R-BayK), selectively blocked one component (R-BayK-sensitive component) without affecting exocytosis, while low concentrations of La(3+) preferentially depressed the other component (La(3+) -sensitive component).

Bicaudal-D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling.

Cargo transport by microtubule-based motors is essential for cell organisation and function. The Bicaudal-D (BicD) protein participates in the transport of a subset of cargoes by the minus-end-directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system.

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.

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.

Synaptic vesicle pools and plasticity of synaptic transmission at the Drosophila synapse.

Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus, this synapse offers an excellent model for studies of the molecular mechanism of synaptic transmission and synaptic plasticity. Two synaptic vesicle (SV) pools have been identified and characterized using a fluorescent styryl dye, FM1-43, to stain SVs.

Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability.

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function.

Repetitive exposures to nicotine induce a hyper-responsiveness via the cAMP/PKA/CREB signal pathway in Drosophila.

Nicotine, in addition to acute effects, has long-lasting effects on mammalian behaviors, such as those leading to addiction. Here we present genetic and pharmacological evidence in Drosophila suggesting that repetitive exposures to nicotine induce a hyper-responsiveness through synthesis of new protein(s) via CREB-mediated gene transcription. Single exposure to volatilized nicotine dose-dependently inhibited the startle-induced climbing response.

Two synaptic vesicle pools, vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction.

Drosophila neuromuscular junctions ( D NMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools.

Ca2+ influx through distinct routes controls exocytosis and endocytosis at drosophila presynaptic terminals.

Endocytosis of synaptic vesicles follows exocytosis, and both processes require external Ca(2+). However, it is not known whether Ca(2+) influx through one route initiates both processes. At larval Drosophila neuromuscular junctions, we separately measured exocytosis and endocytosis using FM1-43.

Selective replenishment of two vesicle pools depends on the source of Ca2+ at the Drosophila synapse.

After synaptic vesicles (SVs) undergo exocytosis, SV pools are replenished by recycling SVs at nerve terminals. At Drosophila neuromuscular synapses, there are two distinct SV pools (i.e.

Extension of glial processes by activation of Ca2+-permeable AMPA receptor channels.

AMPA type-glutamate receptor channels (AMPARs) assembled without the GluR2 (GluR-B) subunit are characterized by high Ca2+ permeability, and are expressed abundantly in cerebellar Bergmann glial cells. Here we show that the morphology of cultured Bergmann glia-like fusiform cells derived from the rat cerebellum was changed by manipulating expression of Ca2+-permeable AMPARs using adenoviral vector-mediated gene transfer. Converting endogenous Ca2+-permeable AMPARs into Ca2+-impermeable channels by viral-mediated transfer of GluR2 gene induced retraction of glial processes.

Synaptic development is controlled in the periactive zones of Drosophila synapses.

A cell-adhesion molecule fasciclin 2 (FAS2), which is required for synaptic growth and still life (SIF), an activator of RAC, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. BetaPS integrin and discs large (DLG), both involved in synaptic development, also decorated the zone. However, shibire (SHI), the Drosophila dynamin that regulates endocytosis, was found in the distinct region.

Tetanic stimulation recruits vesicles from reserve pool via a cAMP-mediated process in Drosophila synapses.

At Drosophila neuromuscular junctions, there are two synaptic vesicle pools, namely the exo/endo cycling pool (ECP) and the reserve pool (RP). We studied the recruitment process from RP using a fluorescent dye, FMI-43. During high-frequency nerve stimulation, vesicles in RP were recruited for release, and endocytosed vesicles were incorporated into both pools, whereas with low-frequency stimulation, vesicles were incorporated into and released from ECP.

Activation of metabotropic glutamate receptors enhances synaptic transmission at the Drosophila neuromuscular junction.

We examined the effects of activation of metabotropic glutamate receptors (mGluRs) on glutamatergic synaptic transmission at the neuromuscular junction of newly hatched Drosophila larvae. In nominally Ca(2+)-free solutions puff-application of low concentrations of glutamate evoked a transient frequency increase of miniature synaptic currents (mSCs). The mean amplitude of mSCs was unaffected, suggesting that this effect was presynaptic.

The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.

According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool.

Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire.

In a temperature-sensitive Drosophila mutant, shibire, synaptic vesicles are completely depleted in nerve terminals after stimulation at 34 degrees C, but upon returning to 22 degrees C, endocytosis resumes. In this study, synaptic vesicles in the boutons of nerve terminals at the mutant neuromuscular junction were loaded with a fluorescent dye, FM1-43, during vesicle reformation at 22 degrees C after complete depletion at 34 degrees C. We found two distinct pools of synaptic vesicles, namely an exo/endo cycling pool, located in the periphery of the bouton, and a reserve pool, located in its center.

Altered bcl-2 and bax expression and intracellular Ca2+ signaling in apoptosis of pancreatic cells and the impairment of glucose-induced insulin secretion.

Apoptosis is the process of cellular self-destruction, and genes such as bcl-2 and bax are known to inhibit and promote apoptosis, respectively. In this study, we show that apoptosis can be induced in pancreatic beta-cell lines, and we investigate the apoptotic pathways through the bcl-2 and bax genes and intracellular Ca2+. Serum deprivation induces apoptosis in the MIN6 and RINm5F pancreatic beta-cell lines, and alters the bcl-2 messenger RNA (mRNA) and protein.

An inhibitory role of calcineurin in endocytosis of synaptic vesicles at nerve terminals of Drosophila larvae.

In this study, we tested a hypothesis that activation of calcineurin, Ca2+/calmodulin-dependent protein phosphatase 2B, is an initiating signal for synaptic vesicle endocytosis. We examined effects of calcineurin inhibitors, cyclosporin A or FK506 and calmodulin inhibitors on stimulus-induced FM1-43 uptake into nerve terminals of Drosophila larvae. Fluorescent FM1-43 labels recycling synaptic vesicles in nerve terminals.

Suramin inhibits glucose-induced Ca2+ response in single rat pancreatic beta cells.

It is reported that glucose releases ATP with insulin from pancreatic beta cells. Suramin (50-200 microM), a purinoceptor blocker, reversibly and dose-dependently inhibited the rise in cytoplasmic calcium concentration ([Ca2+]i) induced by glucose and by ATP in single beta cells. Suramin did not inhibit the glucose-induced increase in NAD(P)H fluorescence.

PACAP/VIP receptors in pancreatic beta-cells: their roles in insulin secretion.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide belonging to the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family. We have isolated a third PACAP receptor subtype, designated PACAPR-3, by molecular cloning. The cDNA encoding PACAPR-3 has been isolated from a mouse insulin-secreting beta-cell line MIN6 cDNA library.

A monitor of secretion from single pancreatic beta-cells.

ATP is known to be coreleased with insulin from pancreatic beta-cells. To monitor insulin secretion from single beta-cells, a single beta-cell was surrounded in culture by Fura 2-loaded calf pulmonary artery endothelium (CPAE) cells, which can detect the ATP. CPAE cells did not respond with an elevation in cytoplasmic calcium concentration ([Ca2+]i) to either tolbutamide (100 mumol/l) or kainate (1 mmol/l) but did respond with an elevation in [Ca2+]i to ATP (0.

Expression and role of ionotropic glutamate receptors in pancreatic islet cells.

Although the excitatory amino acid glutamate and its receptors play crucial roles in many functions of the central nervous system (CNS), their presence in the peripheral tissues has remained unclear. In the present study, we have identified kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) receptor subtype mRNAs in pancreatic islets, using reverse transcriptase polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) measurements and electrophysiological recordings indicate that kainate, AMPA, and NMDA all elicit increases of [Ca2+]i in single pancreatic beta-cells and depolarize them.

Functional neuronal ionotropic glutamate receptors are expressed in the non-neuronal cell line MIN6.

We report that a non-neuronal cell line, MIN6, derived from insulin-secreting pancreatic beta-cells, naturally expresses functional ionotropic glutamate receptors. Electrophysiological recordings show that kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) depolarize single MIN6 cells and evoke inward ionic currents. These agents also increase the intracellular calcium concentration in MIN6 cells.