PubMed Health⌕ Search

Biomedical subjects

L G Wu

Publications and source records attributed to L G Wu.

At least 19 recordsLinked to original sources
PubMed2001

Protein kinase c increases the apparent affinity of the release machinery to Ca2+ by enhancing the release machinery downstream of the Ca2+ sensor.

Modulation of the release probability of releasable vesicles in response to Ca(2+) influx (Prob(Ca)) is involved in mediating several forms of synaptic plasticity, including short-term depression, short-term augmentation, and potentiation induced by protein kinases. Given such an important role, however, the mechanism underlying modulation of the Prob(Ca) is unclear. We addressed this question by investigating how the activation of protein kinase C modulates the Prob(Ca) at a calyx-type nerve terminal in rat brainstem. Various lengths of step depolarization were applied to the nerve terminal to evoke different amounts of Ca(2+) currents and capacitance jumps, the latter of which reflect vesicle release. The relationship between the capacitance jump and the Ca(2+) current integral was sigmoidal and was fit well with a Hill function. The sigmoidal relationship was shifted significantly to the left during the application of the PKC activator 12-myristate 13-acetate (PMA), suggesting that PMA increases the apparent affinity of the release machinery to Ca(2+). This effect was blocked in large part by the application of the PKC inhibitor bisindolylmaleimide, suggesting that the effect is mediated mainly by the activation of PKC. We also found that PMA increased the rate of miniature EPSCs evoked by the application of hypertonic sucrose solution, which triggers release downstream of the Ca(2+) influx. Taken together, our results suggest that PKC enhances the apparent affinity of the release machinery to Ca(2+) by a mechanism downstream of the binding between Ca(2+) and its sensor. These results have provided the first example of the mechanisms underlying modulation of the Prob(Ca).

Animals↗
PubMed2001

Fast kinetics of exocytosis revealed by simultaneous measurements of presynaptic capacitance and postsynaptic currents at a central synapse.

The rate of release from nerve terminals depends on both the number of release sites and the rate of release at each site. The latter remains largely unknown at central synapses. We addressed this issue by simultaneously measuring the nerve terminal membrane capacitance and the postsynaptic current at single calyceal synapses in rat brainstem. We found that a 10 ms presynaptic step depolarization depleted a releasable pool containing 3300-5200 vesicles. Released vesicles were endocytosed with a time constant of a few seconds to tens of seconds. Release of only one third of this pool saturated both postsynaptic AMPA and NMDA receptors. A release site can release more than three vesicles in 10 ms (>300 vesicles per second). We conclude that both a large number of release sites and a fast release rate at each site enable synapses to release at a high rate.

Animals↗
PubMed1999

Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses.

We studied how Ca2+ influx through different subtypes of Ca2+ channels couples to release at a calyx-type terminal in the rat medial nucleus of the trapezoid body by simultaneously measuring the presynaptic Ca2+ influx evoked by a single action potential and the EPSC. Application of subtype-specific toxins showed that Ca2+ channels of the P/Q-, N-, and R-type controlled glutamate release at a single terminal. The Ca2+ influx through the P/Q-type channels triggered release more effectively than Ca2+ influx through N- or R-type channels. We investigated mechanisms that contributed to these differences in effectiveness. Electrophysiological experiments suggested that individual release sites were controlled by all three subtypes of Ca2+ channels. Immunocytochemical staining indicated, however, that a substantial fraction of N- and R-type channels was located distant from release sites. Although these distant channels contributed to the Ca2+ influx into the terminal, they may not contribute to release. Taken together, the results suggest that the Ca2+ influx into the calyx via N- and R-type channels triggers release less effectively than that via P/Q-type because a substantial fraction of the N- and R-type channels in the calyx is localized distant from release sites.

Action Potentials↗
PubMed1999

Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.

The fluorescent probe FM1-43 has been used extensively for imaging vesicle recycling; however, high nonspecific adsorption resulting in elevated background levels has precluded its use in certain tissues, notably brain slices. We have found that a sulfobutylated derivative of beta-cyclodextrin (ADVASEP-7) has a higher affinity for FM1-43 than the plasma membrane. ADVASEP-7 was used as a carrier to remove FM1-43 nonspecifically bound to the outer leaflet of the plasma membrane or extracellular molecules, significantly reducing background staining. This has enabled us to visualize synaptic vesicle recycling in the nematode C. elegans, intact lamprey spinal cord, and rat brain slices.

Animals↗
PubMed1999

The reduced release probability of releasable vesicles during recovery from short-term synaptic depression.

Recovery from synaptic depression is believed to depend mainly on replenishment of the releasable pool of vesicles. We observed that during recovery from depression in a calyx-type synapse, part of the releasable pool was replenished rapidly. Half recovery occurred within 1 s, even in the absence of residual calcium. Vesicles that had recently entered the releasable pool had a 7- to 8-fold lower release probability than those that had been in the pool for more than 30 s. These results suggest that the reduction in the release probability of releasable vesicles contributes greatly to the level of depression. How synapses maintain transmission during repetitive firing is in debate. We propose that during repetitive firing, accumulation of intracellular Ca2+ may facilitate release of the rapidly replenished but reluctant vesicles, making them available for sustaining synaptic transmission.

Action Potentials↗
PubMed1999

Spatial variability in release at the frog neuromuscular junction measured with FM1-43.

We quantified the spatial variability in release properties at different synaptic vesicle clusters in frog motor nerve terminals, using a combination of fluorescence and electron microscopy. Individual synaptic vesicle clusters labeled with FM1-43 varied more than 10-fold in initial intensity (integrated FM1-43 fluorescence) and in absolute rate of dye loss during tetanic electrical nerve stimulation. Most of this variability arose because large vesicle clusters spanned more than one presynaptic active zone (inferred from postsynaptic acetylcholine receptor stripes labeled with rhodamine-conjugated alpha-bungarotoxin); when the rate of dye loss was normalized to the length of receptor stripe covered, variability from spot to spot was greatly reduced. In addition, electron microscopic measurements showed that large vesicle clusters (i.e., those spanning multiple active zones) were also thicker, and the increased depth of vesicles led to increased total spot fluorescence without a corresponding increase in the rate of dye loss during stimulation. These results did not reveal the presence of "hot zones" of secretory activity.

Animals↗
PubMed1998

R-type Ca2+ currents evoke transmitter release at a rat central synapse.

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and gamma-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

Action Potentials↗
PubMed1998

Kinetics of synaptic depression and vesicle recycling after tetanic stimulation of frog motor nerve terminals.

We measured the time courses of two key components of the synaptic vesicle cycle during recovery from synaptic depression under different conditions, and used this and other information to create a kinetic model of the vesicle cycle. End plate potential (EPP) amplitudes were used to follow recovery from synaptic depression after different amounts of tetanic stimulation. This provided an estimate of the time course of vesicle mobilization from the reserve pool to the docked (readily releasable) pool. In addition, FM1-43 was used to measure the rate of membrane retrieval after tetanic stimulation, and the amount of membrane transferred to the surface membrane. This provided a measure of the rate of refilling of the reserve pool with recycled vesicles. The time courses of both synaptic depression and endocytosis were slowed by prolonged tetanic stimulation. This behavior could be fitted by a simple model, assuming a first-order kinetics for both vesicle endocytosis and mobilization. The results show that a nearly 20-fold decrease in the rate constant of endocytosis greatly delays refilling of the depleted reserve pool. However, to fully account for the slower recovery of depression, a decrease in the rate constant of vesicle mobilization from the reserve pool of about sixfold is also required.

Animals↗
PubMed1997

Presynaptic calcium dynamics and transmitter release evoked by single action potentials at mammalian central synapses.

The relationship between presynaptic calcium transients ([Ca2+]t) and transmitter release evoked by a single stimulus was both investigated experimentally and modeled at a mammalian central synapse, the CA3 to CA1 pyramidal cell synapse in guinea pig hippocampal slices. In the present study, we compared the low-affinity calcium indicator furaptra with the higher-affinity indicator fura-2. The 10-90% rise time of the furaptra transient was 2.4 ms compared to 7.8 ms with fura-2; the half-decay time (tau 1/2) was 30 ms for furaptra, compared to 238 ms for fura-2. The half-width of the calcium influx was 1.8 ms with furaptra, which provides an upper limit to the duration of the calcium current (ICa) evoked by an action potential. Modeling the decay time course of the furaptra transients led to the conclusion that the predominant endogenous calcium buffer in these terminals must have relatively slow kinetics (kon < 10(5)/M.s), although the presence of small amounts of fast buffers cannot be excluded. The relationship between the [Ca2+]t measured with furaptra and the postsynaptic response was the same as previously observed with fura-2: the postsynaptic response was proportional to about the fourth power (m approximately 4) of the amplitude of either [Ca2+]t or calcium influx. Thus, although fura-2 may be locally saturated by the high local [Ca2+] responsible for transmitter release, the volume-averaged fura-2 signal accurately reflects changes in this local concentration. The result that both indicators gave similar values for the power m constrains the amplitude of calcium influx in our model: Ica < 1 pA for 1 ms.

Action Potentials↗
PubMed1997

Presynaptic inhibition of elicited neurotransmitter release.

Activation of presynaptic receptors for a variety of neurotransmitters and neuromodulators inhibits transmitter release at many synapses. Such presynaptic inhibition might serve as a means of adjusting synaptic strength or preventing excessive transmitter release, or both. Previous evidence showed that presynaptic modulators inhibit Ca2+ channels and activate K+ channels at neuronal somata. These modulators also inhibit spontaneous transmitter release by mechanisms downstream of Ca2+ entry. The relative contribution of the above mechanisms to the inhibition of elicited release has been debated for a long time. Recent evidence at synapses where the relationship between transmitter release and presynaptic Ca2+ influx has been well characterized suggests that inhibition of presynaptic voltage-dependent Ca2+ channels plays the major role in presynaptic inhibition of elicited neurotransmitter release. In addition, modulation of the release machinery might contribute to inhibition of elicited release.

Animals↗
PubMed1996

Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction.

We used FM1-43 imaging and intracellular recordings of synaptic potentials to measure the time course of endocytosis in frog motor nerve terminals following tetanic nerve stimulation, and we used fura-2 imaging of intraterminal Ca2+ concentration to compare endocytic rate and [Ca2+]i. Following a 30 Hz tetanus, endocytosis declined exponentially with a time constant that depended on the duration of stimulation. The level of [Ca2+]i rose from a resting value of about 100 nM to more than 500 nM during 30 Hz stimulation, and rapidly declined to 200-250 nM after stimulation. [Ca2+]i returned to resting level with a time course that, like endocytosis, depended on the duration of tetanic stimulation. However, the rate of [Ca2+]i recovery was much slower than the rate of endocytosis, leading to the conclusion that endocytic rate is not determined solely by the instantaneous level of [Ca2+]i.

Animals↗
PubMed1995

GABAB receptor-mediated presynaptic inhibition in guinea-pig hippocampus is caused by reduction of presynaptic Ca2+ influx.

1. The hypothesis that activation of GABAB receptors inhibits evoked synaptic transmission by reducing the presynaptic Ca2+ influx was tested using a recently developed technique for simultaneously recording the presynaptic Ca2+ transient ([Ca2+]t) and the field excitatory postsynaptic potential (fEPSP) evoked by a single electrical stimulus at CA3 to CA1 synapses of guinea-pig hippocampus. 2. The GABAB receptor agonist baclofen reversibly blocked, in a dose-dependant manner, both the fEPSP and the presynaptic [Ca2+]t with similar time courses. During application of baclofen, the fEPSP was proportional to about the fourth power of the presynaptic [Ca2+]t, and the presynaptic fibre volley and the resting Ca2+ level did not change. These results are similar to those we previously observed following application of several voltage-dependent Ca2+ channel blockers, suggesting that baclofen inhibits the fEPSP by blocking the presynaptic Ca2+ influx. 3. The inhibition by baclofen of both the fEPSP and the presynaptic [Ca2+]t was blocked by the GABAB receptor antagonist CGP 35348, consistent with the causal relationship between the GABAB receptor-mediated presynaptic inhibition of the [Ca2+]t and the fEPSP. 4. The inhibition by baclofen of the [Ca2+]t was partially occluded by application of the voltage-dependent Ca2+ channel blocker omega-conotoxin-GVIA (omega-CgTX-GVIA), but not omega-agatoxin-IVA (omega-AgaTX-IVA), suggesting that baclofen reduces the presynaptic [Ca2+]t by blocking Ca2+ channels including the omega-CgTX-GVIA-sensitive type. 5. We conclude that baclofen inhibits evoked transmitter release by reducing presynaptic Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Block of multiple presynaptic calcium channel types by omega-conotoxin-MVIIC at hippocampal CA3 to CA1 synapses.

1. The effect of the voltage-dependent Ca channel (VDCC) antagonist omega-conotoxin-MVIIC (omega-CTx-MVIIC) on the presynaptic Ca influx and synaptic transmission was studied in area CA1 of guinea pig hippocampus. The presynaptic Ca transient ([Ca]t) and the field excitatory postsynaptic potential (fEPSP) evoked by a single electrical stimulus were simultaneously recorded at CA3 to CA1 synapses. 2. omega-CTx-MVIIC dose dependently blocked the fEPSP and the presynaptic [Ca]t without affecting the presynaptic fiber volley and the presynaptic resting Ca level. During application of omega-CTx-MVIIC, the decrease of both the fEPSP and the presynaptic [Ca]t had a similar time course, and the initial slope of the fEPSP is proportional to about the fourth power of the amplitude of the presynaptic [Ca]t. These results strongly suggest that omega-CTx-MVIIC inhibits the fEPSP by blocking presynaptic Ca channels at hippocampal CA3 to CA1 synapses. 3. Sequential application of high concentrations of omega-CTx-MVIIC (10 microM) and other VDCC blockers including omega-conotoxin-GVIA (omega-CTx-GVIA, 1 microM) and omega-agatoxin-IVA (omega-Aga-IVA, 1 microM) showed that omega-CTx-MVIIC significantly occludes the effects of omega-CTx-GVIA and omega-Aga-IVA. Combined application of omega-CTx-GVIA (1 microM) and omega-Aga-IVA (1 microM) largely but not completely occluded the effect of omega-CTx-MVIIC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus.

Adenosine is a potent modulator of neuronal activity throughout the nervous system. Previous evidence suggests that adenosine presynaptically inhibits synaptic transmission. The mechanism of presynaptic inhibition is uncertain. Adenosine may inhibit transmitter release by reducing voltage-dependent Ca2+ currents, activating K+ currents, or by mechanisms downstream to Ca2+ influx. By simultaneously recording the presynaptic Ca2+ transient ([Ca]t) and the field excitatory postsynaptic potential (fEPSP) at CA3-CA1 synapses of hippocampal slices, we found that adenosine, through activation of presynaptic A1 receptors, inhibits the fEPSP primarily by reducing the [Ca]t. Reduced [Ca]t was due to inhibition of omega-conotoxin GVIA-sensitive and some unidentified Ca2+ channels, probably including Q-type, but not to omega-agatoxin-IVA-sensitive Ca2+ channels. The adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine enhanced both the [Ca]t and the fEPSP, suggesting a tonic inhibition of the [Ca]t by endogenous adenosine in the hippocampal slice.

Adenosine↗
PubMed1994

Antiplatelet aggregation principles of Dendrobium loddigesii.

The MeOH extract of the stem of Dendrobium loddigesii was found to inhibit the aggregation of rabbit platelets induced by arachidonic acid and collagen. Two active compounds, moscatilin [1] and moscatin [2], were isolated. Moscatilin diacetate [3] also exhibited antiplatelet aggregation activity.

Animals↗
PubMed1994

Presynaptic calcium is increased during normal synaptic transmission and paired-pulse facilitation, but not in long-term potentiation in area CA1 of hippocampus.

We examined the relationship between presynaptic calcium levels and postsynaptic potentials during normal synaptic transmission, paired-pulse facilitation (PPF), and long-term potentiation (LTP) in CA3-CA1 synapses of hippocampus. By selectively loading the presynaptic terminals with the calcium indicator fura-2, we simultaneously recorded a presynaptic calcium (Ca) transient and the corresponding field EPSP evoked by a single stimulus given to the Schaffer collateral-commissural pathway in guinea pig hippocampal slices. A volume average presynaptic Ca influx was obtained by taking the first time derivative of the Ca transient. Our data indicate that the synaptic transmission represented by the initial slope of the field EPSP is approximately proportional to the fourth power of the presynaptic Ca influx, the volume average Ca current. Our results in combination with similar findings at the squid giant synapse (Augustine et al., 1985b; Augustine and Charlton, 1986) suggest that the relationship between Ca influx and transmitter release is well conserved from the molluscan to the mammalian nervous system. A transient increase of the residual Ca level ([Ca]res) is generally thought to be the mechanism underlying PPF (Katz and Miledi, 1968; Charlton et al., 1982); however, the relationship between PPF and the presynaptic [Ca]res had not been examined before. Our results demonstrate that PPF is approximately linearly related to the [Ca]res. This finding further supports the residual Ca hypothesis for PPF. Accumulated evidence from other groups suggests that the presynaptic site contributes to the maintenance of LTP in CA3-CA1 synapses (Bekkers and Stevens, 1990; Malinow and Tsien, 1990); however, our data show that neither an increase of the Ca transient nor a sustained increase of the [Ca]res occurs in the presynaptic terminals during maintenance of LTP. This suggests that the presynaptic mechanism underlying LTP must be downstream to Ca influx.

2-Amino-5-phosphonovalerate↗