Lobster Neuromuscular Synapse Research Articles

Recruitment of presynaptic vesicles and facilitation of transmitter release

Facilitation, an enhancement of neuronal secretion by repetitive stimulation, is due to an increase in the number of vesicles that fuse with the presynaptic membrane and release neurotransmitter in response to an action potential. We performed Monte-Carlo simulation of calcium-dependent priming and release of synaptic vesicles and simulated facilitation of transmitter release. The model incorporated the pools of docked, primed, and released vesicles and stochastic calcium-dependent transitions between these pools. The model was validated by comparison of the time-course of transmitter release, its variability, and the magnitude of facilitation with electrophysiology measurements of transmitter release at the lobster neuromuscular synapse.

Neuromuscular synapses on the dactyl opener muscle of the lobster Homarus americanus

The crustacean dactyl opener neuromuscular system has been studied extensively as a model system that exhibits several forms of synaptic plasticity. We report the ultrastructural features of the synapses on dactyl opener of the lobster (Homarus americanus) as determined by examination of serial thin sections. Several innervation sites supplied by an inhibitory motoneuron have been observed without nearby excitatory innervation, indicating that excitatory and inhibitory inputs to the muscle are not always closely matched. The ultrastructural features of the lobster synapses are generally similar to those described previously for the homologous crayfish muscle, with one major distinction: few dense bars are seen at the presynaptic membranes of these lobster synapses. The majority of the lobster neuromuscular synapses lack dense bars altogether, and the mean number of dense bars per synapse is relatively low. In view of the finding that the physiology of the lobster dactyl opener synapses is similar to that reported for crayfish, these ultrastructural observations suggest that the structural complexity of the synapses may not be a critical factor determining synaptic plasticity.

Recruitment of synapses in the neurosecretory process during long-term facilitation at the lobster neuromuscular junction

We investigated long-term facilitation at the lobster neuromuscular synapse employing a combination of FM1-43 staining of synaptic vesicles, electron microscopy analysis, and electrical recordings of synaptic activity. Synaptic terminals were loaded with the fluorescent dye FM1-43 producing clusters of activity-dependent fluorescent spots. Electron microscopy analysis of synaptic ultrastructure suggested that fluorescent spots represent compartments of synaptic terminals filled with vesicles. Excitatory postsynaptic currents were recorded from the stained synaptic terminals using focal macropatch electrodes. Terminals were stained during the nerve stimulation at a low stimulation frequency (2, 5 or 10Hz) before and after long-term facilitation was elicited by high-frequency stimulation (20 or 30Hz for 5 min). We found that staining after long-term facilitation results in the appearance of new fluorescent spots, as well as in the increase in fluorescence of the spots that appeared before long-term facilitation. This increase in fluorescence accounted for the increase in quantal release. Activation of individual fluorescent spots was found to be non-uniform. In spite of overall increase in fluorescence, some synaptic compartments decreased their staining after long-term facilitation. Thus, our study demonstrates that long-term facilitation produces non-uniform activation of FM1-43 uptake in synaptic compartments that correlates with the increase in quantal neurosecretion.

Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse

At the crustacean neuromuscular junction, facilitation elicited by a repetitive stimulation reaches a plateau level that is proportional to the stimulation frequency. In the present study we demonstrated that plateau facilitation (Fplateau) does not depend on Ca2+ manipulations. We manipulated Ca2+ concentration in the following ways: (1) applying cell permeable chelators BAPTA-AM or EGTA-AM; (2) decreasing Ca2+ concentration in the extracellular media; (3) enhancing Ca2+ influx by 4-aminipyridin. We found that neither Fplateau is decreased by lowering Ca2+ nor it is increased by enhancing Ca2+ influx. In contrast, facilitation elicited by a short train of stimuli (Fgrowth) was altered by Ca2+ manipulations. These results suggested that Fplateau does not result from accumulation of free intracellular Ca2+. We hypothesized that Fplateau results from the accumulation of synaptic vesicles properly activated for transmitter release, the readily releasable pool (RRP). To test this hypothesis, we measured the increase in RRP employing local applications of hypertonic solutions (HS). We found that the size of RRP was significantly increased after Fplateau was induced. Our results suggest that facilitation is mediated by two mechanisms: the increase in the residual Ca2+ and the increase in RRP. Frequency facilitation during continuous stimulation, Fplateau, is primarily controlled by the increase in RRP.

Effect of Ca 2+ on presynaptic facilitation

We investigated and modeled the effect of Ca 2+ on presynaptic facilitation. Facilitation of transmitter release from a presynaptic terminal is believed to arise from the local increase of Ca 2+ concentration. We measured and simulated the effect of Ca 2+ on facilitation at the lobster neuromuscular synapse. Facilitation was measured using quantal analysis of postsynaptic potentials recorded focally during trains of stimuli. We found a facilitation component, which was not affected by Ca 2+ concentration. Transmitter release was simulated using a model of two pools of presynaptic vesicles available for release. The simulation reproduced quantitatively Ca 2+-dependent and Ca 2+-independent components of facilitation.

Syntheses and Biological Activities of Joro Spider Toxin Analogs to Spidamine and Joramine.

In order to study the structure-activity relationships of spidamine and joramine found in the venom of Joro spider, Nephila clavata, we attempted to synthesize various analogs. Six analogs were convergently synthesized according to our previous method for the synthesis of spidamine, N-(3-aminopropyl-β-alanyl)-N'-(2, 4-dihydroxy-phenylacetyl-L-asparaginyl)-1, 5-pentanediamine and joramine, N-(3-aminopropyl-β-alanyl)-N'-(4-hydroxyphenyl-acetyl-L-asparaginyl)-1, 5-pentanediamine. The biological activities of the analogs and four intermediates were compared with those of synthetic spidamine and joramine in three bioassay systems, lobster neuromuscular synapse, cockroaches and mosquito larvae. The glutamate receptors in these systems were inhibited by some analogs, and the D-asparagine- or indoleacetyl-containing analogs were found to be strong inhibitors. These compounds have potential application as insecticides.

Large-Scale Purification and Further Characterization of Spidamine and Joramine from Venom of Spider, Nephila clavata.

We purified a new type of polyamine neurotoxins called spidamine and joramine in the venom of a spider, Nephila clavata, on a large-scale and confirmed their structures by chemical analysis and NMR spectra. Spidamine was N-(2, 4-dihydroxyphenylacetyl-L-asparaginyl)-N'-(3-aminopropyl-β-alanyl)-1, 5-pentanediamine, whereas joramine was N-(4-hydroxyphenylacetyl-L-asparaginyl)-N'-(3-aminopropyl-β-alanyl)-1, 5-pentanediamine.We also studied the toxins biological activity on glutamatergic transmission using lobster neuromuscular synapses. The blocking activity of both toxins on the excitatory post synaptic potentials (EPSPs) in the lobster muscle was about one tenth time less than JSTX-3, the most potent toxin in the venom of Nephila clavata. Interestingly, spidamine which has 2, 4-dihydroxyphenyl group showed an irreversible block of EPSPs, whereas joramine possessing 4-hydroxyphenyl group had a reversible block.

Two structural adaptations for regulating transmitter release at lobster neuromuscular synapses

The distal accessory flexor muscle (DAFM) in the lobster (Homarus americanus) walking leg consists of 5 muscle fiber bundles. All five bundles, one proximal, one distal, and 3 medial, are innervated by one excitatory and one inhibitory motor neuron. Both neurons release more transmitter on the distal bundle than on the proximal bundle. The aim of our studies was to investigate the structural basis of this differentiation. Thin sections cut at 50 microns intervals showed a similar number of excitatory synapses on the two bundles. Freeze-fracture views of excitatory synapses showed that synapse size, active zone number per synapse, and intramembrane particle density in the postsynaptic membrane are similar proximally and distally. Active zones at synapses on the distal bundle are larger and contain about 50% more large intramembrane particles, which are thought to include the voltage-gated Ca2+ channels that couple the action potential to transmitter release, than their counterparts on the most proximal bundle. This difference in channel number appears to produce a disproportionate increase in the probability of transmitter release sufficient to account for most of the proximal-distal disparity in the amplitude of the excitatory postsynaptic potential. In contrast, staining the inhibitor for antibodies to the inhibitory neurotransmitter, GABA, showed that it forms more varicosities on the distal bundle than on the proximal bundle. Because most of the synapses are located in the varicosities, differences in synapse number likely regulate the proximal-distal disparity in the amount of inhibitory transmitter released. Therefore, the regional differentiation in the amount of transmitter released in the DAFM appears to be based on two distinct mechanisms. In the inhibitor, transmitter release appears to be regulated differentially by differences in synapse number. In the excitor, transmitter release appears to be regulated differentially from a similar number of synapses by differences in active zone structure.

Presynaptic glutamate receptors depress inhibitory postsynaptic transmission in lobster neuromuscular synapse.

1. We examined the functional role of GTP-coupled glutamate receptor (GluB-R) in the presynaptic membrane of lobster neuromuscular synapse. 2. Injection of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a hydrolysis-resistant analogue of GTP, into the excitatory axon mimicked the presynaptic glutamate response and effectively suppressed excitatory postsynaptic potentials or excitatory postsynaptic currents (EPSCs). 3. Statistical analysis revealed that the coefficient of variation (standard deviation divided by the mean of EPSC amplitude) was increased after injection of GTP gamma S into the excitatory axon, indicating a presynaptic inhibition of transmitter release. 4. The effect of glutamate on inhibitory postsynaptic potentials (IPSPs) or inhibitory postsynaptic currents (IPSCs) was studied when the postsynaptic glutamate receptors were blocked by the Joro spider toxin (JSTX). Glutamate depressed IPSPs or IPSCs in the JSTX-treated preparation. Furthermore, repetitive stimulation of the excitatory nerve produced effective inhibition of IPSCs. 5. Quisqualate and kainate suppressed IPSCs in a similar way to glutamate. In contrast, N-methyl-D-aspartate, ibotenic acid, trans-D,L-1-amino-1,3-cyclopentanedicarboxyloc acid, and 2-amino-4-phosphonobutanate had no effect on GluB-R. 6. Our results indicate that GluB-R, which exists in both excitatory and inhibitory nerve terminals, regulates transmitter release by a presynaptic inhibitory mechanism.

Spider toxin and the glutamate receptors

A neurotoxin (JSTX) was isolated from the venom of spider ( Nephila clavata). JSTX blocked both the excitatory postsynaptic (EPSPs) and glutamate-induced potentials in lobster neuromuscular synapse and squid giant synapse. In mammalian central nervous system, JSTX blocked the EPSPs in CAI pyramidal neurons resulting from stimulation of Schaffer collateral/commissure input. Pharmacological investigation showed that JSTX preferentially suppressed quisqualate/kainate receptor subtypes but was much less effective on NMDA receptor. Using synthesized spider toxins we studied the structure-activity relationship and found that the 2,4 dihydroxyphenylacetyl asparagine in the toxin structure was responsible for suppressive action, while the remaining part containing a polyamine was related to the agonist binding site with the polycationic part enhancing the toxic activity. Labeling of synthesized JSTX was used for histochemical as well as biochemical studies. Using autoradiography, 125I-JSTX-3 was found to bind at the lobster neuromuscular synapse. Histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat cerebellum and hippocampus, JSTX-3-binding protein was purified from rat brain by affinity chromatography. SDS-PAGE of the affinity purified protein showed at least 4 bands ranging from 40 to 70 kDa.

Suppression of inhibitory postsynaptic potentials by presynaptic glutamate receptors (glutamate B) in the lobster neuromuscular synapse

Suppression of inhibitory postsynaptic potentials by presynaptic glutamate receptors (glutamate B) in the lobster neuromuscular synapse

日本産クモ毒腺中の生物活性アミンの定量および液体クロマトグラフィーによる毒成分の比較

Catecholamine and polyamine as well as serotonin and histamine contained in spider venom glands of nine typical Japanese species were quantified. Spider venoms generally appeared poor in either catecholamine and serotonin but rich in polyamine. However, only Chiracanthium japonicum was distinguishable since large amounts of norepinephrine, epinephrine and serotonin were contained. We also examined chromatographic elution patterns of the venomous substances by using high performance liquid chromatography with a reversed-phase column, and found that a venom fraction from the spiders belonging to genus Agelena blocked lobster neuromuscular synapse in an irreversible manner.

Characterization of Glutamate Receptor by Spider Toxin

AbstractRecent study has shown that the venom of some orb-web spiders contain potent blockers of the glutamate receptors. Joro spider toxin (JSTX) derived from Nephila clavata has been found to block excitatory postsynaptic potentials and glutamate-evoked responses in the neuromuscular synapse of crustacea, the squid giant synapse and the mammalian brain synapse. Structures of the toxins (JSTXs, NSTXs) of spiders belonging to the genus Nephila were determined and it was found that a unique 2,4-dihydroxyphenylacetyl asparaginyl cadaverine part was conserved between all toxins, indicating that this part is intimately involved in the blocking activity.Labeling of synthesized JSTX-3 was used for histological investigation of glutamate receptors. Using autoradiography 125I-JSTX-3 was found to bind at the lobster neuromuscular synapse. A histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat hippocampus and cerebellum. JSTX-3 was used for iso...

G protein is coupled to presynaptic glutamate and GABA receptors in lobster neuromuscular synapse

1. We have examined the effects of L-glutamate and gamma-aminobutyric acid (GABA) on the presynaptic membrane of spiny lobster by the use of intra-axonal recording near the nerve terminals. 2. Application of glutamate to the synaptic region produced hyperpolarization in the presynaptic membrane but depolarization in the postsynaptic membrane. The presynaptic glutamate potential (PGP) is generated by an activation of K+ channels, as evidenced by its dependence on external K+ concentration. 3. The PGP was not affected by a spider toxin (JSTX), which blocks the postsynaptic glutamate receptor. By contrast, pertussis toxin (IAP) effectively blocked the PGP without affecting the resting conductance channels or action potentials in the presynaptic membrane. 4. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a hydrolysis-resistant analogue of GTP, blocked the PGP, suggesting the involvement of a G protein in the generation of K+ current. 5. Application of GABA induced depolarization or hyperpolarization in the presynaptic axon depending on the resting membrane potential. By reducing external Cl-, GABA-induced hyperpolarizations were converted to depolarizations, indicating that they are mainly mediated by Cl-. 6. In contrast to GABA, baclofen consistently induced hyperpolarization in low Cl- solution as well as in normal solution. Baclofen-induced hyperpolarization was blocked by IAP, indicating the mediation of G protein. 7. These results suggest that the presynaptic membrane of lobster neuromuscular synapse has entirely different types of amino-acid receptors from those in the postsynaptic membrane. Both the excitatory and the inhibitory axonal membrane have glutamate ("glutamateB") and GABAB receptors, which activate K+ channels via G protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc ion enhances the blocking potency of synthetic analogs of spider toxin (JSTX) on the glutamate receptor

The effect of synthetic spider toxin analogs containing aza-crown in combination with zinc and copper was studied on the lobster neuromuscular synapse. The suppressive action of N4-type spider toxin analog (N4) on excitatory postsynaptic potentials (EPSPs) was markedly enhanced in the presence of 10(-5)-10(-4) M Zn2+. Cu2+ (10(-4) M) also had a potentiating effect on the suppression of EPSPs by N4 but to a lesser degree than Zn2+. While N6-type spider toxin analog (N6) suppressed EPSPs more effectively than N4, additional suppression was not pronounced in the presence of Zn2+ or Cu2+. The results suggest that chelatable divalent metal ions play an important role in the blocking mechanism of glutamate receptor by the spider toxin.

An autoradiographic study of binding of iodinated spider toxin to lobster muscle

Distribution of the binding sites of Joro spider toxin (JSTX), a specific inhibitor of the glutamate receptors in the crustacean neuromuscular synapse, was studied by using autoradiography. JSTX was synthesized and made radioactive by conjugation with iodine-125. 125I-JSTX irreversibly blocked the excitatory postsynaptic potentials of the lobster neuromuscular synapse in a similar manner as the natural spider toxin. Light microscopic autoradiography of 125I-JSTX treated muscle showed sporadic aggregates of reduced silver grains on the surface of muscles. Electron microscopy of adjoining ultrathin sections revealed that these spots corresponded to the fraction of sarcolemma apposed to axonal terminals with or without synaptic junctional profiles. This finding gives morphological support to the formulation that JSTX binds to the glutamate receptor-ion channel molecules.

Iodinated Joro toxin (JSTX-3). Its structure and binding to the lobster neuromuscular synapse.

Synthetic Joro toxin (JSTX-3) was iodinated by the lactoperoxidase method and the reaction products were purified by high performance liquid chromatoqraphy. Proton-nuclear magnetic resonance spectroscopy revealed that iodination occurred mainly at position 3 of the benzene ring. The radio-iodinated JSTX-3 appeared autoradiographically to be accumulated in the synapse area of the lobster nerve-muscle preparation.

Pertussis toxin blocks presynaptic glutamate receptors — a novel ‘glutamate B’ receptor in the lobster neuromuscular synapse

Topical application of l-glutamate to the neuromuscular synapse of the lobster walking leg induced K +-dependent hyperpolarization in the presynaptic membrane. This presynaptic glutamate potential (PGP) was insensitive to Joro spider toxin (JSTX), a spider toxin which specifically blocks the postsynaptic glutamate receptor, but was blocked by pertussis toxin island activating protein (IAP) in a dose-dependent manner. IAP had little effect on the resting conductance channels in pre- and postsynaptic membranes. GTPγS, a hydrolysis-resistant analogue of GTP, reduced the PGP supporting the involvement of G-protein in generation of K + activation. The results suggest that a new type of glutamate receptor exists in the presynaptic membrane in the crustacean neuromuscular synapse.

Presynaptic glutamate receptor — possible involvement of a K + channel

Intra-axonal recording was made from the excitatory axon of the lobster walking leg near the nerve terminal and the effects of l-glutamate were studied. Topical application of glutamate to the synapse produced hyperpolarization in the presynaptic membrane with increase in conductance. The glutamate-induced hyperpolarization was reversed to a depolarization at about − 100 mV. The reversal potential was not significantly changed by altering the external Cl − but was shifted to more positive values by increasing the external K +. A spider toxin (JSTX), which specifically blocks the postsynaptic glutamate receptor, failed to block the presynaptic glutamate potential. The results suggest that the presynaptic membrane of the lobster neuromuscular synapse has a different type of glutamate receptor from those in the postsynaptic membrane.

Correlation between presynaptic dense bodies and tranmitter output at lobster neuromuscular terminals by serial section electron microscopy

Lobster neuromuscular terminals releasing comparatively small (low-output type) and large (high-output type) amounts of transmitter but arising from the single excitatory motor axon to the proximal accessory flexor muscle were serially sectioned for electron microscopy. The three-dimensional reconstruction showed the two types of terminals to have a complex branching pattern in which thin branches of the motor axon often enlarged into synapse bearing terminal regions. Quantitative comparison showed that the mean surface area of a synapse is similar in the two types of terminals. However, the low-output terminal has a higher synaptic density and devotes a greater part of its surface area to synapses compared to its high-output counterpart suggesting that transmitter output is not directly related to synaptic area. The mean surface area of a presynaptic dense body is not significantly different between low- and high-output synapses, but there is a significantly greater density of these active zones in the high-output terminal. This results in the ratio of mean dense body area to mean synaptic area being approximately 3 × greater in the high-output synapses than the low-output ones. This significant difference in the surface area of presynaptic dense bodies between low- and high-output synapses correlates with the difference in transmitter output at these two synapses, and implicates the dense bodies in the mechanism of transmitter release at lobster neuromuscular synapses.