Lobster Neuromuscular Junction Research Articles

Voltage‐gated sodium channel isoform‐specific effects of pompilidotoxins

Pompilidotoxins (PMTXs, alpha and beta) are small peptides consisting of 13 amino acids purified from the venom of the solitary wasps Anoplius samariensis (alpha-PMTX) and Batozonellus maculifrons (beta-PMTX). They are known to facilitate synaptic transmission in the lobster neuromuscular junction, and to slow sodium channel inactivation. By using beta-PMTX, alpha-PMTX and four synthetic analogs with amino acid changes, we conducted a thorough study of the effects of PMTXs on sodium current inactivation in seven mammalian voltage-gated sodium channel (VGSC) isoforms and one insect VGSC (DmNa(v)1). By evaluating three components of which the inactivating current is composed (fast, slow and steady-state components), we could distinguish three distinct groups of PMTX effects. The first group concerned the insect and Na(v)1.6 channels, which showed a large increase in the steady-state current component without any increase in the slow component. Moreover, the dose-dependent increase in this steady-state component was correlated with the dose-dependent decrease in the fast component. A second group of effects concerned the Na(v)1.1, Na(v)1.2, Na(v)1.3 and Na(v)1.7 isoforms, which responded with a large increase in the slow component, and showed only a small steady-state component. As with the first group of effects, the slow component was dose-dependent and correlated with the decrease in the fast component. Finally, a third group of effects concerned Na(v)1.4 and Na(v)1.5, which did not show any change in the slow or steady-state component. These data shed light on the complex and intriguing behavior of VGSCs in response to PMTXs, helping us to better understand the molecular determinants explaining isoform-specific effects.

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.

Hyperosmolarity reduces facilitation by a Ca(2+)-independent mechanism at the lobster neuromuscular junction: possible depletion of the releasable pool.

1. At the crustacean neuromuscular junction, action potential-evoked neurosecretion increases in proportion to stimulation frequency, a process termed frequency facilitation. In the present study we examined how frequency facilitation is affected by osmotic pressure. 2. Hypertonic solution (HS) was applied by local superfusion of the synaptic area. Quantal release was monitored by focal extracellular recordings of postsynaptic potentials. Several stimulation frequencies (f) in the range from 1 to 10 Hz were employed, and quantal content (m) together with the number of releasable units (n) and release probability (p) was evaluated for each frequency. 3. Osmotic pressure enhanced quantal release at the lowest f tested (1 Hz) but suppressed neurosecretion at higher f (7-10 Hz). Thus, hyperosmolarity enhanced action potential-evoked release but suppressed frequency facilitation. 4. Chelation of intracellular calcium by BAPTA showed that the effect of HS was calcium independent. 5. Binomial analysis of quantal content revealed that HS suppressed the increase in the number of releasable units, which was very pronounced during facilitation under control conditions. Since HS also stimulated asynchronous quantal release, the observed effect of HS on facilitation can be explained by the depletion of the releasable pool of quanta caused by the asynchronous neurosecretion. 6. To test this hypothesis we increased the available pool of vesicles using serotonin and demonstrated that the suppressing effect of HS on facilitation was reversed. 7. The observed effects of HS on facilitated neurosecretion could be described quantitatively using our model for mobilization of vesicles into the releasable pool enhanced by action potentials.

Novel Wasp Toxin Discriminates between Neuronal and Cardiac Sodium Channels

Pompilidotoxins (PMTXs), derived from the venom of solitary wasp has been known to facilitate synaptic transmission in the lobster neuromuscular junction, and a recent further study from rat trigeminal neurons revealed that the toxin slows Na+ channel inactivation without modifying activation process. Here we report that beta-PMTX modifies rat brain type II Na+ channel alpha-subunit (rBII) expressed in human embryonic kidney cells but fails to act on the rat heart alpha-subunit (rH1) at similar concentrations. We constructed a series of chimeric mutants of rBII and rH1 Na+ channels and compared modification of the steady-state Na+ currents by beta-PMTX. We found that a difference in a single amino acid between Glu-1616 in rBII and Gln-1615 in rH1 at the extracellular loop of D4S3-S4 is crucial for the action of beta-PMTX. PMTXs, which are small peptides with 13 amino acids, would be a potential tool for exploring a new functional moiety of Na+ channels.

Facilitation of neurotransmitter release at the spiny lobster neuromuscular junction.

Stimulation-induced facilitation of transmitter release was examined at spiny lobster (Palinurus japonicus) neuromuscular junctions by measuring excitatory junctional potentials (EJPs). We found three components of facilitation with the decay time constants about 16, 200 and 1000 ms, respectively. The decay time constants of the nerve terminal free Ca(2+) concentration after stimulation agreed well with the two slower time constants of facilitation. The relationship among the three components of facilitation and a yet slower component, S, was investigated on the basis of several models. The models that have a multiplicative relationship between any two components of facilitation, and the additive model between all components could not account for the results of experiments. Only the 'unified power model', which assumes that facilitation and S are described by the 3-4th power of the sum of underlying components, could account for both the growth process of EJPs during stimulation and the effects of Ca(2+)-chelators. Loading Ca(2+)-chelators, BAPTA and EGTA, into the presynaptic terminals resulted in reduction, but not elimination, of any component of the facilitation. These results suggest that in the spiny lobster neuromuscular junction, the 'unified power model' can describe the relationship between three components of facilitation and S, and that residual free Ca(2+) enhances all three components of facilitation, although it may not be essential for them.

Ca 2+ dynamics of the presynaptic nerve terminal and short-term plasticity at the spiny lobster neuromuscular junction

Ca 2+ dynamics of the presynaptic nerve terminal and short-term plasticity at the spiny lobster neuromuscular junction

Asynchrony of quantal events in evoked multiquantal responses indicates presynaptic quantal interaction.

Asynchrony of quantal events in evoked multiquantal responses indicates presynaptic quantal interaction. We have analyzed the possibility of quantal interactions by inspecting action potential-evoked postsynaptic multiquantal responses recorded extracellularly from the lobster neuromuscular junction. These recorded responses were compared with simulated multiquantal responses constructed from statistically independent quantal events. The simulated multiquantal responses were generated by random superposition of single quantal responses aligned according to the timing of the action potential. The methods of analysis consisted of 1) the comparison of quantal contents obtained from direct counting or by measuring of the size of the responses and 2) the analysis of distributions of quantal latencies. This analysis revealed a large error in the detection of quantal events for responses simulated with no quantal interaction. In contrast, very few errors in quantal detection were made in the analysis of experimental recordings. Latency histograms of recorded responses demonstrate that the proportion of late quantal events (those with latencies of >/=5 ms) increased as a function of quantal content. This shift in latency histograms was not observed for simulated responses. Our interpretation is that quanta interact presynaptically to cause asynchrony of quantal events in evoked responses.

Cooperative Ca 2+ Removal from Presynaptic Terminals of the Spiny Lobster Neuromuscular Junction

Stimulation-induced changes in presynaptic free calcium concentration ([Ca 2+] i) were examined by fluorescent imaging at the spiny lobster excitor motor nerve terminals. The Ca 2+ removal process in the terminal was analyzed based on a single compartment model, under the assumption that the Ca 2+ removal rate from the terminal cytoplasm is proportional to nth power of [Ca 2+] i. During 100 nerve stimuli at 10–100 Hz, [Ca 2+] i reached a plateau that increased in a less-than-linear way with stimulation frequency, and the power index, n, was about 2. In the decay time course after stimulation, n changed with the number of stimuli from about 1.4 after 10 stimuli to about 2 after 100 stimuli. With the change of n from 1.4 to 2, the rate became larger at high [Ca 2+] i (>1.5 μM), but was smaller at low [Ca 2+] i (<1 μM). These results suggest that a cooperative Ca 2+ removal mechanism of n = 2, such as mitochondria, may play an important role in the terminal. This view is supported by the gradual increase in the [Ca 2+] i plateau during long-term stimulation at 20–50 Hz for 60 s and by the existence of a very slow [Ca 2+] i recovery process after this stimulation, both of which may be due to accumulation of Ca 2+ in the organelle.

Facilitation at the lobster neuromuscular junction: a stimulus-dependent mobilization model.

Frequency facilitation is a process whereby neurosecretion increases as a function of stimulation frequency during repetitive synaptic activity. To examine the physiological basis underlying facilitation, we have estimated the frequency dependence of the synaptic parameters n (number of units capable of responding to a nerve impulse) and P (average probability of responding) at the lobster neuromuscular junction. Both n and P increase as a function of frequency, suggesting that the efficiency of quantal docking and quantal fusion is regulated by repetitive synaptic activity. In experiments in which facilitation is strong and quantal content does not saturate over the frequency range tested, the value of P saturates at low frequencies of stimulation, and increases in quantal content at higher frequencies of stimulation are due to an increase in n. Therefore the value of P does not limit facilitation. We propose that transmitter release is limited by the rates of quantal mobilization and demobilization, and that each excitatory stimulus causes additional mobilization of quanta to dock at the presynaptic release sites. In such a model the binomial parameter n will correspond to the number of quanta docked at the release sites and available for release. We have developed and solved kinetic equations that describe how the number of docked quanta changes as a function of time and of stimulation frequency. The stimulus-dependent mobilization model of facilitation predicts that the reciprocal value of the quantal content depends linearly on the reciprocal product of the stimulation frequency and the probability of release. Fits of the experimental data confirm the accuracy of this prediction, showing that the model proposed here quantitatively describes frequency facilitation. The model predicts that high rates of quantal demobilization will produce strong frequency facilitation.

Intracellular calcium and short term plasticity at presynaptic terminals of the spiny lobster neuromuscular junction

This study used the peptides ω-conotoxin GVIA, ω-agatoxin IVA and ω-conotoxin MVIIC, singly and in combination, to investigate the relative involvement of N-, P- and Q-type voltage-operated calcium channels in the control of striatal dopamine release. Electrically stimulated dopamine release was measured by fast cyclic voltammetry at carbon fibre microelectrodes in rat striatal slices. The contribution of these channel subtypes was compared in dorsolateral and medial neostriatum for ‘regular’ (discrete) and ‘burst’ stimulation modalities. In dorsolateral neostriatum, a role for N-, P- and Q-type channels was demonstrated for discrete stimulations, whilst at least one other unidentified channel was also involved in dopamine release on ‘burst’ stimulations. Similarly, in the medial axis of the neostriatum, N-, P- and Q-type channels were involved in dopamine release for discrete stimulations, and N-, Q- and at least one other channel type for ‘burst’ stimulations. However, blockade of P-type channels had no effect on dopamine release for ‘burst’ stimulations in the medial axis. In both regions and stimulation paradigms, N-type channels played a greater role than P/Q-type channels. In the medial axis of the neostriatum there was a smaller contribution by N- and P-type channels and the unidentified component, but a greater Q-type contribution to DA release. ‘Burst’ stimulations induced a lesser involvement of N- and P-type channels than discrete stimulations, and a greater role of the unidentified component. In summary, this study suggests that there is heterogeneity in the distribution of functional voltage-operated calcium channel subtypes in the neostriatum, and differences in subtype recruitment for different firing patterns.

349 Intracellular calcium and short term plasticity at presynaptic terminals of the spiny lobster neuromuscular junction

349 Intracellular calcium and short term plasticity at presynaptic terminals of the spiny lobster neuromuscular junction

240 Rapid calcium imaging at presynaptic terminals of the spiny lobster neuromuscular junction

240 Rapid calcium imaging at presynaptic terminals of the spiny lobster neuromuscular junction

Protein phosphatase inhibitor okadaic acid enhances transmitter release at neuromuscular junctions.

To test the hypothesis that continual phosphorylation and dephosphorylation of protein components of nerve terminals might be important determinants of synaptic efficacy, the effect of okadaic acid, a potent natural inhibitor of two serine threonine protein phosphatases (phosphatase 1 and phosphatase 2A), was examined on synaptic transmission at frog (cholinergic) and lobster (glutamatergic and GABAergic) neuromuscular junctions. At frog junctions, the addition of 1 microM okadaic acid to the extracellular fluid caused almost a doubling of the amplitude of the end-plate potential. The effect of okadaic acid was reversible. Quantal analysis showed that the augmenting effect was presynaptic, resulting from an increase in the number of quanta of transmitter released by a nerve impulse. Where was no significant change in the amplitude of spontaneously liberated miniature end-plate potentials, but their frequency of release increased in parallel with the increase in amplitude of the nerve-evoked synaptic potential. Similar studies with lobster neuromuscular junctions showed increases in the size of both excitatory and inhibitory synaptic responses that were similar in magnitude to the effects seen in the frog junctions. No significant changes in membrane potential or in input resistance accompanied the increased response size. These results suggest that transmitter release at a variety of junctions using different transmitters is constantly modulated by phosphorylation and dephosphorylation of important protein components within nerve terminals.

The effect of xanthine/xanthine oxidase generated reactive oxygen species on synaptic transmission.

The effect of reactive oxygen species generated by the interaction of xanthine and xanthine oxidase on synaptic transmission was examined at the squid giant synapse and the lobster neuromuscular junction. Exposure of these synaptic regions to xanthine/xanthine oxidase produced a significant depression in evoked release, with no change in either resting membrane properties or in the action potential. Addition of catalase to the xanthine/xanthine oxidase-containing media partially blocked the synaptic depression, indicating that H2O2 contributes to the synaptic changes induced by exposure to xanthine/xanthine oxidase. H2O2 applied directly to the perfusing media also produced a decrease in synaptic efficacy. The results demonstrate that reactive oxygen species, in general, depress evoked synaptic transmission.

Evidence for reduced presynaptic Ca2+ entry in a lobster neuromuscular junction at high pressure.

1. Previous studies have shown that hyperbaric pressure depresses synaptic transmission and have suggested that the effect is primarily on transmitter release. The present study analysed the effects of pressure at a crustacean neuromuscular junction. Changes in pressure were compared to changes in extracellular calcium concentration [Ca2+]o with respect to effects on excitatory junction potential (EJP) amplitude, time course, facilitation and potentiation. 2. The effects of 10.1 MPa pressure on EJP amplitude, facilitation and potentiation, but not time course, were mimicked by reducing [Ca2+]o to approximately one-half the normal level. 3. The effects of pressure and the interaction between compression and calcium concentration were analysed in terms of a model of transmitter release. The model assumes that release is dependent on internal calcium concentration, as modulated by both influx and removal processes; that calcium influx is a saturating function of [Ca2+]o; and that release and removal are saturating functions of [Ca2+]i. 4. The results were consistent with the hypothesis that increased pressure acts primarily to reduce calcium influx into the nerve terminal.

Quantal analysis of synaptic potentials in neurons of the central nervous system.

This review concentrates on the results of quantal analysis at central synapses, particularly where the morphology and the electrophysiology of the same synaptic connection has been investigated. Results from quantal analysis at peripheral synapses are drawn on to help resolve some issues. The major topics covered in this review are the properties of the quantal synaptic potential and the junctional mechanisms involved in its generation

Cyclic AMP only partially mediates the actions of serotonin at lobster neuromuscular junctions

Serotonin (5-HT) has multiple physiological actions at lobster neuromuscular junctions, including facilitation of transmitter release from nerve terminals and an increase in the tone and excitability of muscle fibers. These physiological effects of 5-HT are accompanied by a rise in intracellular levels of cAMP. We have used combined biochemical and physiological approaches to investigate whether cAMP directly mediates the physiological actions of the hormone. Based on the following lines of evidence, we conclude that the postsynaptic increase in muscle tone occurs independently of cAMP and that while the cyclic nucleotide does play a role in the facilitation of transmitter release by 5-HT, there is also a cAMP-independent component to this facilitation. (1) Agents that mimic the action of 5-HT on cAMP levels (forskolin, IBMX, SQ20009, 8-bromo cAMP) fail to mimic the postsynaptic actions of the amine. These agents do facilitate transmitter release, although none of them has as large an effect as does 5-HT. (2) When 5-HT is removed, presynaptic facilitation decays as the sum of 2 exponentials with very different time courses. The rate of the slower process is similar to the rate of breakdown of cAMP, while the faster process and the postsynaptic response decay significantly more rapidly. (3) IBMX retards the breakdown of cAMP and simultaneously retards the decay of the slower presynaptic process, with little or no effect on the other responses. (4) IBMX and forskolin potentiate the effect of 5-HT on cAMP levels and selectively enhance the slowly decaying presynaptic component with little or no effect on the other responses.

Strengthening of synaptic inputs after elimination of a single neurone innervating the same target.

The problem of 'competition' between neurones innervating the same target can be studied in simple neural systems such as the central nervous system of the leech and the lobster neuromuscular junction. Intracellular injection of pronase to kill selectively a single neurone shows that, in the leech, removal of one neurone is a sufficient signal to produce compensatory changes. After removal of a given neurone, only neurones of the same function respond to innervate the 'vacant territory'. This was shown both for a motor neurone (annulus erector) and sensory neurones (T or N). Thus the response is very specific. The lobster neuromuscular junction, with its multiple excitatory and inhibitory innervation, has advantages for the study of changes in synaptic efficacy of the remaining neurones after removal of a defined neurone releasing the same or a different transmitter. Killing the inhibitory neurone produced prolongation of the excitatory synaptic current because of a prolonged channel open time. When an excitatory axon is killed the remaining excitatory axon releases more transmitter. Over a period of 10 days, there is first a strengthening of existing synapses, then the appearance of new release sites and sprouting. Only those terminals of a neurone that innervate a territory with reduced innervation become stronger, while other terminals of the same axon remain normal. Cutting of axons produces different responses from those seen after killing single neurones.

Effect of a spider toxin (JSTX) on excitatory postsynaptic current at neuromuscular synapse of spiny lobster.

1. We studied the blocking properties of a spider (Nephila clavata) toxin (JSTX) purified from venom on the spiny lobster neuromuscular junction. 2. When a small amount of JSTX was applied to the neuromuscular junction, the excitatory postsynaptic potential (EPSP) was partially suppressed. The amplitude of EPSPs remained at a steady level for several hours during the washing of the preparation, showing that the action of JSTX is irreversible. 3. We recorded the excitatory postsynaptic current (EPSC) from synaptic site using a macro-patch electrode. The amplitude of EPSC increased linearly with hyperpolarization of the membrane potential in the presence and absence of JSTX. 4. The decay phase time constant of EPSC and spontaneous EPSC was decreased by hyperpolarizing the membrane potential both in the absence and in the presence of JSTX. The relationship between the decay time constant and the membrane potential was not modified by JSTX. 5. It is suggested that JSTX irreversibly blocks EPSC by acting on the site that is apart from the ionic channel of the glutamate receptor molecule.

Chemoreceptors of Crustaceans: Similarities to Receptors for Neuroactive Substances in Internal Tissues

A description is given of crustacean chemosensory systems and the neurophysiological procedures used to study them. Their response properties and tuning characteristics are discussed. A review is then provided of specific crustacean chemoreceptors that are stimulated selectively by either purine nucleotides, taurine, glutamate, or glycine, all of which have neuroactive properties in internal tissues. Two distinctly different types of purinergic chemoreceptors occur on the antennules of the spiny lobster. P1-like chemoreceptors have a potency sequence of AMP greater than ADP greater than ATP greater than adenosine and show a strict structural requirement for the ribose phosphate moiety. P2-like chemoreceptors have a potency sequence of ATP greater than ADP greater than AMP or adenosine and show a broad sensitivity to nucleotide triphosphates with modifications in both the purine and ribose phosphate moieties. Sensilla containing the dendrites of chemosensory neurons also possess an ectonucleotidase(s) that inactivates excitatory nucleotides to yield adenosine which is subsequently internalized by a sensillar uptake system. Narrowly tuned taurinergic chemoreceptors are present on both the antennules and legs of lobsters. Although taurine itself is the most effective stimulant, the taurine analogs hypotaurine and beta-alanine are also very excitatory. Structure-activity studies indicate these chemoreceptors have marked similarities to taurine-sensitive systems in internal tissues of vertebrates. By contrast, comparative studies of glutamatergic chemoreceptors on the legs of lobsters indicate response spectra different from those of the glutamate receptors in lobster neuromuscular junctions and the three classes of excitatory amino acid receptors identified internally in vertebrates. Crustacean chemoreceptors for glycine, ecdysteroids, and pyridine are also described. The hypothesis that receptors for internal neuroactive agents may have originally evolved as external chemoreceptors of primitive aquatic organisms is discussed.