Squid Giant Synapse Research Articles

A post-docking role for synaptobrevin in synaptic vesicle fusion

We have used the squid giant synapse to determine the role of synaptobrevin, integral membrane proteins of small synaptic vesicles, in neurotransmitter release. The sequence of squid synaptobrevin, deduced by cDNA cloning, is 65%–68% identical to mammalian isoforms and includes the conserved cleavage site for tetanus and botulinum B toxins. Injection of either toxin into squid nerve terminals caused a slow, irreversible inhibition of release without affecting the Ca 2+ signal which triggers release. Microinjection of a recombinant protein corresponding to the cytoplasmic domain of synaptobrevin produced a more rapid and reversible inhibition of release, whereas two smaller peptide fragments were without effect. Electron microscopy of tetanus-injected terminals revealed an increased number of both docked and undocked synaptic vesicles. These data indicate that synaptobrevin participates in neurotransmitter release at a step between vesicle docking and fusion.

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.

Spatiotemporal pattern of quantal release of ATP and noradrenaline from sympathetic nerves: consequences for neuromuscular transmission.

The recent explosive development in research concerning the fundamental mechanisms of synaptic transmission helps put the present paper in context. It is now evident that not all transmitter vesicles in a nerve terminal, not even all those docked at its active zones, are immediately available for release (36). We watch, fascinated, the unraveling of the amazingly complex cellular mechanisms and molecular machinery that determine whether or not a vesicle is "exocytosis-competent" (77,78,39,79). Studies on quantal release in different systems show that neurons are fundamentally similar in one respect: that transmitter release from individual active zones is monoquantal (2). But they also show that active zones in different neurons differ drastically in the probability of monoquantal release and in the number of quanta immediately available for release (3). This implies that one should not extrapolate directly from transmitter release in one set of presynaptic terminals (e.g., in neuromuscular endplate or squid giant synapse) to that in other nerve terminals, especially if they have a very different morphology. As shown here, one should not even extrapolate from transmitter release in sympathetic nerves in one tissue (e.g., rat tail artery) to that in other tissues or species (e.g., mouse vas deferens). It is noteworthy that most studies of quantal release are based on electrophysiological analysis and therefore deal with release of fast, ionotropic transmitters from small synaptic vesicles at the active zones, especially in neurons in which these events may be examined with high resolution (49,48,46,33,32). Such data are useful as general models of the release of both fast and slow transmitters from small synaptic vesicles at active zones in other systems, provided that these transmitters are released in parallel, as are apparently ATP and NA in sympathetic nerves. They tell us little or nothing, however, about the release of transmitters (e.g., neuropeptides) from the large vesicles, nor about the spatiotemporal pattern of monoquantal release from small synaptic vesicles in the many neurons that have boutons-en-passent terminals. They show that the time course of effector responses to fast, rapidly inactivated transmitters such as ACh or ATP is necessarily release related. But they do not even address the possibility that the effector responses to slow transmitters such as NA, co-released from the same terminals, may obey completely different rules and perhaps rather be clearance related (7).(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of calcium concentration microdomains at the active zone in the squid giant synapse.

Localization of calcium concentration microdomains at the active zone in the squid giant synapse.

Postsynaptic activation at the squid giant synapse by photolytic release of L-glutamate from a 'caged' L-glutamate.

1. Pharmacological evidence suggests L-glutamate is a strong candidate as a transmitter at the giant synapse of the squid. Postsynaptic activation at the giant synapse cannot be effected by conventional application of putative neurotransmitters by iontophoresis or perfusion, apparently because the complex structure of the synapse prevents a sufficiently rapid change in concentration at the postsynaptic membrane. Flash photolytic release of L-glutamate from a pharmacologically inert 'caged' L-glutamate pre-equilibrated in the stellate ganglion of Alloteuthis or Loligo was used to determine whether L-glutamate can produce postsynaptic activation when released rapidly in the synaptic clefts. 2. The preparation, reaction mechanism and properties of the caged L-glutamate, N-1-(2-nitrophenyl)ethoxycarbonyl-L-glutamate, are described. The product quantum yield on photolysis was 0.65 (+/- 0.05). On flash photolysis glutamate release followed a single exponential time-course in the pH range 5.5-7.8. The rate constant was proportional to [H+] and was 93 s-1 at pH 5.5 and 16 degrees C in artificial sea water (ionic strength, I = 0.68 M). 3. At pH 7.8 flash photolysis of caged glutamate pre-equilibrated in the synapse caused only a slow depolarization. A second photolytic release of L-glutamate or transsynaptic activation produced no further depolarization, suggesting desensitization and inactivation of postsynaptic mechanisms by the initial pulse of L-glutamate. 4. Synaptic transmission in the giant synapse was normal at pH 5.5. Flash photolysis at pH 5.5 caused rapid production of L-glutamate within the synaptic cleft and a fast postsynaptic depolarization which generated postsynaptic action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

A functional role for GTP-binding proteins in synaptic vesicle cycling.

The squid giant synapse was used to test the hypothesis that guanosine-5'-triphosphate (GTP)-binding proteins regulate the local distribution of synaptic vesicles within nerve terminals. Presynaptic injection of the nonhydrolyzable GTP analog GTP gamma S irreversibly inhibited neurotransmitter release without changing either the size of the calcium signals produced by presynaptic action potentials or the number of synaptic vesicles docked at presynaptic active zones. Neurotransmitter release was also inhibited by injection of the nonhydrolyzable guanosine diphosphate (GDP) analog GDP beta S but not by injection of AIF4-. These results suggest that a small molecular weight GTP-binding protein directs the docking of synaptic vesicles that occurs before calcium-dependent neurotransmitter release. Depletion of undocked synaptic vesicles by GTP gamma S indicates that additional GTP-binding proteins function in the terminal at other steps responsible for synaptic vesicle replenishment.

Theoretical simulation of the calcium action potential in squid giant synapse: Repetitive stimulation

A biophysical model of the experimentally observed calcium action potential (CAP) in squid giant synapse is proposed. Whereas the inclusion of the inward calcium current in the Hodgkin-Huxley model can generate the rising phase of CAP, to account for the observed termination of the action potential, a repolarizing process needs to be introduced. Adding a term representing Ca-activated K current, the observed features of CAP can be reproduced. However, one feature of CAP, namely the gradual shortening of the plateau duration on repetitive stimulation, cannot be simulated by this model. In this paper, it is proved that both the termination of the action potential and the gradual shortening of the plateau cannot be accounted for by inclusion of a single repolarizing process. One more repolarizing process, namely a slow voltage-dependent Ca-inactivation, is therefore proposed to account for all the observed features of CAP.

FMRFamide-related peptides potentiate transmission at the squid giant synapse.

The stellate ganglion of the squid Loligo pealli contains the neuropeptides Phe-Met-Arg-Phe-NH2 (FMRFamide), Phe-Leu-Arg-Phe-NH2 (FLRFamide) and at least one N-terminally extended FMRFamide-related peptide that is yet to be fully characterized. Both local application and arterial perfusion of FLRFamide potentiate transmission at the giant synapse. The N-terminally related peptide Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) produced a similar effect. The threshold for both the tetra- and the hepta-peptides was less than 10 microM. Potentiation could be detected as an increase in rate of rise of the EPSPs, as an increase in amplitude of the EPSP in the absence of spikes, or under voltage clamp as an increase in the EPSC. The effect was most pronounced when the synapse was fatigued by high frequency stimulation. Another molluscan peptide, eledoisin and also leucine enkephalin were without effect. In the absence of any detectable effects of FLRFamide on the resting membrane potential of either pre- or postsynaptic terminals or on the presynaptic spike, it is suggested that the peptide influences transmitter mobilization. However, the peptide could also exert small changes in preterminal calcium currents, which so far we have been unable to detect.

Oxygen dependency of synaptic transmission at the squid Loligo pealei giant synapse

1. 1. Synaptic transmission at the squid giant synapse was blocked in 45 min by exposure to 0.02 atm oxygen but was maintained for more than 90 min in air (0.21 atm oxygen) or pure oxygen (1 atm). 2. 2. Excitatory post-synaptic potential amplitude decreased in 0.02 atm oxygen but did not change in either 1 or 0.21 atm oxygen. Fast facilitation was increased in 0.02 atm oxygen only. 3. 3. Post-synaptic resting membrane potential ( V m and input resistance ( R o) remained unchanged in 1 or 0.21 atm oxygen but R o was increased in 0.02 atm oxygen. 4. 4. Our results suggest that severe hypoxia decreased the release of transmitter from the pre-synaptic terminal.

Presynaptic calcium concentration microdomains and transmitter release

n-Aequorin J, a luminescent protein which responds to calcium concentration changes in the order of several hundred micromoles, was injected into the preterminal fiber in the squid giant synapse. The activation of the presynaptic terminal leading to release of transmitter was accompanied by light emission at well-defined sites at the active zone in the presynaptic terminal. Location of these light emission sites was very much the same from one stimulus to the next, indicating that light emission was triggered by the inward calcium current occurring at specific and invariant locations. The distribution, size and number of these QEDs (quantum emission domains) coincides well with the location and number of active zones in the presynaptic terminal. The results imply that transmitter release is triggered by very well-localized calcium concentration changes that may be as high as several hundred micromoles.

Role of residual calcium in synaptic depression and posttetanic potentiation: Fast and slow calcium signaling in nerve terminals

Trains of action potentials evoked rises in presynaptic Ca 2+ concentration ([Ca 2+] i) at the squid giant synapse. These increases in [Ca 2+] i were spatially nonuniform during the trains, but rapidly equilibrated after the trains and slowly declined over hundreds of seconds. The trains also elicited synaptic depression and augmentation, both of which developed during stimulation and declined within a few seconds afterward. Microinjection of the Ca 2+ buffer EGTA into presynaptic terminals had no effect on transmitter release or synaptic depression. However, EGTA injection effectively blocked both the persistent Ca 2+ signals and augmentation. These results suggest that transmitter release is triggered by a large, brief, and sharply localized rise in [Ca 2+] i, while augmentation is produced by a smaller, slower, and more diffuse rise in [Ca 2+] i.

Inhibitory Effects of Nonhydrolyzable Guanine Nucleotides on Neurotransmitter Release at the Squid Giant Synapse.

We are examining the role of GTP-binding regulatory proteins in the control of neurotransmitter release. To this end, we injected two nonhydrolyzable guanine nucleotides into the giant presynaptic terminal of the most distal synapse of the stellate ganglion of the squid, Loligo pealei, and studied their effects on transmitter release evoked by action potentials. As an assay of release, the maximum rate of rise of postsynaptic potentials (PSPs) produced by intracellular stimulation of the presynaptic neuron was measured. The terminal was also pre-loaded with the Ca-sensitive dye Furato perform dual wavelength measurements of presynaptic free Ca2+ ion concentration, ([Ca*+]J. Iontophoretic injection of GTPyS into the giant presynaptic terminal caused a marked depression of synaptic transmission. The rate of rise of PSPs began to decrease within a few minutes after the onset of injection and continued progressively for several tens of minutes after injection has been stopped. One hundred microCoulomb injections inhibited transmission by 60-80%. An upper estimate of the intraterminal concentration of injected nucleotide is in the high micromolar range. Additional injections did not seem to add to the inhibition. Inhibition of synaptic transmission was not accompanied by any significant changes in the electrical properties of the presynaptic terminal: neither presynaptic resting nor action potentials were affected by GTPyS injections. Further, the changes in the presynaptic [Ca2’li induced by a train of 250 action potentials (5s 50 Hz) were not affected by injection of GTPyS. There was also no steady depolarization of the postsynaptic membrane potential, suggesting that GTPyS did not increase the tonic release of MPSPs. Therefore, GTPrS acts by altering the amount of transmitter release evoked by a given rise in intracellular calcium, rather than by regulating Ca2+ entry into the terminal. We also evaluated the possibility that a low molecular weight GTP-binding protein is involved in this inhibition. A nonhydrolyzable analogue of GDP, GDPPS, was therefore injected into the terminal. GDPS i.e., visible inhibition of synaptic transmission required injections lasting about five-fold longer than those of GTPyS (about 30 min). This could be due to the lower electric charge of GDPpS (-3 as compared to -4 for GTP$S) causing a smaller quantity of the nucleotide being inotophoretitally injected. Alternatively, GDPpS could be weaker or slower in inhibiting release. Like GTPyS, GDP/IS had no significant effect on presynaptic potentials or [Ca2+]i signals. 1.4 I I

Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse

A number of calcium buffers were examined for their ability to reduce evoked transmitter release when injected into the presynaptic terminal of the squid giant synapse. Injection of EGTA was virtually ineffective at reducing transmitter release, even at estimated intracellular concentrations up to 80 mM. Conversely, the buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), which has an equilibrium affinity for calcium similar to that of EGTA at pH 7.2, produced a substantial reduction in transmitter release when injected presynaptically. This effect of BAPTA was reversible, presumably because the buffer diffused out of the terminal and into uninjected regions of the presynaptic axon. BAPTA derivatives with estimated intracellular calcium dissociation constants (Kd) ranging from 0.18 to 4.9 microM were effective at reducing transmitter release at similar estimated concentrations. A BAPTA derivative with an estimated intracellular Kd of 31 mM was less effective. BAPTA did not affect presynaptic action potentials or calcium spikes in ways that could explain its ability to reduce transmitter release. The relative effects of presynaptic injections of BAPTA and derivatives are consistent with the calcium-buffering capability of these compounds if the presynaptic calcium transient that triggers release is hundreds of microM or larger. The superior potency of BAPTA compared to EGTA apparently results from the faster calcium-binding kinetics of BAPTA and suggests that the calcium-binding molecule that triggers release binds calcium in considerably less than 200 microsec and is located very close to calcium channels.

Regulation by synapsin I and Ca(2+)‐calmodulin‐dependent protein kinase II of the transmitter release in squid giant synapse.

1. Presynaptic or simultaneous pre- and postsynaptic voltage-clamp protocols were implemented in the squid giant synapse in order to determine the magnitude and time course of the presynaptic calcium current (ICa) and its relation to transmitter release before and after presynaptic injection of proteins. These included several forms of synapsin I, calcium-calmodulin-dependent protein kinase II (CaM kinase II) and avidin. 2. The quantities and location of these proteins were monitored by fluorescence video-enhanced microscopy during the electrophysiological measurements. 3. Presynaptic injection of dephosphorylated synapsin I inhibited synaptic transmission with a time course consistent with diffusion of the protein through the terminal and action at the active release zone. A mathematical model relating the diffusion of synapsin I into the terminal with transmitter release was developed to aid in the interpretation of these results. 4. Synapsin I inhibition of transmitter release was reversible. 5. The action of synapsin I was highly specific, as phosphorylation of the tail region only or head and tail regions prevented synapsin I from inhibiting release. 6. Injections of heat-treated synapsin I or of avidin, a protein with a size and isoelectric point similar to those of synapsin I, had no effect on transmitter release. 7. CaM kinase II injected presynaptically was found to facilitate transmitter release. This facilitation, which could be as large as 700% of the control response, was related to the level of penetration of the enzyme along the length of the preterminal A mathematical model of this facilitation indicates a reasonable fit between the distribution of CaM kinase II within the terminal and the degree of facilitation. 8. The overall shape of the postsynaptic response was not modified by either synapsin I or CaM kinase II injection. 9. The data suggest that, in addition to releasing transmitter, calcium also penetrates the presynaptic cytosol and activates CaM kinase II. When activated, CaM kinase II phosphorylates synapsin I, which reduces its binding to vesicles and/or cytoskeletal structures, enabling more vesicles to be released during a presynaptic depolarization. The amplitude of the postsynaptic response will then be both directly and indirectly regulated by depolarization induced Ca2+ influx. This model provides a molecular mechanism for synaptic potentiation.

Theoretical simulation of the calcium action potential in squid giant synapse: The plateau termination

A biophysical model is proposed for the simulation of the experimentally observed calcium action potential at the squid giant synapse. It is observed that while Ca activation at the synapse is responsible for the generation of the upstroke of the action potential, a repolarizing process needs to be invoked to simulate the plateau termination and other long-time effects. Out of the likely candidates, the Ca-activated K current has been chosen as the most plausible repolarizing process. The model can reproduce all the observed features of calcium action potential excepting its behaviour after repetitive stimulation.

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.

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.

Regulation of transmitter release at the squid giant synapse by presynaptic delayed rectifier potassium current.

1. The three-microelectrode voltage clamp technique and pharmacological agents were used to examine the properties and functions of potassium currents in squid giant presynaptic terminals. 2. Outward currents consisted of two components: a slow component which activated over hundreds of milliseconds and was blocked by extracellular application of tetraethylammonium (TEA) ions and a more rapidly activating component which was relatively insensitive to extracellular TEA. 3. The more rapid component was studied in isolation by treating presynaptic terminals with extracellular TEA, as well as tetrodotoxin (to block sodium channel currents) and manganese (to block calcium channel currents). The magnitude of this current component was 1-2 mA cm-2 at 0 mV. Rates of activation and deactivation were voltage dependent and little evidence of inactivation was seen for depolarizations less than several seconds in duration. 4. The reversal potential of the current was -70 to -80 mV in normal saline and became more positive with elevated extracellular potassium concentrations, suggesting that potassium is the primary permeant ion. Accumulation of extracellular potassium appeared to be marked during depolarizations that produced significant activation of the current. 5. Extracellular application of 3,4-diaminopyridine (DAP) blocked the current with an apparent dissociation constant of 7 microM at 0 mV. Intracellular applications of DAP and TEA also were effective in reducing this current. These treatments, but not extracellular TEA application, broadened presynaptic action potentials and increased the magnitude and time-to-peak of postsynaptic currents elicited by the broadened presynaptic action potentials. Postsynaptic currents were a sensitive and linear function of action potential duration; a 30% increase in action potential duration increased postsynaptic current amplitude by 190%. 6. Estimation of the magnitude and time course of the presynaptic calcium current, based on previous measurements of calcium channel gating, indicated that action potential broadening produces a large increase in calcium current magnitude. These calculations predict that a 30% increase in presynaptic action potential duration will increase the peak amplitude of the calcium current by approximately 170% and the total amount of calcium entry by approximately 230%. This implies a linear relationship between transmitter release and calcium entry during an action potential and can be explained by assuming that calcium co-operatively triggers release within intracellular domains that do not overlap.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of synapsin I and calcium/calmodulin-dependent protein kinase II on spontaneous neurotransmitter release in the squid giant synapse.

The molecular events that control synaptic vesicle availability in chemical synaptic junctions have not been fully clarified. Among the protein molecules specifically located in presynaptic terminals, synapsin I and calcium/calmodulin-dependent protein kinase II (CaM kinase II) have been shown to modulate evoked transmitter release in the squid giant synapse. In the present study, analysis of synaptic noise in this chemical junction was used to determine whether these proteins also play a role in the control of spontaneous and enhanced spontaneous transmitter release. Injections of dephosphorylated synapsin I into the presynaptic terminal reduced the rate of spontaneous and enhanced quantal release, whereas injection of phosphorylated synapsin I did not modify such release. By contrast CaM kinase II injection increased enhanced miniature release without affecting spontaneous miniature frequency. These results support the view that dephosphorylated synapsin I "cages" synaptic vesicles while CaM kinase II, by phosphorylating synapsin I, "decages" these organelles and increases their availability for release without affecting the release mechanism itself.

Classification of presynaptic calcium channels at the squid giant synapse: neither T-, L- nor N-type

We examined both pharmacological and functional characteristics of the calcium channels which trigger transmitter secretion from giant nerve terminals of squid. These calcium channels are insensitive to organic calcium channel blockers such as dihydropyridines and ω-conotoxin GVIA, moderately sensitive to cadmium, activated by very small depolarizations and slowly inactivating. We conclude that the characteristics of these presynaptic channels do not correspond to the properties of T-, L-, or N-type calcium channels found in other cells.