Squid Giant Synapse Research Articles

Calcium entry and transmitter release at voltage-clamped nerve terminals of squid.

Presynaptic and post-synaptic cells of the squid giant synapse were voltage-clamped simultaneously to study the relationship between presynaptic Ca current and transmitter-induced post-synaptic current (p.s.c.). Local Ca application was used to restrict Ca current and transmitter release to a limited region of the presynaptic terminal and thus minimize errors due to spatial heterogeneity of presynaptic membrane potential. Presynaptic terminals were depolarized by brief (3-6 ms) voltage-clamp pulses of varying amplitude to collect graded series of presynaptic Ca current and p.s.c. records. During presynaptic depolarization at 14 degrees C, Ca current activation preceded initial onset of p.s.c. (on-p.s.c.) by an interval of approximately 1 ms. The main component of on-p.s.c. followed Ca current activation by about 2 ms. The delay between a brief Ca tail current and peak response of the p.s.c. produced after pulse termination (off-p.s.c.) was also approximately 2 ms. Curves relating both Ca current and p.s.c. magnitudes to presynaptic potential were bell shaped with peaks near -10 mV, but the p.s.c. curve showed stronger voltage dependence on both sides of the peak. With very small and very large presynaptic command pulses, Ca current could be observed without measureable p.s.c. Synaptic transfer curves, plotting p.s.c. as a function of presynaptic Ca current, resembled third-power functions. On the average, p.s.c.s fit a curve representing the 2.9 power of Ca current (range 2.4-3.5 in eighteen experiments). Transfer curves consisted of two limbs: one from presynaptic pulses below -10 mV and the other from more positive pulses. These two limbs were similar and generally resembled power functions of identical exponent. It is thus likely that the third-power function accurately reflects synaptic current transfer, rather than interference from some other voltage-dependent process. Power functions fitting small-pulse and large-pulse limbs of some transfer curves had different scale coefficients, even though exponent values were the same. Consideration of synaptic transmission kinetics suggests that the voltage dependence of Ca channel opening rates can probably explain the difference in transfer curve limbs. Our experiments provide no evidence for an intrinsic voltage dependence of the transmitter release process.

Evidence for l-glutamate as the neurotransmitter of the squid giant synapse

The effect of a spider toxin (JSTX), a specific blocker of glutamate receptors, was studied in the squid stellate ganglion. JSTX irreversibly blocked the excitatory postsynaptic potential in a dose-dependent manner. The toxin neither affected the spike in the postsynaptic nor the presynaptic fibers. Spontaneous miniature potentials recorded from thin stellate nerves were suppressed by the toxin. Iontophoretically applied l-glutamate depolarized the postsynaptic membrane of the giant axon and this potential was also blocked by JSTX. Kainic acid also depolarized the postsynaptic membrane but this was partially blocked by JSTX, indicating that JSTX differentiated kainate receptor from glutamate one. The results strongly suggest that l-glutamate is the neurotransmitter in the giant synapse of squid.

Intraterminal injection of synapsin I or calcium/calmodulin-dependent protein kinase II alters neurotransmitter release at the squid giant synapse.

Synapsin I and calcium/calmodulin-dependent protein kinase II were pressure-injected into the preterminal digit of the squid giant synapse to test directly the possible regulation of neurotransmitter release by these substances. Neurotransmitter release was determined by measuring the amplitude, rate of rise, and latency of the postsynaptic potential generated in response to presynaptic depolarizing steps under voltage clamp conditions. Injection of dephosphosynapsin I decreased the amplitude and rate of rise of the postsynaptic potential, whereas injection of either phosphosynapsin I or heat-treated dephosphosynapsin I was without effect. Conversely, injection of calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, increased the rate of rise and amplitude and decreased the latency of the postsynaptic potential. The effects of these proteins were observed without any detectable change in the initial phase of the presynaptic calcium current. A synapsin I-like protein and calcium/calmodulin-dependent protein kinase II were demonstrated by biochemical and immunochemical techniques to be present in squid nervous tissue. The data support the hypothesis that synapsin I regulates the availability of synaptic vesicles for release; we propose that calcium entry into the nerve terminal activates calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, dissociating it from the vesicles and thereby removing a constraint in the release process.

Dependence of cytoplasmic calcium transients on the membrane potential in isolated nerve endings of the guinea pig

The relation of changes in internal, free Ca 2+, measured with arsenazo III, to the membrane potential, measured with the cyanine dye di-SC 2(5) or 86Rb + distribution ratio, was studied in isolated guinea pig cortical nerve endings. Depolarization of the plasma membrane with veratridine or gramicidin as well as addition of ionophore A23187 led to an increase in cytosolic Ca 2+. Only the response to veratridine was inhibited by tetrodotoxin. The dependence of the depolarization-induced increase in intraterminal, free Ca 2+ on the membrane potential between about −50 to 0 mV was sigmoidal. A maximal increase in cytosolic Ca 2+ was reached when the membrane potential was depolarized from the resting level, about −64 mV, to about −40 mV. These results show that in isolated nerve endings the activation of voltage-sensitive Ca 2+ channels concomitantly leads to an increase in cytosolic, free Ca 2+. Comparison of the results of the present study with the previous electrophysiological observations indicate that Ca 2+ channels in synaptosomes, presynaptic nerve terminals of the squid giant synapse and cardiac cells have essentially similar voltage dependency.

Transmission at voltage-clamped giant synapse of the squid: evidence for cooperativity of presynaptic calcium action.

Synaptic transmission was studied at the squid giant synapse with voltage clamp control of both presynaptic and postsynaptic cells. Because presynaptic voltage gradients can complicate interpretation of electrophysiological data obtained from this preparation, we used local Ca application to restrict Ca influx and transmitter release to a short and relatively isopotential portion of the elongated presynaptic terminal. Under these conditions, we found that postsynaptic current varies approximately as the third power of presynaptic Ca current. This finding is consistent with the hypothesis that several Ca ions cooperate in triggering secretion of a single transmitter quantum.

Relationship between presynaptic membrane potential and acetylcholine release in synaptosomes from Torpedo electric organ.

The membrane potential of purely cholinergic synaptosomes isolated from Torpedo electric organ was monitored with fluorescent carbocyanine dyes. An increased fluorescence was associated with depolarization and a quenching with hyperpolarization. Fluorescence data provided evidence that Torpedo synaptosomes have a membrane potential mainly driven by a K+ diffusion potential and a membrane potential of about -50 mV could be estimated after calibration of fluorescence signals with ionophore antibiotics. The release of acetylcholine (ACh) from Torpedo synaptosomes was monitored continuously by measuring the light emitted by a chemiluminescent method (Israël & Lesbats, 1981 a). Using fluorescence data, the release of ACh was expressed as a function of membrane potential. The relationship between presynaptic potential and transmitter release as determined by biochemical methods at cholinergic nerve endings showed striking similarities to that observed at the squid giant synapse. Several substances were also tested with regard to their depolarizing and releasing properties and it was found that the toxin isolated from the venom of the annelid Glycera convoluta, which induced a large increase in quantal release of transmitter (Manaranche, Thieffry, & Israël, 1980) promoted a depolarization of Torpedo synaptosomes in addition to ACh release.

The action of cholinergic agonists on the squid stellate ganglion giant synapse

Although the giant synapse in the squid stellate ganglion has served as a model in the understanding of the ionic and electrical changes that occur during the release of transmitter from nerve terminals, little is known about the pharmacology of this synapse or the identity of its neurotransmitter. In the present study, the suggestion that acetylcholine (ACh) is the excitatory transmitter at this synapse was tested by exploring the actions of cholinergic agents on the pre- and postsynaptic giant axons and on the excitatory postsynaptic potential (EPSP). A novel arterial perfusion technique that circumvents the diffusion barrier from the bathing medium to the synapse has been used to demonstrate a depolarizing action of ACh and its agonist carbachol on the post- but not the presynaptic axon. The cholinergic receptors are distinct from receptors activated by amino acids, such as glutamate, have a reversal potential of about -48 mV, are anion-permeable, and desensitize without blocking the EPSP. It is concluded that these receptors are involved in an inhibitory input onto the postsynaptic giant axon and, therefore, that ACh is most probably not the transmitter at the squid giant synapse.

Divalent cations differentially support transmitter release at the squid giant synapse.

The ability of Ca, Sr and Ba ions to support transmitter release was studied at the squid giant synapse by examining their respective actions on presynaptic current and post-synaptic responses. Transmitter-induced post-synaptic currents were smaller in Sr- than in Ca- containing solutions, and much smaller in Ba-containing solutions. The time course and amplitude of spontaneous miniature post-synaptic potentials were similar in the presence of all three divalent ions. Sr or Ba substitution has little effect on the resting potential of presynaptic terminals. In Sr-containing solutions, action potentials were similar in amplitude and time course to those recorded in Ca. Ba slightly prolonged action potential duration but had no effect on amplitude. Voltage-clamped presynaptic terminals exhibited inward Ca, Sr or Ba currents which were apparently carried through Ca channels. These currents were similar in amplitude and time course in all three ions, being somewhat larger in Ba. Although presynaptic currents were similar in these ions, transmitter release induced by these currents depended upon the divalent species entering the presynaptic terminal. Release was greatest in response to presynaptic current carried by Ca and smallest in response to current carried by Ba. Transfer curves relating presynaptic current to post-synaptic potential were sigmoidal in all three ions, and exhibited limiting slopes of approximately 2. Divalent cations differentially support transmitter release at the squid giant synapse in the sequence Ca greater than Sr much greater than Ba. The differential efficacy of the divalent cations is not due to post-synaptic alterations, presynaptic potential changes or differences in presynaptic divalent cation conductances. This sequence may reflect the cation selectivity of the exocytotic process responsible for transmitter release.

Blockade of synaptic transmission in the squid giant synapse by a spider toxin (JSTX)

We studied the effect of spider toxin (JSTX)--specific blocker of glutamate receptor--on giant synapse of the squid stellate ganglion. JSTX irreversibly blocked the excitatory postsynaptic potential (EPSP) without affecting the presynaptic action potential or the antidromic action potential. L-glutamate depolarized the postsynaptic membrane and suppressed EPSP which may be due to desensitization. The action of glutamate was completely blocked in the presence of JSTX. The results suggest that glutamate is the transmitter at the giant synapse of squid.

Maintained depolarization of synaptic terminals facilitates nerve-evoked transmitter release at a crayfish neuromuscular junction.

Presynaptic and postsynaptic potentials were examined by intracellular recording at a crayfish neuromuscular junction. During normal synaptic transmission, the action potentials were recorded in the terminal region of the excitatory axon and postsynaptic responses were obtained in the muscle fibers. We found that it was possible to modify the synaptic transmission by applying depolarizing or hyperpolarizing currents through the presynaptic intracellular electrode. Typically, a 7-15 mV depolarization lasting longer than 50 msec leads to a large (500%) enhancement of transmitter release, even though the preterminal action potential is reduced in amplitude. Hyperpolarization increases the amplitude of the action potential, but slightly reduces the transmitter release. These results are different from those reported for other neuromuscular synapses and the squid giant synapse, but are similar in many respects to the results reported for several invertebrate central synapses. We conclude, first, that different synapses may have markedly different responses to conditioning by membrane polarization and, secondly, that maintained low-level depolarization may induce a potentiated state in the nerve terminal, perhaps brought about by slow entry of calcium.

Presynaptic calcium diffusion and the time courses of transmitter release and synaptic facilitation at the squid giant synapse

At the squid giant synapse, a presynaptic action potential is accompanied by an influx of calcium ions that continue to be detectable with arsenazo III microspectrophotometry for several seconds. Nevertheless, transmitter release occurs phasically, lasting only about 2 msec. If a second action potential follows within about 100 msec after the first, it releases more transmitter. In this paper, we present a mathematical model of intracellular calcium diffusion with binding to fixed cytoplasmic sites, active extrusion at the surface, and influx during an action potential, to predict the distribution of intracellular calcium following an action potential. With a square law relation between submembrane calcium and transmitter release, the model predicts the phasic release of transmitter and the magnitude and time course of synaptic facilitation following an action potential, as well as the relatively long persistence of free intracellular calcium.

Postsynaptic factors controlling the shape of potentials at the squid giant synapse

The roles of rectification and cable properties of the squid giant axon in determining the shape of synaptic potentials generated at the giant synapse were investigated. Excitatory postsynaptic potentials were recorded in response to selective stimulation of the main presynaptic axon at various temperatures. Excitatory postsynaptic potentials elicited at low temperatures (<18°C) exhibited a marked after-hyperpolarization or undershoot, while those recorded at higher temperatures did not. The postsynaptic current, recorded under voltage clamp conditions, did not show an undershoot. Furthermore, intracellular injection of tetraethylammonium chloride, to block the voltage-dependent rise in potassium conductance, also eliminated the undershoot of the excitatory postsynaptic potential. These results indicate that the duration of synaptic potentials at the squid giant synapse is reduced by rectification due to a delayed rise in potassium conductance. Computer simulations of these synaptic potentials suggested that the effects of rectification will be more prominent in spherical (isopotential) cells than in cells with more complicated geometries.

Transmission by presynaptic spike-like depolarization in the squid giant synapse.

Synaptic transmission produced by artificially generated presynaptic depolarizations, having the same waveform as normal presynaptic action potentials, was studied in the squid giant synapse. These stimulated spikes produced synaptic transmission virtually indistinguishable from that obtained with the original presynaptic spike activation. The technique allows a direct determination of the onset and amplitude of the Ca current, ICa, triggered by the presynaptic action potential and the relationship between ICa and postsynaptic response. The results closely resemble theoretical predictions by a previously published model for synaptic transmission in this synapse and demonstrate that ICa occurs at the falling phase of the presynaptic spike.

Role of presynaptic calcium ions and channels in synaptic facilitation and depression at the squid giant synapse.

1. The roles of presynaptic calcium influx and calcium accumulation in synaptic facilitation and depression were explored at the giant synapse in the stellate ganglion of the squid. 2. Calcium currents were recorded in the presynaptic terminal, using a three-electrode voltage clamp and blocking sodium and potassium currents pharmacologically. The calcium influx was constant during pairs or trains of brief depolarizing pulses that elicited facilitating or depressing excitatory post-synaptic potentials (e.p.s.p.s). 3. The relationship between calcium influx and transmitter release during brief depolarizing pulses of varying amplitude resembled a power function with exponent of about 2. 4. Presynaptic calcium concentration transients were measured by injecting the dye arsenazo III and detecting absorbance changes microspectrophotometrically. Increments in intracellular free calcium accompanying single action potentials appeared constant for repeated action potentials that elicited facilitating e.p.s.p.s. 5. The presynaptic calcium concentration remains elevated for several seconds following action potentials. 6. Presynaptic injection of calcium ions by interbarrel ionophoresis evokes a postsynaptic depolarization, apparently reflecting a large increase in miniature e.p.s.p. frequency. Presynaptic action potentials remain unaffected by this treatment, but e.p.s.p.s triggered by them are facilitated for several seconds, and then depressed. 7. The results are consistent with the hypothesis that synaptic facilitation is due to the action of residual calcium or a calcium complex remaining in the presynaptic terminal after electrical activity. The late depression of release during calcium injection may be a result of the continual release of transmitter and consequent depletion of a presynaptic store.

Are the presynaptic membrane particles the calcium channels?

The number of large intramembrane particles associated with sites of synaptic vesicle release at the squid giant synapse was determined and compared to the average maximal presynaptic calcium current in order to derive an estimate of the conductance each particle would have if it were a calcium channel. This value, 0.21 pS, compares favorably with conductances of calcium channels in other preparations, substantiating the idea that the large intramembrane particles, which are concentrated at "active zones," represent calcium channels.

Calcium transients recorded with arsenazo III in the presynaptic terminal of the squid giant synapse.

Transient changes in free intracellular Ca2+ concentration were monitored in the presynaptic terminal of the giant synapse of the squid, by means of the Ca2+-sensitive dye arsenazo III. Calibration experiments showed a linear relation between the amount of Ca2+ injected by iontophoresis into the terminal, and the peak size of the arsenazo light absorbance record. A light signal could be detected on tetanic stimulation of the presynaptic axon bathed in sea water containing 45 mM Ca2+. During a 1 s tetanus the light signal rose approximately linearly, even though transmitter release declined rapidly and the light signal subsequently declined with a half-time of 2-6 s. The Ca2+ transient elicited by single nerve impulses was recorded by signal averaging, and showed a time course very much slower than the duration of transmitter release.

Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse

The relationship between calcium current and transmitter release was studied in squid giant synapse. It was found that the voltage-dependent calcium current triggers the release of synaptic transmitter in direct proportion to its magnitude and duration. Transmitter release occurs with a delay of approximately 200 mus after the influx of calcium. A model is presented which describes these relations formally.

The non-impulsive stretch-receptor complex of the crab: a study of depolarization-release coupling at a tonic sensorimotor synapse

A new preparation for the study of synaptic transmission is described from the thoracic ganglion of the crabCallinectes sapidus. The central anatomy of the nonimpulsive stretch-receptor neurons of the thoracic-coxal joint and that of the promotor motoneurons with which they form synaptic junctions was studied with intracellular cobalt staining and light and electron microscopy. Attention was centred on the interaction of the stretch-receptor T-fibre and the four large motoneurons supplying the promotor muscle which have their cell-bodies on the dorsal surface of the ganglion. The presynaptic terminal region of the T-fibre appeared to be a simple cylinder in form with a diameter of 40-60 μm and containing large stores of synaptic vesicles at its periphery, opposite the complex of motoneuron dendrites. The transmission characteristics of the junctions between receptor cell and motoneurons were studied by transmembrane current injection into the isolated T-fibre by means of a sucrose gap and simultaneous intracellular recording with microelectrodes from the presynaptic terminal and the somata of postsynaptic cells. It was shown that depolarization-release coupling in the T-fibre has similar properties to those that have been demonstrated in the squid giant synapse, with the same values for ‘threshold’, peak release and ‘suppression potential’. The crab synapses differ from that of the squid in that they normally transmit prolonged, graded depolarizations (i.e. receptor potentials) which are decrementally conducted from the periphery. Consistent with this role, the junctions were found to be capable of continuous tonic transmission over many seconds without the strong depletion seen in more phasic synapses. In a study of the relation between the synaptic properties and the stretch reflex it was shown that some of the time- and amplitude-dependent behaviour of the overall reflex can be encoded at the level of the synaptic transmission, largely through the parameter of transmitter availability. Conduction of electrical signals in the proximal and presynaptic part of the sensory fibre was also investigated. Transient responses to step depolarizing currents in the fibre indicate the existence of a mechanism for the partial compensation of capacitative distortion in the decrementally conducted receptor potential. This is the first example of intracellular recording from presynaptic terminals of nonimpulsive neurons with simultaneous monitoring of postsynaptic potential changes, allowing for a direct analysis of depolarization-release coupling characteristics. The use of the preparation for further study of synaptic physiology and sensorimotor systems is discussed.

The effect of repetitive stimulation on the passive electrical properties of the presynaptic terminal of the squid giant synapse.

The resting electrical properties of the presynaptic terminal of the squid giant synapse have been determined by using constant current pulses. After short periods of repetitive stimulation, the terminal resistance, time constant and capacitance are found to be increased. These changes are absent in terminals bathed in artificial sea water containing no calcium, and sea water containing 5 mM cobalt. It seems likely that these changes are associated with transmitter release.

Presynaptic potentials and facilitation of transmitter release in the squid giant synapse.

Presynaptic potentials were studied during facilitation of transmitter release in the squid giant synapse. Changes in action potentials were found to cause some, but not all, of the facilitation during twin-pulse stimulation. During trains of action potentials, there were no progressive changes in presynaptic action potentials which could account for the growth of facilitation. Facilitation could still be detected in terminals which had undergone conditioning depolarization or hyperpolarization. Facilitation could be produced by small action potentials in low [Ca++]o and by small depolarizations in the presence of tetrodotoxin. Although the production of facilitation varied somewhat with presynaptic depolarization, nevertheless, approximately equal amounts of facilitation could be produced by depolarizations which caused the release of very different amounts of transmitter.