A study of synaptic transmission in the absence of nerve impulses

1. The axo-axonic giant synapse in the stellate ganglion of the squid has been used to study synaptic transmission.2. When nerve impulses have been eliminated with tetrodotoxin, synaptic transfer of potential changes can still be obtained by applying brief depolarizing pulses to the presynaptic terminal.3. Suitably matched pulses are as effective as the normal presynaptic spike in evoking post-synaptic potentials. The synaptic delay and the time course of the post-synaptic potential are very similar to that in the normal preparation.4. The synaptic transfer (input/output) characteristic has been studied under different experimental conditions. With brief (1-2 msec) current pulses, post-synaptic response becomes detectable when the presynaptic depolarization exceeds about 30 mV. The post-synaptic potential increases about tenfold with 10 mV increments of presynaptic depolarization.5. Calcium increases, magnesium reduces the slope of the synaptic transfer curve. The influences on this curve of (i) duration of the pulse, (ii) preceding level of membrane potential, (iii) position of recording electrode, (iv) rate of repetitive stimulation are described.6. After loading the synaptic terminal with tetraethylammonium ions, large inside-positive potentials can be produced in the terminal and maintained for many milliseconds.7. By raising the internal potential to a sufficiently high level, synaptic transfer becomes suppressed during the pulse, and the post-synaptic response is delayed until the end of the pulse.8. This observation is in accord with a prediction of the ;calcium hypothesis', viz. that inward movement of a positively charged Ca compound, or of the calcium ion itself, constitutes one of the essential links in the ;electro-secretory' coupling process of the axon terminal.     

Presynaptic membrane potential and transmitter release at the crayfish neuromuscular junction

Release of transmitter was evoked at neuromuscular junctions of the crayfish opener muscle by passage of current through an intracellular electrode impaling a branch of the motor axon close to a muscle fiber. Membrane-potential changes in the presynaptic axon branch were monitored, together with postsynaptic potentials. Depolarization of impaled secondary axonal branches by more than 10 mV led to an increase in asynchronous transmitter release. The release was facilitated by prolonged (50-500 ms) depolarizations and it decayed rapidly when depolarization was terminated. Ca2+ was essential for facilitated release; however, no indication of a Ca spike was found at the recording site. Input-output curves for the synapse were obtained by applying depolarizing pulses of varying amplitude to the axon branch. Transmitter output was strongly influenced by both amplitude and duration of the applied depolarization. During normal synaptic transmission, propagated Na+-dependent action potentials were recorded in the secondary axonal branches but there was no evidence for a calcium-dependent component for these action potentials. Evoked release was dependent on Ca2+ and was steeply dependent on the amplitude of the action potential, which could be made variable in size by application of tetrodotoxin (TTX). Prolonged depolarization of axonal branches resulted in enhancement of transmitter release evoked by an action potential. The enhancement occurred in spite of a simultaneous reduction of the amplitude of the action potential. Morphological features of the terminals were investigated after injection of lucifer yellow into the axon. An electrical model incorporating the morphological features suggests that membrane-potential changes set up in the main axon reach the nearest terminals with 30-40% attenuation, while events originating in the terminals would be severely attenuated in the main axon. Comparison of the crayfish synapse with other frequently studied synapses shows both similarities and differences, suggesting that it is not possible to apply findings made in one synapse to all others.     

The action of calcium on neuronal synapses in the squid

1. The isolated stellate ganglion of the squid (L. pealii) was studied with intracellular and extracellular micro-electrodes. Three or four nerve fibres in the preganglionic nerve establish synaptic relations with the giant axon in the last stellar nerve. Accordingly, 1-3 small presynaptic spikes (< 1 mV) could be recorded from within the post-synaptic axon.2. A micro-electrode was inserted in the presynaptic fibre and used to polarize and record simultaneously. In the distal (giant) synapse, hyperpolarization of the ending produced an increase in the size of the presynaptic action potential and post-synaptic potential (PSP). Depolarization had the opposite effect. These effects of polarization took more than 10 sec to develop fully, and declined with a similar time course at the end of polarization. Analogous results were obtained with two other preganglionic fibres, which make contacts in the proximal synaptic region.3. The second of a pair of preganglionic impulses evoked a PSP larger than the first. This facilitation of PSP was sometimes accompanied by a small increase in the size of the second action potential in the presynaptic axon. At some shorter intervals, the second presynaptic action potential was reduced in amplitude, but the PSP was still increased. Hyperpolarization of the presynaptic terminal increased the size of both PSPs in a pair and abolished the facilitation. With stronger hyperpolarization the second PSP was even smaller than the first.4. Removing or reducing the Ca in the bathing fluid reversibly abolished the post-synaptic response. The small presynaptic spikes remained practically unaffected. In these conditions a nerve impulse still invaded the ending and normal action potentials could be recorded from the pre-synaptic terminal. This shows that electrical coupling between pre- and post-synaptic axons is insufficient to account for synaptic transmission.5. In low-Ca solution synaptic transmission could be restored locally by extracellular ionophoretic application of Ca to a small portion of the synapse. At sensitive spots a post-synaptic current (recorded with the Ca pipette) and PSP could be detected earlier than 1 sec after commencing the application of Ca.6. Ca was ineffective when injected intracellularly into the presynaptic fibre at a spot where extracellular ionophoresis of Ca restored the PSP.7. The results indicate that synaptic transmission in the squid stellate ganglion is not electrical but due to the release of an unidentified transmitter. Release of this transmitter by the presynaptic nerve impulse requires the presence of Ca in the external medium. During the impulse Ca would combine with a ;Ca-receptor' in the membrane and initiate the reactions which lead to transmitter release. It appears that the ;Ca-receptor' is only accessible from the outside of the membrane.     

Extracellular potassium and trasmitter release at the giant synapse of squid.

1. The effects of changes in extracellular K concentration, [K]0, on synaptic transmission were studied at the squid giant synapse with intracellular recording from the presynaptic terminal and post-synaptic axon. 2. The amplitudes of both the presynaptic spike and the e.p.s.p. varied inversely with [K]0. On the average, a 10 mV change in spike height was accompanied by a 3-1 mV change in e.p.s.p. amplitude. 3. The amplitude of the presynaptic spike after-hyperpolarization (AH) varied inversely with [K]0. On the average, increasing [K]0 resulted in a 20% change in e.p.s.p. amplitude per mV change in presynaptic spike AH. 4. Repetitive antidromic stimulation of the post-synaptic giant axon resulted in an exponential decline in the post-synaptic spike AH, a depolarization of the presynaptic membrane potential and a reduction in the AHs of presynaptic spikes. This suggests that the K which accumulates in the extracellular spaces around the post-synaptic axon also affects the presynaptic terminal. 5. Repetitive antidromic stimulation of the post-synaptic axon resulted in a reduction in the amplitude of e.p.s.p.s. elicted by stimulation of the presynaptic axon. The reduction in e.p.s.p. amplitude relative to the change in presynaptic spike AH was quantitatively close to the change produced by increasing [K]0, suggesting that the reduction in e.p.s.p. amplitude is due to the accumulation of extracellular K at the presynaptic terminal. 6. Repetitive stimulation of the presynaptic axon reduced the amplitudes of the e.p.s.p. and the presynaptic spike AH. On the average, a 1 mV change in presynaptic spike AH was accompanied by a 204% change in e.p.s.p. amplitude, suggesting that K accumulation may only contribute to a small extent, under these conditions, to the depression of transmitter release.     

Synaptic depression related to presynaptic axon conduction block.

1. The depression of synaptic transmission, which occurs during prolonged repetitive activation, was examined in the opener muscle of the crayfish walking leg. 2. Excitatory post-synaptic potentials (e.p.s.p.s) initially facilitated but then declined to low amplitudes after about 4000 stimulus pulses had been delivered; this depression is presynaptic in origin; 3. Axon conduction blocks occured at points of bifurcation along the entire length of the presynaptic nerve. This resulted in failure of the nerve impulse to invade some branches of the terminal arborization. 4. Nerve terminal invasion failure caused either intermittent or complete inactiviation of some synaptic release sites; this was associated with depression of the post-synaptic response. 5. The statistics of transmitter release during prolonged repetitive stimulation were examined by focal extracellular recording methods. Transmitter release could be described by binomial statistics, and depression involved a drop in m, n and p. 6. The rate of spontaneous quantal release did not decrease, however, arguing against transmitter depletion. 7. It is concluded that repetitive stimulation eventually leads to depolarization of the axon membrane. This causes impulse propagation failure which reduces the number of synaptic release sites that are activated and mimics a drop in the effective stimulation rate; both effects cause synaptic depression.     

Single-bouton-mediated synaptic transmission: postsynaptic conductance changes in their relationship with presynaptic calcium signals

Most central neurons contact their dendritic targets at several sites. However, it is not known whether all synapses formed by a single parent axon make the same contribution to the postsynaptic response. In order to answer this question it is necessary to isolate the synaptic currents generated by individual axon terminals. This paper describes a method that was designed to activate transmitter release from solitary synaptic boutons in culture. Neurons from the embryonic rat superior colliculus were grown at low density and double-loaded with a fluorescent marker of synaptic vesicles (FM1-43 or RH414) and a fluorescent Ca2+ indicator (Fura-2, Mag-fura-2, Oregon Green BAPTA-1 or Oregon Green BAPTA-5N). Action potential generation was blocked by tetrodotoxin. Appropriate synaptic boutons were selected under phase-contrast and fluorescence illumination at a magnification of 1000. They were activated by short electrical pulses via a fine-tipped glass pipette filled with bath solution. Presynaptic Ca2+ transients were measured in a region delineated by the FM1-43/RH414 fluorescence. By simultaneous presynaptic Ca2+ imaging and whole-cell recording of postsynaptic responses to single depolarizing pulses, the quantitative relationships between pre- and postsynaptic parameters of synaptic strength in a small synapse of central origin could, for the first time, be analysed. The experiments showed that the average postsynaptic currents depend strongly on the size of the presynaptic Ca2+ transients. However, at any level of presynaptic Ca2+ concentration postsynaptic responses fluctuated in amplitude.     

Presynaptic calcium channels and the depletion of synaptic cleft calcium ions

The entry of calcium ions (Ca(2+)) through voltage-gated calcium channels is an essential step in the release of neurotransmitter at the presynaptic nerve terminal. Because the calcium channels are clustered at the release sites, the flux of Ca(2+) into the terminal inevitably removes the ion from the adjacent extracellular space, the synaptic cleft. We have used the large calyx-type synapse of the chick ciliary ganglion to test for synaptic cleft Ca(2+) depletion. The terminal was voltage clamped at a holding potential (V(H)) of -80 mV and a depolarizing pulse was applied to a range of potentials (-60 to +60 mV). The voltage pulse activated a sustained inward calcium current and was followed, on return of the membrane potential to V(H), by an inward calcium tail current. The amplitude of the tail current reflects both the number of open calcium channels at the end of the voltage pulse and the Ca(2+) electrochemical gradient. External barium was substituted for calcium as the charge-carrying ion because initial experiments demonstrated calcium-dependent inactivation of the presynaptic calcium channels. Tail current recruitment was compared in calyx nerve terminals that remained attached to the postsynaptic neuron and therefore retained a synaptic cleft, with terminals that had been fully isolated. In isolated terminals, the tail currents exhibited recruitment curves that could be fit by a Boltzmann distribution with a mean V(1/2) of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed to depolarized potentials with a mean V(1/2) of 11.9 mV and a slope factor of 12.0. The degree of skew of the recruitment curve in the attached calyces correlated with the amplitude of the inward current evoked by the step depolarization. The simplest interpretation of these findings is that during the depolarizing pulse Ba(2+) is removed from the synaptic cleft faster than it is replenished, thus reducing the tail current by reducing the driving force for ion entry. Ca(2+) depletion during presynaptic calcium channel activation is likely to be a general property of chemical transmission at fast synapses that sets a functional limit to the duration of sustained secretion. The synapse may have evolved to minimized cleft depletion by developing a calcium-efficient mechanism to gate transmitter release that requires the concurrent opening of only a few low conductance calcium channels.     

Localization and electrical characteristics of a giant synapse in the spinal cord of the lamprey.

1. Physiological and morphological experiments were carried out to determine the characteristics of a giant synapse in the lamprey spinal cord. The presynaptic element is a Müller fibre, running the length of the spinal cord, and the post-synaptic element is a lateral interneurone. 2. Injection of the interneurone with fluorescent dye revealed several dendritic processes extending into the region of the Müller fibres and spreading over a longitudinal distance of about 150 mum. Electron microscopic examination of the Müller fibres confirmed that they do not send out processes to form synapses. Thus, the synapse is between the cylindrical fibre and one or more dendritic branches of the interneurone. 3. Measurements with intracellular electrodes showed the Müller fibres to have input resistances of about 1 Momega and space constants of 1-0-1-7 mm. The space constant was larger for hyperpolarizing pulses than for depolarizing pulses because of delayed recitification. The interneurones had input resistances of about 6 Momega. 4. The neurones were electrically as well as chemically coupled. When a current-passing electrode was placed in the fibre and hyperpolarizing pulses applied, the amplitude of the electrical coupling potential recorded from the interneurone was maximal at one position of the current-passing electrode and decreased as the electrode was moved away from the optimal position. The decrease in amplitude with electrode displacement indicated that the region of synaptic contact was very restricted. 5. The electrical synapse was highly rectifying, the forward resistance being about nine-times smaller than the backward resistance.     

Membrane ultrastructure of the giant synapse of the squidLoligo pealei

The ultrastructure of the ‘giant synapse’ of the stellate ganglion of the squid was studied with freeze-fracture and thin-sectioning techniques. A sheath of glial cells separates the pre- and post-synaptic axons. At intervals, round-topped processes of the postsynaptic axon pierce the sheath to contact the presynaptic axon. This area of synaptic contact is marked by a widened intercellular cleft containing electron-dense material and by a cluster of synaptic vesicles within the presynaptic cytoplasm. The number of synaptic vesicles in such clusters was greatly reduced by electrical stimulation of the synapse during fixation. Freeze-fracture reveals a roughly circular patch (0.3 μm diameter) of 10 nm particles on the cytoplasmic leaflet of the presynaptic membrane. A similar patch of particles lies on the external leaflet of the apposed postsynaptic membrane.The squid giant synapse thus consists of multiple small pre- and postsynaptic active zones where neurotransmitter is released from the presynaptic terminal and sensed by postsynaptic receptors. Comparison of the structure of these postsynaptic active zones with those at synapses where the transmitter or transmitter action is known suggests that the excitatory transmitter at this synapse is an amino acid.Presumptive gap junctions, marked by particles in the cytoplasmic leaflet, are found between small-diameter axons in the stellate ganglion but not at the giant synapse. Glial-cell membranes contain aggregates of particles and pits suggestive of gap junctions. The aggregates of pits are embedded within linear arrays of particles which somewhat resemble tight junctions.     

Interaction experiments on the responses evoked in Purkinje cells by climbing fibres

1. The uniquely powerful excitatory synaptic action of a single climbing fibre on a Purkinje cell in the cerebellum of the cat was tested during the intense and prolonged inhibitory action produced by the parallel fibre, basket and stellate cell system. There was depression of the later spike discharges, but the initial discharge was never suppressed.2. With intracellular recording the excitatory post-synaptic potential was depressed during the initial phase (about 10 msec) of the inhibitory action, but there was a later increase with a time course resembling the latter part of the inhibitory hyperpolarization. An explanation of these and other effects is given in terms of conventional ideas of excitatory and inhibitory synaptic interaction.3. These observations on single Purkinje cells, particularly with intracellular recording, have helped in formulating a provisional explanation of the finding that during inhibition there is an increase in the negative field potential evoked by a climbing fibre volley.4. The excitatory action of a climbing fibre synapse is shown to be greatly depressed immediately after a preceding activation and recovery takes hundreds of milliseconds. By the collision technique it is shown that the same climbing fibre is activated by inferior olive and juxta-fastigial stimulation.5. With rapid repetitive activation there was initially a progressive decline in the effectiveness of each successive impulse, but a steady level was soon reached. On cessation of a tetanus of twenty or more impulses there was a delayed recovery of the depolarization, which suggests a continued action of the accumulated transmitter.6. With extracellular recording repetitive spike initiation continued with stimulation frequencies as high as 100/sec, but at still higher frequencies spikes were depressed by the intense synaptically evoked depolarization. On cessation of the stimulation after-discharge often developed as the depolarization declined. The prolonged after-discharges following severe tetani suggest that there is a very effective accumulation of the transmitter.     

Synapsin I injected presynaptically into goldfish mauthner axons reduces quantal synaptic transmission

1. Synapsin I was injected into a vertebrate presynaptic axon to analyze its action on quantal synaptic transmission. Two microelectrodes were used for simultaneous intracellular recording from pairs of identified neurons in the goldfish brain. The postsynaptic electrode was placed in a cranial relay neuron (CRN) within 100 microns of its synapse with the Mauthner neuron. The presynaptic electrode impaled the Mauthner axon (M-axon) 50-200 microns from the first electrode. 2. Spontaneous miniature excitatory postsynaptic potentials (mEPSPs) and evoked postsynaptic potentials (EPSPs) were recorded at steady states before and after synapsin I was microinjected into the presynaptic M-axon. Responses were digitized and subsequently analyzed by computer for quantal parameters. 3. In 12 experiments, injection of synapsin I resulted in a reduction in transmission. The decrease in EPSP amplitude began approximately 30 s after the injection, reached a plateau within 10 min, and appeared to be reversible and dose dependent. 4. Injection of synapsin I decreased quantal content (m), with no effect on postsynaptic receptor sensitivity or on amount of transmitter per quantum. Further analysis based on the simplest binomial model for quantal release revealed that synapsin I consistently reduced the number of quantal units available for release (n) although the probability of release (p) was either unchanged or slightly increased. Injected synapsin I may thus bind to presynaptic vesicles and prevent transmitter quanta from entering a pool subject to evoked release.     

Spontaneous synaptic potentials and quantal release of transmitter in the stellate ganglion of the squid

1. Several kinds of synapses have been studied in the stellate ganglion of the squid.2. A small electric coupling was found between giant fibres in different stellar nerves.3. Post-synaptic potentials recorded from the cells of small axons are composite, indicating that there are converging inputs from several pre-ganglionic fibres.4. Spontaneous miniature synaptic potentials were recorded from all types of synapses. Miniature potentials in the cells of small axons had a slower time course than those in the giant fibre system.5. Tetrodotoxin abolished nerve impulses in the ganglion but did not prevent the spontaneous quantal release of transmitter from the terminals, or its action on the post-synaptic membrane; nor did it prevent the increase in rate of release produced by depolarization of the presynaptic fibre.6. Glutamate depolarized the giant fibre when applied iontophoretically to the synaptic region. Similar doses applied intracellularly were without effect.     

The effect of prolonged depolarization on synaptic transfer in the stellate ganglion of the squid

1. Depolarization of the giant axon terminal of the squid causes local calcium influx which gives rise to transmitter release and post-synaptic response, and which under certain experimental conditions leads to a regenerative action potential in the presynaptic terminal itself.2. There has been conflicting evidence in the literature on the question whether the calcium permeability change in the terminal is rapidly inactivated, or whether it can persist with little diminution for hundreds of milliseconds during a depolarizing voltage step.3. Results are presented which show that there is little ;calcium inactivation', even when very large depolarizing steps are imposed on the terminal and maintained for periods of 1-2 sec.4. Contrary indications are examined and found to be attributable to an increase of potassium conductance, rather than direct inactivation of calcium conductance.     

Depolarization dependence of the kinetics of phasic transmitter release at the crayfish neuromuscular junction

Quantal synaptic currents were recorded at nerve terminals on the crayfish opener muscle by means of a macro-patch-clamp electrode. Release could be elicited by graded depolarization pulses through the recording electrode. At low temperature, distributions of delays of single quantal currents from the onset of depolarization were determined for depolarizations varying from threshold to saturation range. This time course of release was little affected by the amplitude of depolarization: There was a tendency for release to start earlier and to rise faster for larger depolarizations, while the termination of release showed no significant variations. The time course of release after an action potential in the motor axon was similar to that of release after a depolarization pulse. It is concluded that the time course of quantal release is rather independent of amplitude of depolarization and of the amount of calcium (Ca) inflow, which seems to rule out the control of the release after a depolarization by the time course of [Ca]_i.     

Switching between transient and sustained signalling at the rod bipolar-AII amacrine cell synapse of the mouse retina

At conventional synapses, invasion of an action potential into the presynaptic terminal produces a rapid Ca²⁺ influx and ultimately the release of synaptic vesicles. However, retinal rod bipolar cells (RBCs) generally do not produce action potentials, and the rate of depolarization of the axon terminal is instead governed by the rate of rise of the light response, which can be quite slow. Using paired whole-cell recordings, we measured the behaviour of the RBC-AII amacrine cell synapse while simulating light-induced depolarizations either by slowly ramping the RBC voltage or by depolarizing the RBC with the mGluR6 receptor antagonist ( R , S )-α-cyclopropyl-4-phosphonophenylglycine (CPPG). Both voltage ramps and CPPG evoked slow activation of presynaptic Ca²⁺ currents and severely attenuated the early, transient component of the AII EPSC compared with voltage steps. We also found that the duration of the transient component was limited in time, and this limitation could not be explained by vesicle depletion, inhibitory feedback, or proton inhibition. Limiting the duration of the fast transient insures the availability of readily releasable vesicles to support a second, sustained component of release. The mGluR6 pathway modulator cGMP sped the rate of RBC depolarization in response to puffs of CPPG and consequently potentiated the transient component of the EPSC at the expense of the sustained component. We conclude that the rod bipolar cell is capable of both transient and sustained signalling, and modulation of the mGluR6 pathway by cGMP allows the RBC to switch between these two time courses of transmitter release.     

Force generation examined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres

The modulation of synaptic transmission by presynaptic ionotropic and metabotropic receptors is an important means to control and dynamically adjust synaptic strength. Even though synaptic transmission and plasticity at the hippocampal mossy fibre synapse are tightly controlled by presynaptic receptors, little is known about the downstream signalling mechanisms and targets of the different receptor systems. In the present study, we identified the cellular signalling cascade by which adenosine modulates mossy fibre synaptic transmission. By means of electrophysiological and optical recording techniques, we found that adenosine activates presynaptic A₁ receptors and reduces Ca²⁺ influx into mossy fibre terminals. Ca²⁺ currents are directly modulated via a membrane-delimited pathway and the reduction of presynaptic Ca²⁺ influx can explain the inhibition of synaptic transmission. Specifically, we found that adenosine modulates both P/Q- and N-type presynaptic voltage-dependent Ca²⁺ channels and thereby controls transmitter release at the mossy fibre synapse.     

Further study of the role of calcium in synaptic transmission

1. The effect of calcium on synaptic transmission has been studied by intracellular recording of pre- and post-synaptic potential changes in the stellate ganglion of the squid.2. For a given presynaptic ;input' (propagated spike, or local depolarizing pulse after tetrodotoxin treatment), the post-synaptic response increases with external calcium concentration [Ca](o) in a highly non-linear fashion, indicating that transmitter output varies with more than the second power of [Ca](o) over a certain concentration range.     

Synaptic transmission without action potentials: input-output properties of a nonspiking presynaptic neuron.

1. Input-output properties of the inhibitory synaptic connection between non-spiking neurons (EX1) and gastric mill (GM) neurons were examined in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. Current was injected into and the voltage was recorded during current injection, two independent microelectrodes were used. 2. The EX1-GM synaptic connection is a conductance-increase inhibitory type, with an input-output curve that resembles the curve for the squid giant synapse. There is a threshold level of depolarization for transmitter release from the presynaptic cell. Beyond that threshold, increasing presynaptic depolarization causes increasing postsynaptic hyperpolarization (and inhibition). 3. A long presynaptic current step always causes a postsynaptic response with an initial peak of hyperpolarization followed by a decay to a less hyperpolarized plateau level. The plateau level is maintained, in most cells, for the duration of the presynaptic depolarization even over long periods (30 s). 4. The peak, but not the plateau, part of the postsynaptic response is sensitive to the past history of the synaptic connection. If a large conditioning pulse is applied to the presynaptic cell causing a large postsynaptic hyperpolarization, then the postsynaptic response to a later presynaptic test depolarization will have a reduced peak, leaving the plateau component unchanged.     

Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarizing pulses

1. Experiments were conducted to test the hypothesis that facilitation of transmitter release in response to repetitive stimulation of the exciter motor axon to the crayfish claw opener muscle is due to an increase in the amplitude or duration of the action potential in presynaptic terminals. No consistent changes were found in the nerve terminal potential (n.t.p.) recorded extracellularly at synaptic sites on the surface of muscle fibres.2. Apparent changes in n.t.p. are attributed to three causes.(i) Some recordings are shown to be contaminated by non-specific muscle responses which grow during facilitation.(ii) Some averaged n.t.p.s exhibit opposite changes in amplitude and duration which suggest a change in the synchrony of presynaptic nerve impulses at different frequencies.(iii) Some changes in n.t.p. are blocked by gamma-methyl glutamate, an antagonist of the post-synaptic receptor, which suggests that these changes are caused by small muscle movements.3. The only change in n.t.p. believed to represent an actual change in the intracellular signal is a reduction in n.t.p. amplitude to the second of two stimuli separated by a brief interval.4. Tetra-ethyl ammonium ions increase synaptic transmission about 20% and prolong the n.t.p. about 15%. This result suggests that an increase in n.t.p. large enough to increase transmission by the several hundred per cent occurring during facilitation would be detected.5. The nerve terminals are electrically excitable, and most synaptic sites have a diphasic or triphasic n.t.p., which suggests that the motor neurone terminals are actively invaded by nerve impulses.6. When nerve impulses are blocked in tetrodotoxin, depolarization of nerve terminals increases the frequency of miniature excitatory junctional potentials (e.j.p.s), and a phasic e.j.p. can be evoked by large, brief depolarizing pulses. Responses to repetitive or paired depolarizations of constant amplitude and duration exhibit a facilitation similar to that of e.j.p.s evoked by nerve impulses.7. It is concluded that facilitation in the crayfish claw opener is not due to a change in the presynaptic action potential, but is due to some change at a later step in the depolarization-secretion process.     

Depression and recovery of transmission at the squid giant synapse.

1. The process of synaptic depression and recovery were studied in the squid (Loligo pealii) giant synapse with intracellular recording and stimulating electrodes in the prescence of tetrodotoxin (10-minus 7 M). 2. When the synapse was stimulated at 50 Hz, depression occurred rapidly. Recovery after the tetanus was a first-order process with an average recovery time constant of 4-9 sec. The rate of recovery was independent of the amplitude of the post-synaptic potential (p.s.p.) or the degree of depression. 3. For the first five to seven p.s.p.s in the train there was a linear relationship between depression and the total amount of transmitter previously released. This may indicate that depression in this preparation was caused by the depletion of the presynaptic store of transmitter (S). 4. Assuming that this interpretation was correct, we could show that recovery from depression during the tetanus (i.e. 'mobilization') proceeded about 10 times faster than after the end of the tetanus. 5. When the amplitude of the p.s.p. was varied by changing the bathing calcium concentration, [Ca], the degree of depression was correlated to the amplitude of the p.s.p. 6. When the amplitude of the p.s.p. was increased by increasing pre-synaptic depolarization, synaptic depression was found to increase as well. However, synaptic depression increased less than the amplitude of the p.s.p., the relationship between these two measures being non-linear. 7. This finding is interpreted to indicate that the transmitter stores, S, are closely related to the area of the presynaptic membrane which is sufficiently depolarized to release transmitter.