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.     

Cumulative and persistent effects of nerve terminal depolarization on transmitter release

1. Following focal depolarization of rat motor nerve terminals there could often be observed an ;after-discharge' of m.e.p.p.s with transient frequencies of up to 1000/sec. This after-discharge was graded with intensity and duration of the previous depolarization.2. Following pulses which were relatively short (about 1 sec) and not too large (< -100 mV local extracellular potential field) the logarithm of m.e.p.p. frequency fell exponentially. With larger or longer pulses there was a tail to the after-discharge which could persist for several minutes.3. M.e.p.p. frequency during an after-discharge was not inhibited appreciably by nerve terminal hyperpolarization, raised [Ca] (8 mM) or lowered pH.4. Measured as a multiplication of spontaneous m.e.p.p. frequency after-discharge was depressed in solution containing no Ca(2+) and added 1 mM-MgEDTA but equal in 0.125 mM-Ca(2+) or 2 mM-Sr(2+) to that in 2 mM-Ca(2+) or 8 mM-Ca(2+).5. During an after-discharge the multiplication of m.e.p.p. frequency by focal nerve terminal depolarization or raised K(+) was reduced. This phenomenon was termed ;uncoupling'.6. It was concluded that the after-discharge is not caused by a persistent rise of K(+) concentration in the synaptic cleft, nor by a maintained nerve terminal depolarization.7. In preparations depolarized by raised K(+) m.e.p.p. frequency during a relatively small focal depolarizing pulse rose continuously, after an initial rapid rise, and after the pulse there was a tail of increased m.e.p.p. frequency. The magnitude of the rise during the pulse and the tail after it were similar on, a logarithmic basis; during both processes the logarithm of m.e.p.p. frequency usually followed (approximately) an exponential time course.8. The relative magnitude of the slow effect of depolarization, as compared with the fast effect, was increased by lowering [Ca] or increasing [Mg], and the slow effect of depolarization in contrast to the fast effect was found to exist in the presence of Ca reduced to about 10(-7)M, but only during pulses. At this [Ca] there was no rapid response to depolarization. At [Ca] about 10(-10)M, there was no response at all of m.e.p.p. frequency to nerve terminal depolarization.9. The results are discussed, and compared with similar data referring to ;facilitation' and ;post-tetanic potentiation'. It is concluded that these and the slow effect of depolarization represent the same phenomenon, a response of the transmitter release system which can be distinguished from the fast response in terms of ionic requirement as well as time course.     

Synaptic transfer at a vertebrate central nervous system synapse.

1. The relation between presynaptic depolarization and transmitter release was examined at a synapse between a Müller axon and a lateral interneurone in the spinal cord of the lamprey. Two micro-electrodes, one for passing current and the other for recording the resulting voltage change, were placed in the presynaptic axon; a single electrode for recording the post-synaptic potential produced by release of transmitter was placed in the post-synaptic cell. 2. When action potentials were blocked with tetrodotoxin, brief depolarizing pulses in the presynaptic fibre were as effective as the action potential had been in producing transmitter release. 3. The release process had an apparent threshold depolarization of 40-50 mV and saturated at presynaptic depolarizations of the order of 100 mV. Increasing the duration of the presynaptic pulse increased the maximum level of release. 4. Displacing the presynaptic voltage recording electrode from the position of synaptic contact toward the current passing electrode increased the apparent depolarization required to produce a given level of transmitter release. This shift in the input-output relation was consistent in magnitude with the voltage attenuation between the presynaptic recording electrode and the synapse expected from the space constant of the fibre. 5. The effect of conditioning hyperpolarization and depolarization of the presynaptic fibre on subsequent transmitter release by brief depolarizing pulses was examined. No effect was observed when the presynaptic recording electrode was in the region of synaptic contact. When the presynaptic electrode was not so positioned, conditioning effects were observed which depended on electode position and could be attributed to changes in the space constant of the presynaptic fibre. No conditioning effects were observed on transmitter release by the action potential.     

Inactivation of the asymmetrical displacement current in giant axons of Loligo forbesi.

1. Asymmetrical displacement currents ('gating currents') have been recorded in intracellularly perfused squid giant axons by averaging the currents associated with depolarizing and hyperpolarizing pulses. The relation between 'gating current' and Na inactivation was studied by investigating the effect of pulse duration and conditioning pulses. 2. Increasing the pulse duration from 0-3-1 msec to 10-20 msec reduced the off-response of the 'gating current' to 50-70% of its normal size; the time constant was 5 msec at +20 mV and 8 degrees C. The decrease of the Na current during a 10-20 msec pulse was stronger and faster; it decayed to 10-26% with a time constant of 1-35 msec. 3. The effect of pulse duration could also be demonstrated by using only depolarizing pulses. The charge displacement at the end of single or averaged depolarizing pulses was smaller for long pulse durations than for short. A long depolarizing pulse was followed by a small long-lasting tail of inward current. 4. A conditioning depolarizing pulse of 10-20 msec duration to a potential of -30 or +10 mV, followed by a short recovery period at -70 mV, decreased the on-response of the 'gating current'. Its size was reduced to 46-71% and 61-94%, respectively, for a recovery interval of 1-75 and 5 msec at 2-3 degrees C. The reduction of the Na current, measured under similar conditions, was more pronounced; the Na current was decreased to less than 50% of its normal value. 5. The observations about the effect of pulse duration and conditioning pulses on the 'gating current' are qualitatively consistent with those of Bezanilla & Armstrong (1974, 1975) and support the view that part of the asymmetrical charge displacement is inactivated during a 10-20 msec depolarization.     

Ion conductance changes associated with spike adaptation in the rapidly adapting stretch receptor of the crayfish

The time course of the repetitive impulse discharges has been investigated for two high intensities of maintained depolarizing currents, 30 nA and 50 nA, for which the receptor adaptation was complete within 70 msec. The changes in sodium and potassium conductance associated with the decline in spike activity have been analyzed at different instances of time by interrupting in successive experiments the various action potentials in the pulse trains either at the early phase by holding the potential at about -60 mV and recording the inward current (upstroke-gNa) or by evaluating the delayed outward current flowing as the result of a depolarizing voltage pulse which at the end of the action potential re-increased the membrane potential by mV (after potentialgK). At the higher current intensity of 50 nA the discharge frequency was increased, while larger reductions in upstroke-gNa and after potential-gK during receptor adaptation became apparent. The progressive decrease in pulse amplitude from 99 mV to 63 or 55 mV is paralleled by a gradual reduction in upstroke-gNa from 97 mmho/cm-2 to 37 or 27.5 mmho/cm-2 and in after potential-gK from 11.5 mmho/cm-2 to about 7 mmho/cm-2. When under a stimulus of 30 nA the sodium conductance decreases to an average value of 37 mmho/cm-2 only a distorted spike can be elicited, while the spike activity was completely suppressed at upstroke-gNa equals 27.5 mmho/cm-2 was essentially the same under both conditions. The results have been interpreted in terms of the model for impulse generation formulated by Michaelis and Chaplain (1973). According to the model both sodium and potassium inactivation reduce the pulse amplitude. However, while Na-inactivation reduces the frequency of impulse discharge, the K-inactivation actually leads to an increase in spike frequency. As the frequency of the short train of pulses recorded under high-intensity current stimulation remained essentially unaltered, it is suggested that the coupling between Na- and K-inactivation actually leads to an increase in spike frequency. As the frequency of the short train of pulses recorded under high-intensity current stimulation remained essentially unaltered, it is suggested that the coupling between Na- and K-inactivation ensures a constancy of the information-carrying parameter, i.e. the average impulse density.     

Depolarization initiates phasic acetylcholine release by relief of a tonic block imposed by presynaptic M2 muscarinic receptors

The role of presynaptic muscarinic autoreceptors in the initiation of phasic acetylcholine (ACh) release at frog and mouse neuromuscular junctions was studied by measuring the dependency of the amount (m) of ACh release on the level of presynaptic depolarization. Addition of methoctramine (a blocker of M2 muscarinic receptors), or of acetylcholinesterase (AChE), increased release in a voltage-dependent manner; enhancement of release declined as the depolarizing pulse amplitude increased. In frogs and wild-type mice the slope of log m/log pulse amplitude (PA) was reduced from about 7 in the control to about 4 in the presence of methoctramine or AChE. In M2 muscarinic receptor knockout mice, the slope of log m/log PA was much smaller (about 4) and was not further reduced by addition of either methoctramine or AChE. The effect of a brief (0.1 ms), but strong (-1.2 microA) depolarizing prepulse on the dependency of m on PA was also studied. The depolarizing prepulse had effects similar to those of methoctramine and AChE. In particular, it enhanced release of test pulses in a voltage-dependent manner and reduced the slope of log m/log PA from about 7 to about 4. Methoctramine + AChE occluded the prepulse effects. In knockout mice, the depolarizing prepulse had no effects. The cumulative results suggest that initiation of phasic ACh release is achieved by depolarization-mediated relief of a tonic block imposed by presynaptic M2 muscarinic receptors.     

Properties of a facilitating calcium current in pace-maker neurones of the snail, Helix pomatia.

1. Simultaneous measurements of local voltage clamp currents from patches of soma membrane and K activity at the soma surface were used to analyse the time and voltage dependence of the slow inward current in bursting pace-maker neurones of the snail (Helix pomatia). 2. At low levels of depolarization (less than or equal to mV) a net inward current is recorded simultaneously with an efflux of K ions from the cell. 3. With larger depolarizations (20-170 mV from holding potential of -50 mV) the deficit in net outward charge transfer compared with K efflux and the appearance of inward-going tail currents following repolarization, reveal a persistent inward-going current also under these conditions. This inward current is carried primarily by Ca ions, as demonstrated by its voltage dependence (a minimum at about + 115 mV) and its disappearance in Co-Ringer. It is identified with the slow inward Ca current Iin slow (Eckert & Lux, 1976). 4. The inward current predicted from comparisons of current trajectories reaches a maximum at 15-20 msec (for depolarizations from -50 to 0 mV) and gradually declines with sustained depolarization. 5. Partial inactivation is removed by repolarization to -50 mV and the Ca dependent deficit is greater in the sum of repeated voltage clamp pulses than during sustained depolarization. It is largest for pulses of 25-100 msec duration, decreasing as pulse duration increases. 6. Responses to repeated activation with 100 msec pulses with different repolarization intervals reveal a minimum Iin slow at short intervals (e.g. 20 msec) due to failure to remove partial inactivation. At intermediate intervals (e.g. 200-400 msec) Iin slow shows facilitation. This is revealed in calculations of the net charge transfer and current deficits and is also shown in the tail currents following repolarization. The deficit increases progressively with repetitive stimulation. With longer intervals (e.g. 800-1000 msec) defacilitation during repeated stimulation after the first two pulses is revealed in calculations of deficits, current trajectories and in the tail currents. 7. Although facilitation depends on duration of repolarization between pulses, increasing intermediate hyperpolarizations from the holding potential of -50 mV are usually ineffective in increasing Iin slow. Strong preceding hyperpolarization can even decrease the magnitude of Iin slow and prevent its facilitation with repetitive stimulation,whereas preceding depolarizing pulses can increase Iin slow without preventing its facilitation with repetitive stimulation. 8. The properties of Iin slow are contrasted with previously described membrane conductances and compared with properties attributed to Ca fluxes in other systems.     

Asymmetrical displacement currents in the membrane of frog myelinated nerve: Early time course and effects of membrane potential

1. Asymmetrical displacement currents were studied in myelinated nerve fibres from Rana esculenta with a voltage clamp technique. 2. For brief pulses symmetrical with respect to a holding potential of--97mV, the asymmetry current flowing during pulses (on-response) exhibited a rising phase to a peak followed by an approximately exponential decline. After the pulses the rising phase in the off-response could not be resolved; the time constant varied about 2-fold with either size or duration of the pulse. 3. For longer pulses a second slower component could be detected both in on- and off-responses. 4. The rapidly declining on- and off-responses associated with brief pulses carried about the same charges Qon and Qoff. Increasing the duration of the pulse reduced Qoff. For all pulses tested Qoff approached about one fifth of Qmax. The reduction of Qoff was roughly characterised by time constants ranging between 1.5 and 0.5 ms for potentials between--25 and + 23 mV. Analysis of individual membrane currents confirmed that the capacity current after depolarizing pulses decreased with pulse length. 5. The effects of membrane potential on asymmetry current were studied by varying the level from which pulses were applied during 46.9ms prepulses in the range from--97 to--29mV. The fast and slow components of asymmetry current were affected differently. For potentials more positive than--90mV the fast on-response was reduced and reversed its sign at a potential 25mV more negative than the potential estimated from the steady-state charge distribution measured from--97mV.     

Block of potassium channels of the nodal membrane by 4-aminopyridine and its partial removal on depolarization

1. Voltage clamp experiments were done on single myelinated nerve fibres of the frog, Rana esculenta. 2. 53 muM 4-aminopyridine (4-AP) reduced IK to about one-fifth if tested with infrequent (1/min) and short (10 ms) depolarizing pulses; the onset time constant under these circumstances was ca. 160 s (14-15 degrees C). After prolonged treatment the effect was virtually irreversible. 3. At equilibrium with 4-AP, increasing the frequency of short pulses removed part of the block, the block removal accelerating with increasing pulse duration and frequency. 4. In 53 muM 4-AP unblocking of K channels during long (0.8 s) depolarizing pulses proceeded with a time constant, taur, of ca. 0.2 s. Restoration of block at the resting potential proceeded with a much larger time constant, tau'r, of ca. 1 min. 5. The stationary fraction, rinfinity, of K channels conducting in 53 muM 4-AP was 0.66, 0.41, and 0.24 at V = 120, 50, and 0 mV, respectively. 6. In a series of experiments with [4-AP] varying between 13.3 and 848 muM, taur decreased from 0.25 to 0.10 s (V = 130 mV, ca. 17 degrees C) while rinfinity followed the empirical relation 1/rinfinity = 1 + ct + cv exp(-0.77 EF/RT) with E = V - 70 mV. ct and cv are dimensionless quantities that increase with [4-AP] and reflect the voltage-independent and voltage-dependent component, respectively, of block. 7. Block of K channels and partial removal are also observed with inward IK at raised [K+]O. Removal proceeds on depolarization even if IK is additionally but temporarily suppressed by tetraethylammonium. Hence neither direction nor amplitude of IK but only the pulse potential seems to determine the extent of block for a given [4-AP].     

Isolation and characterization of slow, depolarizing responses of cardiac ganglion neurons in the crab, Portunus sanguinolentus.

1. Tetrodotoxin-resistant, active responses to depolarization of the large cardiac ganglion cells were studied in semi-isolated preparations from the crab, Portunus sanguinolentus. Impulse activity was monitored with extracellular electrodes, simultaneous recordings from two or three large cells were made with intracellular electrodes, and current was passed via a bridge or second intracellular electrode. Preparations were continuously perfused with saline containing 3 x 10(-7) M tetrodotoxin (TTX). 2. About 20 min after introduction of TTX, small-cell impulses and resultant EPSPs in large cells cease, while rhythmic, spontaneous bursting of large cells continues. A pacemaker depolarization between bursts and slow depolarizations underlying the impulse bursts are prominent at this time. Shortly after, spontaneous burst rate slows, and at ca. 25 min, the ganglion becomes electrically quiescent. 3. In the quiescent, TTX-perfused ganglion, injection of depolarizing current into any one of the large cells results in active responses. At current strengths of sufficient intensity and duration (e.g., 20 nA, 20 ms; 5 nA, 500 ms) to depolarize a large cell by ca. 10 mV from resting potential (-53 mV, avg), the graded responses become regenerative and of constant form, provided the stimulation rate is less thna 0.15/s. Such responses have been termed "driver potentials." At more rapid rates, thresholds are increased and responses reduced. 4. Driver potentials of anterior large cells reach peak amplitudes of ca. 20 mV (to -32 mV), have maximum rates of rise of 0.45 V/s and of fall of 0.2 V/s, and a duration of ca. 250 ms. They are followed by hyperpolarizing afterpotentials, a rapidly decaying one (1 s) to -58 mV, followed by a slowly decaying one (7.5 s), -55 mV. Responses of posterior large cells are smaller (16 mV) and slower; the site of active response may be at a distance from the soma. 5. The ability of elicit near-synchronous responses and the identity of amplitude and form of responses among anterior cells and of posterior cells, regardless of which cell receives depolarizing current, indicates that all cells undergo active responses and are stimulated by electrotonic spread of depolarization. 6. The responses involve a conductance increase since memses during a driver potential are much reduced. 7. Depolarization by steady current increases the absolute threshold, decreases the maximum depolarization of the peak, and slows rates of rise and fall. Hyperpolarization increases rates of rise and fall; the absolute value reached by the peak depolarization is unchanged. Hyperpolarization reduces the amplitude of the rapid after-potential relative to the displaced resting potential. 8. Hyperpolarizing current pulses imposed during the rise and peak of driver-potential responses are followed by redevelopment of a complete response. Sufficiently strong hyperpolarization can terminate a response. The current strength needed to terminate a response decreases the later during the response the pulse is given...     

The interaction of presynaptic polarization with calcium and magnesium in modifying spontaneous transmitter release from mammalian motor nerve terminals

1. The relationship between motor terminal polarization and miniature end-plate potential (m.e.p.p.) frequency was examined in the presence of various Ca, Mg and K concentrations ([Ca], [Mg] and [K]) and also at modified bathing osmolarity levels. The polarization changes were obtained with ;electrotonic' and ;focal' polarizing currents and with rapid changes in bathing [K].2. M.e.p.p. frequency increased exponentially with electrotonic depolarizing currents, but failed to decrease similarly with hyperpolarizing currents. An increase in bathing [K] to 15 mM increased the sensitivity of the terminals to presynaptic hyperpolarization.3. The slope, on semilogarithmic coordinates, of the function relating m.e.p.p. frequency to electrotonic polarizing currents (the release-current function) was unchanged when bathing [Ca] was raised from 2 to 8 mM. When [Ca] was reduced to 0.5 mM the slope of this function was reduced initially but eventually approached the same slope as in control [Ca]. A similar effect was also found in the presence of 15 mM-KCl.4. The relationship between m.e.p.p. frequency and log [K], at various [Ca], resembled the relationships between m.e.p.p. frequency and presynaptic polarizing currents.5. An increase in bathing [Mg] or osmolarity had a similar effect to a reduction of [Ca].6. Tetrodotoxin (TTX) at a concentration of 10(-6) g/ml. was found to reduce m.e.p.p. frequency, at various [K], by a constant fraction of about 30%.7. In some of the junctions ;anodic break-down' was observed. An examination of this phenomenon with focal polarizing currents disclosed an unusual type of ;anodic break-down', with rapid ;on' and ;off' responses. This phenomenon may indicate that release depends on the influx of positively charged particles into the nerve terminals.8. It is concluded that nerve terminal depolarization accelerates exponentially the activity of a membrane component bearing three Ca molecules, the rate of acceleration being independent of bathing [Ca].     

Pancreatic acinar cells: ionic dependence of acetylcholine-induced membrane potential and resistance change.

1. Intracellular recordings of membrane potential, input resistance and time constant have been made in vitro from the exocrine acinar cells of the mouse pancreas using glass micro-electrodes. The acinar cells were stimulated by acetylcholine (ACh). In some cases ACh was simply directly added to the tissue superfusion bath, in other experiments ACh was applied locally to pancreatic acini by micro-iontophoresis. 2. Current-voltage relations were investigated by injecting rectangular de- or hyperpolarizing current pulses through the recording micro-electrode. Within a relatively wide range (-20 to -70 mV) there was a linear relation between injected current and change in membrane potential. The slope of such linear curves corresponded to an input resistance of about 3-8 M omega. The membrane time constant was about 5-10 msec. 3. ACh depolarized the cell membrane and caused a marked reduction of input resistance and time constant. The minimum latency of the ACh-induced depolarization (microiontophoretic application) was 100-300 msec. Maximal depolarization was about 20 mV. The effect of this local ACh application was abolished by atropine (1-4 x 10-6 M). The blocking effect of atropine was fully reversible. 4. Stimulating with ACh during the passage of large depolarizing current pulses made it possible simultaneously to observe the effect of ACh at two different levels of resting potential (RP). At the spontaneous RP of about minus 40 mV ACh evoked a depolarization of usual magnitude (15-20 mV) while at the artificially displaced level of about -10 mV a small hyperpolarization (about 5 mV) was observed. It therefore appears that the reversal potential of the transmitter equilibrium potential is about -20 mV. 5. Replacement of the superfusion fluid C1 by sulphate or methylsulphate caused an initial short-lasting depolarization, thereafter the normal resting potential was reassumed...     

The slow wave in the circular muscle of the guinea-pig stomach.

The slow wave in the circular muscle of guinea-pig stomach was investigated with the double sucrose-gap method. 2. The amplitude of the slow wave was reduced by depolarization, and it was increased by a small hyperpolarization (5-10 mV). With hyperpolarization greater than 15 mV the amplitude decreased, and the slow wave became reduced, and less dependent on polarization. This residual was not abolished by strong hyperpolarizing current pulses. 3. The frequency of the slow waves was not much affected by membrane polarization. The change was only 15-20% by depolarization or hyperpolarization of 12mV. 4. Rythmic inward currents could be recorded under voltage-clamp conditions. The frequency of the inward currents was the same as that of the slow wave. The intensity of inward current was little affected by membrane polarization. 5. Lowering the temperature reduced the frequency of the slow wave. The rates of rise and fall of the component which remained during strong hyperpolarization were similarly decreased by lowering the temperature. The Q10 of the frequency was about 2-7. 6. It is suggested that the slow wave consists of two different components. One is generated by a potential independent process, and triggers the second component which is potential dependent. The first component may be controlled by some metabolic process.     

A low-voltage activated,transient calcium current is responsible for the time-dependent depolarizing inward rectification of rat neocortical neurons in vitro

Intracellular recordings were obtained from rat neocortical neurons in vitro. The current-voltage-relationship of the neuronal membrane was investigated using current- and single-electrode-voltage-clamp techniques. Within the potential range up to 25 mV positive to the resting membrane potential (RMP: –75 to –80 mV) the steady state slope resistance increased with depolarization (i.e. steady state inward rectification in depolarizing direction). Replacement of extracellular NaCl with an equimolar amount of choline chloride resulted in the conversion of the steady state inward rectification to an outward rectification, suggesting the presence of a voltage-dependent, persistent sodium current which generated the steady state inward rectification of these neurons. Intracellularly injected outward current pulses with just subthreshold intensities elicited a transient depolarizing potential which invariably triggered the first action potential upon an increase in current strength. Single-electrode-voltage-clamp measurements reveled that this depolarizing potential was produced by a transient calcium current activated at membrane potentials 15–20 mV positive to the RMP and that this current was responsible for the time-dependent increase in the magnitude of the inward rectification in depolarizing direction in rat neocortical neurons. It may be that, together with the persistent sodium current, this calcium current regulates the excitability of these neurons via the adjustment of the action potential threshold.     

The relation of membrane changes to contraction in twitch muscle fibres

1. Contractile responses in short twitch-type snake muscle fibres have been studied. These fibres are sufficiently short to allow fairly uniform changes in membrane potential along their length when current is passed through an intracellular micropipette. Active sodium permeability changes were blocked with tetrodotoxin (TTX), procaine, or by using solutions low in sodium. Current and voltage micropipettes were used to voltage-clamp these fibres. Depolarization steps to about -40 mV evoked contractile responses, maximal tension being developed between -10 and 0 mV. The relation between contraction and membrane potential was sigmoid.

2. Depolarization beyond a critical threshold produced an increment of outward current which inactivated with time. The threshold for this delayed rectification was normally similar to the threshold for contractile activation. Fibres exposed to high potassium showed a reversal of this inactivating current to slightly super-threshold depolarizing pulses. At membrane potentials near 0 mV, no inactivating current was noted, while stronger depolarizing pulses produced an inactivating current in the normal direction. Fibres in high potassium show the same threshold for initiation of contraction as in normal solution.

3. Thiocyanate, nitrate, and caffeine shifted the relation between membrane potential and contraction toward higher levels of membrane potential. The threshold for inactivating rectifying current failed to shift to a corresponding extent, although some shift in rectification which did not inactivate was evident.

4. When depolarization was maintained, contractile tension was maximal for several seconds, then gradually disappeared. The rate of this contractile inactivation depended upon the level of depolarization.

     

Postsynaptic control of the induction of long-term changes in efficacy of transmission at neocortical synapses in slices of rat brain

1. Long-term potentiation (LTP) is an enduring, activity-induced increase in the efficacy of synaptic transmission, which has been considered as a possible neural substrate for learning. Recent experiments have shown that LTP can be induced in hippocampal CA1 neurons when a presynaptic volley is paired repetitively with depolarization of the postsynaptic cell, brought about with intracellularly applied depolarizing current pulses (20, 33). We have repeated these experiments in neocortical neurons, in transverse slices of rat sensorimotor cortex in vitro. 2. Stable intracellular recordings were obtained from 28 neurons (mean resting potential -78 mV, mean spike amplitude 95 mV, mean input resistance 41 M omega) mostly in layers V and VI. Two different afferent pathways were stimulated alternately at 0.2 Hz to evoke subthreshold composite excitatory postsynaptic potentials (EPSPs). One micromolar bicuculline methiodide was added to the bathing medium in most experiments. 3. Repetitive pairing of one afferent volley with a coincident intracellular depolarizing current pulse (100-200 ms long) of a magnitude sufficient to make the neuron fire 6 to 13 action potentials/pulse, gave rise after 30-50 pairings in 4 neurons to a significant enduring increase in the amplitude of the paired EPSP. The increase persisted without decrement for as long as the recording continued (range 15-50 min after the pairing ended) but the amplitude of the unpaired EPSP was unchanged. During the LTP, the membrane potential and the apparent input resistance of the postsynaptic neurons were also unchanged. 4. In two cells a significant prolonged depression of the paired EPSP was induced while the unpaired EPSP was unaffected. Membrane potential and input resistance were not changed. In the remaining 22 cells neither the paired nor the unpaired EPSP was altered. 5. Brief, tetanic stimulation was applied to one afferent pathway in 11 of the neurons in which postsynaptic stimulation had been ineffective. A variety of effects was produced (LTP, depression, or posttetanic potentiation). All the effects of tetanic stimulation were confined to the stimulated pathway. 6. We conclude that LTP can be produced in some neocortical neurons by pairing a presynaptic volley with postsynaptic depolarization, in an experimental paradigm that conforms to Hebb's (17) model of associative conditioning. Depression of the paired EPSP was produced in other cells with the same experimental design.(ABSTRACT TRUNCATED AT 400 WORDS)     

The timing of calcium action during neuromuscular transmission

1. When a nerve-muscle preparation is paralysed by tetrodotoxin, brief depolarizing pulses applied to a motor nerve ending cause packets of acetylcholine to be released and evoke end-plate potentials (e.p.p.s), provided calcium ions are present in the extracellular fluid.2. By ionophoretic discharge from a 1 M-CaCl(2) pipette, it is possible to produce a sudden increase in the local calcium concentration at the myoneural junction, at varying times before or after the depolarizing pulse.3. A brief application of calcium facilitates transmitter release if it occurs immediately before the depolarizing pulse. If the calcium pulse is applied a little later, during the period of the synaptic delay, it is ineffective.4. It is concluded that the utilization of external calcium ions at the neuromuscular junction is restricted to a brief period which barely outlasts the depolarization of the nerve ending, and which precedes the transmitter release itself.5. The suppressing effect of magnesium on transmitter release was studied by a similar method, with ionophoretic discharges from a 1 M-MgCl(2)-filled pipette. The results, though not quite as clear as with calcium, indicate that Mg pulses also are only effective if they precede the depolarizing pulses.     

Long-lasting modulation of synaptic input to Purkinje neurons by Bergmann glia stimulation in rat brain slices

Information processing in the nervous system is achieved primarily at chemical synapses between neurons. Recent evidence suggests that glia-neuron interactions contribute in multiple ways to the synaptic process. In the present study we used the frequency of spontaneous postsynaptic currents (sPSC) in Purkinje neurons in acute cerebellar brain slices from juvenile rats (13-19 days old) as a measure of synaptic activity. Following 50 depolarizing pulses to an adjacent Bergmann glial cell (50 mV; duration 0.5 s; 1 Hz) the sPSC frequency of the Purkinje neuron was reduced to 65 ± 7 % of control values within 10 min after glial stimulation and remained depressed for at least 40 min. Depolarizing pulses to 0 mV had a comparable effect (70 ± 5 % of control). The frequency of miniature PSCs, as recorded in 300 n m TTX, was not modulated after glial stimulation. Blockade of ionotropic glutamate receptors (iGluRs) with kynurenic acid (1 m m ) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 μ m ) suppressed the reduction of neuronal activity induced by glial depolarization, whereas the glial modulation of synaptic activity was not inhibited by a block of N -methyl- d -aspartate iGluRs, metabotropic glutamate receptors, cannabinoid receptors or GABA_B receptors. Fluorometric measurements of the intraglial Ca²⁺ concentration revealed no glial Ca²⁺ transients during the depolarization series, and glial cell stimulation reduced the neuronal sPSC frequency even after loading the glial cell with 20 m m of the Ca²⁺ chelator BAPTA. Our results indicate a glia-induced long-lasting depression of neuronal communication mediated by iGluRs.     

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.     

Subthreshold contribution of N-methyl-d-aspartate receptors to long-term potentiation induced by low-frequency pairing in rat hippocampal CA1 pyramidal cells

Long-term potentiation (LTP) is a use-dependent and persistent enhancement of synaptic strength. In the CA1 region of the hippocampus, LTP has Hebbian characteristics and requires precisely timed interaction between presynaptic firing and postsynaptic depolarization. Although depolarization is an absolute requirement for plasticity, it is still not clear whether the postsynaptic response during LTP induction should be subthreshold or suprathreshold for the generation of somatic action potential. Here, we use the whole-cell patch-clamp technique and different pairing protocols to examine systematically the postsynaptic induction requirements for LTP. We induce LTP by changes only in membrane potential while keeping the afferent stimulation constant and at minimal levels. This approach permits differentiation of two types of LTP: LTP induced with suprathreshold synaptic responses (LTP(AP)) and LTP induced with subthreshold excitatory postsynaptic current (EPSCs; LTP(EPSC)). We found that LTP(AP) (>40%) required pairing of depolarization (V(m)>or=-40 mV, for 40-60 s) with four to six (0.1 Hz) single synaptically initiated action potentials. LTP(EPSC) was of smaller magnitude (<30%) and required pairing of depolarization to -50 mV (60 s) with six subthreshold EPSCs. The N-methyl-d-aspartate receptor (NMDAR) antagonists aminophosphonovaleric acid and 7-chlorokynurenic acid consistently blocked LTP(EPSC) but were ineffective in preventing LTP(AP). Robust, NMDAR-independent LTP is obtained by stronger postsynaptic depolarization that converts the EPSCs to suprathreshold somatic action potentials. Purely NMDAR-dependent LTP is obtained by pairing mild somatic depolarization with subthreshold afferent pulses to the postsynaptic cell. Our results indicate that the degree of postsynaptic depolarization in the presence of single afferent pulses determines the type and magnitude of LTP.