The effect of calcium ions and temperature on the binomial parameters that control acetylcholine release by a nerve impulse at amphibian neuromuscular synapses.

1. A study has been made of the effects of changing the external calcium concentration, [Ca](o), and the temperature on both the number of quanta available for release by the nerve impulse (n) as well as the increase in release probability of a quantum p(t) during the release period (from 0 to T) following a nerve impulse at synapses in amphibian striated muscle.2. When [Ca](o) was increased in the low range from 0.25 to 0.4 mM at 18 degrees C, the average quantal content of the e.p.p. (m) increased as the fourth power of [Ca](o) and this was primarily due to a third power dependence of n on [Ca](o); the dissociation constants and power dependence of n on calcium determined in the [Ca](o) range from 0.25 to 1.0 mM were successfully used to predict the changes in size of the e.p.p. in the very high [Ca](o) range from 1 to 10 mM. When the temperature was increased from 7 to 18 degrees C in a [Ca](o) of 0.6 mM or 0.35 mM, n increased with a Q(10) of 2.5.3. When [Ca](o) was increased in the range from 0.25 to 1.0 mM at 18 degrees C, the probability that a quantum initially available for release is released during the release period (p(T)) was very sensitive to [Ca](o), increasing as the third power of [Ca](o) and with a dissociation constant of 0.13 mM. When the temperature was increased from 7 to 18 degrees C in a [Ca](o) of 0.6 mM or 0.35 mM, p(T) decreased.4. The histograms of latencies of individual quanta following a nerve impulse was very temperature dependent: the time to peak of the histograms (i.e. the interval in which most quanta fell) had a Q(10) of over 4 as did the time constant of decline of the histograms in the temperature range from 7 to 18 degrees C.5. The average number of quanta released up to time t during the release period following a nerve impulse, namely np(t), was well described by a stochastic process in which p(t) was determined by two reactions; one of these reactions released available quanta from the nerve terminal whilst the other made some of the available quanta unavailable for release by the nerve impulse.     

The effects of calcium ions on the binomial parameters that control acetylcholine release during trains of nerve impulses at amphibian neuromuscular synapses.

1. A study has been made of the effects of changing the external calcium concentration [Ca](o) on the binomial parameters p and n that control the average quantal content (m) of the end-plate potential (e.p.p.) during trains of nerve impulses at synapses in amphibian striated muscle.2. In high external calcium concentrations (0.4 mM 相似文献   

Dual effect of external calcium on the frequency of miniature synaptic potentials in frog sympathetic ganglion cells

The effect of calcium on spontaneous transmitter release and on the release induced by tetanic stimulation and by raising the external potassium concentration ([K]0) was studied in sympathetic ganglion cells of Rana esculenta. 1. In standard Ringer's solution the frequency of miniature excitatory postsynaptic potentials (mepsp) ranged from 0.05--2.0 s-1 (0.05 +/- 0.09 s-1, n = 37) at room temperature. 2. At a [K]0 of 2.5 mM mepsp frequency was approximately linearly related to the logarithm of the external calcium concentration (log [Ca]0) (0.1 mM less than or equal to [Ca]0 less than or equal to 20 mM). 3. Duration and amplitude of the potentiation of transmitter release after tetanic preganglionic stimulation increased depending on [Ca]0. 4. Mepsp frequency was strongly dependent on [K]0 between 10 and 20 mM; the frequency being increased to about 40 times control level at a [K]0 of 20 mM. 5. Raising [Ca]0 up to 1.8 mM in high K solutions resulted in an increase in mepsp frequency followed by a decrease at higher [Ca]0. 6. These results are consistent with the idea that the effect of calcium on mepsp frequency depends on: (a) the driving force for calcium entry, (b) the effect of Ca ions on the potential gradient within the nerve membrane.     

Electrical activity in pancreatic islet cells: effect of ions

1. Intracellular micro-electrode recording techniques have been used to study the effects of varying the external ion concentration on the membrane potential and glucose-induced electrical activity in cells from mouse islets of Langerhans.2. Increasing [K](o) to 47 mM depolarized islet cells without inducing electrical activity. Normal action potentials were generated in response to glucose after removal of [K](o) for 60 min.3. Reduction of [Cl](o) to 12 mM did not affect the membrane potential and glucose still induced electrical activity.4. Reduction of [Na](o) to 26 mM increased the amplitude of action potentials; subsequently the cells depolarized.5. Removal of [Ca](o) caused cells, already firing action potentials intermittently in response to glucose, to change their pattern of discharge to one of continuous firing. The time constant of the action potentials was also increased. Depletion of calcium for 60 min before addition of glucose prevented the appearance of electrical activity.6. Increasing [Ca](o) threefold to 7.7 mM in the presence of glucose, 11.1 mM, increased action potential amplitude to 12 mV, but a tenfold increase of [Ca](o) to 25.6 mM completely blocked action potential discharge.7. Exposure of islet cells simultaneously to reduced [Na](o) (26 mM) and increased [Ca](o) (7.7 mM) increased the amplitude of glucose-induced action potentials to about 20 mV.8. Strontium, 2.56 mM, was an effective substitute for [Ca](o), 2.56 mM, and maintained a normal pattern of electrical activity in response to glucose.9. Increasing the magnesium concentration tenfold to 11.3 mM did not block electrical activity whereas manganese, 2 mM, blocked glucose-induced action potentials and depolarized islet cells.10. It was concluded that the action potential induced by glucose in islet beta-cells is due predominantly to calcium entry and that sodium ions tend to repress this calcium influx.     

The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coli

1. Spontaneous spike activity and action potentials evoked by external field stimulation were recorded, intracellularly and with the double sucrose gap method, from the smooth muscle of guinea-pig taenia coli.2. Replacement of external NaCl with sucrose (leaving 10 mM-Na in the buffer) caused hyperpolarization and stopped spontaneous activity within 10 min. Spikes could, however, be evoked for 2-3 hr. The amplitude, the overshoot and rate of rise of the spike were increased.3. In 10 mM-[Na](o) the intracellular Na concentration was reduced from 35 to 24 mM, shifting the Na-equilibrium potential from +34 to -22 mV.4. Excess Ca (12.5 mM) caused hyperpolarization and increased membrane conductance. The amplitude and the rate of rise of the spike were increased, the threshold was raised and the latency of the spike evoked by threshold stimulation became shorter.5. The effect of reducing the external Ca concentration depended on the Na concentration present, being greater with higher external [Na](o). When the membrane was depolarized and spikes deteriorated in low Ca (0.2-0.5 mM) reduction of Na to 10 mM caused repolarization and recovery of the action potential.6. Mn (0.5-1.0 mM) blocked spontaneous spike discharge after 20 min. Higher concentrations (more than 2.0 mM) were required to block the evoked action potential.7. The results indicate that the smooth muscle spike in taenia is due to Ca-entry and that Na influences spike activity indirectly by competing with Ca in controlling the membrane potential.     

Penetration-induced hyperpolarization as evidence for Ca2+ activation of K+ conductance in isolated smooth muscle cells

Single smooth muscle cells, freshly isolated by enzymatic digestion of the stomach muscularis of the toad Bufo marinus were studied under direct microscopic observation using standard electrophysiological techniques. Following penetration with a microelectrode, a hyperpolarization lasting many seconds occurred before the membrane depolarized to a steady-state level. The following lines of evidence indicate that the penetration-induced hyperpolarization results from an increase in K+ conductance caused by Ca2+ that enters the cell at the time of penetration: 1) The cell contracted at the time of penetration indicating that [Ca2+]i was elevated even though no action potential had occurred; the cell subsequently relaxed. 2) The input resistance was much lower during the hyperpolarization than during the steady-state resting potential. In the steady state all cells displayed outward-going rectification. 3) At constant [Ca2+]0, the amplitude of the hyperpolarization varied with log[K+]0 (1.3-56 mM) to a much greater degree than did the steady-state potential. Tetraethylammonium chloride (TEA) (18.2 mM) reduced the hyperpolarization. 4) At constant [K+]0, the amplitude of the hyperpolarization increased as the [Ca2+]0 was raised (1.8-52.1 mM). 5) With [Ca2+]0 low (less than or equal to 0.16 mM), the hyperpolarization was almost completely abolished in the presence of a high concentration of Ba2+ (80 mM) or Mn2+ (79.2 mM); this was not the case with Sr2+.     

Correlation of presynaptic and postsynaptic events during establishment of long-term facilitation at crayfish neuromuscular junction

Repetitive stimulation (10-20 Hz) of the motor axon supplying the opener muscle in the crayfish leg produces long-lasting enhancement of excitatory postsynaptic potentials. This long-term facilitation (LTF) was investigated by recording simultaneously from the presynaptic nerve terminal and from the innervated muscle fiber with intracellular microelectrodes. On cessation of stimulation, the facilitated postsynaptic potential declines in amplitude when monitored with low-frequency test stimuli. A rapid decline (phase I) occurs over the first 30 s and is succeeded by a more gradual decline lasting several minutes (phase II). Finally, a residual potentiation with a very slow decay (phase III) persists for several hours. Simultaneous pre- and postsynaptic recordings were made during induction of LTF with stimuli delivered at 20 Hz for 10 min. During the tetanus, excitatory postsynaptic potentials were enhanced 20-fold, while action potentials in the presynaptic terminal declined in amplitude from 108.6 to 97.2 mV, and the presynaptic membrane became hyperpolarized by 6.4 mV. The Na+ pump inhibitor ouabain (0.5-1.0 mM) abolished the hyperpolarization, indicating that the latter resulted from activation of an electrogenic Na+ pump. The reduction in amplitude of the presynaptic action potential was consistent with a reduced transmembrane concentration gradient for Na+. Thus, it is suggested that a significant accumulation of Na+ occurs during repetitive stimulation of crayfish motor axons. Decay of phase II of LTF, but not of phases I or III, had approximately the same time course as the decay of Na+ accumulation in the terminals, monitored by changes in the presynaptic action potential. Thus it is probable that in crayfish this phase of LTF is linked to an increased intraterminal Na+ concentration. Injection of Na+ from a microelectrode into the presynaptic terminal produced enhancement of the excitatory postsynaptic potential lasting for many minutes, as well as changes in presynaptic membrane potential and action potential similar to those seen during repetitive stimulation. The results provide the first direct measurements of electrical and ionic changes in axonal terminals during prolonged periods of activity leading to LTF, and support the hypothesis that accumulation of intraterminal Na+ is associated with one phase of LTF.     

The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres

1. The intracellular Na activity of sheep heart Purkinje fibres has been measured using recessed-tip Na(+)-sensitive glass micro-electrodes.2. The internal Na activity was 7.2 +/- 2.0 mM (mean +/- S.D., n = 32) at the normal external Na concentration, [Na](o), in these experiments of 140 mM (equivalent to an external Na activity of 105 mM). The equilibrium potential for Na across the fibre membrane was therefore approximately + 70 mV.3. When the [K](o) was altered the internal Na activity changed, reaching a new level within about 20 min. Increasing the [K](o) from 4 to 25 mM decreased the internal Na by approximately 30%, while decreasing the [K](o) from 4 to 1 mM increased internal Na by 20%.4. The removal of external K produced an easily reversible increase in the internal Na with an initial rate equivalent to a concentration change of 0.24 +/- 0.07 m-mole/min (mean +/- S.D., n = 8).5. Ouabain produced increases in the internal Na activity that were only very slowly reversible. The threshold concentration for producing an increase was approximately 10(-7)M.6. When [Na](o) was reduced the internal Na activity fell rapidly with a single exponential time course (time constant 3.3 +/- 0.8 min, mean +/- S.D., n = 16) to a new, relatively stable level. The recovery of internal Na on return to the normal [Na](o) did not have a simple time course. It was normally complete within 10-30 min.7. The relationship of the stabilized level of the internal Na activity to the [Na](o) was approximately linear over the range 140-14 mM-[Na](o). When [Na](o) was reduced from 140 to 14 mM the internal Na activity fell by 72 +/- 5% (mean +/- S.D., n = 21).8. When the [Na](o) was reduced, the decrease in the internal Na activity was partially inhibited by Mn or by removal external Ca.9. When the [Ca](o) was altered over the range 0.2-16 mM the internal Na activity was reduced by approximately 50% for a tenfold increase in the [Ca](o).10. The relationship between internal Na and contractility is discussed.     

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.     

Calcium regulates L-Type Ca2+ channel expression in rat skeletal muscle cells

Primary skeletal muscle cells were cultured in a normal- (1.8 mM) or high- (4.8 mM) Ca2+ culture medium to determine whether Ca2+ modulates the number of L-type Ca2+ channels. Skeletal myoballs cultured in a normal medium showed, when exposed to a high extracellular [Ca2+], ([Ca2+]e) a transient increase in intracellular [Ca2+] ([Ca2+]i) from a resting concentration of 60 to 160 nM. By day 3, however, when the experiments were made, [Ca2+]i no longer differed from control (pre-exposure to high Ca2+). The maximum charge movements in myoballs incubated in 1.8 and 4.8 mM were 16.4+/-1.05 (n=56) and 24.1+/-1.18 nC/microF (n=58; P<0.01), respectively, and peak Ca2+ currents at 20 mV were -10.8+/-1.09 (n=46) and -12.8+/-0.75 nA/microF (n=82), respectively (P>0.05). The tail current amplitudes in 1.8 and 4.8 mM Ca2+-treated cells were -9.3+/-1.23 and -14.2+/-1.37 nA/microF (P<0.05), respectively, at 10 mV and -15.3+/-1.76 and -23.6+/-2.02 nA/microF (P<0.05), respectively at 60 mV. The maximum binding of [3H]PN200-110 (a radioligand specific for L-type Ca2+ channel alpha1 subunits) in myoballs cultured in 1.8 and 4.8 mM [Ca2+]e was 1.34+/-0.23 and 3.2+/-0.63 pmol/mg protein (n=8; P<0.02), respectively. The increase in [Ca2+]i associated with the increases in charge movements, tail currents and the number of L-type Ca2+ channel alpha1 subunits in skeletal muscle cells cultured in high [Ca2+]e support the concept that extracellular Ca2+ influx modulates the expression of L-type Ca2+ channels in skeletal muscle cells.     

Pre- and post-synaptic mechanisms of synaptic strength homeostasis revealed by slowpoke and shaker K+ channel mutations in Drosophila

We report naturally occurring, systematic variations in synaptic strength at neuromuscular junctions along the dorsal-ventral (D-V) axis of the Drosophila larval body wall. These gradual changes were correlated with differences in presynaptic neurotransmitter release regulated by nerve terminal excitability and in postsynaptic receptor composition influencing miniature excitatory junctional potential (mEJP) amplitude. Surprisingly, synaptic strength and D-V differentials at physiological Ca(2+) levels were not significantly altered in slowpoke (slo) and Shaker (Sh) mutants, despite their defects in two major repolarizing forces, Ca(2+)-activated Slo (BK) and voltage-activated Sh currents, respectively. However, lowering [Ca(2+)](o) levels revealed greatly altered synaptic mechanisms in these mutants, indicated by drastically enhanced excitatory junctional potentials (EJPs) in Sh but paradoxically reduced EJPs in slo. Removal of Sh current in slo mutants by 4-aminopyridine blockade or by combining slo with Sh mutations led to strikingly increased synaptic transmission, suggesting upregulation of presynaptic Sh current to limit excessive neurotransmitter release in the absence of Slo current. In addition, slo mutants displayed altered immunoreactivity intensity ratio between DGluRIIA and DGluRIIB receptor subunits. This modified receptor composition caused smaller mEJP amplitudes, further preventing excessive transmission in the absence of Slo current. Such compensatory regulations were prevented by rutabaga (rut) adenylyl cyclase mutations in rut slo double mutants, demonstrating a novel role of rut in homeostatic plasticity, in addition to its well-established function in learning behavior.     

Novel mechanism for presynaptic inhibition: GABA(A) receptors affect the release machinery

Presynaptic inhibition is produced by increasing Cl(-) conductance, resulting in an action potential of a smaller amplitude at the excitatory axon terminals. This, in turn, reduces Ca(2+) entry to produce a smaller release. For this mechanism to operate, the "inhibitory" effect of shunting should last during the arrival of the "excitatory" action potential to its terminals, and to achieve that, the inhibitory action potential should precede the excitatory action potential. Using the crayfish neuromuscular preparation which is innervated by one excitatory axon and one inhibitory axon, we found, at 12 degrees C, prominent presynaptic inhibition when the inhibitory action potential followed the excitatory action potential by 1, and even 2, ms. The presynaptic excitatory action potential and the excitatory nerve terminal current (ENTC) were not altered, and Ca(2+) imaging at single release boutons showed that this "late" presynaptic inhibition did not result from a reduction in Ca(2+) entry. Since 50 microM picrotoxin blocked this late component of presynaptic inhibition, we suggest that gamma-aminobutyric acid-A (GABA(A)) receptors reduce transmitter release also by a mechanism other than affecting Ca(2+) entry.     

L-glutamate as an excitatory transmitter at the Drosophila larval neuromuscular junction.

The possibility that L-glutamate is the excitatory transmitter at the Drosophila larval neuromuscular junction and the ionic basis of its action on the muscle membrane are examined. 2. Iontophoretically applied L-glutamate causes muscle depolarization (L-glutamate potential) if and only if the L-glutamate pipette is within a few mum of the nerve ending. D-glutamate, substance P, ACh and GABA are ineffective. 3. Bath-applied L-glutamate produces similar changes in the time course and amplitude of miniature excitatory junctional potential (m.e.j.p.), excitatory junctional potential (e.j.p.) and the L-glutamate potential. 4. Neuromuscular transmission and excitation-contraction coupling are operative in a haemolymph-like solution containing 1 mM L-glutamate. 5. The reversal potentials of the e.j.p. and the L-glutamate potential are identical to each other, changing similarly with changes in the ionic compositions of the external medium (twelve solutions). 6. The ionic dependence of the reversal potentials is predicted from an extended constant-field equation using a ratio of sodium:potassium permeabilities of PNa/PK=1-3, and a ratio of magnesium:potassium permeabilities of PMg/PK=4-7. 7. It is concluded that L-glutamate is, or is an agonist of, the excitatory transmitter at certain Drosophila larval neuromuscular junctions.     

Effect of extracellular [Ca(2+)] on Ca(2+) release from sarcoplasmic reticulum in rat ventricular myocytes

The effect of external [Ca(2+)] ([Ca(2+)](o)) on Ca(2+) release from the sarcoplasmic reticulum (SR) was examined with rested-state twitches in rat ventricular myocytes. The magnitude of transient rise of intracellular [Ca(2+)] ([Ca(2+)](i)) relative to the resting one, F/F(o), as measured with fluo-3, was 1.75+/-0.07 (mean+/-SEM, n=9) and 1.86+/-0.13 (n=9) at 0.3 and 1.8 mM [Ca(2+)](o), respectively; the difference was insignificant. The time from onset to peak and the rate of rise of the [Ca(2+)](i) transient were 0.107+/-0.017 s (n=9) and 18.8+/-3.38 F/F(o)/s, respectively, at 0.3 mM [Ca(2+)](o), they were 0.064+/-0.005 s (n=9) and 31.1+/-0.03 F/F(o)/s, respectively, at 1.8 mM [Ca(2+)](o). The difference in the corresponding values at the two [Ca(2+)](o) was significant (t-test, p<0.05). The half decay time of the [Ca(2+)](i) transient was 0.217+/-0.016 s (n=8) at 0.3 mM [Ca(2+)](o) and was similar to the value of 0.230+/-0.022 s (n=8) at 1.8 mM [Ca(2+)](o), indicating that the rate of decrease of [Ca(2+)](i) is independent of the [Ca(2+)](o). The duration of action potential was similar at 0.3 and 1.8 mM [Ca(2+)](o) as examined with papillary muscle. The results suggest that a lowering of [Ca(2+)](o), i.e., reducing the Ca(2+) influx, slows the rate of Ca(2+) release from the SR fully loaded with Ca(2+) with little effect on the total amount of the Ca(2+) release. An instantaneous relationship between the [Ca(2+)](i) and the myocyte shortening at 0.3 and 1.8 mM [Ca(2+)](o) suggested that the time course of unloaded contraction is related not only to the magnitude, but also to the rate of rise of [Ca(2+)](i).     

Early activation of the primary somatosensory cortex without conscious awareness of somatosensory stimuli in tumor patients

While G-proteins are involved in the synaptic release machinery and also can mediate inhibition of presynaptic Ca2+ channels, we find that pertussis toxin (PTX) does not affect the amount and the time course of quantal release from motor nerve terminals on crayfish or mouse muscle. Monoquantal excitatory currents (qEPSCs) were recorded that were elicited by constant depolarisation pulses to a terminal by means of a perfused macro-patch electrode. Although presynaptic effects of PTX on output and time course of release of quanta were absent, postsynaptically the rise time of qEPCs was increased and their decay time constant reduced. Adenosine (Ad) is known to inhibit quantal release in vertebrate motor nerve terminals via PTX sensitive G-proteins, and Ad is generated during nicotinic synaptic transmission by breakdown of the co-transmitter adenosine triphosphate (ATP). As reported by others, we found in mouse muscle inhibition of quantal release after application of Ad, but in addition late facilitation. Both these effects of Ad were blocked when the muscle was pre-incubated with PTX.     

Excitatory potentials induced by stimulation of the inhibitory axon at the crustacean neuromuscular junction.

Synaptic events in a chloride-deficient condition were studied to elucidate functional aspects of presynaptic inhibitory synapses. The extracellular junctional potentials and nerve terminal potentials were concurrently recorded from a synaptic region. Inhibitory stimulation produced repetitive spikes on the inhibitory nerve terminal and then the excitatory nerve terminal, which resulted in the extracellular excitatory junctional potentials. Excitatory stimulation did not produce repetitive spikes on the inhibitory nerve terminal, indicating one-way signal transmission in this axo-axonal synapse from inhibitory to excitatory axon. The interval required for an inhibitory stimulation to produce the first response in the postsynaptic muscle membrane ranged widely from 10 to 800 msec. When gamma-aminobutyric acid (GABA, 1 times 10-minus 4 M) was added in these experimental conditions, the muscle membrane was transiently depolarized by about 10 mV. The action of GABA mimics that of the neurotransmitter at presynaptic inhibitory synapses. The experimental observations may be well explained by the concept of synapses on synapses, i.e., presynaptic inhibition, where the neurotransmitter may be GABA and chloride ions may be playing essential roles as in the case of postsynaptic inhibition.     

High rates of excitatory miniature currents in crayfish claw opener muscle evoked by high concentrations of gamma-aminobutyric acid (GABA) in normal and Ca2+-deficient superfusions

High concentrations (0.5 mol/l) of the neutral amino acid GABA were used to evoke release of transmitter quanta from excitatory terminals at voltage clamped crayfish muscle fibres in normal and Ca²⁺-deficient superfusions. An experiment in which the release of transmitter quanta proceeded at high rates in both normal and Ca²⁺-deficient superfusion was analyzed in detail indicating a Ca²⁺-independent mechanism of release. In the normal superfusion, on application of GABA, the release rates ñ increased within a few seconds up to about 6000 quanta/s and thereafter declined exponentially with a time constant $\text{τ}{}_{\mathrm{<mtext>q</mtext>}}\text{) = 18.5}\text{s}$, most likely due to depletion of a readily releasable store of transmitter in the excitatory nerve terminals comprising at least 110,000 quanta per muscle fibre. Assuming that about 1900 excitatory synapses exist per muscle fibre [9], it results that about 58 quanta can be associated with each synapse in agreement with morphological data [15] which show that between 47–117 vesicles exist in a single glutamatergic synapse of crayfish.     

The effect of calcium ions on the binomial statistic parameters that control acetylcholine release at preganglionic nerve terminals.

1. A study has been made of the effects of changing [Ca]O and [Mg]O on the binomial statistic parameters p and n that control the average quantal content (m) of the excitatory post-synaptic potential (e.p.s.p.) due to acetylcholine release at preganglionic nerve terminals. 2. When [Ca]O was increased in the range from 0-2 to 0-5 mM, p increased as the first power of [Ca]O whereas n increased as the 0-5 power of [Ca]O; when [Mg]O was increased in the range from 5 to 200 mM, p decreased as the first power of [Mg]O whereas n decreased as the 0-5 power of [Mg]O. 3. The increase in quantal release of a test impulse following a conditioning impulse was primarily due to an increase in n; the increase in quantal content of successive e.p.s.p.s in a short train was due to an increase in n and p, and the increase in n was quantitatively described in terms of the accumulation of a Ca-receptor complex in the nerve terminal. 4. The decrease in quantal content of successive e.p.s.p.s during long trains of impulses over several minutes was primarily due to a decrease in n. These results are discussed in terms of an hypothesis concerning the physical basis of n and p in the release process.     

Synaptic modulation by a neuropeptide depends on temperature and extracellular calcium

The crayfish neuropeptide DRNFLRFamide increases transmitter release from synaptic terminals onto muscle cells. As temperature decreases from 20 to 8 degrees C, the size of excitatory junctional potentials (EJPs) decreases, and the peptide becomes more effective at increasing EJP amplitude. The goal of the present study was to determine whether the enhanced effectiveness of the peptide is strictly a temperature-related effect, or whether it is related to the fact that the EJPs are smaller at low temperature, allowing a greater range for EJP amplitude to increase. Decreasing temperature reduced the number of quanta of transmitter released per nerve impulse (assessed by recording synaptic currents) and increased input resistance in muscle fibers. As in earlier work, the ability of the peptide to increase EJP amplitude was enhanced by decreasing temperature. However, the peptide was also more effective at increasing EJP amplitude when transmitter output was lowered by reducing the ratio of calcium to magnesium ions in the bath. Thus the effectiveness of the peptide may be related to the level of output from the synaptic terminals.     

Tonic depolarization of excitatory nerve terminals in crayfish muscle by high concentrations of extracellular potassium

At voltage-clamped fibres of the claw opener muscle of small crayfish, spontaneous quantal release of excitatory transmitter elicited by raising extracellular K+ to 100 mM was investigated. On application of the high K+ concentration, the rates of quantal release increased to n = 10,000-25,000 quanta/s within 10 s, and thereafter declined exponentially, either with a single (tau congruent to 15-40 s) or with two (tau 1 congruent to 15-40 s, tau 2 greater than 70 s) time constants. The total number of quanta released per trial ranged from s = 200,000 to 800,000 quanta. The results were derived by means of the fluctuation analysis technique.