Level Of Transmitter Release Research Articles

Involvement of Calcium Ions in Regulation of the Kinetics of the Release of Acetylcholine Quanta Forming Multiquantum Postsynaptic Responses

Experiments on frog neuromuscular synapses using the “sequential subtraction” method developed in our laboratory were performed to investigate the quantum composition of endplate currents and the time parameters of the release of acetylcholine quanta forming multiquantum postsynaptic responses in the presence of varying calcium ion levels. These experiments showed that at physiological levels of transmitter release in response to application of single stimuli of the motor nerve, quanta were released asynchronously, as indicated by the nature of fluctuations in the latent periods of single-quantum responses forming the complete quantum signal. Different reactions in terms of the quantum composition of endplate currents and the kinetics of quantum release were seen when the extracellular calcium ion concentration was increased: quantum composition increased exponentially with increases in calcium concentration from 0.4 to 1.8 mM, while the synchronicity of quantum release reached a peak at a calcium concentration of 1.0 mM. These data lead to the conclusion that changes in the synchronicity of transmitter quantum release in response to each nerve impulse in conditions of modified calcium influx into nerve terminals can be regarded as one of the mechanisms mediating synaptic plasticity.

Synapse–glia interactions are governed by synaptic and intrinsic glial properties

It is believed that glial cell activation and their interactions with synapses are predominantly dependent upon the characteristics of synaptic activity and the level of transmitter release. Because synaptic properties vary from one type of synapse to another, synapse-glia interactions should differ accordingly. The goal of this work was to examine how glial cell activation is dependent upon the properties of their respective synapses as well as the level of synaptic activity. We contrasted Ca 2+ responses of perisynaptic Schwann cells (PSCs) at neuromuscular junctions (NMJs) with different synaptic properties; the slow-twitch soleus (SOL) and the fast-twitch levator auris longus (LAL) muscles. Amplitude of PSC Ca 2+ responses elicited by repeated motor nerve stimulation at 40, 50 and 100 Hz were larger and their kinetics faster at LAL NMJs and this, at all frequencies examined. In addition, a greater number of PSCs per NMJ was activated by sustained synaptic transmission at NMJs of LAL in comparison to SOL. Differences in PSC activation could not be explained solely by differences in levels of transmitter release but also by intrinsic PSC properties since increasing transmitter release with tetraethylammonium chloride (TEA) did not increase their responsiveness. As a whole, these results indicate that PSC responsiveness at NMJs of slow- and fast-twitch muscles differ not only according to the level of activity of their synaptic partner but also in accordance with inherent glial properties.

Converging Effects ofGinkgo bilobaExtract at the Level of Transmitter Release, NMDA and Sodium Currents and Dendritic Spikes

In this study, an attempt was made to integrate the effects of GINKGO BILOBA extract (GBE) in different experimental systems (IN VITRO cochlea, brain slice preparations and cortical cell culture) to elucidate whether these processes converge to promote neuroprotection or interfere with normal neural function. GBE increased the release of dopamine in the cochlea. NMDA-evoked currents were dose-dependently inhibited by rapid GBE application in cultured cortical cells. GBE moderately inhibited Na+ channels at depolarised holding potential in cortical cells. These inhibitory effects by GBE may sufficiently contribute to the prevention of excitotoxic damage in neurons. However, these channels also interact with memory formation at the cellular level. The lack of effect by GBE on dendritic spike initiation in neocortical layer 5 pyramidal neurons indicates that the integrative functions may remain intact during the inhibitory actions of GBE.

Interactions of chemostimuli at the single cell level: studies in a model system.

The responses of afferent chemosensory fibres of the carotid body to individual chemostimuli have long been established. However, the mechanisms underlying the multiplicative interactions of these stimuli (i.e. how the combined effects of hypoxia and hypercapnia exert a greater effect on afferent nerve discharge than the sum of their individual effects) have not been elucidated. Using the membrane hypothesis for carotid body chemoreception, in which chemostimuli inhibit type I cell K+ channels, leading to depolarization, voltage-gated Ca2+ entry and hence the triggering of exocytosis, this article considers data acquired in isolated type I carotid body cells and model chemoreceptor (PC12) cells to attempt to explain stimulus interactions. Whilst stimulus interactions are not clearly evident at the level of K+ channel inhibition or rises of [Ca2+]i, they are apparent at the level of transmitter release. Thus, it is clear that individual chemoreceptor cells can sense multiple stimuli, and that interactions of these stimuli can produce greater than additive effects in terms of transmitter release.

Study of the inhibitor of the crayfish neuromuscular junction by presynaptic voltage control.

The inhibitor of the crayfish opener muscle was investigated by a presynaptic voltage control method. Two microelectrodes were inserted into the inhibitor and the amplitude and duration of presynaptic depolarization were controlled by a voltage-clamp amplifier. The inhibitory postsynaptic potential (IPSP) was measured from a muscle fiber located near the presynaptic voltage electrode. Nonlinear summation of IPSP amplitudes was corrected after chloride equilibrium potential was measured. With the use of 5-ms presynaptic pulses, the depolarization-release coupling (D-R) curve constructed from IPSP peak amplitudes (IPSPcor) had a threshold of about -35 mV and reached its maximal level at -5 to -10 mV. Depolarization beyond the maximum led to a suppression of neurotransmitter release. When transmitter release during a presynaptic pulse was completely suppressed, IPSPs activated by tail current could be identified with an average synaptic delay of 2.5 ms. Transmitter secretion triggered by a calcium current activated during the 5-ms pulses (IPSPon) was also measured on the rising phase of an IPSP, at 2.5 ms after the end of the 5-ms pulses. D-R coupling plots measured from IPSPon exhibited a more pronounced suppression than that obtained from IPSPcor. The effect of presynaptic pulse duration on the level of transmitter release was analyzed. Transmitter release increased with increasing duration and was nearly saturated by 20-ms pulses depolarized to 0 mV. The following conditions were identified as necessary to obtain a consistent D-R curve with a clear suppression: 1) small animals, 3.8 cm head to tail, 2) 15 degrees C, 3) 40 mM tetraethylammonium and 1 mM 4-aminopyridine, 4) an extracellular calcium concentration of < or = 10 mM. In addition, a consistent correlation was found among the branching pattern of the inhibitor, the placement of the presynaptic electrode, and the characteristics of the D-R curves. An ideal presynaptic electrode configuration involved placing the voltage electrode in a secondary branch, approximately 100 microns from the main branch point, and placing the current electrode at the branch point. Postsynaptically, optimal recordings were obtained from muscle fibers innervated by a single branch of the inhibitor that originated from a point near the presynaptic voltage electrode. A cable-release model was constructed to evaluate the relationship between the shape of the D-R coupling curves and the space constants of the presynaptic terminals. A comparison between the model and the D-R coupling curves suggested that the space constant of an inhibitor branch on a muscle fiber is > or = 8 times longer than its actual length. Therefore the upper limit estimate of the space constant of a typical preparation is approximately 3 mm. Results reported here outline morphological and physiological conditions needed to achieve optimal control of the presynaptic branch of the crayfish inhibitor. The cable-release model quantitatively defines the extent of presynaptic voltage control.

Hippocampal CA1 lacunosum-moleculare interneurons: modulation of monosynaptic GABAergic IPSCs by presynaptic GABAB receptors

1. Whole cell patch-clamp recordings were employed to characterize monosynaptic inhibitory postsynaptic currents (IPSCs) in morphologically and electrophysiologically identified interneurons located in the stratum lacunosum moleculare, or near the border of the stratum radiatum (LM interneurons), in the CA1 region of hippocampal slices taken from 3- to 4-wk-old rats. Monosynaptic IPSCs, evoked in the presence of glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 microM) and D-2-amino-5-phosphopentanoate (APV; 50 microM) were biphasic. The gamma-aminobutyric acid-A (GABAA) receptor antagonist, bicuculline (20 microM), blocked the fast IPSC, and the slow IPSC was blocked by the GABAB receptor antagonist CGP35348 (500 microM). 2. Monosynaptic IPSCs were evoked by electrical stimulation in several distant regions including the stratum radiatum, the stratum oriens, the stratum lacunosum-moleculare, and the molecular layer of dentate gyrus, suggesting an extensive network of inhibitory interneurons in the hippocampus. In paired recordings of CA1 interneurons and pyramidal cells, IPSCs were evoked by electrical stimulation of most of these distal regions with the exception of the molecular layer of dentate gyrus, which evoked an IPSC only in LM interneurons. 3. Frequent (> 0.1 Hz) stimulation depressed the evoked IPSCs. With a paired-pulse protocol, the second IPSC was depressed and the maximal depression (40-50%) was observed with an interstimulus interval of 100-200 ms. 4. The GABAB receptor agonist baclofen (1 microM) reduced the amplitude of evoked IPSCs and the paired-pulse depression of the second IPSC. The GABAB receptor antagonist CGP35348 (0.5-1 mM) had no significant effect on the amplitude of isolated IPSCs. However, CGP35348 reduced but did not fully block paired-pulse depression, suggesting that this depression is partly due to the activation of presynaptic GABAB receptors. 5. The paired-pulse depression depended on the level of transmitter release. Potentiation of synaptic release of GABA, by increasing the extracellular Ca2+ concentration to 4 mM and reducing the extracellular Mg2+ concentration to 0.1 mM, enhanced the depression. Reduction of transmitter release by increasing extracellular Mg2+ concentration to 7 mM diminished the paired-pulse depression of IPSCs. After potentiation of transmitter release, CGP35348 was less efficient in reducing the paired-pulse depression, suggesting that enhancement of depression by high-calcium/low-magnesium medium was preferentially due to the potentiation of a GABAB-independent component. 6. In summary, monosynaptic IPSCs recorded in LM interneurons show similar features to those recorded in pyramidal cells. The strong correlation between the level of transmitter release and the degree of paired-pulse depression may have important physiological consequences, because in synapses with a high level of activity and a high level of GABA release, inhibition is powerful, but depression can develop more readily.

Desensitization of AMPA receptors upon multiquantal neurotransmitter release

We have investigated the role of AMPA receptor desensitization during transmission at a calyceal synapse. Cyclothiazide blocked the rapid desensitization of AMPA receptors and markedly prolonged the decay time of the evoked excitatory postsynaptic current (PSC). This effect was greater than what would be expected from a simple prolongation of channel open time. Additionally, the drug reduced the depression of PSCs evoked at brief intervals. The effects of cyclothiazide on the PSC were reduced when the level of transmitter release was lowered. These data indicate that AMPA receptors are desensitized by neurotransmitter and that this desensitization depends on the number of quanta in the synaptic cleft. We propose that release of transmitter from many closely spaced sites prolongs the time of receptor-transmitter contact and thereby promotes desensitization. Desensitization may therefore contribute to synaptic depression and prevent the interaction of transmitter quanta within the synaptic cleft.

Calcium-dependent modulation of the facilitation of transmitter release at neuromuscular junctions of Drosophila

The relationship between external Ca and twin pulse facilitation of transmitter release has been investigated in neuromuscular junctions of Drosophila larvae using external electrodes to record excitatory junctional currents (ejcs). The degree of synaptic facilitation decreases in inverse relation to [Ca 2+0, even at levels of transmitter release that do not cause depletion of quanta. This reverse-dependecy of facilitation to external calcium is abolished by the slo mutation which suppreses the transient Ca-dependent K current (I CF). A likely explanation for these observations is that the activation of I CF by residual Ca shortens the action potential duration resulting in a reduced release of transmitter to the test stimulus. This represents a negative feed-back mechanism of synaptic function which opposes synaptic facilitation.

Ultrastructure of botulinum type-A poisoned frog motor nerve terminals after enhanced quantal transmitter release caused by carbonyl cyanide m-chlorophenylhydrazone

The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on spontaneous quantal transmitter release and nerve terminal ultrastructure were studied on isolated cutaneous pectoris nerve-muscle preparations from frogs that were completely paralysed by a single sublethal dose of Clostridium botulinum type A toxin (BoTx). CCCP enhanced miniature endplate potential frequency at poisoned junctions and caused a reduction in the density of clear synaptic vesicles and of large dense core vesicles in motor nerve terminals. However, the intensity of these effects was much less important than that previously reported at unpoisoned junctions. The moderate depletion of synaptic vesicles can be related to the low levels of transmitter release detected with CCCP at BoTx-poisoned terminals.

Endothelial cells inhibit the vascular response to adrenergic nerve stimulation by a receptor-mediated mechanism

Rabbit central ear arteries, with and without endothelium, were perfused at a constant flow rate and the perfusion pressure was measured as an index of the vessel resistance. Transmural nerve stimulation (TNS) induced a frequency-dependent increase in perfusion pressure in all vessels that was blocked by tetrodotoxin, phentolamine, and prazosin. Removal of endothelium significantly enhanced contractions induced by TNS. The inhibitory effect of endothelium was not modified by indomethacin but was abolished by hemoglobin, indicating that endothelium-derived relaxant factor (EDRF) was the vasodilator involved. The endothelium-dependent inhibitory effect (rubbed vessel minus control vessel contractions) increased with time during the first 10-20 s after the beginning of TNS, and was frequency dependent and inhibited by low doses of phentolamine, which suggest a receptor-mediated mechanism. To analyze whether amine neurotransmitters are able to permeate the artery wall and contact the endothelial cell membrane, the passage of [3H]acetylcholine from the abluminal side to the lumen was studied in intact vessels. [3H]acetylcholine readily permeated the vessel wall, as assessed by radioautography, and appeared in the perfusion fluid at a concentration that explains the relaxation induced by perivascular acetylcholine. These data suggest that endothelial cells modulate the effect of perivascular neurotransmitters by a receptor-mediated mechanism. In the case of the sympathetic innervation, such modulation would be more relevant at low levels of transmitter release and would be minimized during intense sympathetic stimulation.

Effects of ethimizol on frog neuromuscular junction

The effects were studied of ethimizol, a substance activating memory processes, on features of synaptic transmission during experiments on frog cutaneous pectoris muscle. It was found that the presynaptic action of ethimizol consists of raising the frequency of miniature potentials, when used at a concentration of 0.5–10 mM, and modulating quantal content of synaptic transmission due to changes in binomial quantal release parameters p and n when 0.5–2 mM ethimizol was used. This substance facilitated transmission at synapses with a low initial level of transmitter release. This substance facilitated transmission at synapses with a low initial level of transmitter release. Ethimizol was also found to have a postsynaptic action, consisting of reducing amplitude at a concentration of 5–10 mM and prolonging synaptic currents and potentials when concentrations of 0.5–10 mM were used. The latter effect produced a considerable increase in the time integral of endplate potentials. The postsynaptic action of ethimizol is perhaps seen in its effects on features of postsynaptic ionic channels. The effects of ethimizol are discussed with a view to how it may act within the central nervous system as a nonspecific modulator.

Structure of axon terminals and active zones at synapses on lizard twitch and tonic muscle fibers

The freeze-fracture technique was used to study differences in membrane structure which could explain differences in the number of quanta released from axon terminals on twitch and tonic muscle fibers in Anolis intercostal muscles. The protoplasmic leaflets of axon terminals facing lizard twitch muscle fibers have intramembrane particle specializations characterized by two parallel linear particle arrays each composed of two particle rows which lie perpendicular to the axis of shallow ridges in the axolemma. During K+ depolarization, vesicles open between the arrays, confirming that these structures are the active zones for synaptic vesicle opening. Active zones at axon terminals on tonic fibers are defined by one linear particle array composed of two parallel particle rows oriented along the axis of a shallow presynaptic ridge; vesicles open beside these arrays. Thus, there are more particles near active zone vesicles in terminals on twitch fibers. Even though terminals on twitch and tonic muscle fibers seem to have similar numbers of synaptic vesicles associated with their active zones, a presynaptic action potential is reported to release at least 10 times more quanta from terminals on twitch fibers. We postulate that the differences in quantal output are related to the observed differences in the number of active zone particles flanking synaptic vesicles at the active zone. Indeed, the correlation between the distribution of these particles and the level of transmitter release provides additional support for the idea that they are the calcium channels which couple transmitter release to the action potential.

Inverse relationship between transmitter release and terminal length in synapses on frog muscle fibers of uniform input resistance

Transmitter release at frog neuromuscular junctions is known to be related positively to nerve terminal length. However, the correlation is inexact, with a wide scatter of data. We have analyzed the endplate potentials (EPPs) of identified frog cutaneous pectoris muscle fibers, correlating release with terminal size and fiber input resistance (Rin). Transmitter release was assayed by quantal analysis of endplate activity in low Ca2+ Ringer solution and by measurement of the EPPs evoked in normal Ringer solution and curare. For fibers of approximately the same Rin, there is an inverse relationship between the level of transmitter release per unit length and total terminal length. Terminals with high levels of release per unit length tend to be shorter than do those which release relatively less transmitter per unit length. Furthermore, if the analysis is restricted similarly to fibers with nearly identical Rin, the total transmitter release of the largest endplates is usually less than that of the shorter terminals in the sample. These findings do not contradict the overall trend of greater release and longer terminals on larger muscle fibers (with lower Rin). Instead, they help explain the variability in measurement of release versus terminal length. The relationship that we find is consistent with the hypothesis that, in the cutaneous pectoris, terminals are induced to grow until an adequate safety factor for transmission is achieved.

Effects of botulinum toxin on neuromuscular transmission in the rat.

1. Botulinum toxin (BoTx) type A partially blocks spontaneous transmitter release from nerve terminals in the rat. Minature end-plate potentials (m.e.p.p.s) are present at all end-plates, initially with a low frequency but increasing with time after posoning. Their amplitude distribution is at first skew with a predominace of very small m.e.p.p.s but, after a few days, larger than normal m.e.p.p.s appear. 2. Tetanic nerve stimulation, Black Widow Spider Venom, the Caionophore A 23187 or mechanical damage to nerve terminals increases the frequency of m.e.p.p.s and alters the amplitude distribution of m.e.p.p.s towards a normal Gaussian one; the m.e.p.p. size approaches that seen at normal end-plates. This was seen at any time after poisoning. 3. Nerve stimulation gives rise to end-plate potentials (e.p.p.s) of low amplitude and high failure rate. Statistical analysis indicates that evoked release is quantal in nature and follows Poisson statistics, quantum size being initially very small, but after a few days approaching normal size. Short-term tetanic nerve stimulation reversibly increases the quantum content of e.p.p.s and during early stages of paralysis long-term (2 hr) stimulation causes an apparently permanent increase in quantum size. 4. Raising the extracellular Ca concentration from 2 to 16 mM increases the frequency of m.e.p.p.s in normal muscle but not in BoTx poisoned ones. K-free medium or ouabain, which are believed to raise the intracellular Ca concentration in nerve terminals, similarly increases m.e.p.p. frequency in normal but not in poisoned muscles. When the Ca-ionophore A 23187 is used together with high extracellular Ca (greater than 4 mM) massive release of transmitter occurs from poisoned terminals. 5. The extracellular Ca concentration which causes a certain level of transmitter release in reponse to nerve impulses is considerably higher at BoTx poisoned end-plates than at normal ones. The slope value for Ca dependence of transmitter release is about 1-5 compared with about 3 at normal end-plates. 6. Tetraethylammonium (TEA) greatly increases the amount of transmitter released by nerve impulses and restores neuromuscular transmission during all stages of poisoning, although it has not effect on spontaneous transmitter release. In the presence of TEA the power relation between Ca concentration and quantum content at the BoTx poisoned end-plate is similar to that seen at normal end-plates. 7. It is suggested that in BoTx poisoning the mechanism for transmitter release has a reduced sensitivity to Ca, and the level for activation by intracellular Ca is elevated. Once the intracellular concentration of Ca is raised to this level, by tetanic nerve stimulation, mechanical injury to nerve terminals, the Ca-ionophore or the prolongation of the nerve action potential with TEA, augmented transmitter release occurs, similar to that which occurs in normal nerve terminals at a lower level of Ca.

Suppression of transmission at foreign synapses in adult newt muscle involves reduction in quantal content

IN the search for the principles underlying the orderly development of the nervous system, it is interesting to know whether vertebrate neurones or muscle fibres can distinguish synaptic contacts of the axons which normally innervate them from the input of incorrect (or foreign) nerves. This possibility has been considered by asking whether foreign synapses induced on a denervated muscle fibre are displaced or suppressed when the muscle is reinnervated by its normal nerve. Observations of oculomotor reflexes in goldfish after experimental cross innervation of extraocular muscles led to the proposal that correct reinnervation suppressed foreign synapses1. Attempts to reproduce these results in the same system2, however, led to the opposite conclusion—that foreign transmission continued unabated after reinnervation by the correct nerve. A similar persistence of foreign synaptic function was found in reinnervated perch gill muscle3, frog fast muscle4, mammalian skeletal muscle5,6 and sympathetic ganglion cells7. Adult salamander muscle is the one other system in which suppression of incorrect synapses has been indicated; by the criterion of muscle fibre contraction monitored during foreign innervation and correct reinnervation, Cass et al.8 concluded that return of the correct nerve caused inactivation of foreign synaptic input. The present investigation examines this phenomenon at the level of transmitter release from individual synapses, and provides evidence that transmission by foreign nerve terminals declines in quantal content when adult newt muscle is reinnervated by its correct nerve.

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.