Reduced neuromuscular quantal content with normal synaptic release time course and depression in canine motor neuron disease

Hereditary canine spinal muscular atrophy is an autosomal dominant version of motor neuron disease in which motor units exhibit extensive dysfunction before motor terminal or axonal degeneration appear. We showed in a previous paper that motor endplate currents (EPCs) are reduced and that failures of nerve-evoked EPCs appear in the homozygote medial gastrocnemius (MG) muscle in which failing motor units are also found, suggesting a presynaptic deficit of ACh release. To examine this further, we performed a detailed analysis of synaptic release properties in the MG muscle of homozygotes and compared the results with data from genetically normal control animals. We found that the amplitude of miniature EPCs (mEPC) did not differ between homozygote and normal synapses, indicating that quantal content is reduced at homozygote motor terminals. Consistent with this, deconvolution analysis showed that the maximum release rates at homozygote motor terminals were significantly reduced relative to normal. This analysis also demonstrated that the time course of quantal release at homozygote synapses did not differ from normal. The extent of quantal release depression during high-frequency activation in homozygotes did not differ from normal despite the significant reduction of quantal content and maximum release rate. Surprisingly, the absolute amount of posttetanic potentiation was not decreased at homozygotes motor terminals despite the differences in quantal content. We conclude that failure of homozygote motor unit force during repetitive activity is due to a unique combination of low quantal content and normal release depression and suggest that the primary deficit in homozygote motor terminals is a reduced supply of readily releasable quanta.     

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

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

Quantal secretion and loss of vesicles induced by veratridine at the crayfish neuromuscular junction

Experiments were conducted in nerve-muscle preparations of small young crayfish (Austropotamobius torrentium, Astacus astacus). Application of veratridine in the superfusate induced strong quantal release of transmitter. After about 5 min when quantal release had declined to a low level preparations were fixed for electron microscopy. Unlike control preparations, veratridine-treated preparations revealed nerve terminals which were largely depleted of their synaptic vesicles. Our findings suggest that in the presence of veratridine the decline of quantal secretion results from the loss of vesicles caused by tonic nerve terminal depolarization. Moreover, our results indicate that during or after excessive quantal release triggered by veratridine synaptic vesicles may fuse with both the presynaptic membrane and each other.     

Intrinsic differences in sensitivity to 5-HT between high- and low-output terminals innervating the same target

The differential action of neuromodulators on synapses of various efficacy provides additional fine tuning of synaptic regulation beyond frequency induced plasticity. We used the well-characterized high- and low-output motor nerve terminals, of the tonic and phasic neuromuscular junctions (NMJs) in the walking leg extensor muscle of the crayfish, to investigate differential actions of serotonin (5-HT) since both terminals innervate the same target. The excitatory postsynaptic potentials of the tonic NMJ are enhanced to a greater extent than for the phasic NMJs during exposure to 5-HT (100 nM). Macropatch current recordings at identified sites along the motor nerve terminals and quantal analysis indicate that mean quantal content is substantially increased by 5-HT. The overall probability of vesicular release increases to a greater extent at tonic terminals than at phasic terminals when exposed to 100 nM 5-HT. Measures in the area (i.e. charge) of spontaneous quantal currents indicate no difference in postsynaptic receptivity to the glutamatergic synaptic transmission upon exposure to 5-HT. The results provide new details concerning differential modulation of low- and high-output synapses present on the same target tissue.     

alpha-Neurexins are required for efficient transmitter release and synaptic homeostasis at the mouse neuromuscular junction

Neurotransmission at chemical synapses of the brain involves alpha-neurexins, neuron-specific cell-surface molecules that are encoded by three genes in mammals. Deletion of alpha-neurexins in mice previously demonstrated an essential function, leading to early postnatal death of many double-knockout mice and all triple mutants. Neurotransmitter release at central synapses of newborn knockouts was severely reduced, a function of alpha-neurexins that requires their extracellular sequences. Here, we investigated the role of alpha-neurexins at neuromuscular junctions, presynaptic terminals that lack a neuronal postsynaptic partner, addressing an important question because the function of neurexins was hypothesized to involve cell-adhesion complexes between neurons. Using systems physiology, morphological analyses and electrophysiological recordings, we show that quantal content, i.e. the number of acetylcholine quanta released per nerve impulse from motor nerve terminals, and frequency of spontaneous miniature endplate potentials at the slow-twitch soleus muscle are reduced in adult alpha-neurexin double-knockouts, consistent with earlier data on central synapses. However, the same parameters at diaphragm muscle neuromuscular junctions showed no difference in basal neurotransmission. To reconcile these observations, we tested the capability of control and alpha-neurexin-deficient diaphragm neuromuscular junctions to compensate for an experimental reduction of postsynaptic acetylcholine receptors by a compensatory increase of presynaptic release: Knockout neuromuscular junctions produced significantly less upregulation of quantal content than synapses from control mice. Our data suggest that alpha-neurexins are required for efficient neurotransmitter release at neuromuscular junctions, and that they may perform a role in the molecular mechanism of synaptic homeostasis at these peripheral synapses.     

Muscarinic inhibition of quantal transmitter release from the magnesium-paralysed frog sartorius muscle

The existence of presynaptic muscarinic acetylcholine receptors on motor nerve terminals of the isolated frog sartorius muscle was investigated. The modulatory role of these receptors was studied by observing the effects of muscarinic ligands on the frequency of miniature endplate potentials and on the quantal content of endplate potentials. The agonist oxotremorine reduced in concentration-dependent fashion the frequency of spontaneous potentials and the amplitude of evoked potentials. Also, high concentrations of oxotremorine depolarized the postsynaptic membrane and reduced the amplitude of the miniature endplate potentials. The depolarizing action of the drug was blocked byd-tubocurarine. The muscarinic antagonist atropine attenuated agonist-induced reductions in endplate potential amplitude and miniature endplate potential frequency but did not affect the depression in amplitude of the spontaneous potentials evoked by oxotremorine. It is concluded that activation of presynaptic muscarinic receptors inhibits the release of acetylcholine from motor nerve terminals.

Atropine itself had no effect on the quantal content of evoked potentials or on the frequency of spontaneous potentials suggesting that the nerve terminal is not affected by non-quantal acetylcholine.     

Post-synaptic potentiation: interaction between quanta of acetylcholine at the skeletal neuromuscular synapse.

1. Post-synaptic responses to acetylcholine (ACh) released from nerve terminals and from iontophoretic micropipettes were investigated in skeletal muscle fibres of the snake. Each fibre has a compact end-plate consisting of fifty to seventy synaptic boutons. The fibres were voltage clamped, and synaptic currents were recorded from visually identified end-plates. 2. When acetylcholinesterase (AChE) is inhibited, a potentiating interaction is observed between two or more quanta that are released close to each other from a synaptic bouton and act upon partially overlapping postsynaptic areas. The potentiation is expressed as a prolongation of the synaptic current. This potentiation also occurs under normal conditions of release when about 300 quanta are distributed over the entire end-plate, so thet the presynaptic release sites are separated by an average of 2 mum. An analogous potentiating interaction is observed when micropipettes, closely apposed to the subsynaptic membrane, substitute for quantal release sites. ACh from one pipette potentiates the response to ACh from another pipette less than 2 mum away. 3. In contrast, with AChE fully active no post-synaptic potentiation is seen when the normal complement of quanta is released over the entire end-plate. The time course of the synaptic currents in response to a single quantum or to 300 quanta is similar. It is concluded that functionally the quanta act independently of each other, because AChE isolates each quantum from its neighbours by limiting the lifetime of ACh and its lateral diffusion in the synaptic cleft. The estimated area over which a quantum normally acts is less than 2mum2. 4. Post-synaptic receptors are not saturated by the ACh in a quantum, since the peak of the quantal response adds linearly to the response produced by an appropriate background concentration of ACh from a pipette. This conclusion is supported by the observation that upon inhibition of AChE the peak amplitude of the quantal current response increases by about 20% with no change in its time to peak. 5. It is suggested that post-synaptic potentiation between quanta may play a role in signalling at synapses in which non-linear dose-response characteristics have been observed and where transmitter is not as repidly inactivated as the neuromuscular synapse.     

Role of NSF in neurotransmitter release: a peptide microinjection study at the crayfish neuromuscular junction

Peptides that inhibit the SNAP-stimulated ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF-2, NSF-3) were injected intra-axonally to study the role of this protein in the release of glutamate at the crayfish neuromuscular junction. Macropatch recording was used to establish the quantal content and to construct synaptic delay histograms. NSF-2 or NSF-3 injection reduced the quantal content, evoked by either direct depolarization of a single release bouton or by axonal action potentials, on average by 66 +/- 12% (mean +/- SD; n = 32), but had no effect on the time course of release. NSF-2 had no effect on the amplitude or shape of the presynaptic action potential nor on the excitatory nerve terminal current. Neither NSF-2 nor NSF-3 affected the shape or amplitude of single quantal currents. Injection of a peptide with the same composition as NSF-2, but with a scrambled amino acid sequence, failed to alter the quantal content. We conclude that, at the crayfish neuromuscular junction, NSF-dependent reactions regulate quantal content without contributing to the presynaptic mechanisms that control the time course of release.     

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

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

Development of neuromuscular synapses

Quantal secretion at nerve terminals in mature muscles depends on the number of terminal branches and the size of release sites (sect. VB4). The physical length of SBL determines the length of terminal branch that can be laid down in a reinnervation experiment (sect. IVA4). A limit is set on the total length of terminal branches formed by a motoneuron; this limit is determined by the amount of TF (sect. IVB) made available from the neuron soma to the peripheral branches of the neuron (sect. VC). As a result of this limit, not all SBL needs to be occupied at a site by terminal branches. The SBL eventually disappears if it is not occupied by terminal branches (sect. IVA2). If a muscle is relatively inactive, it synthesizes and releases at synaptic sites additional amounts of NGF, which stimulates the growth of additional terminal branches. These may secrete sufficient amounts of AF to induce the formation of new SRs with associated SBL. In these circumstances a new synaptic site is formed or an extension of an existing site is created. If the size of a motor unit is decreased, the enhanced release of TF at the remaining terminals ensures that each occupies all the SBL at the synaptic site. Furthermore the enhanced release of AF per terminal induces more SBL, allowing additional terminal branches on the muscle cells to be established. Neither of these changes occurs unless the threshold amount of NGF is available from the muscle to stabilize the terminals. If this condition is met, an increase in quantal release per terminal occurs after reducing the size of a motor unit (sect. VC). An increase in quantal release per terminal also occurs after inactivation of a muscle. Such inactivation leads to an enhanced release of NGF per synaptic site (sect. VA4). Extra terminals may then form if sufficient TF is available; these may innervate existing but empty synaptic sites. In rare circumstances the extra terminal may induce SBL and innervate these new sites if sufficient AF is available. In both cases the quantal release per terminal increases. During development the secretory capacity of the axon terminal depends on the muscle cells with which it synapses. This secretory capacity can be enhanced either by increasing the number of terminal branch pairs or by increasing the secretory capacity of individual release sites. If two terminals innervate a synaptic site, their individual secretory capacity is reduced--in these circumstances the terminal's secretory capacity depends on the amount of NGF available to the terminal; two terminals must share their NGF.     

Synaptic vesicle distribution and release at rat diaphragm neuromuscular junctions

Synaptic vesicle release at the neuromuscular junction (NMJ) is highly reliable and is vital to the success of synaptic transmission. We examined synaptic vesicle number, distribution, and release at individual type-identified rat diaphragm NMJ. Three-dimensional reconstructions of electron microscopy images were used to obtain novel measurements of active zone distribution and the number of docked synaptic vesicles. Diaphragm muscle-phrenic nerve preparations were used to perform electrophysiological measurements of the decline in quantal content (QC) during repetitive phrenic nerve stimulation. The number of synaptic vesicles available for release vastly exceeds those released with a single stimulus, thus reflecting a relatively low probability of release for individual docked vesicles and at each active zone. There are two components that describe the decline in QC resulting from repetitive stimulation: a rapid phase (<0.5 s) and a delayed phase (<2.5 s). Differences in the initial rapid decline in QC were evident across type-identified presynaptic terminals (fiber type classification based on myosin heavy chain composition). At terminals innervating type IIx and/or IIb fibers, the initial decline in QC during repetitive stimulation matched the predicted depletion of docked synaptic vesicles. In contrast, at terminals innervating type I or IIa fibers, a faster than predicted decline in QC with repetitive stimulation suggests that a decrease in the probability of release at these terminals plays a role in addition to depletion of docked vesicles. Differences in QC decline likely reflect fiber-type specific differences in activation history and correspond with well-described differences in neuromuscular transmission across muscle fiber types.     

Multiple types of calcium channels mediate transmitter release during functional recovery of botulinum toxin type A-poisoned mouse motor nerve terminals

The involvement of different types of voltage-dependent calcium channels in nerve-evoked release of neurotransmitter was studied during recovery from neuromuscular paralysis produced by botulinum toxin type A intoxication. For this purpose, a single subcutaneous injection of botulinum toxin (1 IU; DL50) on to the surface of the mouse levator auris longus muscle was performed. The muscles were removed at several time-points after injection (i.e. at one, two, three, four, five, six and 12 weeks). Using electrophysiological techniques, we studied the effect of different types of calcium channel blockers (nitrendipine, omega-conotoxin-GVIA and omega-agatoxin-IVA) on the quantal content of synaptic transmission elicited by nerve stimulation. Morphological analysis using the conventional silver impregnation technique was also made. During the first four weeks after intoxication, sprouts were found at 80% of motor nerve terminals, while at 12 weeks their number was decreased and the nerve terminals were enlarged. The L-type channel blocker nitrendipine (1 microM) inhibited neurotransmitter release by 80% and 30% at two and five weeks, respectively, while no effects were found at later times. The N-type channel blocker omega-conotoxin-GVIA (1 microM) inhibited neurotransmitter release by 50-70% in muscles studied at two to six weeks, respectively, and had no effect 12 weeks after intoxication. The P-type channel blocker omega-agatoxin-IVA (100 nM) strongly reduced nerve-evoked transmitter release (>90%) at all the time-points studied. Identified motor nerve terminals were also sensitive to both nitrendipine and omega-conotoxin-GVIA. This study shows that multiple voltage-dependent calcium channels were coupled to transmitter release during the period of sprouting and consolidation, suggesting that they may be involved in the nerve ending functional recovery process.     

Endo-exocytotic images and changes in synaptic transmission induced by diamide at a cholinergic junction

Small tissue fragments excised from the electric organ of Torpedo marmorata were treated with diamide, a penetrating thiol oxidizing agent, until synaptic transmission was blocked. At this stage, we found an unexpected number of exo-endocytotic images in the presynaptic plasmalemma. Omega-shaped profiles, some of them coated, were seen in thin sections of fixed tissue and pits opened in the P-face of the presynaptic membrane in freeze-fracture replicas from rapidly-frozen preparations. Diamide-treated specimens were frozen at 1 ms time intervals before, during and after a single electrical stimulus. This stimulation did not result in a further increase in the density of presynaptic pits, not in any change affecting the density or size distribution of intramembrane particles. This result is in contrast with what is observed in untreated specimens where transmission of a nerve impulse is accompanied by a momentary rise in the number of large particles. The density of synaptic vesicles--especially that of a subpopulation of small size vesicles--transiently increased within the first 2 h of diamide treatment. During the first stages of intoxication, diamide prolonged the time course of postsynaptic potentials--both spontaneous and evoked--probably by altering the gating properties of receptors (acetyl-cholinesterase activity was not impaired). Later on, all evoked responses were blocked. The spontaneous transmitter release greatly increased, first in the form of quantal miniature potentials. These then subsided whereas a class of very small potentials was generated at a high frequency. Also under the action of diamide, calcium progressively accumulated in the tissue but the number of synaptic vesicles containing calcium deposits was reduced. It is concluded that diamide causes a marked increase in the number of exo-endocytotic images in the presynaptic membrane, suppresses quantal but not subquantal release, and interferes with calcium sequestration in and extrusion from terminals.     

The kinetics of transmitter release at the frog neuromuscular junction

1. Fluctuations in the latency of focally recorded end-plate currents were analysed to determine the time course of the probabilistic presynaptic process underlying quantal release evoked after single nerve stimuli at the frog neuromuscular junction.

2. The early falling phase of the presynaptic probability function can be fitted by a single exponential over two orders of magnitude of quantal release rate. The time constant of the early falling phase is about 0·5 msec at 11° C, and increases with decreasing temperature with a Q₁₀ of at least 4 over the range 1-12° C.

3. After this early exponential fall, quantal release probability returns to control levels with a much slower time course.

4. Conditioning nerve stimuli increase the magnitude and slightly prolong the early time course of release evoked by a test stimulus. When facilitation is calculated for matched time intervals following the conditioning and testing stimuli, it is found that the magnitude of the small, late residual tail of release is facilitated by a greater percentage than the magnitude of larger, early portions of release.

5. These results are discussed in terms of the hypothesis (Katz & Miledi, 1968) that evoked release and facilitation are mediated by a common presynaptic factor which activates release in a non-linear manner.

     

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.     

Tonic activation of presynaptic GABAB receptors in the opener neuromuscular junction of crayfish

Release of excitatory transmitter from boutons on crayfish nerve terminals was inhibited by (R,S)-baclofen, an agonist at GABAB receptors. Baclofen had no postsynaptic actions as it reduced quantal content without affecting quantal amplitude. The effect of baclofen increased with concentration producing 18% inhibition at 10 microM; EC50, 50% inhibition at 30 microM; maximal inhibition, 85% at 100 microM and higher. There was no desensitization, even with 200 or 320 microM baclofen. Phaclofen, an antagonist at GABAB receptors, competitively antagonized the inhibitory action of baclofen (KD = 50 microM, equivalent to a pA2 = 4.3 +/- 0.1). Phaclofen on its own at concentrations below 200 microM had no effect on release, whereas at 200 microM phaclofen itself increased the control level of release by 60%, as did 2-hydroxy-saclofen (200 microM), another antagonist at GABAB receptors. This increase was evidently due to antagonism of a persistent level of GABA in the synaptic cleft, since the effect was abolished by destruction of the presynaptic inhibitory fiber, using intra-axonal pronase. We conclude that presynaptic GABAB receptors, with a pharmacological profile similar to that of mammalian GABAB receptors, are involved in the control of transmitter release at the crayfish neuromuscular junction.     

Rab3a deletion reduces vesicle docking and transmitter release at the mouse diaphragm synapse

Rab3a is a small GTP binding protein associated with presynaptic vesicles that is thought to regulate vesicle targeting to active zones. Although this rab3a function implies that vesicle docking and action potential-evoked release might be inhibited in rab3a gene-deleted synapses, such inhibition has never been demonstrated. To investigate vesicle docking at the neuromuscular junction of rab3a gene-deleted (rab3a(-)) mice, we performed electron microscopy analysis of the diaphragm slow-fatigue (type I) synapses. We found a significant (26%) reduction in the number of vesicles docked to the presynaptic membrane in rab3a(-) terminals, although intraterminal vesicles were not affected. Aiming to detect possible changes in quantal release due to rab3a gene deletion, we minimized the variability between preparations employing focal recordings of synaptic responses from visualized type I endplates. We found a significant decrease in both evoked (27% reduction in quantal content) and spontaneous (28% reduction in mini frequency) quantal release. The decrease in the evoked release produced by rab3a deletion was most pronounced at reduced extracellular Ca(2+) concentrations (over 50% decrease at 0.5 and 0.2 mM Ca(2+)). By manipulating extracellular calcium, we demonstrated that calcium cooperativity is not altered in rab3a(-) synapses, however calcium sensitivity of quantal release is affected. Thus, we demonstrated that rab3a positively regulates docking and basal quantal release at the mouse neuromuscular junction. This result is consistent with the proposed role of rab3a in trafficking and targeting vesicles to the active zones.     

Ultrastructural correlates of experimentally altered transmitter release efficacy in frog motor nerve terminals

After experimentally inducing long term changes in transmitter release, a series of frog neuromuscular junctions were studied with intracellular recording and then semi-serially sectioned and examined in the electron microscope. Transmitter release per unit length of motor nerve terminal was well correlated with several measures of the length of individual presynaptic active zones and with the number of mitochondria per terminal. Total release from each terminal correlated with estimates of the total amount of active zone. This study of neuromuscular junctions in sartorius muscles of the frog Rana pipiens was undertaken to search for ultrastructural correlates of the increase in transmitter release efficacy that follows denervation of the contralateral sartorius. This treatment typically results in greatly enhanced release at some synapses while others appear unaffected. In the present study, nine identified junctions with known physiological properties were sectioned every 6 micron throughout much of their length to yield 40-105 cross-sectional profiles per junction. Overall, these 9 synapses showed a 33-fold range in quantal transmitter release and an 18-fold range in release per unit nerve terminal. Release correlated with estimates of active zone size. No correlations were found between release and the density of synaptic vesicles adjacent to active zones. Our results suggest that active zones in motor nerve terminals are plastic structures, and that changes in active zone size may be the structural basis of long term changes in transmitter release and synaptic efficacy.     

Cholinergic regulation of the evoked quantal release at frog neuromuscular junction

The effects of cholinergic drugs on the quantal contents of the nerve-evoked endplate currents (EPCs) and the parameters of the time course of quantal release (minimal synaptic latency, main modal value of latency histogram and variability of synaptic latencies) were studied at proximal, central and distal regions of the frog neuromuscular synapse. Acetylcholine (ACh, 5 × 10⁻⁴ m ), carbachol (CCh, 1 × 10⁻⁵ m ) or nicotine (5 × 10⁻⁶ m ) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90–120 μm from the last node of Ranvier), where the synchronization of transmitter release was the most pronounced. The parameters of focally recorded motor nerve action potentials were not changed by either ACh or CCh. The effects of CCh and nicotine on quantal dispersion were reduced substantially by 5 × 10⁻⁷ m (+)tubocurarine (TC). The muscarinic agonists, oxotremorine and the propargyl ester of arecaidine, as well as antagonists such as pirenzepine, AF-DX 116 and methoctramine, alone or in combination, did not affect the dispersion of the release. Muscarinic antagonists did not block the dispersion action of CCh. Cholinergic drugs either decreased the quantal content m _o (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased m _o and dispersed the release (ACh, CCh and nicotine). The effects on m _o were not related either to the endplate region or to the initial level of release dispersion. It follows that the mechanisms regulating the amount and the time course of transmitter release are different and that, among other factors, they are altered by presynaptic nicotinic receptors.     

Effects of NH4+ on reflexes in cat spinal cord

1. In deeply barbiturate-anesthetized animals. NH4+ decreases spinal excitatory synaptic transmission by neuronal depolarization and subsequent block of conduction of action potentials into presynaptic terminals of low-threshold (presumably Ia-) afferents. Because barbiturates by themselves depress excitatory synaptic transmission and may have modified the effects of NH4+, this study examines the effect of NH4+ on excitatory synaptic transmission in the unanesthetized animal. 2. The effects of NH4+ on monosynaptic and polysynaptic excitatory reflexes as well as di- and polysynaptic inhibition were investigated in the spinal cord of the decerebrate and unanesthetized cat in vivo. 3. The monosynaptic excitatory reflex (MSR) elicited by muscle nerve stimulation and polysynaptic excitatory reflexes elicited by muscle (MSR-PSR) or cutaneous nerve stimulation (Cut-PSR) were recorded from the ventral roots L7 or S1. The P-wave was recorded from the cord dorsum. Di- and polysynaptic inhibition was elicited by muscle nerve stimulation and measured as decrease of the MSR. 4. Intravenous infusion of ammonium acetate (AA) decreased MSR and the monosynaptic motoneuron pool excitatory postsynaptic potential (EPSP) recorded from the ventral root (VR-EPSP). Decrease of MSR and VR-EPSP was accompanied by an increase of the intraspinal conduction time in presynaptic terminals. The maximal decrease of the MSR was preceded by a period of transient increase of the MSR and reflex discharges from previously subthreshold VR-EPSPs. 5. The effects of NH4+ on MSR and VR-EPSP are consistent with those in barbiturate-anesthetized animals and suggest that NH4+ also decreases monosynaptic excitation in unanesthetized animals by depolarization and subsequent conduction block for action potentials in presynaptic terminals. 6. Decrease of the MSR was accompanied by a decrease of the P-wave, indicating that NH4+ simultaneously decreases mono- and oligosynaptic excitatory synaptic transmission as well as presynaptic inhibition. 7. Decrease of the MSR was accompanied by increases of MSR-PSR and Cut-PSR and decreases of di- and polysynaptic postsynaptic inhibition. 8. The neuronal circuits underlying MSR-PSR and Cut-PSR include presynaptic inhibition of group I and II afferents as well as postsynaptic inhibition of motoneurons. It is suggested that increases of MSR-PSR and Cut-PSR are contributed to by decreases of pre- and postsynaptic inhibition and neuronal depolarization by NH4+. These effects increase afferent input to motoneurons, permit uncontrolled discharge of motoneurons, and initiate reflex discharges by previously subthreshold excitatory postsynaptic potentials.