Motor Nerve Endings Research Articles

Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

Modification of synaptic transmission and sodium channel inactivation by the insect-selective scorpion toxin LqhalphaIT.

The peptide LqhalphaIT is an alpha-scorpion toxin that shows significant selectivity for insect sodium channels over mammalian channels. We examined the symptoms of LqhalphaIT-induced paralysis and its neurophysiological correlates in the house fly (Musca domestica). Injection of LqhalphaIT into fly larvae produced hyperactivity characterized by continuous, irregular muscle twitching throughout the body. These symptoms were correlated with elevated excitability in motor units caused by two physiological effects of the toxin: 1) increased transmitter release and 2) repetitive action potentials in motor nerves. Increased transmitter release was evident as augmentation of neurally evoked synaptic current, and this was correlated with an increased duration of action potential-associated current (APAC) in loose patch recordings from nerve terminals. Repetitive APACs were observed to invade nerve endings. The toxin produced marked inhibition of sodium current inactivation in fly central neurons, which can account for increased duration of the APAC and elevated neurotransmitter release at the neuromuscular junction. Steady-state inactivation was shifted significantly to more positive potentials, whereas voltage-dependent activation of the channels was not affected. The shift in steady-state inactivation provides a mechanism for inducing repetitive activity in motoneurons. The effects of LqhalphaIT on sodium channel inactivation in motor nerve endings can account both for increased transmitter release and repetitive activity leading to hyperactivity in affected insects.

Factors influencing the low-frequency associated nicotinic ACh autoreceptor-mediated depression of ACh release from rat motor nerve terminals.

1. We have studied the inhibitory autoreceptor control of acetylcholine (ACh) release from rat motor nerve endings using an electrophysiological technique to quantify evoked ACh release in isolated hemidiaphragm muscles. Quantal ACh release (m) was estimated from the ratio of amplitudes of nerve evoked endplate currents and spontaneously occurring miniature endplate currents. 2. The nicotinic ACh receptor agonist cytisine (1 microM) decreased m at 0.5 Hz by around 20% but had no effect on m at 50 Hz. Changing the extracellular Ca(2+) concentration from 1.8 mM to either 0.45 or 3.6 mM abolished the effect of cytisine on m at 0.5 Hz. The nicotinic ACh receptor antagonist hexamethonium (200 microM) increased m at 0.5 Hz by 15 - 20%. 3. The effects of cytisine and hexamethonium on m at 0.5 Hz were blocked by 10 microM verapamil, which itself significantly increased m. However, the effects of cytisine and hexamethonium on m at 0.5 Hz were not sensitive to 10 microM of the calmodulin antagonist, W-7. This concentration of W-7 attenuates effects on ACh release mediated by facilitatory prejunctional nicotinic ACh autoreceptors. 4. Our present observations are suggestive of actions of cytisine and hexamethonium to activate and inhibit respectively negative-feedback prejunctional nicotinic ACh autoreceptors. Further, they strengthen the case for the existence of two separate and independent autoregulatory mechanisms for the control of ACh release from motor nerve terminals and give a preliminary insight into the cellular mechanism involved in the autoinhibition of ACh release.

Effects of hindlimb unloading on neuromuscular development of neonatal rats

We hypothesized that hindlimb suspension unloading of 8-day-old neonatal rats would disrupt the normal development of muscle fiber types and the motor innervation of the antigravity (weightbearing) soleus muscles but not extensor digitorum longus (EDL) muscles. Five rats were suspended 4.5 h and returned 1.5 h to the dam for nursing on a 24 h cycle for 9 days. To control for isolation from the dam, the remaining five littermates were removed on the same schedule but not suspended. Another litter of 10 rats housed in the same room provided a vivarium control. Fibers were typed by myofibrillar ATPase histochemistry and immunostaining for embryonic, slow, fast IIA and fast IIB isomyosins. The percentage of multiple innervation and the complexity of singly-innervated motor terminal endings were assessed in silver/cholinesterase stained sections. Unique to the soleus, unloading accelerated production of fast IIA myosin, delayed expression of slow myosin and retarded increases in standardized muscle weight and fiber size. Loss of multiple innervation was not delayed. However, fewer than normal motor nerve endings achieved complexity. Suspended rats continued unloaded hindlimb movements. These findings suggest that motor neurons resolve multiple innervation through nerve impulse activity, whereas the postsynaptic element (muscle fiber) controls endplate size, which regulates motor terminal arborization. Unexpectedly, in the EDL of unloaded rats, transition from embryonic to fast myosin expression was retarded. Suspension-related foot drop, which stretches and chronically loads EDL, may have prevented fast fiber differentiation. These results demonstrate that neuromuscular development of both weightbearing and non-weightbearing muscles in rats is dependent upon and modulated by hindlimb loading.

Effects of ammodytin L on miniature and endplate potentials in neuromuscular junction of frog m. cutaneus pectoris.

Neurotoxic effects of ammodytin L (AtnL), a potent phospholipase A2 homologue, was studied in frog neuromuscular preparation m. cutaneus pectoris by measuring the influence of the toxin on the amplitude and the frequency of miniature and endplate potentials (MEPPs, EPPs). AtnL, in 100 nM concentration, significantly increases spontaneous quantal acetylcholine release from the motor nerve endings, observed as the increase in MEPPs frequency. At 100 nM or higher concentration the toxin decreases EPPs amplitude and the membrane potential (MP) simultaneously. No significant effect of AtnL on EPPs was observed at any concentration bellow 100 nM. Our results indicate that in frog AtnL shows the typical myotoxic effects, but it also exerts presynaptic effects.

Antagonism of calcium currents and neurotransmitter release by barium ions at frog motor nerve endings.

1. The effects of Ba(2+) (0.1 - 2 mM) on the component of the perineural voltage change associated with nerve terminal calcium currents (prejunctional Ca(2+) currents) were compared with the effects of this ion to antagonize calcium-dependent acetylcholine (ACh) release. These experiments were made on isolated neuromuscular junctions of the frog. 2. In the presence of sufficient concentrations of K(+) channel blockers to eliminate measurable prejunctional K(+) currents, low concentrations of Ba(2+) selectively antagonized prejunctional Ca(2+) currents in normal Ca(2+) solutions. Higher concentrations of Ba(2+) also substantially reduced the Na(+) component of the perineural waveform. 3. Ba(2+) inhibited the prolonged prejunctional Ca(2+) currents that developed in the presence of higher concentrations of K(+) channel blockers. 4. Simultaneous measurements of the prejunctional Ca(2+) currents and the electrophysiological correlates of ACh release (i.e. end-plate potentials, EPPs) were made under conditions of modest K(+) channel blockade. Under these conditions, Ba(2+) generally produced simultaneous decreases in both Ca(2+) currents and EPP amplitudes. In some instances, a prolongation of prejunctional Ca(2+) currents and a transient increase in EPP amplitudes preceded the decreases in both electrophysiological events. 5. These results suggest that Ba(2+) ions can antagonize the entry of calcium into motor nerve endings and this effect is likely to be responsible for the inhibitory effects of Ba(2+) on evoked ACh release.

Impairment of neuromuscular transmission in a subgroup of migraine patients

Neuronal voltage-dependent P/Q Ca 2+ channels are genetically abnormal in many cases of familial hemiplegic migraine and possibly associated with the more common forms of migraine with and without aura. Besides the brain, these channels are found in motor nerve endings where they control stimulation-induced acetylcholine release. Using single fiber EMG recordings we were able to demonstrate subclinical abnormalities of neuromuscular transmission in a subgroup of patients suffering from migraine with aura. This could be related to genetic abnormalities of P/Q Ca 2+ channels in certain patients suffering from migraine with aura, which needs to be explored by proper genetic analyses.

Multiple types of spontaneous excitatory synaptic currents in salamander retinal ganglion cells

Spontaneous and light-evoked excitatory postsynaptic currents (sEPSCs and leEPSCs) in retinal ganglion cells of the larval tiger salamander were recorded under voltage clamp conditions from living retinal slices. sEPSCs were isolated from the spontaneous inhibitory postsynaptic currents (sIPSCs) by application of 100 M picrotoxin+1 μM strychnine. In addition to the previously reported sEPSCs [K. Matsui, N. Hosoi, M. Tachibana, Excitatory synaptic transmission in the inner retina: pair recordings of bipolar cells and neurons of the ganglion cell layer, J. Neurosci. 18 (1998) 4500–4510; W.R. Taylor, E. Chen, D.R. Copenhagen, Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells, J. Physiol. 486 (1995) 207–221] [which are equivalent to our fast AMPA receptor-mediated sEPSCs (fAMPAsEPSCs)], we found another type of AMPA receptor-mediated sEPSC with slower rise and decay time courses and larger peak amplitudes (sAMPAsEPSCs), and the NMDA receptor-mediated sEPSCs (NMDAsEPSCs) in ON–OFF ganglion cells. The frequency of all three types of sEPSCs is greatly reduced by cobalt (with zero calcium) and increased by hyperosmotic solution, suggesting that these events are mediated by calcium-dependent exocytosis of glutamatergic synaptic vesicles. The amplitude histograms of sEPSCs do not show multiple peaks, suggesting that larger events are not discrete multiples of elementary events, or quanta, of similar neurotransmitter contents, as in the neuromuscular junction [P. Fatt, B. Katz, Spontaneous subthreshold activity at motor nerve endings, J. Physiol. 117 (1952) 109–128]. The average I– V relations of the fAMPAsEPSCs and sAMPAsEPSCs were outward rectified with reversal potentials at −12.2 mV and −10.8 mV, and that of the NMDAsEPSCs was N-shaped with a reversal potential at −5.8 mV. The average conductance increase associated with a single fAMPAsEPSC, a single sAMPAsEPSC, and a single NMDAsEPSC were 163.26±51.02 pS, 233.33±163.64 pS, and 37.5±50.0 pS at −110 mV; 241.67±22.92 pS, 444.90±469.94 pS, and 25.93±70.37 pS at −60 mV; and 440.48±183.33 pS, 1,192.68±651.22 pS, and 517.71±238.24 pS at +30 mV, respectively. The average frequency of the three sEPSCs at +30 mV were 15 Hz, 3.7 Hz and 3.6 Hz, respectively. The rise time (time to peak) of fAMPAsEPSCs was 1.5±1.05 ms and the decay time could be fitted with a single exponential with an average time constant of 3.4±4.1 ms. The rise and decay time course of the sAMPAsEPSCs and NMDAsEPSCs were much slower and sawtooth-shaped, and each `sawtooth' had time course and amplitude similar to those of individual fAMPAsEPSCs. We propose that each fAMPAsEPSC is mediated by single or synchronized multiples of glutamatergic synaptic vesicles from bipolar cells, and each sAMPAsEPSC or NMDAsEPSC is mediated by larger clusters of synaptic vesicles triggered by spontaneous calcium spikes in bipolar cell axon terminals [J. Burrone, L. Lagnado, Electrical resonance and calcium influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina, J. Physiol. 505 (1997) 571–584; D. Zenisek, G. Matthews, Calcium action potentials in retinal bipolar neurons, Vis. Neurosci. 15 (1998) 69–75].

The glutamate and carbachol effects on the early post-denervation depolarization in rat diaphragm are directed towards furosemide-sensitive chloride transport

The membrane potentials of denervated muscle fibres of the rat diaphragm kept in a tissue culture medium are depolarized by about 8–10 mV (10–12%) within 3 h after denervation. This early post-denervation depolarization (EPD) is substantially reduced (2–3 mV) when muscle strips are bathed with 1 mM l-glutamate (GLU) which is found in motor nerve endings, or with 5×10 −8 M carbachol (CCh), which mimics the effect of nonquantally released acetylcholine (ACh). The hyperpolarizing effects of GLU and CCh on EPD are not influenced by ouabain, an active sodium transport inhibitor, but are absent when Cl − transport is augmented by increased osmolarity (500 mosmol/l) produced by addition of sucrose or NaCl. The EPD and the effect of hyperosmolarity are effectively prevented by the Cl − transport inhibitor furosemide (1×10 −4 M) or by a chloride-free bathing medium. It is suggested that the post-denervation cessation of nonquantal ACh release, and probably also GLU release, from nerve endings leads to the activation of the furosemide-sensitive Cl − transport in the sarcolemma, which is responsible for the early post-denervation depolarization.

Chapter 12 Quantal ATP release from motor nerve endings and its role in neurally mediated depression

Publisher Summary This chapter reviews the mechanisms of presynaptic neurotransmitter modulation within the framework of a physiologically relevant behavior—namely, the process of neurally mediated prejunctional depression at the skeletal neuromuscular junction. At the frog and mouse neuromuscular junctions, depression appears to be because of the release of endogenous ATP that after degradation to adenosine acts back on the nerve ending to inhibit the subsequent release of the neurotransmitter acetylcholine. The chapter also presents the evidence that ATP is released synchronously from motor nerve endings within milliseconds of a solitary nerve impulse and in the appropriate concentration range to mediate neuromuscular depression. The data suggest that, in the frog, adenosine mediates the inhibition of both spontaneous and evoked ACh release and by an action at the secretory apparatus and the downstream of calcium entry.

Three-dimensional structure of the synaptic contact of the neuromuscular junction in the rat lumbrical muscle.

This study examined the three-dimensional structures of the synaptic contact in rat lumbrical muscles by scanning electron microscopy using three different methods: the aldehyde prefix-osmium-dimethyl sulfoxide-osmium method (A-O-D-O method), the cell-extraction method, and the NaOH-digestion method. These three methods visualized the motor nerve endings, subneural basal lamina and postsynaptic sarcolemma, respectively. The motor nerve endings were composed of a cluster of spherical and cylindrical terminals. Pores on the presynaptic membrane were considered openings of exocytotic vesicles. The postsynaptic side of the subneural basal lamina showed numerous ridges, corresponding to junctional folds. Most of the ridges rose vertically from their base. The ridges showed widening, narrowing, and branching. The subneural basal lamina appeared to be composed of small granular substances. The basal lamina of the primary synaptic clefts had pores 25-30 nm in diameter, which may facilitate the transport of acetylcholine (ACh) without being hydrolyzed by ACh esterase in the lamina. On the outer surface of the postsynaptic sarcolemma in a sole plate, the primary synaptic clefts were composed of a mixture of depressions and gutters; so far as we know, this represents the only example of such a phenomenon. These depressions and gutters seem to fit respectively into the spherical and cylindrical terminals of the motor nerve endings. The openings of the junctional folds consisted of a mixture of many slits and a few pits in the primary synaptic clefts.

Passive transfer of Lambert-Eaton myasthenic syndrome induces dihydropyridine sensitivity of ICa in mouse motor nerve terminals.

Mice were injected for 30 days with plasma from three patients with Lambert-Eaton Myasthenic Syndrome (LEMS). Recordings were made from the perineurial sheath of motor axon terminals of triangularis sterni muscle preparations. The objective was to characterize pharmacologically the identity of kinetically distinct, defined potential changes associated with motor nerve terminal Ca2+ currents (ICa) that were affected by LEMS autoantibodies. ICa elicited at 0.01 Hz were significantly reduced in amplitude by approximately 35% of control in LEMS-treated nerve terminals. During 10-Hz stimulation, ICa amplitude was unchanged in LEMS-treated motor nerve terminals, but was depressed in control. During 20- or 100-Hz trains, facilitation of ICa occurred in LEMS-treated nerve terminals whereas in control, no facilitation occurred during the trains at 20 Hz and marked depression occurred at 100 Hz. Saturation for amplitude and duration of ICa in control terminals occurred at 2 and 4-6 mM extracellular Ca2+, respectively; in LEMS-treated terminals, the extracellular Ca2+ concentration had to increase by two to three times of control to cause saturation. Amplitude of the two components of ICa observed when the preparation was exposed to 50 microM 3,4-diaminopyridine and 1 mM tetraethylammonium were both reduced by LEMS plasma treatment. The fast component (ICa,s) was reduced by 35%, whereas the slow component (ICa, s) was reduced by 37%. omega-Agatoxin IVA (omega-Aga-IVA; 0.15 microM) and omega-conotoxin-MVIIC (omega-CTx-MVIIC; 5 microM) completely blocked ICa in control motor nerve terminals. The same concentrations of toxins were 20-30% less effective in blocking ICa in LEMS-treated terminals. The residual ICa remaining after treatment with omega-Aga-IVA or omega-CTx-MVIIC was blocked by 10 microM nifedipine and 10 microM Cd2+. Thus LEMS plasma appears to downregulate omega-Aga-IVA-sensitive (P-type) and/or omega-CTx-MVIIC-sensitive (Q-type) Ca2+ channels in murine motor nerve terminals, whereas dihydropyridine (DHP)-sensitive (L-type) Ca2+ channels are unmasked in these terminals. Acute exposure (90 min) of rat forebrain synaptosomes to LEMS immunoglobulins (Igs; 4 mg/ml) did not alter the binding of [3H]-nitrendipine or [125I]-omega-conotoxin-GVIA (-omega-CgTx GVIA) when compared with synaptosomes incubated with an equivalent concentration of control Igs. Conversely, LEMS Igs significantly decreased the Bmax for [3H]-verapamil to approximately 45% of control. The apparent affinity of verapamil (KD) for the remaining receptors was not significantly altered. Thus acute exposure of isolated central nerve terminals to LEMS Igs does not increase DHP sensitivity, whereas it reduces the number of binding sites for verapamil but not for nitrendipine or omega-CgTx-GVIA. These results suggest that chronic but not acute exposure to LEMS Igs either upregulates or unmasks DHP-sensitive Ca2+ channels in motor nerve endings.

Single fibre electromyography in multifocal motor neuropathy with persistent conduction blocks

OBJECTIVETo study the process of denervation-reinnervation in multifocal motor neuropathy with persistent conduction blocks in clinically affected and unaffected muscles.METHODVolitional single fibre electromyography (SFEMG) was performed in the extensor digitorum...

Prejunctional effects of the nicotinic ACh receptor agonist dimethylphenylpiperazinium at the rat neuromuscular junction

We have studied the effects of the nicotinic acetylcholine (ACh) receptor agonist dimethylphenylpiperazinium (DMPP) on the evoked release of ACh from motor terminals in the rat isolated hemidiaphragm using an electrophysiological approach. DMPP (1–4 μM) had no effect on the rate of spontaneous quantal ACh release but increased the number of quanta of ACh released per impulse during 50 Hz stimulation. The DMPP‐induced increase in evoked ACh release was dependent on the frequency of stimulation, being absent when it was reduced to 0.5 Hz, but was not Ca2+ dependent, being unaffected at 50 Hz by a 4‐fold decrease in the extracellular Ca2+ concentration. The facilitation of evoked ACh release at 50 Hz by 2 μM DMPP was abolished by 10 μM of the calmodulin antagonist W7 (N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide hydrochloride) and, in the presence of W7, 2 μM DMPP depressed evoked ACh release at 0.5 Hz. The ability of the nicotinic ACh receptor antagonist vecuronium (1 μM) to depress evoked ACh release at 50 Hz was also abolished by 10 μM W7. The present findings demonstrate, using an electrophysiological technique, that DMPP can produce changes in the evoked ACh release from rat motor nerve terminals that are consistent with the existence of facilitatory nicotinic ACh receptors on the motor nerve endings. Further, they indicate a role for calmodulin‐dependent systems in this facilitatory effect of the compound.

Bradykinin effect on conduction of rhythmic series of pulses through the neuromuscular synapse

Bradykinin did not change the amplitude and duration of growth and decrease of individual currents of the terminal plate, but potentiated the depression of amplitudes of consecutive currents during stimulation of the motor nerve at a frequency of 10–60 Hz under conditions of two-electrode fixation of potential on a frog neuromuscular preparation. The origin of this effect is presynaptic, because it does not depend on the activity of acetylcholinesterase and is associated with a longer increase in the terminal plate currents which are synchronous with the amplitude depression. The deconvolution method revealed a slower kinetics of the mediator secretion under the effect of bradykinin. Bradykinin impairs the conduction of rhythmic pulse series through the synapse by augmenting the asynchronism of the transmitter quantum secretion from the motor nerve endings, which may underlie the development of muscular asthenia observed in inflammatory and allergic reactions.

Therapeutic and research uses of botulinum toxins

Seven serotypes of botulinum neurotoxin (BoNT, A–G) are produced by Clostridium botulinum; these are large homologous proteins (Mr∼ 150 kDa) composed of a heavy and light chain linked by a disulphide and non-covalent bonds. The characteristic multi-functional activities of each serotype are being exploited effectively for clinical and research purposes. Success in treating human dystonias and dysphonias with type A toxin is due to: (1) its targeting to cholinergic nerve endings by binding avidly via its heavy chain to ecto-acceptors located exclusively on their presynaptic membrane; (2) endocytosis and translocation to the cytosol (steps requiring the inter-chain disulphide bond); and (3) irreversible blockade of acetylcholine release due to Zn2+ dependent proteolytic cleavage of SNAP-25 by the light-chain. Eventual return of spasm to the treated muscles depends on toxin-induced nerve sprouting and the formation of functional synapses; this fascinating process has been demonstrated in vivo for the first time, using image analysis of mouse sternomastoid muscle. Thus BoNT is instrumental in attempts to decipher the molecular basis of synapse remodelling. Another potential therapeutic application has been created from the expression of an enzymically-inactive mutant of BoNT A. As the latter retains the targeting and trafficking abilities of the native toxin, it has been found to serve as an innocuous transporter for delivering a prototype drug inside motor nerve endings in biologically-active form. On the other hand, insights into the roles of SNAP-25 in exocytosis has been gleaned from the subtly different protease activities of BoNT A and E; their cleavage of SNAP-25 at distinct sites revealed that adjacent C-terminal domains contribute to pre- and post-priming stages of Ca2+-elicited catecholamine secretion. Finally, the use of types B and C1 in studies on cultured adipocytes has provided evidence for the involvement of cellubrevin and syntaxin in insulin-stimulated trafficking of vesicles containing glucose transporter-4, while the contrasting lack of inhibition of insulin-induced glucose uptake by BoNT A led to detection in these cells of the homologue SNAP-23.

The Pharmacology of Physostigmine

The Pharmacology of Physostigmine

Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate

1. Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve-diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+-aequorin luminescence. 2. ACh (0.1-3 mM, 20 microl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration-dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 mM) of applied ACh. 3. A pulse of nicotinic agonists, (-)-nicotine (10 mM) and 1,1-dimethyl-4-phenyl-piperazinium (10 mM), but not a muscarinic agonist pilocarpine (10 mM), also elicited a biphasic Ca2+ signal. 4. Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 microg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca2+ component caused by ACh application was observed. 5. These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca2+ signal reflecting Ca2+ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.

Enhanced neurotransmitter release is associated with reduction of neuronal branching in a Drosophila mutant overexpressing frequenin.

Frequenin is a Drosophila Ca2+ binding protein whose overexpression causes a chronic facilitation of transmitter release at the larval neuromuscular junction and multiple firing of action potentials. These functional abnormalities are similar to those found in other hyperexcitable mutants (Shaker, ether-a-gogo, Hyperkinetic) which, in turn, exhibit increased branching at the motor nerve endings. We report here that mutants which overexpress frequenin have motor nerve terminals with reduced number and length of branches as well as number of synaptic boutons. Similar defects are observed in transgenic flies which have additional copies of the frequenin gene indicating that the phenotype can be adscribed to the overexpression of the protein. The ultrastructure of boutons, however, appears indistinguishable from wild type. In addition, we show here that frequenin overexpression leads also to a down regulation of Shaker proteins expression. The contrast between the observations in frequenin and the other hyperexcitable mutants indicates that nerve terminal morphology and enhanced transmitter release do not have a direct causal relationship.

Firing patterns of isolated goldfish retinal ganglion cells and their regulation by a Ba2 +-sensitive K + current

Spontaneous and light-evoked excitatory postsynaptic currents (sEPSCs and leEPSCs) in retinal ganglion cells of the larval tiger salamander were recorded under voltage clamp conditions from living retinal slices. sEPSCs were isolated from the spontaneous inhibitory postsynaptic currents (sIPSCs) by application of 100 M picrotoxin+1 μM strychnine. In addition to the previously reported sEPSCs [K. Matsui, N. Hosoi, M. Tachibana, Excitatory synaptic transmission in the inner retina: pair recordings of bipolar cells and neurons of the ganglion cell layer, J. Neurosci. 18 (1998) 4500–4510; W.R. Taylor, E. Chen, D.R. Copenhagen, Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells, J. Physiol. 486 (1995) 207–221] [which are equivalent to our fast AMPA receptor-mediated sEPSCs (fAMPAsEPSCs)], we found another type of AMPA receptor-mediated sEPSC with slower rise and decay time courses and larger peak amplitudes (sAMPAsEPSCs), and the NMDA receptor-mediated sEPSCs (NMDAsEPSCs) in ON–OFF ganglion cells. The frequency of all three types of sEPSCs is greatly reduced by cobalt (with zero calcium) and increased by hyperosmotic solution, suggesting that these events are mediated by calcium-dependent exocytosis of glutamatergic synaptic vesicles. The amplitude histograms of sEPSCs do not show multiple peaks, suggesting that larger events are not discrete multiples of elementary events, or quanta, of similar neurotransmitter contents, as in the neuromuscular junction [P. Fatt, B. Katz, Spontaneous subthreshold activity at motor nerve endings, J. Physiol. 117 (1952) 109–128]. The average I–V relations of the fAMPAsEPSCs and sAMPAsEPSCs were outward rectified with reversal potentials at −12.2 mV and −10.8 mV, and that of the NMDAsEPSCs was N-shaped with a reversal potential at −5.8 mV. The average conductance increase associated with a single fAMPAsEPSC, a single sAMPAsEPSC, and a single NMDAsEPSC were 163.26±51.02 pS, 233.33±163.64 pS, and 37.5±50.0 pS at −110 mV; 241.67±22.92 pS, 444.90±469.94 pS, and 25.93±70.37 pS at −60 mV; and 440.48±183.33 pS, 1,192.68±651.22 pS, and 517.71±238.24 pS at +30 mV, respectively. The average frequency of the three sEPSCs at +30 mV were 15 Hz, 3.7 Hz and 3.6 Hz, respectively. The rise time (time to peak) of fAMPAsEPSCs was 1.5±1.05 ms and the decay time could be fitted with a single exponential with an average time constant of 3.4±4.1 ms. The rise and decay time course of the sAMPAsEPSCs and NMDAsEPSCs were much slower and sawtooth-shaped, and each `sawtooth' had time course and amplitude similar to those of individual fAMPAsEPSCs. We propose that each fAMPAsEPSC is mediated by single or synchronized multiples of glutamatergic synaptic vesicles from bipolar cells, and each sAMPAsEPSC or NMDAsEPSC is mediated by larger clusters of synaptic vesicles triggered by spontaneous calcium spikes in bipolar cell axon terminals [J. Burrone, L. Lagnado, Electrical resonance and calcium influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina, J. Physiol. 505 (1997) 571–584; D. Zenisek, G. Matthews, Calcium action potentials in retinal bipolar neurons, Vis. Neurosci. 15 (1998) 69–75].