Acetylcholine synthesis is modulated by acetylcholine content of cytosolic fraction but not by that of releasable fraction.

Synthesis and release of acetylcholine (ACh) in the rat hippocampal slices were examined to clarify the mechanism of modulation of ACh synthesis. Treatment with 2-(4-phenylpiperidino)cyclohexanol (AH5183, 50 microM), an inhibitor of ACh transport from cytosol to synaptic vesicles, inhibited the increase in ACh content of the membrane-bound fraction which is readily releasable, but did not affect the cytosolic ACh content. Under these conditions, the total ACh content reached a plateau value. These results indicate that ACh synthesis is modulated by cytosolic ACh content but not by the vesicular fraction.     

The inhibitory effect of adenosine and related nucleotides on the release of acetylcholine

Isolated Auerbach's plexus-longitudinal muscle preparations from guinea-pig ileum and slices of the rat cerebral cortex have been used to study the effect of adenine nucleotides on the release of acetylcholine. The release of acetylcholine evoked by cholecystokinin was completely inhibited by adenosine. The effect of nucleotides on neuro-effector transmission of electrically stimulated longitudinal muscle strip was also studied. Adenosine and adenosine triphosphate reduced the release of acetylcholine provided a low frequency of stimulation was applied. While the three adenine nucleotides (adenosine mono-, di- and triphosphate) dose-dependently reduced neuro-effector transmission in Auerbach's plexus-longitudinal muscle preparation, adenine, guanosine triphosphate and dibutyryl-cyclic AMP had no effect. Theophylline, an adenosine receptor antagonist, prevented the inhibitory effect of the nucleotides. In addition, theophylline alone enhanced the release of acetylcholine both from Auerbach's plexus and from the nerve terminals of the cortical slice. This indicates that there might be a continuous control of ACh release by an adenine nucleotide.These results are discussed in relation to the release of adenosine triphosphate from purinergic nerves in the intestine and of adenosine from slices of cerebral cortex; the possibility is raised that adenine nucleotides released from nerves might act as a type of presynaptic inhibitory transmitter on cholinergic neurons. Furthermore, if some of the released nucleotide originates from the synaptic vesicles of cholinergic neurons, it might serve as a negative feed-back transmitter for acetylcholine release.The inhibitory effect of adenosine and related nucleotides on the cholecystokinin-induced release of acetylcholine from the gut might be of physiological importance since gastrointestinal polypeptides play a very important role in maintaining gastrointestinal motility.     

Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.     

Distribution of soluble N-ethylmaleimide fusion protein attachment proteins (SNAPs) in the rat nervous system.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) plays an essential role in vesicular transport and the release of neurotransmitters and hormones through associations with NSF and SNAP receptors (SNAREs). Three isoforms (alpha, beta and gamma) of SNAP are expressed in mammals. We have generated isoform-specific antibodies and studied the expression and distribution of these SNAP isoforms in the rat nervous system. Each antibody specifically recognized alpha-, beta- or gamma-SNAP in an isoform-specific manner in immunoblots of brain homogenate. Alpha- and gamma-SNAP were ubiquitously expressed in various tissues, whereas beta-SNAP was expressed only in brain. After subcellular fractionation of brain homogenates, all three isoforms were recovered in both soluble and particulate fractions. Immunohistochemistry revealed that alpha- and beta-SNAP were generally differentially distributed both in synaptic and non-synaptic regions, including brain white matter. The presynaptic location of both alpha- and beta-SNAP was confirmed by immunoelectron microscopy. At the neuromuscular junction, immunoreactive alpha-SNAP was identified in synaptic vesicles, while in the cerebellum, beta-SNAP was present in the presynaptic membranes of basket neuron and mossy fiber terminals.From these results we suggest that both alpha- and beta-SNAP may play an important role in neurotransmitter release as well as in constitutive vesicular transport.     

The linoleic acid derivative FR236924 facilitates hippocampal synaptic transmission by enhancing activity of presynaptic alpha7 acetylcholine receptors on the glutamatergic terminals

The present study aimed at understanding the effect of FR236924, a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on hippocampal synaptic transmission in both the in vitro and in vivo systems. FR236924 increased the rate of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, triggered by nicotine in CA1 pyramidal neurons of rat hippocampal slices, that is inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor or alpha-bungarotoxin, an inhibitor of alpha7 acetylcholine (ACh) receptors. FR236924 stimulated glutamate release from rat hippocampal slices and in the hippocampus of freely behaving rats, and the effect was also inhibited by GF109203X or alpha-bungarotoxin. FR236924 induced a transient huge potentiation followed by a long-lasting potentiation in the slope of field excitatory postsynaptic potentials recorded from the CA1 region of rat hippocampal slices, and the latter effect was blocked by GF109203X or alpha-bungarotoxin. Likewise, the compound persistently facilitated hippocampal synaptic transmission in the CA1 region of the intact rat hippocampus. It is concluded from these results that FR236924 stimulates glutamate release by functionally targeting presynaptic alpha7 ACh receptors on the glutamatergic terminals under the influence of PKC, responsible for the facilitatory action on hippocampal synaptic transmission. This may provide evidence for a link between cis-unsaturated free fatty acids and presynaptic alpha7 ACh receptors in hippocampal synaptic plasticity.     

The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone

Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3(-/-) mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.     

Spontaneous and evoked release of acetylcholine and a cholinergic false transmitter from brain slices: comparison to true and false transmitter in subcellular stores

Rat cerebral cortex slices were incubated with [¹⁴Ccholine and [³Hhomocholine to allow synthesis of [¹⁴Cacetylcholine and [³Hacetylhomocholine; release of the acetylated compounds was tested in the presence of eserine and hemicholinium-3. Both [¹⁴Cacetylcholine and [³Hacetylhomocholine were released spontaneously by a Ca²⁺-independent mechanism; exposure of the tissue to a high K⁺ medium resulted in a Ca²⁺-dependent increase in [¹⁴Cacetylcholine release (30-fold) and in [³Hacetylhomocholine release (5 to 7-fold). Thus, spontaneous and K⁺-evoked transmitter release could be distinguished on the basis of their molar ratios of true to false transmitters and these ratios were compared to the molar ratios of the transmitters in subcellular fractions prepared from the incubated tissue. The molar ratio of acetylcholine to acetylhomocholine released from the tissue spontaneously differed from the ratio in subcellular fractions containing occluded transmitters. The molar ratio of acetylcholine to acetylhomocholine released by K⁺ differed from the ratio in a fraction (H) containing occluded transmitters but was similar to the ratio in the fraction (D) containing monodisperse synaptic vesicles and the fraction (0) containing the majority of a soluble cytoplasmic marker. Comparison of the transmitter contents of subcellular fractions from unstimulated and K⁺-stimulated tissue showed that the three fractions (D, H and O) lost equal proportions of both transmitters as a result of K⁺ stimulation.It is concluded that acetylhomocholine may be released from brain slices both spontaneously and in response to stimulation via mechanism similar to those that release acetylcholine, but that there must be some differences in specificity of the acetylcholine storage and/or release processes. The results also indicate that spontaneous transmitter release originates from extravesicular stores; the results are consistent with a vesicular site of origin of evoked transmitter release but do not distinguish between nerve terminal vesicles and cytoplasm as the immediate source of evoked transmitter release.     

The relationship of transmitter release and storage to fine structure in a sympathetic ganglion

Summary Cat sympathetic ganglia were prepared for electron microscopy by perfusion fixation with glutaraldehyde in the presence of Mg⁺⁺. At resting boutons de passage the populations of synaptic vesicles were 6400 per bouton. The vesicle distributions displayed many of the features of spheres in close-packing. Calculations based on a vesicle close-packing hypothesis gave a figure of 8000 vesicles per bouton. In ganglia stimulated for 20 min at 20/s the vesicle populations were reduced to 25%, and to 28.5% in ganglia in which acetylcholine (ACh) synthesis was inhibited by hemicholinium (HC-3). The reduction was to 34% when stimulation was for 1 min. In ganglia stimulated for 20 min at 1/s and 4/s the vesicle populations were reduced to 44% and 46% respectively. Even following 1 min stimulation at 4/s over half the boutons showed significant loss of vesicles. ACh stores in ganglia are known not to be depleted by any of these procedures except stimulation in the presence of HC-3. The fraction of ganglionic ACh stores known to be released by stimulation for 1 min at 20/s or 4/s and by 20 min stimulation at 1/s is substantially less than the fraction of vesicles lost. The observations therefore were not readily accounted for by the vesicle theory of transmitter storage and release. They were consistent with the idea the ACh is stored in vesicles at rest, but that during maintained activity over half the bouton ACh is free in the cytoplasm. The concentration of cytoplasmic ACh was calculated to be approximately 50–150 mm 1^–1. Examination of the hypothesis that ACh may be released from this cytoplasmic pool during synaptic activation indicated an efflux of approximately 1.5–3.0 × 10^–12 M Ach cm^–2 synapsing membrane/impulse.Medical Research Associate of the Medical Research Council of Canada.     

Correlated release of acetylcholine and protein from the neuromuscular junction.

Transmitter release at adrenergic synapses is accompanied by release of chromogranin proteins, which are contained in synaptic vesicles. To determine if a similar phenomenon occurs at the neuromuscular junction, correlated release of acetylcholine (ACh) and protein was investigated using in vitro neuromuscular preparations (phrenic nerve-diaphragm muscle of the rat and mouse, sciatic nerve-sartorius muscle of Rana pipiens and R. catesbeiana). Nerve stimulation of curare-paralyzed preparations increased the rate of efflux of Lowry-reactive material relative to control values. Stimulus-specific responses outlasted the period of neural stimulation. Stimulus-induced release of Lowry-reactive material was correlated with ACh release since it was Ca2+ dependent and Mg2+ antagonized. Conditions that potentiate spontaneous ACh release also significantly increased the rate of efflux of Lowry-reactive material. Most of the Lowry-reactive material released with ACh is not a secretory product of synaptic vesicles because the amount released exceeds the contents of synaptic vesicles that undergo exocytosis. It is concluded that ACh release from the neuromuscular junction is accompanied by release of proteinaceous material that is not entirely derived from synaptic vesicles.     

Characterization of ouabain-induced noradrenaline and acetylcholine release from in situ cardiac autonomic nerve endings

Aim: Although ouabain modulates autonomic nerve ending function, it is uncertain whether ouabain‐induced releasing mechanism differs between in vivo sympathetic and parasympathetic nerve endings. Using cardiac dialysis, we examined how ouabain induces neurotransmitter release from autonomic nerve ending. Methods: Dialysis probe was implanted in left ventricle, and dialysate noradrenaline (NA) or acetylcholine (ACh) levels in the anaesthetized cats were measured as indices of neurotransmitter release from post‐ganglionic autonomic nerve endings. Results: Locally applied ouabain (100 μm ) increased in dialysate NA or ACh levels. The ouabain‐induced increases in NA levels remained unaffected by cardiac sympathetic denervation and tetrodotoxin (Na⁺ channel blocker, TTX), but the ouabain‐induced increases in ACh levels were attenuated by TTX. The ouabain‐induced increases in NA levels were suppressed by pretreatment with desipramine (NA transport blocker) and augmented by reserpine (vesicle NA transport blocker). In contrast, the ouabain‐induced increases in ACh levels remained unaffected by pretreatment with hemicholinium‐3 (choline transport blocker) but suppressed by vesamicol (vesicle ACh transport blocker). The ouabain‐induced increases in NA levels were suppressed by pretreatment with ω‐conotoxin GVIA (N‐type Ca²⁺ channel blocker), verapamil (L‐type Ca²⁺ channel blocker) and TMB‐8 (intracellular Ca²⁺ antagonist). The ouabain‐induced increases in ACh levels were suppressed by pretreatment with ω‐conotoxin MVIIC (P/Q‐type Ca²⁺ channel blocker), and TMB‐8. Conclusions: Ouabain‐induced NA release is attributable to the mechanisms of regional exocytosis and/or carrier‐mediated outward transport of NA, from stored NA vesicle and/or axoplasma, respectively, while the ouabain‐induced ACh release is attributable to the mechanism of exocytosis, which is triggered by regional depolarization. At both sympathetic and parasympathetic nerve endings, the regional exocytosis is because of opening of calcium channels and intracellular calcium mobilization.     

Stimulation, by inhibition of (Na + -K + -Mg 2+ )-activated ATP-ase, of acetylcholine release in cortical slices from rat brain

1. A study has made of the effect of (Na(+)-K(+)-Mg(2+))-activated membrane ATP-ase inhibitors on the acetylcholine release from the terminals of enteric nerves and from cortical slices.2. The resting output of acetylcholine from slices of rat cortex was not affected by tetrodotoxin or by noradrenaline, indicating the lack of propagated activity during rest. Furthermore, there was an output of acetylcholine in the absence of Ca.3. The resting acetylcholine output from cortical slices was increased by (a) addition of ouabain or (b) administration of sodium p-hydroxymercuribenzoate (PHMB), (c) sodium withdrawal and (d) Ca replacement by Ba(+).4. Omission of Ca in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethyl-ether)-N,N'-tetra-acetic acid (EGTA) did not affect the increase of acetylcholine release by the inhibition of (Na(+)-K(+)-Mg(2+))-activated ATP-ase induced by ouabain or by PHMB, but reduced that due to Na removal.5. Ouabain increased acetylcholine release promptly.6. Mg-excess (9.3 mM), noradrenaline and adrenaline were capable of reducing the increase of acetylcholine release from cortical slices evoked by ouabain, PHMB or by Ca replacement by Ba, but not by Na removal.7. A possible role for (Na(+)-K(+)-Mg(2+))-activated ATP-ase in the release of acetylcholine is discussed. It is suggested that the effect of Ca and Mg ions on acetylcholine release might be attributed to their ability to inhibit and activate the membrane ATP-ase, respectively.     

5'-triphosphate recycles independently of acetylcholine in cholinergic synaptic vesicles.

The effect of hemicholinium-3 (HC-3) on vesicular contents in acetylcholine (ACh) and 5-triphosphate (ATP) and the vesicular incorporation of 14C-label derived from [14C]choline ,nd 3H-label derived from [3H]adenosine was investigated after low frequency stimulation (with a subsequent rest period) of the Torpedo electric organ. HC-3 (100 microM) caused an increased depletion of vesicular ACh and blocked the incorporation of 14C-label whereas contents in vesicular ATP and 3H-incorporation were identical with and without HC-3. HC-3 also blocked the recovery of electrical response of the tissue after stimulation but did not cause a change in vesicle numbers. The result suggest that synaptic vesicles continue to recycle ATP in the absence of recycling of ACh and that vesicular uptake and storage of the two components are not coupled to each other.     

Vesicle size and transmitter release at the frog neuromuscular junction when quantal acetylcholine content is increased or decreased

We investigated whether the synaptic vesicles at the neuromuscular junction change size when their acetylcholine (ACh) content is altered. The size of the miniature endplate potential (MEPP) increased 3- or 4-fold in preparations pre-treated in a hypertonic solution in which the anion was gluconate. We measured the dimensions of synaptic vesicles in such preparations and in controls. The size of the vesicles and size distribution were indistinguishable. Quanta contained about half of the usual amount of ACh in preparations stimulated in the presence of hemicholinium-3, an inhibitor of choline uptake, or in NH₄⁺, which diminishes the proton gradient for ACh uptake into the vesicles. Neither treatment changed the size of the synaptic vesicles. ACh content and vesicle size were both decreased in preparations stimulated in (-)-vesamicol, an inhibitor of ACh uptake in vesicles. Since the other inhibitors decreased ACh content by a similar amount without altering vesicle size, (-)-vesamicol may decrease vesicle size by acting on another target. We also found that a hypertonic solution in which the anion was aspartate increased quantal size similar to gluconate. Both anions have high hydration energy and a large volume. When these treatments increased quantal size the mean 20-80 % rise time of MEPPs recorded with an extracellular electrode was 170 μs. In the controls it was 97 μs. Perhaps some of the added ACh is bound within the vesicles, which slows the rise. Our major conclusion is that ACh content can change notably without any change in the size of the synaptic vesicles.     

Loading and recycling of synaptic vesicles in the Torpedo electric organ and the vertebrate neuromuscular junction

In vertebrate motor nerve terminals and in the electromotor nerve terminals of Torpedo there are two major pools of synaptic vesicles: readily releasable and reserve. The electromotor terminals differ in that the reserve vesicles are twice the diameter of the readily releasable vesicles. The vesicles contain high concentrations of ACh and ATP. Part of the ACh is brought into the vesicle by the vesicular ACh transporter, VAChT, which exchanges two protons for each ACh, but a fraction of the ACh seems to be accumulated by different, unexplored mechanisms. Most of the vesicles in the terminals do not exchange ACh or ATP with the axoplasm, although ACh and ATP are free in the vesicle interior. The VAChT is controlled by a multifaceted regulatory complex, which includes the proteoglycans that characterize the cholinergic vesicles. The drug (-)-vesamicol binds to a site on the complex and blocks ACh exchange. Only 10-20% of the vesicles are in the readily releasable pool, which therefore is turned over fairly rapidly by spontaneous quantal release. The turnover can be followed by the incorporation of false transmitters into the recycling vesicles, and by the rate of uptake of FM dyes, which have some selectivity for the two recycling pathways. The amount of ACh loaded into recycling vesicles in the readily releasable pool decreases during stimulation. The ACh content of the vesicles can be varied over eight-fold range without changing vesicle size.     

Abnormal association of mutant huntingtin with synaptic vesicles inhibits glutamate release

In Huntington disease (HD), polyglutamine expansion causes the disease protein huntingtin to aggregate and accumulate in the nucleus and cytoplasm. The cytoplasmic huntingtin aggregates are found in axonal terminals and electrophysiological studies show that mutant huntingtin affects synaptic neurotransmission. However, the biochemical basis for huntingtin-mediated synaptic dysfunction is unclear. Using electron microscopy on sections of HD mouse brains, we found that axonal terminals containing huntingtin aggregates often had fewer synaptic vesicles than did normal axonal terminals. Subcellular fractionation and electron microscopy revealed that mutant huntingtin is co-localized with huntingtin-associated protein-1 (HAP1) in axonal terminals in the brains of HD transgenic mice. Mutant huntingtin binds more tightly to synaptic vesicles than does normal huntingtin, and it decreases the association of HAP1 with synaptic vesicles in HD mouse brains. Brain slices from HD transgenic mice that had axonal aggregates showed a significant decrease in [(3)H]glutamate release, suggesting that neurotransmitter release from synaptic vesicles was impaired. Taken together, these findings suggest that mutant huntingtin has an abnormal association with synaptic vesicles and this association impairs synaptic function.     

Origin of transmitters released by electrical stimulation from a small metabolically very active vesicular pool of cholinergic synapses in guinea-pig cerebral cortex.

The turnover of true and false transmitters was measured to find out from which subcellular pool in guinea-pig cerebral cortex acetylcholine is released after electrical stimulation. [N-Me-¹⁴C]choline was injected along with [N-Me-³H]pyrrolidinecholine or [N-Me-³H]homocholine into cortical tissue. Both choline analogues were acetylated, although two to five times less favourably than choline. All acetylated forms were incorporated into synaptic vesicles; this vesicular uptake was enhanced by low-frequency electrical stimulation (0.1 Hz) and occurred preferentially into a metabolically very active subpopulation of synaptic vesicles closely attached to the presynaptic membrane which can be isolated at the interphase between 0.8 M and 1.0 M sucrose (fraction H), using the sucrose density gradient separation technique. Acetylhomocholine was preferentially localized within the cytoplasmic compartment and taken up into synaptic vesicles to a smaller extent than acetylpyrrolidinecholine.Locke's solution was placed in Perspex cylinders (cups) applied to the surface of the cortex and acetylcholine as well as acetylpyrrolidinecholine and acetylhomocholine were released into these cups by diffusion. High-frequency stimulation (30 Hz) approximately doubled the amount of transmitters released. The molar ratios of the transmitters ([¹⁴C]acetylcholine/[³H]acetylpyrrolidinecholine or [¹⁴C]acetylcholine/[³H]acetylhomocholine) released during stimulation closely approximated those found in fraction H but differed significantly from the molar ratios determined in the monodisperse synaptic vesicle fraction (fraction D) at the 0.4 M sucrose level and also from that in the cytoplasmic compartment. Moreover, the specific activity of the released transmitter was nearly identical with that in fraction H.It is concluded that electrical stimulation releases acetylcholine from a distinct, metabolically very active vesicular pool in the guinea-pig cortex and that synaptic activation in some way facilitates the vesicular uptake of transmitters.     

Cd2+- and K+-evoked ACh release induce different synaptophysin and synaptobrevin immunolabelling at the frog neuromuscular junction

Summary Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd²⁺- or K⁺-induced ACh release at the frog neuromuscular junction. ACh release was stimulated in cutaneous pectoris nerve-muscle preparations by: (1) 1 mM Cd²⁺ in Ca²⁺-free medium for a period of 3 h, (2) 25 or 40mM K⁺ in normal Ringer's solution. Synaptophysin and synaptobrevin were immunolabelled in single fibres teased from fixed muscles using rabbit antisera raised against synaptophysin and synaptobrevin revealed with fluoresceinconjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated -bungarotoxin. Unstimulated and K⁺-stimulated preparations showed synaptophysin and synaptobrevin immunolabelling only after membrane permeabilization with 0.1% Triton X-100. In preparations stimulated with Cd²⁺ in Ca²⁺-free medium, the immunofluorescence was also observed in non Triton X-100 treated muscle fibres. Confocal laser scanning microscopy analysis revealed that in unstimulated and K⁺-stimulated preparations, synaptophysin and synaptobrevin immunofluorescence appears as bands regularly spaced along the permeabilized nerve terminals and that their distribution corresponds to clusters of synaptic vesicles. After Cd²⁺ stimulation in Ca²⁺-free medium, labelling for both proteins is irregularly distributed, being more intense at the lateral margins of swollen nerve terminals, suggesting a translocation of synaptic vesicle proteins to the axolemma. At the electron microscopic level, Cd²⁺ stimulation in Ca²⁺-free medium produces nerve terminal swelling and synaptic vesicle depletion. The results show that when ACh release is stimulated under an impairment of synaptic vesicle recycling, which leads to synaptic vesicle depletion, synaptophysin and synaptobrevin translocation occurs. These findings are in favour of a permanent incorporation of synaptic vesicle membrane into the axolemma. In contrast, after K⁺ stimulation, the immunofluorescence and the normal synaptic vesicle population observed, suggest that a double process of synaptic vesicle exo-endocytosis rapidly occurs, without incorporation of synaptic vesicle components into the axolemma.     

Muscular dystrophy with laminin deficiency decreases the content of butyrylcholinesterase tetramers in sciatic nerves of Lama2dy mice

In this paper, the effect of the alpha-scorpion toxin tityustoxin (TsTX) in the release of gamma-[(3)H]aminobutyric acid ([(3)H]GABA) from rat brain cortical slices is described. The TsTX-stimulatory effect on the release of [(3)H]GABA was dependent on incubation time and TsTX concentration, having an EC(50) of 0.33 microM. Tetrodotoxin (TTX) completely inhibited the TsTX action on [(3)H]GABA release. The scorpion toxin effect was calcium-dependent and involves P/Q calcium channels. beta-Alanine also induces the release of [(3)H]GABA that was not inhibited by TTX but was additive in the presence of TsTX. The data suggest a neuronal origin for the release of [(3)H]GABA by TsTX.     

2-(4-Phenylpiperidino) cyclohexanol (AH5183) decreases quantal size at the frog neuromuscular junction

AH5183 was studied because it inhibits acetylcholine transport into synaptic vesicles. The drug apparently is a slow-acting anti-cholinesterase, so further experiments were performed with this enzyme inhibited. Soaking for hours in AH5183 in Ringer does not decrease quantal size. However, a few minutes of tetanic nerve stimulation results in a marked decrease in quantal size. Quantal size also decreased after hours in a hypertonic Ringer containing the drug.     

Impairment of synaptic vesicle exocytosis and recycling during neuromuscular weakness produced in mice by 2,4-dithiobiuret

Chronic treatment of rodents with 2,4-dithiobiuret (DTB) induces a neuromuscular syndrome of flaccid muscle weakness that mimics signs seen in several human neuromuscular disorders such as congenital myasthenic syndromes, botulism, and neuroaxonal dystrophy. DTB-induced muscle weakness results from a reduction of acetylcholine (ACh) release by mechanisms that are not yet clear. The objective of this study was to determine if altered release of ACh during DTB-induced muscle weakness was due to impairments of synaptic vesicle exocytosis, endocytosis, or internal vesicular processing. We examined motor nerve terminals in the triangularis sterni muscles of DTB-treated mice at the onset of muscle weakness. Uptake of FM1-43, a fluorescent marker for endocytosis, was reduced to approximately 60% of normal after either high-frequency nerve stimulation or K(+) depolarization. Terminals ranged from those with nearly normal fluorescence ("bright terminals") to terminals that were poorly labeled ("dim terminals"). Ultrastructurally, the number of synaptic vesicles that were labeled with horseradish peroxidase (HRP) was also reduced by DTB to approximately 60%; labeling among terminals was similarly variable. A subset of DTB-treated terminals having abnormal tubulovesicular profiles in their centers did not respond to stimulation with increased uptake of HRP and may correspond to dim terminals. Two findings suggest that posttetanic "slow endocytosis" remained qualitatively normal: the rate of this type of endocytosis as measured with FM1-43 did not differ from normal, and HRP was observed in organelles associated with this pathway- coated vesicles, cisternae, as well as synaptic vesicles but not in the tubulovesicular profiles. In DTB-treated bright terminals, end-plate potential (EPP) amplitudes were decreased, and synaptic depression in response to 15-Hz stimulation was increased compared with those of untreated mice; in dim terminals, EPPs were not observed during block with D-tubocurarine. Nerve-stimulation-induced unloading of FM1-43 was slower and less complete than normal in bright terminals, did not occur in dim terminals, and was not enhanced by alpha-latrotoxin. Collectively, these results indicate that the size of the recycling vesicle pool is reduced in nerve terminals during DTB-induced muscle weakness. The mechanisms by which this reduction occurs are not certain, but accumulated evidence suggests that they may include defects in either or both exocytosis and internal vesicular processing.