Continuous Determination by a Chemiluminescent Method of Acetylcholine Release and Compartmentation in <i>Torpedo</i> Electric Organ Synaptosomes

Israël, Maurice; Lesbats, B.

doi:10.1111/j.1471-4159.1981.tb06317.x

Cited by 189 publications

(80 citation statements)

References 14 publications

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“…Slices were subsequently maintained separately in a solution, constantly bubbled with 95% 02/5% C02, containing 6 ml of "standard" Ringer solution (13) (pH 7.5), 20 ,d of 10 mM luminol (Sigma), 20 1.l of choline oxidase at 50 units/ml (Boehringer Mannheim), 20 ,ul of microperoxidase at 10 mg/ml (Sigma; MP II). The composition of this solution is modified from that used in a chemiluminescent reaction developed to detect acetylcholine (AcCho) (12). Choline produced by the hydrolysis of AcCho by AcChoEase is oxidized to betaine with the generation of H202.…”

Section: Methodsmentioning

confidence: 99%

“…However, dilution of released AcChoEase by perfusate limited sensitivity of the assays and restricted time resolution to periods vastly exceeding the time course of the release mechanism. To study release of AcChoEase on a time scale corresponding more to that of true neuronal events, we report the adaptation of a chemiluminescent reaction (12) for immediate and sensitive detection of AcChoEase and the development of optical probes for both on-line visualization and also measurement of luminescence, directly on the surface of nigral tissue.…”

mentioning

confidence: 99%

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On-line visualization of dendritic release of acetylcholinesterase from mammalian substantia nigra neurons.

Llinás¹,

Greenfield²

1987

Proc. Natl. Acad. Sci. U.S.A.

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This study presents, to our knowledge, the first on-line measurement of acetylcholinesterase (AcChoEase) release from brain tissue. It is now well established that a soluble form of the enzyme is released from central nervous system neurons, and it has been proposed on indirect grounds that such release may occur not only from presynaptic terminals but also from the dendrites of dopamine-containing nigrostriatal neurons. We have used a chemiluminescent reaction to examine the real-time release of AcChoEase from the substantia nigra in vitro in brainstem slices. The light emission was captured by two fiber optic systems, one in direct contact with the brain slice from below and the second 4-mm above the slice, allowing simultaneous imaging of the emitted light and quantitative photometry. It was determined that the light signals are not due to the spontaneous hydrolysis of acetylcholine or the presence of free choline, but are caused by the enzymatic action of AcChoEase. Using this technique, it can be directly shown that AcChoEase is spontaneously released from the soma or dendrites of nigral neurons. The release of the enzyme, which is stored in the subcisternal dendritic compartment, is resistant to blockade of voltage-dependent sodium conductances, is calcium dependent, and can be increased by addition of potassium to the bathing solution. The procedure we describe here will make it possible to study the release of endogenous AcChoEase on a time-scale close to that over which it functions.Ultrastructural (1), neurochemical (2, 3), electrophysiological (4, 5), and behavioral (6) findings have suggested that acetylcholinesterase (AcChoEase) is released from nigrostriatal somata and dendrites. It has been proposed (7) that dopamine is also released from the dendrites of these neurons. This release of transmitter and protein has been shown to play a significant role in nigrostriatal cell function (8).However, a precise understanding of the cellular mechanism underlying the release of large molecules has been hampered by technical limitations making it impossible to demonstrate dendritic release in a direct manner. To date, the methods used have entailed in vitro (9) or in vivo (3) perfusion (via push-pull cannulae) followed by spectrophotometric (10) or fluorometric (11) analysis of perfusates. However, dilution of released AcChoEase by perfusate limited sensitivity of the assays and restricted time resolution to periods vastly exceeding the time course of the release mechanism. To study release of AcChoEase on a time scale corresponding more to that of true neuronal events, we report the adaptation of a chemiluminescent reaction (12) for immediate and sensitive detection of AcChoEase and the development of optical probes for both on-line visualization and also measurement of luminescence, directly on the surface of nigral tissue.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

On-line visualization of dendritic release of acetylcholinesterase from mammalian substantia nigra neurons.

Llinás¹,

Greenfield²

1987

Proc. Natl. Acad. Sci. U.S.A.

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“…The total release, i.e. the sum of the 'leak' and the quantal components, is measurable by biochemical means (Gorio, Hurlbut & Ceccarelli, 1978;Israel & Lesbats, 1981). The 'leak', or non-quantal component of release, is difficult to measure directly since its major known effect is to produce a small, steady depolarization at an end-plate when acetylcholinesterase has been thoroughly inhibited.…”

Section: Introductionmentioning

confidence: 99%

Effect of alpha‐latrotoxin on the frog neuromuscular junction at low temperature.

Ceccarelli¹,

Hurlbut²,

Iezzi³

1988

The Journal of Physiology

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SUMMARY1. a-Latrotoxin (a-LTx) was applied to frog cutaneus pectoris muscles bathed at 1-3°C in either Ringer solution, Ca2"-free Ringer solution with 1 mM-EGTA and 4 mM-Mg2+ or Ringer solution plus 4 mM-Mg2+, and its effects on miniature end-plate potential (MEPP) frequency, nerve terminal ultrastructure and uptake of horseradish peroxidase (HRP) were studied.2. Large concentrations (2 ,ug/ml) of a-LTx increased MEPP rates to levels above 100/s at all junctions, but the time course of the increases depended upon the divalent cation content of the bathing solution. However, similar numbers of MEPPs (0-3-407 x 106) were recorded at all junctions during 2 h of secretion.3. Nerve terminals exposed to a-LTx for 2 h lost 60-75 % of their synaptic vesicles and were swollen; their presynaptic membranes were deeply infolded and they often contained many large vesicular structures. Terminals in Ringer solution retained the largest number of synaptic vesicles; terminals in Ringer solution plus Mg2+ swelled the least and contained the largest number of coated vesicles. The average number of synaptic vesicles lost was approximately equal to the average number of MEPPs recorded.4. Few vesicles became loaded with HRP when this extracellular tracer was present in the bathing solution and the muscles were fixed near the peak of secretion.5. When the terminals were warmed to 20°C, those in the Caa2+-free solution with Mg2+ secreted additional quanta and lost almost all their residual vesicles; those in Ringer solution without Mg2+ secreted few additional quanta and retained most of their residual vesicles.6. These results suggest that recycling was blocked at these terminals and that for each quantum secreted a vesicle became permanently incorporated into the axolemma.

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“…Cholinergic synaptosomes isolated from the electric organ of Torpedo are a useful model for studying the mechanisms of ACh release, since the synthesis of neurotransmitter, uptake of its precursors [1|, induced release of ACh [2][3][4] and calcium fluxes [5,6] have been thoroughly characterized in these nerve endings.…”

Section: Introductionmentioning

confidence: 99%

Botulinum neurotoxin inhibits depolarization‐stimulated protein phosphorylation in pure cholinergic synaptosomes

et al. 1987

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Botulinum neurotoxin, a strong blocker of acetylcholine release at peripheral cholinergic synapses, inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata. Moreover, tetrodotoxin has the same effect on protein phosphorylation when cholinergic synaptosomes are depolarized by veratridine. Correlation between presynaptic protein phosphorylation and acetylcholine release is suggested by the fact that botulinum neurotoxin blocks specifically neurotransmitter release without affecting membrane depolarization and calcium fluxes in our synaptosomal preparation.

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Continuous Determination by a Chemiluminescent Method of Acetylcholine Release and Compartmentation in Torpedo Electric Organ Synaptosomes

Cited by 189 publications

References 14 publications

On-line visualization of dendritic release of acetylcholinesterase from mammalian substantia nigra neurons.

On-line visualization of dendritic release of acetylcholinesterase from mammalian substantia nigra neurons.

Effect of alpha‐latrotoxin on the frog neuromuscular junction at low temperature.

Botulinum neurotoxin inhibits depolarization‐stimulated protein phosphorylation in pure cholinergic synaptosomes

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