Regulation of single quantal efficacy at the snake neuromuscular junction.

The Journal of Physiology

2005

At a variety of fast chemical synapses, spent synaptic vesicles are recycled via a large 'reserve' vesicle pool at high stimulus frequencies, and via fast 'local cycling' near release sites (e.g. 'kiss and run' transmitter release) at low stimulus frequencies. We have investigated recycling at the snake neuromuscular junction (NMJ), specifically seeking evidence for local cycling. Activity-dependent staining and destaining of the endocytic probe FM1-43 were directly compared to transmitter release over a range of stimulus frequencies. We found a fixed proportionality between staining/destaining and summed endplate potentials (EPPs) representing total transmitter release. There was no direct dependence of staining or destaining on stimulus frequency, as would be expected if local cycling (and consequent altered FM1-43 retention) were more prevalent at one frequency than another. In other experiments the drug vesamicol was used to abolish refilling of vesicles with transmitter, thereby blocking EPPs contributed by recycled vesicles. Control and vesamicol-treated NMJs had identical quantal content for the first 10 min of 1 Hz stimulation. Afterwards EPP amplitudes at vesamicol-treated NMJs declined at a rate consistent with use of a large pool containing ∼130 000 vesicles. Finally, calibrated paired stimulations show that regenerated vesicles have poorer than random probability of re-release. Our findings are inconsistent with local cycling and suggest that the snake motor terminal utilizes exclusively a single large vesicle pool.

Section: Methodsmentioning

confidence: 99%

Vesicles in snake motor terminals comprise one functional pool and utilize a single recycling strategy at all stimulus frequencies

Lin

Teng

The Journal of Physiology

2005

“…The centre muscle in each preparation was stimulated, with the two adjacent muscles serving as unstimulated controls for activity‐dependent staining, as described below. Intracellular recording of endplate potentials (EPPs) in curarized (30 μM) preparations and subsequent analyses were performed using methods described elsewhere (Wilkinson et al 1992); endplate sites were visualized using differential interference contrast optics. Sulphorhodamine 101 (SR101, 160 μg ml −1 ; Lichtman et al 1985; Teng et al 1999) was the endocytotic probe.…”

Section: Methodsmentioning

confidence: 99%

'Delayed' endocytosis is regulated by extracellular Ca2+ in snake motor boutons

Teng

2003

J Physiology

When cooled below ~7°C, recently endocytosed vesicles in the motor terminals of the garter snake fail to shed their clathrin coats. Perhaps as a result, the terminals complete only about one-half of the compensatory endocytosis expected after a given period of stimulation. Upon return to room temperature (RT), endocytosis resumes immediately and is complete within minutes. This 'delayed' endocytosis following release from cold block provides an opportunity to study clathrindependent endocytotic mechanisms in temporal isolation from those events, such as Ca 2+ entry and consequent exocytosis, that are normally associated with the activation of nerve terminals. We have taken advantage of clathrin decoating blockade to examine the rate, temperature dependence and extracellular Ca 2+ dependence of endocytosis at the snake nerve-muscle synapse. Endocytosis was fast at RT (complete in < 1 min) and markedly faster still at 35°C. Moreover, the rate of endocytosis varied significantly with change in [Ca 2+ ] o ; the rate at 7.2 mM (single exponential time constant, 3 s) was approximately double that at 0 mM (single exponential time constant, ~7 s). Thus, membrane retrieval via clathrin is rapid and, due to its dependence on [Ca 2+ ] o , potentially regulated by changes in the milieu of the synaptic cleft during neural activity.

“…Care was taken to set the threshold level of the amplitude discriminator used to detect MEPPs so that small and/or slow-rising MEPPs, if present, were acquired. Details of recording methods are presented elsewhere (Wilkinson, Lunin & Stevermer, 1992). Boutons were stimulated extracellularly using either of two methods.…”

Section: Preparation Of One-bouton Nmjsmentioning

confidence: 99%

Release properties of isolated neuromuscular boutons of the garter snake.

The Journal of Physiology

Son

Lunin

1996

Self Cite

1. Motor nerve terminals innervating fibres in the transversus abdominis muscle of the garter snake comprise discrete boutons. Using a combination of enzymatic digestion and mechanical manipulation, individual boutons were removed from living terminals for study in isolation. 2. Boutons freed from terminals were usually allowed to remain in their original location on the endplate ('attached' one-bouton synapse). Alternatively, they were removed from the endplate, and then placed on the same or another vacant endplate site to form a 'reconstructed' one-bouton synapse. When removed from the endplate, boutons were 2-4 ,sm in diameter and nearly spherical in shape, in contrast to the variety of complex shapes seen among boutons still in contact with muscle fibre endplates. 3. Transmitter release was assessed by intracellular recording from the postsynaptic fibre.Boutons produced spontaneous miniature endplate potentials (MEPPs) of nearly normal amplitude; extracellular stimulation elicited endplate potentials (EPPs) which resembled MEPPs. Typical EPP amplitudes fluctuated between zero and five quanta per stimulus. For low-frequency stimulation under normal physiological conditions, mean quantal content, m, averaged 1-4; the binomial number of release sites, n, averaged 2-4; and the binomial probability of release, p, averaged 0 57. Statistics of the quantal fluctuations recorded from single boutons agreed only approximately with predictions of simple binomial theory, the discrepancy being that the apparent number of quanta released exceeded n in 5 % of the events.4. In separate experiments, activity-dependent probes were used to locate rare naturally occurring nerve terminals comprising a single bouton. Activation of these small synapses evoked quantal responses similar to those synapses described above.