Properties of "reconstructed" motor synapses of the garter snake

Wilkinson, Robert S.; Lunin, S D

doi:10.1523/jneurosci.14-05-03319.1994

Cited by 11 publications

(16 citation statements)

References 31 publications

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“…Evidence for this strong adhesion comes from the requirement of harsh acid or proteolytic treatments to strip nerve terminals from muscle fibers (Kuffler and Yoshikami, 1975;Fahim et al, 1983;Wilkinson and Lunin, 1994). We also consider the fact that the elongation and widening of muscle fibers during normal growth are associated with intercalary membrane addition throughout the muscle fiber, as seen in the intercalary enlargement of the AChR distribution as muscle fibers grow (Balice-Gordon and .…”

Section: Discussionmentioning

confidence: 99%

From Plaque to Pretzel: Fold Formation and Acetylcholine Receptor Loss at the Developing Neuromuscular Junction

2000

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Although there has been progress in understanding the initial steps in the formation of synapses, less is known about their subsequent maturation (Sanes and Lichtman, 1999). Two alterations on the postsynaptic side of the mammalian neuromuscular junction occur during early postnatal life: acetylcholine receptors (AChRs) disappear from parts of the developing junction as all but one axonal inputs are removed, and the topography of the postsynaptic membrane becomes more complicated as gutters and folds are established. We have studied the maturation of the AChR distribution and postsynaptic topography simultaneously by imaging labeled AChRs at the mouse neuromuscular junction in a new way, using reflected light confocal microscopy. At birth postsynaptic receptors were localized in irregular patches within a spoon-shaped plaque. Beginning several days later, receptor regions within a single endplate were divided into differentiated and less organized compartments. Folds generally oriented orthogonal to the long axis of the muscle fiber were seen in developing gutters, although the orientation of the gutters seemed to be imposed by the branching pattern of the nerve. Eventually, superficial regions lacking AChR labeling were apparent in all junctions. In junctions denervated in the neonatal period both gutter formation and the disappearance of superficial receptors regions were prevented. We suggest that tension between growing muscle fibers and the relatively inelastic synaptic terminals that adhere to them causes the topographic features of the postsynaptic membrane. This view provides a mechanical explanation for gutters, folds, and the location of folds at sites of neurotransmitter release. Key words: reflected light confocal microscopy; postsynaptic membrane topography; acetylcholine receptor distribution; neuromuscular junction development; synapse elimination; active zonesThe use of fluorescently tagged ␣-bungarotoxin to label acetylcholine receptors (AChRs) at the neuromuscular junction has shown that there is a dramatic maturational alteration in receptor distribution in early postnatal life. At birth, AChRs are arranged in an oval plaque in what appears to be a relatively uniform density. Over several weeks this plaque perforates (Nyström, 1968;Steinbach, 1981;Slater, 1982; such that the regions that maintain AChRs become the pretzel-shaped branches of the mature singly innervated neuromuscular junction, whereas regions that lose membraneassociated AChRs also lose nerve terminal innervation during the process of synapse elimination (Balice-Gordon and Lichtman, 1993; Gan and Lichtman, 1998). Although fluorescence labeling of AChRs allows for the study of the overall distribution of receptors during development, this technique has not been useful in studying alterations in the three-dimensional topography of the muscle fiber membrane as neuromuscular junctions mature. Hence, the formation of gutters (primary synaptic clefts) and the elaboration of folds within the gutters could not be related to the changes ...

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Section: Discussionmentioning

confidence: 99%

From Plaque to Pretzel: Fold Formation and Acetylcholine Receptor Loss at the Developing Neuromuscular Junction

2000

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“…Collagenase treatment detaches pre‐synaptic terminals from muscle and leaves post‐synaptic receptors intact (Robitaille et al. 1990; Wilkinson and Lunin 1994). Therefore, we assume that we do not have much post‐synaptic contamination.…”

Section: Methodsmentioning

confidence: 99%

“…Protein sample preparation After stimulation to induce LFD at phasic synapses or treatment for 20 min with regulators of protein phosphorylation (FK-506, Rp-cAMPS, PMA, GF, staurosporine, in saline) at concentrations used previously for pharmacological studies (Silverman-Gavrila et al 2005) the preparations were treated with collagenase (type I, 1 mg/ mL in saline, Calbiochem) for 30 min at 22°C (Kanai et al 1998). Collagenase treatment detaches pre-synaptic terminals from muscle and leaves post-synaptic receptors intact (Robitaille et al 1990;Wilkinson and Lunin 1994). Therefore, we assume that we do not have much post-synaptic contamination.…”

Section: Phospho-proteomicsmentioning

confidence: 99%

Calcineurin and cytoskeleton in low‐frequency depression

Silverman‐Gavrila

Charlton

2009

Journal of Neurochemistry

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kinase C; PMA, phorbol 12-myristate13-acetate; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2B, protein phosphatase 2B; Rp-cAMPS, adenosine 3¢,5¢-cyclic monophosphorothioate Rp-isomer triethylammonium salt; SDS, sodium dodecyl sulphate. AbstractTransmitter release at high probability phasic synapses of crayfish neuromuscular junctions depresses by over 50% in 60 min when stimulated at 0.2 Hz. Inhibition of the protein phosphatase calcineurin by intracellular pre-synaptic injection of autoinhibitory peptide inhibited low-frequency depression (LFD) and resulted in facilitation of transmitter release. Since this inhibitor had no major effects when injected into the postsynaptic cell, only pre-synaptic calcineurin activity is necessary for LFD. To examine changes in phosphoproteins during LFD we performed a phosphoproteomic screen on proteins extracted from motor axons and nerve terminals after LFD induction or treatment with various drugs that affect kinase and phosphatase activity. Proteins separated by PAGE were stained with phospho-specific/total protein ratio stains (Pro-Q Diamond/SYPRO Ruby) to identify protein bands for analysis by mass spectrometry. Phosphorylation of actin and tubulin decreased during LFD, but increased when calcineurin was blocked. Tubulin and phosphoactin immunoreactivity in presynaptic terminals were also reduced after LFD. The actin depolymerizing drugs cytochalasin and latrunculin and the microtubule stabilizer taxol inhibited LFD. Therefore, dephosphorylation of pre-synaptic actin and tubulin and consequent changes in the cytoskeleton may regulate LFD. LFD is unlike long-term depression found in mammalian synapses because the latter requires in most instances postsynaptic calcineurin activity.Thus, this simpler invertebrate synapse discloses a novel pre-synaptic depression mechanism.

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“…We developed previously a technique for removing entire living nerve terminals from endplate sites; the terminals were then replaced onto their original endplate site, or placed onto another vacant endplate site, to form 'reconstructed' NMJs. The method comprised treatment of the entire preparation with bath-applied collagenase, followed by application of protease to a single NMJ via a microperfusion system (details in Wilkinson & Lunin, 1994). Protease solution was perfused until a slight increase in Nomarski contrast of the NMJ was noted.…”

Section: Preparation Of One-bouton Nmjsmentioning

confidence: 99%

“…Unlike NMJs of mammals or amphibians, those of the snake contain clusters of about twenty (tonic) or about sixty (twitch) discrete boutons, similar to mammalian CNS boutons (see Kuffler & Yoshikami, 1975). By adapting a recent technique for removing and reattaching nerve terminals at snake NMJs (synaptic 'reconstruction'; Wilkinson & Lunin, 1994), we have devised a method to remove all terminal boutons innervating an endplate save one, thus creating a onebouton NMJ. Electrophysiological study of this small synapse permits analysis of release from sites within a single bouton while maintaining the advantages associated with the study of NMJs.…”

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confidence: 99%

Release properties of isolated neuromuscular boutons of the garter snake.

Wilkinson

Son

Lunin

1996

The Journal of Physiology

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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.

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Properties of "reconstructed" motor synapses of the garter snake

Cited by 11 publications

References 31 publications

From Plaque to Pretzel: Fold Formation and Acetylcholine Receptor Loss at the Developing Neuromuscular Junction

From Plaque to Pretzel: Fold Formation and Acetylcholine Receptor Loss at the Developing Neuromuscular Junction

Calcineurin and cytoskeleton in low‐frequency depression

Release properties of isolated neuromuscular boutons of the garter snake.

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