Quantal Analysis of Endplate Potentials in Mouse Flexor Digitorum Brevis Muscle

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

The isolated flexor digitorum brevis (FDB) muscle from mice is extremely well suited to rapid acquisition of data and analysis of neurotransmitter release and action at neuromuscular junctions, because the muscle and its tibial nerve supply are simple to dissect and its constituent muscle fibers are short (<1 mm) and isopotential along their length. Methods are described here for dissection of FDB, stimulation of the tibial nerve, microelectrode recording from individual muscle fibers, and quantal analysis of endplate potentials (EPPs) and miniature endplate potentials (MEPPs). Curr. Protoc. Mouse Biol. 1:429?444 © 2011 by John Wiley & Sons, Inc.

Keywords: neuromuscular junction; endplate potential; intracellular recording; electrophysiology; quantal analysis

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Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1:

Materials

Physiological saline solution (see recipe )
Mouse (any laboratory strain of any age/gender; e.g., C57B16)
µ‐conotoxin GIIIB (Bachem)
4 M potassium acetate

Dissection tools (iris scissors, spring scissors, fine forceps)
Sylgard‐lined Petri dish (see recipe )
Fine minutien pins (Fine Science Tools)
Suction electrode (see recipe )
Micromanipulators (e.g., Leica or Sutter Instruments)
Recording chamber: the author's laboratory uses a Perspex chamber made in‐house, but a Sylgard‐lined Petri dish (see recipe ) will suffice
1‐mm glass capillary tubing containing and internal welded glass filament (1.5 mm O.D., 0.84 mm I.D. standard wall borosilicate glass with filament; World Precision Instruments, cat. no. 1B150F‐6)
Electrode puller: Among the most popular electrode pullers with electrophysiologists is the Brown‐Fleming puller manufactured by Sutter Instruments
Syringe with tip drawn out after warming the plastic in a Bunsen burner flame
Microelectrode amplifier (Axoclamp 2B, Axon Instruments)
Silver‐silver chloride wire or pellet (0.8 mm diameter × 20 mm Ag/AgCl electrode; World Precision Instruments, cat. no. EP08)
Mains interference filter (Digitimer Humbug; optional)
Low‐pass (<2 kHz) filters (Neurolog, Digitimer, UK)
Pulse train generator (Digitimer D4030 Programmer)
Isolated stimulator (Digitimer DS2)
Dissecting microscope
Light source for dissecting microscope (dark‐field condenser or flexible fiber‐optic light)
Recording display device (oscilloscope, or computer running WinWCP)
WinWCP software program (Strathclyde Electrophysiology Software; (http://spider.science.strath.ac.uk/sipbs/showPage.php?page=software_ses)
Data acquisition unit (CED micro 1401, Cambridge Electronic Design, http://www.ced.co.uk/)

Additional reagents and equipment for sacrifice of the mouse (Donovan and Brown, )

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Figures

Figure 1. Isolated tibial nerve/FDB muscle preparation and intracellular recordings of EPPs. (A ) Typical appearance of a satisfactory working dissection of the FDB muscle and its attached tibial nerve supply. The tendon of origin at the heel (top) and the three distal tendons are pinned to a Sylgard‐lined dish with minutien pins. (B ) Superimposed digital sweeps during nerve stimulation at about 1/sec from an approximate location in the muscle indicated in A. Dotted lines and arrows show properties of the EPP that are routinely measured, for instance, using WinWCP software. The recording was made after blocking muscle fiber action potentials using µ‐conotoxin GIIIB.

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Figure 2. Typical quantal analysis of EPPs and MEPPs. (A ) Slow scan of resting membrane potential in an FDB muscle fiber showing random, spontaneous MEPPs occurring with unpredictable intervals between events. (B ) Histogram of inter‐MEPP intervals, by frequency, from the data shown in A. (C ) average of about 30 MEPPs from A, on a fast time base, showing principle characteristics and double‐exponential curve fit to the decay time. (D ) MEPP amplitude frequency histogram of the individual records. (E ) Quantized fluctuations, including failures, recorded in an FDB muscle fiber from a preparation bathed in physiological saline with reduced Ca²⁺ and increased Mg²⁺ . (F ) EPP amplitude histogram from 30 successive EPPs elicited at 0.5 Hz from a preparation bathed in physiological saline with reduced Ca²⁺ and increased Mg² (different fiber from E). There were two ‘failures' in this series, indicating a mean quantal content of 2.71. The variance method indicated a mean quantal content of 3.11 and the direct method based on a mean MEPP amplitude of 0.4 mV gave 2.84 as the mean quantal content.

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Literature Cited

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