Measuring Neuromuscular Junction Functionality

Summary

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Abstract

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.

Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1^G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

Introduction

The neuromuscular junction (NMJ) is a chemical synapse formed by the connection between the motor endplate of the muscle fiber and the motor neuron axon terminal. The NMJ has been shown to play a crucial role when communication between muscle and nerve is impaired, as occurs in aging or amyotrophic lateral sclerosis (ALS). As muscle and nerve communicate in a bidirectional way^1,2, being able to measure NMJ defects separately from muscle defects may provide new insights into their physiopathological interplay. Indeed, this functional evaluation may help to assess whether morphological or biochemical alterations....

Protocol

All the animal experiments were approved by the ethics committee of Sapienza University of Rome-Unit of Histology and Medical Embryology and were performed in accordance with the current version of the Italian Law on the Protection of Animals.

1. Experimental set-up

1. Set-up the experimental system composed of 1 actuator/transducer, 2 stimulators, 1 in-vitro muscle apparatus, 1 preparatory tissue bath, 1 suction electrode, 1 digital oscilloscope, 1 stereomicroscope, 1 cold l.......

Discussion

The experimental protocol described above provides an ideal way of measuring and discriminating any functional alterations that have occurred directly in the muscle or indirectly at the neuromuscular junction level. Since this technique is based on an indirect measurement of NMJ functionality, it cannot be used to establish if any defect is related to morphological changes or to biochemical changes. By contrast, it does provide an effective way of determining whether any morphological or biochemical alterations have redu.......

Materials

Name	Company	Catalog Number	Comments
Dual-Mode Lever System	Aurora Scientific Inc.	300B	actuator/transducer
High-Power Bi-Phase Stimulator	Aurora Scientific Inc.	701B	pulse stimulator (nerve)
High-Power Bi-Phase Stimulator	Aurora Scientific Inc.	701C	pulse stimulator (muscle)
In vitro Muscle Apparatus	Aurora Scientific Inc.	800A
Preparatory tissue bath	Radnoti	158400
Monopolar Suction Electrode	A-M Systems	573000	with a home-made reference
Oscilloscope	Tektronix	TDS2014
Stereomicroscope	Nikon	SMZ 800
Cold light illuminator	Photonic Optics	PL 3000
Acquisition board	National Instruments	NI PCIe-6353
Connector block	National Instruments	NI 2110
Personal computer	AMD Phenom II x4 970		Processor 3.50 Ghz with Windows 7
LabView 2012 software	National Instruments
Krebs-Ringer Bicarbonate Buffer	Sigma-Aldrich	K4002	physiological buffer
Sodium bicarbonate	Sigma-Aldrich	S5761
Calcium chloride CaCl2	Sigma-Aldrich	C4901	anhydrous, powder, ≥97%
Potassium dihydrogen phosphate KH₂PO₄	AnalaR	7778-77-0
Magnesium sulphate MgSO₄	AnalaR	7487-88-9
Buffer HEPES	Sigma-Aldrich	H3375	≥99.5% (titration)
Dishes 60mm x 15mm	Falcon	353004	Polystyrene
Silicone	Sylgard	184 Silicone	Elastomer Kit  0.5Kg.
Thermostat	Dennerle	DigitalDuomat 1200
Pump	Newa Mini	MN 606	for aquarium
Heat resistance Thermocable	Lucky Reptile	61403-1	50/60Hz 50W
Bucket	any 10 liters		Polypropylene
O2 + 5%CO2	siad		Mix gas
#5 Forceps	Fine Science Tools	11252-20	2 items
Spring Scissors - 8 mm Blades	Fine Science Tools	15024-10	nerve excision
Sharp Scissors	Fine Science Tools	14059-11	muscle removal
Delicate Scissors	Wagner	02.06.32	external of the animal
Student Scalpel Handle #3	Fine Science Tools	91003-12
Scalpel Blades #10	Fine Science Tools	10010-00
Scalpel Blades #11	Fine Science Tools	10011-00
nylon wire Ø0.16 mm	any