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 SOD1G93A 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 way1,2, 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 reduce neurotransmission signaling functionality.
The comparison of muscle contractile response elicited by nerve stimulation and the response of the same muscle evoked by direct stimulation of its membrane has been proposed as an indirect measurement of NMJ functionality. Indeed, since membrane stimulation by-passes neurotransmission signaling, any differences in the two contractile responses may be ascribed to changes in the NMJ. This approach has been extensively proposed for rats3,4,5,6,7, and also used to gather information on mouse models8,9,10,11,12.
Here, we describe in detail a procedure to excise and test two muscle-nerve preparations, i. e. the soleus-sciatic and diaphragm-phrenic preparations. Using a custom-made software, we designed a continuous testing protocol that combines the measurement of several parameters that characterize both NMJ and muscle functionality, thereby yielding a comprehensive evaluation of NMJ damage separately from that of muscle. In particular, the protocol measures the twitch force, the muscle kinetics, the force-frequency curve for direct and nerve stimulations, the neurotransmission failure13 for both a firing and the tetanic frequencies, and the intratetanic fatigue7.
Protocol
Representative Results
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
Work in the authors’ laboratory was supported by Fondazione Roma and Telethon (grant no. GGP14066).
Materials
| 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 |
Referenzen
- Dadon-Nachum, M., Melamed, E., Offen, D. The "dying-back" phenomenon of motor neurons in ALS. J Mol Neurosci. 43 (3), 470-477 (2011).
- Dobrowolny, G., et al. Skeletal Muscle Is a Primary Target of SOD1G93A-Mediated Toxicity. Cell Metab. 8 (5), 425-436 (2008).
- Mantilla, C. B., Zhan, W. Z., Sieck, G. C. Neurotrophins improve neuromuscular transmission in the adult rat diaphragm. Muscle Nerve. 29 (3), 381-386 (2004).
- Prakash, Y. S., Miyata, H., Zhan, W. Z., Sieck, G. C. Inactivity-induced remodeling of neuromuscular junctions in rat diaphragmatic muscle. Muscle Nerve. 22 (3), 307-319 (1999).
- Sieck, D. C., Zhan, W. Z., Fang, Y. H., Ermilov, L. G., Sieck, G. C., Mantilla, C. B. Structure-activity relationships in rodent diaphragm muscle fibers vs. neuromuscular junctions. Respir Physiol Neurobiol. 180 (1), 88-96 (2012).
- Van Lunteren, E., Moyer, M. Effects of DAP on diaphragm force and fatigue, including fatigue due to neurotransmission failure. J Appl Physiol. 81 (5), 2214-2220 (1996).
- Van Lunteren, E., Moyer, M., Kaminski, H. J. Adverse effects of myasthenia gravis on rat phrenic diaphragm contractile performance. J Appl Physiol. 97 (3), 895-901 (2004).
- Lee, Y., Mikesh, M., Smith, I., Rimer, M., Thompson, W. Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular development even in the absence of failure in neuromuscular transmission or loss of motor neurons. Dev Biol. 356 (2), 432-444 (2011).
- Personius, K. E., Sawyer, R. P. Variability and failure of neurotransmission in the diaphragm of mdx mice. Neuromuscular Disord. 16 (3), 168-177 (2006).
- Röder, I. V., Petersen, Y., Choi, K. R., Witzemann, V., Hammer, J. A., Rudolf, R. Role of myosin Va in the plasticity of the vertebrate neuromuscular junction in vivo. PLoS ONE. 3 (12), e3871 (2008).
- Chevessier, F., et al. A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions. Hum Mol Genet. 17 (22), 3577-3595 (2008).
- Farchi, N., Soreq, H., Hochner, B. Chronic acetylcholinesterase overexpression induces multilevelled aberrations in mouse neuromuscular physiology. J Physiol. 546 (1), 165-173 (2002).
- Kuei, J. H., Shadmehr, R., Sieck, G. C. Relative contribution of neurotransmission failure to diaphragm fatigue. J Appl Physiol (Bethesda, Md: 1985). 68 (1), 174-180 (1990).
- Del Prete, Z., Musarò, A., Rizzuto, E. Measuring mechanical properties, including isotonic fatigue, of fast and slow MLC/mIgf-1 transgenic skeletal muscle. Ann Biomed Eng. 36 (7), 1281-1290 (2008).
- Lynch, G. S., Hinkle, R. T., Chamberlain, J. S., Brooks, S. V., Faulkner, J. A. Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old. J Physiol. 535 (2), 591-600 (2001).
- Brooks, S. V., Faulkner, J. A. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol. 404, 71-82 (1988).
- Lynch, G. S., Hinkle, R. T., Faulkner, J. A. Force and power output of diaphragm muscle strips from mdx and control mice after clenbuterol treatment. Neuromuscul Disord. 11 (2), 192-196 (2001).
- Rizzuto, E., Pisu, S., Musar, A., Del Prete, Z. Measuring Neuromuscular Junction Functionality in the SOD1 G93A Animal Model of Amyotrophic Lateral Sclerosis. Ann Biomed Eng . 43, (2015).
- Soliani, L. . Manuale di statistica per la ricerca e la professione statistica univariata e bivariata parametrica e non-parametrica per le discipline ambientali e biologiche. , (2004).
- Gurney, M. E., Pu, H., et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science (New York, N.Y). 264 (5166), 1772-1775 (1994).
