This method allows the recording of the force of twitch and tetanic contractions and action potentials in three types of motor units in the rat medial gastrocnemius muscle. The functional isolation of a single motor unit is induced by electrical stimulation of the axon.
This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in hindlimb muscles (such as the medial gastrocnemius, soleus, or plantaris muscle) in experimental rats. A crucial element of the method is the application of electrical stimuli delivered to a motor axon isolated from the ventral root. The stimuli may be delivered at constant or variable inter-pulse intervals. This method is suitable for experiments on animals at varying stages of maturity (young, adult or old). Moreover, this protocol can be used in experiments studying variability and plasticity of motor units evoked by a large spectrum of interventions. The results of these experiments may both augment basic knowledge in muscle physiology and be translated into practical applications. This procedure focuses on the surgical preparation for the recording and stimulation of MUs, with an emphasis on the necessary steps to achieve preparation stability and reproducibility of results.
Motor units (MUs) are the smallest functional units of skeletal muscles. Therefore, understanding their function, plasticity and contractile properties, as well as the mechanisms of their force regulation, is crucial for progress in muscle physiology. The basic contractile properties of MUs and the proportions of their physiological types have been documented for numerous muscles, predominantly the hindlimb muscles in experimental animals. However, both the plasticity of MU properties and the mechanisms of MU force regulation are still not fully understood.
The principle of the described method is extensive denervation of the hindlimb muscles except the investigated one and laminectomy at the lumbar vertebrae in order to prepare thin ventral rootlets, each one containing a single "functional" motor axon, stimulated electrically to record the force and action potential of the MU. Using the technique described in this paper, it is possible to isolate more than half of the MUs of the medial gastrocnemius muscle in a successful experiment. The rat medial gastrocnemius is composed of on average 52 MUs (females) or 57 MUs (males) of three physiological types: S (slow), FR (fast resistant) and FF (fast fatigable)1,2, and have variable contractile properties3. For experiments comparing mean values for MUs in the control and experimental groups, isolation and recording of 10-30 MUs for each of these groups are necessary. Critically, individual MUs may be accessible for stimulation for time periods exceeding one hour. Moreover, since this technique allows for recording both MU force and action potentials, this method is suitable for studying phenomena associated with force production, assessing the effect of fatigue, and observing the relationship between the force and action potentials.
Previous studies have confirmed that MU contractile properties are plastic and may be modulated by numerous interventions. Experiments using the technique described here have been performed on rat medial gastrocnemius4 or other hindlimb muscles of the rat5,6 as well as on cat muscles7, using a similar method of single MU isolation. Another series of experiments using trains of stimuli delivered at variable inter-pulse intervals provided observations concerning motor control processes, and the results in general turn attention to the history of stimulation, including considerable effects of a shift in time scale of even one stimulus, crucial for force production8,9.
MUs may also be studied using alternative methods. First, one method is direct stimulation of motoneurons. Burke used intracellular stimulation of motoneurons in cat medial gastrocnemius and soleus with glass microelectrodes used in parallel to determine the electrophysiological properties of these neurons1,10. Other methods have been proposed to study MUs in human muscles, which require considerably lower intervention. For all these methods, the stimulating and recordings electrodes are inserted into the muscle or nerve, and force is recorded from the finger or from the foot. The first of these methods was used to study MUs in the first dorsal interosseous muscle. For this muscle, contracting with a low force, in the electromyogram recorded with the needle electrode inserted into the muscle the action potentials of only one active motor unit were identified. Then the fragments of a muscle force recorded in parallel and following each action potential were averaged (spike-triggered averaging). This method enables extraction of the force of one motor unit from the muscle force recording11. However, the methodological weakness of this procedure is that no single twitch force but rather fragments of tetanic contractions were averaged. Human MUs may also be studied using the second method of intramuscular electrical microstimulation using an electrode inserted into the muscle12, which stimulates a fragment of an axonal tree, leading to activation of a single motor unit. The third method is microstimulation with an electrode inserted into the nerve. When the electrode activates only one motor axon in the nerve, only one motor unit contracts13. These last methods have some limitations, including stability and quality of the recording, ethical restrictions and access to the experimental material. This protocol has been extensively used in cats in the 70's and 80's14.
All procedures need to be approved by the local ethics committee and adhered to the European Union guidelines on animal care as well as the national law on the protection of animals.
NOTE: Each experimenter involved in this procedure must be trained in basic surgical procedures and has to obtain a valid license for performing animal experiments.
1. Anesthesia
2. Surgery
3. Preparation for the recording and stimulation
4. Motor unit recordings
5. Electronic apparatus
NOTE: The custom-made computer program controls the stimulator, providing the possibility to create variable patterns of stimulation, including those indicated in step 4.4. The program cooperates with the analog-to-digital converter (at least 10 kHz for the MUAP and force recordings).
Parameters of motor unit contractions and action potentials can be calculated on the basis of recordings when stable conditions of recordings are ensured. Figure 1 presents a representative recording of the single twitch of a fast MU. The upper trace shows the motor unit action potential. The delay between stimulus delivery and onset of the motor unit action potential is due to conduction time from ventral root to muscle. Figure 2 shows a representative recording of the unfused tetanus force of a fast MU and a train of motor unit action potentials.
Figure 1: A representative recording of the single twitch of a fast MU. Over the force track, there is motor unit action potential. Please click here to view a larger version of this figure.
Figure 2: A representative recording of the unfused tetanus force of a fast MU (middle recording), a train of motor unit action potentials (upper recording) and a time position of a train of applied stimuli (below). Please click here to view a larger version of this figure.
If performed correctly by experienced scientists, the surgical component of the described protocol should be completed within approximately two hours. One should take particular care to maintain stable physiological conditions of the animal during the surgery, particularly body temperature and depth of anesthesia, which should be systematically controlled by assessing pinna and withdrawal reflexes. Following the surgery, it should be possible to maintain stable recording conditions for at least six hours.
The crucial experimental procedure begins with the splitting of the ventral root into very thin filaments leading to isolation of a single motor axon to the studied muscle. In fact, the thin filaments of ventral roots contain groups of axons innervating different muscles of the hindlimb; however, because all muscles except the studied one are denervated, when the stimulated bundle of axons contains only one axon to the studied medial gastrocnemius it is possible to evoke the single MU contraction only in this studied muscle. Following successful identification of the evoked activity as single MU contraction, it is possible to record a set of force recordings (single twitch, the unfused tetanus, the fatigue test) crucial for a classification of MU as one of three physiological types. The advantage of this technique is the ability to record up to 30 units in one experiment; additionally, MUs can be immediately classified as fast or slow types on the basis of “sag” presence1,3. Furthermore, MUs can be classified as fast-fatigable or fast-resistant with very high accuracy based on a profile of the unfused tetanic contraction force recording16. This last method may be used when the classical fatigue test cannot be performed. It is also worth noting that fast/slow MU classification can be also done with a 20 Hz index17.
The proposed stimulation protocol (step 4.4) may be adapted to needs of the study. This particular set of stimulations enable to record twitch (to calculate basic twitch parameters including the twitch force, contraction as well as relaxation time), the maximum tetanus (therefore it is possible to calculate the twitch-to-tetanus ratio), unfused tetanic contractions at a set of stimulation frequencies (to classify an MU as slow or fast basing on sag presence or 20 Hz index, as well as to calculate the force-frequency curve) and the fatigue test (necessary to calculate the fatigue index). The fatigue index calculation is a basic method to classify MUs as fatigable or resistant. This method is open to being modified to produce various stimulation patterns; however, a possible limitation is the computer program that generates the time distribution of stimuli delivered to the axon. Moreover, some additional modifications may be introduced to answer specific research questions, such as several stimulating electrodes to activate several MUs in parallel18, an additional laser sensor to record a mechanomyogram (MMG) from the muscle surface19 or a recording electrode on a nerve branch to the muscle to calculate nerve conduction velocity20.
However, it is important to be aware of the limitations and challenges of this procedure. First, a considerable part of the experimental setup is custom-made (i.e., clamps for the limb and the vertebrae segments, a plate for ventral roots and electrodes). The experimental setup includes a solid metal table with plate (thickness 30 mm) for all supporting metal bars (necessary for animal immobilization and the force transducer) to enable stable conditions for the isometric force recording. The application of this method also requires both extensive training in surgery as well as preparation of a complex experimental setup including an electronic apparatus and a computer program.
The authors have nothing to disclose.
This work was supported by Polish National Research Centre grant 2018/31/B/NZ7/01028.
Force transducer | custom-made | ||
Forceps | Fine Science Tools | No. 11255-20 | Dumont #55 with extra light and fine shanks |
Forceps | Fine Science Tools | No. 11150-10 | Extra Fine Greafe Forceps |
Forceps | Fine Science Tools | No. 11026-15 | Special cupped pattern for superior grip |
Forceps | Fine Science Tools | No. 11023-10 | Slim 1×2 teeth |
Forceps | Fine Science Tools | No. 11251-20 | Dumont #5 |
Hemostats | Fine Science Tools | No. 13003-10 | Hartman |
Isolation Unit | Grass Instruments | S1U5A | |
Low Noise Bioamplifer | World Precision Instruments | Order code 74030 | |
Needle holders | Fine Science Tools | No. 12503-15 | With tungsten carbide jaws |
Rongeurs | Fine Science Tools | No. 16021-14 | Friedman-Pearson |
Scissors | Fine Science Tools | No. 14101-14 | Straight sharp/blunt with large finger loops |
Scissors | Fine Science Tools | No. 14075-11 | Curved blunt/blunt |
Scissors | Fine Science Tools | No. 14084-08 | Extra fine bonn |
Scissors | Fine Science Tools | No. 15000-00 | Straight, ideal for cutting nerves |
Stimulator | Grass Instruments | S88 | Dual Output Square Pulse Stimulator |